Microorganisms

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 without frontiers?
Bensasson D, Boore JL, Nielsen KM.
For bacteria, the primary genetic barrier against the genetic exchange of DNA that is not self-transmissible is dissimilarity in the bacterial DNA sequences concerned . Genetic exchange by homologous recombination is frequent among close bacterial relatives and recent experiments have shown that it can enable the uptake of closely linked nonhomologous foreign DNA . Artificial vectors are mosaics of mobile DNA elements from free-living bacterial isolates and so bear a residual similarity to their ubiquitous natural progenitors . This homology is tightly linked to the multitude of different DNA sequences that are inserted into synthetic vectors . Can homology between vector and bacterial DNA enable the uptake of these foreign DNA inserts? In this review we investigate pUC18 as an example of an artificial vector and consider whether its homology to broad host-range antibiotic resistance transposons and plasmid origins of replication could enable the uptake of insert DNA in the light of studies of homology-facilitated foreign DNA uptake . We also discuss the disposal of recombinant DNA, its persistence in the environment and whether homologies to pUC18 may exist in naturally competent bacteria . Most DNA that is inserted into the cloning site of artificial vectors would be of little use to a bacterium, but perhaps not all.

J Biol Chem, 2004 Jul 30, 279(31), 32151 - 8 Epub 2004 May 25.
Leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus recognizes minihelices; Xu MG et al.; Aminoacylation of the minihelix mimicking the amino acid acceptor arm of tRNA has been demonstrated in more than 10 aminoacyl-tRNA synthetase systems . Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase (LeuRS) is unable to charge the cognate minihelix or microhelix, we show here that minihelix(Leu) is efficiently charged by Aquifex aeolicus synthetase, the only known heterodimeric LeuRS (alpha beta-LeuRS) . Aminoacylation of minihelices is strongly dependent on the presence of the A73 identity nucleotide and greatly stimulated by destabilization of the first base pair as reported for the E . coli isoleucyl-tRNA synthetase and methionyl-tRNA synthetase systems . In the E . coli LeuRS system, the anticodon of tRNA(Leu) is not important for recognition by the synthetase . However, the addition of RNA helices that mimic the anticodon domain stimulates minihelix(Leu) charging by alpha beta-LeuRS, indicating possible domain-domain communication within alpha beta-LeuRS . The leucine-specific domain of alpha beta-LeuRS is responsible for minihelix recognition . To ensure accurate translation of the genetic code, LeuRS functions to hydrolyze misactivated amino acids (pretransfer editing) and misaminoacylated tRNA (posttransfer editing) . In contrast to tRNA(Leu), minihelix(Leu) is unable to induce posttransfer editing even upon the addition of the anticodon domain of tRNA . Therefore, the context of tRNA is crucial for the editing of mischarged products . However, the minihelix(Leu) cannot be misaminoacylated, perhaps because of the tRNA-independent pretransfer editing activity of alpha beta-LeuRS.

Ned Tijdschr Geneeskd, 2004 May 8, 148(19), 916 - 8
{Health Council of the Netherlands advisory report 'Vaccination against pertussis'--time for a new vaccine}; Visser HK; An advisory report on vaccination against pertussis by the National Vaccination Programme Review Committee of the Health Council of the Netherlands makes recommendations on improving pertussis vaccination in the Netherlands . Since 1996, between 4000 and 8000 cases of pertussis have been reported each year, mainly in young children who have already been vaccinated . The main cause of this increase, apart from decreasing immunity in older children and adults, seems to be diminished vaccine effectiveness due to the occurrence of non-vaccine related strains of the pertussis bacterium in the Netherlands . The cellular vaccine used in the Netherlands contains low levels of the major antigens pertussis toxin and pertactin . The Health Council recommends the fastest possible transition to the use of an acellular combination vaccine . Such a vaccine will be effective and will have considerably fewer side effects than the one currently in use . The Committee recommends that research is done into the sources of pertussis infections in young infants.

J Cell Sci, 2004 May 15, 117(Pt 12), 2579 - 90
Matrix-mediated canal formation in primmorphs from the sponge Suberites domuncula involves the expression of a CD36 receptor-ligand system; Muller WE et al.; Sponges (Porifera), represent the phylogenetically oldest metazoan phylum still extant today . Recently, molecular biological studies provided compelling evidence that these animals share basic receptor/ligand systems, especially those involved in bodyplan formation and in immune recognition, with the higher metazoan phyla . An in vitro cell/organ-like culture system, the primmorphs, has been established that consists of proliferating and differentiating cells, but no canals of the aquiferous system . We show that after the transfer of primmorphs from the demosponge Suberites domuncula to a homologous matrix (galectin), canal-like structures are formed in these 3D-cell aggregates . In parallel with the formation of these structures a gene is expressed whose deduced protein falls into the CD36/LIMPII receptor family . The receptor was cloned and found to be strongly expressed after adhesion to the galectin matrix . This process was suppressed if primmorphs were co-incubated with a homologous polypeptide containing the CSVTCG domain, as found in thrombospondin-1 (and related) molecules of vertebrates . In situ hybridization studies revealed that the S . domuncula CD36/LIMPII receptor is localized in the pinacocytes that surround the canals of the sponge . Furthermore, a secondary metabolite from a sponge-associated bacterium was isolated and characterized, the 2-methylthio-1,4-naphthoquinone (MTN) . MTN causes inhibition of cell proliferation of vertebrate tumor cells at concentrations of >80 ng/ml . However, doses of only 2 ng are required to potently inhibit angiogenesis in the chick chorio-allantoic membrane assay . At concentrations of 10 ng/ml this compound was also found to suppress the expression of the S . domuncula CD36/LIMPII; this result is a first indication that this secondary metabolite has a conserved functional activity: the suppression of the formation of the circulation system, from sponges to vertebrates.

Biochim Biophys Acta, 2004 Jun 1, 1699(1-2), 163 - 71
Mapping of functional sites on the primary structure of the contractile tail sheath protein of bacteriophage T4 by mutation analysis; Takeda S et al.; In order to determine the functional roles of amino acid residues in gp18 (gp: gene product), the contractile tail sheath protein of bacteriophage T4, the mutation sites and amino acid replacements of available and newly created missense mutants with distinct phenotypes were determined . Amber mutants were also utilized for amino acid insertion by host amber suppressor cell strains . It was found that mutants that gave rise to a particular phenotype were mapped in a particular region along the polypeptide chain . Namely, all amino acid replacements in the cold-sensitive mutants (cs, which grows at 37 degrees C, but not at 25 degrees C) and the heat-sensitive mutant (hs, lose viability by incubation at 55 degrees C for 30 min) except for one hs mutant were mapped in a limited region in the C-terminal domain . On the other hand, all the temperature-sensitive mutants (ts, grow at 30 degrees C, but not at 42 degrees C) and carbowax mutants (CBW, can adsorb to the host bacterium in the presence of high concentrations of polyethylene glycol, where wild-type phage cannot) were mapped in the N-terminal protease-resistant domain, except for one ts mutant . The results suggested that the C-terminal region of gp18 is important for contraction and assembly, whereas the N-terminal protease-resistant domain constitutes the protruding part of the tail sheath.

Immunol Lett, 2004 May 15, 93(2-3), 137 - 42
Phagocytosis of M . paratuberculosis fails to activate expression of NADH dehydrogenase and nucleolin-related protein in bovine macrophages; Tooker BC et al.; Mycobacterium avium subspecies paratuberculosis (M . paratuberculosis) is a facultative intracellular bacterium and causal agent of Johne's disease in cattle . Following phagocytosis, M . paratuberculosis resides and replicates in macrophage phagosomes that fail to mature . Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used as a high throughput initial screen to begin to test the hypothesis that macrophage gene expression patterns would be differentially affected by M . paratuberculosis when compared to readily degraded bacteria or non-degradable latex beads . Gene expression profiles from immortalized bovine macrophage cells (BOMAC) exposed to M . paratuberculosis were compared to gene expression profiles for BOMAC cells exposed to Escherichia coli, latex beads or PBS . Amplicons representing genes specifically activated or repressed during M . paratuberculosis phagocytosis were cloned for further investigation . Northern blot hybridizations preformed using DDRT-PCR-derived amplicons 3-1-4, 5-2-10, 5-4-2 and 4-1-6 confirmed stimuli dependent differential gene expression . Expression pattern observed for amplicon 3-1-4 represents genes that are up-regulated following phagocytosis of E . coli or latex beads, but not M . paratuberculosis . Amplicon 5-2-10 exhibited a pattern of expression representative of genes that are up-regulated strongly following phagocytosis of E . coli or latex beads but only moderately following M . paratuberculosis phagocytosis . Expression pattern of the gene for amplicon 5-4-2 was representative of genes that are specifically suppressed following M . paratuberculosis phagocytosis, while the amplicon 4-1-6 gene expression pattern represented genes that are generally suppressed following phagocytosis of any of the three stimuli . DNA sequencing and Genbank database analysis of these amplicons revealed that amplicon 3-1-4, whose expression failed to activate following M . paratuberculosis phagocytosis, had high levels of similarity to a Rattus norvegicus nucleolin-related protein (NRP) . Amplicon 5-2-10, which increased expression moderately following M . paratuberculosis phagocytosis, was a near perfect match to bovine nicotinamide adenine dinucleotide dehydrogenase (FNADH dehydrogenase) subunit 1 (ND1) . Failure to activate these two genes at levels observed following phagocytosis of either E . coli or latex beads may uncover new mechanisms for the survival of M . paratuberculosis within bovine macrophage cells.

Biochemistry, 2004 Jun 1, 43(21), 6387 - 92
Identifying latent enzyme activities: substrate ambiguity within modern bacterial sugar kinases; Miller BG et al.; The ability of enzymes to catalyze the transformation of multiple, structurally related substrates could empower the natural evolution of new catalytic functions . The prevalence of such substrate ambiguity in modern catalysts, however, is largely unknown . To search for ambiguous sugar kinases, we generated a bacterium incapable of performing the first step of the glycolytic pathway, the phosphorylation of glucose . This organism cannot survive with glucose as its sole source of carbon . Within its genome, we find three DNA sequences that, when transcribed from a powerful extrachromosomal promoter, can complement the auxotrophy of the organism . These sequences contain the nanK, yajF, and ycfX genes . In vitro, the NanK, YajF, and YcfX proteins function as rudimentary glucokinases with ambiguous substrate specificites, displaying k(cat)/K(m) values for the phosphorylation of glucose that are 10(4)-fold lower than the k(cat)/K(m) value of endogenous bacterial glucokinase . Our findings suggest that modern genomes harbor a wealth of latent enzyme activities and that extant metabolic pathways are equivocal, in contrast to their usual depiction.

Mol Biol Cell, 2004 Aug, 15(8), 3520 - 9 Epub 2004 May 21.
Enteropathogenic Escherichia coli use redundant tyrosine kinases to form actin pedestals; Swimm A et al.; Enteropathogenic Escherichia coli (EPEC) are deadly contaminants in water and food and induce protrusion of actin-rich membrane pedestals beneath themselves upon attachment to intestinal epithelia . EPEC then causes intestinal inflammation, diarrhea, and, among children, death . Here, we show that EPEC uses multiple tyrosine kinases for formation of pedestals, each of which is sufficient but not necessary . In particular, we show that Abl and Arg, members of the Abl family of tyrosine kinases, localize and are activated in pedestals . We also show that pyrido{2,3-d}pyrimidine (PD) compounds, which inhibit Abl, Arg, and related kinases, block pedestal formation . Finally, we show that Abl and Arg are sufficient for pedestal formation in the absence of other tyrosine kinase activity, but they are not necessary . Our results suggest that additional kinases that are sensitive to inhibition by PD also can suffice . Together, these results suggest that EPEC has evolved a mechanism to use any of several functionally redundant tyrosine kinases during pathogenesis, perhaps facilitating its capacity to infect different cell types . Moreover, PD compounds are being developed to treat cancers caused by dysregulated Abl . Our results raise the possibility that PD may be useful in treating EPEC infections, and because PD affects host and not bacterium, selecting resistant strains may be far less likely than with conventional antibiotics.

J Biol Chem, 2004 Jul 30, 279(31), 32545 - 53 Epub 2004 May 19.
Two distinct binding sites for high potential iron-sulfur protein and cytochrome c on the reaction center-bound cytochrome of Rubrivivax gelatinosus; Alric J et al.; The photosynthetic cyclic electron transfer of the purple bacterium Rubrivivax gelatinosus, involving the cytochrome bc(1) complex and the reaction center, can be carried out via two pathways . A high potential iron-sulfur protein (HiPIP) acts as the in vivo periplasmic electron donor to the reaction center (RC)-bound cytochrome when cells are grown under anaerobic conditions in the light, while cytochrome c is the soluble electron carrier for cells grown under (8)aerobic conditions in the dark . A spontaneous reversion of R . gelatinosus C244, a defective mutant in synthesis of the RC-bound cytochrome by insertion of a Km(r) cassette leading to gene disruption with a slow growth rate, restores the normal photosynthetic growth . This revertant, designated C244-P1, lost the Km(r) cassette but synthesized a RC-bound cytochrome with an external 77-amino acid insertion derived from the cassette . We characterized the RC-bound cytochrome of this mutant by EPR, time-resolved optical spectroscopy, and structural analysis . We also investigated the in vivo electron transfer rates between the two soluble electron donors and this RC-bound cytochrome . Our results demonstrated that the C244-P1 RC-bound cytochrome is still able to receive electrons from HiPIP, but it is no longer reducible by cytochrome c(8) . Combining these experimental and theoretical protein-protein docking results, we conclude that cytochrome c(8) and HiPIP bind the RC-bound cytochrome at two distinct but partially overlapping sites.

J Bacteriol, 2004 Jun, 186(11), 3609 - 20
The Sinorhizobium meliloti fur gene regulates, with dependence on Mn(II), transcription of the sitABCD operon, encoding a metal-type transporter; Chao TC et al.; Sinorhizobium meliloti is an alpha-proteobacterium able to induce nitrogen-fixing nodules on roots of specific legumes . In order to propagate in the soil and for successful symbiotic interaction the bacterium needs to sequester metals like iron and manganese from its environment . The metal uptake has to be in turn tightly regulated to avoid toxic effects . In this report we describe the characterization of a chromosomal region of S . meliloti encoding the sitABCD operon and the putative regulatory fur gene . It is generally assumed that the sitABCD operon encodes a metal-type transporter and that the fur gene is involved in iron ion uptake regulation . A constructed S . meliloti sitA deletion mutant was found to be growth dependent on Mn(II) and to a lesser degree on Fe(II) . The sitA promoter was strongly repressed by Mn(II), with dependence on Fur, and moderately by Fe(II) . Applying a genome-wide S . meliloti microarray it was shown that in the fur deletion mutant 23 genes were up-regulated and 10 genes were down-regulated when compared to the wild-type strain . Among the up-regulated genes only the sitABCD operon could be associated with metal uptake . On the other hand, the complete rhbABCDEF operon, which is involved in siderophore synthesis, was identified among the down-regulated genes . Thus, in S . meliloti Fur is not a global repressor of iron uptake . Under symbiotic conditions the sitA promoter was strongly expressed and the S . meliloti sitA mutant exhibited an attenuated nitrogen fixation activity resulting in a decreased fresh weight of the host plant Medicago sativa.

Int J Med Microbiol, 2004 Apr, 293(7-8), 589 - 97
The multiple cellular activities of the VacA cytotoxin of Helicobacter pylori; de Bernard M et al.; Helicobacter pylori has elaborated a unique set of virulence factors that allow it to colonize the stomach wall . These factors include urease, helicoidal shape, flagella, adhesion and pro-inflammatory molecules . Here we discuss the molecular and cellular mechanisms of action of the vacuolating cytotoxin VacA . Its activities are discussed in terms of tissue alterations which promote the release of nutrients necessary to the growth and survival of the bacterium in its nutrient-poor ecological niche . This toxin also shows some pro-inflammatory and immunosuppressive activities which may be functional to the establishment of a chronic type of inflammation.

Mol Genet Genomics, 2004 Jun, 271(5), 545 - 53 Epub 2004 May 18.
Mutational analysis of the binding affinity and transport activity for N-acetylglucosamine of the novel ABC transporter Ngc in the chitin-degrader Streptomyces olivaceoviridis; Saito A et al.; The highly differentiated bacterium Streptomyces olivaceoviridis efficiently hydrolyses chitin, a highly abundant natural polysaccharide, to low molecular weight products including N-acetylglucosamine (NAG) and N,N' -diacetylchitobiose (chitobiose) . NAG is taken up by a PTS (phosphoenolpyruvate-dependent phosphotransferase system) which includes the PtsC2 protein, and via the ABC (ATP-binding cassette) transporter Ngc, which itself includes the substrate-binding protein NgcE . This is at present the only ABC transporter which is known to mediate specific uptake of NAG (K(m) 0.48 microM, V(max) 1.3 nmol/min/mg dry weight) and is competitively inhibited by chitobiose (K(i) 0.68 microM) . The latter finding suggests that the Ngc system transports both NAG and chitobiose efficiently . To identify amino acid residues required for the function of NgcE, either the wild-type or one of several mutant forms of the ngcE gene was introduced into the strain S . olivaceoviridis DeltaNgcE/DeltaPtsC1/DeltaPtsC2, which lacks both functional transport systems for NAG, and chromosomal recombinants were selected . Based on the in vivo transport parameters of the recombinants, and the in vitro binding characteristics of the corresponding purified proteins, the following conclusions can be drawn . (1) Replacement of the C-terminally located residue Y396 by A (Y396A) has little effect on ligand-binding or transport parameters . The W395A mutation also induced little change in the substrate affinity in vitro, but it led in vivo to a marked increase (11 fold) in K(m), and enhanced V(max) (by 1.5 fold) . (2) The amino acids Y201 and W280 both contribute (51% and 38%) to the ligand-binding capacity of NgcE . They are both very important for the in vivo function of the complete transport apparatus; strains expressing either Y201A or W280A show drastically (100 or 150 times) enhanced K(m) values . (3) The concomitant presence of either Y200 and W280 or Y201 and W280 is essential for the function of NgcE . (4) Y201 is located within a tyrosyl-rich motif . This has been found to share some features with the ligand-binding site of amelogenins (enamel matrix proteins), which interact with NAG residues in glycoconjugates . In addition, it is distantly related to the ligand-binding site(s) in the plant-lectins UDA ( Urtica dioicaagglutinin, specific for NAG and its oligomers) and WGA (wheat germ agglutinin, which recognises a motif comprising three consecutive NAG residues).

Int J Biochem Cell Biol, 2004 Aug, 36(8), 1624 - 34
Cellular stress-related protein expression in Helicobacter pylori-infected gastric epithelial AGS cells; Lim JW et al.; Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulceration, and gastric carcinoma . Moreover, H . pylori may induce disease-specific protein expression in gastric epithelial cells . The present study was aimed at determining differentially expressed proteins in H . pylori-infected gastric epithelial AGS cells . AGS cells were treated with H . pylori at a bacterium/cell ratio of 300:1 for 12 h . Altered protein patterns as separated by two-dimensional electrophoresis using pH gradients of 4-7 were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests . Four differentially expressed proteins, whose expression levels were increased by more than two-fold in H . pylori-infected cells, were analyzed . These proteins (14-3-3 protein alpha/beta, cullin homolog 3, alpha-enolase, ezrin) are known to be related to cell proliferation, cell adhesion, and carcinogenesis, and may be mediated by cellular stress, such as reactive oxygen species . In conclusion, the identification of these differentially expressed proteins provide valuable information for the understanding of the pathophysiologic mechanisms of H . pylori-induced gastric diseases, and may be useful as prognostic indices of H . pylori-related gastric disorders.

BMC Biochem . 2004 May 17;5(1):7.
Characterization of a nudix hydrolase from Deinococcus radiodurans with a marked specificity for (deoxy)ribonucleoside 5'-diphosphates; Fisher DI et al.; BACKGROUND: Nudix hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives . The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix hydrolases, an unexpectedly large number . These may contribute to radioresistance by removing mutagenic oxidised and otherwise damaged nucleotides . Characterisation of these hydrolases is necessary to understand the reason for their presence . Here, we report the cloning and characterisation of the DR0975 gene product, a nudix hydrolase that appears to be unique to this organism . RESULTS: The DR0975 gene was cloned and expressed as a 20 kDa histidine-tagged recombinant product in Escherichia coli . Substrate analysis of the purified enzyme showed it to act primarily as a phosphatase with a marked preference for (deoxy)nucleoside 5'-diphosphates (dGDP > ADP > dADP > GDP > dTDP > UDP > dCDP > CDP) . Km for dGDP was 110 microM and kcat was 0.18 s-1 under optimal assay conditions (pH 9.4, 7.5 mM Mg2+) . 8-Hydroxy-2'-deoxyguanosine 5'-diphosphate (8-OH-dGDP) was also a substrate with a Km of 170 microM and kcat of 0.13 s-1 . Thus, DR0975 showed no preference for 8-OH-dGDP over dGDP . Limited pyrophosphatase activity was also observed with NADH and some (di)adenosine polyphosphates but no other substrates . Expression of the DR0975 gene was undetectable in logarithmic phase cells but was induced at least 30-fold in stationary phase . Superoxide, but not peroxide, stress and slow, but not rapid, dehydration both caused a slight induction of the DR0975 gene . CONCLUSION: Nucleotide substrates for nudix hydrolases conform to the structure NDP-X, where X can be one of several moieties . Thus, a preference for (d)NDPs themselves is most unusual . The lack of preference for 8-OH-dGDP over dGDP as a substrate combined with the induction in stationary phase, but not by peroxide or superoxide, suggests that the function of DR09075 may be to assist in the recycling of nucleotides under the very different metabolic requirements of stationary phase . Thus, if DR0975 does contribute to radiation resistance, this contribution may be indirect.

Vet Microbiol, 2004 Jun 3, 100(3-4), 233 - 40
Identification of Ehrlichia ruminantium (Gardel strain) IFN-gamma inducing proteins after vaccination with a killed vaccine; Esteves I et al.; IFN-gamma is considered as a key factor in protection against heartwater of ruminants, caused by the obligate intracellular bacterium Ehrlichia ruminantium . In this study, a better definition of the molecular masses of IFN-gamma inducing proteins of the Gardel strain of E . ruminantium was obtained by the use of continuous flow electrophoresis (CFE) and sensitized polyclonal lymphocytes . Out of 15 E . ruminantium CFE fractions tested within the 14-39 kDa region, eight were commonly reacted to by all goats . Interestingly, half of these fractions fall within the 23-29 kDa region, shown previously to contain polymorphic B-cell epitopes . Thus, the results suggest that this region also contains T-cell epitopes potentially involved in protection . Also, several proteins were found to be more immunogenic than the serologically immunodominant MAP1 protein . Finally, high activity within the 15-19 kDa region was observed, which confirms previous work done with CD4+ T-cell lines obtained from cattle immunized with a South African strain of E . ruminantium . The proteins falling within the molecular weight ranges defined in this study may have potential as vaccine antigens.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 961 - 8
Characterization of a 'Bacteroidetes' symbiont in Encarsia wasps (Hymenoptera: Aphelinidae): proposal of 'Candidatus Cardinium hertigii'; Zchori-Fein E et al.; Previously, analysis of 16S rDNA sequences placed a newly discovered lineage of bacterial symbionts of arthropods in the 'Bacteroidetes' . This symbiont lineage is associated with a number of diverse host reproductive manipulations, including induction of parthenogenesis in several Encarsia parasitoid wasps (Hymenoptera: Aphelinidae) . In this study, electron microscopy and phylogenetic analysis of the 16S rRNA and gyrB genes of symbionts from Encarsia hispida and Encarsia pergandiella are used to describe and further characterize these bacteria . Phylogenetic analyses based on these two genes showed that the Encarsia symbionts are allied with the Cytophaga aurantiaca lineage within the 'Bacteroidetes', with their closest described relative being the acanthamoeba symbiont 'Candidatus Amoebophilus asiaticus' . The Encarsia symbionts share 97 % 16S rDNA sequence similarity with Brevipalpus mite and Ixodes tick symbionts and 88 % sequence similarity with 'Candidatus A . asiaticus' . Electron microscopy revealed that many of the bacteria found in the ovaries of the two Encarsia species contained a regular, brush-like array of microfilament-like structures that appear to be characteristic of the symbiont . Finally, the role of this bacterium in parthenogenesis induction in E . hispida was confirmed . Based on phylogenetic analyses and electron microscopy, classification of the symbionts from Encarsia as 'Candidatus Cardinium hertigii' is proposed.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 851 - 5
Herbaspirillum chlorophenolicum sp . nov., a 4-chlorophenol-degrading bacterium; Im WT et al.; A 4-chlorophenol-degrading bacterial strain, formerly designated as a strain of Comamonas testosteroni, was reclassified as a member of the genus Herbaspirillum based on its phenotypic and chemotaxonomic characteristics, as well as phylogenetic analysis using 16S rDNA sequences . Phylogenetic inference based on 16S rDNA sequences showed that strain CPW301(T) clusters in a phylogenetic branch that contains Herbaspirillum species . 16S rDNA sequence similarity of strain CPW301(T) to species of the genus Herbaspirillum with validly published names is in the range 98.7-98.9 % . Despite the considerably high 16S rDNA sequence similarity, strain CPW301(T) could be distinguished clearly from type strains of Herbaspirillum species with validly published names by DNA-DNA relatedness values, which were <15.7 % . The genomic DNA G+C content of strain CPW301(T) is 61.3 mol% . The predominant ubiquinone is Q-8 and the major cellular fatty acids are C(16 : 0) and cyclo-C(17 : 0) . The strain does not fix nitrogen and is not plant-associated . It is an aerobic rod with one unipolar flagellum . On the basis of these characteristics, a novel Herbaspirillum species, Herbaspirillum chlorophenolicum sp . nov., is proposed . The type strain of the novel species is strain CPW301(T) (=KCTC 12096(T)=IAM 15024(T)).

Water Res, 2004 May, 38(9), 2230 - 9
Predicting the rate and extent of cadmium and copper desorption from soils in the presence of bacterial extracellular polymer; Jensen-Spaulding A et al.; The movement of cationic transition metals through the subsurface is strongly retarded by sorption to the porous media . However, dissolved organic ligands can compete with soil surfaces by providing binding sites for metals in solution . An extracellular polymer produced by a bacterium isolated from soil was used in this study to observe and model the influence of a naturally occurring ligand on the release of adsorbed metals from two test soils . Experimental results show that the presence of dissolved extracellular polymer enhanced the rate and extent of desorptive release of soil-bound cadmium and copper . A kinetic model that uses a gamma distribution of rate constants to account for the physical and chemical heterogeneity of the soil matrix was employed to describe the release of cadmium and copper in batch experiments . Model parameters describing soil, metal and extracellular polymer interactions were obtained through separate experiments . With these parameters the model successfully predicted the influence of dissolved polymer on the rate and extent of release of cadmium and copper from soil in independent batch experiments . These results suggest that the presence of natural metal-binding ligands such as bacterial extracellular polymers can act to increase the driving force for desorption by lowering the aqueous concentration of free unbound metals in solution.

J Biol Chem, 2004 Jul 16, 279(29), 30073 - 80 Epub 2004 May 11.
Thermotoga maritima-Escherichia coli chimeric topoisomerases . Answers about involvement of the carboxyl-terminal domain in DNA topoisomerase I-mediated catalysis; Viard T et al.; Bacterial topoisomerases I are generally composed of two domains as follows: a core domain, which contains all the conserved motifs involved in the trans-esterification reactions, and a carboxyl-terminal domain, highly variable in size and sequence . In the present work, we have addressed the question of the respective roles of the two domains in the different steps of the topoisomerization cycle . For this purpose, we prepared various recombinant topoisomerases from two model enzymes: topoisomerase I from the hyperthermophilic bacterium Thermotoga maritima and topoisomerase I from Escherichia coli . We compared the properties of the two core domains to that of the topoisomerases formed by combining the core domain of one enzyme to the carboxyl-terminal domain of the other . We found that, contrary to E . coli (Lima, C . D., Wang, J . C., and Mondragon, A . (1993) J . Mol . Biol . 232, 1213-1216), the core domain from T . maritima (TmTop65) is able to sustain by itself a complete topoisomerization cycle, although with low efficiency . Fusion of TmTop65 to the entire carboxyl-terminal domain from E . coli considerably increases binding efficiency, thermal stability, and DNA relaxation activity . Moreover, the chimera predominantly acquires the cleavage specificity of E . coli full-length topoisomerase . For the chimera obtained by fusion of the T . maritima carboxyl-terminal domain to the core EcTop67, very low DNA relaxation activity and binding are recovered, but formation of a covalent DNA adduct is impaired . Taken together, our results show that the presence and the nature of the carboxyl-terminal domain of bacterial topoisomerases I strongly determine their DNA binding efficiency and cleavage specificity but is not strictly required for strand passage.

BMC Genet . 2004 May 12;5(1):10.
The sex ratio distortion in the human head louse is conserved over time; Perotti MA et al.; BACKGROUND: At the turn of the 19th century the first observations of a female-biased sex ratio in broods and populations of the head louse, Pediculus humanus capitis, had been reported . A study by Buxton in 1940 on the sex ratio of lice on prisoners in Ceylon is still today the subject of reanalyses . This sex ratio distortion had been detected in ten different countries . In the last sixty years no new data have been collected, especially on scalp infestations under economically and socially more developed conditions . RESULTS: Here we report a female bias of head lice in a survey of 480 school children in Argentina . This bias is independent of the intensity of the pediculosis, which makes local mate competition highly unlikely as the source of the aberrant sex ratio; however, other possible adaptive mechanisms cannot be discounted . These lice as well as lice from pupils in Britain were carrying several strains of the endosymbiotic bacterium Wolbachia pipientis, one of the most wide spread intracellular sex ratio distorters . Similar Wolbachia strains are also present in the pig louse, Haematopinus suis, suggesting that this endosymbiont might have a marked influence on the biology of the whole order . The presence of a related obligate nutritional bacterium in lice prevents the investigation of a causal link between sex ratio and endosymbionts . CONCLUSIONS: Regardless of its origin, this sex ratio distortion in head lice that has been reported world wide, is stable over time and is a remarkable deviation from the stability of frequency-dependent selection of Fisher's sex ratio . A female bias first reported in 1898 is still present over a hundred years and a thousand generations later.

Clin Diagn Lab Immunol, 2004 May, 11(3), 577 - 80
Flow cytometry study of lymphocyte subsets in malnourished and well-nourished children with bacterial infections; Najera O et al.; Protein-energy malnutrition is the primary cause of immune deficiency in children across the world . It has been related to changes in peripheral T-lymphocyte subsets . The aim of the present study was to evaluate the effects of infection and malnutrition on the proportion of peripheral-lymphocyte subsets in well-nourished non-bacterium-infected (WN), well-nourished bacterium-infected (WNI), and malnourished bacterium-infected (MNI) children by flow cytometry . A prospectively monitored cohort of 15 MNI, 12 WNI, and 17 WN children was studied . All the children were 3 years old or younger and had only bacterial infections . Results showed a significant decrease in the proportion of T CD3(+) (P < 0.05 for relative and P < 0.03 for absolute values), CD4(+) (P < 0.01 for relative and absolute values), and CD8(+) (P < 0.05 for relative values) lymphocyte subsets in WNI children compared to the results seen with WN children . Additionally, B lymphocytes in MNI children showed significant lower values (CD20(+) P < 0.02 for relative and P < 0.05 for absolute values) in relation to the results seen with WNI children . These results suggest that the decreased proportions of T-lymphocyte subsets observed in WNI children were associated with infection diseases and that the incapacity to increase the proportion of B lymphocyte was associated with malnutrition . This low proportion of B lymphocytes may be associated with the mechanisms involved in the immunodeficiency of malnourished children.

J Environ Sci (China), 2004, 16(2), 343 - 7
Characterization of a strain of Sphingobacterium sp . and its degradation to herbicide mefenacet; Ye YF et al.; A bacterium (designated strain Y1) degrading acetanilide herbicide mefenacet was isolated from aerobic sludge . Based on the analyses of partial 16S rRNA gene, cellular fatty acid and BIOLOG-GN, and general physiological and biochemical characteristics, strain Y1 was identified as Sphingobacterium multivolum . Strain Y1 was able to degrade mefenacet used as sources of carbon and energy . Degradation of mefenacet was accompanied by producing the metabolites N-methylaniline and an unidentified compound with molecular weight 205, indicating a metabolic pathway of mefenacet initiated by hydrolysis of amido bond.

Biopolymers, 2004 May-Jun 5, 74(1-2), 96 - 9
Anomalous acceleration of the photocycle in photosynthetic reaction centers inhibited on the acceptor side; Gerencser L et al.; The rate of the photocycle (quinone reduction cycle) was measured under continuous light excitation in an isolated reaction center protein of the photosynthetic bacterium Rhodobacter sphaeroides . The rate is determined by the slowest step of the photocycle, which could be the photochemistry (charge separation), the quinone/quinol and cytochrome c(2+)/c(3+) exchanges, or proton delivery to the secondary quinone . The photocycle was driven by high light intensity of a laser diode (5 W/cm(2) at 808 nm) to avoid light limitation of the observed rate . The fast turnover of the reaction center (up to 10(3) s(-1)) was slowed down by inhibition of the proton delivery to the secondary quinone by transition metal ions (Cd(2+) and Ni(2+)), by mutation of a key protonatable group (L213Asp --> Asn), or by use of low-affinity ubiquinone (UQ(0)) to the secondary quinone binding site . Although in all of these cases the rate of turnover was 2-3 orders of magnitude less than that of the primary photochemistry, marked light intensity dependence was observed . The rate of the photocycle increased from 7 s(-1) (Ni(2+), low light intensity) to 27 s(-1) (high light intensity) at pH 8.4 . The anomalous reacceleration is due to alternative events on the acceptor side induced by continuous excitation . We argue that the continuous excitation of the protein trapped in the reduced acceptor (Q(A)(-)Q(B)(-)) state produces short-lived reduced bacteriopheophytin (I(-)) that delivers activation energy to anomalous changes on the acceptor side as second interquinone electron transfer before proton uptake or increase of the quinone dissociation constant .

Biopolymers, 2004 May-Jun 5, 74(1-2), 92 - 5
Delayed fluorescence from the photosynthetic reaction center measured by electronic gating of the photomultiplier; Filus Z et al.; The decay of the delayed fluorescence (920 nm) of reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides R26 in the P(+)Q(A)(-) charge-separated state (P and Q(A) are the primary donor and quinone, respectively) has been monitored in a wide (100 ns to 100 ms) time range . The photomultiplier (Hamamatsu R3310-03) was protected from the intense prompt fluorescence by application of gating potential pulses (-280 V) to the first, third, and fifth dynodes during the laser pulse . The gain of the photomultiplier dropped transiently by a factor of 1 x 10(6) . The delayed fluorescence showed a smooth but nonexponential decay from 100 ns to 1 ms that was explained by the relaxation of the average free energy between P* and P(+)Q(A)(-) changing from -580 to -910 meV . This relaxation is due to the slow protein response to charge separation and can be described by a Kohlrausch relaxation function with time constant of 65 micros and a stretching exponent of alpha = 0.45 .

Biopolymers, 2004 May-Jun 5, 74(1-2), 64 - 8
Structural characterization of glutamine synthetase from Azospirillum brasilense; Kamnev AA et al.; CD spectroscopic study of the secondary structure of partly adenylylated glutamine synthetase (GS) of the bacterium Azospirillum brasilense showed both the native and cation-free (EDTA-treated) enzyme to be highly structured (58 and 49% as alpha-helices, 10 and 20% as beta-structure, respectively) . Mg(2+), Mn(2+), or Co(2+), when added to the native GS, had little effect on its CD spectrum, whereas their effects on the cation-free GS were more pronounced . Emission ((57)Co) Mossbauer spectroscopic (EMS) study of (57)Co(2+)-doped cation-free GS in frozen solution and in the dried state gave similar spectra and Mossbauer parameters for the corresponding spectral components, reflecting the ability of the Co(2+)-enzyme complex to retain its properties upon drying . The EMS data show that (a) A . brasilense GS has 2 cation-binding sites per active center and (b) one site has a higher affinity to Co(2+) than the other, in line with the data on other bacterial GSs .

Proc Natl Acad Sci U S A, 2004 May 18, 101(20), 7600 - 5 Epub 2004 May 07.
Bacteriophage capsids: tough nanoshells with complex elastic properties; Ivanovska IL et al.; The shell of bacteriophages protects the viral DNA during host-to-host transfer and serves as a high-pressure container storing energy for DNA injection into a host bacterium . Here, we probe the mechanical properties of nanometer-sized bacteriophage phi 29 shells by applying point forces . We show that empty shells withstand nanonewton forces while being indented up to 30% of their height . The elastic response varies across the surface, reflecting the arrangement of shell proteins . The measured Young's modulus (approximately 1.8 GPa) is comparable with that of hard plastic . We also observe fatigue and breakage of capsids after probing them repetitively . These results illustrate the mechanoprotection that viral shells provide and also suggest design principles for nanotechnology.

J Eukaryot Microbiol, 2003 Jul-Aug, 50(4), 293 - 8
The endosymbiotic bacterium Holospora obtusa enhances heat-shock gene expression of the host Paramecium caudatum; Hori M et al.; The bacterium Holospora obtusa is a macronuclear-specific symbiont of the ciliate Paramecium caudatum . H . obtusa-bearing paramecia could survive even after the cells were quickly heated from 25 degrees C to 35 degrees C . To determine whether infection with H . obtusa confers heat shock resistance on its host, we isolated genes homologous to the heat shock protein genes hsp60 and hsp70 from P . caudatum . The deduced amino acid sequences of both cDNAs were highly homologous to hsp family sequences from other eukaryotes . Competitive PCR showed that H . obtusa-free paramecia expressed only trace amounts of hsp60 and hsp70 mRNA at 25 degrees C, but that expression of hsp70 was enhanced immediately after the cells were transferred to 35 degrees C . H . obtusa-bearing paramecia expressed high levels of hsp7O mRNA even at 25 degrees C and the level was further enhanced when the cells were incubated at 35 degrees C . In contrast, the expression pattern of hsp60 mRNA was the same in H . obtusa-bearing as in H . obtusa-free paramecia . These results indicate that infection with its endosymbiont can confer a heat-shock resistant nature on its host cells.

J Clin Microbiol, 2004 May, 42(5), 2036 - 42
Clonal diversity and stability of subgingival Eikenella corrodens; Fujise O et al.; Eikenella corrodens is a commensal subgingival bacterium commonly found in both periodontally nondiseased and diseased subjects . The present study examined the clonal diversity and stability of subgingival E . corrodens over time . Ninety-five subjects were enrolled at the baseline examination, including 44 periodontally nondiseased subjects and 51 subjects with aggressive periodontitis . Twenty-two nondiseased subjects and 11 subjects with aggressive periodontitis were subsequently reexamined after an average interval of 14 months . Two subgingival plaque samples were obtained from each subject to determine the total cultivable bacteria . In addition, multiple E . corrodens isolates from each sample were recovered for clonal analysis by arbitrarily primed PCR . The mean numbers of distinct E . corrodens clones harbored by nondiseased subjects and subjects with aggressive periodontitis were 1.3 and 3.0, respectively . Thirty-nine percent of the nondiseased subjects and 63% of the subjects with aggressive periodontitis harbored multiple clones of E . corrodens . The numbers of distinct E . corrodens clones increased significantly (Mann-Whitney ranking test, P < 0.05) in sites from patients with aggressive periodontitis, in sites with pocket depths of 4 mm or greater, in sites with a clinical attachment loss of 2 mm or greater, and in sites coinfected with Porphyromonas gingivalis . Comparison of E . corrodens clones recovered at the baseline and those recovered at the follow-up examination showed that E . corrodens colonization was not stable . Thirty-eight of the 66 follow-up samples (58%) showed a complete change (including de novo colonization of the sites or complete elimination of the organism from the sites) of the subgingival E . corrodens clonal types between the baseline and the follow-up examinations . Our results suggest a complexity of subgingival microbiota not seen previously.

Proc R Soc Lond B Biol Sci, 2004 Mar 7, 271(1538), 509 - 15
Natural interspecific and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia in Trichogramma wasps; Huigens ME et al.; The intracellular bacterium Wolbachia is one of the most common symbionts in arthropods and, because of its manipulative effects on host reproduction, is assumed to be an important factor in several evolutionary processes . These bacteria are mainly vertically transmitted from mother to daughter through the egg cytoplasm, and horizontal transmission is generally assumed to be rare . Here, we show natural inter- and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia between parasitoid wasps of the genus Trichogramma . Horizontal transfer was observed when infected and uninfected larvae shared the same host egg . This is the first report, to our knowledge, on interspecific horizontal transfer of Wolbachia between closely related sympatric species . Some originally uninfected immature wasps acquired Wolbachia while inside the host egg, but not all of these newly infected females exhibited the parthenogenesis phenotype . In general, intraspecific horizontal transfer was more successful than interspecific transfer . Wolbachia underwent vertical transmission in the new species but the infection tended to be lost within several generations . Our results have important implications for understanding the evolution of Wolbachia-host associations.

Appl Environ Microbiol, 2004 May, 70(5), 3163 - 6
Degradation of polycyclic aromatic hydrocarbons by a newly discovered enteric bacterium, Leclercia adecarboxylata; Sarma PM et al.; A bacterial strain, PS4040, capable of degrading polycyclic aromatic hydrocarbons for use as the sole carbon source was isolated from oily-sludge-contaminated soil . The 16S rRNA gene showed 98.8% homology to that of Leclercia adecarboxylata . Comparative molecular typing with the clinical strain of L . adecarboxylata revealed that there were few comigrating and few distinct amplimers among them.

Appl Environ Microbiol, 2004 May, 70(5), 2941 - 51
Viral activity in two contrasting lake ecosystems; Bettarel Y et al.; For aquatic systems, especially freshwaters, there is little data on the long-term (i.e., >6-month period) and depth-related variability of viruses . In this study, we examined virus-induced mortality of heterotrophic bacteria over a 10-month period and throughout the water column in two lakes of the French Massif Central, the oligomesotrophic Lake Pavin and the eutrophic Lake Aydat . Concurrently, we estimated nonviral mortality through heterotrophic nanoflagellate and ciliate bacterivory . Overall, viral infection parameters were much less variable than bacterial production . We found that the frequency of visibly infected cells (FVIC), estimated using transmission electron microscopy, peaked in both lakes at the end of spring (May to June) and in early autumn (September to October) . FVIC values were significantly higher in Lake Pavin (mean {M} = 1.6%) than in Lake Aydat (M = 1.1%), whereas the opposite trend was observed for burst sizes, which averaged 25.7 and 30.2 virus particles bacterium(-1), respectively . We detected no significant depth-related differences in FVIC or burst size . We found that in both lakes the removal of bacterial production by flagellate grazing (M(Pavin) = 37.7%, M(Aydat) = 18.5%) was nearly always more than the production removed by viral lysis (M(Pavin) = 16.2%, M(Aydat) = 19%) or ciliate grazing (M(Pavin) = 2.7%, M(Aydat) = 8.8%) . However, at specific times and locations, viral lysis prevailed over protistan grazing, for example, in the anoxic hypolimnion of Lake Aydat . In addition, viral mortality represented a relatively constant mortality source in a bacterial community showing large variations in growth rate and subject to large variations in loss rates from grazers . Finally, although viruses did not represent the main agent of bacterial mortality, our data seem to show that their relative importance was higher in the less productive system.

Appl Environ Microbiol, 2004 May, 70(5), 2880 - 5
Assembly of G protein-coupled receptors onto nanosized bacterial magnetic particles using Mms16 as an anchor molecule; Yoshino T et al.; G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery . GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation . In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1 . A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies . Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region . D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule . D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants . After washing, the complexes were ready to use for analysis . This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption.

Appl Environ Microbiol, 2004 May, 70(5), 2596 - 602
A novel alpha-Proteobacterium resides in the mitochondria of ovarian cells of the tick Ixodes ricinus; Beninati T et al.; An intracellular bacterium from Ixodes ricinus ticks collected in Italy was characterized by electron microscopy (EM), PCR sequencing of the 16S rRNA gene, molecular phylogenetic analysis, and in situ hybridization (ISH) . This bacterium was shown by EM to be present in the cytoplasm, as well as in the mitochondria of ovarian cells . When universal 16S rRNA bacterial primers were used, PCR amplification of ovarian DNA followed by cloning and sequencing resulted in the same sequence being found in each sample . Phylogenetic analysis of this sequence showed that the bacterium from which it was derived, tentatively designated IricES1, is part of a novel clade in the alpha subdivision of the Proteobacterium: ISH and PCR assays of various tissues performed with oligonucleotides specific for the IricES1 16S rRNA showed that IricES1 is restricted to ovarian cells . Based on the results obtained, we inferred that the bacteria seen by EM in ovarian cells are a single type of bacteria, corresponding to IricES1 . PCR screening of 166 ticks from various parts of Italy and one site in England showed that IricES1 was present in 96% of adult females and 44% of nymphs (unsexed) . No adult males were found to be infected . Despite the apparent parasitism of host mitochondria by IricES1, the available information suggests that the bacterium has an obligate relationship with its host, although this must be confirmed.

J Clin Gastroenterol, 2004 Mar, 38(3), 248 - 51
Ideology of Helicobacter pylori prevalence in peptic ulcer disease in an inner-city minority population; Kalaghchi B et al.; GOALS AND BACKGROUND: The prevalence of Helicobacter pylori infection among patients with peptic ulcer disease has been reported to range from 61 to 94% . Recent studies show a reduction in the prevalence of H . pylori infection in patients with peptic ulcer disease . This study was conducted to determine the prevalence of H . pylori infection in peptic ulcer disease in an inner-city hospital in Washington, DC . METHODS: Medical records of all patients who had undergone upper gastrointestinal endoscopy from July 1997 through June 1999 were reviewed . All patients who had gastric ulcer and/or duodenal ulcer on upper gastrointestinal endoscopy were studied . Demographic characteristics, history of nonsteroidal antiinflammatory drug ingestion, alcohol consumption, and associated diseases were studied . H . pylori was considered to be present if CLOtest and/or histopathology were positive for H . pylori . Patients with negative pathology for H . pylori or negative pathology and CLOtest were considered negative for H . pylori . RESULTS: One-hundred fifty-six patients were found to have gastric and/or duodenal ulcers . Fifty-one ulcer patients did not meet the inclusion criteria and were excluded . Among the 105 patients who were included in the study, gastric ulcers were found in 48 patients (45.7%), duodenal ulcers were found in 46 patients (43.8%), and both gastric and duodenal ulcers were found in 11 patients (10.5%) . H . pylori was present in 66.7% of gastric ulcer patients and in 69.5% of duodenal ulcer patients . Antral histology and CLOtest were in agreement 96% of the time . CONCLUSIONS: At the District of Columbia General Hospital, an inner-city hospital serving predominantly an African-American community, the prevalence of H . pylori in ulcer patients compares similarly to other more recent studies that have found a decreased prevalence of this bacterial infection in ulcer patients . This suggests that the treatment of H . pylori in minority patients is reducing the proportion of ulcers due to this bacterium, as has been seen with the majority population.

Biotechnol Lett, 2004 Mar, 26(6), 479 - 86
Biotransformation of fluorene, diphenyl ether, dibenzo-p-dioxin and carbazole by Janibacter sp; Yamazoe A et al.; Fluorene, diphenyl ether, dibenzo-p-dioxin, and carbazole were used by a dibenzofuran-utilizing Janibacter sp . strain YY-1 . Metabolites were identified by GC-MS . Angular dioxygenation was the major pathway for degradation of fluorene, diphenyl ether, and dibenzo-p-dioxin but not for carbazole . Lateral dioxygenation of all tested compounds was indicated by the detection of mono- or di-hydroxylated compounds . The bacterium also catalyzed the monooxygenation of fluorene at the C9 position.

J Bacteriol, 2004 May, 186(10), 3143 - 52
Involvement of the C-terminal extension of the alpha polypeptide and of the PucC protein in LH2 complex biosynthesis in Rubrivivax gelatinosus; Steunou AS et al.; The facultative phototrophic nonsulfur bacterium Rubrivivax gelatinosus exhibits several differences from other species of purple bacteria in the organization of its photosynthetic genes . In particular, the puc operon contains only the pucB and pucA genes encoding the beta and alpha polypeptides of the light-harvesting 2 (LH2) complex . Downstream of the pucBA operon is the pucC gene in the opposite transcriptional orientation . The transcription of pucBA and pucC has been studied . No pucC transcript was detected either by Northern blotting or by reverse transcription-PCR analysis . The initiation site of pucBA transcription was determined by primer extension, and Northern blot analysis revealed the presence of two transcripts of 0.8 and 0.65 kb . The half-lives of both transcripts are longer in cells grown semiaerobically than in photosynthetically grown cells, and the small transcript is the less stable . It was reported that the alpha polypeptide, encoded by the pucA gene, presents a C-terminal extension which is not essential for LH2 function in vitro . The biological role of this alanine- and proline-rich C-terminal extension in vivo has been investigated . Two mutants with C-terminal deletions of 13 and 18 residues have been constructed . Both present the two pucBA transcripts, while their phenotypes are, respectively, LH2+ and LH2-, suggesting that a minimal length of the C-terminal extension is required for LH2 biogenesis . Another important factor involved in the LH2 biogenesis is the PucC protein . To gain insight into the function of this protein in R . gelatinosus, we constructed and characterized a PucC mutant . The mutant is devoid of LH2 complex under semiaerobiosis but still produces a small amount of these antennae under photosynthetic growth conditions . This conditional phenotype suggests the involvement of another factor in LH2 biogenesis.

Mol Biol (Mosk), 2004 Mar-Apr, 38(2), 317 - 22
{Model of aggregation of pigments in the chlorosomal antenna of the green bacteria Chloroflexus aurantiacus}; Mauring K et al.; Independent experimental and theoretical evaluation was performed for the adequacy of our previously proposed general molecular model of structural organization of light-harvesting pigments in chlorosomal bacteriochlorophyll (BChl) c/d/e-containing superantenna of different green bacteria . Simultaneous measurement of hole burning in the optical spectra of chlorosomal BChl c and temperature dependence of steady-state fluorescence spectra of BChl c was accomplished in intact cells of photosynthetic green bacterium Chloroflexus aurantiacus; this allows unambiguous determination of the structure of exciton levels of BChl c oligomers in this natural antenna, which is a fundamental criterion for adequacy of any molecular model for in vivo aggregation of antenna pigments . Experimental data were shown to confirm our model of organization of oligometric pigments in chlorosomal BChl c antenna of green bacterium Chloroflexus aurantiacus . This model, which is based on experimental data and our theory of spectroscopy of oligomeric pigments, implies that the unit building block of BChl c antenna is a cylindrical assembly containing six excitonically coupled linear pigment chains whose exciton structure with intense upper levels provides for the optimal spectral properties of the light-harvesting antenna.

Prikl Biokhim Mikrobiol, 2004 Mar-Apr, 40(2), 201 - 9
{Dynamics of activity of the key enzymes of polyhydroxyalkanoate metabolism in Ralstonia eutropha}; Volova TG et al.; The dynamics of accumulation of polyhydroxybutyrate (PHB) and the activities of the key enzymes of PHB metabolism (beta-ketothiolase, acetoacetyl-CoA reductase, PHA synthase, D-hydroxybutyrate dehydrogenase, and PHA depolymerase) in the hydrogen bacterium Ralstonia eutropha B5786 were studied under various conditions of carbon nutrition and substrate availability . The highest activities of beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase were recorded at the stage of acceleration of PHB synthesis . The activities of enzymes catalyzing PHB depolymerization (PHB depolymerase and D-hydroxybutyrate dehydrogenase) were low, being expressed only at stimulated endogenous PHB degradation . The change of carbon source (CO2 or fructose) did not cause any marked changes in the time course of enzyme activity.

J Mol Biol, 2004 May 21, 339(1), 103 - 16
Structural studies of the Nudix hydrolase DR1025 from Deinococcus radiodurans and its ligand complexes; Ranatunga W et al.; We have determined the crystal structure, at 1.4A, of the Nudix hydrolase DR1025 from the extremely radiation resistant bacterium Deinococcus radiodurans . The protein forms an intertwined homodimer by exchanging N-terminal segments between chains . We have identified additional conserved elements of the Nudix fold, including the metal-binding motif, a kinked beta-strand characterized by a proline two positions upstream of the Nudix consensus sequence, and participation of the N-terminal extension in the formation of the substrate-binding pocket . Crystal structures were also solved of DR1025 crystallized in the presence of magnesium and either a GTP analog or Ap(4)A (both at 1.6A resolution) . In the Ap(4)A co-crystal, the electron density indicated that the product of asymmetric hydrolysis, ATP, was bound to the enzyme . The GTP analog bound structure showed that GTP was bound almost identically as ATP . Neither nucleoside triphosphate was further cleaved.

J Mol Biol, 2004 May 21, 339(1), 41 - 51
Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA; Rangan P et al.; Self-splicing RNAs must evolve to function in their specific exon context . The conformation of a group I pre-tRNA(ile) from the bacterium Azoarcus was probed by ribonuclease T(1) and hydroxyl radical cleavage, and by native gel electrophoresis . Biochemical data and three-dimensional models of the pre-tRNA showed that the tRNA is folded, and that the tRNA and intron sequences form separate tertiary domains . Models of the active site before steps 1 and 2 of the splicing reaction predict that exchange of the external G-cofactor and the 3'-terminal G is accomplished by a slight conformational change in P9.0 of the Azoarcus group I intron . Kinetic assays showed that the pre-tRNA folds in minutes, much more slowly than the intron alone . The dependence of the folding kinetics on Mg(2+) and the concentration of urea, and RNase T(1) experiments showed that formation of native pre-tRNA is delayed by misfolding of P3-P9, including mispairing between residues in P9 and the tRNA . Thus, although the intron and tRNA sequences form separate domains in the native pre-tRNA, their folding is coupled via metastable non-native base-pairs . This could help prevent premature processing of the 5' and 3' ends of unspliced pre-tRNA.

J Infect Dis, 2004 May 15, 189(10), 1921 - 5 Epub 2004 Apr 26.
Anaplasma phagocytophilum ligation to toll-like receptor (TLR) 2, but not to TLR4, activates macrophages for nuclear factor-kappa B nuclear translocation; Choi KS et al.; Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils and causes human anaplasmosis (formerly human granulocytic ehrlichiosis) . Interferon (IFN)- gamma causes immunopathology in A . phagocytophilum infection models . Plasma IFN- gamma levels are elevated 4 h after infection in experimentally infected mice, which indicates innate immunity and possible Toll-like receptors (TLRs) . The ability of A . phagocytophilum to trigger proinflammatory responses via nuclear factor (NF)- kappa B was tested in TLR2- and TLR4-transfected cell lines and in primary murine macrophages devoid of TLR2 or TLR4 . NF- kappa B was activated only through TLR2, which suggests its role in innate immune induction with A . phagocytophilum infections . The role of innate immunity in human anaplasmosis immunopathology requires more study.

J Biotechnol, 2004 May 27, 110(2), 181 - 99
Modeling and experimental validation of the signal transduction via the Escherichia coli sucrose phospho transferase system; Sauter T et al.; Bacterial signal processing was investigated concerning the sucrose phosphotransferase system (sucrose PTS) in the bacterium Escherichia coli as an example . The about 20 different phosphotransferase systems (PTSs) of the cell fulfill besides the transport of various carbohydrates, also the function of one signal processing system . Extra- and intracellular signals are converted within the PTS protein chain to important regulatory signals affecting, e.g . carbon metabolism and chemotaxis . A detailed dynamical model of the sucrose PTS was developed describing transport and signal processing function . It was formulated using a detailed description of complex formation and phosphate transfer between the chain proteins . Model parameters were taken from literature or were identified with own experiments . Simulation studies together with experimental hints showed that the dynamic behavior of phosphate transfer in the PTS runs within 1 s . Therefore a description of steady state characteristics is sufficient for describing the signaling properties of the sucrose PTS . A steady state characteristic field describes the degree of phosphorylation of the PTS protein EIIACrr as a function of the input variables extracellular sucrose concentration and intracellular phosphoenolpyruvate (PEP):pyruvate ratio . The model has been validated with different experiments performed in a CSTR using a sucrose positive E . coli W3110 derivative . A method for determining intracellular metabolite concentrations has been developed . A sample preparation technique using a boiling ethanol buffer solution was successfully applied . The PTS output signal degree of phosphorylation of EIIACrr was also measured . Steady state conditions with varying dilution rate and dissolved oxygen concentration and dynamical variations applying different stimuli to the culture were considered . Pulse, and stop feeding experiments with limiting sucrose concentrations were performed . Simulation and experimental results matched well . The same holds for the expanded sucrose PTS and glycolysis model.

Ann Trop Med Parasitol, 2004 Apr, 98(3), 271 - 8
High mortality among patients with bacterial meningitis in a rural hospital in Tanzania; Wiersinga WJ et al.; Although the disease is an important cause of mortality in the region, most published reports on bacterial meningitis in East Africa are from urban referral hospitals . Poor laboratory facilities make diagnosis difficult in the area and treatment is limited to inexpensive antibiotics . The case-fatality 'rate' in one rural hospital in Tanzania, the Ndala Mission Hospital (NMH), appears to have increased dramatically over recent years, perhaps as the result of increasing resistance to ampicillin and chloramphenicol . The aim of the present study, which was partially retrospective and partially prospective, was to review the number, characteristics and outcome of children admitted to this hospital with bacterial meningitis and to investigate possible resistance of the causative micro-organisms to the antibiotics used . Data from the 181 children who were admitted with bacterial meningitis {confirmed by the examination of Gram-stained smears of cerebrospinal fluid (CSF)} between 1999 and 2002 were retrospectively reviewed . The overall mortality among these children was 51% . No seasonal pattern was observed in the number of cases . In a 2-month prospective study in 2002, CSF samples from 19 consecutive cases were collected in Trans-Isolate medium and shipped to the Academic Medical Center in Amsterdam for culture and analysis of antibiotic susceptibility . For only eight (42%) of the cases was there agreement between the species of bacterium identified, by Gram-staining, in Tanzania and that identified, by culture, in The Netherlands . As there was no evidence of resistance to ampicillin and the antibiotics used in the NMH were found to be of good quality, the cause of the high mortality in the NMH remains uncertain . Poor laboratory testing, long doctor-patient delays and/or poor drug administration on the wards may all be contributory factors . Attempts will now be made to address each of these problems.

Eur Arch Otorhinolaryngol . 2004 Apr 30; {Epub ahead of print}
Investigation of Helicobacter pylori colonization in laryngeal neoplasia; Akbayir N et al.; Helicobacter pylori has been investigated in several other organ systems and localizations such as the oral cavity, but has not been investigated extensively in squamous cell carcinoma of the larynx, a region that could be directly exposed to the bacterium by the oral route or gastro-esophageal reflux . Only serological studies are available regarding the relation between H . pylori and laryngeal cancer, yielding conflicting results . To our knowledge, there is no study investigating the presence of H . pylori in laryngeal squamous cell carcinoma tissue . The purpose of this study was to investigate the presence of H . pylori in laryngeal squamous cell carcinoma tissue and to investigate the possible role of this organism in the etiopathogenesis of laryngeal cancer . Specimens from 50 patients with laryngeal cancer who underwent total or partial laryngectomy between March 1999 and December 2002 were examined by histopathological and immunohistochemical methods to detect H . pylori . The presence of H . pylori was also investigated histopathologically in 50 benign laryngeal biopsy specimens . In our study, we demonstrated that H . pylori was not present in laryngeal squamous cell carcinoma tissue or in the benign lesions . We could not find any evidence indicating that H . pylori played a role at the tissue level in the pathogenesis of laryngeal carcinoma.

Hum Mol Genet, 2004 Jun 15, 13(12), 1249 - 55 Epub 2004 Apr 28.
Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli; Schwarz G et al.; Substitution therapies for orphan genetic diseases, including enzyme replacement methods, are frequently hampered by the limited availability of the required therapeutic substance . We describe the isolation of a pterin intermediate from bacteria that was successfully used for the therapy of a hitherto incurable and lethal disease . Molybdenum cofactor (Moco) deficiency is a pleiotropic genetic disorder characterized by the loss of the molybdenum-dependent enzymes sulphite oxidase, xanthine oxidoreductase and aldehyde oxidase due to mutations in Moco biosynthesis genes . An intermediate of this pathway-'precursor Z'-is more stable than the cofactor itself and has an identical structure in all phyla . Thus, it was overproduced in the bacterium Escherichia coli, purified and used to inject precursor Z-deficient knockout mice that display a phenotype which resembles that of the human deficiency state . Precursor Z-substituted mice reach adulthood and fertility . Biochemical analyses further suggest that the described treatment can lead to the alleviation of most symptoms associated with human Moco deficiency.

Photochem Photobiol, 2004 Mar, 79(3), 280 - 5
The role of carotenoids in the photoadaptation of the brown-colored sulfur bacterium Chlorobium phaeobacteroides; Hirabayashi H et al.; The brown-colored sulfur bacterium Chlorobium (Cb.) phaeobacteroides 1549 (new name, Chlorobaculum limnaeum 1549) contains many kinds of carotenoids as well as bacteriochlorophyll (BChl) e . These carotenoids were identified with C18-high-performance liquid chromatography, absorption, mass and proton nuclear magnetic resonance spectroscopies and were divided into two groups: the first is carotenoid with one or two phi-end groups such as isorenieratene and beta-isorenieratene and the second is carotenoid with one or two beta-end groups such as p-zeacarotene, beta-carotene and 7,8-dihydro-beta-carotene . The latter 7,8-dihydro-beta-carotene was found to be a novel carotenoid in nature . OH-gamma-Carotene glucoside laurate and OH-chlorobactene glucoside laurate were also found as minor components . The distribution of BChl e homologs in Cb . phaeobacteroides cultivated under various light intensities did not change, but the carotenoid to BChl e ratio changed markedly: carotenoid with the phi-end group maintained the same ratio to BChl e, whereas that with the beta-end group increased with increasing light intensity . The cells cultured under low-light intensity contained more phi-end carotenoids than beta-end . In Cb . phaeobacteroides the wavelength of the Qy band of BChl e aggregates did not change . We suggested that Cb . phaeobacteroides photoadapts to light intensity by changing the carotenoid composition.

Southeast Asian J Trop Med Public Health, 2003 Dec, 34(4), 723 - 6
Recombinant expression of Toxocara canis excretory-secretory antigen TES-120 in Escherichia coli; Fong MY et al.; The gene encoding the excretory-secretory antigen TES-120 of dog ascarid worm Toxocara canis was cloned into the bacterium Escherichia coli . The specificity of the recombinant TES-120 antigen produced by the bacterium was investigated . A total of 45 human serum samples from patients infected with differenthelminthes and protozoa, including 8 cases of toxocariasis, were tested against the recombinant antigens in immunoblot assays . The results from the assays revealed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only . This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.

J Clin Pathol, 2004 May, 57(5), 499 - 503
In vivo and in vitro studies on Anaplasma phagocytophilum infection of the myeloid cells of a patient with chronic myelogenous leukaemia and human granulocytic ehrlichiosis; Bayard-Mc Neeley M et al.; AIMS: The occurrence of human granulocytic ehrlichiosis (HGE) in a patient with chronic myelogenous leukaemia (CML) provided an opportunity to study whether Anaplasma phagocytophilum, the aetiological agent of HGE, infects mature or immature cells, both in vivo and in vitro . METHODS: Diagnosis of HGE was confirmed by culture, polymerase chain reaction (PCR), detection of intragranulocytic inclusions, and serology . The infection rates of different myelogenous stages of granulocytic differentiation were determined by microscopy . Anaplasma phagocytophilum infection of the bone marrow was analysed by PCR, culture, and microscopy . In addition, the in vitro growth of A phagocytophilum in the patient's granulocytes and in HL-60 cells (a promyelocytic leukaemia cell line) was compared . RESULTS: Pretreatment blood smears showed that mature granulocytic cells had a higher infection rate with A phagocytophilum than did immature cells . In the original inoculation of the patient's cells into HL-60 cells to isolate A phagocytophilum, the bacterium grew faster in the patient's leukaemic cells than in HL-60 cells . Anaplasma phagocytophilum inclusions were rarely seen in bone marrow granulocytes and PCR was negative . In vitro, two A phagocytophilum isolates grew faster in the patient's granulocytes than in HL-60 cells . CONCLUSIONS: The superior growth in CML cells compared with HL-60 cells suggests that A phagocytophilum preferentially infects mature granulocytes . The higher infection rate of the patient's mature versus immature granulocytes before treatment and the minimal level of infection of the patient's bone marrow support this . It is possible that the primary site of infection in HGE is the peripheral mature granulocytic population.

Biophys J, 2004 May, 86(5), 2650 - 9
Dynamic receptor team formation can explain the high signal transduction gain in Escherichia coli; Albert R et al.; Evolution has provided many organisms with sophisticated sensory systems that enable them to respond to signals in their environment . The response frequently involves alteration in the pattern of movement, either by directed movement, a process called taxis, or by altering the speed or frequency of turning, which is called kinesis . Chemokinesis has been most thoroughly studied in the peritrichous bacterium Escherichia coli, which has four helical flagella distributed over the cell surface, and swims by rotating them . When rotated counterclockwise the flagella coalesce into a propulsive bundle, producing a relatively straight "run," and when rotated clockwise they fly apart, resulting in a "tumble" which reorients the cell with little translocation . A stochastic process generates the runs and tumbles, and in a chemoeffector gradient, runs that carry the cell in a favorable direction are extended . The cell senses spatial gradients as temporal changes in receptor occupancy and changes the probability of counterclockwise rotation (the bias) on a fast timescale, but adaptation returns the bias to baseline on a slow timescale, enabling the cell to detect and respond to further concentration changes . The overall structure of the signal transduction pathways is well characterized in E . coli, but important details are still not understood . Only recently has a source of gain in the signal transduction network been identified experimentally, and here we present a mathematical model based on dynamic assembly of receptor teams that can explain this observation.

Int J Parasitol, 2004 May, 34(6), 733 - 46
Construction of bacterial artificial chromosome libraries from the parasitic nematode Brugia malayi and physical mapping of the genome of its Wolbachia endosymbiont; Foster JM et al.; The parasitic nematode, Brugia malayi, causes lymphatic filariasis in humans, which in severe cases leads to the condition known as elephantiasis . The parasite contains an endosymbiotic alpha-proteobacterium of the genus Wolbachia that is required for normal worm development and fecundity and is also implicated in the pathology associated with infections by these filarial nematodes . Bacterial artificial chromosome libraries were constructed from B . malayi DNA and provide over 11-fold coverage of the nematode genome . Wolbachia genomic fragments were simultaneously cloned into the libraries giving over 5-fold coverage of the 1.1 Mb bacterial genome . A physical framework for the Wolbachia genome was developed by construction of a plasmid library enriched for Wolbachia DNA as a source of sequences to hybridise to high-density bacterial artificial chromosome colony filters . Bacterial artificial chromosome end sequencing provided additional Wolbachia probe sequences to facilitate assembly of a contig that spanned the entire genome . The Wolbachia sequences provided a marker approximately every 10 kb . Four rare-cutting restriction endonucleases were used to restriction map the genome to a resolution of approximately 60 kb and demonstrate concordance between the bacterial artificial chromosome clones and native Wolbachia genomic DNA . Comparison of Wolbachia sequences to public databases using BLAST algorithms under stringent conditions allowed confident prediction of 69 Wolbachia peptide functions and two rRNA genes . Comparison to closely related complete genomes revealed that while most sequences had orthologs in the genome of the Wolbachia endosymbiont from Drosophila melanogaster, there was no evidence for long-range synteny . Rather, there were a few cases of short-range conservation of gene order extending over regions of less than 10 kb . The molecular scaffold produced for the genome of the Wolbachia from B . malayi forms the basis of a genomic sequencing effort for this bacterium, circumventing the difficult challenge of purifying sufficient endosymbiont DNA from a tropical parasite for a whole genome shotgun sequencing strategy.

Bioelectrochemistry, 2004 Jun, 63(1-2), 73 - 7
Electron transfer kinetics in photosynthetic reaction centers embedded in polyvinyl alcohol films; Francia F et al.; The coupling between electron transfer and protein dynamics has been studied at room temperature in isolated reaction centers (RCs) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in polyvinyl alcohol (PVA) films of different water/RC ratios . The kinetic analysis of charge recombination shows that dehydration of RC-containing PVA films causes reversible, inhomogeneous inhibition of electron transfer from the reduced primary quinone acceptor (Q(A)(-)) to the secondary quinone Q(B) . A more extensive dehydration of solid PVA matrices accelerates electron transfer from Q(A)(-) to the primary photooxidized electron donor P(+) . These effects indicate that incorporation of RCs into dehydrated PVA films hinders the conformational dynamics gating Q(A)(-) to Q(B) electron transfer at room temperature and slows down protein relaxation which stabilizes the primary charge-separated state P(+)Q(A)(-) . A comparison with analogous effects observed in trehalose-coated RCs suggests that protein motions are less severely reduced in PVA films than in trehalose matrices at comparable water/RC ratios.

Int J Food Microbiol, 2004 Apr 15, 92(2), 121 - 7
The use of chlorine dioxide to control Alicyclobacillus acidoterrestris spores in aqueous suspension and on apples; Lee SY et al.; Alicyclobacillus acidoterrestris, a thermoacidophilic, spore-forming bacterium, has been identified as a spoilage organism in commercial, pasteurized fruit juices . This study was undertaken to evaluate chlorine dioxide for reducing numbers of A . acidoterrestris spores on laboratory media and on apples . A . acidoterrestris spores in aqueous suspension and on apple surfaces of four different cultivars were treated with several concentrations of chlorine dioxide . Spores in aqueous suspension treated with 40 ppm for 5 min were reduced by more than 4 log . Treatment with 80 ppm for 1 min and 120 ppm for 30 s resulted in about 1.8 log and 4.8 log reductions of spore viability, respectively, and treatment at 80 and 120 ppm for 5 min reduced spore viability to undetectable levels (<0.7 log CFU/ml) . When applied to the surfaces of four different apple cultivars ('Red Delicious', 'Golden Delicious', 'Gala', and 'Fuji'), 40 ppm free chlorine dioxide reduced A . acidoterrestris spore numbers by 1.5, 3.2, 4.5, >4.8 log after 1-, 2-, 3-, and 4-min treatments, respectively . Spore numbers were reduced by >4.8 log with 120 ppm free chlorine dioxide after only 1-min treatment . However, there was no significant difference between apple cultivars (P>0.05) on spore reduction . These results show the great effectiveness of chlorine dioxide in controlling A . acidoterrestris spores both in aqueous suspension and on apple surfaces . There was no synergistic effect on spore reduction when chlorine dioxide treatment of aqueous suspension was followed by heat.

Extremophiles, 2004 Jun, 8(3), 237 - 42 Epub 2004 Apr 23.
Purification and some properties of superoxide dismutase from Deinococcus radiophilus, the UV-resistant bacterium; Yun YS et al.; The superoxide dismutase (SOD, EC 1.15.1.1) of Deinococcus radiophilus, a bacterium extraordinarily resistant to UV, ionizing radiations, and oxidative stress, was purified 1,920-fold with a 58% recovery yield from the cell-free extract of stationary cells by steps of ammonium sulfate fractionation and Superdex G-75 gel-filtration chromatography . A specific activity of the purified enzyme preparation was ca . 31,300 U mg(-1) protein . D . radiophilus SOD is Mn/FeSOD, judging by metal analysis and its insensitivity to cyanide and a partial sensitivity to H2O2 . The molecular weights of the purified enzyme estimated by gel chromatography and polyacrylamide gel electrophoresis are 51.5+/-1 and 47.1+/-5 kDa, respectively . The SOD seems to be a homodimeric protein with a molecular mass of 26 +/- 0.5 kDa per monomer . The purified native SOD showed very acidic pI of ca . 3.8 . The enzyme was stable at pH 5.0-11.0, but quite unstable below pH 5.0 . SOD was thermostable up to 40 degrees C, but a linear reduction in activity above 50 degrees C . Inhibition of the purified SOD activity by beta-naphthoquinone-4-sulfonic acid, rho-diazobenzene sulfonic acid, and iodine suggests that lysine, histidine, and tyrosine residues are important for the enzyme activity . The N-terminal peptide sequence of D . radiophilus Mn/FeSOD (MAFELPQLPYAYDALEPHIDA(> D) is strikingly similar to those of D . radiodurans MnSOD and Aerobacter aerogenes FeSOD .

Infect Immun, 2004 May, 72(5), 2889 - 98
Helicobacter pylori induces apoptosis of macrophages in association with alterations in the mitochondrial pathway; Menaker RJ et al.; Helicobacter pylori is a gastric bacterial pathogen that evades host immune responses in vivo and is associated with the development of gastritis, peptic ulcer disease, and gastric cancers . Induction of macrophage apoptosis is a method employed by multiple pathogens to escape host immune responses . Therefore, we hypothesized that H . pylori induces apoptosis of infected macrophages . RAW 264.7 cells were infected with H . pylori strain 60190, and apoptosis was assessed . Transmission electron microscopy and fluorescence microscopy showed that infected macrophages displayed morphological features characteristic of apoptosis . Quantification by acridine orange-ethidium bromide fluorescent-dye staining showed that apoptosis was dose and time dependent, and apoptosis was further confirmed by increased binding of annexin V-fluorescein isothiocyanate (FITC) to externalized phosphatidylserine of infected but not of control macrophages . Macrophages infected with isogenic mutants of H . pylori strain 60190 deficient in either cagA or vacA induced significantly less apoptosis than the parental strain, as assessed by increased binding of annexin V-FITC . Western blot analysis of whole-cell protein lysates revealed that infection with strain 60190 induced a time-dependent increase in cleavage of procaspase 8 and disappearance of full-length Bid compared with uninfected cells . Furthermore, pharmacological inhibition of caspase 8 caused a decrease in levels of apoptosis . Finally, infection caused a time-dependent increase in mitochondrial-membrane permeability and release of cytochrome c into the cytosol . These results suggest that H . pylori induces apoptosis of macrophages in association with alterations in the mitochondrial pathway . Elimination of this key immunomodulatory cell may represent a mechanism employed by the bacterium to evade host immune responses.

Infect Immun, 2004 May, 72(5), 2803 - 9
Impact of methoxymycolic acid production by Mycobacterium bovis BCG vaccines; Belley A et al.; BCG vaccines are a family of closely related daughter strains of an attenuated isolate of Mycobacterium bovis derived by in vitro passage from 1908 to 1921 . During subsequent laboratory propagation of the vaccine strain until its lyophilization in 1961, BCG Pasteur underwent at least seven further genomic mutations . The impact of these mutations on the properties of the vaccine is currently unknown . One mutation, a glycine-to-aspartic acid substitution in the mmaA3 gene, occurred between 1927 and 1931 and impairs methoxymycolic acid synthesis in BCG strains obtained from the Pasteur Institute after this period . Mycolic acids of the cell wall are classified into three functional groups (alpha-, methoxy-, and ketomycolic acids), and together these lipids form a highly specialized permeability barrier around the bacterium . To explore the impact of methoxymycolic acid production by BCG strains, we complemented the functional gene of mmaA3 into BCG Denmark and tested a number of in vitro and in vivo phenotypes . Surprisingly, restoration of methoxymycolic acids alone had no effect on cell wall permeability, resistance to antibiotics, or growth in cultured macrophages and C57BL/6 mice . Our results demonstrate that the loss of methoxymycolic acid production did not apparently affect the virulence of BCG strains.

Infect Immun, 2004 May, 72(5), 2698 - 702
Essential role for the gtfA gene encoding a putative glycosyltransferase in the adherence of Porphyromonas gingivalis; Narimatsu M et al.; Porphyromonas gingivalis, an oral bacterium, might play a role in the pathogenesis or progression of adult periodontitis . In this study, we isolated from P . gingivalis a putative glycosyltransferase gene, designated gtfA, which had a consensus domain for glycosyltransferase in its N terminus . GtfA consisted of 248 amino acids and its predicted molecular mass was 28 kDa; however, as the molecular mass of endogenous GtfA protein was around 40 kDa, this suggested that GtfA had undergone some posttranslational modifications . To reveal the role of the gtfA gene in P . gingivalis, we established gtfA-deficient strains by allelic replacement . Morphologically, gtfA-deficient P . gingivalis lacked mature fimbriae . gtfA-deficient P . gingivalis also showed a very low ability for autoaggregation, and its ability to attach to epithelial cells was severely impaired . Thus, the results indicate that the gtfA gene is required for P . gingivalis autoaggregation as well as attachment to epithelial cells . These results suggest that GtfA might have an important role in the pathogenicity of P . gingivalis by regulating adhesion.

Infect Immun, 2004 May, 72(5), 2564 - 73
Complement protein C3 binding to Mycobacterium tuberculosis is initiated by the classical pathway in human bronchoalveolar lavage fluid; Ferguson JS et al.; In high concentrations of fresh nonimmune human serum, Mycobacterium tuberculosis activates the alternative pathway of complement and binds C3 protein, resulting in enhanced phagocytosis by complement receptors on human alveolar macrophages . Yet in the lung, the alternative pathway of complement is relatively inactive compared to the classical pathway . To begin to determine whether C3 opsonophagocytosis of M . tuberculosis by alveolar macrophages can occur in the lung of the immunologically naive host, we characterized the binding of C3 to M . tuberculosis in different concentrations of fresh nonimmune human serum and concentrated human bronchoalveolar lavage fluid . Here we show that in human serum, C3 binding to M . tuberculosis is rapid, initiated by either the alternative pathway or the classical pathway, depending on the concentration of serum, and occurs by covalent linkages between the bacterial surface and the C3 cleavage products, C3b or C3bi . Human bronchoalveolar lavage fluid contains C3 protein and functional classical pathway activity that mediates the binding of C3 to the surface of M . tuberculosis . These studies provide evidence that when M . tuberculosis is first inhaled into the lungs of the human host, the bacterium is opsonized by C3 cleavage via classical pathway activation within the alveolus, providing a C3-dependent entry pathway into resident alveolar macrophages.

Genet Mol Res, 2004 Mar 31, 3(1), 85 - 91
Genetic analysis of violacein biosynthesis by Chromobacterium violaceum; Antonio RV et al.; Chromobacterium violaceum presents a distinctive phenotypic characteristic, the production of a deep violet pigment named violacein . Although the physiological function of this pigment is not well understood, the sequencing of the genome of this bacterium has given some insight into the mechanisms and control of violacein production . It was found that erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, is produced by the non-oxidative portion of the hexose monophosphate pathway, since it lacks 6-phosphogluconate dehydrogenase . All genes leading from E4P plus phosphoenolpyruvate to tryptophan are present in the genome . Nevertheless, these genes are not organized in an operon, as in E . coli, indicating that other mechanisms are involved in expression . The sequencing data also indicated the presence and organization of an operon for violacein biosynthesis . Three of the four gene products of this operon presented similarity with nucleotide-dependent monooxygenases and one with a limiting enzyme polyketide synthase . As previously suggested, genes encoding proteins involved in quorum sensing control by N-hexanoyl-homoserine-lactone, an autoinducer signal molecule, are present in the bacterial genome . These data should help guide strategies to increase violacein biosynthesis, a potentially useful molecule.

Genet Mol Res, 2004 Mar 31, 3(1), 64 - 75
Gene expression in Chromobacterium violaceum; Silva R et al.; The repertoire of 4,431 open reading frames (ORFs), eight rRNA operons and 98 tRNA genes of Chromobacterium violaceum must be expressed in a regulated manner for successful adaptation to a wide variety of environmental conditions . To accomplish this feat, the organism relies on protein machineries involved in transcription, RNA processing and translation . Analysis of the C . violaceum genome showed that transcription initiation, elongation and termination are performed by the five well-known RNA polymerase subunits, five categories of sigma 70 factors, one sigma 54 factor, as well as six auxiliary elongation and termination factors . RNA processing is performed by a variety of endonucleases and exonucleases, such as ribonuclease H, ribonuclease E, ribonuclease P, and ribonuclease III, in addition to poly(A) polymerase and specific methyltransferases and pseudouridine synthases . ORFs for all ribosomal proteins, except S22, were found . Only 19 aminoacyl-tRNA synthetases were found, in addition to three aminoacyl-tRNA synthetase-related proteins . Asparaginyl-tRNA (Asn) is probably obtained by enzymatic modification of a mischarged aminoacyl-tRNA . The translation factors IF-1, IF-2, IF-3, EF-Ts, EF-Tu, EF-G, RF-1, RF-2 and RF-3 are all present in the C . violaceum genome, although the absence of selB suggests that C . violaceum does not synthesize selenoproteins . The components of trans-translation, tmRNA and associated proteins, are present in the C . violaceum genome . Finally, a large number of ORFs related to regulation of gene expression were also found, which was expected, considering the apparent adaptability of this bacterium.

Biochim Biophys Acta, 2004 Apr 12, 1655(1-3), 71 - 6
Novel cyanide inhibition at cytochrome c1 of Rhodobacter capsulatus cytochrome bc1; Osyczka A et al.; Oxidized cytochrome c(1) in photosynthetic bacterium Rhodobacter capsulatus cytochrome bc(1) reversibly binds cyanide with surprisingly high, micromolar affinity . The binding dramatically lowers the redox midpoint potential of heme c(1) and inhibits steady-state turnover activity of the enzyme . As cytochrome c(1), an auxiliary redox center of the high-potential chain of cytochrome bc(1), does not interact directly with the catalytic quinone/quinol binding sites Q(o) and Q(i), cyanide introduces a novel, Q-site independent locus of inhibition . This is the first report of a reversible inhibitor that manipulates the energetics and electron transfers of the high-potential redox chain of cytochrome bc(1), while maintaining quinone substrate catalytic sites in an intact form.

Vet Res, 2004 Mar-Apr, 35(2), 233 - 41
Seroprevalence of Bartonella infection in American free-ranging and captive pumas (Felis concolor) and bobcats (Lynx rufus); Chomel BB et al.; Bartonella henselae is the main agent of cat scratch disease in humans and domestic cats are the main reservoir of this bacterium . We conducted a serosurvey to investigate the role of American wild felids as a potential reservoir of Bartonella species . A total of 479 samples (439 serum samples and 40 Nobuto strips) collected between 1984 and 1999 from pumas (Felis concolor) and 91 samples (58 serum samples and 33 Nobuto strips) collected from bobcats (Lynx rufus) in North America, Central America and South America were screened for B . henselae antibodies . The overall prevalence of B . henselae antibodies was respectively 19.4% in pumas and 23.1% in bobcats, with regional variations . In the USA, pumas from the southwestern states were more likely to be seropositive for B . henselae (prevalence ratio (PR) = 2.82, 95% confidence interval (CI) = 1.55, 5.11) than pumas from the Northwest and Mountain states . Similarly, adults were more likely to be B . henselae seropositive than juveniles and kittens (PR = 1.77, 95% CI = 1.07, 2.93) . Adult pumas were more likely to have higher B . henselae antibody titers than juveniles and kittens (p = 0.026) . B . henselae antibody prevalence was 22.4% (19/85) in bobcats from the USA and 33.3% (2/6) in the Mexican bobcats . In the USA, antibody prevalence varied depending on the geographical origin of the bobcats . In California, the highest prevalence was in bobcats from the coastal range (37.5%) . These results suggest a potential role of wild felids in the epidemiological cycle of Bartonella henselae or closely related Bartonella species.

J Exp Med, 2004 Apr 19, 199(8), 1077 - 87
Potentiation of C1 esterase inhibitor by StcE, a metalloprotease secreted by Escherichia coli O157:H7; Lathem WW et al.; The complement system is an essential component of host defense against pathogens . Previous research in our laboratory identified StcE, a metalloprotease secreted by Escherichia coli O157:H7 that cleaves the serpin C1 esterase inhibitor (C1-INH), a major regulator of the classical complement cascade . Analyses of StcE-treated C1-INH activity revealed that surprisingly, StcE enhanced the ability of C1-INH to inhibit the classical complement-mediated lysis of sheep erythrocytes . StcE directly interacts with both cells and C1-INH, thereby binding C1-INH to the cell surface . This suggests that the augmented activity of StcE-treated C1-INH is due to the increased concentration of C1-INH at the sites of potential lytic complex formation . Indeed, removal of StcE abolishes the ability of C1-INH to bind erythrocyte surfaces, whereas the proteolysis of C1-INH is unnecessary to potentiate its inhibitory activity . Physical analyses showed that StcE interacts with C1-INH within its aminoterminal domain, allowing the unaffected serpin domain to interact with its targets . In addition, StcE-treated C1-INH provides significantly increased serum resistance to E . coli K-12 over native C1-INH . These data suggest that by recruiting C1-INH to cell surfaces, StcE may protect both E . coli O157:H7 and the host cells to which the bacterium adheres from complement-mediated lysis and potentially damaging inflammatory events.

Appl Microbiol Biotechnol, 2004 Sep, 65(4), 457 - 64 Epub 2004 Apr 17.
Death of Escherichia coli during rapid and severe dehydration is related to lipid phase transition; Beney L et al.; This study reports the effects of exposure to increasing osmotic pressure on the viability and membrane structure of Escherichia coli . Changes in membrane structure after osmotic stress were investigated by electron transmission microscopy, measurement of the anisotropy of the membrane fluorescent probe DPH (1,6-diphenyl-1,3,5-hexatriene) inserted in E . coli, and Fourier infrared spectroscopy (FTIR) . The results show that, above a critical osmotic pressure of 35 MPa, the viability of the bacterium is drastically reduced (2 log decrease in survivors) . Electron micrographs revealed a severe contraction of the cytoplasm and the formation of membrane vesicles at 40 MPa . Changes in DPH anisotropy showed that osmotic dehydration to 40 MPa promoted a decrease in the membrane fluidity of integral cells of E . coli . FTIR measurements showed that at 10-40 MPa a transition from lamellar liquid crystal to lamellar gel among the phospholipids extracted from E . coli occurred . Bacterial death resulting from dehydration can be attributed to the conjunction between membrane deformation, caused by the volumetric contraction, and structural changes of the membrane lipids . The influence of the latter on the formation of membrane vesicles and on membrane permeabilization at lethal osmotic pressure is discussed, since vesiculation is hypothetically responsible for cell death.

Biochem Biophys Res Commun, 2004 May 14, 317(4), 1183 - 8
Isolation and characterization of the bacteriophage WO from Wolbachia, an arthropod endosymbiont; Fujii Y et al.; Wolbachia is a group of obligate symbiotic bacteria found in many insects and other arthropods . The presence of Wolbachia alters reproduction in the host, but the mechanisms are unknown . Molecular biological studies of Wolbachia have delayed significantly, and one of the reasons is the lack of transformation techniques of this bacterium . In the present study, bacteriophage particles were isolated from Wolbachia for the first time . The purified phage had an isometric head that was approximately 40 nm in diameter and contained linear double-stranded DNA of approximately 20 kbp . Partial sequence information (total of 20,484 bp) revealed that there were 24 open reading frames including a structural gene module, and genes for replication and lysogenic conversion . This bacteriophage is the only known mobile genetic element potentially used for transformation of Wolbachia.

Environ Pollut, 1990, 67(4), 361 - 74
Effect of lead, mercury and cadmium on a sulphate-reducing bacterium; Loka Bharathi PA et al.; A sulphate-reducing bacterial strain isolated from the south-west coast of India resembling Desulfosarcina in its physiology was tested for its behaviour towards HgCl(2), CdSO(4) and Pb(NO(3))(2) . The order of toxicity to growth of these metal salts in a lactate-based medium at 50 microg ml(-1) concentrations was Cd>Pb>Hg and to respiration Pb>Cd>Hg . Inhibitory concentrations (viz . 100 microg ml(-1) of HgCl(2) and 200 microg ml(-1) of Pb(NO(3)(2)) had a stimulatory effect when the substrate was changed to acetate . With sodium acetate at 0.1% concentration, Hg and Pb had maximum stimulatory effect for growth and sulphide production . Experiments conducted directly with sediment slurries amended with lactate showed that all three metals (at levels below their inhibitory concentrations, i.e . 50 microg ml(-1) of metal salt for Cd and Hg and 100 microg ml(-1) for Pb) inhibited sulphate-reducing activity (SRA) with Pb decreasing the peak production by 68% . The order of toxicity in both lactate and acetate-amended slurry was Pb>Cd>Hg and Pb>Hg>Cd, respectively . With acetate, SRA in the presence of Cd and Hg was stimulated 110% and 27%, respectively . Pb inhibited SRA by 11% . There is a general reduction in the inhibition of sulphide production in slurries as compared with pure culture of the isolate.

J Biol Chem, 2004 Jun 25, 279(26), 27799 - 806 Epub 2004 Apr 16.
Membrane topology of the DrrB protein of the doxorubicin transporter of Streptomyces peucetius; Gandlur SM et al.; Daunorubicin and doxorubicin, two commonly used anticancer agents, are produced by the soil bacterium Streptomyces peucetius . Self-resistance to these antibiotics in S . peucetius is conferred by the drrAB locus that codes for two proteins, DrrA and DrrB . DrrA is an ATP-binding protein . It belongs to the ABC family of transporters and shares sequence and functional similarities with P-glycoprotein of cancer cells . DrrB is an integral membrane protein that might function as a transporter for the efflux of daunorubicin and doxorubicin . Together, DrrA and DrrB are believed to form an ATP-driven pump for the efflux of these drugs . The drrAB locus has been cloned, and the two proteins have been expressed in a functional form in Escherichia coli . A topological analysis of the DrrB protein was performed using gene fusion methodology . Random and site-directed fusions of the drrB gene to lacZ, phoA, or gfp reporter genes were created . Based on the fusion data, a topological model of the DrrB protein is proposed in which the protein has eight membrane-spanning domains with both the N terminus and the C terminus in the cytoplasm.

Genetica, 2004 Mar, 120(1-3), 41 - 50
Mitochondrial DNA in the Drosophila melanogaster complex; Solignac M; Mitochondrial DNA in the complex Drosophila melanogaster was among the first studied in metazoans . The variability of the molecule was extensively studied using restriction enzymes, gene sequence and recently sequence of the whole coding region . Within the complex, seven major haplotypes have been described, one (me) in D . melanogaster, three in D . simulans (siI, siII, siIII), two in D . mauritiana (maI, maII), and one in D . sechellia (se) . The molecular distance between the haplotypes is comprised between 1 and 5%, except for siII and maI, which are virtually identical . The nucleotide diversity within each of these haplotypes is very low, varying from 0 to 0.0005 . Most of the cytoplasms are infected by the bacterium Wolbachia and different bacterial strains infect cytoplasms harboring different mtDNA types . mtDNA polymorphism is discussed in relation with Wolbachia, nuclear polymorphism and speciation events.

J Physiol Pharmacol, 2004 Mar, 55(1 Pt 1), 85 - 98
Platelet-activating factor mediates Porphyromonas gingivalis lipopolysaccharide interference with salivary mucin synthesis via phosphatidylinositol 3-kinase-dependent constitutive nitric-oxide synthase activation; Slomiany BL et al.; Platelet -activating factor (PAF), a phospholipid-derived messenger molecule, is now recognized as the most proximal mediator of cellular events triggered by bacterial lipopolysaccharide (LPS) stimulation . In this study, we assessed the role of PAF in the disturbances in salivary mucin synthesis evoked by LPS of periodontopathic bacterium, P . gingivalis . Using primary culture of mucous acinar cells of sublingual salivary gland, we show that a specific PAF antagonist, BN52020, prevents in a dose-dependent fashion (up to 83.7%) the LPS-induced reduction in mucin synthesis, and the effect is reflected in a marked decrease in the LPS-induced apoptosis (74.8%), NO generation (82.6%), and the expression of TNF-alpha (76.1%) . The impedance by BN52020 of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), which also obviated the inhibitory effect of BN52020 on the LPS-induced upregulation in apoptosis, TNF-alpha, and NO . A potentiation in the impedance by BN52020 of the LPS detrimental effect on mucin synthesis was however attained with NOS-2 inhibitor, 1400W, while cNOS inhibitor, L-NNA caused a reduction in the impedance effect of BN52020 . However, while 1400W and BN52020 countered the potentiating effect of wortmannin on the LPS-induced decrease in mucin synthesis, a further exacerbation of the effect of wortmannin occurred in the presence of L-NNA . The findings implicate PAF as a pivotal factor affecting the extent of pathological consequences of P . gingivalis infection on salivary glands capacity for mucin production, and suggest that its release in response to the LPS serves as a negative regulator of PI3K controlling the pathway of cNOS activation.

Biochemistry, 2004 Apr 20, 43(15), 4431 - 8
Fluorescence spectral fluctuations of single LH2 complexes from Rhodopseudomonas acidophila strain 10050; Rutkauskas D et al.; We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope . The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between quasi-stable levels differing by up to 30 nm . These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum . The frequency and size of these fluorescence peak movements were found to increase linearly with excitation intensity . Using the modified Redfield theory, changes in the realization of the static disorder accounted for the observed changes in spectral shape and intensity . Long lifetimes of the quasi-stable states suggest large free energy barriers between the different realizations.

Microbiology, 2004 Apr, 150(Pt 4), 897 - 910
AstR-AstS, a new two-component signal transduction system, mediates swarming, adaptation to stationary phase and phenotypic variation in Photorhabdus luminescens; Derzelle S et al.; Photorhabdus luminescens is an insect-pathogenic bacterium that forms a symbiosis with specific entomopathogenic nematodes . In this bacterium, a symbiosis-'deficient' phenotypic variant (known as the secondary variant or form II) arises at a low frequency during prolonged incubation . A knock-out mutant was generated of the regulator of a newly identified two-component regulatory system, designated AstR-AstS . Interestingly, this mutation altered the timing of phenotypic switching . Variant cells arose in the mutant strain several days before they did in the wild-type population, suggesting that AstRS is directly or indirectly involved in the genetic mechanism underlying variant cell formation . This mutation also affected motility and antibiotic synthesis . To identify AstRS-regulated genes, a comparative analysis using two-dimensional gel electrophoresis was performed . Seventeen proteins with modified synthesis in stationary phase were identified by mass spectrometry and shown to be involved in electron-transport systems, energy metabolism, iron acquisition and stress responses . The results imply that AstRS is involved in the adaptation of cells to the stationary phase, whilst negatively affecting the competitive advantage of form I cells . The link between AstRS-dependent stationary-phase adaptation and phenotypic variation is discussed.

BMC Microbiol . 2004 Apr 08;4(1):14.
Bioinformatic identification of novel regulatory DNA sequence motifs in Streptomyces coelicolor; Studholme DJ et al.; BACKGROUND: Streptomyces coelicolor is a bacterium with a vast repertoire of metabolic functions and complex systems of cellular development . Its genome sequence is rich in genes that encode regulatory proteins to control these processes in response to its changing environment . We wished to apply a recently published bioinformatic method for identifying novel regulatory sequence signals to gain new insights into regulation in S . coelicolor . RESULTS: The method involved production of position-specific weight matrices from alignments of over-represented words of DNA sequence . We generated 2497 weight matrices, each representing a candidate regulatory DNA sequence motif . We scanned the genome sequence of S . coelicolor against each of these matrices . A DNA sequence motif represented by one of the matrices was found preferentially in non-coding sequences immediately upstream of genes involved in polysaccharide degradation, including several that encode chitinases . This motif (TGGTCTAGACCA) was also found upstream of genes encoding components of the phosphoenolpyruvate phosphotransfer system (PTS) . We hypothesise that this DNA sequence motif represents a regulatory element that is responsive to availability of carbon-sources.Other motifs of potential biological significance were found upstream of genes implicated in secondary metabolism (TTAGGTtAGgCTaACCTAA), sigma factors (TGACN19TGAC), DNA replication and repair (ttgtCAGTGN13TGGA), nucleotide conversions (CTACgcNCGTAG), and ArsR (TCAGN12TCAG) . A motif found upstream of genes involved in chromosome replication (TGTCagtgcN7Tagg) was similar to a previously described motif found in UV-responsive promoters . CONCLUSIONS: We successfully applied a recently published in silico method to identify conserved sequence motifs in S . coelicolor that may be biologically significant as regulatory elements . Our data are broadly consistent with and further extend data from previously published studies . We invite experimental testing of our hypotheses in vitro and in vivo.

Transgenic Res, 2004 Feb, 13(1), 29 - 39
Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds; Hong CY et al.; Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals . Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive . Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and Escherichia coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds . Phytase expression was controlled by a germination inducible alpha-amylase gene (alphaAmy8) promoter, and extracellular phytase secretion directed by an betaAmy8 signal peptide sequence . The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally controlled and tissue-specific manner . No adverse effect on plant development or seed formation was observed . Up to 0.6 and 1.4 U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 46-60 times of phytase activities compared to the non-transformant . The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0-5.5 and 2.0-6.0, respectively . Phytase levels and inheritance of transgenes in one highly expressing plant were stable over four generations . Germinated transgenic rice seeds, which produce a highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals.

Arch Microbiol, 2004 May, 181(5), 345 - 51 Epub 2004 Apr 06.
Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic Cobetia marina strain L-2; Yumoto I et al.; A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5 degrees C as minimum growth temperatures in complex and defined media, respectively, was isolated . On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina . The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species . When the bacterium was grown at 5-15 degrees C in a defined medium, it produced a high amount of trans-unsaturated fatty acids . By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids . In the complex medium at 5 degrees C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium . Following a temperature shift from 11 to 5 degrees C, strain L-2 grew better in complex than in defined medium . Furthermore, when the growth temperature was shifted from 0 to 5 degrees C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced . These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism . The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5 degrees C were also studied . The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium . These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.

J Biol Chem, 2004 Jun 11, 279(24), 25632 - 7 Epub 2004 Apr 02.
Glycine 176 affects catalytic properties and stability of the Synechococcus sp . strain PCC6301 ribulose-1,5-bisphosphate carboxylase/oxygenase; Smith SA et al.; A previously described system for biological selection of randomly mutagenized ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) employing the phototrophic bacterium Rhodobacter capsulatus was used to select a catalytically altered form of a cyanobacterial (Synechococcus sp . strain PCC6301) enzyme . This mutant Rubisco, in which conserved glycine 176 was replaced with an aspartate residue, was not able to support CO(2)-dependent growth of the host strain . Site-directed mutant proteins were also constructed, e.g . asparagine and alanine residues replaced the native glycine with the result that these mutant proteins either greatly reduced the ability of R . capsulatus to support growth or had little effect, respectively . Growth phenotypes were consistent with the Rubisco activity levels associated with these proteins, and this was also borne out with purified recombinant proteins . Despite being catalytically challenged, the G176D and G176N mutant proteins were found to exhibit a more favorable interaction with CO(2) than the wild type protein but exhibited a reduced affinity for the substrate ribulose 1,5-bisphosphate . The G176A enzyme differed little from the wild type protein in these properties . None of the mutants had CO(2)/O(2) specificities that differed markedly from the wild type . Further studies taken from the known structure of the Synechococcus Rubisco indicated that substitutions at Gly-176 affected associations between large subunits . Supporting experimental data included an unusual protein concentration-dependent effect on in vitro activity, differences in thermal stability relative to the wild type protein, and aberrant migration on nondenaturing polyacrylamide gels . From these results, it is apparent that residues not directly located within the active site but near large subunit interfaces can affect key kinetic properties of Rubisco . These results suggest that further bioselection protocols (using these proteins as starting material) might yield novel mutant forms of Rubisco that relate to key functional properties.

Curr Opin Microbiol, 2004 Apr, 7(2), 192 - 7
Setting the pace: mechanisms tying Caulobacter cell-cycle progression to macroscopic cellular events; McGrath PT et al.; The bacterium Caulobacter crescentus divides asymmetrically, producing daughter cells with differing polar structures, different cell fates and asymmetric regulation of the initiation of chromosome replication . Complex intracellular signaling is required to keep the organelle developmental processes at the cell poles synchronized with other cell cycle events . Two recently characterized switch mechanisms controlling cell cycle progress are triggered by relatively large-scale developmental events in the cell: the progress of the DNA replication fork and the physical compartmentalization of the cell that occurs well before division . These mechanisms invoke rapid, precisely timed and even spatially differentiated regulatory responses at important points in the cell cycle.

J Microbiol Methods, 2004 May, 57(2), 207 - 18
Assessment of 35mer amino-modified oligonucleotide based microarray with bacterial samples; Calevro F et al.; Parallel quantification of a large number of messenger RNA transcripts, using microarray technology, promises to provide unsuspected information about many cellular processes . Although experimental protocols on microarray applications are available, only limited methodological information on glass-slide manufacturing and signal interpretation has been published . The aim of this paper is to provide new insights into the practical aspects of the construction and hybridization of oligonucleotide-based microarrays . The intracellular symbiotic bacterium of aphids, Buchnera aphidicola, is used here as a model organism . The first part of the work is devoted to the optimization of procedures for printing slides, labeling of cDNA targets and hybridization . In the second part, based on a statistical analysis of the results, we discuss the influence of the probe attachment chemistry, of the labeling method, of the oligonucleotide position and of the concentration of a spotted oligonucleotide on signal intensity . The problem of signal specificity is also addressed, based on the calculation of the fluorescent ratio for each probe to its corresponding mismatch control probe . Lastly, the selection of internal spiked RNAs appropriate to our bacterial samples and useful for the data normalization step is presented.

Biochemistry (Mosc), 2004 Mar, 69(3), 262 - 9
Intracellular alginolytic enzymes of the marine bacterium Pseudoalteromonas citrea KMM 3297; Alekseeva SA et al.; The marine bacterium Pseudoalteromonas citrea KMM 3297 is an associate of the holothurian Apostichopus japonicus . When grown in a medium containing glucose, the strain produces two intracellular alginolytic enzymes, AlI and AlII . Fucoidan from the brown alga Fucus evanescens induces synthesis of one more alginolytic enzyme, AlIII . These enzymes were separated using anion-exchange chromatography . The alginate lyase AlI completely retains its activity at 35 degrees C, AlII and AlIII being stable at 45 degrees C . The alginate lyases exhibit maximal activities in the range of pH 7-8 . The molecular weights of AlI, AlII, and AlIII determined by gel filtration are 25, 79, and 61 kD, respectively . All the investigated enzymes are endo-type alginate lyases . They catalyze degradation of polyguluronate (poly-G) and polymannuronate (poly-M) yielding oligosaccharides of the polymerization degree of 5 > or = n > or = 3 with the unsaturated bond between the C4 and C5 atoms of the non-reducing terminus . A mixture of these three enzymes exhibits synergism while acting on the polymeric substrate . The Km values of the alginate lyase AlI for poly-G and poly-M are 24 and 34 micro g/ml, respectively . Alginate lyase AlIII exhibits less affinity to poly-M (Km = 130.0 microg/ml) than to poly-G (Km = 40.0 microg/ml) . NaCl (0.2 M), MgCl2 and MgSO4 (0.01 M) activate all three enzymes more than twofold . The presence of several alginolytic enzymes of different specificity provides efficient destruction of alginic acids of brown algae by the strain P . citrea KMM 3297.

J Med Entomol, 2004 Mar, 41(2), 239 - 48
Human behaviors elevating exposure to Ixodes pacificus (Acari: Ixodidae) nymphs and their associated bacterial zoonotic agents in a hardwood forest; Lane RS et al.; Epidemiological evidence suggests that the nymph of the western black-legged tick, Ixodes pacificus Cooley and Kohls, is the primary vector of the Lyme disease spirochete, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, and Brenner, to humans in northwestern California . In spring 2002, six different human behaviors were evaluated as potential risk factors for acquiring I . pacificus nymphs in a deciduous woodland in Mendocino County, California . Also, the prevalence of B . burgdorferi sensu lato (s.l.) and the causative agents of human granulocytic (Anaplasma phagocytophilum {Foggie} Dumler, Barbet, Bekker, Dasch, Palmer, Ray, Rikihisa, and Rurangirwa) and monocytic ehrlichioses (Ehrlichia chaffeensis Anderson, Dawson, Jones, and Wilson) was determined in nymphs that had been collected from subjects or by dragging leaf litter . Activities involving a considerable degree of contact with wood resulted in greater acquisition of nymphs than those involving exposure solely to leaf litter . Time-adjusted tick-acquisition rates demonstrated that sitting on logs was the riskiest behavior, followed, in descending rank, by gathering wood, sitting against trees, walking, stirring and sitting on leaf litter, and just sitting on leaf litter . The number of ticks acquired appeared to be unrelated to the type of footwear worn (hiking boots, hiking sandals, or running shoes) . Overall, 3.4% (n = 234) of the nymphs were infected with A . phagocytophilum, 3.9% (n = 181) with B . burgdorferi s.l., and none (n = 234) with E . chaffeensis . Of 13 nymphs infected with either A . phagocytophilum or B . burgdorferi s.l., 2 (15.4%) were coinfected with both bacteria, as were 1.3% of 158 nymphs obtained from leaf litter, the first report of coinfection in this life stage of I . pacificus . Four unattached, infected nymphs were removed from subjects, including two acquired while sitting on logs that contained A . phagocytophilum, another with the same bacterium obtained while walking, and one acquired while gathering wood that was infected with B . burgdorferi s.l . Despite the use of extreme personal preventive measures by both subjects, two attached, uninfected nymphs were removed from one of them > or = 1-2 d postexposure . The public health implications of these findings are discussed.

Curr Microbiol, 2004 May, 48(5), 383 - 8
Characterization of chimeric isocitrate dehydrogenases of a mesophilic nitrogen-fixing bacterium, Azotobacter vinelandii, and a psychrophilic bacterium, Colwellia maris; Yoneta M et al.; Several properties of chimeric enzymes between a mesophilic isocitrate dehydrogenase (IDH) from a nitrogen-fixing bacterium, Azotobacter vinelandii, and a cold-adapted IDH isozyme (IDH-II) from a psychrophilic bacterium, Colwellia maris, were examined . Each of the genes encoding the IDHs was divided into four regions of almost equal lengths, and each region was ligated with different combinations to construct various chimeric genes . The resultant wild-type and chimeric genes were overexpressed in Escherichia coli . The wild-type and chimeric IDHs were classified into three groups based on optimum temperatures for activity of 20 degrees, 30 degrees, and 40 degrees C . The IDHs with a lower optimum temperature were more thermolabile . The optimum temperature and thermostability of the chimeric enzymes decreased on increasing the proportion derived from the cold-adapted IDH-II of C . maris . Furthermore, the C-terminal region of the C . maris IDH-II was suggested to be responsible for its psychrophilic characteristics.

Curr Microbiol, 2004 May, 48(5), 379 - 82
Expression of Treponema denticola oligopeptidase B in Escherichia coli; Lee SY et al.; Treponema denticola is a small anaerobic spirochete often isolated from periodontal lesions and closely associated with periodontal diseases . This bacterium possesses a particular arginine peptidase activity (previously called "BANA-peptidase" or "trypsin-like enzyme") that is common to the three cultivable bacterial species most highly associated with severe periodontal disease . We recently reported the identification of the opdB locus that encodes the BANA-peptidase activity of T . denticola through DNA sequencing and mutagenesis studies . In the present study, we report expression of T . denticola OpdB peptidase in Escherichia coli . The opdB PCR product was cloned into pET30b and then transformed into the E . coli BL21 (DE3)/pLysS expression strain . Assays of enzymatic activities in E . coli containing T . denticola opdB showed BANA-peptidase activity similar to that of T . denticola . Availability of this recombinant expression system producing active peptidase will facilitate characterization of the potential role of this peptidase in periodontal disease etiology.

Curr Microbiol, 2004 May, 48(5), 368 - 72
Xylella fastidiosa cultivation on a minimal solid defined medium; Almeida RP et al.; A simple defined solid medium containing citrate and succinate, three amino acids (L-glutamine, L-asparagine, and L-cysteine), hemin chloride, potato starch, gellan gum (GelRite), and mineral salts supported the growth of grape strains of Xylella fastidiosa, the bacterial pathogen that causes Pierce's disease of grape . Isolation efficiency from infected grape plant samples, determined by the number of colony forming units recovered, on the defined medium was slightly less ( approximately 10-fold) or indistinguishable from two standard rich media used for culturing X . fastidiosa, PWG and PD3, respectively . The bacterium also grew on media with citrate and L-glutamine as the only carbon and nitrogen sources . Potato starch was not essential for bacterial growth, but no growth was observed on media without hemin chloride . Agar inhibited bacterial growth when used as the gelling agent.

Nature, 2004 Apr 1, 428(6982), 574 - 8
From molecular noise to behavioural variability in a single bacterium; Korobkova E et al.; The chemotaxis network that governs the motion of Escherichia coli has long been studied to gain a general understanding of signal transduction . Although this pathway is composed of just a few components, it exhibits some essential characteristics of biological complexity, such as adaptation and response to environmental signals . In studying intracellular networks, most experiments and mathematical models have assumed that network properties can be inferred from population measurements . However, this approach masks underlying temporal fluctuations of intracellular signalling events . We have inferred fundamental properties of the chemotaxis network from a noise analysis of behavioural variations in individual bacteria . Here we show that certain properties established by population measurements, such as adapted states, are not conserved at the single-cell level: for timescales ranging from seconds to several minutes, the behaviour of non-stimulated cells exhibit temporal variations much larger than the expected statistical fluctuations . We find that the signalling network itself causes this noise and identify the molecular events that produce it . Small changes in the concentration of one key network component suppress temporal behavioural variability, suggesting that such variability is a selected property of this adaptive system.

Curr Microbiol, 2004 Mar, 48(3), 219 - 23
The effect of cellobiose, glucose, and cellulose on the survival of Fibrobacter succinogenes A3C cultures grown under ammonia limitation; Thomas S et al.; The ruminal, cellulolytic bacterium, Fibrobacter succinogenes A3C, grew rapidly on cellulose, cellobiose, or glucose, but it could not withstand long periods of energy source starvation . If ammonia was limiting and either cellobiose or glucose was in excess, the viability declined even faster . The carbohydrate-excess, ammonia-limited cultures did not spill energy, but they accumulated large amounts of cellular polysaccharide . Cultures that were carbohydrate-limited had approximately 4 nmol ATP mg cell protein(-1), but ATP could not be detected in cultures that had an excess of soluble carbohydrates . However, if F . succinogenes A3C was provided with excess cellulose and ammonia was limiting, ATP did not decline, and the cultures digested the cellulose soon after additional nitrogen sources were added . From these results, it appears that excess soluble carbohydrates can promote the death of F . succinogenes, but cellulose does not.

Insect Mol Biol, 2004 Apr, 13(2), 147 - 53
Diversity, distribution and specificity of WO phage infection in Wolbachia of four insect species; Gavotte L et al.; The bacteriophage WO was recently characterized in Wolbachia, a strictly intracellular bacterium that causes several reproductive alterations in its arthropod hosts . To gain insights into the phage-Wolbachia relationships, we studied the phage presence among Wolbachia infecting four insect species sharing several Wolbachia strains, two Drosophila and two of their parasitoid wasps . Based on the phage sequence of ORF7, we identified five different phages in six Wolbachia strains . Among these five bacteriophages, some are specific for a given bacterial strain whereas others are not, but globally phage infection appears stable on a large geographical scale and across insect generations . Their specificity contrasts with the absence of congruence between Wolbachia and phage phylogenies, suggesting phage exchanges between different Wolbachia lineages.

Can J Microbiol, 2001 Jan, 47(1), 77 - 80
Determination of 1-aminocycopropane-1-carboxylic acid (ACC) to assess the effects of ACC deaminase-containing bacteria on roots of canola seedlings; Penrose DM et al.; Previously, it was proposed that plant growth-promoting bacteria that possess the enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, can reduce the amount of ethylene produced by a plant and thereby promote root elongation . To test this model, canola seeds were imbibed in the presence of the chemical ethylene inhibitor, 2-aminoethoxyvinyl glycine (AVG), various strains of plant growth-promoting bacteria, and a psychrophilic bacterium containing an ACC deaminase gene on a broad host range plasmid . The extent of root elongation and levels of ACC, the immediate precursor of ethylene, were measured in the canola seedling roots . A modification of the Waters AccQ.Tag Amino Acid Analysis Method was used to quantify ACC in the root extracts . It was found that, in the presence of the ethylene inhibitor, AVG, or any one of several ACC deaminase-containing strains of bacteria, the growth of canola seedling roots was enhanced and the ACC levels in these roots were lowered.

Microbes Infect, 2004 Feb, 6(2), 229 - 37
Human infection with Photorhabdus asymbiotica: an emerging bacterial pathogen; Gerrard J et al.; The three currently recognised Photorhabdus species are bioluminescent bacteria that are pathogenic to insects . P . luminescens and P . temperata form a symbiotic relationship with nematodes that infect insects . P . asymbiotica, on the other hand, has only been isolated from human clinical specimens from the USA and Australia . The bacterium has been associated with locally invasive soft tissue and disseminated bacteraemic infections . An invertebrate vector for P . asymbiotica has not yet been identified.

Wiad Lek, 2003, 56(9-10), 491 - 2
{Discitis caused by Kingella kingae--case report}; Maciejewski M et al.; The patient with acute infection and the presence of bacterium Kingella kingae cultured from cerebrospinal fluid is presented . The analysis of clinical picture and the results of all performed tests did not allow to diagnose cerebrospinal meningitis . Despite of being not sure if Kingella kingae was an etiopathogenetic factor in the described subject, we decided to discuss this interesting case.

Anal Bioanal Chem, 1996 Jun, 355(5-6), 739 - 41
Spectroscopic characterization of mineral crystals produced by the bacterium Azospirillum brasilense; Kamnev AA et al.; The results of spectroscopic structural and trace elemental analyses of mineral crystals produced by the soil nitrogen-fixing bacterium Azospirillum brasilense cultivated in a synthetic medium are presented and discussed . The mineral formed is shown to have a structure close to struvite (MgNH(4)PO(4) x 6H(2)O; ASTM file No . 15-762) with some differences which may be attributed to the presence of isomorphic admixtures of other cations (struvite is known to have a variety of forms) . AAS/AES and ion chromatography analyses for a number of biologically important microelements and their role in the formation of the crystal structure, as well as some questions related to biomineralization are also discussed.

Glycobiology, 2004 Jun, 14(6), 511 - 9 Epub 2004 Mar 24.
Carbohydrates act as receptors for the periodontitis-associated bacterium Porphyromonas gingivalis: a study of bacterial binding to glycolipids; Hellstrom U et al.; In this study we show for the first time the use of carbohydrate chains on glycolipids as receptors for the periodontitis-associated bacterium Porphyromonas gingivalis . Previous studies have shown that this bacterium has the ability to adhere to and invade the epithelial lining of the dental pocket . Which receptor(s) the adhesin of P . gingivalis exploit in the adhesion to epithelial cells has not been shown . Therefore, the binding preferences of this specific bacterium to structures of carbohydrate origin from more than 120 different acid and nonacid glycolipid fractions were studied . The bacteria were labeled externally with (35)S and used in a chromatogram binding assay . To enable detection of carbohydrate receptor structures for P . gingivalis, the bacterium was exposed to a large number of purified total glycolipid fractions from a variety of organs from different species and different histo-blood groups . P . gingivalis showed a preference for fractions of human and pig origin for adhesion . Both nonacid and acid glycolipids were used by the bacterium, and a preference for shorter sugar chains was noticed . Bacterial binding to human acid glycolipid fractions was mainly obtained in the region of the chromatograms where sulfated carbohydrate chains usually are found . However, the binding pattern to nonacid glycolipid fractions suggests a core chain of lactose bound to the ceramide part as a tentative receptor structure . The carbohydrate binding of the bacterium might act as a first step in the bacterial invasion process of the dental pocket epithelium, subsequently leading to damage to periodontal tissue and tooth loss.

FEMS Microbiol Lett, 2004 Apr 1, 233(1), 165 - 72
Differences in malate dehydrogenases from the obligately piezophilic deep-sea bacterium Moritella sp . strain 2D2 and the psychrophilic bacterium Moritella sp . strain 5710; Saito R et al.; The gene encoding malate dehydrogenase (MDH) of the obligately piezophilic deep-sea bacterium Moritella sp . strain 2D2 was cloned and sequenced . There were two positions {close to the active site (Ala-180) and in the subunit interaction site (His-229)} with 2D2-specific substitutions . The MDH genes of strain 2D2 and a psychrophilic bacterium Moritella sp . strain 5710 exhibiting the highest sequence similarity were overexpressed in Escherichia coli . The 2D2 MDH was more heat-stable than the 5710 MDH . The apparent Km value at 62.1 MPa for NADH of the 2D2 MDH was higher than that of the 5710 MDH . The 2D2 MDH in which a His-Gln substitution was introduced at position 229 decreased the thermal stability and Km value at 62.1 MPa . The 5710 MDH that was substituted Gln-229 with His increased the thermal stability and Km value at 62.1 MPa . These results indicate that the His residue at position 229 of the 2D2 MDH may play a role in the thermal stability and the MDH function at high pressure.

FEMS Microbiol Lett, 2004 Apr 1, 233(1), 29 - 35
Involvement of caspase activation through release of cytochrome c from mitochondria in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans; Kasai H et al.; We previously reported that infection with the periodontopathic bacterium Actinobacillus actinomycetemcomitans induced apoptosis in a mouse macrophage cell line J774.1 . In the present study, we examined the involvement of cytochrome c and caspases in the induction of apoptosis in A . actinomycetemcomitans-infected J774.1 cells . Following infection, the expression levels of cytochrome c, and cleaved forms of caspase-3 and caspase-9 in the cells were examined using immunoblot analysis . Cytochrome c was released from mitochondria into the cytoplasm after A . actinomycetemcomitans-infected J774.1 cells were cultured for 6 h, and caspase-3 and caspase-9 were found to be cleaved forms in the cells . Further, caspase-9 activity was markedly increased, and phosphorylated p53 was detected in the cells 30 h following infection . These results suggest that apoptosis in A . actinomycetemcomitans-infected J774.1 cells is regulated by the release of cytochrome c from mitochondria into cytoplasm and the subsequent activation of caspases through phosphorylation of p53.

Curr Biol, 2004 Mar 23, 14(6), R245 - 6
Evolution: bacterial mutation in stationary phase; Sniegowski P; A recent study indicates that the genomic mutation rate of the gut bacterium Escherichia coli is substantially higher in nongrowing than growing cultures . These findings are important in the light of the ongoing controversy over the generality and robustness of stationary phase mutagenesis and its evolutionary implications.

World J Gastroenterol, 2004 Mar 15, 10(6), 852 - 5
Establishment of Helicobacter pylori infection model in Mongolian gerbils; Yan J et al.; AIM: To establish a stable and reliable model of Helicobacter pylori infection model in Mongolian gerbils and to observe pathological changes in gastric mucosa in infected animals . METHODS: Mongolian gerbils were randomly divided into 18 groups; 6 groups were infected with H pylori clinical strain Y06 (n=6, groups Y), 6 groups were infected with H pylori strain NCTC11637 (n=6, groups N), and 6 uninfected groups as negative controls (n=4, groups C) . H pylori suspensions at the concentrations of 2X10(8) and 2X10(9) CFU/mL of strain NCTC11637 and strain Y06 were prepared . The animals in three groups N and in three groups Y were orally challenged once with 0.5 mL of the low concentration of the bacterial suspension . The animals in another three groups N and in another three groups Y were orally challenged with 0.5 mL of the high concentration of the bacterial suspension for 3 times at the intervals of 24 h, respectively . For the negative controls, the animals in six groups C were orally given with the same volume of Brucella broth at the corresponding inoculating time . The animals were killed after 2nd, 4(th) and 6(th) week after the last challenge and the gastric mucosal specimens were taken for urease test, bacterial isolation, pathological and immunohistochemical examinations . RESULTS: Positive isolation rates of H pylori in the animals of groups Y at the 2nd, 4(th) and 6(th) week after one challenge were 0%, 16.7% and 66.7%, while in the animals of groups N were 0%, 0% and 16.7%, respectively . Positive isolation rates of H pylori in the animals of groups Y at the 2nd, 4(th) and 6(th) week after three challenges were 66.7%, 100% and 100%, while in the animals of groups N were 66.7%, 66.7% and 100%, respectively . In animals with positive isolation of H pylori, the bacterium was found to colonized on the surface of gastric mucosal cells and in the gastric pits, and the gastric mucosal lamina propria was infiltrated with inflammatory cells . CONCLUSION: By using H pylori suspension at high concentration of 2X10(9) CFU/mL for multiple times, the orally challenged Mongolian gerbils can be used as a stable and reliable H pylori infection model . The 2 strains of H pylori can colonize in gastric mucosa of the infected animals and cause mild inflammation reaction.

Med Sci Monit, 2004 Apr, 10(4), CR133 - 6
Prevalence of Helicobacter pylori in patients with portal hypertensive gastropathy--a study from south India; Batmanabane V et al.; BACKGROUND: This study was done to ascertain the prevalence of Helicobacter pylori in patients with PHG and determine whether it contributed to the severity of the disease . MATERIAL/METHODS: A total of thirty-seven consecutive patients who presented with portal hypertensive gastropathy were included in the study . H . pylori status was determined by urease test and histology . The presence of at least one positive test was considered as a positive H . pylori state . Correlation analyses of H . pylori status with age, gender, alcohol consumption, and the site and severity of lesion were done . RESULTS: Sixteen of the 37 patients were positive for H . pylori . A linear trend with age was seen in H . pylori - infected patients . H . pylori positivity was higher in the second and third decades of life, although this did not reach statistical significance . The linear trend with age was similar to that of the control group . There was no association between H . pylori status and alcohol intake or the site of lesion . Twenty-sevenlpatients had endoscopic evidence of mill PHG, 9 had moderate and 1 severe . The H . pylori status was 52%, 22%, and 0% in patients with mild, moderate, and severe gastropathy, respectively, indicating an inverse relatioinship of severity of PHG with H . pylori colonization . CONCLUSIONS: Portal hypertensive gastropathy does not provide a favorable environment for the colonization of H . pylori . The decline in H . pylori positivity with the severity of PHG suggests that this bacterium is unlikely to contribute in the pathogenesis of congestive gastropathy and that hence there might be no need for its routine eradication in patients with PHG.

Infect Immun, 2004 Apr, 72(4), 2075 - 80
Phagocytosis of apoptotic cells increases the susceptibility of macrophages to infection with Coxiella burnetii phase II through down-modulation of nitric oxide production; Zamboni DS et al.; Coxiella burnetii, the agent of Q fever in humans and coxiellosis in other mammals, is an obligate intracellular bacterium which is sheltered and multiplies within typically large phagolysosome-like replicative vacuoles (LRVs) . We have previously shown that, compared with fibroblasts, mouse resident peritoneal macrophages control the development of LRVs and bacterial multiplication within these vacuoles . Earlier experiments with the nitric oxide (NO) synthase inhibitor aminoguanidine (AG) revealed that the control is exerted by NO induced by the bacteria . We report here that phagocytosis of apoptotic-like, but not of aldehyde-killed, lymphocytes by the macrophages reduced the production of NO induced by the bacteria and increased the development of LRVs and, therefore, the total bacterial load in the cultures . Experiments with macrophages from mice deficient for inducible NO synthase (iNOS(-)/(-)) confirmed the involvement of NO in the control of infection, since neither apoptotic lymphocytes nor AG affected the development of LRVs in these phagocytes . Since macrophages are important for the clearance of apoptotic bodies and C . burnetii is able to induce apoptosis in human monocytes, the phenomenon shown here may be biologically relevant to the development of Q fever and coxiellosis.

Infect Immun, 2004 Apr, 72(4), 1856 - 65
Mechanisms involved in apoptosis of human macrophages induced by lipopolysaccharide from Actinobacillus actinomycetemcomitans in the presence of cycloheximide; Suzuki T et al.; Actinobacillus actinomycetemcomitans is a major periodontopathic bacterium with multiple virulence factors, including lipopolysaccharide (LPS) . Previous reports have demonstrated that LPS induced apoptosis in a murine macrophage-like cell line, J744.1, as well as in peritoneal macrophages from C3H/HeN mice in the presence of cycloheximide (CHX) . However, the detailed molecular mechanisms involved in the apoptosis of macrophages induced by LPS and CHX are not well known . To clarify the possible role of LPS in the induction of macrophage apoptosis, we investigated cell death induced by LPS from A . actinomycetemcomitans and CHX in human macrophage-like U937 cells, which were differentiated by 12-O-tetradecanoylphorbol 13-acetate (TPA), and also assessed the molecular mechanisms involved in the process . We found that TPA-differentiated U937 cells usually showed resistance to LPS-induced apoptosis . However, in the presence of CHX, LPS induced release of cytochrome c without modifying steady-state levels of Bcl-2, Bcl-xL, Bax, and Bak . Treatment with LPS in the presence of CHX also led to activation of caspase-3 and apoptosis via, in part, the CD14/toll-like receptor 4 (TLR4) . The induction of cytochrome c release may have been due to dephosphorylation of Akt and Bad, which were cooperatively induced by CHX and LPS . However, endogenous tumor necrosis factor alpha- and Fas-induced signals, extracellular signal-regulated kinase kinase/mitogen-activated protein kinases and I-kappa B alpha/nuclear factor-kappa B (NF-kappa B) were not required for caspase-3-dependent apoptosis . These results emphasize the possible important role of the mitochondrial apoptotic pathway leading to caspase-3 activation in LPS-induced apoptosis of human macrophages in the presence of CHX.

Int J Parasitol, 2004 Feb, 34(2), 191 - 203
Mapping the presence of Wolbachia pipientis on the phylogeny of filarial nematodes: evidence for symbiont loss during evolution; Casiraghi M et al.; Wolbachia pipientis is a bacterial endosymbiont associated with arthropods and filarial nematodes . In filarial nematodes, W . pipientis has been shown to play an important role in the biology of the host and in the immuno-pathology of filariasis . Several species of filariae, including the most important parasites of humans and animals (e.g . Onchocerca volvulus, Wuchereria bancrofti and Dirofilaria immitis) have been shown to harbour these bacteria . Other filarial species, including an important rodent species (Acanthocheilonema viteae), which has been used as a model for the study of filariasis, do not appear to harbour these symbionts . There are still several open questions about the distribution of W . pipientis in filarial nematodes . Firstly the number of species examined is still limited . Secondly, it is not clear whether the absence of W . pipientis in negative species could represent an ancestral characteristic or the result of a secondary loss . Thirdly, several aspects of the phylogeny of filarial nematodes are still unclear and it is thus difficult to overlay the presence/absence of W . pipientis on a tree representing filarial evolution . Here we present the results of a PCR screening for W . pipientis in 16 species of filariae and related nematodes, representing different families/subfamilies . Evidence for the presence of W . pipientis is reported for five species examined for the first time (representing the genera Litomosoides, Litomosa and Dipetalonema); original results on the absence of this bacterium are reported for nine species; for the remaining two species, we have confirmed the absence of W . pipientis recently reported by other authors . In the positive species, the infecting W . pipientis bacteria have been identified through 16S rDNA gene sequence analysis . In addition to the screening for W . pipientis in 16 species, we have generated phylogenetic reconstructions based on mitochondrial gene sequences (12S rDNA; COI), including a total of 28 filarial species and related spirurid nematodes . The mapping of the presence/absence of W . pipientis on the trees generated indicates that these bacteria have possibly been lost during evolution along some lineages of filarial nematodes.

Curr Opin Microbiol, 2004 Feb, 7(1), 67 - 70
Wolbachia pipientis: intracellular infection and pathogenesis in Drosophila; McGraw EA et al.; Wolbachia pipientis is a vertically transmitted, obligate intracellular symbiont of arthropods . The bacterium is best known for its ability to manipulate host reproductive biology where it can induce cytoplasmic incompatibility, parthenogenesis, feminization and male-killing . In addition to the various reproductive phenotypes it generates through interaction with host reproductive tissue it is also known to infect somatic tissues . However, relatively little is known about the consequences of infection of these tissues with the exception that in some hosts Wolbachia acts as a classical mutualist and in others a pathogen, dramatically shortening adult insect lifespan . Manipulation experiments have demonstrated that the severity of Wolbachia-induced effects on the host is determined by a combination of host genotype, Wolbachia strain, host tissue localization, and interaction with the environment . The recent completion of the whole genome sequence of Wolbachia pipientis wMel strain indicates that it is likely to use a type IV secretion system to establish and maintain infection in its host . Moreover, an unusual abundance of genes encoding proteins with eukaryotic-like ankyrin repeat domains suggest a function in the various described phenotypic effects in hosts.

Arch Microbiol, 2004 May, 181(5), 362 - 70 Epub 2004 Mar 19.
Novel transcriptional regulators of Legionella pneumophila that affect replication in Acanthamoeba castellanii; Lebeau I et al.; Legionella pneumophila is commonly found in freshwater environments and is able to invade and replicate within amoebae and ciliated protozoa . Moreover, this bacterium is also able to replicate within human alveolar macrophages causing a severe form of pneumonia, designated Legionnaires' disease . L . pneumophila pathogenesis is not yet completely understood, but the genes responsible for infection and intracellular replication are becoming known . Nonetheless, knowledge as to how these genes are controlled is still very limited . The partially sequenced genome of L . pneumophila was searched for open reading frames encoding proteins with sequence similarity to members of the LuxR family of transcriptional regulators . These were designated LpnR1, LpnR2, LpnR3, and LpnR4 . Although these proteins could not be identified as true LuxR proteins, they do act as regulators, as illustrated in this report . LpnR1 negatively affected rpoS expression, whereas LpnR2 and LpnR3 positively affected flagellin expression . Furthermore, LpnR2 proved to be necessary for efficient invasion of Acanthamoeba castellanii and LpnR3 for intracellular replication in this protozoan host . LpnR4 was recently identified as LetA.

Ann N Y Acad Sci, 2003 Dec, 1010, 568 - 72
NF-kappaB and Bcl-2 in Helicobacter pylori-induced apoptosis in gastric epithelial cells; Chu SH et al.; Helicobacter pylori (H . pylori) has been considered as an important pathogen of gastroduodenal inflammation and gastric carcinogenesis . However, the pathogenic mechanisms including H . pylori-induced apoptosis and subsequent molecular mechanisms have not been clarified yet . The present study examined the role of Bcl-2 and its relation to NF-kappaB in H . pylori-induced apoptosis in human gastric epithelial AGS cells . AGS cells were cultured in the presence of H . pylori, at a bacterium/cell ratio of 300:1, for the determination of apoptosis, NF-kappaB activation with IkBa degradation, and Bcl-2 level . AGS cells were transfected with a control vector (pCMV cells) or a full-length human Bcl-2 expression vector (Bcl-2 cells) . H . pylori-induced apoptosis and NF-kappaB activation were compared in the wild-type cells and the transfected cells . As a result, H . pylori increased apoptotic cells with chromatin condensation and reduced Bcl-2 levels, which were accompanied with NF-kappaB activation . H . pylori induced sixfold increase in the number of apoptotic cells in wild-type cells and pCMV cells . H . pylori-induced increment of apoptotic cells were relatively lower in Bcl-2 cells than pCMV cells . H . pylori-induced NF-kappaB activation and IkBa degradation were not different in the wild-type cells, pCMV cells, and Bcl-2 cells . In conclusion, the reduced gastric Bcl-2 level may be the main pathogenic mechanism of H . pylori-induced apoptosis in gastric epithelial cells.

FEMS Microbiol Lett, 2004 Mar 19, 232(2), 165 - 72
Annotation of the pRhico plasmid of Azospirillum brasilense reveals its role in determining the outer surface composition; Vanbleu E et al.; The plant growth-promoting soil bacterium Azospirillum brasilense enhances growth of economically important crops, such as wheat, corn and rice . In order to improve plant growth, a close bacterial association with the plant roots is needed . Genes encoded on a 90-MDa plasmid, denoted pRhico plasmid, present in A . brasilense Sp7, play an important role in plant root interaction . Sequencing, annotation and in silico analysis of this 90-MDa plasmid revealed the presence of a large collection of genes encoding enzymes involved in surface polysaccharide biosynthesis . Analysis of the 90-MDa plasmid genome provided evidence for its essential role in the viability of the bacterial cell.

Eur J Biochem, 2004 Apr, 271(7), 1372 - 81
Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp . SIB1 in cold-adaptation; Suzuki Y et al.; A psychrotrophic bacterium Shewanella sp . strain SIB1 was grown at 4 and 20 degrees C, and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis . Comparison of these patterns showed that the cellular content of a protein with a molecular mass of 28 kDa and an isoelectric point of four greatly increased at 4 degrees C compared to that at 20 degrees C . Determination of the N-terminal amino acid sequence, followed by the cloning and sequencing of the gene encoding this protein, revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56% to Escherichia coli FKBP22 . This protein was overproduced in E . coli in a His-tagged form, purified, and analyzed for peptidyl-prolyl cis-trans isomerase activity . When this activity was determined by the protease coupling assay using N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide as a substrate at various temperatures, the protein exhibited the highest activity at 10 degrees C with a k(cat)/K(m) value of 0.87 micro m(-1) x s(-1) . When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T(1) refolding assay at 10 and 20 degrees C, the protein exhibited higher activity at 10 degrees C with a k(cat)/K(m) value of 0.50 micro m(-1) x s(-1) . These k(cat)/K(m) values are lower but comparable to those of E . coli FKBP22 . We propose that a FKBP family protein is involved in cold-adaptation of psychrotrophic bacteria.

J Agric Food Chem, 2004 Mar 24, 52(6), 1495 - 503
Glycosylated compounds from okra inhibit adhesion of Helicobacter pylori to human gastric mucosa; Lengsfeld C et al.; In Asian medicine the fruit of the okra plant, Abelmoschus esculentus (L.) Moench., is used as a mucilaginous food additive against gastric irritative and inflammative diseases . To find a rational basis for its use against these diseases, several crude and purified carbohydrate-containing fractions from immature okra fruits were isolated and analyzed, and their effects against Helicobacter pylori in an in situ adhesion model on sections of human gastric mucosa were determined . Pretreatment of the bacteria with a fresh juice preparation inhibited the bacterial adhesion almost completely . Lyophilization and reconstitution of an extract solution led to a reduction of this effect . A crude polysaccharide (RPS) isolated from the fresh juice by ethanolic precipitation showed strong inhibitory effects . Further fractionation of RPS revealed a purified, highly acidic subfraction (AF III) with high antiadhesive qualities . Carbohydrate analysis revealed the presence of rhamnogalacturonans with a considerable amount of glucuronic acid, whereas other inactive subfractions contained little glucuronic acid or were glucuronic acid-free . After heat denaturation of the fresh juice or protein precipitation with 5% TCA the antiadhesive activity of the fresh extract was reduced, indicating that besides polysaccharides, protein fractions also exhibited antiadhesive properties . SDS-PAGE analysis of the precipitate revealed several bands of glycosylated proteins between 25 and 37 kDa that were almost diminished in the nonactive supernatant . Preincubations of gastric tissue with any of the active fractions did not lead to reduced bacterial binding . The antiadhesive activity is therefore due to the blocking capacity of specific Helicobacter surface receptors that coordinate the interaction between host and bacterium . Neither of the active fractions showed inhibitory effects on bacterial growth in vitro . The antiadhesive qualities of okra were assumed to be due to a combination of glycoproteins and highly acidic sugar compounds making up a complex three-dimensional structure that is fully developed only in the fresh juice of the fruit.

J Bacteriol, 2004 Apr, 186(7), 2200 - 5
Evidence for multiple levels of regulation of Oenococcus oeni clpP-clpL locus expression in response to stress; Beltramo C et al.; A locus containing the clpP and clpL genes in the lactic acid bacterium Oenococcus oeni was studied . Real-time reverse transcription-PCR analysis revealed different induction factors involved in expression of these genes during stress . According to the conditions, clpP and clpL genes could be transcripted as two distinct transcripts or cotranscripted . The clpP promoter depended on the CtsR regulator, but surprisingly the clpL promoter did not . The amount of the clpL transcript depended on mRNA stability . This clp ATPase gene is at least controlled at the posttranscriptional level.

Tsitologiia, 2003, 45(12), 1227 - 33
Initial steps of infection of the ciliate Paramecium with bacteria Holospora sp; Fokin SI et al.; New light and electron microscope data on the initial steps of endocytobiosis establishment between the ciliate Paramecium and specific intranuclear bacteria Holospora are provided . At the cytoplasmic step of infection bacteria of all Holospora species are found in a vesicle originating from the membrane of the host cell phagosome . The association between host cell microfilaments and the bacterium bearing vesicle may suggest a possible involvement of the ciliate cytoskeleton in the transportation of bacteria to the host cell nucleus . The authors subdivide the process of infection into 6 steps . Some strains of P . caudatum never take up infectious Holospora bacteria in the course of phagocytosis.

J Biol Chem, 2004 May 14, 279(20), 21394 - 400 Epub 2004 Mar 16.
Roles of heme axial ligands in the regulation of CO binding to CooA; Yamashita T et al.; CooA is a CO-dependent transcription factor of the bacterium Rhodospirillum rubrum that contains a six-coordinate heme . It has as its heme axial ligands Pro(2) and Cys(75) in the ferric state and Pro(2) and His(77) in the ferrous state . To probe the regulation of CO binding and the ligand switching mechanism in CooA, we have prepared site-directed mutants in which the residues contributing the axial ligands are substituted . The properties of these mutants were investigated by resonance Raman and CO titration methods . Wild-type CooA binds CO with a modest dissociation constant (K(d)) of 11 microm, this value being typical for gas-sensing heme proteins . The K(d) value was greatly decreased in the P2H mutant, indicating that Pro(2) coordination fine tunes CO sensing in CooA . The bound CO in P2H gives rise to a nu(Fe-CO) stretching Raman line at 490 cm(-1), which is similar to that in wild-type CooA . Thus, Pro(2) is the ligand that is replaced by exogenous CO . In the H77A mutant, equilibrium CO binding is biphasic, and at high CO pressures two CO molecules occupy both axial sites . The nu(Fe-CO) stretching Raman line for the first CO molecule was observed at 497 cm(-1) . Some of the His(77) mutants showed an additional nu(Fe-CO) line at 525 cm(-1) . The binding affinity of the second CO molecule correlates with the five-coordinate component in the ferrous His(77) mutants and also with the acidity of the side chain at position 77 . Thus, we propose the Cys(75)-His(77) ligand switch is controlled by His(77) acting as a proton reservoir.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 543 - 9
'Candidatus Rhabdochlamydia porcellionis', an intracellular bacterium from the hepatopancreas of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda); Kostanjsek R et al.; Intracellular bacteria were observed in the hepatopancreas of the terrestrial isopod Porcellio scaber . Comparative 16S rRNA gene sequence analysis and electron microscopic observations were used to determine the taxonomic position of these intracellular bacteria . Phylogenetic analysis and a complex developmental cycle affiliate these bacteria to the order Chlamydiales, within which they form a distinctive lineage, close to the family Simkaniaceae . They share <92 % 16S rRNA gene sequence similarity with their closest relative and <88 % similarity with other members of the order Chlamydiales . A specific signature oligonucleotide sequence was identified and used as a probe, enabling the identification of intracellular bacteria in infected hepatopancreatic tissue . According to the distinctive morphology of their elementary bodies, which are rod-shaped rather than spherical and contain translucent oblong structures, their genomic properties and their crustacean host, the name 'Candidatus Rhabdochlamydia porcellionis' is proposed for intracellular bacteria in the hepatopancreas of P . scaber.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 385 - 8
Ancylobacter rudongensis sp . nov., isolated from roots of Spartina anglica; Xin YH et al.; A curved, ring-like bacterium, strain AS 1.1761(T), isolated from the roots of Spartina anglica, was studied by a polyphasic approach . According to phylogenetic analysis, strain AS 1.1761(T) belongs to the genus Ancylobacter, with 99.21 % 16S rDNA sequence similarity to Ancylobacter aquaticus, the only species described so far in this genus . However, strain AS 1.1761(T) had no significant DNA-DNA binding with the type strain of A . aquaticus . In addition, strain AS 1.1761(T) differed from A . aquaticus in many phenotypic features . Based on molecular and phenotypic data, a novel species, Ancylobacter rudongensis sp . nov., is proposed . The type strain is AS 1.1761(T) (=JCM 11671(T)).

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 365 - 71
Mycoplasma ovis comb . nov . (formerly Eperythrozoon ovis), an epierythrocytic agent of haemolytic anaemia in sheep and goats; Neimark H et al.; Eperythrozoon ovis, an erythrocytic agent that causes haemolytic anaemia in sheep and goats, occurs worldwide and is currently thought to be a rickettsia . To determine the relationship between this agent and other haemotrophic bacterial parasites, the 16S rRNA gene of this organism was sequenced . Phylogenetic analysis revealed that this wall-less bacterium is not a rickettsia, but a mycoplasma . This mycoplasma is related closely to several other uncultivated, epierythrocytic mycoplasmas that comprise a recently identified group, the haemotrophic mycoplasmas (haemoplasmas) . The haemoplasma group is composed of former Eperythrozoon and Haemobartonella species, as well as newly identified epierythrocytic mycoplasmas . Haemoplasmas parasitize the surface of erythrocytes of a wide variety of vertebrate animal hosts and are transmitted mainly by blood-feeding arthropod vectors . Recognition that E . ovis is a mycoplasma provides a new approach to dealing with this bacterium . It is proposed that E . ovis should be reclassified as Mycoplasma ovis comb . nov.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 313 - 6
Asaia krungthepensis sp . nov., an acetic acid bacterium in the alpha-Proteobacteria; Yukphan P et al.; Three bacterial strains were isolated from flowers collected in Bangkok, Thailand, by an enrichment-culture approach for acetic acid bacteria . Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were located in the lineage of the genus Asaia but constituted a cluster separate from the type strains of Asaia bogorensis and Asaia siamensis . The DNA base composition of the isolates was 60.2-60.5 mol% G+C, with a range of 0.3 mol% . The isolates constituted a taxon separate from Asaia bogorensis and Asaia siamensis on the basis of DNA-DNA relatedness . The isolates had morphological, physiological, biochemical and chemotaxonomic characteristics similar to those of the type strains of Asaia bogorensis and Asaia siamensis, but the isolates grew on maltose . The major ubiquinone was Q(10) . On the basis of the results obtained, the name Asaia krungthepensis sp . nov . is proposed for the isolates . The type strain is isolate AA08(T) (=BCC 12978(T)=TISTR 1524(T)=NBRC 100057(T)=NRIC 0535(T)), which had a DNA G+C content of 60.3 mol% and was isolated from a heliconia flower ('paksaasawan' in Thai; Heliconia sp.) collected in Bangkok, Thailand.

BMC Bioinformatics . 2004 Jan 30;5(1):10.
Aggregation of topological motifs in the Escherichia coli transcriptional regulatory network; Dobrin R et al.; BACKGROUND: Transcriptional regulation of cellular functions is carried out through a complex network of interactions among transcription factors and the promoter regions of genes and operons regulated by them . To better understand the system-level function of such networks simplification of their architecture was previously achieved by identifying the motifs present in the network, which are small, overrepresented, topologically distinct regulatory interaction patterns (subgraphs) . However, the interaction of such motifs with each other, and their form of integration into the full network has not been previously examined . RESULTS: By studying the transcriptional regulatory network of the bacterium, Escherichia coli, we demonstrate that the two previously identified motif types in the network (i.e., feed-forward loops and bi-fan motifs) do not exist in isolation, but rather aggregate into homologous motif clusters that largely overlap with known biological functions . Moreover, these clusters further coalesce into a supercluster, thus establishing distinct topological hierarchies that show global statistical properties similar to the whole network . Targeted removal of motif links disintegrates the network into small, isolated clusters, while random disruptions of equal number of links do not cause such an effect . CONCLUSION: Individual motifs aggregate into homologous motif clusters and a supercluster forming the backbone of the E . coli transcriptional regulatory network and play a central role in defining its global topological organization.

Carbohydr Res, 2004 Feb 25, 339(3), 649 - 54
Characterization 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, was shown to produce a lipopolysaccharide in which the antigenic O-polysaccharide component was found by chemical, 1H and 13C NMR and MS analyses to be an unbranched neutral linear polymer of a repeating tetrasaccharide unit composed of 2-acetamido-2-deoxy-D-galacturonamide (D-GalNAcAN) and 2,4-diacetamido-2,4,6-trideoxy-D-glucose (D-Qui2NAc4NAc, di-N-acetylbacillosamine) residues (3:1) and had the structure: -->4)-alpha-D-GalNAcAN-(1-->4)-alpha-D-GalNAcAN-(1-->4)-alpha-D-GalNAcAN-(1-->3)-alpha-D-QuiNAc4NAc-(1--> . With polyclonal murine antibody, the F . novicida O-antigen did not show serological cross-reactivity with the O-antigen of F . tularensis despite the occurrence of a common -->4)-D-GalpNAcAN-(1-->4)-alpha-D-GalpNAcAN-(1--> disaccharide unit in their respective O-antigens . Thus, O-PS serology offers a practical way to distinguish between the two Francisella species.

Carbohydr Res, 2004 Feb 25, 339(3), 477 - 82
Structure of the O-polysaccharide of Idiomarina zobellii KMM 231T containing two unusual amino sugars with the free amino group, 4-amino-4,6-dideoxy-D-glucose and 2-amino-2-deoxy-L-guluronic acid; Kilcoyne M et al.; Mild acid degradation of the lipopolysaccharide of the bacterium Idiomarina zobellii, type strain KMM 231T, with aq 2% HOAc at 100 degrees C, yielded an oligosaccharide, which represents one repeating unit of the O-polysaccharide . A polysaccharide was obtained by mild base degradation of the lipopolysaccharide . The following structure of the O-polysaccharide was elucidated by 1H and 13C NMR spectroscopy of the oligosaccharide and base-degraded lipopolysaccharide, including COSY, TOCSY, ROESY, 1H, 13C HSQC, HSQC-TOCSY and HMBC experiments: {-->3)-alpha-D-Quip4N-(1-->4)-alpha-D-GlcpA-(1-->6)-alpha-D-GlcpNAc-(1-->4)-alpha-L-GulpNA-(1-->3)-beta-D-FucpNAc-(1-->} The O-polysaccharide is distinguished by the presence of two unusual amino sugars, 4-amino-4,6-dideoxy-D-glucose (D-Qui4N) and 2-amino-2-deoxy-L-guluronic acid (L-GulNA), both having the free amino group . The unexpectedly high acid lability of the glycosidic linkage of 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc) could be associated with the presence of a free amino group adjacent to the site of attachment of FucNAc to Qui4N.

Annu Rev Plant Physiol Plant Mol Biol, 1999 Jun, 50, 245 - 276
CELLULOSE BIOSYNTHESIS: Exciting Times for A Difficult Field of Study; Delmer DP; The past few decades have witnessed exciting progress in studies on the biosynthesis of cellulose . In the bacterium Acetobacter xylinum, discovery of the activator of the cellulose synthase, cyclic diguanylic acid, opened the way for obtaining high rates of in vitro synthesis of cellulose . This, in turn, led to purification of the cellulose synthase and for the cloning of genes that encode the catalytic subunit and other proteins that bind the activator and regulate its synthesis and degradation, or that control secretion and crystallization of the microfibrils . In higher plants, a family of genes has been discovered that show interesting similarities and differences from the gene in bacteria that encodes the catalytic subunit of the synthase . Genetic evidence now supports the concept that members of this family encode the catalytic subunit in these organisms, with various members showing tissue-specific expression . Although the cellulose synthase has not yet been purified to homogeneity from plants, recent progress in this area suggests that this will soon be accomplished.

J Evol Biol, 2004 Mar, 17(2), 322 - 30
What maintains noncytoplasmic incompatibility inducing Wolbachia in their hosts: a case study from a natural Drosophila yakuba population; Charlat S et al.; Cytoplasmic incompatibility (CI) allows Wolbachia to invade hosts populations by specifically inducing sterility in crosses between infected males and uninfected females . In some species, non-CI inducing Wolbachia, that are thought to derive from CI-inducing ancestors, are common . In theory, the maintenance of such infections is not possible unless the bacterium is perfectly transmitted to offspring--and/or provides a fitness benefit to infected females . The present study aims to test this view by investigating a population of Drosophila yakuba from Gabon, West Africa . We did not find any evidence for CI using wild caught females . Infected females from the field transmitted the infection to 100% of their offspring . A positive effect on female fecundity was observed one generation after collecting, but this was not retrieved five generations later, using additional lines . Similarly, the presence of Wolbachia was found to affect mating behaviour, but the results of two experiments realized five generations apart were not consistent . Finally, Wolbachia was not found to affect sex ratio . Overall, our results would suggest that Wolbachia behaves like a neutral or nearly neutral trait in this species, and is maintained in the host by perfect maternal transmission.

Appl Environ Microbiol, 2004 Mar, 70(3), 1405 - 12
Vanadium(V) reduction by Shewanella oneidensis MR-1 requires menaquinone and cytochromes from the cytoplasmic and outer membranes; Myers JM et al.; The metal-reducing bacterium Shewanella oneidensis MR-1 displays remarkable anaerobic respiratory plasticity, which is reflected in the extensive number of electron transport components encoded in its genome . In these studies, several cell components required for the reduction of vanadium(V) were determined . V(V) reduction is mediated by an electron transport chain which includes cytoplasmic membrane components (menaquinone and the tetraheme cytochrome CymA) and the outer membrane (OM) cytochrome OmcB . A partial role for the OM cytochrome OmcA was evident . Electron spin resonance spectroscopy demonstrated that V(V) was reduced to V(IV) . V(V) reduction did not support anaerobic growth . This is the first report delineating specific electron transport components that are required for V(V) reduction and of a role for OM cytochromes in the reduction of a soluble metal species.

Biochemistry, 2004 Mar 16, 43(10), 2915 - 25
Chemo- and regioselective peptide cyclization triggered by the N-terminal fatty acid chain length: the recombinant cyclase of the calcium-dependent antibiotic from Streptomyces coelicolor; Grunewald J et al.; Here we report the first biochemical characterization of a recombinant nonribosomal peptide cyclase of a streptomycete, the model actinomycete Streptomyces coelicolor A3(2) . This bacterium produces the calcium-dependent antibiotic (CDA), which is a branched cyclic macrolactone belonging to the group of acidic lipopeptides . The recombinant CDA3 cyclase from CDA synthetase efficiently catalyzes ring formation of linear peptidyl thioester substrates based on a sequence analogous to natural CDA . Four leaving groups were attached to the C-terminus of the undecapeptide: coenzyme A (CoA), phosphopantetheine, N-acetylcysteamine (SNAC), and thiophenol . The best rates for cyclization were determined for the thiophenol substrate, revealing that chemical reactivity is more important than cofactor recognition . The cyclase catalyzes the formation of two regioisomeric macrolactones, which arise from simultaneous nucleophilic attack of the two adjacent Thr(2) and Ser(1) residues onto the C-terminus of the acyl-enzyme intermediate . This relaxed regioselectivity has not been observed for any other recombinant NRPS or PKS cyclases so far . Substitution of either Ser(1) or Thr(2) by alanine led to selective formation of a decapeptide or undecapeptide lactone ring . In contrast to that, CDA3 cyclase strictly retains stereoselectivity for both nucleophiles, accepting only l-configured Ser(1) and Thr(2) for cyclization . Further, our studies provide evidence for the crucial role of N-terminal fatty acyl groups of lipopeptides in controlling the regio- and chemoselectivity of enzyme-catalyzed macrocyclization . Elongation of the fatty acyl group of our thioester substrate from C(2) to C(6) as in CDA turned the relaxed regioselectivity into a strict regioselectivity, yielding solely the decapeptide lactone ring with a significantly improved cyclization-to-hydrolysis ratio.

J Immunol, 2004 Mar 15, 172(6), 3695 - 703
Lipopolysaccharide from Coxiella burnetii is involved in bacterial phagocytosis, filamentous actin reorganization, and inflammatory responses through Toll-like receptor 4; Honstettre A et al.; The role of Toll-like receptors (TLRs) in the recognition of extracellular and facultative intracellular bacteria by the innate immune system has been extensively studied, but their role in the recognition of obligate intracellular organisms remains unknown . Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that specifically inhabits monocytes/macrophages . We showed in this study that C . burnetii LPS is involved in the uptake of virulent organisms by macrophages but not in that of avirulent variants . The uptake of virulent organisms was dependent on TLR4 because it was reduced in macrophages from TLR4(-/-) mice . In addition, LPS was responsible for filamentous actin reorganization induced by virulent C . burnetii, which was prevented in TLR4(-/-) macrophages . In contrast, the intracellular fate of C . burnetii was not affected in TLR4(-/-) macrophages, suggesting that TLR4 does not control the maturation of C . burnetii phagosome and the microbicidal activity of macrophages . These results are consistent with in vivo experiments because the pattern of tissue infection and the clearance of C . burnetii were similar in wild-type and TLR4(-/-) mice . We also showed that the number of granulomas was decreased in the liver of infected TLR4(-/-) mice, and the formation of splenic granulomas was only transient . The impaired formation of granulomas was associated with decreased production of IFN-gamma and TNF . Taken together, these results demonstrate that TLR4 controls early events of C . burnetii infection such as macrophage phagocytosis, granuloma formation, and cytokine production.

Mod Pathol, 2004 May, 17(5), 489 - 95
Histologic and molecular diagnosis of tularemia: a potential bioterrorism agent endemic to North America; Lamps LW et al.; Francisella tularensis (FT), a zoonotic bacterium that causes tularemia, has received attention as a possible bioterrorism threat . We developed a PCR assay for use in fixed, processed tissues, which are safer to handle and allow archival testing . PCR analysis for a 211-bp fragment of the FT lipoprotein gene was performed on tissues from 16 cases of tularemia . In all, 14/15 cases with intact DNA (93%) were positive for FT by PCR . Frequent histologic findings in PCR-positive tissues included irregular microabscesses and granulomas in liver, spleen, kidney, and lymph nodes, and necrotizing pneumonia . Unusual cases featuring suppurative leptomeningitis and gastrointestinal ulcers were also seen . As this disease is endemic in North America, and has been identified as a potential bioterroristic threat, awareness of the clinicopathologic spectrum of disease and available detection methods is increasingly important . This PCR assay, the first designed for use in processed tissues, is an excellent method for diagnosis of tularemia.

Biochemistry (Mosc), 2004 Feb, 69(2), 164 - 9
Properties of stable hydrogenase from the purple sulfur bacterium Lamprobacter modestohalophilus; Zadvorny OA et al.; Some properties of a hydrogenase from the recently isolated phototrophic sulfur bacterium Lamprobacter modestohalophilus strain Syvash and its resistance to a number of inactivating factors have been investigated . The enzyme consists of two subunits, 64 and 30 kD; pI = 4.5 . The optimal pH was 8.5-9.5 for hydrogen uptake and 4.0 for H2 evolution . Hydrogenase preparations were resistant to the effects of O2, CO, and temperature, revealing high stability under storage . A considerable inactivation of the enzyme was observed at temperatures above 80 degrees C; the temperature optimum of methyl viologen reduction by H2 was 85 degrees C . Inhibitory effects of Ni2+, Cd2+, and Mg2+ on the hydrogenase activity were shown to be reversible and competitive with respect to methyl viologen in the hydrogen oxidation reaction.

Cell Mol Life Sci, 2004 Feb, 61(4), 417 - 36
Oxygen intervention in the regulation of gene expression: the photosynthetic bacterial paradigm; Zeilstra-Ryalls JH et al.; The means by which oxygen intervenes in gene expression has been examined in considerable detail in the metabolically versatile bacterium Rhodobacter sphaeroides . Three regulatory systems are now known in this organism, which are used singly and in combination to modulate genes in response to changing oxygen availability . The outcome of these regulatory events is that the molecular machinery is present for the cell to obtain energy by means that are best suited to prevailing conditions, while at the same time maintaining cellular redox balance . Here, we explore the dangers associated with molecular oxygen relative to the various metabolisms used by R . sphaeroides, and then present the most recent findings regarding the features and operation of each of the three regulatory systems which collectively mediate oxygen control in this organism.

J Econ Entomol, 2003 Apr, 96(2), 264 - 71
Transmission of Xylella fastidiosa to grapevines by Homalodisca coagulata (Hemiptera: Cicadellidae); Almeida RP et al.; Pierce's disease (PD) of grapevines is caused by a xylem-limited bacterium Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner) that is transmitted to plants by xylem sap-feeding insects . The introduction of the sharpshooter leafhopper Homalodisca coagulata (Say) into California has initiated new PD epidemics in southern California . In laboratory experiments, the major characteristics of H . coagulata's transmission of X . fastidiosa to grapevines were the same as reported for other vectors: short or absent latent period; nymphs transmitted but lost infectivity after molting and regained infectivity after feeding on infected plants; and infectivity persisted in adults . Adult H . coagulata acquired and inoculated X . fastidiosa in <1 h of access time on a plant . Inoculation rates increased with access time, but acquisition efficiency (20% per individual) did not increase significantly beyond 6-h access . Estimated inoculation efficiency per individual per day was 19.6, 17.9, and 10.3% for experiments where plant access was 1, 2, and 4 d, respectively . Freshly molted adults and nymphs acquired and transmitted X . fastidiosa more efficiently than did older, field-collected insects . H . coagulata transmitted X . fastidiosa to 2-yr-old woody tissues of grapevines as efficiently as to green shoots . H . coagulata transmitted X . fastidiosa 3.5 mo after acquisition, demonstrating persistence of infectivity in adults . About half (14/29) of the H . coagulata from which we failed to culture X . fostidiosa from homogenized heads (with a detection threshold of 265 CFU/head) transmitted the pathogen to grape, and 17 of 24 from which we cultured X . fastidiosa transmitted.

Microb Ecol, 2004 Apr, 47(3), 300 - 4 Epub 2004 Mar 04.
Subfreezing growth of the sea ice bacterium "Psychromonas ingrahamii"; Breezee J et al.; "Psychromonas ingrahamii," named for John L . Ingraham, was isolated from sea ice from off Point Barrow, Alaska . This large rod-shaped bacterium belongs to the gamma-Proteobacteria . "P . ingrahamii" is a psychrophilic, heterotrophic bacterium that is gas vacuolate and nonmotile . "P . ingrahamii" is notable in that it grows at a temperature of -12 degrees C with a generation time of 240 h . This is the lowest growth temperature of any organism authenticated by a growth curve.

Microbiology, 2004 Mar, 150(Pt 3), 727 - 34
Groupings of highly similar major surface protein (p44)-encoding paralogues: a potential index of genetic diversity amongst isolates of Anaplasma phagocytophilum; Casey AN et al.; Anaplasma phagocytophilum is a tick-borne bacterium that is zoonotic in the USA and southern Europe, but although the bacterium is endemic in the UK, no cases of clinical human disease have yet been detected in that country . Potential genomic differences amongst UK and USA isolates were investigated by comparing partial 16S rRNA gene and p44 paralogue sequences amplified by PCR from 10 UK ruminant or tick isolates, with published sequences from USA isolates . No significant clustering among the isolates was resolved by phylogenetic analysis of alignments containing 16S rRNA gene sequences . The structure of predicted proteins encoded by p44 paralogues, amplified from 81 clones obtained from the UK isolates, was similar to that described previously for paralogues from USA isolates . Paralogue sequences did not obviously cluster by country, host species or isolate, but most paralogues were 30-70 % similar, making meaningful alignments difficult . Some p44 paralogues from different isolates formed clusters of sequences that were more than 90 % similar to one another ('similarity groups') . The paralogues in each cluster were particularly similar in gene regions most likely to code for ligands . In the sample studied, 95 % of the similarity groups comprised paralogues from either USA or UK isolates only and occurred with greater frequency amongst paralogues from USA rather than UK isolates . These findings raise the hypothesis that sequences of paralogues in similarity groups may provide an index of adaptation of different 'strains' of A . phagocytophilum to specific reservoir hosts in different geographical locations, and any associations with infectivity for different species including humans.

Extremophiles, 2004 Jun, 8(3), 185 - 92 Epub 2004 Feb 26.
Growth kinetics of haloalkaliphilic, sulfur-oxidizing bacterium Thioalkalivibrio versutus strain ALJ 15 in continuous culture; Banciu H et al.; The chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio versutus strain ALJ 15, isolated from a soda lake in Kenya, was grown in a continuous culture, with thiosulfate or polysulfide as growth-limiting energy source and oxygen as electron acceptor, at pH 10 and at pH 0.6, 2 M and 4 M total sodium . The end product of the sulfur-compound oxidation was sulfate . Elemental sulfur and a cell-bound, polysulfide-like compound appeared as intermediates during substrate oxidation . In the thiosulfate-limited culture, the biomass yields and maximum specific growth rates decreased two and three times, respectively, with increasing sodium concentration . The apparent affinity constant measured for thiosulfate and polysulfide was in the micromolar range (Ks = 6 +/- 3 microM) . The maintenance requirement (ms = 8 +/- 5 mmol S2O3(2)/g dry weight h(-1)) was in the range of values found for other autotrophic sulfur-oxidizing bacteria . The organism had a comparable maximum specific rate of oxygen uptake with thiosulfate, polysulfide, and sulfide, while elemental sulfur was oxidized at a lower rate . Glycine betaine was the main organic compatible solute . The respiration rates with different species of polysulfides (Sn2-) were tested . All polysulfide species were completely oxidized at high rates to sulfate . Overall data demonstrated efficient growth and sulfur compounds oxidation of haloalkaliphilic chemolithoautotrophic bacteria from soda lakes .

FEMS Immunol Med Microbiol, 2004 Mar 8, 40(2), 129 - 37
Identification of an in vivo CD4+ T cell-mediated response to polymorphic membrane proteins of Chlamydia pneumoniae during experimental infection; Mygind T et al.; Chlamydia pneumoniae is an obligate intracellular bacterium that causes upper and lower respiratory tract infection in humans . C . pneumoniae harbors the polymorphic membrane protein (Pmp) family with 21 different proteins with a molecular mass around 100 kDa . The Pmps are species-specific, abundant and, together with major outer membrane protein and outer membrane protein 2, the dominant proteins in the C . pneumoniae outer membrane complex . Nevertheless, it is unknown whether Pmps are recognized by the cell-mediated immune response . To address this issue, C57BL/6J mice were infected intranasally with C . pneumoniae and the immune response to primary infection was investigated . We demonstrate, as expected, that the primary response is of the Th1 type by IgG2a- and IgG1-specific sELISA (Medac) on serum . In vivo-primed spleen lymphocytes were found to be reactive to Pmp8, Pmp20 and Pmp21 in an interferon-gamma ELISpot assay . The responses were shown to be mediated by CD4(+) T cells . To our knowledge, this is the first identification of antigens recognized by CD4(+) T cells during murine C . pneumoniae infection.

J Biol Inorg Chem, 2004 Apr, 9(3), 256 - 68 Epub 2004 Feb 19.
Cyanide as a copper and quinone-directed inhibitor of amine oxidases from pea seedlings ( Pisum sativum) and Arthrobacter globiformis: evidence for both copper coordination and cyanohydrin derivatization of the quinone cofactor; Shepard EM et al.; The interactions of cyanide with two copper-containing amine oxidases (CuAOs) from pea seedlings (PSAO) and the soil bacterium Arthrobacter globiformis (AGAO) have been investigated by spectroscopic and kinetic techniques . Previously, we rationalized the effects of azide and cyanide for several CuAOs in terms of copper coordination by these exogenous ligands and their effects on the internal redox equilibrium TPQ(amr)-Cu(II) right harpoon over left harpoon TPQ(sq)-Cu(I) . The mechanism of cyanide inhibition was proposed to occur through complexation to Cu(I), thereby directly competing with O(2) for reoxidation of TPQ . Although cyanide readily and reversibly reacts with quinones, no direct spectroscopic evidence for cyanohydrin derivatization of TPQ has been previously documented for CuAOs . This work describes the first direct spectroscopic evidence, using both model and enzyme systems, for cyanohydrin derivatization of TPQ . K(d) values for Cu(II)-CN(-) and Cu(I)-CN(-), as well as the K(i) for cyanide inhibition versus substrate amine, are reported for PSAO and AGAO . In spite of cyanohydrin derivatization of the TPQ cofactor in these enzymes, the uncompetitive inhibition of amine oxidation is determined to arise almost exclusively through CN(-) complexation of Cu(I).

Nature, 2004 Feb 26, 427(6977), 839 - 43
Global organization of metabolic fluxes in the bacterium Escherichia coli; Almaas E et al.; Cellular metabolism, the integrated interconversion of thousands of metabolic substrates through enzyme-catalysed biochemical reactions, is the most investigated complex intracellular web of molecular interactions . Although the topological organization of individual reactions into metabolic networks is well understood, the principles that govern their global functional use under different growth conditions raise many unanswered questions . By implementing a flux balance analysis of the metabolism of Escherichia coli strain MG1655, here we show that network use is highly uneven . Whereas most metabolic reactions have low fluxes, the overall activity of the metabolism is dominated by several reactions with very high fluxes . E . coli responds to changes in growth conditions by reorganizing the rates of selected fluxes predominantly within this high-flux backbone . This behaviour probably represents a universal feature of metabolic activity in all cells, with potential implications for metabolic engineering.

Am J Pathol, 2004 Mar, 164(3), 947 - 57
Epithelia under metabolic stress perceive commensal bacteria as a threat; Nazli A et al.; The normal gut flora has been implicated in the pathophysiology of inflammatory bowel disease and there is increased interest in the role that stress can play in gut disease . The chemical stressor dinitrophenol (DNP, uncouples oxidative phosphorylation) was injected into the ileum of laparotomized rats and mitochondria structure, epithelial permeability, and inflammatory cell infiltrate were examined 6 and 24 hours later . Monolayers of human colonic epithelial cells (T84, HT-29) were treated with DNP +/- commensal Escherichia coli, followed by assessment of epithelial permeability, bacterial translocation, and chemokine (ie, interleukin-8) synthesis . Delivery of DNP into rat distal ileum resulted in disruption of epithelial mitochondria; similar changes were noted in mildly inflamed ileal resections from patients with Crohn's disease . Also, DNP-treated ileum displayed increased gut permeability and immune cell recruitment . Subsequent studies revealed deceased barrier function, increased bacterial translocation, increased production of interleukin-8, and enhanced mobilization of the transcription factor AP-1 in the model epithelial cell lines exposed to commensal bacteria (E . coli strains HB101 or C25), but only when the monolayers were pretreated with DNP (0.1 mmol/L) . These data suggest that enteric epithelia under metabolic stress perceive a normally innocuous bacterium as threatening, resulting in loss of barrier function, increased penetration of bacteria into the mucosa, and increased chemokine synthesis . Such responses could precipitate an inflammatory episode and contribute to existing enteric inflammatory disorders.

Virology, 2004 Feb 20, 319(2), 274 - 9
Construction of carrier state viruses with partial genomes of the segmented dsRNA bacteriophages; Sun Y et al.; The cystoviridae are bacteriophages with genomes of three segments of dsRNA enclosed within a polyhedral capsid . Two members of this family, Phi6 and Phi8, have been shown to form carrier states in which the virus replicates as a stable episome in the host bacterium while expressing reporter genes such as kanamycin resistance or lacalpha . The carrier state does not require the activity of all the genes necessary for phage production . It is possible to generate carrier states by infecting cells with virus or by electroporating nonreplicating plasmids containing cDNA copies of the viral genomes into the host cells . We have found that carrier states in both Phi6 and Phi8 can be formed at high frequency with all three genomic segments or with only the large and small segments . The large genomic segment codes for the proteins that constitute the inner core of the virus, which is the structure responsible for the packaging and replication of the genome . In Phi6, a carrier state can be formed with the large and middle segment if mutations occur in the gene for the major structural protein of the inner core . In Phi8, carrier state formation requires the activity of genes 8 and 12 of segment S.

Analyst, 2004 Mar, 129(3), 265 - 9 Epub 2004 Jan 27.
An automated turbidimetric method for the identification of certain antibiotic groups in incurred kidney samples; Myllyniemi AL et al.; An automated turbidimetric method was developed for group level identification of penicillinase sensitive penicillins, tetracyclines and fluoroquinolones in kidney samples . A sample pretreatment procedure was elaborated for the extraction of incurred residues from kidney tissue in a translucent solution to enable the measurement of changes in optical density . The method was comprised of three pairs, one for each antibiotic group: a sensitive test bacterium strain and a resistant strain for the identification of fluoroquinolones and tetracyclines, and a sensitive strain with and without penicillinase for the identification of penicillinase sensitive penicillins . The algorithm employed compared the areas under the OD vs . time curves; threshold values and experimentally observed intra-test criteria were also included in the algorithm . Antibiotics were reliably identified to group level, and no false identifications were obtained with antibiotics belonging to groups not included in the reference panel . Incurred penicillin G, oxytetracycline and enrofloxacin-ciprofloxacin residues were identified at or below the MRL levels for kidney tissue . The graphically determined shortest possible identification times varied between 2 and 7 h . The method developed could furthermore easily be diversified to include other antibiotic groups by adding new "sensitive-resistant" bacterium and medium combinations.

Infect Immun, 2004 Mar, 72(3), 1530 - 6
Populations of human T lymphocytes that traverse the vascular endothelium stimulated by Borrelia burgdorferi are enriched with cells that secrete gamma interferon; Gergel EI et al.; Some diseases are characterized by prevalence in the affected tissues of type 1 T lymphocytes, which secrete gamma interferon (IFN-gamma) and other proinflammatory cytokines . For example, type 1 T cells predominate in the lesions of patients with Lyme disease, which is caused by the bacterium Borrelia burgdorferi . We used an in vitro model of the blood vessel wall to test the premise that the vascular endothelium actively recruits circulating type 1 T cells to such lesions . When T lymphocytes isolated from human peripheral blood were examined, the populations that traversed monolayers of resting human umbilical vein endothelial cells (HUVEC) or HUVEC stimulated by interleukin-1beta or B . burgdorferi were markedly enriched for T cells that produced IFN-gamma compared to the initially added population of T cells . No enrichment was seen for cells that produced interleukin-4, a marker for type 2 T lymphocytes . Very late antigen-4 and CD11/CD18 integrins mediated passage of the T cells across both resting and stimulated HUVEC, and the endothelium-derived chemokine CCL2 (monocyte chemoattractant protein 1) was responsible for the enhanced migration of T cells across stimulated HUVEC . These results suggest that the vascular endothelium may contribute to the selective accumulation of type 1 T cells in certain pathological lesions, including those of Lyme disease.

Front Biosci, 2004 May 01, 9, 1228 - 339
Main features on tailed phage, host recognition and DNA uptake; Letellier L et al.; Phage nucleic acid transport is atypical among membrane transport and thus poses a fascinating problem: transport is unidirectional; it concerns a unique molecule the size of which may represent 50 times that of the bacterium . The rate of DNA transport can reach values as high as 3 to 4 thousands base pairs/sec . This raises many questions, which will be addressed in this review . Is there a single mechanism of transport for all types of phages? How does the phage genome overcome the hydrophobic barrier of the host envelope? Is DNA transported as a free molecule or in association with proteins? Is such transport dependent on phage and/or host cell components? What is the driving force for transport? Data will be presented for a few selected tailed phages, which are the most common type of phages and for which DNA transport has been most extensively studied . Part of the review is devoted to recent in vitro data which have allowed to partly decipher the mechanism of phage T5 DNA transport.

J Bacteriol, 2004 Mar, 186(5), 1330 - 6
Dimerization of the RamC morphogenetic protein of Streptomyces coelicolor; Hudson ME et al.; RamC is required for the formation of spore-forming cells called aerial hyphae by the bacterium Streptomyces coelicolor . This protein is membrane associated and has an amino-terminal protein kinase-like domain, but little is known about its mechanism of action . In this study we found that the presence of multiple copies of a defective allele of ramC inhibits morphogenesis in S . coelicolor, consistent with either titration of a target or formation of inactive RamC multimers . We identified a domain in RamC that is C terminal to the putative kinase domain and forms a dimer with a K(d) of approximately 0.1 micro M . These data suggest that RamC acts as a dimer in vivo.

J Bacteriol, 2004 Mar, 186(5), 1448 - 61
Transcriptional profiling of Caulobacter crescentus during growth on complex and minimal media; Hottes AK et al.; Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source . Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media . The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity . Amino acid degradation pathways constituted the largest class of genes induced in PYE . In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine . Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C . crescentus for growth on glucose . Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism . A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression . Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C . crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation.

Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 2782 - 7 Epub 2004 Feb 17.
Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum; Zhang Y et al.; The P(II) regulatory protein family is unusually widely distributed, being found in all three domains of life . Three P(II) homologs called GlnB, GlnK, and GlnJ have been identified in the photosynthetic bacterium Rhodospirillum rubrum . These have roles in at least four distinct functions, one of which is activation of the nitrogen fixation-specific regulatory protein NifA . The activation of NifA requires only the covalently modified (uridylylated) form of GlnB . GlnK and GlnJ are not involved . However, the basis of specificity for different P(II) homologs in different processes is poorly understood . We examined this specificity by altering GlnJ to support NifA activation . A small number of amino acid substitutions in GlnJ were important for this ability . Two (affecting residues 45 and 54) are in a loop called the T-loop, which contains the site of uridylylation and is believed to be very important for contacts with other proteins, but other critical residues lie in the C terminus (residues 95-97 and 109-112) and near the N terminus (residues 3-5 and 17) . Because many of the residues important for P(II)-NifA interaction lie far from the T-loop in the known x-ray crystal structures of P(II) proteins, our results lead to the hypothesis that the T-loop of GlnB is flexible enough to come into proximity with both the C- and N-terminal regions of the protein to bind NifA . Finally, the results show that the level of P(II) accumulation is also an important factor for NifA activation.

Pol Merkuriusz Lek, 2003 Nov, 15(89), 428 - 31
{Helicobacter pylori infection and its influence on Parkinson disease}; Wlodarek D et al.; The aim of this study was to determine the frequency of Helicobacter pylori (H . pylori) infection and its influence on the symptoms of Parkinson's disease . The study group consisted of 58 people with Parkinson's disease aged 49 to 83 years old, and 26 of their spouses . Percentage of people infected by H . pylori was similar between people with Parkinson's disease and their spouses . In the same time in this study percentage of people infected by this bacterium was lower than in other studies of polish population . There was no relation between Parkinson's disease and H . pylori infection in the investigated group of patients suffering from Parkinson's disease.

Environ Sci Technol, 2004 Feb 1, 38(3), 769 - 74
Role of Leptothrix discophora in mediating metal uptake in the filter-feeding bivalve Mytilus trossulus (edulis); Widmeyer JR et al.; The potential for filter-feeding bivalves to accumulate metals from a wide range of food sources is an important consideration when examining trophic transfer of metals up the food chain . The objective of this study was to determine the role of Leptothrix discophora in mediating metal uptake in the filter-feeding bivalve Mytilus trossulus . The bacterium L . discophora SP-6 was cultured in the absence or presence of Mn, allowing for a naturally formed Mn oxide sheath to develop . Secondary metals (Cd and Pb) were then added to the cultures, allowing for potential Cd and Pb adsorption to the Mn oxide sheath . Resulting bacterial aggregates of known diameter were then fed to the bivalve M . trossulus using a flow-through system . Initial concentrations of both Pb and Cd on the bacterium did not differ significantly in the presence or absence of the Mn oxide; conversely both Pb (F = 7.39, p < 0.0001) and Cd (F= 33.65, p < 0.0001) were found at lower concentrations in the mussel tissue when the Mn oxide was present . To determine whether these differences in metal uptake could be attributed to sorting by the mussel based on food quality, nutritional analysis was performed . Bacterial food matrixes containing Mn oxides were found to have significantly lower levels of carbon (F = 256, p < 0.0001) . Particle clearance rates for the various food matrixes were positively correlated with organic content (R2 = 0.852, p > 0.008) . The results of our study suggest that metal uptake in M . trossulus was significantly decreased for Cd with a similar trend for Pb when the SP-6 sheath contained Mn oxides . The mechanism mediating this differential uptake is best explained by food quality, in that a higher quality food source enhanced metal uptake due to an increased clearance rate of organic-rich particles by M . trossulus.

Syst Biol, 2004 Feb, 53(1), 95 - 110
Host-symbiont stability and fast evolutionary rates in an ant-bacterium association: cospeciation of camponotus species and their endosymbionts, candidatus blochmannia; Degnan PH et al.; Bacterial endosymbionts are widespread across several insect orders and are involved in interactions ranging from obligate mutualism to reproductive parasitism . Candidatus Blochmannia gen . nov . (Blochmannia) is an obligate bacterial associate of Camponotus and related ant genera (Hymenoptera: Formicidae) . The occurrence of Blochmannia in all Camponotus species sampled from field populations and its maternal transmission to host offspring suggest that this bacterium is engaged in a long-term, stable association with its ant hosts . However, evidence for cospeciation in this system is equivocal because previous phylogenetic studies were based on limited gene sampling, lacked statistical analysis of congruence, and have even suggested host switching . We compared phylogenies of host genes (the nuclear EF-1alphaF2 and mitochondrial COI/II) and Blochmannia genes (16S ribosomal DNA {rDNA}, groEL, gidA, and rpsB), totaling more than 7 kilobases for each of 16 Camponotus species . Each data set was analyzed using maximum likelihood and Bayesian phylogenetic reconstruction methods . We found minimal conflict among host and symbiont phylogenies, and the few areas of discordance occurred at deep nodes that were poorly supported by individual data sets . Concatenated protein-coding genes produced a very well-resolved tree that, based on the Shimodaira-Hasegawa test, did not conflict with any host or symbiont data set . Correlated rates of synonymous substitution (d(S)) along corresponding branches of host and symbiont phylogenies further supported the hypothesis of cospeciation . These findings indicate that Blochmannia-Camponotus symbiosis has been evolutionarily stable throughout tens of millions of years . Based on inferred divergence times among the ant hosts, we estimated rates of sequence evolution of Blochmannia to be approximately 0.0024 substitutions per site per million years (s/s/MY) for the 16S rDNA gene and approximately 0.1094 s/s/MY at synonymous positions of the genes sampled . These rates are several-fold higher than those for related bacteria Buchnera aphidicola and Escherichia coli . Phylogenetic congruence among Blochmannia genes indicates genome stability that typifies primary endosymbionts of insects.

Protein Pept Lett, 2004 Feb, 11(1), 93 - 6
Crystallization and MAD data collection of high-molecular weight cytochrome c from Desulfovibrio vulgaris Miyazaki F; Shibata N et al.; Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized . X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method . The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.

MMW Fortschr Med, 2003 Dec 4, 145(49), 42 - 5
{Guidelines for the diagnosis and treatment of H . pylori infection}; Malfertheiner P; In accordance with our current understanding, infection with the pathogen Helicobacter pylori is an important, but readily diagnosable and treatable cause of various gastroduodenal diseases . A lasting cure is achieved when the bacterium is eradicated with the aid of an established therapeutic regimen . Furthermore, eradication is a rational preventive measure even in asymptomatic patients, since current clinical data show that it offers protection, in particular to high-risk groups, from gastric carcinoma, but also from other complications (e.g . peptic ulcer) . The upgraded European Guidelines on the diagnosis and treatment of H . pylori infection put out by the European Helicobacter Pylori Study Group (EHPSG) in Maastricht take account of the present state of our knowledge of the significance of H . pylori eradication.

Mar Biotechnol (NY), 2001 Mar, 3(2), 96 - 9
Genetic transformation system for a psychrotrophic deep-sea bacterium: isolation and characterization of a psychrotrophic plasmid; Kurusu Y et al.; A versatile system that permits genetic manipulation of a psychrotrophic deep-sea bacterium, Pseudoalteromonas sp . PS1M3, has been developed . A cryptic indigenous plasmid, pPS1M3, of 3.1 kb from the above strain was isolated and characterized . The nucleotide sequence analysis of plasmid pPS1M3 revealed the presence of one open reading frame, and its deduced amino acid sequence was identified as the essential protein for plasmid maintenance . Transformation with the pPS1M3 harboring antibiotic resistance genes by electroporation was fully successful using the pPS1M3-cured strain as a host . This plasmid was quite stable under nonselective culture conditions for about 100 generations at 4 degrees C . The copy number of this plasmid in the cell was about 5 copies per chromosome.

Environ Microbiol, 2004 Mar, 6(3), 191 - 7
Cloning and functional analysis of alkB genes in Alcanivorax borkumensis SK2; Hara A et al.; Alcanivorax is an alkane-degrading marine bacterium which propagates and becomes predominant in crude-oil-containing seawater when nitrogen and phosphorus nutrients are supplemented . To identify the genes responsible for alkane degradation in this organism, two putative genes for alkane hydroxylases were cloned from Alcanivorax borkumensis SK2 . They were named alkB1 and alkB2 . These genes were subsequently disrupted in A . borkumensis SK2, and the growth phenotypes of the disruptants were examined . The results indicate that the alkB1 gene is responsible for the degradation of short-chain n-alkanes . A double mutant defective in both alkB1 and alkB2 was still able to grow on medium-chain n-alkanes, indicating that genes other than alkB1 and alkB2 are also involved in n-alkane hydroxylation by A . borkumensis SK2.

FEMS Microbiol Lett, 2004 Feb 9, 231(1), 39 - 43
Oxalate metabolism by the acetogenic bacterium Moorella thermoacetica; Daniel SL et al.; Whole-cell and cell-extract experiments were performed to study the mechanism of oxalate metabolism in the acetogenic bacterium Moorella thermoacetica . In short-term, whole-cell assays, oxalate consumption was low unless cell suspensions were supplemented with CO(2), KNO(3), or Na(2)S(2)O(3) . Cell extracts catalyzed the oxalate-dependent reduction of benzyl viologen . Oxalate consumption occurred concomitant to benzyl viologen reduction; when benzyl viologen was omitted, oxalate was not appreciably consumed . Based on benzyl viologen reduction, specific activities of extracts averaged 0.6 micromol oxalate oxidized min(-1) mg protein(-1) . Extracts also catalyzed the formate-dependent reduction of NADP(+); however, oxalate-dependent reduction of NADP(+) was negligible . Oxalate- or formate-dependent reduction of NAD(+) was not observed . Addition of coenzyme A (CoA), acetyl-CoA, or succinyl-CoA to the assay had a minimal effect on the oxalate-dependent reduction of benzyl viologen . These results suggest that oxalate metabolism by M . thermoacetica requires a utilizable electron acceptor and that CoA-level intermediates are not involved.

Appl Environ Microbiol, 2004 Feb, 70(2), 1040 - 50
Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense; Grunberg K et al.; We analyzed the biochemical composition of the magnetosome membrane (MM) in Magnetospirillum gryphiswaldense . Isolated magnetosomes were associated with phospholipids and fatty acids which were similar to phospholipids and fatty acids from other subcellular compartments (i.e., outer and cytoplasmic membranes) but were present in different proportions . The binding characteristics of MM-associated proteins were studied by selective solubilization and limited proteolysis . The MM-associated proteins were further analyzed by various proteomic approaches, including one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Edman and mass spectrometric (electrospray ionization-mass spectrometry-mass spectrometry) sequencing, as well as capillary liquid chromatography-mass spectrometry-mass spectrometry of total tryptic digests of the MM . At least 18 proteins were found to constitute the magnetosome subproteome, and most of these proteins are novel for M . gryphiswaldense . Except for MM22 and Mms16, all bona fide MM proteins (MMPs) were encoded by open reading frames in the mamAB, mamDC, and mms6 clusters in the previously identified putative magnetosome island . Eight of the MMPs display homology to known families, and some of them occur in the MM in multiple homologues . Ten of the MMPs have no known homologues in nonmagnetic organisms and thus represent novel, magnetotactic bacterium-specific protein families . Several MMPs display repetitive or highly acidic sequence patterns, which are known from other biomineralizing systems and thus may have relevance for magnetite formation.

Appl Environ Microbiol, 2004 Feb, 70(2), 722 - 8
Cyanobacterial-type, heteropentameric, NAD+-reducing NiFe hydrogenase in the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina; Rakhely G et al.; Structural genes coding for two membrane-associated NiFe hydrogenases in the phototrophic purple sulfur bacterium Thiocapsa roseopersicina (hupSL and hynSL) have recently been isolated and characterized . Deletion of both hydrogenase structural genes did not eliminate hydrogenase activity in the cells, and considerable hydrogenase activity was detected in the soluble fraction . The enzyme responsible for this activity was partially purified, and the gene cluster coding for a cytoplasmic, NAD+-reducing NiFe hydrogenase was identified and sequenced . The deduced gene products exhibited the highest similarity to the corresponding subunits of the cyanobacterial bidirectional soluble hydrogenases (HoxEFUYH) . The five genes were localized on a single transcript according to reverse transcription-PCR experiments . A sigma54-type promoter preceded the gene cluster, suggesting that there was inducible expression of the operon . The Hox hydrogenase was proven to function as a truly bidirectional hydrogenase; it produced H2 under nitrogenase-repressed conditions, and it recycled the hydrogen produced by the nitrogenase in cells fixing N2 . In-frame deletion of the hoxE gene eliminated hydrogen evolution derived from the Hox enzyme in vivo, although it had no effect on the hydrogenase activity in vitro . This suggests that HoxE has a hydrogenase-related role; it likely participates in the electron transfer processes . This is the first example of the presence of a cyanobacterial-type, NAD+-reducing hydrogenase in a phototrophic bacterium that is not a cyanobacterium . The potential physiological implications are discussed.

Cell Microbiol, 2004 Mar, 6(3), 255 - 67
Helicobacter pylori gamma-glutamyltranspeptidase upregulates COX-2 and EGF-related peptide expression in human gastric cells; Busiello I et al.; Gastric mucosa responds to Helicobacter pylori-induced cell damage by increasing the expression of COX-2 and EGF-related peptides . We sought to investigate the bacterial virulence factor/s and the host cellular pathways involved in the upregulation of COX-2, HB-EGF and amphiregulin in MKN 28 and AGS gastric mucosal cells . H . pylori strain CCUG 17874 was grown in Brucella broth supplemented with 0.2% (2,6-dimethyl)-beta-cyclodextrins . The soluble proteins released in the culture medium by the bacterium were fractionated by exclusion size and anion exchange chromatography . A single peak retaining the ability to upregulate COX-2 and HB-EGF mRNA and protein expression was obtained . SDS-PAGE analysis of the peak showed two peptides with an apparent molecular weight of 38 and 22 kDa, which were identified by automated Edman degradation analysis as the N-terminal and C-terminal peptides of H . pylori gamma-glutamyltranspeptidase respectively . Acivicin, a selective gamma-glutamyltranspeptidase inhibitor, counteracted H . pylori-induced upregulation of COX-2 and EGF-related peptide mRNA expression . An H . pylori isogenic mutant gamma-glutamyltranspeptidase-deficient strain did not exert any effect on COX-2, HB-EGF and amphiregulin mRNA expression . Blockade of phosphatidylinositol-3 kinase and p38 kinase, but not MAP kinase kinase, inhibited H . pylori gamma-glutamyltranspeptidase-induced upregulation of COX-2 and EGF-related peptide mRNA expression.

Proc Natl Acad Sci U S A, 2004 Feb 17, 101(7), 2106 - 11 Epub 2004 Feb 03.
Modification of Helicobacter pylori outer membrane protein expression during experimental infection of rhesus macaques; Solnick JV et al.; Clinical isolates of Helicobacter pylori show marked diversity, which may derive from genomic changes that occur during the often lifelong association of the bacterium with its human host . We used the rhesus macaque model, together with DNA microarrays, to examine genomic changes in H . pylori that occur early during experimental infection . Microarray analysis showed that H . pylori recovered from challenged macaques had deleted babA, a member of a large family of paralogous outer membrane proteins (OMPs) that mediates attachment of H . pylori to the Lewis B blood group antigen on gastric epithelium . In some cases the babA gene was replaced by babB, an uncharacterized OMP that is closely related to babA . In other cases the babA gene was present but was not expressed because of alteration in dinucleotide CT repeats in the 5' coding region . In either case, strains lacking babA did not adhere to Lewis B, which is expressed on macaque gastric epithelium . Absence of babA and duplication of babB was also seen in H . pylori isolates derived from human clinical samples, suggesting that this gene conversion event is not unique to experimentally infected rhesus monkeys . These results demonstrate in real time with a relevant animal model that H . pylori regulates OMP expression in vivo by using both antigenic variation and phase variation . We suggest that changes in babA and babB after experimental infection of macaques represent a dynamic response in the H . pylori outer membrane that facilitates adherence to the gastric epithelium and promotes chronic infection.

J Bacteriol, 2004 Feb, 186(4), 1021 - 928
OppA, the substrate-binding subunit of the oligopeptide permease, is the major Ecto-ATPase of Mycoplasma hominis; Hopfe M et al.; Most ATPases, involved in energy-driven processes, act in the cytoplasm . However, external membrane-bound ATPases have also been described in parasites and eukaryotic cells . In Mycoplasma hominis, a bacterium lacking a cell wall, the surface-exposed substrate-binding protein OppA of an oligopeptide permease (Opp) contains an ATP binding P-loop structure in the C-terminal region . With ATP affinity chromatography and tryptic digestion in the presence or absence of ATP, the functionality of the Mg(2+)-dependent ATP binding site is demonstrated . In addition to ATP, ADP also could bind to OppA . The presence of an ATPase activity on the surface of M . hominis is indicated by the inactivation of ATP hydrolyzing activity of intact mycoplasma cells by the impermeable ATPase inhibitor 4',4'-diisothiocyanostilbene-2',2'-disulfonic acid and influenced by the ATP analog 5'-fluorosulfonyl-benzoyladenosine . Comparing equimolar amounts of OppA in intact mycoplasma cells and in the purified form indicated that more than 80% of the surface-localized ATPase activity is derived from OppA, implying that OppA is the main ATPase on the surface of mycoplasma cells . Together, these data present the first evidence that the cytoadhesive substrate binding protein OppA of the oligopeptide permease also functions as an ecto-ATPase in Mycoplasma hominis.

J Bacteriol, 2004 Feb, 186(4), 1001 - 8
Myxococcus xanthus chemotaxis homologs DifD and DifG negatively regulate fibril polysaccharide production; Black WP et al.; The extracellular matrix fibrils of Myxococcus xanthus are essential for the social lifestyle of this unusual bacterium . These fibrils form networks linking or encasing cells and are tightly correlated with cellular cohesion, development, and social (S) gliding motility . Previous studies identified a set of bacterial chemotaxis homologs encoded by the dif locus . It was determined that difA, difC, and difE, encoding respective homologs of a methyl-accepting chemotaxis protein, CheW, and CheA, are required for fibril production and therefore S motility and development . Here we report the studies of three additional genes residing at the dif locus, difB, difD, and difG . difD and difG encode homologs of chemotaxis proteins CheY and CheC, respectively . difB encodes a positively charged protein with limited homology at its N terminus to conserved bacterial proteins with unknown functions . Unlike the previously characterized dif genes, none of these three newly studied dif genes are essential for fibril production, S motility, or development . The difB mutant showed no obvious defects in any of the processes examined . In contrast, the difD and the difG mutants were observed to overproduce fibril polysaccharides in comparison with production by the wild type . The observation that DifD and DifG negatively regulate fibril polysaccharide production strengthens our hypothesis that the M . xanthus dif genes define a chemotaxis-like signal transduction pathway which regulates fibril biogenesis . To our knowledge, this is the first report of functional studies of a CheC homolog in proteobacteria . In addition, during this study, we slightly modified previously developed assays to easily quantify fibril polysaccharide production in M . xanthus.

Chemosphere, 2004 Apr, 55(2), 147 - 56
Recombinant luminescent bacterial sensors for the measurement of bioavailability of cadmium and lead in soils polluted by metal smelters; Ivask A et al.; Environmental hazard of heavy metals in soils depends to a large extent on their bioavailability . The approach used in this study enables the determination of bioavailable metals in solid-phase samples . Two recombinant bacterial sensors, one responding specifically to cadmium and the other to lead and cadmium by increase of luminescence (firefly luciferase was used as a reporter) were used to determine the bioavailability of these metals in soil-water suspensions (a contact assay) and respective particle-free extracts . Fifty agricultural soils sampled near zinc and lead smelters in the Northern France containing up to (mg/kg) 20.1 of Cd, 1050 of Pb and 1390 of Zn were analysed . As the soil matrix interferes with the assay, recombinant luminescent control bacteria lacking the metal recognizing protein and corresponding promoter (thus, being not metal-inducible) but otherwise comparable to the sensor bacteria (the same host bacterium and plasmid encoding luciferase) were used in parallel to take into account the possible quenching and/or stimulating effects of the sample on the luminescence of the sensor bacteria . Both, chemical and sensor analysis showed that only microg/l levels of metals were extracted from the soil into the water phase (0.1% of the total Cd, 0.07% of Pb and 0.5% of Zn) . However, 115-fold more Cd and 40-fold more Pb proved bioavailable if the sensor bacteria were incubated in soil suspensions (i.e., in the contact assay) . The bioavailability of metals in different soils varied (depending probably on soil type) ranging from 0.5% to 56% for cadmium and from 0.2% to 8.6% for lead.

Rev Med Chir Soc Med Nat Iasi, 2003 Jan-Mar, 107(1), 59 - 65
Helicobacter pylori infection in patients with diabetes mellitus; Stanciu OG et al.; Data concerning the prevalence of Helicobacter pylori (H . pylori) in diabetic patients are scanty and controversial . The aim of this study was to evaluate the prevalence of H . pylori infection in patients with diabetes mellitus (DM), and to assess whether the presence of bacterium was associated with severity of dyspeptic symptoms and endoscopic findings in such patients . The study involved 42 patients (19 men, 23 women; mean age 55 years, range 34-75 years) with DM and dyspeptic symptoms . Sixteen patients (38%) were classified as having type 1 diabetes and 26 (62%) patients as having type 2 diabetes . All patients had chronic dyspepsia, and each patient has completed a self-report questionnaire to obtain information concerning the presence and severity of upper gastrointestinal tract symptoms . H . pylori status was confirmed by 13C-urea breath test (13C-UBT), and diabetic patients underwent upper gastrointestinal endoscopy . Twenty-six (61.9%) of patients with DM were positive for 13C-UBT . There were no statistically significant differences in the infection rate between patients with type 1 and type 2 diabetes, and the prevalence of H . pylori infection was not associated with the known duration of diabetes . There was no significant difference in the symptoms score between H . pylori-positive and H . pylori-negative diabetic patients, and endoscopic findings in patients with DM were in the same range with those found in dyspeptic subjects from the same region . In conclusion, H . pylori infection is not associated with DM, duration of diabetes, or severity of dyspeptic symptoms in patients with DM.

Electrophoresis, 2004 Feb, 25(3), 519 - 31
Biochemical characterization of symbiosome membrane proteins from Medicago truncatula root nodules; Catalano CM et al.; The symbiosome membrane represents a specialized plant membrane that forms both a structural and a functional interface between the legume plant and its bacterial counterpart . In this study, the symbiosome membrane protein profile from the model system Medicago truncatula and the corresponding bacterium Sinorhizobium meliloti was examined using two-dimensional electrophoresis and microcapillary high-performance liquid chromatography (HPLC) tandem mass spectrometry . The identities of 51 proteins were obtained and these proteins were categorized into functional classes to indicate biochemical roles . Symbiosome membrane proteins include an H(+)-ATPase, ENOD16, ENOD8, nodulin-25, BiP, HSP70, PDI, multifunctional aquaporin, a putative syntaxin, and other proteins of known and unknown identity and function . The majority of the proteins identified were involved with protein destination and storage . These results allow us to understand better the biochemical composition of the symbiosome membrane and thus provide a basis to hypothesize mechanisms of symbiosome membrane formation and function.

Inorg Chem, 2004 Feb 9, 43(3), 1137 - 52
Bacterial iron transport: coordination properties of azotobactin, the highly fluorescent siderophore of Azotobacter vinelandii; Palanche T et al.; Azotobacter vinelandii, a nitrogen-fixing soil bacterium, secretes in iron deficiency azotobactin delta, a highly fluorescent pyoverdin-like chromopeptidic hexadentate siderophore . The chromophore, derived from 2,3-diamino-6,7 dihydroxyquinoline, is bound to a peptide chain of 10 amino acids: (L)-Asp-(D)-Ser-(L)-Hse-Gly-(D)-beta-threo-HOAsp-(L)-Ser-(D)-Cit-(L)-Hse-(L)-Hse lactone-(D)-N(delta)-Acetyl, N(delta)-HOOrn . Azotobactin delta has three different iron(III) binding sites which are one hydroxamate group at the C-terminal end of the peptidic chain (N(delta)-Acetyl, N(delta)-HOOrn), one alpha-hydroxycarboxylic function in the middle of the chain (beta-threo-hydroxyaspartic acid), and one catechol group on the chromophore . The coordination properties of its iron(III) and iron(II) complexes were measured by spectrophotometry, potentiometry, and voltammetry after the determination of the acid-base functions of the uncomplexed free siderophore . Strongly negatively charged ferric species were observed at neutral p{H}'s corresponding to a predominant absolute configuration Lambda of the ferric complex in solution as deduced from CD measurements . The presence of an alpha-hydroxycarboxylic chelating group does not decrease the stability of the iron(III) complex when compared to the main trishydroxamate siderophores or to pyoverdins . The value of the redox potential of ferric azotobactin is highly consistent with a reductive step by physiological reductants for the iron release . Formation and dissociation kinetics of the azotobactin delta ferric complex point out that both ends of this long siderophore chain get coordinated to Fe(III) before the middle . The most striking result provided by fluorescence measurements is the lasting quenching of the fluorophore in the course of the protonation of the ferric azotobactin delta complex . Despite the release of the hydroxyacid and of the catechol, the fluorescence remains indeed quenched, when iron(III) is bound only to the hydroxamic acid, suggesting a folded conformation at this stage, around the metal ion, in contrast to the unfolded species observed for other siderophores such as ferrioxamine or pyoverdin PaA.

Am J Prev Med, 2004 Feb, 26(2), 135 - 40
Risk perceptions regarding ticks and Lyme disease: a national survey; Herrington JE; BACKGROUND: Lyme disease (LD) is caused by the tickborne bacterium Borrelia burgdorferi and, in 2000, accounted for >90% of all reported cases of vectorborne illness in the United States . Aside from anecdotal and indirect evidence, little empirical evidence exists regarding what the U.S . public knows, says, or does about preventing LD . OBJECTIVES: To examine knowledge, perceptions, and practices regarding prevention of tick bites and LD . METHODS: In 1998, a random-digit-dial frame was used to collect a cross-sectional sample (n =1500) from the 48 coterminous states plus the District of Columbia, and an over-sample (n =250) from six states with the highest incidence of LD . RESULTS: Forty percent of respondents reported doing something to avoid being bitten by ticks . Less than half (41%) used insect repellent . Ninety-two percent of those who had heard about LD stated their likelihood of ever getting the disease was </=50 on a 100-point scale (mean=29; standard deviation, 23.5) . Being somewhat to very concerned about being bitten by ticks was strongly associated with taking preventive measures (odds ratio, 8.34; 95% confidence interval, 6.29-11.06) . CONCLUSIONS: Having seen ticks, being concerned about being bitten, perceiving insect repellent to be effective, having heard about LD, and knowing someone who had LD are the factors most predictive of specific tick-bite preventive behaviors (p <0.001) . However, greater efforts are needed in promoting the effectiveness and safety of DEET-containing insect repellents.

Indian J Med Res, 2003 Jul, 118, 1 - 24
Shiga toxin producing Escherichia coli infection: current progress & future challenges; Khan A et al.; Shiga toxin producing Escherichia coli (STEC) is a newly emerged pathogen that has been the focus of immense international research effort driven by its recognition as a major cause of large scale epidemics and thousands of sporadic cases of gastrointestinal illness . It produces a severe bloody diarrhoea that is clinically distinct from other types of diarrhoeal diseases caused by other enteric pathogens . One of the most important areas of current exploration concerns how STEC enters our food chain, an investigational avenue that begins with the ecology of STEC in animals and in the environment . A variety of foods have been identified as vehicles of STEC-associated illness and this makes the organism one of the most serious threats to the food industry in recent years . The pathogenesis of STEC is multifactorial and involves several levels of interaction between the bacterium and the host . STEC strains carry a set of virulence genes that encode the factors for attachment to host cells, elaboration of effective molecules and production of two different types of Shiga toxins . These genes are found in the locus of enterocyte effacement (LEE), lamboid phages, and a large virulence associated plasmid . The publication of the complete genome sequence of Esch . coli O157:H7 chromosome offers a unique resource that will help to identify additional virulence genes, to develop better methods of strain detection and in the understanding of the evolution of Esch . coli through comparison with the genome of the non-pathogenic laboratory strain Esch . coli K-12 . These research efforts in turn, should lead to development of new potent and cost effective anti-Stx therapies or vaccines and thereby major improvement in human health world-wide.

Biochim Biophys Acta, 2004 Jan 20, 1676(2), 172 - 81
Cloning and expression of the enolase gene from Desulfovibrio vulgaris (Miyazaki F); Kitamura M et al.; The gene encoding an enolase from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli . A 2.1-kb DNA fragment, isolated from D . vulgaris (Miyazaki F) by double digestion with PstI and BamHI, contained an enolase gene (eno) and part of the methylenetetrahydrofolate dehydrogenase gene (folD) . The nucleotide sequence of eno indicates that the protein monomer is composed of 434 amino acids . An expression system for eno under control of the T7 promoter was constructed in E . coli . The purified His-tagged enolase formed a homooctamer and was active in the formation of phosphoenolpyruvate (PEP) as well as in the reverse reaction, the formation of D-(+)-2-phosphoglyceric acid (2-PGA) . The pH dependence and kinetic properties of the recombinant enolase from the sulfate-reducing bacterium were also studied . The amounts of eno mRNA when the bacterium was grown on glycerol or glucose were compared to that when D . vulgaris was grown on lactate.

J Ind Microbiol Biotechnol, 2004 Jan, 31(1), 41 - 7 Epub 2004 Jan 24.
Production of an endoglucanase by the shipworm bacterium, Teredinobacter turnirae; Ahuja SK et al.; The nutritional behavior of a cellulolytic nitrogen-fixing shipworm bacterium, Teredinobacter turnirae, is described, with respect to various carbon and nitrogen sources, in terms of endoglucanase production . Also, the effects of various surfactants on enzyme production are reported . Among the carbon sources, sucrose results in the maximum enzyme production, followed by cellulose . Ammonium phosphate proves to be the best nitrogen source for endoglucanase production . Various surfactants enhance the enzyme titers, with Triton X-100 yielding the best results . A combination of the above-mentioned components improves the enzyme production by 3.6-fold.

Biochem J, 2004 May 1, 379(Pt 3), 633 - 40
Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens; Marokhazi J et al.; A proteolytic enzyme, Php-B ( Photorhabdus protease B), was purified from the entomopathogenic bacterium, Photorhabdus luminescens . The enzyme is intracellular, and its molecular mass is 74 kDa . Tested on various peptide and oligopeptide substrates, Php-B hydrolysed only oligopeptides, with significant activity against bradykinin and a 2-furylacryloyl-blocked peptide, Fua-LGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat=3.6x10(2) s(-1), K(m)=5.8x10(-5) M(-1), pH optimum approx . 7.0) . The p K(a1) and the p K(a2) values of the enzyme activity (6.1 and 7.9 respectively), as well as experiments with enzyme inhibitors and bivalent metal ions, suggest that the activity of Php-B is dependent on histidine and cysteine residues, but not on serine residues, and that it is a metalloprotease, which most probably uses Zn2+ as a catalytic ion . The enzyme's ability to cleave oligopeptides that contain a sequence similar to collagen repeat (-Pro-Xaa-Gly-), bradykinin and Fua-LGPA (a synthetic substrate for bacterial collagenases and collagen peptidases), but not native collagens (types I and IV) or denatured collagen (gelatin), indicates that Php-B is probably a collagen peptidase, the first enzyme of this type to be identified in an insect pathogen, that might have a role in the nutrition of P . luminescens by degrading small collagen fragments . For the determination of enzyme kinetic constants, we fitted a numerically integrated Michaelis-Menten model to the experimental progress curves . Since this approach has not been used before in the characterization of proteases that are specific for the P1'-P4' substrate sites (e.g . collagenolytic enzymes), we present a comparison of this method with more conventional ones . The results confirm the reliability of the numerical integration method in the kinetic analysis of collagen-peptide-hydrolysing enzymes.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 93 - 7
Polaromonas naphthalenivorans sp . nov., a naphthalene-degrading bacterium from naphthalene-contaminated sediment; Jeon CO et al.; Strain CJ2T, capable of growth on naphthalene as a sole carbon and energy source, was isolated from coal-tar-contaminated freshwater sediment . The Gram reaction of strain CJ2T was negative . The cells were non-spore-forming, non-motile cocci (without flagella) . The isolate was found to be an aerobic heterotroph capable of utilizing glucose and other simple sugars . Growth was observed between 4 and 25 degrees C (optimum, 20 degrees C) and between pH 6.0 and 9.0 (optimum, pH 7.0-7.5) . The G+C content of the genomic DNA was 61.5 mol% and the major quinone was ubiquinone-8 . The peptidoglycan of strain CJ2T was determined as belonging to type A1-gamma, meso-diaminopimelic acid . The major fatty acids of strain CJ2T were 16:1omega7c (67.0%), 16:0 (19.6%), 18:1omega7c (approximately 7.9%) and 10:0 3-OH (approximately 2.5%) . The polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol . Mycolic acid and glycolipids could not be detected . Comparative 16S rDNA analysis indicated that strain CJ2T is related to the family Comamonadaceae and that the nearest phylogenetic relative was Polaromonas vacuolata 34-PT (97.1% similarity) . On the basis of the physiological and molecular properties, the naphthalene-degrading isolate was designated Polaromonas naphthalenivorans sp . nov . The type strain is CJ2T (=ATCC BAA-779T=DSM 15660T).

Environ Sci Technol, 2004 Jan 1, 38(1), 187 - 93
Inhibition of biological reductive dissolution of hematite by ferrous iron; Royer RA et al.; Bacterial dissimilatory iron reduction is self-inhibited by the production of ferrous {Fe(II)} iron resulting in diminished iron reduction as Fe(II) accumulates . Experiments were conducted to investigate the mechanisms of Fe(II) inhibition employing the dissimilatory metal-reducing bacterium Shewanella putrefaciens strain CN32 under nongrowth conditions in a system designed to minimize precipitation of ferrous iron minerals . After an initial period (ca . 1 day) of relatively rapid iron reduction, hematite reduction rates were controlled by mass transfer of Fe(II) . Experiments in which hematite was equilibrated with Mn(II) prior to inoculation indicated that the observed inhibition was not due to Fe(II) sorption . At longer times, soluble Fe(II) accumulated such that the reaction was slowed due to a decreased thermodynamic driving force . The thermodynamic evaluation also supported the prior conclusion that hydrated hematite surface sites may yield substantially more energy during bioreduction than "bulk" hematite . For well-mixed conditions, the rates of hematite reduction were directly proportional to the biologically available reaction potential.

Appl Microbiol Biotechnol, 2004 Aug, 65(2), 211 - 8 Epub 2004 Jan 22.
Degradation of polycyclic aromatic hydrocarbons by a newly isolated dibenzofuran-utilizing Janibacter sp . strain YY-1; Yamazoe A et al.; The dibenzofuran (DF)-utilizing bacterium strain YY-1 was newly isolated from soil . The isolate was identified as Janibacter sp . with respect to its 16S rDNA sequence and fatty acid profiles, as well as various physiological characteristics . In addition to DF, strain YY-1 could grow on fluorene and dibenzothiophene as sole sources of carbon and energy . It was also able to cometabolize a variety of polycyclic aromatic hydrocarbons including dibenzo- p-dioxin, phenanthrene, and anthracene . The major metabolites formed from DF, biphenyl, dibenzothiophene, and naphthalene were identified by using gas chromatography-mass spectrometry as 2,3,2'-trihydroxybiphenyl, biphenyl-dihydrodiol, dibenzothiophene 5-oxide, and coumarin, respectively . These results indicate that strain YY-1 can catalyze angular dioxygenation, lateral dioxygenation, and sulfoxidation.

Protein Sci, 2004 Feb, 13(2), 342 - 50
High-temperature solution NMR structure of TmCsp; Jung A et al.; Cold shock proteins (Csps) are assumed to play a central role in the regulation of gene expression under cold shock conditions . Acting as single-stranded nucleic acid-binding proteins, they trigger the translation process and are therefore involved in the compensation of the influence of low temperatures (cold shock) upon the cell metabolism . However, it is unknown so far how Csps are switched on and off as a function of temperature . The aim of the present study is the study of possible structural changes responsible for this switching process . (1)H-(15)N HSQC spectra recorded at different temperatures and chemical-shift analysis have indicated subtle conformational changes for the cold-shock protein from the hyperthermophilic bacterium Thermotoga maritima (TmCsp) when the temperature is elevated from 303 K to its physiological temperature (343 K) . The three-dimensional structure of TmCsp was determined by nuclear magnetic resonance (NMR) spectroscopy at 343 K to obtain quantitative information concerning these structural changes . By use of residual dipolar couplings, the loss of NOE information at high temperature could be compensated successfully . Most pronounced conformational changes compared with room-temperature conditions are observed for amino acid residues closely neighbored to two characteristic beta-bulges and a well-defined loop region of the protein . Because the residues shown to be responsible for the interaction of TmCsp with single-stranded nucleic acids can almost exclusively be found within these regions, nucleic acid-binding activity might be down-regulated with increasing temperature by the described conformational changes.

Mol Biol Evol, 2004 Apr, 21(4), 652 - 8 Epub 2004 Jan 22.
Bacterial proteins predisposed for targeting to mitochondria; Lucattini R et al.; Mitochondria evolved from an endosymbiotic proteobacterium in a process that required the transfer of genes from the bacterium to the host cell nucleus, and the translocation of proteins thereby made in the host cell cytosol into the internal compartments of the organelle . According to current models for this evolution, two highly improbable events are required to occur simultaneously: creation of a protein translocation machinery to import proteins back into the endosymbiont and creation of targeting sequences on the protein substrates themselves . Using a combination of two independent prediction methods, validated through tests on simulated genomes, we show that at least 5% of proteins encoded by an extant proteobacterium are predisposed for targeting to mitochondria, and propose we that mitochondrial targeting information was preexisting for many proteins of the endosymbiont . We analyzed a family of proteins whose members exist both in bacteria and in mitochondria of eukaryotes and show that the amino-terminal extensions occasionally found in bacterial family members can function as a crude import sequence when the protein is presented to isolated mitochondria . This activity leaves the development of a primitive translocation channel in the outer membrane of the endosymbiont as a single hurdle to initiating the evolution of mitochondria.

J Biol Inorg Chem, 2004 Mar, 9(2), 224 - 30 Epub 2004 Jan 20.
NMR-validated structural model for oxidized Rhodopseudomonas palustris cytochrome c(556); Bertini I et al.; The structure of oxidized Rhodopseudomonas palustris cytochrome c(556) has been modeled after that of high-spin cytochrome c' from the same bacterium, the latter being the protein with the greatest sequence identity (35%) among all sequenced proteins in the genomes . The two proteins differ in the number of ligands to iron and in spin state, the former being six-coordinate low-spin and the latter five-coordinate high-spin . In order to validate this modeled structure, several structural restraints were obtained by performing a restricted set of NMR experiments, without performing a complete assignment of the protein signals . The aim was to exploit the special restraints arising from the paramagnetism of the metal ion . A total of 43 residual-dipolar-coupling and 74 pseudocontact-shift restraints, which together sampled all regions of the protein, were used in conjunction with over 40 routinely obtained NOE distance restraints . A calculation procedure was undertaken combining the program MODELLER and the solution structure determination program PARAMAGNETIC DYANA, which includes paramagnetism-based restraints . The directions and magnitude of the magnetic susceptibility anisotropy tensor were also calculated . The approach readily provides useful results, especially for paramagnetic metalloproteins of moderate to large dimensions.

J Immunol, 2004 Feb 1, 172(3), 1786 - 800
Overproduction of TNF-alpha by CD8+ type 1 cells and down-regulation of IFN-gamma production by CD4+ Th1 cells contribute to toxic shock-like syndrome in an animal model of fatal monocytotropic ehrlichiosis; Ismail N et al.; Human monocytotropic ehrlichiosis (HME) is an emerging, life-threatening, infectious disease caused by Ehrlichia chaffeensis, an obligate intracellular bacterium that lacks cell wall LPS . We have previously developed an animal model of severe HME using a strain of Ehrlichia isolated from Ixodes ovatus ticks (IOE) . To understand the basis of susceptibility to severe monocytotropic ehrlichiosis, we compared low and high doses of the highly virulent IOE strain and the less virulent Ehrlichia muris strain that are closely related to E . chaffeensis in C57BL/6 mice . Lethal infections caused by high or low doses of IOE were accompanied by extensive liver damage, extremely elevated levels of TNF-alpha in the serum, high frequency of Ehrlichia-specific, TNF-alpha-producing CD8(+) T cells in the spleen, decreased Ehrlicha-specific CD4(+) T cell proliferation, low IL-12 levels in the spleen, and a 40-fold decrease in the number of IFN-gamma-producing CD4(+) Th1 cells . All groups contained negligible numbers of IL-4-producing cells in the spleen . Transfer of Ehrlichia-specific polyclonal Abs and IFN-gamma-producing Ehrlichia-specific CD4(+) and CD8(+) type 1 cells protected naive mice against lethal IOE challenge . Interestingly, infection with high dose E . muris provided protection against rechallenge with a lethal dose of IOE . Cross-protection was associated with substantial expansion of IFN-gamma-producing CD4(+) and CD8(+) cells, but not TNF-alpha-producing CD8(+) T cells, a high titer of IgG2a, and a low serum level of TNF-alpha . In conclusion, uncontrolled TNF-alpha production by CD8(+) T cells together with a weak CD4(+) Th1 cell response are associated with immunopathology and failure to clear IOE in the fatal model of HME.

Biochemistry, 2004 Jan 27, 43(3), 759 - 72
Cloning, expression, and characterization of a cis-3-chloroacrylic acid dehalogenase: insights into the mechanistic, structural, and evolutionary relationship between isomer-specific 3-chloroacrylic acid dehalogenases; Poelarends GJ et al.; The gene encoding the cis-3-chloroacrylic acid dehalogenase (cis-CaaD) from coryneform bacterium strain FG41 has been cloned and overexpressed, and the enzyme has been purified to homogeneity and subjected to kinetic and mechanistic characterization . Kinetic studies show that cis-CaaD processes cis-3-haloacrylates, but not trans-3-haloacrylates, with a turnover number of approximately 10 s(-1) . The product of the reaction is malonate semialdehyde, which was confirmed by its characteristic 1H NMR spectrum . The enzyme shares low but significant sequence similarity with the previously studied trans-3-chloroacrylic acid dehalogenase (CaaD) and with other members of the 4-oxalocrotonate tautomerase (4-OT) family . While 4-OT and CaaD function as homo- and heterohexamers, respectively, cis-CaaD appears to be a homotrimeric protein as assessed by gel filtration chromatography . On the basis of the known three-dimensional structures and reaction mechanisms of CaaD and 4-OT, a sequence alignment implicated Pro-1, Arg-70, Arg-73, and Glu-114 as important active-site residues in cis-CaaD . Subsequent site-directed mutagenesis experiments confirmed these predictions . The acetylene compounds, 2-oxo-3-pentynoate and 3-bromo- and 3-chloropropiolate, were processed by cis-CaaD to products consistent with an enzyme-catalyzed hydration reaction previously established for CaaD . Hydration of 2-oxo-3-pentynoate afforded acetopyruvate, while the 3-halopropiolates became irreversible inhibitors that modified Pro-1 . The results of this work revealed that cis-CaaD and CaaD have different primary and quaternary structures, and display different substrate specificity and catalytic efficiencies, but likely share a highly conserved catalytic mechanism . The mechanism may have evolved independently because sequence analysis indicates that cis-CaaD is not a 4-OT family member, but represents the first characterized member of a new family in the tautomerase superfamily that probably resulted from an independent duplication of a 4-OT-like sequence . The discovery of a fifth family of enzymes within this superfamily further demonstrates the diversity of activities and structures that can be created from 4-OT-like sequences.

Proteomics, 2004 Jan, 4(1), 151 - 79
Global analysis of the Ralstonia metallidurans proteome: prelude for the large-scale study of heavy metal response; Noel-Georis I et al.; A proteome map of Ralstonia metallidurans strain CH34 was constructed using two-dimensional (2-D) gel electrophoresis in combination with automated Edman degradation and mass spectrometry (MS) . R . metallidurans CH34 is the type-strain of a family of highly related strains characterized by their multiple resistance to millimolar amounts of heavy metals, conferred by two large plasmids . The protein content of this bacterium grown in minimal medium was separated by 2-D gel electrophoresis using various pH gradients . Protein identification was carried out via N-terminal amino acid sequencing, matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) and tandem MS . So far, 224 different proteins were characterized from 352 protein spots . Although the proteome map is still not complete, one could appraise the importance of proteomics for genome analyses through (i) . the identification of previously undetected open reading frames, (ii) . the identification of proteins not encoded by the already sequenced genome fragments, (iii) . the characterization of protein-encoding genes spanning two different contigs, enabling their merging, and (iv) . the precise delineation of the N-terminus of several proteins . Finally, this map will prove a useful tool in the identification of proteins differentially expressed in the presence of different heavy metals.

Mar Biotechnol (NY), 2003 Nov-Dec, 5(6), 536 - 44 Epub 2003 Jul 17.
Structures of oligosaccharides derived from Cladosiphon okamuranus fucoidan by digestion with marine bacterial enzymes; Sakai T et al.; A fucoidan-utilizing marine bacterium, Fucophilus fucoidanolyticus, was cultivated in medium containing fucoidan from Cladosiphon okamuranus . The C . okamuranus fucoidan was digested into oligosaccharides with the intracellular enzymes of F . fucoidanolyticus, and their structures were determined by nuclear magnetic resonance analyses . Some of their structures are represented by one general structural formula, (-3 L-Fuc palpha1-3 L-Fuc p(4- O-sulfate)alpha1-3 L-Fuc p(4- O-sulfate)alpha1-3( D-Glc pUAalpha1-2) L-Fuc palpha1)(m)-3 L-Fuc palpha1-3 L-Fuc p(4- O-sulfate)alpha1-3 L-Fuc p(4- O-sulfate) alpha1-3 L-Fuc p ( m = 0, 1, 2, or 3) . We concluded that all oligosaccharides obtained were derived from a sulfated-fucose-containing polysaccharide of C . okamuranus, which has a repeating unit of (-3 L-Fuc palpha1-3 L-Fuc p(4- O-sulfate)alpha1-3 L-Fuc p(4- O-sulfate)alpha1-3( D-Glc pUAalpha1-2) L-Fuc palpha1-).

J Bacteriol, 2004 Feb, 186(3), 646 - 53
Nine mutants of Chlorobium tepidum each unable to synthesize a different chlorosome protein still assemble functional chlorosomes; Frigaard NU et al.; Chlorosomes of the green sulfur bacterium Chlorobium tepidum comprise mostly bacteriochlorophyll c (BChl c), small amounts of BChl a, carotenoids, and quinones surrounded by a lipid-protein envelope . These structures contain 10 different protein species (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) but contain relatively little total protein compared to other photosynthetic antenna complexes . Except for CsmA, which has been suggested to bind BChl a, the functions of the chlorosome proteins are not known . Nine mutants in which a single csm gene was inactivated were created; these mutants included genes encoding all chlorosome proteins except CsmA . All mutants had BChl c contents similar to that of the wild-type strain and had growth rates indistinguishable from or within approximately 90% (CsmC(-) and CsmJ(-)) of those of the wild-type strain . Chlorosomes isolated from the mutants lacked only the protein whose gene had been inactivated and were generally similar to those from the wild-type strain with respect to size, shape, and BChl c, BChl a, and carotenoid contents . However, chlorosomes from the csmC mutant were about 25% shorter than those from the wild-type strain, and the BChl c absorbance maximum was blue-shifted about 8 nm, indicating that the structure of the BChl c aggregates in these chlorosomes is altered . The results of the present study establish that, except with CsmA, when the known chlorosome proteins are eliminated individually, none of them are essential for the biogenesis, light harvesting, or structural organization of BChl c and BChl a within the chlorosome . These results demonstrate that chlorosomes are remarkably robust structures that can tolerate considerable changes in protein composition.

Tissue Cell, 2004 Feb, 36(1), 43 - 53
A symbiont of the tick Ixodes ricinus invades and consumes mitochondria in a mode similar to that of the parasitic bacterium Bdellovibrio bacteriovorus; Sacchi L et al.; We have recently performed molecular characterisation of an intracellular alpha-proteobacterium, named IricES1, which resides in the ovarian tissue of female Ixodes ricinus ticks from Italy . A unique characteristic of this bacterium is its ability to invade the mitochondria of the cells in which it resides . Although some ultrastructural studies have been performed on close relatives of this bacterium from I . ricinus in England and Switzerland, a number of questions remain about its movement within ovarian tissues and mitochondria . We have performed the first detailed ultrastructural examination of IricES1 in engorged female adult I . ricinus . Among our findings was that the bacterium enters mitochondria in a similar way to that employed by the 'predatory' bacterium Bdellovibro bacteriovorus, that is, between the inner and outer membranes . It then appears to multiply, with the new 'colony' consuming the mitochondrial matrix . Despite having many of their mitochondria consumed, oocytes appear to develop normally, and the bacteria are likely to be vertically transferred to all eggs.

Proc R Soc Lond B Biol Sci, 2003 Dec 22, 270(1533), 2543 - 50
Changing partners in an obligate symbiosis: a facultative endosymbiont can compensate for loss of the essential endosymbiont Buchnera in an aphid; Koga R et al.; Almost all aphids harbour an endosymbiotic bacterium, Buchnera aphidicola, in bacteriocytes . Buchnera synthesizes essential nutrients and supports growth and reproduction of the host . Over the long history of endosymbiosis, many essential genes have been lost from the Buchnera genome, resulting in drastic genome reduction and the inability to live outside the host cells . In turn, when deprived of Buchnera, the host aphid suffers retarded growth and sterility . Buchnera and the host aphid are often referred to as highly integrated almost inseparable mutualistic partners . However, we discovered that, even after complete elimination of Buchnera, infection with a facultative endosymbiotic gamma-proteobacterium called pea aphid secondary symbiont (PASS) enabled survival and reproduction of the pea aphid . In the Buchnera-free aphid, PASS infected the cytoplasms of bacteriocytes that normally harbour Buchnera, establishing a novel endosymbiotic system . These results indicate that PASS can compensate for the essential role of Buchnera by physiologically and cytologically taking over the symbiotic niche . By contrast, PASS negatively affected the growth and reproduction of normal host aphids by suppressing the essential symbiont Buchnera . These findings illuminate complex symbiont-symbiont and host-symbiont interactions in an endosymbiotic system, and suggest a possible evolutionary route to novel obligate endosymbiosis by way of facultative endosymbiotic associations.

Appl Microbiol Biotechnol, 2004 Jun, 64(5), 651 - 8 Epub 2004 Jan 16.
Liquid culture mass production of biocontrol nematodes, Heterorhabditis bacteriophora (Nematoda: Rhabditida): improved timing of dauer juvenile inoculation; Johnigk SA et al.; Heterorhabditis bacteriophora is used in biological control of soil-borne insect pests in horticulture and turf . Mass production is carried out in monoxenic liquid cultures pre-incubated with the symbiont of the nematodes, the bacterium Photorhabdus luminescens, before nematode dauer juveniles (DJ) are inoculated . As a response to bacterial food signals, the DJ recover from the developmentally arrested dauer stage, grow to adults and produce DJ offspring . Variable DJ recovery after inoculation into cultures of P . luminescens often causes process failure due to low numbers of adult nematodes in the medium . In order to enhance DJ recovery, improve nematode population management and increase yields, the optimal timing for DJ inoculation was sought . The process parameter pH and respiration quotient (RQ) were recorded in order to test whether changes can be used to identify the best moment for DJ inoculation . When DJ were inoculated during the lag and early logarithmic growth phases of P . luminescens cultures, DJ recovery was low and almost no nematode reproduction was obtained . High populations of P . luminescens phase variants were recorded . Recovery and yields increased when DJ were inoculated during the latter log phase during which the RQ dropped to values <0.8 and the pH reached a maximum . The highest DJ recovery and yields were observed in cultures that were inoculated during the late stationary growth phase . This period started with the increase of the pH after its distinct minimum at pH <8.0 . Thus optimal timing for DJ inoculation can be defined through monitoring of the pH in the P . luminescens culture.

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