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  E.coli news vol. 8  2004.11.16
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This is a digest of daily PubMed searching of E.coli new finding.

***this year finding***

 >>>yhcS, yhcR, yhcQ, and yhcP->aaeR, aaeX, aaeA, and aaeB, aromatic carboxylic acids efflux pump<<<

J Bacteriol. 2004 Nov;186(21):7196-204.
Characterization of the Escherichia coli AaeAB efflux pump: a metabolic relief valve?
Van Dyk TK, Templeton LJ, Cantera KA, Sharpe PL, Sariaslani FS.
DuPont Company CR&D, Rt. 141 and Powdermill Road, P.O. Box 80173, Wilmington, DE 19880-0173, USA.

Treatment of Escherichia coli with p-hydroxybenzoic acid (pHBA) resulted in upregulation of yhcP, encoding a protein of the putative efflux protein family. Also upregulated were the adjacent genes yhcQ, encoding a protein of the membrane fusion protein family, and yhcR, encoding a small protein without a known or suggested function. The function of the upstream, divergently transcribed gene yhcS, encoding a regulatory protein of the LysR family, in regulating expression of yhcRQP was shown. Furthermore, it was demonstrated that several aromatic carboxylic acid compounds serve as inducers of yhcRQP expression. The efflux function encoded by yhcP was proven by the hypersensitivity to pHBA of a yhcP mutant strain. A yhcS mutant strain was also hypersensitive to pHBA. Expression of yhcQ and yhcP was necessary and sufficient for suppression of the pHBA hypersensitivity of the yhcS mutant. Only a few aromatic carboxylic acids of hundreds of diverse compounds tested were defined as substrates of the YhcQP efflux pump. Thus, we propose renaming yhcS, yhcR, yhcQ, and yhcP, to reflect their role in aromatic carboxylic acid efflux, to aaeR, aaeX, aaeA, and aaeB, respectively. The role of pHBA in normal E. coli metabolism and the highly regulated expression of the AaeAB efflux system suggests that the physiological role may be as a "metabolic relief valve" to alleviate toxic effects of imbalanced metabolism.

PMID: 15489430

 >>>gadY, smallRNA<<<

J Bacteriol. 2004 Oct;186(20):6698-705.
GadY, a small-RNA regulator of acid response genes in Escherichia coli.
Opdyke JA, Kang JG, Storz G.
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

A previous bioinformatics-based search for small RNAs in Escherichia coli identified a novel RNA named IS183. The gene encoding this small RNA is located between and on the opposite strand of genes encoding two transcriptional regulators of the acid response, gadX (yhiX) and gadW (yhiW). Given that IS183 is encoded in the gad gene cluster and because of its role in regulating acid response genes reported here, this RNA has been renamed GadY. We show that GadY exists in three forms, a long form consisting of 105 nucleotides and two processed forms, consisting of 90 and 59 nucleotides. The expression of this small RNA is highly induced during stationary phase in a manner that is dependent on the alternative sigma factor sigmaS. Overexpression of the three GadY RNA forms resulted in increased levels of the mRNA encoding the GadX transcriptional activator, which in turn caused increased levels of the GadA and GadB glutamate decarboxylases. A promoter mutation which abo lished gadY expression resulted in a reduction in the amount of gadX mRNA during stationary phase. The gadY gene was shown to overlap the 3' end of the gadX gene, and this overlap region was found to be necessary for the GadY-dependent accumulation of gadX mRNA. We suggest that during stationary phase, GadY forms base pairs with the 3'-untranslated region of the gadX mRNA and confers increased stability, allowing for gadX mRNA accumulation and the increased expression of downstream acid resistance genes.

PMID: 15466020

 >>>yabN->sgrR, transcriptional activator and ryaA, smallRNA<<<

Mol Microbiol. 2004 Nov;54(4):1076-89.
Involvement of a novel transcriptional activator and small RNA in post-transcriptional regulation of the glucose phosphoenolpyruvate phosphotransferase system.
Vanderpool CK, Gottesman S.
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Summary RyaA is a small non-coding RNA in Escherichia coli that was identified by its ability to bind tightly to the RNA chaperone Hfq. This study reports the role of RyaA in mediating the cellular response to glucose-specific phosphoenolypyruvate phosphotransferase system (PTS)-dependent phosphosugar stress. Aiba and co-workers have shown that a block in the metabolism of glucose 6-phosphate causes transient growth inhibition and post-transcriptional regulation of ptsG, encoding the glucose-specific PTS transporter. We found that RyaA synthesis was induced by a non-metabolizable glucose phosphate analogue and was necessary for relief of the toxicity of glucose phosphate stress. Expression of RyaA was sufficient to cause a rapid loss of ptsG mRNA, probably reflecting degradation of the message mediated by RyaA:ptsG pairing. The ryaA gene was renamed sgrS, for sugar transport-related sRNA. Expression of sgrS is regulated by a novel transcriptional activator, SgrR (f ormerly YabN), which has a putative DNA-binding domain and a solute-binding domain similar to those found in certain transport proteins. Our results suggest that under conditions of glucose phosphate accumulation, SgrR activates SgrS synthesis, causing degradation of ptsG mRNA. Decreased ptsG mRNA results in decreased production of glucose transport machinery, thus limiting further accumulation of glucose phosphate.

PMID: 15522088

 >>>ydaO -> ttcA, Related protein of thiolation of cytidine 32 of tRNA<<<

J Bacteriol. 2004 Feb;186(3):750-7. 
The conserved Cys-X1-X2-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium.
Jager G, Leipuviene R, Pollard MG, Qian Q, Bjork GR.
Department of Molecular Biology, Umea University, S-90187 Umea, Sweden.

The modified nucleoside 2-thiocytidine (s(2)C) has so far been found in tRNA from organisms belonging to the phylogenetic domains Archaea and BACTERIA: In the bacteria Escherichia coli and Salmonella enterica serovar Typhimurium, s(2)C is present in position 32 of only four tRNA species-, and. An in-frame deletion of an S. enterica gene (designated ttcA, for "two-thio-cytidine") was constructed, and such a mutant has no detectable s(2)C in its tRNA. The TtcA protein family is characterized by the existence of both a PP-loop and a Cys-X(1)-X(2)-Cys motif in the central region of the protein but can be divided into two distinct groups based on the presence and location of additional Cys-X(1)-X(2)-Cys motifs in terminal regions of the sequence. Mutant analysis showed that both cysteines in this central conserved Cys-X(1)-X(2)-Cys motif are required for the formation of s(2)C. The DeltattcA1 mutant grows at the same rate as the congenic wild-type strain, and no growth disadvantage caused by the lack of s(2)C was observed in a mixed-population experiment. Lack of s(2)C32 did not reduce the selection rate at the ribosomal aminoacyl-tRNA site (A-site) for at any of its cognate CGN codons, whereas A-site selection at AGG by was dependent on the presence of s(2)C32. The presence of s(2)C32 in peptidyl- or in peptidyl- interfered with decoding in the A-site. The presence of s(2)C32 in decreased the rate of translation of the CGA codon but not that of the CGU codon.

PMID: 14729701

 >>>EnvC (YibP), ?<<<

Mol Microbiol. 2004 Jun;52(5):1255-69. 
Screening for synthetic lethal mutants in Escherichia coli and identification of EnvC (YibP) as a periplasmic septal ring factor with murein hydrolase activity.
Bernhardt TG, de Boer PA.
Case Western Reserve University, School of Medicine, W239, Department of Molecular Biology and Microbiology, 10900 Euclid Ave., Cleveland, OH 44106, USA.

Bacterial cytokinesis is driven by the septal ring apparatus, the assembly of which in Escherichia coli is directed to mid-cell by the Min system. Despite suffering aberrant divisions at the poles, cells lacking the minCDE operon (Min(-)) have an almost normal growth rate. We developed a generally applicable screening method for synthetic lethality in E. coli, and used it to select for transposon mutations (slm) that are synthetically lethal (or sick) in combination with DeltaminCDE. One of the slm insertions mapped to envC (yibP), proposed to encode a lysostaphin-like, metallo-endopeptidase that is exported to the periplasm by the general secretory (Sec) pathway. Min(-) EnvC(-) cells showed a severe division defect, supporting a role for EnvC in septal ring function. Accordingly, we show that an EnvC-green fluorescent protein fusion, when directed to the periplasm via the twin-arginine export system, is both functional and part of the septal ring apparatus. Using an in-gel assay, we also present evidence that EnvC possesses murein hydrolytic activity. Our results suggest that EnvC plays a direct role in septal murein cleavage to allow outer membrane constriction and daughter cell separation. By uncovering genetic interactions, the synthetic lethal screen described here provides an attractive new tool for studying gene function in E. coli.

PMID: 15165230

 >>>yhdJ, DNA methyltransferase<<<

J Bacteriol. 2004 Apr;186(7):2061-7. 
A DNA adenine methyltransferase of Escherichia coli that is cell cycle regulated and essential for viability.
Kossykh VG, Lloyd RS.
Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1071, USA.

DNA sequence analysis revealed that the putative yhdJ DNA methyltransferase gene of Escherichia coli is 55% identical to the Nostoc sp. strain PCC7120 gene encoding DNA methyltransferase AvaIII, which methylates adenine in the recognition sequence, ATGCAT. The yhdJ gene was cloned, and the enzyme was overexpressed and purified. Methylation and restriction analysis showed that the DNA methyltransferase methylates the first adenine in the sequence ATGCAT. This DNA methylation was found to be regulated during the cell cycle, and the DNA adenine methyltransferase was designated M.EcoKCcrM (for "cell cycle-regulated methyltransferase"). The CcrM DNA adenine methyltransferase is required for viability in E. coli, as a strain lacking a functional genomic copy of ccrM can be isolated only in the presence of an additional copy of ccrM supplied in trans. The cells of such a knockout strain stopped growing when expression of the inducible plasmid ccrM gene was shut off. Overe xpression of M.EcoKCcrM slowed bacterial growth, and the ATGCAT sites became fully methylated throughout the cell cycle; a high proportion of cells with an anomalous size distribution and DNA content was found in this population. Thus, the temporal control of this methyltransferase may contribute to accurate cell cycle control of cell division and cellular morphology. Homologs of M.EcoKCcrM are present in other bacteria belonging to the gamma subdivision of the class Proteobacteria, suggesting that methylation at ATGCAT sites may have similar functions in other members of this group.

PMID: 15028690

 >>>cadaverine-lysine antiporter<<<

Mol Microbiol. 2004 Mar;51(5):1401-12. 
Excretion and uptake of cadaverine by CadB and its physiological functions in Escherichia coli.
Soksawatmaekhin W, Kuraishi A, Sakata K, Kashiwagi K, Igarashi K.
Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.

The functions of the putative cadaverine transport protein CadB were studied in Escherichia coli. CadB had both cadaverine uptake activity, dependent on proton motive force, and cadaverine excretion activity, acting as a cadaverine-lysine antiporter. The Km values for uptake and excretion of cadaverine were 20.8 and 303 microM respectively. Both cadaverine uptake and cadaverine-lysine antiporter activities of CadB were functional in cells. Cell growth of a polyamine-requiring mutant was stimulated slightly at neutral pH by the cadaverine uptake activity and greatly at acidic pH by the cadaverine-lysine antiporter activity. At acidic pH, the operon containing cadB and cadA, encoding lysine decarboxylase, was induced in the presence of lysine. This caused neutralization of the extracellular medium and made possible the production of CO(2) and cadaverine and aminopropylcadaverine instead of putrescine and spermidine. The induction of the cadBA operon also generated a proton motive force. When the cadBA operon was not induced, the expression of the speF-potE operon, encoding inducible ornithine decarboxylase and a putrescine-ornithine antiporter, was increased. The results indicate that the cadBA operon plays important roles in cellular regulation at acidic pH.

PMID: 14982633

 >>>iscA localization<<<

Proc Natl Acad Sci U S A. 2004 Jan 20;101(3):835-40.
Evidence for polar positional information independent of cell division and nucleoid occlusion.
Janakiraman A, Goldberg MB.
Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital/Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA.

We present evidence that, in Escherichia coli, polar positional information is present at midcell independent of known cell division factors. In filamented cells, IcsA, which is normally polar, localizes at or near potential cell division sites. Because the cell pole is derived from the septum, the sites to which IcsA localizes in filaments correspond to future poles. IcsA localization to these sites is independent of FtsZ, MinCDE, septation, and nucleoid occlusion, indicating that positional information for the future pole is independent of cell division and chromosome positioning. Upon IcsA localization to these sites, septation is inhibited, suggesting that IcsA recognition of this polar positional information may influence cell division.

PMID: 14715903

 >>>Fructoselysine 3-epimerase<<<

Biochem J. 2004 Mar 15;378(Pt 3):1047-52. 
Fructoselysine 3-epimerase, an enzyme involved in the metabolism of the unusual Amadori compound psicoselysine in Escherichia coli.
Wiame E, Van Schaftingen E.
Laboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology and Universite Catholique de Louvain, Avenue Hippocrate 75, B-1200 Brussels, Belgium.

The frl (fructoselysine) operon encodes fructoselysine 6-kinase and fructoselysine 6-phosphate deglycase, allowing the conversion of fructoselysine into glucose 6-phosphate and lysine. We now show that a third enzyme encoded by this operon catalyses the metal-dependent reversible interconversion of fructoselysine with its C-3 epimer, psicoselysine. The enzyme can be easily assayed through the formation of tritiated water from [3-3H]fructoselysine. Psicoselysine supports the growth of Escherichia coli, causing the induction of the three enzymes of the frl operon. No growth on fructoselysine or psicoselysine was observed with Tn5 mutants in which the putative transporter (FrlA) or fructoselysine 6-phosphate deglycase (FrlB) had been inactivated, indicating the importance of the frl operon for the metabolism of both substrates. The ability of E. coli to grow on psicoselysine suggests the occurrence of this unusual Amadori compound in Nature.

PMID: 14641112

 >>>membrane chaperon?<<<

EMBO J. 2004 Jan 28;23(2):294-301.
Escherichia coli YidC is a membrane insertase for Sec-independent proteins.
Serek J, Bauer-Manz G, Struhalla G, van den Berg L, Kiefer D, Dalbey R, Kuhn A.
Institute of Microbiology and Molecular Biology, University of Hohenheim, Stuttgart, Germany.

YidC is a recently discovered bacterial membrane protein that is related to the mitochondrial Oxa1p and the Alb3 protein of chloroplasts. These proteins are required in the membrane integration process of newly synthesized proteins that do not require the classical Sec machinery. Here we demonstrate that YidC is sufficient for the membrane integration of a Sec-independent protein. Microgram amounts of the purified single-spanning Pf3 coat protein were efficiently inserted into proteoliposomes containing the purified YidC. A mutant Pf3 coat protein with an extended hydrophobic region was inserted independently of YidC into the membrane both in vivo and in vitro, but its insertion was accelerated by YidC. These results show that YidC can function separately from the Sec translocase to integrate membrane proteins into the lipid bilayer.

PMID: 14739936

***additional finding***

 >>>NADP(H)-dependent aldo-keto reductase<<<

Obmolova G, Teplyakov A, Khil PP, Howard AJ, Camerini-Otero RD, Gilliland GL.
Crystal structure of the Escherichia coli Tas protein, an NADP(H)-dependent aldo-keto reductase.
Proteins. 2003 Nov 1;53(2):323-5. No abstract available.
PMID: 14517983