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

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.

A putative azoreductase gene is involved in the Shewanella oneidensis response to heavy metal stress.

The Shewanella oneidensis MR-1 gene SO3585, which is annotated as a putative flavin mononucleotide-dependent azoreductase, shares 28% sequence identity with Bacillus subtilis azoreductase and Pseudomonas putida ChrR, a soluble flavoprotein exhibiting chromate reductase activity. Reverse transcription polymerase chain reaction demonstrated that the SO3585 gene is co-transcribed with two downstream open reading frames: SO3586 (a glyoxalase family protein) and SO3587 (a predicted membrane-associated hypothetical protein). The transcriptional start site of the so3585 transcript was localized using 5' rapid amplification of complementary DNA ends analysis. To investigate the cellular function of SO3585, an in-frame deletion of the so3585 locus was generated in MR-1, and the phenotype of the resulting mutant was characterized. The so3585 deletion mutant was comparable to the parental strain in its ability to decolorize two sulfonated azo dyes (Orange II, Direct Blue 15) under aerobic conditions. By contrast, growth of the so3585 deletion mutant was sensitive to different exogenous transition heavy metals [Cr(VI), Cd(II), Cu(II), and Zn(II)], while the most severe growth deficiencies were observed in the presence of Cd(II) and Cu(II). In addition, the rate of extracellular chromate disappearance by the deletion strain was initially impaired, although both the so3585 mutant and MR-1 wild type reduced Cr(VI) within the same time period.

Enumeration of aromatic oxygenase genes to evaluate biodegradation during multi-phase extraction at a gasoline-contaminated site.

Multi-phase extraction (MPE) is commonly used at petroleum-contaminated sites to volatilize and recover hydrocarbons from the vadose and saturated zones in contaminant source areas. Although primarily a physical treatment technology, the induced subsurface air flow can potentially increase oxygen supply and promote aerobic biodegradation of benzene, toluene, ethylbenzene, and xylenes (BTEX), the contaminants of concern at gasoline-contaminated sites. In this study, real-time PCR enumeration of aromatic oxygenase genes and PCR-DGGE profiles were used to elucidate the impact of MPE operation on the aquifer microbial community structure and function at a gasoline-contaminated site. Prior to system activation, ring-hydroxylating toluene monooxygenase (RMO) and naphthalene dioxygenase (NAH) gene copies were on the order of 10(6) to 10(10)copies L(-1) in groundwater samples obtained from BTEX-impacted wells. Aromatic oxygenase genes were not detected in groundwater samples obtained during continuous MPE indicating decreased populations of BTEX-utilizing bacteria. During periods of pulsed MPE, total aromatic oxygenase gene copies were not significantly different than prior to system activation, however, shifts in aromatic catabolic genotypes were noted. The consistent detection of RMO, NAH, and phenol hydroxylase (PHE), which catabolizes further oxidation of hydroxylated BTEX metabolites indicated the potential for aerobic biodegradation of dissolved BTEX during pulsed MPE.

Transcriptome profiling of Shewanella oneidensis gene expression following exposure to acidic and alkaline pH.

The molecular response of Shewanella oneidensis MR-1 to variations in extracellular pH was investigated based on genomewide gene expression profiling. Microarray analysis revealed that cells elicited both general and specific transcriptome responses when challenged with environmental acid (pH 4) or base (pH 10) conditions over a 60-min period. Global responses included the differential expression of genes functionally linked to amino acid metabolism, transcriptional regulation and signal transduction, transport, cell membrane structure, and oxidative stress protection. Response to acid stress included the elevated expression of genes encoding glycogen biosynthetic enzymes, phosphate transporters, and the RNA polymerase sigma-38 factor (rpoS), whereas the molecular response to alkaline pH was characterized by upregulation of nhaA and nhaR, which are predicted to encode an Na+/H+ antiporter and transcriptional activator, respectively, as well as sulfate transport and sulfur metabolism genes. Collectively, these results suggest that S. oneidensis modulates multiple transporters, cell envelope components, and pathways of amino acid consumption and central intermediary metabolism as part of its transcriptome response to changing external pH conditions.