Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296

Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae. From one of these strains (X. nematophilus PMFI296) a cosmid genome library was prepared in Escherichia coli and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E. coli (50% lethal concentration [LC(50)] of 2 to 6 microg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P. brassicae. One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E. coli clone. Although multiple genes may be needed for full activity, E. coli cells expressing the xptA1 gene from the bacteriophage lambda P(L) promoter were shown to have insecticidal activity (LC(50) of 112 microg of total protein/g of diet). This is contrary to the toxin genes identified in P. luminescens, which were not insecticidal when expressed individually in E. coli. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E. coli clone expressing xptA1. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X. nematophilus PMFI296.     

Complementation of mutations in Escherichia coli and Bordetella pertussis by B. pertussis DNA cloned in a broad-host-range cosmid vector

A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24.9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.     

An Escherichia coli-Mycobacterium shuttle cosmid vector, pMSC1.

A shuttle cosmid vector, pMSC1, has been constructed which replicates in Escherichia coli and Mycobacterium smegmatis. The vector was mainly derived from the lambda ori cosmid, Lawrist4, and the Mycobacterium fortuitum cryptic plasmid, pAL5000, which replicates in M. smegmatis and Mycobacterium bovis BCG. The vector contains two cos sites which facilitates library construction, unique BamHI and HindIII sites for cloning, and a kanamycin-resistance-encoding gene for selection in mycobacteria. After packaging, the vector sequences comprise 10.3 kb, so that the theoretical size limits for inserts are 30-42 kb. A genomic library from M. smegmatis was constructed in E. coli; clones from this library were transferred into M. smegmatis by electroporation, and back again to E. coli, without any apparent rearrangements. This vector will be useful in cloning genes encoding complex pathways in mycobacteria.     

Cloning and expression in Escherichia coli of Haemophilus influenzae fimbrial genes establishes adherence to oropharyngeal epithelial cells.

In this report the first example of functional expression of a fimbrial gene cluster of a non-enteric human pathogen in Escherichia coli is described. This is shown for Haemophilus influenzae fimbriae which mediate adherence to oropharyngeal epithelial cells. A genomic library of H.influenzae type b, strain 770235f+bo, was constructed using a cosmid vector and screened with a synthetic oligonucleotide probe derived from the N-terminal sequence of the fimbrial subunit of H.influenzae. Four cosmid clones were found which hybridized to this oligonucleotide probe. Escherichia coli strains harbouring these clones expressed the H.influenzae fimbriae at their cell surface, as was demonstrated in a whole-cell ELISA and by immunogold electron microscopy using a monoclonal antibody specific for the H.influenzae fimbriae. Surface expression could be maintained during subcloning until a minimal H.influenzae DNA insert of approximately 8.1 kb was obtained. Escherichia coli strains harbouring the 8.1 kb H. influenzae DNA were able to cause a mannose-resistant adherence to oropharyngeal epithelial cells and a mannose-resistant haemagglutination of human AnWj-positive erythrocytes. The nucleotide sequence of hifA, the gene encoding the major fimbrial subunit, was determined. The predicted amino acid sequence shows a significant homology with a number of E.coli fimbrial subunits.     

Construction and preliminary analysis of a metagenomic library from a deep-sea sediment of east Pacific Nodule Province

The Pacific Nodule Province is a unique ocean area containing an abundance of polymetallic nodules. To explore more genetic information and discover potentially industrial useful genes of the microbial community from this particular area, a cosmid library with an average insert of about 35 kb was constructed from the deep-sea sediment. The bacteria in the cosmid library were composed mainly of Proteobacteria including Alphaproteobacteria, Gammaproteobacteria and Deltaproteobacteria. The end sequences of some cosmid clones were determined and the complete insert sequences of two cosmid clones, 10D02 and 17H9, are presented. 10D02 has a length of 40.8 kb and contains 40 predicted encoding genes. It contains a partial 16S rRNA gene of Alphaproteobacteria. 17H9 is 36.8 kb and predicted to have 31 encoding genes and a 16S-23S-5S rRNA gene operon. Phylogenetic analysis of 16S and 23S rRNA gene sequence on the 17H9 both reveals that the inserted DNA from 17H9 came from a novel Alphaproteobacteria and is closely related to Magnetospirillum species. The predicted proteins of ORF 1-11 also have high identity to those of Magnetospirillum species, and the organization of these genes is highly conserved among known Magnetospirillum species. The data suggest that the retrieved DNA in 17H9 might be derived from a novel Magnetospirillum species.     

Characterization of DNA fragments encoding fimbriae of the uropathogenic Escherichia coli strain KS71

Recombinant plasmids were constructed that expressed the KS71A, KS71B and KS71C fimbrial antigens of the pyelonephritogenic Escherichia coli strain KS71 (O4:K12) in E. coli HB101. The KS71C-encoding genes were located on a 6.4 kb HindIII-XhoI fragment obtained from the recombinant cosmid pKTH145 that expresses this antigen. Spontaneous KS71C-mutants were isolated that contained a 0.8 kb insert in a specific restriction fragment of KS71C-encoding recombinant plasmids. The KS71B-encoding segment was located on a 11.5 kb deletable DNA fragment of recombinant cosmid pKTH144. A DNA fragment encoding the KS71A fimbria was obtained on a 12 kb EcoRI fragment of the recombinant cosmid expressing this antigen in E. coli HB101 and closely resembled the KS71B-encoding fragment. In the recombinant cosmid, the KS71B-expressing region was flanked by homologous DNA segments. A similar stretch of DNA was found close to the KS71A-expressing DNA region.     

克雷伯氏菌gdrA和gdrB基因的克隆、协同表达与功能鉴定

1,3-丙二醇是一种重要的化工原料,其生物法生产的研究越来越受到广泛的关注。以克雷伯氏菌的总DNA为模板,通过PCR分别扩增出约1．8kb的gdrA和0．4kb的gdrB的两个基因片段,随后,将此两基因以多顺反子的方式与pSE380相连构建表达载体,并在大肠杆菌中进行了高效表达,表达量约占总蛋白的30％。将高效表达的激活因子用金属亲合层析和分子筛进行了纯化,得到电泳纯级的激活因子,SDS-PAGE分析显示：大、小亚基分子量约为64kDa和12kDa;非变性胶分析显示：全酶的分子量约为150kDa,扫描分析激活因子是以勉＆方式结合的。以克雷伯氏菌甘油脱水酶为研究对象,进行激活实验,结果证实该激活因子具备甘油脱水酶激活因子的功能。该研究为进一步阐明甘油脱水酶的激活机制及1,3一丙二醇的高效生产奠定了基础。     

Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase.

Two genes, xynD and gluB, encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase (lichenase) from Bacillus polymyxa have been cloned and expressed in Escherichia coli and Bacillus subtilis. A sequenced DNA fragment of 4,466 bp contains both genes, which are separated by 155 bp. The xynD and gluB genes encode proteins of 67.8 kDa (XYND) and 27 kDa (GLUB). Two peptides with molecular masses of 62 and 53 kDa appear in cell extracts of E. coli and culture supernatants of B. subtilis clones containing the xynD gene. Both peptides show xylanase activity in zymogram analysis. The XYND enzyme also shows alpha-L-arabinofuranosidase activity. The XYND peptide and the xylanase XYNZ from Clostridium thermocellum (O. Grépinet, M. C. Chebrou, and P. Béguin, J. Bacteriol. 170:4582-4588, 1988) show 64% homology in a stretch of about 280 amino acids.     

Identification and characterization of coenzyme B12-dependent glycerol dehydratase- and diol dehydratase-encoding genes from metagenomic DNA libraries derived from enrichment cultures

To isolate genes encoding coenzyme B(12)-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

Isolation and characterization of beta- and gamma-crystallin genes from rat genomic cosmid libraries

Two libraries, together containing about 10(6) colonies, have been constructed by cloning different size fractions of a partial Sau3A digest of rat genomic DNA in the cosmid vector pTM. Upon screening with two cDNA clones, one containing alpha A2-crystallin and one containing beta B1-crystallin sequences, 14 cosmid clones were isolated which were beta B1-crystallin-specific; none was found which contained alpha A2-crystallin sequences. The inserts of the beta B1 clones, which range from 35 to 45 kb in length, contain overlapping DNA segments covering more than 60 kb of rat genomic DNA. The composite BamHI restriction map of this region shows a single beta B1-crystallin gene, which is interrupted by several intronic sequences. Five recombinants hybridizing with two different rat lens gamma-crystallin cDNA clones were also isolated from these libraries. Four of these contain 31- to 41-kb inserts, whereas the fifth recombinant contains a 12.2-kb insert. Hybridization analysis with 5' and 3'-specific cDNA fragments indicates that altogether these inserts contain six gamma-crystallin genes, three of which are located on one insert of only 31 kb.     

Methylation of inorganic and organic selenium by the bacterial thiopurine methyltransferase

Escherichia coli cells expressing the tpm gene encoding the bacterial thiopurine methyltransferase (bTPMT) are shown to methylate selenite and (methyl)selenocysteine into dimethylselenide (DMSe) and dimethyldiselenide (DMDSe). E. coli cells expressing tpm from a gene library cosmid clone (harboring a Pseudomonas syringae insert of about 20 kb) also methylated selenate into DMSe and DMDSe. bTPMT is the first methyltransferase shown to be involved in the methylation of these selenium derivatives.     

Cosmid cloning and sample sequencing of the genome of the uncultivable mollicute,Western X-disease phytoplasma,using DNA purified by pulsed-field gel electrophoresis

Western X-disease (WX) phytoplasma is an uncultivable, intracellular pathogen of plants and insects with an AT-rich genome of 670 kb. As part of the genome sequencing project of WX phytoplasma, we have cloned approximately 50% of its genome into the pcosRW2 cosmid vector using DNA purified from pulsed-field gels. One of the cosmid clones with an insert of 24.6 kb was sequenced, which along with the cosmid end sequences, represents 60 kb of unique sequence from the WX phytoplasma genome. The putative genes identified in this sequence represent a wide variety of functions and many had not been previously identified in a phytoplasma.     

Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1.

A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0.     

Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1

A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0.     

Cloning and detailed mapping of the fra-ymt region of the Yersinia pestis pFra plasmid]

The genetical libraries of the pFra plasmid of Yersinia pestis genes were obtained by insertion into the PstI, SalGI, EcoRI, XhoI restriction sites of the cosmid vector pHC79. The immunochemical analysis of the recombinant clones has revealed the clones synthesizing the antigen Fl (fraction I) and mouse toxin (Ymt--Yersinia pestis murine toxin). The restriction analysis of the plasmids from antigen synthesizing clones has permitted to construct the detailed physical map of the fra-ymt region of the pFra plasmid the size of 22 kb. The recombinant F1 positive clones of Escherichia coli are able to form at 37 degrees C the capsule-like structure peculiar for Yersinia pestis. The antigen F1 and the mouse toxin were isolated, purified and characterized. The antigen F1 is an 1-2 Md polymer containing a 16 kDa protein subunit. The mouse toxin a 240 kDa protein consisting of 61 kDa subunits. The nucleotide sequence of ymt gene has been defined.     

1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon.

The dha regulon in Klebsiella pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.     

1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon.

The dha regulon in Klebsiella pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.     

环境样品中DNA的分离纯化和文库构建

采用研磨 /冻融和SDS/蛋白酶K热处理等理化方法 ,直接从性质不同的环境样品中提取和纯化混合基因组DNA。所获得纯品DNA的产量为每克样品 2～ 1 6μg。对纯品DNA进行限制性内切酶处理后 ,构建了以pUC1 8为载体的DNA文库。建库效率为从每克环境样品获得约 1 0 3～ 1 0 4 个含 3～ 8kb外源随机插入片段的克隆。通过DNA序列测定和基因注释 ,对从文库中随机选取的克隆进行了分析 ,发现外源插入片段均含序列未见报道的新基因。本文所做的尝试对于保存、研究和开发未培养微生物基因资源具有意义     

Molecular cloning of a gene coding for a Vibrio cholerae haemagglutinin

Recombinant plasmids encoding a Vibrio cholerae haemagglutinin were isolated from the highly virulent V. cholerae strain C5 by cosmid cloning. Both Escherichia coli HB101 containing the recombinant plasmids and V. cholerae C5 were able to agglutinate a variety of erythrocytes from human and animal origin; this haemagglutination was not inhibited by D-mannose or L-fucose. Subcloning of the recombinant cosmid DNA revealed that a 1.3 kb DNA fragment was sufficient for haemagglutinin production in E. coli HB101. Under direction of this 1.3 kb Vibrio DNA fragment, two proteins were made in E. coli minicells, of 27 and 10 kDa. Haemagglutinin-encoding sequences were not detected in every V. cholerae strain.     

Molecular cloning and expression in E. coli of a Salmonella typhi porin gene

Immunoscreening of a Salmonella typhi cosmid library in E. coli allowed the detection of clones producing a 36 kDa porin from S. typhi. The gene is efficiently expressed in an E. coli porin-less mutant and the protein is exported to the outer membrane envelope. Two clones which markedly differ in their level of expression have been isolated.