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| J Bacteriol, 2000 Mar, 182(6), 1702 - 5 Dual roles of Bradyrhizobium japonicum nickelin protein in nickel storage and GTP-dependent Ni mobilization; Olson JW et al.; The hydrogenase accessory protein HypB, or nickelin, has two functions in the N(2)-fixing, H(2)-oxidizing bacterium Bradyrhizobium japonicum . One function of HypB involves the mobilization of nickel into hydrogenase . HypB also carries out a nickel storage/sequestering function in B . japonicum, binding nine nickel ions per monomer . Here we report that the two roles (nickel mobilization and storage) of HypB can be separated in vitro and in vivo using molecular and biochemical approaches . The role of HypB in hydrogenase maturation is completely dependent on its intrinsic GTPase activity; strains which produce a HypB protein that is severely deficient in GTPase activity but that fully retains nickel-sequestering ability cannot produce active hydrogenase even upon prolonged nickel supplementation . A HypB protein that lacks the nickel-binding polyhistidine region near the N terminus lacks only the nickel storage capacity function; it is still able to bind a single nickel ion and also retains complete GTPase activity. Biophys J, 2000 Mar, 78(3), 1570 - 7 Spectroscopy of individual light-harvesting 2 complexes of Rhodopseudomonas acidophila: diagonal disorder, intercomplex heterogeneity, spectral diffusion, and energy transfer in the B800 band; van Oijen AM et al.; This paper reports a detailed spectroscopic study of the B800 absorption band of individual light-harvesting 2 (LH2) complexes of the photosynthetic purple bacterium Rhodopseudomonas acidophila at 1 . 2 K . By applying single-molecule detection techniques to this system, details and properties can be revealed that remain obscured in conventional ensemble experiments . For instance, from fluorescence-excitation spectra of the individual complexes a more direct measure of the diagonal disorder could be obtained . Further spectral diffusion phenomena and homogeneous linewidths of individual bacteriochlorophyll a (BChl a) molecules are observed, revealing valuable information on excited-state dynamics . This work demonstrates that it is possible to obtain detailed spectral information on individual pigment-protein complexes, providing direct insight into their electronic structure and into the mechanisms underlying the highly efficient energy transfer processes in these systems. Infect Immun, 2000 Mar, 68(3), 1735 - 9 Bordetella pertussis virulence factors affect phagocytosis by human neutrophils; Weingart CL et al.; The interaction between human neutrophils and wild-type Bordetella pertussis or mutants expressing altered lipopolysaccharide or lacking virulence factors-pertussis toxin, adenylate cyclase toxin, dermonecrotic toxin, filamentous hemagglutinin (FHA), pertactin, or BrkA-was examined . In the absence of antibodies, the wild-type strain and the mutants, with the exception of mutants lacking FHA, attached efficiently to neutrophils . The addition of opsonizing antibodies caused a significant reduction (approximately 50%) in attachment of the wild-type strain and most of the mutants expressing FHA, suggesting that bacterium-mediated attachment is more efficient than Fc-mediated attachment . Phagocytosis was also examined . In the absence of antibodies, about 12% of the wild-type bacteria were phagocytosed . Opsonization caused a statistically significant reduction in phagocytosis (to 3%), possibly a consequence of reduced attachment . Phagocytosis of most of the mutants was similar to that of the wild type, with the exception of the mutants lacking adenylate cyclase toxin . About 70% of the adenylate cyclase toxin mutants were phagocytosed, but only in the presence of opsonizing antibody, suggesting that Fc receptor-mediated signaling may be needed for phagocytosis . These studies indicate that FHA mediates attachment of B . pertussis to neutrophils, but adenylate cyclase toxin blocks phagocytosis. Infect Immun, 2000 Mar, 68(3), 1480 - 4 Influence of the bcg locus on natural resistance to primary infection with the facultative intracellular bacterium Francisella tularensis in mice; Kovarova H et al.; The implication of the Bcg locus in the control of natural resistance to infection with a live vaccine strain (LVS) of the intracellular pathogen Francisella tularensis was studied . Analysis of phenotypic expression of natural resistance and susceptibility was performed using mouse strains congenic at the Bcg locus . Comparison of the kinetics of bacterial colonization of spleen showed that B10.A.Bcg(r) mice were extremely susceptible during early phases of primary sublethal infection, while their congenic C57BL/10N {Bcg(s)} counterparts could be classified as resistant to F . tularensis LVS infection according to the 2-log-lower bacterial CFU within the tissue as long as 5 days after infection . Different phenotypes of Bcg congenic mice were associated with differential expression of the cytokines tumor necrosis factor alpha, interleukin-10, and gamma interferon and production of reactive oxygen intermediates . These results strongly suggest that the Bcg locus, which is close or identical to the Nramp1 gene, controls natural resistance to infection by F . tularensis and that its effect is the opposite of that observed for other Bcg-controlled pathogens. Infect Immun, 2000 Mar, 68(3), 1441 - 9 Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes; Katz J et al.; Porphyromonas gingivalis is considered among the etiological agents of human adult periodontitis . Although in vitro studies have shown that P . gingivalis has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known . Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (beta1-integrin) transmembrane proteins . These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells . In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier . Using the MDCK cell system, we examined the effect of P . gingivalis on epithelial barrier function . Exposure of the basolateral surfaces of MDCK cells to P . gingivalis (>10(9) bacteria/ml) resulted in a decrease in transepithelial resistance . Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and beta1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateral P . gingivalis . Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure . Cell viability was not affected by either basolateral or apical exposure to P . gingivalis . Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and beta1-integrin in lysates derived from MDCK cells exposed to P . gingivalis . Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by P . gingivalis, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins . Collectively, these data suggest that P . gingivalis is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium . These results also indicate the importance of a critical threshold concentration of P . gingivalis to initiate epithelial barrier destruction. Infect Immun, 2000 Mar, 68(3), 1391 - 9 Role of decay-accelerating factor domains and anchorage in internalization of Dr-fimbriated Escherichia coli; Selvarangan R et al.; Dr-fimbriated Escherichia coli capable of invading epithelial cells recognizes human decay-accelerating factor (DAF) as its cellular receptor . The role of extracellular domains and the glycosylphosphatidylinositol anchor of DAF in the process of internalization of Dr(+) E . coli was characterized in a cell-cell interaction model . Binding of Dr(+) E . coli to the short consensus repeat 3 domain of DAF expressed by Chinese hamster ovary cells was critical for internalization to occur . Deletion of short consensus repeat 3 domain or replacement of Ser(165) by Leu in this domain, or the use of a monoclonal antibody to this region abolished internalization . Replacing the glycosylphosphatidylinositol anchor of DAF with the transmembrane anchor of membrane cofactor protein or HLA-B44 resulted in abolition or reduction of internalization respectively . Cells expressing glycosylphosphatidylinositol-anchored DAF but not the transmembrane-anchored DAF internalized Dr(+) E . coli through a glycolipid pathway, since the former cells were more sensitive to inhibition by methyl-beta-cyclodextrin, a sterol-chelating agent . Electron microscopic studies revealed that the intracellular vacuoles containing the internalized Dr(+) E . coli were morphologically distinct between the anchor variants of DAF . The cells expressing glycosylphosphatidylinositol-anchored DAF contained a single bacterium in tight-fitting vacuoles, while the cells expressing transmembrane-anchored DAF contained multiple (two or three) bacteria in spacious phagosomes . This finding suggests that distinct postendocytic events operate in the cells expressing anchor variants of DAF . We provide direct evidence for the DAF-mediated internalization of Dr(+) E . coli and demonstrate the significance of the glycosylphosphatidylinositol anchor, which determines the ability and efficiency of the internalization event. Clin Ter, 1999 Sep-Oct, 150(5), 343 - 6 {The effects of eradication therapy in patients with chronic atrophic gastritis and seropositivity for anti-HP antibodies and histological negativity for Helicobacter pylori}; Leri O et al.; PURPOSE: The present study was undertaken to analyze both whether the elevated Helicobacter pylori levels in patients with atrophic gastritis without histologic evidence of Helicobacter pylori would be a sign of an ongoing infection and the effects of eradication on gastric atrophy . PATIENTS AND METHODS: Twenty patients (10 M e 10 F; mean age 57.25 SD 12.19) with atrophic gastritis and elevated Helicobacter pylori titers without histological evidence for Helicobacter-like organisms were included in the study . Ten patients were randomized into eradication group (Group 1) (amoxicillin at 500 mg twice a day for 14 days, metronidazole at 500 mg twice a day for 10 days and omeprazole at 20 mg twice a day for 20 days) and 10 patients were randomized into the control group (Group 2) . For all subjects, serum samples and duplicate biopsy specimens (obtained endoscopically) were collected prior the study period and approximately 6 months after the therapy or the follow-up for serum samples and 8 weeks for biopsy specimens . RESULTS: In the Group 1, the Helicobacter pylori antibody titers dropped significantly in 73.39% of the patients (p < 0.0001), while in the Group 2, the antibody titers declined only in a patient who received antibiotics during the study period (p < 0.00006) . In both groups, no significant improvement of atrophic gastritis was observed . CONCLUSIONS: In conclusion, in patients with atrophic gastritis, the only histological evaluation of Helicobacter-like organisms colonization in gastric biopsy specimens, appeared in our study to underestimate the true prevalence of current HP infection and the importance of the bacterium in the pathogenesis and progression of such disease . Since HP infection is often associated with an increase of proliferative index, the eradication of HP could induce a mucosal protective effect against the other carcinogen factors, although it is extremely unlikely that it can promote the regeneration of a normal gastric mucosa. Genetika, 1999 Dec, 35(12), 1718 - 20 {Spontaneous mutants of Alteromonas espejiana, resistant to kanamycin and bleomycin}; Gonikberg EM et al.; An attempt was made to induce insertions in marine bacterium Alteromonas espejiana Bal-31 (Ae) using the TnphoA transposon . The Ae mutants selected on a kanamycin-containing medium after conjugation of the Ae with the transposon donor, Escherichia coli SM10(pRt291), were resistant not only to kanamycin (Kn), but also to bleomycin (Bm), and were sensitive to tetracycline . Although the mutants were phenotypically similar to insertion mutants, the mutations appeared to be spontaneous . The sensitivity of spontaneous Kmr Ae mutants selected at various Km concentrations to Bm was investigated . The mutants selected at low Km concentrations were resistant to Bm, whereas those selected at high Km concentrations were sensitive to Bm . The possible mechanisms underlying the dual resistance to Bm and aminoglycosides in bacteria are discussed. J Mol Biol, 2000 Mar 3, 296(4), 969 - 77 Antirestriction protein Ard (Type C) encoded by IncW plasmid pSa has a high similarity to the "protein transport" domain of TraC1 primase of promiscuous plasmid RP4; Belogurov AA et al.; The IncW plasmid pSa contains the gene ard encoding an antirestriction function that is specific for type I restriction and modification systems . The nucleotide sequence of ard was determined and an appropriate polypeptide of about 33 kDa was identified in Escherichia coli T7 expression system . Analysis of deduced amino acid sequence of Ard encoded by pSa revealed that this protein has no significant similarities with the known Ard proteins (ArdA and ArdB types) except the "antirestriction" motif (14 amino acid residues in length) conserved for all known Ard proteins . This finding suggests that pSa Ard may be classified as a new type of Ard proteins which we designated ArdC . The remarkable feature of ArdC is that it has a high degree of similarity (about 38 % identity) to the N-terminal region of RP4 TraC1 primase which includes about 300 amino acid residues and seems to be essential for binding to the single-stranded DNA and TraC1 protein transport to the recipient cells during the conjugal transfer of plasmid DNA . ArdC also binds to single-stranded DNA . In addition, this protein is able in vitro to protect the single-stranded but not double-stranded plasmid DNA against the activity of type II restriction endonuclease HhaI that cleaves both single and double-stranded DNA . We suggest that like TraC1, ArdC would be transported as a result of their interaction with the single-stranded DNA of transferred plasmid strand during conjugative passage through the cell envelope to the recipient bacterium . Such properties of ArdC protein might be useful to protect immediately the incoming single-stranded DNA from the host endonucleases . J Periodontal Res, 1999 Oct, 34(7), 400 - 5 The rag locus of Porphyromonas gingivalis: a novel pathogenicity island; Curtis MA et al.; Previous studies in our laboratories of the serum IgG antibody response of periodontal patients have demonstrated the presence of an immunodominant surface antigen (Mr 55 kDa) in the outer membrane of Porphyromonas gingivalis W50 . Genetic analysis of this antigen revealed that the corresponding gene forms part of a small operon which may have arisen via horizontal gene transfer into the genome of this strain . On the basis of sequence homology, the 55 kDa antigen (RagB) and the product of a cotranscribed gene (RagA) may act in concert at the surface of the bacterium to facilitate active transport, mediated through the periplasmic spanning protein, TonB, or form part of a signal transduction system in this organism . The rag locus is present in only a proportion of P . gingivalis laboratory strains and clinical isolates . Analysis of the distribution of ragB in subgingival samples by PCR demonstrated that rag+ P . gingivalis are more frequently detected in deep periodontal pockets than shallow sites in periodontal patients . These findings indicate that the rag genes may influence the virulence potential of P . gingivalis strains which harbour this locus and may thus be considered a novel pathogenicity island . Furthermore, horizontal gene transfer between organisms in subgingival plaque may represent a significant force in the evolution of these bacteria with ramifications for both diagnosis and targeted treatment of periodontal disease. Res Vet Sci, 2000 Feb, 68(1), 23 - 6 Detection of Lawsonia intracellularis in the tonsils of pigs with proliferative enteropathy; Jensen TK et al.; The presence of Lawsonia intracellularis, the obligate intracellular bacterium causing proliferative enteropathy (PE), in the tonsils of pigs as a locus for infection or extraintestinal occurrence of the bacterium was investigated by PCR and immunohistochemistry . Tonsillar occurrence of L . intracellularis could be part of the pathogenesis of PE and an important risk factor in the spread of the disease . L . intracellularis was detected by only PCR in the tonsils of 2/32 pigs without PE at necropsy but with a clinical history of diarrhoea and detection of the bacterium in faeces 1 to 3 weeks prior to necropsy but not in four pigs with moderate PE lesions . However, L . intracellularis was detected in the tonsils of 4/9 pigs with PE complicated with necroses and in 4/4 pigs with proliferative haemorrhagic enteropathy in which L . intracellularis antigen also was demonstrated in tonsillar macrophages and as intact bacteria in the lumen of the crypts . The results show that L . intracellularis is detectable in the tonsils of pigs and that the tonsillar presence of L . intracellularis appears to be correlated to the severity of the intestinal lesions possibly as a result of local retention and not as part of the pathogenesis of PE . Nucleic Acids Res, 2000 Mar 15, 28(6), 1447 - 54 Ligation reaction specificities of an NAD(+)-dependent DNA ligase from the hyperthermophile Aquifex aeolicus; Tong J et al.; An NAD(+)-dependent DNA ligase from the hyperthermophilic bacterium Aquifex aeolicus was cloned, expressed in Escherichia coli and purified to homogeneity . The enzyme is most active in slightly alkaline pH conditions with either Mg(2+)or Mn(2+)as the metal cofactor . Ca(2+)and Ni(2+)mainly support formation of DNA-adenylate intermediates . The catalytic cycle is characterized by a low k (cat)value of 2 min(-1)with concomitant accumulation of the DNA - adenylate intermediate when Mg(2+)is used as the metal cofactor . The ligation rates of matched substrates vary by up to 4-fold, but exhibit a general trend of T/A < or = G/C < C/G < A/T on both the 3'- and 5'-side of the nick . Consistent with previous studies on Thermus ligases, this Aquifex ligase exhibits greater discrimination against a mismatched base pair on the 3'-side of the nick junction . The requirement of 3' complementarity for a ligation reaction is reaffirmed by results from 1 nt insertions on either the 3'- or 5'-side of the nick . Furthermore, most of the unligatable 3' mismatched base pairs prohibit formation of the DNA-adenylate intermediate, indicating that the substrate adenylation step is also a control point for ligation fidelity . Unlike previously studied ATP ligases, gapped substrates cannot be ligated and intermediate accumulation is minimal, suggesting that complete elimination of base pair complementarity on one side of the nick affects substrate adenylation on the 5'-side of the nick junction . Relationships among metal cofactors, ligation products and intermediate, and ligation fidelity are discussed. FEMS Immunol Med Microbiol, 2000 Mar, 27(3), 241 - 6 Isolation and characterization of a human neutrophil aggregation defective mutant of Fusobacterium nucleatum; Guo M et al.; Fusobacterium nucleatum is known to adhere to human polymorphonuclear neutrophils (PMNs) and cause them to aggregate . In this study, we isolated a spontaneously occurring aggregation defective (AGG(-)) mutant and this mutant will be used for future study of the interactions between this bacterium and human PMN . Genomic DNA fingerprinting by random-primed polymerase chain reaction method revealed a difference between the parent strain and the AGG(-) mutant . This mutant also showed an altered phenotype in both microbicidal and phagocytic assays, suggesting that the bacterial factor involved in the aggregation may also be very important for the phagocytosis and, subsequently, the killing by human PMNs . Further study of this mutant may help to clarify the molecular mechanisms of the interaction between this pathogen and human PMNs. J Biotechnol, 2000 Feb 17, 77(2-3), 219 - 34 Engineering of a proteolytically stable human beta 2-adrenergic receptor/maltose-binding protein fusion and production of the chimeric protein in Escherichia coli and baculovirus-infected insect cells; Hampe W et al.; The hydrophobic human beta 2 adrenergic receptor was produced in fusion to the hydrophilic maltose-binding protein (MalE) in Escherichia coli . Photoaffinity labeling with the adrenergic ligand {125I}cyanopindolole-diazirine indicated that the majority of the protein was proteolyzed in the intergenic region between the fusion partners after production in E . coli . The simple and fast genetics of the bacterium enabled us to engineer a linker with an increased proteolytic stability . The fusion protein produced in E . coli was fully functional with respect to binding of adrenergic ligands and coupling to stimulatory GTP-binding protein . The production level with 3 pmol receptor fusion protein per mg membrane protein in a crude membrane preparation was significantly higher than those reported for other beta 2 adrenergic receptor constructs in E . coli . After solubilization with dodecanoyl sucrose, the fusion protein was purified to near homogeneity by affinity chromatography on immobilized Ni2+ ions (binding to a C-terminal His6-tag) and on crosslinked amylose (binding to the MalE) . In order to achieve higher production levels, the fusion protein preceded by an insect signal peptide was produced in baculovirus-infected insect cells . As expected, the production level with about 17 pmol receptor per mg membrane protein was higher in the insect cells than in E . coli . The receptor fusion protein produced in the insect cells bound adrenergic ligands and activated heterotrimeric GTP-binding proteins with biochemical properties comparable to that of the unfused receptor. Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 302 - 5 Deficiency of current methods in assaying endochitinase activity; Ren Y et al.; Since chitin is degraded by a combination of both endo- and exochitinases, it is likely that both enzymes will be present in a crude extract . Currently used substrates for detecting endochitinase activity suffer from the fact that they could easily be digested by the contaminating exochitinase, thus giving either a false-positive or an inaccurate reading of the endochitinase activity . Using Photorhabdus luminescens, a bacterium symbiotically associated with insect-parasitic nematode Heterorhabditis bacteriophora as an exemplary system, we have identified these two chitinases by a simple "fluorimetric zymography" procedure . The exochitinase is a metalloenzyme and its activity is inhibited by 1,10-phenanthroline . Once the exochitinase activity is detected in a crude extract, its contribution must be eliminated before accurate determination of the endochitinase activity can be carried out . Specific properties of these enzymes including the pH activity profile, the requirement of metal ions for activity, and the molecular weight of the enzymes are among the factors to be considered in developing assaying procedures for endochitinase . J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 245 - 51 Pullulanase from the hyperthermophilic bacterium Thermotoga maritima: purification by beta-cyclodextrin affinity chromatography; Kriegshauser G et al.; This is the first report about the isolation of a type I pullulanase from a hyperthermophilic bacterium, Thermotoga maritima strain MSB8 . Purification of the enzyme from a cleared cell-free extract was achieved by anion-exchange chromatography and beta-cyclodextrin affinity chromatography . Using this convenient two-step method we have purified the pullulanase 406-fold with a 26% yield . The purified enzyme displayed maximum pullulan hydrolysis at pH 5.9 and 90 degrees C (15-min assay) and was remarkably resistant against thermoinactivation, having a half-life at 90 degrees C of about 3.5 h . To our knowledge, the T . maritima pullulanase is the most thermostable type I pullulanase known to date . The affinity-based purification protocol described here may be useful for the efficient isolation of other pullulanases. Ann Surg, 2000 Feb, 231(2), 153 - 8 Eradication of Helicobacter pylori prevents recurrence of ulcer after simple closure of duodenal ulcer perforation: randomized controlled trial; Ng EK et al.; OBJECTIVE: In this randomized trial, the authors sought to determine whether eradication of Helicobacter pylori could reduce the risk of ulcer recurrence after simple closure of perforated duodenal ulcer . BACKGROUND DATA: Immediate acid-reduction surgery has been strongly advocated for perforated duodenal ulcers because of the high incidence of ulcer relapse after simple patch repair . Although H . pylori eradication is now the standard treatment of uncomplicated and bleeding peptic ulcers, its role in perforation remains controversial . Recently a high prevalence of H . pylori infection has been reported in patients with perforations of duodenal ulcer . It is unclear whether eradication of the bacterium confers prolonged ulcer remission after simple repair and hence obviates the need for an immediate definitive operation . METHODS: Of 129 patients with perforated duodenal ulcers, 104 (81%) were shown to be infected by H . pylori . Ninety-nine H . pylori-positive patients were randomized to receive either a course of quadruple anti-helicobacter therapy or a 4-week course of omeprazole alone . Follow-up endoscopy was performed 8 weeks, 16 weeks (if the ulcer did not heal at 8 weeks), and 1 year after hospital discharge for surveillance of ulcer healing and determination of H . pylori status . The endpoints were initial ulcer healing and ulcer relapse rate after 1 year . RESULTS: Fifty-one patients were assigned to the anti-Helicobacter therapy and 48 to omeprazole alone . Nine patients did not undergo the first follow-up endoscopy . Of the 90 patients who did undergo follow-up endoscopy, 43 of the 44 patients in the anti-Helicobacter group and 8 of the 46 in the omeprazole alone group had H . pylori eradicated; initial ulcer healing rates were similar in the two groups (82% vs . 87%) . After 1 year, ulcer relapse was significantly less common in patients treated with anti-Helicobacter therapy than in those who received omeprazole alone (4.8% vs . 38.1%) . CONCLUSIONS: Eradication of H . pylori prevents ulcer recurrence in patients with H . pylori-associated perforated duodenal ulcers . Immediate acid-reduction surgery in the presence of generalized peritonitis is unnecessary. Biochim Biophys Acta, 1999 Dec 23, 1489(2-3), 281 - 92 Characterization of the Azoarcus ribozyme: tight binding to guanosine and substrate by an unusually small group I ribozyme; Kuo LY et al.; We report novel chemical properties of the ribozyme derived from the smallest group I intron (subgroup IC3) that comes from the pre-tRNA(Ile) of the bacterium Azoarcus sp . BH72 . Despite the small size of the Azoarcus ribozyme (195 nucleotides (nt)), it binds tightly to the guanosine nucleophile (Kd = 15 +/- 3 microM) and exhibits activity at high temperatures (approximately 60-70 degrees C) . These features may be due to the two GA3 tetraloop interactions postulated in the intron and the high GC content of the secondary structure . The second order rate constant for the Azoarcus ribozyme, ((k(cat)/Km)S = 8.4 +/- 2.1 x 10(-5) M(-1) min(-1)) is close to that found for the related ribozyme derived from the pre-tRNA(Ile) of the cyanobacterium Anabaena PCC7120 . pH dependence studies and kinetic analyses of deoxy-substituted substrates suggest that the chemical cleavage step is the rate-determining process in the Azoarcus ribozyme . This may be due to the short 3-nt guide sequence-substrate pairing present in the Azoarcus ribozyme . Finally, the Azoarcus ribozyme shares features conserved in other group I ribozymes including the pH profile, the stereospecificity for the Rp-phosphorothioate at the cleavage site and the 1000-fold decrease in cleavage rate with a deoxyribonucleoside leaving group. Res Microbiol, 1999 Nov-Dec, 150(9-10), 711 - 24 Short repeats and IS elements in the extremely radiation-resistant bacterium Deinococcus radiodurans and comparison to other bacterial species; Makarova KS et al.; Computer analysis of the complete genome of Deinococcus radiodurans R1 has shown that the number of insertion sequences (ISs) and small noncoding repeats (SNRs) it contains is very high, and comparable with those of Escherichia coli . IS elements and several families of SNRs are described, together with their possible function in the D . radiodurans genome. Helicobacter, 2000 Mar, 5(1), 1 - 12 Analysis of iceA1 transcription in Helicobacter pylori; Donahue JP et al.; BACKGROUND: Transcription of the Helicobacter pylori iceA1 gene is induced following adherence of the bacterium to gastric epithelial cells in vitro, suggesting that this gene might be involved in H . pylori pathogenesis . Consequently, the current studies were undertaken to characterize iceA1 transcription and to define the structure of iceA1-containing transcripts to evaluate the potential of this gene to encode functional proteins . MATERIALS AND METHODS: Northern blots and primer extension of RNA isolated from broth-grown cultures of various H . pylori strains was done to analyze iceA1-specific gene transcription . Reverse transcriptase (RT)-PCR was used to determine the levels of iceA1 transcripts derived from readthrough transcription that was initiated upstream of iceA1 within the 5'-flanking cysE gene . RESULTS: Three major transcripts were detected and each was initiated from a common promoter, designated PI . Two of these transcripts were comprised of iceA1 sequence, while a third transcript was dicistronic and included the downstream gene, hpyIM . In addition, 10-fold lower levels of iceA1 transcripts were initiated upstream of PI, either within or immediately downstream of cysE . CONCLUSIONS: The present analysis suggests that iceA1 does not encode a functional protein in the majority of H . pylori strains . However, transcription of hpyIM, which encodes a highly conserved DNA adenine methyltransferase, is linked to iceA1 transcription . Therefore, iceA1 may affect H . pylori virulence in vivo through transcriptional regulation of hpyIM expression levels, which may result in specific variations in DNA methylation patterns leading to alteration in the expression of genes involved in virulence or pathogenesis. J Biol Chem, 2000 Feb 18, 275(7), 4679 - 86 Regulation of cytochrome bd expression in the obligate aerobe Azotobacter vinelandii by CydR (Fnr) . Sensitivity to oxygen, reactive oxygen species, and nitric oxide; Wu G et al.; Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant nitrogen fixation requires cytochrome bd . Regulation of cytochrome bd expression is achieved by CydR (an Fnr homologue), which represses transcription of the oxidase genes cydAB . cydAB mRNA was mapped by primer extension; the transcriptional start site was determined, and putative -10 and -35 regions were deduced . Two "CydR boxes," one at the +1 site and one upstream of the -35 region, were identified . Transcriptionally inactive, purified CydR was converted, by adding NifS, cysteine, and Fe(2+), into an active form possessing acid-labile sulfide and spectra suggesting a {4Fe-4S}(2+) cluster . Reconstituted CydR specifically bound both CydR boxes cooperatively, with higher affinity for the nearer consensus +1 site . Low concentrations of O(2) or NO ({O(2)}/{{CydR} or {NO}/{CydR} = 0.1-0 . 6) elicited loss of the 420 nm absorbance attributed to the {4Fe-4S}(2+) cluster, formation of a 315 nm species, and loss of ability to retard DNA migration . Retardation by reconstituted CydR was enhanced by superoxide dismutase and/or catalase, suggesting a role for reactive oxygen species in CydR inactivation . The role of CydR in regulating cydAB expression in the supposedly anoxic cytoplasm of A . vinelandii and similarities to cydAB regulation by Fnr in Escherichia coli are discussed. J Bacteriol, 2000 Mar, 182(5), 1442 - 7 Cytochrome c' from Rhodobacter capsulatus confers increased resistance to nitric oxide; Cross R et al.; We report the cloning and sequencing of the gene containing cytochrome c' (cycP) from the photosynthetic purple bacterium Rhodobacter capsulatus and the regions flanking that gene . Mutant strains unable to synthesize cytochrome c' had increased sensitivity to nitrosothiols and to nitric oxide (which binds to the heme moiety of cytochrome c'). J Bacteriol, 2000 Mar, 182(5), 1208 - 14 Enhanced nitrogenase activity in strains of Rhodobacter capsulatus that overexpress the rnf genes; Jeong HS et al.; In the photosynthetic bacterium Rhodobacter capsulatus, a putative membrane-bound complex encoded by the rnfABCDGEH operon is thought to be dedicated to electron transport to nitrogenase . In this study, the whole rnf operon was cloned under the control of the nifH promoter in plasmid pNR117 and expressed in several rnf mutants . Complementation analysis demonstrated that transconjugants which integrated plasmid pNR117 directed effective biosynthesis of a functionally competent complex in R . capsulatus . Moreover, it was found that strains carrying pNR117 displayed nitrogenase activities 50 to 100% higher than the wild-type level . The results of radioactive labeling experiments indicated that the intracellular content of nitrogenase polypeptides was marginally altered in strains containing pNR117, whereas the levels of the RnfB and RnfC proteins present in the membrane were four- and twofold, respectively, higher than the wild-type level . Hence, the enhancement of in vivo nitrogenase activity was correlated with a commensurate overproduction of the Rnf polypeptides . In vitro nitrogenase assays performed in the presence of an artificial electron donor indicated that the catalytic activity of the enzyme was not increased in strains overproducing the Rnf polypeptides . It is proposed that the supply of reductants through the Rnf complex might be rate limiting for nitrogenase activity in vivo . Immunoprecipitation experiments performed on solubilized membrane proteins revealed that RnfB and RnfC are associated with each other and with additional polypeptides which may be components of the membrane-bound complex. J Bacteriol, 2000 Mar, 182(5), 1200 - 7 Role of the H protein in assembly of the photochemical reaction center and intracytoplasmic membrane in Rhodospirillum rubrum; Cheng YS et al.; Rhodospirillum rubrum is a model for the study of membrane formation . Under conditions of oxygen limitation, this facultatively phototrophic bacterium forms an intracytoplasmic membrane that houses the photochemical apparatus . This apparatus consists of two pigment-protein complexes, the light-harvesting antenna (LH) and photochemical reaction center (RC) . The proteins of the photochemical components are encoded by the puf operon (LHalpha, LHbeta, RC-L, and RC-M) and by puhA (RC-H) . R . rubrum puf interposon mutants do not form intracytoplasmic membranes and are phototrophically incompetent . The puh region was cloned, and DNA sequence determination identified open reading frames bchL and bchM and part of bchH; bchHLM encode enzymes of bacteriochlorophyll biosynthesis . A puhA/G115 interposon mutant was constructed and found to be incapable of phototrophic growth and impaired in intracytoplasmic membrane formation . Comparison of properties of the wild-type and the mutated and complemented strains suggests a model for membrane protein assembly . This model proposes that RC-H is required as a foundation protein for assembly of the RC and highly developed intracytoplasmic membrane . In complemented strains, expression of puh occurred under semiaerobic conditions, thus providing the basis for the development of an expression vector . The puhA gene alone was sufficient to restore phototrophic growth provided that recombination occurred. Proc R Soc Lond B Biol Sci, 2000 Jan 7, 267(1438), 69 - 73 Sex-ratio-distorting Wolbachia causes sex-role reversal in its butterfly host; Jiggins FM et al.; Sex-role-reversed mating systems in which females compete for males and males may be choosy are usually associated with males investing more than females in offspring . We report that sex-role reversal may also be caused by selfish genetic elements which distort the sex ratio towards females . Some populations of the butterflies Acraea encedon and Acraea encedana are extremely female biased because over 90% of females are infected with a Wolbachia bacterium that is maternally inherited and kills male embryos . Many females in these populations are virgins suggesting that their reproductive success may be limited by access to males . These females form lekking swarms at landmarks in which females exhibit behaviours which we interpret as functioning to solicit matings from males . The hypothesis that female A . encedon swarm in order to mate is supported by the finding that, in release recapture experiments, mated females tend to leave the swarm while unmated females remained . This behaviour is a sex-role-reversed form of a common mating system in insects in which males form lekking swarms at landmarks and compete for females . Female lekking swarms are absent from less female-biased populations and here the butterflies are instead associated with resources in the form of the larval food plant. J Med Microbiol, 2000 Feb, 49(2), 139 - 47 Ultrastructural study of Mycobacterium avium infection of HT-29 human intestinal epithelial cells; Sangari FJ et al.; Mycobacterium avium is a common pathogen in AIDS patients and, in a large percentage of those patients, M . avium infection appears to be acquired via the gastrointestinal tract . M . avium is able to bind to and enter human and murine intestinal epithelial cells in vitro and in vivo . The invasion by and intracellular fate of M . avium in the HT-29 intestinal epithelial cell line was examined in an ultrastructural study . Bacterial contact with polarised cells was observed 10-15 min after monolayer infection and in polarised monolayers this always occurred in areas lacking microvilli . Contact with HT-29 cells did not appear to take place in a preferential area on the bacterial cell . Following invasion, M . avium was encountered within vacuoles containing either single or multiple bacteria; the latter evolved to contain only an individual bacterium . Vacuoles containing more than one bacterium were seen early in the infection and eventually underwent segmentation, with each bacterium occupying a vacuole . No bacteria were observed outside vacuoles up to 5 days after infection. Acta Crystallogr D Biol Crystallogr, 2000 Jan, 56 ( Pt 1), 3 - 13 The three-dimensional structure of a Trichoderma reesei beta-mannanase from glycoside hydrolase family 5; Sabini E et al.; The crystal structure of the catalytic core domain of beta-mannanase from the fungus Trichoderma reesei has been determined at a resolution of 1.5 A . The structure was solved using the anomalous scattering from a single non-isomorphous platinum complex with two heavy-metal sites in space group P2(1) . The map computed with the experimental phases was enhanced by the application of an automated model building and refinement procedure using the amplitudes and experimental phases as observations . This approach is expected to be of more general application . The structure of the native enzyme and complexes with Tris-HCl and mannobiose are also reported: the mannobiose binds in subsites +1 and +2 . The structure is briefly compared with that of the homologous beta-mannanase from the bacterium Thermomonospora fusca. Acta Crystallogr D Biol Crystallogr, 2000 Feb, 56 ( Pt 2), 215 - 7 Crystallization and preliminary X-ray analysis of a membrane-bound nitrite reductase from Desulfovibrio desulfuricans ATCC 27774; Dias JM et al.; Nitrite reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is a multihaem (type c) membrane-bound enzyme that catalyzes the dissimilatory conversion of nitrite to ammonia . Crystals of the oxidized form of this enzyme were obtained using PEG and CaCl(2) as precipitants in the presence of 3--(decylmethylammonium)propane-1-sulfonate and belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 78.94, b = 104.59, c = 143.18 A . A complete data set to 2.30 A resolution was collected using synchrotron radiation at the ESRF . However, the crystals may diffract to beyond 1.7 A and high-resolution data will be collected in the near future. Chemosphere, 2000 Feb, 40(3), 319 - 25 The effect of EDTA and fulvic acid on Cd, Zn, and Cu toxicity to a bioluminescent construct (pUCD607) of Escherichia coli; Campbell CD et al.; The hypothesis, that metal toxicity is dominated by free ion activity, was tested by comparing calculated metal activities with measured toxic responses to a genetically modified, luminescent bacterium, Escherichia coli . The toxicity of Cd, Cu, and Zn sulphate salts in the presence of EDTA and fulvic acid in well-defined solutions was measured . Good agreement between free metal activity and toxicity was found for Cu but not for Zn and Cd . The toxicity relationships were altered by glucose addition to the organism . Stable chloride complexes may have contributed to the toxicity of Cd under the test conditions . The results suggest that there is not always a simple relationship between toxicity and free-ion metal concentration and that further account should be taken of competitive interactions between living cells and ligands and the physiological status of the organism. Trends Biochem Sci, 2000 Feb, 25(2), 74 - 9 Lesions in DNA: hurdles for polymerases; Baynton K et al.; Translesion synthesis (TLS) is one of the DNA damage tolerance strategies, which have evolved to enable organisms to replicate their genome despite the presence of unrepaired damage . The process of TLS has the propensity to produce mutations, a potential origin of cancer, and is therefore of medical interest . Significant progress in our understanding of TLS has come primarily from studies of the bacterium Escherichia coli, the budding yeast Saccharomyces cerevisiae and, more recently, human cells . Results from these analyses indicate that the fundamental mechanism of TLS and the proteins involved have been conserved throughout evolution from bacteria to humans. Cell Signal, 1999 Dec, 11(12), 863 - 9 Activation of arachidonate release and cytosolic phospholipase A2 via extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in macrophages stimulated by bacteria or zymosan; Hiller G et al.; The mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK) and p38, can both contribute to the activation of cytosolic phospholipase A2 (cPLA2) . We have investigated the hypothesis that ERK and p38 together or independent of one another play roles in the regulation of cPLA2 in macrophages responding to the oral bacterium Prevotella intermedia or zymosan . Stimulation with bacteria or zymosan beads caused arachidonate release and enhanced in vitro cPLA2 activity of cell lysate by 1.5- and 1.7-fold, respectively, as well as activation of ERK and p38 . The specific inhibitor of MAP kinase kinase, PD 98059, and the inhibitor of p38, SB 203580, both partially inhibited cPLA2 activation and arachidonate release induced by bacteria and zymosan . Together, the two inhibitors had additive effects and completely blocked cPLA2 activation and arachidonate release . The present results demonstrate that ERK and p38 both have important roles in the regulation of cPLA2 and together account for its activation in P . intermedia and zymosan-stimulated mouse macrophages. Adv Exp Med Biol, 1999, 473, 207 - 14 Coiling phagocytosis is the predominant mechanism for uptake of the colonic spirochetosis bacterium Serpulina pilosicoli by human monocytes; Cheng X et al.; Serpulina pilosicoli is a newly identified pathogenic spirochete that establishes persistent colonic infections in human beings and animals . Macrophages are one of the key defenses against invasion of mucosal surfaces by bacterial pathogens . Macrophages engulf many bacteria by conventional phagocytosis; however recent studies indicate coiling phagocytosis as a new and important mechanism for internalization of Legionella pneumophila and spirochetes of the genus Borrelia, Leptospira, and Treponema . In this study, THP-1 human monocytic cells were incubated with the human S . pilosicoli strain SP16 and the contribution of coiling and conventional phagocytosis to the total number of phagocytic events were determined by sequential ultrastructural examination between 5 and 45 minutes . The frequency of phagocytosis increased over time from 5.1% after 5 minutes up to 21.9% after 45 minutes with greater than 70% of the events involving coiling phagocytosis . The data indicate that coiling phagocytosis may be a universal mechanism for uptake of pathogenic spirochetes. Mil Med, 2000 Jan, 165(1), 21 - 7 Association of Helicobacter pylori infection with gastric cancer; Alexander GA et al.; BACKGROUND: Helicobacter pylori has generated public health interest since its identification in 1983 . Past studies have suggested that the bacterium plays a role in the pathogenesis of gastric cancer . More recent studies support the conclusion that the association of H . pylori with gastric cancer is causal . The purpose of this article is to review the available evidence supporting the association of H . pylori with gastric cancer . METHODS: We performed a critical review of the relevant literature published in the English language on H . pylori and gastric cancer using MEDLINE, Index Medicus for the years 1985 to 1997 . The reference lists of selected articles also were reviewed to capture citations for further pertinent studies . RESULTS: H . pylori is thought to be the major cause of chronic atrophic gastritis . H . pylori gastritis is worldwide in distribution . H . pylori is now categorized by the International Agency for Cancer Research as a group 1 carcinogen, i.e., an agent that is carcinogenic to humans . Several reports from the United States have found the highest frequencies of gastric cancer in geographic areas and populations with the highest rates of acquisition of H . pylori infection . The high prevalence of H . pylori infection has been documented most notably in blacks and Hispanics, who also are at high risk for gastric cancer . CONCLUSIONS: New studies that focus on the epidemiology and pathology of H . pylori improve our understanding of its relationship with gastric cancer and advance the development of gastric cancer prevention and control strategies that are proposed. Mol Plant Microbe Interact, 2000 Jan, 13(1), 6 - 13 Resistance of tomato line Hawaii7996 to Ralstonia solanacearum Pss4 in Taiwan is controlled mainly by a major strain-specific locus; Wang JF et al.; Bacterial wilt caused by the soilborne bacterium Ralstonia solanacearum attacks hundreds of plant species, including many agriculturally important crops . Natural resistance to this disease has been found in some species and is usually inherited as a polygenic trait . In tomato, a model crop plant, genetic analysis previously revealed the involvement of several QTL (quantitative trait loci) controlling resistance and, in all of these studies with different strains of the pathogen, loci on chromosome 6 played the predominant role in controlling this trait . Using quantitative data collected from a greenhouse test F3 population, we identified a new locus on chromosome 12 that appears to be active specifically against a race 1 biovar 3 Pss4 bacterial strain endemic to Taiwan . Chromosome 6 still contributes significantly to the control of the resistance, and weaker associations of the trait to other regions of the genome are observed . These results are discussed in the context of current molecular knowledge about the strain specificity of disease resistance genes. Eur J Gastroenterol Hepatol, 1999 Dec, 11(12), 1371 - 7 Antral gastric permeability to antigens in mice is altered by infection with Helicobacter felis; Matysiak-Budnik T et al.; OBJECTIVE: Gastric inflammation is observed not only during Helicobacter pylori infection but also after eradication of the bacterium . The hypothesis that an altered gastric permeability could be involved was tested using a model of mice infected with Helicobacter felis . DESIGN: The antral and corpus gastric permeability during infection and after eradication of bacteria was studied . METHODS: Gastric fragments from the antrum and corpus of healthy mice, mice infected with H . felis, or mice after bacterial eradication, were mounted in Ussing chambers, and fluxes of sodium (JNa), mannitol (JMan) and horseradish peroxidase (HRP) under intact (JHRPi) and degraded (JD) form were measured . RESULTS: In healthy mice, JNa, JMan, JHRPi and JD, respectively, were greater in the antrum (6.5 +/- 0.5 microEq/h.cm2; 0.137 +/- 0.016 micromol/h.cm2; 30.4 +/- 7.4 ng/h.cm2 and 852 +/- 173 ng/h.cm2) than in the corpus (5.0 +/- 0.3 microEq/h.cm2; 0.085 micro 0.013 micromol/h.cm2; 9.5 +/- 2.8 ng/h.cm2 and 434 +/- 139 ng/h.cm2) . In H . felis-infected mice, HRP fluxes in the antrum were increased (JHRPi = 182 +/- 86, JD = 948 +/- 94 ng/h.cm2) as compared to controls (JHRPi = 10.3 +/- 2.6, JD = 458 +/- 98 ng/h.cm2) . Bacterial eradication led to the reduction of intact (JHRPi = 53 +/- 26 ng/h.cm) but not of degraded (JD = 844 +/- 213 ng/h.cm) HRP fluxes . After eradication, degraded HRP fluxes returned to normal in mice without inflammation (JD = 558 +/- 36 ng/h.cm2) but not in those with persistent inflammation (JD = 987 +/- 310 ng/h.cm2) . CONCLUSIONS: The results suggest that during H . felis infection, bacterial colonization and inflammation lead to an increased gastric permeability along the direct and degradative pathways, respectively . Such an increased antigenic load could contribute to the perpetuation of gastric inflammation after bacterial eradication, and possibly to food protein sensitization. Appl Environ Microbiol, 2000 Feb, 66(2), 671 - 7 Cytochrome c(3) mutants of Desulfovibrio desulfuricans; Rapp-Giles BJ et al.; To explore the physiological role of tetraheme cytochrome c(3) in the sulfate-reducing bacterium Desulfovibrio desulfuricans G20, the gene encoding the preapoprotein was cloned, sequenced, and mutated by plasmid insertion . The physical analysis of the DNA from the strain carrying the integrated plasmid showed that the insertion was successful . The growth rate of the mutant on lactate with sulfate was comparable to that of the wild type; however, mutant cultures did not achieve the same cell densities . Pyruvate, the oxidation product of lactate, served as a poor electron source for the mutant . Unexpectedly, the mutant was able to grow on hydrogen-sulfate medium . These data support a role for tetraheme cytochrome c(3) in the electron transport pathway from pyruvate to sulfate or sulfite in D . desulfuricans G20. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 55 - 61 Gene cloning, nucleotide sequence and biochemical properties of a cytoplasmic cyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius, reclassification of a group of enzymes; Matzke J et al.; A gene encoding a cyclomaltodextrinase (neopullulanase) was cloned from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius ATCC27009 and its nucleotide sequence was determined . The encoded CdaA protein lacked an N-terminal signal sequence and aligned well with a family of bacterial proteins described as maltogenic alpha-amylases, neopullulanases or cyclomaltodextrinases . Escherichia coli cells harboring the cloned cdaA gene produced a 66-kDa protein that degraded pullulan in a sodium dodecyl sulfate-polyacrylamide gel . A . acidocaldarius cells grown on maltose, soluble starch or pullulan synthesized the same protein . Neopullulanase activity of the protein was cytoplasmic and its pH optimum of 5.5 was close to the pH value of the cytoplasm . CdaA degraded cyclomaltodextrins rapidly and pullulan (to panose) more slowly . It is proposed that CdaA functions as a cytoplasmic cyclomaltodextrinase (EC 3.2.1.54). Biochemistry (Mosc), 1999 Dec, 64(12), 1418 - 26 Phenoptosis: programmed death of an organism; Skulachev VP; Programmed cell death (apoptosis) is well-established in many multicellular organisms . Apoptosis purifies a tissue from cells that became useless or even harmful for the organism . Similar phenomena are already described also at subcellular level (suicide of mitochondria, i.e., mitoptosis) as well as at supracellular level (degradation of some organs temporarily appearing in the course of ontogenesis and then disappearing by means of apoptosis of the organ-composing cells) . Following the same logic, one may put a question about programmed death of an organism as a mechanism of purification of a kin, community of organisms, or population from individuals who became unwanted for this kin, etc . A putative mechanism of such kind is proposed to be coined "phenoptosis" by analogy with apoptosis and mitoptosis . In a unicellular organism (the bacterium Escherichia coli), three different biochemical mechanisms of programmed death are identified . All of them are actuated by the appearance of phages inside the bacterial cell . This may be regarded as a precedent of phenoptosis which prevents expansion of the phage infection among E . coli cells . Purification of a population from infected individuals looks like an evolutionary invention useful for a species . Such an invention has high chances to be also employed by multicellular organisms . Most probably, septic shock in animals and humans serves as an analog of the phage-induced bacterial phenoptosis . It is hypothesized that the stress-induced ischemic diseases of brain and heart as well as carcinogenesis if they are induced by repeated stresses also represent phenoptoses that, in contrast to sepsis, are age-dependent . There are interrelations of programmed death phenomena at various levels of complexity of the living systems . Thus, extensive mitoptosis in a cell leads to apoptotic death of this cell and extensive apoptosis in an organ of vital importance results in phenoptotic death of an individual . In line with this logic, some cases are already described when inhibition of apoptosis strongly improves the postischemic state of the organism. J Bacteriol, 2000 Feb, 182(4), 1158 - 61 Cell cycle arrest in archaea by the hypusination inhibitor N(1)-guanyl-1,7-diaminoheptane; Jansson BP et al.; Hypusination is an essential posttranslational modification unique to archaeal and eukaryotic protein synthesis initiation factor 5A (aIF5A and eIF5A, respectively) . We have investigated the effect of the efficient hypusination inhibitor N(1)-guanyl-1,7-diaminoheptane (GC(7)) on four archaeal and one bacterial species . We found that (i) archaea are sensitive to GC(7), whereas the bacterium Escherichia coli is not, (ii) GC(7) causes rapid and reversible arrest of growth of the archaeon Sulfolobus acidocaldarius, and (iii) the growth arrest is accompanied by a specific reversible arrest of the cell cycle prior to cell division . Our findings establish a link between hypusination and sustained growth of archaea and thereby provide the framework to study molecular details of archaeal cell cycle in connection with in vivo functions of hypusine and of aIF5A and eIF5A. J Bacteriol, 2000 Feb, 182(4), 983 - 92 Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum; Zhang Y et al.; Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase-dinitrogenase reductase-activating glycohydrolase) system . We report here the characterization of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation . Two mutants which affect glnB (structural gene for P(II)) were constructed . While P(II)-Y51F showed a lower nitrogenase activity than that of wild type, a P(II) deletion mutant showed very little nif expression . This effect of P(II) on nif expression is apparently the result of a requirement of P(II) for NifA activation, whose activity is regulated by NH(4)(+) in R . rubrum . The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P(II) might exist in R . rubrum and regulate the modification of GS . P(II) also appears to be involved in the regulation of DRAT activity, since an altered response to NH(4)(+) was found in a mutant expressing P(II)-Y51F . The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH(4)(+) and darkness treatments. Structure Fold Des, 1999 Dec 15, 7(12), 1505 - 15 Conformational changes induced by phosphorylation of the FixJ receiver domain; Birck C et al.; BACKGROUND: A variety of bacterial adaptative cellular responses to environmental stimuli are mediated by two-component signal transduction pathways . In these phosphorelay cascades, histidine kinases transphosphorylate a conserved aspartate in the receiver domain, a conserved module in the response regulator superfamily . The main effect of this phosphorylation is to alter the conformation of the response regulator in order to modulate its biological function . The response regulator FixJ displays a typical modular arrangement, with a phosphorylatable N-terminal receiver domain and a C-terminal DNA-binding domain . In the symbiotic bacterium Sinorhizobium meliloti, phosphorylation of this response regulator activates transcription of nitrogen-fixation genes . RESULTS: The crystal structures of the phosphorylated and of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) were solved at 2.3 A and 2.4 A resolution, respectively . They reveal the environment of the phosphoaspartate in the active site and the specific conformational changes leading to activation of the response regulator . Phosphorylation of the conserved aspartate induces major structural changes in the beta 4-alpha 4 loop, and in the signaling surface alpha 4-beta 5 that mediates dimerization of the phosphorylated full-length response regulator . A site-directed mutant at this protein-protein interface decreases the affinity of the phosphorylated response regulator for the fixK promoter tenfold . CONCLUSIONS: The cascade of phosphorylation-induced conformational changes in FixJN illustrates the role of conserved residues in stabilizing the phosphoryl group in the active site, triggering the structural transition and achieving the post-phosphorylation signaling events . We propose that these phosphorylation-induced conformational changes underly the activation of response regulators in general. C R Acad Sci III, 1999 Nov, 322(11), 973 - 8 Vaccination against infections by Chlamydia pneumoniae; Puolakkainen M et al.; Chlamydia pneumoniae is an intracellularly growing bacterium that causes respiratory infections and is strongly associated with atherosclerosis . Antibodies against C . pneumoniae are frequently encountered in the adult population, indicating past exposure to the micro-organism . Immunity to reinfection is, however, only partial and does not prevent development of sequelae . Infections caused by and associated with C . pneumoniae are a major cause of morbidity and mortality world wide . Development of a vaccine capable of protecting against infections due to C . pneumoniae and their sequelae would prevent up to 10% of community-acquired pneumonias in adults and add a new dimension to the prevention of atherosclerosis and coronary heart disease. Vet Pathol, 2000 Jan, 37(1), 47 - 53 Specific in situ hybridization of Haemobartonella felis with a DNA probe and tyramide signal amplification; Berent LM et al.; Haemobartonella felis is an epierythrocytic bacterium suspected to be the causative agent of feline infectious anemia . Previous studies with a polymerase chain reaction assay have identified a mycoplasmal 16S rRNA gene sequence that coincides with clinical disease and the presence of organisms in the blood . Tissues from a cat experimentally infected with H . felis were used for in situ hybridization studies to physically link this 16S rRNA gene to the organisms on the red cells . A biotin-labeled probe was used in conjunction with tyramide signal amplification to visualize the hybridization signal . This study clearly demonstrates a specific hybridization signal on the red cells in the tissues of the H . felis-infected cat . This in situ hybridization study is the final step in fulfilling the molecular guidelines for disease causation and proves that H . felis, a mycoplasmal organism, is the causative agent of feline infectious anemia. Infect Immun, 2000 Feb, 68(2), 779 - 90 Helicobacter felis infection is associated with lymphoid follicular hyperplasia and mild gastritis but normal gastric secretory function in cats; Simpson KW et al.; The relationship of Helicobacter felis, a bacterium observed in the stomachs of cats, to gastric disease is unclear . The objective of this study was to determine if H . felis infection alters gastric histopathology, proinflammatory cytokine expression, and secretory function and evokes a humoral immune response in cats . Five specific-pathogen-free (SPF) Helicobacter-free cats were studied before and for 1 year after oral inoculation with H . felis (ATCC 49179) . Four SPF H . felis-uninfected cats served as controls . The stomachs of all five H . felis-inoculated cats became colonized, as determined by urease activity, histopathology, PCR, culture, and transmission electron microscopy of serial gastric biopsies at 0, 3, 5, 8, and 12 months . Uninoculated cats remained Helicobacter free . Lymphoid follicular hyperplasia, atrophy, and fibrosis were observed primarily in the pylorus of infected cats . Mild mononuclear inflammation was detected in both infected and uninfected cats, but was more extensive in infected cats, with pangastric inflammation, eosinophilic infiltrates, and cardia gastritis observed only in infected cats . No upregulation of antral mucosal interleukin 1alpha (IL-1alpha), IL-1beta, or tumor necrosis factor alpha was detected by reverse transcription-PCR in any cat . The gastric secretory axes, assessed by fasting plasma gastrin, antral mucosal gastrin and somatostatin immunoreactivity, and pentagastrin-stimulated gastric acid secretion, were similar in both infected and uninfected cats . Gradual seroconversion (immunoglobulin G) was observed in four of five infected cats, with enzyme-linked immunosorbent assay values reaching 4x to 12x baseline 12 months postinfection . These findings indicate that H . felis infection in cats induces lymphoid follicular hyperplasia, mild gastritis, and seroconversion, but is associated with normal gastric secretory function. Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 859 - 64 Genetic analysis of a bacterial genetic exchange element: the gene transfer agent of Rhodobacter capsulatus; Lang AS et al.; An unusual system of genetic exchange exists in the purple nonsulfur bacterium Rhodobacter capsulatus . DNA transmission is mediated by a small bacteriophage-like particle called the gene transfer agent (GTA) that transfers random 4.5-kb segments of the producing cell's genome to recipient cells, where allelic replacement occurs . This paper presents the results of gene cloning, analysis, and mutagenesis experiments that show that GTA resembles a defective prophage related to bacteriophages from diverse genera of bacteria, which has been adopted by R . capsulatus for genetic exchange . A pair of cellular proteins, CckA and CtrA, appear to constitute part of a sensor kinase/response regulator signaling pathway that is required for expression of GTA structural genes . This signaling pathway controls growth-phase-dependent regulation of GTA gene messages, yielding maximal gene expression in the stationary phase . We suggest that GTA is an ancient prophage remnant that has evolved in concert with the bacterial genome, resulting in a genetic exchange process controlled by the bacterial cell. Nucleic Acids Res, 2000 Feb 1, 28(3), 706 - 9 Structural analysis of DNA sequence: evidence for lateral gene transfer in Thermotoga maritima; Worning P et al.; The recently published complete DNA sequence of the bacterium Thermotoga maritima provides evidence, based on protein sequence conservation, for lateral gene transfer between Archaea and Bacteria . We introduce a new method of periodicity analysis of DNA sequences, based on structural parameters, which brings independent evidence for the lateral gene transfer in the genome of T.maritima . The structural analysis relates the Archaea-like DNA sequences to the genome of Pyrococcus horikoshii . Analysis of 24 complete genomic DNA sequences shows different periodicity patterns for organisms of different origin . The typical genomic periodicity for Bacteria is 11 bp whilst it is 10 bp for Archaea . Eukaryotes have more complex spectra but the dominant period in the yeast Saccharomyces cerevisiae is 10.2 bp . These periodicities are most likely reflective of differences in chromatin structure. Mol Microbiol, 2000 Jan, 35(1), 90 - 100 Initial events in the degradation of the polycistronic puf mRNA in Rhodobacter capsulatus and consequences for further processing steps; Heck C et al.; Individual segments of the polycistronic puf mRNA of Rhodobacter capsulatus exhibit extremely different half-lives contributing to the stoichiometry of light-harvesting and reaction centre complexes of this facultative phototrophic bacterium . While earlier investigations shed light on the processes leading to the degradation of the 2.7 kb pufBALMX mRNA and, consequently, to the formation of the highly stable 0.5 kb pufBA mRNA processing product, we have now investigated the initial events in the degradation of the highly unstable 3.2 kb pufQBALMX primary transcript . Sequence modifications of two putative RNase E recognition sites within the pufQ coding region provide strong evidence that RNase E-mediated cleavage of a sequence at the 3' end of pufQ is involved in rate-limiting cleavage of the primary pufQBALMX transcript in vivo . The putative RNase E recognition sequence at the 5' end of pufQ is cleaved in vitro but does not contribute to rate-limiting cleavage in vivo . Analysis of the decay of puf mRNA segments transcribed from wild-type and mutated puf DNA sequences in R . capsulatus and Escherichia coli reveal that RNase E-mediated cleavage within the pufQ mRNA sequence also affects the stability of the 0.5 kb pufBA processing product . These findings demonstrate that the stability of a certain mRNA segment depends on the pathway of processing of its precursor molecule. Eur J Biochem, 2000 Jan, 267(2), 450 - 6 Pigments and proteins in green bacterial chlorosomes studied by matrix-assisted laser desorption ionization mass spectrometry; Persson S et al.; We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum . By applying a small volume (1 microL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c . The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample . The same result was also obtained when whole cells of Chl . tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll homologs in a small amount of green bacterial cells . In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins . Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB. J Invertebr Pathol, 2000 Jan, 75(1), 55 - 8 Pathogenicity, development, and reproduction of Heterorhabditis bacteriophora and Steinernema carpocapsae under axenic in vivo conditions; Han R et al.; Galleria mellonella larvae cultured axenically were treated with axenic dauer juveniles of Heterorhabditis bacteriophora and Steinernema carpocapsae . After 3 days S . carpocapsae had killed all insects, with 9.4 +/- 4.3 nematodes per larva . H . bacteriophora were unable to kill G . mellonella, although 13.3 +/- 6.4 nematodes per Galleria were found in the hemocoel . Invading nematodes of both strains recovered from the dauer stage . H . bacteriophora developed into hermaphrodites with eggs and J1 in the uterus and in the hemolymph of the living insects . Development beyond the J1 stage was not recorded . An injection of supernatants from different Photorhabdus luminescens cultures killed the insects but could not provide nutrients to support a further development . Only the injection of bacterial cells supported production of dauers in the axenic insects . Axenic S . carpocapsae developed to adults and produced offspring . After 3 weeks an average of 5275 nematodes per larva were counted, of which 6.7% were dauer juveniles, 39.2% other juvenile stages, 11.9% males, and 42.2% females . Compared to in vivo reproduction in the presence of the symbiotic bacterium Xenorhabdus nematophilus the dauer juvenile yields were low . Even after 5 weeks the percentage of dauer juveniles did not surpass 10% . J Invertebr Pathol, 2000 Jan, 75(1), 47 - 54 Pathogenicity caused by high virulent and low virulent strains of Steinernema carpocapsae to Galleria mellonella; Simoes N et al.; Steinernema carpocapsae is an entomopathogenic nematode associated with a symbiotic bacterium, Xenorhabdus nematophilus . Both components of the complex participate in a pathogenic process in insects . This has raised two questions: how much does each one participate, and what mechanisms are involved? In this paper we compare the virulence of two strains of S . carpocapsae: a high virulent strain (Breton) and a low virulent strain (Az27), both of which are free of symbiotic bacteria . Breton and Az27 strains each one have similar ability to invade Galleria mellonella with median infectious times of 3.9 and 3.2 h, respectively . However, the LD(50) of the Breton and Az27 strains are 48.6 and 894.5 infective juveniles per insect, respectively . Breton strain takes 38 h to kill 100% of exposed insects, whereas Az27 takes three times longer . The lethal time of the low virulent strain in G . mellonella larvae is highly dependent on the number of nematodes which have penetrated the hemocelium, whereas it is not on the high virulent strain . Hemolymph patterns in SDS-PAGE of insects parasitized by the high virulent strain showed important differences in respect to the low virulent strain and control . Secretion/excretion products of the high virulent strain have important proteolytic activity as well as alpha-mannosidase and alpha-fucosidase activities, whereas, in secretion/excretion products of the avirulent strain, proteolytic activity was lower and alpha-mannosidase and alpha-fucosidase activities were undetected . Dig Dis Sci, 1999 Dec, 44(12), 2397 - 404 Potential relevance of agmatine as a virulence factor of Helicobacter pylori; Molderings GJ et al.; The polyamine agmatine is able to increase gastric acid secretion . Therefore, we investigated whether Helicobacter pylori is able to form and release agmatine in vitro and in the human stomach in vivo, and if so, whether a relationship exists among agmatine concentration in gastric juice, H . pylori infection, and gastroduodenal lesions . Agmatine was determined by means of HPLC . In the supernatant of H . pylori cultures, agmatine concentrations up to 1500 ng/ml (approximately 12 microM) were determined, depending on the number of the bacteria in the individual cultures . Agmatine concentration in gastric juice from H . pylori-positive patients was higher than in that from H . pylori-negative patients . Gastrin in blood was elevated in H . pylori-positive patients compared with H . pylori-negative patients . Agmatine concentration in gastric juice and serum gastrin level appeared to be related . In conclusion, H . pylori is able to form and to release agmatine in vitro and in vivo . This may be assumed to be relevant in vivo, since higher amounts of agmatine are present in gastric juice from H . pylori-positive than from H . pylori-negative patients . Accordingly, agmatine produced by H . pylori may be a virulence factor of this bacterium and may be involved in the pathogenesis of gastroduodenal lesions. J Bacteriol, 2000 Jan, 182(2), 286 - 94 Biochemical and molecular characterization of phenylacetate-coenzyme A ligase, an enzyme catalyzing the first step in aerobic metabolism of phenylacetic acid in Azoarcus evansii; El-Said Mohamed M; Phenylacetate-coenzyme A ligase (PA-CoA ligase; AMP forming, EC 6.2 . 1.30), the enzyme catalyzing the first step in the aerobic degradation of phenylacetate (PA) in Azoarcus evansii, has been purified and characterized . The gene (paaK) coding for this enzyme was cloned and sequenced . The enzyme catalyzes the reaction of PA with CoA and MgATP to yield phenylacetyl-CoA (PACoA) plus AMP plus PPi . The enzyme was specifically induced after aerobic growth in a chemically defined medium containing PA or phenylalanine (Phe) as the sole carbon source . Growth with 4-hydroxyphenylacetate, benzoate, adipate, or acetate did not induce the synthesis of this enzyme . This enzymatic activity was detected very early in the exponential phase of growth, and a maximal specific activity of 76 nmol min(-1) mg of cell protein(-1) was measured . After 117-fold purification to homogeneity, a specific activity of 48 micromol min(-1) mg of protein(-1) was achieved with a turnover number (catalytic constant) of 40 s(-1) . The protein is a monomer of 52 kDa and shows high specificity towards PA; other aromatic or aliphatic acids were not used as substrates . The apparent K(m) values for PA, ATP, and CoA were 14, 60, and 45 microM, respectively . The PA-CoA ligase has an optimum pH of 8 to 8.5 and a pI of 6.3 . The enzyme is labile and requires the presence of glycerol for stabilization . The N-terminal amino acid sequence of the purified protein showed no homology with other reported PA-CoA ligases . The gene encoding this enzyme is 1, 320 bp long and codes for a protein of 48.75 kDa (440 amino acids) which shows high similarity with other reported PA-CoA ligases . An amino acid consensus for an AMP binding motif (VX2SSGTTGXP) was identified . The biochemical and molecular characteristics of this enzyme are quite different from those of the isoenzyme catalyzing the same reaction under anaerobic conditions in the same bacterium. Res Microbiol, 2000 Nov, 151(9), 747 - 54 Characterization of the 16S rRNA gene V2 region and the rrn operons of Gardnerella vaginalis; Nath K et al.; Ribosomal RNA (rRNA) gene polymorphism was apparent when Gardnerella vaginalis DNA restriction profiles were hybridized with nonradioactively labeled total rRNA isolated from this bacterium . In contrast, use of a polymerase chain reaction (PCR)-based 16S rRNA gene V2 region resulted in a 118-bp V2-PCR amplicon that was specific and common in all 30 tested G . vaginalis isolates . In addition to providing a G . vaginalis-specific fingerprint, when the V2-PCR amplicon along with total rRNA were utilized as probes, a partial rRNA gene restriction map could be constructed . G . vaginalis contains two rrn operons with an EcoRI fragment of 1.6 kb common to both. Ned Tijdschr Geneeskd, 1999 Dec 11, 143(50), 2505 - 7 {Eradication of Helicobacter pylori possibly followed by gastroesophageal reflux disease}; Loffeld RJ; Colonisation with Helicobacter pylori exerts several effects on the production of acid . The bacterium does not play a direct role in the pathogenesis of gastroesophageal reflux disease (GORD) . Successful eradication of H . pylori in patients with ulcer disease appears to be followed by an increase in incidence of GORD . It has been postulated that the infection could protect against the development of GORD . H . pylori ulcers and gastritis should be treated by eradication therapy . It is unclear yet what is the best treatment in patients with H . pylori infection without an ulcus or gastritis. Biochemistry, 1999 Nov 30, 38(48), 15779 - 90 Comparison of the binding sites for high-potential iron-sulfur protein and cytochrome c on the tetraheme cytochrome subunit bound to the bacterial photosynthetic reaction center; Osyczka A et al.; A tetraheme cytochrome subunit bound to the photosynthetic reaction center (RC) of purple bacterium, Rubrivivax gelatinosus, interacts with two types of soluble electron donors, cytochromes c and high-potential iron-sulfur protein (HiPIP), at a binding domain in the vicinity of low-potential heme 1, the fourth heme from the special pair of bacteriochlorophyll . To clarify the mechanism of the interaction, the domain around heme 1 was examined using site-directed mutants that changed the surface charge in the region within 20 A from the heme edge . In the case of the interaction with soluble cytochrome c, a strong dependence on the sign of the introduced charge was observed in all mutants: positive charge inhibited the reaction rate, whereas additional negative charge accelerated it . This confirmed the electrostatic nature of the binding . Interaction with HiPIP was inhibited by a limited number of mutations at the close vicinity of heme 1, and no acceleration was observed (the effects of some mutations were independent of the sign of the introduced charge) . The acidic residues which were critically important for the binding of cytochrome c showed much less contribution to the binding of HiPIP . The binding site for HiPIP appears to be mostly formed by uncharged and hydrophobic residues, occupying a significantly smaller area than the cytochrome-c-binding site . It is proposed that the docking of HiPIP to the RC in Rvi . gelatinosus is primarily controlled by hydrophobic contacts between protein surfaces, thus differing from the electrostatic mode of the RC-cytochrome c interaction. J Mol Biol, 2000 Jan 14, 295(2), 279 - 88 Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties; Wassenberg D et al.; Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C . As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima . At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C . Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C . At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization . Compared to mesophilic MBP from E . coli as a reference, this value is increased by about 60 kJ mol(-1) . At temperatures around the optimal growth temperature of T . maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity . TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E . coli and the hyperthermophilic archaeon Thermococcus litoralis . Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified . Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1) . Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM . This result is in agreement with data reported for the MBPs from E . coli and T . litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states . FEMS Microbiol Lett, 2000 Jan 15, 182(2), 259 - 64 A role for the bacterial outer membrane in the pathogenesis of Helicobacter pylori infection; Keenan J et al.; Helicobacter pylori infection in humans is associated with diverse of clinical outcomes which are partly attributed to bacterial strain differences . Secreted bacterial products are thought to be involved in the pathogenesis caused by this non-invasive bacterium . Electron microscopy of gastric biopsies from infected individuals revealed blebbing of the H . pylori outer membrane, similar to the process of outer membrane vesicle shedding which occurs when the bacterium is grown in broth . Porins, a class of proinflammatory proteins, were observed in the outer membrane vesicles . The VacA cytotoxin, which is produced by 50-60% of H . pylori strains and associated with increased pathogenesis of infection, was also found to be vesicle-associated and biologically active . This supports the hypothesis that these vesicles represent a vehicle for the delivery of damaging bacterial products to the gastric mucosa. Biochem J, 2000 Jan 15, 345 Pt 2, 255 - 62 Proteins isolated from lucerne roots by affinity chromatography with sugars analogous to Nod factor moieties; Minic Z et al.; Nod factors are important elicitors in legume-bacterium symbiosis . Any candidate plant receptor(s) for these lipo-oligosaccharides can be expected to show some lectin-like properties . A novel protein (P60), a native tetramer with 60 kDa monomers, has been isolated from a membrane fraction of Medicago sativa (lucerne, alfalfa) roots by using affinity chromatography with either GlcNAc or N,N', N"-triacetyl-(1-->4)-beta-d-chitotriose {(GlcNAc)(3)} grafted to agarose beads as the matrix and, in a second step, Sephadex G-200 gel filtration . With (GlcNAc)(3)-agarose an additional protein of 78 kDa was isolated . P60 showed haemagglutination activity with specificity for GalNAc, GalN, GlcNAc and GlcN . Binding experiments with radioactive GlcNAc gave a K(d) of 95 nM and one binding site per monomer of P60; Nod factor competed strongly for this binding . In native PAGE, protein incubated with O-sulphated Nod factors had a higher electrophoretic mobility as a consequence of binding . However, the largest modification was observed with a natural mixture of Nod factors, containing the O-acetylated and O-sulphated tetrasaccharidic NodRm-IV(Ac,S) (in which Ac stands for an O-acetylated group at the non-reducing end and S for O-sulphation at the reducing end) in addition to the non-O-acetylated NodRm-IV(S) (which alone had little effect) and NodRm-V(S) . The native PAGE study was also performed with known lectins from other sources, but only the 34 kDa lectin of Phytolacca americana (pokeweed) showed any such interaction, although without discrimination between Nod factors . Finally, one peptide of each isolated protein was sequenced; the peptide from P60 showed some similarity with dihydrolipoamide dehydrogenase and ferric leghaemoglobin reductase, whereas the peptide from P78 was identical with an analogous region of 70 kDa heat shock protein. FEMS Microbiol Ecol, 2000 Jan 1, 31(1), 61 - 71 Molecular detection of Gluconacetobacter sacchari associated with the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell) and the sugarcane leaf sheath microenvironment by FISH and PCR; Franke IH et al.; Molecular tools for the detection of the newly described acetic acid bacterium Gluconacetobacter sacchari from the pink sugarcane mealybug, Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae), and in the sugarcane leaf sheath microenvironment were developed . G . sacchari specific 16S rRNA-targeted oligonucleotide primers were designed and used in PCR amplification of G . sacchari DNA directly from mealybugs, and in a nested PCR to detect low numbers of the bacteria from sugarcane leaf sheath fluid and cane internode scrapings . A sensitivity level of detection of 40-400 cells/reaction was obtained using PCR from exponentially grown bacterial cultures and of 1-10 cells in cane internode scrapings and leaf sheath fluid samples using nested PCR . The specificity of the primer set was demonstrated by the lack of amplification product formation in PCR by closely related acetic acid bacteria, including Gluconacetobacter liquefaciens, and Gluconacetobacter diazotrophicus . A Cy3 labeled probe for G . sacchari was designed and shown to be specific for the species . Investigation of the mealybug microenvironment by whole cell fluorescent in situ hybridization revealed that G . sacchari appears to represent only a minor proportion of the population of the microbiota in the mealybugs tested . This study has shown the usefulness of 16S rRNA-based molecular tools in the identification and detection of G . sacchari from environmental samples and will allow these tools to be used in further ecological research. J Clin Microbiol, 2000 Jan, 38(1), 460 - 1 Isolation of Pantoea agglomerans in two cases of septic monoarthritis after plant thorn and wood sliver injuries; De Champs C et al.; Arthritis after plant injury is often apparently aseptic . We report two cases due to Pantoea agglomerans . In one case, the bacterium was isolated only from the pediatric blood culture media, BACTEC Peds Plus, monitored in BACTEC 9240, and not from the other media inoculated with the joint fluid . This procedure could help improve the diagnosis of septic arthritis. Mol Cell, 1999 Nov, 4(5), 683 - 94 Differential localization of two histidine kinases controlling bacterial cell differentiation; Wheeler RT et al.; The bacterium C . crescentus coordinates cellular differentiation and cell cycle progression via a network of signal transduction proteins . Here, we demonstrate that the antagonistic DivJ and PleC histidine kinases that regulate polar differentiation are differentially localized as a function of the cell cycle . The DivJ kinase localizes to the stalked pole in response to a signal at the G1-to-S transition, while the PleC kinase is localized to the flagellar pole in swarmer and predivisional cells but is dispersed throughout the cell in the stalked cell . PleC, which is required for DivJ localization, may provide the cue at the G1-to-S transition that directs the polar positioning of DivJ . The dynamic positioning of signal transduction proteins may contribute to the regulation of polar differentiation at specific times during the bacterial cell cycle. Appl Environ Microbiol, 2000 Jan, 66(1), 105 - 12 Induction of a futile Embden-Meyerhof-Parnas pathway in Deinococcus radiodurans by Mn: possible role of the pentose phosphate pathway in cell survival; Zhang YM et al.; Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium Deinococcus radiodurans . Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation . Mn also facilitated glucose interactions with other nutrient components . These predictions were verified by in vivo and in vitro experiments . TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP) . Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light . The presence of glucose also enhanced the radioresistance of the culture . Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in D . radiodurans . The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio . Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO(2) . Mn-treated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light . Mn-treated cells were also more sensitive to UV light . Results suggested that metabolites of glucose generated from the PPP enhanced the survival of D . radiodurans . Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance. J Bacteriol, 2000 Jan, 182(1), 107 - 15 flhDC, the flagellar master operon of Xenorhabdus nematophilus: requirement for motility, lipolysis, extracellular hemolysis, and full virulence in insects; Givaudan A et al.; Xenorhabdus is a major insect pathogen symbiotically associated with nematodes of the family Steinernematidae . This motile bacterium displays swarming behavior on suitable media, but a spontaneous loss of motility is observed as part of a phenomenon designated phase variation which involves the loss of stationary-phase products active as antibiotics and potential virulence factors . To investigate the role of one of the transcriptional activators of flagellar genes, FlhDC, in motility and virulence, the Xenorhabdus nematophilus flhDC locus was identified by functional complementation of an Escherichia coli flhD null mutant and DNA sequencing . Construction of X . nematophilus flhD null mutants confirmed that the flhDC operon controls flagellin expression but also revealed that lipolytic and extracellular hemolysin activity is flhDC dependent . We also showed that the flhD null mutant displayed a slightly attenuated virulence phenotype in Spodoptera littoralis compared to that of the wild-type strain . Thus, these data indicated that motility, lipase, hemolysin, or unknown functions controlled by the flhDC operon are involved in the infectious process in insects . Our investigation expands the view of the flagellar regulon as a checkpoint coupled to a major network involving bacterial physiological aspects as well as motility. Electrophoresis, 1999 Dec, 20(18), 3514 - 20 Classifying symbiotic proteins from Bradyrhizobium japonicum into functional groups by proteome analysis of altered gene expression levels; Dainese-Hatt P et al.; The advent of whole genome sequences has brought with it a vast number of new potential proteins whose function is unknown . We describe an approach to sorting proteins into functional groups by comparative two-dimensional (2-D) gel mapping of cells grown under different physiological conditions . Computerized image analysis selects the proteins whose expression levels change significantly for subsequent identification by mass spectrometry . The protein groupings are further subdivided by directed alteration of their expression levels (e.g., by gene inactivation) and following the changes in the expression pattern of the mutants . We have applied this approach to study the regulation of micro- and anaerobically induced genes including the genes involved in nitrogen fixation in the symbiotic bacterium Bradyrhizobium japonicum . The results obtained show that in addition to the two known regulons controlled by the transcription factors NifA and FixK2, a third set of proteins may exist in B . japonicum which are induced by anaerobic conditions and are regulated independently . The approach can be applied generally and can be used to build up functional relationship maps of genomes . Protein identification by mass spectrometry was shown to be vital to the interpretation of the expression analysis since 15% of the 2-D gel spots contained more than one protein. J Infect Dis, 2000 Jan, 181(1), 195 - 202 Borrelia burgdorferi--induced oxidative burst, calcium mobilization, and phagocytosis of human neutrophils are complement dependent; Suhonen J et al.; When Borrelia burgdorferi, the spirochete causing Lyme disease, is transmitted to a human, the complement system is among the first challenges facing the bacterium . Neutrophils are crucial leukocytes in the first line of host defense against bacterial infections . To investigate the role of complement in the Borrelia-induced activation of human neutrophils, oxidative burst, calcium mobilization, and phagocytosis induced by three subspecies of B . burgdorferi were studied . Each subspecies induced all observed neutrophil functions in a complement-dependent manner . Serum-derived factors bound to the surface of B . burgdorferi were found to be essential for the induction of the oxidative burst . The CD11b chain of CR3 was found to participate in the oxidative burst and calcium mobilization induced by B . burgdorferi. J Infect Dis, 2000 Jan, 181(1), 188 - 94 Repeated pregnancies in BALB/c mice infected with Coxiella burnetii cause disseminated infection, resulting in stillbirth and endocarditis; Stein A et al.; Q fever is a widespread zoonosis caused by the obligate intracellular bacterium Coxiella burnetii . Although this highly virulent organism is most concentrated in mammals during parturition, there are few reports on the manifestations of perinatal Q fever in the human and animal host . The affinity of C . burnetii to pregnancy and its abortifacient potential were investigated in a murine animal model . Intraperitoneal infection of female BALB/c mice with C . burnetii, followed by repeated pregnancies over a 2-year period, resulted in persistent infection associated with abortion and perinatal death, with a statistically significant decrease in viable offspring . In addition, endocarditis occurred in 2 of the adult animals, and C . burnetii antigen and DNA were detected in their heart valves . Taken together, these results demonstrate the abortifacient potential of C . burnetii and the increased risk of persistent infection and endocarditis in pregnant mice, probably related to a decline in cellular immunity during pregnancy. Arch Immunol Ther Exp (Warsz), 1999, 47(6), 347 - 53 Genomic catastrophism and the origin of vertebrate immunity; Hughes AL; Genomic catastrophism is the belief that unique genetic events, unlike those observed in recent evolutionary history, played a key role in the origin of vertebrate adaptations . Catastrophist hypotheses have been particularly popular is accounting for the origin of vertebrate specific immunity . Two major such hypotheses involve genome duplication by polyploidization and horizontal gene transfer . Recent analyses lead to decisive rejection of the widely cited hypothesis that the vertebrate genome underwent two rounds of genome duplication, and theoretical considerations suggest that genome duplication is unlikely to lead to new adaptive advances . Likewise, the evidence that key elements of the vertebrate immune system arose by horizontal transfer from a bacterium or by incorporation of a transposable element into the vertebrate genome remains relatively weak . Thus, at present, a uniformitarian view of the origin of the vertebrate immune system seems more reasonable, especially given the longer time-frame for vertebrate evolution indicated by molecular data. Microbes Infect, 1999 Apr, 1(5), 367 - 76 Clinical and biological aspects of infection caused by Ehrlichia chaffeensis; Rikihisa Y; Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects the monocyte-macrophage . E . chaffeensis, which is transmitted to humans by ticks primarily from infected deer, causes human monocytic ehrlichiosis, an acute febrile systemic illness . This paper reviews current knowledge of clinical and biological aspects of infections caused by E . chaffeensis. Scand J Immunol, 1999 Dec, 50(6), 651 - 6 Antibodies given orally in the neonatal period can affect the immune response for two generations: evidence for active maternal influence on the newborn's immune system; Lundin BS et al.; Two day old Wistar rats were tube fed with 1 or 10 micrograms of a mouse IgG1 monoclonal anti-idiotypic (a-Id) antibody that was directed against an anti-Escherichia coli-K13 capsular polysaccharide antibody . A control group was given 10 micrograms of an unrelated control antibody . Six weeks after the administration of antibodies, the rats were intestinally colonised with an ovalbumin (OVA)-producing E . coli O6K13 strain . At 8 weeks of age, the male rats (first generation) and the offsprings of the female rats (second generation), were parenterally immunised with OVA and dead wild type E . coli O6K13, and the immune response was followed . In the rats of the first generation, there were no major differences between the groups in the immune response to the bacterium . However, the offspring of the neonatally a-Id administered rats had a profoundly affected immune response to the idiotypically connected antigen K13, but also to other antigens on the bacteria . Thus, a-Id treatment in the first generation gave, in the second generation, a greatly enhanced serum antibody response to the spatially related antigens OVA and O6 LPS, as well as to the idiotypically connected antigen K13 . Concurrently, the in vitro spleen cell proliferative response to both OVA and the wild type bacterium was lowered . Overall, greater effects were seen with the higher dose of a-Id . In conclusion, our results demonstrate that by giving monoclonal antibodies idiotypically connected to a single bacterial component to neonatal rats, one profoundly influence the immune response also to other-spatially related-bacterial antigens in their offsprings. Biospectroscopy, 1999, 5(6), 338 - 45 Certain species of the Proteobacteria possess unusual bacteriochlorophyll a environments in their light-harvesting proteins; Gall A et al.; In this work, we have examined, using Fourier-transform Raman (FT-R) spectroscopy, the bacteriochlorophyll a (BChl a) binding sites in light-harvesting (LH) antennae from different species of the Proteobacteria that exhibit unusal absorption properties . While the LH1 complexes from Erythromicrobium (E.) ramosum (RC-B871) and Rhodospirillum centenum (B875) present classic FT-R spectra in the carbonyl high-frequency region, we show that in the blue-shifted LH1 complex, absorbing at 856 nm, from Roseococcus thiosulfatophilus, as well as in the B798-832 LH2 from E . ramosum, or in the B830 complex from the obligate phototrophic bacterium Chromatium purpuratum, some H-bonds between the acetyl carbonyl of the BChl a and the surrounding protein are missing . The molecular mechanisms responsible for the unusual absorption of these complexes are thus similar to those responsible for tuning of the absorption of the LH2 complexes between 850 and 820 nm . Furthermore, our results suggest that the binding pocket of the monomeric BChl in the LH2 from E . ramosum is different from that of Rps . acidphila or Rb . sphaeroides . The FT-R spectra of Chromatium purpuratum indicate that, in contrast with every LH2 complex previously studied by FT-R spectroscopy, no free-from-interaction keto groupings exist in this complex. Infect Immun, 2000 Jan, 68(1), 125 - 32 Host and bacterial factors involved in the innate ability of mouse macrophages to eliminate internalized unopsonized Escherichia coli; Hamrick TS et al.; In an effort to better understand genetic and cellular factors that influence innate immunity, we examined host and bacterial factors involved in the nonopsonic phagocytosis and killing of Escherichia coli K-12 by mouse macrophages . Unelicited (resident) peritoneal macrophages from five different mouse strains, BALB/c, C57BL/6, CD-1, C3H/HeJ, and C3H/HeN, were employed . Additional macrophage populations were obtained from CD-1 mice (bone marrow-derived macrophages) . Also, for BALB/c and C57BL/6 mice, peritoneal macrophages elicited with either thioglycolate or proteose peptone, bone marrow-derived macrophages, and macrophage-like cell lines derived from the two strains were employed . Two E . coli K-12 strains that differed specifically in their abilities to produce type 1 pili containing the adhesive protein FimH were examined . The parameters used to assess macrophage bacteriocidal activity were (i) the killing of internalized (gentamicin-protected) E . coli during the approximately 4-h assay and (ii) the initial rate at which internalized E . coli were eliminated . Data on these parameters allowed the following conclusions: (i) unelicited or proteose peptone-elicited peritoneal macrophages were significantly better at eliminating internalized bacteria than thioglycolate-elicited peritoneal macrophages, bone marrow-derived macrophages, or macrophage cell lines; (ii) the host genetic background had no significant effect upon the ability of unelicited peritoneal macrophages to kill E . coli (even though the mouse strains differ widely in their in vivo susceptibilities to bacterial infection); and (iii) the FimH phenotype had no significant effect upon E . coli survival once the bacterium was inside a macrophage . Additionally, there was no correlation between the bacteriocidal effectiveness of a macrophage population and the number of bacteria bound per macrophage . However, macrophage populations that were the least bacteriocidal tended to bind higher ratios of FimH(+) to FimH(-) E . coli . The effect of gamma interferon, fetal calf serum, and the recombination proficiency of E . coli were examined as factors predicted to influence intracellular bacterial killing . These had no effect upon the rate of E . coli elimination by unelicited peritoneal macrophages. FEBS Lett, 1999 Dec 10, 463(1-2), 169 - 74 Identification of a hydrogen bond in the phe M197-->Tyr mutant reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides by X-ray crystallography and FTIR spectroscopy; Kuglstatter A et al.; In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D) . An X-ray structure analysis of the Phe M197-->Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 A resolution suggests the formation of a hydrogen bond as postulated by Wachtveitl et al . {Biochemistry 32, 12875-12886, 1993} between the Tyr M197 hydroxy group and one of the 2a-acetyl carbonyls of D . In combination with electrochemically induced FTIR difference spectra showing a split band of the pi-conjugated 9-keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond. J Bacteriol, 1999 Dec, 181(24), 7464 - 9 Functional domains present in the mycobacterial hemagglutinin, HBHA; Delogu G et al.; Identification and characterization of mycobacterial adhesins and complementary host receptors required for colonization and dissemination of mycobacteria in host tissues are needed for a more complete understanding of the pathogenesis of diseases caused by these bacteria and for the development of effective vaccines . Previous investigations have demonstrated that a 28-kDa heparin-binding mycobacterial surface protein, HBHA, can agglutinate erythrocytes and promote mycobacterial aggregation in vitro . In this study, further molecular and biochemical analysis of HBHA demonstrates that it has three functional domains: a transmembrane domain of 18 amino acids residing near the N terminus, a large domain of 81 amino acids consistent with an alpha-helical coiled-coil region, and a Lys-Pro-Ala-rich C-terminal domain that mediates binding to proteoglycans . Using His-tagged recombinant HBHA proteins and nickel chromatography we demonstrate that HBHA polypeptides which contain the coiled-coil region form multimers . This tendency to oligomerize may be responsible for the induction of mycobacterial aggregation since a truncated N-terminal HBHA fragment containing the coiled-coil domain promotes mycobacterial aggregation . Conversely, a truncated C-terminal HBHA fragment which contains Lys-Pro-Ala-rich repeats binds to the proteoglycan decorin . These results indicate that HBHA contains at least three distinct domains which facilitate intercalation into surface membranes, promote bacterium-bacterium interactions, and mediate the attachment to sulfated glycoconjugates found in host tissues. Curr Microbiol, 2000 Feb, 40(2), 132 - 4 Isolation of intracytoplasmic membrane from the methanotrophic bacterium Methylomicrobium album BG8; Brantner CA et al.; Methane-oxidizing bacteria, including Methylomicrobium album BG8, form an intracytoplasmic membrane in addition to the cytoplasmic and outer membranes of the cell envelope . Techniques to isolate the intracytoplasmic membrane of M . album BG8 were developed . An intracytoplasmic membrane fraction was separated from a cell envelope fraction on the basis of sedimentation velocity in sucrose density gradients . Proteins associated with the particulate methane monooxygenase were found in both membrane fractions. J Immunol Methods, 1999 Nov 19, 230(1-2), 121 - 30 Fluobodies: green fluorescent single-chain Fv fusion proteins; Griep RA et al.; An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed . This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selected recombinant antibodies by flow cytometry or fluorescent cell staining . Two different single-chain variable fragment antibodies, both directed against the lipopolysaccharide of the bacterium Ralstonia solanacearum have been genetically fused to a red-shifted green fluorescent protein and the produced fusion protein tested for usefulness . These fluobodies can be produced in cultures of bacterial cells and purified using immobilized metal affinity chromatography . They function well in flow cytometry and immunofluorescent cell staining, are specific for their target antigens and, unlike FITC-conjugated antibodies, they do not fade upon illumination. J Microbiol Methods, 1999 Nov, 38(3), 183 - 9 Measurement of charge transfer during bacterial adhesion to an indium tin oxide surface in a parallel plate flow chamber; Poortinga AT et al.; An experimental method is described for the measurement of charge transfer during bacterial adhesion in situ to a transparent, semiconducting indium tin oxide (ITO) coated glass plate in a parallel plate flow chamber . Bacterial adhesion is measured simultaneously with either the electric potential or the capacitance of the surface . Initial bacterial adhesion was accompanied by a change in electric potential of the surface with no measurable change in capacitance . Consequently, it can be assumed that the change in electric potential of the surface is due to charge transfer between bacteria and the surface, and it can be calculated that, on average, a charge of about 10(-14) C per bacterium is exchanged during initial adhesion, which corresponds to only several percent of the total surface charge of a bacterium . Charge transfer could either be to or from the bacterial cell surface, dependent on the bacterial strain involved and the ionic strength used. Mutat Res, 1999 Nov 29, 430(1), 75 - 85 Intraclonal polymorphism in the bacterium Streptomyces ambofaciens ATCC23877: evidence for a high degree of heterogeneity of the wild type clones; Martin P et al.; In Streptomyces ambofaciens a genetic instability generates a high degree of polymorphism consisting of four main phenotypes: pigmented colonies (Pig(+) qualified as WT phenotype), pigment-defective colonies, pigmented colonies with pigment-defective sector and pigmented colonies with pigment-defective papillae . Molecular analysis of Pig(col)(-) and Pig(sec)(-) (pigment-defective mutant derived from a colony and a sector, respectively) produced by genetic instability and isolated in five Pig(+) subclones progenies revealed a new aspect of polymorphism in S . ambofaciens ATCC23877 . Frequencies of Pig(col)(-) and Pig(sec)(-) mutants deleted at the chromosome ends varied from one WT progeny to another . Two main types of deleted mutants were observed: deleted for one or both chromosomal extremities . The relative proportion of these two categories differed according to the WT progeny . These results argue for heterogeneity of the WT clones, i.e., Pig(+) colonies, originated from S . ambofaciens ATCC23877. Mol Gen Genet, 1999 Oct, 262(3), 501 - 7 A plasmid-based vector system for the cloning and expression of Helicobacter pylori genes encoding outer membrane proteins; Fischer W et al.; Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin . We observed that the functional expression of such proteins is deleterious to Escherichia coli, the host bacterium used for gene cloning . Therefore, a general method was developed for the functional expression of such genes on a shuttle vector in H . pylori, which has been termed SOMPES (Shuttle vector-based Outer Membrane Protein Expression System) . The intact, active gene is reconstituted by recombination in H . pylori from partial gene sequences cloned on an E . coli-H . pylori shuttle vector . This system was established in an H . pylori strain carrying a precise, unmarked chromosomal deletion of the vacA gene, which was constructed by adapting the streptomycin sensitivity system to H . pylori . It is based on the expression of the H . pylori rpsL gene as a counterselectable marker in the genetic background of an rpsL mutant . The utility of this approach is demonstrated by the expression of a recombinant gene encoding vacuolating cytotoxin (vacA) and a recombinant gene encoding an adherence-associated outer membrane protein (alpA) in H . pylori. Microbiology, 1999 Nov, 145 ( Pt 11), 3245 - 53 Novel Helicobacter pylori alpha1,2-fucosyltransferase, a key enzyme in the synthesis of Lewis antigens; Wang G et al.; Helicobacter pylori lipopolysaccharides (LPS) contain complex carbohydrates known as Lewis antigens which may contribute to the pathogenesis and adaptation of the bacterium . Involved in the biosynthesis of Lewis antigens is an alpha1,2-fucosyltransferase (FucT) that adds fucose to the terminal betaGal unit of the O-chain of LPS . Recently, the H . pylori (Hp) alpha1,2-FucT-encoding gene (fucT2) was cloned and analysed in detail . However, due to the low level of expression and instability of the protein, its enzymic activity was not demonstrated . In this study, the Hp fucT2 gene was successfully overexpressed in Escherichia coli . Sufficient amounts of the protein were obtained which revealed alpha1,2-fucosyltransferase activity to be associated with the protein . A series of substrates were chosen to examine the acceptor specificity of Hp alpha1,2-FucT, and the enzyme reaction products were identified by capillary electrophoresis . In contrast to the normal mammalian alpha,2-FucT (H or Se enzyme), Hp alpha1,2-FucT prefers to use Lewis X {betaGal1-4(alphaFuc1-3)betaGlcNAc} rather than LacNAc {betaGal1-4betaGIcNAc} as a substrate, suggesting that H . pylori uses a novel pathway (via Lewis X) to synthesize Lewis Y . Hp alpha1,2-FucT also acts on type 1 acceptor {betaGal1-3betaGlcNAc} and Lewis a {betaGal1-3(alphaFuc1-4)betaGIcNAc}, which provides H . pylori with the potential to synthesize H type 1 and Lewis b epitopes . The ability to transfer fucose to a monofucosylated substrate (Lewis X or Lewis a) makes Hp alpha1,2-FucT distinct from normal mammalian alpha1,2-FucT. Vet Immunol Immunopathol, 1999 Nov 30, 71(3-4), 173 - 9 A DNA fragment of Leptospira interrogans encodes a protein which shares epitopes with equine cornea; Lucchesi PM et al.; Horses infected with Leptospira interrogans present several clinical disorders, one of them being recurrent uveitis . An antigenic relationship between this bacterium and equine cornea has been described in previous studies . With the aim to make progress on defining the molecular basis and pathogenesis of equine recurrent uveitis, here we describe the cloning of one DNA fragment from a Leptospira interrogans serovar pomona genomic lambda gt11 library . Although there are references of transcription of leptospiral genes in E . coli from their own leptospiral promoters, in this recombinant construction the leptospiral DNA was located under the control of lacZ promoter since no expression could be detected in the absence of IPTG . This clone, isolated by expression screening with polyclonal serum raised against equine corneal proteins, encodes a 90 kDa protein of L . interrogans which crossreacts with equine cornea as proved Western-blotting . Antibodies directed against this leptospiral protein strongly recognised a 66 kDa equine corneal protein, one of those recognised by an anti-equine cornea serum . Our findings suggest that an immune response to 90 kDa protein participates in pathogenesis of equine uveitis. Appl Environ Microbiol, 1999 Dec, 65(12), 5532 - 40 Initial reactions in anaerobic alkane degradation by a sulfate reducer, strain AK-01; So CM et al.; An alkane-degrading, sulfate-reducing bacterial strain, AK-01, isolated from a petroleum-contaminated sediment was studied to elucidate its mechanism of alkane metabolism . Total cellular fatty acids of AK-01 were predominantly C even when it was grown on C-even alkanes and were predominantly C odd when grown on C-odd alkanes, suggesting that the bacterium anaerobically oxidizes alkanes to fatty acids . Among these fatty acids, some 2-, 4-, and 6-methylated fatty acids were specifically found only when AK-01 was grown on alkanes, and their chain lengths always correlated with those of the alkanes . When {1,2-(13)C(2)}hexadecane or perdeuterated pentadecane was used as the growth substrate, (13)C-labeled 2-Me-16:0, 4-Me-18:0, and 6-Me-20:0 fatty acids or deuterated 2-Me-15:0, 4-Me-17:0, and 6-Me-19:0 fatty acids were recovered, respectively, confirming that these monomethylated fatty acids were alkane derived . Examination of the (13)C-labeled 2-, 4-, and 6-methylated fatty acids by mass spectrometry showed that each of them contained two (13)C atoms, located at the methyl group and the adjacent carbon, thus indicating that the methyl group was the original terminal carbon of the {1, 2-(13)C(2)}hexadecane . For perdeuterated pentadecane, the presence of three deuterium atoms, on the methyl group and its adjacent carbon, in each of the deuterated 2-, 4-, and 6-methylated fatty acids further supported the hypothesis that the methyl group was the terminal carbon of the alkane . Thus, exogenous carbon appears to be initially added to an alkane subterminally at the C-2 position such that the original terminal carbon of the alkane becomes a methyl group on the subsequently formed fatty acid . The carbon addition reaction, however, does not appear to be a direct carboxylation of inorganic bicarbonate . A pathway for anaerobic metabolism of alkanes by strain AK-01 is proposed. Res Microbiol, 1999 Oct, 150(8), 507 - 20 Membrane topology of CadA homologous P-type ATPase of Helicobacter pylori as determined by expression of phoA fusions in Escherichia coli and the positive inside rule; Melchers K et al.; The only experimental data available on the membrane topology of transition metal ATPases are from in vitro studies on two distinct P-type ATPases (CadA and CopA) of a gastric bacterium, Helicobacter pylori, both postulated to contain eight transmembrane domains (H1 to H8) . In this study, H . pylori CadA ATPase was subjected to analysis of membrane topology in vivo by expression of ATPase-alkaline phosphatase (AP) hybrid proteins in Escherichia coli using a novel vector, pBADphoA . This vector contains an inducible arabinose promoter and unique restriction sites for fusion of DNA fragments to phoA . The phoA gene lacking sequences encoding its N-terminal signal peptide was linked to the C-terminal regions of the postulated five cytoplasmic and four periplasmic segments of the H . pylori pump . The results obtained by heterologous expression of ATPase-AP hybrid proteins showed consistence with a model of eight transmembrane domains . They also demonstrated that the H . pylori ATPase sequences are well assembled in the cytoplasmic membrane of E . coli, a neutralophilic bacterium . Cloning and amino acid sequence analysis of the homologous ATPase of Helicobacter felis further verified the topological model for the H . pylori pump analyzed here, although the degree of amino acid sequence identity varied between the corresponding transmembrane segments, from 25% for H1 up to 100% for H6 . It was found that the topology of ATPase follows the 'positive inside rule' . With respect to the bioenergetic capacities of H . pylori, we discuss here the membrane potential as a possible factor directing insertion of ATPases in the cytoplasmic membrane of gastric bacteria. Res Microbiol, 1999 Oct, 150(8), 499 - 505 Phage DNA transport across membranes; Letellier L et al.; Phage nucleic acid transport is atypical in bacterial membrane transport: it is unidirectional and concerns a unique molecule the size of which may represent 50 times that of the bacterium . The rate of DNA transport, although it varies from one phage to another, can reach values as high as 3000 bp s(-1) . This raises the following questions which will be discussed in this review . Is there a single mechanism of transport for all types of phages? Does the phage genome cross the outer and inner membranes by a unique mechanism? Is it transported as a free molecule or in association with proteins? How does it avoid periplasmic nucleases? Is such transport dependent on phage and/or host cell components? What is the driving force for transport? Recent cryoelectron microscopy experiments will be presented which show that it is possible to encapsulate a phage genome (121000 bp) into unilamellar liposomes . The interest of such a model system in gene delivery and in the study of the mechanisms of DNA compaction will be discussed. Biotechnol Appl Biochem, 1999 Dec, 30 ( Pt 3), 209 - 12 Photoproduction of L-tryptophan from indole and glycine by Rhodobacter sphaeroides OU5; Rajasekhar N et al.; A purple non-sulphur anoxygenic phototrophic bacterium Rhodobacter sphaeroides could synthesize L-tryptophan from indole and glycine with intermediate formation of D,L-alanine and L-serine . Presence of externally supplied keto acids has enhanced the rate and yields of L-tryptophan photoproduction. Int J Food Microbiol, 1999 Oct 15, 51(2-3), 145 - 58 Growth and enumeration of the meat spoilage bacterium Brochothrix thermosphacta; Rattanasomboon N et al.; Brochothrix thermosphacta is a common meat spoilage bacterium . The morphology of this bacterium changes from coccobacilli and short rods to chains during growth, which may give a false estimation in numbers using some enumeration techniques . Methods for the quantification of this bacterium have been compared . Turbidimetric readings showed good agreement with cell dry weight indicating that the former provides a good measure of the change in cell mass during growth . The turbidimetric method also correlated well with bacterial numbers determined by plate counts, flow cytometry and manual counts (by microscope) over a limited range of 10(7)-10(9) cells/ml . Flow cytometry and manual counts gave a linear relationship over a wider range of 10(5)-10(9) cells/ml . The sensitivity of analysis, growth rates and lag time attained using these methods were also compared . As a consequence of changes in bacterial cell size during growth, turbidimetry over-estimated the growth rate . The plate count method proved unable to detect the difference between bacteria existing as chains or single cells . The sensitivity of analysis and the calculated growth related parameters were similar for flow cytometry and manual counts . This suggests that flow cytometry is capable of counting individual cells in a chain . Further investigation showed that passage of B . thermosphacta cells through the flow cytometer resulted in the breakage of chains into single cells . The reliability, low error and rapidity of this technique make it attractive for bacterial enumeration, something which has been demonstrated using B . thermosphacta, a bacterium which exhibits complex morphologies. J Bacteriol, 1999 Dec, 181(23), 7356 - 62 The basis of ammonium release in nifL mutants of Azotobacter vinelandii; Brewin B et al.; In Azotobacter vinelandii, nitrogen fixation is regulated at the transcriptional level by an unusual two-component system encoded by nifLA . Certain mutations in nifL result in the bacterium releasing large quantities of ammonium into the medium, and earlier work suggested that this occurs by a mechanism that does not involve NifA, the activator of nif gene transcription . We have investigated a number of possible alternative mechanisms and find no evidence for their involvement in ammonium release . Enhancement of NifA-mediated transcription, on the other hand, by either elimination of nifL or overexpression of nifA, resulted in ammonium release, correlating with enhanced levels of nifH mRNA, raised levels of nitrogenase and acetylene-reducing activity, and increased concentrations of intracellular ammonium . Up to 35 mM ammonium can accumulate in the medium . Where measured, intracellular levels exceeded extracellular levels, indicating that rather than being actively transported, ammonium is lost from the cell passively, possibly by reversal of an NH(4)(+) uptake system . The data also indicate that in the wild type the bulk of NifA is inactivated by NifL during steady-state growth on dinitrogen. J Bacteriol, 1999 Dec, 181(23), 7248 - 55 Physical mapping and functional assignment of the geranylgeranyl-bacteriochlorophyll reductase gene, bchP, of Rhodobacter sphaeroides; Addlesee HA et al.; The bacteriochlorophyll of the purple photosynthetic bacterium Rhodobacter sphaeroides is esterified with phytol . The presence of this alcohol moiety is essential for the correct assembly of the photosynthetic apparatus . Despite this, and the fact that R . sphaeroides is widely used for the study of structure-function relationships in photosynthesis, the molecular genetics of the steps in which the alcohol is added and modified have not previously been investigated in this organism . Sequencing near the center of the photosynthesis gene cluster has now revealed the existence of an open reading frame encoding a putative 394-amino-acid polypeptide displaying strong homology with the products of a number of genes from other photosynthetic organisms, each proposed to be responsible for the reduction of the alcohol moiety of (bacterio)chlorophyll to phytol . An R . sphaeroides transposon mutant in this gene, bchP, possessed a structurally modified photosystem assembled with bacteriochlorophyll esterified with geranylgeraniol, rather than with phytol, implying that the product of this gene was geranylgeranyl-bacteriochlorophyll reductase . This identification was confirmed by the performance of in vitro assays using heterologously expressed protein, providing the first direct demonstration of the activity of a bchP gene product. J Immunol, 1999 Dec 1, 163(11), 6078 - 85 Subversion of monocyte functions by coxiella burnetii: impairment of the cross-talk between alphavbeta3 integrin and CR3; Capo C et al.; Several intracellular pathogens exploit macrophages as a niche for survival and replication . The success of this strategy requires the subversion or the avoidance of microbicidal functions of macrophages . Coxiella burnetii, the agent of Q fever, is a strictly intracellular bacterium that multiplies in myeloid cells . The survival of C . burnetii may depend on the selective use of macrophage receptors . Virulent C . burnetii organisms were poorly internalized but survived successfully in human monocytes, whereas avirulent variants were efficiently phagocytosed but were also rapidly eliminated . The uptake of avirulent organisms was mediated by leukocyte response integrin (alphavbeta3 integrin) and CR3 (alphaMbeta2 integrin), as demonstrated by using specific Abs and RGD sequence-containing peptides . The phagocytic efficiency of CR3 depends on its activation via alphavbeta3 integrin and integrin-associated protein . Indeed, CR3-mediated phagocytosis of avirulent C . burnetii was abrogated in macrophages from integrin-associated protein-/- mice . In contrast, the internalization of virulent C . burnetii organisms involved the engagement of alphavbeta3 integrin but not that of CR3 . The pretreatment of monocytes with virulent C . burnetii organisms prevented the CR3-mediated phagocytosis of zymosan particles and CR3 activation assessed by the expression of the 24 neo-epitope . We conclude that the virulence of C . burnetii is associated with the engagement of alphavbeta3 integrin and the impairment of CR3 activity, which probably results from uncoupling alphavbeta3 integrin from integrin-associated protein . This study describes a strategy not previously reported of phagocytosis modulation by intracellular pathogens. Infect Immun, 1999 Dec, 67(12), 6418 - 23 Involvement of protein kinase C in Rickettsia rickettsii-induced transcriptional activation of the host endothelial cell; Sahni SK et al.; Our laboratory has reported on a biphasic pattern of nuclear factor kappaB (NF-kappaB) activation in cultured human umbilical vein endothelial cells during infection with Rickettsia rickettsii, an obligate, intracellular bacterium, and the etiologic agent of Rocky Mountain spotted fever . Transcriptional activation of the tissue factor (TF) gene during this infection has been shown to involve NF-kappaB . To further understand the signal transduction events underlying these phenomena, we studied the role of protein kinase C (PKC), a ubiquitous family of phospholipid-dependent enzymes implicated in the regulation of a variety of cell signaling pathways . Two inhibitors of PKC, namely, bisindolylmaleimide I hydrochloride (BM-1) and calphostin C, which exhibit different inhibitory properties towards various isozymes of PKC, were used . Infection of cells with R . rickettsii in the presence of BM-1 (50 nM) did not significantly affect NF-kappaB, whereas calphostin C (2.5 microM) completely blocked the early phase of NF-kappaB activation . The late, sustained phase also was not affected by treatment with BM-1 . Downregulation of phorbol ester-sensitive PKCs by long-term treatment with phorbol 12-myristate 13-acetate (PMA) did not inhibit NF-kappaB activation . Likewise, this downregulation had no effect on induction of TF activity . The activity of TF was, however, sensitive to BM-1 and calphostin C, whereas expression of TF mRNA was inhibited only by calphostin C . Overall, these results suggest the lack of involvement of classical PKC pathways in R . rickettsii-induced NF-kappaB activation but the possible involvement of a non-PMA-responsive PKC isoform in the posttranscriptional control of TF expression. Science, 1999 Nov 19, 286(5444), 1571 - 7 Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1; White O et al.; The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284, 156 base pairs . Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D . radiodurans to survive under conditions of starvation, oxidative stress, and high amounts of DNA damage were identified . Deinococcus radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell. J Clin Microbiol, 1999 Dec, 37(12), 3822 - 7 Development of a universal intimin antiserum and PCR primers; Batchelor M et al.; Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E . coli (EHEC) constitute a significant risk to human health worldwide . A hallmark of both pathogens is their ability to produce characteristic attaching-and-effacing (A/E) lesions in intestinal epithelial cells . Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE) . Intimin, an LEE-encoded bacterial adhesion molecule, mediates the intimate bacterium-host cell interaction characteristic of A/E lesions . On the basis of characterization of the C-terminal 280-amino-acid cell binding domain of intimin (Int280(661-939)), four distinct Int280 types (types alpha, beta, gamma, and delta) have been identified . Importantly, Int280alpha and Int280beta antisera specifically recognized their respective intimin types . Using a conserved region of the intimin molecule (Int(388-667)) and primers synthesized to generate the recombinant Int(388-667), we have now generated universal intimin antiserum and PCR primers that are reactive with the different intimin types expressed by both human and animal A/E lesion-forming strains . Use of immunogold electron microscopy to visualize intimin on the surfaces of EPEC and EHEC strains revealed, in general, a uniform distribution on the bacterial cell surface . However, a filamentous staining pattern was observed with a few strains expressing intimin gamma . Cloning of the intimin eae gene from one such strain (strain ICC57) into strain CVD206, an EPEC strain which harbors a null deletion in eae, produced a uniform intimin staining pattern indicating that, if the filamentous staining pattern defines a filamentous form of intimin gamma, it is dependent upon the genetic background of the strain and is not a feature of the intimin molecule. Biochemistry, 1999 Nov 16, 38(46), 15238 - 44 Dark aerobic growth conditions induce the synthesis of a high midpoint potential cytochrome c8 in the photosynthetic bacterium Rubrivivax gelatinosus; Menin L et al.; In several strains of the photosynthetic bacterium Rubrivivax gelatinosus, the synthesis of a high midpoint potential cytochrome is enhanced 4-6-fold in dark aerobically grown cells compared with anaerobic photosynthetic growth . This observation explains the conflicting reports in the literature concerning the cytochrome c content for this species . This cytochrome was isolated and characterized in detail from Rubrivivax gelatinosus strain IL144 . The redox midpoint potential of this cytochrome is +300 mV at pH 7 . Its molecular mass, 9470 kDa, and its amino acid sequence, deduced from gene sequencing, support its placement in the cytochrome c8 family . The ratio of this cytochrome to reaction center lies between 0.8 and 1 for cells of Rvi . gelatinosus grown under dark aerobic conditions . Analysis of light-induced absorption changes shows that this high-potential cytochrome c8 can act in vivo as efficient electron donor to the photooxidized high-potential heme of the Rvi . gelatinosus reaction center. J Biol Chem, 1999 Nov 19, 274(47), 33594 - 600 Magnesium insertion by magnesium chelatase in the biosynthesis of zinc bacteriochlorophyll a in an aerobic acidophilic bacterium Acidiphilium rubrum; Masuda T et al.; To elucidate the mechanism for formation of zinc-containing bacteriochlorophyll a in the photosynthetic bacterium Acidiphilium rubrum, we isolated homologs of magnesium chelatase subunits (bchI, -D, and -H) . A . rubrum bchI and -H were encoded by single genes located on the clusters bchP-orf168-bchI-bchD-orf320-crtI and bchF-N-B-H-L as in Rhodobacter capsulatus, respectively . The deduced sequences of A . rubrum bchI, -D, and -H had overall identities of 59 . 8, 40.5, and 50.7% to those from Rba . capsulatus, respectively . When these genes were introduced into bchI, bchD, and bchH mutants of Rba . capsulatus for functional complementation, all mutants were complemented with concomitant synthesis of bacteriochlorophyll a . Analyses of bacteriochlorophyll intermediates showed that A . rubrum cells accumulate magnesium protoporphyrin IX monomethyl ester without detectable accumulation of zinc protoporphyrin IX or its monomethyl ester . These results indicate that a single set of magnesium chelatase homologs in A . rubrum catalyzes the insertion of only Mg(2+) into protoporphyrin IX to yield magnesium protoporphyrin IX monomethyl ester . Consequently, it is most likely that zinc-containing bacteriochlorophyll a is formed by a substitution of Zn(2+) for Mg(2+) at a step in the bacteriochlorophyll biosynthesis after formation of magnesium protoporphyrin IX monomethyl ester. Mol Microbiol, 1999 Oct, 34(2), 365 - 76 Dual control by regulatory gene fdsR of the fds operon encoding the NAD+-linked formate dehydrogenase of Ralstonia eutropha; Oh JI et al.; The transcriptional regulator gene fdsR was identified 150 bp upstream of the divergently oriented fdsGBACD operon encoding the soluble, NAD+-linked formate dehydrogenase in the chemoautotrophic bacterium Ralstonia eutropha H16 . Its deduced product, FdsR, displays a basal sequence similarity to the regulatory proteins of the LysR family . The carboxy-terminal domain of FdsR contains a short region that is conserved in formate dehydrogenases . Deletion of fdsR revealed a dual regulatory effect of FdsR on the fds operon by acting as transcriptional activator in the presence of formate or as repressor in the absence of formate . Studies with fdsR transcriptional fusions also suggested a negative autoregulation of the gene . A promoter structure resembling sigma70-dependent promoters from Escherichia coli was identified upstream of the fdsR transcriptional start site . FdsR purified to homogeneity after overexpression of fdsR in E . coli is a 130 kDa homotetramer binding to the fds control region located between the fdsR and fdsG genes . Formate significantly increased the binding affinity of FdsR for this region . Two FdsR binding sites characterized by the inverted-repeat structure ATANG-N10-CNTAT were identified . The regulatory pattern found in R . eutropha was also observed in the heterologous host E . coli and results from a novel mode of control of formate dehydrogenase genes. Mol Microbiol, 1999 Oct, 34(2), 257 - 67 Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis; Camacho LR et al.; Tuberculosis remains the greatest cause of death worldwide due to a single pathogen . In order to identify the genes required for the pathogenicity of Mycobacterium tuberculosis, a functional genomic approach was developed . A library of signature-tagged transposon mutants of this bacterium was constructed and screened for those affected in their multiplication within the lungs of mice . From 1927 mutants tested, 16 were attenuated for their virulence . The insertions harboured by the selected mutants were mapped on the M . tuberculosis genome and most of the mutated loci appeared to be involved in lipid metabolism or transport across the membrane . Four independent mutations identified a cluster of virulence genes located on a 50 kb chromosomal region . These genes might be involved in the production of phthiocerol and phenolphthiocerol derivatives, a group of molecules restricted to eight mycobacterial species, seven of them being either strict or opportunistic pathogens . The interaction of five mutant strains with mouse bone marrow macrophages was investigated . These five mutants were still able to multiply in this cell type . However, in three cases, there was a growth defect in comparison with the wild-type strain . The other two strains exhibited no clear difference from the virulent strain, MT103, in this model . This study, which is the first global research of virulence factors of M . tuberculosis, opens the way to a better understanding of the molecules that are key players in the interaction of this pathogen with its host. Biochim Biophys Acta, 1999 Sep 21, 1421(1), 53 - 63 Correlation between anti-bacterial activity and pore sizes of two classes of voltage-dependent channel-forming peptides; Beven L et al.; Anti-bacterial activities were compared for two series of voltage-dependent pore-formers: (i) alamethicin (Alm) and its synthetic analogs (Alm-dUL) where alpha-amino-isobutyric acid residues (Aibs) were replaced by leucines and selected key residues substituted and (ii) homologous voltage sensors of the electric eel sodium channel (repeats S4L45 (III) and S4L45 (IV)) . Spiroplasma melliferum, a bacterium related to the mycoplasmas, was used as a target cell . The data show that with respect to growth inhibition, cell deformation and plasma membrane depolarization, the highest efficient peptide remained natural Alm although the minimal inhibitory concentrations of its Leu analogs were within the same range as the parent molecule, except for Alm-dUL P14A . Thus, as for the pore-forming activity observed in artificial membranes and for the toxicity towards mammalian cells, proline-14 proved to be a critical residue for the anti-bacterial activity of alamethicin . Regarding the sodium voltage sensors, their anti-bacterial efficiency was at least 10 times lower although they promoted spiroplasma cell agglutination . The anti-bacterial activities of the peptides were correlated with their pore-forming properties, especially with the apparent and mean number of monomers per conducting aggregate (<N>) when both peptide families were considered and, secondly, with mean open times (tau(o)) within each family . This suggests that although they may form 'raft-like' structures, the mechanism underlying anti-bacterial activity of Alm and its active analogs, as well as the S4L45 voltage sensors with the S . melliferum plasma membrane, is predominantly through pore-formation according to the 'barrel-stave' mechanism. Med Res Rev, 1999 Nov, 19(6), 543 - 58 Novel macrolides through genetic engineering; Katz L et al.; Erythromycin, a complex polyketide antibiotic belonging to the macrolide class, is produced as a natural product by the bacterium Saccharopolyspora erythraea . The genes encoding the enzymes responsible for the synthesis of the antibiotic have been cloned and sequenced, revealing that the polyketide backbone of the molecule in produced by a polyketide synthase (PKS) composed of multifunctional proteins that contain discrete functional domains for each step of synthesis . Genetic manipulation of the PKS-encoding genes can result in predictable changes in the structure of the polyketide component of erythromycin, many of which are not easily achievable through standard chemical derivatization or synthesis . Many of the changes can be combined to lead to the further generation of navel structures . Whereas genetic engineering of the erythromycin structure has been practiced for a number of years, the re cent and continuing discoveries of modular PKSs for the synthesis of many other important complex polyketides has raised the possibility of generating novel structures in these molecules by genetic manipulation, as well . FEMS Microbiol Lett, 1999 Nov 15, 180(2), 279 - 86 Phylogenetic analysis of the 16S rDNA of the cytoplasmic bacterium Wolbachia from the novel host Folsomia candida (Hexapoda, Collembola) and its implications for wolbachial taxonomy; Vandekerckhove TT et al.; Wolbachia pipientis are intracellular, transovarially inherited alpha-Proteobacteria in invertebrates . Four major Wolbachia groups exist: A, B (contained in divergent arthropods), C and D (harbored by Nematoda) . By means of transmission electron microscopy, we observed Wolbachia-like bacteria in a primitive insect, Folsomia candida (Hexapoda, Collembola, Isotomidae) . 16S rDNA analysis proved them to constitute a novel lineage, henceforth named group E, in the wolbachial phylogenetic tree . It shares 97.8% 16S rDNA homology with its nearest neighbors, groups A and B, which diverged from it more recently . We propose (i) a new taxon E for the Wolbachia strain in F . candida, (ii) that the single-described Wolbachia pipientis fall apart into at least three species: C, D and the large E-A-B complex . F . candida's group E Wolbachia rekindle the question about invasive capacities of free-living ancestral wolbachiae and horizontal transfer. FEMS Microbiol Lett, 1999 Nov 15, 180(2), 249 - 54 Differentiation of Coxiella burnetii isolates by sequence determination and PCR-restriction fragment length polymorphism analysis of isocitrate dehydrogenase gene; Nguyen SV et al.; The isocitrate dehydrogenase (icd) gene of Coxiella burnetii was cloned and sequenced to differentiate between isolates with various geographic origins and phenotypic properties . Based on the gene sequences all 19 isolates studied could be divided into three groups . Group 1 contained isolates originating from acute cases of Q fever, ticks and cows . Groups 2 and 3 included isolates from chronic Q fever patients and a prototype strain from an aborted goat . Although the icd gene profiles were different among isolates of the latter two groups, there were two base differences common for both groups which could be used as markers to distinguish them from group 1 isolates . Based on one of the markers a simple method using PCR-restriction fragment length polymorphism analysis was developed for rapid differentiation of C . burnetii isolates as well as for direct detection and differentiation of the bacterium in human serum samples . Taken together, the study results suggest that the icd-based differentiation method may be useful in clinical investigation of Coxiella infections. Biochim Biophys Acta, 1999 Nov 10, 1413(3), 172 - 80 Association of bacteriochlorophyll a with the CsmA protein in chlorosomes of the photosynthetic green filamentous bacterium Chloroflexus aurantiacus; Sakuragi Y et al.; The protein assumed to be associated with bacteriochlorophyll (BChl) a in chlorosomes from the photosynthetic green filamentous bacterium Chloroflexus aurantiacus was investigated by alkaline treatment, proteolytic digestion and a new treatment using 1-hexanol, sodium cholate and Triton X-100 . Upon alkaline treatment, only the 5.7 kDa CsmA protein was removed from the chlorosomes among six proteins detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, concomitantly with the disappearance of BChl a absorption at 795 nm . Trypsin treatment removed two proteins with molecular masses of 11 and 18 kDa (CsmN and CmsM), whereas the spectral properties of BChl a and BChl c were not changed . By the new hexanol-detergent (HD) treatment, most BChl c and all of the detected proteins except CsmA were removed from the chlorosomes without changing the BChl a spectral properties . Subsequent proteinase K treatment of these HD-treated chlorosomes caused digestion of CsmA and a simultaneous decrease of the BChl a absorption band . Based on these results, we suggest that CsmA is associated with BChl a in the chlorosomes . This suggestion was supported by the measured stoichiometric ratio of BChl a to CsmA in isolated chlorosomes, which was estimated to be between 1.2 and 2.7 by amino acid analysis of the SDS-PAGE-resolved protein bands. Biochim Biophys Acta, 1999 Nov 10, 1413(3), 108 - 16 Exogenous quinones inhibit photosynthetic electron transfer in Chloroflexus aurantiacus by specific quenching of the excited bacteriochlorophyll c antenna; Frigaard N et al.; In the photosynthetic green filamentous bacterium Chloroflexus aurantiacus, excitation energy is transferred from a large bacteriochlorophyll (BChl) c antenna via smaller BChl a antennas to the reaction center . The effects of substituted 1,4-naphthoquinones on BChl c and BChl a fluorescence and on flash-induced cytochrome c oxidation were studied in whole cells under aerobic conditions . BChl c fluorescence in a cell suspension with 5.4 microM BChl c was quenched to 50% by addition of 0.6 microM shikonin ((R)-2-(1-hydroxy-4-methyl-3-pentenyl)-5,8-dihydroxy-1, 4-naphthoquinone), 0.9 microM 5-hydroxy-1,4-naphthoquinone, or 4 microM 2-acetyl-3-methyl-1,4-naphthoquinone . Between 25 and 100 times higher quinone concentrations were needed to quench BChl a fluorescence to a similar extent . These quinones also efficiently inhibited flash-induced cytochrome c oxidation when BChl c was excited, but not when BChl a was excited . The quenching of BChl c fluorescence induced by these quinones correlated with the inhibition of flash-induced cytochrome c oxidation . We concluded that the quinones inhibited electron transfer in the reaction center by specifically quenching the excitation energy in the BChl c antenna . Our results provide a model system for studying the redox-dependent antenna quenching in green sulfur bacteria because the antennas in these bacteria inherently exhibit a sensitivity to O(2) similar to the quinone-supplemented cells of Cfx . aurantiacus. J Mol Biol, 1999 Nov 19, 294(1), 181 - 91 Antigenic properties of peptidic mimics for epitopes of the lipopolysaccharide from Brucella; De Bolle X et al.; The lipopolysaccharide (LPS) is up to now the only identified major virulence determinant of Brucella . This bacterium is responsible for brucellosis in animals and for Malta fever in humans . Several monoclonal antibodies (mAbs) directed against various LPS epitopes have been characterized . Two mAbs, named A15-6B3 and B66-2C8, directed against distinct LPS epitopes have been used to select peptides from 11 phage display libraries . The sequences of the selected peptides contain an overrepresentation of either proline or tryptophan residues when selected with either A15-6B3 or B66-2C8 mAbs, respectively . For the best binding peptides, competition with LPS for the binding to the mAb is detected, which suggests that the peptides bind to the paratope of the mAb . The phages selected from the libraries were used to immunise mice, and a weak antibody response directed against LPS has been observed . These data suggest that a subset of the selected peptides are mimotopes of the LPS epitopes . J Biol Inorg Chem, 1999 Aug, 4(4), 478 - 94 Nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774:primary sequence determination, crystallographic refinement at 1.8 and modelling studies of its interaction with the tetrahaem cytochrome c3; Matias PM et al.; A monomeric nine-haem cytochrome c (9Hcc) with 292 amino acid residues was isolated from cells of the sulfate- and nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 grown under both nitrate- and sulfate-respiring conditions . The nucleotide sequence encoding the 292 residues was determined, allowing the correction of about 10% of the previous primary structure, determined from 1.8 A electron density maps . The refinement at 1.8 A resolution of the structural model was completed, giving an R-value of 16.5% . The nine haem groups are arranged into two tetrahaem clusters, located at both ends of the molecule, with Fe-Fe distances and local protein fold very similar to tetrahaem cytochromes c3, and the extra haem is located asymmetrically between the two regions . The new primary sequence determination confirmed the 39% sequence homology found between this cytochrome and the C-terminal region (residues 229-514) of the high-molecular-weight cytochrome c (Hmc) from D . vulgaris Hildenborough, providing strong evidence of structural similarity between 9Hcc and the C-terminal region of Hmc . The interaction between 9Hcc and the tetrahaem cytochrome c3 from the same organism was studied by modelling methods, and the results suggest that a specific interaction is possible between haem 4 of tetrahaem cytochrome c3 and haem 1 or haem 2 of 9Hcc, in agreement with previous kinetic experiments which showed the catalytic effect of the tetrahaem cytochrome c3 upon the reduction of 9Hcc by the {NiFe} hydrogenase from D . desulfuricans ATCC 27774 . These studies suggest a role for 9Hcc as part of the assembly of redox proteins involved in recycling the molecular hydrogen released by the cell as a result of substrate oxidation. Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1873 - 7 Actinomyces bowdenii sp . nov., isolated from canine and feline clinical specimens; Pascual C et al.; Four strains of a previously undescribed Actinomyces-like bacterium were isolated from canine and feline clinical specimens . Phenotypic studies indicated the strains were members of the genus Actinomyces, and most closely resembled Actinomyces viscosus serotype I and Actinomyces slackii . Comparative 16S rRNA gene sequencing studies demonstrated the unknown bacterium constitutes a new subline within a group of Actinomyces species, which includes Actinomyces bovis, the type species of the genus . Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium be classified as Actinomyces bowdenii sp . nov . The type strain of Actinomyces bowdenii is CCUG 37421T. Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1801 - 8 Description of Desulfotomaculum sp . Groll as Desulfotomaculum gibsoniae sp . nov; Kuever J et al.; Strain GrollT, isolated from fresh water, is a mesophilic, spore-forming, sulfate-reducing bacterium that uses a large variety of substrates as electron donors ranging from simple organic compounds to long-chain fatty acids and several aromatic compounds . Sulfate, thiosulfate and sulfite are used as electron acceptors . Homoacetogenic growth occurs under sulfate-free conditions . Substrate oxidation is usually complete, leading to CO2, but acetate or other fatty acids can accumulate at high substrate concentrations . The G + C content of the DNA is 54.8 mol% . Strain GrollT was found to be phenotypically and phylogenetically different from known members of the genus Desulfotomaculum . 16S rRNA gene sequence analyses show that this organism falls within the radiation of the genus Desulfotomaculum cluster and has < 96% sequence similarity to previously described species . The name Desulfotomaculum gibsoniae sp . nov . is proposed for this strain; the type strain is GrollT (= DSM 7213T). Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1785 - 91 Gordonia polyisoprenivorans sp . nov., a rubber-degrading actinomycete isolated from an automobile tyre; Linos A et al.; A rubber-degrading bacterium (strain Kd2T) was isolated from fouling tyre water inside a deteriorated automobile tyre . The strain was aerobic, Grampositive, produced elementary branching hyphae which fragmented into rod/coccus-like elements and showed chemotaxonomic markers which were consistent with the classification of Gordonia, i.e . meso-diaminopimelic acid, N-glycolyl muramic acid, arabinose and galactose as diagnostic sugars, a fatty acid pattern composed of unbranched saturated and monounsaturated fatty acids with a considerable amount of tuberculostearic acid, and mycolic acids comprising 58-66 carbon atoms with two principal mycolic acids C60 and C62 counting for over 60% . Results of 16S rDNA analyses as well as chemotaxonomic results, led to the conclusion that Gordonia sp . strain Kd2T (= DSM 44302T) represents a new species within the genus Gordonia for which the name Gordonia polyisoprenivorans is proposed. Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1681 - 93 Description of Gluconacetobacter sacchari sp . nov., a new species of acetic acid bacterium isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug; Franke IH et al.; A new species of the genus Gluconacetobacter, for which the name Gluconacetobacter sacchari sp . nov . is proposed, was isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug, Saccharicoccus sacchari, found on sugar cane growing in Queensland and northern New South Wales, Australia . The nearest phylogenetic relatives in the alpha-subclass of the Proteobacteria are Gluconacetobacter liquefaciens and Gluconacetobacter diazotrophicus, which have 98.8-99.3% and 97.9-98.5% 16S rDNA sequence similarity, respectively, to members of Gluconacetobacter sacchari . On the basis of the phylogenetic positioning of the strains, DNA reassociation studies, phenotypic tests and the presence of the Q10 ubiquinone, this new species was assigned to the genus Gluconacetobacter . No single phenotypic characteristic is unique to the species, but the species can be differentiated phenotypically from closely related members of the acetic acid bacteria by growth in the presence of 0.01% malachite green, growth on 30% glucose, an inability to fix nitrogen and an inability to grow with the L-amino acids asparagine, glycine, glutamine, threonine and tryptophan when D-mannitol was supplied as the sole carbon and energy source . The type strain of this species is strain SRI 1794T (= DSM 12717T). Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1573 - 6 Characterization of a novel Atopobium isolate from the human vagina: description of Atopobium vaginae sp . nov; Rodriguez Jovita M et al.; Phenotypic and phylogenetic studies were performed on a hitherto undescribed micro-organism isolated from the human vagina . Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strain constituted a new subline within the genus Atopobium . The unknown bacterium was readily distinguished from other Atopobium species by biochemical tests and electrophoretic analysis of whole-cell proteins . Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Atopobium vaginae sp . nov . The type strain of Atopobium vaginae is CCUG 38953T. Arch Microbiol, 1999 Nov, 172(5), 313 - 20 A heme-C-containing enzyme complex that exhibits nitrate and nitrite reductase activity from the dissimilatory iron-reducing bacterium Geobacter metallireducens; Martinez Murillo F et al.; Nitrate reduction in the dissimilatory iron-reducing bacterium Geobacter metallireducens was investigated . Nitrate reductase and nitrite reductase activities in nitrate-grown cells were detected only in the membrane fraction . The apparent K(m )values for nitrate and nitrite were determined to be 32 and 10 microM, respectively . Growth on nitrate was not inhibited by either tungstate or molybdate at concentrations of 1 mM or less, but was inhibited by both at 10 and 20 mM . Nitrate and nitrite reductase activity in the membrane fraction was not, however, affected by dialysis with 20 mM tungstate . An enzyme complex that exhibited both nitrate and nitrite reductase activity was solubilized from membrane fractions with CHAPS and was partially purified by preparative gel electrophoresis . It was found to be composed of four different polypeptides with molecular masses of 62, 52, 36, and 16 kDa . The 62-kDa polypeptide {a low-midpoint potential (-207 mV), multiheme cytochrome c} exhibited nitrite reductase activity under denaturing conditions . No molybdenum was detected in the complex by plasma-emission mass spectrometry. Clin Diagn Lab Immunol, 1999 Nov, 6(6), 787 - 90 Complement fixation test to assess humoral immunity in cattle and sheep vaccinated with Brucella abortus RB51; Adone R et al.; The live attenuated Brucella abortus strain RB51 is a rifampin-resistant, lipopolysaccharide (LPS) O-chain-deficient mutant of virulent B . abortus 2308 . The reduced O-chain content in RB51 prevents this bacterium from inducing antibodies detectable by the conventional serologic tests for bovine brucellosis diagnosis that mainly identify antibodies to LPS . The absence of available serologic tests for RB51 also complicates the diagnosis of possible RB51 infections in humans exposed to this strain . The purpose of this study was to evaluate the suitability of a complement fixation (CF) test performed with the rough strain B . abortus RB51, previously deprived of anticomplementary activity, in detecting anti-B . abortus RB51 antibodies in cattle and sheep experimentally vaccinated with this strain . The results of this study showed that a CF test with RB51 as the antigen is able to specifically detect antibodies following RB51 vaccination in cattle and sheep . In addition, this method could be a useful tool for detecting B . abortus RB51 infection in humans. Appl Environ Microbiol, 1999 Nov, 65(11), 5017 - 22 Protein-mediated adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to hydrous ferric oxide; Caccavo F Jr; The rate and extent of bacterial Fe(III) mineral reduction are governed by molecular-scale interactions between the bacterial cell surface and the mineral surface . These interactions are poorly understood . This study examined the role of surface proteins in the adhesion of Shewanella alga BrY to hydrous ferric oxide (HFO) . Enzymatic degradation of cell surface polysaccharides had no effect on cell adhesion to HFO . The proteolytic enzymes Streptomyces griseus protease and chymotrypsin inhibited the adhesion of S . alga BrY cells to HFO through catalytic degradation of surface proteins . Trypsin inhibited S . alga BrY adhesion solely through surface-coating effects . Protease and chymotrypsin also mediated desorption of adhered S . alga BrY cells from HFO while trypsin did not mediate cell desorption . Protease removed a single peptide band that represented a protein with an apparent molecular mass of 50 kDa . Chymotrypsin removed two peptide bands that represented proteins with apparent molecular masses of 60 and 31 kDa . These proteins represent putative HFO adhesion molecules . S . alga BrY adhesion was inhibited by up to 46% when cells were cultured at sub-MICs of chloramphenicol, suggesting that protein synthesis is necessary for adhesion . Proteins extracted from the surface of S . alga BrY cells inhibited adhesion to HFO by up to 41% . A number of these proteins bound specifically to HFO, suggesting that a complex system of surface proteins mediates S . alga BrY adhesion to HFO. FEMS Microbiol Lett, 1999 Nov 1, 180(1), 21 - 9 The amo operon in marine, ammonia-oxidizing gamma-proteobacteria; Alzerreca JJ et al.; While there is an extensive database of genes encoding ammonia monooxygenase (amo) found in the ammonia-oxidizing beta-proteobacteria, few amo sequences are available representing the gamma-proteobacteria . We sequenced the complete amo operon (amoCAB) for Nitrosococcus oceani (ATCC 19707), a marine, autotrophic, ammonia-oxidizing bacterium belonging to the gamma-subdivision of the proteobacteria . An additional autotrophic, ammonia-oxidizing bacterium isolated from a marine environment (strain C-113) was identified as belonging to the Nitrosococcus genus by 16S rDNA analysis and its amo operon was sequenced . This is the first report of a full-length sequence for the amo operon from a gamma-subdivision autotrophic ammonia-oxidizing bacterium . The N . oceani and C-113 amo genes were 88-90% identical to each other, 49-53% identical to the pmo genes encoding the related particulate methane monooxygenase of Methylococcus capsulatus (Bath), and 39-42% identical to the amo genes of the beta-subdivision autotrophic ammonia-oxidizing bacteria . In both Nitrosococcus strains, the amo operon was found as a single copy and contained three genes, amoC, amoA, amoB, with intergenic spacer regions between amoC and amoA (286 bp) and between amoA and amoB (65 bp) . We conclude that the amo genes will allow for a finer scale phylogenetic differentiation than 16S rDNA within the gamma-subdivision AOB. Protein Expr Purif, 1999 Nov, 17(2), 299 - 304 Construction and characterization of histidine-tagged haloalkane dehalogenase (LinB) of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26; Nagata Y et al.; The linB gene product (LinB), which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26, is a member of haloalkane dehalogenases with a broad range of substrate specificity . Elucidation of the factors determining its substrate specificity is of interest . Aiming to facilitate purification of recombinant LinB protein for site-directed mutagenesis analysis, a 6-histidyl tail was added to the C-terminus of LinB . The His-tagged LinB was specifically bound with Ni-NTA resin in the buffer containing 10 mM imidazole . After elution with 500 mM imidazole, quantitative recovery of protein occurred . The steady-state kinetic parameters of the His-tagged LinB for four substrates were in good agreement with that of wild-type recombinant LinB . Although the His-tagged LinB expressed in an average of 80% of the activity of the wild type LinB for 10 different substrates, the decrease was very similar for different substrates with the standard deviation of 5.5% . The small activity reduction is independent of the substrate shape, size, or number of substituents, indicating that the His-tagged LinB can be used for further mutagenesis studies . To confirm the suitability of this system for mutagenesis studies, two mutant proteins with substitution in putative halide binding residues (W109 and F151) were constructed, purified, and tested for activity . As expected, complete loss in activity of W109L and sustained activity of F151W were observed . Appl Environ Microbiol, 1999 Nov, 65(11), 5163 - 8 Effect of various ions, pH, and osmotic pressure on oxidation of elemental sulfur by Thiobacillus thiooxidans; Suzuki I et al.; The oxidation of elemental sulfur by Thiobacillus thiooxidans was studied at pH 2.3, 4.5, and 7.0 in the presence of different concentrations of various anions (sulfate, phosphate, chloride, nitrate, and fluoride) and cations (potassium, sodium, lithium, rubidium, and cesium) . The results agree with the expected response of this acidophilic bacterium to charge neutralization of colloids by ions, pH-dependent membrane permeability of ions, and osmotic pressure. Appl Environ Microbiol, 1999 Nov, 65(11), 5089 - 99 Natural communities of Achromatium oxaliferum comprise genetically, morphologically, and ecologically distinct subpopulations; Gray ND et al.; The diversity and ecology of natural communities of the uncultivated bacterium Achromatium oxaliferum were studied by use of culture-independent approaches . 16S rRNA gene sequences were PCR amplified from DNA extracted from highly purified preparations of cells that were morphologically identified as A . oxaliferum present in freshwater sediments from three locations in northern England (Rydal Water, Jenny Dam, Hell Kettles) . Cloning and sequence analysis of the PCR-amplified 16S rRNA genes revealed that multiple related but divergent sequences were routinely obtained from the A . oxaliferum communities present in all the sediments examined . Whole-cell in situ hybridization with combinations of fluorescence-labelled oligonucleotide probes revealed that the divergent sequences recovered from purified A . oxaliferum cells corresponded to genetically distinct Achromatium subpopulations . Analysis of the cell size distribution of the genetically distinct subpopulations demonstrated that each was also morphologically distinct . Furthermore, there was a high degree of endemism in the Achromatium sequences recovered from different sediments; identical sequences were never recovered from different sampling locations . In addition to ecological differences that were apparent between Achromatium communities from different freshwater sediments, the distribution of different subpopulations of Achromatium in relation to sediment redox profiles indicated that the genetically and morphologically distinct organisms that coexisted in a single sediment were also ecologically distinct and were adapted to different redox conditions . This result suggests that Achromatium populations have undergone adaptive radiation and that the divergent Achromatium species occupy different niches in the sediments which they inhabit. Appl Environ Microbiol, 1999 Nov, 65(11), 4734 - 40 Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum; Kessi J et al.; The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum . Anoxic photosynthetic cultures were able to completely reduce as much as 1 . 5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM . The presence of selenite in the culture medium strongly affected cell division . In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density . The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium . Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells . Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells . This demonstrated that R . rubrum expels elemental selenium across the plasma membrane and the cell wall . Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase. Biochim Biophys Acta, 1999 Oct 28, 1447(2-3), 357 - 65 Identification and characterization of five cspA homologous genes from Myxococcus xanthus; Yamanaka K et al.; Escherichia coli contains a large CspA family consisting of nine homologues, in which four are cold-shock inducible and one is stationary-phase inducible . Here, we demonstrate that Myxococcus xanthus possesses at least five CspA homologues, CspA to CspE . Hydrophobic residues forming a hydrophobic core, and aromatic residues, which are included in functional motifs RNP-1 and RNP-2 involved in binding to RNA and ssDNA, are well conserved . These facts suggest that M . xanthus CspA homologues have a similar structure and function as E . coli CspA . However, in contrast to the E . coli CspA family, the expression of M . xanthus csp genes as judged by primer extension analysis is not significantly regulated by temperature changes, except for cspB of which expression was reduced to less than 10% upon heat shock at 42 degrees C . Such constitutive expression of the csp genes may be important for M . xanthus, a soil-dwelling bacterium, to survive under conditions of exposure to various environmental changes in nature. J Bacteriol, 1999 Nov, 181(21), 6788 - 96 Structural characterization of the symbiotically important low-molecular-weight succinoglycan of Sinorhizobium meliloti; Wang LX et al.; The production of succinoglycan by Sinorhizobium meliloti Rm1021 is required for successful nodule invasion by the bacterium of its host plant, alfalfa . Rm1021 produces succinoglycan, an acidic exopolysaccharide composed of an octasaccharide repeating unit modified with acetyl, succinyl, and pyruvyl moieties, in both low- and high-molecular-weight forms . Low-molecular-weight (LMW) succinoglycan, previously thought to consist of monomers, trimers, and tetramers of the repeating unit, has been reported as being capable of promoting the formation of nitrogen-fixing nodules by succinoglycan-deficient derivatives of strain Rm1021 . We have determined that the three size classes of LMW succinoglycan species are in fact monomers, dimers, and trimers of the repeating unit and that the trimer is the species active in promoting nodule invasion . A detailed structural analysis of the components of LMW succinoglycan by using various chromatographic techniques, along with nuclear magnetic resonance analyses, has revealed that there is considerable heterogeneity within the LMW succinoglycan oligomers in terms of noncarbohydrate substitutions, and we have determined the structural basis of this heterogeneity. J Appl Microbiol, 1999 Sep, 87(3), 410 - 7 Reduced toxicity of expression, in Escherichia coli, of antipollutant antibody fragments and their use as sensitive diagnostic molecules; Strachan G et al.; Single-chain antibody fragments (scAb), specific for the chlorophenoxy acid herbicide mecoprop, have been expressed and purified from the bacterium Escherichia coli . Co-expression with the colE1-compatible, arabinose-inducible, skp expression vector pHELP1 prevented bacterial lysis and significantly increased both total and functional expression yield . The periplasmic protein, SKP, may have a role as a generic detoxification protein . Surface plasmon resonance (BIAcore 2000) analysis confirmed that the purified scAb retained similar binding kinetics to the monoclonal antibody (Mab) from which it was cloned . In competition ELISA, the bacterial scAb showed the same specificity for mecoprop and a related herbicide, MCPA, as the Mab but an increase in sensitivity for free antigen in all ELISA formats . Bacterially expressed antibody fragments provide a simple, sensitive and cost-effective alternative to the traditional production of diagnostic Mabs via tissue culture. Proc Natl Acad Sci U S A, 1999 Oct 26, 96(22), 12833 - 8 Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization; Wilson M et al.; Tuberculosis is a chronic infectious disease that is transmitted by cough-propelled droplets that carry the etiologic bacterium, Mycobacterium tuberculosis . Although currently available drugs kill most isolates of M . tuberculosis, strains resistant to each of these have emerged, and multiply resistant strains are increasingly widespread . The growing problem of drug resistance combined with a global incidence of seven million new cases per year underscore the urgent need for new antituberculosis therapies . The recent publication of the complete sequence of the M . tuberculosis genome has made possible, for the first time, a comprehensive genomic approach to the biology of this organism and to the drug discovery process . We used a DNA microarray containing 97% of the ORFs predicted from this sequence to monitor changes in M . tuberculosis gene expression in response to the antituberculous drug isoniazid . Here we show that isoniazid induced several genes that encode proteins physiologically relevant to the drug's mode of action, including an operonic cluster of five genes encoding type II fatty acid synthase enzymes and fbpC, which encodes trehalose dimycolyl transferase . Other genes, not apparently within directly affected biosynthetic pathways, also were induced . These genes, efpA, fadE23, fadE24, and ahpC, likely mediate processes that are linked to the toxic consequences of the drug . Insights gained from this approach may define new drug targets and suggest new methods for identifying compounds that inhibit those targets. Proc Natl Acad Sci U S A, 1999 Oct 26, 96(22), 12356 - 61 Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity; Carmen AA et al.; Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes . These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription . To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function . In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and Escherichia coli, and has intrinsic activity when produced in the bacterium . Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification . We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity in vitro for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11 . We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity. J Gastroenterol, 1999 Oct, 34(5), 560 - 70 The vast majority of gastric T cells are polarized to produce T helper 1 type cytokines upon antigenic stimulation despite the absence of Helicobacter pylori infection; Itoh T et al.; Helicobacter pylori infection is associated with chronic infiltration by various cell types, including T cells, whose cytokine production may regulate the inflammatory reaction as well as local immune response to the bacterium . We prospectively analyzed the constituents of the cellular infiltrates and the cytokines produced by T cells in antral biopsies obtained from 73 subjects with and without H . pylori infection, before and after eradication therapy, and compared them with a histological grade of gastritis . We found that T cells predominated in cell number, followed by granulocytes/monocytes and plasma cells in both H . pylori-infected and H . pylori-uninfected subjects . Despite the absence of H . pylori infection, more than 70% of gastric CD4-positive T cells obtained from uninfected tissue produced interferon-gamma (IFN-gamma) in the cytosol . Upon receptor cross-linking of a CD3 and a CD28 molecule, T cells in both infected and uninfected tissue continuously secreted a far greater amount of IFN-gamma than those in peripheral blood mononuclear cell controls for a period of cell culture, whereas the increase in interleukin-4 (IL-4) was very small, and no increase in IL-2 secretion was seen . In H . pylori-infected patients, IFN-gamma secretion was correlated with the grade of mononuclear cell infiltration and decreased to an uninfected control level after eradication therapy . We did not see the effect of eradication on IL-4 secretion . Anti-H . pylori antibody of the IgG2 subclass was remarkably increased in H . pylori-infected subjects . These results together suggest that gastric T cells are already differentiated to produce a large amount of IFN-gamma by a mechanism unrelated to H . pylori infection . H . pylori infection appeared to activate T cells to secrete even more IFN-gamma, which may contribute to maintaining a perpetual inflammation in H . pylori-infected stomach. J Periodontol, 1999 Oct, 70(10), 1202 - 8 Arbitrarily primed-polymerase chain reaction for identification and epidemiologic subtyping of oral isolates of Fusobacterium nucleatum; Avila-Campos MJ et al.; BACKGROUND: Fusobacterium nucleatum is the most frequently isolated bacterium in periodontal disease and plays an important role in serious infections in other parts of the body . Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to construct primers for specific identification and subtyping of F . nucleatum . Subtypes may differ in virulence and, hence, are important as periodontal pathogens . Subtypes also may differ in antibiotic susceptibility; therefore, knowing the subtypes may influence choice of treatment . METHODS: We analyzed 70 DNA samples of F . nucleatum isolated from patients with periodontal disease (PD) (N = 32) or AIDS-related PD (N = 8) and from healthy carriers (N = 30) . From 90 AP-PCR primers screened, five amplification products were selected, cloned in pCR II vector, and sequenced . These sequences were used to design new pairs of specific primers . Sequences were compared to GenBank entries with BLAST and showed no significant matches . RESULTS: Three primer pairs produced bands of approximately 1 Kb (primer 5059S) or 0.5 Kb (primers FN5047 or M1211) with all F . nucleatum DNAs tested . PCR amplification using primer pair M8171 produced a 1 Kb band with isolates from 7 (22%) PD and 5 (63%) PD-AIDS patients and 9 (30%) healthy controls . Using the same primer pair, 2 other bands of approximately 0.5 Kb and 0.4 Kb were observed with DNA from isolates from 2 (6%) PD and all PD-AIDS patients, but were not observed with DNA samples from healthy controls (P<0.0001) . All the primer pairs produced no or different amplicon profiles with DNA samples from bacterial species other than F . nucleatum . CONCLUSIONS: Our results suggest that PCR primer pairs 5059S, FN5047 or M1211 can be used to specifically identify F . nucleatum isolates and distinguish them from other bacteria . The primer pair M8171 could also be used to differentiate F . nucleatum isolated from periodontal patients or healthy individuals . These specific primers can be used in PCR analysis for specific identification of F . nucleatum and to distinguish it from other bacteria associated with human periodontitis . These approaches appear promising in facilitating laboratory identification, molecular subtyping, and taxonomy of putative periodontopathogens. Mol Biol Rep, 1999 Aug, 26(3), 195 - 9 An unusual arrangement of pur and lpx genes in the photosynthetic purple sulfur bacterium Allochromatium vinosum; Chen YL et al.; The nucleotide sequence of a 1634 bp DNA fragment from the photosynthetic purple sulfur bacterium Allochromatium vinosum contains one complete and two partial open reading frames . Sequence comparisons to genes from other organisms suggest that this A . vinosum DNA fragment contains, starting from the 5' end, the following: (1) 234 bp at the 3' end of the A . vinosum purH gene, coding for 78 amino acids at the C-terminus of the bi-functional 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide formyltransferase/IMP cyclohydrolase (EC 2.1.2.3), an enzyme involved in de novo purine biosynthesis; (2) 777 bp of the A . vinosum lpxA gene, coding for all 259 amino acids of the UDP-N-acetylglucosamine-O-acyltransferase, an enzyme involved in lipid A biosynthesis; and (3) 567 bp at the 5' end of the A . vinosum purD gene, coding for 189 amino acids at the N-terminus of 5'-phosphoribosyl glycinamide synthetase (EC 6.3.4.13), a second enzyme involved in de novo purine biosynthesis . The presence of a gene coding for an enzyme involved in lipid A biosynthesis between two genes coding for enzymes of the de novo purine biosynthesis pathway represents a unique arrangement of these genes. Infect Immun, 1999 Nov, 67(11), 6002 - 7 Importance of B cells, but not specific antibodies, in primary and secondary protective immunity to the intracellular bacterium Francisella tularensis live vaccine strain; Elkins KL et al.; Although there appears to be little if any role for specific antibodies in protection against intracellular bacteria, such as the model pathogen F . tularensis live vaccine strain (LVS), the role of B cells themselves in primary and secondary infection with such bacteria has not been examined directly . We show here that mice deficient in mature B cells and antibodies (B-cell knockout mice) are marginally compromised in controlling primary sublethal infection but are 100-fold less well protected against secondary lethal challenge than are their normal counterparts . This defect in optimal specific protective immunity was readily reconstituted by the transfer of primed, and to a lesser degree, unprimed B cells, but not by the transfer of specific antibodies . The results indicate a previously unappreciated role for B cells in secondary immunity to intracellular pathogens through a function other than antibody production. Infect Immun, 1999 Nov, 67(11), 5972 - 8 Bordetella bronchiseptica-mediated cytotoxicity to macrophages is dependent on bvg-regulated factors, including pertactin; Forde CB et al.; The effect of Bordetella bronchiseptica infection on the viability of murine macrophage-like cells and on primary porcine alveolar macrophages was investigated . The bacterium was shown to be cytotoxic for both cell types, particularly where tight cell-to-cell contacts were established . In addition, bvg mutants were poorly cytotoxic for the eukaryotic cells, while a prn mutant was significantly less toxic than wild-type bacteria . B . bronchiseptica-mediated cytotoxicity was inhibited in the presence of cytochalasin D or cycloheximide, an inhibitor of microfilament-dependent phagocytosis or de novo eukaryotic protein synthesis, respectively . The mechanism of eukaryotic cell death was examined, and cell death was found to occur primarily through a necrotic pathway, although a small proportion of the population underwent apoptosis. Clin Infect Dis, 1999 Sep, 29(3), 617 - 20 Pigeon pneumonia in provence: a bird-borne Q fever outbreak; Stein A et al.; Q fever is a widespread zoonosis caused by Coxiella burnetii, an obligate intracellular bacterium, which humans usually acquire through the inhalation of infected dust from subclinically infected mammals . Human infection commonly takes place when an infected mammal gives birth, since high concentrations of the organism are found in the products of conception . Worldwide, cattle, sheep, and goats are the most common reservoirs for C . burnetii . A few investigators have also reported parturient cats and dogs as the sources of human outbreaks of Q fever . During a 10-day period in May 1996, all five members of one family living on a farm in Provence, in the south of France, became ill with fever, general malaise, and cough . All of them had acute Q fever . An epidemiological investigation suggested that this outbreak resulted from exposure to contaminated pigeon feces and ticks. Biochemistry, 1999 Oct 5, 38(40), 13216 - 22 Dynamics of energy conversion in reaction center core complexes of the green sulfur bacterium Prosthecochloris aestuarii at low temperature; Neerken S et al.; Excited-state and electron-transfer dynamics at cryogenic temperature in reaction center core (RCC) complexes of the photosynthetic green sulfur bacterium Prosthecochloris aestuarii were studied by means of time-resolved absorption spectroscopy, using selective excitaton of bacteriochlorophyll (BChl) a and of chlorophyll (Chl) a 670 . The results indicate that the BChls a of the RCC complex form an excitonically coupled system . Relaxation of the excitation energy within the ensemble of BChl a molecules occurred within 2 ps . A time constant of about 25 ps was ascribed to charge separation . Absorption changes in the 670 nm region, where Chl a 670 absorbs, were fairly complicated . They showed various time constants and were dependent on the wavelength of excitation and they did not lead to a simple picture of the electron acceptor reaction . Energy transfer from Chl a 670 to BChl a occurred with a time constant of 1.5 ps . However, upon excitation of Chl a 670 the amount of oxidized primary electron donor, P840(+), formed relative to that of excited BChl a was considerably larger than upon direct excitation of BChl a . This indicates the existence of an alternative pathway for charge separation which does not involve excited BChl a. Clin Ter, 1999 May-Jun, 150(3), 221 - 4 {Helicobacter pylori and gastric cancer}; Tomaselli G et al.; Several unresolved issues still cast doubts on the epidemiological data which point to an association between infection from Helicobacter pylori (Hp) and gastric cancer . These are: a) the male/female ratio of gastric cancer ranges from 4 to 1.5 in all studies, whereas the prevalence of Hp infection is the same in both sexes; b) the prevalence of Hp infection is as high as 90% in several developing countries where the frequency of gastric cancer is very low; c) 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, on the average, 10/100,000; d) the incidence of cancer in patients with a duodenal ulcer is half of that of the general population but Hp infects up to 100% of these patients . In the sequence of events that lead 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 which lead to 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 ones expressing the cagA gene . In a study we conducted in an area with a low incidence of gastric cancer (Latina), the prevalence of Hp infection was equal to 78.6% and, among the positives, 81% resulted cagA positive . This data, if compared with a similar research that took place in another area with a high incidence of gastric cancer (San Marino) where the prevalence of Hp infection was 51% and cagA 69%, further demonstrates the inconsistency of associating Hp and cancer of the stomach. FEBS Lett, 1999 Oct 15, 459(3), 395 - 8 PcpA, which is involved in the degradation of pentachlorophenol in Sphingomonas chlorophenolica ATCC39723, is a novel type of ring-cleavage dioxygenase; Ohtsubo Y et al.; The pentachlorophenol (PCP) mineralizing bacterium Sphingomonas chlorophenolica ATCC39723 degrades PCP via 2,6-dichlorohydroquinone (2,6-DCHQ) . The pathway converting PCP to 2,6-DCHQ has been established previously; however, the pathway beyond 2,6-DCHQ is not clear, although it has been suggested that a PcpA plays a role in 2, 6-DCHQ conversion . In this study, PcpA expressed in Escherichia coli was purified to homogeneity and shown to have novel ring-cleavage dioxygenase activity in conjunction with hydroquinone derivatives, and converting 2,6-DCHQ to 2-chloromaleylacetate. EMBO J, 1999 Oct 15, 18(20), 5453 - 62 Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold; Eichinger A et al.; Gingipains are cysteine proteinases acting as key virulence factors of the bacterium Porphyromonas gingivalis, the major pathogen in periodontal disease . The 1.5 and 2.0 A crystal structures of free and D-Phe-Phe-Arg-chloromethylketone-inhibited gingipain R reveal a 435-residue, single-polypeptide chain organized into a catalytic and an immunoglobulin-like domain . The catalytic domain is subdivided into two subdomains comprising four- and six-stranded beta-sheets sandwiched by alpha-helices . Each subdomain bears topological similarities to the p20-p10 heterodimer of caspase-1 . The second subdomain harbours the Cys-His catalytic diad and a nearby Glu arranged around the S1 specificity pocket, which carries an Asp residue to enforce preference for Arg-P1 residues . This gingipain R structure is an excellent template for the rational design of drugs with a potential to cure and prevent periodontitis . Here we show the binding mode of an arginine-containing inhibitor in the active-site, thus identifying major interaction sites defining a suitable pharmacophor. Periodontol 2000, 1999 Jun, 20, 65 - 81 Oral ecology and person-to-person transmission of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis; Asikainen S et al.; The ecological characteristics of the oral cavity are dissimilar for A . actinomycetemcomitans and for P . gingivalis, as judged by differences in their colonization preferences and patterns, associations with periodontal disease parameters, relationships with the subgingival microbiota and the type of periodontitis and their clonal persistence in the oral cavity . These features also suggest that as a periodontal pathogen, A . actinomycetemcomitans is different from P . gingivalis . Probably in most infected individuals, low levels of A . actinomycetemcomitans can persist for years in equilibrium with the host and the resident oral microbiota . However, it is well established that A . actinomycetemcomitans can cause disease in some individuals or in some circumstances when the regulatory mechanisms are unable to maintain homeostasis in the ecosystem . Elevated A . actinomycetemcomitans proportions of the biota can be regarded as a sign of ecological imbalance, leading to increased risk of periodontal destruction . There is also evidence showing elevated pathogenic potential of certain A . actinomycetemcomitans clones . Although A . actinomycetemcomitans seems to be relatively rarely transmitted between cohabiting adults, transmission can occur to periodontally healthy children of A . actinomycetemcomitans-positive parents . Parents and children may share factors that promote successful oral colonization of A . actinomycetemcomitans, or the window of opportunity is in childhood . Therefore, to prevent parent-child transmission of A . actinomycetemcomitans, bacterium-positive parents of young children are optimal targets for enhanced information and treatment . In selected populations, screening for specific clones of A . actinomycetemcomitans has been employed in prevention of peridontitis . Future research aiming at finding the reasons which cause the changes in the oral homeostasis to allow the growth of A . actinomycetemcomitans may give insight into novel prevention strategies for A . actinomycetemcomitans-associated periodontitis . Compared with A . actinomycetemcomitans, P . gingivalis shows a different pattern of coexistence with the host . In periodontal health or in children, P . gingivalis is absent or only rarely detected . When present, P . gingivalis is commonly recovered in high numbers from dentitions exhibiting inflamed periodontitis and poor oral hygiene . Contrary to A . actinomycetemcomitans, the data on the vertical transmission of P . gingivalis are limited . The major infection route of P . gingivalis seems to be between adults, indicating that P . gingivalis commonly colonizes in an established oral microbiota . These characteristics suggest that the degree of tolerance between P . gingivalis and the host is inferior to that between A . actinomycetemcomitans and the host . It appears that the association of P . gingivalis with disease is a rule rather than an accidental incident . On these grounds, it seems that the host-P . gingivalis relationship approaches antibiosis . Since P . gingivalis infection is related to a typical periodontal eco-pathology, the susceptibility to person-to-person transmission of this pathogen may be controlled by periodontal treatment and emphasizing the significance of high standard oral hygiene. J Biol Chem, 1999 Oct 22, 274(43), 30672 - 8 Crystal structures of intermediates in the dehalogenation of haloalkanoates by L-2-haloacid dehalogenase; Ridder IS et al.; The L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoates to their corresponding D-2-hydroxyalkanoates, with inversion of the configuration at the C(2) atom . The structure of the apoenzyme at pH 8 was refined at 1.5-A resolution . By lowering the pH, the catalytic activity of the enzyme was considerably reduced, allowing the crystal structure determination of the complexes with L-2-monochloropropionate and monochloroacetate at 1.7 and 2.1 A resolution, respectively . Both complexes showed unambiguous electron density extending from the nucleophile Asp(8) to the C(2) atom of the dechlorinated substrates corresponding to a covalent enzyme-ester reaction intermediate . The halide ion that is cleaved off is found in line with the Asp(8) Odelta1-C(2) bond in a halide-stabilizing cradle made up of Arg(39), Asn(115), and Phe(175) . In both complexes, the Asp(8) Odelta2 carbonyl oxygen atom interacts with Thr(12), Ser(171), and Asn(173), which possibly constitute the oxyanion hole in the hydrolysis of the ester bond . The carboxyl moiety of the substrate is held in position by interactions with Ser(114), Lys(147), and main chain NH groups . The L-2-monochloropropionate CH(3) group is located in a small pocket formed by side chain atoms of Lys(147), Asn(173), Phe(175), and Asp(176) . The size and position of the pocket explain the stereospecificity and the limited substrate specificity of the enzyme . These crystallographic results demonstrate that the reaction of the enzyme proceeds via the formation of a covalent enzyme-ester intermediate at the nucleophile Asp(8). FEMS Microbiol Lett, 1999 Oct 15, 179(2), 473 - 7 Effects of antibiotics on the early infection process of a macronuclear endosymbiotic bacterium Holospora obtusa of Paramecium caudatum; Dohra H et al.; We examined the effects of antibiotics involved in bacterial DNA, RNA and protein synthesis and host protein synthesis on the early infection process of the bacterium Holospora obtusa, a macronucleus-specific symbiont of the ciliate Paramecium caudatum . Infection of the host macronucleus by the bacterium was not inhibited by mitomycin C, rifampicin and chloramphenicol . However, ingestion of the bacterium into the host digestive vacuoles and escape of the bacterium from the vacuoles to the host cytoplasm were significantly arrested with emetine . The results suggest that newly synthesized host proteins play an important role in the early infection process. J Infect Dis, 1999 Nov, 180(5), 1713 - 7 Effect of nitric oxide on Helicobacter pylori morphology; Cole SP et al.; Helicobacter pylori causes chronic gastritis punctuated with fluctuating episodes of acute distress that can lead to peptic ulcer disease . Several factors produced by the bacterium have been shown to initiate the inflammatory response, but mechanisms potentially involved in the down-regulation of inflammation have not been described . We show that nitric oxide (NO) released from synthetic NO generators causes a rapid and dose-dependent morphologic conversion of H . pylori from the replicating spiral form to the nonreplicating, but viable, coccoid form . Because only spiral organisms-and not coccoid forms-are capable of inducing interleukin-8 secretion by epithelial cells, this conversion could result in down-regulation of the inflammatory response . These data suggest that the increase in NO synthase activity observed during gastritis results in morphologic conversion to a potentially dormant but viable H . pylori. J Bacteriol, 1999 Oct, 181(20), 6396 - 402 A novel role for Escherichia coli endonuclease VIII in prevention of spontaneous G-->T transversions; Blaisdell JO et al.; In the bacterium Escherichia coli, oxidized pyrimidines are removed by two DNA glycosylases, endonuclease III and endonuclease VIII (endo VIII), encoded by the nth and nei genes, respectively . Double mutants lacking both of these activities exhibit a high spontaneous mutation frequency, and here we show that all of the mutations observed in the double mutants were G:C-->A:T transitions; no thymine mutations were found . These findings are in agreement with the preponderance of C-->T transitions in the oxidative and spontaneous mutational databases . The major oxidized purine lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is processed by two DNA glycosylases, formamidopyrimidine DNA glycosylase (Fpg), which removes 8-oxoG opposite C, and MutY DNA glycosylase, which removes misincorporated A opposite 8-oxoG . The high spontaneous mutation frequency previously observed in fpg mutY double mutants was significantly enhanced by the addition of the nei mutation, suggesting an overlap in the substrate specificities between endo VIII and Fpg/MutY . When the mutational specificity was examined, all of the mutations observed were G:C-->T:A transversions, indicating that in the absence of Fpg and MutY, endo VIII serves as a backup activity to remove 8-oxoG . This was confirmed by showing that, indeed, endo VIII can recognize 8-oxoG in vitro. J Biol Chem, 1999 Oct 15, 274(42), 30094 - 100 Exonuclease X of Escherichia coli . A novel 3'-5' DNase and Dnaq superfamily member involved in DNA repair; Viswanathan M et al.; DNA exonucleases are critical for DNA replication, repair, and recombination . In the bacterium Escherichia coli there are 14 DNA exonucleases including exonucleases I-IX (including the two DNA polymerase I exonucleases), RecJ exonuclease, SbcCD exonuclease, RNase T, and the exonuclease domains of DNA polymerase II and III . Here we report the discovery and characterization of a new E . coli exonuclease, exonuclease X . Exonuclease X is a member of a superfamily of proteins that have homology to the 3'-5' exonuclease proofreading subunit (DnaQ) of E . coli DNA polymerase III . We have engineered and purified a (His)(6)-exonuclease X fusion protein and characterized its activity . Exonuclease X is a potent distributive exonuclease, capable of degrading both single-stranded and duplex DNA with 3'-5' polarity . Its high affinity for single-strand DNA and its rapid catalytic rate are similar to the processive exonucleases RecJ and exonuclease I . Deletion of the exoX gene exacerbated the UV sensitivity of a strain lacking RecJ, exonuclease I, and exonuclease VII . When overexpressed, exonuclease X is capable of substituting for exonuclease I in UV repair . As we have proposed for the other single-strand DNA exonucleases, exonuclease X may facilitate recombinational repair by pre-synaptic and/or post-synaptic DNA degradation. J Mol Biol, 1999 Oct 15, 293(1), 117 - 24 Calorimetric analysis of the Ca(2+)-binding betagamma-crystallin homolog protein S from Myxococcus xanthus: intrinsic stability and mutual stabilization of domains; Wenk M et al.; The betagamma-crystallin superfamily consists of a class of homologous two-domain proteins with Greek-key fold . Protein S, a Ca(2+)-binding spore-coat protein from the soil bacterium Myxococcus xanthus exhibits a high degree of sequential and structural homology with gammaB-crystallin from the vertebrate eye lens . In contrast to gammaB-crystallin, which undergoes irreversible aggregation upon thermal unfolding, protein S folds reversibly and may therefore serve as a model in the investigation of the thermodynamic stability of the eye-lens crystallins . The thermal denaturation of recombinant protein S (PS) and its isolated domains was studied by differential scanning calorimetry in the absence and in the presence of Ca(2+) at varying pH . Ca(2+)-binding leads to a stabilization of PS and its domains and increases the cooperativity of their equilibrium unfolding transitions . The isolated N-terminal and C-terminal domains (NPS and CPS) obey the two-state model, independent of the pH and Ca(2+)-binding; in the case of PS, under all conditions, an equilibrium intermediate is populated . The first transition of PS may be assigned to the denaturation of the C-terminal domain and the loss of domain interactions, whereas the second one coincides with the denaturation of the isolated N-terminal domain . At pH 7.0, in the presence of Ca(2+), where PS exhibits maximal stability, the domain interactions at 20 degrees C contribute 20 kJ/mol to the overall stability of the intact protein . Emerg Infect Dis, 1999 Sep-Oct, 5(5), 694 - 700 Diphtheria antitoxin levels in the Netherlands: a population-based study; de Melker HE et al.; In a population-based study in the Netherlands, diphtheria antitoxin antibodies were measured with a toxin-binding inhibition assay in 9, 134 sera from the general population and religious communities refusing vaccination . The Dutch immunization program appears to induce long-term protection against diphtheria . However, a substantial number of adults born before the program was introduced had no protective diphtheria antibody levels . Although herd immunity seems adequate, long-term population protection cannot be assured . As more than 60% of orthodox reformed persons have antibody levels lower than 0.01 IU/ml, introduction of diphtheria into religious communities refusing vaccination may constitute a danger of spread of the bacterium. Microbios, 1999, 99(393), 95 - 104 Adherence of Fusobacterium necrophorum subspecies necrophorum to different animal cells; Okada Y et al.; The adherence of Fusobacterium necrophorum subsp . necrophorum to the surfaces of animal cells was studied in order to elucidate the differences between the bacterial appearance in clinical specimens from various animals . The bacterial cells had a strong affinity for murine and rabbit cheek cell surfaces . The bacterium showed a moderate affinity for goat cells, whereas it adhered not so well to canine, feline, human or porcine cells . Treatment of the bacterial cells with haemagglutinin antiserum prior to the binding assay reduced the degree of attachment to murine and rabbit cells . Scanning electron microscopy revealed that the adherent fusobacteria often penetrated into murine and rabbit cell membranes . These observations indicate that the bacterial attachment contributes to the establishment of the infection in mice and rabbits . It is suggested that the weak binding ability resulted in a low incidence of the bacterium in canine, feline and porcine lesions. J Ind Microbiol Biotechnol, 1999 Aug, 23(2), 123 - 126 Expression of multiple complex polysaccharide-degrading enzyme systems by marine bacterium strain 2-40; Ensor L et al.; Saprophytic marine bacterium strain 2-40 (2-40) can degrade numerous complex polysaccharides (CP) including agar, alginic acid, carrageenan, carboxymethylcellulose, chitin, beta-glucan, laminarin, pectin, pullulan, starch, and xylan . The growth of 2-40 was assessed in minimal media containing one of 16 CP or simple carbohydrates, with the result that all supported growth . Each of the carbohydrase systems was elicited at highest levels by the homologous substrate . Each, excluding amylase, was repressed when 2-40 was cultured in glucose minimal synthetic media . Cyclic adenosine monophosphate alleviated the repression . Agarose as sole carbon source supported the synthesis of the most heterologous complex carbohydrase systems, although, generally, at a lower level of activity than the homologous CP. Mol Cell Probes, 1999 Oct, 13(5), 373 - 9 Detection and identification of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of the beta operon; Hocquellet A et al.; Huanglongbing, previously called greening, is a destructive disease of citrus in Asia and Africa . It is caused by an uncultured, phloem-restricted bacterium for which two species Candidatus Liberobacter asiaticum and Candidatus Liberobacter africanum have been characterized . In 1996, a polymerase chain reaction (PCR) detection method based on the amplification of the 16S ribosomal operon was developed and proved to be efficient for the detection of both liberobacter species . However, in order to distinguish between Candidatus Liberobacter asiaticum and Candidatus Liberobacter africanum, digestion of the amplified DNA with XbaI was required . We have now developed a new PCR detection method based on the amplification of ribosomal protein genes which allows direct identification of the liberobacter species by the size of the amplified DNA . This PCR method is as specific, and at least as sensitive as the previously described one for detection of the two liberobacter species Biochemistry, 1999 Sep 14, 38(37), 12124 - 37 Electron transfer kinetics in purified reaction centers from the green sulfur bacterium Chlorobium tepidum studied by multiple-flash excitation; Kusumoto N et al.; Reaction center preparations from the green sulfur bacterium Chlorobium tepidum, which contain monoheme cytochrome c, were studied by flash-absorption spectroscopy in the near-UV, visible, and near-infrared regions . The decay kinetics of the photooxidized primary donor P840(+), together with the amount of photooxidized cytochrome c, were analyzed along a series of four flashes spaced by 1 ms: 95% of the P840(+) was reduced by cytochrome c with a t(1/2) of approximately 65 micros after the first flash, 80% with a t(1/2) of approximately 100 micros after the second flash, and 23% with a t(1/2) of approximately 100 micros after the third flash; after the fourth flash, almost no cytochrome c oxidation occurred . The observed rates, the establishment of redox equilibrium after each flash, and the total amount of photooxidizable cytochrome c are consistent with the presence of two equivalent cytochrome c molecules per photooxidizable P840 . The data are well fitted assuming a standard free energy change DeltaG degrees of -53 meV for electron transfer from one cytochrome c to P840(+), DeltaG degrees being independent of the oxidation state of the other cytochrome c . These observations support a model with two monoheme cytochromes c which are symmetrically arranged around the reaction center core . From the ratio of menaquinone-7 to the bacteriochlorophyll pigment absorbing at 663 nm, it was estimated that our preparations contain 0.6-1.2 menaquinone-7 molecules per reaction center . However, no transient signal due to menaquinone could be observed between 360 and 450 nm in the time window from 10 ns to 4 micros . No recombination reaction between the primary partners P840(+) and A(0)(-) could be detected under normal conditions . Such a recombination was observed (t(1/2) approximately 19 ns) under highly reducing conditions or after accumulation of three electrons on the acceptor side during a series of flashes, showing that the secondary acceptors can stabilize three electrons . From our data, there is no evidence for involvement of menaquinone in charge separation in the reaction center of green sulfur bacteria. Appl Environ Microbiol, 1999 Oct, 65(10), 4701 - 4 A phase variant of Azospirillum lipoferum lacks a polar flagellum and constitutively expresses mechanosensing lateral flagella; Alexandre G et al.; Flagellation of a nonswimming variant of the mixed flagellated bacterium Azospirillum lipoferum 4B was characterized by electron microscopy, and polyclonal antibodies were raised against polar and lateral flagellins . The variant cells lacked a polar flagellum due to a defect in flagellin synthesis and constitutively expressed lateral flagella . The variant cells were able to respond to conditions that restricted the rotation of lateral flagella by producing more lateral flagella, suggesting that the lateral flagella, as well as the polar flagellum, are mechanosensing. Orv Hetil, 1999 Sep 5, 140(36), 1985 - 9 {Detection of Helicobacter pylori in tissue samples of stomach cancer}; Palatka K et al.; The role of Helicobacter pylori in the carcinogenesis of the stomach has been recognised both in intestinal and diffuse forms . The occurrence of the bacterium was studied in this report, with various methods in biopsy samples from the cancerous stomach, as well as the presence of associated gastritis and metaplasia related to the histological type . Retrospective histological examination were performed on endoscopic biopsy samples from 124 patients with distal stomach cancer using haematoxillin-eosin and Giemsa staining and immunohistochemical tests . Out of the 124 samples 69 (55.64%) was positive: 48 with Giemsa staining and further 21 samples showed immunohistochemical positivity on atrophic gastritis samples despite negative Giemsa staining . In view of the presence of gastritis and metaplasia significant difference (p < 0.001) was found between the positive and negative cases . The ratio of the Helicobacter pylori positive samples was high both for intestinal and diffuse type carcinomas . Our results suggest that the presence of Helicobacter pylori infection is important in the development of both types of carcinoma, nevertheless, the hystological type of the tumor is also decisively influenced by the onset of action of other more direct local eliciting factors. Eur J Gastroenterol Hepatol, 1999 Aug, 11 Suppl 2, S69 - 71; discussion S73 Should we go further and screen and treat? Forman D. Helicobacter pylori is probably the most common human bacterial infection in the world, and, in a minority of infected individuals, it can cause life-threatening disease . As testing procedures and treatment regimens become cheaper and more reliable, is the screening of asymptomatic populations to identify and treat infected individuals justified? Evidence on which to base an answer to this question is sparse, but there are several reasons to believe that a screen-and-treat strategy for H . pylori infection might constitute a viable public health intervention . Screening can be sensitive, H . pylori eradication regimens are increasingly effective, and both screening for and eradicating the bacterium are relatively inexpensive . There are, however, a number of concerns that can be addressed only through further research . Decisive evidence concerning all the risks and benefits of a screen-and-treat strategy will be derived only from large, long-term, randomized, controlled trials. J Biochem (Tokyo), 1999 Oct, 126(4), 731 - 7 Identification of the gene encoding esterase, a homolog of hormone-sensitive lipase, from an oil-degrading bacterium, strain HD-1; Mizuguchi S et al.; The gene encoding an esterase (HDE) was cloned from an oil-degrading bacterium, strain HD-1 . HDE is a member of the hormone-sensitive lipase family and composed of 317 amino acid residues with a molecular weight of 33,633 . The HDE-encoding gene was expressed in Escherichia coli, and the recombinant protein was purified and characterized . Amino acid sequence analysis indicated that the methionine residue was removed from its NH(2)-terminus . The good agreement of the molecular weights estimated by SDS-PAGE (35,000) and gel filtration (38,000) suggests that it acts in a monomeric form . HDE showed hydrolytic activity towards p-nitrophenyl esters of fatty acids with an acyl chain length of 2 to 14 and tributyrin, whereas it showed little hydrolytic activity towards p-nitrophenyl oleate (C(18)), tricaprylin and triolein . Determination of the kinetic parameters for the hydrolyses of the p-nitrophenyl substrates from C(2) to C(14) indicated that HDE shows a relatively broad substrate specificity . However, comparison of the k(cat)/K(m) values indicated that the C(10)-C(14) substrates are the most preferred ones . Such a preference for substrates with long acyl chains may be a characteristic of HDE. Oral Microbiol Immunol, 1999 Jun, 14(3), 194 - 6 Serum antibody response to oral infection precedes but does not prevent Porphyromonas gingivalis-induced alveolar bone loss in mice; Baker PJ et al.; The purpose of this study was to determine whether humoral immunity prevents bacterially induced alveolar bone loss . BALB/cByJ mice were orally infected with the human periodontopathic bacterium Porphyromonas gingivalis, and were compared with sham-infected mice . Specific serum antibody titers to P . gingivalis were measured by enzyme-linked immunosorbent assay . Alveolar bone levels were measured as the distance from the cementoenamel junction to the alveolar bone crest and bone loss was defined as a change in bone levels over time or between infected and sham-infected animals . The specific humoral response was predominantly of the IgG isotype, although low levels of specific serum IgA were also present . Antibody titers in the infected animals were significantly different from those in the sham-infected animals by 18 days and remained at maximal levels at 47 days . Bone loss became significant by 26 days and continued to progress at 47 days . Thus the serum antibody response to oral infection with P . gingivalis preceded detectable bone loss and remained elevated while bone loss increased . The presence of specific antibody did not prevent the onset or progression of bone loss. Electrophoresis, 1999 Aug, 20(11), 2196 - 203 Cross-species characterisation of abundantly expressed Ochrobactrum anthropi gene products; Wasinger VC et al.; The identity of 45 protein spots representing 32 orthologues within the Ochrobactrum anthropi proteome within a gradient of pH 4-7, and mass range 5-90 kDa were determined across species boundaries . These proteins could be classified into 13 functional categories and establish metabolic, regulatory and translatory systems including amino acid biosynthesis, electron transport and the potential for plant symbiosis in a molecularly understudied organism . Amino acid composition and/or peptide mass fingerprinting were employed as a means to search the Swiss-Prot and OWL protein sequence databases for similarity within a broad taxonomic class of bacteria . Candidate matches from database searches could be compared and a simple multiplication matrix based on co-occurrence and rank within the top 96 most similar entries was used to provide statistical confidence . This mathematical matrix was evaluated with respect to the characterisation of O . anthropi, an unsequenced and understudied bacterium, in the light of the recent influx of DNA sequence information. Avian Dis, 1999 Jul-Sep, 43(3), 553 - 63 Consequences to chicks hatched from Escherichia coli-inoculated embryos; Montgomery RD et al.; An Escherichia coli causing negligible mortality in embryonated chicken eggs was adapted to grow in media containing nalidixic acid . This isolate (EcNAL) was inoculated into 12-day-old embryonated eggs . Additional embryos inoculated with tryptose phosphate broth (TPB) served as controls . Six days later, all surviving eggs were moved to hatching units . One hatcher contained half of the TPB-inoculated eggs; the chicks hatching from these eggs served as negative controls . The EcNAL-inoculated eggs and the remaining TPB-inoculated eggs were moved to a second hatcher and allowed to hatch together; chicks hatching from these TPB-inoculated eggs served as contact controls . On day of hatch and at intervals thereafter, chicks from each of the treatment groups were sampled . Their body and yolk weights were recorded, and various tissues were cultured for the presence of the EcNAL bacterium . Hatchability of the EcNAL-inoculated embryos was markedly lower than that of either control group . Chicks from EcNAL-inoculated embryos also had low but detectable levels of mortality, lowered body weights, and increased yolk-to-body weight ratios . These same chicks had persistently high levels of EcNAL in the yolk and lower but detectable levels of the organism in the lungs and tracheas, which lasted a few days . The contact controls, on the other hand, were similar to the negative controls as far as having negligible mortality, steadily increasing body weights, and declining yolk-to-body weight ratios . However, in contrast to the negative controls, EcNAL was recovered primarily from the respiratory tract of the contact controls for a brief period of 3-4 days after hatch. Biochemistry, 1999 Sep 21, 38(38), 12205 - 11 Structural determination and biosynthetic studies of the O-antigenic polysaccharide from the enterohemorrhagic Escherichia coli O91 using 13C-enrichment and NMR spectroscopy; Kjellberg A et al.; The structure of the O-antigenic polysaccharide from the enterohemorrhagic Escherichia coli O91 has been determined using primarily NMR spectroscopy on the (13)C-enriched polysaccharide . The O-antigen is composed of pentasaccharide repeating units with the following structure: -->4)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->4)-beta-D-GlcpA-6-N- Gly -(1-->3)-beta-D-GlcpNAc-(1-->4)-alpha-D-Quip-3-N-{(R)-3-hydroxy butyra mido}-(1--> . The bacterium was grown with D-{UL-(13)C}glucose in the medium which resulted in an overall degree of labeling of approximately 65% in the sugar residues and approximately 50% in the N-acyl substituents, indicating some metabolic dilution in the latter . The (13)C-enrichment of the polysaccharide proved valuable since NMR assignments could be made on the basis of (13)C, (13)C-connectivity in uniformly labeled residues . The biosynthesis of the (R)-3-hydroxybutyramido substituent via C(2) fragments was identified by NMR spectroscopy . The (R)-configuration at C3 is in accord with fatty acid biosynthesis . Additional cultures with specifically labeled D-{1-(13)C}glucose or D-{6-(13)C}glucose corroborated the direct incorporation of glucose as the building block for the hexose skeletons in the polysaccharide and the biosynthesis of acyl substituents occurring via the triose pool followed by decarboxylation to give acetyl building blocks labeled with (13)C at the methyl group. Eur J Biochem, 1999 Oct 1, 265(1), 290 - 9 Characterization of the rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinant Escherichia coli; Bolt EL et al.; The hemA and hemT genes encoding 5-aminolaevulinic acid synthase (ALAS) from the photosynthetic bacterium Rhodobacter sphaeroides, were cloned to allow high expression in Escherichia coli . Both HemA and HemT appeared to be active in vivo as plasmids carrying the respective genes complemented an E . coli hemA strain (glutamyl-tRNA reductase deficient) . The over-expressed isoenzymes were isolated and purified to homogeneity . Isolated HemA was soluble and catalytically active whereas HemT was largely insoluble and failed to show any activity ex vivo . Pure HemA was recovered in yields of 5-7 mg x L-1 of starting bacterial culture and pure HemT at 10 mg x L-1 x HemA has a final specific activity of 13 U x mg-1 with 1 unit defined as 1 micromol of 5-aminolaevulinic acid formed per hour at 37 degrees C . The Km values for HemA are 1.9 mM for glycine and 17 microM for succinyl-CoA, with the enzyme showing a turnover number of 430 h-1 . In common with other ALASs the recombinant R . sphaeroides HemA requires pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis . Removal of this cofactor resulted in inactive apo-ALAS . Similarly, reduction of the HemA-PLP complex using sodium borohydride led to > 90% inactivation of the enzyme . Ultraviolet-visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5'-phosphate that was not observed when HemT was incubated with the cofactor . HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates . Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross-react with mouse ALAS, indicating that all of the proteins have conserved epitopes. Nature, 1999 Sep 9, 401(6749), 188 - 93 Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors; Finnin MS et al.; Histone deacetylases (HDACs) mediate changes in nucleosome conformation and are important in the regulation of gene expression . HDACs are involved in cell-cycle progression and differentiation, and their deregulation is associated with several cancers . HDAC inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), have anti-tumour effects, as they can inhibit cell growth, induce terminal differentiation and prevent the formation of tumours in mice models, and they are effective in the treatment of promyelocytic leukemia . Here we describe the structure of the histone deacetylase catalytic core, as revealed by the crystal structure of a homologue from the hyperthermophilic bacterium Aquifex aeolicus, that shares 35.2% identity with human HDAC1 over 375 residues, deacetylates histones in vitro and is inhibited by TSA and SAHA . The deacetylase, deacetylase-TSA and deacetylase-SAHA structures reveal an active site consisting of a tubular pocket, a zinc-binding site and two Asp-His charge-relay systems, and establish the mechanism of HDAC inhibition . The residues that make up the active site and contact the inhibitors are conserved across the HDAC family . These structures also suggest a mechanism for the deacetylation reaction and provide a framework for the further development of HDAC inhibitors as antitumour agents. Orv Hetil, 1999 Apr 18, 140(16), 887 - 90 {Helocobacter heilmannii (Gastrospirillum hominis)-associated gastritis: first reported cases in Hungary}; Tiszlavicz L et al.; We found a tightly coiled bacterium, Helicobacter heilmannii that is distinct from Helicobacter pylori in the gastric mucosal biopsy of a 55-year-old man and a 30-year-old woman, both of whom had previous history of intermittent epigastric pain . The organisms were distributed in the antral mucosa, which was endoscopically interpreted as demonstrating chronic gastritis in one case, and being normal in the other . Histologically, the affected mucosa presented a mild chronic gastritis without activity and a moderate chronic gastritis with marker activity, respectively . The bacteria seen on haematoxylin eosin stained slides were strongly Giemsa positive, and were also visualized with Warthin-Starry stain . They were negative with the anti-Helicobacter pylori immunohistochemistry, although a few spots of staining could represent some cross reaction . Electron microscopy revealed fragments of bacterium, with its characteristic spiral shape and length of 4-7 mm in the plane of the section . One of the patient was treated medically and the control examination revealed normal antral mucosa without spiral bacteria . Detection of these organisms may provide a new insight into the pathogenesis of human gastritis. Biochem Biophys Res Commun, 1999 Sep 16, 263(1), 58 - 62 Overlapping sequences with high homology to functional proteins coexist on complementary strands of DNA in the rumen bacterium Prevotella albensis; Walker ND et al.; The potential for two complementary fragments of DNA from a clone from the ruminal bacterium Prevotella albensis to encode sequences with homology to at least part of functional proteins is described . One strand contains a sequence with high homology to dnaK, a member of the hsp70 family, and the other strand contains a sequence with some homology to glutamate dehydrogenase genes . Overlapping of these two genes on opposite strands has been reported in eukaryotic species, and is now reported for the first time in a bacterial species . Further investigation of previously described dnaK genes demonstrates that it is more widespread than might be anticipated, with all thirty other dnaK genes investigated also retaining long sequences encoding at least part of a sequence with high homology to a glutamate dehydrogenase gene . FEMS Microbiol Lett, 1999 Sep 1, 178(1), 123 - 8 Cloning of two cold shock genes, cspA and cspG, from the deep-sea psychrophilic bacterium Shewanella violacea strain DSS12; Fujii S et al.; We cloned and characterized two cold shock inducible genes from the deep-sea psychrophilic bacterium Shewanella violacea strain DSS12 . The cloned genes, designated cspA and cspG, encode proteins each consisting of 70 amino acid residues which show 62 and 67% sequence identity with Escherichia coli CspA and CspG, respectively . AT-rich UP elements were found immediately upstream of the promoter region and the cspA and cspG mRNA contained unusually long 5' untranslated regions like that in the E . coli cspA, cspB, cspG and cspI genes . Following a temperature downshift to 4 degrees C or -1 degree C, the levels of cspA and cspG mRNA increased and the level of expression of cspG was greater than that of cspA both before and after cold shock . These results suggest that CspA and CspG may function as RNA chaperones, the mRNAs encoded by these two genes may be regulated post-transcriptionally and they may function as regulators of other cold shock inducible genes like in E . coli. Bone Marrow Transplant, 1999 Sep, 24(5), 551 - 4 Mycobacterium tuberculosis infection in allogeneic bone marrow transplantation patients; Aljurf M et al.; Bone marrow transplant (BMT) recipients are prone to bacterial, viral and fungal infections . Mycobacterium tuberculosis infection can occur in these patients, but the incidence is lower than that of other infections . This report describes four patients with Mycobacterium tuberculosis infection identified from 641 adult patients who received a BMT over a 12-year period (prevalence 0.6%) . The pre-transplant diagnosis was AML in two patients and CML in the other two . Pre-transplant conditioning consisted of BU/CY in three patients and CY/TBI in one . Graft-versus-host disease (GVHD) prophylaxis was MTX/CsA in three patients and T cell depletion of the graft in one patient . Sites of infection were lung (two), spine (one) and central nervous system (one) . Onset of infection ranged from 120 days to 20 months post BMT . Two patients had co-existing CMV infection . One patient had graft failure . The two patients who received anti-tuberculous (TB) therapy recovered from the infection . Although the incidence of tuberculosis in BMT patients is not as high as in patients with solid organ transplants, late diagnosis due to the slow growth of the bacterium can lead to delay in instituting anti-TB therapy . A high index of suspicion should be maintained, particularly in endemic areas. J Bacteriol, 1999 Sep, 181(18), 5711 - 7 Analysis of fusion junctions of circularized chromosomes in Streptomyces griseus; Kameoka D et al.; A filamentous soil bacterium, Streptomyces griseus 2247, carries a 7 . 8-Mb linear chromosome . We previously showed by macrorestriction analysis that mutagenic treatments easily caused deletions at both ends of its linear chromosome and changed the chromosome to a circular form . In this study, we confirmed chromosomal circularization by cloning and sequencing the junction fragments from two deletion mutants, 404-23 and N2 . The junction sequences were compared with the corresponding right and left deletion end sequences in the parent strain, 2247 . No homology and a 6-bp microhomology were found between the two deletion ends of the 404-23 and N2 mutants, respectively, which indicate that the chromosomal circularization was caused by illegitimate recombination without concomitant amplification . The circularized chromosomes were stably maintained in both mutants . Therefore, the chromosomal circularization might have occurred to prevent lethal deletions, which otherwise would progress into the indispensable central regions of the chromosome. FEMS Microbiol Lett, 1999 Oct 1, 179(1), 91 - 99 Identification of cytochrome c oxidase in the alkaliphilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium 'Thioalcalomicrobium aerophilum' strain AL 3; Sorokin DY et al.; Cytochrome c oxidase from the novel alkaliphilic autotrophic sulfur bacterium 'Thioalcalomicrobium aerophilum' strain AL 3 was isolated and purified 87-fold . Spectroscopic analysis revealed the presence of both c- and b-type hemes as well as copper in a ratio of 3:2:1 . The purified enzyme consists of three subunits with apparent molecular masses of 41, 34 and 32 kDa . The two small subunits contain covalently bound heme c . With TMPD as a substrate the pH optimum was determined to be pH 8.0 . In the presence of monovalent cations the specific activity of the purified oxidase increased significantly . The enzyme was not able to oxidize external cytochrome c, but accepted electron from its native electron donor . The latter was separated from the other membrane cytochromes during anion-exchange chromatography and was identified as a high potential cytochrome c(551) . Overall the data indicate that the cytochrome c oxidase from this alkaliphilic autotrophic bacterium belongs to the heme-copper oxidase superfamily; regarding its subunit composition and content of prosthetic groups, the enzyme is similar in many aspects to the cbb(3)-type cytochrome c oxidases described for several neutrophilic bacteria, including anaerobic phototrophic and aerobic sulfur-oxidizing bacteria. Biol Pharm Bull, 1999 Aug, 22(8), 787 - 93 Influence of various bile acids on the metabolism of glycyrrhizin and glycyrrhetic acid by Ruminococcus sp . PO1-3 of human intestinal bacteria; Akao T; Ruminococcus sp . PO1-3, an intestinal bacterium isolated from human feces, metabolized glycyrrhizin (GL) to glycyrrhetic acid (GA) and GA to 3-oxo-glycyrrhetic acid (3-oxo-GA) and possessed GL beta-D-glucuronidase and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) involved in the metabolism of GL . This bacterial growth was enhanced by GL at a concentration of 0.4 mm and was suppressed by GA at concentration of 1.0 mM . Chenodeoxycholic acid, deoxycholic acid and lithocholic acid among the bile acids added to this bacterium suppressed the growth and GL beta-D-glucuronidase activity and 3beta-HSD activity incident to it at a concentration of 1.0 mM, while cholic acid, hyodeoxycholic acid and glycine and taurin conjugates of cholic acid, chenodeoxycholic acid, deoxycholic acid and lithocholic acid had almost no effect on this bacterium at a concentration of 0.2 to 1.0 mm . However, these enzyme activities of this sonicated bacteria were inhibited by all of these bile acids . Although each bile acid and GL added to bacteria at the same time suppressed the growth and the amount of metabolite GA by all bile acids used except cholic acid, taurocholic acid and taurodeoxycholic acid with GL, a combination of each bile acid and GA eased the growth inhibition caused by GA at a concentration of 0.2 mM and enhanced the amount of metabolite 3-oxo-GA by the glycine conjugate of bile acids with GA . GL or GA added after 6 h culture with each of these bile acids and bacteria was metabolized to a relatively large amount of GA by chenodeoxycholic acid and lithocholic acid and their glycine and taurine conjugates, glycocholic acid and taurodeoxycholic acid, or had almost no effect on the amount of metabolite 3-oxo-GA, respectively . These results showed that although GL added after the exposure to bile acid and GA and bile acid added at the same time as bacteria had different bile acid action, these conditions enhanced the amount of metabolite GA from GL and metabolite 3-oxo-GA from GA. J Wildl Dis, 1999 Jul, 35(3), 600 - 2 Plague in free-ranging mammals in western North Dakota; Dyer NW et al.; From July through October of 1996, 48 blood samples were collected from coyotes (Canis latrans), badgers (Taxidea taxus), and raccoons (Procyon lotor) in western North Dakota (USA) for the purposes of determining antibody titers to the plague bacterium, Yersniia pestis . The passive hemagglutination paper-strip blood-sampling technique was utilized with hemagglutination inhibition controls . Two positive samples were obtained from McKenzie county, one from a coyote with a titer of 1:64 and one from a badger with a titer of 1:256 . Considering coyote and badger population dynamics, this study documents plague in western North Dakota. Biospectroscopy, 1999, 5(4), 229 - 36 Pharmacologic application of FTIR spectroscopy: effect of ascorbic acid-induced free radicals on Deinococcus radiodurans; Melin AM et al.; Fourier transform infrared (FTIR) spectroscopy was used as a convenient and easy-to-run method to monitor radical-induced damage on the radiation-resistant Deinococcus radiodurans strain . Increasing concentrations of ascorbic acid added to the culture medium during the stationary phase produced striking changes in the infrared spectra . These changes especially occurred in the 1700-900 cm(-1) region, which is spectroscopically assigned to the amide I and II components, nucleotide bases, phosphodiester backbone and sugar rings, and were correlated with the oxidant effect of ascorbic acid . Thus, FTIR analysis allows a rapid characterization of the changes induced by ascorbic acid in the cell environment, which can be correlated in part with the generation of free radicals . Beyond a critical ascorbic acid concentration of 40 mM, these free radicals can cause severe damage to the biomolecular components, as soon as the antioxidant defenses of the bacterium are overwhelmed. Food Chem Toxicol, 1999 Jun, 37(6), 655 - 61 The effects of vitamin E on arylamine N-acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients; Chung JG; Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) and p-aminobenzoic acid (PABA) were determined in the bacterium Helicobacter pylori . Cytosols or suspensions of H . pylori, with or without specific concentrations of vitamin E co-treatment, showed different percentages of 2-AF acetylation . The data indicated that there was increased NAT activity associated with increased levels of vitamin E in H . pylori cytosols and intact bacteria . For the cytosol and intact bacteria examinations, the apparent values of Km and Vmax were increased when vitamin E was added to the reaction mixtures for 2-AF and PABA acetylation, respectively . This report is the first demonstration to show that antioxidant agents (vitamin E) can promote H . pylori N-acetyltransferase activity. Science, 1999 Sep 3, 285(5433), 1558 - 62 Whole-genome shotgun optical mapping of Deinococcus radiodurans; Lin J et al.; A whole-genome restriction map of Deinococcus radiodurans, a radiation-resistant bacterium able to survive up to 15,000 grays of ionizing radiation, was constructed without using DNA libraries, the polymerase chain reaction, or electrophoresis . Very large, randomly sheared, genomic DNA fragments were used to construct maps from individual DNA molecules that were assembled into two circular overlapping maps (2.6 and 0.415 megabases), without gaps . A third smaller chromosome (176 kilobases) was identified and characterized . Aberrant nonlinear DNA structures that may define chromosome structure and organization, as well as intermediates in DNA repair, were directly visualized by optical mapping techniques after gamma irradiation. Nature, 1999 Aug 26, 400(6747), 841 - 7 Placement of protein and RNA structures into a 5 A-resolution map of the 50S ribosomal subunit; Ban N et al.; We have calculated at 5.0 A resolution an electron-density map of the large 50S ribosomal subunit from the bacterium Haloarcula marismortui by using phases derived from four heavy-atom derivatives, intercrystal density averaging and density-modification procedures . More than 300 base pairs of A-form RNA duplex have been fitted into this map, as have regions of non-A-form duplex, single-stranded segments and tetraloops . The long rods of RNA crisscrossing the subunit arise from the stacking of short, separate double helices, not all of which are A-form, and in many places proteins crosslink two or more of these rods . The polypeptide exit channel was marked by tungsten cluster compounds bound in one heavy-atom-derivatized crystal . We have determined the structure of the translation-factor-binding centre by fitting the crystal structures of the ribosomal proteins L6, L11 and L14, the sarcin-ricin loop RNA, and the RNA sequence that binds L11 into the electron density . We can position either elongation factor G or elongation factor Tu complexed with an aminoacylated transfer RNA and GTP onto the factor-binding centre in a manner that is consistent with results from biochemical and electron microscopy studies. Appl Environ Microbiol, 1999 Sep, 65(9), 4268 - 70 Poly(aspartic acid) degradation by a Sphingomonas sp . isolated from freshwater; Tabata K et al.; A poly(aspartic acid) degrading bacterium (strain KT-1 {JCM10459}) was isolated from river water and identified as a member of the genus Sphingomonas . The isolate degraded only poly(aspartic acid)s of low molecular masses (<5 kDa), while the cell extract hydrolyzed high-molecular-mass poly(aspartic acid)s of 5 to 150 kDa to yield aspartic acid monomer. Appl Environ Microbiol, 1999 Sep, 65(9), 3929 - 35 Purification and characterization of the soluble methane monooxygenase of the type II methanotrophic bacterium Methylocystis sp . strain WI 14; Grosse S et al.; Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation . A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity . Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis . The sMMO is expressed only during growth under copper limitation (<0.1 microM) and with ammonium or nitrate ions as the nitrogen source . The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics . It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold . The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively . The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an alpha(2)beta(2)gamma(2) structure . We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry . sMMO is strongly inhibited by Hg(2+) ions (with a total loss of enzyme activity at 0.01 mM Hg(2+)) and Cu(2+), Zn(2+), and Ni(2+) ions (95, 80, and 40% loss of activity at 1 mM ions) . The complete sMMO gene sequence has been determined . sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp . strain M, and Methylococcus capsulatus (Bath). Appl Environ Microbiol, 1999 Sep, 65(9), 3843 - 9 Distribution and diversity of sulfur-oxidizing Thiomicrospira spp . at a shallow-water hydrothermal vent in the Aegean Sea (Milos, Greece); Brinkhoff T et al.; A shallow-water hydrothermal vent system in the Aegean Sea close to the island of Milos (Greece) was chosen to study the diversity and distribution of the chemolithoautotrophic sulfur-oxidizing bacterium Thiomicrospira . Cell numbers in samples from different regions around a solitary vent decreased toward the center of the vent (horizontal distribution), as well as with depth (vertical distribution), corresponding to an increase in temperature (from ca . 25 to 60 degrees C) and a decrease in pH (from ca . pH 7 to 5) . Thiomicrospira was one of the most abundant culturable sulfur oxidizers and was even dominant in one region . Phylogenetic analysis of Thiomicrospira spp . present in the highest most-probable-number (MPN) dilutions revealed that most of the obtained sequences grouped in two new closely related clusters within the Thiomicrospira branch . Two different new isolates, i.e., Milos-T1 and Milos-T2, were obtained from high-dilution (10(-5)) enrichments . Phylogenetic analysis indicated that isolate Milos-T1 is related to the recently described Thiomicrospira kuenenii and Hydrogenovibrio marinus, whereas isolate Milos-T2 grouped with the MPN sequences of cluster 2 . The predominance of strain Milos-T2 was indicated by its identification in several environmental samples by hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns and by sequencing of one of the corresponding bands, i.e., ML-1, from the DGGE gel . The results shown in this paper support earlier indications that Thiomicrospira species are important members of hydrothermal vent communities. J Biochem (Tokyo), 1999 Sep, 126(3), 578 - 83 Phosphocarrier proteins in an intracellular symbiotic bacterium of aphids; Matsumoto K et al.; A GroEL homolog produced by Buchnera, an intracellular symbiotic bacterium of aphids, is not only a molecular chaperone but also a novel phosphocarrier protein, suggesting that this protein plays a role in a signal transducing system specific to bacteria living in an intracellular environment . This prompted us to look into phosphocarrier proteins of Buchnera that may be shared in common with other bacteria . As a result, no evidence was obtained for the presence of sensor kinases of the two-component system in Buchnera, which are found in many bacteria . It is possible that the lack of sensor kinases is compensated for by the mulitifunctional GroEL homolog in this symbiotic bacteria . In contrast, we successfully identified three phosphotransferase system genes, ptsH, ptsI, and crr in Buchnera, and provide evidence for their active expression . While the deduced amino acid sequences of these gene products, histidine-containing phosphocarrier protein, Enzyme I, and Enzyme III were similar to their counterparts in Escherichia coli, the predicted isoelectric points of the Buchnera proteins were strikingly higher . It was also suggested that Buchnera Enzyme I, when produced in E . coli, is able to accept the phosphoryl group from phosphoenolpyruvate, but not from ATP. J Bacteriol, 1999 Sep, 181(17), 5505 - 8 First evidence for the presence of a hydrogenase in the sulfur-reducing bacterium Desulfuromonas acetoxidans; Brugna M et al.; Hydrogenases, which are ubiquitous in sulfate-reducing bacteria, were previously thought to be absent from Desulfuromonas acetoxidans . For the first time, a hydrogenase from the strict anaerobic sulfur-respiring bacterium D . acetoxidans, grown on ethanol-malate, was detected and enriched . To assay the role of the hydrogenase in the energetic metabolism of D . acetoxidans, we examined the reactivity of the enzyme with polyheme cytochromes from the same bacterium. J Bacteriol, 1999 Sep, 181(17), 5409 - 13 Two different types of dehalogenases, LinA and LinB, involved in gamma-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26 are localized in the periplasmic space without molecular processing; Nagata Y et al.; gamma-Hexachlorocyclohexane (gamma-HCH) is one of several highly chlorinated insecticides that cause serious environmental problems . The cellular proteins of a gamma-HCH-degrading bacterium, Sphingomonas paucimobilis UT26, were fractionated into periplasmic, cytosolic, and membrane fractions after osmotic shock . Most of two different types of dehalogenase, LinA (gamma-hexachlorocyclohexane dehydrochlorinase) and LinB (1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase), that are involved in the early steps of gamma-HCH degradation in UT26 was detected in the periplasmic fraction and had not undertaken molecular processing . Furthermore, immunoelectron microscopy clearly showed that LinA and LinB are periplasmic proteins . LinA and LinB both lack a typical signal sequence for export, so they may be secreted into the periplasmic space via a hitherto unknown mechanism. Arch Microbiol, 1999 Sep, 172(3), 150 - 6 Aerobic turnover of dimethyl sulfide by the anoxygenic phototrophic bacterium thiocapsa roseopersicina Jonkers HM, Jansen M, Van der Maarel MJ, Van Gemerden H. This is the first report describing the complete oxidation of dimethyl sulfide (DMS) to sulfate by an anoxygenic, phototrophic purple sulfur bacterium . Complete DMS oxidation was observed in cultures of Thiocapsa roseopersicina M11 incubated under oxic/light conditions, resulting in a yield of 30.1 mg protein mmol(-1) . No oxidation of DMS occurred under anoxic/light conditions . Chloroform, methyl butyl ether, and 3-amino-1,2,4-triazole, which are specific inhibitors of aerobic DMS oxidation in thiobacilli and hyphomicrobia, did not affect DMS oxidation in strain M11 . This could be due to limited transport of the inhibitors through the cell membrane . The growth yield on sulfide as sole electron donor was 22.2 mg protein mmol(-1) under anoxic/light conditions . Since aerobic respiration of sulfide would have resulted in yields lower than 22 mg protein mmol(-1), the higher yield on DMS under oxic/light conditions suggests that the methyl groups of DMS have served as an additional carbon source or as an electron donor in addition to the sulfide moiety . The kinetic parameters V(max) and K(m) for DMS oxidation under oxic/light conditions were 12.4 +/- 1.3 nmol (mg protein)(-1) min(-1) and 2 &mgr;M, respectively . T . roseopersicina M11 also produced DMS by cleavage of dimethylsulfoniopropionate (DMSP) . Specific DMSP cleavage rates increased with increasing initial substrate concentrations, suggesting that DMSP lyase was only partly induced at lower initial DMSP concentrations . A comparison of T . roseopersicina strains revealed that only strain M11 was able to oxidize DMS and cleave DMSP . Both strain M11 and strain 5811 accumulated DMSP intracellularly during growth, while strain 1711 showed neither of these characteristics . Phylogenetic comparison based on 16S rRNA gene sequence revealed a similarity of 99.0% between strain M11 and strain 5811, and 97.6% between strain M11 and strain 1711 . DMS and DMSP utilization thus appear to be strain-specific. Microb Pathog, 1999 Aug, 27(2), 81 - 91 Outer membrane vesicles of Porphyromonas gingivalis inhibit IFN-gamma-mediated MHC class II expression by human vascular endothelial cells; Srisatjaluk R et al.; Porphyromonas gingivalis is thought to be one of the major pathogenic organisms of adult periodontitis . Of the several virulence factors associated with the pathology it causes, evidence is now presented suggesting that outer membrane vesicles, which form from blebbing of the outer membrane, may also contribute to the pathogenesis of this bacterium . To evaluate this possibility, outer membrane vesicles were isolated from cultures of P . gingivalis and tested for their ability to promote inflammation and for their effects on the biosynthesis of E-selectin and ICAM-1 adhesion molecules and MHC class II glycoproteins . The results indicate that these vesicles are capable of inducing acute inflammation characterized by the accumulation of a large number of neutrophils in the connective tissue . This cellular response corresponds to the vesicle-mediated biosynthesis and surface membrane expression of E-selectin and ICAM-1 by vascular endothelial cells . In contrast, IFN-gamma-dependent synthesis of MHC class II molecules was found to be inhibited by vesicles . Inhibition of HLA-DR expression occurred regardless of whether vesicles were added at the same time as, 24 h before, or 24 h after IFN-gamma stimulation of endothelial cells, suggesting that the inhibitory effects occur at both the membrane and intracellular level . These findings, taken together, indicate that P . gingivalis membrane vesicles are capable of inducing and regulating cellular responses involved in inflammation and initiation of acquired immunity . Membrane vesicles are composed of muramyl peptides, periplasmic proteins and outer membrane constituents . The combination of these components probably contribute to the immune regulatory functions reported herein. FEBS Lett, 1999 Aug 6, 456(2), 239 - 42 Exciton levels structure of antenna bacteriochlorophyll c aggregates in the green bacterium Chloroflexus aurantiacus as probed by 1.8-293 K fluorescence spectroscopy; Mauring K et al.; We have demonstrated temperature-dependence of the steady-state fluorescence lineshape of the bacteriochlorophyll (BChl) c band measured for intact cells of the green bacterium Chloroflexus aurantiacus over the 1.8-293 K range . The measured temperature-dependence has been shown to be in good agreement with the theoretical one, calculated for our original model of pigment organization in the chlorosomal oligomeric antenna of green photosynthetic bacteria based on spectral hole-burning studies (Fetisova, Z.G . et al . (1996) Biophys . J . 71, 995-1010) . This model implies that the BChl c antenna unit is a tubular aggregate of six exciton-coupled linear pigment chains having the exciton level structure with strongly allowed higher levels. Biochem J, 1999 Sep 1, 342 ( Pt 2), 439 - 48 Characterization of a flavocytochrome that is induced during the anaerobic respiration of Fe3+ by Shewanella frigidimarina NCIMB400; Dobbin PS et al.; A 63.9 kDa periplasmic tetrahaem flavocytochrome c(3), designated Ifc(3), was found to be expressed in Shewanella frigidimarina NCIMB400 grown anaerobically with ferric citrate or ferric pyrophosphate as the sole terminal electron acceptor, but not in anaerobic cultures of the bacterium with other respiratory substrates . Ifc(3) was purified to homogeneity and revealed by biochemical, spectroscopic and primary structure analyses to contain four low-spin bis-His-ligated c(3)-haems, with midpoint reduction potentials of -73, -141, -174 and -259 mV . A low-potential flavin was present in the form of non-covalently bound FAD; the protein possessed a unidirectional fumarate reductase activity . Disruption of the chromosomal gene encoding Ifc(3), ifcA, did not lead to a significant change in the rate of Fe(3+) reduction in batch culture . However, during such growth the Ifc(3)-deficient mutant produced both a 35 kDa periplasmic c-type cytochrome and a 45 kDa membrane-associated c-type cytochrome at markedly higher levels than did the parent strain . Nucleotide sequencing data from directly upstream of ifcA indicated the presence of an open reading frame encoding a putative outer-membrane beta-barrel protein of 324 amino acid residues. Acta Biochim Pol, 1999, 46(1), 155 - 62 A human putative Suv3-like RNA helicase is conserved between Rhodobacter and all eukaryotes; Dmochowska A et al.; We have cloned and sequenced a cDNA of the human homologue of the Saccharomyces cerevisiae Suv3 putative RNA helicase which is indispensable for mitochondrial function in yeast . The human Suv-3-like protein has a typical mitochondrial leader sequence . Northern blot data and analysis of ESTs in the data banks indicate that this human gene (SUPV3L1) is expressed in practically all tissues, though at different levels . Sequence homology analysis has shown a strong conservation of the protein in a number of eukaryotic organisms -- plants, mammals and fungi, but no close homologues exist in bacteria with the exception of the purple bacterium Rhodobacter sphaeroides . This gene is thus ubiquitously present in all eukaryotic organisms. Can J Microbiol, 1999 Jun, 45(6), 458 - 63 Cresol metabolism by the sulfate-reducing bacterium Desulfotomaculum sp . strain Groll; Londry KL et al.; The metabolism of cresols under sulfate-reducing conditions was investigated in Desulfotomaculum sp . strain Groll . This strain grows on a variety of aromatic compounds, including para- and meta- but not ortho-cresol . Degradation of p-cresol proceeded by oxidation reactions of the methyl group to yield p-hydroxybenzoate, which was then dehydroxylated to benzoate . The aromatic intermediates expected for this pathway, p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, p-hydroxybenzoate, and benzoate, were readily metabolized by strain Groll . Utilization of these intermediates generally preceded and inhibited the degradation of p-cresol . p-Hydroxybenzoate and benzoate were detected in culture fluid as metabolites of p-cresol . p-Hydroxybenzaldehyde and p-hydroxybenzoate were detected in cultures degrading p-hydroxybenzyl alcohol . Enzyme activities responsible for utilization of p- and m-cresol, induced by growth on the respective cresol, were detected in cell-free extracts of strain Groll . The compounds detected in culture fluids and the enzyme activities detected in cell-free extracts indicate that the pathways for the degradation of p- and m-cresol converge on benzoate, followed by metabolism to benzoyl-coenzyme A (CoA) . Strain Groll can utilize both cresol isomers under sulfate-reducing conditions by similar reactions, but the enzyme activities catalyzing these transformations of the two isomers appear distinct. Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9757 - 62 Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes; Makovets S et al.; ClpXP-dependent proteolysis has been implicated in the delayed detection of restriction activity after the acquisition of the genes (hsdR, hsdM, and hsdS) that specify EcoKI and EcoAI, representatives of two families of type I restriction and modification (R-M) systems . Modification, once established, has been assumed to provide adequate protection against a resident restriction system . However, unmodified targets may be generated in the DNA of an hsd(+) bacterium as the result of replication errors or recombination-dependent repair . We show that ClpXP-dependent regulation of the endonuclease activity enables bacteria that acquire unmodified chromosomal target sequences to survive . In such bacteria, HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP . A mutation that blocks only the modification activity of EcoKI, leaving the cell with approximately 600 unmodified targets, is not lethal provided that ClpXP is present . Our data support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage. Int J Food Microbiol, 1999 Jun 1, 48(3), 191 - 202 Effect of rapid cooling and acidic pH on cellular homeostasis of Pectinatus frisingensis, a strictly anaerobic beer-spoilage bacterium; Chihib NE et al.; Pectinatus frisingensis is a strictly anaerobic mesophilic bacterium involved in bottled beer spoilage . Cellular volume, adenylate energy charge, intracellular pH and intracellular potassium concentration measurements were performed in late exponential-phase cell suspensions placed in different physiological conditions, to evaluate the capability of this bacterium to maintain cellular homeostasis . The intracellular pH was calculated from the intracellular accumulation of a {carboxyl-14C}benzoic acid . Optimum physiological conditions were the presence of a carbon source and pH of 6.2, hostile conditions were a pH 4.5, absence of a carbon source, and rapid cooling treatment . The cell was able to maintain a higher intracellular pH than the external pH under all conditions . Intracellular volume was lower at pH 4.5 than at pH 6.2 . A low net potassium efflux rate was routinely measured in starving cells, while glucose addition promoted immediate net potassium uptake from the medium . Cooling treatment resulted in sudden net potassium efflux from the cell, a decrease of the intracellular pH, and low modifications of the adenylate energy charge in metabolizing-glucose cell suspensions . Thus, cold treatment perturbs the P . frisingensis homeostasis but the bacteria were able to restore their homeostasis in the presence of a carbon source, and under warm conditions. J Periodontol, 1999 Jul, 70(7), 772 - 8 Soluble CD14-dependent intercellular adhesion molecular-1 induction by Porphyromonas gingivalis lipopolysaccharide in human gingival fibroblasts; Masaka T et al.; BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) is involved in the accumulation and activation of leukocytes in inflammatory sites through binding to beta2 integrins expressed on leukocytes . We investigated whether or not lipopolysaccharide (LPS) derived from the periodontopathic bacterium Porphyromonas gingiualis affects ICAM-1 expression on human gingival fibroblasts (HGF) . CD14 is a receptor for LPS on monocytes and macrophages and is also present in serum as a soluble protein . We further examined the effects of serum and soluble CD14 (sCD14) on ICAM-1 expression in HGF stimulated with P . gingivalis LPS . METHODS: HGF were prepared from explants of human gingival tissues and incubated in 96-well culture plates before LPS stimulation . LPS derived from Escherichia coli O55:B5 and P . gingivalis ATCC 33277 LPS were employed . sCD14 was purified from normal human serum (NHS) by affinity chromatography using an anti-CD14 monoclonal antibody . ICAM-1 expression on HGF was measured by a cell enzyme-linked immunosorbent assay . RESULTS: P . gingivalis LPS induced ICAM-1 on HGF in a dose-dependent manner in the presence of either 10% fetal calf serum or 2% NHS . The ability of P . gingivalis LPS to induce ICAM-1 was comparable to that of LPS from E . coli at high LPS concentrations . In the absence of NHS, ICAM-1 induction was negligible in HGF stimulated with P . gingivalis LPS, reaching a maximum at 2% NHS . The ICAM-1 expression induced by P . gingivalis LPS was inhibited by a monoclonal antibody to CD14 . Supplementation of serum-free medium with sCD14 alone restored the capacity of HGF to respond to P . gingivalis LPS . CONCLUSIONS: These results indicate that P . gingivalis LPS induces ICAM-1 expression in HGF in an sCD14-dependent manner . The overexpression of ICAM-1 on fibroblasts in gingiva induced by P . gingivalis LPS seems to be involved in the retention of inflammatory cells in periodontitis lesions. Microbiology, 1999 Jul, 145 ( Pt 7), 1743 - 8 Evidence for the presence of the reductive pentose phosphate cycle in a filamentous anoxygenic photosynthetic bacterium, Oscillochloris trichoides strain DG-6; Ivanovsky RN et al.; Studies on autotrophic CO2 fixation by the filamentous anoxygenic photosynthetic bacterium Oscillochloris trichoides strain DG-6 demonstrated that, unlike other green bacteria, this organism metabolized CO2 via the reductive pentose phosphate cycle . Both key enzymes of this cycle--ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase--were detected in cell extracts . The main product of ribulose 1,5-bisphosphate-dependent CO2 fixation was 3-phosphoglyceric acid . KCN, which is known to be a competitive inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase, completely inhibited the CO2 assimilation by whole cells as well as by cell extracts of O . trichoides . The 13C/12C carbon isotope fractionation during photoautotrophic growth of O . trichoides was -19.7/1000, which is close to that obtained for autotrophic organisms that use ribulose-1,5-bisphosphate carboxylase as the primary carboxylation enzyme . Cell extracts of O . trichoides contained all the enzymes of the tricarboxylic acid cycle except 2-oxoglutarate dehydrogenase . No activity of isocitrate lyase, a key enzyme of the glyoxylate shunt, was found in cell extracts of O . trichoides DG-6. J Bacteriol, 1999 Aug, 181(16), 5017 - 23 In Thermoanaerobacterium thermosulfurigenes EM1 S-layer homology domains do not attach to peptidoglycan; Brechtel E et al.; Three exocellular enzymes of Thermoanaerobacterium thermosulfurigenes EM1 possess a C-terminal triplicated sequence related to a domain of bacterial cell surface proteins (S-layer proteins) . At least one copy of this sequence, named the SLH (for S-layer homology) domain, is also present at the N terminus of the S-layer protein of this bacterium . The hypothesis that SLH domains serve to anchor proteins to the cell surface was investigated by using the SLH domain-containing xylanase . This enzyme was isolated from T . thermosulfurigenes EM1, and different forms with and without SLH domains were synthesized in Escherichia coli . The interaction of these proteins with isolated components of the cell envelope was determined to identify the attachment site in the cell wall . In addition, a polypeptide consisting of three SLH domains and the N terminus of the S-layer protein of T . thermosulfurigenes EM1 were included in these studies . The results indicate that SLH domains are necessary for the attachment of these proteins to peptidoglycan-containing sacculi . Extraction of the native sacculi with hydrofluoric acid led to the conclusion that not peptidoglycan but accessory cell wall polymers function as the adhesion component in the cell wall . Our results provide further evidence that attachment of proteins via their SLH domains represents an additional mode to display polypeptides on the cell surfaces of bacteria. J Bacteriol, 1999 Aug, 181(16), 4825 - 33 Transformations in flagellar structure of Rhodobacter sphaeroides and possible relationship to changes in swimming speed; Armitage JP et al.; Rhodobacter sphaeroides is a photosynthetic bacterium which swims by rotating a single flagellum in one direction, periodically stopping, and reorienting during these stops . Free-swimming R . sphaeroides was examined by both differential interference contrast (DIC) microscopy, which allows the flagella of swimming cells to be seen in vivo, and tracking microscopy, which tracks swimming patterns in three dimensions . DIC microscopy showed that when rotation stopped, the helical flagellum relaxed into a high-amplitude, short-wavelength coiled form, confirming previous observations . However, DIC microscopy also revealed that the coiled filament could rotate slowly, reorienting the cell before a transition back to the functional helix . The time taken to reform a functional helix depended on the rate of rotation of the helix and the length of the filament . In addition to these coiled and helical forms, a third conformation was observed: a rapidly rotating, apparently straight form . This form took shape from the cell body out and was seen to form directly from flagella that were initially in either the coiled or the helical conformation . This form was always significantly longer than the coiled or helical form from which it was derived . The resolution of DIC microscopy made it impossible to identify whether this form was genuinely in a straight conformation or was a low-amplitude, long-wavelength helix . Examination of the three-dimensional swimming pattern showed that R . sphaeroides changed speed while swimming, sometimes doubling the swimming speed between stops . The rate of acceleration out of stops was also variable . The transformations in waveform are assumed to be torsionally driven and may be related to the changes in speed measured in free-swimming cells . The roles of and mechanisms that may be involved in the transformations of filament conformations and changes in swimming speed are discussed. Vet Immunol Immunopathol, 1999 May, 68(2-4), 159 - 68 Early cytokine induction in mouse P388D1 macrophages infected by Coxiella burnetii; Tujulin E et al.; Q-fever is caused by Coxiella burnetii, which is an obligate intracellular bacterium with a broad spectrum of host cells, including macrophages . Cytokines produced from macrophages infected by intracellular bacteria play a critical role in the expression of innate immune responses as well as in the subsequent triggering of protective acquired cell-mediated immunity . We followed the induction and secretion of the pro-inflammatory cytokines interleukin 1 alpha (IL-1alpha), tumor necrosis factor alpha (TNF-alpha), and interleukin 12 (IL-12) in the macrophage-like mouse cell line P388D1 during the initial phase of an in vitro infection by virulent C . burnetii Nine Mile . Secretion of IL-1alpha and TNF-alpha were observed within 3 h post-inoculation . IL-12, however, was not detected in cell supernatants . Two forms of C . burnetii exist, virulent phase I and avirulent phase II organisms . To determine whether the cytokine response was dependent on the form of C . burnetii, the induction of IL-1alpha and TNF-alpha in infected P388D1 cells was compared . Both cytokines were produced by macrophages early after infection with Phase I bacteria . A similar induction of TNF-alpha was observed after infection with the avirulent Phase II bacteria, but no IL-1alpha induction could be detected . As the only difference identified between the two forms of C . burnetii is the composition of their lipopolysaccharides (LPS), the ability of each of the purified LPS from the two variants to induce IL-1alpha was investigated . Purified C . burnetii LPS induced a moderate IL-1alpha response in comparison to that induced by the efficient stimulator E . coli LPS . Furthermore, there was no significant difference in action between Phase I and Phase II LPS preparations . We thus postulate that factors other than LPS differ between the two variants of C . burnetii, and these differences may account for differences in IL-1alpha induction. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 8884 - 9 Crystal structure and mechanism of CO dehydrogenase, a molybdo iron-sulfur flavoprotein containing S-selanylcysteine; Dobbek H et al.; CO dehydrogenase from the aerobic bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO with H(2)O, yielding CO(2), two electrons, and two H(+) . Its crystal structure in the air-oxidized form has been determined to 2.2 A . The active site of the enzyme, which contains molybdenum with three oxygen ligands, molybdopterin-cytosine dinucleotide and S-selanylcysteine, delivers the electrons to an intramolecular electron transport chain composed of two types of {2Fe-2S} clusters and flavin-adenine dinucleotide . CO dehydrogenase is composed of an 88.7-kDa molybdoprotein (L), a 30 . 2-kDa flavoprotein (M), and a 17.8-kDa iron-sulfur protein (S) . It is organized as a dimer of LMS heterotrimers and resembles xanthine dehydrogenase/oxidase in many, but not all, aspects . A mechanism based on a structure with the bound suicide-substrate cyanide is suggested and displays the necessity of S-selanylcysteine for the catalyzed reaction. Genetics, 1999 Aug, 152(4), 1669 - 77 Mitochondrial DNA polymorphism, sex ratio distorters and population genetics in the isopod Armadillidium vulgare; Rigaud T et al.; Two maternally inherited sex ratio distorters (SRD) impose female-biased sex ratios on the wood louse Armadillidium vulgare by feminizing putative males . These SRD are (i) an intracytoplasmic bacterium of the genus Wolbachia, and (ii) another non-Mendelian element of unknown nature: the f element . Mitochondrial DNA variation was investigated in A . vulgare field populations to trace the evolution of host-SRD relationships and to investigate the effect of SRD on host cytoplasmic polymorphism . The Wolbachia endosymbionts showed no polymorphism in their ITS2 sequence and were associated with two closely related mitochondrial types . This situation probably reflects a single infection event followed by a slight differentiation of mitochondria . There was no association between the f element and a given mitochondrial type, which may confirm the fact that this element can be partially paternally transmitted . The spreading of a maternally inherited SRD in a population should reduce the mitochondrial diversity by a hitchhiking process . In A . vulgare, however, a within-population mtDNA polymorphism was often found, because of the deficient spread of Wolbachia and the partial paternal inheritance of the f element . The analysis of molecular variance indicated that A . vulgare populations are genetically structured, but without isolation by distance. FEMS Microbiol Lett, 1999 Jul 15, 176(2), 357 - 66 Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli; Guo L et al.; We developed an efficient method for isolation of novel dnaA mutations based on PCR mutagenesis in the presence of manganese ion and shuffling of dnaA-carrying plasmids in a dnaA deletion host bacterium . Using this system, we obtained 30 cold-sensitive mutants from 4000 clones carrying plasmids with a mutagenized dnaA gene . All 27 cold-sensitive mutants analyzed were defective in DNA replication; none had a DnaAcos (over-initiation) phenotype . Nucleotide sequencing revealed that novel 15 alleles (mutations in 14 amino acid residues) are responsible for the cold-sensitive phenotype and are all located in the carboxy-terminal half of the DnaA protein. FEMS Microbiol Lett, 1999 Jul 15, 176(2), 339 - 44 Identification of a novel periplasmic catalase-peroxidase KatA of Legionella pneumophila; Amemura-Maekawa J et al.; A gene katA that encodes a novel catalase-peroxidase was cloned from the chromosome of Legionella pneumophila . The nucleotide sequence revealed that KatA was highly homologous to members of the bacterial bifunctional catalase-peroxidase family . In addition, KatA has a N-terminal signal sequence and was considered to be present in the periplasm of the bacterium. FEMS Microbiol Lett, 1999 Jul 15, 176(2), 327 - 32 Protein kinase activity in Helicobacter pylori; Grangeasse C et al.; Based on the predictive analysis of the cellular protein content from the complete genome sequence of Helicobacter pylori, discrepant results were previously reported concerning the occurrence of a protein kinase in this bacterium . To solve this ambiguity, we have directly assayed cellular extracts for their capacity of phosphorylating endogenous proteins . At least eight different proteins, ranging from 24 to 200 kDa, were found to be phosphorylated to a varying extent . Individual measurement of their phosphoamino acid composition showed that they all were modified at serine residues . These data indicate that H . pylori does contain a protein-serine kinase activity. Appl Environ Microbiol, 1999 Aug, 65(8), 3757 - 60 Physicochemical parameters for growth of the sea ice bacteria Glaciecola punicea ACAM 611(T) and Gelidibacter sp . strain IC158; Nichols DS et al.; The water activity and pH ranges for growth of Glaciecola punicea (a psychrophile) were extended when this organism was grown at suboptimal rather than optimal temperatures . No such extension was observed for Gelidibacter sp . strain IC158 (a psychrotolerant bacterium) at analogous temperatures . Salinity and pH may be primary physicochemical parameters controlling bacterial community development in sea ice. Appl Environ Microbiol, 1999 Aug, 65(8), 3660 - 7 Distribution and evolution of the xylanase genes xynA and xynB and their homologues in strains of Butyrivibrio fibrisolvens; Dalrymple BP et al.; The ruminal bacterium Butyrivibrio fibrisolvens is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants . However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of B . fibrisolvens . In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of Butyrivibrio fibrisolvens have been investigated . Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49 xynA gene . However, these four xynA-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to xynA (subfamily I) . Homologues of xynB, a second previously described xylanase gene from B . fibrisolvens, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains . However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of xynA . The distribution of genes and the nucleotide sequence relationships between the members of the two xynA subfamilies are consistent with the progenitor of all strains of B . fibrisolvens having contained a xynA subfamily I gene . Since many xylanolytic strains of B . fibrisolvens did not contain members of either of the xynA subfamilies or of the xynB family, at least one additional xylanase gene family remains to be identified in B . fibrisolvens. Appl Environ Microbiol, 1999 Aug, 65(8), 3588 - 93 A novel mineral flotation process using Thiobacillus ferrooxidans; Nagaoka T et al.; Oxidative leaching of metals by Thiobacillus ferrooxidans has proven useful in mineral processing . Here, we report on a new use for T . ferrooxidans, in which bacterial adhesion is used to remove pyrite from mixtures of sulfide minerals during flotation . Under control conditions, the floatabilities of five sulfide minerals tested (pyrite, chalcocite, molybdenite, millerite, and galena) ranged from 90 to 99% . Upon addition of T . ferrooxidans, the floatability of pyrite was significantly suppressed to less than 20% . In contrast, addition of the bacterium had little effect on the floatabilities of the other minerals, even when they were present in relatively large quantities: their floatabilities remained in the range of 81 to 98% . T . ferrooxidans thus appears to selectively suppress pyrite floatability . As a consequence, 77 to 95% of pyrite was removed from mineral mixtures while 72 to 100% of nonpyrite sulfide minerals was recovered . The suppression of pyrite floatability was caused by bacterial adhesion to pyrite surfaces . When normalized to the mineral surface area, the number of cells adhering to pyrite was significantly larger than the number adhering to other minerals . These results suggest that flotation with T . ferrooxidans may provide a novel approach to mineral processing in which the biological functions involved in cell adhesion play a key role in the separation of minerals. Appl Environ Microbiol, 1999 Aug, 65(8), 3373 - 85 Genetic structure of natural populations of Escherichia coli in wild hosts on different continents; Souza V et al.; Current knowledge of genotypic and phenotypic diversity in the species Escherichia coli is based almost entirely on strains recovered from humans or zoo animals . In this study, we analyzed a collection of 202 strains obtained from 81 mammalian species representing 39 families and 14 orders in Australia and the Americas, as well as several reference strains; we also included a strain from a reptile and 10 from different families of birds collected in Mexico . The strains were characterized genotypically by multilocus enzyme electrophoresis (MLEE) and phenotypically by patterns of sugar utilization, antibiotic resistance, and plasmid profile . MLEE analysis yielded an estimated genetic diversity (H) of 0.682 for 11 loci . The observed genetic diversity in this sample is the greatest yet reported for E . coli . However, this genetic diversity is not randomly distributed; geographic effects and host taxonomic group accounted for most of the genetic differentiation . The genetic relationship among the strains showed that they are more associated by origin and host order than is expected by chance . In a dendrogram, the ancestral cluster includes primarily strains from Australia and ECOR strains from groups B and C . The most differentiated E . coli in our analysis are strains from Mexican carnivores and strains from humans, including those in the ECOR group A . The kinds and numbers of sugars utilized by the strains varied by host taxonomic group and country of origin . Strains isolated from bats were found to exploit the greatest range of sugars, while those from primates utilized the fewest . Toxins are more frequent in strains from rodents from both continents than in any other taxonomic group . Strains from Mexican wild mammals were, on average, as resistant to antibiotics as strains from humans in cities . On average, the Australian strains presented a lower antibiotic resistance than the Mexican strains . However, strains recovered from hosts in cities carried significantly more plasmids than did strains isolated from wild mammals . Previous studies have shown that natural populations of E . coli harbor an extensive genetic diversity that is organized in a limited number of clones . However, knowledge of this worldwide bacterium has been limited . Here, we suggest that the strains from a wide range of wild hosts from different regions of the world are organized in an ecotypic structure where adaptation to the host plays an important role in the population structure. J Am Acad Dermatol, 1999 Aug, 41(2 Pt 2), 338 - 40 Primary cutaneous nocardiosis in a husband and wife; Angelika J et al.; A 62-year-old woman suffered from acute purulent skin disease with multiple subcutaneous abscesses . At the same time, her 65-year-old husband presented with multiple subcutaneous nodules along the lymphatic vessels of his right arm . Both had a history of a minor scratch by a thorn of a bush at the site of infection . Nocardia was identified as causative bacterium from the woman's lesions . Therefore the rare phenomenon of 2 different forms of acute primary cutaneous nocardiosis after simultaneous infection, lymphocutaneous infection, and superficial skin infection was diagnosed. Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1125 - 8 Aerococcus christensenii sp . nov., from the human vagina; Collins MD et al.; Phenotypic and phylogenetic studies were performed on two strains of a hitherto undescribed Aerococcus-like organism isolated from the human vagina . Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strains constitute a new subline within the genus Aerococcus . The unknown bacterium was readily distinguished from the two currently recognized Aerococcus species, Aerococcus viridans and Aerococcus urinae, by biochemical tests and electrophoretic analysis of whole-cell proteins . On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Aerococcus christensenii sp . nov . The type strain of A . christensenii is CCUG 28831T. Eur J Med Res, 1999 Jul 28, 4(7), 286 - 92 Effect of immunization on gastrin release in rat antrum mucosa; Andress HJ et al.; Helicobacter pylori (H.P.) infection is the main cause of chronic active gastritis and is closely associated with peptic ulcer disease . Chronic hypergastrinaemia is known to be induced in patients infected with this pathogen . Gastrin is the most potent stimulator of gastric acid secretion . In animals infected with H.P . or H . felis vaccination against H.P.can eliminate the bacterium and alleviate associated gastritis . However little is known on the influence of immunization regimes on gastrin release . The aim of this study was to evaluate the effect of systemic immunization on gastrin release in a rat model, using a well defined but for the rat unknown protein . OVA-immunized and non immunized animals received OVA intragastrically in vivo, and serum gastrin was measured . Stimulation with bovine serum albumin (BSA) served as control . In vitro single cell suspensions of the antrum mucosa and mucosal fragments of immunized and non immunized animals were also incubated with OVA and BSA . The results show specific significant gastrin suppression after immunization and antigen feeding . The same results were obtained in the model of mucosa fragments but not in single cell suspensions . This hypothesized that gastric suppression could be mediated by mast cells or T-helper two cells (TH2) in the mucosal layer of the stomach . A single cell suspension is not suitable for studying immunologically mediated gastrin release because direct cell contact is necessary . Further studies with these animal models have to show whether effects seen by vaccination could be mediated by induction of gastrin suppression and studies on human tissues are necessary to show possible similar effects with H.P. Mol Reprod Dev, 1999 Sep, 54(1), 86 - 91 Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization; Luciano AM et al.; Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP . The intracellular levels of cAMP in cattle cumulus-oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis . High concentrations of iAC (1 or 5 microgram/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 microgram/ml) resulted in high rates of maturation to the MII stage (92.6 +/- 2.5 and 98.5 +/- 1.4% respectively) . The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 +/- 4.2 and 45.1 +/- 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 +/- 2.9%) . Finally, the addition of 0.1 microgram/ml iAC and 0.5 mM 3-isobutyl 1-methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 microgram/ml iAC (31.9 +/- 5.5 vs . 12.1 +/- 1.6 and 45.5 +/- 2.9 vs . 19.1 +/- 2.3% respectively) . It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones . Mol . Reprod . Dev . 54:86-91,1999 . Protein Sci, 1999 Jul, 8(7), 1536 - 45 Structural studies by X-ray diffraction on metal substituted desulforedoxin, a rubredoxin-type protein; Archer M et al.; Desulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio gigas, is a small homodimeric (2 x 36 amino acids) protein . Each subunit contains a high-spin iron atom tetrahedrally bound to four cysteinyl sulfur atoms, a metal center similar to that found in rubredoxin (Rd) type proteins . The simplicity of the active center in Dx and the possibility of replacing the iron by other metals make this protein an attractive case for the crystallographic analysis of metal-substituted derivatives . This study extends the relevance of Dx to the bioinorganic chemistry field and is important to obtain model compounds that can mimic the four sulfur coordination of metals in biology . Metal replacement experiments were carried out by reconstituting the apoprotein with In3+, Ga3+, Cd2+, Hg2+, and Ni2+ salts . The In3+ and Ga3+ derivatives are isomorphous with the iron native protein; whereas Cd2+, Hg2+, and Ni2+ substituted Dx crystallized under different experimental conditions, yielding two additional crystal morphologies; their structures were determined by the molecular replacement method . A comparison of the three-dimensional structures for all metal derivatives shows that the overall secondary and tertiary structures are maintained, while some differences in metal coordination geometry occur, namely, bond lengths and angles of the metal with the sulfur ligands . These data are discussed in terms of the entatic state theory. J Bacteriol, 1999 Aug, 181(15), 4509 - 16 Characterization of an atypical superoxide dismutase from Sinorhizobium meliloti; Santos R et al.; Sinorhizobium meliloti Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants . A single detectable superoxide dismutase (SOD) was found in free-living growth conditions . The corresponding gene was isolated from a genomic library by using a sod fragment amplified by PCR from degenerate primers as a probe . The sodA gene was located in the chromosome . It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical M(r) of 22,430 and pI of 5 . 8 . S . meliloti SOD complemented a deficient E . coli mutant, restoring aerobic growth of a sodA sodB recA strain, when the gene was expressed from the synthetic tac promoter but not from its own promoter . Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H(2)O(2) . The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg . Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer) . The apoenzyme was prepared from crude extracts of S . meliloti . Activity was restored by dialysis against either MnCl(2) or Fe(NH(4))(2)(SO(4))(2), demonstrating the cambialistic nature of the S . meliloti SOD . The recovered activity with manganese was sevenfold higher than with iron . Both reconstituted enzymes were resistant to H(2)O(2) . Sequence comparison with 70 FeSODs and MnSODs indicates that S . meliloti SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H(2)O(2) of this unusual Fe-type SOD. J Bacteriol, 1999 Aug, 181(15), 4476 - 84 Role of TolR N-terminal, central, and C-terminal domains in dimerization and interaction with TolA and tolQ; Journet L et al.; The Tol-PAL system of Escherichia coli is a multiprotein system involved in maintaining the cell envelope integrity and is necessary for the import of some colicins and phage DNA into the bacterium . It is organized into two complexes, one near the outer membrane between TolB and PAL and one in the cytoplasmic membrane between TolA, TolQ, and TolR . In the cytoplasmic membrane, all of the Tol proteins have been shown to interact with each other . Cross-linking experiments have shown that the TolA transmembrane domain interacts with TolQ and TolR . Suppressor mutant analyses have localized the TolQ-TolA interaction to the first transmembrane domain of TolQ and have shown that the third transmembrane domain of TolQ interacts with the transmembrane domain of TolR . To get insights on the composition of the cytoplasmic membrane complex and its possible contacts with the outer membrane complex, we focused our attention on TolR . Cross-linking and immunoprecipitation experiments allowed the identification of Tol proteins interacting with TolR . The interactions of TolR with TolA and TolQ were confirmed, TolR was shown to dimerize, and the resulting dimer was shown to interact with TolQ . Deletion mutants of TolR were constructed, and they allowed us to determine the TolR domains involved in each interaction . The TolR transmembrane domain was shown to be involved in the TolA-TolR and TolQ-TolR interactions, while TolR central and C-terminal domains appeared to be involved in TolR dimerization . The role of the TolR C-terminal domain in the TolA-TolR interaction and its association with the membranes was also demonstrated . Furthermore, phenotypic studies clearly showed that the three TolR domains (N terminal, central, and C terminal) and the level of TolR production are important for colicin A import and for the maintenance of cell envelope integrity. FEMS Microbiol Lett, 1999 Jul 1, 176(1), 125 - 30 Organization of the genes involved in the ribulose monophosphate pathway in an obligate methylotrophic bacterium, Methylomonas aminofaciens 77a; Sakai Y et al.; The 4.4-kb PstI fragment harboring the gene encoding 3-hexulose-6-phosphate synthase, rmpA, which was previously cloned from the chromosome of an obligate methylotroph, Methylomonas aminofaciens 77a, was investigated in detail . In addition to the rmpA gene, the fragment contained three open reading frames encoding transaldolase (rmpD), IS10-R (rmpI), and 6-phospho-3-hexuloisomerase (PHI) (rmpB) . The rmpB gene product was overproduced in Escherichia coli cells, purified to homogeneity, and then enzymatically identified as PHI . The gene organization of the ribulose monophosphate pathway enzymes together with a transposon, IS10-R, is discussed from both evolutionary and regulatory aspects. Infect Immun, 1999 Aug, 67(8), 3816 - 23 Variable carbohydrate modifications to the catalytic chains of the RgpA and RgpB proteases of Porphyromonas gingivalis W50; Curtis MA et al.; Proteases of Porphyromonas gingivalis are considered to be important virulence determinants of this periodontal bacterium . Several biochemical isoforms of arginine-specific proteases are derived from rgpA and rgpB . HRgpA is a heterodimer composed of the catalytic alpha chain noncovalently associated with a beta adhesin chain derived from the C terminus of the initial full-length translation product . The catalytic alpha chain is also present as a monomer (RgpA) either free in solution or associated with membranes . rgpB lacks the coding region for the adhesin domain present in rgpA and yields only monomeric forms (RgpB) which again may be soluble or membrane associated . In this study, the catalytic chains of this unusual group of enzymes are shown to be differentially modified by the posttranslational addition of carbohydrate . A monoclonal antibody (MAb 1B5) raised to the monomeric RgpA did not react with the corresponding recombinant RgpA alpha chain expressed in Escherichia coli but was immunoreactive with P . gingivalis lipopolysaccharide . MAb 1B5 also reacted with the membrane-associated forms of RgpA and RgpB but not with the heterodimeric HRgpA and the soluble form of RgpB . RgpA treated with denaturants was capable of binding to MAb 1B5 whereas treatment with periodate abolished this binding, suggesting the presence of carbohydrate residues within the epitope . Chemical deglycosylation abolished immunoreactivity with MAb 1B5 and caused a approximately 30% reduction in the size of the membrane-associated enzymes . Monosaccharide analysis of HRgpA and RgpA demonstrated 2.1 and 14.4%, respectively, carbohydrate by weight of protein . Furthermore, distinct differences were detected in their monosaccharide compositions, indicating that these protease isoforms are modified not only to different extents but also with different sugars . The variable nature of these additions may have a significant effect on the structure, stability, and immune recognition of these protease glycoproteins. Plasmid, 1999 Jul, 42(1), 13 - 9 The first detection of the insertion sequence ISW1 in the intracellular reproductive parasite Wolbachia; Masui S et al.; Wolbachia are maternally inherited intracellular rickettsia-like bacteria known to infect a wide range of arthropods . They are associated with a number of different reproductive phenotypes in their hosts, such as cytoplasmic incompatibility, parthenogenesis, and feminization . We report on a novel insertion sequence (IS), ISW1, which was identified in the region downstream of groEL of a Wolbachia strain, wTai . The 573-bp-long ISW1 sequence is the first IS element observed in this organism, displays significant similarity to IS200, and lacks terminal inverted repeats . There were more than 20 copies of ISW1 on the chromosome of wTai . Sequence analysis of nine distinct ISW1 copies and their flanking regions showed that the copies were identical and suggested that ISW1 has no preference for its insertion sites . Possible roles of ISW1 in the adaptation of Wolbachia to intracellular environments and in various reproductive alterations caused by this bacterium are discussed . Biochemistry, 1999 Jul 20, 38(29), 9435 - 9 Substrate specificity of Deinococcus radiodurans Fpg protein; Senturker S et al.; A DNA repair enzyme has recently been isolated from the ionizing radiation-resistant bacterium Deinococcus radiodurans {Bauche, C., and Laval, J . (1999) J . Bacteriol . 181, 262-269} . This enzyme is a homologue of the Fpg protein of Escherichia coli . We investigated the substrate specificity of this enzyme for products of oxidative DNA base damage using gas chromatography/isotope-dilution mass spectrometry and DNA substrates, which were either gamma-irradiated or treated with H(2)O(2)/Fe(III)-EDTA/ascorbic acid . Excision of purine lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 8-hydroxyguanine (8-OH-Gua) was observed among 17 lesions detected in damaged DNA substrates . The extent of excision was determined as a function of enzyme concentration, time, and substrate concentration . FapyGua and FapyAde were excised with similar specificities from three DNA substrates, whereas 8-OH-Gua was the least preferred lesion . The results show that D . radiodurans Fpg protein and its homologue E . coli Fpg protein excise the same modified DNA bases, but the excision rates of these enzymes are significantly different . Formamidopyrimidines are preferred substrates of D . radiodurans Fpg protein over 8-OH-Gua, whereas E . coli Fpg protein excises these three lesions with similar efficiencies from various DNA substrates . Substrate specificities of these enzymes were also compared with that of Saccharomyces cerevisiae Ogg1 protein, which excises FapyGua and 8-OH-Gua, but not FapyAde. Biochemistry, 1999 Jul 13, 38(28), 9169 - 78 Stability and folding of dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima; Dams T et al.; Dihydrofolate reductase (DHFR) has been a well-established model system for protein folding . The enzyme DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR) displays distinct adaptations toward high temperatures at the level of both structure and stability . The enzyme represents an extremely stable dimer; no isolated structured monomers could be detected in equilibrium or during unfolding . The equilibrium unfolding strictly follows the two-state model for a dimer (N(2) right harpoon over left harpoon 2U), with a free energy of stabilization of DeltaG = -142 +/- 10 kJ/mol at 15 degrees C . The two-state model is applicable over the whole temperature range (5-70 degrees C), yielding a DeltaG vs T profile with maximum stability at around 35 degrees C . There is no flattening of the stability profile . Instead, the enhanced thermostability is characterized by shifts toward higher overall stability and higher temperature of maximum stability . TmDHFR unfolds in a highly cooperative manner via a nativelike transition state without intermediates . The unfolding reaction is much slower (ca . 10(8) times) compared to DHFR from Escherichia coli (EcDHFR) . In contrast to EcDHFR, no evidence for heterogeneity of the native state is detectable . Refolding proceeds via at least two intermediates and a burst-phase of rather low amplitude . Reassociation of monomeric intermediates is not rate-limiting on the folding pathway due to the high association constant of the dimer. Mol Microbiol, 1999 Jul, 33(2), 350 - 62 Cloning and allelic exchange mutagenesis of two flagellin genes of Helicobacter felis; Josenhans C et al.; Helicobacter felis has been used extensively in animal model studies of gastric Helicobacter infections . Attempts to manipulate H . felis genetically have, however, been unsuccessful and, consequently, little is known about the pathogenic mechanisms of this bacterium . In common with other Helicobacter spp., H . felis is a highly motile organism . To characterize the flagellar structures responsible for this motility, we cloned and sequenced the two flagellin-encoding genes, flaA and flaB, from H . felis . These genes encode two flagellin proteins that are expressed simultaneously under the control of putative sigma28 and sigma54 promoters respectively . Isogenic mutants of H . felis in flaA and flaB were generated by electroporation-mediated allelic disruption and replacement, showing for the first time that H . felis could be manipulated genetically . Both types of H . felis flagellin mutants exhibited truncated flagella and were poorly motile . H . felis flaA mutants were unable to colonize the gastric mucosa in a mouse infection model. Science, 1999 Jul 16, 285(5426), 406 - 9 Bacterial photoreceptor with similarity to photoactive yellow protein and plant phytochromes; Jiang Z et al.; A phytochrome-like protein called Ppr was discovered in the purple photosynthetic bacterium Rhodospirillum centenum . Ppr has a photoactive yellow protein (PYP) amino-terminal domain, a central domain with similarity to phytochrome, and a carboxyl-terminal histidine kinase domain . Reconstitution experiments demonstrate that Ppr covalently attaches the blue light-absorbing chromophore p-hydroxycinnamic acid and that it has a photocycle that is spectrally similar to, but kinetically slower than, that of PYP . Ppr also regulates chalcone synthase gene expression in response to blue light with autophosphorylation inhibited in vitro by blue light . Phylogenetic analysis demonstrates that R . centenum Ppr may be ancestral to cyanobacterial and plant phytochromes. Microbiology, 1999 Jun, 145 ( Pt 6), 1289 - 98 A novel tRNA-associated locus (trl) from Helicobacter pylori is co-transcribed with tRNA(Gly) and reveals genetic diversity; Dundon WG et al.; To date several genes have been identified in Helicobacter pylori that are expressed in only a proportion of strains, some of which are correlated with the pathogenicity of the bacterium . With this in mind, the present study was undertaken to identify other genes that are not expressed in all clinical isolates of H . pylori . Using arbitrarily primed PCR of RNA, a cDNA fragment of 187 bp (designated trl for transfer RNA-associated locus) was identified that was expressed in only one of two clinical isolates being tested . The fragment was purified, cloned and sequenced . A search of public databases prior to the release of the complete genome sequence of H . pylori strain 26695 showed no similarity with any other known genes or gene products . Inverse PCR was used to obtain further nucleotide sequence information surrounding the trl locus . A DNA probe derived from the trl locus hybridized with 32 (50%) of 64 clinical H . pylori isolates tested . Comparison of the nucleotide sequences of a trl-positive and trl-negative isolate showed that the locus is situated between two tRNA genes, tRNA(Gly) and tRNA(Leu), in H . pylori . Primer extension analysis showed that the trl locus is co-transcribed with tRNA(Gly) . Analysis of the region between tRNA(Gly) and tRNA(Leu) in trl-negative isolates revealed additional genetic diversity among these isolates. J Leukoc Biol, 1999 Jul, 66(1), 113 - 9 Differential iron transport into phagosomes isolated from the RAW264.7 macrophage cell lines transfected with Nramp1Gly169 or Nramp1Asp169; Kuhn DE et al.; The transport of iron by RAW264.7 macrophage cell lines transfected with either Nramp1Gly169 (resistant) or Nramp1ASp169 (susceptible) alleles was assessed . We found no difference between resistant and susceptible cells in the rate of Fe import or export when Fe transport was measured in intact cells . In contrast, the rate of Fe import by latex-bead phagosomes isolated from resistant cells was more than double the rate by latex-bead phagosomes from susceptible cells . Similarly, phagosomes isolated from resistant cells that had been pre-labeled with 55Fe-citrate before phagocytosis contained up to four times as much Fe as the corresponding phagosomes from susceptible cells . Phagocytosis of Mycobacterium avium was accompanied by an increase in the production of hydroxyl radicals by Nramp1cGly169-transfected macrophages but not by macrophages transfected with the susceptible allele . These results are consistent with the hypothesis that Nramp1 functions to transport Fe into the bacterium-containing phagosome where it serves as a catalyst for the Haber-Weiss reaction, which accounts for the increased capacity of these cells to limit mycobacterial growth. J Biol Chem, 1999 Jul 23, 274(30), 21234 - 43 A novel mechanism for the regulation of photosynthesis gene expression by the TspO outer membrane protein of Rhodobacter sphaeroides 2.4.1; Yeliseev AA et al.; A bacterial homolog of the mammalian mitochondrial benzodiazepine receptor, the tryptophan-rich sensory protein (TspO) has been previously demonstrated to negatively affect the transcriptional expression of several photosynthesis genes of Rhodobacter sphaeroides . To identify components of the signal transduction pathway from the outer membrane-localized TspO to the DNA-active transcription factor(s), we examined the involvement of TspO in the regulation of tetrapyrrole metabolism in R . sphaeroides . By analyzing the tetrapyrrole pigments accumulated by resting cell suspensions of R . sphaeroides, we demonstrated that TspO negatively regulates the activity of coproporphyrinogen III oxidase in this bacterium . hemN, encoding one of the isoenzymes of coproporphyrinogen III oxidase of R . sphaeroides, provided in trans to the wild type strain, produced a TSPO1 mutant phenotype by abolishing the negative effect of TspO on the transcription of the photosynthesis genes, crtI and puc . It is proposed that TspO, by regulating the exit of certain tetrapyrrole intermediates of the heme/bacteriochlorophyll biosynthetic pathways in R . sphaeroides in response to the availability of molecular oxygen, causes the accumulation of a biosynthetic intermediate that serves as a corepressor for both specific pigment gene transcription and the puc operon . The relationship between the bacterial TspO and the mitochondrial peripheral benzodiazepine receptor is discussed. Can J Microbiol, 1999 Mar, 45(3), 273 - 8 Survival of insect pathogenic and human clinical isolates of Photorhabdus luminescens in previously sterile soil; Bleakley BH et al.; Most characterized strains of the bacterium Photorhabdus luminescens are symbionts of entomopathogenic nematodes, whereas other strains have been isolated from human clinical specimens . The ability of P . luminescens strains to survive and grow in soil has received limited attention, with some studies indicating these bacteria have little or no ability to persist in soil . Survival and (or) growth of P . luminescens strains in previously sterilized soil, and examination of different soil amendments on their numbers in soil, have not been previously reported . Entomopathogenic P . luminescens (ATCC 29999) and a human clinical isolate (ATCC 43949) were introduced into a soil that had been sterilized by autoclaving, with or without different soil amendments, and bacterial numbers were estimated over time by viable plate count . In the previously sterilized soil receiving no exogenous amendments, numbers fell drastically over a week's time, followed by an increase in numbers by day 30 . Treatments involving the addition of calcium carbonate and gelatin or casamino acids to soil usually resulted in higher bacterial numbers . For some sampling dates and soil treatments, there were statistically significant differences between the numbers of the two bacterial strains recovered from soil . The two strains of P . luminescens used in this study were able to survive and grow after being inoculated into previously sterilized soil. Glycobiology, 1999 Aug, 9(8), 765 - 78 Fingerprinting of large oligosaccharides linked to ceramide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: highly heterogeneous polyglycosylceramides of human erythrocytes with receptor activity for Helicobacter pylori; Karlsson H et al.; Highly microheterogeneous polyglycosylceramides (PGCs) of human erythrocytes with an average composition of about 25 monosaccharides linked to ceramide were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) . The human gastric pathogen Helicobacter pylori was earlier shown to bind this glycosphingolipid mixture by thin-layer chromatogram binding assay . The receptor activity was present along the whole nonresolved chromatographic interval . Mass spectra of intact PGCs were compared with corresponding spectra of oligosaccharides enzymatically released from the ceramides . Two subfractions of PGCs containing less than one and more than one sialic acid residue per molecule were used . MALDI-MS spectra were recorded in both linear and reflectron mode with the accuracies of </=0.08% and </=0.02%, respectively, which allowed determination of the constituent parts of the detected ions in form of ceramide and number of hexoses, N-acetylhexosamines, fucoses and sialic acids . Molecular species were found based on ceramide with mainly sphingosine and fatty acids 24:0 and 24:1 (with less amounts of 22:0), and with a total number of monosaccharides ranging from 11 (neutral, m/z = 2605 for {M+Na}(+)) to 41 (one sialic acid, m/z = 8057 for {M-H}(-)) . The saccharide composition obtained supported a successively extended and branched N -acetyllactosamine core with substitutions of fucoses (0 up to 8) and sialic acid (0 to 1) . The reliable molecular analysis of large oligosaccharides linked to ceramide using this approach will be of great help for the further structure analysis in order to define the epitope for the sialic acid-dependent binding by the bacterium. J Clin Microbiol, 1999 Aug, 37(8), 2456 - 60 Helicobacter pylori infection in indigenous families of Central America: serostatus and oral and fingernail carriage; Dowsett SA et al.; Helicobacter pylori infection remains one of the most common in humans, but the route of transmission of the bacterium is still uncertain . This study was designed to elucidate possible sources of infection in an isolated, rural population in Guatemala . A total of 242 subjects in family units participated in the study . A medical history, including a history of dyspepsia, was taken by a physician and immunoglobulin G antibodies to H . pylori were detected with the QuickVue (Quidel, San Diego, Calif.) onsite serology test . Overall, 58% of subjects were seropositive, with a positive relationship between mother and child (P = 0.02) and a positive correlation between the serostatuses of siblings (intraclass correlation coefficient = 0.63) . There was no association between serostatus and gastric symptoms . Oral H . pylori was detected from periodontal pockets of various depths and the dorsum of the tongue by nested PCR . Eighty-seven percent of subjects had at least one oral site positive for H . pylori, with the majority of subjects having multiple positive sites . There was no association between periodontal pocket depth and the detection of H . pylori . Nested PCR was also used to detect H . pylori from beneath the nail of the index finger of each subject's dominant hand . Overall, 58% of subjects had a positive fingernail result, with a significant positive relationship between fingernail and tongue positivity (P = 0.002) . In conclusion, the results of this study suggest that oral carriage of H . pylori may play a role in the transmission of infection and that the hand may be instrumental in transmission. J Antimicrob Chemother, 1999 Jun, 43(6), 753 - 8 The role of the rdxA gene in the evolution of metronidazole resistance in Helicobacter pylori; Jenks PJ et al.; It was recently demonstrated that inactivation of the rdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase, is associated with the development of resistance to metronidazole by Helicobacter pylori . In order to further evaluate the contribution of rdxA to metronidazole resistance, the sequence of the rdxA gene was determined for a series of metronidazole-sensitive and -resistant isolates derived from a single, metronidazole-sensitive strain using an H . pylori mouse model . These strains were cultured from the stomachs of mice experimentally infected with H . pylori strain SS1 and then treated orally with metronidazole . The sequence of the rdxA gene of all 10 sequenced metronidazole-sensitive and two (7%) of the 27 metronidazole-resistant isolates was identical to that of the parental strain . In contrast, the rdxA gene of the other 25 metronidazole-resistant isolates contained between one and three frameshift or missense mutations . This suggests that while the development of metronidazole resistance in H . pylori is frequently associated with mutational inactivation of the rdxA gene, other mechanisms of resistance are likely to exist in this bacterium. Appl Biochem Biotechnol, 1999 Spring, 77-79, 511 - 20 Bioconversion of fumarate to succinate using glycerol as a carbon source; Ryu HW et al.; In this study, a facultative bacterium that converts fumarate to succinate at a high yield was isolated . The yield of bioconversion was enhanced about 1.2 times by addition of glucose into culture medium at an initial concentration of 6 g/L . When the initial cell density was high (2 g/L), the succinate produced at pH 7.0 for initial fumarate concentrations of 30, 50, 80, and 100 g/L were 29.3, 40.9, 63.6, and 82.5 g/L, respectively, showing an increase with the initial fumarate concentration . The high yield of 96.8%/mole of fumarate in just 4 h was obtained at the initial fumarate concentration of 30 g/L . Comparing these values to those obtained with low cell culture (0.2 g/L), we found that the amount of succinate produced was similar, but the production rate in the high cell culture was about three times higher than was the case in the low cell culture . This strain converted fumarate to succinate at a rate of 3.5 g/L.h under the sparge of CO2. J Mol Biol, 1999 Jul 23, 290(4), 851 - 8 The reaction center complex from the green sulfur bacterium Chlorobium tepidum: a structural analysis by scanning transmission electron microscopy; Remigy HW et al.; The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution . The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein . Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit . An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes . Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis . Averages of the major classes indicated that different morphologies of the elongated particles (length=19 nm, width=8 nm) resulted from a rotation around the long axis . The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer . A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex . Pathologica, 1999 Feb, 91(1), 18 - 24 {Helicobacter heilmannii: anatomo-clinical study of 14 new cases}; Foschini MP et al.; INTRODUCTION: Helicobacter pylori is one of the most common causes of human gastritis . Recently, a new agent has been isolated, which also causes a gastritis . It has been initially named Gastrospirillum hominis and renamed Helicobacter heilmannii (Hh) . Hh is extremely rare . In spite of the rarity it is important to recognize and diagnose it, as it requires a proper therapy, different from Hp therapy . Clinical presentation and serological results of Hh are superimposable to those of HP . Therefore differential diagnosis resides on histological grounds . PURPOSE of the present paper is to report 14 new cases of Hh gastritis, which constitutes the first italian series . RESULTS: Cases constituted 0.01% of all gastric biopsies seen in the period 1994-1998 . Nine patients were male and five were female; age ranged from 32 to 76 years (50 years on average) . All patients presented a mild to moderate gastritis . Hh is a spiral bacterium, being about 10 micra in length, localized in single or small groups in the glandular mucus . Two cases were associated with Hp . One case was associated with gastric adenocarcinoma . Two cases were diagnosed during the follow-up of duodenal ulcer . In CONCLUSION, the incidence of Hh gastritis in the present series seems consistent with that from other European countries . In all cases the presence of Hh was associated with features of gastritis . This confirms the pathogenetic role of Hh. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 8190 - 5 Discovery of virulence genes of Legionella pneumophila by using signature tagged mutagenesis in a guinea pig pneumonia model; Edelstein PH et al.; Legionella pneumophila is the cause of Legionnaires' disease, which is a form of potentially fatal pneumonia . To identify genes required for virulence of the bacterium, a library of 1,386 L . pneumophila signature tagged transposon mutants was studied for guinea pig virulence . The mutants were screened in pools of 96 each in a guinea pig model of L . pneumophila pneumonia . Sixteen unique mutant clones were determined to have attenuated virulence after being screened twice in the animal model . All 16 mutants failed to multiply in both lungs and spleens . Four of the sixteen had no apparent defect for intracellular multiplication in macrophages . Partial DNA sequences of the interrupted genes adjacent to the transposon insertions showed that six of them had mutations in five known L . pneumophila virulence genes: dotB, dotF/icmG, dotO/icmB, icmX, and proA . Three of the sequenced clones contained mutations in genes without known homology to other published bacterial genes, and seven clones appeared to be homologous to five different known bacterial genes but are still being characterized . With this methodology, we demonstrate the existence of L . pneumophila genes responsible for non-macrophage-related virulence . The discovery of L . pneumophila virulence genes indicates the utility of the signature tagged mutagenesis technique for pulmonary pathogens. Biochim Biophys Acta, 1999 Jun 30, 1412(2), 108 - 17 Oxygen uncouples light absorption by the chlorosome antenna and photosynthetic electron transfer in the green sulfur bacterium chlorobium tepidum Frigaard NU, Matsuura K. In photosynthetic green sulfur bacteria excitation energy is transferred from large bacteriochlorophyll (BChl) c chlorosome antennas via small BChl a antennas to the reaction centers which then transfer electrons from cytochrome c to low-potential iron-sulfur proteins . Under oxidizing conditions a reversible mechanism is activated in the chlorosomes which quenches excited BChl c . We used flash-induced cytochrome c oxidation to investigate the effect of this quenching on photosynthetic electron transfer in whole cells of Chlorobium tepidum . The extent of cytochrome c photooxidation under aerobic conditions decreased to approx . 3% of that under anaerobic conditions when BChl c was excited under light-limiting conditions . Photooxidation obtained by excitation of BChl a was similar under aerobic and anaerobic conditions . We interpret this drastic decrease in energy transfer from BChl c to the reaction center as a consequence of the quenching mechanism which is activated by O2 . This reversible uncoupling of the chlorosome antenna might prevent formation of toxic reactive oxygen species from photosynthetically produced reductants under aerobic conditions . The green filamentous bacterium Chloroflexus aurantiacus also contains chlorosomes but energy transfer from the BChl c and BChl a antennas to the reaction center in this species was not affected by O2. Biophys J, 1999 Jul, 77(1), 597 - 603 Temperature dependence of switching of the bacterial flagellar motor by the protein CheY(13DK106YW); Turner L et al.; The behavior of the bacterium Escherichia coli is controlled by switching of the flagellar rotary motor between the two rotational states, clockwise (CW) and counterclockwise (CCW) . The molecular mechanism for switching remains unknown, but binding of the response regulator CheY-P to the motor component FliM enhances CW rotation . This effect is mimicked by the unphosphorylated double mutant CheY13DK106YW (CheY**) . To learn more about switching, we measured the fraction of time that a motor spends in the CW state (the CW bias) at different concentrations of CheY** and at different temperatures . From the CW bias, we computed the standard free energy change of switching . In the absence of CheY, this free energy change is a linear function of temperature ( . Biophys . J . 71:2227-2233) . In the presence of CheY**, it is nonlinear . However, the data can be fit by models in which binding of each molecule of CheY** shifts the difference in free energy between CW and CCW states by a fixed amount . The shift increases linearly from approximately 0.3kT per molecule at 5 degrees C to approximately 0.9kT at 25 degrees C, where k is Boltzmann's constant and T is 289 Kelvin (= 16 degrees C) . The entropy and enthalpy contributions to this shift are about -0 . 031kT/ degrees C and 0.10kT, respectively. Biophys J, 1999 Jul, 77(1), 424 - 30 Exciton delocalization in the B808-866 antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy; Novoderezhkin V et al.; A model of pigment organization in the B808-866 bacteriochlorophyll a antenna of the green photosynthetic bacterium Chloroflexus aurantiacus based on femtosecond pump-probe studies is proposed . The building block of the antenna was assumed to be structurally similar to that of the B800-850 light-harvesting 2 (LH2) antenna of purple bacteria and to have the form of two concentric rings of N strongly coupled BChl866 pigments and of N/2 weakly coupled BChl808 monomers, where N = 24 or 32 . We have shown that 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 . Using the exciton model, we have obtained a quantitative fit of the pump-probe spectra of the B866 and B808 bands . The anomalously high bleaching value of the B866 band with respect to the B808 monomeric band provided the direct evidence for a high degree of exciton delocalization in the BChl866 ring antenna . The coherence length of the steady-state exciton wave packet corresponds to five or six BChl866 molecules at room temperature. FEBS Lett, 1999 Jun 11, 452(3), 223 - 7 The structure of chromatophores from purple photosynthetic bacteria fused with lipid-impregnated collodion films determined by near-field scanning optical microscopy; Shinkarev VP et al.; Lipid-impregnated collodion (nitrocellulose) films have been frequently used as a fusion substrate in the measurement and analysis of electrogenic activity in biological membranes and proteoliposomes . While the method of fusion of biological membranes or proteoliposomes with such films has found a wide application, little is known about the structures formed after the fusion . Yet, knowledge of this structure is important for the interpretation of the measured electric potential . To characterize structures formed after fusion of membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides with lipid-impregnated collodion films, we used near-field scanning optical microscopy . It is shown here that structures formed from chromatophores on the collodion film can be distinguished from the lipid-impregnated background by measuring the fluorescence originating either from endogenous fluorophores of the chromatophores or from fluorescent dyes trapped inside the chromatophores . The structures formed after fusion of chromatophores to the collodion film look like isolated (or sometimes aggregated, depending on the conditions) blisters, with diameters ranging from 0.3 to 10 microm (average approximately 1 microm) and heights from 0.01 to 1 microm (average approximately 0.03 microm) . These large sizes indicate that the blisters are formed by the fusion of many chromatophores . Results with dyes trapped inside chromatophores reveal that chromatophores fused with lipid-impregnated films retain a distinct internal water phase. FEMS Microbiol Lett, 1999 Jun 15, 175(2), 255 - 60 Green fluorescent protein as a detection marker for Coxiella burnetii transformation; Lukacova M et al.; The molecular biological study of the obligate intracellular bacterium Coxiella burnetii is hampered because of the lack of an efficient DNA transformation system . We used expression of the green fluorescent protein (GFP) in addition to ampicillin resistance as a selection marker for detection of transformed C . burnetii cells . Fluorescent microscopy studies revealed that transformed C . burnetii cells can be detected easily inside the host cell line . A high level of GFP expression was reached with the strong Escherichia coli trc (trp/lac) promoter . The use of GFP not only provides a convenient marker for transformation of C . burnetii, but also allows detection of this obligate intracellular pathogen inside host eukaryotic cells . Possible applications for GFP in the study of host-pathogen interactions are discussed. FEMS Microbiol Lett, 1999 Jun 15, 175(2), 205 - 10 Purification and characterization of triheme cytochrome c7 from the metal-reducing bacterium, Geobacter metallireducens; Afkar E et al.; A soluble c-type cytochrome was first purified from Geobacter metallireducens to an electrophoretically homogeneous state . The purified cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form . Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate allowed us to calculate the molecular mass at 9.5 kDa . It contained 3 mol of heme c per molecule of the protein on the basis of heme c and protein concentration . The mid-point redox potential at pH 7.0 was determined to be -190 mV . Although the N-terminal amino acid sequence of the first 17 residues was similar to that of Desulfuromonas acetoxidans cytochrome c7, G . metallireducens cytochrome c did not show Fe(III)-reducing activity. J Bacteriol, 1999 Jul, 181(13), 3935 - 41 The "green" form I ribulose 1,5-bisphosphate carboxylase/oxygenase from the nonsulfur purple bacterium Rhodobacter capsulatus; Horken KM et al.; Form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of the Calvin-Benson-Bassham cycle may be divided into two broad phylogenetic groups, referred to as red-like and green-like, based on deduced large subunit amino acid sequences . Unlike the form I enzyme from the closely related organism Rhodobacter sphaeroides, the form I RubisCO from R . capsulatus is a member of the green-like group and closely resembles the enzyme from certain chemoautotrophic proteobacteria and cyanobacteria . As the enzymatic properties of this type of RubisCO have not been well studied in a system that offers facile genetic manipulation, we purified the R . capsulatus form I enzyme and determined its basic kinetic properties . The enzyme exhibited an extremely low substrate specificity factor, which is congruent with its previously determined sequence similarity to form I enzymes from chemoautotrophs and cyanobacteria . The enzymological results reported here are thus strongly supportive of the previously suggested horizontal gene transfer that most likely occurred between a green-like RubisCO-containing bacterium and a predecessor to R . capsulatus . Expression results from hybrid and chimeric enzyme plasmid constructs, made with large and small subunit genes from R . capsulatus and R . sphaeroides, also supported the unrelatedness of these two enzymes and were consistent with the recently proposed phylogenetic placement of R . capsulatus form I RubisCO . The R . capsulatus form I enzyme was found to be subject to a time-dependent fallover in activity and possessed a high affinity for CO2, unlike the closely similar cyanobacterial RubisCO, which does not exhibit fallover and possesses an extremely low affinity for CO2 . These latter results suggest definite approaches to elucidate the molecular basis for fallover and CO2 affinity. DNA Res, 1999 Apr 30, 6(2), 109 - 15 Cloning of cellulose synthase genes from Acetobacter xylinum JCM 7664: implication of a novel set of cellulose synthase genes; Umeda Y et al.; Three sets of cellulose synthase genes were cloned from a cellulose-producing bacterium Acetobacter xylinum JCM 7664 . One set of genes (bcsAI/bcsBI/bcsCI/bcsDI) were highly conserved with the well-established type I genes in other strains of A . xylinum, while the other two (bcsABII-A, bcsABII-B) were homologous to the known type II (acsAII) . Unexpectedly, they were immediately followed by a gene cluster of bcsX/bcsY/bcsCII/ORF569, likely forming an operon . Western blotting demonstrated that the BcsY protein accumulated in cells . Since BcsY showed striking similarities to a number of membrane-bound transacylases, it was hypothesized that the type II cellulose synthase produces acylated cellulose, which might be anchored on the cytoplasmic membrane . An insertion sequence of IS1380-type was found just upstream of the one type II gene (bcsABII-B), suggestive of nonfunctioning. Helicobacter, 1999 Jun, 4(2), 77 - 81 Invasiveness of Helicobacter pylori into human gastric mucosa; Ko GH et al.; BACKGROUND: Helicobacter pylori has generally been observed only in the gastric mucous layer or in the spaces between gastric mucus-secreting cells and not in the gastric epithelial cells or in the lamina propria . The purpose of this study is to determine whether H . pylori invades the gastric mucosa, using an immunoelectron microscopical examination of human gastric mucosa infected with H . pylori . MATERIALS AND METHODS: Five hundred gastric antral biopsy specimens were fixed in a periodate-lysin-paraformaldehyde solution, embedded in Lowicryl, sectioned, and examined with a light microscope . One hundred specimens moderately or severely infected with H . pylori were selected and were incubated with polyclonal rabbit anti-H . pylori antibody . The specimens were washed, incubated with 20 nm of colloidal gold-conjugated goat anti-rabbit IgG, stained with uranyl acetate and lead citrate, and observed with a transmission electron microscope . RESULTS: In one case, a bacterium was observed within the cytoplasm of a gastric mucus-secreting cell; in another case, a few bacteria were observed within the cytoplasm of a stromal cell in the lamina propria . The bacteria could be differentiated from degenerated intracellular organelles by gold particles attached to the bacteria . CONCLUSION: H . pylori rarely invade the lamina propria and gastric cells. Biosci Biotechnol Biochem, 1999 May, 63(5), 859 - 65 Cloning of a gene encoding hydroxyquinol 1,2-dioxygenase that catalyzes both intradiol and extradiol ring cleavage of catechol; Murakami S et al.; Two Escherichia coli transformants with catechol 1,2-dioxygenase activity were selected from a gene library of the benzamide-assimilating bacterium Arthrobacter species strain BA-5-17, which produces four catechol 1,2-dioxygenase isozymes . A DNA fragment isolated from one transformant contained a complete open reading frame (ORF) . The deduced amino acid sequence of the ORF shared high identity with hydroxyquinol 1,2-dioxygenase . An enzyme expressed by the ORF was purified to homogeneity and characterized . When hydroxyquinol was used as a substrate, the purified enzyme showed 6.8-fold activity of that for catechol . On the basis of the sequence identity and substrate specificity of the enzyme, we concluded that the ORF encoded hydroxyquinol 1,2-dioxygenase . When catechol was used as a substrate, cis,cis-muconic acid and 2-hydroxymuconic 6-semialdehyde, which were products by the intradiol and extradiol ring cleavage activities, respectively, were produced . These results showed that the hydroxyquinol 1,2-dioxygenase reported here was a novel dioxygenase that catalyzed both the intradiol and extradiol cleavage of catechol. Pac Symp Biocomput . 1999;:65-76. Analysis of the stabilizing effect of Rom on the genetic network controlling ColE1 plasmid replication; Goss PJ et al.; A stochastic model of ColE1 plasmid replication is presented . It is implemented by using UltraSAN, a simulation tool based on an extension of stochastic Petri nets (SPNs) . It allows an exploration of the variation in plasmid number per bacterium, which is not possible using a deterministic model . In particular, the rate at which plasmid-free bacteria arise during bacterial division is explored in some detail since spontaneous plasmid loss is a widely observed empirical phenomenon . The rate of spontaneous plasmid loss provides an evolutionary explanation for the maintainance of Rom protein . The presence of Rom acts to reduce variance in plasmid copy number, thereby reducing the rate of plasmid loss at bacterial division . The ability of stochastic models to link biochemical function with evolutionary considerations is discussed. APMIS, 1999 Jun, 107(6), 566 - 76 An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole; Brorson O et al.; The aim of this study was to examine the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole . Because B . burgdorferi is a microaerobic bacterium like Helicobacter pylori, metronidazole (MZ) was chosen in the susceptibility test . For both microaerobic and aerobic incubation the normal mobile spirochetes were resistant to this antibiotic with an MBC > or = 512 microg/ml . Conversion of mobile spirochetes to cystic forms was not observed when they were incubated with MZ . When they were incubated under microaerobic conditions, the biologically active cystic forms had an MBC > or = 4 microg/ml, but the MBC was > or = 32 microg/ml with aerobic incubation at 37 degrees C . Staining with acridine orange (AO), dark field microscopy (DFM), and transmission electron microscopy (TEM) revealed that the contents of the cysts were degraded when the concentration of MZ was > or = MBC . Some cysts were also ruptured . When incubated with a sufficient concentration of MZ, core structures did not develop inside the cysts, and AO revealed less RNA in the cysts . Our observations may help efforts to treat resistant infections caused by B . burgdorferi with a combination of MZ and other antibiotics in order to eradicate both cystic and mobile forms of B . burgdorferi. FEMS Immunol Med Microbiol, 1999 Jun, 24(2), 169 - 74 Characterization of the respiratory chain of Helicobacter pylori; Chen M et al.; The respiratory chain of Helicobacter pylori has been investigated . The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium . NADPH-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H . pylori . Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase . The activity of succinate-cytochrome c reductase was inhibited by antimycin, implying the presence of a classical pathway from complex II to complex III in this bacterium . The presence of NADH-fumarate reductase (FRD) was demonstrated in H . pylori and fumarate could reduce H2O2 production from NADH, indicating fumarate to be an endogenous substrate for accepting electrons from NADH . The activity of NADH-FRD was inhibited by 2-thenoyltrifluoroacetone . A tentative scheme for the electron transfer pathway in H . pylori is proposed, which may be helpful in clarifying the pathogenesis of H . pylori and in opening new lines for chemotherapy against this bacterium. Parasitol Today, 1999 Jul, 15(7), 295 - 300 Immune responses to Dermatophilus congolensis infections; Ambrose N et al.; Complex mechanisms underly the establishment of dermatophilosis, an exudative and proliferative skin disease of ruminants . This multicomponent system involves the bacterium Dermatophilus congolensis, transmission by various routes including flies, host genetic factors and immunosuppression by Amblyomma variegatum ticks . Here, Nick Ambrose and colleagues summarize recent evidence for an association between A . variegatum and severe chronic dermatophilosis in cattle . Breed-based differences in resistance to dermatophilosis are probably related to immunity to ticks or resistance to the immunosuppressive effects of ticks . Immunity to dermatophilosis might involve non-classic responses mediated by CD1 antigen presentation and gammadelta T cells . Progress towards vaccination is further complicated by strain-specific acquired immunity to D . congolensis. Cancer, 1999 Jun 15, 85(12), 2506 - 11 Patients younger than 40 years with gastric carcinoma: Helicobacter pylori genotype and associated gastritis phenotype; Rugge M et al.; BACKGROUND: In the general population, Helicobacter pylori (H . pylori), particularly the cagA positive strain, has been associated with intestinal-type gastric carcinoma . Gastric carcinomas are rarely observed in patients age < or = 40 years . Host-related factors have been thought to be more important than environmental agents in these early-onset cancers . The aim of this study was to ascertain the possible role of H . pylori infection and that of cagA positive strains in the development of gastric carcinoma in these young patients . METHODS: In this case-control study, 105 gastric carcinoma patients (male-to-female ratio = 1.1; mean age, 34.4 years; range, 16-40 years) and an equal number of controls (matched for gender and age) were retrospectively selected from the same geographic area . The phenotypes of gastritis and H . pylori were histologically assessed, and the presence of the ureC gene, which is indicative of H . pylori infection, and the cagA genotype were determined by polymerase chain reaction . Gastric carcinoma risk was calculated by both univariate and multivariate statistical methods, taking into account the cancer phenotype, the gastritis phenotype detected in both patients and controls, and the H . pylori genotype . RESULTS: For 74 diffuse and 31 intestinal gastric carcinomas, multivariate logistic regression analysis produced results consistent with those of univariate statistical tests, showing a significant association between gastric carcinoma and both H . pylori infection (odds ratio {OR} = 2.79; 95% confidence interval {CI} = 1.52-5.11) and cagA positive status (OR = 2.94; 95% CI = 1.56-5.52) . CONCLUSIONS: In young Italian patients with gastric carcinoma, the significant association with cagA positive H . pylori infection suggests that the bacterium has an etiologic role in both diffuse-type and intestinal-type gastric carcinoma. Microbiology, 1999 May, 145 ( Pt 5), 1235 - 44 Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath; Stolyar S et al.; Genes for the subunits of particulate methane monooxygenase, PmoABC, have been sequenced from the gamma-proteobacterial methanotroph Methylococcus capsulatus Bath . M . capsulatus Bath contains two complete copies of pmoCAB, as well as a third copy of pmoC . The two pmoCAB regions were almost identical at the nucleotide sequence level, differing in only 13 positions in 3183 bp . At the amino acid level, each translated gene product contained only one differing residue in each copy . However, the pmoC3 sequence was more divergent from the two other pmoC copies at both the far N-terminus and far C-terminus . Chromosomal insertion mutations were generated in all seven genes . Null mutants could not be obtained for pmoC3, suggesting that it may play an essential role in growth on methane . Null mutants were obtained for pmoC1, pmoC2, pmoA1, pmoA2, pmoB1 and pmoB2 . All of these mutants grew on methane, demonstrating that both gene copies were functional . Copy 1 mutants showed about two-thirds of the wild-type whole-cell methane oxidation rate, while copy 2 mutants showed only about one-third of the wild-type rate, indicating that both gene copies were necessary for wild-type particulate methane monooxygenase activity . It was not possible to obtain double null mutants that were defective in both pmo copies, which may indicate that some expression of pMMO is important for growth. Microbiology, 1999 May, 145 ( Pt 5), 1217 - 26 gdhB, a gene encoding a second quinoprotein glucose dehydrogenase in Pantoea citrea, is required for pink disease of pineapple; Pujol CJ et al.; The pink disease of pineapple, caused by the bacterium Pantoea citrea, is characterized by a dark coloration on fruit slices after canning . A glucose dehydrogenase (Gdh) encoded by the gdhA gene has been implicated in the colour formation activity of P . citrea . In this paper it has been shown that P . citrea contains a second, homologous gdh gene and its product, GdhB, represents the main source of Gdh activity in this organism . Unlike gdhA, gdhB is constitutively expressed during the exponential phase of growth and is induced in stationary phase . A previously isolated chemical mutant, CMC6, which is deficient in Gdh activity and pink disease formation, failed to express gdhB during the stationary phase of growth . The CMC6 mutant can be complemented by a 54 bp DNA fragment located upstream of gdhA . This fragment, which contains an operator-like 11 bp inverted repeat, strongly enhances the expression of gdhA, probably by titrating away a negative effector of its expression . These results illustrate the complex interplay operating between the two gdh genes and emphasize the role of glucose metabolism in the pathway leading to pink disease. Microb Pathog, 1999 Jul, 27(1), 25 - 41 Complement resistance in Borrelia burgdorferi strain 297: outer membrane proteins prevent MAC formation at lysis susceptible sites; Patarakul K et al.; Two variants of Borrelia burgdorferi strain 297, complement-resistant wild-type (WT297) and complement-sensitive mutant (MUT297), were used as a model to study the mechanism of resistance to the alternative complement pathway in this organism . No difference in the quantity of membrane attack complex (MAC) deposition on WT297 and MUT297 was observed after 2 h incubation with normal human serum (NHS), at which time 4% of WT297 and 95% of MUT297 were killed . The polymerization of C9 bound to WT297 and MUT297 was demonstrated by immunoblotting using an anti-C9 polyclonal antibody . Immunofluorescence and thin-section immunoelectron microscopy showed MAC to be diffusely distributed on the outer membrane of both variants . Furthermore, MAC appeared to be tightly bound to the surface of both variants as demonstrated by elution studies . Protease treatment rendered WT297 susceptible to killing by NHS, suggesting that outer membrane proteins may be associated with complement resistance of WT297 . One- and two-dimensional gel electrophoreses showed that proteins of 20 and 30 kDa, and 66 kDa were present in WT297 but were absent or sparse in trypsin-treated WT297 and MUT297 . Interestingly, immunoblotting using a polyclonal antibody against C3 showed that C3 fragments appeared to bind different acceptors on WT297 than on trypsin-treated WT297, or MUT297 . Therefore, the binding of C3 fragments to acceptors on WT297, in contrast to MUT297, may not direct the formation of the MAC to lysis-susceptible sites on the surface of the bacterium, resulting in the complement resistance of WT297 . Structure Fold Des, 1999 Jan 15, 7(1), 65 - 79 Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods; Dias JM et al.; BACKGROUND: The periplasmic nitrate reductase (NAP) from the sulphate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is induced by growth on nitrate and catalyses the reduction of nitrate to nitrite for respiration . NAP is a molybdenum-containing enzyme with one bis-molybdopterin guanine dinucleotide (MGD) cofactor and one {4Fe-4S} cluster in a single polypeptide chain of 723 amino acid residues . To date, there is no crystal structure of a nitrate reductase . RESULTS: The first crystal structure of a dissimilatory (respiratory) nitrate reductase was determined at 1.9 A resolution by multiwavelength anomalous diffraction (MAD) methods . The structure is folded into four domains with an alpha/beta-type topology and all four domains are involved in cofactor binding . The {4Fe-4S} centre is located near the periphery of the molecule, whereas the MGD cofactor extends across the interior of the molecule interacting with residues from all four domains . The molybdenum atom is located at the bottom of a 15 A deep crevice, and is positioned 12 A from the {4Fe-4S} cluster . The structure of NAP reveals the details of the catalytic molybdenum site, which is coordinated to two MGD cofactors, Cys140, and a water/hydroxo ligand . A facile electron-transfer pathway through bonds connects the molybdenum and the {4Fe-4S} cluster . CONCLUSIONS: The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase . The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity. Clin Ter, 1999 Jan-Feb, 150(1), 67 - 76 {Dyspepsia and Helicobacter pylori}; Carella AM et al.; Since Helicobacter pylori (Hp) was first isolated in 1983, much work has been carried out on the pathogenic effects of this organism . Hp infection is common in humans and currently is the most important etiologic agent in the development of chronic active gastritis, gastric and duodenal ulcers, carcinoma and Malt-lymphoma of the stomach . Moreover Hp infection has also been associated with various extradigestive diseases . At present, a role of Hp infection in dyspepsia is discussed . Dyspepsia is defined by persistence of pain, burning or discomfort localised to the upper abdomen; some authors include in dyspepsia symptoms such as belching, bloating, alitosis, nausea, postprandial repletion, vomiting and regurgitation . In absence of any underlying pathologies, such as peptic ulcer, gastroesophageal reflux, pancreatitis, biliary tract disease or others, dyspepsia is defined as functional or idiopathic dyspepsia . Functional dyspepsia may be distinct in ulcer, reflux or dysmotility-like dyspepsia and unspecified dyspepsia . Hp infection is common in dyspeptic patients and a role of this bacterium has been postulated mostly in ulcer-like dyspepsia . Mechanisms by when Hp induces dyspeptic symptoms are uncertain; bacterial cytotoxins, phlogosis mediators, activity of chronic gastritis Helicobacter-related and host immune response probably play an important role in pathogenesis of functional dyspepsia . However, dyspepsia is not present only in infected patients; therefore other pathogenic factors may be implicated in expression of dyspeptic symptoms in uninfected subjects, such as gastric dysmotility, modifications of gastric output or altered visceral sensibility, psychological factors, gastroesophageal reflux and irritable bowel. J Biol Chem, 1999 Jun 18, 274(25), 17828 - 36 Multiple p44 genes encoding major outer membrane proteins are expressed in the human granulocytic ehrlichiosis agent; Zhi N et al.; Human granulocytic ehrlichiosis (HGE) is caused by infection with an obligatory intracellular bacterium, the HGE agent . We previously cloned a gene encoding HGE agent 44-kDa major outer membrane protein and designated it p44 . In this study, we (i) identified five different mRNAs that are transcribed from p44-homologous genes in the HGE agent cultivated in HL-60 cells; (ii) cloned genes corresponding to the mRNAs from the genomic DNA of the HGE agent; (iii) showed that the genes being expressed were not clustered in the HGE agent genome; (iv) estimated that a minimum copy number of the p44-homologous genes in the genome is 18; (v) detected two different P44-homologous proteins expressed by the HGE agent; and (vi) demonstrated existence of antibodies specific to the two proteins in sera from patients with HGE . These findings showed that p44 multigenes have several active expression sites and the expression is regulated at transcriptional level, suggesting a potentially unique mechanism for generating the diversity in major antigenic outer membrane proteins of the HGE agent . Characterization of p44-homologous genes expressed by the HGE agent in a tissue culture would assist in understanding a role of the p44 multigene family in pathogenesis and immune response in HGE. Curr Opin Struct Biol, 1999 Jun, 9(3), 353 - 7 Peptide nucleic acids as therapeutic agents; Nielsen PE; Peptide nucleic acids (PNAs) have been around for more than seven years and it was hoped, at their introduction, that they would quickly enter the fields of antisense and antigene technology and drug development . Despite their extremely favorable hybridization and stability properties, as well as the encouraging antisense and antigene activity of PNA in cell-free systems, progress has been slow and experiments on cells in culture and in animals have been lacking . Judging from the very promising results published within the past year, however, there is every reason to believe that both PNA antisense and, possibly, PNA antigene research will strongly pick up momentum again . Specifically, it has been demonstrated that certain peptide-PNA conjugates are taken up very efficiently by, at least some, eukaryotic cells and that antisense down regulation of target genes in nerve cells in culture is attainable using such PNA conjugates . Perhaps even more exciting is that antisense-compatible effects have been reported using PNAs injected into the brain of rats . Finally, it has been shown that the bacterium Escherichia coli is susceptible to antisense gene regulation using PNA. Cell Mol Life Sci, 1999 Apr, 55(4), 617 - 38 Glutamate synthase: a complex iron-sulfur flavoprotein; Vanoni MA et al.; Glutamate synthase is a complex iron-sulfur flavoprotein that forms L-glutamate from L-glutamine and 2-oxoglutarate . It participates with glutamine synthetase in ammonia assimilation processes . The known structural and biochemical properties of glutamate synthase from Azospirillum brasilense, a nitrogen-fixing bacterium, will be discussed in comparison to those of the ferredoxin-dependent enzyme from photosynthetic tissues and of the eukaryotic reduced pyridine nucleotide-dependent form of glutamate synthase in order to gain insight into the mechanism of the glutamate synthase reaction . Sequence analyses also revealed that the small subunit of bacterial glutamate synthase may be the prototype of a novel class of flavin adenine dinucleotide- and iron-sulfur-containing oxidoreductase widely used as an enzyme subunit or domain to transfer reducing equivalents from NAD(P)H to an acceptor protein or protein domain. Biochim Biophys Acta, 1999 May 26, 1412(1), 47 - 55 Cytochrome c" from the obligate methylotroph Methylophilus methylotrophus, an unexpected homolog of sphaeroides heme protein from the phototroph Rhodobacter sphaeroides; Klarskov K et al.; The complete primary structure of an unusual soluble cytochrome c isolated from the obligate methylotrophic bacterium Methylophilus methylotrophus has been determined to contain 124 amino acids and to have an average molecular mass of 14293.0 Da . The sequence has two unusual features: firstly, the location of the heme-binding cysteines is far downstream from the N-terminus, namely at positions 49 and 52; secondly, an extra pair of cysteine residues is present near the C-terminus . In both respects, cytochrome c" is similar to the oxygen-binding heme protein SHP from the purple phototrophic bacterium Rhodobacter sphaeroides . In contrast to SHP, cytochrome c" changes from low-spin to high-spin upon reduction, due to dissociation of a sixth heme ligand histidine which is identified as His-95 by analogy to the class I cytochromes c . The distance of His-95 from the heme (41 residues) and the presence of certain consensus residues suggests that cytochrome c" is the second example of a variant class I cytochrome c. Clin Chem Lab Med, 1999 Mar, 37(3), 223 - 9 Polymorphonuclear oxidative burst after Helicobacter pylori water extract stimulation is not influenced by the cytotoxic genotype but indicates infection and gastritis grade; Basso D et al.; H . pylori-associated gastric mucosal inflammation is characterized by the presence of polymorphonuclear (PMN) leukocyte infiltrate, which is more severe when the infecting strain is cagA positive . After appropriate stimuli, such as bacterial products, PMN release large amounts of oxygen derived free radicals and proteases, to kill the bacterium . H . pylori seems to be particularly resistant to the oxidative machinery of PMN, which can in turn damage the host gastric mucosa . We evaluated peripheral PMN oxidative burst response after stimulation with water extracts from cagA positive (WEcagA+) or negative (WEcagA-) H . pylori strains in infected (n=31) and non-infected patients (n=32) in comparison with healthy controls (n=16); the influence of gastric mucosal inflammatory infiltrate and activity grade on PMN oxidative burst were also assessed . PMN oxidative burst was measured by FACS analysis . H . pylori water extracts were obtained from bacterial culture . H . pylori genotype was determined by means of the polymerase chain reaction . The PMN oxidative burst in H . pylori infected patients was significantly higher than that in H . pylori negative or healthy controls, no differences being found when the results following WEcagA+ and WEcagA- stimulation were compared . The difference in PMN oxidative burst obtained after WEcagA- and E . coli (standard stimulus for PMN oxidative burst) stimulation discriminated H . pylori infected from non-infected patients with a sensitivity of 90% and a specificity of 97% . The grade of PMN oxidative burst correlated with PMN infiltration grade of the gastric mucosa . Our findings allow to conclude that PMN oxidative burst activation by H . pyloriWE is species- but not strain-correlated . PMN priming, probably consequent to the action of soluble mediators released by mononuclear cells, makes PMN hyper-responsive to H . pylori products, thus favoring the release in the gastric mucosa of infected patients of large amounts of oxygen-derived free radicals, which are not enough to eliminate the infection, but may contribute to damaging the gastric mucosa itself . Peripheral PMN oxidative burst response to H . pyloriWE might furthermore be of help in diagnosing H . pylori infection. J Bacteriol, 1999 Jun, 181(11), 3452 - 61 A functional 4-hydroxysalicylate/hydroxyquinol degradative pathway gene cluster is linked to the initial dibenzo-p-dioxin pathway genes in Sphingomonas sp . strain RW1; Armengaud J et al.; The bacterium Sphingomonas sp . strain RW1 is able to use dibenzo-p-dioxin, dibenzofuran, and several hydroxylated derivatives as sole sources of carbon and energy . We have determined and analyzed the nucleic acid sequence of a 9,997-bp HindIII fragment downstream of cistrons dxnA1A2, which encode the dioxygenase component of the initial dioxygenase system of the corresponding catabolic pathways . This fragment contains 10 colinear open reading frames (ORFs), apparently organized in one compact operon . The enzymatic activities of some proteins encoded by these genes were analyzed in the strain RW1 and, after hyperexpression, in Escherichia coli . The first three ORFs of the locus, designated dxnC, ORF2, and fdx3, specify a protein with a low homology to bacterial siderophore receptors, a polypeptide representing no significant homology to known proteins, and a putative ferredoxin, respectively . dxnD encodes a 69-kDa phenol monooxygenase-like protein with activity for the turnover of 4-hydroxysalicylate, and dxnE codes for a 37-kDa protein whose sequence and activity are similar to those of known maleylacetate reductases . The following gene, dxnF, encodes a 33-kDa intradiol dioxygenase which efficiently cleaves hydroxyquinol, yielding maleylacetate, the ketoform of 3-hydroxy-cis,cis-muconate . The heteromeric protein encoded by dxnGH is a 3-oxoadipate succinyl coenzyme A (succinyl-CoA) transferase, whereas dxnI specifies a protein exhibiting marked homology to acetyl-CoA acetyltransferases (thiolases) . The last ORF of the sequenced fragment codes for a putative transposase . DxnD, DxnF, DxnE, DxnGH, and DxnI (the activities of most of them have also been detected in strain RW1) thus form a complete 4-hydroxysalicylate/hydroxyquinol degradative pathway . A route for the mineralization of the growth substrates 3-hydroxydibenzofuran and 2-hydroxydibenzo-p-dioxin in Sphingomonas sp . strain RW1 thus suggests itself. J Bacteriol, 1999 Jun, 181(11), 3358 - 67 Maltose metabolism in the hyperthermophilic archaeon Thermococcus litoralis: purification and characterization of key enzymes; Xavier KB et al.; Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis . Maltose was degraded by the concerted action of 4-alpha-glucanotransferase and maltodextrin phosphorylase (MalP) . The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate . Phosphoglucomutase activity was also detected in T . litoralis cell extracts . Glucose derived from the action of 4-alpha-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway . The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an alpha-glucosidase, a p-nitrophenyl-alpha-D-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose . A PNPG-hydrolyzing activity was also detected in T . litoralis, but maltose was not a substrate for this enzyme . The two key enzymes in the pathway for maltose catabolism in T . litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose . The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da) . The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98 degrees C), of 66 U/mg . A Km of 0.46 mM was determined with heptaose as the substrate at 60 degrees C . The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E . coli (13%) . The consensus binding site for pyridoxal 5'-phosphate is conserved in the T . litoralis enzyme. J Bacteriol, 1999 Jun, 181(11), 3321 - 9 Cell cycle expression and transcriptional regulation of DNA topoisomerase IV genes in caulobacter; Ward DV et al.; DNA replication and differentiation are closely coupled during the Caulobacter crescentus cell cycle . We have previously shown that DNA topoisomerase IV (topo IV), which is encoded by the parE and parC genes, is required for chromosomal partitioning, cell division, and differentiation in this bacterium (D . Ward and A . Newton, Mol . Microbiol . 26:897-910, 1997) . We have examined the cell cycle regulation of parE and parC and report here that transcription of these topo IV genes is induced during the swarmer-to-stalked-cell transition when cells prepare for initiation of DNA synthesis . The regulation of parE and parC expression is not strictly coordinated, however . The rate of parE transcription increases ca . 20-fold during the G1-to-S-phase transition and in this respect, its pattern of regulation is similar to those of several other genes required for chromosome duplication . Transcription from the parC promoter, by contrast, is induced only two- to threefold during this cell cycle period . Steady-state ParE levels are also regulated, increasing ca . twofold from low levels in swarmer cells to a maximum immediately prior to cell division, while differences in ParC levels during the cell cycle could not be detected . These results suggest that topo IV activity may be regulated primarily through parE expression . The presumptive promoters of the topo IV genes display striking similarities to, as well as differences from, the consensus promoter recognized by the major Caulobacter sigma factor sigma73 . We also present evidence that a conserved 8-mer sequence motif located in the spacers between the -10 and -35 elements of the parE and parC promoters is required for maximum levels of parE transcription, which raises the possibility that it may function as a positive regulatory element . The pattern of parE transcription and the parE and parC promoter architecture suggest that the topo IV genes belong to a specialized subset of cell cycle-regulated genes required for chromosome replication. Rev Biol Trop, 1998 Sep, 46(3), 829 - 32 {The relation of Helicobacter pylori with dysplasia and stomach neoplasms in Costa Rica}; Miranda M et al.; Occurrence of the bacterium Helicobacter pylori was compared for two Costa Rican sites with contrasting levels of gastric cancer incidence, Poas (incidence 15.13%) and Puriscal (83.53%) . A sample of 185 adults of similar age and sex proportions was studied in each site, using both H . pylori antiserum tests and gastroscopy to collect two biopsies per case . No clear association between H . pylori and gastric cancer was found. J Biol Chem, 1999 Jun 4, 274(23), 16343 - 8 Autophosphorylation, phosphotransfer, and DNA-binding properties of the RegB/RegA two-component regulatory system in Rhodobacter capsulatus; Bird TH et al.; In the purple, photosynthetic bacterium, Rhodobacter capsulatus, the RegB/RegA two-component system is required for activation of several anaerobic processes, such as synthesis of the photosynthetic apparatus and assimilation of CO2 and N2 . It is believed that RegB is an integral membrane histidine kinase that monitors the external environment . Under anaerobic growth conditions, it transduces a signal through phosphorylation of the response regulator, RegA, which then induces target gene expression . We used an in vitro assay to characterize the phosphorylation of wild-type RegA and a mutant variant (RegA*) that is responsible for abnormally high photosynthesis gene expression under both aerobic and anaerobic growth conditions . Phosphorylation assays indicate that phosphorylated RegA* (RegA* approximately P) is much more stable than RegA approximately P, indicating that it may be locked in a conformation that is resistant to dephosphorylation . DNase I footprint assays also indicate that unphosphorylated RegA* has a much higher affinity for specific DNA binding sites than the wild-type protein . Phosphorylation of RegA* increases DNA binding 2 . 5-fold, whereas phosphorylation of RegA increases DNA binding more than 16-fold . Collectively, these results support the hypothesis that RegA* is a constitutively active variant that does not require phosphorylation to assume a structural conformation required to bind DNA. Appl Environ Microbiol, 1999 Jun, 65(6), 2356 - 62 Quantitative immunofluorescence of regulated eps gene expression in single cells of Ralstonia solanacearum; Kang Y et al.; Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes . Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester . In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10(7) cells/ml) . To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells . For R . solanacearum, QIF was used to determine the amount of beta-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele . When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression . QIF analysis of R . solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture . These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R . solanacearum in culture . Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined. Cancer Res, 1999 May 15, 59(10), 2277 - 81 Helicobacter pylori inhibits the G1 to S transition in AGS gastric epithelial cells; Shirin H et al.; Infection with the bacterium Helicobacter pylori is associated epidemiologically with development of gastric cancer . To better understand the role of H . pylori in carcinogenesis, we examined the effects of H . pylori on cell cycle-related events in the AGS gastric cancer cell line . During coculture, wild-type, toxigenic, cagA-positive H . pylori induced both apoptosis and inhibition of cell cycle progression at G1-S in AGS cells . These effects were most apparent in AGS cells synchronized by serum-deprivation and then stimulated to progress through the cell cycle by refeeding . An isogenic cagA-negative mutant H . pylori, produced similar effects . In contrast to changes induced by 5-fluorouracil, the inhibition of cell cycle progression from G1 to S caused by H . pylori was not accompanied by sustained changes in p53 or p21cip1, but was associated with reduced expression of p27kip1 and inhibition of transcriptional activation of the serum-response element of c-fos . Our results indicate that H . pylori inhibits cell cycle progression at G1-S and induces apoptosis, associated with reduced expression of p27kip1 in AGS gastric cancer cells . In vivo, similar effects as a result of H . pylori infection may lead to potentially deleterious compensatory hyperproliferation by nonneoplastic gastric epithelial cells.
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