Bacillus subtilis contains seven extracytoplasmic-function factors that activate partially overlapping regulons. We here identify four additional members of the X regulon, pbpX (penicillin-binding protein), ywnJ, the dlt operon (D-alanylation of teichoic acids), and the pss ybfM psd operon (phosphatidylethanolamine biosynthesis). Modification of teichoic acids by esterification with D-alanine and incorporation of phosphatidylethanolamine into the cell membrane have a common consequence: in both cases positively charged amino groups are introduced into the cell envelope. The resulting reduction in the net negative charge of the cell envelope has been previously implicated as a resistance mechanism specific for cationic antimicrobial peptides. Consistent with this notion, we find that both sigX and dltA mutants are more sensitive to nisin than wild-type cells. We conclude that activation of the X regulon serves to alter cell surface properties to provide protection against antimicrobial peptides.Bacillus subtilis encodes seven extracytoplasmic-function (ECF) factors. Most studies to date have focused on three:X , W , and M (reviewed in reference 19). 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). These results suggested that X modulates aspects of cell envelope metabolism. Interestingly, most X -controlled genes are also transcribed by other forms of holoenzyme. For example, csbB has an additional B -dependent promoter, lytR and rapD both have additional A -dependent promoters, and sigX itself is preferentially transcribed from an upstream A -dependent site in addition to the X -dependent autoregulatory promoter (20,22). Moreover, in some cases (e.g., abh and divIC) the promoter activated by the E X holoenzyme can also be recognized by the E W holoenzyme at least in vitro (21). Similarly, the recently defined bcrC gene (a bacitracin resistance gene) is transcribed from a promoter that is recognized by either X or M (7, 38). The unknown function of many X -controlled genes makes it difficult to predict a phenotype for the sigX mutant. This challenge is exacerbated by the fact that many of the genes are expressed from multiple promoters or by multiple holoenzyme forms activating the same promoter. The latte...