Global Analysis of Cellular Factors and Responses Involved inPseudomonas aeruginosaResistance to Arsenite

Abstract

The impact of arsenite [As(III)] on several levels of cellular metabolism and gene regulation was examined in Pseudomonas aeruginosa. P. aeruginosa isogenic mutants devoid of antioxidant enzymes or defective in various metabolic pathways, DNA repair systems, metal storage proteins, global regulators, or quorum sensing circuitry were examined for their sensitivity to As(III). Mutants lacking the As(III) translocator (ArsB), superoxide dismutase (SOD), catabolite repression control protein (Crc), or glutathione reductase (Gor) were more sensitive to As(III) than wild-type bacteria. The MICs of As(III) under aerobic conditions were 0.2, 0.3, 0.8, and 1.9 mM for arsB, sodA sodB, crc, and gor mutants, respectively, and were 1.5-to 13-fold less than the MIC for the wild-type strain. A two-dimensional gel/matrix-assisted laser desorption ionization-time of flight analysis of As(III)-treated wild-type bacteria showed significantly (>40-fold) increased levels of a heat shock protein (IbpA) and a putative allo-threonine aldolase (GlyI). Smaller increases (up to 3.1-fold) in expression were observed for acetyl-coenzyme A acetyltransferase (AtoB), a probable aldehyde dehydrogenase (KauB), ribosomal protein L25 (RplY), and the probable DNA-binding stress protein (PA0962). In contrast, decreased levels of a heme oxygenase (HemO/PigA) were found upon As(III) treatment. Isogenic mutants were successfully constructed for six of the eight genes encoding the aforementioned proteins. When treated with sublethal concentrations of As(III), each mutant revealed a marginal to significant lag period prior to resumption of apparent normal growth compared to that observed in the wild-type strain. Our results suggest that As(III) exposure results in an oxidative stress-like response in P. aeruginosa, although activities of classic oxidative stress enzymes are not increased. Instead, relief from As(III)-based oxidative stress is accomplished from the collective activities of ArsB, glutathione reductase, and the global regulator Crc. SOD appears to be involved, but its function may be in the protection of superoxide-sensitive sulfhydryl groups.Arsenic is present in numerous disturbed and natural ecosystems and is a top-priority national pollutant. It can exist in multiple oxidation states, with the most common being arsenite [As(III)] and arsenate [As(V)]. As(V) is an analogue of phosphate, and its toxicity is due to the disruption of critical cellular functions or synthesis of essential building blocks. These include uncoupling of ATP phosphorylation that would directly impact energy flow, as well as nucleic acid and phospholipid synthesis. As(III) toxicity is thought to be due predominantly to its ability to covalently bind protein sulfhydryl groups. Of the two species, As(III) is considered the most toxic (16,68).Although some microorganisms can utilize As(V) for anaerobic respiration (81) or oxidize As(III) as a sole energy source (37, 75), arsenic generally is toxic to most microorganisms. Arsenic resistance in bacteria is due, in part,...

A Na + :H + Antiporter and a Molybdate Transporter Are Essential for Arsenite Oxidation in Agrobacterium tumefaciens

“…We were unable to demonstrate increased As(III) sensitivity in the mutants isolated in this study, although it is important to note that the screening methodology selected for mutants that were tolerant of 1 mM As(III) [lower As(III) concentrations in the AgNO 3 screening technique gave inconsistent staining results on MMN agar medium (results not shown)]. This may have biased our results by eliminating mutants that were exquisitely As(III) sensitive (e.g., as we have found with Pseudomonas aeruginosa [40]), but the fact remains that the complete loss of As(III) activity due to non-regulatorytype mutations did not yield an As(III)-sensitive phenotype.…”

Aldehyde dehydrogenases in cellular responses to oxidative/electrophilicstress

“…Arsenite (As 3+ ) exposure induces oxidative stress by disrupting the citric acid cycle and releasing unbound Fe 2+ which then elicits ROS generation via the Fenton reaction. Exposure of Pseudomonas aeruginosa to arsenite causes an up-regulation of several proteins, including the predicted ALDH KauB (ALDH1P1) [46]. The potential bioremediation bacterium Acidothiobacillus ferrooxidans up-regulates an unknown ALDH (ALD1Q1) (NCBI Entrez gene ID: 198283897) when exposed to bornite (Cu 5 FeS 4 ), an agent that induces oxidative stress through the release of copper ions into media during solubilization [47].…”

A proteome analysis of the response of a Pseudomonas aeruginosa oxyR mutant to iron limitation

“…In another study involving proteome analysis it was found that exposure of P. aeruginosa cells to arsenite (AsIII) generates an oxidative stress and the induction of both IbpA and Dps (Parvatiyar et al 2005), suggesting a role of these two proteins in defense against oxidative stress like in E. coli (Kitagawa et al 2002;Matuszewska et al 2008). PA0941 encodes a small hypothetical protein annotated as a glutaredoxin.…”