Defective cell wall synthesis in Streptococcus pneumoniae R6 depleted for the essential PcsB putative murein hydrolase or the VicR (YycF) response regulator.
PcsB is a protein of unknown function(s) that influences the cell morphology of several pathogenic species of streptococcus. PcsB contains a CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain found in bacterial murein hydrolases; however, direct links between steps in cell wall biosynthesis and PcsB function(s) have not been demonstrated. We show here that pcsB is essential in the human respiratory pathogen, Streptococcus pneumoniae, that depletion of PcsB is bacteriostatic and that alanine substitutions in the conserved cysteine and histidine residues of the CHAP domain appear to be lethal. We stained wild-type parent and mutant bacteria deficient in expression of PcsB with fluorescent vancomycin and DAPI to determine patterns of cell wall synthesis and nucleoid segregation respectively. The wild-type parent strain exhibited ordered, simultaneous septal and equatorial cell wall synthesis. In contrast, reduced expression of PcsB resulted in formation of long chains of cells in which peptidoglycan synthesis occurred at nearly every division septum and cell equator. Severe depletion of PcsB led to abnormal, uncontrolled cell wall synthesis at misplaced septa and around large cells. Together, these physiological properties are consistent with a role for PcsB as a murein hydrolase that balances the extent of cell wall synthesis in S. pneumoniae. Finally, we show that the defects in morphology and cell wall synthesis that result from depletion of PcsB strongly resemble those caused by depletion of the essential VicRK two component regulatory system (TCS). This result and the essentiality of pcsB support the hypothesis that the essentiality of the VicRK TCS results from its positive regulation of PcsB expression.
Department of Biology, Indiana University, Bloomington, IN 47405, USA.
Wai-Leung
WL
Ng
Krystyna M
KM
Kazmierczak
Malcolm E
ME
Winkler
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020043:Genes, Essential; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; C422465:PcsB protein, Streptococcus agalactiae; C119939:YycF protein, Bacteria; D009238:N-Acetylmuramoyl-L-alanine Amidase