Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in
            <i>Escherichia coli</i>

Richard, Christelle; Drider, Djamel; Elmorjani, Khalil; Marion, Didier; Prévost, Hervé

doi:10.1128/jb.186.13.4276-4284.2004

Cited by 71 publications

(58 citation statements)

References 67 publications

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“…In this particular case of extensively modified thurincin H, the sulfur-to-␣-carbon bonds were very unique and to date are reported to exist in only three other bacterial peptides (24)(25)(26). These bonds are not reported in proteins expressed by commonly used expression hosts, such as E. coli (27) or lactic acid bacteria (11), rendering the deletion mutant a logical host for expression of site-directed mutants. The pGW131 complementation experiments indicated that the thurincin H prepeptide translated from the plasmids was correctly modified by the chromosomally encoded radical SAM enzyme, processed, and exported to the extracellular environment.…”

Section: Discussionmentioning

confidence: 97%

“…In homologous expression of less extensively modified bacteriocins, only the mature bacteriocin-encoding sequence was incorporated into the expression vector (9,27). However, the leader peptide of the thurincin H precursor was included in our con- structs, since the leader peptide of the bacteriocins can play essential roles in the formation of lanthionine/methyllanthionine in lantibiotics, as well as keep the bacteriocin prepeptide in an inactive state and facilitate exportation (31).…”

Section: Discussionmentioning

confidence: 99%

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Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361

Wang

Manns

Churey

et al. 2014

Appl Environ Microbiol

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Thurincin H is an antimicrobial peptide produced by Bacillus thuringiensis SF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-␣-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1, thnA2, and thnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designated B. thuringiensis SF361 ⌬thnA1 ⌬thnA2 ⌬thnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter, thnA1, and a Cry protein terminator into the Escherichia coli-B. thuringiensis shuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in the thnA1 gene, were generated and separately transformed into B. thuringiensis SF361 ⌬thnA1 ⌬thnA2 ⌬thnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities of B. thuringiensis SF361 ⌬thnA1 ⌬thnA2 ⌬thnA3 carrying different pGW133 variants against three different indicator strains were subsequently compared. Bacteriocins are ribosomally synthesized, antimicrobial peptides or proteins produced by bacteria that usually have a narrow inhibitory spectrum, although notable exceptions exist (1). Bacteriocins produced by lactic acid bacteria (LAB) have been extensively studied and used as natural food preservatives. Due to the fact that LAB have a generally recognized as safe (GRAS) status, they have been investigated as potential agents for preventing spoilage and enhancing the safety of foods. The LAB bacteriocins can be divided into three main classes: the class I lantibiotics, containing a lanthionine thioether bond linking the sulfur atom of cysteines with the ␤ carbon of other amino acids; the class II nonlanthionine-containing bacteriocins; and the class III heat-labile large proteins (2). In recent years, bacteriocins produced by Bacillus spp. have gained increasing research interest since many of them exhibit a broader antimicrobial spectrum than most lactic acid bacteriocins, and potential applications in the food, agricultural, and pharmaceutical industries in controlling various spoilage and pathogenic microorganisms are anticipated (3, 4). One Bacillus bacteriocin, thurincin H, is an antimicrobial peptide produced by Bacillus thuringiensis SF361, a strain originally isolated from U.S. domestic sunflower honey. I...

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361

Wang

Manns

Churey

et al. 2014

Appl Environ Microbiol

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“…As the roles of the disulfide bonds, of the consensus sequence YGNGVXCX 4 C, and of the positively charged residues mainly located in the N-terminal domain have been studied previously, here we designed variants with single amino acid replacements located in the central and C-terminal parts of the molecule, using a genetic approach which previously has been proven to be efficient (47). We also took advantage of our knowledge of the production of DvnRV41, a recombinant form of this two-disulfide-bond class IIa bacteriocin which retained the antibacterial activity of the natural bacteriocin (44,60), in order to develop an efficient production system for the desired variants. In order to determine the precise role of the amino acid side chains, each selected amino acid was replaced by another amino acid belonging to a similar polarity group, in agreement with the amino acid Venn diagram (51).…”

Section: Discussionmentioning

confidence: 99%

Insights into Structure-Activity Relationships in the C-Terminal Region of Divercin V41, a Class IIa Bacteriocin with High-Level Antilisterial Activity

Rihakova

Petit

Demnerova

et al. 2009

Appl Environ Microbiol

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Divercin V41 (DvnV41) is a class IIa bacteriocin with potent antilisterial activity isolated from Carnobacterium divergens V41. Previously, we expressed from a synthetic gene, in Escherichia coli Origami, a recombinant DvnV41 designated DvnRV41, which possesses four additional amino acids (AMDP) in the N-terminal region that result from enzymatic cleavage and retains the initial DvnV41 activity. To unravel the relationship between the structure of DvnRV41 and its particularly elevated activity, we produced by site-directed mutagenesis eight variants in which a single amino acid replacement was specifically introduced into the sequence. The point mutations were designed to change either conserved residues in class IIa bacteriocins or residues specific to DvnV41 located mainly in the C-terminal region. The fusion proteins were purified from the cytosoluble fractions by immobilized affinity chromatography. DvnRV41 and its variants were released from the fusion proteins by enzymatic cleavage, using enterokinase. The purity of DvnRV41 and of the variants was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance liquid chromatography, and mass spectrometry. The antibacterial activity of DvnRV41 and its variants was assessed using different indicator strains, including Listeria monocytogenes EGDe and Enterococcus faecalis JH2-2. The activity of all of the variants appeared to be less than the activity of DvnRV41. The decrease in activity did not appear to be related to a global conformational change, as determined by circular dichroism. Overall, the variants of DvnRV41 produced in the present study provide interesting insights into structure-activity relationships of class IIa bacteriocins.Lactic acid bacterium (LAB) bacteriocins are ribosomally synthesized antimicrobial peptides that could be useful as natural and nontoxic food preservatives. Most bacteriocins produced by LAB are known to be active only against grampositive bacteria. A few exceptions to this general rule, including bacteriocins from Lactobacillus species (5, 31, 35, 49, 52), AS-48 from Enterococcus faecalis (1), enterocin E50-52 from Enterococcus faecium (50), and thermophylin produced by Streptococcus thermophilus (26), have been described; these bacteriocins exhibit various degrees of activity against gram-negative bacteria.A classification of bacteriocins produced by LAB was first proposed by Klaenhammer (30) and then was modified by Nes and Holo (39) and further updated by Cotter et al. (9). Class I and class II bacteriocins are the most abundant and thoroughly studied bacteriocins. Class I bacteriocins, namely lantibiotics, have been widely studied, and one of them, nisin, is routinely used by the food industry as a preservative due to its high level of inhibitory activity against a wide range of bacterial pathogens. These bacteriocins are characterized by the presence in their primary structure of posttranslationally modified amino acid residues (lanthionine and methylanthionine). Bacteriocin class II is composed...

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“…Another interesting system allowing large-scale production of pure recombinant piscicolin 126 at 26 mg/liter from a native DNA was reported with E. coli AD494(DE3) (72). Divercin V41 and piscicolin 126 have been expressed as bacteriocinthioredoxin chimeric soluble proteins requiring, respectively, enzymatic or chemical treatment to release the recombinant bacteriocin (72,155). Importantly, both systems provide ease of purification and a high yield of bacteriocins with correct disulfide bond formation.…”

Section: Purification and Heterologous Production Of Class Iia Bactermentioning

confidence: 99%

The Continuing Story of Class IIa Bacteriocins

Drider

Fimland

Héchard

et al. 2006

Microbiol Mol Biol Rev

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646

543

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SUMMARY Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.

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Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in Escherichia coli

Cited by 71 publications

References 67 publications

Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361

Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361

Insights into Structure-Activity Relationships in the C-Terminal Region of Divercin V41, a Class IIa Bacteriocin with High-Level Antilisterial Activity

The Continuing Story of Class IIa Bacteriocins

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