Molecular Cloning of a Gene Affecting the Autolysin Level and Flagellation in Bacillus subtilis

Abstract

A 2.8 kb PstI fragment of Bacillus subtilis 168W DNA has been cloned into Escherichia coli HBlOl and B. subtilis AG5 using pAC3 as a shuttle plasmid. The new plasmid (pBRGl), of 10.2 kb, complemented _PaD mutations which show reduced production of autolysin(s), filamentation and non-motility (deficiency of flagella). Deletion experiments showed that the suppressive gene is located between the Hind111 and XbaI sites (1.0 kb apart) in pBRG 1. The integration of a plasmid having chloramphenicol resistance closely linked to the$aD gene into the B. subtilis AC703 chromosome and its genetic analysis indicated that the cloned fragment contained the_PaD gene itself. A high-copy-number plasmid carrying the cloned gene did not lead to an increase in autolysin production above the wild-type level, but it changed the colony morphology from smooth to rough. Among several autolysin-deficient mutations, lyt-151 was suppressed only by the high-copy-number plasmid carrying the cloned gene.

High-level transcription of the major Bacillus subtilis autolysin operon depends on expression of the sigma D gene and is affected by a sin (flaD) mutation

“…Some mutations of these factors [e.g., sacU(Hy) and sin (flaD, lyt)] cause an increase in the expression of extracellular enzymes (2,6,11,12,47,50) and a decrease in the amount of autolysin(s) (6,9,45,46). In this report, we present evidence that the sin (flaDI) mutation greatly reduces expression of the cwlB operon at the transcriptional level.…”

“…pMC1871 DNA was digested with SmaI and Sall, and a 3.1-kb SmaI (blunt-ended)-SalI fragment containing the 3-galactosidase gene was recovered by using Gene Clean. These two fragments and Sail-digested pUD1 DNA (45) were ligated and then used to transform E. coli competent cells. The resultant sigD-lacZ translational fusion plasmid (pSD3), an integrative vector for B. subtilis, is shown in Fig.…”

“…Recently, we demonstrated that the flaD (lyt) gene is identical to the sin (sporulation inhibition) gene (11,45,46). Smith and colleagues found that Sin is a 14-kDa dual-function DNA-binding protein which has a negative function in sporulation (spoIL4, spoIIF, and spoIIG) and in aprE (major alkaline protease gene) expression but also has a positive role in the development of competence, motility, and autolysin production (11,28,49).…”

SinI modulates the activity of SinR, a developmental switch protein of Bacillus subtilis, by protein-protein interaction.

“…Previous work has shown that SinR is a repressor of aprE, the gene coding for the secreted protease, subtilisin, and it also inhibits sporulation (Gaur et al 1986 It has recently been shown that SinR exerts its repressive effects on sporulation immediately before stage II, by inhibiting the expression of spolIA, spolIE, and spoHG (Mandic-Mulec et al 1992}. On the other hand, null mutations in the sinR gene prevent development of competence and motility and the production of growth-related autolysins (Gaur et al 1986;Sekiguchi et al 1988}. It has been shown that its requirement for the development of motility is at the level of motility reguIon gene expression (M. Predich, M. Zelic, and I.…”

Cloning and sequencing of a 40.6 kb segment in the 73 -76 region of the Bacillus subtilis chromosome containing genes for trehalose metabolism and acetoin utilization

“…B. subtilis AC327 (purl3 his-1 smo-1) (Sekiguchi et al, 1988) was used as the source of chromosomal DNA to construct a I library.…”