Cloning, expression, and nucleotide sequence of a lipase gene from Pseudomonas fluorescens B52.

In this study, we report the cloning and expression of lipase gene from Pseudomonas fluorescens B52, a psychrotrophic spoilage bacterium isolated from refrigerated raw milk. Sequence analysis revealed one major open reading frame of 1,428 nucleotides that was predicted to encode a protein with a molecular weight of 50,241. The predicted enzyme was found to contain an amino acid sequence highly homologous to the putative substrate-binding domain present within all lipases examined to date.     

Cloning, expression, and nucleotide sequence of a lipase gene from Pseudomonas fluorescens B52.

In this study, we report the cloning and expression of lipase gene from Pseudomonas fluorescens B52, a psychrotrophic spoilage bacterium isolated from refrigerated raw milk. Sequence analysis revealed one major open reading frame of 1,428 nucleotides that was predicted to encode a protein with a molecular weight of 50,241. The predicted enzyme was found to contain an amino acid sequence highly homologous to the putative substrate-binding domain present within all lipases examined to date.     

Influence of iron(III) and pyoverdine on extracellular proteinase and lipase production by Pseudomonas fluorescens B52

Factors associated with the production of extracellular lipase and proteinase by Pseudomonas fluorescens B52 during the late-log, early-stationary phase of grown were examined. Active lipase production by resting cell suspensions was observed when cells were harvested during the log phase (A₆₀₀ of 0.3–0.9) Resting suspensions of younger cells (A₆₀₀<0.1) synthesized lipase after a significant lag. Addition of cells of the proteinase-and lipasedeficient mutant P. fluorescens RM14 to B52 cells at low density resulted in stimulation of lipase and proteinase production. Similar results were found using cell-free culture fluid of RM14. Gel filtration on Biogel P2 revealed that the stimulatory factor co-chromatographed with the iron(III) siderophore, pyoverdine. Partially purified pyoverdine stimulated enzyme synthesis at a concentration of 6 M while having no effect on activity of preformed enzyme. Production of pyoverdine and extracellular enzymes was also stimulated by transferrin, a strong iron(III) binding protein. Growth of B52 in deferrated media was limited to 27% of that found with untreated media. Maximum pyoverdine, proteinase and lipase synthesis was obtained at a final iron(III) concentration of 5.75 M. Growth was maximal in 8.75 M iron(III) while synthesis of pyoverdine, proteinase and lipase was reduced to 3.6, 6.6 and 30% respectively in 23.75 M iron(III). Lipase activity in cell-free culture fluid was slightly inhibited by the addition of up to 400 M iron(III) while proteinase activity was unaffected. In dilute cell suspensions, lipase synthesis was more sensitive to iron(III) than was proteinase (50% inhibition at 1.6 M and a maximum of 40% inhibition at 5.0 M, respectively). In the case of lipase, added pyoverdine was able to partially protect enzyme production from the effects of iron(III). The results are consistent with a role for iron(III) in the regulation of extracellular lipase and proteinase synthesis by P. fluorescens.Contribution No. 677 from the Food Research Centre     

Developmental and gene expression analyses of a cotton naked seed mutant

Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis

A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR. When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI. Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins. The gene encoded a sequence of 1,177 amino acids compared to 1,155 amino acids encoded by all the other 16 cry1Ab genes reported so far. An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family. Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.     

Cloning and expression of gene, and activation of an organic solvent-stable lipase from Pseudomonas aeruginosa LST-03

Organic solvent-tolerant Pseudomonas aeruginosa LST-03 secretes an organic solvent-stable lipase, LST-03 lipase. The gene of the LST-03 lipase (Lip9) and the gene of the lipase-specific foldase (Lif9) were cloned and expressed in Escherichia coli. In the cloned 2.6 kbps DNA fragment, two open reading frames, Lip9 consisting of 933 nucleotides which encoded 311 amino acids and Lif9 consisting of 1,020 nucleotides which encoded 340 amino acids, were found. The overexpression of the lipase gene (lip9) was achieved when T7 promoter was used and the signal peptide of the lipase was deleted. The expressed amount of the lipase was greatly increased and overexpressed lipase formed inclusion body in E. coli cell. The collected inclusion body of the lipase from the cell was easily solubilized by urea and activated by using lipase-specific foldase of which 52 or 58 amino acids of N-terminal were deleted. Especially, the N-terminal methionine of the lipase of which the signal peptide was deleted was released in E. coli and the amino acid sequence was in agreement with that of the originally-produced lipase by P. aeruginosa LST-03. Furthermore, the overexpressed and solubilized lipase of which the signal peptide was deleted was more effectively activated by lipase-specific foldase.     

Molecular cloning and characterization of a novel WRKY gene from Brassica chinensis

A new WRKY gene was cloned from Brassica chinensis by rapid amplification of cDNA ends (RACE). The full-length cDNA of BcWRKY was 1175 bp long and contained a 924-bp open reading frame (ORF) encoding a putative W-box-binding protein of 308 amino acids. The predicted BcWRKY protein was found to have a potential bipartite nuclear localization sequence (NLS-PB) in its N-terminal region followed by a WRKY DNA-binding domain. Bioinformatic analysis revealed that BcWRKY resembled other WRKY domain-containing proteins from Arabidopsis thaliana (AtWRKY18), tobacco (WIZZ), parsley (PcWRKY4), and wild oat (ABF2). Expression of the BcWRKY gene could be induced by salicylic acid (SA) and influenced by Pseudomonas syringae pv. tomato strain DC3000 infection and wounding treatment. Our study implies that BcWRKY might have similar functions possessed by other WRKY genes, such as inducing the expression of some defense-related genes and increasing plants’ disease resistance ability. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 816–824. The text was submitted by the authors in English.     

Cloning, heterologous expression, and functional characterization of the nicotinate dehydrogenase gene from Pseudomonas putida KT2440

6-Hydroxynicotinate can be used for the production of drugs, pesticides and intermediate chemicals. Some Pseudomonas species were reported to be able to convert nicotinic acid to 6-hydroxynicotinate by nicotinate dehydrogenase. So far, previous reports on NaDH in Pseudomonas genus were confused and contradictory each other. Recently, Ashraf et al. reported an NaDH gene cloned from Eubacterium barkeri and suggested some deducted NaDH genes from other nine bacteria. But they did not demonstrate the activity of recombinant NaDH and did not mention NaDH gene in Pseudomonas. In this study we cloned the gene of NaDH, ndhSL, from Pseudomonas putida KT2440. NdhSL in P. putida KT2440 is composed of two subunits. The small subunit contains [2Fe2S] iron sulfur domain, while the large subunit contains domains of molybdenum cofactor and cytochrome c. Expression of recombinant ndhSL in P. entomophila L48, which lacks the ability to produce 6-hydroxynicotinate, enabled the resting cell and cell extract of engineering P. entomophila L48 to hydroxylate nicotinate. Gene knockout and recovery studies further confirmed the ndhSL function.     

棉花GhZIP4基因的克隆及表达分析

根据EST拼接的序列设计引物,利用RT-PCR和PCR方法,从陆地棉‘苏棉18’-cDNA和基因组DNA中分别克隆获得了GhZIP4基因片段.结果表明:(1)GhZIP4基因cDNA序列全长1 487 bp,包含1 269 bp ORF,编码422个氨基酸残基,其氨基酸序列具有典型的ZIP蛋白特征,预测具有8个跨膜结构域,第Ⅲ和第Ⅳ跨膜结构域间存在可变区,在可变区有2个富含His的结构域“HRHSHPHG”和“HSHGHGHD”.(2)氨基酸进化树分析显示,GhZIP4同拟南芥ZIP家族AtZIP4的相似性较高.(3)GhZIP4 DNA序列编码区全长1 778 bp,包含4个外显子和3个内含子,所有外显子/内含子交接点都遵从gt/ag剪接规则.(4)半定量分析显示,GhZIP4基因在茎中表达量最高,表明该基因有可能在某些金属离子地上部和根部的动态平衡分布过程中具有重要作用.     

Cloning, sequencing and heterologous expression of a new chitinase gene, chi92, from Streptomyces olivaceoviridis ATCC 11238

A new chitinase gene, chi92, encoding the largest known chitinase from Streptomyces olivaceoviridisATCC 11238 was sequenced by means of different PCR-methods. The cloned gene was expressed in E. coliand the recombinant protein could be detected by Western-blot analysis. The multiplicity of chitinolytic enzymes of this strain is discussed.     

The nucleotide sequence of a carboxymethylcellulase gene from Pseudomonas fluorescens subsp. cellulosa

Summary The complete nucleotide sequence of the gene coding for one of the carboxymethycellulases (CMCase), expressed by Pseudomonas fluorescens subsp. cellulosa, has been determined. The structural gene consists of an open reading frame, commencing with an ATG start codon, of 2886 base pairs followed by a TAA stop codon. The gene was shown to code for a signal peptide which closely resembles the signal peptides of other secreted proteins. Unlike most pseudomonas genes, the CMCase sequence does not have a high G+C (51%) content and there is no marked preference for codons ending in G or C. Upstream of the structural gene there are no sequences which bear a strong resemblance to consensus Escherichia coli promoters. A sequence is present, however, which exhibits homology to the consensus DNA sequence that binds the catabolic activator protein (CAP). Bal31 deletions of the structural gene revealed the extent by which the gene could be modified and still encode a functional CMCase. Subclones of the cellulase gene have been constructed in pUC18 and pUC19. One of the resultant plasmids, pJHS1 directs a 20-fold increase in CMCase synthesis, when compared to the original construct, pJHH2. Analysis of cells harbouring pJHS1 showed the cellulase polypeptide to have a molecular weight of 106000. This is in close agreement with the predicted size of the enzyme deduced from the nucleotide sequence data.Abbreviations CMCase carboxymethylcellulase - PAGE polyacrylamide gel electrophoresis - IPTG isopropyl--D-thiogalactoside - CAT chloramphenicol acetyl transferase     

Revised structures of the pyoverdins from Pseudomonas putida CFBP 2461 and from Pseudomonas fluorescens CFBP 2392

Several suggestions for structures of the siderophores (pyoverdins) from Pseudomonas spp. can be found in the literature which are based on a FAB mass spectrometric analysis only. Availability of two original strains of two Pseudomonas spp. allowed to re-investigate the structure of their pyoverdins. In both cases the amino acid sequence had to be corrected. In addition, d- and l-amino acids could be identified and located in the peptide chain. The knowledge of the correct structures is important in view of an ongoing study to establish relationships between the nature of the peptide chains of pyoverdins and their recognition by outer membrane proteins.     

Cloning, gene expression and characterization of a novel bacterial NAD-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Neisseria meningitidis strain Z2491

Alignment of the amino acid sequence of some archaeal, bacterial and eukaryotic non-phosphorylating glyceraldehydes-3-phosphate dehydrogenases (GAPNs) and aldehyde dehydrogenases (ALDHs) with the sequence of a putative GAPN present in the genome of the Gram-negative bacterium Neisseria meningitidis strain Z2491 demonstrated the conservation of residues involved in the catalytic activity. The predicted coding sequence of the N. meningitidis gapN gene was cloned in Escherichia coli XL1-blue under the expression of an inducible promoter. The IPTG-induced GAPN was purified ca. 48-fold from E. coli cells using a procedure that sequentially employed conventional ammonium sulfate fractionation as well as anion-exchange and affinity chromatography. The purified recombinant enzyme was thoroughly characterized. The protein is a homotetramer with a 50-kDa subunit, exhibiting absolute specificity for NAD and a broad spectrum of aldehyde substrates. Isoelectric focusing analysis with the purified fraction showed the presence of an acidic polypeptide with an isoelectric point of 6.3. The optimum pH of the purified enzyme was between 9 and 10. Studies on the effect of increasing temperatures on the enzyme activity revealed an optimal value ca. 64 °C. Molecular phylogenetic data suggest that N. meningitidis GAPN has a closer relationship with archaeal GAPNs and glyceraldehyde dehydrogenases than with the typical NADP-specific GAPNs from Gram-positive bacteria and photosynthetic eukaryotes.     

Cloning, sequencing and expression of a recA gene from Amycolatopsis mediterranei

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter.     

Cloning and expression of the polyhydroxyalkanote depolymerase gene from Pseudomonas putida, and characterization of the gene product

A polyhydroxyalkanote (PHA) depolymerase gene ( pha Z) was cloned by PCR from Pseudomonas putida and over-expressed in Escherichia coli as inclusion bodies. Nucleotide sequence analysis predicted an 852 bp open reading frame encoding a protein of 283 amino acids with a predicted molecular weight of 31283 Da. The deduced amino acid sequence had at least 80% homology to the PHA depolymerase from other Pseudomonas strains and consisted a conserved lipase box-like sequence (G-X-S(102)-X-G). The inclusion bodies were refolded and biochemically characterized. The depolymerase activity was optimal at 40 degrees C and pH 8.     

棉花磷脂酶C基因的克隆及参与油脂代谢的功能分析

以陆地棉胚珠和纤维为实验材料,利用RT-PCR技术克隆得到磷脂酶C(Phospholipase C,PLC)基因(GhPLC,GenBank登录号:KR154219),将棉花磷脂酶C基因转化拟南芥,分析其在油脂代谢过程中的重要作用。测序鉴定显示,GhPLC基因的全长开放读码框为1 524bp,编码包含508个氨基酸的蛋白质,理论分子量约为55kD。序列比对分析显示,GhPLC属于典型的碱性磷酸酶超家族的磷脂酶。利用pET32a-GhPLC原核表达获得分子量约为55kD左右的重组蛋白GhPLC;酶活力分析显示,重组GhPLC蛋白具有较高的将卵磷脂(PC)催化为二酰甘油(DAG)的酶活力。半定量RT-PCR结果表明,GhPLC基因参与棉花种子和纤维发育过程。构建植物过量表达载体35S∷GhPLC并转化哥伦比亚野生型拟南芥,转基因拟南芥中GhPLC基因的表达和PLC酶活力显著提高,且转基因拟南芥种子的油脂含量提高了5.3%。     

Cloning,expression and characterization of a beta-agarase gene from a marine bacterium,Pseudomonas sp. SK38

A gene (pagA) encoding -agarase from Pseudomonas sp. SK38 was cloned and expressed in Escherichia coli. The structural gene consists of 1011 bp encoding 337 amino acids with a predicted molecular weight of 37326 and has a signal peptide of 18 amino acids. The deduced amino acid sequence showed 57% and 58% homology to -agarase from Pseudoalteromonas atalntica and Aeromonas sp., respectively. The recombinant enzyme was purified and biochemically characterized. The enzyme had maximum activity at pH 9 and 30 °C. It was stable at pHs from 8 to 9 and below 37 °C.     

Degumming of vegetable oils by a novel phospholipase B from Pseudomonas fluorescens BIT-18

Pseudomonas fluorescens BIT-18 was isolated from soil near a vegetable oil factory and shown to produce a B-type phospholipase. The enzyme was partially purified by ammonium sulfate precipitation. Gas chromatography demonstrated that the enzyme preparation hydrolyzed both the 1- and 2-ester bonds of phosphatidylcholine. When degumming of soybean, rapeseed, and peanut oil was performed with this enzyme preparation, oils with phosphorous contents lower than 5 mg/kg were obtained after 5 h of enzyme treatment at 40 °C. The enzyme preparation did not show lipase activity, thus free fatty acids were only generated from the phospholipids. Therefore, this novel phospholipase B is potentially useful for the refining of high-quality oils with attractive yields.