Isolation of a Variant of Subtilosin A with Hemolytic Activity

Huang, Tai Huang; Geng, Hao; Miyyapuram, Venugopal R.; Sit, Clarissa S.; Vederas, John C.; Nakano, Michiko

doi:10.1128/jb.00541-09

Cited by 57 publications

(36 citation statements)

References 37 publications

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“…The operon is induced under anaerobic conditions and is controlled by the transition state regulatory protein AbrB [4]. It shows antibacterial activity against Bacillus spp., E. faecalis , Gardnerella vaginalis and Listeria monocytogenes by targeting their membranes and forming pores [71–73]. …”

Section: Resultsmentioning

confidence: 99%

Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species

2016

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BackgroundGram-positive bacteria of the Bacillales are important producers of antimicrobial compounds that might be utilized for medical, food or agricultural applications. Thanks to the wide availability of whole genome sequence data and the development of specific genome mining tools, novel antimicrobial compounds, either ribosomally- or non-ribosomally produced, of various Bacillales species can be predicted and classified. Here, we provide a classification scheme of known and putative antimicrobial compounds in the specific context of Bacillales species.ResultsWe identify and describe known and putative bacteriocins, non-ribosomally synthesized peptides (NRPs), polyketides (PKs) and other antimicrobials from 328 whole-genome sequenced strains of 57 species of Bacillales by using web based genome-mining prediction tools. We provide a classification scheme for these bacteriocins, update the findings of NRPs and PKs and investigate their characteristics and suitability for biocontrol by describing per class their genetic organization and structure. Moreover, we highlight the potential of several known and novel antimicrobials from various species of Bacillales.ConclusionsOur extended classification of antimicrobial compounds demonstrates that Bacillales provide a rich source of novel antimicrobials that can now readily be tapped experimentally, since many new gene clusters are identified.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3224-y) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species

2016

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“…Genetic engineering has been used as a tool to construct bacteriocin mutants with new features. For example, replacing the threonine at residue 6 with isoleucine in subtilosin A not only enhanced its bactericidal activity but also rendered the mutant hemolytic (36). Deferred antagonism assays were conducted to evaluate the bacteriocin activities of B. thuringiensis SF361 ⌬thnA1 ⌬thnA2 ⌬thnA3 carrying different pGW133 variants with mutated thurincin H structural genes.…”

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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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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“…The peptide consists of 35 amino acids, covalently attached by amide linkages between an aspargine residue at the N-terminus and a glycine residue at the C-terminus. An atypical inter-residue thioester bridge is formed between the sulfurs of two cysteines and the α-carbon of two phenylalanine residues, while the sulfur of a third cysteine residue is attached to the α-carbon of a threonine residue (Kawulka et al 2004;Huang et al 2009). Subtilosin has a net charge of −2 due to the presence of lysine, two aspartates, and one glutamate (Thennarasu et al 2005).…”

Section: Introductionmentioning

confidence: 98%

Subtilosin A production by Bacillus subtilis KATMIRA1933 and colony morphology are influenced by the growth medium

Nikiforova

Klykov²,

Volski

et al. 2015

Ann Microbiol

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Bacillus subtilis KATMIRA1933, cultured in modified MRS (de Man, Rogosa, and Sharpe) broth without peptone (animal-free [AF]-MRS), produced subtilosin A at levels similar to, or even higher, than when cultured in MRS broth with peptone. AF-MRS medium contained 2.5 % (w/v) Martone or 2.5 % (w/v) cottonseed hydrolysate instead of peptone and 0.25 % (w/v) bacteriological grade yeast extract instead of normal yeast extract. An increase in cell numbers, accompanied by an increase in subtilosin activity, was recorded when cells were grown in AF-MRS supplemented with 0.4 % (w/v) K 2 HPO 4 and 0.02 % (w/v) MgSO 4 . Subtilosin production increased from 30 arbitrary units (AU) mL −1 in a static culture to 320 AU mL −1 when cells were agitated on an orbital shaker at 300 rpm. A further increase in subtilosin production, from 150 AU mL −1 to 240 AU mL −1 , was recorded when cells were cultured in AF-MRS supplemented with 2.0 % (w/v) amylopectin or 2.0 % (w/v) maltodextrin. Slightly higher cell densities were recorded in the presence of maltodextrin. Two colony types, one with a flat (Btypical^), sprawling morphology and the other elevated (Batypical^), were isolated from cells grown in AF-MRS broth. Higher subtilosin levels (213 AU mL −1 ) were recorded from cells grown in AF-MRS broth supplemented with MgSO 4 as compared to cells grown in the absence of MgSO 4 (150 AU mL −1 ).

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Isolation of a Variant of Subtilosin A with Hemolytic Activity

Cited by 57 publications

References 37 publications

Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species

Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species

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

Subtilosin A production by Bacillus subtilis KATMIRA1933 and colony morphology are influenced by the growth medium

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