Novel shuttle vector pGMβ1 for conjugative chromosomal manipulation of Lactobacillus delbrueckii subsp. bulgaricus.

Content Provider	Europe PMC
Author	IWAMOTO, Daiki ISHIZAKI, Megumi MIURA, Taiki SASAKI, Yasuko
Copyright Year	2022
Abstract	Lactobacillus delbrueckii subsp. bulgaricus (L.bulgaricus) is widely used as a starter for yogurt and cheese worldwide.Despite the economic importance of this bacterium in the dairy industry, there have beenfew genetic studies involving knockout or overexpression mutants to identify the functionsof L. bulgaricus genes. One of the main reasons for this gap is the lowtransformation efficiency of available L. bulgaricus chromosome-integrating vectors upon performing conventional electroporation. We previouslyproposed the conjugal plasmid pAMβ1 as an integration vector for L.bulgaricus, as conjugation could avert the need for a restriction modificationsystem; pAMβ1 does not replicate and integrate into the chromosome of L.bulgaricus. Here, we describe an effective chromosomal manipulation systeminvolving a novel shuttle vector pGMβ1, which could improve the operability of the broadhost-range conjugal plasmid pAMβ1. We further developed an enhanced filter-mating methodfor conjugation. To validate this system, the effectiveness of conversion of the lactatedehydrogenase gene D-ldh of L. bulgaricus to theL-ldh form of Streptococcus thermophilus was examined.As pGMβ1 and pAMβ1 are unable to replicate in L. delbrueckii subsp.delbrueckii, they were chromosomally integrated. However, theseplasmids could replicate in L. delbrueckii subsp.indicus and sunkii. This integration system couldunearth important gene functions in L. bulgaricus and thus improve itsapplications in the dairy industry. Moreover, this conjugation system could be used as astable vector for the transformation of long cluster genes in several species of lacticacid bacteria.
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ISSN	21866953
Journal	Bioscience of Microbiota, Food and Health [Biosci Microbiota Food Health]
Volume Number	41
DOI	10.12938/bmfh.2021-014
PubMed Central reference number	PMC8727053
Issue Number	1
PubMed reference number	35036250
e-ISSN	21863342
Language	English
Publisher	BMFH Press
Publisher Date	2021-10-18
Access Restriction	Open
Rights License	This is an open-access article distributed under the terms of the CreativeCommons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0:https://creativecommons.org/licenses/by-nc-nd/4.0/) ©2022 BMFH Press
Subject Keyword	Lactobacillus delbrueckii subsp. bulgaricus recalcitrant strains chromosomal manipulation conjugal shuttle vector lactic acid bacteria
Content Type	Text
Resource Type	Article
Subject	Gastroenterology Immunology Microbiology Food Science Applied Microbiology and Biotechnology