Transcriptomic and phenotypic analysis of paralogous spx gene function in Bacillus anthracis Sterne

Abstract

Spx of Bacillus subtilis is a redox-sensitive protein, which, under disulfide stress, interacts with RNA polymerase to activate genes required for maintaining thiol homeostasis. Spx orthologs are highly conserved among low %GC Gram-positive bacteria, and often exist in multiple paralogous forms. In this study, we used B. anthracis Sterne, which harbors two paralogous spx genes, spxA1 and spxA2, to examine the phenotypes of spx null mutations and to identify the genes regulated by each Spx paralog. Cells devoid of spxA1 were sensitive to diamide and hydrogen peroxide, while the spxA1 spoxA2 double mutant was hypersensitive to the thiol-specific oxidant, diamide. Bacillus anthracis Sterne strains expressing spxA1DD or spxA2DD alleles encoding protease-resistant products were used in microarray and quantitative real-time polymerase chain reaction (RT-qPCR) analyses in order to uncover genes under SpxA1, SpxA2, or SpxA1/SpxA2 control. Comparison of transcriptomes identified many genes that were upregulated when either SpxA1DD or SpxA2DD was produced, but several genes were uncovered whose transcript levels increased in only one of the two SpxADD-expression strains, suggesting that each Spx paralog governs a unique regulon. Among genes that were upregulated were those encoding orthologs of proteins that are specifically involved in maintaining intracellular thiol homeostasis or alleviating oxidative stress. Some of these genes have important roles in B. anthracis pathogenesis, and a large number of upregulated hypothetical genes have no homology outside of the B. cereus/thuringiensis group. Microarray and RT-qPCR analyses also unveiled a regulatory link that exists between the two spx paralogous genes. The data indicate that spxA1 and spxA2 are transcriptional regulators involved in relieving disulfide stress but also control a set of genes whose products function in other cellular processes.Bacillus anthracis harbors two paralogs of the global transcriptional regulator of stress response, SpxA. SpxA1 and SpxA2 contribute to disulfide stress tolerance, but only SpxA1 functions in resistance to peroxide. Transcriptome analysis uncovered potential SpxA1 and SpxA2 regulon members, which include genes activated by both paralogs. However, paralog-specific gene activation was also observed. Genes encoding glutamate racemase, CoA disulfide reductase, and products functioning in bacillithiol biosynthesis, are among the genes activated by the SpxA paralogs.

Evidence that Oxidative Stress Induces spxA2 Transcription in Bacillus anthracis Sterne through a Mechanism Requiring SpxA1 and Positive Autoregulation

“…Vector plasmid pSB24 was constructed by first amplifying the =spoVG=lacZ promoterless fragment using the primers oSB86 and oSB87 (see Table S3 in the supplemental material) and pDG793 (47) as the template. The fragment was cleaved with EcoRI and HindIII and inserted by ligation into pJMA402 (40) that was cleaved with the same enzymes. The resulting pSB24 plasmid was cleaved with EcoRI and PstI and ligated with EcoRI/PstI cleaved PCR fragments of the spxA2 promoter region that were amplified using the primer oSB97 and several primers listed in Table S3 in the supplemental material.…”

“…As with several Gram-positive species, B. anthracis produces two paralogous forms of Spx, SpxA1 and SpxA2 (40). SpxA1 resembles Spx of B. subtilis, both in terms of the primary structure and in terms of the genetic context specifying its expression, although spxA1 transcript levels do not increase in response to oxidative stress.…”

“…spxA2 is the first gene of a four-gene operon that includes saiR (suppressor of spxA1), encoding a repressor of spxA2 transcription (41). Earlier transcriptomic microarray data indicate that SpxA1 and SpxA2 govern overlapping regulons, many of which are orthologous genes corresponding to those activated by B. subtilis Spx (40). However, some differences in regulon composition were noted, suggesting distinct promoter sequence recognition properties.…”

“…However, some differences in regulon composition were noted, suggesting distinct promoter sequence recognition properties. The concentration of both SpxA1 and SpxA2 increase upon encounters with toxic oxidants (40), likely due in part to downregulation of YjbH/ClpXP proteolysis, the orthologous components of which are encoded in the B. anthracis genome.…”

“…Treatment with the thiol-specific oxidant, diamide, results in elevated spxA2 transcript levels (40). Earlier work showed that derepression of spxA2 in response to stress required the spxA2 gene product, suggesting a mechanism of positive autoregulation (41).…”