Characterization of ywhE, which encodes a putative high-molecular-weight class A penicillin-binding protein in Bacillus subtilis

• Published in: Gene Vol. 246; no. 1; pp. 187 - 196
• Main Authors: Pedersen, L.B., Ragkousi, K., Cammett, T.J., Melly, E., Sekowska, A., Schopick, E., Murray, T., Setlow, P.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 04.04.2000
• Subjects: Amino Acid Sequence; Bacillus subtilis; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Bacillus subtilis - physiology; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Division - genetics; Escherichia coli; Escherichia coli - cytology; Escherichia coli - genetics; Gene Expression Regulation, Bacterial; Hexosyltransferases; Lac Operon - genetics; lacZ gene; Molecular Sequence Data; Molecular Weight; Muramoylpentapeptide Carboxypeptidase - chemistry; Muramoylpentapeptide Carboxypeptidase - genetics; Muramoylpentapeptide Carboxypeptidase - metabolism; Mutagenesis, Insertional; Mutation; Penicillin-binding protein; Penicillin-Binding Proteins; Peptidyl Transferases; Promoter Regions, Genetic - genetics; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Sigma Factor - physiology; sigma super(F) Factor; sigma super(G) factor; Spores, Bacterial - genetics; Sporulation; ywhE gene; σ F-dependent expression; aa, amino acid(s); Δ2–41, YwhE lacking aa 2–41; kan, kanamycin resistance gene; amyE, structural gene for amylase; CDS, coding sequence; bp, base pair(s); HMW, high-molecular-weight; SDS, sodium dodecyl sulfate; lacZ, structural gene for β-galactosidase; Sporulation; PAGE, polyacrylamide gel electrophoresis; Spo +, sporogenous; ywhEΔ2–41, gene encoding Δ2–41; N-terminal, amino-terminal; PBP, penicillin-binding protein; Penicillin-binding protein; MW, molecular weight; erm, erythromycin resistance gene; bla, Ap r gene; IPTG, isopropyl-β- D-thiogalactopyranoside; kDa, kilodalton(s); σ F-dependent expression; PMSF, phenylmethylsulfonyl fluoride; RBS, ribosome binding site; Ap r, ampicillin resistance; Cm r, chloramphenicol resistance; nt, nucleotide(s); P spac, IPTG-inducible spac promoter; PCR, polymerase chain reaction; cat, Cm r gene; OD 600, optical density at 600 nm
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(00)00084-6

The Bacillus subtilis genome sequencing project [Kunst et al., Nature 390 (1997) 249–256] identified ywhE as a gene that potentially encodes a high-molecular-weight class A penicillin-binding protein. Analysis of the expression of a translational ywhE–lacZ fusion showed that ywhE expression is sporulation-specific, and is controlled predominantly by the forespore-specific σ factor σ F, and to a lesser extent by σ G. Primer extension analysis identified two transcription start sites located 26 and 27 nucleotides upstream of the ywhE translational initiation codon. Sequences located in the −10 and −35 regions relative to the transcription start sites showed good homology to the consensus sequences for promoter elements of σ F-dependent genes. An insertional mutation in ywhE had no significant effect on growth, morphology, and sporulation, and ywhE spores had normal heat-resistance, cortex structure, and germination and outgrowth properties. However, overexpression of ywhE in Escherichia coli resulted in cell lysis.