Cloning, heterologous expression, and sequencing of the Proteus vulgaris glnAntrBC operon and implications of nitrogen control on heterologous urease expression

Abstract The glnAntrBC operon of Proteus vulgaris was cloned and heterologously expressed in Escherichia coli . The nucleotide sequence was determined. An open reading frame of 1407 bp was identified as the glnA gene and the deduced amino acid sequence showed 82% identity with the E. coli glutamine synthetase protein. Heterologous expression of the glnA gene in E. coli restored glutamine synthetase (GS) activity in a GS-negative mutant and a 52 kDa protein was detected and addressed as the GS subunit of P. vulgaris . Adjacent to the glnA gene the regulatory genes ntrB and ntrC were identified. Their coding regions comprised 1053 and 1452 bp, respectively, and the deduced gene products NR_II (NtrB) and NR_I (NtrC) shared 72% identity with the corresponding E. coli proteins. Heterologous expression in E. coli revealed only a 54 kDa protein which was shown to be NR_I. NR_II was not detectable using the methods employed.     

Cloning and sequencing of the gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough

Abstract The gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough (148 amino acid residues), the first flavoprotein for which a three-dimensional structure has been determined, was cloned with the use of two synthetic oligonucleotides, designed to recognize the coding sequence for amino acid residues 11–19 and 98–103, respectively. The two oligonucleotides were used to screen a library of 900 λ-clones of the D. vulgaris chromosome. A single clone, λFL1, reacting with both probes was isolated. The entire structural gene for flavodoxin is contained in the 15 kb insert of λFL1 as found by nucleic acid sequencing. The codon usage in the flavodoxin gene is strongly biased towards G or C in the third codon position. A table in which codon usage information from all genes of D. vulgaris sequenced to date is combined is presented and should facilitate further gene cloning with oligonucleotide probes.     

Cloning and sequencing of a gene encoding the 69-kDa extracellular chitinase of Janthinobacterium lividum

Abstract Janthinobacterium lividum secretes a major 56-kDa chitinase and a minor 69-kDa chitinase. A chitinase gene was defined on a 3-kb fragment of clone pRKT10, by virtue of fluorescent colonies in the presence of 4-methylumbelliferyl-β-d-N,N',N"-chitotrioside. Nucleotide sequencing revealed an 1998-bp open reading frame with the potential to encode a 69 716-Da protein with amino acid sequences similar to those in other chitinases, suggesting it encodes the minor chitinase (Chi69). Chitinase activity of Escherichia coli (pRKTIO) lysates was detected mainly in the periplasmic fraction and immunoblotting detected a 70-kDa protein in this fraction. Chi69 has an N-terminal secretory leader peptide preceding two probable chitin-binding domains and a catalytic domain. These functional domains are separated by linker regions of proline-threonine repeats. Amino acid sequencing of cyanogen bromide cleavage-derived peptides from the major 56-kDa chitinase suggested that Chi69 may be a precursor of Chi56. In addition, an N-terminally truncated version of Chi69 retained chitinase activity as expected if in vivo processing of Chi69 generates Chi56.     

普通变形杆菌位置非特异性脂肪酶基因的克隆及在大肠杆菌中的表达

【目的】本文拟克隆普通变形杆菌(Proteus vulgaris)脂肪酶基因,并实现其在大肠杆菌中的高效表达,并检验外源表达脂肪酶的催化性质。【方法】通过PCR方法,从P.vulgaris基因组中扩增其脂肪酶基因(PVL),并将其开放读码框区域连接到表达载体pET-DsbA及pMBP-P上,在大肠杆菌中用IPTG诱导表达。我们对培养基组分及培养条件进行优化,以获得最高的酶产量。用Ni柱亲和层析法对所得重组脂肪酶进行纯化,并考察其酶学性质。【结果】PVL基因编码区含864个碱基,编码含287个氨基酸的酶蛋白。该序列在GenBank的登录号为FJ643627。PVL基因在大肠杆菌BL21内诱导表达的最佳条件为:在pH8.5的LB培养基中添加15g/L葡萄糖及200mg/L氨苄青霉素,在培养至OD600为1.2时加入100mg/LIPTG,15℃诱导15h,最高酶活达到192.2U/mL。通过亲和层析纯化了重组脂肪酶,得到一个约31kDa的蛋白条带。外源表达的脂肪酶的催化特性与野生菌脂肪酶相似,具有催化的位置非特异性,对长链脂肪酸酯亲和性最高。【结论】PVL基因在大肠杆菌中的高效表达为P.vulgaris脂肪酶的进一步研究与应用奠定基础。     

Cloning and sequencing of genes encoding the beta subunits of the ATP-synthases from Enterobacter aerogenes and Flavobacterium ferrugineum

Abstract The genes encoding the β-subunit of the ATPase from Enterobacter aerogenes and Flavobacterium ferrugineum were cloned and their sequences determined. The predicted amino acid sequences were compared with the corresponding proteins from other eubacteria. Homology values of 58–98% confirmed the highly conserved character of the ATPase β-subunit. The enterobacterial ( Escherichia coli, E. aerogenes ) β-subunits represent the shortest sequences, whereas the corresponding F. ferrugineum protein exhibits an additional 33 amino acid residues as insertions at three different locations.     

Characterization of an alkaline lipase from Proteus vulgaris K80 and the DNA sequence of the encoding gene

Abstract A facultatively anaerobic bacterium producing an extracellular alkaline lipase was isolated from the soil collected near a sewage disposal plant in Korea and identified to be a strain of Proteus vulgaris . The molecular mass of the purified lipase K80 was estimated to be 31 kDa by SDS-PAGE. It was found to be an alkaline enzyme having maximum hydrolytid activity at pH 10, while fairly stable in a wide pH range from 5 to 11. The gene for lipase K80 was cloned in Escherichia coli . Sequence analysis showed an open reading frame of 861 bp coding for a polypeptide of 287 amino acid residues. The deduced amino acid sequence of the lipase gene had 46.3% identity to the lipase from Pseudomonas fragi .     

乳粉中普通变形杆菌和奇异变形杆菌复合PCR-DHPLC检测技术的建立

应用复合PCR结合变性高效液相色谱(DHPLC)技术,建立乳粉中普通变形杆菌和奇异变形杆菌的高通量检测方法。根据普通变形杆菌的blaA和blaB基因及奇异变形杆菌的ureR基因序列分别设计特异性引物,复合PCR扩增的产物经DHPLC技术进行快速检测,并以37株参考菌株做特异性试验。试验结果表明,该方法具有很好的特异性,经复合PCR-DHPLC可同时检测乳粉中普通变形杆菌和奇异变形杆菌。该方法可以快速、准确、高通量检测普通变形杆菌和奇异变形杆菌,是乳粉中致病菌高通量检测的新技术。     

Cloning and expression of various staphylococcal genes encoding urease in Staphylococcus carnosus

The urease genes from Staphylococcus xylosus C2a, Staphylococcus aureus U500, and S. aureus Newman were cloned in Staphylococcus carnosus using the plasmid vectors pCA43 and pCA44. The resulting respective recombinant plasmids pUra 402, pUraUH66, and pUra17 contained chromosomal DNA fragments with sizes of 5.6, 5.8, and 6.8 kb, respectively. Investigations on urease expression of the donor and recombinant strains in media with various nitrogen sources revealed that S. xylosus C2a produced urease constitutively at the highest specific activity. All of the recombinant strains had significantly lower urease activities than their DNA-donor strains. The nickel-dependence of urease was demonstrated in S. aureus U500 by a plate diffusion assay.     

Nickel availability and urease expression in Proteus mirabilis

Cells of Proteus mirabilis, previously grown in nutrient broth (NB), exhibited an increase in urease activity during subsequent incubation in mineral medium even when protein biosynthesis was inhibited. During growth in NB, degradation of amino acids obviously led to the formation of nickel-complexing metabolites, and nickel ions were therefore inavailable for maximal expression of enzymatically active urease; this inhibition of urcase biosynthesis was overcome by the addition of nickel to the growth medium, and also by added glucose. Experiments concerning the incorporation of radioactive nickel into urease finally indicated that the observed increase in urease activity was caused by posttranslational insertion of nickel into preformed apourease.     

斑点叉尾鮰源普通变形杆菌的分离、鉴定及药敏特性

【目的】对患病斑点叉尾鮰进行病原菌分离、鉴定及药敏实验,为斑点叉尾鮰肠道坏死病的防控提供参考。【方法】从患病斑点叉尾鮰病灶、肝、脾和肾分离纯化病原菌,经理化特性测定及16S rRNA基因序列分析对其进行鉴定,开展人工感染试验,并利用纸片扩散法进行药敏特性分析。【结果】分离菌株k1为本次引发斑点叉尾鮰病害的致病菌,其对斑点叉尾鮰的LD50为2.82×10~5 CFU/g。菌株k1理化特性与普通变形杆菌Proteus vulgaris基本一致,16S rRNA基因序列与普通变形杆菌相似性最高,综合判定分离菌株为普通变形杆菌。分离菌株k1对环丙沙星、头孢唑林及头孢拉定等12种抗生素高度敏感,对苯唑西林、阿莫西林及痢特灵等7种抗生素耐药。【结论】分离菌株k1是斑点叉尾鮰病原菌,养殖时可选用庆大霉素及氟苯尼考等药物进行防控。     

牛蛙致病变形菌的鉴定及其敏感药物筛选

【目的】分离鉴定引起牛蛙(Rana catesbeiana)皮肤溃疡病症的致病菌,筛选抗菌药物。【方法】采用无菌解剖组织划线分离法分离致病菌,通过人工感染试验、菌体和菌落形态观察、API20E系统鉴定、16S r RNA基因序列分析,确定分离菌的致病性及分类地位,并对该菌进行药物敏感性试验。【结果】从患病濒死牛蛙体内分离细菌NWG20141026,通过形态特征观察、生理生化试验、API 20E系统鉴定和16S r RNA基因序列相似性分析,认为NWG20141026菌株为普通变形菌(Proteus vulgaris)。人工感染试验显示,NWG20141026菌株不仅可以通过皮肤伤口感染引起蛙体表溃疡溃烂病症,也可通过消化道感染引起蛙肠炎病。药敏实验结果表明,氟苯尼考、四环素、多西环素、萘啶酸、磺胺异噁唑、恩诺沙星、红霉素等7种药物对普通变形菌具有较好的抑杀菌作用。【结论】引起牛蛙(R.catesbeiana)皮肤溃疡病症的致病菌NWG20141026为普通变形菌(P.vulgaris),所患疾病命名为牛蛙变形菌病。普通变形菌对健康牛蛙具有较强致病性,氟苯尼考、四环素、多西环素、萘啶酸、磺胺异噁唑等5种药可作为防治牛蛙变形菌病的候选药物。     

幽门螺杆菌尿素酶B亚单位的克隆和序列分析

幽门螺杆菌是消化道疾病的主要致病菌。其有效抗原成份尿素酶B亚单位(UreB)可刺激机体产生保护性的免疫反应。用高保真PCR扩增系统扩增出UreB基因片段,将其克隆至质粒pUC19中,对其全序列进行了测定。克隆的UreB基因序列与报道的序列完全一致。这一研究获得了序列正确的UreB基因,为将来以UreB分子为抗原的疫苗研制工作打下了良好的基础。     

Characterization of the genes encoding urease activity of Klebsiella pneumoniae

Abstract The genes encoding urease activity of Klebsiella pneumoniae were cloned and expressed in Escherichia coli . Transformants containing recombinant plasmids were selected by the antibiotic resistance phenotype and the production of urease in a Urease-test agar. Deletion derivatives of the parental recombinant plasmid were construced, and the relative position of six genes, necessary for urease production, was determined. Using a colorimetric assay it was demonstrated that some of the transformants exhibited ureolytic activity up to six-times greater than that of the original K pneumoniae isolate. Dot-blot DNA hybridization analysis revealed that the urease gene cluster of K. pneumoniae possesses no significant homology with those of Proteus species and Morganella morganii .     

Enhanced phenylpyruvic acid production with Proteus vulgaris in fed-batch and continuous fermentation

Phenylpyruvic acid is a deaminated form of phenylalanine and is used in various areas such as development of cheese and wine flavors, diagnosis of phenylketonuria, and to decrease excessive nitrogen accumulation in the manure of farm animals. However, reported phenylpyruvic acid fermentation studies in the literature have been usually performed at shake-flask scale with low production. In this study, phenylpyruvic acid production was evaluated in bench-top bioreactors by conducting fed-batch and continuous fermentation for the first time. As a result, maximum phenylpyruvic acid concentrations increased from 1350 mg/L (batch fermentation) to 2958 mg/L utilizing fed-batch fermentation. Furthermore, phenylpyruvic acid productivity was increased from 48 mg/L/hr (batch fermentation) to 104 and 259 mg/L/hr by conducting fed-batch and continuous fermentation, respectively. Overall, this study demonstrated that fed-batch and continuous fermentation significantly improved phenylpyruvic acid production in bench-scale bioreactor production.     

Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum

Abstract A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-α-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.     

Cloning and sequencing of the gene encoding the beta subunit of the sodium ion translocating ATP synthase of Propionigenium modestum

Abstract The gene coding for the β-subunit of the sodium ion translocating ATP synthase from Propionigenium modestum was cloned and sequenced. The predicted amino acid sequence resembles that of the β-subunits from proton-translocating ATP synthases. The same conserved regions are found in both types of ATP synthases. This is a good indication that the β-subunits of the proton and sodium ion translocating ATP synthases have evolved from a common ancestor.     

Cloning, expression and sequencing of Helicobacter felis urease genes

Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid cione led to the construction of plLL205 (9.5 kb) which conferred a urease activity of 1.2±0.5 μmole urea min^-1 mg^-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on plLL205 which were necessary for the expression of an urease-positive phenotype in E. coii clones. To localize the putative structural genes of H. felis on plLL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant cione did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had caicuiated moiecuiar masses of 26 074 and 61 663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.     

Organization and nucleotide sequence of genes encoding core components of the phycobilisomes from Synechococcus 6301

Summary Cyanobacteria possess specialized organelles, called phycobilisomes, which collect and transfer light energy to the reaction centres of photosystem II, in the photosynthetic membrane. Phycobilisomes consist of a central core, mainly composed of allophycocyanin, from which six rods radiate. We report here the isolation, for the first time, of three genes that encode core components of cyanobacterial phycobilisomes. The genes coding for the -and -subunit apoproteins of allophycocyanin (apcA and apcB) were cloned from Synechococcus PCC 6301 and subjected to nucleotide sequence analysis. Dowstream of apcB, we found a third open reading frame (apcC) which, by comparison with known amino acid sequences, was assigned to L _c ^7.8 , a linker polypeptide associated with phycobiliproteins within the core of the phycobilisomes. Homologies between amino acid sequences deduced from the nucleotide sequence of the Synechococcus PCC 6301 apc genes and the amino acid sequences published for corresponding proteins either from cyanobacteria or chloroplast-like organelles of eukaryotic organisms, are 75% or more. The genetic organization of this photosynthetic gene cluster relative to that observed in the cyanelle genome of the flagellate Cyanophora paradoxa is discussed.     

Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B

A 3 kb DNA fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coli and its nucleotide sequence determined. Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus flavus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs. A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same operon in T. aquaticus B. Expression of the T. aquaticus B mdh gene in E. coli was found to be at a relatively low level. A simple method for purification of thermostable MDH from the E. coli clone containing the T. aquaticus B mdh gene is presented.