Dataset Information

Occurrence and Diversity of CRISPR Loci in Lactobacillus casei Group.

ABSTRACT: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system that resists foreign genes through nuclease targeting in bacteria and archaea. In this study, we analyzed 68 strains of Lactobacillus casei group from the NCBI GenBank database, and bioinformatic tools were used to investigate the occurrence and diversity of CRISPR system. The results showed that a total of 30 CRISPR loci were identified from 27 strains. Apart from three strains which contained double loci with distinguishable distributed sites, most strains contained only one CRISPR locus. The analysis of direct repeat (DR) sequences showed that all DR could form stable RNA secondary structures. The CRISPR spacers showed diversity, and their origin and evolution were revealed through the investigation of their spacer sequences. In addition, a large number of CRISPR spacers showed perfect homologies to phage and plasmid sequences. Collectively, our results would contribute to researches of resistance in L. casei group, and also provide a new vision on the diversity and evolution of CRISPR/Cas system.

SUBMITTER: Yang L

PROVIDER: S-EPMC7156538 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Occurrence and Diversity of CRISPR Loci in <i>Lactobacillus casei</i> Group.

Yang Lan L Li Weixun W Ujiroghene Obaroakpo Joy OJ Yang Yang Y Lu Jing J Zhang Shuwen S Pang Xiaoyang X Lv Jiaping J

Frontiers in microbiology 20200408

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system that resists foreign genes through nuclease targeting in bacteria and archaea. In this study, we analyzed 68 strains of <i>Lactobacillus casei</i> group from the NCBI GenBank database, and bioinformatic tools were used to investigate the occurrence and diversity of CRISPR system. The results showed that a total of 30 CRISPR loci were identified from 27 strains. Apart from three strains which contained ...[more]

PMID: 32322250

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Project description:Lactobacillus casei 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 L. casei 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 L. casei genome engineering based on CRISPR-Cas9D10A In addition to the P23-Cas9D10A and Pldh-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 LC2W_1628 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 L. casei, and its potential application in other lactic acid bacteria (LAB) is also discussed in this study.IMPORTANCE The lack of efficient genetic tools has limited the investigation and biotechnological application of many LAB. The CRISPR-Cas9D10A nickase-based genome editing in Lactobacillus casei, 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 L. casei 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 L. casei As a broad-host-range plasmid, pLCNICK has the potential to be adapted to other Lactobacillus species for genome editing.

Project description:Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus are phenotypically and genotypically closely related, and together comprise the L. casei group. Although the strains of this group are commercially valuable as probiotics, the taxonomic status and nomenclature of the L. casei group have long been contentious because of the difficulties in identifying these three species by using the most frequently used genotypic methodology of 16S rRNA gene sequencing. Long used as the gold standard for species classification, DNA-DNA hybridization is laborious, requires expert skills, and is difficult to use routinely in laboratories. Currently, genome-based comparisons, including average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), are commonly applied to bacterial taxonomy as alternatives to the gold standard method for the demarcating phylogenetic relationships. To establish quick and accurate methods for identifying strains in the L. casei group at the species and subspecies levels, we developed species- and subspecies-specific identification methods based on housekeeping gene sequences and whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral pattern analysis. By phylogenetic analysis based on concatenated housekeeping gene sequences (dnaJ, dnaK, mutL, pheS, and yycH), 53 strains were separated into four clusters corresponding to the four species: L. casei, L. paracasei and L. rhamnosus, and Lactobacillus chiayiensis sp. nov. A multiplex minisequencing assay using single nucleotide polymorphism (SNP)-specific primers based on the dnaK gene sequences and species-specific primers based on the mutL gene sequences provided high resolution that enabled the strains at the species level to be identified as L. casei, L. paracasei, and L. rhamnosus. By MALDI-TOF MS analysis coupled with an internal database and ClinProTools software, species- and subspecies-level L. casei group strains were identified based on reliable scores and species- and subspecies-specific MS peaks. The L. paracasei strains were distinguished clearly at the subspecies level based on subspecies-specific MS peaks. This article describes the rapid and accurate methods used for identification and classification of strains in the L. casei group based on housekeeping gene sequences and MALDI-TOF MS analysis as well as the novel speciation of this group including L. chiayiensis sp. nov. and 'Lactobacillus zeae' by genome-based methods.

Dataset Information

Occurrence and Diversity of CRISPR Loci in Lactobacillus casei Group.

Publications

Occurrence and Diversity of CRISPR Loci in <i>Lactobacillus casei</i> Group.

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