Microbiology (2014), 160, 2607–2617

DOI 10.1099/mic.0.081398-0

Proteome of Geobacter sulfurreducens in the presence of U(VI) Roberto Orellana,1 Kim K. Hixson,2 Sean Murphy,1 Tu¨nde Mester,3 Manju L. Sharma,1 Mary S. Lipton2 and Derek R. Lovley1 Correspondence

1

Roberto Orellana

2

[email protected]

Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA Environmental Molecular Sciences Laboratory and Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA

3

Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Kellogg Eye Center, Ann Arbor, MI 48105, USA

Received 3 June 2014 Accepted 29 September 2014

Geobacter species often play an important role in the in situ bioremediation of uraniumcontaminated groundwater, but little is known about how these microbes avoid uranium toxicity. To evaluate this further, the proteome of Geobacter sulfurreducens exposed to 100 mM U(VI) acetate was compared to control cells not exposed to U(VI). Of the 1363 proteins detected from these cultures, 203 proteins had higher abundance during exposure to U(VI) compared with the control cells and 148 proteins had lower abundance. U(VI)-exposed cultures expressed lower levels of proteins involved in growth, protein and amino acid biosynthesis, as well as key central metabolism enzymes as a result of the deleterious effect of U(VI) on the growth of G. sulfurreducens. In contrast, proteins involved in detoxification, such as several efflux pumps belonging to the RND (resistance–nodulation–cell division) family, and membrane protection, and other proteins, such as chaperones and proteins involved in secretion systems, were found in higher abundance in cells exposed to U(VI). Exposing G. sulfurreducens to U(VI) resulted in a higher abundance of many proteins associated with the oxidative stress response, such as superoxide dismutase and superoxide reductase. A strain in which the gene for superoxide dismutase was deleted grew more slowly than the WT strain in the presence of U(VI), but not in its absence. The results suggested that there is no specific mechanism for uranium detoxification. Rather, multiple general stress responses are induced, which presumably enable Geobacter species to tolerate high uranium concentrations.

INTRODUCTION Uranium contamination of sediments and ground/surface water has become a serious environmental concern, especially at many former uranium mining and processing facilities (Bradford et al., 1990; Wall & Krumholz, 2006; Schnug & Haneklaus, 2008). One strategy for preventing the spread of uranium in the subsurface is to take advantage of the ability of some micro-organisms to reduce soluble U(VI) to poorly soluble U(IV) (Anderson et al., 2003; Finneran et al., 2002; Holmes et al., 2007, 2009; Lovley et al., 1991; Abbreviations: AMT, accurate mass and time; FT-ICR, Fourier transform ion cyclotron resonance; NET, normalized elution time; PMT, putative mass and time; RND, resistance–nodulation–cell division; TFA, trifluoroacetic acid; UMC, unique mass class. The Peptide Atlas repository accession number for the crude proteomics data is PASS00563. Two supplementary figures and 20 supplementary tables are available with the online Supplementary Material.

081398 G 2014 The Authors

Printed in Great Britain

Snoeyenbos-West et al., 2000; Vrionis et al., 2005). This approach has been investigated in a diversity of subsurface sites (Converse et al., 2013; Newsome et al., 2014; Wall & Krumholz, 2006; Williams et al., 2013). In many instances, stimulation of dissimilatory metal reduction with organic electron donors specifically enriched Geobacter species which were highly effective in U(VI) reduction (Lovley et al., 2011; Williams et al., 2013). Geobacter sulfurreducens has served as the primary model organism to elucidate the physiological capabilities of Geobacter species (Lovley et al., 2011; Mahadevan et al., 2011). Gene deletion and uraninite localization studies have suggested that G. sulfurreducens reduces U(VI) at the outer cell surface with a diversity of c-type cytochromes (Orellana et al., 2013; Shelobolina et al., 2007). However, some uranium may enter the cell and little is known about the physiological response to this uranium. Unlike essential metals that can be imported or extruded depending on the requirements of the cell (Nies, 2013), uranium is not 2607

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expected to be a required nutrient and is likely to be toxic because U(VI) can denature proteins, inactivate functional groups of enzymes, disrupt the cell envelope and damage DNA (Ca´novas et al., 2003; Nies, 1999). There is not yet evidence for U(VI)-specific transport or a protective mechanism in any bacteria, suggesting that physiological systems designed for handling other toxic materials may also deal with U(VI) toxicity. In this study we employed a genome-scale proteomic analysis and targeted gene deletions to gain insights into the impact of uranium exposure on the physiology of G. sulfurreducens. The results suggested that rather than a U(VI)-specific detoxification system, G. sulfurreducens utilizes a combination of mechanisms to cope with stress produced by U(VI).

METHODS Strains and culturing. G. sulfurreducens strain DL-1 (Coppi et al., 2001) as well as DsodA (Strycharz et al., 2011), DGSU2212 (Tran, 2009), DGSU2213 (Tran, 2009) and DBESTZ (Voordeckers et al., 2010) were obtained from our laboratory culture collection. Cells were routinely grown in anaerobic medium with acetate as the electron donor and fumarate as the electron acceptor (Caccavo et al., 1994).

For proteomic analysis of the impact of U(VI) exposure, 100 mM U(VI) in the form of uranyl acetate was added to mid-exponentialphase cultures. Cells were collected by centrifugation at 9000 g for 15 min at 4 uC, washed with 50 mM Tris/HCl containing 10 mM MgCl2 and protease inhibitors, and stored at 220 uC until use. Controls received no U(VI) additions. Almost all U(VI) added into the cultures remained as U(VI) during the incubation (Fig. S1, available in the online Supplementary Material). The concentration of U(VI) and total uranium in the aqueous fraction were measured using a Kinetic Phosphorescence Analyser (Chemcheck) as described previously (Orellana et al., 2013). Cellular protein extraction and digestion. Frozen cells were

washed once with NH4HCO3 buffer (pH 8, 100 mM) and resuspended in the same buffer. Cells were lysed by bead beating with 0.1 mm zirconia/silica beads in a Bullet Blender (Next Advance) set at speed 8 for 3 min. Samples were centrifuged at 4500 g for 5 min at 4 uC to remove cell debris and the resulting supernatant was harvested. An additional 200 ml NH4HCO3 buffer (pH 8, 100 mM) was added to the tube followed by brief vortexing to wash beads and centrifugation for 5 min, and then combined with the previous supernatant fraction. Proteins in this cell lysate and the cell-free fraction (see above) were denatured and reduced by incubating with urea (7 M), thiourea (2 M) and dithiothreitol (5 mM) at 60 uC for 30 min. After incubation, samples were diluted 10 times with the same NH4HCO3 buffer. CaCl2 was added to the diluted samples with the final concentration of 1 mM and then the total proteins were digested for 4 h at 37 uC with sequencing-grade trypsin (Promega) at a concentration of 1 U (50 U protein)–1. The digested samples were desalted using C-18 SPE columns (Supelco) according to the manufacturer’s instructions. The collected peptides in 1 ml 80 % acetonitrile plus 0.1 % trifluoroacetic acid (TFA) were concentrated by a SpeedVac concentrator (GMI) to a final volume of 502100 ml. Protein concentrations were measured with a BCA Protein Assay kit (Thermo Fisher). Tandem MS/MS and putative peptide identification. The

capillary liquid chromatography system consisted of a pair of syringe 2608

pumps (100 ml; model 100DM; ISCO) and a controller (series D; ISCO), and an in-house manufactured mixer, capillary column selector and sample loop for manual injections. Separations were achieved with a 5000 p.s.i. reversed-phase in-house packed capillary (150 mm6360 mm; Polymicro Technologies) by using two mobile phase solvents consisting of 0.2 % acetic acid and 0.05 % TFA in water (A) and 0.1 % TFA in 90 % acetonitrile/10 % water (B). The mobile phase selection valve was switched from position A to B 10 min after injection, creating an exponential gradient as mobile phase B displaced A in the mixer. Flow through the capillary HPLC column was ~1.8 ml min21 when equilibrated to 100 % mobile phase A. Sample eluate from the HPLC was infused into a conventional iontrap mass spectrometer (LCQ; ThermoFinnigan) operating in a datadependent MS/MS mode over a range of 400–2000 m/z. For each cycle, the three most abundant ions from MS analysis were selected for MS/MS analysis by using a collision energy setting of 30 %. Dynamic exclusion was used to discriminate against previously analysed ions. The collision-induced dissociation spectra from the conventional ion-trap mass spectrometer were analysed using SEQUEST (Eng et al., 1994) and the genome sequence of G. sulfurreducens (Mehta et al., 2006). Initial peptide identifications [i.e. putative mass and time (PMT) tags] were based on a minimum cross-correlation (Xcorr) score of 1.5 for all peptides identified at least twice in all MS/ MS experiments. For peptides only identified once, Xcorr values had to be a minimum of 1.9, 2.2 and 3.5 for charge states of 1+, 2+ and 3+, respectively. All peptides conformed to a tryptic cleavage state on at least one of their termini. The false-discovery rate of peptide identifications was 4 %, which was calculated utilizing a decoy database methodology (Qian et al., 2005) whereby peptides identified using SEQUEST to search against the protein sequence database were compared to those identified using SEQUEST to search against a decoy protein database containing reversed sequences. Using the same liquid chromatography conditions, each sample was further analysed in triplicate by FT-ICR (Fourier transform ion cyclotron resonance)-MS. The FT-ICR mass spectrometers developed at our laboratory use electrospray ionization interfaced with an electrodynamic ion funnel assembly coupled to a radiofrequency quadruple for collisional ion focusing, and highly efficient ion accumulation and transport to a cylindrical FT-ICR cell for analysis (Harkewicz et al., 2002). Determination of accurate mass and time (AMT) tags. The

peptide library (containing peptide sequence information, elution time information and theoretical mass) was then compared to the high-resolution, high-accuracy peptide mass and elution time obtained from the FT-ICR-MS runs. The peptides that were matched and verified in this manner were then deemed accurate mass and time (AMT) tags. AMT tag validation has been described previously (Lipton et al., 2002). Briefly, the resultant FT-ICR data were processed using the PRISM data analysis system – a series of software tools developed in-house. The first step involved de-isotoping the MS data, giving the monoisotopic mass, charge and intensity of the major peaks in each mass spectrum. Following this, the data were examined in a 2D fashion to find the groups of mass spectral peaks that were observed in sequential spectra. Each group, known as a unique mass class (UMC), has a median mass, central normalized elution time (NET) and abundance estimate, computed by summing the intensities of the MS peaks that comprise the UMC. The identity of the UMCs was determined by comparing the mass and NET of each UMC to the masses and NETs of all identified peptides ascertained from all prior MS/MS analyses performed on G. sulfurreducens. Search tolerances were ±6 p.p.m. for the mass and ±5 % of the total run time for the elution time. Relative abundance values for each peptide were determined from the summed ion current value of all MS scans that detected the peptide eluting. Protein Microbiology 160

Proteome of G. sulfurreducens in the presence of U(VI)

RESULTS

values were represented by the most abundant peptide values observed for each protein.

In order to evaluate how bacterial cells respond to the presence of U(VI), G. sulfurreducens was grown anaerobically to mid-exponential phase, exposed to 100 mM uranyl acetate for 4 h and then harvested for proteomic analysis. A total of 1363 proteins were detected in cells from these cultures. This represented ~40 % of the 3469 predicted protein-encoding ORFs in the genome of G. sulfurreducens (Methe´ et al., 2003). There were 203 proteins detected with higher abundance during exposure to U(VI) compared with the control cells not exposed to U(VI) and 148 proteins with lower abundance (Tables S1 and S2). This accounted for 26 % of the total proteins detected, indicating that protein expression was significantly affected by the presence of U(VI).

The abundance values for proteins detected in each analysis were transformed into a z score (also called the standard row function) to determine change significance. The z score was obtained as previously described (Ding et al., 2006). Briefly, z scores were calculated by using the mean value of each protein across both conditions, subtracted from each individual protein abundance value and divided by the SD of the values. z scores between samples were considered significantly different if the difference was ¢1.5. Impact of mutations on growth in the presence of U(VI). We

monitored the growth of the aforementioned strains under two conditions: ‘environmental relevant stress’ (in the presence of 100 mM U(VI) in the form of uranyl acetate) and ‘severe U(VI) stress’ (in the presence of 1 mM U(VI) in the form of uranyl acetate). Cells were grown in acetate/fumarate medium in anaerobic pressure tubes (Coppi et al., 2001). Each culture was inoculated with 5 % midexponential-phase cells. Uranium was added to a final concentration of 100 mM and 1 mM from a concentrated stock of uranyl acetate (20 mM) dissolved in bicarbonate buffer. An equivalent volume of bicarbonate buffer (41 mM) was added to uranium-free control cultures. During incubation, culture tubes were shaken horizontally to minimize the attachment of cells on the glass. Cell numbers were determined with epifluorescence microscopy utilizing cells stained with acridine orange (0.01 %) as described previously (Lovley & Phillips, 1988). In order to have a statistically relevant description of growth, more than eight fields were recorded for each time point in three independent replicate cultures. Images were taken digitally with SimplePCI version 5.3 (C-Imaging Systems) and cells were quantified using ImageJ (http://rsb.info.nih.gov/ij/).

Proteins with differential expression in the presence of U(VI) were classified under 17 categories according to their annotation function in the genome (Fig. 1, Tables S3–S19). The number in parentheses after each category is the number of proteins that showed differential expression patterns. Proteins associated with energy conservation (n526) had the highest number of proteins with greater abundance following uranium exposure, other than proteins with unknown function and hypothetical proteins (n558). The majority of proteins that were in lower abundance in the uranium-exposed cells were also annotated as hypothetical proteins (n529), proteins of unknown function (n524) and

Transcription

3

DNA metabolism

5

3

Fatty acid and phospholipid metabolism

1

Signal transduction

5

Nucleotide metabolism

4

Central intermediary metabolism Protein fate

21

8

10

Cellular processes

3

Cell envelope

3 6

Amino acid biosynthesis

6 6

14

Biosynthesis of cofactors

4 6

2

Protein synthesis

9 10 10

6

Transport and binding proteins

13

Regulatory functions

5

Unknown function

13 18

20

Energy metabolism Hypothetical proteins

2

21 26 25

24 29

33 Number of proteins

Fig. 1. Changes in the protein profile as a result of U(VI) exposure. Left (open bars), number of proteins with lower relative abundance. Right (shaded bars), number of proteins with increased relative abundance. The proteins are grouped according to functional class as defined by The Institute for Genomic Research annotation. http://mic.sgmjournals.org

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proteins involved in energy conservation (n520) (Fig. 1, Tables S1 and S2). Proteins involved in growth Exposure to U(VI) slightly reduced the growth rate of G. sulfurreducens (Fig. 2a) and many proteins associated with the central metabolism were in lower abundance in cells exposed to U(VI). For example, the expression of citrate synthase (GltA, GSU1106) that catalyses the condensation of acetyl-CoA and oxaloacetate to citric acid in the TCA cycle and, therefore, directly correlated with metabolic rates of G. sulfurreducens (Holmes et al., 2005; Wilkins et al., 2011; Yun et al., 2011), was lower in the presence of U(VI) compared with the untreated control (Table 1), suggesting that metabolism was slower in the presence of U(VI). Phosphoenolpyruvate synthase (PpsA, GSU0803), which activates pyruvate to phosphoenolpyruvate (Mahadevan et al., 2006), and two subunits of ATP synthase (GSU0108 and GSU0111) were also less abundant when U(VI) was present (Table 1).

(a)

Uranium has high affinity for organic molecules and can form strong bonds with functional groups in proteins (Matsuda & Nakajima, 2012). The uranium binding can produce conformational changes in proteins (Gray, 1994;

8.0×108 6.0×108 4.0×108

1.2×109

Growth (cells ml–1)

1.0×109

0

50 Time (h)

No U(VI)m = 0.069 h–1 m 100 mM U(VI) = 0.062 h–1 1 mM U(VI)m = 0.057 h–1

(d)

4.0×108

1.2×109 1.0×109

8.0×108 6.0×108 4.0×108

0

50 Time (h)

100

No U(VI)m = 0.068 h–1 100 mM U(VI)m = 0.066 h–1 1 mM U(VI)m = 0.058 h–1

8.0×108 6.0×108 4.0×108 2.0×108

2.0×108 0

6.0×108

0

100

Growth (cells ml–1)

(c)

8.0×108

2.0×108

2.0×108 0

No U(VI)m = 0.064 h–1 m 100 mM U(VI) = 0.058 h–1 1 mM U(VI)m = 0.042 h–1

1.2×109 1.0×109

Growth (cells ml–1)

Growth (cells ml–1)

1.0×109

Protein and DNA damage

(b)

No U(VI)m = 0.070 h–1 m 100 mM U(VI) = 0.067 h–1 1 mM U(VI)m = 0.060 h–1

1.2×109

It also appeared that protein biosynthesis was less important in the presence of U(VI), which was reflected in the lower abundance of proteins involved in translation, such as GSU1920 (elongation factor Ts), GSU0102 (selenocysteine-specific translation elongation factor) and GSU1516 (translation initiation factor IF-3), as well as several ribosomal proteins, such as RpsG, RplR, RpsT, YfiA, RplX, RpsS, RpsA, RplF, RpsK and RpsP, and proteins involved in ribosome biogenesis, ObgE and EngB (Table 1). Proteins involved in amino acid biosynthesis, such as GSU1061 (aspartate aminotransferase), GSU3099 (histidinol-phosphate aminotransferase), GSU3095 (imidazole glycerol phosphate synthase subunit HisF) and GSU1828 (chorismate mutase) were also less abundant (Table 1).

0

50 Time (h)

100

0

0

50 Time (h)

100

Fig. 2. Effect of U(VI) on the growth of (a) WT, (b) DsodA, (c) DGSU2212 and (d) DGSU2213: &, cultures grown in the absence of U(VI); h, cultures grown in the presence of 100 mM U(VI); #, cultures grown in the presence of 1 mM U(VI). Data represent the mean±SD from three independent replicate cultures. Bars designate one standard deviation of the mean. 2610

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Table 1. Selected proteins with lower relative abundance during exposure to U(VI) compared with the control cells Locus ID

Gene annotation

Proteins involved in growth Central metabolism and energy conservation GSU1106 Citrate synthase GSU0803 Phosphoenolpyruvate synthase GSU0108 ATP synthase F0, b subunit, putative GSU0111 ATP synthase F1, a subunit Translation and protein synthesis GSU1920 Translation elongation factor Ts GSU0102 Selenocysteine-specific translation elongation factor GSU1516 Translation initiation factor IF-3 GSU2861 Ribosomal protein S7 GSU2841 Ribosomal protein L18 GSU2206 Ribosomal protein S20 GSU1886 Ribosomal subunit interface-associated sigma-54 modulation protein GSU2846 Ribosomal protein L24 GSU2853 Ribosomal protein S19 GSU2603 Ribosomal protein S1 GSU2842 Ribosomal protein L6 GSU2833 Ribosomal protein S11 GSU0643 Ribosomal protein S16 Ribosome biogenesis GSU3213 Ribosome biogenesis GTPase ObgE GSU3013 GTPase EngB Amino acid biosynthesis GSU1061 Aspartate aminotransferase GSU3099 Histidinol-phosphate aminotransferase GSU3095 Imidazole glycerol phosphate synthase, cyclase subunit GSU1828 Chorismate mutase DNA repair GSU0145 RecA protein

Ghosh & Rosen, 2002; Yazzie et al., 2003). Uranium ions can generate ligands with functional groups of thiolates as well as carboxylate from acidic amino acids, such as aspartate or glutamate (Ghosh & Rosen, 2002; Van Horn & Huang, 2006; Zobel & Beer, 1961). Enzymes in G. sulfurreducens that assist in protein folding may help to avoid these potential deleterious effects. For example, exposure to U(VI) resulted in higher expression of the chaperonin GroES (GSU3339), the DnaJ-related molecular chaperone (GSU0014) and the DnaJ adenine nucleotide exchange factor (GrpE, GSU0032) that is involved in the protection and renaturation of heat-labile proteins (Table 2). This is in agreement with a former study that reported that transcripts of DnaJ and GrpE were found to be expressed in higher abundance in cells of the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1 exposed to 200 mM U(VI) (Wen, 2008). The expression of several proteins related to peptide secretion and trafficking was also more abundant in the presence of U(VI). For example, SecE and SecF (GSU2869 and GSU2616), which belong to the general Sec system, and PulQ (GSU1778) and GspK (GSU0322), which are http://mic.sgmjournals.org

Gene name

z score difference [zU(VI)–zcontrol]

gltA ppsA atpA

–1.65 –1.62 –1.59 –1.74

tsf selB infC rpsG rplR rpsT yfiA rplX rpsS rpsA rplF rpsK rpsP

–1.65 –1.70 –1.51 –1.51 –1.53 –1.54 –1.55 –1.57 –1.57 –1.67 –1.69 –1.72 –1.73

obgE engB

–1.74 –1.79

hisC hisF

21.53 21.63 21.64 21.66

recA

21.60

part of the type II secretion system, were in higher abundance in the presence of U(VI) (Table 2). Previous studies have suggested that the type II secretion system has an essential role in localizing several metal-containing proteins on the outer surface of the cell (Lovley et al., 2011; Mehta et al., 2006). Uranium has a high affinity for DNA, which can result in DNA strand breakage and inhibition of DNA–protein interactions (Hartsock et al., 2007; Matsuda & Nakajima, 2012; Yazzie et al., 2003; Zobel & Beer, 1961). Thus, exposure to U(VI) could be expected to result in DNA damage. However, only three proteins involved in DNA metabolism, DnaA (GSU3470), DNA topoisomerase I TopA (GSU2549) and single-strand binding protein Ssb-2 (GSU3117), were more abundant following U(VI) exposure (Table 2), and surprisingly RecA (GSU0145), an essential protein for the repair and maintenance of DNA, was in lower abundance in the presence of U(VI) (Table 1). This suggested that although some uranium could have reached the cytoplasm, uranium scavenging enzymes were highly efficient, preventing the subsequent DNA damage. 2611

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Table 2. Selected proteins with higher relative abundance during exposure to U(VI) compared with the control cells Locus ID

Gene annotation

Protein and DNA damage Protein folding GSU3339 Chaperonin GroES GSU0014 DnaJ-related molecular chaperone GSU0032 DnaJ adenine nucleotide exchange factor GrpE Peptide secretion and trafficking GSU2869 Preprotein translocase, SecE subunit GSU2616 Protein-export membrane protein SecF GSU1778 Type II secretion system secretin lipoprotein PulQ GSU0322 Type II secretion system protein GspK DNA protection GSU3470 Chromosomal replication initiator protein DnaA GSU2549 DNA topoisomerase I GSU3117 Single-strand binding protein Detoxification and membrane damage Detoxification GSU2695 Efflux pump, RND family, outer-membrane protein GSU1482 Efflux pump, RND family, outer-membrane protein GSU2136 Efflux pump, RND family, membrane fusion protein GSU2781 Efflux transporter, RND family, MFP subunit GSU0496 Efflux transporter, RND family, MFP subunit GSU2700 Tungstate ABC transporter, periplasmic tungstate-binding protein, putative GSU1678 Cation-transport ATPase, E1-E2 family Polyphosphate metabolism GSU0728 Polyphosphate kinase GSU2559 Exopolyphosphatase Lipoproteins GSU1817 Outer membrane lipoprotein, Slp family GSU0457 Outer membrane lipoprotein LolB, putative GSU0157 Lipoprotein, putative Peptidoglycan and cell wall biosynthesis D-Alanine–D-alanine ligase GSU3066 GSU2923 Glutamate racemase Oxidative stress response Peroxiredoxins and glutaredoxins GSU0352 Peroxiredoxin, atypical 2-Cys subfamily GSU3246 Peroxiredoxin, typical 2-Cys subfamily GSU1155 Glutaredoxin family protein Rhodonase-like proteins GSU0505 Rhodanese homology domain superfamily protein GSU2516 Rhodanese homology domain pair protein Reduction/oxidation of superoxide GSU0720 Superoxide reductase GSU1158 Superoxide dismutase Extracellular matrix proteins Chemotaxis GSU2212 Chemotaxis protein CheY GSU2213 GAF domain protein c-type cytochromes Periplasmic cytochromes GSU0357 Cytochrome c family protein GSU2801 Cytochrome c, 5 haem-binding sites GSU1648 Cytochrome c, 5 haem-binding sites Gene regulation and/or signal transduction function GSU3292 Transcriptional regulator, Fur family

2612

Gene name

z-score difference [zU(VI)–zcontrol]

groES grpE

1.54 1.82 1.63

secE secF pulQ gspK

1.73 1.50 1.65 1.54

dnaA topA ssb-2

1.78 1.68 1.67

tupA mgtA

1.77 1.81 1.71 1.64 1.56 1.77 1.54

ppk-2

1.55 1.64 1.79 1.76 1.65

ddl murI

1.74 1.66

prx-3 prx-2

1.66 1.80 1.77 1.66 1.77

sodA

cheY-5

macC

1.78 1.72

1.71 1.82

1.74 1.72 1.73 1.79

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Table 2. cont. Locus ID GSU2698 GSU2666 GSU2625

Gene annotation

Gene name

z-score difference [zU(VI)–zcontrol]

Transcriptional regulator, TetR family Transcriptional regulator, TetR family Transcriptional regulator, ArsR family

1.51 1.77 1.55

RND, resistance–nodulation–cell division.

Detoxification and membrane damage Although there are no known uranium-specific detoxification systems in micro-organisms, metal efflux pumps for other toxic metals exist (Nies, 2013) and could conceivably play a role in preventing uranium toxicity. Several efflux pumps in the RND (resistance–nodulation–cell division) family, which confer metal tolerance by extruding a wide spectrum of metals, were more abundant in cells exposed to U(VI) (Table 2). For example, RND family proteins associated with the CzcABC complex were triggered by the presence of U(VI). GSU2695 (RND-type efflux pump) and GSU1482 (CzcC, RND-type efflux pump), which correspond to outer membrane proteins able to transport heavy metals across the outer membrane, were more abundant in cells exposed to U(VI). The three membrane fusion proteins, GSU2136 (RND-type efflux pump), GSU2781 (RND-type efflux pump) and GSU0496 (RND-type efflux pump), which span the periplasmic space and funnel cations across it (Kim et al., 2011), were also in higher abundance in the presence of U(VI) (Table 2). These enzymes belonging to the RND family have been shown to convey resistance against many other metals, such as Co2+, Zn2+, Cd2+ and Ni2+, in the metal-resistant microbe Cupriavidus metallidurans (Nies, 2003). Many other proteins related to the binding and transport of metals were also significantly more abundant in the presence of U(VI), such as the putative periplasmic tungstate ABC transporter (TupA, GSU2700), which is part of the tungstate transport complex, and MgtA (GSU1678) commonly involved in Mg2+ transport (Table 2). Another strategy for heavy metal detoxification is precipitation (Gadd, 2010; Lloyd & Lovley, 2001; Wall & Krumholz, 2006). Several micro-organisms are known to use phosphate derived from polyphosphate to precipitate uranium (Merroun & Selenska-Pobell, 2008). The polyphosphate kinase (Ppk-2, GSU0728), which catalyses the transfer of phosphate from ATP to form a long-chain polyphosphate, and the exopolyphosphatase (GSU2559), which irreversibly hydrolyses polyP to form phosphates, were both more abundant in cells exposed to uranium, suggesting a potential role in uranium detoxification (Table 2). Moreover, polyphosphates can indirectly affect the efflux of other heavy metals, regenerating ATP, which can then be used to activate efflux ATPases (Seufferheld et al., 2008). For instance, cadmium tolerance of Escherichia coli mainly relies on polyphosphate metabolism (Keasling & Hupf, 1996). Also, http://mic.sgmjournals.org

the level of intracellular polyphosphate directly correlates with resistance to cadmium in Anacystis nidulans (Keyhani et al., 1996) and nickel is compartmentalized by polyphosphate in Staphylococcus aureus (Gonzalez & Jensen, 1998). The lipid bilayer of the outer membrane is the most external barrier before the peptidoglycan in G. sulfurreducens. This layer is rich in phosphate and carboxylate groups, which may strongly bind U(VI) (Merroun & Selenska-Pobell, 2008). Many lipoproteins, such as GSU1817 (outer membrane lipoprotein, Slp family), GSU0457 (outer membrane lipoprotein LolB) and GSU0157 (lipoprotein), were more abundant in the presence of U(VI). A similar response was observed with proteins involved in peptidoglycan and cell wall biosynthesis, such as D-alanine–D-alanine ligase Ddl (GSU3066) and glutamate racemase MurI (GSU2923), respectively (Table 2), that has been previously observed in Shewanella strain MR-1 exposed to chromium (Chourey et al., 2006). Proteins involved in oxidative stress G. sulfurreducens is an aerotolerant anaerobe, with effective mechanisms for dealing with oxidative stress (Lin et al., 2004; Van Horn & Huang, 2006). Many proteins that are involved in the typical oxidative stress response in bacteria are also induced in response to other environmental stimuli, such as heat (Geslin et al., 2001; Privalle & Fridovich, 1987), high salt concentrations (Bernhardt et al., 1997) and heavy metals stress (Bernhardt et al., 1997; Chourey et al., 2006; Geslin et al., 2001; Roux & Cove´s, 2002). A number of proteins associated with the oxidative stress response were more abundant in cells exposed to uranium (Table 2). A possible explanation for this is that glutathione can reduce U(VI) that enters the cells, producing oxidized bisglutathione and hydrogen peroxide (Hu et al., 2005; Pourahmad et al., 2006; Smirnova & Oktyabrsky, 2005). This reduction could potentially be catalysed by two proteins encoding typical 2Cys subfamily peroxiredoxins (GSU0352 and GSU3246) and glutaredoxin (GSU1155), which were more highly expressed in cells grown in the presence of U(VI) (Table 2). Homologues of rhodanese-like proteins (GSU0505 and GSU2516) that are involved in the oxidative stress response of E. coli (Farr & Kogoma, 1991) were also expressed in higher abundance when U(VI) was present (Table 2). Exposing G. sulfurreducens to U(VI) resulted in a higher abundance of both superoxide dismutase (SodA, GSU1158) 2613

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and superoxide reductase (GSU0720) (Table 2). A former transcriptional study of the Geobacter species that predominated during in situ uranium bioremediation at a field study site in Rifle (CO, USA) reported that the gene encoding the superoxide dismutase was highly expressed despite the presence of a highly reduced environment (Mouser et al., 2009). Another study that evaluated the transcriptional expression of the Geobacter uraniireducens, an isolate from the Rifle site (Shelobolina et al., 2008), also found that the sodA gene was upregulated when the isolate was grown in the contaminated subsurface sediments (Holmes et al., 2009). Both results suggested that the expression of the superoxide dismutase could not only be triggered as a result of oxygen stress, but also by other factors in the sediments. Furthermore, the gene encoding the superoxide dismutase was upregulated when cells of the highly uraniumtolerant oligotroph, Caulobacter crescentus, were exposed to uranium, cadmium, chromate and dichromate (Hu et al., 2005), suggesting that this enzyme is involved in the response to a wide range of heavy metals. In order to evaluate the role of superoxide dismutase in response to U(VI) stress, the growth of a SodA-deficient strain in the presence U(VI) was evaluated (Fig. 2b). In the absence of U(VI) the growth of the SodA-deficient strain was comparable to that of the WT (Fig. 2a). However, in the presence of 100 mM U(VI), the SodA-deficient strain grew more slowly than WT (Fig. 2b). The impact of the loss of superoxide dismutase was even more apparent in the presence of 1 mM U(VI) (Fig. 2b). Extracellular matrix proteins Two regulatory proteins (GSU2212 and GSU2213) related to the che5 gene cluster, which has been shown to participate in the synthesis of extracellular matrix and biofilm formation (Tran, 2009), were more abundant in cells exposed to uranium (Table 2). In order to evaluate the potential role of these proteins in response to uranium toxicity, cultures in which one of the genes for these proteins was deleted were grown in the presence of uranium (Fig. 2c, d). However, deletion of these genes did not significantly inhibit growth in the presence of uranium, suggesting that these regulatory proteins were not essential for the response to uranium toxicity. c-type cytochromes Three c-type cytochromes (GSU0357, GSU1648 and GSU2801) were expressed at higher abundance when cells were exposed to U(VI) (Table 2). GSU0357 is predicted to be a nitrite reductase. The function of GSU2801 is unknown. GSU2801 is not essential for Fe(III) oxide reduction in G. sulfurreducens (Smith et al., 2013), but its homologue in Geobacter metallireducens had higher transcript abundance in cells grown on Fe(III) oxide than in Fe(III) citrate-grown cells (Smith et al., 2013). GSU 1648 (MacC) is predicted to be periplasmic. The gene encoding 2614

a MacC homologue was more highly expressed in G. uraniireducens grown in a U(VI)-contaminated subsurface than in culture medium (Holmes et al., 2009). A number of G. sulfurreducens outer surface cytochromes appear to contribute to U(VI) reduction (Orellana et al., 2013; Shelobolina et al., 2007). The reduction of U(VI) at the outer surface might be expected to be one mechanism for reducing uranium toxicity because poorly soluble U(IV) is unlikely to enter the cell. To test this concept, studies were conducted with the previously described quintuple mutant (Voordeckers et al., 2010) in which the genes for the outer surface c-type cytochromes OmcB, OmcE, OmcS, OmcT and OmcZ were deleted. Although cell suspensions of this quintuple mutant reduced U(VI) at a rate of only 18 % that of WT cells (Orellana et al., 2013), this strain grew as well as the WT strain in the presence of U(VI) (Fig. S2). Therefore, this result suggested that the enzymic reduction of U(VI) at the cell surface may not be an important mechanism for reducing enzyme toxicity. Regulatory response Many proteins involved in the regulation of gene expression in response to changes in the environment were differentially expressed when cells were exposed to U(VI) (Table 1). For instance, the transcriptional regulator that belongs to the Fur family (GSU3292) was expressed in higher abundance when U(VI) was present (Table 2). The Fur regulon is important in the regulation of the iron uptake pathway (Andrews et al., 2003), suggesting increased iron uptake under U(VI) exposure. Two of the nine G. sulfurreducens TetR family transcriptional regulatory proteins that coordinate expression of efflux pumps (GSU2666 and GSU2698) were also expressed in higher abundance (Table 2). The TetR family is involved in the regulation of efflux pumps and tolerance to toxic compounds, amongst other functions in Geobacter species (Krushkal et al., 2011). Also, ArsR family transcriptional regulator (GSU2625) was expressed in higher abundance (Table 2). The ArsR family of transcriptional regulators responds to metal ion stress (Summers, 2009), and modulates the transcription of genes involved in metal efflux, sequestration and detoxification (Tottey et al., 2005). RpoH is a sigma factor that regulates gene expression under heat shock (Ueki & Lovley, 2007) and a homologue of this gene was found to be upregulated when Caulobacter crescentus was exposed to U(VI) (Hu et al., 2005). Although the protein encoded by the rpoH homologue present in G. sulfurreducens (GSU0655) was not detected by our proteomic analysis, 33 proteins encoded by genes that contained RpoH-binding sites were expressed in higher abundance under U(VI) exposure (Table S20).

IMPLICATIONS The ability of G. sulfurreducens (this study) and other Geobacter species (Lovley et al., 1991) to grow in the Microbiology 160

Proteome of G. sulfurreducens in the presence of U(VI)

presence of millimolar quantities of uranium is remarkable because it is unlikely that there has ever been any major evolutionary pressure on these organisms to deal with such high concentrations of uranium in natural environments. The differential expression of proteins in the presence of U(VI) did not reveal a specific U(VI) detoxification system. Rather, resistance to U(VI) appears to be accomplished by multiple stress response systems and regulatory networks that facilitate fast adaptation to rapidly changing conditions, including the increased expression of enzymes involved in DNA and protein protection, multiple efflux pumps capable of transporting heavy metals across the outer membrane, and enzymes involved in the oxidative stress response. The ability of Geobacter species to cope with potential U(VI) toxicity in this manner may be one of the reasons that these species are often one of the most abundant genera of micro-organisms during in situ uranium bioremediation.

ACKNOWLEDGEMENTS This research was supported by the Office of Science (Office of Biological and Environmental Research), US Department of Energy (award number DE-SC0006790). R. O. was supported by a Fulbright– CONICYT Equal Opportunities Scholarship. Portions of this research were supported by the US Department of Energy Office of Biological and Environmental Research Genome Sciences Program under the Pan-omics project. Work was performed in the Environmental Molecular Science Laboratory, a US Department of Energy national scientific user facility at Pacific Northwest National Laboratory in Richland, WA.

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