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A survey of wild-type Caulobacter strains revealed naturally occurring plasmids in three species. Further analysis showed instances of naturally occurring antibiotic resistance and bacteriocin production.
Carnobacterium piscicola LV17 isolated from vacuum-packed meat produces bacteriocin(s) that is active against closely related lactic acid bacteria, Enterococcus spp., and a strain of Listeria monocytogenes but not against gram-negative bacteria. The bacteriocin has a bactericidal mode of action, is heat resistant, and is stable over a wide range of pH but is inactivated by proteolytic enzymes. Sensitive and resistant cells were shown to adsorb the bacteriocin, but cell death depended on contact of the bacteriocin with the cell membrane. Bacteriocin production is detected early in the growth cycle of the organism in APT broth, but it is not produced in APT broth adjusted to pH 5.5. Bacteriocin production and resistance to the bacteriocin produced are associated with two plasmids of 40 and 49 megadaltons. The possibility that two bacteriocins are produced is indicated because the inhibitory substances of the mutant strains containing either the 40- or 49-megadalton plasmids have different antimicrobial spectra.
AIMS: To determine the production of bacteriocin by Shigella flexneri strains, to relate their production to the presence of dysenteric diarrhoea and to asses the genetic determination of the bacteriocin. METHODS AND RESULTS: One hundred and sixteen strains of Sh. flexneri were isolated from patients with diarrhoea and 49 of them produced bacteriocin active against several Escherichia coli and abacteriocinogenic Sh. flexneri strains. The extrachromosomal DNA isolated from bacteriocinogenic Sh. flexneri strains were used as a substrate to transform E. coli HB-101 cells by means of electroporation. CONCLUSIONS: Only the Sh. flexneri strains isolated from dysenteric diarrhoea produced bacteriocin. It was demonstrated that a plasmid of approx. 3 kb was responsible for the genetic determination of these anti-bacterial substances. SIGNIFICANCE AND IMPACT OF THE STUDY: A 3-kb plasmid that harboured information for the production of bacteriocin by Sh. flexneri strains was described. The production of this bacteriocin may be related to dysenteric diarrhoea produced by these bacterial strains.
AIMS: To characterize bacteriocin production, antimicrobial spectrum and plasmid content in bacteriocinogenic enterococci from foods. METHODS AND RESULTS: Bacteriocinogenic Enterococcus faecium (14 isolates) and Enterococcus faecalis (three isolates) showed two different patterns of bacteriocin production in liquid broth: exponential-phase and stationary-phase production. Bacteriocin concentrates from all enterococci were inactivated by trypsin, but seldom by heat (100-117 degrees C), extremes of pH (2.0 to 9.0) or reducing agents (such as dithiothreitol). All bacteriocin concentrates were active against Listeria innocua and Listeria monocytogenes, and most were also active against many Ent. faecalis and Ent. faecium isolates. Enterococci clustered in three main groups according to their plasmid content (which included plasmids from 2.0 to 53 kb). Several isolates from different foods showed almost identical plasmid profiles. The enterocin P structural gene (entP) was detected by hybridization on plasmids of c. 19, 26 and/or 35-38 kb. CONCLUSIONS: Enterococci from food show different patterns of bacteriocin production and different plasmid content in spite of carrying similar bacteriocin-encoding genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the diversity of bacteriocinogenic enterococci from food sources carrying apparently similar enterocin genes.
Enterococcus faecalis 226 NWC, isolated from natural whey cultures utilized as starter in water-buffalo Mozzarella cheese manufacture, produces a bacteriocin, designated Enterocin 226 NWC, which is inhibitory to Listeria monocytogenes. Plasmid analysis of E. faecalis 226 NWC showed a single 5.2-kb plasmid, pEF226. In conjugation experiments, pEF226 was transferred into a plasmid-free strain of E. faecalis JH2-2. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The identity of conjugation was confirmed by digestion with SmaI restriction endonuclease and subsequent pulsed-field gel electrophoresis (PFGE) of the genomic DNA of E. faecalis 226, E. faecalis JH2-2 and of the isolates after the mating. The data indicate that the ability of E. faecalis 226 NWC to produce the bacteriocin is linked to the 5.2-kb conjugative plasmid pEF226.
The conjugative 63-kb lactococcal plasmid pMRC01 encodes bacteriophage resistance and production of and immunity to a novel broad-spectrum bacteriocin, designated lacticin 3147 (M.P. Ryan, M.C. Rea, C. Hill, and R.P. Ross, Appl. Environ. Microbiol. 62:612-619, 1996). The phage resistance is an abortive infection mechanism which targets the phage-lytic cycle at a point after phage DNA replication. By using the genetic determinants for bacteriocin immunity encoded on the plasmid as a selectable marker, pMRC01 was transferred into a variety of lactococcal starter cultures to improve their phage resistance properties. Selection of resulting transconjugants was performed directly on solid media containing the bacteriocin. Since the starters exhibited no spontaneous resistance to the bacteriocin as a selective agent, this allowed the assessment of the transfer of the naturally occurring plasmid into a range of dairy starter cultures. Results demonstrate that efficient transfer of the plasmid was dependent on the particular recipient strain chosen, and while high-frequency transfer (10(-3) per donor) of the entire plasmid to some strains was observed, the plasmid could not be conjugated into a number of starters. In this study, transconjugants for a number of lactococcal starter cultures which are phage resistant and bacteriocin producing have been generated. This bacteriocin-producing phenotype allows for control of nonstarter flora in food fermentations, and the phage resistance property protects the starter cultures in industry. The 63-kb plasmid was also successfully transferred into Lactococcus lactis MG1614 cells via electroporation.
Production of bacteriocin activity designated pediocin PA-1 was associated with the presence of a 6.2-megadalton plasmid in Pediococcus acidilactici PAC1.0. The bacteriocin exhibited activity against P. acidilactici, P. pentosaceus, Lactobacillus plantarum, L. casei, L. bifermentans, and Leuconostoc mesenteroides subsp. dextranicum. Partial characterization of pediocin PA-1 is described. The molecular weight of pediocin PA-1 was ca. 16,500. Additionally, strain PAC1.0 was found to contain a 23-megadalton plasmid associated with sucrose-fermenting ability.
Production of bacteriocin Bc-48 by Enterococcus faecalis S-48 is encoded by the conjugative plasmid pMB1, which is approximately 90 kb and also responds to sex pheromones of E. faecalis OG1X. Mutants harboring deleted forms of this plasmid (pMB1-del, 75 kb) have lost both the phenotype Bc-48 (production and immunity) and the clumping response. The conjugal transfer of pMB1 to E. faecalis OG1X results in the acquisition by this strain of both bacteriocin production and immunity and also the clumping response. In the transconjugants isolated, the bacteriocinogenic trait is associated with a smaller plasmid (52 kb), which we call pMB1-1. The relationship among plasmids pMB1, pMB1-del, and pMB1-1 has been demonstrated by DNA hybridization. Plasmid pMB1-1 has been transferred with high frequency to E. faecalis mutants cured of Bc-48 production (carrying pMB1-del), conferring to them the Bc-48 trait and clumping response. In the transconjugants from a second mating, pMB1-1 and pMB1-del coexist without appreciable segregation.
Four bacteriocin (enterocin) producing strains of Streptococcus faecium were examined for extrachromosomal DNA. Three strains proved to harbor minicircular plasmid DNA of uniform length as evidenced by CsCl ethidium bromide density centrifugation and electron microscopy. Molecular weight determination obtained through contour length measurements showed 2.4 X 10(6) dalton for S. faecium 25, 3.4 X 10(6) dalton for S. faecium 3 and 3.8 X 10(6) dalton for S. faecium 59. Elimination experiments for bacteriocin production with intercalating dyes proved sterile. The role of these plasmids remains for the time being unknown.
The complete sequence of the 10.9-kbp bacteriocinogenic plasmid pBL1 from Lactococcus lactis subsp. lactis IPLA 972 has been determined. Thirteen ORFs were encountered, of which 5 were incomplete. pBL1 proved to be a narrow-host-range plasmid which replicates neither in Bacilus subtilis nor in Lactobacillus spp. The structural organization of the pBL1 replication region was highly similar to other well-known theta-replicating plasmids of lactococci, at both the untranslated (the replication origin) and the translated (repB and orfX) sequences. As in other plasmids, the product of orfX was not necessary for plasmid replication. However, it was shown to be involved in plasmid stability. Three genes organized in an operon-like structure encompassed, most likely, the bacteriocin-encoding region. Upstream of the origin of replication a nicking site (oriT) was found. This oriT sequence proved to be functional by mobilization of plasmids wearing it. One complete and several partial IS elements were identified on pBL1.
Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C2/C18 reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI. entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1.
The complete sequence of pMRC01, a large conjugative plasmid from Lactococcus lactis ssp. lactis DPC3147, has been determined. Using a shotgun sequencing approach, the 60,232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundancy). Sixty-four open reading frames (ORFs) were identified. Analysis of the gene organization of pMRC01 suggests that the plasmid can be divided into three functional domains, with each approximately 20 kb region separated by insertion sequence (IS) elements. The three regions are (i) the conjugative transfer region, including a 16-gene Tra (transfer) operon; (ii) the bacteriocin production region, including an operon responsible for the synthesis of the novel bacteriocin lacticin 3147; and (iii) the phage resistance and plasmid replication region of the plasmid. The complete sequence of pMRC01 provides important information about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large gram-positive conjugative plasmids in general. The completely sequenced pMRC01 plasmid should also provide a useful framework for the design of novel plasmids to be incorporated into starter strain improvement programmes for the dairy industry.
A Lactobacillus plantarum strain, LTF154, isolated from a fermented sausage, produces a bacteriocin, designated plantacin 154. Plantacin 154 was stable to heat treatment, and its activity was sensitive to proteolytic enzymes. The molecular mass, as indicated by activity detection after SDS-PAGE, was estimated to be 3.0 kDa or less. A plasmid-curing experiment and transformation analysis indicated that a 9.5-MDa plasmid, pLP1542, may be involved in the production of plantacin 154.
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Each of three isolates of Streptococcus faecalis subsp. zymogenes harbored three R plasmids and a hemolysin-bacteriocin plasmid. The plasmids carried by one of these strains were physically characterized after their conjugative transfer. In each strain one of the plasmids carried genetic markers for resistance to gentamicin, kanamycin, sisomicin, netilmicin, and tobramycin.
A total of 203 lactic acid bacteria isolated from raw goat's milk and artisanal cheese were tested for antibacterial activity. Only two strains of Lactococcus lactis, one strain of Enterococcus faecalis and one strain of Lactobacillus curvatus were shown to produce a bacteriocin-like substance. Lactobacillus curvatus IFPL105 produced a heat-stable bacteriocin, which was hydrolysed by alpha-chymotrypsin, proteinase K and pancreatin and exhibited a broad spectrum of inhibitory activity. The bactericidal activity of the bacteriocin was more potent when sensitive strains were in the logarithmic growth phase, inducing cell lysis, as observed by decreases in optical density and release of intracellular marker enzymes. Curing experiments resulted in variants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of the parental strain and the bacteriocin-negative variants indicated that a plasmid of about 46 kbp may be involved in bacteriocin production and immunity to this antibacterial compound.
Streptococcus faecalis strain DS16 harbors the conjugative hemolysin-bacteriocin plasmid pAD1 (35 megadaltons) and the nonconjugative R-plasmid pAD2 determining resistance to streptomycin, kanamycin, and erythromycin; a tetracycline resistance (Tetr) determinant is located on the chromosome. When strain DS16 was mated (on membrane filters) with the plasmid-free strain JH2-2, Tetr transconjugants could be obtained at a frequency of about 10(-6) per recipient. Analyses of transconjugants showed that some contained the Tetr determinant linked to pAD1. Subsequent studies showed that the Tetr determinant was located on a 10-megaldalton transposon, designated Tn916, which could insert into two hemolysin plasmids: pAM gamma 1 and pOB1. In addition, derivatives of DS16 devoid of pAD1 were capable of transferring Tetr to recipient strains. Transconjugants (plasmid-free) from such matings could subsequently act as donors in the transfer of Tetr. Both transposition and transfer were found to be rec independent.
A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.