Molecular cloning and functional expression of d-arabitol dehydrogenase gene from Gluconobacter oxydans in Escherichia coli

A NADP-dependent d-arabitol dehydrogenase gene was cloned from Gluconobacter oxydans CGMCC 1.110 and functionally expressed in Escherichia coli. With d-arabitol as sole carbon source, E. coli transformants grew rapidly in minimal medium, and produced d-xylulose. The enzymatic properties of the 29kDa enzyme were documented. The DNA sequence surrounding the gene suggested that it is part of an operon with several components of a sugar alcohol transporter system, and the d-arabitol dehydrogenase gene belongs to the short-chain dehydrogenase family.     

利用山梨糖脱氢酶活性筛选氧化葡糖杆菌PQQ合成基因簇

【目的】利用山梨糖脱氢酶醌酶活性从氧化葡糖杆菌H24中分离PQQ生物合成基因簇。【方法】利用ptsG位点整合sdh基因的大肠杆菌JM109作为宿主菌构建了氧化葡糖杆菌H24的基因组DNA文库。通过山梨糖脱氢酶活性检测,从文库中筛选具有PQQ合成能力的单菌落并进行亚克隆。【结果】从氧化葡糖杆菌H24的基因组文库中筛选得到一株具有山梨糖脱氢酶活性的单菌落,亚克隆后序列分析显示插入片段全长5400bp,对应5个编码框(pqqABCDE),与其他细菌PQQ生物合成基因簇有很高的序列同源性。【结论】利用山梨糖脱氢酶醌酶活性成功从氧化葡糖杆菌H24中分离克隆得到了PQQ生物合成基因簇pqqABCDE。     

基于重组氧化葡萄糖酸杆菌生物合成吡咯喹啉醌

吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)是一种重要的氧化还原酶辅基,具有多种生理生化功能,在食品、医药卫生及农业等领域具有广泛的应用。文中采用重组氧化葡萄糖酸杆菌生物合成吡咯喹啉醌。首先构建丙酮酸脱羧酶基因GOX1081敲除的重组菌G. oxydans T1,减少副产物乙酸的形成。然后利用筛选的内源性组成型启动子P0169融合表达pqqABCDE基因簇及tldD基因,构建重组菌G. oxydans T2。最后对发酵培养基添加物和发酵条件进行优化。结果显示重组菌G. oxydans T1、G. oxydans T2生物量较野生菌分别提高43.02%和38.76%,而PQQ的产量分别是野生菌的4.82倍和20.5倍。进一步优化G. oxydans T2碳源及培养条件,最终PQQ产量达(51.3241±0.8997)mg/L,是野生菌的345.62倍。通过基因工程手段,可以有效提高氧化葡萄糖酸杆菌的生物量和合成PQQ的产量,为改善PQQ生物合成效率奠定基础。     

Pyrroloquinoline quinone, a chemotactic attractant for Escherichia coli.

Escherichia coli is attracted by pyrroloquinoline quinone (PQQ), and chemotaxis toward glucose is enhanced by the presence of PQQ. A ptsI mutant showed no chemotactic response to either glucose or PQQ alone but did show a chemotactic response to a mixture of glucose and PQQ. A strain lacking the methylated chemotaxis receptor protein Tar showed no response to PQQ.     

Cloning of Escherichia coli lacZ and lacY genes and their expression in Gluconobacter oxydans and Acetobacter liquefaciens

An efficient transformation protocol for Gluconobacter oxydans and Acetobacter liquefaciens strains was developed by preparation of electrocompetent cells grown on yeast extract-ethanol medium. Plasmid pBBR122 was used as broad-host-range vector to clone the Escherichia coli lacZY genes in G. oxydans and A. liquefaciens. Although both lac genes were functionally expressed in both acetic acid bacteria, only a few transformants were able to grow on lactose. However, this ability strictly depended on the presence of a plasmid expressing both lac genes. Mutations in the plasmids and/or in the chromosome were excluded as the cause of growth ability on lactose.     

Pyrroloquinoline quinone attenuates iNOS gene expression in the injured spinal cord

Pyrroloquinoline quinone (PQQ) is a naturally occurring redox cofactor that acts as an essential nutrient, antioxidant, and redox modulator. PQQ has been demonstrated to oxidize the redox modulatory site of N-methyl-d-aspartic acid (NMDA) receptors. Such agents are known to be neuroprotective in experimental stroke models. Therefore, we examined the possible ameliorating effect of PQQ on spinal cord injury (SCI) in adult rats. Intraperitoneal administration of PQQ effectively promoted the functional recovery of SCI rats after hemi-transection, which was preceded by the attenuation of the expression of inducible nitric oxide (NO) synthase (iNOS) mRNA in the injury site. NO is involved in the secondary detrimental mechanisms and has been implicated in NMDA receptor-mediated neurotoxicity. In fact, administration of PQQ induced significantly decreased lesion size and increased axon density adjoining the lesion area. These observations suggest that PQQ protects against the secondary damage by reducing iNOS expression following primary physical injury to the spinal cord.     

Conjugation and heterologous gene expression in Gluconobacter oxydans ssp. suboxydans

Abstract: Conjugal transfer of a series of incompatibility group P and Q plasmids has been studied in the acetic acid bacterium, Gluconobacter oxydans ssp. suboxydans . Transfer frequencies for the IncP/Q vectors ranged from 10⁻⁵−10⁻⁹ exconjugants per recipient cell. It was found in the case of the IncP vector, pRK290, that Bgl II insert constructs displayed increased conjugal transfer frequencies over pRK290 per se, the parent plasmid. A gentamycin-resistant encoding pRK290 vector which was constructed offers considerable potential as a versatile gene delivery system for Gluconobacter . The lactose transposon, Tn951, was used as a model to examine heterologous gene expression in G. oxydans ssp. suboxydans . The expression level of Tn951 encoded β-galactosidase in this strain was found to be less than 5% of that found in the parent Escherichia coli strain, JC3272.     

Study of the pathways regulating the biosynthesis of Gluconobacter oxydans levansucrase

The synthesis of levansucrase is derepressed during the growth of Gluconobacter oxydans L-1 in media with mannitol, sorbitol or fructose. The level of levansucrase activity under these conditions is 20-30 times higher than in cultures growing in the presence of xylite, galactose or glucose. Addition of mannitol or sucrose to the culture grown in a medium with xylite increases the differential rate of levansucrase synthesis. Addition of glucose at a concentration of 1% to the culture growing in a medium with mannitol at constant pH represses the synthesis of levansucrase only for a short period of time (15-20 min). The mechanism regulating the activity of levansucrase in the bacterial culture is susceptible to changes in the pH of the medium: the differential rate of levansucrase synthesis is three-fold higher when the culture is grown at pH 5.7 cf. pH 4.7.     

Cloning of Escherichia coli lacZ and lacY Genes and Their Expression in Gluconobacter oxydans and Acetobacter liquefaciens

An efficient transformation protocol for Gluconobacter oxydans and Acetobacter liquefaciens strains was developed by preparation of electrocompetent cells grown on yeast extract-ethanol medium. Plasmid pBBR122 was used as broad-host-range vector to clone the Escherichia coli lacZY genes in G. oxydans and A. liquefaciens. Although both lac genes were functionally expressed in both acetic acid bacteria, only a few transformants were able to grow on lactose. However, this ability strictly depended on the presence of a plasmid expressing both lac genes. Mutations in the plasmids and/or in the chromosome were excluded as the cause of growth ability on lactose.     

Construction of expression vectors for protein production in Gluconobacter oxydans

The characteristic ability of Gluconobacter oxydans to incompletely oxidize numerous sugars, sugar acids, polyols, and alcohols has been exploited in several biotechnological processes, for example vitamin C production. The genome sequence of G. oxydans 621H is known but molecular tools are needed for the characterization of putative proteins and for the improvement of industrial strains by heterologous and homologous gene expression. To this end, promoter regions for the genes encoding G. oxydans ribosomal proteins L35 and L13 were introduced into the broad-host-range plasmid pBBR1MCS-2 to construct two new expression vectors for gene expression in Gluconobacter spp. These vectors were named pBBR1p264 and pBBR1p452, respectively, and have many advantages over current vectors for Gluconobacter spp. The uidA gene encoding β-D-glucuronidase was inserted downstream of the promoter regions and these promoter-reporter fusions were used to assess relative promoter strength. The constructs displayed distinct promoter strengths and strong (pBBR1p264), moderate (pBBR1p452) and weak (pBBR1MCS-2 carrying the intrinsic lac promoter) promoters were identified.     

A complete view of the genetic diversity of the Escherichia coli O-antigen biosynthesis gene cluster

The O antigen constitutes the outermost part of the lipopolysaccharide layer in Gram-negative bacteria. The chemical composition and structure of the O antigen show high levels of variation even within a single species revealing itself as serological diversity. Here, we present a complete sequence set for the O-antigen biosynthesis gene clusters (O-AGCs) from all 184 recognized Escherichia coli O serogroups. By comparing these sequences, we identified 161 well-defined O-AGCs. Based on the wzx/wzy or wzm/wzt gene sequences, in addition to 145 singletons, 37 serogroups were placed into 16 groups. Furthermore, phylogenetic analysis of all the E. coli O-serogroup reference strains revealed that the nearly one-quarter of the 184 serogroups were found in the ST10 lineage, which may have a unique genetic background allowing a more successful exchange of O-AGCs. Our data provide a complete view of the genetic diversity of O-AGCs in E. coli showing a stronger association between host phylogenetic lineage and O-serogroup diversification than previously recognized. These data will be a valuable basis for developing a systematic molecular O-typing scheme that will allow traditional typing approaches to be linked to genomic exploration of E. coli diversity.     

Gluconobacter oxydans WD膜结合山梨醇脱氢酶基因在E.coli中的克隆表达

本研究构建了四株含有氧化葡萄糖酸杆菌山梨醇脱氢酶基因的重组大肠杆菌,并初步探究SldB和SldA亚基在山梨醇脱氢酶转化甘油反应中的作用。将pET28a、pETduet与PCR扩增的目的基因连接,构建单启动子调控重组质粒pET28a-sldB、pET28a-sldA、pET28a-sldBA和双启动子调控重组质粒pETduet-sldB'-sldA'。只有含pET28a-sldBA和pETduet-sldB'-sldA'的重组菌具有转化甘油的活性,表明G.oxydans WD的山梨醇脱氢酶催化甘油脱氢需要SldB和SldA亚基的共同作用。串联基因sldBA的蛋白表达结果与双启动子控制sldB和sldA基因蛋白表达结果基本相同,表明位于sldB基因末端的sldA的RBS序列可被E.coli C43的核糖体识别。     

弱氧化葡糖杆菌ddsA基因在大肠杆菌不同宿主菌中的表达

泛醌（辅酶Q）在生物体氧化呼吸链中作为重要的质子和电子传递物质。聚十异戊烯焦磷酸合成酶催化辅助酶Q10的侧链的生物合成。为了获得高产辅助酶Q10的菌株,将选择了10种不同大肠杆菌宿主菌用于弱氧化葡糖杆菌的聚十异戊烯焦磷酸合成酶基因ddsA的表达,通过产物分析证实该基因能在大肠杆菌中表达出有活性的聚十异戊烯焦磷酸合成酶,使大肠杆菌合成了辅酶Q10。此外,还发现在Escherichia coli HB101这一菌株中,ddsA的表达使辅酶Q10的产量略超过了在野生型中占主导地位的辅酶Q8的产量。该结果证明了利用大肠杆菌大规模发酵生产辅酶Q10的可能性。     

Cloning and expression of the rfe–rff gene cluster of Escherichia coli

We have cloned a 13 kb Escherichia coli DNA fragment which complemented the rfe mutation to recover the biosynthesis of E. coli O9 polysaccharide. Using Tn5 insertion inactivation, the rfe gene was localized at the 1.5 kb HindIII-EcoRI region flanking the rho gene. We constructed an rfe-deficient E. coli K-12 mutant by site-directed inactivation using a DNA fragment of the cloned 1.5 kb rfe gene. This also confirmed the presence of the rfe gene in the 1.5 kb region. By simultaneous introduction of both the rfe plasmid and the plasmid of our previously cloned E. coli O9 rfb into this rfe mutant, we succeeded in achieving in vivo reconstitution of O9 polysaccharide biosynthesis. From sequence analysis of the rfe gene, a putative promoter followed by an open reading frame (ORF) was identified downstream of the rho gene. This ORF coincided with the position of the rfe gene determined by Tn5 analysis and site-directed mutagenesis. Furthermore, we identified the rff genes in the 10.5 kb DNA flanking the rfe gene. We recognized at least two functional domains on this cloned rff region. Region I complemented a newly found K-12 rff mutant, A238, to synthesize the enterobacterial common antigen (ECA). Deletion of region II resulted in the synthesis of ECAs with shorter sugar chains. When the 10.5 kb rff genes of the plasmid were inactivated by either deletion or Tn5 insertion, the plasmid lost its ability to give rise to transformants of the rfe mutants.     

Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.     

Cloning of the ARO cluster gene of Neurospora crassa and its expression in Escherichia coli

Summary We have constructed a phage, Ncl, which comprises a 4.0 kb HindIII insert of Neurospora DNA into the immunity region of the vector 598. Ncl complements the aroD6 mutation of E. coli, permitting the formation of galaxy plaques on medium lacking aromatic supplements, and transforms an aro-9 qa-2 Neurospora mutant to prototrophy at a low frequency. Low levels of 5-dehydroquinate hydrolyase (E.C.4.2.1.10.), with properties unlike those of the catabolic isoenzyme that is coded by qa-2, are present in E. coli aroD6 cell lysates following infection with Ncl. Ncl does not hybridize with qa-2 DNA and it is concluded that it contains at least the aro-9 region of the pentafunctional aro cluster gene.     

Regulatory gene mutations affecting arginine biosynthesis in Escherichia coli

Summary Mutants of Escherichia coli with altered regulation of arginine biosynthesis were isolated. The alterations in all of the mutants with increased levels of the biosynthetic enzymes were found to map in the argR locus. The mutants were grouped into three classes based on their effect on the regulatory behavior. Complementation studies with stable merodiploid strains demonstrated that the derepressed synthesis in the mutants was recessive to wild-type regulation.     

Characterization and sequence of the Escherichia coli panBCD gene cluster

Abstract A metabolic key enzyme malate dehydrogenase (MDH) was purified from a deep-sea psychrophilic bacterium, Vibrio sp. strain no. 5710. The enzyme displayed an optimal activity shifted toward lower temperature and a pronounced heat lability. A gene encoding this enzyme was isolated and cloned. Recombinant Escherichia coli cells harboring the isolated clone expressed MDH activity with temperature stability identical to that of the parental psychrophile. Nucleotide sequencing of the gene revealed that its primary sequence was similar to that of a mesophile E. coli MDH (78% amino acid identity), for which the three-dimensional structure is known. The enzyme is thus suitable for the analysis of molecular adaptations to low temperatures.     

Characterization of Gluconobacter oxydans immobilized in calcium alginate

Summary Gluconobacter oxydans cells were immobilized in calcium alginate and the preparation was used for the oxidation of glycerol to dihydroxyacetone. The characterization was done according to the guidelines given by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The pH optimum of the preparation was found to be 5.0 and the temperature optimum was 40°C. However, the operational stability was better at 30°C. The glycerol concentration required to obtain half the maximal reaction rate was about 5 mM for both immobilized and free cells. At low concentrations of glycerol and high concentrations of dihydroxyacetone a slight inhibition was noted. No loss of activity of the immobilized preparation was observed after storage for 68 days at +4°C. Investigation of the operational stability revealed a half-life of 5 days. Studies of the influence of particle size and cell densities as well as that of oxygen concentration revealed that the oxygen supply was the rate limiting step.