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  E.coli news vol. 11  2005.8.9
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This is a digest of daily PubMed searching of E.coli new finding.

***this year finding***

 >>>sequence-specific endoribonuclease that cleaves mRNAs<<<

J Biol Chem. 2005 May 18; [Epub ahead of print] Characterization of ChpBK, an mRNA interferase from Escherichia coli.
Zhang Y, Zhu L, Zhang J, Inouye M.
Biochemistry Dept., Robert Wood Johnson Medical School, Piscataway, NJ 08904.

Escherichia coli contains a number of antitoxin-toxin modules on its chromosome, which are responsible for cell growth arrest and possible cell death. ChpBK is a toxin encoded by the ChpBIK antitoxin-toxin module. This module consists of a pair of genes, chpBI and chpBK encoding antitoxin ChpBI and toxin ChpBK, respectively. ChpBK consists of 116 amino acid residues and its sequence shows 35% identity and 52% similarity to MazF, another E. coli toxin. MazF has been shown to be a sequence-specific (ACA) endoribonuclease that cleaves cellular mRNAs and effectively blocks protein synthesis and is thus termed as an mRNA interferase. Here we demonstrate that ChpBK is another mRNA interferase in E. coli whose induction effectively blocks cell growth in a manner similar to that of MazF. The protein synthesis as judged by incorporation of [35S]-methionine was, however, reduced by only 60% upon ChpBK induction. We demonstrate that ChpBK is a new sequence-specific endoribonu clease that cleaves mRNAs both in vivo and in vitro at the 5' or 3' side of the A residue in ACY sequences (Y is U, A or G). The ChpBK cleavage of a synthetic RNA substrate generated a 2', 3'- cyclic phosphate group at the 3' end of the 5'-end product, and a 5'-OH group at the 5' end of the 3'-end product in a manner identical to that of MazF.

PMID: 15901733

 >>>new aminopeptidases<<<
J Bacteriol. 2005 Jun;187(11):3671-7.
Characterization of two new aminopeptidases in Escherichia coli.
Zheng Y, Roberts RJ, Kasif S, Guan C.
Bioinformatics Graduate Program, Boston University, Massachusetts 02215, USA.

Two genes in the Escherichia coli genome, ypdE and ypdF, have been cloned and expressed, and their products have been purified. YpdF is shown to be a metalloenzyme with Xaa-Pro aminopeptidase activity and limited methionine aminopeptidase activity. Genes homologous to ypdF are widely distributed in bacterial species. The unique feature in the sequences of the products of these genes is a conserved C-terminal domain and a variable N-terminal domain. Full or partial deletion of the N terminus in YpdF leads to the loss of enzymatic activity. The conserved C-terminal domain is homologous to that of the methionyl aminopeptidase (encoded by map) in E. coli. However, YpdF and Map differ in their preference for the amino acid next to the initial methionine in the peptide substrates. The implication of this difference is discussed. ypdE is the immediate downstream gene of ypdF, and its start codon overlaps with the stop codon of ypdF by 1 base. YpdE is shown to be a metallo aminopeptidase and has a broad exoaminopeptidase activity.

PMID: 15901689

 >>>4th glutaredoxin<<<
J Biol Chem. 2005 Apr 15;
A novel monothiol glutaredoxin (GRX4) from Escherichia coli can serve as a substrate for thioredoxin reductase.
Fernandes AP, Fladvad M, Berndt C, Andresen C, Lillig CH, Neubauer P, Sunnerhagen M, Holmgren A, Vlamis-Gardikas A.
Department of Biochemistry, Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm SE17177.

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli (E. coli) has three glutaredoxins (Grx1, Grx2 and Grx3), all containing the classical dithiol active site CPYC. We report the cloning, expression and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knockout techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl- disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained 3 thiols and treatment wit h GSSG resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase while the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific ELISA, and most likely regulated by guanosine-3 ,5 -tetraphosphate (ppGpp) upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.

PMID: 15833738

 >>><<<

***additional finding***

J Bacteriol. 2004 Oct;186(20):6714-20.
Global transcriptional effects of a suppressor tRNA and the inactivation of the regulator frmR.
Herring CD, Blattner FR.
Laboratory of Genetics, University of Wisconsin-Madison, 445 Henry Mall, Madison, WI 53706, USA.

Expression of an amber suppressor tRNA should result in read-through of the 326 open reading frames (ORFs) that terminate with amber stop codons in the Escherichia coli genome, including six pseudogenes. Abnormal extension of an ORF might alter the activities of the protein and have effects on cellular physiology, while suppression of a pseudogene could lead to a gain of function. We used oligonucleotide microarrays to determine if any effects were apparent at the level of transcription in glucose minimal medium. Surprisingly, only eight genes had significantly different expression in the presence of the suppressor. Among these were the genes yaiN, adhC, and yaiM, forming a single putative operon whose likely function is the degradation of formaldehyde. Expression of wild-type yaiN was shown to result in repression of the operon, while a suppression-mimicking allele lacking the amber stop codon and extended 7 amino acids did not. The operon was shown to be induced by formaldehyde, and the genes have been renamed frmR, frmA, and frmB, respectively.

PMID: 15466022

 >>>yggB->mscS, mechanosensitive ion channel<<<

 J Biol Chem. 2003 Mar 28;278(13):11237-45.
C termini of the Escherichia coli mechanosensitive ion channel (MscS) move apart upon the channel opening.
Koprowski P, Kubalski A.
Department of Cell Biology, Nencki Institute of Experimental Biology; 3, Pasteur Street, 02-093 Warsaw, Poland.

Heptameric YggB is a mechanosensitive ion channel (MscS) from the inner membrane of Escherichia coli. We demonstrate, using the patch clamp technique, that cross-linking of the YggB C termini led to irreversible inhibition of the channel activities. Application of Ni(2+) to the YggB-His(6) channels with the hexahistidine tags added to the ends of their C termini also resulted in a marked but reversible decrease of activities. Western blot revealed that YggB-His(6) oligomers are more stable in the presence of Ni(2+), providing evidence that Ni(2+) is coordinated between C termini from different subunits of the channel. Intersubunit coordination of Ni(2+) affecting channel activities occurred in the channel closed conformation and not in the open state. This may suggest that the C termini move apart upon channel opening and are involved in the channel activation. We propose that the as yet undefined C-terminal region may form a cytoplasmic gate of the channel. The re sults are discussed and interpreted based on the recently released quaternary structure of the channel.

PMID: 12551944

Biophys J. 2002 Jul;83(1):290-8.
Purification of the small mechanosensitive channel of Escherichia coli (MscS): the subunit structure, conduction, and gating characteristics in liposomes.
Sukharev S.
Department of Biology, University of Maryland, College Park, Maryland 20742, USA.

The small mechanosensitive channel, MscS, is a part of the turgor-driven solute efflux system that protects bacteria from lysis in the event of osmotic downshift. It has been identified in Escherichia coli as a product of the orphan yggB gene, now called mscS (Levina et al., 1999, EMBO J. 18:1730). Here I show that that the isolated 31-kDa MscS protein is sufficient to form a functional mechanosensitive channel gated directly by tension in the lipid bilayer. MscS-6His complexes purified in the presence of octylglucoside and lipids migrate in a high-resolution gel-filtration column as particles of approximately 200 kDa. Consistent with that, the protein cross-linking patterns predict a hexamer. The channel reconstituted in soybean asolectin liposomes was activated by pressures of 20-60 mm Hg and displayed the same asymmetric I-V curve and slight anionic preference as in situ. At the same time, the single-channel conductance is proportional to the buffer conductivity  in a wide range of salt concentrations. The rate of channel activation in response to increasing pressure gradient across the patch was slower than the rate of closure in response to decreasing steps of pressure gradient. Therefore, the open probability curves were recorded with descending series of pressures. Determination of the curvature of patches by video imaging permitted measurements of the channel activity as a function of membrane tension (gamma). Po(gamma) curves had the midpoint at 5.5 +/- 0.1 dyne/cm and gave estimates for the energy of opening DeltaG = 11.4 +/- 0.5 kT, and the transition-related area change DeltaA = 8.4 +/- 0.4 nm(2) when fitted with a two-state Boltzmann model. The correspondence between channel properties in the native and reconstituted systems is discussed.

PMID: 12080120