Stable integration and expression of heterologous genes in several lactobacilli using an integration vector constructed from the integrase and attP sequences of phage ΦAT3 isolated from Lactobacillus casei ATCC 393.

An integration vector capable of stably integrating and maintaining in the chromosomes of several lactobacilli over hundreds of generations has been constructed. The major integration machinery used is based on the ΦAT3 integrase (int) and attP sequences determined previously. A novel core sequence located at the 3' end of the tRNA(leu) gene is identified in Lactobacillus fermentum ATCC 14931 as the integration target by the integration vector though most of such sequences found in other lactobacilli are similar to that determined previously. Due to the lack of an appropriate attB site in Lactococcus lactis MG1363, the integration vector is found to be unable to integrate into the chromosome of the strain. However, such integration can be successfully restored by cotransforming the integration vector with a replicative one harboring both attB and erythromycin resistance sequences into the strain. Furthermore, the integration vector constructed carries a promoter region of placT from the chromosome of Lactobacillus rhamnosus TCELL-1 which is used to express green fluorescence and luminance protein genes in the lactobacilli studied.

Characterization of putative class II bacteriocins identified from a non-bacteriocin-producing strain Lactobacillus casei ATCC 334.

Several putative class II bacteriocin-like genes were identified in Lactobacillus casei ATCC 334, all of which might encode peptides with a double-glycine leader. Six peptides encoded by these genes were heterologously expressed in Escherichia coli and then partially purified in order to test their bacteriocin activity. The results revealed that the mature LSEI_2163 peptide was a class IId bacteriocin that exhibited antimicrobial activity against some lactobacilli and several Listeria species. Similarly, mature LSEI_2386 was a putative pheromone peptide that also had significant bacteriocin activity against several Listeria species. The activities of both peptides tolerated 121°C for 30 min but not treatment with proteinase K or trypsin. The two Cys residues located at positions 4 and 24 in the mature LSEI_2163 peptide were shown by mass spectrometry to form a disulfide bridge, which was required for optimal antibacterial activity. However, replacement of one or both Cys with Ser would cause significant reduction of the antibacterial activity, the reduction being greater when only one of the Cys residues (C4S) was replaced than when both (C4S/C24S) were replaced.

Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac- to Lac+.

Lactose metabolism is a changeable phenotype in strains of Lactobacillus casei. In this study, we found that L. casei ATCC 27139 was unable to utilize lactose. However, when exposed to lactose as the sole carbon source, spontaneous Lac(+) clones could be obtained. A gene cluster (lacTEGF-galKETRM) involved in the metabolism of lactose and galactose in L. casei ATCC 27139 (Lac(-)) and its Lac(+) revertant (designated strain R1) was sequenced and characterized. We found that only one nucleotide, located in the lacTEGF promoter (lacTp), of the two lac-gal gene clusters was different. The protein sequence identity between the lac-gal gene cluster and those reported previously for some L. casei (Lac(+)) strains was high; namely, 96-100 % identity was found and no premature stop codon was identified. A single point mutation located within the lacTp promoter region was also detected for each of the 41 other independently isolated Lac(+) revertants of L. casei ATCC 27139. The revertants could be divided into six classes based on the positions of the point mutations detected. Primer extension experiments conducted on transcription from lacTp revealed that the lacTp promoter of these six classes of Lac(+) revertants was functional, while that of L. casei ATCC 27139 was not. Northern blotting experiments further confirmed that the lacTEGF operon of strain R1 was induced by lactose but suppressed by glucose, whereas no blotting signal was ever detected for L. casei ATCC 27139. These results suggest that a single point mutation in the lacTp promoter was able to restore the transcription of a fully functional lacTEGF operon and cause a phenotype switch from Lac(-) to Lac(+) for L. casei ATCC 27139.

Formation of an inverted repeat junction in the transposition of insertion sequence ISLC3 isolated from Lactobacillus casei.

An insertion sequence, ISLC3, of 1351 bp has been isolated from Lactobacillus casei. Formation of IS circles containing a 3 bp spacer (complete junction) or deletion of 25 bp at the left inverted repeat (IRL) between the abutted IS ends of the ISLC3 junction region (deleted junction) was also discovered in the lactobacilli and Escherichia coli system studied. We found that the promoter formed by the complete junction P(jun) was more active than that formed by the 25 bp deleted junction P(djun) or the indigenous promoter P(IRL). The corresponding transcription start sites for both promoter P(jun) and P(IRL) as well as P(djun) were subsequently determined using a primer extension assay. The activity of transposase OrfAB of ISLC3 was also assayed using an in vitro system. It was found that this transposase preferred to cleave a single DNA strand at the IRR over the IRL end in the transposition process, suggesting that attack of one end by the other was oriented from IRR to IRL.

Complete genomic sequence of the temperate bacteriophage PhiAT3 isolated from Lactobacillus casei ATCC 393.

The complete genomic sequence of a temperate bacteriophage PhiAT3 isolated from Lactobacillus (Lb.) casei ATCC 393 is reported. The phage consists of a linear DNA genome of 39,166 bp, an isometric head of 53 nm in diameter, and a flexible, noncontractile tail of approximately 200 nm in length. The number of potential open reading frames on the phage genome is 53. There are 15 unpaired nucleotides at both 5' ends of the PhiAT3 genome, indicating that the phage uses a cos-site for DNA packaging. The PhiAT3 genome was grouped into five distinct functional clusters: DNA packaging, morphogenesis, lysis, lysogenic/lytic switch, and replication. The amino acid sequences at the NH2-termini of some major proteins were determined. An in vivo integration assay for the PhiAT3 integrase (Int) protein in several lactobacilli was conducted by constructing an integration vector including PhiAT3 int and the attP (int-attP) region. It was found that PhiAT3 integrated at the tRNAArg gene locus of Lactobacillus rhamnosus HN 001, similar to that observed in its native host, Lb. casei ATCC 393.

Homology modeling of the central catalytic domain of insertion sequence ISLC3 isolated from Lactobacillus casei ATCC 393.

The tertiary structure of the central catalytic domain of insertion sequence ISLC3 isolated from Lactobacillus casei ATCC 393 was predicted using the homology modeling approach. The novel insertion sequence was isolated by us from the template bacteriophage phiA3 of L.casei ATCC 393. The number of amino acid residues of the ISLC3 central catalytic domain was 116 and was treated as the query sequence. There were five Web-available threading methods used to find some primary structure templates for the query sequence. These primary templates were further screened using the SWISS-MODEL Protein Modeling Server and the default parameter settings therein to give six final structure templates. All of these final structure templates were the integrase (IN) protein of retroviruses. Multiple sequence alignment using these IN sequences against the query one revealed the signature DDE motif. Based on the structures of these final templates, the structure of the query sequence was constructed using the InsightII/Discover/Homology programs. A metal ion, Mg(2+), was inserted into the center of the putative catalytic pocket formed by the DDE residues of the predicted structure in the final rounds of refinement by molecular dynamics (MD) simulations. The structure with a metal ion included was designated with Mg and that without a metal ion was designated free Mg. The average exposed surface area of some hydrophobic residues of both the predicted free Mg and with Mg structures were computed and compared with those computed for the six structure templates. Whereas the predicted with Mg structure was slightly more exposed than the predicted free Mg structure, the former appeared to be more stable than the latter, as revealed by the lower conformation energy recorded for the former during the structure refinement by MD simulations. To verify further the predicted structures, the coordinates of both predicted structures were fed into the ERRAT Protein Verification Server. It was found that the quality of the predicted with Mg structure was much better than that of the free Mg structure. The validation results also indicated that regions of the predicted with Mg structure that can be rejected at the 95% confidence level were approximately 20% whereas those which can be rejected at the same level for the six structure templates were approximately 10%. The predicted with Mg structure was also docked into a short oligonucleotide representing the substrate of the ISLC3 transposase using the DOCK_4.0.2 program. It was found that both Glu140 and Asp68 residues of the DDE motif of the predicted with Mg structure were able to form hydrogen bonds with the DNA substrate, which was similar to what was observed in a docking study using the retrovirus IN 1asu and its DNA substrate.