Construction and screening of metagenomic libraries derived from enrichment cultures: generation of a gene bank for genes conferring alcohol oxidoreductase activity on Escherichia coli

Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C(2) to C(4)) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any other known gene product.     

Isolation of genes required for hydrogenase synthesis in Escherichia coli

A mutant strain of Escherichia coli, strain AK23, is devoid of hydrogenase activity when grown anaerobically on glucose and cannot grow on H2 plus fumarate. From E. coli chromosomal DNA library, a plasmid, pAK23, was isolated which restored hydrogenase activity in this strain. Two smaller plasmids, pAK23C and pAK23S, containing different parts of the insert DNA fragment of plasmid pAK23, were isolated. The former plasmid restored activity in strain AK23 while the latter did not. The smallest active DNA fragment in plasmid pAK23C was 0.9 kb. This gene is designated hydE. Plasmids pAK23 and pAK23S restored activity in another hydrogenase-negative strain, SE-3-1 (hydB), while plasmid pAK23C did not, suggesting that plasmid pAK23 contains two genes required for hydrogenase expression. Strain AK23 was also devoid of formate hydrogenlyase and formate dehydrogenase activities and these activities were restored by some of the plasmids. Hydrogenase and formate-related activities in strain AK23 were restored by growth of cells in a high concentration of nickel. Plasmid pAK23C led to synthesis of a polypeptide of subunit molecular mass 36 kDa and plasmid pAK23S led to synthesis of polypeptides of subunit molecular masses 30 and 41 kDa.     

肺炎链球菌体内诱导基因的筛选及初步鉴定

为筛选鉴定肺炎链球菌宿主体内诱导的基因,寻找潜在的抗生素作用靶点和疫苗候选者,应用体内表达技术,以肺炎链球菌荚膜合成的关键基因galU作为体内报告基因,利用其缺陷体不能合成荚膜多糖,从而不能在宿主体内存活的特点,筛选鉴定肺炎链球菌体内诱导基因。首先,把肺炎链球菌基因组DNA的随机酶切片段(200~500bp)克隆到含有体内、体外双重报告基因(galU-lacZ)的报告载体pEVP3-galU的BglⅡ位点,将获得的质粒库转化肺炎链球菌galU缺陷菌株,得到肺炎链球菌体内启动子诱捕文库,将此文库去感染BALB/c小鼠,经过两轮体内筛选,在涂布有X-gal的TSA血清平板上得到了165个白色菌落,对插入的随机片段进行测序及生物信息学分析,共证实15个不同的体内诱导基因片段,8个为单独的ORF,7个为含有多个ORFs的操纵子结构,它们分别参与细菌在宿主体内的定植与粘附、能量代谢、物质转运、转录调节、DNA复制与重组、细胞壁合成等,另外还包括功能不明的假想蛋白。其中部分ORFs可能与细菌毒力相关,可以作为候选疫苗和药物的靶标。     

Involvement of Streptococcus gordonii beta-glucoside metabolism systems in adhesion, biofilm formation, and in vivo gene expression

Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA). To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed. To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions. Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides. Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants. Esculin and oligochitosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci. Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium. Therefore, genes associated with beta-glucoside metabolism may regulate S. gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization.     

Construction and Screening of Metagenomic Libraries Derived from Enrichment Cultures: Generation of a Gene Bank for Genes Conferring Alcohol Oxidoreductase Activity on Escherichia coli

Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C₂ to C₄) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any other known gene product.     

Ralstonia solanacearum genes induced during growth in tomato: an inside view of bacterial wilt

The phytopathogen Ralstonia solanacearum has over 5000 genes, many of which probably facilitate bacterial wilt disease development. Using in vivo expression technology (IVET), we screened a library of 133 200 R. solanacearum strain K60 promoter fusions and isolated approximately 900 fusions expressed during bacterial growth in tomato plants. Sequence analysis of 307 fusions revealed 153 unique in planta-expressed (ipx) genes. These genes included seven previously identified virulence genes (pehR, vsrB, vsrD, rpoS, hrcC, pme and gspK) as well as seven additional putative virulence factors. A significant number of ipx genes may reflect adaptation to the host xylem environment; 19.6%ipx genes are predicted to encode proteins with metabolic and/or transport functions, and 9.8%ipx genes encode proteins possibly involved in stress responses. Many ipx genes (18%) encode putative transmembrane proteins. A majority of ipx genes isolated encode proteins of unknown function, and 13% were unique to R. solanacearum. The ipx genes were variably induced in planta; beta-glucuronidase reporter gene expression analysis of a subset of 44 ipx fusions revealed that in planta expression levels were between two- and 37-fold higher than in culture. The expression of many ipx genes was subject to known R. solanacearum virulence regulators. Of 32 fusions tested, 28 were affected by at least one virulence regulator; several fusions were controlled by multiple regulators. Two ipx fusion strains isolated in this screen were reduced in virulence on tomato, indicating that gene(s) important for bacterial wilt pathogenesis were interrupted by the IVET insertion; mutations in other ipx genes are necessary to determine their roles in virulence and in planta growth. Collectively, this profile of ipx genes suggests that in its host, R. solanacearum confronts and overcomes a stressful and nutrient-poor environment.     

Comparison of transformation protocols in Streptococcus gordonii and evaluation of native promoter strength using a multiple-copy plasmid

An active area of research in the development of Streptococcus gordonii for use as a bacterial commensal vector involves the identification and utilization of strong promoters for high-level expression of heterologous products. Escherichia coli plasmid vectors containing different streptococcal promoters often fail to become established in E. coli for unknown reasons. Therefore, it is desirable at times to transform S. gordonii, which is naturally competent, with small quantities of nascently ligated DNA without using E. coli first to amplify or screen the product. By comparing the efficiency of two methods used to induce competence in S. gordonii, it was shown that the use of a synthetic competence stimulating peptide substantially enhanced plasmid uptake by S. gordonii. We amplified the amylase-binding protein (abpA) promoter from the S. gordonii genome and, using a synthetic peptide to induce competence, directly introduced plasmid DNA containing this promoter into S. gordonii as an unamplified product of ligation. This plasmid facilitated abundant secretion of a heterologous product by S. gordonii. By assessing the levels of heterologous product secreted by two plasmid constructs, it was possible to evaluate the relative strength of two native promoters.     

IVET-based identification of virulence factors in Vibrio vulnificus MO6-24/O

Vibrio vulnificus is an opportunistic pathogen that causes septicemia in humans. To identify the genes associated with its pathogenicity, in vivo expression technology (IVET) was used to select genes specifically expressed in a host, yet not significantly in vitro. Random lacZ-fusions in the genome of V. vulnificus strain MO6-24/O were constructed using an IVET vector, pSG3, which is a suicide vector containing promoterless-aph and -lacZ as reporter genes. A total of approximately 18,000 resulting library clones were then intraperitoneally injected into BALB/c mice using a colony forming unit (CFU) of 1.6 x 10(6). Two hours after infection, kanamycin was administered at 200 microg per gram of mouse weight. After two selection cycles, 11 genes were eventually isolated, which were expressed only in the host. Among these genes, VV20781 and VV21007 exhibiting a homology to a hemagglutinin gene and tolC, respectively, were selected based on having the highest frequency. When compared to wild-type cells, mutants with lesions in these genes showed no difference in the rate of growth rate, yet a significant decrease in cytotoxicity and the capability to form a biofilm.     

一种基于NanoLuc荧光素酶报告布鲁菌基因启动子活性质粒的构建及应用

[背景]布鲁菌病是由布鲁菌感染引起的一种人兽共患传染病,对畜牧业发展和人类健康有着巨大的威胁。利用新型报告基因NanoLuc荧光素酶构建一种可以检测布鲁菌基因启动子活性的质粒,对于研究布鲁菌毒力基因的调控表达具有重要意义。[目的]制备NanoLuc荧光素酶多克隆抗体,构建一种基于NanoLuc荧光素酶报告布鲁菌基因启动子活性的质粒,并通过测定bcsp31基因启动子和virB启动子活性验证该方法的可行性。[方法]构建NanoLuc荧光素酶原核表达载体pET-Nluc,纯化蛋白免疫新西兰大白兔制备多克隆抗体;以广宿主质粒pBBR1MCS为骨架,构建质粒pNluc、pBcsp31-Nluc和pVirB-Nluc,通过电转化构建S2308(Nluc)、S2308(Bcsp31-Nluc)和S2308(VirB-Nluc)重组菌株,在体外培养条件下测定bcsp31基因启动子和virB启动子活性;比较分析virB启动子在胞内感染条件下和体外培养条件下的活性。[结果]通过原核表达获得NanoLuc荧光素酶重组蛋白,并制备得到效价高于1:100 000的多克隆抗体;成功构建pNluc、pBcsp31-Nluc和pVirB-Nluc质粒以及S2308(Nluc)、S2308(Bcsp31-Nluc)和S2308(VirB-Nluc)重组菌株;体外培养条件下测定bcsp31基因启动子和virB启动子活性,结果显示pNluc质粒可以精确报告其活性;测定virB启动子在胞内诱导条件下和体外培养条件下的活性,结果显示virB启动子活性在胞内感染条件下明显增强。[结论]构建了基于NanoLuc荧光素酶报告布鲁菌基因启动子活性的质粒,并验证其可以精确反映布鲁菌基因启动子活性,为研究布鲁菌毒力基因以及揭示其致病机制奠定了基础。     

Use of a novel mobilizable vector to inactivate the scrA gene of Streptococcus sobrinus by allelic replacement.

The virulence factors of the cariogenic bacterium Streptococcus sobrinus have been difficult to assess because of a lack of tools for the genetic manipulation of this organism. The construction of an Escherichia coli-Streptococcus shuttle vector, pDL289, that can be mobilized into S. sobrinus by the conjugative plasmid pAM beta 1 was described in a previous report. The vector contains pVA380-1 for replication and mobilization in streptococci, the pSC101 replicon for maintenance in E. coli, a kanamycin resistance marker that functions in both hosts, and the multiple cloning site and lacZ from pGEM7Zf(-). pDL289 is stable with or without selection in several species of Streptococcus. In this study, a derivative with a deletion in the minus origin of the pVA380-1 component of pDL289 was constructed. This derivative, pDL289 delta 202, was less stable than pDL289 in Streptococcus gordonii Challis, Streptococcus mutans, and S. sobrinus. Both pDL289 and pDL289 delta 202 were mobilizable by pAM beta 1 into S. sobrinus, with frequencies of 3 x 10(-6) and 1 x 10(-7) transconjugants per recipient CFU, respectively. The cloned scrA gene of S. sobrinus 6715-10 coding for the EIISuc of the sucrose-specific phosphoenolpyruvate phosphotransferase system was interrupted by the insertion of a streptococcal spectinomycin resistance gene active in E. coli and streptococci. The interrupted scrA gene was subcloned into both pDL289 and pDL289 delta 202. Each recombinant plasmid was introduced into the DL1 strain of S. gordonii Challis, which was then used as a recipient for the conjugative transfer of pAM beta 1. The latter plasmid was used to mobilize each recombinant plasmid from S. gordonii Challis DL1 to S. sobrinus 6715-10RF. Subsequently, recombinants derived from a double-crossover event were isolated on the basis of resistance to spectinomycin and susceptibility to kanamycin. Recombinational events were confirmed by Southern hybridization, and the inactivation of the EII Suc in double crossovers was confirmed by phosphotransferase system assays. This is the first report of allelic replacement in S. sobrinus.     

Identification of Lactobacillus reuteri genes specifically induced in the mouse gastrointestinal tract

Lactobacilli are common inhabitants of the gastrointestinal tracts of mammals and have received considerable attention due to their putative health-promoting properties. Little is known about the traits that enhance the ability of these bacteria to inhabit the gastrointestinal tract. In this paper we describe the development and application of a strategy based on in vivo expression technology (IVET) that enables detection of Lactobacillus reuteri genes specifically induced in the murine gut. A plasmid-based system was constructed containing 'ermGT (which confers lincomycin resistance) as the primary reporter gene for selection of promoters active in the gastrointestinal tract of mice treated with lincomycin. A second reporter gene, 'bglM (beta-glucanase), allowed differentiation between constitutive and in vivo inducible promoters. The system was successfully tested in vitro and in vivo by using a constitutive promoter. Application of the IVET system with chromosomal DNA of L. reuteri 100-23 and reconstituted lactobacillus-free mice revealed three genes induced specifically during colonization. Two of the sequences showed homology to genes encoding xylose isomerase (xylA) and peptide methionine sulfoxide reductase (msrB), which are involved in nutrient acquisition and stress responses, respectively. The third locus showed homology to the gene encoding a protein whose function is not known. Our IVET system has the potential to identify genes of lactobacilli that have not previously been functionally characterized but which may be essential for growth of these bacteria in the gastrointestinal ecosystem.     

谷氨酸棒杆菌/大肠杆菌穿梭型启动子探测载体构建

从质粒pXZ10145和pUC19出发,构建了一个谷氨酸棒杆菌/大肠杆菌穿梭载体pAK6。pAK6的大小为5684bp,带有卡那霉素和氨苄青霉素抗性选择标记,以及多克隆位点。在pAK6基础上,构建了以氯霉素乙酰转移酶为报告基因的启动子探测载体pAKC6,pAKC6的大小为6474bp。采用鸟枪法,将经Sau3AI消化的谷氨酸棒杆菌基因组片段连入pAKC6;根据谷氨酸棒杆菌对氯霉素的抗性,从中分离出两个具有启动子功能的插入片段。通过测定报告基因氯霉素乙酰转移酶的活性,对两个启动子片段在谷氨酸棒杆菌中的强度进行了初步的判断;测序后,用启动子预测软件对其结构进行了预测,证实了启动子序列的存在。     

Conjugative transfer of multiple antibiotic resistance markers in Streptococcus pneumoniae.

Two antibiotic-resistant isolates of Streptococcus pneumoniae were investigated for conjugative transfer of their drug resistance markers into streptococcal (groups B and D) and pneumococcal (encapsulated and non-encapsulataed) recipients. Of these, 7 wild-type donor pneumococci transferred all their resistance markers (except Pc [penicillin], Su [sulfonamide], and Tp [trimethoprim]) into group D Streptococcus and non-encapsulated S. pneumoniae recipients at a low frequency (10(-5) to 10(-6)). The resistance markers transferred were Tc (tetracycline); Tc and Cm (chloramphenicol); Tc and MLS (macrolides, lincosamides, and streptogramin B); Tc, MLS, Km (kanamycin), and Cm. The transconjugants obtained retransferred their resistance markers into appropriate streptococcal or pneumococcal recipients or both. The resistance markers of streptococcal transconjugants could not be cured by chemical agents. All attempts to detect extra-chromosomal deoxyribonucleic acid from pneumococcal or streptococcal transconjugants were unsuccessful. The molecular weight of a streptococcal conjugative R plasmid (pIP501) was investigated after transfer into the non-encapsulated S. pneumoniae recipient and was found to be similar to that of the wild-type group B Streptococcus host (20 x 10(6)).     

IS1-dependent generation of high-copy-number replicons from bacteriophage P1 Ap Cm as a mechanism of gene amplification.

Mutant P1 Ap Cm lysogens were isolated in which the drug resistance genes resident on the plasmid prophage P1 Ap Cm are amplified by a novel mechanism. The first step required for amplification is IS1-mediated rearrangement of the P1 Ap Cm prophage. The drug resistance genes are amplified from the rearranged P1 Ap Cm prophage by the formation of a plasmid (P1dR) which contains the two resistance genes. The P1dR plasmid is an independent replicon about one-half the size of P1 Ap Cm that can be maintained at a copy number eightfold higher than that at which P1 Ap Cm can be maintained. It contains no previously identified replication origin and is dependent on the Rec+ function of the host.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

Identification of novel staphylococcal virulence genes by in vivo expression technology

We have applied in vivo expression technology (IVET) to the study of staphylococcal virulence. Using a promoter trap that relies on genetic recombination as a reporter of gene expression, we identified 45 staphylococcal genes that are induced during infection in a murine renal abscess model. Of these, only six were known previously; 11 others have homology to known non-staphylococcal genes. The known staphylococcal genes include agrA , part of a key locus regulating numerous virulence products, and a glycerol ester hydrolase, which may enhance staphylococcal survival in abscesses. We constructed 11 strains containing mutations in previously unknown ivi genes. Of these strains, seven were significantly attenuated in virulence compared with the wild-type parent. The mutagenized ivi genes may encode novel staphylococcal virulence factors.