J Bacteriol, 2002 Nov, 184(21), 5898 - 902 Hydroxylamine reductase activity of the hybrid cluster protein from Escherichia coli; Wolfe MT et al.; The hybrid cluster protein (HCP; formerly termed the prismane protein) has been extensively studied due to its unique spectroscopic properties . Although the structural and spectroscopic characteristics are well defined, its enzymatic function, up to this point, has remained unidentified . While it was proposed that HCP acts in some step of nitrogen metabolism, a specific role for this enzyme remained unknown . Recent studies of HCP purified from Escherichia coli have identified a novel hydroxylamine reductase activity . These data reveal the ability of HCP to reduce hydroxylamine in vitro to form NH(3) and H(2)O . Further biochemical analyses were completed in order to determine the effects of various electron donors, different pH levels, and the presence of CN(-) on in vitro hydroxylamine reduction. J Bacteriol, 2002 Nov, 184(21), 5871 - 9 Truncation analysis of TatA and TatB defines the minimal functional units required for protein translocation; Lee PA et al.; The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli . C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation . Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function . Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport . These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity . A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted. J Bacteriol, 2002 Nov, 184(21), 5862 - 70 Molecular characterization of the growth phase-dependent expression of the lsrA gene, encoding levansucrase of Rahnella aquatilis; Seo JW et al.; Expression of the lsrA gene from Rahnella aquatilis, encoding levansucrase, is tightly regulated by the growth phase of the host cell; low-level expression was observed in the early phase of cell growth, but expression was significantly stimulated in the late phase . Northern blot analysis revealed that regulation occurred at the level of transcription . The promoter region was identified by primer extension analysis . Two opposite genetic elements that participate in the regulation of lsrA expression were identified upstream of the lsrA gene: the lsrS gene and the lsrR region . The lsrS gene encodes a protein consisting of 70 amino acid residues (M(r), 8,075), which positively activated lsrA expression approximately 20-fold in a growth phase-dependent fashion . The cis-acting lsrR region, which repressed lsrA expression about 10-fold, was further narrowed to two DNA regions by deletion analysis . The concerted action of two opposite regulatory functions resulted in the growth phase-dependent activation of gene expression in Escherichia coli independent of the stationary sigma factor sigma(S). J Bacteriol, 2002 Nov, 184(21), 5855 - 61 Transcription activation by FNR: evidence for a functional activating region 2; Blake T et al.; The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia via the assembly-disassembly of oxygen-labile iron-sulfur clusters . Previous work identified patches of surface-exposed amino acids (designated activating regions 1 and 3 {AR1 and AR3, respectively}) of FNR which allow it to communicate with RNA polymerase (RNAP) and thereby activate transcription . Previously it was thought that FNR lacks a functional activating region 2 (AR2), although selecting for mutations that compensate for defective AR1 or a miscoordinated iron-sulfur cluster can reactivate AR2 . Here we show that the substitution of two surface-exposed lysine residues (Lys49 and Lys50) of FNR impaired transcription from class II (FNR box centered at -41.5) but not class I (FNR box centered at -71.5) FNR-dependent promoters . The degree of impairment was greater when a negatively charged residue (Glu) replaced either Lys49 or Lys50 than when uncharged amino acid Ala was substituted . Oriented heterodimers were used to show that only the downstream subunit of the FNR dimer was affected by the Lys-->Ala substitutions at a class II promoter . Site-directed mutagenesis of a negatively charged patch ((162)EEDE(165)) within the N-terminal domain of the RNAP alpha subunit that interacts with the positively charged AR2 of the cyclic AMP receptor protein suggested that Lys49 and Lys50 of FNR interact with this region of the alpha subunit of RNAP . Thus, it was suggested that Lys49 and Lys50 form part of a functional AR2 in FNR. J Bacteriol, 2002 Nov, 184(21), 5842 - 7 Study of second-site suppression in the pheP gene for the phenylalanine transporter of Escherichia coli; Pi J et al.; Site-directed mutagenesis was used to investigate a region of the PheP protein corresponding to the postulated consensus amphipathic region (CAR) in the GabP protein . Whereas some critical residues are conserved in both proteins, there are major differences between the two proteins which may reflect different functions for this region . Replacement of R317, Y313, or P341 by a number of other amino acids destroyed the PheP function . An R317E-E234R double mutant exhibited low levels of PheP transport activity, indicating that there is a possible interaction between these two residues in the wild-type protein . E234 is highly conserved in members of the superfamily of amino acid-polyamine-organocation transporters and also is critical for PheP function in the wild-type protein . Second-site suppressors were isolated for mutants with mutations in E234, Y313, R317, and P341 . Most suppressor mutations were found to cluster towards the extracellular face of spans III, IX, and X . Some mutations, such as changes at M116, were able to suppress each of the primary changes at positions E234, Y313, R317, and P341 but were unable to restore function to a number of other primary mutants . The possible implications of these results for the tertiary structure of the protein are discussed. J Bacteriol, 2002 Nov, 184(21), 5833 - 41 Escherichia coli insertion sequence IS150: transposition via circular and linear intermediates; Haas M et al.; IS150, a member of the widespread IS3 family, contains two consecutive out-of-phase open reading frames, orfA and orfB, that partially overlap . These open reading frames encode three proteins, InsA, InsB, and the InsAB protein, which is jointly encoded by both open reading frames by means of programmed translational frameshifting . We demonstrate that the InsAB protein represents the IS150 element's transposase . In vivo, the wild-type IS150 element generates circular excision products and linear IS150 molecules . Circular and linear species have previously been detected with mutant derivatives of other members of the IS3 family . Our finding supports the assumption that these products represent true transposition intermediates of members of this family . Analysis of the molecular nature of these two species suggested that the circular forms are precursors of the linear molecules . Elimination of InsA synthesis within the otherwise intact element led to accumulation of large amounts of the linear species, indicating that the primary role of InsA may be to prevent abortive production of the linear species and to couple generation of these species to productive insertion events. J Biol Chem, 2002 Dec 20, 277(51), 49935 - 44 Epub 2002 Oct 08. Phosphoinositide binding by the pleckstrin homology domains of Ipl and Tih1; Saxena A et al.; The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions . This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice . However, its biochemical function is unknown . The closest paralogue of Ipl is Tih1, another small PH domain protein . By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily . Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins . Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C delta1, and dual adaptor for phosphoinositides and phosphotyrosine 1 . In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm . Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1 . Site-directed mutagenesis defines amino acids in the beta1-loop1-beta2 regions of Ipl and Tih1 as essential for growth rescue in this assay . Thus, Ipl and Tih1 are bona fide PH domain proteins, with broad specificity and moderate affinity for PIPs. J Biol Chem, 2002 Dec 13, 277(50), 48241 - 7 Epub 2002 Oct 08. The Aes protein and the monomeric alpha-galactosidase from Escherichia coli form a non-covalent complex . Implications for the regulation of carbohydrate metabolism; Mandrich L et al.; Aes, a 36-kDa acetylesterase from Escherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon . The activity of MalT is depressed through a direct protein-protein interaction with Aes . Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive . To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E . coli and purified . At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column . A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected . By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio . The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected . The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa . Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the alpha-galactosidase from E . coli . The E . coli cells harboring plasmid pT7-SCII-aes and, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source . Because alpha-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E . coli. J Biol Chem, 2002 Dec 13, 277(50), 48276 - 81 Epub 2002 Oct 08. Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase; Yaoi K et al.; A novel oligoxyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH), with a molecular mass of 97 kDa and a pI of 6.1, was isolated from the fungus Geotrichum sp . M128 . Analysis of substrate specificity using various xyloglucan oligosaccharide structures revealed that OXG-RCBH had exoglucanase activity . It recognized the reducing end of oligoxyloglucan and released two glucosyl residue segments from the main chain . The full-length cDNA encoding OXG-RCBH was cloned and sequenced, and it had a 2436-bp open reading frame encoding an 812amino acid protein . The deduced protein showed approximately 35% identity to members of glycoside hydrolase family 74 . The cDNA encoding OXG-RCBH was then expressed in Escherichia coli . Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant OXG-RCBH was obtained. EMBO J, 2002 Oct 15, 21(20), 5577 - 85 Holliday junction resolution in human cells: two junction endonucleases with distinct substrate specificities; Constantinou A et al.; Enzymatic activities that cleave Holliday junctions are required for the resolution of recombination intermediates and for the restart of stalled replication forks . Here we show that human cell-free extracts possess two distinct endonucleases that can cleave Holliday junctions . The first cleaves Holliday junctions in a structure- and sequence-specific manner, and associates with an ATP-dependent branch migration activity . Together, these activities promote branch migration/resolution reactions similar to those catalysed by the Escherichia coli RuvABC resolvasome . Like RuvC-mediated resolution, the products can be religated . The second, containing Mus81 protein, cuts Holliday junctions but the products are mostly non-ligatable . Each nuclease has a defined substrate specificity: the branch migration-associated resolvase is highly specific for Holliday junctions, whereas the Mus81-associated endonuclease is one order of magnitude more active upon replication fork and 3'-flap structures . Thus, both nucleases are capable of cutting Holliday junctions formed during recombination or through the regression of stalled replication forks . However, the Mus81-associated endonuclease may play a more direct role in replication fork collapse by catalysing the cleavage of stalled fork structures. EMBO J, 2002 Oct 15, 21(20), 5323 - 30 Ionic regulation of MscK, a mechanosensitive channel from Escherichia coli; Li Y et al.; Three gene products that form independent mechanosensitive channel activities have been identified in Escherichia coli . Two of these, MscL and MscS, play a vital role in allowing the cell to survive acute hypotonic stress . Much less is known of the third protein, MscK (KefA) . Here, we characterize the MscK channel activity and compare it with the activity of its structural and functional homologue, MscS . While both show a slight anionic preference, MscK appears to be more sensitive to membrane tension . In addition, MscK, but not MscS activity appears to be regulated by external ionic environment, requiring not only membrane tension but also high concentrations of external K(+), NH(4)(+), Rb(+) or Cs(+) to gate; no activity is observed with Na(+), Li(+) or N-methyl-D-glucamine (NMDG) . An MscK gain-of-function mutant gates spontaneously in the presence of K(+) or similar ions, and will gate in the presence of Na(+), Li(+) and NMDG, but only when stimulated by membrane tension . Increased sensitivity and the highly regulated nature of MscK suggest a more specialized physiological role than other bacterial mechanosensitive channels. Electrophoresis, 2002 Sep, 23(19), 3289 - 99 Escherichia coli single-stranded DNA-binding protein, a molecular tool for improved sequence quality in pyrosequencing; Ehn M et al.; Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle . Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis . In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction . In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones . More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms . Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates . Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity . Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated. Biol Neonate, 2002, 82(3), 188 - 96 Detrimental effects of nicotine and endotoxin in the newborn piglet brain during severe hypoxemia; Froen JF et al.; Hypoxia-ischemia is a major cause of perinatal brain damage, but evidence shows that brain injury also is associated with intrauterine infections and maternal smoking . The mechanisms are not known, and we therefore explored the effects of experimental inflammation or nicotine on perinatal brain metabolism and injury during severe hypoxemia . Twenty-eight 1-week-old piglets were anesthetized and instrumented with microdialysis probes in the striatum and brainstem . We studied three pretreatment groups: (1) 20 microg/kg i.v . nicotine (n = 9); (2) 1 microg/kg i.v . endotoxin from Escherichia coli (n = 11), or (3) control (n = 8) . The piglets were subsequently exposed to 30 min of hypoxemia (6% O(2)) . In order to minimize any ischemic component and increase survival, this was abrupted for 1 min if blood pressure fell to 30 mm Hg . During hypoxemia, both the pretreatment with endotoxin and nicotine induced higher levels of extracellular lactate and peak lactate/pyruvate ratio compared with controls (54.7 +/- 9.6 (p < 0.01) and 65.2 +/- 13.1 (p < 0.02) vs . 15.9 +/- 7.4, respectively), reflecting a deterioration of the metabolic status in these groups . The two pretreated groups reached significantly higher peak levels of extracellular glycerol (30.9 +/- 4.1 vs . 77.9 +/- 12.7 and 89.4 +/- 14.2 micromol/l, respectively, p = 0.01), indicating a higher level of cellular membrane disintegration or leakage . In addition, 3 endotoxin piglets and 4 nicotine piglets died during reoxygenation, while all controls survived (p = 0.13 and p < 0.04, respectively) . Mortality was associated with a rise in extracellular glutamate at the end of hypoxemia/start reoxygenation (p = 0.02) . These findings contribute in explaining how nicotine and inflammatory response to bacterial toxins could act as cofactors for hypoxic-ischemic neurologic injury in the immature brain . J Biomed Sci, 2002, 9(6 Pt 1), 542 - 8 Dysfunction of the mitochondrial respiratory chain in the rostral ventrolateral medulla during experimental endotoxemia in the rat; Chuang YC et al.; We investigated the functional changes in the mitochondrial respiratory chain at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor tone, in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome . In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of Escherichia coli lipopolysaccharide (LPS; 30 mg/kg) induced a reduction (Phase I), followed by an augmentation (Phase II) and a secondary decrease (Phase III) in the power density of vasomotor components (0-0.8 Hz) in systemic arterial pressure signals . LPS also elicited progressive hypotension, and death ensued within 4 h . Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochrome c reductase (Complexes I + III) and cytochrome c oxidase (Complex IV) in the RVLM during all three phases of endotoxemia . On the other hand, the activity of succinate cytochrome c reductase (Complexes II + III) remained unaltered . We conclude that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM, whose neuronal activity is intimately related to the death process, is closely associated with fatal endotoxemia in the rat . J Biol Chem, 2002 Dec 6, 277(49), 47878 - 84 Epub 2002 Oct 07. Phosphorylation of the catalytic subunit of protein kinase A . Autophosphorylation versus phosphorylation by phosphoinositide-dependent kinase-1; Moore MJ et al.; The identification of phosphoinositide-dependent kinase-1 (PDK-1) as an activating kinase for members of the AGC family of kinases has led to its implication as the activating kinase for cAMP-dependent protein kinase . It has been established in vitro that PDK-1 can phosphorylate the catalytic (C) subunit (), but the Escherichia coli-expressed C-subunit undergoes autophosphorylation . To assess which of these mechanisms occurs in mammalian cells, a set of mutations was engineered flanking the site of PDK-1 phosphorylation, Thr-197, on the activation segment of the C-subunit . Two distinct requirements appeared for autophosphorylation and phosphorylation by PDK-1 . Autophosphorylation was disrupted by mutations that compromised activity (Thr-201 and Gly-200) or altered substrate recognition (Arg-194) . Conversely, only residues peripheral to Thr-197 altered PDK-1 phosphorylation, including a potential hydrophobic PDK-1 binding site at the C terminus . To address the in vivo requirements for phosphorylation, select mutant proteins were transfected into COS-7 cells, and their phosphorylation state was assessed with phospho-specific antibodies . The phosphorylation pattern of these mutant proteins indicates that autophosphorylation is not the maturation mechanism in the eukaryotic cell; instead, a heterologous kinase with properties resembling the in vitro characteristics of PDK-1 is responsible for in vivo phosphorylation of PKA. J Biol Chem, 2002 Dec 13, 277(50), 48199 - 204 Epub 2002 Oct 07. In vivo interactions between gene products involved in the final stages of molybdenum cofactor biosynthesis in Escherichia coli; Magalon A et al.; The final stages of bacterial molybdenum cofactor (Moco) biosynthesis correspond to molybdenum chelation and nucleotide attachment onto an unique and ubiquitous structure, the molybdopterin . Using a bacterial two-hybrid approach, here we report on the in vivo interactions between MogA, MoeA, MobA, and MobB implicated in several distinct although linked steps in Escherichia coli . Numerous interactions among these proteins have been identified . Somewhat surprisingly, MobB, a GTPase with a yet unclear function, interacts with MogA, MoeA, and MobA . Probing the effects of various mo . mutations on the interaction map allowed us (i) to distinguish Moco-sensitive interactants from insensitive ones involving MobB and (ii) to demonstrate that molybdopterin is a key molecule triggering or facilitating MogA-MoeA and MoeA-MobA interactions . These results suggest that, in vivo, molybdenum cofactor biosynthesis occurs on protein complexes rather than by the separate action of molybdenum cofactor biosynthetic proteins. J Biol Chem, 2002 Dec 6, 277(49), 47885 - 90 Epub 2002 Oct 07. Zinc mediates assembly of the T1 domain of the voltage-gated K channel 4.2; Jahng AW et al.; An intermolecular Zn(2+)-binding site was identified in the structure of the T1 domain of the Shaw-type potassium channels (aKv3.1) . T1 is a BTB/POZ-type domain responsible for the ordered assembly of voltage-gated potassium channels and interactions with other macromolecules . In this structure, a Zn(2+) ion was found to be coordinated between each of the four assembly interfaces of the T1 tetramer by three Cys and one His encoded in the sequence motif (HX(5)CX(20)CC) of the T1 domain . This sequence motif is conserved among all non-Shaker-type voltage-dependent potassium (Kv) channels, but not in Shaker-type channels . The presence of this conserved Zn(2+)-binding site is a primary molecular determinant that distinguishes the tetrameric assembly of non-Shaker Kv channel subunits from that of Shaker channels . We report here that tetramerization of the Shal (rKv4.2) T1 in solution requires the presence of Zn(2+), and the addition/removal of Zn(2+) reversibly switches the protein between a stable tetrameric or monomeric state . We further show that the conversion from tetramers to monomers is profoundly pH-dependent: as the solution pH gets lower, the dissociation rate increases significantly . The unfolding energy of the T1 tetramer as a measure of the conformational stability of the structure is also pH-dependent . Surprisingly, at a lower pH we observe a distinctly altered conformational state of the T1 tetramer trapped during the process of unfolding of the T1 tetramer in the presence of Zn(2+) . The conformational alteration may be responsible for increased rate of dissociation at lower pH by allowing Zn(2+) to be removed more effectively by EDTA . The ability of the T1 domain to adopt stable alternative conformations may be essential to its function as a protein-protein interaction/signaling domain to modulate the ion conduction properties of intact full-length Kv channels. FEBS Lett, 2002 Oct 9, 529(2-3), 332 - 6 Recombinant Escherichia coli biotin synthase is a {2Fe-2S}(2+) protein in whole cells; Cosper MM et al.; EPR and Mossbauer spectroscopies have been used to determine the type and properties of the iron-sulfur clusters present in homologously expressed recombinant Escherichia coli BioB in whole cells prior to purification . Difference EPR spectra of samples of whole cells from a strain over-expressing E . coli BioB and a strain containing the same plasmid but without the bioB insertion showed an axial S=1/2 resonance that was attributed to the {2Fe-2S}(+) cluster of the E . coli iron-sulfur cluster assembly 2Fe ferredoxin, based on principal g-values, linewidths and relaxation behavior . Comparison of the Mossbauer spectra of whole cells with and without the bioB insertion revealed that the E . coli cells with over-expressed BioB contain an additional species that exhibits a spectrum identical to that of the {2Fe-2S}(2+) cluster in purified recombinant BioB . The concentration of this {2Fe-2S}(2+) species in the whole cell sample was quantified using a Mossbauer standard and found to be approximately 260 microM, which was comparable to the BioB protein concentration estimated for the cell paste . The results demonstrate that the {2Fe-2S}(2+) cluster found in purified samples of recombinant BioB is not an artifact of the protein purification procedure, and indicate that recombinant BioB is over-expressed in an inactive form during aerobic growth. FEBS Lett, 2002 Oct 9, 529(2-3), 237 - 42 Thermal inactivation of reduced ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli; Jarrett JT et al.; Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) is an essential enzyme that supplies electrons from NADPH to support flavodoxin-dependent enzyme radical generation and enzyme activation . FNR is a monomeric enzyme that contains a non-covalently bound FAD cofactor . We report that reduced FNR from Escherichia coli is subject to inactivation due to unfolding of the protein and dissociation of the FADH(2) cofactor at 37 degrees C . The inactivation rate is temperature-dependent in a manner that parallels the thermal unfolding of the protein and is slowed by binding of ferredoxin or flavodoxin . Understanding factors that minimize inactivation is critical for utilizing FNR as an accessory protein for S-adenosyl-L-methionine-dependent radical enzymes and manipulating FNR as an electron source for biotechnology applications. FEBS Lett, 2002 Oct 9, 529(2-3), 225 - 31 3'-end processing of precursor M1 RNA by the N-terminal half of RNase E; Sim S et al.; M1 RNA, the catalytic component of Escherichia coli RNase P, is derived from the 3'-end processing of precursor M1 RNA, a major transcript of the rnpB gene . In this study, we investigated the mechanism of 3'-end processing of M1 RNA using the recombinant N-terminal half RNase E . The cleavage site preference of RNase E differed from that of the 40% ammonium sulfate precipitate (ASP-40), a partially purified cell extract containing processing activity . However, the addition of a trace amount of ASP-40 changed the cleavage site preference of RNase E to that of ASP-40 suggesting the involvement of a soluble factor in cleavage site preference. FEBS Lett, 2002 Oct 9, 529(2-3), 151 - 6 HU: promoting or counteracting DNA compaction? Dame RT, Goosen N. The role of HU in Escherichia coli as both a protein involved in DNA compaction and as a protein with regulatory function seems to be firmly established . However, a critical look at the available data reveals that this is not true for each of the proposed roles of this protein . The role of HU as a regulatory or accessory protein in a number of systems has been thoroughly investigated and in many cases has been largely elucidated . However, almost 30 years after its discovery, convincing evidence for the proposed role of HU in DNA compaction is still lacking . Here we present an extensive literature survey of the available data which, in combination with novel microscopic insights, suggests that the role of HU could be the opposite as well . The protein is likely to play an architectural role, but instead of being responsible for DNA compaction it could be involved in antagonising compaction by other proteins such as H-NS. Biochem Biophys Res Commun, 2002 Oct 11, 297(5), 1096 - 101 Change of product specificity of hexaprenyl diphosphate synthase from Sulfolobus solfataricus by introducing mimetic mutations; Hemmi H et al.; The introduction of several sets of amino acid substitutions into the region around a substrate-binding site of a medium-chain (all-E) prenyl diphosphate synthase, hexaprenyl diphosphate synthase from a thermoacidophilic archaeon Sulfolobus solfataricus, to mimic the product determination mechanisms of various kinds of short-chain enzymes revealed that the structure around the region of the medium-chain enzyme resembles those of eukaryotic farnesyl diphosphate synthases but not those of the other short-chain enzymes, reflecting the evolutional relationships among these enzymes. J Hosp Infect, 2002 Sep, 52(1), 43 - 51 Genetic relationship between Escherichia coli strains isolated from the intestinal flora and those responsible for infectious diseases among patients hospitalized in intensive care units; Mereghetti L et al.; The exact origin of strains of Escherichia coli responsible for infectious diseases in intensive care units (ICUs) remains partly unknown . Our aim was to determine the nature of the link between strains from the intestinal flora of hospital staff, strains from the intestinal flora of patients hospitalized in ICUs and strains isolated from ICU patients with invasive diseases . For this purpose, 77 strains of E . coli were genetically characterized by exploring their entire genomes by random amplified polymorphism of DNA (RAPD), and by determining their phylogenetic position in ECOR (E . coli reference) groups, the virulence factors harboured (pap, sfa, afa, hly, aer and cnf) and their ability to mutate . The strains isolated from the intestinal flora of hospital staff were found to constitute a genetically heterogeneous population compared with the strains isolated from ICU carriers, which were highly clustered . The latter strains harboured numerous virulence factors, and 80% belonged to the group ECOR B2 . The strains isolated from infected patients harboured fewer virulence factors than those from the ICU carriers, and only half belonged to ECOR B2 . Moreover, these strains were more genetically related to strains from hospital staff than to strains from ICU carriers . Thus, the exogenous origin of the E . coli strains is probably almost as important as translocation from intestinal flora in ICUs . Moreover, a strong mutator phenotype had a minor, or no, role in the rapid adaptation to modifications in the ecological environment. Cell, 2002 Oct 4, 111(1), 129 - 40 Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing; Lancaster L et al.; Ribosome recycling factor (RRF) disassembles posttermination complexes in conjunction with elongation factor EF-G, liberating ribosomes for further rounds of translation . The striking resemblance of its L-shaped structure to that of tRNA has suggested that the mode of action of RRF may be based on mimicry of tRNA . Directed hydroxyl radical probing of 16S and 23S rRNA from Fe(II) tethered to ten positions on the surface of E . coli RRF constrains it to a well-defined location in the subunit interface cavity . Surprisingly, the orientation of RRF in the ribosome differs markedly from any of those previously observed for tRNA, suggesting that structural mimicry does not necessarily reflect functional mimicry. Cell, 2002 Oct 4, 111(1), 105 - 15 Crystal structure and functional analysis of the histone methyltransferase SET7/9; Wilson JR et al.; Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure . The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity . We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain . Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3 . Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate. J Am Chem Soc, 2002 Oct 16, 124(41), 12144 - 53 Solution NMR techniques for large molecular and supramolecular structures; Riek R et al.; Transverse relaxation-optimized spectroscopy (TROSY) or generation of heteronuclear multiple quantum coherences during the frequency labeling period and TROSY during the acquisition period have been combined either with cross-correlated relaxation-induced polarization transfer (CRIPT) or cross-correlated relaxation-enhanced polarization transfer (CRINEPT) to obtain two-dimensional (2D) solution NMR correlation spectra of (15)N,(2)H-labeled homo-oligomeric macromolecules with molecular weights from 110 to 800 kDa . With the experimental conditions used, the line widths of the TROSY-components of the (1)H- and (15)N-signals were of the order of 60 Hz at 400 kDa, whereas, for structures of size 800 kDa, the line widths were about 75 Hz for (15)N and 110 Hz for (1)H . This paper describes the experimental schemes used and details of their setup for individual measurements . The performance of NMR experiments with large structures depends critically on the choice of the polarization transfer times, the relaxation delays between subsequent recordings, and the water-handling routines . Optimal transfer times for 2D {(15)N,(1)H}-CRIPT-TROSY experiments in H(2)O solutions were found to be 6 ms for a molecular weight of approximately 200 kDa, 2.8 ms for 400 kDa, and 1.4 ms for 800 kDa . These data validate theoretical predictions of inverse proportionality between optimal transfer time and size of the structure . The proton longitudinal relaxation times in H(2)O solution were found to be of the order of 0.8 s for structure sizes around 200 kDa, 0.4 s at 400 kDa, and 0.3 s at 800 kDa, which enabled the use of recycle times below 1 s . Since improper water handling results in severe signal loss, the water resonance was kept along the z-axis during the entire duration of the experiments by adjusting each water flip-back pulse individually. Oncogene, 2002 Oct 10, 21(46), 7126 - 30 Elevated mutant frequencies and predominance of G:C to A:T transition mutations in Msh6(-/-) small intestinal epithelium; Mark SC et al.; The DNA mismatch repair (MMR) system is primarily responsible for purging newly synthesized DNA of errors incurred during semi-conservative replication . Lesion recognition is initially carried out by one of two heterodimeric protein complexes, MutS(alpha) or MutS(beta) . While the former, comprised of MSH2 and MSH6, recognizes mispairs as well as short (1-2 nucleotide) insertions/deletions (IDLs), the latter, made up of MSH2 and MSH3, is primarily responsible for recognizing 2-6 nucleotide IDLs . As most of the functional information on these heterodimers is derived from in vitro studies, it was of interest to study the in vivo consequences of a lack of MutS(alpha) . To this end, Big Blue( trade mark ) mice, that carry a lacI(+) transgenic lambda shuttle-phage mutational reporter, were crossed with Msh6(-/-) mice to evaluate the specific contribution of MutS(alpha) to genome integrity . Consistent with the importance of MutS(alpha) in lesion surveillance, small intestine epithelial cell DNA derived from lacI(+) Msh6(-/-) mice exhibited striking increases (average of 41-fold) in spontaneous mutant frequencies . Furthermore, the lacI gene mutation spectrum was dominated by G:C to A:T transitions, highlighting the critical importance of the MutS(alpha) complex in suppressing this frequently observed type of spontaneous mutation. Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13538 - 43 Epub 2002 Oct 07. Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior; Adelman K et al.; By using single-molecule measurements, we demonstrate that the elongation kinetics of individual Escherichia coli RNA polymerase molecules are remarkably homogeneous . We find no evidence of distinct elongation states among RNA polymerases . Instead, the observed heterogeneity in transcription rates results from statistical variation in the frequency and duration of pausing . When transcribing a gene without strong pause sites, RNA polymerase molecules display transient pauses that are distributed randomly in both time and distance . Transitions between the active elongation mode and the paused state are instantaneous within the resolution of our measurements (<1 s) . This elongation behavior is compared with that of a mutant RNA polymerase that pauses more frequently and elongates more slowly than wild type. Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13533 - 7 Epub 2002 Oct 07. Lipid-protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy; Fernandez C et al.; Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and dihexanoylphosphatidylcholine (DHPC) provided a detailed description of protein-detergent interactions . The NOEs were measured in 3D (15)N- and (13)C-resolved {(1)H,(1)H}-NOESY spectra recorded with selectively methyl-protonated and otherwise uniformly (2)H,(13)C,(15)N-labeled OmpX in micelles of DHPC at natural isotope abundance . In these mixed micelles the NMR structure of OmpX consists of an eight-stranded antiparallel beta-barrel . The OmpX surface area covered with intermolecular NOEs to the DHPC hydrophobic tails forms a continuous cylinder jacket of approximately 28 A in height, which is centered about the middle of the long axis through the beta-barrel . In addition, some intermolecular NOEs with methyl groups of the DHPC polar head were identified along both boundaries of this cylinder jacket . The experimental data suggest that the hydrophobic surface areas of OmpX are covered with a monolayer of DHPC molecules, which appears to mimic quite faithfully the embedding of the beta-barrel in a double-layer lipid membrane. Mol Cell Biol, 2002 Nov, 22(21), 7701 - 11 A human mitochondrial GTP binding protein related to tRNA modification may modulate phenotypic expression of the deafness-associated mitochondrial 12S rRNA mutation; Li X et al.; Human mitochondrial 12S rRNA A1555G mutation has been found to be associated with deafness . However, putative nuclear modifier gene(s) has been proposed to regulate the phenotypic expression of this mutation . In yeast cells, mutant alleles of MSS1, encoding a mitochondrial GTP-binding protein, manifest a respiratory-deficient phenotype only when coupled with mitochondrial 15S rRNA P(R)(454) mutation corresponding to human A1555G mutation . This suggests that an MSS1-like modifier gene may influence the phenotypic expression of the A1555G mutation . We report here the identification and characterization of human MSS1 homolog, GTPBP3, the first identified vertebrate gene related to mitochondrial tRNA modification . The Gtpbp3 is the mitochondrial GTPase evolutionarily conserved from bacteria to mammals . Functional conservation of this protein is supported by the observation that isolated human GTPBP3 cDNA can complement the respiratory-deficient phenotype of yeast mss1 cells carrying P(R)(454) mutation . GTPBP3 is ubiquitously expressed in various tissues as multiple transcripts, but with a markedly elevated expression in tissues of high metabolic rates . We showed that Gtpbp3 localizes in mitochondrion . These observations suggest that the human GTPBP3 is a structural and functional homolog of yeast MSS1 . Thus, allelic variants in GTPBP3 could, if they exist, modulate the phenotypic manifestation of human mitochondrial A1555G mutation. Mol Cell Biol, 2002 Nov, 22(21), 7524 - 34 High-resolution mapping of changes in histone-DNA contacts of nucleosomes remodeled by ISW2; Kassabov SR et al.; The imitation switch (ISWI) complex from yeast containing the Isw2 and Itc1 proteins was shown to preferentially slide mononucleosomes with as little as 23 bp of linker DNA from the end to the center of DNA . The contacts of unique residues in the histone fold regions of H4, H2B, and H2A with DNA were determined with base pair resolution before and after chromatin remodeling by a site-specific photochemical cross-linking approach . The path of DNA and the conformation of the histone octamer in the nucleosome remodeled or slid by ISW2 were not altered, because after adjustment for the new translational position, the DNA contacts at specific sites in the histone octamer had not been changed . Maintenance of the canonical nucleosome structure after sliding was also demonstrated by DNA photoaffinity labeling of histone proteins at specific sites within the DNA template . In addition, nucleosomal DNA does not become more accessible during ISW2 remodeling, as assayed by restriction endonuclease cutting . ISW2 was also shown to have the novel capability of counteracting transcriptional activators by sliding nucleosomes through Gal4-VP16 bound initially to linker DNA and displacing the activator from DNA. J Biol Chem, 2002 Dec 13, 277(50), 48657 - 63 Epub 2002 Oct 04. Escherichia coli glutamyl-tRNA reductase . Trapping the thioester intermediate; Schauer S et al.; In the first step of tetrapyrrole biosynthesis in Escherichia coli, glutamyl-tRNA reductase (GluTR, encoded by hemA) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde . Soluble homodimeric E . coli GluTR was made by co-expressing the hemA gene and the chaperone genes dnaJK and grpE . During Mg(2+)-stimulated catalysis, the reactive sulfhydryl group of Cys-50 in the E . coli enzyme attacks the alpha-carbonyl group of the tRNA-bound glutamate . The resulting thioester intermediate was trapped and detected by autoradiography . In the presence of NADPH, the end product, glutamate-1-semialdehyde, is formed . In the absence of NADPH, E . coli GluTR exhibited substrate esterase activity . The in vitro synthesized unmodified glutamyl-tRNA was an acceptable substrate for E . coli GluTR . Eight 5-aminolevulinic acid auxotrophic E . coli hemA mutants were genetically selected, and the corresponding mutations were determined . Most of the recombinant purified mutant GluTR enzymes lacked detectable activity . Based on the Methanopyrus kandleri GluTR structure, the positions of the amino acid exchanges are close to the catalytic domain (G7D, E114K, R314C, S22L/S164F, G44C/S105N/A326T, G106N, S145F) . Only GluTR G191D (affected in NADPH binding) revealed esterase but no reductase activity. Protein Pept Lett, 2002 Oct, 9(5), 419 - 26 Cloning and high-level expression of scorpion toxin BmKITa1 in Escherichia coli and insect cells; Liu Z et al.; BmK ITa1 cDNA was cloned and highly expressed in E . coli and insect cell . SDS-PAGE and western blot analysis revealed that subunit molecular weight of expression products is about 40 kDa and 10 kDa respectively . The expression product purified by a Ni(2+)-IDA-sepharose 6B column was toxic for insect, which indicated that it was biologically activity . Furthermore, Quantitative estimation show that the biological activity of recombinant BmK ITa1 from Tn cells was more powerful than from E . coli. Curr Protein Pept Sci, 2002 Aug, 3(4), 451 - 60 Structures and interactions of proteins involved in the coupling function of the protonmotive F(o)F(1)-ATP synthase; Gaballo A et al.; The mitochondrial F(1)F(o) ATP synthase complex has a key role in cellular energy metabolism . The general architecture of the enzyme is conserved among species and consists of a globular catalytic moiety F(1), protruding out of the inner side of the membrane, a membrane integral proton translocating moiety F(o), and a stalk connecting F(1) to F(o) . The X-ray crystallographic analysis of the structure of the bovine mitochondrial F(1) ATPase has provided a structural basis for the binding-change rotary mechanism of the catalytic process in F(1), in which the gamma subunit rotates in the central cavity of the F(1) alpha3/beta3 hexamer . Rotation of gamma and eta subunits in the E . coli enzyme and of, gamma and delta subunits in the mitochondrial enzyme, is driven, during ATP synthesis, by proton motive rotation of an oligomer of c subunits (10-12 copies) within the F(o) base piece . Average analysis of electron microscopy images and cross-linking results have revealed that, in addition to a central stalk, contributed by gamma and delta/eta subunits, there is a second lateral one connecting the peripheries of F(o) and F(1) . To gain deeper insight into the mechanism of coupling between proton translocation and catalytic activity (ATP synthesis and hydrolysis), studies have been undertaken on the role of F(1) and F(o) subunits which contribute to the structural and functional connection between the catalytic sector F(1) and the proton translocating moiety F(o) . These studies, which employed limited proteolysis, chemical cross-linking and functional analysis of the native and reconstituted F(1)F(o) complex, as well as isolated F(1), have shown that the N-terminus of alpha subunits, located at the top of the F(1) hexamer is essential for energy coupling in the F(1)F(o) complex . The alpha N-terminus domain appears to be connected to F(o) by OSCP (F(o) subunit conferring sensitivity of the complex to oligomycin) . In turn, OSCP contacts F(o)I-PVP(b) and d subunits, with which it constitutes a structure surrounding the central gamma and delta rotary shaft . Cross-linking of F(o)I-PVP(b) and gamma subunits causes a dramatic enhancement of downhill proton translocation decoupled from ATP synthesis but is without effect on ATP driven uphill proton transport . This would indicate the existence of different rate-limiting steps in the two directions of proton translocation through F(o) . In mitochondria, futile ATP hydrolysis by the F(1)F(o) complex is inhibited by the ATPase inhibitor protein (IF(1)), which reversibly binds at one side of the F(1)F(o) connection . The trans-membrane deltapH component of the respiratory deltap displaces IF(1) from the complex; in particular the matrix pH is the critical factor for IF(1)association and its related inhibitory activity . The 42L-58K segment of the IF(1) has been shown to be the most active segment of the protein; it interacts with the surface of one alpha/beta pairs of F(1), thus inhibiting, with the same pH dependence as the natural IF(1), the conformational interconversions of the catalytic sites involved in ATP hydrolysis . IF(1) has a relevant physiopathological role for the conservation of the cellular ATP pool in ischemic tissues . Under these conditions IF(1), which appears to be over expressed, prevents dissipation of the glycolytic ATP. Mini Rev Med Chem, 2001 Sep, 1(3), 293 - 306 Aerobic nitroreduction by flavoproteins: enzyme structure, mechanisms and role in cancer chemotherapy; Skelly JV et al.; NQO1 (DT-diaphorase) and its truncated isoenzyme, the metalloenzyme NQO2, can reduce quinone substrates by two-electron transfer . While NQO1 is a known detoxification enzyme, the function of NQO2 is less well understood . Both rat NQO1 and human NQO2 reductively bioactivate the dinitroarene CB 1954 to a cytotoxic product that behaves as a difunctional DNA-crosslinking species with potent anti-tumour activity, although human NQO1 is much less effective . A FMN-dependent nitroreductase from E . coli B also reduces quinones and reductively bioactivates CB 1954 . However, this enzyme reduces CB 1954 to the 2- and 4-hydroxylamines in equivalent yield, whereas NQO1 and NQO2 generate only the 4-isomer . The reduction profile is a key factor in the development of anti-tumour prodrugs, where distinct delivery strategies are being evaluated: prodrug therapy, antibody-, macromolecule and gene-directed enzyme prodrug therapy (ADEPT, MDEPT or GDEPT) . The flavoprotein enzymes are explored in terms of structure and bioreduction mechanism, particularly for use in the design of novel prodrugs with potential application as chemotherapeutic agents. Curr Protein Pept Sci, 2001 Sep, 2(3), 227 - 44 Chaperone-assisted protein folding in the cell cytoplasm; Houry WA; Folding of polypeptides in the cell typically requires the assistance of a set of proteins termed molecular chaperones . Chaperones are an essential group of proteins necessary for cell viability under both normal and stress conditions . There are several chaperone systems which carry out a multitude of functions all aimed towards insuring the proper folding of target proteins . Chaperones can assist in the efficient folding of newly-translated proteins as these proteins are being synthesized on the ribosome and can maintain pre-existing proteins in a stable conformation . Chaperones can also promote the disaggregation of preformed protein aggregates . Many of the identified chaperones are also heat shock proteins . The general mechanism by which chaperones carry out their function usually involves multiple rounds of regulated binding and release of an unstable conformer of target polypeptides . The four main chaperone systems in the Escherichia coli cytoplasm are as follows . (1) Ribosome-associated trigger factor that assists in the folding of newly-synthesized nascent chains . (2) The Hsp 70 system consisting of DnaK (Hsp 70), its cofactor DnaJ (Hsp 40), and the nucleotide exchange factor GrpE . This system recognizes polypeptide chains in an extended conformation . (3) The Hsp 60 system, consisting of GroEL (Hsp 60) and its cofactor GroES (Hsp 10), which assists in the folding of compact folding intermediates that expose hydrophobic surfaces . (4) The Clp ATPases which are typically members of the Hsp 100 family of heat shock proteins . These ATPases can unfold proteins and disaggregate preformed protein aggregates to target them for degradation . Several advances have recently been made in characterizing the structure and function of all of these chaperone systems . These advances have provided us with a better understanding of the protein folding process in the cell. Biochemistry, 2002 Oct 15, 41(41), 12520 - 8 GTPase activation of elongation factors Tu and G on the ribosome; Mohr D et al.; The GTPase activity of elongation factors Tu and G is stimulated by the ribosome . The factor binding site is located on the 50S ribosomal subunit and comprises proteins L7/12, L10, L11, the L11-binding region of 23S rRNA, and the sarcin-ricin loop of 23S rRNA . The role of these ribosomal elements in factor binding, GTPase activation, or functions in tRNA binding and translocation, and their relative contributions, is not known . By comparing ribosomes depleted of L7/12 and reconstituted ribosomes, we show that, for both factors, interactions with L7/12 and with other ribosomal residues contribute about equally and additively to GTPase activation, resulting in an overall 10(7)-fold stimulation . Removal of L7/12 has little effect on factor binding to the ribosome . Effects on other factor-dependent functions, i.e., A-site binding of aminoacyl-tRNA and translocation, are fully explained by the inhibition of GTP hydrolysis . Based on these results, we propose that L7/12 stimulates the GTPase activity of both factors by inducing the catalytically active conformation of the G domain . This effect appears to be augmented by interactions of other structural elements of the large ribosomal subunit with the switch regions of the factors. Biochemistry, 2002 Oct 15, 41(41), 12421 - 6 A positive charge preservation at position 116 of alpha A-crystallin is critical for its structural and functional integrity; Bera S et al.; An autosomal dominant congenital cataract associated with a missense mutation, Arg-116 to Cys (R116C), in the coding sequence of human alphaA-crystallin has been reported . Subsequent study of this mutant, generated by site-directed mutagenesis, showed significant changes in secondary and tertiary structures, partial loss of chaperone activity, and substantially increased oligomeric size . The study presented here aims to show whether these changes are due to the loss of a positive charge at this position or due to the presence of an extra Cys . To show this, Arg-116 in alphaA-crystallin was mutated to Lys (R116K), Cys (R116C), Gly (R116G), and Asp (R116D) and expressed in Escherichia coli cells . The wild-type (alphaA-wt) and mutant proteins were purified by size exclusion chromatography and characterized by measurements of circular dichroism, intrinsic tryptophan fluorescence, and TNS fluorescence and by determination of molecular masses and chaperone function which was assessed as the ability to suppress target protein aggregation or enhance target protein refolding . Mutation of Arg-116 to a Cys or Gly showed very similar changes in structure, oligomerization, and chaperone function which suggest that the presence of this Cys per se is not the cause of the changes . The R116K mutant, on the other hand, had nearly the same structure, oligomeric size, and chaperone function as alphaA-wt, whereas the mutant with an acidic amino acid in this position, R116D, showed drastic changes in protein structure . Thus, a positive charge must be preserved at this position for the structural and functional integrity of alphaA-crystallin. Biochemistry, 2002 Oct 15, 41(41), 12284 - 96 NMR solution structure of ATTp, an Arabidopsis thaliana trypsin inhibitor; Zhao Q et al.; The three-dimensional structure of the precursor form of the Arabidopsis thaliana trypsin inhibitor (ATT(p), GenBank entry Z46816), a 68-residue (approximately 7.5 kDa) rapeseed class proteinase inhibitor, has been determined in solution at pH 5.0 and 25 degrees C by multinuclear magnetic resonance spectroscopy . The protein contains one alpha-helix and two strands of antiparallel beta-sheet, with a type IV beta-turn connecting the two strands . The alpha-helix and the inhibitory loop are connected to the beta-sheet through three disulfide bridges; a fourth disulfide bridge connects the N- and C-termini . The overall structural topology of ATT(p) is similar to those of the sweet tasting protein brazzein (rmsd of 3.0 A) and the antifungal protein Rs-Afp1 {a knottin protein from radish (Raphanus sativus), rmsd of 2.7 A} . The precursor segment in ATT(p) is disordered, as visualized by the final 20-conformer ensemble and as confirmed by (15)N heteronuclear NOE analysis . The overall fold of ATT(p) is distinct from those of other classes of serine proteinase inhibitors except in the inhibitor loop; therefore, it represents a new inhibitor fold. Plant Mol Biol, 2002 Oct, 50(3), 511 - 21 Cloning and functional expression of two plant thiol methyltransferases: a new class of enzymes involved in the biosynthesis of sulfur volatiles; Attieh J et al.; Glucosinolates are defensive compounds found in several plant families . We recently described five distinct isoforms of a novel plant enzyme, thiol methyltransferase (TMT), which methylate the hydrolysis products of glucosinolates to volatile sulfur compounds that have putative anti-insect and anti-pathogen roles . In the work presented here, two cDNAs encoding these enzymes (cTMT1 and cTMT2) were isolated by screening a cabbage cDNA library with an Arabidopsis EST showing high sequence homology to one TMT isoform . The genomic clone of cTMT1 was subsequently amplified by PCR . Both cDNAs encoded polypeptides of identical lengths (227 amino acids) and similar predicted masses (ca . 25 kDa), but differing in 13 residues . The cDNAs contained the typical methyltransferase signatures, but were otherwise distinct from conventionally known N-, O- or S-methyltransferases . A chloride methyl transferase was the only gene with an assigned function that shared significant similarity with the TMT cDNAs . Southern analysis indicated single copy for each TMT gene . The two cDNAs were expressed in Escherichia coli . The substrate range, kinetic properties and molecular sizes of the purified recombinant proteins were comparable to those of the native enzyme . These data, together with the detection of the sequenced amino acid motif of one native TMT peptide in the cDNAs, confirmed that the latter were authentic TMTs . The expression pattern of the TMTs in various cabbage tissues was consistent with their association with glucosinolates . The cloning of this new class of plant genes furnishes crucial molecular tools to understand the role of this metabolic sector in plant defenses against biotic stress. Plant Mol Biol, 2002 Oct, 50(3), 393 - 403 Molecular analysis of de novo pyrimidine synthesis in solanaceous species; Giermann N et al.; The de novo synthesis of pyrimidine nucleotides in plants has been analysed on a molecular level with special focus on cDNA cloning and structure analysis of all genes involved and their expression pattern during development . The exhaustive cloning of all cDNAs resulted from screening with heterologous cDNAs or by using complementation strategies with Escherichia coli mutants and subsequent enzyme activity measurements . Southern hybridization and comparison with the Arabidopsis genome reveals plant specific aspects and a simple genomic organization of pyrimidine synthesis in plants, which is superimposed by the postulated, complex subcellular compartmentalization . Northern hybridization evinces coordinated expression of all genes under developmental control during tobacco leaf growth. Nat Struct Biol, 2002 Nov, 9(11), 862 - 9 The catalytic mechanism of the ESA1 histone acetyltransferase involves a self-acetylated intermediate; Yan Y et al.; Yeast ESA1 is a member of the MYST subfamily of histone acetyltransferases (HATs), which use acetyl-coenzyme A (CoA) to acetylate specific Lys residues within histones to regulate gene expression . The structure of an ESA1-CoA complex reveals structural similarity to the catalytic core of the GCN5/PCAF subfamily of HAT proteins . Here we report additional structural and functional studies on ESA1 that demonstrate that histone acetylation proceeds through an acetyl-cysteine enzyme intermediate . This Cys residue is strictly conserved within the MYST members, suggesting a common mode of catalysis by this HAT subfamily . However, this mode of catalysis differs dramatically from the GCN5/PCAF subfamily, which mediate direct nucleophilic attack of the acetyl-CoA cofactor by the enzyme-deprotonated substrate lysine of the histone . These results demonstrate that different HAT subfamilies can use distinct catalytic mechanisms, which have implications for their distinct biological roles and for the development of HAT-specific inhibitors. Nat Biotechnol, 2002 Nov, 20(11), 1140 - 5 Epub 2002 Oct 07. Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and gamma-glutamylcysteine synthetase expression; Dhankher OP et al.; We have developed a genetics-based phytoremediation strategy for arsenic in which the oxyanion arsenate is transported aboveground, reduced to arsenite, and sequestered in thiol-peptide complexes . The Escherichia coli arsC gene encodes arsenate reductase (ArsC), which catalyzes the glutathione (GSH)-coupled electrochemical reduction of arsenate to the more toxic arsenite . Arabidopsis thaliana plants transformed with the arsC gene expressed from a light-induced soybean rubisco promoter (SRS1p) strongly express ArsC protein in leaves, but not roots, and were consequently hypersensitive to arsenate . Arabidopsis plants expressing the E . coli gene encoding gamma-glutamylcysteine synthetase (gamma-ECS) from a strong constitutive actin promoter (ACT2p) were moderately tolerant to arsenic compared with wild type . However, plants expressing SRS1p/ArsC and ACT2p/gamma-ECS together showed substantially greater arsenic tolerance than gamma-ECS or wild-type plants . When grown on arsenic, these plants accumulated 4- to 17-fold greater fresh shoot weight and accumulated 2- to 3-fold more arsenic per gram of tissue than wild type or plants expressing gamma-ECS or ArsC alone . This arsenic remediation strategy should be applicable to a wide variety of plant species. Plant Cell, 2002 Oct, 14(10), 2325 - 38 Rose scent: genomics approach to discovering novel floral fragrance-related genes; Guterman I et al.; For centuries, rose has been the most important crop in the floriculture industry; its economic importance also lies in the use of its petals as a source of natural fragrances . Here, we used genomics approaches to identify novel scent-related genes, using rose flowers from tetraploid scented and nonscented cultivars . An annotated petal EST database of approximately 2100 unique genes from both cultivars was created, and DNA chips were prepared and used for expression analyses of selected clones . Detailed chemical analysis of volatile composition in the two cultivars, together with the identification of secondary metabolism-related genes whose expression coincides with scent production, led to the discovery of several novel flower scent-related candidate genes . The function of some of these genes, including a germacrene D synthase, was biochemically determined using an Escherichia coli expression system . This work demonstrates the advantages of using the high-throughput approaches of genomics to detail traits of interest expressed in a cultivar-specific manner in nonmodel plants . EST sequences were submitted to the GenBank database (accession numbers BQ 103855 to BQ 106728). Microbiology, 2002 Oct, 148(Pt 10), 3265 - 75 Intragenic suppressors of a mutation in the aspartate chemoreceptor gene that abolishes binding of the receptor to methyltransferase; Shiomi D et al.; In the chemotaxis of Escherichia coli, receptor methylation is the key process of adaptation . The methyltransferase CheR binds to the carboxy-terminal NWETF sequence of major chemoreceptors . The substitution of Ala for Trp of this sequence (W550A) of the aspartate chemoreceptor (Tar) abolishes its CheR-binding ability . In this study, six independent intragenic suppressors of the mutation were isolated . They were divided into two classes . Tar carrying the class I suppressors (G278A-L488M, T334A, G278A, G278C and A398T) showed signal biases toward tumbling, corresponding to increased activities of the receptor-associated histidine kinase CheA . These suppressors further reduced the unstimulated methylation level of Tar-W550A, but allowed slight but significant stimulation of methylation by aspartate . Some other CheA-activating mutations were also found to serve as class I suppressors . These results suggest that the class I suppressors compensate for the signal bias of Tar-W550A caused by its low methylation level and that the NWETF sequence is required primarily to maintain an appropriate level of methylation by increasing the local concentration of CheR around the receptor . The class II suppressor was a mutation in the termination codon (Op554W) resulting in the addition of 11 residues containing an xWxxF motif . This revertant Tar supported chemotaxis and was methylated almost as effectively as wild-type Tar . This effect was reversed by introducing a mutation in the xWxxF motif . These results reinforce the importance of the xWxxF motif and suggest that the motif does not have to be located at the extreme carboxy terminus. Microbiology, 2002 Oct, 148(Pt 10), 3213 - 22 Effects of the Min system on nucleoid segregation in Escherichia coli; Akerlund T et al.; The Min system of Escherichia coli directs cell division to the mid-cell by a mechanism that involves the dynamic localization of all of its three constituent proteins, MinC, MinD and MinE . Both the Min system and the nucleoid regulate cell division negatively and strains of E . coli lacking a functional Min system can divide at nucleoid-free cell poles in addition to the nucleoid-free region between newly segregated nucleoids . Interestingly, E . coli strains with a defective Min system have disturbed nucleoid segregation and the cause for this disturbance is not known . It is reported here that growth conditions promoting a higher frequency of polar divisions also lead to a more pronounced disturbance in nucleoid segregation . In strains with an intact Min system, expression of MinE, but not of MinD, from an inducible promoter was followed by impaired nucleoid segregation . These results suggest that the disturbed nucleoid segregation in min mutants is not caused by polar divisions per se, nor by impaired resolution of chromosome dimers in min mutants, leaving open the possibility that the Min system has a direct effect on nucleoid segregation . It is also shown how the disturbed nucleoid segregation can explain in part the unexpected finding that the clear majority of cells in min mutant populations contain 2(n) (n=0, 1, 2.) origins of replication. J Virol, 2002 Nov, 76(21), 10598 - 607 Nuclear localization of human immunodeficiency virus type 1 preintegration complexes (PICs): V165A and R166A are pleiotropic integrase mutants primarily defective for integration, not PIC nuclear import; Limon A et al.; Retroviral replication requires the integration of reverse-transcribed viral cDNA into a cell chromosome . A key barrier to forming the integrated provirus is the nuclear envelope, and numerous regions in human immunodeficiency virus type 1 (HIV-1) have been shown to aid the nuclear localization of viral preintegration complexes (PICs) in infected cells . One region in integrase (IN), composed of Val-165 and Arg-166, was reportedly essential for HIV-1 replication and nuclear localization in all cell types . In this study we confirmed that HIV-1(V165A) and HIV-1(R166A) were replication defective and that less mutant viral cDNA localized to infected cell nuclei . However, we present three lines of evidence that argue against a specific role for Val-165 and Arg-166 in PIC nuclear import . First, results of transient transfections revealed that V165A FLAG-tagged IN and green fluorescent protein-IN fusions carrying either V165A or R166A predominantly localized to cell nuclei . Second, two different strains of previously described class II IN mutant viruses displayed similar nuclear entry profiles to those observed for HIV-1(V165A) and HIV-1(R166A), suggesting that defective nuclear import may be a common phenotype of replication-defective IN mutant viruses . Third, V165A and R166A mutants were defective for in vitro integration activity, when assayed both as PICs isolated from infected T-cells and as recombinant IN proteins purified from Escherichia coli . Based on these results, we conclude that HIV-1(V165A) and HIV-1(R166A) are pleiotropic mutants primarily defective for IN catalysis and that Val-165 and Arg-166 do not play a specific role in the nuclear localization of HIV-1 PICs in infected cells. J Biol Chem, 2002 Dec 6, 277(49), 47770 - 8 Epub 2002 Oct 03. Non-canonical transit peptide for import into the chloroplast; Miras S et al.; The large majority of plastid proteins are nuclear-encoded and, thus, must be imported within these organelles . Unlike most of the outer envelope proteins, targeting of proteins to all other plastid compartments (inner envelope membrane, stroma, and thylakoid) is strictly dependent on the presence of a cleavable transit sequence in the precursor N-terminal region . In this paper, we describe the identification of a new envelope protein component (ceQORH) and demonstrate that its subcellular localization is limited to the inner membrane of the chloroplast envelope . Immunopurification, microsequencing of the natural envelope protein and cloning of the corresponding full-length cDNA demonstrated that this protein is not processed in the N-terminal region during its targeting to the inner envelope membrane . Transient expression experiments in plant cells were performed with truncated forms of the ceQORH protein fused to the green fluorescent protein . These experiments suggest that neither the N-terminal nor the C-terminal are essential for chloroplastic localization of the ceQORH protein . These observations are discussed in the frame of the endosymbiotic theory of chloroplast evolution and suggest that a domain of the ceQORH bacterial ancestor may have evolved so as to exclude the general requirement of an N-terminal plastid transit sequence. Genome Res, 2002 Oct, 12(10), 1523 - 32 Factors influencing the identification of transcription factor binding sites by cross-species comparison; McCue LA et al.; As the number of sequenced genomes has grown, the questions of which species are most useful and how many genomes are sufficient for comparison have become increasingly important for comparative genomics studies . We have systematically addressed these questions with respect to phylogenetic footprinting of transcription factor (TF) binding sites in the gamma-proteobacteria, and have evaluated the statistical significance of our motif predictions . We used a study set of 166 Escherichia coli genes that have experimentally identified TF binding sites upstream of the gene, with orthologous data from nine additional gamma-proteobacteria for phylogenetic footprinting . Just three species were sufficient for approximately 74.0% of the motif predictions to correspond to the experimentally reported E . coli sites, and important characteristics to consider when choosing species were phylogenetic distance, genome size, and natural habitat . We also performed simulations using randomized data to determine the critical maximum a posteriori probability (MAP) values for statistical significance of our motif predictions (P = 0.05) . Approximately 60% of motif predictions containing sites from just three species had average MAP values above these critical MAP values . The inclusion of a species very closely related to E . coli increased the number of statistically significant motif predictions, despite substantially increasing the critical MAP value. Am J Pathol, 2002 Oct, 161(4), 1475 - 84 Lipopolysaccharide induces overexpression of MUC2 and MUC5AC in cultured biliary epithelial cells: possible key phenomenon of hepatolithiasis; Zen Y et al.; Bacterial infection, bile stasis, mucin hypersecretion, and an alteration of the mucin profile such as an aberrant expression of gel-forming apomucin (MUC2 and MUC5AC) in the intrahepatic biliary tree are thought to be important in the lithogenesis of hepatolithiasis . So far, there have been no detailed studies linking bacterial infection to altered mucus secretion of biliary epithelium . In this study, the influence of lipopolysaccharide (LPS), a bacterial component, on apomucin expression in cultured murine biliary epithelial cells was examined with emphasis on the participation of tumor necrosis factor (TNF)-alpha . It was found that LPS up-regulated the expression of MUC2 and MUC5AC in cultured murine biliary epithelial cells . LPS also induced the expression of TNF-alpha in biliary epithelial cells and its secretion into the culture medium . The up-regulation of these apomucins was inhibited by pretreatment with TNF-alpha antibody . TNF-alpha alone also induced the overexpression of MUC2 and MUC5AC in cultured biliary epithelial cells . This overexpression was inhibited by pretreatment with calphostin C, an inhibitor of protein kinase C . These findings suggest that LPS can induce overexpression of MUC2 and MUC5AC in biliary epithelial cells via synthesis of TNF-alpha and activation of protein kinase C . This mechanism might be involved in the lithogenesis of hepatolithiasis. Toxicon, 2002 Oct, 40(10), 1383 - 7 Bordetella pertussis adenylate cyclase toxin: a versatile screening tool; Dautin N et al.; The calmodulin-activated adenylate cyclase (AC) toxin is an essential virulence factor of Bordetella pertussis, the causative agent of whooping cough . This toxin has been exploited to devise screening techniques for investigating diverse biological processes . This mini-review describes several such applications . First, AC has been utilized as a selective reporter for protein translocation from bacteria to eukaryotic cells, in particular to study protein targeting by type III secretion machinery . More recently, AC has been used as a signal transducer in Escherichia coli to elaborate genetic screens for protein-protein interactions ("bacterial two-hybrid system") or site-specific proteolytic activities . Trends Biochem Sci, 2002 Oct, 27(10), 489 - 92 Nanotransducers in cellular redox signaling: modification of thiols by reactive oxygen and nitrogen species; Cooper CE et al.; The control of signal transduction involves post-translational modification of proteins at key amino acids . Cysteine residues are important in the control of 'redox' cell-signaling pathways, as thiol chemistry offers the possibility of modification by structurally diverse species, including those derived from oxidized lipids, peroxides or nitric oxide . An important and provocative study of the modification of thiols in the transcription factor OxyR recently extended this hypothesis . The findings offer the enticing possibility that the cell can distinguish between different degrees of oxidant and nitrosative exposure by modification at a single site on a signaling molecule. J Virol Methods, 2002 Oct, 106(1), 141 - 51 Generation of recombinant fowlpox virus using the non-essential F11L orthologue as insertion site and a rapid transient selection strategy; Boulanger D et al.; Avipoxviruses show an abortive replication phenotype in mammalian cells and are under evaluation as safe vectors for vaccination . Non-essential gene sequences located in highly conserved regions of virus genomes are considered particularly useful to integrate heterologous DNA . Fowlpox virus F11L orthologue is described in this paper as a suitable locus for insertion into fowlpox virus genome . Disruption of the F11L coding sequence by integration of an expression cassette for the Escherichia coli lacZ and guanine phosphoribosyltransferase marker genes resulted in the isolation of replication competent knockout viruses . Growth of F11L-knockout viruses in primary chicken embryo fibroblasts was unimpaired in comparison to wild type-virus . To test the generation of vector viruses, an insertion plasmid was constructed that contains F11L-specific sequences for homologous recombination, the E . coli lacZ and gpt genes as transient selectable marker, and the vaccinia virus early/late promoter P7.5 for transcriptional control of target gene expression . The coding sequence of the melanoma-associated antigen tyrosinase was chosen as model recombinant gene . Isolation of tyrosinase-recombinant viruses, which produced stably the insert, demonstrated the usefulness of the F11L-insertion site for the generation of fowlpox vectors . Rapid isolation of those recombinants was achieved by using a double selective system and linearising the vector plasmid before transfection. Antiviral Res, 2002 Nov, 56(2), 99 - 114 Biochemical characterization of rhinovirus RNA-dependent RNA polymerase; Hung M et al.; Human rhinoviruses (HRV) represent the single most important causative agent of the common cold . The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) designated 3D polymerase that is required for replication of the HRV RNA genome . We have expressed and purified recombinant HRV-16 3D polymerase to near homogeneity from Escherichia coli transformed with an expression plasmid containing the full-length 460 amino acid HRV-16 3D sequence with a methionine at the N-terminus and a glycine-serine linker followed by a 6-histidine affinity tag at the C-terminus . The purified recombinant protein has rifampicin-resistant activity in a poly(A)-dependent poly(U) polymerase assay while corresponding fractions similarly purified from E . coli transformed with an expression plasmid without the HRV-16 3D sequence showed no activity . The optimal conditions for temperature, pH, divalent cations Mg(2+) and Mn(2+), and KCl were determined . The recombinant protein has RNA polymerase activity on homopolymeric templates poly(A) and poly(C) and heteropolymeric RNA templates primed with either RNA or DNA oligonucleotide primers or self-primed by a copy-back mechanism . A unique, secondary structureless heteropolymeric RNA template that is an efficient substrate was developed to facilitate kinetic characterizations of the enzyme . In the presence of Mg(2+), the enzyme displayed strong base and sugar specificity . However, when Mg(2+) was replaced by Mn(2+) specificity for ribonucleotides was lost, utilization of deoxynucleotides became possible and primer-independent activity was observed on the poly(C) template . Zn(2+) was found to inhibit HRV-16 3D polymerase with an IC(50) as low as 0.6 microM by a mechanism distinct from the magnesium ion stimulation . The activity of this 6His-tagged HRV-16 3D polymerase was compared with that of a recombinant HRV-16 3D polymerase expressed without the 6His-tag and was found to be identical . The availability of recombinant rhinovirus RdRp in a purified form will facilitate the structure-function analysis of this enzyme as well as the identification of specific inhibitors to the rhinovirus 3D polymerase that have therapeutic value in the treatment of the common cold . J Mol Biol, 2002 Oct 4, 322(5), 1135 - 46 Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes; de Leeuw E et al.; The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane . The integral membrane proteins TatA, TatB and TatC are essential components of this pathway . Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif . Here we have systematically examined the Tat complexes that can be purified from overproducing strains . Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein . The latter complex is shown to be capable of binding a Tat signal peptide . Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component. J Mol Biol, 2002 Oct 4, 322(5), 1039 - 52 Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide phage display libraries; Rodi DJ et al.; Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries . These libraries behave statistically as though they correspond to populations containing roughly 4.0+/-1.6% of the random dodecapeptides and 7.9+/-2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations . Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis . There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues . The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site . Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a beta-turn conformation . The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences . These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship. J Mol Biol, 2002 Oct 4, 322(5), 983 - 95 Fur-DNA interactions at the bidirectional fepDGC-entS promoter region in Escherichia coli; Lavrrar JL et al.; The transcriptional repressor Fur binds to a 19-bp consensus sequence, 5'-GATAATGATAATCATTATC-3', under high iron conditions . The fepDGC-entS promoter of Escherichia coli contains two Fur-binding sites (FBS) offset by 6bp . Genetic studies of this promoter region revealed two mutations that exhibited a loss of iron regulation in vivo . One mutation altered the upstream portion of FBS 1, whereas the other, originally created to improve entS promoter strength, inadvertently altered the downstream portion of FBS 2 . In both cases, there remains a 19-bp sequence that by current models should be sufficient for Fur binding . The effect of these mutations on Fur binding was examined using in vitro gel retardation assays and DNase I footprinting experiments . Though Fur bound wild-type DNA with high affinity, its affinity for the mutants was reduced, suggesting that both sites are required . In addition, gel shift studies demonstrated that the Fur-promoter complexes exhibit a unique hierarchy of binding, with distinct species forming at increasing concentrations of Fur . The DNA sequences bound in each gel-shifted species were determined using a coupled gel shift/footprint technique . The data presented here, with previously published data, suggest a new model for Fur-DNA interactions similar to that seen with the transcriptional repressor, DtxR . The model predicts that the 19-bp consensus Fur operator is configured as overlapping 13-mer sequences, and that two Fur dimers interact with these sequences from opposite faces of the helix. Mol Microbiol, 2002 Oct, 46(1), 245 - 56 Acidic phospholipids inhibit the DNA-binding activity of DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli; Makise M et al.; In order to initiate chromosomal DNA replication in Escherichia coli, the DnaA protein must bind to both ATP and the origin of replication (oriC) . Acidic phospholipids are known to inhibit DnaA binding to ATP, and here we examine the effects of various phospholipids on DnaA binding to oriC . Among the phospholipids in E . coli membrane, cardiolipin showed the strongest inhibition of DnaA binding to oriC . Synthetic phosphatidylglycerol containing unsaturated fatty acids inhibited binding more potently than did synthetic phosphatidylglycerol containing saturated fatty acids, suggesting that membrane fluidity is important . Thus, acidic phospholipids seem to inhibit DnaA binding to both oriC and adenine nucleotides in the same manner . Adenine nucleotides bound to DnaA did not affect the inhibitory effect of cardiolipin on DnaA binding to oriC . A mobility-shift assay re-vealed that acidic phospholipids inhibited formation of a DnaA-oriC complex containing several DnaA molecules . DNase I footprinting of DnaA binding to oriC showed that two DnaA binding sites (R2 and R3) were more sensitive to cardiolipin than other DnaA binding sites . Based on these in vitro data, the physiological relevance of this inhibitory effect of acidic phospholipids on DnaA binding to oriC is discussed. Mol Microbiol, 2002 Oct, 46(1), 215 - 22 Sequence-specific interaction of nascent antiterminator RNA with the zinc-finger motif of Escherichia coli RNA polymerase; Sen R et al.; The N-terminal Zn-finger motif of the beta' subunit of RNA polymerase contains two pairs of invariant cysteines flanking a moderately well-conserved segment of 13 amino acids that is rich in basic residues . Previous work showed that replacement of certain Zn-finger residues prevented transcription antitermination in response to phage HK022 put sites . Nascent put RNA binds to and modifies transcribing polymerase, so that it becomes resistant to termination . To characterize the Zn finger further, we replaced each of the basic residues with alanine and determined the effects of the substitutions on termination, antitermination and cell viability . All the mutants were defective in put-mediated antitermination . The severity of the defect depended on the mutant and on the sequence of the upstream stem-loop of put RNA . Some, but not all, mutants distinguished between put variants that differed in this region . This suggests that the Zn-finger motif interacts directly and specifically with put RNA . All the mutants in the basic residues complemented a temperature-sensitive beta' mutant for cell growth at a non-permissive temperature, and those mutant enzymes that were tested transcribed and terminated normally in vitro on a template that lacked a put site. Mol Microbiol, 2002 Oct, 46(1), 203 - 14 Physiological role of the GlnK signal transduction protein of Escherichia coli: survival of nitrogen starvation; Blauwkamp TA et al.; Escherichia coli contains two PII-like signal trans-duction proteins, PII and GlnK, involved in nitrogen assimilation . We examined the roles of PII and GlnK in controlling expression of glnALG, glnK and nac during the transition from growth on ammonia to nitrogen starvation and vice versa . The PII protein exclusively controlled glnALG expression in cells adapted to growth on ammonia, but was unable to limit nac and glnK expression under conditions of nitrogen starvation . Conversely, GlnK was unable to limit glnALG expression in cells adapted to growth on ammonia, but was required to limit expression of the glnK and nac promoters during nitrogen starvation . In the absence of GlnK, very high expression of the glnK and nac promoters occurred in nitrogen-starved cells, and the cells did not reduce glnK and nac expression when given ammonia . Thus, one specific role of GlnK is to regulate the expression of Ntr genes during nitrogen starvation . GlnK also had a dramatic effect on the ability of cells to survive nitrogen starvation and resume rapid growth when fed ammonia . After being nitrogen starved for as little as 10 h, cells lacking GlnK were unable to resume rapid growth when given ammonia . In contrast, wild-type cells that were starved immediately resumed rapid growth when fed ammonia . Cells lacking GlnK also showed faster loss of viability during extended nitrogen starvation relative to wild-type cells . This complex phenotype resulted partly from the requirement for GlnK to regulate nac expression; deletion of nac restored wild-type growth rates after ammonia starvation and refeeding to cells lacking GlnK, but did not improve viability during nitrogen starvation . The specific roles of GlnK during nitrogen starvation were not the result of a distinct function of the protein, as expression of PII from the glnK promoter in cells lacking GlnK restored the wild-type phenotypes. Mol Microbiol, 2002 Oct, 46(1), 113 - 24 IHF and HU stimulate assembly of pre-replication complexes at Escherichia coli oriC by two different mechanisms; Ryan VT et al.; Pre-replication complexes (pre-RC) assemble on replication origins and unwind DNA in the presence of chromatin proteins . As components of Escherichia coli pre-RC, two histone-like proteins HU and IHF (integration host factor), stimulate initiator DnaA-catalysed unwinding of the chromosomal replication origin, oriC . Using in vivo footprint analysis just before DNA synthesis initiates, we detect IHF binding coincident with a shift of DnaA to weaker central oriC sites . Integration host factor redistributed pre-bound DnaA to identical sites in vitro . HU did not redistribute DnaA, but suppressed binding specifically at I3 . These results suggest that different pathways mediated by bacterial chromatin proteins exist to regulate pre-RC assembly and unwind oriC. Mol Microbiol, 2002 Oct, 46(1), 63 - 74 The P1 plasmid is segregated to daughter cells by a 'capture and ejection' mechanism coordinated with Escherichia coli cell division; Li Y et al.; The fate of the P1 plasmid of Escherichia coli was followed by time-lapse photomicroscopy . A GFP-ParB fusion marked the plasmid during partition (segregation) to daughter cells at slow growth rate . The process differs from that previously inferred from statistical analysis of fixed cells . A focus of plasmid copies is captured at the cell centre . Immediately before cell division, the copies eject bidirectionally along the long axis of the cell . Cell division traps one or more plasmid copies in each daughter . They are not directed to a prescribed position but are free to move, associate and disassociate . Later, they are captured to the new cell centre to restart the cycle . A null P1 par mutant associates to form a focus, but it is neither captured nor ejected . A dominant negative ParB protein forms a plasmid focus that attaches to the cell centre but never ejects . It remains captive at the centre and blocks host cell division . The cells elongate . Eventually the intact focus is pushed to one side and the cells divide simultaneously in several places at the same time . This suggests that the wild-type plasmid imposes a regulatory node on the host cell cycle, preventing cell division until its own segregation is completed. Plant J, 2002 Oct, 32(1), 93 - 103 Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid beta-oxidation; Fulda M et al.; Post-germinative growth of oilseeds is dependent on the breakdown of the stored lipid reserves . Long-chain acyl-CoA synthetase activities (LACS) are critically involved in this process by activating the released free fatty acids and thus feeding the beta-oxidation cycle in glyoxysomes . Here we report on the identification of two LACS genes, AtLACS6 and AtLACS7 from Arabidopsis thaliana coding for peroxisomal LACS proteins . The subcellular localization was verified by co-expression studies of spectral variants of the green fluorescent protein (GFP) . While AtLACS6 is targeted by a type 2 (PTS2) peroxisomal targeting sequence, for AtLACS7 a functional PTS1 as well as a PTS2 could be demonstrated . Possible explanations for this potentially redundant targeting information will be discussed . Expression studies of both genes revealed a strong induction 1 day after germination resembling the expression pattern of other genes involved in beta-oxidation . Analysis of the substrate specificities of the two LACS proteins demonstrated enzymatic activity for both enzymes with the whole spectrum of fatty acids found in stored lipid reserves . These results suggest that both LACS proteins might have overlapping functions and are able to initiate beta-oxidation in plant peroxisomes. Clin Exp Pharmacol Physiol, 2002 Nov, 29(11), 990 - 5 Nitric oxide inhibits renal cytochrome P450-dependent epoxygenases in the rat; Oyekan A; 1 . Nitric oxide (NO), or peroxynitrite, is known to inhibit haemoproteins, including cytochrome P450 mono-oxygenases . The present study explores the functional correlates of the inhibition by NO of renal epoxygenase on the vascular responses to arachidonic acid (AA) in the perfused kidney . 2 . Control kidneys produce measurable amounts of epoxyeicosatrienoic acids (epoxides), which were increased from 0.6 +/- 0.2 to 1.8 +/- 0.9 ng/min (P < 0.05) following the addition of AA 5 micro g . Sodium nitroprusside (SNP; 100 micro mol/L), an NO donor, blunted the basal and AA-stimulated efflux of epoxides . 3 . Sodium nitroprusside at 10 and 100 micro mol/L inhibited renal microsomal conversion of {14C}-AA to epoxides and its hydration products dihydroxyeicosatrienoic acid (diols) . Microsomes harvested from rats 3 h after treatment with Escherichia coli endotoxin (lipopolysaccharide; LPS) also inhibited renal epoxygenase activity (81 +/- 8%; P < 0.05) . 4 . In the phenylephrine-preconstricted and indomethacin (2.8 micro mol/L)-treated kidney, AA at 5, 10 and 25 micro g elicited vasodilation that was blunted by miconazole (2 micro mol/L), 80 mmol/L KCl, tetraethylammonium (10 mmol/L), a K+ channel blocker, or SNP (100 micro mol/L) . 5 . Vasodilation induced by AA, but not 5,6-epoxide, was reduced in rats treated with LPS, an effect that was abolished by Nomega-nitro-l-arginine (100 mg/kg in drinking water for 10 days) . 6 . These data suggest that NO inhibits renal epoxygenase activity and inhibits epoxide-mediated AA-induced vasodilation in the rat kidney. Phys Rev E Stat Nonlin Soft Matter Phys . 2002 Sep;66(3 Pt 1):031910 . Epub 2002 Sep 24. Recognition of an organism from fragments of its complete genome; Anh VV et al.; This paper considers the problem of matching a fragment to an organism using its complete genome . Our method is based on the probability measure representation of a genome . We first demonstrate that these probability measures can be modeled as recurrent iterated function systems (RIFS) consisting of four contractive similarities . Our hypothesis is that the multifractal characteristics of the probability measure of a complete genome, as captured by the RIFS, is preserved in its reasonably long fragments . We compute the RIFS of fragments of various lengths and random starting points, and compare with that of the original sequence for recognition using the Euclidean distance . A demonstration on five randomly selected organisms supports the above hypothesis. Pharmacology, 2002 Oct, 66(2), 57 - 60 In vitro effect of fluticasone propionate on interleukin 8 production by monocytes obtained from patients affected by moderate-severe allergic asthma; Gangemi S et al.; BACKGROUND: Interleukin-8 (IL-8), a potent chemotactic and activating factor for neutrophils and eosinophils, may be a crucial factor in severe asthma . The aim of this study was to evaluate the effect of fluticasone propionate (FP), a corticosteroid with potent anti-inflammatory activity, on the in vitro release of IL-8 by monocytes obtained from asthmatic patients . METHODS: Monocytes from 15 non-atopic healthy donors and from 15 patients affected by moderate-severe allergic asthma were isolated and incubated (37 degrees C, 5% CO(2)) for 24 h with varying combinations of lipopolysaccharide (LPS) from Escherichia coli (1 microg/ml) and FP (100 nmol/l) . IL-8 concentration in the culture supernatant was measured by an immuno-enzymatic method (ELISA) . RESULTS: A highly significant inverse correlation between FEV1 (forced expiratory volume) values before withdrawal and in vitro IL-8 production by unstimulated monocytes from asthmatic patients was observed (Rho = -0.787; p = 0.0032) . IL-8 production by either LPS-stimulated or unstimulated monocytes from asthmatic subjects was statistically increased compared to monocytes from healthy donors (p < 0.05) . FP addition reduced IL-8 production by monocytes from asthmatic patients and also from healthy donors (p < 0.05) . CONCLUSIONS: The partial IL-8 inhibition by FP could be closely related to the anti-inflammatory activity of this corticosteroid . Based on these results, we propose that the clinical improvement of asthma, observed following FP administration, may depend, at least in part, on the ability of this drug to modulate cytokine production by monocytes . Nucleic Acids Res, 2002 Oct 1, 30(19), 4250 - 63 RNA canonical and non-canonical base pairing types: a recognition method and complete repertoire; Lemieux S et al.; The problem of systematic and objective identification of canonical and non-canonical base pairs in RNA three-dimensional (3D) structures was studied . A probabilistic approach was applied, and an algorithm and its implementation in a computer program that detects and analyzes all the base pairs contained in RNA 3D structures were developed . The algorithm objectively distinguishes among canonical and non-canonical base pairing types formed by three, two and one hydrogen bonds (H-bonds), as well as those containing bifurcated and C-H.X...H-bonds . The nodes of a bipartite graph are used to encode the donor and acceptor atoms of a 3D structure . The capacities of the edges correspond to probabilities computed from the geometry of the donor and acceptor groups to form H-bonds . The maximum flow from donors to acceptors directly identifies base pairs and their types . A complete repertoire of base pairing types was built from the detected H-bonds of all X-ray crystal structures of a resolution of 3.0 A or better, including the large and small ribosomal subunits . The base pairing types are labeled using an extension of the nomenclature recently introduced by Leontis and Westhof . The probabilistic method was implemented in MC-Annotate, an RNA structure analysis computer program used to determine the base pairing parameters of the 3D modeling system MC-Sym. Nucleic Acids Res, 2002 Oct 1, 30(19), 4158 - 65 Investigating the endonuclease activity of four Pyrococcus abyssi inteins; Saves I et al.; Among the 14 inteins of the Pyrococcus abyssi genome, 10 harbour the LAGLIDADG motifs of dodecapeptide endonucleases . Four of these were cloned, expressed in Escherichia coli and purified to assay their potential endonuclease activity . PabRIR1-2 and PabRIR1-3 are specific endonucleases, named PI-PabI and PI-PabII, respectively, cleaving the sequence spanning their homing site . This is consistent with their size and with the relative positions and sequences of their endonuclease motifs . However, PI-PabI is 10-fold more active than PI-PabII and a discrepancy of the DNA recognition and cleavage mechanisms was observed between the two inteins . In particular, analysis of the DNA cleavage reactions by MALDI-TOF highlighted that while the cleavage of DNA by PI-PabI consists of two steps corresponding to the cleavage of each DNA strand, PI-PabII processes the two DNA strands simultaneously . Furthermore, the two inteins interact differently with DNA . In addition, we did not detect any endonuclease activity for PabLon and PabRIR1-1 . Deletions in the intein sequences and mutations in the putative endonuclease motifs probably abolish this activity . Hence, inteins from the same archaebacteria, even if contained in the same host protein, did not evolve uniformly and are presumably at different stages of the invasion cycle. Protein Eng, 2002 Aug, 15(8), 635 - 42 A major IgE epitope-containing grass pollen allergen domain from Phl p 5 folds as a four-helix bundle; Maglio O et al.; Phl p 5, a 29 kDa major allergen from timothy grass pollen, is one of the most reactive members of group 5 allergens . Its sequence comprises two repeats of a novel alanine-rich motif (AR) whose structure and allergenic response are still mostly unknown . We report here a structural characterization of an immunodominant fragment of Phl p 5, Phl p 5(56-165) which comprises the first AR repeat . Recombinant (r)Phl p 5(56-165) was expressed in Escherichia coli, purified to homogeneity and shown to be sufficient to react with serum IgE from 90% of grass pollen allergic patients . Using NMR spectroscopy, we show conclusively that the fragment forms a compact globular domain which is, however, prone to degradation with time . The rPhl p 5(56-165) fold consists of a four-helix bundle held together by hydrophobic interactions between the aromatic rings and aliphatic side chains . This evidence gives clear indications about the structure of the full-length Phl p 5 and provides a rational basis for finding ways to stabilize the fold and designing therapeutic vaccines against grass pollen allergy. Protein Eng, 2002 Aug, 15(8), 627 - 33 Structural consequences of replacement of an alpha-helical Pro residue in Escherichia coli thioredoxin; Rudresh et al.; While it is well known that introduction of Pro residues into the interior of protein alpha-helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein alpha-helix . We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1-1.5 kcal/mol in the temperature range 280-330 K . This paper describes the structure of the P40S mutant at a resolution of 1.8 A . In wild-type thioredoxin, P40 is located in the interior of helix two, a long alpha-helix that extends from residues 32 to 49 with a kink at residue 40 . Structural differences between the wild-type and P40S are largely localized to the above helix . In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36 . The helix remains kinked . In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39 . However, these are absent in P40S . Instead, these main chain atoms are hydrogen bonded to water molecules . The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin. Circ Res, 2002 Oct 4, 91(7), 618 - 25 Adrenomedullin reduces endothelial hyperpermeability; Hippenstiel S et al.; Endothelial hyperpermeability induced by inflammatory mediators is a hallmark of sepsis and adult respiratory distress syndrome . Increased levels of the regulatory peptide adrenomedullin (ADM) have been found in patients with systemic inflammatory response . We analyzed the effect of ADM on the permeability of cultured human umbilical vein endothelial cell (HUVEC) and porcine pulmonary artery endothelial cell monolayers . ADM dose-dependently reduced endothelial hyperpermeability induced by hydrogen peroxide (H2O2), thrombin, and Escherichia coli hemolysin . Moreover, ADM pretreatment blocked H2O2-related edema formation in isolated perfused rabbit lungs and increased cAMP levels in lung perfusate . ADM bound specifically to HUVECs and porcine pulmonary artery endothelial cells and increased cellular cAMP levels . Simultaneous inhibition of cAMP-degrading phosphodiesterase isoenzymes 3 and 4 potentiated ADM-dependent cAMP accumulation and synergistically enhanced ADM-dependent reduction of thrombin-induced hyperpermeability . However, ADM showed no effect on endothelial cGMP content, basal intracellular Ca2+ levels, or the H2O2-stimulated, thrombin-stimulated, or Escherichia coli hemolysin-stimulated Ca2+ increase . ADM diminished thrombin- and H2O2-related myosin light chain phosphorylation as well as stimulus-dependent stress fiber formation and gap formation in HUVECs, suggesting that ADM may stabilize the barrier function by cAMP-dependent relaxation of the microfilament system . These findings identify a new function of ADM and point to ADM as a potential interventional agent for the reduction of vascular leakage in sepsis and adult respiratory distress syndrome. J Biol Chem, 2002 Dec 20, 277(51), 50155 - 9 Epub 2002 Oct 02. Mechanism of action of RNase T . I . Identification of residues required for catalysis, substrate binding, and dimerization; Zuo Y et al.; Escherichia coli RNase T, an RNA-processing enzyme and a member of the DEDD exonuclease superfamily, was examined using sequence analysis and site-directed mutagenesis . Like other DEDD exonucleases, RNase T was found to contain three conserved Exo motifs that included four invariant acidic residues . Mutagenesis of these motifs revealed that they are essential for RNase T activity, indicating that they probably form the RNase T catalytic center in a manner similar to that found in other DEDD exonucleases . We also identified by sequence analysis three short, but highly conserved, sequence segments rich in positively charged residues . Site-directed mutagenesis of these regions indicated that they are involved in substrate binding . Additional analysis revealed that residues within the C-terminal region of RNase T are essential for RNase T dimerization and, consequently, for RNase T activity . These data define the domains necessary for RNase T action, and together with information in the accompanying article, have led to the formulation of a detailed model for the structure and mechanism of action of RNase T. J Biol Chem, 2002 Dec 20, 277(51), 50160 - 4 Epub 2002 Oct 02. Mechanism of action of RNase T . II . A structural and functional model of the enzyme; Zuo Y et al.; A detailed structural and functional model of E . coli RNase T was generated based on sequence analysis, homology modeling, and experimental observation . In the accompanying article, three short sequence segments (nucleic acid binding sequences (NBS)) important for RNase T substrate binding were identified . In the model, these segments cluster to form a positively charged surface patch . However, this patch is on the face of the RNase T monomer opposite the DEDD catalytic center . We propose that by dimerization, the NBS patch from one subunit is brought to the vicinity of the DEDD center of the second monomer to form a fully functional RNase T active site . In support of this model, mutagenetic studies show that one NBS1 residue, Arg(13), sits at the catalytic center despite being on the opposite side of the monomer . Second, the complementarity of the RNase T subunits through the formation of homodimers was demonstrated by reconstitution of partial RNase T activity from monomers derived from two inactive mutant proteins, one defective in catalysis and one in substrate binding . These data explain why RNase T must dimerize to function . The model provides a detailed framework on which to explain the mechanism of action of RNase T. Biotechnol Prog, 2002 Sep-Oct, 18(5), 1054 - 9 Protein purification via aqueous two-phase extraction (ATPE) and immobilized metal affinity chromatography . Effectiveness of salt addition to enhance selectivity and yield of GFPuv; Li Y et al.; This study illustrates the compatibility and complementary nature of aqueous two-phase extraction (ATPE) and immobilized metal affinity chromatography (IMAC) in a general recovery scheme . The purification of green fluorescent protein (GFPuv) from extracts of Eschericia coli was investigated using a combination of these two techniques . High molarity of sodium chloride was found effective in increasing selectivity, with the promotion of hydrophobic interaction the probable mechanism that drove the target protein to a particular phase in ATPE, as well as that which enhanced GFPuv adsorption in IMAC . Moreover, the similar salt condition allows the direct application of the GFPuv-containing phase to the IMAC column without additional adjustment step . A simple screen of conditions was therefore performed to generate a favorable two-step purification scheme for GFP leading to an overall high purity. J Comput Aided Mol Des, 2002 Mar, 16(3), 167 - 79 Investigation of the metal binding site in methionine aminopeptidase by density functional theory; Jorgensen AT et al.; All methionine aminopeptidases exhibit the same conserved metal binding site . The structure of this site with either Co2+ ions or Zn2+ ions was investigated using density functional theory . The calculations showed that the structure of the site was not influenced by the identity of the metal ions . This was the case for both of the systems studied; one based on the X-ray structure of the human methionine aminopeptidase type 2 (hMetAP-2) and the other based on the X-ray structure of the E . coli methionine aminopeptidase type I (eMetAP- 1) . Another important structural issue is the identity of the bridging oxygen, which is part of either a water molecule or a hydroxide ion . Within the site of hMetAP-2 the results strongly indicate that a hydroxide ion bridges the metal ions . By contrast, the nature of the oxygen bridging the metal ions within the metal binding site of eMetAP-1 cannot be determined based on the results here, due to the similar structural results obtained with a bridging water molecule and a bridging hydroxide ion. Oral Dis, 2002 Sep, 8(5), 261 - 7 Effect of high-density lipoprotein on lipopolysaccharide-induced alveolar bone resorption in rats; Jonarta AL et al.; OBJECTIVE: To determine whether treatments with high-density lipoprotein (HDL) may alter the lipopolysaccharide (LPS)-induced alveolar bone resorption in rats . MATERIALS AND METHODS: Rats were injected with 500 microg of LPS from Escherichia coli at the alveolar mucosa of lower right first molar once every 2 days for 8 days . The negative and positive control were injected with phosphate buffered saline (PBS) and LPS alone, respectively . In HDL-treated animals various concentration of HDL were injected immediately before, after the third or the final LPS injection . The bone sections were stained with tartrate-resistant acid phosphatase (TRAP) and the numbers of both osteoclasts and preosteoclasts and the levels of alveolar bone resorption were assessed . RESULTS: The numbers of both osteoclasts and preosteoclasts and the levels of alveolar bone resorption in animals treated with HDL before or during LPS injections were lower than those in the positive control, but higher than those in the negative control, regardless of HDL doses . Similar results were also observed in animals treated with 250 and 500 microg of HDL after the final LPS injection . Only treatments with 1000 microg of HDL after LPS injections completely reduced the number of both osteoclasts and preosteoclasts, but only partially decreased the alveolar bone resorption . CONCLUSION: HDL treatments partially reduced the LPS-induced alveolar bone resorption in vivo in rats, suggesting that HDL may neutralize the ability of LPS to induce alveolar bone resorption. Microb Drug Resist, 2002 Fall, 8(3), 179 - 85 Inactivation of the acrA gene is partially responsible for chloramphenicol sensitivity of Escherichia coli CM2555 strain expressing the chloramphenicol acetyltransferase gene; Potrykus J et al.; An Escherichia coli CM2555 strain, sensitive to chloramphenicol when expressing the cat gene and producing active chloramphenicol acetyltransferase (CAT), was described recently . It was proposed that this sensitivity is due to decreased levels of acetyl coenzyme A (Acetyl CoA) in cat-expressing CM2555 cells in the presence of chloramphenicol . CAT catalyzes transfer of the acetyl moiety from Acetyl CoA to a chloramphenicol molecule . Thus, a very efficient acetylation of chloramphenicol may cause deprivation of Acetyl CoA and cell death . A specific mutation causing the chloramphenicol sensitivity phenotype of CM2555 was not reported to date . Therefore, we aimed to identify a genetic defect causing this phenotype . Here, we found that overexpression of the acrEF genes, encoding a transmembrane pump, or the acrE gene alone, results in restoration of chloramphenicol-resistance of cat-expressing CM2555 strain . Although no mutation exists in the CM2555 acrE locus, a nonsense mutation in the 67th codon of the acrA gene, which encodes a component of another transmembrane pump, has been found . Although introduction of the deltaacrAB allele into CM732, a parental strain of CM2555, and into some other commonly used E . coli strains led to their chloramphenicol sensitivity in the presence of CAT, the same genetic manipulation did not result in such a phenotype in other genetic backgrounds, including "wild-type" E . coli MG1655 . These results suggest that the acrA dysfunction is one of more mutations responsible fo