Transposition in Lactobacillus delbrueckii subsp. bulgaricus: identification of two thermosensitive replicons and two functional insertion sequences

Serror, Pascale; Ilami, Golnar; Chouayekh, Hichem; Ehrlich, S. Dusko; Maguin, Emmanuelle

doi:10.1099/mic.0.25827-0

Cited by 12 publications

(7 citation statements)

References 54 publications

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“…For some lactobacilli, poor transformation efficiency is a genetic barrier if a nonreplicative integration plasmid has to be employed (33). Even though poor transformation can be addressed by using conditionally replicating vectors, the process of single crossover and subsequent double crossover selection is still cumbersome and time-consuming (34)(35)(36). Prophage recombinaseassisted double-stranded DNA (dsDNA) recombineering can be used for both deletion and insertion of large DNA fragments effectively.…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 ^D10A Nickase-Assisted Genome Editing in Lactobacillus casei

Song

Huang

Xiong

et al. 2017

Appl Environ Microbiol

149

125

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has drawn increasing attention as a health-promoting probiotic, while effective genetic manipulation tools are often not available, e.g., the single-gene knockout in still depends on the classic homologous recombination-dependent double-crossover strategy, which is quite labor-intensive and time-consuming. In the present study, a rapid and precise genome editing plasmid, pLCNICK, was established for genome engineering based on CRISPR-Cas9 In addition to the P-Cas9 and P-sgRNA (single guide RNA) expression cassettes, pLCNICK includes the homologous arms of the target gene as repair templates. The ability and efficiency of chromosomal engineering using pLCNICK were evaluated by in-frame deletions of four independent genes and chromosomal insertion of an enhanced green fluorescent protein (eGFP) expression cassette at the locus. The efficiencies associated with in-frame deletions and chromosomal insertion is 25 to 62%. pLCNICK has been proved to be an effective, rapid, and precise tool for genome editing in, and its potential application in other lactic acid bacteria (LAB) is also discussed in this study. The lack of efficient genetic tools has limited the investigation and biotechnological application of many LAB. The CRISPR-Cas9 nickase-based genome editing in , an important food industrial microorganism, was demonstrated in this study. This genetic tool allows efficient single-gene deletion and insertion to be accomplished by one-step transformation, and the cycle time is reduced to 9 days. It facilitates a rapid and precise chromosomal manipulation in and overcomes some limitations of previous methods. This editing system can serve as a basic technological platform and offers the possibility to start a comprehensive investigation on As a broad-host-range plasmid, pLCNICK has the potential to be adapted to other species for genome editing.

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Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 ^D10A Nickase-Assisted Genome Editing in Lactobacillus casei

Song

Huang

Xiong

et al. 2017

Appl Environ Microbiol

149

125

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“…The primary cause for the limited genetic work on Lb. bulgaricus is the lack of effective and reproducible methods for chromosome manipulation [5].…”

Section: Introductionmentioning

confidence: 99%

Novel shuttle vector pGMβ1 for conjugative chromosomal manipulation of <i>Lactobacillus delbrueckii</i> subsp. <i>bulgaricus</i>

Iwamoto

Ishizaki

Miura

et al. 2022

Bioscience of Microbiota, Food and Health

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Lactobacillus delbrueckii subsp. bulgaricus (Lb. 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 been few genetic studies involving knockout or overexpression mutants to identify the functions of Lb. bulgaricus genes. One of the main reasons for this gap is the low transformation efficiency of available Lb. bulgaricus chromosome-integrating vectors upon performing conventional electroporation. We previously proposed the conjugal plasmid pAMβ1 as an integration vector for Lb. bulgaricus, as conjugation could avert the need for a restriction 2 modification system; pAMβ1 does not replicate and integrate into the chromosome of Lb. bulgaricus. Here, we describe an effective chromosomal manipulation system involving a novel shuttle vector pGMβ1, which could improve the operability of the broad host-range conjugal plasmid pAMβ1. We further developed an enhanced filter-mating method for conjugation. To validate this system, the effectiveness of conversion of the lactate dehydrogenase gene D-ldh of Lb. bulgaricus to the L-ldh form of Streptococcus thermophilus was examined. As pGMβ1 and pAMβ1 are unable to replicate in Lb. delbrueckii subsp. delbrueckii, they were chromosomally integrated.However, these plasmids could replicate in Lb. delbrueckii subsp. indicus and sunkii.This integration system could unearth important gene functions in Lb. bulgaricus and thus improve its applications in the dairy industry. Moreover, this conjugation system could be used as a stable vector for the transformation of long cluster genes in several species of lactic acid bacteria.

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“…The discovery of functional IS-elements in lactobacilli should aid in the development of functional mutagenesis and insertional vectors for a variety of intestinal lactobacilli. The insertion of IS1223 into pSA3 (157), and IS1223 or IS1201 into pIP501 (158) exemplify the successful usage of IS elements for random mutagenesis in lactobacilli. The addition of the lactococcal ISS1 to the thermosensitive replicon pG + host allowed construction of a food grade random mutagenesis system which has been employed for generating stable mutants in L. plantarum (159) and other gram-positive bacteria (160).…”

Section: Random Mutagenesis Systemsmentioning

confidence: 99%

Genetic tools for investigating the biology of commensal lactobacilli

Fang

O’Toole²

2009

Front Biosci

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Lactobacilli belong to the genus Lactobacillus, the largest genus among the lactic acid bacteria (LAB). They are abundant in plant material and food resources, or they may inhabit niches in or on the bodies of humans and animals, as commensals. Lactobacilli of food origin are commercially important in the production of dairy products, fermented meats, vegetables, and sourdough, and many of their properties have been well studied. Commensal lactobacilli are good candidates for development as probiotics. In recent years, the general biology and host interaction mechanisms of commensal lactobacilli have attracted great interest. Although the metabolic pathways, predicted gene functions, and some phenotypic traits, of commensal lactobacilli can be inferred or deduced to an extent by the growing number of Lactobacillus genome sequencing project, various genetic tools are still required to confirm their phenotypic properties and biological traits. The current state of the art with respect to the available complement of genetic tools including genomic resources, and more traditional approaches to investigate the biology of commensal lactobacilli, will now be reviewed

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Transposition in Lactobacillus delbrueckii subsp. bulgaricus: identification of two thermosensitive replicons and two functional insertion sequences

Cited by 12 publications

References 54 publications

CRISPR-Cas9 ^D10A Nickase-Assisted Genome Editing in Lactobacillus casei

CRISPR-Cas9 ^D10A Nickase-Assisted Genome Editing in Lactobacillus casei

Novel shuttle vector pGMβ1 for conjugative chromosomal manipulation of <i>Lactobacillus delbrueckii</i> subsp. <i>bulgaricus</i>

Genetic tools for investigating the biology of commensal lactobacilli

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Transposition in Lactobacillus delbrueckii subsp. bulgaricus: identification of two thermosensitive replicons and two functional insertion sequences

Cited by 12 publications

References 54 publications

CRISPR-Cas9 D10A Nickase-Assisted Genome Editing in Lactobacillus casei

CRISPR-Cas9 D10A Nickase-Assisted Genome Editing in Lactobacillus casei

Novel shuttle vector pGMβ1 for conjugative chromosomal manipulation of <i>Lactobacillus delbrueckii</i> subsp. <i>bulgaricus</i>

Genetic tools for investigating the biology of commensal lactobacilli

Contact Info

Product

Resources

About

CRISPR-Cas9 ^D10A Nickase-Assisted Genome Editing in Lactobacillus casei

CRISPR-Cas9 ^D10A Nickase-Assisted Genome Editing in Lactobacillus casei