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 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 . Using two-dimensional gel electrophoretic analysis, Ackerley et al.  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 . We previously reported that S. oneidensis SO4651, a homolog of OscA , was up-regulated at both the transcript and protein levels during the entry response to Cr(VI) stress . Viti et al.  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 . As already mentioned, chromate is a competitive inhibitor of sulfate uptake . 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  and the Alphaproteobacterium Caulobacter crescentus  but not for S. oneidensis MR-1 cells subjected to an acute chromate treatment . 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 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 . 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 . 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 , 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 . 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 . 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  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 . 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 . 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.