Proc Natl Acad Sci U S A, 2001 Jun 5, 98(12), 6725 - 9 Epub 2001 May 22. Evidence for clonal propagation in natural isolates of Plasmodium falciparum from Venezuela; Urdaneta L et al.; We have analyzed 75 isolates of Plasmodium falciparum, collected in Venezuela during both the dry (November) and rainy (May-July) seasons, with a range of genetic markers including antigen genes and 14 random amplified polymorphic DNA (RAPD) primers . Thirteen P . falciparum stocks from Kenya and four other Plasmodium species are included in the analysis for comparison . Cross-hybridization shows that the 14 RAPD primers reveal 14 separate regions of the parasite's genome . The P . falciparum isolates are a monophyletic clade, significantly different from the other Plasmodium species . We identify three RAPD characters that could be useful as "tags" for rapid species identification . The Venezuelan genotypes fall into two discrete genetic subdivisions associated with either the dry or the rainy season; the isolates collected in the rainy season exhibit greater genetic diversity . There is significant linkage disequilibrium in each seasonal subsample and in the full sample . In contrast, no linkage disequilibrium is detected in the African sample . These results support the hypothesis that the population structure of P . falciparum in Venezuela, but not in Africa, is predominantly clonal . However, the impact of genetic recombination on Venezuelan P . falciparum seems higher than in parasitic species with long-term clonal evolution like Trypanosoma cruzi, the agent of Chagas' disease . The genetic structure of the Venezuelan samples is similar to that of Escherichia coli, a bacterium that propagates clonally, with occasional genetic recombination. J Agric Food Chem, 2001 May, 49(5), 2298 - 301 Herbicidal properties of the thaxtomin group of phytotoxins; King RR et al.; The thaxtomins are a group of phytotoxins generated by the bacterium Streptomyces scabies (the main causal organism of potato common scab) . Available members of the group were assessed for herbicidal activity by a variety of standard tests . Test results indicated that thaxtomin A, the predominant member, was also the most physiologically active . Injury symptoms in most instances were similar to those caused by known cellulose biosynthetic inhibitors such as dichlobenil and isoxaben . Although test results indicated that the thaxtomins had many of the biological properties desirable in a commercial herbicide, they nevertheless lacked the systemic phytotoxicity critical to deliver reliable weed control at low rates. Biometals, 2001 Mar, 14(1), 13 - 22 Influence of divalent cations on the catalytic properties and secondary structure of unadenylylated glutamine synthetase from Azospirillum brasilense; Antonyuk LP et al.; Fully unadenylylated glutamine synthetase (GS) from the endophytic bacterium Azospirillum brasilense Sp245 was isolated and purified . The enzyme was electrophoretically homogeneous and contained strongly bound metal ions, which could not be removed by dialysis . Mn2+, Mg2+, and Co2+ were found to be effective in supporting biosynthetic activity of the A . brasilense GS . Some kinetic properties of Mn2+-activated and Mg2+-activated unadenylylated GS were characterized . Circular dichroism analysis of the enzyme showed that the A . brasilense GS is a highly structured protein: 59% of its residues form alpha-helices and 13% beta-strands . Removal of the metal ions from the A . brasilense GS by treatment with EDTA resulted in alterations in the enzyme secondary structure. Prehospital Disaster Med, 2001 Jan-Mar, 16(1), 14 - 7 Aseptic efficacy of prefilled syringes in a polluted environment; Ninomiya N et al.; INTRODUCTION: To evaluate the aseptic efficacy of prefilled syringes compared with ampules when used in a polluted environment similar to that at a disaster site . METHODS: The researchers tested epinephrine, 0.1%, atropine sulfate, 0.05%, and lidocaine hydrochloride solutions, 2% (Group A) as well as lidocaine hydrochloride, 10%, sodium bicarbonate, 8.4%, and glucose solutions, 50% (Group B), that frequently are used for intravenous injection and intravenous infusion respectively in Disaster Medicine . Each of these solutions in 10 prefilled syringes (PFSs) and 10 ampules was placed in a box of contaminated soil along with needles and empty syringes for ampules . In the box, each was taken out of its package, all syringes were connected with a needle, and empty syringes were filled with a solution . After this procedure, all syringes were taken out of the box to check their contents for bacterial contamination . RESULTS: No bacterium was observed in any of the 10 PFS samples of Group A and B solutions . In contrast, out of 10 ampule samples, six of the 10 samples containing epinephrine, nine of the 10 containing atropine sulfate, all 10 samples containing lidocaine hydrochloride, 2%, and all of the ampule samples containing Group B solutions tested positive for bacteria . A statistically significant difference was observed between the PFS and ampule samples in all six solutions . CONCLUSION: Results indicate that, in environments with airborne contaminants, the use of prefilled syringes may be useful for preventing bacterial contamination of the medicine inside. Science, 2001 May 18, 292(5520), 1360 - 3 Bacterial recognition of mineral surfaces: nanoscale interactions between Shewanella and alpha-FeOOH; Lower SK et al.; Force microscopy has been used to quantitatively measure the infinitesimal forces that characterize interactions between Shewanella oneidensis (a dissimilatory metal-reducing bacterium) and goethite (alpha-FeOOH), both commonly found in Earth near-surface environments . Force measurements with subnanonewton resolution were made in real time with living cells under aerobic and anaerobic solutions as a function of the distance, in nanometers, between a cell and the mineral surface . Energy values {in attojoules (10(-18) joules)} derived from these measurements show that the affinity between S . oneidensis and goethite rapidly increases by two to five times under anaerobic conditions in which electron transfer from bacterium to mineral is expected . Specific signatures in the force curves suggest that a 150-kilodalton putative iron reductase is mobilized within the outer membrane of S . oneidensis and specifically interacts with the goethite surface to facilitate the electron transfer process. Environ Sci Technol, 2001 Jan 1, 35(1), 182 - 91 Ferrographic tracking of bacterial transport in the field at the narrow channel focus area, Oyster, VA; Johnson WP et al.; The first results from an innovative bacterial tracking technique, ferrographic capture, applied to bacterial transport in groundwater are reported in this paper . Ferrographic capture was used to analyze samples during an October 1999 bacterial injection experiment at the Narrow Channel focus area of the South Oyster site, VA . Data obtained using this method showed that the timing of bacterial breakthrough was controlled by physical (hydraulic conductivity) heterogeneity in the vertical dimension as opposed to variation in sedimentsurface or aqueous chemical properties . Ferrographic tracking yielded results that compared well with results from other tracking techniques over a concentration range of 8 orders of magnitude and provided a low detection limit relative to most other bacterial tracking techniques . The low quantitation limit of this method (approximately 20 cells/mL) allowed observation of transport of an adhesion-deficient bacterium over distances greater than 20 m in the fine sand aquifer underlying this site. Environ Sci Technol, 2001 Jan 1, 35(1), 127 - 32 The influence of sulfide on solid-phase mercury bioavailability for methylation by pure cultures of Desulfobulbus propionicus (1pr3); Benoit JM et al.; To help understand the mechanism and control of Hg uptake in Hg-methylating bacteria, we investigated the effect of sulfide on Hg methylation by pure cultures of the sulfate-reducing bacterium Desulfobulbus propionicus (1pr3) . Our previous research in natural sediments has suggested that Hg methylation occurs most rapidly when sulfide concentrations favor formation of neutral dissolved Hg-S species . In this study, the chemical speciation of Hg in culture media was manipulated by growing D . propionicus across a range of sulfide concentrations, with inorganic Hg (HgI) added in the form of ground ores . A solid-phase, rather than a dissolved source of Hg, was used to simulate the controls on Hg partitioning between solid and aqueous phases found in natural sediments . Methylmercury (MeHg) production by cultures was not related to the absolute solid-phase concentration of Hg in the ores, and it was only weakly related to the dissolved HgI concentration in the medium . However, MeHg production was linearly related to the calculated concentration of the dominant neutral complex in solution, HgS degrees . Furthermore, the diffusive membrane permeability of HgS degrees, as estimated from its octanol-water partitioning coefficient, was found to be sufficient to support MeHg production by cells . The present paper expands on our previous work by providing experimental support of our hypothesis that sulfide influences methylation by affecting the speciation of dissolved HgI and its uptake via passive diffusion. J Mol Biol, 2001 May 11, 308(4), 765 - 82 Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901; Campos-Olivas R et al.; AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tu901 . Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases . No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g . agglutinins, lectins, hevein) is observed . AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown . Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy . The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich . This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity . A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean) . AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily . The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data . We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor . After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein . Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1 . The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction . Infect Immun, 2001 Jun, 69(6), 4055 - 64 Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis; Daniell SJ et al.; Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol . However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system . We have previously shown that in enteropathogenic and enterohemorrhagic Escherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell . Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD . The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability . Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays . Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic E . coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells . Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells . These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation. FEMS Microbiol Rev, 2001 May, 25(3), 309 - 33 The virulence factors of Bordetella pertussis: a matter of control; Smith AM et al.; Bordetella pertussis is the causative agent of whooping cough, a contagious childhood respiratory disease . Increasing public concern over the safety of whole-cell vaccines led to decreased immunisation rates and a subsequent increase in the incidence of the disease . Research into the development of safer, more efficacious, less reactogenic vaccine preparations was concentrated on the production and purification of detoxified B . pertussis virulence factors . These virulence factors include adhesins such as filamentous haemagglutinin, fimbriae and pertactin, which allow B . pertussis to bind to ciliated epithelial cells in the upper respiratory tract . Once attachment is initiated, toxins produced by the bacterium enable colonisation to proceed by interfering with host clearance mechanisms . B . pertussis co-ordinately regulates the expression of virulence factors via the Bordetella virulence gene (bvg) locus, which encodes a response regulator responsible for signal-mediated activation and repression . This strict regulation mechanism allows the bacterium to express different gene subsets in different environmental niches within the host, according to the stage of disease progression. Eur J Oral Sci, 2001 Apr, 109(2), 109 - 13 Monoclonal antibody against Porphyromonas gingivalis hemagglutinin inhibits hemolytic activity; Hosogi Y et al.; Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis . This bacterium possesses hemagglutinating and hemolytic activities to attach and lyse erythrocytes . Hemolysis by this oral pathogen functions to provide heme-containing molecules for growth in the periodontal pocket . We previously constructed a monoclonal antibody using P . gingivalis vesicles as the immunogen, designated as MAb-Pg-vc, which inhibited vesicle-associated hemagglutinating activity . Furthermore, we cloned the gene encoding 130-kDa hemagglutinin (130-kDa HAG) and identified its functional motif for attachment to erythrocytes . Generally, bacterial cell attachment to erythrocytes is an important initial step for expressing hemolysis activity . In the present study, we examined the effect of MAb-Pg-vc on the hemolytic activity of P . gingivalis cells . The MAb-Pg-vc significantly inhibited the hemolytic activity and, further, this inhibitory activity was reduced by the synthetic peptide of the 130-kDa HAG functional motif. Dtsch Med Wochenschr, 2001 Apr 12, 126(15), 431 - 3 {Mitral valve endocarditis caused by Erysipelothrix rhusiopathiae}; Heidrich JP et al.; HISTORY AND CLINICAL FINDINGS: A 67 year-old country woman was admitted to the hospital because of a four weeks history of continuous catarrh, arthralgia and fever . Recently, she had also developed upper abdominal pain after oral ibuprofen treatment . The clinical examination showed a patient of impaired general condition . The heart and lungs were auscultatory normal and there were no signs of dyspnea, cyanosis or inflammatory skin lesions . EXAMINATIONS: Physical examination of heart and lung, electrocardiography and transthoracic echocardiography were without pathological findings . CLINICAL COURSE: Gastroscopy revealed acute antral gastritis and duodenitis with presence of Helicobacter pylori . Eradication therapy resolved the abdominal symptoms but fever returned after the antibiotic therapy was stopped . The patient developed a severe endocarditis with progressive mitral regurgitation within a few days . Erysipelothrix rhusiopathiae was isolated from blood cultures and identified by conventional and molecular methods . The patient was treated successfully with 3 x 2 g ampicillin daily, applied parenterally for six weeks, and a mitral valve replacement . CONCLUSION: This was an unusual manifestation of systemic Erysipelothrix rhusiopathiae infection . The bacterium Erysipelothrix rhusiopathiae has still to be considered in the diagnosis and treatment of endocarditis in patients with increased risk of exposure (e.g . farmers, butchers and fishermen). J Mol Evol, 2001 Apr, 52(4), 333 - 41 Horizontal transfer of the photosynthesis gene cluster and operon rearrangement in purple bacteria; Igarashi N et al.; A 37-kb photosynthesis gene cluster was sequenced in a photosynthetic bacterium belonging to the beta subclass of purple bacteria (Proteobacteria), Rubrivivax gelatinosus . The cluster contained 12 bacteriochlorophyll biosynthesis genes (bch), 7 carotenoid biosynthesis genes (crt), structural genes for photosynthetic apparatuses (puf and puh), and some other related genes . The gene arrangement was markedly different from those of other purple photosynthetic bacteria, while two superoperonal structures, crtEF-bchCXYZ-puf and bchFNBHLM-lhaA-puhA, were conserved . Molecular phylogenetic analyses of these photosynthesis genes showed that the photosynthesis gene cluster of Rvi . gelatinosus was originated from those of the species belonging to the alpha subclass of purple bacteria . It was concluded that a horizontal transfer of the photosynthesis gene cluster from an ancestral species belonging to the alpha subclass to that of the beta subclass of purple bacteria had occurred and was followed by rearrangements of the operons in this cluster. Bioelectrochemistry, 2001 Mar, 53(2), 233 - 41 Effect of D2O and cryosolvents on the redox properties of bacteriochlorophyll dimer and electron transfer processes in Rhodobacter sphaeroides reaction centers; Paschenko VZ et al.; Effects of environmental changes on the reaction pattern of excitation energy trapping and transformation into the "stable" radical pair P+Q(A)-, have been analyzed in isolated reaction centers of the anoxygenic purple bacterium Rhodobacter sphaeroides . The following results were obtained: (a) replacement of exchangeable protons by deuterons significantly retarded the electron transfer steps of primary charge separation, leading to the radical pair P+I- and of the subsequent reoxidation of I- by the quinone acceptor Q(A) but has virtually no effect on the midpoint potential of P/P+ that was found to be 430+/-20 mV; (b) addition of 70% (v/v) glycerol causes a shift of Em by about 30 mV towards higher values whereas the kinetics of the electron transfer reactions remain almost unaffected; (c) in the presence of the cryoprotectant DMSO, a combined effect arises, i.e . a retardation of the electron transfer kinetics comparable to that induced by H/D exchange and simultaneously an upshift of the Em value to 475+/-20 mV, resembling the action of glycerol . These results are discussed within the framework of effects on the midpoint potential due to the dielectric constant of the medium and changes of the charge distribution in the vicinity of the redox groups and the influence of relaxation processes on electron transfer reactions. Trends Genet, 2001 May, 17(5), 235 - 7 Plagiarized bacterial genes in the human book of life; Ponting CP; The initial analysis of the human genome draft sequence reveals that our 'book of life' is multi-authored . A small but significant proportion of our genes owes their heritage not to antecedent eukaryotes but instead to bacteria . The publicly funded Human Genome Project study indicates that about 0.5% of all human genes were copied into the genome from bacterial sources . Detailed sequence analyses point to these 'horizontal gene transfer' events having occurred relatively recently . So how did the human 'book of life' evolve to be a chimaera, part animal and part bacterium? And what was the probable evolutionary impact of such gene plagiarism? Biochim Biophys Acta, 2001 Jun 1, 1505(2-3), 209 - 19 Heterologous expression and properties of the gamma-subunit of the Fe-only hydrogenase from Thermotoga maritima; Verhagen MF et al.; Thermotoga maritima is a hyperthermophilic bacterium that contains a complex, heterotrimeric (alpha(beta)gamma) Fe-only hydrogenase . Sequence analysis indicates that the gene encoding the smallest subunit (gamma), hydC, contains a predicted iron-sulfur cluster binding motif . However, characterization of the native gamma-subunit has been hampered by interference from and the inability to separate intact gamma-subunit from the other two subunits (alpha and beta) . To investigate the function and properties of the isolated gamma-subunit, the gene encoding HydG was expressed in Escherichia coli . Two forms of the recombinant protein were obtained with molecular masses of 10 and 18 kDa, respectively . Both contained a single {2Fe-2S} cluster based on metal analysis, EPR and UV-visible spectroscopy . NH2-terminal sequencing revealed that the 10 kDa protein is a truncated form of the intact gamma-subunit and lacks the first 65 amino acid residues . The midpoint potential of the 18 kDa form was -356 mV at pH 7.0 and 25 degrees C, as measured by direct electrochemistry, and was pH dependent with a pK(ox) of 7.5 and a pK(red) of 7.7 . The oxidized, recombinant gamma-subunit was stable at 80 degrees C under anaerobic conditions with a half-life greater than 24 h, as judged by the UV-visible spectrum of the {2Fe-2S} cluster . In the presence of air the protein was less stable and denatured with a half-life of approx . 2.5 h . The recombinant gamma-subunit was electron transfer competent and was efficiently reduced by pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus, with a Km of 5microM and a Vmax of 9 U/mg . In contrast, native T . maritima hydrogenase holoenzyme and its separated alpha-subunit were much less effective electron donors for the gamma-subunit, with a V(max) of 0.01 U/mg and 0.1 U/mg, respectively. Enferm Infecc Microbiol Clin, 2001 Feb, 19(2), 49 - 52 {Helicobacter pylori infections: antigen detection in stool samples}; Gonzalez-Cuevas A et al.; BACKGROUND: The aim of this study is to evaluate a new diagnostic test to detect Helicobacter pylori antigen in stool samples (HpSA), and compare the results with those obtained by standard techniques (rapid urease test,culture, histological examination of gastric biopsy specimens,13C-urea breath test and serology), in a paediatric population with gastrointestinal symptomatology . PATIENTS AND METHODS: Sixty patients with dyspeptic symptoms (37 females and 23 males;mean age 10.9 years) attending the Gastroenterology Service were included in the study . Exclusion criterium was previous treatment with proton pump inhibitors, bismuth compounds or antibiotics . Rapid urease test, culture and histologic study of gastric biopsies,13C-urea breath test and serology, as well as HpSA, were performed to all patients . RESULTS: Forty seven patients were considered infected by H.pylori on the basis of bacterium isolation and 13C-urea breath test positivity . HpSAwas detected in 45 of the 47 H.pylori positive patients(95.7%) . There were no HpSA false positive . CONCLUSION: Our results show that this new test is highly sensitive (95%) and specific(100%) for detection of H . pylori infection . It has some advantages over other non invasive techniques: it is easy to perform,requires no blood samples and its cost is lower than that of 13C-urea breath test. Proc Natl Acad Sci U S A, 2001 May 8, 98(10), 5910 - 5 Epub 2001 May 01. Two enzymes of diacylglyceryl-O-4'-(N,N,N,-trimethyl)homoserine biosynthesis are encoded by btaA and btaB in the purple bacterium Rhodobacter sphaeroides; Klug RM et al.; Betaine lipids are ether-linked, nonphosphorous glycerolipids that resemble the more commonly known phosphatidylcholine in overall structure . Betaine lipids are abundant in many eukaryotes such as nonseed plants, algae, fungi, and amoeba . Some of these organisms are entirely devoid of phosphatidylcholine and, instead, contain a betaine lipid such as diacylglyceryl-O-4'-(N,N,N,-trimethyl)homoserine . Recently, this lipid also was discovered in the photosynthetic purple bacterium Rhodobacter sphaeroides where it seems to replace phosphatidylcholine under phosphate-limiting growth conditions . This discovery provided the opportunity to study the biosynthesis of betaine lipids in a bacterial model system . Mutants of R . sphaeroides deficient in the biosynthesis of the betaine lipid were isolated, and two genes essential for this process, btaA and btaB, were identified . It is proposed that btaA encodes an S-adenosylmethionine:diacylglycerol 3-amino-3-carboxypropyl transferase and btaB an S-adenosylmethionine-dependent N-methyltransferase . Both enzymatic activities can account for all reactions of betaine lipid head group biosynthesis . Because the equivalent reactions have been proposed for different eukaryotes, it seems likely that orthologs of btaA/btaB may be present in other betaine lipid-containing organisms. J Biol Chem, 2001 Jul 6, 276(27), 24781 - 9 Epub 2001 Apr 30. Vitreoscilla hemoglobin . Intracellular localization and binding to membranes; Ramandeep et al.; The obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions . Expression of VHb in heterologous hosts often enhances growth and product formation . A role in facilitating oxygen transfer to the respiratory membranes is one explanation of its cellular function . Immunogold labeling of VHb in both Vitreoscilla and recombinant Escherichia coli bearing the VHb gene clearly indicated that VHb has a cytoplasmic (not periplasmic) localization and is concentrated near the periphery of the cytosolic face of the cell membrane . OmpA signal-peptide VHb fusions were transported into the periplasm in E . coli, but this did not confer any additional growth advantage . The interaction of VHb with respiratory membranes was also studied . The K(d) values for the binding of VHb to Vitreoscilla and E . coli cell membranes were approximately 5-6 microm, a 4-8-fold higher affinity than those of horse myoglobin and hemoglobin for these same membranes . VHb stimulated the ubiquinol-1 oxidase activity of inverted Vitreoscilla membranes by 68% . The inclusion of Vitreoscilla cytochrome bo in proteoliposomes led to 2.4- and 6-fold increases in VHb binding affinity and binding site number, respectively, relative to control liposomes, suggesting a direct interaction between VHb and cytochrome bo. Biochemistry, 2001 May 8, 40(18), 5573 - 8 A bacteriochlorophyll a antenna complex from purple bacteria absorbing at 963 nm; Permentier HP et al.; A recently isolated species of the photosynthetic purple sulfur bacteria, provisionally called strain 970, was investigated with respect to its antenna function by means of various spectroscopic techniques, including fluorescence and pump-probe absorption difference spectroscopy . The bacterium contains bacteriochlorophyll a and an as yet unidentified carotenoid, perhaps 3,4,3',4'-tetrahydrospirilloxanthin . It has a single antenna complex of the LH1 type, with a Q(y) absorption band situated at the unusually long wavelength of 963 nm at room temperature and 990 nm at 6 K . In contrast to many other species, the reaction center showed two well-separated absorption bands of bacteriopheophytin at 6 K, located at 747 and 762 nm . The primary electron donor showed a bleaching band centered at 925 nm upon photooxidation . Thus, the energy gap between LH1 and the primary electron donor is quite large in this strain: 425 cm(-1) . Nevertheless, trapping occurred with a time constant of 65 +/- 5 ps, similar to the rates observed in other purple bacteria . As in other species, no back-transfer from the reaction center to the antenna was observed . Our results show that strain 970 is a unique subject for the study of antenna and reaction center function and organization. Biosci Biotechnol Biochem, 2001 Mar, 65(3), 690 - 3 Piezoresponse of the cyo-operon coding for quinol oxidase subunits in a deep-sea piezophilic bacterium, Shewanella violacea; Nakasone K et al.; We have isolated the genes for quinol oxidase from a deep-sea piezophilic bacterium, Shewanella violacea . Analysis of the deduced amino acid sequences of the cyo subunits showed that this oxidase has high similarity to Escherichia coli bo-type quinol oxidase . Northern blot analysis showed that these genes are expressed at a high level when the bacterium is grown at elevated pressure . Upstream in the cyo-operon, a sigma54-binding motif and an octamer sequence unit were found, suggesting that these elements may play a role in regulation of expression of the cyo-operon in response to changes in pressure. Biosci Biotechnol Biochem, 2001 Mar, 65(3), 666 - 9 Characterization of the gene encoding the beta-lactamase of the psychrophilic marine bacterium Moritella marina strain MP-1; Tanaka M et al.; The beta-lactamase gene (mbla) of the psychrophilic marine bacterium Moritella marina strain MP-1 was identified in a previously isolated genomic DNA fragment and it was expressed in Escherichia coli cells . The mbla gene encoded a protein consisting of 287 amino acid residues . Its predicted amino acid sequence showed approximately 50% identity with that of a number of class A beta-lactamases, especially with that of CARB/PSE type of beta-lactamases (carbenicillinases) . E . coli transformed with the plasmid containing mbla grew on an ampicillin-containing plate at 37 degrees C but not at 42 degrees C, suggesting that the beta-lactamase of this bacterium is heat-labile. Biochemistry, 2001 Feb 20, 40(7), 2210 - 9 Distinct reactions catalyzed by bacterial and yeast trans-aconitate methyltransferases; Cai H et al.; The trans-aconitate methyltransferase from the bacterium Escherichia coli catalyzes the monomethyl esterification of trans-aconitate and related compounds . Using two-dimensional (1)H/(13)C nuclear magnetic resonance spectroscopy, we show that the methylation is specific to one of the three carboxyl groups and further demonstrate that the product is the 6-methyl ester of trans-aconitate (E-3-carboxy-2-pentenedioate 6-methyl ester) . A similar enzymatic activity is present in the yeast Saccharomyces cerevisiae . Although we find that yeast trans-aconitate methyltransferase also catalyzes the monomethyl esterification of trans-aconitate, we identify that the methylation product of yeast is the 5-methyl ester (E-3-carboxyl-2-pentenedioate 5-methyl ester) . The difference in the reaction catalyzed by the two enzymes may explain why a close homologue of the E . coli methyltransferase gene is not found in the yeast genome and furthermore suggests that these two enzymes may play distinct roles . However, we demonstrate here that the conversion of trans-aconitate to each of these products can mitigate its inhibitory effect on aconitase, a key enzyme of the citric acid cycle, suggesting that these methyltransferases may achieve the same physiological function with distinct chemistries. Phys Rev Lett, 2001 Apr 30, 86(18), 4167 - 70 Exciton delocalization probed by excitation annihilation in the light-harvesting antenna LH2; Trinkunas G et al.; Singlet-singlet annihilation is used to study exciton delocalization in the light harvesting antenna complex LH2 (B800-B850) from the photosynthetic purple bacterium Rhodobacter sphaeroides . The characteristic femtosecond decay constants of the high intensity isotropic and the low intensity anisotropy kinetics of the B850 ring are related to the hopping time tau(h) and the coherence length N(coh) of the exciton . Our analysis yields N(coh) = 2.8+/-0.4 and tau(h) = 0.27+/-0.05 ps . This approach can be seen as an extension to the concept of the spectroscopic ruler. Biochemistry, 2001 Feb 27, 40(8), 2387 - 96 Solution structure of the ribosome recycling factor from Aquifex aeolicus; Yoshida T et al.; The solution structure of ribosome recycling factor (RRF) from hyperthermophilic bacterium, Aquifex aeolicus, was determined by heteronuclear multidimensional NMR spectroscopy . Fifteen structures were calculated using restraints derived from NOE, J-coupling, and T1/T2 anisotropies . The resulting structure has an overall L-shaped conformation with two domains and is similar to that of a tRNA molecule . The domain I (corresponding to the anticodon stem of tRNA) is a rigid three alpha-helix bundle . Being slightly different from usual coiled-coil arrangements, each helix of domain I is not twisted but straight and parallel to the main axis . The domain II (corresponding to the portion with the CCA end of tRNA) is an alpha/beta domain with an alpha-helix and two beta-sheets, that has some flexible regions . The backbone atomic root-mean-square deviation (rmsd) values of both domains were 0.7 A when calculated separately, which is smaller than that of the molecule as a whole (1.4 A) . Measurement of 15N-{1H} NOE values show that the residues in the corner of the L-shaped molecule are undergoing fast internal motion . These results indicate that the joint region between two domains contributes to the fluctuation in the orientation of two domains . Thus, it was shown that RRF remains the tRNA mimicry in solution where it functions. Biochemistry, 2001 Feb 13, 40(6), 1850 - 60 Retardation of proton transfer caused by binding of the transition metal ion to the bacterial reaction center is due to pKa shifts of key protonatable residues; Gerencser L et al.; Transition metal ions bind to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides and slow the light-induced electron and proton transfer to the secondary quinone, Q(B) . We studied the properties of the metal ion-RC complex by measuring the pH dependence of the dissociation constant and the stoichiometry of proton release upon ligand formation . We investigated the mechanism of inhibition by measuring the stoichiometry and kinetics of flash-induced proton binding, the transfer of (first and second) electrons to Q(B), and the rate of steady-state turnover of the RC in the absence and presence of Cd(2+) and Ni(2+) on a wide pH range . The following results were obtained . (1) The complexation of transition metal ions Cd(2+) and Ni(2+) with the bacterial RC showed strong pH dependence . This observation was explained by different (pH-dependent) states of the metal-ligand cluster: the complex formation was strong when the ligand (Asp and His residues) was deprotonated and was much weaker if the ligand was partly (or fully) protonated . A direct consequence of the model was the pH-dependent proton release upon complexation . (2) The retardation of transfer of electrons and protons to Q(B) was also strongly pH-dependent . The effect was large in the neutral pH range and decreased toward the acidic and alkaline pH values . (3) Steady-state turnover measurements indicated that the rate of the second proton transfer was much less inhibited than that of the first one, which became the rate-limiting step in continuous turnover of the RC . (4) Sodium azide partly recovered the proton transfer rate . The effect is not due to removal of the bound metal ion by azide but probably by formation of a proton-transporting azide network similarly as water molecules may build up proton pathways . (5) We argue that the inhibition comes mainly from pK(a) shifts of key protonatable residues that control the proton transfer along the H-bond network to Q(B) . The electrostatic interaction between the metal ion and these residues may result in acidic pK(a) shifts between 1.5 and 2.0 that account for the observed retardation of the electron and proton transfer. Biochemistry, 2001 Feb 13, 40(6), 1587 - 95 A refined model of the chlorosomal antennae of the green bacterium Chlorobium tepidum from proton chemical shift constraints obtained with high-field 2-D and 3-D MAS NMR dipolar correlation spectroscopy; van Rossum BJ et al.; Heteronuclear 2-D and 3-D magic-angle spinning NMR dipolar correlation spectroscopy was applied to determine solid-state (1)H shifts for aggregated bacteriochlorophyll c (BChl c) in uniformly (13)C-enriched light harvesting chlorosomes of the green photosynthetic bacterium Chlorobium tepidum . A complete assignment of 29 different observable resonances of the 61 protons of the aggregated BChl c in the intact chlorosomes is obtained . Aggregation shifts relative to monomeric BChl c in solution are detected for protons attached to rings I, II, and III/V and to their side chains . The 2(1)-H(3), 3(2)-H(3), and 3(1)-H resonances are shifted upfield by -2.2, -1, and -3.3 ppm, respectively, relative to monomeric BChl c in solution . Although the resonances are inhomogeneously broadened and reveal considerable global structural heterogeneity, the 5-CH and the 7-Me responses are doubled, which provides evidence for the existence of at least two relatively well-defined structurally different arrangements . Ab initio quantum chemical modeling studies were performed to refine a model for the self-assembled BChl c with two different types of BChl stacks . The BChl in the stacks can adopt either anti- or syn-configuration of the coordinative bond, where anti and syn designate the relative orientation of the Mg-OH bond relative to the direction of the 17-17(1) bond . The analogy between aggregation shifts for BChl c in the chlorosome and for self-assembled chlorophyll a/H(2)O is explored, and a bilayer model for the tubular supra-structure of sheets of BChl c is proposed, from a homology modeling approach. J Clin Microbiol, 2001 May, 39(5), 1746 - 50 iceA genotypes of Helicobacter pylori strains isolated from Brazilian children and adults; Ashour AA et al.; Data concerning the geographic distribution of iceA alleles are scarce, and information on the association of the gene with the disease is rare and still controversial . Furthermore, no such study has been developed in Brazil, where duodenal ulcer and gastric adenocarcinoma are very common . We investigated, by PCR, the frequency of iceA alleles and cagA status in Helicobacter pylori strains isolated from 142 patients (62 children and 80 adults; 66 female; mean age, 30.0 years; age range, 3 to 78 years) with gastritis, duodenal ulcer, or gastric adenocarcinoma . iceA was identified in bacterium samples obtained from all patients . Eleven (7.7%) of them were infected with multiple strains . Among the patients with nonmixed infection, iceA2 allele was detected in 118 (90.1%) . iceA2 allele was associated with ulcer (P = 0.02) and with carcinoma (P = 0.001) . iceA2 amplicons of 229, 334, or 549 bp were detected, but none of them was associated with the patient's disorder . iceA2 strains were more frequent in patients older than 7 years (P = 0.001) . The gene was also more frequent in strains obtained from males (P = 0.02) . cagA was more common in strains obtained from carcinoma (P = 0.0008) and ulcer patients (P < 0.006) . cagA-positive strains were more frequent in children older than 7 years (P < 0.003) . No association between cagA status and sex was found (P = 0.28) . In conclusion, we think iceA should not be used as a reliable marker for predicting the clinical outcome of H . pylori infection. J Biol Chem, 2001 Jul 13, 276(28), 25791 - 6 Epub 2001 Apr 26. Structural determinants of cold adaptation and stability in a large protein; D'Amico S et al.; The heat-labile alpha-amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry . Mutants of this enzyme were produced, carrying additional weak interactions found in thermostable alpha-amylases . It is shown that single amino acid side chain substitutions can significantly modify the melting point T(m), the calorimetric enthalpy Delta H(cal), the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters k(cat) and K(m) . The correlation between thermal inactivation and unfolding reversibility displayed by the mutants also shows that stabilizing interactions increase the frequency of side reactions during refolding, leading to intramolecular mismatches or aggregations typical of large proteins . Although all mutations were located far from the active site, their overall trend is to decrease both k(cat) and K(m) by rigidifying the molecule and to protect mutants against thermal inactivation . The effects of these mutations indicate that the cold-adapted alpha-amylase has lost a large number of weak interactions during evolution to reach the required conformational plasticity for catalysis at low temperatures, thereby producing an enzyme close to the lowest stability allowing maintenance of the native conformation. J Bacteriol, 2001 May, 183(10), 3204 - 10 A homolog of the CtrA cell cycle regulator is present and essential in Sinorhizobium meliloti; Barnett MJ et al.; During development of the symbiotic soil bacterium Sinorhizobium meliloti into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes . Regulatory genes controlling the early stages of this process have not been identified . As a first step in the search for regulators of these events, we report the isolation and characterization of a ctrA gene from S . meliloti . We show that the S . meliloti CtrA belongs to the CtrA-like family of response regulators found in several alpha-proteobacteria . In Caulobacter crescentus, CtrA is essential and is a global regulator of multiple cell cycle functions . ctrA is also an essential gene in S . meliloti, and it is expressed similarly to the autoregulated C . crescentus ctrA in that both genes have complex promoter regions which bind phosphorylated CtrA. J Bacteriol, 2001 May, 183(10), 3169 - 75 Promoter cloning in the radioresistant bacterium Deinococcus radiodurans; Meima R et al.; Deinococcus radiodurans is a highly radiation-resistant bacterium that is classed in a major subbranch of the bacterial domain . Since very little is known about gene expression in this bacterium, an initial study of promoters was undertaken . In order to isolate promoters and study promoter function, a series of integrative vectors for stable chromosomal insertion in D . radiodurans were developed . These vectors are based on Escherichia coli replicons that are unable to replicate autonomously in D . radiodurans and carry homologous sequences for replacement recombination in the D . radiodurans chromosome . The resulting integration vectors were used to study expression of reporter genes fused to a number of putative promoters that were amplified from the D . radiodurans R1 genome . Further analysis of these and other putative promoters was performed by Northern hybridization and primer extension experiments . In contrast to previous reports, the -10 and -35 regions of these promoters resembled the sigma(70) consensus sequence of E . coli. J Bacteriol, 2001 May, 183(10), 3142 - 8 The N terminus of FliM is essential to promote flagellar rotation in Rhodobacter sphaeroides; Poggio S et al.; FliM is part of the flagellar switch complex . Interaction of this protein with phospho-CheY (CheY-P) through its N terminus constitutes the main information relay point between the chemotactic system and the flagellum . In this work, we evaluated the role of the N terminus of FliM in the swimming behavior of Rhodobacter sphaeroides . Strains expressing the FliM protein with substitutions in residues previously reported in Escherichia coli as being important for interaction with CheY showed an increased stop frequency compared with wild-type cells . In accordance, we observed that R . sphaeroides cells expressing FliM lacking either the first 13 or 20 amino acids from the N terminus showed a stopped phenotype . We show evidence that FliMDelta13 and FliMDelta20 are stable proteins and that cells expressing them allow flagellin export at levels indistinguishable from those detected for the wild-type strain . These results suggest that the N-terminal region of FliM is required to promote swimming in this bacterium . The role of CheY in controlling flagellar rotation in this organism is discussed. J Bacteriol, 2001 May, 183(10), 3117 - 26 A Phosphopantetheinyl transferase homolog is essential for Photorhabdus luminescens to support growth and reproduction of the entomopathogenic nematode Heterorhabditis bacteriophora; Ciche TA et al.; The bacterium Photorhabdus luminescens is a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora . The nematode requires the bacterium for infection of insect larvae and as a substrate for growth and reproduction . The nematodes do not grow and reproduce in insect hosts or on artificial media in the absence of viable P . luminescens cells . In an effort to identify bacterial factors that are required for nematode growth and reproduction, transposon-induced mutants of P . luminescens were screened for the loss of the ability to support growth and reproduction of H . bacteriophora nematodes . One mutant, NGR209, consistently failed to support nematode growth and reproduction . This mutant was also defective in the production of siderophore and antibiotic activities . The transposon was inserted into an open reading frame homologous to Escherichia coli EntD, a 4'-phosphopantetheinyl (Ppant) transferase, which is required for the biosynthesis of the catechol siderophore enterobactin . Ppant transferases catalyze the transfer of the Ppant moiety from coenzyme A to a holo-acyl, -aryl, or -peptidyl carrier protein(s) required for the biosynthesis of fatty acids, polyketides, or nonribosomal peptides . Possible roles of a Ppant transferase in the ability of P . luminescens to support nematode growth and reproduction are discussed. J Med, 2001, 32(1-2), 97 - 112 Different effects of H . pylori water extracts on cytokines, pepsinogen C and gastrin mucosal release in patients with or without duodenal ulcer; Basso D et al.; In the present study we ascertained whether cagA positive and negative H . pylori strains release water soluble products that can influence the production of gastric mucosal cytokines and endocrine (gastrin) or exocrine (pepsinogen C) secretion in 23 H . pylori positive and 19 H . pylori negative patients . Antral biopsies were obtained to classify inflammation, activity, atrophy, intestinal metaplasia and H . pylori density grade . The cagA gene was identified by means of the polymerase chain reaction (PCR) in H . pylori positive colonies after culture of mucosal samples . Three antral biopsies from each patient were incubated with (1.) Water extracts from cagA positive, (2.) Water extracts from cagA negative strains or (3.) H2O (control) at 37 degrees C in a CO2 incubator for 24 hrs . Gastrin, pepsinogen C, IL-1 beta, IL-8, GMCSF, and TNF alpha were measured in the supernatants and mucosal homogenates . H . pylori infection was significantly associated with an increased antral inflammation and activity (chi 2 = 21.7, p < 0.001 and chi 2 = 42.0, p < 0.001), and increased mucosal levels of IL-1 beta, IL-8 and TNF alpha . Water extracts from cagA positive strains enhanced the release of PGC in mucosal biopsy supernatants (p < 0.05) when patients were considered overall and the release of TNF alpha (p < 0.05) when only patients with duodenal ulcer were considered . Water extracts from cagA negative strains stimulated gastrin secretion (p < 0.05) . None of the remaining cytokines were influenced by H . pylori water extracts . In conclusion, pepsinogen C and TNF alpha can be induced by cagA positive water extracts and may contribute to damage the gastric and duodenal mucosa . Our findings indicate that in patients with H . pylori infection the increase of the mucosal levels of IL-1 beta and IL-8 does not depend on H . pylori water soluble products, but probably depends on the entire bacterium. J Physiol Pharmacol, 2001 Mar, 52(1), 153 - 64 Heat shock protein 70 (HSP70) in gastric adaptation to aspirin in Helicobacter pylori infection; Konturek JW et al.; We have recently shown that adaptation of gastric mucosa to aspirin (ASA) is disturbed in Helicobacter pylori (H . pylori)-infected human stomach, but can be restored by eradication of the bacterium . The aim of this study was 1) to evaluate the influence of H . pylori on expression of heat shock protein 70 (HSP70) during ASA ingestion in these subjects and in mice model and 2) to evaluate, whether altered HSP70 expression might be associated with different adaptation to ASA in H . pylori-positive and eradicated subjects . The gastric mucosal HSP 70 gene expression was determined by quantitative RT-PCR and Western blot and immunohistochemistry during 14 days of ASA ingestion (1 g bid) in the same 8 subjects before and 3 months after successful eradication of H . pylori . In addition, HSP70 mRNA and protein expression were examined in 30 mice without and with H . pylori infection and eradication . During 14 days of ASA treatment, human H . pylori-infected mucosa revealed a decrease of HSP70 expression, while after eradication a higher expression and further increase of HSP70 expression during ASA ingestion were observed . Mice inoculated with H . pylori also exhibited decreased gastric mucosal HSP70 mRNA expression that was restored after eradication therapy . Decreased basal and ASA-induced expression of HSP70 may partly be responsible for impaired gastric adaptation to ASA in H . pylori-positive subjects . We conclude that 1 . The HSP70 gene and protein expression is reduced during infection with H . pylori in men and mice and that gastric adaptation to ASA in H . pylori eradicated subjects is accompanied by increased HSP70 expression; 2 . It is reasonable to assume that decreased HSP70 expression might contribute to disturbed gastric adaptation in H . pylori infection in humans and 3 . The expression of HSP70 plays an important role in the mechanism of gastric adaptation to ASA and that H . pylori infection interferes with this adaptation due to decrease of HSP70 expression in gastric mucosal cells. Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 679 - 82 Actinomyces catuli sp . nov., from dogs; Hoyles L et al.; An Actinomyces-like bacterium was recovered from two dogs . Based on cellular morphology and biochemical criteria, the unknown bacterium resembled the genus Actinomyces but it did not appear to correspond to any of the currently recognized species of this genus . PAGE analysis of whole-cell proteins confirmed that the strain was phenotypically distinct from all other Actinomyces species and comparative 16S rRNA gene sequencing showed that the bacterium represents an unknown sub-line within the genus . Based on phenotypic and phylogenetic evidence, it is proposed that the bacterium from dogs be classified as a new species of the genus Actinomyces, Actinomyces catuli . The type strain of Actinomyces catuli is CCUG 41709T (= CIP 106507T). Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 559 - 63 Asaia siamensis sp . nov., an acetic acid bacterium in the alpha-proteobacteria; Katsura K et al.; Five bacterial strains were isolated from tropical flowers collected in Thailand and Indonesia by the enrichment culture approach for acetic acid bacteria . Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were located within the cluster of the genus Asaia . The isolates constituted a group separate from Asaia bogorensis on the basis of DNA relatedness values . Their DNA G+C contents were 58.6-59.7 mol%, with a range of 1.1 mol%, which were slightly lower than that of A . bogorensis (59.3-61.0 mol%), the type species of the genus Asaia . The isolates had morphological, physiological and biochemical characteristics similar to A . bogorensis strains, but the isolates did not produce acid from dulcitol . On the basis of the results obtained, the name Asaia siamensis sp . nov . is proposed for these isolates . Strain S60-1T, isolated from a flower of crown flower (dok rak, Calotropis gigantea) collected in Bangkok, Thailand, was designated the type strain ( = NRIC 0323T = JCM 10715T = IFO 16457T). Pathol Biol (Paris), 2001 Mar, 49(2), 128 - 37 {Contribution of the laboratory to the epidemiologic study of bacterial infections}; Biron M et al.; The laboratory plays a significant role in the epidemiologic investigations by the comparative analysis of the bacterial strains involved in the outbreaks . Recently, the use of molecular analysis methods provided better performance than traditional phenotypic methods which are still used as preliminary tests because of their relatively low cost and technical simplicity . These analyses deal with either the whole chromosome of the bacterium, plasmids or particular genes . The classification of these methods runs up against the lack of consensus concerning their nomenclature . A clearer denomination based upon the technique responsible for revealing the polymorphisms of these various targets, makes it possible to divide these methods in two principal groups: methods of RFLP (based on the fragments resulting from digestion with restriction enzymes) and methods of AFLP (based on the products of amplification by PCR) . The knowledge of the typability of the strains and the qualities of these various methods, particularly their discriminatory power, is essential to the accuracy of the laboratory analysis in the investigations of outbreaks. BMC Biotechnol . 2001;1(1):1 . Epub 2001 Apr 18. Inhibition of spontaneous induction of lambdoid prophages in Escherichia coli cultures: simple procedures with possible biotechnological applications; Czyz A et al.; BACKGROUND: Infections of bacterial cultures by bacteriophages are serious problems in biotechnological laboratories . Apart from such infections, prophage induction in the host cells may also be dangerous . Escherichia coli is a commonly used host in biotechnological production, and many laboratory strains of this bacterium harbour lambdoid prophages . These prophages may be induced under certain conditions leading to phage lytic development . This is fatal for further cultivations as relatively low, though still significant, numbers of phages may be overlooked . Thus, subsequent cultures of non-lysogenic strains may be infected and destroyed by such phage . RESULTS: Here we report that slow growth of bacteria decreases deleterious effects of spontaneous lambdoid prophage induction . Moreover, replacement of glucose with glycerol in a medium stimulates lysogenic development of the phage after infection of E . coli cells . A plasmid was constructed overexpressing the phage 434 cI gene, coding for the repressor of phage promoters which are necessary for lytic development . Overproduction of the cI repressor abolished spontaneous induction of the lambda(imm434) prophage . CONCLUSIONS: Simple procedures that alleviate problems with spontaneous induction of lambdoid prophage and subsequent infection of E . coli strains by these phages are described . Low bacterial growth rate, replacement of glucose with glycerol in a medium and overproduction of the cI repressor minimise the risk of prophage induction during cultivation of lysogenic bacteria and subsequent infection of other bacterial strains. Mikrobiologiia, 2000 Sep-Oct, 69(5), 717 - 21 {Evaluation of the effect of ecologically hazardous pollutants on the bacteriolytic activity of the predatory bacterium Bdellovibrio}; Markelova NIu et al.; We studied the effect of various concentrations of ecologically hazardous pollutants, urea, phenol, diuron, and cadmium ions, on the physiological activity and survival of the parasitic bacterium Bdellovibrio . Experiments showed that the survival of bdellovibrios in the presence of the pollutants was two times higher when they were cultivated on agar than when they were cultivated in liquid medium . The data obtained are in agreement with the recent concept of the surface-associated state as a survival strategy of bdellovibrios in various ecosystems. Can J Microbiol, 2001 Mar, 47(3), 206 - 12 Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus; Yakunin AF et al.; The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness . This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG) . Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions . Here we show that four different strains of Rba . sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba . capsulatus strains possess this ability . Introduction of Rba . capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba . sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium . However, transfer of draTG from Rba . capsulatus was sufficient to confer on Rba . sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness . These data suggest that Rba . sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins. J Agric Food Chem, 2001 Mar, 49(3), 1200 - 2 Analysis of canthaxanthin and related pigments from Gordonia jacobaea mutants; de Miguel T et al.; A collection of 43 mutant strains of the bacterium Gordonia jacobaea was obtained by means of ethyl methanesulfonate treatment, and the strains were selected for their different pigmentation with respect to the wild-type strain . None of the mutants showed auxotrophy . They all showed good genetic stability and a growth rate similar to that of the parental strain . Canthaxanthin and other carotenoids from these mutants were extracted with acetone and ethanol and separated by high-performance liquid chromatography (HPLC) . These HPLC analyses, together with spectrophotometric detection at 480 nm, revealed variations in the pigment contents of the different mutant strains. Mol Microbiol, 2001 Apr, 40(2), 387 - 96 Non-heritable change of a spirochaete's phenotype by decoration of the cell surface with exogenous lipoproteins; Bunikis J et al.; Genetic transformation of Borrelia spp . is limited in development and has found application in only one species . For a non-genetic approach for manipulating the phenotype of these spirochaetes, we determined whether exogenous recombinant lipoproteins would incorporate in the cell's outer membrane . Using unlabelled or 125I-labelled Osp proteins, Osp-specific monoclonal antibodies, proteinase K and formaldehyde as reagents, we found that decoration of spirochaetes had the following characteristics . (i) Purified recombinant OspA or OspD lipoproteins associated with Borrelia burgdorferi and B . hermsii cells that lacked abundant lipoproteins of their own . (ii) This decoration of the cells with exogenous OspA did not affect cell's viability . (iii) The decoration was concentration and temperature dependent and stable for at least 24 h . (iv) Like native OspA, the recombinant OspA decorating the cells was accessible to antibodies and proteases and could be cross-linked to the integral outer membrane protein, P66 . (v) Decoration of viable B . burgdorferi and B . hermsii with OspA rendered the cells susceptible to killing by OspA-specific antiserum . Such non-genetic alteration of the surface of a bacterium may be used to study functions and properties of lipoproteins in situ. Fish Shellfish Immunol, 2001 Feb, 11(2), 143 - 53 Response of haemocyte lysosomes to bacterial inoculation in the oysters Ostrea edulis L . and Crassostrea gigas (Thunberg) and the scallop Pecten maximus (L); Hauton C et al.; Data are presented that demonstrate the application of the neutral red retention assay (NRR) to monitor the effects of a bacterial inoculation on the haemocyte lysosomes of the European flat oyster Ostrea edulis, Pacific oyster Crassostrea gigas and scallop Pecten maximus . Bivalves were acclimated to three temperature regimes (5, 15 and 25 degrees C), at constant salinity for 7 days in the laboratory . Once baseline responses to acclimation temperature had been established, the effects of an in vivo inoculation on haemocyte lysosomal stability were assessed using the NRR assay . Lysosomal membrane stability was reduced in the presence of bacteria for all three species of bivalve, but destabilisation of C . gigas haemocyte lysosomes appeared to be most sensitive to the presence of the bacterium Listonella anguillarum . For all three bivalve species, the reduction in lysosomal stability appeared to be proportional to the growth of the bacterial inoculate . Using appropriate controls, the NRR assay was demonstrated to have great potential as a tool with which to make rapid initial assessments of the immune status of bivalve molluscs. Scand J Infect Dis, 2001, 33(3), 163 - 74 An update on Helicobacter pylori microbiology and infection for the new millennium; Enroth H et al.; The finding of the bacterium Helicobacter pylori in patients with symptomatic gastric diseases was a breakthrough for both treatment of peptic ulcer disease and studies of other infectious diseases . Helicobacter pylori infection is rare among the young, indicating that improved childhood living conditions have halted the transmission of the bacterium within families, with a parallel decrease in symptomatic gastroduodenal diseases . Extensive strain variation in H . pylori has been demonstrated at both the genomic and the protein level, and the interstrain variation is higher than in any other bacterium studied so far . Pathogenic markers in H . pylori and host genetics are both of importance for disease outcome . Genotypic or phenotypic markers of H . pylori strains may be used to discriminate patients who should undergo eradication therapy from those who might not benefit from it . Possible positive effects of the infection are still under investigation, and several hypotheses regarding the etiology of diseases in different parts of the stomach have been proposed . To be able to separate the disease-causing infections from the silent infections is a real challenge for the new millennium, and one of the most important issues for therapy and prevention, in the research field of H . pylori. Extremophiles, 2001 Feb, 5(1), 53 - 60 Liposome-mediated DNA uptake and transient expression in Thermotoga; Yu JS et al.; We report here the successful application of a PCR-based method to detect genetic transformation of Thermotoga neapolitana and Thermotoga maritima . Plasmid vectors were constructed using pRQ7, an 846-bp plasmid found in Thermotoga species strain RQ7, which replicates by a rolling circle mechanism . The vector pJY1 was constructed by placing a gene encoding a thermostable chloramphenicol acetyltransferase from Stacphylococcus aureus under the control of the tac promoter and joining this with pRQ7 in a pBluescript vector . A second vector, pJY2, was similarly constructed using a gene encoding a kanamycin nucleotidyltransferase previously engineered for thermostability . Genetic transformation of T . neapolitana and T . maritima spheroplasts was achieved using cationic liposomes . The transforming DNA was detected in cells grown in liquid cultures using polymerase chain reaction amplification of the cat or kan genes . T . neapolitana could maintain pJY1 for at least 25 generations in liquid medium containing chloramphenicol . The pJY2 vector conferred kanamycin resistance to T . maritima cells grown in liquid culture . Isolation of stable transformants on solid media after 2-3 days of incubation at 77 degrees C was not possible with either vector, probably because of the instability of both vectors and antibiotics under these conditions . However, this transformation procedure provides, for the first time, a method to introduce DNA into this hyperthermophilic bacterium for potential applications such as targeted gene disruption analyses. Tidsskr Nor Laegeforen, 2001 Mar 10, 121(7), 805 - 6 {Human granulocytic ehrlichiosis in Norway}; Kristiansen BE et al.; BACKGROUND: The bacterium that causes human granulocytic ehrlichiosis may be transmitted by ticks . MATERIAL AND METHODS: We describe two patients with human granulocytic ehrlichiosis . During the summer of 1998, both patients were bitten by ticks . Four to 7 days later they developed influenza-like symptoms with fever, headache and myalgia . After 4 and 21 days, respectively, both patients were given doxycycline for suspected bacterial respiratory diseases, and recovered . RESULTS: Blood samples for human granulocytic ehrlichiosis antibodies showed a fourfold increase in titer in one patient and a remaining high titer in the other . Both patients had a positive polymerase chain reaction with primers specific for the Ehrlichia phagocytophilae genogroup . INTERPRETATION: The two patients fulfill the human granulocytic ehrlichiosis diagnostic criteria set by Centers for Disease Controls and Prevention, and are the first two human granulocytic ehrlichiosis cases described in Norway. Plant Physiol, 2001 Apr, 125(4), 1585 - 90 Expression of bar in the plastid genome confers herbicide resistance; Lutz KA et al.; Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides . Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme . We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT . We also describe a second bacterial bar gene (b-bar2) and a codon-optimized synthetic bar (s-bar) gene with significantly elevated levels of expression in plastids (>7% of total soluble cellular protein) . Although these genes are expressed at a high level, direct selection thus far did not yield transplastomic clones, indicating that subcellular localization rather than the absolute amount of the enzyme is critical for direct selection of transgenic clones . The codon-modified s-bar gene is poorly expressed in Escherichia coli, a common enteric bacterium, due to differences in codon use . We propose to use codon usage differences as a precautionary measure to prevent expression of marker genes in the unlikely event of horizontal gene transfer from plastids to bacteria . Localization of the bar gene in the plastid genome is an attractive alternative to incorporation in the nuclear genome since there is no transmission of plastid-encoded genes via pollen. Mol Microbiol, 2001 Apr, 40(1), 257 - 69 BldD is a direct regulator of key developmental genes in Streptomyces coelicolor A3(2); Elliot MA et al.; BldD is a transcription factor required for aerial hyphae formation in the filamentous bacterium Streptomyces coelicolor . Three targets of BldD regulation were discovered by a number of means, including examination of bld gene interdependence, selective enrichment of chromosomal DNA fragments bound by BldD and searching the promoter regions of known developmental genes for matches to a previously characterized BldD binding site . The three BldD targets identified were the developmental sigma factor genes, whiG and bldN, and a previously uncharacterized gene, designated bdtA, encoding a putative transcription factor . In each target gene, the sequences bound by BldD were characterized by electrophoretic mobility shift and DNase I footprinting assays, and their alignment suggested AGTgA (n)m TCACc as a consensus BldD operator . The in vivo effect of mutation in bldD on the expression of these three target genes was assessed using S1 nuclease protection assays . In each case, target gene expression was upregulated during early colony development in the bldD background, suggesting that, in the wild type, BldD acts to repress premature expression of whiG, bldN and bdtA during vegetative growth. Biochemistry, 2001 Mar 27, 40(12), 3737 - 47 Excitation trap approach to analyze size and pigment-pigment coupling: reconstitution of LH1 antenna of Rhodobacter sphaeroides with Ni-substituted bacteriochlorophyll; Fiedor L et al.; Replacement of the central Mg in chlorophylls by Ni opens an ultrafast (tens of femtoseconds time range) radiationless de-excitation path, while the principal ground-state absorption and coordination properties of the pigment are retained . A method has been developed for substituting the native bacteriochlorophyll a by Ni-bacteriochlorophyll a ({Ni}-BChl) in the light harvesting antenna of the core complex (LH1) from the purple bacterium, Rhodobacter (Rb.) sphaeroides, to investigate its unit size and excited state properties . The components of the complex have been extracted with an organic solvent from freeze-dried membranes of an LH1-only strain of Rb . sphaeroides and transferred into the micelles of n-octyl-beta-glucopyranoside (OG) . Reconstitution was achieved by solubilization in 3.4% OG, followed by dilution, yielding a complex nearly identical to the native one, in terms of absorption, fluorescence, and circular dichroism spectra as well as energy transfer efficiency from carotenoid to bacteriochlorophyll . By adding increasing amounts of {Ni}-BChl to the reconstitution mixture, a series of LH1 complexes was obtained that contain increasing levels of this efficient excitation trap . In contrast to the nearly unchanged absorption, the presence of {Ni}-BChl in LH1 markedly affects the emission properties . Incorporation of only 3.2 and 20% {Ni}-BChl reduces the emission by 50% and nearly 100%, respectively . The subnanosecond fluorescence kinetics of the complexes were monoexponential, with the lifetime identical to that of the native complex, and its amplitude decreasing in parallel with the steady-state fluorescence yield . Quantitative analysis of the data, based on a Poisson distribution of the modified pigment in the reconstituted complex, suggests that the presence of a single excitation trap per LH1 unit suffices for efficient emission quenching and that this unit contains 20 +/- 1 BChl molecules. FEMS Microbiol Ecol, 2001 Apr, 35(2), 137 - 144 Biological activity and colonization pattern of the bioluminescence-labeled plant growth-promoting bacterium Kluyvera ascorbata SUD165/26; Ma W et al.; Kluyvera ascorbata SUD165/26 is a spontaneous siderophore-overproducing mutant of K . ascorbata SUD165, which was previously isolated from nickel-contaminated soil and shown to significantly enhance plant growth in soil contaminated with high levels of heavy metals . To develop a better understanding of the functioning of K . ascorbata SUD165/26 in the environment, and to trace its distribution in the rhizosphere, isolates of this bacterium were labeled with either green fluorescent protein or luciferase . When the plant growth-promoting activities of the labeled strains were assayed and compared with the activities of the unlabeled strain, none of the monitored parameters had changed to any significant extent . When the spatial colonization patterns of the labeled bacteria on canola roots were determined after seed application, it was observed that the bacterium was tightly attached to the surface of both roots and seeds, and formed aggregates . The majority of the bacterial population inhabited the upper two thirds of the roots, with no bacteria detected around the root tips. Vet Microbiol, 2001 May 21, 80(2), 149 - 62 Rapid and accurate typing of Dichelobacter nodosus using PCR amplification and reverse dot-blot hybridisation; Zhou H et al.; Here we describe an approach to genotyping D . nodosus, based on variation in the fimbrial subunit gene (fimA), which uses polymerase chain reaction (PCR) amplification and hybridisation to immobilised oligonucleotides (PCR/oligotyping).The variable region of D . nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip . A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane . After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments hybridising were detected with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (SCIP) . The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D . nodosus fimA sequences that had a single base difference . DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing {Vet . Microbiol . 71 (2000) 113}, was assayed using the PCR/oligotyping technique . All types of D . nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure . However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D . nodosus . Further PCR amplification using type-specific primers, confirmed that these types of the bacterium were present in the footrot samples . These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples . In combination with a rapid DNA extraction protocol, D . nodosus strains present in a footrot sample can be accurately identified in less than 2 days. Epidemiol Infect, 2001 Feb, 126(1), 147 - 52 Presence of Legionellaceae in warm water supplies and typing of strains by polymerase chain reaction; Zietz B et al.; Outbreaks of Legionnaire's disease present a public health challenge especially because fatal outcomes still remain frequent . The aim of this study was to describe the abundance and epidemiology of Legionellaceae in the human-made environment . Water was sampled from hot-water taps in private and public buildings across the area of Gottingen, Germany, including distant suburbs . Following isolation, we used polymerase chain reaction in order to generate strain specific banding profiles of legionella isolates . In total, 70 buildings were examined . Of these 18 (26%) had the bacterium in at least one water sample . Legionella pneumophila serogroups 1, 4, 5 and 6 could be identified in the water samples . Most of the buildings were colonized solely by one distinct strain, as proven by PCR . In three cases equal patterns were found in separate buildings . There were two buildings in this study where isolates with different serogroups were found at the same time. Epidemiol Infect, 2001 Feb, 126(1), 129 - 33 Survival of Shiga toxin-producing Escherichia coli O157 in marine water and frequent detection of the Shiga toxin gene in marine water samples from an estuary port; Miyagi K et al.; Shiga toxin-producing Escherichia coli (STEC) O157 was investigated with respect to its halotolerance and whether it can survive in marine water . STEC O157 could multiply in a medium containing 5% NaCl and in sterilized marine water, and could survive in unsterilized marine water for at least 15 days . On the basis of these results, we postulated that STEC O157 may survive in natural marine water, and attempted to isolate the bacterium and Shiga toxin gene (stx) from marine water in Japan . The stx, comprising stx1 and stx2, was detected from marine water samples by PCR . STEC and other stx-positive bacteria, however, could not be isolated from these samples in this study . These results indicate that stx-positive bacteria may survive in marine water and suggest the necessity of a survey. Infect Immun, 2001 May, 69(5), 3502 - 6 Characterization of proteins in the outer membrane preparation of a murine pathogen, Helicobacter bilis; Ge Z et al.; Helicobacter bilis is a bacterial pathogen associated with multifocal hepatitis and inflammatory bowel disease in certain strains of mice . This bacterium colonizes the liver, bile, and lower intestine in mice and has also been isolated from a wide spectrum of laboratory animals . In this study, proteins present in the outer membrane preparation (OMP) of four H . bilis strains isolated from a mouse, a dog, a rat, and a gerbil were characterized and compared with that of Helicobacter pylori, a human gastric pathogen . All four H . bilis strains had similar OMP protein profiles that were distinct from those of H . pylori . Immunoblotting demonstrated that OMP proteins from H . bilis and H . pylori have little cross-reactivity, except for their flagellins . Nine major immunogenic polypeptides were present in the H . bilis OMPs . By using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five heat-modifiable proteins with molecular masses of 82, 66, 52, 47 and 37 kDa were identified . The N-terminal sequences of the 46- and 47-kDa OMP proteins had no homology with protein sequences available in public databases . These results indicate that H . bilis has a conserved, unique OMP protein profile that is distinct from those of H . pylori. Infect Immun, 2001 May, 69(5), 3323 - 34 P13, an integral membrane protein of Borrelia burgdorferi, is C-terminally processed and contains surface-exposed domains; Noppa L et al.; To elucidate antigens present on the bacterial surface of Borrelia burgdorferi sensu lato that may be involved in pathogenesis, we characterized a protein, P13, with an apparent molecular mass of 13 kDa . The protein was immunogenic and was expressed in large amounts during in vitro cultivation compared to other known antigens . An immunofluorescence assay, immunoelectron microscopy, and protease sensitivity assays indicated that P13 is surface exposed . The deduced sequence of the P13 peptide revealed a possible signal peptidase type I cleavage site, and computer analysis predicted that P13 is an integral membrane protein with three transmembrane-spanning domains . Mass spectrometry, in vitro translation, and N- and C-terminal amino acid sequencing analyses indicated that P13 was posttranslationally processed at both ends and modified by an unknown mechanism . Furthermore, p13 belongs to a gene family with five additional members in B . burgdorferi sensu stricto . The p13 gene is located on the linear chromosome of the bacterium, in contrast to five paralogous genes, which are located on extrachromosomal plasmids . The size of the p13 transcript was consistent with a monocistronic transcript . This new gene family may be involved in functions that are specific for this spirochete and its pathogenesis. Rev Sci Tech, 2001 Apr, 20(1), 219 - 51 Mycobacterium leprae and Mycobacterium lepraemurium infections in domestic and wild animals; Rojas-Espinosa O et al.; Mycobacterium leprae, the aetiological agent of leprosy in humans, gives rise to a chronic granulomatous disease that affects primarily the skin and peripheral nerves, and secondarily some internal organs such as the testis and the eye; viscera are seldom involved . Depending on host resistance, leprosy may present as a benign disease (tuberculoid leprosy) or as a malignant disease (lepromatous leprosy), with a spectrum of intermediate stages appearing between the two . Immunity against leprosy depends on the cell-mediated immunity of the host, and this is severely compromised in the malignant (lepromatous) form of leprosy . Although culture of M . leprae has never been achieved in artificial media, the bacterium may be grown in several experimental animals, including the armadillo, non-human primates, and to a certain extent, rodents . Naturally acquired leprosy has been reported in wild nine-banded armadillos (Dasypus novemcinctus) and in three species of non-human primates (chimpanzees {Pan troglodytes}, sooty mangabey monkeys {Cercocebus atys} and cynomolgus macaques {Macaca fascicularis}), thus qualifying leprosy as a zoonosis . Murine leprosy is a leprosy-like disease of rats and mice, caused by Mycobacterium lepraemurium . The disease affects primarily viscera and the skin, and very rarely peripheral nerves . Depending on the host strain, rodent leprosy may also evolve as 'lepromatous' or 'tuberculoid' leprosy, and strains of mouse that develop intermediate forms of the disease may exist . Growth of M . lepraemurium on conventional media for mycobacteria is not successful, but the bacterium has been cultured on an egg yolk-based medium . Naturally acquired murine leprosy has been observed in rats, mice and cats, but not in humans or any other species . Thus, in contrast to human leprosy, murine leprosy is not a zoonosis. Orv Hetil, 2001 Mar 11, 142(10), 509 - 14 {Determination of cagA, vacA genotypes of Helicobacter pylori with real-time PCR-method}; Ruzsovics A et al.; Presence of cagA gene of Helicobacter pylori (H . pylori) increases proliferation of stomach mucosa and it is an index of raised virulence of the bacteria . The vacA gene of H . pylori induces a serious inflammation of stomach . The purpose of this study was to determine cagA and vacA genotypes of H . pylori using real-time polymerase chain reaction (PCR) method with the double strain DNA-(dsDNA) binding SYBR Green I . dye . Results were compared with those of two immunohistochemical methods . 43 patients' paraffin embedded biopsy tissue samples were examined by histology, epidermal growth factor receptor (EGFR), proliferating cell nuclear antigen (PCNA) immunohistochemistry and melting curve analysis of real-time PCR using LightCycler instrument . Results of histology and real-time PCR from gastric biopsies correlated in 57% of cag acases and in 58% of vac cases . Significant difference was detected between normal and gastritis cases in the presence of cagA gene (p = 0.003) and between normal epithelial and intestinal metaplasia cases in the presence of vacA gene (p = 0.045) by investigation of association of histology and genotype of bacterium . Statistically significant difference (p = 0.02) was found between increased cell proliferation and the presence of gastritis . Significant correlation was found between the presence of cagA gene and EGFR expression in intestinal metaplasia cases (p = 0.0418) . Results underlie the statistics that infection with cagA positive H . pylori strain increases the cell proliferation on the stomach mucosa and raises the chance of development of intestinal metaplasia . Infection with vacA positive H . pylori inhibits the signal-transduction pathway of EGFR, which influences mechanisms of mucosa repair . The role of EGFR and H . pylori infection is yet unclear in intestinal metaplasia and cancer . The authors' method seem to be suitable for determination of genotypes of H . pylori. Proc Natl Acad Sci U S A, 2001 Apr 10, 98(8), 4681 - 6 Epub 2001 Apr 03. Proteomic analysis of the bacterial cell cycle; Grunenfelder B et al.; A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus . Approximately one-fourth (979) of the estimated C . crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns . Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups . Pattern analysis revealed that coexpression groups were functionally clustered . A total of 48 proteins were rapidly degraded in the course of one cell cycle . More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle . This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control. Biochemistry, 2001 Apr 10, 40(14), 4253 - 60 X-ray structure analyses of photosynthetic reaction center variants from Rhodobacter sphaeroides: structural changes induced by point mutations at position L209 modulate electron and proton transfer; Kuglstatter A et al.; The structures of the reaction center variants Pro L209 --> Tyr, Pro L209 --> Phe, and Pro L209 --> Glu from the photosynthetic purple bacterium Rhodobacter sphaeroides have been determined by X-ray crystallography to 2.6-2.8 A resolution . These variants were constructed to interrupt a chain of tightly bound water molecules that was assumed to facilitate proton transfer from the cytoplasm to the secondary quinone Q(B) {Baciou, L., and Michel, H . (1995) Biochemistry 34, 7967-7972} . However, the amino acid exchanges Pro L209 --> Tyr and Pro L209 --> Phe do not interrupt the water chain . Both aromatic side chains are oriented away from this water chain and interact with three surrounding polar side chains (Asp L213, Thr L226, and Glu H173) which are displaced by up to 2.6 A . The conformational changes induced by the bulky aromatic rings of Tyr L209 and Phe L209 lead to unexpected displacements of Q(B) compared to the wild-type protein . In the structure of the Pro L209 --> Tyr variant, Q(B) is shifted by approximately 4 A and is now located at a position similar to that reported for the wild-type reaction center after illumination {Stowell, M . H . B., et al . (1997) Science 276, 812-816} . In the Pro L209 --> Phe variant, the electron density map reveals an intermediate Q(B) position between the binding sites of the wild-type protein in the dark and the Pro L209 --> Tyr protein . In the Pro L209 --> Glu reaction center, the carboxylic side chain of Glu L209 is located within the water chain, and the binding site of Q(B) remains unchanged compared to the wild-type structure. J Biochem Mol Toxicol, 2001, 15(2), 67 - 75 Crystal structure-based studies of cytosolic sulfotransferase; Yoshinari K et al.; Sulfation is a widely observed biological reaction conserved from bacterium to human that plays a key role in various biological processes such as growth, development, and defense against adversities . Deficiencies due to the lack of the ubiquitous sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) are lethal in humans . A large group of enzymes called sulfotransferases catalyze the transfer reaction of sulfuryl group of PAPS to the acceptor group of numerous biochemical and xenochemical substrates . Four X-ray crystal structures of sulfotransferases have now been determined: cytosolic estrogen, hydroxysteroid, aryl sulfotransferases, and a sulfotransferase domain of the Golgi-membrane heparan sulfate N-deacetylase/N-sulfotransferase 1 . These have revealed the conserved core structure of the PAPS binding site, a common reaction mechanism, and some information concerning the substrate specificity . These crystal structures introduce a new era of the study of the sulfotransferases . Microbiology, 2001 Apr, 147(Pt 4), 1045 - 57 Defining the mycoplasma 'cytoskeleton': the protein composition of the Triton X-100 insoluble fraction of the bacterium Mycoplasma pneumoniae determined by 2-D gel electrophoresis and mass spectrometry; Regula JT et al.; After treating Mycoplasma pneumoniae cells with the nonionic detergent Triton X-100, an undefined, structured protein complex remains that is called the 'Triton X-100 insoluble fraction' or 'Triton shell' . By analogy with eukaryotic cells and supported by ultrastructural analyses it is supposed that this fraction contains the components of a bacterial cytoskeleton-like structure . In this study, the composition of the Triton X-100 insoluble fraction was defined by electron microscopic screening for possible structural elements, and by two-dimensional (2-D) gel electrophoresis and MS to identify the proteins present . Silver staining of 2-D gels revealed about 100 protein spots . By staining with colloidal Coomassie blue, about 50 protein spots were visualized, of which 41 were identified by determining the mass and partial sequence of tryptic peptides of individual proteins . The identified proteins belonged to several functional categories, mainly energy metabolism, translation and heat-shock response . In addition, lipoproteins were found and most of the proteins involved in cytadherence that were previously shown to be components of the Triton X-100 insoluble fraction . There were also 11 functionally unassigned proteins . Based on sequence-derived predictions, some of these might be potential candidates for structural components . Quantitatively, the most prevalent proteins were the heat-shock protein DnaK, elongation factor Tu and subunits alpha and beta of the pyruvate dehydrogenase complex (PdhA, PdhB), but definite conclusions regarding the composition of the observed structures can only be drawn after specific proteins are assigned to them, for example by immunocytochemistry. Microbiology, 2001 Apr, 147(Pt 4), 995 - 1006 Rapid phenotypic change and diversification of a soil bacterium during 1000 generations of experimental evolution; Riley MS et al.; Evolutionary pathways open to even relatively simple organisms, such as bacteria, may lead to complex and unpredictable phenotypic changes, both adaptive and non-adaptive . The evolutionary pathways taken by 18 populations of Ralstonia strain TFD41 while they evolved in defined environments for 1000 generations were examined . Twelve populations evolved in liquid media, while six others evolved on agar surfaces . Phenotypic analyses of these derived populations identified some changes that were consistent across all populations and others that differed among them . The evolved populations all exhibited morphological changes in their cell envelopes, including reductions of the capsule in each population and reduced prostheca-like surface structures in most populations . Mean cell length increased in most populations (in one case by more than fourfold), although a few populations evolved shorter cells . Carbon utilization profiles were variable among the evolved populations, but two distinct patterns were correlated with genetic markers introduced at the outset of the experiment . Fatty acid methyl ester composition was less variable across populations, but distinct patterns were correlated with the two physical environments . All 18 populations evolved greatly increased sensitivity to bile salts, and all but one had increased adhesion to sand; both patterns consistent with changes in the outer envelope . This phenotypic diversity contrasts with the fairly uniform increases in competitive fitness observed in all populations . This diversity may represent a set of equally probable adaptive solutions to the selective environment; it may also arise from the chance fixation of non-adaptive mutations that hitchhiked with a more limited set of beneficial mutations. J Biol Chem, 2001 Jun 22, 276(25), 22095 - 9 Epub 2001 Mar 29. Interaction of cytochrome bd with carbon monoxide at low and room temperatures: evidence that only a small fraction of heme b595 reacts with CO; Borisov VB et al.; Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant dinitrogen fixation requires cytochrome bd . This oxidase comprises two polypeptide subunits and three hemes, but no copper, and has been studied extensively . However, there remain apparently conflicting reports on the reactivity of the high spin heme b(595) with ligands . Using purified cytochrome bd, we show that absorption changes induced by CO photodissociation from the fully reduced cytochrome bd at low temperatures demonstrate binding of the ligand with heme b(595) . However, the magnitude of these changes corresponds to the reaction with CO of only about 5% of the heme . CO binding with a minor fraction of heme b(595) is also revealed at room temperature by time-resolved studies of CO recombination . The data resolve the apparent discrepancies between conclusions drawn from room and low temperature spectroscopic studies of the CO reaction with cytochrome bd . The results are consistent with the proposal that hemes b(595) and d form a diheme oxygen-reducing center with a binding capacity for a single exogenous ligand molecule that partitions between the hemes d and b(595) in accordance with their intrinsic affinities for the ligand . In this model, the affinity of heme b(595) for CO is about 20-fold lower than that of heme d. Appl Environ Microbiol, 2001 Apr, 67(4), 1783 - 7 Purification, characterization, and gene cloning of purine nucleosidase from Ochrobactrum anthropi; Ogawa J et al.; A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides . The nucleosidase was purified to homogeneity . The enzyme has a molecular weight of about 170,000 and consists of four identical subunits . It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K(m) values being 11.8 to 56.3 microM . The optimal activity temperature and pH were 50 degrees C and pH 4.5 to 6.5, respectively . Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme . The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K(i) and K(i)' values of 0.455 to 11.2 microM . Metal ion chelators inhibited activity, and the addition of Zn(2+) or Co(2+) restored activity . A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli . The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH(2)-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites . The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases. J Biol Chem, 2001 Jun 1, 276(22), 18984 - 91 Epub 2001 Mar 16. Activation of human prothrombin by arginine-specific cysteine proteinases (Gingipains R) from porphyromonas gingivalis; Imamura T et al.; The effect of 95- (HRgpA) and 50-kDa gingipain R (RgpB), arginine-specific cysteine proteinases from periodontopathogenic bacterium Porphyromonas gingivalis on human prothrombin activation was investigated . Each enzyme released thrombin from prothrombin in a dose- and time-dependent manner with the former enzyme, containing adhesion domains, being 17-fold more efficient than the single chain RgpB . A close correlation between the generation of fibrinogen clotting activity and amidolytic activity indicated that alpha-thrombin was produced by gingipains R, and this was confirmed by SDS-polyacrylamide gel electrophoresis, thrombin active site labeling, and amino-terminal sequence analysis of prothrombin digestion fragments . Significantly, the catalytic efficiency of HRgpA to generate thrombin (k(cat)/K(m) = 1.2 x 10(6) m(-)1 s(-)1) was 100-fold higher than that of RgpB (k(cat)/K(m) = 1.2 x 10(4) m(-)1 s(-)1) . The superior prothrombinase activity of HRgpA over RgpB correlates with the fact that only the former enzyme was able to clot plasma, and kinetic data indicate that prothrombin activation can occur in vivo . At P . gingivalis-infected periodontitis sites HRgpA may be involved in the direct production of thrombin and, therefore, in the generation of prostaglandins and interleukin-1, both have been found to be associated with the development and progression of the disease . Furthermore, by taking into account that the P . gingivalis bacterium has been immunolocalized in carotid atherosclerotic plaques at thrombus formation sites (Chiu, B . (1999) Am . Heart J . 138, S534-S536), our results indicate that bacterial proteinases may potentially participate in the pathogenesis of cardiovascular disease associated with periodontitis. J Mol Biol, 2001 Mar 30, 307(3), 951 - 63 Target recognition by EcoKI: the recognition domain is robust and restriction-deficiency commonly results from the proteolytic control of enzyme activity; O'Neill M et al.; We report a genetic and biochemical analysis of a target recognition domain (TRD) of EcoKI, a type I restriction and modification enzyme . The TRDs of type I R-M systems are within the specificity subunit (HsdS) and HsdS confers sequence specificity to a complex endowed with both restriction and modification activities . Random mutagenesis has revealed that most substitutions within the amino TRD of EcoKI, a region comprising 157 amino acid residues, have no detectable effect on the phenotype of the bacterium, even when the substitutions are non- conservative . The structure of the TRD appears to be robust . All but one of the six substitutions that confer a restriction-deficient, modification-deficient (r(-)m(-)) phenotype were found to be in the interval between residues 80 and 110, a region predicted by sequence comparisons to form part of the protein-DNA interface . Additional site-directed mutations affecting this interval commonly impair both restriction and modification . However, we show that an r(-) phenotype cannot be taken as evidence that the EcoKI complex lacks endonuclease activity; in response to even a slightly impaired modification efficiency, the endonuclease activity of EcoKI is destroyed by a process dependent upon the ClpXP protease.Enzymes from mutants with an r(-)m(-) phenotype commonly retain some sequence-specific activity; methylase activity can be detected on hemimethylated DNA substrates and residual endonuclease activity is implied whenever the viability of the r(-)m(-) bacterium is dependent on ClpXP . Conversely, the viability of ClpX(-) r(-)m(-) bacteria can be used as evidence for little, or no, endonuclease activity . Of 14 mutants with an r(-)m(-) phenotype, only six are viable in the absence of ClpXP . The significance of four of the six residues (G91, G105, F107 and G141) is enhanced by the finding that even conservative substitutions for these residues impair modification, thereby conferring an r(-)m(-) phenotype . Biosci Biotechnol Biochem, 2001 Jan, 65(1), 63 - 71 Purification and some properties of ubiquinol oxidase from obligately chemolithotrophic iron-oxidizing bacterium, Thiobacillus ferrooxidans NASF-1; Kamimura K et al.; Ubiquinol-oxidizing activity was detected in an acidophilic chemolithotrophic iron-oxidizing bacterium, T . ferrooxidans . The ubiquinol oxidase was purified 79-fold from plasma membranes of T . ferrooxidans NASF-1 cells . The purified oxidase is composed of two polypeptides with apparent molecular masses of 32,600 and 50,100 Da, as measured by gel electrophoresis in the presence of sodium dodecyl sulfate . The absorption spectrum of the reduced enzyme at room temperature showed big peaks at 530 and 563, and a small broad peak at 635 nm, indicating the involvement of cytochromes b and d . Characteristic peaks of cytochromes a and c were not observed in the spectrum at around 600 and 550 nm, respectively . This enzyme combined with CO, and its CO-reduced minus reduced difference spectrum showed peaks at 409 nm and 563 nm and a trough at 431 nm . These results indicated that the oxidase contained cytochrome b, but the involvement of cytochrome d was not clear . The enzyme catalyzed the oxidations of ubiquinol-2 and reduced N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride . The ubiquinol oxidase activity was activated by the addition of albumin and lecithin to the reaction mixture and inhibited by the respiratory inhibitors KCN, HQNO, NaN3, and antimycin A1, although the enzyme was relatively resistant to KCN, and the divalent cation, Zn2+, compared with ubiquinol oxidases of E . coli. Biosci Biotechnol Biochem, 2001 Jan, 65(1), 133 - 42 Purification and characterization of bifunctional alginate lyase from Alteromonas sp . strain no . 272 and its action on saturated oligomeric substrates; Iwamoto Y et al.; A marine bacterium (strain No . 272) isolated from sea mud in Omura Bay produced an alginate lyase and was classified as an Alteromonas species . The enzyme was purified from the culture medium of the bacterium by DEAE-Cellulofine, Sephadex G-100 gel chromatography to an electrophoretically homogeneous state in the presence and absence of SDS . The molecular mass of the enzyme was 23 and 33.9 kDa on Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis, respectively, with an isoelectric point of 3.8 . The predominant secondary structure of the enzyme was found to be most likely beta-structure by circular dichroism . The enzyme was most active at pH 7.5-8.0 and stable around pH 5-11 . The enzyme was more labile in Tris-HCI buffer (pH 7.0) to heat treatment, than in phosphate buffer (pH 7.0) . No of metal ions significantly affected the enzyme activity . The enzyme acted on sodium alginate in an endo-type manner and on two components of alginate, poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate, as judged by routine ultraviolet assay (235 nm) and circular dichroic spectral changes of the substrates . However, the coexisting poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate apparently interacted with the enzyme in a competitive manner . Although the enzyme depolymerized alginate in an endo-type, it did not act on trimeric guluronate and mannuronate, but on the tetramers or more . The kinetic analyses showed that kcat/Km for each oligomer was larger for the guluronate oligomers than for the mannuronate ones, and that the subsite structure of the enzyme most likely consisted of six binding sites from the intrinsic reaction rate constant (kint) and intrinsic substrate binding constant (Kint). Electrophoresis, 2000 Nov, 21(17), 3833 - 42 Proteome analysis demonstrates complex replicon and luteolin interactions in pSyma-cured derivatives of Sinorhizobium meliloti strain 2011; Chen H et al.; Sinorhizobium meliloti was studied by proteomic analysis to investigate the contribution made by plasmid-encoded functions on the intracellular regulation of this bacterium . Protein profiles of strain 2011 were compared with those from its mutant strains which were either cured of their pRme2011a (also called pSyma) plasmid (strain 818), or contained an extensive deletion of this plasmid (strain SmA146) . Plasmid pSyma contains the nodulation and nitrogen fixation genes and is 1.4 Mbp with an estimated coding potential of 1,400 proteins . However, under the growth conditions used we could detect 60 differences between the parent strain and its pSyma-cured derivative, strain 818 . While the majority of these differences were due to regulatory changes, such as up- and downregulation, some proteins were totally missing in some strains . These 60 proteins were classified into 21 subgroups, A to U, based on their measured protein levels when the cells were grown in the presence or absence of luteolin . Comparisons were made between the different strains to assess the possible interactions of the different proteins of the subgroups and plasmid pSyma . These results suggest that pSyma has a role in the regulation of the expression of genes from the other replicons (3.5 Mbp chromosome and the 1.7 Mbp pSymB plasmid) present in the S . meliloti cells . Proteome analysis provides a sensitive tool to examine the functional organisation of the S . meliloti genome and the intracellular gene interactions between replicons and will provide a powerful analytical tool to complement the genome sequencing of strain 1021. Electrophoresis, 2000 Nov, 21(17), 3765 - 80 Towards a two-dimensional proteome map of Mycoplasma pneumoniae; Regula JT et al.; A Proteome map of the bacterium Mycoplasma pneumoniae was constructed using two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry (MS) . M . pneumoniae is a human pathogen with a known genome sequence of 816 kbp coding for only 688 open reading frames, and is therefore an ideal model system to explore the scope and limits of the current technology . The soluble protein content of this bacterium grown under standard laboratory conditions was separated by 1-D or 2-D gel electrophoresis applying various pH gradients, different acrylamide concentrations and buffer systems . Proteins were identified using liquid chromatography-electrospray ionization ion trap and matrix-assisted laser desorption/ionization-MS . Mass spectrometric protein identification was supported and controlled using N-terminal sequencing and immunological methods . So far, proteins from about 350 spots were characterized with MS by determining the molecular weights and partial sequences of their tryptic peptides . Comparing these experimental data with the DNA sequence-derived predictions it was possible to assign these 350 proteins to 224 genes . The importance of proteomics for genome analysis was shown by the identification of four proteins, not annotated in the original publication . Although the proteome map is still incomplete, it is already a useful reference for comparative analyses of M . pneumoniae cells grown under modified conditions. Electrophoresis, 2000 Nov, 21(17), 3740 - 56 Towards the proteome of Mycobacterium tuberculosis; Rosenkrands I et al.; Human tuberculosis is caused by the intracellular pathogen Mycobacterium tuberculosis . Sequencing of the genome of M . tuberculosis strain H37Rv has predicted 3924 open reading frames, and enabled identification of proteins from this bacterium by peptide mass fingerprinting . Extracellular proteins from the culture medium and proteins in cellular extracts were examined by two-dimensional gel electrophoresis using immobilized pH gradient technology . By mass spectrometry and immunodetection, 49 culture filtrate proteins and 118 lysate proteins were identified, 83 of which were novel . To date, 288 proteins have been identified in M . tuberculosis proteome studies, and a list is presented which includes all identified proteins (available at The information obtained from the M . tuberculosis proteome so far is discussed in relation to the information obtained from the complete genome sequence. Carbohydr Res, 2001 Feb 15, 330(3), 325 - 33 Structure and conformation of a novel genetically engineered polysaccharide P2; Colquhoun IJ et al.; A new exocellular polysaccharide (P2) has been produced by the manipulation of a glycosyl transferase gene (aceP) involved in the biosynthesis of the polysaccharide acetan by the bacterium Acetobacter xylinum strain CKE5 . The P2 polysaccharide has been studied by methylation analysis, reductive cleavage, and 1H and 13C NMR spectroscopy . The data are consistent with the structure predicted when the aceP gene is deactivated: {Molecular structure: see text} . The effect of cooling on proton NMR line width indicates a coil-helix transition in P2 at about 70 degrees C. Dig Dis Sci, 2001 Jan, 46(1), 46 - 53 Effect of Helicobacter pylori infection on gastric emptying and gastrointestinal hormones in dyspeptic and healthy subjects; Chiloiro M et al.; There is no general agreement as regards the effect of Helicobacter pylori infection on gastric emptying in patients with functional dyspepsia . Food releases several gastrointestinal hormones, and some of these are known to contribute to the regulation of gastric emptying . The aim of this study was to investigate the influence of H . pylori on gastric emptying in dyspeptic and healthy subjects and to verify whether different hormone secretion patterns are affected by the presence of the bacterium . Twenty-seven patients affected by functional dyspepsia and 30 asymptomatic healthy subjects entered the study . H . pylori presence was assessed in controls by IgG antibodies to H . pylori and {13C} urea breath test, and that in patients by Warthin-Starry stain on gastric biopsies . After ingesting a standard solid-liquid meal, an ultrasound examination of gastric emptying was performed . Plasma concentrations of gastrin, cholecystokinin, and pancreatic polypeptide were measured in the fasting and postprandial period for 4 hours . The incidence of H . pylori infection was not higher in functional dyspepsia patients than in controls . As regards gastric emptying, no difference was detected between patients and controls with and without H . pylori infection . On the contrary, the presence of H . pylori infection determined alterations in gastrin levels, which were higher in controls than in patients . Basal CCK levels were higher in the H . pylori-negative patients than H . pylori-positive patients and controls . In conclusion, H . pylori infection seems not to cause alterations in gastric emptying, but rather alterations in gastrin levels . In contrast, the altered levels of CCK account for its involvement in the pathophysiology of H . pylori-negative dyspepsia. Curr Microbiol, 2001 Mar, 42(3), 151 - 4 Adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to crystalline Fe(III) oxides; Das A et al.; Shewanella alga BrY adhesion to hydrous ferric oxide, goethite, and hematite was examined . Adhesion to each oxide followed the Langmuir adsorption model . No correlation between adhesion and Fe(III) oxide surface area or crystallinity was observed . Zeta potential measurements suggested that electrostatic interactions do not influence S . alga BrY adhesion to these minerals . Cell adhesion does not appear to explain the recalcitrance of crystalline Fe(III) oxides to bacterial reduction. Hepatogastroenterology, 2001 Jan-Feb, 48(37), 104 - 6 Synchronous gastric adenocarcinoma and MALT lymphoma in a patient with H . pylori infection . Could the two neoplasms share a common pathogenesis? Cammarota G, Larocca LM, D'Ugo D, Persiani R, Cianci R, Nocente R, Picciocchi A, Gasbarrini G. Low-grade primary MALT (mucosa-associated lymphoid tissue) lymphoma of the stomach is a neoplasm with an indolent course and a good prognosis . Patients with this type of neoplasm seem to have a higher risk for other neoplasms . Of interest is the association of gastric MALT lymphoma with gastric adenocarcinoma of intestinal type . We report the case of a patient, with a history of H . pylori-related gastritis, in whom a diagnosis of synchronous gastric adenocarcinoma of intestinal type and low-grade MALT lymphoma, occurring as collision tumors, was made . The stage procedures confirmed the presence of a locally advanced gastric tumor staged as T3 N1 . The patient underwent two cycles