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.     

The Product of the cysK Gene of Bacillus stearothermophilus V Mediates Potassium Tellurite Resistance in Escherichia coli

The nucleotide sequence of a 4,539 bp fragment of Bacillus stearothermophilus V mediating tellurite resistance in Escherichia coli was determined. Four ORFs of more than 150 amino acids encoding polypeptides of 244, 258, 308, and 421 residues were found in the restriction fragment. E. coli cells harboring a recombinant plasmid containing the Te^r determinant express, when challenged with tellurite, a 32 kDa protein with an amino terminal sequence identical to the ten first residues of the 308 ORF. This ORF shows great similarity with the cysteine synthase gene (cysK) of a number of organisms. Recombinant clones carrying the active cysK gene have minimal inhibitory concentrations to K₂TeO₃ that were tenfold higher than those determined for the host strain or that of clones carrying ORFs 244, 258, and 421. Introduction of the B. stearothermophilus V cysK gene into a cysK strain of Salmonella typhimurium LT2 resulted in complementation of the mutation as well as transfer of tellurite resistance. Received: 28 March 2001 / Accepted: 17 April 2001     

Identification, cloning and sequence of the Streptococcus faecium infB (translational initiation factor IF2) gene

Summary The structural gene for translational initiation factor IF2 (infB) from Streptococcus faecium was identified by cross-hybridization with DNA probes derived from the corresponding gene of Bacillus stearothermophilus. The entire infB gene (ca. 2.8 kb) was cloned and sequenced. The amino acid sequence deduced from the nucleotide sequence shows that S. faecium initiation factor IF2 (785 amino acids, M_r 86,415) displays extensive homology (ca. 69% and 53%) with the region comprising three-quarters of the molecule from the carboxy-terminus of B. stearothermophilus and Escherichia coli IF2, respectively. The region comprising one-quarter of the molecule from the amino-terminus, on the other hand, does not display any significant homology.     

Molecular cloning and sequence of theBacillus stearothermophilus translational initiation factor IF2 gene

Summary The structural gene for theBacillus stearothermophilus initiation factor IF2 was localized to a 6 kbHindIII restriction fragment by cross-hybridization with theSstI-SmaI fragment of theEscherichia coli infB gene. This fragment corresponds to the central region of the molecule containing the GTP-binding domain which is homologous inE. coli IF2, EF-Tu, EF-G and the humanras1 oncogene protein. After cloning into pACYC177, theHindIII fragment was further analysed by restriction mapping and cross-hybridization. A smaller (2.2 kb)SphI-HindIII fragment, which showed cross-hybridization, was subcloned into M13 phage and sequenced by the dideoxy chain-terminating method. This fragment was found to contain the entire IF2 gene except for the region coding for the N-terminus. This remaining region, coding for 45 amino acids, was located by homologous hybridization on an overlappingClaI-SstI fragment which was also subcloned and sequenced. Overall, theB. stearothermophilus IF2 gene codes for a protein of 742 amino acids (M_r=82,043) whose primary sequence displays extensive homology with the C-terminal two-thirds (but little or no homology with the N-terminal one-third) of the correspondingE. coli IF2 molecule. When cloned into an expression vector under the control of the λP_L promoter, theB. stearothermophilus IF2 gene, reconstituted by ligation of the two separately cloned pieces, could be expressed at high levels inE. coli cells.     

滇水金凤花距发育相关基因ABP的克隆及表达分析

为探究滇水金凤(Impatiens uliginosa)ABP基因的结构和表达特征,该研究以滇水金凤为材料,采用RT-PCR 技术对滇水金凤ABP基因进行克隆,运用DNAMAN和MEGA对其所编码的蛋白序列进行同源性分析和系统进化分析,并利用qRT-PCR分析ABP基因的时空表达模式。结果表明:(1)滇水金凤ABP基因的cDNA 全长为627 bp,编码208 aa,命名为IuABP基因,其蛋白具有Cupin超家族蛋白的典型结构。(2)同源性分析表明滇水金凤ABP基因的氨基酸序列与喜马拉雅凤仙花(I. glandulifera)、月季(Rose chinensis)、木薯(Manihot esculenta)等物种的同源性均达71%; 系统进化分析表明IuABP与喜马拉雅凤仙花(Impatiens glandulifera)聚为一支,亲缘关系最近。(3)qRT-PCR分析表明IuABP基因在滇水金凤花距发育的3个时期及2个部位均有表达。随着花距的发育,IuABP基因在滇水金凤花距檐部的表达量呈先下降后上升的趋势,在盛花期时达最高,而在花距距部的表达量逐渐下降。以上结果为进一步研究滇水金凤ABP基因在花距发育中的功能及其表达调控机制提供了一定的理论参考。     

The complete primary structure of ribosomal protein L1 fromThermus thermophilus

The primary structure of the 23S rRNA binding ribosomal protein L1 from the 50S ribosomal subunit ofThermus thermophilus ribosomes has been elucidated by direct protein sequencing of selected peptides prepared by enzymatic and chemical cleavage of the intact purified protein. The polypeptide chain contains 228 amino acids and has a calculated molecular mass of 24,694 D. A comparison with the primary structures of the corresponding proteins fromEscherichia coli andBacillus stearothermophilus reveals a sequence homology of 49% and 58%, respectively. With respect to both proteins, L1 fromT. thermophilus contains particularly less Ala, Lys, Gln, and Val, whereas its content of Glu, Gly, His, Ile, and Arg is higher. In addition, two fragments obtained by limited proteolysis of the intact, unmodified protein were characterized.     

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene

Summary The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely –35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.     

地衣芽孢杆菌E7氨肽酶的异源表达及其在高效蛋白水解中的应用

【目的】将地衣芽孢杆菌(Bacilluslicheniformis)E7氨肽酶基因pepN克隆到大肠杆菌(Escherichia coli) BL21中,实现氨肽酶Ec PepN的异源表达,研究重组酶的酶学性质及其与碱性蛋白酶协同作用,高效水解大豆蛋白和酪蛋白,产生小分子活性肽和游离氨基酸。【方法】以地衣芽孢杆菌E7基因组DNA为模板,将氨肽酶基因pepN克隆到载体pET28a中,构建重组表达载体pET28-pepN,转化到大肠杆菌BL21感受态细胞中,经DNA测序验证,获得重组菌E. coli BL21/pET28-pepN。利用镍离子亲和层析柱对重组酶进行分离纯化,研究纯酶的pH和温度稳定性、半衰期和NaCl的耐受性等酶学性质。以商品化氨肽酶与碱性蛋白酶协同作用为对照,重组酶Ec PepN与碱性蛋白酶协同水解大豆蛋白和酪蛋白,测定水解产物中小分子活性肽和游离氨基酸的组成。【结果】Ec PepN在大肠杆菌BL21中可溶性表达,SDS-PAGE分析表明纯化的重组酶在52kDa左右显示单一条带。在7种测定底物中,Ec PepN的最适底物为Ala-pNA。在最适条件(pH 9.0和50°C...     

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis

Summary The ptsG gene of Bacillus subtilis encodes Enzyme II^G1c of the phosphoenolpyruvate: glucose phosphotransferase system. The 3 end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes III^Glc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5 end. These results, combined with the sequence of the 3 end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes II^Glc and II^Nag of E. coli but which are present in the II^G1c-like protein encoded by the E. coli malX gene.     

Cysteine metabolism-related genes and bacterial resistance to potassium tellurite

Tellurite exerts a deleterious effect on a number of small molecules containing sulfur moieties that have a recognized role in cellular oxidative stress. Because cysteine is involved in the biosynthesis of glutathione and other sulfur-containing compounds, we investigated the expression of Geobacillus stearothermophilus V cysteine-related genes cobA, cysK, and iscS and Escherichia coli cysteine regulon genes under conditions that included the addition of K₂TeO₃ to the culture medium. Results showed that cell tolerance to tellurite correlates with the expression level of the cysteine metabolic genes and that these genes are up-regulated when tellurite is present in the growth medium.     

Cloning, sequencing, and expression of a β-1,4-endoxylanase gene from Indonesian Bacillus licheniformis strain I5 in Escherichia coli

The gene encoding an endo-β-1,4-xylanase from an Indonesian indigenous Bacillus licheniformis strain I5 was amplified using PCR, cloned, and expressed in Escherichia coli. The nucleotide sequence of a 642 bp DNA fragment was determined, revealing one open reading frame that encoded a xylanase. Based on the nucleotide sequence, calculated molecular mass of the enzyme was 23 kDa. This xylanase has a predicted typical putative signal peptide; however, in E. coli, the active protein was located mainly in intracellular form. Neither culture supernatant of recombinant E. coli nor periplasmic fraction has significantly detectable xylanase activity. The deduced amino acid of the gene has 91% identity with that of Bacillus subtilis endoxylanase. Optimal activity of the recombinant enzyme was at pH 7 and 50°C     

<Emphasis Type="Italic">Marinococcus halophilus</Emphasis> DSM 20408<Superscript>T </Superscript>encodes two transporters for compatible solutes belonging to the betaine-carnitine-choline transporter family: identification and characterization of ectoine transporter EctM and glycine betaine transporter BetM

In response to osmotic stress, the halophilic, Gram-positive bacterium Marinococcus halophilus accumulates compatible solutes either by de novo synthesis or by uptake from the medium. To characterize transport systems responsible for the uptake of compatible solutes, a plasmid-encoded gene bank of M. halophilus was transferred into the transport-deficient strain Escherichia coli MKH13, and two genes were cloned by functional complementation required for ectoine and glycine betaine transport. The ectoine transporter is encoded by an open reading frame of 1,578 bp named ectM. The gene ectM encodes a putative hydrophobic, 525-residue protein, which shares significant identity to betaine-carnetine-choline transporters (BCCTs). The transporter responsible for the uptake of glycine betaine in M. halophilus is encoded by an open reading frame of 1,482 bp called betM. The potential, hydrophobic BetM protein consists of 493 amino acid residues and belongs, like EctM, to the BCCT family. The affinity of whole cells of E. coli MKH13 for ectoine (K_s=1.6 M) and betaine (K_s=21.8 M) was determined, suggesting that EctM and BetM exhibit a high affinity for their substrates. An elevation of the salinity in the medium resulted in an increased uptake of ectoine via EctM and glycine betaine via BetM in E. coli MKH13 cells, demonstrating that both systems are osmoregulated.Communicated by W.D. Grant     

Replacement of potassium ions by ammonium ions in different micro-organisms grown in potassium-limited chemostat culture

The biomass concentration extant in potassiumlimited cultures of either Klebsiella pneumoniae or Bacillus stearothermophilus (when growing at a fixed temperature and dilution rate in a glucose/ammonium salts medium) increased progressively as the medium pH value was raised step-wise from 7.0 to 8.5. Because the macromolecular composition of the organisms did not vary significantly, this increase in biomass could not be attributed to an accumulation of storage-type polymers but appeared to reflect a pH-dependent decrease in the cells' minimum K⁺ requirement. Significantly, this effect of pH was not eviden with cultures in which no ammonium salts were present and in which either glutamate or nitrate was added as the sole nitrogen source; however, it was again manifest when various concentrations of NH₄Cl were added to the glutamate-containing medium. This suggested a functional replacement of K⁺ by NH ₄ ⁺ , a proposition consistent with the close similarity of the ionic radii of the potassium ion (1.33 Å) and the ammonium ion (1.43 Å). At pH 8.0, and with a medium containing both glutamate (30 mM) and NH₄Cl (100 mM), cultures of B. stearothermophilus would grow without added potassium at a maximum rate of 0.7 h^-1. Under these conditions the cells contained maximally 0.1% (w/w) potassium (derived from contaminating amounts of this element in the medium constituents), a value which should be compared with one of 1.4% (w/w) for cells growing in a potassiumlimited medium containing initially 0.5 mM K⁺. Qualitatively similar findings were made with cultures of K. pneumoniae; and whereas one may not conclude that NH ₄ ⁺ can totally replace K⁺ in the growth of these bacteria, it can clearly do so very extensively.     

Expression of Bacillus stearothermophilus LV cadmium resistance genes in Escherichia coli causes hypersensitivity to cadmium chloride

Determination of the nucleotide sequence of a 4.5-kb chromosomal DNA fragment of Bacillus stearothermophilus LV revealed two open reading frames (ORFs) of 121 and 727 amino acids (aa) that exhibit a high degree of similarity with the cadC and cadA cadmium resistance genes of a number of microorganisms. Transfer and expression of the B. stearothermophilus LV cadA or cadC/cadA genes in E. coli caused increased cadmium chloride susceptibility in the bacterial host. Transfer of cadC alone did not result in any detectable phenotypic change in E. coli. Received: 26 November 2001 / Accepted: 21 December 2001     

致犊牛脑膜炎大肠杆菌新疆分离株ibeB基因的特性分析北大核心CSCD

【目的】分析致犊牛脑膜炎大肠杆菌分离株ibeB基因的分子生物学信息。【方法】以自脑炎死亡犊牛脑组织、肝组织分离鉴定的O161-K99-STa致病性大肠杆菌牛-EN株和牛-EG分离株为材料。根据GenBank中公布的脑膜炎大肠杆菌K1株RS218 ibeB基因序列设计1对引物,采用PCR方法,从分离株中成功克隆ibe B基因,比较分离株ibeB基因与不同来源大肠杆菌ibeB基因的部分生物信息学特性。【结果】分离株ibeB基因序列全长1500 bp,包含1371 bp开放阅读框,共编码457个氨基酸;生物信息学分析显示,牛-EN株与致人脑膜炎大肠杆菌K1 RS218的核苷酸和氨基酸同源性分别为90.5%和96.9%,牛-EG株与大肠杆菌K12的核苷酸和氨基酸同源性分别为99.4%和100.0%;ibeB蛋白为亲水性蛋白,分子质量为50.26 kDa,理论等电点为6.05;该蛋白无跨膜区,但具有信号肽序列;亚细胞定位显示,分泌信号通路位点(SP)占比例为0.939,说明该蛋白属于分泌型蛋白。【结论】从致脑膜炎大肠杆菌分离株中成功克隆ibeB基因,该基因与致人脑膜炎大肠杆菌K1 RS218 ibeB基因有较高的同源性,均有相似的生物学特性,属肠外致病性大肠杆菌。     

Cloning of theHelicobacter pylori recA gene and functional characterization of its product

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ⁷⁰ promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ⁻) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.     

Cloning and characterization of a secY homolog from Chlamydia trachomatis

Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc- ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50 195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SceY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.     

Primary structure of the tms and prs genes of Bacillus subtilis

Summary The nucleotide sequence was determined of a 3211 nucleotide pair EcoRI-PvuII DNA fragment containing the tms and prs genes as well as a part of the ctc gene of Bacillus subtilis. The prs gene encodes phosphoribosylpyrophosphate (PRPP) synthetase, whereas the functioning of the tms and ctc gene products remains to be established. The prs gene contains an open reading frame of 317 codons resulting in a subunit M_r of 34828. An open reading frame comprising the tms gene contained 456 codons resulting in a putative translation product with an M_r of 49554. Comparison of the deduced B. subtilis PRPP synthetase amino acid sequence with PRPP synthetases from Escherichia coli and rat liver showed extensive similarity. The deduced Tms amino acid sequence was found to be 43% similar to the deduced amino acid sequence of ecourfl, a gene of E. coli with unknown function.