Identification and Molecular Analysis of PcsB, a Protein Required for Cell Wall Separation of Group B Streptococcus

Abstract

Group B streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis in neonates. N-terminal sequencing of major proteins in the culture supernatant of a clinical isolate of GBS identified a protein of about 50 kDa which could be detected in all of 27 clinical isolates tested. The corresponding gene, designated pcsB, was isolated from a GBS cosmid library and subsequently sequenced. The deduced PcsB polypeptide consists of 447 amino acid residues (M r , 46,754), carries a potential N-terminal signal peptide sequence of 25 amino acids, and shows significant similarity to open reading frames of unknown function from different organisms and to the murein hydrolase P45 from Listeria monocytogenes. Northern blot analysis revealed a monocistronic transcriptional organization for pcsB in GBS. Insertional inactivation of pcsB in the genome of GBS resulted in mutant strain Sep1 exhibiting a drastically reduced growth rate compared to the parental GBS strain and showing an increased susceptibility to osmotic pressure and to various antibiotics. Electron microscopic analysis of GBS mutant Sep1 revealed growth in clumps, cell separation in several planes, and multiple division septa within single cells. These data suggest a pivotal role of PcsB for cell division and antibiotic tolerance of GBS.Group B streptococcus (GBS), also known as Streptococcus agalactiae, is part of the normal human flora colonizing the respiratory, gastrointestinal, and urogenital tracts. It is also the leading cause of bacterial sepsis and meningitis in neonates in the United States and western Europe, and it is a major cause of endocarditis and fever in parturient women (2). In the last decade, the incidence of GBS infections has increased especially in the elderly and in immunocompromised persons (58), but despite its clinical importance, GBS is only poorly understood on the molecular level.Bacterial cell division is a complex interplay of topological processes, biosynthetic reactions, and cleavage mechanisms. Septum formation is initiated by the cooperative action of division inhibitors, topological specificity factors, and proteins constituting the cell division apparatus both found in gramnegative and gram-positive bacteria (reviewed in reference 5, 31, and 42). In globular cells, like streptococci, the division septum can be arranged in several orientations, resulting in the generation of two daughter cells of the same size and shape. The choice of the division plane determines the three-dimensional organization of the multicellular arrays of progeny cells that are formed. In GBS, the plane of division is approximately the same in each division cycle, resulting in the formation of frequently long chains (20). The division septum that is formed between the daughter cells is composed of newly synthesized membrane and peptidoglycan components. Peptidoglycan biosynthesis is accomplished by the insertion of peptide-carrying disaccharide units into the existing murein sacculus by transglycosylation and transpeptidation (34). Cell divi...

Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae

“…To investigate whether full-length PcsB has muralytic activity, we performed zymography with purified recombinant PcsB and pneumococcal cells mixed into the separating SDS-PAGE gel as substrate. Consistent with the results reported by others 8,11,12 , no muralytic activity was detected. Next, we expressed only the C-terminal CHAP domain of PcsB.…”

“…Despite the presence of this putative catalytic domain in the PcsB sequence, attempts to demonstrate hydrolytic activity of full-length PcsB have been unsuccessful 8,11,12 . Recently, it was discovered that PcsB interacts with the membrane-embedded protein FtsX through its N-terminal CC domain.…”

“…PcsB is a secreted putative murein hydrolase that localizes to the septal area. It has been characterized to various degrees in S. pneumoniae 8,9 , Streptococcus mutans (where it is called GbpB) 10 , and Streptococcus agalactiae 11 . In all cases, it has been shown to be required for normal growth and cell division.…”

Two-component signal transduction systems in oral bacteria

“…Consistently, atypical long-chain phenotypes are found in v icK isogenic mutants obtained in S. pnemoniae , S. mutans , and S. sanguinis [90,105,106,111]. In VicR regulons, the most conserved target gene encodes PcsB (protein required for cell separation of group B Streptococcus ) that was first identified in group B streptococci [114] and orthologue GbpB (glucan-binding protein B in S. mutans ) [115,116]. Table 2 shows comparisons of VicR gene targets among streptococcal species of the oral cavity and oropharynx.…”

Is the GehD Lipase from Staphylococcus epidermidis a Collagen Binding Adhesin?

“…Additional proteins with adhesive functions located on the surface without LPXTG motifs include SEN (27), a surface enolase of S. pyogenes, SDH (28), a surface dehydrogenase of group A streptococci, and the S. epidermidis autolysin AtlE, which specifically binds to vitronectin (11). Anchorless proteins with other biological functions have also been described (29,30). Clearly, the number of anchorless adhesins identified in Gram-positive bacteria will increase in the future, but it is not clear if these proteins are virulence factors.…”