CsiA is a bacterial cell wall synthesis inhibitor contributing to DNA translocation through the cell envelope.
Conjugation is a widely spread mechanism allowing bacteria to adapt and evolve by acquiring foreign DNA. The chromosome of Lactococcus lactis MG 1363 contains a 60 kb conjugative element called the sex factor capable of high-frequency DNA transfer. Yet, little is known about the proteins involved in this process. Comparative genomics revealed a close relationship between the sex factor and elements found in Gram-positive pathogenic cocci. Among the conserved gene products, CsiA is a large protein that contains a highly conserved domain (HCD) and a C-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain in its C-terminal moiety. Here, we show that CsiA is required for DNA transfer. Surprisingly, increased expression of CsiA affects cell viability and the cells become susceptible to lysis. Point mutagenesis of HCD reveals that this domain is responsible for the observed phenotypes. Growth studies and electron microscope observations suggest that CsiA is acting as a cell wall synthesis inhibitor. In vitro experiments reveal the capacity of CsiA to bind d-Ala-d-Ala analogues and to prevent the action of penicillin binding proteins. Our results strongly suggest that CsiA sequesters the peptidoglycan precursor and prevents the final stage of cell wall biosynthesis to enable the localized assembly of the DNA transfer machinery through the cell wall.
Stentz
Régis
R
Commensals and Microflora (G2), Institute of Food Research, Norwich, UK. regis.stentz@bbsrc.ac.uk
Wegmann
Udo
U
Parker
Mary
M
Bongaerts
Roy
R
Lesaint
Laurie
L
Gasson
Mike
M
Shearman
Claire
C
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors; D002473:Cell Wall / Q000378:metabolism*; D055028:Comparative Genomic Hybridization; D003227:Conjugation, Genetic; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005144:F Factor / Q000235:genetics / Q000378:metabolism*; D013294:Lactococcus lactis / Q000235:genetics*; D050296:Microbial Viability; D046529:Microscopy, Electron, Transmission; D005810:Multigene Family; D016296:Mutagenesis; D010457:Peptidoglycan / Q000378:metabolism; D017354:Point Mutation
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010457:Peptidoglycan; D002268:Carboxypeptidases