Elaboration of chloramphenicol acetyltransferase by cells of E. coli K-12 under conditions altering the intracellular concentration of cyclic adenosine-3',5'-monophosphate]

The influence of cAMP, ACTH and glucose on the stimulation of chloramphenicol acetyl transferase synthesis in the cells of E. coli CSH-2/R222 and E. coli WZ-78/R222. (cya855) was examined. Glucose proved to decrease the enzyme synthesis in E. coli CSH-2/R222, causing catabolite repression; 5 mM of cAMP and 1000 mug/ml ACTH overcame the latter. The enzyme synthesis in E. coli WZ-78/R222 was insensitive to the catabolite repression; as to ACTH - it failed to cause stimulation ofchloramphenicol acetyl transferase synthesis in E. coli WZ-78/R222.     

Functioning of the lactose operon of E. coli K-12 under the influence of non-specific regulators]

A possible role played by cAMP in the stimulating action of ACTH and hydrocortisone on lactose E. coli K-12 operon was studied. It was shown that ACTH caused no effect in the E. coli WZ-78/F'lac (cya855) and E. coli CA8001 (L1) strains with destroyed positive cAMP control system of the lactose operon function, at the same time producing a stimulating effect on the lactose operon in the strains of wild type, i.e. E coli 200PS/F'lac and E. coli 3000. Hydrocortisone stimulated the lactose operon function both in E. coli 3000 and in the mutant E. coli CA8001 (L1). It was supposed that the accelerating effect of ACTH on the lactose operon was mediated through cAMP; as to hydrocortisone--it stimulated the lactose operon function independently of cAMP.     

A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase from Rahnella aquatilis with Significantly Reduced Glyphosate Sensitivity

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroA(R.aquatilis), was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroA(E.coli)), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroA(R.aquatilis) were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroA(E.coli). To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious.     

Molecular Recombination Between R-Factor Deoxyribonucleic Acid Molecules in Escherichia coli Host Cells

THREE PREVIOUSLY STUDIED R FACTORS WERE USED: 222/R4, controlling transmissible resistance to sulfonamide, streptomycin, chloromycetin, and tetracycline (SU(r) SM(r) CM(r) TC(r)); 222/R3, a derivative of 222/R4 (now termed 222/R3W) having lost TC(r); and R15, controlling infectious resistance to SU and SM only. Two types of derivative R factors were isolated from 222/R4 by serial subculture in Salmonella species. One derivative, termed 222/R1, lost resistance to SU, SM, and CM, and the other, termed 222/R3N, lost only TC(r). Each factor was transferred to a standard Escherichia coli K-12 host. Recombinant factors of 222/R4 phenotype were isolated by selection after mixed culture of E. coli (222/R1)(+) and (222/R3N)(+) strains. Density-gradient equilibrium centrifugation of lysates of E. coli R(+) hosts in the presence of ethidium bromide separated R-factor deoxyribonucleic acid (DNA) as a heavy satellite peak which was subjected to electron microscopy or analytical density gradient centrifugation. Each DNA comprised a unimolecular species of circular DNA. The contour of R15 measured 22.3 mum [equivalent to 46 x 10(6) atomic mass units (AMU)], and that of 222/R4 measured 33.6 mum (70 x 10(6) AMU). 222/R3W appeared to be a point mutant or small deletion of 222/R4 with an almost identical size, whereas 222/R3N had lost a DNA segment of about 3 mum, and measured 30.3 mum or 63 x 10(6) AMU. The 222/R1 factors also appeared to have arisen by loss of DNA from 222/R4, 222/R1A being 22.3 mum or 46 x 10(6) AMU, whereas all other 222/R1 factors appeared to be duplicates, measuring 25.6 mum or 53 x 10(6) AMU. The DNA from six recombinant factors of R4 phenotype was indistinguishable in size and configuration from the parental 222/R4. In most cases, the number of R-factor copies (present as covalently closed circular molecules) per copy of the E. coli chromosome was less than 2, ranging from 1.2 to 3.3.     

Identification of a cross-reactive epitope widely present in lipopolysaccharide from enterobacteria and recognized by the cross-protective monoclonal antibody WN1 222-5

Septic shock due to infections with Gram-negative bacteria is a severe disease with a high mortality rate. We report the identification of the antigenic determinants of an epitope that is present in enterobacterial lipopolysaccharide (LPS) and recognized by a cross-reactive monoclonal antibody (mAb WN1 222-5) regarded as a potential means of treatment. Using whole LPS and a panel of neoglycoconjugates containing purified LPS oligosaccharides obtained from Escherichia coli core types R1, R2, R3, and R4, Salmonella enterica, and the mutant strain E. coli J-5, we showed that mAb WN1 222-5 binds to the distal part of the inner core region and recognizes the structural element R1-alpha-d-Glcp-(1-->3)-[l-alpha-d-Hepp-(1-->7)]-l-alpha-d-Hepp 4P-(1-->3)-R2 (where R1 represents additional sugars of the outer core and R2 represents additional sugars of the inner core), which is common to LPS from all E. coli, Salmonella, and Shigella. WN1 222-5 binds poorly to molecules that lack the side chain heptose or lack phosphate at the branched heptose. Also molecules that are substituted with GlcpN at the side chain heptose are poorly bound. Thus, the side chain heptose and the 4-phosphate on the branched heptose are main determinants of the epitope. We have determined the binding kinetics and affinities (KD values) of the monovalent interaction of E. coli core oligosaccharides with WN1 222-5 by surface plasmon resonance and isothermal titration microcalorimetry. Affinity constants (KD values) determined by SPR were in the range of 3.6 x 10-5 to 3.2 x 10-8 m, with the highest affinity being observed for the core oligosaccharide from E. coli F576 (R2 core type) and the lowest KD values for those from E. coli J-5. Affinities of E. coli R1, R3, and R4 oligosaccharides were 5-10-fold lower, and values from the E. coli J-5 mutant were 29-fold lower than the R2 core oligosaccharide. Thus, the outer core sugars had a positive effect on binding.     

R Factor Deoxyribonucleic Acid in Chromosomeless Progeny of Escherichia coli

The deoxyribonucleic acid (DNA) of resistance (R) factor 222 carried by Escherichia coli strain P678-54 was found in the normally chromosomeless progeny (minicells) of that strain. The entry of the R222 DNA into minicells appears to be via segregation at the time of their formation from normal cells. The R222 DNA can replicate in minicells although the extent of its replication appears to be limited. An analysis of the R222 DNA structure indicates that it exists in minicells as double-stranded linear, open circular, and twisted circular monomers (molecular weight, about 6.2 x 10(7) daltons). The monomers visualized by electron microscopy are 31.0 +/- 0.5 mum in length. An examination of the effect of acridine orange on the replication of R222 and colicin E1 DNA indicates the dye intereferes with plasmid DNA replication.     

CFA/I-ST plasmids: comparison of enterotoxigenic Escherichia coli (ETEC) of serogroups O25, O63, O78, and O128 and mobilization from an R factor-containing epidemic ETEC isolate

Colonization factor antigen I (CFA/I) plays an important role in the pathogenesis of diarrhea due to enterotoxigenic Escherichia coli. In this study, we examined 11 CFA/I+ enterotoxigenic E. coli from serogroups O25, O63, O78, and O128 and found that with all strains, spontaneous loss of CFA/I was associated with the loss of heat-stable toxin (ST) and with the loss of a single plasmid ranging in size from 54 to 60 megadaltons; when heat-labile toxin was lost, this was associated with the loss of another plasmid. The R factor of one strain, TX432 (O78:H12:CFA/I+; ST+), was found to mobilize the CFA/I-ST plasmid into E. coli K-12 at a frequency of 20%. These studies provide further evidence that CFA/I production is plasmid mediated in enterotoxigenic E. coli belonging to serogroups O25, O63, O78, and O128.     

Acceptance and transfer of plasmid Rts1 by bacteria of the genus Erwinia]

The ability of 13 Erwinia strains to accept, to inherit and to transmit the Rts1 factor by conjugation was studied. 11 strains accepted the Rts1 factor from Escherichia coli K-12 CSH-2 with the frequency of about 10(-7)--10(-3). The Rts1 factor was genetically stable in the Erwinia cells and was not eliminated by acriflavine and under the temperature of 37 and 42 degrees C. All the R+ exconjugants were characterized with more high degree of the resistance of kanamycin than E. coli cells harbouring the same R factor. Erwinia strains harbouring the Rts1 plasmid transferred it by conjugation into homologic (Erwinia) and heterologic (E. coli) bacteria. The study of kinetics of the transfer of the Rts1 factor in different mating systems showed that the transfer of this plasmid from R+ Erwinia into R- Erwinia and R- E. coli--in the liquid medium. It is concluded that Erwinia can be the host and the donor of the Rts1 factor.     

Improved penicillin amidase production using a genetically engineered mutant of Escherichia coli ATCC 11105

Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, we constructed various recombinant E. coli HB101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic and (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the Hindlll fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene. (c) 1993 John Wiley & Sons, Inc.     

Superoxide dismutase and catalase levels in halophilic vibrios.

Superoxide dismutase (SOD) and catalase (CAT) levels were determined for several aerobically grown halophilic vibrios and compared with those found in aerobically grown Escherichia coli K-12. The SOD levels ranged from 25 to 103.6 U/mg of protein for the vibrios compared with 44.6 U/mg of protein for E. coli. The CAT levels ranged from 2.1 to 32.1 U/mg of protein. Electrophoretic analysis of cell extracts revealed that the halophilic vibrios tested possessed only one detectable SOD enzyme, except one strain which possessed two distinct enzymes, as compared with the three SOD enzymes in aerobically grown E. coli K-12. A comparison of anaerobically and aerobically grown vibrios revealed a three- to fourfold increase in SOD activity in the aerobic cells, suggesting that oxygen acts as an inducer for SOD in the vibrios as has been reported for E. coli. In one strain, Vibrio parahaemolyticus 27519, both SOD enzymes were observed in low levels in anaerobic and at higher levels in aerobically grown cells as compared with only one SOD enzyme in anaerobically grown E. coli. This suggests that differences in SOD regulation occur between the two genera. Our results indicate that halophilic vibrios possess SOD, which could enhance viruulence by allowing the organisms to survive in oxygenated environments.     

Identification of the diacylglycerol kinase structural gene of Rhizobium meliloti 1021.

The cyclic beta-1,2-glucans of Rhizobium may function during legume nodulation. These molecules may become highly substituted with phosphoglycerol moieties from the head group of phosphatidylglycerol; diglyceride is a by-product of this reaction (K. J. Miller, R. S. Gore, and A. J. Benesi, J. Bacteriol. 170:4569-4575, 1988). We recently reported that R. meliloti 1021 produces a diacylglycerol kinase (EC 2.7.1.107) activity that shares several properties with the diacylglycerol kinase enzyme of Escherichia coli (W. P. Hunt, R. S. Gore, K. J. Miller, Appl. Environ. Microbiol. 57:3645-3647, 1991). A primary function of this rhizobial enzyme is to recycle diglyceride generated during cyclic beta-1,2-glucan biosynthesis. In the present study, we report the cloning and initial characterization of a single-copy gene from R. meliloti 1021 that encodes a diacylglycerol kinase homolog; this homolog can complement a diacylglycerol kinase deficient strain of E. coli. The sequence of the rhizobial diacylglycerol kinase gene was predicted to encode a protein of 137 amino acids; this protein shares 32% identity with the E. coli enzyme. Analysis of hydropathy and the potential to form specific secondary structures indicated a common overall structure for the two enzymes. Because diglyceride metabolism and cyclic beta-1,2-glucan biosynthesis are metabolically linked, future studies with diacylglycerol kinase mutants of R. meliloti 1021 should further elucidate the roles of the cyclic beta-1,2-glucans in the Rhizobium-legume symbiosis.     

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA.方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径.结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆...     

The purification and properties of the trimethoprim-resistant dihydrofolate reductase mediated by the R-factor, R388.

The R-factor R388 mediates the production of a trimethoprim-resistant dihydrofolate reductase. This enzyme has a different molecular weight and pH profile to the trimethoprim-sensitive enzyme of the Escherichia coli host. The R-factor mediated enzyme was separated completely from the host E. coli enzyme by DEAE-cellulose ion-exchange chromatography. The purified R-factor enzyme was about 20 000 times less susceptible to trimethoprim than the E. coli enzyme and although it was inhibited competitively by trimethoprim, its inhibitor constant (Ki) was 20 000 times greater than that of the host enzyme. The R388 and E. coli enzymes also differed in their substrate specificity requirements. In addition, the R388 enzyme suprisingly conferred high level resistance to the broad spectrum dihydrofolate reductase inhibitor, amethopterin. The possible origins of the R388 enzyme are discussed.     

Silver accumulation and resistance in Escherichia coli R1

E. coli R1 contains at least 2 large plasmids (83 and 77 kb) while E. coli S1 was previously cured of the 83 kb plasmid. Transformation using artificial competence, high-voltage electroporation, and plasmid mobilization experiments with the mobilizing plasmid RP4, failed to ascertain the 83 kb plasmid was responsible for Ag(+)-resistance. Silver accumulation by an Ag(+)-sensitive E. coli S1 strain was 5-fold higher than an Ag(+)-resistant E. coli R1 strain. The Ag(+)-resistant E. coli R1 strain produced 33% more H2S and 32% more intracellular acid labile SH than the Ag(+)-sensitive E. coli R1 strain when grown in the absence of AgNO3. Hydrophobic interaction chromatography revealed E. coli R1 displayed higher cell surface hydrophobicity than E. coli S1. HPLC protein analysis of cell-free extracts also revealed differences between protein fractions in E. coli R1 and S1 strains.     

Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain

A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library of Escherichia coli K-12 DNA by complementation to methionine prototrophy in an E. coli strain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE, metHH). Maxicell expression of a series of plasmids containing deletions in the metH structural gene was employed to map the position and orientation of the gene on the cloned DNA fragment. A 6.3-kilobase EcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides. The enzyme was purified from an overproducing strain of E. coli harboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-type E. coli. Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay. These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240-1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain of E. coli B (Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H., and Matthews, R. G. (1988) Biochemistry 27, 8458-8465). Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight. This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.     

Enhanced production of (R)-1,2-propanediol by metabolically engineered Escherichia coli

1,2-Propanediol (1,2-PD) is a major commodity chemical currently derived from propylene. Previously, we have demonstrated the production of enantiomerically pure (R)-1,2-propanediol from glucose by an engineered E. coli expressing genes for NADH-linked glycerol dehydrogenase and methylglyoxal synthase. In this work, we investigate three methods to improve 1,2-PD in E. coli. First, we investigated improving the host by eliminating production of a byproduct, lactate. To do this, we constructed strains with mutations in two enzymes involved in lactate production, lactate dehydrogenase and glyoxalase I. (Surprisingly, when mutations were made in its ability to produce lactate, one strain of E. coli [MM294], produced a small amount of 1,2-PD without any added genes.) Second, we constructed a complete pathway to 1,2-PD from the glycolytic intermediate, dihydroxyacetone phosphate. Our previous 1, 2-PD producing strains relied on at least one endogenous E. coli activity and only produced 0.7 g/L of 1,2-PD. The complete pathway involved the coexpression of methylglyoxal synthase (mgs), glycerol dehydrogenase (gldA), and either yeast alcohol dehydrogenase (adhI) or E. coli 1,2-propanediol oxidoreductase (fucO). Third, we investigated bioprocessing improvements by carrying out a fed-batch fermentation with the best engineered strain (expressing mgs, gldA, and fucO). A final titer of 4.5 g/L of (R)-1,2-PD was produced, with a final yield of 0.19 g of 1,2-PD per gram of glucose consumed. This work provides a basis for further strain and process improvement.     

近平滑假丝酵母(R)-羰基还原酶在大肠杆菌中的外泌表达及高效转化(R)-苯基乙二醇

克隆了近平滑假丝酵母(Candida parapsilosis)(R)-羰基还原酶基因rcr,构建胞外表达工程茵Escherichia coli BL21(DE3)/pET20b-rcr,实现了(R)-羰基还原酶在大肠杆菌中高效外泌表达,周质空间和发酵液酶的比活力分别达0.68 U/mg和0.26 U/mg,与大肠杆菌的胞内体系重组酶相比,酶的比活力提高了近两倍。为了更好地促进该重组酶的外分泌于大肠杆菌细胞外,通过添加温和型化学渗透剂甘氨酸,改善细胞壁的透性,(R)-羰基还原酶的活力提高至1.99 U,与添加甘氨酸前相比,酶活力提高了12.4倍,比活提高了4.3倍。浓缩后的发酵液催化2-羟基苯乙酮,产生(R)-苯基乙二醇,产率为88.1%,e.e.值为93.9%。与胞内重组酶相比,产率和光学纯度分别提高了44.4%和15.9%。本研究通过构建(R)-羰基还原酶的大肠杆菌分泌表达体系,大幅度提高了(R)-羰基还原酶的比活和生物转化手性醇的效率。     

Identification of arginine residues important for the activity of Escherichia coli signal peptidase I

Escherichia coil signal peptidase I (leader peptidase, SPase I) is an integral membrane serine protease that catalyzes the cleavage of signal (leader) peptides from pre-forms of membrane or secretory proteins. We previously demonstrated that E. coil SPase I was significantly inactivated by reaction with phenylglyoxal with concomitant modification of three to four of the total 17 arginine residues in the enzyme. This result indicated that several arginine residues are important for the optimal activity of the enzyme. In the present study, we have constructed 17 mutants of the enzyme by site-directed mutagenesis to investigate the role of individual arginine residues in the enzyme. Mutation of Arg127, Arg146, Arg198, Arg199, Arg226, Arg236, Arg275, Arg282, and Arg295 scarcely affected the enzyme activity in vivo and in vitro. However, the enzymatic activity toward a synthetic substrate was significantly decreased by replacements of Arg77, Arg222, Arg315, or Arg318 with alanine/lysine. The kcat values of the R77A, R77K, R222A, R222K, R315A, R318A, and R318K mutant enzymes were about 5.5-fold smaller than that of the wild-type enzyme, whereas the Km values of these mutant enzymes were almost identical with that of the wild-type. Moreover, the complementing abilities in E. Arg222, Arg315, coil IT41 were lost completely when Arg77, or Arg318 was replaced with alanine/lysine. The circular dichroism spectra and other enzymatic properties of these mutants were comparable to those of the wild-type enzyme, indicating no global conformational changes. However, the thermostability of R222A, R222K, R315A, and R318K was significantly lower compared to the wild type. Therefore, Arg77, Arg222, Arg315, and Arg318 are thought to be important for maintaining the proper and stable conformation of SPase I.     

异戊二烯合成酶(IspS)在大肠杆菌中的表达及其产异戊二烯的研究

将来源于银白杨的异戊二烯合成酶基因按照大肠杆菌密码子偏爱性进行优化,克隆到表达载体pACYCDu-et-1上,在大肠杆菌BL21(DE3)中异源表达,采用镍柱亲和层析纯化重组蛋白并测定其异戊二烯合成酶活性,通过摇瓶发酵实验对重组菌产异戊二烯进行进一步研究。结果显示:银白杨异戊二烯合成酶在大肠杆菌中能够高效表达,经过镍柱纯化后,电泳检测到特异性表达条带;该重组异戊二烯合成酶能够催化异戊二烯的合成,重组菌的异戊二烯产量可达到60μg/L。