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Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.

Pang XY, Cui WM, Liu L, Zhang SW, Lv JP - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Agro-Food Processing and Quality Control, Institute of Agro-Food Science and Technology, Chinese Academy of Agricultural Science, Beijing, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus

Light microscopic observation of different cellular morphology of Ljj-6 and engineering bacteria.A: L. bulgaricus Ljj-6 (wild type); B: L. bulgaricus K12(pUC19-mur::EryBII); C: L. bulgaricus O3(pMG76e-mur). Magnification: ×1000.
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pone-0104829-g009: Light microscopic observation of different cellular morphology of Ljj-6 and engineering bacteria.A: L. bulgaricus Ljj-6 (wild type); B: L. bulgaricus K12(pUC19-mur::EryBII); C: L. bulgaricus O3(pMG76e-mur). Magnification: ×1000.

Mentions: Autolysis differences of lactic acid bacteria can result in significantly different variation on cell morphology. As shown in Figure 9, mycelial morphology showed a uniform rod and the boundary was clear between the cells after the wild type L. bulgaricus was cultured for 16 h in MRS broth at 37°C. However, for the mur knockout strain K12, cells were connected and became filamentous, and the boundary was blurred between the cells. For the mur overexpressed strain O3, the growth speed of cells was significantly slower, and the size and density of cells were smaller than wild type cells.


Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.

Pang XY, Cui WM, Liu L, Zhang SW, Lv JP - PLoS ONE (2014)

Light microscopic observation of different cellular morphology of Ljj-6 and engineering bacteria.A: L. bulgaricus Ljj-6 (wild type); B: L. bulgaricus K12(pUC19-mur::EryBII); C: L. bulgaricus O3(pMG76e-mur). Magnification: ×1000.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4128740&req=5

pone-0104829-g009: Light microscopic observation of different cellular morphology of Ljj-6 and engineering bacteria.A: L. bulgaricus Ljj-6 (wild type); B: L. bulgaricus K12(pUC19-mur::EryBII); C: L. bulgaricus O3(pMG76e-mur). Magnification: ×1000.
Mentions: Autolysis differences of lactic acid bacteria can result in significantly different variation on cell morphology. As shown in Figure 9, mycelial morphology showed a uniform rod and the boundary was clear between the cells after the wild type L. bulgaricus was cultured for 16 h in MRS broth at 37°C. However, for the mur knockout strain K12, cells were connected and became filamentous, and the boundary was blurred between the cells. For the mur overexpressed strain O3, the growth speed of cells was significantly slower, and the size and density of cells were smaller than wild type cells.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Agro-Food Processing and Quality Control, Institute of Agro-Food Science and Technology, Chinese Academy of Agricultural Science, Beijing, P.R. China.

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.

No MeSH data available.


Related in: MedlinePlus