Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis. .
DNA sequencing of a region upstream of the mms223 gene of Bacillus subtilis showed the presence of two open reading frames, orf1 and orf2, which may encode 18- and 27-kDa polypeptides, respectively. The predicted amino acid sequence of the latter shows high similarity to a major autolysin of B. subtilis, CwlB, with 35% identity over 191 residues, as well as to other autolysins (CwlC, CwlM, and AmiB). The gene was tentatively named cwlD. Bright spores produced by a B. subtilis mutant with an insertionally inactivated cwlD gene were committed to germination by the addition of L-alanine, and spore darkening, a slow and partial decrease in A580, and 72% dipicolinic acid release compared with that of the wild-type strain were observed. However, degradation of the cortex was completely blocked. Spore germination of the cwlD mutant measured by colony formation after heat treatment was less than 3.7 x 10(-8). The germination deficiency of the cwlD mutant was only partially removed when the spores were treated with lysozyme. Analysis of the chromosomal transcription of cwlD demonstrated that a transcript (RNA2) appearing 3 h after initiation of sporulation may have originated from an internal sigma E-dependent promoter of the cwlD operon, and a longer transcript (RNA1) appearing 4.5 h after sporulation may have originated from a sigma G-dependent promoter upstream of the orf1 gene. The cwlD mutant harboring a B. subtilis vector plasmid containing the intact cwlD gene recovered germination at a frequency 26% of the wild-type level.
Sekiguchi
J
J
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan.
Akeo
K
K
Yamamoto
H
H
Khasanov
F K
FK
Alonso
J C
JC
Kuroda
A
A
D000409:Alanine / Q000494:pharmacology; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002473:Cell Wall / Q000201:enzymology; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D009113:Muramidase / Q000494:pharmacology; D016254:Mutagenesis, Insertional; D009238:N-Acetylmuramoyl-L-alanine Amidase; D016366:Open Reading Frames; D010848:Picolinic Acids / Q000378:metabolism; D012329:RNA, Bacterial / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013171:Spores, Bacterial / Q000187:drug effects / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D010848:Picolinic Acids; D012329:RNA, Bacterial; D012333:RNA, Messenger; D009113:Muramidase; C095875:CwlD protein, Bacillus subtilis; D009238:N-Acetylmuramoyl-L-alanine Amidase; D000409:Alanine; C004860:dipicolinic acid