Abstract

Background

Pseudomonas putida is a ubiquitous gram-negative, saprophytic bacterium belonging to the gamma class of the Proteobacteria. Endowed with a remarkable environmental adaptability, P. putida strain F1 [1], for example, has been investigated most extensively as a model organism for the microbial degradation of such xenobiotic aromatic compounds as toluene, benzene, and ethylbenzene [2]. Considerably less scientific focus has been devoted to elucidating the molecular basis of heavy metal resistance functions exhibited by P. putida strains. Genomic analysis of P. putida strain KT2440 revealed an unexpectedly large repertoire of genes predicted to be involved in metal homeostasis, tolerance, and resistance, inferring that the organism is habituated to heavy metal exposure in its environment [3]. P. putida strains, for instance, have been isolated from the effluent of previously operated mercury-reducing bioreactors [4], and Pseudomonas spp. were reported to be dominant members of heavy metal-contaminated U.S. Department of Energy (DOE) sites, including a subsurface paleosol microbial community [5] and uranium-contaminated groundwater environment [6].

Hexavalent chromium [Cr(VI)], in the form of chromate (CrO₄^2-) or dichromate (Cr₂O₇^2-), is a widely distributed environmental contaminant due to its prevalent use in industrial and military defense applications [7,8]. Microbially mediated reduction of soluble, toxic Cr(VI) to sparingly soluble, less toxic Cr(III) is a potentially promising strategy for the in situ remediation of Cr(VI)-contaminated subsurface environments. Priester et al. [9] demonstrated that unsaturated biofilms of P. putida strain mt-2 on membranes overlaying iron-deficient solid media either containing dichromate or solid dichromate-coated hematite were able to completely reduce Cr(VI) under both conditions. Additionally, efficient and rapid reduction of Cr(VI) to Cr(III) has been measured for a soluble flavoprotein (ChrR) purified and kinetically characterized from P. putida MK1 [10,11] and for soluble crude fractions prepared from P. putida PRS2000 [12], suggesting that P. putida, if present in the indigenous microbial community, is likely an important contributor to the bioreduction of chromate in polluted soil environments. Surprisingly, there is a paucity of information on the physiological and global molecular response of P. putida to exogenous metals such as chromate, despite the fact that this soil bacterium has been used as a model organism for vadose zone bioremediation in various studies [9,13,14]. The availability of the genome sequences for P. putida strains F1 [DOE Joint Genome Institute (JGI); http://genome.jgi-psf.org/finished_microbes/psepu/psepu.home.html] and KT2440 [15] provides an opportunity to access its metabolic versatility and explore the molecular basis for its enhanced ability to adapt to the various environmental conditions present in pristine and metal-contaminated soils.

Here we define the growth sensitivity of P. putida F1 to chromate when cultivated in complex (Luria Bertani) medium compared to defined minimal medium containing three different single carbon and energy sources (glucose, lactate, or acetate). Using a liquid-chromatography mass spectrometry-based "bottom-up" proteomic approach, we analyzed the global proteome profiles of F1 in an attempt to provide insight into the molecular systems underlying the cellular stress response to an acute exposure of chromate. In general, the chromate-perturbed F1 proteome was markedly different depending on whether challenged cells were cultivated in LB broth or minimal medium with lactate as the single carbon substrate. A core protein response to chromate, irrespective of the nutritional environment tested, is described and may provide potential in situ biomarkers for environmental biomonitoring of heavy metal stress in natural microbial communities.

Results

Impact of nutritional environment on P. putida growth and Cr(VI) reduction

To assess the physiological impact of nutrient conditions on P. putida F1 sensitivity to Cr(VI), we determined the minimal inhibitory concentrations (MICs) of chromate for F1 in LB and M9 minimal media supplemented with 50 mM glucose (M9G), 50 mM sodium lactate (M9L), or 67 mM sodium acetate (M9A) as the sole carbon source in the presence of K₂CrO₄ at concentrations ranging from 0.04 to 14 mM. F1 cells exhibited markedly different tolerance responses to chromate depending on the nutritional environment. The MIC of Cr(VI) for LB-propagated F1 cells was >4 mM (Figure ​(Figure1A),1A), an approximately 40-fold greater concentration than that observed for F1 cells grown in M9 media irrespective of carbon source (MIC > 0.1 mM) (Figure ​(Figure1B).1B). Among the different minimal media, the nature of the carbon source influenced the growth ability of F1 in the presence of chromate. Higher ODs were measured for M9G in the absence of and at the lowest Cr(VI) concentration (0.04 mM) tested than for M9L and M9A. As Cr(VI) concentrations were increased, however, M9L supported growth to higher cell densities than either M9G or M9A, and at the highest levels of chromate tested, 0.08 mM and 0.1 mM, cell densities in M9L surpassed those in M9G by 3- and 9-fold, respectively (Figure ​(Figure1B).1B). Thus, there was a carbon-source-dependent hierarchy in chromate-amended M9 media (lactate > glucose >> acetate) that cannot be explained simply on the basis of better growth medium energetics since glucose supported higher cell densities than lactate in the absence of Cr(VI), yet as Cr(VI) levels increased lactate supported higher cell densities than glucose. Therefore, M9L was chosen as the minimal medium for subsequent growth studies.

Open in a separate window

Figure 1

Effect of carbon source on MIC of chromate for P. putida F1. Optical densities (OD₆₀₀) of F1 cultures after 48 h of growth in the presence of varying concentrations of chromate (K₂CrO₄) are shown. (A) F1 growth in LB broth. (B) F1 growth in M9 minimal media supplemented with 50 mM glucose (grey bars), 50 mM sodium lactate (white bars), or 67 mM sodium acetate (black bars) as the sole source of carbon. All cultures were grown in triplicate. Error bars denote the standard deviation of replicate measurements.

To determine whether F1 is capable of chromate reduction, cells were grown in LB broth containing K₂CrO₄ and chromate reduction was measured for up to 31.5 h. F1 reduced 0.3 mM Cr(VI) to 0.05 mM or less within 25 h (Figure ​(Figure2).2). The ability of F1 to carry out this reduction was largely independent of the initial cell density of the culture as evidenced by the fact that both a culture grown to mid-log phase (OD = 0.5) prior to chromate exposure and a newly inoculated culture (OD = 0.07) both reduced chromate to similar final concentrations (Figure ​(Figure2).2). In addition, we tested reduction ability at 1 mM chromate by mid-log cells and found that there was no reduction within the first 75 min (initial chromate concentration and concentration at 75 min measured at 0.93 ± 0.01 and 0.92 ± 0.04 mM, respectively); furthermore, at this higher initial concentration there was less reduction over a 24 h period (final concentration = 0.77 mM ± 0.03 mM).

Open in a separate window

Figure 2

Chromate reduction by P. putida F1. The progression of reduction of 0.3 mM K₂CrO₄ by P. putida F1 in LB broth is shown over a 31.5 h period. At T = 0, F1 cells were either freshly inoculated into the chromate-containing growth medium from an overnight culture to an OD of 0.07 (open triangles) or chromate was added to a culture of F1 already in mid-log phase (open squares). The abiotic control is also shown (red open circles). Each data point represents the mean of three replicate measurements. Error bars indicate one standard deviation.

A chromate level of 1 mM was selected for challenging mid-log-phase LB- and M9L-grown cells, because it represented the lowest concentration tested for which cell toxicity was observed under LB conditions. Because we were interested in delineating the early molecular response to Cr(VI) exposure, the 75-min time interval following chromate exposure was selected for harvest and analysis of F1 proteomes. Figure ​Figure33 shows the growth differences between the Cr(VI)-treated and untreated cultures for the two different nutrient conditions at the collection time interval (75 min post-chromate addition) for shotgun proteomics analysis. The effect of Cr(VI) addition was greater on M9L cells, which showed a ~34% reduction in cell density at the harvest point, while the growth difference between Cr(VI)-treated and untreated control cells in LB liquid media was ~10%. No measurable Cr(VI) reduction by F1 was detected under either growth condition within the 75-min period of exposure, although this strain of P. putida was shown to reduce 0.3 mM chromate to ~0 mM over 24 h (Figure ​(Figure22).

Open in a separate window

Figure 3

Impact of chromate challenge on P. putida F1 growth in LB versus M9L media. The optical densities (OD₆₀₀) of F1 cultures grown aerobically under LB (closed symbols) versus defined minimal M9L media (open symbols) in either the absence or presence of 1 mM K₂CrO₄ are shown: LB-grown cells with no chromate (closed squares), LB-grown cells with chromate added to 1 mM at the mid-log point (closed circles), M9L-grown cells with no chromate (open squares), and M9L-grown cells with chromate added to 1 mM at the mid-log point (open circles). The point of Cr(VI) addition and cell harvesting for proteomic analyses is presented. Each data point represents the mean of three replicate samples. Error bars indicate one standard deviation.

Global analysis of P. putida F1 proteomes under different growth conditions

Protein abundance measurements were compared for Cr(VI)-challenged and untreated P. putida F1 cells grown aerobically in either complex (LB) medium or defined minimal (M9L) medium containing lactate as the sole carbon/energy source in order to assess growth condition-dependent differences in the molecular response to acute chromate exposure as well as proteomic signatures indicative of heavy metal stress irrespective of the nutritional milieu. In general, a total of 2140 (41%) and 2383 (45%) functionally diverse, non-redundant proteins out of 5252 predicted proteins were detected in P. putida F1 cultivated in LB or M9L media, respectively, with and without chromate. By pooling all of the non-redundant proteins across both minimal and LB media samples, a total of 2,681 proteins were detected, which is ~50% of the predicted proteome just from these four growth conditions. The entire list of identified proteins with their spectral counts and associated statistical analyses can be found in Additional file 1. A total of 946 proteins were found in all growth states and technical replicates (Additional file 2). Further analysis indicated that 257 proteins were exclusively expressed in LB-grown cell samples, while 485 proteins were unique to M9L-grown cells (Additional file 3).

Statistically significant proteins, i.e., those exhibiting at least a two-fold change in expression and an FDR < 0.01, in LB- and M9L-cultivated control cells had a broad functional distribution (Figure ​(Figure44 and Additional file 4). Not unexpectedly, control cells propagated in M9L medium, which is much less nutritionally robust than LB, showed a much higher proportion of protein expression in the COG categories of cell wall membrane and envelope biogenesis (m), amino acid transport and metabolism (e), and energy production and conversion (c) (Figure ​(Figure4).4). Enzymes and transport proteins, specifically D-lactate dehydrogenase (cytochrome; Pput 4603) and L-lactate transporter (permease; Pput 4601), related to lactate utilization were only identified in cell samples grown with this carbon source (Additional file 1), which was a mixture of the D and L stereoisomers. Lactate is converted to pyruvate by lactate dehydrogenase and then is further metabolized via the tricarboxylic acid (TCA) cycle. In addition, pyruvate dehydrogenase (Pput 0594), involved in catalyzing the oxidative decarboxylation of pyruvate to acetyl-CoA and CO₂, and two other enzymes of the TCA cycle, NADP-dependent isocitrate dehydrogenase (Pput 1822) and malate dehydrogenase (Pput 4958), had substantially decreased abundance levels under chromate conditions in M9L medium. The reduced synthesis of these key primary metabolic enzymes is consistent with the more severe Cr(VI)-induced growth defects observed for F1 cells in M9L media (Figure ​(Figure33).

Open in a separate window

Figure 4

Functional category distribution of proteins identified from F1 cells grown in LB or M9L media in the absence of chromate. Letters along the x-axis refer to the following functional role categories: (v) defense mechanisms; (u) intracellular trafficking, secretion and vesicular transport; (t) signal transduction mechanisms; (s) unknown function; (r) general function prediction; (q) secondary metabolite biosynthesis, transport and catabolism; (p) inorganic ion transport and metabolism; (o) post-translational modification, protein turnover, and chaperones; (n) cell motility; (m) cell wall membrane and envelope biogenesis; (l) DNA replication, recombination and repair; (k) transcription; (j) translation of ribosomal structure and biogenesis; (i) lipid transport and metabolism; (h) coenzyme transport and metabolism; (g) carbohydrate transport and metabolism; (f) nucleotide transport and metabolism; (e) amino acid transport and metabolism; (d) cell cycle control, cell division and chromosome partitioning; (c) energy production and conversion; and (none) no specific function. The number of proteins identified for each COG category under the different growth conditions is presented within the split vertical bars.

Hypothetical proteins (s) and poorly characterized proteins [i.e., those with general functions (r) or no specific function (none)] constituted the largest proportion of the two control proteome datasets, with 31% for the LB samples and 32% for the M9L samples (Figure ​(Figure4).4). For those genes showing no similarity to sequences with known functions, this study provides evidence of their actual expression and suggests clues to their possible biological roles in F1. Expression of proteins involved in cell motility (n) characterized the global protein profile of M9L-grown control cells but were not represented in the LB sub-proteome. Similarly, proteins belonging to the following COG categories were not identified in LB control samples: defense mechanisms (v); intracellular trafficking, secretion and vesicular transport (u); and secondary metabolite biosynthesis, transport and catabolism (q) (Figure ​(Figure44).

Discussion

This global proteomic study was undertaken to interrogate growth media-based differences in the P. putida F1 molecular response to the stress of the oxyanion chromate, a widespread anthropogenic contaminant and a current focus of bioremediation efforts by the DOE. The ultimate aim was to identify proteome changes reflective of, although not necessarily specific to, early chromate stress in microbial cells irrespective of the nutritional environment. Our physiological investigations indicated that the impact of chromate toxicity was largely dependent on the growth medium. Rich (LB) medium, for example, was able to support F1 growth at input chromate concentrations at least 40-fold greater than any of the defined minimal M9-based media. Presently, it is not known whether this observation was due to differences in growth rate as a function of the medium [the initial growth rate of F1 was 3 to 5 times faster in LB than in M9 media (data not shown)], a higher bioavailability of Cr(VI) in minimal media, or a lack of sequestration of toxic reduced chromium forms by organic molecules in the media. Comparative analysis of the proteomic profiles generated in this study revealed that acute chromate exposure affects, either directly or indirectly, a wide range of cellular processes and functions, with some of the most profound changes in expression occurring among proteins with annotated functions in inorganic ion transport, amino acid transport and metabolism, cell wall membrane and envelope biogenesis, and energy production.

Certain hallmark features of the bacterial molecular response to chromate challenge are emerging with the increasing availability of transcriptomic and proteomic descriptions of Cr(VI)-challenged microorganisms. Comparative analysis of the LB- and M9L-derived proteomes described here revealed that an adaptive strategy employed by P. putida F1 in response to acute chromate exposure was up-regulation of proteins involved in sulfate transport (sulfate ABC transporter Pput 1565), cysteine biosynthesis (Pput 4421 and 4422 encoding sulfate adenylyltransferase large and small subunits, respectively), and the uptake and utilization of alternative sulfur sources such as aliphatic sulfonates (taurine dioxygenase Pput 0190 and ABC-type nitrate/sulfonate/bicarbonate transport systems protein Pput 0191) (Additional file 1 and Table ​Table2).2). Because of its structural similarity to SO₄^2-, exogenous chromate (CrO₄^2-) competes with sulfate for transport across bacterial cell membranes via sulfate anion transport systems [21-24], leading to low internal levels of sulfur. Up-regulation of genes/proteins with annotated functions in sulfur transport and metabolism has been observed in other chromate-stressed Gammaproteobacteria [25-27] as well as in the highly chromate-resistant Arthrobacter sp. strain FB24, a high G + C actinobacterium [28]. Using two-dimensional gel electrophoretic analysis, Ackerley et al. [25] showed that chromate-challenged nonadapted E. coli K-12 cells contained increased abundance levels of CysK and CysN, while challenged pre-adapted cells expressed alkane sulfonate monooxygenase, which functions by converting alkane sulfonates to sulfite and aldehyde. Similarly, multidimensional HPLC-MS/MS analysis of Shewanella oneidensis MR-1 exposed to different sub-lethal concentrations of chromate demonstrated increased abundance of cysteine synthase A (CysK), sulfate adenylyltransferase (CysD and CysN), adenylylsulfate kinase (CysC), sulfite reductase (CysI), and periplasmic sulfate-binding protein (Sbp) [26,27]. A recent study investigating chromate resistance in Pseudomonas corrugata 28, a Cr(VI)-hyper-resistant (MIC, 40 mM K₂CrO₄) bacterium, demonstrated that Cr(VI) susceptibility was attributed to insertional inactivation of oscA, which encodes a hypothetical small protein of unknown function and is located in a gene cluster with components of the sulfate ABC transporter system [29]. We previously reported that S. oneidensis SO4651, a homolog of OscA [29], was up-regulated at both the transcript and protein levels during the entry response to Cr(VI) stress [26]. Viti et al. [29] showed that the P. corrugata oscA-sbp transcriptional unit was strongly overexpressed after chromate exposure, thus lending further support for the link between sulfate starvation and Cr(VI) stress response. Sufficient cysteine availability is critical not only for protein biosynthesis but also in the defense against metal-induced oxidative stress by maintaining cellular redox homeostasis through the production of protective thiol-containing compounds such as glutathione. Therefore, the up-regulation of genes/proteins involved in sulfur transport and metabolism constitutes a major and conserved cellular response among Gammaproteobacteria to the oxidative burden imposed by chromate challenge.

Reduced P. putida F1 growth rates and higher sensitivity to chromate in M9L medium may be attributable to stress resulting from low intracellular sulfate levels as well as high intracellular chromate levels. The sole source of sulfate in M9L medium is MgSO₄, which is likely transported into the cell by the sulfate active transport system. This transport system has been shown in other Pseudomonas species to be principally responsible for the uptake of chromate [24]. As already mentioned, chromate is a competitive inhibitor of sulfate uptake [24]. Reduced sulfate uptake is compensated for by increased uptake of cysteine and cysteine-containing compounds (as well as other sulfur-containing compounds) through alternative transporter systems not competitively inhibited by chromate. Cysteine is freely available in complex LB medium but not in M9L medium. The reduced availability of cysteine in M9L medium could have resulted in lower levels of intracellular cysteine needed for important cellular biosynthetic processes, leading to the more striking reduced growth rates and chromate sensitivity of cells cultivated in M9L medium compared to LB medium.

Chromate toxicity has been attributed primarily to oxidative stress generated by the intracellular reduction of Cr(VI) to the transient highly reactive radical Cr(V), which redox cycles and thereby creates reactive oxygen species (ROS) [22,25,30]. ROS production leads to the damage of such cellular components as DNA and proteins. Presumably to counter Cr(VI)-induced oxidative stress, synthesis of the antioxidant defense protein Mn/Fe-binding superoxide dismutase (Sod; Pput 0985), which catalyzes decomposition of superoxide anion (O₂·^-) to hydrogen peroxide and molecular oxygen, was detected only in Cr(VI)-exposed LB- and M9L-grown P. putida F1 cells. Similarly, the up-regulation of Sod at the transcript or protein level was observed for Cr(VI)-stressed E. coli [25] and the Alphaproteobacterium Caulobacter crescentus [31] but not for S. oneidensis MR-1 cells subjected to an acute chromate treatment [26]. Instead, katG-1 (catalase/peroxidase hydroperoxidase) and katB (catalase) were preferentially induced at Cr(VI) exposure time intervals of 60 and 90 min. Based on our proteomic analyses, the enhancement of free-radical detoxifying activities constitutes a vital cellular defense mechanism against chromate toxicity in P. putida F1 regardless of the growth medium, whereas cellular defenses against H₂O₂ (i.e., increased expression of catalase) were not required during the early stages of chromate exposure in contrast to that observed for S. oneidensis MR-1.

Located immediately upstream of the F1 sod gene is fumC (Pput 0984), which also was identified only in Cr(VI)-stressed cells under both growth media conditions (Table ​(Table3).3). The gene synteny is similar (but not identical) to that found in the genome of Pseudomonas aeruginosa, in which an O₂·^- resistant isoform of fumarase (or fumarate hydratase/lyase) is linked to downstream genes orfX (of unknown function) and sodA (encodes Mn-cofactored Sod) in an iron-responsive operon [32]. Fumarase catalyzes the reversible conversion of fumarate to malate in the TCA cycle and has been shown to be up-regulated under iron-limiting conditions in P. aeruginosa [33]. It remains to be determined whether expression of P. putida F1 FumC exclusively in Cr(VI)-challenged cells is part of an iron starvation response.

As previously demonstrated for S. oneidensis MR-1 [26,27] and C. crescentus [31], another prominent characteristic of the cellular response to acute chromate exposure is the up-regulation of genes/proteins with functions in iron acquisition and homeostasis, namely TonB-dependent receptors and siderophore biosynthesis proteins. Similarly, TonB-dependent receptors for high-affinity iron chelators were a conspicuous feature of up-regulated, Cr(VI)-perturbed P. putida sub-proteomes, particularly under LB growth conditions. The increased abundance of four such outer membrane receptors under LB-Cr(VI) conditions is likely connected with the concomitant up-regulation of five amino acid adenylation domains. The adenylation (A) domains represent the active core of each modular unit comprising large multifunctional enzymes termed nonribosomal peptide synthetases [34]. The A domain functions by recognizing a specific amino or hydroxyl acid substrate and activating it as aminoacyl adenylate via ATP hydrolysis. While nonribosomal peptide synthetases can produce peptides of broad and sometimes obscure biological activity, these multidomain enzymes are known to be involved in the assembly of aryl-capped peptide and peptide-polyketide siderophores from Pseudomonas spp. [35,36]. Furthermore, expression of a TonB-dependent hemoglobin/transferrin/lactoferrin family receptor (Pput 1043) as part of the media-independent core molecular response to chromate exposure points to iron availability and homeostasis as playing an important role in the cellular adaptation of F1 to chromate stress.

Earlier reports revealed a distinct link between cell sensitivity to chromate and iron availability. It was observed, for example, that tonB mutants of E. coli exhibited increased sensitivity to chromium salts [37-39], which could be relieved by adding iron to the growth medium [38]. More recently, siderophores produced by certain Pseudomonas species were shown to bind exogenous transition metals other than Fe(III) with appreciable affinity [40,41], thus invoking conditions of low internal iron. The siderophore pyridine-2,6-bis(thiocarboxylic acid) (pdtc) from Pseudomonas stutzeri KC, for example, has been shown to detoxify extracellular chromium(VI), selenium, and tellurium oxyanions, suggesting that pdtc functions not only in iron acquisition but also in an initial line of defense against metal toxicity [42,43]. Research is required to delineate the physiological basis for the increased expression of iron acquisition receptors in response to acute chromate stress.

The Cr(VI)-responsive proteomic subset shared between the two different growth conditions consisted mostly of structural proteins with the exception of one regulatory protein, annotated as a two-component response regulator (Pput 0287) belonging to the winged helix-turn-helix family of DNA-binding regulators. Prototypical two-component systems, which constitute the predominant mechanism used by bacteria for coupling environmental signals to specific adaptive responses, comprise a sensor histidine kinase and a cognate response regulator [44,45]. The regulatory targets for the P. putida response regulator have not previously been identified. A similar observation was noted for Cr(VI)-stressed S. oneidensis MR-1 cells in which a DNA-binding response regulator (designated SO2426), also of the OmpR/PhoB subfamily [46] of winged-helix DNA-binding domains, was detected at the protein level only in chromate-challenged cells [26,27]. Further in-depth functional analysis using a Δso2426 mutant strain found that this two-component response regulator likely is involved in the activation of genes required for siderophore-mediated iron acquisition [47]. A DNA-binding response regulator consisting of a CheY-like receiver domain also was up-regulated (at the transcript level) in Cr(VI)-treated C. crescentus [31]. At this point, it is not known whether expression of Pput 0287 is part of a cellular regulatory system for sensing external toxic metals or a secondary effect of Cr(VI) challenge.

Conclusions

In summary, effective bioremediation strategies should encompass an understanding of the fundamental physiological and molecular processes extant in the remediating populations. Ideally, one would like to perform such studies in the most ecologically relevant context, but the trade-off is that in situ it is difficult to control enough variables to make the results interpretable. As described here, we have monitored growth and proteome expression in the laboratory under disparate but controlled nutrient conditions ranging from the less (a rich medium) to the more ecologically relevant (minimal medium). We have shown that a strain of P. putida, F1, well-known for its ability to degrade aromatic xenobiotics, is also resistant to low mM levels of Cr(VI) and is capable of reducing hexavalent chromium. Furthermore, the proteome expressed in response to acute chromate challenge is dependent upon factors other than just the presence of external chromate. Most importantly, we have identified in this study a core set of proteins that are differentially expressed in response to Cr(VI) challenge that appear to be media-independent, and this core protein subset will serve as a baseline for comparison with future proteomic analyses of P. putida-containing microbial communities in naturally occurring Cr(VI)-contaminated soils.

Methods

Authors' contributions

DKT and GSW primarily conceived the study, helped prepare cell samples for proteomic analyses, supervised all physiological experiments, and performed the majority of the data interpretation and manuscript writing. KC performed the proteomic measurements, participated in the data interpretation, and helped to develop the proteomic datasets along with MS and NCV. SBT, ATM, and MAR carried out all of the physiological studies and chromate reduction assays for P. putida F1. BZ conducted the statistical analyses of the proteomic data and provided text for this portion of the manuscript. NCV and RLH coordinated and supervised the mass spectrometry studies. All authors read and approved the final manuscript.

Supplementary Material

References