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.     

Sequence Analysis and Molecular Characterization of the Lactococcus lactis Temperate Bacteriophage BK5-T

The Lactococcus lactis temperate bacteriophage BK5-T is one of twelve type phages that define L. lactis phage species. This paper describes the nucleotide sequence and analysis of a 21-kbp region of the BK5-T genome and completes the nucleotide sequence of the genome of this phage. The 40,003-nucleotide linear genome encodes 63 open reading frames. Sequence runoff experiments showed that the cohesive ends of the BK5-T genome contained a 12-bp 3′ single-stranded overhang with the sequence 5′-CACACACATAGG-3′. Two major BK5-T structural proteins, of approximately 30 and 20 kDa, were identified, and N-terminal sequence analysis determined that they were encoded by orf7 and orf12, respectively. A 169-bp fragment containing a 37-bp direct repeat and several smaller repeat sequences conferred resistance to BK5-T infection when introduced in trans to the host cell and is likely a part of the BK5-T origin of replication (ori).     

Sequence analysis and molecular characterization of the temperate lactococcal bacteriophage r1t

The temperate lactococcal bacteriophage r1t was isolated from its lysogenic host and its genome was subjected to nucleotide sequence analysis. The linear r1t genome is composed of 33 350 bp and was shown to possess 3′ staggered cohesive ends. Fifty open reading frames (ORFs) were identified which are, probably, organized in a life-cycle-specific manner. Nucleotide sequence comparisons, N-terminal amino acid sequencing and functional analyses enabled the assignment of possible functions to a number of DNA sequences and ORFs. In this way, ORFs specifying regulatory proteins, proteins involved in DNA replication, structural proteins, a holin, a lysin, an integrase, and a dUTPase were putatively identified. One ORF seems to be contained within a self-splicing group I intron. In addition, the bacteriophage att site required for site-specific integration into the host chromosome was determined.     

Phenotypic and molecular characterization of Lactococcus lactis from milk and plants

AIMS: The aim of this study was to obtain new Lactococcus lactis strains from nondairy materials for use as milk fermentation starters. The genetic and phenotypic traits of the obtained strains were characterized and compared with those of L. lactis strains derived from milk. It was confirmed that the plant-derived bacteria could be used as milk fermentation starters. METHODS AND RESULTS: About 2600 lactic acid bacteria were subjected to screening for L. lactis with species-specific PCR. Specific DNA amplification was observed in 106 isolates. Forty-one strains were selected, including 30 strains of milk-derived and 11 of plant-derived, and their phenotypic traits and genetic profiles were determined. The plant-derived strains showed tolerance for high salt concentration and high pH value, and fermented many more kinds of carbohydrates than the milk-derived strains. There were no remarkable differences in the profiles of enzymes, such as lipases, peptidases and phosphatases. Isolates were investigated by cluster analysis based on randomly amplified polymorphic DNA profiles. There were no significant differences between isolates from milk and those from plant. The L. lactis subsp. cremoris strains were clustered into two distinct groups, one composed of the strains having the typical cremoris phenotype and the other composed of strains having a phenotype similar to subsp. lactis. Fermented milk manufactured using the plant-derived strains were not inferior in flavour to that manufactured using the milk-derived strains. CONCLUSIONS: Plant-derived L. lactis strains are genetically close to milk-derived strains but have various additional capabilities, such as the ability to ferment many additional kinds of carbohydrates and greater stress-tolerance compared with the milk-derived strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The lactic acid bacteria obtained from plants in this study may be applicable for use in the dairy product industry.     

Phenotypic and genetic characterization of the bacteriophage abortive infection mechanism AbiK from Lactococcus lactis.

The natural plasmid pSRQ800 isolated from Lactococcus lactis subsp. lactis W1 conferred strong phage resistance against small isometric phages of the 936 and P335 species when introduced into phage-sensitive L. lactis strains. It had very limited effect on prolate phages of the c2 species. The phage resistance mechanism encoded on pSRQ800 is a temperature-sensitive abortive infection system (Abi). Plasmid pSRQ800 was mapped, and the Abi genetic determinant was localized on a 4.5-kb EcoRI fragment. Cloning and sequencing of the 4.5-kb fragment allowed the identification of two large open reading frames. Deletion mutants showed that only orf1 was needed to produce the Abi phenotype. orf1 (renamed abiK) coded for a predicted protein of 599 amino acids (AbiK) with an estimated molecular size of 71.4 kDa and a pI of 7.98. DNA and protein sequence alignment programs found no significant homology with databases. However, a database query based on amino acid composition suggested that AbiK might be in the same protein family as AbiA. No phage DNA replication nor phage structural protein production was detected in infected AbiK+ L. lactis cells. This system is believed to act at or prior to phage DNA replication. WHen cloned into a high-copy vector, AbiK efficiency increased 100-fold. AbiK provides another powerful tool that can be useful in controlling phages during lactococcal fermentations.     

Purification and characterization of the lytic activity induced by the prolate-headed bacteriophage P001 in Lactococcus lactis

The lytic activity induced by the lactococcal bacteriophage P001 was isolated from phage lysates of Lactococcus lactis by a four-step purification procedure. Two proteins lytic for L. lactis were identified with molecular weights of 28 kDA and 8 kDa, respectively. The N-terminal amino acid sequences of the two proteins were determined and degenerated oligonucleotide probes corresponding to these sequences were synthesized. DNA hybridization experiments with phage P001-DNA and lactococcal DNA revealed that both proteins were apparently encoded by a single lysin gene located on the phage P001 genome. This was confirmed by alignment of the determined N-terminal amino acid sequences with nucleotide sequences which were deduced from cloned Lactococcus bacteriophage lysin genes.     

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.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was purified to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (alpha and beta) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known 'double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the purified alpha and beta peptides in the residues serine, threonine, cysteine and alanine. Post-translational modification, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are flanked by IS946 elements, suggesting an en bloc transposition. The findings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.     

Purification and characterization of pullulanase from Lactococcus lactis

This paper describes a simple and efficient method of isolation of a plullulanase type I from amylolytic lactic acid bacteria (ALAB). Extracellular pullulanase type I was purified from a cell-free culture supernatant of Lactococcus lactis IBB 500 by using ammonium sulfate fractionation and dialysis (instead of ultrafiltration), and ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step. A final purification factor of 14.36 was achieved. The molecular mass of the enzyme was estimated as 73.9 kD. The optimum temperature for the enzyme activity was 45°C and the optimum pH was 4.5. Pullulanase activity was increased by addition Co(2+) and completely inhibited by Hg(2+). The enzyme activity was specifically directed toward α-1,6 glycosidic linkages of pullulan giving maltotriose units. Enzymatic hydrolysis of starch and amylose produced a mixture of maltose and maltotriose.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

Genome analysis of the obligately lytic bacteriophage 4268 of Lactococcus lactis provides insight into its adaptable nature

Analysis of the complete nucleotide sequence of the lactococcal phage 4268, which is lytic for the cheese starter Lactococcus lactis DPC4268, is presented. Phage 4268 has a linear genome of 36,596 bp, which is modularly organised and encompasses 49 open reading frames. Putative functions were assigned to approximately 45% of the predicted products of these open reading frames based on sequence similarity with known proteins, N-terminal sequence analysis and identification of conserved domains. Significantly, a segment of the genome has homology to the recently sequenced lysogenic module in lactococcal phage phi31 that contains a lytic switch but no phage integrase or attachment site. This suggests that it is derived from a prophage. A phage 4268-encoded and a host-encoded methylase were found to be highly similar, having only two nucleotide mismatches, suggesting that the phage acquired the methylase gene to protect it from a host endonuclease. Comparative genomic analysis revealed significant homology between phage 4268 and the lactococcal phage BK5-T. The comparative analysis also supported the classification of phage 4268 and other BK5-T-related phage as separate from the proposed P335 species of lactococcal phage.