Citation
Molecular investigation of cadmium response in an environmental pseudomonas isolate

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Title:
Molecular investigation of cadmium response in an environmental pseudomonas isolate
Creator:
Atchison, Katie Ann
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
Publication Date:
Language:
English
Physical Description:
ix, 83 leaves : ; 28 cm.

Subjects

Subjects / Keywords:
Soils -- Cadmium content ( lcsh )
Cadmium -- Environmental aspects ( lcsh )
Bioremediation ( lcsh )
Soil remediation ( lcsh )
Pseudomonas ( lcsh )
Bioremediation ( fast )
Cadmium -- Environmental aspects ( fast )
Pseudomonas ( fast )
Soil remediation ( fast )
Soils -- Cadmium content ( fast )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (M.A.)--University of Colorado at Denver, 2004. Biology
Bibliography:
Includes bibliographical references (leaves 73-83).
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Katie Ann Atchison.

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Source Institution:
University of Colorado Denver
Holding Location:
|Auraria Library
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
60608819 ( OCLC )
ocm60608819

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MOLECULAR INVESTIGATION OF CADMIUM RESPONSE IN AN ENVIRONMENTAL PSEUDOMONAS ISOLATE by Katie Ami Atchison B.S., University of Colorado at Denver, 2001 A thesis submitted to the University of Colorado at Denver in partial fulfillment of the requirements for the degree of Master of Science Biology 2004

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This thesis for the Master of Science degree by Katie Ann Atchison has been approved by Gerald Audesirk !cl/fjrl-/ Date

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Atchison, Katie Ann (M.S. Biology) Molecular Investigation of Cadmium Response in an Environmental Pseudomonas Isolate Thesis directed by Assistant Professor Timberley M. Roane ABSTRACT Cadmium is toxic to organisms at low soluble levels, and cadmium contamination in the environment is of increasing concern due to anthropogenic activities as well as natural processes. Metal resistant microorganisms capable of remediating metal-contaminated sites are being investigated as a solution for cleanup of soils. In environments of atypical metal concentrations, many bacteria have been isolated as possible candidates for bioremediation. This study looked at the potential of one such environmental isolate, a Pseudomonas sp. strain S8A, to resist the heavy metal cadmium. S8A was isolated from a heavily contaminated mining soil in Northern Idaho containing up to 4.9 mg Kg-1 total cadmium and 385 mg Kg-1 total lead. Previous studies have shown that this bacterium can survive in metal concentrations of up to 200 mg L-1 soluble Cd. One-dimensional SDS-PAGE showed a marked expression of a 28 kDa protein. N-terrninal sequencing identified the protein as a putative CpxP protein (92% .identity to P. fluorescens Pf0-1 ), an important envelope stress protein. The Cpx stress response has not previously been studied in pseudomonads nor ever been associated with metal stress. Transcriptional analysis using RT-PCR confirmed the upregulation of the putative cpxP gene when exposed to cadmium. Transformation of the gene into E. coli did not confer increased cadmium resistance, yet knockout of the cpxP gene in S8A indicates an altered growth pattern when exposed to cadmium. This abstract accurate! y represents the content of the candidate's thesis. I recommend its publication. lll

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DEDICATION I dedicate this thesis work to my mother Alicia and my sister Erika (a.k.a. Pooh Bear). Thank you so much for all of your support and love ... I know I've been a wild child. You both mean the world to me and I never would have succeeded without both of you to pick me up through the tough times. All my love back at you ...

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ACKNOWLEDGEMENT Dearest thanks to all of the friends that I've made in my time at CU-Denver, especially those that I've worked closely with ... Carol, Lisa, Jacinta, Biewenga, Adam, Julie J., James, Drew, Deana, Leigh and Jeff Boon. You've all inspired me greatly to become the person that I am today and I thank you for all of your support and patience with me. Aida and Duri you have always been my big sister and brother. I love you both very much ... I appreciate you getting into trouble so I knew what NOT to do to upset the boss I definitely missed you when you left but know that I am so proud of both of your accomplishments. Thank you Dr. Audesirk for being a part of my thesis and comp committees and for allowing me to work in your lab as well. Huge hugs to my other committee members, Dr. Timberley Roane and Dr. Martin Gonzalez (a.k.a. Thing 1 and Thing 2). You have both been absolutely amazing to work for and endless thank yous for the long chats and giving me this wonderful opportunity. May the last bacterium standing be either E. coli or Pseudomonas ... although we already know who it will be. Thank you to my surrogate family: Marty, Kris, Danny, Steven and Jo Jo ... you taught me patience and the importance of what truly matters in life. A big shout out to all the students, faculty and staff at Southwestern University for welcoming me and being so interested in this research project. Lastly I would like to thank my husband for supporting women in science. You're amazing lover ... stay cool forever.

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CONTENTS Figures ......................................................................................................... viii Tables ............................................................................................................ ix CHAPTER 1. LITERA. TURE REVIEW ......................................................................... 1 What are Metals? ................................................................................ 1 Microbial Metal Resistance Mechanisms ........................................... 9 Characteristics and Microbial Remediation of Cadmium ................ 18 Bacterial Envelope Stress Responses ............................................... 22 Soil Remediation Challenges and Processes .................................... 30 Metals in Soil Systems ......................................................... 30 Traditional Metal Remediation.-34 Biological Remediation ........................................................ 38 Conclusion ........................................................................................ 40 Project Summary .............................................................................. 42 2. MANUSCRIPT ...................................................................................... 44 Introduction ...................................................................................... 44 vi

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Methods ............................................................................................ 48 Bacterial Strains, Plasmids and Growth Conditions ............ .48 One-dimensional Protein Profile .......................................... 50 Primer Design and PCR Amplification ................................ 51 Plasmid Constructions and Strategy for Cd Gene Knockout ..................................................................... 52 RNA Isolation and Transcriptional Analysis ....................... 53 Mating of S8A with SMlOA.pir pMKT/pKJH ....................... 56 Determination of Metal Tolerance ....................................... 57 Results ......................................... ; .................................................... 57 One-Dimensional Protein Expression .................................. 57 Transcriptional Analyses of Putative Cd Resistance Gene .. 58 S8A cpxP Disruption ............................................................ 61 Cadmium Resistance of S8A Knockout ............................... 61 Discussion ......................................................................................... 63 3. FUTURE DIRECTION .......................................................................... 69 APPENDIX A. UNSUCCESSFUL PROTOCOLS .......................................................... 71 REFERENCES ......................................................................................................... 73 vii

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FIGURES Figure 1.1 Proposed Microbial Metal Resistance Mechanisms ........................................... 12 1.2 The Czc Anti porter ............................................................................................. 17 1.3 The
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TABLES Table 1.1 Standard Concentrations of Various Metal Elements Found in Environmental Systems ............................................................................... 6 2.1 Bacterial Strains and Plasmids Used ................................................................. .49 2.2 Comparisons of DNA and Amino Acid (aa) Sequence Similarity to Homo logs of the CpxP Protein Between Pseudomonads and E. coli .................. 64 ix

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CHAPTER 1 LITERATURE REVIEW What are Metals? The lithosphere is the crust-associated mass of the earth that contains many of the metals useful to society. Concentrated metal deposits, or ores, are located within the lithosphere and are typically found as solid inorganic compounds, e.g. the mineral bauxite is aluminum oxide (Ah03), the mineral greenockite is cadmium sulfide (CdS) and the mineral cinnabar is mercuric sulfide (HgS). These minerals and others are the main sources of commercially useful metals, such as lead (Pb), copper (Cu), titanium (Ti), zinc (Zn), cadmium (Cd), mercury (Hg), gold (Au), silver (Ag) and iron (Fe), and are commonly found as metal oxides, sulfides and carbonates. Metallurgic processes, such as smelting and leaching, extract pure metals from their natural crude sources for commercial purposes. Many commercial metals are actually alloys, or mixtures of metals. For example, bismuth (Bi) is the primary metal in the alloy Wood's metal, a fusible metal used in sprinkler systems and frre extinguishers that also contains trace amounts of lead, cadmium and tin (Sn). Stainless steel is often composed of iron, nickel (Ni) and chromium (Cr), while brass metal is typically a mixture of copper and zinc. 1

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In general, elemental metals are malleable, ductile, lustrous solids that are good conductors of heat and electricity. With the exception of mercury, metals are solids at room temperature, and in aqueous solutions they tend to form cations, or positively charged ions. With a characteristic specific gravity greater than 5 g/cm3 heavy metals (e.g. Cu, Cd, Pb), are differentiated from other metals. Most heavy metals have incompletely filled d-orbitals, which allow them to form complexes that are redox active and Lewis acids (Nies and Brown, 1998). Semimetals, commonly called metalloids, are those elements with properties of both metals and nonmetals, for example silicon (Si), arsenic (As) and tellurium (Te). They are lustrous like metals, yet like nonmetals are not efficient at conduction or insulation and tend to form anions or oxyanions in aqueous solutions. To date, there are 86 known metal/metalloid elements in the periodic table, all of which can be further classified based on their biological necessity as essential, toxic or nonessential and non-toxic. Essential metals are those that are nutritionally required in trace amounts (microgram to milligram amounts per day) for organism survival. These include copper, zinc, iron, cobalt, nickel, chromium, calcium (Ca), potassium (K), sodium (Na), magnesium (Mg), molybdenum (Mo), manganese (Mn) and selenium (Se). These metals form complexes with ligands and chelating agents, which are important for cellular processes including photosynthesis (Cornah et al. 2003), gene regulation (O'Halloran, 1993) and a multitude of bacterial enzymes and processes (W ackett et al., 1989). Many metabolic processes depend on the formation of these 2

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metal:chelate compounds in order to function properly; some enzymes require. two essential metals for catalytic function (Gaetke and Chow, 2003). Essential metals also help maintain osmotic balance within the cell. Metals with no known biological function or purpose and that have demonstrated dangerous properties to biological systems are deemed toxic. Toxic metals include Cd, Pb, Ag, Cu and Hg, which can inhibit metabolic processes, modify physicochemical properties of the cell surface and alter microbial community structure (Shannmuganathan et al. 2004; Akagi et al., 2000; Roane and Kellogg, 1996; Gupta and Singhal, 1995; McEldowney, 1994). Toxic metals and metalloids resemble essential metal elements. For example, toxic divalent cations are similar in size to essential ions and can therefore enter cells through essential ion uptake systems. Because of the similarities between essential metal elements, uptake systems are not always highly specific due to the energy expense of influx, and therefore do not differentiate between essential and toxic ions. In addition, oxyanions like chromate and arsenate resemble the macronutrients sulfate and phosphate, respectively (Nies, 1999; Silver and Walderhaug, 1992), leading to the disruption of protein and DNA structure. Toxic metals include heavy metals and some metalloids, but even essential metals may be toxic to cells at increased biologically available levels. For example, copper is important for maintaining protein structure and function, yet when copper is overly abundant, non-chelated reactive copper can cause oxidative stress and damage due to radical formation 3

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(Gaetke and Chow, 2003). Recent research has also indicated that multiple glycolytic proteins in Saccharomyces cerevisiae are oxidatively damaged when exposed to 170 ppm of copper (Shannmuganathan et al. 2004 ). The nonessential-nontoxic metals have no apparent function within cells, and even when found to accumulate in cells demonstrate no known toxic effect (Roane and Pepper, 2000). Such metals include titanium (Ti) and cesium (Cs). Although considered nontoxic, the Agency for Toxic Substances and Disease Registry (ATSDR) has indicated that certain non-toxic metal compounds, such as titanium tetrachloride, can have some human health risks associated with them. The use of metals, predominantly copper and gold, dates back to 6000 B.C. These two metals were readily found in their "free" form instead of chemical compounds and could be collected as nuggets from the beds of rivers and streams. With time, humans recognized that the metals could be hammered and manipulated into different shapes and sizes for use as tools, weapons and ornaments, soon replacing those made of clay and stone. Smelting sites resembling pottery kilns have been discovered dating back to 5000 B.C. and mining for gold is thought to have occurred before 3000 B.C., revealing the advancement of metallurgy. Other metals of interest included lead, silver, iron and tin (used to make the alloy bronze) and many civilizations, including Egyptian dynasties and Roman empires, actively mined and smelted metal ores into their pure form (Aitchison, 1960). Rapid and active use of the versatile elements markedly expanded with the Industrial 4

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Revolution and has yet to cease. Today's technologically advancing society uses metals for manufacturing major exported and imported goods, including automobiles and computers. Metals are also components of agricultural pesticides, medicines, tobacco products, piping, paints, jewelry, coinage, batteries, buildings, surgical tools, dishes, artwork, appliances and many other products without which our society could not operate. While multifaceted and important worldwide past and present, metals pose a threat when they interact with biological systems at elevated levels. Interestingly, its been proposed that the fall of the Roman Empire was due to lead poisoning (Nriagu, 1983b), a common metal used to line pots and dishware, and that Napoleon died of chronic arsenic poisoning at St. Helena (Forshufvud and Weider, 1978). Anthropogenic influences have greatly increased the release of bioavailable metals into the environment, far beyond natural processes. According to the ATSDR, 25-30 thousand tons oftotal Cd are released per year, with only about half being due to natural processes. Mining and fossil fuel burning specifically are responsible for 4-13 thousand tons of this total release. Prior to the ban of leaded gasoline in 1996, 94.6 million Kg of lead was released from car exhaust alone in 1979 and while drastically decreased to 2.2 million Kg by 1989, lead is still actively used in industry, e.g. batteries, ammunition, pipes, explosives, etc. Natural processes such as erosion, volcanic eruptions and forest fires have additionally contributed to global metal pollution. Smichowski et al. (2003) detected emissions of metals and 5

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metalloids including Cd, Cr, Cu, Hg, Pb, Zn, nickel (Ni), antimony (Sb ), uranium (U), vanadium (V) and arsenic (As) from Copahue volcano in Argentina. Forest fires in South Korea have contributed to the release of the heavy metal cadmium, found in surface soil, litter and the ash of burned trees in quantities exceeding 790 ppm (Shin et al., 2002). Metals are additionally released from deep sea hydrothermal vents (Metz and Trefry, 2000), often in the form of sulfate and sulfite compounds. As metals are released into the environment, they pollute drinking water and agricultural soils and their mobility creates concerns globally since they are carried in the atmosphere and waterways. Table 1.1 shows background environmental concentrations of select toxic metals found in soil, water and air. Table 1.1 Standard Concentrations of Various Metal Elements Found in Environmental Systems. Metallic Soil1 Water1 Atmosphere1 Element (part per million) (part per billion) (part per billion) Arsenic 1-40 1 1-2000 Cadmium 0.25 <1 <1-40 Chromium <0.002 ND2 10-30 Mercury 0.02-0.265 <5 <6-20 Nickel 4-80 <10 2.2 Lead ND2 <5 ND2 Zinc ND2 3 <1 1Detennined by the Agency for Toxic Substances and Disease Registry (ATSDR) 2Not determined 3Present in most drinking water sources. 6

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Metals in the environment can be found in insoluble and soluble forms. Insoluble metals are complexed, immobile and typically considered biologically unavailable. On the other hand, soluble metals are mobile and biologically available, or bioavailable, and therefore considered toxic to living cells. Because both metal forms make up the total environmental metal concentration of a system, it is important to understand that total concentrations do not necessarily reflect the toxicity of the system. For example, cadmium in its mineral form CdS is biologically unavailable to most organisms, but soluble Cd2+ is chemically reactive and can displace essential metals such as Zn2+ within the cell creating nonfunctional enzymes, thereby disturbing metabolic processes. While total environmental metal concentrations are of concern, metal speciation is particularly important when discussing biologically available levels of metal. Though found naturally in the environment, metals do not generally exhibit lethal properties while complexed; however, upon natural weathering of rock formations, metals are released into soil and water systems where they can transform into several different chemical species dependent upon environmental conditions, e.g. pH, temperature, oxygen gradients and organic matter (Collins and Stotzky, 1989). Depending on the type of metal and the particular metal compound formed, metals will react differently with biological systems. Different metal types will also exert different toxic effects, if any, depending on the bioavailable metal concentration. 7

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Three factors detennine the biological significance of metals: the abundance of the metal in the lithosphere, the toxicity of the metal and the solubility of the metal ion in water (Nies and Brown, 1998). Metals with increasing atomic mass are generally less abundant in the lithosphere and therefore do not occur in ecosystems at high concentrations. Most biologically important metals are widespread in the environment. Metals with increasing mass also tend to be more toxic to cells due to their increased affinity for sulfur compounds, including amino acids. Such metals, including Cd, Pb, Hg and Ag, bind strongly to sulfides and are too toxic to play a physiological role in the cell. While some essential metals fulfill these characteristics, they are only mildly toxic and only at increased concentrations, e.g. Cu and Zn. Otherwise, at appropriate concentrations they are important enzymatic ligands. In addition, many metal cations are nearly insoluble at neutral pH, e.g. some divalent and many tri-or tetravalent metals (Nies and Brown, 1998). Because biological systems are predominantly water-based, these metals are unavailable. Although toxic metals are detrimental at elevated bioavailable levels, organisms from all kingdoms have developed metal-tolerance and resistance mechanisms for survival in environments with increased metals. Mechanisms can be grouped as metal-independent, e.g. production of a slime layer, and metal dependent, e.g. efflux pumps; both groups can help alleviate metal stress. 8

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Microbial Metal Resistance Mechanisms It is important to understand that while metals are toxic at elevated concentrations, they are typically most toxic when in a soluble form. Soluble metal ions tend to be most chemically reactive in biological systems and cross cell membranes more readily, whereas insoluble, bound metal compounds are less bioavailable. When metals are bound they have a difficult time crossing the membrane, in part due to the size of the metal compound as compared to the ionic form. Microorganisms, as well as some plant and animal cells, have developed resistance mechanisms that alter the chemical state of toxic metals and metalloids. While not fully understood, we do know that proposed microbial metal resistance mechanisms can be classified into two categories: (1) nonspecific or metal independent and (2) metal-dependent (Figure 1.1). Nonspecific mechanisms include those that are primarily involved in cellular functions and only secondarily in metal resistance, for example exopolysaccharide (BPS) production, biosurfactant production, and metal salt production. BPS comprises extracellular slime layers and is associated with the formation of biofilms, as well as adhesion and cellular protection against dessication. Slime layers include carbohydrates and protein monomers with various functional groups that may result in sequestration or ionic binding of metal ions rendering the metal less reactive, and therefore, less biologically available. In a study by Loaec et al. (1998), BPS (0.1% w/v) from hydrothermal vent bacteria was found to chelate lead, 9

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cadmium and zinc differently depending on the content of the slime layer and the specific metal ion characteristics. Lead ions proved to bind more readily to the strong electron donors carboxylate and N-acetyl hexosamines of the BPS due to their larger radii (112 pm) with a binding capacity of up to 316 parts per million, as compared to cadmium (97 pm) and zinc (74 pm) which demonstrated respective binding capacities of 154 parts per million and 77 parts per million. Mohamed (2001) shows evidence of the cyanobacterium Gloeothece magna adsorbing 38-43% of cadmium (in up to 1 mg L-1 total Cd) and 18-47% of manganese (in up to 5 mg L-1 total Mn) via exoand capsular-polysaccharide extracted from G. magna at concentrations of 9J.Lg mL-1 and 15.5 J.Lg mg-1 respectively. Studies have also shown that bacteria embedded in biofilms are protected against copper toxicity, displaying sequestration of the metal by the BPS (feitzel and Parsek, 2003). Biosurfactants are compounds produced by microorganisms that alter conditions at interfaces, such as decreasing the surface tension of water, to increase nutrient availability. Biosurfactants are amphipathic molecules that act as emulsifying agents and can be used to increase the degradability of organic compounds (Maier, 2000; Mulligan, 2005). Multiple surfactants have been discovered, including rhamnolipid and surfactin from the bacteria Pseudomonas aeruginosa and Bacillus subtilis, respectively, and sophorolipid from the yeast Torulopsis bombicola. No studies to date have shown that surfactant is produced in response to metal; however, the natural production of surfactant has minimized the 10

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toxicity of various metals by nonspecific binding of the metal via their hydrophilic portion, trapping the metal in micelles, which are too large to cross cell membranes. For example, Mulligan et al. (2001) demonstrated the use of rhamnolipid, sophorolipid and surfactin to remove the metals Cu and Zn from contaminated soil sediments. Single washes removed 65% Cd and 18% Zn (rhamnolipid), 25% Cu and 60% Zn (sophorolipid) and 15% Cd and 6% Zn (surfactin), and the mechanism of removal included sorption of the surfactant onto the soil particles and binding to the metal, followed by detachment of the metal from the soil particles and association with the surfactant micelles. Studies have also indicated that the addition of rhamnolipid to cultures of a cadmium sensitive Burholderia sp. allowed enhanced degradation of naphthalene in the presence of cadmium at equimolar or greater concentrations of rhamnolipid to cadmium, due to complexation of free cadmium to the surfactant (Sandrin et aL, 2000). The release of lipopolysaccharide (LPS) from the cells also increased with increasing rhamnolipid concentration, making the cell surface more hydrophobic and less sensitive to cadmium uptake. Byproducts of microbial metabolism can also complex metals making them insoluble, for example sulfides and phosphates produced during anaerobic respiration and chemolithotrophy. Sharma et al. (2000) have shown that a Klebsiella planticola strain isolated from a salt marsh is able to grow in up to 1700 ppm total CdCh and, when grown with thiosulfate, precipitates ca. 20% of the cadmium as CdS. Desulfotomaculum auripigmentum, when grown in the presence of 1 mM 11

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Reduct1on Hg_. Hg Cu-+Cu Cd Cd CH Hg (CH 1 Hg Metallothionem-llke protem production -(cys..<:ys) Cd Prec as metal salts Cd _. CdS /EPS sequestration Cd CdCdCd Cd Pb Pb Pb' Pb Pb Pb Pb Pb Pb Pb Intracellular sequestrabon Cd -+ CdPO Figure 1.1 Proposed Microbial Metal Resistance Mechanisms (from Roane and Pepper, 2000). 12

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sulfur (VI) and 1 mM arsenic (V), precipitates arsenic trisulfide (As2S3) both intra and extracellularly within a week (Newman et al., 1997). Another sulfate-reducing bacterium, Desulfomicrobium norvegicum, removed 200 J!M.selenite (Seol, from solution upon precipitation with bacterially produced sulfide (Hockin and Gadd, 2003). Recently, it has also been shown that the presence of 100 mM copper stimulated the degradation of cellular polyphosphate and subsequent efflux of the metal-phosphate compound out of the bacterium Acidithiobacillus ferrooxidans (formerly Thiobacillus ferroandoxidans), indicating a combination of metal independent and dependent tolerance mechanisms (Alvarez and Jerez, 2004). Metal-dependent mechanisms are utilized in response to a specific metal species and include sequestration of the metal within the cell, elimination of the metal from the cell and reduction of toxic metal species to a less toxic, reactive form. Metallothionein and metallothionein-related proteins, i.e. glutathione and phytochelatin, are low molecular weight, cysteine-rich proteins with the ability to sequester specific metals intracellularly. Various species including bacteria, yeast, plant and animal cells utilize these proteins, typically in response to copper, zinc and cadmium toxicity (Higham and Sadler, 1984; Cavet et al., 2003; Gupta et al., 1995; Memon et al., 2001); an extensive review by Coyle et al. (2002) discusses the importance of metallothionein in mediating metal and drug toxicity of eukaryotic cells. While thought to be important in zinc homeostasis, metallothionein can also be regulated by increased levels of cadmium and copper. The smtA metallothionein 13

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gene in cyanobacteria, well-characterized in Synechococcus PCC 7942, is upregulated in the presence of zinc, cadmium and copper and when deleted from the genome has shown to decrease bacterial cadmium resistance (Cavet et al., 2003; Ybarra and Webb, 1999). The yeast Saccharomyces cerevisiae also produces a metallothionein-like protein CRS5 in response to elevated copper levels, and when the gene was deleted, yeast sensitivity to copper increased (Culotta et al., 1994). Microbial species can transform some metals into a gaseous state, specifically mercury, arsenic and selenium, via the addition of methyl or ethyl groups to the metal ions. While more toxic and able to cross cell barriers more easily in a methylated form due to their lipophilic nature, methylated metal compounds are capable of diffusing away from the cell resulting in a less toxic environment, at least in the immediate area surrounding the cell. For example, Pak and Bartha (1998) showed that pure cultures of Desulfovibrio desulfuricans LS were able to produce methylmercury when grown in the presence of HgC}z or CH3Hgl. Pseudomonas fluorescens has also been shown to methylate selenite under anaerobic conditions (Eriksen et al., 1999). Mercury methylation was observed in the sulfate-reducing bacteria Desulfobacterium sp. strain BG33 and Desulfovibrio desulfuricans, producing greater than 647 pg mL-1 CH3Hg when grown in 100 ng mL-1 inorganic mercuric nitrate. While less than 1% of the total mercuric nitrate was transformed, this may be sufficient methylation to reduce mercury toxicity immediately surrounding the cells (King et al., 2000). 14

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Efflux systems, both energy-dependent and independent, decrease intracellular metal concentrations by pumping metal ions out of the cell. Metal efflux is attributed to multiple metal-exporting protein families, including the RND (for metal resistance, root nodulation, and cell .Q.ivision) family, the CDF (ationic .Q.iffusion facilitator) family and the soft metal-transporting ATPases (or CPx-type ATPases) (Nies, 2003). Proteins in the RND family are part of a proton-driven antiporter and may be involved in the transport of heavy metals (Cu, Cd, Zn, Co, Ni, Ag), hydrophobic compounds (namely antibiotics) and nodulation factors. They are capable of exporting their substrates from the cytoplasm, cytoplasmic membrane or periplasm. An example is the well-characterized CzcA protein of Ralstonia metallidurans CH34 which increases resistance to Zn, Co and Cd by up to 50-fold (Nies, 2003). Figure 1.2 depicts the Czc efflux system. As protons are formed during respiration and released into the periplasm, they are then imported through the RND protein, in this case CzcA, while substrates such as metal ions are expelled. Legatzki et al. (2003) have shown that the Czc system in R. metallidurans AE104 increased zinc resistance 240-fold (from an MIC of to 12,000 and cadmium resistance 50-fold (from an MIC of 1 J.1M to CDF family transporters are driven by a concentration gradient and most are involved in Zn resistance (others include Co, Ni, Cd and Fe). The CPx-type pumps are energy dependent and substrates include essential and toxic metals such as Ca, Mg, Ag, Zn and Cd. A recent study has shown that the presence of the CPx-type Cad (for 15

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cadmium) efflux system could elevate Cd resistance 350-fold (from an MIC of I p.M to 350 p.M) in R. metallidurans AEI04, and in conjunction with the Czc system AEI04 acquired a 3000-fold increase (from an MIC of I J.LM to 3000 p.M) in cadmium resistance (Legatski et al., 2003). Studies indicate that ATPases are also involved in vacuolar sequestration of arsenic in the yeast Saccharomyces cerevisiae (Ghosh et al., I999). While they are important for ridding cells of toxic metal ions, CPx-type systems are also the entrance point for toxic metal ions. For example, Cd2+ ions can enter the cytoplasm through the essential Mn(ll) and Mg(ll) uptake systems in bacteria and yeast and the Ca(ll) uptake system in plants. While some efflux systems are activated by one metal species, others can be turned on by multiple metals, as observed with the Czc system. A fmal metal-dependent resistance mechanism includes the reduction of toxic metal species to less reactive forms. For example, organisms harboring mercury resistance, usually in the form of the mer operon, can reduce toxic mercuric ions (Hg2l and sometimes organomercurials to their elemental form (Hg0). Bacteria containing the mer operon transport Hg2+ into the cytoplasm (MerP, MerT and MerC proteins) where it is reduced by mercuric reductase MerA to Hg0 all regulated by the protein MerR (Silver and Walderhaug, I992; Nies, I999). Various forms of the mer determinant have been isolated and are found on plasmids, transposons and the chromosomes of both Gram-positive and Gramnegative 16

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out .. Peri.' Figure 1.2 The Czc Antiporter (modified from released into the periplasm during respiration n exported from the cell interior. 17

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bacteria. The toxic oxyanion selenite (SeOl") can be bacterially reduced into elemental selenium (Se0 ) as well. A recent study by Kessi et al. (1999) shows a complete reduction of up to 1.5 mM selenite into Se0 by the phototroph Rhodospirillum rubrum. Strong evidence suggests that the selenium is exported out of the cell upon reduction and remains stable in this form. Microbial metal resistance has been studied extensively in hopes of utilizing microorganisms for remediation purposes (Mergeay, 1991; Silver, 1996; Nies, 1999; Bruins et al., 2000; Malik, 2004). Specifically, bacteria have adapted to surviving in metal-contaminated environments, like those of mining soils or industrial wastewaters. We would like to take advantage of these known resistance mechanisms in order to detoxify the environment in hopes of reclaiming the waters and lands that have become detrimental to organism diversity and dispersal (Turpeinen et al., 2004; Larison et al., 2000). Thousands of acres of metal contaminated soils may once again be used for non-food crops and recreational areas, instead of lying barren and idle. Characteristics and Microbial Remediation of Cadmium Cadmium is a transition metal located within Group 2B of the periodic table with atomic number 48 and a molecular weight of 112.4 g mor1 This metal has a density of about 8.65 g/cm3 classifying it as a heavy metal (>5 g/cm3). Cadmium serves no known biological function and is thought to be a physiological toxin 18

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because of the nonspecific complexes that it can form within cells, especially at increased concentrations (Nies, 1999). In their respective reviews of heavy metal toxicity, Nies (1999) and Bruins (2000) describe how non-biologically relevant metals like cadmium tend to become toxic once they have crossed cell barriers because they resemble essential metal ions structurally, thereby disrupting protein and nucleic acid structure and function. For example, cadmium tends to bind sulfhydryl (-SH) groups of enzymes, which can induce conformational changes of proteins or inhibit important physiological roles of essential ions, namely zinc (Zn), which is an important component of DNA binding proteins. Because cadmium is toxic to most organisms at low soluble levels (Nriagu, 1983a), it remains one of the top metals of concern in the environment. Cadmium is associated with zinc ores, often a primary component of contaminated mining soils, and is widely used by industry. Major uses of cadmium include electroplating (Morrow, 2000), pigmentation of paints (Feller, 1986), pesticides and fertilizers (Taylor, 1997; Norwood ill and Tate, 1992), battery proquction (Putois, 1995) and computer chip manufacture (Y annai and Meshulam, 1996). With increased levels of these industrial activities, cadmium has become a contaminant of the surrounding soil, water and atmosphere, leading to bioaccumulation of the heavy metal in plants (Asami, 1984) and animals (Vahteristo et al., 2003; Saeki et al., 2000; Endo et al., 19

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2002; Endo et al., 2004), in addition to the inhibition of microbial biogeochemical cycling (Sandrin and Maier, 2003; Ganguly and Jana, 2002). While intolerable to most organisms, cadmium resistance has been observed in various bacterial species. Both Gram-positive and Gram-negative bacteria have exhibited cadmium resistance, utilizing both non-specific and metal-mediated resistance mechanisms. Cadmium sequestration via metallothionein proteins has been observed in the cyanobacterium Synechococcus (Daniels et al., 1998). The most prominent form of cadmium resistance is that of efflux, both energy-dependent P-type ATPases and RND antiporters, as described earlier (Oger et al,. 2003; Legatzki et al., 2003; Lee et al., 2001; Hassan et al., 1999; Nies, 1995; Nucifora et al., 1989). Two well-characterized efflux systems associated with cadmium resistance include the Cad system, originally found in the Gram-positive bacterium Staphylococcus aureus, and the Czc system, in the Gram-negative bacterium Ralstonia metallidurans CH34 (formerly Alcaligenes eutrophus), also involved in zinc and cobalt efflux. The Cad system is energy-dependent while the Czc system is a cation-proton antiporter. Both systems, however, decrease cytoplasmic concentrations of cadmium within the cell (Nies and Silver, 1989; Legatzki et al., 2003; Nies, 2003), and cloned strains of E. coli harboring the Czc operon have been shown to decrease internal cadmium concentrations as well (Nies, 1995). Figure 1.2 depicts the Czc pump system as demonstrated by Rensing et al. (1997). The CzcA 20

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transmembrane protein transports the metal ions across the cytoplasmic membrane, while CzcB and CzcC remove the ions from the periplasm and through the outer membrane, respectively. The Cad system is a CPx-type efflux system that decreases intracellular accumulations of cadmium within bacterial cells by actively pumping cadmium out of the cell as it enters through other pump systems, e.g. the manganese transport system. Homologs of the cad and czc genes have been isolated from bacteria of multiple genera and species, including Bacillus, Halobacillus, Staphylococcus, Pseudomonas, Micrococcus, and Listeria (Oger et al., 2003; Lebrun,.1994; Hassan et al., 1999) and are both plasmidand chromosomally encoded. Other studies have shown the importance of nonspecific factors, such as biosurfactant production (Sandrin et al., 2000; AlTahhan et al., 2000) or cadmium sulfide production (Sharma et al., 2000), to be critical for cadmium resistance; both nonspecific mechanisms decrease metal toxicity. These incredible cadmium resistance mechanisms have played an important role in cell survival and are of interest to scientists and industry for bioremediation purposes. Some bacteria, due to their ability to decrease metal solubility and mobility, are currently being used for remediation of metal-contaminated wastewaters and soils (Malik, 2004; Yannai and Meshulam, 1996). However, the need for additional organisms is necessary in order to establish metal-resistant communities so that we can utilize abandoned contamination sites once again. There 21

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is also a need to better understand current and unidentified mechanisms of cadmium resistance among microorganisms. Bacterial Envelope Stress Responses Prokaryotic cells are often faced with the challenges of environmental conditions due to their size and simplicity. The only protective barrier is the bacterial envelope, which will encounter the external environment directly. The envelope of Gram-positive bacteria consists of a cell wall, while that of Gram negative bacteria consists of a cell wall with an additional outer membrane. Multiple structural and functional proteins are located within the bacterial envelope, both integral and peripheral. For example, extracellular appendages used for motility and attachment, as well as proteins important for nutrient transport and oxidative phosphorylation are located within the envelope. Depending on the specific environmental stress encountered, i.e. temperature, pH, salinity, pressure, radiation, etc., conditions within the envelope may no longer be suitable for proper protein function due to denaturation or misfolding of the protein. Because of such stressors, more resilient enzymes called foldases and proteases help combat such disturbances. Foldases assist in proper protein folding while proteases degrade abnormally folded proteins (Raivio and Silhavy, 2000). These specific enzymes are under the regulation of certain envelope stress response systems, and these systems are turned on in response to changes in envelope protein folding due to environmental changes. 22

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Two main envelope stress response systems have been identified and characterized in Gram-negative bacteria, namely Escherichia coli, and are reviewed in detail by Duguay and Silhavy (2004). The first system is regulated by an alternative sigma factor crE (or cr24), termed pathway, and was identified when studying the heat shock response in E. coli. Similar to the cytoplasmic heat shock sigma factor cr32 crE transcribes the gene product DegP, a protease important for degradation of misfolded proteins, in response to heat and ethanol (Figure 1.3) (Duguay and Silhavy, 2004; Erickson and Gross, 1989). However, DegP is localized in the periplasm when functioning as compared to the cytoplasm with cr32 The system responds to misfolded and overproduced outer membrane (OM) proteins as they are shuttled from the cytoplasm externally (Mecsas et al., 1993). The RseB and RseA proteins are responsible for the regulation of crE gene products. RseA is an integral protein of the cytoplasmic membrane that interacts with crE in the cytoplasm, possibly as an antisigma factor, and RseB in the periplasm. As misfolded proteins concentrate in the cytoplasm under stressful conditions, it is hypothesized that RseB is titrated away from RseA and is subsequently released, allowing for transcription of its gene targets. The second envelope stress response is the Cpx pathway, which resembles response but been has shown to respond distinctly to different stresses, and is 23

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Normal conditions / Fo111inlfii<:IDn r* I dtil' }-l'roleale .,oHI'J rpoEP2 IJQ Misfoldcd envelope proteins (1JI proteins a larJtel Figure 1.3 The aE Pathway of Escherichia coli (from Raivio and Silhavy, 2004). RseB and aE bind RseA at the inner membrane (IM) under normal conditions. As outer membrane proteins (OMPs) accumulate within the periplasm or external conditions become stressful, RseB is titrated from RseA releasing aE, which can now transcribe its gene targets. 24

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regulated via a two-component system. This system responds to overproduction of P pilin subunits within the periplasm (Duguay and Silhavy, 2004; Jones et al., 1997). As pilin subunits are shuttled externally through the cytoplasmic membrane, they are secreted with a chaperone protein that will assist in final protein assembly. However, if the chaperone is absent, the subunits cannot assemble properly and the Cpx pathway is activated. Attachment of cells to surfaces has also shown to activate the Cpx response (Otto and Silhavy, 2002). The outer membrane lipoprotein NlpE, specifically, is important for proper cell:surface interactions, and studies have shown that the Cpx system is immediately induced upon adhesion due to overproduction of NlpE (DiGiuseppe and Silhavy, 2003). The Cpx system is a two-component regulatory system composed of a sensor histidine kinase, CpxA, and a response regulator, CpxR. Under stress conditions, CpxA is autophosphorylated and in tum phosphorylates CpxR, which leads to the transcription of gene targets including DegP and the Cpx operon (Figure 1.4) (Raivio and Silhavy, 2004; Raivio et al., 1999). Another member, CpxP, acts very similar to RseB of the c!system. CpxP is located within the peri plasm and binds CpxA, preventing autophosphorylation and therefore the Cpx response. As pilin subunits or the NlpE proteins become concentrated, it is hypothesized that CpxP is titrated away from CpxA, inducing autophosphorylation. Studies have shown that cpxP mutants are sensitive to alkaline pH (Danese and Silhavy, 1998) 25

PAGE 35

and that CpxP could also function as a chaperone when titrated from CpxA (DiGiuseppe and Silhavy, 2003). Although there is some overlap, both of these envelope stress response systems are turned on by distinctly different mechanisms. Envelope maintenance is ongoing due to its direct contact with the external environment. Factors such as heat and alkalinity have shown to induce the cl or Cpx pathway, respectively, and both produce the important protease DegP when activated (Danese and Silhavy, 1998; De Las Penas et al., 1997; Erickson et al., 1989). As mentioned, these responses are induced by the concentration of misfolded proteins within the periplasm. Other environmental factors that have been shown to affect the bacterial envelope include metal toxicity and biosurfactant production, yet neither has been shown to induce the crE or Cpx pathway. Metals and surfactant compounds have been shown to affect adhesion and the physicochemical properties of the bacterial envelope. Collins and Stotzky (1992) ----report changes in cell surface charge of various bacteria and yeast when exposed to different heavy metals (Cd, Cr, Cu, Hg, Pb, Zn, Na and Mg). In most instances, the net charge of the cell surface changed from positive (at low pH) to negative (at neutral pH) and back to positive (at high pH), demonstrating the importance of pH in metal speciation and cell:metal interactions. They suggest thatthis charge reversal was a result of the adsorption affinity of different metal species for binding sites on the cell surface changing the net charge of the cell. McEldowney (1994) 26

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demonstrates the effects of cadmium and zinc on physicochemical properties of the Pseudomonas fluorescens H2 envelope. Cell surface hydrophobicity increased substantially (up to 80%) in the presence of cadmium, but not zinc; additionally, anionic surface charges increased in the presence of both cadmium and zinc (up to 63% and 64% respectively) and cationic surface charges increased in the presence of cadmium (up to 41% ). Interestingly, cell adhesion to glass correlated with the increase in surface hydrophobicity. As discussed earlier, biosurfactant can induce release of LPS from a Gram negative Burkholderia sp. altering cell surface properties (Sandrin et al., 2000). The biosurfactant rhamnolipid has additionally shown to induce removal of LPS from the outer membrane of P. aeruginosa strains creating a more hydrophobic surface, hypothesized to occur via binding of the surfactant to Mg2+ ions which are crucial to LPS stability (AITahhan et al., 2000). Depending on the bacterial strain, growth substrate (glucose or hexadecane) and growth phase of the bacteria. cell hydrophobicity returned to normal levels in most cases, indicating possible regeneration of LPS. Modifications to the cell surface may very well induce bacterial stress responses, since these modifications may include outer membrane proteins that must be shuttled through the periplasm, e.g. NlpE and P pilin subunits, which could potentially concentrate in the periplasm if external conditions are altered. In addition, altering cell surface properties may affect interactions between 27

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microorganisms and surfaces, such as soil particles, or bioftlm formation if important surface proteins are removed or altered. Otto and Silhavy (2002) have demonstrated that the Cpx pathway is important for adhesion of cells via the lipoprotein NlpE. Because bacterial cells are directly involved with their external environment, they must overcome unsuitable conditions in orcler to maintain cell surface properties and community structure and function. 28

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/ Poldiq l'rol
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Soil Remediation Challenges and Processes Metals in Soil Systems Metals, unlike organic pollutants, cannot be degraded. Their persistence in the environment is challenging to metal remediation and requires that a biologically available metal be transformed to a less toxic, unavailable form or that the metal be physically removed. In aquatic systems, metals are easily manipulated, for example, by way of changing the pH of the system to increase Qower pH) or decrease (higher pH) metal solubility in order to recover the metals. Remediation of soils, however, is difficult due to their heterogeneous nature; soil is a porous medium containing solid, liquid and gas phases, in addition to the soil biota (see Figure 1.5) (Maier and Pepper, 2000). Metal compounds are associated with all aspects of the soil and are distributed throughout the environment via the soil solution, or liquid phase (Roane and Pepper, 2000). Depending on soil type and characteristics, metals can take on multiple forms, including gaseous, ionic and precipitated metal salts. While we can never rid the environment completely of toxic metals, we can decrease metal bioavailability and mobility, and a variety of methods have been developed in order to achieve this common goal. 30

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0 II I D Sand Silt Clay Cocci bacteria Bacilli bacteria Soil solution Organic matter Fung a l hyphae ...... . .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. . . . . . . . . . . . . . . . Figure 1.5 Illustration of Soil Heterogeneity. A soil aggregate comprises the solid matter of soil : sand, silt, clay, organic material and biota. Soil type and composition will dictate the porosity of the soil. Note that bacteria are found within soil aggregates as well as externally. Fungal hyphae and plant roots are important constituents that help maintain soil structure. 31

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Because contaminated soils are vast, remediation techniques have been aimed at metal detoxification, decreasing metal mobility and solubility, instead of removal. However, soils are complicated and metal species are not fiXed. Soil composition and type play an important role in determining the bioavailability of metals, which are determined by the status of the soil ingredients. The solid phase of soil contains the physical particles of sand, silt and clay, as well as organic matter. These particles have negative charges associated with them and dictate nutrient, water and microbial movement and availability in the system. Another phenomenon known as cation exchange capacity (CEC) is equally important to nutrient and metal availability. The affinity of cations to sorb soil particles is dependent on cation size and charge. Smaller, highly charged cations (Al3+) have greater adsorption affmities than do larger, monovalent cations (Kl (Maier and Pepper, 2000). Soils containing more organic matter or clay particles tend to have an increased overall negative charge associated with them and therefore sorb cations more readily. Because metals located in soils of high CEC remain bound tightly to soil particles, these soils accumulate metals. Still, the overall total toxicity of the soil is low compared to the total metal concentration because most of the metal is biologically unavailable. Soluble nutrients, minerals, organics and metals are located within the soil solution. Water is the main constituent of the soil solution and plays a major role in the concentration and movement of these solutes, as well as microbial growth. Soil particles are also an important aspect of water movement; larger sand particles do 32

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not hold water as efficiently as smaller clay particles, again due to the greater negative charge of clay particles. Water movement due to rainfall, irrigation of soils, groundwater movement and gravitational forces alters nutrient and metal availability, and the flux of these solutes has a tremendous influence on soil pH and microbial community structure and processes. The pH of the soil can alter metal speciation as well. For example, saturated soils tend to favor anaerobic growth and many anaerobic bacteria produce acidic waste products contributing to the pH of the soil solution. Decreased soil pH increases the solubility and mobility of many metals and can also lead to leaching of important nutrients, e.g. Ca2+. Alkaline soils, on the other hand, can lead to the formation of multiple hydroxylated metal species [M(OH)+, M(OH)2, M(OH)J-, M(OH)/-]. The formation of hydroxylated metals varies with metal type and pH, and depending on species will affect the sorption affinity to a charged surface, e.g. soil particles or cell surfaces (Collins and Stotzky, 1989). The gas phase, or soil atmosphere, contains oxygen, nitrogen, and carbon dioxide, which diffuse through the soil as a consequence of biological activity. In well-aerated soils, the soil atmosphere is similar to the gas composition of air; gas exchange occurs at the soil: air interface of soil. However, in subsurface soils exchange with the atmosphere is minimal, so biological activity plays an important role in soil atmosphere composition. Oxygen composition is typically low due to depletion by aerobically respiring organisms, which in tum elevates carbon dioxide 33

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levels. Atmospheric nitrogen does not change as often as other gases due to the limited number of organisms that can utilize gaseous nitrogen. Changes in gas composition further alter redox conditions within the soil, affecting the availability of certain terminal electron acceptors for anaerobic microorganisms, in addition to metal availability and toxicity. Reducing environments have a negative oxidation reduction potential (Eh), while oxidizing environments have a positive Eh (Collins and Stotzky, 1989). Metals located within reducing soils can bind available sulfides and phosphates (produced via microbial metabolism) to form insoluble metal salts; in addition, Eh also affects the valence of metal species. For example, chromium can exist as c2+ or Cr6+ and the toxicity of each differs. Metal remediation is challenging because of the heterogeneous nature of soil. There are many aspects that must be addressed in order to successfully detoxify heavy metal-contaminated soils. Additionally, microbial activity within the soil system will be important for establishing useful and efficient bioremediation strategies. Traditional Metal Remediation Metal remediation methods are targeted at (1) preventing further movement of the metals through the soil beyond the already existing contaminated source and (2) physically removing the contaminants from the soil. Traditional means of physically and/or chemically altering metal-impacted soils include excavation or 34

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removal via soil washing, immobilization or precipitation via chelating agents, and incineration. However, these methods are labor intensive and costly to industry, especially considering that many metal-contaminated soils cover large areas of land, e.g. mining soils, and are damaging to soil structure, composition and fertility. Upon physical removal, storage of contaminated soils in landfills is another issue of increasing concern. Physically removing metal species from the soil or removing the metal contaminated soil altogether is achieved via soil washing and excavation, respectively. Soil washing is a method whereby solubilizing chemicals are added to the soil to facilitate metal removal. For example, Natasohn et al. (1992) demonstrated that concentrated hydrochloric acid (HCl) dissolved up to 80% the total mass of tungsten (W) ore tailings, including the trace metals iron (Fe), manganese (Mn) and scandium (Sc). They developed a sequential process to remove, separate and recover these metals in purified forms for industrial recycling. However, toxic residues may be left over after treatment still containing trace metal compounds. Furthermore, soil microbiota and soil structure are destroyed in the process. An alternative for removal is excavation in which soil is hauled from the contaminated site to a hazardous landfill. Still, depending on the area and depth of soil to be removed, excavation may be impractical and storage space is limited. Immobilization and precipitation of metals can be achieved via multiple methods. Physical isolation, or capping, of contaminated soil with other materials, 35

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such as clean soils or plastic lining, decreases downward movement of metals into groundwater or upward movement into pelagic and benthic ecosystems from contaminated sediments. The capping materials, typically containing organic materials, are formulated to trap metals that escape the contaminated soil (Thoma et al., 1993). Metals tend to accumulate in the soil-litter, which is rich in organic matter (Odendaal and Reinecke, 1999), and the addition of organic material to contaminated soils can also increase metal precipitation via electrostatic interactions of the metal and organic compounds, another reason they are utilized in capping materials. The addition of chelating agents has shown promising efficiency at removing lead, cadmium, copper, mercury and zinc from contaminated soils and fertilizers (Peters and Shem, 1992; Norwood ill and Tate, 1992). One study demonstrated that concentrations of ethylenediaminetetraacetic acid (EDT A) ranging from 0.01 to 0.1M were able to remove lead (500 to 10,000 mg Kg-1 ) by more than 60% in silty and clayey soils within one hour (Peters and Shem, 1992). Chelators, for example trithiocarbonate (Na2CS3), have also been used to treat phosphate fertilizers containing metal impurities, remnants of fertilizer processing (Norwood m and Tate, 1992). Despite the formation of stable metal complexes upon amendment with chelators, soil composition, i.e. organic and clay content, is of great importance for facilitation of this process so metals do not resolubilize. 36

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Chelators should also sustain biodegradation without banning the environment. A downfall, however, is that chelators will also sequester essential elements. Burning soil, or incineration, allows removal of metals through volatilization. However, the metals are only removed from the immediate area, to be redeposited elsewhere. A form of incineration called vitrification is an intense burning of the soil ( 11 00-1450C), followed by freezing to form a glass like substance (Staley, 1995). While the metal is no longer mobile, these processes permanently transform the soil physically, chemically and biologically so that it is neither functional nor useful, and in some instances must still be excavated to a hazardous landfill. Staley (1995) discusses six vitrification technologies that have been developed for permanent isolation of metal and organic contaminants in soil, all requiring atrocious amounts of energy but generating solid byproducts that passed the Toxicity Characteristic Leaching Procedure (TCLP) tests for metals. One technology, the Horsehead Resources Flame Reactor, was developed in 1991 and has shown to recover greater than 80% of cadmium, lead and zinc. Soil heated to 2000C separates the volatile metals from the rest of the soil, termed slag. The non volatile metals remaining in the slag are considered inert and the volatile metals are oxidized after recovery, which can be reused in industry. Of these six technologies, only one has been designed for in site treatment. The soil is melted in the ground using electrical resistance heating, and gases are collected in a hood above the treatment area. While efficient at removing metal and organic impurities, soils are 37

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destroyed in the process and excavation pretreatment is still an issue for some incineration methods. Biological Remediation In lieu of traditional remediation strategies, inoculation of soils with microorganisms (and often plant species) or amendment of soils with biological constituents has been proposed and is actively being researched (see review by Malik, 2004 ). This approach has been termed bioremediation. Microorganisms have demonstrated their ability to survive in the presence of heavy metals and degrade organic pollutants, and more importantly are native to the soil environment. Microbial populations, including bacteria, have been implemented in wastewater treatment for decades, generating reusable water and agricultural fertilizers and compost. Bioremediation efforts aim at utilizing known mechanisms of heavy metal resistance and organic degradation, offering a cost-effective and practical approach to reclaiming contaminated soils. Microbial bioremediation tactics include (1) stimulation of indigenous soil microorganisms via the addition of specific nutrients, pH optimization, etc.; .(2) addition or inoculation with non-native microorganisms; (3) amendment with biological components, such as enzymes or surfactants; and (4) combinations of the three (Bollag et al, 1994). In order for these tactics to be implemented, microbial soil populations and their mechanisms of heavy metal resistance must be researched 38

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and characterized. As described earlier, many mechanisms of metal resistance may enhance transformation of the toxic metal species into a less biologically available form, yet others may not. For example, the efflux pump only protects individual cells, whereas a cell sequestering metal intracellularly or extracellularly via metallothionein proteins or EPS, respectively, would benefit the surrounding community, including higher organisms, by reducing overall bioavailable metal concentrations. Metal removal via volatilization is ideal because there would be no risk of remobilization, as would be with other mechanisms, although accumulation elsewhere is still a recurring issue. We have to be acquainted with our system and the microbial populations within the system in order to formulate efficient bioremediation strategies. Treatment of soils can occur outside of the soil system, ex situ, or directly within the soil system, in situ, utilizing all of the aforementioned tactics. Ex situ remediation allows for control and optimization of treatments within a closed system, in addition to containment of the microorganisms within the system. Treatment of soils ex situ requires that the soil be excavated prior to treatment and typically occurs in bioreactors, contained vessels used to create optimal conditions for a particular microorganism and to enhance contact between the pollutant and the microorganism (Roane et al. 2001). On the other hand, in situ remediation takes place directly within the contaminated soil, wherein indigenous population growth is enhanced via amendment of the soil with nutrients, oxygen, etc. or non-native 39

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microorganisms are added to enhance detoxification and degradation (Maier, 2000; Roane and Pepper, 2000). For either method, however, microbial resistance is typically demonstrated in a laboratory setting prior to enhancement or addition of the microorganisms. Intrinsic bioremediation has also been employed allowing natural detoxification/degradation processes to occur. For example, bacteria in the sediments of Boston Harbor have dramatically decreased mercury levels by 50% since 1978 (http://marine.usgs.gov). While natural remediation can be successful, in situ and ex situ treatments tend to be more efficient and proceed at a considerably faster rate. Conclusion As a leading country in technology and industry, the United States produces more hazardous waste than any other developed country. Over 1200 Superfund sites have been proposed for cleanup, equaling greater than $30 billion dollars (Bollag et al., 1994 ). Many of these sites are contaminated soils from pre-regulation acts and standards, leaving many sites without responsible parties. In Washington state between the years of 1905-1947, lead arsenate was used as a pesticide in fruit orchards and traces of lead and arsenic are still detected in soils and homes located within these sites (Wolz et al., 2003). Many mining areas have been closed for decades as well, leaving thousands of acres of land highly contaminated and without responsible parties. 40

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Human risk associated with living on or near contaminated sites and consumption of contaminated water and food is increasing. Soils are a sink for metal accumulation due to the binding properties of soil particles. Soils with a higher cation exchange capacity (CEC), or ability to sorb metals, include clayey soils and soils high in organic matter, as discussed earlier. Risk associated with these soils is connected to the potential of these metals to leach into groundwater and other water sources used for irrigation of agricultural fields or fishing, a huge staple in Asian countries. Both biotic and abiotic factors determine bioavailable metal concentrations in soil and therefore overall toxicity of metals in the environment. Studies conducted in neighborhoods on or near old lead and gold processing plants have demonstrated impacts on children including gingival discoloration, dermatologic abnormalities and blood metal levels exceeding those considered toxic by the Centers for Disease Control (Akagi et al., 2000; Dfaz-Barriga et al., 1997; Jarosinska et al., 2004). Accumulation of metals, particularly mercury and cadmium, in fish and rice paddies has also been a concern in other technologically advanced countries like Japan and China (Asami, 1984; Endo et al., 2002; Endo et al., 2004). Dickman et al. (1998) have shown a possible correlation between mercury exposure and subfertility in men, while V ahter et al. (2002) provide a review on the health effects of metals in women, including increased bone damage. We have to find low cost, feasible methods in order to detoxify our environment and the use of biological systems and components is a practical and efficient approach. 41

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A better understanding of microbial metal resistance mechanisms will allow for biological remediation. Project Summary Cadmium toxicity is detrimental to the well-being of organisms, disrupting metabolic activity of cells and bioaccumulating in higher plants and animals. Traditional and current methods of remediation are being employed as a means of reclaiming water and soil systems contaminated with this extremely toxic heavy metal. Methods such as excavation physically remove metals from a contaminated site; however, this strategy in addition to other traditional methods is not efficient or feasible for larger quantities of contaminated material and is costly to industry. Bioremediation technologies, which can include inoculation of water and soil with metal-detoxifying bacteria, are economically and environmentally sound, as traditional techniques can destroy soil properties and result in residual pollution. Bioremediation technologies manipulate microbial metal resistance mechanisms in order to detoxify contaminants of interest, both organic and metal pollutants. This study demonstrates a potentially novel cadmium resistance mechanism utilized by the soil bacterium Pseudomonas sp. strain S8A isolated from a heavily metal-impacted mining soil in Northern Idaho. Previous research conducted in our lab shows that S8A can survive in metal concentrations of up to 200 mg L"1 soluble cadmium and 300 mg L-1 soluble lead. The production of BPS 42

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and biosurfactant has been observed in this isolate; both are possible nonspecific mechanisms of cadmium resistance. We have additionally found the expression of a 28 kDa protein when grown in the presence of cadmium. The overall objective of this study was to determine the role of this protein in cadmium resistance. The long term goal of this research is to elucidate the mechanism of metal resistance utilized by this Pseudomonas sp. and to examine the potential use of this novel mechanism in bioremediation of metal-impacted sites. 43

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CHAPTER2 MANUSCRIPT Introduction The bacterial envelope is in direct contact with the environment and is the only protective barrier between the cell and its external surroundings. The Gram positive envelope consists of a thick cell wall, while that of Gram-negative bacteria consists of a thin cell wall and an additional outer membrane. The periplasm that exists between the cell wall and inner membrane of each envelope type is dense with proteins and cell constituents being shuttled to the external cell surface, e.g. pilin and peptidoglycan. External environmental factors, such as temperature, pH, nutrient availability and redox potential, play a critical role in determining the fate of bacterial populations (Collins and Stotzky, 1989). As the dynamic of the environment is altered, multiple physicochemical and physiological changes take place on and in the cell, which in turn affect gene regulation and expression. A diverse arrangement of proteins, polymers and lipids exists on the bacterial cell surface, e.g. teichoic acids, lipopolysaccharide (LPS), autolysins and phospholipids; all are exposed to external perturbations, and surface composition will vary with external conditions and the type of bacterium involved (Doyle, 1989; 44

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Ferris, 1989). It is not unexpected, then, that certain proteins are induced when envelope stress is sensed in order to maintain envelope and cellular integrity. Two main envelope stress responses have been identified and characterized in Gram-negative bacteria, namely Escherichia coli: the ifand Cpx stress response systems (Duguay and Silhavy, 2004). Both systems respond to accumulation of misfolded proteins in the periplasm, yet distinct proteins induce each response. ifis induced via misfolded outer membrane proteins (OMPs) (Mecsas et al., 1993), while Cpx has shown to be induced in response to accumulation of P pilin subunits and the lipoprotein NlpE (DiGiuseppe and Silhavy, 2003; Jones et al., 1997); Additionally, ethanol and heat stress stimulate the crE response, and alkaline conditions stimulate the Cpx response (Danese and Silhavy, 1998; Duguay and Silhavy, 2004; Erickson and Gross, 1989). While quite interesting, neither pathway has ever been studied in response to heavy metal exposure or biosurfactant production, a nonspecific mechanism of heavy metal resistance, both which can alter cell surface properties. Additionally, neither pathway has been characterized in pseudomonads. As a consequence of the presence of carboxyl, amino and phosphodiester groups on the cell surface, cells have a charge associated with them. Because most cells live in a pH above their surface isoelectric point (PI), they tend to have a net negative charge (Collins and Stotzky, 1989); however, it has been documented that heavy metal exposure and biosurfactant production can alter the physicochemical 45

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properties of cell surfaces (Sandrin et al., 2000; Al-Tahhan et al., 2000; McEldowney, 1994; Collins and Stotzky, 1992; Falla and Block, 1993). Collins and Stotzky (1992) have shown that the electrokinetic properties of various microorganisms were altered when exposed to both toxic and nontoxic metal salts. In most instances, the net charge of the cell surface changed from positive (at low pH) to negative (at neutral pH) and back to positive (at high pH), demonstrating the importance of pH in metal speciation and cell:metal interactions. Research by Falla and Block (1993) demonstrates the ability to chemically modify charges on the bacterial envelope, thereby altering metal binding sites and binding affinities of nickel, copper, zinc and cadmium to the envelope. Studies also report an increase in surface hydrophobicity by Pseudomonas fluorescens H2 when exposed to the heavy metals zinc and cadmium, which consequently affected cell attachment to glass substrata (McEldowney, 1994). Metallic ions, such as Ca2+ and Mg2+, are important for the stabilization of multiple cell surface constituents and the activity of surface enzymes, e.g. peptidoglycan synthetases, teichoic acids, and LPS (Doyle, 1989; Ferris, 1989). Biosurfactant, an amphipathic compound produced by microorganisms to increase the availability of organic materials for degradation, can induce release ofLPS (an important determinant of surface charge) from a Gram-negative Burkholderia sp. and Pseudomonas aeruginosa strains, increasing cell surface hydrophobicity (Sandrin et al., 2000; Al-Tahhan et al., 2000). Release is hypothesized to occur via 46

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interactions of the surfactant with Mg2+ ions, crucial to LPS stability. EDTA has shown to induce cell surface hydrophobicity as well, most likely in a similar manner, chelating the essential ions (Johnson et al., 1996). Surfactant production is common in various genera of microorganisms (Mulligan, 2005) and, as a result, may alter cell surface properties. Although no evidence suggests that surfactant is produced directly in response to metal toxicity, surfactant can nonspecifically decrease the toxicity of heavy metals via entrapment of the metal in micelles. Microorganisms producing surfactant may be at an advantage since metals are not only trapped by surfactant, but surface hydrophobicity increases thereby potentially decreasing cationic metal uptake by the cell. This study shows that an environmental soil bacterium Pseudomonas sp. S8A, isolated from a mining soil in Northern Idaho, produces a putative CpxP protein involved in the Cpx envelope stress response system when grown in the presence of cadmium. S8A produces exopolysaccharide, in addition to an unknown biosurfactant; both are nonspecific mechanisms of metal resistance. The protein is 92%,57%,58% and 65% identical to putative CpxP proteins of Pseudomonas jluorescens Pf0-1, P. putida KT2440, P. aeruginosa and P. syringae, respectively, but the Cpx response has not been characterized in this genus. Of the three pseudomonads that have been completely sequenced (P. aeruginosa PA0-1, P. putida KT2440 and P. syringae pv. tomato str. DC3000), only one demonstrates an identified member of the Cpx regulon, CpxR (www.ncbi.nlm.nih.gov). Multiple 47

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approaches were utilized, including transformation of the putative cpxP gene into E. coli and mutation of the gene within S8A, in order to determine the significance of this gerte product in cadmium resistance. We demonstrated that strain S8A produces a 28 kDa protein in response to cadmium, in addition to increased expression of the putative cpxP gene. Evidence suggests that the Cpx stress response or a Cpx-like response is induced when the bacterium is exposed to the heavy metal cadmium. Methods Bacterial Strains, Plasmids and Growth Conditions Bacterial strains and plasmids are listed in Table 2.1. Pseudomonas sp. strains were cultured at 28C in minimal salts medium (MSM) amended with total cadmium as CdCh in concentrations ranging from 0 to 200 mg mL-1 (0-1.8 mM) when appropriate. CdChis completely soluble in this medium at least up to 168 h. The MSM contained the following: 0.5 g of sodium citrate (C6HsNa307), 0.1 g of magnesium sulfate (MgS04 7Hz0), 1.0 g of ammonium sulfate [(Nl4)zS04], 1.0 g of glucose (C6H1206), 0.1 g of sodium pyrophosphate and 100 mL of 0.1 M MES (2-(4-morpholino-ethane sulfonic acid)), adjusted to pH 6.0 with IOMNaOH. E. coli strains were cultured at 37C on Luria-Bertani (LB) agar or in LB broth supplemented with the appropriate antibiotics. Antibiotic concentrations used 48

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Table 2.1 Bacterial Strains and Plasmids Used. Strains and Plasmids Strain E. coli DH5a SM10A.pir Pseudomonas sp. S8A TRr2 P. fluorescens ATCC 13525 Plasmid pMAL-p2X pACYC177 pATCH pATCH2 pATCH3 pATCH4 pMRS101 pMKT pKJH Genotype or description Plasmid free thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu Km A.pir WT S8A cpxP::kan ATCC strain 13525 Cloning vector; AmpR Containing Kan cassette Ndei-EcoRl S8A cpxP PCR fragment ( 450 bp) inserted at pMAL-p2X Ndei-EcoRl site; under control of tac promotor Stul Kan fragment (1340 bp) of pACYC177 inserted at Eagl site of pATCH Ndel site of cpxP BAL31 digested (61 nt) to remove start codon in pATCH Stul Kan fragment (1340 bp) of pACYC177 inserted at Xmnl site of pATCH3 sacB strAB Ap ORIT R6KORI; a pKNG101 sucide vector derivative Pvull cpxP::kan fragment (2318 bp) of pATCH4 inserted at BamHI site of pMRS 101 Deletion of Notl fragment (1800 bp) from pMKT 1 Belgian Co-oridinated Collections of Micro-organisms 49 Source/Reference Invitrogen BCCM/LMBP1 This study This study New England Biolabs New England Biolabs This study This study This study This study This study This study

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are as follows: ampicillin, 100 ug mL-1 ; kanamycin, 50 ug mL-1 ; and streptomycin, 25 ug mL-1 To construct deletion mutants, LB medium was used and supplemented with antibiotics when required for selection. One-dimensional Protein Profile Cells were harvested in exponential phase (OD6oo 0.08 for S8AffRr2 and OD6oo 0.4-0.5 for DH5a.) by centrifugation at 14,000 x g for 10 min (4C) and resuspended in 45 Jll molecular grade water and 15 J.114X Laerrunli loading buffer (25 mL 4X upper buffer [45.4 g Tris, 1.0 g SDS up to 250 mL, pH 8.0], 20 mL glycerol, 4.0 g SDS, 3.1 g DTT, 0.2 g bromophenol blue, made up to 50 mL with dl). Samples were boiled at 95C for 10 min and stored at -80C until use. Cells were quantified via optical density prior to harvesting and samples (60 Jll) were subjected to 16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and run at a total of 140 rnA on a Hoefer SE400 vertical gel unit. Gels were stained with Coomassie Blue R-250 for 1 h followed by destaining in 10% methanol. Protein size was determined using a Rainbow marker (Amersham; Piscataway, NJ) with high range molecular weight (14,300-220,000 MW) or Kaleidoscope Prestained Standards (BIO-RAD; Hercules, CA). When transformed with pATCH (see plasmid construction Figure 2.1 and 2.2), DH5a. cells were grown in LB supplemented with ampicillin. To over-express our protein, DH5a. cells were induced with 1 mM IPTG upon reaching an OD6oo of 50

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0.4 and incubated for 1 h at 37C. Cells were then subjected to SDS-PAGE as described. Primer Design and PCR Amplification The putative cadmium resistance gene was amplified by PCR using unique primers Fs8a (5' ctg age tee ata tgc gca aga etc tta tc 3') and Rs8a (5' ctg age tcg aat tct tat tgc gee ttt tgc gg 3 '), designed with flanking Ndel and EcoRI restriction sites, respectively. These primers were designed to the gene coding sequence of our protein as identified in Pseudomonas fluorescens Pf0-1 by GeneBank (www.ncbi.nlm.nih.gov), subsequently used to amplify the gene coding sequence in SSA. PCR conditions were as follows (per 50J.ll volume): 5 J.lllOX Pfu Buffer (Stratagene; La Jolla, CA), 3 J.ll MgCh (3 mM final concentration), 10 Jtl deoxynucleoside triphosphates (0.2 mM of each), 5 Jtl DMSO (dimethyl sulfoxide), 2.5 Jtl of each primer (0.5 JiM final concentration), 2 Jtl bovine serum albumin (0.16 mg mL-1 final concentration), 1 J.ll Pfu polymerase (2.5 U), 17 J.ll DNase/RNase-free H20, and 2 Jtl DNA template (not quantified). To decrease interference by EPS (exopolysaccharide), a 50 Jtl sample of culture was centrifuged (10,000 x g), and the pellet was resuspended in sterile diH20, repeated twice. Cell lysis was achieved by heat lysing the washed cells for 10 min at 95C in a Perkin Elmer GeneAmp PCR System 2400 thermal cycler (Norwalk, CT). 51

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The PCR conditions included 30 cycles of 95C for 1 min, 57C for 1 min, and 72C for I.5 min. The resulting PCR reactions were then incubated at 72C for 5 min and then kept at 4C. PCR products were electrophoresed on a I% agarose Tris Borate EDT A ( disodium ethylenediamine tetraacetate dihydrate) gel at 120 V, containing a fmal concentration of 0.2 J.Lg mL"1 ethidium bromide. A 123-bp ladder was used to determine PCR product size along with a I Kb ladder for quantification. Upon successful amplification, PCR products were purified using the Wizard PCR Preps DNA Purification System (Promega; Madison, WI) and sequenced (University of Colorado Health Sciences Center Core DNA Sequencing Lab). DNA sequences were analyzed with the BLASTN program provided by the National Center for Biotechnology Information. Plasmid Constructions and Strategy for Cd Gene Knockout To construct the cpxP expressing-vector pATCH, the cpxP gene was amplified from SSA as a 450 bp PCR (described above) and cloned into pMAL-p2X (New England Biolabs; Beverly, MA) after digestion with EcoRI and Ndel restriction endonucleases (New England Biolabs; Beverly, MA), thereby placing the cpxP gene under control of the IPTG inducible tac promotor. The recombinant plasmid was then transformed into E. coli DH5a. Standard cloning methods were used. 52

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To ensure disruption of cpxP in the event of a single recombination crossover into the S8A chromosome, pATCH was BAL31 digested at the Ndel site for 1 min to remove the start codon and shine delgarno sequence of the subcloned cpxP gene, generating pATCH3. pATCH4 was obtained upon insertion of the kanamycin resistance cassette (KanR) from pACYC177 (Stul fragment) into the Xmnl site within the cpxP gene located in pATCH3 (Figure 2.1). A 2318 bp Pvull fragment, containing the cpxP gene truncated at the S'end and interrupted by a kanamycin cassette, was used for ligation into the vector pMR.SlOl, resulting in pMKT (Figure 2.2a). The vector pMRS101 is a pKNG101 suicide vector derivative used in order to force a recombination event of the mutant cpxP allele into the S8A chromosome. Upon removal of the Notl fragment from pMKT, the suicide vector pKJH was created containing the cpxP disruption cassette subsequently used for electroporation and mating into S8A (Figure 2.2b ). RNA Isolation and Transcriptional Analysis Cells of S8A were cultured in the presence of increasing cadmium concentrations (0-50 mg L-1 total Cd) to induce transcription of the target mRNA. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Inc.; Valencia, CA) at an OD6oo of 0.08 in MSM medium. The use of RNase-free DNase I (Qiagen, Inc.; Valencia, CA) removed genomic contamination. 53

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0 100 200 300 400 500 bp Pvull Ndel Xmnl EcoRI Pvull pATCH pATCH3 KanR (1340 bp) pATCH4 Figure 2.1 Construction of pATCH Plasmids. cpxP was obtained as a 450 bp PCR product and cloned into the expression vector pMAL-p2X, generating pATCH. Digest with BAL31 removed the Ndel site (and the start codon) of cpxP (61 nt) constructing pATCH3. The kanamycin cassette was inserted at the Xmnl site of pA TCH3, and pA TCH4 was subsequently used to create the suicide vector pKJH (a pKNG 101 derivative). Restriction sites shown are those used to construct the mutant allele, as described in Methods. The Pviii sites are located on pMAL-p2X. 54

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Pvull EcoRI Pvull 5' cpxP kan cpxP3' BamHI B SBA wild type pMKT --sacB AmpR Str41 TRr2 Figure 2.2 Knockout of cpxP Wild Type Allele. (A) A 2318 bp PvuiT fragment from pATCH4 was cloned into the BamHI site of the suicide vector derivative pMRS 1 01 to create pMK.T. Removal of the Not I fragment from pMKT resulted in pKJH, subsequently used for electroporation and mating into S8A (B) Homologous recombination by the 5' end of the mutant allele in pMK.T/pKJH initiated allelic substitution with the wild type allele to create strain TRr2. 55

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RT-PCR was performed with the OneStep RT-PCR kit (Qiagen, Inc.; Valencia, CA) using 0.5 J.lg total RNA per 50 J.1l reaction. Fs8a (5' sense primer) and Rs8a (3' antisense primer for putative cpxP gene) described earlier were used at a final concentration of 0.5 J.lM each. PCR conditions were as follows: reverse transcription for 30 min at 50C; Taq activation for 15 min at 95C; 30 cycles of 1 min at 94C, 1 min at 57C, and 1 min at 72C; and final extension for 10 min at 72C. Samples (10 J.ll) were visualized on a 1% agarose gel stained with ethidium bromide as described previously. Mating of SSA with SM10A.pir pMKT/pKJH Plasmid pMKT/pKJH was transferred from SMlOA.pir (donor) into SSA (recipient) using an agar plate mating technique with a 1:10 ratio of recipient to donor. Both donor and recipient were spotted onto an LB agar plate and incubated for 24 h at 30C. Cells were suspended in sterile dl water and plated onto LB containing both ampicillin (100 J.lg mL-1 ) and kanamycin (50 J.lg mL-1). Recombinants were confirmed by PCR analysis using the following conditions (per 50 J.ll reaction): 5 J.ll10X Pfx Buffer (Invitrogen; Carlsbad, CA), 5 J.ll10X Pfx enhancer (provided with polymerase), 3 J.ll MgS04 (provided with polymerase), 15 J.ll deoxynucleoside triphosphates (0.2 mM of each), 2.5 J.ll respective primer (0.5 J.lM final concentration), 1 J.ll platinum Pfx polymerase (2.5 U), 14 J.1l DNase!RNase free H20, and 2 J.ll DNA template (not quantified). Primer TR210 (5' tag tcg aga agt 56

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ace tgg aaa aac tc 3 ') was designed for knockout strains and used with primer Rs8a. PCR conditions included 30 cycles of 95C for 1 min, 55C for 1 min, and 72C for 2 min. After cycling, PCR reactions were incubated at 72C for 5 min and then kept at 4C. PCR products were electrophoresed as described previously. Determination of Metal Tolerance To examine survival of S8A in cadmium upon disruption of the cpxP gene, cells (105 cells mL-1 ) were amended with aqueous cadmium chloride (CdCf) to final concentrations of 0, 10 and 20 mg L-1 total Cd. All cultures were incubated at 28C at 180 rpm. Growth was determined by diluting and plating onto MSM medium every 24 h for 168 h. Soluble metal analyses were performed simultaneously using a Perkin Elmer 5000 Atomic Absorption (AA) Spectrophotometer. Samples (5 mL) were centrifuged at 2,500 x g for 10 min to remove particulates prior to AA analysis. Results One-dimensional Protein Expression When grown in various concentrations of cadmium (0-50 mg L-1 total Cd), S8A expresses a protein with molecular weight 28 kDa, which is not evident in cells grown in the absence of cadmium. We have shown that this protein is expressed in 57

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as little as 10 mg L-1 total Cd (data not shown). Our lab has previously isolated and sequenced this protein, identifying the protein as a putative CpxP homolog, a member of the Cpx envelope stress response. Determination of the complete 149 amino acid sequence predicts a molecular weight of 17 kDa. Upon induction of the cpxP gene in pATCH transformed DH5a, protein expression was observed at 17 kDa (Figure 2.3), the predicted molecular weight of the cpxP gene product. However, no increased cadmium resistance was observed with a maximum inhibitory concentration (MIC) of 50 mg L-1 total Cd. Transcriptional Analyses of Putative Cd Resistance Gene We examined the effects of cadmium exposure on in vivo mRNA transcription of the putative cpxP gene in SSA. Reverse transcription of the target mRNA with primers Fs8a and Rs8a demonstrated a linear increase in expression in SSA cultures amended with 0-50 mg L-1 total Cd (Figure 2.4). Northern analysis was unsuccessful due to minimal amounts of RNA isolated ( -5-10 J.lg), therefore transcript size could not be determined. Expressional increase was also observed in P. fluorescens 13525 upon exposure to cadmium (data not shown). 58

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Figure 2.3 SDS-PAGE of E. coli DH5a pATCH. The cpxP gene in pATCH is inducible under the tac promotor by IPTG. Lanes 1, DH5a pATCH (no induction); 2, DH5a pATCH (induction); M, High range Rainbow protein standard. Protein extracts were subjected to 16% SDS-PAGE at a total of 140 rnA. 59

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500 Figure 2.4 Detection of the in vivo Transcripts of cpxP by RT -PCR of S8A to Cadmium. RT-PCR products were electrophoresed in 1 containing EtBr at 120 V. Lanes: M1, 1 Kb marker; 1, 0 mg L-1 tota L-1 total Cd; 3, 20 mg L-1 total Cd; 4, 30 mg L-1 total Cd; 5, 50 mg 1 100 bp marker. 60

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SSA cpxP Disruption To examine the role of the putative cpxP gene in cadmium resistance, we disrupted the wild-type gene with a kanamycin resistance cassette. The cpxP gene was amplified from the SSA chromosome and disrupted by inserting a kanamycin resistance cassette at the 3' end of the gene and chewing back the start site of the gene, exactly 61 nt (Figure 2.1). The gene knockout was inserted into vector pMRSlOl (a pKNG101 suicide vector derivative) for allele replacement by homologous recombination with the wild-type SSA cpxP gene. Recombination was forced to occur at the 5' end of the gene target. Putative SSA strains carrying the cpxP knockout (designated TRr2) were readily obtained using the pMKT plasmid (29 colonies), but not the pKJH plasmid for reasons unclear. As a result, this gene is not essential for cell viability. PCR analysis of the putative cpxP::kan recombination verified integration of the suicide vector into the host chromosomal copy of cpxP (Figure 2.5). Cadmium Resistance of SSA Knockout Figure 2.6 depicts SSA and TRr2 strains grown in the presence of various CdCh concentrations (0-20 mg L-1 total Cd). As compared with SSA, the mutant strain TRr2 appears to have an altered growth pattern when exposed to cadmium. SSA has repeatedly demonstrated increased resistance to higher concentrations of cadmium; however, TRr2 does not seem to follow this pattern indicating 61

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Figure 2.5 PCR Analysis of cpxP::kan Mutant Allele Upon Mati1 SMlOA.pir. PCR products were electrophoresed in 1% agarose co1 120 V. Lanes: 1, SMlOA.pir; 2, SM10A.pir pMKTf; 3, SMlOA.pir p Pseudomonas jluorescens 13525; 5, S8A wild type; 6, S8A post-1 SMlOA.pir pMKTr; 7, S8A post-mating with SMlOA.pir pMKTf; 62

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possible effects of the cpxP mutant allele. Metal solubility was not substantially affected in either strain (data not shown). Discussion Upon amplification and sequencing of the putative cadmium resistance gene in isolate Pseudomonas sp. strain SSA, 92% identity to a putative cpxP protein of P. fluorescens Pf0-1 was observed (see Table 2.2). This protein is a member of a known envelope stress response, the Cpx two-component system (Duguay and Silhavy, 2004; Danese and Silhavy, 1998; Raivio et al., 1999; DiGiuseppe and Silhavy, 2003). During envelope stress, the Cpx system is activated via phosphotransfer reactions of Cpx.A and CpxR (Figure 1.4). Cpx.A is an autophosphorylated kinase that, when activated, phosphorylates CpxR resulting in transcription of Cpx gene targets, such as the protease DegP. CpxP, a periplasmic repressor, binds CpxA at the periplasmic terminus repressing the Cpx response. During stressful conditions, CpxP separates from CpxA leading to response induction; the exact mechanism of detachment is unknown. Transcriptional analyses of SSA indicated that this gene is, in fact, strongly induced with increasing cadmium concentration (Figure 2.4). Because of the similarity of the protein to P.fluorescens Pf0-1, we additionally examined another P. fluorescens strain (ATCC 13525), and as hypothesized, we did observe induction (data not shown). We propose that cadmium is either (a) interacting with the protein 63

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Table 2.2 Comparisons of DNA and Amino Acid (aa) Sequence Similarity to Homo logs of the CpxP Protein Between Pseudomonads and E. coli. Species E. coli K12 P. aeruginosa PA01 P. putida KT2440 P. syringae pv. tomato strain DC3000 P. fluorescens Pf0-1 1 No significant similarity found. 2 16% gaps in sequence. 3 No gaps in sequence. DNA sequence aa sequence identity identity <10% gap ___ 1 29% (29/97 aa)" 72% (162/225 nt) 54% (671122 aa) 76% (1011132 nt) 58% (651111 aa) 73% (197/267 nt) 64% (66/102 aa) 89% (347/386 nt) 92% (117/126 aaf 64

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directly, diminishing repressor activity of the putative CpxP protein or (b) initiating surface changes, as has been documented (McEldowney, 1994; Collins and Stotzky, 1992; Falla and Block, 1993), thereby inducing the Cpx response. As observed with one-dimensional SDS-PAGE, a 28 kDa protein was expressed in the presence of cadmium; however, the amino acid sequence of the putative CpxP protein predicts a molecular weight of 17 kDa, as was also observed when the gene was transformed into E. coli (Figure 2.3). The size discrepancy could be due to direct interaction of cadmium with the protein or post-translational modification of the protein, e.g. phosphorylation. Although shifts in protein size due to metal binding have not been observed to our knowledge, interaction of cadmium and the protein seems plausible. Similar to the accumulation of misfolded proteins titrating CpxP from CpxA to induce the Cpx response (Duguay and Silhavy, 2004), cadmium could be interacting with the peri plasmic protein inducing titration, and therefore the stress response. The frequent mode of cadmium resistance is efflux via soft metal transporting ATPases or proton-driven antiporters (Nies and Silver, 1989; Rensing et al., 1997), and few studies have illustrated bacterial sequestration of cadmium intracellularly (Higham and Sadler, 1984; Cavet et al., 2003; Turner et al., 1996; Roane et al., 2001). Metalloproteins involved in intracellular sequestration are typically rich in amino acid cysteine, e.g. metallothionein and glutathione, but our protein contains no cysteine residues. In a study by Cha and Cooksey (1991), 65

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three proteins of the cop operon have shown to reside within the peri plasm and outer membrane where they bind copper ions. The proteins CopA and CopC are not abundant in cysteine, yet they demonstrated that each protein could bind 11 atoms and 1 atom of copper, respectively, per protein. Therefore, it is plausible that cadmium is interacting with the protein in a different manner. Further investigation will be necessary in order to determine if cadmium is accumulated by the putative Cpx.P protein, or if the size differential could be due to post-translational modification, which studies have not previously addressed with CpxP. Additionally, cadmium or the production ofbiosurfactant, as has been observed with this isolate, could be altering the physicochemical properties of the bacterial envelope, potentially inducing an envelope stress response. Cadmium has a great affinity for sulfhydryl ( -SH) groups of proteins, altering protein conformation and function. As such, misfolded surface and periplasmic proteins may be accumulating within the periplasm due to the toxic effects of cadmium. Surfactant, on the other hand, has shown to increase surface hydrophobicity of cells decreasing cellular uptake of cadmium, most likely through interactions with essential elements like Mg2+ on the cell surface that are important for envelope integrity (AlTahann et al., 2000; Sandrin et al., 2000). Whether biosurfactant production induces surface changes or cadmium is directly responsible for inducing an envelope stress response will be of interest to future studies. 66

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Knockout of the putative cpxP allele in S8A appeared to have an effect on growth when cultured in the presence of cadmium. A characteristic growth pattern repeatedly observed in S8A was no longer observed in the knockout strain TRr2, although TRr2 was able to reach stationary phase within 96 hours as also observed in S8A (Figure 2.6). S8A has shown to have increased resistance to higher concentrations of cadmium; however, decreased growth of TRr2 is observed with increasing cadmium concentration. Future studies will need to address the ability of TRr2 to grow at increased levels of cadmium, as previously observed with S8A. Cadmium solubility did not appear to change throughout the 168 h growth period. A common mechanism of metal resistance in pseudomonads is efflux (Cervantes and Silver, 1996) and we have previously shown that S8A produces an unidentified biosurfactant, which may sequester cadmium decreasing the bioavailability of the metal, although still remaining soluble. Because no detectable decrease in cadmium solubility was observed in either strain, we believe that surfactant production and efflux may be the predominant cadmium resistance mechanisms utilized by this bacterium. However, sequestration by CpxP cannot be excluded as decrease in cadmium solubility may only be in the part per billion range, which is undetectable by our method of metal analysis. Future work will determine cadmium:cell interactions. 67

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Growth of SSA in Cd Growth of TRr2 in Cd le+9 le+9 le+8 le+8 ...1 le+7 le+7 le+6 le+6 U) le+S le+S u le+4 le+4 le+3 le+3 0\ 1a...L'1 1a-.L'1 I'Y'>

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CHAPTER3 FUTURE DIRECTION Additional characterization of this putative cadmium resistance protein is necessary in order to understand the interaction, whether direct or indirect, between the putative CpxP protein and cadmium. Current research has demonstrated that the protein is defmitely induced in response to cadmium; however, the exact mechanism of interaction is unclear. Because of the difficulty in discerning one-dimensional protein gels, 2-D electrophoresis would be greatly helpful so as to detect other gene products of the Cpx stress response (or other responses) that are induced during exposure of SSA to cadmium. Elucidation of cell:metal interactions via the use of transmission electron microscopy will determine whether cadmium is sequestered near or on the cell envelope, possibly leading to induction of the Cpx response. It will also be necessary to examine whether the putative CpxP protein is in fact phosphorylated, so the size discrepancy of the protein can be resolved. Results of the present research indicate that this organism is an unlikely candidate for metal remediation because decreases in metal solubility are not observed, one of the current goals of bioremediation. Nevertheless, an 69

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understanding of how this bacterium deals with cadmium toxicity may lead to novel approaches for cadmium remediation. 70

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APPENDIX A.UNSUeeESSFULPROTOeOLS Northern Blot For northern blots, RNA was extracted from S8A cells grown in LB amended with edeh to isolate sufficient quantities RNA that could not be achieved when grown in MSM; however, sufficient RNA could not be extracted (10-20 Jlg) using our protocol. Total RNA was separated using forrnaldehyde-agarose gel electrophoresis and transferred onto nylon membrane using the TURBOBLOTTER Rapid Downward Transfer System under alkaline conditions (Schleicher and Schnell, Germany) for at least 4 h. Nucleic acid was cross-linked to the membrane with a UV Stratalinker 1800 (Stratagene; La Jolla, eA). After prehybridization at 42e for 1 h with DIG Easy Hyb (Roche; Indianapolis, IN), blots were hybridized with 100 ng rnL-1 of denatured digoxigenin-labeled probe (5' -GeG GGA eAG GTe eAG eTG GeT GTA eGG Gee-3') at least 18h at 42e. Blots were washed at minimal stringency due to the high Ge content of the probe (2 X 5 min with 2X SSe, 2 X 30 min with 0.5 SSe at 50e, and 1 min with phosphate-buffered saline (PBS)). After blocking in PBS containing 2% non-fat powdered milk for 30 min, blots were 71

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developed with 1:15,000 dilution of Anti-Digoxin-AP (Jackson Immunoresearch Laboratories, Inc.; West Grove, PA) for 1 h. The membrane was then washed 3 X 10 min in PBS before applying CSPD (Roche; Indianapolis, IN) for 5 min and exposing the blot to X-ray film (Pierce; Rockford, IL) for 5 min, depending on signal intensity. Electroporation S8A cells were transformed with pMKT via electroporation with a modified method ofFarinha and Kopinski (1990) using GenePulser (BioRad; Hercules, CA) with 0.2 em electrode gap cuvettes. Cells were grown in 50 mL LB broth at 30C to an OD 0.8 (-108 cells mL-1 ) and chilled on ice for 15 min. All reagents and equipment were chilled on ice prior to use. Cells were centrifuged for 10 min at 3,500 x g (4C) and washed 1X with 20 mL sterile water and 2X with 20 mL sterile 10% glycerol solution. The final pellet was resuspended in 0.4 mL 10% glycerol solution and 40 J.Ll was added to a tube containing 1J.Lg DNA. After transfer to the cuvette, cells were subjected to a total electrical pulse of 2.5kV over 5.5 msec. LB (containing 20mM MgCh) was immediately added to the cuvette and allowed to recover for one minute. The cells were then transferred to prewarmed (30C) LB and incubated for 1 h and then plated onto LB supplemented with 50 J.Lg mL-1 kanamycin for selection of putative Cd gene knockouts. 72

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