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| J Bacteriol, 1989 May, 171(5), 2795 - 802 A maturation protein is essential for production of active forms of Lactococcus lactis SK11 serine proteinase located in or secreted from the cell envelope; Vos P et al.; The complete nucleotide sequence of a gene located immediately upstream of the Lactococcus lactis subsp . cremoris SK11 prtP gene encoding the cell envelope-attached proteinase was determined . This gene, designated prtM, was found to be transcribed from the same promotor region as was the proteinase gene but in the opposite direction . The prtM gene directed the expression in Escherichia coli of a protein with a size similar to the expected value of 33 kilodaltons, as deduced from the nucleotide sequence data . The derived amino acid sequence of the PrtM protein indicated the presence of a consensus lipoprotein signal sequence at the N terminus, which suggested that PrtM is a lipoprotein . Plasmids containing the prtM gene, the prtP gene, or both were constructed . Expression studies of L . lactis clones containing these plasmids showed that the prtM gene encodes a trans-acting activity involved in the maturation of cell envelope-located and -secreted forms of the SK11 proteinase. J Clin Microbiol, 1989 Apr, 27(4), 731 - 4 Identification of Enterococcus species isolated from human infections by a conventional test scheme; Facklam RR et al.; Streptococci (206 cultures) previously identified as enterococci were retrieved from storage and reidentified by using tests designed to identify species of the genus Enterococcus . Of these 188, 91% were correctly identified as Enterococcus species . Of the remaining strains, nine (4%) were unidentified and six (3%) and 3 (1.5%) were identified as Leuconostoc sp . and Lactococcus sp., respectively . Two new Enterococcus species were discovered: E . raffinosus and E . solitarius . DNA-DNA hybridizations were performed on selected strains to assure correct identification . Cultures representing 10 of the 12 Enterococcus species were among the 188 strains identified . An identification system based on the grouping of key reactions of 20 phenotypic characteristics of Enterococcus species is described. J Bacteriol, 1989 Mar, 171(3), 1453 - 8 Transport of basic amino acids by membrane vesicles of Lactococcus lactis; Driessen AJ et al.; The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system . In the presence of ascorbate N,N,N'N'-tetramethylphenylenediamine-cytochrome c as the electron donor, these fused membranes accumulated lysine but not ornithine or arginine under aerobic conditions . The mechanism of energy coupling to lysine transport was examined in membrane vesicles of L . lactis subsp . cremoris upon imposition of an artificial electrical potential (delta psi) or pH gradient or both and in fused membranes of these vesicles with cytochrome c oxidase liposomes in which the delta psi and delta pH were manipulated with ionophores . Lysine uptake was shown to be coupled to the PMF and especially to the delta psi, suggesting a proton symport mechanism . The lysine carrier appeared to be specific for L and D isomers of amino acids with a guanidine or NH2 group at the C6 position of the side chain . Uptake of lysine was blocked by p-chloromercuribenzene sulfonic acid but not by maleimides . Counterflow of lysine could not be detected in L . lactis subsp . cremoris, but in the arginine-ornithine antiporter-containing L . lactis subsp . lactis, rapid counterflow occurred . Homologous exchange of lysine and heterologous exchange of arginine and lysine were mediated by this antiporter . PMF-driven lysine transport in these membranes was noncompetitively inhibited by arginine, whereas the uptake of arginine was enhanced by lysine . These observations are compatible with a model in which circulation of lysine via the lysine carrier and the arginine-ornithine antiporter leads to accumulation of arginine. Appl Environ Microbiol, 1989 Mar, 55(3), 604 - 10 Genetic transformation of intact Lactococcus lactis subsp . lactis by high-voltage electroporation; McIntyre DA et al.; To apply recombinant DNA techniques for genetic manipulation of the industrially important lactococci, an efficient and reliable high-frequency transformation system must be available . High-voltage electric pulses have been demonstrated to enhance uptake of DNA into protoplasts and intact cells of numerous gram-negative and gram-positive microorganisms . The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp . lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit (BTX Transfector 100; BTX, Inc., San Diego, Calif.) . Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse . Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 microseconds to 1 s) . Transformation efficiencies of 10(3) transformants per microgram of DNA were obtained when dense suspensions (final concentration, 5 x 10(10) CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA . Dilution of porated cells in broth medium followed by an expression period of 2 h at 30 degrees C was beneficial in enhancing transformation efficiencies . Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure. Appl Environ Microbiol, 1989 Feb, 55(2), 394 - 400 Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp . lactis; Leenhouts KJ et al.; Integrable vectors were constructed based on the plasmid pHV60, which is essentially a pBR322 replicon carrying a chloramphenicol resistance marker, by inserting 1.3-kilobase chromosomal fragments of Lactococcus lactis subsp . lactis MG1363 into this plasmid . Three constructs as well as pHV60 were electroporated to strain MG1363 . Transformants were obtained with all constructs, and also with pHV60 (albeit with low frequency) . By using Southern hybridizations, it appeared that pHV60 showed homology with the chromosome of MG1363, and that it most probably uses this homology to integrate in a Campbell-like manner . The presence of chromosomal sequences in pHV60 stimulated insertion elsewhere in the chromosome by a factor of 5 to 100 . In all cases the integrated plasmids were amplified, at a selective pressure of 5 micrograms of chloramphenicol per ml, to a level of approximately 15 copies per chromosome . Although the amplification was gradually lost under nonselective conditions, one copy remained stably integrated in the chromosome . The results show that a Campbell-like integration strategy can be used to improve the accessibility of the lactococcal chromosome for genetic analysis and is potentially useful in stabilizing unstable genes in lactococci. Crit Rev Microbiol, 1989, 16(6), 419 - 76 Bioenergetics and solute transport in lactococci; Konings WN et al.; During the last few years the studies about the physiology and bioenergetics of lactic acid bacteria during growth and starvation have evolved from a descriptive level to an analysis of the molecular events in the regulation of various processes . Considerable progress has been made in the understanding of the modes of metabolic energy generation, the mechanism of homeostasis of the internal pH, and the mechanism and regulatory processes of transport systems for sugars, amino acids, peptides, and ions . Detailed studies of these transport processes have been performed in cytoplasmic membrane vesicles of these organisms in which a foreign proton pump has been introduced to generate a high proton motive force. Appl Environ Microbiol, 1989 Jan, 55(1), 224 - 8 Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp . lactis; van de Guchte M et al.; A pair of vectors for expression of heterologous genes in Lactococcus lactis was constructed . In addition to an origin of replication that has a broad host range, these vectors contain a multiple cloning site flanked by gene expression signals originating from L . lactis subsp . cremoris Wg2 . The two vectors, about 3.7 kilobase pairs in size, differ only in the type of antibiotic resistance they confer to their hosts . pMG36 carries a kanamycin resistance marker, which was replaced by an erythromycin resistance marker in pMG36e . As an example of the use of these vectors, the hen egg white lysozyme-coding sequence was inserted . A fusion protein of the expected size was detected in a transformed L . lactis subsp . lactis strain by using Western blotting (immunoblotting). J Bacteriol, 1989 Jan, 171(1), 292 - 8 Mechanism and energetics of dipeptide transport in membrane vesicles of Lactococcus lactis; Smid EJ et al.; Alanyl-alpha-glutamate transport has been studied in Lactococcus lactis ML3 cells and in membrane vesicles fused with liposomes containing beefheart cytochrome c oxidase as a proton-motive-force-generating system . The uptake of Ala-Glu observed in de-energized cells can be stimulated 26-fold upon addition of lactose . No intracellular dipeptide pool could be detected in intact cells . In fused membranes, a 40-fold accumulation of Ala-Glu was observed in response to a proton motive force . Addition of ionophores and uncouplers resulted in a rapid efflux of the accumulated dipeptide, indicating that Ala-Glu accumulation is directly coupled to the proton motive force as a driving force . Ala-Glu uptake is an electrogenic process and the dipeptide is transported in symport with two protons . In both fused membranes and intact cells the same affinity constant (0.70 mM) for Ala-Glu uptake was found . Accumulated Ala-Glu is exchangeable with externally added alanyl-glutamate, glutamyl-glutamate, and leucyl-leucine, while no exchange occurred upon addition of the amino acid glutamate or alanine . These results indicate that the Ala-Glu transport system has a broad substrate specificity. Biochimie, 1988 Apr, 70(4), 535 - 42 Utilization of dipeptides by Lactococcus lactis ssp . cremoris; van Boven A et al.; Different strains of Lactococcus lactis ssp . cremoris hydrolyze peptides at different rates while the cell-free extracts of these strains all show the same or much higher rates of hydrolysis . These observations indicate that the uptake of peptides is the rate-limiting step in peptide hydrolysis . Utilization of leucyl-leucine by non-growing cells is competitively inhibited by the structurally related dipeptide alanyl-alanine . After hydrolysis of peptides, the amino acids are released into the medium and only a small fraction is accumulated and/or incorporated . This hydrolysis is independent of the synthesis of proteases indicating that the synthesis of proteases and peptidases are regulated differently . The specific growth rate of L . lactis ssp . cremoris E8 depends upon the amino acid source in the medium . No significant differences have been observed in the intracellular peptidase activities and the rates of peptide uptake between L . lactis ssp . cremoris E8 cells grown in different media, indicating that this growth rate is determined by the availability of amino acids in free amino acids or peptides. Biochimie, 1988 Apr, 70(4), 519 - 21 New nomenclature of the non-rod-shaped lactic acid bacteria; Sandine WE; Bacteria of the Lactococcus (formerly lactic Streptococcus) genus are described to emphasize certain less well-known facts of importance in their successful use as dairy starters . Other streptococci (non-rod forms) are briefly characterized to emphasize their usefulness in industrial milk fermentations. Biochimie, 1988 Mar, 70(3), 451 - 6 Loss of phage resistance encoded by plasmid pSK112 in chemostat cultures of Lactococcus lactis ssp . cremoris SK110; Sterkenburg A et al.; In cultures of L . lactis ssp . cremoris SK110, phage SK11G-resistant through the presence of pSK112, phage-sensitive variants segregated spontaneously that lacked the plasmid . In overnight batch culture these comprised up to 1% of the total population . Upon prolonged incubation in chemostat culture, a further loss of resistance was observed after a lag period . At high growth rates (0.7 h-1) this period amounted to approximately 35 generations, whereas cultures grown at rates of 0.4 and 0.1 h-1 remained resistant for 55 and 70 generations, respectively . At average-to-high growth rate, characteristics of the partially mixed populations that evolved were comparable to those of pure cultures of L . lactis ssp . cremoris SK110 . However, in the culture fluid of the mixed populations that occurred at growth rate 0.1 h-1, higher acetate and formate concentrations were found than in the fluid of pure cultures of L . lactis ssp . cremoris SK110 . This indicated that the former metabolized lactose more efficiently . Competition experiments between the resistant strain and a cured, sensitive derivative, L . lactis ssp . cremoris SK112, gave stable mixed populations . It is concluded that at average-to-high growth rates, loss of resistance from cultures of L . lactis ssp . cremoris SK110 had occurred due to instability of the plasmid and not to a competitive disadvantage of the resistant strain towards emerging sensitive variants. Biochimie, 1988 Mar, 70(3), 437 - 42 Plasmid-encoded functions of ropy lactic acid streptococcal strains from Scandinavian fermented milk; Neve H et al.; Ropy Streptococcus (Lactococcus) cremoris strains isolated from a ropy Swedish sour milk ("longfil") and a ropy Finnish milk product ("Viili") were screened for their plasmid-encoded functions . Curing experiments strongly indicated that the ropy phenotype was linked to a 17-Md plasmid in the Swedish strains and to a 30 Md plasmid in the Finnish strains . Comparative restriction endonuclease analysis and DNA/DNA-hybridization studies revelated that plasmids from both strain families shared homologous DNA regions . Though be Swedish ropy strains harbored a conjugative 45-Md lactose plasmid, no cotransfer of the 17 Md plasmid occurred.
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