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  E.coli news vol. 9  2005.2.7
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This is a digest of daily PubMed searching of E.coli new finding.

***this year finding***

 >>>ycjJKL,ordL,aldH, novel putrescine utilization pathway<<<

 J Biol Chem. 2004 Dec 8; [Epub ahead of print] A novel putrescine utilization pathway involves gamma-glutamylated intermediates of Escherichia coli K-12.
Kurihara S, Oda S, Kato K, Kim HG, Koyanagi T, Kumagai H, Suzuki H.
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto 606-8502.

A novel bacterial putrescine utilization pathway was discovered. Seven genes, the functions of whose products were not known, are involved in this novel pathway. Five of them encode enzymes which catabolize putrescine, one encodes a putrescine importer, and the other, a transcriptional regulator. This novel pathway involves six sequential steps: 1) import of putrescine, 2) ATP-dependent gamma-glutamylation of putrescine, 3) oxidization of gamma-glutamyl-putrescine (gamma-Glu-Put), 4) dehydrogenation of gamma-glutamyl-gamma-aminobutyraldehyde, 5) hydrolysis of the gamma-glutamyl linkage of gamma-glutamyl-gamma-aminobutyrate (gamma-Glu-GABA), and 6) transamination of gamma-aminobutyrate (GABA) to form the final product of this pathway, succinate semialdehyde, which is the precursor of succinate.

PMID: 15590624

 >>>hrpA, ATP-dependent DEAH-box RNA helicase, involved in mRNA processing of a fimbrial operon.<<<

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

 >>>cedA, ycaL -> RapBC, RNAP assosiated protein B and C<<<

Nature. 2005 Feb 3;433(7025):531-7.
Interaction network containing conserved and essential protein complexes in Escherichia coli.
Butland G, Peregrin-Alvarez JM, Li J, Yang W, Yang X, Canadien V, Starostine A, Richards D, Beattie B, Krogan N, Davey M, Parkinson J, Greenblatt J, Emili A.
Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6, Canada.

Proteins often function as components of multi-subunit complexes. Despite its long history as a model organism, no large-scale analysis of protein complexes in Escherichia coli has yet been reported. To this end, we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames (approximately 23% of the genome). A total of 857 proteins, including 198 of the most highly conserved, soluble non-ribosomal proteins essential in at least one bacterial species, were tagged successfully, whereas 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry. An interaction network of protein complexes involved in diverse biological processes was uncovered and validated by sequential rounds of tagging and purification. This network includes many new interactions as well as interactions predicted based solely on genomic inference or limited phenotypic data.
This study provides insight into the function of previously uncharacterized bacterial proteins and the overall topology of a microbial interaction network, the core components of which are broadly conserved across Prokaryota.

PMID: 15690043

 >>>ygfE, FtsZ-regulating proteins ZapA homolog<<<

Genes Dev. 2005 Jan 1;19(1):127-37.
Premature targeting of a cell division protein to midcell allows dissection of divisome assembly in Escherichia coli.
Goehring NW, Gueiros-Filho F, Beckwith J.
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

Cell division in Escherichia coli requires the recruitment of at least 10 essential proteins to the bacterial midcell. Recruitment of these proteins follows a largely linear dependency pathway in which depletion of one cell division protein leads to the absence from the division site of "downstream" proteins in the pathway. Analysis of events that underlie this pathway is complicated by the fact that a protein's ability to recruit "downstream" proteins is dependent on its own recruitment by "upstream" proteins. Hence, one cannot separate the individual contributions of various upstream proteins to any specific recruitment step. Here we present a method-premature targeting-for bypassing the normal localization requirements of a cell division protein and apply it to FtsQ, a protein recruited midway through the pathway. We fused FtsQ to the FtsZ-binding protein ZapA such that FtsQ was targeted to FtsZ rings independently of proteins FtsA and FtsK, which are normally r equired for FtsQ localization. Analysis of the resulting ZapA-FtsQ fusion suggests that FtsQ associates with a large complex of cell division proteins and that premature targeting of FtsQ can restore localization of this complex under conditions in which neither FtsQ nor the associated proteins would normally be localized.

PMID: 15630023

J Bacteriol. 2004 Apr;186(8):2418-29.
ZipA is required for targeting of DMinC/DicB, but not DMinC/MinD, complexes to septal ring assemblies in Escherichia coli.
Johnson JE, Lackner LL, Hale CA, de Boer PA.
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4960, USA.

The MinC division inhibitor is required for accurate placement of the septal ring at the middle of the Escherichia coli cell. The N-terminal domain of MinC ((Z)MinC) interferes with FtsZ assembly, while the C-terminal domain ((D)MinC) mediates both dimerization and complex formation with either MinD or DicB. Binding to either of these activators greatly enhances the division-inhibitory activity of MinC in the cell. The MinD ATPase plays a crucial role in the rapid pole-to-pole oscillation of MinC that is proposed to force FtsZ ring formation to midcell. DicB is encoded by one of the cryptic prophages on the E. coli chromosome (Qin) and is normally not synthesized. Binding of MinD or DicB to (D)MinC produces complexes that have high affinities for one or more septal ring-associated targets. Here we show that the FtsZ-binding protein ZipA is required for both recruitment of the (D)MinC/DicB complex to FtsZ rings and the DicB-inducible division block normally seen in
MinC(+) cells. In contrast, none of the known FtsZ-associated factors, including ZipA, FtsA, and ZapA, appear to be specifically required for targeting of the (D)MinC/MinD complex to rings, implying that the two MinC/activator complexes must recognize distinct features of FtsZ assemblies. MinD-dependent targeting of MinC may occur in two steps of increasing topological specificity: (i) recruitment of MinC from the cytoplasm to the membrane, and (ii) specific targeting of the MinC/MinD complex to nascent septal ring assemblies on the membrane. Using membrane-tethered derivatives of MinC, we obtained evidence that both of these steps contribute to the efficiency of MinC/MinD-mediated division inhibition.

PMID: 15060045

Genes Dev. 2002 Oct 1;16(19):2544-56.
A widely conserved bacterial cell division protein that promotes assembly of the tubulin-like protein FtsZ.
Gueiros-Filho FJ, Losick R.
Department of Molecular and Cellular Biology, Harvard University, Cambridge Massachusetts 02138, USA.

Cell division in bacteria is mediated by the tubulin-like protein FtsZ, which assembles into a structure known as the Z ring at the future site of cytokinesis. We report the discovery of a Z-ring-associated protein in Bacillus subtilis called ZapA. ZapA was found to colocalize with the Z ring in vivo and was capable of binding to FtsZ and stimulating the formation of higher-order assemblies of the cytokinetic protein in vitro. The absence of ZapA alone did not impair cell viability, but the absence of ZapA in combination with the absence of a second, dispensable division protein EzrA caused a severe block in cytokinesis. The absence of ZapA also caused lethality in cells producing lower than normal levels of FtsZ or lacking the division-site-selection protein DivIVA. Conversely, overproduction of ZapA reversed the toxicity of excess levels of the division inhibitor MinD. In toto, the evidence indicates that ZapA is part of the cytokinetic machinery of the cell and acts by promoting Z-ring formation. Finally, ZapA is widely conserved among bacteria with apparent orthologs in many species, including Escherichia coli, in which the orthologous protein exhibited a strikingly similar pattern of subcellular localization to that of ZapA. Members of the ZapA family of proteins are likely to be a common feature of the cytokinetic machinery in bacteria.

PMID: 12368265

 >>>MicC<<<
J Bacteriol. 2004 Oct;186(20):6689-97.
MicC, a second small-RNA regulator of Omp protein expression in Escherichia coli.
Chen S, Zhang A, Blyn LB, Storz G.
IBIS Therapeutics, ISIS Pharmaceuticals, Inc., Carlsbad, California, USA.

In a previous bioinformatics-based search for novel small-RNA genes encoded by the Escherichia coli genome, we identified a region, IS063, located between the ompN and ydbK genes, that encodes an approximately 100-nucleotide small-RNA transcript. Here we show that the expression of this small RNA is increased at a low temperature and in minimal medium. Twenty-two nucleotides at the 5' end of this transcript have the potential to form base pairs with the leader sequence of the mRNA encoding the outer membrane protein OmpC. The deletion of IS063 increased the expression of an ompC-luc translational fusion 1.5- to 2-fold, and a 10-fold overexpression of the small RNA led to a 2- to 3-fold repression of the fusion. Deletion and overexpression of the IS063 RNA also resulted in increases and decreases, respectively, in OmpC protein levels. Taken together, these results suggest that IS063 is a regulator of OmpC expression; thus, the small RNA has been renamed MicC. The an tisense regulation was further demonstrated by the finding that micC mutations were suppressed by compensatory mutations in the ompC mRNA. MicC was also shown to inhibit ribosome binding to the ompC mRNA leader in vitro and to require the Hfq RNA chaperone for its function. We suggest that the MicF and MicC RNAs act in conjunction with the EnvZ-OmpR two-component system to control the OmpF/OmpC protein ratio in response to a variety of environmental stimuli.

PMID: 15466019

***additional finding***

 >>>YbgF, TolA binding protien<<<
Mol Microbiol. 2002 May;44(3):695-708.
The Tol/Pal system function requires an interaction between the C-terminal domain of TolA and the N-terminal domain of TolB.
Walburger A, Lazdunski C, Corda Y.
Laboratoire d'Ingenierie des Systemes Macromoleculaires, Institut de Biologie Structurale et Microbiologie, CNRS 31, Chemin Joseph Aiguier, Marseille, France.

The Tol/Pal system of Escherichia coli is composed of the YbgC, TolQ, TolA, TolR, TolB, Pal and YbgF proteins. It is involved in maintaining the integrity of the outer membrane, and is required for the uptake of group A colicins and DNA of filamentous bacteriophages. To identify new interactions between the components of the Tol/Pal system and gain insight into the mechanism of colicin import, we performed a yeast two-hybrid screen using the different components of the Tol/Pal system and colicin A. Using this system, we confirmed the already known interactions and identified several new interactions. TolB dimerizes and the periplasmic domain of TolA interacts with YbgF and TolB. Our results indicate that the central domain of TolA (TolAII) is sufficient to interact with YbgF, that the C-terminal domain of TolA (TolAIII) is sufficient to interact with TolB, and that the amino terminal domain of TolB (D1) is sufficient to bind TolAIII. The TolA/TolB interaction was c onfirmed by cross-linking experiments on purified proteins. Moreover, we show that the interaction between TolA and TolB is required for the uptake of colicin A and for the membrane integrity. These results demonstrate that the TolA/TolB interaction allows the formation of a trans-envelope complex that brings the inner and outer membranes in close proximity.

PMID: 11994151 [PubMed - indexed for MEDLINE]

 >>>Ribosome-associated cold shock response protein<<<

 Biochem Biophys Res Commun. 2002 Dec 20;299(5):710-4.
Solution structure of the ribosome-associated cold shock response protein Yfia of Escherichia coli.
Rak A, Kalinin A, Shcherbakov D, Bayer P.
Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Str.11, 44227 Dortmund, Germany.

The solution structure of the ribosome-associated cold shock response protein Yfia of Escherichia coli was determined by nuclear magnetic resonance with a RMSD of 0.6A. Yfia shows a global beta-alpha-beta-beta-beta-alpha folding topology similar to its homologue HI0257 of Haemophilus influenzae and the double-strand-binding domain of Drosophila Staufen protein. Yfia and HI0257 differ in their surface charges and in the composition of their flexible C-termini, indicating their specificity to different target molecules. Both proteins exhibit a hydrophobic and polar region, which probably functions as interaction site for protein complex formation. Despite their similarity to the dsRBD fold, Yfia does not bind to model fragments of 16S ribosomal RNA as determined by NMR titration and gel shift experiments.

PMID: 12470636

 >>>yhbH, Ribosome-associated factor Y<<<

Eur J Biochem. 2002 Nov;269(21):5182-91.
Ribosome-associated factor Y adopts a fold resembling a double-stranded RNA binding domain scaffold.
Ye K, Serganov A, Hu W, Garber M, Patel DJ.
Cellular Biochemistry & Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, USA.

Escherichia coli protein Y (pY) binds to the small ribosomal subunit and stabilizes ribosomes against dissociation when bacteria experience environmental stress. pY inhibits translation in vitro, most probably by interfering with the binding of the aminoacyl-tRNA to the ribosomal A site. Such a translational arrest may mediate overall adaptation of cells to environmental conditions. We have determined the 3D solution structure of a 112-residue pY and have studied its backbone dynamic by NMR spectroscopy. The structure has a betaalphabetabetabetaalpha topology and represents a compact two-layered sandwich of two nearly parallel alpha helices packed against the same side of a four-stranded beta sheet. The 23 C-terminal residues of the protein are disordered. Long-range angular constraints provided by residual dipolar coupling data proved critical for precisely defining the position of helix 1. Our data establish that the C-terminal region of helix 1 and the loop link ing this helix with strand beta2 show significant conformational exchange in the ms- micro s time scale, which may have relevance to the interaction of pY with ribosomal subunits. Distribution of the conserved residues on the protein surface highlights a positively charged region towards the C-terminal segments of both alpha helices, which most probably constitutes an RNA binding site. The observed betaalphabetabetabetaalpha topology of pY resembles the alphabetabetabetaalpha topology of double-stranded RNA-binding domains, despite limited sequence similarity. It appears probable that functional properties of pY are not identical to those of dsRBDs, as the postulated RNA-binding site in pY does not coincide with the RNA-binding surface of the dsRBDs.

PMID: 12392550

 >>>YfiA and YhbH, associated with resting ribosomes<<<

Genes Cells. 2000 Dec;5(12):965-74.
Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli.
Maki Y, Yoshida H, Wada A.
Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan.

BACKGROUND: Ribosomes in Escherichia coli change their composition and conformation in the stationary phase. Ribosome modulation factor (RMF) and ribosomal protein S22 are known to be associated with stationary phase ribosomes. RMF association causes the loss of translational activity and the dimerization of 70S ribosomes into 100S ribosomes, which may increase cell survival in the stationary phase. RESULTS: Two weakly acidic proteins having related amino acid sequences were found to be associated with E. coli ribosomes in the stationary phase. These proteins are the products of ORFs named yfiA and yhbH. The sum of the copy numbers of their product proteins, YfiA and YhbH, in the ribosomal particles was low in the log phase, but increased to nearly one in the stationary phase. YfiA was found in the 70S ribosomal fraction rather than the 100S. On the other hand, YhbH was detected exclusively in the 100S ribosomal fraction. When the stationary phase cells were transf erred to fresh medium, YfiA and YhbH were found in the 70S ribosomal fraction, but not in the polysome fraction. CONCLUSIONS: Two proteins, YfiA and YhbH, associated with E. coli ribosomes were found to accumulate in the stationary phase, leading to the formation of several types of ribosomes. They are not likely to have roles in the elongation step of the translation in log phase cells, but are likely to be involved in the stabilization and preservation of ribosomes in the stationary phase, which might be necessary for cell survival.

***additional finding (homolog)***

 >>>ybgC may be hydrolase of short-chain acyl-CoA thioesters<<<
PMID: 11168583

FEBS Lett. 2002 Apr 10;516(1-3):161-3.
The YbgC protein encoded by the ybgC gene of the tol-pal gene cluster of Haemophilus influenzae catalyzes acyl-coenzyme A thioester hydrolysis.
Zhuang Z, Song F, Martin BM, Dunaway-Mariano D.
Department of Chemistry, University of New Mexico, Albuquerque, NM 87131, USA.

This paper examines the catalytic function of the protein YbgC, encoded by the ybgC gene of the tol-pal gene cluster in Haemophilus influenzae. The YbgC protein, a homologue of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-coenzyme A thioesterase, conserves the active site Asp residue associated with thioesterase activity. The H. influenzae ybgC gene was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and tested for thioesterase activity towards acyl-CoA and acyl-N-acetylcysteamine thioesters. The YbgC protein catalyzes the hydrolysis of short chain aliphatic acyl-CoA thioesters, while the D18N YbgC mutant protein (prepared to serve as a control) does not.

PMID: 11959124