A heme-deficient strain of Escherichia coli has a three-base pair deletion in a "hotspot" in hemA

The key regulatory step in heme biosynthesis in Escherichia coli is at the level of glutamyl-tRNA reductase (GTR), an enzyme which is encoded by hemA. A strain, HU227, with a spontaneous in-frame mutation in hemA has no GTR activity. The mutation is shown to be a three-base deletion at a "hotspot" in the gene. The amino acid sequence in this region is highly conserved.     

Escherichia coli RecBC pseudorevertants lacking chi recombinational hotspot activity

Pseudorevertants of an Escherichia coli exonuclease V (RecBC enzyme)-negative mutant have been isolated after ethyl methane sulfonate mutagenesis of a recC73 (presumed missense) mutant. The remedial mutations in each of the four pseudorevertants studied in detail map and complement as recC mutations. By several criteria, such as recombination proficiency, support of phage growth, RecBC nuclease activity, and cell viability, the pseudorevertants appear to have regained partially or completely various aspects of RecBC activity. However, chi recombinational hotspots, which stimulate exclusively the RecBC pathway of recombination, have no detectable activity in lambda vegetative crosses in the pseudorevertants. The properties of these mutants, in which the RecBC pathway of recombination is active yet in which chi is not active, are consistent with the hypothesis that wild-type RecBC enzyme directly interacts with chi sites; alternatively, the mutants may block or bypass the productive interaction of another recombinational enzyme with chi.     

A novel outer-membrane-associated protease in Escherichia coli.

Human gamma interferon produced by recombinant Escherichia coli was degraded by endogenous protease after cell disruption. Specific cleavages took place at the center of two pairs of basic amino acids (Lys-131-Arg-132 and Arg-142-Arg-143) in the C-terminal region, giving rise to products with molecular weights of 17,500 and 16,000. The proteolytic activity was associated with the outer membrane of E. coli. It was insensitive to the protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, tosyl-L-lysine chloro-methyl ketone, EDTA, and p-chloromercuribenzoate. Benzamidine and the bivalent cations Zn2+ and Cu2+ inhibited the activity. Dynorphin A(1-13) (Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys) was a good substrate and was preferentially cleaved at the center of Arg-6-Arg-7. Neither the amino nor carboxyl sides of Arg-9 and Lys-11 were digested. These results indicate that the protease specifically cleaves the peptide bond between consecutive basic residues and therefore is different from the known membrane enzymes, proteases IV, V, and VI. We have designated this new enzyme protease VII.     

Metagenomic DNA fragments that affect Escherichia coli mutational pathways

A multicopy cloning approach was used to search for metagenomic DNA fragments that affect Escherichia coli mutational pathways. Soil metagenomic expression libraries were constructed with DNA samples prepared directly from soil samples collected from the UCLA Botanical Garden. Using frameshift mutator screening, we obtained a total of 26 unique metagenomic fragments that stimulate frameshift rates in an E. coli wild-type host. Mutational enhancer strains such as an ndk-deficient strain and a temperature sensitive mutS strain (mutS60) were used to further verify the mutator phenotype. We found that the presence of multiple copies of certain types of metagenomic DNA sequence repeats cause general genome instability in the wild-type E. coli host and the effect can be suppressed by overproducing a DNA mismatch component MutL. In addition, we identified nine metagenomic mutator genes (designated as smu genes) that encode proteins that have not been linked to mutator phenotypes prior to this study including a putative RNA methyltransferase Smu10A. The strain overproducing Smu10A displays one prominent base substitution hotspot in the rpoB gene, which coincides with the base substitution hotspot we have observed in cells that are partially deficient in the proofreading function carried out by the DNA polymerase III epsilon subunit. Based on the structural conservation of DNA replication/recombination/repair machineries among microorganisms, this approach would allow us to both identify new mutational pathways in E. coli and to find genes involved in DNA replication, recombination or DNA repair from vast unculturable microbes.     

A novel,cupin-type phosphoglucose isomerase in Escherichia coli

BackgroundEscherichia coli cells contain a homolog of presumed 5-keto-4-deoxyuronate isomerase (KduI) from pectin-degrading soil bacteria, but the catalytic activity of the E. coli protein (o-KduI) was never demonstrated.MethodsThe known three-dimensional structure of E. coli o-KduI was compared with the available structures of sugar-converting enzymes. Based on the results of this analysis, sugar isomerization activity of recombinant o-KduI was tested against a panel of D-sugars and their derivatives.ResultsThe three-dimensional structure of o-KduI exhibits a close similarity with Pyrococcus furiosus cupin-type phosphoglucose isomerase. In accordance with this similarity, o-KduI was found to catalyze interconversion of glucose-6-phosphate and fructose-6-phosphate and, less efficiently, conversion of glucuronate to fructuronate. o-KduI was hexameric in crystals but represented a mixture of inactive hexamers and active dimers in solution and contained a tightly bound Zn²⁺ ion. Dilution, substrate binding and Zn²⁺ removal shifted the hexamer ⇆ dimer equilibrium to the dimers.ConclusionsOur findings identify o-KduI as a novel phosphosugar isomerase in E. coli, whose activity may be regulated by changes in oligomeric structure.General SignificanceMore than 5700 protein sequences are annotated as KduI, but their enzymatic activity has not been directly demonstrated. E. coli o-KduI is the first characterized member of this group, and its enzymatic activity was found to be different from the predicted activity.     

Common deletion of SMAD4 in juvenile polyposis is a mutational hotspot

Juvenile polyposis (JP) is an autosomal dominant syndrome in which affected patients develop upper- and/or lower-gastrointestinal (GI) polyps. A subset of families with JP have germline mutations in the SMAD4 (MADH4) gene and are at increased risk of GI cancers. To date, six families with JP have been described as having the same SMAD4 deletion (1244-1247delAGAC). The objective of the present study is to determine whether this deletion is a common ancestral mutation or a mutational hotspot. DNA from members of four families with JP, from Iowa, Mississippi, Texas, and Finland, that had this 4-bp deletion was used to genotype 15 simple tandem repeat polymorphism (STRP) markers flanking the SMAD4 gene, including 2 new STRPs within 6.3 and 70.9 kb of the deletion. Haplotypes cosegregating with JP in each family were constructed, and the distances of the closest markers were determined from the draft sequence of the human genome. No common haplotype was observed in these four families with JP. A 14-bp region containing the deletion had four direct repeats and one inverted repeat. Because no common ancestor was suggested by haplotype analysis and the sequence flanking the deletion contains repeats frequently associated with microdeletions, this common SMAD4 deletion in JP most likely represents a mutational hotspot.     

(6R)-5,6,7,8-tetrahydro-L-monapterin from Escherichia coli, a novel natural unconjugated tetrahydropterin

The structure of the major tetrahydropterin in Escherichia coli was determined as (6R)-5,6,7,8-tetrahydro-L-monapterin, i. e. (6R)-2-amino-5,6,7,8-tetrahydro-6-[(1S,2S)-1,2,3-trihydroxypropyl]pteridin-4(3H)-one. Although the stereochemical structure of the trihydroxypropyl side chain has been determined previously by fluorescence detected circular dichroism analysis on its aromatic derivative, the most important configuration at C(6) has not been clarified. The major difficulties for the determination of the chirality were instability toward air oxidation and very low concentration of the tetrahydropterin derivative. In the present study, the C(6)-configuration was determined as R by comparing its stable hexaacetyl derivative with authentic (6R)- and (6S)-hexaacetyl-5,6,7,8-tetrahydro-L-monapterins by high performance liquid chromatography (HPLC) and HPLC-mass spectrometry (LC-MS). (6R)-5,6,7,8-Tetrahydro-L-monapterin is a new unconjugated tetrahydropterin from natural sources.     

A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi

In wild-type Escherichia coli, recognition of the recombination hotspot, chi (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombination. However, in the recC* class of RecBCD mutants, stimulation of recombination by the canonical chi sequence is not detectable, but the levels of homologous recombination are nearly wild-type. In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of chi, termed chi* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi. Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of chi-recognition. In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the recombination function of RecBCD enzyme.     

A novel replicon occurring naturally in Escherichia coli is a phage-plasmid hybrid.

A novel DNA replicon in Escherichia coli was identified. It is the smallest natural isolate (1282 bp) found so far. In the presence of phage M13 it grows as a filamentous single-stranded DNA phage. Contrary to previously identified mini-phages this replicon displays sequence homology only to parts of the M13 viral and complementary strand origin. In the absence of M13 this DNA replicates autonomously. The only gene (arp) of the replicon encodes a 32-kd protein, which is essential for autonomous replication. The host rep gene required for replication of single-stranded DNA phages is dispensable. Distinct replication mechanisms are thus involved during growth as defective phage or as autonomous plasmid.     

A novel endoribonuclease, RNase LS, in Escherichia coli

The dmd gene of bacteriophage T4 is required for the stability of late-gene mRNAs. When this gene is mutated, late genes are globally silenced because of rapid degradation of their mRNAs. Our previous work suggested that a novel Escherichia coli endonuclease, RNase LS, is responsible for the rapid degradation of mRNAs. In this study, we demonstrated that rnlA (formerly yfjN) is essential for RNase LS activity both in vivo and in vitro. In addition, we investigated a role of RNase LS in the RNA metabolism of E. coli cells under vegetative growth conditions. A mutation in rnlA reduced the decay rate of many E. coli mRNAs, although there are differences in the mutational effects on the stabilization of different mRNAs. In addition, we found that a 307-nucleotide fragment with an internal sequence of 23S rRNA accumulated to a high level in rnlA mutant cells. These results strongly suggest that RNase LS plays a role in the RNA metabolism of E. coli as well as phage T4.     

A novel ATP-driven glucose transport system in Escherichia coli.

In Escherichia coli wild-type cells and in ATPase-deficient cells (unc mutants), glucose was found to be transported mainly by an ATP-driven system. The evidence is based on experiments involving interference at different sites of energy metabolism with the use of uncouplers, arsenate, and starved cells. Furthermore, addition of succinate to starved cells increased glucose uptake only in the wild-type cells, where ATP could be regenerated. Glucose transport was also ATP-dependent in cells deficient in methyl-beta-galactoside transport (a system that carries glucose specificity). It was found to be shock-sensitive in all strains tested. The NOVEL ATP-driven glucose transport is a high-affinity (Km 3-10 microM) and high-capacity (V 240-330 Mmol . min-1 . mg cell protein-1) uptake system.     

A novel family of Escherichia coli toxin-antitoxin gene pairs

Bacterial toxin-antitoxin protein pairs (TA pairs) encode a toxin protein, which poisons cells by binding and inhibiting an essential enzyme, and an antitoxin protein, which binds the toxin and restores viability. We took an approach that did not rely on sequence homology to search for unidentified TA pairs in the genome of Escherichia coli K-12. Of 32 candidate genes tested, ectopic expression of 6 caused growth inhibition. In this report, we focus on the initial characterization of yeeV, ykfI, and ypjF, a novel family of toxin proteins. Coexpression of the gene upstream of each toxin restored the growth rate to that of the uninduced strain. Unexpectedly, we could not detect in vivo protein-protein interactions between the new toxin and antitoxin pairs. Instead, the antitoxins appeared to function by causing a large reduction in the level of cellular toxin protein.     

Esre: a novel essential non-coding RNA in Escherichia coli

YigP gene (GeneID: 948915) locates between ubiquinone biosynthetic genes ubiE and ubiB in Escherichia coli. GeneBank annotates yigP as a putative protein-coding gene. In this study, we found a new essential sRNA gene, esre, locates within the region of yigP. The E. coli strain with inactive esre must rely on a complementary plasmid to survive. Moreover, RACE experiments showed esre encodes an RNA molecule of 252 nt. Further experiments revealed esre gene is immune to frame shift mutations and the function of esre depends mostly on the RNA secondary structure, which are typical traits of sRNA. Since it is difficult to predict the target of an essential sRNA, more research is needed to reveal the function and mechanism of esre.     

Escherichia coli is naturally transformable in a novel transformation system

A novel transformation system, in which neither a nonphysiological concentration of Ca2+ and temperature shifts nor electronic shocks were required, was developed to determine whether Escherichia coli is naturally transformable. In the new protocol, E. coli was cultured normally to the stationary phase and then cultured statically at 37 degrees C in Luria-Bertani broth. After static culture, transformation occurred in bacteria spread on Luria-Bertani plates. The protein synthesis inhibitor chloramphenicol inhibited this transformation process. The need for protein synthesis in plated bacteria suggests that the transformation of E. coli in this new system is regulated physiologically.     

A novel inhibitor protein of N-myristoyltransferase from Escherichia coli

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal of the glycine residue of various eukaryotic and viral proteins of diverse functions. Earlier, we have demonstrated that NMT activity is elevated in colon and gall bladder cancer. Attenuation of NMT activity may prove a novel therapeutic protocol for cancer. We report here a novel inhibitor protein of NMT being expressed in Escherichia coli cells containing the human NMT gene on increasing the incubation period from 5 to 24h. The inhibitor protein was purified by SP-Sepharose column chromatography, heat treatment, ammonium sulfate precipitation, and Superose 12 HR/30 FPLC column chromatography. The inhibitor protein had an apparent molecular mass of 10kDa by gel filtration. It inhibited human NMT in a concentration-dependent manner with 50% inhibition at 640+/-4.68nM. The inhibitor protein showed no direct interaction with myristoyl-CoA and demonstrated no demyristoylase or protease activity. Therefore, we conclude that the inhibitor protein acts directly on NMT.