Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a proteobacterial lipase family and an analogous lipase chaperone family

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5kDa) and 347 aa (38.6kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.     

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 .     

Circular dichroism of the O-specific polysaccharide of Vibrio cholerae O1 and some related derivatives

The O-specific polysaccharide (O-SP) of Vibrio cholerae O1 is a homopolymer of α-(1 → 2)-linked 4-amino-4,6-dideoxy- -mannopyranose whose amino group is acylated with 3-deoxy- -glycero-tetronic acid [

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. The circular dichroism (CD) of the O-SP as well as of a number of N-acyl (formyl, acetyl, 4-hydroxybutyl, 3-deoxy- and -glycero-tetronyl) derivatives of methyl α-glycosides of 4-amino-4,6-dideoxy- -mannopyranose (methyl α- -perosaminide) has been studied for solutions in water, acetonitrile and 1,1,1-trifluoroethanol. The strong solvent dependence of the sign and intensity of the CD observed for the monosaccharide amides bearing achiral acyl groups is explained by solvent-mediated change of the orientation of the amido group relative to the proximal hydroxyl group at C-3. A change in the population of the nonplanar conformers with a pyramidal arrangement of bonds at the amido nitrogen has also been considered. The effect of solvents upon the CD spectra of compounds bearing chiral N-acyl substituents is less pronounced than that of their counterparts bearing achiral N-acyl substituents. The sign of the CD for the O-SP was found negative in all solvents used. This result is in agreement with the negative sign of the CD of the n → π electron transition observed, independent of solvent, for the monosaccharide derivative containing the group, and the positive sign found for its -glycero-counterpart.     

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

Role of the calcium ion and the disulfide bond in the Burkholderia glumae lipase

The role of the Ca²⁺ ion that is present in the structure of Burkholderia glumae lipase was investigated. Previously, we demonstrated that the denatured lipase could be refolded in vitro into an active enzyme in the absence of calcium. Thus, an essential role for the ion in catalytic activity or in protein folding can be excluded. Therefore, a possible role of the Ca²⁺ ion in stabilizing the enzyme was considered. Chelation of the Ca²⁺ ion by EDTA severely reduced the enzyme activity and increased its protease sensitivity, however, only at elevated temperatures. Furthermore, EDTA induced unfolding of the lipase in the presence of urea. From these results, it appeared that the Ca²⁺ ion in B. glumae lipase fulfils a structural role by stabilizing the enzyme under denaturing conditions. In contrast, calcium appears to play an additional role in the Pseudomonas aeruginosa lipase, since, unlike B. glumae lipase, in vitro refolding of this enzyme was strictly dependent on calcium. Besides the role of the Ca²⁺ ion, also the role of the disulfide bond in B. glumae lipase was studied. Incubation of the native enzyme with dithiothreitol reduced the enzyme activity and increased its protease sensitivity at elevated temperatures. Therefore, the disulfide bond, like calcium, appears to stabilize the enzyme under detrimental conditions.     

Cloning, expression and characterization of a novel thermal stable and short-chain alcohol tolerant lipase from Burkholderia cepacia strain G63

A lipase gene lipA and its chaperone gene lipB were cloned from Burkholderia cepacia strain G63. The lipA was composed of 1092 bp, encoding 363 amino acid residues, and the lipB composed of 1035 bp, corresponding to 344 amino acid residues. The significant amino acid similarity with Pseudomonas cepacia lipase revealed that this enzyme could be classified into the lipolytic subfamily I.2. The lipA and lipB genes were cloned into pBBR1Tp vector and conjugated into B. cepacia strains G63 with the help of pRK2013. The recombinant strain was fermented in 10 l bioreactor and the lipase was purified by a combination of ammonium sulfate fractionation, DEAE ion-exchange chromatography and gel filtration. The purified lipase kept stable at a temperature range of 40–70 °C. After incubated at 70 °C, the optimal temperature of this enzyme, for 10 h it remained 86.1% of its activity. The enzyme was also highly tolerant to a series of organic solution. Incubated in 50% methanol solution up to 48 h, the enzyme still kept 98.3% of its activity. The transesterification activity of soybean oil to fatty acid methyl esters (FAMEs) reached 87.8% after 72 h, indicating that it is a potential biocatalyzer for biodiesel production.     

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans

Summary A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cm^s), arginine auxotrophic (Arg^–) mutant of Streptomyces lividan 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cm^s Arg^– mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.     

解脂耶氏酵母(Yarrowia lipolytica) ATCC30162脂肪酶基因Yllip1和Yllip2的结构及表达特征

以目前报道油脂产量最高的解脂耶氏酵母菌株(Yarrowia lipolytica)ATCC 30162为对象,采用逆转录PCR扩增到脂肪酶编码基因Yllip1和Yllip2,编码产物分别为816和549个氨基酸。保守结构域预测表明,Yllip1包含Patatin类磷脂酶和功能未知的DUF3336结构域,而Yllip2包含lipase_3类脂肪酶结构域,且这两个蛋白都具有1～4个跨膜区域。与不同物种来源的脂肪酶同源蛋白的多序列比对表明Yllip1和Yllip2分别包含8和6个保守区域,这些生物信息学分析表明这两个来源于解脂耶氏酵母的脂肪酶作用底物可能分别为细胞内膜磷脂和酰基甘油酯。荧光定量PCR分析表明:培养基中添加油酸在短期内(6 h)诱导了这两个脂肪酶基因Yllip1和Yllip2的显著上调表达,表明它们可能参与了酵母分解利用油酸的生化过程。     

Cloning, sequence analysis, and expression of ansB from Pseudomonas fluorescens, encoding periplasmic glutaminase/asparaginase

A gene (ansB) encoding a class II glutaminase/asparaginase has been cloned from Pseudomonas fluorescens and characterized by DNA sequencing, promoter analysis and heterologous expression in Escherichia coli. We show that ansB is monocistronic and depends on the alternate sigma factor sigma 54 for expression. A second open reading frame located downstream of ansB is highly homologous to a number of bacterial genes that encode secreted endonucleases of unknown function.     

华癸中慢生根瘤菌7653R 3个脂质转运蛋白基因的克隆、突变及共生表型北大核心CSCD

【目的】脂类转移家族蛋白基因编码一类参与脂类转运及代谢的蛋白。本研究旨在构建华癸中慢生根瘤菌3个脂质转运家族蛋白基因的突变株,检测及分析突变体与紫云英共生条件下的表型及功能。【方法】利用生物信息学分析与预测转脂蛋白的结构特征及功能,采用荧光定量技术检测目标基因在自生和共生条件下的表达特性,通过插入突变技术构建目标基因突变株,并进行植物盆栽实验考察其共生表型。【结果】MCHK-5577、MCHK-2172和MCHK-2779基因编码蛋白属于START/RHO alpha_C/PITP/Bet_v1/Cox G/Cal C(SRPBCC)超家族,包含脂类转移结构域,参与脂类转运或代谢,与百脉根等中慢生根瘤菌相应基因的序列相似性达95%以上。这3个基因在共生条件下的表达水平都增高。分别构建了MCHK-5577、MCHK-2172和MCHK-2779基因突变菌株,与野生型菌株7653R相比,接种突变株MCHK-2172mut、MCHK-2779mut和MCHK-5577mut后的植株地上部分生物量和根瘤固氮酶活性显著降低。【结论】华癸中慢生根瘤菌脂质转移家族蛋白基因在共生互作过程中发挥重要作用,突变后明显影响共生固氮表型。本文的实验结果为深入研究脂类转移蛋白在共生固氮作用中的功能机制奠定了基础。     

Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea

Summary A gene from Magnaporthe grisea was cloned using a cDNA clone of the Colletotrichum gloeosporioides cutinase gene as a heterologous probe; the nucleotide sequence of a 2 kb DNA segment containing the gene has been determined. DNA hybridization analysis shows that the M. grisea genome contains only one copy of this gene. The predicted polypeptide contains 228 amino acids and is homologous to the three previously characterized cutinases, showing 74% amino acid similarity to the cutinase of C. gloeosporioides. Comparison with previously determined cutinase sequences suggests that the gene contains two introns, 115 and 147 bp in length. The gene is expressed when cutin is the sole carbon source but not when the carbon source is cutin and glucose together or glucose alone. Levels of intracellular and extracellular cutinase activity increase in response to growth in the presence of cutin. The activity level is higher in a transformant containing multiple copies of the cloned gene than in the parent strain. Non-denaturing polyacrylamide gels stained for esterase activity show a single major band among intracellular and extracellular proteins from cutin-grown cultures that is not present among intracellular and extracellular proteins prepared from glucose-grown or carbon-starved cultures. This band stains more intensely in extracts from the multicopy transformant than in extracts from the parent strain. We conclude that the cloned DNA contains a M. grisea gene for cutinase, which we have named CUT1.     

Cloning and analysis of the mating type genes from Cochliobolus heterostrophus

Cochliobolus heterostrophus, a heterothallic Ascomycete, has a single mating type locus with two alternate forms called MAT-1 and MAT-2. MAT-1 was cloned by complementing a MAT-2 strain using a cosmid library from a MAT-1 strain and screening for a homothallic transformant. The cosmid recovered from this transformant was able to re-transform a MAT-2 strain to homothallism and MAT identity was proven by restriction fragment length polymorphism and conventional genetic mapping. All homothallic transformants could mate with either MAT-1 or MAT-2 strains, although the number of ascospores produced by self matings or crosses to MAT-2 strains was low. Progeny of selfed homothallic transformants were themselves homothallic. MAT-2 was cloned by probing a cosmid library from a MAT-2 strain with a fragment of insert DNA from the MAT-1 cosmid. A 1.5 kb subclone of either MAT-containing cosmid was sufficient to confer mating function in transformants. Examination of the DNA sequence of these subclones revealed that MAT-1 and MAT-2 contain 1297 by and 1171 bp, respectively, of completely dissimilar DNA flanked by DNA common to both mating types. Putative introns were found (one in each MAT gene) which, when spliced out, would yield open reading frames (ORFs) that occupied approximately 90% of the dissimilar DNA sequences. Translation of the MAT-1 ORF revealed similarity to the Neurospora crassa MATA, Podospora anserina mat–, and Saccharomyces cerevisiae MAT1 proteins; translation of the MAT-2 ORF revealed similarity to the N. crassa MATa, P. anserina mat+, and Schizosaccharomyces pombe mat-Mc proteins. These gene products are all proven or proposed DNA binding proteins. Those with similarity to MAT-2 are members of the high mobility group.The first three authors contributed equally to the work     

北美鹅掌楸LtAGO1基因的克隆、表达及其启动子分析

为深入探究叶原基分化成叶器官的形态建成机制,该研究以北美鹅掌楸为材料,采用RT-PCR和RACE克隆技术获得LtAGO1的cDNA全长和启动子序列并预测其功能,通过RT-qPCR分析LtAGO1在鹅掌楸属中的组织表达模式。同时,经抗性筛选和DNA鉴定获得ProAGO1 ∷ GUS的转基因拟南芥株系,并进一步对T2代阳性植株进行表型和GUS组织化学染色分析。结果表明:(1) LtAGO1基因包含3 300 bp的开放阅读框,编码1 100个氨基酸,分子量为122.14 kD,理论等电点(pI)为9.36。(2)氨基酸序列分析显示LtAGO1含Gly-rich-AGO1和Piwi两个典型的AGO基因结构域,同源性分析显示LtAGO1蛋白与沉水樟AGO1蛋白(RWR84608.1)亲缘关系最近。(3)组织表达特异性分析显示LtAGO1在北美鹅掌楸不同组织间的相对表达量为雄蕊>花芽>花瓣>花萼>叶片>雌蕊>叶芽>茎,LtAGO1在北美鹅掌楸叶片不同发育阶段的相对表达量为叶芽萌动期>幼叶期>衰老期>成熟期,AGO1在鹅掌楸属叶缘的表达量高于叶片的其他部位且北美鹅掌楸叶凹陷部位的表达量高于叶尖部位。(4)获得叶中-侧轴向和基-顶轴向的极性缺失、叶缘锯齿、重瓣花型的转化株系,GUS组织染色显示ProAGO1启动GUS基因在叶芽顶端稳定表达且在新分化的叶柄上表达较强,在成熟期的茎、叶、花和果的维管束中均特异表达。LtAGO1启动子的GUS活性强度为叶顶芽>花>维管束,这与实时定量PCR结果相一致。综上认为,LtAGO1基因在顶端分生组织特异表达且受到多种途径的调控而参与到叶和花器官的发育进程中。该研究结果为进一步了解北美鹅掌楸LtAGO1基因的基本功能及其调控叶形发育机制提供了理论基础。     

Cloning, sequence, and expression of a lipase gene from Pseudomonas cepacia: lipase production in heterologous hosts requires two Pseudomonas genes.

The lipA gene encoding an extracellular lipase from Pseudomonas cepacia was cloned and sequenced. Downstream from the lipase gene an open reading frame was identified, and the corresponding gene was named limA. lipA was well expressed only in the presence of limA. limA exerts its effect both in cis and in trans and therefore produces a diffusible gene product, presumably a protein of 344 amino acids. Replacement of the lipA expression signals (promoter, ribosome-binding site, and signal peptide-coding sequences) by heterologous signals from gram-positive bacteria still resulted in limA-dependent lipA expression in Escherichia coli, Bacillus subtilis, and Streptomyces lividans.     

Genomic characterization of antibiotic resistance-encoding genes in clinical isolates of Vibrio cholerae non-O1/non-O139 strains from Kolkata,India: generation of novel types of genomic islands containing plural antibiotic resistance genes

Non-O1/non-O139 nontoxigenic Vibrio cholerae associated with cholera-like diarrhea has been reported in Kolkata, India. However, the property involved in the pathogenicity of these strains has remained unclear. The character of 25 non-O1/non-O139 nontoxigenic V. cholerae isolated during 8 years from 2007 to 2014 in Kolkata was examined. Determination of the serogroup showed that the serogroups O6, O10, O35, O36, O39, and O70 were represented by two strains in each serogroup, and the remaining isolates belonged to different serogroups. To clarify the character of antibiotic resistance of these isolates, an antibiotic resistance test and the gene analysis were performed. According to antimicrobial drug susceptibility testing, 13 strains were classified as drug resistant. Among them, 10 strains were quinolone resistant and 6 of the 13 strains were resistant to more than three antibiotics. To define the genetic background of the antibiotic character of these strains, whole-genome sequences of these strains were determined. From the analysis of these sequences, it becomes clear that all quinolone resistance isolates have mutations in quinolone resistance-determining regions. Further research on the genome sequence showed that four strains possess Class 1 integrons in their genomes, and that three of the four integrons are found to be located in their genomic islands. These genomic islands are novel types. This indicates that various integrons containing drug resistance genes are spreading among V. cholerae non-O1/non-O139 strains through the action of newly generated genomic islands.     

Cloning and sequence analysis of the arginine deiminase gene from Mycoplasma arginini

Summary A deletion mutant of Rhizobium leguminosarum biovar viciae lacking the host-specific nodulation (nod) gene region (nodFEL nodMNT and nodO) but retaining the other nod genes (nodD nodABCIJ) was unable to nodulate peas or Vicia hirsuta, although it did induce root hair deformation. The mutant appeared to be blocked in its ability to induce infection threads and could be rescued for nodulation of V. hirsuta in mixed inoculation experiments with an exopolysaccharide deficient mutant (which is also Nod^–). The nodulation deficiency of the deletion mutant strain could be partially restored by plasmids carrying the nodFE, nodFEL or nodFELMNT genes but not by nodLMN. Surprisingly, the mutant strain could also be complemented with a plasmid that did not carry any of the nodFELMNT genes but which did carry the nodO gene on a 30 kb cloned region of DNA. Using appropriate mutations it was established that nodO is essential for nodulation in the absence of nodFE. Thus, either of two independent nod gene regions can complement the deletion mutant for nodulation of V. hirsuta. Similar observations were made for pea nodulation except that nodL was required in addition to nodO for nodulation in the absence of the nodFE genes. These observations show that nodulation can occur via either of two pathways encoded by non-homologous genes.Dedicated to the memory of the late Dr. David Goodchild     

独行菜LaMCT基因的克隆、序列分析与原核表达

强心苷作为药用植物独行菜( Lepidium apetalum)的活性成分,其化学和药理学研究已有良好的基础,但其生物合成途径目前仍不清楚。该研究以独行菜幼苗为材料,通过分析独行菜转录组数据,设计特异性引物,PCR扩增得到了强心苷生物合成MEP途径的关键酶2-C-甲基赤藓醇-4-磷酸胞苷酰转移酶( MCT)基因的开放阅读框( ORF),命名为LaMCT( Genbank注册号KT832554),并进行序列分析和原核表达。序列分析结果表明：LaMCT基因ORF全长为912 bp,编码304个氨基酸。亚细胞定位和保守结构域分析结果表明：LaMCT蛋白位于叶绿体中,不含信号肽,没有跨膜区,含有类异戊二烯合成酶保守结构域( isoprenoid synthase domain)。系统进化树结果表明：LaMCT蛋白与拟南芥的MCT蛋白具有94％的序列相似性,亲缘关系较近。通过构建pET-32a-LaMCT原核表达载体,成功在大肠杆菌BL21( DE3)菌株中诱导表达LaMCT重组蛋白,并得到了纯化的LaMCT重组蛋白。该研究首次从独行菜中克隆了LaMCT基因,建立其稳定的原核表达体系,为LaMCT蛋白抗体的制备以及研究LaMCT基因在独行菜强心苷类化合物生物合成途径中的功能奠定了基础。     

枸骨IcFPS1基因的克隆、表达及生物信息学分析

法尼基焦磷酸合酶(farnesyl diphosphate synthase,FPS)是三萜皂苷生物合途径的一个关键酶,为研究FPS基因在枸骨中的功能,该研究采用PCR技术将一个FPS基因的cDNA序列从枸骨叶中分离出来,并命名为IcFPS1。结果表明:根据测序结果分析发现扩增获得的IcFPS1基因cDNA长度为1 591 bp,包含一个完整的开放阅读框,大小为1 029 bp。通过序列分析发现枸骨IcFPS1基因编码342个氨基酸,分子量和等电点分别为39.58 kDa和5.18。通过理化性质预测分析发现IcFPS1蛋白不含信号肽,不含有跨膜区域,该IcFPS1蛋白为亲水性蛋白质。通过序列多重比对发现IcFPS1蛋白质与其他植物的FPS蛋白质高度同源,有共同的保守区域和氨基酸序列,其中与西洋参FPS序列的相似性高达89%。通过系统进化树分析发现枸骨FPS蛋白与同属于被子植物的五加科植物FPS蛋白亲缘关系较近,说明FPS基因在进化过程中相对比较保守。根据蛋白调控网络预测分析结果发现该蛋白可能与IPP1、IPP2、GGPS3、GGPS6和ERA1相互作用,参与类异戊二烯的合成代谢过程。通过实时荧光定量PCR分析发现IcFPS1基因在枸骨各个组织部位中均有表达,其中在枸骨根中表达量最高,在茎和雌花中表达量最低。     

Cloning and comparative sequence analysis of PUM1 and PUM2 genes,human members of the Pumilio family of RNA-binding proteins

Drosophila gene Pumilio (Pum) is a founder member of an evolutionarily conserved family of RNA-binding proteins that are present from yeast to mammals, and act as translational repressors during embryo development and cell differentiation. The human genome contains two Pumilio related genes, PUM1 and PUM2, that encode 127 and 114 kDa proteins with evolutionarily highly conserved Pum RNA-binding domain (86 and 88% homology with the fly Pum protein). PUM1 and PUM2 proteins share 83% overall similarity, with RNA-binding domain being 91% identical. Both PUM1 and PUM2 show relatively widespread and mostly overlapping expression in human tissues, and are very large genes with highly conserved gene structure. PUM1 consists of 22 exons, spanning about 150 kb on chromosome 1p35.2, whereas PUM2 consists of 20 exons and spans at least 80 kb on chromosome 2p23-24. Extremely high evolutionary conservation of the RNA-binding domain from yeast to humans, and conserved function of Pumilio proteins in invertebrates and lower vertebrates suggest that mammalian Pumilio proteins could also play an important role in translational regulation of embryogenesis and cell development and differentiation.