Gene Knockout and Overexpression Analysis Revealed the Role of N-Acetylmuramidase in Autolysis of Lactobacillus delbrueckii subsp. bulgaricus Ljj-6

Abstract

Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur), was cloned and sequenced (GenBank accession number: KF157911). Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.

Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase

“…S2A and B). Previous research has demonstrated that Lactobacillus species can undergo autolysis in the late stationary phase, especially in response to carbon starvation (25,26). BHI medium only contains 0.2% (wt/vol) glucose, so we tested whether glucose limitation induced autolysis under these conditions.…”

Is Acrylamide as Harmful as We Think? A New Look at the Impact of Acrylamide on the Viability of Beneficial Intestinal Bacteria of the Genus Lactobacillus

“…It is well known that some lactic acid bacteria can undergo enzymatic cleavage of cell wall peptidoglycans by peptidoglycan hydrolases present in the bacterial cells and that the autolysis depends on factors, such as carbon source, temperature, osmotic concentration, and pH [56]. It has also been demonstrated that N-acetylmuramidase has a critical function in Lactobacillus bulgaricus autolysis [57] as one of the major degraders of the cell wall. In our experiment, the percentage of live L. plantarum cells was significantly lower after 48 h than at the beginning or after 24 h. Moreover, significant differences in L. plantarum morphology were observed.…”

A fibronectin-binding protein (FbpA) of Weissella cibaria inhibits colonization and infection of Staphylococcus aureus in mammary glands

“…To delete the fbpA gene from the genome of W. cibaria isolate LW1, a homologous recombination procedure was performed employing the suicide vector pUC19 (Pang, Cui, Liu, Zhang, & Lv, ). DNA fragments for 630 bp upstream and 670 bp downstream of fbpA gene were amplified using the specific primer pairs fbpAUp‐F/fbpAUp‐R and fbpADown‐F/fbpADown‐R, respectively, (Table ).and cloned into pUC19 with digestion and ligation.…”