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

AbstractThere 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 protein therapeutics. We previously reported the discovery of plantacyclin B21AG, a novel 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 the WCFS1 (pTRKH2) indicator strain, indicating functional expression of plantacyclin B21AG. 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 may result in similar inhibitory zones observed in both wild type and recombinant hosts despite the low copy number of pCycB21.

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Differential expression of Zymomonas mobilis sucrase genes (sacB and sacC) in Escherichia coli and sucrase mutants of Zymomonas mobilis

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89132004000300001 ◽

2004 ◽

Vol 47 (3) ◽

pp. 329-338 ◽

Cited By ~ 2

Author(s):

Sangiliyandi Gurunathan ◽

Paramasamy Gunasekaran

Keyword(s):

Copy Number ◽

Zymomonas Mobilis ◽

Northern Blot Analysis ◽

Shuttle Vector ◽

Transcript Levels ◽

E Coli ◽

Low Copy Number ◽

Genes Encoding ◽

Sacb Gene ◽

In Cells

The sacB and sacC genes encoding levansucrase and extracellular sucrase respectively were independently subcloned in pBluescript (high copy number) and in Z. mobilis-E. coli shuttle vector, pZA22 (low copy number). The expression of these genes were compared under identical background of E. coli and Z. mobilis host. The level of sacB gene expression in E. coli was almost ten fold less than the expression of sacC gene, irrespective of the growth medium or the host strain. In Z. mobilis the expression of sacB and sacC genes was shown to be subject to carbon source dependent regulation. The transcript of sacB and sacC was three fold higher in cells grown on sucrose than in cells grown on glucose/fructose. Northern blot analysis revealed that the transcript levels of sacC was approximately 2-3 times higher than that of sacB. These results suggested that the expression of sacC gene was more pronounced than sacB.

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

PLoS ONE ◽

10.1371/journal.pone.0232806 ◽

2020 ◽

Vol 15 (8) ◽

pp. e0232806

Author(s):

Mian Chee Gor ◽

Aida Golneshin ◽

Thi Thu Hao Van ◽

Robert J. Moore ◽

Andrew T. Smith

Keyword(s):

Lactobacillus Plantarum ◽

Functional Expression ◽

Food Grade ◽

Probiotic Lactobacillus ◽

Circular Bacteriocin

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Plantaricyclin A, a Novel Circular Bacteriocin Produced by Lactobacillus plantarum NI326: Purification, Characterization, and Heterologous Production

Applied and Environmental Microbiology ◽

10.1128/aem.01801-17 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 24

Author(s):

Juan Borrero ◽

Eoin Kelly ◽

Paula M. O'Connor ◽

Philip Kelleher ◽

Colm Scully ◽

...

Keyword(s):

Amino Acid ◽

Antimicrobial Peptide ◽

Gene Cluster ◽

Lactobacillus Plantarum ◽

Leader Peptide ◽

Alicyclobacillus Acidoterrestris ◽

Content Type ◽

Circular Bacteriocin ◽

Spoilage Bacterium

ABSTRACTBacteriocins from lactic acid bacteria (LAB) are of increasing interest in recent years due to their potential as natural preservatives against food and beverage spoilage microorganisms. In a screening study for LAB, we isolated from olives a strain,Lactobacillus plantarumNI326, with activity against the beverage-spoilage bacteriumAlicyclobacillus acidoterrestris. Genome sequencing of NI326 enabled the identification of a gene cluster (designatedplc) encoding a putative circular bacteriocin and proteins involved in its modification, transport, and immunity. This novel bacteriocin, named plantaricyclin A (PlcA), was grouped into the circular bacteriocin subgroup II due to its high degree of similarity with other gassericin A-like bacteriocins. Purification of PlcA from the supernatant ofLb. plantarumNI326 resulted in an active peptide with a molecular mass of 5,570 Da, corresponding to that predicted from the (processed) PlcA amino acid sequence. Theplcgene cluster was cloned and expressed inLactococcus lactisNZ9000, resulting in the production of an active 5,570-Da bacteriocin in the supernatant. PlcA is believed to be produced as a 91-amino-acid precursor with a 33-amino-acid leader peptide, which is predicted to be removed, followed by joining of the N and C termini via a covalent linkage to form the mature 58-amino-acid circular bacteriocin PlcA. We report the characterization of a circular bacteriocin produced byLb. plantarum. The inhibition displayed againstA. acidoterrestrishighlights its potential use as a preservative in food and beverages.IMPORTANCEIn this work, we describe the purification and characterization of an antimicrobial peptide, termed plantaricyclin A (PlcA), produced by aLactobacillus plantarumstrain isolated from olives. This peptide has a circular structure, and all genes involved in its production, circularization, and secretion were identified. PlcA shows antimicrobial activity against different strains, includingAlicyclobacillus acidoterrestris, a common spoilage bacterium, which causes substantial economic losses in the beverage industry every year. In this study, we describe a circular antimicrobial peptide, PlcA, for aLactobacillus plantarumstrain.

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Low Copy Number of the Salivary Amylase Gene (AMY1) Is Associated with Obesity, Dyslipidemia, and Chronic Low-Grade Inflammation but Not Insulin Sensitivity and Secretion

Diabetes ◽

10.2337/db18-58-lb ◽

2018 ◽

Vol 67 (Supplement 1) ◽

pp. 58-LB

Author(s):

BARBORA DE COURTEN ◽

AYA MOUSA ◽

NEGAR NADERPOOR

Keyword(s):

Insulin Sensitivity ◽

Copy Number ◽

Low Grade ◽

Amylase Gene ◽

Salivary Amylase ◽

Low Copy Number ◽

Low Grade Inflammation

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Faculty Opinions recommendation of Counting low-copy number proteins in a single cell.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1059023.512628 ◽

2007 ◽

Author(s):

Russ Hille

Keyword(s):

Single Cell ◽

Copy Number ◽

Low Copy Number

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Infection with antiretroviral-susceptible HIV in an individual adherent to pre-exposure prophylaxis: strategies for treatment initiation

International Journal of STD & AIDS ◽

10.1177/0956462420971125 ◽

2021 ◽

pp. 095646242097112

Author(s):

Jessica M Hughes ◽

Darrell HS Tan ◽

Peter Anderson ◽

Janani Bodhinayake ◽

Paul A MacPherson

Keyword(s):

Viral Load ◽

Copy Number ◽

Case Reports ◽

Pharmacy Records ◽

Treatment Regimens ◽

Viral Copy Number ◽

Low Copy Number ◽

Viral Copy ◽

Exposure Prophylaxis ◽

Tenofovir Diphosphate

HIV pre-exposure prophylaxis (PrEP) is effective at preventing sexual acquisition of HIV, and failures in clinical trials are largely attributable to medication nonadherence. We report here a case of infection with a fully susceptible strain of HIV in an individual adherent to PrEP as demonstrated by pharmacy records and intracellular tenofovir diphosphate levels. At diagnosis, the viral load was 90 copies/mL precluding initial genotype testing due to low copy number. While PrEP failure is rare, this case underscores the importance of regular HIV testing for patient on PrEP and prompts discussion regarding the approach to treatment following failure where an initial genotype is not yet available or not possible due to low viral load. Few other case reports of PrEP failure exist in the literature and approaches to treatment varied widely. We suggest the initial viral copy number may guide next steps and discuss the risks and benefits of stopping PrEP, escalating therapy with integrase inhibitors or boosted protease inhibitors, or switching to non-nucleoside antiretroviral treatment regimens.

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