A ViableBacillus subtilisStrain without Functional Extracytoplasmic Function Sigma Genes

Abstract

We constructed a Bacillus subtilis Marburg strain that harbors deletion mutations in all seven extracytoplasmic function (ECF) sigma genes. The strain shows wild-type growth at 37°C both in a complex and in a synthetic medium and exhibits wild-type sporulation. ECF sigma genes of B. subtilis are dispensable as long as no stress is imposed, although they seem to be required for quick response to stresses.Since the assignment of sigma E of Streptomyces coelicolor to a subfamily of eubacterial RNA polymerase sigma factors (14), many sigma factors belonging to this family have been described (3, 4, 9, 18, 22). These sigma factors were called extracytoplasmic function (ECF) sigma factors, as they were found to direct the expression of proteins functioning in the outer membrane or in periplasmic space (14). Further analysis, however, revealed that these sigma factors direct expression of proteins functioning in cytoplasmic space, as well (2,5,9,21,28).The numbers of ECF sigma genes vary among bacterial species. Some bacteria have lost ECF sigma genes, and some bacteria possess a lot of them (9). These ECF sigma genes are believed to be nonessential and auxiliary in nature, although inactivation of rpoE in Escherichia coli and sigG in Synechocystis sp. strain PCC6803 was proven to be lethal (8, 11). In Bacillus subtilis W23, simultaneous inactivation of sigX and sigM causes a growth defect (17). However, the exact roles of those sigma genes in the maintenance of growth have still not been elucidated, and there is no study in which all stress sigma genes have been systematically inactivated in an organism. For this study, we successively interrupted all ECF sigma genes of B. subtilis to construct an ECF sigmaless strain in order to examine whether the genes are indispensable or merely auxiliary.B. subtilis has seven ECF sigma genes (sigM, sigV, sigW, sigX, sigY, sigZ, and ylaC), which are negatively regulated by antisigma proteins, except for sigZ (2,6,7,9,13,16,25,26,27). Inactivation of these sigma genes one at a time does not affect viability because the sigma factors seem to overlap functionally (6,9,10,16,26). A quadruple mutant (⌬sigV ⌬sigY ⌬sigZ ⌬ylaC) of B. subtilis was constructed and was found to be viable (15).In order to examine whether there is a minimal set of ECF sigma genes required for B. subtilis growth under nonstress conditions, we constructed deletion mutations in all seven ECF sigma genes on the genome of B. subtilis Marburg 168 (trpC2) as outlined in Fig. 1. Only one construction pathway is shown. There are many other possible pathways and many combinations of multiple disruptions, and we constructed a large number of different multiple disruptants (data not shown). During successive disruptions of ECF sigma genes, sufficient quantities of wild-type size transformants or segregants were obtained to suggest that there were no suppressor mutations, concerning colony size on LB agar, associated with the disruption of the ECF sigma genes. The sigW gene was disrupted at the end of the procedure becaus...

Transcriptomic and Phenotypic Characterization of aBacillus subtilisStrain without Extracytoplasmic Function σ Factors

“…As the ⌬7ECF strain carries unmarked de- letions (2), this strain did not display these resistances. Relative to the wt strain and to the ⌬MWX strain, the ⌬7ECF strain displayed similar susceptibility to sisomicin (aminoglycoside) and higher susceptibility to chlorpromazine (phenothiazine) and chelerythrine (protein kinase C inhibitor).…”

“…The following antibiotics were used for selection when necessary: spectinomycin, 100 g/ml; kanamycin, 10 g/ml; chloramphenicol, 10 g/ml; tetracycline, 5 g/ml; and erythromycin, 1 g/ml, with lincomycin (25 g/ml) (macrolide-lincomycinstreptogramin B [mls] resistance). The ⌬7ECF strain contains unmarked deletions of all seven ECF factor-encoding genes as previously described (2).…”

The Bacillus subtilis Extracytoplasmic Function σ Factor σ V Is Induced by Lysozyme and Provides Resistance to Lysozyme

“…V is the only lysozyme-inducible ECF factor. The seven ECF factors of B. subtilis have been shown to induce their own expression (2). Several other ECF factors are induced by cell envelope stresses, including sigX, sigW, and sigM (9,11,32).…”

“…factor redundancy masks a critical role of V in resistance to lysozyme. B. subtilis encodes six ECF factors in addition to V (2). Several of these ECF factors are induced by compounds which damage the PG (9)(10)(11)32).…”

Bacillus subtilis σ V Confers Lysozyme Resistance by Activation of Two Cell Wall Modification Pathways, Peptidoglycan O-Acetylation and d -Alanylation of Teichoic Acids

“…To define the V regulon, we induced the expression of V in a strain devoid of all other ECF factors (⌬7ECF) (1,43). We used DNA microarray hybridization to monitor transcriptional changes 20 min after the induction of V to selectively detect direct effects and thereby define promoters activated by V RNAP.…”