Temperate bacteriophages and lysogeny in lactic acid bacteria.

Lysogeny is widespread in the lactic acid bacteria. The majority of lysogens can be induced by UV irradiation or treatment with mitomycin C, but indicator strains which allow lytic growth of the induced phage are often not easy to identify. A few temperate phages have been shown to transduce chromosomal and/or plasmid markers. Information about the molecular biology of the temperate phages from lactic acid bacteria is sparse and needs significant supplementation in order that these potentially valuable phages might be utilized more efficiently as tools for improving existing starter strains in dairy fermentations.

Construction of plasmid vectors for the detection of streptococcal promoters.

Plasmid vectors have been constructed for detecting DNA fragments that exhibit promoter activity in Streptococcus sanguis. The plasmids are able to replicate in both S. sanguis and Escherichia coli, and contain an erythromycin resistance marker which is expressed in both hosts. Selection for promoter activity is dependent upon the insertion of appropriate DNA fragments upstream from a promoterless chloramphenicol acetyl transferase gene (cat) from Staphylococcus aureus. To facilitate this insertion, a pair of vectors, pMU1327 and pMU1328, were constructed with the polylinker from M13mp 18 in either orientation. The to transcriptional terminator of phage lambda is present downstream from cat. Translation stop codons in all reading frames are located between the polylinker and the initiation codon of cat. These plasmids have been used to isolate DNA fragments from S. sanguis, S. lactis and S. cremoris that exhibit promoter activity in S. sanguis.

Analysis of the cos region of the Lactococcus lactis bacteriophage sk1.

The location, structure and nature of the cos site of the Lactococcus lactis bacteriophage sk1 was determined using a Taq DNA polymerase runoff sequencing technique. The cos site contains a single-stranded 3' overhang of 11 nucleotides. The region surrounding cos contains several features which may be involved in the binding and catalytic action of a phage terminase. These include four putative terminase-binding sites which show some homology to lambda R-sites, an 11-bp direct repeat, a 10-bp inverted repeat, a string of eight consecutive C residues and six copies of the pentanucleotide, AATCT. The spacing between adjacent copies of the pentanucleotides would place them on the same side of the DNA helix.

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.

Identification and characterization of a cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363.

This paper describes the nucleotide sequence of a gene encoding cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363, its overexpression in Escherichia coli and some functional characteristics of the purified recombinant protein.

The metabolism of [14C]bicarbonate by Streptococcus lactis: the synthesis of succinic acid.

Whole cells of Streptococcus lactis C10, when incubated with an energy source, converted fumarate to succinate and malate to lactate. Cell-free extracts of Str. lactis C10 contained fumarate reductase, but no aspartase, adenylosuccinate synthetase and lyase or argininosuccinate synthetase and lyase activity could be detected. Cells grown in the presence of [14C]bicarbonate produced labelled succinate during the synthesis of purine bases. However, the amount of succinate produced by this pathway only accounted for approximately one-sixth of the succinate produced by the cells.

The metabolism of [14C]bicarbonate by Streptococcus lactis: the fixation of [14C]bicarbonate by pyruvate carboxylase.

The fixation of [14C]bicarbonate into aspartate by Streptococcus lactis C10 was achieved by the combined reactions of pyruvate carboxylase (E.C. 6.4.1.1) and glutamate-oxaloacetate transaminase (E.C. 2.6.1.1). The pyruvate carboxylase from Str. lactis C10, which was most active at pH 8.0, was activated by the divalent metal ions Mn2+, Mg2+ and Co2+, and inhibited by sulphydryl reagents. The enzyme was inhibited non-competitively by aspartic acid and competitively by oxaloacetate.

The nature of the stimulation of the growth of Streptococcus lactis by yeast extract.

Yeast extract was fractionated on Sephadex G-25 into 7 fractions. The fraction most stimulatory to the growth of Streptococcus lactis C10 contained over 70% of the amino N present in yeast extract and consisted of a wide variety of free amino acids and a small amount of peptide material. Examination of possible replacement factors for this fraction revealed that the amino -acid material present was largely responsible for the stimulation of Str. lactis C10. Purine and pyrimidine bases and inorganic constituents also contributed to the stimulation. In addition, yeast extract contained a component which decomposed H2O2, a metabolite which accumulates in the growth medium under aerobic conditions and inhibits growth. The nature of the stimulation was studied by isolating slow and fast acid-producing colonies of Str. lactis C10. It appeared that yeast extract and other amino-acid supplements prevented an observed inhibition of the growth of the slow variants below pH 6.0, apparently by satisfying a nutritional deficiency caused by a drop in pH.

Molecular characterization of promoters of the Lactococcus lactis subsp. cremoris temperate bacteriophage BK5-T and identification of a phage gene implicated in the regulation of promoter activity.

DNA fragments from the temperate lactococcal bacteriophage BK5-T were cloned into the promoter-detecting plasmid pMU1328. Five DNA fragments conferring promoter activity were selected by transformation of Streptococcus sanguis and were functional in Escherichia coli, S. sanguis, and Lactococcus lactis subspp. lactis and cremoris. The nucleotide sequences of these fragments were determined, and primer extension analysis was used to locate the site of initiation of transcription from each promoter in both E. coli and S. sanguis. Transcription was initiated from the same nucleotide in these two organisms, and the promoters contained -10 and -35 regions similar to the consensus sequence for E. coli promoters. The activities of three of the five promoters were decreased two- to threefold when a compatible plasmid containing a 3.8-kilobase-pair EcoRI fragment (EcoRI-f) of BK5-T was coresident with the promoter-containing plasmid in either L. lactis subsp. cremoris or E. coli. Data from Tn5 mutagenesis, subcloning experiments, and DNA sequence analysis indicate that this decrease in promoter activity requires a region of EcoRI-f that contains a 621-base-pair open reading frame. This region has been designated bpi (for BK5-T promoter inhibitor).

Circular Permutation of the Genome of a Temperate Bacteriophage from Streptococcus cremoris BK5.

The temperate bacteriophage BK5-T was isolated from Streptococcus cremoris BK5 by induction with mitomycin C. Electron microscopy revealed that BK5-T DNA consists of linear molecules, ranging in size from 39.7 to 46 kilobase pairs. Restriction analysis of self-ligated BK5-T DNA showed that the ends of the DNA were not cohesive. The EcoRI restriction fragments of the phage genome were cloned into pACYC184. Restriction enzyme analysis of both the phage DNA and the cloned EcoRI fragments with EcoRI, BstEII, PstI, ClaI, and XbaI yielded a 37.6-kilobase-pair-long circular restriction map for the phage genome. It was concluded that the BK5-T DNA molecules in the population differ in their sequence by a circular permutation and that individual DNA molecules are terminally redundant. The map location of the sites at which packaging of BK5-T DNA into phage heads is initiated (pac) and at which the phage integrates into the bacterial chromosome (att) were established.

Temporal transcription map of the Lactococcus lactis bacteriophage sk1.

Bacteriophage sk1 is a small isometric-headed lytic phage that infects Lactococcus lactis. The phage has a linear double-stranded DNA genome of 28 kbp, with cohesive ends. RNA was prepared from phage-infected L. lactis cells harvested at various intervals after infection, and the RNA molecules were resolved by electrophoresis. Northern blots of these gels were hybridized with sk1 DNA probes and the results obtained from these experiments, together with the results of primer extension analyses, enabled a transcription map of the phage genome to be prepared. Three classes of phage transcripts, designated as early, middle or late based on their time of appearance, were detected. Seven partially overlapping early transcripts were detected; these were transcribed from a 10 kbp region of the phage. The nine middle transcripts were derived from a 2 kbp region, limited by cos at one end and the start of the early transcripts at the other. The early and middle transcripts were transcribed divergently from a region mapping at 26 kbp on the sk1 physical map. The four late transcripts were derived from a 16 kbp region of the phage limited at one end by cos. The late transcripts were transcribed in the opposite direction to the early transcripts and three of the late transcripts terminated in the same region of the phage genome as three of the early transcripts.

Transport and metabolism of lactose, glucose, and galactose in homofermentative lactobacilli.

A number of species of lactobacilli were examined for their ability to ferment both the glucose and galactose moieties of lactose. Lactobacillus helveticus strains metabolized both the glucose and galactose moieties, whereas L. bulgaricus, L. lactis, and L. acidophilus strains metabolized only the glucose moiety and released galactose into the growth medium. All four species tested contained beta-galactosidase activity, and no significant phospho-beta-galactosidase activity was observed. L. bulgaricus and L. helveticus had a phosphoenolpyruvate (PEP):glucose phosphotransferase system for the uptake of glucose, but no evidence for a PEP:lactose phosphotransferase or PEP:galactose phosphotransferase system was obtained.

Conjugal transfer in Lactococcus lactis of a 68-kilobase-pair chromosomal fragment containing the structural gene for the peptide bacteriocin nisin.

Nisin-producing transconjugants were generated by mating nisin-producing strains of Lactococcus lactis subsp. lactis with derivatives of L. lactis subsp. lactis LM0230. The sucrose-utilizing ability and reduced bacteriophage sensitivity were also transferred with the nisin-producing character. Pulsed-field gel electrophoretic analysis of genomic DNA from donor, recipient, and nisin-producing transconjugants indicated that 68 kbp of DNA was transferred from the chromosome of the donor into the chromosome of the recipient in the conjugation process. The location of the transferred nisin structural gene spaN in the transconjugant HID500 was not stable, and cultures of strain HID500 were a mixture of different genotypes in which spaN was located at different positions in the chromosome on different SmaI fragments. ApaI, BglI, BssHII, NciI, SalI, and SmaI digests of genomic DNA were used to map the location of spaN in a donor (DL11) and a nisin-producing transconjugant (HID504).

Cloning, nucleotide sequence, expression, and chromosomal location of ldh, the gene encoding L-(+)-lactate dehydrogenase, from Lactococcus lactis.

A gene (designated ldh) that encodes fructose-1,6-bisphosphate-activated L-(+)-lactate dehydrogenase was cloned from Lactococcus lactis subsp. lactis. Plasmids containing ldh conferred fructose-1,6-bisphosphate-activated L-(+)-lactate dehydrogenase activity on Escherichia coli cells. This activity was conferred only when a promoter had been introduced into the plasmid to express the cloned ldh. The nucleotide sequence of ldh predicted a chain length of 324 amino acids and a subunit molecular weight of 34,910 for the enzyme, after removal of the N-terminal methionine residue. Northern analyses of L. lactis subsp. lactis RNA showed that a 4.1-kb transcript hybridized strongly with ldh and that 1.2- and 1.1-kb transcripts hybridized to much lesser extents. Promoter- and terminator-cloning studies in which we used the vectors pGKV210 and pGKV259 in L. lactis subsp. lactis revealed that the 5' flanking DNA of ldh is devoid of transcription initiation signals and that transcription entering the 3' flanking DNA from either direction is efficiently terminated. These data and the data from Northern analyses led to the conclusion that ldh is expressed as the 3' gene of the 4.1-kb transcript and suggested that posttranscriptional processing yielded the shorter transcripts. We determined that ldh is located on the L. lactis subsp. lactis chromosome between coordinates 1.619 and 1.669 of the previously reported physical map (D. L. Tulloch, L. R. Finch, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 173:2768-2775, 1991).

Lactococcus lactis glyceraldehyde-3-phosphate dehydrogenase gene, gap: further evidence for strongly biased codon usage in glycolytic pathway genes.

The gene gap, encoding glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), was isolated from a genomic library of Lactococcus lactis LM0230 DNA. Plasmids containing the L. lactis gene were able to complement a gap mutant of Escherichia coli. The nucleotide sequence of gap predicted a polypeptide chain of 337 amino acids for the enzyme and a subunit molecular mass of 36,043. The codon usage in gap and four other glycolytic genes from L. lactis showed a high degree of bias, when compared with 84 other chromosomal genes. Northern blot analysis of total L. lactis RNA showed that gap hybridized strongly with a 1.3 kb transcript. The 5' end of the transcript was determined by primer extension analysis to be a C located 35 bp upstream from the gap start codon. These transcript analyses, and the orientation of the open reading frames in the DNA flanking gap, indicated that in L. lactis gap is expressed on a monocistronic transcript. Nucleotide sequencing indicated that the DNA adjacent to gap did not encode other glycolytic pathway enzymes. The DNA sequence flanking gap contained two open reading frames (ORF156 and ORF211) of unknown function. The 3' end of a clpA homologue was identified in the sequence upstream of ORF156. The location of gap on the L. lactis DL11 chromosome map was determined to be between map coordinates 0.530 and 0.660.

Sequence analysis of the Lactococcus lactis temperate bacteriophage BK5-T and demonstration that the phage DNA has cohesive ends.

The Lactococcus lactis temperate bacteriophage BK5-T is a type phage in the lactococcal phage classification (A. W. Jarvis, G. F. Fitzgerald, M. Mata, A. Mercenier, H. Neve, I. B. Powell, C. Ronda, M. Saxelin, and M. Teuber, Intervirology 32:2-9, 1991). The nucleotide sequence of 18,935 bp of the genome of BK5-T was determined and analyzed for the presence of open reading frames and other structural features. Thirty-two open reading frames longer than 60 codons were identified, and these appeared to be grouped into at least seven transcriptional units. A search of the nucleotide sequence for restriction sites identified a small number of discrepancies with the previously published physical map of the BK5-T genome (G. Lakshmidevi, B. E. Davidson, and A. J. Hillier, Appl. Environ. Microbiol. 54:1039-1045, 1988). Subsequent analysis of restriction digests of BK5-T DNA which were heated prior to electrophoresis indicated that BK5-T DNA was not terminally redundant as previously reported but contained cohesive ends.

Identification of prophage genes expressed in lysogens of the Lactococcus lactis bacteriophage BK5-T.

Bacteriophage BK5-T is a small isometric-headed temperate phage that infects Lactococcus lactis subsp. cremoris. Northern (RNA) analysis of mRNA produced by lysogenic strains containing BK5-T prophage revealed four major BK5-T transcripts that are 0.8, 1.3, 1.8, and 1.8 kb in size and enabled a transcription map of the prophage genome to be prepared. The position and size of each transcript corresponded closely to the position and size of open reading frames predicted from the nucleotide sequence of BK5-T. Analysis of the transcripts suggested that one of them was derived from the gene encoding the BK5-T integrase and another was from the gene encoding the BK5-T homolog of the lambda cI repressor. Computer analysis of the nucleotide sequence upstream of the BK5-T cI homolog predicted the presence of a pair of divergent promoters and three inverted repeat sequences, features characteristic of temperature-phage immunity regions. By analogy with lambda, the three inverted repeat sequences could be binding sites for cI or Cro homologs and the two divergent promoters could initiate transcription through the BK5-T equivalents of cI and cro.

Spontaneous deletion mutants of the Lactococcus lactis temperate bacteriophage BK5-T and localization of the BK5-T attP site.

Spontaneous deletion mutants of the temperate lactococcal bacteriophage BK5-T were obtained when the phage was grown vegetatively on the indicator strain Lactococcus lactis subsp. cremoris H2. One deletion mutant was unable to form stable lysogens, and analysis of this mutant led to the identification of the BK5-T attP site and the integrase gene (int). The core sequences of the BK5-T attP and host attB regions are conserved in a number of lactococcal phages and L. lactis strains.