The Vitreoscilla hemoglobin gene: Molecular cloning, nucleotide sequence and genetic expression in Escherichia coli

Summary Vitreoscilla hemoglobin is involved in oxygen metabolism of this bacterium, possibly in an unusual role for a microbe. We have isolated the Vitreoscilla hemoglobin structural gene from a pUC19 genomic library using mixed oligodeoxy-nucleotide probes based on the reported amino acid sequence of the protein. The gene is expressed in Escherichia coli from its natural promoter as a major cellular protein. The nucleotide sequence, which is in complete agrecment with the known amino acid sequence of the protein, suggests the existence of promoter and ribosome binding sites with a high degree of homology to consensus E. coli upstream sequences. In the case of at least some amino acids, a codon usage bias can be detected which is different from the biased codon usage pattern in E. coli. The down-stream sequence exhibits homology with the 3 end sequences of several plant leghemoglobin genes. E. coli cells expressing the gene contain greater than fivefold more heme than controls.     

十字花科黑腐病菌一个新的III型效应物的鉴定

【目的】在全基因组范围内鉴定十字花科黑腐病菌Xcc 8004中新的Ⅲ型效应物(type Ⅲ secreted effector,T3SE)基因。【方法】通过构建与AvrBs1_59-445整合的Tn5转座子系统,进行文库筛选。在avrBs1缺失突变体背景下生成突变文库,在辣椒ECW-10R上进一步筛选。【结果】大规模HR测定筛选出7个具有明显过敏反应(hypersensitive response,HR)的克隆。通过质粒拯救和测序发现除了插入已知T3SE基因的3个突变体外,还鉴定了一个位于XC_0438和XC_0439之间且在Xcc 8004中未注释的新基因。结合生物信息学,我们将其命名为一个新的基因XC_0438a。易位实验证实XC_0438a信号区可引导报告蛋白AvrBs1的分泌和易位,并诱导辣椒ECW-10R产生HR反应。β-葡萄糖醛酸酶(β-glucuronidase,GUS)活性测定,XC_0438a在基本培养基中诱导表达,并由关键调控蛋白HrpG和HrpX激活。然而,在我们的实验条件下,XC_0438a对Xcc的致病力没有显著的贡献。【结论】我们鉴定了一种新的依赖于Ⅲ型分泌系统的效应基因XC_0438a。     

野油菜黄单胞菌中3-羟基脂酰ACP脱水酶FabZ的生理功能

【背景】野油菜黄单胞菌(Xanthomonas campestris pv. campestris, Xcc)引起十字花科植物黑腐病,在全球范围内造成经济损失,亟须深入研究其致病机理,开发新的黑腐病防控措施。细菌脂肪酸合成系统不仅为细胞膜合成提供原料,其中间代谢产物还是许多生物活性分子合成的底物,具有重要的生理功能,也是抗菌药物筛选的重要靶标。【目的】研究XccfabZ对扩散信号分子(diffusible signal factor, DSF)类信号产量、致病力、胞外酶、胞外多糖和运动性等方面的影响。【方法】利用报告菌株检测法分析了不同替换突变株的DSF类群体感应信号产量。利用同源重组原理,在DSF类信号高产菌株中获得替换突变株,利用高效液相色谱(highperformanceliquid chromatography, HPLC)法测定DSF类信号产量。利用剪叶法检测替换突变株对寄主植物甘蓝的致病力,并分析了不同菌株的胞外多糖、胞外酶和运动性差异。【结果】报告菌株检测法和HPLC法都证明大肠杆菌fabZ替换突变株(XccΔfabZ/pSRK-EcfabZ)中DSF类信号产量显著下降。...     

Use of oligonucleotide probes to identify members of two-component regulatory systems in Xanthomonas campestris pathovar campestris

Summary Two-component regulatory systems comprising a sensor and a regulator protein, both with highly conserved amino acid domains, and commonly genetically linked, have been described in a range of bacterial species and are involved in sensing environmental stimuli. We used two oligonucleotide probes matching the postulated coding regions for domains of sensor and regulator proteins respectively in Xanthomonas campestris pathovar campestris (Xcc) to identify possible two-component regulatory systems in Xcc. Two different fragments of Xcc DNA with homology to both of these probes were cloned. The DNA sequence of part of one of these fragments encompassed a potential open reading frame (ORF), the predicted amino acid sequence of which had extensive homology with regulator proteins of two-component regulatory systems. Analysis of the predicted amino acid sequence for the 3 end of an adjacent ORF revealed a very high level of homology with the C-terminal end of sensor proteins. Strains of Xcc with Tn5-induced mutations in the regulator gene were affected in extracellular polysaccharide production, and also in resistance to salt and chloramphenicol. No effects of mutation in the second clone were observed.     

Molecular cloning of a maltose transport gene from Bacillus stearothermophilus and its expression in Escherichia coli K-12

Genes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli. The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E. coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region). Transport studies in E. coli and B. stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B. stearothermophilus ATCC7953. Nucleotide sequence analysis of a 3.6 kb fragment of pAM 1750 revealed three open reading frames (ORFs). One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments. MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MaIG protein, a member of a binding protein-dependent transport system in E. coli. The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E. coli mutants. Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E. coli, the protein was very poorly expressed and could not be identified.     

毒力基因yqeH功能分析及对禽致病性大肠杆菌致病性的影响

【背景】禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)可引起禽类急性或亚急性感染,在近年新发现的大肠杆菌Ⅲ型分泌系统2 (Escherichia coli type III secretion system 2,ETT2)中,毒力基因yqeH对其致病性的影响尚不明确。【目的】探究yqeH在APEC致病过程中的作用,为后期深入研究ETT2致病机制奠定基础。【方法】利用Red同源重组技术构建yqeH缺失株ΔyqeH及其回复株CΔyqeH,通过运动性、生物被膜形成能力、抗逆性、抗血清杀菌能力等试验分析yqeH对APEC生物学功能的影响,并通过细胞黏附、侵袭试验、致病力测定及荧光定量PCR检测细胞炎性因子转录水平,探究yqeH对APEC感染宿主的影响。【结果】构建了缺失株ΔyqeH和回复株CΔyqeH;生物学特性试验结果表明,与野生株APEC81相比,缺失株ΔyqeH生物被膜形成能力、运动能力降低,对酸、碱、渗透压、氧化休克的耐受力降低,抗血清杀菌能力及致病力显著降低;与野生株APEC81相比,缺失株ΔyqeH对鸡气管黏膜上皮细胞的黏附及侵袭能...     

The cloning of the rorB gene of Escherichia coli

Summary A segment of the Escherichia coli genome which complements the ionising radiation sensitivity of the rorB mutation was cloned into pBR322. This DNA segment also complements the mitomycin C sensitivity of the rorB mutation. The gene was subcloned until defined in a fragment of 1.05 kb. Only one gene product, a protein of approximately 16.5 kDa, was found on maxicell analysis of the various subclones. Iso-electric focusing of this gene product suggests it may function in a complex.     

Identification of a pathogenicity locus in Xanthomonas campestris pv. vesicatoria

Summary Mutants of a tomato strain ofXanthomonas campestris pv.vesicatoria (XCV), causal agent of bacterial spot of tomato and pepper, were produced using the transposon Tn5 carried in the suicide plasmid pGS9. One prototrophic mutant, M461, was isolated which caused no visible reaction on tomato or pepper, but maintained the wild-type ability to induce a hypersensitive reaction (HR) on tobacco. This mutant showed similar growth characteristics to the wild-type in culture, but growth in planta was reduced. A genomic library of wild-type XCV was constructed in the broad host range cosmid vector pLAFR3. Clone p6AD4 restored pathogenicity to M461 on tomato and the ability to induce a HR on pepper. This clone contained ca. 22 kb of XCV DNA. The insertion in M461 was in a site corresponding to a 1.1 kbEcoRI fragment of p6AD4.     

十字花科黑腐病菌III型效应物XopXccR1植物亚细胞定位

[目的] 植物病原细菌通过III型分泌系统（type III secretion system,T3SS）将III型效应物（type III secreted effectors,T3SEs）分泌转运到宿主细胞的不同位点上,进而行使不同的致病功能。本研究旨在确定Xcc 8004 III型效应物中分子量最大的蛋白XopXccR1在植物中的亚细胞定位。[方法] 利用生物信息学方法分析XopXccR1的跨膜信息。通过同源重组方法将XopXccR1全长、N端（1–1220 aa）和C端（1221–2030 aa）分别克隆到植物表达载体pCAMBIA-2300-35S：：EGFP上,利用根癌农杆菌介导的瞬时表达浸染本生烟,通过激光共聚焦显微镜观察亚细胞定位结果。[结果] XopXccR1全长和N端定位在本生烟细胞膜上,而C端定位在细胞质中。[结论] XopXccR1的N端与C端可能分别存在定位信号,N端信号主导全长蛋白的最终定位。     

草菇过氧化氢酶基因(VvCAT1)异源表达及耐温度胁迫功能分析

[背景] 过氧化氢酶（catalase,CAT）参与真菌的生长发育,逆境胁迫时保护真菌免受氧化损伤。[目的] 实现草菇过氧化氢酶基因（VvCAT1）的异源表达,分析VvCAT1耐温度胁迫的功能。[方法] 克隆VvCAT1,构建过表达载体pBAR GPE1/VvCAT1,转化到大肠杆菌（Escherichia coli）菌株Stbl3中,异源表达草菇过氧化氢酶。测定温度胁迫后重组菌（pBAR GPE1/VvCAT1/Stbl3）与对照菌（pBAR GPE1/Stbl3）的过氧化氢酶活性和生长情况,验证VvCAT1的功能。[结果] 重组菌的CAT酶活性显著提高,生长情况显著优于对照菌。[结论] VvCAT1的导入及表达显著提高了大肠杆菌Stbl3的耐温度胁迫功能。     

双组分系统rcsC基因影响禽致病性大肠杆菌的致病性及相关生物学特性

【目的】双组分系统Rcs感受外界环境变化,并调控细菌的适应性及生存等。本文探讨Rcs双组分系统传感器激酶RcsC对禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)相关生物学特性及致病性的影响。【方法】采用Red同源重组的方法构建rcsC基因缺失株,并利用互补质粒构建互补株,然后比较野生株、基因缺失株与互补株的生长特性、运动性、生物被膜、凝集沉淀能力、致病力及毒力基因转录水平的差异。【结果】rcsC基因缺失不影响APEC的生长速度,然而,缺失RcsC导致APEC的运动能力升高、生物被膜形成能力降低和凝集能力增强。凝集试验结果显示rcsC基因有助于APEC的凝集沉降。细胞黏附入侵结果表明,rcsC在APEC侵袭DF-1细胞过程中发挥作用,而对黏附能力无影响。动物感染试验结果表明rcsC基因缺失能显著降低APEC的毒力。荧光定量PCR检测结果表明,rcsC基因缺失株中ompA、aatA、fyuA和luxS基因的转录水平均显著降低,而fimC和tsh基因的转录水平显著升高。【结论】RcsC参与调控APEC的运动性、生物被膜形成、凝集沉降和致病力。     

A novel method for the cloning of chromosomal mutations in a single step: Isolation of two mutant alleles of envZ, an osmoregulatory gene from Escherichia coli

Summary We have developed a simple, rapid and powerful method for the cloning of chromosomal mutations from total cellular DNA in a single step using a plasmid carrying the clined wild-type locus of interest and a convenient selectable marker such as antibiotic resistance. This method relies upon the ability of the cloned wild-type gene to form a heteroduplex with the mutant chromosomal locus. The plasmid from primary transformants can be screened rapidly by size; more than 50% of plasmids of the correct size contained the mutant locus. When this method was used to clone two chromosomal mutations in the envZ gene of Escherichia coli, a locus which encodes a membrane-bound sensory protein involved in the osmoregulation of outer membrane porin biosynthesis, more than 50% of the retransformants from the plasmids selected by size were found to exhibit the mutant phenotype. Preliminary characterization of these mutant alleles is discussed. This novel and powerful method should be generally applicable in any system where the cloned locus is available.This work was presented at the 86th Annual Meeting of the American Society for Microbiology, March 1986, Washingnton, D.C.     

Expression of the recA gene of Escherichia coli in several species of gram-negative bacteria

Summary A broad host range plasmid containing an operon fusion between the recA and lacZ genes of Escherichia coli was introduced into various aerobic and facultative gram-negative bacteria — 30 species belonging to 20 different genera — to study the expression of the recA gene after DNA damage. These included species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Vibrionaceae, Neisseriaceae, Rhodospirillaceae and Azotobacteraceae. Results obtained show that all bacteria tested, except Xanthomonas campestris and those of the genus Rhodobacter, are able to repress and induce the recA gene of E. coli in the absence and in the presence of DNA damage, respectively. All these data indicate that the SOS system is present in bacterial species of several families and that the LexA-binding site must be very conserved in them.     

米曲霉（Aspergillus oryzae）果胶酸内切水解酶（PGA）在原核系统中重组表达

果胶酶具有广阔的商业用途,在食品工业上主要用于果汁和酒类的澄清、提高植物油的提取率、提高水果的硬度和植物纤维脱胶。米曲霉（Aspergillus oryzae）一直用于传统发酵食品的生产,自然条件下其果胶酶的产量较低。文献报道的果胶酶的重组表达成功的例子较少,且活性较低。通过RT-PCR 的方法,获得不含信号肽的果胶酸内切水解酶A(polygalacturonase A, PGA) 的cDNA,PGA cDNA 连入pET-28a (+)载体, 构建 pET-28a (+)-pga 质粒。pET-28a (+)-pga 转化Turner (DE3) placⅠ细胞,得到转化子pET-28a (+)-pga-Turner (DE3) placⅠ,首次实现了米曲霉PGA在大肠杆菌系统中过表达,进一步对PGA在大肠杆菌系统中表达的条件进行了研究。在37℃、220 r/min条件培养pET-28a (+)-pga-Turner (DE3) placⅠ细胞,OD₆₀₀至 0.8左右时,用500μmol/L isopropyl β-D-thiogalactogalactopyranoside (IPTG)进行诱导表达,在15℃和170r/min条件下继续培养24 h,表达效果最好,相对于每毫升培养基而言,产酶可达到70u/mL,是米曲霉自然条件产酶量的87.5倍,远优于文献报道的重组表达的PGA酶活     

米曲霉（Aspergillus oryzae）果胶酸内切水解酶（PGA）在原核系统中重组表达

果胶酶具有广阔的商业用途,在食品工业上主要用于果汁和酒类的澄清、提高植物油的提取率、提高水果的硬度和植物纤维脱胶。米曲霉（Aspergillus oryzae）一直用于传统发酵食品的生产,自然条件下其果胶酶的产量较低。文献报道的果胶酶的重组表达成功的例子较少,且活性较低。通过RT-PCR 的方法,获得不含信号肽的果胶酸内切水解酶A(polygalacturonase A, PGA) 的cDNA,PGA cDNA 连入pET-28a (+)载体, 构建 pET-28a (+)-pga 质粒。pET-28a (+)-pga 转化Turner (DE3) placⅠ细胞,得到转化子pET-28a (+)-pga-Turner (DE3) placⅠ,首次实现了米曲霉PGA在大肠杆菌系统中过表达,进一步对PGA在大肠杆菌系统中表达的条件进行了研究。在37℃、220 r/min条件培养pET-28a (+)-pga-Turner (DE3) placⅠ细胞,OD₆₀₀至 0.8左右时,用500μmol/L isopropyl β-D-thiogalactogalactopyranoside (IPTG)进行诱导表达,在15℃和170r/min条件下继续培养24 h,表达效果最好,相对于每毫升培养基而言,产酶可达到70u/mL,是米曲霉自然条件产酶量的87.5倍,远优于文献报道的重组表达的PGA酶活     

pUB307 mobilizes resistance plasmids from Escherichia coli into Neisseria gonorrhoeae

Summary The plasmid pUB307, a derivative of RP1, is a conjugative, broad-host-range plasmid. We have shown that this element mobilizes gonococcal resistance plasmids from Escherichia coli to Neisseria gonorrhoeae, thus providing evidence that extrachromosomal elements can efficiently enter gonococci by conjugation. Furthermore, pUB307 can also be used as a helper element to mobilize the cloning vector pLES2 into N. gonorrhoeae. This finding significantly increases the usefulness of pLES2 as a shuttle vector between E. coli and gonococcus.     

Transposable elements ofXanthomonas campestris pv.citri originating from indigenous plasmids

Summary After introduction of the broad host range plasmid RP4 inXanthomonas campestris pv.citri strain XAS4501 twoXanthomonas transposable elements, ISXC4 and ISXC5, were isolated. These elements were found to be capable of transposition inEscherichia coli. Restriction analysis, DNA hybridization and heteroduplex experiments revealed that ISXC4 and ISXC5 are about 5.55 and 6.95 kb in size, respectively, possess inverted repeats about 50±18 bp in length and share DNA homology in their left (5.0 kb) and right (0.6 kb) ends. ISXC4 and ISXC5 were found to originate from plasmids pXW45N and pXW45J, which are indigenous replicons inX. campestris pv.citri strain XW45.