Cloning and characterization of the gene encoding an esterase from Spirulina platensis

The gene encoding a 23 kDA serine esterase from the cyanobacterium Spirulina platensis has been identified, cloned, characterized and expressed in Escherichia coli. The primary structure of the esterase deduced from the DNA sequence displayed 32% sequence identity with the carboxylesterase (esterase II) encoded by estB of Pseudomonas fluorescens; the highest degree of homology is found in a stretch of 11 identical or highly conserved amino acid residues corresponding to the GXSXG consensus motif found in the catalytic site of many serine proteases, lipases and esterases.     

螺旋藻有效成分超临界萃取与分离研究

使用超临界CO2萃取技术对钝顶螺旋藻粉进行萃取与分离,结果表明:超临界CO2萃取的最优条件为压力20Mpa,夹带剂使用量100mL/100g,温度50℃,萃取时间2h;利用凯氏定氮法及气相色谱法分别对超临界萃取处理过的螺旋藻中蛋白质和多糖进行研究与分析,结果表明:螺旋藻粉中蛋白质含量为49.39%,螺旋藻多糖中的单糖含量占多糖粗品的9.25%,主要组成为鼠李糖、木糖、甘露糖、葡萄糖、半乳糖等。     

钝顶螺旋藻低中温适应型新品系的选育及RAPD分析

【目的】选育低中温适应型螺旋藻新品系,显著拓展螺旋藻工厂化培植的地理范围及时间、提高产量和降低成本。【方法】以用于工厂化培植的钝顶螺旋藻(Spirulina platensis) ZJU0116为出发品系,用组织匀浆和离心分离法制得其单细胞或原生质球,先以0.6%甲基磺酸乙酯(ethyl methanesulfonate, EMS)处理30 min,再用2.4 kGy的⁶⁰Co γ射线辐照,进行低温逆境筛选、5次冷/热(12 ℃/38 ℃)骤变交替处理、藻丝单体分离培养、温度适应面构建和蛋白质含量等检测及生产培植试验。【结果】获得了1株蛋白质含量与ZJU0116的相当、而温度适应面和平均日产量依次提高10.7%和10.9%的低中温适应型突变体,命名为ZJU0116(LMTA)。光学显微形态与随机扩增的多态性DNA (randomly amplified polymorphic DNA, RAPD)分析结果显示,与其亲本ZJU0116相比,ZJU0116(LMTA)藻丝的螺旋数和长度依次减少54.4%和42.1%,螺距增加28.8%;基因组DNA在随机引物S30的扩增产物中多了1条约470 bp的条带。【结论】ZJU0116(LMTA)在工厂化培植中温度适应性好、生产性状稳定,干藻粉产量可增加10%以上,可为当前螺旋藻产业深度融合“双碳”目标、迈向高质量发展新阶段提供必要支撑。     

A regulatory element within a gene of a ribosomal protein operon of Escherichia coli negatively controls expression by decreasing the translational efficiency

Summary The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kDa protein (21K) of unknown function, the tRNA(m¹G37)methyltransferase (TrmD), and r-protein L19, in this order. Previously we have shown that the steady-state amount of the two r-proteins exceeds that of the 21K and TrmD proteins 12- and 40-fold, respectively, and that this differential expression is solely explained by translational regulation. Here we have constructed translational gene fusions of the trmD operon and lacZ. The expression of a lacZ fusion containing the first 18 codons of the 21K protein gene is 15-fold higher than the expression of fusions containing 49 or 72 codons of the gene. This suggests that sequences between the 18th and the 49th codon may act as a negative element controlling the expression of the 21K protein gene. Evidence is presented which demonstrates that this regulation is achieved by reducing the efficiency of translation.     

Cloning and characterization of chloroplast ribosomal protein-encoding genes, rpl16 and rps3, of the marine macro-algae, Gracilaria tenuistipitata

In Gracilaria tenuistipitata, a highly differentiated multicellular member of the marine red algae, Rhodophyta, chloroplast (cp) DNA can be separated as a satellite band from the nuclear DNA in a CsCl gradient. Using a heterologous probe from Chlamydomonas, the ribosomal protein-encoding gene, rpl16, was located on a 4.5-kb EcoRI fragment of cp DNA. The fragment was cloned and a 1365-bp region around rpl16 was sequenced. The gene order around rpl16, 5′ rpl22-rps3-rpl16, is identical to that detected in the chloroplast DNA of liverwort, tobacco and maize. Both the nucleotide sequence and the amino-acid sequence of rpl16 are more conserved than that of rps3. The rpl16 gene contains no intron, a feature which shows more similarity to the unicellular green algae, Chlamydomonas, than the other land plants. Sequences that may form a stable stem-loop structure were detected within the coding sequence of rpl16.     

高沥水性钝顶螺旋藻新品系的选育及超微结构与RAPD分析

[目的]选育高沥水性的螺旋藻新品系,显著降低藻粉生产中干燥的能耗。[方法]以用于工厂化培植的钝顶螺旋藻ZJU0115为出发品系,用组织匀浆-离心沉降法制得其原生质球,并先以0.6%EMS处理30 min再用2.4 kGy的~(60)Coγ射线辐照,经含0.02%黄原胶(xanthan gum)的Zarrouk's培养液筛选、藻丝单体分离培养、藻泥持水率和胞外多糖(EPS)等检测及生产培植试验。[结果]获得了一株产量、蛋白质和多糖含量与ZJU0115相当,而藻泥持水率和EPS含量分别下降5.9%和29.7%的突变体,命名为ZJU0115(HD)。超微结构与随机扩增多态性DNA标记(random amplified polymorphic DNA,RAPD)分析结果显示,与其亲本ZJU0115相比,ZJU0115(HD)藻丝表面更光滑,可能为酸性多糖的乳白状粘附物也更少;ZJU0115(HD)细胞内的多磷酸盐颗粒显著变小且呈弥散状;基因组DNA在随机引物S90的扩增产物中显示出多态性差异。[结论]ZJU0115(HD)在工厂化培植中生产性状好、高沥水性能稳定,藻泥的干燥能耗降低了近50%,它的育成与应用,有助于推进当前螺旋藻产业迈向高效、节能、绿色、环保发展的新阶段。     

Distribution of sequences homologous to the impCAB operon of TP110 among bacterial plasmids of different incompatibility groups

Summary Mutagenic DNA repair is a function of many naturally occurring plasmids belonging to several different incompatibility groups. A DNA probe corresponding to the impCAB operon of the IncIl plasmid TP110, which encodes such functions, was used to investigate the distribution of homologous sequences in both related and unrelated plasmids. Southern blotting was used to demonstrate considerable sequence conservation amongst a number of plasmid types, with imp-related sequences being found on plasmids belonging to the I1, I1/B, B and FIV incompatibility groups. However, no homology was detected amongst plasmids of the N and L/M incompatibility groups, many of which carry functionally similar gene clusters. It appears that sequences determining mutagenic repair functions have been largely conserved within any one incompatibility group, but that significant divergent evolution has occurred between groups.     

The nifEN genes participating in FeMo cofactor biosynthesis and genes encoding dinitrogenase are part of the same operon in Bradyrhizobium species

Summary The nucleotide sequences of genes homologous to the Klebsiella pneumoniae nifEN genes have been determined in Bradyrhizobium japonicum 110. The coding regions for the nifE and nifN consist, respectively, of 1641 and 1407 nucleotides. The nifD gene (coding for the -subunit of dinitrogenase) and nifE are linked, and separated by 95 nucleotides. In the region of 12 nucleotides that separates nifE from nifN the stop codon for nifE overlaps the putative ribosome binding site for nifN. In contrast to Klebsiella and Azotobacter vinelandii, the B. japonicum nifEN genes are linked to the nifDK genes in the same operon. Comparison of dinitrogenase polypeptides (nifDK products) and the polypeptides of the nifE and nifN genes reveals considerable homology between nifD and nifE, and between nifK and nifN. Several protein domains, containing highly conserved cysteine residues, are conserved among the gene products of nifD, nifK, nifE and nifN. This result allows us to propose a probable evolutionary pathway for the common origin of these genes.     

The meta cleavage operon of TOL degradative plasmid pWWO comprises 13 genes

Summary The meta-cleavage operon of TOL plasmid pWWO of Pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to Krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol. The genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kilodaltons) are The xyIXYZ genes encode three subunits of toluate 1,2-dioxygenase. The xylL, xyIE, xyIG, xylF, xylJ, xylK, xylI and xylH genes encode 1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylate dehydrogenase, catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase and 4-oxalocrotonate tautomerase, respectively. The functions of xyIT and xylQ are not known at present. The comparison of the coding capacity and the sizes of the products of the meta-cleavage operon genes indicated that most of the DNA between xyIX and xyIH consists of coding sequences.     

Amino acid sequence of the ribosomal protein HS23 from the halophilicHaloarcula marismortui and homology studies to other ribosomal proteins

The ribosomal protein HS23 from the 30S subunit of the extreme halophilicHaloarcula marismortui, belonging to the group of archaea, was isolated either by RP-HLPLC or two-dimensional polyacrylamide gel electrophoresis. The complete amino acid sequence was determined by automated N-terminal microsequencing. The protein consists of 123 residues with a corresponding molecular mass of 12,552 Da as determined by electrospray mass spectroscopy; the pI is 11.04. Homology studies reveal similarities to the eukaryotic ribosomal protein S8 fromHomo sapiens, Rattus norvegicus, Leishmania major, andSaccharomyces cerevisiae.Abbreviations H. marismortui Haloarcula marismortui - PVDF polyvinylidene difluoride - PTH phenylthiohydantoin - RP-HPLC reversed-phase high-performance liquid chromatography - TFA trifluoro acetic acid - TP30 total protein mixture from the 30S ribosomal subunit ofH. marismortui     

Characterization of three genes in the dam-containing operon of Escherichia coli

The dam-containing operon in Escherichia coli is located at 74 min on the chromosomal map and contains the genes aroK, aroB, a gene called urf74.3, dam and trpS. We have determined the nucleotide sequence between the dam and trpS genes and show that it encodes two proteins with molecular weights of 24 and 27 kDa. Furthermore, we characterize the three genes urf74.3, 24kDa, 27kDa and the proteins they encode. The predicted amino acid sequences of the 24 and 27 kDa proteins are similar to those of the CbbE and CbbZ proteins, respectively, of the Alcaligenes eutrophus cbb operon, which encodes enzymes involved in the Calvin cycle. In separate experiments, we have shown that the 24 kDa protein has d-ribulose-5-phosphate epimerase activity (similar to CbbE), and we call the gene rpe. Similarly, the 27 kDa protein has 2-phosphoglycolate phosphatase activity (similar to CbbZ), and we name the gene gph. The Urf74.3 protein, with a predicted molecular weight of 46 kDa, migrated as a 70 kDa product under denaturing conditions. Overexpression of Urf74.3 induced cell filamentation, indicating that Urf74.3 directly or indirectly interferes with cell division. We present evidence for translational coupling between aroB and urf74.3 and also between rpe and gph. Proteins encoded in the dam superoperon appear to be largely unrelated: Dam, and perhaps Urf74.3, are involved in cell cycle regulation, AroK, AroB, and TrpS function in aromatic amino acid biosynthesis, whereas Rpe and Gph are involved in carbohydrate metabolism.     

苏云金芽胞杆菌G03 spoIVF操纵子敲除对芽胞和晶体形成的影响

spoIVF是一个普遍存在于芽胞杆菌中的操纵子。在枯草芽胞杆菌中,它编码的两个蛋白是芽胞形成所必需的。采用基因重组技术敲除了苏云金芽胞杆菌G03菌株中的spoIVF操纵子,构建了spoIVF缺失株G03(spoIVF-)。研究表明:该突变株丧失了形成芽胞和晶体的能力。lacZ基因与cry1Aa基因的启动子融合表达分析发现:突变株中的cry1Aa基因的活性严重降低。利用载体pSTK携带spoIVF操纵子在突变株中的表达,使突变株部分恢复了产胞和形成杀虫晶体蛋白的能力。这说明spoIVF操纵子是所必需的,同时该操纵子还影响σ^E因子控制的cry1Aa基因表达。