A practical random mutagenesis system for probiotic Lactobacillus casei using Tn5 transposition complexes.

AIMS: Despite the fact that the entire genome sequence of probiotic Lactobacillus casei has recently been available, their mechanisms of beneficial effects are poorly clarified, probably because of the lack of an efficient mutagenesis system. The aim of this study was to establish a practical random mutagenesis system of L. casei using the Tn5 transposome complexes. METHODS AND RESULTS: We optimized the conditions for transformation using a plasmid pUCYIT356-1-Not2 and then transposition reaction using Tn5 transposome system for L. casei ATCC 27139. Tn5 insertion library of this strain being consisted of 9408 mutants was constructed by repeating the mutagenesis procedure. To examine the utility of this mutagenesis system, we screened a panel of insertion mutants for nutrient requirements. Six auxotrophic mutants were isolated and their Tn5 insertion sites were determined by inverse PCR, which demonstrated that insertions occur randomly throughout the whole bacterial genome. CONCLUSIONS: Tn5 transposome system functioned efficiently to generate transposon insertion mutants of L. casei and enabled to construct useful L. casei Tn5 insertion library at optimized conditions for transformation and transposition. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of this system facilitates the study of the mechanisms of beneficial effects of L. casei for human health.

BinI: a new site-specific endonuclease from Bifidobacterium infantis.

A new restriction endonuclease, BinI, from Bifidobacterium infantis 659 has been isolated. By mapping and sequencing of the cleavage sites on SV40 DNA, it was deduced that BinI recognizes the asymmetric pentanucleotide sequence (formula; see text) and cleaves at the sites indicated by the arrows, generating mononucleotide 5'-terminal extensions. BinI is a member of a unique class of Type-II restriction endonucleases which recognize a specific but asymmetric sequence and cleave at a site several bases away.

Insertion of bacteriophage phiFSW into the chromosome of Lactobacillus casei strain Shirota (S-1): characterization of the attachment sites and the integrase gene.

The integrase gene (int) on the genome of φFSW, which is a temperate bacteriophage of Lactobacillus casei strain Shirota (formerly denoted as S-1), and the four attachment sites on the genomes of the phage and its host were characterized by sequencing. The φFSW integrase was found to belong to the integrase family of site-specific tyrosine recombinase. The attachment sites shared a 40bp common core within which an integrative site-specific recombination occurs. The common core was flanked on one side by an additional segment of high sequence similarity. An integration plasmid, consisting of int, the phage attachment site (attP), and a selectable marker, inserted stably into the bacterial attachment site (attB) within the common core, as did the complete prophage genome at a frequency of more than 10(3)/microg of plasmid DNA. This plasmid was used as a test system for a preliminary mutational analysis of int and attP. The attB common core was located within and near the end of an open reading frame that appears to encode a homolog to glucose 6-phosphate isomerase, an enzyme of the glycolytic pathway. It is unlikely that the prophage integration inactivates this protein, since a change of only the C-terminal amino acid is predicted because of the sequence similarity between attP and attB.

Two site-specific endonucleases BinSI and BinSII from Bifidobacterium infantis.

Two site-specific endonucleases, BinSI and BinSII, were isolated from Bifidobacterium infantis S76e. BinSI was found to be an isoschizomer of EcoRII, while BinSII was shown to have the same sequence and cutting specificity as BbeI, 5'-GGCGC decreases C-3'. Both BinSII- and BbeI-generated DNA fragments could be ligated with HaeII-generated DNA fragments.

ISL1: a new transposable element in Lactobacillus casei.

The genome structures of a temperate Lactobacillus phage, phi FSW, and its virulent mutants, phi FSVs, were examined by restriction, heteroduplex and nucleotide-sequence analyses. The results showed that two out of three phi FSVs had the same 1.3 kbp insertion (designated as ISL1) at different positions in the phi FSW sequence. ISL1 was 1,256 bp long and contained at least two long open reading frames of 279 and 822 bases on one strand. Inverted repeats were found at the termini of the ISL1 which was bracketed by 3 bp direct repeats of the phi FSW sequence. From this evidence, we concluded that ISL1 was a transposable element in Lactobacillus casei.

Molecular characterization of a cell wall-associated proteinase gene from Streptococcus lactis NCDO763.

Streptococcus lactis NCDO763 harbours a plasmid designated pLP763. The cells harbouring pLP763 are able to grow to a higher density in milk because of their proteinase-positive phenotype (Prt+). The 6.2 kb HindIII-PstI fragment from pLP763 was found to be responsible for the Prt+ phenotype. The DNA fragment contains an incomplete large open reading frame (ORF). Further sequence analysis downstream from the PstI site revealed that the ORF consists of 5706 bases. It was found that the deduced amino acid sequence consisting of 1902 amino acid residues was extremely similar to that of the Wg2 proteinase, a serine protease from Streptococcus cremoris, suggesting that both genes were derived from a common ancestral gene.