Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases.     

Dissolution of xylose metabolism in Lactococcus lactis

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl(-). Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl(+) strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose.     

Genetic and transcriptional analysis of a novel plasmid-encoded copper resistance operon from Lactococcus lactis

A plasmid-borne copper resistance operon (lco) was identified from Lactococcus lactis subsp. lactis LL58-1. The lco operon consists of three structural genes lcoABC. The predicted products of lcoA and lcoB were homologous to chromosomally encoded prolipoprotein diacylglyceral transferases and two uncharacterized proteins respectively, and the product of lcoC is similar to several multicopper oxidases, which are generally plasmid-encoded. This genetic organization represents a new combination of genes for copper resistance in bacteria. The three genes are co-transcribed from a copper-inducible promoter, which is controlled by lcoRS encoding a response regulator and a kinase sensor. The five genes are flanked by two insertion sequences, almost identical to IS-LL6 from L. lactis. Transposon mutagenesis and subcloning analysis indicated that the three structural genes were all required for copper resistance. Copper assay results showed that the extracellular concentration of copper of L. lactis LM0230 containing the lco operon was significantly higher than that of the host strain when copper was added at concentrations from 2 to 3 mM. The results suggest that the lco operon conferred copper resistance by reducing the intracellular accumulation of copper ions in L. lactis.     

Identification of Lactococcus lactis genes required for bacteriophage adsorption

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and phi 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that phi645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that phi 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and phi 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and phi 645 that showed no homology in the C-terminal part.     

Duplication of the pepF gene and shuffling of DNA fragments on the lactose plasmid of Lactococcus lactis.

The gene corresponding to the lactococcal oligopeptidase PepF1 (formerly PepF [V. Monnet, M. Nardi, A. Chopin, M.-C. Chopin, and J.-C. Gripon, J. Biol. Chem. 269:32070-32076, 1994]) is located on the lactose-proteinase plasmid of Lactococcus lactis subsp. cremoris NCDO763. Use of the pepF1 gene as a probe with different strains showed that pepF1 is present on the chromosome of Lactococcus lactis subsp. lactis IL1403, whereas there is a second, homologous gene, pepF2, on the chromosome of strain NCDO763. From hybridization, PCR amplification, and sequencing experiments, we deduced that (i) pepF1 and pepF2 exhibit 80% identity and encode two proteins which are 84% identical and (ii) pepF2 is included in an operon composed of three open reading frames and is transcribed from two promoters. The protein, encoded by the gene located downstream of pepF2, shows significant homology with methyltransferases. Analysis of the sequences flanking pepF1 and pepF2 indicates that only a part of the pepF2 operon is present on the plasmid of strain NCDO763, while the operon is intact on the chromosome of strain IL1403. Traces of several recombination events are visible on the lactose-proteinase plasmid. This suggests that the duplication of pepF occurred by recombination from the chromosome of an L. lactis subsp. lactis strain followed by gene transfer. We discuss the possible functions of PepF and the role of its amplification.     

Structure and expression of the Lactococcus lactis gene for phospho-beta-galactosidase (lacG) in Escherichia coli and L. lactis

The Lactococcus lactis subsp. lactis 712 lacG gene encoding phospho-beta-galactosidase was isolated from the lactose mini-plasmid pMG820 and cloned and expressed in Escherichia coli and L. lactis. The low phospho-beta-galactosidase activity in L. lactis transformed with high-copy-number plasmids containing the lacG gene contrasted with the high activity found in L. lactis containing the original, low-copy-number lactose plasmid pMG820, and indicated that the original lactose promoter was absent from the cloned DNA. In E. coli the phospho-beta-galactosidase could be overproduced using the strong inducible lambda PL promoter, which allowed a rapid purification of the active enzyme. The complete nucleotide sequence of the L. lactis lacG gene and its surrounding regions was determined. The deduced amino acid sequence was confirmed by comparison with the amino acid composition of the purified phospho-beta-galactosidase and its amino-terminal sequence. This also allowed the exact positioning of the lacG gene and identification of its characteristic Gram-positive translation initiation signals. The homologous expression data and the sequence organization of the L. lactis lacG gene indicate that the gene is organized into a large lactose operon which contains an intergenic promoter located in an inverted repeat immediately preceding the lacG gene. The organization and sequence of the L. lactis lacG gene were compared with those of the highly homologous lacG gene from Staphylococcus aureus. A remarkable bias for leucine codons was observed in the lacG genes of these two species. Heterogramic homology was observed between the deduced amino acid sequence of the L. lactis phospho-beta-galactosidase, that of the functionally analogous E. coli phospho-beta-glucosidase, and that of an Agrobacterium beta-glucosidase (cellobiase).     

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis.

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.     

Divergence of Genomic Sequences between Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris

Relatedness between Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis was assessed by Southern hybridization analysis, with cloned chromosomal genes as probes. The results indicate that strains of the two subspecies form two distinct groups and that the DNA sequence divergence between L. lactis subsp. lactis and L. lactis subsp. cremoris is estimated to be between 20 and 30%. The previously used phenotypic criteria do not fully discriminate between the groups; therefore, we propose a new classification which is based on DNA homology. In agreement with this revised classification, the L. lactis subsp. lactis and L. lactis subsp. cremoris strains from our collection have distinct phage sensitivities.     

Comparative phenotypic and molecular genetic profiling of wild Lactococcus lactis subsp. lactis strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes, isolated from starter-free cheeses made of raw milk

Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of the cremoris genotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely, atpA, rpoA, pheS, pepN, bcaT, pepX, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into the cremoris or lactis genotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in the cremoris group showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identical cremoris genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the cremoris genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the lactis and cremoris genotypes of phenotypic Lactococcus lactis subsp. lactis actually represent true subspecies. Some strains of the two subspecies in this study appear to be good starter candidates.     

Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes.

The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp. lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined. The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. The cloned thyA gene was strongly expressed in L. lactis subsp. lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain. In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria. In transformation experiments with thyA mutants of E. coli and conjugation experiments with thyA mutants of R. meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline. Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon. This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.