Isolation and characterization of the Escherichia coli mutH gene product

The Escherichia coli mutH gene product has been isolated in near homogeneous form using an in vitro complementation assay for DNA mismatch correction (Lu, A.-L., Clark, S., and Modrich, P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4639-4643) which is dependent on mutH function. The protein has a subunit Mr of 25,000, and purified preparations contain a Mg2+-dependent endonuclease activity which cleaves 5' to the dG of d(GATC) sequences to generate 5'-phosphoryl and 3'-hydroxyl termini. Symmetrically methylated d(GATC) sites are resistant to the endonuclease, hemimethylated sequences are cleaved on the unmethylated strand, and unmethylated d(GATC) sites are usually subject to scission on only one DNA strand. Although this endonuclease activity is extremely weak (less than 1 scission/h/mutH monomer equivalent) and cleavage at a d(GATC) site does not depend on the presence of a mismatched base pair within the DNA substrate, the activity does not appear to be a contaminant of mutH preparations. d(GATC) endonuclease activity and mutH complementing activity co-purify through multiple column steps without change in relative specific activities, and both activities co-electrophorese under native conditions. These findings suggest that the mutH product functions at the strand discrimination stage of mismatch correction and that this stage of the reaction involves scission of the unmethylated DNA strand.     

Dominant negative mutator mutations in the mutL gene of Escherichia coli.

The mutL gene product is part of the dam-directed mismatch repair system of Escherichia coli but has no known enzymatic function. It forms a complex on heteroduplex DNA with the mismatch recognition MutS protein and with MutH, which has latent endonuclease activity. An N-terminal hexahistidine-tagged MutL was constructed which was active in vivo. As a first stop to determine the functional domains of MutL, we have isolated 72 hydroxylamine-induced plasmid-borne mutations which impart a dominant-negative phenotype to the wild-type strain for increased spontaneous mutagenesis. None of the mutations complement a mutL deletion mutant, indicating that the mutant proteins by themselves are inactive. All the dominant mutations but one could be complemented by the wild-type mutL at about the same gene dosage. DNA sequencing indicated that the mutations affected 22 amino acid residues located between positions 16 and 549 of the 615 amino acid protein. In the N-terminal half of the protein, 12 out of 15 amino acid replacements occur at positions conserved in various eukaryotic MutL homologs. All but one of the sequence changes affecting the C-terminal end of the protein are nonsense mutations.     

Structural and biochemical characterization of the Escherichia coli argE gene product.

The DNA sequence of a 2,100-bp region containing the argE gene from Escherichia coli has been determined. The nucleotide sequence of the ppc-argE intergenic region was also solved and shown to contain six tandemly repeated REP sequences. Moreover, the oxyR gene has been mapped on the E. coli chromosome and shown to flank the arg operon. The codon responsible for the translation start of argE was determined by using site-directed mutants. This gene spans 1,400 bp and encodes a 42,350-Da polypeptide. The argE3 allele and a widely used argE amber gene have also been cloned and sequenced. N-Acetylornithinase, the argE product, has been overproduced and purified to homogeneity. Its main biochemical and catalytic properties are described. Moreover, we demonstrate that the protein is composed of two identical subunits. Finally, the amino acid sequence of N-acetylornithinase is shown to display a high degree of identity with those of the succinyldiaminopimelate desuccinylase from E. coli and carboxypeptidase G2 from a Pseudomonas sp. It is proposed that this carboxypeptidase might be responsible for the acetylornithinase-related activity found in the Pseudomonas sp.     

Specificity of Escherichia coli mutD and mutL mutator strains

The products of the mutD and mutL genes of Escherichia coli are involved in proofreading by DNA polymerase III and DNA adenine MTase (Dam)-dependent mismatch repair, respectively. We have used the plasmid-borne bacteriophage P22 mnt gene as a target to determine the types of mutations produced in mutL25 and mutD5 strains. Of 60 mutations identified from mutL25 cells, 52 were transition mutations and of these the AT----GC subset predominated (40 out of 52). The majority of AT----GC mutations were found at the same three sites (hotspots). In contrast, transversion mutations (47 out of 76) were found about twice as frequently as transitions (28 out of 76) from mutD5 bacteria. Two hotspots were identified but at different sites than those in the mutL25 cells. These results suggest that the proofreading function of DNA polymerase III primarily repairs potential transversion mutations while Dam-dependent mismatch repair rectifies potential transition mutations.     

Isolation and characterization of the Escherichia coli htrD gene, whose product is required for growth at high temperatures.

Those genes in Escherichia coli defined by mutations which result in an inability to grow at high temperatures are designated htr, indicating a high temperature requirement. A new htr mutant of E. coli was isolated and characterized and is designated htrD. The htrD gene has been mapped to 19.3 min on the E. coli chromosome. Insertional inactivation of htrD with a mini-Tn10 element resulted in a pleiotropic phenotype characterized by a severe inhibition of growth at 42 degrees C and decreased survival at 50 degrees C in rich media. Furthermore, htrD cells were sensitive to H2O2. Growth rate analysis revealed that htrD cells grow very slowly in minimal media supplemented with amino acids. This inhibitory effect has been traced to the presence of cysteine in the growth medium. Further studies indicated that the rate of cysteine transport is higher in htrD cells relative to the wild type. All of these results, taken together, indicate that the htrD gene product may be required for proper regulation of intracellular cysteine levels and that an increased rate of cysteine transport greatly affects the growth characteristics of E. coli.     

Hyperexpression of a Bacillus circulans xylanase gene in Escherichia coli and characterization of the gene product

A 4.0-kilobase (kb) fragment of Bacillus circulans genomic DNA inserted into pUC19 and encoding endoxylanase activity was subjected to a series of subclonings. A 1.0-kb HindIII-HincII subfragment was found to code for xylanase activity. Maximum expression levels were observed with a subclone that contained an additional 0.3-kb sequence upstream from the coding region. Enhancer sequences in the upstream region are thought to be responsible for these high expression levels. Southern hybridization analyses revealed that the cloned gene hybridized with genomic DNA from Bacillus subtilis and Bacillus polymyxa. Xylanase activity expressed by Escherichia coli harboring the cloned gene was located primarily in the intracellular fraction. Levels of up to 7 U/ml or 35 mg/liter were obtained. The protein product was purified by ion exchange and gel permeation chromatography. The xylanase had a molecular weight of 20,500 and an isoelectric point of 9.0.     

Purification and characterization of the Escherichia coli Kl neuB gene product N-acetylneuraminic acid synthetase

Escherichia coli K1 produces a capsular polysaccharide of     

Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product.

We attempted to clone an inosine kinase gene of Escherichia coli. A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning. A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine. The fragment was sequenced, and one protein of 434 amino acids long was found. This protein was overexpressed. The overexpressed protein was purified and characterized. The enzyme had both inosine and guanosine kinase activity. The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively. The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively. This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate. These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine. The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W. Harlow, P. Nygaard, and B. Hove-Jensen, J. Bacteriol. 177:2236-2240, 1995).     

Hyperexpression of a Bacillus circulans xylanase gene in Escherichia coli and characterization of the gene product.

A 4.0-kilobase (kb) fragment of Bacillus circulans genomic DNA inserted into pUC19 and encoding endoxylanase activity was subjected to a series of subclonings. A 1.0-kb HindIII-HincII subfragment was found to code for xylanase activity. Maximum expression levels were observed with a subclone that contained an additional 0.3-kb sequence upstream from the coding region. Enhancer sequences in the upstream region are thought to be responsible for these high expression levels. Southern hybridization analyses revealed that the cloned gene hybridized with genomic DNA from Bacillus subtilis and Bacillus polymyxa. Xylanase activity expressed by Escherichia coli harboring the cloned gene was located primarily in the intracellular fraction. Levels of up to 7 U/ml or 35 mg/liter were obtained. The protein product was purified by ion exchange and gel permeation chromatography. The xylanase had a molecular weight of 20,500 and an isoelectric point of 9.0.     

Isolation and characterization of the rabbit prt gene product

The product of the rabbit prt gene (PRT), a gene linked to the immunoglobulin κ-light chain gene ab, was purified from rabbit serum by precipitation with ammonium sulfate and by chromotography on DEAE-Sephadex and Sephacryl S300. Analysis of PRT indicated that it was associated rabbit hemopexin; the molecular weight of PRT (i.e., 68,000), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar to the reported molecular weight of rabbit hemopexin; the PRT phenotypes correlated with the phenotypes of a hematin binding protein; PRT itself bound hematin; and the amino acid composition of PRT was similar to the amino acid composition of rabbit hemopexin. The prt gene, however, need not be the structural gene for hemopexin; it may encode a glycosyl transferase responsible in part for the carbohydrate associated with the protein.     

Isolation and characterization of an aldehyde dehydrogenase encoded by the aldB gene of Escherichia coli

An aldehyde dehydrogenase was detected in crude cell extracts of Escherichia coli DH5alpha. Growth studies indicated that the aldehyde dehydrogenase activity was growth phase dependent and increased in cells grown with ethanol. The N-terminal amino acid sequence of the purified enzyme identified the latter as an aldehyde dehydrogenase encoded by aldB, which was thought to play a role in the removal of aldehydes and alcohols in cells that were under stress. The purified enzyme showed an estimated molecular mass of 220 +/- 8 kDa, consisting of four identical subunits, and preferred to use NADP and acetaldehyde. MgCl2 increased the activity of the NADP-dependent enzyme with various substrates. A comparison of the effect of Mg2+ ions on the bacterial enzyme with the effect of Mg2+ ions on human liver mitochondrial aldehyde dehydrogenase revealed that the bacterial enzyme shared kinetic properties with the mammalian enzyme. An R197E mutant of the bacterial enzyme appeared to retain very little NADP-dependent activity on acetaldehyde.     

Characterization of the gltF gene product of Escherichia coli

The glt operon of Escherichia coli comprises the structural genes for the glutamate synthase subunits (gltB and gltD) and gltF, whose product was previously suggested to have regulatory functions. The A/T-rich region between gltD and gltF contains a weak promoter and a translation initiation site for gltF. The GltF protein is preceded by a signal peptide, which is cleaved off during export into the periplasmic space. A gltF::Km(R) insertion mutant was constructed and shown here to have no detectable phenotype with respect to amino acid utilization or ammonium transport. Thus, GltF is apparently not involved in regulation of nitrogen catabolism.     

Mutation rate is reduced by increased dosage of mutL gene in Escherichia coli K-12

A variable but substantial proportion of wild Escherichia coli isolates present consistently lower mutation frequencies than that found in the ensemble of strains. The genetic mechanisms responsible for the hypo-mutation phenotype are much less known than those involved in hyper-mutation. Changes in E. coli mutation frequencies derived from the gene-copy effect of mutS, mutL, mutH, uvrD, mutT, mutY, mutM, mutA, dnaE, dnaQ, and rpoS are explored. When present in a very high copy number ( approximately 300 copies cell(-1)), mutL, mutH, and mutA gene copies yielded >/=twofold decrease in mutation rates determined by Luria-Delbrück fluctuation tests. Nevertheless, when the copy number was not such high ( approximately 15 copies cell(-1)), only mutL results in a consistent twofold decrease in the mutation rate. This reduction seems to be independent from the RecA background, phase of growth, or from the presence of proficient MutS. An increase in mutL gene copies was also able to partially compensate the hypermutator phenotype of a mutS-defective E. coli derivative.     

Isolation and characterization of pyridoxine auxotrophs of Escherichia coli

Dempsey, Walter B. (University of Florida, Gainesville), and P. F. Pachler. Isolation and characterization of pyridoxine auxotrophs of Escherichia coli. J. Bacteriol. 91:642-645. 1966.-Pyridoxine auxotrophs of Escherichia coli B have been consistently produced by a modified penicillin enrichment method. This modified penicillin technique included a 6-hr growth period in the absence of any pyridoxine followed by a 2-hr treatment with penicillin at 1,000 units per ml. Penicillin was removed from these cultures by membrane filtration and the process was repeated. A minimum of three cycles was found necessary to isolate auxotrophs. Different phenotypes within the group of pyridoxine auxotrophs were distinguished by their responses to feeding with the various forms of pyridoxine, as well as by cross-feeding tests. Two auxotrophs were also differentiated by their frequency of reversion to prototrophy. Cross-feeding tests indicated that seven of the phenotypes fed in a linear sequence. These phenotypes had a very low frequency of reversion. The auxotrophs with a high frequency of reversion cross-fed in a linear sequence and fed the first five of the other seven auxotrophs. One of the auxotrophs isolated was a pyridoxal auxotroph.     

Isolation and characterization of Escherichia coli birA intragenic suppressors

Summary The biotin (bio) operon in Escherichia coli is negatively regulated by BirA, a bifunctional protein with both repressor and biotin-activating functions. Twenty-five heatresistant revertants of three temperature-sensitive birA alleles (birA 85, bir A 104 and bir A 879) were isolated and categorized into five growth and six repression classes. The revertants appear to increase biotin activation by raising the specific activity of BirA and/or, increasing the number of enzyme molecules. The 19 bir A 85 revertants displayed a broad range of activity for both enzyme and repressor functions, and may represent intragenic second-site suppressor mutations. The bir A 85 revertants included a novel class of bio superrepressor mutations. Repressor titration experiments suggested that many of the bir A 85 revertants increase BirA concentrations above wild-type levels because the repressors were not competed from the chromosomal bio operator by multicopy bio operator plasmids. The majority of the bir A 104 revertants resulted in both wild-type repressor and enzyme activity; they are possibly true revertants in which the amino acid residue altered by the bir A 104 mutation has been substituted by the wild-type or a chemically similar amino acid.     

Isolation and characterization of nucleoid proteins from Escherichia coli

Summary Of the molecular species of proteins associated with the nucleoids of Escherichia coli cells, those with relatively high affinity to bind to DNA were isolated and characterized. Seven classes of nucleoid proteins with molecular weights of 9,000, 17,000 (two molecular species), 22,000, 24,000, 27,000 and 28,000 were isolated at more than 90% purity or were partially purified. On the basis of its amino acid composition and other chemical properties, the 9,000 dalton protein was identified as HLP II (or HU protein or BH2) (Pettijohn 1982: Rouvière-Yaniv and Gros 1975; Varshavsky et al. 1978). The 17 K protein consisted of two molecular species and one of these, 17 K (a) protein, seemed to be identical with HLPI (or protein 1 or BH1) reported previously (Pettijohn 1982; Varshavsky et al. 1977; Varshavsky et al. 1978). The 26 K protein was identical to the 22 K protein (Kishi et al. 1982). The 27 K protein showed immunological cross-reactivity with the antibody for histone H2A and was thus identified as the H protein reported previously (Hübscher et al. 1980). Two basic proteins, 9 K and 17 K(a), showed relatively high binding affinities to DNA, while the 28 K protein showed moderate binding affinity. The biological significance of these nucleoid proteins, which constitute a family of proteins participating in formation of the nucleoid structure, is discussed.