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fermentations of pectin-rich biomass with recombinant bacteria to produce fuel ethanol.pectin-rich residues from sugar beet processing contain significant carbohydrates and insignificant amounts of lignin. beet pulp was evaluated for conversion to ethanol using recombinant bacteria as biocatalysts. hydrolysis of pectin-rich residues followed by ethanolic fermentations by yeasts has not been productive because galacturonic acid and arabinose are not fermentable to ethanol by these organisms. the three recombinant bacteria evaluated in this study, escherichia coli strain ko11, klebs ...200010849785
secretion of nucleoside diphosphate kinase by mucoid pseudomonas aeruginosa 8821: involvement of a carboxy-terminal motif in secretion.nucleoside diphosphate kinase (ndk) is a ubiquitous enzyme which functions in balancing the nucleotide pool of the cell. we have recently reported that in addition to being intracellular in both mucoid and nonmucoid pseudomonas aeruginosa, ndk is also secreted into the extracellular environment by mucoid p. aeruginosa cells. this secreted ndk has biochemical activity similar to the intracellular ndk and is 16 kda in size. to demonstrate that ndk is indeed secreted and to localize the secretion m ...200010851000
a regulatory rna (prrb rna) modulates expression of secondary metabolite genes in pseudomonas fluorescens f113.the gacs-gaca two-component signal transduction system, which is highly conserved in gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in pseudomonas spp. screening of a pseudomonas fluorescens f113 gene bank led to the isolation of a previously undefined locus which could restore secondary metabolite production to both gacs and gaca mutants of f113. sequence analysis of this locus demonstrated that it did not contain any obvious pseudomonas protein-c ...200010869066
influence of deletions within domain ii of exotoxin a on its extracellular secretion from pseudomonas aeruginosa.pseudomonas aeruginosa is a gram-negative bacterium that secretes many proteins into the extracellular medium via the xcp machinery. this pathway, conserved in gram-negative bacteria, is called the type ii pathway. the exoproteins contain information in their amino acid sequence to allow targeting to their secretion machinery. this information may be present within a conformational motif. the nature of this signal has been examined for p. aeruginosa exotoxin a (pe). previous studies failed to id ...200010869085
overproduction in escherichia coli of the pectin methylesterase a from erwinia chrysanthemi 3937: one-step purification, biochemical characterization, and production of polyclonal antibodies.pectin methylesterase a (ec 3.1.1.11), one of the pathogenicity factors of erwinia chrysanthemi strain 3937, was purified to homogeneity using one-step chromatography on cross-linked pectate. the purified protein showed maximum activity at ph 8-9, 50 degrees c, 50-100 mm monovalent cations or 5-10 mm divalent cations, and on a 50% esterified pectin. a particular effect of ca2+ and zn2+ on pmea activity, due to the formation of a pectin gel, was observed. a km value of 0.03% and 0.051% was determ ...200010872083
a novel beta-glucoside-specific pts locus from streptococcus mutans that is not inhibited by glucose.a regulon from streptococcus mutans that plays a role in the utilization of beta-glucosides has been isolated, sequenced and subjected to sequence analysis. this regulon encodes a beta-glucoside-specific enzyme ii (eii) component (bglp) of the phosphoenolpyruvate-dependent phosphotransferase system (pts) and a phospho-beta-glucosidase (bgla) which is responsible for the breakdown of the phospho-beta-glucosides within the cell. both the bglp and bgla gene products have significant similarity with ...200010878120
cloning and sequencing of pel gene responsible for cmcase activity from erwinia chrysanthemi py35.the phytopathogenic bacterium erwinia chrysanthemi secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanase. we cloned genomic dna from erwinia chrysanthemi py35. one of the e. coli xl1-blue clones contained a 5.1-kb bamhi fragment and hydrolyzed carboxymethyl cellulose and polygalacturonic acid. by subsequent subcloning, we obtained a 2.9-kb fragment (ppy100) that contained the pel gene responsible for cmcase and pectate lyase activities. the pel ...200010879460
cloning and sequencing of cel5z gene from erwinia chrysanthemi py35.the phytopathogenic bacterium erwinia chrysanthemi (ech) secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanases. we cloned genomic dna from ech py35 digested with sau3ai and ligated into pbluescript ii sk+. one of the e. coli xl1-blue clones had the ability to hydrolyze carboxymethyl cellulose and polygalacturonic acid. by subsequent subcloning from this 2.9 kb fragment, we obtained a 2.0 kb (ppy401), designated cel5z, which had the activity of ...200010901164
crystal structure of escherichia coli cyay protein reveals a previously unidentified fold for the evolutionarily conserved frataxin family.friedreich ataxia is an autosomal recessive neurodegenerative disease caused by defects in the frda gene, which encodes a mitochondrial protein called frataxin. frataxin is evolutionarily conserved, with homologs identified in mammals, worms, yeast, and bacteria. the cyay proteins of gamma-purple bacteria are believed to be closely related to the ancestor of frataxin. in this study, we have determined the crystal structure of the cyay protein from escherichia coli at 1.4-a resolution. it reveals ...200010908679
identification and characterization of a membrane permease involved in iron-hydroxamate transport in staphylococcus aureus.staphylococcus aureus was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desferrioxamine. an s. aureus mutant defective in the ability to transport ferric hydroxamate complexes was isolated from a tn917-ltv1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin. chromosomal dna flanking the tn917-ltv1 insertion was identified by sequencing of chromosomal dna isolated from ...200010913070
functional characterization of the hasa(pf) hemophore and its truncated and chimeric variants: determination of a region involved in binding to the hemophore receptor.hemophores are secreted by several gram-negative bacteria (serratia marcescens, pseudomonas aeruginosa, pseudomonas fluorescens, and yersinia pestis) and form a family of homologous proteins. unlike the s. marcescens hemophore (hasa(sm)), the p. fluorescens hemophore hasa(pf) has an additional region of 12 residues located immediately upstream from the c-terminal secretion signal. we show that hasa(pf) undergoes a c-terminal cleavage which removes the last 21 residues when secreted from p. fluor ...200010913071
regulation of expression of the yiaklmnopqrs operon for carbohydrate utilization in escherichia coli: involvement of the main transcriptional factors.the yiaklmnopqrs (yiak-s) gene cluster of escherichia coli is believed to be involved in the utilization of a hitherto unknown carbohydrate which generates the intermediate l-xylulose. transcription of yiak-s as a single message from the unique promoter found upstream of yiak is proven in this study. the 5' end has been located at 60 bp upstream from the atg. expression of the yiak-s operon is controlled in the wild-type strain by a repressor encoded by yiaj. no inducer molecule of the yiak-s op ...200010913096
a genomic sample sequence of the entomopathogenic bacterium photorhabdus luminescens w14: potential implications for virulence.photorhabdus luminescens is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. after invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. however, the bacterial virulence factors associated with this "symbiosis of pathogens" remain largely obscure. in order to identify genes encoding potential virulence factors, we performed approximately 2,00 ...200010919786
structure and function of pectic enzymes: virulence factors of plant pathogens.the structure and function of erwinia chrysanthemi pectate lysase c, a plant virulence factor, is reviewed to illustrate one mechanism of pathogenesis at the molecular level. current investigative topics are discussed in this paper.200010922032
pseudomonas syringae hrp type iii secretion system and effector proteins.pseudomonas syringae is a member of an important group of gram-negative bacterial pathogens of plants and animals that depend on a type iii secretion system to inject virulence effector proteins into host cells. in p. syringae, hrp/hrc genes encode the hrp (type iii secretion) system, and avirulence (avr) and hrp-dependent outer protein (hop) genes encode effector proteins. the hrp/hrc genes of p. syringae pv syringae 61, p. syringae pv syringae b728a, and p. syringae pv tomato dc3000 are flanke ...200010922033
plants and animals share functionally common bacterial virulence factors.by exploiting the ability of pseudomonas aeruginosa to infect a variety of vertebrate and nonvertebrate hosts, we have developed model systems that use plants and nematodes as adjuncts to mammalian models to help elucidate the molecular basis of p. aeruginosa pathogenesis. our studies reveal a remarkable degree of conservation in the virulence mechanisms used by p. aeruginosa to infect hosts of divergent evolutionary origins.200010922040
a new high-alkaline and high-molecular-weight pectate lyase from a bacillus isolate: enzymatic properties and cloning of the gene for the enzyme.a pectate lyase (pel; pectate transeliminase: ec4.2.2.2.), designated pel-15h, was found in an alkaline culture of bacillus sp. strain ksm-p15 and purified to homogeneity by sequential column chromatographies. the molecular weight of the enzyme determined by sds-polyacrylamide gel electrophoresis was approximately 70,000 and the pi was around ph 4.6. pel-15h randomly trans-eliminated polygalacturonate in the presence of ca2+ ions, and the maximum activity was observed at ph 11.5 and at 55 degree ...200010923781
achromobactin, a new citrate siderophore of erwinia chrysanthemi.the structure of a citrate siderophore named achromobactin isolated from the culture medium of erwinia chrysanthemi was elucidated by spectroscopic methods and chemical degradation.200010928541
characterization of a tonb mutation in erwinia chrysanthemi 3937: tonb(ech) is a member of the enterobacterial tonb family.the pectinolytic enterobacterium erwinia chrysanthemi 3937 causes a systemic disease in its natural host, the african violet (saintpaulia: ionantha). it produces two structurally unrelated siderophores, chrysobactin and achromobactin. chrysobactin makes a large contribution to invasive growth of the bacterium in its host. insertion mutants of a chrysobactin-defective strain were constructed and screened on the universal cas-agar medium used for siderophore detection. a set of mutants affected in ...200010931909
external ph: an environmental signal that helps to rationalize pel gene duplication in erwinia chrysanthemi.the phytopathogenic bacterium erwinia chrysanthemi produces five major pectate lyases that are key virulence factors in soft-rot disease development. using transcriptional fusions, we studied the regulation of pela, peld, and pele gene expression as a function of variation of the external ph. pela and peld were expressed when bacteria were grown in an acidic medium while pele was transcribed only in basic medium. using phenol red, we observed that, in chicory leaves, ph value of infected tissue ...200010939260
up-regulation of both intimin and eae-independent adherence of shiga toxigenic escherichia coli o157 by ler and phenotypic impact of a naturally occurring ler mutation.shiga toxigenic escherichia coli (stec) strains are important human pathogens which are capable of causing diarrhea, hemorrhagic colitis, and the potentially fatal hemolytic-uremic syndrome (hus). an important virulence trait of certain stec strains, such as those belonging to serogroup o157, is the capacity to produce attaching and effacing (a/e) lesions on enterocytes, a property encoded by the locus for enterocyte effacement (lee). lee contains the eae gene, which encodes intimin, an outer me ...200010948164
the redox-sensitive transcriptional activator oxyr regulates the peroxide response regulon in the obligate anaerobe bacteroides fragilis.the peroxide response-inducible genes ahpcf, dps, and katb in the obligate anaerobe bacteroides fragilis are controlled by the redox-sensitive transcriptional activator oxyr. this is the first functional oxidative stress regulator identified and characterized in anaerobic bacteria. oxyr and dps were found to be divergently transcribed, with an overlap in their respective promoter regulatory regions. b. fragilis oxyr and dps proteins showed high identity to homologues from a closely related anaer ...200010960088
plant genome complexity may be a factor limiting in situ the transfer of transgenic plant genes to the phytopathogen ralstonia solanacearum.the development of natural competence by bacteria in situ is considered one of the main factors limiting transformation-mediated gene exchanges in the environment. ralstonia solanacearum is a plant pathogen that is also a naturally transformable bacterium that can develop the competence state during infection of its host. we have attempted to determine whether this bacterium could become the recipient of plant genes. we initially demonstrated that plant dna was released close to the infecting ba ...200010966449
erwinia chrysanthemi strains cause death of human gastrointestinal cells in culture and express an intimin-like protein.the bacterium erwinia chrysanthemi is a model plant pathogen, responsible for causing cell death in plant tissue. cell-wall depolymerizing enzymes and avirulence proteins essential for parasitism by this bacterium utilize dedicated type ii and type iii secretion systems, respectively. although e. chrysanthemi is not recognized as a mammalian pathogen, we have observed that the bacterium can adhere to, cause an oxidative stress response in and kill cultured human adenocarcinoma cells. these bacte ...200010981694
gene conservation and loss in the muts-rpos genomic region of pathogenic escherichia coli.the extent and nature of dna polymorphism in the muts-rpos region of the escherichia coli genome were assessed in 21 strains of enteropathogenic e. coli (epec) and enterohemorrhagic e. coli (ehec) and in 6 strains originally isolated from natural populations. the intervening region between muts and rpos was amplified by long-range pcr, and the resulting amplicons varied substantially in length (7.8 to 14.2 kb) among pathogenic groups. restriction maps based on five enzymes and sequence analysis ...200010986240
synergistic hydrolysis of carboxymethyl cellulose and acid-swollen cellulose by two endoglucanases (celz and cely) from erwinia chrysanthemi.erwinia chrysanthemi produces a battery of hydrolases and lyases which are very effective in the maceration of plant cell walls. although two endoglucanases (celz and cely; formerly egz and egy) are produced, celz represents approximately 95% of the total carboxymethyl cellulase activity. in this study, we have examined the effectiveness of cely and celz alone and of combinations of both enzymes using carboxymethyl cellulose (cmc) and amorphous cellulose (acid-swollen cellulose) as substrates. s ...200011004164
alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the erwinia chrysanthemi cel5 cellulase (ex-egz) via the type ii system.cel5 (formerly known as endoglucanase z) of erwinia chrysanthemi is secreted by the out type ii pathway. previous studies have shown that the catalytic domain (cd), linker region (lr) and cellulose-binding domain (cbd) each contain information needed for secretion. the aim of this work was to further investigate the secretion-related information present in the cbd(cel5). firstly(, )deleting a surface-exposed flexible loop had no effect on secretion. this indicated that some structural freedom is ...200011023779
pseudobactin biogenesis in the plant growth-promoting rhizobacterium pseudomonas strain b10: identification and functional analysis of the l-ornithine n(5)-oxygenase (psba) gene.pseudobactin(b10), the fluorescent siderophore produced by the rhizobacterium pseudomonas strain b10, contains the hydroxamate ligand d-n(5)-hydroxyornithine (d-n(5)-oh-orn). we cloned the l-orn n(5)-oxygenase (psba) gene from a genomic library of pseudomonas strain b10 and demonstrated that psba is involved in the conversion of l-orn to its n(5)-oh derivative. psba shows significant similarity to microbial omega-amino acid hydroxylases containing flavin adenine dinucleotide and nadp cofactor-bi ...200011029447
functional implications of the beta-helical protein fold: differences in chemical and thermal stabilities of erwinia chrysanthemi ec16 pectate lyases b, c, and e.colonization of plant tissue by the phytopathogen erwinia chrysanthemi ec16 is aided by the activities of the pectate lyase isozymes (pls), which depolymerize the polygalacturonic acid component (pga) of plant cell walls. the bacterium secretes four pectate lyases (pla, plb, plc, and ple), two of which, plc and ple, have been shown to fold into a similar domain motif, the beta-helix. to understand the rationale behind the evolution and retention of these isoforms, the susceptibilities of pectate ...200011032414
molecular cloning and analysis of a putative siderophore abc transporter from staphylococcus aureus.from a mass-excised staphylococcus aureus lambdazapii expression library, we cloned an operon encoding a novel abc transporter with significant homology to bacterial siderophore transporter systems. the operon encodes four genes designated ssta, -b, -c, and -d encoding two putative cytoplasmic membrane proteins (ssta and sstb), an atpase (sstc), and a membrane-bound 38-kda lipoprotein (sstd). the sst operon is preceded by two putative fur boxes, which indicated that expression of the sst operon ...200011035736
complete nucleotide sequence of ubiquitous plasmid pea29 from erwinia amylovora strain ea88: gene organization and intraspecies variation.the complete sequence of plasmid pea29 from erwinia amylovora strain ea88 consists of 28,185 bp with a 50.2% g+c content. as deletions and insertions were detected in other derivatives of pea29, its size actually varied from 27.6 to 34.9 kb. thirteen open reading frames that encoded predicted proteins with similarities to known proteins from other bacteria were identified along with two open reading frames related to hypothetical proteins found in genbank and six open reading frames with no simi ...200011055941
purification and properties of an enzyme capable of degrading the sheath of sphaerotilus natans.microorganisms which can degrade and grow on the purified sheath of a sheathed bacterium sphaerotilus natans were collected from soil and river water. two bacterial strains were isolated from the soil and designated strains tb and tk. both strains are rod shaped, negatively stained by gram staining, facultatively anaerobic, and formed ellipsoidal endospores. these characteristics suggested that the isolates belong to the genus paenibacillus, according to ash et al. (c. ash, f. g. priest, and m. ...200011055955
molecular signals required for type iii secretion and translocation of the xanthomonas campestris avrbs2 protein to pepper plants.strains of xanthomonas campestris pv. vesicatoria (xcv) carrying avrbs2 are specifically recognized by bs2 pepper plants, resulting in localized cell death and plant resistance. agrobacterium-mediated transient expression of the xcv avrbs2 gene in plant cells results in bs2-dependent cell death, indicating that the avrbs2 protein alone is sufficient for the activation of disease resistance-mediated cell death in planta. we now provide evidence that avrbs2 is secreted from xcv and that secretion ...200011078519
biological role of xanthomonadin pigments in xanthomonas campestris pv. campestris.previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. we used a xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. although xanthomonadin mutant strains were comparable to the parent st ...200011097878
fatty acid competition as a mechanism by which enterobacter cloacae suppresses pythium ultimum sporangium germination and damping-off.interactions between plant-associated microorganisms play important roles in suppressing plant diseases and enhancing plant growth and development. while competition between plant-associated bacteria and plant pathogens has long been thought to be an important means of suppressing plant diseases microbiologically, unequivocal evidence supporting such a mechanism has been lacking. we present evidence here that competition for plant-derived unsaturated long-chain fatty acids between the biological ...200011097912
a functional-phylogenetic classification system for transmembrane solute transporters.a comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the international union of biochemistry and molecular biology. this system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. almost all of the more than 250 identified families of transporters in ...200010839820
roles of dna adenine methylation in regulating bacterial gene expression and virulence. 200111705888
type ii secretion and pathogenesis. 200111349009
isolation and characterization of escherichia coli tolc mutants defective in secreting enzymatically active alpha-hemolysin.this study describes the isolation and characterization of a unique class of tolc mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. treatment of the secreted toxin with guanidine hydrochloride significantly restored ...200111698380
direct process integration of cell disruption and fluidised bed adsorption in the recovery of labile microbial enzymes.the practical feasibility and generic applicability of the direct integration of cell disruption by bead milling with the capture of intracellular products by fluidised bed adsorption has been demonstrated. pilot-scale purification of the enzyme l-asparaginase from unclarified erwinia chrysanthemi disruptates exploiting this novel approach yielded an interim product which rivalled or bettered that produced by the current commercial process employing discrete operations of alkaline lysis, centrif ...200111787801
quorum sensing in vibrio fischeri: analysis of the luxr dna binding region by alanine-scanning mutagenesis.luxr is the transcriptional activator for quorum-sensing control of luminescence in vibrio fischeri. a series of alanine-scanning mutants spanning a predicted helix-turn-helix region in the dna binding domain of luxr was constructed, and the activity of each of the luxr mutant proteins in recombinant escherichia coli was investigated. the region covered by the mutagenesis spanned residues 190 to 224. about half of the alanine-scanning mutants showed activities similar to that of the wild-type lu ...200111114939
relative effects on virulence of mutations in the sap, pel, and hrp loci of erwinia chrysanthemi.we constructed strains of erwinia chrysanthemi ec16 with multiple mutations involving three virulence systems in this bacterium, namely pel (coding for the major pectate lyases pelabce), hrp (hypersensitive response and pathogenicity), and sap (sensitivity to antimicrobial peptides). the relative effects on virulence of those mutations have been analyzed on potato tubers and chicory leaves. in potato tubers, the sap mutation (bt105) had a greater effect in the reduction of the virulence than the ...200111277436
pyruvated galactose and oligosaccharides from erwinia chrysanthemi ech6 extracellular polysaccharide.the acidic extracellular polysaccharide of ech6 was depolymerized by fuming hcl. the pyruvated sugars were isolated and characterized by methods that included a combination of low-pressure gel-filtration and high-ph anion-exchange chromatographies, methylation linkage analyses, mass (gc-ms and maldi-tof ms) and 1h nmr (1d and 2d) spectroscopies. the following pyruvated sugars were obtained: 4,6-o-(1-carboxyethylidene)-d-galp; 4,6-o-(1-carboxyethylidene)- alpha-d-galp-(1-->4)-beta-d-glcap-(1-->3) ...200111284505
expression of bar in the plastid genome confers herbicide resistance.phosphinothricin (ppt) is the active component of a family of environmentally safe, nonselective herbicides. resistance to ppt in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. we report here expression of a bacterial bar gene (b-bar1) in tobacco (nicotiana tabacum cv petit havana) plastids that confers field-level tolerance to liberty, an herbicide containing ppt. we also describe a second bacterial ...200111299340
cloning of two pectate lyase genes from the marine antarctic bacterium pseudoalteromonas haloplanktis strain ant/505 and characterization of the enzymes.a marine antarctic psychrotolerant bacterium (strain ant/505), isolated from sea ice-covered surface water from the southern ocean, showed pectinolytic activity on citrus pectin agar. the sequencing of the 16s rrna of isolate ant/505 indicates a taxonomic affiliation to pseudoalteromonas haloplanktis. the supernatant of this strain showed three different pectinolytic activities after growth on citrus pectin. by activity screening of a genomic dna library of isolate ant/505 in escherichia coli, t ...200111302501
the cela gene, encoding a glycosyl hydrolase family 3 beta-glucosidase in azospirillum irakense, is required for optimal growth on cellobiosides.the cela beta-glucosidase of azospirillum irakense, belonging to glycosyl hydrolase family 3 (ghf3), preferentially hydrolyzes cellobiose and releases glucose units from the c(3), c(4), and c(5) oligosaccharides. the growth of a deltacela mutant on these cellobiosides was affected. in a. irakense, the ghf3 beta-glucosidases appear to be functional alternatives for the ghf1 beta-glucosidases in the assimilation of beta-glucosides by other bacteria.200111319128
the pecm protein of the phytopathogenic bacterium erwinia chrysanthemi, membrane topology and possible involvement in the efflux of the blue pigment indigoidine.the pecs regulatory locus negatively modulates the expression of many virulence genes in erwinia chrysanthemi. this locus consists of two genes, pecs and pecm, divergently transcribed. previous studies have shown that pecs down-regulates the expression of both pecsand pecmgenes and that pecm is required for full pecs activity. computer-aided hydropathy analysis of pecm predicted the presence of between 8 to 10 potential transmembrane segments. we analyzed the membrane topology of pecm using the ...200111321588
osmoregulated periplasmic glucans of erwinia chrysanthemi.we report the initial characterization of the osmoregulated periplasmic glucans (opgs) of erwinia chrysanthemi. opgs are intrinsic components of the bacterial envelope necessary to the pathogenicity of this phytopathogenic enterobacterium (f. page, s. altabe, n. hugouvieux-cotte-pattat, j.-m. lacroix, j. robert-baudouy and j.-p. bohin, j. bacteriol. 183:0000-0000, 2001 [companion in this issue]). opgs were isolated by trichloracetic acid treatment and gel permeation chromatography. the synthesis ...200111325941
osmoregulated periplasmic glucan synthesis is required for erwinia chrysanthemi pathogenicity.erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. osmoregulated periplasmic glucans (opgs) are intrinsic components of the gram-negative bacterial envelope. we cloned the opggh operon of e. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of opgs, by complementation of the homologous locus mdogh of escherichia coli. opgg and opgh show a high level of similarity with mdog and mdoh, respectively, and mutations in ...200111325942
the pdz domain of outc and the n-terminal region of outd determine the secretion specificity of the type ii out pathway of erwinia chrysanthemi.the plant pathogens erwinia chrysanthemi and erwinia carotovora secrete multiple exoproteins by a type ii pathway, the out system. secretion in erwinia is species-specific: exoproteins of one species cannot be secreted by the other. we analysed the role of two components of the out system, the bitopic inner membrane protein outc and the secretin outd, in the specific recognition of secreted proteins. we demonstrated that the pdz domain of outc determines its secretion specificity towards certain ...200111327762
structural basis for the activity and substrate specificity of erwinia chrysanthemi l-asparaginase.bacterial l-asparaginases, enzymes that catalyze the hydrolysis of l-asparagine to aspartic acid, have been used for over 30 years as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. other substrates of asparaginases include l-glutamine, d-asparagine, and succinic acid monoamide. in this report, we present high-resolution crystal structures of the complexes of erwinia chrysanthemi l-asparaginase (era) with the products of such reactions that also can serve as substr ...200111341830
combined, functional genomic-biochemical approach to intermediary metabolism: interaction of acivicin, a glutamine amidotransferase inhibitor, with escherichia coli k-12.acivicin, a modified amino acid natural product, is a glutamine analog. thus, it might interfere with metabolism by hindering glutamine transport, formation, or usage in processes such as transamidation and translation. this molecule prevented the growth of escherichia coli in minimal medium unless the medium was supplemented with a purine or histidine, suggesting that the hishf enzyme, a glutamine amidotransferase, was the target of acivicin action. this enzyme, purified from e. coli, was inhib ...200111344143
biochemical and x-ray crystallographic studies on shikimate kinase: the important structural role of the p-loop lysine.shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (nmp) kinase family, which includes adenylate kinase. in this paper we have explored the roles of residues in the p-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. in common with many members of the p-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of th ...200111369852
beta-elimination of glucosyluronic residues during methylation of an acidic polysaccharide from erwinia chrysanthemi cu 643.the erwinia chrysanthemi cu643 eps has a linear hexasaccharide repeating unit in which a 4-linked uronic acid residue is present. the eps was methylated by either the naoh-me2so-mei or li-dimsyl procedure. maldi-tof ms analysis of the methylated products indicates that the beta-eliminative degradation occurs during the methylation, as characterized by serial fragments of the hexasaccharide repeating units. the degradation was clearly defined from the methylation of a glucosyluronic-containing py ...200111376611
essential role of superoxide dismutase on the pathogenicity of erwinia chrysanthemi strain 3937.the soda gene from erwinia chrysanthemi strain 3937 was cloned by functional complementation of an escherichia coli soda sodb mutant and sequenced. we identified a 639-bp open reading frame, which encodes a protein that is 85% identical to the e. coli manganese-containing superoxide dismutase mnsod. promoter elements of this gene were identified by transcriptional mapping experiments. we constructed an e. chrysanthemi deltasoda mutant by reverse genetics. the deltasoda mutation resulted in the a ...200111386371
control of exut activity for galacturonate transport by the negative regulator exur in erwinia chrysanthemi ec16.the negative regulatory protein exur in erwinia chrysanthemi regulates expression of the galacturonate uptake (exut) and utilization (uxaa, uxab, uxac) genes. we cloned and determined the nucleotide sequence of the exur gene from e. chrysanthemi ec16. analysis of the deduced amino acid sequence indicates that this protein possesses a helix-turn-helix motif and belongs to the gntr family of transcriptional repressors. northern blot analysis and studies with transcriptional fusions of exut in wild ...200111386378
role of the nucleoid-associated protein h-ns in the synthesis of virulence factors in the phytopathogenic bacterium erwinia chrysanthemi.the ability of the enterobacterium erwinia chrysanthemi to induce pathogenesis in plant tissue is strongly related to the massive production of plant-cell-wall-degrading enzymes (pectinases, cellulases, and proteases). additional factors, including flagellar proteins and exopolysaccharides (eps), also are required for the efficient colonization of plants. production of these virulence factors, particularly pectate lyases, the main virulence determinant, is tightly regulated by environmental cond ...200111194867
mexr repressor of the mexab-oprm multidrug efflux operon of pseudomonas aeruginosa: identification of mexr binding sites in the mexa-mexr intergenic region.the mexr repressor of the mexab-oprm multidrug efflux operon of pseudomonas aeruginosa was purified as a c-terminal histidine-tagged protein by metal chelate affinity chromatography. the purified protein was shown to bind ca. 200 bp upstream of mexa, at two sites, each of which contains a repeat of the nucleotide sequence gttga in inverse orientation. dna sequence analysis identified mexa and mexr promoters within the mexr binding regions, consistent with the previously observed negative regulat ...200111208776
effects exerted by transcriptional regulator pcau from acinetobacter sp. strain adp1.protocatechuate degradation is accomplished in a multistep inducible catabolic pathway in acinetobacter sp. strain adp1. the induction is brought about by the transcriptional regulator pcau in concert with the inducer protocatechuate. pcau, a member of the new iclr family of transcriptional regulators, was shown to play a role in the activation of transcription at the promoter for the structural pca genes, leaving open the participation of additional activators. in this work we show that there i ...200111208784
exchange of xcp (gsp) secretion machineries between pseudomonas aeruginosa and pseudomonas alcaligenes: species specificity unrelated to substrate recognition.pseudomonas aeruginosa and pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type ii or general secretory pathway, which requires at least 12 xcp gene products (xcpa and xcpp to -z). despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. based on results obtained with erwinia, it has been proposed that the xcpp and/or xcpq homologs determine this secretion specificity (m. linderberg, g. ...200111208795
effect of sequences of the active-site dipeptides of dsba and dsbc on in vivo folding of multidisulfide proteins in escherichia coli.we have examined the role of the active-site cxxc central dipeptides of dsba and dsbc in disulfide bond formation and isomerization in the escherichia coli periplasm. dsba active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsba promoter by integration of the respective alleles into the bacterial chromosome. the dsba alleles gave significant differences in the yield of active murine urokinase, a prote ...200111208797
molecular characterization of global regulatory rna species that control pathogenicity factors in erwinia amylovora and erwinia herbicola pv. gypsophilae.rsmb(ecc) specifies a nontranslatable rna regulator that controls exoprotein production and pathogenicity in soft rot-causing erwinia carotovora subsp. carotovora. this effect of rsmb(ecc) rna is mediated mostly by neutralizing the function of rsma(ecc), an rna-binding protein of e. carotovora subsp. carotovora, which acts as a global negative regulator. to determine the occurrence of functional homologs of rsmb(ecc) in non-soft-rot-causing erwinia species, we cloned the rsmb genes of e. amylovo ...200111222584
functional characterization of a novel xylanase from a corn strain of erwinia chrysanthemi.a beta-1,4-xylan hydrolase (xylanase a) produced by erwinia chrysanthemi d1 isolated from corn was analyzed with respect to its secondary structure and enzymatic function. the ph and temperature optima for the enzyme were found to be ph 6.0 and 35 degrees c, with a secondary structure under those conditions that consists of approximately 10 to 15% alpha-helices. the enzyme was still active at temperatures higher than 40 degrees c and at phs of up to 9.0. the loss of enzymatic activity at tempera ...200111222610
mobilization function of the pbhr1 plasmid, a derivative of the broad-host-range plasmid pbbr1.the pbhr1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pbbr1, which was isolated from the gram-negative bacterium bordetella bronchiseptica (r. antoine and c. locht, mol. microbiol. 6:1785-1799, 1992). plasmid pbbr1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. we show that the mob protein specifically recognizes a 52-bp sequence ...200111222611
phase-variable expression of an operon encoding extracellular alkaline protease, a serine protease homolog, and lipase in pseudomonas brassicacearum.the rhizobacterium pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity. the operon encoding these enzymes, a serine protease homolog, and a type i secretion machinery was characterized. transcriptional lacz gene fusions revealed that the expression of the operon is under the control of phase variation.200111222613
structures of two highly homologous bacterial l-asparaginases: a case of enantiomorphic space groups.quasi-enantiomorphic crystals of the y25f mutant of escherichia coli l-asparaginase and of the native erwinia chrysanthemi l-asparaginase were obtained in the hexagonal space groups p6(5)22 and p6(1)22, respectively. the structures of these highly homologous enzymes were solved by molecular replacement and were refined with data extending to 2.2-2.5 a. these structures were compared with each other, as well as with other l-asparaginase structures previously observed with different crystal packin ...200111223513
effect of protracted high-dose l-asparaginase given as a second exposure in a berlin-frankfurt-münster-based treatment: results of the randomized 9102 intermediate-risk childhood acute lymphoblastic leukemia study--a report from the associazione italiana ematologia oncologia pediatrica.to assess in a randomized study the therapeutic effect of the addition of high-dose l-asparaginase (hd asp) in the context of a berlin-frankfurt-münster (bfm)-based chemotherapy regimen for intermediate risk (ir) childhood acute lymphoblastic leukemia (all).200111230471
soxr-dependent response to oxidative stress and virulence of erwinia chrysanthemi: the key role of sufc, an orphan abc atpase.erwinia chrysanthemi causes soft-rot disease in a great variety of plants. in addition to the depolymerizing activity of plant cell wall-degrading enzymes, iron acquisition and resistance to oxidative stress contribute greatly to the virulence of this pathogen. here, we studied the pin10 locus originally thought to encode new virulence factors. the sequence analysis revealed six open reading frames that were homologous to the escherichia coli sufa, sufb, sufc, sufd, sufs and sufe genes. sequence ...200111251816
type ii protein secretion is a subset of the pild-dependent processes that facilitate intracellular infection by legionella pneumophila.previously, we had demonstrated that a legionella pneumophila prepilin peptidase (pild) mutant does not produce type iv pili and shows reduced secretion of enzymatic activities. moreover, it displays a distinct colony morphology and a dramatic reduction in intracellular growth within amoebae and macrophages, two phenotypes that are not exhibited by a pilin (pile(l)) mutant. to determine whether these pild-dependent defects were linked to type ii secretion, we have constructed two new mutants of ...200111254562
identification of the exported proteins of the oral opportunistic pathogen actinobacillus actinomycetemcomitans by using alkaline phosphatase fusions.a phoa fusion library of actinobacillus actinomycetemcomitans genomic dna has been screened to identify genes encoding exported and secreted proteins. a total of 8,000 colonies were screened, and 80 positive colonies were detected. from these, 48 genes were identified with (i) more than half having homology to known or hypothetical haemophilus influenzae genes, (ii) 14 having no ascribed function, and (iii) 4 having very limited or no homology to known genes. the proteins encoded by these genes ...200111254647
pectate lyase a, an enzymatic subunit of the clostridium cellulovorans cellulosome.clostridium cellulovorans uses not only cellulose but also xylan, mannan, pectin, and several other carbon sources for its growth and produces an extracellular multienzyme complex called the cellulosome, which is involved in plant cell wall degradation. here we report a gene for a cellulosomal subunit, pectate lyase a (pela), lying downstream of the engy gene, which codes for cellulosomal enzyme engy. pela is composed of an orf of 2,742 bp and encodes a protein of 914 aa with a molecular weight ...200111259664
an inner membrane platform in the type ii secretion machinery of gram-negative bacteria.the type ii secretion machinery allows most gram-negative bacteria to deliver virulence factors into their surroundings. we report that in erwinia chrysanthemi, gspe (the putative ntpase), gspf, gspl and gspm constitute a complex in the inner membrane that is presumably used as a platform for assembling other parts of the secretion machinery. the gspe-gspf-gspl-gspm complex was demonstrated by two methods: (i) co-immunoprecipitation of gspe-gspf-gspl with antibodies raised against either gspe or ...200111266368
characterization of vibrio cholerae o1 el tor galu and gale mutants: influence on lipopolysaccharide structure, colonization, and biofilm formation.recently we described the isolation of spontaneous bacteriophage k139-resistant vibrio cholerae o1 el tor mutants. in this study, we identified phage-resistant isolates with intact o antigen but altered core oligosaccharide which were also affected in galactose catabolism; this strains have mutations in the galu gene. we inactivated another gal gene, gale, and the mutant was also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal lipopolysaccharid ...200111119535
gene integration and expression and extracellular secretion of erwinia chrysanthemi endoglucanase cely (cely) and celz (celz) in ethanologenic klebsiella oxytoca p2.the development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. one promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. by starting with an ethanologenic derivative (strain p2) of klebsiella oxytoca m5a1 with the native ability to metabolize cellobiose, the need for supplemental beta- ...200111133422
cloning and characterization of a periplasmic nuclease of vibrio vulnificus and its role in preventing uptake of foreign dna.we have cloned a nuclease gene, vvn, from vibrio vulnificus, an estuarine bacterium that causes wound infections and septicemia in humans and eels. the gene contained a 696-bp open reading frame encoding 232 amino acids (aa), including a signal sequence of 18 aa. the deduced amino acid sequence of the mature nuclease predicted a molecular mass of 25 kda, which was confirmed by vital stain, and a pi of 8.6. vvn was produced in the periplasm of either v. vulnificus or recombinant escherichia coli ...200111133431
direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia.enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. we have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cu ...200111133432
pantoea agglomerans strain eh318 produces two antibiotics that inhibit erwinia amylovora in vitro.pantoea agglomerans (synonym: erwinia herbicola) strain eh318 produces through antibiosis a complex zone of inhibited growth in an overlay seeded with erwinia amylovora, the causal agent of fire blight. this zone is caused by two antibiotics, named pantocin a and b. using a genomic library of eh318, two cosmids, pcpp702 and pcpp704, were identified that conferred on escherichia coli the ability to inhibit growth of e. amylovora. the two cosmids conferred different antibiotic activities on e. col ...200111133457
involvement of the xpsn protein in formation of the xpsl-xpsm complex in xanthomonas campestris pv. campestris type ii secretion apparatus.the xps gene cluster is required for the second step of type ii protein secretion in xanthomonas campestris pv. campestris. deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. by analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the xpsl and the xpsm proteins. the xpsl protein was undetectable in total lysate prepared from the xpsm mutant strain, and vice versa. introducti ...200111133946
ihfa gene of the bacterium myxococcus xanthus and its role in activation of carotenoid genes by blue light.myxococcus xanthus responds to blue light by producing carotenoids. several regulatory genes are known that participate in the light action mechanism, which leads to the transcriptional activation of the carotenoid genes. we had already reported the isolation of a carotenoid-less, tn5-induced strain (mr508), whose mutant site was unlinked to the indicated regulatory genes. here, we show that omegamr508::tn5 affects all known light-inducible promoters in different ways. it blocks the activation o ...200111133949
dsba and dsbc affect extracellular enzyme formation in pseudomonas aeruginosa.dsba and dsbc proteins involved in the periplasmic formation of disulfide bonds in pseudomonas aeruginosa were identified and shown to play an important role for the formation of extracellular enzymes. mutants deficient in either dsba or dsbc or both genes were constructed, and extracellular elastase, alkaline phosphatase, and lipase activities were determined. the dsba mutant no longer produced these enzymes, whereas the lipase activity was doubled in the dsbc mutant. also, extracellar lipase p ...200111133952
disulfide bond in pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase.pseudomonas aeruginosa secretes a 29-kda lipase which is dependent for folding on the presence of the lipase-specific foldase lif. the lipase contains two cysteine residues which form an intramolecular disulfide bond. variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. wild-type and variant lipase genes fused to the ...200111133953
hrpz(psph) from the plant pathogen pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro.the hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. some hrp gene products constitute elements of the type iii secretion system, by which effector proteins are exported and delivered into plant cells. here, we show that the hrpz gene product from the bean halo-blight pathogen, pseudomonas syringae pv. phaseolicola (hrpz(psph)), is secreted in an hrp-dependent manner in p. syringae pv. ...200111134504
hrpz(psph) from the plant pathogen pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro.the hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. some hrp gene products constitute elements of the type iii secretion system, by which effector proteins are exported and delivered into plant cells. here, we show that the hrpz gene product from the bean halo-blight pathogen, pseudomonas syringae pv. phaseolicola (hrpz(psph)), is secreted in an hrp-dependent manner in p. syringae pv. ...200111134504
analysis of the type iv fimbrial-subunit gene fima of xanthomonas hyacinthi: application in pcr-mediated detection of yellow disease in hyacinths.a sensitive and specific detection method was developed for xanthomonas hyacinthi; this method was based on amplification of a subsequence of the type iv fimbrial-subunit gene fima from strain s148. the fima gene was amplified by pcr with degenerate dna primers designed by using the n-terminal and c-terminal amino acid sequences of trypsin fragments of fima. the nucleotide sequence of fima was determined and compared with the nucleotide sequences coding for the fimbrial subunits in other type iv ...200111157222
molecular characterization of the iron-hydroxamate uptake system in staphylococcus aureus.to investigate iron uptake, a chromosomal locus containing three consecutive open reading frames, designated fhuc, fhub, and fhud, was identified in staphylococcus aureus. whereas the fhuc gene encodes an atp-binding protein, fhub and fhud code for ferrichrome permeases and thus resemble an atp-binding cassette transporter. a fhub knockout mutant showed impaired uptake of iron bound to the siderophores but not of ferric chloride, suggesting that this operon is specific for siderophore-mediated i ...200111157278
disulfide bond formation in secreton component pulk provides a possible explanation for the role of dsba in pullulanase secretion.when expressed in escherichia coli, the 15 klebsiella oxytoca pul genes that encode the so-called pul secreton or type ii secretion machinery promote pullulanase secretion and the assembly of one of the secreton components, pulg, into pili. besides these pul genes, efficient pullulanase secretion also requires the host dsba gene, encoding a periplasmic disulfide oxidoreductase, independently of disulfide bond formation in pullulanase itself. two secreton components, the secretin pilot protein pu ...200111157944
subset of hybrid eukaryotic proteins is exported by the type i secretion system of erwinia chrysanthemi.erwinia chrysanthemi exports degradative enzymes by using a type i protein secretion system. the proteases secreted by this system lack an n-terminal signal peptide but contain a c-terminal secretion signal. to explore the substrate specificity of this system, we have expressed the e. chrysanthemi transporter system (prtdef genes) in escherichia coli and tested the ability of this abc transporter to export hybrid proteins carrying c-terminal fragments of e. chrysanthemi protease b. the c terminu ...200111157948
cloning, sequences, and characterization of two chitinase genes from the antarctic arthrobacter sp. strain tad20: isolation and partial characterization of the enzymes.arthrobacter sp. strain tad20, a chitinolytic gram-positive organism, was isolated from the sea bottom along the antarctic ice shell. arthrobacter sp. strain tad20 secretes two major chitinases, chia and chib (archia and archib), in response to chitin induction. a single chromosomal dna fragment containing the genes coding for both chitinases was cloned in escherichia coli. dna sequencing analysis of this fragment revealed two contiguous open reading frames coding for the precursors of archia (8 ...200111160110
three-dimensional structure of erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site.most structures of neutral lipases and esterases have been found to adopt the common alpha/beta hydrolase fold and contain a catalytic ser-his-asp triad. some variation occurs in both the overall protein fold and in the location of the catalytic triad, and in some enzymes the role of the aspartate residue is replaced by a main-chain carbonyl oxygen atom. here, we report the crystal structure of pectin methylesterase that has neither the common alpha/beta hydrolase fold nor the common catalytic t ...200111162105
mxim and mxij, base elements of the mxi-spa type iii secretion system of shigella, interact with and stabilize the mxid secretin in the cell envelope.the type iii secretion pathway is broadly distributed across many parasitic bacterial genera and serves as a mechanism for delivering effector proteins to eukaryotic cell surface and cytosolic targets. while the effectors, as well as the host responses elicited, differ among type iii systems, they all utilize a conserved set of 9 to 11 proteins that together form a bacterial envelope-associated secretory organelle or needle complex. the general structure of the needle complex consists of a trans ...200111717255
characterization of pseudomonas aeruginosa chitinase, a gradually secreted protein.the gram-negative bacterium pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type i, ii, and iii secretion systems. in this study, a gene, chic, coding for an extracellular chitinolytic enzyme, was identified. the chic gene encodes a polypeptide of 483 amino acid residues, without a typical n-terminal signal sequence. nevertheless, an n-terminal segment of 11 residues was found to be cleaved off in the secreted protein. the protein shows sequence similarit ...200111717261
complex regulatory network controls initial adhesion and biofilm formation in escherichia coli via regulation of the csgd gene.the escherichia coli ompr/envz two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation. up mutations in the ompr gene, such as the ompr234 mutation, stimulate laboratory strains of e. coli to grow as a biofilm community rather than in a planktonic state. in this report, we show that the ompr234 protein promotes biofilm formation by binding the csgd promoter region and stimulating its transcription. the csgd gene encodes the transcription regulator ...200111717281
crystallization and preliminary x-ray diffraction analysis of shikimate kinase from mycobacterium tuberculosis in complex with mgadp.shikimate kinase (sk) from mycobacterium tuberculosis (mt) was overexpressed in escherichia coli, purified and cocrystallized with mgadp in hanging drops using the vapor-diffusion procedure with peg 4000 and 2-propanol as precipitants at ph 7.5. the crystal of mtsk-mgadp, which diffracted to 2.2 a resolution, belonged to space group p3(2)21 or p3(1)21, with unit-cell parameters a = b = 64.01, c = 92.41 a. there was one mtsk molecule in the asymmetric unit. molecular-replacement trials with the c ...200111717501
the conserved methionine residue of the metzincins: a site-directed mutagenesis study.the metalloprotease clan of the metzincins derive their name from the presence of a conserved methionine residue that is located on the c-terminal side of the zinc-binding consensus sequence hexxhxxgxxh. this methionine residue is located in a rather divergent part of the primary sequence but is structurally very well conserved. it is located under the pyramidal base of the three histidine residues that coordinate the catalytic zinc ion and is not involved in any direct contact with the metal no ...200111718552
protease c of erwinia chrysanthemi: the crystal structure and role of amino acids y228 and e189.prtc, a metallo-protease secreted by erwinia chrysanthemi, is a member of the serralysin family and hence belongs to the metzincin superfamily. while the crystal structures of representatives of all metzincin subfamilies have been elucidated in the past, there is still some controversy about the reaction mechanism and the role of certain characteristic amino acids in the active centre. in this study, we probed the role of tyr228 and glu189 by site-directed mutagenesis and x-ray crystallography. ...200111718553
structure of human dipeptidyl peptidase i (cathepsin c): exclusion domain added to an endopeptidase framework creates the machine for activation of granular serine proteases.dipeptidyl peptidase i (dppi) or cathepsin c is the physiological activator of groups of serine proteases from immune and inflammatory cells vital for defense of an organism. the structure presented shows how an additional domain transforms the framework of a papain-like endopeptidase into a robust oligomeric protease-processing enzyme. the tetrahedral arrangement of the active sites exposed to solvent allows approach of proteins in their native state; the massive body of the exclusion domain fa ...200111726493
molecular cloning and sequencing of the gene encoding an exopolygalacturonase of a bacillus isolate and properties of its recombinant enzyme.an exopolygalacturonase (exo-pgase; ec 3.2.1.82) was found in the culture broth of a bacillus isolate. the gene encoding the exo-pgase, pehk, was cloned by polymerase chain reaction using mixed primers designed from n-terminal and internal amino acid (aa) sequences of the enzyme (pehk). the determined nucleotide (nt) sequence of pehk revealed a 2940 bp open reading frame (980 aa) that encoded a putative signal sequence (27 aa) and a mature protein (953 aa; 103810 da). the recombinant enzyme was ...200111750764
betawrap: successful prediction of parallel beta -helices from primary sequence reveals an association with many microbial pathogens.the amino acid sequence rules that specify beta-sheet structure in proteins remain obscure. a subclass of beta-sheet proteins, parallel beta-helices, represent a processive folding of the chain into an elongated topologically simpler fold than globular beta-sheets. in this paper, we present a computational approach that predicts the right-handed parallel beta-helix supersecondary structural motif in primary amino acid sequences by using beta-strand interactions learned from non-beta-helix struct ...200111752429
do bacterial l-asparaginases utilize a catalytic triad thr-tyr-glu?the structures of erwinia chrysanthemi l-asparaginase (era) complexed with the l- and d-stereoisomers of the suicide inhibitor, 6-diazo-5-oxy-norleucine, have been solved using x-ray crystallography and refined with data extending to 1.7 a. the distances between the calpha atoms of the inhibitor molecules and the hydroxyl oxygen atoms of thr-15 and tyr-29 (1.20 and 1.60 a, respectively) clearly indicate the presence of covalent bonds between these moieties, confirming the nucleophilic role of th ...200111755201
analysis of the pilq secretin from neisseria meningitidis by transmission electron microscopy reveals a dodecameric quaternary structure.pilq is a member of the secretin family of outer membrane proteins and is specifically involved in secretion of type iv pili in neisseria meningitidis, neisseria gonorrhoeae, and pseudomonas aeruginosa. the quaternary structure of pilq from n. meningitidis was analyzed by transmission electron microscopy by using a negative stain. single particle averaging was carried out with a total data set of 650 individual particles, which produced a projection map generated from 296 particles at an estimat ...200111395444
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