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1.
Expression of a putative plant viral gene in Escherichia coli   总被引:2,自引:0,他引:2  
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2.
Porphyrin-Accumulating Mutants of Escherichia coli   总被引:17,自引:9,他引:8       下载免费PDF全文
Four mutants (pop-1, pop-6, pop-10, and pop-14) which accumulate a red water-insoluble pigment were obtained in Escherichia coli K-12 AB1621. For each mutant, the red pigment was shown to be protoporphyrin IX, a late precursor of heme. Mutagenic treatment of mutant pop-1 yielded a secondary mutant, pop-1 sec-20, which accumulated a brown water-soluble pigment. The brown pigment was shown to be coproporphyrin III. Mutant pop-1 resembled the parental strain in its cytochrome absorption spectrum, catalase activity, and ability to grow on nonfermentable carbon and energy sources; therefore, its ability to produce and utilize heme was unimpaired. Judged on the same criteria, the secondary mutant, pop-1 sec-20, was partially heme and respiratory deficient. Growth in anaerobic conditions decreased by 25% the accumulation of protoporphyrin by pop-1; under the same conditions, pop-1 sec-20 did not accumulate coproporphyrin or coproporphyrinogen. The mutations causing protoporphyrin accumulation in all four pop mutants were found to map in the lac to purE (10-13 min) region of the E. coli chromosome. In the case of mutant pop-1, the mutation was shown to be strongly linked to the tsx locus (12 min). In mutant pop-1 sec-20, the second mutation causing coproporphyrin accumulation was co-transducible with the gal locus at a frequency of 88 to 96%. The mechanism of porphyrin accumulation by the mutants is discussed.  相似文献   

3.
Heme molecules play important roles in electron transfer by redox proteins such as cytochromes. In addition, a structural role for heme in protein folding and the assembly of enzymes has been suggested. Previous results obtained using Escherichia coli hemA mutants, which are unable to synthesize 5-aminolevulinic acid, a precursor of porphyrins and hemes, have demonstrated a requirement for heme biosynthesis in the assembly of a functional succinate-ubiquinone reductase (SQR or complex II), which is a component of the aerobic respiratory chain. In the present study, in order to investigate the role of the heme in the assembly of E. coli SQR, we used a hemH (encodes ferrochelatase) mutant that lacks the ability to insert iron into the porphyrin ring. The hemH mutant failed to insert functional SQR into the cytoplasmic membrane, and the catalytic portion of SQR [the flavoprotein subunit (Fp) and the iron-sulfur protein subunit (Ip)] was localized in the cytoplasm of the cell. It is of interest to note that protoporphyrin IX accumulated in the mutant cells and inactivated the cytoplasmic succinate dehydrogenase (SDH) activity associated with the catalytic Fp-Ip complex. In contrast, SQR was assembled into the membrane of a heme-permeable hemH double mutant when hemin was present in the culture. Only a low level of SQR activity was found in the membrane when hemin was replaced by non-iron metalloporphyrins: Mn-, Co-, Ni-, Zn- and Cu-protoporphyrin IX, or protoporphyrin IX These results indicate that heme iron is indispensable for the functional assembly of SQR in the cytoplasmic membrane of E. coli, and provide a new insight into the biological role of heme in the molecular assembly of the multi-subunit enzyme complex.  相似文献   

4.
During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Oxygen-dependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a Km value of 2.6 microm for coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the enzymatic reaction. Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2. Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored coproporphyrinogen III oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. One catalytically important tryptophan residue was localized in position 274. None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity. Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity. Two alternative routes for HemF-mediated catalysis, one metal-dependent, the other metal-independent, are proposed.  相似文献   

5.
The cytochrome o complex is one of two ubiquinol oxidases in the aerobic respiratory system of Escherichia coli. This enzyme catalyzes the two-electron oxidation of ubiquinol-8 which is located in the cytoplasmic membrane, and the four-electron reduction of molecular oxygen to water. The purified oxidase contains at least four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and has been shown to couple electron flux to the generation of a proton motive force across the membrane. In this paper, the DNA sequence of the cyo operon, containing the structural genes for the oxidase, is reported. This operon is shown to encode five open reading frames, cyoABCDE. The gene products of three of these, cyoA, cyoB, and cyoC, are clearly related to subunits II, I, and III, respectively, of the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. This family of cytochrome c oxidases contain heme a and copper as prosthetic groups, whereas the E. coli enzyme contains heme b (protoheme IX) and copper. The most striking sequence similarities relate the large subunits (I) of both the E. coli quinol oxidase and the cytochrome c oxidases. It is likely that the sequence similarities reflect a common molecular architecture of the two heme binding sites and of a copper binding site in these enzymes. In addition, the cyoE open reading frame is closely related to a gene denoted ORF1 from Paracoccus dentrificans which is located in between the genes encoding subunits II and III of the cytochrome c oxidase of this organism. The function of the ORF1 gene product is not known. These sequence relationships define a superfamily of membrane-bound respiratory oxidases which share structural features but which have different functions. The E. coli cytochrome o complex oxidizes ubiquinol but has no ability to catalyze the oxidation of reduced cytochrome c. Nevertheless, it is clear that the E. coli oxidase and the aa3-type cytochrome c oxidases must have very similar structures, at least in the vicinity of the catalytic centers, and they are very likely to have similar mechanisms for bioenergetic coupling (proton pumping).  相似文献   

6.
7.
A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s).  相似文献   

8.
Nucleotide sequence of calf prorennin cDNA cloned in Escherichia coli   总被引:2,自引:0,他引:2  
The nucleotide sequence of prorennin (prochymosin) cDNA cloned in E. coli was determined by the technique of Maxam and Gilbert. The longest prorennin cDNA insert in pTACR1 contained the putative signal sequence and the coding sequence for the peptide from the 1st amino acid, Ala (NH2 terminal), to the 296th, Ser, and the other clone pTACR9 contained the coding sequence from the 258th, Asp, to the 365th, Ile (COOH terminal), and the TGA termination codon followed by the 3'-untranslated region. Thus, the whole coding sequence for prorennin was obtained in the pair of pTACR1 and pTACR9.  相似文献   

9.
A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s).  相似文献   

10.
The cytochromes of Escherichia coli   总被引:1,自引:0,他引:1  
Abstract Escherichia coli contains numerous heme-containing proteins when grown either aerobicaly or anaerobically. These cytochrome species are distributed in the cytoplasm, the periplasm, or are bound to the cytoplasmic membrane. They are involved in various physiological functions, including electron transport, oxidative phosphorylation, assimilatory metabolism and detoxification. One dozen unique cytochrome species have been biochemically and/or genetically characterized. They contain one or more of the four heme groups which E. coli is known to produce: protoheme IX, heme c , heme d , and siroheme. The purpose of this articles is to summarize what we know about the structure and function of this collection of heme proteins.  相似文献   

11.
A Fibrobacter succinogenes S85 gene that encodes endoglucanase hydrolysing CMC and xylan was cloned and expressed in Escherichia coli DH5 by using pUC19 vector. Recombinant plasmid DNA from a positive clone hydrolysing CMC and xylan was designated as pCMX1, harboring 2,043 bp insert. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The nucleotide sequence accession number of the cloned gene sequence in Genbank is U94826. The endoglucanase gene cloned in this study does not have amino sequence homology to the other endoglucanase genes from F. succinogenes S85, but does show sequence homology to family 5 (family A) of glycosyl hydrolases from several species. The ORF encodes a polypeptide of 654 amino acids with a measured molecular weight of 81.3 kDa on SDS-PAGE. Putative signal sequences, Shine-Dalgarno-type ribosomal binding site and promoter sequences (-10) related to the consensus promoter sequences were deduced. The recombinant endoglucanase by E. coli harboring pCMX1 was partially purified and characterized. N-terminal sequences of endoglucanase were Ala-Gln-Pro-Ala-Ala, matched with deduced amino sequences. The temperature range and pH for optimal activity of the purified enzyme were 55 approximately 65 degrees C and 5.5, respectively. The enzyme was most stable at pH 6 but unstable under pH 4 with a K(m) value of 0.49% CMC and a V(max) value of 152 U/mg.  相似文献   

12.
13.
The 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form.  相似文献   

14.
15.
K Nakamura  Y Iwasaki  T Hattori 《Gene》1986,44(2-3):347-351
An Escherichia coli expression vector designed for the efficient synthesis and identification of a full-length cDNA clone is constructed. The vector allows the synthesis of double-stranded cDNAs downstream from the tandem lac control regions employing the vector-primer and linker procedure of Okayama and Berg [Mol. Cell Biol. 2 (1982) 161-170]. Full-length cDNA clones carrying the 5'-noncoding region in addition to the entire coding and 3'-noncoding regions can be expressed in E. coli cells without fusing their coding region to that of E. coli proteins; these clones are identified by colony immunoassay. The entire cDNA insert can be easily excised from the plasmid, since the multiple cloning sites in the vector are duplicated at both ends of the cDNA insert during its synthesis.  相似文献   

16.
Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat-shock genes, the groESL heat-shock operon from a Vibrio cholerae TSI-4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1,632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature-sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.  相似文献   

17.
18.
蔡勤  何志勇  龚毅  杨胜利 《遗传》1999,21(5):1-4
利用RT-PCR技术从中华眼镜蛇毒腺组织中成功地克隆了短链神经毒素CDNA。测序结果表明,该基因开放阅读框架编码83个氨基酸残基,其中对个为信号肽,成熟肽为62个氨基酸残基。该基因与GenBank报道的相同物种的神经毒素基因有相当的同源性,不同物种之间的信号肽序列十分保守。将短链神经毒素CDNA再经PCR扩增除去信号肽序列,克隆到pT7ZZ表达质粒中,转化E.coliBL21(DE3)后,经IPTG诱导可高效表达分子量为23kDa②左右的融合蛋白。表达产物占菌体总蛋白的25%左右。  相似文献   

19.
Cranberry juice has long been believed to benefit the prevention and treatment of urinary tract infections (UTIs). As the first step in the development of infection, bacterial adhesion is of great research interest, yet few studies have addressed molecular level adhesion in this context. P-fimbriated Escherichia coli play a major role in the development of a serious type of UTI, acute pyelonephritis. Experiments were conducted to investigate the molecular-scale effects of cranberry juice on two E. coli strains: HB101, which has no fimbriae, and the mutant HB101pDC1 which expresses P-fimbriae. Atomic force microscopy (AFM) was used to investigate both bacterial surface characteristics and adhesion forces between a probe surface (silicon nitride) and the bacteria, providing a direct evaluation of bacterial adhesion and interaction forces. Cranberry juice affected bacterial surface polymer and adhesion behavior after a short exposure period (<3 h). Cranberry juice affected the P-fimbriated bacteria by decreasing the adhesion forces between the bacterium and tip and by altering the conformation of the surface macromolecules on E. coli HB101pDC1. The equilibrium length of polymer (P-fimbriae) on this bacterium decreased from approximately 148 to approximately 48 nm upon being exposed to cranberry juice. Highly acidic conditions were not necessary for the prevention of bacterial adhesion, since neutralization of cranberry juice solutions to pH = 7.0 allowed us to observe differences in adhesion between the E. coli strains. Our results demonstrate molecular-level changes in the surfaces of P-fimbriated E. coli upon exposure to neutralized cranberry juice.  相似文献   

20.
The cytochrome o terminal oxidases from the bacteria Vitreoscilla and Escherichia coli are structurally and functionally related. They have similar optical spectra, both exhibit ubiquinol-1 oxidase activity and are inhibited similarly. Both enzymes contain four subunits by SDS-polyacrylamide gel electrophoresis analysis and contain protoheme IX and Cu2+ prosthetic groups. Antibodies raised against the oxidase purified from E. coli crossreact with the Vitreoscilla oxidase.  相似文献   

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