转座因子IS10一个新的插入靶位点的序列分析

转座因子在生物体内广泛存在,它在研究基因的重组机理以及生物染色体的进化方面有着重要意义。IS10是细菌中的一种转座因子,它既能单独作为插入序列,也能作为Tn10的一部分进行转座。利用含sacB基因的质粒pXT3sacB,获得了由转座因子IS10插入而导致sacB基因失活的突变体。通过对插入突变体质粒DNA的序列测定（GenBank登记号为AY580883．1）,结果表明IS10两端分别包括22bp倒置重复区CTGAGAGATCCCCTCATAATTT和AAATCATTAGGGGATTCATCAG,这与前人的报道一致;而IS10两端的插入靶位点序列为TGCTTGGTT,该9bp靶位点序列与前人报道的序列NGCTNAGCN不同。根据文献资料,本研究中的靶位点序列是首次报道。此外,通过Southern blot杂交分析,插入sacB基因中的IS10来源于宿主大肠杆菌DH5α染色体DNA,并且IS10在DH5α染色体中为两个拷贝。此外,本研究利用sacB基因捕获到转座因子IS10,该方法为研究其他插入序列提供了一个有益的体系。     

Sequence Analysis of a Novel Insertion Site of Transposon IS10

A sacB mutant was obtained by transposon IS10 inactivation of a plasmid pXT3sacB carrying the sacB gene. Sequencing of this mutant plasmid DNA (GenBank accession No. AY580883.1) showed that the IS10 flanking the 22 bp inverted repeats were 5′-CTGAGAGATCCCCTCATAATTT-3′ and 5′-AAATCATTAGGGGATTCATCAG-3′, which were the similar to those published in reports previously. However, the target sequence adjacent to IS10 was 5′-TGCTTGGTT-3′ instead of the previously reported 5′-NGCTNAGCN-3′. To our knowledge, this is the first report on the novel insertion site of IS10. In addition, Southern blot hybridization confirmed that the mobile IS10 originated from the chromosomal DNA of the host strain Escherichia coli DH5α and that there were two copies in the DH5α genome.     

The properties of citrate transport catalyzed by CitP of Lactococcus lactis ssp. lactis biovar diacetylactis

Abstract The SC3 hydrophobin gene of Schizophyllum commune was disrupted by homologous integration of an SC3 genomic fragment interrupted by a phleomycin resistance cassette. The phenotype of the mutant was particularly clear in sealed plates in which formation of aerial hyphae was blocked. In non-sealed plates aerial hyphae did form but these were hydrophilic and not hydrophobic as in wild-type strains. Complementation with a genomic SC3 clone restored formation of hydrophobic aerial hyphae in sealed plates. In a dikaryon homozygous for the SC3 mutation normal sporulating fruiting bodies were produced but aerial hyphae were hydrophilic.     

Higher titer hyaluronic acid production in recombinant Lactococcus lactis

Abstract

Hyaluronic acid (HA) is a natural biopolymer and has long been attracting the attention of biotechnology industry due to its various biological functions. HA production with natural producer Streptococcus equi subsp. zooepidemicus has not been preferred because it has many drawbacks due to its pathogenicity. Therefore, in the present study, Streptococcal hyaluronan synthase gene (hasA) was introduced and expressed in Lactococcus lactis, through the auto inducible NICE system and the effect of nisin amount on the production of HA was examined. Newly constructed plasmid was transformed into L. lactis CES15, produced 6.09 g/l HA in static flask culture after three hours of induction period with initial 7.5 ng/ml nisin concentration within total six hours of incubation. The highest HA titer value ever was reported for recombinant HA-producing L. lactis by examining the effect of initial nisin concentration. We have shown that initial nisin concentration, which used to initiate the auto-inducing mechanism of NICE system and consequently hyaluronan synthase expression, has a direct and significant effect on the produced HA amount. Recently constructed recombinant L. lactis CES15 strain provide significant advantages for industrial HA production than those in literature in terms of production time, energy demand, carbon usage, and safety status.     

Gene expression in Lactococcus lactis

Abstract Lactic acid bacteria are of major economic importance, as they occupy a key position in the manufacture of fermented foods. A considerable body of research is currently being devoted to the development of lactic acid bacterial strains with improved characteristics, that may be used to make fermentations pass of more efficiently, or to make new applications possible. Therefore, and because the lactococci are designated 'GRAS' organisms ('generally recognized as safe') which may be used for safe production of foreign proteins, detailed knowledge of homologous and heterologous gene expression in these organisms is desired. An overview is given of our current knowledge concerning gene expression in Lactococcus lactis . A general picture of gene expression signals in L. lactis emerges that shows considerable similarity to those observed in Escherichia coli and Bacillus subtilis . This feature allowed the expression of a number of L. lactis -derived genes in the latter bacterial species. Several studies have indicated, however, that in spite of the similarities, the expression signals from E. coli, B. subtilis and L. lactis are not equally efficient in these three organisms.     

乳链菌肽前体基因（nisZ）在乳酸乳球菌中的克隆和表达

用PCR技术从克隆有完整乳链菌肽生物合成基因簇（来自于乳链菌肽高产菌株L.lactis AL2)的重组噬菌体λHJ-3中扩增了编码乳链菌肽的前体基因,与pMG36e连接得到重组质粒pHJ201,用电击转化法将pHJ201转化到L.lactis NZ9800中,经活性测定和Tricine－SDS－PAGE电泳证实乳链菌肽前体基因获得了功能表达。DNA序列分析表明乳链菌肽高产菌株L.lactis AL2产生的是NisinZ。发现pHJ201d L.lactis NZ9800 中有良好的稳定性。     

Cloning and Production of a Novel Bacteriocin, Lactococcin K, from Lactococcus lactis Subsp. lactis MY23

A gene encoding the antimicrobial peptide, lactococcin K, was isolated from Lactococcus lactis subsp. lactis MY23 then cloned and expressed in Escherichia coli. Because the expressed lactococcin K was formed as an inclusion body in recombinant E. coli, a fusion protein containing lactococcin K and maltose-binding protein (MBP) was produced in a soluble form. For high-level production of lactococcin K, we performed a pH-stat fed-batch culture to produce 43,000 AU lactococcin K ml⁻¹ in 12 h. Revisions requested 3 November 2005; Revisions received 7 December 2005     

Insertion sequences in the IS1111 family that target the attC recombination sites of integron-associated gene cassettes

Members of the recently identified IS 1111 family differ from the majority of insertion sequences (IS) in that they target specific sites in an orientation-specific manner. However, the way in which target selection is achieved is not known. ISKpn4 is representative of a new subgroup of the IS 1111 family whose members are found in the attC sites (59-be) of the gene cassettes associated with integrons. The transposases of this subgroup are closely related (over 75% identity), confirming that closely related IS usually share a common target. However, among more distant relatives encoding a transposase <45% identical to those of the ISKpn4 group, one IS, ISPa25, was found that also targets attC sites. It appears that the targeting determinant of the ISKpn4 group has become associated with a transposase gene from a different group, and this allowed us to localize the region that is likely to be required for target selection to a long noncoding region found downstream of the transposase gene in all IS 1111 family members. This region may determine an RNA used to guide the IS to its specific target.     

细菌插入序列的转座酶和转座机制

插入序列（insertion sequence, IS）是细菌中最简单的移动遗传因子,由两端的反向重复序列（inverted repeats, IR）和中间的转座酶 (transposase)编码序列组成。在细菌中,因为插入序列的转座酶催化活性中心氨基酸序列不同,所以将其转座酶分为DDE转座酶、DEDD转座酶、HUH转座酶和丝氨酸转座酶。在转座过程中,根据插入序列是否有复制,将插入序列的转座分为复制型转座（replicative -ansposition）和非复制型转座（non-replicative transposition）,而将形成夏皮罗中间体（Shapiro intermediate）的非复制型转座称为保守型转座（conservative transposition）。此外,插入序列通过不同的转座机制插入到基因编码区导致基因突变、缺失和倒置;或者插入到基因上游,通过自身启动子或与基因形成杂交启动子来影响插入序列下游基因的表达,从而帮助细菌抵抗复杂的环境变化。本文主要围绕细菌插入序列的特征、转座酶、转座机制和转座影响展开综述,以期为进一步研究插入序列的机制和插入序列在细菌中所起的作用提供参考。     

IS946-mediated integration of heterologous DNA into the genome of Lactococcus lactis subsp. lactis.

The lactococcal insertion sequence IS946 was used to construct suicide vectors for insertion of heterologous DNA into chromosomal and plasmid sequences of Lactococcus lactis subsp. lactis. Electroporation of L. lactis strains, including the recombination-deficient strain MMS362, with the suicide vector pTRK145 yielded 10(1) to 10(3) transformants per micrograms of DNA. pTRK145 insertions occurred primarily in the chromosome, with one insertion detected in a resident plasmid. Vector-specific probes identified junction fragments that varied among transformants, indicating random insertions of pTRK145.     

IS946-mediated integration of heterologous DNA into the genome of Lactococcus lactis subsp. lactis.

The lactococcal insertion sequence IS946 was used to construct suicide vectors for insertion of heterologous DNA into chromosomal and plasmid sequences of Lactococcus lactis subsp. lactis. Electroporation of L. lactis strains, including the recombination-deficient strain MMS362, with the suicide vector pTRK145 yielded 10(1) to 10(3) transformants per micrograms of DNA. pTRK145 insertions occurred primarily in the chromosome, with one insertion detected in a resident plasmid. Vector-specific probes identified junction fragments that varied among transformants, indicating random insertions of pTRK145.     

Secretion of biologically active murine interleukin-10 by Lactococcus lactis

We investigated the ability of Lactococcus lactis to secrete biologically active, murine interleukin-10 (mIL-10). mIL-10 was synthesized as a fusion protein, consisting of the mature part of the eukaryotic protein fused to the secretion signal of the lactococcal Usp45 protein. The secreted protein was analyzed by PAGE, ELISA and bioassay.We show that L. lactis can efficiently secrete biologically active, murine IL-10. Determination of the N-terminal amino acid sequence confirmed correct processing of the fusion polypeptide by the lactococcal signal peptidase. The amount of mIL-10, accumulating in the medium, could be increased by a factor of ten by growing the cells in an optimized medium, buffered at near-neutral pH. Under these conditions, up to 30 mg of mIL-10 was obtained from a 10-litre fermentation.     

Transposition of a bacterial insertion sequence in chloroplasts

Bacterial transposable elements (IS elements, transposons) represent an important determinant of genome structure and dynamics, and are a major force driving genome evolution. Here, we have tested whether bacterial insertion sequences (IS elements) can transpose in a prokaryotic compartment of the plant cell, the plastid (chloroplast). Using plastid transformation, we have integrated different versions of the Escherichia coli IS element IS 150 into the plastid genome of tobacco ( Nicotiana tabacum ) plants. We show that IS 150 is faithfully mobilized inside the chloroplast, and that enormous quantities of transposition intermediates accumulate. As synthesis of the IS 150 transposase is dependent upon programmed ribosomal frame shifting, our data indicate that this process also occurs in chloroplasts. Interestingly, all insertion events detected affect a single site in the plastid genome, suggesting that the integration of IS 150 is highly sequence dependent. In contrast, the initiation of the transposition process was found to be independent of the sequence context. Finally, our data also demonstrate that plastids lack the capacity to repair double-strand breaks in their genomes by non-homologous end joining, a finding that has important implications for genome stability, and which may explain the peculiar immunity of the plastid to invading promiscuous DNA sequences of nuclear and mitochondrial origin.     

sacA and nisA genes are not always linked in Lactococcus lactis subsp. lactis strains

Sixty-seven lactococcal strains arising from dairy habitat were screened for the presence of the sucrose 6-phosphate hydrolase gene by polymerase chain reaction. Of the strains tested, 35.8% were able to ferment sucrose as well as to harbour the sucrose-6-phosphate hydrolase gene, even though they were unable to produce nisin as well as to show the nisin structural gene. After pulsed-field gel electrophoresis and hybridisation all Suc⁺Nis⁻ strains exhibited physical linkage between sacA gene and the left end of lactococcal transposons (Tn5276 or Tn5301) without linkage to nisin genes. However, we were unable to transfer the sacA gene as well as to detect Suc⁻ derivatives from Suc⁺Nis⁻ strains after conjugation and curing experiments.     

Transposition of IS1181 in the genomes of Staphylococcus and Listeria

The recombinant plasmid pIP1713 was constructed to analyse the transpositional activity of the insertion sequence IS1181 in Staphylococcus aureus RN4220, Staphylococcus carnosus TM300 and Listeria monocytogenes EGD. This 11.3-kb plasmid contains two genetically different elements: (i) a pE194ts-derived replicon, the ermC gene of which confers resistance to erythromycin in Gram-positive bacteria of several species, and (ii) a copy of IS1181, cloned from S. aureus BM3121, in which the tetracycline resistance gene, tet(T), has been inserted between the transposase-encoded gene and the downstream inverted repeat. When introduced by electroporation into the three bacterial hosts, pIP1713 delivered IS1181Ωtet(T) to various chromosomal sites. Cointegrate structures between pIP1713 and the host chromosome were occasionally detected. Transposition was associated with 8-bp repeats at the insertion sites. IS1181Ωtet(T) could be used for random mutagenesis in Gram-positive bacteria.     

Screening for the presence of the insertion sequence IS1 in the genus Bacteroides

Abstract A specific DNA probe, containing a conserved region of the insertion sequence IS1, was hybridised to dot blots of total genomic DNA from 2 oral and 5 intestinal Bacteroides spp. Using Escherichia coli K12 as a positive control and Pseudomonas aeruginosa as a negative control, DNA homologous to the probe could not be detected in Bacteroides corporis, Bacteroides intermedius, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron or 2 strains of Bacteroides fragilis . The total DNA included plasmid DNA of 30.2, 42.7 and 42.7 MDa from B. fragilis, B. intermedius and B. corporis , respectively.
IS1 is commonly found in members of the Enterobacteriaceae, and it was concluded that the 2 groups of bacteria are not closely related.