Regulation of the thdF gene, which is involved in thiophene oxidation by Escherichia coli K-12

The thdF gene of Escherichia coli encodes a 48 kD protein which is involved in the oxidation of derivatives of the sulphur-containing heterocycle thiophene and which appears to be induced during stationary phase. In this work the upstream regulatory region of the thdF gene was isolated by polymerase chain reaction and inserted in front of the lacZ structural gene. Examination of the resulting thdF-lacZ operon fusions showed that expression of the thdF gene increased as E. coli entered the stationary phase. However, the expression of thdF was not dependent on RpoS (KatF), the stationary phase sigma factor. The thdF gene was subject to substantial catabolite repression by glucose and its expression was also greatly decreased in the absence of oxygen. The thdF-lacZ fusions were not significantly affected by elevated temperature or medium of high osmolarity, nor by mutations in thdA, fadR, arcA, arcB, or fnr. Both multicopy, plasmid-borne fusions and single-copy fusions gave similar results in all of the above cases except that the plasmid-borne fusions still showed substantial expression in the absence of oxygen. The heterocyclic compounds thiophene carboxylic acid, furan carboxylic acid and proline increased expression of the thdF gene by 2- to 3-fold, but only during the stationary phase. Tryptophan, indole, and several indole derivatives had no effect.     

Molecular cloning and nucleotide sequence of the gene for Escherichia coli leucyl-tRNA synthetase.

The gene for Escherichia coli leucyl-tRNA synthetase leuS has been cloned by complementation of a leuS temperature sensitive mutant KL231 with an E.coli gene bank DNA. The resulting clones overexpress leucyl-tRNA synthetase (LeuRS) by a factor greater than 50. The DNA sequence of the complete coding regions was determined. The derived N-terminal protein sequence of LeuRS was confirmed by independent protein sequencing of the first 8 aminoacids. Sequence comparison of the LeuRS sequence with all aminoacyl-tRNA synthetase sequences available reveal a significant homology with the valyl-, isoleucyl- and methionyl-enzyme indicating that the genes of these enzymes could have derived from a common ancestor. Sequence comparison with the gene product of the yeast nuclear NAM2-1 suppressor allele curing mitochondrial RNA maturation deficiency reveals about 30% homology.     

Molecular cloning and nucleotide sequence of the HU-1 gene of Escherichia coli

Summary The Escherichia coli HU-1 was cloned by use of mixed synthetic oligonucleotides (17-mer) predicted from a portion of its amino acid sequence. The amino acid sequence of the HU-1 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream from the translational initiation codon (GUG) of the HU-1 gene.     

Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of enterobacterial common antigen. Molecular cloning of the rfe-rff gene cluster.

The genetic determinants of enterobacterial common antigen (ECA) include the rfe and rff genes located between ilv and cya near min 85 on the Escherichia coli chromosome. The rfe-rff gene cluster of E. coli K-12 was cloned in the cosmid pHC79. The cosmid clone complemented mutants defective in the synthesis of ECA due to lesions in the rfe, rffE, rffD, rffA, rffC, rffT, and rffM genes. Restriction endonuclease mapping combined with complementation studies of the original cosmid clone and six subclones revealed the order of genes in this region to be rfe-rffD/rffE-rffA/rffC-rffT-rffM . The rfe gene was localized to a 2.54-kilobase ClaI fragment of DNA, and the complete nucleotide sequence of this fragment was determined. The nucleotide sequencing data revealed two open reading frames, ORF-1 and ORF-2, located on the same strand of DNA. The putative initiation codon of ORF-1 was found to be 570 nucleotides downstream from the termination codon of rho. ORF-1 and ORF-2 specify putative proteins of 257 and 348 amino acids with calculated Mr values of 29,010 and 39,771, respectively. ORF-1 was identified as the rfe gene since ORF-1 alone was able to complement defects in the synthesis of ECA and 08-side chain synthesis in rfe mutants of E. coli. Data are also presented which suggest the possibility that the rfe gene is the structural gene for the tunicamycin sensitive UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase that catalyzes the synthesis of GlcNAc-pyrophosphorylundecaprenol (lipid I), the first lipid-linked intermediate involved in ECA synthesis.     

Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III

Escherichia coli contains two type 1 topoisomerases, topoisomerase I and III. Although topoisomerase III can be purified as a potent decatenase, its role in DNA metabolism is unclear. In order to address this issue, the gene encoding topoisomerase III from E. coli has been molecularly cloned and its DNA sequence determined. The cloned fragment of DNA contains an open reading frame that can encode a polypeptide of 73.2 kDa. The first 20 amino acids of this open reading frame are identical to those of topoisomerase III as determined by amino-terminal gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame, using a bacteriophage T7 transient expression system, results in the accumulation of a 74-kDa polypeptide. Soluble extracts prepared from cells overexpressing this gene product show a dramatic increase in topoisomerase activity when compared with control extracts. We propose that this gene be designated topB.     

Molecular cloning of the ecotin gene in Escherichia coli

The nucleotide sequence of a 876 bp region in E. coli chromosome that encodes Ecotin was determined. The proposed coding sequence for Ecotin is 486 nucleotides long, which would encode a protein consisting of 162 amino acids with a calculated molecular weight of 18,192 Da. The deduced primary sequence of Ecotin includes a 20-residue signal sequence, cleavage of which would give rise to a mature protein with a molecular weight of 16,099 Da. Ecotin does not contain any consensus reactive site sequences of known serine protease inhibitor families, suggesting that Ecotin is a novel inhibitor.     

Molecular cloning of the Escherichia coli B L-fucose-D-arabinose gene cluster.

To metabolize the uncommon pentose D-arabinose, enteric bacteria often recruit the enzymes of the L-fucose pathway by a regulatory mutation. However, Escherichia coli B can grow on D-arabinose without the requirement of a mutation, using some of the L-fucose enzymes and a D-ribulokinase that is distinct from the L-fuculokinase of the L-fucose pathway. To study this naturally occurring D-arabinose pathway, we cloned and partially characterized the E. coli B L-fucose-D-arabinose gene cluster and compared it with the L-fucose gene cluster of E. coli K-12. The order of the fucA, -P, -I, and -K genes was the same in the two E. coli strains. However, the E. coli B gene cluster contained a 5.2-kb segment located between the fucA and fucP genes that was not present in E. coli K-12. This segment carried the darK gene, which encodes the D-ribulokinase needed for growth on D-arabinose by E. coli B. The darK gene was not homologous with any of the L-fucose genes or with chromosomal DNA from other D-arabinose-utilizing bacteria. D-Ribulokinase and L-fuculokinase were purified to apparent homogeneity and partially characterized. The molecular weights, substrate specificities, and kinetic parameters of these two enzymes were very dissimilar, which together with DNA hybridization analysis, suggested that these enzymes are not related. D-Arabinose metabolism by E. coli B appears to be the result of acquisitive evolution, but the source of the darK gene has not been determined.     

Molecular cloning of a gene which regulates the adaptive response to alkylating agents in Escherichia coli

Summary The ada ⁺ gene of E. coli is a regulatory gene of the adaptive response to simple alkylating agents. ada mutants are sensitive to both the mutagenicity and toxicity of alkylating agents, and are unable to induce O⁶-methylguanine DNA methyltransferase and 3-methyladenine DNA glycosylase II. The ada ⁺ gene was cloned from wild type E. coli B by ligating bacterial DNA partially digested with Sau3A into the cosmid vector pJB8. The hybrid cosmid, pCS33, conveyed N-methyl-N-nitro-N-nitrosoguanidine resistance to ada mutants of E. coli B and E. coli K12, and resulted in the constitutive synthesis of the two DNA repair enzymes at high levels. An alk mutation, which results in a deficiency of only the DNA glycosylase, was not complemented by this cosmid. It was concluded that the product of the ada ⁺ gene is a positive regulator of the adaptive response. The cosmid insert DNA was subcloned into the plasmid vector pAT153, and the ada ⁺ plasmids pCS42 and pCS58 selected. The ada ⁺ gene located in PCS58 by transposon mutagenesis and subcloning. Two polypeptides of Mr 37,000 and 27,000, were identified in maxicells as products of the ada ⁺ gene(s). It is as yet unclear whether they represent different forms of the same gene product, or are encoded by separate ada ⁺ genes within the same operon.     

Molecular characterization of the Escherichia coli htrD gene: cloning, sequence, regulation, and involvement with cytochrome d oxidase.

The Escherichia coli htrD gene was originally isolated during a search for new genes required for growth at high temperature. Insertional inactivation of htrD leads to a pleiotropic phenotype characterized by temperature-sensitive growth in rich medium, H2O2 sensitivity, and sensitivity to cysteine. The htrD gene was cloned and sequenced, and an htrD::mini-Tn10 insertion mutation was mapped within this gene. The htrD gene was shown to encode a protein of approximately 17.5 kDa. Expression of the htrD gene was examined by using an phi (htrD-lacZ) operon fusion. It was found that htrD is not temperature regulated and therefore is not a heat shock gene. Further study revealed that htrD expression is increased under aerobic growth conditions. Conversely, under anaerobic growth conditions, htrD expression is decreased. In addition, a mutation within the nearby cydD gene was found to drastically reduce htrD expression under all conditions tested. These results indicate that htrD is somehow involved in aerobic respiration and that the cydD gene product is necessary for htrD gene expression. In agreement with this conclusion, htrD mutant bacteria are unable to oxidize the cytochrome d-specific electron donor N,N,N',N'-tetramethyl-p-phenylenediamine.     

Molecular cloning of gltS and gltP, which encode glutamate carriers of Escherichia coli B.

Two genes encoding distinct glutamate carrier proteins of Escherichia coli B were cloned into an E. coli K-12 strain by using a cosmid vector, pHC79. One of them was the gltS gene coding for a glutamate carrier of an Na+-dependent, binding protein-independent, and glutamate-specific transport system. The content of the glutamate carrier was amplified about 25-fold in the cytoplasmic membranes from a gltS-amplified strain. The gltS gene was located in a 3.2-kilobase EcoRI-MluI fragment, and the gene product was identified as a membrane protein with an apparent Mr of 35,000 in a minicell system. A gene designated gltP was also cloned. The transport activity of the gltP system in cytoplasmic membrane vesicles from a gltP-amplified strain was driven by respiratory substrates and was independent of the concentrations of Na+, K+, and Li+. An uncoupler, carbonylcyanide m-chlorophenylhydrazone, completely inhibited the transport activities of both systems, whereas an ionophore, monensin, inhibited only that of the gltS system. The Kt value for glutamate was 11 microM in the gltP system and 3.5 microM in the gltS system. L-Aspartate inhibited the glutamate transport of the gltP system but not that of the gltS system. Aspartate was taken up actively by membrane vesicles from the gltP-amplified strain, although no aspartate uptake activity was detected in membrane vesicles from a wild-type E. coli strain. These results suggest that gltP is a structural gene for a carrier protein of an Na+-independent, binding protein-independent glutamate-aspartate transport system.     

Molecular cloning of the Escherichia coli miaA gene involved in the formation of delta 2-isopentenyl adenosine in tRNA.

Escherichia coli mia strains were shown to lack delta 2-isopentenylpyrophosphate transferase activity, the first step in the synthesis of the 2-methylthio derivative of 6-(delta 2-isopentenyl) adenosine (ms2i6A). A double mutant, rpsL (Smp) miaA, was streptomycin dependent. The wild-type miaA gene was cloned by selecting for lambda recombinant bacteriophage which eliminated the streptomycin-dependent phenotype and was subsequently recloned into plasmid vectors. The cloned miaA gene restored the ms2i6A modification to tRNA. The miaA gene mapped to 95 min on the E. coli map, and we propose the order mutL-miaA-hflA-purA.     

Molecular cloning and sequencing of pheU, a gene for Escherichia coli tRNAPhe.

A recombinant plasmid (designated pID2) carrying the E. coli gene for tRNAPhe has been isolated from a plasmid bank constructed by the ligation of a total EcoRI digest of E. coli K12 DNA into the EcoRI site of pACYC184 DNA. The plasmid was selected by virtue of its ability to complement a temperature-sensitive lesion in the gene (PheS) for the alpha-subunit of phenylalanyl-tRNA synthetase. Crude tRNA isolated from such transformants exhibited elevated levels of phenylalanine acceptor activity. The tRNAPhe gene has been localized within the first 300 base pairs of a 3.6 kb SalI fragment of pID2. The sequence of the gene and its flanking regions is presented.     

Molecular cloning and primary structure of the Escherichia coli methionyl-tRNA synthetase gene.

The intact metG gene was cloned in plasmid pBR322 from an F32 episomal gene library by complementation of a structural mutant, metG83. The Escherichia coli strain transformed with this plasmid (pX1) overproduced methionyl-tRNA synthetase 40-fold. Maxicell analysis showed that three major polypeptides with MrS of 76,000, 37,000, and 29,000 were expressed from pX1. The polypeptide with an Mr of 76,000 was identified as the product of metG on the basis of immunological studies and was indistinguishable from purified methionyl-tRNA synthetase. In addition, DNA-DNA hybridization studies demonstrated that the metG regions were homologous on the E. coli chromosome and on the F32 episome. DNA sequencing of 642 nucleotides was performed. It completes the partial metG sequence already published (D. G. Barker, J. P. Ebel, R. Jakes, and C. J. Bruton, Eur. J. Biochem. 127:449-451, 1982). Examination of the deduced primary structure of methionyl-tRNA synthetase excludes the occurrence of any significant repeated sequences. Finally, mapping of mutation metG83 by complementation experiments strongly suggests that the central part of methionyl-tRNA synthetase is involved in methionine recognition. This observation is discussed in the light of the known three-dimensional crystallographic structure.     

Molecular cloning and DNA sequence of dniR, a gene affecting anaerobic expression of the Escherichia coli hexaheme nitrite reductase.

A gene responsible for increased synthesis of hexaheme nitrite reductase (cytochrome c552) of Escherichia coli K-12 was cloned into pBR322 by the direct immunological screening method using antiserum against the purified enzyme. The cloned gene was mapped at 5 min on the chromosomal linkage map as the dni gene (related to increased synthesis of the dissimilatory nitrite reductase) by conjugation and transduction. The dni gene was subcloned into pUC118 and was shown to be on a 2.6-kilobase-pair PstI-BamHI fragment by immunoblotting analysis of the expressed enzyme. The nucleotide sequence of this fragment was determined. A plausible open-reading frame corresponding to 222 amino acids was detected. Analysis of a dni deletion mutant by immunoblotting demonstrated that this mutant expressed a greatly reduced amount of the nitrite reductase. Thus, the dni gene is suggested to have a positive regulatory action on induced synthesis of the nitrite reductase, and was designated as dniR.     

Molecular cloning and characterization of the alkB gene of Escherichia coli

Summary Using methods of in vitro recombination we constructed hybrid plasmids that can suppress the increased methylmethane sulfonate sensitivity caused by alkB mutation. Since the cloned DNA fragment was mapped at 47 min on the Escherichia coli K12 genetic map, an area where the alkB gene is located, we concluded that the cloned DNA fragment contains the alkB gene itself but not other genes that suppress alkB mutation. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the alkB codes for a polypeptide with a molecular weight of about 27,000. Introduction of a small deletion into the alkB region of the bacterial chromosome resulted in inactivation of both the alkB and ada genes, thereby suggesting that the two genes are adjacent on the E. coli chromosome.Abbreviations Ap ampicillin - Cm chloramphenicol - HPLC high performance liquid chromatography - kb kilobases - kd kilodaltons - MMS methylmethane sulfonate - MNU methylnitrosourea - MNNG N-methyl-N-nitro-N-nitrosoguanidine - Tc tetracycline - SDS sodium dodecyl sulfate     

Characterization of a gene, glnL, the product of which is involved in the regulation of nitrogen utilization in Escherichia coli.

DNA was prepared from a strain of Escherichia coli bearing a mutation which confers the GlnC phenotype (inability to reduce the expression of glnA and other nitrogen-regulated operons in response to ammonia in the growth medium). A fragment of this DNA carrying glnA, the structural gene for glutamine synthetase, was cloned on plasmid pBR322. By using recombination in vitro, we mapped the GlnC mutation to a region between glnA and glnG. This region defines a gene, glnL, which codes for a trans-acting product; the GlnC mutant produces an altered product. The glnL product plays a key role in the communication of information concerning the quality and abundance of the nitrogen source in the growth medium to a destination responsible for the regulation of glnA and other genes for enzymes responsible for nitrogen utilization.