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.     

The cost and benefit of quorum sensing‐controlled bacteriocin production in Lactobacillus plantarum

Bacteria eliminate competitors via ‘chemical warfare’ with bacteriocins. Some species appear to adjust bacteriocin production conditionally in response to the social environment. We tested whether variation in the cost and benefit of producing bacteriocins could explain such conditional behaviour, in the bacteria Lactobacillus plantarum. We found that: (a) bacterial bacteriocin production could be upregulated by either the addition of a synthetic autoinducer peptide (PLNC8IF; signalling molecule), or by a plasmid which constitutively encodes for the production of this peptide; (b) bacteriocin production is costly, leading to reduced growth when grown in poor and, to a lesser extent, in rich media; (c) bacteriocin production provides a fitness advantage, when grown in competition with sensitive strains; and (d) the fitness benefits provided by bacteriocin production are greater at higher cell densities. These results show how the costs and benefits of upregulating bacteriocin production can depend upon abiotic and biotic conditions.     

Genetic analysis of bacteriocin 43 of vancomycin-resistant Enterococcus faecium

A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan were tested for bacteriocin production. Of the 277 (44%) bacteriocinogenic strains, 21 were active against E. faecalis, E. faecium, E. hirae, E. durans, and Listeria monocytogenes. Of those 21 strains, a representative bacteriocin of strain VRE82, designated bacteriocin 43, was found to be encoded on mobilizable plasmid pDT1 (6.2 kbp). Nine open reading frames (ORFs), ORF1 to ORF9, were presented on pDT1 and were oriented in the same direction. The bacteriocin 43 locus (bac43) consists of the bacteriocin gene bacA (ORF1) and the immunity gene bacB (ORF2). The deduced bacA product is 74 amino acids in length with a putative signal peptide of 30 amino acids at the N terminus. The bacB gene encodes a deduced 95-amino-acid protein without a signal sequence. The predicted mature BacA protein (44 amino acids) showed sequence homology with the membrane-active class IIa bacteriocins of lactic acid bacteria and showed 86% homology with bacteriocin 31 from E. faecalis YI717 and 98% homology with bacteriocin RC714. Southern analysis with a bac43 probe of each plasmid DNA from the 21 strains showed hybridization to a specific fragment corresponding to the 6.2-kbp EcoRI fragment, suggesting that the strains harbored the pDT1-like plasmid (6.2 kb) which encoded the bacteriocin 43-type bacteriocin. The bac43 determinant was not identified among non-VRE clinical isolates.     

Highly efficient Staphylococcus carnosus mutant selection system based on suicidal bacteriocin activation

Strains from various staphylococcal species produce bacteriocin peptides, which are thought to play important roles in bacterial competition and offer interesting biotechnological avenues. Many bacteriocins are secreted as inactive prepeptides with subsequent activation by specific proteolytic cleavage. By deletion of the protease gene gdmP in Staphylococcus gallinarum Tü3928, which produces the highly active lanthionine-containing bacteriocin gallidermin (lantibiotic), a strain was created producing inactive pregallidermin. On this basis, a new suicidal mutant selection system in the food-grade bacterium Staphylococcus carnosus was developed. Whereas pregallidermin was inactive against S. carnosus, it exerted potent bactericidal activity toward GdmP-secreting S. carnosus strains. To take advantage of this effect, gdmP was cloned in plasmid vectors used for random transposon mutagenesis or targeted allelic replacement of chromosomal genes. Both mutagenesis strategies rely on rare recombination events, and it has remained difficult and laborious to identify mutants among a vast majority of bacterial clones that still contain the delivery vectors. The gdmP-expressing plasmids pGS1 and pGS2 enabled very fast, easy, and reliable identification of transposon and gene replacement mutants, respectively. Mutant selection in the presence of pregallidermin caused suicidal inactivation of all clones that had retained the plasmids and allowed growth of only plasmid-cured mutants. Efficiency of mutant identification was several magnitudes higher than standard screening for the absence of plasmid-encoded antibiotic resistance markers and reached 100% specificity. Thus, the new pregallidermin-based mutant selection system represents a substantial improvement of staphylococcal mutagenesis methodology.     

Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius

Streptococcus salivarius strains commonly produce bacteriocins as putative anticompetitor or signalling molecules. Here we report that bacteriocin production by the oral probiotic strain S. salivarius K12 is encoded by a large (ca. 190 kb) plasmid. Oral cavity transmission of the plasmid from strain K12 to a plasmid-negative variant of this bacterium was demonstrated in two subjects. Tests of additional S. salivarius strains showed large (up to ca. 220 kb) plasmids present in bacteriocin-producing isolates. Various combinations (up to 3 per plasmid) of loci encoding the known streptococcal lantibiotics salivaricin A, salivaricin B, streptin and SA-FF22 were localised to these plasmids. Since all bacteriocin-producing strains of S. salivarius tested to date appear to harbour plasmids, it appears that they may function as mobile repositories for bacteriocin loci, especially those of the lantibiotic class.     

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.     

Distinct groups of plasmids correlated with bacteriocin production in Staphylococcus aureus

The genetic basis of bacteriocin (Bac) production by six strains of Staphylococcus aureus was examined. Gene transfer experiments (in which the plasmids were tagged with the erythromycin resistance transposon Tn551) and plasmid-elimination experiments by growth at 43 degrees C associated bacteriocin production with a particular plasmid in each strain. The Bac plasmids could be separated into two distinct groups: the first comprised plasmids larger than 40 kb, which did not specify immunity to bacteriocins; the second comprised small plasmids (8.0-10.4 kb) which also specified immunity to bacteriocins. The sequence relations among the small plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) were investigated by comparing restriction enzyme digest patterns and by hybridization. Plasmids pRJ10 and pRJ11 were indistinguishable and very closely related to plasmid pRJ9. Plasmid pRJ6, although different from the others, shared regions of sequence homology with them. No homology was found between plasmids pRJ6 or pRJ9 and the large Bac plasmids.     

Isolation and identification of a bacteriocin with antibacterial and antibiofilm activity from Citrobacter freundii

Multi- and pan-antibiotic-resistant bacteria area major health challenge in hospital settings. Furthermore,when susceptible bacteria establish surface-attached biofilm populations, they become recalcitrant to antimicrobial therapy. Therefore, there is a need for novel antimicrobials that are effective against multi-drug-resistant and surface-attached bacteria. A screen to identify prokaryote-derived antimicrobials from a panel of over 100 bacterial strains was performed. One compound isolated from Citrobacter freundii exhibited antimicrobial activity against a wide range of Gram-negative bacteria and was effective against biofilms. Random transposon mutagenesis was performed to find mutants unable to produce the antimicrobial compound.Transposons mapped to a bacteriocin gene located on a small plasmid capable of replication in Escherichia coli. The plasmid was sequenced and found to be highly similar to a previously described colicinogenic plasmid.Expression of the predicted bacteriocin immunity gene conferred bacteriocin immunity to E. coli. The predicted bacteriocin gene, colA-43864, expressed in E. coli was sufficient to generate anti-microbial activity, and purified recombinant ColA-43864 was highly effective in killing E. coli, Citrobacter species, and Klebsiella pneumoniae cells in a planktonic and biofilm state. This study suggests that bacteriocins can be an effective way to control surface-attached pathogenic bacteria.     

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.     

Interactions between oral bacteria: inhibition of Streptococcus mutans bacteriocin production by Streptococcus gordonii

Streptococcus mutans has been recognized as an important etiological agent in human dental caries. Some strains of S. mutans also produce bacteriocins. In this study, we sought to demonstrate that bacteriocin production by S. mutans strains GS5 and BM71 was mediated by quorum sensing, which is dependent on a competence-stimulating peptide (CSP) signaling system encoded by the com genes. We also demonstrated that interactions with some other oral streptococci interfered with S. mutans bacteriocin production both in broth and in biofilms. The inhibition of S. mutans bacteriocin production by oral bacteria was stronger in biofilms than in broth. Using transposon Tn916 mutagenesis, we identified a gene (sgc; named for Streptococcus gordonii challisin) responsible for the inhibition of S. mutans bacteriocin production by S. gordonii Challis. Interruption of the sgc gene in S. gordonii Challis resulted in attenuated inhibition of S. mutans bacteriocin production. The supernatant fluids from the sgc mutant did not inactivate the exogenous S. mutans CSP as did those from the parent strain Challis. S. gordonii Challis did not inactivate bacteriocin produced by S. mutans GS5. Because S. mutans uses quorum sensing to regulate virulence, strategies designed to interfere with these signaling systems may have broad applicability for biological control of this caries-causing organism.     

Epidemiological Differentiation of Serratia marcescens: Typing by Bacteriocin Sensitivity

Strains of Serratia marcescens were compared and differentiated by a new method. Bacteriocin lysates were prepared from mitomycin-induced S. marcescens and added to lawns of test strains. From 100 bacteriocin producers, 12 were chosen with the aid of computer analysis as the most useful in differentiation. Uniform drops of the 12 standard bacteriocins were added simultaneously with a bacteriocin-bacteriophage dropper to each strain to be typed. All 93 strains of S. marcescens tested were typable and were differentiated into 79 different sensitivity patterns. One pattern had three strains, 12 patterns had two strains each, and 66 patterns had only one strain. The bacteriocins also inhibited Shigella, Klebsiella, and Enterobacter, but no other Enterobacteriaceae. Bacteriocin sensitivity was less stable as an epidemiological marker than bacteriocin production. Several colonial mutants had sensitivity patterns different from the wild types, but most mutants were identical. In three different instances when cross-infection had been shown by other methods, bacteriocin sensitivity also gave the correct epidemiological results. Until the significance and frequency of genetic variations are known, a more stable epidemiological technique should be used in conjunction with bacteriocin sensitivity.     

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.     

Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen-fixing nodules by Rhizobium leguminosarum

A system which allows direct selection for curing of plasmids in Gram-negative bacteria was used to generate derivatives of Rhizobium leguminosarum VF39 cured of each of six plasmids present in this strain. Phenotypes could be correlated with the absence of five of the six plasmids. The smallest plasmid, pRleVF39a, carries genes for the production of a melanin-like pigment as has been previously reported. Plasmid pRleVF39d carries nodulation and nitrogen fixation genes. Curing of the plasmids pRleVF39c and pRleVF39e gave rise to strains which formed Fix- nodules on peas, lentils, and faba beans. The nodules formed by the strains cured of pRleVF39c contained few, if any, bacteria. Analysis of washed cells by SDS-PAGE showed that this strain is defective in lipopolysaccharide (LPS) production; the defect could be complemented by introducing plasmids from several other R. leguminosarum strains, and by the R. leguminosarum biovar phaseoli LPS gene clones pCos126 and pDel27. The nodules formed by the strain cured of pRleVF39e had a reduced symbiotic zone, an enlarged senescence zone, and an abundance of starch granules. This strain grew at a much slower rate than the wild type, was unable to grow on minimal medium, and no longer produced melanin. These defects could be complemented by at least one other Rhizobium plasmid, pRle336e, a plasmid of strain 336 which is distinct from the nodulation plasmid (pRle336c) and the plasmid (pRle336d) which could complement the LPS defect associated with the loss of pRleVF39c. This demonstrates that genes necessary for symbiosis can be carried on at least three different plasmids in R. leguminosarum.     

Selection of strains cured of the Rhizobium leguminosarum Sym-plasmid pRL1JI by using small bacteriocin

Abstract Fast growing Rhizobia are usually insensitive to a low M _r (small) bacteriocin. Introduction of the Sym-plasmid pRL1JI into these strains results in sensitivity towards small bacteriocin. Of such strains, small bacteriocin insensitive mutants (Sbs⁻) were selected.
A high percentage of these Sbs⁻ mutants appeared to be cured of pRL1JI. This selection of cured strains was feasible as well for the wild-type plasmid pRL1JI, as for a variety of transposon marked derivatives in several bacterial backgrounds.     

Interactions between Oral Bacteria: Inhibition of Streptococcus mutans Bacteriocin Production by Streptococcus gordonii

Streptococcus mutans has been recognized as an important etiological agent in human dental caries. Some strains of S. mutans also produce bacteriocins. In this study, we sought to demonstrate that bacteriocin production by S. mutans strains GS5 and BM71 was mediated by quorum sensing, which is dependent on a competence-stimulating peptide (CSP) signaling system encoded by the com genes. We also demonstrated that interactions with some other oral streptococci interfered with S. mutans bacteriocin production both in broth and in biofilms. The inhibition of S. mutans bacteriocin production by oral bacteria was stronger in biofilms than in broth. Using transposon Tn916 mutagenesis, we identified a gene (sgc; named for Streptococcus gordonii challisin) responsible for the inhibition of S. mutans bacteriocin production by S. gordonii Challis. Interruption of the sgc gene in S. gordonii Challis resulted in attenuated inhibition of S. mutans bacteriocin production. The supernatant fluids from the sgc mutant did not inactivate the exogenous S. mutans CSP as did those from the parent strain Challis. S. gordonii Challis did not inactivate bacteriocin produced by S. mutans GS5. Because S. mutans uses quorum sensing to regulate virulence, strategies designed to interfere with these signaling systems may have broad applicability for biological control of this caries-causing organism.     

Bacteriocin Typing of Burkholderia (Pseudomonas) solanacearum Race 1 of the French West Indies and Correlation with Genomic Variation of the Pathogen

Burkholderia solanacearum race 1 isolates indigenous to the French West Indies were characterized by bacteriocin typing and two genomic fingerprinting methods: pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction endonucleases (RC-PFGE) and PCR with primers corresponding to repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and BOX elements (collectively known as rep-PCR). The survey comprised 24 reference strains and 65 isolates obtained from a field trial in Guadeloupe in 1993. Comparison of the data identified RC-PFGE as the most discriminatory method, delineating 17 pulsed-field gel profile types. rep-PCR and bacteriocin typing identified nine rep-PCR profile types and nine bacteriocin groups. Independent determination of similarity coefficients and clustering of RC-PFGE and rep-PCR data identified six groups common to both sets of data that correlated to biovar and bacteriocin groups. Further study of bacteriocin production in planta gave results consistent with in vitro bacteriocin typing. It was observed that spontaneous bacteriocin-resistant mutants exhibited a cross-resistance to other bacteriocins as identified by the typing scheme and that such mutants possessed a selective advantage for growth over isogenic nonmutants in the presence of a bacteriocin. The results are significant in the search for biological control of disease by nonpathogenic mutants of the wild-type organism.     

A C-terminal disulfide bridge in pediocin-like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum

Several lactic acid bacteria produce so-called pediocin-like bacteriocins that share sequence characteristics, but differ in activity and target cell specificity. The significance of a C-terminal disulfide bridge present in only a few of these bacteriocins was studied by site-directed mutagenesis of pediocin PA-1 (which naturally contains the bridge) and sakacin P (which lacks the bridge). Introduction of the C-terminal bridge into sakacin P broadened the target cell specificity of this bacteriocin, as illustrated by the fact that the mutants were 10 to 20 times more potent than the wild-type toward certain indicator strains, whereas the potency toward other indicator strains remained essentially unchanged. Like pediocin PA-1, disulfide-containing sakacin P mutants had the same potency at 20 and 37 degrees C, whereas wild-type sakacin P was approximately 10 times less potent at 37 degrees C than at 20 degrees C. Reciprocal effects on target cell specificity and the temperature dependence of potency were observed upon studying the effect of removing the C-terminal disulfide bridge from pediocin PA-1 by Cys-->Ser mutations. These results clearly show that a C-terminal disulfide bridge in pediocin-like bacteriocins contributes to widening of the antimicrobial spectrum as well as to higher potency at elevated temperatures. Interestingly, the differences between sakacin P and pediocin PA-1 in terms of the temperature dependency of their activities correlated well with the optimal temperatures for bacteriocin production and growth of the bacteriocin-producing strain.     

Plasmid content and bacteriocin production by five strains of Lactococcus lactis isolated from semi-hard homemade cheese

In this study, the plasmid content and bacteriocin production of natural isolates of lactococci were investigated. Five bacteriocin producing lactococcal strains (Lactococcus lactis subsp. lactis BGMN1-2, BGMN1-3, BGMN1-5, BGMN1-6, and BGMN2-7) were isolated as nonstarter microflora of semi-hard homemade cheese and characterized. All isolates contained a number of plasmids. It was shown that lcnB structural genes for bacteriocin lactococcin B were located on large plasmids in all isolates. In the strains BGMN1-3 and BGMN1-5 proteinase prtP genes collocated with lcnB. Furthermore, these strains produced two additional bacteriocins (LsbA and LsbB) with genes responsible for their production and immunity located on the small rolling circle-replicating plasmid pMN5. Using deletion experiments of pMN5, minimal replicon of the plasmid and involvement of a bacteriocin locus in plasmid maintenance were identified. In addition, plasmid curing experiments showed that genes for catabolism or transport of 10 carbohydrates in the strain BGMN1-5 were plasmid located.     

Plant lectin-like bacteriocin from a rhizosphere-colonizing Pseudomonas isolate

Rhizosphere isolate Pseudomonas sp. strain BW11M1, which belongs to the Pseudomonas putida cluster, secretes a heat- and protease-sensitive bacteriocin which kills P. putida GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a TnMod mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into Escherichia coli cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in E. coli. This gene was designated llpA (lectin-like putidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the llpA gene also conveyed bacteriocin production to several Pseudomonas fluorescens strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described Pseudomonas bacteriocins.