BlpC-regulated bacteriocin production in Streptococcus thermophilus

Streptococcus thermophilus B59671 produces a bacteriocin with anti-pediococcal activity, but genes required for its production are not characterized. Genome sequencing of S. thermophilus has identified a genetic locus encoding a quorum sensing (QS) system that regulates production of class II bacteriocins. However, in strains possessing this gene cluster, production of bacteriocin like peptides (Blp) was only observed when excess pheromone was provided. PCR analysis revealed this strain possessed blpC, which encodes the 30-mer QS pheromone. To investigate if BlpC regulates bacteriocin production in S. thermophilus B59671, an integrative vector was used to replace blpC with a gene encoding for kanamycin resistance and the resulting mutant did not inhibit the growth of Pediococcus acidilactici. Constitutive expression of blpC from a shuttle vector restored the bacteriocin production, confirming the blp gene cluster is essential for bacteriocin activity in S. thermophilus B59671.     

Coordinated Bacteriocin Expression and Competence in Streptococcus pneumoniae Contributes to Genetic Adaptation through Neighbor Predation

Streptococcus pneumoniae (pneumococcus) has remained a persistent cause of invasive and mucosal disease in humans despite the widespread use of antibiotics and vaccines. The resilience of this organism is due to its capacity for adaptation through the uptake and incorporation of new genetic material from the surrounding microbial community. DNA uptake and recombination is controlled by a tightly regulated quorum sensing system that is triggered by the extracellular accumulation of competence stimulating peptide (CSP). In this study, we demonstrate that CSP can stimulate the production of a diverse array of blp bacteriocins. This cross stimulation occurs through increased production and secretion of the bacteriocin pheromone, BlpC, and requires a functional competence regulatory system. We show that a highly conserved motif in the promoter of the operon encoding BlpC and its transporter mediates the upregulation by CSP. The accumulation of BlpC following CSP stimulation results in augmented activation of the entire blp locus. Using biofilm-grown organisms as a model for competition and genetic exchange on the mucosal surface, we demonstrate that DNA exchange is enhanced by bacteriocin secretion suggesting that co-stimulation of bacteriocins with competence provides an adaptive advantage. The blp and com regulatory pathways are believed to have diverged and specialized in a remote ancestor of pneumococcus. Despite this, the two systems have maintained a regulatory connection that promotes competition and adaptation by targeting for lysis a wide array of potential competitors while simultaneously providing the means for incorporation of their DNA.     

Bacteriocin-mediated competition in cystic fibrosis lung infections

Bacteriocins are toxins produced by bacteria to kill competitors of the same species. Theory and laboratory experiments suggest that bacteriocin production and immunity play a key role in the competitive dynamics of bacterial strains. The extent to which this is the case in natural populations, especially human pathogens, remains to be tested. We examined the role of bacteriocins in competition using Pseudomonas aeruginosa strains infecting lungs of humans with cystic fibrosis (CF). We assessed the ability of different strains to kill each other using phenotypic assays, and sequenced their genomes to determine what bacteriocins (pyocins) they carry. We found that (i) isolates from later infection stages inhibited earlier infecting strains less, but were more inhibited by pyocins produced by earlier infecting strains and carried fewer pyocin types; (ii) this difference between early and late infections appears to be caused by a difference in pyocin diversity between competing genotypes and not by loss of pyocin genes within a lineage over time; (iii) pyocin inhibition does not explain why certain strains outcompete others within lung infections; (iv) strains frequently carry the pyocin-killing gene, but not the immunity gene, suggesting resistance occurs via other unknown mechanisms. Our results show that, in contrast to patterns observed in experimental studies, pyocin production does not appear to have a major influence on strain competition during CF lung infections.     

Genomic analyses of pneumococci reveal a wide diversity of bacteriocins – including pneumocyclicin,a novel circular bacteriocin

Background

One of the most important global pathogens infecting all age groups is Streptococcus pneumoniae (the ‘pneumococcus’). Pneumococci reside in the paediatric nasopharynx, where they compete for space and resources, and one competition strategy is to produce a bacteriocin (antimicrobial peptide or protein) to attack other bacteria and an immunity protein to protect against self-destruction. We analysed a collection of 336 diverse pneumococcal genomes dating from 1916 onwards, identified bacteriocin cassettes, detailed their genetic composition and sequence diversity, and evaluated the data in the context of the pneumococcal population structure.

Results

We found that all genomes maintained a blp bacteriocin cassette and we identified several novel blp cassettes and genes. The composition of the ‘bacteriocin/immunity region’ of the blp cassette was highly variable: one cassette possessed six bacteriocin genes and eight putative immunity genes, whereas another cassette had only one of each. Both widely-distributed and highly clonal blp cassettes were identified. Most surprisingly, one-third of pneumococcal genomes also possessed a cassette encoding a novel circular bacteriocin that we called pneumocyclicin, which shared a similar genetic organisation to well-characterised circular bacteriocin cassettes in other bacterial species. Pneumocyclicin cassettes were mainly of one genetic cluster and largely found among seven major pneumococcal clonal complexes.

Conclusions

These detailed genomic analyses revealed a novel pneumocyclicin cassette and a wide variety of blp bacteriocin cassettes, suggesting that competition in the nasopharynx is a complex biological phenomenon.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1729-4) contains supplementary material, which is available to authorized users.     

Expression of Streptococcus pneumoniae Bacteriocins Is Induced by Antibiotics via Regulatory Interplay with the Competence System

Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra-species competition within the human host. However, the triggers of pneumocin expression are poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39. Furthermore, by analogy with pneumococcal competence, we show that several antibiotics activate the blp-genes. Using real-time gene expression measurements we show that while the promoter driving expression of the two-component regulatory system blpR/H is constitutive, the remaining blp-promoters that control pneumocin expression, immunity and the inducer peptide BlpC, are pH-dependent and induced in the late exponential phase. Intriguingly, competence for genetic transformation, mediated by the paralogous ComD/E two-component quorum system, is induced by the same environmental cues. To test for interplay between these regulatory systems, we quantified the regulatory response to the addition of synthetic BlpC and competence-stimulating peptide (CSP). Supporting the idea of such interplay, we found that immediately upon addition of CSP, the blp-promoters were activated in a comD/E-dependent manner. After a delay, blp-expression was highly induced and was strictly dependent on blpRH and blpC. This raised the question of the mechanism of BlpC export, since bioinformatic analysis showed that the genes encoding the putative exporter for BlpC, blpAB, are not intact in strain D39 and most other strains. By contrast, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. Consistent with the idea that comAB mediate BlpC export, we finally show that high-level expression of the blp-genes requires comAB. Together, our results demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might therefore have unpredictable consequences on pneumococcal colonization dynamics by activating genes that mediate intra-specific interference competition.     

Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac^R). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac^R derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac^R isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac^R strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.     

Identification of a New Plasmid-Encoded sec-Dependent Bacteriocin Produced by Listeria innocua 743

Listeria innocua 743 produces an inhibitory activity demonstrating broad-spectrum inhibition of Listeria monocytogenes isolates. Gel-electrophoretic analysis of culture supernatants indicated that two inhibitors with different molecular weights were produced by this strain. Insertion of Tn917 into a 2.9 Kb plasmid (pHC743) generated mutants with either an impaired ability or a loss in ability to produce one of the inhibitors. Sequence analysis of the transposon insertion regions revealed the presence of two continuous open reading frames, the first encoding a new pediocin-like bacteriocin (lisA) and the second encoding a protein homologous with genes involved in immunity toward other bacteriocins (lisB). Translation of the bacteriocin gene (lisA) initiates from a noncanonical start codon and encodes a 71-amino-acid prebacteriocin which lacked the double glycine leader peptidase processing site common in other type II bacteriocins. Alignment of the sequence with the processed N termini of related bacteriocins suggests that the mature bacteriocin consists of 43 amino acids, with a predicted molecular mass of 4,484 Da. Mutants containing insertions into lisA were sensitive to the inhibitor, indicating that lisAB forms a single operon and that lisB represents the immunity protein. Cloning of an amplicon containing the lisAB operon into Escherichia coli resulted in expression and export of the bacteriocin. This finding confirms that the phenotype is dependent on the structural and immunity gene only and that export of this bacteriocin is sec dependent. This is the first confirmation of bacteriocin production in a Listeria spp., and it is of interest that this bacteriocin is closely related to the pediocin family of bacteriocins produced by lactic acid bacteria.     

Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae

The production of bacteriocins can be favorable for colonization of the host by eliminating other bacterial species that share the same environment. In Streptococcus pneumoniae, the pnc (blp) locus encoding putative bacteriocins, immunity, and export proteins is controlled by a two-component system similar to the comCDE system required for the induction of genetic competence. A detailed comparison of the pnc clusters of four genetically distinct isolates confirmed the great plasticity of this locus and documented several repeat sequences. Members of the multiple-antibiotic-resistant Spain23F-1 clone, one member of the Spain9V-3 clone, sensitive 23F strain 2306, and the TIGR4 strain produced bactericidal substances active against other gram-positive bacteria and in some cases against S. pneumoniae as well. However, other strains did not show activity against the indicator strains despite the presence of a bacteriocin cluster, indicating that other factors are required for bacteriocin activity. Analysis of strain 2306 and mutant derivatives of this strain confirmed that bacteriocin production was dependent on the two-component regulatory system and genes involved in bacteriocin transport and processing. At least one other bacteriocin gene, pncE, is located elsewhere on the chromosome and might contribute to the bacteriocin activity of this strain.     

Characterization of a heat stable anti-listerial bacteriocin produced by vancomycin sensitive Enterococcus faecium isolated from idli batter

Lactic acid bacteria (LAB) are known to produce various types of bacteriocins, ribosomally synthesized polypeptides, which have antibacterial spectrum against many food borne pathogens. Listeria monocytogenes, a pathogenic bacterium, is of particular concern to the food industry because of its ability to grow even at refrigeration temperatures and its tolerance to preservative agents. Some of the bacteriocins of LAB are known to have anti-listerial property. In the present study, the bacteriocin produced by vancomycin sensitive Enterococcus faecium El and J4 isolated from idli batter samples was characterized. The isolates were found to tolerate high temperatures of 60°C for 15 and 30 min and 70°C for 15 min. The bacteriocin was found to be heat stable and had anti-listerial activity. The bacteriocin did not lost anti-listerial activity when treated at 100°C for 30 min or at 121°C for 15 min. The bacteriocin lost its antimicrobial activity after treating with trypsin, protinase-K, protease and peptidase.     

Synergistic Mode of Action of Mesenterocins 52A and 52B Produced by <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> subsp. <Emphasis Type="Italic">mesenteroides</Emphasis> FR 52

Few studies have been published on the effects of two bacteriocins combinations and particularly on combinations of two bacteriocins with different structures produced by the same strain. In this work, the actions of mesenterocin 52A (class IIa) and mesenterocin 52B (class II), produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52, were studied on strains susceptible to only one bacteriocin or to both. In broth, combination of mesenterocins enhanced the adaptation time of the strain susceptible to the both mesenterocins (48 h vs 17 h with only one bacteriocin). In agar medium, mesenterocins displayed, as expected, a synergistic effect on this strain (FIC_index < 1), but also on the two strains susceptible to only one mesenterocin. This original result was probably due to membrane composition modifications induced by the mesenterocin that enhanced bacteriocin action. Thus, this hurdle technique seems to be interesting in food preservation in terms of minimizing bacteriocin concentrations.     

Bacterioides fragilis: production and sensitivity to bacteriocins

Bacteriocin production and sensitivity to bacteriocins have been successfully applied as an epidemiological tool in several species of bacteria. However, little work has been carried out on the bacteriocins produced by Bacteroides fragilis, which is the most frequently isolated anaerobe species from clinical specimens. Thirty two clinical isolates of B. fragilis grown anaerobically on a 0.22 microm membrane filter spotted on an agar plate, were tested for bacteriocin production and used in a screen for bacteriocin sensitivity. Sensitivity to at least one bacteriocin was found in 94% of the strains, 62.5% were sensitive to two bacteriocins, whereas 34.4% were sensitive to three or more and finally one strain was found sensitive to 17 bacteriocins. Of the strains studied, 94% inhibited at least one strain, 66% inhibited two strains, and 30% inhibited at least three strains or more. Finally, one strain was extremely active by inhibiting the growth of 17 strains. Bacteriocin types are characterised by geographic variation, and their epidemiological investigation by a simple method could be promoted.     

Competitive interactions in Escherichia coli populations: the role of bacteriocins

Explaining the coexistence of competing species is a major challenge in community ecology. In bacterial systems, competition is often driven by the production of bacteriocins, which are narrow-spectrum proteinaceous toxins that serve to kill closely related species, providing the producer better access to limited resources. Bacteriocin producers have been shown to competitively exclude sensitive, nonproducing strains. However, the dynamics between bacteriocin producers, each lethal to its competitor, are largely unknown. In this study, we used in vitro, in vivo and in silico models to study competitive interactions between bacteriocin producers. Two Escherichia coli strains were generated, each carrying a DNA-degrading bacteriocin (colicins E2 and E7). Using reporter-gene assays, we showed that each DNase bacteriocin is not only lethal to its opponent but, at lower doses, can also induce the expression of its opponent''s toxin. In a well-mixed habitat, the E2 producer outcompeted its adversary; however, in structured environments (on plates or in mice colons), the two producers coexisted in a spatially ‘frozen'' pattern. Coexistence occurred when the producers were initiated with a clumped spatial distribution. This suggests that a ‘clump'' of each producer can block invasion of the other producer. Agent-based simulation of bacteriocin-mediated competition further showed that mutual exclusion in a structured environment is a relatively robust result. These models imply that colicin-mediated colicin induction enables producers to successfully compete and defend their niche against invaders. This suggests that localized interactions between producers of DNA-degrading toxins can lead to stable coexistence of heterogeneously distributed strains within the bacterial community and to the maintenance of diversity.     

Bacteriocin production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> in complex growth media

Objective

To test if the production of bacteriocins by Streptococcus thermophilus is influenced when grown in various complex media commonly used for the culturing of lactic acid bacteria.

Results

Forty-one strains of S. thermophilus were screened for the production of bacteriocins in tryptone/yeast extract/lactose (TYL), M17-lactose (M17L), M17-glucose (M17G) and MRS media. Two strains, ST144 and ST145, were identified as novel bacteriocin producers, with constitutive production observed only in M17G. Strains ST110, ST114 and ST134 constitutively produced bacteriocins in all growth media but ST114 required growth in MRS for its antimicrobial activity to persist in a 24 h culture. The addition of a synthetic quorum sensing peptide (BlpC) induced bacteriocin production by ST106 in all media tested; and by ST118 in TYL and M17L. Strain ST109, which constitutively produced a bacteriocin in TYL and M17 broths, required BlpC induction when grown in MRS. Real-time PCR analysis showed that the natural expression of blpC in ST109 was lower when grown in MRS, suggesting that something in medium interfered with the blp quorum sensing system.

Conclusion

As the choice of growth medium influences both bacteriocin production and peptide stability, several types of production media should be tested when screening for novel bacteriocin-producing strains of S. thermophilus.

     

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.     

Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.     

Bacteriocin plasmids ofPediococcus acidilactici

Summary Pediococcus acidilactici strains E, F and H isolated from fermented sausages produced bacteriocins which were protein in nature and inhibitory to a variety of spoilage and pathogenic microorganisms often encountered in foods. These strains harbored two to three plasmids ranging in size from 7.4 to 40.2 megadaltons. Curing experiments and plasmid profile analysis indicated the involvement of plasmid DNA with bacteriocin activity in all three strains. Carbohydrate fermentation and antibiotic resistance phenotypes did not appear to be associated with bacteriocin plasmids. Both bacteriocin activity and resistance determinants were linked in strain H and mediated by a 7.4-megadalton plasmid, whereas in strains E and F these two traits were not linked.     

Characterization of a new organization of the plantaricin locus in the inducible bacteriocin-producing<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> J23 of grape must origin

Lactobacillus plantarum J23 was previously characterized as a bacteriocin-producer-strain when it was cocultured with other lactic acid bacteria. In this work, the genetic organization of the pln locus in the J23 strain was studied and compared with those of previously described L. plantarum C11, WCFS1 and NC8 strains. A new organization of the plantaricin locus was detected in the J23 strain. The sequenced fragment (20,266 bp) comprised plnJLR, plnMNOP, plnEFI, plnGHSTUVWXY, and plNC8IF-plNC8HK-plnD operons, as well as a new region that includes three new orfs (GenBank accession number DQ323671). When the J23 pln gene sequences were compared with those included in the GenBank database, the identity of the putative encoded proteins was in the range 67.1–100%. The regulatory system and the repertoire of putative bacteriocins of the J23 pln locus presented important differences with respect to the ones of C11, WCFS1 and NC8, such as the absence of plnK and the presence of a larger plnJ gene than the previously described for the other L. plantarum strains. The pln locus in L. plantarum strains seems to be a mosaic-like structure with different modules and reorganizations that presents highly conserved regions related to transport and bacteriocin maturation and variable regions related to regulation and bacteriocin production.     

Bioassay for the rapid detection of bacteriocins in fermentation broth

A bioassay for the rapid detection of bacteriocins by detecting efflux of K⁺ ions from a bacteriocin-sensitive indicator strain was developed. There was a rapid increase in concentration of K⁺ in the medium from approximately 1.6 to 9.5 mM when the crude bacteriocin was added to the sensitive strain. A bacteriocin activity of 8.25 AU ml^–1 produced the lowest detectable concentration of K⁺ (0.179 mM) and the smallest inhibition zone (0.4 mm). The smallest cell dry mass of the indicator strain capable of releasing a detectable level of K⁺ (0.102 mM) was 0.76 mg. Detection of the bacteriocin by measuring the efflux of K⁺ compares well with the conventional agar well diffusion assay. Low activities of bacteriocin and low amounts of the sensitive indicator strain biomass can be used with this bioassay.     

Bacteriocin production by members of the genusKlebsiella

Bacteriocin production was tested in 36Klebsiella and 3Enterobacter aerogenes strains. Bacteriocins produced byK. pneumoniae were found to be active on most strains ofK. edwardsi, K. aerogenes, K. rhinoscleromatis andE. aerogenes. The bacteriocin produced byE. aerogenes 37 is also active onK. pneumoniae andK. ozaenae. The bacteriocins produced byK. rhinoscleromatis, K. edwardsi andK. aerogenes are active on only a few strains. The activity spectra of the bacteriocins of a number of strains were similar. The method of classification used for colicins could not be applied to these bacteriocins as mutants resistant to one bacteriocin were nearly always resistant to all other bacteriocins. One mutant, though resistant, still adsorbed the bacteriocin to which it was resistant and it is very likely that the same applies for all other resistant mutants. The hypothesis is made that allKlebsiella bacteriocins have the same biochemical target, or more likely, possess a common transmission mechanism.