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  E.coli news vol. 10  2005.4.13
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This is a digest of daily PubMed searching of E.coli new finding.

***this year finding***

 >>>ybhE, 6-phosphogluconolactonase<<<
FEMS Microbiol Lett. 2005 Mar 15;244(2):275-80.
Escherichia coli ORF ybhE is pgl gene encoding 6-phosphogluconolactonase (EC 3.1.1.31) that has no homology with known 6PGLs from other organisms.
Zimenkov D, Gulevich A, Skorokhodova A, Biriukova I, Kozlov Y, Mashko S.
Ajinomoto-Genetika Research Institute, 1st Dorozhny proezd, Moscow 117545, Russian Federation.

The pentose-phosphate pathway (PPP) is an important part of central metabolism in many organisms. A pgl(-) mutation that decreases the efficiency of the second stage of PPP has been described and mapped at approx. 17.2 min of the Escherichia coli chromosome more than 30 years ago. Although it has recently been shown that deletion of ORF ybhE leads to earlier detected Pgl(-) phenotype for E. coli mutant strain, 6-phosphogluconolactonase from this organism has not been characterized. In the present, independent investigation we show that the Pgl(-) phenotype of DeltaybhE MG1655 could be complemented by insertion of the well-characterized pgl gene from Pseudomonas putida whose protein product has no visible homology with E. coli YbhE. Moreover, a final confirmation that ybhE actually encodes 6PGL in E. coli was obtained through overexpression of the cloned gene, purification of the protein product, followed by direct determination of its enzymatic activity in vitro.

PMID: 15766779

 >>>ryhB block degradation of mRNA of sodB<<<

Nucleic Acids Res. 2005 Mar 21;33(5):1678-89.
Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB.
Afonyushkin T, Vecerek B, Moll I, Blasi U, Kaberdin VR.
Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, University Departments at the Vienna Biocenter Dr. Bohrgasse 9/4, A-1030 Vienna, Austria.

Previous work has demonstrated that iron-dependent variations in the steady-state concentration and translatability of sodB mRNA are modulated by the small regulatory RNA RyhB, the RNA chaperone Hfq and RNase E. In agreement with the proposed role of RNase E, we found that the decay of sodB mRNA is retarded upon inactivation of RNase E in vivo, and that the enzyme cleaves within the sodB 5'-untranslated region (5'-UTR) in vitro, thereby removing the 5' stem-loop structure that facilitates Hfq and ribosome binding. Moreover, RNase E cleavage can also occur at a cryptic site that becomes available upon sodB 5'-UTR/RyhB base pairing. We show that while playing an important role in facilitating the interaction of RyhB with sodB mRNA, Hfq is not tightly retained by the RyhB-sodB mRNA complex and can be released from it through interaction with other RNAs added in trans. Unlike turnover of sodB mRNA, RyhB decay in vivo is mainly dependent on RNase III, and its cleavage b y RNase III in vitro is facilitated upon base pairing with the sodB 5'-UTR. These data are discussed in terms of a model, which accounts for the observed roles of RNase E and RNase III in sodB mRNA turnover.

PMID: 15781494

*comment: This process may be functional analog of siRNA/miRNA-AGO complex in eukaryotes cell.

 >>>YgiN, quinol monooxygenase/mdaB, menadione reductase<<<
J Biol Chem. 2005 Mar 4;280(9):8358-63.
Structural and biochemical evidence for an enzymatic quinone redox cycle in Escherichia coli: identification of a novel quinol monooxygenase.
Adams MA, Jia Z.
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.

Naturally synthesized quinones perform a variety of important cellular functions. Escherichia coli produce both ubiquinone and menaquinone, which are involved in electron transport. However, semiquinone intermediates produced during the one-electron reduction of these compounds, as well as through auto-oxidation of the hydroxyquinone product, generate reactive oxygen species that stress the cell. Here, we present the crystal structure of YgiN, a protein of hitherto unknown function. The three-dimensional fold of YgiN is similar to that of ActVA-Orf6 monooxygenase, which acts on hydroxyquinone substrates. YgiN shares a promoter with "modulator of drug activity B," a protein with activity similar to that of mammalian DT-diaphorase capable of reducing mendione. YgiN was able to reoxidize menadiol, the product of the "modulator of drug activity B" (MdaB) enzymatic reaction. We therefore refer to YgiN as quinol monooxygenase. Modulator of drug activity B is reported to
be involved in the protection of cells from reactive oxygen species formed during single electron oxidation and reduction reactions. The enzymatic activities, together with the structural characterization of YgiN, lend evidence to the possible existence of a novel quinone redox cycle in E. coli.

PMID: 15613473 [PubMed - in process]

 >>>Antisense regulated gene candidates list<<<
Nucleic Acids Res. 2005 Feb 17;33(3):1040-50.
Detection of 5'- and 3'-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli.
Kawano M, Reynolds AA, Miranda-Rios J, Storz G.
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development Building 18T, Room 101, Bethesda, MD 20892-5430, USA.

Evidence is accumulating that small, noncoding RNAs are important regulatory molecules. Computational and experimental searches have led to the identification of approximately 60 small RNA genes in Escherichia coli. However, most of these studies focused on the intergenic regions and assumed that small RNAs were >50 nt. Thus, the previous screens missed small RNAs encoded on the antisense strand of protein-coding genes and small RNAs of <50 nt. To identify additional small RNAs, we carried out a cloning-based screen focused on RNAs of 30-65 nt. In this screen, we identified RNA species corresponding to fragments of rRNAs, tRNAs and known small RNAs. Several of the small RNAs also corresponded to 5'- and 3'-untranslated regions (UTRs) and internal fragments of mRNAs. Four of the 3'-UTR-derived RNAs were highly abundant and two showed expression patterns that differed from the corresponding mRNAs, suggesting independent functions for the 3'-UTR-derived small RNAs. We
 also detected three previously unidentified RNAs encoded in intergenic regions and RNAs from the long direct repeat and hok/sok elements. In addition, we identified a few small RNAs that are expressed opposite protein-coding genes and could base pair with 5' or 3' ends of the mRNAs with perfect complementarity.

PMID: 15718303

 >>>Hfq binding to poly(A) tails<<<
FEBS J. 2005 Jan;272(2):454-63.
Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails.
Folichon M, Allemand F, Regnier P, Hajnsdorf E.
UPR CNRS 9073, conventionnee avec l'Universite Paris 7 - Denis Diderot, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France.

The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3' end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I. In contrast, formation of the Hfq-RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).

PMID: 15654883

***additional finding***

 >>>ybhE, 6-phosphogluconolactonase<<<
J Bacteriol. 2004 Dec;186(24):8248-53.
Identification of the Escherichia coli K-12 ybhE gene as pgl, encoding 6-phosphogluconolactonase.
Thomason LC, Court DL, Datta AR, Khanna R, Rosner JL.
Gene Regulation and Chromosome Biology Laboratory, Building 539, Room 243, National Cancer Institute-Frederick, Frederick, MD 21702, USA. lthomason@ncifcrf.gov

We report identification of the Escherichia coli ybhE gene as the pgl gene that encodes 6-phosphogluconolactonase. A tentative identification was first made based on the known approximate location of the pgl gene and the similarity of the presumptive ybhE-encoded protein sequence to a known Pgl enzyme. To test this notion, the ybhE gene was deleted and replaced with a drug marker. Like previously characterized pgl mutants, the ybhE deletion mutant had a Blu- phenotype (dark-blue staining with iodine due to accumulation of starch after growth on minimal maltose) and demonstrated impaired growth on minimal glucose medium when combined with a pgi mutation. Biochemical assay of crude extracts for 6-phosphogluconolactonase enzymatic activity showed that ybhE encodes this activity. The ybhE gene was transferred from the E. coli chromosome to an expression vector. This ybhE clone complemented both the precise deletion of the ybhE gene and a larger deletion, pglDelta8, for
 the Blu- phenotype and for phosphogluconolactonase activity, confirming that ybhE is the pgl gene. A newly observed phenotype of pgl strains is a lowered frequency of appearance of Bgl+ mutants that can utilize the beta-glucoside salicin. This is likely due to poor growth of Bgl+ pgl strains on salicin due to the accumulation of 6-phosphogluconolactone.

PMID: 15576773

*comment: missing link filled !

 >>>SurE, YfbR, and YjjG, nucleotidases<<<
J Biol Chem. 2004 Dec 24;279(52):54687-94.
General enzymatic screens identify three new nucleotidases in Escherichia coli. Biochemical characterization of SurE, YfbR, and YjjG.
Proudfoot M, Kuznetsova E, Brown G, Rao NN, Kitagawa M, Mori H, Savchenko A, Yakunin AF.
Banting and Best Department of Medical Research, University of Toronto, Ontario M5G 1L6, Canada.

To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. These screens identified the presence of nucleotidase activity in three uncharacterized E. coli proteins, SurE, YfbR, and YjjG, that belong to different enzyme superfamilies: SurE-like family, HD domain family (YfbR), and haloacid dehalogenase (HAD)-like superfamily (YjjG). The phosphatase activity of these proteins had a neutral pH optimum (pH 7.0-8.0) and was strictly dependent on the presence of divalent metal cations (SurE: Mn(2+) > Co(2+) > Ni(2+) > Mg(2+); YfbR: Co(2+) > Mn(2+) > Cu(2+); YjjG: Mg(2+) > Mn(2+) > Co(2+)). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate vari
ous ribo- and deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates with highest affinity to 3'-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P(20-25)). YfbR was strictly specific to deoxyribonucleoside 5'-monophosphates, whereas YjjG showed narrow specificity to 5'-dTMP, 5'-dUMP, and 5'-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di- and triphosphates: YfbR was equally sensitive to both di- and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.

PMID: 15489502

 >>>yiaK, 2,3-diketo-L-gulonate reductase<<<
J Biol Chem. 2004 Mar 26;279(13):13148-55.
A novel NAD-binding protein revealed by the crystal structure of 2,3-diketo-L-gulonate reductase (YiaK).
Forouhar F, Lee I, Benach J, Kulkarni K, Xiao R, Acton TB, Montelione GT, Tong L.
Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027, USA.

Escherichia coli YiaK catalyzes the reduction of 2,3-diketo-L-gulonate in the presence of NADH. It belongs to a large family of oxidoreductases that is conserved in archaea, bacteria, and eukaryotes but shows no sequence homology to other proteins. We report here the crystal structures at up to 2.0-A resolution of YiaK alone and in complex with NAD-tartrate. YiaK has a new polypeptide backbone fold and a novel mode of recognizing the NAD cofactor. In addition, NAD is bound in an unusual conformation, at the interface of a dimer of the enzyme. The crystallographic analysis unexpectedly revealed the binding of tartrate in the active site. Enzyme kinetics studies confirm that tartrate and the related D-malate are inhibitors of YiaK. In contrast to most other enzymes where substrate binding produces a more closed conformation, the binding of NAD-tartrate to YiaK produces a more open active site. The free enzyme conformation is incompatible with NAD binding. His(44) is
likely the catalytic residue of the enzyme.

PMID: 14718529

*comment, assay method is A340 for NADH

 >>>yiaMNO, L-xylulose tri-partite ATP-independent periplasmic (TRAP) transporter component.<<<
Mol Membr Biol. 2004 Jan-Feb;21(1):51-7.
Functional characterization of the Escherichia coli K-12 yiaMNO transport protein genes.
Plantinga TH, Van Der Does C, Badia J, Aguilar J, Konings WN, Driessen AJ.
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands.

The yiaMNO genes of Escherichia coli K-12 encode a binding protein-dependent secondary, or tri-partite ATP-independent periplasmic (TRAP), transporter. Since only a few members of this family have been functionally characterized to date, we aimed to identify the substrate for this transporter. Cells that constitutively express the yiaK-S gene cluster metabolized the rare pentose L-xylulose, while deletion of the yiaMNO transporter genes reduced L-xylulose metabolism. The periplasmic substrate-binding protein YiaO was found to bind L-xylulose, and stimulated L-xylulose uptake by spheroplasts. These date indicate that the yiaMNO transporter mediates uptake of this rare pentose.

PMID: 14668138

>>>yahH may be misspredicted<<<

Proteomics. 2004 Jun;4(6):1597-613.
Fine-tuning the prediction of sequences cleaved by signal peptidase II: a curated set of proven and predicted lipoproteins of Escherichia coli K-12.
Gonnet P, Rudd KE, Lisacek F.
Institute of Computational Science, Swiss Federal Institute of Technology, Zurich, Switzerland.

A curated set of 81 proven and 44 predicted lipoproteins of Escherichia coli K-12 was defined with the combined use of a literature survey, a variety of predictive tools and human expertise. The well-documented Gram-negative proteome of E. coli K-12 was chosen to assess how the different approaches complement each other and to ensure a stable definition of a consistent set of lipoproteins. The results of detailed analysis of such proteins at the level of a single proteome are presented, corroborated and rationalized.

PMID: 15174130

 >>>Hfq-RNA binding<<<
Nat Struct Mol Biol. 2004 Dec;11(12):1206-14.
Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs.
Mikulecky PJ, Kaw MK, Brescia CC, Takach JC, Sledjeski DD, Feig AL.
Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, Indiana 47405, USA.

The bacterial Sm-like protein Hfq facilitates RNA-RNA interactions involved in post-transcriptional regulation of the stress response. Specifically, Hfq helps pair noncoding RNAs (ncRNAs) with complementary regions of target mRNAs. To probe the mechanism of this pairing, we generated a series of Hfq mutants and measured their affinity for RNAs like those with which Hfq must associate in vivo. We tested the mutants' DsrA-dependent activation of rpoS, and their ability to stabilize DsrA ncRNA against degradation in vivo. Our results suggest that Hfq has two independent RNA-binding surfaces. In addition to a well-known site around the core of the Hfq hexamer, we observe interactions with the distal face of Hfq, a new locus with which mRNAs and poly(A) sequences associate. Our model explains how Hfq can simultaneously bind a ncRNA and its mRNA target to facilitate the strand displacement reaction required for Hfq-dependent translational regulation.

Mol Microbiol. 2004 Nov;54(4):905-20.
The Sm-like protein Hfq regulates polyadenylation dependent mRNA decay in Escherichia coli.
Mohanty BK, Maples VF, Kushner SR.
Department of Genetics, University of Georgia, Athens, GA 30602, USA.

In Escherichia coli, the post-transcriptional addition of poly(A) tails by poly(A) polymerase I (PAP I, pcnB) plays a significant role in cellular RNA metabolism. However, many important features of this system, including its regulation and the selection of polyadenylation sites, are still poorly understood. Here we show that the inactivation of Hfq (hfq), an abundant RNA-binding protein, leads to the reduction in the ability of PAP I to add poly(A) tails at the 3' termini of mRNAs containing Rho-independent transcription terminators even though PAP I protein levels remain unchanged. Those poly(A) tails that are synthesized in the absence of Hfq are shorter in length, even in the absence of polynucleotide phosphorylase (PNPase), RNase II and RNase E. In fact, the biosynthetic activity of PNPase in the hfq single mutant is enhanced and it becomes the primary polynucleotide polymerase, adding heteropolymeric tails almost exclusively to 3' truncated mRNAs. Surprisingl
y, both PNPase and Hfq co-purified with His-tagged PAP I under native conditions indicating a potential complex among these proteins. Immunoprecipitation experiments using PNPase- and Hfq-specific antibodies confirmed the protein-protein interactions among PAP I, PNPase and Hfq. Analysis of mRNA half-lives in hfq, deltapcnB and hfq deltapcnB mutants suggests that Hfq and PAP I function in the same mRNA decay pathway.

PMID: 15522076

Biochem Biophys Res Commun. 2004 Oct 22;323(3):1017-23.
Functional effects of variants of the RNA chaperone Hfq.
Sonnleitner E, Napetschnig J, Afonyushkin T, Ecker K, Vecerek B, Moll I, Kaberdin VR, Blasi U.
Max F. Perutz Laboratories, Department of Microbiology and Genetics, University Departments at the Vienna Biocenter, Dr. Bohrgasse 9/4, 1030 Vienna, Austria.

The ring-shaped RNA chaperone Hfq has recently received much attention owing to its multiple roles in RNA metabolism. In this study we have performed a mutational analysis of the Escherichia coli hfq gene, and have studied the effects of amino acid substitutions at several positions in the Hfq protein as well as of C-terminal truncations on its role in phage Qbeta replication, in repression of a target mRNA, and on the stability of the small regulatory RNA DsrA. These functional studies provided insights into the interaction of Hfq with RNA and suggested a role for the C-terminus of Hfq in DsrA stability.

PMID: 15381101

 >>>YegE and YaiC, GGDEF domain protein, putative diguanylate cyclase<<<
J Mol Microbiol Biotechnol. 2003;5(1):11-6.
MASE1 and MASE2: two novel integral membrane sensory domains.
Nikolskaya AN, Mulkidjanian AY, Beech IB, Galperin MY.
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.

Escherichia coli proteins YegE and YaiC contain N-terminal integral membrane regions, followed by the putative diguanylate cyclase (GGDEF, DUF1) domains. The membrane domains of these proteins, named MASE1 (membrane-associated sensor) and MASE2, respectively, were found in other bacterial signaling proteins, such as histidine kinases (MASE1) and an adenylate cyclase (MASE2). Although the nature of the signals sensed by MASE1 and MASE2 is still unknown, MASE1-containing receptors appear to play important roles in bacteria, including iron and/or oxygen sensing by hemerythrine-containing proteins in the sulfate-reducing bacterium Desulfovibrio vulgaris.

PMID: 12673057