Cloning and analysis of the sfrB (sex factor repression) gene of Escherichia coli K-12.

The sfrB gene of Escherichia coli K-12 and the rfaH gene of Salmonella typhimurium LT2 are homologous, controlling expression of the tra operon of F and the rfa genes for lipopolysaccharide synthesis. We have determined a restriction map of the 19-kilobase ColE1 plasmid pLC14-28 which carries the sfrB gene of E. coli. After partial Sau3A digestion of pLC14-28, we cloned a 2.5-kilobase DNA fragment into the BamHI site of pBR322 to form pKZ17. pKZ17 complemented mutants of the sfrB gene of E. coli and the rfaH gene of S. typhimurium for defects of both the F tra operon and the rfa genes. pKZ17 in minicells determines an 18-kilodalton protein not determined by pBR322. A Tn5 insertion into the sfrB gene causes loss of complementing activity and loss of the 18-kilodalton protein in minicells, indicating that this protein is the sfrB gene product. These data indicate that the sfrB gene product is a regulatory element, since the single gene product elicits the expression of genes for many products for F expression and lipopolysaccharide synthesis.     

New gene in Escherichia coli K-12 (drpA): does its product play a role in RNA synthesis?

The mutation drpA1 defines a new gene in Escherichia coli K-12 that maps at about 5.2 min. This mutation was obtained after enriching a population of cells for temperature sensitive dna mutations with the [3H]thymidine "suicide" technique followed by screening for mutants defective in transposon Tn5 precise excision. When growing cells carrying the drpA1 allele were shifted to the nonpermissive temperature, we showed that DNA, RNA, and protein syntheses shut off quickly, with the cessation of RNA synthesis occurring first. A recombinant plasmid between pBR322 and an HindIII fragment from wild-type E. coli restores the growth defect in drpA1 mutants. Using transposon Tn5 mutagenesis of this plasmid, we have been able to correlate the presence of a 68-kilodalton protein, as observed with the maxicell technique, with the ability of this plasmid to restore growth to drpA1 mutants.     

Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

Cloning and characterization of the ferric enterobactin receptor gene (pfeA) of Pseudomonas aeruginosa.

Pseudomonas aeruginosa K407, a mutant lacking a high-affinity 80,000-molecular-weight ferric enterobactin receptor protein (80K protein), exhibited poor growth (small colonies) on iron-deficient succinate minimal medium containing ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and enterobactin. The gene encoding the ferric enterobactin receptor was cloned by complementation of this growth defect. The complementing DNA was subsequently localized to a 7.1-kilobase-pair (kb) SstI-HindIII fragment which was able to restore synthesis of the 80K protein in strain K407 and also to direct the synthesis of high levels of a protein of the same molecular weight in the outer membranes of Escherichia coli fepA strains MT912 and IR20. Moreover, the fragment complemented the fepA mutation in MT912, restoring both growth in EDDHA-containing medium and enterobactin-dependent uptake of 55Fe3+. Expression of the P. aeruginosa receptor in E. coli IR20 was shown to be regulated by both iron and enterobactin. The complementing DNA was further localized to a 5.3-kb SphI-SstI fragment which was then subjected to deletion analysis to obtain the smallest fragment capable of directing the synthesis of the 80K protein in the outer membrane of strain K407. A 3.2-kb DNA fragment that restored production of the receptor in strain K407 was subsequently isolated. The fragment also directed synthesis of the protein in E. coli MT912 but at levels much lower than those previously observed. Nucleotide sequencing of the fragment revealed an open reading frame (designated pfeA for Pseudomonas ferric enterobactin) of 2,241 bp capable of encoding a 746-amino-acid protein with a molecular weight of 80,967. The PfeA protein showed more than 60% homology to the E. coli FepA protein. Consistent with this, the two proteins showed significant immunological cross-reactivity.     

DNA synthesis in Escherichia coli cells infected with gene H mutants of bacteriophage phi X174.

Escherichia coli cells infected with gene H mutants of bacteriophage phi X174 produce two types of particles. The 110S particles contain single-stranded circular DNA; the 110S particles are not infectious, although their DNA is infectious for E. coli spheroplasts. The second type of particles, 70S particles, contain a fragment of single-stranded DNA ranging from 0.2 to 0.5 genome in length. This fragment DNA anneals only to restriction enzyme fragments of replicative-form DNA from the portion of the molecule corresponding to the origin and early region of phi X174 single-stranded synthesis, although full-round single-stranded DNA synthesis is occurring in the H mutant-infected cells. Different H mutant phages produce different proportions of 70S to 110S particles; those mutants producing the most 70S also exhibit the largest amount of degradation of intracellularly labeled DNA during infection. These results suggest that in H mutant-infected cells, full-length single-stranded DNA is synthesized; varying amounts of degradation of the single-stranded material occur, and the resulting fragment DNA is subsequently incorporated into 70S particles.     

An improved method for the purification of DNA-dependent RNA polymerase from Escherichia coli

DNA-dependent RNA polymerase from Escherichia coli was purified further by elution through heparin-Sepharose CL-6B column after the enzyme was obtained, partially purified, using Burgess and Jendrisak's method [(1975)Biochemistry 14, 4634] The total yield of the pure protein was 10 mg from 50 g of E.coli cells. The method was found to be very reproducible and convenient. The enzyme preparation had 60% active molecules and the elongation rate of RNA synthesis by this enzyme was measured to be 11 bases/s over delta D111 T7 DNA.     

寡聚核苷酸诱导突变法改造φ×174噬菌体A基因蛋白的DNA识别序列

 为了进一步研究φX174噬菌体A基因蛋白的复制功能与其所识别的30核苷酸保守序列的关系,我们采用寡聚核苷酸诱导的定点突变法成功地改造了这30核苷酸保守序列。将此保守序列重组到M_(13)mp9噬菌体后,以其单链为模板,在14或16寡聚核苷酸的诱导下,合成共价闭环DNA。经转化到E.coli JM103菌株,用点印迹(Dot blot)杂交法筛选,得到两种重组突变株。一种突变株其30核苷酸保守序列正链的第22碱基由A改为G。另一突变株为其第10碱基A改为C,第11碱基T改为A。突变效率约为5％。制备了此突变株单链及双链DNA,分别做了双脱氧末端终止法及Maxam和Gilbert法序列分析鉴定。     

大肠杆菌O157:H7的sRNA伴侣蛋白Ufq的基因克隆、表达及纯化

目的:克隆、表达并纯化肠出血性大肠杆菌(EHEC)O157:H7的sRNA伴侣蛋白Hfq.方法:利用PCR方法从EHEC O157:H7基因组中扩增出基因hfq,并插入含6xHis标签序列的原核表达载体pET28a(+)的多克隆位点中,构建重组表达质粒pET28a(+)-hfq,以重组质粒转化大肠杆菌BL21(DE3)...     

Molecular cloning and isolation of a cyanobacterial gene which increases the UV and methyl methanesulphonate survival of recA strains of Escherichia coli K12

The unicellular cyanobacterium Gloeocapsa alpicola contains both photoreactivation and excision repair mechanisms for correcting UV-induced damage to its cellular DNA. An 11.5 kb EcoRI fragment was isolated from a cosmid bank of G. alpicola and was shown to complement a recA deletion in Escherichia coli S.17 and JC10289. These recA strains showed increased survival to UV and methyl methanesulphonate (MMS) when transformed with the cyanobacterial DNA fragment, and also showed filamentation in response to UV irradiation. Preliminary analysis of the protein encoded by the cyanobacterial DNA fragment indicated a major protein of 39,000 Da; this is very similar in size to the recA protein of E. coli.     

Expression and regulation of a Vibrio alginolyticus sucrose utilization system cloned in Escherichia coli.

A halotolerant collagenolytic Vibrio alginolyticus strain isolated from salted hides had intracellular sucrase activity and did not secret sucrase into the medium. The strain actively transported sucrose by a sucrose-inducible, Na+-independent process. A 10.4-kilobase DNA fragment of V. alginolyticus DNA was cloned into Escherichia coli. The recombinant E. coli(pVS100) could utilize sucrose as a sole carbon source. In contrast to V. alginolyticus, the recombinant E. coli produced both intra- and extracellular sucrase activities. Up to 20% of the total sucrase activity was in the supernatant. Sucrase synthesis in E. coli(pVS100) was inducible and was subject to glucose repression, which was relieved by cyclic AMP. Sucrose was actively transported by a sucrose-inducible, Na+-independent system in E. coli(pVS100). Sucrose uptake was inhibited by the addition of a proton conductor. The maximum velocity and apparent Km values of sucrose uptake for the V. alginolyticus strain and E. coli(pVS100) were 130 nmol/mg of protein per min and 50 microM and 6 nmol/mg of protein per min and 275 microM, respectively.     

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.     

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.     

Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).     

立氏立克次体外膜蛋白H 基因片段的克隆表达与免疫原性分析

目的:克隆表达立氏立克次体(Rickettsia rickettsii)外膜蛋白H基因(ompH)片段并对其进行免疫原性分析。方法:采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果:获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论:pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。     

Effect of inhibitors on the viability and protein and nucleic acid synthesis of Escherichia coli

The object of this work was to study how the synthesis of protein, RNA and DNA in Escherichia coli M17 and its viability were influenced by chloramphenicol (50 and 300 micrograms/ml) an inhibitor of protein biosynthesis, and sodium azide (200 and 2000 microM) and aminazine (50 micrograms/ml), inhibitors of respiration. The exposed were inhibitors with the bacteria for 60 min at room temperature and for 1-4 months at -10 degrees C. The inhibition of the E. coli viability by chloramphenicol was shown to be reversible. The respiration inhibitors stabilized its viability upon storage at -10 degrees C for one month. The inhibitors were found to produce a different effect on the synthesis of RNA and protein in E. coli. The rates of DNA synthesis hardly changed. No correlation was established between changes in the synthesis of protein and nucleic acids by E. coli after the action of the inhibitors and its viability.     

Role of Gene 52 in Bacteriophage T4 DNA Synthesis

In an attempt to elucidate the mechanism of delayed DNA synthesis in phage T4, Escherichia coli B cells were infected with H17 (an amber mutant defective in gene 52 possessing a "DNA-delay" phenotype). The fate of (14)C-labeled H17 parental DNA after infection was followed: we could show that this DNA sediments more slowly in neutral sucrose than wild-type DNA 3 min postinfection. In pulse-chase experiments progeny DNA was found to undergo detachment from the membrane at 12 min postinfection. Reattachment to the membrane was found to be related to an increase in rate of DNA synthesis. A nucleolytic activity that is absent from cells infected by wild-type phage and from uninfected cells could be detected in extracts prepared from mutant-infected cells. In contrast, degradation of host DNA was found to be less extensive in am H17 compared with wild-type infected cells. Addition of chloramphenicol to mutant-infected cells 10 min postinfection inhibited the appearance of a nuclease activity on one hand and suppressed the "DNA-delay" phenotype on the other hand. We conclude that the gene 52 product controls the activity of a nuclease in infected cells whose main function may be specific strand nicking in association with DNA replication. This gene product might directly attack both E. coli and phage T4 DNA, or indirectly determine their sensitivity to degradation by another nuclease.     

Gene 1.2 protein of bacteriophage T7. Effect on deoxyribonucleotide pools

The gene 1.2 protein of bacteriophage T7, a protein required for phage T7 growth on Escherichia coli optA1 strains, has been purified to apparent homogeneity and shown to restore DNA packaging activity of extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants (Myers, J. A., Beauchamp, B. B., White, J. H., and Richardson, C. C. (1987) J. Biol. Chem. 262, 5280-5287). After infection of E. coli optA1 by T7 gene 1.2 mutant phage, under conditions where phage DNA synthesis is blocked, the intracellular pools of dATP, dTTP, and dCTP increase 10-40-fold, similar to the increase observed in an infection with wild-type T7. However, the pool of dGTP remains unchanged in the mutant-infected cells as opposed to a 200-fold increase in the wild-type phage-infected cells. Uninfected E. coli optA+ strains contain severalfold higher levels of dGTP compared to E. coli optA1 cells. In agreement with this observation, dGTP can fully substitute for purified gene 1.2 protein in restoring DNA packaging activity to extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants. dGMP or polymers containing deoxyguanosine can also restore packaging activity while dGDP is considerably less effective. dATP, dTTP, dCTP, and ribonucleotides have no significant effect. The addition of dGTP or dGMP to packaging extracts restores DNA synthesis. Gene 1.2 protein elevates the level of dGTP in these packaging extracts and restores DNA synthesis, thus suggesting that depletion of a guanine deoxynucleotide pool in E. coli optA1 cells infected with T7 gene 1.2 mutants may account for the observed defects.     

Molecular cloning and expression of a synthetic DNA coding for the antimicrobial protein of bull seminal plasma

A DNA carrying the coding sequence for the antimicrobial protein from bull seminal plasma (SAP) was obtained by enzymic ligation of six synthetic oligonucleotides. The 162 bp synthetic DNA fragment was cloned into the C-terminal part of the lacZ-gene employing the vector pUR289. Expression in E. coli in the presence of the inducer isopropylthiogalactoside (IPTG) led to the formation of a fusion protein, which was shown by immuno-blotting to contain immuno-reactive antimicrobial protein. Approximately 90 min after induction, the cells stopped growing and the culture was found to contain no viable cells 3 h after induction. We conclude from this observation that the beta-galactosidase-antimicrobial protein fusion product was toxic for the E. coli cell and that the SAP-residue attached to beta-galactosidase was responsible for the cytotoxicity.     

Studies on the inhibition of protein synthesis by selenodiglutathione.

Amino acid incorporation in a cell-free system derived from rat liver has previously been found to be inhibited by GSSeSG (selenodiglutathione). In the present experiments the effect of GSSeSG on protein synthesis in 3T3-f cells, on growth and protein synthesis in Escherichia coli, and on amino acid incorporation in a cell-free system derived from E. coli, was studied. GSSeSG inhibits the incorporation of [3H]leucine into protein by 3T3-f cells. This inhibition cannot be reversed by removing GSSeSG and is correlated with the uptake of GSSeSG. Sodium selenite (Na2SeO3) and oxidized glutathione had no inhibitory effect in this system. [3H]Uridine or [3H]thymidine incorporation into RNA or DNA was not inhibited, indicating that the primary action of GSSeSG was on protein synthesis. GSSeSG did not influence the growth of E. coli in a synthetic medium, although enhanced amino acid incorporation was observed. In the cell-free system derived from E. coli, amino acid incorporation was not changed by GSSeSG, indicating that elongation factor G, in contrast to elongation factor 2 of mammalian cell systems, is not blocked by GSSeSG.     

费氏中华根瘤菌与耐盐有关的DNA片段的亚克隆和测序

将费氏中华根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ ｆｒｅｄｉｉ）ＫＴ１９与耐盐有关的２３ｋｂ ＤＮＡ片段用ＢａｍＨⅠ酶切成大小不同的长度,分别与质粒ｐＭＬ１２２连接,然后转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）Ｓ１７－１,筛选出３个转化子。以这些转化子为供体,ＲＴ１９的盐敏感突变株ＲＣ３－３为受体,分别进行二亲本杂交,筛选到接合子ＢＲ２,得到４．４ｋｂ与耐盐有关的ＤＮＡ片段。根据其物理图谱,酶