The Bacillus subtilis sigma(X) protein is an extracytoplasmic function sigma factor contributing to survival at high temperature

Abstract

The sigX gene, identified as part of the international effort to sequence the Bacillus subtilis genome, has been proposed to encode an alternative factor of the extracytoplasmic function (ECF) subfamily. The sigX gene is cotranscribed with a downstream gene, ypuN, during logarithmic and early stationary phases of growth. We now report that strains lacking X are impaired in the ability to survive at high temperature whereas a ypuN mutant has increased thermotolerance. We overproduced and purified X from Escherichia coli and demonstrate that in vitro, both A and X holoenzymes recognize promoter elements within the sigX-ypuN control region. However, they have distinct salt optima such that A -dependent transcription predominates at low salt while X -dependent transcription predominates at high salt. A 54-bp region upstream of sigX suffices as a X -dependent promoter in vivo, demonstrating that sigX is at least partially under positive autoregulatory control. Mutation of ypuN increases expression from the X -dependent promoter in vivo, suggesting that ypuN may encode a negative regulator of X activity.Alternative sigma factors provide a powerful regulatory mechanism whereby a subset of RNA polymerase (RNAP) can be reprogrammed to activate new regulons (13,14,17). This mechanism is prominently featured in the regulatory cascade governing sporulation in Bacillus subtilis (26), in the synthesis of flagellar motility systems in diverse bacteria (16), and in both stationary-phase and heat shock adaptations (12,15,23). In Escherichia coli, the heat shock response is controlled by an alternative factor, 32, which is itself activated by a second heat-inducible factor, E (12). The E protein is a highly diverged member of the 70 family of proteins (30, 31). Recently, a large family of regulatory proteins, many of which had not previously been recognized as factors, were shown to be similar in sequence to E and proposed to coordinate the expression of extracytoplasmic functions (ECF subfamily of factors) (24).The B. subtilis sigX gene (originally defined as ORFX20) was identified during the genome sequencing project as a possible regulatory locus encoding a protein 26% identical to E. coli FecI, a regulator of ferric citrate transport (38). ORFX20 is cotranscribed with a downstream gene (ORFX21; ypuN) of unknown function (2). ORFX20 was renamed sigX because the corresponding gene product is related to the ECF family of factors (24). ECF factors control a variety of functions ranging from heat shock genes in E. coli, alginate biosynthesis in Pseudomonas aeruginosa, iron uptake in E. coli and Pseudomonas spp., carotenoid biosynthesis in Myxococcus xanthus, agarase secretion in Streptomyces coelicolor, plant pathogenicity in Pseudomonas syringae, nickel and cobalt efflux in Alcaligenes eutrophus, and outer membrane protein synthesis in Photobacterium sp. strain SS9 (7,24).In this report, we demonstrate that sigX encodes a polypeptide with the predicted factor activity, X . Reconstituted X holoenzyme (E X ) initiates transcription...

Interaction of Bacillus subtilis extracytoplasmic function (ECF) sigma factors with the N-terminal regions of their potential anti-sigma factors

“…and sigX, our transcriptional activation experiment for sigV and sigW, as well as previous reports on sigX, indicate that these three sigma factors are negatively regulated by the second gene products of the cognate operon (Table 3, and Huang et al, 1997and Huang et al, , 1998. On the other hand, we find the hexa-cistronic sigY operon to be rather complicated.…”

“…Among them, SigX has been reported to contribute in survival at high temperature (Huang et al, 1997). Brutsche & Braun (1997) demonstrated and confirmed the anti-sigma activity and the predicted membrane localization of RsiX (YpuN), the product of the downstream gene.…”

The Bacillus subtilis Extracytoplasmic-Function σ X Factor Regulates Modification of the Cell Envelope and Resistance to Cationic Antimicrobial Peptides

“…sigX and its downstream gene rsiX (encoding the anti-X factor) were originally observed to be homologous (but not orthologous) to Escherichia coli fecI and fecR, which are involved in expression of ferric citrate transport genes (37). Although expression of sigX in E. coli can complement a fecI mutant (4), the B. subtilis sigX mutant is not affected in any known ferri-siderophore uptake systems (20).To understand the function of X , we identified several X -regulated genes using a consensus promoter search method (22). In these initial studies, we characterized six genes that are preceded by promoters recognized by X : sigX, abh (an AbrB homolog), csbB (a membrane-bound glucosyl transferase) (2), divIC (a membrane-bound cell-division initiation protein), lytR (a negative regulator of autolysin) (30), and rapD (a response regulator aspartate phosphatase) (43).…”

Dynamic localization of penicillin-binding proteins during spore development in Bacillus subtilis

“…A sigX-null mutant is impaired in its ability to survive at high temperature (Huang et al, 1997). To determine whether this effect is mediated by pbpX, the survival of the wild-type and the DpbpX strain was tested, after transferring exponentially growing cells to 54 uC for 30 min.…”