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  E.coli news vol. 7  2004.9.8
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This is a digest of daily PubMed searching of E.coli new finding.

***this year finding***

 >>>alpha-xylosidase and alpha-glucosidase<<<

Protein Expr Purif. 2004 Sep;37(1):170-9. 

Overexpression and characterization of two unknown proteins, YicI and YihQ, originated from Escherichia coli.
Okuyama M, Mori H, Chiba S, Kimura A.
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.

The proteins encoded in the yicI and yihQ gene of Escherichia coli have similarities in the amino acid sequences to glycoside hydrolase family 31 enzymes, but they have not been detected as the active enzymes. The functions of the two proteins have been first clarified in this study. Recombinant YicI and YihQ produced in E. coli were purified and characterized. YicI has the activity of alpha-xylosidase. YicI existing as a hexamer shows optimal pH at 7.0 and is stable in the pH range of 4.7-10.1 with incubation for 24h at 4 degrees C and also is stable up to 47 degrees C with incubation for 15min. The enzyme shows higher activity against alpha-xylosyl fluoride, isoprimeverose (6- [Formula: see text] -alpha-xylopyranosyl-glucopyranose), and alpha-xyloside in xyloglucan oligosaccharides. The alpha-xylosidase catalyzes the transfer of alpha-xylosyl residue from alpha-xyloside to xylose, glucose, mannose, fructose, maltose, isomaltose, nigerose, kojibiose, sucrose, and trehalose. YihQ exhibits the hydrolysis activity against alpha-glucosyl fluoride, and so is an alpha-glucosidase, although the natural substrates, such as alpha-glucobioses, are scarcely hydrolyzed. alpha-Glucosidase has been found for the first time in E. coli.

PMID: 15294295 [PubMed - in process]

 >>>menaquinone-dependent disulfide bond formation pathway<<<

 J Biol Chem. 2004 Aug 30 [Epub ahead of print]

Characterization of menaquinone-dependent disulfide bond formation pathway of Escherichia coli.
Takahashi YH, Inaba K, Ito K.
Institute for Virus Research, Kyoto University, Kyoto, Kyoto 606-8507.

In the protein disulfide-introducing system of E. coli, plasma membrane-integrated DsbB oxidizes peiplasmic DsbA, the primary disulfide donor. While the DsbA-DsbB system utilizes oxidizing power of ubiquinone under aerobic conditions, menaquinone is believed to function as an immediate electron acceptor under anaerobic conditions. Here, we characterized menaquinone reactivities with DsbB. In the absence of ubiquinone, DsbB was complexed with menaquinone-8 in the cell. In vitro studies showed that, by binding to DsbB in a manner competitive with ubiquinone, menaquinone oxidizes specifically the Cys41 and Cys44 residues of DsbB and activates its catalytic function to oxidize reduced DsbA. In contrast, menadione used in some earlier studies proved to be a more non-specific oxidant of DsbB. During the catalysis, menaquinone-8 undergoes a spectroscopic transition to develop visible violet color (lambd max, 550 nm), which required a reduced state of Cys44 as shown previo usly for the ubiquinone color development (lambda max, 500 nm) on DsbB. In an in vitro reaction system of menaquinone-8 dependent oxidation of DsbA at 30 degrees C, two reaction components were observed, one completing within minutes and the other taking more than one hour. Both of these modes of reactions were accompanied by the transition state of menaquinone, among which the slower reaction proceeded through disulfide-linked DsbA-DsbB-menaquinone intermediate. The menaquinone-dependent pathway provides opportunities to further dissect the quinone-depedent DsbB-DsbA redox reactions.

PMID: 15347648

 >>>Zinc phosphodiesterase<<<

Biochem J. 2004 Aug 23;Pt. [Epub ahead of print]

Zinc and iron dependent cytosolic metallo-beta-lactamase domain proteins exhibit similar zinc binding affinities, independently of an atypical glutamate at the metal binding site.
Schilling O, Vogel A, Kostelecky B, Natal Da Luz H, Spemann D, Spath B, Marchfelder A, Troger W, Meyer-Klaucke W.

Zinc phosphodiesterase (ZiPD, synonyms are ElaC, ecoZ, RNaseZ, 3' tRNase) and the iron dependent redox enzyme flavorubredoxin (FlRd) from Escherichia coli represent prototypical cases of proteins sharing the metallo-beta-lactamase fold that require strict metal selectivity for catalytic activity; yet their metal selectivity has only been partially understood. In contrast to hydrolytic metallo-beta-lactamase proteins, iron dependent FlRd-like enzymes have an atypical glutamate ligand which replaces one otherwise conserved histidine ligand. X-ray absorption spectroscopy revealed that the FlRd metallo-beta-lactamase domain is capable of incorporating two zinc ions into the binuclear metal binding site. Zinc dissociation constants, determined by isothermal titration calorimetry are similar for zinc binding to E. coli ZiPD (K (d) (1) = 2.2 +/- 0.2 microM, K (d) (2) = 23.0 +/- 0.6 microM) and to the E. coli FlRd metallo-beta-lactamase domain (K (d) (1) = 0.7 +/- 0.1 micr oM, K (d) (2) = 26.0 +/- 0.1 microM). In good correspondence, apo-ZiPD requires incubation with 10 microM zinc for full reconstitution of the phosphodiesterase activity. Accordingly, metal selectivity of ZiPD and FlRd only partially relies on first shell metal ligands. Back mutation of the atypical glutamate in FlRd to a histidine unexpectedly resulted in an elevated first zinc dissociation constant (K (d) (1) = 30 +/- 4 microM and K (d) (2) = 23 +/- 2 microM). In combination with a recent mutational study on ZiPD (Vogel, A., Schilling, O., Meyer-Klaucke, W., Biochemistry, in press) we conclude that the atypical glutamate does not guide metal selectivity of the FlRd metallo-beta-lactamase domain but suppresses possible hydrolytic cross activity.

PMID: 15324305

 >>>DEAH-box RNA helicase in mRNA processing<<<

Mol Microbiol. 2004 Jun;52(6):1813-26. 

HrpA, a DEAH-box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli.
Koo JT, Choe J, Moseley SL.
Department of Microbiology, University of Washington, Box 357242, Seattle, WA 98195-7242, USA.

Endonucleolytic cleavage of mRNA in the daa operon of Escherichia coli is responsible for co-ordinate regulation of genes involved in F1845 fimbrial biogenesis. Cleavage occurs by an unidentified endoribonuclease, is translation dependent and involves a unique recognition mechanism. Here, we present the results of a genetic strategy used to identify factors involved in daa mRNA processing. We used a reporter construct consisting of the daa mRNA processing region fused to the gene encoding green fluorescent protein (GFP). A mutant defective in daa mRNA processing and expressing high levels of GFP was isolated by flow cytometry. To determine the location of mutations, two different genetic approaches, Hfr crosses and P1 transductions, were used. The mutation responsible for the processing defect was subsequently mapped to the 32 min region of the E. coli chromosome. A putative DEAH-box RNA helicase-encoding gene at this position, hrpA, was able to restore the ability  of the mutant to cleave daa mRNA. Site-directed mutagenesis of the hrpA regions predicted to encode nucleotide triphosphate binding and hydrolysis functions abolished the ability of the gene to restore the processing defect in the mutant. We propose that HrpA is a novel enzyme involved in mRNA processing in E. coli.

PMID: 15186427

 >>>PTS system for 2-O-alpha-mannosyl-D-glycerate<<<

J Biol Chem. 2004 Feb 13;279(7):5537-48. Epub 2003 Nov 25.

Phosphotransferase-mediated transport of the osmolyte 2-O-alpha-mannosyl-D-glycerate in Escherichia coli occurs by the product of the mngA (hrsA) gene and is regulated by the mngR (farR) gene product acting as repressor.
Sampaio MM, Chevance F, Dippel R, Eppler T, Schlegel A, Boos W, Lu YJ, Rock CO.
Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal.

2-O-alpha-mannosyl-D-glycerate (MGs) has been recognized as an osmolyte in hyperthermophilic but not mesophilic prokaryotes. We report that MG is taken up and utilized as sole carbon source by Escherichia coli K12, strainMC4100. Uptake is mediated by the P-enolpyruvate-dependent phosphotransferase system with the MG-inducible HrsA (now called MngA) protein as its specific EIIABC complex. The apparent Km of MG uptake in induced cells was 10 microm, and the Vmax was 0.65 nmol/min/10(9) cells. Inverted membrane vesicles harboring plasmid-encoded MngA phosphorylated MG in a P-enolpyruvate-dependent manner. A deletion mutant in mngA was devoid of MG transport but is complemented by a plasmid harboring mngA. Uptake of MG in MC4100 also caused induction of a regulon specifying the uptake and the metabolism of galactarate and glucarate controlled by the CdaR activator. The ybgG gene (now called mngB) the gene immediately downstream of mngA encodes a protein with alpha-mann osidase activity. farR, the gene upstream of mngA (now called mngR) had previously been characterized as a fatty acyl-responsive regulator; however, deletion of mngR resulted in the up-regulation of only two genes, mngA and mngB. The mngR deletion caused constitutive MG transport that became MG-inducible after transformation with plasmid expressed mngR. Thus, MngR is the regulator (repressor) of the MG transport/metabolism system. Thus, the mngR mngA mngB gene cluster encodes an MG utilizing system.
PMID: 14645248

 >>>tRNA 4-thiouridine modification enzyme<<<

J Biol Chem. 2004 May 28;279(22):23022-9.

Substrate specificity for 4-thiouridine modification in Escherichia coli.
Lauhon CT, Erwin WM, Ton GN.
School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705, USA.

The biosynthesis of 4-thiouridine (s4U) in Escherichia coli tRNA requires the action of both the thiamin pathway enzyme ThiI and the cysteine desulfurase IscS. IscS catalyzes sulfur transfer from l-cysteine to ThiI, which utilizes Mg-ATP to activate uridine 8 in tRNA and transfers sulfur to give s4U. In this work, we show through deletion analysis of unmodified E. coli tRNA(Phe) that the minimum substrate for s4U modification is a mini-helix comprising the stacked acceptor and T stems containing an internal bulged region. The size of the bulged loop must be at least 4 nucleotides and contain the target uridine as the first nucleotide. Replacement of the T loop sequence with a tetraloop in the deletion substrate increases activity and shows that the TpsiC primary sequence is not a recognition element. An unmodified tRNA(Phe) transcript in which the 3'-terminal ACCA sequence is removed to give a blunt terminus has <0.1% activity, although the addition of a single ove rhanging base essentially restores activity. In addition, reducing the distance of the 3' terminus relative to U8 by as little as 1 bp severely impairs activity. By dissecting a minimal RNA substrate in the T loop region, a two-piece system consisting of a substrate RNA and a "guide" RNA is efficiently modified. Our results indicate that outside of the modified U8, there is no primary sequence requirement for substrate recognition. However, the secondary and tertiary structure restrictions appear sufficient to explain why s4U modification is limited in the cell to tRNA.

PMID: 15037613

J Biol Chem. 2004 May 7;279(19):19551-8.

Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo.
Lauhon CT, Skovran E, Urbina HD, Downs DM, Vickery LE.
School of Pharmacy, University of Wisconsin, Madison, WI 53705, USA.

IscS catalyzes the fragmentation of l-cysteine to l-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys(328) resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys(328) identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s(2)C and ms(2)i(6)A) yet showed wild type levels of Fe-S-independent thionucleosides (s(4)U and mnm(5)s(2)U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfura se activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

PMID: 14978044

J Biol Chem. 2004 Apr 23;279(17):17054-62.
Thiamine biosynthesis in Escherichia coli: in vitro reconstitution of the thiazole synthase activity.
Leonardi R, Roach PL.
School of Chemistry, University of Southampton, Southampton SO17 1BJ, United Kingdom.

The biosynthesis of thiamine in Escherichia coli requires the formation of an intermediate thiazole from tyrosine, 1-deoxy-d-xylulose-5-phosphate (Dxp), and cysteine using at least six structural proteins, ThiFSGH, IscS, and ThiI. We describe for the first time the reconstitution of thiazole synthase activity using cell-free extracts and proteins derived from adenosine-treated E. coli 83-1 cells. The addition of adenosine or adenine to growing cultures of Aerobacter aerogenes, Salmonella typhimurium, and E. coli has been shown previously to relieve the repression by thiamine of its own biosynthesis and increase the expression levels of the thiamine biosynthetic enzymes. By exploiting this effect, we show that the in vitro thiazole synthase activity of cleared lysates or desalted proteins from E. coli 83-1 cells is dependent upon the addition of purified ThiGH-His complex, tyrosine (but not cysteine or 1-deoxy-d-xylulose-5-phosphate), and an as yet unidentified inte rmediate present in the protein fraction from these cells. The activity is strongly stimulated by the addition of S-adenosylmethionine and NADPH.

PMID: 14757766 [PubMed - indexed for MEDLINE]

 >>>aminoacylate glutamyl-tRNA synthetase<<<

Proc Natl Acad Sci U S A. 2004 May 18;101(20):7530-5.
Comment in:
Proc Natl Acad Sci U S A. 2004 May 18;101(20):7493-4.
An aminoacyl-tRNA synthetase-like protein encoded by the Escherichia coli yadB gene glutamylates specifically tRNAAsp.
Dubois DY, Blaise M, Becker HD, Campanacci V, Keith G, Giege R, Cambillau C, Lapointe J, Kern D.

Departement de Biochimie et Microbiologie, Faculte de Sciences et de Genie, Centre de Recherche sur la Fonction, la Structure et l'Ingenierie des Proteines, Universite Laval, Quebec, QC, Canada G1K 7P4.

The product of the Escherichia coli yadB gene is homologous to the N-terminal part of bacterial glutamyl-tRNA synthetases (GluRSs), including the Rossmann fold with the acceptor-binding domain and the stem-contact fold. This GluRS-like protein, which lacks the anticodon-binding domain, does not use tRNA(Glu) as substrate in vitro nor in vivo, but aminoacylates tRNA(Asp) with glutamate. The yadB gene is expressed in wild-type E. coli as an operon with the dksA gene, which encodes a protein involved in the general stress response by means of its action at the translational level. The fate of the glutamylated tRNA(Asp) is not known, but its incapacity to bind elongation factor Tu suggests that it is not involved in ribosomal protein synthesis. Genes homologous to yadB are present only in bacteria, mostly in Proteobacteria. Sequence alignments and phylogenetic analyses show that the YadB proteins form a distinct monophyletic group related to the bacterial and organella r GluRSs (alpha-type GlxRSs superfamily) with ubiquitous function as suggested by the similar functional properties of the YadB homologue from Neisseria meningitidis.

PMID: 15096594 [PubMed - indexed for MEDLINE]

 >>>tRNA pseudouridine synthase<<<

Acta Crystallogr D Biol Crystallogr. 2004 Apr;60(Pt 4):775-6.

Expression, purification, crystallization and preliminary diffraction studies of the tRNA pseudouridine synthase TruD from Escherichia coli.
Ericsson UB, Andersson ME, Engvall B, Nordlund P, Hallberg BM.
Department of Biochemistry and Biophysics, Stockholm University, Roslagstullsbacken 15, SE-114 21 Stockholm, Sweden.

Pseudouridine, the 5-ribosyl isomer of uridine, is the most common modification of structural RNA. The recently identified pseudouridine synthase TruD belongs to a widespread class of pseudouridine synthases without significant sequence homology to previously known families. TruD from Escherichia coli was overexpressed, purified and crystallized. The crystals diffract to a minimum Bragg spacing of 2.4 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 63.4, b = 108.6, c = 111.7 A.

PMID: 15039583 [PubMed]

 >>>GadE<<<

Microbiology. 2004 Jan;150(Pt 1):61-72. 

GadE (YhiE): a novel activator involved in the response to acid environment in Escherichia coli.
Hommais F, Krin E, Coppee JY, Lacroix C, Yeramian E, Danchin A, Bertin P.
Unite de Genetique des Genomes Bacteriens, URA CNRS 2185, Institut Pasteur, France.

In several Gram-positive and Gram-negative bacteria glutamate decarboxylases play an important role in the maintenance of cellular homeostasis in acid environments. Here, new insight is brought to the regulation of the acid response in Escherichia coli. Overexpression of yhiE, similarly to overexpression of gadX, a known regulator of glutamate decarboxylase expression, leads to increased resistance of E. coli strains under high acid conditions, suggesting that YhiE is a regulator of gene expression in the acid response. Target genes of both YhiE (renamed GadE) and GadX were identified by a transcriptomic approach. In vitro experiments with GadE purified protein provided evidence that this regulator binds to the promoter region of these target genes. Several of them are clustered together on the chromosome and this chromosomal organization is conserved in many E. coli strains. Detailed structural (in silico) analysis of this chromosomal region suggests that the prom oters of the corresponding genes are preferentially denatured. These results, along with the G+C signature of the chromosomal region, support the existence of a fitness island for acid adaptation on the E. coli chromosome.

PMID: 14702398

***additional finding***

 >>>tRNA pseudouridylate synthase<<<

RNA. 2003 Jun;9(6):711-21. 

A novel unanticipated type of pseudouridine synthase with homologs in bacteria, archaea, and eukarya.
Kaya Y, Ofengand J.
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, USA.

Putative pseudouridine synthase genes are members of a class consisting of four subgroups that possess characteristic amino acid sequence motifs. These genes have been found in all organisms sequenced to date. In Escherichia coli, 10 such genes have been identified, and the 10 synthase gene products have been shown to function in making all of the pseudouridines found in tRNA and ribosomal RNA except for tRNA(Glu) pseudouridine13. In this work, a protein able to make this pseudouridine was purified by standard biochemical procedures. Amino-terminal sequencing of the isolated protein identified the synthase as YgbO. Deletion of the ygbO gene caused the loss of tRNA(Glu) pseudouridine13 and plasmid-borne restoration of the structural gene restored pseudouridine13. Reaction of the overexpressed gene product, renamed TruD, with a tRNA(Glu) transcript made in vitro also yielded only pseudouridine13. A search of the database detected 58 homologs of TruD spanning all thre e phylogenetic domains, including ancient organisms. Thus, we have identified a new wide-spread class of pseudouridine synthase with no sequence homology to the previously known four subgroups. The only completely conserved sequence motif in all 59 organisms that contained aspartate was GXKD, in motif II. This aspartate was essential for in vitro activity.

PMID: 12756329

 >>>barA/uvrY->csrAB signal transduction<<<

J Bacteriol. 2003 Feb;185(3):843-53. 

The Escherichia coli BarA-UvrY two-component system is needed for efficient switching between glycolytic and gluconeogenic carbon sources.
Pernestig AK, Georgellis D, Romeo T, Suzuki K, Tomenius H, Normark S, Melefors O.
Microbiology and Tumorbiology Center, Karolinska Institutet, SE-17177 Stockholm, Sweden.

The Escherichia coli BarA and UvrY proteins were recently demonstrated to constitute a novel two-component system, although its function has remained largely elusive. Here we show that mutations in the sensor kinase gene, barA, or the response regulator gene, uvrY, in uropathogenic E. coli drastically affect survival in long-term competition cultures. Using media with gluconeogenic carbon sources, the mutants have a clear growth advantage when competing with the wild type, but using media with carbon sources feeding into the glycolysis leads to a clear growth advantage for the wild type. Results from competitions with mutants in the carbon storage regulation system, CsrA/B, known to be a master switch between glycolysis and gluconeogenesis, led us to propose that the BarA-UvrY two-component system controls the Csr system. Taking these results together, we propose the BarA-UvrY two-component system is crucial for efficient adaptation between different metabolic path ways, an essential function for adaptation to a new environment.

PMID: 12533459 [PubMed - indexed for MEDLINE]

 >>>Zinc phosphodiesterase<<<

J Biol Chem. 2002 Aug 9;277(32):29078-85. Epub 2002 May 23.

ElaC encodes a novel binuclear zinc phosphodiesterase.
Vogel A, Schilling O, Niecke M, Bettmer J, Meyer-Klaucke W.
European Molecular Biology Laboratory Outstation Hamburg, Notkestr. 85, 22603 Hamburg, Germany.

ElaC is a widespread gene found in eubacteria, archaebacteria, and mammals with a highly conserved sequence. Two human ElaC variants were recently associated with cancer (Tavtigian, S. V., Simard, J., Teng, D. H., Abtin, V., Baumgard, M., Beck, A., Camp, N. J., Carillo, A. R., Chen, Y., Dayananth, P., Desrochers, M., Dumont, M., Farnham, J. M., Frank, D., Frye, C., Ghaffari, S., Gupte, J. S., Hu, R., Iliev, D., Janecki, T., Kort, E. N., Laity, K. E., Leavitt, A., Leblanc, G., McArthur-Morrison, J., Pederson, A., Penn, B., Peterson, K. T., Reid, J. E., Richards, S., Schroeder, M., Smith, R., Snyder, S. C., Swedlund, B., Swensen, J., Thomas, A., Tranchant, M., Woodland, A. M., Labrie, F., Skolnick, M. H., Neuhausen, S., Rommens, J., and Cannon-Albright, L. A. (2001) Nat. Genet. 27, 172-180; Yanaihara, N., Kohno, T., Takakura, S., Takei, K., Otsuka, A., Sunaga, N., Takahashi, M., Yamazaki, M., Tashiro, H., Fukuzumi, Y., Fujimori, Y., Hagiwara, K., Tanaka, T., and Yoko ta, J. (2001) Genomics 72, 169-179). Analysis of the primary sequence indicates homology to an arylsulfatase and predicts a metallo-beta-lactamase fold. At present, no ElaC gene product has been investigated. We cloned the Escherichia coli ElaC gene and purified the recombinant gene product. An enzymatic analysis showed that ElaC does not encode an arylsulfatase but rather encodes a phosphodiesterase that hydrolyzes bis(p-nitrophenyl)phosphate with a k(cat) of 59 s(-1) and K' of 4 mm. Kinetic analysis of the dimeric enzyme revealed positive cooperativity for the substrate bis(p-nitrophenyl)phosphate with a Hill coefficient of 1.6, whereas hydrolysis of the substrate thymidine-5'-p-nitrophenyl phosphate followed Michaelis-Menten kinetics. Furthermore, the enzyme is capable of binding two zinc or two iron ions. However, it displays phosphodiesterase activity only in the zinc form. The metal environment characterized by zinc K-edge x-ray absorption spectroscopy was mo deled with two histidine residues, one carboxylate group, and 1.5 oxygen atoms. This corresponds to the coordination found in other metallo-beta-lactamase domain proteins. Phosphodiesterase activity is strongly dependent on the presence of zinc. These results identify the currently unassigned gene product ElaC to be a novel binuclear zinc phosphodiesterase.

PMID: 12029081

 >>>Pseudouridylate synthase<<<

Ribosomal large subunit pseudouridine synthase F (EC 4.2.1.70) (Pseudouridylate synthase) (Uracil hydrolyase).
tRNA pseudouridine synthase A (EC 4.2.1.70)

RNA. 2001 Nov;7(11):1603-15. 
Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli.
Del Campo M, Kaya Y, Ofengand J.
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.

There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, respectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseudouridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(Ile1) and tRNA(Asp). Deletion of each of these synthase genes individually had no effect on exponential growth in rich media at 25 degrees C, 37 degrees C, or 42 degrees C. A strain lacking RluB and RluF also showed no grow th defect under these conditions. Mutation of a conserved aspartate in a common sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, also caused a loss of in vivo activity in all four of the synthases studied in this work.

PMID: 11720289