Identification and phenotypic characterization of the cell-division protein CdpA.
Analysis of the automated computer annotation of the early draft phase genome of Lactobacillus acidophilus NCFM revealed the previously discovered S-layer gene slpA and an additional partial ORF with weak similarities to S-layer proteins. The entire gene was sequenced to reveal a 1799-bp gene coding for 599 amino acids with a calculated molecular mass of 64.8 kDa. No transcription or translation signals could be determined in close proximity to the 5'-region. However, a strong putative terminator with a free energy of -16.84 kcal/mol was identified directly downstream of the gene. A PSI-Blast analysis showed similarities to members of S-layer proteins, cell-wall associated proteinases and hexosyl-transferases. Calculation of an unrooted phylogenetic tree with other examples of S-layer proteins and proteinases placed the deduced protein separately from both groups. A derivative of L. acidophilus NCFM was constructed by targeted integration into the gene. SDS-PAGE analysis of non-covalently linked proteins of the cell wall of the mutant, compared to the wild type, revealed the loss of a cell-surface protein. Phenotypic analyses of the mutant revealed significant changes in cell morphology, altered responses to various environmental stresses, and lowered cell adhesion. Based on the in silico and functional analyses, we ascertained that this protein plays a role in cell-wall processing during the growth and cell-cell separation and designated the gene as cell-division protein, cdpA.
Altermann
Eric
E
Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, 339 Schaub Hall, Box 7624, Raleigh, NC 27695-7624, USA.
Buck
Logan B
LB
Cano
Raul
R
Klaenhammer
Todd R
TR
D001422:Bacterial Adhesion / Q000235:genetics / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D018938:Caco-2 Cells; D018797:Cell Cycle Proteins / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D002473:Cell Wall / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000431:Ethanol / Q000494:pharmacology; D005615:Freezing; D020868:Gene Silencing; D006801:Humans; D007779:Lactobacillus acidophilus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D008562:Membrane Glycoproteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D008562:Membrane Glycoproteins; C055764:S-layer proteins; D000431:Ethanol; D012965:Sodium Chloride