Microorganisms

J Bacteriol, 2003 Jul, 185(14), 4172 - 85
Mycobacterium tuberculosis chaperonin 10 heptamers self-associate through their biologically active loops; Roberts MM et al.; The crystal structure of Mycobacterium tuberculosis chaperonin 10 (cpn10(Mt)) has been determined to a resolution of 2.8 A . Two dome-shaped cpn10(Mt) heptamers complex through loops at their bases to form a tetradecamer with 72 symmetry and a spherical cage-like structure . The hollow interior enclosed by the tetradecamer is lined with hydrophilic residues and has dimensions of 30 A perpendicular to and 60 A along the sevenfold axis . Tetradecameric cpn10(Mt) has also been observed in solution by dynamic light scattering . Through its base loop sequence cpn10(Mt) is known to be the agent in the bacterium responsible for bone resorption and for the contribution towards its strong T-cell immunogenicity . Superimposition of the cpn10(Mt) sequences 26 to 32 and 66 to 72 and E . coli GroES 25 to 31 associated with bone resorption activity shows them to have similar conformations and structural features, suggesting that there may be a common receptor for the bone resorption sequences . The base loops of cpn10s in general also attach to the corresponding chaperonin 60 (cpn60) to enclose unfolded protein and to facilitate its correct folding in vivo . Electron density corresponding to a partially disordered protein subunit appears encapsulated within the interior dome cavity of each heptamer . This suggests that the binding of substrates to cpn10 is possible in the absence of cpn60.

Res Microbiol, 2003 Jun, 154(5), 353 - 9
Characterization and identification of an iron-oxidizing, Leptospirillum-like bacterium, present in the high sulfate leaching solution of a commercial bioleaching plant; Romero J et al.; Most copper bioleaching plants operate with a high concentration of sulfate salts, caused by the continuous addition of sulfuric acid and the recycling of the leaching solution . Since the bacteria involved in bioleaching have been generally isolated at low sulfate concentrations, the bacterial population present in the high-sulfate (150 gl(-1)) leaching solution, employed in a copper production plant, was investigated . The iron-oxidizing bacteria able to grow in the leaching solution were enriched by several batch cultivations and, after serial dilution, an abundant bacterial strain was isolated . This strain, called LA, exhibited a relatively constant rate of iron-oxidation in media containing sulfate ions at concentrations ranging from 10 to 150 gl(-1) . Culture collection strains of Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans showed limited abilities to grow at sulfate ion concentrations higher than 70 gl(-1) . In spite of its tolerance to high sulfate concentrations, strain LA was as sensitive to NaCl as A . ferrooxidans . Comparative sequence analysis of the 16S rRNA gene of strain LA indicated that it is phylogenetically related to strains described as Leptospirillum ferrooxidans . Bacterial community DNA restriction patterns of 16S rRNA genes suggested that strain LA was a minor component of the bacterial population present in leaching solution, but is abundant in ore leached with this solution.

J Struct Funct Genomics, 2000, 1(1), 15 - 25
NMR structure determination and structure-based functional characterization of conserved hypothetical protein MTH1175 from Methanobacterium thermoautotrophicum; Cort JR et al.; The solution structure of MTH1175, a 124-residue protein from the archaeon Methanobacterium thermoautotrophicum has been determined by NMR spectroscopy . MTH1175 is part of a family of conserved hypothetical proteins (COG1433) with unknown functions which contains multiple paralogs from all complete archaeal genomes and the archaeal gene-rich bacterium Thermotoga maritima . Sequence similarity indicates this protein family may be related to the nitrogen fixation proteins NifB and NifX . MTH1175 adopts an alpha/beta topology with a single mixed beta-sheet, and contains two flexible loops and an unstructured C-terminal tail . The fold resembles that of Ribonuclease H and similar proteins, but differs from these in several respects, and is not likely to have a nuclease activity.

Biosci Biotechnol Biochem, 2003 May, 67(5), 1172 - 6
Escherichia coli tRNAs are resistant to the hyperprocessing reaction of homologous E . coli ribonuclease P ribozyme; Tanaka T et al.; Bacterial ribonuclease P RNA ribozyme can do the hyperprocessing reaction, the internal cleavage reaction of some floppy eukaryotic tRNAs . The hyperprocessing reaction can be used as a detection tool to examine the stability of the cloverleaf shape of tRNA . Until now, the hyperprocessing reaction has been observed in the heterologous combination of eukaryotic tRNAs and bacterial RNase P enzymes . In this paper, we examined the hyperprocessing reaction of Escherichia coli tRNAs by homologous E . coli RNase P, to find that these homologous tRNAs were resistant to the toxic hyperprocessing reaction . Our results display the evidence for molecular co-evolution between homologous tRNAs and RNase P in the bacterium E . coli.

Acta Crystallogr D Biol Crystallogr, 2003 Jul, 59(Pt 7), 1280 - 2 Epub 2003 Jun 27.
Purification, crystallization and preliminary X-ray analysis of an unusual thioredoxin from the gastric pathogen Helicobacter pylori; Filson H et al.; Thioredoxin-2 (HP1458) from Helicobacter pylori is a member of the thioredoxin family, but possesses the unusual active-site motif CPDC (compared with CGPC in other thioredoxins) . H . pylori is deficient in the glutaredoxin system, making the thioredoxin system the sole reduction system in the bacterium and critical for its ability to survive oxidative stress . The recombinant protein has been overexpressed, purified and crystallized . This is the first reported crystallization of a thioredoxin possessing this unusual active site . Single crystals have been obtained using the sitting-drop technique . Crystals diffract to 2.4 A resolution and belong to space group P4(1), with unit-cell parameters a = b = 40.21, c = 64.65 A . Molecular replacement using AMoRe proved unsuccessful; however, implementation of the program BEAST gave successful molecular-replacement solutions.

Genes Dev, 2003 Jul 15, 17(14), 1727 - 40 Epub 2003 Jun 27.
The chaplins: a family of hydrophobic cell-surface proteins involved in aerial mycelium formation in Streptomyces coelicolor; Elliot MA et al.; The filamentous bacterium Streptomyces coelicolor differentiates by forming specialized, spore-bearing aerial hyphae that grow into the air . Using microarrays, we identified genes that are down-regulated in a mutant unable to erect aerial hyphae . Through this route, we identified a previously unknown layer of aerial mycelium surface proteins (the "chaplins") . The chaplins share a hydrophobic domain of approximately 40 residues (the "chaplin domain"), and all have a secretion signal . The five short chaplins (ChpD,E,F,G,H) have one chaplin domain, whereas the three long chaplins (ChpA,B,C) have two chaplin domains and a C-terminal "sorting signal" that targets them for covalent attachment to the cell wall by sortase enzyme . Expression of the two chaplin genes examined (chpE, chpH) depended on aerial hyphae formation but not sporulation, and egfp fusions showed their expression localized to aerial structures . Mass spectrometry of cell wall extracts confirmed that the short chaplins localized to the cell surface . Deletion of chaplin genes caused severe delays in aerial hyphae formation, a phenotype rescued by exogenous application of chaplin proteins . These observations implicate the chaplins in aerial mycelium formation, and suggest that coating of the envelope by the chaplins is required for aerial hyphae to grow out of the aqueous environment of the substrate mycelium into the air.

Comp Biochem Physiol B Biochem Mol Biol, 2003 Jul, 135(3), 511 - 9
Lysozyme of the beet armyworm, Spodoptera exigua: activity induction and cDNA structure; Bae S et al.; Lysozyme of the beet armyworm, Spodoptera exigua, was characterized in its up-regulation pattern, and its cDNA was cloned by RT-PCR using degenerate primers designed from some conserved amino acid regions shared with related lepidopteran species . Lysozyme activity of the non-immunized S . exigua had developmental variation, with the highest level in the fifth instar larvae . The basal level of the lysozyme activity was significantly enhanced by the injection of laminarin or lipopolysaccharide (LPS) . Among different LPSs tested, the extract from an entomopathogenic bacterium, Xenorhabdus nematophilus, proved to be the most potent . Fat body was the major tissue to express the lysozyme in S . exigua . Even though there was a significantly elevated level of lysozyme in the hemolymph at 12 h after laminarin injection, the transcript ( approximately 1.1 kbp) was found in the fat body as early as 6 h after injection . The cDNA of the lysozyme was cloned as 602 bp with a deduced 141-amino-acid residue open reading frame containing two introns . Except for a signal peptide with 20 amino acid residues, the estimated molecular weight and isoelectric point of the lysozyme was 14313.83 Da and 8.59, respectively . Only a single copy gene of the lysozyme was found in S . exigua genome from Southern analysis . The amino acid sequence of S . exigua lysozyme showed higher similarity (88.7%) with noctuid species compared to other lepidopteran species.

Am J Kidney Dis, 2003 Jul, 42(1), 1 - 11
The association of nephrolithiasis with cystic fibrosis; Gibney EM et al.; BACKGROUND: There is a growing body of evidence regarding the association between cystic fibrosis (CF) and nephrolithiasis and the role that Oxalobacter formigenes may have in that association . METHODS: We performed a MEDLINE search of "cystic fibrosis and nephrolithiasis" and "Oxalobacter formigenes." Epidemiological and experimental evidence and possible mechanisms explaining the association were critically reviewed . RESULTS: Of patients with CF, 3.0% to 6.3% are affected with nephrolithiasis, a percentage greater than that of age-matched controls without CF, in whom the rate is 1% to 2% . Studies have suggested possible mechanisms for the association, including hyperuricosuria, hyperoxaluria, primary defects in calcium handling caused by mutation of the CF transmembrane regulator (CFTR), hypocitraturia, and lack of colonization with O formigenes, an enteric oxalate-degrading bacterium . The absence of colonization could be related to frequent courses of antibiotics . CONCLUSION: Although the incidence of stones in patients with CF may be increased compared with controls without CF, many possible mechanisms are implicated . The relative contributions of these mechanisms remain uncertain . Future directions may include specific identification of lithogenic risks and therapy aimed at stone prevention in this population.

J Nat Prod, 2003 Jun, 66(6), 883 - 4
A new cyclic peptide from a marine-derived bacterium of the genus Nocardiopsis; Shin J et al.; A new cyclic tetrapeptide (1) was isolated from the culture broth of an actinomycete of the genus Nocardiopsis collected from the Pacific deep-sea sediment . The structure of this compound was determined to be cyclo-(L-isoleucyl-L-prolyl-L-leucyl-L-prolyl) on the basis of combined chemical and spectral methods.

Trends Microbiol, 2003 Jun, 11(6), 239 - 42
Taxing questions in development; Armitage JP; Bacteria use taxis-controlled movement to reach their optimum environment . Chemotaxis is probably the best understood behavioural system in biology, biasing the normal random movement of bacteria using a phospho-relay pathway from receptors to the motility organelles . The pathways are typified by signal recognition and receptor adaptation, enabling bacteria to sense and respond to changing environments . Models have been derived from the single chemosensory pathway of Escherichia coli but the sequencing of an increasing number of bacterial genomes is revealing genes that apparently encode multiple chemosensory pathways . Recently, data have accumulated suggesting that some of these pathways might not control motility, although the mechanisms by which this might happen remain unclear . Information from the soil bacterium Myxococcus xanthus could lead the way to an understanding of such mechanisms.

Med Vet Entomol, 2003 Jun, 17(2), 232 - 4
The poultry red mite, Dermanyssus gallinae, a potential vector of Erysipelothrix rhusiopathiae causing erysipelas in hens; Chirico J et al.; Erysipelas is a bacterial disease caused by Erysipelothrix rhusiopathiae, which may infect swine as well as several other species of mammals and birds, including domestic fowl . In poultry, erysipelas may cause sudden high mortality due to septicemia . This communication describes the first isolation of E . rhusiopathiae from the haematophagous poultry red mite, Dermanyssus gallinae DeGeer (Acari: Dermanyssidae), that was collected on three farms where hen erysipelas was diagnosed . The bacteria were isolated from the integument as well as from the interior of the mites . Serotypes 1a and 1b of E . rhusiopathiae found in the mites corresponded with those isolated from the diseased birds . These findings imply that D . gallinae is a potential vector of E . rhusiopathiae . The current lack of effective measures to control D . gallinae causes recurring mite problems in poultry facilities once afflicted by this parasite . Consequently, mites containing E . rhusiopathiae may act as reservoir hosts of this bacterium, allowing it to persist in the poultry house between flock cycles as a source of infection for the replacement pullets . The zoonotic potentials of both E . rhusiopathiae and D . gallinae should also be considered.

Infect Immun, 2003 Jul, 71(7), 4067 - 78
Soluble extracts from Helicobacter pylori induce dome formation in polarized intestinal epithelial monolayers in a laminin-dependent manner; Terres AM et al.; Helicobacter pylori colonizes the stomach at the interface between the mucus layer and the apical pole of gastric epithelial cells . A number of secreted and shed products from the bacteria, such as proteins and lipopolysaccharide, are likely to have a role in the pathogenesis at the epithelial level . To determine the physiological response of transporting polarized epithelia to released soluble factors from the bacterium, we used the T84 cell line . Monolayers of T84 cells were exposed to soluble extracts from H . pylori . The extracts induced rapid "dome" formation as well as an immediate decrease in transepithelial electrical resistance . Domes are fluid-filled blister-like structures unique to polarized epithelia . Their formation has been linked to sodium-transporting events as well as to diminished adherence of the cells to the substrate . H . pylori-induced dome formation in T84 monolayers was exacerbated by amiloride and inhibited by ouabain . Furthermore, it was associated with changes in the expression of the laminin binding alpha 6 beta 4 integrin and the 67-kDa laminin receptor . Domes formed primarily on laminin-coated filters, rather than on fibronectin or collagen matrices, and their formation was inhibited by preincubating the bacterial extract with soluble laminin . This effect was specific to H . pylori and independent of the urease, vacA, cagA, and Lewis phenotype of the strains . These data indicate that released elements from H . pylori can alter the physiological balance and integrity of the epithelium in the absence of an underlying immune response.

Infect Immun, 2003 Jul, 71(7), 4018 - 25
Major surface protein 2 of Anaplasma phagocytophilum facilitates adherence to granulocytes; Park J et al.; Anaplasma phagocytophilum is an obligate intracellular bacterium that infects myeloid cells in the mammalian host . Msp2 (p44) is the major immunodominant outer-membrane protein of these bacteria . We hypothesized that Msp2 acts as an adhesin for A . phagocytophilum entry into granulocytes . This potential role was investigated by blocking binding with Msp2 monoclonal antibodies and by antagonizing binding and propagation with recombinant Msp2 (rMsp2) in vitro . With HL-60 cells, fresh human peripheral blood neutrophils, and a cell line devoid of the fucosylated platelet selectin glycoprotein ligand 1 (PSGL-1) receptor for A . phagocytophilum or one that was transfected to express this ligand, Msp2 monoclonal antibody and rMsp2 used as the antagonist caused concentration-dependent reductions in bacterial adhesion (P < 0.007 and P < 0.02, respectively) and propagation (P < 0.05 and P < 0.001), although inhibition of adhesion or propagation was moderate and incomplete . Likewise, rMsp2 bound to surfaces of the transfected cell at a level similar to that of extracellular A . phagocytophilum and significantly (P < 0.05) beyond that of nontransfected cells . Moreover, a dose-dependent reduction (P < 0.019) in PSGL-1 monoclonal antibody binding to HL-60 cells was elicited with rMsp2 . We conclude that Msp2s of A . phagocytophilum are involved in bacterial adhesion to ligands on host myeloid cells before intracellular infection.

Immunity, 2003 Jun, 18(6), 722 - 4
Intracellular pathogens and antigen presentation-new challenges with Legionella pneumophila; Unanue ER; In this issue of Immunity, examine the intracellular life of Legionella pneumophila in dendritic cells (DC) and macrophages, as well as the presentation of its antigens to CD4 T cells . Legionella is a particularly interesting bacterium because of the peculiarities inherent in its intracellular sojourn in phagocytes: it resides in an unusual vesicle characterized by ribosomes studded along its walls . In this compartment, Legionella proteins encoded by the dot gene inhibit phagosome-lysosome fusion and endosomal acidification, yielding a vesicular structure conducive to the multiplication of Legionella, poor in lysosomal contents, and in MHC molecules.

Philos Transact A Math Phys Eng Sci, 2003 Jun 15, 361(1807), 1089 - 99
Molecular autonomous agents; Kauffman S; I consider an autonomous agent to be a physical system able to act on its own behalf, such as a bacterium swimming up a glucose gradient . I tentatively define an autonomous agent to be a system capable of self-reproduction and at least capable of performing one thermodynamic work cycle . I give a hypothetical chemical example . I then explore the increasingly odd implications of this definition.

J Neurosurg, 2003 Jun, 98(6), 1198 - 202
Clinical application of a physically and chemically processed human substitute for dura mater; Dufrane D et al.; OBJECT: Allogenic human fascia lata used in neurosurgery as a dura mater substitute can be associated with the risk of virus and bacterium transmission and with a delay in its incorporation due to immunological and inflammatory reactions . The authors review their preliminary experience with a chemically and physically processed fascia lata graft . METHODS: Grafts that had been treated with solvent detergents, freeze-dried for conservation, and gamma irradiated (25,000 Gy) for sterilization were placed into 17 patients during neurosurgical procedures performed to treat brain tumors, cerebral malformations, trigeminal neuralgia, and posttraumatic lesions . The handling properties of the material, surgical wound features, and hematological parameters were evaluated . The average follow-up period was 23.8 +/- 2.2 months (mean +/- standard deviation) . The handling properties and biocompatibility of these human dural substitutes were highly satisfactory and no major complications were observed . Postoperative computerized tomography or magnetic resonance images obtained in 13 patients revealed no abnormal findings at the site of fascia lata implantation . In one patient who underwent a second surgery performed 12 months after the initial operation, this dural substitute was found to have been recolonized by host fibroblastic cells and replaced by autologous collagenous tissue . CONCLUSIONS: Human fascia lata that has been rendered safe by applying physical and chemical treatment is a fully biocompatible alternative to the dural graft materials currently available.

Izv Akad Nauk Ser Biol, 2003 May-Jun, (3), 301 - 5
{Isolation, purification, and properties of malate dehydrogenases from sulfur bacteria Beggiatoa leptomitiformis}; Eprintsev AT et al.; Malate dehydrogenase (E.C . 1.1.1.37) from bacterium Beggiatoa leptomitiformis was isolated and purified 123 times using a five-step purification procedure including the enzyme extraction, ammonium sulfate protein fractionation, gel filtration, ion exchange chromatography, and gel chromatography . The enzyme was homogenous according to the electrophoresis data; its activity was 20.43 U/mg proteins . This malate dehydrogenase is a homotetramer (Mr = 172 kDa) . The catalytic and thermodynamic properties, as well as the analysis of the published data suggest that the tetrameric structure of the enzyme allows it to participate in constructive metabolism supplying the cell with organic acids as a source of carbon.

Microbes Infect, 2003 Jul, 5(8), 741 - 8
Animal models of Helicobacter pylori infection and disease; O'Rourke JL et al.; The acceptance of Helicobacter pylori as a major human pathogen has necessitated the development of animal models to help elucidate the pathogenic mechanisms of this bacterium and aid in the development of improved strategies for the treatment of gastric disease . Appropriate models, utilising a range of animal species, have been developed to examine factors such as the influence of host responses and bacterial factors in disease development and the success of new therapeutic regimens, including vaccination, to cure infection.

Microbes Infect, 2003 Jul, 5(8), 715 - 21
Molecular and cellular mechanisms of action of the vacuolating cytotoxin (VacA) and neutrophil-activating protein (HP-NAP) virulence factors of Helicobacter pylori; Montecucco C et al.; Helicobacter pylori has elaborated a unique set of virulence factors that allow it to colonise the stomach wall . These factors include urease, helicoidal shape, flagella and adhesion molecules . Here we discuss the molecular characteristics and mechanisms of action of the vacuolating cytotoxin, VacA, and the neutrophil-activating protein, HP-NAP . Their activities are discussed in terms of tissue alterations, which promote the release of nutrients necessary for the growth and survival of the bacterium in its nutrient-poor ecological niche.

Cell Microbiol, 2003 Jul, 5(7), 469 - 80
Maturation of the Coxiella burnetii parasitophorous vacuole requires bacterial protein synthesis but not replication; Howe D et al.; This study examined whether protein synthesis and replication are required for maturation and fusogenicity of the lysosomal-like, large and spacious parasitophorous vacuole (PV) of Coxiella burnetii, an obligate intracellular bacterium . Large and spacious PV with multiple non-replicating C . burnetii were observed by phase microscopy in Vero cells infected at a multiplicity of infection of ten and treated with a bacteriostatic concentration of nalidixic acid or carbenicillin, antimicrobics that inhibit DNA and cell wall biosynthesis respectively . Conversely, large and spacious PV were not observed in cells treated with a bacteriostatic concentration of the protein synthesis inhibitor chloramphenicol . Rather, fluorescence microscopy of individual cells revealed multiple, acidic PV harbouring a single organism tightly bounded by a LAMP-1 positive vacuolar membrane . These vacuoles homotypically fused to form a large and spacious PV upon removal of the drug . Chloramphenicol also inhibited trafficking of latex beads to large and spacious PV and caused mature PV to collapse . Collectively, these results demonstrate that C . burnetii protein synthesis, but not replication, is required for fusion between nascent C . burnetii PV and latex bead phagosomes, and also for formation and maintenance of large and spacious, replicative PV . However, transit of nascent PV through the endocytic pathway to ultimately acquire lysosomal markers appears to occur irrespective of Coxiella protein synthesis.

J Bacteriol, 2003 Jul, 185(13), 3780 - 7
Common and distinguishing regulatory and expression characteristics of the highly related KorB proteins of streptomycete plasmids pIJ101 and pSB24.2; Ducote MJ et al.; The conjugative plasmid pIJ101 of the spore-forming bacterium Streptomyces lividans contains a regulatory gene, korB, whose product is required to repress potentially lethal expression of the pIJ101 kilB gene . The KorB protein also autoregulates korB gene expression and may be involved in control of pIJ101 copy number . KorB (pIJ101) is expressed as a 10-kDa protein in S . lividans that is immediately processed to a mature 6-kDa repressor molecule . The conjugative Streptomyces cyanogenus plasmid pSB24.1 is deleted upon entry into S . lividans to form pSB24.2, a nonconjugative derivative that contains a korB gene nearly identical to that of pIJ101 . Previous evidence that korB of pSB24.2 is capable of overriding pIJ101 kilB-associated lethality supported the notion that pIJ101 and pSB24.2 encode highly related, perhaps even identical conjugation systems . Here we show that KorB (pIJ101) and KorB (pSB24.2) repress transcription from the pIJ101 kilB promoter equally well, although differences exist with respect to their interactions with kilB promoter sequences . Despite high sequence and functional similarities, KorB (pSB24.2) was found to exist as multiple stable forms ranging in size from 10 to 6 kDa both in S . lividans and S . cyanogenus . Immediate processing of KorB (pIJ101) exclusively to the 6-kDa repressor form meanwhile was conserved between the two species . A feature common to both proteins was a marked increase in expression or accumulation upon sporulation, an occurrence that may indicate a particular need for increased quantities of this regulatory protein upon spore germination and resumption of active growth of plasmid-containing cells.

J Environ Qual, 2003 May-Jun, 32(3), 751 - 9
Leaching characteristics of heavy metals from sewage sludge by Acidithiobacillus thiooxidans MET; Ryu HW et al.; An acidophilic, sulfur-oxidizing Acidithiobacillus thiooxidans MET bacterium was isolated from anaerobically digested, dewatered sewage sludge . This bacterium showed sulfur-oxidizing ability at both acidic and neutral conditions, and allowed metal leaching even at a high (130 g L(-1)) sludge solids concentration . We found that low metal leaching efficiency at high solids concentration was mainly due to an increase in buffering capacity resulting in retardation of pH reduction . Therefore, metal leaching was mainly influenced not by sludge solids concentration, but by the pH (or sulfate concentration per unit sludge mass) of the sludge solutions . The relationship between the pH of the sludge solution and the efficiency of metal leaching was obtained by quantitatively investigating the effect of pH reduction or the amount of sulfate produced per unit sludge mass on leaching of each metal . Furthermore, the relationship between total metal content in the sludge and metal leached to the solution was obtained for each metal . Such a relationship allowed estimation of leachable metal at various amounts of total metal content in sludge.

J Cell Sci, 2003 Jul 15, 116(Pt 14), 3017 - 26
Stimulation of MMP-7 (matrilysin) by Helicobacter pylori in human gastric epithelial cells: role in epithelial cell migration; Wroblewski LE et al.; Epithelial cell responses to bacterial infection include induction of matrix metalloproteinase 7 (MMP-7) . Here, we identify increased MMP-7 expression in the gastric epithelium in response to the oncogenic bacterium Helicobacter pylori, and report on the mechanisms and consequences for gastric epithelial cell migration . In patients infected with H . pylori, there was increased MMP-7 in gastric biopsies detected by western blot . MMP-7 was localized to the advancing edge of migrating gastric epithelial cell colonies, including lamellipodia . Rates of spreading of gastric gland cells were higher in H . pylori-infected cultures compared with control, and this was inhibited by antisense oligonucleotides to MMP-7 . Complementary data were obtained in a gastric cancer cell line (AGS cells) . In the latter, H . pylori induced expression of an MMP-7-luciferase promoter/reporter vector through mechanisms that involved activation of Rho and Rac . RhoA acted through activation of both NF-kappaB and AP-1, whereas Rac activated NF-kappaB but not AP-1 . MMP-7 is commonly upregulated in gastric cancer; since H . pylori is a recognized gastric carcinogen, the data suggest a new mechanism by which the bacterium might predispose towards gastric neoplasia.

Commun Dis Intell, 2003, 27 Suppl, S127 - 31
Surveillance for antibiotic resistance in veterinary pathogens from the perspective of a regional diagnostic laboratory; Stephens CP; The Toowoomba Veterinary Laboratory tests for antibiotic resistance through passive surveillance of bacterial pathogens from diseased, frequently intensively managed, animals . Testing is carried out on the basis of the number of animals involved, the nature and severity of the disease and the identity and significance of the bacterium, the results guiding the submitting veterinarian in implementing appropriate treatment . The antibiotics chosen for testing are those that are currently registered for veterinary use and are considered effective in the given situation . Testing is carried out according to the current National Committee for Clinical Laboratory Standards Approved Standard for Disc Susceptibility Tests . This paper presents some results of testing bacterial pathogens from cattle and pigs.

Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 787 - 93
Description of Sulfurospirillum halorespirans sp . nov., an anaerobic, tetrachloroethene-respiring bacterium, and transfer of Dehalospirillum multivorans to the genus Sulfurospirillum as Sulfurospirillum multivorans comb . nov; Luijten ML et al.; An anaerobic, halorespiring bacterium (strain PCE-M2(T) = DSM 13726(T) = ATCC BAA-583(T)) able to reduce tetrachloroethene to cis-dichloroethene was isolated from an anaerobic soil polluted with chlorinated aliphatic compounds . The isolate is assigned to the genus Sulfurospirillum as a novel species, Sulfurospirillum halorespirans sp . nov . Furthermore, on the basis of all available data, a related organism, Dehalospirillum multivorans DSM 12446(T), is reclassified to the genus Sulfurospirillum as Sulfurospirillum multivorans comb . nov.

Genome Res, 2003 Jul, 13(7), 1665 - 74 Epub 2003 Jun 12.
Systematic cloning of Treponema pallidum open reading frames for protein expression and antigen discovery; McKevitt M et al.; A topoisomerase-based method was used to clone PCR products encoding 991 of the 1041 open reading frames identified in the genome sequence of the bacterium that causes syphilis, Treponema pallidum subsp . pallidum . Cloning the open reading frames into the univector plasmid system permitted the rapid conversion of the original clone set to other functional vectors containing a variety of promoters or tag sequences . A computational prediction of signal sequences identified 248 T . pallidum proteins that are potentially secreted from the cell . These clones were systematically converted into vectors designed to express the encoded proteins as glutathione-S-transferase fusion proteins . To test the potential of the clone set for novel antigen discovery, 85 of these fusion proteins were expressed from Escherichia coli, partially purified, and tested for antigenicity by using sera from rabbits infected with T . pallidum . Twelve of the 85 proteins bound significant levels of antibody . Of these 12 proteins, seven had previously been identified as T . pallidum antigens, and the remaining five represent novel antigens . These results demonstrate the potential of the T . pallidum clone set for antigen discovery and, more generally, for advancing the biology of this enigmatic spirochete.

Blood, 2003 Oct 1, 102(7), 2692 - 4 Epub 2003 Jun 12.
Persistent Mycobacterium avium infection following nonmyeloablative allogeneic peripheral blood stem cell transplantation for interferon-gamma receptor-1 deficiency; Horwitz ME et al.; Interferon-gamma receptor-1 (IFNgammaR1) deficiency is a rare inherited immunodeficiency . We performed a nonmyeloablative allogeneic stem cell transplantation on a boy with complete IFNgammaR1 deficiency and refractory disseminated Myco- bacterium avium infection . Despite the patient's profound immune defect, early donor stem cell engraftment was low . Full donor engraftment was accomplished only following multiple donor lymphocyte infusions . Detection of IFNgammaR1 expression on peripheral blood monocytes and neutrophils corresponded with establishment of stable, complete donor hematopoietic chimerism . However, expression of, and signaling through IFNgammaR1 disappeared shortly thereafter . Disseminated Mycobacterium avium infection persisted and the patient died . Coculture of Myco- bacterium avium with normal myeloid cells resulted in an IFNgamma signaling defect similar to that observed in vivo . Active disseminated Mycobacterium avium infection may significantly compromise normal immune reconstitution following allogeneic stem cell transplantation . Patients with IFNgammaR1 deficiency should receive transplants before developing refractory mycobacterial infections.

FEBS Lett, 2003 Jun 19, 545(2-3), 120 - 6
Structural and functional analysis of a lycopene beta-monocyclase gene isolated from a unique marine bacterium that produces myxol; Teramoto M et al.; A gene coding for lycopene beta-monocyclase, which metabolizes lycopene (psi,psi-carotene) to gamma-carotene (beta,psi-carotene), was isolated for the first time from a unique marine bacterium strain P99-3 that produces myxol (a gamma-carotene derivative) . This lycopene beta-monocyclase gene (designated crtYm) was included in the gene cluster which contained carotenoid biosynthetic gene (crtI, crtB, crtZ, crtY, and crtA) homologs . CrtYm, the CrtY homolog, metabolized lycopene to gamma-carotene, which was confirmed by deletion/expression analysis of the crtYm and by subsequent analysis of the metabolites from lycopene based on the retention times on high-performance liquid chromatography, UV-visible absorption spectra, and mass spectrometry.

Lett Appl Microbiol, 2003, 37(1), 21 - 5
Overlapping role of the outer membrane cytochromes of Shewanella oneidensis MR-1 in the reduction of manganese(IV) oxide; Myers JM et al.; AIM: To determine if the outer membrane (OM) cytochromes OmcA and OmcB of the metal-reducing bacterium Shewanella oneidensis MR-1 have distinct or overlapping roles in the reduction of insoluble manganese(IV) oxide . METHODS AND RESULTS: The gene replacement mutant (OMCA1) which lacks OmcA was partially deficient in Mn(IV) reduction . Complementation of OMCA1 with a vector (pVK21) that contains omcB but not omcA restored Mn(IV) reduction to levels that were even greater than those of wild-type . Examination of the OM of OMCA1/pVK21 revealed greater than wild-type levels of OmcB protein and specific haem content . CONCLUSIONS: Overexpression of OmcB can compensate for the absence of OmcA in the reduction of insoluble Mn(IV) oxides . Therefore, there is at least a partial overlap in the roles of these OM cytochromes in the reduction of insoluble Mn(IV) oxide . SIGNIFICANCE: The overlapping roles of these two cytochromes has important implications for understanding the mechanism by which MR-1 reduces insoluble metal oxides . There is no obligatory sequential electron transfer from one cytochrome to the other . They could both potentially serve as terminal reductases for extracellular electron acceptors.

Arch Biochem Biophys, 2003 Jul 1, 415(1), 87 - 93
Unique metal dependency of cytosolic alpha-mannosidase from Thermotoga maritima, a hyperthermophilic bacterium; Nakajima M et al.; A putative cytosolic alpha-mannosidase gene from a hyperthermophilic marine bacterium Thermotoga maritima was cloned and expressed in Escherichia coli . The purified recombinant enzyme appeared to be a homodimer of a 110-kDa subunit . The enzyme showed metal-dependent ability to hydrolyze p-nitrophenyl-alpha-D-mannopyranoside . In the absence of a metal, the enzyme was inactive . Cobalt and cadmium supported high activity (60 U/mg at 70 degrees C), while the activity with zinc and chromium was poor . Cobalt (0.8 mol) bound to 1 mol monomer with a K(d) of 70 microM . The optimum pH and temperature were 6.0 and 80 degrees C, respectively . The activity was inhibited by swainsonine, but not by 1-deoxymannojirimycin, which is in agreement with the features of cytosolic alpha-mannosidase.

Crit Rev Oral Biol Med, 2003, 14(3), 226 - 33
Oral Helicobacter pylori: can we stomach it?
Dowsett SA, Kowolik MJ.
Helicobacter pylori infection is one of the most common in man . The bacterium primarily resides in the human stomach, where it plays a significant role in gastric disease . If the spread of H . pylori is to be prevented, an understanding of the transmission process is essential . The oral cavity has been proposed as a reservoir for gastric H . pylori, which has been detected by culture and PCR in both dental plaque and saliva . This review will discuss the evidence for the role of the oral cavity in the transmission of gastric H . pylori . Moreover, the difficulties encountered in addressing this topic, possible directions for future research, and the implications for the dental profession are discussed.

Vaccine, 2003 Jun 20, 21(21-22), 2911 - 22
Porcine Ig isotypes: function and molecular characteristics; Crawley A et al.; In pigs, protection against the toxigenic extra-cellular bacterium Actinobacillus pleuropneumoniae was correlated with an increased IgG(1):IgG(2) ratio of haemolytic toxin-specific antibodies . In all species so far studied, IgG isotype expression is controlled by Type 1 (IFN-gamma, IL-12) and Type 2 (IL-4, IL-10) cytokines which dictate immune response polarization to cell-mediated (CMI) or antibody-mediated immunity (AMI), respectively . Thus, immunoglobulin (Ig) isotypes reflect Type 1 or Type 2 immune responses . Immunoglobulin isotype production by porcine B-cells cultured in the presence of recombinant porcine (rp) cytokines varies by individual, however pigs tend to generate a high IgG(1):IgG(2) ratio in response to rp IL-10 and the inverse in response to rp IFN-gamma or rp IL-12 . Differential Ig isotype production should favor an isotype with a functional advantage to control the inciting infection and disease . However, functions of porcine Ig isotypes have not been described . To compare function of porcine IgM, IgG(1) and IgG(2) of defined specificity for hen eggwhite lysozyme (HEWL), Ig isotypes were affinity purified from serum by HEWL specificity and by isotype-specific mouse monoclonal antibodies . Their ability to activate complement (C') and to opsonize was tested in vitro . Porcine IgG(2) had greater guinea pig C' activating ability than did IgG(1) . Neither isotype opsonized HEWL-conjugated sheep erythrocytes in vitro . Amino acid sequence analysis of IgG isotypes revealed that all subclasses have putative C' binding sites but that IgG(2a), IgG(2b) and IgG(4) were more flexible in the middle hinge region than IgG(1) and IgG(3) and would likely activate C' more efficiently . Thus, porcine IgG isotypes associated with resistance and susceptibility to disease also differ in their actual and predicted biological functions.

J Biol Chem, 2003 Sep 12, 278(37), 35384 - 93 Epub 2003 Jun 09.
Activation mechanism of the CO sensor CooA . Mutational and resonance Raman spectroscopic studies; Coyle CM et al.; CooA is a CO-dependent heme protein transcription factor of the bacterium Rhodospirillum rubrum . CO binding to its heme causes CooA to bind DNA and activate expression of genes for CO metabolism . To understand the nature of CO activation, several CooA mutational variants have been studied by resonance Raman spectroscopy, in vivo activity measurements, and DNA binding assays . Analysis of the Fe-C and C-O stretching Raman spectroscopy bands permits the conclusion that when CO displaces the Pro2 heme ligand, the protein forms a hydrophobic pocket in which the C-helix residues Gly117, Leu116, and Ile113 are close to the bound CO . The displaced Pro2 terminus is expelled from this pocket, unless the pH is raised above the pKa, in which case the terminus remains in H-bond contact . The pKa for this transition is 8.6, two units below that of aqueous proline, reflecting the hydrophobic nature of the pocket . The proximal Fe-His bond in Fe{II}CooA is as strong as it is in myoglobin but is weakened by CO binding, an effect attributable to loss of an H-bond from the proximal His77 ligand to the adjacent Asn42 side chain . A structural model is proposed for the position of the CO-bound heme in the active form of CooA, which has implications for the mechanism of CO activation.

J Am Acad Dermatol, 2003 Jun, 48(6), 966 - 9
Culture and immunohistochemical evidence of Chlamydia pneumoniae infection in ulcerative pyoderma gangrenosum; Sams HH et al.; A potentially contributing factor to the development and chronicity of pyoderma gangrenosum is infection with the relatively recently characterized human pathogen, Chlamydia pneumoniae . C pneumoniae is an obligate intracellular bacterium that can infect endothelial, monocyte, and smooth muscle cells and is associated with cardiopulmonary diseases . A case of serologically, polymerase chain reaction-positive, immunohistochemically, and culture-documented viable C pneumoniae organisms in a chronic pyoderma gangrenosum ulcer is reported, a finding that has not been described previously.

Appl Environ Microbiol, 2003 Jun, 69(6), 3646 - 9
Characterization of the first molluscicidal lipopolysaccharide from Moraxella osloensis; Tan L et al.; Moraxella osloensis is a bacterium that is mutualistically associated with Phasmarhabditis hermaphrodita, a nematode that has potential for the biocontrol of mollusk pests, especially the slug Deroceras reticulatum . We discovered that purified M . osloensis lipopolysaccharide (LPS) possesses a lethal toxicity to D . reticulatum when administered by injection but no contact or oral toxicity to this slug . The toxicity of the LPS resides in the lipid A moiety . M . osloensis LPS was semiquantitated at 6 x 10(7) endotoxin units per mg . The LPS is a rough-type LPS with an estimated molecular weight of 5,300 . Coinjection of galactosamine with the LPS increased the LPS's toxicity to the slug two- to four-fold . The galactosamine-induced sensitization of the slug to the LPS was reversed completely by uridine.

Appl Environ Microbiol, 2003 Jun, 69(6), 3093 - 102
Genes involved in the biosynthesis of photosynthetic pigments in the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina; Kovacs AT et al.; A pigment mutant strain of the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina BBS was isolated by plasposon mutagenesis . Nineteen open reading frame, most of which are thought to be genes involved in the biosynthesis of carotenoids, bacteriochlorophyll, and the photosynthetic reaction center, were identified surrounding the plasposon in a 22-kb-long chromosomal locus . The general arrangement of the photosynthetic genes was similar to that in other purple photosynthetic bacteria; however, the locations of a few genes occurring in this region were unusual . Most of the gene products showed the highest similarity to the corresponding proteins in Rubrivivax gelatinosus . The plasposon was inserted into the crtD gene, likely inactivating crtC as well, and the carotenoid composition of the mutant strain corresponded to the aborted spirilloxanthin pathway . Homologous and heterologous complementation experiments indicated a conserved function of CrtC and CrtD in the purple photosynthetic bacteria . The crtDC and crtE genes were shown to be regulated by oxygen, and a role of CrtJ in aerobic repression was suggested.

J Biochem Mol Biol, 2003 May 31, 36(3), 282 - 7
Production of superoxide dismutase by Deinococcus radiophilus; Yun YS et al.; The production of superoxide dismutase (SOD) varied in Deinococcus radiophilus, the UV resistant bacterium, depending upon different phases of growth, UV irradiation, and superoxide treatment . A gradual increase in total SOD activity occurred up to the stationary phases . The electrophoretic resolution of the SOD in cell extracts of D . radiophilus at each growth phase revealed the occurrence of MnSOD throughout the growth phases . The SOD profiles of D . radiophilus at the exponential phase received oxidative stress by the potassium superoxide treatment or UV irradiation also revealed the occurrence of a single SOD . However, these treatments caused an increase in SOD activity . The data strongly suggest that D . radiophilus has only one species of SOD as a constitutive enzyme, which seems to be a membrane-associated protein.

Mol Microbiol, 2003 Jun, 48(5), 1317 - 23
The DNA excision repair system of the highly radioresistant bacterium Deinococcus radiodurans is facilitated by the pentose phosphate pathway; Zhang YM et al.; Deinococcus radiodurans is highly resistant to radiation and mutagenic chemicals . Mutants defective in the putative glucose-6-phosphate dehydrogenase gene (zwf-) and the aldolase gene (fda-) were generated by homologous recombination . These mutants were used to test the cells' resistance to agents that cause dimer formation and DNA strand breaks . The zwf - mutants were more sensitive to agents that induce DNA excision repair, such as UV irradiation and H2O2, but were as resistant to DNA strand break-causing agents such as methylmethanesulphonic acid (MMS) and mitomycin C (MMC) as the wild-type cells . Analysis of the cytoplasmic fraction of zwf- cells showed that the concentrations of inosine monophosphate (IMP) and uridine monophosphate (UMP) were only 30% of those found in the wild-type cells . The fda- mutants were slightly more resistant to UV light and H2O2 . Results suggested that the deinococcal pentose phosphate pathway augmented the DNA excision repair system by providing cells with adequate metabolites for the DNA mismatch repair.

Aliment Pharmacol Ther, 2003 Jun, 17 Suppl 2, 75 - 81
Review article: molecular basis of gastric carcinogenesis; Nardone G; Gastric cancer is constituted by two histomorphological entities 'intestinal' and 'diffuse', however lesions with similar morphologies may differ in biological aggressiveness and response to therapy . Two distinct molecular pathways have been identified in gastric carcinogenesis: the microsatellite mutator phenotype and a phenotype associated with chromosomal and intrachromosomal instability . Mounting evidence suggests that microsatellite mutator phenotype alterations and expression of the products of cancer-related genes are early markers of cell transformation, and may serve to identify the gastric carcinoma histotypes . The lack of a clear genetic basis, lends weight to the notion that gastric cancer is not a monomorphic entity but may be affected by environmental factors . Helicobacter pylori is the most important environmental risk factor associated with sporadic gastric cancer . Exposure of gastric epithelial cells to bacterium results in the generation of reactive oxygen species and inducible nitric oxide synthase that in turn may cause genetic alterations leading to cancer in a subset of subjects . Thus, gastric cancer may be considered the result of an interplay between host genetic profile and environmental toxic agents . The new technologies of molecular analysis will help to establish an individual's risk of developing gastric cancer and will lead to novel biological therapeutic strategies.

Environ Sci Technol, 2003 May 15, 37(10), 2173 - 83
Role of lipopolysaccharides in the adhesion, retention, and transport of Escherichia coli JM109; Abu-Lail NI et al.; The role of lipopolysaccharides (LPS) in bacterial adhesion was investigated via atomic force microscopy (AFM) . Adhesion between a silicon nitride tip and Escherichia coli JM109 was measured in water and 0.01 M phosphate-buffered saline (PBS) on untreated cells and on a sample of E . coli treated with 100 mM ethylenediaminetetraacetic acid (EDTA), which removes approximately 80% of the LPS molecules . LPS removal decreased the adhesion affinity between the bacterial cells and the AFM tip from -2.1 +/- 1.8 to -0.40 +/- 0.36 nN in water and from -0.74 +/- 0.44 to -0.46 +/- 0.23 nN in 0.01 M PBS (statistically different, Mann-Whitney rank sum test, P < 0.01) . The distributions of adhesion affinities between E . coli LPS macromolecules and the AFM tip could be described by gamma distribution functions . Direct measurements of the adhesive force between E . coil and a surface were compared with adhesion in batch and column experiments, and agreement was observed between the influences of LPS on adhesion in each system . Bacterial batch retention to glass or in packed beds to quartz sand decreased after LPS removal . When interaction forces were measured during the approach of the AFM tip to a bacterium, steric repulsive forces were seen for both treated and untreated cells, but the repulsion was greater when the LPS was intact A model for steric repulsion predicted a reduction of the equilibrium length of the surface polymers from 242 to 64 nm in water and from 175 to 81 nm in buffer, after removal of a portion of the LPS . DLVO calculations based on conventional and soft-particle DLVO theories predicted higher energy barriers to adhesion for all surfaces after LPS removal, consistent with experimental findings . Adhesion forces between the AFM tip and bacterial polymers were correlated with bacterial attachment and retention, while measurements of interaction forces during the approach of the AFM tip to the bacterium did not correlate with subsequent adhesion behavior to glass or quartz sand.

Curr Microbiol, 2003 Jul, 47(1), 1 - 4
Monitoring the uptake of protein-derived peptides by Porphyromonas gingivalis with fluorophore-labeled substrates; Yoshioka M et al.; The aim of this study was to develop a simple method to quantify peptide uptake by the periodontopathogenic bacterium Porphyromonas gingivalis . After incubation of bacterial cells with self-quenched fluorescent bovine serum albumin (DQ Green BSA), the fluorescence measured in the supernatant of the assay mixture indicated the degree of protein degradation, whereas the fluorescence associated with the lysate of washed cells indicated the amount of BSA-derived fragments incorporated by the bacteria . The optimal conditions for uptake of fluorophore-labeled albumin fragments were found to be mid-log grown cells, 150 m M NaCl in phosphate buffer, pH 7, 37 degrees C, and anaerobiosis . Among the protease inhibitors tested, 4-(2-aminoethyl)-benzene sulfonyl-fluoride hydrochloride (AEBSF) and cathepsin B inhibitor II caused a significant inhibition of the uptake of BSA-derived peptides . This assay was applicable for other commercially available fluorescent substrates . This simple method may be useful to investigate protein processing in proteolytic bacteria and for studying the effects of environmental parameters or cell treatments on the peptide uptake.

Biochemistry, 2003 Jun 10, 42(22), 6726 - 34
Spectroscopic and mutational analysis of the blue-light photoreceptor AppA: a novel photocycle involving flavin stacking with an aromatic amino acid; Kraft BJ et al.; The flavoprotein AppA is a blue-light photoreceptor that functions as an antirepressor of photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides . Heterologous expression studies show that FAD binds to a 156 amino acid N-terminal domain of AppA and that this domain is itself photoactive . A pulse of white light causes FAD absorption to be red shifted in a biphasic process with a fast phase occurring in <1 micros and a slow phase occurring at approximately 5 ms . The absorbance shift was spontaneously restored over a 30 min period, also in a biphasic process as assayed by fluorescence quenching and electronic absorption analyses . Site-directed replacement of Tyr21 with Leu or Phe abolished the photochemical reaction implicating involvement of Tyr21 in the photocycle . Nuclear magnetic resonance analysis of wild-type and mutant proteins also indicates that Tyr21 forms pi-pi stacking interactions with the isoalloxazine ring of FAD . We propose that photochemical excitation of the flavin results in strengthening of a hydrogen bond between the flavin and Tyr 21 leading to a stable local conformational change in AppA.

Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1012 - 5 Epub 2003 May 23.
Solving the phase problem for carbohydrate-binding proteins using selenium derivatives of their ligands: a case study involving the bacterial F17-G adhesin; Buts L et al.; The Escherichia coli adhesin F17-G is a carbohydrate-binding protein that allows the bacterium to attach to the intestinal epithelium of young ruminants . The structure of the 17 kDa lectin domain of F17-G was determined using the anomalous dispersion signal of a selenium-containing analogue of the monosaccharide ligand N-acetyl-d-glucosamine in which the anomeric oxygen was replaced by an Se atom . A three-wavelength MAD data set yielded good experimental phases to 2.6 A resolution . The structure was refined to 1.75 A resolution and was used to solve the structures of the ligand-free protein and the F17-G-N-acetyl-d-glucosamine complex . This selenium-carbohydrate phasing method could be of general use for determining the structures of carbohydrate-binding proteins.

Microbiology, 2003 Jun, 149(Pt 6), 1423 - 35
The senX3-regX3 two-component regulatory system of Mycobacterium tuberculosis is required for virulence; Parish T et al.; Two-component regulatory systems have been widely implicated in bacterial virulence . To investigate the role of one such system in Mycobacterium tuberculosis, a strain was constructed in which the senX3-regX3 system was deleted by homologous recombination . The mutant strain (Tame15) showed a growth defect after infection of macrophages and was attenuated in both immunodeficient and immunocompetent mice . Competitive hybridization of total RNA from the wild-type and mutant strains to a whole-genome microarray was used to identify changes in gene expression resulting from the deletion . One operon was highly up-regulated in the mutant, indicating that regX3 probably has a role as a repressor of this operon . Other genes which were up- or down-regulated were also identified . Many of the genes showing down-regulation are involved in normal growth of the bacterium, indicating that the mutant strain is subject to some type of growth slow-down or stress . Genes showing differential expression were further grouped according to their pattern of gene expression under other stress conditions . From this analysis 50 genes were identified which are the most likely to be controlled by RegX3 . Most of these genes are of unknown function and no obvious motifs were found upstream of the genes identified . Thus, it has been demonstrated that the senX3-regX3 two-component system is involved in the virulence of M . tuberculosis and a number of genes controlled by this system have been identified.

Vet Hum Toxicol, 2003 Jun, 45(3), 160 - 2
Poisoning of livestock in oregon in the 1940s to 1960s attributed to corynetoxins produced by Rathayibacter in nematode galls in chewings fescue (Festuca nigrescens); Riley IT et al.; Tunicaminyluracil antibiotics, similar to the corynetoxins produced by Rathayibacter toxicus in Australia and South Africa, were found in old nematode seed-galls from Festuca nigrescens from New Jersey (USA) and New Zealand (NZ) . The toxin profiles from the NZ and USA galls were similar to each other, but differed from those produced by R toxicus from Australia and South Africa, suggesting that a geographical variant of R toxicus or closely related species may be involved . The NZ galls gave a positive response to a R toxicus-specific monoclonal antibody assay, albeit a considerably weaker response than that seen with Australian R toxicus galls, but the older USA galls were negative, possibly due to deterioration of the antigen . From these findings, it is postulated that livestock deaths associated with the feeding of nematode and bacterial infected screenings of F nigrescens in Oregon, USA, in the 1940s to 1960s were caused by corynetoxin-like toxins produced by the bacterium.

Biochem J, 2003 Sep 1, 374(Pt 2), 529 - 35
Hydride transfer during catalysis by dihydrofolate reductase from Thermotoga maritima; Maglia G et al.; DHFR (dihydrofolate reductase) catalyses the metabolically important reduction of 7,8-dihydrofolate by NADPH . DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR), which shares similarity with DHFR from Escherichia coli, has previously been characterized structurally . Its tertiary structure is similar to that of DHFR from E . coli but it is the only DHFR characterized so far that relies on dimerization for stability . The midpoint of the thermal unfolding of TmDHFR was at approx . 83 degrees C, which was 30 degrees C higher than the melting temperature of DHFR from E . coli . The turnover and the hydride-transfer rates in the kinetic scheme of TmDHFR were derived from measurements of the steady-state and pre-steady-state kinetics using absorbance and stopped-flow fluorescence spectroscopy . The rate constant for hydride transfer was found to depend strongly on the temperature and the pH of the solution . Hydride transfer was slow (0.14 s(-1) at 25 degrees C) and at least partially rate limiting at low temperatures but increased dramatically with temperature . At 80 degrees C the hydride-transfer rate of TmDHFR was 20 times lower than that observed for the E . coli enzyme at its physiological temperature . Hydride transfer depended on ionization of a single group in the active site with a p K(a) of 6.0 . While at 30 degrees C, turnover of substrate by TmDHFR was almost two orders of magnitude slower than by DHFR from E . coli; the steady-state rates of the two enzymes differed only 8-fold at their respective working temperatures.

Biochemistry (Mosc), 2003 Apr, 68(4), 385 - 90
Ion transport coupled to terminal oxidase functioning in the extremely alkaliphilic halotolerant bacterium Thioalkalivibrio; Grischuk YV et al.; Proton transport in the terminal part of the respiratory chain in the extremely alkaliphilic halotolerant bacterial strain Thioalkalivibrio versutus was studied under near-optimum growth conditions (pH 9.0-9.5) . Under these conditions, bacterial cells generated electric potential with the negative charge being inside the cells . When only the terminal part of the respiratory chain functioned, it was found that: 1) unlike other bacteria known, this bacterium did not acidify the medium in the presence of K(+) and valinomycin; 2) in the presence of an uncoupler, CCCP, but in the absence of valinomycin, reversible alkalinization of the medium occurred as a result of proton influx into the cells . Cyanide prevented this alkalinization . The difference spectra indicate that cell membranes contained cytochromes c and (b+o), some of which reacted with CO . The respiratory activity of membranes in the terminal part of the respiratory chain was optimal at pH 9.5 and specifically depended on sodium ions (C(1/2) = 10 mM) . The data suggest the presence of a Na(+)-pump in the terminal part of the respiratory chain of the studied strain which can pump Na(+) out of the cells.

Appl Microbiol Biotechnol, 2003 Sep, 62(4), 407 - 13 Epub 2003 May 23.
Antifungal mechanism of an anti-Pythium protein (SAP) from the marine bacterium Streptomyces sp . strain AP77 is specific for Pythium porphyrae, a causative agent of red rot disease in Porphyra spp; Woo JH et al.; Previously we reported an antifungal protein specific to Pythium porphyrae, a causative agent of red rot disease afflicting seaweed Porphyra spp . This study was carried out to identify the antifungal mechanism of the antifungal protein to P . porphyrae . When we first examined the effect of an anti- Pythium protein (SAP) on the P . porphyrae cell walls, SAP did not decompose the six structural polysaccharides in Pythium cell walls . However, hyphal growth was significantly inhibited in Pythium cells treated with 50 microg/ml of SAP by MTT assay . Protoplasmic leakage was observed in P . porphyrae hyphae treated with SAP for 1 h, followed by hyphal swelling and disintegration, using SYTOX Green, and SAP permeabilized the membrane of P . porphyrae in a dose-dependent manner . Treating P . porphyrae cells with SAP in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a membrane-depolarizing agent, significantly reduced the membrane permeability to SYTOX Green . Moreover, a similar effect was observed when the P . porphyrae cells were treated with SAP in the presence of MgCl2 . In contrast, identical treatment in the presence of KCl significantly increased the membrane permeability to SYTOX Green . These results suggested that anti- Pythium mechanism of SAP was related to alteration of the membrane permeability in P . porphyrae.

J Biochem (Tokyo), 2003 Jan, 133(1), 51 - 8
Thermostable aspartase from a marine psychrophile, Cytophaga sp . KUC-1: molecular characterization and primary structure; Kazuoka T et al.; We found that a psychrophilic bacterium isolated from Antarctic seawater, Cytophaga sp . KUC-1, abundantly produces aspartase {EC4.3.1.1}, and the enzyme was purified to homogeneity . The molecular weight of the enzyme was estimated to be 192,000, and that of the subunit was determined to be 51,000: the enzyme is a homotetramer . L-Aspartate was the exclusive substrate . The optimum pH in the absence and presence of magnesium ions was determined to be pH 7.5 and 8.5, respectively . The enzyme was activated cooperatively by the presence of L-aspartate and by magnesium ions at neutral and alkaline pHs . In the deamination reaction, the K(m) value for L-aspartate was 1.09 mM at pH 7.0, and the S(1/2) value was 2.13 mM at pH 8.5 . The V(max) value were 99.2 U/mg at pH 7.0 and 326 U/mg at pH 8.5 . In the amination reaction, the K(m) values for fumarate and ammonium were 0.797 and 25.2 mM, respectively, and V(max) was 604 U/mg . The optimum temperature of the enzyme was 55 degrees C . The enzyme showed higher pH and thermal stabilities than that from mesophile: the enzyme was stable in the pH range of 4.5-10.5, and about 80% of its activity remained after incubation at 50 degrees C for 60 min . The gene encoding the enzyme was cloned into Escherichia coli, and its nucleotides were sequenced . The gene consisted of an open reading frame of 1,410-bp encoding a protein of 469 amino acid residues . The amino acid sequence of the enzyme showed a high degree of identity to those of other aspartases, although these enzymes show different thermostabilities.

Infect Immun, 2003 Jun, 71(6), 3645 - 7
Involvement of nicotinic acetylcholine receptors in controlling Chlamydia pneumoniae growth in epithelial HEp-2 cells; Yamaguchi H et al.; Nicotinic acetylcholine receptors (nAChRs) play an essential role in neurotransmission . Recent studies have indicated that nAChRs may be involved in the regulation of some bacterial infections through immunological mechanisms in macrophages . However, the regulation of infection with Chlamydia pneumoniae, which is a ubiquitous pneumonia-causing bacterium, by an nAChR-mediated mechanism is still unclear . In the present study, it was found that stimulation of nAChRs with ligands such as nicotine and acetylcholine altered the growth of C . pneumoniae in epithelial HEp-2 cells . Thus, the results revealed a possible pathophysiological role of nAChRs in the regulation of intracellular bacterial infection.

Infect Immun, 2003 Jun, 71(6), 3529 - 39
pH-regulated gene expression of the gastric pathogen Helicobacter pylori; Merrell DS et al.; Colonization by the gastric pathogen Helicobacter pylori has been shown to be intricately linked to the development of gastritis, ulcers, and gastric malignancy . Little is known about mechanisms employed by the bacterium that help it adapt to the hostile environment of the human stomach . In an effort to extend our knowledge of these mechanisms, we utilized spotted-DNA microarrays to characterize the response of H . pylori to low pH . Expression of approximately 7% of the bacterial genome was reproducibly altered by shift to low pH . Analysis of the differentially expressed genes led to the discovery that acid exposure leads to profound changes in motility of H . pylori, as a larger percentage of acid-exposed bacterial cells displayed motility and moved at significantly higher speeds . In contrast to previous publications, we found that expression of the bacterial virulence gene cagA was strongly repressed by acid exposure . Furthermore, this transcriptional repression was reflected at the level of protein accumulation in the H . pylori cell.

Infect Immun, 2003 Jun, 71(6), 3010 - 9
A recombinant beta-1,3-glucanosyltransferase homolog of Coccidioides posadasii protects mice against coccidioidomycosis; Delgado N et al.; Coccidioides posadasii is a fungal respiratory pathogen which is responsible for recurrent epidemics of San Joaquin Valley fever (coccidioidomycosis) in desert regions of the southwestern United States . Numerous studies have revealed that the cell wall of the parasitic phase of the fungus is a reservoir of immunoreactive macromolecules and a potential source of a vaccine against this mycosis . A 495-bp fragment of a C . posadasii gene which encodes a putative wall-associated, glycosylphosphatidylinositol (GPI)-anchored beta-1,3-glucanosyltransferase was identified by computational analysis of the partially sequenced genome of this pathogen . The translated, full-length gene (GEL1) showed high sequence homology to a reported beta-1,3-glucanosyltransferase of Aspergillus fumigatus (70% identity, 90% similarity) and was selected for further study . The GEL1 mRNA of C . posadasii was detected at the highest level during the endosporulation stage of the parasitic cycle, and the mature protein was immunolocalized to the surface of endospores . BALB/c or C57BL/6 mice were immunized subcutaneously with the bacterium-expressed recombinant protein (rGel1p) to evaluate its protective efficacy against a lethal challenge of C . posadasii by either the intraperitoneal or intranasal route . In both cases, rGel1p-immune mice infected with the pathogen showed a significant reduction in fungal burden and increased survival compared to nonimmune mice . The recombinant beta-1,3-glucanosyltransferase is a valuable addition to an arsenal of immunoreactive proteins which could be incorporated into a human vaccine against coccidioidomycosis.

Antimicrob Agents Chemother, 2003 Jun, 47(6), 1972 - 5
Chlamydia pneumoniae resists antibiotics in lymphocytes; Yamaguchi H et al.; Chlamydia pneumoniae infection of lymphocytes in blood has been well documented, and it is apparent that control of this pathogen in these cells may be critical in the development of chronic inflammatory diseases associated with infection by this bacterium . The activity of antibiotics against C . pneumoniae in lymphocytes was assessed in this study by utilizing an in vitro infection model with lymphoid cells . The results obtained indicated that although all of the antibiotics tested showed remarkable activity against bacterial growth in epithelial cells, C . pneumoniae in lymphocytes was less susceptible to antibiotics than was bacterial growth in epithelial cells, which are widely used for the evaluation of anti-C . pneumoniae antibiotics.

Environ Microbiol, 2003 Jun, 5(6), 503 - 9
Marinobacterium sp . strain DMS-S1 uses dimethyl sulphide as a sulphur source after light-dependent transformation by excreted flavins; Hirano H et al.; Marinobacterium sp . strain DMS-S1 is a unique marine bacterium that can use dimethyl sulphide (DMS) as a sulphur source only in the presence of light . High-performance liquid chromatography (HPLC) analyses of the culture supernatant revealed that excreted factors, which could transform DMS to dimethyl sulphoxide (DMSO) under light, are FAD and riboflavin . In addition, FAD appeared to catalyse the photolysis of DMS to not only DMSO but also methanesulphonate (MSA), formate, formaldehyde and sulphate . As strain DMS-S1 can use sulphate and MSA as a sole sulphur source independently of light, the excretion of flavins appeared to support the growth on DMS under light . Furthermore, three out of 12 marine bacteria from IAM culture collection were found to be able to grow on DMS with the aid of photolysis by the flavins excreted . This is the first report that bacteria can use light to assimilate oceanic organic sulphur compounds outside the cells by excreting flavins as photosensitizers.

Eur J Biochem, 2003 Jun, 270(11), 2476 - 85
Two W-containing formate dehydrogenases (CO2-reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidans; de Bok FA et al.; Two formate dehydrogenases (CO2-reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans . Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger . The purified enzymes exhibited extremely high formate-oxidation and CO2-reduction rates, and low Km values for formate . For the enzyme designated FDH-1, a specific formate oxidation rate of 700 U.mg-1 and a Km for formate of 0.04 mm were measured when benzyl viologen was used as an artificial electron acceptor . The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 U.mg-1 and a Km of 0.01 mm for formate with benzyl viologen as electron acceptor . The specific CO2-reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 U.mg-1 and 89 U.mg-1, respectively . From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 +/- 3, 56 +/- 2 and 19 +/- 1 kDa) and has a native molecular mass of approximately 350 kDa . FDH-2 appeared to be a heterodimer composed of a 92 +/- 3 kDa and a 33 +/- 2 kDa subunit . Both enzymes contained tungsten and selenium, while molybdenum was not detected . EPR spectroscopy suggested that FDH-1 contains at least four {2Fe-2S} clusters per molecule and additionally paramagnetically coupled {4Fe-4S} clusters . FDH-2 contains at least two {4Fe-4S} clusters per molecule . As both enzymes are produced under all growth conditions tested, but with differences in levels, expression may depend on unknown parameters.

Int J Med Microbiol, 2003 Apr, 293(1), 69 - 76
Mucosal immune response to Tropheryma whipplei; Ring S et al.; Whipple's disease is a rare infectious disease caused by the ubiquitously occurring Tropheryma whipplei in predisposed persons . Genetic or acquired defects in the mucosal and peripheral immune system become apparent as diminished Th1 immune functions with decreased production of IL-12 and IFN-gamma accompanied by an increased secretion of IL-4 . These defects may enable T . whipplei to survive and replicate . The recently established cultivation of the bacterium in HEL cells and the isolation from infected intestinal biopsies enable a multitude of experimental possibilities which may lead to an improved diagnosis as well as understanding of the etiology and pathogenesis of Whipple's disease.

Prikl Biokhim Mikrobiol, 2003 May-Jun, 39(3), 322 - 8
{Growth of bacteria degrading naphthalene and salicylate at low temperatures}; Grishchenkov VG et al.; A total of 58 bacterial strains degrading naphthalene and salicylate were isolated from soil samples polluted with oil products, collected in different regions of Russia during winter and summer . The isolates were assessed for their ability to grow at low temperatures (4, 8, and 15 degrees C); bacteria growing at 4 degrees C in the presence of naphthalene or salicylate accounted for 65% and 53%, respectively, of the strains isolated . The strains differed in the temperature dependence of their growth rates . It was demonstrated that the type of expression of Nah+ phenotype at low temperatures depended on the combination of the host bacterium and the plasmid.

J Bacteriol, 2003 Jun, 185(11), 3352 - 60
Genomic analysis and initial characterization of the chitinolytic system of Microbulbifer degradans strain 2-40; Howard MB et al.; The marine bacterium Microbulbifer degradans strain 2-40 produces at least 10 enzyme systems for degrading insoluble complex polysaccharides (ICP) . The draft sequence of the 2-40 genome allowed a genome-wide analysis of the chitinolytic system of strain 2-40 . The chitinolytic system includes three secreted chitin depolymerases (ChiA, ChiB, and ChiC), a secreted chitin-binding protein (CbpA), periplasmic chitooligosaccharide-modifying enzymes, putative sugar transporters, and a cluster of genes encoding cytoplasmic proteins involved in N-acetyl-D-glucosamine (GlcNAc) metabolism . Each chitin depolymerase was detected in culture supernatants of chitin-grown strain 2-40 and was active against chitin and glycol chitin . The chitin depolymerases also had a specific pattern of activity toward the chitin analogs 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside (MUF-diNAG) and 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside (MUF-triNAG) . The depolymerases were modular in nature and contained glycosyl hydrolase family 18 domains, chitin-binding domains, and polycystic kidney disease domains . ChiA and ChiB each possessed polyserine linkers of up to 32 consecutive serine residues . In addition, ChiB and CbpA contained glutamic acid-rich domains . At 1,271 amino acids, ChiB is the largest bacterial chitinase reported to date . A chitodextrinase (CdxA) with activity against chitooligosaccharides (degree of polymerization of 5 to 7) was identified . The activities of two apparent periplasmic (HexA and HexB) N-acetyl-beta-D-glucosaminidases and one cytoplasmic (HexC) N-acetyl-beta-D-glucosaminidase were demonstrated . Genes involved in GlcNAc metabolism, similar to those of the Escherichia coli K-12 NAG utilization operon, were identified . NagA from strain 2-40, a GlcNAc deacetylase, was shown to complement a nagA mutation in E . coli K-12 . Except for the GlcNAc utilization cluster, genes for all other components of the chitinolytic system were dispersed throughout the genome . Further examination of this system may provide additional insight into the mechanisms by which marine bacteria degrade chitin and provide a basis for future research on the ICP-degrading systems of strain 2-40.

J Bacteriol, 2003 Jun, 185(11), 3249 - 58
Inactivation of Mg chelatase during transition from anaerobic to aerobic growth in Rhodobacter capsulatus; Willows RD et al.; The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism . As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue . A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg(2+) to form Mg-protoporphyrin IX . Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R . capsulatus . We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts . BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX . Recombinant BchH is rapidly inactivated by light in the presence of O(2), and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX . When photosynthetically growing R . capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions . The light- and O(2)-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.

FEBS Lett, 2003 May 22, 543(1-3), 184 - 9
Plant polyphenols inhibit VacA, a toxin secreted by the gastric pathogen Helicobacter pylori; Tombola F et al.; VacA is a major virulence factor of the widespread stomach-dwelling bacterium Helicobacter pylori . It causes cell vacuolation and tissue damage by forming anion-selective, urea-permeable channels in plasma and endosomal membranes . We report that several flavone derivatives and other polyphenols present in vegetables and plants inhibit ion and urea conduction and cell vacuolation by VacA . Red wine and green tea, which contain many of the compounds in question, also potently inhibit the toxin . These observations suggest that polyphenols or polyphenol derivatives may be useful in the prevention or cure of H . pylori-associated gastric diseases.

Oral Microbiol Immunol, 2003 Jun, 18(3), 135 - 9
Distribution of Actinobacillus actinomycetemcomitans serotypes and Porphyromonas gingivalis in Japanese adults; Yoshida Y et al.; Strains of the bacterium Actinobacillus actinomycetemcomitans found in the human oral cavity are divided into five serotypes, a, b, c, d, and e . In this study, A . actinomycetemcomitans serotypes and Porphyromonas gingivalis were isolated from 656 subgingival sites in systemically healthy Japanese adults . A . actinomycetemcomitans was detected in 19.5% of 328 Japanese subjects, while 27.1% of subjects were positive for P . gingivalis . Of 75 A . actinomycetemcomitans-positive sites, only one serotype was detected in 39 sites (52.0%) . The numbers of sites in which two different serotypes and three different serotypes were detected were 18 (25.0%) and 7 (9.3%), respectively . A . actinomycetemcomitans serotype c was detected more frequently in sites that were positive for both P . gingivalis and A . actinomycetemcomitans (76.9%) than in sites that were P . gingivalis-negative and A . actinomycetemcomitans-positive (33.9%) . In addition, serotype c was detected much more frequently than the other serotypes (<16%) in sites that were positive for both P . gingivalis and A . actinomycetemcomitans . These findings suggest that the characteristics of serotype c may differ from those of the other serotypes . This report is the first to use PCR to describe the distribution of A . actinomycetemcomitans serotypes in humans and to examine the association between the distribution of A . actinomycetemcomitans serotypes and the presence of P . gingivalis.

J Periodontal Res, 2003 Jun, 38(3), 318 - 23
The herpesvirus-Porphyromonas gingivalis-periodontitis axis; Slots J et al.; OBJECTIVES AND BACKGROUND: Members of the herpesvirus family have accumulated considerable support for a role in severe types of periodontitis . This study aimed to examine whether human cytomegalovirus (HCMV), Epstein-Barr virus type 1 (EBV-1) or herpes simplex virus (HSV) together with the major periodontopathic bacterium Porphyromonas gingivalis might interact in the pathogenesis of periodontal breakdown . METHODS: Sixteen subjects each contributed paper point samples from two progressing and two stable periodontitis lesions, as determined by ongoing loss of probing attachment . Polymerase chain reaction methodology was used to identify subgingival herpesviruses, P . gingivalis and other bacterial pathogens . Chi-squared tests and multivariate logistic regression were employed to identify statistical associations between herpesviruses, periodontopathic bacteria and clinical variables . RESULTS: HCMV and HSV were both significant predictors of the presence of subgingival P . gingivalis . In turn, P . gingivalis was positively associated with periodontitis active disease, probing attachment level, probing pocket depth, gingival bleeding upon probing and patient age . EBV-1 was not linked to P . gingivalis, although the virus was predictive of periodontitis active disease . The periodontitis disease risk associated with herpesvirus-P . gingivalis combinations depended on both site-specific and subject-specific factors . CONCLUSION: The present data of aggressive periodontitis implicate HCMV, HSV and P . gingivalis as either cofactors in its etiology or triggers of relapses . Further studies are needed to determine the spectrum of periodontopathogenicity of herpesviruses and effective management of these viruses in periodontal sites.

Eur J Biochem, 2003 May, 270(10), 2218 - 27
Accessory proteins functioning selectively and pleiotropically in the biosynthesis of {NiFe} hydrogenases in Thiocapsa roseopersicina; Maroti G et al.; There are at least two membrane-bound (HynSL and HupSL) and one soluble (HoxEFUYH) {NiFe} hydrogenases in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium . Genes coding for accessory proteins that participate in the biosynthesis and maturation of hydrogenases seem to be scattered along the chromosome . Transposon-based mutagenesis was used to locate the hydrogenase accessory genes . Molecular analysis of strains showing mutant phenotypes led to the identification of hupK (hoxV ), hypC1, hypC2, hypD, hypE, and hynD genes . The roles of hynD, hupK and the two hypC genes were investigated in detail . The putative HynD was found to be a hydrogenase-specific endoprotease type protein, participating in the maturation of the HynSL enzyme . HupK plays an important role in the formation of the functionally active membrane-bound {NiFe} hydrogenases, but not in the biosynthesis of the soluble enzyme . In-frame deletion mutagenesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme . The lack of either HypC protein drastically reduced the activity of every hydrogenase . Hence both HypCs might participate in the maturation of {NiFe} hydrogenases . Homologous complementation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H2 metabolism of T . roseopersicina.

Sao Paulo Med J, 2003 Jan 2, 121(1), 15 - 8 Epub 2003 Jul 04.
Therapeutic efficacy of ranitidine bismuth citrate with clarithromycin for seven days in the eradication of Helicobacter pylori in Brazilian peptic ulcer patients; Eisig JN et al.; CONTEXT: The curative treatment of peptic ulcer is made available nowadays through the eradication of the bacterium Helicobacter pylori, which is associated with it, but the best therapeutic regimen is yet to be determined . OBJECTIVE: To assess the efficacy of a therapeutic regimen with 400 mg ranitidine bismuth citrate associated with 500 mg clarithromycin given twice a day for seven days in a cohort of Brazilian patients with peptic ulcer . TYPE OF STUDY: Cross-sectional study . SETTING: Tertiary-care hospital . PATIENTS: One hundred and twenty nine outpatients, with active or healed peptic ulcers infected by Helicobacter pylori, diagnosed via endoscopy with confirmation via the urease test and histological examination, who had never undergone a regimen for the eradication of the bacterium . PROCEDURE: Administration of 400 mg ranitidine-bismuth and 500 mg clarithromycin twice a day, for seven days . MAIN MEASUREMENTS: Efficacy of the treatment, with a check on the cure done via another endoscopy eight weeks after drug administration . The eradication of the bacterium was determined via the urease test and histological examination . Patients who were negative for both were considered to be cured . RESULTS: Eight patients failed to complete the study . The eradication rate according to intention to treat was 81% (104/129) and per protocol was 86% (104/121) . CONCLUSION: The bismuth ranitidine compound associated with clarithromycin used for one week was shown to be a simple, effective and well-tolerated therapeutic regimen for the eradication of Helicobacter pylori.

DNA Seq, 2003 Feb, 14(1), 53 - 9
Isolation, sequencing, and characterization of the cytochrome bo operon from Vitreoscilla; Hwang KW et al.; The entire operon encoding the sodium pumping cytochrome bo from the bacterium Vitreoscilla was isolated and sequenced, and this sequence was analyzed by blast and hydropathy plots . There are fairly similar phylogenetic relationships which apply to all five proteins, but overall greater similarity to members of the gamma subdivision than the beta subdivision of the Proteobacteria . Hydropathy plots of all five Cyo proteins show near identity with those of the corresponding E . coli subunits, indicating that the similarity extends from sequence to structure . The operon appears to have a typical Shine-Dalgarno sequence, an E . coli-like promoter, and several possible binding sites for regulatory proteins . The Vitreoscilla Cyo B subunit (the probable Na+ pump) is almost identical to E . coli Cyo B at 18 key amino acids; thus, there are no obvious changes in Vitreoscilla Cyo B that hint at the details of its Na+ pumping ability.

Genetics, 2003 May, 164(1), 5 - 12
Male-killing Wolbachia and mitochondrial DNA: selective sweeps, hybrid introgression and parasite population dynamics; Jiggins FM; Mitochondrial DNA (mtDNA) sequences are widely used as neutral genetic markers in insects . However, patterns of mtDNA variability are confounded by the spread of maternally transmitted parasites, which are genetically linked to the mitochondria . We have investigated these effects in the butterflies Acraea encedon (which is host to two strains of male-killing Wolbachia bacteria) and A . encedana (which is host to one strain) . Within a population, the mitochondria are in linkage disequilibrium with the different male-killers . Furthermore, there has been a recent selective sweep of the mtDNA, which has led to the loss of mitochondrial variation within populations and erased any geographical structure . We also found that one of the male-killers, together with the associated mtDNA, has introgressed from A . encedana into A . encedon within the last 16,000 years . Interestingly, because butterflies are female heterogametic, this will presumably have also led to the introgression of genes on the W sex chromosome . Finally, in A . encedon the mitochondria in uninfected females are unaltered by the spread of the male-killer and have diverse, geographically structured mtDNA . This means we can reject the hypothesis that the male-killer is at a stable equilibrium maintained by imperfect transmission of the bacterium . Instead, some other form of balancing selection may be maintaining uninfected females in the population and preventing the species from going extinct due to a shortage of males.

Syst Appl Microbiol, 2003 Mar, 26(1), 3 - 12
Analysis of conserved non-rRNA genes of Tropheryma whipplei; Maiwald M et al.; The causative agent of Whipple's disease, Tropheryma whipplei, is a slow-growing bacterium that remains poorly-understood . Genetic characterization of this organism has relied heavily upon rRNA sequence analysis . Pending completion of a complete genome sequencing effort, we have characterized several conserved non-rRNA genes from T . whipplei directly from infected tissue using broad-range PCR and a genome-walking strategy . Our goals were to evaluate its phylogenetic relationships, and to find ways to expand the strain typing scheme, based on rDNA sequence comparisons . The genes coding for the ATP synthase beta subunit (atpD), elongation factor Tu (tuf), heat shock protein GroEL (groEL), beta subunit of DNA-dependent RNA polymerase (rpoB), and RNase P RNA (rnpB) were analyzed, as well as the regions upstream and downstream of the rRNA operon . Phylogenetic analyses with all non-rRNA marker molecules consistently placed T . whipplei within the class, Actinobacteria . The arrangement of genes in the atpD and rpoB chromosomal regions was also consistent with other actinomycete genomes . Tandem sequence repeats were found upstream and downstream of the rRNA operon, and downstream of the groEL gene . These chromosomal sites and the 16S-23S rRNA intergenic spacer regions were examined in the specimens of 11 patients, and a unique combination of tandem repeat numbers and spacer polymorphisms was found in each patient . These data provide the basis for a more discriminatory typing method for T . whipplei.

Arch Biochem Biophys, 2003 Jun 1, 414(1), 51 - 8
Heterologous expression, purification, and enzymatic characterization of the acyclic carotenoid 1,2-hydratase from Rubrivivax gelatinosus; Steiger S et al.; The carotenoid 1,2-hydratase CrtC from Rubrivivax gelatinosus has been expressed in Escherichia coli in an active form and purified by affinity chromatography . The enzyme catalyzes the conversion of various acyclic carotenes including 1-hydroxy derivatives . This broad substrate specificity reflects the participation of CrtC in 1'-HO-spheroidene and in spirilloxanthin biosynthesis . Enzyme kinetic studies including the determination of substrate specificity constants indicate that among the alternative biosynthetic routes to 1'-HO-spheroidene the one via spheroidene is the dominating pathway . In contrast to CrtC from Rvi . gelatinosus, the equivalent enzyme from Rhodobacter capsulatus, a closely related bacterium which lacks the biosynthetic branch to spirilloxanthin and accumulates spheroidene instead of substantial amounts of 1'-HO-spheroidene, is extremely poor in converting 1-HO-carotenoids . The individual catalytic properties of both carotenoid 1,2-hydratases reflect the in situ carotenogenic pathways in both purple photosynthetic bacteria.

Parasitol Res, 2003 May, 90(1), 38 - 47 Epub 2003 Jan 29.
An aspartate aminotransferase of Wolbachia endobacteria from Onchocerca volvulus is recognized by IgG1 antibodies from residents of endemic areas; Fischer P et al.; Wolbachia are intracellular alpha-proteobacteria, closely related to Rickettsia, that infect various arthropods and filarial parasites . In the present study, the cDNA encoding the aspartate aminotransferase (AspAT) of Wolbachia from the human pathogenic filarial parasite Onchocerca volvulus (Ov-WolAspAT) was identified . At the amino acid level, the identity of the Ov-WolAspAT was 56% to Rickettsia prowazekii AspAT and 54% to the AspAT of the nitrogen-fixing bacterium Sinorhizobium meliloti, but the highest degree of identity was found to the putative AspAT of Wolbachia from Brugia malayi and Drosophila melanogaster (85%) . All of these bacterial AspATs are members of the AspAT subclass Ib . A 35 kDa fragment of the Ov-WolAspAT was expressed in Escherichia coli, and immunolocalization using polyclonal antibodies against this antigen revealed that Ov-WolAspAT is present in a considerable proportion of the Wolbachia from O . volvulus, as well as in the endobacteria of several other filarial parasites . Western blot analysis using recombinant Ov-WolAspAT as antigen showed that IgG1 antibodies were present in 70 (51%) individuals living in areas endemic for O . volvulus, B . malayi or Wuchereria bancrofti and no IgG4 or IgE antibodies were found . Among 40 sera of persons from Uganda and Liberia who were putatively not infected with human filarial parasites, 11 (28%) individuals presented IgG1 antibodies, while none of the 33 sera from healthy Europeans and none of the 14 sera from patients with proven Rickettsia or Brucella infections reacted with the antigen . These results also show that an intracellular protein of Wolbachia endobacteria (WolAspAT) acts as antigen in human filariasis.

Transplant Proc, 2003 May, 35(3 Suppl), 227S - 230S
Molecular actions of sirolimus: sirolimus and mTor; Kirken RA et al.; Recent therapeutic strategies to combat organ allograft rejection have focused on T-cell signaling pathways and the molecules that comprise them . The macrolide antibiotic produced by the bacterium Streptomyces hygroscopicus, known as sirolimus or rapamycin, has shown great therapeutic potential in the transplant setting . Sirolimus alone or in combination with other immunosuppressive agents can block acute rejection, chronic graft destruction, and promote permanent allograft acceptance . Sirolimus targets a unique serine-threonine kinase, mammalian target of rapamycin (mTor), which plays a key role in mitogenic and nutritional cells signals . Within T cells, mTor regulates a number of proteins likely dependent on T cell growth factors such as interleukin 2 . This review is focused on the molecular mechanisms by which mTor may regulate T-cell signaling cascades and affect T-cell responsiveness, and how sirolimus likely uncouples this activity.

Biomacromolecules, 2003 May-Jun, 4(3), 850 - 5
Directed capture of enzymes and bacteria on bioplastic films; Koepsel RR et al.; The development of smart coatings for a variety of uses depends on the ability of the coating material to perform specific functions . We have used water dispersible polyurethane preparations for the immobilization of binding proteins under mild conditions . In these experiments, antibodies against the enzyme beta-galactosidase or the bacterium Escherichia coli were immobilized in polyurethane coatings and then used to effectively capture their cognate antigen . Further, a second, more general, capture protocol was developed which involves the incorporation of the protein avidin in the plastics . This system efficiently captures biotinylated beta-galactosidase . Biotinylated anti-E . coli antibody captured by avidin bioplastics resulted in a nearly 5-fold increase in the number of bound bacteria when compared to blank polyurethane . The use of avidin in a bioplastic allows any biotinylated antibody to be applied to all or part of the surface resulting in a patterning of capture agents on a preformed surface.

Drugs Today (Barc), 2001 Nov, 37(11), 767 - 781
Esomeprazole: A significant advance beyond omeprazole in the treatment of acid-related disease; Rabasseda X et al.; The second-generation proton pump inhibitor (PPI) esomeprazole is a new chemical entity consisting of an optical isomer of omeprazole, which for many years has been acknowledged as the gold standard therapy in gastric acid-related disorders . Esomeprazole has demonstrated a unique pharmacokinetic profile and enhanced efficacy over omeprazole, with improved inter-patient pharmacokinetic consistency and a similar safety profile . Esomeprazole has been tested as a therapeutic agent in the management of erosive esophagitis, symptomatic gastroesophageal reflux disease (GERD) and, in combination with appropriate antibiotic therapy, for the eradication of the Helicobacter pylori bacterium and healing of H . pylori-associated duodenal ulcers . In clinical studies, esomeprazole has shown greater efficacy than omeprazole with a comparable low incidence of adverse events . (c) 2001 Prous Science . All rights reserved.

FEMS Immunol Med Microbiol, 2003 May 25, 36(3), 127 - 34
Interactions of the gastrotropic bacterium Helicobacter pylori with the leukocyte-endothelium adhesion molecules, the selectins--a preliminary report; Galustian C et al.; The deleterious effects of Helicobacter pylori infection of the stomach are largely the result of a vigorous chronic inflammatory response, and include chronic gastritis, peptic ulceration and gastric cancer . We are exploring the possibility that carbohydrate components on H . pylori contribute to the persistent inflammation through interactions with leukocyte-endothelial adhesion molecules of the host . Lipopolysaccharides of most H . pylori strains contain sequences related to the Lewis (Le(x) or Le(a)) antigens . Carbohydrate sequences of this family encompass ligands for the leukocyte-endothelium adhesion molecules of the host, namely, the E- and P-selectins, which are expressed on inflamed endothelia, and L-selectin, which is constitutively expressed on leukocytes . Here we investigate H . pylori isolates from patients with chronic gastritis, duodenal ulcer and gastric cancer for their interactions with the selectins . Our results provide unequivocal evidence of interactions of isolates from each of the diagnostic groups with E- and L-selectins.

Structure (Camb), 2003 May, 11(5), 547 - 55
The structure of Acidithiobacillus ferrooxidans c(4)-cytochrome: a model for complex-induced electron transfer tuning; Abergel C et al.; The study of electron transfer between the copper protein rusticyanin (RCy) and the c(4)-cytochrome CYC(41) of the acidophilic bacterium Acidithiobacillus ferrooxidans has evidenced a remarkable decrease of RCy's redox potential upon complex formation . The structure of the CYC(41) obtained at 2.2 A resolution highlighted a specific glutamate residue (E121) involved in zinc binding as potentially playing a central role in this effect, required for the electron transfer to occur . EPR and stopped-flow experiments confirmed the strong inhibitory effect of divalent cations on CYC(41):RCy complex formation . A docking analysis of the CYC(41) and RCy structure allows us to propose a detailed model for the complex-induced tuning of electron transfer in agreement with our experimental data, which could be representative of other copper proteins involved in electron transfer.

Ear Nose Throat J, 2003 Apr, 82(4), 263 - 5
Tularemia of the head and neck: a possible sign of bioterrorism; Stupak HD et al.; Recent bioterror attacks and other world events have focused the medical community's attention on agents that might be used in biological warfare . One of these potential biological weapons is Francisella tularensis, a gramnegative coccobacillus that is one of the most infectious bacteria known . F tularensis can cause severe, even fatal, systemic tularemia . Under normal circumstances, F tularensis is transmitted by infected ticks, insects, and other animals . As a weapon of terrorism, the bacterium would likely be disseminated as an aerosol and contracted by inhalation . Because many cases of tularemia are characterized by head and neck symptoms, otolaryngologists should be familiar with the diagnosis and management of this disease . In this article, we describe a case of zoonotic tularemia that manifested as a neck mass, and we review the pathophysiology, diagnosis, and treatment of tularemia . We also summarize what is known about its potential as a biological weapon.

J Clin Microbiol, 2003 May, 41(5), 1869 - 74
Evaluation of Coxiella burnetii antibiotic susceptibilities by real-time PCR assay; Brennan RE et al.; Coxiella burnetii is an obligate intracellular bacterium . The inability to cultivate this organism on axenic medium has made calculation of infectious units challenging and prevents the use of conventional antibiotic susceptibility assays . A rapid and reliable real-time PCR assay was developed to quantify C . burnetii cells from J774.16 mouse macrophage cells and was applied to antibiotic susceptibility testing of C . burnetii Nine Mile, phase I . For calculation of bacterial replication, real-time PCR performed equally as well as immunofluorescent-antibody (IFA) assay when J774.16 cells were infected with 10-fold serial dilutions of C . burnetii and was significantly (P < 0.05) more repeatable than IFA when 2-fold dilutions were used . Newly infected murine macrophage-like J774.16 cells were treated with 8 microg of chloramphenicol per ml, 4 microg of tetracycline per ml, 4 microg of rifampin per ml, 4 microg of ampicillin per ml, or 1 microg of ciprofloxacin per ml . After 6 days of treatment, tetracycline, rifampin, and ampicillin significantly (P < 0.01) inhibited the replication of C . burnetii, while chloramphenicol and ciprofloxacin did not . In general, these results are consistent with those from prior reports on the efficacy of these antibiotics against C . burnetii Nine Mile, phase I, and indicate that a real-time PCR-based assay is an appropriate alternative to the present methodology for evaluation of the antibiotic susceptibilities of C . burnetii.

Genome Biol . 2003;4(5):213 . Epub 2003 Apr 28.
Discovering human history from stomach bacteria; Disotell TR; Recent analyses of human pathogens have revealed that their evolutionary histories are congruent with the hypothesized pattern of ancient and modern human population migrations . Phylogenetic trees of strains of the bacterium Helicobacter pylori and the polyoma JC virus taken from geographically diverse groups of human beings correlate closely with relationships of the populations in which they are found.

Curr Microbiol, 2003 May, 46(5), 329 - 33
Nitrite as an energy-conserving electron sink for the acetogenic bacterium Moorella thermoacetica; Seifritz C et al.; Nitrite served as an energy-conserving electron acceptor for the acetogenic bacterium Moorella thermoacetica . Growth occurred in an undefined (0.1% yeast extract) medium containing 20 m M glyoxylate and 5 m M nitrite and was essentially equivalent to that observed in the absence of nitrite . In the presence of nitrite, acetate (the normal product of glyoxylate-derived acetogenesis) was not detected during growth . Instead, growth was coupled to nitrite dissimilation to ammonium, and acetogenesis was limited to the stationary phase . Furthermore, membranes from glyoxylate-grown cells under nitrite-dissimilating conditions were deficient in the b-type cytochrome that is typically found in the membranes of acetogenic cells . Unlike glyoxylate, other acetogenic substrates (fructose, oxalate, glycolate, vanillin, and hydrogen) were not growth supportive in the undefined medium containing nitrite, and glyoxylate-dependent growth did not occur in a nitrite-supplemented, basal (without yeast extract) medium . Glyoxylate-dependent growth by Moorella thermoautotrophica was not observed in the undefined medium containing nitrite.

J Biol Chem, 2003 Jul 18, 278(29), 27059 - 67 Epub 2003 May 05.
Crystal structure of fungal lectin: six-bladed beta-propeller fold and novel fucose recognition mode for Aleuria aurantia lectin; Wimmerova M et al.; Aleuria aurantia lectin is a fungal protein composed of two identical 312-amino acid subunits that specifically recognizes fucosylated glycans . The crystal structure of the lectin complexed with fucose reveals that each monomer consists of a six-bladed beta-propeller fold and of a small antiparallel two-stranded beta-sheet that plays a role in dimerization . Five fucose residues were located in binding pockets between the adjacent propeller blades . Due to repeats in the amino acid sequence, there are strong similarities between the sites . Oxygen atoms O-3, O-4, and O-5 of fucose are involved in hydrogen bonds with side chains of amino acids conserved in all repeats, whereas O-1 and O-2 interact with a large number of water molecules . The nonpolar face of each fucose residue is stacked against the aromatic ring of a Trp or Tyr amino acid, and the methyl group is located in a highly hydrophobic pocket . Depending on the precise binding site geometry, the alpha- or beta-anomer of the fucose ligand is observed bound in the crystal . Surface plasmon resonance experiments conducted on a series of oligosaccharides confirm the broad specificity of the lectin, with a slight preference for alphaFuc1-2Gal disaccharide . This multivalent carbohydrate recognition fold is a new prototype of lectins that is proposed to be involved in the host recognition strategy of several pathogenic organisms including not only the fungi Aspergillus but also the phytopathogenic bacterium Ralstonia solanacearum.

Appl Environ Microbiol, 2003 May, 69(5), 2906 - 13
Isolation and characterization of novel psychrophilic, neutrophilic, Fe-oxidizing, chemolithoautotrophic alpha- and gamma-proteobacteria from the deep sea; Edwards KJ et al.; We report the isolation and physiological characterization of novel, psychrophilic, iron-oxidizing bacteria (FeOB) from low-temperature weathering habitats in the vicinity of the Juan de Fuca deep-sea hydrothermal area . The FeOB were cultured from the surfaces of weathered rock and metalliferous sediments . They are capable of growth on a variety of natural and synthetic solid rock and mineral substrates, such as pyrite (FeS(2)), basalt glass ( approximately 10 wt% FeO), and siderite (FeCO(3)), as their sole energy source, as well as numerous aqueous Fe substrates . Growth temperature characteristics correspond to the in situ environmental conditions of sample origin; the FeOB grow optimally at 3 to 10 degrees C and at generation times ranging from 57 to 74 h . They are obligate chemolithoautotrophs and grow optimally under microaerobic conditions in the presence of an oxygen gradient or anaerobically in the presence of nitrate . None of the strains are capable of using any organic or alternate inorganic substrates tested . The bacteria are phylogenetically diverse and have no close Fe-oxidizing or autotrophic relatives represented in pure culture . One group of isolates are gamma-Proteobacteria most closely related to the heterotrophic bacterium Marinobacter aquaeolei (87 to 94% sequence similarity) . A second group of isolates are alpha-Proteobacteria most closely related to the deep-sea heterotrophic bacterium Hyphomonas jannaschiana (81 to 89% sequence similarity) . This study provides further evidence for the evolutionarily widespread capacity for Fe oxidation among bacteria and suggests that FeOB may play an unrecognized geomicrobiological role in rock weathering in the deep sea.

Int J Hematol, 2003 Apr, 77(3), 239 - 44
Can eradication therapy for Helicobacter pylori really improve the thrombocytopenia in idiopathic thrombocytopenic purpura? Our experience and a literature review; Ando K et al.; Helicobacter pylori has recently been postulated to play a role in the pathogenesis of autoimmune diseases, including idiopathic thrombocytopenic purpura (ITP) . We investigated the prevalence of H pylori infection and the effects of its eradication in 61 patients with ITP . H pylori infection was found in 50 patients (83%), an incidence significantly higher than not only healthy volunteers in Japan (60%) but also subjects in other reported ITP series (approximately 43%-71%) . In our study, the mean age of H pylori-positive ITP patients (58.0 years) was significantly higher than that of H pylori-negative ITP patients (40.5 years) . Bacterium eradication efforts were performed in 29 infected ITP patients and succeeded in 27 patients (93%) . The 29 patients with eradicated H pylori infections showed significant increases in platelet counts compared with patients with uneradicated infections or who were H pylori-negative . During the follow-up period (median, 11.0 months), 16 (55%) of 29 patients achieved a major or a minor response . The patients who achieved a major response had not received previous prednisolone therapy, suggesting a relationship between prednisolone therapy and the response to eradication efforts . The assessment of H pylori infection and its eradication should be attempted in cases of ITP, because this approach may be a good new strategy for treating some ITP patients, especially elderly Japanese patients . Some regional factors have been suggested as causes of H pylori-associated ITP.

J Bacteriol, 2003 May, 185(10), 3223 - 7
The single superoxide dismutase of Rhodobacter capsulatus is a cambialistic, manganese-containing enzyme; Tabares LC et al.; The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SOD(Rc)) homologous to iron-containing superoxide dismutase enzymes . Recombinant SOD(Rc), however, displayed higher activity after refolding with Mn(2+), especially when the pH of the assay mixture was raised . SOD(Rc) isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures . Therefore, SOD(Rc) behaves as a Mn-containing dismutase with cambialistic properties.

J Bacteriol, 2003 May, 185(10), 3031 - 5
S-adenosylmethionine transport in Rickettsia prowazekii; Tucker AM et al.; Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells . Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm . Analysis of the R . prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet) . Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter . We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified . The influx of AdoMet into rickettsiae was a saturable process with a K(T) of 2.3 micro M . Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine . Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide . AdoMet transporters with similar properties were also identified in the Breinl strain of R . prowazekii and in Rickettsia typhi . By screening Escherichia coli clone banks for AdoMet transport, the R . prowazekii gene coding for a transporter, RP076 (sam), was identified . AdoMet transport in E . coli containing the R . prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae . The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.

Protein Expr Purif, 2003 May, 29(1), 33 - 41
An exotype alginate lyase in Sphingomonas sp . A1: overexpression in Escherichia coli, purification, and characterization of alginate lyase IV (A1-IV); Miyake O et al.; Sphingomonas sp . A1 (strain A1) cells contain three kinds of endotype alginate lyases {A1-I, A1-II, and A1-III}, all of which are formed from a common precursor through posttranslational processing . In addition to these lyases, another type of lyase (A1-IV) that acts on oligoalginates exists in the bacterium . A1-IV was overexpressed in Escherichia coli cells through control of its gene under the T7 promoter . The expression level of the enzyme in E . coli cells was 8.6U/L-culture, which was about 270-fold higher than that in strain A1 cells . The enzyme was purified to homogeneity through three steps with an activity yield of 10.9% . The optimal pH and temperature, thermal stability, and mode of action of the purified enzyme were similar to those of the native enzyme from strain A1 cells . A1-IV exolytically degraded oligoalginates, which were produced from alginate through the reaction of A1-I, A1-II, or A1-III, into monosaccharides, indicating that the cooperative actions of these four enzymes cause the complete depolymerization of alginate in strain A1 cells.

FEMS Microbiol Lett, 2003 Apr 25, 221(2), 191 - 6
Novel precursor substrates for polythioesters (PTE) and limits of PTE biosynthesis in Ralstonia eutropha; Lutke-Eversloh T et al.; A novel class of biopolymers referred to as polythioesters (PTE) was recently detected when the polyhydroxyalkanoate (PHA) accumulating bacterium Ralstonia eutropha was cultivated in the presence of 3-mercaptopropionic acid (3MP) or 3,3'-thiodipropionic acid (TDP) . In this study, 3,3'-dithiodipropionic acid (DTDP) and 3-mercaptovaleric acid (3MV) were identified as two additional precursor carbon sources for in vivo biosynthesis of PTE in R . eutropha . Biosynthesis of copolymers of 3-hydroxybutyrate (3HB) and 3MP, which contributed 19-25% of cell dry matter, was compared referring to the different precursor substrates . Using DTDP as carbon source, which is probably cleaved into two molecules 3MP, yielded an about 2.3-fold higher molar 3MP content of the copolyester than TDP, which is probably cleaved into only one molecule 3MP . Furthermore, cultivation of R . eutropha in the presence of 3MV resulted in biosynthesis of copolymers consisting predominantly of 3HB with low amounts of 3MV and 3-hydroxyvalerate, each contributing less than 5 mol% of the constituents . In contrast, 4-mercaptobutyric acid could be not incorporated into PHAs, although - as documented in this study - five different strategies, various precursor substrates, R . eutropha and also a recombinant strain of Escherichia coli were employed . Therefore, this study not only extended the range of substrates suitable for PTE biosynthesis and also the range of PTE constituents in R . eutropha, it also demonstrates limits for PTE biosynthesis in this bacterium.

Methods, 2003 Mar, 29(3), 236 - 47
Use of combinatorial peptide libraries for T-cell epitope mapping; Sospedra M et al.; T lymphocytes play important roles not only in infectious diseases and autoimmunity, but also in immune responses against tumors . For many of these disorders, the relevant target antigens are not known . Designing effective methods that allow the search for T-cell epitopes is therefore an important goal in the areas of infectious diseases, oncology, vaccine development, and numerous other biomedical specialties . So far, the strategies used to examine T-cell recognition have been based largely on mapping T-cell epitopes with overlapping peptides from known proteins or with entire proteins, e.g., from a specific virus, bacterium, or human tissue . These approaches are tedious and have a number of limitations . It is, for example, almost impossible to isolate T cells that infiltrate an organ or infectious site and identify their specificity unless one already has a concept as to which antigens may be relevant . During recent years, a number of laboratories have developed less biased approaches that employ either the selection of putative T-cell epitopes based on the prediction of binding to certain major histocompatibilty complex (MHC) molecules and peptide or protein libraries that have been generated in expression systems, e.g . phage, or rely on combinatorial peptide chemistry . The latter technique has been refined by a number of laboratories including ours . Bead-bound or, preferably, positional scanning synthetic and soluble combinatorial peptide libraries allow the identification of T-cell epitopes within complex mixtures of proteins even for T cells that have been expanded from an organ infiltrate with a polyclonal stimulus . The practical steps that are involved in the latter method are described in this article.

Prikl Biokhim Mikrobiol, 2003 Mar-Apr, 39(2), 189 - 93
{Kinetic parameters of a culture of the hydrogen-oxidizing Ralstonia eutropha, grown under the regimen of biosynthesis of polyhydroxybutyrate}; Volova TG et al.; Kinetic parameters of a culture of the hydrogen-oxidizing bacterium Ralstonia eutropha, grown on a gas substrate under the conditions favoring autotrophic biosynthesis of polyhydroxybutyrate, were studied . The following parameters, making it possible to control and optimize the process in industrial situations, were determined: specific rate of substrate consumption, physical properties of culture medium, and coefficients of heat emission and mass transfer.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 247 - 54
Saccharification of marine microalgae using marine bacteria for ethanol production; Matsumoto M et al.; The saccharification of marine microalgae using amylase from marine bacteria in saline conditions was investigated . An amylase-producing bacterium, Pseudoalterimonas undina NKMB 0074 was isolated and identified . The green microalga NKG 120701 was determined to have the highest concentration of intracellular carbohydrate and was found from our algal culture stocks . P . undina NKMB 0074 was inoculated into suspensions containing NKG 120701 cells and increasingly reduced suspended sugars with incubation time . Terrestrial amylase and glucoamylase were inactive in saline suspension . Therefore, marine amylase is necessary in saline conditions for successful saccharification of marine microalgae.

Dis Aquat Organ, 2003 Mar 17, 54(1), 43 - 8
Experimental infection of Penaeus vannamei by a rickettsia-like bacterium (RLB) originating from P . monodon; Nunan LM et al.; A rickettsia-like bacterium (RLB), which caused severe mortalities of commercially farmed Penaeus monodon in the southwest region of Madagascar, was investigated to determine whether the organism would produce the same disease in P . vannamei . Two series of bioassays were performed to determine whether this RLB could be transmitted to P . vannamei through injection and per os exposure . The first series of challenge bioassays used frozen, RLB-infected P . monodon tissue from Madagascar as the inoculum and feed for the injection, and per os bioassays with specific pathogen free (SPF) P . vannamei . In the second series of bioassays, frozen RLB-infected P . vannamei tissue derived from the first series of injection bioassays was used as the inoculum to challenge by injection and per os SPF P . vannamei . Disease status was determined through standard histological techniques and by in situ hybridization assays with a digoxigenin-labeled probe specific for this RLB . The results indicated that P . vannamei did develop the RLB infection when injected with either RLB infected P . monodon or P . vannamei tissue homogenates . This contrasts with results from the per os exposure to the RLB in which the disease could not be reproduced.

Can J Microbiol, 2003 Feb, 49(2), 92 - 100
Algicidal activity and gliding motility of Saprospira sp . SS98-5; Furusawa G et al.; A marine bacterium, Saprospira sp . SS98-5, which was isolated from Kagoshima Bay, Japan, was able to kill and lyse the cells of the diatom Chaetoceros ceratosporum . The multicellular filamentous cells of this bacterium captured the diatom cells, formed cell aggregates, and lysed them in an enriched sea water (ESS) liquid medium . Strain SS98-5 also formed plaques on double layer agar plates incorporating diatom cells . The diatom cell walls were partially degraded at the contact sites with the bacteria, the bacteria invaded from there into the diatom cells, and then the diatom cells were completely lysed . The strain possessed gliding motility and grew as spreading colonies on ESS agar plates containing lower concentrations of polypeptone (below 0.1%) while forming nonspreading colonies on ESS agar plates containing 0.5% polypeptone . Electron micrographs of ultrathin sections demonstrated that microtubule-like structures were observable only in gliding motile cells . Both the gliding motility and the microtubule-like structures were diminished by the addition of podophyllotoxin, an inhibitor of microtubule assembly, suggesting that the microtubule-like structures observed in these bacterial cells are related to their gliding motility.

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 603 - 6
Actinomyces vaccimaxillae sp . nov., from the jaw of a cow; Hall V et al.; A previously undescribed Actinomyces-like bacterium was isolated from a lesion in the jaw of a cow . Based on its cellular morphology and the results of biochemical testing, the organism was tentatively identified as a member of the genus Actinomyces . Comparative 16S rRNA gene sequencing studies showed that the bacterium represents a hitherto unknown species within the genus Actinomyces, and is related to a group of species that includes Actinomyces turicensis and its close relatives . It is proposed that the unknown organism be classified as Actinomyces vaccimaxillae sp . nov . (the type strain is CCUG 46091T =CIP 107423T).

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 527 - 32
Psychromonas profunda sp . nov., a psychropiezophilic bacterium from deep Atlantic sediments; Xu Y et al.; A psychropiezophilic bacterium, strain 2825T (=LMG 21260T =JCM 11437T), isolated from deep Atlantic sediments at a depth of 2770 m and a temperature of 2 degrees C, was found by polyphasic analysis to represent a novel species of the genus Psychromonas, Psychromonas profunda sp . nov . It is a strict psychrophile and a moderate piezophile, whose degree of piezophily is increased markedly when the temperature is raised to 10 degrees C . The piezophily of P . profunda is intermediate between that of the type species, Psychromonas antarctica, which is not piezophilic, and that of Psychromonas kaikoae, which is an obligate piezophile.

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 473 - 7
Sphingopyxis chilensis sp . nov., a chlorophenol-degrading bacterium that accumulates polyhydroxyalkanoate, and transfer of Sphingomonas alaskensis to Sphingopyxis alaskensis comb . nov; Godoy F et al.; The taxonomic position of a chlorophenol-degrading bacterium, strain S37T, was investigated . The 16S rDNA sequence indicated that this strain belongs to the genus Sphingopyxis, exhibiting high sequence similarity to the 16S rDNA sequences of Sphingomonas alaskensis LMG 18877T (98.8%), Sphingopyxis macrogoltabida LMG 17324T (98.2%), Sphingopyxis terrae IFO 15098T (95%) and Sphingomonas adhaesiva GIFU 11458T (92%) . These strains (except Sphingopyxis terrae IFO 15098T, which was not investigated) and the novel isolate accumulated polyhydroxyalkanoates consisting of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from glucose as carbon source . The G + C content of the DNA of strain S37T was 65.5 mol% . The major cellular fatty acids of this strain were octadecenoic acid (18 : 1omega7c), heptadecenoic acid (17 : 1omega6c) and hexadecanoic acid (16 : 0) . The results of DNA-DNA hybridization experiments and its physiological characteristics clearly distinguished the novel isolate from all known Sphingopyxis species and indicated that the strain represents a novel Sphingopyxis species . Therefore, the species Sphingopyxis chilensis sp . nov . is proposed, with strain S37T (=LMG 20986T =DSM 14889T) as the type strain . The transfer of Sphingomonas alaskensis to the genus Sphingopyxis as Sphingopyxis alaskensis comb . nov . is also proposed.

Biosens Bioelectron, 2003 May, 18(5-6), 599 - 603
Rapid and specific detection of herbicides using a self-assembled photosynthetic reaction center from purple bacterium on an SPR chip; Nakamura C et al.; In this study, a direct detection system for herbicides inhibiting photosynthetic electron transfer was developed using the photosynthetic reaction center (RC) from the purple bacterium, Rhodobacter sphaeroides, and surface plasmon resonance (SPR) apparatus . The heavy-subunit-histidine-tagged RCs (HHisRCs) were immobilized on an SPR sensor chip via nickel chelation chemistry as a binder for one of the triazine herbicides, atrazine . Immediately after injection of atrazine solution on the HHisRCs-immobilized chip, the SPR responses increased and reached plateaus within 1 min . The SPR signals were proportional to the sample concentrations of atrazine in the range 1-100 microg/ml . To evaluate the binding specificity to atrazine, chlorinated aromatic herbicides, DCMU and MCPP, were investigated using the HHisRCs-immobilized chip . An RC inhibitor, DCMU, could also be detected with a higher detection limit of 20 microg/ml than atrazine (1 microg/ml) . MCPP showed no signals because its inhibition mechanism against plants is different from that of atrazine and DCMU . These results indicated that the sensor chip immobilized RCs could be used for the specific detection of photosynthetic inhibitors.

Biosens Bioelectron, 2003 May, 18(5-6), 571 - 7
Monitoring and classification of PAH toxicity using an immobilized bioluminescent bacteria; Lee HJ et al.; An immobilized recombinant bioluminescent Escherichia coli strain, harboring a lac::luxCDABE fused plasmid, which shows lower bioluminescence levels when cellular metabolism is inhibited, was used to monitor the cellular toxicity of polycyclic aromatic hydrocarbons (PAHs) . PAHs, classified as pericondensed (PCPAHs) or catacondensed (CCPAHs) according to their molecular structures, were differentiable according to the response of this biosensor . Only CCPAHs were found to cause cellular toxicity, resulting in a dose-dependent decrease in the bioluminescent output . The induction of cellular toxicity by CCPAHs and PCPAHs was compared with acute toxicity predictions obtained using the quantitative structure-activity relationship (QSAR) model . A good relationship was obtained between the toxicities determined with the bioluminescent response of the immobilized bacterium GC2 and the QSAR model . It was also found that the present study offers a new method of predicting the cellular toxicities of CCPAHs or PCPAHs using this biosensor.

Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5455 - 60 Epub 2003 Apr 18.
Complete genome sequence of the Q-fever pathogen Coxiella burnetii; Seshadri R et al.; The 1,995,275-bp genome of Coxiella burnetii, Nine Mile phase I RSA493, a highly virulent zoonotic pathogen and category B bioterrorism agent, was sequenced by the random shotgun method . This bacterium is an obligate intracellular acidophile that is highly adapted for life within the eukaryotic phagolysosome . Genome analysis revealed many genes with potential roles in adhesion, invasion, intracellular trafficking, host-cell modulation, and detoxification . A previously uncharacterized 13-member family of ankyrin repeat-containing proteins is implicated in the pathogenesis of this organism . Although the lifestyle and parasitic strategies of C . burnetii resemble that of Rickettsiae and Chlamydiae, their genome architectures differ considerably in terms of presence of mobile elements, extent of genome reduction, metabolic capabilities, and transporter profiles . The presence of 83 pseudogenes displays an ongoing process of gene degradation . Unlike other obligate intracellular bacteria, 32 insertion sequences are found dispersed in the chromosome, indicating some plasticity in the C . burnetii genome . These analyses suggest that the obligate intracellular lifestyle of C . burnetii may be a relatively recent innovation.

Infect Immun, 2003 May, 71(5), 2643 - 55
Gene expression profiling of Helicobacter pylori reveals a growth-phase-dependent switch in virulence gene expression; Thompson LJ et al.; The global pattern of growth-phase-dependent gene expression of Helicobacter pylori during in vitro culture was analyzed by using a high-density DNA microarray . To detect consistent coordinated gene expression in this bacterium, temporal changes in transcription were assessed in two independent time courses . Cluster analysis of the expression profiles highlighted a major switch in gene expression during the late log-to-stationary phase transition that we have termed the Log-Stat switch . Statistical analysis of the genes that were significantly induced or repressed during the Log-Stat switch revealed that many of these genes were related to virulence . Among these, expression of the genes for the neutrophil activating protein (napA) and the major flagellin subunit (flaA) were significantly induced . Additionally, the expression of a number of genes involved in iron homeostasis changed dramatically at this switch; the gene for the iron-storage protein, pfr, was induced, while the genes for two putative iron uptake proteins, fecA and frpB, were significantly repressed . These data suggest that the late log phase may correspond to the most virulent phase of growth in H . pylori and may be intimately related to its pathogenesis . The use of microarrays to analyze the kinetics of the transcriptional response of a bacterial pathogen to a changing environment has enabled the discovery of previously unappreciated relationships between genes by elucidation of coordinated gene expression profiles.

Infect Immun, 2003 May, 71(5), 2394 - 403
lvgA, a novel Legionella pneumophila virulence factor; Edelstein PH et al.; Several novel Legionella pneumophila virulence genes were previously discovered by use of signature-tagged mutagenesis (P . H . Edelstein, M . A . Edelstein, F . Higa, and S . Falkow, Proc . Natl . Acad . Sci . 96:8190-8195, 1999) . One of these mutants appeared to be defective in multiplication in guinea pig lungs and spleens, yet it multiplies normally in guinea pig alveolar macrophages . Here we report further characterization of the mutated gene and its protein and the virulence role of the gene . The complete sequence of the gene, now called lvgA, is 627 bp long, and its protein product is approximately 27 kDa in size . lvgA was present in all 50 strains of L . pneumophila tested . No significant nucleic acid or protein homology was found in the GenBank database for the gene, nor were any distinctive motifs discovered in a search of other databases . The expression of both DotA and IcmX in the lvgA mutant was normal . Subcellular fractionation studies localized LvgA to the outer membrane fraction, and protease digestion studies suggested that at least some of the protein is surface expressed . No change in bacterial lipopolysaccharide composition or reactivity to serogroup-specific antisera was detected in the mutant . Growth competition studies with alveolar macrophages showed that the mutant was outcompeted by its parent 3-fold in 24 h and 24-fold in 48 h, in contrast to what was observed with the null phenotype in parallel testing with alveolar macrophages or with the A549 alveolar epithelial cell line . This macrophage defect of the mutant bacterium was due to slower growth, as the mutant invaded alveolar macrophages normally . Electron microscopy showed that the mutant bacterium resided in a ribosome-studded phagosome in alveolar macrophages, with no distinction from its parent . The lvgA mutant was outcompeted by its parent about sixfold in guinea pig lungs and spleens; prolonged observation of infected animals showed no late-onset virulence of the mutant . Transcomplementation of the mutant restored the parental phenotype in guinea pigs . The lvgA mutant was twofold more susceptible to killing by human beta-defensin 2 but not to killing by other cationic peptides, serum complement, or polymorphonuclear neutrophils . lvgA is a novel virulence gene that is responsible for pleiotropic functions involving both extracellular and intracellular bacterial resistance mechanisms.

Glycobiology, 2003 Sep, 13(9), 635 - 40 Epub 2003 Apr 17.
Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4; Volpi N; Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues . The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) (K4d) . K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase . The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE . Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material . Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4 . As a consequence, a mixture of uniform- size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography . These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, -->4)-GlcUA-beta(1-->3)GalNAc-beta(1-->, will be available for investigating important biological functions of this polymer.

J Clin Microbiol, 2003 Apr, 41(4), 1454 - 7
Distribution of Chlamydia pneumoniae DNA in atherosclerotic carotid arteries: significance for sampling procedures; Cochrane M et al.; Despite extensive efforts to confirm a direct association between Chlamydia pneumoniae and atherosclerosis, different laboratories continue to report a large variability in detection rates . In this study, we analyzed multiple sections from atherosclerotic carotid arteries from 10 endartectomy patients to determine the location of C . pneumoniae DNA and the number of sections of the plaque required for analysis to obtain a 95% confidence of detecting the bacterium . A sensitive nested PCR assay detected C . pneumoniae DNA in all patients at one or more locations within the plaque . On average, 42% (ranging from 5 to 91%) of the sections from any single patient had C . pneumoniae DNA present . A patchy distribution of C . pneumoniae in the atherosclerotic lesions was observed, with no area of the carotid having significantly more C . pneumoniae DNA present . If a single random 30- microm-thick section was tested, there was only a 35.6 to 41.6% (95% confidence interval) chance of detecting C . pneumoniae DNA in a patient with carotid artery disease . A minimum of 15 sections would therefore be required to obtain a 95% chance of detecting all true positives . The low concentration and patchy distribution of C . pneumoniae DNA in atherosclerotic plaque appear to be among the reasons for inconsistency between laboratories in the results reported.

Gastroenterol Clin Biol, 2003 Mar, 27(3 Pt 2), 440 - 52
{Could Helicobacter pylori treatment reduce stomach cancer risk?}; Bretagne JF; Despite its dramatic decline in incidence in developed countries, gastric cancer is a major public health issue in the world . Accumulating evidence for considering H . pylori as a causal factor for gastric cancer comes from recent epidemiologic studies, the advent of an animal model of gastric cancer and from new insights into the biological mechanisms for gastric carcinogenesis . The stomach cancer risk for people infected with H . pylori is rather low, inferior to 1% . It depends on genotypic polymorphisms of both the bacterium and the host . Environmental risk factors such as smoking habits, salt intake, and the amount of antioxidants in diet may interfere with H . pylori and modify the cancer risk . There is no definite clinical evidence of the benefit of eradication on cancer risk in humans due to the lack of randomized controlled studies in large populations . The occurrence of gastric adenocarcinomas in patients after complete remission of gastric MALT lymphoma induced by H . pylori eradication suggests also the limits of the preventive strategy against gastric cancer . Furthermore, the effectiveness of eradication to reverse precancerous gastric lesions such as severe atrophy and intestinal metaplasia is questionable . For many reasons discussed in our review, population-based screening and routine eradication of H . pylori infection seem to be an unrealistic goal and cannot be recommended in France . By waiting for effective anti-H . pylori vaccine, public health measures such as dietary modification should be promoted to further decrease the gastric cancer incidence . On the individual basis the specialist has a role in the diagnosis of gastric precancerous lesions by endoscopy and also in the prevention of gastric cancer by selecting indications for H . pylori therapy.

Gastroenterol Clin Biol, 2003 Mar, 27(3 Pt 2), 401 - 8
{Virulence factors of Helicobacter pylori: what are they?}; Contreras M et al.; The pathogenic properties of Helicobacter pylori are due to the ability of this bacterium to survive in the acidic gastric juice, to freely move and multiply within the mucus, to colonize the gastric mucosa and persist as an extracellular bacterium for decades despite the strong immune local and cellular responses they trigger . Every H . pylori isolate expresses properties that permit them colonization and persistence . In contrast, isolates may or not be expressing properties that potentially contribute to the genesis of tissue lesions due to the direct action of deleterious bacterial products or to the induction and/or modulation of the inflammatory response associated with the expression of specific antigens . Those are designated as pathogenic factors and might be useful to discriminate isolates.

Gastroenterol Clin Biol, 2003 Mar, 27(3 Pt 2), 391 - 400
{What are the gastric modifications induced by acute and chronic Helicobacter pylori infection?}; Lamarque D et al.; H . pylori colonisation of the stomach causes the recruitment of the inflammatory cells by the adherence of the bacteria with the epithelium and the release of factors of virulence either to the contact (oipA or other soluble factors) or in the cell by translocation (CagA) . Such contact triggers interleukin 8 expression in the epithelial cell and attracts lymphocytes and monocytes into the chorion . Bacterial lipopolysaccharide and urease support the activation of these inflammatory cells . The lymphocytes produce pro-inflammatory cytokines, which direct the immune response towards the Th1 pathway . The variability of the inflammatory response depends on hereditary factors of the host such as the interleukin 1 genotypes, which determine the level of the pro-inflammatory cytokine expression, and of bacterial factors such as the cag pathogenicity island, the lipopolysaccharide and the vacuolating toxin, vacA . The mucosal inflammation provokes apoptosis and atrophy of the epithelial cells through the effect of pro-inflammatory cytokines and free radicals . Epithelial proliferation is a consequence of excessive apoptosis caused by the infection . It is stimulated by the expression of inducible cyclo-oxygenase and inducible nitric oxide synthase . The development of atrophic gastritis towards cancer is supported by nitric oxide which has a mutagenic effect on DNA and inhibits p53 protein and by the bacterium itself which decreases DNA mismatch repairing activity . The gastritis induced by Helicobacter pylori changes acid secretion according to the prevalent location of the gastritis in the antrum or in the gastric body . Prevalent gastritis in the gastric body causes hypochlorhydria by reducing the release of histamin from ECL cells and inhibiting the parietal cells through the effect of tumor necrosis factor and interleukin 1-beta . Hypochlorhydria is more marked among patients having a pro-inflammatory genotype for interleukin 1-beta and those infected by bacteria with virulence factors . In the event of antrum predominant gastritis, the pro-inflammatory cytokines cause a reduction of somatostatin and gastrin releases from the D and the G cells, respectively . The result of all is increased maximal acid output and the meal-stimulated acid secretion.

Gastroenterol Clin Biol, 2003 Mar, 27(3 Pt 2), 367 - 73
{Helicobacter pylori and the others}; Lehours P; Since the discovery of H . pylori, several new Helicobacter species have been isolated from man and mainly from animals . Helicobacter species can be broadly grouped according to whether they colonize the gastric or enterohepatic niche . H . pylori is a bacterium of great clinical importance, essentially in the domain of gastroenterology . H . pylori infection is the first chronic infection known to give rise to cancer in man (gastric carcinoma and MALT lymphoma) . The pathogenesis of H . pylori is now well studied . Two H . pylori strains have been sequenced: the first one isolated from a patient with gastric ulcer, the second one associated with gastritis . Global analysis of the gene content of H . pylori strains gives insight into the extent of its genetic diversity . Substantial evidence attests to certain extragastric Helicobacter species playing a role in the pathogenesis of enteric, hepatic and biliary disorders, especially for H . hepaticus which have just been sequenced . But isolation of non-pylori Helicobacter species continues to be a major problem, substantially limiting a better understanding of their prevalence and role . Therefore, animal models are of interest because of their value for modeling human disease and testing therapeutic strategies such as vaccines.

Biol Bull, 2003 Apr, 204(2), 215 - 20
Plants, mycorrhizal fungi and endobacteria: a dialog among cells and genomes; Bonfante P; This review focuses on mycorrhizas, which are associations between fungi and the roots of 90% of terrestrial plants . These are the most common symbioses in the world; they involve about 6000 species of fungi distributed through all the fungal phyla and about 240000 species of plants, including forest and crop plants . Thanks to mycorrhizal symbiosis and nutrient exchanges, regulated by complex molecular signals, the plant improves its vegetative growth, while the fungus accomplishes its life cycle . Molecular and cellular analyses demonstrate that during colonization the cellular organization of the two eukaryotes is completely remodeled . For example, in cortical cells, structural modifications involve both the host and the microbiont . Recent studies revealed that in arbuscular mycorrhizas (AM), system complexity is increased by the presence of a third symbiont: a bacterium living inside the fungus . The presence of this resident genome makes the investigation of the molecular dialogues among the symbiotic partners even more complex . Molecular analysis showed that the bacterium has genes involved in the acquisition of mineral nutrients . The experimental data support the current view that mycorrhizal symbioses are often tripartite associations.

Mikrobiologiia, 2003 Jan-Feb, 72(1), 33 - 9
{The 1(2)-dehydrogenation of steroid substrates by Nocardioides simplex VKM Ac-2033D}; Fokina VV et al.; The bacterium formerly known as Arthrobacter globiformis 193 has high 1(2)-dehydrogenase activity toward pharmaceutically important steroids, 9(11)-dehydrocortexolone in particular . The complex analysis of the morphostructural, physiological, biochemical, and phylogenetic properties of this bacterium allowed us to reclassify it into Nocardioides simplex (N . simplex VKM Ac-2033D).

J Infect Dis, 2003 Apr 15, 187(8), 1165 - 77 Epub 2003 Apr 02.
Intracellular and interstitial expression of Helicobacter pylori virulence genes in gastric precancerous intestinal metaplasia and adenocarcinoma; Semino-Mora C et al.; Gastric intestinal metaplasia (IM) and gastric cancer are associated with Helicobacter pylori, but the bacterium often is undetectable in these lesions . To unravel this apparent paradox, IM, H . pylori presence, and the expression of H . pylori virulence genes were quantified concurrently using histologic testing, in situ hybridization, and immunohistochemistry . H . pylori was detected inside metaplastic, dysplastic, and neoplastic epithelial cells, and cagA and babA2 expression was colocalized . Importantly, expression of cagA was significantly higher in patients with IM and adenocarcinoma than in control subjects . The preneoplastic "acidic" MUC2 mucin was detected only in the presence of H . pylori, and MUC2 expression was higher in patients with IM, dysplasia, and cancer . These novel findings are compatible with the hypothesis that all stages of gastric carcinogenesis are fostered by persistent intracellular expression of H . pylori virulence genes, especially cagA inside MUC2-producing precancerous gastric cells and pleomorphic cancer cells.

Mol Ecol, 2003 May, 12(5), 1207 - 15
Cryptic species, cryptic endosymbionts, and geographical variation in chemical defences in the bryozoan Bugula neritina; McGovern TM et al.; Molecular markers often offer the only means to discriminate between species and to elucidate the specificity of many community interactions, both of which are key to the understanding of ecological patterns . Western Atlantic populations of the bryozoan Bugula neritina vary in the palatability of their larvae to predators: individuals south of Cape Hatteras produce chemical deterrents to fish predators that are absent in more northern individuals . We use mitochondrial cytochrome oxidase c subunit I (COI) sequences to show that the differences in palatability between populations correlate with the geographical distributions of two cryptic species within B . neritina . Furthermore, these cryptic species differ in their associations with bacteria that may confer chemical resistance to predation . Small subunit rRNA primers specific to a subset of gamma-proteobacteria amplified only the bacterium Endobugula sertula from the southern cryptic species . Endobugula sertula produces a family of chemical compounds (bryostatins) that may deter predators of its animal host . In contrast, the same primers amplified an array of gamma-proteobacteria from the unprotected northern cryptic bryozoan species, but never E . sertula . In combination, these findings suggest that the geographical variation in palatability observed in the larvae of B . neritina is not the result of local adaptation of a single species to regions of differing predation pressure, but rather results from the comparison of cryptic species that differ in the presence or absence of a bacterium that may provide protection against predators . The ability to identify the cryptic Bugula species and their differing relationships with bacterial associates provides an example of the important role molecular techniques may play in addressing ecological questions.

Biochemistry (Mosc), 2003 Feb, 68(2), 172 - 6
Purification and physicochemical properties of malate dehydrogenase from bacteria of the genus Beggiatoa; Eprintsev AT et al.; Homogeneous malate dehydrogenase (MDH) with a specific activity of 20-24 units per mg protein was purified from the sulfur bacterium Beggiatoa leptomitiformis strain D-402 grown organotrophically and lithotrophically and from the organotrophic bacterium Beggiatoa alba . MDHs from the B . leptomitiformis strain D-402 grown under organotrophic conditions and from B . alba are homodimers with the subunit molecular weight of 40 kD . Tetrameric MDH is formed in B . leptomitiformis strain D-402 grown under lithotrophic conditions . The dimeric and tetrameric forms of MDH from B . leptomitiformis D-402 display some differences in kinetic properties.

Scand J Infect Dis, 2003, 35(2), 146 - 7
Postoperative spondylodiskitis due to Stomatococcus mucilaginosus in an immunocompetent patient; Bureau-Chalot F et al.; A case is reported of postoperative spondylodiskitis due to Stomatococcus mucilaginosus in an immunocompetent woman . The route of infection remains unknown . Intravenous treatment with cefotaxime and fosfomycin was given, followed by oral administration of rifampin and pristinamycin until resolution of infection . This report shows that this bacterium can cause severe infections in immunocompetent patients.

Phys Rev Lett . 2003 Mar 28;90(12):128102 . Epub 2003 Mar 27.
Pattern formation inside bacteria: fluctuations due to the low copy number of proteins; Howard M et al.; We examine fluctuation effects due to the low copy number of proteins involved in pattern-forming dynamics within a bacterium . We focus on a stochastic model of the oscillating MinCDE protein system regulating accurate cell division in E . coli . We find that, for some parameter regions, the protein concentrations are low enough that fluctuations are essential for the generation of patterns . We also examine the role of fluctuations in constraining protein concentration levels.

Biodegradation, 2002, 13(5), 307 - 16
Induction characteristics of reductive dehalogenation in the ortho-halophenol-respiring bacterium, Anaeromyxobacter dehalogenans; He Q et al.; Anaeromyxobacter dehalogenans strain 2CP-C dehalogenates ortho-substituted di- and mono-halogenated phenols and couples this activity to growth . Reductive dehalogenation activity has been reported to be inducible, however, this process has not been studied extensively . In this study, the induction of reductive dehalogenation activity by strain 2CP-C is characterized . Constitutive 2-chlorophenol dechlorination activity occurs in non-induced fumarate-grown cells, with rates averaging 0.138 micromol of Cl- h(-1) mg of protein(-1) . Once induced, these cultures dechlorinate 2- chlorophenol (2-CP) at rates as high as 116 micromol of Cl(-1) h(-1) mg of protein(-1) . Dechlorination of 2-CP is induced by phenol, 2-chlorophenol, 2,4-dichlorophenol, 2,5-dichlorophenol, 2,6-dichlorophenol, and 2-bromophenol . Of the substrates tested, 2-bromophenol shows the highest induction potential, yielding double the 2-chlorophenol dechlorination rate when compared to other inducing substrates . No induced dechlorination is observed at concentrations less than 5 microM 2-CP . When fumarate cultures were diluted 100-fold, fumarate reduction rates were reduced roughly according to the dilution factor, while dechlorination rates were similar in fumarate grown cells amended with 2-CP and cells diluted 100-fold prior to the addition of chlorophenol . This indicates that the majority of the fumarate-grown cells in late log phase were not induced when exposed to inducing substrates such as 2-CP . This observation may have ramifications on the success of bioaugmentation using halorespiring bacteria, which traditionally relies on growing cultures using more readily utilized substrates . The rapid dechlorination rate and unique induction pattern also make strain 2CP-C a promising model organism for understanding the regulation of reductive dehalogenation at the enzymatic level.

An Pediatr (Barc), 2003 Apr, 58(4), 309 - 15
{Culture-confirmed whooping cough in a tertiary center over a twelve-year period}; Ferrer Marcelles A et al.; OBJECTIVE: To study the characteristics of patients diagnosed with whooping cough at a tertiary center in Barcelona, Spain . MATERIAL AND METHODS: We performed a retrospective study of patients aged less than 18 years treated for pertussis-like cough or clinically-suspected whooping cough over a 12-year period (1989-2000) . Only patients with isolated Bordetella spp . were included . The variables of age, sex, vaccination status, hospitalization, clinical manifestations, severity, and lethality were analyzed . RESULTS: One hundred sixty-one patients with positive Bordetella spp . culture were identified . Of these, complete information was available in 149 (79 boys and 70 girls) with a median age of 3 months (range: 13 days-17 years); 77.2 % were aged 6 months or less . All the isolated strains corresponded to B . pertussis except three that corresponded to B . parapertussis . Three epidemic cycles (in 1989, 1992 and 2000) were observed during the study period . A total of 72.5 % of cases occurred between May and September . Bordetella spp . was associated with other bacteria in 28.2 % of the patients, viruses in 13.4 % and a bacterium and a virus in 4.7 % . One hundred twenty-one patients required hospitalization, of which 14.9 % were admitted to the intensive care unit . Age was the only factor associated with risk for hospitalization, which was more frequent in younger infants (p < 0.0001) . Paroxysmal cough with cyanosis was present in 53.4 % of the patients, leucocytosis with lymphocytosis occurred in 67.5 % and apneas were present in 21.5 % . Chest X-ray revealed atelectasis in 34.1 % . The mean length of hospital stay was 11 days (range: 1-70 days) . Three boys aged less than 3 months with malignant pertussis syndrome died (lethality: 2 %) . More than half the patients (59.7 %) were not vaccinated (55.4 % for being under the age of 3 months) and only 16 % had received three or more vaccination doses . CONCLUSIONS: Whooping cough continues to be a severe disease in infants, with a high admission rate during the first 6 months of life . New preventive strategies are required to protect infants who have not yet developed full immunity to this infection.

Trends Genet, 2003 Apr, 19(4), 217 - 23
Evolutionary consequences of Wolbachia infections; Charlat S et al.; The past decade has revealed the bacterium Wolbachia as the most widespread symbiont of arthropods and nematodes . Behind this evolutionary success is an remarkable variety of effects on host biology, ranging from manipulation of reproduction in favor of females to more classical mutualistic interactions . Here we discuss the potential of Wolbachia for promoting evolutionary changes in its hosts.

Trends Genet, 2003 Apr, 19(4), 176 - 80
Why are the genomes of endosymbiotic bacteria so stable?
Silva FJ, Latorre A, Moya A.
The comparative analysis of three strains of the endosymbiotic bacterium Buchnera aphidicola has revealed high genome stability associated with an almost complete absence of chromosomal rearrangements and horizontal gene transfer events during the past 150 million years . The loss of genes involved in DNA uptake and recombination in the initial stages of endosymbiosis probably underlies this stability . Gene loss, which was extensive during the initial steps of Buchnera evolution, has continued in the different Buchnera lineages since their divergence.

Trends Genet, 2003 Apr, 19(4), 172 - 6
Potential genomic determinants of hyperthermophily; Makarova KS et al.; We searched for genes that could be important for hyperthermophily using a flexible approach to phyletic pattern analysis . We identified 290 clusters of orthologous groups of proteins (COGs) that are preferentially present in archaeal and bacterial hyperthermophiles . Of these, 58 COGs include proteins from at least one bacterium and two archaea, and these were considered to be the best candidates for a specific association with the hyperthermophilic phenotype . Detailed sequence and genome-context analysis of these COGs led to functional predictions for several previously uncharacterized protein families, including a novel group of putative molecular chaperones and a unique transcriptional regulator.

Evolution Int J Org Evolution, 2003 Feb, 57(2), 249 - 60
Parasite-mediated selection in experimental Daphnia magna populations; Capaul M et al.; It has been suggested that parasites are a strong selecting force for their hosts and therefore may alter the outcome of competition among host genotypes . We tested the extent to which parasite-mediated selection by different parasite species influenced competition among clones of the cyclic parthenogen Daphnia magna . We monitored clone frequency changes in laboratory microcosm populations consisting of 21 D . magna clones . Parasite treatments (two microsporidians, Glugoides intestinalis and Ordospora colligata) and a parasite-free control treatment were followed over a nine-month period . A further treatment with the bacterium Pasteuria ramosa failed . We found significant differences in clonal success among the treatments: the two parasite treatments differed from the control treatment and from each other . Additionally, we measured the clone-specific population carrying capacity, competitive ability against tester clones, and reproductive success of infected and uninfected females to test whether they correlate with clonal success in the microcosms . The clone-specific competitive ability was a good predictor of clonal success in the microcosms, but clonal carrying capacity and host reproductive success were not . Our study shows that parasite-mediated selection can strongly alter the outcome of clonal competition . The results suggest that parasites may influence microevolution in Daphnia populations during periods of asexual reproduction.

Mol Biol Evol, 2003 May, 20(5), 686 - 93 Epub 2003 Apr 02.
Arsenite oxidase, an ancient bioenergetic enzyme; Lebrun E et al.; Operons coding for the enzyme arsenite oxidase have been detected in the genomes from Archaea and Bacteria by Blast searches using the amino acid sequences of the respective enzyme characterized in two different beta-proteobacteria as templates . Sequence analyses show that in all these species, arsenite oxidase is transported over the cytoplasmic membrane via the tat system and most probably remains membrane attached by an N-terminal transmembrane helix of the Rieske subunit . The biochemical and biophysical data obtained for arsenite oxidase in the green filamentous bacterium Chloroflexus aurantiacus allow a structural model of the enzyme's membrane association to be proposed . Phylogenies for the two constituent subunits (i.e., the molybdopterin-containing and the Rieske subunit) of the heterodimeric enzyme and their respective homologs in DMSO-reductase, formate dehydrogenase, nitrate reductase, and the Rieske/cytb complexes were calculated from multiple sequence alignments . The obtained phylogenetic trees indicate an early origin of arsenite oxidase before the divergence of Archaea and Bacteria . Evolutionary implications of these phylogenies are discussed.

J Bioenerg Biomembr, 2002 Dec, 34(6), 413 - 21
Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus; Fernandes AS et al.; The rotenone sensitive NADH:menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized . Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I . The optimum conditions for NADH dehydrogenase activity are 50 degrees C and pH 8.1, and the enzyme presents a KM of 9 microM for NADH . The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified . When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP . The purified enzyme contains 13.5 +/- 3.5 iron atoms and approximately 3.7 menaquinone per FMN . At least five iron-sulfur centers are observed by EPR spectroscopy: two {2Fe-2S}(2+/1+) and three {4Fe-4S}(2+/1+) centers . By fluorescence spectroscopy a still unidentified chromophore was detected in R . marinus complex I.

Dev Biol (Basel), 2002, 111, 153 - 8
Batch potency testing of inactivated erysipelas vaccines by ELISA--development, validation and implementation; Rosskopf-Streicher U et al.; Inactivated erysipelas vaccines are widely used to protect pigs against erysipelas disease caused by the bacterium Erysipelothrix (E.) rhusiopathiae . Quality control tests for this vaccine are laid down in the European Pharmacopoeia (Ph.Eur.) Monograph No . 64 . A laboratory animal model using a vaccination-challenge procedure is currently required as batch potency test . More than 10 years ago we initiated the first studies to develop an alternative ELISA potency model to replace this regulatory challenge test in mice . A short retrospective outline of the various steps from the development of the method until implementation into the regulatory requirements is described.

Appl Environ Microbiol, 2003 Apr, 69(4), 2395 - 8
Wide geographic distribution of bacteriophages that lyse the same indigenous freshwater isolate (Sphingomonas sp . strain B18); Wolf A et al.; An indigenous freshwater bacterium (Sphingomonas sp . strain B18) from Lake Plubetasee (Schleswig-Holstein, Germany) was used to isolate 44 phages from 13 very different freshwater and brackish habitats in distant geographic areas . This bacterial strain was very sensitive to a broad spectrum of phages from different aquatic environments . Phages isolated from geographically distant aquatic habitats, but also those from the same sample, were diverse with respect to morphology and restriction pattern . Some phages were widely distributed, while different types coexisted in the same sample . It was concluded that phages could be a major factor in shaping the structure of bacterial communities and maintaining a high bacterial diversity.

Appl Environ Microbiol, 2003 Apr, 69(4), 2032 - 7
Insecticidal activity associated with the outer membrane vesicles of Xenorhabdus nematophilus; Khandelwal P et al.; Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane . Transmission electron micrographs of X . nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium . The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X . nematophilus and analyzed . Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm . A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present . The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type . Live cells of X . nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera . The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs . The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H . armigera neonatal larvae . The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay . The OMV proteins showed chitinase activity . This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X . nematophilus into the extracellular medium.

Appl Environ Microbiol, 2003 Apr, 69(4), 1890 - 7
For the insect pathogen Photorhabdus luminescens, which end of a nematode is out?
Ciche TA, Ensign JC.
The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae . The dauer juvenile (DJ) stage nematode selectively retains P . luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host . We report the results of studying the transmission of the bacteria by its nematode vector . Cells of P . luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus . Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria . Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth . This regurgitation reaction was induced by a low-molecular-weight, heat- and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures . Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph . The ability to visualize P . luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae . This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study.

Am Nat, 2003 Feb, 161(2), 254 - 66
Genetic conflicts over sex ratio: mite-endosymbiont interactions; Vala F et al.; Nucleocytoplasmic genetic conflicts arise as a result of asymmetric transmission of cytoplasmic and nuclear genes . Spread of a cytoplasmic element promoting female-biased sex ratios creates selection on nuclear genes for mechanisms that decrease the bias . Here we investigate the conflict over sex ratio between the cytoplasmic bacterium Wolbachia and the two-spotted spider mite Tetranychus urticae Koch . We show that, first, infected females produce significantly more female-biased sex ratios than uninfected (cured) females . Second, this effect is not due to parthenogenesis, male killing, or feminization, phenotypes commonly associated with infection by Wolbachia . Third, sex ratio is a trait with a heritable component in this species; thus, it can evolve under selection . Fourth, the sex ratio produced by uninfected (cured) females changes over time, approaching the sex ratio produced by females from the infected culture . On the basis of these results, we suggest that after sex ratio manipulation by Wolbachia, a host compensatory mechanism evolved that allows infected females to produce the sex ratio favored by nuclear genes . We discuss the evolution of "mutualism" with respect to the evolution of host mechanisms that compensate for effects induced by vertically transmitted "parasites."

J Physiol Pharmacol, 2003 Mar, 54(1), 3 - 15
Activation of peroxisome proliferator-activated receptor gamma impedes Porphyromonas gingivalis lipopolysaccharide interference with salivary mucin synthesis through phosphatidylinositol 3-kinase/erk pathway; Slomiany BL et al.; Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the superfamily of nuclear receptor transcription factors, plays a critical role in the regulation of the expression of genes associated with inflammation . Using mucous acinar cells of sublingual salivary gland, we investigated the effect of PPARgamma activation on the disturbances in salivary mucin synthesis evoked by lipopolysaccharide (LPS) of periodontopathic bacterium, P . gingivalis . Exposure of the acinar cells to the LPS led to a dose-dependent decrease (up to 58.4%) in mucin synthesis, accompanied by a massive enhancement in apoptosis and NO production, and an induction in inducible nitric oxide synthase (NOS-2) activity . Activation of PPARgamma with a specific synthetic agonist, ciglitazone, prevented in a dose-dependent fashion the LPS-induced reduction in mucin synthesis, and the effect was reflected in a marked decrease in apoptosis, NO generation, and the expression of NOS-2 activity . The impedance by ciglitazone of the LPS-induced changes in mucin synthesis was blocked by PD98059, an inhibitor of extracellular signal regulated kinase (ERK), as well as wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) . Moreover, both agents caused further enhancement in the LPS-induced nitric oxide generation and countered the inhibitory effect of ciglitazone on the LPS-induced upregulation in NOS-2 . The findings suggest that the impedance of P . gingivalis LPS inhibition of salivary, mucin synthesis by PPARgamma agonist, ciglitazone, involves activation of ERK pathway by PI3K.

J Mol Microbiol Biotechnol, 2003, 5(1), 11 - 6
MASE1 and MASE2: two novel integral membrane sensory domains; Nikolskaya AN et al.; Escherichia coli proteins YegE and YaiC contain N-terminal integral membrane regions, followed by the putative diguanylate cyclase (GGDEF, DUF1) domains . The membrane domains of these proteins, named MASE1 (membrane-associated sensor) and MASE2, respectively, were found in other bacterial signaling proteins, such as histidine kinases (MASE1) and an adenylate cyclase (MASE2) . Although the nature of the signals sensed by MASE1 and MASE2 is still unknown, MASE1-containing receptors appear to play important roles in bacteria, including iron and/or oxygen sensing by hemerythrine-containing proteins in the sulfate-reducing bacterium Desulfovibrio vulgaris .

Anal Biochem, 2003 Apr 1, 315(1), 106 - 13
Optical imaging fiber-based live bacterial cell array biosensor; Biran I et al.; A live cell array biosensor was fabricated by immobilizing bacterial cells on the face of an optical imaging fiber containing a high-density array of microwells . Each microwell accommodates a single bacterium that was genetically engineered to respond to a specific analyte . A genetically modified Escherichia coli strain, containing the lacZ reporter gene fused to the heavy metal-responsive gene promoter zntA, was used to fabricate a mercury biosensor . A plasmid carrying the gene coding for the enhanced cyan fluorescent protein (ECFP) was also introduced into this sensing strain to identify the cell locations in the array . Single cell lacZ expression was measured when the array was exposed to mercury and a response to 100nM Hg(2+) could be detected after a 1-h incubation time . The optical imaging fiber-based single bacterial cell array is a flexible and sensitive biosensor platform that can be used to monitor the expression of different reporter genes and accommodate a variety of sensing strains.

Genome Res, 2003 Apr, 13(4), 570 - 8
Microarray analyses of Xylella fastidiosa provide evidence of coordinated transcription control of laterally transferred elements; Nunes LR et al.; Genetically distinct strains of the plant bacterium Xylella fastidiosa (Xf) are responsible for a variety of plant diseases, accounting for severe economic damage throughout the world . Using as a reference the genome of Xf 9a5c strain, associated with citrus variegated chlorosis (CVC), we developed a microarray-based comparison involving 12 Xf isolates, providing a thorough assessment of the variation in genomic composition across the group . Our results demonstrate that Xf displays one of the largest flexible gene pools characterized to date, with several horizontally acquired elements, such as prophages, plasmids, and genomic islands (GIs), which contribute up to 18% of the final genome . Transcriptome analysis of bacteria grown under different conditions shows that most of these elements are transcriptionally active, and their expression can be influenced in a coordinated manner by environmental stimuli . Finally, evaluation of the genetic composition of these laterally transferred elements identified differences that may help to explain the adaptability of Xf strains to infect such a wide range of plant species.

J Biol Chem, 2003 Jun 6, 278(23), 20687 - 94 Epub 2003 Mar 31.
The Treponema pallidum tro operon encodes a multiple metal transporter, a zinc-dependent transcriptional repressor, and a semi-autonomously expressed phosphoglycerate mutase; Hazlett KR et al.; The Treponema pallidum tro operon encodes an ABC transporter (TroABCD), a transcriptional repressor (TroR), and the essential glycolytic enzyme phosphoglycerate mutase (Gpm) . The apparently discordant observations that the solute binding protein (TroA) binds Zn2+, whereas DNA binding by TroR in vitro is Mn2+-dependent, have generated uncertainty regarding the identities of the ligand(s) and co-repressor(s) of the permease . Moreover, this operonic structure suggests that Gpm expression, and hence glycolysis, the sole source of ATP for the bacterium, would be suspended during TroR-mediated repression . To resolve these discrepancies, we devised an experimental strategy permitting a more direct assessment of Tro operon function and regulation . We report that (i) apo-TroA has identical affinities for Zn2+ and Mn2+; (ii) the Tro transporter expressed in Escherichia coli imports Zn2+, Mn2+, and possibly iron; (iii) TroR represses transporter expression in E . coli at significantly lower concentrations of Zn2+ than of Mn2+; and (iv) TroR-mediated repression causes a disproportionately greater down-regulation of the transporter genes than of gpm . The much higher concentrations of Zn2+ than of Mn2+ in human body fluids suggests that Zn2+ is both the primary substrate and co-repressor of the permease in vivo . Our data also indicate that Gpm expression and, therefore, glycolysis would not be abrogated when T . pallidum encounters high Zn2+ levels.

Biophys J, 2003 Apr, 84(4), 2483 - 91
The ring structure and organization of light harvesting 2 complexes in a reconstituted lipid bilayer, resolved by atomic force microscopy; Stamouli A et al.; The main function of the transmembrane light-harvesting complexes in photosynthetic organisms is the absorption of a light quantum and its subsequent rapid transfer to a reaction center where a charge separation occurs . A combination of freeze-thaw and dialysis methods were used to reconstitute the detergent-solubilized Light Harvesting 2 complex (LH2) of the purple bacterium Rhodopseudomonas acidophila strain 10050 into preformed egg phosphatidylcholine liposomes, without the need for extra chemical agents . The LH2-containing liposomes opened up to a flat bilayer, which were imaged with tapping and contact mode atomic force microscopy under ambient and physiological conditions, respectively . The LH2 complexes were packed in quasicrystalline domains . The endoplasmic and periplasmic sides of the LH2 complexes could be distinguished by the difference in height of the protrusions from the lipid bilayer . The results indicate that the complexes entered in intact liposomes . In addition, it was observed that the most hydrophilic side, the periplasmic, enters first in the membrane . In contact mode the molecular structure of the periplasmic side of the transmembrane pigment-protein complex was observed . Using Foster's theory for describing the distance dependent energy transfer, we estimate the dipole strength for energy transfer between two neighboring LH2s, based on the architecture of the imaged unit cell.

IUBMB Life, 2002 Dec, 54(6), 315 - 21
Degradation of mRNA in Escherichia coli; Jain C; Degradation of messenger RNAs (mRNAs) is a universal process that occurs in every cell and has important implications for nucleotide metabolism and gene expression . One organism in which mRNA degradation has been thoroughly studied is the bacterium Escherichia coli (E . coli) . In this review I describe what is presently known about the different processes involved in the conversion of mRNAs from high molecular weight species to mononucleotides in E . coli . The ribonucleases and accessory factors involved in mRNA degradation, and features on mRNAs that make them resistant or sensitive to degradation will also be described . At the conclusion of this review, some of the anticipated directions of future research on this topic will be discussed.

Eur J Oral Sci, 2002 Oct, 110(5), 366 - 73
Inhibited proliferation of human periodontal ligament cells and gingival fibroblasts by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin; Belibasakis G et al.; Actinobacillus actinomycetemcomitans can inhibit fibroblast proliferation . The objective of this study was to characterize the early proliferative responses of human periodontal ligament cells (PDLC) and gingival fibroblasts (GF) to A . actinomycetemcomitans components and to investigate the possible involvement of the cytolethal distending toxin (cdt) produced by this bacterium . The PDLC and GF were challenged with surface components of A . actinomycetemcomitans . Both DNA and protein synthesis as well as cell lysis or apoptosis were assayed for a 6-h period after addition of the bacterial extract . Unlike the controls, inhibition of DNA synthesis had already occurred in the challenged cells at the end of the initial 3- to 6-h period . No lysis or apoptosis was detected, and the total protein synthesis remained unaffected . The persistence of the effect on cell growth was confirmed after a 72-h period of challenge, during which the cells remained viable but exhibited an elongated and distended cell body . No significant differences were observed between PDLC and GF . When a cdt-knockout strain of A . actinomycetemcomitans was used almost no inhibitory effect on cell proliferation was observed . It was concluded that A . actinomycetemcomitans causes a non-lethal inhibition of proliferation in PDLC and GF as a result of an early arrest of DNA synthesis . Cytolethal distending toxin is responsible for most of this effect . This bacterial property may compromise tissue homeostasis in the periodontium.

Extremophiles, 2003 Apr, 7(2), 145 - 57 Epub 2003 Jan 23.
{NiFe} hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus: properties, function, and phylogenetics; Brugna-Guiral M et al.; Genes potentially coding for three distinct {NiFe} hydrogenases are present in the genome of Aquifex aeolicus . We have demonstrated that all three hydrogenases are expressed under standard growth conditions of the organism . Two hydrogenases were further purified to homogeneity . A periplasmically oriented hydrogenase was obtained in two forms, i.e., as a soluble enzyme containing only the two essential subunits and as a detergent-solubilized complex additionally containing a membrane-integral b-type cytochrome . The second hydrogenase purified was identified as a soluble cytoplasmic enzyme . The isolated enzymes were characterized with respect to biochemical/biophysical parameters, activity, thermostability, and substrate specificity . The phylogenetic positioning of all three hydrogenases was analyzed . A model for the metabolic roles of the three enzymes is proposed on the basis of the obtained results.

Arch Microbiol, 2003 May, 179(5), 354 - 62 Epub 2003 Mar 28.
Characterization of unusual hydroxy- and ketocarotenoids in Rubrivivax gelatinosus: involvement of enzyme CrtF or CrtA; Pinta V et al.; Carotenoids are widely spread terpenoids found in photosynthetic organisms and a number of non-photosynthetic fungi and bacteria . The photosynthetic non-sulfur purple bacterium Rubrivivax gelatinosus produces carotenoids by both the spheroidene and the normal spirilloxanthin pathways . The characteristics of two carotenogenesis enzymes, spheroidene monooxygenase CrtA and O-methyltransferase CrtF, were investigated . Disruption of the corresponding genes by insertional mutagenesis affected carotenoid species in both pathways, and the genetic evidence indicated that both genes are involved in the two pathways . In these mutants, several unusual hydroxy- and ketocarotenoids were identified by spectroscopic and chemical methods . Moreover, the carotenoid analyses demonstrated that a large number of different carotenoid intermediates are accepted as substrates by the CrtA enzyme . The combined manipulation of crtF and crtA allowed new carotenoids to be produced and broadened the diversity of structurally different carotenoids synthesized by Rvi . gelatinosus . Methylated carotenoids, such as spheroidene and spirilloxanthin, are known to function as accessory pigments in the light-harvesting and reaction-center complexes of purple bacteria; the demethylated carotenoids described here were able to fulfill the same functions in the mutants.

J Infect Dis, 2003 Apr 1, 187(7), 1107 - 15 Epub 2003 Mar 13.
Chlamydia pneumoniae infection of alveolar macrophages: a model; Haranaga S et al.; Chlamydia (Chlamydophila) pneumoniae is a common respiratory pathogen, and it seems likely that alveolar macrophages may have an important role in infection with this bacterium . In the present study, we examined the usefulness of a continuous cell line of murine alveolar macrophages, designated "MH-S," as an in vitro C . pneumoniae infection model . Infection of MH-S cells with C . pneumoniae resulted in the development of typical inclusion bodies in the cells, similar to that seen in primary alveolar macrophages . However, we noted that, although the number of bacteria in the cultures increased during the infection, there was a restricted production of infective elementary bodies . The analysis of bacterial messenger RNA in the cultures showed that the message levels for the omcB gene were present only at a moderate level, but the levels of hsp60 messages increased markedly during infection . Neutralization of tumor necrosis factor (TNF)-alpha induced by inoculation with antibody significantly enhanced the infection, but omcB message levels were still inhibited . These results indicate that the growth of C . pneumoniae in alveolar macrophages may be restricted . Endogenous TNF-alpha may be one of the factors responsible for such restriction, but other factors also may be involved.

J Parasitol, 2003 Feb, 89(1), 14 - 20
Tick salivary gland extract accelerates proliferation of Francisella tularensis in the host; Krocova Z et al.; Accelerated proliferation of the tick-borne bacterial pathogen Francisella tularensis was demonstrated in mice when the bacterium was injected together with salivary gland extract from Ixodes ricinus ticks . A significant increase in the numbers of bacteria was recorded in the dermal site of infection,the draining lymph nodes, and the spleen . Analysis of the expression of cytokine messenger ribonucleic acids showed polarization toward a Th2 profile . Salivary gland extract-mediated suppression of interleukin-12 and interferon-gamma, the cytokines required for the expression of the protective immunity against tularemic infection, apparently contributed to the decreased resistance against this tick-transmitted pathogen.

Appl Microbiol Biotechnol, 2003 Mar, 61(1), 44 - 54 Epub 2002 Dec 19.
Phthalate catabolic gene cluster is linked to the angular dioxygenase gene in Terrabacter sp . strain DBF63; Habe H et al.; Phthalate is a metabolic intermediate of the pathway of fluorene (FN) degradation via angular dioxygenation . A gene cluster responsible for the conversion of phthalate to protocatechuate was cloned from the dibenzofuran (DF)- and FN-degrading bacterium Terrabacter sp . strain DBF63 and sequenced . The genes encoding seven catabolic enzymes, oxygenase large subunit of phthalate 3,4-dioxygenase (phtA1), oxygenase small subunit of phthalate 3,4-dioxygenase (phtA2), cis-3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase (phtB), {3Fe-4S} or {4Fe-4S} type of ferredoxin (phtA3), ferredoxin reductase (phtA4), 3,4-dihydroxyphthalate decarboxylase (phtC) and putative regulatory protein (phtR), were found in the upstream region of the angular dioxygenase gene (dbfA1A2), encoded in this order . Escherichia coli carrying phtA1A2BA3A4 genes converted phthalate to 3,4-dihydroxyphthalate, and the 3,4-dihydroxyphthalate decarboxylase activity by E . coli cells carrying phtC was finally detected with the introduction of a Shine-Dalgarno sequence in the upstream region of its initiation codon . Homology analysis on the upstream region of the pht gene cluster revealed that there was an insertion sequence (IS) (ISTesp2; ORF14 and its flanking region), part of which was almost 100% identical to the orf1 and its flanking region adjacent to the extradiol dioxygenase gene ( bphC1) involved in the DF degradation of Terrabacter sp . strain DPO360 {Schmid et al . (1997) J Bacteriol 179:53-62} . This suggests that ISTesp2 plays a role in the metabolism of aromatic compounds in Terrabacter sp . strains DBF63 and DPO360.

Acta Crystallogr D Biol Crystallogr, 2003 Apr, 59(Pt 4), 644 - 53 Epub 2003 Mar 25.
Structure of dimeric cytochrome c3 from Desulfovibrio gigas at 1.2 A resolution; Aragao D et al.; The structure of dimeric cytochrome c(3) from the sulfate-reducing bacterium Desulfovibrio gigas, diDg, obtained by ab initio methods was further refined to 1.2 A resolution, giving final reliability factors of R(free) = 14.8% and R = 12.4% . This cytochrome is a dimer of tetraheme cytochrome c(3) molecules covalently linked by two solvent-accessible disulfide bridges, a characteristic unique to members of the cytochrome c(3) superfamily . Anisotropic analysis using the semi-rigid TLS method shows different behaviour for analogous loops in each monomer arising from their different packing environments . A detailed sequence and structural comparison with all other known cytochrome c(3) domains in single- and multi-domain cytochromes c(3) shows the presence of structurally conserved regions in this family, despite the high variability of the amino-acid sequence . An internal water molecule is conserved in a common structural arrangement in all c(3) tetraheme domains, indicating a probable electron-transfer pathway between hemes I and II . Unique features of diDg are an internal methionine residue close to heme I and to one of the axial ligands of heme III, where all other structures of the cytochrome c(3) superfamily have a phenylalanine, and a rather unusual CXXXCH heme-binding motif only found so far in this cytochrome.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 183 - 7
Kocuria polaris sp . nov., an orange-pigmented psychrophilic bacterium isolated from an Antarctic cyanobacterial mat sample; Reddy GS et al.; Strain CMS 76orT, an orange-pigmented bacterium, was isolated from a cyanobacterial mat sample from a pond located in McMurdo Dry Valley, Antarctica . On the basis of chemotaxonomic and phylogenetic properties, strain CMS 76orT was identified as a member of the genus Kocuria . It exhibited a 16S rDNA similarity of 99.8% and DNA-DNA similarity of 71% with Kocuria rosea (ATCC 186T) . Phenotypic traits confirmed that strain CMS 78orT and K . rosea were well differentiated . Furthermore, strain CMS 76orT could be differentiated from the other reported species of Kocuria, namely Kocuria kristinae (ATCC 27570T), Kocuria varians (ATCC 15306T), Kocuria rhizophila (DSM 11926T) and Kocuria palustris (DSM 11025T), on the basis of a number of phenotypic features . Therefore, it is proposed that strain CMS 76orT (= MTCC 3702T = DSM 14382T) be assigned to a novel species of the genus Kocuria, as Kocuria polaris.

Eur J Gastroenterol Hepatol, 2003 Apr, 15(4), 395 - 401
How Helicobacter pylori urease may affect external pH and influence growth and motility in the mucus environment: evidence from in-vitro studies; Sidebotham RL et al.; BACKGROUND: Survival of Helicobacter pylori is dependent upon urease in the cytoplasm and at the bacterial surface . We have sought to clarify how alkaline ammonium salts, released from urea by this enzyme, might alter mucus pH and so affect growth and motility of the bacterium in the gastric mucus environment . METHODS: Experiments were conducted in vitro to determine how the growth and motility of H . pylori are affected by changes in external pH, and how the bacterium, by hydrolysing urea, alters the pH of the bicarbonate buffer that occurs at the gastric mucosal surface . These data were fitted into experimental models that describe how pH varies within the mucus layer in the acid-secreting stomach . RESULTS: H . pylori was motile between pH 5 and 8, with optimal motility at pH 5 . It grew between pH 6 and 8, with optimal growth at pH 6 . The bacterium had urease activity between pH 2.7 and 7.4, as evidenced by pH rises in bicarbonate-buffered solutions of urea . Changes in buffer pH were dependent upon initial pH and urea concentration, with the greatest rate of pH change occurring at pH 3 . Modelling experiments utilizing these data indicated that (1) in the absence of urease, H . pylori growth and motility in the mucus layer would be restricted severely by low mucus pH in the acid-secreting stomach, and (2) urease will sometimes inhibit H . pylori growth and motility in the mucus layer by elevating the pH of the mucus environment above pH 8 . CONCLUSIONS: Urease is essential to the growth and motility of H . pylori in the mucus layer in the acid-secreting stomach, but, paradoxically, sometimes it might suppress colonization by raising the mucus pH above 8 . This latter effect may protect the bacteria from the adverse consequences of overpopulation.

Infect Immun, 2003 Apr, 71(4), 2218 - 25
Cloning and characterization of an Ehrlichia canis gene encoding a protein localized to the morula membrane; Teng CH et al.; A gene encoding a 23.5-kDa ehrlichial morula membrane protein designated MmpA was cloned by screening an Ehrlichia canis expression library with convalescent dog sera, which resulted in three positive clones . Sequence analysis of the insert DNAs from all three clones indicated an open reading frame with a size of 666 bp that encodes MmpA . The structural analysis of MmpA indicated that it is a transmembrane protein with extreme hydrophobicity . Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated the presence of a single copy of the mmpA gene in E . canis and Ehrlichia chaffeensis but not in the human granulocytic ehrlichiosis agent . The mmpA gene was amplified, cloned, and expressed as a fusion protein . Polyclonal antibodies to the recombinant protein (rMmpA) were raised in rabbits . Western blot analysis of E . canis and E . chaffeensis lysates with the anti-rMmpA serum resulted in the presence of an MmpA band only in E . canis, not in E . chaffeenesis . Sera from dogs which were either naturally or experimentally infected with E . canis recognized the recombinant protein . Double immunofluorescence confocal microscopy studies demonstrated that MmpA was localized mainly on the morula membrane of E . canis . Since the morula membrane is the interface between the ehrlichial growing environment and the host cytoplasm, MmpA may play a role in bacterium-host cell interactions.

DNA Seq, 2002 Dec, 13(6), 375 - 81
Cloning and characterization of a gene encoding glutathione-regulated potassium-efflux system protein KefKL from the endosymbiont Wolbachia; Kang L et al.; The maternally inherited intracellular symbiont Wolbachia is well known for inducing a variety of reproductive and developmental abnormalities in the diverse arthropod hosts it infects . It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, feminization of genetic males and male killing in different hosts . However, the molecular mechanisms by which this fastidious bacterium causes these abnormalities have not yet been determined . In our study, representational difference analysis (RDA) was used to analyze the genomic difference between different Wolbachia strains . A gene encoding glutathione-regulated potassium-efflux system protein KefKL from Wolbachia in Drosophila simulans Riverside (w Ri) was isolated . The homologous genes from Wolbachia in Drosophila melanogaster yw67c23 (wMel) and Wolbachia in Drosophila melanogaster CantonS (wMelCS) were also cloned and sequenced . Sequence analysis showed that these deduced amino acid sequences contained two important motifs: Na+/H+ antiportor and NAD binding domain, which shared conserved sequences among different strains . Considering the crucial function of KefKL for ionic homeostasis, this gene might play an important role in Wolbachia physiology . Further study indicated that there was no homologue detected from Wolbachia in Drosophila simulans DSW/Mau (wMa) and Wolbachia in Drosophila simulans Noumea (wNo) . Whether Wolbachia contained KefKL (or the homologous gene) was consistent with the phylogenetic studies using wsp sequences, which showed that wMa and wNo were grouped into one branch, while w Ri, wMel and wMelCS were more closely related.

Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 4191 - 6 Epub 2003 Mar 21.
Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation; Liu Y et al.; Deinococcus radiodurans R1 (DEIRA) is a bacterium best known for its extreme resistance to the lethal effects of ionizing radiation, but the molecular mechanisms underlying this phenotype remain poorly understood . To define the repertoire of DEIRA genes responding to acute irradiation (15 kGy), transcriptome dynamics were examined in cells representing early, middle, and late phases of recovery by using DNA microarrays covering approximately 94% of its predicted genes . At least at one time point during DEIRA recovery, 832 genes (28% of the genome) were induced and 451 genes (15%) were repressed 2-fold or more . The expression patterns of the majority of the induced genes resemble the previously characterized expression profile of recA after irradiation . DEIRA recA, which is central to genomic restoration after irradiation, is substantially up-regulated on DNA damage (early phase) and down-regulated before the onset of exponential growth (late phase) . Many other genes were expressed later in recovery, displaying a growth-related pattern of induction . Genes induced in the early phase of recovery included those involved in DNA replication, repair, and recombination, cell wall metabolism, cellular transport, and many encoding uncharacterized proteins . Collectively, the microarray data suggest that DEIRA cells efficiently coordinate their recovery by a complex network, within which both DNA repair and metabolic functions play critical roles . Components of this network include a predicted distinct ATP-dependent DNA ligase and metabolic pathway switching that could prevent additional genomic damage elicited by metabolism-induced free radicals.

Microbes Infect, 2003 Feb, 5(2), 135 - 42
Innate and adaptive immune responses to an intracellular bacterium, Francisella tularensis live vaccine strain; Elkins KL et al.; The immune response to intracellular bacterium, Francisella tularensis, which causes tularemia and is proposed to be a potential bioterrorism pathogen, has been studied in mice using the attenuated live vaccine strain (LVS) . Here we review this infection model, which provides a convenient means of studying protective immune mechanisms not only for Francisella, but also for the large and important class of intracellular pathogens.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Apr 25, 787(2), 405 - 13
Liquid chromatography-electrospray mass spectrometry study of cysteine-10 S-glutathiolation in recombinant glutathione S-transferase of Ochrobactrum anthropi; Celli N et al.; Glutathione S-transferase of Ochrobactrum anthropi (OaGST), a bacterium isolated from soils contaminated by xenobiotic pollutants, was recently purified, cloned and characterised in our laboratories . The recombinant OaGST (rOaGST), highly expressed in Escherichia coli, when purified by glutathione-affinity chromatography and then analysed by electrospray ionisation mass spectrometry (ESI-MS), has evidenced a disulphide bond with glutathione (S-glutathiolation), which was removable by reduction with 2-mercaptoethanol . Enzymatic digestion of rOaGST with endoproteinase Glu-C, followed by liquid chromatography (LC)-ESI-MS analyses of the peptide mixtures under both reducing and not reducing conditions, have shown that glutathione was covalently bound to the Cys10 residue of rOaGST . Furthermore, LC-ESI-MS analyses of overexpressed rOaGST in Escherichia coli crude extracts, with and without incubation with glutathione, have not shown any S-glutathiolation of the recombinant enzyme.

Trends Microbiol, 2003 Mar, 11(3), 118 - 23
Will the enigma of Francisella tularensis virulence soon be solved?
Titball RW, Johansson A, Forsman M.
Francisella tularensis is one of the most infectious bacterial pathogens known and is the causative agent of the zoonotic disease tularemia . In spite of the importance of this pathogen little is known about its virulence mechanisms . However, it is clear that the bacterium is an intracellular pathogen, replicating mainly in macrophages, with replication in amoebae also having been reported . The genome sequence of a high virulence strain of F . tularensis is close to completion and when available, will stimulate further research into virulence mechanisms.

J Immunol, 2003 Apr 1, 170(7), 3819 - 27
The resolution of relapsing fever borreliosis requires IgM and is concurrent with expansion of B1b lymphocytes; Alugupalli KR et al.; The rate of pathogen clearance is a critical determinant of morbidity and mortality . We sought to characterize the immune response responsible for the remarkably rapid clearance of individual episodes of bacteremia caused by the relapsing fever bacterium, Borrelia hermsii . SCID or Rag(-/-) mice were incapable of resolving B . hermsii infection, indicating a critical role for T and/or B cells . TCR(-/-) mice, which lack T cells, and IL-7(-/-) mice, which are deficient in both T cells and follicular B cells, but not in B1 cells and splenic marginal zone (MZ) B cells, efficiently cleared B . hermsii . These findings suggested that B1 cells and/or MZ B cells, two B cell subsets that are known to participate in rapid, T-independent responses, might be involved . The efficient resolution of the episodes of moderate level bacteremia by splenectomized mice suggested that MZ B cells do not play the primary role in clearance of this bacterium . In contrast, xid mice, which are deficient in B1 cells, suffered more severe episodes of bacteremia than wild-type mice . The hypothesis that B1 cells are critical for clearance of B . hermsii was further supported by a selective expansion of the B1b (i.e., IgM(high), IgD(-/low), Mac1(+) CD23(-), and CD5(-)) cell subset in infected xid mice, which coincided with the eventual resolution of infection . Finally, mice selectively incapable of secreting IgM, the dominant isotype produced by B1 cells, were completely unable to clear B . hermsii . Together these results support the model that B1b cells generate the T-independent IgM required for the control and resolution of relapsing fever borreliosis.

J Bacteriol, 2003 Apr, 185(7), 2338 - 45
The Streptomyces coelicolor developmental transcription factor sigmaBldN is synthesized as a proprotein; Bibb MJ et al.; bldN is one of a set of genes required for the formation of specialized, spore-bearing aerial hyphae during differentiation in the mycelial bacterium Streptomyces coelicolor . Previous analysis (M . J . Bibb et al., J . Bacteriol . 182:4606-4616, 2000) showed that bldN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that translation from the most strongly predicted start codon (GTG(1)) would give rise to a sigma factor having an unusual N-terminal extension of ca . 86 residues . Here, by using a combination of site-directed mutagenesis and immunoblot analysis, we provide evidence that all bldN translation arises from initiation at GTG(1) and that the primary translation product is a proprotein (pro-sigma(BldN)) that is proteolytically processed to a mature species (sigma(BldN)) by removal of most of the unusual N-terminal extension . A time course taken during differentiation of the wild type on solid medium showed early production of pro-sigma(BldN) and the subsequent appearance of mature sigma(BldN), which was concomitant with aerial mycelium formation and the disappearance of pro-sigma(BldN) . Two genes encoding members of a family of metalloproteases that are involved in the regulated proteolytic processing of transcription factors in other organisms were identified in the S . coelicolor genome, but their disruption did not affect differentiation or pro-sigma(BldN) processing.

Lett Appl Microbiol, 2003, 36(4), 227 - 9
Proteolytic activity of a yeast cell wall lytic Arthrobacter species; Adamitsch BF et al.; AIMS: To investigate properties of the proteolytic activity of a yeast cell wall lytic soil bacterium identified as an Arthrobacter species . METHODS AND RESULTS: The organism was grown at pH 7.5 and 30 degrees C in shake flasks on media with different complex subtrates . Highest proteolytic activity assayed with azocaseine was detected in media with wheat gluten . In addition, l-leucine, l-alanine exopeptidase activity and esterase activity were found . The proteolytic activity showed stability up to pH 12, with a maximum at pH 11 . The temperature optimum was at 55 degrees C, but there was a loss in enzyme activity of 50% within 2 h . The proteolytic activity was inhibited by 3,4-dichloroisocumarin, whereas there was little or no effect with EDTA, pepstatin A or E64 . CONCLUSIONS: The proteolytic activity is highly alkaline stable . The formation of the enzyme can be induced by media with high protein content.

J Protein Chem, 2002 Nov, 21(8), 523 - 7
Effects of different buffers on the thermostability and autolysis of a cold-adapted protease MCP-01; Chen XL et al.; A cold-adapted protease MCP-01 was obtained from deep-sea psychrotrophic bacterium Pseudoaltermonas sp . SM9913 . The effects of four different buffers, all at 50 mmol/l concentration, on its thermostability and autolysis were studied . The autolysis process of MCP-01 was studied by capillary electrophoresis . The thermostability of MCP-01 increased successively in the following order: carbonate < Tris < phosphate < borate . The optimum temperature for casein hydrolysis also increased in the same order . This suggested that the conformation of MCP-01 was flexible and its autolytic susceptibility was affected by some factors in the buffers such as charge and ionic species . The results also showed that different buffers, in addition to affecting the autolysis speed, gave different patterns of autolysis products . In carbonate buffer, Tris buffer, phosphate buffer and borate buffer, the autolysis patterns of MCP-01 were different . These results suggested that protease MCP-01 probably have different conformations in different buffers, thus exposing different autolysis sites on the enzyme surface . In addition, the loss of activity correlated with the speed of autolysis in the four different buffers, showing that autolysis may be a reason for the low thermostability of the enzyme.

Exp Anim, 2003 Jan, 52(1), 77 - 80
Spontaneous development of dermatitis in DS-Nh mice under specific pathogen-free conditions; Watanabe A et al.; Spontaneous development of dermatitis in DS-Nh mice under specific pathogen-free conditions was examined to verify the hypothesis {Exp . Anim . 46: 225-229, 1997} that Stapylococcus aureus (S . aureus) infection is causally associated with the dermatitis . Observation of the mice up to 28 weeks of age indicated that obvious dermatitis does occur under S . aureus-free conditions, though the incidence was low (six of 42 females and two of 90 males) . Skin lesions in the absence of this bacterium showed histological changes very similar to those that can be observed under conventional conditions . In addition, hyperproduction of serum IgE was demonstrated in the dermatitis-positive mouse . These findings suggested that the dermatitis is triggered by IgE-mediated allergic reactions.

Phys Rev E Stat Nonlin Soft Matter Phys . 2003 Feb;67(2 Pt 1):021913 . Epub 2003 Feb 27.
Evolution equation of population genetics: relation to the density-matrix theory of quasiparticles with general dispersion laws; Bezak V; The Waxman-Peck theory of population genetics is discussed in regard of soil bacteria . Each bacterium is understood as a carrier of a phenotypic parameter p . The central objective is the calculation of the probability density with respect to p, Phi(p,t;p(0)), of the carriers living at time t>0, provided that initially at t(0)=0, all bacteria carried the phenotypic parameter p(0)=0 . The theory involves two small parameters: the mutation probability mu and a parameter gamma involved in a function w(p) defining the fitness of the bacteria to survive the generation time tau and give birth to an offspring . The mutation from a state p to a state q is defined by a Gaussian with a dispersion sigma(2)(m) . The author focuses our attention on a function phi(p,t) which determines uniquely the function Phi(p,t;p(0)) and satisfies a linear equation (Waxman's equation) . The Green function of this equation is mathematically identical with the one-particle Bloch density matrix, where mu characterizes the order of magnitude of the potential energy . (In the x representation, the potential energy is proportional to the inverted Gaussian with the dispersion sigma(2)(m)) . The author solves Waxman's equation in the standard style of a perturbation theory and discusses how the solution depends on the choice of the fitness function w(p) . In a sense, the function c(p)=1-w(p)/w(0) is analogous to the dispersion function E(p) of fictitious quasiparticles . In contrast to Waxman's approximation, where c(p) was taken as a quadratic function, c(p) approximately gammap(2), the author exemplifies the problem with another function, c(p)=gamma{1-exp(-ap(2))}, where gamma is small but a may be large . The author shows that the use of this function in the theory of the population genetics is the same as the use of a nonparabolic dispersion law E=E(p) in the density-matrix theory . With a general function c(p), the distribution function Phi(p,t;0) is composed of a delta-function component, N(t)delta(p), and a blurred component . When discussing the limiting transition for t--> infinity, the author shows that his function c(p) implies that N(t)-->N( infinity ) not equal 0 in contrast with the asymptotics N(t)-->0 resulting from the use of Waxman's function c(p) approximately p(2).

Indian J Exp Biol, 2002 Mar, 40(3), 369 - 72
Influence of cell wall degrading enzymes on colonization of N2 fixing bacterium, Azorhizobium caulinodans in rice; Buvana R et al.; In rice, nodule like structures were formed by inoculation of A . caulinodans combined with growth regulators and enzymes . Among the treatments, combination of cell wall degrading enzyme mixture and NAA with A . caulinodans induced more number of paranodules in rice . Total nitrogen content also increased in treated plants compared to uninoculated control.

Heredity, 2003 Feb, 90(2), 157 - 61
Wolbachia segregation dynamics and levels of cytoplasmic incompatibility in Drosophila sechellia; Charlat S et al.; In Drosophila sechellia, the endocellular bacterium Wolbachia induces cytoplasmic incompatibility (CI): in crosses involving infected males, a partial or complete embryonic mortality occurs unless the female bears the same Wolbachia . D . sechellia is known to harbour two Wolbachia variants, namely wSh and wSn, closely related to wHa and wNo, respectively, two strains infecting the populations of D . simulans from the Seychelles archipelago and New Caledonia . Strikingly, the two species show similar infection patterns: in D . sechellia, wSh can be present on its own or in double infection with wSn, but individuals carrying wSn only do not occur; in D . simulans, wHa can be present on its own or in double infection with wNo, but individuals carrying wNo only do not occur, or occur at very low frequency . Previous experiments on D . simulans showed that lines singly infected by wNo can be obtained by segregation, and stably maintained . Here we investigate this issue in D . sechellia through an 18 generation experiment, and show that wSn and wSh singly infected lines can arise by segregation . Using singly infected lines obtained in this experiment, we estimate the CI intensities of wSh and wSn in D . sechellia, and compare these to the CI intensities of the same Wolbachia injected into D . simulans . Our results do not suggest any consistent effect of the host species on the CI induced by wSh . On the contrary, it seems that wSn expression is repressed by host factors in D . sechellia.

Heredity, 2003 Mar, 90(3), 236 - 46
Incomplete sexual isolation in sympatry between subspecies of the butterfly Danaus chrysippus (L.) and the creation of a hybrid zone; Lushai G et al.; Subspecies chrysippus, dorippus and alcippus of the butterfly Danaus chrysippus differ at three biallelic colour gene loci . They have partially vicariant distributions, but their ranges overlap over a substantial part of central and East Africa, where hybridism is commonplace . We now report that the West African subspecies alcippus differs from other subspecies, not only in nuclear genotype but also in mitochondrial haplotype in both allopatry and sympatry . The maintenance of concordant nuclear and cytoplasmic genetic differences in sympatry, and in the face of hybridisation, is prima facie evidence for sexual isolation . Other evidence that suggests alcippus may be isolated from chrysippus and dorippus include differences in sex ratio (SR), heterozygote deficiency at one site and deduced differences in patterns of migration . We suggest that, within the hybrid zone, differential infection of subspecies by a male-killing Spiroplasma bacterium causes SR differences that restrict female choice, triggering rounds of heterotypic mating and consequent heterozygote excess that is largely confined to females . The absence of these phenomena from hybrid populations that test negative for Spiroplasma supports the hypothesis . The incomplete sexual isolation and partial vicariance of alcippus suggests that it is a nascent species.

Microbiology, 2003 Mar, 149(Pt 3), 665 - 72
Catalase (KatA) and KatA-associated protein (KapA) are essential to persistent colonization in the Helicobacter pylori SS1 mouse model; Harris AG et al.; Helicobacter pylori infects the human gastric mucosa and elicits an aggressive inflammatory response . Despite the severity of the inflammatory response, the bacterium is able to persist and cause a chronic infection . It is believed that antioxidant defence mechanisms enable this organism to persist . Wild-type H . pylori strain SS1, and KatA- and KapA-deficient mutants, were used to infect C57/BL6 mice to test this hypothesis . Neither KatA nor KapA was essential for the initial colonization of H . pylori SS1 in the murine model of infection . The wild-type SS1 colonized the gastric mucosa at significantly higher levels than both mutants throughout the 24-week experiment . Neither KatA- nor KapA-deficient mutants were able to maintain consistent ongoing colonization for the 24-week period, indicating the necessity of both KapA and KatA in sustaining a long-term infection . At 24 weeks, 5/10 mice inoculated with the KatA mutant and 2/10 mice inoculated with the KapA mutant were colonized, compared with 10/10 of the mice inoculated with the wild-type SS1 . An increase in the severity of inflammation in the wild-type-inoculated mice appeared to correlate with the decline in colonization of animals inoculated with the mutants, suggesting that increased oxidative stress militated against continued infection by the mutants . These data indicate that KapA may be of equal or greater importance than KatA in terms of sustained infection on inflamed gastric mucosae.

J Med Invest, 2003 Feb, 50(1-2), 48 - 54
Long-term follow-up of gastric metaplasia after eradication of Helicobacter pylori; Urakami Y et al.; BACKGROUNDS AND AIMS: There is no commonly accepted view concerning changes in gastric metaplasia after the eradication of Helicobacter pylori . The aim of this study was to evaluate the long-term course of gastric metaplasia after the eradication of this bacterium . METHODS: The subjects were 59 patients with duodenal ulcer who were positive for Helicobacter pylori . Forty patients were classified as the eradication group . Gastric metaplasia was endoscopically and histologically evaluated before and after eradication of this bacterium . The follow-up period was 2-7.1 years . In the other 19 patients in the non-eradication group, gastric metaplasia was evaluated before and after treatment of the ulcer . Gastric metaplasia was evaluated in terms of its extent and type in all patients . RESULTS: Gastric metaplasia showed the incomplete type before eradication but changed to the complete type after eradication, which persisted for a long period . The extent of gastric metaplasia increased after eradication . In the non-eradication group, gastric metaplasia infrequently changed to the complete type during the scarring period of ulcer . CONCLUSION: Gastric metaplasia changed to the complete type after the eradication of Helicobacter pylori, which persisted for a long period.

Biochemistry, 2003 Mar 18, 42(10), 2806 - 15
A flavodiiron protein and high molecular weight rubredoxin from Moorella thermoacetica with nitric oxide reductase activity; Silaghi-Dumitrescu R et al.; A five-gene "oxidative stress protection" cluster has recently been described from the strictly anaerobic, acetogenic bacterium, Moorella thermoacetica {Das, A., et al . (2001) J . Bacteriol . 183, 1560-1567} . Within this cluster are two cotranscribed genes, fprA (for A-type flavoprotein) and hrb (for high molecular weight rubredoxin) whose encoded proteins have no known functions . Here we show that FprA and Hrb are expressed in M . thermoacetica under normal anaerobic growth conditions and report characterizations of the recombinant FprA and Hrb . FprA contains flavin mononucleotide (FMN) and a non-heme diiron site . Mossbauer spectroscopy shows that the irons of the diferric site are antiferromagnetically coupled, implying a single-atom, presumably solvent, bridge between the irons . Hrb contains FMN and a rubredoxin-like {Fe(SCys)4} site . NADH does not directly reduce either the FMN or the diiron site in FprA, whereas Hrb functions as an efficient NADH:FprA oxidoreductase . Substitution of zinc for iron in Hrb completely abolished this activity . The observation that homologues of FprA from other organisms show O2 and/or anaerobic NO consumption activity prompted an examination of these activities for M . thermoacetica FprA . The Hrb/FprA combination does indeed have both NADH:O2 and NADH:NO oxidoreductase activities . The NO reductase activity, however, was significantly more efficient due to a lower Km for NO (4 M) and to progressive and irreversible inactivation of FprA during O2 reductase turnover but retention of activity during NO reductase turnover . Substitution of zinc for iron in FprA completely abolished these reductase activities . The stoichiometry of 1 mol of NADH oxidized:2 mol of NO consumed implies reduction to N2O . Fits of an appropriate rate law to the kinetics data are consistent with a mechanism in which 2NO's react at each FprA active site in the committed step . Expression of FprA in an Escherichia coli strain deficient in NO reductase restored the anaerobic growth phenotype of cultures exposed to otherwise toxic levels of exogenous NO . The accumulated results indicate that Hrb/FprA is fully capable of functioning in nitrosative stress protection in M . thermoacetica.

Glycobiology, 2003 Jun, 13(6), 471 - 8 Epub 2003 Feb 20.
The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x; van Die I et al.; Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S . mansoni . We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) . Monoclonal antibodies against the carbohydrate antigens Lewisx (Lex) and GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDNF) inhibit binding of DC-SIGN to SEAs, suggesting that these glycan antigens may be critically involved in binding . In a solid-phase adhesion assay, DC-SIGN-Fc binds polyvalent neoglycoconjugates that contain the Lex antigen, whereas no binding was observed to Galbeta1-4GlcNAc, and binding to neoglycoconjugates containing only alpha-fucose or oligosaccharides with a terminal alpha1-2-linked fucose is low . These data indicate that binding of DC-SIGN to Lex antigen is fucose-dependent and that adjacent monosaccharides and/or the anomeric linkage of the fucose are important for binding activity . Previous studies have shown that DC-SIGN binds HIV gp120 that contains high-mannose-type N-glycans . Site-directed mutagenesis within the carbohydrate recognition domain (CRD) of DC-SIGN demonstrates that amino acids E324 and E347 are involved in binding to HIV gp120, Lex, and SEAs . By contrast, mutation of amino acid Val351 abrogates binding to SEAs and Lex but not HIV gp120 . These data suggest that DC-SIGN recognizes these ligands through different (but overlapping) regions within its CRD . Our data imply that DC-SIGN not only is a pathogen receptor for HIV gp120 but may also function in pathogen recognition by interaction with the carbohydrate antigens Lex and possibly LDNF, which are found on important human pathogens, such as schistosomes and the bacterium Helicobacter pylori.

J Laryngol Otol, 2003 Feb, 117(2), 118 - 21
The prevalence of Helicobacter pylori infection in malignant and premalignant conditions of the head and neck; Rubin JS et al.; Helicobacter pylori is an accepted cause of chronic active gastritis and has a major causative role in peptic ulceration . It is a gastric carcinogen . Its role in non-ulcer dyspepsia (NUD) is less clear; yet 50 per cent of patients with NUD are infected with H pylori . H pylori has been investigated in several other organ systems, but has not been investigated extensively in squamous cell carcinoma of the upper aerodigestive tract, a region which could be directly exposed to the bacterium by gastro-oesophageal reflux (GOR) . In this study 61 patients with severe laryngeal dysplasia or frank carcinoma of the head and neck are striated by age, investigated for the presence of antibodies to H pylori and compared to age and sex matched controls . In the age group of 46-61 years, the presence of H pylori antibodies was marginally greater in the experimental (63.0 per cent) than the control group (40.7 per cent) (Pearson Chi square p = 0.055, Fisher 2-sided exact test p = 0.066) . When combining this age group with the younger age group and thereby creating two roughly equal groups (n = 31 and n = 30) there was also a statistical trend towards increased positivity in the experimental group . These findings are discussed in the light of other studies with gastro-oesophageal reflux disease (GORD).

BMC Microbiol . 2003 Feb 06;3(1):3.
New knowledge from old: in silico discovery of novel protein domains in Streptomyces coelicolor; Yeats C et al.; BACKGROUND: Streptomyces coelicolor has long been considered a remarkable bacterium with a complex life-cycle, ubiquitous environmental distribution, linear chromosomes and plasmids, and a huge range of pharmaceutically useful secondary metabolites . Completion of the genome sequence demonstrated that this diversity carried through to the genetic level, with over 7000 genes identified . We sought to expand our understanding of this organism at the molecular level through identification and annotation of novel protein domains . Protein domains are the evolutionary conserved units from which proteins are formed . RESULTS: Two automated methods were employed to rapidly generate an optimised set of targets, which were subsequently analysed manually . A final set of 37 domains or structural repeats, represented 204 times in the genome, was developed . Using these families enabled us to correlate items of information from many different resources . Several immediately enhance our understanding both of S . coelicolor and also general bacterial molecular mechanisms, including cell wall biosynthesis regulation and streptomycete telomere maintenance . DISCUSSION: Delineation of protein domain families enables detailed analysis of protein function, as well as identification of likely regions or residues of particular interest . Hence this kind of prior approach can increase the rate of discovery in the laboratory . Furthermore we demonstrate that using this type of in silico method it is possible to fairly rapidly generate new biological information from previously uncorrelated data.

Microbiology, 2003 Feb, 149(Pt 2), 445 - 50
Catalytic properties of an endogenous beta-lactamase responsible for the resistance of Azospirillum lipoferum to beta-lactam antibiotics; Boggio SB et al.; Azospirillum lipoferum RG20, a nitrogen-fixing bacterium found in all kind of soils, was found to be naturally resistant to penicillins and cephalosporins . 6-beta-Bromopenicillanic acid, an irreversible inhibitor of serine-beta-lactamases, completely abolished this resistance . A beta-lactamase was purified 518-fold from a cell-free extract of A . lipoferum RG20 . A single band on SDS-PAGE (apparent molecular mass 31000 Da) and on isoelectric focussing (pI9.35) was observed with the purified protein . The enzyme hydrolysed benzylpenicillin, ampicillin, cephalothin and cephaloridine with comparable k(cat) values and catalytic efficiencies . However, carbenicillin and cefotaxime were hydrolysed with significantly lower kinetic parameters and oxacillin was hydrolysed at a rate 100 times slower . The purified beta-lactamase was inhibited by clavulanic acid and sulbactam but not by EDTA or aztreonam . Its substrate and inhibitor profiles are consistent with those of the broad-spectrum beta-lactamases inhibited by clavulanic acid (group 2b of the Bush-Jacoby-Medeiros scheme) . The effect of pH on k(cat) and K(m) values for benzylpenicillin hydrolysis was studied . The dependence of k(cat) on pH suggests that the enzyme-substrate (ES) complex must be in at least three protonation states: two with k(cat) values equal to 2800 and 1450 s(-1) and a third inactive one {pK(1(ES)) 4.7 and pK(2(ES)) 7.9} . Similarly, the dependence of k(cat)/K(m) on pH can be explained by postulating that the enzyme free form can be at least in three different protonation states: two of them with k(cat)/K(m) values equal to 2.7 x 10(6) and 3.7 x 10(8) M(-1) s(-1) and a third one unable to productively bind substrate . Interestingly, the dependence of k(cat)/K(m) on pH is consistent with positive cooperativity for proton binding to the enzyme free form {pK(1(E)) 8.5 and pK(2(E)) 7.2}.

Microbiology, 2003 Feb, 149(Pt 2), 419 - 30
Thioredoxin 2 is involved in oxidative stress defence and redox-dependent expression of photosynthesis genes in Rhodobacter capsulatus; Li K et al.; Thioredoxins are small ubiquitous proteins that display different functions mainly via redox-mediated processes . The facultatively photosynthetic bacterium Rhodobacter capsulatus harbours at least two genes for thioredoxin 1 and 2, trxA and trxC . It is demonstrated that thioredoxin 2 of R . capsulatus can partially replace the thioredoxin 1 function as a hydrogen donor for methionine sulfoxide reductase but cannot replace thioredoxin 1 as a subunit of phage T7 DNA polymerase . By inactivating the trxC gene in R . capsulatus, it is shown that thioredoxin 2 is involved in resistance against oxidative stress . As thioredoxin 1 of Rhodobacter sphaeroides, R . capsulatus thioredoxin 2 affects the oxygen-dependent expression of photosynthesis genes, albeit in an opposite way . The trxC mutant of R . capsulatus shows a stronger increase in photosynthesis gene expression after a decrease in oxygen tension than the isogenic wild-type strain . The expression of the trxC gene is downregulated by oxygen.

Microb Pathog, 2003 Feb, 34(2), 57 - 63
Infection of human fibroblast-like synovial cells with Chlamydia trachomatis results in persistent infection and interleukin-6 production; Hanada H et al.; Recent studies have shown that the urogenital pathogen Chlamydia trachomatis to be a major bacterium triggering reactive arthritis (ReA), and is able to induce interleukin-6 (IL-6) production in human fibroblast-like synovial cells (FSC) in vitro . In the present study, we examined the correlation between IL-6 production and multiplication of chlamydia in FSC . All FSC from five patients secreted highly increased quantities of IL-6 in a dose-dependent and time-dependent fashion . Heat and UV inactivated chlamydia failed to enhance production of IL-6 . When azithromycin was added to infected cultures of FSC at 0 or 48 h after infection, the level of IL-6 production was very low . Transmission electron microscopy of such infected cultures revealed many abnormal forms of chlamydia within the inclusions in FSC . From one step-growth curve experiments, it was suggested that C . trachomatis hardly multiplied in FSC . In contrast, in C . trachomatis infected HeLa 229 cells, chlamydia multiplied as usual, but little IL-6 production were found . These observations indicated that live chlamydia and the persistence of chlamydia may be essential for stimulating the synthesis of IL-6 in FSC.

Drug Discov Today, 2003 Mar 15, 8(6), 239 - 40
Scientists expand the genetic code; Foubister V; A bacterium has been created that synthesizes an unnatural amino acid and incorporates it into proteins with a fidelity and efficiency that rivals that of the 20 natural amino acids.

Microb Pathog, 2003 Jan, 34(1), 17 - 25
Interleukin-1beta responses to Mycoplasma pneumoniae infection are cell-type specific; Yang J et al.; Interleukin-1beta (IL-1beta) is a major proinflammatory cytokine that is involved in many important cellular functions such as proliferation, differentiation, and activation of different cell types . Its mature form is released from the cells in response to various bacterial and viral infections, and it plays a significant role in host defense . Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans following attachment to respiratory epithelium, as well as extrapulmonary infections . Very little is known about the role of cytokines in pathogenesis or the response of target cells to M.pneumoniae attachment . The purpose of this study was to investigate the ability of M . pneumoniae to induce IL-1beta in human lung epithelial carcinoma A549 and in human monocytic U937 cell lines . Following M . pneumoniae infection, both IL-1beta mRNA and protein were induced in A549 cells vs . no induction in uninfected cells; however, the protein remained inside the A549 cells . Similarly, M . pneumoniae infection strongly increased mRNA and extracellular protein levels in U937 cells, which unlike A549 cells did exhibit baseline constitutive levels . De novo IL-1beta protein expression was verified by cycloheximide studies . M . pneumoniae infection did not affect constitutive caspase-1 mRNA or protein levels in either cell line . Reduced caspase-1 activity in A549 cell lysates suggests the presence of an endogenous caspase-1 inhibitory component in the A549 cells . These collective data confirm previous studies that show that M . pneumoniae is a potent inducer of cytokines following adherence to host target cells, and establish that IL-1beta release in response to M . pneumoniae infection is cell-type specific, thus emphasizing the importance of carefully considering multiple cell types in M . pneumoniae pathogenesis studies involving both immune cells and cytokine release patterns.

J Bacteriol, 2003 Mar, 185(6), 2022 - 5
Plasmid R16 ArdA protein preferentially targets restriction activity of the type I restriction-modification system EcoKI; Thomas AT et al.; The ArdA antirestriction protein of the IncB plasmid R16 selectively inhibited the restriction activity of EcoKI, leaving significant levels of modification activity under conditions in which restriction was almost completely prevented . The results are consistent with the hypothesis that ArdA functions in bacterial conjugation to allow an unmodified plasmid to evade restriction in the recipient bacterium and yet acquire cognate modification.

Curr Opin Microbiol, 2003 Feb, 6(1), 66 - 71
Virulence determinants and protective antigens of Francisella tularensis; Sjostedt A; Very little is known about virulence mechanisms of the highly virulent bacterium Francisella tularensis . Specific genetic features of F . tularensis have been obstacles for the development of effective tools for genetic manipulation . However, recent genomic sequencing and large-scale proteomic work have resulted in a substantial increase in the knowledge of F . tularensis . There is also a paucity of information on potential vaccine candidates . Recent work assessing the protective efficacy of the F . tularensis lipopolysaccharide has resulted in important contributions to the understanding of host-protective mechanisms . T-cell-mediated immunity appears to be crucial to protect against virulent F . tularensis strains . Few other vaccine candidates have been identified.

Diagn Microbiol Infect Dis, 2003 Feb, 45(2), 107 - 15
In vitro Bartonella quintana infection modulates the programmed cell death and inflammatory reaction of endothelial cells; Liberto MC et al.; Bartonella quintana is an epicellular bacterium, which in vivo as well as in vitro, invades endothelial cells and develops within them inducing proliferative effects that play a pivotal role in neovascular manifestation of this disease . We investigated the effect of live Bartonella quintana and its LPS on apoptosis and inflammatory response in HUVEC-C, an endothelial cell line . The kinetics of the programmed cell death of Bartonella quintana-infected HUVEC-C showed a peculiar course . Even if early during infection apoptosis reached a peak after 6 h, later on apoptosis was inhibited . Such apoptosis inhibition was not observed during Bartonella quintana lipopolysaccharide treatment because LPS-stimulated HUVEC-C did progress to cell death . Evaluation of multiple cell signal transduction pathways revealed an overexpression of Apaf 1 and caspase 8 in HUVEC-C after 2 h of infection, and of bcl-2 starting from 10 h post Bartonella quintana infection . Moreover, Bartonella quintana and its LPS showed a different effect on the activation of genes involved in inflammatory response as revealed by molecular analysis of host cells . Bartonella quintana appears to be able to inhibit programmed cell death, inducing intracellular signals leading to survival and proliferation through the bcl-2 gene, despite the early increase of inflammatory status induced in endothelial cells . This mechanism, together with a poor endotoxin ability to stimulate strong inflammatory response, could contribute to the capability of the bacteria to persist intracellularly, causing chronic disease and producing neovascular manifestations.

Biomed Instrum Technol, 2003 Jan-Feb, 37(1), 49 - 50
How to safely maintain equipment where hazardous materials may lurk; Jones I; The best protection is preparation . Assess any equipment/device that requires repair or maintenance for potential contamination or source of injury, such as sharp edges . Know where your protective apparel is located and use it . Review decontamination procedures and keep disinfectants available . Know your employee report of injury program and seek medical care whenever you have concerns regarding potential injury or exposure . Know your policies and procedures and where to find them if you need further information . Your infection control staff should be available 24 hours a day . The standard personal protective equipment that your employer is required to make available to you should include gloves, masks, eye protection, and gowns . In addition, if you are expected to enter a negative pressure room while a patient is in Airborne Precautions, you must be properly fit tested with an N95 respirator prior to entering the room . This respirator is very similar to a normal mask, but is able to filter out particles such as the TB bacterium . Infection control boils down to 2 commandments: 1 . Wash your hands! 2 . Use your head/common sense: If it looks soiled--clean it . If you have concerns--ask for clarification . If you think you have been exposed--seek medical assistance.

Arch Microbiol, 2003 Mar, 179(3), 191 - 6 Epub 2003 Feb 04.
Saccharin as a sole source of carbon and energy for Sphingomonas xenophaga SKN; Schleheck D et al.; A bacterium, strain SKN, that was able to utilize saccharin as the sole source of carbon and energy for aerobic growth, was enriched and isolated from communal sewage . The isolate was identified as a strain of Sphingomonas xenophaga . Saccharin was quantitatively converted to cell material, sulfate, ammonium and, presumably, CO(2) . The specific rate of saccharin-dependent oxygen uptake during growth reached a maximum before the culture entered the stationary phase and then fell to undetectable levels . Saccharin was degraded only in the presence of molecular oxygen . Catechol was detected as an intermediate during degradation of saccharin in whole cells and catechol 1,2-dioxygenase was expressed inducibly during growth with saccharin . There was an apparent requirement of 2 mol O(2)/mol saccharin to remove the substituents on the ring and to cleave the ring . We presume that S . xenophaga SKN synthesizes a multi-component saccharin dioxygenase that simultaneously cleaves off both vicinal substituents from the aromatic ring to yield catechol and the undefined precursor of CO(2) as well as sulfate and ammonium ions.

Dis Aquat Organ, 2003 Jan 22, 53(1), 15 - 23
Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea); Nunan LM et al.; Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar . Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P . monodon tissue as the template . The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene . These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples . The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P . vannamei tissue and several other strains of bacteria . The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested . The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays . This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative . The probe was specific for both natural and experimental rickettsial infections . Hybridization with this probe required a stringent temperature of 65 degrees C, otherwise cross-reactivity was observed with other types of bacteria.

Nippon Rinsho, 2003 Jan, 61(1), 137 - 45
{The present status and problems of Helicobacter pylori eradication therapy with a special reference to gastric cancer}; Shimoyama T et al.; Since the eradication therapy of Helicobacter pylori(H . pylori) for peptic ulcer was covered by Japanese health insurance at 2000 November, this therapy has been generalized in Japan . However, some problems have cropped up at the time, for example, emergence of clarithromycin resistant bacterium etc . On the other hand, investigation of relation between H . pylori and gastric cancer is making progress . Uemura, et al . demonstrated H . pylori had an important role for gastric carcinogenesis by the elegant prospective study in 2001 . Under the present circumstances, the Japanese society of Helicobacter Research has to reconsider the guideline for eradication therapy . In this paper, we would like to state the present status and problems of eradication therapy of the current guideline for eradication therapy, especially focusing on gastric cancer.

FEBS Lett, 2003 Feb 27, 537(1-3), 161 - 5
Photoreduction of the quinone pool in the bacterial photosynthetic membrane: identification of infrared marker bands for quinol formation; Mezzetti A et al.; The photoreduction of the quinone (Q) pool in the photosynthetic membrane of the purple bacterium Rhodobacter sphaeroides was investigated by steady-state and time-resolved Fourier transform infrared difference spectroscopy . The results are consistent with the existence of a homogeneous Q pool inside the chromatophore membrane, with a size of around 20 Q molecules per reaction center . IR marker bands for the quinone/quinol (Q/QH(2)) redox couple were recognized . QH(2) bands are identified at 1491, 1470, 1433 and 1388-1375 cm(-1) . The 1491 cm(-1) band, which is sensitive to (1)H/(2)H exchange, is assigned to a C-C ring mode coupled to a C-OH mode . A feature at approximately 1743/1720 cm(-1) is tentatively related to a perturbation of the carbonyl modes of phospholipid head groups induced by QH(2) formation . Complex conformational changes of the protein in the amide I and II spectral ranges are also apparent during reduction and reoxidation of the Q pool.

J Biol Chem, 2003 May 9, 278(19), 16488 - 93 Epub 2003 Feb 25.
Complete spectra of the far-red chemiluminescence of the oxygenase reaction of Mn2+-activated ribulose-bisphosphate carboxylase/oxygenase establish excited Mn2+ as the source; Lilley RM et al.; Chemiluminescence emitted by Mn(2+)-activated ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) while catalyzing oxygenation was analyzed to clarify the source of the emission . Using dual detectors capturing radiation over a wide range of visible and infrared wavelengths, we tested for radiation from singlet O(2) decay and found it to be essentially absent (less than 0.1% of the total luminescence intensity) . Spectra were determined between 647 and 885 nm with a very sensitive, charge-coupled detector-based spectrograph to detect differences in the emission spectra between rubiscos from bacterial and higher plant sources . All Mn(2+)-activated rubiscos emitted a broad, smooth spectrum of chemiluminescence, unchanging as the reaction progressed . The spectra from higher plant rubiscos (spinach and both the wild type and an L335V mutant from tobacco), all exhibited maxima at about 800 nm . However, Mn(2+)-activated rubisco from the bacterium, Rhodospirillum rubrum, emitted at shorter wavelengths (760 nm peak), demonstrating host ligand-field influences arising from aminoacyl residue differences and/or conformational changes caused by the absence of small subunits . The findings provide strong evidence that the chemiluminescence arises from an excited state of the active-site Mn(2+) that is produced during oxygenation . We propose that the Mn(2+) becomes excited by a one-electron exchange mechanism of oxygenation that is not available to Mg(2+)-activated rubisco.

Emerg Infect Dis, 2003 Feb, 9(2), 204 - 10
Applying network theory to epidemics: control measures for Mycoplasma pneumoniae outbreaks; Ancel Meyers L et al.; We introduce a novel mathematical approach to investigating the spread and control of communicable infections in closed communities . Mycoplasma pneumoniae is a major cause of bacterial pneumonia in the United States . Outbreaks of illness attributable to mycoplasma commonly occur in closed or semi-closed communities . These outbreaks are difficult to contain because of delays in outbreak detection, the long incubation period of the bacterium, and an incomplete understanding of the effectiveness of infection control strategies . Our model explicitly captures the patterns of interactions among patients and caregivers in an institution with multiple wards . Analysis of this contact network predicts that, despite the relatively low prevalence of mycoplasma pneumonia found among caregivers, the patterns of caregiver activity and the extent to which they are protected against infection may be fundamental to the control and prevention of mycoplasma outbreaks . In particular, the most effective interventions are those that reduce the diversity of interactions between caregivers and patients.

Helicobacter, 2003 Feb, 8(1), 72 - 8
Statistical model of the interactions between Helicobacter pylori infection and gastric cancer development; Welin M et al.; BACKGROUND: The bacterium Helicobacter pylori is associated with a number of gastrointestinal diseases, such as gastric ulcer, duodenal ulcer and gastric cancer . Several histological changes may be observed during the course of infection; some may influence the progression towards cancer . The aim of this study was to build a statistical model to discover direct interactions between H . pylori and different precancerous changes of the gastric mucosa, and in what order and to what degree those may influence the development of the intestinal type of gastric cancer . METHODS: To find direct and indirect interactions between H . pylori and different histological variables, log-linear analyses were used on a case-control study . To generate mathematically and biologically relevant statistical models, a designed algorithm and observed frequency tables were used . RESULTS: The results show that patients with H . pylori infection need to present with proliferation and intestinal metaplasia to develop gastric cancer of the intestinal type . Proliferation and intestinal metaplasia interacted with the variables atrophy and foveolar hyperplasia . Intestinal metaplasia was the only variable with direct interaction with gastric cancer . Gender had no effect on the variables examined . CONCLUSION: The direct interactions observed in the final statistical model between H . pylori, changes of the mucosa and gastric cancer strengthens and supports previous theories about the progression towards gastric cancer . The results suggest that gastric cancer of the intestinal type may develop from H . pylori infection, proliferation and intestinal metaplasia, while atrophy and foveolar hyperplasia interplay with the other histological variables in the disease process.

Pac Symp Biocomput . 2003;:41-52.
MOPAC: motif finding by preprocessing and agglomerative clustering from microarrays; Ganesh R et al.; We propose a novel strategy for discovering motifs from gene expression data . The gene expression data in our experiments comes from DNA Microarray analysis of the bacterium E . coli in response to recovery from nutrient starvation . We have annotated the data and identified the upregulated genes . Our interest is to find common regulatory motifs that are responsible for the upregulation of these specific genes . We assume that a common motif that a regulatory protein can bind to will be present in the upstream region of the upregulated genes and will not be present in the upstream regions of genes that showed a constant level of expression over time . Our objective is to find the common motifs that are present in at least some of the upstream sequences of upregulated genes and not present in the control set, which is the set of genes whose expression remained the same . Because it is possible that there could be several subsets of co-regulated genes under different control mechanisms among the co-expressed genes, we do not want to require motifs to be present in all upregulated sequences . Therefore, we propose a new algorithm for finding such motifs through stages of pre-processing, denoising, agglomerative clustering and consensus checking . Through this process, we have found some motifs that are good candidates for further validation.

DNA Res, 2002 Dec 31, 9(6), 189 - 97
Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110; Kaneko T et al.; The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined . The genome of B . japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1% . No plasmid was detected . The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes . Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes . The remaining 18% had no apparent similarity to reported genes . Thirty-four percent of the B . japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti, while 23% were unique to this species . A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by Gottfert et al., was identified . Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced . A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome . It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island . DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes . These observations suggest plasticity of the B . japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.

Indian J Exp Biol, 2002 Aug, 40(8), 967 - 70
Photobiodegradation of pyridine by Rhodopseudomonas palustris JA1; Ramana ChV et al.; A purple non-sulfur bacterium isolated from dairy effluent was identified as Rps . palustris JA1 . This organism was able to grow on pyridine as sole source of carbon in a light dependent anaerobic process with a doubling time of 30 h . Intermediates of pyridine photobiodegradation were identified as glycine and malonate, produced in stoichiometric molar ratios with simultaneous utilization, yielding biomass.

Dtsch Tierarztl Wochenschr, 2002 Dec, 109(12), 507 - 9
{Pathogenesis and immune reactions of paratuberculosis}; Valentin-Weigand P; Mycobacterium avium subspecies paratuberculosis (M . ptb) is known as the cause of paratuberculosis for over a century but the knowledge on biology of the organism and pathogenesis of the disease is still limited . There are several reasons for the present lack of progress, these are (i) the extremely slow growth of the bacterium, a feature which has also protected the organism against researchers, (ii) confusion over its taxonomy and identification, (iii) limited possibilities for the application of molecular biology techniques, and (iv) the extremely long incubation period in natural infection for which no suitable laboratory model exists . Despite these discouraging facts, recent research efforts have led to important findings, which have shown that a better understanding of the disease may contribute to the improvement of control strategies . This presentation focuses mainly on the unique nature of M . ptb within the mycobacteria and the central role of the macrophage in pathogenesis and immune response . More details can be found in a number of excellent recent reviews (see list of references).

Dtsch Tierarztl Wochenschr, 2002 Dec, 109(12), 504 - 6
{Paratuberculosis: the pathogen and routes of infection}; Gerlach GF; Paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (M . paratuberculosis), is a chronic and incurable enteritis of ruminants with economic importance worldwide . The infectious agent is an acid-fast rod defined solely based on its mycobactin-dependent growth in vitro and the presence of insertion element IS900 . The bacterium, which is difficult to culture primarily due to its extremely slow growth, occurs not only in cattle but also in other ruminant . In addition, it has been isolated from non-ruminant species . Despite its wide spectrum of potential hosts the contact between adult cattle and calves is the predominant route of infection within a herd as well as among herds . To interrupt this route of infection hygienic measures, primarily for the housing and feeding of calves, as well as diagnostic measures prior to trading of cattle are urgently required.

Bioessays, 2003 Mar, 25(3), 274 - 82
AraC protein: a love-hate relationship; Schleif R; In the bacterium Escherichia coli, the AraC protein positively and negatively regulates expression of the proteins required for the uptake and catabolism of the sugar L-arabinose . This essay describes how work from my laboratory on this system spanning more than thirty years has aided our understanding of positive regulation, revealed DNA looping (a mechanism that explains many action-at-a-distance phenomena) and, more recently, has uncovered the mechanism by which arabinose shifts AraC from a state where it prefers to bind to two well-separated DNA half-sites and form a DNA loop to a state where it binds to two adjacent half-sites and activates transcription . This work required learning how to assay, purify, and work with a protein possessing highly uncooperative biochemical properties . Present work is focussed on understanding arabinose-responsive mechanism in atomic detail and is also directed towards understanding protein structure and function well enough to be able to engineer the allosteric mechanism seen in AraC onto other proteins .

Bioessays, 2003 Mar, 25(3), 259 - 65
On the mechanism of Wolbachia-induced cytoplasmic incompatibility: confronting the models with the facts; Poinsot D et al.; The endocellular bacterium Wolbachia manipulates the reproduction of its arthropod hosts for its own benefit by various means, the most widespread being cytoplasmic incompatibility (CI) . To date, the molecular mechanism involved in CI has not been elucidated . We examine here three different CI models described in previous literature, namely, the "lock-and-key", "titration-restitution" and "slow-motion" models . We confront them with the full range of CI patterns discovered so far, including the most complex ones such as multiple infections, asymmetrical and partial compatibility relationships and the existence of Wolbachia variants that can rescue the host from CI but not induce it . We conclude that the lock-and-key model is the most parsimonious of the models and fits the observations best . The two other models cannot be categorically invalidated, but they encounter some difficulties that make additional hypotheses necessary .

Arch Pharm (Weinheim), 2002, 335(11-12), 511 - 25
Antitubercular isoniazid and drug resistance of Mycobacterium tuberculosis--a review; Scior T et al.; Isoniazid is one of the most potent drugs available for tuberculosis treatment . As a pro-drug it requires activation, which is performed by catalase/peroxidase . The active principle, whose identity has not yet been determined unambiguously, then acts on at least one target molecule, the enoyl-acyl carrier protein, required for the synthesis of the vital mycolic acids present in the cell wall of the bacterium . Some other targets have been proposed in order to explain the unusual potency of isoniazid; however, the supporting data are still controversial . We thoroughly discuss the action of isoniazid, resistance mechanisms, and the possible active product, which includes an isonicotinic acid-NADH adduct as well as a meta-isomer of NADH . Both structures have been probed positively in a 3D modeling analysis.

Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 529 - 31 Epub 2003 Feb 21.
Crystallization and preliminary X-ray analysis of an alkaline serine protease from Nesterenkonia sp; Bakhtiar S et al.; A novel calcium-independent serine protease from an alkaliphilic bacterium, Nesterenkonia sp . AL20, has been purified and crystallized at 296 K using sodium formate as the main precipitant . This enzyme is optimally active at pH 10, exhibits high stability towards autolytic digestion and its stability is not affected by the presence of EDTA or detergents . The triangular prism-shaped crystals diffracted X-rays to beyond 1.5 A at a synchrotron beamline, with space group R3 and unit-cell parameters a = b = 92.26, c = 137.88 A . A complete data set has been collected to 1.39 A resolution . The asymmetric unit is estimated and confirmed by self-rotation function calculation to contain two molecules, giving a crystal Volume per protein mass (V(M)) of 2.68 A(3) Da(-1) and a solvent content of 54%.

Infect Immun, 2003 Mar, 71(3), 1316 - 20
Reduced glutathione is required for pertussis toxin secretion by Bordetella pertussis; Stenson TH et al.; The abilities of cysteine-containing compounds to support growth of Bordetella pertussis and influence pertussis toxin transcription, assembly, and secretion were examined . Cysteine is an essential amino acid for B . pertussis and must be present for protein synthesis and bacterial growth . However, cysteine can be metabolized to sulfate, and high concentrations of sulfate can selectively inhibit transcription of the virulence factors, including pertussis toxin, via the BvgAS two-component regulatory system in a process called modulation . In addition, pertussis toxin possesses several disulfide bonds, and the cysteine-containing compound glutathione can influence oxidation-reduction reactions and perhaps disulfide bond formation . Bacterial growth was not observed in the absence of a source of cysteine . Oxidized glutathione, as a sole source of cysteine, also did not support bacterial growth . Cysteine, cystine, and reduced glutathione did support bacterial growth, and none of these compounds caused modulation at the concentrations tested . Similar amounts of periplasmic pertussis toxin were detected regardless of the source of cysteine; however, in the absence of reduced glutathione, pertussis toxin was not efficiently secreted . Addition of the reducing agent dithiothreitol was unable to compensate for the lack of reduced glutathione and did not promote secretion of pertussis toxin . These results suggest that reduced glutathione does not affect the accumulation of assembled active pertussis toxin in the periplasm but plays a role in efficient pertussis toxin secretion by the bacterium.

Biochimie, 2002 Nov, 84(11), 1047 - 59
Complex restriction enzymes: NTP-driven molecular motors; Bourniquel AA et al.; Survival is assuredly the prime directive for all living organisms either as individuals or as a species . One of the main challenges encountered by bacterial populations is the danger of bacteriophage attacks, since infection of a single bacterium may rapidly propagate, decimating the entire population . In order to protect themselves against this acute threat, bacteria have developed an array of defence mechanisms, which range from preventing the infection itself via interference with bacteriophage adsorption to the cell surface and prevention of phage DNA injection, to degradation of the injected phage DNA . This last defence mechanism is catalysed by the bacterial restriction-modification (R-M) systems, and in particular, by nucleoside 5'-triphosphate (NTP)-dependent restriction enzymes, e.g . type I and type III R-M systems or the modification-dependent endonucleases . Type I and type III restriction systems have dual properties . They may either act as methylases and protect the host's own DNA against restriction by methylating specific residues, or they catalyse ATP-dependent endonuclease activity so that invading foreign DNA lacking the host-specific methylation is degraded . These defence mechanism systems are further complemented by the presence of methylation-dependent, GTP-dependent endonucleases, that restricts specifically methylated DNA . Although all three types of endonucleases are structurally very different, they share a common functional mechanism . They recognise and bind to specific DNA sequences but do not cleave DNA within those target sites . They belong to the general class of DNA motor proteins, which use the free energy associated with nucleoside 5'-triphosphate hydrolysis to translocate DNA so that the subsequent DNA cleavage event occurs at a distance from the endonuclease recognition site . Moreover, DNA cleavage appears to be a random process triggered upon stalling of the DNA translocation process and requiring dimerisation of the bound endonucleases for a concerted break of both DNA strands . In this review, we present a detailed description and analysis of the functional mechanism of the three known NTP-dependent restriction systems: type I and type III restriction-modification enzymes, as well as the methylation-dependent McrBC endonuclease.

Avian Pathol, 2002 Dec, 31(6), 619 - 24
The effect of Ornithobacterium rhinotracheale vaccination of broiler breeder chickens on the performance of their progeny; Cauwerts K et al.; The effect of Ornithobacterium rhinotracheale vaccination of broiler breeders on antibody titres and performance of breeders and broilers was investigated . O . rhinotracheale antibody titres and performance data were recorded from 16 different broiler breeder flocks and from 79 of their broiler progeny flocks . Eight breeder flocks were vaccinated with an inactivated O . rhinotracheale vaccine while the other eight breeder flocks were left unvaccinated against this bacterium . Following vaccination, mean O . rhinotracheale antibody titres in the breeders rose to a 6.5 log(2) units higher value than in unvaccinated breeders, and remained at a mean titre of 15 log(2) units during the entire production period . This resulted in significantly higher maternal antibody titres against O . rhinotracheale in the broiler progeny of vaccinated breeder flocks compared with the offspring of unvaccinated flocks . Statistical analyses revealed no differences in performance between vaccinated and unvaccinated breeders . There was a significantly lower mean mortality rate and higher mean production index in the broilers derived from vaccinated breeders.

J Periodontol, 2003 Jan, 74(1), 111 - 8
The role of gingipains in the pathogenesis of periodontal disease; Imamura T; Gingipains are trypsin-like cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis . HRgpA (95 kDa) and RgpB (50 kDa), products of 2 distinct but related genes, rgpA and rgpB, respectively, are specific for Arg-Xaa peptide bonds . Kgp, a product of the kgp gene, is specific for Lys-Xaa bonds . HRgpA and Kgp are non-covalent complexes containing separate catalytic and adhesion/ hemagglutinin domains, while RgpB has only a catalytic domain with a primary structure essentially identical to that of the catalytic subunit of HRgp . HRgpA and RgpB induce vascular permeability enhancement through activation of the kallikrein/kinin pathway and activate the blood coagulation system, which, respectively, are potentially associated with gingival crevicular fluid production and progression of inflammation leading to alveolar bone loss in the periodontitis site . Kgp is the most potent fibrinogen/fibrin degrading enzyme of the 3 gingipains in human plasma and is involved in the bleeding tendency at the diseased gingiva . HRgpA activates coagulation factors and degrades fibrinogen/fibrin more efficiently than RgpB due to the adhesion/hemagglutinin domains, which have affinity for phospholipids and fibrinogen . Gingipains degrade macrophage CD14, thus inhibiting activation of the leukocytes through the lipopolysaccharide (LPS) receptor, and thereby facilitating sustained colonization of P . gingivalis . Gingipains play a role in bacterial housekeeping and infection, including amino acid uptake from host proteins and fimbriae maturation . Based on the important activities of gingipains in the bacterial infection and the pathogenesis of periodontitis, the bacterial proteinases can be targets for periodontal disease therapy . Immunization with RgpB, HRgpA, or a portion of HRgpA catalytic domain attenuated P . gingivalis induced disorders in mice . In addition, a trypsin-like proteinase inhibitor retarded P . gingivalis growth specifically . Gingipains are potent virulence factors of P . gingivalis, and are likely to be associated with the development of periodontitis . It is, therefore, suggested that gingipain inhibition by vaccination and gingipain-specific inhibitors is a useful therapy for adult periodontitis caused by P . gingivalis infection.

Lijec Vjesn, 2002 Sep, 124 Suppl 1, 23 - 8
{Serodiagnosis of Helicobacter pylori infection}; Presecki V et al.; Infection with Helicobacter pylori induces antibodies, but these are not able to eradicate the bacterium from the gastric mucosa . Enzyme linked immunosorbent assay is the laboratory based method and most commonly used to measure qualitatively and quantitatively anti-Helicobacter pylori antibodies of different immunoglobulin classes in almost all infected patients . Quantitative serological tests are useful in the follow-up of eradication therapy . Serology is the method of choice in population studies and in the retrospective analysis of stored serum samples to study the natural course of this chronic infection.

Lijec Vjesn, 2002 Sep, 124 Suppl 1, 1 - 5
{Helicobacter pylori--introduction and review of research}; Katicic M et al.; The discovery of Helicobacter pylori has revolutionized the pathophysiological and clinical approach to gastric and duodenal ulcer . Since the first paper identifying H . pylori was published only 17 years ago, it has been found out that this bacterium causes probably the commonest human infection . Numerous papers published so far have confirmed causal relationship between H . pylori infection and gastritis, duodenal ulcer, gastric ulcer and gastric cancer . If any recent achievement in the world of medicine is to be called revolutionary, then it is the discovery of the role of a spiral bacterium in the etiopathogenesis of gastritis, gastric ulcer, duodenal ulcer and gastric cancer . The discovery of the role of Helicobacter pylori has entirely changed our views and approach to the treatment of patients with stomach disorders . Not only do these discoveries change our actions, but above all our way of thinking . Almost routine diagnostics and treatment of gastritis, gastric ulcer or cancer has been replaced by studies in epidemiology, isolation and eradication of a single bacterium.

Arch Biochem Biophys, 2003 Mar 1, 411(1), 129 - 35
Mechanism of strong resistance of Helicobacter pylori respiration to nitric oxide; Park AM et al.; The aim of the present work is to elucidate the mechanism by which the respiration of Helicobacter pylori but not of Escherichia coli shows a strong resistance to nitric oxide (NO) . Nitric oxide strongly but reversibly inhibited the oxygen consumption by sonicated membranes from H . pylori and Triton X-100-treated cells . Although the sensitivity of the H . pylori respiration to cyanide was low, it also increased after the treatment with Triton X-100 . Kinetic analyses revealed that NO was rapidly degraded by E . coli and the Triton X-100-treated H . pylori, but not by the intact H . pylori . Thus, the low sensitivity to NO might reflect the low affinity of the cytochrome c oxidase for this radical within the membrane/lipid bilayers of H . pylori . Such properties of the oxidase in H . pylori membranes may, at least in part, underlie the mechanism by which this bacterium thrives in NO-enriched gastric juice.

J Clin Gastroenterol, 2003 Mar, 36(3), 204 - 8
Family history of gastric cancer: should we test and treat for Helicobacter pylori?
Niv Y.
A close link has been established between infection and gastric cancer . In this article, we suggest that using a risk stratification technique (like that for colorectal cancer), the high-risk group of first-degree relatives of patients with gastric cancer can be separated out for testing and treatment . This would be more manageable and more cost-effective than screening the whole population, in which the mortality from distal gastric cancer has declined concomitant with the eradication of infection . Support for the feasibility of this approach is derived from studies showing that the family is the core unit of transmission and that childhood colonization, especially with a virulent strain, is apparently a major risk factor for disease progression to the neoplastic stage . When there is a case of gastric cancer in the family, first-degree relatives, who might be infected by a bacterium with an identical genetic fingerprint, are at higher risk than normal for developing gastric cancer . Furthermore, genetic and epidemiologic studies based on the Correa model have shown that both primary and secondary prevention of gastric cancer is possible . Calculations done in high-risk populations, such as Japanese-Americans, confirm the savings in cost and the safety of the test-and-treat strategy . Considering that eradication should be done as early as possible, at a point in the cascade when the changes are still reversible, and that gastric cancer is associated with a high mortality rate, we suggest that this strategy be applied to this high-risk population.

J Biol Inorg Chem, 2003 Feb, 8(3), 360 - 70 Epub 2002 Dec 19.
A novel iron centre in the split-Soret cytochrome c from Desulfovibrio desulfuricans ATCC 27774; Abreu IA et al.; The facultative sulfate/nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 harbours a split-Soret cytochrome c . This cytochrome is a homodimeric protein, having two bis-histidinyl c-type haems per monomer . It has an unique architecture at the haem domain: each haem has one of the coordinating histidines provided by the other monomer, and in each monomer the haems are parallel to each other, almost in van der Waals contact . This work reports the cloning and sequencing of the gene encoding for this cytochrome and shows, by transcriptional analysis, that it is more expressed in nitrate-grown cells than in sulfate-grown ones . In addition, the gene-deduced amino acid sequence revealed two new cysteine residues that could be involved in the binding of a non-haem iron centre . Indeed, the presence of a novel type of an iron-sulfur centre (possibly of the {2Fe-2S} type) was demonstrated by EPR spectroscopy, and putative models for its localization and structure in the cytochrome molecule are proposed on the basis of the so-far-known 3D crystallographic structure of the aerobically purified split-Soret cytochrome, which lacks this centre.

J Biol Inorg Chem, 2003 Feb, 8(3), 306 - 17 Epub 2003 Jan 04.
A thin-film electrochemical study of the "blue" copper proteins, auracyanin A and auracyanin B, from the photosynthetic bacterium Chloroflexus aurantiacus: the reduction potential as a function of pH; Rooney MB et al.; The reversible formal potentials of auracyanin A and auracyanin B, two closely related "blue" copper proteins from the photosynthetic bacterium Chloroflexus aurantiacus, have been determined by protein film voltammetry in the range 4<or=pH<or=9 . At pH 7 in 0.1 M NaCl, the values of for auracyanin A and auracyanin B are 205+/-7 mV and 215+/-7 mV, respectively, versus the standard hydrogen electrode . In both cases there is a smooth but non-sigmoidal change in from approximately 190 mV at pH 9 to approximately 240 mV at pH 4 . The small changes in as a function of pH indicate that auracyanin A and auracyanin B differ from those "blue" copper proteins in which the Cu site in the reduced (Cu(I)) state switches to a redox-inhibited form at low pH . For auracyanin A, the results obtained by protein film voltammetry are closely similar to those obtained by the conventional spectroelectrochemical method . The findings are discussed in relation to the putative role of auracyanin in biological electron transfer.

FEMS Microbiol Lett, 2003 Jan 28, 218(2), 371 - 5
Siderophore production by the magnetic bacterium Magnetospirillum magneticum AMB-1; Calugay RJ et al.; Siderophore production by the magnetic bacterium Magnetospirillum magneticum AMB-1 is elicited by sufficient iron rather than by iron starvation . In order to clarify this unusual pattern, siderophore production was monitored in parallel to iron assimilation using the chrome azurol sulfonate assay and the ferrozine method respectively . Iron concentration lowered approximately five times less than its initial concentration only within 4 h post-inoculation, rendering the medium iron deficient . A concentration of at least 6 microM Fe(3+) is required to initiate siderophore production . The propensity of M . magneticum AMB-1 for the assimilation of large amounts of iron accounts for the rapid depletion of iron in the medium, thereby triggering siderophore excretion . M . magneticum AMB-1 produces both hydroxamate and catechol siderophores.

FEMS Microbiol Lett, 2003 Jan 28, 218(2), 345 - 9
Involvement of a quinoprotein (PQQ-containing) alcohol dehydrogenase in the degradation of polypropylene glycols by the bacterium Stenotrophomonas maltophilia; Tachibana S et al.; Previous work has shown that when the bacterium Stenotrophomonas maltophilia is grown on polypropylene glycol, different dye-linked polypropylene glycol dehydrogenase (PPG-DH) activities are induced during growth . Here the purification and characterization of the dehydrogenase activity induced in the stationary phase, and present in the periplasmic space, is described . The homogeneous enzyme preparation obtained consists of a homodimeric protein with a molecular mass of about 123 kDa and an isoelectric point of 5.9 . The cofactor of the enzyme appeared to be pyrroloquinoline quinone (PQQ), no heme c was present, and holo-enzyme contained two PQQ molecules per enzyme molecule . In these respects, PPG-DH described here is similar to already known quinoprotein alcohol dehydrogenases, but in other respects, it is different . Therefore, it is suggested that PPG-DH could be a new type of quinoprotein alcohol dehydrogenase . Based on its strong preference for polyols, PPG-DH seems well fitted to carry out the first step in the degradation of PPGs, synthetic polymers containing a variety of hydroxyl groups.

Biochim Biophys Acta, 2003 Feb 17, 1610(1), 3 - 10
Assembly and overexpression of membrane proteins in Escherichia coli; Drew D et al.; The bacterium Escherichia coli is one of the most popular model systems to study the assembly of membrane proteins of the so-called helix-bundle class . Here, based on this system, we review and discuss what is currently known about the assembly of these membrane proteins . In addition, we will briefly review and discuss how E . coli has been used as a vehicle for the overexpression of membrane proteins .

Anal Chem, 2003 Feb 1, 75(3), 580 - 5
Combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria; Neufeld T et al.; Here, we describe a novel electrochemical method for the rapid identification and quantification of pathogenic and polluting bacteria . The design incorporates a bacteriophage, a virus that recognizes, infects, and lyses only one bacterial species among mixed populations, thereby releasing intracellular enzymes that can be monitored by the amperometic measurement of enzymatic activity . As a model system, we used virulent phage typing and cell-marker enzyme activity (beta-D-galactosidase), a combination that is specific for the bacterial strain Escherichia coli (K-12, MG1655) . Filtration and preincubation before infecting the bacteria with the phage enabled amperometric detection at a wide range of concentrations, reaching as low as 1 colony-forming unit/100 mL within 6-8 h . In principle, this electrochemical method can be applied to any type of bacterium-phage combination by measuring the enzymatic marker released by the lytic cycle of a specific phage.

Nucleic Acids Res, 2003 Feb 15, 31(4), 1197 - 207
Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli; Akama K et al.; The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA(Ser)(CGA), an Arabidopsis intron-containing tRNA(Tyr)(GTA) and an Arabidopsis intron-containing tRNA(Met)(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants . This versatile system allows the detection of beta-glucuronidase (GUS) activity by histochemical and enzymatic analyses . The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene . Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA(Ser) gene and the GUS reporter gene resulted in approximately 10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene . Amber suppressor tRNAs derived from intron-containing tRNA(Tyr) or tRNA(Met) genes were functional in vivo only after some additional gene manipulations . The G3:C70 base pair in the acceptor stem of tRNA(Met)(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity . The inability of amber suppressor tRNA(Tyr) to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence . The improved amber suppressors tRNA(Tyr) and tRNA(Met) were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.

Extremophiles, 2003 Feb, 7(1), 17 - 28 Epub 2002 Oct 01.
GroEL from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125: molecular characterization and gene cloning; Tosco A et al.; The heat shock response of the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 (PhTAC 125) gives rise to the production of several inducible proteins . Among these, the protein corresponding to a 55-kDa band on SDS-PAGE was purified to homogeneity and identified as a GroEL-like protein . The gene coding for this protein (PhGroEL) was cloned and sequenced; the deduced amino acid sequence shows 82% sequence identity to GroEL from Escherichia coli (EcGroEL) . The ORF found in the 5' upstream region codes for a homologue of the GroES from E . coli (PhGroES, 71% sequence identity to EcGroES) . PhGroEL shows a chaperone activity and can use GroES from E . coli as a co-chaperone . PhGroEL melting temperature, 6 degrees C lower than that of EcGroEL, and equilibrium unfolding experiments in urea showed a less stable protein architecture for the psychrophilic GroEL . The data herein reported demonstrate that PhGroEL cold adaptation consists in a shift of the protein properties toward lower temperatures without increasing catalytic efficiency at low temperatures . Primary extension analysis depicted a complex organization of regulative elements for the operon containing the genes coding for PhgroES and PhgroEL (PhgroE), suggesting that a fine-tuning of transcription can also be involved in thermal adaptation of PhTAC 125.

Scand J Infect Dis, 2002, 34(11), 800 - 3
Ochrobactrum anthropi bacteraemia in immunocompetent children; Galanakis E et al.; Ochrobactrum anthropi is an emerging pathogen in immunocompromised patients but infections with the bacterium have very rarely been documented in normal hosts . We report the characteristics of O . anthropi bacteraemia in 11 immunocompetent children, aged 2 months to 7 y, hospitalized in a general hospital during a 5-y period . Children commonly presented with fever, non-specific respiratory or gastrointestinal manifestations, leukocytosis and neutrophilia and had a rapid recovery, even when they did not receive a specific treatment . In 10 cases positive blood cultures were obtained on admission and in all cases subsequent cultures were sterile . In conclusion, O . anthropi may cause bacteraemia in immunocompetent hosts, although further studies are required to clarify whether these isolates represent pseudobacteraemia or whether O . anthropi is a potential pathogen of low virulence.

Proteins, 2003 Mar 1, 50(4), 526 - 30
Crystal structure of a hypothetical protein, TM841 of Thermotoga maritima, reveals its function as a fatty acid-binding protein; Schulze-Gahmen U et al.; We determined the three-dimensional (3D) crystal structure of protein TM841, a protein product from a hypothetical open-reading frame in the genome of the hyperthermophile bacterium Thermotoga maritima, to 2.0 A resolution . The protein belongs to a large protein family, DegV or COG1307 of unknown function . The 35 kDa protein consists of two separate domains, with low-level structural resemblance to domains from other proteins with known 3D structures . These structural homologies, however, provided no clues for the function of TM841 . But the electron density maps revealed clear density for a bound fatty-acid molecule in a pocket between the two protein domains . The structure indicates that TM841 has the molecular function of fatty-acid binding and may play a role in the cellular functions of fatty acid transport or metabolism .

Microbiology, 2003 Jan, 149(Pt 1), 121 - 9
Role of the thioredoxin system and the thiol-peroxidases Tpx and Bcp in mediating resistance to oxidative and nitrosative stress in Helicobacter pylori; Comtois SL et al.; Helicobacter pylori possesses two distinct thioredoxin proteins (Trx1 and Trx2) which may play important roles in the ability of this bacterium to survive oxidative stress . Trx1 has previously been shown to be an electron donor in vitro for alkyl-hydroperoxide reductase (AhpC), one of three members of the peroxiredoxin family of antioxidant peroxidases present in H . pylori . In this study, mutants in the trxA1 and trxA2 genes encoding Trx1 and Trx2, respectively, and in the tpx and bcp genes, which encode the remaining two members of the H . pylori peroxiredoxin family, were constructed in order to determine their roles in resistance to damage by reactive oxygen and nitrogen species . Mutation of trxA1 led to a pronounced increase in sensitivity to oxygen, hydrogen peroxide and the superoxide generator paraquat, as well as to the nitric oxide (NO) releasers sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO), consistent with an in vivo role for Trx1 as a reductant for AhpC . A trxA2 single mutant grew normally in an atmosphere of 2 % (v/v) O(2) but grew very poorly in 10 % (v/v) O(2) . It showed slight increases in killing by hydrogen peroxide, paraquat, SNP and GSNO compared to the wild-type, but was significantly more sensitive to cumene hydroperoxide in disc-diffusion assays . A trxA1 trxA2 double mutant was very sensitive to all of the oxidative and nitrosative stresses applied . Comparison of the phenotypes of the tpx and bcp mutants showed that Tpx plays a significant role in peroxide and superoxide resistance in H . pylori, while the role of Bcp is minimal . No evidence was obtained for a role for either Tpx or Bcp in resistance to the toxic effects of NO . The results show that a functional thioredoxin system is necessary for both oxidative and nitrosative stress resistance in H . pylori but, surprisingly, is not essential for viability despite the absence of glutathione and a glutaredoxin system in this bacterium.

Mol Plant Microbe Interact, 2003 Feb, 16(2), 115 - 22
Sulfated fucan oligosaccharides elicit defense responses in tobacco and local and systemic resistance against tobacco mosaic virus; Klarzynski O et al.; Sulfated fucans are common structural components of the cell walls of marine brown algae . Using a fucan-degrading hydrolase isolated from a marine bacterium, we prepared sulfated fucan oligosaccharides made of mono- and disulfated fucose units alternatively bound by alpha-1,4 and alpha-1,3 glycosidic linkages, respectively . Here, we report on the elicitor activity of such fucan oligosaccharide preparations in tobacco . In suspension cell cultures, oligofucans at the dose of 200 microg ml(-1) rapidly induced a marked alkalinization of the extracellular medium and the release of hydrogen peroxide . This was followed within a few hours by a strong stimulation of phenylalanine ammonia-lyase and lipoxygenase activities . Tobacco leaves treated with oligofucans locally accumulated salicylic acid (SA) and the phytoalexin scopoletin and expressed several pathogenesis-related (PR) proteins, but they displayed no symptoms of cell death . Fucan oligosaccharides also induced the systemic accumulation of SA and the acidic PR protein PR-1, two markers of systemic acquired resistance (SAR) . Consistently, fucan oligosaccharides strongly stimulated both local and systemic resistance to tobacco mosaic virus (TMV) . The use of transgenic plants unable to accumulate SA indicated that, as in the SAR primed by TMV, SA is required for the establishment of oligofucan-induced resistance.

J Immunol, 2003 Feb 15, 170(4), 1939 - 48
Constrained intracellular survival of Mycobacterium tuberculosis in human dendritic cells; Tailleux L et al.; Dendritic cells (DCs) are likely to play a key role in immunity against Mycobacterium tuberculosis, but the fate of the bacterium in these cells is still unknown . Here we report that, unlike macrophages (Mphis), human monocyte-derived DCs are not permissive for the growth of virulent M . tuberculosis H37Rv . Mycobacterial vacuoles are neither acidic nor fused with host cell lysosomes in DCs, in a mode similar to that seen in mycobacterial infection of Mphis . However, uptake of the fluid phase marker dextran, and of transferrin, as well as accumulation of the recycling endosome-specific small GTPase Rab11 onto the mycobacterial phagosome, are almost abolished in infected DCs, but not in Mphis . Moreover, communication between mycobacterial phagosomes and the host-cell biosynthetic pathway is impaired, given that <10% of M . tuberculosis vacuoles in DCs stained for the endoplasmic reticulum-specific proteins Grp78/BiP and calnexin . This correlates with the absence of the fusion factor N-ethylmaleimide-sensitive factor onto the vacuolar membrane in this cell type . Trafficking between the vacuoles and the host cell recycling and biosynthetic pathways is strikingly reduced in DCs, which is likely to impair access of intracellular mycobacteria to essential nutrients and may thus explain the absence of mycobacterial growth in this cell type . This unique location of M . tuberculosis in DCs is compatible with their T lymphocyte-stimulating functions, because M . tuberculosis-infected DCs have the ability to specifically induce cytokine production by autologous T lymphocytes from presensitized individuals . DCs have evolved unique subcellular trafficking mechanisms to achieve their Ag-presenting functions when infected by intracellular mycobacteria.

Environ Health Perspect . 2003 Feb;111(2):A77; author reply A77.
Chronic Lyme disease: it's not all in our heads; Morgenstern RG; Those of us with chronic Lyme disease are not at all confused, as suggested by Sigal and Hassett (2002) . We know from years of experience that we have real, specific symptoms that are usually painful and disabling and include severe headaches, crippling arthritis, and heart palpitations, which lead to serious heart disease . Many of us know that our symptoms are kept in check while we are on antibiotics, but they painfully reappear when the antibiotics are withdrawn . Just because the medical community cannot detect a specific causative bacterium and managed health care companies want to maximize profits doesn't mean that those of us afflicted with this terrible condition are delusional and not truly benefiting from antibiotic treatment . We are not all crazy; we are sick and we should not be required to prove it to get medical care.

Curr Pharm Des, 2003, 9(1), 61 - 5
Role of type I cytokines in host defense against Mycobacterium avium infection; Danelishvilli L et al.; Mycobacterium avium is a human pathogen that causes infection in immunocompetent as well as immunocompromised patients . Infection is acquired both by the respiratory and gastrointestinal routes, and bacterial invasion of mucosal epithelial cells is characteristic . M . avium crosses the mucosal barrier without triggering substantial inflammatory response . Once in the intestinal submucosa or in the alveolar space M . avium infects macrophages . Intracellular bacteria block the production of cytokines involved in the host response against the infection, such as TNF-alpha and IL-12, and suppress antigen presentation by the macrophage . Innate response against the infection is effective to certain extent but the ability of the bacterium to remain "silent" for a period of time prevents neutrophil and NK cells from effectively controlling the establishing of the infection . CD4+ T cells as well as CD8+ T cells are activated, although only CD4+ T cells appear to be effective in inducing anti-M . avium activity in macrophages . M . avium-specific CD8+ T cells undergo apoptosis early in the infection . Therefore, the immune mechanisms of the host and bacterial strategies for survival are complex and fascinating.

Curr Microbiol, 2003 Mar, 46(3), 157 - 62
Comparison between NaCl tolerance response and acclimation to cold temperature in Shewanella putrefaciens; Leblanc L et al.; Two strains of the spoiling bacterium S . putrefaciens showed an adaptation capacity to hyperosmotic shock when they were pretreated with a sublethal concentration of NaCl . The maximal tolerance factor for the CIP 69.29 strain was obtained when cells were incubated for 1 h in the presence of 1.5% NaCl, whereas for the J13.1 strain, an incubation of 15 min in the presence of 1% NaCl seemed to be the optimal conditions to harden the cells against a subsequent lethal salt treatment . During NaCl adaptation and growth at low temperatures (2 degrees C), 37 and 32 polypeptides were induced respectively . Interestingly, 11 proteins were common between the two different stress responses . These proteins and the corresponding genes seem to play a key role in the observed cross-protection towards the NaCl challenge induced by growth of the cultures at 2 degrees C . One of the overlapping proteins has been identified to correspond to the alkyl hydroperoxide reductase (AhpC) of S . putrefaciens . Northern blot analysis showed that induction of this enzyme was accompanied by accumulation of the corresponding transcript under both conditions.

Laryngoscope, 2003 Feb, 113(2), 270 - 5
Bacteria detected by culture and 16S rRNA sequencing in maxillary sinus samples from intensive care unit patients; Westergren V et al.; OBJECTIVES/HYPOTHESIS: In critically ill patients, the occurrence of artificial ventilation-acquired sinus disease is common . A possible sinus bacterial infection is occult and is combined with diagnostic difficulties . Culture as a method has limited capacity to verify the presence of bacteria and leaves unanswered the question of a possible infective agent because bacteria are difficult to grow when killed or suppressed by current antibiotic therapy . Hitherto unidentified micro-organisms are also possible in the microenvironments of maxillary sinuses . STUDY DESIGN: Prospective case series . METHODS: Twenty maxillary sinus samples (17 aspirates and 3 lavages) from nine critically ill patients with possible infectious disease were investigated by broad-range 16S rRNA polymerase chain reaction followed by sequencing . These results were compared with the previous culture results from gingiva (passage route), maxillary sinus absorption, and mucosa samples . RESULTS: The contaminations were rare (2 of 20) and corresponded well to culture results . One previously undiagnosed bacterium was found . Two aspirates were negative on both culture and polymerase chain reaction, whereas the corresponding maxillary sinus mucosal cultures had been positive . CONCLUSIONS: Applying a low-contamination maxillary sinuses sampling technique makes 16S rRNA sequencing clinically useful . The polymerase chain reaction and culture results were generally comparable . However, by polymerase chain reaction, bacteria were found that were missed by culturing . Some aspirates were free of bacterial remnants on 16S rRNA polymerase chain reaction, but corresponding mucosal cultures were positive . This could indicate that infection is induced within the tissue . It also indicates that infectious agents introduced into the sinuses may have routes other than the ostium . Further clinical use of 16S rRNA sequencing is required to enlarge our knowledge in applied microbiology and paranasal sinus disease.

Biochemistry, 2003 Feb 11, 42(5), 1354 - 64
An Isc-type extremely thermostable {2Fe-2S} ferredoxin from Aquifex aeolicus . Biochemical, spectroscopic, and unfolding studies; Mitou G et al.; Analysis of the genome of the hyperthermophilic bacterium Aquifex aeolicus has revealed the presence of a previously undetected gene potentially encoding a plant- and mammalian-type {2Fe-2S} ferredoxin . Expression of that gene in Escherichia coli has yielded a novel thermostable {2Fe-2S} ferredoxin (designated ferredoxin 5) whose sequence is most similar to those of ferredoxins involved in the assembly of iron-sulfur clusters (Isc-Fd) . It nevertheless differs from the latter proteins by having deletions near its N- and C-termini, and no cysteine residues other than those involved in {2Fe-2S} cluster coordination . Resonance Raman, low-temperature MCD and EPR studies show close spectral similarities between ferredoxin 5 and the Isc-Fd from Azotobacter vinelandii . Mossbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian and interpreted in the framework of the ligand field model proposed by Bertrand and Gayda . The redox potential of A . aeolicus ferredoxin 5 (-390 mV) is in keeping with its relatedness to Isc-Fd . Unfolding experiments showed that A . aeolicus ferredoxin 5 is highly thermostable (T(m) = 106 degrees C at pH 7), despite being devoid of features (e.g., high content of charged residues) usually associated with extreme thermal stability . Searches for genes potentially encoding plant-type {2Fe-2S} ferredoxins have been performed on the sequenced genomes of hyperthermophilic organisms . None other than the two proteins from A . aeolicus were retrieved, indicating that this otherwise widely distributed group of proteins is barely represented among hyperthermophiles.

J Bacteriol, 2003 Feb, 185(4), 1245 - 52
Analysis of DNA binding and transcriptional activation by the LysR-type transcriptional regulator CbbR of Xanthobacter flavus; van Keulen G et al.; The LysR-type transcriptional regulator CbbR controls the expression of the cbb and gap-pgk operons in Xanthobacter flavus, which encode the majority of the enzymes of the Calvin cycle required for autotrophic CO2 fixation . The cbb operon promoter of this chemoautotrophic bacterium contains three potential CbbR binding sites, two of which partially overlap . Site-directed mutagenesis and subsequent analysis of DNA binding by CbbR and cbb promoter activity were used to show that the potential CbbR binding sequences are functional . Inverted repeat IR1 is a high-affinity CbbR binding site . The main function of this repeat is to recruit CbbR to the cbb operon promoter . In addition, it is required for negative autoregulation of cbbR expression . IR3 represents the main low-affinity binding site of CbbR . Binding to IR3 occurs in a cooperative manner, since mutations preventing the binding of CbbR to IR1 also prevent binding to the low-affinity site . Although mutations in IR3 have a negative effect on the binding of CbbR to this site, they result in an increased promoter activity . This is most likely due to steric hindrance of RNA polymerase by CbbR since IR3 partially overlaps with the -35 region of the cbb operon promoter . Mutations in IR2 do not affect the DNA binding of CbbR in vitro but have a severe negative effect on the activity of the cbb operon promoter . This IR2 binding site is therefore critical for transcriptional activation by CbbR.

J Bacteriol, 2003 Feb, 185(4), 1153 - 60
Molecular analysis of the gene encoding a novel cold-adapted chitinase (ChiB) from a marine bacterium, Alteromonas sp . strain O-7; Orikoshi H et al.; The chitinase B (ChiB) secreted by Alteromonas sp . strain O-7 was purified, and the corresponding gene (chiB) was cloned and sequenced . The open reading frame of the chiB gene encodes a protein of 850 amino acids with a calculated molecular mass of 90,223 Da . ChiB is a modular enzyme consisting of two reiterated domains and a catalytic domain belonging to chitinase family 18 . The reiterated domains are composed of chitin-binding domain (ChtBD) type 3 and two fibronectin type III (Fn3)-like domains . Expression plasmids coding for ChiB or deletion derivatives thereof were constructed in Escherichia coli . Deletion analysis showed that the ChtBD of ChiB plays an important role in efficient hydrolysis of insoluble chitin . The optimum pH and temperature of ChiB were 6.0 and 30 degrees C, respectively . The enzyme showed relatively high catalysis, even at low temperatures close to 0 degrees C, and remarkable thermal lability compared to ChiA and ChiC, which are the mesophilic chitinases of the same strain . The kca)/Km value for the ChiB reaction at 10 degrees C was about 4.7 times higher than that of ChiC . These results suggest that ChiB is a cold-adapted enzyme . The RNA transcript of chiB was induced by 1% GlcNAc, and along with a rise in temperature, the RNA transcript showed a tendency to decrease . Thus, among the ChiA, ChiB, and ChiC chitinases, production of ChiB may be advantageous for the strain, allowing it to easily acquire nutrients from chitin and to survive in cold environments.

Aliment Pharmacol Ther, 2003 Feb, 17(3), 421 - 8
Helicobacter pylori strains and histologically-related lesions affect the outcome of triple eradication therapy: a study from southern Italy; Russo F et al.; BACKGROUND: Certain evidence suggests that Helicobacter pylori strains expressing genes for cytotoxin production show a higher sensitivity than non-cytotoxic organisms to eradication treatment . No data are available on the involvement of bacterium-related lesions in different therapeutic outcomes . AIMS: (i) To investigate whether differences in eradication rates may be related to the different expression of virulent strains (cagA, vacA, iceA) in patients undergoing proton pump inhibitor-based triple therapy, and (ii) to evaluate whether therapeutic outcome may be affected by bacterium-induced gastric lesions . METHODS: One hundred and ten H . pylori-positive subjects were enrolled . H . pylori was genotyped by polymerase chain reaction . Treatment consisted of lansoprazole-amoxicillin-clarithromycin, twice daily for 1 week . Eradication was checked by urea breath test . RESULTS: The eradication rate was 70%, and the absence of cagA was associated with unsuccessful treatment . No difference between the groups with successful and unsuccessful eradication was found with regard to vacA and iceA . Lympho-epithelial lesions and fibrosis were associated with unsuccessful treatment . CONCLUSIONS: The present data confirm the importance of cagA (but not vacA and iceA) as a predictor of successful eradication . When fibrosis and lympho-epithelial lesions are present, therapy appears to be less effective . Therefore, these histological features may be involved in an unsuccessful therapeutic outcome.

Carbohydr Res, 2003 Feb 14, 338(5), 459 - 62
Structure of an acidic polysaccharide from the marine bacterium Pseudoalteromonas flavipulchra NCIMB 2033(T); Muldoon J et al.; An acidic polysaccharide was isolated from Pseudoalteromonas flavipulchra type strain NCIMB 2033(T) and found to consist of 6-deoxy-L-talose (L-6dTal), D-galactose and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) . The identities of the monosaccharides were ascertained by sugar analysis and 1D 1H and 13C NMR spectroscopy in conjunction with 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments, which enabled determination of the following structure of the trisaccharide repeating unit of the polysaccharide:-->3)-alpha-L-6dTalp4Ac-(1-->3)-beta-D-Galp-(1-->7)-alpha-Kdop-(2-->.

Theriogenology, 2003 Apr 1, 59(7), 1651 - 60
Lack of association of Mycobacterium avium subsp . paratuberculosis with oocytes and embryos from moderate shedders of the pathogen; Kruip TA et al.; Paratuberculosis is a chronic and progressive disease of the intestine in ruminants caused by Mycobacterium avium subsp . paratuberculosis (Map) . The bacterium is transmitted to young animals, becomes manifest in adulthood and leads to economic losses . The aim of this study is to investigate if cows shedding Map possess oocytes and embryos that are carriers of the bacterium . New genetical material can enter the dairy farm using embryo transfer but the question as to whether this technique is safe with respect to transmission of paratuberculosis has yet to be addressed . We selected and bought 16 cows, all proven to be moderate shedders of the bacterium in the faeces immediately prior to the experiment but none were clinically sick . One sample of uterine content was collected from each animal by flushing the uterus on the day of heat and five samples of homogenised uterine tissue were collected on the eighth day of the same cycle by biopsy . In addition, 217 cumulus-oocyte complexes (COCs), ranging from 3 to 35 COCs per animal, were collected using ultrasound guided transvaginal puncture of the ovarian follicles (OPU) . On the seventh day of the subsequent cycle 31 embryos were obtained using the classic technique of super ovulation induction, artificial insemination (AI), followed by flushing of the uterus . These embryos have been washed and trypsinised . Fourteen of the 16 cows were treated again for super ovulation in the subsequent cycle and 19 foetuses were collected by opening of the uterus after euthanasia on Days 35-49 of the cycle . All samples were cultured for presence of Map and checked every 2 months during 1 year for bacterial growth . None of the samples showed growth of Map after 12 months of culture . Pathological examination of the cows revealed different degrees of severity of pathological alterations of the intestinal tract and mesenteric lymph nodes . However, the results suggest that neither in vivo embryo's nor oocytes are carriers of the bacteria and do not form an extra risk at transfer . However, due to the limited size of the experiment (sample size of 16 cows), a certain margin for error remains.

Res Microbiol, 2002 Dec, 153(10), 653 - 8
Presence in bovine enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli of genes encoding for putative adhesins of human EHEC strains; Szalo IM et al.; Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) infections are characterised by the formation of attaching and effacing lesions on intestinal epithelial cells . The first step of EPEC and EHEC pathogenesis involves the initial adherence of the bacterium to the intestinal epithelium . A collection of bovine EPEC and EHEC strains belonging to different serogroups was tested by colony blot hybridization with gene probes for putative adhesins (BFPA, LPFA, IHA, LIFA) of human EPEC and EHEC, and also for fimbrial and afimbrial adhesins (AFA8, F17, Cs31A) of bovine necrotoxigenic E . coli (NTEC) . In the bovine EPEC and EHEC strains tested, sequences homologous to lifA, ihA, and lpfA genes were detected, sometimes in association with particular serogroups . Bovine 026 EPEC also possessed a sequence homologous to a gene of the c/p operon, coding for the CS31A adhesin, associated with bovine NTEC . Overall results showed that different genes encoding for putative adhesins of human EHEC strains are present in bovine EPEC and EHEC strains, but not one of them is present in all strains.

J Egypt Soc Parasitol, 2001 Apr, 31(1), 295 - 325
Effects of the entomopathogenic nematode, Heterorhabditis bacteriophora HP 88 and azadirachtin on the immune defence response and prophenoloxidase of Parasarcophaga surcoufi larvae (Diptera: Sarcophagidae); Ayaad TH et al.; Each third larval instar (155-175 mg) of the fleshfly, Parasarcophaga surcoufi was injected with 20 and 40 infective juveniles (IJs) of entomopathogenic nematode, Heterorhabditis bacteriophora, and with 0.5 and 1 microg of azadirachtin . Injection with 20 IJs decreased the total haemocyte count (THC) up to 40 h post-injection, except for its increase at 20 h . On the contrary, injection with 40 IJs increased the THC during the hours post-injection, except for its decrease at 40 h . The injection of nematode (40 IJs) decreased, the percentage of differential haemocyte count of P . surcoufi larvae, specially the granulocytes and plasmatocytes at 40 h . Whereas, prohaemocytes and oenocytoids were increased at 40 h post-infection . The granulocytes and plasmatocytes were engaged with encapsulation of the nematode, H . bacteriophora . The released symbiotic bacterium, Xenorhabdus luminescens was poorly phagocytosed by the granulocytes . After injection of the larvae with azadirachtin, THC and the percentage of the number of oenocytoids were particularly increased after 40 h of injection with azadirachtin; plasmatocytes and granulocytes were decreased at 10 h post-injection with 0.5 microg and at 40 h post-injection with 1 microg of azadirachtin . The most prominent haemocyte deformities in P . surcoufi larvae treated with azadirachtin were the release of cytoplasmic components from granulocytes together with bulging or lysis of plasmatocytes . Maximal haemolymph phenoloxidase (PO) activity was obtained at pH 6.2 and 6.6 . This activity increased with increasing the pre-incubation time using laminarin, alpha-chymotrypsin and trypsin as activators . Injection of both nematode and azadirachtin significantly suppressed the haemolymph PO activity even in the presence of the activators laminarin, alpha-chymotrypsin and methanol . This suppression was dose-dependent and reached maximum at 30 h post-injection . The electrophoretic haemolymph protein profile was time-dependent as indicated by appearance and disappearance of protein bands . After 40 h post-injection with the nematode all the protein bands were replaced by new ones (probably containing immune protein) . However, at this time injection with azadirachtin (1 microg) lead to appearance of 10 new protein bands & disappearance of an equal number of the present band.

Wei Sheng Wu Xue Bao, 2002 Jun, 42(3), 348 - 53
{Purification and properties of thermostable catalase in engineered E . coli}; Wang F et al.; A thermostable catalase in engineered bacterium E . coli was purified to electrophoretic homogenenity by heat treatment, ammonium sulfate fractionation precipitation, DEAE-A50 ion exchange chromatography, HiPrep 16/10 Phenyl hydrophobic interaction chromatography and Superdex200 HR 10/30 size exclusion chromatography with 187.2-fold purification and 9.8% recovery . The optimum reaction temperature and pH of this recombinant catalase were 70 degrees C and 7.0 respectively . The catalase is stable below 60 degrees C and at pH range 3-8 . The residual activity of the catalase was about 60% after treated at 70 degrees C for 60 minutes and 80 degrees C for 10 minutes . The apprant Km and Vmax value of the catalase were 7.75 mmol/L and 27.8 mmol.min-1.mg-1 respectively . The affects of some metal ions and compounds on this enzyme were shown . Zn2+, Ba2+, Mn2+ of 1 mmol/L could completely inactivate the enzyme, EDTA of 50 mmol/L had no affect on activity.

J Biol Chem, 2003 Apr 4, 278(14), 12574 - 8 Epub 2003 Jan 27.
The NudA protein in the gastric pathogen Helicobacter pylori is an ubiquitous and constitutively expressed dinucleoside polyphosphate hydrolase; Lundin A et al.; The gastric pathogen Helicobacter pylori harbors one Nudix hydrolase, NudA, that belongs to the nucleoside polyphosphate hydrolase subgroup . In this work, the enzymatic activity of purified recombinant NudA protein was analyzed on a number of nucleoside polyphosphates . This predicted 18.6-kDa protein preferably hydrolyzes diadenosine tetraphosphate, Ap(4)A at a k(cat) of 0.15 s(-1) and a K(m) of 80 microm, resulting in an asymmetrical cleavage of the molecule into ATP and AMP . To study the biological role of this enzyme in H . pylori, an insertion mutant was constructed . There was a 2-7-fold decrease in survival of the mutant as compared with the wild type after hydrogen peroxide exposure but no difference in survival after heat shock or in spontaneous mutation frequency . Western blot analyses revealed that NudA is constitutively expressed in H . pylori at different growth stages and during stress, which would indicate that this protein has a housekeeping function . Given that H . pylori is a diverse species and that all the H . pylori strains tested in this study harbor the nudA gene and show protein expression, we consider NudA to be an important enzyme in this bacterium.

Wei Sheng Wu Xue Bao, 2000 Apr, 40(2), 204 - 9
{Studies on the trehalose-producing by Colloides CB39}; Zheng Y et al.; A bacterium which had the ability of producing sugar produced sugar at low temperature (18 degrees C) was selected from the water of Tianchi in Changbai mountain . The sugar was identified to be trehalose by methods of thin-layer chromatography, reaction of producing osazyone, capillary electrophoresis and infrared spectrum . It was also been found that the trehalose produced by this bacterium (identified to be a new species of Colloides Sp . CB39) was exocrine . At 18 degrees C, its trehalose content in culture solution was 256 mg/g dry weight of the strain . This characteristic is different from that of other strains, which had been proved to have the ability of producing trehalose . then the strain CB39 was induced by ultraviolet in order to abstain mutants which can produce trehalose in high level at usual temperature . The mutant strain 5 that could produce trehalose in high level at usual temperature (25 degrees C) was selected from all the mutant strains . The value of its productivity of trehalose is 416.7 mg/g dry weight of the strain . (At the same temperature, trehalose productivity of mutant strain 5 was eight times of that of the wild type strain CB39).

Wei Sheng Wu Xue Bao, 2000 Apr, 40(2), 126 - 31
{Cloning and expression of a resistant Culex gene in Escherichia coli}; Yan Y et al.; Recombinant plasmid pRLB1 was constructed from detoxifying gene B1 of pesticide resistant Culex pipiens quinquefasciatus and from plasmid pRL439 contained the strong promoter PpsbA . The positive clone was identified by digestion and Southern analysis . Expression of recombinant plasmid containing esterase gene was detected . An engineered bacterium possessing high enzyme activity was obtained and immobilized . It can effectively degrade the specific substrate alpha-naphthyl acetate (alpha-NA) and beta-naphthyl of esterase enzyme . Assays showed that pesticide acetofenate (7504 an organic choride pesticide) was degraded by the immobilized cells within one hour.

Biochem Soc Trans, 2003 Feb, 31(Pt 1), 98 - 103
Regulation, secretion and activity of type III-secreted proteins of enterohaemorrhagic Escherichia coli O157; Roe AJ et al.; Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 causes gastrointestinal disease with the potential for life-threatening sequelae . Although Shiga-like toxins are responsible for much of the serious pathology in humans, the bacterium also possesses a type III protein secretion system that is responsible for intimate attachment to host intestinal mucosa . This sophisticated interaction requires co-ordination that is governed by environmental and genetic factors . Ongoing research supports the following model for how EHEC enables and controls this process: (i) specific environmental cues that are present in the host result in the expression of a number of adhesins, including fimbriae, which allow the initial binding to the mucosal surface . The same conditions support the expression of the basal type III secretion apparatus; (ii) targeting and assembly of the translocon requires both an mRNA signal and chaperones, with coupled translation and secretion of translocon proteins, EspA, B and D; (iii) opening up of a conduit between the bacterium and host cell releases a cytoplasmic pool of effector proteins . A consequence of this is increased expression of particular effector proteins . Potentially, different proteins could be released into the cell at different times or have activities modulated with time; (iv) intimate contact between the translocated intimin receptor (Tir) and the bacterial surface factor intimin requires translocon expression to be down-regulated and translocon filaments to be lost . Fluorescent protein fusions allow contact-mediated regulation and protein targeting through the type III secretion system to be studied in detail.

Biotechniques, 2003 Jan, 34(1), 106 - 10, 112-5
High-sensitivity quantitative PCR platform; DeGraves FJ et al.; Real-time PCR methods have become widely used within the past few years . However, real-time PCR is rarely used to study chronic diseases with low pathogen loads, presumably because of insufficient sensitivity . In this report, we developed an integrated nucleic acid isolation and real-time PCR platform that vastly improved the sensitivity of the quantitative detection of the intracellular bacterium, Chlamydia spp., by fluorescence resonance energy transfer real-time PCR . Determinants of the overall detection sensitivity were analyzed by extracting nucleic acids from bovine milk specimens spiked with low amounts of chlamydial organisms . Nucleic acids were optimally preserved and recovered by collection in guanidinium stabilization buffer, binding to a matrix of glass fiber fleece, and elution in low volume . Step-down thermal cycling and an excess of hot-start Taq polymerase vastly improved the robustness and sensitivity of the real-time PCR while essentially maintaining 100% specificity . The amplification of Chlamydia 23S rRNA allowed for the differentiation of chlamydial species and was more robust at low target numbers than amplification of the omp1 gene . The best combined method detected single targets per a 100-microL specimen equivalent in a 5-microL real-time PCR input . In an initial application, this high-sensitivity real-time PCR platform demonstrated a high prevalence of chlamydial infection in cattle.

Microb Ecol, 2003 Feb, 45(2), 109 - 18 Epub 2003 Jan 28.
Sunscreen products increase virus production through prophage induction in marine bacterioplankton; Danovaro R et al.; Classical pollutants (e.g., hydrocarbon, pesticides) have been recently recognized to induce lytic cycle in lysogenic bacteria, but information on micro-pollutants is almost completely lacking . We investigated the effects of cosmetic sun products (sunscreen and solar oil) on viral abundance and bacterial activity . We found that both sunscreen and solar oil acted as pollutants, inducing viral development and controlling bacterial abundance and production, thus leading to an increase of the virus to bacterium ratio . Short-term experiments revealed that sunscreen supplementation induced the lytic cycle in a large fraction of total bacterial abundance (13-24% of bacteria, at low and high concentrations, respectively), whereas solar oil had a lower impact (6-9%) . A synchronized development of the phage-host system was observed only after sunscreen addition . The addition of sunscreen, even at low concentrations, had a significant impact on all enzymatic activities (aminopeptidase, glucosidase, and phosphatase), which increased significantly . However, when enzymatic activities were normalized per cell, a selective enhancement was observed for certain enzymes (e.g., aminopeptidase) and inhibition for others (e.g., glucosidase) . These results indicate that sunscreen products can modify C, N, and P biogeochemical cycling in seawater and increase virus abundance through prophage induction in marine bacterioplankton.

Blood Coagul Fibrinolysis, 2003 Jan, 14(1), 15 - 8
Viral load and disease progression as responsible for endothelial activation and/or injury in human immunodeficiency virus-1-infected patients; de Larranaga GF et al.; The endothelium participates in haemostasis, inflammation, blood pressure regulation and other physiological systems . Consequently, endothelial dysfunction has been related to hypertension, thrombosis and atherosclerosis . Both von Willebrand factor (vWF) and tissue-type plasminogen activator (t-PA) are synthesized by the endothelium and their plasma levels increased during endothelium activation or injury . So far, they are well-known markers of endothelial cell function . Many circumstances activate or damage the endothelium, such as viruses, bacterium and inflammation . Circulating vWF and t-PA were studied in 92 unselected human immunodeficiency virus-1 (HIV-1)-infected patients {27 patients with and 65 patients without acquired immunodeficiency syndrome (AIDS)} and correlated with plasma levels of pro-inflammatory cytokines (tumour necrosis factor-alpha, interleukin-6), viral load, CD4 T-cell count and infectious status . HIV-1-infected patients had significantly higher plasma levels of vWF (152 versus 90%), tumour necrosis factor-alpha (31.3 versus 9.0 pg/ml) and interleukin-6 (3.5 versus 1.9 pg/ml) but not t-PA (5.9 versus 4.2 ng/ml) than the control group . These two endothelial markers correlated significantly with viral load and interleukin-6 levels in HIV-1-infected patients . The highest levels of vWF and t-PA were found in patients with AIDS . In conclusion, endothelial cell perturbation is present in HIV infection and may be a consequence of different mechanisms such as viral load, cytokines and advanced diseases .

J Intern Med, 2003 Feb, 253(2), 102 - 19
Helicobacter pylori: resurrection of the cancer link; Bjorkholm B et al.; Helicobacter pylori is one of the most common pathogenic bacterial infections, colonizing an estimated half of all humans . In a subset of individuals, the infection leads to serious gastroduodenal disease such as peptic ulcers and gastric adenocarcinoma . The factors contributing to skewing this, in most cases benign, relationship into disease development are largely unknown . However, factors emanating from the bacterium, host and the environment have been shown to affect the risk for disease, although no factor can be singled out to be most important . The known factors are associated with affecting the risk of disease, and are not absolute . Virulence of H . pylori is affected by the existence and regulation of certain genes present in the bacterial population in a stomach . The effects of H . pylori on gastric cancer development have been challenged and the risk associated with infection with virulent (i.e . Cag PAI positive) H . pylori has likely been underestimated.

Se Pu, 2002 May, 20(3), 197 - 201
{Study on the production of trehalose by bacterium D-97 endocellular enzymes using HPLC/RI and HPLC/ESI-MS}; Rong SF et al.; The mechanism in which trehalose is produced from dextrin or starch hydrolyzate by endocellular enzymes of bacterium D-97 can be elucidated high performance liquid chromatography (HPLC) with differential refraction detection (RI) basically, including the effect of the different carbon sources on the endocellular trehalose-producing enzymes in bacterium D-97 and the possibility or ability of the endocellular enzymes to produce trehalose using maltooligosaccharides of different chain lengths . After purification of endocellular enzymes of bacterium D-97, two enzymes (called Enzyme A and Enzyme B) related to trehalose synthesis were found . The unknown oligosaccharides produced by Enzyme A were analyzed with the HPLC/RI and HPLC/ESI-MS (electrospray ionization mass spectrometry) . The results showed the relative molecular masses of the unknown oligosaccharides were the same as those of the enzymatic reaction substances (maltotriose, maltotetraose and maltopentaose) respectively . In combining with other results of biological experiments, these unknown oligosaccharides had been identified basically . There was no reduction power in these unknown oligosaccharides and only one trehalose residue exited in the molecular chain of these unknown oligosaccharides.

Rapid Commun Mass Spectrom, 2003, 17(3), 257 - 63
Detection of Escherichia coli using immunomagnetic separation and bacteriophage amplification coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Madonna AJ et al.; The application of whole cell analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has emerged as a valuable tool for rapidly identifying/detecting bacteria . This technique requires minimal sample preparation and is simple to perform, but is generally limited to purified samples of bacteria at concentrations greater than 1.0 x 10(6) cells/mL . In this paper, we describe a bacterial detection method that integrates immunomagnetic separation with bacteriophage amplification prior to MALDI-MS analysis . The developed method consists of three main stages: (1) isolation of a target bacterium by immunomagnetic separation; (2) infection of the immuno-captured bacterium with a lytic bacteriophage; and (3) assay of infected medium for bacteriophage progeny using MALDI-MS to produce a molecular weight signal for the virus capsid protein . With this technique, the presence of Escherichia coli in broth was determined in less then 2 h total analysis time at a concentration of approximately 5.0 x 10(4) cells/mL .

J Am Chem Soc, 2003 Jan 29, 125(4), 935 - 9
Generation of a bacterium with a 21 amino acid genetic code; Mehl RA et al.; We have generated a completely autonomous bacterium with a 21 amino acid genetic code . This bacterium can biosynthesize a nonstandard amino acid from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon . The biosynthetic pathway for the amino acid p-aminophenylalanine (pAF) as well as a unique pAF synthetase and cognate tRNA were added to Escherichia coli . Denaturing gel electrophoresis and mass spectrometric analysis show that pAF is incorporated into myoglobin with fidelity and efficiency rivaling those of the common 20 amino acids . This and other such organisms may provide an opportunity to examine the evolutionary consequences of adding new amino acids to the genetic repertoire, as well as generate proteins with new or enhanced biological functions.

Hunan Yi Ke Da Xue Xue Bao, 2001 Feb 28, 26(1), 89 - 91
{Using L-form bacterium ATP bioluminescence assay for rapid testing L-form bacterial susceptibility}; Tang Y et al.; Adenosine triphosphate is a kind of necessary metabolites in living cells . The authors detected ATP contents by using bioluminescence method in 111 strains of L-form bacteria after exposing to 5 kinds of antibiotics . The results showed that the mean value of CPM was less than (35 +/- 10.2)%.s-1 . Thus, the value could be acted as a critical concentration between susceptibility and resistance . The conincidence rate of this method and K-B method was 95.3% . It indicates that the bioluminescence method has a high sensitivity . It can be used to detect L-form bacterium-drug susceptibility quickly and may play an important role for choosing the antibiotics.

Mol Microbiol, 2003 Feb, 47(3), 657 - 69
Motility modes of Spiroplasma melliferum BC3: a helical, wall-less bacterium driven by a linear motor; Gilad R et al.; Spiroplasma are members of the Mollicutes (Mycoplasma, Acholeplasma and Spiroplasma) - the simplest, minimal, free-living and self-replicating forms of life . The mollicutes are unique among bacteria in completely lacking cell walls and flagella and in having an internal, contractile cytoskeleton, which also functions as a linear motor . Spiroplasma are helical, chemotactic and viscotactic active swimmers . The Spiroplasmal cytoskeleton is a flat ribbon composed of seven pairs of fibrils . The ribbon is attached to the inner side of the cell membrane along its innermost (shortest) helical line . The cell's geometry and dynamic helical parameters, and consequently motility, can be controlled by changing differentially and in a co-ordinated manner, the length of the fibrils . We identified several consistent modes of cell movements and motility originating, most likely, as a result of co-operative or local molecular switching of fibrils: (i) . regular extension and contraction within the limits of helical symmetry (this mode also includes straightening, beyond what is allowed by helical symmetry, and reversible change of helical sense); (ii) . spontaneous and random change of helical sense originating at random sites along the cell (these changes propagate along the cell in either direction and hand switching is completed within approximately 0.08 second); (iii) . forming a deformation on one of the helical turns and propagating it along the cell (these helical deformations may travel along the cell at a speed of up to approximately 40 microm s-1); (iv) . random bending, flexing and twitching (equivalent to tumbling) . In standard medium (viscosity = 1.147 centipoise) the cells run at approximately 1.5 microm s-1, have a Reynolds number of approximately 3.5 x 10-6 and consume approximately 30 ATP molecules s-1 . Running velocity, duration, persistence and efficiency increase with viscosity upon adding ficoll, dextran and methylcellulose to standard media . Relative force measurements using optical tweezers confirm these findings.

J Biol Chem, 2003 Mar 21, 278(12), 10458 - 64 Epub 2003 Jan 17.
Sequential autolytic processing activates the zymogen of Arg-gingipain; Mikolajczyk J et al.; Most proteases are synthesized as inactive precursors to protect the synthetic machinery of the cell and allow timing of activation . The mechanisms used to render latency are varied but tend to be conserved within protease families . Proteases belonging to the caspase family have a unique mechanism mediated by transitions of two surface loops, and on the basis of conservation of mechanism one would expect this to be preserved by caspase relatives . We have been able to express the full-length precursor of the Arg-specific caspase relative from the bacterium Porphyromonas gingivalis, Arg-gingipain-B, and we show that it contains N- and C-terminal extensions that render a low amount of latency, meaning that the zymogen is substantially active . Three sequential autolytic processing steps at the N and C terminus are required for full activity, and the N-propeptide may serve as an intramolecular chaperone rather than an inhibitory peptide . Each step in activation requires the previous step, and an affinity probe reveals that incremental activity enhancements are achieved in a stepwise manner.

Insect Biochem Mol Biol, 2002 Nov, 32(11), 1429 - 38
The major protein in the midgut of teneral Glossina morsitans morsitans is a molecular chaperone from the endosymbiotic bacterium Wigglesworthia glossinidia; Haines LR et al.; Molecules in the midgut of the tsetse fly (Diptera: Glossinidiae) are thought to play an important role in the life cycle of African trypanosomes by influencing their initial establishment in the midgut and subsequent differentiation events that ultimately affect parasite transmission . It is thus important to determine the molecular composition of the tsetse midgut to aid in understanding disease transmission by these medically important insect vectors . Here, we report that the most abundant protein in the midguts of teneral (unfed) Glossina morsitans morsitans is a 60 kDa molecular chaperone of bacterial origin . Two species of symbiotic bacteria reside in the tsetse midgut, Sodalis glossinidius and Wigglesworthia glossinidia . To determine the exact origin of the 60 kDa molecule, a protein microchemical approach involving two-dimensional (2-D) gel electrophoresis and mass spectrometry was used . Peptide mass maps were compared to virtual peptide maps predicted for S . glossinidius and W . glossinidia 60 kDa chaperone sequences . Four signature peptides were identified, revealing that the source of the chaperone was W . glossinidia . Comparative 2-D gel electrophoresis and immunoblotting further revealed that this protein was localized to the bacteriome and not the distal portion of the tsetse midgut . The possible function of this highly abundant endosymbiont chaperone in the tsetse midgut is discussed.

Keio J Med, 2002 Dec, 51 Suppl 2, 69 - 73
Helicobacter pylori and gastric carcinoma--from the view point of animal model; Fujioka T et al.; Many epidemiological studies have shown a strong association between chronic Helicobacter pylori infection and subsequent development of gastric carcinoma in humans . To confirm this link more clearly, it is necessary to use this bacterium in experimental studies to develop gastric carcinoma in suitable experimental animals . Persistent H . pylori infection has recently been achieved in the Japanese Monkeys and Mongolian gerbil models, with results demonstrating that the sequential histopathological changes in the gastric mucosa are closely mimic the gastric mucosal changes caused by H . pylori infection in humans . Gastric mucosa infected with H . pylori exhibited significantly higher gastritis score, reduction in glandular height, increase in the number of Ki-67 positive cells and over expression of p53 protein and p53 gene mutation in the Japanese Monkey Model . In the Mongolian gerbil model, H . pylori infection enhances gastric carcinogenesis in combination with known carcinogens such as MNU and MNNG, and also demonstrated that H . pylori infection alone can result in the development of gastric carcinoma . However, diagnostic criteria of gastric carcinoma in animal models remain in the great discussion . These important results provide a starting point for further studies to clarify the mechanism of gastric carcinogenesis as a result of H . pylori infection and assist the planning of eradication therapy to prevent gastric carcinoma.

Keio J Med, 2002 Dec, 51 Suppl 2, 15 - 9
Growth cycle of Helicobacter pylori in gastric mucous layer; Nakazawa T; Helicobacter pylori bacterium is characterized by its strong urease activity . Our studies on the role of H . pylori urease revealed; (i) it is essential for colonization, (ii) exogenous urea is required for acid resistance, (iii) the bacteria have the ability to move toward urea and sodium bicarbonate, (iv) urea hydrolysis accelerates chemotactic locomotion, and (v) decay of urease mRNA to accomplish the active center is pH-regulated; i.e., the mRNA is stabilized and destabilized under acidic and neutral conditions, respectively . Based on the above results, I propose the growth cycle of H . pylori in gastric mucous layer . H . pylori bacteria proliferate on the epithelial cell surface by utilizing nutrients derived from degraded cells . Proliferated bacteria leave the cell surface to pH-variable region where they encounter strong acid . Urease is activated with simultaneous opening of UreI channel so that urea is hydrolyzed to neutralize acid . Chemotaxis of H . pylori toward urea and sodium bicarbonate that are abundant on the cell surface is accelerated by urea hydrolysis so that the bacteria go back to the cell surface for the next round of proliferation . This growth cycle may allow the bacteria to infect persistently in the stomach.

Nucleic Acids Res, 2003 Jan 15, 31(2), 551 - 5
Mutational analysis of the Chlamydia trachomatis dnaK promoter defines the optimal -35 promoter element; Schaumburg CS et al.; A long-standing question in the biology of the intracellular bacterium, Chlamydia, has been the structure of the promoter recognized by its RNA polymerase . The 'RNA polymerase sigma subunit paradox' refers to the difficulty reconciling the conservation between the RNA polymerases of Chlamydia and Escherichia coli, especially at the level of the promoter-recognition sigma subunit, with the general lack of homology between chlamydial promoters and the E.coli sigma(70) consensus promoter . While the -10 promoter element appears to be conserved between Chlamydia and E.coli, the structure of the chlamydial -35 promoter element has not been defined . We have investigated the structure of the -35 element of the Chlamydia trachomatis dnaK promoter by measuring the effects of single base pair substitutions on in vitro promoter activity . Most substitutions produced large decreases in promoter activity, which allowed us to define the optimal -35 sequence in the context of the dnaK promoter . We found that the optimal chlamydial -35 promoter sequence is identical to the E.coli sigma(70) consensus -35 promoter element (TTGACA) . These results indicate that the optimal promoter specificities of the major form of chlamydial RNA polymerase and E.coli sigma(70) RNA polymerase are in fact highly conserved . A further implication of our results is that many chlamydial promoters have a suboptimal promoter structure . We hypothesize that these chlamydial promoters are intrinsically weak promoters that can be regulated during the chlamydial developmental cycle by additional transcription factors.

Clin Microbiol Rev, 2003 Jan, 16(1), 37 - 64
Ehrlichia chaffeensis: a prototypical emerging pathogen; Paddock CD et al.; Ehrlichia chaffeensis is an obligately intracellular, tick-transmitted bacterium that is maintained in nature in a cycle involving at least one and perhaps several vertebrate reservoir hosts . The moderate to severe disease caused by E . chaffeensis in humans, first identified in 1986 and reported for more than 1,000 patients through 2000, represents a prototypical "emerging infection." Knowledge of the biology and natural history of E . chaffeensis, and of the epidemiology, clinical features, and laboratory diagnosis of the zoonotic disease it causes (commonly referred to as human monocytic ehrlichiosis {HME}) has expanded considerably in the period since its discovery . In this review, we summarize briefly the current understanding of the microbiology, pathogenesis, and clinical manifestations associated with this pathogen but focus primarily on discussing various ecological factors responsible for the recent recognition of this important and potentially life-threatening tick-borne disease . Perhaps the most pivotal element in the emergence of HME has been the staggering increases in white-tailed deer populations in the eastern United States during the 20th century . This animal serves as a keystone host for all life stages of the principal tick vector (Amblyomma americanum) and is perhaps the most important vertebrate reservoir host for E . chaffeensis . The contributions of other components, including expansion of susceptible human populations, growth and broadening geographical distributions of other potential reservoir species and A . americanum, and improvements in confirmatory diagnostic methods, are also explored.

J Protein Chem, 2002 Oct, 21(7), 455 - 63
Primary structure and chemical modification of some amino acid residues of bifunctional alginate lyase from a marine bacterium Pseudoalteromonas sp . strain no . 272; Iwamoto Y et al.; The entire amino acid sequence of bifunctional alginate lyase from Pseudoalteromonas sp . strain No . 272 were determined by two approaches, Edman degradation of the peptides obtained from protease digestion of the enzyme protein and analysis of PCR products of the structural gene . The former resulted in incomplete amino acid sequence in the entire sequence, due to lacking of the proper peptides from the protease digestion . To compensate for this lack of sequences we applied the method of PCR of the structural gene that was initially elucidated from the primers designed from N- and C-terminal amino acid sequences of the enzyme . The results of the amino acid sequences from these two approaches showed good agreement . The enzyme consisted of 233 amino acid residues with a molecular mass of 25,549.5, including the sole W and cystine residue . The sequence homology search among the other alginate lyases from different origins indicated that they were very weakly homologous, with the exception of the sequence homology (80.3%) of Pseudoalteromonas elyakovii alginate lyase . The consensus sequence, YFKhG + Y-Q (Wong, T . Y., Preston, L . A., and Schiller, N . L . 2000 . Annu . Rev . MicrobioL 54: 289-340) in the C-terminal regions was conserved . The kinetic analyses of chemical modification of some amino acid residues of the enzyme showed that W, K, and Y appeared to be important in the enzyme function.

Heredity, 2003 Jan, 90(1), 49 - 55
Characterization of non-cytoplasmic incompatibility inducing Wolbachia in two continental African populations of Drosophila simulans; Charlat S et al.; Wolbachia is an endocellular bacterium infecting arthropods and nematodes . In arthropods, it invades host populations through various mechanisms, affecting host reproduction, the most common of which being cytoplasmic incompatibility (CI) . CI is an embryonic mortality occurring when infected males mate with uninfected females or females infected by a different Wolbachia strain . This phenomenon is observed in Drosophila simulans, an intensively studied Wolbachia host, harbouring at least five distinct bacterial strains . In this study, we investigate various aspects of the Wolbachia infections occurring in two continental African populations of D . simulans: CI phenotype, phylogenetic position based on the wsp gene and associated mitochondrial haplotype . From the East African population (Tanzania), we show that (i) the siIII mitochondrial haplotype occurs in continental populations, which was unexpected based on the current views of D . simulans biogeography, (ii) the wKi strain (that rescues from CI while being unable to induce it) is very closely related to the CI-inducing strain wNo, (iii) wKi and wNo might not derive from a unique infection event, and (iv) wKi is likely to represent the same entity as the previously described wMa variant . In the West African population (Cameroon), the Wolbachia infection was found identical to the previously described wAu, which does not induce CI . This finding supports the view that wAu might be an ancient infection in D . simulans.

Science, 2003 Jan 10, 299(5604), 254 - 6
Ringlike structure of the Deinococcus radiodurans genome: a key to radioresistance?
Levin-Zaidman S, Englander J, Shimoni E, Sharma AK, Minton KW, Minsky A.
The bacterium Deinococcus radiodurans survives ionizing irradiation and other DNA-damaging assaults at doses that are lethal to all other organisms . How D . radiodurans accurately reconstructs its genome from hundreds of radiation-generated fragments in the absence of an intact template is unknown . Here we show that the D . radiodurans genome assumes an unusual toroidal morphology that may contribute to its radioresistance . We propose that, because of restricted diffusion within the tightly packed and laterally ordered DNA toroids, radiation-generated free DNA ends are held together, which may facilitate template-independent yet error-free joining of DNA breaks.

Mol Microbiol, 2003 Jan, 47(2), 539 - 47
Penicillin-binding protein PBP2 of Escherichia coli localizes preferentially in the lateral wall and at mid-cell in comparison with the old cell pole; Den Blaauwen T et al.; The localization of penicillin-binding protein 2 (PBP2) in Escherichia coli has been studied using a functional green fluorescent protein (GFP)-PBP2 fusion protein . PBP2 localized in the bacterial envelope in a spot-like pattern and also at mid-cell during cell division . PBP2 disappeared from mid-cell just before separation of the two daughter cells . It localized with a preference for the cylindrical part of the bacterium in comparison with the old cell poles, which are known to be inert with respect to peptidoglycan synthesis . In contrast to subunits of the divisome, PBP2 failed to localize at mid-cell when PBP3 was inhibited by the specific antibiotic aztreonam . Therefore, despite its dependency on active PBP3 for localization at mid-cell, it seems not to be an integral part of the divisome . Cells grown for approximately half a mass doubling time in the presence of the PBP2 inhibitor mecillinam synthesized nascent cell poles with an increased diameter, indicating that PBP2 is required for the maintenance of the correct diameter of the new cell pole.

Biochem J, 2003 Apr 15, 371(Pt 2), 473 - 83
Treponema denticola cystalysin exhibits significant alanine racemase activity accompanied by transamination: mechanistic implications; Bertoldi M et al.; To obtain information on the reaction specificity of cystalysin from the spirochaete bacterium Treponema denticola, the interaction with L- and D-alanine has been investigated . Binding of both alanine enantiomers leads to the appearance of an external aldimine absorbing at 429 nm and of a band absorbing at 498 nm, indicative of a quinonoid species . Racemization and transamination reactions were observed to occur with both alanine isomers as substrates . The steady-state kinetic parameters for racemization, k (cat) and K (m), for L-alanine are 1.05+/-0.03 s(-1) and 10+/-1 mM respectively, whereas those for D-alanine are 1.4+/-0.1 s(-1) and 10+/-1 mM . During the reaction of cystalysin with L- or D-alanine, a time-dependent loss of beta-elimination activity occurs concomitantly with the conversion of the pyridoxal 5'-phosphate (PLP) coenzyme into pyridoxamine 5'-phosphate (PMP) . The catalytic efficiency of the half-transamination of L-alanine is found to be 5.3x10(-5) mM(-1) x s(-1), 5-fold higher when compared with that of D-alanine . The partition ratio between racemization and half-transamination reactions is 2.3x10(3) for L-alanine and 1.4x10(4) for D-alanine . The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with p K values of 6.5-6.6, which must be unprotonated for catalysis . Addition of pyruvate converts the PMP form of the enzyme back into the PLP form and causes the concomitant recovery of beta-elimination activity . In contrast with other PLP enzymes studied so far, but similar to alanine racemases, the apoform of the enzyme abstracted tritium from C4' of both (4' S)- and (4' R)-{4'-(3)H}PMP in the presence of pyruvate . Together with molecular modelling of the putative binding sites of L- and D-alanine at the active site of the enzyme, the implications of these studies for the mechanisms of the side reactions catalysed by cystalysin are discussed.

J Am Chem Soc, 2003 Jan 15, 125(2), 379 - 87
Assay for the enantiomeric analysis of {2H1}-fluoroacetic acid: insight into the stereochemical course of fluorination during fluorometabolite biosynthesis in streptomyces cattleya; O'Hagan D et al.; A sensitive method for the configurational analysis of (R)- and (S)-{2H1}-fluoroacetate has been developed using 2H{1H}-NMR in a chiral liquid crystalline solvent . This has enabled biosynthetic experiments to be conducted which reveal stereochemical details on biological fluorination occurring during the biosynthesis of fluoroacetate and 4-fluorothreonine in the bacterium Streptomyces cattleya . In particular, feeding experiments to S . cattleya with isotopically labeled (1R, 2R)- and (1S, 2R)-{1-2H1}-glycerol 3d and 3e and {2,3-2H(4)}-succinate 4a gave rise to samples of enantiomerically enriched {2-2H1}-fluoroacetates 1a . The predominant enantiomer resulting from each experiment suggests that the stereochemical course of biological fluorination takes place with an overall retention of configuration between a glycolytic intermediate and fluoroacetate 1 . Consequently, this outcome suggests that the stereochemical course of the recently identified fluorinase enzyme which mediates a reaction between fluoride ion and S-adenosyl-l-methionine (SAM), occurs with an inversion of configuration.

Minerva Med, 2002 Dec, 93(6), 447 - 51
{Whipple's disease: progress in the diagnosis and review of the literature}; Ghittoni G et al.; Whipple's disease is a rare, chronic, multisystemic disease characterized by the presence of fever, diarrhea, weight loss and malabsorption, preceded by arthritis . Although Whipple's disease almost always includes involvement of the small intestine and the presence of malabsorption, it commonly affects other organs, especially the heart, brain, eyes and joints . Whipple's disease greately mimics other diseases and is caused by a cultivation-resistant bacterium . The disease is fatal unless patients are treated with antibiotics . The diagnosis of Whipple's disease can be made by histologic analysis of small-intestinal biopsy specimens . Identification of Whipple bacterium, Tropheryma whippelii, has led to the development of the polymerase chain reaction This technique can be used to detect the bacterium in many organs and fluids, including synovial tissue and fluid . Affected patients tend to have dilated intestinal villi that are infiltrated with foamy macrophages.

Biochemistry, 2003 Jan 14, 42(1), 222 - 30
A novel binuclear {CuSMo} cluster at the active site of carbon monoxide dehydrogenase: characterization by X-ray absorption spectroscopy; Gnida M et al.; The structurally characterized molybdoenzyme carbon monoxide dehydrogenase (CODH) catalyzes the oxidation of CO to CO2 in the aerobic bacterium Oligotropha carboxidovorans . The active site of the enzyme was studied by Mo- and Cu-K-edge X-ray absorption spectroscopy . This revealed a bimetallic {Cu(I)SMo(VI)(double bond O)2} cluster in oxidized CODH which was converted into a {Cu(I)SMo(IV)(double bond O)OH2} cluster upon reduction . The Cu...Mo distance is 3.70 A in the oxidized form and is increased to 4.23 A upon reduction . The bacteria contain CODH species with the complete and functional bimetallic cluster along with enzyme species deficient in Cu and/or bridging S . The latter are precursors in the posttranslational biosynthesis of the metal cluster . Cu-deficient CODH is the most prominent precursor and contains a {HSMo(double bond O)OH2} cluster . Se-K-edge X-ray absorption spectroscopy demonstrates that Se is coordinated by two C atoms at 1.94-1.95 A distance . This is interpreted as a replacement of the S in methionine residues . In contrast to a previous report {Dobbek, H., Gremer, L., Meyer, O., and Huber, R . (1999) Proc . Natl . Acad . Sci . U.S.A . 96, 8884-8889} Se was not identified in the active site of CODH.

Appl Environ Microbiol, 2003 Jan, 69(1), 600 - 6
Survival and growth of Francisella tularensis in Acanthamoeba castellanii; Abd H et al.; Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals . The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it . To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F . tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium . GFP fluorescence within A . castellanii was then monitored by flow cytometry and fluorescence microscopy . In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies . Electron microscopy was used to determine the intracellular location of F . tularensis in A . castellanii, and viable counts were obtained for both extracellular and intracellular bacteria . The results showed that many F . tularensis cells were located intracellularly in A . castellanii cells . The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells . F . tularensis was found in intact trophozoites, excreted vesicles, and cysts . Furthermore, F . tularensis grew faster in cocultures with A . castellanii than it did when grown alone in the same medium . This increase in growth was accompanied by a decrease in the number of A . castellanii cells . The interaction between F . tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F . tularensis.

Appl Environ Microbiol, 2003 Jan, 69(1), 504 - 8
Gene Cloning, purification, and characterization of a phosphodiesterase from Delftia acidovorans; Tehara SK et al.; A novel phosphodiesterase (PdeA) was purified from Delftia acidovorans, the gene encoding the enzyme was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to apparent homogeneity and characterized . PdeA is an 85-kDa trimer that exhibits maximal activity at 65 degrees C and pH 10 even though it was isolated from a mesophilic bacterium . Although PdeA exhibited both mono- and diesterase activity, it was most active on the phosphodiester bis(p-nitrophenyl)phosphate with a K(m) of 2.9 +/- 0.1 mM and a k(cat) of 879 +/- 73 min(-1) . The enzyme showed sequence similarity to cyclic AMP (cAMP) phosphodiesterase and cyclic nucleotide phosphodiesterases and exhibited activity on cAMP in vivo when the gene was expressed in E . coli . The IS1071 transposon insertion sequence was found downstream of pdeA.

J Bacteriol, 2003 Jan, 185(2), 399 - 404
Characterization of benzoyl coenzyme A biosynthesis genes in the enterocin-producing bacterium "Streptomyces maritimus"; Xiang L et al.; The novel benzoyl coenzyme A (benzoyl-CoA) biosynthesis pathway in "Streptomyces maritimus" was investigated through a series of target-directed mutations . Genes involved in benzoyl-CoA formation were disrupted through single-crossover homologous recombination, and the resulting mutants were analyzed for their ability to biosynthesize the benzoyl-CoA-primed polyketide antibiotic enterocin . Inactivation of the unique phenylalanine ammonia-lyase-encoding gene encP was previously shown to be absolutely required for benzoyl-CoA formation in "S . maritimus" . The fatty acid beta-oxidation-related genes encH, -I, and -J, on the other hand, are necessary but not required . In each case, the yield of benzoyl-CoA-primed enterocin dropped below wild-type levels . We attribute the reduced benzoyl-CoA formation in these specific mutants to functional substitution and cross-talk between the products of genes encH, -I, and -J and the enzyme homologues of primary metabolism . Disruption of the benzoate-CoA ligase encN gene did not perturb enterocin production, however, demonstrating that encN is extraneous and that benzoic acid is not a pathway intermediate . EncN rather serves as a substitute pathway for utilizing exogenous benzoic acid . These experiments provide further support that benzoyl-CoA is formed in a novel bacterial pathway that resembles the eukaryotic assembly of benzoyl-CoA from phenylalanine via a beta-oxidative path.

What Is Botulism?, What Is Amino Acid?, What Is Biofilm?, What Is Protein?, What Is Fermentation?, n, Bacteriology, i, Microbes, a, Bacterium, c, Microorganism, r, Microbiology, s, Prokaryotes, a, Microbial, r, Staphylococcus, i, Bacillus subtilis, a, Escherichia coli, c, Erythromycin, i, Escherichia coli, r, Multidrug resistant, n, Microbial, a, Salmonella typhimurium, o, Corynebacter, i, S. cerevisiae, e, Escherichia coli, r, Multidrug resistant, n, S. cerevisiae, s, Escherichia coli, r, Kluyveromyces, n, Anaerobes, i, Salmonella, e, Yeasts, i, Escherichia coli

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005