Effective plasmid pX3 transduction in Lactobacillus delbrueckii by bacteriophage LL-H.

High-frequency plasmid transductions in Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus strains mediated by pac-type bacteriophages were observed and further investigated. The frequency of plasmid transduction by phages LL-H and LL-S attained levels of from 0.10 to about 1 with plasmid p X 3, but only about 2 x 10(-2) with plasmid pJK650. Infection of L. delbrueckii subsp. lactis strain LKT(pX3) or ATCC 15808(pX3) with phage LL-H resulted in intensive concatemerization of plasmid pX3, and most progeny phage particles contained concatemers of plasmid DNA instead of phage LL-H DNA. The synthesis of phage LL-H DNA was depressed. No evident homology or recombination was observed between phage LL-H DNA and plasmid pX3. The unusually high frequency of plasmid pX3 transduction by phage LL-H could be considered to result from specific interaction(s) between a particular phage and plasmid. These interactions may include pX3-mediated blockage of phage LL-H DNA replication and effective use of a particular pac-like site located about 1 kb from BglII in the smaller NdeI-BglII fragment of plasmid pX3. Phage LL-H together with plasmid vector pX3 could be used as effective plasmid transduction tools for genetic engineering of L. delbrueckii subsp. lactis and subsp. bulgaricus strains.

Three new insertion sequence elements ISLdl2, ISLdl3, and ISLdl4 in Lactobacillus delbrueckii: isolation, molecular characterization, and potential use for strain identification.

A group of new insertion sequence (IS) elements, ISLdl2, ISLdl3, and ISLdl4, from Lactobacillus delbrueckii subsp. lactis ATCC 15808 was isolated, characterized, and used for strain identification together with ISLdl1, recently characterized as an L. delbrueckii IS element belonging to the ISL3 family. ISLdl2 was 1367 bp in size and had a 24 bp IR and an 8 bp DR. The single ORF of ISLdl2 encoded a protein of 392 aa similar to transposases of the IS256 family. ISLdl3 had a single ORF encoding a protein of 343 aa similar to transposases of the IS30 family. Finally, ISLdl4 had a single ORF encoding a protein of 406 aa and displayed homology to the transposases of the IS110 family. ISLdl4 was only slight different from ISL4 (Accession No. AY040213). ISLdl1, ISLdl2, and ISLdl4 were present in all of the 10 L. delbrueckii subsp. lactis and subsp. delbrueckii strains tested, as well as in three of the 11 L. delbrueckii subsp. bulgaricus strains tested. ISLdl3 was present only in four closely related strains of L. delbrueckii subsp. lactis. These IS elements were not observed in Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, or Lactobacillus plantarum. A cluster of IS elements, ISLdl1, ISLdl2, ISLdl3, ISLdl4, and ISL6, was observed in L. delbrueckii subsp. lactis strain ATCC 15808. Within this cluster, ISLdl4 was inserted into ISLdl1 between the left IR and the start codon of ORF455, encoding a putative transposase. Most of the integration sites of the IS elements were strain-specific. We have observed that IS elements can migrate from one strain to another as integral parts of bacterial DNA by using phage LL-H as a vehicle. We demonstrate for the first time that inverse PCR and vectorette PCR methods with primers based on sequences of the IS elements could be used for identification of L. delbrueckii strains.

A new insertion sequence element, ISLdl1, in Lactobacillus delbrueckii subsp. lactis ATCC 15808.

A new insertion sequence element designated ISLdl1 has been isolated and characterized from Lactobacillus delbrueckii subsp. lactis ATCC 15808. It is the first IS element of L. delbrueckii subsp. lactis described. ISLdl1 is a 1508 bp element flanked by 26 bp imperfect inverted repeats, and generates an 8 bp AT-rich target duplication upon insertion. It contains one ORF encoding a protein of 455 amino acids. This protein shows significant homology to the transposases of the ISL3 family and to other bacterial transposases and putative transposases, and no homology to other proteins. Based on these structural features, ISLdl1 belongs to the ISL3 family. ISLdl1 is present in about 10-12 copies in the genome of ATCC 15808 based on Southern hybridization analysis. Location sites of eight ISLdl1 copies have been determined in more detail by cloning and sequencing one or both of the flanking regions of each ISLdl1 copy. ISLdl1 or ISLdl1-like IS elements were found exclusively in Lactobacillus delbrueckii species and in all strains of subsp. lactis tested. The nucleotide sequence of ISLdl1 is deposited under the accession number AJ302652.

A conserved C-terminal region in Gp71 of the small isometric-head phage LL-H and ORF474 of the prolate-head phage JCL1032 is implicated in specificity of adsorption of phage to its host, Lactobacillus delbrueckii.

Thirty-five phage-resistant mutants of Lactobacillus delbrueckii subsp. lactis ATCC 15808 were selected. Thirty-three of these mutants were assigned to the Bes group, while the remaining two were grouped under the Ads designation. Bes group mutants adsorbed phage LL-H but did not allow efficient phage development. Preliminary evidence suggests that these strains exhibit a mutation that changes the DNA specificity of a restriction-modification system. The Ads group mutants did not adsorb the small isometric-head phage LL-H. The results suggest that there are at least three different types of phage receptors in L. delbrueckii: two that are specific for small isometric-head phages and one that is specific for prolate-head phage JCL1032. Five LL-H host-range mutants which could overcome the adsorption block (a-type mutants) were selected and investigated by sequencing the genes g71 and g17, which encode minor and major tail proteins, respectively. Each of the a-type mutants carried a nucleotide change at the 3' end of gene g71. No mutations were observed in gene g17. Comparison of the gene product of g71 of phage LL-H with its homolog in JCL1032 (ORF474) showed that these proteins had very similar C-terminal regions. No similarities were found at the N-terminal part of the proteins. We conclude that the C-terminal portion of the protein encoded by g71 of phage LL-H and its homolog in phage JCL1032 determines the adsorption specificities of these phages on L. delbrueckii.