Proteomics reveals a core molecular response of Pseudomonas putida F1 to acute chromate challenge

Abstract

Background: Pseudomonas putida is a model organism for bioremediation because of its remarkable metabolic versatility, extensive biodegradative functions, and ubiquity in contaminated soil environments. To further the understanding of molecular pathways responding to the heavy metal chromium(VI) [Cr(VI)], the proteome of aerobically grown, Cr(VI)-stressed P. putida strain F1 was characterized within the context of two disparate nutritional environments: rich (LB) media and minimal (M9L) media containing lactate as the sole carbon source.

Results: Growth studies demonstrated that F1 sensitivity to Cr(VI) was impacted substantially by nutrient conditions, with a carbon-source-dependent hierarchy (lactate > glucose >> acetate) observed in minimal media. Two-dimensional HPLC-MS/MS was employed to identify differential proteome profiles generated in response to 1 mM chromate under LB and M9L growth conditions. The immediate response to Cr(VI) in LB-grown cells was up-regulation of proteins involved in inorganic ion transport, secondary metabolite biosynthesis and catabolism, and amino acid metabolism. By contrast, the chromate-responsive proteome derived under defined minimal growth conditions was characterized predominantly by up-regulated proteins related to cell envelope biogenesis, inorganic ion transport, and motility. TonB-dependent siderophore receptors involved in ferric iron acquisition and amino acid adenylation domains characterized up-regulated systems under LB-Cr(VI) conditions, while DNA repair proteins and systems scavenging sulfur from alternative sources (e.g., aliphatic sulfonates) tended to predominate the up-regulated proteome profile obtained under M9L-Cr(VI) conditions.

Conclusions: Comparative analysis indicated that the core molecular response to chromate, irrespective of the nutritional conditions tested, comprised seven up-regulated proteins belonging to six different functional categories including transcription, inorganic ion transport/metabolism, and amino acid transport/metabolism. These proteins might potentially serve as indicators of chromate stress in natural microbial communities.

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.

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.

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.

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.