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  E.coli news vol. 12  2005.12.1
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This is a digest of daily PubMed searching of E.coli new finding.

***this year finding***

 >>>ZupT also use Fe++/Co++<<<
J Bacteriol. 2005 Mar;187(5):1604-11.
The metal permease ZupT from Escherichia coli is a transporter with a broad substrate spectrum.
Grass G, Franke S, Taudte N, Nies DH, Kucharski LM, Maguire ME, Rensing C.
Institut fur Mikrobiologie, Martin-Luther-Universitat, Halle, Germany.

The Escherichia coli zupT (formerly ygiE) gene encodes a cytoplasmic membrane protein (ZupT) related to members of the eukaryotic ZIP family of divalent metal ion transporters. Previously, ZupT was shown to be responsible for uptake of zinc. In this study, we show that ZupT is a divalent metal cation transporter of broad substrate specificity. An E. coli strain with a disruption in all known iron uptake systems could grow in the presence of chelators only if zupT was expressed. Heterologous expression of Arabidopsis thaliana ZIP1 could also alleviate iron deficiency in this E. coli strain, as could expression of indigenous mntH or feoABC. Transport studies with intact cells showed that ZupT facilitates uptake of 55Fe2+ similarly to uptake of MntH or Feo. Other divalent cations were also taken up by ZupT, as shown using 57Co2+. Expression of zupT rendered E. coli cells hypersensitive to Co2+ and sensitive to Mn2+. ZupT did not appear to be metal regulated: expressio n of a Phi(zupT-lacZ) operon fusion indicated that zupT is expressed constitutively at a low level.

PMID: 15716430

 >>>YrdC, a putative ribosome maturation factor<<<

Biochim Biophys Acta. 2005 Feb 14;1727(2):87-96.
The YrdC protein--a putative ribosome maturation factor.

Kaczanowska M, Ryden-Aulin M.

Department of Genetics, Microbiology and Toxicology (GMT), University of Stockholm, S-106 91 Stockholm, Sweden.

Release factor one (RF1) terminates protein synthesis in response to stop codons UAG and UAA. A mutant allele of RF1 causes temperature sensitive growth at 42 degrees C. We have earlier described the isolation of a suppressor of the temperature sensitive phenotype. The suppressor mutation is a small deletion in the open reading frame yrdC, and we have shown that the DeltayrdC mutation leads to immature 30S subunits and, as a consequence, to fewer translating ribosomes. YrdC is a small conserved protein with a dsRNA-binding surface. Here, we have characterized the YrdC protein. We show that the deletion leads to no production of functional protein, and we have indications that the YrdC protein might be essential in a wild type background. The protein is needed for the maturation of 16S rRNA, even though it does not interact tightly with either of the ribosomal subunits, or the 70S particles. The less effective maturation of rRNA affects the ribosomal feedback contro l, leading to an increase in expression from P1rrnB. We suggest that the function of the YrdC protein is to keep an rRNA structure needed for proper processing of 16S rRNA, especially at lower temperatures. This activity may require other factor(s). We suggest the gene be renamed rimN, and the mutant allele rimN141.

PMID: 15716138

 >>>yliABCD, glutathione importer<<<
J Bacteriol. 2005 Sep;187(17):5861-7.
The yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a novel glutathione importer with an ATP-binding cassette.
Suzuki H, Koyanagi T, Izuka S, Onishi A, Kumagai H.
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. hideyuki@lif.kyoto-u.ac.jp

Glutathione protects cells and organisms from oxygen species and peroxides and is indispensable for aerobically living organisms. Moreover, it acts against xenobiotics and drugs by the formation and excretion of glutathione S conjugates. In this study, we show that the yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a glutathione transporter with the ATP-binding cassette. The transporter imports extracellular glutathione into the cytoplasm in an ATP-dependent manner. This transporter, along with gamma-glutamyltranspeptidase, has an important role in E. coli growth with glutathione as a sole sulfur source.

PMID: 16109926

 >>>yfgL, Outer membrane protein assembly protein<<<

Cell. 2005 Apr 22;121(2):307-17.
Chemical conditionality: a genetic strategy to probe organelle assembly.
Ruiz N, Falcone B, Kahne D, Silhavy TJ.
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.

The assembly of the Escherichia coli outer membrane (OM) is poorly understood. Although insight into fundamental cellular processes is often obtained from studying mutants, OM-defective mutants have not been very informative because they generally have nonspecific permeability defects. Here we show that toxic small molecules can be used in selections employing strains with permeability defects to create particular chemical conditions that demand specific suppressor mutations. Suppressor phenotypes are correlated with the physical properties of the small molecules, but the mutations are not in their target genes. Instead, mutations allow survival by partially restoring membrane impermeability. Using "chemical conditionality," we identified mutations in yfgL, and, here and in the accompanying paper by Wu et al. published in this issue of Cell (Wu et al., 2005), we show that YfgL is part of a multiprotein complex involved in the assembly of OM beta barrel proteins. We  posit that panels of toxic small molecules will be useful for generating chemical conditionalities that enable identification of genes required for organelle assembly in other organisms.

PMID: 15851036

Cell. 2005 Apr 22;121(2):235-45.
Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli.
Wu T, Malinverni J, Ruiz N, Kim S, Silhavy TJ, Kahne D.
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.

Gram-negative bacteria have an outer membrane (OM) that functions as a barrier to protect the cell from toxic compounds such as antibiotics and detergents. The OM is a highly asymmetric bilayer composed of phospholipids, glycolipids, and proteins. Assembly of this essential organelle occurs outside the cytoplasm in an environment that lacks obvious energy sources such as ATP, and the mechanisms involved are poorly understood. We describe the identification of a multiprotein complex required for the assembly of proteins in the OM of Escherichia coli. We also demonstrate genetic interactions between genes encoding components of this protein assembly complex and imp, which encodes a protein involved in the assembly of lipopolysaccharides (LPS) in the OM. These genetic interactions suggest a role for YfgL, one of the lipoprotein components of the protein assembly complex, in a homeostatic control mechanism that coordinates the overall OM assembly process.

PMID: 15851030

J Bacteriol. 2005 Apr;187(7):2286-96.
Strong decrease in invasive ability and outer membrane vesicle release in Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 with the yfgL gene deleted.
Rolhion N, Barnich N, Claret L, Darfeuille-Michaud A.
Pathogenie Bacterienne Intestinale, Laboratoire de Bacteriologie, Universite d'Auvergne, CBRV, 28 Place Henri Dunant, 63001 Clermont-Ferrand, France.

Adherent-invasive Escherichia coli strain LF82 recovered from a chronic lesion of a patient with Crohn's disease is able to invade cultured intestinal epithelial cells. Three mutants with impaired ability to invade epithelial cells had the Tn5phoA transposon inserted in the yfgL gene encoding the YfgL lipoprotein. A yfgL- negative isogenic mutant showed a marked decrease both in its ability to invade Intestine-407 cells and in the amount of the outer membrane proteins OmpA and OmpC in the culture supernatant, as shown by analysis of the culture supernatant protein contents by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Transcomplementation of the LF82-DeltayfgL isogenic mutant with the cloned yfgL gene restored invasion ability and outer membrane protein release in the culture supernatant. The outer membrane proteins in the culture supernatant of strain LF82 resulted fro m the formation of vesicles. This was shown by Western blot analysis of periplasmic and outer membrane fraction markers typically found in outer membrane vesicles and by transmission electron microscopic analysis of ultracentrifuged cell-free LF82 supernatant pellets, indicating the presence of vesicles with a bilayered structure surrounding a central electron-dense core. Thus, deletion of the yfgL gene in strain LF82 resulted in a decreased ability to invade intestinal epithelial cells and a decreased release of outer membrane vesicles.

PMID: 15774871

 >>>yadB, aminoacyl-tRNA synthetase that glutamylates tRNA(Asp) anticodon<<<

Biochimie. 2005 Sep-Oct;87(9-10):847-61.
Glu-Q-tRNA(Asp) synthetase coded by the yadB gene, a new paralog of aminoacyl-tRNA synthetase that glutamylates tRNA(Asp) anticodon.
Blaise M, Becker HD, Lapointe J, Cambillau C, Giege R, Kern D.
Departement Machineries Traductionnelles, UPR 9002 du CNRS, Institut de Biologie Moleculaire et Cellulaire, 15, rue Rene Descartes, 67084 Strasbourg cedex, France.

Analysis of the completed genome sequences revealed presence in various bacteria of an open reading frame (ORF) encoding a polypeptide chain presenting important similarities with the catalytic domain of glutamyl-tRNA synthetases but deprived of the C-terminal anticodon-binding domain. This paralog of glutamyl-tRNA synthetases, the YadB protein, activates glutamate in the absence of tRNA and transfers the activated glutamate not on tRNA(Glu) but instead on tRNA(Asp). It has been shown that tRNA(Asp) is able to accept two amino acids: aspartate charged by aspartyl-tRNA synthetase and glutamate charged by YadB. The functional properties of YadB contrast with those of the canonical glutamyl-tRNA synthetases, which activate Glu only in presence of the cognate tRNA before aminoacylation of the 3'-end of tRNA. Biochemical approaches and mass spectrometry investigations revealed that YadB transfers the activated glutamate on the cyclopenthene-diol ring of the modified nuc leoside queuosine posttranscriptionally inserted at the wobble position of the anticodon-loop to form glutamyl-queuosine. Unstability of the ester bond between the glutamate residue and the cyclopenthene-diol (half-life 7.5 min) explains why until now this modification escaped detection. Among Escherichia coli tRNAs containing queuosine in the wobble position, only tRNA(Asp) is substrate of YadB. Sequence comparison reveals a structural mimicry between the anticodon-stem and loop of tRNA(Asp) and the amino acid acceptor-stem of tRNA(Glu). YadB, renamed glutamyl-Q-tRNA(Asp) synthetase, constitutes the first enzyme structurally related to aminoacyl-tRNA synthetases which catalyzes a hypermodification in tRNA, and whose function seems to be conserved among prokaryotes. The discovery of glutamyl-Q-tRNA(Asp) synthetase breaks down the current paradigm according to which the catalytic domain of aminoacyl-tRNA synthetases recognizes the amino acid acceptor-stem of tRNA an d aminoacylates the 3'-terminal ribose. The evolutionary significance of the existence of an aminoacyl-tRNA synthetase paralog dedicated to the hypermodification of a tRNA anticodon will be discussed.

PMID: 16164993

 >>>queCF, Queuosine biosynthesis protein<<<

J Bacteriol. 2005 Oct;187(20):6893-901.
Genetic analysis identifies a function for the queC (ybaX) gene product at an initial step in the queuosine biosynthetic pathway in Escherichia coli.
Gaur R, Varshney U.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.

Queuosine (Q), one of the most complex modifications occurring at the wobble position of tRNAs with GUN anticodons, is implicated in a number of biological activities, including accuracy of decoding, virulence, and cellular differentiation. Despite these important implications, its biosynthetic pathway has remained unresolved. Earlier, we observed that a naturally occurring strain of Escherichia coli B105 lacked Q modification in the tRNAs. In the present study, we developed a genetic screen to map the defect in E. coli B105 to a single gene, queC (renamed from ybaX), predicted to code for a 231-amino-acid-long protein with a pI of 5.6. As analyzed by mobility of tRNA(Tyr) on acid urea gels and two-dimensional thin-layer chromatography of the modified nucleosides, expression of QueC from a plasmid-borne copy confers a Q+ phenotype to E. coli B105. Further, analyses of tRNA(Tyr) from E. coli JE10651 (queA mutant), its derivative generated by deletion of chromosomal queC (queA deltaqueC), and E. coli JE7325, deficient in converting preQ0 to preQ1, have provided the first genetic evidence for the involvement of QueC at a step leading to production of preQ0, the first known intermediate in the generally accepted pathway that utilizes GTP as the starting molecule. In addition, we discuss the possibilities of collaboration of QueC with other cellular proteins in the production of preQ0.

PMID: 16199558

 >>>GutQ, D-arabinose 5-phosphate isomerase<<<

J Bacteriol. 2005 Oct;187(20):6936-42.
Identification of GutQ from Escherichia coli as a D-arabinose 5-phosphate isomerase.
Meredith TC, Woodard RW.
Department of Medicinal Chemistry, University of Michigan, Ann Arbor 48109-1065, USA.

The glucitol operon (gutAEBDMRQ) of Escherichia coli encodes a phosphoenolpyruvate:sugar phosphotransferase system that metabolizes the hexitol D-glucitol (sorbitol). The functions for all but the last gene, gutQ, have been previously assigned. The high sequence similarity between GutQ and KdsD, a D-arabinose 5-phosphate isomerase (API) from the 3-deoxy-D-manno-octulosonate (KDO)-lipopolysaccharide (LPS) biosynthetic pathway, suggested a putative activity, but its role within the context of the gut operon remained unclear. Accordingly, the enzyme was cloned, overexpressed, and characterized. Recombinant GutQ was shown to indeed be a second copy of API from the E. coli K-12 genome with biochemical properties similar to those of KdsD, catalyzing the reversible aldol-ketol isomerization between D-ribulose 5-phosphate (Ru5P) and D-arabinose 5-phosphate (A5P). Genomic disruptions of each API gene were constructed in E. coli K-12. TCM11[(deltakdsD)] was capable of sustai ning essential LPS synthesis at wild-type levels, indicating that GutQ functions as an API inside the cell. The gut operon remained inducible in TCM7[(deltagutQ)], suggesting that GutQ is not directly involved in d-glucitol catabolism. The conditional mutant TCM15[(deltagutQdeltakdsD)] was dependent on exogenous A5P both for LPS synthesis/growth and for upregulation of the gut operon. The phenotype was suppressed by complementation in trans with a plasmid encoding a functional copy of GutQ or by increasing the amount of A5P in the medium. As there is no obvious obligatory role for GutQ in the metabolism of d-glucitol and there is no readily apparent link between D-glucitol metabolism and LPS biosynthesis, it is suggested that A5P is not only a building block for KDO biosynthesis but also may be a regulatory molecule involved in expression of the gut operon.

PMID: 16199563

 >>>glgX, isoamylase-type debranching enzyme<<<

J Bacteriol. 2005 Feb;187(4):1465-73.
Role of the Escherichia coli glgX gene in glycogen metabolism.
Dauvillee D, Kinderf IS, Li Z, Kosar-Hashemi B, Samuel MS, Rampling L, Ball S, Morell MK.
CSIRO UMR8576, Cite Scientifique, Villeneuve d'Ascq, France.

A role for the Escherichia coli glgX gene in bacterial glycogen synthesis and/or degradation has been inferred from the sequence homology between the glgX gene and the genes encoding isoamylase-type debranching enzymes; however, experimental evidence or definition of the role of the gene has been lacking. Construction of E. coli strains with defined deletions in the glgX gene is reported here. The results show that the GlgX gene encodes an isoamylase-type debranching enzyme with high specificity for hydrolysis of chains consisting of three or four glucose residues. This specificity ensures that GlgX does not generate an extensive futile cycle during glycogen synthesis in which chains with more than four glucose residues are transferred by the branching enzyme. Disruption of glgX leads to overproduction of glycogen containing short external chains. These results suggest that the GlgX protein is predominantly involved in glycogen catabolism by selectively debranching  the polysaccharide outer chains that were previously recessed by glycogen phosphorylase.

PMID: 15687211

 >>>rcsF, Alternative sensor kinase for RcsB., yojN/rscD<<<

J Bacteriol. 2005 Oct;187(19):6770-8.
Role of RcsF in signaling to the Rcs phosphorelay pathway in Escherichia coli.
Majdalani N, Heck M, Stout V, Gottesman S.
National Cancer Institute, 9000 Rockville Pike, Bldg. 37, Bethesda, MD 20892, USA.

The rcs phosphorelay pathway components were originally identified as regulators of capsule synthesis. In addition to the transmembrane sensor kinase RcsC, the RcsA coregulator, and the response regulator RcsB, two new components have been characterized, RcsD and RcsF. RcsD, the product of the yojN gene, now renamed rcsD, acts as a phosphorelay between RcsC and RcsB. Transcription of genes for capsule synthesis (cps) requires both RcsA and RcsB; transcription of other promoters, including that for the small RNA RprA, requires only RcsB. RcsF was described as an alternative sensor kinase for RcsB. We have examined the role of RcsF in the activation of both the rprA and cps promoters. We find that a number of signals that lead to activation of the phosphorelay require both RcsF and RcsC; epistasis experiments place RcsF upstream of RcsC. The RcsF sequence is characteristic of lipoproteins, consistent with a role in sensing cell surface perturbation and transmitting t his signal to RcsC. Activation of RcsF does not require increased transcription of the gene, suggesting that modification of the RcsF protein may act as an activating signal. Signals from RcsC require RcsD to activate RcsB. Sequencing of an rcsC allele, rcsC137, that leads to high-level constitutive expression of both cps and rprA suggests that the response regulator domain of RcsC plays a role in negatively regulating the kinase activity of RcsC. The phosphorelay and the variation in the activation mechanism (dependent upon or independent of RcsA) provide multiple steps for modulating the output from this system.

PMID: 16166540

 >>>yecG, yiiT, ynaF, ybdQ, ydaA, -> uspCDEFG, Universal stress protein<<<

J Bacteriol. 2005 Sep;187(18):6265-72.
Differential roles of the universal stress proteins of Escherichia coli in oxidative stress resistance, adhesion, and motility.
Nachin L, Nannmark U, Nystrom T.
Department of Cell and Molecular Biology, Goteborg University, Medicinaregatan 9C, 413 90 Goteborg, Sweden.

The universal stress protein (UspA) superfamily encompasses a conserved group of proteins that are found in bacteria, archaea, and eukaryotes. Escherichia coli harbors six usp genes--uspA, -C, -D, -E, -F, and -G--the expression of which is triggered by a large variety of environmental insults. The uspA gene is important for survival during cellular growth arrest, but the exact physiological role of the Usp proteins is not known. In this work we have performed phenotypic characterization of mutants with deletions of the six different usp genes. We report on hitherto unknown functions of these genes linked to motility, adhesion, and oxidative stress resistance, and we show that usp functions are both overlapping and distinct. Both UspA and UspD are required in the defense against superoxide-generating agents, and UspD appears also important in controlling intracellular levels of iron. In contrast, UspC is not involved in stress resistance or iron metabolism but is es sential, like UspE, for cellular motility. Electron microscopy demonstrates that uspC and uspE mutants are devoid of flagella. In addition, the function of the uspC and uspE genes is linked to cell adhesion, measured as FimH-mediated agglutination of yeast cells. While the UspC and UspE proteins promote motility at the expense of adhesion, the UspF and UspG proteins exhibit the exact opposite effects. We suggest that the Usp proteins have evolved different physiological functions that reprogram the cell towards defense and escape during cellular stress.

PMID: 16159758

 >>>YdgT, oriC-binding protien<<<
J Bacteriol. 2005 Oct;187(20):6998-7008.
Cnu, a novel oriC-binding protein of Escherichia coli.
Kim MS, Bae SH, Yun SH, Lee HJ, Ji SC, Lee JH, Srivastava P, Lee SH, Chae H, Lee Y, Choi BS, Chattoraj DK, Lim HM.
Department of Biology, School of Biological Sciences and Biotechnology, Chungnam National University, Taejon, 305-764 Korea.

We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

PMID: 16199570

 >>>dos, Heme-Regulated Phosphodiesterase.<<<

J Bacteriol. 2005 Oct;187(19):6678-82.
DOS(Ec), a heme-regulated phosphodiesterase, plays an important role in the regulation of the cyclic AMP level in Escherichia coli.
Yoshimura-Suzuki T, Sagami I, Yokota N, Kurokawa H, Shimizu T.
Department of Molecular Cell Signalling, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan. yoshimura_tokiko@hotmail.com

Heme-regulated phosphodiesterase from Escherichia coli (DOS(Ec)) catalyzes the hydrolysis of cyclic AMP (cAMP) in vitro and is regulated by the redox state of the bound heme. Changes in the redox state result in alterations in the three-dimensional structure of the enzyme, which is then transmitted to the functional domain to switch catalysis on or off. Because DOS(Ec) was originally cloned from E. coli genomic DNA, it has not been known whether it is actually expressed in wild-type E. coli. In addition, the turnover number of DOS(Ec) using cAMP as a substrate is only 0.15 min(-1), which is relatively low for a physiologically relevant enzyme. In the present study, we demonstrated for the first time that the DOS(Ec) gene and protein are expressed in wild-type E. coli, especially under aerobic conditions. We also developed a DOS(Ec) gene knockout strain (Deltados). Interestingly, the knockout of dos caused excess accumulation of intracellular cAMP (26-fold higher th an in the wild-type strain) under aerobic conditions, whereas accumulation of cAMP was not observed under anaerobic conditions. We also found differences in cell morphology and growth rate between the mutant cells and the wild-type strain. The changes in the knockout strain were partially complemented by introducing an expression plasmid for dos. Thus, the present study revealed that expression of DOS(Ec) is regulated according to environmental O2 availability at the transcriptional level and that the concentration of cAMP in cells is regulated by DOS(Ec) expression.

PMID: 16166529

Biochemistry. 2005 Jul 19;44(28):9598-605.
Investigation of the relationship between protein-protein interaction and catalytic activity of a heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) by protein microarray.
Sasakura Y, Kanda K, Yoshimura-Suzuki T, Matsui T, Fukuzono S, Shimizu T.
Bio-Medical Center, R&D Division, Nanotechnology Product Business Group, Hitachi High-Technologies Corporation, Hitachinaka, Ibaraki, 312-8504, Japan.

Ec DOS, a heme-regulated phosphodiesterase from Escherichia coli, is composed of an N-terminal heme-bound PAS domain and a C-terminal phosphodiesterase domain. The heme redox state in the PAS domain regulates Ec DOS phosphodiesterase activity. Interestingly, the isolated heme-bound PAS fragment enhances phosphodiesterase activity of full-length Ec DOS. The enhancement is also regulated by the heme redox state of the isolated PAS domain. In the present study, we used a newly developed protein microarray system to examine the relationship between catalytic activity and the interaction of full-length Ec DOS and the isolated PAS fragment. Adenosine 3',5'-cyclic monophosphate (cAMP), a substrate of the Ec DOS phosphodiesterase, was found to be indispensable for the interaction between Ec DOS and the PAS fragment, and two phosphodiesterase inhibitors, 3-isobutyl-methyl-xanthine and etazolate hydrochloride, hindered the interaction. In addition, an enzyme with a mutation in the putative cAMP-binding sites (H590 and H594) was unable to interact with Ec DOS and lacked enzymatic activity. These results strongly suggest a close relationship between Ec DOS phosphodiesterase activity and interaction with the isolated PAS fragment. Therefore, this study provides insights into the mechanism of how the isolated PAS domain activates Ec DOS, which has important implications for the general role of the isolated PAS domain in cells. Moreover, we found that multiple microscale analyses using the protein microarray system had several advantages over conventional affinity column methods, including the quantity of protein needed, the sensitivity, the variability of immobilized protein, and the time required for the experiment.

PMID: 16008345

 >>>yqhE,yafB,yghZ,yeaE,yajO->Aldo-keto reductase<<<

J Bacteriol. 2005 Aug;187(16):5782-9.
Conversion of methylglyoxal to acetol by Escherichia coli aldo-keto reductases.
Ko J, Kim I, Yoo S, Min B, Kim K, Park C.
Department of Life Sciences, Korea Advanced Institute of Science and Technology, Yusong-Ku, Taejon 305-701, Republic of Korea.

Methylglyoxal (MG) is a toxic metabolite known to accumulate in various cell types. We detected in vivo conversion of MG to acetol in MG-accumulating Escherichia coli cells by use of (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopy. A search for homologs of the mammalian aldo-keto reductases (AKRs), which are known to exhibit activity to MG, revealed nine open reading frames from the E. coli genome. Based on both sequence similarities and preliminary characterization with (1)H-NMR for crude extracts of the corresponding mutant strains, we chose five genes, yafB, yqhE, yeaE, yghZ, and yajO, for further study. Quantitative assessment of the metabolites produced in vitro from the crude extracts of these mutants and biochemical study with purified AKRs indicated that the yafB, yqhE, yeaE, and yghZ genes are involved in the conversion of MG to acetol in the presence of NADPH. When we assessed their in vivo catalytic activities by creating double mutants, all of t hese genes except for yqhE exhibited further sensitivities to MG in a glyoxalase-deficient strain. The results imply that the glutathione-independent detoxification of MG can occur through multiple pathways, consisting of yafB, yqhE, yeaE, and yghZ genes, leading to the generation of acetol.

PMID: 16077126

*substrate specificity were assessed.

***additional finding***

 >>>YdgT, hns/stpA binding protien<<<
Mol Microbiol. 2004 Oct;54(1):251-63.
YdgT, the Hha paralogue in Escherichia coli, forms heteromeric complexes with H-NS and StpA.
Paytubi S, Madrid C, Forns N, Nieto JM, Balsalobre C, Uhlin BE, Juarez A.
Departament de Microbiologia, Universitat de Barcelona, Avda Diagonal 645, 08028 Barcelona, Spain.

In enteric bacteria, proteins of the Hha/YmoA family play a role in the regulation of gene expression in response to environmental factors. Interaction of both Hha and YmoA with H-NS has been reported, and an Hha/H-NS complex has been shown to modulate expression in Escherichia coli of the haemolysin operon of plasmid pHly152. In addition to the hns gene, the chromosome of E. coli and other enteric bacteria also includes the stpA gene that encodes the StpA protein, an H-NS paralogue. We report here the identification of the Hha paralogue in E. coli, the YdgT protein. As Hha paralogue, YdgT appears to fulfil some of the functions reported for StpA as H-NS paralogue: YdgT is overexpressed in hha mutants and can compensate, at least partially, some of the hha-induced phenotypes. We also demonstrate that YdgT interacts both with H-NS and with StpA. Protein cross-linking studies showed that YdgT/H-NS heteromeric complexes are generated within the bacterial cell. The Stp A protein, which is subjected to Lon-mediated turnover, was less stable in the absence of Hha or YdgT. Our findings suggest that Hha, YdgT and StpA may form complexes in vivo.

PMID: 15458420

 >>>yojN, Sensor-like histidine kinase for phosphorelay between RcsC and RcsB (EC 2.7.3.-).<<<

 J Bacteriol. 2003 Oct;185(19):5735-46.
Genome-wide analyses revealing a signaling network of the RcsC-YojN-RcsB phosphorelay system in Escherichia coli.
Hagiwara D, Sugiura M, Oshima T, Mori H, Aiba H, Yamashino T, Mizuno T.
Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.

In Escherichia coli, capsular colanic acid polysaccharide synthesis is regulated through the multistep RcsC-->YojN-->RcsB phosphorelay. By monitoring a hallmarked cps::lacZ reporter gene, we first searched for physiological stimuli that propagate the Rcs signaling system. The expression of cps::lacZ was activated when cells were grown at a low temperature (20 degrees C) in the presence of glucose as a carbon source and in the presence of a relatively high concentration of external zinc (1 mM ZnCl(2)). In this Rcs signaling system, the rcsF gene product (a putative outer membrane-located lipoprotein) was also an essential signaling component. Based on the defined signaling pathway and physiological stimuli for the Rcs signaling system, we conducted genome-wide analyses with microarrays to clarify the Rcs transcriptome (i.e., Rcs regulon). Thirty-two genes were identified as putative Rcs regulon members; these genes included 15 new genes in addition to 17 of the prev iously described cps genes. Using a set of 37 two-component system mutants, we performed alternative genome-wide analyses. The results showed that the propagation of the zinc-responsive Rcs signaling system was largely dependent on another two-component system, PhoQ/P. Considering the fact that the PhoQ/P signaling system responds to external magnesium, we obtained evidence which supports the view that there is a signaling network that connects the Rcs system with the PhoQ/P system, which coordinately regulates extracellular polysaccharide synthesis in response to the external concentrations of divalent cations.

PMID: 13129944

Mol Microbiol. 2001 Apr;40(2):440-50.
A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC --> YojN --> RcsB signalling pathway implicated in capsular synthesis and swarming behaviour.
Takeda S, Fujisawa Y, Matsubara M, Aiba H, Mizuno T.
Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.

In this study, we re-investigated the previously characterized RcsC (sensor His-kinase) --> RcsB (response regulator) phosphorelay system that is involved in the regulation of capsular polysaccharide synthesis in Escherichia coli. The previously proposed model hypothesized the occurrence of a direct phosphotransfer from RcsC to RcsB in response to an unknown external stimulus. As judged from the current general view as to the His --> Asp phosphorelay, this RcsC --> RcsB framework is somewhat puzzling, because RcsC appears to contain both a His-kinase domain and a receiver domain, but not a histidine (His)-containing phosphotransmitter domain (e.g. HPt domain). We thus suspected that an as yet unknown mechanism might be underlying in this particular His --> Asp phosphorelay system. Here, we provide several lines of in vivo and in vitro evidence that a novel and unique His-containing phosphotransmitter (named YojN) is essential for this signalling system. A revised m odel is proposed in which the multistep RcsC --> YojN --> RcsB phosphorelay is implicated. It was also demonstrated that this complex signalling system is somehow involved in the modulation of a characteristic behaviour of E. coli cells during colony formation on the surface of agar plates, namely swarming.

PMID: 11309126