Salivaricin A2 and the novel lantibiotic salivaricin B are encoded at adjacent loci on a 190-kilobase transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12

The commercial probiotic Streptococcus salivarius strain K12 is the prototype of those S. salivarius strains that are the most strongly inhibitory in a standardized test of streptococcal bacteriocin production and has been shown to produce the 2,368-Da salivaricin A2 (SalA2) and the 2,740-Da salivaricin B (SboB) lantibiotics. The previously uncharacterized SboB belongs to the type AII class of lantibiotic bacteriocins and is encoded by an eight-gene cluster. The genetic loci encoding SalA2 and SboB in strain K12 have been fully characterized and are localized to nearly adjacent sites on pSsal-K12, a 190-kb megaplasmid. Of 61 strongly inhibitory strains of S. salivarius, 19 (31%) were positive for the sboB structural gene. All but one (strain NR) of these 19 strains were also positive for salA2, and in each of these cases of double positivity, the two loci were separated by fewer than 10 kb. This is the first report of a single streptococcus strain producing two distinct lantibiotics.     

Extended Safety Data for the Oral Cavity Probiotic Streptococcus salivarius K12

Previous studies of the bacteriocin-producing Streptococcus salivarius K12 monitored a variety of intrinsic strain characteristics of potential relevance to its application as an oral probiotic in humans. These included the content of antibiotic resistance and virulence determinants, the production of deleterious metabolic by-products and its genetic stability. In the present study, we examined additional safety factors including the responses of rats to either short- or long-term oral dosing with strain K12 preparations. In addition, the potential genotoxicity of strain K12 was tested using a bacterial reverse mutation assay. To determine the occurrence and concentrations in human saliva of S. salivarius having the same bacteriocin phenotype as strain K12, saliva samples from 780 children were evaluated. The level of dosing with strain K12 required to achieve oral cavity colonization levels similar to those occurring naturally for this type of bacteriocin-producing S. salivarius was established using 100 human subjects. Following the oral instillation of lyophilized S. salivarius K12 cells in these subjects, its persistence was not at levels higher than those found naturally for this type of bacterium. The various sets of data obtained in this study showed no evidence of genotoxicity and no acute or subacute toxicity effects associated with strain K12. Based on the previously published data, the long history of use by humans and the information presented here, it is concluded that S. salivarius K12 is safe for human consumption.     

Development of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> LL441 and its plasmid-cured derivatives in cheese

A wild Lactobacillus plantarum strain and two of its plasmid-cured derivatives were separately used as adjunct cultures in the manufacture of a Gouda-like traditional Spanish cheese. The wild strain, LL441, harbours seven plasmids and produces a lantibiotic-like bacteriocin. The LL441-B2 derivative has lost plasmids of 40 and 80 kb and the bacteriocin-producing capability. The LL441-B11 derivative has lost in addition a 70 kb plasmid encoding active α- and β-galactosidases. All three strains could be used as adjunct cultures as none of the technological and biochemical parameters of the cheeses was affected. Both the wild-type and the two derivatives were recovered from experimental cheeses up to 30 days after manufacture at similar rates of nearly 20%. Thus, the phenotypic traits under examination were not essential for L. plantarum to grow into the cheese matrix. Electronic Publication     

Streptococcal Bacteriocin-Like Inhibitory Substances: Some Personal Insights into the Bacteriocin-Like Activities Produced by Streptococci Good and Bad

The background to the discovery and commercial development of the first Streptococcus salivarius probiotic is documented. A 40-year search of the genus Streptococcus for a harmless natural antagonist of Streptococcus pyogenes had as its operational basis a simple deferred antagonism “fingerprinting” procedure, the application of which results in each tested strain being accorded an inhibitor production (P)-type and inhibitor sensitivity (S)-type profile. Systematic application of this schema has opened a “Pandora’s Box” of novel streptococcal bacteriocin-like inhibitory substances (BLIS). The numerically prominent commensal S. salivarius is proposed to have a pivotal population-modulating role within the oral microbiota of humans. The probiotic strain S. salivarius K12 produces several megaplasmid-encoded BLIS including the lantibiotics salivaricin A and salivaricin B. Strain K12 and other BLIS-producing S. salivarius are currently in use or under development for application to the control of a variety of common maladies and infections of the human oral cavity.     

Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) streptococci.

Thirteen bacteriocin-producing strains of group N (lactic acid) streptococci were screened for their potential to transfer this property by conjugation to Streptococcus lactis subsp. diacetylactis Bu2-60. Bacteriocin production in three strains was plasmid encoded as shown by conjugal transfer and by analysis of cured, bacteriocin-negative derivatives of the donor strains and the transconjugants. With Streptococcus cremoris strains 9B4 and 4G6 and S. lactis subsp. diacetylactis 6F7 as donors, bacteriocin-producing transconjugants were isolated with frequencies ranging from ca. 2 X 10(-2) to 2 X 10(-1) per recipient cell. Bacteriocin-producing transconjugants had acquired a 39.6-megadalton plasmid from the donor strains 9B4 and 4G6, and a 75-megadalton plasmid from the donor strain 6F7. As shown by restriction endonuclease analysis, the plasmids from strains 9B4 and 4G6 were almost identical. The plasmid from strain 6F7 yielded some additional fragments not present in the two other plasmids. In hybridization experiments any of the three plasmids strongly hybridized with each other and with some other bacteriocin but nontransmissible plasmids from other S. cremoris strains. Homology was also detected to a variety of cryptic plasmids in lactic acid streptococci.     

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.     

Salivaricin P, One of a Family of Two-Component Antilisterial Bacteriocins Produced by Intestinal Isolates of Lactobacillus salivarius

Lactobacillus salivarius DPC6005, a porcine intestinal isolate, produces a two-component bacteriocin, salivaricin P, with homology to ABP-118 produced by a human probiotic L. salivarius strain. Indeed, molecular characterization revealed that while the peptides Sln1 and ABP-118α are identical, their companion peptides (Sln2 and ABP-118β, respectively) differ by two amino acids. This observation suggests that two-component bacteriocins may be a common feature of intestinal L. salivarius strains.     

Plasmids in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2

Summary Five strains of Streptomyces ambofaciens were examined for their plasmid content. Among these strains, four belong to the same lineage (strains B) and the other was isolated independently (strain A). A large plasmid (ca. 80 kb), called pSAM1 in this paper and already described, was present in all B strains, and absent in strain A. A second plasmid, not described before, was found as covalently closed circular DNA in two of the four B strains. This plasmid with a size 11.1 kb was called pSAM2. A restriction map for 14 enzymes was established. Hybridization experiments showed that a unique sequence homologous to this plasmid is integrated in a larger replicon, which is not pSAM1 and is probably the chromosome, in all B strains and not in strain A. It seems probable that the integrated se1uence is the origin of the free plasmid found in two strains of the B family. It is noteworthy that the integrated form and the free plasmid may be found together. Transformation experiments proved that pSAM2 may be maintained autonomously in S. ambofaciens strain A and in S. lividans. pSAM2 is a self-transmissible plasmid, able to elicit the lethal zygosis reaction. pSAM2 was compared to the plasmids SLP1, pIJ110 and pIJ408, which all come from integrated sequences in three Streptomyces species and are found as autonomous plasmids after transfer to S. lividans. If pSAM2 resembles these plasmids in its origin, it does not appear to be related directly to them. Concerning their plasmid content, the two isolates of S. ambofaciens are very different. One of them contains neither pSAM1 not pSAM2. As this isolate produces spiramycin, these plasmids probably do not play an important role in spiramycin production. Apart from its intrinsic biological interest, pSAM2 may be useful in the construction of cloning vectors for S. ambofaciens. Very stable transformants might be obtained in certain strains of S. ambofaciens, because of the possibility of integration of the pSAM2 derivative vector.     

Survey of plasmid profiles of <Emphasis Type="Italic">Shigella</Emphasis> species isolated in Malaysia during 1994–2000

Summary Plasmid profiling was used to characterize 219 strains of Shigellaspecies isolated from sporadic cases of shigellosis in Malaysia during the period 1994–2000. Heterogeneous plasmid patterns were observed in all Shigella spp. There was a correlation between plasmid patterns and serotypes of S. flexneri, S. dysenteriaeand S. sonnei. Five common small plasmids (>20.0 kb) were observed in S. flexneri1b and 2a, whereas six common small plasmids were found in serotype 3a. Some of these plasmids appeared to maintain their existence stably in each individual serotype. Plasmids of size 11.40 and 4.20 kb were present only in S. flexneri2a isolates, whereas the 4.40 kb plasmid was unique for serotype 3a. Large (>150 kb) or mid-range plasmid (20.0–150 kb) was not observed from any S. flexneri1b isolates. Eighty-nine percent of S. flexneriof various serotypes harboured the plasmid of 3.20 kb. All S. dysenteriaetype 2 isolates harboured the 9.00 kb plasmid, while four common small plasmids were found in S. sonneiisolates. The 2.10 kb plasmid was only seen in S. sonnei. Streptomycin resistance in S. dysenteriaetype 2 and multi-drug resistance in S. sonneimay be associated with the 9.00 and 14.8 kb plasmids, respectively. Plasmid profiling provided a further discrimination beyond serotyping and a useful alternative genotypic marker for differentiation of Shigellaspecies. To the best of our knowledge, this is the first report on the plasmid prevalence of the Malaysian Shigellaspecies.     

Plasmid content of salt stress-tolerant <Emphasis Type="Italic">Rhizobium</Emphasis> strains from Egyptian soils nodulating common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Plasmid profile analysis is useful to characterize Rhizobium strains within the same species. Among the 16 Rhizobium strains examined, 14 had distinct plasmid profiles. The size of plasmids ranged from 40 to 650 kb, and three plasmids of 650, 510 and 390 kb were common to several strains. Plasmid analysis revealed that Rhizobium etli contained a mega-plasmid, similar in size to Rhizobium tropici. All the salt-tolerant strains examined had a plasmid of 250 kb, except for strain EBRI 29. This suggests that this plasmid may play an important adaptive role under salt stress conditions.     

Bacteriocin production by Enterococcus faecium NA01 from 'wara'—a fermented skimmed cow milk product from West Africa

An Enterococcus faecium strain from Nigerian fermented skimmed cow milk ('wara') produced bacteriocin inhibitory towards Lactobacillus, Enterococcus and Listeria strains. The bacteriocin (designated enterocin 01) was inactivated by proteases, heat-stable at 100°C and active at pH 2.0–6.0. The Ent. faecium isolate harboured plasmids of ca 36.3 and 23.1 kb. Curing experiments with ethidium bromide resulted in a bacteriocin-negative mutant which had not lost immunity to the bacteriocin. Slight differences in plasmid profiles between wild-type and mutant indicated a possible plasmid-coded bacteriocin production.     

A kinetic study on the plasmid stability of three <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Absract The plasmid stability of three wild type Lactococcus lactis strains and their mutants was investigated at different incubation time and temperatures in two different media [M17 broth and reconstituted skim milk (RSM)]. The results showed that both incubation times and temperature are effective on plasmid loss. The plasmid profiles of wild type strains exhibited 8 to 9 distinct plasmid species with molecular weights from 2.1 to 24.0 kb. Lactose fermentation ability was found to be encoded by 22.2 (strain U70), 23.6 (strain U29) and 24.0 (strain U52) kb plasmids in the wild type strains, respectively. The stabilities of the plasmids were explained by applying a second-order polynomial modeling system. Reasonable fittings were obtained for the model and the adjusted regression coefficients (R ² _adj) were between 0.76 and 0.99 for the overall data. Overall, it was found that incubation time had the most profound effect on plasmid stability, with plasmid loss occurring after 72 h, while temperatures in the range of 15–40°C also induced plasmid instability.     

In vitro Inhibition of Clinical Isolates of Otitis Media Pathogens by the Probiotic Streptococcus salivarius BLIS K12

Otitis media is a common childhood infection, frequently requiring antibiotics. With high rates of antibiotic prescribing and increasing antibiotic resistance, new strategies in otitis media prevention and treatment are needed. The aim of this study was to assess the in vitro inhibitory activity Streptococcus salivarius BLIS K12 against otitis media pathogens. Efficacy of the bacteriocin activity of S. salivarius BLIS K12 against the otitis media isolates was assessed using the deferred antagonism test. Overall, 48% of pathogenic isolates exhibited some growth inhibition by S. salivarius BLIS K12. S. salivarius BLIS K12 can inhibit the in vitro growth of the most common pathogens.

     

Development and application of a multiple typing system for Clostridium difficile

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

Development and application of a multiple typing system for Clostridium difficile.

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

Proteinase PI and lactococcin A genes are located on the largest plasmid in Lactococcus lactis subsp. lactis bv. diacetylactis S50

Lactococcus lactis subsp. lactis bv. diacetylactis S50 produces a lactococcin A-like bacteriocin named bacteriocin S50, and cell envelope-associated PI-type proteinase activity. This strain harbours 3 small size plasmids: pS6 (6.3 kb), pS7a (7.31 kb), and pS7b (7.27 kb). Plasmid curing using a combination of novobiocin treatment (10 microg.mL-1) and sublethal temperature (40 degrees C) resulted in a very low yield (0.17%) of Prt-, Bac-, Bacs derivatives, which retained all 3 small size resident plasmids. Pulsed-field gel electrophoresis of DNA isolated from the strain S50 and cured derivatives in combination with restriction enzyme analysis and DNA-DNA hybridization revealed that S50 contains 2 additional large plasmids: pS140 (140 kb) and pS80 (80 kb). Conjugation experiments using strain S50 as a donor and various lactococcal recipients resulted in Prt+, Bac+, Bacr transconjugants. Analysis of these transconjugants strongly indicated that plasmid pS140 harbours the prt and bac genes encoding proteinase and bacteriocin production, and immunity to bacteriocin, since each Prt+, Bac+, Bacr tranconjugant contained pS140. Accordingly, none of the Prt-,Bac-, Bacs transconjugants contained this plasmid. pS140 was a self-transmissible conjugative plasmid regardless of the host lactococcal recipient used in the test. Frequency of conjugation of plasmid pS140 did not depend on either the donor or recipient strain.     

Analysis of R-primes demonstrates that genes for broad host range nodulation of Rhizobium strain NGR234 are dispersed on the Sym plasmid

Summary R-prime plasmids carrying regions of the symbiotic (Sym) plasmid of the broad host range Rhizobium strain NGR234 were isolated in intergeneric matings with Escherichia coli K12. Three R-primes carrying approximately 180 kb (pMN23), 220 kb (pMN31) and 330 kb (pMN49) of Sym DNA were characterized in more detail. Restriction enzyme analysis and hybridization studies showed that these R-primes carried large overlapping regions of the Sym plasmid, and had the symbiotic genes (two copies of nifH, D and K; nodA, B, C and D; region II; host specific nodulation (hsn) genes) located over half of the 470 kb Sym plasmid. Only the largest of these R-primes (pMN49) contained the complete nodulation host range of the original parent strain NGR234. This broad host range was shown to be present on plasmid pMN49 by being expressed in Agrobacterium tumefaciens strain A136. Furthermore the R-prime plasmids were shown to contain different regions of distinctive host specific nodulation (hsn) for tropical legume infection and for the nodulation of the non-legume Parasponia. Nodulation of soybeans, however, required an additional region that was not essential for the nodulation of other tropical legumes. Strain NGR234 was also found to nodulate the stem and roots of the tropical legume Sesbania rostrata at a very low efficiency. However, the R-prime mini Sym plasmid constructions enabled a greater efficiency of nodulation of Sesbania rostrata to occur.     

Unusual genome complexity in Lactobacillus salivarius JCM1046

Background

Lactobacillus salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable and is often mediated by plasmids or integrative and conjugative elements. L. salivarius strains typically have multireplicon genomes including circular megaplasmids that encode strain-specific traits for intestinal survival and probiotic activity. Linear plasmids are less common in lactobacilli and show a very limited distribution in L. salivarius. Here we present experimental evidence that supports an unusually complex multireplicon genome structure in the porcine isolate L. salivarius JCM1046.

Results

JCM1046 harbours a 1.83 Mb chromosome, and four plasmids which constitute 20% of the genome. In addition to the known 219 kb repA-type megaplasmid pMP1046A, we identified and experimentally validated the topology of three additional replicons, the circular pMP1046B (129 kb), a linear plasmid pLMP1046 (101 kb) and pCTN1046 (33 kb) harbouring a conjugative transposon. pMP1046B harbours both plasmid-associated replication genes and paralogues of chromosomally encoded housekeeping and information-processing related genes, thus qualifying it as a putative chromid. pLMP1046 shares limited sequence homology or gene synteny with other L. salivarius plasmids, and its putative replication-associated protein is homologous to the RepA/E proteins found in the large circular megaplasmids of L. salivarius. Plasmid pCTN1046 harbours a single copy of an integrated conjugative transposon (Tn6224) which appears to be functionally intact and includes the tetracycline resistance gene tetM.

Conclusion

Experimental validation of sequence assemblies and plasmid topology resolved the complex genome architecture of L. salivarius JCM1046. A high-coverage draft genome sequence would not have elucidated the genome complexity in this strain. Given the expanding use of L. salivarius as a probiotic, it is important to determine the genotypic and phenotypic organization of L. salivarius strains. The identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-771) contains supplementary material, which is available to authorized users.     

Isolation and characterization of Rhodobacter capsulatus strains lacking endogenous plasmids

Phodobacter capsulatus (formerly Rhodopseudomonas capsulata) strain B10 was found to contain a single plasmid of molecular weight 86×10⁶. Strains lacking this plasmids were isolated by various methods from strains containing the mutant R plasmid, pTH10. With the exception of two strains, which were found to contain chromosomal insertions of R plasmid DNA, strains lacking the endogenous plasmid appeared to be unaffected in any of the following metabolic or genetic functions: photosynthetic, autotrophic, diazotrophic, and dark, anaerobic growth; the production of bacteriocin; homologous recombination; the restriction of foreign DNA; and the production of gene transfer agent. DNA-DNA hybridization experiments confirmed that the plasmid had been eliminated from these strains and not become integrated into the chromose. However, sequences homologous to those of the endogenous plasmid were found to be present in the chromosome of R. capsulatus B10. This suggests, among other possibilities, that the endogenous plasmid may have originated in the chromosome, and might serve to duplicate certain chromosomal functions.Abbreviations kb kilobase-pair - GTA gene transfer agent - Cma chromosome mobilizing ability     

Distribution of Plasmid-Borne Stress Protein Genes in Streptococcus thermophilus and Other Lactic Acid Bacteria

The presence of heat stress protein genes (hsp) was tested by Southern hybridization analysis in total DNA extracts from species of the genus Streptococcus (47 strains), Lactobacillus (34 strains), Lactococcus (24 strains), and Leuconostoc (5 strains). The biotinylated hsp16.4 probe prepared from an ORF2 fragment of pER341 (2.8 kb) tested positively with restricted DNA extracts of seven Streptococcus thermophilus strains and a single strain of Lactococcus lactis subsp. cremoris. In all positive S. thermophilus strains, the hsp was located on plasmids ranging from ca. 2.8 kb to 11 kb in size, while hsp was present in a 7.5-kb plasmid in Lactococcus lactis subsp. cremoris. Southern blots with a rep probe showed that all hsp16.4 ⁺ plasmids in S. thermophilus strains also shared homology with the replication function (rep) of pER341, suggesting the common origin of these plasmids. Received: 18 July 1998 / Accepted: 19 August 1998