Discovery and characterisation of circular bacteriocin plantacyclin B21AG from Lactiplantibacillus plantarum B21.

Lactiplantibacillus plantarum B21 isolated from Vietnamese sausage (nem chua) has previously displayed broad antimicrobial activity against Gram-positive bacteria including foodborne pathogens Listeria monocytogenes and Clostridium perfringens. This study successfully identified the antimicrobial agent as plantacyclin B21AG, a 5668 Da circular bacteriocin demonstrating high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. We report a reverse genetics approach to identify and characterise plantacyclin B21AG from first principles. The bacteriocin was purified from culture supernatant by a three-step process consisting of concentration, n-butanol extraction and cation exchange chromatography. A de novo peptide sequencing using LC-MS/MS techniques identified two putative peptide fragments which were mapped to the genome sequence of L. plantarum B21. This revealed an ORF corresponding to a putative circular bacteriocin with a 33-amino acid leader peptide and a 58-amino acid mature peptide encoded on a native plasmid pB21AG01. The bacteriocin is shown to be a small cationic predominantly α-helical protein (69%). The corresponding gene cluster, consisted of seven genes associated with post-translational circularisation, immunity and secretion. Whilst plantacyclin B21AG is 86% identical to the newly published plantaricyclin A it is more highly cationic having a net charge of +3 due to an additional basic residue in the putative membrane interaction region. This and other substitutions may well go some way to explaining functional differences. The robust nature of plantacyclin B21AG, its antimicrobial activity and associated machinery for cyclisation make it an interesting biotechnological target for development, both as a food-safe antimicrobial or potentially a platform technology for recombinant protein circularisation.

Cloning and functional expression of a food-grade circular bacteriocin, plantacyclin B21AG, in probiotic Lactobacillus plantarum WCFS1.

There is an increasing consumer demand for minimally processed, preservative free and microbiologically safe food. These factors, combined with risks of antibiotic resistance, have led to interest in bacteriocins produced by lactic acid bacteria (LAB) as natural food preservatives and as potential protein therapeutics. We previously reported the discovery of plantacyclin B21AG, a circular bacteriocin produced by Lactobacillus plantarum B21. Here, we describe the cloning and functional expression of the bacteriocin gene cluster in the probiotic Lactobacillus plantarum WCFS1. Genome sequencing demonstrated that the bacteriocin is encoded on a 20 kb native plasmid, designated as pB21AG01. Seven open reading frames (ORFs) putatively involved in bacteriocin production, secretion and immunity were cloned into an E. coli/Lactobacillus shuttle vector, pTRKH2. The resulting plasmid, pCycB21, was transformed into L. plantarum WCFS1. The cell free supernatants (CFS) of both B21 and WCFS1 (pCycB21) showed an antimicrobial activity of 800 AU/mL when tested against WCFS1 (pTRKH2) as the indicator strain, showing that functional expression of plantacyclin B21AG had been achieved. Real-time PCR analysis revealed that the relative copy number of pB21AG01 was 7.60 ± 0.79 in L. plantarum B21 whilst pCycB21 and pTRKH2 was 0.51 ± 0.05 and 25.19 ± 2.68 copies respectively in WCFS1. This indicates that the bacteriocin gene cluster is located on a highly stable low copy number plasmid pB21AG01 in L. plantarum B21. Inclusion of the native promoter for the bacteriocin operon from pB21AG01 results in similar killing activity being observed in both the wild type and recombinant hosts despite the lower copy number of pCycB21.

Draft Genome Sequence of Lactobacillus plantarum Strain A6, a Strong Acid Producer Isolated from a Vietnamese Fermented Sausage (Nem Chua).

Lactobacillus plantarum strain A6, a strong acid producer, was isolated from a Vietnamese fermented sausage (nem chua). Here, we report the genome sequence of this strain (3,368,579 bp).

Exploiting the intrinsic microbial degradative potential for field-based in situ dechlorination of trichloroethene contaminated groundwater.

Bioremediation of trichloroethene (TCE) polluted groundwater is challenging, with limited next generation sequencing (NGS) derived information available on microbial community dynamics associated with dechlorination. Understanding these dynamics is important for designing and improving TCE bioremediation. In this study, biostimulation (BS), biostimulation-bioaugmentation (BS-BA) and monitored natural attenuation (MNA) approaches were applied to contaminated groundwater wells resulted in ≥ 95% dechlorination within 7 months. Vinyl chloride's final concentrations in stimulated wells were between 1.84 and 1.87 µg L(-1), below the US EPA limit of 2.0 µg L(-1), compared to MNA (4.3 µg L(-1)). Assessment of the groundwater microbial community with qPCR showed up to ∼ 50-fold increase in the classical dechlorinators' (Geobacter and Dehalococcoides sp.) population post-treatment. Metagenomic assays revealed shifts from Gammaproteobacteria (pre-treatment) to Epsilonproteobacteria and Deltaproteobacteria (post-treatment) only in stimulated wells. Although stimulated wells were functionally distinct from MNA wells post-treatment, substantial dechlorination in all the wells implied some measure of redundancy. This study, one of the few NGS-based field studies on TCE bioremediation, provides greater insights into dechlorinating microbial community dynamics which should be useful for future field-based studies.

Complete Genome Sequence of Lactobacillus plantarum Strain B21, a Bacteriocin-Producing Strain Isolated from Vietnamese Fermented Sausage Nem Chua.

Lactobacillus plantarum strain B21 was isolated from Vietnamese sausage (nem chua) and demonstrated broad antimicrobial activity due to the production of bacteriocins. Here, we report the complete genome sequence of this strain (3,284,260 bp).