title: The putative penicillin-binding proteins 1 and 2 are important for viability, growth and cell morphology of Brucella melitensis.
abstract: The penicillin-binding proteins (PBPs) are enzymes that regulate the assembly of the peptidoglycan layer of the bacterial cell wall. The genome of Brucella melitensis strain 16M possesses seven pbp genes: three in pbp-1 family (designated as 1A, 1B, and 1C); one in pbp-2 family; and three in pbp-6 family (designated as 6A, 6B, and 6C). We investigated the importance of pbp-1 and pbp-2 genes to viability, cell morphology and infectivity of B. melitensis. A recombinant B. melitensis strain (designated 16MDeltapbp1C) was generated by disrupting the pbp-1C of strain 16M by allelic exchange. This strain produced nearly 20% smaller colonies on trypticase soy agar plates, and grew slower in trypticase soy broth compared to the strain 16M. Electron microscopy revealed that strain 16M exhibited native cocco-bacillus morphology, while 16MDeltapbp1C possessed a spherical morphology. Strain 16MDeltapbp1C did not differ from strain 16M in terms of recovery from infected mouse macrophage cell line J774.1, or recovery from spleens of infected BALB/c mice, suggesting that pbp-1C is dispensable for intracellular persistence of B. melitensis. Expression of mRNA of fixR, the gene downstream of pbp-1C was similar between the strains 16M and 16MDeltapbp1C suggesting that disruption of pbp-1C did not induce any polar effects. Multiple attempts to mutate pbp-1A, pbp-1B, or pbp-2 genes failed, most probably because these genes are indispensable for viability of B. melitensis. Our findings suggest that pbp-1C regulates in vitro growth and cell morphology, whereas pbp-1A, pbp-1B, and pbp-2 are essential for viability of B. melitensis.
authors.affiliation: Department of Biomedical Sciences & Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0910, USA. abandara@vt.edu
authors.forename: Aloka B
authors.initials: AB
authors.lastname: Bandara
authors.forename: Gerhardt G
authors.initials: GG
authors.lastname: Schurig
authors.forename: Nammalwar
authors.initials: N
authors.lastname: Sriranganathan
authors.forename: Rajeev
authors.initials: R
authors.lastname: Prasad
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Boyle
mesh_terms: D000818:Animals; D017347:Brucella melitensis / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008264:Macrophages; D051379:Mice; D008807:Mice, Inbred BALB C; D046915:Penicillin-Binding Proteins / Q000378:metabolism*
chemical_list: D046915:Penicillin-Binding Proteins