門多 真理子

ホスホケトラーゼ(PK)はヘテロ乳酸発酵を担うが、Enterococcus mundtii QU 25株ではその遺伝子xfpAはホモ乳酸発酵条件においても恒常的に転写されていた。PK活性の制御メカニズムを知るため、ホモ乳酸発酵条件とヘテロ乳酸発酵条件の細胞中の酵素タンパク質XfpAの量をウエスタンブロッティングで調べた。その結果XfpAの量は両方の条件で類似していた。また、XfpAはどちらの条件でもホモダイマーを形成していると推定された。以上のことから、PK活性の制御メカニズムは翻訳後であると示唆された。

エジプトの土壌から分離され、バンコマイシンには中程度耐性を示すEnterococcus faecium QU50の完全ゲノムを報告する。このゲノムは2,535,796塩基対の環状染色体と、196,595塩基対と17,267塩基対の大小2つのプラスミドから成っていた。 IS1062様の配列は見つけることが出来なかった。

Enterococcus mundtii QU 25, a non-dairy lactic acid bacterium, produces optically pure L-lactic acid (>= 99.9%) via homo-fermentation when cultured in the presence of xylose at high concentrations. However, as the xylose concentration decreases, a metabolic shift to hetero-lactic fermentation occurs in this strain. Furthermore, this strain preferentially metabolizes glucose when cultured in medium containing high concentrations of both glucose and xylose, indicating that a previously uncharacterized carbon-catabolite repression system may govern the regulation of these processes. Therefore, to increase the productivity of pure L-lactate by QU 25, it is necessary to investigate this regulatory process. In this study, we performed transcriptional analyses, including RNA sequencing to analyze the transcriptome of QU 25 cultivated in the presence of various glucose and/or xylose concentrations. Our results demonstrate that there was a gradual reduction in the expression of several genes in the xylose gene cluster as the glucose concentration increased, and that there was robust transcription of the genes involved in hetero-lactic fermentation under homo-lactic fermentation conditions. The former result indicates that transcriptional regulation of genes in the xylose gene cluster is involved in the catabolite repression observed in QU 25. The latter results show that the metabolic shift between homo-and hetero-lactic fermentation in QU 25 is not caused by the transcriptional regulation of related genes under the conditions tested. We therefore propose that a yet uncharacterized transcriptional regulation process is involved in the observed catabolite repression.

Enterococcus mundtii QU 25, a non-dairy bacterial strain of ovine faecal origin, can ferment both cellobiose and xylose to produce L-lactic acid. The use of this strain is highly desirable for economical L-lactate production from renewable biomass substrates. Genome sequence determination is necessary for the genetic improvement of this strain. We report the complete genome sequence of strain QU 25, primarily determined using Pacific Biosciences sequencing technology. The E. mundtii QU 25 genome comprises a 3 022 186-bp single circular chromosome (GC content, 38.6%) and five circular plasmids: pQY182, pQY082, pQY039, pQY024, and pQY003. In all, 2900 protein-coding sequences, 63 tRNA genes, and 6 rRNA operons were predicted in the QU 25 chromosome. Plasmid pQY024 harbours genes for mundticin production. We found that strain QU 25 produces a bacteriocin, suggesting that mundticin-encoded genes on plasmid pQY024 were functional. For lactic acid fermentation, two gene clusters were identified-one involved in the initial metabolism of xylose and uptake of pentose and the second containing genes for the pentose phosphate pathway and uptake of related sugars. This is the first complete genome sequence of an E. mundtii strain. The data provide insights into lactate production in this bacterium and its evolution among enterococci.

Lactococcus lactis IO-1 (JCM7638) produces L-lactic acid predominantly when grown at high xylose concentrations, and its utilization is highly desired in the green plastics industry. Therefore it is worthwhile studying its genomic traits. In this study, we focused on (i) genes of possible horizontal transfer derivation (prophages, the nisin-sucrose transposon, and several restriction-modification systems), and (ii) genes for the synthetic pathways of amino acids and vitamins in the IO-1 genome. In view of the results of this analysis, we consider their meanings in strain IO-1.

L型乳酸生産に優れた<I>Lactococcus lactis</I> IO-1株のグルコースおよびキシロースを糖源とした合成培地を編んだ。またIO-1株の栄養要求性について調べたところ、2つのビタミンおよび６つのアミノ酸を要求し、核酸塩基の要求性は無かった。

We report the complete genome sequence of Lactococcus lactis IO-1 (= JCM7638). It is a nondairy lactic acid bacterium, produces nisin Z, ferments xylose, and produces predominantly L-lactic acid at high xylose concentrations. From ortholog analysis with other five L. lactis strains, IO-1 was identified as L. lactis subsp. lactis.

We have developed a method to improve the transformation efficiency in genome-sequenced bacteria, using Plasmid Artificial Modification (PAM), using the hosts own restriction system. In this method, a shuttle vector was pre-methylated in Escherichia coli cells, which carry all the putative genes encoding the DNA modification enzymes of the target microorganism, before electroporation was performed. In the case of Bifidobacterium adolescentis ATCC15703 and pKKT427 (3.9 kb E. coli-Bifidobacterium shuttle vector), introducing two Type II DNA methyltransferase genes lead to an enhancement in the transformation efficiency by five orders of magnitude. This concept was also applicable to a Type I restriction system. In the case of Lactococcus lactis IO-1, by using PAM with a putative Type I methyltransferase system, hsdMS1, the transformation efficiency was improved by a factor of seven over that without PAM.

Some of the lactic acid bacteria (LAB) have been considered to contribute to human health and are used in foods and pharmaceutical products as probiotics. Basic studies on the specific effects of these bacteria and their mechanisms of action are indispensable to the verification of their effectiveness. The development of molecular biological techniques plays an important role in the advancement of these studies.Lactobacillus caseistrain Shirota is a lactic acid bacterium isolated from the human intestine and has been used industrially as a probiotic strain, whose beneficial effects on humans and animals, including an immunomodulatory function, are well documented. This report summarizes the developmental situation of genetic manipulation inL. caseistrain Shirota as follows: 1) transformation and plasmid vectors, 2) a system using an integration-excision vector to maintain desired sequences safely and stably on its chromosome, and 3) experimental display of a chimeric protein on the cell surface of the strain Shirota.

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 φFSW 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 φFSW, within which the site-specific recombination took place with the attB of φFSW on the recipient chromosome through the φFSW 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. © 2001 Elsevier Science B.V. All rights reserved.

The integrase gene (int) on the genome of phi FSW, 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 phi FSW integrase was found to belong to the integrase family of site-specific tyrosine recombinase. The attachment sites shared a 40 bp 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)/mu g 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. (C) 2000 Published by Elsevier Science B.V. All rights reserved.

乳酸菌における遺伝および育種研究の方法論に関する総説。生態系や食品等への利用上、および研究材料としての乳酸菌の特徴、乳酸菌研究の歴史、乳酸菌の遺伝子構成や構造上の特徴、遺伝子発現機構の解析法、古典的な変異株や育種株の取得方法からここ 10年程で急速に進歩しつつある組換えDNAの方法について解説した。これらの研究は、系統分類学の方法を表現型による分類から遺伝子型による分類へと変えるのに大きく貢献している。

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.

T. WATANABE, H. KUMATA, M. SASAMOTO AND M. SHIMIZU‐KADOTA. 1992. Hybridization was used to investigate the distribution of enterococcal plasmid sequences among 306 strains of Enterococcus and Streptococcus spp. isolated from faeces of humans of various ages. As DNA probes for the survey three plasmids, whose DNAs did not hybridize each other and designated as pMS13, pTW34 and pHK30, were selected from plasmids borne in Ent. faecalis. pTW34 DNA hybridized only with DNAs from enterococci, with high frequency in Ent. faecalis and low frequency in Ent. faecium. pMS13 DNA hybridized with DNAs of all Enterococcus spp. tested and with Strep. bovis, Strep. equinus and Strep. salivarius. Eighty‐five percent of Ent. faecium isolates had sequences homologous to pMS13 but in the other species the values were less than 60%. Some enterococci had DNAs which hybridized with the pHK30 probe. The different distribution of the three DNA sequences indicates the possibility that plasmid DNAs encode advantageous phenotypes for the colonization of bacteria in the lumen of the bowel. Copyright © 1992, Wiley Blackwell. All rights reserved

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-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.

A restriction endonuclease <I>Bbe</I>I (R.<I>Bbe</I>eI) produced by <I>Bifidobacterium breve</I> YIT4006 recognizes a hexagonal DNA sequence, GGCGCC. A experiment of the DNA protection from R.<I>Bbe</I>I digestion using commercial methylases and synthetic oligonucleotide shows that YIT4006 is expected to have a modification methylase, <I>Bbe</I>I (M.<I>Bbe</I>I), which methylates the cytosine residue in the DNA sequence of GGmCGCC(mC stands for the cytosine residue to be methylated).

The insertion element ISL<I>1</I> , originally isolated from <I>Lactobacillus casei</I> S-1, was found to have an extremely restricted host range. By Southern filter hybridization, it was found that only 3 of 19 <I>L. casei</I> strains contained the sequences. In two of these, the hybridizing sequences were found on lactose plasmids. No homologous sequences were detected in a survey of 14 other <I>Lactobacillus</I> strains (9 specis) and 15 strains of other bacteria (8 genera, 12 species).

Two of three virulent mutants of a temperate <I>Lactobacillus</I> phage phiFSW had the same 1.3 kbp insertion designated as ISL<I>1</I> at different positions in the phiFSW genome. ISL<I>1</I> was 1256 bp long and contained at least two long open reading frames. Inverted repeats were found at the termini of the ISL<I>1</I> which was bracketed by 3 bp direct repeats of the phiFSW sequence. From this evidence, we conclude that ISL<I>1</I> was a transposable element in <I>Lactobacillus casei</I>.

乳酸桿菌(<I>Lactobacillus</I>)属の細菌細胞に溶菌酵素を作用させて作製したスフェロプラストとリン脂質でファージ DNAを包んだリポソームを融合させることによりDNA感染が起きることが明らかとなったので、ファージベクターを開発して、このDNA感染法を組換えDNA分子を細胞内に導入することに適用させれば、乳酸桿菌（<I>Lactobacillus</I>)属の細菌において宿主－ベクター系を開発し、遺伝子操作技術を用いて菌の改良が可能と考えられた。

Attempts were made to obtain prophage-cured derivatives from <I>Lactobacillus casei</I> lysogenic and industrial strain S-1. A thermoinducible mutant lysogen was isolated from mutagenized strain S-1. 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 fermentations.

A new restriction endonuclease, SlaI,was found and partially purified from <I>Streptomyces lavendulae</I> ATCC8664. The 5'- mononucleotide present after cleavage with SlaI was mostly dTMP determined by chromatography on PEI-cellulose thin layer. The recognition sequence of SlaI was identified by electrophoreses on cellulose-acetate film, followed by homochromatography on a DEAE-cellulose thin-layer plate. The determined recognition sequence was 5'-C-G-A-G-3'.