Physiological, genetic and proteomic analysis of chromate resistance in Arthrobacter sp strain FB24

Chromium is an abundant metal within the environment, with trivalent and hexavalent chromium being the most stable oxidation states. Hexavalent chromium, which is often present as the oxyanion, chromate, is regarded as highly toxic and more soluble than the trivalent form. Due to its widespread use in industry, chromate is a frequent contaminant in the environment. Microorganisms have evolved mechanisms to resist chromate toxicity, including efflux of the chromate ion from the intracellular space. Arthrobacter sp strain FB24 was isolated from soils that have experienced long-term insult with elevated levels of hydrocarbons and metals, including chromium. FB24 was found to be resistant to high levels of chromate and putative chromate resistance genes were identified on an apparent 96 kb plasmid through genome sequence analysis. Loss of plasmid DNA from strain FB24 resulted in the chromate sensitive derivative, strain D11. Chromate resistance was recovered in strain D11 through mating with FB24, further supporting the presence of a plasmid-borne chromate resistance determinant in strain FB24. A 10.6 kb cluster of 8 genes located on the 96 kb FB24 plasmid was cloned and expressed in strain D11, and conferred chromate resistance in the sensitive strain. The genes comprising this cluster encode a putative chromate efflux protein, ChrA (Arth_4248), putative chromate resistance regulatory proteins (Arth_4249, 4253, 4254), a malate:quinone oxidoreductase (Arth_4255), a WD40 repeat domain protein (Arth_4252) and a lipoprotein (Arth_4247). Quantitative reverse transcriptase PCR demonstrated increased expression of each gene in response to increasing concentrations of chromate. This is the first example of the association of WD40 domain proteins, malate:quinone oxidoreductases and lipoproteins in bacterial chromate resistance. Global proteomic analysis of chromate-exposed FB24 cultures using a combined two-dimensional gel electrophroesis and LC-MS/MS approach provided coverage of at least 71% of the predicted proteome of FB24. The majority of proteins exhibiting altered expression profiles in response to chromate fell within the functional categories of energy production, carbon metabolism and amino acid uptake and metabolism. Proteins potentially involved in the sulfate limitation response exhibited some of the highest fold changes under chromate stress. This is consistent with competition of the chromate ion for the sulfate transporter, leading to intracellular sulfur deprivation