Conjugal plasmid transfer between lactococci on solid surface matings and during cheese making

Abstract The concept of deliberate use of genetically enginereed microorganisms in dairy products requires a clear understanding of their behaviour and of the dissemination of introduced DNA in these strains. Thus, transfer of a self-transmissible plasmid and a non-self-transmissible but mobilizable plasmid from an engineered strain of Lactococcus lactis subsp. lactis IL 1403 to wild-type strains of L. lactis subsp. lactis and subsp. cremoris of technological interest was studied on standard solid surface matings and in cheese during manufacture. On solid surface matings, transfer of the conjugative plasmid occurred at frequencies ranging from < 2.3 × 10⁻⁹ to 2.8 × 10⁻⁴. Mobilization of the non-conjugative plasmid was observed at a lower frequency (ca. 10⁻⁵) in only one recipient which was then selected along with another recipient strain (presenting intermediate transfer frequencies) for making Camembert cheese. During cheese making, only the transfer of the self-transmissible plasmid was observed. It occurred in the early stages of manufacturing. The transfer frequencies were 7.0 × 10⁻⁸ or 7.6 × 10⁻¹ 1, depending upon the recipient strain. These were about 3 to 4 orders of magnitude lower than on solid surface matings. Mobilization of the non-conjugative plasmid was never detected in cheese.     

Transduction of concatemeric plasmids containing the cos site of Lactococcus lactis bacteriophage sk1

Lactococcus lactis bacteriophage sk1 can transduce plasmids containing the phage cos site and surrounding DNA sequences at frequencies as high as 2x10(-3) transductants per PFU. Deletion analysis demonstrated that the presence of phage DNA spanning cos and putative R sites were the most important for efficient plasmid transduction. Inserts of 440 bp containing cos and the R sites were sufficient to induce transduction frequencies of 10(-4) transductants per PFU. The role of the R1 site was investigated by altering 14 of the 19 bases in the site. This resulted in a two-fold decrease in transduction frequency compared to a 26-fold decrease in transduction following deletion of the entire site. It was demonstrated that transducing plasmids were packaged as linear trimeric concatemers commencing at the cos site.     

Plasmid involvement in the formation of a spontaneous bacteriophage insensitive mutant of Lactococcus lactis

Lactococcus lactis subsp. lactis biovar. diacetylactis DPC721 is a spontaneous bacteriophage insensitive mutant of strain DPC220, isolated after challenge with an industrial bacteriophage, phi D1. Plasmid analysis demonstrated that the bacteriophage insensitivity was associated with the absence of two native DPC220 plasmids (pAH82 and pAH33), and the presence of a novel plasmid (pAH90) in DPC721. The plasmids were transferred by conjugative mobilization to a plasmid free background where it was confirmed by restriction mapping that pAH90 is a co-integrate formed by the precise recombination of pAH82 and pAH33. The resistance phenotype encoded by pAH90 was also active against two bacteriophage homologous for the plasmid-free strain. Plasmid pAH90 was shown to encode at least two independent resistance mechanisms, including an adsorption-inhibition mechanism and a restriction and modification system. The adsorption-inhibition mechanism encoded by the co-integrate plasmid was specific for one of the phage used in this study.     

Distribution of insertion sequence IS1 in multiple-antibiotic resistant clinical Enterobacteriaceae strains

The presence of insertion sequence IS1 in 70 multiple-antibiotic resistant clinical strains was determined. This 70-strain collection comprised 46 Escherichia coli, 18 Salmonella and 6 Shigella strains. The presence of IS1 was detected in the chromosome and plasmids of 73% and 63% of the strains, respectively, and 51% of the strains carried IS1 in both. The frequency of IS1 was higher in Salmonella than in E. coli and Shigella strains. A total of 31 strains carried large plasmids with IS1; 10 of these strains (32.3%) were able to transfer all or some of the antibiotic resistance markers to E. coli K12 or S. typhimurium recipient strains. Resistance markers of all clinical strains were maintained stably after several generations of growth. The presence of IS1 in a relatively high percentage of plasmids of multiple-antibiotic resistant clinical isolates, suggests a role for this sequence in the dissemination of genes which code for antibiotic resistance.     

Mapping of insertion element IS30 in the Escherichia coli K12 chromosome

Summary We identified seven phage clones containing the insertion element IS30 in a phage library mini-set, which includes 476 clones carrying chromosomal segments that cover almost the entire chromosome ofEscherichia coli K12 W3110 (Kohara et al. 1987). We could assign locations and orientations to four copies of IS30 (namedis30A tois30D) on the W3110 chromosome by restriction analysis of phage DNAs containing them. These IS30s were present at the same locations in chromosomes of both W3110 and anotherE. coli K12 strain JE5519, and thus are assumed to be present in otherE. coli K12 derivatives, including early isolates. Among the IS30 copies found, one (is30B) contained a large deletion and possessed only a 181 by stretch of the right terminal region of IS30.EMBL Accession Number: The EMBL accession number of the sequence reported in this paper is X17345     

Characterisation of IS1126 from Porphyromonas gingivalis W83: a new member of the IS4 family of insertion sequence elements

Abstract The nucleotide sequence of IS 1126 , the only insertion sequence so far isolated from the oral pathogen Porphyromonas gingivalis , has been determined. It had a nucleotide sequence of 1338 base pair (bp) flanked by 12 bp perfect inverted repeats and generated a 5 bp target site duplication. The single major open reading frame encoded a predicted protein of 361 amino acids and molecular mass of 41 kDa. The gene encoding the transpsosase was subcloned into pUC18 and the transposase expressed in Escherichia coli minicells. The predicted amino acid sequence of the transposashad homology to putative transposases of IS 1106 and IS 1186 both of which belong to the IS 5 group within the IS₄ super-family of insertion elements. On the basis of this homology we propose that IS 1126 should also be included in the IS 5 group. Southern-blot analysis of a number of P. gingivalis strains using IS 1126 as a probe revealed a unique pattern of hybridisation for each strain and the absence of IS 1126 from other closely related Porphyromonas species. This should allow IS 1126 to be used as a rapid epidemiological tool in studying oral infections by P. gingivalis .     

Simultaneous conjugal transfer in Lactococcus to genes involved in bacteriocin production and reduced susceptibility to bacteriophages

Conjugal matings were performed between Lactococcus lactis DRC1 (a lactose-fermenting (Lac+), bacteriocin-producing (Bac+) strain) and L. lactis HID113 (Lac- and Bac-). Transconjugant derivatives of HID113 were identified on the basis of lactose fermentation, resistance to the DRC1 bacteriocin (dricin) or reduced sensitivity to phage sk1. Regardless of how they were identified, all transconjugants gave fewer and smaller plaques with phages c2 and sk1 than did HID113. All but one of 275 transconjugants tested also produced dricin, suggesting some functional relationship or close genetic linkage between the reduced phage sensitivity and dricin production and resistance. Some transconjugants were also Lac+, but this property was unstable.     

A new insertion sequence from Sinorhizobium meliloti with homology to IS1357 from Methylobacterium sp. and IS1452 from Acetobacter pasteurianus

The insertion sequence ISRm8 was identified by sequence analysis of the cryptic plasmid pRmeGR4b of Sinorhizobium meliloti GR4. ISRm8 is 1451 bp in length and carries 22/24-bp terminal imperfect inverted repeats with seven mismatches and a direct target site duplication of 3 bp. ISRm8 carries a unique open reading frame whose putative protein showed significant similarity to the insertion sequences IS1357 and IS1452, isolated from Methylobacterium sp. and Acetobacter pasteurianus, respectively. Two copies of this IS element were found in strain GR4; one of them is linked to plasmid pRmeGR4b, whereas the other is localized out of the non-pSym plasmids. In S. meliloti field populations ISRm8 shows a limited distribution (50% of the strains tested carry the IS element), with a copy number ranging from 1 to 6.     

Physical analysis of in vivo pCI301 fusion plasmids in Lactococcus lactis subsp. lactis

Abstract In vivo fusion plasmids identified following conjugative mobilization of pCI301, the 75-kilobase (kb) lactose-proteinase plasmid of Lactococcus lactis subsp. lactis UC317, were characterized. These plasmids (95 kb) were generated from fusion-deletion events involving pCI301 and the 38-kb UC317-derived cryptic plasmid, pCI303. Recombinant plasmids were separable into distinct classes based on their associated phenotypes and restriction maps. The formation of pCI301: : pCI303 composite plasmids within strain UC317 was also demonstrated.     

Genetic variability of the frameshift region in IS911 transposable elements from Escherichia coli clinical isolates

The IS911 bacterial transposable element has been analyzed for its mechanism of transposition and for the way it controls the expression of its genes by programmed -1 translational frameshifting. In the present study the prevalence of IS911 has been determined in the Enterobacteriaceae family and in other Gram-negative bacilli. Three variants, found in Escherichia coli clinical isolates and having mutations in the region implicated in frameshifting, were functionally characterized. All three were altered in their frameshifting and transposition abilities, suggesting that the frameshift region of IS911 may constitute a target for mutations reducing the transposition frequency of this mobile element in natural populations of E. coli.     

Nucleotide sequence of IS1542, an insertion sequence identified within VanA glycopeptide resistance elements of enterococci

IS1542 is an insertion sequence-like element which was originally identified in the orf2-vanR intergenic region of VanA resistance elements of glycopeptide-resistant Enterococcus faecium from hospital patients and from non-human sources in the UK. The nucleotide sequence of IS1542 was determined. It showed 81% homology with IS256 and contained an open reading frame sufficient to encode a 390 amino acid peptide predicted to have 87.2% identity with the transposase of IS256. By PCR, IS1542 was detected in the genomes of 26 of 28 (93%) VanA enterococci isolated from poultry in the UK and Ireland, but in only two of 65 (3%) glycopeptide-sensitive or VanC enterococci from hospital patients in the UK and Brazil. IS1542 may be a useful marker for evolutionary and epidemiological studies of VanA glycopeptide resistance in enterococci.     

Characterization of extrachromosomal replicons present in the extended host range Rhizobium sp. LPU83

In several rhizobia, bacteria that inhabit the soil in free-living conditions and associate in symbiosis with the root of legumes as nitrogen-fixing organisms, plasmid DNA can constitute a high percentage of the genome. We have characterized acid-tolerant isolates of rhizobia-here represented by the strain Rhizobium sp. LPU83-that have an extended nodulation-host range including alfalfa, the common bean, and Leucena leucocephala. In this study we analyzed the plasmids of R. sp. LPU83 in order to characterize their role in the evolution of Medicago symbionts and their involvement in symbiotic behavior. The pLPU83a plasmid was found to be transmissible with no associated phenotypic traits. The symbiotic plasmid pLPU83b could be transferred at very low frequencies under laboratory conditions only when pLPU83a was present; could restore nodulation to a strain cured of its symbiotic plasmid, S. meliloti A818; but could not restore the full nitrogen fixation associated with alfalfa.     

Novel insertion sequence-like elements in phytoplasma strains of the aster yellows group are putative new members of the IS3 family

Novel insertion sequence (IS)-like elements were isolated and characterized from phytoplasma strains in the aster yellows (AY) group (16SrI). The IS-like elements were cloned from phytoplasma strains AY1 and NJAY or PCR-amplified from 15 additional strains representing nine subgroups in the AY group using primers based on sequences of the putative transposases (Tpases). All IS-like elements contained sequences encoding similar Tpases of 321 amino acids (320 for strain CPh). Substantial amino acid sequence variability suggested multiple species of Tpases or IS-like elements exist in the AY phytoplasma group. These Tpases have an identical DDE motif that is most similar to the DDE consensus of Tpases in the IS3 family.     

Variability in the distribution and composition of insertion sequence-like elements in strains of Mycoplasma fermentans

Mycoplasma fermentans has been reported to be pathogenic for man. All fourteen strains tested contain an insertion sequence-like element (ISLE) which may be present in multiple copies. To determine whether ISLE copies are similarly distributed in different strains of M. fermentans, restriction enzyme digest fragments of genomic DNA from 14 isolates, from a variety of sources, were separated by electrophoresis, blotted and hybridized to a biotin labelled polymerase chain reaction (PCR) amplified fragment of ISLE. A range of patterns was observed suggesting that the element has a tendency to undergo rearrangement within the genome. Analysis of ISLE sequences revealed inter- and intra-strain polymorphisms.