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Genome Analysis of Lactobacillus plantarum LL441 and Genetic Characterisation of the Locus for the Lantibiotic Plantaricin C.

ABSTRACT: Bacteriocins are ribosomally synthesized peptides produced by bacteria with antimicrobial activity. The bacteriocins produced by lactic acid bacteria (LAB) may inhibit food-borne pathogens and spoilage organisms, and therefore have potential as natural preservatives. Lactobacillus plantarum LL441 produces a lantibiotic bacteriocin known as plantaricin C, a pore-forming antimicrobial peptide containing modified amino acids that inhibits cell wall synthesis by forming a complex with the peptidoglycan precursor lipid II. The present work describes the genome sequencing of L. plantarum LL441 and the characterisation of the plantaricin C locus. The draft genome sequence of L. plantarum LL441 consisted of 170 contigs and had a total 3,124,603 bp; the GC content was 44.52%. The plantaricin C locus was found in an 18 kbp-long contig, and consisted of six genes organized in an operon-like arrangement. This locus included the bacteriocin structural gene (plnC), followed by a gene encoding a LanM-like protein thought to be involved in the maturation of plantaricin C, and four downstream genes encoding ABC-type transporter components, probably belonging to its putative immunity and export machinery. plnC encodes a precursor of the bacteriocin, i.e., a 58-amino acid peptide containing a 31-amino acid double-glycine leader peptide and a 27-amino acid core peptide. In silico analysis and hybridisation experiments placed the plantaricin C locus to be located on pLL441-1, a large plasmid of L. plantarum LL441. Joining up the gaps between the contigs by conventional PCR, sequencing of the amplicons, and sequence assemblage, allowed the complete 55.3 kbp pLL441-1 molecule to be established. A portion of pLL441-1 larger than 34 kbp, which included the plantaricin C region, was identified in a plasmid-derived contig from the L. plantarum Nizo 3893 genome. Further, the plantaricin C coding locus (about 8.7 kbp) was shown to share 91% nucleotide identity with a portion of the plasmids pPECL-6 from Pediococcus claussenii ATCC BAA-344 and pL11995-4 from Lactobacillus paracollinoides TMW 1.1995. Knowledge of the sequence of the plantaricin C coding region will help in studying its molecular components and allow their involvement in bacteriocin synthesis to be investigated, facilitating the use of the bacteriocin or its genetic elements in new biotechnological applications.

SUBMITTER: Florez AB

PROVIDER: S-EPMC6107846 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundWe evaluated the functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit and to compete among the gut microbiota in vivo. Considering the probiotic potential of Lb. brevis SF9B, this study aims to investigate the antibacterial activity of Lb. plantarum SF9C and their potential for in vivo colonisation in rats, which could be the basis for the investigation of their synergistic functionality.ResultsA plantaricin-encoding cluster was identified in Lb. plantarum SF9C, a strain which efficiently inhibited the growth of Listeria monocytogenes ATCC® 19111™ and Staphylococcus aureus 3048. Homology-based three-dimensional (3D) structures of SF9C plantaricins PlnJK and PlnEF were predicted using SWISS-MODEL workspace and the helical wheel representations of the plantaricin peptide helices were generated by HELIQUEST. Contrary to the plantaricin-producing SF9C strain, the S-layer-carrying SF9B strain excluded Escherichia coli 3014 and Salmonella enterica serovar Typhimurium FP1 from the adhesion to Caco-2 cells. Finally, PCR-DGGE analysis of the V2-V3 regions of the 16S rRNA gene confirmed the transit of the two selected lactobacilli through the gastrointestinal tract (GIT). Microbiome profiling via the Illumina MiSeq platform revealed the prevalence of Lactobacillus spp. in the gut microbiota of the Lactobacillus-treated rats, even on the 10th day after the Lactobacillus application, compared to the microbiota of the healthy and AlCl3-exposed rats before Lactobacillus treatment.ConclusionThe combined application of Lb. plantarum SF9C and Lb. brevis SF9B was able to influence the intestinal microbiota composition in rats, which was reflected in the increased abundance of Lactobacillus genus, but also in the altered abundances of other bacterial genera, either in the model of healthy or aberrant gut microbiota of rats. The antibacterial activity and capacity to withstand in GIT conditions contributed to the functional aspects of SF9C and SF9B strains that could be incorporated in the probiotic-containing functional foods with a possibility to positively modulate the gut microbiota composition.

Project description:BACKGROUND: Lactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for in situ delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality. RESULTS: We have constructed a library of 76 Sec-type signal peptides from L. plantarum WCFS1 that were predicted to be cleaved by signal peptidase I. SP functionality was studied using staphylococcal nuclease (NucA) as a reporter protein. 82% of the SPs gave significant extracellular NucA activity. Levels of secreted NucA varied by a dramatic 1800-fold and this variation was shown not to be the result of different mRNA levels. For the best-performing SPs all produced NucA was detected in the culture supernatant, but the secretion efficiency decreased for the less well performing SPs. Sequence analyses of the SPs and their cognate proteins revealed four properties that correlated positively with SP performance for NucA: high hydrophobicity, the presence of a transmembrane helix predicted by TMHMM, the absence of an anchoring motif in the cognate protein, and the length of the H+C domain. Analysis of a subset of SPs with a lactobacillal amylase (AmyA) showed large variation in production levels and secretion efficiencies. Importantly, there was no correlation between SP performance with NucA and the performance with AmyA. CONCLUSION: This is the first comprehensive experimental study showing that predicted SPs in the L. plantarum genome actually are capable of driving protein secretion. The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted. Several SPs stand out as promising candidates for efficient secretion of heterologous proteins in L. plantarum. The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult. The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.

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Genome Analysis of Lactobacillus plantarum LL441 and Genetic Characterisation of the Locus for the Lantibiotic Plantaricin C.

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