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 a nucleotide sequence conserved in Lactococcus lactis bacteriophages

A genetic element which is conserved in the genomes of numerous Lactococcus lactis bacteriophage isolates has been identified and its nucleotide sequence determined. Approximately 95-99% of all L. lactis bacteriophages collected over a period of six years from two geographically distinct sources carry this conserved DNA fragment. Genetic variation in other regions of the genomes of these bacteriophages is exhibited by changes in the overall restriction patterns. The complete nt sequence for a 1.6-kb region from nine independent L. lactis bacteriophage isolates was determined and only five changes in the nt sequence were observed within a span of 1536 bp. This region has a single large 1356-bp open reading frame (ORF) coding for a 51-kDa protein. Three out of the five changes occur in a 187-bp region, 5' to this large ORF. The two additional changes are found within the 1356-bp ORF, which results in two amino acid substitutions that do not, however, change the net charge of the protein. The encoded protein is extremely charged and shares some homology with yeast translation initiation factor. In addition, there is a potential zinc-binding domain within this protein, similar to those observed in genes from bacteriophages T4 and T7.     

Genetic analysis of chromosomal regions of Lactococcus lactis acquired by recombinant lytic phages

Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi(+)) with phage phi31. HindIII restriction maps of the variants (phi31.1, phi31.2, phi31.7, and phi31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage phi31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, phi31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the phi31.1 DNA, which replaced the phi31 origin. Three separate HindIII fragments, accounting for most of the recombinant region of phi31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.     

Sec-Mediated Secretion of Bacteriocin Enterocin P by Lactococcus lactis

Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase.     

Sec-mediated secretion of bacteriocin enterocin P by Lactococcus lactis

Most lactic acid bacterium bacteriocins utilize specific leader peptides and dedicated machineries for secretion. In contrast, the enterococcal bacteriocin enterocin P (EntP) contains a typical signal peptide that directs its secretion when heterologously expressed in Lactococcus lactis. Signal peptide mutations and the SecA inhibitor azide blocked secretion. These observations demonstrate that EntP is secreted by the Sec translocase.     

Homing of a group II intron from Lactococcus lactis subsp. lactis ML3.

Ll.ltrB is a functional group II intron located within a gene (ltrB) encoding a conjugative relaxase essential for transfer of the lactococcal element pRSO1. In this work, the Ll.ltrB intron was shown to be an independent mobile element capable of inserting into an intronless allele of the ltrB gene. Ll.ltrB was not observed to insert into a deletion derivative of the ltrB gene in which the intron splice site was removed. In contrast, a second vector containing a 271-nucleotide segment of ltrB spanning the Ll.ltrB splice site was shown to be a proficient recipient of intron insertion. Efficient homing was observed in the absence of a functional host homologous recombination system. This work demonstrates that the Ll.ltrB intron is a novel site-specific mobile element in lactococci and that group II intron self-transfer is a mechanism for intron dissemination among bacteria.     

Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment

Group II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli, retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonuclease-dependent pathway. However, in retrotransposition in L. lactis, the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonuclease-independent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli, using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis, in E. coli, Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonuclease-independent pathway, and, as in L. lactis, such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Additionally, growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.     

Milk contamination and resistance to processing conditions determine the fate of Lactococcus lactis bacteriophages in dairies

Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments. Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%). However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples. This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72 degrees C for 15 s). The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment. The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination. Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages. Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains. This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent.     

The ability of the plasmid-encoded restriction and modification system LlaBIII to protect Lactococcus lactis against bacteriophages

AIMS: To investigate the potential of the plasmid-encoded restriction and modification (R/M) system LlaBIII to protect Lactococcua lactis against bacteriophages during milk fermentations. METHODS AND RESULTS: The R/M system LlaBIII on plasmid pJW566 was cloned with a chloramphenicol cassette, resulting in plasmid pJK1. When introduced into L. lactis strains, pJK1 conferred increased phage resistance against the three most common lactococcal phage species 936, c2, and P335 and three unclassified industrial phages. The growth of the strains in RSM was not affected by the presence of plasmid pJK1. CONCLUSIONS: The plasmid-encoded R/M system LlaBIII has great ability to protect L. lactis strains against bacteriophages in milk fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evaluates the ability of the LlaBIII R/M system to function as a phage defence mechanism which is an essential step prior to considering utilizing it for improving starter cultures.     

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.