The Global Transcriptional Responses of Bacillus anthracis Sterne (34F ₂ ) and a Δ sodA1 Mutant to Paraquat Reveal Metal Ion Homeostasis Imbalances during Endogenous Superoxide Stress

Abstract

Microarray analyses were conducted to evaluate the paraquat-induced global transcriptional response of Bacillus anthracis Sterne (34F 2 ) to varying levels of endogenous superoxide stress. Data revealed that the transcription of genes putatively involved in metal/ion transport, bacillibactin siderophore biosynthesis, the glyoxalase pathway, and oxidoreductase activity was perturbed most significantly. A B. anthracis mutant lacking the superoxide dismutase gene sodA1 (⌬sodA1) had transcriptional responses to paraquat similar to, but notably larger than, those of the isogenic parental strain. A small, unique set of genes was found to be differentially expressed in the ⌬sodA1 mutant relative to the parental strain during growth in rich broth independently of induced oxidative stress. The bacillibactin siderophore biosynthetic genes were notably overexpressed in Sterne and ⌬sodA1 cells after treatment with paraquat. The bacillibactin siderophore itself was isolated from the supernatants and lysates of cells grown in iron-depleted medium and was detected at lower levels after treatment with paraquat. This suggests that, while transcriptional regulation of these genes is sensitive to changes in the redox environment, additional levels of posttranscriptional control may exist for bacillibactin biosynthesis, or the enzymatic siderophore pipeline may be compromised by intracellular superoxide stress or damage. The ⌬sodA1 mutant showed slower growth in a chelated iron-limiting medium but not in a metal-depleted medium, suggesting a connection between the intracellular redox state and iron/metal ion acquisition in B. anthracis. A double mutant lacking both the sodA1 and sodA2 genes (⌬sodA1 ⌬sodA2) was attenuated for growth in manganese-depleted medium, suggesting a slight level of redundancy between sodA1 and sodA2, and a role for the sod genes in manganese homeostasis.

Cellular Iron Distribution in Bacillus anthracis

“…3E, right) suggests that SodA2, predicted to be manganese dependent, is catalytically active when cofactored with iron. These results conflict with previous publications predicting that SodA1 and SodA2 were manganese-dependent enzymes (28,31), and the metal ion bound at the active site in published crystal structures of both enzymes was previously modeled as Mn 2ϩ (9).…”

“…3D, left). PMF using MALDI-TOF-MS identified this protein as SodA1 (ORF BA4499; see Table S1 in the supplemental material) (predicted MW ϭ 22,664; pI ϭ 5.31), one of four SOD enzymes encoded by the B. anthracis genome (31), which is predicted to utilize Mn 2ϩ as a cofactor (9,27,28). A number of unmatched peptides in this sample suggested that a mixture of proteins was present.…”

“…Thus, we have been investigating the responses of B. anthracis to such stresses (30,41). Transcriptomic studies have shown the induction of a number of genes encoding putative iron import and siderophore biosynthetic systems during PQ treatment (28,30), which contrasts with the response of E. coli, which represses iron transport in favor of manganese import (3). In light of our identification of the major cytosolic iron pools, we investigated whether this induced gene expression gave rise to increased iron import and whether iron occupancy of these known iron pools was altered under superoxide stress.…”

“…A number of unmatched peptides in this sample suggested that a mixture of proteins was present. The mass spectrum was therefore reanalyzed after removal of the peaks that were matched to SodA1, and this revealed the presence of SodA2 (ORF BA5696) (predicted MW ϭ 24,023; pI ϭ 5.40), which is also predicted to utilize Mn 2ϩ as a cofactor (9,27,28). The presence of a mixture of SodA1 and SodA2 was confirmed by LC-ESI-MS (see Table S1 in the supplemental material).…”

Study of exogenous oxidative stress response in Escherichia coli, Pseudomonas spp., Bacillus spp., and Salmonella spp.

“…based on the available information about their survival mechanisms has yet to be undertaken (Christman et al, 1985;Inaoka et al, 1990;Farr and Kogoma, 1991;Givskov et al, 1994;Nitta et al, 2000;Sabra et al, 2002;Kabir et al, 2004;Passalacqua et al, 2006Passalacqua et al, , 2007Den Besten et al, 2009;Noor et al, 2009aNoor et al, , 2009bZuber, 2009;Pohl et al, 2011;Huillet et al, 2012;Murata et al, 2012;Den Besten et al, 2013;Nagamitsu et al, 2013). In relation to current studies on bacterial oxidative stress responses, the present study employed preliminary experiments to resolve the impact of increased concentration of the oxidant.…”