Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

Restriction fragments hybridizing to phage HP1c1 DNA were identified in digests of DNA from lysogenic strains of Haemophilus influenzae. The results showed that the cohesive ends of the mature phage DNA were joined in lysogens and that the phage genome was covalently linked to the host DNA, indicating that lysogeny involves recombination between specific sites on the phage and host chromosomes. The site on the phage chromosome at which this recombination occurred was between 110 and 750 base pairs of the left end on the mature phage genome.     

Molecular characterization of polyphosphate kinase (ppk) gene from Serratia marcescens

To understand the mechanism of phosphate accumulation, a gene encoding polyphosphate kinase (PPK) was cloned from the genomic library of Serratia marcescens by Southern hybridization. From the nucleotide sequence of a 4 kb DNA fragment, an open reading frame of 2063 nucleotides was identified encoding a protein of 686 amino acids with molecular mass of 70 kDa. The potential CRP binding site and pho box sequence were found upstream of the putative promoter in the regulatory region. The expression of PPK resulted in the formation of inclusion bodies and the product was active at low temperature. The E. coli strain harboring plasmid pSPK5 with ppk gene increased enzyme activity of polyphosphate kinase, resulting in increased accumulation of polyphosphate in E. coli.     

Overexpression of polyphosphate kinase gene (ppk) increases bioinsecticide production by Bacillus thuringiensis

Polyphosphate (polyP), synthesized by polyP kinase (PPK) using the terminal phosphate of ATP as substrate, performs important functions in every living cell. The present work reports on the relationship between polyP metabolism and bioinsecticide production in Bacillus thuringiensis subsp. israelensis (Bti). The ppk gene of Bti was cloned into vector pHT315 and the effect of its overexpression on endotoxin production was determined. Endotoxin production by the recombinant strain was found to be consistently higher than that by the wild type strain and the strain that carried the empty plasmid. The toxicity of the recombinant mutant strain (LC₅₀ 5.8 ± 0.6 ng ml^?1) against late 2nd instar Culex quinquefasciatus was about 7.7 times higher than that of Bti (LC₅₀ 44.9 ± 7 ng ml^?1). To our knowledge this is the first reported study which relates polyP metabolism with bioinsecticide biosynthesis.     

Crystallization and characterization of polyphosphate kinase from Escherichia coli

Linear polyphosphate chains have been found to play a key role in bacterial responses to stresses and nutritional depletion, and are necessary for host infection of various pathogens. Polyphosphate kinase (PPK) is a critical enzyme responsible for polyphosphate synthesis in bacteria. PPK knockout mutations in several Gram-negative pathogens identify PPK as an ideal drug target for the development of a new class of antibacterial drugs. To reveal the catalytic mechanism and provide a structural basis for drug discovery, we have purified and crystallized full-length Escherichia coli PPK and its complex with AMP-PNP. The crystals diffract to a resolution of 2.5A and belong to the space group P4(2)2(1)2 with unit-cell parameters a=152.0, b=152.0, and c=150.0 A. Crystal structure of PPK is being determined by the Se-Met MAD experiment.     

Escherichia coli glutaminyl-tRNA synthetase. I. Isolation and DNA sequence of the glnS gene

We have isolated a lambda-transducing phage carrying the gene (glnS) for Escherichia coli glutaminyl-tRNA synthetase. The location of the glnS gene within the 13.5-kilobase E. coli DNA transducing fragment was determined by genetic means. The glnS gene was recloned into plasmid pBR322 and its nucleotide sequence was established. The DNA sequence translates to a protein of 550 amino acids.     

Nucleotide sequence of the gene ompA coding the outer membrane protein II of Escherichia coli K-12.

A nucleotide sequence of 2271 basepairs has been determined from cloned E. coli DNA which contains ompA. Withing that sequence, starting at nucleotide 1037, an open translational reading frame encodes a protein of 367 amino acids which starting with amino acid 22 agrees with the primary structure of protein II. The preceeding 21 amino acids constitute a typical signal sequence. There is a non-translated region of 360 nucleotides in front of the translational start. The insertion point of an IS1 element 110 nucleotides upstream from the start codon and an amber codon at the position of amino acid residue 28 have been localized in the DNA from two ompA mutants.     

Chromosomal location of the structural gene for glycerol kinase in Escherichia coli

Cozzarelli, N. R. (Harvard Medical School, Boston, Mass.), and E. C. C. Lin. Chromosomal location of the structural gene for glycerol kinase in Escherichia coli. J. Bacteriol. 91:1763-1766. 1966.-A glycerol kinase mutant site has been mapped by transduction and sexual conjugation. Three-factor crosses with the two procedures yielded the following gene order: arginine-1-methionine-1-glycerol kinase-isoleucine, valine-16. An additional 13 independent glycerol kinase mutant sites mapped in the same region. Since some of the mutants were able to produce a protein serologically indistinguishable from the wild-type enzyme, it is concluded that the region mapped represents the structural gene for the kinase.     

Ribosomal protein gene sequence changes in erythromycin-resistant mutants of Escherichia coli.

The genes for ribosomal proteins L4 and L22 from two erythromycin-resistant mutants of Escherichia coli have been isolated and sequenced. In the L4 mutant, an A-to-G transition in codon 63 predicted a Lys-to-Glu change in the protein. In the L22 strain, a 9-bp deletion removed codons 82 to 84, eliminating the sequence Met-Lys-Arg from the protein. Consistent with these DNA changes, in comparison with wild-type proteins, both mutant proteins had reduced first-dimension mobilities in two-dimensional polyacrylamide gels. Complementation of each mutation by a wild-type gene on a plasmid vector resulted in increased erythromycin sensitivity in the partial-diploid strains. The fraction of ribosomes containing the mutant form of the protein was increased by growth in the presence of erythromycin. Erythromycin binding was increased by the fraction of wild-type protein present in the ribosome population. The strain with the L4 mutation was found to be cold sensitive for growth at 20 degrees C, and 50S-subunit assembly was impaired at this temperature. The mutated sequences are highly conserved in the corresponding proteins from a number of species. The results indicate the participation of these proteins in the interaction of erythromycin with the ribosome.     

Nucleotide sequence of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.

The nucleotide sequence of a 2220-base-pair fragment containing the btuB gene of Escherichia coli was determined. There was a single open reading frame which was translated into a 614-amino-acid polypeptide, the first 20 amino acids of which comprised a typical leader sequence. The putative mature or processed form had a molecular weight (66,400) and a composition in close agreement with that determined for the purified receptor. The distribution of amino acids in the receptor protein was similar to that of other outer membrane proteins, showing a fairly even distribution of charged residues and the absence of extensive hydrophobic stretches. The btuB451 mutation appears to alter the receptor to eliminate its ability to function in vitamin B12 uptake without affecting its ligand binding properties. The sequence of the DNA from this mutant was determined and revealed a leucine-to-proline (C-to-T transition) change in the eighth amino acid of the mature form.     

Nucleotide sequence of the Escherichia coli mutH gene.

The complete nucleotide sequence of mutH gene from E. coli has been determined. Based on the deduced amino acid sequence, the MutH protein has a molecular weight of 25.4 kdaltons in agreement with the previous estimates based on SDS-polyacrylamide gel electrophoresis of the purified protein. Deletion analysis of the DNA sequences upstream of mutH has identified the promoter region for this gene. Two independently isolated temperature sensitive alleles of the mutH gene have also been sequenced. One mutation results in an amino acid change at position 27 (thr to leu) while the other occurs at position 156 (asp to asn).     

Cloning and location of the dgsA gene of Escherichia coli.

The dgsA locus of Escherichia coli was isolated on plasmids obtained from the library of L. Clarke and J. Carbon (Cell 9:91-99, 1976). Restriction fragment analysis and further subcloning demonstrated that the gene is located at kilobase 425 on the Bouché physical map of the terminus region (J. P. Bouché, J. Mol. Biol., 154:1-20, 1982). This corresponds to 35.2 min on the Bachmann genetic map (B. J. Bachmann, Microbiol. Rev. 47:180-230, 1983).     

Genetic map location of the Escherichia coli dnaG gene.

The dnaG locus of Escherichia coli K-12 has been mapped at about 60 min on the genetic map by three-factor crosses using P1 transduction. In crosses selecting for dnaG+, the cotransduction frequency with the tolC marker is 15% and that with the uxaC marker is 49%. The gene order is tolC dnaG uxaC.     

Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12

Summary In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of M_r 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.     

The nucleotide sequence of the Escherichia coli rts gene

The nucleotide sequence of rts, an essential Escherichia coli gene, has been determined. Transformation of an rts mutant with the plasmid, pJAF1, containing the rts gene resulted in rescue of the defect. The transformation experiments indicate that the rts gene is distinct from the flanking birA, tRNA and tufB genes.     

DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.

The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.