METAL RESISTANCE AND DETOXIFICATION OF AN ENVIRONMENTAL
Duried Mawaheb Kassab
B.S., University of Colorado at Denver, 2000
B. A., University of Colorado at Denver, 2000
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
This thesis for the Master of Arts
Duried Mawaheb Kassab
has been approved
Charles A. Ferguson
Kassab, Duried Mawaheb (M.A., Biology)
Metal Resistance and Detoxification of an Environmental Pseudomonas putida
Thesis directed by Assistant Professor Timberley M. Roane
The overall objective of this study was to identify mechanisms of resistance
amenable to metal remediation. This study examined the microbial populations in
metal-impacted soils collected from the Northern Idaho Silver Valley Mining
Region. While this soil has been characterized and several microbial populations
were isolated, one isolate in particular, Isolate S8A, later identified as,
Pseudomonas putida, exhibited a bimodal growth response in the presence of metal,
prompting further investigation of this organism. Pseudomonas putida S8A was
resistant in up to 200mg/L soluble cadmium and 300mg/L soluble lead. S8A (from
106 to 109 cells/ml) in the presence of up to 80mg/L cadmium reduced soluble
cadmium concentrations from 26% to 38%, while reducing soluble lead
concentrations up to 44mg/L by 45% to 83%. During growth experiments, this
Pseudomonas putida exhibited two distinct colony morph-o-types: one runny
(unusual) and the other convex and round (usual). This prompted an investigation
into the two morph-o-types to identify any physiological and genetic differences
between the two. Standard screening tests, including cell morphology, gram
reaction, motility, metabolic and genetic fingerprinting, production of an exo-
polymer, and finally 16s rDNA identification, were conducted comparing the two
colonies. All of the tests indicated that the morph-o-types were identical genetically
and physiologically, with one notable exception in biosurfactant production. The
runny morphology reduced the surface tension of water by 62%, indicative of strong
biosurfactant production, whereas the round morphology reduced the surface
tension of water by 26%, also indicative of surfactant production but to a lesser
degree. Based on the appearance of the morph-o-types, during exposure to
cadmium and lead, we suspect that this isolate uses two different mechanisms in
response to cadmium and lead. One-dimensional SDS-PAGE analysis found a 28-
kDa protein that is expressed only under cadmium exposure and not under no metal
and lead stress.
This abstract accurately represents the content of the candidates thesis. I
recommend its publication.
'imberley M. Roane
The most beautiful thing we can experience is the mysterious. It is the source of
all true art and science.
I dedicate this thesis to those lucky few that are willing and able fall into
something unexpected and different and just go with it. An experience where you
encounter these rare people in the world, people that exude brilliance, inspire us,
teach us, and support us, taking it as it come to see where it takes you and who you
are after it is all done. This thesis goes out to those lucky few that have been there
like myself, and those few still to come.
My most heartfelt thanks and appreciation goes first and foremost to oh captain my
captain, my advisor, Dr Roane for her unbelievable support, time, insight and
patience since she had been in my life. My other wonderfhl committee members:
Dr. G, Charlie, and Dr. Lanning, for their tireless support and effort throughout this
endeavor. The Silver Valley Mining Company in Northern Idaho for providing soil
samples. Jeff Boon and the Center for Environmental Sciences for his help in the
analytical processes. Dr. Raina Maier, in the SWES Department at the University of
Arizona for her help with the biosurfactant assay. Last but not least, my parent of
the unconditional love and support, all my friends and family for their friendship,
their support, their motivation and their understanding through the years. The girls,
Katie and Aida, for keeping me smiling, not hesitating to help when I needed it,
sharing this experience, the vocabulary we developed and all the fun times and late
night poster building. Finally, I would also like to acknowledge the 6 Ps, when I
A University of Colorado Faculty Grant Award provided financial support for this
1. INTRODUCTION AND BACKGROUND...............................1
Metals and Their Sources...............................4
Metal Toxicity to Biological Systems Including
Mechanisms of Microbial Resistance.....................9
Which of these Mechanisms are Good for Remediation
What is Known about Cadmium and Lead Resistance
Advantage of Microbial Remediation over Traditional
Methods for Objective 1: Isolation of Metal Resistant
Organisms from Metal Contaminated Soils......................20
S oil Moisture Content................................ 20
Culturable Bacterial Numbers...........................21
Total Cell Counts......................................21
Isolation of Metal Resistant Microorganisms............22
Methods for Objective 2: Characterization of a Metal
Resistant Isolate.......................................... 23
Metal Resistance of Isolate S8 A...................... 23
Effect on Metal Solubility.............................24
Methods for Objective 3: Elucidation of Mechanisms of
Cadmium and Lead Resistance...................................29
One-Dimensional Protein Analysis...............29
Sample Collection and Preparation for PAGE
3. RESULTS ..................:..............................33
Results for Objective 1: Isolation of Metal Resistant
Organisms from Metal Contaminated Soils..............33
Results for Objective 2: Characterization of a Metal
Results for Objective 3: Elucidation of Mechanisms of
Cadmium and Lead Resistance....'.................... 38
4. DISCUSSION............................................ 72
5. FUTURE DIRECTION.........................................77
REFERENCES ...................................................... 81
Figure 1.1 Soil Aggregate ...............................................3
Figure 1.2 Metal Toxicity on Cellular Processes...........................9
Figure 1.3 Biosurfactant Complex.........................................12
Figure 1.4 Microbial Metal Resistance Mechanisms.........................15
Figure 3.1 Bimodal Growth................................................42
Figure 3.2 Cell Growth in Cd Over Time...................................43
Figure 3.3a Changes in Soluble Cadmium Concentration Upon Growth
of Isolate S8A in the Presence of 20 mg/L Total Cadmium...............44
Figure 3.3b Changes in Soluble Cadmium Concentration Upon Growth
of Isolate S8A in the Presence of 40 mg/L Total Cadmium...............45
Figure 3.3c Changes in Soluble Cadmium Concentration Upon Growth
of Isolate S8A in the Presence of 60 mg/L Total Cadmium...............46
Figure 3.3d Changes in Soluble Cadmium Concentration Upon Growth
of Isolate S8A in the Presence of 80 mg/L Total Cadmium...............47
Figure 3.4 Changes in Soluble Lead Concentrations
Upon Growth of Isolate S8A...............................................48
Figure 3.5 Two Colony Morph-o-types......................................49
Figure 3.6 Twitching Motility............................................50
Figure 3.7 Genetic Fingerprinting........................................55
Figure 3.8 Isolation Identification......................................56
Figure 3.9 Plasmid Extraction
Figure 3.10 Changes in Colony Morphology of P.putida
Upon Growth Under No Metal Stress......................................58
Figure 3.11a Changes in Colony Morphology of P. putida
and Lead Solubility Upon Growth in 20mg/L Lead......................... 59
Figure 3.11b Changes in Colony Morphology of P. putida
and Lead Solubility Upon Growth in 40mg/L Lead.........................60
Figure 3.11c Changes in Colony Morphology of P. putida
and Lead Solubility Upon Growth in 60mg/L Lead.........................61
Figure 3.1 Id Changes in Colony Morphology of P. putida
and Lead Solubility Upon Growth in 80mg/L Lead.........................62
Figure 3.11 e Changes in Colony Morphology of P. putida
and Lead Solubility Upon Growth in lOOmg/L Lead........................63
Figure 3.12a Changes in Colony Morphology of P. putida
and Cadmium Solubility Upon Growth in 20mg/L Cadmium................... 64
Figure 3.12b Changes in Colony Morphology ofP. putida
and Cadmium Solubility Upon Growth in 40mg/L Cadmium...................65
Figure 3.12c Changes in Colony Morphology of P. putida
and Cadmium Solubility Upon Growth in 60mg/L Cadmium...................66
Figure 3.12d Changes in Colony Morphology of P. putida
and Cadmium Solubility Upon Growth in 80mg/L Cadmium...................67
Figure 3.12e Changes in Colony Morphology of P. putida
and Cadmium Solubility Upon Growth in lOOmg/L Cadmium..................68
Figure 3.13 One-Dimensional Protein Profile............................70
Figure 3.14 Protein Sequence...........................................71
Table 3.1 Soil Moisture..........................................................40
Table 3.2 Soil Characterization..................................................41
Table 3.3 a Growth of Isolate S8A on Sugar Substrates............................51
Table 3.3b Growth of Isolate S8A on Amino Acids..................................52
Table 3.3c Growth of Isolate S8A on Organic Acids................................53
Table 3.3d Growth of Isolate S8A on Other Substrates............................54
Table 3 .4 Morph-o-type Characteristics Summary.................................69
INTRODUCTION AND BACKGROUND
The lithosphere, composed of soil and rock, makes up 29% of the Earths
total surface area. Of the Earths land surface, 80% of that is soil, where as exposed
rock only makes up about 5%. Soil is defined by Websters dictionary as: Finely
divided rock mixed with decayed vegetable or animal matter, constituting that
portion of the surface of the earth in which plants grow (Websters Ninth
Collegiate Dictionary, 1995). A soil scientist would define soil as, the earths
surface layer exploited by plant roots, (Paul and Clark, 1996). Soil scientists use
this definition to refer to undisturbed soils that manifest major terrestrial
ecosystems, and when soils are managed, they may offer arable land and/or other
resources (Paul and Clark, 1996). However, it is known that soils are often
disturbed, e g., by urban activities, mining, industry, etc., affecting the types of
microorganisms a soil may harbor. Maier and Pepper (2000) include that
terrestrial environments are arguably the richest and most complex of all microbial
environments and all soils teem with activity and diversity of microorganisms,
regardless of varied environments, even in soils affected by pollution.
The soil habitat includes mineral particles of various shapes, sizes and
chemical composition, soil gases, soil water and dissolved minerals. Soil is partially
composed of the particles sand, silt and clay, each more weathered than the prior.
The weathering of these particles can include physical, chemical, and biochemical
forces. To name a few examples, physical weathering can include abrasive forces
between soil particles or fracturing from soil freezing and thawing. Chemical
weathering can be due to acidic erosion via acid deposition (Paul and Clark, 1996).
Soil particles themselves are negatively charge, and as weathering increases,
more negatively charged edges become exposed, increasing the surface area of the
particle. Therefore, a clay particle will have the more negative character when
compared to other particles, and will be important when metal interactions with soil
are examined more closely.
The composition of a soil aggregate, the collection or combination of
different soil particles, will affect a soils character (Figure 1.1). Aggregates are
found in soils and the chemical and biological composition within each aggregate
can vary. For example, waste product accumulations, oxygen depletion, pH, etc., all
select for the growth of specific microorganisms within the pore space.
Furthermore, soil contaminants, such as metals, can also accumulate within the pore
space selecting for metal resistant microorganisms.
Figure 1.1 Soil Aggregate: A soil aggregate composed of various soil particles,
with bacteria attached to the particle surfaces. Note that environmental conditions
within the aggregate pore space can vary substantially influencing microbial growth.
Since the day of Sergi Winogradsky (1856-1953), the Father of Soil
Microbiology, soils have been known to harbor many microorganisms, including
indigenous populations of various groups of bacteria, algae, fungi, protozoa, and
viruses. Bacteria are considered the most abundant and diverse of all the
microorganisms found in soils. Examples of different types of bacteria found in soil
include Gram positive and Gram negative, aerobic and anaerobic, autotrophic and
heterotrophic. Dominant soil bacterial genera include Arthrobacter, Bacillus,
Pseudomonas, and Streptomyces (Maier and Pepper, 2000). Many of these bacteria
play a role in nutrient cycling and degradation, such as Pseudomonas and Bacillus
species in organic degradation. Others will fix nitrogen for plants to grow and
survive, such as Rhizobium species, and others even produce antibiotics, used in the
pharmaceutical industry, such as Streptomyces species. Unfortunately, some of
these bacteria found in soils are also pathogenic, including Pseudomonas
aeruginosa, a human pathogen, and Xanthomonas campestis, a plant pathogen. In
addition to these traditional roles in soils, some bacteria have been shown to degrade
and detoxify pollutants released into the environment. For example, an important
nitrogen fixing actinomycete, Frankia, has demonstrated metal resistance (Richards
et al., 2002), as well as Bacillus subtilis, which has been shown to bind metals, such
as mercury, to their cells wall (Roane, 1994; Beveridge and Doyle, 1989).
Metals and Their Sources
Metals put simply are a specific class of chemical elements. Their properties
include being a good conductor of heat and electricity and form lustrous solids.
There are three main classes of metals: essential metals, metalloids, and heavy
metals. Essential metals include essential elements, like the earth metals and alkali
earth metals, such as magnesium, sodium, calcium, copper, iron, and potassium.
The metalloids are a class of metals that exhibit both metal and non-metal
characteristics meaning metalloids look like metals but their ability to conduct heat
and electricity is between metals and non-metals. Silicon, boron, and arsenic are
examples of metalloids. Heavy metals are so named because of their high specific
gravity (> 5g/ml) and their potential toxic effect on ecological and biological
systems (Briuns et al, 2000; Hughes and Poole, 1989; Poole and Gadd, 1989, Ji and
Silver, 1995). Heavy metals include the metals lead, cadmium, and mercury, to
name a few. Interestingly, any metal at high enough concentrations can be toxic to
any system, even if the metal is a vital component to that system (Bruins et al.,
Heavy metal contamination, such as mining, is one of the most common
forms of soil metal pollution. Maier et al. (2000) in Environmental Microbiology
states that anthropogenic emission of heavy metals, like lead, cadmium, zinc, etc.,
exceed natural sources by 100 fold, and that these metal contaminants can cause
serious health and environmental risks. Metal pollution can be coupled with human
activity that supercedes natural biogeochemical processes (Roane and Pepper, 1996,
2000; Ji and Silver, 1995). For example, lead poisoning was an undetected problem
in Roman times due to the use of lead pipes in the water distribution system.
However, some pollution problems have only recently been recognized. An
example is the Gold Rush. Leaving mounds of the useless unearthed ore
exposed to the air and rain was not a concern and the effects of exposed metal
remained unnoticed until recent studies identified acid mine drainage. Not only is
acid mine drainage toxic to biological systems but is also a highly mobile form of
metal resulting in soil metal accumulation and contaminated aquatic systems.
Cadmium is naturally found in the lithosphere at concentrations ranging
from 0.01 to 1 8mg/Kg and is usually associated with zinc ores (Bruins et al., 2000;
Trevors et al., 1986; Nies, 1992; Williams and Coleman, 1992). The Environmental
Bureau of Investigation (EBI) reports that cadmium is readily emitted into the air
during the production of other ore metals, such as lead and zinc. The largest source
of cadmium release into the environment is via incineration of municipal waste
materials (Bruins et al., 2000). Cadmium also enters soil and water systems via
industrial and household wastewater dumping and agricultural use.
Lead is a malleable and corrosion resistant metal and an easily mined ore,
even though lead only makes up about 0.0013% of the earths crust. Lead is usually
ored as galena (PbS), minum (PbsCL), cerussite (PbCOs), and anglesite (PbSCL).
Lead is also one of the first metals used by man, in fact, leads use dates back as far
as 4,000 B .C. Interestingly, the decline of the Roman Empire has been credited, in
part, to lead concentrations accumulating in the water supply due to lead plumbing
(Roane and Pepper, 2000; Freedman, 1989). Until recently, lead was used for
pigmentation in paints, as a fuel additive in gasoline, and in shielding and battery
production (Roane and Pepper, 2000; Freedman, 1989).
Lead is a very persistent metal in the environment. Because of the use of
lead in industrial and commercial products, and the waste generated from the
manufacturing, lead and lead compounds have been continually released into the
environment. Interestingly, lead makes up one of the most hazardous contaminants
found in approximately 70% of US Superfund sites. Lead also readily accumulates
in most biological systems. The primary source of lead pollution used to be from
automobile emissions, battery manufactures, and smelters (Walter, 1980). The
TNRCC (Texas Natural Resource Conservation Commission) claims that
transportation alone contributed about 80% of the annual emissions of lead. The
primary source of current lead pollution in the environment is from the mining
When looking at metals and their interactions with soils, it is important to
understand (1) the forms of metals that are found in soil systems, and (2) what
factors affect metal:soil interactions. Metal concentrations are typically divided into
two groups: bioavailable and non-bioavailable. Roane and Pepper (2000) define
bioavailable metals as the soluble, nonsorbed and mobile forms of metal, usually in
the metals free ionic form, that is toxic to any biological system. Non-bioavailable
metal is described as the metal concentration that is insoluble or found as a
precipitant, sorbed and not mobile. There are many factors in soils, both biotic and
abiotic, that determine whether a metal is bioavailable or non-bioavailable. Some of
these factors include the concentrations of clays and organics found in the soil that
can adsorb the metal (the cation exchange capacity), redox potential, pH, and the
types of microorganisms present (Roane and Pepper, 1996, 2000, Babich and
Stotzky, 1980; 1985; Bernhard et al., 1986; Morrison et al., 1989; Alloway, 1990).
The most prevalent property effecting metal bioavailability in soils is the
cation exchange capacity (CEC). The CEC is the ability of a soil particle and the
associated organic compounds in the soil to exchange cations, which indicates how
tightly things will bind, thus determining constituent composition and
bioavailability of minerals, nutrients and metals (Van Loon and Duffy, 2000).
Clayey soils typically have a higher CEC than sandy soils, thereby making metals in
clayey soils less bioavailable. Metals are also generally more bioavailable under
acidic pHs, higher redox potentials and low organic matter
Metal Toxicity to Biological Systems Including Microorganisms
As mentioned, metals in their ionic form are more bioavailable, and it is the
strong ionic character of metals that makes them toxic. In biological systems,
metals will bind to cellular ligands and displace essential metals from their normal
binding sites (Bruins et al., 2000; Hughes and Poole, 1989; Poole and Gadd, 1989;
Gadd, 1986). An example is how arsenate has been shown to replace phosphate in
the cell (Bruins et al., 2000; Nies and Silver, 1995). Figure 1.2 summarizes the
toxic effects of metals on cells, affecting all cellular processes.
These toxic effects on biological systems do not exclude microorganisms.
Metal-microbial interactions seen at the individual and community level result in
decreased cell numbers, inhibition of biochemical processes and abnormal
morphology changes (Beveridge and Doyle, 1989). For example, studies showing
the effects of metals on microbial soil communities and activity see a decrease in
community size and activity in the presence of metal contamination (Roane et al.,
1996; Duxbury, 1981;Baath, 1989).
Protein denaturation and
inhibition of enzyme activity
Cell division inhibition and cell
Figure 1.2 Metal Toxicity on Cellular Processes: The various ways metals can
disrupt cellular processes.
Mechanisms of Microbial Resistance
As with many stresses, microorganisms have been able to successfully adapt
to their immediate environment in elaborate and ingenious ways (Williams and
Silver, 1984). One model as to how microorganisms have evolved metal resistance
centers around the environmental conditions of early earth when life began (Bruins
et al., 2000; Ji and Silver, 1995). Conditions of early earth included high
concentrations of toxic metals, presumably forcing early microorganisms to develop
metal resistance. Some mechanisms of which may be retained in todays
microorganisms. Others have proposed that metal resistance had evolved more
recently when metal contamination became increasingly prevalent, due to increasing
mining and industrial waste being released into the environment (Bruins et al., 2000,
Rouch et al., 1995, Ehrlich 1990).
The goal of microbial metal resistance is to overcome the detrimental effect
of metals on cells. Moreover, many of the genes that code for metal resistance have
been found on plasmids (Bruins et al., 2000, Silver and Misra, 1984; ji and Silver,
1995, Williams and Silver, 1984; Mergeay, 1991) possibly explaining the spread of
metal resistance in the environment. Some examples of plasmid encoded metal
resistance include the cad operon that encodes for three cad genes, cadA, cadB, and
cadC, providing a bacterium cadmium resistance via an efflux pump (Bruins et al.,
2000; Mergeay, 1991, Smith and Novick, 1972), and the mer operon that encodes
for seven mer genes, merR, merT, merP, merC, merA, merB, and merD, providing
bacteria the ability to enzymatically detoxify mercury via volatilization (Bruins et
al., 2000; Mergeay, 1991; Misra, 1992; OHalioran, 1993; Ji and Silver, 1995).
Microbial mechanisms of metal resistance can be categorized into two types:
general mechanisms of metal resistance and metal dependent mechanisms of metal
General mechanisms of metal resistance include those mechanisms that may
have another function to the organism, yet can also protect the organism from metal
toxicity. An example of a general mechanism is the production of an
exopolysaccharide (EPS) that sorbs metals to keep the metals from interacting with
important intercellular components. Exopolysaccharides are also important in
microbial surface adhesion and biofilm formation (Bruins et al., 2000; Scott et al.,
1988; Scott and Palmer, 1990). Other general mechanisms include biosurfactant
production and metal reduction by metabolic by-products (Roane and Pepper,
2000). Biosurfactants are compounds that have a polar head and a non-polar tail,
produced by some microorganisms that can be secreted by the microorganisms to
complex metals like cadmium, zinc and lead. Rhamnolipid is an example of a
described biosurfactant produced by Pseudomonas aeruginosa (Figure 1.3).
Biosurfactants do not immobilize the metal, however, the metal complex is no
longer chemically reactive and bioavailable. Metal reduction is process in which
the microorganisms metabolic by-products will change the solubility of a metal via
conversion to a metal salt, such as phosphidic and sulfidic salts (Roane and Pepper,
2000). These metal salts are immobile and biologically unavailable.
~v ^ C
'OCH-CH-CO-CHtH C-0 * OCCH C^OMTH-CHO
< CH. J /CH.V. CH-^TCH.: > CH CH,f" /ch' CH a
j CH; I j CH CH; j j CH. i
, O'- ^ 1 CH. i i CH i ; CH.
; CH; i 1 CH f Non-polar tails ^ CH. ; CH: 1
; CH i CH. 1 I CH. ; CH.
CH CH ch; CH
Complex: A Known Microbial Bio
fr '' o\
... aw sT
Rhamnolipid and an Interaction with Cadmium. Image from Roane. T.M. and I.L. Pepper,
2000. Microorganisms and Metal Pollutants, in Environmental Microbiology. Maier, R.M., I.L. Pepper, C.P.
Gerba (eds). Academic Press. San Diego, CA. Chapter 17, pg. 411
Metal dependent mechanisms of metal resistance are reliant on the specific
metal for activation. An example of a metal dependent mechanism is a metal efflux
pump, which is an energy-expending mechanism that removes metals from the cell
by actively pumping them out (Bruins et al., 2000; Silver et al., 1989; Nies and
Silver, 1995). Studies have shown that these pumps include ATPases in Gram
positive bacteria and chemiosmotic ion/proton pumps in Gram negative bacteria.
The metals usually associated with efflux pumps include arsenate, chromium and
cadmium (Roane and Pepper, 2000; Horitsu et al., 1986; Laddaga et al., 1987;
Hughes and Poole, 1989; Rani and Mahadevan, 1989; Nies, 1992; Silver, 1992).
Other metal dependent mechanisms include metallothioneins and metallothionein-
like proteins and methylation (Roane and Pepper, 2000). Metallothioneins and
metallothionein-like proteins are produced within the cell to sequester metals, thus
detoxifying the metal. Methylation is a process in which a microorganism will
volatilize the metal, making the metal an organometal, such as methyl- or
dimethylmercury. While organometals are generally more toxic to the cell,
volatilized metals readily diffuse away from the cell, reducing the concentration of
metal. Volaltilzation has also been observed with lead, arsenic, tin, and selenium
(Roane and Pepper, 2000). Figure 1.4 illustrates and summarizes the various known
metal resistant mechanisms.
Which of These Mechanisms are Good for
Interestingly, while all mechanisms of metal resistance protect the cell from
metal toxicity, not all metal resistance mechanisms are amendable to metal
remediation. Some metal resistance mechanisms, such as efflux pumps and
biosurfactants, may not change the bioavailability of the metal or may not
immobilize the metal. Both are goals of metal remediation. Metal resistance
mechanisms being studied for remediation purposes include exopolymer production,
metal reduction and metallothionein complexation, all of which reduce toxicity and
mobility of metals.
For example, biosurfactants, a general metal resistance mechanism, have
been studied for their ability to complex metals like cadmium, zinc and lead (Miller,
1995). However, the metal-biosurfactant complex does not reduce metal solubility,
in fact, biosurfactants generally increase the solubility of the metal. Nonetheless,
this metal-biosurfactant complex is no longer toxic to the organism and may be used
in soil washing to remove metals from soils.
Metallothioneins and metallothionein-like proteins, which are among the
metal dependent resistance mechanisms, are proteins that are cysteine-rich and have
a low molecular weight. These cysteine-rich regions of these proteins give the
protein a high affinity for mercury, zinc, cadmium, copper, and silver due to the
thio-group on cysteine. These methallothioneins are usually produced by higher
organisms like plants and yeast, but studies have shown that metallothionein-like
proteins have been utilized by some bacteria like Pseudomonas putida (Roane and
Pepper, 2000; Gupta et al., 1993). This metal dependent mechanism detoxifies the
metal intracellularly and immobilizes the metal as an accumulation in the cellular
Figure 1.4 Microbial Metal Resistance Mechanisms: Known metal resistance
mechanisms used by microorganisms. Image from Roane, T.M and LL. Pepper, 2000.
Microorganisms and Metal Pollutants, in Environmental Microbiology. Maier, R.M., LL. Pepper, C.P. Gerba
(eds). Academic Press. San Diego, CA. Chapter 17, pg. 410
What is Known about Cadmium and Lead
Cadmium will enter a cell through divalent ion transport, driven by
membrane potentials, usually coupled with manganese, via co-transportation, which
would normally transport essential metals like phosphates and magnesium for
cellular function (Bruins et al., 2000; Archibald and Duong, 1984; Laddaga et al.,
1985; Laddaga and Silver, 1985). Once inside the cell, cadmium will interfere with
protein function by binding to sulfhydryl groups on these proteins (Bruins et al.,
2000; Nies, 1992; Lebrun et al., 1994b) and can cause single strand DNA breakage
(Bruins et al., 2000; Trevors et al., 1986). To remove the cadmium from inside the
cell the organism excretes the cadmium out through a cadmium/hydrogen antiporter
pump (Roane and Pepper, 2000). The best studied specific mechanism of cadmium
resistance includes plasmid encoded efflux systems (Smith and Novick, 1972; Nies,
1992; Williams and Coleman, 1992; Bruins et al., 2000; Mergeay, 1991; Poole and
Gadd, 1989; Ji and Silver, 1994), and intercellular accumulation via metallothionein
and metallothionein-like proteins, as seen in bacteria, such as Pseudomonas putida
(Bruins et al., 2000; Pouch et al., 1995; Silver and Phung, 1996; Trevors et al.,
1986; Mergeay, 1991).
Lead resistance, on the other hand, is not as well understood. Although
many lead resistant organisms have been isolated, little is known of their
mechanism of metal resistance. However, nonspecific and surface binding
mechanisms have been shown to reduce soluble lead concentrations. The best
known mechanism for lead resistance is biotransformation via methylation (Hughes
and Poole, 1989; Reisinger et al., 1981; Thayer and Brinckman, 1982), which is
thought to be plasmid borne (Mergeay, 1991).
Advantage of Microbial Remediation over
Traditional Remediation Methods
Traditional or conventional methods of remediating metal contaminated soils
include physical and chemical processes of clean up. The ultimate goal of
traditional remediation, in general, is removal of the metal contaminants, to keep the
metal from spreading into the surrounding soil areas and ground water.
Physical methods include excavation and incineration. Excavation is the
process where the impacted soils are literally dug out and hauled off to a hazardous
waste landfill or incinerated. This technique does nothing to detoxify or destroy the
contaminated soil. Incineration is a process that thermally treats soils. The process
itself is very costly and cannot handle large quantities of soil. Furthermore, by
thermally treating metal contaminated soils, metals are released into the atmosphere.
Moreover, the thermal treatment also treats the important components of soil,
destroying the nutrients in the soils as well as the important soil flora.
Soils can be remediated chemically by collecting these contaminated soils
and washing the metals to remove them. Metal solubility can change by adjusting
the pH of the soil or by adding an organic, such as a chelator, that can interact and
sorb the metal. A problem with washing soils using chelators or acidic solutions is
the toxic residues left over by these washing solutions.
Microbial remediation offers an alternative to these conventional methods of
remediating metal contaminated soils. The goals of microbial remediation are
similar to the traditional methods, i.e. removal of metal toxicity and mobility, but
microbial remediation exploits microbial mechanisms of metal resistance and
detoxification. Microbial remediation has the potential to treat large volumes of soil
and can be administered in situ reducing costs associated with traditional
remediation methods. However, much still remains in understanding microbial
metal resistance and detoxification mechanisms, and their potential use in metal
remediation (Bruins et al., 2000).
Metal contamination of soils is a serious issue that needs to be addressed and
because of the continual increase in metal contamination and the ineffectiveness of
the conventional methods of remediating contaminated soils (mostly due to the sizes
of these contaminated areas exceeding the remediating capacity of these
techniques), an alternative method of remediation of contaminated soils must be
developed. Bioremediation is a young field and plenty is left to learn with respect to
microbial metal resistance and detoxification that can provide remediation potential,
as well as how to apply the growing technology.
Therefore, our goal for this project was to examine and elucidate indigenous
microbial metal resistance mechanisms that may be useful in the remediation and
detoxification of metal contaminated soils. Using metal contaminated soils,
provided by the Sunshine Mining Company in the Silver Valley mining area of
Northern Idaho, our approach in this study was to address three specific objectives,
which included: (1) isolate metal resistant organisms from the metal contaminated
soils, (2) characterize the metal resistant isolates and (3) elucidate mechanisms of
cadmium and lead resistance. In this study, our model metals were cadmium and
lead, which are two of the top ten metals of environmental concern as determined by
the Environmental Protection Agency (EPA). Furthermore, not much is known
regarding microbial resistance mechanisms to lead and cadmium in environmental
Methods for Objective 1: Isolation of Metal
Resistant Organisms from Metal Contaminated Soils
Metal resistant isolates were obtained from soils collected from the Silver
Valley Mining Region in Northern Idaho, by the Sunshine Mining Company. Soil
samples were aseptically collected from the top 2-3 inches of the soil surface
horizon and shipped overnight. All soils were stored at 4C until analysis. The
following soils were provided: S4, S6, S8, S9, S10, SI 1, Upper yard and North
yard. The following parameters were provided for each soil: soil pH and total soil
metal concentrations. Upon arrival, the following analyses were immediately
performed: soil moisture content, culturable bacterial numbers and total microbial
Soil Moisture Content
Soil moisture content was determined using aluminum weighing dishes. A
wet weight was measured out using a level counter top laboratory digital scale. The
soil was then baked in a laboratory oven for 24 hours at 110C and reweighed to
determine the dry weight of the soil. The difference between the two weights
reflected the soil moisture content.
Culturable Bacterial Numbers
For each soil, the number of culturable bacterial numbers was determined
using the standard plate counts procedure of Koch (1981). Serial dilutions of soil
slurries (5g soil to 20ml 0.1% glycerophosphate), mixed on a rotary shaker at
130rpm for 30 minutes at 28C, were spread plated onto Minimal Salts Medium
(MSM) agar. Agar plates were incubated for 48 hours at 28C, upon which
individual colonies were counted. The MSM contained per liter: 0.5g sodium
citrate (C6H5Na307), 0. lg magnesium sulfate (MgS04 H2O), l.Og ammonium
sulfate ([(NFLO2SO4]), l.Og glucose (C6H12O6); O.lg sodium pyrophosphate
(Na4P207 IOH2O); 100ml 0.1M MES (buffer), 900ml dlT^O, final pH adjusted to
6.0 with 10M sodium hydroxide (NaOH).
Total Cell Counts
Acridine orange direct microscopic counts for total bacterial numbers were
performed for each soil sample based on the method of Hobbie et al. (1977).
Bacteria were extracted from each soil using 0.1% glycerophosphate, to neutralize
soil charge, and mixed on a rotary shaker for 30 minutes at 150rpm. Dilutions of
each soil slurry were performed with sterile distilled water to achieve 10-100 cells
per field using fluorescence microscopy. A fraction of each dilution (1 8ml) was
mixed with 0.2% sterile acridine orange (0.2ml).
Following a 2 minute incubation, the cell:acridine orange mixture was
filtered through a 25mm black polycarbonate 0.2pm filter. Sterile distilled water
(2ml) was used to rinse excess acridine orange from the filter The filter was then
placed (cell side up) on a microscope slide with a drop of immersion oil and covered
with a coverslip. The stained filters were examined at lOOOx magnification using
epifluorescence microscopy with a Zeiss microscope equipped with a BEX420 -490
excitation filter and Y-50NF barrier filter. Twenty fields were counted per slide,
and the total number of bacteria were calculated according to:
Where N= total number of bacteria per ml
a= effective filter area of filter, mm2
n= mean count
m= area of optical field, mm2
v= volume of water sampled (ml)
Isolation of Metal Resistant Microorganisms
Bacteria tolerant of cadmium and lead toxicity were isolated using MSM
broth amended with 0, 10, 20, and 50mg/L soluble cadmium or 0, 10, 20, 50mg/L
soluble lead. Cadmium chloride (CdCh) was used for the cadmium amendments,
while lead acetate (Pb(C2H302)2) was used for the lead amendments Replicate
125ml flasks containing 25ml of metal-amended broth were inoculated with 200ml
of soil slurry (prepared as above), and incubated at 28C on a rotary shaker at
130rpm until stationary phase was reached (ranging from 24 hours to 9 days
depending on the metal concentrations). Dilution and plating onto MSM agar was
used to enumerate the metal resistant bacterial isolates.
Methods for Objective 2: Characterization of
a Metal Resistant Isolate
Metal Resistance of Isolate S8A
The next step was to survey (1) the maximum resistance of both cadmium
and lead and (2) examine how the growth of the organism effects the solubility of
the respective metals.
For determining the maximum resistance of cadmium and lead,
concentrations of the metals were increased until the organism could no longer grow
or in the case of the lead, lead saturation occurred and increasing soluble metal
could not be achieved. The cadmium concentrations examined ranged from Omg/L
to 250mg/L, while lead concentrations ranged from Omg/L to 300mg/L.
Effect on Metal Solubility
To assess how the growth of S8A influenced metal solubility, growth
experiments were set up wherein 25ml MSM broth was amended with various metal
concentrations and inoculated with 106 cells/ml of isolate S8A. Each culture was
incubated at 28C at 130rpm. Aliquots were taken every 24 hours, including a time
zero. Aliquots were diluted and plated onto MSM agar to establish culturable cell
counts. Aliquots were also removed every 24 hours for flame atomic absorption
(AA) analysis (Perkin-Elmer 5000) to determine soluble metal concentrations. Prior
to AA analysis, all samples were centrifuged at 2500 x g.
For controls, blanks containing media and metal were run simultaneously
without inoculum during the growth experiments and during the maximum
resistance experiments to monitor abiotic metal effects.
Cell morphology and gram reaction was determined by running a gram stain
and visualizing with a light microscope (Olympus GH-2, under lOOOx
Motility was assessed using (1) wet mounts of young broth cultures and (2)
modified MSM agar stabs. Wet mounts were examined under lOOOx magnification
with light microscopy for flagellar cell motility.
Agar plate stabs were done using MSM agar plates modified with the
addition of 0.05% TTC (triphenlytetrazolium chloride). Each plate was stab
inoculated from a plate colony and incubated at 30 and 37C for 18 hours, because
the results rely on metabolism, it was important to find optimal metabolism rates
(modified from Semmler et al., 1999). Agar stabs were visualized using an inverted
microscope (Olympus Phase Contrast model# UCWCD 0.30). Halo formation and
satellite colonies in the interstitial layer indicated a positive result for twitching
A type of Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) was
used to determine the genetic fingerprint of the isolate. Each reaction consisted of
the following: 5.0p.l 10X TBE (tris base, boric acid, and EDTA) buffer (see
Appendix A), 5.0p.l dNTP mixture (2.5mM each), 6.0|o.l MgCl2(25mM), l.Opl
primer (40p.M), 27.5pl nuclease free dIH20, 0.5pl Taq polymerase (5u/p.l), and
5.0pxl DNA template, which was extracted by heat lysing the cells at 98C for 10
minutes (modified from Versalovic et al., 1994). The PCR cycle was as follows: 1
cycle involving denaturation at 94C for 3 minutes, primer annealing at 37C for 1
minute, and extension at 72C for 1 minute; 30 cycles involving denaturation at
94C for 1 minute and 30 seconds, primer annealing at 55C for 1 minute and
extension at 72C for 1 minute; 1 cycle of final extension at 72C for 7 minutes and
a fixed 4C reaction storage. PCR products were run on a Tris-borate-EDTA-1.2%
agarose gel at lOOV/cm, stained with ethidium bromide (l|j.g/ml), and viewed under
Primer sequence: 5- ATG TAA GCT CCT GGG GAT TCA C -3
16s rDNA amplification and sequencing was done with each reaction
consisting of the following. 5.0pl Buffer B (Appendix A), 3.0pl 25mM MgCl2,
lO.Opl dNTP (2.5mM each), 5.0pl DMSO (dimethyl sulfoxide), 2.0pl (4mg/ml)
bovine serum albumin, l.Opl Taq polymerase (5u/pl), 16.0p.l nuclease free dIH20,
2.0pl 63F(orward) primer (40pM), 2.0p.l 1387R(everse) primer (40pM), and 2.0pl
DNA template. The PCR cycle was as follows: 30 cycles involving denaturation at
95C for 1 minute, primer annealing at 55C for 1 minute, and extension at 72C for
1 minute and 30 seconds; 1 cycle of final extension at 72C for 5 minutes and a
fixed 4C reaction storage. The PCR reactions were purified using the Promega
Wizard PCR Kit, and sequenced at the University of Colorado Health Sciences
Center, Cancer Center DNA Sequencing Core. Sequences were then checked
against the NCBI Bacterial Identification Database, www.ncbi.nlm.nih.gov. to
identify the isolates. Primer sequences:
63F: 5 CAG GCC TAA CAC ATG CAA GTC 3
1387R: 5 GGG CGG WGT GTA CAA GGC 3
In order to characterize the metabolic flexibility of the Isolate S8A, GN2
Biolog (Hayward, CA) plates were used. These are 96-well plates with each well
containing a different metabolic substrate. Each well also contains a tetrazolium
dye that changes color when reduced due to metabolic activity The plates were
inoculated with cultures suspended in sterile dIH20 with 63% transmission. The
Labsystems Multiskan Ascent Automated Plate Reader (model # 354) was used to
measure the optical density of each well at 560nm for the color formation after
incubation at 28C for 24 hours. Differential well coloration yielded a metabolic
fingerprint for the isolate.
The alkaline lysis procedure of Kado and Liu (1981) was used to isolate and
purify plasmid DNA. Cultures were grown in the presence and absence of metal to
turbidity, and optical density was determined at 600nm on a UV spectrophotometer
(Milton Roy Company, Spectronic 1001). An absorbance of at least of 0.8 was used
(some required concentration to achieve the absorbance). Cells were then spun
down at 2500 x g at 4C for 7 minutes. The supernant was poured off and the pellet
was resuspended in 1ml E buffer (Appendix A). To lyse the cells, 2ml of 0.2pm
filtered Lysis Buffer (Appendix A) was added and the cells were incubated at 65C
for 20 minutes. Two volumes of phenol/chloroform solution (50:50 v/v) was added
and inverted several times to emulsify. To separate the DNA from the other cellular
material, the emulsification was spun down at 2500 x g at 4C for 15 minutes. The
aqueous layer containing the plasmid was collected without disturbing the cellular
layer using cut P-1000 tips. Aliquots of the plasmid samples were then put into
1.5ml microcentrifuge tubes and stored at -20C. A 0.7% agarose gel was made
using E buffer. Sample (35pl) was mixed with lOpl of 6X Loading Dye and sample
was split in half and loaded onto the gel. The gel was run for one hour at 120 volts.
The gel was then stained with ethidium bromide (lpg/ml) for 20 minutes and
destained in fresh diLLO bath for 20 minutes. The gel was visualized under UV
Methods for Objective 3: Elucidation of
Mechanisms of Cadmium and Lead Resistance
Exopolymer production was determined using Sudan Black B, which is a
lipophilic stain, to flood 40-hour-old colonies on both Tryptic soy agar (TSA) and
MSM agar plates for 10 minutes. The stain was then discarded, and each plate was
washed with absolute ethanol (200 proof) and swirled for 2 minutes. The alcohol
was discarded and the colonies on the plates were observed. Black-colored colonies
meant the isolate was negative for exopolymer production (Liu et al., 1998).
For testing biosurfactant production, isolated colonies on TSA plates were
sealed with para-film, labeled blindly, and sent for testing to Dr. Raina Maiers lab
in the Department of Soil, Water and Environmental Science at the University of
Arizona for analysis. A reduction of the surface tension of water reflected
biosurfactant production (Bodour and Miller, 1998).
One-Dimensional Protein Analysis
To assess protein expression, the organism was grown under no metal stress,
cadmium stress and lead stress in amended MSM broth. Samples were taken from
each culture at exponential phase and stationary phase. Stationary phase sample
volumes were adjusted so that the equivalent numbers of cells were used in each
assay. Proteins were extracted using 2.0% SDS (lauryl sulfate) and a series of
freezing and thawing steps. Protein extractions were kept at -80C until analysis.
Protein expression in each sample was determined by running the extractions using
15 and 16% SDS-Polyacrylamide Gel Electrophoresis (PAGE).
Sample Collection and Preparation for PAGE Analysis
Samples (1.5ml) of exponential growth cultures were collected and placed in
eppendorf tubes. Stationary phase sample collection differed with an adjusted
volume of culture taken per sample so pellet sizes between exponential phase and
stationary phase were similar sizes. The samples were spun down at 14,000 x g for
10 minutes. Following centrifugation, the supernatent was then poured off
(discarding as much media as much as possible without disturbing the pellet). The
pellet was then resuspended in 90|il of sterile water. Once the pellet was
resuspended, 30pf of loading buffer (Appendix A) was added and the sample was
vortexed to mix thoroughly. The samples were then ready for lysis.
Samples were placed in liquid nitrogen for two minutes and then
immediately transferred to a 37C water bath for two minutes. The freeze/thaw
cycle was repeated at least two more times (3 times total). Following the final
freeze/thaw cycle, the cell lysate were kept at -80C until SDS-PAGE analysis.
A large acrylamide separating gel was prepared by mixing together 7.0ml
4X lower gel buffer (Appendix A), 5.0ml diF^O, and 16ml of acrylamide. The cross
linker, which included 120p.l of 10% APS (ammonium persulphate) and 8.0pl of
TEMED, was added just prior to pouring. The gel was then poured immediately
and over-layed with diF^O. The gel was left to polymerize for at least 30 minutes.
Then once the lower layer was polymerized, the overlay was poured off and a 4%
stacking gel was added. The stacking gel consisted of 1,5ml of 4X upper gel buffer
(Appendix A), 3.6ml of diF^O, and 0.81ml of acrylamide. Then the cross linker
solution, 18[il 10% APS and 8.0pl of TEMED was added last. The comb was set in
and the gel was poured. The gel was left to polymerize for at least 30 minutes.
Following polymerization, the comb was removed and the wells washed with IX
running buffer (Appendix A). The wells were then filled with IX running buffer,
and 25-1 OOpil of the prepared protein samples were loaded into the wells. The gel
was run at 9mA for 16 hours.
The gel was then stained with Coomassie Stain (Appendix A) for one hour
on a rotary shaker. The Coomassie was decanted and the gel destained with 250ml
10% methanol over night. Following destaining, the gel was visually inspected for
differential protein expression. Dr. Martin Gonzalez, University of Colorado at
Denver, Dept, of Biology, provided the SDS-PAGE protocol.
Protein sequencing was done directly from the polyacrylamide gel. The
expressed protein bands of interest were cut out of the gel as clean and accurately as
possible using a #11 disposable scalpel blade. The cut bands were then transferred
into a sterile 1.5 ml microcentrifuge tube, remained open, covered loosely with
aluminum foil, and placed in a laminar flow hood to dry the sample overnight. The
tube was then sealed and placed in a 15ml falcon tube for shipping. Protein samples
were sent to Stanford Universitys Beckman Center for sequencing. Edman
degradation and HPLC-Mass Spectrometry was used for the sequencing. The
sequences were then blasted on the NCBI Entrez Protein index,
http://ww.ncbi.nlm,nih.gov/entrez/query. to identify the protein.
Results for Objective 1: Isolation of Metal
Resistant Organisms from Metal Contaminated Soils
All of the soils collected were sandy with moisture contents ranging from
19.5% to 34%, resulting in relatively moist soils (Table 3.1). Soil pHs ranged from
3.3 (site S10) to 4.9 (site S4). Soil pH, total metal concentrations, total cell counts
and culturable cell counts are summarized in Table 3 .2. Because of the acidic pH,
the metals would be relatively soluble and, thus, bioavailable in these soils.
Information on total metal concentrations of each soil was sent with the soil samples
and the most contaminated soils came from site S4 and the North Yard with metal
concentrations of 3313 and 4176mg/kg lead and 17.1 and 48.2mg/kg cadmium,
When soil samples were treated with acridine orange to determine the total
number of cells in each soil, the soils that had the highest total cell numbers
included soils S8 and S6 with 6.3xlOn 2.7xlOn and 5.5xlOn 2.8x10',
respectively. When culturable cells were determined by dilution and plating
extractions from the soils, the Upper Yard soil and soil S9 had the highest culturable
cell counts with 1.2xl06 + 2.2xl04 and l.OxlO6 4.0xl05, respectively. The
presumed bioavailability of metal in conjunction with the culturable counts
suggested the presence of metal resistant microorganisms.
When individual isolates were grown in the presence of metal, several metal
resistant isolates were obtained. One isolate in particular, Isolate S8A,
demonstrated an interesting growth pattern when grown in the presence of
cadmium. The growth of S8A showed a bimodal growth pattern wherein growth
decreased with increasing cadmium concentrations, as would be expected, however,
with exposure to yet higher metal concentrations, growth recovered and actually
increased with the increased metal exposure (Figure 3.1). The bimodal resistance is
demonstrated in Figure 3.2, in which Isolate S8A grew better in 80mg/L of
cadmium versus 40 and 60mg/L of cadmium at 48 and 72 hours. Because of this
interesting growth pattern, the specific mechanisms of metal resistance used by
Isolate S8A were examined.
Results for Objective 2: Characterization
of a Metal Resistant Isolate
In order to better assess the metal resistance shown by S8A, the isolate was
grown in increasingly higher concentrations of lead or cadmium until concentrations
were reached wherein the organism could no longer grow. It was then found that
S8A was resistant up to 200mg/L soluble cadmium and 300mg/L soluble lead,
although the range for the metal resistance may be much greater with respect to the
lead due to solution saturation (metal precipitation in the medium).
In addition to determining the maximum metal resistance level, the ability of
S8A to reduce the soluble metal concentrations (a goal for remediation of metals)
was evaluated. The growth of S8A (from 106 to 109 cells/ml) in concentrations up
to 80mg/L cadmium reduced soluble cadmium concentrations by 26 38%,
although a longer lag period was observed with increasing metal concentrations
(Figure 3.3). Likewise, soluble lead concentrations up to 44mg/L were reduced by
45 83% (Figure 3.4). Although growth under lead conditions was not monitored,
75% of the cadmium remained soluble in the absence of inoculum, while 83% of the
lead remained soluble in the absence of the inoculum.
Throughout the metal growth studies with S8A, a second colony
morphology continually appeared. This second colony morphology appeared to be
flat and runny, which was very different from the convex and round colony
morphology originally associated with S8A (Figure 3.5). To determine whether or
not this second morph-o-type was a contaminant or a subset of the population
responding differently, comparative assays were run to compare the runny and
round colony morphologies. The tests included cell morphology, gram reaction,
motility, metal resistance characteristics, metabolic profiling, genetic fingerprinting,
and 16s rDNA sequencing. Cell morphology and gram reaction were determined by
running several gram stains of the two morph-o-types finding that both colony
morphologies were gram-negative bacilli.
When motility was looked at using wet mounts of the two morph-o-types,
flagellarmotility was not observed. However, halo and satellite colony formations
were seen in the interstitial layer of the plate (the area between the bottom of the
agar and the base surface of the plate) in agar stabs, indicative of twitching motility.
There was no observed difference in twitching between the round and runny colony
morphologies. Figure 3.6 shows an example of the halo and satellite formations
observed with each morph-o-type.
Metabolic fingerprinting was further used to characterize the two morph-o-
types. According to the Biolog plate results, which were used to determine the
metabolic fingerprints of the two colonies, the two colony morphologies had similar
metabolic fingerprints. It was also ascertained that Isolate S8A did not have stored
carbon sources for growth in minimal conditions, indicated by a lack of growth in
the no substrate well. The metabolic fingerprint also showed that the two morph-o-
types favored amino acids and weak organic acids as substrates with a few
disaccharides also used (Table 3.3).
When the two morph-o-types were compared genetically via AP-PCR, their
genomic fingerprint as seen in Figure 3.7 showed that the two colony morphologies
had similar genomic fingerprints. When the 16s rDNA sequences were compared,
they were 98% similar, identifying both morph-o-types as Pseudomonas putida
Finally, the now identified Pseudomonas putida isolate was examined for
the presence of plasmids. A general plasmid extraction showed the presence of a
large plasmid, >23Kb (Figure 3.9).
When the two colony morphologies were confirmed to be one organism, it
was now important to find out why two colony morphologies were appearing. A
new growth experiment was run with monitoring changes in colony morphology
under no metal and metal stress. All of the growth experiments were started with
the runny morph-o-type as the inoculum. Figure 3.10 illustrates that under the no
metal and Figure 3.11 illustrates that under lead stress, the growth and colony
morphology patterns do not differ Both conditions showed a reversion in colony
morphology back to the round morphology upon inoculation, within the first 12
hours of growth, and a switch to the runny morphology in stationary phase.
However, in the presence of cadmium (Figure 3.12), the runny morphology was
maintained through exponential phase with reversion to the round morphology in
stationary phase, prompting the examination into the mechanisms of cadmium and
lead resistance used by this Pseudomonas putida.
Results for Objective 3: Elucidation of Mechanisms
of Cadmium and Lead Resistance
Pseudomonas putida was examined for known mechanisms of metal
resistance. The production of an exopolymer was examined using the lipophilic
stain Sudan Black B Pseudomonas putida did not take up the stain, indicating the
presence of a polar glycocalyx (sugar) layer surrounding the cells. Another known
mechanism of metal resistance is the production of biosurfactants. Interestingly,
this metal resistance mechanism could result in a change in the morphology of a
colony. After both morph-o-types were examined by Dr. Raina Maiers Lab
(University of Arizona), the results showed that the runny colony morphology was
able to reduce the surface tension of water more so than the round colony
morphology, 62% versus 26%, respectively. A summary of the characteristics of
the two colony morph-o-type is provided in Table 3.4.
To identify additional mechanisms of resistance, protein expression patterns
(one-dimensional) were examined in the presence and absence of metal.
Interestingly, the protein expression patterns in Pseudomonas putida grown in the
presence of lead and in the absence of any metal were similar. However, in the
presence of cadmium, a 28kDa protein was apparent and was missing under the no
metal and lead conditions (Figure 3.13). This cadmium induced protein was
isolated and sequenced, and was identified as a hypothetical protein found in the
genomes of Pseudomonas fluorescens, Pseudomonas syringae, and Pseudomonas
putida. The sequence of the protein is illustrated in Figure 3.14. Further studies are
currently underway to identify the function of this protein and its role in cadmium
Table 3.1: Soil Moisture: Cadmium and Lead Contaminated Soils from Northern Idaho
Site Wet Weight (g) Dry Weight (g) %
S6 1.72 1.4605 19.5
S4 1.1475 0.9155 27.5
Upper 3.512 2.6255 34
North 1.499 1.108 34
S8 2.623 2.091 25.5
S9 2.407 1.943 23.5
S10 2.659 2.0585 29
Sll 2.972 2.219 34
Wet weight-Dry weight x 100 =% soil moisture content
Table 3.2: Soil Characterization:
Soil pH Cadmium1 (mgflcg) Lead1 (mg/kg) Total Cell #s2 (Cells g'1) Culturable Cell #s (CFUg1)
S4 4.9 17.1 3313 SixlO'^UxlO11 S.dxlO^l^xlO5
S6 4.8 5.0 103 5.5xl0ll2.8xl011 s.exitfio
S8 3.4 4.9 385 6.3xlOn2.7xlO" 7.6x1051.6x105
S9 3.6 5.3 426 2.7xl010i2.3xlOto l.OxlO^.OxlO5
S10 3.3 18.3 207 4.9xl011+4.2xl011 6.0xl04+2.4xl04
Sll 3.5 5.4 798 3.7xl0ll1.6xl011 4.8x105+2.2x104
North 4.3 48.2 4176 2.3xlO"1.8xlOn S-OxlO+^xlO4
Upper 3.6 0.6 91 4.0xl011+1.8xl011 1.2x106+2.2x104
1 Metal concentrations were determined using EPA Method
3050 and atomic absorption.
^otal cell numbers were determined using acridine orange
Figure 3.1 Bimodal Growth: Bimodal growth curve with cells at the higher and lower
metal concentrations versus intermediate concentrations showing better growth.
Figure 3.2 Cell Growth in Cd Over Time: An image of the growth of Isolate S8A in
various metal concentrations over time. Isolate S8A grew better at 80mg/L of cadmium
versus 40 and 60mg/L cadmium.
0 hr 24 hr 48 hr 72 hr 96 hr 120 hr 144 hr
Sol. Cd No Inoculum
Q Sol. Cd Inoculated
A Cell Density
Figure 3.3a Changes in Soluble Cadmium Concentration Upon Growth of Isolate S8A in
the Presence of 20 mg/L Total Cadmium
48 hr 72 hr 96 hr
120 hr 144 hr
Sol. Cd No Inoculum
Sol. Cd Inoculated
A Cell Density
Figure 3.3b Changes in Soluble Cadmium Concentration Upon Growth of Isolate S8A in
the Presence of 40 mg/L Total Cadmium
Ohr 24 hr 48 hr 72 hr 96 hr 120 hr 144 hr
Sol. Cd No Inoculum
B Sol. Cd Inoculated
A Cell Density
Figure 3.3c Changes in Soluble Cadmium Concentration Upon Growth of Isolate S8A in
the Presence of 60 mg/L Total Cadmium
0 hr 24 hr 48 hr 72 hr 96 hr
120 hr 144 hr
Sol. Cd No Inoculum
H Sol. Cd Inoculated
A Cell Density
Figure 3.3d Changes in Soluble Cadmium Concentration Upon Growth of Isolate S8A in
the Presence of 80 mg/L Total Cadmium: An example of the overall soluble cadmium
reduction by S8A. An increase in lag time with increasing cadmium concentration from
0 mg/L to 80mg/L was observed and the soluble cadmium concentrations were reduced
by 26 38 %. (a) shows the effects of growth on 20mg/L cadmium, (b) at 40mg/L
cadmium, (c) at 60mg/L cadmium, and (d) at 80mg/L cadmium.
11 22 33 44
Total [Pb] mg/L
Figure 3.4 Changes in Soluble Lead Concentrations Upon Growth of Isolate S8A: The
amount of soluble lead was reduced by 3.03, 6.86, 8.77, and 33.84mg/L within 48 hours
at lead concentrations 20, 40, 60 mg/L, respectively, upon growth of S8A from 106 to 109
cells/ml. A remarkable reduction in soluble lead from 28.68mg/L to 4.81 mg/L occurred
within 120 hours at a total lead concentration of 33mg/L.
Figure 3.5 Two Colony Morph-o-types: This picture shows examples of the two
colony morph-o-types. The first plate (left) was the runny colony morphology
with the second plate (right) was the round colony morphology.
Figure 3.6 Twitching Motility: At the end of the gel stab the circular spreading
around the bottom of the gel stab was a halo formation (a), indicative of twitching. A
second indicator of twitching was the satellite colony formations in the interstitial
layer of the plate outside of the halo (b).
Table 3.3a: Growth of Isolate S8A on Sugar Substrates
Well Substrate Result Well Substrate Result
2 a-Cyclodextrin N 21 Lactulose N
3 Dextrin N 22 Maltose N
4 Glycogen N 23 D-Mannitol SP
7 N-Acetyl-D-Galactosamine N 24 D-Mannose WP
8 N-Acetyl-D-Glucosamine WP 25 D-Melibiose N
9 Adonitol N 26 P-Methyl-D-Glucoside N
10 L-Arabinose SP 27 D-Psicose N
11 D-Arabitol SP 28 D-Raffinose N
12 D-Cellobiose N 29 L-Rhamnose N
13 I-Erythritol N 30 D-Sorbitol N
14 D-Fructose WP 31 Sucrose N
15 L-Fucose N 32 D-Trehalose N
16 D-Galactose SP 33 Turanose N
17 Gentiobiose N 34 Xylitol N
18 a-D-Glucose SP 35 Pyruvic Acid Methyl Ester WP
19 m-Inositol N 95 a-D-Glucose-1 -Phosphate N
20 a-D- Lactose N 96 D-Glucose-6-Phosphate N
Key: Strongly Positive (SP), Positive (P), Weakly Positive (WP), Negative (N).
Table 3.3b: Well Growth of Isolate S8A on Amino Acids Substrate Result
64 L-Alaninamide P
65 D-Alanine SP
66 L-Alanine SP
67 L-Alanyl-Glycine SP
68 L-Asparagine SP
69 L-Aspartic Acid SP
70 L-Glutamic Acid SP
71 Glycyl-L-Aspartic Acid N
72 Glycyl-L-Glutamic Acid WP
73 L-Histidine N
74 Hydroxy-L-Proline SP
75 L-Leucine WP
76 L-Omithine WP
77 L-Phenylalanine N
78 L-Proline WP
79 L-Pyroglutamic Acid WP
80 D-Serine N
81 L-Serine WP
82 L-Threonine N
83 D,L-Caihitine WP
84 Y-Aminobutyric Acid WP
85 Urocanic Acid WP
Key: Strongly Positive (SP), Positive (P), Weakly Positive (WP), Negative (N).
Table 3.3c: Growth of Isolate S8A on Organic Acids
Well Substrate Result
36 Succinic Acid Mono-Methyl Ester N
37 Acetic Acid WP
38 Cis-Aconitic Acid SP
39 Citric Acid SP
40 Formic Acid WP
41 D-Galactonic Acid Lactone WP
42 D-Galaturonic Acid SP
43 D-Gluconic Acid WP
44 D-Glucosaminic Acid WP
45 D-Glucuronic Acid WP
46 a-Hydroxybutyric Acid N
47 P-Hydroxybutyric Acid SP
48 y-Hydroxybutyric Acid N
49 p-Hydroxyphenylacetic Acid N
50 Itaconic Acid N
51 cx-Ketobutyric Acid N
52 a-Ketoglutaric Acid N
53 a-Ketovaleric Acid WP
54 D,L-Lactic Acid SP
55 Malonic Acid WP
56 Propionic Acid WP
57 Quinic Acid WP
58 D-Saccharic Acid SP
59 Sebacic Acid N
60 Succinic Acid WP
61 Bromosuccinic Acid N
62 Succinamic Acid N
63 Glucuronamide N
Strongly Positive (SP). Positive (P), Weakly Positive (WP), Negative (N).
Table 3.3d: Growth of Isolate S8A on Other Substrates
Well Substrate Result
1 Water N
5 Tween 40 WP
6 Tween 80 WP
86 Inosine SP
87 Uridine N
88 Thymidine N
89 Phenylethylamine N
90 Putrescine WP
91 2-Aminoethanol WP
92 2,3-Butanediol N
93 Glycerol WP
94 D.L. a-Glycerol Phosphate N
Key: Strongly Positive (SP), Positive (P), Weakly Positive (WP), Negative (N).
2 3 4 5 6
1. 123 bp ladder
2. morphology 1
3. morphology 2
4. colony 1 (1->10)
5. colony 2 (1-MO)
6. E. coli
Figure 3.7 Genetic Fingerprinting: The image shows the various genetic fingerprints
(banding patterns) of Isolate S8A colony morphology one and two and E. coli (the
positive control). Similarities were seen between lanes 2-5 indicating this banding
pattern was the same, and that the AP-PCR worked because of the presence of E.
colis genetic fingerprint.
12 3 4 5
1. 123 bp ladder
2. SSApunny (diluted 1:1)
3. S8Around (diluted 1:1)
4. SSArunnj (undiluted)
5. S8Around (undiluted)
Figure 3.8 Isolate Identification: The image shows successful isolation of the 16s
rDNA from each colony morphology, with the diluted samples more intense than the
12 3 4
1. P. putida, no metal
2. P. putida, no metal
3. No sample loaded
4. 1 Kb ladder
Figure 3.9 Plasmid Extraction: Gel shows successful isolation of a large plasmid
from Pseudomonas putida. The first two lanes show the isolated plasmids, from a
duplicate extraction. The fourth lane was a 1Kb ladder with the top band being 10Kb.
The plasmid is estimated to be >23Kb.
0 12 24 36 48 60 72 84 96 108 120 132 144 156
Figure 3.10 Changes in Colony Morphology of P.putida Upon Growth Under No Metal
Stress: The growth of Pseudomonas putida and its colony morphology changing over time.
In no metal, a complete reversion to the round morphology was seen immediately. A
switch to the flat morphology was not seen until the onset of stationary phase.
o> 20 +
Ohr 12 24 36 48 60 72 84 96 108 120 132 144 156
hr hr hr hr hr hr hr hr hr hr hr hr hr
Figure 3.11a Changes in Colony Morphology of P. putida and Lead Solubility Upon
Growth in 20mg/L Lead.
Figure 3.11b Changes in Colony Morphology of P. putida and Lead Solubility Upon
Growth in 40mg/L Lead.
Ohr 12 24 36 48 60 72 84 96 108 120 132 144 156
hr hr hr hr hr hr hr hr hr hr hr hr hr
Figure 3.11c Changes in Colony Morphology of P. putida and Lead Solubility Upon
Growth in 60mg/L Lead
Ohr 12 24 36 48 60 72 84 96 108 120 132 144 156
hr hr hr hr hr hr hr hr hr hr hr hr hr
Figure 3.1 Id Changes in Colony Morphology of P. putida and Lead Solubility Upon
Growth in 80mg/L Lead
Ohr 12 24 36 48 60 72 84 96 108 120 132 144 156
hr hr hr hr hr hr hr hr hr hr hr hr hr
Figure 3.1 le Changes in Colony Morphology of P. putida and Lead Solubility Upon
Growth in lOOmg/L Lead: The growth of Pseudomonas putida and its colony
morphology change over time in the presence of lead. It is seen here that similar to no
metal, an immediate reversion to the normal round morphology was observed and a
switch to the flat morphology was not seen until well into stationary phase. The metal
concentrations used were: (a) 20mg/L, (b) 40mg/L, (c) 60mg/L, (d) 80 mg/L and (e)
lOOmg/L of soluble lead.
0 12 24 36 48
60 72 84 96 108 120 132 144
Figure 3.12a Changes in Colony Morphology of P. putida and Cadmium Solubility Upon
Growth in 20mg/L Cadmium
0 12 24 36 48 60 72 84 96 108 120 132 144
Figure 3.12b Changes in Colony Morphology of P. putida and Cadmium Solubility Upon
Growth in 40mg/L Cadmium
0 12 24 36 48 60 72 84 96 108 120 132 144
Figure 3.12c Changes in Colony Morphology of P. putida and Cadmium Solubility Upon
Growth in 60mg/L Cadmium
g = intermediate
0 12 24 36 48 60 72 84 96 108 120 132 144
Figure 3.12d Changes in Colony Morphology of P. putida and Cadmium Solubility Upon
Growth in 80mg/L Cadmium
I = intermediate
Figure 3.12e Changes in Colony Morphology of P. putida and Cadmium Solubility Upon
Growth in lOOmg/L Cadmium: The growth of Pseudomonas putida and its colony
morphology change over time in the presence of cadmium. Unlike the growth pattern of
the no metal and lead, in the presence of cadmium, the starting morphology shape, flat,
was maintained through exponential phase and a reversion to the normal round
morphology was not seen until the onset of stationary phase. The metal concentrations
examined were: (a) 20mg/L, (b) 40mg/L, (c) 60mg/L, (d) 80 mg/L and (e) lOOmg/L of
Table 3.4: Morph-o-type Characteristics Summary:
Max cadmium resistance 200 mg/L 200 mg/L
Max lead resistance 300 mg/L 300 mg/L
Biosurfactant production strongly positive(1) less positive(2)
Exopolysaccharide positive positive
Gram reaction negative negative
Cell morphology bacillus bacillus
Same genetic fingerprint yes yes
16S rDNA gene sequence(3) Pseudomonas putida Pseudomonas putida
Motility twitching twitching
(1) Reduced the surface tension of water by 62%
(2) Reduced the surface tension of water by 26%
(3) Of the 700 base pair sequence the two morphologies displayed a 98% similarity.
12 3 4 5 6 7 8
1. 60 mg/L (Cd) Stationary
2. 60 mg/L (Cd) Exponential
3. No metal
4. No metal
5. 60 mg/L (Pb) Stationary
6. 60 mg/L (Pb) Exponential
7. No metal
8. No metal
9. Protein Ladder
Figure 3.13 One-Dimensional Protein Profile: An SDS-PAGE of proteins extracted
from the Pseudomonas putidci isolate grown under no metal, cadmium, and lead
stress. The first two lanes show the protein expression in the presence of cadmium.
Note the presence of a 28kDa protein under cadmium exposure. The expression of
the protein was noted in exponential phase and intensified during stationary phase.
Figure 3.14 Protein Sequence: The determined sequence from Edman degradation
and HPLC-Mass Spectrometry of the cadmium induced protein. Included is a key
of the single letter symbols of the amino acids.
B-Asparagine or Aspartic acid
In this study, highly metal resistant bacteria were isolated from metal-
contaminated soils collected in Northern Idaho. One isolate in particular, S8A,
identified as Pseudomonas putida, revealed an interesting growth response in the
presence of cadmium and lead. Bimodal metal resistance in which S8A was better
able to grow in the lower concentrations (10 and 20mg/L) and higher concentrations
(60 and 80mg/L) of metal versus intermediate concentrations, with cell
concentrations similar to that of no cadmium was observed. Because of this
bimodal response, we attempted to identify mechanisms of metal resistance in the
Pseudomonas putida. In growing this organism in the presence of cadmium and
lead, this Pseudomonas putida reduced soluble cadmium concentrations by up to
38% and soluble lead concentrations by up to 83%. The maximum level of
resistance where growth was still observed was 200mg/L soluble cadmium and
300mg/L soluble lead, both extremely high concentrations of bioavailable metal.
Interestingly, two distinct colony morphologies were observed in the growth
studies. The second or less common colony morphology (the runny morphology)
was initially thought to be a contaminant, which had prompted the characterization
of both morph-o-types to determine if they represented the same or different
bacteria physiologically and genetically. Both morph-o-types were physiologically
and genetically identical with both resistant to 200mg/L cadmium and 300mg/L
lead, and both being gram-negative bacilli that produced an exopolysaccharide.
Both had the same metabolic fingerprint, and both demonstrated twitching motility.
Where they differed was in the production of biosurfactant, the runny morphology
producing more biosurfactant than the round morphology.
Having confirmed that both morph-o-types were the same isolate, the
appearance of the second (runny) colony morphology was more closely
investigated. To do this, the appearance of the different colony morphologies
during growth in the presence and absence of metal was examined. No differences
in the appearance of the colony morphologies were found between the no metal and
the lead cultures. In these cultures, the appearance of the second colony
morphology was not seen until stationary phase versus exponential phase of growth.
Because the runny colony morphology did not show up until stationary phase of
growth may suggest that the second morphology may be in response to a stressor
other than lead, and that the lead was not toxic enough to initiate the appearance of
the second morph-o-type. The cadmium cultures, on the other hand, showed the
second morphology during exponential phase of growth. We hypothesize, based on
the differential patterns of colony morphologies, that exposure to cadmium was
more stressful for the organism, initiating the appearance of the runny morphology
Based on the data thus far, we suspect that a subset of the Pseudomonas
putida population used exopolymer production and biosurfactant production (both
known metal-complexing agents) to represent two metal resistance mechanisms.
Which mechanism is used over the other or if both are used simultaneously is not
clear at this point. However, the bimodal pattern of metal resistance observed
during the growth studies may reflect the use of different metal resistance
mechanisms. For example, while an overall reduction in soluble metal
concentrations often resulted from the growth of the isolate, the variability in the
reductions may be due to the presence of the biosurfactant. Biosurfactants are
excreted outside of the cell and can readily bind to metals rendering them less
chemically reactive (and so less toxic) even though the metal remains soluble.
Interestingly, the production of biosurfactants can cause changes in colony
morphology. As the surface tension of water surrounding the colony decreases, the
result is usually a runny colony (Sandrin et al., 2000; Al-Tahhan et al., 2000;
Zhang and Miller, 1994). While both colony morph-o-types produce surfactant, the
colony with the runny appearance produced more surfactant than the normal type.
Once surfactant is bound, therefore becoming less chemically reactive itself, a
reversion of the runny colony to the normal type could occur. Since the runny
morph-o-type was only apparent during stationary phase in the presence of lead
versus in exponential phase in the presence of cadmium, we suspect that
Pseudomonas putida S8A uses a different mechanism of resistance in the presence
of lead versus cadmium.
In order to better assess if this organism uses a different resistance
mechanism for the lead versus cadmium, we analyzed protein expression patterns
under each condition. A 28-kDa protein was evident using one-dimensional gel
electrophoresis when the isolate was grown in cadmium. The protein was present in
both exponential and stationary phase growth, and was not apparent in no lead or
Following sequencing, this protein was found in the genomes of
Pseudomonas fiuorescens, Pseudomonas aeruginosa, and Pseudomonas putida, as
an unidentified protein. While specific mechanisms of metal resistance are not fully
understood, it is known that such mechanisms can be complex involving many
different proteins. For example, the cad operon found in primarily Staphylococcus
species involves three so far identified proteins. The cadmium efflux system (coded
for by the cad operon) does not affect cadmium solubility, like other metal
resistance mechanisms can, but protects the cell from cadmium toxicity by
constantly pumping intracellular cadmium outside of the cell. Interestingly, a
number of the cadmium efflux proteins have hydrophobic regions, indicative of
their placement in the cytoplasmic membrane. The 28-kDa unidentified protein
found in this study has a hydrophobic region; possibly indicating a membrane bound
function. Studies will continue to confirm the relationship between this protein and
cadmium exposure, as well as attributing a function to the protein.
A lot of ground work was laid down with this project and because of the
newness that had come with the results plenty can be built off this study. The
finding of the unidentified protein that appears to be associated with cadmium
resistance is exciting. This protein maybe associated with a new mechanism of
resistance not previously documented or may contribute a new component to an
existing, known mechanism. Particularly important would be if this protein is
associated with a mechanism of metal resistance that could prove useful in metal
remediation. At this point, we do not know if this protein detoxifies cadmium.
Further studies to confirm its expression under cadmium stress should be done as
well as assessment of the function of the protein.
What's more, it would be valuable to assess whether or not the plasmid
found with this Pseudomonas putida is linked to metal resistance. If the plasmid is
important for metal resistance then the plasmid could be examined further, for
example, in gene transfer studies to establish metal detoxifying microbial
populations in metal remediation. In gene transfer based remediation, a donor
organism donates its DNA to indigenous soil microorganisms, with the hope that
receiving populations now obtain metal resistance.
Regardless of whether or not the plasmid plays a role in metal resistance, it
would nonetheless be important to determine whether or not biosurfactant
production is related to the appearance of the second colony morph-o-type. If so,
this will be one of the only documented cases of a subset of a microbial population
responding to metal stress.
Overall, this study has perhaps raised more questions than it answered
making this work a foundation for a number of future projects.
4X Lower Buffer:
pH to 8.8 with HC1
H20 to 250ml
4X Upper Buffer:
pH tO 6.8 with HC1
l .Og SDS
H20 to 250ml
4X Laemmli Loading Buffer:
25ml 4X Upper Buffer
3. lg DTT
0.2g Bromophenol Blue
H20 to 50ml
10X Running Buffer:
H20 to 1.0 liter
55.0g Boric acid
40ml 0.5M EDTA
H20 to 1.0 liter
Wizard PCR Preps
DNA purification System
Cat. # A7170
10g Coomassie R-250
1 3g Cupric Acetate
75 ml Acetic Acid
H20 to 1.0 liter
40mM Tris Acetate
(adjust accordingly to vol.)
pH to 12.6 with NaOH
(adjust accordingly to vol.)
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