In order to explore the effect of peptidoglycan hydrolase on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease, five peptidoglycan hydrolase genes (lytC, lytD, lytE, lytF and lytG) of B. amyloliquefaciens TCCC111018 were knocked out individually. The viable cell counts of the bacteria and their alkaline protease activities before and after gene deletion were determined. The viable cell counts of the knockout mutants BA ΔlytC and BA ΔlytE achieved 1.67×10⁶ CFU/mL and 1.44×10⁶ CFU/mL respectively after cultivation for 60 h, which were 32.5% and 14.3% higher than that of the control strain BA Δupp. Their alkaline protease activities reached 20 264 U/mL and 17 265 U/mL, respectively, which were 43.1% and 27.3% higher than that of the control strain. The results showed that deleting some of the peptidoglycan hydrolase genes effectively maintained the viable cell counts of bacteria and increased the activity of extracellular enzymes, which may provide a new idea for optimization of the microbial host for production of industrial enzymes.
Bacillus amyloliquefaciens/genetics* ; Bacterial Proteins ; Cell Count ; Endopeptidases/genetics* ; N-Acetylmuramoyl-L-alanine Amidase/genetics*

Cell autolysis plays important physiological roles in the life cycle of clostridial cells. Understanding the genetic basis of the autolysis phenomenon of pathogenic Clostridium or solvent producing Clostridium cells might provide new insights into this important species. Genes that might be involved in autolysis of Clostridium acetobutylicum, a model clostridial species, were investigated in this study. Twelve putative autolysin genes were predicted in C. acetobutylicum DSM 1731 genome through bioinformatics analysis. Of these 12 genes, gene SMB_G3117 was selected for testing the in tracellular autolysin activity, growth profile, viable cell numbers, and cellular morphology. We found that overexpression of SMB_G3117 gene led to earlier ceased growth, significantly increased number of dead cells, and clear electrolucent cavities, while disruption of SMB_G3117 gene exhibited remarkably reduced intracellular autolysin activity. These results indicate that SMB_G3117 is a novel gene involved in cellular autolysis of C. acetobutylicum.
Autolysis ; genetics ; Clostridium acetobutylicum ; genetics ; metabolism ; Computational Biology ; Genes, Bacterial ; N-Acetylmuramoyl-L-alanine Amidase ; genetics ; metabolism ; Temperature

BACKGROUND: Resistance to penicillin of Streptococcus pneumoniae in clinical isolates has occurred by the development of altered penicillin-binding proteins (PBPs) that have greatly decreased affinity for antibiotics and has been encountered with increasing frequency in Korea. In this study, the identification of altered PBPs of S. pneumoniae in clinical isolates by using polymerase chain reaction (PCR) and relationship of PCR with the conventional antibiotic susceptibility test of penicillin were evaluated. METHODS: Thirty isolates of S. pneumoniae from clinical specimens were used. Four sets of PCR primers of penicillin-sensitive and -resistant S. pneumoniae were designed to amplify (i) PBP 2BS: a 360 base pair fragment of the PBP 2B gene, (ii) PBP 2BCA: a 350 base pair fragment of the class A mutations present in PBP 2B gene, (iii) PBP 2BCB: a 295 base pair fragment of the class B mutations present in PBP 2B gene, and (iv) PBP 1AR: a 434 base pair fragment of the PBP 1A gene. In addition, a set of primers that amplify 273 base pair of the autolysin gene (ALY) was applied in combination with the above to identify S. pneumoniae. PCR results were compared with antibiotic susceptibility test (disk diffusion test and penicillin MIC). RESULT: Among 30 clinical isolates tested, 80% of isolates were penicillin resistant. The results of antibiotic susceptibility test were same as those of PCR methods. Among 24 penicillin resistant isolates detected by PCR methods, 5 isolates revealed PBP 2BCB gene, but 19 isolates revealed both of PBP 2BCB and 1AR genes. Five isolates with PBP 2BCB gene showed lower range of penicillin MIC (0.19 ~ 1.0 g/mL) than 19 isolates with PBP 2BCB and 1AR genes (0.75 ~ 4.0 g/mL). CONCLUSION: Detection of altered PBP genes of S. pneumoniae by PCR may be performed for the study of penicillin resistance. This study indicates that more altered PBPs in 1AR genes are related with higher MICs.
Anti-Bacterial Agents ; Base Pairing ; Diffusion ; Korea ; N-Acetylmuramoyl-L-alanine Amidase ; Penicillin Resistance ; Penicillin-Binding Proteins ; Penicillins ; Pneumonia ; Polymerase Chain Reaction* ; Streptococcus pneumoniae* ; Streptococcus*