Cloning of the Escherichia coli K-12 hemB gene.

An Escherichia coli heme-requiring, heme-permeable mutant had no detectable 5-aminolevulinate dehydratase or porphobilinogen deaminase activities. The gene which complemented this mutation was cloned to a high-copy-number plasmid, and porphobilinogen deaminase activity was restored to normal levels, but the synthesis of 5-aminolevulinate dehydratase increased 20- to 30-fold. A maxicell procedure confirmed that the gene cloned was hemB.     

The Non-Random Pattern of Insertion of IS2 into the hemB Gene of Escherichia coli

The hemB gene of Escherichia coli has been identified as a hot spot for the insertion of the transposable element IS2. The insertional specificity of IS2 is still unclear. This study reports on the attempt to sequence a statistically significant number of insertions in hemB, in order to determine whether there might be a basis for future studies to determine a molecular basis of IS2 insertional specificity. The results indicate that IS2 inserts in a non-random manner into a 240 bp segment at the 5′ end of the gene (region I). Twenty-one of 24 insertions occurred in region I. Three insertions have been identified in the two middle 250 bp segments of the 975 bp gene, and none in the 3′ terminal segment. A seventeen bp sequence showing 88.2% identity with a segment of IS2, 221 bp from the 3′ terminus has been identified in region I. Four instances of repeated insertion between the same pair of nucleotides have been observed at four different sites.     

rhs gene family of Escherichia coli K-12.

Two additional members of a novel Escherichia coli gene family, the rhs genes, have been cloned and characterized. The structures of these loci, rhsC and rhsD, have been compared with those of rhsA and rhsB. All four loci contain a homologous 3.7-kilobase-pair core. Sequence comparison of the first 300 nucleotides of the cores showed that rhsA, rhsB, and rhsC are closely related, with only 1 to 2% sequence divergence, whereas rhsD is 18% divergent from the others. The beginning of the core coincides with the initiation of an open reading frame that extends beyond the 300 nucleotides compared. Whether a protein product is produced from this open reading frame has not been established. However, nucleotide substitutions which differentiate the cores have highly conservative effects on the predicted protein products; this suggests that products are made from the open reading frame and are under severe selection. The four rhs loci have been placed on both the genetic and restriction maps of E. coli K-12. A fifth rhs locus remains to be characterized. In terms of size, number, and sequence conservation, the rhs genes make up one of the most significant repetitions in E. coli, comparable to the rRNA operons.     

The structure of the Escherichia coli hemB gene

The Escherichia coli hemB gene, which encodes 5-aminolevulinic acid dehydratase, and was cloned into pTZ18U, a multicopy plasmid, was sequenced. The hemB insert was double-digested with restriction enzymes and recloned back into pTZ18U and pTZ19U to allow for sequencing in two directions. In a second procedure, used to fill in gaps and to confirm the sequence derived from the first procedure, the whole insert was cloned into M13 phages. A nested set of deletions was constructed and recloned into M13. Both the double-digested fragments cloned into plasmids pTZ18U and pTZ19U and the overlapping fragments contained in M13 phages were sequenced using the dideoxy procedure with [35S]dATP. Computer software was used to identify coding regions and the correct reading frame. Two promoter regions, two Shine-Dalgarno sequences and two possible start sites were identified. Extensive homologies with yeast (36%), human liver (40%) and rat liver (40%) amino-acid (aa) sequences were observed, especially in the 16-aa Zn-binding region (75%) and the 4 aa surrounding the essential lysine at the active site (100% for rat and human proteins). Computer analysis of promoter strength and two independent analyses of codon usage indicated that the hemB gene is moderately expressed.     

Multiple IS insertion sequences near the replication terminus in Escherichia coli K-12.

In order to assess the feasibility of semi-automatic procedures for large genome sequencing, a fragment of 9.4 kb of Escherichia coli chromosomal DNA isolated at random was sequenced. It was found to map at 30 min on the chromosome map and to harbour two insertion sequences (IS2 and IS30) as well as several putative coding sequences which had no feature in common with known proteins.     

Mapping of IS1 elements flanking the argF gene region on the Escherichia coli K-12 chromosome

Summary Two directly-repeated IS1 elments have been mapped on the Escherichia coli K-12 chromosome at positions 23.2 kb and 34.5 kb counterclockwise of the IS3 element ₃₃ by using F-prime plasmids (including the F lac ^- proAB⁺ plasmid F128) that carry different portions of the bacterial chromosome in the purE to proA region. Mapping was accomplished in part by construction of EcoRI, BamHI, and BglII restriction enzyme cleavage maps. Electron microscope heteroduplex and hybridization studies indicate that the chromosomal region flanked by these IS1 elements is completely homologous to the IS1-argF-IS1 region (Tn2901) on the P1argF5 transducing phage (York and Stodolsky, 1981), which suggests that the argF gene region in the usual E. coli K-12 strains has a transposon-like structure.     

The Escherichia coli K-12 lexA2 gene encodes a hypocleavable repressor.

LexA2 repressor was partially inactivated after mitomycin C or UV light treatment in a recA+ or recA85(Prtc) (protease constitutive) host background. LexA2 protein was cleaved, but the reaction was slower than that observed for LexA+ repressor. lexA2 had a C-to-T transition at nucleotide 461 (Thr-154 to Ile).     

Mutation of the N-acetylmuramyl-L-alanine amidase gene of Escherichia coli K-12.

Mutants of Escherichia coli with very low N-acetylmuramyl-L-alanine amidase activity were isolated. The gene amiA responsible for most of this enzyme activity was mapped at 51 min on the E. coli chromosome, with the most plausible gene order assumed to be amiA pts(H or I) purC. The mutant phenotype was recessive and physiologically undiscernible.     

Cloning and expression of the Escherichia coli K-12 sad gene.

The Escherichia coli K-12 sad gene, which encodes an NAD-dependent succinic semialdehyde dehydrogenase, was cloned into a high-copy-number vector. Minicells carrying a sad+ plasmid produced a 55,000-dalton peptide, the probable sad gene product.     

Penetration of nalidixic acid into Escherichia coli K-12 cells

Transport of nalidixic acid (NAL) into Escherichia coli cells subjected to osmotic shock, permeabilised with toluene or treated with DNP, CCCP or EDTA, was studied. It was found that osmotic shock and protonophores do not inhibit the transport of [3H]NAL, however, the transport of [3H]DAP and [3H]glucose is reduced. EDTA and toluene enhance penetration of [3H]NAL. This effect is, however, abolished in the presence of Mg++ ions. It is suggested that NAL penetrates into the cell by simple or facilitated diffusion and that the outer membrane of E. coli is the penetration barrier for the drug.     

Insertion mutations in the dam gene of Escherichia coli K-12

The dam gene of E. coli can be inactivated by insertion of Tn9 or Mud phage. Strains bearing these mutations are viable indicating that the dam gene product is dispensable.     

Cloning and characterization of Escherichia coli K-12 regulator gene tyrR.

The Escherichia coli K-12 regulator gene tyrR was cloned into the multicopy plasmid pBR322 from a lambda(Tn10)tyrR+ specialized transducing phage. Further subcloning localized the gene within a 2.1-kilobase region. Analysis of plasmid-coded proteins showed that the tyrR gene codes for a 63,000-dalton polypeptide.     

Molecular cloning of the fdhF gene of Escherichia coli K-12

Abstract The fdhF gene of Escherichia coli , coding for at least one component of benzyl viologen-linked formate dehydrogenase (FDH-BV) activity, was isolated on a ColE1- fdhF hybrid plasmid from the Clarke and Carbon colony bank.
Endonuclease restriction maps of this plasmid and its pBR322-subcloned derivative, pLW06, were constructed. Various hybrid plasmids were further obtained by deletion of endonuclease-cleaved fragments from pLW06 DNA. Their complementation pattern was analyzed after introduction into different fdhF mutant strains. The fdhF gene was shown to be located on a 5.5 kb Bam HI- Pvu II-DNA fragment, which restored FDH-BV activity to the wild-type level.     

Cloning of the adenylate cyclase gene of Escherichia coli K-12

The adenylate cyclase gene of Escherichia coli has been cloned on the plasmid vector pBR325. The hybrid plasmid pTH4 obtained has a molecular weight of 6,4 megadalton and represents pBR325 plasmid with the insertion of 2,8 megadalton in the Pst1 site. The cya mutant bacteria carrying pTH4 recover their ability to utilize mannitol, lactose and other carbohydrates as carbon sources, and lose this ability again in the case of rare spontaneous excision of the DNA insert from the Pst1 site. The phenotypical effect of pTH4 in cya mutants can be only seen in the crp+ genome. The strains carrying pTH4 are also characterized by the ability of beta-galactosidase induction under conditions of catabolite repression. Besides, the bacteria containing cya+ allele on the plasmid do not grow on glycerol, which seems to be caused by toxic concentrations of methylglyoxal formed as a result of the increased intracellular level of cyclic adenosine monophosphate.