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

ABSTRACT: 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.

SUBMITTER: Durmaz E

PROVIDER: S-EPMC91919 | biostudies-literature | 2000 Mar

REPOSITORIES: biostudies-literature

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

Durmaz E E Klaenhammer T R TR

Applied and environmental microbiology 20000301 3

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 ...[more]

PMID: 10698748

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Project description:The DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage phi31 was determined, and its regulatory elements were investigated. The identification of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that phi31 was derived from a temperate bacteriophage. Regulation of the two early promoters was analyzed by primer extension and transcriptional promoter fusions to a lacLM reporter. The regulatory behavior of the promoter region differed significantly from the genetic responses of temperate Lactococcus lactis phages. The cro gene homologue regulates its own production and is an efficient repressor of cI gene expression. No detectable cI gene expression could be measured in the presence of cro. cI gene expression in the absence of cro exerted minor influences on the regulation of the two promoters within the genetic switch. Homology comparisons revealed a replication module which is most likely expressed from the promoter located upstream of the cro gene homologue. The replication module encoded genes with strong homology to helicases and primases found in several Streptococcus thermophilus phages. Downstream of the primase homologue, an AT-rich noncoding origin region was identified. The characteristics and location of this region and its ability to reduce the efficiency of plaquing of phi31 10(6)-fold when present at high copy number in trans provide evidence for identification of the phage origin of replication. Phage phi31 is an obligately lytic phage that was isolated from commercial dairy fermentation environments. Neither a phage attachment site nor an integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a temperate phage ancestor. Several regions showing extensive DNA and protein homologies to different temperate phages of Lactococcus, Lactobacillus, and Streptococcus were also discovered, indicating the likely exchange of DNA cassettes through horizontal gene transfer in the dynamic ecological environment of dairy fermentations.

Project description:Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano-/microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. In this study, the food-grade, generally-regarded-as-safe gram-positive organism Lactococcus lactis was engineered to recombinantly produce the biopolyester poly(3-hydroxybutyrate) and the respective intracellular inclusions. The codon-optimized polyhydroxybutyrate biosynthesis operon phaCAB from Cupriavidus necator was expressed using the nisin-controlled gene expression system. Recombinant L. lactis accumulated up to 6% (wt/wt) poly(3-hydroxybutyrate) of cellular dry weight. Poly(3-hydroxybutyrate) granules were isolated and analyzed with respect to bound proteins using biochemical methods and with respect to shape/size using transmission electron microscopy. The immunoglobulin G (IgG) binding ZZ domain of Staphylococcus aureus protein A was chosen as an exemplary functionality to be displayed at the granule surface by fusing it to the N terminus of the granule-associated poly(3-hydroxybutyrate) synthase. The presence of the fusion protein at the surface of isolated granules was confirmed by peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight (mass spectrometry). The functionality of the ZZ domain-displaying granules was demonstrated by enzyme-linked immunosorbent assay and IgG affinity purification. In both assays, the ZZ beads from recombinant L. lactis performed at least equally to ZZ beads from Escherichia coli. Overall, in this study it was shown that recombinant L. lactis can be used to manufacture endotoxin-free poly(3-hydroxybutyrate) beads with surface functionalities that are suitable for biomedical applications.

Project description:Cysticercosis is a cosmopolitan zoonotic parasitic disease infected by larval of Taenia solium (T. solium). Several drugs for the treatment of cysticercosis, such as praziquantel, albendazole, and mebendazole, have certain toxicity and side effects. Considering that there is no vaccine available, we studied a new vaccine for cysticercosis in this study. The complete TSOL18 gene and the optimized SP-TSOL18 gene fragments were obtained using PCR-based accurate synthesis method. The secretory and intracellular recombinant pMG36e-SP-TSOL18/Lactococcus lactis (L. lactis) and pMG36e-TSOL18/L. lactis vaccines of T. solium were prepared. Immune responses in mice orally immunized with these two recombinant L. lactis vaccines were analyzed by the determination of specific antibodies (IgG, IgG1, IgG2a, and sIgA) in serum, spleen lymphocyte proliferation, and cytokines (IL-2, IFN-?, IL-4, and IL-10) in spleen lymphocyte culture supernatant. Our results showed that, after the first immunization, in these two recombinant L. lactis vaccine groups, the levels of serum specific IgG, IgG2a, and IgG1 increased on 14-56?d and reached the highest level on days 42, 42, and 28, respectively. The level of specific sIgA of intestinal mucosa also increased on 14-56?d and reached the highest level on day 42. The level of spleen lymphocyte proliferation increased on 14-56?d and reached the highest level on day 42. The levels of IL-2, IFN-?, IL-4, and IL-10 in spleen lymphocyte culture supernatant increased on 14-56?d and reached the highest level on days 42, 42, 28, and 28, respectively. These results indicated that the recombinant pMG36e-SP-TSOL18/L. lactis and pMG36e-TSOL18/L. lactis vaccines can induce specific cellular, humoral, and mucosal immune responses in mice with oral vaccination. More importantly, the recombinant pMG36e-SP-TSOL18/L. lactis vaccine has a better immune effect. In summary, these results demonstrated the possibility of using L. lactis strain as a vector to deliver protective antigens of T. solium.

Dataset Information

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

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

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