Biomedical subjects

M Shimizu-Kadota

Publications and source records attributed to M Shimizu-Kadota.

13 recordsLinked to original sources

PubMed2001

A method to maintain introduced DNA sequences stably and safely on the bacterial chromosome: application of prophage integration and subsequent designed excision.

By application of prophage integration and subsequent intended excision, a method to maintain an introduced DNA sequence stably onto a bacterial chromosome has been proposed. Recently-constructed integration plasmids using Campbell-type prophage integration system in Lactobacillus casei strain Shirota and its temperate phage phi FSW was modified for this purpose and a chloramphenicol (Cm)-resistance gene was used as a model passenger DNA. On the integration plasmid having an erythromycin (Em)-resistance gene as a selection marker, N- and C-terminally-truncated Cm-resistance genes were inserted into both sides of the attP of phi FSW, within which the site-specific recombination took place with the attB of phi FSW on the recipient chromosome through the phi FSW integrase. Primary integrants of the modified plasmid (integration-excision vector) exhibiting Em-resistant and Cm-sensitive phenotype generated Em-sensitive and Cm-resistant derivatives under the nonselective conditions. Sequence analyses showed that one copy of the complete Cm-resistance gene resided at the attachment site on the host chromosome and the other vector-derived sequences were excised probably by endogenous homologous recombination in the host cells to derive final integrants. The Cm-resistant phenotype of the final integrants was stable for more than 50 generations under non-selective conditions. Frequency of the homologous recombination suggests that negative selection is also adoptable. Thus, this method using the integration-excision vector gives a stable and safe derivatives of the strain and is likely to be applicable to various bacteria, since Campbell-type prophage integration system and homologous recombination are prevalent among bacteria.

Bacteriophages↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed1993

Copy number and location of insertion sequences ISS1 and IS981 in lactococci and several other lactic acid bacteria.

Genomic DNA from 49 lactococcal strains was screened by Southern hybridization for the presence and relative copy number of lactococcal insertion sequence ISS1: ISS1 was found in 47 of 49 strains giving 1 to 20 hybridizing bands per strain. Southern hybridizations of undigested plasmid DNA from 17 lactococcal strains probed with ISS1 and IS981 showed that ISS1 was present on plasmids in all 17 strains, whereas IS981 was present on plasmids in 14 of the 17 strains. Both insertion sequences were present primarily on larger plasmids (> 25 kb), and some plasmids contained copies of both insertion sequences. When probed with ISS1, Southern hybridizations of DNA isolated from Lactococcus lactis ssp. lactis ML3 frozen stock culture and from isolated colonies showed that the stock culture consisted of a mixture of cells having different ISS1-hybridizing bands, indicating that stock cultures may contain cells with varying locations of ISS1 sequences. The number of copies and their widespread distribution among lactococcal strains establish that insertion sequences will contribute significantly to genotypic and phenotypic events that may affect the industrial performance and stability of lactococcal strains.

Blotting, Southern↗

PubMed1991

Shuttle plasmid vectors for Lactobacillus casei and Escherichia coli with a minus origin.

Recombinant plasmids which can be used as shuttle vectors between Escherichia coli and the industrially used strains of Lactobacillus casei were constructed. They have replication regions closely related to those of pUB110 and are likely to replicate by a rolling-circle mechanism via a plus-strand-specific DNA intermediate in L. casei. Both orientations of palA from the staphylococcal plasmid pC194 and those of the intergenic region from coliphage M13 are identified as active minus origins in L. casei, in contrast to the pAM alpha 1 delta 1-derived BA3 minus origin which does not function in L. casei. Stability of the plasmids increased in L. casei when one of these two active minus origins was inserted. All the DNA sequences of the constructed vectors were known.

Base Sequence↗

PubMed1989

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.

Amino Acid Sequence↗

PubMed1988

Cloning and expression of the phospho-beta-galactosidase genes on the lactose plasmid and the chromosome of Lactobacillus casei C257 in Escherichia coli.

A 4.7 kb BglII fragment of lactose plasmid pLY101 of Lactobacillus casei C257, which hybridized weakly to a fragment including a streptococcal phospho-beta-galactosidase (P-beta-gal) gene, was cloned into plasmid vectors of Escherichia coli. Transformant cells expressed P-beta-gal activity, confirming the presence of a P-beta-gal gene on pLY101. Deletion mapping showed that the structural gene of P-beta-gal was located on the 2.4 kb PvuI-EcoRV fragment. The direction of transcription was determined using an expression vector. L. casei MSK248 is a non-lactose-utilizing, pLY101-cured derivative of C257 and no P-beta-gal activity was detected. From MSK248, spontaneous lactose-utilizing revertants having P-beta-gal activity were isolated. Using the pLY101-derived P-beta-gal gene as a probe, a homologous sequence was detected in the chromosomal DNAs of MSK248 and MSK256, and cloned into plasmid vectors of E. coli. Both transformants carrying the homologous sequence from MSK248 and MSK256 expressed P-beta-gal activity. Restriction maps of the chromosomal P-beta-gal genes from MSK248 and MSK256 were identical with that derived from pLY101.

Chromosome Mapping↗

PubMed1988

Evidence that Lactobacillus casei insertion element ISL1 has a narrow host range.

The 1.3-kilobase-pair insertion element ISL1, originally isolated from Lactobacillus casei S-1, was found to have an extremely restricted host range. By DNA-DNA hybrizations performed with Southern transfers by using a cloned internal fragment of ISL1 as a molecular probe, it was found that only 3 of 19 L. casei strains examined contained sequences that hybridized to the ISL1 probe. In two of these, the hybridizing sequences were found on lactose plasmids. No homologous sequences were detected in a survey of 14 other Lactobacillus strains (9 species) and 15 strains of other bacteria (8 genera, 12 species).

Blotting, Southern↗

PubMed1987

Properties of Lactose Plasmid pLY101 in Lactobacillus casei.

A starter strain, Lactobacillus casei C257, was found to carry a lactose plasmid, pLY101. Restriction mapping showed that pLY101 DNA was 68.2 kilobases long. Since a non-lactose-utilizing variant of C257, MSK248, lost phospho-beta-galactosidase (P-beta-gal) activity and pLY101 DNA had a sequence(s) homologous to the streptococcal fragment including a P-beta-gal gene, pLY101 is likely to encode a P-beta-gal gene required for lactose metabolism in C257. MSK248 grew in galactose medium at a rate identical to that of C257 and retained phosphoenolpyruvate-dependent phosphotransferase system activity for lactose similar to that of C257. Therefore, the C257 chromosome appears to encode a complete set of genes for the lactose-phosphotransferase system and the predominant galactose metabolic pathway in C257. pLY101 DNA had a sequence homologous to a lactobacillus insertion sequence, ISL1, which mapped more than 12 kilobases from the sequence homologous to the streptococcal P-beta-gal fragment.

Journal Article↗

PubMed1987

Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3.

In Streptococcus lactis ML3, the lactose plasmid (pSK08) forms cointegrates with a conjugal plasmid (pRS01). It has been proposed that cointegration is mediated by insertion sequences (IS) present on pSK08 (D. G. Anderson and L.L. McKay, J. Bacteriol. 158:954-962, 1984). We examined the junction regions of the cointegrate pPW2 and the corresponding regions of pSK08 (donor) and pRS01 (target) and identified a new IS element on pSK08 (ISS1S) which was involved in and duplicated during formation of pPW2. ISS1S was 808 base pairs (bp) in size, had 18-bp inverted repeats (GGTTCTGTTGCAAAGTTT) at its ends, contained a single long open reading frame encoding a putative protein of 226 amino acids, and generated 8-bp direct repeats of target DNA during cointegrate formation. An iso-IS element, ISS1T, which is duplicated in some other cointegrate plasmids, was also found on pSK08. ISS1T was also 808 bp in size and was identical to ISS1S in sequence except for 4 bp, none of which altered the inverted repeats or amino acid sequence of the open reading frame. Comparison of ISS1 with gram-negative IS26 revealed strong homologies in size (820 bp), sequence of inverted repeats (GGCACTGTTGCAAA), size of direct repeats generated after cointegration (8 bp), and number, size, and amino acid sequence (44.5% identical) of the open reading of frame.

Amino Acid Sequence↗

PubMed1985

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.

Bacteriophages↗

PubMed1984

Physical mapping of the virion and the prophage DNAs of a temperate Lactobacillus phage phi FSW.

We analysed the physical structure of the DNA of phi FSW, which is a temperate phage of Lactobacillus casei S-1. A circular restriction map of the virion DNA has been constructed with three restriction endonucleases, BamHI, SalI and XhoI. Other data indicated that the phage genome was circularly permuted. In lysogens, the DNA of the prophage was found to be linearized at a specific site and integrated into a specific locus of the host genome, with the same orientation in each case, as evidenced by Southern filter hybridization. We compared the physical structure of phi FSW with its three virulent mutants. One of them had a restriction map indistinguishable from that of phi FSW and two of them contained host-derived DNA sequence(s) in a specific region of the phi FSW genome (V-region). The prophage integration site was mapped on a different segment of the phage genome to the V-region. Derivation of virulent mutants from phi FSW is discussed in relation to the physical structure of the phage genome.

Bacteriophages↗

PubMed1983

Prophage Origin of a Virulent Phage Appearing on Fermentations of Lactobacillus casei S-1.

For protection from the abnormal fermentation of Lactobacillus casei S-1 caused by contamination of a virulent phage, phiFSV, the origin of this phage was studied. Morphologies, viral structural proteins, and DNA structures of three independent isolates of phiFSV were compared with those of phiFSW, which is lysogenized in strain S-1. The results showed (i) that the morphology of phiFSV phages is indistinguishable from that of phiFSW and (ii) that all viral structural components found in phiFSW are present in the particles of phiFSV's. In addition, restriction endonuclease analyses of viral DNA showed that the HindIII-digested fragments of phiFSW DNA, the sum of which covered at least 94.7% of this phage genome, were conserved in the phiFSV DNA digests. Results of Southern filter hybridization of the S-1 and prophage-cured cell (C239) DNAs with phiFSV DNA as a probe revealed that C239 had lost most of the phiFSV DNA sequence, whereas S-1 had about one copy of the phiFSV DNA sequence. These results indicate that virulent phage phiFSV is derived from the lysogenized phage phiFSW. Therefore, the appearance of phiFSV can be eliminated by using the prophage-cured derivative of S-1.

Journal Article↗

PubMed1982

Prophage Curing in Lactobacillus casei by Isolation of a Thermoinducible Mutant.

To eliminate the occurrence of virulent phage in industrial fermentation, attempts were made to obtain prophage-cured derivatives from Lactobacillus casei lysogenic strain S-1. A thermoinducible mutant lysogen was isolated from mutagenized strain S-1, since S-1 cannot be induced under laboratory conditions. The mutation responsible for thermoinducibility was located on the prophage. Prophage-cured strains were selected after heat induction of the mutant. These cured strains did not produce the virulent phage and should be valuable for industrial fermentation.

Journal Article↗