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Recombinant yersinia pestis yspl protein principally produces N-(3-oxooctanoyl)-L-homoserine lactone during quorum sensing signal generation

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Title:
Recombinant yersinia pestis yspl protein principally produces N-(3-oxooctanoyl)-L-homoserine lactone during quorum sensing signal generation
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Kirwan, J. Paul
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English
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xii, 88 leaves : illustrations ; 28 cm

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Subjects / Keywords:
Yersinia pestis ( lcsh )
Lactones ( lcsh )
Genetic regulation ( lcsh )
Cellular signal transduction ( lcsh )
Cellular signal transduction ( fast )
Genetic regulation ( fast )
Lactones ( fast )
Yersinia pestis ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 84-88).
Thesis:
Department of Chemistry
Statement of Responsibility:
by J. Paul Kirwan.

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|University of Colorado Denver
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ocm57507592
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Full Text
RECOMBINANT Yersiniapestis YspI PROTEIN
PRINCIPALLY PRODUCES
N-(3-oxooctanoyl)-L-homoserine lactone
DURING
QUORUM SENSING SIGNAL GENERATION
by
J. Paul Kirwan
B.S., Benedictine College, 1999
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Chemistry


This thesis for the Master of Science
Degree by
J. Paul Kirwan
has been approved
by

Date


Kirwan, J. Paul (M.S., Chemistry)
Recombinant Yersinia pest is Yspl Protein Principally Produces
N-(3-oxooctanoyl)-L-homoserine lactone During Quorum Sensing Signal Generation
Thesis composition directed by Professor Douglas F. Dyckes
Thesis research directed by Associate Professor Mair E. A. Churchill
(Department of Pharmacology, University of Colorado Health Science Center)
ABSTRACT
The Yersinia pestis Yspl protein is a member of the family of N-acyl-
homoserine-L-lactone synthases (AHL-synthases), the signal producing machinery
for N-acylhomoserine lactone (AHL) mediated gene regulation in gram negative
bacteria. Known as quorum sensing, this well established phenomenon of bacterial
cell-to-cell signaling, is gradually redefining our understanding of gene regulation in
bacteria. In many cases, quorum sensing coordinates the transition from individual
cell growth to collective virulence. The sub-cloning of a pET28a-yspl DNA
construct, followed by transformation into E. coli, allowed overexpression of the Yspl
protein and provided a means for studying AHL signal generation in Yersinia pestis,
the organism responsible for the plague. Assays of culture supernatant extracts of E.
t-o//(BL21 )-pET28a-yspl, confirmed the activity of recombinant Yspl. Further
analysis of these extracts by LC-Tandem Mass Spectrometry revealed a profile of
AHL signals, in which N-(3-oxooctanoyl)-L-homoserine lactone was the most
abundantly produced. Identification of the principal AHL signal produced in Yersinia
m


pestis quorum sensing is a step toward determining the role of the mechanism in the
organism's virulence. A thorough understanding of quorum sensing in pathogenic
bacterial species could lead to the design of inhibitory drugs, and the eradication of
diseases like the plague.
This abstract accurately represents the content of the candidate's thesis. I recommend
its publication.
Signed
IV


DEDICATION
To Amy, for giving me true happiness as a person, and appreciating the enjoyment 1
get from being a scientist
To our son Elliott
To my parents, for giving me the opportunity to succeed academically, for teaching
me the merit in hard work, and for encouraging me to believe in my abilities


ACKNOWLEDGEMENT
1 thank my research advisor Dr. Mair Churchill, for giving me the opportunity to
study in her laboratory and work in an environment of advanced learning. The
experience 1 gained was invaluable and instrumental in my growth as a scientist.
Thanks to my committee chairman Dr. Douglas Dyckes, for being open to research
opportunities outside the CU Denver Department of Chemistry, and for assisting me
with departmental research and thesis requirements.
Thanks to Dr. Robert Damrauer for serving on my committee.
I would like to thank current and former members of the Churchill Lab: Linda Farb,
Susan Fouts, Ty Gould, Emily Hoagland, Janet Klass, Purnima Mungalachetty, Bill
Watson, and Don Zapien, for their helpful suggestions during the course of my study.
Thanks also to Matt Cheever, Schoen Kruse, and Erik MacLaren for their input on
various experiments.
Special thanks to:
Scott Beardon, Herbert Schweizer, and colleagues, for providing me with the
pCR2.1-yspl construct, without which this study would not have been possible.
Ty Gould, for his advice and assistance throughout my time in the Churchill Lab, and
particularly with the implementation of the LC-MS experiments performed for this
study.


CONTENTS
Figures........................................................................x
Tables.......................................................................xii
Chapter
1. Quorum Sensing..............................................................1
1.1 The Components of Quorum Sensing Systems..................................2
1.2 The Model Quorum Sensing System...........................................4
1.3 Catalytic Function of the AHL-Synthase Esal...............................7
1.4 Hierarchical Regulation in Pseudomonas aeruginosa.........................11
1.5 Quorum Sensing in the Yersinia Genus......................................13
1.6 Objectives for the Study of Yspl..........................................18
2. Sub-Cloning of a pET28a-yspl DNA Construct.................................20
2.1 Introduction..............................................................20
2.2 Results..................................................................22
2.2.1. Preparation of pET28a plasmid and yspl Gene Insert.....................22
2.2.2 Ligation, Transformation, and Sequencing of E. eoli (DH5u)-pET28a-yspl..27
2.2.3 Yspl Protein Purification...............................................34
2.3 Discussion..............................................................40
3. Demonstration of Recombinant Yspl Activity
42


3.1 Introduction
42
3.2 Results.....................................................................46
3.3 Discussion..................................................................49
4. AHL Signal Profile for Yspl.................................................52
4.1 Introduction................................................................52
4.2 Results.....................................................................53
4.3 Discussion..................................................................62
5. Conclusions and Future Studies..............................................66
5.1 Conclusions..................................................................66
5.2 Future Studies..............................................................68
6. Methods......................................................................69
6.1 Molecular Cloning...........................................................69
6.1.1 Plasmid DNA Purification by Miniprcp.......................................69
6.1.2 Phenol Extraction Ethanol Precipitation of Plasmid DNA...................70
6.1.3 Plasmid DNA Purification by G50 Spin Column..............................71
6.1.4 Ultraviolet/Visible Spectroscopy..........................................71
6.1.5 Restriction Endonuclease Digestion of pET28a..............................72
6.1.6 Restriction Endonuclease Digestion of pCR2.1-yspl.........................73
6.1.7 Gel Extraction of yspl DNA Insert........................................73
6.1.8 Phosphatase and DNA Ligase Reactions......................................74


6.1.9 Transformation by Electroporation........................................75
6.1.10 Polymerase Chain Reaction (PCR)........................................76
6.1.11 Transformation by Heat Shock...........................................77
6.2 Protein Purification......................................................78
6.2.1 Protein Expression.......................................................78
6.2.2 Cell Lysis and Ni-NTA Affinity Chromatography............................78
6.2.3 Size Exclusion Chromatography............................................79
6.3 Yspl Activity............................................................80
6.3.1 AHL Extraction...........................................................80
6.3.2 Activity Assay of AHL Extract............................................81
6.4 AHL Profile...............................................................82
6.4.1 LC-MS Sample Prep: Solid Phase Extraction................................82
6.4.2 LC-MS Analysis...........................................................83
References.....................................................................84
IX


FIGURES
Figure
1.1 AHL Quorum Sensing Molecules............................................3
1.2 Sequence and Structural Alignment of Selected AHL Synthases.............6
1.3 Proposed Mechanism of Acyl Transfer.....................................9
1.4 Variable Temperature TLC Overlay Data..................................16
2.1 Ndel and Hindi 11 Digestion of pET28a DNA Plasmid......................24
2.2 Ndel and Hindi 11 Digestion of pCR2.1-yspl DNA Construct...............26
2.3 PCR Screen of Plasmid Samples 1-8......................................30
2.4 PCR Screen of Plasmid Samples 9-16....................................31
2.5 YspI Sequence Data....................................................33
2.6 Elution of Protein from a Ni-NTA Column...............................35
2.7 SDS-PAGE of Eluted Fractions from a Ni-NTA Column.....................36
2.8 Size Exclusion Chromatogram of Fractions Eluted from Ni-NTA...........38
2.9 SDS-PAGE of Fractions from Size Exclusion Chromatography..............39
3.1 Recombinant Ytbl and YpsI TLC Overlay Data.............................44
3.2 Results of Miller Assay to Determine Yspl Activity.....................48
4.1 Representative Chromatogram of AHL Extract.............................55
4.2 Precursor Ion Mass Spectrum for LC Peak at 10.6 Minutes...............56
x


4.3 Precursor Ion Mass Spectrum for LC Peak at 21.8 Minutes....................57
4.4 Precursor Ion Mass Spectrum for LC Peak at 27.9 Minutes....................58
4.5 Precursor Ion Mass Spectrum for LC Peak at 28.6 Minutes....................59
xi


TABLES
Table
2.1 Results of the Transformation of pET28a-yspl into E. coli (DH5a) Cells..28
4.1 Peak Area Data..........................................................61
xii


1. Quorum Sensing
Over the past thirty years, scientists have made an increasing effort to
understand the ability of microorganisms to respond to environmental stimuli.
Mechanisms of intercellular communication have been identified in several bacterial
species. Now known as quorum sensing, the ability to communicate enables bacteria
to detect the size of their local population through the production of and response to
low molecular weight signal molecules (1,2). The concentration of signal molecules
in the surrounding environment is proportional to the population density of
neighboring microbes. As the population density increases, a threshold signal
concentration is achieved, stimulating transcription at specific gene opcrons in
bacterial cells throughout the quorum (1,2). Depending on the species, the activated
genes range from those which code for enzymes involved in bioluminescence to those
which express virulence factors and initiate pathogenic bacterial lifestyles (1,2).
Regardless of the resulting phenotype, coordinated expression of genes which
enhance survival is advantageous, in that a greater impact may be made by a
multitude of cells than by an individual (2). Researchers endeavor to understand the
mechanisms of cell-to-cell communication in bacteria, particularly for those species
which utilize the phenomenon to cause disease.


1.1 The Components of Quorum Sensing Systems
Quorum sensing systems rely on two proteins for proper function; one that
produces the signal and another that serves as the signal receptor. In Gram-negative
bacteria, the signal molecules are predominantly N-acyl-homoserine lactones (AHLs)
(1,2). Asa result, the quorum sensing enzyme that produces the AHL signal is
commonly referred to as an AHL-synthase. AHL-synthases catalyze the synthesis of
AHLs from the substrates S-adenosylmethionine (SAM) and acyl-acyl carrier protein
(acyl-ACP) (3, 4). SAM, a product of amino acid metabolism that typically functions
as a biological methylating agent, is converted into the lactone ring of the signal
molecule (5). Acyl-ACP, more commonly a precursor for fatty acid biosynthesis, is
acquired from cellular pools and provides acyl chain variation for the AHL structure
(6). The N-acyl chain of the resulting AHL signal may be short or long, saturated or
unsaturated, substituted or unsubstituted at the C3 position, or a combination of these
(Figure 1.1) (1,2). Most AHL signals freely diffuse into and out of the neighboring
bacterial cells of a growing quorum (1,2). The signal binds to the receptor protein
inside these cells, creating a complex which behaves as a transcription factor,
triggering transcription of specific gene operons (1,2). Among the genes activated is
the gene encoding AHL-synthase.
?


Figure 1.1 AHL Quorum Sensing Molecules.
AHL structures, produced by various AHL-synthases, display acyl chains different in
length, saturation, and substitution at the C3 position. (A) N-butanoyl-L-homoserine
lactone (C4-AHL) (B) N-(3-hydroxybutanoyl)-L-homoserine lactone (3-hydroxy-C4-
AHL) (C) N-hexanoyl-L-homoserine lactone (C6-AHL) (D) N-(3-oxohexanoyl-L-
homoserine lactone (3-oxo-C6-AHL) (E) N-octanoyl-L-homoserine lactone (C8-
AHL) (F) N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-AHL) (G) N-(3-
hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone (3-hydroxy-7-ene-C 14-AHL)
(H) N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-AHL).
Adapted from Whitehead, et al., 2001.
3


Consequently, as AHL signal coneentration increases in the environment of a
growing quorum and binds more receptor proteins, expression of AHL-synthase also
increases, leading to even more signal production. The identification of this positive
feedback characterizes the process as one of autoinduction (1,2). AHL-synthases,
then, are frequently referred to as autoinducer synthases, since they produce the
autoinducer (AHL signal) which further enhances the response. Since the receptor
proteins regulate the response to AHL signal, they are typically called response
regulators. Through the coordinated function of AHL-synthase and response
regulator, quorum sensing provides many bacterial species w ith efficient energy
management, ensuring that specialized gene products are only expressed when they
can achieve maximum effect.
1.2 The Model Quorum Sensing System
Quorum sensing was first identified in Vibrio fisheri, a seawater microbe
known to luminesce at high cell densities. It is not uncommon for this organism to
form symbiotic relationships w ith a variety of marine life (1,2). Included in the
bioluminescence gene cluster of V. fisheri are the genes luxR and luxI. The products
of these genes, LuxR and Luxl, were found to regulate luminescence in V. fisheri as
part of a quorum sensing system (1,2). LuxR and Luxl are considered the archetype
response regulator and AHL-synthase, respectively. Accordingly, response regulators
and AHL-synthases are also known as R proteins and 1 proteins. The discovery of the
mechanism of quorum sensing was initially regarded as little more than an unusual
4


means of gene regulation employed by a peculiar sea dwelling bacterial species.
However, the recent efforts of researchers have established its significance, as several
bacterial species are now known to use quorum sensing to regulate a variety of genes,
many of which initiate pathogenicity (1,2). These species have been identified by
sequence comparisons of their suspected quorum sensing proteins with LuxR or Luxl
(Figure 1.2). Proteins that reflect homology with established critical sequence
residues in LuxR or Luxl are accepted as homologues and designated as part of a
quorum sensing system for that bacterial species. The discovery of numerous quorum
sensing systems, combined with their common involvement in virulence gene
expression, has generated an influx of interest into the structural and functional
characteristics of quorum sensing proteins. The phenomenon depends entirely upon
the proper function of response regulator and AHL-synthase. Without AHL signal
production, initiation of the mechanism never occurs. Without AHL signal detection,
critical genes arc not activated. Both circumstances impede or prevent proper
virulence factor expression in pathogenic quorum sensing species, limiting or
eliminating their ability to cause disease. To this end, the potential reward for the
development of inhibitory drugs is tremendous and certainly warrants investigation.
5


LuxI
Era!
Lasl
Rhii
Yp el
YpsT
YspX
Esal :
Lasl:
LUX I
Esal
Lasl
Rhii
YpeT
YpsI
YspI
Esal:
Lasl :
Figure 1.2. Sequence and Structural Alignment of Selected AHL-Synthases. Sequence homology is shown
between various AHL-synthases and the archetype LuxI. Grey shaded regions are conserved sequence blocks
that constitute an enzyme's "signature sequence." Residues are colored red to indicate acidic character, blue for
basic, and orange for hydrophobic. Residues shaded in solid color are absolutely conserved among AHL-
synthases. Boxed residues are homologous among AHL-synthases. The topologies of Esal and Lasl, which are
compared to each other at the bottom of the alignment, provide insight into the relationship between sequence
position and secondary structure for the displayed AHL-synthases. This alignment does not include all known
AHL-synthase sequences.
AHL-synthase identification: LuxI (Vibrio fisheri), Esal (Pantoea stewartii), Lasl (Pseudomonas aeruginosa),
Rhll (Pseudomonas aeruginosa), Ypel (Yersiniapestis), YpsI (Yersiniapseudotuberculosis), and YspI (Yersinia
pest is).
Adapted from Watson, et al., 2002.
Lasl alignment and topology constructed by Ty Gould prior to publication.
MT'IMIKKfSDFLAIPSEEYKGILS
mleledvsyeelqttrseelyk:
MIVQIGRREEFDKXLLGEMHK
MIELLSESI.EGLSAAMIAELGR
ML K VFNVNFDRMSENKLDEI FT:
MLKVFNVNFLRMSENK LDEIFT:
MLFTFDVRYDEI TDIFSEDLYK
il(WENNLE'S--j
EtylCSQGKES---
teYIDEMEI---
tVETSRVRDQ:
IDGKES---
'CIDGKES---
E^CSNGME-----F
,CLDTE- -tfvfc!
CEGE----V
QEDTPEA^IV
MSRQG-
;tiodt- -
TI DDTir^.
I YOG*' -lil
LLPTTGDY
KSVKPt5LLGvVSAPKDPNI\
SLDRPty ijTHTFQHCFSDV -TLPAYG
lILDTTGPYl
LPTTDAi
EMQYPTtfcf
IELHLFI'
ITFPELLHOKKAPCSPH lb l.
DVFAYLCSETP- PSDPSW
ipfapyfrdt.d-lpidgf:
L3PFAPYFRDLD-LPIDGFI
1TFNALFDDVA LPKRGY 2
102
AVGXNS5KINNSASEI1MK LFEAIYXHAVSQGITKYV1 yTSTAfe^RF:
rVTDKARARALLGEMYPlSQVLFLAMVMWAQIWAYGNIYl EVS RAW/KILT
AINEGQKGSLGFSDCTLE- -AMRALARYSLQNDIQTLVI \KTVGWpKMMI
AASAADD------PQLAMK IFWSSLCCAWYLGASSWJft ^TTaI^.RYFVH
FVFKALARDKLGNf'JGSI SAILFLSMVNYARNRGYXGI h'l vjvSRCffTILK
FVEKALARDKLGNtJGSLSA ILFLSMVNYARNCGYKGIL1 ^SEGiirT2
FVDKTRAKLLFGL'HYPISYLEFLSIINYSRHNGYTGIYTlivSRAki.TILK
IKVPCHRIGDKEIKVLGDTKSVVLSMPINEQFKKAVLN
:KVIKEAFLTEXEP:YLLTLPAGQDDKC-CLGGpVrVSRTGCPPVAL^TTWPLTLFV
AGLDVSRFGPIU.KIGIERAVALRIELNAKTQTALYGGVLVEQRLAVS
VILQRLG P PQKVKGETLVAISF P A Y^ERGLEML-L-R YHPEWLOCiVP LSMAV
KGbAiilTVINQGESEKNEr/IYLLKLSIDSNSOQQLIPKigRVHNIDTHTLASWFLVYPSMTK
SGWGITVINQGESEKNEVTYLLHLSIDSNSwQQLIRKIQRVHKIDTHTLESWPLVVPSMTK
XGWO'/EVIKEAKITEKEP.IYLLHLPIDRDNQARLLLQVNQRLODPCSVLSTWPISLFVMPESA
3*>#
a5
-^ao-


1.3 Catalytic Function of the AHL-synthase Esal
Although several pathogenic bacterial species utilize quorum sensing, a
relatively small number of them have been studied. Among the more recently studied
species, Pantoea stewartii subspecies stewartii is the pathogen responsible for
Stewarts wilt in sweet corn and maize (7, 8). Stewart's wilt results from the
production of an extracellular polysaccharide (EPS) virulence factor (7, 8). One of
the functions of EPS is to provide bacterial cells w ith protection from host defense
responses (9). Transcription of the genes required for EPS biosynthesis is triggered by
N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-AHL), the product of the AHL-
synthase Esal (10, 11). As previously described, a complex of AHL signal and
response regulator usually behaves as a transcriptional activator for specific gene
operons. However, in the case of P. s. subsp. stewartii, the response regulator EsaR
acts as a transcriptional repressor rather than an activator (12). In order to be
activated, genes repressed by EsaR binding at their promoter site require a threshold
concentration of 3-oxo-C6-AHL, which binds to EsaR, interrupts its binding to DNA,
and derepresses the operon. This functional difference implies a structural
uniqueness for EsaR, in that it must assume a favorable DNA-binding conformation
in the absence of 3-oxo-C6-AHL and an unfavorable conformation when bound to the
signal (12). LuxR homologues typically behave in the opposite manner. The unique
7


function of EsaR demonstrates the efficiency of quorum sensing. P. s. subsp.
stewartii more effectively infects its host if EPS biosynthesis is delayed rather than
constitutively expressed, a feature illustrated by the avirulence of EsaR mutant strains
of P. s. subsp. stewartii (12). The systematic regulation portrayed, underscores the
significance of quorum sensing to the life cycle of disease causing bacteria.
Appropriate genes must be expressed at the proper time, not only to aid, but to avoid
hindering the mechanism of pathogenesis (12).
An increasing effort has been made to obtain more detailed information about
specific quorum sensing systems in order to understand the phenomenon more
completely. In a recent study of Esal, structural data provided a powerful foundation
for the explanation of protein function. The study reported the crystal structure of
Esal and the recognition that its phosphopantetheine binding fold is structurally
related to the catalytic fold of the N-acetyltransferase family of enzymes (13).
Modeling of the Esal-phosphopantetheine complex based on this comparison, along
w ith the mutagenesis of key amino acid residues, provided a substantial insight into
the mechanism of AHL synthesis and a basis for substrate specificity (13). The
model supported a proposed mechanism in which Ser99 or Glu97 stabilizes a
catalytically active FfO molecule that deprotonates the SAM amine group, enabling
nucleophilic attack of the Cl carbonyl carbon of acyl-ACP (Figure 1.3), and sets the
stage for internal lactonization via the carboxylate oxygen of SAM (13).
8


Adoo sC

Ad*nolna
Adanotln*
V^s
Ad#no*li
NH ---- HM -----------* NH H6
V/Ys|/Ss''/ s>^
n n r\ C"i n r.
O .0
f^H
JL
o \0
M 'M
JL. ^*kv
o o
/v
u H
JL ^
Figure 1.3 Proposed Mechanism of Acyl Transfer
A) Esal acylation cleft with relevant residues shown in grey, a modeled phosphopantetheine group in cyan,
and ordered water molecules depicted as red spheres. B) Proposed N-acylation reaction is catalyzed by
nucleophilic attack on the Cl carbonyl carbon of acyl-ACP by the amine lone pair electrons of SAM made
available after proton abstraction by a water molecule. The resulting hydronium ion (not shown) is stabilized
by Glu97 or Ser99.
Watson, et al., 2002 (13)


Site directed mutagenesis, performed at the Ser 99 and Glu97 residues, resulted in
little or no AHL production according to measurements obtained from two TLC
overlay bioassays (13, 14, 15). The bioassays confirmed the contribution of residues
designated as critical by the Esal-acyl-phosphopantetheine model and strongly
supported the predictions of their mechanistic role. The model also identified a
region in the active site consistent with an acyl chain binding pocket which would
provide specificity for the synthesis of a C3 substituted AHL (13). Mutations were
made in this region to determine its contribution to enzyme activity. The mutation
F123M, w hich would increase the size of the acyl chain binding pocket, made little
impact on enzyme activity or substrate specificity (13). The mutation T140V, which
would decrease the acyl chain binding pocket, resulted in a mutant with limited
function (13). But, a T140A mutation yielded an active enzyme which produced
more unsubstituted AHL signal than the wild type protein. This evidence led to the
conclusion that the identity of the residue at position 140 affects the recognition of C3
substituted acyl-ACP substrate. More specifically, the results suggest that AHL-
synthases, having a threonine at residue 140, prefer acyl-ACP oxidized at the C3
position (13). In support of the hypothesis, Esal, Luxl, and LasI have a conserved
threonine at residue 140 and preferentially react with 3-oxo-acyl-ACPs to produce 3-
oxo-AHLs (13). The detailed study of the Esal active site, described in Watson et ah,
resulted in a theory for AHL-synthase substrate specificity and provided a greater
understanding of quorum sensing. In addition, the established modeling of Esal and
10


phosphopantetheine applies to the entire AHL-synthase family, including AHL
synthases that regulate bacterial diseases in humans. With the new perspective
obtained from the study of Esal, the potential for effective drug design has increased,
along with the hope for successful treatment of these diseases.
1.4 Hierarchical Regulation in Pseudomonas aeruginosa
One of the more notorious human pathogens known to health workers is
Pseudomonas aeruginosa, an opportunistic organism responsible for both chronic and
life threatening infection in patients suffering from Cystic Fibrosis, burn related
immune deficiency, or those fitted with various intrusive medical devices (16,17).
Quorum sensing allows P. aeruginosa to regulate its ability to form biofilms,
extracellular polysaccharide matrices, which can attach to implanted objects like
catheters or stents, or grow in tissues as in the lungs of cystic fibrosis patients (16,
17). Biofilms are complex structures composed of interspersed channels that provide
circulation of water and nutrients, but are naturally resistant to antimicrobial
treatment (16, 17). These properties, combined with the ability of P. aeruginosa to
continually multiply w ithin biofilms, makes the organism a source of chronic
infection once colonization is established (16,17). P. aeruginosa is able to regulate
biofilm formation, among other virulence factors, through the use of two quorum
sensing systems, lasR/lasI and rhlR/rhll (18). The former primarily synthesizes and
detects N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-AHL) while the
latter predominantly produces and responds to N-butanoyl-L-homoserine lactone (C4-


AHL) (16, 17, 18). The las genes code for multiple extracellular virulence factors,
including the enzyme elastase, which collectively cause extensive tissue damage in
humans, especially cystic fibrosis patients and burn victims (1, 19). The rhl genes are
responsible for rhamnolipid production, a hemolysin which contributes to tissue
invasion, and activation of rpoS, a sigma factor or promoter specificity factor that is
essential for transcription of additional virulence genes (18). Yet, the most interesting
characteristic of the P. aeruginosa quorum sensing systems is their integrated
function. Evidence indicates that the las system not only regulates certain virulence
factors, but also controls the rhl system (18). In addition, a study conducted by Singh
et al. (17) measured a high concentration ratio of C4-AHL to 3-oxo-C12-AHL in
biofilm material obtained from growth on silicone tubing, as well as cystic fibrosis
sputum, indicating that the rhl system is more active during advanced infection (17).
These results create an image of the disease course in P. aeruginosa infections.
During the initial stages of infection, the las system controls the production of
virulence factors that prepare the environment for biofilm proliferation and
multiplication of the organism. As cell density and 3-oxo-C 12-AHL concentration
increases, the rhl system is eventually activated for additional virulence factor
production and biofilm differentiation (17), leading to advanced disease and the
presence of large amounts of C4-AHL. Future studies will attempt to delineate the
actual pathogenesis of P. aeruginosa infection, but the hierarchical relationship
12


between las and rhl is clear (18) and applies to other bacterial species possessing
multiple quorum sensing systems.
1.5 Quorum Sensing in the Yersinia Genus
Another human pathogen, having international notoriety and serving as the
subject of this study, is Yersinia pestis. Commonly referred to as a subspecies of
Yersinia pseudotuberculosis, Y. pestis is the causative agent of the bubonic,
septicemic, and pneumonic forms of the plague (20, 21). Plague is a zoonosis that is
typically transmitted from animal reservoirs to humans by flea bites (20). The disease
has caused three pandemics affecting Africa, Europe, and Asia, beginning in the 6th
century, and continuing in widespread areas of the world today (20, 22). The global
and lasting effects of the plague have fueled scientific studies of the disease and its
source throughout history. In light of the current times, the terrifying concept of the
potential use of plague as a biological warfare agent has further heightened scientific
interest. In 2001, an effort to understand the fundamental characteristics of plague
virulence was initiated through a project to sequence the genome of Y. pestis (22).
Among many other things, the sequencing project led to the identification of tw o
genes whose transcription products are homologous to the Luxl family of AHL-
synthases (23). The genes, now known as yspl and ypel, encode the proteins Yspl
and Ypel, respectively. Perhaps an illustration of the close relationship between the
species, Yspl is identical in amino acid sequence to the AHL-synthase Ytbl of Y.
pseudotuberculosis. The established hierarchical relationships between the quorum
13


sensing systems of P. aeruginosa (18) and Y. pseudoiuberculosis (24) insinuate a
similar interaction between YspR/YspI and YpeR/Ypel of Y. pest is, though no studies
have yet confirmed the hypothesis.
Very little research has been performed on the quorum sensing system of Y.
pestis, but the disease-causing machinery of the organism is relatively well
documented. The source of virulence in the three Yersinia species
{pseudotuberculosis, pestis, and enteroco/itica) is an ~70 kb plasmid which directs
expression of the Yersinia Outer Membrane Proteins (Yops) (25). Most of the yop
genes have been sequenced and are virtually identical between the Yersinia species
(25). Despite what their name implies, experiments have demonstrated that the Yops
could be recovered from culture supernatants, indicating that they are secreted, and
not simply associated with the cell membrane (25). Further investigation led to the
identification of a new secretion pathway, type III, which requires the Ysc (protein)
apparatus, also encoded in the virulence plasmid (25). As a result of these findings,
the Yops can be categorized into two groups: Yops that form a delivery apparatus that
inserts into the membrane of a macrophage, and Yops that are delivered through this
channel as intracellular effectors (25).
Considering the identification of LuxI homologues in all of the species of the
Yersinia genus, quorum sensing regulation of virulence in Yersinia becomes
immediately interesting. Although the mechanism is unclear in Y. pestis, studies
have been performed for Y. pseudotuberculosis which offer insight. In a study by
14


Atkinson et al., two Y. pseudotuberculosis mutants were constructed in order to
characterize the ypsR/I quorum sensing system (24). The ypsR mutant was
designated by a 299 bp deletion encompassing the ypsR gene. The ypsl mutant was
designated by a 190 bp deletion which spanned the ypsl gene. Within the deletion
sites of both mutants, a kanamycin resistance cassette was inserted for experimental
selectivity (24). Both mutants still contained unaltered ytbR/I quorum sensing
systems. Dichloromethane extracts of culture supernatants of the wild type protein
and both mutants, grown at different temperatures, were subjected to a
Chromobacterium violaceum TLC overlay bioassay (14, 24). The assay, which
detects the presence of AHLs in the extract, revealed that the mutants yielded AHL
profiles that varied depending on the temperature (14, 24). Solvent extracts were
made from culture supernatants of ypsl mutant, ypsR mutant, and wild type,
incubated at different temperatures (24). AHL profiles obtained from each extract
were compared. The AHL profile for the ypsl mutant differed from the wild type at
28C, but was the same at 22C and 37C (24, 26). The AHL profile for the ypsR
mutant differed from the wild type at 22C, but was the same at 28C and 37C (24,
26) (Figure 1.4). In addition to the changes in AHL profile with respect to
temperature, light microscopy of ypsR mutant cultures, grown at 28C and 37C,
displayed clumping when compared to ypsl mutant and wild type cultures (24).
5


I
i
I
.Voxo-Ct* HSL
Cft HSi
C8-HSL
Wt \pU .p\ft
Vk, ,fiK
Figure 1.4 Variable Temperature TLC Overlay Data
Chromatograms of the AHL profiles from wild type, ypsl mutant, and ypsR mutant
culture supernatant extracts detected using the C. violaceum CV026 overlay assay.
TLC was performed on culture supernatant extracts from LB broth cultures grown at
37HC (A), 28C (B), and 22C (C). For A-C, lane 1 contains AHL extracted from 1 L
of supernatant from wild type Yersinia pseudotuberculosis; lane 2 contains AHL
extracted from 1 L of supernatant from the ypsl mutant; lane 3 contains AHL
extracted from 1 L of supernatant from the ypsR mutant; lane 4 contains 2.2 x l()'x
mol of synthetic C8-AHL standard; lane 5 contains 5.0 x 10"8 mol of synthetic C6-
AHL standard; lane 6 contains 1.4 x 1()'8 mol of synthetic 3-oxo-C6-AHL standard.
At 37C, 3-oxo-C6-AHL, C6-AHL, and C8-AHL are produced by the wild type, ypsl
mutant, and ypsR mutant (A). At 28C, the ypsl mutant is deficient for 3-oxo-C6-
AHL (B). At 22C, the ypsR mutant is deficient for C8-AHL (C).
Atkinson, et al., 1999 (24)
16


SDS-PAGE of the ypsR mutant culture identified several up-regulated proteins,
including a 42 kD polypeptide bearing N-terminal sequence identity with the major
flagellin structural proteins, FleA and FleB, of Y. enterocolitica (24). The
significance of these discoveries, is that quorum sensing not only appears to regulate
expression of membrane surface proteins associated with clumping in Y.
pseudotuberculosis, but also the motility of the organism as well (24). The above
studies make a case for the arguement that Y. pseudotuberculosis adapts to changing
temperature by using the quorum sensing system (ypsR/I or ytbR/l) that activates the
genes which give the organism the best chance for survival in that environment. The
ability to regulate clumping and motility is advantageous as well. When surviving in
the soil (20, 24), cell clumping may increase the chances for the organism to infect a
passing animal or human. However, when multiplying within a host, limited cell
clumping and enhanced motility could better the odds of avoiding phagocytosis by
immune polymorphonuclear leukocytes and decrease detection by immune cells that
stimulate the inflammatory response through cytokine up-regulation (25). Again,
quorum sensing is elegantly implemented in a manner that complements the lifestyle
of the organism.
The demonstration that temperature is an important environmental regulating
factor in Yersinia, brought about further investigation. In consideration of the
chemistry of lactone rings, the hypothesis was proposed that AHLs may be
hydrolyzed in alkaline conditions. Yates et al. showed that a Y. pseudotuberculosis
17


culture at 37C has a pH that fluctuates from 7.1 to 8.5 over a growth period of 24
hours (26). As a result, nonenzymatic lactonolysis was possible as the growth time
increased. Temperature, then, may simply increase the rate of lactonolysis under
basic pH conditions, which could also account for the changes in AHL profile at
varying temperatures (26). However, the stability of AHLs varies from one to the
next. nC-NMR experiments revealed that AHLs having longer acyl chains are more
resistant to ring opening, while acyl chains with electronegative substituents have
reduced resistance (26). Perhaps under higher temperatures Y. pseudotuberculosis
alternates to a quorum sensing system that utilizes a more stable signal. Regardless
of the true connection between AHL profile and temperature for Y.
pseudotuberculosis, the temperature of the environment does dictate which AHLs
w ill predominate. The temperatures at different sites within a host may then help to
create a "preferred" region of infection for the organism.
1.6 Objectives for the Study of Yspl
The characteristics of Y. pseudotuberculosis identified through previous
studies may provide some perspective of the function of other Yersinia species, but
the only way to develop a true understanding of an organism is to study it directly. A
great deal of scientific interest surrounds Y. pestis, due to its relevance to w orld-wide
health. The progress made in the study of quorum sensing in other bacterial species
has created excitement for the potential development of drug therapies to treat serious
diseases like the plague. In the chapters that follow', initial characterization of the
18


LuxI homologue YspI, of Yersinia pestis is discussed. First, successful cloning of a
pET28a-yspl DNA construct is demonstrated. The construct will be used to express
recombinant Yspl protein in E. coli cultures as part of an effort to study the enzyme's
function. Second, activity assays are presented to show that the recombinant Yspl
protein produces AHL signal. Third, LC-MS analysis of a culture supernatant extract
reveals a profile of AHL signal molecules produced by recombinant Yspl, and
establishes N-(3-oxooctanoyl)-L-homoserine lactone as the predominant signal
produced by the enzyme. This identification indicates that 3-oxo-C8-ACP is the
primary substrate for Yspl and should be used if accurate kinetic studies are to be
performed. To date, information regarding quorum sensing regulation in the Yersinia
genus is limited, and no data is currently available on the function of this
phenomenon in K pestis specifically. The data generated throughout this study has
laid the groundwork for a greater understanding of Y. pestis virulence.
19


2. Sub-Cloning of a pET28a-yspl DNA Construct
2.1 Introduction
Advances in molecular biology have led to the development of several
techniques that have broadened the seope of investigation for researchers of many
disciplines. Gene cloning, in particular, has provided structural biologists and
biochemists with the opportunity to study specific enzymes in their native
conformation and function. Cloning is used to produce a genetically identical group
of cells or organisms for study. The process sometimes involves the copying and
arrangement of a gene of interest into DNA associated with a completely different
organism. A common example is the cloning of foreign genes into E. coli for
overexpression of a specific gene product. Before E. coli can be utilized, a gene must
be cloned out of its organism and into a form that will allow its introduction to the
bacterial cells. This can be accomplished by inserting the gene into a plasmid, an
extrachromosomal ring of bacterial DNA, that serves as a deliverable DNA
compartment or vector. Insertion of the gene into a plasmid, allows the gene to be
taken up with the plasmid by bacterial cells during transformation. The genetic
information encoded in the plasmid DNA may then be expressed in those cells. One
approach to the insertion of a gene into a plasmid is to use restriction endonucleases,
enzymes that cleave DNA at specific nucleotide sequences. These enzymes can be
20


used to "cut" a gene out of a plasmid, as well as to cut a space in a plasmid where the
gene can be inserted. The ability to transfer a gene from one plasmid to another is
desirable because plasmids are engineered to yield different advantages for the cells
that incorporate them. For instance, plasmids often contain a multiple cloning site in
which several different restriction enzymes can be used to insert a gene, but the sites
differ from one plasmid to the next so that different genes can be accommodated.
Plasmids also contain antibiotic resistance genes that allow for the selection of
transformed cells from nontransformed cells, through growth of the cells in an
environment of the appropriate antibiotic. Some plasmids, know n as expression
plasmids, contain the lac operator upstream of the gene insert site. For these
plasmids, expression of the inserted gene's product can be strategically activated and
exaggerated by the addition of the allolactose analog, isopropylthiogalactopyranoside
(1PTG). It is w ith this type of treatment that an expression plasmid like pET28a is
made to coordinate the mass production of an inserted gene product. The following
describes the construction of an E. tY;//(BL21 )-pET28a-yspl clone, an expression
system designed to produce YspI protein. A gene sequence of the pET28a plasmid
containing the yspl gene insert is presented for verification. Expression and
purification of a protein consistent with Yspl is also demonstrated. The creation of
the pET28a-yspI construct enabled the study of quorum sensing signal generation in
Y. pestis.
21


2.2 Results
2.2.1 Preparation of pET28a plasmid and
yspl Gene Insert
Prior to the commencement of this study, the yspl gene was cloned out of the
Yersiniapestis genome and into the DNA plasmid pCR2.1 by Scott Beardon and
colleagues, collaborating scientists at the Centers for Disease Control and Prevention.
Beginning with the pCR2.1-yspI construct, the initial objective for the study of Yspl
was to clone the yspl gene into a DNA plasmid suitable for overexpression of the
protein. Although there are different ways to accomplish this goal, the approach used
here involved subcloning the yspl gene segment from the pCR2.1 plasmid to the
pET28a plasmid using the restriction endonucleases Ndel and Hindi 11. These
enzymes were chosen because the specific DNA sequences at which they cleave were
present both in the multiple cloning site of pET28a as well as the surrounding,
noncoding region of the yspl gene in pCR2.1.
Overnight cultures of E. coli (DH5u, transformation strain) cells containing
pCR2.1 -yspl or pET28a were grown and the plasmids were isolated by miniprep.
After purification of the plasmids by phenol extraction/ethanol precipitation and
passage through a Sephadex G50 (Amersham Pharmacia) size exclusion spin column,
the concentrations of the resulting 40 pL plasmid solutions were determined by
UVAVis spectroscopy to be 1714 ng/pLand 166.5 ng/pL, respectively.
22


Before the yspl gene insert could be cloned into pET28a, the plasmid had to
be digested with Ndel and Hindi 11 to create the proper DNA overhangs for successful
ligation of the gene. The digest of pET28a was performed sequentially by incubating
the plasmid with Ndel, follow ed by incubation with Hindlll. Small portions of the
Ndel digested plasmid and Hindlll control digest, without Ndel, were analyzed by
electrophoresis through a 0.7% agarose gel containing ethidium bromide. Ethidium
bromide binds to DNA and fluoresces when exposed to ultraviolet light, visualizing
DNA fragments present in the gel. The analysis was performed to ensure that both
enzymes were cleaving the pET28a DNA individually. Figure 2.1 shows the
ethidium bromide stained gel with DNA bands visualized by ultraviolet light. Since
supercoiled DNA, nicked DNA (single strand cleavage), and cut DNA (double strand
cleavage) migrate through agarose gel at different rates, digested DNA can be
identified through a comparison of DNA in different conditions. Lane 5 shows a faint
band of nicked pET28a DNA running slower than the lower supercoiled pET28a
DNA band. Lanes 7 and 9 show bands from individual pET28a digestions with Ndel
and Hindlll running slower than supercoiled DNA but faster than nicked DNA. This
is characteristic of DNA cut by a restriction enzyme, and indicates that the pET28a
plasmid was cut effectively by both enzymes. The sequential digest was completed
by incubating the Ndel cleaved pET28a with Hindlll. Upon completion of the second
half of the digest, Calf Intestine Alkaline Phosphatase (MB1 Fermentas) w as added to
prevent the digested pET28a plasmid from reannealing to itself.
23


Figure 2.1 Ndel and HindllI Digestion of pET28a DNA Plasmid
0.7% agarose gel verifying cleavage of pET28a plasmid by Ndel and Hindi 11
enzymes individually. The contents of the gel are: Lane 3 400 ng 1 kb DNA ladder;
Lane 5 250 ng uncut pET28a; Lane 7 274 ng Ndel cleaved pET28a; Lane 9 250
ng Hindi 11 cleaved pET28a. Lane 5 shows a faint upper band of nicked pET28a and
an intense lower band of supercoiled pET28a. Lanes 7 and 9 display pET28a cut with
Ndel and Hindll 1 respectively.
24


The digested, phosphatase-treated pET'28a was then purified by phenol
extraction/ethanol precipitation and passage through a G50 spin column. The
resulting 40 pL solution had a concentration of 18.5 ng/pL as determined by UV/Vis
spectroscopy.
To obtain the yspl insert separated from the pCR2.1 plasmid, the pCR2.1-yspI
construct was sequentially digested with Ndel and Hindi 11 and the entire sample was
analyzed by electrophoresis through a 1.2% agarose gel containing ethidium bromide.
Figure 2.2 shows the visualized DNA bands resulting from the digest. Lane 4
contains nicked and supercoiled pCR2.1 -yspl. Lane 6 clearly shows a separate band
running just above the 500 bp marker, w hich is consistent with the approximate size
of the yspl gene insert of 650 bp. With the insert separated from pCR2.1, the gel
surrounding the yspl DNA was physically removed with a razor blade and purified by
treatment with a QIAquick gel extraction kit (Qiagen). The insert was further
purified by passage through a G50 spin column and the resulting 85 pL solution was
measured to be 18.5 ng/pL by UV/Vis spectroscopy.
25


6
Figure 2.2 Ndel and H ind 111 Digestion of pC'R2.1-yspI DNA Construct
1.2% agarose gel displaying yspl digested out of the pCR2.1 plasmid.
The contents of the gel are: Lane 2 400 ng 500 bp DNA ladder; Lane 4 1714 ng
uncut pCR2.1-yspl; Lane 6 10284 ng Ndel and Hindi 11 digested pCR2.1 -yspl. Lane
4 shows nicked (upper band) and supercoilcd (lower band) pCR2.1 -yspl. Lane 6
contains an upper band of Ndel-Hindlll cut pCR2.1 and a lower band of yspl insert
separated from the plasmid.
26


2.2.2 Ligation, Transformation, and Sequencing of
E. coli(DH5a)-pET28a-yspI
With the pET28a plasmid and yspl insert digested, purified, and isolated, two
reactions were prepared to ligate the insert into the plasmid. Reaction A contained
equal amounts of insert and vector, while reaction B contained a 2:1 insert to vector
ratio. DNA ligase, an enzyme that repairs single strand DNA breaks, was included in
both reactions. Different ratios of insert to vector are often prepared for ligation
reactions due to the unpredictability of the ensuing transformation experiments. A
negative control reaction containing no yspl insert, was also carried out as a
reference. Grow th from a transformation experiment with this type of negative
control would indicate that the plasmids were not cleaved properly. After incubation
at room temperature for 1.5 hours, reaction A, reaction B, the negative control, and a
positive control of supercoiled pET28a were transformed into E. coli (DH5a) cells by
the heat shock method. The transformed cells were then plated onto agar/kanamycin
plates and incubated over night. Cells that successfully incorporated the pET28a-yspl
construct also gained resistance to kanamycin and were able to grow' selectively on
the plates. The volumes of plated cells included 50 pL and 100 pL of the transformed
cells spread onto agar/kanamycin plates after incubation. For the concentrated cell
volumes plated, the remainder of the transformed cells was centrifuged dow n and all
but 100 pL of supernatant w as removed. The concentrated cells were then


resuspended in that volume and the entire amount spread onto an agar/kanamycin
plate. The results are summarized in Table 2.1.
Number of Colonies Present After Incubation
Volume of Plated Cells Positive Control Negative Control Reaction A Reaction B
50 pL 2 2 2 2
100 pL 7 1 9 4
100 pL Concentrate ~250 1 -75 -50
Table 2.1. Results of the Transformation of pET28a-yspl into E. coli (DH5a) Cells
The table indicates the number of bacterial colonies present from transformations
with the indicated ligation reaction for a particular volume of cells plated after
incubation at 37C for 16-20 hours on agar/kanamycin plates. The values are
reported with respect to the volume of cells plated following the transformation
experiment.
The number of colonies present in the 100 pL concentrate positive control clearly
proved that transformation occurred. Although the transformation efficiencies for
reactions A and B w ere not as high as the positive control, the number of colonies
yielded from the reactions was large compared to the negative control, suggesting that
the preceding ligation reactions were successful, as was the transformation. Thus, it
was likely that E. coli (DH5a)-pET28a-yspl clones had been created, although there
was no way to know conclusively w ithout sequencing the DNA samples. In order to
identify promising samples of potential pET28a-yspl construct for sequencing, a
Polymerase Chain Reaction (PCR) screen was performed for several plasmids
obtained from overnight cultures of a number of the transformed colonies. Small
28


amounts of each plasmid were added to PCR experiments designed to amplify the
DNA present in their cloning sites. The sizes of the resulting DNA fragments were
assessed by electrophoresis through 2.0% agarose gels. Figures 2.3 and 2.4 show the
ethidium bromide stained gels with visualized DNA bands. The size of the DNA
encompassing the cloning site in the pET28a plasmid, from T7 promoter to T7
terminator, is 280 bp. Plasmids yielding amplified DNA fragments that
electrophorese near 280 bp are indicative of a cloning site that does not contain yspl
insert. The PCR control (supercoiled pET28a with no insert) in Figure 2.3 Lane 4
shows a band near 300 bp, slightly larger than the theoretical 280 bp. Still, the
presence of the band near 280 bp demonstrates that the PCR reaction occurred. With
insert present in the cloning site, the resulting amplified fragment should be the size
of the DNA from promoter to terminator, plus the size of the insert. Since the yspl
insert is 650 bp, plasmids that yield DNA fragments running near 930 bp (280+650)
were likely to contain yspl insert in their cloning site. The plasmid samples in Figure
2.3 Lanes 6-13, and Figure 2.4 Lanes 4-11, all display intensely visualized DNA
fragments running between 900-1000 bp, suggesting the presence of pET28a-yspI
construct in each. The same respective lanes in both figures also display less intense
bands just above 600 bp. These bands are likely to be free yspl insert released by
some of the cells as foreign DNA. It is not uncommon for a construct to be absent
from all but a few of the chosen colonies grow n from transformed cells.
29


Figure 2.3 PCR Screen of Plasmid Samples 1-8
2.0% agarose gel displaying segments of DNA cloned from the pET28a insert site in
PCR reactions of multiple transformed colonies. The gel contents are: Lane 3 400
ng 100 bp DNA ladder; Lane 4 PCR control supercoiled pET28a; Lane 5 negative
control (ligation without insert); Lanes 6-13 PCR reactions for transformed colonies
1-8; Lane 14 400 ng 100 bp DNA ladder. Lane 4 displays amplified DNA fragment
near 280 bp, expected for the size of T7 promoter plus T7 terminator but no insert.
Lanes 6-13 all display amplified DNA fragments near 930 bp, the expected size of the
T7 promoter plus the T7 terminator plus the yspl insert. All colonics were
successfully transformed.
30


Figure 2.4 PCR Screen of Plasmid Samples 9-16
2.0% agarose gel displaying segments of DNA amplified out of the pET28a insert site
in PCR reactions of multiple transformed colonies. The gel contents are: Lane 3 -
400 ng 100 bp DNA ladder; Lanes 4-1 1 PCR reactions for transformed colonies 9-
16; Lane 12 400 ng 100 bp DNA ladder. Lanes 4-11 all display amplified DNA
fragments near 930 bp, the expected size of the T7 promoter plus the T7 terminator
plus the yspl insert. All colonies were successfully transformed.
31


The fact that each colony removed from a plate and cultured yielded plasmid DNA
consistent with pET28a-yspI was remarkable. Regardless of the number of
apparently successful constructs created, only one of the samples needed to contain
the construct so that it could be transformed into an E. coli expression strain such as
BL21.
The final task in the effort to prove that yspl insert was successfully cloned
into pET28a plasmid, involved selecting one of the 16 plasmids obtained from
transformed cells and having it sequenced by an automated version of the chain
terminator (dideoxy) method (27). Typically, one would choose the plasmid whose
PCR product of the insert site produced the most promising fragment size. In this
case, however, the plasmid solution #13 was chosen at random since every colony
appeared to contain the desired clone. The #13 plasmid solution was sent to the
Molecular Biology Core facility at the Barbara Davis Center for Childhood Diabetes
for the sequencing process. Upon completion of the necessary reactions the result of
the sequencing effort was received as a DNA sequence. Using the DNA Stridcr
software, the DNA sequence was translated to protein for comparison to established
sources. Figure 2.5 displays the sequence. The sequence data confirmed the
successful construction of a pET28a-yspl clone. The sequence obtained from plasmid
#13 was compared to the documented sequence of Yspl according to the databank of
the National Centers for Biotechnology Information (23) and was found to match
exactly.
32


mm DNA Strider 1.3f9 ### Thursday, July 17, 2003 5:10:32 PM
03-26-03 pET28a-yspl #13 seq -> Translate 1-frame DNA sequence 1477 bp A*G*G***CGG* ... **GC**GG 1/1 31/11 A*G *G* **C GG* TAA ACA ATT *CC CCT *CT A*A AAT AAT TTT *C GTT linear TAA CTT TAA G*A AGG 61/21 AG *AT ATA CCA TOG G*C AGC AGC CAT CAT
XXX X * T I X P X X N N F V * L * X R XXI P W X S S B H
91/31 CAT CAT CAT CAC AGC AGC GGC CTG GTC CCG 121/41 CGC GGC AGC CAT ATC TTA GAA ATT TTC GAT 151/51 GTC AGA TAC GAC GAG TTC ACC GAT ATA CGG
H H H H S S G L V P R G S H M L E I F D V R Y D E L T D I R
181/61 TCT GAG GAC CTG TAT AAA TTA AGG AAA AAG 211/71 ACG TIT AAG GAT CGT CTT AAC TCG GAG GTC 241/81 AAT TCC AGC AAC GGC ATC GAG TTT GAT GAA
S E D L Y K L R X X T F K D R L N W E V N C S N G M E F D E
271/91 TAT GAT AAT TCG GAT ACA car TAT CTC CTT 301/101 GGT ATT TAC CAA GGT CAA CTA ATC TCC AGT 331/111 GTC CGT TTT ATA GAA CTT CAC CTC CCT AAT
Y D N S D T R Y L L G I Y Q G Q L I C S V R F I E L H L P N
361/121 ATS ATC ACT CAT ACC TTC AAT GCA CTC TTC 391/131 GAT GAC GTC GCA CTA CCT AAG AGG GGC TAT 421/141 ATA GAA TCG AGC CGT TTC TTC GTT GAT AAA
MIT H T F N A L F D D V A L P K R G Y I E S S R F F V D K
451/151 ACC CGT GCC AAG TTG CTT TTT GGT AAC CAT 481/161 TAC CCC ATA AGC TAC TTA TTC TTT TTA TCG 511/171 ATT ATT AAT TAC TCC CGA CAT AAT GGC TAC
T R A K L L F G N H Y P I S Y L F F L S I I N Y S R H N G Y
541/181 ACA GGT ATT TAT ACT ATC GTC AGC CGA GCA 571/191 ATC TTC ACC ATC CTT AAA CGA TCA GGC TCG 601/201 CAG GTT GAG GTC ATT AAA GAA GCA CAT ATT
TGI Y T I V S R A M L T I L K R S Q W Q V E V 1 K E A H I
631/211 ACC GAA AAA GAA AGG ATA TAC TTA CTC CAT 661/221 CTC CCG ATA GAT CGG GAC AAT CAA GCA CGG 691/231 CTC CTT TTA CAG GTT AAT CAG CGT TTG CAG
T E K E R I Y L L H L P I D R D N Q A R L L L Q V N Q R L Q
721/241 GAT CCA TCT TCG GTA TTA AGT ACA TCG CCT 751/251 ATA TCG TTG CCC GTT ATC CCA GAA TCG GCT 781/261 TAA TAG GAT CCA AAG CCG AAT TCC AGC ACA
D P C S V I> S T W P I S L p V M P E S A * D P K P N S S T
811/271 CTG GOG GCC GTT ACT AGT GGA TCC GAG CTC 841/201 GGT ACC AAG CTT GCG GCC GCA CTC GAG CAC 871/291 CAC CAC CAC CAC CAC TGA GAT CCG GCT GCT
L A A V T S G S E L G T K L A A A L E a H H H H K * D P A A
901/301 AAC AAA GCC OGA AAG GAA GCT GAG TTC GCT 931/311 GCT GCC ACC GCT GAG CAA TAC CTA GCA TAA 961/321 CCC CTT GGG GGC CTC TAA ACG GGT CTT GAG
N K A R K E A E L A AAT A E Q Y L A * P L G G L * T G L E
991/331 GGG TTT TIG CTG AAA GGA GGA ACT ATA TCC 1021/341 GGA TTC GCG AAT GGG ACG CGC CTG TAG CGG 1051/351 CGC ATT AAG CGC GGC GGG TC* GGG GTT CCC
G F L L K G G T I S G L A N G T R L * R R I K R G G X G V p
1081/361 CCA **G *GA C*G TTA ACT *QC AG* GCC TA* 1111/371 CGC CCG TCC TIT G*T T*T TCC TTC *TT TCG 1141/381 C*C GT* GCG GTT *CC G** AGC TTA ATC GGC
P X X X L T X X A X R P S F X X S F X S X X A V X X S L I G
1171/391 GCT CCT *AG GTC AT TAG GTT CG* *CT G*C 1201/401 CAA ACT GTT GGG A*G TCC GA* GGC **c CCG 1231/411 AAA *GT TTC CTT GCT G** C** TTT T*A GGC
A P X V X * V X X X Q T V G X s X 0 X P X X F L A X X F X G
1261/421 TTG TCA *GA ACC TAG TP *G* *T* TTA TTA 1291/431 GG* T*C *** GCT TCT AAG GCG TAA A*T CGA 1321/441 TTA A** C*T CTC G*C TTG GAG *GG CCT TTT
L S X T Y X X X L L XXX A C K A * X R L X X L X L E X P F
1351/451 TTA C*A *GC C* A *TA ACC *CA AAC GTT TCT 1381/461 *CT T*A CTT GA *cc GC GG* CC* G** CCC 1411/471 *C CT* CAA G* * A'* G** GG* TTT **c C*A
L X X X X T X N V C XXL X X X X X X p X X Q X X X X F X X
Figure 2.5 YspI Sequence Data
The sequence data obtained for plasmid from transformant colony #13, translated by
the DNA Strider software, display the Yspl protein sequence. The sequence begins
just beyond residue 41 at MLEIFD... and concludes just before residue 261 at
...VMPESA (bold numbers represent protein residue number). The sequence matches
exactly with the documented Yspl sequence in the National Center for Biotechnology
Information databank, accession NP 40461.
National Centers for Biotechnology Information (23)
33


2.2.3 YspI Protein Purification
Confirmation of the successful construction of pET28a-yspl allowed an initial
attempt to purify the Yspl protein. A 6 L culture of E. eo//-pET28a-yspl was grown
to '0.6 OD, induced with 0.2 mM IPTG, and incubated for an additional hour. The
cultures were centrifuged to force the cells into a pellet and the supernatant was
discarded. Varying amounts of cells were available for lysis depending on the
purpose. In this example, a cell pellet from 500 mL of culture was lysed with 5 mL
of buffer, sonicated, and centrifuged at high speed to remove the cellular debris. The
supernatant was then allowed to incubate with 1 mL of 50/50 Ni-NTA slurry for 2
hours. After incubation, the Ni-NTA beads were manually washed with wash buffer
several times and then loaded into a C-10 column (Amersham Biosciences). After
rinsing the column of Ni-NTA beads with wash buffer, bound Yspl protein was then
eluted off the beads into fraction tubes using a gradient of 100% w ash buffer / 0%
elution buffer (high imidazole concentration) to 0% wash buffer / 100% elution buffer
over 40 minutes. The chromatogram is shown in Figure 2.6. The protein did not
elute off the column near the middle of the gradient. The result was about a 5 minute
delay in elution after solvent B was at 100%. Under this type of condition, the eluted
protein may not by as pure as possible by Ni-affinity, but further purification should
produce protein of sufficient purity for crystal trials.
34


iiiA I
Figure 2.6 Elution of Protein from a Ni-NTA Column
The chromatogram shows the elution of protein from 1 mL of 50/50 slurry of Ni-NTA beads. The Ni-NTA
beads were manually packed into a Pharmacia CIO column after incubation with E. cr;//-pET28a-yspI culture
lysate. The protein was eluted with a gradient of 100% wash buffer (10% glycerol, 20 mM Tris, 500 mM NaCl,
10 mM Imidazole) / 0% Elution buffer (10% glycerol, 20 mM Tris pH 8.0, 500 mM NaCl, 250 mM imidazole, 1
mM p-mercaptoethanol) to 0% wash buffer / 100% Elution buffer over 15 minutes. 250 pL fractions were
collected. Fractions 38-41 were pooled for further purification. The chromatography was performed on an Akta
Purifier (Amersham Biosciences) at a flow rate of 1 mL/min.


1 2
- *r
?5kD MMPn*
50kD^_
37 kD
25kD 15kD
3 4 5
6 7 8
t
Figure 2.7 SDS-PAGE of Eluted Fractions from a Ni-NTA Column
The gel shows the contents of samples before and after Ni-NTA affinity purification.
Lane 1 contains 5pL of Precision Standard (BioRad) with applicable masses indicated
(Yspl ~ 26.5kD). Lane 2 contains 3 pL of resuspended insoluble fraction from E.
co//-pET28a-yspI culture cell lysis. A small amount of insoluble protein is visible
just above the 25kD marker. Lane 3 contains 5 pL of unbound protein in culture
supernatant after incubation with Ni-NTA beads. Very little protein is visible just
above the 25kD marker, indicating that it bound the Ni-NTA. Lanes 4-7 contain 30
pL of fractions 38-41 from the Ni-NTA column elution. Thick bands of eluted
protein are visible in these lanes just above the 25kD marker, consistent with Yspl.
Lane 8 contains 20 pL of the rinse of the Ni-NTA beads after heating them in SDS.
A thin band of protein is visible near 25 kD, a portion that did not elute off the Ni-
NTA column. Most of the protein in excess did elute off the Ni-NTA beads.
36


Fractions 38-41 were collected and analyzed by SDS-PAGE (Figure 2.7). Lane 2
contained a portion of the insoluble fraction from the culture lysate. Some protein is
present just above the 25 kD marker. But, considering the eluted fractions, most of
this protein remained soluble upon lysis. Lane 3 contains protein that remained
unbound to the Ni-NTA beads after incubation. Very little protein of ~26 kD mass
remained unbound, indicating that enough Ni-NTA material was added to the culture
supernatant to bind that protein. For having lysed only half of a typical cell pellet, a
considerable amount of desirable protein was present in the fractions of the Ni-NTA
eluted sample, lanes 4-7. Lane 8 contains the remaining material on the Ni-NTA
beads after heating them in SDS. Relatively little protein had remained on the Ni-
NTA beads after elution. The Ni affinity purification was effective and the eluted
protein was ready for further purification. Fractions 38-41 were pooled and injected
onto a Superdex 75 (Amersham Biosciences) size exclusion column to separate the
proteins present in the pool (lanes 4-7 of Figure 2.7). The size exclusion
chromatogram is shown in Figure 2.8. The three peaks in the figure show adequate
separation. Portions of fractions 26, 33, 38, 39, 40, 41,42, 59, and 70 were analyzed
by SDS-PAGE (Figure 2.9). The fractions surrounding the peak at fraction 39, lanes
4-8, all appeared to contain significant amounts of protein consistent with Yspl. Of
these, lanes 5-8 contain particularly pure protein. Fractions 39-42 were pooled an
stored at -80D for future concentration. These results were obtained from a small
scale lysis of£. tW/-pET28a-yspl.
37



Size Exclusion SD75
Injection Vol: 1 niL
Flow Rate: 1 inL/min
Full Scale: 0.2 AU
Figure 2.8 Size Exclusion Chromatogram of Fractions Eluted from Ni-NTA
The chromatogram was generated from a 1 mL injection of pooled fractions, eluted
from a Ni-NTA column, onto a Superdex 75 (Amersham Pharmaceia) size exclusion
column. The chromatographic system used was a Pharmacia FPLC.
Flow Rate: 1 mL/min, Full Scale: 0.2 AU.
38


2 3 4 5 6 ~ H 9 10
50 kD ,
37 kD
15 kD
Figure 2.9 SDS-PAGE of Fractions from Size Exclusion Chromatography
The gel shows the purity of individual fractions obtained from size exclusion
chromatography. Lane 1 contains 5 pL of Precision Standard (BioRad) with
applicable masses indicated (Yspl ~ 26.5kD). Lanes 2-10 contained 30 pL of size
exclusion fractions 26, 33, 38, 39, 40, 41,42, 59, and 70, respectively (Figure 5.3).
All fractions but 59 and 70 contained detectable amounts of protein consistent with
Yspl, but fractions 39-42 in lanes 5-8 contained the purest and most highly
concentrated amounts. These fractions could be pooled, concentrated and potentially
used in kinetic assays or crystal trials, assuming that the isolated protein is Yspl.
39


It would follow that a lysis of 1-3 cell pellets, from 1 L of culture each, could yield a
very large amount of desirable protein. The protein isolated by Ni-NTA affinity
chromatography and further purified by size exclusion chromatography is consistent
with Yspl. The above described approach appears to be an effective way to obtain
very pure protein. Considering amount of protein recovered from this analysis and
the possibility of scaling up the process, this approach would be valuable for
preparation of a significant amount Yspl protein for crystallography trials.
2.3 Discussion
The sequencing results for the pET28a-yspl clone not only confirmed the
presence of the yspl gene insert, but also revealed an interesting property of the
encoded protein. According to the National Center for Biotechnology Information
(25), the Yspl protein of Y. pest is is identical in amino acid sequence to the Ytbl
protein of Y. pseudotuberculosis. The identity reflects the relationship between Y.
pestis and Y. pseudotuberculosis and provides a basis for comparison of the yspR/l
and ytbR/l systems. A study by Atkinson et al. (24), identified altered AHL profiles
in Y. pseudotuberculosis and explained their result as part of a temperature regulation
pathway. The study established an AHL profile for the ytbR/l system. Considering
the sequence data, one would expect a yspR/l system to yield the same AHL profile.
AHL signals extracted from a culture supernatant of E. coli (DH5u) pET28a-yspl
would test this hypothesis. Now' that sequence data have confirmed construction of
40


E. coli pET28a-yspI, the stage is set for a study of the AHL profile of Yspl. Although
the protein expression and purification results presented were promising,
crystallographic trials were beyond the scope of this study, and accurate kinetic
studies were not possible due to the lack of pure acyl-ACP substrate. As a result,
activity of the protein w ill be demonstrated via biochemical assay to verify that the
construct directs the expression of a functional product.
41


3. Demonstration of Recombinant YspI Activity
3.1 Introduction
There are a number of AHL reporter systems available for the evaluation of
AHL signal production. Some detection systems rely on measurement of [)-
galactosidase activity as a reflection of AHL signal production in culture supernatant
extracts (15). Included in this category are detection systems that have been
developed for the quorum sensing systems luxR/1 of Vibrio fisher i (34) and traR/I of
Agrobacterium tumefaciens (35). These systems provide a general response to the
presence of AHL signal in an assay sample but do not indicate which specific signals
are present. The ability to assess changes in AHL signal production is vital for
mutational studies of quorum sensing proteins and for the effort to understand critical
regions involved in enzyme activity. The interest in a broad spectrum screen for
AHLs led to the development of an assay that employs the Chromobacterium
violaeeum mutant CV026 (14). C. violaceum is a gram negative bacterium that
inhabits soil and water and uses quorum sensing to regulate production of the purple
pigment violacein (14). The CV026 mutant cannot synthesize its AHL signal to
activate violacein production, but was found to produce the pigment when exposed to
AHL signals with acyl chains of four to eight carbons in length (14). However, the
assay responds weakly to 3-oxo substituted AHLs, and did not respond to AHL
42


signals with acyl chains of ten to fourteen carbons in length (14). The utility of
CV026 was combined with the established separation of AHLs by thin layer
chromatography (TLC) to create TLC overlay assays (14, 36). For these assays,
solvent extracted AHLs, from a free cell culture supernatant, are separated on TLC
plates and then overlaid with molten agar containing CV026. After overnight
incubation of the overlaid plates, purple spots form above the AHL signals.
Atkinson, et al., performed this assay for Y. pseudotuberculosis extracts and
compared the resulting profile to the profiles of recombinant Ypsl and Ytbl in E. eo/i
(Figure 3.1). One of the conclusions made for this particular experiment was that
recombinant Ytbl produced C8-AHL and C6-AHL, but did not produce 3-oxo-C6-
AHL. However, interpretation of the assay is obscure, as circular spots are supposed
to be indicative of unsubstituted AHLs, while tear drop shaped spots reflect the
presence of 3-oxo substituted AHLs. These features are difficult to distinguish. Also,
spots in lanes 3 and 4 migrating between C8-AHL and C6-AHL in Figure 3.1 could
not be identified. The response of the assay is definitely questionable for AHLs of
longer acyl chain length, as well as 3-oxo substituted AHLs. Despite the efficiency
and practicality that the CV026 TLC overlay assay provides, identification of AHL
signals is termed tentative when based solely on this method (24).
As mentioned above, other assays have been developed to exploit the action
of [3-galactosidase. (Tgalactosidase is the product of the lacZ gene of E. coli and is
responsible for the hydrolysis of P-glycosidic bonds.
i
i
i
43


Figure 3.1 Recombinant Ytbl and Ypsl TLC Overlay Data
Chromatogram of the AHLs present in cell free culture supernatant extracts of wild
type Y. pseudotuherculosis, E coli JM 109 (pYtbV), and E. coli JM109 (pYpslV)
clones grown at 37C. Lanes 1 and 2 contain AHL extracted from 1 L of supernatant
from wild type Y. psendotuberculosis; lane 3 contains AHL extracted from 1 L of
supernatant from E. coli JM 109 (pYtbV); lane 4 contains AHL extracted from 1 L of
supernatant from E. coli JM 109 (pYpslV); lane 5 contains 2.2 x 1(FX mol of synthetic
C8-AHL standard; lane 6 contains 5.0 x 10'9 mol of synthetic C6-AHL standard; lane
7 contains 1.4 x 10 x mol of synthetic 3-oxo-C6-AHL standard. The conclusions
reached were that E. coli JM109 (pYpslV) produces 3-oxo-C6-AHL and C6-AHL,
while E. coli JM 109 (pYtbV) produces C6-AH L and C8-AH L.
Atkinson, et al., 1999 (24)
44


The colorimetric Miller assay measures this function through the addition of the
chromogenic substrate o-nitrophenyl-fTD-galactopyranoside (ONPG), which is
converted by P-galactosidase to glucose, galactose, and the o-nitrophenolate anion
(28). Spectrophotometric measurement at 420 nrn (A.nBX) indicates the amount of o-
nitrophenolate anion present in an assay sample and corresponds to a proportional
amount of P-galactosidase activity (28). The Miller assay became more applicable to
the study of AHL-synthases with the construction of E. coli strain MG4/pKDT17,
which contains a lasB::lacZ detection system and a lasR gene under control of the lac
operon (29, 30). LasR is then constitutively expressed and serves as a general
receptor for AHLs. Although LasR will bind both 3-oxo substituted and
nonsubstituted AHLs with side chains from 8-14 carbons in length (29, 30, 31), the
sensitivity of the detection system is less for AHLs other than N-(3-oxododecanoyl)-
L-homoserine lactone, and undetermined for 3-hydroxy substituted AHLs (29).
Incubation of MG4/pKDT17 in the presence of AHLs results in AHL-LasR binding,
transcriptional activation of lacZ, and production of P-galactosidase. With this
approach, the Miller assay can measure AHL-synthase activity in samples of culture
supernatant AH L extract after incubation with MG4 pKDT 17. AH L-synthase
activity, reflected in Miller Units, is then proportional to P-galactosidase production,
which is measured by timed reactions with ONPG and spectrophotometric analysis of
the samples. Miller Units represent the increase in o-nitrophenolate anion per minute
per bacterium for an assay sample and are calculated using equation 3.1.
45


MU = 1000X
<>D.75XOC 551 Iron )
limey- vo/umex()D(Mhim
(3.1)
The OD value at 600 nm measures cell density before the assay. The OD values at
420 nm and 550 nm are measurements of the absorbance of the o-nitrophenolate
anion and the absorbance due to light scattering in the yellow region of the spectrum
for the samples after completion of the assay. The reaction time (minutes) and the
volume of diluted MG4/pKDTI7 culture added to the sample (milliliters) are also
entered into equation 3.1. Measurement of AHL-synthase activity in this manner is
more general than the CV026 assay, but was employed to confirm AH L signal
production by recombinant Yspl generated from E. co//(BL21 )-pET28a-yspl. The
data presented in this chapter establish the presence of AHL signal in E. coli(BL21 )-
pET28a-yspl culture supernatant and shows that the signal was produced by
recombinant Yspl protein. A specific AHL profile for Yspl is then presented in
Chapter 4 for comparison with the results reported in Atkinson, et al., 1999.
3.2 Results
In order to determine the activity of Yspl protein, culture supernatants were
analyzed for the presence of AHL signal. Two E. call cultures were grown in LB at
37C, one with pET28a plasmid and the other w-ith pET28a-yspl construct, and
induced with isopropylthiogalactopyranoside (IPTG). After induction, the cultures
46


were centrifuged and the supernatant was decanted and filtered. AHLs present in the
culture supernatant were extracted twice with acidified ethyl acetate (0.1% acetic
acid). The extracts were evaporated to dryness and reconstituted in 100 pL of
methanol. Two dilutions of both extracts were made, 1:50 and 1:100 in methanol, to
observe the range of response of the MG4/pKDT17 lasR-lasB::lacZ detection system.
5 pL of each extract dilution was added to triplicate samples for incubation w ith 100
pL, 300 pL, and 500 pL of grown MG4 pKDTl 7 culture to find the optimal assay
conditions for the expected levels of [Tgalactosidase production (28). After
incubating the samples at 30C for 5.5 hours and measuring their ODfioomnValues, the
Miller assay was performed by adding ONPG to each sample, recording the time
required for the samples to turn yellow, and stopping the reactions with
After the assay, OD420nm and OD^onm values were measured for each sample and the
collected data were entered into a spreadsheet for Miller Unit calculations. Figure 3.2
shows the results of the activity assays for the two E. co/i cultures, labeled yspl(-) and
yspl(+). The most prominent result is the Miller Unit value for the yspl(+)undiluted-
100 sample. This sample demonstrates a saturated response for the lasR-lasB::lacZ
detection system and indicates a high relative concentration of AHL signal in the E.
coli(BL21 )-pET28a-yspI culture supernatant. In addition, the yspl(+) samples all
yielded Miller Unit values outside the margin of error defined by their respective
negative controls.
47


Figure 3.2 Results of Miller Assay to Determine YspI Activity
The above data display Yspl activity in terms of Miller Units. Miller Units are a
measure of the increase of o-nitrophenol per minute per bacterium, which is
proportional to the translation and function of P-galactosidase, which is proportional
to Yspl activity. AHLs were extracted from yspl(-) and yspl(+) culture supernatants
with acidified ethyl acetate (0.1% acetic acid), evaporated to dryness, and
reconstituted in 100 pL of methanol. 1:50 and 1:100 dilutions were made of both
extracts. 5 pL from each extract dilution w'as added to three assay samples prepared
with 100 pL, 300 pL, and 500 pL volumes of MG4/pKDT17 culture, and diluted to 1
mL with Z buffer (see methods). Sample names indicate the source of the extract, a
yspl(-) culture or a yspl(+) culture, the dilution of extract used, and the volume
(microliters) of MG4/pKDTl 7 culture included in the 1 mL assay sample volume.
Theses samples were assayed along with negative controls prepared with 5 pL of
methanol and the indicated MG4/pKDT17 culture volume. The graph shows that
extracts of yspl(-) cultures yield Miller unit values comparable to the negative
controls. Extracts of yspl(+) cultures display increasing activity with increasing
extract concentration. These results show that assay activity is due to Yspl and not
attributable to E. coli or pET28a plasmid alone.
48


Inconsistencies in the data do exist in the comparison of values between the three
subsets of data. For example, the samples yspl(+) 1:50-100, yspl(+) 1:50-300, and
yspl(+)l :50-500 should all have similar Miller Unit values, with the differences in
MG4/pKDT17 culture volume accounted for in the time dependence of each assay
reaction included in equation 3.1. Considering that the reactions are stopped
arbitrarily when "sufficient" yellow color is present, and that light cleaves the (U
glycosidic bond of ONPG, it is understandable that data exhibit some deviation. The
consequence of this is poor reproducibility between samples assayed on different
occasions. However, the relative differences in samples assayed together provide a
reliable comparison. Perhaps the most telling trend in the data presented above is in
the Miller Unit values for the ysp(-) samples. All show values nearly identical to or
less than their respective negative control samples. These values demonstrate that
there is no individual contribution from E. coli or the pET28a plasmid to the AHL
concentration in the culture supernatant. Since the only difference between the
yspl(-) and yspl(+) cultures is the absence or presence of the recombinant Yspl
protein, there is no doubt that the pET28a-yspI construct produces an active product.
3.3 Discussion
The Miller assay is a well established method for detection of (5-galactosidase
activity as a measure of transcriptional activation of a gene of interest fused to the
lacZ gene (29, 30, 31). However, there are no studies with which to compare the
above presented results. The approach of using the lasR-lasB::lacZ detection system
49


of MG4/pKDTl 7 to produce |3-galactosidase in response to AHL signal was reported
as a useful technique for the evaluation of AHL-synthase activity (30). But, the
method has not been used to measure AHL-synthase activity in any Yersinia species
other than what has been presented for Yersinia pesiis YspI protein. Therefore, it is
more pertinent to discuss the potential problems with the use of MG4/pKDTl 7 under
these circumstances. Two factors that address the sensitivity of the detection system,
are LasR-AHL binding and LasR conformational change as a result of AHL binding.
The first factor is the questionable ability of LasR to bind AHLs other than 3-oxo-
C12-AHL. Passador, et al. established the significance of acyl chain length of an
AHL in binding to LasR (31). Effective binding, resulting in the induction of
lasB::lacZ, was not limited to its primary LasR signal 3-oxo-C12-AHL (31). AHLs
having acyl chain lengths of at least eight carbons, and as many as fourteen,
demonstrated an ability to induce transcription of the fusion construct (31). Still, the
response was less sensitive for signals with greater divergence in acyl chain length
from the optimal 3-oxo-C12-AHL (31). Acyl chain length was determined to have
more impact on LasR-AHL binding than C3 substitution or carbon-carbon double
bonds in the acyl chain (31). One step beyond the initial binding of LasR and AHL is
the second factor for consideration, the conformational change of LasR. The
established dimerization in LuxR and TraR that occurs upon binding AHL signal,
suggests the possibility of a similar conformational change in LasR, should AHL
binding occur (2, 32, 33). For the situation in which an AHL other than 3-oxo-C12-
50


AHL is binding LasR, the resulting conformational change may not be sufficient for
dimerization with another LasR-AHL complex, thus diminishing or preventing
transcriptional activation of the fusion construct. However, the actual impact of an
interaction between LasR and a less than optimal AHL is really uncertain, as there is
limited structural understanding of R protein-AHL binding. Despite these aspects of
concern surrounding the use of the MG4/pKDTl 7 for assessment of Yspl activity, the
data presented still offer a relative measure of activity. Controlled responses were
demonstrated in the assay between extracts of two fold concentration difference and
up to the point of a saturated response. The specificity of LasR for AHL signals
produced by Yspl is of minimal importance, as long as some induction occurs, which
clearly was the case. Even though the data produced here would not compare to the
same assay of Lasl, their relative reflection of activity is qualitatively accurate. The
results show that recombinant Yspl produces AHL signal independently.
51


4. AHL Signal Profile for Yspl
4.1 Introduction
The application of tandem mass spectrometry (MS) to the analysis of
biomolecules is well established, particularly since the development of Electrospray
ionization (37). Considering its potential for detailed analysis, and the compatibility
between AHLs and liquid chromatography (LC), LC-MS could serve as an alternative
to the AHL reporter bioassays already discussed (38). The specific advantage gained
by using LC-MS for AHL detection and identification is that the method may have no
bias for signals of particular acyl chain length or substitution. The instrument would
simply identify the contents of a particular culture supernatant extract. For instance, a
triple quadrupole mass spectrometer can perform precursor ion scanning experiments,
in which, the first mass selector (Q1) identifies precursor ions that fragment in Q2 to
a particular product ion detected in Q3. Data of this sort would characterize the
contents of a specific peak for samples initially analyzed by liquid chromatography
and introduced into an MS/MS/MS instrument through electrospray ionization (ESI).
Precursor ion scanning data collected for extracts of E. co//-pET28a-yspl culture
supernatants are presented in this chapter. The data provide an AHL profile for Yspl
and indicate that the protein predominantly produces N-(3-oxooctanoyl)-L-
homoserine lactone (3-oxo-C8-AHL) in E. coli.
52


4.2 Results
Culture AHL extractions of E. co//-pET28a-yspl were prepared for LC-MS
analysis to identify the AHLs produced by Yspl. Two 100 mL cultures of E. coli-
pET28a-yspl were grown in LB at 37C to log phase growth and induced with 0.2
mM 1PTG. After induction, the cultures were centrifuged and the supernatant was
decanted and passed through a 0.22 pm filter. AHLs present in the culture
supernatants were extracted tw o times each with 100 mL portions of acidified ethyl
acetate (0.1% acetic acid). The extracts were dried with MgSC>4, filtered, and
evaporated using a rotary evaporator and a speed vac. The extracts (A and B) were
consolidated to glass vials and then evaporated to dryness. Both extracts were
reconstituted in 1 mL of methanol. Three samples were prepared for both extracts
(A1-A3 and B1-B3) through purification by solid phase extraction. 50 pL of extract
per sample were passed through silica separation packs (Sigma) in 50/50 hexane/ether
and eluted with acidified ethyl acetate. The samples were evaporated to dryness in
autosampler vials, reconstituted in 50 pL of methanol and analyzed by LC-El-MS-
MS-MS (Perkin Elmer). After chromatography with a gradient of 20% methanol (1%
acetic acid) / 80% Water (1% acetic acid) to 95% methanol (1% acetic acid) over 30
minutes, the contents of each peak are analyzed by precursor ion scanning with Q3
set to detect the mass of homoserine lactone product ions of m/z = 102. Upon
53


detection of a product ion of m/z = 102, the m/z of the corresponding precursor ion
from Q1 is identified based on the time required for an ion to travel from Q1 to Q3,
and the rf frequency of Q1 when the precursor ion was filtered. The results of this
analysis are depicted in Figures 4.1 -4.5. Figure 4.1 displays a chromatogram
representative of the six samples analyzed. The chromatogram show s four distinct
AHL signals present in the culture extract. The precursor ion spectrum in Figure 4.2
indicates that the first peak in the chromatogram is primarily composed of material
with mass 214, which is consistent with N-(3-oxohexanoyl)-L-homoserine lactone.
The relatively brief retention time of the peak also supports this conclusion, as the
short carbon chain and of 3-oxo-C6-AHL would have limited interaction with the
Cl8 stationary phase. The C3 substitution contributes to a shorter retention time due
to the decreased hydrophobicity of the compound. The spectrum in Figure 4.3
displays a mass value of 242, which indicates the presence of N-(3-oxooctanoyl)-L-
homoserine lactone in the second peak of the chromatogram in Figure 4.1. Although
the C3 substitution would tend to limit the interaction of the compound with Cl 8, the
additional carbon atoms on the acyl chain begin to counter the tendency, leading to
the increased retention. Figure 4.4 shows a spectrum w ith a major mass value of 228,
suggesting that the third peak in the chromatogram of Figure 4.1 is composed of N-
octanoyl-L-homoserine lactone. The extended retention time is characteristic of a
substantially hydrophobic molecule like the unsubstituted C8-AHL
54


| < 1C of *Pr=c <102 20^ ftom SvPCfie 6 i A3l of 02113 pul2 wW
Ms< 2 2e7ci>s
7rw Figure 4.1 Representative Chromatogram of AHL Extract
A 5 pL injection of sep pack purified sample of AHL Extract was
chromatographically separated into four peaks, not including the peak shoulder at 26
minutes. The peak intensities are reflected in total ion current. The method used a
gradient of 20% methanol to 95 % methanol (both with 1% acetic acid) over 30
minutes. The peak labels indicate software calculated peak areas before manual
integrations were completed. Manually integrated peak area data are presented
below. Precursor ion scans w ere performed for the contents of each peak. The spectra
are shown in the follow ing figures.
55


| Piet; (102 20) 10.677 mm from Sample 6 (A3) of 03113_pul2 Max 9.9e5ops
Figure 4.2 Precursor Ion Mass Spectrum for LC Peak at 10.6 minutes
The mass spectrum indicates that the peak at 10.6 minutes in Figure 4.1 is composed
of material with m/z = 214. This mass is consistent with N-(3-oxohexanoyl)-L-
honioserine lactone.
56


| *Prec *102 20) 21 85$ nvn horn Sample 6 Mtx 2 0e cps
mtz arrvti
Figure 4.3 Precursor Ion Mass Spectrum for LC Peak at 21.8 minutes
The mass spectrum indicates that the peak at 21.8 minutes in Figure 4.1 is composed
of material with m/z = 242. This mass is consistent with N-(3-oxooctanoyl)-L-
homoserine lactone.
57


| -PecpQ2 20) 27 545 ">10 from Ssmpl 6 < A3) of 03113_pa-ji2 vtfT
Mdi 3 2e5 cp>
mi*£. amu
Figure 4.4 Precursor Ion Mass Spectrum for Peak at 27.9 minutes
The mass spectrum indicates that the peak at 27.9 minutes in Figure 4.1 is composed
of material with m/z = 228. This mass is consistent with N-octanoyl-L-homoserine
lactone.
58


I *Pec *102.20) 28 962 srun from Sample 6< A3V of 03113_p9'-i2 vt3
Max 3 0e5 cps
Figure 4.5 Precursor Ion Mass Spectrum for Peak at 28.6 minutes
The mass spectrum indicates that the peak at 28.6 minutes in Figure 4.1 is composed
of material with m/z = 270. This mass is consistent with N-(3-oxodecanoyl)-L-
homoserine lactone.
59


The mass spectrum in Figure 4.5, identifying the contents of the last peak of the
chromatogram in Figure 4.1, presents a mass of 270, the mass of N-(3-oxodecanoyl)-
L-homoserine lactone. In this case, the hydrophobicity of the chain greatly reduces
the effect of C3 substitution, leading to substantial retention by Cl8. Among the four
signals displayed in the chromatogram in Figure 4.1, the first and second peaks, 3-
oxo-C6-AHL and 3-oxo-C8-AF!L, clearly stand out as the major signals produced by
YspI in E. coli. To establish some consistent treatment of the data from one
chromatogram to the next, the Analyst software was used to perform manual
integrations on these two peaks for all six extract samples analyzed (manually
integrated peaks not shown). Table 4.1 displays the data, along with standard
deviations calculated using Microsoft Excel. Table 4.1 presents a comparison of the
average peak areas of 3-oxo-C6-AHL and 3-oxo-C8-AHL, along with standard
deviations. The samples analyzed from both extracts A and B yielded average peak
area values for 3-oxo-C8-AFlL that were two times greater than the average peak area
values for 3-oxo-C6-AFlL. In addition, standard deviation calculations indicated that
the ranges of error for the mean peak area values did not overlap. The data indicate
that Yspl, expressed in E. coli, produces 3-oxo-C8-AHL more abundantly than 3-oxo-
C6-AHL.
60


Extract Sample Component Peak Area Mean Standard Deviation
A1 3-oxo-C8 7.29E+08
A2 3-oxo-C8 7.61E+08 8.24E+08 1.38E+08
A3 3-oxo-C8 9.82E+08
A1 3-oxo-C6 2.70E+08
A2 3-oxo-C6 3.99E+08 3.59E+08 7.72E+07
A3 3-oxo-C6 4.08E+08
B1 3-oxo-C8 7.47E+08
B2 3-oxo-C8 7.85E+08 8.05 E+08 7.07E+07
B3 3-oxo-C8 8.84E+08
B1 3-oxo-C6 3.05E+08
B2 3-oxo-C6 4.25E+08 3.89E+08 7.27E+07
B3 3-oxo-C6 4.36E+08
Table 4.1 Peak Area Data
The Analyst software was used to manually integrate the first two peaks of the
chromatograms for each extract sample. The average peak areas of 3-oxo-C6-AHL
and 3-oxo-C8-AHL are compared along with their standard deviations. For both the
extract A and extract B sets of data, the mean peak area of 3-oxo-C8-AHL is two
times greater than the mean peak area of 3-oxo-C6-AHL. Their standards of
deviation do not overlap. The data indicate that YspI produces 3-oxo-C8-AHL more
abundantly than 3-oxo-C6-AHL when expressed in E. tali.
61


4.3 Discussion
To date, there is one documented reference that comments briefly on the topic
of quorum sensing in Yersinia peslis (39). Aside from this, two studies were
published that discuss quorum sensing in Yersinia pseudotuberculosis (24, 26) and
apply to Y. pestis. Both of these studies involve the Yersinia pseudotuberculosis
AHL-synthase Ytbl, which is known to be identical in protein sequence to Yspl of
Yersinia pestis (23). The results presented above for AHL signal production in Yspl,
differ from that documented for Ytbl (24, 26). Atkinson, et al. (24) reported that E.
coli (JM 109)-ytbl clones yielded culture supernatant extracts containing C6-AHL and
C8-AHL, as detected by CV026 TLC overlay assay (14). The precursor ion scanning
data, presented above, did not detect the presence of C6-AHL, although C8-AHL was
present in a significant amount. Atkinson, et al. (24) also reported a TLC spot
migrating between C6-AHL and C8-AHL that went unidentified. A compound that
migrates in this manner is consistent with 3-oxo-C8-AHL, which was certainly
present in E. co//-pET28a-yspl culture supernatant extracts, but only detected at
minor levels with the CV026 TLC overlay bioassay (24). Differences between E. coli
JM 109 cells and E. coli BL21 cells are not enough to account for the disparity
between the AHL signal profile obtained for Ytbl and that obtained for Yspl.
62


The mass spectrometry data presented in Figures 4.2 4.5 are critical to the
conclusion that N-(3-oxooctanoyl)-L-homoserine lactone is the predominantly
produced AHL signal in E. co//-pET28a-yspl cultures. The molecular assignments
made for the separated peaks of the chromatogram in Figure 4.1 are based on the m/z
values (mass 214 corresponds to 3-oxo-C6-AFIL, etc.) obtained from this data.
Precursor ion scanning provides confidence beyond a simple matching of m/z values
with a consistent AHL structure. The scan in Q3 for the product ion of m/z = 102 is
more specific, in that a Q1 detected mass is only recorded if it later fragments to the
homoserine lactone mass of 102. The possibility does exist that a precursor ion,
present in an extract sample, could have the mass of a common AHL and fragment to
an m/z of 102, but not actually be an AHL. But, this level of coincidence is unlikely.
There are, however, three issues that influence the quantitative interpretation of the
data presented in section 4.2. The first issue is the ionization of the AHLs. Although
the mechanism of ionization is not known, the ionized form in which AHLs enter Q1
may involve an oxonium ion at the Cl carbonyl of the acyl chain. This ionized form
may be more stabilized in 3-oxo substituted AHLs due to the presence of the C3
carbonyl. As a result, fewer unsubstituted AHL ions may exist due to their
instability. Less effective ionization of unsubstituted AHLs would lead to a
decreased detection of their presence and an exaggerated response for 3-oxo
substituted AHLs. The second issue is the fragmentation of AHL signals upon
entering Q2. Since the peaks displayed in Figure 4.1 are associated with specific m/z
63


ratios, their composition is firmly established. Their individual intensities, though,
are a measure of total ion current for detected product ions of m/z = 102. If all four of
the reported AHLs fragment to yield the same relative amount of product ion with
m/z = 102 in a mole per mole fashion, the intensities shown are authentic. In
contrast, if the AHLs do not fragment with the same efficiency, the resulting
intensities may not be indicative of their actual abundance. Thus, it must be
demonstrated that 1 mol of 3-oxo substituted AHL fragments to yield the same
amount of product ion m/z = 102, as 1 mol of unsubstituted AHL. The third issue is a
consideration of both the solvent extraction and the solid phase extraction of AHL
signal. The AHLs contained in an extract and identified by LC-MS, should
accurately reflect the AHLs present in the culture supernatant under scrutiny. It is
possible that culture supernatant extraction with ethyl acetate may bias the subsequent
analysis to detect signals that are more soluble in ethyl acetate. In a similar manner,
purification of AHL extract by silica solid phase extraction, may lead to loss of
certain AHLs during washing steps with hexane, creating another bias in AHL
detection. These issues can only be addressed through control experiments to identify
the bias and correct for it. Since no such experiments were presented here, the
relative amounts of the different AHLs reported are not firmly established and cannot
be thoroughly compared to other published results.
The difference noted between the results reported for this study of YspI and
other related studies of Ytbl is distinct. The key difference presented here, is the
64


presence and apparent abundance of N-(3-oxooctanoyl)-L-homoserine lactone
detected in E. co//-pET28a-yspI culture supernatant extracts. The results obtained
from the analysis of culture supernatant extracts of E. coli (JM 109)-ytbl using the
CV026 TLC overlay bioassay, indicated an abundance of C6-AHL and the presence
of C8-AHL. The environment in which AHL-synthases are produced could have an
impact on the generated AHL profile. AHL-synthases expressed in different strains
of E. coli, versus those expressed in their native organism, may have access to
different concentrations and derivatives of substrate in their respective acyl-ACP
pools. However, the differences between E. coli strains JM109 and BL21 are few, and
so could not significantly affect the AHL profiles of Ytbl or YspI, under comparison
here. Control experiments assessing the impact of LC-MS response and potential loss
of AHL signal during extraction and sample preparation would help to plainly
establish the AHL profile in YspI. Still, an excess of evidence points to the
conclusion drawn from the data in section 4.2 that N-(3-oxooctanoyl)-L-homoserine
lactone is the primary AHL signal produced by YspI.
65


5. Conclusions and Future Studies
5.1 Conclusions
The molecular cloning efforts, discussed in Chapter 2, led to the successful
construction of an E. co//-pET28a-yspl clone. Although it was used primarily as an
AHL generator in support of LC-MS determination of the AHL signal profile for
Yspl, the same construct was needed to overexpress the YspI protein for purification.
Purified protein is required, and at reasonably high concentration, for the preparation
of crystal trials. The protein purification results presented in Chapter 2 demonstrated
that very pure protein was attainable and was consistent with Yspl. The process
needs only to be scaled up for higher concentrations of pure protein.
The results presented in chapter 3 are significant in their own right, as the E.
co//-pET28a-yspl construct was shown to produce independently active Yspl protein.
The activity assay employed both the use of the MG4/pKDTl 7 reporter strain and the
Miller assay for measurement of P-galactosidase activity. The specific AHL signals
produced were not identified with this assay, but the association of Yspl with AHL
signal production was established. An extract of culture grown from the construct
containing the yspl gene yielded Miller Unit values above background levels. An
extract of culture grown from E. ro//-pET28a without the yspl gene yielded Miller
Unit values in the background level. Tlius, presence of the yspl gene in the construct
66


corresponded to activity and indicated that its product, YspI, must be involved in
signal production.
The most substantial result for this study was the data obtained by LC-MS
analysis of the E. co//-pET28a-yspI culture supernatant extracts, which showed an
abundance of N-(3-oxooctanoyl)-L-homoserine lactone. Previous studies of the
closely related Ytbl of Yersinia pseudotuberculosis, reported the production of
different AHLs as quorum sensing signals, although Ytbl has sequence identity to
YspI (23, 24, 26). An AHL profile established for Yspl from LC-MS analysis was
identified from a culture supernatant extract of E. coli BL2 l-pET28a-yspI. This
profile did not match the profile reported for recombinant Ytbl in E. coli JM109. The
most likely explanation for the differing results is that LC-MS analysis of culture
supernatant extracts is a more objective method for detecting the presence of different
AHL signals. The amounts of the AHLs reported to be present in the YspI extract
indicated that 3-oxo-C8-AHL is the most abundant signal produced by YspI in E.
coli. Until control experiments are performed to account for response due to
fragmentation and potential loss of AHL during extraction, the results presented must
be viewed qualitatively. Yet, when considering 3-oxo-C8-AHL and 3-oxo-C6-AHL,
it is difficult to imagine their fragmentation patterns differing by a significant value.
The likelihood that these tw o signals fragment to similar amounts of product ion m/z
= 102 is high, considering that they only differ by two carbon atoms. Regardless of
the accuracy of the AHL intensities displayed in Figure 4.1, 3-oxo-C8-AHL is
67


definitely present and C6-AHL does not appear to be present. The results presented
here have helped to identify the next stages of research that will continue to increase
general knowledge of quorum sensing and further the understanding of this
phenomenon in Yersinia pestis.
5.2 Future Studies
An initial goal in continued studies would be to identify the purified protein as
Yspl by performing a western blot. The purification procedure could then be scaled
up to produce excess pure Yspl for crystal trials and kinetic assays. Another goal
would be to identify an AHL profile of Yspl in Y. pestis culture. Theoretically, the
pool of available acyl-AC'P molecules in E. coli might be different from the available
pool in Yersinia pestis. Supernatant extracts from Yersinia pestis culture, subjected to
LC-MS precursor ion scanning analysis, would go a long way toward establishing the
significance of the available acyl-ACP pool to the resulting AHL profile. For this
reason, the LC-MS analysis performed for the E. co//-pET28a-yspl culture
supernatant extracts should also be performed for extracts from Yersinia pestis, in
order to identify an accurate AHL profile. To address the uncertainty of instrument
response during LC-MS analysis, known amounts of synthetic AHL standards should
be analyzed by LC-MS in order to assess the potential differences in fragmentation of
AHL molecules, especially 3-oxo substituted AHLs versus unsubstituted AHLs.
These goals would allow for a more complete characterization of Yspl.
68


6. Methods
6.1 Molecular Cloning
6.1.1 Plasmid DNA Purification by Miniprep
An overnight culture off", coli containing the plasmid of interest was grown
in Luria-Bertani (LB) medium, with lOOpg/mL ampicillin or 50 pg/mL kanamycin, at
37C. One miniprep was performed for each 5 mL aliquot of overnight culture. The
culture was transferred to tubes and centrifuged to pellet the cells. After discarding
the supernatant, the pellet was resuspended in 250 pL of buffer PI (RNAse A
[primary ingredient], Qiagen). Next, 250 pL of buffer P2 (sodium hydroxide
[primary ingredient], Qiagen) was added to lvse the cells and the tube was inverted
gently to mix. Within 1 minute, 350 pL of buffer N3 (guanidine hydrochloride, acetic
acid [primary ingredients], Qiagen) was added to precipitate the cell debris in the
lysate, followed by immediate inversion to mix. The lysate was then centrifuged for
10 minutes at 16.1 kref to pellet the debris. The resulting supernatant was added to a
QIAprep (Qiagen) spin column (silica gel membrane) and centrifuged for 1 minute at
16.1 kref. The flow through was discarded. The spin column was then washed by
adding 500 pL of buffer PB (guanidine hydrochloride, isopropanol [primary
ingredients], Qiagen) and centrifuging for 1 minute at 16.1 kref. The flow through
was discarded. The spin column was washed a second time by adding 750 pL of
69


buffer PE (Ethanol [primary ingredient], Qiagen) and centrifuging for 1 minute. The
flow through was discarded and the spin column was centrifuged for an additional 1
minute to remove residual wash buffer. The plasmid DNA was eluted from the spin
column into a clean 1.5 mL mierofuge tube by adding 50 pL of buffer EB (10 mM
Tris-Cl, pH 8.5, Qiagen), allowing to stand for 1 minute, and centrifuging for 1
minute.
6.1.2 Phenol Extraction / Ethanol Precipitation of
Plasmid DNA
To plasmid samples obtained from minipreps, TE (lOmM Tris pH 7.4, 1 mM
EDTA) buffer was added to a total volume of 150 pL. 150 pL of
phenol:chloroform:isoamyl alcohol (25:24:1) was also added to the sample, which
was vortexed well and centrifuged for 3 minutes at 16.1 krcf. The top layer
(aqueous), containing isolated plasmid DNA, was removed and delivered to a
separate mierofuge tube labeled "aqueous". An additional 100 pL of TE (lOmM Tris
pH 7.4, 1 mM EDTA) was added to the original sample tube, which was vortexed and
centrifuged as before. Again, the top layer was removed and delivered to the
"aqueous" mierofuge tube.
400 pL of ether was added to the "aqueous" mierofuge tube to trap residual
phenol. The tube was inverted gently and centrifuged for 1 minute at 16.1 krcf. The
top layer (organic) was aspirated off with a needle. Sample treatment with ether was
repeated two more times.
70


After treatment with ether, 1/10 the sample volume of 3 M sodium acetate was
added to the sample. Next, 3 times the sample volume of 100% ethanol was added,
followed by gentle inversion, to precipitate the plasmid DNA. The sample was
centrifuged for 10 minutes at 16.1 kref. Following the spin, all but 50 pL of
supernatant was carefully removed. 400 pL of 80% ethanol was then added and the
sample was centrifuged again for 10 minutes at 16.1 krcf. After the second spin, all
but 50 pL of supernatant was removed. Finally, the sample was completely dried
down in a speed-vac (Savant) and resuspended in 50 pL of TE (10 mM Tris pH 7.4
and 1 mM EDTA).
6.1.3 Plasmid DNA Purification by G50 Spin Column
750 pL of Sephadex G50 (Amersham Pharmacia) / water slurry was delivered
to the filter top of a spin-x tube and centrifuged at 700 ref for 1 minute. The resin
was washed tw ice with 375 pL volumes of Milli-Q water, centrifuging at 700 ref for
1 minute after the first wash. After the second wash the centrifuge time was 3
minutes to remove residual water. The DNA to be purified was added to the washed
resin in the filter top of the spin-x tube, centrifuged at 700 ref for 3 minutes, and
recovered in the filtrate. For most purifications performed in this manner, the volume
of the DNA sample was increased slightly by residual Milli-Q water.
6.1.4 Llftraviolet/Visible Spectroscopy
100 pL of concentrated or diluted sample DNA was loaded into a 100 pL
cuvette. An absorbance scan was performed from 320 nm to 220 nm using a DU 520
71


spectrophotometer (Beckman). The absorbance value at 320 nm was subtracted from
the absorbance value at 260 nm and difference was multiplied by 50 ng/'pL (average
concentration per absorbance unit of double stranded DNA). If a dilution was made,
the product of A260nm and 50 ng/pL was multiplied by the dilution factor.
6.1.5 Restriction Endonuclease Digestion of pET28a
Overnight cultures of plasmid DNA vector, pET28a, grown in Terrific Broth
(TB) medium at 37C with 50 pg/mL kanamycin, were purified by miniprep and
phenol extraction / ethanol precipitation to 166.5 ng/pL in 40 pL. 21 pL of pET28a
was incubated in a microfuge tube with 2.5 pL of lOx NE Buffer 2(10 mM Tris-HCl,
10 niM MgCb, 50 mM NaCl, 1 mM Dithiothreitol, pH 7.9, New England BioLabs)
and 2 pL of Ndel restriction endonuclease for 1.5 hours at 37C. A "HindiII control"
(1.5 pL pET28a, 1 pL lOx NE Buffer 2, 1 pL Hindi 11 restriction endonuclease, and
6.5 pL Milli-Q water) and an "uncut control" (1.5 pL pET28a, 1 pL lOx NE Buffer 2,
and 7.5 pL Milli-Q water) were incubated in separate microfuge tubes along with the
digest. After incubation, 2 pL of the digest was removed to a separate tube for
comparison w ith the two controls by agarose gel electrophoresis. Additional amounts
of 2.5 pL of 1 Ox NE Buffer 2, 1 pL of Ndel, 1 pi of H ind III, and 20 pL of Milli-Q
water were added to the digest, which was incubated for 1 hour more. Finally, the
solution of digested pET28a was heated to 65C for 20 minutes, to inactivate the
restriction enzymes, and stored at -20C.
72


6.1.6 Restriction Endonuclease Digestion of pCR2.1-yspl
Overnight cultures of plasmid DNA containing the yspl insert, grown in
Terrific Broth (TB) medium at 37C with 100 pg-'mL ampicillin, were purified by
miniprep and phenol extraction / ethanol precipitation to 1714 ng/'pL in 40 pL. 6 pL
of pCR2.1-yspI was incubated in a microfuge tube with 2.5 pL of 10.x NE Buffer 2
(New England BioLabs), 2 pL of Ndel restriction endonuclease, and 14.5 pL of Milli-
Q water for 1.5 hours at 37C. An "uncut control" (1 pL pET28a, 1 pL 1 Ox NE
Buffer 2, and 8 pL Milli-Q water) was incubated in a separate microfuge tube along
with the digest. After incubation, additional amounts of 2.5 pL of lOx NE Buffer 2, 1
pL of Ndel, 1 pi of Hindlll, and 20.5 pL of Milli-Q water were added to the digest,
which was incubated for 1 hour more.
6.1.7 Gel Extraction of yspl DNA Insert
The solution containing Ndel-Hindlll digested plasmid DNA, pCR2.1, and
DNA insert, yspl, was loaded onto a 1.2% agarose gel containing ethidium bromide
and electrophoresed. The DNA insert band was visualized in the gel via an
ultraviolet light source and excised with a razor blade. The cut segment of gel
containing the DNA insert was placed in a tared microfuge tube and weighed. 300
pL of buffer QG (guanidine thiocyanate [primary ingredient], Qiagen) per 100 mg of
gel was added to the tube. A maximum of 400 mg of gel w as allowed for one
73


QIAquick (Qiagen) spin column. The tube was then incubated for 10 minutes at 50C
and vortexed until completely soluble in a yellow solution. If the color was orange or
violet, lOpL of 3 M sodium acetate, pH 5, was added to yield a yellow solution. The
color of the solution reflected its pH, which should be < 7.5 for efficient binding of
the DNA to the spin column. Next, 100 pL of isopropanol per 100 mg of gel was
added to the sample to maximize DNA yield. The DNA sample was loaded onto a
QIAquick (Qiagen) spin column and centrifuged for 1 minute at 16.1 krcf. The flow
through was discarded. To remove residual agarose, 500 pL of buffer QG (Qiagen)
was loaded onto the spin column and centrifuged for 1 minute at 16.1 krcf. To wash
the DNA, 750 pL of buffer PE (Qiagen) was loaded onto the spin column and
centrifuged for 1 minute 16.1 krcf. The flow through was discarded and the column
was centrifuged for an additional 1 minute to remove residual wash buffer. The DNA
was eluted into a clean niicrofuge tube by adding 50 pL of buffer EB (10 rnM Tris-
HC1, pH 8.5) and centrifuging for I minute at 16.1 krcf. The eluted yspl insert was
then purified by passage through a G50 spin column. A final concentration of 18.5
ng/pL in 85 pL was determined by UV/Vis absorbance. The solution was stored in
EB at -20C.
6.1.8 Phosphatase and DNA Ligase Reactions
The solution containing Ndcl-Hindl 11 digested plasmid DNA, pET28a, was
removed from -20C storage and thawed on ice. 10 pL of lOx Calf Intestine Alkaline
Phosphatase reaction buffer (0.1 M Tris-HCl pH 7.5, 0.1 M MgCE, MB1 Fermentas),
74


1 pL of Calf Intestine Alkaline Phosphatase (MBI Fermentas), and 31 pL of Milli-Q
water were added to the solution, which was then incubated at 37C for 30 minutes.
The phosphatase treated pET28a was purified by phenol extraction / ethanol
precipitation and passage through a G50 spin column. A final concentration of 18.5
ng/pL in 40 pL was determined by UV/Vis absorbance.
Ligation of yspl insert into pET28a was performed in two reactions with
different amounts. Reaction A contained 6 pL of pET28a, 6 pL of yspl, 1 pL of lOx
ligase buffer (300 mM Tris-HCl pH 7.8, 100 mM MgCL, 100 mM Dithiothreitol, 10
mM ATP, Fisher), and 1 pL of T4 DNA Ligase (Fisher). Reaction B contained 4 pL
of pET28a, 8 pL of yspl, 1 pL of lOx ligase buffer, and 1 pL of ligase. A negative
control was also prepared and contained 4 pL of pET28a, 1 pL of lOx ligase buffer, 1
pL of ligase, and 8 pL of Milli-Q water. The reactions and the control were
incubated at room temperature for 1.5 hours.
6.1.9 Transformation by Electroporation
The plasmid DNA to be transformed into E. coli strain DH5a was diluted in
water to ~1 ng/pL. In the case where the plasmid DNA construct to be transformed
was obtained from the ligation reaction of yspl and digested, phosphatase treated
pET28a vector, a 1:5 dilution of the ligation reaction was made. Electrocompetent E.
coli (DH5a) cells were removed from -80C storage and briefly thawed on ice. 1 pL
of diluted DNA construct was added to the electrocompetent cells. The cells were
transferred from an o-ring tube to a chilled electroporation cuvette using a transfer
75


pipette. Care was taken to ensure that no air bubbles were introduced, and that the
cells were spread evenly at the bottom of the cuvette. The conductive sides of the
cuvette were positioned between the two leads of a (BioRad Gene Pulser 1652076)
and the cell contents were pulsed with a potential of 2.5 V. 900 pL of chilled SOB (In
1 L Milli-Q water: 20 g tryptone, 5 g yeast extract, and 0.5 g of NaCl) medium was
immediately added to the cuvette. The entire contents were then transferred back to
the original o-ring tube and placed in an incubator at 37C, 225 rpni, for 1 hour. After
incubation, 100 pL of cells in SOB were streaked onto agar plates containing 50
pg/mL kanamycin. The plates were incubated at 37 for 16-20 hours.
6.1.10 Polymerase Chain Reaction (PCR)
Overnight cultures were grown from sixteen individual colonies obtained
from the transformation of pET28a-yspI into E. coli (DH5a). Eighteen microfuge
tubes were prepared containing 12.5 pLof 2x Taq Master Mix (Taq DNA
polymerase, KC1, (NHQSO^ MgCb, dNTPs, to final concentrations 1.5 mM MgCE
and 200 pM dNTPs, Qiagen), 0.625 pL of 40 pM T7 Promoter Primer (5'-
TAATACGACTCACTATAGGG-3', Qiagen), 2.5 pL of T7 Terminator Primer (5'-
GCTAGTCATTGCTCAGCGG-3Qiagen), and 6.375 pL of distilled water. 3 pL of
DNA construct from each miniprep was added to a prepared tube. Two tubes were
reserved for negative and positive controls, to which no DNA and 3 pL supercoilcd
pET28a were added, respectively. 25 pL of mineral oil was overlayed in each sample
and control to prevent rapid boiling. The samples and controls were placed in a
76


Programmable Thermal Controller (MJ Research, Inc.) and incubated through a series
of temperature cycles according to the TOUCH 69 program [(1) 10 min at 94 (2)
1.25 min at 94 (3) 1.25 min at 69 (4) 1 min at 72 (5) return once to step "2" (6) 1.25
min at 94 (7) 1.25 min at 68 (8) 1 min at 72 (9) return once to step "6"...etc....(87)
1.25 min at 59 (88) 1 min at 72] After incubation, portions of each sample were
visualized by electrophoresis on a 2% agarose gel.
6.1.11 Transformation by Heat Shock
An overnight culture of pET28a-yspI in E. coli (DH5a) cells was grown. The
DNA construct was isolated and purified by miniprep. Chemically competent cells of
E. coli strain BL21 were removed from -80C storage and briefly thawed on ice. 1
pL of the construct in solution was added to the chemically competent cells. The
cells were incubated on ice for 1 hour. After incubation, the cells were place in a
42C water bath for 1.5 minutes for heat shock. The cells were immediately placed
on ice for 2 minutes. 1 mL of SOC (SOB + 10 mM MgS04, 10 mM MgCb, and 20
mM Glucose) medium was added to the cells, which w'ere then incubated at 37C,
225 rpm for 1 hour. 100 pL of the incubated cells were streaked onto agar plates
containing antibiotic (50 pg/mL kanamycin or 100 pg/mL ampicillin). The plates
were incubated at 37 for 16-20 hours.
77


6.2 Protein Purification
6.2.1 Protein Expression
An overnight culture was initiated in Luria Broth (LB) rich media from a
frozen glycerol stock of E. coli(BL21 )-pET28a-yspl and incubated at 37, 200 rpm.
Kanamycin was added to the culture at a concentration of 50 pg/mL for selectivity.
After 12-18 hours of growth, six 1 L volumes of LB were each inoculated with 10 mL
of the overnight culture. The 1 L cultures were grown at 37C until reaching an
A600nm of 0.6. Expression of Yspl was induced with 0.2 mM isopropyl-|TD-
thiogalactopyranoside (IPTG) in each culture and incubation for 1 hour at 25C, 175
rpm. The cultures were transferred to bottles and centrifuged at 8000 rpm for 10
minutes to form cell pellets. The supernatant was decanted and the pellets were ready
for lysis (or temporary storage at -20C).
6.2.2 Cell Lysis and Ni-NTA Affinity Chromatography
A cell pellet from 500 mL of culture was resuspended in 5 mL of lysis buffer
(10% glycerol, 20mM Tris pH 8, 500 mM NaCl, 1 mM (Tmercaptoethanol) with 1
mg/mL lysozyme and 40 pg/mL DNAse. The resuspended cells were incubated on
ice for 30 minutes and then sonicated 3 times for 30 seconds. The lysate was
centrifuged at 18k rpm for 25 minutes and the resulting supernatant was decanted into
a new tube. 750 pL of Ni-NTA bead slurry (Qiagen) was added to the lysate
78


supernatant and the mixture was agitated for 2 hours at 4C. After incubation, the
beads were centrifuged at 700 ref for 5 minutes and the supernatant containing
unbound protein was draw n off. 10 mL of wash buffer (10% glycerol, 20 mM Tris
pH 8, 500 mM Nad, 10 mM imidazole) was added to the beads, which were
resuspended and centrifuged as before. The bead wash supernatant was removed and
the wash was repeated four more times. After the final manual wash, the beads were
resuspended in wash buffer and transferred to a C-IO column (Amersham Pharmacia).
The column of Ni beads was washed for 15 minutes with wash buffer. Bound protein
was then eluted from the Ni beads with a linear gradient of 0% B to 100% B over 15
minutes (A: wash buffer / B: elution buffer (10% glycerol, 20 mM Tris pH 8, 500
mM NaCl, 250 mM imidazole, 1 mM p-mercaptoethanol)) and collected in 250 pL
fractions.
6.2.3 Size Exclusion Chromatography
Fractions containing protein eluted from the column of Ni-NTA beads were
pooled, filtered using a spin-x (Corning) microfuge tube, and injected onto a
Superdex 75 (Amersham Pharmacia) size exclusion column for isocratic separation in
Superdex 75 buffer (10% glycerol, 20 mM Tris pH 8, 500 mM NaCl, 10 mM
Dithiothreitol) at 1 mL'min flow rate.
79


6.3 YspI Activity
6.3.1 AHL Extraction
A culture of E. coli(BL21 )-pET28a-yspI in 10 mL Luria-Bertani (LB)
medium with 50 pg/rnL kanamycin was prepared from a frozen glycerol stock and
incubated overnight at 37C and 225 rpm. After 12-18 hours of incubation, 2 mL of
the overnight culture was added to 100 mL of LB w ith 50 pg/mL kanamycin. After
5.75 hours of incubation, the 100 mL culture reached an OD of 0.603. The culture
was induced with 0.2 mM isopropylthiogalactopyranoside (IPTG) and incubated at
37C for 1 hour. After induction, the culture was transferred to centrifuge tubes and
centrifuged at 4000 rpm for 10 minutes. The culture supernatant was decanted and
passed through a 0.22 pm disposable filter. AHLs present in the E. coli(BL21 )-
pET28a-yspl culture growth were extracted twice from the filtered culture
supernatant with 100 mL of ethyl acetate acidified with 0.01% acetic acid. The
extract was dried with anhydrous MgS04, poured through filter paper to remove
MgS04, and collected in a round bottom flask. The extract was evaporated to dryness
using a rotary evaporator (Buchi Rotovapor 210), leaving an oily residue on the inside
of the round bottom flask. 6 mL of acidified ethyl acetate was added to the flask to
reconstitute the extracted AHLs. The reconstituted AHLs were transferred to fraction
tubes and dried down in a speed vac. This process was repeated two additional times.
80


During the final cycle, the reconstituted AHLs were consolidated in a screw cap
autosampler vial before complete evaporation, and then dried completely in the vial
using a speed vac. The dried AHL extract was stored at -20C. All glassware used
for the extraction was washed with concentrated HC1 and rinsed with Milli-Q water
and ethanol prior to use.
6.3.2 Activity Assay of AHL Extract
A 10 mL culture of E. coli strain MG4/pkDT17A in LB, with 100 pg/mL
ampicillin, was prepared from a frozen glycerol stock and incubated overnight at
30C and 225 rpm. After 12-18 hours of incubation, the overnight culture was diluted
to an optical density (OD) of 0.109 with A medium (60 mM K2HPO4, 33 mM
KH2PO4, 7.5 mM (NH4)2S04, 1.7 mM sodium citrate, 0.4% glucose, 0.05% yeast
extract, 1 mM MgSCL (added after autoclaving) ). The AHL extract was
reconstituted with 1 mL of methanol. From this concentrated solution, 1:50 and
1:100 dilutions of extract were made in methanol. 5 pL of the 1:50 and 1:100 extract
dilutions were added to separate tubes in triplicate and evaporated to dryness.
Negative controls were prepared by adding 5 pL of methanol to each of three tubes
and evaporating to dryness. Three samples were prepared in the tubes of dried
material from each solution and the negative control. 100 pL of diluted overnight
culture and 900 pL of Z buffer (0.06 M Na2HP04-7H20, 0.04 M NaH2P04 H20, 0.01
M KC1, 0.001 M MgS04-7H20, 0.05 M P-mercaptoethanol, adjusted to pH 7) were
added to one tube. 300 pL of diluted overnight culture and 700 pL of Z buffer were
81


added to a second tube. 500 f.iL of diluted overnight culture and 500 pL of Z buffer
were added to a third tube. A concentrated sample was prepared by adding 5pL of
undiluted extract to a tube, evaporating to dryness, and adding 100 pL of diluted
overnight culture plus 900 pL of Z buffer. The ten samples were incubated at 30C
for 5.5 hours and 225 rpm. After incubating, the OD of each sample was measured at
600 nrn. 2 drops of chloroform and 1 drop of 0.1% SDS were added to each of the
ten tubes, followed by vortex mixing. The assay reactions were started by adding
200 pL of 4 mg/mL o-nitrophenyl-fl-D-galactopyranoside (ONPG) to each tube.
After noticeable yellow color appeared, the reaction was stopped with 500 pL of 1 M
NaiCCb and the time of the reaction was recorded. The OD of each sample was then
measured at 420 nm and 550 nm.
6.4 AHL Profile
6.4.1 LC-MS Sample Prep: Solid Phase Extraction
A 50/50 hexane/ether mixture was prepared for solid phase extraction of Yspl
extract. The hexane was first passed through silica solid phase extraction columns
(sep pack), using a vacuum manifold, to remove any trace contaminants from the
solvent. The pure hexane was then added to an equal amount of ether. New silica
sep packs were then washed with 7 mL hexane/ether, 7 mL acidified ethyl acetate
(with 0.1% acetic acid), and 7 mL of hexane/ether prior to application of Yspl extract
sample. One sep pack was washed for each sample to be purified.
82


100 pL of the reconstituted Yspl AHL extract described in section 6.2.2 was
mixed with 400 pL of hexane/ether and added to a washed sep pack along w ith 2 mL
hexane/ether. After allowing the sample to bind, the sep pack was washed with 7 mL
of hexane/ether. The bound sample was then eluted from the silica into a clean
collection tube with 7.5 mL acidified ethyl acetate. The contents of the collection
tube were dried down in a speed vac to -500 pL, and then transferred to an
autosampler vial. The sample was then evaporated to dryness and reconstituted with
50 pL methanol.
6.4.2 LC-MS Analysis
Prepared samples were analyzed by LC-Tandem Mass Spectrometry on a
triple quadrupole instrument (Perkin Elmer). Injected samples were
chromatographically separated using a gradient of 20% B to 95% B over 30 minutes
(Mobile Phase A: milli-Q water, 1% acetic acid / Mobile Phase B: methanol, 1%
acetic acid) at a 50 pL/min flow' rate through a C18 column. Components eluting
from the HPLC were ionized by electrospray ionization (ESI) and identified by
precursor ion scanning with Q3 set to detect product ions with m/z of 102.
83


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