Microb Ecol, 2004 Jan, 47(1), 68 - 79 Analysis of diversity among 3-chlorobenzoate-degrading strains of Rhodopseudomonas palustris; Oda Y et al.; The phenotypic and genetic characteristics of 14 strains of the purple nonsulfur bacterium Rhodopseudomonas palustris were studied to assess diversity within this species . While all strains had certain phenotypic characteristics in common, including the ability to metabolize benzoate and degrade 2- and 3-chlorobenzoate, there were also significant differences among the strains such as the rate of growth in media containing benzoate as a carbon source . Genetic characterization of the strains revealed there were three divergent lineages in the species . Based on 16S rRNA gene sequences, the 14 strains could be grouped into three distinct clusters (A, B, and C), and this clustering was congruent with that based on gene sequences of form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) . Although BOX-PCR genomic DNA fingerprints of all 14 strains exhibited differences, analysis of the fingerprint images and UPGMA/product-moment analysis of similarities showed there were three groupings that were entirely consistent with clusters based on other characteristics of the strains . Thus, regardless of the method of analysis used, strains in groups A and B consistently clustered together and were separate from those of group C . These results suggest that strains in groups A-B and C represent phylogenetically related clones that have diverged from one another . This indicates that at least three lineages of Rhodopseudomonas palustris exist among the strains included in this study, and that each may be particularly well adapted to a distinct ecological niche. Microb Ecol, 2004 Jan, 47(1), 48 - 58 Predator/prey interaction between Pfiesteria piscicida and Rhodomonas mediated by a marine alpha proteobacterium; Alavi MR; The dinoflagellate Pfiesteria piscicida coexists with bacteria in aquatic environments and as such, may interact with them at the physiological level . This study was designed to investigate the influence of bacteria, present in a clonal culture of Pfiesteria piscicida, on the predator/prey relationship of this dinoflagellate with the alga Rhodomonas . A series of replenishment experiments with bacteria isolated from P . piscicida clonal culture and the bacteria-free P . piscicida derived from the same culture were carried out . In the presence of bacteria, the number of P . piscicida increased significantly when incubated with alga Rhodomonas . This enhanced growth was almost entirely due to the increased consumption rate of Rhodomonas by P . piscicida since in bacteria-free (axenic) cultures Rhodomonas were consumed at significantly reduced rates relative to cultures with bacteria . Subsequent replenishment experiments with individual bacterial isolates showed that a single isolate was responsible for the increased predation rate of P . piscicida . The presence or absence of this specific bacterium determined the outcome of the interaction between P . piscicida and Rhodomonas . Partial sequence analysis of the 16S rDNA of this isolate indicated that it was a novel marine alpha proteobacterium with sequence similarities to a Roseobacter sp . and a bacterium recently isolated from a toxic dinoflagellate Alexandrium sp. J Pathol, 2004 Aug, 203(4), 896 - 903 Gastric B-cell mucosa-associated lymphoid tissue (MALT) lymphoma in an animal model of 'Helicobacter heilmannii' infection; O'Rourke JL et al.; While Helicobacter pylori is accepted as the dominant human gastric bacterial pathogen, a small percentage of human infections have been associated with another organism, commonly referred to as 'Helicobacter heilmannii', which is more prevalent in a range of animal species . This latter bacterium has been seen in association with the full spectrum of human gastric diseases including gastritis, peptic ulceration, and gastric carcinomas, including gastric B-cell mucosa-associated lymphoid tissue (MALT) lymphoma . This study describes an analysis of the pathogenic potential of a number of 'H heilmannii' isolates in an animal model of gastric MALT lymphoma . BALB/c mice were infected with ten different 'H heilmannii' isolates originating from both human and animal hosts . The animals were examined at various time points for up to 28 months after infection . The infected animals initially developed a chronic inflammatory response within 6 months . This histological response increased in severity with the length of infection, with the development of overt lymphoma in some animals 18 months after infection . MALT lymphomas were detected in up to 25% of the infected animals . The prevalence of lymphoma was dependent on the length of infection and the origin of the infecting isolates . A range of other histological features accompanied the lymphocytic infiltration, including invaginations of the gastric epithelium and associated hyperplastic tissue, mucus metaplasia, and a small number of diffuse large B-cell lymphomas . The ability to manipulate experientially the presence of the bacterium in the animal model will allow further studies examining the role of antigen drive in the development of Helicobacter-associated MALT lymphoma . Bull Soc Pathol Exot, 2004 May, 97(2), 95 - 6 {Contribution of gene amplification in Mycobacterium ulcerans detection in exudates and cutaneous biopsies in Côte d'Ivoire}; Ekaza E et al.; Mycobacterium ulcerans skin ulceration is a major issue of public health in Cote d'Ivoire . The diagnosis of M . ulcerans infection is hampered by the slow growth of the bacterium in culture, implying a delay of several weeks before a specific diagnosis can be obtained . In Cote d'Ivoire the diagnosis of Buruli ulcer is almost based on clinical features . During the last decade, many studies have demonstrated the extremely high capacity of PCR for rapidly and specifically detecting bacteria and genes of interest . That ability has revealed PCR as a powerful tool in clinical microbiology studies . In this study we evaluated the M . ulcerans detection in specimens of exudates and biopsies collected from patients clinically suspected of Buruli ulcer and treated in "Raoul Follereau" centre of Manikro in the North-central region of Cote d'Ivoire . The microscopic research of BAAR in 185 swabs loaded with skin lesions collected from these patients showed a positive rate of 14.6% . The PCR detection in 48 h or 72 h of the M . ulcerans IS2404 and IS2606 in the swabs and in the 26 biopsies, from these patients, showed positive rates of 15.7% and 84.6% respectively and in the same samples . These results obtained with PCR detection of M . ulcerans insertions sequences suggest that this technique performed with exudates and biopsy can be used to confirm a routine specific diagnosis of M . ulcerans and early screening of Buruli ulcer in Cote d'Ivoire. J Cell Physiol, 2004 Sep, 200(3), 334 - 42 Effects of Helicobacter pylori infection on cell cycle progression and the expression of cell cycle regulatory proteins; De Luca A et al.; Helicobacter pylori lives in the stomach lumen adhering and specifically interacting with gastric epithelial cells . H . pylori infection can cause a broad range of diseases . Although most infected individuals only develop a chronic inflammation of the stomach, some patients progress to chronic gastritis, duodenal ulceration, or, rarely, cancer . H . pylori is able to send and to receive signals from the gastric epithelium, allowing host and bacteria to become linked in a dynamic equilibrium . Several studies have demonstrated that H . pylori infection induces morphological changes of gastric epithelial cells other than cell proliferation, increase of mitosis and mutations . It has also been demonstrated that H . pylori may predispose to cancer by altering gastric epithelial cell turnover acting specifically on transcription factors . Although H . pylori is able to induce several host responses, it specifically perturbs the delicate balance of those factors that usually help to maintain cell homeostasis . The study of mechanisms of interaction between the bacterium and gastric cells will surely help to prevent the increase and diffusion of malignancies all over the world . FEBS Lett, 2004 Jul 16, 570(1-3), 184 - 8 1-Hydroxy monocyclic carotenoid 3,4-dehydrogenase from a marine bacterium that produces myxol; Teramoto M et al.; A crtD (1-HO carotenoid 3,4-dehydrogenase gene) homolog from marine bacterium strain P99-3 included in the gene cluster for the biosynthesis of myxol (3',4'-didehydro-1',2'-dihydro-beta, psi-carotene-3,1',2'-triol) was functionally identified . The P99-3 CrtD was phylogenetically distant from the other CrtDs . A catalytic feature was its high activity for the monocyclic carotenoid conversion: 1'-HO-torulene (3',4'-didehydro-1',2'-dihydro- beta, psi-caroten-1'-ol) was prominently formed from 1'-HO-gamma-carotene (1',2'-dihydro-beta, psi-caroten-1'-ol) in Escherichia coli with P99-3 CrtD, indicating that this enzyme has been highly adapted to myxol biosynthesis . This unique type of crtD is a valuable tool for obtaining 1'-HO-3',4'-didehydro monocyclic carotenoids in a heterologous carotenoid production system. FEMS Microbiol Lett, 2004 Jul 15, 236(2), 175 - 81 Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306; Choi HP et al.; The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306 . The deduced protein (ALAS) of this gene contained 409 amino acids . The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21 . The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease . The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively . The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA . The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+ . The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis . Removal of this cofactor led to complete loss of the activity . Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP. Biochem Biophys Res Commun, 2004 Aug 6, 320(4), 1112 - 7 Protein recycling is a major component of post-irradiation recovery in Deinococcus radiodurans strain R1; Joshi B et al.; Exposure to 6kGy dose of (60)Co gamma-rays resulted in immediate growth arrest, followed by complete recovery of Deinococcus radiodurans strain R1 cells . Selective degradation and resynthesis of several predicted highly expressed proteins (including major chaperones, key TCA cycle enzymes, and few stress proteins) and several hypothetical proteins marked the lag period, preceding resumption of growth . A major exercise in protein recycling appears to be an integral component of post-irradiation recovery in D . radiodurans and complements the extensive DNA repair, characteristic of this extremely radioresistant bacterium. J Gen Appl Microbiol, 2004 Apr, 50(2), 55 - 63 Molecular characterization and transcriptional regulation of nitrate reductase in a ruminal bacterium, Selenomonas ruminantium; Asanuma N et al.; Nitrate reductase (NaR) of a strain of Selenomonas ruminantium was purified, and the gene encoding NaR (nar) was sequenced . The 6.4 kbp nar gene consisted of narG, H, J, and I in this order . The deduced amino acid sequences of these subunits resembled those of membrane-bound nitrate reductase-A reported for Escherichia coli . It was shown that narG, H, J, and I are transcribed as a single polycistronic message (nar operon) . The level of intracellular nar-mRNA was higher when S . ruminantium was grown with nitrate than when grown without nitrate, suggesting that nar transcription is enhanced by nitrate . The level of nar-mRNA, which was in parallel to the amount of NaR per cellular nitrogen, was suggested to be enhanced in response to the deficiency of energy and electron supply . Therefore, NaR synthesis in S . ruminantium appeared to be regulated at the transcriptional level in response to the availability of energy and electrons . S . ruminantium reduced nitrate and fumarate simultaneously with no significant effect of fumarate on nar transcription . Addition of fumarate stimulated nitrate reduction, which was caused by increased cell growth because of increased acquirement of ATP via electron transport phosphorylation coupled with fumarate reduction. Endoscopy, 2004 Jul, 36(7), 659 - 62 Refractory Whipple's disease with anaemia: first lessons from capsule endoscopy; Fritscher-Ravens A et al.; Whipple's disease is a chronic multisystem disorder caused by infection with the rod-shaped bacterium, Tropheryma whippelii . We report the case of a 65-year-old woman with intestinal Whipple's disease that had been refractory to monotherapy with a number of antibiotics over a 2-year period . The patient then presented with watery diarrhoea, cachexia (body mass index 18 kg/m (2)) and chronic anaemia (haemoglobin 7.6 g/dl) . Wireless capsule endoscopy showed that the disease affected the entire small intestine . Focal occult areas of bleeding were observed in different parts of the jejunum . The capsule's transit time through the small intestine was 2 hours 43 minutes . Capsule endoscopy allows novel insights into the pathophysiology of Whipple's disease. J Clin Microbiol, 2004 Jul, 42(7), 3371 - 3 Prosthetic mitral valve endocarditis due to Ochrobactrum anthropi: case report; Romero Gomez MP et al.; We describe a case of infective endocarditis in a prosthetic mitral valve due to Ochrobactrum anthropi . Although O . anthropi is an emerging pathogen in immunocompromised patients, infections with the bacterium have very rarely been documented in healthy hosts, and endocarditis is rare . To our knowledge, only two cases of O . anthropi endocarditis have been reported in the medical literature. J Clin Microbiol, 2004 Jul, 42(7), 3219 - 24 Immunohistostaining assays for detection of Chlamydia pneumoniae in atherosclerotic arteries indicate cross-reactions with nonchlamydial plaque constituents; Hoymans VY et al.; Detection of Chlamydia pneumoniae antigens in PCR-negative atheromata by immunohistochemistry assays has given rise to controversies regarding a link between the bacterium and atherosclerosis . One hundred ninety-seven human arterial segments removed surgically were examined for C . pneumoniae DNA by conventional PCR with three different primer pairs and by real-time PCR in two different laboratories . No C . pneumoniae DNA was detected . Eighty atherosclerotic lesions were studied by immunohistochemistry assays . Immunoreactivity for C . pneumoniae was frequently present but was not related to the extent of atherosclerosis . Mammary arteries showed immunoreactivity . Serial sections of 17 atheromata were analyzed by Western blotting, histological staining, and UV fluorescence microscopy . Chlamydial proteins were not detected . The sites with positive results by C . pneumoniae immunohistostaining assays precisely matched the sites with autofluorescent ceroid deposits . Immunoblotting and antigenic staining for C . pneumoniae were negative in tests with fetal aortas . The absence of C . pneumoniae DNA in human atherosclerotic lesions, together with negative results for C . pneumoniae proteins by Western blotting analysis, and the perfect matching of C . pneumoniae immunoreactive sites with sites with autofluorescent ceroid deposits suggest a nonspecific reactivity of antichlamydial antibodies with plaque constituents . On the basis of the results of the present study, there are no arguments for an etiologic role of C . pneumoniae in atherosclerosis. J Clin Microbiol, 2004 Jul, 42(7), 2966 - 76 Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7; Kuhne SA et al.; Intimin, Tir, and EspA proteins are expressed by attaching-effacing Escherichia coli, which include enteropathogenic and enterohemorrhagic E . coli pathotypes . EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed intimin docks the bacterium to the translocated Tir . This intimate attachment leads to attaching and effacing lesions . Recombinant forms of these effector proteins from enterohemorrhagic E . coli O157:H7 were produced by using E . coli expression vectors . Binding of intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragment containing amino acid residues 258 to 361 . Recombinant intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced . Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or intimin . Antibodies recognizing different C-terminal epitopes on intimin bound specifically to the gamma intimin of O157:H7 and not to other classes of intimin . Antibodies recognizing EspA from E . coli O157 also recognized the protein from the eae-deficient O157 mutant DM3 and from E . coli O111 . Anti-intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of gamma intimin . The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential. Clin Diagn Lab Immunol, 2004 Jul, 11(4), 752 - 7 Use of bispecific antibodies in molecular velcro assays whose specificity approaches the theoretical limit of immunodetection for Bordetella pertussis; Tang XL et al.; A bispecific monoclonal antibody (bsMAb) that detects Bordetella pertussis, the causative agent of whooping cough, and horseradish peroxidase (HRPO) has been developed by use of the quadroma technology . A quadroma, P123, was produced by fusing two well-characterized hybridomas against the bacterium and the enzyme and was subcloned to obtain a stable bsMAb-secreting cell line . The quadroma was theoretically expected to produce up to 10 different molecular species of immunoglobulins, so secreted bispecific antibody was complexed with excess HRPO and the HRPO-bsMAb complex was purified in one step by benzhydroxamic acid-agarose affinity cochromatography . An ultrasensitive homosandwich molecular "velcro" enzyme-linked immunosorbent assay for the detection of B . pertussis whole bacteria with HRPO-bsMAb was established in both microplate and nasopharyngeal swab formats . This assay demonstrates a high sensitivity that approaches the theoretical limit of detection of one bacterium . This new nanoprobe can be used to develop a new generation of assays that are simple, inexpensive alternatives to quantitative PCR and that can be used by clinical laboratories . This strategy of homosandwich assays with solid-phase monospecific antibodies and solution-phase bsMAb with specificity for the same repeating surface determinants can be applied to generate ultrasensitive immunodiagnostic assays for viruses and bacteria. Lett Appl Microbiol, 2004, 39(2), 181 - 6 Characterization of a protease of a feather-degrading Microbacterium species; Thys RC et al.; AIMS: To characterize a new feather-degrading bacterium . METHODS AND RESULTS: The strain kr10 producing a high keratinolytic activity when cultured on native feather broth was identified as Microbacterium sp., based on phenotypical characteristics and 16S rDNA sequence . The bacterium presented optimum growth and feather-degrading activity at pH 7.0 and 30 degrees C . Complete feather degradation was achieved during cultivation . The keratinase was partially purified by gel filtration chromatography . It was optimally active at pH 7.0 and 55 degrees C . The enzyme was inhibited by 1,10-phenanthroline, EDTA, p-chloromercuribenzoic acid, 2-mercaptoethanol and metal ions like Hg(2+), Cu(2+) and Zn(2+) . SIGNIFICANCE AND IMPACT OF THE STUDY: A new Microbacterium sp . strain was characterized presenting high feather-degrading activity, which appears to be associated to a metalloprotease-type keratinase . This micro-organism has enormous potential for use in biotechnological processes involving keratin hydrolysis. Appl Environ Microbiol, 2004 Jul, 70(7), 4177 - 86 Chloromethane-dependent expression of the cmu gene cluster of Hyphomicrobium chloromethanicum; Borodina E et al.; The methylotrophic bacterium Hyphomicrobium chloromethanicum CM2 can utilize chloromethane (CH(3)Cl) as the sole carbon and energy source . Previously genes cmuB, cmuC, cmuA, and folD were shown to be essential for the growth of Methylobacterium chloromethanicum on CH(3)Cl . These CH(3)Cl-specific genes were subsequently detected in H . chloromethanicum . Transposon and marker exchange mutagenesis studies were carried out to identify the genes essential for CH(3)Cl metabolism in H . chloromethanicum . New developments in genetic manipulation of Hyphomicrobium are presented in this study . An electroporation protocol has been optimized and successfully applied for transformation of mutagenesis plasmids into H . chloromethanicum to generate stable CH(3)Cl-negative mutants . Both transposon and marker exchange mutageneses were highly applicable for genetic analysis of Hyphomicrobium . A reliable and reproducible selection procedure for screening of CH(3)Cl utilization-negative mutants has also been developed . Mutational inactivation of cmuB, cmuC, or hutI resulted in strains that were unable to utilize CH(3)Cl or to express the CH(3)Cl-dependent polypeptide CmuA . Reverse transcription-PCR analysis indicated that cmuB, cmuC, cmuA, fmdB, paaE, hutI, and metF formed a single cmuBCA-metF operon and were coregulated and coexpressed in H . chloromethanicum . This finding led to the conclusion that, in cmuB and cmuC mutants, impaired expression of cmuA was likely to be due to a polar effect of the defective gene (cmuB or cmuC) located upstream (5') of cmuA . The detrimental effect of mutation in hutI on the upstream (5')-located cmuA is not clear but indicated that all the genes located within the cmuBCA-metF operon are coordinately expressed . Expression of the cmuBCA-metF transcript was also shown to be strictly CH(3)Cl inducible and was not repressed by the alternative C(1) substrate methanol . Sequence analysis of a transposon mutant (D20) led to the discovery of the previously undetected hutI and metF genes located 3' of the paaE gene in H . chloromethanicum . MetF, a putative methylene-tetrahydrofolate reductase, had 27% identity to MetF from M . chloromethanicum . Mutational and transcriptional analysis data indicated that, in H . chloromethanicum, CH(3)Cl is metabolized via a corrinoid-specific (cmuA) and tetrahydrofolate-dependent (metF, purU, folD) methyltransfer system. Appl Environ Microbiol, 2004 Jul, 70(7), 4096 - 102 Replication of the endosymbiotic bacterium Blochmannia floridanus is correlated with the developmental and reproductive stages of its ant host; Wolschin F et al.; The dynamics of replication of the intracellular endosymbiotic bacterium Blochmannia floridanus was determined during the larval development of its host ant Camponotus floridanus by real-time quantitative PCR . The bacteria were found to proliferate during pupation and immediately after the eclosion of the imagines (adult ants) . In older workers the number of bacteria present in the midgut bacteriocytes decreased significantly . In contrast, the bacterial population in the ovaries was dependent on the reproductive state of the animal . An age-dependent degeneration of the midgut bacteriocytes was also investigated by microscopic techniques in males and female castes of the closely related ant species C . herculeanus and C . sericeiventris, respectively, with similar results and supports the concept of age-dependent degeneration of the midgut bacteriocytes in all castes. Cell Microbiol, 2004 Aug, 6(8), 743 - 51 Anaplasma phagocytophilum AnkA binds to granulocyte DNA and nuclear proteins; Park J et al.; Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum . The bacterium infects, survives, propagates in, and alters neutrophil phenotype, indicating unique survival mechanisms . AnkA is the only known A . phagocytophilum component that gains access beyond neutrophil vacuoles and is transported to the infected host cell nucleus . The ability of native and recombinant AnkA to bind DNA and nuclear proteins from host HL-60 cells was assessed by the use of immunoprecipitation after cis-diamminedichloroplatinum (cis-DDP) DNA-protein crosslinking, by probing uninfected HL-60 cell nuclear lysates for AnkA binding, and by recovery and sequence analysis of immunoprecipitated DNA . AnkA binds HL-60 cell DNA as well as nuclear proteins of approximately 86, 53 and 25 kDa, whereas recombinant A . phagocytophilum Msp2 or control proteins do not . DNA immunoprecipitation reveals AnkA binding to a variety of target genes in the human genome, including genes that encode proteins with ATPase, tyrosine phosphatase and NADH dehydrogenase-like functions . These data indicate that AnkA could exert some effect on cells through binding to protein:DNA complexes in neutrophil nuclei . Whether AnkA binding leads to neutrophil functional alterations, and how such alterations might occur will depend upon definitive identification of binding partners and associated metabolic and biochemical pathways. APMIS, 2004 Apr-May, 112(4-5), 239 - 47 Detection of Anaplasma phagocytophilum in animals by real-time polymerase chain reaction; Hulinska D et al.; The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animals and to identify the phylogenetic relationships of different strains of this bacterium . We adapted six published conventional methods targeting 16S fragments for real-time polymerase chain reaction . Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed with several different primers and a TaqMan probe . We also performed DNA quantification and melting curve analysis . The nucleic acid of Anaplasma sp . was detected in a higher percentage of cases in members of the deer family, hares, bank voles and mice (12.5 approximately 15%) than in foxes, boars, cows, and horses (around 4 approximately 6%) . We also performed blood analysis of cows, horses, mice, and ticks removed from animals, evaluating the presence of antibodies against granulocytic Anaplasma sp . Finally, we subjected 11 randomly selected PCR amplified products to direct sequencing and we constructed the corresponding phylogenetic tree with respect to the Ehrlichia equi sequence, homologous to the human granulocytic ehrlichiosis agent . Mutual identity of the sequencing ranged from 99% to 100%. Lakartidningen, 2004 Jun 3, 101(23), 2014 - 5 {Helicobacter pylori can in rare cases be the cause of iron and vitamin B 12 deficiency . No increased risk of iron and vitamin B 12 deficiency due to proton pump inhibitors}; Mattsson N et al.; This article reviews iron and vitamin B12 malabsorption due to the use of proton pump inhibitors (PPI) and infection with Helicobacter pylori . The bacterium is in some studies associated with low serum values of both ferritin and cobalamin and has in several cases been shown to cause reversible deficiency of these nutrients . PPI depresses absorption of vitamin B12, but only one case of deficiency has been reported in standard reflux therapy . Case reports exist of PPI-related iron deficiency, but studies have not confirmed these risks . General substitution with iron or B12 supplements in PPI therapy can't be advocated . The safety of long-term use of PPI is well documented, but it is still unclear whether PPI accelerates the development of atrophic corpus gastritis in the presence of H pylori. J Bacteriol, 2004 Jul, 186(14), 4748 - 58 Construction and validation of the Rhodobacter sphaeroides 2.4.1 DNA microarray: transcriptome flexibility at diverse growth modes; Pappas CT et al.; A high-density oligonucleotide DNA microarray, a genechip, representing the 4.6-Mb genome of the facultative phototrophic proteobacterium, Rhodobacter sphaeroides 2.4.1, was custom-designed and manufactured by Affymetrix, Santa Clara, Calif . The genechip contains probe sets for 4,292 open reading frames (ORFs), 47 rRNA and tRNA genes, and 394 intergenic regions . The probe set sequences were derived from the genome annotation generated by Oak Ridge National Laboratory after extensive revision, which was based primarily upon codon usage characteristic of this GC-rich bacterium . As a result of the revision, numerous missing ORFs were uncovered, nonexistent ORFs were deleted, and misidentified start codons were corrected . To evaluate R . sphaeroides transcriptome flexibility, expression profiles for three diverse growth modes--aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis--were generated . Expression levels of one-fifth to one-third of the R . sphaeroides ORFs were significantly different in cells under any two growth modes . Pathways involved in energy generation and redox balance maintenance under three growth modes were reconstructed . Expression patterns of genes involved in these pathways mirrored known functional changes, suggesting that massive changes in gene expression are the major means used by R . sphaeroides in adaptation to diverse conditions . Differential expression was observed for genes encoding putative new participants in these pathways (additional photosystem genes, duplicate NADH dehydrogenase, ATP synthases), whose functionality has yet to be investigated . The DNA microarray data correlated well with data derived from quantitative reverse transcription-PCR, as well as with data from the literature, thus validating the R . sphaeroides genechip as a powerful and reliable tool for studying unprecedented metabolic versatility of this bacterium . Adv Exp Med Biol, 2004, 547, 31 - 46 Genome function--a virus-world view; Yin J; By studying viruses one may begin to understand how static genomes can define dynamic processes of development . This talk will describe some of the approaches we are taking, using computer simulations and laboratory experiments, to account for the many molecular-level processes and interactions that occur when a common bacterium, E . coli, is infected by one of its viruses, phage T7 . We accounted for processes of phage genome entry, transcription, translation, and DNA replication, including protein-DNA and protein-protein regulatory interactions, and we predicted the dynamics of phage progeny formation . The simulations have enabled us to identify limiting host-cell resources in phage growth, discover novel anti-viral strategies, and suggest frameworks for mining data from global mRNA and protein studies. Mol Microbiol, 2004 Jul, 53(1), 167 - 82 Functional consequences of single:double ring transitions in chaperonins: life in the cold; Ferrer M et al.; The cpn60 and cpn10 genes from psychrophilic bacterium, Oleispira antarctica RB8, showed a positive effect in Escherichia coli growth at low temperature, shifting its theoretical minimal growth temperature from +7.5 degrees C to -13.7 degrees C {Ferrer, M., Chernikova, T.N., Yakimov, M., Golyshin, P.N., and Timmis, K.N . (2003) Nature Biotechnol 21: 1266-1267} . To provide experimental support for this finding, Cpn60 and 10 were overproduced in E . coli and purified to apparent homogeneity . Recombinant O.Cpn60 was identical to the native protein based on tetradecameric structure, and it dissociates during native PAGE . Gel filtration and native PAGE revealed that, in vivo and in vitro, (O.Cpn60)(7) was the active oligomer at 4-10 degrees C, whereas at > 10 degrees C, this complex was converted to (O.Cpn60)(14) . The dissociation reduces the ATP consumption (energy-saving mechanism) and increases the refolding capacity at low temperatures . In order for this transition to occur, we demonstrated that K468 and S471 may play a key role in conforming the more advantageous oligomeric state in O.Cpn60 . We have proved this hypothesis by showing that single and double mutations in K468 and S471 for T and G, as in E.GroEL, produced a more stable double-ring oligomer . The optimum temperature for ATPase and chaperone activity for the wild-type chaperonin was 24-28 degrees C and 4-18 degrees C, whereas that for the mutants was 45-55 degrees C and 14-36 degrees C respectively . The temperature inducing unfolding (T(M)) increased from 45 degrees C to more than 65 degrees C . In contrast, a single ring mutant, O.Cpn60(SR), with three amino acid substitutions (E461A, S463A and V464A) was as stable as the wild type but possessed refolding activity below 10 degrees C . Above 10 degrees C, this complex lost refolding capacity to the detriment of the double ring, which was not an efficient chaperone at 4 degrees C as the single ring variant . We demonstrated that expression of O.Cpn60(WT) and O.Cpn60(SR) leads to a higher growth of E . coli at 4 degrees C ( micro (max), 0.22 and 0.36 h(-1) respectively), whereas at 10-15 degrees C, only E . coli cells expressing O.Cpn60 or O.Cpn60(DR) grew better than parental cells (-cpn) . These results clearly indicate that the single-to-double ring transition in Oleispira chaperonin is a wild-type mechanism for its thermal acclimation . Although previous studies have also reported single-to-double ring transitions under many circumstances, this is the first clear indication that single-ring chaperonins are necessary to support growth when the temperature falls from 37 degrees C to 4 degrees C. Mol Microbiol, 2004 Jul, 53(1), 9 - 18 The impact of prophages on bacterial chromosomes; Canchaya C et al.; Prophages were automatically localized in sequenced bacterial genomes by a simple semantic script leading to the identification of 190 prophages in 115 investigated genomes . The distribution of prophages with respect to presence or absence in a given bacterial species, the location and orientation of the prophages on the replichore was not homogeneous . In bacterial pathogens, prophages are particularly prominent . They frequently encoded virulence genes and were major contributors to the genetic individuality of the strains . However, some commensal and free-living bacteria also showed prominent prophage contributions to the bacterial genomes . Lysogens containing multiple sequence-related prophages can experience rearrangements of the bacterial genome across prophages, leading to prophages with new gene constellations . Transfer RNA genes are the preferred chromosomal integration sites, and a number of prophages also carry tRNA genes . Prophage integration into protein coding sequences can lead to either gene disruption or new proteins . The phage repressor, immunity and lysogenic conversion genes are frequently transcribed from the prophage . The expression of the latter is sometimes integrated into control circuits linking prophages, the lysogenic bacterium and its animal host . Prophages are apparently as easily acquired as they are lost from the bacterial chromosome . Fixation of prophage genes seems to be restricted to those with functions that have been co-opted by the bacterial host. Parasite Immunol, 2004 Feb, 26(2), 95 - 103 Analysis of Ehrlichia ruminantium-specific T1/T2 responses during vaccination with a protective killed vaccine and challenge of goats; Esteves I et al.; Ehrlichia ruminantium is an obligate intracellular bacterium that causes heartwater in ruminants and for which T-cell-mediated immunity is believed to play an important role in protection . To better characterize protective cellular immunity, E . ruminantium-specific IFN-gamma and IL-4 recall responses in major T-cell subsets were analysed by flow cytometry during immunization of goats with a killed vaccine and following a virulent challenge . The killed vaccine elicited both CD8+ and CD4+ subsets to produce cytoplasmic IFN-gamma in the absence of IL-4, thus indicating a biased T1 response . The relative capacity of CD8+ T-cells to produce IFN-gamma was significantly higher than CD4+ T-cells but the final contribution of both subsets was comparable . Circulating ER-specific CD4 and CD8 effectors substantially decreased in numbers after the booster injection and could not be detected in most animals during challenge, which warrants further investigation in immune compartments other than blood . Since IFN-gamma inhibits the growth of the pathogen in target cells, the information provided in this study on E . ruminantium-specific T1 responses will be valuable to develop cellular tools for the identification of potential protective antigens. Environ Sci Technol, 2004 Jun 1, 38(11), 3063 - 7 Abiotic transformation of toxaphene by superreduced vitamin B12 and dicyanocobinamide; Ruppe S et al.; Toxaphene is a complex organochlorine pesticide mixture, residues of which are widespread in the environment . Previous studies with the isolated bacterium Sulfurospirillum (formerly Dehalospirillum) multivorans resulted in an effective anaerobic biotransformation of toxaphene . Since the bacterium contains a corrinoid derivative in the active center of the tetrachloroethene dehalogenase, we attempted to use superreduced corrinoids for abiotic transformation of toxaphene . The two corrinoids studied were dicyanocobinamide and cyanocobalamin (vitamin B12) . Superreduced dicyanocobinamide mediated a rapid transformation of toxaphene . More than 90% of the initial pool was transformed within 6 h . The transformation was nonselective, and even the most persistent metabolite in environmental samples, the so-called dead-end metabolite 2-exo,3-endo,6-exo,8,9,10-hexachlorobornane (B6-923 or Hx-Sed) was transformed within hours . Superreduced cyanocobalamin was also able to transform toxaphene albeit at significantly lower velocity . The lack of transformation products detectable in gas chromatograms of hexanes-extracted fractions of the assays suggests rapid, sequential dehalogenation and/or destruction of the C10-hydrocarbon backbone of the compounds of technical toxaphene. Med Res Rev, 2004 Sep, 24(5), 621 - 38 Bionanotechnology based on silica nanoparticles; Tan W et al.; We have developed uniform core/shell nanoparticles, consisting of a silica layer coating and pigments or magnetite core, using a water-in-oil microemulsion method . The nanoparticles are highly luminescent and photostable with the size ranging from 5 nm to 400 nm . Bioconjugation of these silica nanoparticles adds unique biofunctions with various molecules such as enzymes, antibodies, and DNA molecules . Significant advantages have been shown in using bioconjugated nanoparticles for biosensing and bioimaging, such as cell staining, DNA detection and separation, rapid single bacterium detection, and biotechnological application in DNA protection . Proteomics, 2004 Jul, 4(7), 1859 - 72 Sinorhizobium meliloti metabolism in the root nodule: a proteomic perspective; Djordjevic MA; The proteome of the model symbiotic bacterium, Sinorhizobium meliloti was examined to determine the enzymatic reactions and cell processes that occur when S . meliloti occupies the root nodules of Medicago truncatula and Melilotus alba . The proteomes of the nodule bacteria were compared to that of S . meliloti grown under laboratory cultured conditions as an additional control . All the detectable protein spots on the two-dimensional (2-D) gels between pH 4-7 were analyzed . In total, the identity of proteins in 1545 spots from 2-D gels was determined using peptide mass fingerprinting . There were clear differences in the proteome of nodule bacteria and cultured bacteria and putative nodule-specific and nodule suppressed proteins were identified . The data were analyzed using metabolic pathway prediction programs and used to review the biochemical and genetic studies that had been done previously on S . meliloti over several decades . There was a broad congruency between the proteomic and biochemical data when the overall pathways of central carbon and nitrogen metabolism were considered . A selective suite of ABC-type transporters was present in nodule bacteria that were biased towards the transport of amino acids and inorganic ions (P and Fe) suggesting that a highly specialized nutrient exchange was occurring between the nodule bacteria and the host . Proteins prominent in nodule bacteria were those involved in the pathways for vitamin synthesis and stress-related processes (chaperoning, heat shock, detoxification of reactive oxygen species, regulation of stress and osmo-regulation) . Some of these proteins were found only in nodule bacteria . These results show the extent of the shift in metabolism that occurs when S . meliloti invades legume plants and establishes a nitrogen fixing symbiosis. Carbohydr Res, 2004 Jul 12, 339(10), 1813 - 6 Structure of the O-polysaccharide of the lipopolysaccharide of Azospirillum irakense KBC1; Fedonenko YP et al.; The O-polysaccharide was isolated from the lipopolysaccharide of the plant-growth-promoting bacterium Azospirillum irakense KBC1 and studied by sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy, including 1H, 13C HSQC and NOESY experiments for linkage and sequence analysis . The following structure of the branched hexasaccharide repeating unit of the O-polysaccharide with an unusually long side chain was established: {carbohydrate structure: see text}. Adv Biochem Eng Biotechnol, 2004, 89, 1 - 45 Molecular components of physiological stress responses in Escherichia coli; Wick LM et al.; In order to survive under and adapt to different conditions Escherichia coli has evolved elaborate systems that are able to sense and respond to environmental stimuli . Very often, different stresses act on a bacterium simultaneously and a variety of stresses have similar effects on cellular molecules and processes . Therefore, the various stress response systems have to interact (cross talk) with each other . A complex network of global regulatory systems with a multitude of molecular components ensures a coordinated and effective answer . Such regulatory components include DNA, mRNAs, sRNAs, proteins, such as DNA-and RNA binding proteins, alternative sigma factors and two-component systems, as well as small molecular weight molecules, as for example (p)ppGpp . These regulatory systems govern the expression of a plethora of further effectors that aim at maintaining stability of the cellular equilibrium under the various conditions . Using five of the most important stress response systems, we will discuss the roles and mechanisms of such regulatory and effector molecules in more detail . The heat shock response, controlled by the sigma factor sigma32, and the envelope stress response, controlled by the sigma factor sigmaE and the Cpx two-component system, both result in an increased expression of chaperones and proteases in response to misfolded proteins . The cold shock response governs expression of RNA chaperones and ribosomal factors, ensuring accurate translation at low temperatures . The general stress response depends on the sigma factor sigmaS, which controls the expression of more than 50 genes conferring resistance to many different stresses . The (p)ppGpp-dependent stringent response reduces the cellular protein synthesis capacity and controls further global responses upon nutritional downshift . A lot has been learned in recent years about the mechanisms of action of single components . However, the main challenge for the future is to achieve an understanding of the interactions of these components under different physiological conditions. Cryo Letters, 2004 May-Jun, 25(3), 195 - 204 Purification and characterization of uridine phosphorylase from the ice-nucleating bacterium, Pantoea agglomerans NBRC12686; Obata H et al.; The ice-nucleating bacterium, Pantoea agglomerans NBRC12686 responds to a decrease in temperature with the induction of proteins, which are classified as cold-induced proteins . When the temperature of the strain NBRC12686 culture was lowered from 30 degree C to 12 degree C, the viability after freezing treatment significantly improved . By the use of SDS-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC), we analyzed the cold acclimation response in strain NBRC12686 . After a shift from 30 degree C to 12 degree C, several proteins and saccharides were synthesized . After 48 h of cold acclimation, the induction level of proteins increased . In addition, ribose-1-phosphate was fractionated by HPLC using a TSK gel Sugar AXG column . Cell-free extracts were prepared from a cold acclimation culture (30 degree C to 12 degree C) and a non-cold acclimation culture (30 degree C), and then subjected to SDS-PAGE . A protein of approximately 29.7-kDa was present in the cold acclimation culture but was not present in the non-cold acclimation culture . The 29.7-kDa protein was purified by various chromatographies . We found that apparent molecular mass of the protein was approximately 119-kD constructed of 4 subunits of 29.7-kDa each . Based on the analysis of the N-terminal amino acid sequences of proteins, the 29.7-kDa protein had 83 percent identity with that of uridine phosphorylase (UPase) obtained from Escherichia coli K-12 . We confirmed that the 29.7-kDa protein was novel, judged by molecular mass different from the already-known UPase or cryoprotectants . The cryoprotective activity of UPase of 29.7-kDa protein for LDH was approximately 30 percent at 5.0 microgram per ml of the protein . Furthermore, UPase had a high level of cryoprotective activity even after treating at 70 degree C for 30 min, but had no activity after treating at 100 degree C . We could elucidate that UPase from strain NBRC12686 had a cryoprotective activity as well as an enzyme activity, and it seems that UPase works in two different mechanisms for freezing tolerance. Exp Mol Pathol, 2004 Aug, 77(1), 49 - 56 Linking chronic wasting disease to scrapie by comparison of Spiroplasma mirum ribosomal DNA sequences; Bastian FO et al.; Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative diseases of man and animals and are transmitted by a filterable pathogen whose identity is currently unresolved . Our data indicates that Spiroplasma, a wall-less bacterium, is involved in the pathogenesis of TSE . We searched for Spiroplasma ribosomal gene sequences in 10 scrapie-infected sheep brains and 10 normal sheep brains, 7 cervid samples infected with chronic wasting disease (CWD), and 7 normal cervid brains . DNA was extracted from these tissue samples and amplified by polymerase chain reaction (PCR) using primers specific for Spiroplasma-specific 16S rDNA . Specificity of the amplicon was determined by Southern blotting and DNA sequence analyses . Spiroplasma 16S rDNA was found in 8 of 10 scrapie-infected sheep brains and 6 of 7 CWD-infected tissue samples . All normal animal brain samples were negative . Spiroplasma 16S rDNA was also found in two human Creutzfeldt-Jakob diseased (CJD) brains but not in two age-matched normal human brains . DNA sequence analyses of the amplified PCR products from human and animal TSE cases revealed greater than 99% nucleotide sequence homology with Spiroplasma mirum . The presence of Spiroplasma DNA in TSE-infected tissues supports our hypothesis that Spiroplasma may be involved in the pathogenesis of these diseases. Clin Microbiol Infect, 2004 Jul, 10(7), 598 - 614 Lyme borreliosis: from infection to autoimmunity; Singh SK et al.; Lyme borreliosis in humans is an inflammatory disease affecting multiple organ systems, including the nervous system, cardiovascular system, joints and muscles . The causative agent, the spirochaete Borrelia burgdorferi, is transmitted to the host by a tick bite . The pathogenesis of the disease in its early stages is associated largely with the presence of viable bacteria at the site of inflammation, whereas in the later stages of disease, autoimmune features seem to contribute significantly . In addition, it has been suggested that chronic persistence of B . burgdorferi in affected tissues is of pathogenic relevance . Long-term exposure of the host immune system to spirochaetes and/or borrelial compounds may induce chronic autoimmune disease . The study of bacterium-host interactions has revealed a variety of proinflammatory and also immunomodulatory-immunosuppressive features caused by the pathogen . Therapeutic strategies using antibiotics are generally successful, but chronic disease may require immunosuppressive treatment . Effective and safe vaccines using recombinant outer surface protein A have been developed, but have not been propagated because of fears that autoimmunity might be induced . Nevertheless, new insights into the modes of transmission of B . burgdorferi to the warm-blooded host have been generated by studying the action of these vaccines. Lett Appl Microbiol, 2004, 38(4), 333 - 8 Bacteriophages that infect the cellulolytic ruminal bacterium Ruminococcus albus AR67; Klieve AV et al.; AIM: To isolate bacterial viruses that infect the ruminal cellulolytic bacterium Ruminococcus albus . METHODS: Four phages infecting R . albus AR67 were isolated under anaerobic conditions using the soft-agar overlay technique . The phages were characterized on morphology, solvent stability, nucleic acid type and digestion characteristics . Two phages, phiRa02 and phiRa04 comprised icosahedral virions with linear double-stranded DNA and appeared to belong to the family Tectiviridae . The other two phages are most likely filamentous phages with circular single-stranded DNA of the family Inoviridae . SIGNIFICANCE OF THE STUDY: Viruses of the families Tectiviridae and Inoviridae have not previously been isolated from rumen bacteria . The phages isolated in this study are the first phages shown to infect the cellulolytic bacteria of the rumen . This suggests that the cellulolytic populations of the rumen are subject to lytic events that may impact on the ability of these bacteria to degrade plant fibre and on the nutrition of the animal. Syst Appl Microbiol, 2004 May, 27(3), 290 - 300 Xylella fastidiosa subspecies: X . fastidiosa subsp piercei, subsp . nov., X . fastidiosa subsp . multiplex subsp . nov., and X . fastidiosa subsp . pauca subsp . nov; Schaad NW et al.; Xylella fastidiosa, a fastidious bacterium causing disease in over 100 plant species, is classified as a single species, although genetic studies support multiple taxons . To determine the taxonomic relatedness among strains of X . fastidiosa, we conducted DNA-DNA relatedness assays and sequenced the 16S-23S intergenic spacer (ITS) region using 26 strains from 10 hosts . Under stringent conditions (Tm -15 degrees C), the DNA relatedness for most X . fastidiosa strains was *70% . However, at high stringency (Tm -8 degrees C), three distinct genotypes (A, B, and C) were revealed . Taxon A included strains from cultivated grape, alfalfa, almond (two), and maple, interrelated by 85% (mean); taxon B included strains from peach, elm, plum, pigeon grape, sycamore, and almond (one), interrelated by 84%; and taxon C included only strains from citrus, interrelated by 87% . The mean reciprocal relatedness between taxons A and B, A and C, and B and C, were 58, 41, and 45%, respectively . ITS results also indicated the same grouping; taxons A and B, A and C, and B and C had identities of 98.7, 97.9, and 99.2%, respectively . Previous and present phenotypic data supports the molecular data . Taxon A strains grow faster on Pierce's disease agar medium whereas B and C strains grow more slowly . Taxon B and C strains are susceptible to penicillin and resistant to carbenicillin whereas A strains are opposite . Each taxon can be differentiated serologically as well as by structural proteins . We propose taxons A, B, and C be named X . fastidiosa subsp . piercei, subsp . nov, subsp . multiplex, subsp . nov., and subsp . pauca, subsp . nov., respectively . The type strains of the subspecies are subsp . piercei ICPB 50025 (= ATTC 35879T and ICMP 15197), subsp . multiplex ICPB 50039 (= ATTC 35871 and ICMP 15199), and subsp . pauca ICPB 50031 (= ICMP 15198). Tsitologiia, 2004, 46(3), 208 - 20 {Structural organization and distribution of symbiotic bacteria Wolbachia in early embryos and ovaries of Drosophila melanogaster and D . simulans}; Dudkina NV et al.; Electron microscopic and morphometric analyses of Wolbachia distribution in early embryos of Drosophila flies have demonstrated that the number of bacteria in the embryo remains constant from fertilization to blastoderm, and that afterwards the symbionts could be observed only in the polar cells . Each bacterium has a three-layer envelope, makes contacts with microtubules and moves through the cytoplasm following the actively dividing nuclei . It has been found for the first time that Wolbachia could produce secretory vacuoles in the cytoplasm of early embryos . The relative volume of Wolbachia was five times as much in the embryos of Drosophila simulans as in those of D . melanogaster (Canton S), while the survival rate of D . simulans was half as much as that of D . melanogaster . It was shown that Wolbachia could form spore-like structures in D . simulans embryos . Ultrastructural investigations of Drosophila ovaries suggest that the bacteria may be present in all ovariol cells, including the oocyte, within whose cytoplasm they are delivered to the host . The highest number of symbionts was observed in germarium cells . In ovariol cells, the bacteria gradually decrease in number as oogenesis progresses . It has been determined for the first time that the symbionts are located closely to membranes of rough endoplasmatic reticulum in follicular and nurse cells of D . melanogaster . The data obtained suggest that Wolbachia may be involved in the regulation of oocyte maturation. Eur J Immunol, 2004 Jul, 34(7), 1789 - 97 Frontline: control of Anaplasma phagocytophilum, an obligate intracellular pathogen, in the absence of inducible nitric oxide synthase, phagocyte NADPH oxidase, tumor necrosis factor, Toll-like receptor (TLR)2 and TLR4, or the TLR adaptor molecule MyD88; von Loewenich FD et al.; Anaplasma phagocytophilum is an obligate intracellular bacterium that is related to rickettsial organisms and replicates in the hostile environment of neutrophils . Previous studies with SCID mice suggested that T and/or B cells are required for its control in vivo . Here, we used mice deficient for Toll-like receptor (TLR)2 and TLR4, MyD88, tumor necrosis factor, inducible nitric oxide synthase, or phagocyte NADPH oxidase (gp91(phox-/-)) to define the pathways that are critical for the recognition and the killing of this pathogen . Whereas SCID mice developed a 60-fold higher bacterial load in the blood compared to wild-type mice and succumbed to infection, all other gene-deficient mouse strains were fully capable in overcoming a systemic infection with A . phagocytophilum . From these data we conclude that effector mechanisms that are crucial to the defense against numerous other intracellular pathogens are dispensable for the control of A . phagocytophilum. Eur J Immunol, 2004 Jul, 34(7), 1783 - 8 Commentary: adaptive immunity in the absence of innate immune responses? The un-Tolled truth of the silent invaders; Ehlers S; The development and expression of effective adaptive immunity is currently thought to hinge entirely upon inductor and effector mechanisms furnished by cells of the innate immune system . An obligate intracellular bacterium, the causative agent of human granulocytic anaplasmosis, apparently defies this dogma: in a mouse model of infection, Anaplasma phagocytophilum is controlled by specific lymphocyte immunity even in the absence of Toll-like receptor (TLR)2, TLR4, the TLR-adaptor protein MyD88, inducible nitric oxide synthase or the gp91 component of the NADPH oxidase complex . A . phagocytophilum infection biology raises some interesting questions about the development of resistance to innate defense strategies by vector-borne pathogens, and challenges our current bias concerning the relative importance and the mode of interaction of the innate and adaptive arms of infection control in vertebrates. Infect Immun, 2004 Jul, 72(7), 4200 - 9 Mycobacterium bovis BCG urease attenuates major histocompatibility complex class II trafficking to the macrophage cell surface; Sendide K et al.; We have previously shown that Mycobacterium tuberculosis attenuates cell surface expression of major histocompatibility complex class II molecules in response to gamma interferon (IFN-gamma) by a mechanism dependent on intracellular sequestration of alpha,beta dimers . In this study we examined whether intracellular alkalinization due to mycobacterial urease could account for the defect in intracellular trafficking of class II molecules . Phagocytosis of wild-type Mycobacterium bovis BCG was associated with secretion of ammonia intracellularly, which increased substantially upon addition of exogenous urea to the culture medium . Increased intracellular ammonia, due to urea degradation by the bacterium, correlated with inhibition of class II surface expression . Conversely, no ammonia was detected in cells infected with a urease-negative mutant strain of M . bovis BCG, which also displayed a reduced effect on surface expression of class II molecules . A direct cause-effect relationship between urease and class II molecule trafficking was established with experiments where cells ingesting beads coated with purified urease showed an increased ammonia level and decreased surface expression of class II in response to IFN-gamma . In contrast to BCG, infection of macrophages with Mycobacterium smegmatis, which expresses relatively greater urease activity in cell-free culture, had a marginal effect on both the intracellular level of ammonia and class II expression . The limited effect of M . smegmatis was consistent with a failure to resist intracellular killing, suggesting that urease alone is not sufficient to resist macrophage microbicidal mechanisms and that this is required for a more distal effect on cell regulation . Our results demonstrate that alkalinization of critical intracellular organelles by pathogenic mycobacteria expressing urease contributes significantly to the intracellular retention of class II dimers. Infect Immun, 2004 Jul, 72(7), 4109 - 13 Protection afforded by heat shock protein 60 from Francisella tularensis is due to copurified lipopolysaccharide; Hartley MG et al.; Heat shock proteins (Hsps) have attracted significant attention as protective antigens against a range of diseases caused by bacterial pathogens . However, more recently there have been suggestions that the protective response is due to the presence of peptide components other than Hsps . We have shown that mice that had been immunized with purified heat shock protein 60 (Hsp60) isolated from Francisella tularensis were protected against a subsequent challenge with some strains of the bacterium . However, this protection appeared to be due to trace amounts of lipopolysaccharide, which were too low to be detected by using the Limulus amoebocyte lysate assay . This finding raises the possibility that the protection afforded by other bacterial Hsp60 proteins may be due to trace quantities of polysaccharide antigens carried by and acting in conjunction with the Hsps. Dis Aquat Organ, 2004 Apr 21, 59(1), 27 - 33 Renibacterium salmoninarum: effect of hypochlorite treatment, and survival in water; Hirvela-Koski V; The effect of different concentrations of sodium hypochlorite on Renibacterium salmoninarum and the survival of the bacterium in autoclaved river water and groundwater were examined . The disinfection trial was performed using R . salmoninarum ATCC 33209 . The concentrations of free chlorine were 10, 50, 100 and 200 mg 1(-1), the contact times were 5, 15, and 30 min and 24 h, and the test suspensions were subcultured both on Kidney disease medium (KDM2) agar and in 3 parallel KDM2 broths, which were then subcultured on KDM2 and selective KDM (SKDM) agar . The survival of the bacterium in river water and groundwater was studied using 4 isolates of R . salmoninarum including ATCC 33209 . Treatment with sodium hypochlorite effectively reduced the number of culturable cells of R . salmoninarum, but use of the recovery broth showed that small numbers of cells remained viable at all concentrations of free chlorine . The numbers of R . salmoninarum decreased to an undetectable level after 4 wk incubation in the survival trials, but low numbers of colonies were again found in the subculture after 5 wk incubation . Viable cells of R . salmoninarum were still detected in subcultures of all strains after 20 wk of incubation in river water. Dis Aquat Organ, 2004 May 5, 59(2), 125 - 30 Prevalence and diagnosis of bacterial kidney disease (BKD) in Scotland between 1990 and 2002; Bruno DW; Bacterial kidney disease (BKD) is a notifiable disease for salmonids under United Kingdom and European Union legislation . Within the UK, legislation and the control of infected fish with BKD has been operating for 25 yr . Infection by the bacterium Renibacterium salmoninarum results in a chronic, debilitating infection and mortality . Records of BKD outbreaks and the detection of R . salmoninarum were monitored from 1990 through to 2002 for Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss reared in Scottish waters . The test methods included ELISA, culture and light microscopy . New outbreaks of BKD in salmon in seawater declined during this period, but with year-to-year variation . Only 1 record of BKD has occurred in freshwater-reared salmon (prevalence 1.04) . BKD in farmed rainbow trout in seawater is uncommon and was only identified in 1993 and between 1998 and 2000 . The number of active designated area orders (DAOs) for outbreaks in salmon has fallen since 1990, but has remained relatively constant for trout over the period of study. Proc Natl Acad Sci U S A, 2004 Jun 29, 101(26), 9722 - 7 Epub 2004 Jun 21. Computational inference of scenarios for alpha-proteobacterial genome evolution; Boussau B et al.; The alpha-proteobacteria, from which mitochondria are thought to have originated, display a 10-fold genome size variation and provide an excellent model system for studies of genome size evolution in bacteria . Here, we use computational approaches to infer ancestral gene sets and to quantify the flux of genes along the branches of the alpha-proteobacterial species tree . Our study reveals massive gene expansions at branches diversifying plant-associated bacteria and extreme losses at branches separating intracellular bacteria of animals and humans . Alterations in gene numbers have mostly affected functional categories associated with regulation, transport, and small-molecule metabolism, many of which are encoded by paralogous gene families located on auxiliary chromosomes . The results suggest that the alpha-proteobacterial ancestor contained 3,000-5,000 genes and was a free-living, aerobic, and motile bacterium with pili and surface proteins for host cell and environmental interactions . Approximately one third of the ancestral gene set has no homologs among the eukaryotes . More than 40% of the genes without eukaryotic counterparts encode proteins that are conserved among the alpha-proteobacteria but for which no function has yet been identified . These genes that never made it into the eukaryotes but are widely distributed in bacteria may represent bacterial drug targets and should be prime candidates for future functional characterization. J Immunol, 2004 Jul 1, 173(1), 586 - 93 Helicobacter pylori arginase inhibits T cell proliferation and reduces the expression of the TCR zeta-chain (CD3zeta); Zabaleta J et al.; Helicobacter pylori infects approximately half the human population . The outcomes of the infection range from gastritis to gastric cancer and appear to be associated with the immunity to H . pylori . Patients developing nonatrophic gastritis present a Th1 response without developing protective immunity, suggesting that this bacterium may have mechanisms to evade the immune response of the host . Several H . pylori proteins can impair macrophage and T cell function in vitro through mechanisms that are poorly understood . We tested the effect of H . pylori extracts and live H . pylori on Jurkat cells and freshly isolated human normal T lymphocytes to identify possible mechanisms by which the bacteria might impair T cell function . Jurkat cells or activated T lymphocytes cultured with an H . pylori sonicate had a reduced proliferation that was not caused by T cell apoptosis or impairment in the early T cell signaling events . Instead, both the H . pylori sonicate and live H . pylori induced a decreased expression of the CD3zeta-chain of the TCR . Coculture of live H . pylori with T cells demonstrated that the wild-type strain, but not the arginase mutant rocF(-), depleted L-arginine and caused a decrease in CD3zeta expression . Furthermore, arginase inhibitors reversed these events . These results suggest that H . pylori arginase is not only important for urea production, but may also impair T cell function during infection. Oral Microbiol Immunol, 2004 Aug, 19(4), 277 - 80 Prevalence of Helicobacter pylori detected by polymerase chain reaction in the oral cavity of periodontitis patients; Gebara EC et al.; Helicobacter pylori is an important gastrointestinal pathogen associated with gastritis, peptic ulcers, and an increased risk of gastric carcinoma . The oral cavity has been indicated as a possible H . pylori reservoir, and may therefore be involved in the reinfection of the stomach which sometimes follows treatment of H . pylori infection . The objective of the present study was to evaluate the prevalence of H . pylori as detected by polymerase chain reaction (PCR) in the oral cavity of periodontitis patients testing positive for this bacterium in the stomach . Thirty adult patients with alterations of the superior digestive tract, testing urease positive after endoscopy and biopsy, were selected . A full-mouth periodontal examination was performed in every patient and the subjects were allocated to two groups: gingivitis (15 patients) and chronic periodontitis (15 patients) . Plaque and saliva samples collected from each patient were stored in 0.5 ml of TE buffer . DNA was extracted from the samples by the boiling method and was evaluated for the presence of H . pylori using the PCR method . JW 22/23 primers were used . The DNA of ATCC H . pylori 43629 (positive control) and water (negative control) were used for controlling the reactions . Of the 30 evaluated patients, 13 (43.3%) harbored H . pylori in the mouth . The bacterium was not found on the dorsum of the tongue of any patient, but was found in saliva in three patients (10%), in the supragingival plaque in six patients (20%), and in the subgingival plaque in eight patients (26.6%) . The presence of H . pylori was similar in the gingivitis and chronic periodontitis groups . In conclusion, a high percentage of patients harbored H . pylori in their mouth . The bacterium was detected in saliva, supragingival and subgingival plaque, suggesting that these sites may be considered reservoirs for H . pylori in urease-positive patients. Water Res, 2004 Jun, 38(11), 2757 - 63 Photoreactivation of Legionella pneumophila after inactivation by low- or medium-pressure ultraviolet lamp; Oguma K et al.; Photoreactivation of Legionella pneumophila after the inactivation by low-pressure (LP) or medium-pressure (MP) UV lamp was investigated in comparison with that of Escherichia coli . An endonuclease sensitive site (ESS) assay was used to determine the number of UV-induced pyrimidine dimers in the genome DNA of L . pneumophila or E . coli, while the survival ratio of each bacterium was also investigated by cultivation methods . L . pneumophila performed photoreactivation with almost complete repair of pyrimidine dimers associated with the quick recovery of survival ratio . A 3 log inactivation of L . pneumophila by LP or MP UV lamp was, respectively, resulted in 0.5 log or 0.4 log inactivation when photoreactivation was completed . Interestingly, L . pneumophila performed equivalent photoreactivation after LP and MP UV lamp exposures while photoreactivation of E . coli was significantly repressed after the inactivation by MP UV lamp . This study indicated that an attention would be required to design and operate a UV disinfection system targeting L . pneumophila . It was further implied that E . coli would not correctly indicate the fate of L . pneumophila in UV disinfection systems when photoreactivation takes place. Eur J Gastroenterol Hepatol, 2004 Jul, 16(7), 643 - 7 The role of cagA Helicobacter pylori strains in gastro-oesophageal reflux disease; Pereira-Lima JC et al.; BACKGROUND AND AIMS: The role of Helicobacter pylori infection in gastro-oesophageal reflux disease is controversial . The aim of this study was to evaluate the prevalence of colonization by cagA-positive and cagA-negative H . pylori strains in the spectrum of gastro-oesophageal reflux disease . METHODS: A total of 108 patients (50 male/58 female; mean age, 50.3 years) with dyspepsia and peptic ulcer or erosive gastritis/duodenitis were categorized into patients without reflux and patients with reflux oesophagitis graded from I to IV . All patients underwent upper endoscopy with biopsies of the antrum . H . pylori was detected by histology, urease test and polymerase chain reaction . The cagA status was diagnosed in the gastric biopsy by polymerase chain reaction . RESULTS: The overall prevalence of H . pylori colonization in patients with reflux was 68.6% and was 70.2% in those without oesophageal disease (P = 0.862) . Colonization by cagA-positive strains was also not statistically different between the two groups (31.4% versus 40.4%, P = 0.332) . However, patients with grades II-IV reflux oesophagitis were less colonized by the bacterium (36.4%) than patients with grade I oesophagitis (77.5%) (P = 0.009) . H . pylori cagA-positive strains were also less likely to colonize the stomach of patients with grades II-IV oesophagitis (0%), than grade I reflux oesophagitis (40%) patients and controls (40.4%) . CONCLUSIONS: Infection of the stomach by H . pylori and especially by H . pylori cagA strains may play a protective role against the development of the most severe forms of gastro-oesophageal reflux disease. Mikrobiologiia, 2004 Mar-Apr, 73(2), 175 - 9 {Deuterium oxide as a stress factor to the methylotrophic bacterium Methylophilus sp.}; Nevo AN et al.; The adaptation of the methylotrophic bacterium Methylophilus sp . B-7741 to growth in highly deuterated media was studied . For the first time, we showed the cross adaptation of bacterial cells to deuterated media and oxidative and osmotic stresses . The activity at catalase in deuterated cells was higher than in the control cells . Deuterated cell-free culture liquids showed protective effects on the growth of Methylophilus sp . B-7741 in deuterated media, which was manifested as an increase in the deuterated biomass yield . These data and the data available in the literature suggest that the mechanisms of bacterial cell adaptation to heavy water and to oxidative and osmotic stresses are similar. Mikrobiologiia, 2004 Mar-Apr, 73(2), 169 - 74 {The aeration-dependent effect of vitamin B12 on DNA biosynthesis in Methylobacterium dichloromethanicum}; Danilova IV et al.; The effect of vitamin B12 (cobalamin) on DNA biosynthesis in Methylobacterium dichloromethanicum was studied . When cultivated in media with methanol or dichloromethane, the bacterium produced approximately 10 micrograms corrinoids per g dry biomass, compared to about 7 micrograms/g when cultivated on ethanol or succinate . Exogenous adenosylcobalamin (AdoCbl) stimulated DNA biosynthesis in M . dichloromethanicum cells grown under poor aeration, the effect being mediated by AdoCb1-linked ribonucleotide reductase . In vitro studies showed that M . dichloromethanicum also has AdoCbl-independent ribonucleotide reductase . Under good aeration, exogenous AdoCbl had no effect on DNA biosynthesis, while hydroxyurea suppressed it . These data suggest that AdoCbl-independent ribonucleotide reductase, which is likely to be activated by oxygen, plays an important part in DNA biosynthesis when M . dichloromethanicum is cultured with good aeration, whereas AdoCbl-dependent ribonucleotide reductase is active under the conditions of poor aeration. Mol Plant Microbe Interact, 2004 Jun, 17(6), 696 - 706 Flagellin is not a major defense elicitor in Ralstonia solanacearum cells or extracts applied to Arabidopsis thaliana; Pfund C et al.; The phytopathogenic bacterium Ralstonia solanacearum requires motility for full virulence, and its flagellin is a candidate pathogen-associated molecular pattern that may elicit plant defenses . Boiled extracts from R . solanacearum contained a strong elicitor of defense-associated responses . However, R . solanacearum flagellin is not this elicitor, because extracts from wild-type bacteria and fliC or flhDC mutants defective in flagellin production all elicited similar plant responses . Equally important, live R . solanacearum caused similar disease on Arabidopsis ecotype Col-0, regardless of the presence of flagellin in the bacterium or the FLS2-mediated flagellin recognition system in the plant . Unlike the previously studied flg22 flagellin peptide, a peptide based on the corresponding conserved N-terminal segment of R . solanacearum, flagellin did not elicit any response from Arabidopsis seedlings . Thus recognition of flagellin plays no readily apparent role in this pathosystem . Flagellin also was not the primary elicitor of responses in tobacco . The primary eliciting activity in boiled R . solanacearum extracts applied to Arabidopsis was attributable to one or more proteins other than flagellin, including species purifying at approximately 5 to 10 kDa and also at larger molecular masses, possibly due to aggregation . Production of this eliciting activity did not require hrpB (positive regulator of type III secretion), pehR (positive regulator of polygalacturonase production and motility), gspM (general secretion pathway), or phcA (LysR-type global virulence regulator) . Wild-type R . solanacearum was virulent on Arabidopsis despite the presence of this elicitor in pathogen extracts. Microb Pathog, 2004 Jul, 37(1), 29 - 33 Anaerobiosis, growth phase and Actinobacillus pleuropneumoniae RTX toxin production; Jarma E et al.; Actinobacillus pleuropneumoniae is the causative agent of an economically significant form of porcine pleuropneumonia . This bacterium produces four distinct RTX toxins (ApxI-ApxIV) that play a key role in its pathogenesis, but further characterization of these hemolytic toxins is needed to identify the environmental signals and genes that affect their production during infection . In this report, we examined the effect of oxygen limitation on the production of ApxI and ApxII, the two RTX toxins that are produced by all highly virulent strains in North America . Batch cultures of ApxI- and ApxII-producing strains were grown in heart infusion broth supplemented with NAD, and samples were prepared throughout the growth curve . We compared batch cultures with normal oxygen levels to those that were maintained in an oxygen-depleted state . ApxI and ApxII hemolytic activity were nearly identical under both growth conditions . The level of toxin activity was confirmed by examination of extracellular ApxI concentrations and apxII mRNA levels . In addition, toxin activity examined on blood agar plates grown aerobically or anaerobically showed no significant difference . These results have important implications for the disease state where high-density growth is likely to result in local anaerobic or microaerophilic environments. Cell Biol Int, 2004, 28(5), 411 - 9 Intracellular phenotype of Mycobacterium avium enters macrophages primarily by a macropinocytosis-like mechanism and survives in a compartment that differs from that with extracellular phenotype; Bermudez LE et al.; Mycobacterium avium uptake by human macrophages differs between the phenotypes of bacterium grown in laboratory media (extracellular growth, EG) and bacterium grown within macrophages (intracellular growth, IG) . Studies in vivo have confirmed that, when spreading, pathogenic mycobacteria enter macrophages by a complement receptor 3-independent pathway, in contrast to mycobacteria uptake in vitro . M . avium, grown in macrophages (IG) for 3 or more days, invade fresh macrophages by a macropinocytosis-like mechanism, in contrast to bacteria grown in media (EG), confirmed by the inhibitory effect of wortmannin, an inhibitor of phosphoinoside-3-kinase, on the uptake of IG, but not EG, by macrophages . The IG phenotype was seen in vacuoles with lower pH than those inhabited by the EG phenotype . Incubation of macrophages with bafilomycin A1, an inhibitor of vacuole acidification, decreased the viability of intracellular IG, but not EG, phenotype, suggesting the importance of an acidic environment for the regulation of IG genes . In addition, the percentage of vacuoles that incorporate and retain LAMP-1 is smaller with EG than with IG bacteria . The formation of M . avium macropinosomes was also shown to be independent of microtubules . These data suggest that uptake of extracellular fluid is part of M . avium IG phenotype uptake by macrophages, and that the IG phenotype inhabits a slightly different vacuole than that of EG. Biophys J, 2004 Jun, 86(6), 4094 - 109 The of ATP synthase: ohmic conductance (10 fS), and absence of voltage gating; Feniouk BA et al.; The membrane portion of F(0)F(1)-ATP synthase, F(0), translocates protons by a rotary mechanism . Proton conduction by F(0) was studied in chromatophores of the photosynthetic bacterium Rhodobacter capsulatus . The discharge of a light-induced voltage jump was monitored by electrochromic absorption transients to yield the unitary conductance of F(0) . The current-voltage relationship of F(0) was linear from 7 to 70 mV . The current was extremely proton-specific (>10(7)) and varied only slightly ( approximately threefold) from pH 6 to 10 . The maximum conductance was approximately 10 fS at pH 8, equivalent to 6240 H(+) s(-1) at 100-mV driving force, which is an order-of-magnitude greater than of coupled F(0)F(1) . There was no voltage-gating of F(0) even at low voltage, and proton translocation could be driven by deltapH alone, without voltage . The reported voltage gating in F(0)F(1) is thus attributable to the interaction of F(0) with F(1) but not to F(0) proper . We simulated proton conduction by a minimal rotary model including the rotating c-ring and two relay groups mediating proton exchange between the ring and the respective membrane surface . The data fit attributed pK values of approximately 6 and approximately 10 to these relays, and placed them close to the membrane/electrolyte interface. Biophys J, 2004 Jun, 86(6), 4049 - 58 The fast tumble signal in bacterial chemotaxis; Khan S et al.; We have analyzed repellent signal processing in Escherichia coli by flash photorelease of leucine from photolabile precursors . We found that 1) . response amplitudes of free-swimming cell populations increased with leucine jump concentration, with an apparent Hill coefficient of 1.3 and a half-maximal dose of 14.4 microM; 2) . at a 0-0.5 mM leucine concentration jump sufficient to obtain a saturation motile response, the swimming cell response time of approximately 0.05 s was several-fold more rapid than the motor response time of 0.39 +/- 0.18 s measured by following the rotation of cells tethered by a single flagellum to quartz coverslips; and 3) . the motor response time of individual cells was correlated with rotation bias but not cell size . These results provide information on amplification, rate-limiting step, and flagellar bundle mechanics during repellent signal processing . The difference between the half-maximal dose for the excitation response and the corresponding value reported for adaptation provides an estimate of the increase in the rate of formation of CheYP, the phosphorylated form of the signal protein CheY . The estimated increase gives a lower limit receptor kinase coupling ratio of 6.0 . The magnitude and form of the motor response time distribution argue for it being determined by the poststimulus switching probability rather than CheYP turnover, diffusion, or binding . The temporal difference between the tethered and swimming cell response times to repellents can be quantitatively accounted for and suggests that one flagellum is sufficient to cause a measurable change of direction in which a bacterium swims. Mol Ecol, 2004 Jul, 13(7), 2009 - 16 Distribution of the bacterial symbiont Cardinium in arthropods; Zchori-Fein E et al.; Abstract 'Candidatus Cardinium', a recently described bacterium from the Bacteroidetes group, is involved in diverse reproduction alterations of its arthropod hosts, including cytoplasmic incompatibility, parthenogenesis and feminization . To estimate the incidence rate of Cardinium and explore the limits of its host range, 99 insect and mite species were screened, using primers designed to amplify a portion of Cardinium 16S ribosomal DNA (rDNA) . These arthropods were also screened for the presence of the better-known reproductive manipulator, Wolbachia . Six per cent of the species screened tested positive for Cardinium, compared with 24% positive for Wolbachia . Of the 85 insects screened, Cardinium was found in four parasitic wasp species and one armoured scale insect . Of the 14 mite species examined, one predatory mite was found to carry the symbiont . A phylogenetic analysis of all known Cardinium 16S rDNA sequences shows that distantly related arthropods can harbour closely related symbionts, a pattern typical of horizontal transmission . However, closely related Cardinium were found to cluster among closely related hosts, suggesting host specialization and horizontal transmission among closely related hosts . Finally, the primers used revealed the presence of a second lineage of Bacteroidetes symbionts, not related to Cardinium, in two insect species . This second symbiont lineage is closely allied with other arthropod symbionts, such as Blattabacterium, the primary symbionts of cockroaches, and male-killing symbionts of ladybird beetles . The combined data suggest the presence of a diverse assemblage of arthropod-associated Bacteroidetes bacteria that are likely to strongly influence their hosts' biology. Antonie Van Leeuwenhoek, 2000 Apr, 77(3), 271 - 80 Overexpression, purification and immunodetection of DsrD from Desulfovibrio vulgaris Hildenborough; Hittel DS et al.; Dissimilatory sulfite reductase (DsrAB) of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough is an alpha2beta2 tetramer of 180 kDa, encoded by the dsr operon . In addition to the dsrA and dsrB genes, this operon contains a gene (dsrD) encoding a protein of only 78 amino acids . Although, the function of DsrD is currently unknown, the presence of a dsrD gene has been demonstrated in a variety of sulfate-reducing bacteria and archaea . DsrD was expressed in Escherichia coli at a very high level and purified to homogeneity . Protein blotting experiments, using antisera raised against purified DsrD, demonstrated that it is expressed constitutively in D . vulgaris and does not copurify with DsrAB . Spectroscopic analysis of DsrD indicated that it does not bind either sulfite or sulfide, the substrate and product, respectively of the reaction catalyzed by DsrAB . Thus, although the conservation of this protein and its demonstrated presence in D . vulgaris, suggest an essential function in dissimilatory sulfite reduction, this function remains to be elucidated. Proteomics, 2004 May, 4(5), 1247 - 64 Comparison of the proteome of Methylobacterium extorquens AM1 grown under methylotrophic and nonmethylotrophic conditions; Laukel M et al.; Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium that is capable of growing in the presence of methanol as the sole carbon and energy source, but is also able to grow on a limited number of C(2), C(3), and C(4) compounds, for example succinate . This study provides a proteomic view of the cellular adaptation of M . extorquens AM1 to growth on methanol and succinate, respectively . Cytosolic proteins were separated by two-dimensional gel electrophoresis employing overlapping pH ranges and visualized by silver nitrate or fluorescence staining . A proteomic reference map containing 229 different proteins identified by peptide mass fingerprinting of tryptic fragments was established . Comparative proteome profiling of methanol- and succinate-grown cells led to the identification of 68 proteins that are induced under methylotrophic growth conditions in comparison to growth on succinate . This group includes most proteins known to be directly involved in methanol oxidation to CO(2) and in assimilation of one carbon units by the serine cycle as well as 18 proteins without any assigned function and two proteins with a predicted regulatory function . Furthermore, the proteome analysis revealed putative isoenzymes for formaldehyde-activating enzyme Fae, malyl-CoA lyase, malate-dehydrogenase, and fumarase, that need to be characterized functionally in future studies. Methods Mol Biol, 2004, 274, 325 - 40 Gene inactivation in the cyanobacterium Synechococcus sp . PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation; Frigaard NU et al.; Inactivation of a chromosomal gene is a useful approach to study the function of the gene in question and can be used to produce a desired phenotype in the organism . This chapter describes how to generate such mutants of the cyanobacterium Synechococcus sp . PCC 7002 and the green sulfur bacterium Chlorobium tepidum by natural transformation with synthetic DNA constructs . Two alternative methods to generate the DNA constructs, both performed entirely in vitro and based on the polymerase chain reaction (PCR), are also presented . These methods are ligation of DNA fragments with T4 DNA ligase, and megaprimer PCR. Appl Microbiol Biotechnol, 2004 Aug, 65(3), 356 - 62 Epub 2004 Jun 08. Coprecipitation of Th(4+) and the purified extracellular polysaccharide produced by bacterium Bradyrhizobium (Chamaecytisus) BGA-1; Diaz-Marrero AR et al.; The soil bacterium Bradyrhizobium (Chamaecytisus) strain BGA-1 produces an extracellular polymeric substance (EPS) that, in the presence of Fe(3+), Al(3+) or Th(4+) solutions, forms a gel-like precipitate composed of polysaccharide, protein, lipopolysaccharide and the metal . Precipitation of the main component of the EPS, the extracellular polysaccharide, and thorium was studied . The precipitate was stable, but redissolved at pH values below 3.0 or in the presence of 10 mM EDTA . In the precipitate, the ratio thorium/basic repeating unit of the polysaccharide ranged from 0.4 to 0.8 mol/mol . Soluble polysaccharide-thorium complexes were not found, and larger polysaccharide molecules were precipitated in preference to smaller ones . Kinetic studies showed a non-linear dependence of the precipitate on the concentrations of both thorium and polysaccharide . The behaviors of the purified polysaccharide and of whole EPS with the thorium-containing precipitate were compared . The results suggested that EPS components other than polysaccharide are able to modify the precipitating ability of the polysaccharide . Thus, whole EPS is a better substrate than the purified polysaccharide for the removal of thorium from its solutions. J Med Microbiol, 2004 Jul, 53(Pt 7), 585 - 93 Switches, cross-talk and memory in Escherichia coli adherence; Holden NJ et al.; Escherichia coli is a successful commensal and pathogen . Its pathogenic diversity stems from the acquisition and expression of multiple virulence-associated loci . Many of the key virulence factors are surface structures involved in adherence and motility . These are important antigens and their expression is limited by phase-variable genetic switches that are considered to act randomly . This review considers the possibility that such stochastic expression within a bacterial population belies sequential or co-ordinate control at the level of the individual bacterium . Co-ordinated expression or cross-talk between virulence loci can lead to a programmed set of events within a bacterium analogous to a simple form of electronic memory that is of benefit during infection. J Clin Microbiol, 2004 Jun, 42(6), 2366 - 71 Nocardia arthritidis sp . nov., a new pathogen isolated from a patient with rheumatoid arthritis in Japan; Kageyama A et al.; Two different bacterial strains with different drug susceptibilities were isolated from the sputum and an inflammatory discharge from a swelling in the left thigh of a patient with rheumatoid arthritis . Both bacterial strains were provisionally assigned to the genus Nocardia on the basis of their morphological and chemotaxonomic characteristics and were further studied in order to establish their taxonomic status . One strain (IFM 10034) was identified as Nocardia farcinica on the basis of its physiological characteristics . The other strain, which was designated Nocardia sp . strain IFM 10035(T), revealed a unique pattern of phenotypic properties that distinguished it from other representatives of established Nocardia species . Comparative 16S rRNA gene sequence studies of Nocardia sp . strain IFM 10035(T) also showed that the bacterium was closely related to the species Nocardia beijingensis . Determination of DNA-DNA relatedness, however, indicated that Nocardia sp . strain IFM 10035(T) could be delineated from N . beijingensis . The genotypic and phenotypic data combined indicated that the bacterium merits description as a new Nocardia species . The name proposed for the new species is Nocardia arthritidis sp . nov., the type strain being IFM 10035(T) (NBRC 100137(T), JCM 12120(T), DSM44731(T)) . The present study suggests that Nocardia infections can be caused by multiple species of the bacterium. Appl Environ Microbiol, 2004 Jun, 70(6), 3785 - 8 Stable carbon isotopic fractionations associated with inorganic carbon fixation by anaerobic ammonium-oxidizing bacteria; Schouten S et al.; Isotopic analyses of Candidatus "Brocadia anammoxidans," a chemolithoautotrophic bacterium that anaerobically oxidizes ammonium (anammox), show that it strongly fractionates against (13)C; i.e., lipids are depleted by up to 47 per thousand versus CO(2) . Similar results were obtained for the anammox bacterium Candidatus "Scalindua sorokinii," which thrives in the anoxic water column of the Black Sea, suggesting that different anammox bacteria use identical carbon fixation pathways, which may be either the Calvin cycle or the acetyl coenzyme A pathway. Appl Environ Microbiol, 2004 Jun, 70(6), 3772 - 5 Novel epibiotic thiothrix bacterium on a marine amphipod; Gillan DC et al.; Comparative analysis of the 16S rRNA gene and fluorescent in situ hybridization (FISH) was used to identify epibiotic filamentous bacteria living on the marine amphipod crustacean Urothoe poseidonis . The epibionts belong to the gamma proteobacteria and represent a novel marine phylotype within the genus Thiothrix . FISH and denaturing gradient gel electrophoresis revealed that the Thiothrix filaments are present on the majority of the amphipods examined. Appl Environ Microbiol, 2004 Jun, 70(6), 3417 - 24 Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7; O'Flynn G et al.; Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura . This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E . coli O157:H7 . Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E . coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro . Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37 degrees C . However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge . All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology . The frequency of BIM formation (10(-6) CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10(-4) CFU) . In addition, BIMs commonly reverted to phage sensitivity within 50 generations . In an initial meat trial experiment, the phage cocktail completely eliminated E . coli O157:H7 from the beef meat surface in seven of nine cases . Given that the frequency of BIM formation is low (10(-6) CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment. Carbohydr Res, 2004 Jun 22, 339(9), 1643 - 8 Characterisation of the core part of the lipopolysaccharide O-antigen of Francisella novicida (U112); Vinogradov E et al.; Francisella novicida (U112), a close relative of the highly virulent bacterium F . tularensis, is known to produce a lipopolysaccharide that is significantly different in biological properties from the LPS of F . tularensis . Here we present the results of the structural analysis of the F . novicida LPS core part, which is found to be similar to that of F . tularensis, differing only by one additional alpha-Glc residue:where R is an O-chain, linked via a beta-bacillosamine (2,4-diamino-2,4,6-trideoxyglucose) residue . The lipid part of F . novicida LPS contains no phosphate substituent and apparently has a free reducing end, a feature also noted in F . tularensis LPS. Biochemistry, 2004 Jun 15, 43(23), 7236 - 43 Identification of a novel protonation pattern for carboxylic acids upon Q(B) photoreduction in Rhodobacter sphaeroides reaction center mutants at Asp-L213 and Glu-L212 sites; Nabedryk E et al.; In the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides, light energy is rapidly converted to chemical energy through coupled electron-proton transfer to a buried quinone molecule Q(B) . Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations that are observable using light-induced Fourier transform infrared (FTIR) difference spectroscopy . Upon formation, Q(B)(-) induces protonation of Glu-L212, located within 5 A of Q(B), resulting in a IR signal at 1728 cm(-1) . However, no other IR signal is observed within the classic absorption range of protonated carboxylic acids (1770-1700 cm(-1)) . In particular, no signal for Asp-L213 is found despite its juxtaposition to Q(B) and importance for proton uptake on the second electron-transfer step . In an attempt to uncover the reason behind this lack of signal, the microscopic electrostatic environment in the vicinity of Q(B) was modified by interchanging Asp and Glu at the L213 and L212 positions . The Q(B)(-)/Q(B) FTIR spectrum of the Asp-L212/Glu-L213 swap mutant in the 1770-1700 cm(-1) range shows several distinct new signals, which are sensitive to (1)H/(2)H isotopic exchange, indicating that the reduction of Q(B) results in the change of the protonation state of several carboxylic acids . The new bands at 1752 and 1747 cm(-1) were assigned to an increase of protonation in response to Q(B) reduction of Glu-L213 and Asp-L212, respectively, based on the effect of replacing them with their amine analogues . Since other carboxylic acid signals were observed, it is concluded that the swap mutations at L212 and L213 affect a cluster of carboxylic acids larger than the L212/L213 acid pair . Implications for the native reaction center are discussed. Biochim Biophys Acta, 2004 Jun 7, 1656(2-3), 114 - 26 Apparent redundancy of electron transfer pathways via bc(1) complexes and terminal oxidases in the extremophilic chemolithoautotrophic Acidithiobacillus ferrooxidans; Brasseur G et al.; Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic bacterium that can grow in the presence of either the weak reductant Fe(2+), or reducing sulfur compounds that provide more energy for growth than Fe(2+) . We have previously shown that the uphill electron transfer pathway between Fe(2+) and NAD(+) involved a bc(1) complex that functions only in the reverse direction {J . Bacteriol . 182, (2000) 3602} . In the present work, we demonstrate both the existence of a bc(1) complex functioning in the forward direction, expressed when the cells are grown on sulfur, and the presence of two terminal oxidases, a bd and a ba(3) type oxidase expressed more in sulfur than in iron-grown cells, besides the cytochrome aa(3) that was found to be expressed only in iron-grown cells . Sulfur-grown cells exhibit a branching point for electron flow at the level of the quinol pool leading on the one hand to a bd type oxidase, and on the other hand to a bc(1)-->ba(3) pathway . We have also demonstrated the presence in the genome of transcriptionally active genes potentially encoding the subunits of a bo(3) type oxidase . A scheme for the electron transfer chains has been established that shows the existence of multiple respiratory routes to a single electron acceptor O(2) . Possible reasons for these apparently redundant pathways are discussed. Biochem Biophys Res Commun, 2004 Jun 25, 319(2), 501 - 5 Endothelial Chlamydia pneumoniae infection promotes oxidation of LDL; Dittrich R et al.; The bacterium Chlamydia pneumoniae chronically infects atheromatous lesions and is linked to atherosclerosis by modifying inflammation, proliferation, and the lipid metabolism of blood monocytes . As continuous LDL modification in the vascular intima is crucial for atherogenesis we investigated the impact of endothelial infection on LDL oxidation . HUVEC were infected with a vascular C . pneumoniae strain . Supernatants of infected cells but not cell lysates increased lipid peroxidation products (6.44 vs 6.14 nmol/ml, p<0.05) as determined by thiobarbituric acid reacting substances assay . Moreover, supernatants rendered human LDL more susceptible to oxidation as shown in a copper-ion catalysed LDL oxidation assay by a 16% reduction of LDL resistance against pro-oxidative stimuli (p<0.05) . Chlamydial infection of vascular endothelial cells releases acellular components that convert LDL to its proatherogenic form and reduce its resistance against oxidation . Foci of chronic endothelial chlamydial infection may thus continuously contribute to the dysregulated lipid metabolism that promotes atherogenesis. J Bacteriol, 2004 Jun, 186(12), 3766 - 76 Mutational analysis of the Myxococcus xanthus Omicron4499 promoter region reveals shared and unique properties in comparison with other C-signal-dependent promoters; Yoder DR et al.; The bacterium Myxococcus xanthus undergoes multicellular development during times of nutritional stress and uses extracellular signals to coordinate cell behavior . C-signal affects gene expression late in development, including that of Omega4499, an operon identified by insertion of Tn5 lac into the M . xanthus chromosome . The Omega4499 promoter region has several sequences in common with those found previously to be important for expression of other C-signal-dependent promoters . To determine if these sequences are important for Omega4499 promoter activity, the effects of mutations on expression of a downstream reporter gene were tested in M . xanthus . Although the promoter resembles those recognized by Escherichia coli sigma(54), mutational analysis implied that a sigma(70)-type sigma factor likely recognizes the promoter . A 7-bp sequence known as a C box and a 5-bp element located 6 bp upstream of the C box have been shown to be important for expression of other C-signal-dependent promoters . The Omega4499 promoter region has C boxes centered at -33 and -55 bp, with 5-bp elements located 7 and 8 bp upstream, respectively . A multiple-base-pair mutation in any of these sequences reduced Omega4499 promoter activity more than twofold . Single base-pair mutations in the C box centered at -33 bp yielded a different pattern of effects on expression than similar mutations in other C boxes, indicating that each functions somewhat differently . An element from about -81 to -77 bp exerted a twofold positive effect on expression but did not appear to be responsible for the C-signal dependence of the Omega4499 promoter . Mutations in sigD and sigE, which are genes that encode sigma factors, reduced expression from the Omega4499 promoter . The results provide further insight into the regulation of C-signal-dependent genes, demonstrating both shared and unique properties among the promoter regions so far examined. J Bacteriol, 2004 Jun, 186(12), 3760 - 5 Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family; Martin C et al.; Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough . ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents . The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents . ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types . In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT . A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents . Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux . Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE . These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures . Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux . Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux . Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers. J Bacteriol, 2004 Jun, 186(12), 3703 - 11 The decrease in FlaA observed in a flaB mutant of Borrelia burgdorferi occurs posttranscriptionally; Motaleb MA et al.; The Lyme disease bacterium Borrelia burgdorferi is a motile spirochete with a flat-wave morphology . The periplasmic flagella, which are situated between the outer membrane sheath and cell cylinder, are essential for both the cell's wavy shape and motility . Here we focus on the structure and regulation of its periplasmic flagella . Previous studies have suggested that the periplasmic flagella consist of a polymer of the major filament protein FlaB and a minor protein, FlaA . We used immunoprecipitation methodology to present further evidence that FlaA is indeed a flagellar protein . In addition, in contrast to FlaA of the spirochete Brachyspira hyodysenteriae, B . burgdorferi FlaA did not impact the overall helical shape of the periplasmic flagella . We have previously shown that B . burgdorferi lacks the sigma factor-dependent cascade control of motility gene transcription found in other bacteria . To begin to understand motility gene regulation in B . burgdorferi, we examined the effects of an insertion mutation in flaB on the amounts of proteins encoded by motility genes . Of several motility gene-encoded proteins examined, only the amount of FlaA was decreased in the flaB mutant; it was 13% compared to the wild-type amount . Real-time reverse transcriptase PCR analysis indicated that this inhibition was not the result of a decrease in flaA mRNA . In addition, protein stability analysis suggested that FlaA was turned over in the flaB mutant . Our results indicate that the lack of FlaB negatively influences the amount of FlaA found in the cell and that this effect is at the level of either translational control or protein turnover. Neurobiol Aging, 2004 May-Jun, 25(5), 619 - 27 Infiltration of the brain by pathogens causes Alzheimer's disease; Itzhaki RF et al.; Despite very numerous studies on Alzheimer's disease (AD), especially on amyloid plaques and neurofibrillary tangles, little information has been obtained thus on the causes of the disease . Evidence is described here that implicates firstly herpes simplex virus type 1 (HSV1) as a strong risk factor when it is present in brain of carriers of the type 4 allele of the gene for apolipoprotein E (APOE-4) . Indirect support comes from studies indicating the role of APOE in several diverse diseases of known pathogen cause . A second putative risk factor is the bacterium, Chlamydia pneumoniae . This pathogen has been identified and localized in AD brain . Current studies aimed at "proof of principle" address the entry of the organism into the CNS, the neuroinflammatory response to the organism, and the role that the organism plays in triggering AD pathology . An infection-based animal model demonstrates that following intranasal inoculation of BALB/c mice with C . pneumoniae, amyloid plaques/deposits consistent with those observed in the AD brain develop, thus implicating this infection in the etiology of AD. J Mol Microbiol Biotechnol, 2004, 7(1-2), 72 - 7 The junctional pore complex and the propulsion of bacterial cells; Wolgemuth CW et al.; Gliding motility is defined as translocation in the direction of the long axis of the bacterium while in contact with a surface . This definition leaves unspecified any mechanism and, indeed, it appears that there is more than one physiological system underlying the same type of motion . Currently, two distinct mechanisms have been discovered in myxobacteria . One requires the extension, attachment, and retraction of type IV pili to pull the cell forwards . Recent experimental evidence suggests that a second mechanism for gliding motility involves the extrusion of slime from an organelle called the 'junctional pore complex' . This review discusses the role of slime extrusion and the junctional pore complex in the gliding motility of both cyanobacteria and myxobacteria . Proteins, 2004 Jul 1, 56(1), 28 - 39 Solution structure of hypothetical Nudix hydrolase DR0079 from extremely radiation-resistant Deinococcus radiodurans bacterium; Buchko GW et al.; Using nuclear magnetic resonance (NMR) based methods, including residual dipolar coupling restraints, we have determined the solution structure of the hypothetical Deinococcus radiodurans Nudix protein DR0079 (171 residues, MW = 19.3 kDa) . The protein contains eight beta-strands and three alpha-helices organized into three subdomains: an N-terminal beta-sheet (1-34), a central Nudix core (35-140), and a C-terminal helix-turn-helix (141-171) . The Nudix core and the C-terminal helix-turn-helix form the fundamental fold common to the Nudix family, a large mixed beta-sheet sandwiched between alpha-helices . The residues that compose the signature Nudix sequence, GX5EX7REUXEEXGU (where U = I, L, or V and X = any amino acid), are contained in a turn-helix-turn motif on the face of the mixed beta-sheet . Chemical shift mapping experiments suggest that DR0079 binds Mg2+ . Experiments designed to determine the biological function of the protein indicate that it is not a type I isopentenyl-diphosphate delta-isomerase and that it does not bind alpha,beta-methyleneadenosine 5'-triphosphate (AMPCPP) or guanosine 5'-{beta,gamma-imido}triphosphate (GMPPNP) . In this article, the structure of DR0079 is compared to other known Nudix protein structures, a potential substrate-binding surface is proposed, and its possible biological function is discussed . Biotechnol Bioeng, 2004 Jun 30, 86(7), 737 - 46 Recombinant antigen from Helicobacter pylori urease as vaccine against H . pylori-associated disease; Fujii R et al.; It is well documented that the enzymatic active site of Helicobacter pylori urease is present in the beta-subunit . An important sequence of 135 amino acids of the beta-subunit was determined from the structure of H . pylori urease and by a homology-based study of the urease of other bacteria and plants . The sequence (UreB) was expressed in Escherichia coli as a recombinant fusion protein with glutathione-S-transferase (GST) . Seventeen monoclonal antibodies, UA-1-17, were produced using the UreB-GST as the immunogen . The obtained monoclonal antibodies showed a high specificity to UreB, and some of the MAbs cross-reacted with Jack bean urease . About 70% of the established MAbs displayed an inhibitory effect on the enzymatic activity of the urease . Among them, UA-15 MAb could reduce the activity by 53% and it immunologically binds to the bacterium infecting the human stomach mucosa . The antiserum induced by immunization with a recombinant UreB-GST into rabbits displayed a specific binding to mucosal surfaces of the human stomach infected with the pathogen H . pylori . Moreover, the antiserum suppressed the enzymatic activity of H . pylori urease, while the purified H . pylori urease could not induce such an antiserum . Heredity, 2004 Jun, 92(6), 483 - 9 Genes witho