Novel sucrose transposons from plant strains of Lactococcus lactis (original) (raw)
Related papers
sacA and nisA genes are not always linked in Lactococcus lactis subsp. lactis strains
FEMS Microbiology Letters, 2000
Sixty-seven lactococcal strains arising from dairy habitat were screened for the presence of the sucrose 6-phosphate hydrolase gene by polymerase chain reaction. Of the strains tested, 35.8% were able to ferment sucrose as well as to harbour the sucrose-6-phosphate hydrolase gene, even though they were unable to produce nisin as well as to show the nisin structural gene. After pulsed-field gel electrophoresis and hybridisation all Suc Nis 3 strains exhibited physical linkage between sacA gene and the left end of lactococcal transposons (Tn5276 or Tn5301) without linkage to nisin genes. However, we were unable to transfer the sacA gene as well as to detect Suc 3 derivatives from Suc Nis 3 strains after conjugation and curing experiments. z : S 0 3 7 8 -1 0 9 7 ( 9 8 ) 0 0 5 7 1 -0
Insertion of Transposon Tn 917 Derivatives into the Lactococcus lactis subsp. lactis Chromosome
Applied and Environmental Microbiology, 1993
Two transposition vectors, pTV32 and pLTV1, containing transposon Tn 917 derivatives TV32 and LTV1, respectively, were introduced into Lactococcus lactis subsp. lactis MG1614. It was found that pTV32 and pLTV1 replicate and that TV32 and LTV1 transpose in this strain. A protocol for production of a collection of Tn 917 insertions in L. lactis subsp. lactis was developed. The physical locations of TV32 on the chromosomal Sma I fragments of 62 independent transpositions were established by pulsed-field gel electrophoresis. These transpositions could be divided into at least 38 different groups that exhibited no Tn 917 -dominating hot spots on the L. lactis subsp. lactis chromosome. A total of 10 of the 62 transpositions resulted in strains that express β-galactosidase. This indicates that there was fusion of the promoterless lacZ of the Tn 917 derivatives to a chromosomal promoter. Thus, the Tn 917 -derived transposons should be powerful genetic tools for studying L. lactis subsp. lac...
Applied and Environmental Microbiology
Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip' Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip' Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10-7 to 10-8 per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip' Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than ifiter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.
Transfer of Sucrose-Fermenting Ability and Nisin Production Phenotype among Lactic Streptococci
Applied and environmental microbiology, 1985
Transfer of sucrose fermentation ability, nisin production, and nisin resistance from Streptococcus lactis to S. lactis and Streptococcus lactis subsp. diacetylactis occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Transconjugants were able to act as donors to transfer the Suc-Nis phenotype in subsequent mating. No changes in sensitivity to lytic phage c2 were noted in S. lactis transconjugants. However, temperature-independent restriction of lytic phage 18-16 was noted in transconjugants of S. lactis subsp. diacetylactis 18-16. Adsorption studies with phage-resistant transconjugants showed that resistance was not due to lack of adsorption by the lytic phage. Physical evidence for the presence of introduced plasmid DNA was not found in lysates of transconjugants.
Journal of Applied Microbiology, 2009
Aims: This paper describes optimization of electrotransformation of Mu transposition complexes into Lactococcus lactis cells and identification of genes affecting nisin production. Methods and Results: The highest transformation efficiency, 1AE1 • 10 2 transformants lg)1 of input transposon DNA, was achieved when cells were grown to an OD 600 of 0AE5 in the presence of 1AE5% of glycine and treated with 20 lg ml)1 ampicillin for 60 min. Three insertions affecting nisin production, which were identified at nisB, fhuR, and rpiA genes, were screened from a library of 2000 erythromycin-resistant transformants using a nisin bioassay method. NisB is part of the nisin biosynthetic machinery, explaining the loss of nisin production in nisB mutant. FhuR is a transcription regulator involved in sulphur acquisition. Inactivation of fhuR presumably results in a low cellular cystein level, which affects nisin biosynthesis that involves utilization of cystein. RpiA is involved in pentose phosphate pathway and carbon fixation. The rpiA mutant showed reduction in nisin production and slow growth rate. Conclusions: The results showed that Mu transposition complex mutagenesis can be used to identify genes in L. lactis. Three genes involved in nisin production were identified. Significance and Impact of the Study: Expanding the Mu transposition-based mutagenesis to Lactococci adds a new tool for studies of industrially important bacteria.
Efficient insertional mutagenesis in Lactococci and other Gram-positive bacteria
Journal of Bacteriology
In lactococci, the study of chromosomal genes and their regulation is limited by the lack of an efficient transposon mutagenesis system. We associated the insertion sequence ISS1 with the thermosensitive replicon pG ؉ host to generate a mutagenic tool that can be used even in poorly transformable strains. ISS1 transposition is random in different lactococcal strains as well as in Enterococcus faecalis and Streptococcus thermophilus. High-frequency random insertion (of about 1%) obtained with this system in Lactococcus lactis allows efficient mutagenesis, with typically one insertion per cell. After ISS1 replicative transposition, the chromosome contains duplicated ISS1 sequences flanking pG ؉ host. This structure allows cloning of the interrupted gene. In addition, efficient excision of the plasmid leaves a single ISS1 copy at the mutated site, thus generating a stable mutant strain with no foreign markers. Mutants obtained by this transposition system are food grade and can thus be used in fermentation processes.
Molecular Microbiology, 1991
In matings between Lactococcus tactis strains, the conjugative transposons Tn9/6and Tn919are found in the chromosome of the transconjugants in the same place as in the chromosome of the donor, indicating that no transposition has occurred. In agreement with this, the frequency of L. lactis transconjugants from intraspecies matings is the same whether the donor contains the wild-type form of the transposon or the mutant 7n916-int1, which has an insertion in the transposon's integrase gene. However, in intergeneric crosses with Baciiius subtiiis or Enterococcus faecaiis donors, Tn916 and Tn919 transpose to different locations on the chromosome of the L. iactis transconjugants. Moreover, Jn916 and Tn919 could not be transferred by conjugation from L. iactis and B. subtiiis, E. faecaiis or Streptococcus pyogenes. This suggests that excision of these elements does not occur in L. iactis. When cloned into E. coii with adjacent chromosomal DNA from L. iactis, the conjugative transposons were able to excise, transpose and promote conjugation. Therefore, the inability of these elements to excise in L. iactis is not caused by a permanent structural alteration in the transposon. We conclude that L. iactis lacks a factor required for excision of conjugative transposons.
Conjugative transfer of raffinose metabolism in Lactococcus lactis
FEMS Microbiology Letters, 1998
Lactococcus lactis subsp. lactis strains isolated from various sprouted seed products were able to transfer the ability to ferment raffinose in conjugation experiments at frequencies between 10 3R and 10 3U per donor cell. There was no evidence of plasmid transfer, but pulsed-field gel electrophoresis analysis showed that all transconjugants had acquired large chromosomal insertions indicative of conjugative transposons. Raffinose transconjugants contained inserts of 45 or 60 kb at one of two chromosomal sites, and these inserts contained two copies of an element related to the lactococcal insertion sequence ISS1. z
Fems Microbiology Letters, 1996
Sixty-four lactococcal strains isolated from natural whey starters were screened for the presence of the nisin structural gene by polymerase chain reaction. Seven of them showed a specific PCR product of 320 bp; only two produced antagonistic activity and were resistant to nisin. Southern blots of SnzaI-digested DNA from PCR-positive strains hybridized with a nisA probe displayed a location of the gene on different SmaI fragments. Among PCR-positive strains. nisin producers showed specific transcript after reverse transcriptase-PCR, as well as some non-nisin-producing strains. The RT-PCR product could not be shown in one non-nisin-producing PCR-positive strain.
Natural DNA transformation is functional in Lactococcus lactis ssp. cremoris KW2
Applied and environmental microbiology, 2017
Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis ssp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the ...