AbiQ, an abortive infection mechanism from Lactococcus lactis.

Lactococcus lactis W-37 is highly resistant to phage infection. The cryptic plasmids from this strain were coelectroporated, along with the shuttle vector pSA3, into the plasmid-free host L. lactis LM0230. In addition to pSA3, erythromycin- and phage-resistant isolates carried pSRQ900, an 11-kb plasmid from L. lactis W-37. This plasmid made the host bacteria highly resistant (efficiency of plaquing <10(-8)) to c2- and 936-like phages. pSRQ900 did not confer any resistance to phages of the P335 species. Adsorption, cell survival, and endonucleolytic activity assays showed that pSRQ900 encodes an abortive infection mechanism. The phage resistance mechanism is limited to a 2.2-kb EcoRV/BclI fragment. Sequence analysis of this fragment revealed a complete open reading frame (abiQ), which encodes a putative protein of 183 amino acids. A frameshift mutation within abiQ completely abolished the resistant phenotype. The predicted peptide has a high content of positively charged residues (pI = 10.5) and is, in all likelihood, a cytosolic protein. AbiQ has no homology to known or deduced proteins in the databases. DNA replication assays showed that phage c21 (c2-like) and phage p2 (936-like) can still replicate in cells harboring AbiQ. However, phage DNA accumulated in its concatenated form in the infected AbiQ+ cells, whereas the AbiQ- cells contained processed (mature) phage DNA in addition to the concatenated form. The production of the major capsid protein of phage c21 was not hindered in the cells harboring AbiQ.

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.

Loss of plasmid-mediated oligopeptide transport system in lactococci: another reason for slow milk coagulation.

Fast milk-coagulating (Fmc+) strains of lactococci are known to segregate slow milk-coagulating (Fmc-) variants, which has been attributed to loss of proteinase (Prt) activity encoded by plasmid DNA. It was found that the Fmc- phenotype could also be due to loss of a plasmid encoding an oligopeptide permease (Opp) system. In Lactococcus lactis subsp. lactis (L. lactis) C2O, lactose metabolism (Lac) and Prt were linked to pJK550 and the Opp system to pJK430. In Lactococcus lactis subsp. cremoris SK11, known to possess Prt on a 78-kb plasmid, DNA sequence analysis of a 7.4-kb region from the Lac plasmid, pSK11L, revealed that it possessed the Opp system. The Lac plasmid in L. lactis C2 encoded both the Prt and Opp systems. Fmc- derivatives of L. lactis C2 were missing the prt genes and had Opp integrated into the chromosome, possibly due to transposition events. Growth studies showed the Opp systems were functional and, in combination with Prt, produced the Fmc+ phenotype.

Characteristics of Tn5307 exchange and intergeneric transfer of genes associated with nisin production.

Transfer of the Lactococcus lactis 11454 nisin-sucrose conjugative transposon, Tn5307, was investigated to develop a methodology for conjugation of this element to other lactic acid bacteria. Tn5307 exchange was sensitive to temperature and pH but was not affected by protease or amylase treatments to donor cells. Moreover, conjugation studies demonstrated that the direct-plate method could be employed to rapidly identify LM2301 transconjugants able to transfer Tn5307 at least ten times more efficiently than 11454. Intergeneric transfer of nisin and sucrose genes between L. lactis and a dairy Enterococcus sp. was also investigated. Erythromycin-resistant Enterococcus sp. recipients were developed by electro-transformation with pGK13 or by conjugal introduction of the broad-host-range plasmid pAM beta 1. Matings between L. lactis 11454 and an Enterococcus sp. recipient that contained pAM beta 1 yielded sucrose-positive, nisin-immune transconjugants at a frequency of 2.3 x 10(-9) transconjugants per donor cfu. Agar-overlay assays for nisin production revealed that enterococcal transconjugants did not produce the bacteriocin, but DNA.DNA hybridization with a nisA-specific probe demonstrated that these bacteria had acquired the nisin structural gene.

Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation.

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.

Nisin inhibits several gram-positive, mastitis-causing pathogens.

Organisms known to cause bovine mastitis, Enterococcus faecalis ssp. liquefaciens ATCC 27959, Staphylococcus aureus ATCC 29740, Streptococcus agalactiae ATCC 27956, Streptococcus equinus ATCC 27960, Streptococcus dysgalactiae ATCC 27957, Streptococcus uberis ATCC 27958, and the neotype Staphylococcus epidermidis ATCC 14990 were examined for their susceptibility to the small peptide antibiotic, nisin. Using a disc assay, minimum inhibitory concentrations of nisin ranged from 10 to 250 micrograms/ml among the strains. Examination of the antimicrobial effect of 50 micrograms/ml nisin in milk showed nisin inhibited all gram-positive pathogens tested.

Plasmid-linked maltose utilization in Lactobacillus ssp.

Five strains of Lactobacillus plantarum and 4 strains of Lactobacillus ssp. isolated from fresh meat contained between 1 and 5 plasmids ranging in Mr from 1.3 to 49 MDa. Plasmid-curing studies suggested that maltose utilization is associated with a 49 MDa plasmid (pML291) in Lactobacillus sp. DB29 and 34.5 MDa plasmids in Lactobacillus ssp. DB27, DB28 and DB31. Restriction digestion of pML291 and a putative plasmid deletion derivative, pML292, isolated from a maltose negative mutant of DB29, generated common restriction fragments. Southern blot DNA-DNA hybridization using pML 291 as a probe indicated that there is strong homology between putative maltose plasmids.

Effect of Proteolytic Enzymes on Transfection and Transformation of Streptococcus lactis Protoplasts.

With both chymotrypsin and mutanolysin used to form protoplasts, consistent transformation frequencies of 10 to 10 transformants and transfectants per mug of DNA were achieved. The procedure was used to transform protoplasts of Streptococcus cremoris CS224 at low frequency (5 transformants per mug of DNA).

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.

Transformation of Streptococcus lactis Protoplasts by Plasmid DNA.

Polyethylene glycol-treated protoplasts prepared from Streptococcus lactis LM3302, a lactose-negative (Lac) derivative of S. lactis ML3, were transformed to lactose-fermenting ability by a transductionally shortened plasmid (pLM2103) coding for lactose utilization.