|
|
|
|
|
| Am J Gastroenterol, 1998 Sep, 93(9), 1425 - 31 Reversal of fundic atrophy after eradication of Helicobacter pylori; Tucci A et al.; OBJECTIVES: We sought to evaluate the effect of Helicobacter pylori eradication in patients with fundic atrophic gastritis . METHODS: Acid secretion, gastric emptying, and histology were evaluated in 20 patients with fundic atrophic gastritis and H . pylori infection . After investigation, 10 patients (Group 1) received an eradicating treatment and 10 (Group 2) did not receive any treatment . One year later, the baseline investigations were repeated . Subsequently, patients in Group 2 received the same treatment given to patients in Group 1 and were reevaluated 12 months later . A further follow-up was performed in both groups 36 months after the treatment . RESULTS: At 1-yr follow-up, all the patients in Group 1 were H . pylori negative whereas all the patients in Group 2 were still infected . In Group 1, there was a significant improvement of both fundic atrophy and acid secretion, compared with baseline (p < 0.01) . In Group 2, no substantial modification of either histological or functional parameters was observed at the first follow-up; conversely, a significant (p < 0.01) improvement of fundic atrophy and acid secretion was detected in these patients 12 months after eradication of the bacterium . Histological pattern remained unchanged at 36 months of follow-up in both groups . Gastric emptying remained, on the average, unaffected by the treatment; however, three patients with delayed gastric emptying at entry had normal gastric emptying after eradication of H . pylori . CONCLUSIONS: Our data suggest that mucosal atrophy can be reduced or even reversed by the eradication of H . pylori, and this is associated with a recovery of gastric function. Int J Syst Bacteriol, 1998 Apr, 48 Pt 2, 431 - 40 Pelistega europaea gen . nov., sp . nov., a bacterium associated with respiratory disease in pigeons: taxonomic structure and phylogenetic allocation; Vandamme P et al.; Twenty-four strains isolated mainly from infected respiratory tracts of pigeons were characterized by an integrated genotypic and phenotypic approach . An extensive biochemical examination using conventional tests and several API microtest systems indicated that all isolates formed a phenotypically homogeneous taxon with a DNA G + C content between 42 and 43 mol% . Whole-cell protein and fatty acid analysis revealed an unexpected heterogeneity which was confirmed by DNA-DNA hybridizations . Four main genotypic sub-groups (genomovars) were delineated . 16S rDNA sequence analysis of a representative strain indicated that this taxon belongs to the beta-subclass of the Proteobacteria with Taylorella equigenitalis as its closest neighbour (about 94.8% similarity) . A comparison of phenotypic and genotypic characteristics of both taxa suggested that the pigeon isolates represented a novel genus for which the name Pelistega is proposed . In the absence of differential phenotypic characteristics between the genomovars, it was preferred to include all of the isolates into a single species, Pelistega europaea, and strain LMG 10982 was selected as the type strain . The latter strain belongs to fatty acid cluster I and protein electrophoretic sub-group 1, which comprise 13 and 5 isolates, respectively . It is not unlikely that the name P . europaea will be restricted in the future to organisms belonging to fatty acid cluster I, or even to protein electrophoretic sub-group 1, upon discovery of differential diagnostic features. BMJ, 1998 Sep 5, 317(7159), 637 - 42 Understanding the culture of prescribing: qualitative study of general practitioners' and patients' perceptions of antibiotics for sore throats; Butler CC et al.; OBJECTIVES: To better understand reasons for antibiotics being prescribed for sore throats despite well known evidence that they are generally of little help . DESIGN: Qualitative study with semi-structured interviews . SETTING: General practices in South Wales . SUBJECTS: 21 general practitioners and 17 of their patients who had recently consulted for a sore throat or upper respiratory tract infection . MAIN OUTCOME MEASURES: Subjects' experience of management of the illness, patients' expectations, beliefs about antibiotic treatment for sore throats, and ideas for reducing prescribing . RESULTS: Doctors knew of the evidence for marginal effectiveness yet often prescribed for good relationships with patients . Possible patient benefit outweighed theoretical community risk from resistant bacteria . Most doctors found prescribing "against the evidence" uncomfortable and realised this probably increased workload . Explanations of the distinction between virus and bacterium often led to perceived confusion . Clinicians were divided on the value of leaflets and national campaigns, but several favoured patient empowerment for self care by other members of the primary care team . Patient expectations were seldom made explicit, and many were not met . A third of patients had a clear expectation for antibiotics, and mothers were more likely to accept non-antibiotic treatment for their children than for themselves . Satisfaction was not necessarily related to receiving antibiotics, with many seeking reassurance, further information, and pain relief . CONCLUSIONS: This prescribing decision is greatly influenced by considerations of the doctor-patient relationship . Consulting strategies that make patient expectations explicit without damaging relationships might reduce unwanted antibiotics . Repeating evidence for lack of effectiveness is unlikely to change doctors' prescribing, but information about risk to individual patients might . Emphasising positive aspects of non-antibiotic treatment and lack of efficacy in general might be helpful. Appl Environ Microbiol, 1998 Sep, 64(9), 3429 - 36 Assessment of reductive acetogenesis with indigenous ruminal bacterium populations and Acetitomaculum ruminis; Le Van TD et al.; The objective of this study was to evaluate the role of reductive acetogenesis as an alternative H2 disposal mechanism in the rumen . H2/CO2-supported acetogenic ruminal bacteria were enumerated by using a selective inhibitor of methanogenesis, 2-bromoethanesulfonic acid (BES) . Acetogenic bacteria ranged in density from 2.5 x 10(5) cells/ml in beef cows fed a high-forage diet to 75 cells/ml in finishing steers fed a high-grain diet . Negligible endogenous acetogenic activity was demonstrated in incubations containing ruminal contents, NaH13CO3, and 100% H2 gas phase since {U-13C}acetate, as measured by mass spectroscopy, did not accumulate . Enhancement of acetogenesis was observed in these incubations when methanogenesis was inhibited by BES and/or by the addition of an axenic culture of the rumen acetogen Acetitomaculum ruminis 190A4 (10(7) CFU/ml) . To assess the relative importance of population density and/or H2 concentration for reductive acetogenesis in ruminal contents, incubations as described above were performed under a 100% N2 gas phase . Both selective inhibition of methanogenesis and A . ruminis 190A4 fortification (>10(5) CFU/ml) were necessary for the detection of reductive acetogenesis under H2-limiting conditions . Under these conditions, H2 accumulated to 4, 800 ppm . In contrast, H2 accumulated to 400 ppm in incubations with active methanogenesis (without BES) . These H2 concentrations correlated well with the pure culture H2 threshold concentrations determined for A . ruminis 190A4 (3,830 ppm) and the ruminal methanogen 10-16B (126 ppm) . The data demonstrate that ruminal methanogenic bacteria limited reductive acetogenesis by lowering the H2 partial pressure below the level necessary for H2 utilization by A . ruminis 190A4. Appl Environ Microbiol, 1998 Sep, 64(9), 3166 - 74 Analysis of Escherichia coli O157:H7 survival in ovine or bovine manure and manure slurry; Kudva IT et al.; Farm animal manure or manure slurry may disseminate, transmit, or propagate Escherichia coli O157:H7 . In this study, the survival and growth of E . coli O157:H7 in ovine or bovine feces under various experimental and environmental conditions were determined . A manure pile collected from experimentally inoculated sheep was incubated outside under fluctuating environmental conditions . E . coli O157:H7 survived in the manure for 21 months, and the concentrations of bacteria recovered ranged from <10(2) to 10(6) CFU/g at different times over the course of the experiment . The DNA fingerprints of E . coli O157:H7 isolated at month 1 and month 12 were identical or very similar . A second E . coli O157:H7-positive ovine manure pile, which was periodically aerated by mixing, remained culture positive for 4 months . An E . coli O157:H7-positive bovine manure pile was culture positive for 47 days . In the laboratory, E . coli O157:H7 was inoculated into feces, untreated slurry, or treated slurry and incubated at -20, 4, 23, 37, 45, and 70 degreesC . E . coli O157:H7 survived best in manure incubated without aeration at temperatures below 23 degreesC, but it usually survived for shorter periods of time than it survived in manure held in the environment . The bacterium survived at least 100 days in bovine manure frozen at -20 degreesC or in ovine manure incubated at 4 or 10 degreesC for 100 days, but under all other conditions the length of time that it survived ranged from 24 h to 40 days . In addition, we found that the Shiga toxin type 1 and 2 genes in E . coli O157:H7 had little or no influence on bacterial survival in manure or manure slurry . The long-term survival of E . coli O157:H7 in manure emphasizes the need for appropriate farm waste management to curtail environmental spread of this bacterium . This study also highlights the difficulties in extrapolating laboratory data to on-farm conditions. Appl Environ Microbiol, 1998 Sep, 64(9), 3134 - 9 Signal transduction in the protozoan host Hartmannella vermiformis upon attachment and invasion by Legionella micdadei; Abu Kwaik Y et al.; The intracellular pathogens Legionella micdadei and Legionella pneumophila are the two most common Legionella species that cause Legionnaires' disease . Intracellular replication within pulmonary cells is the hallmark of Legionnaires' disease . In the environment, legionellae are parasites of protozoans, and intracellular bacterial replication within protozoans plays a major role in the transmission of Legionnaires' disease . In this study, we characterized the initial host signal transduction mechanisms involved during attachment to and invasion of the protozoan host Hartmannella vermiformis by L . micdadei . Bacterial attachment prior to invasion of H . vermiformis by L . micdadei is associated with tyrosine dephosphorylation of multiple host cell proteins, including a 170-kDa protein . We have previously shown that this 170-kDa protein is the galactose N-acetylgalactosamine (Gal/GalNAc)-inhibitable lectin receptor that mediates attachment to and invasion of H . vermiformis by L . pneumophila . Subsequent bacterial entry targets L . micdadei into a phagosome that is not surrounded by the rough endoplasmic reticulum (RER) . In contrast, uptake of L . pneumophila mediated by attachment to the Gal/GalNAc lectin is followed by targeting of the bacterium into an RER-surrounded phagosome . These results indicate that despite similarities in the L . micdadei and L . pneumophila attachment-mediated signal transduction mechanisms in H . vermiformis, the two bacterial species are targeted into morphologically distinct phagosomes in their natural protozoan host. J Immunol, 1998 Sep 1, 161(5), 2407 - 13 B7-1, but not CD28, is crucial for the maintenance of the CD4+ T cell responses in human leprosy; Schlienger K et al.; We used human leprosy as a model to compare patterns of costimulatory molecule expression in respect to the clinical/immunologic spectrum of disease . We found that B7-1, B7-2, and CD28 transcripts dominated in tuberculoid leprosy patients, who have potent T cell responses to Mycobacterium leprae . In contrast, CTLA-4 was more strongly expressed in lesions from lepromatous patients, who manifest specific T cell anergy to the leprosy bacterium . T cell clones from tuberculoid lesions were CD4+CD28+ or CD4+CD28-, and T cell clones from lepromatous lesions were predominantly CD8+CD28- . The M . leprae-specific recall response of CD4+ T cell clones from tuberculoid lesions was blocked by anti-B7-1 mAb, but not by anti-B7-2 mAb or CTLA-Ig . However, anti-CD28 and anti-CTLA-4 mAbs did not block activation of clones from tuberculoid lesions, suggesting that B7-1 may utilize another costimulatory pathway . Peripheral blood T cell responses in the lepromatous form were strongly regulated by CD28 during T cell activation, in contrast to the tuberculoid form . Thus, B7-1 costimulation could play a role in maintaining a strong immune response to the pathogen. Biochemistry, 1998 Sep 1, 37(35), 12293 - 300 Membrane-bound cytochrome cz couples quinol oxidoreductase to the P840 reaction center complex in isolated membranes of the green sulfur bacterium Chlorobium tepidum; Oh-oka H et al.; The reaction of quinol oxidoreductase and membrane-bound c-type cytochromes was studied in chlorosome-depleted membranes isolated from Chlorobium tepidum . Rapid oxidations of c-type cytochromes were detected after flash excitation . Their re-reductions occurred in parallel with the reduction of cytochrome b, especially in the presence of antimycin A, whereas reductions of both cytochromes c and b were suppressed by added stigmatellin . These results indicate the tight coupling between the photosynthetic reaction center and quinol oxidoreductase . Turnovers of two types of cytochromes c were detected . One was assigned to the monoheme-type cytochrome c (designated cytochrome cz), which is known to be tightly bound to the reaction center complex . The other was a new c-type cytochrome, cytochrome c-556, which functions the same as cytochrome c1 . The steps of electron-transfer scheme, menaquinol --> Rieske FeS center --> cytochrom c-556 --> cytochrome cz --> P840, are estimated to have reaction times of 20 ms and 560, 150, and 40 microseconds, respectively . We conclude that quinol oxidoreductase and the reaction center complex in Chlorobium tepidum are linked by two distinct membrane-bound cytochromes, cz and c-556, with no involvement of water-soluble cytochromes. Dig Dis Sci, 1998 Aug, 43(8), 1641 - 5 Helicobacter pylori eradication ameliorates primary Raynaud's phenomenon; Gasbarrini A et al.; Raynaud's phenomenon is defined by an intermittent vasospasm of the arterioles of the distal limbs . Helicobacter pylori infection has been recently associated with Raynaud's phenomenon . The aim of this study was to assess the effects of H . pylori eradication on Raynaud's attacks . Forty-six patients affected by primary Raynaud's phenomenon were evaluated . H . pylori infection was assessed by {13C}urea breath test . Eradication therapy was given to infected patients for seven days . Discomfort and the duration and frequency of attacks of Raynaud's phenomenon per week were assessed . Thirty-six subjects were infected with H . pylori; the bacterium was eradicated in 83% of these after therapy . Attacks of Raynaud's phenomenon completely disappeared in 17% of the patients with H . pylori eradication . Discomfort and the duration and frequency of attacks of Raynaud's phenomenon were significantly reduced in 72% of the remaining patients . Conversely, attacks of Raynaud's disease did not change significantly during the 12-week follow-up period either in the H . pylori-negative patients or in the infected subjects in whom the bacterium was not eradicated by therapy . The study shows that H . pylori eradication causes a significant decrease in clinical attacks of Raynaud's disease . The reduction of vasoactive substances determined by the eradication of the bacterium may be the pathogenetic mechanism underlying the phenomenon. Proc R Soc Lond B Biol Sci, 1998 Aug 7, 265(1404), 1447 - 52 Widespread occurrence of the micro-organism Wolbachia in ants; Wenseleers T et al.; For more than 20 years, sex allocation in hymenopteran societies has been a major topic in insect sociobiology . A recurring idea was that relatedness asymmetrics arising from their haplodiploid sex determination system would lead to various parent-offspring conflicts over optimal reproduction . A possible weakness of existing theory is that only interests of nuclear genes are properly accounted for . Yet, a diversity of maternally transmitted elements manipulate the reproduction of their host in many solitary arthropod groups . The bacterium Wolbachia is a striking example of such a selfish cytoplasmic element, with effects ranging from reproductive incompatibility between host strains, induction of parthenogenesis and feminization of males . This paper reports on a first PCR-based Wolbachia screening in ants . Out of 50 Indo-Australian species, 50% screened positive for an A-group strain . One of these species also harboured a B-group strain in a double infection . Various factors that might explain the unusually high incidence of Wolbachia in ants are discussed . In general, Wolbachia may represent a widespread and previously unrecognized party active in the conflicts of interest within social insect colonies. J Bacteriol, 1998 Sep, 180(17), 4325 - 31 Parallel and divergent genotypic evolution in experimental populations of Ralstonia sp; Nakatsu CH et al.; Genetic rearrangements within a population of bacteria were analyzed to understand the degree of divergence occurring after experimental evolution . We used 18 replicate populations founded from Ralstonia sp . strain TFD41 that had been propagated for 1,000 generations with 2,4-dichlorophenoxyacetic acid (2,4-D) as the carbon source . Genetic divergence was examined by restriction fragment length polymorphism analysis of the incumbent plasmid that carries the 2,4-D catabolic genes and by amplification of random regions of the genome via PCR . In 18 evolved clones examined, we observed duplication within the plasmid, including the tfdA gene, which encodes a 2,4-D dioxygenase that catalyzes the first step in the 2,4-D catabolic pathway . In 71 of 72 evolved clones, a common 2.4-kb PCR product was lost when genomic fingerprints produced by PCR amplification using degenerate primers based on repetitive extragenic palindromic (REP) sequences (REP-PCR) were compared . The nucleotide sequence of the 2.4-kb PCR product has homology to the TRAP (tripartite ATP-independent periplasmic) solute transporter gene family . Hybridization of the 2 . 4-kb REP-PCR product from the ancestor to genomic DNA from the evolved populations showed that the loss of the PCR product resulted from deletions in the genome . Deletions in the plasmid and presence and/or absence of other REP-PCR products were also found in these clones but at much lower frequencies . The common and uncommon genetic changes observed show that both parallel and divergent genotypic evolution occurred in replicate populations of this bacterium. Appl Microbiol Biotechnol, 1998 Jul, 50(1), 55 - 64 Analysis of the gene for beta-fructosidase (invertase, inulinase) of the hyperthermophilic bacterium Thermotoga maritima, and characterisation of the enzyme expressed in Escherichia coli; Liebl W et al.; This is the first report describing the gene structure and the enzymatic properties of a beta-fructosidase of a hyperthermophilic organism . The bfrA gene of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue, polypeptide of about 50 kDa, with significant sequence similarity to other beta-fructosidases . On the basis of its primary structure, BfrA can be assigned to glycosyl hydrolase family 32 . The bfrA gene was expressed in Escherichia coli and the recombinant enzyme was purified and characterised . BfrA was specific for the fructose moiety and the beta-anomeric configuration of the glycosidic linkages of its substrates . The enzyme released fructose from sucrose and raffinose, and the fructose polymer inulin was hydrolysed quantitatively in an exo-type fashion . BfrA displayed similar catalytic efficiencies for the hydrolysis of sucrose and inulin with Kcat/K(m) values (at 75 degrees C, pH 5.5) of about 4.1 x 10(4) M-1S-1 and 3.1 x 10(4) M-1S-1 respectively . BfrA had an optimum temperature of 90-95 degrees C (10-min assay) and was extremely insensitive to thermo-inactivation . During 5 h at temperatures up to 80 degrees C at pH 7, the enzyme retained at least 85% of its initial activity . Thus, BfrA is the most thermostable beta-fructosidase and also the most thermostable inulinase described to date . In conclusion, the T . maritima enzyme can be classified as an exo-beta-D-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase activity . Its catalytic properties along with the extreme thermostability recommend it for use in biotechnology. Microbiology, 1998 Aug, 144 ( Pt 8), 2263 - 9 Phototrophic oxidation of ferrous iron by a Rhodomicrobium vannielii strain; Heising S et al.; Oxidation of ferrous iron was studied with the anaerobic phototrophic bacterial strain BS-1 . Based on morphology, substrate utilization patterns, arrangement of intracytoplasmic membranes and the in vivo absorption spectrum, this strain was assigned to the known species Rhodomicrobium vannielii . Also, the type strain of this species oxidized ferrous iron in the light . Phototrophic growth of strain BS-1 with ferrous iron as electron donor was stimulated by the presence of acetate or succinate as cosubstrates . The ferric iron hydroxides produced precipitated on the cell surfaces as solid crusts which impeded further iron oxidation after two to three generations . The complexing agent nitrilotriacetate stimulated iron oxidation but the yield of cell mass did not increase stoichiometrically under these conditions . Other complexing agents inhibited cell growth . Ferric iron was not reduced in the dark, and manganese salts were neither oxidized nor reduced . It is concluded that ferrous iron oxidation by strain BS-1 is only a side activity of this bacterium that cannot support growth exclusively with this electron source over prolonged periods of time. Microbiology, 1998 Aug, 144 ( Pt 8), 2195 - 202 Lysogenic bacteriophage M1 from Selenomonas ruminantium: isolation, characterization and DNA sequence analysis of the integration site; Cheong JP et al.; Bacteriophage M1 from the ruminal bacterium Selenomonas ruminantium strain ML12 comprises a 30 nm icosahedral capsid, a 25 nm tail and 48 kb of linear dsDNA with cohesive ends . A restriction map of the phage genome has been constructed . The presence of bacteriophage M1 in the rumen has been demonstrated by PCR amplification and Southern blot analysis of DNA from rumen bacterial samples obtained from ten different sheep . Lysogeny was demonstrated by hybridization of M1 DNA to host chromosomal DNA and by identification and cloning of a 2.3 kb region of the phage containing the predicted attP domain which promotes chromosomal integration . DNA sequencing of the attP region demonstrated two major ORFs surrounding the predicted attP site and structural analysis of this region revealed a motif comprising three different inverted repeats surrounding a 12 bp palindrome . Analysis of the translated amino acid sequence upstream of the attP site demonstrated the presence of conserved residues found within integrase proteins of several temperate phages of different bacterial species. Biochim Biophys Acta, 1998 Jul 31, 1393(1), 57 - 62 Novel glycine containing glucolipids from the alkane using bacterium Alcanivorax borkumensis; Abraham WR et al.; The polar lipids from the hydrocarbon using and biosurfactant-producing bacterium Alcanivorax borkumensis were isolated and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy . The biosurfactant produced by this species is an anionic glucose lipid with a tetrameric oxyacyl side chain . The glycolipids extracted from the cell wall consist of this biosurfactant N-terminally esterified with glycine . Ten different derivatives of this lipid type were identified and their structures elucidated by MSMS . They vary by the chain length of one or two of the four beta-hydroxy fatty acids (C6, C8 and C10) and by the location of these different fatty acids within the molecule . All compounds are reported here for the first time . In addition to these glycolipids, three different phosphatidylglycerols were identified . While these lipids were found in all strains of A . borkumensis, the relative abundances of the different lipids vary between the strains. J Food Prot, 1998 Aug, 61(8), 929 - 33 Effect of environmental and substrate factors on survival and growth of Helicobacter pylori; Jiang X et al.; The effect of temperature (4 to 42 degrees C), NaCl concentration (0.5 to 7.5%), NaNO2 concentration (0 to 400 micrograms/ml), water activity (aw level of 0.6 to 0.995), pH (3.5 to 7.3) and urea (8 mM) on the survival and growth of Helicobacter pylori in a nutrient-rich laboratory culture medium was investigated . Under microaerobic conditions (5% O2, 10% CO2, and 85% N2), the organism grew well in brain heart infusion broth supplemented with 7% horse serum and antibiotics (BHI-HS-TVA) in a temperature range of 30 to 37 degrees C with agitation . H . pylori (initial population of ca . 5 x 10(3) CFU/ml) survived for 14 days at 4 degrees C, for 2 days at 25 degrees C, and for less than 1 day at 40 and 42 degrees C . The optimal NaCl concentration for growth of H . pylori was 0.5 to 1.0%; 2.0% NaCl inhibited growth . Up to 400 micrograms of NaNO2 per ml did not prevent growth . The minimum aw (adjusted with glycerol) and pH (acidified with HCl) for growth of H . pylori was 0.98 and 4.5, respectively . The addition of urea to broth greatly enhanced the growth of H . pylori at both pH 4.5 and 5.5 . Although H . pylori did not grow at pH 3.5, the presence of urea in broth enhanced its survival . Considering the apparent fastidious conditions for growth of H . pylori in BHI-HS-TVA broth, H . pylori is unlikely to grow well, if at all, in most foods . The bacterium may, however, survive for extended periods of time in low acid-high moisture environments under refrigerated storage. Infect Immun, 1998 Sep, 66(9), 4450 - 60 Early events in phagosome establishment are required for intracellular survival of Legionella pneumophila; Wiater LA et al.; During infection, the Legionnaires' disease bacterium, Legionella pneumophila, survives and multiplies within a specialized phagosome that is near neutral pH and does not fuse with host lysosomes . In order to understand the molecular basis of this organism's ability to control its intracellular fate, we have isolated and characterized a group of transposon-generated mutants which were unable to kill macrophages and were subsequently found to be defective in intracellular multiplication . These mutations define a set of 20 genes (19 icm {for intracellular multiplication} genes and dotA {for defect in organelle trafficking}) . In this report, we describe a quantitative assay for phagosome-lysosome fusion (PLF) and its use to measure the levels of PLF in cells that have been infected with either wild-type L . pneumophila or one of several mutants defective in different icm genes or dotA . By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L . pneumophila colocalized with host lysosomes prelabeled with rhodamine-dextran . Remarkably, mutations in the six genes that were studied resulted in maximal levels of PLF as quickly as 30 min following infection . These results indicate that several, and possibly all, of the icm and dotA gene products act at an early step during phagosome establishment to determine whether L . pneumophila-containing phagosomes will fuse with lysosomes . Although not ruled out, subsequent activity of these gene products may not be necessary for successful intracellular replication. Infect Immun, 1998 Sep, 66(9), 4050 - 5 Expression of mucin-type glycoprotein K88 receptors strongly correlates with piglet susceptibility to K88(+) enterotoxigenic Escherichia coli, but adhesion of this bacterium to brush borders does not; Francis DH et al.; Three antigenic variants of the K88 fimbrial adhesin exist in nature, K88ab, K88ac, and K88ad . Enterotoxigenic Escherichia coli (ETEC) strains that produce these fimbriae cause life-threatening diarrhea in some but not all young pigs . The susceptibility of pigs to these organisms has been correlated with the adherence of bacteria to isolated enterocyte brush borders . Whether that correlation holds for multiple K88 variants and over a broad genetic base of pigs is unknown and was the impetus for this study . We also desired to examine the correlation of the expression of a porcine intestinal brush border mucin-type glycoprotein (IMTGP) which binds K88ab and K88ac with the susceptibility of piglets to K88(+) ETEC . Of 31 neonatal gnotobiotic pigs inoculated with K88ab+ or K88ac+ ETEC, 13 developed severe diarrhea, became dehydrated, and died or became moribund . Another pig became severely lethargic but not dehydrated . In vitro brush border adherence analysis was not possible for 10 of the severely ill pigs due to colonization by challenge strains . However, of the 17 pigs that did not become severely ill, 8 (47%) had brush borders that supported the adherence of K88ab+ and K88ac+ bacteria in vitro, suggesting a poor correlation between in vitro brush border adherence and piglet susceptibility to K88(+) ETEC . By contrast, the expression of IMTGP was highly correlated with susceptibility to K88(+) ETEC . Of the 12 pigs that produced IMTGP, 11 developed severe diarrhea . The other pig that produced IMTGP became lethargic but not severely diarrheic . Only 2 of 18 pigs that did not produce IMTGP became severely diarrheic . Colonizing bacteria were observed in histologic sections of intestines from all pigs that expressed IMTGP except for the one that did not develop severe diarrhea . However, colonizing bacteria were observed in histologic sections from only one pig that did not produce IMTGP . The bacterial concentration in the jejuna and ilea of pigs expressing IMTGP was significantly greater (P < 0.005) than that in pigs not expressing IMTGP . These observations suggest the IMTGP is a biologically relevant receptor for K88ab+ and K88ac+ E . coli or a correlate for expression for such a receptor. Infect Immun, 1998 Sep, 66(9), 4030 - 5 Route of infection that induces a high intensity of gamma interferon-secreting T cells in the genital tract produces optimal protection against Chlamydia trachomatis infection in mice; Igietseme JU et al.; The induction of local T helper type 1 (Th1)-mediated cellular immunity is crucial for resistance of mice to genital infection by the obligate intracellular bacterium Chlamydia trachomatis . We tested the hypothesis that the route of immunization that elicits relatively high numbers of chlamydia-specific, gamma interferon (IFN-gamma)-secreting T lymphocytes (ISTLs) in the genital tract would induce optimal protective immunity against reinfection . Female BALB/c mice were infected intravaginally (i.v.), intranasally (i.n.), orally (p.o.), or subcutaneously (s.c.) with C . trachomatis . At days 7, 14, 21, and 28 postinfection, T cells isolated from the genital tract tissues were restimulated with chlamydial antigen in vitro, and the amounts of IFN-gamma induced were measured by a sandwiched enzyme-linked immunosorbent assay method . At day 7 postinfection, i.n.- and i.v.-immunized mice had high levels of chlamydia-specific ISTLs in their genital tracts (203.58 +/- 68.1 and 225.5 +/- 12.1 pg/ml, respectively) . However, there were no detectable ISTLs in the genital tracts of p.o.- or s.c.-infected mice . When preinfected mice were challenged i.v . 70 days later, animals preexposed by the i.n . route were highly resistant to reinfection, with greatly reduced chlamydial burden, and suffered an attenuated infection that resolved by day 6 postchallenge . Animals preexposed by the i.v . route were modestly protected, whereas p.o . and s.c . groups were indistinguishable in this regard from control mice . The resistance of i.n.-immunized mice (and to some extent the i.v.-exposed mice) to reinfection was associated with early appearance (within 24 h) of high levels of genital ISTLs compared with mice preinfected by other routes . Furthermore, although i.n . and i.v.-immunized mice had comparable levels of chlamydia-specific immunoglobulin A (IgA) antibodies in their vaginal washes, the levels of IgG2a were four- sixfold higher in i.n.-immunized mice than in any of the other groups . The results suggested that immunization routes that foster rapid induction of vigorous genital mucosal cell-mediated immune (CMI) effectors (e.g., IFN-gamma), the CMI-associated humoral effector, IgG2a, and to some extent secretory IgA produce protective immunity against chlamydial genital infection . Therefore, i.n . immunization is a potential delivery route of choice in the development of a vaccine against Chlamydia. Biochim Biophys Acta, 1998 Jul 20, 1365(3), 435 - 42 Stopped-flow kinetic studies of low potential electron carriers of the photosynthetic bacterium, Rhodobacter capsulatus: ferredoxin I and NifF; Hallenbeck PC et al.; The kinetics of electron-transfer reactions involving nif-specific proteins from Rhodobacter capsulatus; ferredoxin I, NifF, Fe-protein of nitrogenase and dithionite were studied using stopped-flow spectrophotometry . Kinetic evidence was obtained for the formation of a tight (0.44 microM) complex between NifF and Fe-protein . Under the same conditions, FdI interacted only weakly (Kd > 325 microM) with Fe-protein . There was no evidence for complex formation between NifF and FdI since the reaction NifFSQ + FdIred had a bimolecular rate constant of 12.5 +/- 1.2 x 10(3) M-1 s-1 . These results suggest that NifF, which is present in only small quantities in the cell, can make a significant contribution to the overall rate of nitrogen fixation due its high reactivity with Fe-protein . Moreover, the apparent lack of specific interaction between NifF and FdI suggest that they act in vivo in parallel to reduce Fe-protein and not in series. Fold Des, 1998, 3(4), 229 - 38 Fold and function predictions for Mycoplasma genitalium proteins; Rychlewski L et al.; BACKGROUND: Uncharacterized proteins from newly sequenced genomes provide perfect targets for fold and function prediction . RESULTS: For 38% of the entire genome of Mycoplasma genitalium, sequence similarity to a protein with a known structure can be recognized using a new sequence alignment algorithm . When comparing genomes of M . genitalium and Escherichia coli, > 80% of M . genitalium proteins have a significant sequence similarity to a protein in E . coli and there are > 40 examples that have not been recognized before . For all cases of proteins with significant profile similarities, there are strong analogies in their functions, if the functions of both proteins are known . The results presented here and other recent results strongly support the argument that such proteins are actually homologous . Assuming this homology allows one to make tentative functional assignments for > 50 previously uncharacterized proteins, including such intriguing cases as the putative beta-lactam antibiotic resistance protein in M . gentalium . CONCLUSIONS: Using a new profile-to-profile alignment algorithm, the three-dimensional fold can be predicted for almost 40% of proteins from a genome of the small bacterium M . genitalium, and tentative function can be assigned to almost 80% of the entire genome . Some predictions lead to new insights about known functions or point to hitherto unexpected features of M . genitalium. FEBS Lett, 1998 Jul 31, 432(1-2), 27 - 30 The effect of chemical oxidation on the fluorescence of the LH1 (B880) complex from the purple bacterium Rhodobium marinum; Law CJ et al.; The effect of chemical oxidation on the absorption and fluorescence emission spectra of the LH1 complex from Rhodobium marinum was investigated . Mild chemical oxidation of the LH1 complex, by addition of 10 mM potassium ferricyanide, caused a 2-3% bleaching of the 880-nm Qy absorption band . In contrast, at the same ferricyanide concentration, fluorescence emission intensity of the LH1 complex was quenched by about 50% . This result demonstrates that oxidation of very few bacteriochlorophyll (BChl) molecules in the LH1 ring is enough to completely quench its fluorescence. J Membr Biol, 1998 Sep 1, 165(1), 65 - 76 Characterization of fumarate transport in Helicobacter pylori; Mendz GL et al.; The fumarate transport system of the bacterium Helicobacter pylori was investigated employing radioactive tracer analysis . The transport of fumarate at micromolar concentrations was saturable with a KM of 220 +/- 21 micron and Vmax of 54 +/- 2 nmole/min/mg protein at 20 degrees C, depended on temperature between 4 and 40 degrees C, and was susceptible to inhibitors, suggesting the presence of one or more fumarate carriers . The release of fumarate from cells was also saturable with a KM of 464 +/- 71 micron and Vmax of 22 +/- 2 nmol/min/mg protein at 20 degrees C . The rates of fumarate influx at millomolar concentrations increased linearly with permeant concentration, and depended on the age of the cells . The transport system was specific for dicarboxylic acids suggesting that fumarate is taken up via dicarboxylate transporters . Succinate and fumarate appeared to form an antiport system . The properties of fumarate transport were elucidated by investigating the effects of amino acids, monovalent cations, pH and potential inhibitors . The results provided evidence that influx and efflux of fumarate at low concentrations from H . pylori cells was a carrier-mediated secondary transport with the driving force supplied by the chemical gradient of the anion . The anaerobic C4-dicarboxylate transport protein identified in the genome of the bacterium appeared to be a good candidate for the fumarate transporter. J Biol Chem, 1998 Aug 21, 273(34), 21669 - 74 The bacterial irr protein is required for coordination of heme biosynthesis with iron availability; Hamza I et al.; Heme is a ubiquitous macromolecule that serves as the active group of proteins involved in many cellular processes . The multienzyme pathway for heme formation culminates with the insertion of iron into a protoporphyrin ring . The cytotoxicity of porphyrins suggests the need for coordination of its biosynthesis with iron availability . We isolated a mutant strain of the bacterium Bradyrhizobium japonicum that, under iron limitation, accumulated protoporphyrin and showed aberrantly high expression of hemB, an iron-regulated gene encoding the heme synthesis enzyme delta-aminolevulinic acid dehydratase . The strain carries a loss of function mutation in irr, a newly described gene that encodes a putative member of the GntR family of bacterial transcriptional regulators . Irr accumulated only under iron limitation, and turned over rapidly upon an increase in iron availability . A separate role for Irr in controlling the cellular iron level was inferred based on a deficiency in high affinity iron transport activity in the irr strain, and suggests that regulation of the heme pathway is coordinated with iron homeostasis . A high level of protoporphyrin accumulation is not a normal consequence of nutritional iron deprivation, thus a mechanism for iron-dependent control of heme biosynthesis may be present in other organisms. Biochem Biophys Res Commun, 1998 Jul 30, 248(3), 505 - 10 Induction of KDNase Sm, a deaminoneuraminic acid (KDN) residue-specific sialidase from Sphingobacterium multivorum, using synthetic KDN-glycosides; Fuchizawa S et al.; Various aryl and alkyl alpha-glycosides of KDN were synthesized and tested as substrates for their susceptibility to a deaminoneuraminic acid (KDN)-specific sialidase from Sphingobacterium multivorum, designated KDNase Sm . The synthetic KDN-glycosides were all hydrolyzed by the action of KDNase Sm . A hydroxyl group at C-5 position of KDN was required for the recognition by the enzyme, and was shown not to be replaced by an amino- or an acylamino group for the enzymatic recognition . These synthetic KDN-glycosides were also examined for their inducing activity of KDNase in S . multivorum and were shown to induce the KDNase activity effectively when the bacterium was cultured minimum salt medium containing both 0.1% glucose and 0.1% various KDN-glycosides . No KDNase activity was induced by the KDN-glycosides without 0.1% glucose . This is the first case of using synthetic KDN-glycosides as inducers of KDNase Sm. Invasion Metastasis, 1997, 17(3), 138 - 48 Bacterium-assisted invasion of Entamoeba histolytica through human enteric epithelia in two-compartment chambers; Leroy A et al.; Entamoeba histolytica trophozoites initiate amebiasis by invasion into the enteric mucosa . It is the aim of our experiments to understand how bacteria and leukocytes act during amebic invasion through enteric cell layers . Cocultures were established in two-compartment chambers and studied by measurement of transepithelial electrical resistance (TER) and by histological examination . Trophozoites caused a decrease in TER that was followed by formation of holes in the enteric cell layer and transfilter migration of trophozoites . Phagocytosed bacteria activated trophozoites that opened the intracellular junctions and provided access for the invasion of bacteria . Leukocytes had no effect on the different steps of invasion of the trophozoites through the human enteric cell layers . We conclude that trophozoites, eventually assisted by enteric bacteria, disrupt enterocytic tight junctions before they open the enteric cell layer and invade through it. Aliment Pharmacol Ther, 1998 Feb, 12 Suppl 1, 25 - 36 Review article: Relationship between Helicobacter pylori, atrophic gastritis and gastric cancer; Kuipers EJ; Helicobacter pylori causes chronic active inflammation of the gastric mucosa in the majority of infected patients . In a considerable number of them, this will eventually lead to a loss of gastric glands, and thus the establishment of atrophic gastritis . This is associated with the development of intestinal metaplasia and dysplasia . These consecutive conditions increase the risk for gastric cancer . particularly of the intestinal type . We reviewed the evidence that H . pylori plays an important role in this sequence of events that can lead to gastric cancer . This paper focuses on the difficulties in staging atrophic gastritis, the incidence and prevalence of this condition and the relation with H . pylori infection . Furthermore, it describes the evidence for the role of this organism and gastric mucosal atrophy in the aetiology of gastric cancer and focuses on the life-time incidence of gastric cancer in the presence of this bacterium. Dev Comp Immunol, 1998 Jul-Aug, 22(4), 417 - 32 Production of a macrophage growth factor(s) by a goldfish macrophage cell line and macrophages derived from goldfish kidney leukocytes; Neumann NF et al.; We recently established a spontaneously proliferating macrophage cell line from the goldfish (GMCL), and in this report demonstrate the production of a macrophage-specific growth factor(s) (MGFs) by these cells . The supernatants from GMCL cultures induced proliferation and differentiation of macrophage-like cells from kidney hematopoietic tissues of goldfish . Kidney leukocytes cultured at 6.25 x 10(4)cells/ml in the presence of GMCL-derived MGFs proliferated during two weeks of cultivation, whereas those cultured without the MGFs did not . Leukocytes cultured at higher densities (2.5 x 10(5) cells/ml) proliferated in the absence of exogenous growth factor, but not to the same extent as those stimulated with GMCL-derived MGFs, suggesting that kidney leukocytes may produce endogenous MGFs . At higher cell density (1 x 10(6) cells/ml), kidney leukocytes multiplied extensively over a two-week cultivation period in the absence of exogenous GMCL-derived MGFs . The supernatants from these cultures restored the proliferative ability of leukocytes cultured at low densities, providing direct evidence of MGFs production by kidney leukocytes . The predominant cell-type in cultures grown in the presence of GMCL or kidney leukocyte-MGFs was the macrophage based on the following criteria: (1) non-specific esterase staining; (2) morphologic similarity to GMCL; (3) phagocytosis of the bacterium, A . salmonicida; (4) production of reactive oxygen and nitrogen intermediates in response to stimulation with macrophage activating factors and/or bacterial lipopolysaccharide; and (5) flow cytometric analyses . Both in vitro-derived kidney macrophage (IVDKM) and GMCL cultures contained three distinct populations of cells, (determined by flow cytometry), suggesting that these macrophage cultures are comprised of cells arrested at distinct differentiation junctures in macrophage development . Production of MGFs by macrophages and kidney leukocytes may play an important role in regulating macrophage hematopoiesis in fish. Braz J Med Biol Res, 1998 Mar, 31(3), 373 - 6 Mouse inoculation for the detection of non-cultivable gastric tightly spiralled bacteria; Mendes EN et al.; In the present study we compared the inoculation of swine gastric mucus into the stomach of mice, the ureas test and carbolfuchsin-stained smears for the diagnosis of the infection with "Gastrospirillum suis" ("Helicobacter heilmannii" type 1), an uncultivated tightly spiralled gastric bacterium . Fragments obtained from the antral and oxyntic mucosa of the stomach of 50 slaughtered pigs were used for urease test, for carbolfuchsin-stained smears and for obtaining scrapings of mucus for mouse inoculation . The mice were killed by spinal dislocation 10 days after inoculation and fragments of the antral and oxyntic mucosa were used for spiral bacterium identification (urease test and carbolfuchsin-stained smears) . Among the methods employed for the diagnosis of "H . heilmannii" infection, the inoculation of gastric mucus into the stomach of mice was the most sensitive and demonstrated bacterial positivity in 31 (62.0%) swine . Direct examination showed tightly spiralled bacteria in the gastric mucosa of only 4 (8.0%) of the 50 pigs studied . Among them, 3 (6.0%) presented a positive preformed urease test . Spiral bacteria were not seen in the gastric mucosa of any control mice . These results show that the use of the mouse inoculation method improved the detection of "H . heilmannii" in swine. Clin Exp Immunol, 1998 Jul, 113(1), 105 - 10 Characteristics of Prevotella intermedia-specific CD4+ T cell clones from peripheral blood of a chronic adult periodontitis patient; Wassenaar A et al.; Periodontitis is a chronic destructive inflammatory disease associated with periodontopathic bacteria . In addition, autoantigens such as collagen and heat shock proteins (hsp) have been suggested to play a role . Established periodontal lesions are characterized by dense infiltrations of immune cells such as cytokine-producing CD4+ and CD8+ T cells . CD4+ T cells specific for Prevotella intermedia can be isolated from lesional gingiva, suggesting an active role for CD4+ T cells in the response to this bacterium . We therefore investigated the characteristics of a panel of 13 P . intermedia-specific CD4+ T cells generated from the peripheral blood of a patient with chronic adult periodontitis . All 13 P . intermedia-specific CD4+ T cells recognized the antigens in the context of HLA-DR . The T cell clones were mainly classified as Th0, producing comparable amounts of interferon-gamma (IFN-gamma) and IL-4, and Th2, producing high amounts of IL-4 and almost no IFN-gamma . None of the P . intermedia-specific T cell clones recognized antigens of the periodontopathic bacteria Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis and of the autoantigens collagen and hsp . The reactivity profile of the T cell clones to size-fractionated cell envelope antigens of P . intermedia indicated that P . intermedia-specific CD4+ T cell clones recognize probably five different antigen specificities in the context of the MHC class II molecules, DR7 or DR15 . These results suggest that a broad panel of cell-associated protein antigens play a role in the induction of P . intermedia-specific CD4+ T cell response. Microbiology, 1998 Jul, 144 ( Pt 7), 1881 - 94 Sirohaem sulfite reductase and other proteins encoded by genes at the dsr locus of Chromatium vinosum are involved in the oxidation of intracellular sulfur; Pott AS et al.; The sequence of the dsr gene region of the phototrophic sulfur bacterium Chromatium vinosum D (DSMZ 180) was determined to clarify the in vivo role of 'reverse' sirohaem sulfite reductase . The dsrAB genes encoding dissimilatory sulfite reductase are part of a gene cluster, dsrABEFHCMK, that encodes four small, soluble proteins (DsrE, DsrF, DsrH and DsrC), a transmembrane protein (DsrM) with similarity to haem-b-binding polypeptides and a soluble protein (DsrK) resembling {4Fe-4S}-cluster-containing heterodisulfide reductase from methanogenic archaea . Northern hybridizations showed that expression of the dsr genes is increased by the presence of reduced sulfur compounds . The dsr genes are not only transcribed from a putative promoter upstream of dsrA but primary transcripts originating from (a) transcription start site(s) downstream of dsrB are also formed . Polar insertion mutations immediately upstream of dsrA, and in dsrB, dsrH and dsrM, led to an inability of the cells to oxidize intracellularly stored sulfur . The capability of the mutants to oxidize sulfide, thiosulfate and sulfite under photolithoautotrophic conditions was unaltered . Photoorganoheterotrophic growth was also unaffected . 'Reverse' sulfite reductase and DsrEFHCMK are, therefore, not essential for oxidation of sulfide or thiosulfate, but are obligatory for sulfur oxidation . These results, together with the finding that the sulfur globules of C . vinosum are located in the extracytoplasmic space whilst the dsr gene products appear to be either cytoplasmic or membrane-bound led to the proposal of new models for the pathway of sulfur oxidation in this phototrophic sulfur bacterium. Microbiology, 1998 Jul, 144 ( Pt 7), 1869 - 79 Evidence of past recombination events among the genes encoding the Erp antigens of Borrelia burgdorferi; Stevenson B et al.; A single Borrelia burgdorferi bacterium may contain six or more different 32 kb circular plasmids (cp32s) . Although these plasmids are homologous throughout much of their sequences, two loci have been identified at which they can vary significantly . The cp32 plasmids and their relatives each contain two adjacent genes, orfC and orf3, that vary in sequence between plasmids found within clones of individual bacteria . The orfC gene product is homologous to proteins involved in partitioning of bacterial plasmids, and the differences at this locus between plasmids may account for their compatibility . The orfC-orf3 loci are located approximately 5 kb from another variable locus called erp . The orfC-orf3 loci were used as physically linked markers to assess genetic rearrangements in the erp loci; this revealed examples of recombination involving both individual genes and entire erp loci . Recombination of the genes encoding the Erp antigens might contribute to the evasion of the mammalian immune response and could play roles in the establishment and persistence of B . burgdorferi infections in mammalian hosts. Parasitology, 1998 Jul, 117 ( Pt 1), 15 - 9 Horizontal transfer of parasitic sex ratio distorters between crustacean hosts; Dunn AM et al.; Parasitic sex distorters were artificially transferred within and between crustacean host species in order to study the effects of parasitism on host fitness and sex determination and to investigate parasite host specificity . Implantation of Nosema sp . to uninfected strains of its Gammarus duebeni host resulted in an active parasite infection in the gonad of recipient females and subsequent transovarial parasite transmission . The young of artificially infected females were feminized by the parasite, demonstrating that Nosema sp . is a cause of a sex ratio distortion in its host . In contrast, we were unable to cross-infect Armadillidium vulgare with the feminizing microsporidian from G . duebeni or to cross-infect G . duebeni with the feminizing bacterium Wolbachia sp . from A . vulgare. Ophthalmologica, 1998, 212(5), 344 - 6 Bilateral optic papillitis following mycoplasma pneumoniae pneumonia; Milla E et al.; Mycoplasma pneumoniae is an atypical bacterium that can cause a great variety of respiratory infections and be responsible for ocular involvement such as conjunctivitis, anterior uveitis and very rarely optic neuropathy . We report herein an additional case of bilateral optic disc swelling with profound visual loss following Mycoplasma pneumoniae pneumonia and review the world literature on the ocular manifestations associated with this pathogen. Digestion, 1998 Jul-Aug, 59(4), 314 - 20 Helicobacter pylori infection decreases gastric alcohol dehydrogenase activity and first-pass metabolism of ethanol in man; Simanowski UA et al.; BACKGROUND/AIMS: Ethanol is metabolized by alcohol dehydrogenase in the human stomach . This metabolism contributes to the so-called first-pass metabolism of ethanol which is affected by gender, medication, and morphological alterations of the gastric mucosa . Recently, it has been shown that Helicobacter pylori is capable to oxidize ethanol to acetaldehyde in vitro . Since H . pylori also injures gastric mucosa, the present study examines the effect of this bacterium on gastric alcohol dehydrogenase activity and systemic availability of ethanol in vivo . METHODS: Thirteen volunteers (7 men and 6 women, aged 18-52 years) with gastric H . pylori infection diagnosed by a positive CLO test and positive gastric histology received ethanol (0.225 g/kg) either orally or intravenously before and after H . pylori elimination to determine systemic availability of ethanol . In addition, gastric biopsy specimens were taken from all subjects before and after H . pylori elimination for histological assessment of mucosal alterations and determinations of gastric alcohol dehydrogenase activity and phenotype of the enzyme . RESULTS: In the presence of H . pylori the first-pass metabolism of ethanol was found to be significantly reduced (625 +/- 234 vs . 1,155 +/- 114 mg/dl/min, p = 0.046) . This reduction of first-pass metabolism of ethanol was associated with a significant decrease in alcohol dehydrogenase activity (4.8 +/- 1.5 vs . 12.1 +/- 2.3 nmol/mg protein x min, p < 0.05) and an increase in the severity of mucosal damage as determined by a histological score (p < 0.05) . CONCLUSIONS: H . pylori infection leads to gastric mucosal injury which is associated with a decrease in gastric alcohol dehydrogenase activity and first-pass metabolism of ethanol . Ethanol metabolism by H . pylori does not play an important role in vivo . However, gastric morphology is one important factor determining systemic availability of ethanol in man. Biochemistry, 1998 Jul 28, 37(30), 10792 - 7 Excited states and trapping in reaction center complexes of the green sulfur bacterium Prosthecochloris aestuarii; Neerken S et al.; The excited states of bacteriochlorophyll (BChl) a were studied by pump-probe transient absorption spectroscopy in reaction center core (RCC), Fenna-Matthews-Olson (FMO) and FMO-RCC complexes of the green sulfur bacterium Prosthecochloris aestuarii . Excitation at 790 or 835 nm resulted in rapid equilibration of the energy between the BChl a molecules of the RCC complex: within 1 ps, most of the excitations had relaxed to the lowest energy level (835 nm), as a result of strong interactions between the BChls . Excitation of chlorophyll a 670 resulted in energy transfer to BChl a with a time constant of 1.2 ps, followed by thermal equilibration . Independent of the wavelength of excitation, the decay at 835 nm could be fitted with a time constant of about 25 ps, comparable to the 30 ps measured earlier with membrane fragments, which is ascribed to trapping in the reaction centers . Similar results were obtained with the FMO-RCC complex upon excitation at 835 or 670 nm, but the results upon 790 nm excitation were quite different . Again an equilibrium was rapidly reached, but now most of the excitations remained within the FMO complex, with a maximum bleaching at 813 nm, the same as observed in the isolated FMO . Even after 100 ps there was no bleaching at 835 nm and no evidence for charge separation . We conclude that there is no equilibration of the energy between the FMO and the RCC complex and that the efficiency of energy transfer from FMO to the reaction center core is low. Biosci Biotechnol Biochem, 1998 Jun, 62(6), 1138 - 43 Isolation and chemical composition of the sheath of Sphaerotilus natans; Takeda M et al.; A sheathed bacterium, Sphaerotilus natans, was cultured with vigorous shaking in a medium containing peptone . Then the biomass was harvested and treated with lysozyme, sodium dodecyl sulfate, and protease . With treatment, 1.6 mg of sheaths was obtained from 15 mg of biomass . For the preparation of sheaths of high purity, cultivation must be in the absence of glucose with sufficient aeration to prevent poly(3-hydroxybutyrate) accumulation . Carbohydrate (54.1%), protein (12.2%), and lipid (1-3%) were detected in the sheaths by colorimetric reactions and solvent extraction . Gas-liquid chromatography showed glucose and galactosamine to be present in the molar ratio of 1:4 . The most abundant amino acids in the sheath protein were glycine (49.2 mol%) and cysteine (24.6 mol%) . The sheaths were resistant to agents that reduce disulfide bonds (dithiothreitol and 2-mercaptoethanol) and to protease . However, sheathes were degraded completely by hydrazine, and a heteropolysaccharide composed of glucose and galactosamine (1:4) was released . The weight-average molecular weight of the polysaccharide was estimated to be 1.2 x 10(5) by gel filtration chromatography with a low-angle laser-light scattering photometer and a rotation index detector . A ladder of 1.5-kDa peptides separable by sodium dodecyl sulfate gel electrophoresis was obtained by partial hydrolysis of sheaths, suggesting the sheath protein has repeating units of 1.5 kDa. Biosci Biotechnol Biochem, 1998 Jun, 62(6), 1081 - 6 Isolation and some properties of cytochrome c oxidase purified from a bisulfite ion resistant Thiobacillus ferrooxidans strain, OK1-50; Iwahori K et al.; Sulfite ion (HSO3-) is one of the products when elemental sulfur is oxidized by the hydrogen sulfide:ferric ion oxidoreductase of Thiobacillus ferrooxidans AP19-3 . Under the conditions in which HSO3- is accumulated in the cells, the iron oxidase of this bacterium was strongly inhibited by HSO3- . Since cytochrome c oxidase is one of the most important components of the iron oxidase enzyme system in T . ferrooxidans, effects of HSO3- on cytochrome c oxidase activity were studied with the plasma membranes of HSO3(-)-resistant and -sensitive strains of T . ferrooxidans, OK1-50 and AP19-3 . The enzyme activity of AP19-3 compared with OK1-50 was strongly inhibited by HSO3- . To investigate the inhibition mechanism of HSO3- in T . ferrooxidans, cytochrome c oxidases were purified from both strains to an electrophoretically homogeneous state . Cytochrome c oxidase activity of a purified OK1-50 enzyme was not inhibited by 5 mM HSO3- . In contrast, the same concentration of HSO3- inhibited the enzyme activity of AP19-3 50%, indicating that the cytochrome c oxidase of OK1-50 was more resistant to HSO3- than that of AP19-3 . Cytochrome c oxidases purified from both strains were composed of three subunits . However, the molecular weight of the largest subunit differed between OK1-50 and AP19-3 . Apparent molecular weights of the three subunits of cytochrome c oxidases were 53,000, 24,000, and 19,000 for strain AP19-3 and 55,000, 24,000, and 19,000 for strain OK1-50, respectively. Biosci Biotechnol Biochem, 1998 Jun, 62(6), 1068 - 74 alpha-L-rhamnosidase of Sphingomonas sp . R1 producing an unusual exopolysaccharide of sphingan; Hashimoto W et al.; A soil bacterium with alpha-L-rhamnosidase was isolated from a cumulative mixed culture containing a polysaccharide of gellan as a carbon source and identified to be Sphingomonas paucimobilis, known as a potent producer of gellan . The isolate (designated Sphingomonas sp . R1) produced an unusual exopolysaccharide of sphingan (denoted HWR1) distinct from gellan . The rhamnose in gellan was replaced with mannose in HWR1 . The bacterium had a peculiar cell surface covered with many complicated plaits . alpha-L-Rhamnosidase purified from Sphingomonas sp . R1 grown in the presence of naringin was a monomer with a molecular mass of 110 kDa and most active at pH 8.0 and 50 degrees C . The enzyme required divalent metal ions for the activity and released L-rhamnose from various rhamnosyl glycosides. Biosci Biotechnol Biochem, 1998 Jun, 62(6), 1061 - 7 Gene cloning and characterization of an acidic xylanase from Acidobacterium capsulatum; Inagaki K et al.; The gene xynA encoding an acid endo-beta-1,4-xylanase from an acidophilic bacterium, Acidobacterium capsulatum 161, was cloned and expressed in Eschrichia coli . The nucleotide sequence of the 1.6-kb DNA fragment containing xynA was analyzed, revealing an open reading frame of 1,215 bp encoding a peptide of 405 amino acid residues . The deduced amino acid sequence of XynA was very similar to other xylanases that are from the glycosyl hydrolase family 10 . XynA was purified to homogeneity by SDS-polyacrylamide gel electrophoresis from E . coli transformants . The molecular mass and isoelectric point of XynA were 41 kDa and 7.3, respectively . The xylanase activity of the cloned XynA is an endo-acting enzyme that shows optimal activity at pH 5.0 and 65 degrees C, and is stable pH between 3.0 and 8.0 . The K(m) and Vmax with oat spelt xylan as a substrate at pH 5.0 and 30 degrees C are 3.5 mg/ml and 403 mumol/min/mg. Gut, 1998 Jun, 42(6), 768 - 71 Reactive oxygen species activity and lipid peroxidation in Helicobacter pylori associated gastritis: relation to gastric mucosal ascorbic acid concentrations and effect of H pylori eradication; Drake IM et al.; BACKGROUND: Helicobacter pylori is an independent risk factor for gastric cancer, and this association may be due to the bacterium causing reactive oxygen species mediated damage to DNA in the gastric epithelium . High dietary ascorbic acid intake may protect against gastric cancer by scavenging reactive oxygen species . AIMS: To assess reactive oxygen species activity and damage in gastric mucosa in relation to gastric pathology and mucosal ascorbic acid level, and to determine the effect of H pylori eradication on these parameters . PATIENTS: Gastric biopsy specimens were obtained for analysis from 161 patients undergoing endoscopy for dyspepsia . METHODS: Reactive oxygen species activity and damage was assessed by luminol enhanced chemiluminescence and malondialdehyde equivalent estimation respectively . Ascorbic acid concentrations were measured using HPLC . RESULTS: Chemiluminescence and malondialdehyde levels in gastric mucosa were higher in patients with H pylori gastritis than in those with normal histology . Successful eradication of the bacterium led to decreases in both parameters four weeks after treatment was completed . Gastric mucosal ascorbic acid and total vitamin C concentrations were not related to mucosal histology, but correlated weakly with reactive oxygen species activity (chemiluminescence and malodialdehyde levels) . CONCLUSIONS: Data suggest that reactive oxygen species play a pathological role in H pylori gastritis, but mucosal ascorbic acid is not depleted in this condition. Curr Microbiol, 1998 Sep, 37(3), 172 - 6 Anaerobic degradation and transformation of p-toluidine by the sulfate-reducing bacterium Desulfobacula toluolica; Raber T et al.; The ability of the strictly anaerobic sulfate-reducing bacterium Desulfobacula toluolica (strain Tol2) to cometabolically degrade p-toluidine (p-methylaniline) while using toluene as the primary source of carbon and energy has been studied . This organism has been shown to modify and degrade toluidine in dense cell suspensions when no other source of carbon and energy is added . The metabolism led to the formation of a variety of metabolites . From these metabolites a biphenyl-like compound as well as phenylacetic acid have been identified by means of HPLC/MS techniques . The probable conversion of p-toluidine to p-aminophenylacetic acid and phenylacetic acid as dead end products suggested that this organism initiates p-toluidine degradation by the carboxylation of the methyl group . If this could be validated in further experiments, it would be the first time that a toluidine was carboxylated at the methyl moiety by an anaerobic, sulfate-reducing bacterium. FEBS Lett, 1998 Jul 3, 430(3), 323 - 6 Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy; Savikhin S et al.; Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus . Anomalously high values of photoinduced absorption changes were revealed in the BChl c Qy transition band . Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7-8 times greater than those at the bleaching peak in the BChl a band of the chlorosome . This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome. Eur J Biochem, 1998 Jun 15, 254(3), 602 - 9 The hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) exists in several forms as shown by electrospray-ionisation mass spectrometry; Buzy A et al.; The hydroxylase of the soluble methane monooxygenase from the bacterium Methylococcus capsulatus (Bath) has been investigated by means of electrospray-ionisation mass spectrometry (ESI-MS) and liquid chromatography ESI-MS (LC/ESI-MS) . The hydroxylase is a non-heme diiron protein consisting of three pairs of non-identical subunits (alpha approximately 60 kDa, beta approximately 45 kDa and gamma approximately 20 kDa) . Liquid chromatographic separation of the hydroxylase subunits was required before MS analysis in order to detect the alpha-subunit . The masses measured for the three subunits were found to disagree with those calculated from their gene sequences . Experiments involving the use of CNBr and trypsin cleavage followed by LC/ESI-MS and MS/MS analyses permitted the location and correction of errors in the sequences deduced from the use of cDNA . The ESI-MS results also showed that the alpha-subunit of the hydroxylase exists in multiple forms which result from cleavage of the protein . This observation explains a number of enigmatic features of the protein previously reported in the literature and illustrates the pivotal role of ESI-MS in complementing data obtained from molecular biology for the characterisation of the primary sequence of proteins. Eur J Biochem, 1998 Jun 15, 254(3), 480 - 9 Sequence-alignment modelling and molecular docking studies of the epoxygenase component of alkene monooxygenase from Nocardia corallina B-276; Gallagher SC et al.; Whole cells of Nocardia corallina B-276 catalyse the stereoselective epoxygenation of alkenes to chiral epoxides . The bacterium expresses an enzyme, alkene monooxygenase, which catalyses the epoxygenation reaction stereoselectively . The enzyme consists of a terminal oxygenase (epoxygenase), an NADH-dependent reductase (reductase) and a regulatory component (coupling protein) . The epoxygenase component contains a bridged diiron centre similar to that found in the hydroxylase component of soluble methane monooxygenase . Sequence-alignment modelling, supported by chemical modification and fluorescence probing, identified a hydrophobic oxygen/substrate binding site within the epoxygenase . The diiron centre was coordinated by the two His and two Glu residues from two conserved Glu-Xaa-Xaa-His sequences and by two further Glu residues . Molecular docking of substrates and products into the proposed active-site model of the epoxygenase suggested that Ala91 and Ala185 were responsible for the stereoselectivity exerted by AMO . It is proposed that these residues clamped the intermediate and/or product of the reaction, thereby controlling the configuration of the epoxide produced . In soluble methane monooxygenase these residues are replaced by two Gly residues which do not provide sufficient steric hindrance to prevent rotation of the intermediate in the active site and, therefore, the product of the reaction catalysed by this enzyme is achiral. J Exp Med, 1998 Aug 3, 188(3), 505 - 14 Identification of putative cytoskeletal protein homologues in the protozoan host Hartmannella vermiformis as substrates for induced tyrosine phosphatase activity upon attachment to the Legionnaires' disease bacterium, Legionella pneumophila; Venkataraman C et al.; The Legionnaires' disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen that invades and replicates within two evolutionarily distant hosts, free living protozoa and mammalian cells . Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaires' disease . We have recently reported the identification of a galactose/N-acetyl-D-galactosamine (Gal/GalNAc) lectin in the protozoan host Hartmannella vermiformis as a receptor for attachment and invasion by L . pneumophila (Venkataraman, C., B.J . Haack, S . Bondada, and Y.A . Kwaik . 1997 . J . Exp . Med . 186:537-547) . In this report, we extended our studies to the effects of bacterial attachment and invasion on the cytoskeletal proteins of H . vermiformis . We first identified the presence of many protozoan cytoskeletal proteins that were putative homologues to their mammalian counterparts, including actin, pp125(FAK), paxillin, and vinculin, all of which were basally tyrosine phosphorylated in resting H . vermiformis . In addition to L . pneumophila-induced tyrosine dephosphorylation of the lectin, bacterial attachment and invasion was associated with tyrosine dephosphorylation of paxillin, pp125(FAK), and vinculin, whereas actin was minimally affected . Inhibition of bacterial attachment to H . vermiformis by Gal or GalNAc monomers blocked bacteria-induced tyrosine dephosphorylation of detergent-insoluble proteins . In contrast, inhibition of bacterial invasion but not attachment failed to block bacteria-induced tyrosine dephosphorylation of H . vermiformis proteins . This was further supported by the observation that 10 mutants of L . pneumophila that were defective in invasion of H . vermiformis were capable of inducing tyrosine dephosphorylation of H . vermiformis proteins . Entry of L . pneumophila into H . vermiformis was predominantly mediated by noncoated receptor-mediated endocytosis (93%) but coiling phagocytosis was infrequently observed (7%) . We conclude that attachment but not invasion by L . pneumophila into H . vermiformis was sufficient and essential to induce protein tyrosine dephosphorylation in H . vermiformis . These manipulations of host cell processes were associated with, or followed by, entry of the bacteria by a noncoated receptor-mediated endocytosis . A model for attachment and entry of L . pneumophila into H . vermiformis is proposed. Appl Environ Microbiol, 1998 Aug, 64(8), 2882 - 7 Deletion of the rbo gene increases the oxygen sensitivity of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough; Voordouw JK et al.; The rbo gene of Desulfovibrio vulgaris Hildenborough encodes rubredoxin oxidoreductase (Rbo), a 14-kDa iron sulfur protein; forms an operon with the gene for rubredoxin; and is preceded by the gene for the oxygen-sensing protein DcrA . We have deleted the rbo gene from D . vulgaris with the sacB mutagenesis procedure developed previously (R . Fu and G . Voordouw, Microbiology 143:1815-1826, 1997) . The absence of the rbo-gene in the resulting mutant, D . vulgaris L2, was confirmed by PCR and protein blotting with Rbo-specific polyclonal antibodies . D . vulgaris L2 grows like the wild type under anaerobic conditions . Exposure to air for 24 h caused a 100-fold drop in CFU of L2 relative to the wild type . The lag times of liquid cultures of inocula exposed to air were on average also greater for L2 than for the wild type . These results demonstrate that Rbo, which is not homologous with superoxide dismutase or catalase, acts as an oxygen defense protein in the anaerobic, sulfate-reducing bacterium D . vulgaris Hildenborough and likely also in other sulfate-reducing bacteria and anaerobic archaea in which it has been found. Appl Environ Microbiol, 1998 Aug, 64(8), 2806 - 13 Algicidal effects of a novel marine pseudoalteromonas isolate (class Proteobacteria, gamma subdivision) on harmful algal bloom species of the genera Chattonella, Gymnodinium, and Heterosigma; Lovejoy C et al.; During a bacterial survey of the Huon Estuary in southern Tasmania, Australia, we isolated a yellow-pigmented Pseudoalteromonas strain (class Proteobacteria, gamma subdivision), designated strain Y, that had potent algicidal effects on harmful algal bloom species . This organism was identified by 16S rRNA sequencing as a strain with close affinities to Pseudoalteromonas peptidysin . This bacterium caused rapid cell lysis and death (within 3 h) of gymnodinoids (including Gymnodinium catenatum) and raphidophytes (Chattonella marina and Heterosigma akashiwo) . It caused ecdysis of armored dinoflagellates (e.g., Alexandrium catenella, Alexandrium minutum, and Prorocentrum mexicanum), but the algal cultures then recovered over the subsequent 24 h . Strain Y had no effect on a cryptomonad (Chroomonas sp.), a diatom (Skeletonema sp.), a cyanobacterium (Oscillatoria sp.), and two aplastidic protozoans . The algicidal principle of strain Y was excreted into the seawater medium and lost its efficacy after heating . Another common bacterial species, Pseudoalteromonas carrageenovora, was isolated at the same time and did not have these algicidal effects . The minimum concentrations of strain Y required to kill G . catenatum were higher than the mean concentrations found in nature under nonbloom conditions . However, the new bacterium showed a chemotactic, swarming behavior that resulted in localized high concentrations around target organisms . These observations imply that certain bacteria could play an important role in regulating the onset and development of harmful algal blooms. J Appl Toxicol, 1998 May-Jun, 18(3), 179 - 85 Ibuprofen affects arylamine N-acetyltransferase activity in Helicobacter pylori from peptic ulcer patients; Chang SH et al.; Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene and p-aminobenzoic acid were determined in the bacterium Helicobacter pylori collected from peptic ulcer patients . Cytosols or suspensions of H . pylori with or without specific concentrations of ibuprofen co-treatment showed different percentages of 2-aminofluorene and p-aminobenzoic acid acetylation . The data indicate that there was decreased NAT activity associated with increased levels of ibuprofen in H . pylori cytosols . Inhibition of growth studies on H . pylori demonstrated that ibuprofen elicited a dose-dependent bactericide effect in H . pylori cultures, i.e . the greater the concentration of ibuprofen, the greater the inhibition of growth to H . pylori . For the cytosol and intact bacteria examinations, the apparent values of Km and Vmax were decreased after co-treatment with 40 microM ibuprofen . This report is the first demonstration of ibuprofen inhibition of arylamine N-acetyltransferase activity and ibuprofen inhibition of growth in the bacterium H . pylori. FEBS Lett, 1998 Jul 10, 431(1), 34 - 8 The 49-kDa subunit of NADH-ubiquinone oxidoreductase (Complex I) is involved in the binding of piericidin and rotenone, two quinone-related inhibitors; Darrouzet E et al.; Piericidin is a potent inhibitor of the mitochondrial and bacterial type I NADH-ubiquinone oxidoreductases (Complex I) and is considered to bind at or close to the ubiquinone binding site(s) of the enzyme . Piericidin-resistant mutants of the bacterium Rhodobacter capsulatus have been isolated and the present work demonstrates that a single missense mutation at the level of the gene encoding the peripheral 49-kDa/NUOD subunit of Complex I is definitely associated with this resistance . Based on this original observation, we propose a model locating the binding site for piericidin (and quinone) at the interface between the hydrophilic and hydrophobic domains of Complex I. Int J Clin Pract . 1998 Apr-May;52(3):205. Fatal septicaemia in a previously healthy man following a dog bite; Saab M et al.; A rare case of fatal septicaemia after a dog bite in a previously healthy individual is presented and discussed . Capnocytophaga canimorsus (DF-2 bacterium) was isolated from blood cultures . Our case shows the importance of immediate antibiotic treatment for all dog and cat bites, regardless of severity. Dis Aquat Organ, 1998 Jun 19, 33(2), 101 - 9 Pathology attributed to Mycobacterium chelonae infection among farmed and laboratory-infected Atlantic salmon Salmo salar; Bruno DW et al.; This study was promoted following concern over increasing mortality on 2 farms rearing Atlantic salmon Salmo salar in the Shetland Isles, Scotland . A Mycobacterium sp . was isolated from moribund, market-sized Atlantic salmon . Biochemical tests, lipid analysis and PCR (polymerase chain reaction) techniques confirmed the bacterium to be Mycobacterium chelonae . Multiple greyish-white miliary granuloma-like nodules were observed in several tissues . Dense hard-packed nodules contained abundant acid-fast bacteria . Atlantic salmon injected with M . chelonae remained sub-clinically infected, demonstrating the chronic nature of this disease . The source of the pathogen was not identified. Hepatogastroenterology, 1998 May-Jun, 45(21), 765 - 70 Beneficial effects of Helicobacter pylori eradication on migraine; Gasbarrini A et al.; BACKGROUND/AIMS: Migraine is a commonly unilateral throbbing headache, which has been associated with disorders of the vascular tone . Helicobacter pylori, the most relevant cause of gastritis and peptic ulcer, has been recently associated with a typical functional vascular disorder such as primary Raynaud phenomenon . The aim of this study was to assess the prevalence of H . pylori for patients affected by migraine and the effects of H . pylori eradication on migraine symptoms . METHODOLOGY: Two-hundred and twenty-five patients were consecutively enrolled between October 1996 and January 1997 . H . pylori was assessed by 13C-urea breath test . Infected subjects were eradicated of the bacterium; frequency, intensity and duration of attacks of migraine were assessed during a 6 month follow-up period . RESULTS: H . pylori was detected in 40% of the patients . Eighty-three percent of the patients who underwent therapy were eradicated . Intensity, duration and frequency of attacks of migraine were significantly reduced in all eradicated patients . CONCLUSIONS: H . pylori is common in subjects with migraine . Bacterium eradication causes a significant decrease in attacks of migraine . The reduction of vasoactive substances produced during infection may be the pathogenetic mechanism underlying the phenomenon. Clin Immunol Immunopathol, 1998 Jul, 88(1), 80 - 3 Distribution of IgA subclass response to Coxiella burnetii in patients with acute and chronic Q fever; Camacho MT et al.; The progression of Coxiella burnetii infection to acute or chronic Q fever has been attributed to biological characteristics of the bacterium and to the host immune response . We measured whether serum levels of total and specific subclasses IgA1 and IgA2 could be correlated with the course of disease in acute and chronic Q fever infections, and with the occurrence of endocarditis . In patients with chronic infection, total IgA2 levels were significantly increased . Q-fever-specific IgA1 antibodies were detectable in both acute and chronic infections, but only patients with endocarditis had IgA2 antibodies to C . burnetii phase II antigens . These findings indicate that the measurement of IgA subclasses may be a useful aid in the serological diagnosis of Q fever . Our results reinforce the idea that immunologically mediated host factors are important in the pathogenesis of Q fever and in the disease outcome of this infection . FEMS Microbiol Lett, 1998 Jul 15, 164(2), 375 - 82 Properties of aspartate transcarbamylase from TAD1, a psychrophilic bacterial strain isolated from Antarctica; Sun K et al.; TAD1 is a psychrophilic strain isolated from continental frozen water in Antarctica . Study of aspartate transcarbamylase in the bacterium shows an impressive activity of this enzyme at low temperature . At 0 degree C, its activity is up to 26% of its maximal activity observed at 30 degrees C . In comparison with the Escherichia coli enzyme, some of its kinetic properties suggest that this high activity at low temperature results from an increased catalytic efficiency . This property might result from a discrete modification localized at the catalytic site, since this psychrophilic enzyme is as stable as its Escherichia coli homologue at high temperature. Extremophiles, 1997 Nov, 1(4), 183 - 91 Shuttle vectors for hyperthermophilic archaea; Aravalli RN et al.; Progress in understanding the basic molecular, biochemical, and physiological characteristics of archaeal hyperthermophiles has been limited by the lack of suitable expression vectors . Here, we report the construction of versatile shuttle vectors that can be maintained, and selected for, in both archaea and bacteria . The primary construct, pAG1, was produced by ligating portions of the archaeal cryptic plasmid pGT5 and the bacterial plasmid pUC19, both of which exhibit high copy numbers . A second vector construct, pAG2, was generated, with a reduced copy number in Escherichia coli, by introducing the Rom/Rop gene from pBR322 into pAG1 . After transformation, both pAG1 and pAG2 were stably maintained and propagated in the euryarchaeote Pyrococcus furiosus, the crenarchaeote Sulfolobus acidocaldarius, and in Escherichia coli . An archaeal selective marker, the alcohol dehydrogenase gene from Sulfolobus solfataricus, was isolated by polymerase chain reaction (PCR) amplification and cloned into the two constructs . They were stably maintained and expressed in the two archaea and conferred resistance to butanol and benzyl alcohol . However, the vector pAG21, deriving from pAG2, proved the more stable in E . coli probably due to its lower copy number in the bacterium . Conditions are presented for the use of the vectors which, potentially, can be used for other hyperthermophilic archaea. Mol Microbiol, 1998 Jun, 28(6), 1283 - 93 N-terminal characterization of the Bordetella pertussis filamentous haemagglutinin; Lambert-Buisine C et al.; The major adhesin of Bordetella pertussis, filamentous haemagglutinin (FHA), is produced and secreted at high levels by the bacterium . Mature FHA derives from a large precursor, FhaB, that undergoes several post-translational maturations . In this work, we demonstrate by site-directed mutagenesis that the N-terminal signal peptide of FHA is composed of 71 amino acids, including a 22-residue-long 'N-terminal extension' sequence . This sequence, although highly conserved in various other secretory proteins, does not appear to play an essential part in FHA secretion, as shown by deletion mutagenesis . The entire N-terminal signal region of FhaB is removed in the course of secretion by proteolytic cleavage at a site that corresponds to a Lep signal peptidase recognition sequence . After this maturation, the N-terminal glutamine residue is modified to a pyroglutamate residue . This modification is not crucial for heparin binding, haemagglutination or secretion . Interestingly, however, the modification is absent from Escherichia coli secreted FHA derivatives . In addition, it is dependent in B . pertussis on the presence of all three cysteines contained in the signal peptide of FhaB . These observations suggest that it does not occur spontaneously but perhaps requires a specific enzymatic machinery. Biochem Mol Biol Int, 1998 Jun, 45(2), 355 - 62 Pigment organization and exciton dynamics in the B808-866 antenna of the green bacterium Chloroflexus aurantiacus; Novoderezhkin V et al.; The model for the B808-866 antenna of the green photosynthetic bacterium Chloroflexus aurantiacus based on femtosecond pump-probe studies is proposed . The three-dimensional structure of the B808-866 antenna is assumed to be similar to the structure of the B800-850 antenna of purple bacteria, i.e . it has the form of two concentric rings of N strongly coupled BChl866 pigments and of N/2 weakly coupled BChl808 monomers . The Qy transition dipoles of BChl808 and BChl866 molecules form the angles 43 degrees +/- 3 degrees and 8 degrees +/- 4 degrees, respectively, with the plane of the corresponding rings . The lowest limit of the BChl866 aggregate size is N = 18 . The anomalously high bleaching value of the BChl866 band with respect to the monomeric BChl808 band provides evidence for a high degree of exciton delocalization . To account for this phenomenon, the exciton model describing exciton dynamics in the spectrally disordered circular BChl866 aggregate is developed . According to this model, the effective exciton size in this antenna (Neff) is 6-8. J Food Prot, 1998 Jul, 61(7), 910 - 2 Thermal inactivation analysis of mesophiles using the Arrhenius and z-value models; Fujikawa H et al.; The Arrhenius and z-value models were compared for thermal inactivation analysis of a mesophilic bacterium . The models produced a linear curve for the thermal inactivation data . Concerning the rate constant of inactivation, the D and k values predicted by the models at a constant temperature were similar . For extrapolated temperatures the z-value model predicted an insignificant decrease in the survival ratio compared to the Arrhenius model . The dynamic temperature survival curves predicted by the models were similar, and the models characterized the results . These results demonstrated that the models can be used for thermal inactivation analysis of mesophiles at various temperatures. FEMS Microbiol Lett, 1998 Jul 1, 164(1), 215 - 8 Partial purification and characterization of RalF40I, a class II restriction endonuclease from Ruminococcus albus F-40, which recognizes and cleaves 5'-/GATC-3'; Miyagi T et al.; Restriction endonuclease RalF40I was purified from cell-free extracts of the rumen cellulolytic bacterium Ruminococcus albus F-40 heparin-Sepharose chromatography . The preparation was active only on DNA substrates that were not Dammethylated . RalF401 recognizes the 4-bp palindrome, 5'-/GATC-3', and cleaves DNA at the 5' side of G in the sequence, producing 5' tetranucleotide protruding ends . RalF40I is a class II restriction endonuclease and an isoschizomer of MboI and DpnII. Ital J Gastroenterol Hepatol, 1998 Apr, 30(2), 153 - 9 Epithelial-cell apoptosis and proliferation in Helicobacter pylori-related chronic gastritis; Anti M et al.; BACKGROUND: Several studies have shown that Helicobacter pylori infection is associated with enhanced gastric epithelial-cell proliferation, which is thought to be involved in its apparent carcinogenicity . This hyperproliferation is believed to be related to the inflammatory effects of the bacterium . The role of Helicobacter pylori in gastric epithelial apoptosis, however, is less clear . AIM: We attempted to identify the effect of Helicobacter pylori infection on apoptosis in the gastric epithelium and its possible relation to epithelial-cell proliferation and mucosal inflammation . PATIENTS AND METHODS: We studied cell proliferation (via bromodeoxyuridine labelling), apoptosis (using in situ TdT-mediated dUTP-biotin nick end labelling of DNA strand breaks) and mononuclear and polymorphonuclear cell infiltrates (computer-assisted image analysis) in gastric antral biopsies obtained from 37 gastritis patients (20 Helicobacter pylori-positive, 17 Helicobacter pylori-negative) . RESULTS: Helicobacter pylori-positives displayed significantly enhanced proliferation within the gastric epithelium that was positively correlated with both acute and chronic inflammatory-cell densities . Apoptotic indexes were similar in both groups and showed no correlation with any of the parameters under consideration . CONCLUSIONS: Enhanced epithelial cell proliferation and an altered distribution of cycling cells within the gastric glands are a common feature of chronic superficial gastritis caused by Helicobacter pylori . In vivo immunohistochemically detected apoptosis of gastric epithelial cells does not seem to be affected by Helicobacter pylori infection . Further study is needed to clarify the effect of this infection on programmed cell death within gastric glands. J Appl Microbiol, 1998 May, 84(5), 889 - 94 Iron deficiency-induced tetracycline production in submerged cultures by Streptomyces aureofaciens; Bechet M et al.; Streptomyces aureofaciens ATCC 10762 grown in rotary-shaken submerged cultures produced substantial amounts of tetracycline only when the defined medium was deprived of iron . The biosynthesis of tetracycline was inhibited either by free iron at concentrations above 1-2 mumol l-1, or by chelated iron provided by the siderophores of this bacterial strain . Late static iron-containing cultures allowed cell differentiation and sporulation and led to tetracyclines synthesis . A nitrosoguanidine-induced mutant able to synthesize tetracycline in the presence of iron in shaken submerged cultures was isolated and compared to the wild-type strain . However, no constitutive siderophore-mediated iron transport occurred in the mutant . These results suggest the involvement of a putative iron-controlled repressor in the biosynthesis of these secondary metabolites during vegetative growth and primary metabolism of the bacterium. Extremophiles, 1998 May, 2(2), 93 - 9 Purification of a ccb-type quinol oxidase specifically induced in a deep-sea barophilic bacterium, Shewanella sp . strain DB-172F; Qureshi MH et al.; We investigated for the first time the respiratory chain system of a deep-sea barophilic bacterium, Shewanella sp . strain DB-172F . A membrane-bound ccb-type quinol oxidase, from cells grown at 60 MPa pressure, was purified to an electrophoretically homogeneous state . The purified enzyme complex consisted of four kinds of subunits with molecular masses of 98, 66, 18.5, and 15kDa, and it contained 0.96 mol of protoheme and 1.95mol of covalently bound heme c per mol of enzyme . Only protoheme in the enzyme reacted with CO and CN-, and the catalytic activity of the enzyme was 50% inhibited by 4 microM CN- . The isoelectric point of the native enzyme complex was determined to be 5.0 . This enzyme was specifically induced only under conditions of elevated hydrostatic pressure, and high levels were expressed in cells grown at 60 MPa . The membranes isolated from cells grown at atmospheric pressure (0.1 MPa) exhibited high levels of both cytochrome c oxidase and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPDH2)-oxidase activity . These results suggest the presence of two kinds of respiratory chains regulated in response to pressure in the deep-sea bacterium DB-172F. J Mol Biol, 1998 Jul 31, 280(5), 775 - 84 Pathways for transcriptional activation of a glutathione-dependent formaldehyde dehydrogenase gene; Barber RD et al.; The widespread occurrence of glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) suggests that this enzyme serves a conserved function in preventing the cytogenetic and potentially lethal interaction of formaldehyde with nucleic acids, proteins and other cell constituents . Despite this potential role of GSH-FDH, little is known about how its expression is regulated . Here, we identify metabolic and genetic signals that activate transcription of a GSH-FDH gene (adhI) in the bacterium Rhodobacter sphaeroides . Activity of the adhI promoter is increased by both exogenous formaldehyde and metabolic sources of this toxin . Elevated adhI promoter activity in DeltaGSH-FDH mutants implicates formaldehyde or the glutathione adduct that serves as a GSH-FDH substrate, S-hydroxymethylglutathione, as a transcriptional effector . From studying adhI expression in different host mutants, we find that the photosynthetic response regulator PrrA and the trans-acting spd-7 mutation increase function of this promoter . The behavior of a nested set of adhI::lacZ fusions indicates that activation by formaldehyde, PrrA and spd-7 requires only sequences 55 bp upstream of the start of transcription . A working model is presented to explain how GSH-FDH expression responds to formaldehyde and global signals generated from the reduced pyridine nucleotide produced by the activity of this enzyme . Microbios, 1998, 93(374), 7 - 16 Isolation and characterization of S-layer proteins from a vent prosthecate bacterium; Shen N et al.; MWapp 116,000 and 29,000 proteins (p116 and p29), major outer membrane proteins of Hyphomonas jannaschiana reproductive cells, were extracted from cell envelopes by dialysis against EDTA, 2 M urea or distilled water . These proteins were precipitated by divalent cations and resolubilized by EDTA-Na, reflecting alternate monomer, multimer states . From two-dimensional gel electrophoresis it was determined that p116 and p29 had a pl of 4.5 . Both were glycoproteins . Results suggest that p116 and p29 are surface layer (S-layer) proteins, with p116 a tetramer of the p29 . The S-layer could protect the adherent H . jannaschiana reproductive cell from exoenzyme activity, antibiotics and other bacteriocidal molecules produced in the bacterial films formed on many marine surfaces. Biochemistry (Mosc), 1998 Jun, 63(6), 625 - 8 Photophosphorylation in alkalophilic halobacterial cells containing halorhodopsin: chloride-ion cycle? Avetisyan AV, Kaulen AD, Skulachev VP, Feniouk BA. Light-driven ATP synthesis is found in cells of the alkalophilic bacterium Natronobacterium pharaonis containing halorhodopsin but deficient in H+-pumping bacteriorhodopsin . Photophosphorylation occurs with cyanide-inhibited respiratory chain as well as without cyanide in conditions with low C1- concentration in the incubation medium . Increase in C1- concentration from 0.1 to 2.35 M in the incubation medium leads to inhibition of photophosphorylation . Continuous illumination increases membrane Delta Psi if respiration is inhibited by cyanide . This effect is stimulated by DCCD, an ATPase inhibitor . These data can be explained if one suggests that halorhodopsin pumps C1- into the cells whereas C1- release from the cells through C1--ATP-synthase is coupled with the ATP synthesis (chloride-ion cycle). J Mol Biol, 1998 Jul 17, 280(3), 323 - 6 Homology-based fold predictions for Mycoplasma genitalium proteins; Huynen M et al.; Homology search techniques based on the iterative PSI-BLAST method in combination with various filters for low sequence complexity are applied to assign folds to all Mycoplasma genitalium proteins . The resulting procedure (implemented as a web server) is able to predict at least one domain in 37% of these proteins automatically, with an estimated accuracy higher than 98% . Taking structural features such as coiled coil or transmembrane regions aside, folds can be assigned to more than half of the globular proteins in a bacterium just by iterative sequence comparison . FEBS Lett, 1998 Jun 16, 429(3), 295 - 8 Characterisation of a new rubredoxin isolated from Desulfovibrio desulfuricans 27774: definition of a new family of rubredoxins; LeGall J et al.; A new rubredoxin from the sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as terminal electron acceptor, was isolated and characterised . The protein is an 8.5 kDa monomer containing one iron atom per molecule, with a reduction potential of 25 +/- 5 mV at pH 7.6 . Like the recombinant Rdl protein from D . vulgaris, expressed in Escherichia coli {Lumpio, H.L., Shenvi, N.V., Garg, R.P., Summers, A.O . and Kurtz, D.M., J . Bacteriol . 179 (1997) 4607-4615}, it contains an unusual spacing of four amino acids between the first two of the iron coordinating cysteinyl residues . This difference is reflected in the structure of the iron centre, as observed by visible and EPR spectroscopies . All together, these features make these proteins the first members of a new family of rubredoxins. J Biol Chem, 1998 Jul 17, 273(29), 18509 - 13 DNA binding characteristics of RegA . A constitutively active anaerobic activator of photosynthesis gene expression in Rhodobacter capsulatus; Du S et al.; In the purple non-sulfur bacterium Rhodobacter capsulatus, RegA and RegB comprise a two-component regulatory system that is required for maximal anaerobic transcription of key photosynthesis genes . RegB is a sensor kinase that uses ATP to phosphorylate its cognate response regulator, RegA . The mechanism under which RegA approximately P influences transcription of target genes has been unclear given that past attempts to demonstrate DNA binding activity by isolated RegA have failed . This led to a model invoking a role for RegA approximately P as an intermediate in a more complex multicomponent phosphoryl transfer cascade . In the present study, we describe the isolation of a mutant version of RegA (RegA*) which promotes high level expression of photosynthesis genes independent of RegB . DNase I footprint analyses show that purified RegA* binds to the promoters of the puf and puc operons at locations that are consistent with RegA functioning as a transcriptional activator for these operons . We conclude that RegA functions, like most members of the response regulator family, as a DNA-binding protein that directly affects the expression of its target genes. Biochim Biophys Acta, 1998 May 19, 1384(2), 345 - 55 Purification and primary structure analysis of two cytochrome c2 isozymes from the purple phototrophic bacterium Rhodospirillum centenum; Samyn B et al.; The isolation and amino acid sequences of two cytochromes c-552 from the thermotolerant bacterium Rhodospirillum (R.) centenum have been determined . They are very similar to one another with 85% identity . They are homologous to the cytochromes c2 from purple bacteria with approximately 67% identity to that from Rhodopseudomonas (Rps.) palustris compared to only 42% identity with others of the c2 subclass . In addition, they share an unusual six-residue insertion with Rps . palustris cytochrome c2 not found in any other cytochrome . The relationship with Rps . palustris is thus highly significant . The redox potentials of the R . centenum isozymes are 293 and 316 mV . Although the proteins have strongly different iso-electric points, both have three conserved lysine residues at the proposed site of electron transfer . These results suggest that they may be functionally interchangeable. Eur J Biochem, 1998 Apr 15, 253(2), 437 - 44 The reductase RedA2 of the multi-component dioxin dioxygenase system of Sphingomonas sp . RW1 is related to class-I cytochrome P450-type reductases; Armengaud J et al.; The first step in the oxidation of the diaryl ethers dibenzo-p-dioxin and dibenzofuran by the bacterium Sphingomonas sp . RW1 is carried out by an atypical multi-component ring hydroxylating dioxygenase . This heteromeric enzyme requires the participation of a flavoprotein, reductase A2, and an iron-sulfur protein, Fdx1, to mediate the transfer of electrons from NADH to the dioxygenase for oxygen activation {Bunz, P . V . & Cook, A . M . (1993) J . Bacteriol . 175, 6467-6475} . From the type of ferredoxin (Fd) and flavoprotein, this complex is presumed to belong to the class-IIA dioxygenase system which has not been genetically analysed so far . The gene encoding the flavoprotein was identified by screening a genomic library constructed in pLAFR3 with a probe generated by PCR amplification . The nucleotide sequence of a 2.0-kb DNA fragment encompassing the reductase gene, redA2, was determined . The specified protein shares 37-40% identity with class-I cytochrome P450 reductases and 27-35% identity with reductases acting with class-IIB dioxygenases . An FAD-binding amino acid consensus sequence, as well as an NADH-binding site were detected by analogy beginning at residues 10 and 153, respectively . The redA2 gene is not linked to the dioxin dioxygenase cistrons . The rare start codon, GTG, of the reductase was changed to ATG and the modified gene hyperexpressed in Escherichia coli using the strong T7 polymerase promoter . The recombinant reductase was purified to homogeneity with an approximate yield of 3.3 mg/g wet mass cells . Flavin analysis confirmed the presence of 1 FAD/mol protein . The Km values for NADH and Fdx1 are 22 (+/- 3) microM and 3.7 (+/- 0.4) microM, respectively. J Infect Dis, 1998 Jul, 178(1), 274 - 7 High prevalence of Chlamydia pneumoniae DNA in peripheral blood mononuclear cells in patients with cardiovascular disease and in middle-aged blood donors; Boman J et al.; Nested polymerase chain reaction (nPCR) demonstrated the presence of Chlamydia pneumoniae-specific DNA in peripheral blood mononuclear cells (PBMC) . PBMC samples were obtained from 103 consecutive patients (62 male, 41 female) aged 22-85 years (mean, 64) admitted for coronary angiography because of suspected coronary heart disease and from 52 blood donors (43 male, 9 female) aged 40-64 years (mean, 49) . Of the 101 evaluable patients, 60 (59%) were identified by nPCR assay as C . pneumoniae DNA carriers; C . pneumoniae-specific microimmunofluorescence (MIF) serology confirmed exposure to the bacterium in 57 (95%) of the 60 nPCR-positive patients . Among the 52 blood donors, the nPCR assay identified 24 (46%) C . pneumoniae DNA carriers, all of whom were positive by C . pneumoniae-specific serology . Thirty-two patients (32%) and 23 blood donors (44%) were MIF antibody-positive but repeatedly nPCR-negative; Bartonella henselae- or Bartonella quintana-specific antibodies were not detected among any of these subjects . In this study, C . pneumoniae DNA was common in PBMC of patients with coronary heart disease and in middle-aged blood donors. Dig Dis Sci, 1998 Jun, 43(6), 1226 - 9 Beneficial effects of Helicobacter pylori eradication on idiopathic chronic urticaria; Di Campli C et al.; Helicobacter pylori, the most important cause of gastritis and peptic ulcer, recently has been associated with several extradigestive diseases . The aim of this study was to assess the prevalence of Helicobacter pylori infection and the effects of bacterium eradication in 42 consecutive patients affected by idiopathic chronic urticaria . Helicobacter pylori was assessed by {13C}urea breath test . Amoxicillin, clarithromycin, and lansoprazole were given to infected patients for seven days . Urticaria and gastrointestinal symptoms were assessed on enrollment and after eradication . Fifty-five percent of patients proved to be infected by Helicobacter pylori . Prevalence of gastrointestinal symptoms did not differ between infected and uninfected patients . Eighty-eight percent of infected patients in whom the bacterium was eradicated after therapy showed a total or partial remission of urticaria symptoms . Conversely, symptoms remained unchanged in all uninfected patients . In conclusion, Helicobacter pylori affects a high percentage of patients with idiopathic chronic urticaria; however, typical gastrointestinal symptoms do not identify infection status . Bacterium eradication is associated with a remission of urticaria symptoms, suggesting a possible role of Helicobacter pylori in the pathogenesis of this skin disorder. Br J Rheumatol, 1998 May, 37(5), 520 - 4 Bacteria-specific lymphocyte proliferation in peripheral blood in reactive arthritis and related diseases; Fendler C et al.; The cellular immune response seems to be important for the pathogenesis of reactive arthritis (ReA) and a bacteria-specific lymphocyte proliferation (LP) is often found in synovial fluid (SF) of ReA patients . However, the role of the bacteria-specific LP in peripheral blood (PB) is less well defined . In this study, we investigated 215 paired samples of SF and PB from patients with ReA (n = 65), undifferentiated oligoarthritis (n = 133) and undifferentiated spondylarthropathy (n = 17) to analyse the LP in PB and SF in relation to time . In 24 out of 87 patients (27.6%) with a bacteria-specific LP in synovial fluid, a positive LP to the same bacterium was also found in PB . While a positive LP in SF was found most frequently in the first week of the arthritis, a positive LP in PB was detected in 45% of patients when investigated between weeks 2 and 4 after the onset of arthritis, but was rarely found very early and late in the course of the arthritis . The time point seems to be crucial for the investigation of an LP in PB in patients with ReA. Appl Microbiol Biotechnol, 1998 May, 49(5), 552 - 9 Identification of tandemly repeated type VI cellulose-binding domains in an endoglucanase from the aerobic soil bacterium Cellvibrio mixtus; Fontes CM et al.; Cellulose-binding domains (CBD) play a pivotal role during plant cell wall hydrolysis by cellulases and xylanases from aerobic soil bacteria . Recently we have reported the molecular characterisation of a single-domain endoglucanase from Cellvibrio mixtus, suggesting that some cellulases produced by this aerobic bacterium preferentially hydrolyse soluble cellulosic substrates . Here we describe the complete nucleotide sequence of a second cellulase gene, celB, from the soil bacterium C . mixtus . It revealed an open reading frame of 1863 bp that encoded a polypeptide, defined as cellulase B (CelB) with a predicted Mr of 66 039 . CelB contained a glycosyl hydrolase family 5 catalytic domain at its N terminus followed by two repeated domains, which exhibited sequence identity with type VI CBD previously found in xylanases . Full-length CelB bound to cellulose while catalytically active truncated cellulase derivatives were unable to bind the polysaccharide, confirming that CelB is a modular enzyme and that the type VI CBD homologues were functional . Analysis of the biochemical properties of CelB revealed that the enzyme hydrolyses a range of cellulosic substrates, although it was unable to depolymerise Avicel . We propose that type VI CBD, usually found in xylanases, provide an additional mechanism by which cellulases can accumulate on the surface of the plant cell wall, although they do not potentiate cellulase activity directly . The results demonstrate that C . mixtus, in common with other aerobic bacteria, is able to produce cellulases that are directed to the hydrolysis of cellulose in its natural environment, the plant cell wall. MMWR Morb Mortal Wkly Rep, 1998 Jun 19, 47(23), 476 - 80 Statewide surveillance for ehrlichiosis--Connecticut and New York, 1994-1997; DNA analysis of temperate bacteriophage Aa(phi)23 isolated from actinobacillus actinomycetemcomitans; Institute of Preventive Dentistry and Oral Microbiology, Dental Center, University of Basel, SwitzerlandThe DNA of the temperate bacteriophage Aaphi23 isolated from the oral bacterium Actinobacillus actinomycetemcomitans was examined structurally both in the phage head and in the prophage . The DNA in phage particles comprises 44 kb linear molecules with a terminal redundancy of 1.6 kb, which represent circular permutations . Thus, DNA is packaged into phage heads by the headful mechanism . The Aaphi23 prophage is integrated into the host chromosome. Appl Environ Microbiol, 1998 Jul, 64(7), 2380 - 5 Molecular cloning, sequencing, and expression of a chemoreceptor gene from Leptospirillum ferrooxidans; Delgado M et al.; We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans . This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I . This is the first sequence reported for a gene from L . ferrooxidans encoding a protein . The lcrI gene showed both sigma 28-like and sigma 70-like putative promoters . The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains . We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites . The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation . In addition, this 58-kDa protein was expressed in E . coli, being associated with its cytoplasmic membrane fraction . It was not possible to determine a chemotactic receptor function for LcrI expressed in E . coli . This was most likely due to the fact that the periplasmic pH of E . coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain. Tierarztl Prax Ausg K Klientiere Heimtiere, 1998 May, 26(3), 154 - 9 {Relevance of Helicobacter pylori infection in the development of gastric tumors}; Bartram HP; Helicobacter pylori plays an important role in the pathogenesis of peptic ulcer disease . Besides, epidemiological studies indicate that helicobacter may be involved in the development of gastric cancer and MALT-lymphoma by inducing gastritis and accumulation of lymph follicles in the gastric mucosa . The carcinogenic effect of helicobacter pylori can be explained by various pathogenetic factors which are produced by the bacterium itself . Furthermore, influences of helicobacter pylori on the vitamin C content of gastric mucosa might play a role . Currently, Hp-eradication therapies for the treatment of gastric MALT lymphomas are already under investigation in controlled studies . Concerning gastric cancer prevention, however, the available data are not sufficient to warrant a general recommendation for eradication-therapies of all Hp-infected persons . However, further studies must show whether Hp-eradication of high-risk subjects, i.e . members of gastric cancer families, young patients with chronic-active gastritis or with chronic-atrophic gastritis and intestinal metaplasia, are effective in reducing gastric cancer risk. Ital J Gastroenterol Hepatol, 1997 Jun, 29(3), 214 - 9 Effect of Helicobacter pylori eradication on gastric epithelial proliferation . Relationship with ras oncogene p21 expression; Ierardi E et al.; BACKGROUND: Impaired changes in gastric epithelium proliferation have been described in Helicobacter pylori infection, and a progressive increase of proliferating cells has been shown with the progression of mucosal lesions . AIMS: Purpose of this investigation was to study the effect of eradication on bacterium-induced proliferative changes, evaluated by the proliferating cell nuclear antigen labelling index (PCNA LI) and its relationship to the ras oncoprotein p21, involved in early events of gastric carcinogenesis . PATIENTS AND METHODS: This retrospective study was performed, before and after therapy, in five different groups of patients with progressive stages of Helicobacter pylori damage (N: normality; HG: histological gastritis with normal endoscopy; EHG: histological gastritis with endoscopic chronic erosions; CIM: complete intestinal metaplasia; IIM: incomplete intestinal metaplasia) . RESULTS: Six months after eradication, a normalization of PCNA LI was observed in the areas of gastritis, but not in those of intestinal metaplasia, which showed on unchanged type . Moreover, immunohistochemical membrane expression of ras oncoprotein p21 was only associated to intestinal metaplasia . The protein was also expressed in the cytoplasm in 3 patients with incomplete type . CONCLUSIONS: These results suggest that the development of intestinal metaplasia may be associated with an alteration in the control of gastric epithelium proliferation and could represent an initial stage in gastric carcinogenesis . Nevertheless, further genetic changes are necessary for a complete progression to neoplastic disease . A long-term follow-up on extension, type, proliferative situation and oncoprotein expression in areas of intestinal metaplasia may be helpful to explain whether the present data provide new information on the mechanism of Helicobacter pylori induced gastric carcinogenesis. Infection, 1998 May-Jun, 26(3), 178 - 80 Whipple's disease with aortic regurgitation requiring aortic valve replacement; Schneider T et al.; Cardiac involvement in Whipple's disease is well established . However, clinical consequences beside antibiotic therapy have rarely been reported . Our observation of a middle-aged man with increasing dyspnea, fatigue, chest pain, and dizziness leading to admission to a cardiology department demonstrates that cardiac symptoms may represent the main symptoms in patients with Whipple's disease . The diagnosis was not made prior to upper endoscopy, performed because of diarrhea, and revealed Whipple's agent now classified as Tropheryma whippelii, which is a PAS-positive rod-shaped bacterium in the macrophages of the intestinal lamina propria . The aortic valve was replaced after the intestinal symptoms were resolved by antibiotic treatment reducing the number of infectious agents in the duodenal mucosa . Histological analysis of the aortic valve demonstrated the presence of PAS-positive rod shaped material as the most likely cause of aortic insufficiency . Five months after valve replacement, the patient had completely recovered from intestinal and cardiac symptoms . Still under antibiotic treatment 16 months later, no more PAS-positive macrophages were detectable in the intestinal mucosa. J Bacteriol, 1998 Jul, 180(13), 3368 - 74 Enantioselective uptake and degradation of the chiral herbicide dichlorprop {(RS)-2-(2,4-dichlorophenoxy)propanoic acid} by Sphingomonas herbicidovorans MH; Zipper C et al.; Sphingomonas herbicidovorans MH was able to completely degrade both enantiomers of the chiral herbicide dichlorprop {(RS)-2-(2,4-dichlorophenoxy)propanoic acid}, with preferential degradation of the (S) enantiomer over the (R) enantiomer . These results are in agreement with the recently reported enantioselective degradation of mecoprop {(RS)-2-(4-chloro-2-methylphenoxy)propanoic acid} by this bacterium (C . Zipper, K . Nickel, W . Angst, and H.-P . E . Kohler, Appl . Environ . Microbiol . 62:4318-4322, 1996) . Uptake of (R)-dichlorprop, (S)-dichlorporp, and 2,4-D (2,4-dichlorophenoxyacetic acid) was inducible . Initial uptake rates of cells grown on the respective substrate showed substrate saturation kinetics with apparent affinity constants (Kt) of 108, 93, and 117 microM and maximal velocities (Vmax) of 19, 10, and 21 nmol min-1 mg of protein-1 for (R)-dichlorprop, (S)-dichlorprop, and 2,4-D, respectively . Transport of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D was completely inhibited by various uncouplers and by nigericin but was only marginally inhibited by valinomycin and by the ATPase inhibitor N,N'-dicyclohexylcarbodiimine . Experiments on the substrate specificity of the putative transport systems revealed that (R)-dichlorprop uptake was inhibited by (R)-mecoprop but not by (S)-mecoprop, (S)-dichlorprop, or 2,4-D . On the other hand, the (S)-dichlorprop transport was inhibited by (S)-mecoprop but not by (R)-mecoprop, (R)-dichlorprop, or 2,4-D . These results provide evidence that the first step in the degradation of dichlorprop, mecoprop, and 2,4-D by S . herbicidovorans is active transport and that three inducible, proton gradient-driven uptake systems exist: one for (R)-dichlorprop and (R)-mecoprop, another for (S)-dichlorprop and (S)-mecoprop, and a third for 2,4-D. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, 1997, (115), 155 - 60 {Development of a geographical information system and its application to Escherichia coli O-157 patient distribution}; Kaminuma T et al.; The so called Geographical Information System (GIS) is one of the basic tools for wide range of public health applications . We had developed a general purpose GIS and applied it to represent geographical distribution of patients of the bacterium E . coli O-157 which bursted out in Japan last early summer particularly at Sakai City in Osaka Prefecture . The patient record have been supplied from the Food Safety Office of the Ministry of Health and Welfare . These records were handled by EXCEL . The basic geographical data was constructed from the map data provided by Japan Geographical Survey Institute, and ArcView 2 was used as the map system . The maps were converted to Graphics Interchange Format (GIF) files and put on our Web server. Arch Microbiol, 1998 Jul, 170(1), 59 - 68 Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum; Reinartz M et al.; Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180(T)) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro . Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit . The genotype of the flavocytochrome-c-deficient Chr . vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level . The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type . Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system . In accordance with these results, Chr . vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-) . Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors . The enzyme is strictly membrane-bound and is constitutively expressed . The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr . vinosum. Infect Immun, 1998 Jul, 66(7), 3433 - 5 Tube phagocytosis, a novel way for neutrophils to Phagocytize borrelia burgdorferi; Suhonen J et al.; Interactions between human neutrophils and Borrelia burgdorferi, the Lyme disease spirochete, were studied by dark-field microscopy combined with video technology . A previously unrecognized mechanism for neutrophils to phagocytize the spirochete was discovered . During phagocytosis, the spirochete attaches to the neutrophil head-on, the neutrophil forms a thin tubelike protrusion around the bacterium, and the fully covered spirochete is drawn into the cell. Infect Immun, 1998 Jul, 66(7), 3198 - 207 Bioluminescence as a reporter of intracellular survival of Bordetella bronchiseptica in murine phagocytes; Forde CB et al.; The uptake and persistence of Bordetella bronchiseptica was characterized in murine phagocytes by using a novel bioluminescence-based reporter system . A mini-Tn5 promoter probe carrying the intact lux operon from the terrestrial bacterium Photorhabdus luminescens which allowed measurement of light output without the addition of exogenous substrate was constructed . It was used to create a pool of bioluminescent fusion strains of B . bronchiseptica . The internalization and persistence in murine macrophages of a constitutive bioluminescent strain of B . bronchiseptica was monitored by luminometry and by fluorescence and electron microscopy . The number of bacteria internalized, in a microfilament-dependent process, by a mouse macrophage-like cell line after 2 h was approximately 1% of the inoculum for several different multiplicities of infection (MOI) . At an MOI of <500:1 (bacteria to macrophages), viable numbers of intracellular bacteria declined over a 4-day period . However, at an MOI of >/=500:1, long-term survival was enhanced, with viable bacteria recovered up to 4 days postinfection with little decline in numbers, indicating that a critical population size may have been essential for intracellular persistence . No evidence of macrophage killing by intracellular bacteria was detected over the 4-day period . Intracellular bioluminescent B . bronchiseptica organisms in mouse peritoneal cells were detected at 24 and 48 h after intraperitoneal injection of mice . Bioluminescence is shown to act as a convenient real-time technique for monitoring of intracellular survival of B . bronchiseptica in vitro and may provide a suitable means for examining the role of long-term intracellular survival of the bacterium in the host. Science, 1998 Jun 19, 280(5371), 1938 - 40 Bloodstream- versus tick-associated variants of a relapsing fever bacterium; Schwan TG et al.; The relapsing fever spirochete, Borrelia hermsii, alternates infections between a mammal and a tick vector . Whether the spirochete changes phenotypically in the different hosts was examined by allowing the tick vector Ornithodoros hermsi to feed on mice infected with serotype 7 or serotype 8 of B . hermsii . Upon infection of ticks, the spirochetal serotype-specific variable major proteins (Vmps) 7 and 8 became undetectable and were replaced by Vmp33 . This switch from a bloodstream- to tick-associated phenotype could be induced in culture by a decrease in temperature . After tick-bite transmission back to mice, the process was reversed and the spirochetes resumed expression of the same Vmp present in the previous infectious blood meal. Genes Dev, 1998 Jun 15, 12(12), 1884 - 93 A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase; Bowman WC et al.; A commonly accepted view of gene regulation in bacteria that has emerged over the last decade is that promoters are transcriptionally activated by one of two general mechanisms . The major type involves activator proteins that bind to DNA adjacent to where the RNA polymerase (RNAP) holoenzyme binds, usually assisting in recruitment of the RNAP to the promoter . This holoenzyme uses the housekeeping sigma70 or a related factor, which directs the core RNAP to the promoter and assists in melting the DNA near the RNA start site . A second type of mechanism involves the alternative sigma factor (called sigma54 or sigmaN) that directs RNAP to highly conserved promoters . In these cases, an activator protein with an ATPase function oligomerizes at tandem sites far upstream from the promoter . The nitrogen regulatory protein (NtrC) from enteric bacteria has been the model for this family of activators . Activation of the RNAP/sigma54 holoenzyme to form the open complex is mediated by the activator, which is tethered upstream . Hence, this class of protein is sometimes called the enhancer binding protein family or the NtrC class . We describe here a third system that has properties of each of these two types . The NtrC enhancer binding protein from the photosynthetic bacterium, Rhodobacter capsulatus, is shown in vitro to activate the housekeeping RNAP/sigma70 holoenzyme . Transcriptional activation by this NtrC requires ATP binding but not hydrolysis . Oligomerization at distant tandem binding sites on a supercoiled template is also necessary . Mechanistic and evolutionary questions of these systems are discussed. Biochemistry, 1998 Jun 23, 37(25), 8987 - 94 A null lesion in the rhodopin 3,4-desaturase of Rhodospirillum rubrum unmasks a cryptic branch of the carotenoid biosynthetic pathway; Komori M et al.; The carotenoids accumulated by a mutant Rhodospirillum rubrum ST4, containing a single Tn5 lesion in the pathway for carotenoid biosynthesis, were analyzed by HPLC, 1H NMR spectroscopy, and field desorption mass spectrometry . The main carotenoid was identified as 3,4,3',4'-tetrahydrospirilloxanthin, and the four minor carotenoids were identified as rhodopin, 3,4-dihydroanhydrorhodovibrin, 3', 4'-dihydrorhodovibrin, and 1,1'-dihydroxylycopene . The C-3,4 and C-3',4' bonds of all 5 carotenoids are saturated, and they have 11 conjugated double bonds . With the exception of rhodopin, which is a normal intermediate of the wild-type pathway, all of the carotenoids are not naturally occurring . The Tn5 lesion was assigned to rhodopin 3,4-desaturase which is proposed to catalyze dehydrogenation at both ends of the symmetrical spirilloxanthin derivative . An unexpected finding was that the enzymes following rhodopin 3,4-desaturase are still able to end-modify the 3,4-, and 3',4'-saturated precursors and that the order of methylation and hydroxylation is not obligatory . It is proposed that the observed nonnatural carotenoids can be explained by the inclusion of a cryptic branch, unmasked by the absence of rhodopin 3,4-desaturase, in the established linear pathway for spirilloxanthin biosynthesis . This is the first example of latent branching of the carotenoid biosynthesis pathway exhibited by a carotenoid mutant of a phototrophic bacterium. Biophys J, 1998 Jun, 74(6), 3226 - 40 Effects of temperature and deltaGo on electron transfer from cytochrome c2 to the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides; Venturoli G et al.; The kinetics of electron transfer from cytochrome c2 to the primary donor (P) of the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides have been investigated by time-resolved absorption spectroscopy . Rereduction of P+ induced by a laser pulse has been measured at temperatures from 300 K to 220 K in a series of specifically mutated reaction centers characterized by altered midpoint redox potentials of P+/P varying from 410 mV to 765 mV (as compared to 505 mV for wild type) . Rate constants for first-order electron donation within preformed reaction center-cytochrome c2 complexes and for the bimolecular oxidation of free cytochrome c2 have been obtained by multiexponential deconvolution of the kinetics . At all temperatures the rate of the fastest intracomplex electron transfer increases by more than two orders of magnitude as the driving force -deltaGo is varied over a range of 350 meV . The temperature and deltaGo dependences of the rate constant fit the Marcus equation well . Global analysis yields a reorganization energy lambda = 0.96 +/- 0.07 eV and a set of electronic matrix elements, specific for each mutant, ranging from 1.2 10(-4) eV to 2.5 10(-4) eV . Analysis in terms of the Jortner equation indicates that the best fit is obtained in the classical limit and restricts the range of coupled vibrational modes to frequencies lower than approximately 200 cm(-1) . An additional slower kinetic component of P+ reduction, attributed to electron transfer from cyt c2 docked in a nonoptimal configuration of the complex, displays a Marcus type dependence of the rate constant upon deltaGo, characterized by a similar value of lambda (0.8 +/- 0.1 eV) and by an average electronic matrix element smaller by more than one order of magnitude . In all of the mutants, as the temperature is decreased below 260 K, both intracomplex reactions are abruptly inhibited, their rate being negligible at 220 K . The free energy dependence of the second-order rate constant for oxidation of cyt c2 in solution suggests that the collisional reaction is partially diffusion controlled, reaching the diffusion limit at exothermicities between 150 and 250 meV over the temperature range investigated. Biochim Biophys Acta, 1998 May 27, 1364(3), 361 - 72 The influence of structural-dynamic organization of RC from purple bacterium rhodobacter sphaeroides on picosecond stages of photoinduced reactions Paschenko VZ, Gorokhov VV, Grishanova NP, Goryacheva EA, Korvatovsky BN, Knox PP, Zakharova NI, Rubin AB. Effects of the hydrogen bond network on the rate constants of energy migration (km), charge separation (ke), electron transfer to QA (kQ) and P+I- recombination in RC of Rhodobacter sphaeroides were analysed in control and modified RC preparations at different temperatures . Modification of RC were made by the addition of 40% v/v DMSO . The rate constants km, ke, kQ were evaluated from pump-and-probe measurements of the absorption difference kinetics at 665 nm corresponding to BPhL- formation and subsequent electron transfer to QA . For the investigation of P+I- recombination a primary quinone acceptor was pre-reduced in the dark by adding of 1 mg/ml of dithionite and 1 mM sodium ascorbate . Recombination kinetics were measured at 665 and 870 nm . The numerical analysis of the temperature dependence of ke and kQ was performed on the basis of the model proposed by Kakitani and Kakitani (T . Kakitani and H . Kakitani (1981), Biochim . Biophys . Acta, 635, 498-514) . It was found that: (a) in control samples the molecular rate constants km, ke and kQ were about (3.4 ps)-1, (4.5 ps)-1 and (200 ps)-1, respectively; (b) under modification by DMSO these rates decrease up to (5.3 ps)-1, (10.3 ps)-1 and (500 ps)-1, respectively; (c) as the temperature drops from 300 K to 77 K the rate constant km decreases by 1.8 times in control and by 3.2 times in modified samples . In contrast to the observed km changes the increase in ke and kQ values by 2 and more times under cooling was found in control and modified RC; (d) in control preparations with QA acceptor pre-reduced in the dark the lowering of the temperature caused the increase in the time of P+I- recombination from 10 to 20 ns . After DMSO modification the kinetics of charge recombination in RC was biexponential at room temperature with tau=10 ns and tau1=0.8 ns, and at 77 K with tau=20 ns and tau1=0.6 ns, correspondingly . The results obtained reveal that in RC isolated from Rb . sphaeroides the processes of energy migration, charge separation, electron transfer to QA and ion-radical pair P+I- recombination depend on the state of hydrogen bonds of water-protein structure . Fast relaxation processes in RC structure including polarization of H-containing molecules in the surrounding of electron carriers can accept electron energy dissipated at the initial steps of energy and electron transfer . Mol Microbiol, 1998 May, 28(3), 531 - 41 Distal genes of the nuo operon of Rhodobacter capsulatus equivalent to the mitochondrial ND subunits are all essential for the biogenesis of the respiratory NADH-ubiquinone oxidoreductase; Dupuis A et al.; Seven out of the 13 proteins encoded by the mitochondrial genome of mammals (peptides ND1 to ND6 plus ND4L) are subunits of the respiratory NADH-ubiquinone oxidoreductase (complex I) . The function of these ND subunits is still poorly understood . We have used the NADH-ubiquinone oxidoreductase of Rhodobacter capsulatus as a model for the study of the function of these proteins . In this bacterium, the 14 genes encoding the NADH-ubiquinone oxidoreductase are clustered in the nuo operon . We report here on the biochemical and spectroscopic characterization of mutants individually disrupted in five nuo genes, equivalent to mitochondrial genes nd1, nd2, nd5, nd6 and nd4L . Disruption of any of these genes in R . capsulatus leads to the suppression of NADH dehydrogenase activity at the level of the bacterial membranes and to the disappearance of complex I-associated iron-sulphur clusters . Individual NUO subunits can still be immunodetected in the membranes of these mutants, but they do not form a functional subcomplex . In contrast to these observations, disruption of two ORFs (orf6 and orf7), also present in the distal part of the nuo operon, does not suppress NADH dehydrogenase activity or complex I-associated EPR signals, thus demonstrating that these ORFs are not essential for the biosynthesis of complex I. Mol Microbiol, 1998 May, 28(3), 421 - 34 Ordered expression of ftsQA and ftsZ during the Caulobacter crescentus cell cycle; Sackett MJ et al.; The mechanisms by which bacterial cell division and DNA replication are co-ordinated are still unknown . We have used the easily synchronizable bacterium Caulobacter crescentus to determine when the cell division genes ftsQ and ftsA are transcribed during the DNA replication cycle and to compare their transcription with that of ftsZ . Unlike the situation in Escherichia coli, transcription of ftsQ and ftsA does not extend into ftsZ in Caulobacter . ftsQ and ftsA are co-transcribed by a strong promoter, P(QA), present within the end of the ddl gene upstream of ftsQ . Transcription of P(QA) is turned on at the end of the DNA replication period, coincident with the end of the ftsZ transcription period . ftsA is also transcribed by another promoter, P(A), present between ftsQ and ftsA . P(A) transcription is approximately 10 times weaker than P(QA) and occurs during the DNA replication period . Transcription of ftsA by P(A) is sufficient for cell viability, but is not sufficient for normal cell division . When the transcription of ftsA is increased constitutively, cell division is inhibited and stalks are synthesized at aberrant positions . Thus, transcription of ftsA and ftsZ mimics their order of action in Caulobacter and proper transcription of ftsA has to be maintained for normal cell division and differentiation. Arch Microbiol, 1998 Jun, 169(6), 503 - 8 Purification and characterization of a catalase from the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 and its role in the oxidative stress response; Terzenbach DP et al.; When challenged with reactive oxidants, the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 exhibited an oxidative stress response during both phototrophic and chemotrophic growth . Upon preincubation with 100 microM H2O2, catalase activity increased fivefold . Catalase was also induced by other forms of oxidative stress, heat-shock, ethanol treatment, and stationary-phase conditions . Only one band of catalase activity was detected after native and denaturing PAGE . The enzyme was purified 304-fold with a yield of 7% . The purified enzyme displayed a heterodimeric structure with subunits of 75 and 68 kDa, corresponding to a molecular mass of approximately 150 kDa for the native enzyme . The subunits had almost identical amino-terminal peptide sequences, sharing substantial similarity with other bacterial catalases . The enzyme exhibited an apparent Km of 40 mM and a Vmax of 285,000 U (mg protein)-1 . Spectroscopic analysis indicated the presence of protoheme IX . The heme content calculated from pyridine hemochrome spectra was 0.43 mol per mol of enzyme . The enzyme had a broad pH optimum and was inhibited by cyanide, azide, hydroxylamine, 2-mercaptoethanol, and sodium dithionite . These data indicate that this catalase belongs to the class of monofunctional catalases. J Theor Biol, 1998 May 7, 192(1), 117 - 28 Predicting temporal fluctuations in an intracellular signalling pathway; Morton-Firth CJ et al.; We used a newly developed stochastic-based program to predict the fluctuations in numbers of molecules in a chemotactic signalling pathway of coliform bacteria . Specifically, we examined temporal changes in molecules of CheYp, a cytoplasmic protein known to influence the direction of rotation of the flagellar motor . Signalling molecules in the vicinity of a flagellar motor were represented as individual software objects interacting according to probabilities derived from experimentally-observed concentrations rate constants . The simulated CheYp molecules were found to undergo random fluctuations in number about an average corresponding to the deterministically calculated concentration . Both the relative amplitude of the fluctuations, as a proportion of the total number of molecules, and their average duration, increased as the simulated volume was reduced . In a simulation corresponding to 10% of the volume of a bacterium, the average duration of fluctuations was found to be 80.7 ms, which is much shorter than the observed alternations between clockwise and counter clockwise rotations of tethered bacteria (typically 2.6 s) . Our results are therefore not in agreement with a simple threshold-crossing model for motor switching . However, it is possible to filter the CheYp fluctuations to produce temporal distributions closer to the observed swimming behaviour and we discuss the possible implications for the control of motor rotation. FEMS Immunol Med Microbiol, 1998 Apr, 20(4), 257 - 66 Effect of host Lewis and ABO blood group antigen expression on Helicobacter pylori colonisation density and the consequent inflammatory response; Heneghan MA et al.; The Lewis(b) blood group antigen has been implicated as a putative receptor for Helicobacter pylori in the gastric mucosa . Furthermore, an increased prevalence of duodenal ulcer was found in non-secretors and it has been suggested that secretor status may influence bacterial colonisation density . Other investigators have hypothesised that severity of antral gastritis may be related to colonisation density of the bacterium alone, and that a critical bacterial load is necessary for the development of duodenal ulcer . Our objectives were to investigate whether a relationship existed between host Lewis and ABO blood group phenotype and prevalence of H . pylori infection . In addition we investigated whether bacterial colonisation density and the ensuing inflammatory response was influenced by secretor status and ABO blood group phenotype . The Lewis and ABO blood group phenotype of 207 patients undergoing upper endoscopy was determined . Of these, 136 were secretors and 62 were nonsecretors . Forty-five percent of patients were infected with H . pylori . No significant association was found between H . pylori infection and expression of Lewis(a) or Lewis(b) blood group antigen . The mean histological density of H . pylori was 1.8 +/- 0.2 among non-secretors and 1.51 +/- 0.13 among secretors (P = 0.209), with a mean grade of lymphocytic infiltration significantly greater in H . pylori-infected non-secretors (2.23 +/- 0.123 vs 1.8 +/- 0.074; P = 0.003) . In addition, blood group O non-secretors had a significantly higher grade of lymphocyte infiltration of their gastric mucosa compared to non-O non-secretors (2.53 +/- 0.133 vs 1.93 +/- 0.181, P = 0.027) . These results suggest that although no in vivo relationship exists between H . pylori and preferential adhesion to the putative Lewis(b) receptor, bacterial colonisation and the ensuing inflammatory response may be influenced at least in part by host expression of ABO and Lewisa blood group antigens. J Biol Chem, 1998 Jun 12, 273(24), 14788 - 95 Spectinomycin kinase from Legionella pneumophila . Characterization of substrate specificity and identification of catalytically important residues; Thompson PR et al.; The bacterium Legionella pneumophila is the responsible agent for Legionnaires' disease and has recently been shown to harbor a gene encoding a kinase that confers resistance to the aminoglycoside antibiotic spectinomycin (Suter, T . M., Viswanathan, V . K., and Cianciotto, N . P . (1997) Antimicrob . Agents Chemother . 41, 1385-1388) . We report the overproduction, purification, and characterization of this spectinomycin kinase from an expressing system in Escherichia coli . The purified protein shows stringent substrate specificity for spectinomycin with Km = 21.5 microM and kcat = 24.2 s-1 and does not bind other aminoglycosides including kanamycin, amikacin, neomycin, butirosin, streptomycin, or apramycin . Purification of spectinomycin phosphate followed by characterization by mass spectrometry and 1H, 13C, and 31P NMR established the site of phosphorylation to be at the hydroxyl group at position 9 . Thus this enzyme is designated APH(9)-Ia (where APH is aminoglycoside kinase) . The enzyme was inactivated by the electrophilic ATP analogue 5'-{p-(fluorosulfonyl)benzoyl}adenosine, consistent with a nucleophilic residue such as Lys lining the nucleotide binding pocket . Site-directed mutagenesis of Lys-52 and Asp-212 to Ala confirmed that these residues were important for catalysis, with Lys-52 playing a potential role in ATP binding and Asp-212 in phosphoryl transfer . Thio and solvent isotope effect experiments in the presence of either Mg2+ or Mn2+ were consistent with a kinetic mechanism in which phosphate transfer does not contribute significantly to the rate-limiting step . These results establish that APH(9)-Ia is a highly specific antibiotic resistance kinase and provides the requisite mechanistic information for future structural studies. Biochemistry, 1998 Jun 2, 37(22), 8105 - 14 Interactions between the cytochrome b, cytochrome c1, and Fe-S protein subunits at the ubihydroquinone oxidation site of the bc1 complex of Rhodobacter capsulatus; Saribas AS et al.; Ubihydroquinone:cytochrome (cyt) c oxidoreductase (bc1 complex and its plant counterpart b6f complex) is a vital component of energy-transducing systems in most organisms from bacteria to eukaryotes . In the facultative phototrophic (Ps) bacterium Rhodobacter capsulatus, it is constituted by the cyt b, cyt c1, and Rieske Fe-S protein subunits and is essential for Ps growth . Of these subunits, cyt b has two nontransmembrane helices, cd1 and cd2, which are critical for its structure and function . In particular, substitution of threonine (T) at position 163 on cd1 with phenylalanine (F) or proline (P) leads to the absence of the bc1 complex . Here, Ps+ revertants of B:T163F were obtained, and their detailed characterizations indicated that position 163 is important for the assembly of the bc1 complex by mediating subunit interactions at the Qo site . The loss of the hydroxyl group at position 163 of cyt b was compensated for by the gain of either a hydroxyl group at position 182 of cyt b or 46 of the Fe-S protein or a sulfhydryl group at position 46 of cyt c1 . Examination of the mitochondrial bc1 complex crystal structure {Zhang, Z., Huang, L., Shulmeister, V . M., Chi, Y.-I., Kim, K . K., Hung, L.-W., Crofts, A . R., Berry, E . A., and Kim, S.-H . (1998) Nature 392, 677-684} revealed that the counterparts of B:G182 (i.e., G167) and F:A46 (i.e . , A70) are located close to B:T163 (i.e., T148), whereas the C:R46 (i.e., R28) is remarkably far from it . The revertants contained substoichiometric amounts of the Fe-S protein subunit and exhibited steady-state and single-turnover, electron transfer activities lower than that of a wild-type bc1 complex . Interestingly, their membrane supernatants contained a smaller form of this subunit with physicochemical properties identical to those of its membrane-bound form . Determination of the amino-terminal amino acid sequence of this soluble Fe-S protein revealed that it was derived from the wild-type protein by proteolytic cleavage at V44 . This work revealed for the first time that position 163 of cyt b is important both for proper subunit interactions at the Qo site and for inactivation of the bc1 complex by proteolytic cleavage of its Fe-S protein subunit at a region apparently responsible for its mobility during Qo site catalysis. Am Surg, 1998 Jun, 64(6), 545 - 50; discussion 550-1 Early gastric cancer and Helicobacter pylori: 34 years of experience at Charity Hospital in New Orleans; Eckert MW et al.; Good survival rates have been reported for resected early gastric adenocarcinoma (EGC) in patients found via screening procedures . However, the prevalence of Helicobacter pylori in EGC in unscreened populations is unclear . The major purpose of this investigation was to analyze the clinical experience and incidence of H . pylori in unscreened patients presenting with EGC at Charity Hospital over a 34-year period . From 1963 through 1997, the tumor registry at Charity Hospital compiled data on 2497 patients evaluated for gastric carcinoma . Of these patients, 26 (1%) had lesions that were confined to the mucosa or submucosa, i.e., T1N0M0 (American Joint Commission on Cancer classification) . Pathology specimens and medical records were retrieved for confirmation of diagnosis and retrospective analysis for H . pylori . H . pylori was analyzed by Steiner staining and immunohistochemistry using a polyclonal antibody . EGC was detected in 12 men and 14 women with a mean age of 62 years . Upper gastrointestinal X-ray studies were performed on 19 of the 26 patients and failed to conclusively demonstrate a lesion in any case . Endoscopy was performed on 22 patients, and preoperative biopsies were positive in 95 per cent of these . Operative procedures included 2 local excisions and 22 subtotal and 2 total gastrectomies . No extended nodal dissections were performed . Microscopic evaluation revealed lesions limited to the mucosa in 63 per cent of cases and involving the submucosa in 37 per cent of the cases . Of the 14 patients evaluable of H . pylori, 79 per cent were positive for the bacterium . The status of 2 patients is unknown, and only 1 patient died of the original gastric cancer, for a disease-free survival of 96 per cent . The 5-year and 10-year overall survival rates were calculated to be 50 per cent and 21 per cent, respectively, when all causes of death were taken into consideration . Median follow-up of the survivors was 64 months . Resection of early gastric carcinoma in unscreened patients without extended lymphadenectomy yielded excellent results . H . pylori was present in 79 per cent of cases . These data suggest an association between H . pylori and EGC . Whether H . pylori infection is an etiologic factor in gastric cancer remains an area of active research. Ann N Y Acad Sci, 1997 Dec 29, 830, 353 - 60 Mechanisms of recurrent otitis media: importance of the immune response to bacterial surface antigens; Murphy TF et al.; Otitis-prone children experience recurrent episodes of otitis media due to nontypeable H . influenzae (NTHI) . A protective immune response occurs following infection, but this immune response is specific for the infecting strain, leaving the child susceptible to infection by other strains of NTHI . Little is known about the mechanism by which a strain-specific antibody response occurs to nonencapsulated bacteria . To explore the mechanism by which this strain-specific response occurs, animals were inoculated with whole bacterial cells and the antibody response was studied . The antibody response was predominantly directed to a highly strain-specific, immunodominant surface loop on the major outer membrane protein . This exquisitely restricted immune response leaves the host susceptible to recurrent infections by many strains of NTHI . The ability of the bacterium to direct the host to make a strain-specific antibody response has important implications in understanding the immune response to otitis media due to NTHI and in designing strategies for vaccine development. Ital J Gastroenterol Hepatol, 1998 Feb, 30(1), 115 - 8 Vascular and immunological disorders associated with Helicobacter pylori infection; Gasbarrini A et al.; Helicobacter pylori, the main aetiological factor for gastritis and peptic ulcer has been associated with some extradigestive diseases . In particular, vascular disorders, such as ischaemic heart disease, primary Raynaud phenomenon and headache appear to be related to the presence of the bacterium . Moreover, some autoimmune diseases, such as Henoch-Schonlein purpura, Sjogren syndrome and others have been shown to have a link with Helicobacter pylori infection Probably, in subjects with a particular predisposition, the release of substances with a vasoactive and proinflammatory activity, which is determined by Helicobacter pylori infection, may influence some extradigestive districts. J Bacteriol, 1998 Jun, 180(11), 3007 - 12 A Tn7-like transposon is present in the glmUS region of the obligately chemoautolithotrophic bacterium Thiobacillus ferrooxidans; Oppon JC et al.; The region downstream of the Thiobacillus ferrooxidans ATCC 33020 atp operon was examined, and the genes encoding N-acetylglucosamine-1-uridyltransferase (glmU) and glucosamine synthetase (glmS) were found . This atpEFHAGDC-glmUS gene order is identical to that of Escherichia coli . The T . ferrooxidans glmS gene was shown to complement E . coli glmS mutants for growth on minimal medium lacking glucosamine . A Tn7-like transposon, Tn5468, was found inserted into the region immediately downstream of the glmS gene in a manner similar to the site-specific insertion of transposon Tn7 within the termination region of the E . coli glmS gene . Tn5468 was sequenced, and Tn7-like terminal repeat sequences as well as several open reading frames which are related to the Tn7 transposition genes tnsA, tnsB, tnsC, and tnsD were found . Tn5468 is the closest relative of Tn7 to have been characterized to date . Southern blot hybridization indicated that a similar or identical transposon was present in three T . ferrooxidans strains isolated from different parts of the world but not in two Thiobacillus thiooxidans strains or a Leptospirillum ferrooxidans strain . Since T . ferrooxidans is an obligately acidophilic autotroph and E . coli is a heterotroph, ancestors of the Tn7-like transposons must have been active in a variety of physiologically different bacteria so that their descendants are now found in bacteria that occupy very different ecological niches. J Bacteriol, 1998 Jun, 180(11), 2849 - 53 Distal cleavage of 3-chlorocatechol by an extradiol dioxygenase to 3-chloro-2-hydroxymuconic semialdehyde; Riegert U et al.; A 2,3-dihydroxybiphenyl 1,2-dioxygenase from the naphthalenesulfonate-degrading bacterium Sphingomonas sp . strain BN6 oxidized 3-chlorocatechol to a yellow product with a strongly pH-dependent absorption maximum at 378 nm . A titration curve suggested (de)protonation of an ionizable group with a pKa of 4.4 . The product was isolated, purified, and converted, by treatment with diazomethane, to a dimethyl derivative and, by incubation with ammonium chloride, to a picolinic acid derivative . Mass spectra and 1H and 13C nuclear magnetic resonance (NMR) data for these two derivatives prove a 3-chloro-2-hydroxymuconic semialdehyde structure for the metabolite, resulting from distal (1,6) cleavage of 3-chlorocatechol . 3-Methylcatechol and 2,3-dihydroxybiphenyl are oxidized by this enzyme, in contrast, via proximal (2,3) cleavage. Arch Microbiol, 1998 Apr, 169(4), 346 - 52 Investigation of the fumarate metabolism of the syntrophic propionate-oxidizing bacterium strain MPOB; Van Kuijk BL et al.; The growth of the syntrophic propionate-oxidizing bacterium strain MPOB in pure culture by fumarate disproportionation into carbon dioxide and succinate and by fumarate reduction with propionate, formate or hydrogen as electron donor was studied . The highest growth yield, 12.2 g dry cells/mol fumarate, was observed for growth by fumarate disproportionation . In the presence of hydrogen, formate or propionate, the growth yield was more than twice as low: 4.8, 4.6, and 5.2 g dry cells/mol fumarate, respectively . The location of enzymes that are involved in the electron transport chain during fumarate reduction in strain MPOB was analyzed . Fumarate reductase, succinate dehydrogenase, and ATPase were membrane-bound, while formate dehydrogenase and hydrogenase were loosely attached to the periplasmic side of the membrane . The cells contained cytochrome c, cytochrome b, menaquinone-6 and menaquinone-7 as possible electron carriers . Fumarate reduction with hydrogen in membranes of strain MPOB was inhibited by 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO) . This inhibition, together with the activity of fumarate reductase with reduced 2,3-dimethyl-1,4-naphtoquinone (DMNH2) and the observation that cytochrome b of strain MPOB was oxidized by fumarate, suggested that menequinone and cytochrome b are involved in the electron transport during fumarate reduction in strain MPOB . The growth yields of fumarate reduction with hydrogen or formate as electron donor were similar to the growth yield of Wolinella succinogenes . Therefore, it can be assumed that strain MPOB gains the same amount of ATP from fumarate reduction as W . succinogenes, i . e . 0.7 mol ATP/mol fumarate . This value supports the hypothesis that syntrophic propionate-oxidizing bacteria have to invest two-thirds of an ATP via reversed electron transport in the succinate oxidation step during the oxidation of propionate . The same electron transport chain that is involved in fumarate reduction may operate in the reversed direction to drive the energetically unfavourable oxidation of succinate during syntrophic propionate oxidation since (1) cytochrome b was reduced by succinate and (2) succinate oxidation was similarly inhibited by HOQNO as fumarate reduction. Biochemistry, 1998 May 19, 37(20), 7057 - 61 Ultrafast carotenoid band shifts probe structure and dynamics in photosynthetic antenna complexes; Herek JL et al.; We report observations of ultrafast carotenoid band shifts correlated with energy transfer dynamics between bacteriochlorophyll (BChl) molecules within the peripheral light-harvesting complex (LH2) from the photosynthetic bacterium Rhodobacter sphaeroides . Direct excitation of the bacteriochlorophyll Qy bands yielded distinct changes in the carotenoid S2 absorption from 430 to 530 nm . Transient absorption spectra and kinetics were measured in a femtosecond pump-probe experiment, revealing the ultrafast carotenoid response to excited BChl pigments . These data are an indication of a new property of carotenoids that is manifested as a unique ability to detect and report changes in their immediate environment, thereby serving as sensitive probes of local structure and dynamics. Appl Environ Microbiol, 1998 Jun, 64(6), 2232 - 6 Growth of geobacter sulfurreducens with acetate in syntrophic cooperation with hydrogen-oxidizing anaerobic partners Cord-Ruwisch R, Lovley DR, Schink B. Pure cultures of Geobacter sulfurreducens and other Fe(III)-reducing bacteria accumulated hydrogen to partial pressures of 5 to 70 Pa with acetate, butyrate, benzoate, ethanol, lactate, or glucose as the electron donor if electron release to an acceptor was limiting . G . sulfurreducens coupled acetate oxidation with electron transfer to an anaerobic partner bacterium in the absence of ferric iron or other electron acceptors . Cocultures of G . sulfurreducens and Wolinella succinogenes with nitrate as the electron acceptor degraded acetate efficiently and grew with doubling times of 6 to 8 h . The hydrogen partial pressures in these acetate-degrading cocultures were considerably lower, in the range of 0.02 to 0.04 Pa . From these values and the concentrations of the other reactants, it was calculated that in this cooperation the free energy change available to G . sulfurreducens should be about -53 kJ per mol of acetate oxidized, assuming complete conversion of acetate to CO2 and H2 . However, growth yields (18.5 g of dry mass per mol of acetate for the coculture, about 14 g for G . sulfurreducens) indicated considerably higher energy gains . These yield data, measurement of hydrogen production rates, and calculation of the diffusive hydrogen flux indicated that electron transfer in these cocultures may not proceed exclusively via interspecies hydrogen transfer but may also proceed through an alternative carrier system with higher redox potential, e.g., a c-type cytochrome that was found to be excreted by G . sulfurreducens into the culture fluid . Syntrophic acetate degradation was also possible with G . sulfurreducens and Desulfovibrio desulfuricans CSN but only with nitrate as electron acceptor . These cultures produced cell yields of 4.5 g of dry mass per mol of acetate, to which both partners contributed at about equal rates . These results demonstrate that some Fe(III)-reducing bacteria can oxidize organic compounds under Fe(III) limitation with the production of hydrogen, and they provide the first example of rapid acetate oxidation via interspecies electron transfer at moderate temperature. Curr Microbiol, 1998 Jun, 36(6), 327 - 31 Identification and isolation of the indole-3-pyruvate decarboxylase gene from Azospirillum brasilense Sp7: sequencing and functional analysis of the gene locus; Zimmer W et al.; The root-associated bacterium Azospirillum brasilense Sp7 produces the growth-stimulating phytohormone indole-3-acetic acid (= IAA) via the indole-3-pyruvate pathway . The DNA region containing ipdC, the structural gene for indole-3-pyruvate decarboxylase, was identified in a cosmid gene library of strain Sp7 by hybridization and has been sequenced . Upstream of the gene, two other ORF homologous to gltX and cysS were sequenced that are transcribed in the opposite direction . A functional analysis of the cloned ipdC region has been performed . To test the expression of the gene, a lacZ-Km cartridge was introduced into the gene . By this construct, tryptophan-dependent stimulation of gene expression in A . brasilense Sp7 was observed . Evidences for the existence of another copy of the ipdC gene in the Azospirillum genome are also reported. Appl Environ Microbiol, 1998 Jun, 64(6), 2061 - 4 Development of a genetic transformation system for an alga-lysing bacterium; Kato J et al.; Four marine bacteria, Alteromonas sp . strains A27, A28, A29, and A30, that lyse the diatom Skeletonema costatum NIES-324 were isolated from coastal seawater samples . They were also able to lyse the diatoms Thalassiosira sp . and Eucampia zodiacs and the raphidophycean flagellate Chattonella antiqua . Cryptic indigenous plasmids, designated pAS28 and pAS29, were detected in Alteromonas sp . strains A28 and A29, respectively . These plasmids appeared to be similar based on size and restriction site analysis . A shuttle vector that replicates in Escherichia coli and Alteromonas sp . strain A28 was constructed by fusing pAS28 and E . coli vector pCRIIc . The 16-kbp chimeric plasmid, designated pASS1, had the ability to transform strain A28 at a frequency of 10(6) transformants per microg of DNA . Deletion analysis of pASS1 showed that the 4.7-kb EcoRI-HindIII region of pAS28 was essential for plasmid maintenance in strain A28 . This EcoRI-HindIII fragment contained an open reading frame which appeared to encode a 708-amino-acid protein. Appl Environ Microbiol, 1998 Jun, 64(6), 1987 - 90 Mercury methylation by interspecies hydrogen and acetate transfer between sulfidogens and methanogens; Pak K et al.; Cocultures of Desulfovibrio desulfuricans and Methanococcus maripaludis grew on sulfate-free lactate medium while vigorously methylating Hg2+ . Individually, neither bacterium could grow or methylate mercury in this medium . Similar synergistic growth of sulfidogens and methanogens may create favorable conditions for Hg2+ methylation in low-sulfate anoxic freshwater sediments. Arch Microbiol, 1998 May, 169(5), 452 - 9 Two new motile phototrophic consortia: "Chlorochromatium lunatum" and "Pelochromatium selenoides" Abella CA, Cristina XP, Martinez A, Pibernat I I, Vila X. Two new phototrophic consortia, "Chlorochromatium lunatum" and "Pelochromatium selenoides", were observed and collected in the hypolimnion of several dimictic lakes in Wisconsin and Michigan (USA) . The two consortia had the same morphology but different pigment composition . The cells of the photosynthetic components of the consortia were half-moon-shaped . This morphology was used to differentiate them from the previously described motile phototrophic consortia "Chlorochromatium aggregatum" and "Pelochromatium roseum" . These phototrophic cells did not resemble any described unicellular green sulfur bacteria . The predominant pigments detected were bacteriochlorophyll d and chlorobactene for the green-colored "Clc . lunatum", and bacteriochlorophyll e and isorenieratene for the brown-colored "Plc . selenoides" . Their pigment compositions and the presence of chlorosomes attached to the inner face of the cytoplasmic membrane in both kinds of photosynthetic cells confirmed this new half-moon-shaped morphotype as a green sulfur bacterium . Both consortia were found thriving in lakes with low concentrations of sulfide (< 60 &mgr;M), below the layers of "Clc . aggregatum" and "Plc . roseum" . The green consortia were observed in lakes where the oxic-anoxic interface was located at shallow depths (2-7 m), while the brown consortia were found at greater depths (8-16 m) . The two newly described consortia were never detected together at the same depth in any lake. Acta Virol, 1997 Oct, 41(5), 285 - 8 Transcriptional regulation of the gltA and TLC genes in Rickettsia prowazekii growing in a respiration-deficient host cell; Cai J et al.; The regulation of the citrate synthase (gltA) and ATP/ADP translocase (tlc) genes of the obligate intracellular bacterium, Rickettsia prowazekii, was analyzed in rickettsia-infected respiration-deficient G14 cells . The level of the gltA mRNAII and the tlc mRNA was much lower in the total RNA isolated from the infected G14 cells grown in 1 g/l glucose (low glucose, GL) medium than in that from infected G14 cells grown in 4.5 g/l glucose (high glucose, GH) medium . However, the level of the gltA mRNAI relative to 16 S rRNA was the same in GL and GH media . An increase in the level of the gltA mRNAII and the tlc mRNA could be observed as early as 2 hrs after shifting from GL to GH medium . We conclude that, under these experimental conditions, the tlc promoter and the gltA promoter P2, but not gltA promoter P1, were transcriptionally regulated. Clin Diagn Lab Immunol, 1998 May, 5(3), 294 - 8 Use of lytic bacteriophage for control of experimental Escherichia coli septicemia and meningitis in chickens and calves; Barrow P et al.; A lytic bacteriophage, which was previously isolated from sewage and which attaches to the K1 capsular antigen, has been used to prevent septicemia and a meningitis-like infection in chickens caused by a K1+ bacteremic strain of Escherichia coli . Protection was obtained even when administration of the phage was delayed until signs of disease appeared . The phage was able to multiply in the blood . In newly borne colostrum-deprived calves given the E . coli orally, intramuscular inoculation of phage delayed appearance of the bacterium in the blood and lengthened life span . With some provisos there is considerable potential for this approach to bacterial-disease therapy. Br Med Bull, 1998, 54(1), 195 - 205 Current regimens for treatment of Helicobacter pylori infection; Harris A; The aim of treatment of Helicobacter pylori is eradication of the bacterium from the foregut . Treatment is difficult because of the bacterium's habitat and acquired resistance to commonly used antibiotics . Dual therapy, the 2 week combination of omeprazole or ranitidine bismuth citrate and either amoxycillin or clarithromycin, eradicates H . pylori in 50-80% of patients . Classical triple therapy is commonly associated with side effects, is highly dependent on patient's compliance, and is significantly less effective in the presence of metronidazole-resistant strains of H . pylori, where eradication may be 50% . One week, twice daily, proton pump inhibitor (PPI)-based triple therapy regimens eradicate about 90% of H . pylori and are associated with mild side effects . Second line regimens include 7 days treatment with omeprazole and 3 times daily amoxycillin and metronidazole or a PPI-based quadruple therapy regimen . In some cases, the bacterium defeats all attempts at eradication. Br Med Bull, 1998, 54(1), 55 - 62 Clinical science of Helicobacter pylori infection: ulcers and NSAIDs; Calam J; It is now clear that Helicobacter pylori is a major aetiological factor in peptic ulcer disease . About 95% of patients with duodenal ulcers and perhaps 80% of patients with gastric ulcers are infected with this bacterium and its eradication greatly diminishes recurrence of these ulcers . Many patients are still not receiving the benefit of this treatment, however . Most patients who have peptic ulcers without H . pylori infection are taking non-steroidal anti-inflammatory drugs (NSAIDs), although this may not be recognised or admitted . Such patients have to be managed by withdrawal of these agents or by giving protective agents such as misoprostol . If H . pylori is present in a patient who develops an ulcer whilst taking NSAIDs, it remains unclear whether it is beneficial to eradicate the infection, but this seems advisable in case the ulcer is due to the infection in that particular patient. Microb Pathog, 1998 Mar, 24(3), 133 - 43 Identification of iron-regulated proteins of Mycobacterium tuberculosis and cloning of tandem genes encoding a low iron-induced protein and a metal transporting ATPase with similarities to two-component metal transport systems; Calder KM et al.; Iron plays a central role in the pathogenesis of Mycobacterium tuberculosis, the principal causative agent of tuberculosis . To learn more about iron acquisition by this bacterium, its iron regulated proteins (IRPs) were investigated . Seven IRPs were identified - three increased by high iron concentrations, and four by low iron concentrations . The smallest protein induced by low iron, Irp10, is tightly iron regulated as it is virtually absent in bacteria cultured in the presence of high iron concentrations . The gene (irpA ) encoding this protein and an adjacent open reading frame, mtaA, were cloned and sequenced . The protein encoded by mtaA (Mta72) has striking homology to metal transporting P-type ATPases . This study suggests that Irp10 and Mta72 function as a two-component metal transport system in M . tuberculosis . Infect Immun, 1998 Jun, 66(6), 2514 - 20 Protein kinase A-mediated inhibition of gamma interferon-induced tyrosine phosphorylation of Janus kinases and latent cytoplasmic transcription factors in human monocytes by Ehrlichia chaffeensis; Lee EH et al.; Ehrlichia chaffeensis, an obligatory intracellular bacterium of monocytes or macrophages, is the etiologic agent of human monocytic ehrlichiosis . Our previous study showed that gamma interferon (IFN-gamma) added prior to or at early stage of infection inhibited infection of human monocytes with E . chaffeensis; however, after 24 h of infection, IFN-gamma had no antiehrlichial effect . To test whether ehrlichial infection disrupts Janus kinase (Jak) and signal transducer and activator of transcription (Stat) signaling induced by IFN-gamma, tyrosine phosphorylation of Stat1, Jak1, and Jak2 in E . chaffeensis-infected THP-1 cells was examined by immunoprecipitation followed by immunoblot analysis . Viable E . chaffeensis organisms blocked tyrosine phosphorylation of Stat1, Jak1, and Jak2 in response to IFN-gamma within 30 min of infection . Similar results were obtained with human peripheral blood monocytes infected with E . chaffeensis . Heat or proteinase K treatment but not periodate treatment of E . chaffeensis abrogated the inhibitory effect, suggesting that protein factor(s) of E . chaffeensis is responsible for the inhibition of IFN-gamma-induced tyrosine phosphorylation . Preincubation of E . chaffeensis with the Fab fragment of dog anti-E . chaffeensis immunoglobulin G also abrogated the inhibitory effect . On the other hand, monodansylcadaverine, which does not block binding but blocks internalization of ehrlichiae into macrophages, did not have any influence on the tyrosine phosphorylation . These results indicate that ehrlichial binding to host cells is sufficient to inhibit Stat1 tyrosine phosphorylation induced by IFN-gamma . Protein kinase A (PKA) activity in THP-1 cells increased approximately 25-fold within 30 min of infection with E . chaffeensis . In THP-1 cells pretreated with a PKA inhibitor, Rp isomer of adenosine 3',5'-cyclic phosphorothioate, E . chaffeensis-induced inhibition of Stat1 tyrosine phosphorylation was partially abrogated . These results suggest that E . chaffeensis blocks IFN-gamma-induced tyrosine phosphorylation of Jak and Stat through raising PKA activity in THP-1 cells, which may be an important survival mechanism of ehrlichiae within the host cell. EMBO J, 1998 Apr 15, 17(8), 2166 - 76 A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells; Knutton S et al.; Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, employ a type III secretion system to deliver effector proteins across the bacterial cell . In EPEC, four proteins are known to be exported by a type III secretion system_EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor (Tir) protein (formerly Hp90) which is tyrosine-phosphorylated following transfer to the host cell to become a receptor for intimin-mediated intimate attachment and 'attaching and effacing' (A/E) lesion formation . The structural basis for protein translocation has yet to be fully elucidated for any type III secretion system . Here, we describe a novel EspA-containing filamentous organelle that is present on the bacterial surface during the early stage of A/E lesion formation, forms a physical bridge between the bacterium and the infected eukaryotic cell surface and is required for the translocation of EspB into infected epithelial cells. J Clin Microbiol, 1998 Apr, 36(4), 1015 - 9 Culture-confirmed reinfection of a person with different strains of Borrelia burgdorferi sensu stricto; Golde WT et al.; In recent years, the utility of serum-based diagnostic testing for Lyme disease has improved substantially; however, recovery by culture of the bacterium from skin biopsies of suspected patients is still the only definitive laboratory test . Reinfection of patients has been assumed to occur but as yet has not been documented by serial isolates from the same person . We present a case of culture-confirmed reinfection of a patient in Menominee County, Michigan . Borrelia burgdorferi was isolated from the skin punch biopsy specimens during each episode of erythema migrans (EM) and was subjected to molecular strain typing, genetic analysis of two outer surface protein genes, protein profile analysis, and serum antibody response testing . Results show that these isolates are distinct strains of the bacterium and that the two episodes of EM were caused by independent infections . This report describes the documented, culture-confirmed reinfection of a human by two different strains of B . burgdorferi. Gene, 1998 Mar 27, 210(1), 151 - 61 Topoisomerase I of Helicobacter pylori: juxtaposition with a flagellin gene (flaB) and functional requirement of a fourth zinc finger motif; Suerbaum S et al.; Cloning and nucleotide sequence analysis showed that in Helicobacter pylori the gene encoding topoisomerase I (topA) lies about 170 nucleotides upstream from flaB, a gene encoding one of the two flagellin proteins that is required for virulence . The topA and flaB genes are divergently transcribed . The orientation and spatial relationship between flaB and topA are remarkably conserved among strains of a bacterium in which genomic rearrangements are common . The deduced amino acid sequence of topoisomerase I revealed four zinc finger motifs, one more than has been reported previously for the Escherichia coli homologue . The additional motif, which is near the C-terminus of the protein, appears to be essential for function since mutations in that region are lethal . These data show that TopA proteins can be divided into several classes on the basis of zinc finger motifs and raise the interesting possibility that the H . pylori enzyme has local topological effects focussed on a flagellin gene. Mol Microbiol, 1998 Apr, 28(1), 25 - 35 ClpX and ClpP are essential for the efficient acquisition of genes specifying type IA and IB restriction systems; Makovets S et al.; Efficient acquisition of genes that encode a restriction and modification (R-M) system with specificities different from any already present in the recipient bacterium requires the sequential production of the new modification enzyme followed by the restriction activity in order that the chromosome of the recipient bacterium is protected against attack by the restriction endonuclease . We show that ClpX and ClpP, the components of ClpXP protease, are necessary for the efficient transmission of the genes encoding EcoKI and EcoAI, representatives of two families of type I R-M systems, thus implicating ClpXP in the modulation of restriction activity . Loss of ClpX imposed a bigger barrier than loss of ClpP, consistent with a dual role for ClpX, possibly as a chaperone and as a component of the ClpXP protease . Transmission of genes specifying EcoKI was more dependent on ClpX and ClpP than transmission of the genes for EcoAI . Sensitivity to absence of the protease was also influenced by the mode of gene transfer; conjugative transfer and transformation were more dependent on ClpXP than transduction . In the absence of either ClpX or ClpP transfer of the EcoKI genes by P1-mediated transduction was impaired, transfer of the EcoAI genes was not. Mol Microbiol, 1998 Apr, 28(1), 15 - 24 Cytochrome c biogenesis is involved in the transposon Tn5-mediated bleomycin resistance and the associated fitness effect in Escherichia coli; Adam E et al.; The transposon Tn5 ble gene and the Escherichia coli alkylation-inducible aidC locus are co-operatively involved in the resistance to the anti-cancer drug and DNA-cleaving agent bleomycin and enhance fitness of bacteria in the absence of the drug . In this report, we demonstrate that the aidC locus is identical to nrfG, the last gene of the nrf operon involved in the periplasmic formate-dependent nitrite reduction . In the presence of Ble, NrfG expression is specifically induced and restores both bleomycin resistance and its associated beneficial growth effect in an aidC- strain . In vitro DNA protection assays reveal that purified Ble prevents bleomycin-mediated DNA breakage, as do bleomycin-binding proteins . Similarities between haems of the cytochrome c biogenesis nrf pathway and iron bleomycin suggest a DNA repair-independent molecular mechanism for both bleomycin resistance and increased viability . The Ble protein binds bleomycin and prevents DNA breakage . It also induces the nrf locus that may assimilate bleomycin into haem for extracellular transport or inactivate bleomycin . Inactivation of potent DNA oxidants confers a better fitness to the bacterium carrying the transposon, suggesting a symbiotic relationship between host and transposon. J Infect Dis, 1998 May, 177(5), 1326 - 31 Inhibition of Chlamydia pneumoniae replication in HEp-2 cells by interferon-gamma: role of tryptophan catabolism; Mehta SJ et al.; Interferon-gamma (IFN-gamma) induces tryptophan catabolism in HEp-2 cells, possibly via stimulation of host cell indoleamine-2,3-dioxygenase activity, in a dose-dependent (12.5-1600 U/mL) fashion after 24 h, resulting in a 99% conversion to its metabolites at 1600 U/mL . Replication of Chlamydia pneumoniae isolates A-03 and BAL-16 was inhibited in HEp-2 cells following treatment with 50 and 100 U/mL IFN-gamma, respectively; however, addition of excess L-tryptophan (200 microg/mL) to monolayers infected with C . pneumoniae resulted in unrestricted growth of both isolates up to 1600 U/mL IFN-gamma . C . pneumoniae could be recovered from IFN-gamma-treated monolayers, indicating the potential for this bacterium to undergo an altered life cycle, in vitro, analogous to that described in detail for Chlamydia trachomatis . The ability of C . pneumoniae to persist in host tissue despite an immunologic response would be an important attribute in order to cause or exacerbate chronic infections. Nature, 1998 May 7, 393(6680), 85 - 8 Receptor clustering as a cellular mechanism to control sensitivity; Bray D et al.; Chemotactic bacteria such as Escherichia coli can detect and respond to extremely low concentrations of attractants, concentrations of less than 5 nM in the case of aspartate . They also sense gradients of attractants extending over five orders of magnitude in concentration (up to 1 mM aspartate) . Here we consider the possibility that this combination of sensitivity and range of response depends on the clustering of chemotactic receptors on the surface of the bacterium . We examine what will happen if ligand binding changes the activity of a receptor, propagating this change in activity to neighbouring receptors in a cluster . Calculations based on these assumptions show that sensitivity to extracellular ligands increases with the extent of spread of activity through an array of receptors, but that the range of concentrations over which the array works is severely diminished . However, a combination of low threshold of response and wide dynamic range can be attained if the cell has both clusters and single receptors on its surface, particularly if the extent of activity spread can adapt to external conditions . A mechanism of this kind can account quantitatively for the sensitivity and response range of E . coli to aspartate. Circulation, 1998 May 5, 97(17), 1675 - 9 Association of virulent Helicobacter pylori strains with ischemic heart disease; Pasceri V et al.; BACKGROUND: Previous studies have reported an association between chronic Helicobacter pylori infection and ischemic heart disease . However, it is not clear whether this association is really due to the virulence of the bacterium or is merely the result of confounding factors (in particular, age and social class) . METHODS AND RESULTS: We assessed the prevalence of infection by Helicobacter pylori and by strains bearing the cytotoxin-associated gene-A (CagA), a strong virulence factor, in 88 patients with ischemic heart disease (age, 57+/-8 years; 74 men) and in 88 age- and sex-matched controls (age, 57+/-8 years; 74 men) with similar social background . Prevalence of Helicobacter infection was significantly higher in patients than in controls (62% versus 40%; P=.004), with an odds ratio of 2.8 (95% CI, 1.3 to 7.4; P<.001) adjusted for age, sex, main cardiovascular risk factors, and social class . Patients with ischemic heart disease also had a higher prevalence of CagA-positive strains (43% versus 17%; P=.0002), with an adjusted odds ratio of 3.8 (95% CI, 1.6 to 9.1; P<.001) . Conversely, prevalence of CagA-negative strains was similar in patients and controls (19% versus 23%), with an adjusted odds ratio of 0.8 (95% CI, 0.4 to 1.4) . CONCLUSIONS: The association between Helicobacter pylori and ischemic heart disease seems to be due to a higher prevalence of more virulent Helicobacter strains in patients . These results support the hypothesis that Helicobacter pylori may influence atherogenesis through low-grade, persistent inflammatory stimulation. Electrophoresis, 1998 Apr, 19(4), 551 - 3 Illuminating the agent of syphilis: the Treponema pallidum genome project; Norris SJ et al.; As the causative agent of the common sexually transmitted disease syphilis and a fastidious, microaerophilic obligate parasite of humans, Treponema pallidum subsp . pallidum is one of the few prominent infectious agents that has not been cultured continuously in vitro . T pallidum therefore represents an attractive candidate for genomic sequencing . Preliminary sequence results from the 1.13 million base pair genome are consistent with the expected limited metabolic capabilities of this spirochete, but indicate that the bacterium may express toxins and surface proteins that have not been identified previously. Arch Biochem Biophys, 1998 Apr 15, 352(2), 193 - 8 Dicyclohexylcarbodiimide inhibits proton pumping in ubiquinol:cytochrome c oxidoreductase of Rhodobacter sphaeroides and binds to aspartate-187 of cytochrome b; Wang Y et al.; In recent studies we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in ubiquinol:cytochrome c oxidoreductase (cytochrome bc1 complex) from yeast mitochondria where it was bound to aspartate-160 of cytochrome b . In the current study, we report that DCCD and its fluorescent analogue, N-cyclohexyl-N'-{4-(dimethylamino)naphthyl}-carbodiimide (NCD-4), inhibit 50-60% proton pumping in the cytochrome bc1 complex of the bacterium Rhodobacter sphaeroides with a 20% inhibition of electron transfer activity . Radioactive DCCD is bound exclusively to cytochrome b at aspartate-187, which is located at the C-terminal region of the CD loop connecting membrane-spanning helices C and D of cytochrome b . Fluorescent studies with NCD-4 revealed that aspartate-187 is located in a mildly hydrophobic pocket in the bc1 complex at a distance of 2-3 A from the surface of the membrane. Nucleic Acids Symp Ser, 1997, (37), 297 - 8 Characteristic distribution of bases and codons around the initiation and termination codons in whole reading frames in bacteria and yeast genomes; Watanabe H et al.; Recently the complete nucleotide sequence of the entire genome was determined for yeast and a few kinds of bacterium . To see characteristic features of base sequence in the cistron (actually the open reading frame, ORF) and in the regions around a cistron (ORF), the biases of appearance frequency of bases from the base ratio were studied statistically . In the regions before the base biases were observed . The characteristic base distribution patterns were similar to all the cases of bacteria, but different from yeast . The base biases are reflected on the appearance frequency of amino acids near the N-termini and C-termini of proteins . The characteristic biases found in the amino acid sequence of the N-terminal part of bacteria proteins are different from that in yeast proteins. Eur J Gastroenterol Hepatol, 1998 Mar, 10(3), 239 - 41 Non-pylori Helicobacter infections in humans; Kusters JG et al.; The spectrum of human non-pylori Helicobacter infections is expanding . Evidence for the presence of bacteria such as H . heilmannii, H . felis, H . rappini, H . cinaedi, H . fennelliae and H . pullorum has been reported . These bacteria are likely to be associated with different clinical disorders . H . heilmannii is the most commonly described non-pylori Helicobacter in humans . Colonization with this bacterium is usually associated with mild gastritis . In some cases, gastric ulcer disease may occur . H . heilmannii are classified as such on the basis of morphological criteria . Recent phenotypical and genotypical data suggest that this is insufficient . Therefore, for a better understanding of the relation between non-pylori Helicobacter species and disease, there is a need for studies focusing on genetic instead of morphological criteria. Acta Biochim Pol, 1997, 44(4), 849 - 52 A novel restriction endonuclease UnbI, a neoschizomer of Sau96I from an unidentified psychrofilic bacterium from Antarctica is inhibited by phosphate ions; Kawalec M et al.; A novel type II restriction endonuclease UnbI was isolated from an unidentified psychrofilic bacterial strain from Antarctica . UnbI recognizes and cleaves the sequence 5'-GGNCC-3', producing 5 nucleotide long sticky ends . In this respect it differs from its neoschizomer Sau96I and all other restriction enzymes recognizing this sequence . UnbI has a relatively low temperature optimum of 15 degrees C to 20 degrees C and its activity is completely inhibited by inorganic phosphate. Biochemistry, 1998 Apr 14, 37(15), 5046 - 51 A permanent hole burning study of the FMO antenna complex of the green sulfur bacterium Prosthecochloris aestuarii; Franken EM et al.; A permanent hole burning study on the Fenna-Matthews-Olson, or FMO, antenna complex of the green sulfur bacterium Prosthecochloris aestuarii was carried out at 6 K . Excitation resulted not only in relatively sharp features resonant with the burn wavelength but also in broad absorbance changes in the wavelength region of 800-820 nm . The shape of the latter changes was almost independent of the wavelength of excitation . Evidence is given that they are induced by a different mechanism than that which causes the resonant holes and that they may be due to a conformational change of the protein . The original spectrum was restored upon warming to 60 K . The effective dephasing times T2, as obtained from the homogeneous line widths, increased from about 0.5 ps at 803 nm to >/=20 ps at 830 nm and are in good agreement with recent measurements of accumulated photon-echo and time-resolved absorbance changes. J Bacteriol, 1998 May, 180(9), 2373 - 8 Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis; Braig HR et al.; The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects . It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts . The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined . In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it . The functionality of the wsp promoter region was also successfully tested in Escherichia coli . Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability . This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects . As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host. J Bacteriol, 1998 May, 180(9), 2330 - 6 2-Ketocyclohexanecarboxyl coenzyme A hydrolase, the ring cleavage enzyme required for anaerobic benzoate degradation by Rhodopseudomonas palustris; Pelletier DA et al.; 2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria . This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography . The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids . The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins . Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration . This indicates that the native form of the enzyme is a homotetramer . The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 micromol min(-1) mg of protein(-1) . Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate . An R . palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate . These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R . palustris. Infect Immun, 1998 May, 66(5), 2107 - 14 Expansion of Vgamma9 Vdelta2 T cells is triggered by Francisella tularensis-derived phosphoantigens in tularemia but not after tularemia vaccination; Poquet Y et al.; Tularemia is a disease caused by the facultative intracellular bacterium Francisella tularensis . Here we demonstrate that during the first weeks of infection, a significant increase in levels of Vgamma9 Vdelta2 cells occurred in peripheral blood: in 13 patients analyzed 7 to 18 days after the onset of disease, these lymphocytes represented, on average, 30.5% of CD3+ cells and nearly 100% of gammadelta+ T cells . By contrast, after vaccination with the live vaccine strain (LVS) of F . tularensis, only a minor increase occurred . Eleven days after vaccination, gammadelta T cells represented an average of 6.7% and Vgamma9 Vdelta2 cells represented an average of 5.3% of T cells, as in control subjects . Since derivatives of nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vgamma9 Vdelta2 cells, this observation prompted an investigation of phosphoantigens in F . tularensis strains . The F . tularensis phosphoantigens triggered in vitro a proliferative response of human Vgamma9 Vdelta2 peripheral blood leukocytes as well as a cytotoxic response and tumor necrosis factor release from a Vgamma9 Vdelta2 T-cell clone . Quantitatively similar phosphoantigenic activity was detected in acellular extracts from two clinical isolates (FSC171 and Schu) and from LVS . Taken together, the chemical nature of the stimulus from the clinical isolates and the significant increase in levels of Vgamma9 Vdelta2 cells in peripheral blood of tularemia patients indicate that phosphoantigens produced by virulent strains of F . tularensis trigger in vivo expansion of gammadelta T cells in tularemia. Appl Environ Microbiol, 1998 May, 64(5), 1983 - 5 A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae; Govan VA et al.; Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae . This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria . PCR was used selectively to amplify specific rRNA gene sequences of M . pluton from pure culture, from crude cell lysates, and directly from infected bee larvae . The PCR primers were designed from M . pluton 16S rRNA sequence data . The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M . pluton by sequencing in both directions . Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested . This method enabled the rapid and specific detection and identification of M . pluton from pure cultures and infected bee larvae. Appl Environ Microbiol, 1998 May 1, 64(5), 1860 - 3 Improved Dechlorinating Performance of Upflow Anaerobic Sludge Blanket Reactors by Incorporation of Dehalospirillum multivorans into Granular Sludge Horber C, Christiansen N, Arvin E, Ahring BK. Dechlorination of tetrachloroethene, also known as perchloroethylene (PCE), was investigated in an upflow anaerobic sludge blanket (UASB) reactor after incorporation of the strictly anaerobic, reductively dechlorinating bacterium Dehalospirillum multivorans into granular sludge . This reactor was compared to the reference 1 (R1) reactor, where the granules were autoclaved to remove all dechlorinating abilities before inoculation, and to the reference 2 (R2) reactor, containing only living granular sludge . All three reactors were fed mineral medium containing 3 to 57 microM PCE, 2 mM formate, and 0.5 mM acetate and were operated under sterile conditions . In the test reactor, an average of 93% (mole/mole) of the effluent chloroethenes was dichloroethene (DCE), compared to 99% (mole/mole) in the R1 reactor . The R2 reactor, with no inoculation, produced only trichloroethene (TCE), averaging 43% (mole/mole) of the effluent chloroethenes . No dechlorination of PCE was observed in an abiotic control consisting of sterile granules without inoculum . During continuous operation with stepwise-reduced hydraulic retention times (HRTs), both the test reactor and the R1 reactor showed conversion of PCE to DCE, even at HRTs much lower than the reciprocal maximum specific growth rate of D . multivorans, indicating that this bacterium was immobilized in the living and autoclaved granular sludge . In contrast, the R2 reactor, with no inoculation of D . multivorans, only converted PCE to TCE under the same conditions . Immobilization could be confirmed by using fluorescein-labeled antibody probes raised against D . multivorans . In granules obtained from the R1 reactor, D . multivorans grew mainly in microcolonies located in the centers of the granules, while in the test reactor, the bacterium mainly covered the surfaces of granules. Appl Environ Microbiol, 1998 May, 64(5), 1688 - 93 Isolation of an amoeba naturally harboring a distinctive Legionella species; Newsome AL et al.; There are numerous in vitro studies documenting the multiplication of Legionella species in free-living amoebae and other protozoa . It is believed that protozoa serve as host cells for the intracellular replication of certain Legionella species in a variety of environmental settings . This study describes the isolation and characterization of a bacterium initially observed within an amoeba taken from a soil sample . In the laboratory, the bacterium multiplied within and was highly pathogenic for Acanthamoeba polyphaga . Extracellular multiplication was observed on buffered charcoal yeast extract agar but not on a variety of conventional laboratory media . A 16S rRNA gene analysis placed the bacterium within the genus Legionella . Serological studies indicate that it is distinct from previously described species of the genus . This report also describes methods that should prove useful for the isolation and characterization of additional Legionella-like bacteria from free-living amoebae . In addition, the characterization of bacterial pathogens of amoebae has significant implications for understanding the ecology and identification of other unrecognized bacterial pathogens. Mol Med Today, 1998 Apr, 4(4), 166 - 73 Vaccines against Chlamydia: approaches and progress; Stagg AJ; Infections of the eye and genital tract with the bacterium Chlamydia trachomatis are a major cause of morbidity worldwide and are costly to treat . Development of a vaccine capable of protecting against infection or severe disease presents special challenges but would be the most effective long-term option for control of chlamydial disease . Progress has been made in understanding protective and pathological immune mechanisms in these infections, and a number of potential vaccine candidates have been developed. J Invertebr Pathol, 1998 May, 71(3), 268 - 70 Screening mollusks for Wolbachia infection; Schilthuizen M et al.; We screened 38 species of mollusks for infection by Wolbachia, a bacterium that is a common endosymbiont in arthropods, where it induces alterations in reproduction . Using a PCR assay, we could not detect the symbiont in any of the samples, indicating that, in mollusks, it might be absent . J Appl Toxicol, 1998 Mar-Apr, 18(2), 117 - 23 Rhein affects arylamine N-acetyltransferase activity in Helicobacter pylori from peptic ulcer patients; Chung JG et al.; Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene and p-aminobenzoic acid were determined in the bacterium Helicobacter pylori collected from peptic ulcer patients . Cytosols or suspensions of H . pylori with or without specific concentrations of rhein co-treatment showed different percentages of 2-aminofluorene and p-aminobenzoic acid acetylation . The data indicate that there was decreased NAT activity associated with increased levels of rhein in H . pylori cytosols . Inhibition of growth studies from H . pylori demonstrated that rhein elicited dose-dependent bacteriostatic activity in H . pylori cultures: i.e . the greater the concentration of rhein, the greater the inhibition of growth to H . pylori . For the cytosol and intact bacteria examination, the apparent values of Km and Vmax were decreased after co-treatment with 40 microM rhein . This report is the first demonstration of rhein inhibition of arylamine N-acetyltransferase activity and rhein inhibition of growth in the bacterium H . pylori. Anticancer Res, 1998 Jan-Feb, 18(1A), 97 - 106 Antitumor activity of novel octalactin A analogs in murine leukemia cells in vitro; Perchellet JP et al.; Octalactin A and B (code names K1 and K2) are eight-membered-ring lactones from a marine bacterium . K1 is reportedly cytotoxic . Since access to this natural product is severely limited, the entire synthesis of K1 has been achieved in K . Buszek's laboratory, and several of its structural and stereochemical analogs (code names K3-K9) have been tested for their ability to prevent murine L1210 leukemic cells from synthesizing macromolecules and growing in vitro . At 50 microM, K1 is inactive and the eight-membered lactone K4, an oxocene, is the only compound found to inhibit tumor cell growth by about 90% in the L1210 system . The long-term inhibition of L1210 cell growth by K4 is concentration dependent (IC50 around 10 microM) and not reversible following drug removal . The delayed and weaker cytotoxic effects of K4 suggest that the inhibition of tumor cell proliferation observed 1-4 days after K4 treatment is not solely caused by drug cytotoxicity . When compared to a spectrum of representative anticancer drugs, higher concentrations of K4 must be used to maximally inhibit tumor cell growth . In contrast to its antiproliferative activity, 50 microM K4 fails to alter the rates of DNA, RNA and protein synthesis in L1210 cells . This discrepancy between the ability of K4 to inhibit macromolecule synthesis and leukemic cell growth suggests that other molecular targets are involved in the antitumor action of this drug . At 50 microM, K4 inhibits the polymerization of purified tubulin by about 45%, and therefore may be a novel microtubule de-stabilizing drug weaker than vincristine . Even though other mechanisms may be involved in its antitumor action, the ability of K4 to partially disrupt microtubule dynamics indirectly suggests that this synthetic oxocene may be a cell cycle-specific anticancer drug that blocks mammalian cells in M-phase. Chemosphere, 1998 Apr, 36(10), 2321 - 35 Effect of selected environmental factors on degradation and mineralization of biaryl compounds by the bacterium Ralstonia pickettii in soil and compost; Hundt K et al.; By varying selected environmental factors, the degradation and mineralization of biaryl compounds by the bacterium Ralstonia pickettii in soil and compost were investigated . An optimized soil moisture and enhanced bioavailability by using the nonionic surfactant Tween 80 were of great importance for the degradation rates of biaryl compounds like biphenyl and 4-chlorobiphenyl by cells of Ralstonia picketti SBUG 290 inoculated into soil . Additionally, degradation of these compounds by the investigated strain in soil was strongly dependent upon the medium of precultivation . Also the influence of temperature and soil pH-value was tested . In contrast to the used soil, the autochthonous flora of the compost seemed to have a higher physiological activity . All investigated compounds (biphenyl, 4-chlorobiphenyl and dibenzofuran) were degraded quickly in compost . Inoculation with the investigated bacterium did not enhance the degradation rates significantly. Int J Syst Bacteriol, 1998 Jan, 48 Pt 1, 311 - 5 Desulfovibrio aespoeensis sp . nov., a mesophilic sulfate-reducing bacterium from deep groundwater at Aspö hard rock laboratory, Sweden; Motamedi M et al.; A sulfate-reducing bacterium, strain Aspo-2, was isolated from granitic groundwater sampled at a depth of 600 m . This and other strains of SRB frequently occur in the deep granitic rock aquifers studied . On the basis of its morphological, physiological and genotypical properties, and its unique habitat, we propose strain Aspo-2 as a new species of the genus Desulfovibrio, Desulfovibrio aespoeensis (DSM 10631T). FEMS Microbiol Lett, 1998 Apr 1, 161(1), 21 - 7 Lack of protection following immunisation with H . pylori outer membrane vesicles highlights antigenic differences between H . felis and H . pylori; Keenan JI et al.; Helicobacter pylori-induced inflammation is associated with the development of gastritis, peptic ulcer disease and gastric cancer in humans . Immunisation against this bacterium would ultimately have a major impact on H . pylori-related disease, notably global gastric cancer rates . To date, several potential H . pylori vaccine candidates have been identified . In this study, the Helicobacter felis/murine model was used to assess the immunogenicity of a previously undescribed H . pylori outer membrane vesicle fraction in immune protection. Genetics, 1998 Apr, 148(4), 1579 - 85 Antimutator mutants in bacteriophage T4 and Escherichia coli; Schaaper RM; Antimutators are mutant strains that have reduced mutation rates compared to the corresponding wild-type strain . Their existence, along with mutator mutants that have higher mutation rates compared to the wild-type strain, are powerful evidence that mutation rates are genetically controlled . Compared to mutator mutants, antimutators have a very distinguishing property . Because they prevent normally occurring mutations, they, uniquely, are capable of providing insight into the mechanisms of spontaneous mutations . In this review, antimutator mutants are discussed in bacteriophage T4 and the bacterium Escherichia coli, with regard to their properties, possible mechanisms, and implications for the sources of spontaneous mutations in these two organisms. J Rheumatol, 1998 Apr, 25(4), 734 - 42 Synovial Chlamydia trachomatis in patients with reactive arthritis/Reiter's syndrome are viable but show aberrant gene expression; Gerard HC et al.; OBJECTIVE: We used reverse transcription-polymerase chain reaction (RT-PCR) assays to assess expression of genes from Chlamydia trachomatis in synovial tissues of patients with reactive arthritis (ReA)/Reiter's syndrome (RS) to determine viability/metabolic activity of the bacterium in joints of infected patients . METHODS: Synovial biopsies were obtained from 18 patients with ReA, RS, or other arthritides; nucleic acids from 16 samples were PCR positive for chlamydial chromosomal DNA . RT-PCR assays targeting primary transcripts from C . trachomatis rRNA operons, and mRNA from the bacterial omp1, hsp60, glyQS, and r-protein S5 and L5 genes, were used to characterize viability/metabolic activity . Host actin mRNA was assessed as control in each sample preparation . RESULTS: RT-PCR of host cell actin mRNA in the 18 patient samples confirmed the quality of all RNA preparations . RNA from 14/16 PCR positive samples was positive by RT-PCR for chlamydial rRNA primary transcripts . Each of these same 14 samples was also RT-PCR positive in assays targeting glyQS, r-protein S5 and L5, and hsp60 mRNA . However, none of the 14 samples showing chlamydial rRNA and mRNA was positive for omp1 transcripts . CONCLUSION: Synovial chlamydia are viable/metabolically active, since primary rRNA transcripts and mRNA from chlamydial genes specifying components of the bacterial protein synthetic system were present in most patient samples assayed . Expression of omp1, encoding the major outer membrane protein, is strongly attenuated in persistently infecting synovial chlamydia, while that of hsp60, specifying a highly immunogenic heat shock protein of the organism, is not downregulated. Plant Cell Physiol, 1998 Feb, 39(2), 144 - 52 Identification of a novel triterpenoid saponin from Pisum sativum as a specific inhibitor of the diguanylate cyclase of Acetobacter xylinum; Ohana P et al.; A specific and highly potent inhibitor of diguanylate cyclase, the key regulatory enzyme of the cellulose synthesizing apparatus in the bacterium Acetobacter xylinum, was isolated from extracts of etiolated pea shoots (Pisum sativum) . The inhibitor has been purified by a multistep procedure, and sufficient amounts of highly purified compound (3-8 mg) for spectral analysis were obtained . The structure of this compound was established as 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1--> 2)-beta-D-glucuronopyranosyl soyasapogenol B 22-O-alpha-D-glucopyranoside . The structure was elucidated on the basis of susceptibility to various enzymes, chemical and spectral methods, such as GC-MS, FAB-MS, and the following types of 2D-NMR: COSY, ROESY, TOCSEY, HMQC, HMBC analyses . An identical or a very similar compound with identical biological activity was also isolated from A . xylinum, strongly suggesting that at least certain aspects of cellulose synthesis in the bacteria and in higher plants may be regulated in a similar manner . The content of this saponin in etiolated plants was about 0.04 mumol (g fresh tissue)-1. Biochem Genet, 1997 Dec, 35(11-12), 383 - 93 Allozyme polymorphism and geographic variation in the small brown planthopper, Laodelphax striatellus (Homoptera: Delphacidae); Hoshizaki S; The small brown planthopper, Laodelphax striatellus, immigrates annually into Japan over the East China Sea from the Asian mainland . It is not known whether this long-distance dispersal has any effect on the genetic structure of Japanese L . striatellus populations . The dispersal of L . striatellus is suspected to be relevant to the population dynamics of infection with the parasitic bacterium Wolbachia, which causes cytoplasmic incompatibility in L . striatellus . Wolbachia infection has spread within and among Japanese L . striatellus populations due to this cytoplasmic incompatibility . In the present study, the geographic differences among II L . striatellus populations from Japan and Taiwan was investigated using allozyme polymorphism . FST values on three enzyme loci (GPI, PGM, and AK) indicated a geographically differentiated population structure . Significant differentiation was found even among populations located along the course of the long-distance dispersal . The results indicated that long-range dispersal of L . striatellus does not occur regularly over the main islands of Japan and that it does not have a large effect on the population structure of L . striatellus . This conclusion is in agreement with the geographically variable life history of L . striatellus adapted to local climates . The short-term rice stripe epidemic, which is vectored by L . striatellus, in northwestern Kyushu, Japan, during 1985 and 1986 corresponds to these results . Based on the present findings, short-distance dispersal was considered to drive the spatial spread of Wolbachia infection among L . striatellus populations. J Bacteriol, 1998 Apr, 180(8), 2118 - 24 Transformation of Rickettsia prowazekii to rifampin resistance; Rachek LI et al.; Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell . The absence of techniques for genetic manipulation hampers the study of this organism's unique biology and pathogenic mechanisms . To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic exchange in R . prowazekii . Comparison of the rpoB sequences from the rifampin-sensitive (Rifs) Madrid E strain and a rifampin-resistant (Rifr) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of the R . prowazekii RNA polymerase beta subunit . A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rifs Madrid E strain of R . prowazekii by electroporation, and in the presence of rifampin, resistant rickettsiae were selected . Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rifr region of rickettsial rpoB . This is the first successful demonstration of genetic transformation of Rickettsia prowazekii and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen. Structure, 1998 Mar 15, 6(3), 377 - 88 The structure of the Escherichia coli phosphotransferase IIAmannitol reveals a novel fold with two conformations of the active site; van Montfort RL et al.; BACKGROUND: The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) catalyses the cellular uptake and subsequent phosphorylation of carbohydrates . Moreover, the PTS plays a crucial role in the global regulation of various metabolic pathways . The PTS consists of two general proteins, enzyme I and the histidine-containing protein (HPr), and the carbohydrate-specific enzyme II (EII) . EIIs are usually composed of two cytoplasmic domains, IIA and IIB, and a transmembrane domain, IIC . The IIA domains catalyse the transfer of a phosphoryl group from HPr to IIB, which phosphorylates the transported carbohydrate . Knowledge of the structures of the IIA proteins may provide insight into the mechanisms by which the PTS couples phosphorylation reactions with carbohydrate specificity . RESULTS: We have determined the crystal structure of the Escherichia coli mannitol-specific IIA domain, IIAmtl (M(r) 16.3 kDa), by multiple anomalous dispersion analysis of a selenomethionine variant of IIAmtl . The structure was refined at 1.8 A resolution to an R factor of 19.0% (Rfree 24.2%) . The enzyme consists of a single five-stranded mixed beta sheet, flanked by helices on both sides . The phosphorylation site (His65) is located at the end of the third beta strand, in a shallow crevice lined with hydrophobic residues . The sidechains of two conserved active-site residues, Arg49 and His111, adopt two different conformations in the four independent IIAmtl molecules . Using a solution structure of phosphorylated HPr, and a combination of molecular modelling and NMR binding experiments, structural models of the HPr-IIAmtl complex were generated . CONCLUSIONS: The fold of IIAmtl is completely different from the structures of other IIA proteins determined so far . The two conformations of Arg49 and His111 might represent different states of the active site, required for the different phosphoryl transfer reactions in which IIAmtl is involved . A comparison of the HPr-IIAmtl model with models of HPr in complex with other IIA enzymes shows that the overall interaction mode between the two proteins is similar . Differences in the stabilisation of the invariant residue Arg17 of HPr by the different IIA proteins might be part of a subtle mechanism to control the hierarchy of carbohydrate utilisation by the bacterium. Structure, 1998 Mar 15, 6(3), 351 - 61 Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium; Russell RJ et al.; BACKGROUND: The structural basis of adaptation of enzymes to low temperature is poorly understood . Dimeric citrate synthase has been used as a model enzyme to study the structural basis of thermostability, the structure of the enzyme from organisms living in habitats at 55 degrees C and 100 degrees C having previously been determined . Here the study is extended to include a citrate synthase from an Antarctic bacterium, allowing us to explore the structural basis of cold activity and thermostability across the whole temperature range over which life is known to exit . RESULTS: We report here the first crystal structure of a cold-active enzyme, citrate synthase, isolated from an Antarctic bacterium, at a resolution of 2.09 A . In comparison with the same enzyme from a hyperthermophilic host, the cold-active enzyme has a much more accessible active site, an unusual electrostatic potential distribution and an increased relative flexibility of the small domain compared to the large domain . Several other features of the cold-active enzyme were also identified: reduced subunit interface interactions with no intersubunit ion-pair networks; loops of increased length carrying more charge and fewer proline residues; an increase in solvent-exposed hydrophobic residues; and an increase in intramolecular ion pairs . CONCLUSIONS: Enzymes from organisms living at the temperature extremes of life need to avoid hot or cold denaturation yet maintain sufficient structural integrity to allow catalytic efficiency . For hyperthermophiles, thermal denaturation of the citrate synthase dimer appears to be resisted by complex networks of ion pairs at the dimer interface, a feature common to other hyperthermophilic proteins . For the cold-active citrate synthase, cold denaturation appears to be resisted by an increase in intramolecular ion pairs compared to the hyperthermophilic enzyme . Catalytic efficiency of the cold-active enzyme appears to be achieved by a more accessible active site and by an increase in the relative flexibility of the small domain compared to the large domain. Biochim Biophys Acta, 1998 Mar 25, 1363(3), 199 - 208 Redox properties of an H-subunit-depleted photosynthetic reaction center from Rhodopseudomonas viridis; Hara M et al.; Recently, we reported that a H-subunit-depleted photosynthetic reaction center (RC-H) was purified from purple nonsulfer photosynthetic bacterium Rhodopseudomonas viridis (Rps . viridis) using a strong detergent sodium alkyl ether sulfate . We compared the redox properties of a native photosynthetic reaction center (RC) and RC-H of Rps . viridis . In RC-H prepared by our method, secondary quinone (QB) was removed while primary quinone (QA) was retained . Absorption spectrum of RC-H was similar to that of RC . After reconstitution of ubiquinone 10 into QB sites, RC-H showed electron transfer activity that was the same as that for native RC . This is the first report about the redox properties of RC-H of Rps . viridis . Curr Microbiol, 1998 Mar, 36(3), 125 - 30 Evidence for arylamine N-acetyltransferase activity in the Escherichia coli; Chang FC et al.; N-Acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene as substrates were determined in isolates of the bacterium Escherichia coli . The N-acetyltransferase activity was determined by an acetyl CoA recycling assay and high pressure liquid chromatography . The N-acetyltransferase activities from a number of E . coli isolates were found to be 0.67 +/- 0.04 nmole/min/mg protein for 2-aminofluorene, and 0.46 +/- 0.02 nmole/min/mg protein for p-aminobenzoic acid . The apparent Km and Vmax values obtained were 2 . 85 +/- 0.65 mM and 7.51 +/- 0.86 nmol/min/mg protein, respectively, for 2-aminofluorene, and 2.35 +/- 0.39 mM and 9.43 +/- 0.78 nmol/min/mg protein, respectively, for p-aminobenzoic acid . The optimal pH value for the enzyme activity was 7.0 for both substrates tested . The optimal temperature for enzyme activity was 37 degrees C for both substrates . The N-acetyltransferase activity was inhibited by iodoacetamide: at 0.25 mM iodoacetamide, activity was reduced 50%, and at 1.0 mM, more than 90% . Among a series of divalent cations and salts, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors . This report is the first demonstration of acetyl CoA:arylamine N-acetyltransferase activity in E . coli. Genes Dev, 1998 Mar 15, 12(6), 880 - 93 Cell cycle-dependent transcriptional and proteolytic regulation of FtsZ in Caulobacter; Kelly AJ et al.; In the differentiating bacterium Caulobacter crescentus, the cell division initiation protein FtsZ is present in only one of the two cell types . Stalked cells initiate a new round of DNA replication immediately after cell division and contain FtsZ, whereas the progeny swarmer cells are unable to initiate DNA replication and do not contain FtsZ . We show that FtsZ expression is controlled by cell cycle-dependent transcription and proteolysis . Transcription of ftsZ is repressed in swarmer cells and is activated concurrently with the initiation of DNA replication . At the end of the DNA replication period, transcription of ftsZ decreases substantially . We show that the global cell cycle regulator CtrA is involved in the cell cycle control of ftsZ transcription . CtrA binds to a site that overlaps the ftsZ transcription start site . Removal of the CtrA-binding site results in transcription of the ftsZ promoter in swarmer cells . Decreasing the cellular concentration of CtrA increases ftsZ transcription and conversely, increasing the concentration of CtrA decreases ftsZ transcription . Because CtrA is present in swarmer cells, is degraded at the same time as ftsZ transcription begins, and reappears when ftsZ transcription decreases at the end of the cell cycle, we propose that CtrA is a repressor of ftsZ transcription . We show that proteolysis is an important determinant of cell type-specific distribution and cell cycle variation of FtsZ . FtsZ is stable when it is synthesized and assembles into the cytokinetic ring at the beginning of the cell cycle . After the initiation of cell division, the rate of FtsZ degradation increases as both the constriction site and the FtsZ ring decrease in diameter . When ftsZ is expressed constitutively from inducible promoters, the abundance of FtsZ still varies during the cell cycle . The coupling of transcription and proteolysis to cell division ensures that FtsZ is inherited only by the progeny cell that will begin DNA replication immediately after cell division. Scand J Gastroenterol, 1998 Mar, 33(3), 271 - 5 The influence of Helicobacter pylori eradication on the gastric mucosal content of epidermal growth factor, transforming growth factor-alpha, and their common receptor; Russo F et al.; BACKGROUND: The relationship between the expression of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) and that of their receptor (EGF-R) in the Helicobacter pylori-infected gastric mucosa has not been completely elucidated . The aim of this study was to examine the interplay between H . pylori colonization and gastric mucosal growth factor content . METHODS: By means of a solid-phase enzyme-linked immunosorbent assay EGF, TGF-alpha, and EGF-R levels and interleukin-1beta (IL-1beta) content, which is considered a marker of chronic inflammation, were evaluated in the antral mucosa of 24 H . pylori-positive patients before and 8 weeks after eradication therapy . RESULTS: After therapy H . pylori was eradicated in 19 patients . The eradication was accompanied by a significant decrease in IL-1beta content and an increase in EGF and TGF-alpha levels . On the other hand, in the five patients in whom the bacterium was not eradicated EGF, TGF-alpha, and EGF-R levels were quite similar to those assayed before therapy, whereas IL-1beta content was still high . CONCLUSIONS: These results suggest that H . pylori exerts an inhibitory effect on the mucosal expression of EGF and TGF-alpha, which are likely involved in the gastric mucosa repair process. APMIS, 1998 Mar, 106(3), 385 - 8 Evaluation of an immunohistochemical test with polyclonal antibodies raised against mycobacteria used in formalin-fixed tissue compared with mycobacterial specific culture; Carabias E et al.; An immunohistochemical (IH) test (commercially available polyclonal antiserum rabbit anti-Myco-bacterium bovis; DAKO A/S) was used to detect the presence of mycobacteria in 65 formalin-fixed, paraffin-embedded tissue blocks from different organs, showing necrotizing caseous granuloma lesions on hematoxylin and eosin sections from 65 patients . These 65 samples were dyed using an acid-fast fluorescent technique and compared using the immunohistochemical method . Both results were also compared with the mycobacterial cultures . The IH test, compared with the culture, showed a sensitivity (S) of 52%, a specificity (Sp) of 76%, a positive predictive value (PV pos) of 61% and a negative predictive value (PV neg) of 69% . We analyze these data and discuss the possible causes of false-positive and -negative results of the IH test . This rapid test on paraffin embedded tissue seems valuable in the period when waiting for the culture results. Am J Trop Med Hyg, 1998 Mar, 58(3), 305 - 8 Helicobacter pylori serostatus in backpackers following travel to tropical countries; Potasman I et al.; The mode of transmission of Helicobacter pylori is unknown . The seroprevalence of H . pylori and the rate of transmission of feco-oral pathogens in developing countries are both high . Long-term travelers to these regions, who come from developed countries are thus potentially at increased risk of an infection with this bacterium . We studied the H . pylori serology status before and after travel of 104 backpackers who traveled to tropical countries; 76 medical students who did not leave Israel served as controls . Southeast Asia (70%) and South America (24%) were the major destinations, but the area of travel had no effect on the seroconversion rate . The total time spent abroad was 53 person-years . Thirty six of the travelers and 30 controls were positive at the outset . Seropositivity at entry was significantly associated with being a Sepharadic Jew or having a parent with a peptic ulcer disease . The majority of travelers (86.5%) and controls (92.1%) did not change their serostatus . Four travelers seroconverted, but 10 seroreverted, while three controls seroconverted, and three others seroreverted . No significant association with gastroenteritis was found . Serostatus may have been affected by mefloquine use because none of the four seroconverters, but eight of 10 seroreverters used it as malaria prophylaxis . In vitro studies demonstrated that mefloquine has anti-H . pylori activity . Feco-oral transmission is apparently not an important route of transmission of this organism among travelers. Appl Environ Microbiol, 1998 Apr, 64(4), 1532 - 5 Enterohemorrhagic Escherichia coli O157:H7 present in radish sprouts; Itoh Y et al.; Using cultivation, immunofluorescence microscopy, and scanning electron microscopy, we demonstrated the presence of viable enterohemorrhagic Escherichia coli O157:H7 not only on the outer surfaces but also in the inner tissues and stomata of cotyledons of radish sprouts grown from seeds experimentally contaminated with the bacterium . HgCl2 treatment of the outer surface of the hypocotyl did not kill the contaminating bacteria, which emphasized the importance of either using seeds free from E . coli O157:H7 in the production of radish sprouts or heating the sprouts before they are eaten. Appl Environ Microbiol, 1998 Apr, 64(4), 1510 - 3 Extremely barophilic bacteria isolated from the Mariana Trench, Challenger Deep, at a depth of 11,000 meters; Kato C et al.; Two strains of obligately barophilic bacteria were isolated from a sample of the world's deepest sediment, which was obtained by the unmanned deep-sea submersible Kaiko in the Mariana Trench, Challenger Deep, at a depth of 10,898 m . From the results of phylogenetic analysis based on 16S rRNA gene sequences, DNA-DNA relatedness study, and analysis of fatty acid composition, the first strain (DB21MT-2) appears to be most highly similar to Shewanella benthica and close relatives, and the second strain (DB21MT-5) appears to be closely related to the genus Moritella . The optimal pressure conditions for growth of these isolates were 70 MPa for strain DB21MT-2 and 80 MPa for strain DB21MT-5, and no growth was detected at pressures of less than 50 MPa with either strain . This is the first evidence of the existence of an extreme-barophile bacterium of the genus Moritella isolated from the deep-sea environment. Helicobacter, 1998 Mar, 3(1), 1 - 8 How do practicing clinicians manage Helicobacter pylori-related gastrointestinal diseases in Germany? A survey of gastroenterologists and family practitioners; Breuer T et al.; BACKGROUND: Since the bacterium H . pylori was identified in 1982, overwhelming evidence has implicated it as the causal factor in the occurrence and relapse of peptic ulcer disease . The major objective of this study was to examine the extent to which physicians recognize H . pylori as a causal agent in peptic ulcer disease or as potential cofactor in other gastrointestinal diseases, and the extent to which this knowledge has influenced diagnostic and therapeutic practices . MATERIALS AND METHODS: Using a national mail survey in Germany in September 1995, 1197 family practitioners and 1197 gastroenterologists were selected for the study . RESULTS: Of the surveyed physicians, 756 (32%) responded . Family practitioners treated almost 50% of their patients with initial presentation of suspected ulcer disease without ordering further diagnostic tests . More than 25% of the family practitioners and 14% of the gastroenterologists reported that they do not treat diagnosed H . pylori infection in the first presentation of duodenal ulcer . At the time we conducted the study, 22% of responding family practitioners and 5% of responding gastroenterologists treated the first presentation of H . pylori-positive ulcer disease with regimens determined to be ineffective according to the available literature . CONCLUSIONS: Gastroenterologists preferred to treat H . pylori infection when the associated disease was one for which a causal relationship had been more clearly established, while family practitioners showed less discrimination . In order to provide optimal therapy aimed at minimizing the course and consequences of H . pylori-related diseases, researchers in the field must ensure continuous dissemination of current knowledge. Biophys J, 1998 Apr, 74(4), 2069 - 75 Energy transfers in the B808-866 antenna from the green bacterium Chloroflexus aurantiacus; Novoderezhkin VI et al.; Energy transfers within the B808-866 BChl a antenna in chlorosome-membrane complexes from the green photosynthetic bacterium Chloroflexus aurantiacus were studied in two-color pump-probe experiments at room temperature . The steady-state spectroscopy and protein sequence of the B808-866 complex are reminiscent of well-studied LH2 antennas from purple bacteria . B808-->B866 energy transfers occur with approximately 2 ps kinetics; this is slower by a factor of approximately 2 than B800-->B850 energy transfers in LH2 complexes from Rhodopseudomonas acidophila or Rhodobacter sphaeroides . Anisotropy studies show no evidence for intra-B808 energy transfers before the B808-->B866 step; intra-B866 processes are reflected in 350-550 fs anisotropy decays . Two-color anisotropies under 808 nm excitation suggest the presence of a B808-->B866 channel arising either from direct laser excitation of upper B866 exciton components that overlap the B808 absorption band or from excitation of B866 vibronic bands in nontotally symmetric modes. Protein Sci, 1998 Mar, 7(3), 564 - 72 Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor; Aghajari N et al.; Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile . We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution . The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases . AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds . The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264) . These are all involved in firm binding of the Tris moiety . A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation . A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site . We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase. Vet Pathol, 1998 Mar, 35(2), 153 - 6 Specific detection of Lawsonia intracellularis in porcine proliferative enteropathy inferred from fluorescent rRNA in situ hybridization; Boye M et al.; Fluorescent in situ hybridization targeting 16S ribosomal RNA was used for specific detection of the obligate intracellular bacterium Lawsonia intracellularis in enterocytes from pigs affected by proliferative enteropathy . A specific oligonucleotide probe was designed and the specificity of the probe was determined by simultaneous comparison with indirect immunofluorescence assay for detection of L . intracellularis in formalin-fixed tissue samples from 15 pigs affected by porcine proliferative enteropathy . We used 10 tissue samples from pigs without proliferative mucosal changes as negative controls . The results showed that the oligonucleotide probe is specific for L . intracellularis and that fluorescent in situ hybridization targeting ribosomal RNA is a suitable and fast method for specific detection and histological recognition of L . intracellularis in formalin-fixed tissue. Carbohydr Res, 1997 Dec, 305(1), 117 - 22 The production of a new water-soluble polysaccharide by Acetobacter xylinum NCI 1005 and its structural analysis by NMR spectroscopy; Tajima K et al.; A new water-soluble polysaccharide (WSP) was isolated from a culture of Acetobacter xylinum NCI 1005 grown on sucrose . The structure of the WSP was analysed by nuclear magnetic resonance spectroscopy and determined to be a beta-(2-->6)-linked polyfructan, which is structurally different from the polymer synthesized from glucose instead of sucrose by the same strain . The discovery of this new polysaccharide has revealed that the bacterium is able to synthesize two different kinds of water-soluble polysaccharides. Curr Biol, 1998 Feb 26, 8(5), R154 - 7 Bacterial chemotaxis: the five sensors of a bacterium; Grebe TW et al.; The components of the Escherichia coli chemosensory system have been identified and their activities characterized, but how sensory information is processed to give an integrated response remains an open question. Biosci Biotechnol Biochem, 1998 Feb, 62(2), 347 - 53 Molecular cloning and expression of a gene encoding a novel sorbitol oxidase from Streptomyces sp . H-7775; Hiraga K et al.; A gene encoding a novel intracellular sorbitol oxidase of a soil bacterium, Streptomyces sp . H-7775, was cloned and sequenced . The gene consists of an open reading frame of 1,260-bp encoding a protein of 420 amino acids with a molecular weight of 45,148 . Deduced amino acid sequence of the gene has 25.3% identity and 68.1% similarity to that of rat L-gulonolactone oxidase at the overall amino acids . Nucleotide-binding motifs were not found in the deduced amino acid sequence of SOX protein . We succeeded in expressing recombinant sorbitol oxidase with covalently bound FAD in E . coli at about a 4,000-fold higher total enzyme activity than that of the Streptomyces sp . H-7775 . The enzymatic properties of the recombinant SOX were similar to those of the enzyme from Streptomyces sp . H-7775 . This is the first report of the cloning and expression of a newly categorized enzyme, sorbitol oxidase, from Streptomyces sp. FEMS Microbiol Lett, 1998 Mar 15, 160(2), 247 - 52 Segregation pattern of kanamycin resistance marker in Azotobacter vinelandii did not show the constraints expected in a polyploid bacterium; Pulakat L et al.; It was suggested that Azotobacter vinelandii cells contain about 80 copies of their chromosome and when foreign genes are introduced into the cell, it took several generations for them to spread to all 80 chromosomes even in the presence of selection . In contrast, the fact that many recessive mutants can be isolated from A . vinelandii without the constraints expected for a cell that has 80 copies of its chromosome argued against this organism being highly polyploid . We have investigated the segregation of a kanamycin resistant genetic marker under non-selective conditions in A . vinelandii . Plasmid DNA was used to introduce the kanamycin resistance gene onto the A . vinelandii chromosome at the nifY locus by homologous recombination . The transformants were identified from non-transformants with the aid of replica plating, and hence the colonies examined for segregation of the genetic marker were never subjected to kanamycin selection . In spite of growing the transformants in the absence of selection pressure, no segregant that lacked the kanamycin resistance gene was scored . These analyses suggested that the segregation of the kanamycin marker in A . vinelandii did not exhibit any constraints expected in a highly polyploid bacterium. Gastroenterologist, 1998 Mar, 6(1), 16 - 20 Helicobacter pylori and gastric cancer: what is the real risk? Crespi M, Citarda F. Some epidemiological data point to an association between infection from Helicobacter pylori (Hp) and gastric cancer, although several unresolved issues still cast doubts on the real weight of this association . These issues are as follows: the male-to-female ratio of gastric cancer ranges from 4:1 to 1.5:1 in all studies, whereas the prevalence of Hp infection is the same in both sexes; the prevalence of Hp infection is as high as 90% in several developing countries where the frequency of gastric cancer is very low; the acquisition of the infection at a young age, considered very important with regard to the risk for cancer, varies from 4.2% to 83% in several countries in which the mortality for stomach cancer is approximately 10 in 100,000; and the incidence of cancer in patients with a duodenal ulcer is half that of the general population, but Hp infects up to 100% of these patients . In the sequence of events that leads to gastric cancer, Hp appears to play a role only in the very initial steps, as a causative agent of chronic inflammation . The further events that cause gastric atrophy, intestinal metaplasia, dysplasia, and cancer are multifactorial, involving environmental agents and the host response . It is therefore inappropriate to consider Hp a direct carcinogen for humans . This also applies to specific strains of the bacterium such as the cagA gene . In fact, Hp infection is widespread in humans, and only a small minority will ever be affected by peptic ulcer and cancer. Biochim Biophys Acta, 1998 Feb 2, 1369(1), 94 - 102 Lipids in total extracts from Acholeplasma laidlawii A pack more closely than the individual lipids . Monolayers studied at the air-water interface; Andersson AS et al.; Pressure-area curves were obtained at 25, 35 and 45 degrees C for total lipid extracts and four individual glucolipids isolated from Acholeplasma laidlawii strain A-EF22 . The glucolipids are 1,2-diacyl-3-0-(alpha-D-glucopyranosyl)-sn-glycerol (MGlcDAG), 1,2 -diacyl-3-0-{alpha-D-glucopyranosyl-(1-->2)-0-alpha-D-glucopyranosyl} -sn-glycerol (DGlcDAG), 1,2-diacyl-3-0-{alpha-D-glucopyranosyl-(1-->2)-0-(6-0-acyl-alpha-D-gluco pyranosyl)}-sn-glycerol (MADGlcDAG), and 1,2-diacyl-3-0-{glycerophosphoryl-6-0-(alpha-D-glucopyranosyl-(1-- >)-0-alpha-D-glucopyranosyl)}-sn-glycerol (GPDGlcDAG) . The total lipid extracts were obtained from A . laidlawii, grown at 37 degrees C with fatty acids of varying degrees of unsaturation and chain length . The mean surface area per molecule was obtained from these pressure-area curves at surface pressures equal to 10, 20, 30 and 40 mN/m . It was found that the interfacial area of the lipids increases with increasing degree of unsaturation, but is nearly independent of the acyl chain length at constant unsaturation . The surface charge density varied between 4.7 x 10(-3) e-/angstrom(2) and 9.4 x 10(-3) e-/angstrum(2) for the total lipid extracts studied, but did not exhibit any consistent dependence on variations in degree of unsaturation or acyl chain length . The mean area per molecule was found to be smaller for the total lipid extracts than for the individual lipids . It is concluded that the bacterium strives to regulate its lipid composition in such a way that the packing of the lipids in the membrane is appropriately tight, and/or to keep a slight negative spontaneous curvature of the lipid bilayer of the cell membrane ("optimal packing") . This is in accordance with the physico-chemical model for the regulation of the lipid composition in the membrane of A . laidlaiwii previously presented by us (see e.g . Andersson, A.-S., Riffors, L., Bergqvist, M., Persson, S . and Lindblom, G . (1996) Biochemistry 35, 11119-11130). Infect Immun, 1998 Apr, 66(4), 1594 - 600 Maturation of the arginine-specific proteases of Porphyromonas gingivalis W50 is dependent on a functional prR2 protease gene; Aduse-Opoku J et al.; The prpR1 of Porphyromonas gingivalis codes for three distinct enzymes with specificity for arginyl peptide bonds termed RI, RIA, and RIB . These three isoforms comprise the majority of the extracellular, arginine-specific protease activity in P . gingivalis W50 . RI is a heterodimer in which the catalytic alpha chain is noncovalently associated with a second chain involved in adherence phenomena . RIA and RIB are both monomeric species . RIA represents the free alpha chain, and RIB is a highly posttranslationally modified form of the alpha chain which is exclusively vesicle or membrane associated and migrates as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . In previous studies, insertional inactivation of the prpR1 demonstrated that arginine-specific protease activity can also arise from a closely related second gene, prR2 . In the present work, the prR2 was insertionally inactivated in P . gingivalis W50 in order to establish the contribution of this locus to the arginine-specific protease activity of this periodontal bacterium . Loss of prR2 function had several effects on prpR1-derived enzymes . First, the total Arg-X activity was reduced by approximately 50% relative to that of the parent strain . The reduction in total activity was a consequence of decreased concentrations of the monomeric enzymes derived from the prpR1, while the heterodimeric enzyme, RI, was unaffected by this mutation . Second, the chromatographic behavior of both the soluble and vesicle- or membrane-associated monomeric enzymes was radically different from the behavior of RIA and RIB from the parent strain . Finally, the vesicle- or membrane-associated enzyme in the prR2 mutant strain lacked the extensive posttranslational additions which are found on RIB in P . gingivalis W50 . These data suggest that the product(s) of the prR2 plays a significant role in the maturation pathway of prpR1-derived enzymes, and this may contribute to the coconservation of these two genes in P . gingivalis. Infect Immun, 1998 Apr, 66(4), 1570 - 8 Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro; Hicks S et al.; Attaching and effacing (A/E) lesion formation is central to enteropathogenic Escherichia coli (EPEC) pathogenesis . In vitro experiments with human epithelial cell lines have implicated virulence plasmid-encoded bundle-forming pili (BFP) in initial binding and intimin in intimate attachment and A/E lesion formation . This study investigated the role of BFP and intimin in EPEC interactions with pediatric small intestinal biopsy tissue in in vitro organ culture . Organ culture infections (2 to 8 h) were performed with E2348/69 (a wild-type EPEC O127:H6 clinical isolate) and E2348/69 derivatives including CVD206 (eae deficient), CVD206(pCVD438) (eae-complemented CVD206), CVD206(pCVD438/01) (expressing intimin, which is nonfunctional due to a single amino acid substitution), JPN15 (spontaneous EPEC adherence factor virulence plasmid-cured E2348/69), and 31-6-1(1) (E2348/69 with a TnphoA insertion inactivation mutation in the virulence plasmid-encoded bfpA gene) . Scanning and transmission electron microscopy revealed that after 8 h E2348/69 and CVD206 (pCVD438) (both Int+ BFP+) adhered to all specimens, causing A/E lesions with surrounding microvillous elongation . JPN15 and 31-6-1(1) (both Int+ BFP-) adhered and caused A/E lesions although bacteria adhered in "flat," two-dimensional groups . CVD206 and CVD206(pCVD438/01) (both Int- BFP+) did not adhere to any sample, and no pathological tissue changes were seen . Thus, in human intestinal organ culture, BFP do not appear to be involved in the initial stages of EPEC nonintimate adhesion but are implicated in the formation of complex, three-dimensional colonies via bacterium-bacterium interactions . Intimin appears to play an essential role in establishing colonization of EPEC on pediatric small intestinal tissue. Infect Immun, 1998 Apr, 66(4), 1349 - 55 Immune responses of specific-pathogen-free mice to chronic Helicobacter pylori (strain SS1) infection; Ferrero RL et al.; A model permitting the establishment of persistent Helicobacter pylori infection in mice was recently described . To evaluate murine immune responses to H . pylori infection, specific-pathogen-free Swiss mice (n = 50) were intragastrically inoculated with 1.2 x 10(7) CFU of a mouse-adapted H . pylori isolate (strain SS1) . Control animals (n = 10) received sterile broth medium alone . Animals were sacrificed at various times, from 3 days to 16 weeks postinoculation (p.i.) . Quantitative culture of gastric tissue samples from inoculated mice demonstrated bacterial loads of 4.0 x 10(4) to 8 x 10(6) CFU per g of tissue in the animals . Infected mice had H . pylori-specific immunoglobulin M (IgM) and IgG antibodies in serum (at day 3 p.i.) and IgG and IgA antibodies in their gastric contents (weeks 4 and 16 p.i.) and saliva (week 16 p.i.) . Mucosal IgM antibodies were not detected . Histological examination of the gastric mucosae from control and infected mice revealed mild chronic gastritis, characterized by the presence of polymorphoneutrophil cell infiltrates and submucosal lymphoid aggregates, in infected animals at 16 weeks p.i . Differences in the quantities of IgG1 and IgG2a subclass antibodies detected in the sera of mouse strains (Swiss, BALB/c, and C57BL/6) infected by H . pylori suggested that host factors influence the immune responses induced against this bacterium in the host . In conclusion, immune responses to H . pylori infection in mice, like those in chronically infected humans, appear to be ineffective in resolving the infection. Infect Immun, 1998 Apr, 66(4), 1293 - 8 Superoxide dismutase-dependent, catalase-sensitive peroxides in human endothelial cells infected by Rickettsia rickettsii; Hong JE et al.; The generation and intracellular accumulation of reactive oxygen species have been shown to be associated with the infection of human umbilical vein endothelial cells (HUVEC) by Rickettsia rickettsii . In response to the oxidant superoxide, the activity of the enzyme superoxide dismutase (SOD) increases following infection by this obligate intracellular bacterium . Other oxidants which are capable of oxidizing the fluorescent probe 2',7'-dichlorofluorescin (DCFH) also accumulate intracellularly within infected cells . In the study reported here, we show that (i) an inhibitor of SOD, diethyldithiocarbamic acid, reduces the observed rise in SOD activity in infected cells by 40 to 60% and at the same time reduces the degree of intracellular oxidation of DCFH; (ii) catalase-sensitive peroxides can be detected in supernatants of R . rickettsii-infected cells shortly after rickettsial exposure; and (iii) fluorescence-activated cell sorter analysis demonstrates significant intracellular oxidant activity in infected cells within 5 h after exposure to R . rickettsii . The results of these experiments indicate that hydrogen peroxide is a major oxidant associated with infection of HUVEC by R . rickettsii and that intracellular oxidant activity sensitive to SOD inhibition is detectable early and prior to significant rickettsial multiplication and much earlier than the ultrastructural manifestations of cell injury seen by electron microscopy. Eur J Oral Sci, 1998 Feb, 106(1), 576 - 81 LPS from Actinobacillus actinomycetemcomitans and production of nitric oxide in murine macrophages J774; Blix IJ et al.; Nitric oxide (NO) plays a complex role in the modulation of the inflammatory response, having either a pro-inflammatory or a protective role . Actinobacillus actinomycetemcomitans is considered an important etiological agent in localized juvenile periodontitis . We have studied the effect of lipopolysaccharide (LPS) extracted from this periodontopathogenic bacterium on NO synthesis in an in vitro murine macrophage system . LPS from A . actinomycetemcomitans induced a significant production of NO even at concentrations as low as 1 ng/ml, whereas LPS from E . coli had to be added in concentrations of 100 ng/ml to obtain similar effects . Production of NO was blocked by NG-nitro-L-arginine methylester, and pre-treatment of LPS from A . actinomycetemcomitans with polymyxin B abolished the production of NO, while prostaglandin E2 enhanced the synthesis of NO. J Vet Diagn Invest, 1998 Jan, 10(1), 60 - 6 Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmonid ovarian fluid; Pascho RJ et al.; Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR) . On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R . salmoninarum cells . Among the positive fish, the R . salmoninarum concentrations ranged from 25 cells/ml to 4.3 x 10(9) cells/ml . A soluble antigenic fraction of R . salmoninarum was detected in 39% of the fish (40/103) by the ELISA . The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R . salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 x 10(4) cells/ml according to the MF-FAT counts . When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R . salmoninarum, 100% of the 100 samples tested were positive . The results provided strong evidence that R . salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods. Planta Med, 1998 Mar, 64(2), 176 - 8 Aloe-emodin effects on arylamine N-acetyltransferase activity in the bacterium Helicobacter pylori; Wang HH et al.; Arylamine N-acetyltransferase (NAT) activities with p-aminobenzoic acid (PABA) and 2-aminofluorene (AF) were determined in H . pylori collected from peptic ulcer patients . Cytosols or suspensions of H . pylori with or without different concentrations of aloe-emodin co-treatment showed different percentages of AF and PABA acetylation . The data indicate that there was decreased NAT activity associated with increased aloe-emodin in H . pylori cytosols . Inhibition of growth study from H . pylori demonstrated that aloe-emodin elicited dose-dependent growth inhibition in H . pylori cultures . The report is the first finding of aloe-emodin inhibition of arylamine NAT activity in a strain of H . pylori. Am J Gastroenterol, 1998 Mar, 93(3), 306 - 10 Helicobacter pylori--more light, less heat; Moss SF et al.; Several areas of broad agreement exist concerning the management of specific patient groups with clear-cut complications of H . pylori-colonization . Other aspects of this infection remain less well defined . These include the mode of transmission and pathogenesis of H . pylori, the clinical management of patients who do not have ulcer disease, and the approach to populations at risk of the clinical consequences of this bacterium . This review focuses on the unresolved issues of H . pylori infection that are of concern to the clinical gastroenterologist. Arch Microbiol, 1998 Feb, 169(2), 129 - 35 Physiology and tactic response of the phototrophic consortium "Chlorochromatium aggregatum" Frostl JM, Overmann J. The phototrophic consortium "Chlorochromatium aggregatum" was enriched from sediment samples of a eutrophic freshwater lake and was maintained at high numbers in anoxic sulfide-reduced medium . Growth of intact consortia was observed only in the light and in the presence of 2-oxoglutarate as an organic carbon source . Consortia of "C . aggregatum" reached maximum growth rates at light intensities >/= 5 &mgr;mol quanta m-2 s-1 . Of ten compounds tested, sulfide, thiosulfate, 2-oxoglutarate, and citrate served as a chemoattractant for "C . aggregatum" . When incubated in the presence of sulfide and in the light, epibionts reduced the fluorochrome 5-cyano-2, 3-di-4-tolyl-tetrazolium chloride (CTC) . Reduction of CTC was not observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or in the dark, indicating that sulfide serves as an electron donor for the phototrophic epibiont . Motile consortia accumulated scotophobically in microcuvettes at a wavelength of 740 nm . Since this wavelength corresponds to the position of the absorption maximum of bacteriochlorophylls c or d, the photosynthetic pigments are most likely the photoreceptors of the scotophobic response . It is concluded that, within the consortia, a rapid interspecies signal transfer occurs between the nonmotile, green-colored epibiont and the motile, colorless central bacterium. J Bacteriol, 1998 Mar, 180(6), 1460 - 5 Unusual organization of the genes coding for HydSL, the stable {NiFe}hydrogenase in the photosynthetic bacterium Thiocapsa roseopersicina BBS; Rakhely G et al.; The characterization of a hyd gene cluster encoding the stable, bidirectional {NiFe}hydrogenase 1 enzyme in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium belonging to the family Chromatiaceae, is presented . The heterodimeric hydrogenase 1 had been purified to homogeneity and thoroughly characterized (K . L . Kovacs et al., J . Biol . Chem . 266:947-951, 1991; C . Bagyinka et al., J . Am . Chem . Soc . 115:3567-3585, 1993) . As an unusual feature, a 1,979-bp intergenic sequence (IS) separates the structural genes hydS and hydL, which encode the small and the large subunits, respectively . This IS harbors two sequential open reading frames (ORFs) which may code for electron transfer proteins ISP1 and ISP2 . ISP1 and ISP2 are homologous to ORF5 and ORF6 in the hmc operon, coding for a transmembrane electron transfer complex in Desulfovibrio vulgaris . Other accessory proteins are not found immediately downstream or upstream of hydSL . A hup gene cluster coding for a typical hydrogen uptake {NiFe}hydrogenase in T . roseopersicina was reported earlier (A . Colbeau et al . Gene 140:25-31, 1994) . The deduced amino acid sequences of the two small (hupS and hydS) and large subunit (hupL and hydL) sequences share 46 and 58% identity, respectively . The hup and hyd genes differ in the arrangement of accessory genes, and the genes encoding the two enzymes are located at least 15 kb apart on the chromosome . Both hydrogenases are associated with the photosynthetic membrane . A stable and an unstable hydrogenase activity can be detected in cells grown under nitrogen-fixing conditions; the latter activity is missing in cells supplied with ammonia as the nitrogen source . The apparently constitutive and stable activity corresponds to hydrogenase 1, coded by hydSL, and the inducible and unstable second hydrogenase may be the product of the hup gene cluster. Biophys J, 1998 Mar, 74(3), 1135 - 48 Low-temperature electron transfer from cytochrome to the special pair in Rhodopseudomonas viridis: role of the L162 residue; Ortega JM et al.; Electron transfer from the tetraheme cytochrome c to the special pair of bacteriochlorophylls (P) has been studied by flash absorption spectroscopy in reaction centers isolated from seven strains of the photosynthetic purple bacterium Rhodopseudomonas viridis, where the residue L162, located between the proximal heme c-559 and P, is Y (wild type), F, W, G, M, T, or L . Measurements were performed between 294 K and 8 K, under redox conditions in which the two high-potential hemes of the cytochrome were chemically reduced . At room temperature, the kinetics of P+ reduction include two phases in all of the strains: a dominant very fast phase (VF), and a minor fast phase (F) . The VF phase has the following t(1/2): 90 ns (M), 130 ns (W), 135 ns (F), 189 ns (Y; wild type), 200 ns (G), 390 ns (L), and 430 ns (T) . These data show that electron transfer is fast whatever the nature of the amino acid at position L162 . The amplitudes of both phases decrease suddenly around 200 K in Y, F, and W . The effect of temperature on the extent of fast phases is different in mutants G, M, L, and T, in which electron transfer from c-559 to P+ takes place at cryogenic temperatures in a substantial fraction of the reaction centers (T, 48%; G, 38%; L, 23%, at 40 K; and M, 28%, at 60 K), producing a stable charge separated state . In these nonaromatic mutants the rate of VF electron transfer from cytochrome to P+ is nearly temperature-independent between 294 K and 8 K, remaining very fast at very low temperatures (123 ns at 60 K for M; 251 ns at 40 K for L; 190 ns at 8 K for G, and 458 ns at 8 K for T) . In all cases, a decrease in amplitudes of the fast phases is paralleled by an increase in very slow reduction of P+, presumably by back-reaction with Q(A)- . The significance of these results is discussed in relation to electron transfer theories and to freezing at low temperatures of cytochrome structural reorganization. J Biochem (Tokyo), 1998 Jan, 123(1), 115 - 9 A two-dimensional 1H detected 13C NMR investigation of pyruvate metabolism in Halobacterium salinarium; Majumdar A et al.; Two-dimensional 1H detected 13C NMR spectroscopy has been used to study the intracellular metabolism of {3-(13)C}pyruvate in Halobacterium salinarium . The method, resulting in considerable improvement in spectral resolution and signal-to-noise ratio, is well suited for studying transient metabolic intermediates . Pyruvate utilization by the bacterium is a double exponential function with rate constants of 49.13 and 4.67x10(-3) per min . The relative 13C enrichment is the fastest for C-3 glutamate . Glutamate C-4 labeling decreases initially and increases later on during incubation, while glutamine C-3 is high to begin with and exhibits a declining trend . The glutamate labeling indicates a high initial flux through pyruvate carboxylase and extensive randomizing of the label in the tricarboxylic acid cycle. FEMS Microbiol Lett, 1998 Feb 15, 159(2), 247 - 54 Possible involvement of protein kinase C in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans; Nonaka K et al.; We have previously reported the evidence for apoptosis in the mouse macrophage cell line J774.1 by the periodontopathic bacterium Actinobacillus actinomycetemcomitans . In this study, we examined the role of protein kinases in the induction of apoptosis in A . actinomycetemcomitans-infected J774.1 cells by the MTT assay, fluorescence microscopy and flow cytometric analysis . After J774.1 cells were precultured with protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), J774.1 cells infected with A . actinomycetemcomitans showed the increased percentage of apoptotic cells . On the contrary, protein kinase A (PKA) activators, such as forskolin and dibutyryl cAMP, do not mimic the effect of PMA . PKC inhibitors, such as staurosporine, calphostin C, chelerythrine chloride, and H7 were found to suppress apoptotic cell death in J774.1 cells infected with A . actinomycetemcomitans . However, HA1004, known as PKA inhibitor, had no effect on apoptosis in infected macrophages . The results presented here suggest that the signals through PKC may play crucial roles in the modulation of apoptosis in macrophages infected with A . actinomycetemcomitans. J Infect Dis, 1998 Mar, 177(3), 725 - 9 Induction of macrophage foam cell formation by Chlamydia pneumoniae; Kalayoglu MV et al.; Foam cell formation is the hallmark of early atherosclerosis . It was found that the intracellular bacterium Chlamydia pneumoniae induces foam cell formation by human monocyte-derived macrophages . Exposure of macrophages to C . pneumoniae followed by low-density lipoprotein (LDL) caused a marked increase in the number of foam cells and accumulation of cholesteryl esters . Foam cell formation was not inhibited by the antioxidant butylated hydroxytoluene nor fucoidan, suggesting that lipid accumulation did not involve scavenger receptors . In contrast, addition of heparin, which blocks binding of LDL to the LDL receptor, inhibited C . pneumoniae-induced foam cell formation, suggesting that the pathogen induced lipid accumulation by dysregulating native LDL uptake or metabolism (or both) . These data demonstrate that an infectious agent can induce macrophage foam cell formation and implicate C . pneumoniae as a causative factor in atherosclerosis. Curr Microbiol, 1998 Feb, 36(2), 80 - 4 Rickettsial relative associated with papaya bunchy top disease; Davis MJ et al.; The phylogeny of a previously unidentified, obligate laticifer-inhabiting bacterium associated with the papaya bunchy top disease was investigated . Portions of genes corresponding to those for 16S rRNA, the flavoprotein subunit of succinate dehydrogenase (SdhA), citrate synthase (GltA), and the 17-kDa rickettsial common antigen were isolated and sequenced from the non-cultivable bacterium from diseased plants . Comparative sequence analyses consistently indicated that the bacterium is a member of the alpha-subdivision of the Proteobacteria and of the genus Rickettsia . The rickettsia was detected by polymerase chain reaction in diseased, but not healthy, papaya tissues and in the leafhopper vector, Empoasca papayae, providing further evidence of the possible etiological role of the bacterium in the disease . Although Rickettsia have been found naturally in arthropods and can be pathogenic to humans and other vertebrates, this is the first evidence of its kind implicating a Rickettsia as a plant pathogen. J Bacteriol, 1998 Mar, 180(5), 1261 - 9 Insertional inactivation of genes encoding the crystalline inclusion proteins of Photorhabdus luminescens results in mutants with pleiotropic phenotypes; Bintrim SB et al.; The entomopathogenic bacterium Photorhabdus luminescens exhibits phase variation when cultured in vitro . The variant forms of P . luminescens are pleiotropic and are designated phase I and phase II variants . One of the characteristic phenotypes of phase I cells is the production of two types of intracellular protein inclusions . The genes encoding the protein monomers that form these inclusions, designated cipA and cipB, were cloned and characterized . cipA and cipB encode hydrophobic proteins of 11,648 and 11,308 Da, respectively . The deduced amino acid sequences of CipA and CipB have no significant amino acid sequence similarity to any other known protein but have 25% identity and 49% similarity to each other . Insertional inactivation of cipA or cipB in phase I cells of P . luminescens produced mutants that differ from phase I cells in bioluminescence, the pattern and activities of extracellular products, biochemical traits, adsorption of dyes, and ability to support nematode growth and reproduction . In general, the cip mutants were phenotypically more similar to each other than to either phase I or phase II variants. J Bacteriol, 1998 Mar, 180(5), 1174 - 84 Genetic analysis of the nuo locus, which encodes the proton-translocating NADH dehydrogenase in Escherichia coli; Falk-Krzesinski HJ et al.; Complex I (EC 1.6.99.3) of the bacterium Escherichia coli is considered to be the minimal form of the type I NADH dehydrogenase, the first enzyme complex in the respiratory chain . Because of its small size and relative simplicity, the E . coli enzyme has become a model used to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex . To begin dissecting the processes by which E . coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus, which encodes the 14 Nuo subunits comprising E . coli complex I . Here we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits . In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I. J Clin Periodontol, 1998 Feb, 25(2), 99 - 105 The distribution and transmission of Actinobacillus actinomycetemcomitans in families with localized juvenile periodontitis; Tinoco EM et al.; The prevalence and distribution of A . actinomycetemcomitans in families where at least one family member (proband) suffered from localized juvenile periodontitis was investigated . 25 probands with localized juvenile periodontitis (LJP) and their 78 close family members were screened for the presence of A . actinomycetemcomitans . Among these 25 families, 10 contained at least one additional family member colonized with oral A . actinomycetemcomitans . Genomic DNA from subgingival A . actinomycetemcomitans strains from each of the probands and their family members were amplified and characterized by the polymerase chain reaction (PCR) using a single primer known to distinguish A . actinomycetemcomitans strains . The PCR products from each strain were separated by electrophoresis on a 1% submarine agarose gel containing ethidium bromide and visualized by UV light transillumination . The studies showed that 41.2% of the parents and 58% of the siblings in this LJP-based population harbored the bacterium . Comparison of the PCR generated amplitypes showed that there was a wide distribution of amplitypes among the probands and immediate relatives . No clear transmission paths were observed in this specific population. J Eukaryot Microbiol, 1998 Jan-Feb, 45(1), 71 - 9 Structure and expression of a GroE-homologous operon of a macronucleus-specific symbiont Holospora obtusa of the ciliate Paramecium caudatum; Dohra H et al.; The reproductive form of a macronucleus-specific symbiont Holospora obtusa, when harbored by the macronucleus of the ciliate Paramecium caudatum, selectively synthesized a 63-kDa protein which is immunologically related to GroEL, or HSP60, of Escherichia coli . Heat shock treatment of isolated cells of the reproductive and infectious form of the bacterium also induced the synthesis of the GroEL homolog . Immunoblotting showed that the amount of this protein per cell, whether the reproductive or infectious form, is roughly constant . Cloning and sequencing of a gene coding for the GroEL homolog suggested that the protein is 55.2% identical to GroEL of E . coli at the amino acid sequence level, and that the gene is preceded by an open reading frame which encodes a protein 39.6% identical to GroES of E . coli . Northern blot hybridization showed that the groEL homologous gene is highly expressed in the reproductive form, but only in a trace amount in the intermediate and infectious form . Immunoelectron microscopy revealed that the GroEL homolog is localized in the cytoplasm of the reproductive and infectious form. FEMS Microbiol Lett, 1998 Mar 1, 160(1), 97 - 100 Enhanced invasion of Tyzzer's organism into cultured mouse hepatocytes by cytochalasin D; Kawamura S et al.; The effects of cytoskeleton inhibitors on the invasion of Tyzzer's organism, an obligate intracellular bacterium, into cultured mouse hepatocytes were examined by double immunofluorescence observation and plaque assay . The two techniques gave comparable results . Invasion of bacteria was significantly enhanced by cytochalasin D, a microfilament disrupting drug, and markedly suppressed by vinblastine, a microtubule inhibitor . Another microtubule inhibitor, colchicine, did not show any substantial effect . However, the cytoskeletal system of cultured mouse hepatocytes was sensitive to these three drugs, as judged by inhibition of FITC-dextran uptake . These results suggest that Tyzzer's organisms invade host cells by a unique mechanism that is suppressed by the normal functions of host cell microfilaments. Ital J Gastroenterol Hepatol, 1997 Oct, 29(5), 470 - 5 Effect of Helicobacter pylori eradication on intestinal metaplasia and gastric epithelium proliferation; Ierardi E et al.; Gastric epithelial turnover increase in Helicobacter pylori infection has been demonstrated by interventional and non interventional methods for proliferating cell detection . We have observed a progressive hyperproliferation with the progression of Helicobacter pylori-induced mucosal lesions until the development of intestinal metaplasia . A similar result has been reported in other studies in the succession from normal mucosa to gastric carcinoma even if interventional techniques show less conspicuous differences in comparison to non interventional ones, which give an overestimated picture of proliferation . Later studies show that Helicobacter pylori-related hyperproliferation reverses after eradication . We have observed that this reversibility does not occur in areas of intestinal metaplasia, where the oncoprotein ras p21, involved in early gastric carcinogenesis, is expressed . This finding agrees with that demonstrating that hyperproliferation in intestinal metaplasia or gastric cancer is not affected by Helicobacter pylori . Other oncogenetic changes in intestinal metaplasia (i.e., p53 mutation) may further explain the persistently modified proliferative pattern of the epithelium . Recent studies suggest a lack of reversibility of intestinal metaplasia after Helicobacter pylori eradication, but this problem remains controversial . Our experience suggests that the persistence of the bacterium may increase the extent of this lesion . In conclusion the development of intestinal metaplasia is associated with an impaired regulation of gastric epithelial proliferation . Nevertheless, from the biological point of view, the progression towards carcinoma requires further DNA changes . Moreover, many questions need to be answered in order to establish clear guidelines for the clinical management. Microbiology, 1998 Feb, 144 ( Pt 2), 577 - 87 Extracellular enzyme activities potentially involved in the pathogenicity of Mycobacterium tuberculosis; Raynaud C et al.; To evaluate the potential contribution of extracellular enzymes to the pathogenicity of mycobacteria, the presence of selected enzyme activities was investigated in the culture filtrates of the obligate human pathogen Mycobacterium tuberculosis, M . bovis BCG, the opportunistic pathogens M . kansasii and M . fortuitum, and the non-pathogenic species M . phlei and M . smegmatis . For M . tuberculosis and M . bovis, 22 enzyme activities were detected in the culture filtrates and/or cell surfaces, of which eight were absent from the culture fluids of non-pathogens: alanine dehydrogenase, glutamine synthetase, nicotinamidase, isonicotinamidase, superoxide dismutase, catalase, peroxidase and alcohol dehydrogenase . These activities, which correspond to secreted enzymes, formed a significant part (up to 92%) of the total enzyme activities of the bacteria and were absent from the culture fluids and the cell surfaces of the non-pathogenic species M . smegmatis and M . phlei . The extracellular location of superoxide dismutase and glutamine synthetase seemed to be restricted to the obligate pathogens examined . The difference in the enzyme profiles was not attributable to the growth rates of the two groups of bacteria . The presence of the eight enzyme activities in the outermost compartments of obligate pathogens and their absence in those of non-pathogens provides further evidence that these enzymes may be involved in the pathogenicity of mycobacteria . In addition, the eight enzyme activities were demonstrated in the cell extract of M . smegmatis . Stepwise erosion of the cell surface of M . smegmatis to expose internal capsular constituents showed that the various enzyme activities, with the possible exception of superoxide dismutase, were located more deeply in the cell envelope of this bacterium . This suggests that the molecular architecture of the mycobacterial envelopes may play an important role in the pathogenicity of these organisms. Eur J Biochem, 1998 Jan 15, 251(1-2), 65 - 71 Rhodobacter capsulatus HypF is involved in regulation of hydrogenase synthesis through the HupUV proteins; Colbeau A et al.; The photosynthetic bacterium Rhodobacter capsulatus contains a membrane-bound {NiFe}hydrogenase encoded by the hupSL genes . We show in this study that hypF mutants are devoid of hydrogenase activity and lack the HupL protein . We also observed that, in contrast to the wild-type strain B10, transcription of the hupSL genes was not stimulated by H2 in the hypF mutants RS13 and BSE19 . Complementation of the hypF mutants with the plasmid borne hypF gene restored hydrogenase activity to wild-type levels and inducibility by H2 . The R . capsulatus hupU and hupV gene products share significant similarities with the small (HupS) and the large (HupL) hydrogenase subunits, respectively . Active HupUV proteins can catalyze the hydrogen-deuterium exchange reaction . In whole cells, this H-D exchange is distinguishable from the H-D exchange catalyzed by the membrane-bound HupSL proteins by its insensitivity to O2 and to acetylene . By measuring the formation of H2 and HD in exchange with D2 uptake, we demonstrated that the hypF mutants have no active HupUV nor HupSL proteins . H-D exchange activity, of both HupUV and HupSL, was restored by hypF gene complementation . These data indicate that the HypF protein participates not only in the maturation of HupSL, but also in the maturation of the HupUV proteins and that the latter are involved in the cellular response to H2. Mol Gen Genet, 1998 Jan, 257(2), 205 - 12 A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli; Dmitrova M et al.; Interactions between proteins affect a wide variety of biological processes, such as signal transduction and control of gene expression . In order to facilitate the study of protein-protein interactions we have developed a new method for specifically detecting the heterodimerization of two heterologous proteins in the bacterium Escherichia coli . The assay is based on the simultaneous use of protein fusions with an altered specificity and a wild-type LexA repressor DNA-binding domain . We have tested this system with two well known eukaryotic dimerization domains (the Fos and Jun leucine zippers) . The two interacting proteins were, respectively, fused to a wild-type and a mutant LexA DNA-binding domain . Their hetero-association is specifically measured by the transcriptional repression of a reporter gene (lacZ) controlled by a hybrid operator containing a wild-type half-site (CTGT) and a mutated operator half-site (CCGT) . The hybrid operator/lacZ construct was integrated into the chromosome of the reporter strain (SU202) to avoid possible artefacts due to variations in plasmid copy number . This method should be particularly useful in those cases where one or both partners are also able to form homodimers, since the assay described here is sensitive only to the formation of heterodimers . Furthermore, this assay gives rise to a screenable red/white phenotype on MacConkey-lactose indicator plates, allowing for a genetic study of the specificity of the interaction. Eur J Biochem, 1998 Feb 1, 251(3), 641 - 8 A genetically engineered increase in fatty acid unsaturation in Synechococcus sp . PCC 7942 allows exchange of D1 protein forms and sustenance of photosystem II activity at low temperature; Sippola K et al.; Photosystem II (PSII)-reaction-center protein D1 is encoded by three psbA genes in Synechococcus sp . PCC 7942 . The psbAI gene encodes D1 form I (D1:1) and the psbAII and psbAIII genes encode the transiently expressed D1 form II (D1:2) . We have studied the role of membrane-lipid unsaturation in the expression of psbA genes at low temperature, using a Synechococcus transformant with an increased unsaturation level of membrane lipids . Transfer of the cells from 32 degrees C to 25 degrees C under growth light resulted in the exchange of D1:1, the prevailing form, for D1:2 in the wild-type bacterium and the transformant, with no loss of PSII activity . Lowering the temperature further to 18 degrees C caused a drastic decrease in PSII activity in the wild-type bacterium, whereas the transformant was much less affected . Similar decreases in psbAI transcripts and loss of D1:1 occurred in both strains at 18 degrees C, with concomitant accumulation of psbAII/III transcripts, the latter event being especially prominent in the wild-type bacterium . However, the wild-type bacterium was incapable of accumulating D1:2 to compensate for the loss of D1:1, which resulted in disassembly of PSII at low temperature . These results imply translational rather than transcriptional regulation of psbA gene expression in Synechococcus 7942 at low temperature, and demonstrate the crucial role of the degree of membrane-lipid unsaturation in promotion of exchange of the D1 protein forms and thus the sustenance of PSII function at low temperature. Lett Appl Microbiol, 1998 Jan, 26(1), 12 - 6 Nucleotide sequence of the nifH gene coding for nitrogen reductase in the acetic acid bacterium Acetobacter diazotrophicus; Franke IH et al.; The nifH gene sequence of the nitrogen-fixing bacterium Acetobacter diazotrophicus was determined with the use of the polymerase chain reaction and universal degenerate oligonucleotide primers . The gene shows highest pair-wise similarity to the nifH gene of Azospirillum brasilense . The phylogenetic relationships of the nifH gene sequences were compared with those inferred from 16S rRNA gene sequences . Knowledge of the sequence of the nifH gene contributes to the growing database of nifH gene sequences, and will allow the detection of Acet . diazotrophicus from environmental samples with nifH gene-based primers. Arch Microbiol, 1998 Jan, 169(1), 84 - 7 Tetrahydrofolate serves as a methyl acceptor in the demethylation of dimethylsulfoniopropionate in cell extracts of sulfate-reducing bacteria; Jansen M et al.; Tetrahydrofolate was shown to function as a methyl acceptor in the anaerobic demethylation of dimethylsulfoniopropionate to methylthiopropionate in cell extracts of the sulfate-reducing bacterium strain WN . Dimethylsulfoniopropionate-dependent activities were 0.56 micromol methyltetrahydrofolate min-1 (mg protein)-1 and were higher than required to explain the growth rate of strain WN on dimethylsulfoniopropionate . The reaction did not require ATP or reductive activation by titanium(III)-nitrilotriacetic acid . Preincubation of the extract under air significantly decreased the activity (35% loss in 3 h) . Three other dimethylsulfoniopropionate-demethylating sulfate reducers, Desulfobacterium niacini, Desulfobacterium vacuolatum, and Desulfobacterium strain PM4, had dimethylsulfoniopropionate:tetrahydrofolate methyltransferase activities of 0.16, 0.05, and 0.24 micromol min-1 (mg protein)-1, respectively . No methyltransferase activity to tetrahydrofolate was found with betaine as a substrate, not even in extracts of betaine-grown cells of these sulfate reducers . Dimethylsulfoniopropionate demethylation in cell extracts of strain WN was completely inhibited by 0.5 mM propyl iodide; in the light, the inhibition was far less strong, indicating involvement of a corrinoid-dependent methyltransferase. Arch Microbiol, 1998 Jan, 169(1), 36 - 42 Degradation of p-nitrophenol by the phototrophic bacterium Rhodobacter capsulatus; Roldan MD et al.; The phototrophic bacterium Rhodobacter capsulatus detoxified p-nitrophenol and 4-nitrocatechol . The bacterium tolerated moderate concentrations of p-nitrophenol (up to 0.5 mM) and degraded it under light at an optimal O2 pressure of 20 kPa . The bacterium did not metabolize the xenobiotic in the dark or under strictly anoxic conditions or high O2 pressure . Bacterial growth with acetate in the presence of p-nitrophenol took place with the simultaneous release of nonstoichiometric amounts of 4-nitrocatechol, which can also be degraded by the bacterium . Crude extracts from R . capsulatus produced 4-nitrocatechol from p-nitrophenol upon the addition of NAD(P)H, although at a very low rate . A constitutive catechol 1, 2-dioxygenase activity yielding cis,cis-muconate was also detected in crude extracts of R . capsulatus . Further degradation of 4-nitrocatechol included both nitrite- and CO2-releasing steps since: (1) a strain of R . capsulatus (B10) unable to assimilate nitrate and nitrite released nitrite into the medium when grown with p-nitrophenol or 4-nitrocatechol, and the nitrite concentration was stoichiometric with the 4-nitrocatechol degraded, and (2) cultures of R . capsulatus growing microaerobically produced low amounts of 14CO2 from radiolabeled p-nitrophenol . The radioactivity was also incorporated into cellular compounds from cells grown with uniformly labeled 14C-p-nitrophenol . From these results we concluded that the xenobiotic is used as a carbon source by R . capsulatus, but that only the strain able to assimilate nitrite (E1F1) can use p-nitrophenol as a nitrogen source. Immunol Res, 1998, 17(1-2), 191 - 207 Rational approaches to immune regulation; Paterson Y; Our studies are mainly focused on developing strategies of immune regulation . In the case of infectious and neoplastic disease, our approach is to upregulate cell-mediated immunity to viral of tumor antigens using an intracellular bacterium as a vector for targeting these antigens to the major histocompatibility complex (MHC) class I and class II pathways of antigen processing, in addition to exploiting the adjuvant properties of the vector to stimulate innate immunity . In the area of autoimmunity, we are attempting to downregulate the immune response by specific immune intervention directed against autoreactive T cells . In these studies we use murine models for multiple sclerosis . Our approach is to use both rationally designed T cell receptor (TCR) peptide analogs and recombinant viral vectors that express TCR components to regulate the disease. Ital J Gastroenterol Hepatol, 1997 Aug, 29(4), 375 - 82 Host mechanisms: are they the key to the various clinical outcomes of Helicobacter pylori infection? Calam J. It is unclear why Helicobacter pylori produces different diseases in different persons . High and low acid secretion rates probably contribute to duodenal ulceration and gastric carcinogenesis, respectively . Both of these changes seem to be corrected by eradicating Helicobacter pylori . We are therefore exploring the basic mechanisms and asking why patients react differently? Helicobacter pylori products and certain cytokines released in Helicobacter pylori gastritis release gastrin from G-cells but inhibit parietal cells . Also tumour necrosis factor alpha inhibits somatostatin-cells and interleukin 1 beta inhibits enterochromaffin-like cells . The net result is that antral gastritis tends to increase, whilst corpus gastritis tends to decrease acid secretion . Corpus atrophy further lowers acid through loss of parietal cells . Factors postulated to increase corpus gastritis include host genetics, early acquisition of Helicobacter pylori, more aggressive strains, poor general health and diets high in salt or lacking in antioxidant vitamins . Further research should address the interaction of bacterium, host and environment with a view to preventing the serious clinical outcomes. Plasmid, 1998, 39(1), 84 - 8 Structural analysis of a new cryptic plasmid pAR67 isolated from Ruminococcus albus AR67; Ohara H et al.; The complete nucleotide sequence of a new cryptic plasmid pAR67 isolated from a rumen bacterium Ruminococcus albus AR67 has been determined . The plasmid pAR67 was 3419 bp in size with a 45% GC content . Two open reading frames, ORF1 and ORF2, encoding potential polypeptides of 285 and 165 amino acids, with limited sequence similarity to replication and mobilization proteins, respectively, were identified within the sequence . The region upstream of ORF1 had an AT-rich (80%) segment followed by four 19-bp direct repeats, which is similar to the structural organization characteristic of replication origins of some bacterial plasmids. Biochemistry, 1998 Jan 13, 37(2), 530 - 7 Function of a glutathione-dependent formaldehyde dehydrogenase in Rhodobacter sphaeroides formaldehyde oxidation and assimilation; Barber RD et al.; Despite its reactivity with many biological molecules, formaldehyde can be commonly encountered by virtually all cells . The widespread existence of glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) in procaryotes and eucaryotes suggests this enzyme plays a central and universal role in biological formaldehyde oxidation . This work sought to determine the role of GSH-FDH in the facultative phototrophic bacterium Rhodobacter sphaeroides . Growth phenotypes of wild-type and mutant cells, changes in enzyme specific activities, and the pattern of 13C-labeled compounds detected by NMR spectroscopy cumulatively suggest that R . sphaeroides GSH-FDH can play a critical role in formaldehyde metabolism under both photosynthetic and aerobic respiratory conditions . In photosynthetic cells, the data indicate that GSH-FDH generates reducing power, in the form of NADH, and one-carbon skeletons that are oxidized to carbon dioxide for subsequent assimilation by the Calvin-Benson-Bassham cycle . For example, use of methanol as a sole photosynthetic carbon source increases the specific activities of GSH-FDH, an NAD-dependent formate dehydrogenase, and the key Calvin-Benson-Bassham cycle enzyme, ribulose-1,5-bisphosphate carboxylase . This role of GSH-FDH is also supported by the pattern of {13C}formaldehyde oxidation products that accumulate in photosynthetic cells and the inability of defined GSH-FDH or Calvin cycle mutants to use methanol as a sole carbon source . Our data also suggest that GSH-FDH acts in formaldehyde dissimilation when aerobic respiratory cultures cometabolize methanol and succinate. Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 201 - 6 Control of direction of flagellar rotation in bacterial chemotaxis; Scharf BE et al.; The motile behavior of the bacterium Escherichia coli depends on the direction of rotation of its flagellar motors . Binding of the phosphorylated signaling molecule CheY to a motor component FliM is known to enhance clockwise rotation . It is difficult to study this interaction in vivo, because the dynamics of phosphorylation of CheY by its kinase CheA and the hydrolysis of CheY (accelerated by CheZ) are not under direct experimental control . Here, we examine instead the interaction with the flagellar motor of a double mutant CheY13DK106YW that is active without phosphorylation . The behavioral assays were carried out on tethered cells lacking CheA and CheZ . The effects of variation in intracellular concentration of the mutant protein were highly nonlinear . However, they can be explained by a thermal isomerization model in which the free energies of clockwise and counterclockwise states depend linearly on the amount of CheY bound. Arch Microbiol, 1997 Dec, 168(6), 520 - 7 Purification, characterization and gene sequence analysis of a novel cytochrome c co-induced with reductive dechlorination activity in Desulfomonile tiedjei DCB-1; Louie TM et al.; The sulfate-reducing bacterium, Desulfomonile tiedjei DCB-1, conserves energy for growth from reductive dechlorination of 3-chlorobenzoate via halorespiration . To understand this respiratory process better, we examined electron carriers from different cellular compartments of D . tiedjei . A 50-kDa cytochrome from the membrane fraction was found to be co-induced with dechlorination activity . This inducible cytochrome was extracted from the membrane fractions by Tris-HCl buffer containing ammonium sulfate at 35% saturation and was purified to electrophoretic homogeneity by phenyl superose, Mono Q, and hydroxyapatite chromatography . The purified cytochrome had a high-spin absorption spectrum . In a pH titration experiment, the absorption spectrum of the inducible cytochrome shifted to low spin at pH 13.2 . The midpoint potential of the inducible cytochrome at pH 7.0 was -342 mV . The NH2-terminal amino acid sequence of the inducible cytochrome was determined and was used to obtain inverse PCR products containing the sequence of the gene encoding the inducible cytochrome . The ORF was 1398 bp and coded for a protein of 52.6 kDa . Two c-type heme-binding domains were identified in the COOH-terminal half of the protein . A putative signal peptide of 26 residues was found at the NH2-terminal end . The protein sequence was not found to have substantial sequence similarity to any other sequence in GenBank . We conclude that this is a c-type cytochrome substantially different from previously characterized c-type cytochromes. Arch Microbiol, 1997 Dec, 168(6), 513 - 9 Comparative studies on tetrachloroethene reductive dechlorination mediated by Desulfitobacterium sp . strain PCE-S; Miller E et al.; Tetrachloroethene reductive dechlorination was studied with cell extracts of a newly isolated, tetrachloroethene-utilizing bacterium, Desulfitobacterium sp . strain PCE-S . Tetrachloroethene dehalogenase mediated the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with artificial electron donors such as methyl viologen . The chlorinated aromatic compounds tested so far were not reduced . A low-potential electron donor (E0' < -0.4 V) was required for tetrachloroethene reduction . The enzyme in its reduced state was inactivated by propyl iodide and reactivated by light, indicating the involvement of a corrinoid in reductive tetrachloroethene dechlorination. Arch Microbiol, 1997 Dec, 168(6), 480 - 5 Evidence for a tungsten-stimulated aldehyde dehydrogenase activity of Desulfovibrio simplex that oxidizes aliphatic and aromatic aldehydes with flavins as coenzymes; Zellner G et al.; The aldehyde dehydrogenase activity of the sulfate-reducing bacterium Desulfovibrio simplex strain DSM 4141 was characterized in cell-free extracts . Oxygen-sensitive, constitutive aldehyde dehydrogenase activity was found in cells grown on l(+)-lactate, hydrogen, or vanillin with sulfate as the electron acceptor . A 1.83- to 2.6-fold higher specific activity was obtained in cells grown in media supplemented with 1 microM WO42- . The aldehyde dehydrogenase in cell-free extracts catalyzed the oxidation of aliphatic (Km < 20 microM) and aromatic aldehydes (Km < 0.32 mM) using methyl viologen as the electron acceptor . Flavins (FMN and FAD) were also active and are proposed to be the natural cofactors, while no activity was obtained with NAD+ or NADP+ . 185WO42- was incorporated in vivo into D . simplex; it was found exclusively in the soluble fraction (>/= 98%) . Anionic-exchange chromatography demonstrated coelution of 185W with two distinct peaks, the first one containing hydrogenase and formate dehydrogenase activities, and the second one aldehyde dehydrogenase activity. Arch Microbiol, 1997 Dec, 168(6), 448 - 56 Purine metabolism and the microaerophily of Helicobacter pylori; Mendz GL et al.; The requirements for purine nucleotide synthesis, the effects of purine analogues, and the metabolism of adenine in the bacterium Helicobacter pylori were investigated employing cell culture techniques and one-dimensional NMR spectroscopy . Bacterial cells grew and proliferated in media lacking preformed purines, indicating that H . pylori can synthesize purine nucleotides de novo to meet its requirements . Blocking of this pathway in the absence of sufficient preformed purines for salvage nucleotide synthesis led to cell death . Analogues of purine nucleobases and nucleosides taken up by the cells were cytotoxic, suggesting that salvage routes could be exploited for therapy . Adenine or hypoxanthine were able to substitute for catalase in supporting cell growth and proliferation, suggesting a role for these bases in maintaining the microaerophilic conditions essentially required by the bacterium. Carbohydr Res, 1997 Nov 28, 304(3-4), 325 - 33 Fine chemical structure analysis of oligosaccharides produced by an ulvan-lyase degradation of the water-soluble cell-wall polysaccharides from Ulva sp, (Ulvales, Chlorophyta); Lahaye M et al.; A marine bacterium degrading the water-soluble cell wall polysaccharides from Ulva sp . (ulvan) has been isolated . The good correlation between ulvan degradation monitored by reducing-power, UV absorbance and viscosimetry, indicated that the crude enzymatic extract contains essentially an endo-ulvan lyase activity . This activity was rapidly inhibited by the reaction products which consisted of a series of ulvanobiouronic acid A 3-sulfate {-->4)-beta-D-GlcpA-(1-->4)-alpha-L-Rhap 3-sulfate-(1-->}n with 4-deoxy-L-threo-hex-4-enopyranosiduronic acid at the non-reducing end . Other deviant repeating structures with beta-D-Xylp or alpha-IdopA replacing beta-D-GlcpA in the repeating ulvanobiouronic acid disaccharide and the presence of two consecutive (1-->4) linked beta-D-Glc pA demonstrated the great variability and complexity of ulvan chemical structure. Digestion, 1998, 59(1), 53 - 9 Activation of epithelial cells in gastritis; El Kaissouni J et al.; BACKGROUND: Helicobacter pylori is now recognised to be the major cause of antral gastritis and a risk factor for further development of gastric cancer . This infection results in local inflammation and a modification of gastric mucosal epithelial cell characteristics . Systematic investigation for the expression of the secretory component by gastric epithelium in a personal historical series of biopsy specimens showed the expression of this activation marker in 38% of the cases, 19% also showing clear signs of H . pylori infection . AIMS: To further appreciate the activation of epithelial cells in the chronic gastric inflammation associated with H . pylori infection and other types of gastritis without consideration of the grade of gastritis . METHODS: Punch gastric biopsies from 36 patients (10 patients with confirmed H . pylori gastritis, 16 patients with non-H . pylori gastritis and 10 controls) were tested for the expression of ICAM-1/CD54, HLA-DP, -DQ, -DR, secretory component and bcl-2 by immunofluorescence . RESULTS: Up-regulation of most markers was observed both in H . pylori and non-H . pylori gastritis, although secretory component, CD54 and DP expression was more closely associated with H . pylori gastritis . CONCLUSION: H . pylori infection appears to activate gastric epithelial cells more strongly than other types of gastritis . This suggests an active relationship between this bacterium and epithelial cells . Investigation of the up-regulationship between this bacterium and epithelial cells . Investigation of the up-regulation of secretory component, ICAM-1/CD54 or DP in gastric biopsies may serve as extensive markers in search of H . pylori gastric infections. Microbiology, 1998 Jan, 144 ( Pt 1), 219 - 27 Rhodobacter capsulatus genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbLS) and neighbouring genes were acquired by a horizontal gene transfer; Paoli GC et al.; Analysis of the nucleotide sequence of the form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes (cbbL and cbbS) of the non-sulfur purple bacterium Rhodobacter capsulatus indicated that the deduced amino acid sequence of the large subunit was not closely homologous to the large subunit from related organisms . Indeed, phylogenetic analysis suggested that the large subunit protein (CbbL) more closely resembled the enzyme from alpha/beta/gamma purple bacteria and cyanobacteria and is within a 'green-like' radiation of the RubisCO phylogenetic tree, well separated from CbbL of the related organism Rhodobacter sphaeroides . A cbbQ gene was discovered downstream of cbbS in Rh . capsulatus, a gene arrangement which also appears to be limited to certain organisms containing a 'green-like' RubisCO . Upstream, and divergently transcribed from cbbLSQ, is a gene (cbbRI) that encodes a LysR-type transcriptional activator . Phylogenetic analysis of the deduced amino acid sequence of CbbRI also suggests that this protein is quite distinct from the Rh . sphaeroides CbbR protein, and is even distinct from the previously described CbbRII protein, the gene of which is upstream and divergently transcribed from the cbbII operon of Rh . capsulatus . Interestingly, Rh . capsulatus CbbRI is more closely related to CbbR from bacteria whose RubisCO falls within the 'green-like' radiation of the CbbL tree . These studies suggest that the cbbRI-cbbL-cbbS-cbbQ genes were acquired by Rh . capsulatus via horizontal gene transfer from a bacterial species containing a 'green-like' RubisCO. Appl Environ Microbiol, 1998 Feb, 64(2), 795 - 9 Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA; Marchesi JR et al.; We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria . Their specificity and efficacy were tested systematically with a bacterial species and environmental samples . They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used. Appl Environ Microbiol, 1998 Feb, 64(2), 472 - 8 Characterization of chitinase C from a marine bacterium, Alteromonas sp . strain O-7, and its corresponding gene and domain structure; Tsujibo H et al.; One of the chitinase genes of Alteromonas sp . strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined . An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da . Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain . The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases . Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity . Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain. Vet Microbiol, 1997 Dec, 59(1), 37 - 51 Electrophoretic and antigenic characterisation of Dermatophilus congolensis extracellular products; Ambrose NC et al.; Dermatophilus congolensis is the causative agent of bovine dermatophilosis and lumpy wool in sheep . Two field isolates of D . congolensis, one each from a cow in Ghana and a sheep in Scotland, were cultured for 24-72 h in a synthetic medium based on RPMI-1640 . Culture filtrates were examined by SDS-PAGE and considered to contain extracellular products released by growing hyphae and filaments . Electrophoretic profiles of culture filtrates of the two isolates contained common bands and bands that were unique to each isolate . The composition of extracellular products altered with increasing culture periods indicating that specific products were released at different stages of growth . Culture filtrate prepared in the presence of serine protease and metalloprotease inhibitors contained more and better defined bands than that prepared without protease inhibitors indicating the presence of proteases in culture filtrates . Western blot analysis of extracellular products using a panel of sera showed that the two isolates from different host species and distant geographical locations contained cross-reactive antigens . Natural and experimental infections stimulated antibody responses to antigens in culture filtrates, sera from animals that were disease free but in-contact with dermatophilosis-infected animals also contained antibodies to extracellular antigens . The antigens recognised by most sera had molecular weights of 200 kDa in the bovine isolate, 170 kDa in the ovine isolate and 67, 27 and 52-55 kDa in both isolates . The number of antigenic bands of both isolates was positively correlated with the intensity of challenge and the severity of infection: antibodies in sera from disease-free cattle in Ghana recognised more antigens than sera from disease-free sheep in Scotland and more antigens were recognised by sera from chronically-infected Ghanaian cattle than by sera from experimentally-infected calves and sheep . The latter developed antibodies to antigens of 27 and 24 kDa during the course of infection . The electrophoretic profiles of extracellular products of D . congolensis are less complex than those of other structures of the bacterium yet they exhibit differences between the two isolates . Extracellular products contain antigens recognised by sera from naturally exposed and experimentally-infected animals that may be involved in immunity to D . congolensis or immunopathogenesis of dermatophilosis. Protein Eng, 1997 Aug, 10(8), 967 - 73 Recombinant ferritin: modulation of subunit stoichiometry in bacterial expression systems; Rucker P et al.; We describe a strategy for the creation of recombinant ferritin heteropolymers which mimic the natural heterogeneity of this protein . This method entailed the co-expression of cDNA for both ferritin H and ferritin L subunits in a single bacterium using either a bicistronic vector, in which both cDNAs were expressed from the vector, or a dual vector expression strategy, in which each subunit was expressed from a separate compatible plasmid in a single bacterial host . Electron microscopy and sucrose density gradient centrifugation demonstrated that ferritin assembled spontaneously in such bacteria to form catalytically active proteins of the expected size and shape . Isoelectric focusing revealed that protein isolated from any of these bacteria exhibited a restricted heterogeneity in subunit composition . Such multi-subunit recombinant ferritins spontaneously assembled in bacteria may be useful in further studies of ferritin assembly and function . Our results further suggest that varying expression levels is a simple way to alter levels of individual components within a multi-subunit recombinant protein, and that this approach may be of general utility in assessing the contribution of individual components to the function of multi-subunit proteins or protein complexes. Mol Microbiol, 1997 Oct, 26(2), 251 - 9 Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum; Flavier AB et al.; Expression of virulence genes in Ralstonia solanacearum, a phytopathogenic bacterium, is controlled by a complex regulatory network that integrates multiple signal inputs . Production of several virulence determinants is coordinately reduced by inactivation of phcB, but is restored by growth in the presence of a volatile extracellular factor (VEF) produced by wild-type strains of R . solanacearum . The VEF was purified from spent culture broth by distillation, solvent extraction, and liquid chromatography . Gas chromatography and mass spectroscopy identified 3-hydroxypalmitic acid methyl ester (3-OH PAME) as the major component in the single peak of VEF activity . Authentic 3-OH PAME and the purified VEF were active at < or =1 nM, and had nearly equivalent specific activities for stimulating the expression of eps (the biosynthetic locus for extracellular polysaccharide) in a phcB mutant . Authentic 3-OH PAME also increased the production of three virulence factors by a phcB mutant over 20-fold to wild-type levels, restored normal cell density-associated expression of eps and increased expression of eps when delivered via the vapour phase . Reanalysis of the PhcB amino acid sequence suggested that it is a small-molecule S-adenosylmethionine-dependent methyltransferase, which might catalyse synthesis of 3-OH PAME from a naturally occurring fatty acid . Biologically active concentrations of extracellular 3-OH PAME were detected before the onset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild-type R . solanacearum . Other than acyl-homoserine lactones, 3-OH PAME is the only endogenous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long-distance intercellular communication. Arch Microbiol, 1998 Jan, 169(1), 76 - 80 Amino acid degradation by the mesophilic sulfate-reducing bacterium desulfobacterium vacuolatum Rees GN, Harfoot CG, Sheehy AJ. Desulfobacterium vacuolatum strain IbRM was able to grow using casamino acids as a source of carbon, energy and nitrogen . Growth was accompanied by utilization of several amino acids and sulfide production . Proline and glutamate were used preferentially and to the greatest extent . Glycine, serine and alanine were used more slowly and only after proline and glutamate were used . Isoleucine, valine, leucine and aspartate decrease was slowest and occurred in a linear fashion throughout the growth phase . Amino acids used from casamino acids, excluding aspartate, were also used as single carbon, energy and nitrogen sources . As a single amino acid, aspartate could only be used as a nitrogen source . Aspartate was not used as an electron acceptor . No growth occurred on any amino acid in the absence of sulfate . As single substrates, isoleucine, proline and glutamate were oxidized without formation of acetate and with molar yields of 13.1, 9.4 and 7.7 g mol-1, respectively. Gastroenterology, 1998 Feb, 114(2), 245 - 55 Infection of Helicobacter pylori in gastric adaptation to continued administration of aspirin in humans; Konturek JW et al.; BACKGROUND & AIMS: Involvement of Helicobacter pylori in aspirin-induced gastropathy and adaptation to aspirin remains unclear . The aim of this study was to compare gastric damage and adaptation after repeated exposures to acetylsalicylic acid in the same subjects before and after eradication of H . pylori . METHODS: Before and after H . pylori eradication, 8 volunteers were given aspirin, 2 g/day during 14 days . Mucosal damage was evaluated by endoscopy and histological analysis of biopsy samples . Gastric microbleeding, DNA synthesis, prostaglandin E2 generation, and luminal contents of transforming growth factor alpha and its immunohistochemical expression were determined on days 0, 3, 7, and 14 of aspirin course . RESULTS: In all subjects, aspirin-induced gastric damage that reached maximum on day 3 . In H . pylori-positive subjects, this damage was maintained at a similar level up to day 14 . After H . pylori eradication, the damage was significantly lessened both in endoscopy and histology at day 14 and accompanied by increased mucosal expression and luminal release of transforming growth factor alpha . Prostaglandin E2 generation was significantly greater in H . pylori-positive subjects than after H . pylori eradication, but aspirin treatment resulted in >90% reduction of this generation independent of H . pylori status . CONCLUSIONS: Gastric adaptation to aspirin is impaired in H . pylori-positive subjects, but eradication of this bacterium restores this process. FEMS Microbiol Lett, 1998 Jan 1, 158(1), 89 - 93 Aureobacterium resistens sp . nov., exhibiting vancomycin resistance and teicoplanin susceptibility; Funke G et al.; Two similar strains of a coryneform bacterium were isolated from human clinical material . Both strains were resistant to vancomycin but susceptible to teicoplanin . Detailed biochemical, chemotaxonomical, and molecular genetic investigations revealed that both isolates were members of a hitherto undescribed species of genus Aureobacterium . The name Aureobacterium resistens sp . nov . is proposed for the new bacterium and the type strain is CCUG 38312. FEMS Microbiol Lett, 1998 Jan 1, 158(1), 9 - 15 Isolation and analysis of genes for amylolytic enzymes of the hyperthermophilic bacterium Thermotoga maritima; Bibel M et al.; In addition to the previously identified 4-alpha-glucanotransferase gene mgtA and the alpha-amylase gene amyA of Thermotoga maritima strain MSB8 we have now isolated three further genes encoding amylolytic enzymes from a gene library of this ancestral bacterium . The genes code for the extremely thermostable enzymes pullulanase (pulA), maltodextrin phosphorylase (agpA) and alpha-glucosidase (aglA) and have the potential to encode polypeptides with calculated molecular masses of 96.3 kDa, 96.1 kDa and 52.5 kDa, respectively . Comparative amino acid sequence analysis revealed that PulA and AgpA are clearly related to other known enzymes with similar function . AglA, on the other hand, was not related to other alpha-glucosidases but appears to belong to an enzyme family containing alpha-galactosidases and 6-phospho-beta-glucosidases . Enzyme properties are reported which demonstrate the extreme thermostability of these T . maritima enzymes. Science, 1998 Jan 16, 279(5349), 373 - 7 Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging; Ilver D et al.; The bacterium Helicobacter pylori is the causative agent for peptic ulcer disease . Bacterial adherence to the human gastric epithelial lining is mediated by the fucosylated Lewis b (Leb) histo-blood group antigen . The Leb-binding adhesin, BabA, was purified by receptor activity-directed affinity tagging . The bacterial Leb-binding phenotype was associated with the presence of the cag pathogenicity island among clinical isolates of H . pylori . A vaccine strategy based on the BabA adhesin might serve as a means to target the virulent type I strains of H . pylori. Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14433 - 7 Absence of a barrier to backwards rotation of the bacterial flagellar motor demonstrated with optical tweezers; Berry RM et al.; A cell of the bacterium Escherichia coli was tethered covalently to a glass coverslip by a single flagellum, and its rotation was stopped by using optical tweezers . The tweezers acted directly on the cell body or indirectly, via a trapped polystyrene bead . The torque generated by the flagellar motor was determined by measuring the displacement of the laser beam on a quadrant photodiode . The coverslip was mounted on a computer-controlled piezo-electric stage that moved the tether point in a circle around the center of the trap so that the speed of rotation of the motor could be varied . The motor generated approximately 4500 pN nm of torque at all angles, regardless of whether it was stalled, allowed to rotate very slowly forwards, or driven very slowly backwards . This argues against models of motor function in which rotation is tightly coupled to proton transit and back-transport of protons is severely limited. J Mol Biol, 1997 Dec 19, 274(5), 687 - 92 Structural study of the response regulator HupR from Rhodobacter capsulatus . Electron microscopy of two-dimensional crystals on a nickel-chelating lipid; Venien-Bryan C et al.; Two-dimensional crystals of the histidine-tagged-HupR protein, a transcriptional regulator from the photosynthetic bacterium Rhodobacter capsulatus, were obtained upon specific interaction with a Ni2+-chelated lipid monolayer . HupR is a response regulator of the NtrC family; it activates the transcription of the structural genes, hupSLC, of the {NiFe}hydrogenase . The lipid (Ni-NTA-DOGA) uses the metal chelator nitrilotriacetic group as the hydrophilic headgroup and contains unsaturated oleyl tails to provide the fluidity necessary for two-dimensional protein crystallization . A projection map of the full-length protein at 18 A resolution was generated by analysing electron microscopy micrographs of negatively stained crystals . The HupR protein appeared to be dimeric and revealed a characteristic "propeller-like" motif . Each monomer forms an L-shaped structure . J Ind Microbiol Biotechnol, 1997 Nov-Dec, 19(5-6), 360 - 8 Evidence for the evolution of a single component phenol/cresol hydroxylase from a multicomponent toluene monooxygenase; Olsen RH et al.; We have previously reported on the organization of a unique toluene-3-monooxygenase pathway for the degradation of alkyl-substituted petroleum hydrocarbons including characteristics of the second step in the pathway transforming phenols to catechols . In the present work we have focused on the regulation and unusual genetic organization of this metabolic step . In particular, we have sequenced the 3-kb DNA interval between the region encoding the tbuD gene product (phenol/cresol hydroxylase) and part of the toluene-3-monooxygenase operon of strain PKO1 . Then, various regions of this DNA were fused to a LacZ expression system to ascertain the location of the tbuD gene promoter and the binding site for its regulator, TbuT . The 5' end for transcripts for the putative promoter of the tbuD gene was also analyzed using primer extension analysis . Collectively, these results revealed that the promoter was located 2.5-kb upstream of the region encoding the tbuD gene product whose N-terminal region had been previously determined by peptide sequencing . Remarkably, the intervening 2.5-kb region showed sequence identity to results we reported previously for a multi-subunit toluene-2-monooxygenase cloned from a different bacterium, strain JS150, for which phenols are also substrates and effectors . When the DNA sequence for the tbuD gene and its contiguous 2.5-kb upstream region were compared to the entire toluene-2-monooxygenase sequence cloned from strain JS150, a promoter proximal region encoding three reading frames showed 99% identity to subunits for the toluene-2-monooxygenase operon . Within the contiguous tbuD gene region, however, DNA sequence homology was reduced to 64% overall identity and deduced amino acid sequence homology was only 21% similar . Although regions internal to the tbuD gene showed homology to corresponding toluene-2-monooxygenase subunits, domains associated with the putative functions proposed for such subunits were deleted . We believe that these results suggest that through evolution either tbuD was derived from the 2-monooxygenase pathway by deletions and molecular rearrangements, or alternatively the tbuD gene recruited part of the 2-monooxygenase pathway and its regulatory system which is activated by benzene, alkyl-substituted benzenes and phenols. Lett Appl Microbiol, 1997 Dec, 25(6), 426 - 30 Modulation of spore adhesion of the hyperparasitic bacterium Pasteuria penetrans to nematode cuticle; Sharma SB et al.; Monoclonal antibodies (mAb) raised to the cuticule surface of second-stage juveniles (J2) of the nematode Heterodera cajani were partially characterized by immunofluorescence and Western blot analysis . Five antigens with relative molecular weights (M(r)) 55, 80, 110, 180 and 210 kDa were identified with six mAb . Pasteuria spores, originating from the same population of H . cajani to which the antibodies were raised, were tested for their ability to attach to J2, which had been pretreated with each of the mAb . Monoclonal antibody HC/129 was found to reduce spore attachment by 42%, whereas HC/145 increased spore attachment by 124% . This is the first record of an antibody binding to the cuticle and increasing spore attachment, and suggests that components of the cuticle involved in inhibiting spore attachment may be masking the Pasteuria receptor present on the cuticle. Am J Gastroenterol, 1998 Jan, 93(1), 30 - 4 Analysis of the expression of CagA and VacA and the vacuolating activity in 167 isolates from patients with either peptic ulcers or non-ulcer dyspepsia; Takata T et al.; OBJECTIVES: The goals of this study were: 1) to examine the prevalence of cytotoxin-associated protein (CagA), vacuolating cytotoxin (VacA), and the vacuolating cytotoxin activity (VCA) in vitro of infecting Helicobacter pylori isolates and 2) to clarify the relation between the expression of these virulence factors and the occurrence of peptic ulceration . METHODS: One hundred sixty-seven clinical isolates of H . pylori from patients with peptic ulcer disease (gastric ulcer, 62 cases; duodenal ulcer, 48 cases) and nonulcer dyspepsia (57 cases) were studied regarding their genetic and phenotypic properties . RESULTS: Type 1 bacteria, which had both CagA and VCA, and type 2 bacteria, which did not express either CagA or VCA, represented 62.9% and 7.8%, respectively; the remaining 29.4% had an intermediate phenotype, expressing either CagA independent of the presence of VCA (CagA+VCA-) or vice versa (CagA-VCA+) . CagA+VCA- and CagA-VCA+ bacteria represented 17.4 % and 12.0%, respectively, both of which were more numerous than the type 2 category . The proportion of the CagA-positive isolates was significantly higher in both the duodenal ulcer (97.9%) and gastric ulcer (83.9%) patients than in the non-ulcer dyspepsia patients (61.4%) (p < 0.01) . On the other hand, the proportion of VacA/VCA-positive isolates was not significantly different between peptic ulcer disease and non-ulcer dyspepsia . CONCLUSIONS: The currently used classification of this bacterium based on the concomitant expression of CagA and VacA/VCA into the two major types is not adequate . The CagA-positive phenotype thus may be important as a virulence marker for peptic ulcer disease independent of the presence of VacA/VCA. J Chromatogr B Biomed Sci Appl, 1997 Dec 5, 703(1-2), 177 - 83 Quantitative analysis of a bacteria-derived antibiotic in nematode-infected insects using HPLC-UV and TLC-UV methods; Hu K et al.; 3,5-Dihydroxy-4-isopropylstilbene (ST), an antibiotic produced by the bacterial symbiont Photorhabdus luminescens of the nematodes of the genus Heterorhabditis was determined quantitatively in nematode bacterium-infected insects using HPLC or TLC for separation and UV for quantification . Comparable and reproducible results were obtained with both HPLC-UV and TLC-UV methods . Several factors, including solvents for extraction of the antibiotic from the infected insects, eluents for TLC development and programs for HPLC operation, were investigated . Of the four solvents used, namely acetone, methanol, ethyl acetate and diethyl ether, acetone had the highest extraction efficacy, and the ST recovery rate was about 95% . ST can be easily separated from all other bacterial metabolites on a TLC plate using a mixture of chloroform-methanol (98.5:1.5) or by HPLC using acetonitrile and water as the mobile phase. J Immunol Methods, 1997 Nov 10, 209(1), 17 - 24 Dual silver staining to characterise Helicobacter spp . outer membrane components; Keenan JI et al.; Helicobacter pylori is a bacterial pathogen, estimated to infect half the world's population . The bacterium is the aetiological cause of gastritis, the common precursor for peptic ulcer disease and gastric cancer . Immunisation of at-risk individuals is the most cost-effective means of dealing with such a widespread pathogen . Potential vaccine candidates need to be identified and characterised . Conventional silver staining is commonly used for the sensitive detection of bacterial protein components separated by SDS-PAGE . Modified silver stains employing periodate oxidation have also been developed for the analysis of purified bacterial lipopolysaccharide . By using these methods in parallel, as a dual silver stain, bacterial fractions can be characterised in terms of protein and LPS content . Strain differences can also be readily identified by comparing protein and LPS profiles . When combined with differential immunoblotting, the dual silver stain is a useful analytical tool for characterising potential vaccine candidate antigens. Appl Microbiol Biotechnol, 1997 Oct, 48(4), 528 - 33 Isolation and characterization of a marine bacterium capable of utilizing 2-methylphenanthrene; Gilewicz M et al.; A marine bacterium isolated from a coastal hydrocarbon-polluted sediment has been described and attributed on the basis of its phenotypic and genotypic characteristics to the genus Sphingomonas sp . This strain was capable of using an alkylated phenanthrene 2-methylphenanthrene, as sole source of carbon and energy . In experiments, 2-methylphenanthrene (0.2 g/l) was added as crystals to the culture medium . After 5 days of aerobic growth at 30 degrees C, 70% was degraded and the complete dissipation occurred after 20 days . Furthermore, the strain could degrade various kinds of polyaromatic compounds, but failed to grow on aliphatic hydrocarbons. J Clin Periodontol, 1997 Dec, 24(12), 937 - 44 Attachment loss and serum antibody levels against autologous and reference strains of Actinobacillus actinomycetemcomitans in untreated localized juvenile periodontitis patients; Tinoco EM et al.; Immunological data have been suggested to be a potential tool in the diagnosis, classification and monitoring of periodontal diseases . However, the role of circulating antibodies in periodontal patients is poorly understood . Patients suffering from localized juvenile periodontitis (LJP) are often reported to show high titers of serum IgG antibodies against Actinobacillus actinomycetemcomitans (A . actinomycetemcomitans), but several affected patients do not . Most studies use well-known reference strains of the bacterium for testing against the patients' sera . The aim of the present investigation was to study the relationship between serum IgG antibody levels to autologous A . actinomycetemcomitans strains and clinical attachment loss (CAL) . In addition, we wanted to assess the patients' serum titers against 4 well-known reference strains of the bacterium as well as their general potential immunoglobulin response . Intravenous blood samples were taken from 23 LJP patients and 10 healthy individuals, and autologous A . actinomycetemcomitans strains were cultured from 18 of the LJP patients . CAL was measured at 4 different sites around all present teeth and assessed as a % of teeth with at least 1 site moderately > or = 2 < 5 mm) or severely (> or = 5 mm) involved . An enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the serum titers of IgG antibodies to A . actinomycetemcomitans antigens . No significant correlation was found between serum IgG antibody titers to autologous strains and CAL . However, there was a trend that low responders had more moderately affected teeth than had high responders and patients with undetectable A . actinomycetemcomitans levels, which is in agreement with a hypothetically protective role of the antibodies . The total counts of immunoglobulin assessed in all participants showed that the predominant class was IgG and the reference group displayed significantly less (p < 0.05) IgG and IgG1 counts than the LJP patients . Both the reaction pattern against reference and autologous strains varied widely . We conclude that the specific antibody response against A . actinomycetemcomitans shows a weak correlation to clinical attachment levels in LJP patients. Tidsskr Nor Laegeforen, 1997 Nov 10, 117(27), 3937 - 40 {Q-fever imported into Norway}; Jensenius M et al.; Q fever is an important zoonosis that occurs throughout the world . In contrast to most other European countries, there has been no evidence of endemic Q fever in Norway up to now . The disease is caused by Coxiella burnetii, a rickettsia-like bacterium . Humans are infected mainly by inhalation of contaminated aerosols from cattle, sheep and goats . Clinical manifestations are protean, ranging from asymptomatic infection to life-threatening endocarditis . In this article we present the first four cases of serological proven acute Q fever imported into Norway . The patients were Norwegian tourists who had visited Bhutan, the Canary Islands, and Morocco . Two patients had fever with maculopapular exanthema, one had pneumonia, and one had biopsy-proven granulomatous hepatitis . Three were treated with tetracyclines . All four patients recovered well. J Bacteriol, 1998 Jan, 180(2), 274 - 81 Reverse gyrase from the hyperthermophilic bacterium Thermotoga maritima: properties and gene structure; Bouthier de la Tour C et al.; The hyperthermophilic bacterium Thermotoga maritima MSB8 possesses a reverse gyrase whose enzymatic properties are very similar to those of archaeal reverse gyrases . It catalyzes the positive supercoiling of the DNA in an Mg2+- and ATP-dependent process . Its optimal temperature of activity is around 90 degrees C, and it is highly thermostable . We have cloned and DNA sequenced the corresponding gene (T . maritima topR) . This is the first report describing the analysis of a gene encoding a reverse gyrase in bacteria . The T . maritima topR gene codes for a protein of 1,104 amino acids with a deduced molecular weight of 128,259, a value in agreement with that estimated from the denaturing gel electrophoresis of the purified enzyme . Like its archaeal homologs, the T . maritima reverse gyrase exhibits helicase and topoisomerase domains, and its sequence matches very well the consensus sequence for six reverse gyrases now available . Phylogenetic analysis shows that all reverse gyrases, including the T . maritima enzyme, form a very homogeneous group, distinct from the type I 5' topoisomerases of the TopA subfamily, for which we have previously isolated a representative gene in T . maritima (topA) . The coexistence of these two distinct genes, coding for a reverse gyrase and an omega-like topoisomerase, respectively, together with the recent description of a gyrase in T . maritima (O . Guipaud, E . Marguet, K . M . Noll, C . Bouthier de la Tour, and P . Forterre, Proc . Natl . Acad . Sci . USA 94:10606-10611, 1977) addresses the question of the control of the supercoiling in this organism. J Biol Chem, 1997 Dec 19, 272(51), 32121 - 8 Purification to homogeneity and reconstitution of the individual components of the epoxide carboxylase multiprotein enzyme complex from Xanthobacter strain Py2; Allen JR et al.; Epoxide metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by an NADPH- and NAD+-dependent carboxylation reaction that forms beta-keto acids as products . Epoxide carboxylase, the enzyme catalyzing this reaction, was resolved from the soluble fraction of cell-free extracts into four protein components that are obligately required for functional reconstitution of epoxide carboxylase activity . One of these components, component II, has previously been purified and characterized as an NADPH:disulfide oxidoreductase . In the present study, the three additional epoxide carboxylase components have been purified to homogeneity and characterized . These component proteins are as follows: component I, a homohexameric protein consisting of 41.7-kDa subunits; component III, a dimeric protein consisting of 26.0- and 26.2-kDa polypeptides; and component IV, a dimeric protein consisting of a single 25.4-kDa polypeptide . Component I contained 5 mol of tightly bound zinc per mol of protein . Component I was specifically inactivated by methylepoxypropane, a time-dependent irreversible inactivator of epoxide carboxylase activity, suggesting that this component plays an integral role in epoxide binding and activation . No metals or organic cofactors were detected for components III and IV . The molecular weights, N-terminal sequences, and amino acid compositions of the purified epoxide carboxylase components were determined and found to correlate with open reading frames within and adjacent to a cloned fragment of DNA that complements Xanthobacter Py2 mutants defective in epoxide degradation . Using the purified epoxide carboxylase system, epoxide carboxylation was found to be stoichiometrically coupled to the transhydrogenation of pyridine nucleotide cofactors according to the following equation: epoxypropane + CO2 + NADPH + NAD+ --> acetoacetate + H+ + NADP+ + NADH. Eur J Med Res, 1996 Sep 20, 1(11), 537 - 42 Granulocyte activation by Helicobacter pylori; Hatz RA et al.; Helicobacter pylori-associated gastritis (HAG) is characterized by granulocytic and mononuclear cell infiltrates within infected gastric mucosa . Since the bacterium does not invade the epithelial layer, it must be assumed that components or products of the pathogen which permeate the epithelial barrier may initiate chemotaxis and activation of neutrophils . The aim of this study was to evaluate the effect of H . pylori water soluble protein (WSP) components on the induction of granulocyte adherence and activation . The results show that H . pylori WSP led to enhanced expression of the beta 2-integrin CD11b/CD18 on the granulocyte surface . Following upregulation of this adhesion molecule, activated granulocytes demonstrated increased adhesion to human endothelial cells (HUVEC) in culture . These observations support the hypothesis that in vivo neutrophil activation may be a direct result of H . pylori constituents promoting transendothelial migration into the lamina propria of infected gastric mucosa. Eur Arch Otorhinolaryngol, 1997, 254(9-10), 481 - 2 A possible role of Helicobacter pylori infection in the etiology of chronic laryngitis; Borkowski G et al.; In order to study a possible role of Helicobacter pylori infection in chronic laryngitis, we performed endoscopic and histological assessments in addition to a urease test for the bacterium in 35 patients with chronic hoarseness . Six of the patients investigated (17.1%) revealed a positive urease test of the laryngeal biopsy (four male and two female patients) . These H . pylori-positive patients were treated with omeprazole and an antibiotic regimen using clarithromycin and metronidazole . This led to an eradication of the H . pylori and resolution of clinical signs and symptoms . These findings show a possible role of H . pylori infection in the etiology of chronic laryngitis in certain patients and can be important for clinical diagnosis and treatment. Gastroenterologist, 1997 Dec, 5(4), 295 - 305 Helicobacter pylori: beyond peptic ulcer disease; Wisniewski RM et al.; Beyond peptic ulcer disease, Helicobacter pylori infection is associated with intestinal-type gastric cancer and low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma . It is also currently implicated as a possible cause of dyspepsia and extraintestinal disorders such as coronary artery disease, rosacea, chronic urticaria, and delayed growth in children . There are strong epidemiological data from large cohort studies linking H . pylori to gastric adenocarcinoma . Several cofactors, including early childhood acquisition of infection, strain-specific differences, genetic predisposition of the host, and the environment, appear to play a role in the progression of chronic gastritis to gastric cancer . H . pylori infection is seen in over 90% of MALT lymphomas, and about 70% of localized nonbulky tumors will undergo complete histological regression after eradication of the bacterium . Because follow-up data are limited to less than 2 years, those undergoing H . pylori eradication as primary therapy for MALT lymphoma require frequent histological surveillance for tumor recurrence . There are conflicting data from short-term studies regarding the effect of H . pylori eradication on dyspeptic symptoms . The decision to test or not for H . pylori in the dyspeptic patient may become easier when well-controlled studies with longer periods of follow-up become available . Because H . pylori induces a systemic inflammatory response, investigators are beginning to explore possible extraintestinal disease associations with the infection . The global prevalence of both peptic ulcer disease and gastric cancer has led to studies focusing on noninvasive screening for H . pylori in high-risk populations and prevention of primary infection by means of vaccination. Appl Environ Microbiol, 1998 Jan, 64(1), 126 - 32 Heterogeneity in the attachment and uptake mechanisms of the Legionnaires' disease bacterium, Legionella pneumophila, by protozoan hosts; Harb OS et al.; Invasion and intracellular replication of Legionella pneumophila within protozoa in the environment plays a major role in the transmission of Legionnaires' disease . Intracellular replication of L . pneumophila within protozoa occurs in a rough endoplasmic reticulum (RER)-surrounded phagosome (Y . Abu Kwaik, Appl . Environ . Microbiol . 62:2022-2028, 1996) . Since the subsequent fate of many intracellular pathogens is determined by the route of entry, we compared the mechanisms of attachment and subsequent uptake of L . pneumophila by the two protozoa Hartmannella vermiformis and Acanthamoeba polyphaga . Our data provide biochemical and genetic evidence that the mechanisms of attachment and subsequent uptake of L . pneumophila by the two protozoan hosts are, in part, different . First, uptake of L . pneumophila by H . vermiformis is completely blocked by the monovalent sugars galactose and N-acetyl-D-galactosamine, but these sugars partially blocked A . polyphaga . Second, attachment of L . pneumophila to H . vermiformis is associated with a time-dependent and reversible tyrosine dephosphorylation of multiple host proteins . In contrast, only a slight dephosphorylation of a 170-kDa protein of A . polyphaga is detected upon infection . Third, synthesis of H . vermiformis proteins but not of A . polyphaga proteins is required for uptake of L . pneumophila . Fourth, we have identified L . pneumophila mutants that are severely defective in attachment to A . polyphaga but which exhibit minor reductions in attachment to H . vermiformis and, thus, provide a genetic basis for the difference in mechanisms of attachment to both protozoa . The data indicate a remarkable adaptation of L . pneumophila to attach and invade different protozoan hosts by different mechanisms, yet invasion is followed by a remarkably similar intracellular replication within a RER-surrounded phagosome and subsequent killing of the host cell. Appl Environ Microbiol, 1998 Jan, 64(1), 53 - 61 ISD1, an insertion element from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough: structure, transposition, and distribution; Fu R et al.; Insertion element ISD1, discovered when its transposition caused the insertional inactivation of an introduced sacB gene, is present in two copies in the genome of Desulfovibrio vulgaris Hildenborough . Southern blot analysis indicated at least two insertion sites in the sacB gene . Cloning and sequencing of a transposed copy of ISD1 indicated a length of 1,200 bp with a pair of 44-bp imperfect inverted repeats at the ends, flanked by a direct repeat of the 4-bp target sequence . AAGG and AATT were found to function as target sequences . ISD1 encodes a transposase from two overlapping open reading frames by programmed translational frameshifting at an A6G shifty codon motif . Sequence comparison showed that ISD1 belongs to the IS3 family . Isolation and analysis of the chromosomal copies, ISD1-A and ISD1-B, by PCR and sequencing indicated that these are not flanked by direct repeats . ISD1-A is inserted in a region of the chromosome containing the gapdh-pgk genes (encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase) . Active transposition to other loci in the genome was demonstrated, offering the potential of a new tool for gene cloning and mutagenesis . ISD1 is the first transposable element described for the sulfate reducers, a large and environmentally important group of bacteria . The distribution of ISD1 in genomes of sulfate-reducing bacteria is limited . A single copy is present in the genome of D . desulfuricans Norway. Appl Environ Microbiol, 1998 Jan, 64(1), 43 - 52 Purification and properties of ArfI, an alpha-L-arabinofuranosidase from Cytophaga xylanolytica; Renner MJ et al.; An alpha-L-arabinofuranosidase (alpha-L-arabinofuranoside arabinofuranohydrolase {EC 3.2.1.55}; referred to below as ArfI) from Cytophaga xylanolytica XM3 was purified 85-fold by anion-exchange and hydrophobic interaction column chromatography . The native enzyme had a pI of 6.1 and an apparent molecular mass of 160 to 210 kDa, and it appeared to be a trimer or tetramer consisting of 56-kDa subunits . With p-nitrophenyl-alpha-L-arabinofuranoside as the substrate, the enzyme exhibited a K(m) of 0.504 mM and a Vmax of 319 mumol.min-1.mg of protein-1, and it had optimum activity at pH 5.8 and 45 degrees C . ArfI was relatively stable over a pH range of 4 to 10 and at temperatures up to 45 degrees C, and it retained nearly full activity when stored at 4 degrees C for periods as long as 24 months . The enzyme also released arabinose from 4-methylumbelliferyl-alpha-L-arabinofuranoside, as well as from rye, wheat, corn cob, and oat spelt arabinoxylans and sugar beet arabinan, but not from arabinogalactan . ArfI showed no hydrolytic activity toward a range of p-nitrophenyl- or 4-methylumbelliferyl-glycosides other than arabinoside, for which it was entirely specific for the alpha-L-furanoside configuration . ArfI interacted synergistically with three partially purified endoxylanase fractions from C . xylanolytica in hydrolyzing rye arabinoxylan . However, cell fractionation studies revealed that ArfI was largely, if not entirely, cytoplasmic, so its activity in vivo is probably most relevant to hydrolysis of arabinose-containing oligosaccharides small enough to pass through the cytoplasmic membrane . Antibodies prepared against purified ArfI also cross-reacted with arabinofuranosidases from other freshwater and marine strains of C . xylanolytica, as well as with some proteins that did not possess arabinofuranosidase activity . To our knowledge, this is the first alpha-L-arabinofuranosidase to be purified and characterized from any gliding bacterium. Chin J Biotechnol, 1997, 13(3), 193 - 9 Isolation and identification of berberine from cell cultures of Coptis chinensis; Mao T et al.; Calli were formed by the young leaves of Coptis chinensis inoculated onto a 6,7-V solid medium containing 1.0-2.0 mg/L of 2,4-D and 0.1 mg/L of kinetin . The loose calli were chosen and transferred into a liquid medium and free cells and cell aggregates were obtained . Cell lines with a higher content of berberine were selected by irradiation of suspension culture and a plate-screening technique . After 35 subcultures, the selected cell lines were cultured in a larger quantity for the extraction of alkaloids . Yellow crystals were obtained from the extracts and identified as berberine by TCL, UV and IR absorption, and mass spectrography . They have the same molecular structure and anti-bacterium activities as the berberine obtained from natural plants. Mol Microbiol, 1997 Dec, 26(5), 927 - 37 The Rhodobacter capsulatus hupSLC promoter: identification of cis-regulatory elements and of trans-activating factors involved in H2 activation of hupSLC transcription; Toussaint B et al.; The {NiFe}hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus is encoded by the structural hupSLC operon, the expression of which is induced by H2 . H2 activation was no longer observable in chromosomal hupR mutants, an indication that HupR is implicated directly in the activation by H2 of hupS gene expression . The transcriptional start site of the hupS promoter, determined by primer extension mapping, was located 55 nucleotides upstream from the translational start codon of the hupS gene . Regulatory sequences were identified by serial 5' deletions of the 300bp hupS promoter-regulatory region (phupS) and phupS-lacZ translational fusions . Cis-regulatory sequences capable of interacting with two transcription factors, IHF and HupR, a response regulator of the NtrC subfamily, were studied by electrophoretic mobility shift assays (EMSAs) . The R . capsulatus IHF and HupR proteins were overexpressed in Escherichia coli and purified by affinity chromatography . IHF binds to a site, 5'-TCACACACCATTG, centred at -87 nt from the transcription start site . The HupR protein binds to one site within the -162 to -152 nt region, which contains the palindromic sequence 5'-TTG-R5-CAA . By the use of 5' deletions and site-directed mutagenesis of the -162/-152 region, this palindrome was shown to be required for in vivo hupS transcriptional activation by H2. Mol Microbiol, 1997 Dec, 26(5), 897 - 910 Requirement of topoisomerase IV parC and parE genes for cell cycle progression and developmental regulation in Caulobacter crescentus; Ward D et al.; We have identified the parC and parE genes encoding DNA topoisomerase IV (Topo IV) in Caulobacter crescentus . We have also characterized the effect of conditional Topo IV mutations on cell division and morphology . Topo IV mutants of C . crescentus are unlike mutants of Escherichia coli and S . typhimurium, which form long filamentous cells that are defective in nucleoid segregation and divide frequently to produce anucleate cells . Topo IV mutants of C . crescentus are highly pinched at multiple sites (cell separation phenotype) and they do not divide to produce cells lacking DNA . These results suggest unique regulatory mechanisms coupling nucleoid partitioning and cell division in this aquatic bacterium . In addition, distinctive nucleoid-partitioning defects are not apparent in C . crescentus Topo IV mutants as they are in E . coli and S . typhimurium . However, abnormal nucleoid segregation in parE mutant cells could be demonstrated in a genetic background containing a conditional mutation in the C . crescentus ftsA gene, an early cell division gene that is epistatic to parE for cell division and growth . We discuss these results in connection with the possible roles of C . crescentus Topo IV in the regulation of cell division, chromosome partitioning, and late events in polar morphogenesis . Although the ParC and ParE subunits of Topo IV are very similar in sequence to the GyrA and GyrB subunits of DNA gyrase, we have used DNA sequence analysis to identify a highly conserved 'GyrA box' sequence that is unique to the GyrA proteins and may serve as a hallmark of the GyrA protein family. Infect Immun, 1998 Jan, 66(1), 247 - 58 Borrelia burgdorferi induces the production and release of proinflammatory cytokines in canine synovial explant cultures; Straubinger RK et al.; Canine synovial membrane explants were exposed to high- or low-passage Borrelia burgdorferi for 3, 6, 12, and 24 h . Spirochetes received no treatment, were UV light irradiated for 16 h, or were sonicated prior to addition to synovial explant cultures . In explant tissues, mRNA levels for the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), IL-1beta, and IL-8 were surveyed semiquantitatively by reverse transcription-PCR . Culture supernatants were examined for numbers of total and motile (i.e., viable) spirochetes, TNF-like and IL-1-like activities, polymorphonuclear neutrophil (PMN) chemotaxis-inducing activities, and IL-8 . During exposure to synovial explant tissues, the total number of spirochetes in the supernatants decreased gradually by approximately 30%, and the viability also declined . mRNAs for TNF-alpha, IL-1alpha, IL-1beta, and IL-8 were up-regulated in synovial explant tissues within 3 h after infection with untreated or UV light-irradiated B . burgdorferi, and mRNA levels corresponded to the results obtained with bioassays . During 24 h of coincubation, cultures challenged with untreated or UV light-irradiated spirochetes produced similar levels of TNF-like and IL-1-like activities . In contrast, explant tissues exposed to untreated B . burgdorferi generated significantly higher levels of chemotactic factors after 24 h of incubation than did explant tissues exposed to UV light-treated spirochetes . In identical samples, a specific signal for IL-8 was identified by Western blot analysis . High- and low-passage borreliae did not differ in their abilities to induce proinflammatory cytokines . No difference in cytokine induction between untreated and sonicated high-passage spirochetes was observed, suggesting that fractions of the organism can trigger the production and release of inflammatory mediators . The titration of spirochetes revealed a dose-independent cytokine response, where 10(3) to 10(7) B . burgdorferi organisms induced similar TNF-like activities but only 10(7) spirochetes induced measurable IL-1-like activities . The release of chemotactic factors was dose dependent and was initiated when tissues were infected with at least 10(5) organisms . We conclude that intact B . burgdorferi or fractions of the bacterium can induce the local up-regulation of TNF-alpha, IL-1alpha, and IL-1beta in the synovium but that the interaction of viable spirochetes with synovial cells leads to the release of IL-8, which probably is a prime initiator of PMN migration during acute Lyme arthritis. Biochemistry, 1997 Dec 9, 36(49), 15269 - 76 Conformation-activated protonation in reaction centers of the photosynthetic bacterium Rhodobacter sphaeroides; Kalman L et al.; Kinetics and stoichiometry of proton binding/unbinding induced by intense (1 W cm-2) and continuous illumination were measured in the isolated reaction center (RC) protein from photosynthetic purple bacterium Rhodobacter sphaeroides in the absence of an external electron donor . At high ionic strength (100 mM), large proton release (approximately 6 H+ per RC) was observed at pH 6 and substoichiometric H+-ion binding (approximately 0.3 H+ per RC) at pH 8 . These observations together with optical spectroscopy on the oxidized dimer indicate that, at room temperature, two distinct conformations of the RC can be obtained depending on the pH, Eh, and illumination . Acidic pH, a large redox gap between the actual Eh of the solution and the midpoint potential of the acceptor quinone, and strong illumination favor the conversion of the RC from the dark-adapted state to the light-adapted state . These conformations differ greatly in the rates of primary photochemistry, the reoxidation of semiquinone and the rereduction of the oxidized dimer, and the protonation states of the amino acids of the protein . Whereas substoichiometric proton unbinding is observed in the P+Q redox state of the protein in the dark-adapted conformation, much larger H+-ion release is detected in the light-adapted conformation . From the pH dependence of the key processes in the conformational change and reoxidation of semiquinone, we concluded that they are controlled by protonatable groups available in the protein . A simple phenomenological model is presented that relates the rates and equilibrium constants of the electron transfer reactions and the conformational change of the RC. J Bacteriol, 1998 Jan, 180(1), 159 - 62 Dynamics of iron uptake and Fe3O4 biomineralization during aerobic and microaerobic growth of Magnetospirillum gryphiswaldense; Schuler D et al.; Iron uptake and magnetite (Fe3O4) crystal formation could be studied in the microaerophilic magnetic bacterium Magnetospirillum gryphiswaldense by using a radioactive tracer method for iron transport and a differential light-scattering technique for magnetism . Magnetite formation occurred only in a narrow range of low oxygen concentration, i.e., 2 to 7 microM O2 at 30 degrees C . Magnetic cells stored up to 2% iron as magnetite crystals in intracytoplasmic vesicles . This extraordinary uptake of iron was coupled tightly to the biomineralization of up to 60 magnetite crystals with diameters of 42 to 45 nm. J Virol, 1998 Jan, 72(1), 358 - 65 Potato leafroll virus binds to the equatorial domain of the aphid endosymbiotic GroEL homolog; Hogenhout SA et al.; A GroEL homolog with a molecular mass of 60 kDa, produced by the primary endosymbiotic bacterium (a Buchnera sp.) of Myzus persicae and released into the hemolymph, has previously been shown to be a key protein in the transmission of potato leafroll virus (PLRV) . Like other luteoviruses and pea enation mosaic virus, PLRV readily binds to extracellular Buchnera GroEL, and in vivo interference in this interaction coincides with reduced capsid integrity and loss of infectivity . To gain more knowledge of the nature of the association between PLRV and Buchnera GroEL, the groE operon of the primary endosymbiont of M . persicae (MpB groE) and its flanking sequences were characterized and the PLRV-binding domain of Buchnera GroEL was identified by deletion mutant analysis . MpB GroEL has extensive sequence similarity (92%) with Escherichia coli GroEL and other members of the chaperonin-60 family . The genomic organization of the Buchnera groE operon is similar to that of the groE operon of E . coli except that a constitutive promoter sequence could not be identified; only the heat shock promoter was present . By a virus overlay assay of protein blots, it was shown that purified PLRV bound as efficiently to recombinant MpB GroEL (expressed in E . coli) as it did to wild-type MpB GroEL . Mutational analysis of the gene encoding MpB GroEL revealed that the PLRV-binding site was located in the so-called equatorial domain and not in the apical domain which is generally involved in polypeptide binding and folding . Buchnera GroEL mutants lacking the entire equatorial domain or parts of it lost the ability to bind PLRV . The equatorial domain is made up of two regions at the N and C termini that are not contiguous in the amino acid sequence but are in spatial proximity after folding of the GroEL polypeptide . Both the N- and C-terminal regions of the equatorial domain were implicated in virus binding. Harefuah, 1997 Oct 2, 133(7-8), 281 - 3, 335 {Q fever endocarditis and bicuspid aortic valve}; Shovman O et al.; Q fever is caused by the rickettsia Coxiella burnetti, an obligate intracellular bacterium acquired by inhalation of infected dust from subclinically infected animals . Q fever may be acute or chronic; the chronic form mostly presents as endocarditis . Immunocompromised states and underlying heart disease are the most important risk factors . Usually the symptoms of Q fever endocarditis are nonspecific and diagnosis is often established very late . New criteria for diagnosis include a single blood culture positive for Coxiella burnetti, positive Q fever serology and characteristic echocardiographic studies . We describe a 49-year-old man with bicuspid aortic valve admitted with fever, weight loss and a new heart murmur . The diagnosis of Q fever endocarditis was established by positive Q fever serology, and an echocardiogram showing vegetations and valvular dysfunction . This case suggests that Q fever endocarditis should be considered in patients with "sterile" endocarditis. FEMS Microbiol Lett, 1997 Dec 1, 157(1), 195 - 200 Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex; Kuczek K et al.; An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2) . The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase . There is a high similarity with acyltransferase domains from so-called type I polyketide synthases . Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants . The amplified fragment is considered to be a part of a larger gene complex. FEMS Microbiol Lett, 1997 Dec 1, 157(1), 95 - 101 Purification and characterization of 2,4-dichlorophenol hydroxylase isolated from a bacterium of the alpha-2 subgroup of the Proteobacteria; Makdessi K et al.; 2,4-Dichlorophenol hydroxylase (EC 1.14.13.20) was purified to apparent homogeneity from the bacterial strain S1, a member of the alpha-2 subgroup of the Proteobacteria . The molecular masses of the native enzyme and the subunit were determined to be 256 and 64 kDa, respectively, suggesting a homotetrameric structure . The enzyme converted 2,4-dichlorophenol to 3,5-dichlorocatechol . The apparent K(m) values for 2,4-dichlorophenol, NADPH and NADH were 3, 240 and 420 microM, respectively . The enzyme hydroxylated a broad range of halogenated phenols . 3-Chloro-, 2,6-dichloro- and 2,4,6-trichlorophenol acted as 'non-substrate' effectors . The N-terminal sequence revealed 72% identity with the amino acid sequence deduced from the pJP4-encoded tfdB gene. J Mol Biol, 1997 Dec 12, 274(4), 676 - 83 Disruption of an ionic network leads to accelerated thermal denaturation of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima; Pappenberger G et al.; The role of an ionic network of four charged amino acid side-chains in the thermostability of the enzyme D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima (TmGAPDH) has been assessed by site-directed mutagenesis, replacing the central residue of the ionic network, arginine 20, by either alanine (R20A) or asparagine (R20N) . The purified mutant enzymes display no differences to the wild-type enzyme regarding spectroscopic properties and enzymatic activity . However, denaturation kinetics reveal that the resistance towards thermal denaturation is strongly diminished in the mutant enzymes . This is reflected by a decrease in free energy of activation for thermal unfolding of about 4 kJ/mol at 100 degrees C and a shift of temperature of half denaturation after one hour incubation from 96 to 89 degrees C for both mutant enzymes . Due to a large decrease in activation enthalpy, the effects of the mutations are temperature dependent and become even more significant at the physiological temperature of Thermotoga maritima (approximately 80 degrees C) . The importance of the arginine 20 side-chain for kinetic thermal stability is plausible in the light of its key role in the ionic network and the strategic positioning of this ionic network in the context of the overall protein structure. J Cell Biochem, 1998 Jan 1, 68(1), 1 - 7 Magnetic field exposure enhances mRNA expression of sigma 32 in E . coli; Cairo P et al.; The mechanism of interaction between weak electromagnetic fields and cells is not understood . As a result, the health effect(s) induced by exposure to these fields remains unclear . In addition to questions relating to the site of initial magnetic field (MF) interactions, the nature of the cell's response to these perturbations is also unclear . We examined the hypothesis that the cells respond to MFs in a manner similar to other environmental stressors such as heat . Using the bacterium Escherichia coli, we examined the mRNA levels of sigma 32, a protein that interacts with RNA polymerase to help it recognize a variety of stress promoters in the cell . Our data show that the intracellular level of sigma 32 mRNA is enhanced following a 15-min exposure to a 60 Hz, 1.1 mT magnetic field. Jpn J Cancer Res, 1997 Oct, 88(10), 921 - 4 Helicobacter pylori extracts exhibit nicotinamide adenine dinucleotide-derived adenylation but not mono(adenosine 5'-diphosphate-ribosyl)ation of DNA ligase; Nozaki T et al.; The issue of toxins produced by Helicobacter pylori (H . pylori) urgently requires clarification given that the bacterium causes gastric epithelial cell damage which may lead to precancerous and cancerous changes . During an investigation of the possibility of mono(adenosine 5'-diphosphate (ADP)-ribosyl)ation by H . pylori products, as observed for other bacterial toxins, we found that radioactivity of {adenylate-32P}nicotinamide adenine dinucleotide (NAD) is incorporated into an H . pylori protein of 80 kDa after incubation with crude bacterial extract . In contrast, {carbonyl-14C}NAD did not show any radioactivity incorporation . Unexpectedly, treatment of the modified protein with 0.1 N HCl, but not 0.1 N NaOH, released the AMP moiety . Such chemical properties are characteristic of bacterial DNA ligase-AMP complexes . We found that an antibody raised against Escherichia coli DNA ligase {EC 6.5.1.2} immunoprecipitated the modified 80 kDa protein . Our results indicate that incorporation of radioactivity derived from NAD into the 80 kDa protein was due to adenylation, but not mono(ADP-ribosyl)ation, of the DNA ligase of H . pylori. Biochemistry, 1997 Nov 18, 36(46), 14167 - 72 Isolation and properties of photochemically active reaction center complexes from the green sulfur bacterium Prosthecochloris aestuarii; Francke C et al.; A new and rapid procedure was developed for the isolation of the reaction center core (RCC)-complex from the green sulfur bacterium Prosthecochloris aestuarii . Reaction center preparations containing the Fenna Matthews Olson (FMO) protein were also obtained . The procedure involved incubation of broken cells with the detergents Triton X-100 and SB12, sucrose gradient centrifugation and hydroxyapatite chromatography . Three different pigment protein complexes were obtained: one containing (about) three FMO trimers per RCC, one with one FMO per RCC and one consisting of RCC only . The last one contained polypeptides with apparent molecular masses of 64 kDa (pscA) and 35 kDa (pscB, the FA/FB, FeS subunit), but no cytochrome . Bacteriochlorophyll a and the chlorophyll a isomer functioning as primary electron acceptor were present at a ratio of 4.8:1 . The complexes were also characterized spectroscopically and in terms of photochemical activity, at room temperature as well as at cryogenic temperatures . Illumination caused oxidation of the primary donor P840, with the highest activity in the RCC complex (DeltaA840/A810 = 0.06) . At room temperature in the RCC complex essentially all of the P840+ produced in a flash was re-reduced slowly in the dark (several seconds) . At low temperatures (150-10 K) a triplet was formed in a fraction of the reaction centers, presumably by a reversal of the charge separation, whereas in others P840+ formed in the light was re-reduced in 40-50 ms.
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
