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| J Biol Chem, 1989 Aug 15, 264(23), 13579 - 85 Primary structure and organization of the gene for a procaryotic, cell envelope-located serine proteinase; Vos P et al.; We have determined the complete nucleotide sequence of the gene for the cell envelope-located proteinase of Lactococcus lactis SK11 . The gene contains a very AT-rich promoter region followed by the coding sequence of a protein of 1962 amino acids . Comparison of the NH2-terminal amino acid sequence of the mature proteinase and the expected primary translation product of the proteinase gene indicates that the enzyme is probably synthesized as a pre-pro-protein . This is confirmed by expression studies of the proteinase gene in Escherichia coli . The amino acid sequence of the proteinase shows significant homology to a number of serine proteinases of the subtilisin family . Compared with the related proteinase of L . lactis Wg2, the proteinase of L . lactis SK11 contains a 60-amino acids duplication and a total of 44-amino acid substitutions, some of which may account for the different cleavage specificity of both enzymes . Furthermore, a region was identified in the Lactococcus proteinase, which shows homology to the membrane-anchoring domains of a number of proteins from other Gram-positive bacteria. Antonie Van Leeuwenhoek, 1989 Aug, 56(2), 139 - 60 Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria; Driessen AJ; Lactococci are fastidious bacteria which require an external source of amino acids and many other nutrients . These compounds have to pass the membrane . However, detailed analysis of transport processes in membrane vesicles has been hampered by the lack of a suitable protonmotive force (pmf)-generating system in these model systems . A membrane-fusion procedure has been developed by which pmf-generating systems can be functionally incorporated into the bacterial membrane . This improved model system has been used to analyze the properties of amino acid transport systems in lactococci . Detailed studies have been made of the specificity and kinetics of amino acid transport and also of the interaction of the transport systems with their lipid environment . The properties of a pmf-independent, arginine-catabolism specific transport system in lactococci will be discussed. Mol Gen Genet, 1989 Aug, 218(2), 214 - 21 Cloning and DNA sequence analysis of a Lactococcus bacteriophage lysin gene; Shearman C et al.; A gene for the lysin of Lactococcus lactis bacteriphage phi vML3 was cloned using an Escherichia coli/bacteriophage lambda host-vector system . The gene was detected by its expression of antimicrobial activity against L . lactis cells in a bioassay . The cloned fragment was analysed by sub-cloning on to E . coli plasmid vectors and by restriction endonuclease and deletion mapping . Its entire DNA sequence was determined and an open reading frame for the lysin structural gene was identified . The sequenced lysin gene would express a protein of 187 amino acids with a molecular weight of 21,090, which is in good agreement with that of a protein detected after in vitro transcription and translation of DNA encoding the gene . Expression of the lysin gene in E . coli and B . subtilis from an adjacent bacteriophage promoter was readily detected but in L . lactis expression of lysin was found to be lethal . The bacteriophage phi vML3 lysin had sequence homology with protein 15 of B . subtilis bacteriophage PZA . This protein is involved in DNA packaging during bacteriophage maturation rather than in host cell lysis . The cloning and analysis of the phi vML3 lysin gene is of importance in further understanding lactic streptococcal bacteriophages, for the development of positive selection vectors and for biotechnological applications of relevance to the dairy industry. Plasmid, 1989 Jul, 22(1), 44 - 51 Characterization of the genetic element coding for lactose metabolism in Lactococcus lactis subsp . lactis KP3; Steele JL et al.; The Lactococcus lactis subsp . lactis KP3 Lac genetic element was investigated . KP3 is a lactose-positive (Lac+) transconjugant which contains no detectable plasmid DNA . The KP3 Lac genetic element was self-transmissible (Tra+) and encoded a reduced bacteriophage sensitivity (Rbs+) phenotype . Matings of KP3 with a recombination-deficient (Rec-) recipient resulted in Lac+ transconjugants which were phenotypically indistinguishable from KP3 and contained a 96-MDa plasmid (pJS96) . Phenotypic and physical analyses of pJS96 indicated that it was a deletion derivative of a putative pKB32::pJS88 Lac+ Tra+ cointegrate . pKB32 is the Lac plasmid and pJS88 is the Tra+ Rbs+ plasmid in L . lactis subsp . lactis 11007, the donor used in obtaining KP3 . The results presented suggest that pJS96 is an episome, since it appeared to replicate both as a plasmid and as an integrated part of the chromosome . Conjugal transfer of chromosomal DNA mediated by pJS96 was not observed . Conjugal transfer of pJS96 resulted in Lac+ transconjugants containing plasmids ranging in size from 21 to 90 MDa . Only in Rec+ recipients were transconjugants isolated which appeared to contain pJS96 integrated into the host chromosome . Restriction analysis of several plasmids in the 21 to 90 MDa range suggested the deletions were due to intramolecular transposition of a transposable element on pJS96 . This report suggests that a self-transmissible episome exists in KP3 and provides an explanation of how plasmids which vary in size yet encode similar phenotypes may be formed and disseminated. Plasmid, 1989 Jul, 22(1), 32 - 43 Conjugal transfer of genetic material by Lactococcus lactis subsp . lactis 11007; Steele JL et al.; Conjugal transfer of genetic material by Lactococcus lactis subsp . lactis 11007 was examined . A plasmid of 88 MDa (pJS88) was identified in addition to the previously reported conjugally transferred plasmids of 32 (pKB32) and 4.8 MDa . Proteinase activity, reduced bacteriophage sensitivity, bacteriocin resistance, and conjugal transfer ability were encoded by pJS88 . The ability to metabolize lactose (Lac+) was encoded by pKB32, and the 4.8-MDa plasmid was cryptic . When a strain containing both pKB32 and pJS88 was mated with a recipient deficient in host-mediated homologous recombination (Rec-), a plasmid of 40 MDa (pJS40) was observed in approximately 50% of the Lac+ transconjugants . DNA-DNA hybridization results indicated that pJS40 contained homology with both pKB32 and pJS88 . These results indicated that pKB32 was conjugally transferred via conduction and suggested that pJS40 is a deletion derivative of a pKB32::pJS88 cointegrate . A Rec- strain containing pKB32 and pJS88 mediated Lac+ conjugal transfer, suggesting that the pKB32::pJS88 cointegrate could form via a rec-independent event . Resolution of the pKB32::pJS88 cointegrate was observed in both Rec- and Rec+ hosts . Cointegrate formation and resolution via rec-independent mechanisms suggest the involvement of a transposable element in the Tn3 family. Mutagenesis, 1989 Jul, 4(4), 280 - 2 Response of Lactococcus (Streptococcus) lactis to N-methyl-N'-nitro-N-nitrosoguanidine: absence of adaptive response; Auffray Y et al.; Pretreatment of cells of Lactococcus lactis subsp . lactis with low levels of N-methyl-N'-nitro-N-nitrosoguanidine does not reduce the cytotoxic and mutagenic effects caused by high concentration of this agent . This observation indicates that there is no efficient inducible error-free repair system for alkylation damage similar to the 'adaptive response' described in detail for Escherichia coli. Appl Environ Microbiol, 1989 Jul, 55(7), 1769 - 74 Insertion and amplification of foreign genes in the Lactococcus lactis subsp . lactis chromosome; Chopin MC et al.; The plasmid pE194 is unable to replicate in Lactococcus lactis subsp . lactis (formerly Streptococcus lactis) . When linked to resident bacteriophage sequences, pE194 was able to integrate into the L . lactis subsp . lactis chromosome either by Campbell-like recombination or by double crossing over with deletion . Integration occurred into the DNA of the prophage and prevented its multiplication . When a selective pressure was applied to an integrant in which pE194 was flanked by two direct repeats of prophage fragment, amplification of pE194 and the prophage fragment was observed . The pE194 copy number was assessed at six to nine, and amplification was stable upon growth under nonselective conditions. Appl Environ Microbiol, 1989 Jul, 55(7), 1684 - 9 Localization, cloning, and expression of genetic determinants for bacteriophage resistance (Hsp) from the conjugative plasmid pTR2030; Hill C et al.; Genetic determinants for a bacteriophage resistance mechanism (Hsp+) encoded by plasmid pTR2030 (46.2 kilobases {kb}) were localized by mapping an 11.5-kb deletion that accompanied the transition of Lactococcus lactis LMA12-4 transconjugants (M . E . Sanders, P . J . Leonard, W . D . Sing, and T . R . Klaenhammer, Appl . Environ . Microbiol . 52:1001-1007, 1986) from phage resistance to phage sensitivity . The deleted 34.7-kb replicon (pTR2023, Hsp-) retained its conjugative ability, demonstrating that the phage resistance and conjugal transfer determinants were genetically distinct . The Hsp region of pTT2030, which was contained within a 13.6-kb BglII fragment, was cloned into the BamHI site of bacteriophage lambda EMBL3, and Hsp was subcloned into the Escherichia coli-Streptococcus shuttle vector pSA3 . The recombinant plasmids pTK6 and pTK9 were recovered in E . coli HB101 and contained a 13.6-kb insert in opposite orientations . L . Lactis MG1363 transformants carrying pTK6 or pTK9 exhibited a significant reduction in plaque size, in addition to a slight reduction in the efficiency of plaquing for both prolate and small isometric phages . Phenotypic reactions observed for the recombinant plasmids suggest that pTR2030-encoded Hsp acts similarly against both prolate and small isometric phages . Tn5 mutagenesis was used to define the region essential for the expression of the Hsp+ phenotype . Any of four insertions within a 3-kb region resulted in the loss of phage resistance, whereas a further 26 insertions outside this locus had no effect on Hsp expression . In vitro deletion analysis confirmed that the 3-kb region contained all the information necessary for the observed resistance. J Biol Chem, 1989 Jun 25, 264(18), 10361 - 70 Kinetic mechanism and specificity of the arginine-ornithine antiporter of Lactococcus lactis; Driessen AJ et al.; The kinetic mechanism and specificity of the arginine-ornithine antiporter was investigated in membrane vesicles derived from Lactococcus lactis . Membrane vesicles loaded with ornithine, and diluted into an arginine-free medium, rapidly released a limited amount of ornithine during the first seconds of incubation . The amount of ornithine released was independent of the amount initially present on the inside and roughly matched the number of ornithine-binding sites in the membrane . Net flow of ornithine was only observed in membrane vesicles derived from induced cells and blocked by p-chloromercuribenzene sulfonic acid . These results suggest that net flow of ornithine is caused by a single turnover of the antiporter . With saturating concentrations of arginine in the external medium, efflux of ornithine was stoichiometrically coupled to uptake of arginine . Arginine-ornithine exchange and net flow of ornithine are electrically silent and not regulated by the electrical potential . The kinetics of the homologous exchange reactions indicate that the Vmax values for arginine and ornithine uptake are comparable, whereas the apparent Kt values differ . No major sidedness of the apparent Kt values are observed for both surfaces of the cytoplasmic membrane . Various basic amino acid analogues, including optical isomers, are transported as well, albeit with different efficiencies (Vmax/Kt) . Evidence for a competitive character of arginine and ornithine interactions for binding sites on the antiporter are provided by transport and binding measurements . The Vmax and apparent Kt for arginine uptake increases with increasing internal ornithine, with little effect on the ratio of Vmax to apparent Kt . These results are discussed in terms of a simple carrier model in which the substrate-binding site is presented alternately to the two surfaces of the membrane as in a Ping Pong mechanism for enzyme kinetics. Appl Environ Microbiol, 1989 Jun, 55(6), 1483 - 9 Plasmid transformation by electroporation of Leuconostoc paramesenteroides and its use in molecular cloning; David S et al.; In this report, we demonstrate the utility of electroporation as an efficient method for genetic transformation of Leuconostoc paramesenteroides . We optimized several factors which determine the transformation frequency, resulting in transformation efficiencies of up to 4 x 10(3) transformants per micrograms of pNZ12 DNA, which contains the promiscuous Lactococcus lactis pSH71 replicon . Slightly lower efficiencies were obtained with a deletion derivative of the broad-host-range plasmid pAM beta 1 . These plasmids could be stably maintained in L . paramesenteroides NZ6009 for more than 100 generations, even in the absence of selective pressure . In order to show the use of the developed host-vector system, we cloned the Lactococcus lactis gene encoding phospho-beta-galactosidase in L . paramesenteroides . Expression of this heterologous gene in L . paramesenteroides under control of Lactococcus lactis expression signals was evident from the presence, in transformants, of phospho-beta-galactosidase activity and a specific phospho-beta-galactosidase protein band on Western blots (immunoblots) . In addition, we transformed a lactose-deficient derivative of L . paramesenteroides with a plasmid carrying a Lactococcus lactis-Escherichia coli lacZ gene fusion . The resulting transformants synthesized high levels of beta-galactosidase, indicating the efficiency of heterologous gene expression signals in L . paramesenteroides. Appl Environ Microbiol, 1989 May, 55(5), 1187 - 91 Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid; van Belkum MJ et al.; Lactococcus lactis subsp . cremoris 9B4 plasmid p9B4-6 (60 kilobases {kb}), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1 . Two regions on p9B4-6 were identified which specify inhibitory activity on L . lactis indicator strains: one that could be confined to a 1.8-kb ScaI-ClaI fragment with low antagonistic activity and a 15-kb XbaI-SalI fragment specifying high antagonistic activity . The inhibitory substances produced by these two clones were sensitive to proteolysis . A 4-kb HindIII fragment derived from the 15-kb fragment strongly hybridized with the 1.8-kb fragment . The antagonistic activity specified by the 4-kb fragment was somewhat reduced as compared with that of the 15-kb fragment . A 1.3-kb ScaI-HindIII subfragment of the 4-kb fragment contained both the immunity and bacteriocin genes . Inhibition studies showed that the two bacteriocins had different specificities. J Bacteriol, 1989 May, 171(5), 2789 - 94 Identification of a gene required for maturation of an extracellular lactococcal serine proteinase; Haandrikman AJ et al.; Directly upstream of the Lactococcus lactis subsp . cremoris Wg2 proteinase gene is an oppositely directed open reading frame (ORF1) . The complete nucleotide sequence of ORF1, encoding a 33-kilodalton protein, was determined . A protein of approximately 32 kilodaltons was synthesized when ORF1 was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter . L . lactis subsp . lactis MG1363 transformants carrying the proteinase gene but lacking ORF1 were phenotypically proteinase deficient, unlike transformants carrying both the proteinase gene and ORF1 . Synthesis and secretion of proteinase antigen by L . lactis could be detected with proteinase-directed monoclonal antibodies regardless of whether ORF1 was present . The requirement of ORF1 for proteinase activation was reflected in a reduction in the molecular weight of the secreted proteinase . Furthermore, deletion of the 130 C-terminal amino acids of the Wg2 proteinase prevented attachment of the enzyme to lactococcal cells.
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