Molecular characterization of a phage-encoded resistance system in Lactococcus lactis (original) (raw)
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Improvement and Optimization of Two Engineered Phage Resistance Mechanisms in Lactococcus lactis
Applied and Environmental Microbiology, 2001
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Applied and environmental microbiology, 2014
Lactococcus lactis subsp. cremoris strains are used globally for the production of fermented dairy products, particularly hard cheeses. Believed to be of plant origin, L. lactis strains that are used as starter cultures have undergone extensive adaptation to the dairy environment, partially through the acquisition of extrachromosomal DNA in the form of plasmids that specify technologically important phenotypic traits. Here, we present a detailed analysis of the eight plasmids of L. lactis UC509.9, an Irish dairy starter strain. Key industrial phenotypes were mapped, and genes that are typically associated with lactococcal plasmids were identified. Four distinct, plasmid-borne bacteriophage resistance systems were identified, including two abortive infection systems, AbiB and AbiD1, thereby supporting the observed phage resistance of L. lactis UC509.9. AbiB escape mutants were generated for phage sk1, which were found to carry mutations in orf6, which encodes the major capsid protein...
Journal of bacteriology, 1992
The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp. lactis ME2 has been characterized and sequenced. The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system. Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10(-2) to 10(-3) and decreases the plaque size and burst size of the small isometric-headed phage p2 in L. lactis subsp. lactis LM0230. However, normal-size plaques occurred at a frequency of 10(-4) and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host. Prf does not prevent phage adsorption or promote restriction and modification activities, but 90% of Prf+ cells infected with phage p2 die. Thus, phage infections in Prf+ cells are aborted. Prf is effective in both L. lactis subsp. lactis and L. lactis subsp. cremoris strains...
Applied and Environmental Microbiology, 2001
pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A twocomponent food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.
Activation and Transfer of the Chromosomal Phage Resistance Mechanism AbiV in Lactococcus lactis
Applied and Environmental Microbiology, 2009
AbiV is a chromosomally encoded phage resistance mechanism that is silent in the wild-type phage-sensitive strain Lactococcus lactis subsp. cremoris MG1363. Spontaneous phage-resistant mutants of L. lactis MG1363 were analyzed by reverse transcriptase PCR and shown to express AbiV. This expression was related to a reorganization in the upstream region of abiV. Transfer of abiV between two lactococcal strains, most likely by conjugation, was also demonstrated. To our knowledge, this is the first report of natural transfer of a chromosomally encoded phage resistance mechanism.
Bacteriophage Resistance of a thyA Mutant of Lactococcus lactis Blocked in DNA Replication
Applied and Environmental Microbiology, 2002
The thyA gene, which encodes thymidylate synthase (TS), of Lactococcus lactis CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of thyA by gene replacement. The resulting strain, MBP71 Δ thyA , was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced large...
Essentiality of the Early Transcript in the Replication Origin of the Lactococcal Prolate Phage c2
Journal of Bacteriology, 2004
The genome of the prolate-headed lytic lactococcal bacteriophage c2 is organized into two divergently oriented blocks consisting of the early genes and the late genes. These blocks are separated by the noncoding origin of DNA replication. We examined the functional role of transcription of the origin in a plasmid model system. Deletion of the early promoter P E 1 abolished origin function. Introduction of mutations into P E 1 which did not eliminate promoter activity or replacement of P E 1 with an unrelated but functional promoter did not abolish replication. The A-T-rich region upstream of P E 1, which is conserved in prolate phages, was not required for plasmid replication. Replacement of the P E 1 transcript template sequence with an unrelated sequence with a similar G؉C content abolished replication, showing that the sequence encoding the transcript is essential for origin function. Truncated transcript and internal deletion constructs did not support replication except when the deletion was at the very 3 end of the DNA sequence coding for the transcript. The P E 1 transcript could be detected for all replication-proficient constructs. Recloning in a plasmid vector allowed detection of P E 1 transcripts from some fragments that did not support replication, indicating that stability of the transcript alone was not sufficient for replication. The data suggest that production of a transcript of a specific length and with a specific sequence or structure is essential for the function of the phage c2 origin in this model system.
The majority of lactococcal plasmids carry a highly related replicon
Microbiology, 1994
DNA sequence analysis and Southern hybridizations, together with complementation experiments, were used to study relationships between lactococcal plasmid replicons. p W 0 2 , p W 0 4 and p W 0 5 , which co-exist in Lactococcus lactis subsp. cremoris Wg2, and plL7 (isolated from another strain) all contained a functional replication region which appeared to be very similar to that of some known lactococcal plasmids. They contain a gene encoding a highly conserved RepB protein (SO-SO O/ O amino acid identity between pWO2, p W 0 4 and pWOS), which is essential for replication. When supplied in trans, repB of p W 0 2 complemented a repB deficiency of p W 0 5. Upstream of the repB gene, all these plasmids contain a strongly conserved region including a 22 bp sequence tandemly repeated three-and-a-half times, and an M-rich region. The similarity with pWV02, which is known to replicate via a theta mechanism, suggests that all plasmids of this family are capable of theta replication. Southern hybridizations revealed that many lactococcal strains contain plasmids of this family.
Applied and Environmental Microbiology, 2003
In a collection of 122 prolate phages, three distinct, non-cross-hybridizing groups of origins of DNA replication were found. The nonconserved sequence was confined to the template for an untranslated transcript, P E 1-T, 300 to 400 nucleotides in length, while the flanking sequences were conserved. All three origin types, despite the low sequence homology, have the same functional characteristics: they express abundant P E 1-T transcripts and can function as origins of plasmid replication in the absence of phage proteins. Using chimeric constructs, we showed that hybrids of two nonhomologous origin sequences failed to function as replication origins, suggesting that preservation of a particular secondary structure of the P E 1-T transcript is required for replication. This is the first systematic survey of the sequence and function of origins of replication in a group of lactococcal phages.