Selective cloning of genes encoding RNase H from Salmonella typhimurium, Saccharomyces cerevisiae and Escherichia coli rnh mutant

Summary We have cloned genes encoding RNase H from Escherichia coli rnh mutants, Salmonella typhimurium and Saccharomyces cerevisiae. Selection was accomplished by suppression of the temperature-sensitive growth phenotype of Escherichia coli strains containing the rnh-339::cat and either recB270 (Ts) or recC271 (Ts) mutations. RNases H from E. coli and S. typhimurium contained 155 amino acid residues and differed at only 11 positions. The S. cerevisiae and E. coli RNases H were about 30% homologous. A comparison of the amino acid sequences of several RNases H from cellular and retroviral sources revealed some strongly conserved regions as well as variable regions; the carboxyl-terminus was particularly variable. The overlapping, divergent promoter gene organization found in E. coli was observed to be present in S. typhimurium.     

Spontaneous and reversible high-frequency frameshifts originating a phase transition in the carotenoid biosynthesis pathway of the phototrophic bacterium Rhodobacter sphaeroides 2.4.1

Summary The nucleotide sequence of the melB gene coding for the Na⁺(Li⁺)/melibiose symporter of Salmonella typhimurium LT2 was determined, and its amino acid sequence was deduced. It consists of 1428 bp, corresponding to a protein of 476 amino acid residues (calculated molecular weight 52800). The amino acid sequence is homologous to that of the melibiose permease of Escherichia coli K12, with 85% identical residues. All, except one, of the amino acid residues that have been reported to be important for cation or substrate recognition in the melibiose permease of E. coli are conserved in the melibiose permease of S. typhimurium. In addition, part of the sequence resembles the lactose permease of Streptococcus thermophilus, the animal glucose transporter (GLUT1), the plasmid-coded raffinose permease (RafB), and the NADH-ubiquinone oxidoreductase chain 4 (Nuo4) of Aspergillus amstelodami.The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank/EMBL Data Bank with accession number X62101     

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.     

Comparison of sequences from the malB regions of Salmonella typhimurium and Enterobacter aerogenes with Escherichia coli K12: A potential new regulatory site in the interoperonic region

Summary The malE and malK genes from Salmonella typhimurium, and the MalEFG operon and a portion of malK from Enterobacter aerogenes were cloned and sequenced. Plasmid-borne malE genes from both species and the malF and malG genes from E. aerogenes were expressed normally in Escherichia coli, and their products function in maltose transport. This shows that the malB products from the three species are interchangeable, at least in the combinations tested. The general genetic organization of the malB region is conserved. Potential binding sites and distances between them are highly conserved in the regulatory intervals. An unexpected conserved region was detected, which we call the U box, and which could be another target for a regulatory protein. This hypothesis is supported by the presence of the U box in the regulatory, region of the pulA-malX operon in Klebsiella pneumoniae. The intergenic region between malE and malF from S. typhimurium and E. aerogenes, contains inverted repeats similar to the palindromic units (PU or REP) found at the same location in E. coli. The predicted amino acid sequence of the encoded proteins showed 90% or more identity in every pairwise comparison of species.     

Identification of a chloroform-soluble membrane miniprotein in Escherichia coli and its homolog in Salmonella typhimurium

Two homologous 29 amino acid-long highly hydrophobic membrane miniproteins were identified in the Bligh–Dyer lipid extracts of Escherichia coli and Salmonella typhimurium using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The amino acid sequences of the proteins were determined by collision-induced dissociation tandem mass spectrometry, in conjunction with a translating BLAST (tBLASTn) search, i.e., comparing the MS/MS-determined protein query sequence against the six-frame translations of the nucleotide sequences of the E. coli and S. typhimurium genomes. Further MS characterization revealed that both proteins retain the N-terminal initiating formyl-methionines. The methodologies described here may be amendable for detecting and characterizing small hydrophobic proteins in other organisms that are difficult to annotate or analyze by conventional methods.     

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.     

Synthesis and localisation of Escherichia coli UDP-glucose hydrolase (5′-nucleotidase), and demonstration of a cytoplasmic inhibitor of this enzyme in Salmonella typhimurium

A strain of Salmonella typhimurium has been constructed, by transfer of F-13 from Escherichia coli, which synthesises and secretes Escherichia coli UDPglucose hydrolase. Although Salmonella typhimurium does not normally contain a secreted (periplasmic) UDPglucose hydrolase, we find that it contains, like Escherichia coli, an intracellular inhibitor of the enzyme.     

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12

Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.     

Molecular cloning of the Salmonella typhimurium lep gene in Escherichia coli

Summary A system is described which enabled the selection of a heterologous ep gene, encoding signal peptidase I, in Escherichia coli. It is based on complementation of an E. coli mutant, in which the synthesis of signal peptidase I can be regulated. With this system the lep gene of Salmonella typhimurium was cloned and the nucleotide sequence was determined. The S. typhimurium lep gene encodes a protein of 324 amino acids. Expression of the gene in the E. coli mutant resulted in suppression of growth inhibition and in the restoration of processing activity under conditions where synthesis of E. coli signal peptidase I was repressed. The cloned S. typhimurium signal peptidase I had an apparent molecular weight of 36000 daltons, which is in agreement with the calculated molecular weight of 35782 daltons. The system described for selection of the S. typhimurium lep gene may permit the cloning and expression of other heterologous signal peptidase I gen/es.     

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Summary Salmonella typhimurium and S. typhi were transformd with high efficiency by electroporation. Transformation efficiencies of up to 10¹⁰ transformants per g of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a galE mutation slightly improved transformation efficiency in S. typhimurium (< tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.     

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。     

Analysis of chimeric UmuC proteins: identification of regions inSalmonella typhimurium UmuC important for mutagenic activity

UnlikeEscherichia coli, the closely related bacteriumSalmonella typhimurium is relatively unresponsive to the mutagenic effects of DNA-damaging agents. Previous experiments have suggested that these phenotypic differences might result from reduced activity of theS. typhimurium UmuC protein. To investigate this possibility, we have taken advantage of the high degree of homology between the UmuC proteins ofE. coli andS. typhimurium and have constructed a series of plasmid-encoded chimeric proteins. The possibility that the phenotypic differences might be due to differential expression of the respective UmuC proteins was eliminated by constructing chimeric proteins that retained the first 25 N-terminal amino acids of either of the UmuC proteins (and presumably the same translational signals), but substituting the remaining 397 C-terminal amino acids with the corresponding segments from the reciprocal operon. Constructs expressing mostlyE. coli UmuC were moderately proficient for mutagenesis whereas those expressing mostlyS. typhimurium UmuC exhibited much lower frequencies of mutation, indicating that the activity of the UmuC protein ofS. typhimurium is indeed curtailed. The regions responsible for this phenotype were more precisely localized by introducing smaller segments of theS. typhimurium UmuC protein into the UmuC protein ofE. coli. While some regions could be interchanged with few or no phenotypic effects, substitution of residues 212–395 and 396–422 ofE. coli UmuC with those fromS. typhimurium resulted in reduced mutability, while substitution of residues 26–59 caused a dramatic loss of activity. We suggest, therefore, that the primary cause for the poor mutability ofS. typhimurium can be attributed to mutations located within residues 26–59 of theS. typhimurium UmuC protein.     

Molecular Cloning of the Phytase Gene from Citrobacter braakii and its Expression in Saccharomyces cerevisiae

The gene, appA, encoding phytase was cloned from a size-selected genomic library of Citrobacter braakii YH-15 by Southern hybridization using a degenerate probe based on the N-terminal amino acid sequence of the phytase. The deduced amino acid sequence of appA contained the N-terminal RHGXRXP motif and the C-terminal HD motif, which are common in histidine acid phosphatases. It also had significant homology (60% identity) with phytase from Escherichia coli, while the physical mapping analysis of appA revealed that gene organization near appA in C. braakii was similar to that in Salmonella typhimurium genome. C. braakii AppA contained five putative N-glycosylation sites. The recombinant phytases, rAppA_Ec and rAppA_Sc, were produced in E. coli and Saccharomyces cerevisiae, respectively, with both being fused with C-terminal His-tag. After purification, rAppA_Sc was shown to be hyperglycosylated by Endo-H treatment. It had greater thermostability than the wild type phytase and rAppA_Ec.     

Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation

Summary A DNA fragment that codes for the 364 amino-terminal amino acid residues of a putative Bacillus subtilis SecA homologue has been cloned using the Escherichia coli SecA gene as a probe. The deduced amino acid sequence showed 58% identity to the aminoterminus of the E. coli SecA protein. A DNA fragment which codes for 275 amino-terminal amino acid residues of the B. subtilis SecA homologue was expressed in E. coli and the corresponding gene product was shown to be recognized by anti-E. coli SecA antibodies. This polypeptide, although only about 30% the size of the E. coli SecA protein, also restored growth of E. coli MM52 (secA ^ts) at the non-permissive temperature and the translocation defect of proOmpA in this mutant was relieved to a substantial extent.     

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.     

envB mutations confer UV-sensitivity to Salmonella typhimurium and UV-resistance to Escherichia coli

Summary An envB mutation isolated in Salmonella typhimurium LT2 was transferred by conjugation to Escherichia coli K-12. The mutation produced the same alterations in E. coli as in S. typhimurium concerning cell shape, sensitivity to drugs, autolysis, and fermentation of carbohydrates. However, although the mutation conferred sensitivity to UV irradiation in Salmonella, in E. coli it behaved as a genuine envB mutation producing resistance to UV inactivation. The fact that the mutation produced opposite effects in the survival of UV-irradiated S. typhimurium and E. coli discloses an intriguing difference between these closely related species.Career Investigator of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina     

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.     

Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: homologies between OxyR protein and a family of bacterial activator proteins

Summary Treatment of Escherichia coli and Salmonella typhimurium cells with a low dose of hydrogen peroxide induces expression of a large number of genes, and confers resistance to oxidative stresses. The oxyR gene encodes a positive regulatory protein for a subset of these genes involved in the defense against oxidative damage. We cloned a DNA fragment that contains the E. coli oxyR region on a plasmid vector, and analyzed the nucleotide sequence of the gene. The amino acid sequence of OxyR protein, deduced from the nucleotide sequence, shows a high degree of homology to the sequences of a number of bacterial activator proteins including LysR, cysB, IlvY, MetR and NodD. The product of the oxyR gene identified by the maxicell procedure was a 34 kDa protein, which agrees with the size predicted from the nucleotide sequence of the gene.     

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO

Summary The gene braB, encoding the Na^–-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na^–-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated M_r of 45279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with thebrnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.