Cloning and characterization of a gene encoding a new thermostable hemolysin from Vibrio parahaemolyticus

A new thermostable hemolysin (delta-VPH) gene was cloned from a Kanagawa-negative Vibrio parahaemolyticus strain into vector pBR322 in Escherichia coli K12. The nucleotide and amino acid sequences had no homology with those of the thermostable direct hemolysin (TDH) which causes the Kanagawa phenomenon, and of the thermolabile hemolysin (TLH) of V. parahaemolyticus. The gene was present in all V. parahaemolyticus strains tested and also in one strain of V. damsela.     

Cloning and expression of two genes encoding auxin-binding proteins from tobacco

Two genes encoding the auxin-binding protein (ABP1) of tobacco (Nicotiana tabacum L.), both of which possess the characteristics of a luminal protein of the endoplasmic reticulum (ER), were isolated and sequenced. These genes were composed of at least five exons and four introns. The two coding exons showed 95% sequence homology and coded for two precursor proteins of 187 amino acid residues with molecular masses of 21 256 and 21 453 Da. The deduced amino acid sequences were 93% identical and both possessed an amino-terminal signal peptide, a hydrophilic mature protein region with two potential N-glycosylation sites and a carboxyl-terminal sorting signal, KDEL, for the ER. Restriction mapping of the cDNAs encoding tobacco ABP1, previously purified by amplification of tobacco cDNA libraries by polymerase chain reaction (PCR) using specific primers common to both genes, indicated that both genes were expressed, although one was expressed at a higher level than the other. Genomic Southern blot hybridization showed no other homologous genes except for these two in the tobacco genome. The apparent molecular mass of the mature form of tobacco ABP1 was revealed to be 25 kDa by SDS polyacrylamide gel electrophoresis using affinity-purified anti (tobacco ABP1) antibodies raised against a fusion protein with maltose-binding protein. Expression of the recombinant ABP1 gene in transgenic tobacco resulted in accumulation of the 25 kDa protein. A single point mutation of an amino acid residue at either of the two potential N-glycosylation sites resulted in a decrease in the apparent molecular mass and produced a 22 kDa protein. Mutations at both sites resulted in the formation of a 19.3 kDa protein, suggesting that tobacco ABP1 is glycosylated at two asparagine residues.     

一株对羟基苯甲酸酯海洋降解菌的关键酶基因克隆与表达

【背景】对羟基苯甲酸及其酯类常作为合成多种芳香族化合物的前体物质广泛应用于多个领域,但其难以自然降解给环境造成了污染问题,同时这些污染物随着洋流迁移到海洋中破坏海洋生态环境。【目的】从海洋环境中筛选对羟基苯甲酸酯高效降解菌,通过全基因组测序及注释分析,预测对羟基苯甲酸酯代谢通路,确定其代谢过程中的关键酶并进行功能研究。【方法】通过富集培养从海洋环境中分离对羟基苯甲酸酯降解菌,利用基因克隆技术将降解对羟基苯甲酸酯关键酶基因在大肠杆菌中高效表达,探究重组蛋白活性及酶学特征。【结果】从海底泥沙中筛选到一个菌株,经16S rRNA基因测序鉴定为硝化柠檬球菌(Citricoccus nitrophenolicus);该菌株能够利用多种对羟基苯甲酸酯类物质进行生长,在甲酯为碳源条件下生长状态最好;将羧酸酯酶基因和单加氧酶基因在大肠杆菌中进行高效表达,重组表达的羧酸酯酶最适反应条件为：pH 8.0,30℃反应30 min;重组表达的单加氧酶活性表达依赖于辅酶,Mg²⁺、Mn²⁺、Zn²⁺和Fe3+可增强该酶活性;经荧光定量PCR进一步...     

Cloning and antigenic expression of two genes from Chlamydia psittaci avian strain

Abstract: Genes from Chlamydia psittaci P-1041 were cloned into the Bam HI site of pUC19 and were transformed to host Escherichia coli JM109. Two recombinant plasmids that expressed protein antigens of Chlamydia were isolated. The sizes of the DNA fragments were 1350 and 1710 bp, and encoded for polypeptides of M _r 25 and 42 kilodaltons (kDa), respectively. The 25-kDa protein had cross-reactivity with antisera to ten C. psittaci strains and two C. trachomatis strains, whereas the 42-kDa protein reacted only with homologous antiserum to the C. psittaci P-1041 strain. Furthermore, in Southern hybridization analysis these two fragments as probes hybridized with DNA of ten C. psittaci strains and four C. trachomatis strains. These results indicated that the two fragments shared a DNA sequence common to the chlamydial genus.     

Cloning and expression of genes encoding meta-cleavage enzymes from 4,6-dimethyldibenzothiophene-degrading Sphingomonas strain TZS-7

Sphingomonas strain TZS-7 was reported as the first strain to have the ability to degrade 4,6-dimethyldibenzothiophene (4,6-dmDBT) by the ring-destructive pathway. Two genes for meta-cleavage dioxygenases were cloned from strain TZS-7. Expression of each gene showed that one enzyme was specific for 2,3-dihydroxybiphenyl while another was more specific for catechol. The genes for the two enzymes were named dmdC and catA. The analysis of deduced amino acid sequences indicates that CatA falls into the class of meta-cleavage dioxygenases acting on dihydroxylated monocyclic compounds and DmdC falls into the class of meta-cleavage dioxygenases acting on dihydroxylated polycyclic compounds.     

Comparative amino acid sequence analysis of hemolysins produced by Vibrio hollisae and Vibrio parahaemolyticus.

Vibrio hollisae produces a hemolysin (Vh-rTDH) that is related to the thermostable direct hemolysin of Vibrio parahaemolyticus (Vp-TDH). Although both hemolysins are essentially similar biologically and immunologically, they differ markedly in heat stability; Vp-TDH is heat stable, whereas Vh-rTDH is heat labile. To elucidate the relationships between their characteristics and molecular structures, we analyzed the amino acid sequence of Vh-rTDH and compared it with that of Vp-TDH. Vh-rTDH consisted of 165 residues, of which 23 residues, spread over the peptide chain, differed from those of Vp-TDH.     

Isolation of a new drug-resistance plasmid from a strain of Vibrio parahaemolyticus

A new R plasmid, pSA55, with a molecular weight of 112 megadaltons (Md), was isolated from a strain of Vibrio parahaemolyticus with multiple drug resistance. The pSA55 plasmid conferred on its host resistance to chloramphenicol, tetracycline, streptomycin, kanamycin, ampicillin, trimethoprim and 2,4-diamino-6,7-diisopropyl pteridine, and belongs to incompatibility group C. The plasmid was transferable to Escherichia coli, V. parahaemolyticus, V. alginolyticus and NAG bivrio at a frequency of 10(-3) approximately -7, and was stably inherited by the transconjugants of these species. The conjugal transfer of pSA55 plasmid was significantly affected by the growth culture phase. The resistance pattern and resistance levels of transconjugants were the same as those of the donor strain. We did not observe fluctuations in minimal inhibitory concentrations with transfer, unlike the case of V. cholerae. The relationship between the pSA55 plasmid and the Kanagawa phenomenon was not clarified in the present study.     

Comparison of hemolysins of Vibrio cholerae non-O1 and Vibrio hollisae with thermostable direct hemolysin of Vibrio parahaemolyticus

Hemolysin (Vh-rTDH) produced by Vibrio hollisae and hemolysin (NAG-rTDH) produced by Vibrio cholerae non-O1 were characterized and compared with hemolysin (Vp-TDH) produced by Vibrio parahaemolyticus. These three hemolysins are each composed of two subunits and have similar, but not identical, molecular weights. The amino acid compositions of Vp-TDH and NAG-rTDH are similar, but are different from that of Vh-rTDH. The three hemolysins showed similar lethal toxicities to mice. The effects of temperature on hemolysis and the time dependencies of hemolysis by the three hemolysins were similar. The three were concluded to be immunologically related, but not identical, and to have common and also unique antigenic determinants.     

Cloning and sequence analysis of a novel metalloprotease gene from Vibrio parahaemolyticus 04

The metalloprotease gene (vppC) from Vibrio parahaemolyticus 04 has been cloned and sequenced. The vppC gene contains an open reading frame of 2442 nucleotides encoding a polypeptide of 814 amino acids with a calculated molecular mass of 89,833 Da. The predicted amino acid sequence of VppC containing a zinc metalloprotease HEXXH consensus motif displays extensive homology to the collagenase from Vibrio alginolyticus. The activity of the recombinant protease produced in Escherichia coli was examined by gelatin zymography and proteolytic activity assays. The substrate specificity study showed that the type I collagen and synthetic collagenase substrate carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-L-prolyl-L-alanine were the best substrates, indicating that the cloned metalloprotease is indeed a collagenase. Multiple alignment analysis of the amino acid sequences and the enzymatic properties such as molecular mass and substrate specificity revealed three distinct classes of Vibrio metalloproteases. The identification of a new metalloprotease gene expands the role of Vibrio metalloproteases as a virulence factor for host infection.     

Identification and characterization of the sodA genes encoding manganese superoxide dismutases in Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus

Sequencing of Fur titration assay-positive clones obtained from genomic DNA libraries of Vibrio parahaemolyticus, V. mimicus and V. vulnificus revealed open reading frames encoding proteins of 202, 205 and 202 amino acid residues, respectively. Each open reading frame was preceded by a predicted Fur box which overlaps a likely promoter with similarity to the -10 and -35 consensus sequence of Escherichia coli. The deduced amino acid sequences shared considerable homology with bacterial Mn-containing superoxide dismutases (MnSODs). Consistent with this, these Vibrio strains produced proteins with SOD activity resistant to inhibition by H2O2 and KCN only when grown under iron-limiting conditions. Primer extension analysis of the total RNA from these vibrios revealed iron-repressible expression of the genes. Furthermore, when grown under iron-limiting conditions, E. coli carrying a plasmid with each cloned gene overexpressed protein with the same electrophoretic mobility and insensitivity of SOD activity to H2O2 and KCN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing revealed that proteins (MnSODs) having N-terminal amino acid sequences consistent with those deduced from the corresponding genes were present in cell lysates of the vibrios grown under these iron-limited conditions. These results demonstrate that the genes cloned in this study are sodA homologs encoding MnSODs, whose expression is regulated by the iron status of the growth medium. PCR using a primer set based on the V. parahaemolyticus sodA sequence revealed the presence of homologous genes in certain other Vibrio species.     

Cloning, sequencing, and expression of the genes encoding an isocyclomaltooligosaccharide glucanotransferase and an alpha-amylase from a Bacillus circulans strain

The gene for a novel glucanotransferase, isocyclomaltooligosaccharide glucanotransferase (IgtY), involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}] from starch, was cloned from the genome of B. circulans AM7. The IgtY gene, designated igtY, consisted of 2,985 bp encoding a signal peptide of 35 amino acids and a mature protein of 960 amino acids with a calculated molecular mass of 102,071 Da. The deduced amino-acid sequence showed similarities to 6-alpha-maltosyltransferase, alpha-amylase, and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in this enzyme, indicating that this enzyme should be assigned to this family. The DNA sequence of 8,325-bp analyzed in this study contained two open reading frames (ORFs) downstream of igtY. The first ORF, designated igtZ, formed a gene cluster, igtYZ. The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme was purified. The enzyme acted on maltooligosaccharides that have a degree of polymerization (DP) of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme (IgtZ), which has a novel amino-acid sequence, should be assigned to alpha-amylase. It is notable that both IgtY and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to a family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.     

罗尼氏弧菌Vibrio shilonii BY新琼胶酶基因的克隆、序列分析及表达

【目的】从海洋来源的罗尼氏弧菌菌株BY中克隆得到一个具有琼胶酶活性的新基因,并对其进行重组表达。【方法】对实验室保藏的产琼胶酶菌株BY进行16S rRNA基因序列分析,并构建系统发育树。根据已报道的琼胶酶基因序列的同源性,设计简并引物,利用降落PCR (Touch-down PCR)及染色体步移技术扩增琼胶酶基因序列全长,对基因序列进行生物信息学分析。将目的基因插入pET22a(+)载体,转化大肠杆菌BL21(DE3),对重组酶进行表达,利用DNS法测定了重组酶的酶活,对该重组琼胶酶酶学性质进行研究。【结果】克隆得到一条新的琼胶酶基因,命名为Vibrio sp. BY (GenBank登录号：AIW39921.1),Vibrio sp. BY基因序列全长2 232 bp,编码744个氨基酸,理论分子量为85 kD,Vibrio sp. BY的氨基酸序列基因库中与已知的琼胶酶氨基酸序列Vibrio sp. EJY3的相似度为86%。发酵液琼胶酶酶活力为71.73 U/mL,证明表达的蛋白为琼胶酶。酶学性质研究表明重组琼胶酶的最适温度及pH分别为50 °C和7.0,并且具有较好的稳定性。【结论】利用染色体步移技术克隆得到一条新的琼胶酶基因,并在大肠杆菌BL21(DE3)中实现了重组表达,为琼胶酶的应用奠定了基础。     

副溶血性弧菌毒力基因表达时内参基因的选择

[目的]筛选出合适的内参基因用于分析不同环境条件下副溶血性弧菌毒力基因的表达情况.[方法]本研究以虾样品中、海水样品中、过滤海水样品中以及TSB培养条件下的副溶血性弧菌为材料,利用qRT-PCR技术评价了GAPDH、pvuA、pvsA和rpoS4种常用管家基因在不同条件下的表达稳定性.[结果]4种管家基因均能特异扩增,表达稳定性排列顺序为pvuA(2.906)＞pvsA(3.197)＞GAPDH(3.746)＞rpoS(6.512),进一步通过geNorm软件分析,最终选择两个表达最为稳定的内参基因即pvuA和pvsA,以二者的几何平均值作为参照可更为准确地校正目的基因的表达.[结论]pvuA和pvsA可作为环境样品中副溶血性弧菌毒力基因表达变化研究的内参基因.     

Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli

The regulation of internal Na(+) and K(+) concentrations is important for bacterial cells, which, in the absence of Na(+) extrusion systems, cannot grow in the presence of high external Na(+). Likewise, bacteria require K(+) uptake systems when the external K(+) concentration becomes too low to support growth. At present, we have little knowledge of K(+) toxicity and bacterial outward-directed K(+) transport systems. We report here that high external concentrations of K(+) at alkaline pH are toxic and that bacteria require K(+) efflux and/or extrusion systems to avoid excessive K(+) accumulation. We have identified the first example of a bacterial K(+)(specific)/H(+) antiporter, Vp-NhaP2, from Vibrio parahaemolyticus. This protein, a member of the cation : proton antiporter-1 (CPA1) family, was able to mediate K(+) extrusion from the cell to provide tolerance to high concentrations of external KCl at alkaline pH. We also report the discovery of two V. parahaemolyticus Na(+)/H(+) antiporters, Vp-NhaA and Vp-NhaB, which also exhibit a novel ion specificity toward K(+), implying that they work as Na(+)(K(+))/H(+) exchangers. Furthermore, under specific conditions, Escherichia coli was able to mediate K(+) extrusion against a K(+) chemical gradient, indicating that E. coli also possesses an unidentified K(+) extrusion system(s).     

Organization and expression of the two homologous genes encoding the NADP-malate dehydrogenase in Sorghum vulgare leaves

Summary We previously described the isolation and the nucleotide sequence of a nuclear gene from sorghum (NMDHI; 4.6 kb) encoding the NADP-malate dehydrogenase. Further analysis led us to identify a second homologous gene (NMDH II; 4.8 kb) located within the same 12.3 kb genomic clone (LM17); these two genes are tandemly organized, in direct orientation. This second gene was entirely sequenced and comparison with the first gene showed that the positions on the 14 exons and 13 introns are conserved in both genes. The analysis of the genomic organization and copy number in the Sorghum vulgare genome revealed that there are no additional homologues and there is only one copy each of NMDH I and NMDH II. The isolation of two different cDNA clones in a previous work suggested that both genes were probably expressed. Analysis of specific mRNA accumulation during the greening process using synthetic oligonucleotide probes showed that the NMDH I gene is induced in the presence of light while the NMDH II gene seems to be constitutively expressed at low level.Abbreviations Cab chlorophyll a/b binding protein - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide - NADP-MDH NADP-dependent dehydrogenase - RbcS ribulose-1,5-bisphosphate carboxylase small subunit - SSC 0.15 M NaCl, 0.015 M sodium citrate pH 7.6     

The viable but nonculturable state of Kanagawa positive and negative strains of Vibrio parahaemolyticus

Ingestion of shellfish-associated Vibrio parahaemolyticus is the primary cause of potentially severe gastroenteritis in many countries. However, only Kanagawa phenomenon (hemolysin) positive (KP+) strains of V. parahaemolyticus are isolated from patients, whereas >99% of strains isolated from the environment do not produce this hemolysin (i.e. are KP-). The reasons for these differences are not known. Following a temperature downshift, Vibrio parahaemolyticus enters the viable but nonculturable (VBNC) state wherein cells maintain viability but cannot be cultured on routine microbiological media We speculated that KP+ and KP- strains may respond differently to the temperature and salinity conditions of seawater by entering into this state which might account for the low numbers of culturable KP+ strains isolated from estuarine waters. The response of eleven KP+ and KP- strains of V. parahaemolyticus following exposure to a nutrient and temperature downshift in different salinities, similar to conditions encountered in their environment, was examined. The strains included those from which the KP+ genes had been selectively removed or added. Our results indicated that the ability to produce hemolysin did not affect entrance into the VBNC state. Further, VBNC cells of both biotypes could be restored to the culturable state following an overnight temperature upshift.     

Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.