Microorganisms

FEMS Microbiol Rev, 2001 Dec, 25(5), 513 - 29
Survival of enteric bacteria in seawater; Rozen Y et al.; Enteric bacteria exposed to the marine environment simultaneously encounter a variety of abiotic and biotic challenges . Among the former, light appears to be critical in affecting seawater survival; previous growth history plays a major part in preadaptation of the cells, and stationary phase cells are generally more resistant than exponentially growing ones . Predation, mostly by protozoa, is probably the most significant biotic factor . Using Escherichia coli as a model, a surprisingly small number of genes was found that, when mutated, significantly affect seawater sensitivity of this bacterium . Most prominent among those is rpoS, which was also dominant among genes induced upon transfer to seawater.

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 15056 - 61 Epub 2001 Dec 11.
Recombination and mutation during long-term gastric colonization by Helicobacter pylori: estimates of clock rates, recombination size, and minimal age; Falush D et al.; The bacterium Helicobacter pylori colonizes the gastric mucosa of half of the human population, resulting in chronic gastritis, ulcers, and cancer . We sequenced ten gene fragments from pairs of strains isolated sequentially at a mean interval of 1.8 years from 26 individuals . Several isolates had acquired small mosaic segments from other H . pylori or point mutations . The maximal mutation rate, the import size, and the frequency of recombination were calculated by using a Bayesian model . The calculations indicate that the last common ancestor of H . pylori existed at least 2,500-11,000 years ago . Imported mosaics have a median size of 417 bp, much smaller than for other bacteria, and recombination occurs frequently (60 imports spanning 25,000 bp per genome per year) . Thus, the panmictic population structure of H . pylori results from very frequent recombination during mixed colonization by unrelated strains.

J Biol Chem, 2002 Feb 22, 277(8), 5785 - 95 Epub 2001 Dec 07.
Phenotypic variation in molecular mimicry between Helicobacter pylori lipopolysaccharides and human gastric epithelial cell surface glycoforms . Acid-induced phase variation in Lewis(x) and Lewis(y) expression by H . Pylori lipopolysaccharides; Moran AP et al.; Helicobacter pylori is an important gastroduodenal pathogen of humans whose survival in the gastric environment below pH 4 is dependent on bacterial production of urease, whereas above pH 4 urease-independent mechanisms are involved in survival, but that remain to be elucidated fully . Previous structural investigations on the lipopolysaccharides (LPSs) of H . pylori have shown that the majority of these surface glycolipids express partially fucosylated, glucosylated, or galactosylated N-acetyllactosamine (LacNAc) O-polysaccharide chains containing Lewis(x) (Le(x)) and/or Lewis(y) (Le(y)), although some strains also express type 1 determinants, Lewis(a), Lewis(b), and H-1 antigen . In this study, we investigated acid-induced changes in the structure and composition of LPS and cellular lipids of the genome-sequenced strain, H . pylori 26695 . When grown in liquid medium at pH 7, the O-chain consisted of a type 2 LacNAc polysaccharide, which was glycosylated with alpha-1-fucose at O-3 of the majority of N-acetylglucosamine residues forming Le(x) units, including chain termination by a Le(x) unit . However, growth in liquid medium at pH 5 resulted in production of a more complex O-chain whose backbone of type 2 LacNAc units was partially glycosylated with alpha L-fucose, thus forming Le(x), whereas the majority of the nonfucosylated N-acetylglucosamine residues were substituted at O-6 by alpha-D-galactose residues, and the chain was terminated by a Le(y) unit . In contrast, detailed chemical analysis of the core and lipid A components of LPS and analysis of cellular lipids did not show significant differences between H . pylori 26695 grown at pH 5 and 7 . Although putative molecular mechanisms affecting Le(x) and Le(y) expression have been investigated previously, this is the first report identifying an environmental trigger inducing phase variation of Le(x) and Le(y) in H . pylori that can aid adaptation of the bacterium to its ecological niche.

Microbiology, 2001 Dec, 147(Pt 12), 3215 - 29
Twitching motility of Ralstonia solanacearum requires a type IV pilus system; Liu H et al.; Twitching motility is a form of bacterial translocation over firm surfaces that requires retractile type IV pili . Microscopic colonies of Ralstonia solanacearum strains AW1, K60 and GMI1000 growing on the surface of a rich medium solidified with 1.6% agar appeared to exhibit twitching motility, because early on they divided into motile 'rafts' of cells and later developed protruding 'spearheads' at their margins . Individual motile bacteria were observed only when they were embedded within masses of other cells . Varying degrees of motility were observed for 33 of 35 strains of R . solanacearum in a selected, diverse collection . Timing was more important than culture conditions for observing motility, because by the time wild-type colonies were easily visible by eye (about 48 h) this activity ceased and the spearheads were obscured by continued bacterial multiplication . In contrast, inactivation of PhcA, a transcriptional regulator that is essential for R . solanacearum to cause plant disease, resulted in colonies that continued to expand for at least several additional days . Multiple strains with mutations in regulatory genes important for virulence were tested, but all exhibited wild-type motility . Many of the genes required for production of functional type IV pili, and hence for twitching motility, are conserved among unrelated bacteria, and pilD, pilQ and pilT orthologues were identified in R . solanacearum . Colonies of R . solanacearum pilQ and pilT mutants did not develop spearheads or rafts, confirming that the movement of cells that had been observed was due to twitching motility . Compared to the wild-type parents, both pilQ and pilT mutants caused slower and less severe wilting on susceptible tomato plants . This is the first report of twitching motility by a phytopathogenic bacterium, and the first example where type IV pili appear to contribute significantly to plant pathogenesis.

Vaccine, 2001 Dec 12, 20(5-6), 979 - 88
Immunization with a portion of rickettsial outer membrane protein A stimulates protective immunity against spotted fever rickettsiosis; Crocquet-Valdes PA et al.; Two approaches for presentation of a part of the rickettsial outer membrane protein A (OmpA) of Rickettsia rickettsii, namely (1) recombinant Mycobacterium vaccae (rMV) or (2) recombinant DNA vaccine, stimulated protective immunity against a lethal challenge with the closely related bacterium, R . conorii, in mice . After primary immunization with rMV and booster immunization with homologous recombinant protein, 67 and 55% of mice were protected against challenge in two experiments . DNA vaccination with booster recombinant protein immunization protected six out of eight animals from a lethal challenge . Production of IFN-gamma by antigen-exposed T-lymphocytes of DNA vaccine recipients indicated that cellular immunity had been stimulated.

Surv Ophthalmol, 2001 Nov-Dec, 46(3), 209 - 33
Immunopathology of the noninfectious posterior and intermediate uveitides; Boyd SR et al.; The posterior and intermediate uveitides share an underlying immune etiology; however, they can be clinically and immunopathologically distinguished . Although the initiating stimuli for posterior and intermediate uveities are not known, it is believed that an exogenous agent (such as a bacterium or a virus) or an endogenous molecule may induce disease . In either case, T-helper lymphocytes in conjunction with human leukocyte antigens are likely to be involved . This review examines the epidemiology, histology, immunopathology, and theories of pathogenesis of several posterior and intermediate uveitides, including sympathetic ophthalmia, Vogt-Koyanagi-Harada syndrome, Behcet's disease, sarcoidosis, intermediate uveitis, white dot syndromes, and birdshot retinochoroidopathy.

J Clin Periodontol, 2001 Dec, 28(12), 1163 - 71
Distribution of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in an Australian population; Hamlet SM et al.; BACKGROUND, AIM: The present study describes (i) the natural distribution of the three putative periodontopathogens Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in an Australian population and (ii) the relationship between these organisms, pocket depths and supragingival plaque scores . METHODS: Subgingival plaque was collected from the shallowest and deepest probing site in each sextant of the dentition . In total, 6030 subgingival plaque samples were collected from 504 subjects . An ELISA utilising pathogen-specific monoclonal antibodies was used to quantitate bacterial numbers . RESULTS:: A . actinomycetemcomitans was the most frequently detected organism (22.8% of subjects) followed by P . gingivalis and P . intermedia (14.7% and 9.5% of subjects respectively) . The majority of infected subjects (83%) were colonised by a single species of organism . A . actinomycetemcomitans presence was over-represented in the youngest age group but under-represented in the older age groups . Conversely, P . gingivalis and P . intermedia presence was under-represented in the youngest age group but over-represented in the older age groups . Differing trends in the distribution of these bacteria were observed between subjects depending upon the site of the infection or whether a single or mixed infection was present; however, these differences did not reach significance . Bacterial presence was strongly associated with pocket depth for both A . actinomycetemcomitans and P . gingivalis . For A . actinomycetemcomitans, the odds of a site containing this bacterium decrease with deeper pockets . In contrast, for P . gingivalis the odds of a site being positive are almost six times greater for pockets >3 mm than for pockets < or =3 mm . These odds increase further to 15.3 for pockets deeper than 5 mm . The odds of a site being P . intermedia positive were marginally greater (1.16) for pockets deeper than 3 mm . CONCLUSIONS: This cross-sectional study in a volunteer Australian population, demonstrated recognised periodontal pathogens occur as part of the flora of the subgingival plaque . Prospective longitudinal studies are needed to examine the positive relationship between pocket depth and pathogen presence with periodontal disease initiation and/or progression.

Eur J Biochem, 2001 Dec, 268(24), 6559 - 68
Cloning, overproduction and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus; De Smet L et al.; The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over-expressing the BCCP structural gene . BCCP from Rb . capsulatus oxidizes the Rhodobacter cytochrome c2 and reduces hydrogen peroxide, probably functioning as a detoxification mechanism . The enzyme binds two haem c groups covalently . The gene encoding BCCP from Rb . capsulatus was cloned through the construction of a 7-kb subgenomic clone . In comparison with the protein sequence, the sequence deduced from the gene has a 21-amino-acid N-terminal extension with the characteristics of a signal peptide . The purified recombinant enzyme showed the same physico-chemical properties as the native enzyme . Spectrophotometric titration established the presence of a high-potential (Em=+270 mV) and a low-potential haem (between -190 mV and -310 mV) as found in other BCCPs . The enzyme was isolated in the fully oxidized but inactive form . It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme . This activation is associated with the formation of a high-spin state at the low-potential haem . BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c2; the catalytic activities ('turnover number') are 85 800 min(-1) and 63 600 min(-1), respectively . These activities are the highest ever found for a BCCP.

Clin Exp Immunol, 2001 Dec, 126(3), 474 - 81
IFN-gamma synergizes with TNF-alpha but not with viable H . pylori in up-regulating CXC chemokine secretion in gastric epithelial cells; Kraft M et al.; Helicobacter pylori colonizes the gastric epithelial surface and induces epithelial cells to increase production of the neutrophil attractant IL-8 . Little is known about the role of the gastric epithelium in regulating mucosal T cell trafficking . We therefore characterized constitutive and regulated epithelial expression of the CXC chemokines IP-10, I-TAC and Mig, which specifically attract CXCR3 expressing CD4(+) T cells . Human gastric epithelial cell lines (AGS, Kato III, NCI) were used to characterize the constitutive and regulated expression of three CXC chemokines in response to IFN-gamma, TNF-alpha and different H . pylori preparations . Chemokine mRNA and protein production were measured by RT-PCR and ELISA . Gastric epithelial cells constitutively expressed mRNA for IP-10, Mig and I-TAC . IFN-gamma in combination with TNF-alpha strongly induced secretion of those chemokines . Soluble or membranous fractions of H . pylori significantly inhibited IFN-gamma/TNF-alpha induced epithelial cell IP-10 and Mig production . Gastric epithelial cells may contribute to mucosal T cell trafficking . The capacity of H . pylori products to inhibit IP-10 and Mig secretion may explain, at least in part, the failure to induce protective immunity against this bacterium and the ability of H . pylori to affect the presentation of the local inflammation.

Biochem J, 2001 Dec 15, 360(Pt 3), 675 - 81
Bacterial peptide methionine sulphoxide reductase: co-induction with glutathione S-transferase during chemical stress conditions; Tamburro A et al.; Peptide methionine sulphoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins, while the glutathione S-transferases (GSTs) are a major family of detoxification enzymes . A gene homologous to MsrA was identified in a chromosomal fragment from the bacterium Ochrobactrum anthropi, and this gene is located just downstream of a GST gene identified previously (OaGST) {Favaloro, Tamburro, Angelucci, De Luca, Melino, Di Ilio and Rotilio (1998) Biochem . J . 335, 573-579} . This raises the question of whether the products of these two genes may be involved in a common cellular protection function . To test this hypothesis, the hypothetical MsrA protein has been overexpressed in Escherichia coli as a functional 51 kDa GST fusion protein . Following cleavage with thrombin and purification, the soluble 24 kDa protein showed MsrA activity with N-acetylmethionine sulphoxide as substrate, as well as with other sulphoxide compounds . Therefore polyclonal antibodies were raised against the recombinant protein, and the modulation of MsrA in this bacterium, grown in the presence of different stimulants simulating several stress conditions, was investigated . The level of expression of MsrA was detected both by measuring the mRNA level and by immunoblotting experiments, in addition to measuring its catalytic activity . MsrA is a constitutive enzyme which is also inducible by chemical stress involving phenolic compounds such as phenol and 4-chlorophenol . Recently we reported that the GST of this bacterium, like MsrA, is only modulated by toxic chemical compounds {Favaloro, Tamburro, Trofino, Bologna, Rotilio and Heipieper (2000) Biochem . J . 346, 553-559}; therefore this is the first indication of a co-induction of the MsrA and GST enzymes during chemical stress.

BMC Evol Biol . 2001;1(1):10 . Epub 2001 Nov 09.
Wolbachia: evolutionary novelty in a rickettsial bacteria; Anderson CL et al.; BACKGROUND: Although closely related, the alpha-proteobacteria Wolbachia and the Rickettsiaceae (Rickettsia and Ehrlichia), employ different evolutionary life history strategies . Wolbachia are obligate endocellular symbionts that infect an extraordinary host range and, in contrast to the infectious and pathogenic Rickettsia and Ehrlichia, profoundly influence host reproductive biology . RESULTS: Phylogenies of the Rickettsia, Ehrlichia, and Wolbachia were independently inferred from 16S rDNA sequences and GroEL amino acid sequences . Topologies inferred from both sets of sequence data were consistent with one another, and both indicate the genus Wolbachia shared a common ancestor most recently with Ehrlichia . These two genera are a sister group to the genus Rickettsia . Mapping biological properties onto this phylogeny reveals that manipulation of host reproduction, characteristic of Wolbachia strains, is a derived characteristic . This evolutionary novelty is accompanied by the loss of the ability to infect vertebrate hosts . CONCLUSIONS: Because of the contrasting transmission strategies employed by each, Wolbachia is expected to maximize efficiency of vertical transmission, while Ehrlichia and Rickettsia will optimize horizontal transfer of infection . Wolbachia manipulation of host reproduction could thus be viewed as strategy employed by this bacterium to foster its own propagation via vertical transmission.

Vet Pathol, 2001 Nov, 38(6), 667 - 78
Helicobacter pylori-induced gastritis in experimentally infected conventional piglets; Poutahidis T et al.; A conventional nonmutant animal that could be experimentally infected with Helicobacter pylori isolates would be a useful animal model for human H . pylori-associated gastritis . Gnotobiotic and barrier-born pigs are susceptible to H . pylori infection, but attempts to infect conventional pigs with this bacterium have been unsuccessful . In the present study, a litter of eight 20-day-old crossbreed piglets were purchased from a commercial farm . Six of them were orally challenged two to five times at different ages, between 29 and 49 days, with doses of H . pylori inoculum containing approximately 10(9) bacterial cells . Two animals served as controls . The inoculation program began 2 days postweaning when the piglets were 29 days of age . Prior to every inoculation, the piglets were fasted and pretreated with cimetidine, and prior to the first and second inoculation each piglet also was pretreated with dexamethasone . The challenged piglets were euthanasized between 36 and 76 days of age . H . pylori colonized all six inoculated piglets . The pathology of the experimentally induced gastritis was examined macroscopically and by light and electron microscopy . H . pylori induced a severe lymphocytic gastritis in the conventional piglets and reproduced the large majority of the pathologic features of the human disease . Therefore, the conventional piglet represents a promising new model for study of the various pathogenic mechanisms involved in the development of lesions of the human H . pylori-associated gastritis.

FEMS Microbiol Lett, 2001 Nov 13, 204(2), 347 - 53
Zeaxanthin and menaquinone-7 biosynthesis in Sphingobacterium multivorum via the methylerythritol phosphate pathway; Rosa-Putra S et al.; Feeding of {1-(13)C}glucose, {U-(13)C(6)}glucose, {3-(13)C}alanine and {1-(13)C}acetate to Sphingobacterium multivorum showed that this bacterium utilizes the methylerythritol phosphate pathway for the biosynthesis of menaquinone-7 and zeaxanthin, a carotenoid of industrial importance . Differential incorporation of the labeled precursors gave some insight into the preferred carbon sources involved in isoprenoid biosynthesis.

FEMS Microbiol Lett, 2001 Nov 13, 204(2), 341 - 5
Lithoautotrophic growth of the freshwater strain Beggiatoa D-402 and energy conservation in a homogeneous culture under microoxic conditions; Grabovich MY et al.; The freshwater filamentous bacterium Beggiatoa D-402 was shown to grow lithoautotrophically in a homogeneous culture under microoxic conditions only, the growth yield being the highest at 0.1 mg O(2) l(-1) . High activities of the Calvin cycle key enzymes and of the dissimilatory path thiosulfate oxidation enzymes were found in the bacterial cells . The rate of CO(2) fixation above 112 nmol min(-1) (mg protein)(-1), an about 90% increase in the protein carbon at the expense of CO(2) carbon and an increase in the molar yield up to 12 mg dry weight (mmol oxidized thiosulfate)(-1) indicate the bacterial growth was autotrophic . Thiosulfate was oxidized by the strain almost completely into sulfate . The metabolically useful energy was conserved by oxidative phosphorylation that was coupled to oxidation of sulfur compounds . The bacterial membranes were found to contain CO-binding cytochromes b and two cytochromes c with M(r) 23 and 26 kDa, the terminal part of the respiratory chain containing presumably a cbb(3)-type oxidase . A cytochrome c with M(r) 12 kDa was detected in the soluble fraction.

FEMS Microbiol Lett, 2001 Nov 13, 204(2), 215 - 21
Super-channel in bacteria: function and structure of the macromolecule import system mediated by a pit-dependent ABC transporter; Mishima Y et al.; In a soil isolate, Sphingomonas sp . A1, the transport of a macromolecule (alginate: 27 kDa) is mediated by a pit-dependent ATP-binding cassette (ABC) transporter . The transporter is different from other ABC transporters so far analyzed in that its function is dependent on a pit, a mouth-like organ formed on the cell surface only when cells are compelled to assimilate macromolecules, and in that it allows direct import of macromolecules into cells . The ABC transporter coupled with the pit, which functions as a funnel and/or concentrator of macromolecules to be imported, was designated the 'super-channel', and in this review, we discuss the three-dimensional structure and specific function of the 'super-channel' for macromolecule import found for the first time in a bacterium.

Trends Microbiol, 2001 Dec, 9(12), 597 - 605
Mycobacterial persistence: adaptation to a changing environment; Honer zu Bentrup K et al.; Mycobacterium tuberculosis is a bacterial pathogen that can persist within an infected individual for extended periods of time without causing overt, clinical disease, in a state normally referred to as latent or chronic tuberculosis . Although the replicative state of the bacterium during this period is a matter of some conjecture, recent developments have indicated that the bacterium requires the regulated expression of a set of genes and metabolic pathways to maintain a persistent infection in an immunocompetent host . The characterization of these gene products and their role in bacterial metabolism and physiology is starting to provide insights into the mechanisms that M . tuberculosis has evolved to adopt its highly successful mode of pathogenicity.

FEMS Microbiol Lett, 2001 Nov 27, 205(1), 77 - 81
Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis; Kuoppa K et al.; Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages . Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202 . A promoter library was created in F . tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214 . Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro . The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F . tularensis genes that are expressed during infection.

FEMS Microbiol Lett, 2001 Nov 27, 205(1), 37 - 41
Potassium uptake and retention by Oceanomonas baumannii at low water activity in the presence of phenol; Brown GR et al.; Oceanomonas baumannii(T) (ATCC 700832) is a halotolerant bacterium capable of degrading phenol, which requires potassium in order for turgor growth to occur in minimal medium containing 5% NaCl (w/v) . However, at this salinity growth can be inhibited by reduced potassium concentrations . The affinity for potassium (K(S)) was determined to be 219 microM and 408 microM for cultures utilising phenol and succinate respectively as the sole carbon source for growth . Rubidium but not caesium could substitute for potassium in alleviating growth inhibition due to potassium limitation . The effect of elevated phenol on potassium retention was studied, and it was shown that contrary to expectations, as external phenol concentration was increased the levels of intracellular potassium were significantly elevated . This observation correlated with changes in the cytoplasmic membrane, particularly the increase in the saturated:unsaturated fatty acid ratio from 0.47 to 1.44, and the decrease in the zwitterionic:anionic phospholipid ratio from 2.23 to 1.22 . Both these changes promote membrane bilayer configurations and increase lipid ordering of the membrane reducing its permeability and inhibiting cation efflux.

Nat Genet, 2001 Dec, 29(4), 375 - 6
Conjugation between bacterial and mammalian cells; Waters VL; Bacterial conjugation, in which DNA is transferred from one bacterium to another, was first reported in 1946 and found to be mediated by the F factor . Although the F and RK2/RP4 prototypic plasmids can mediate the transfer of DNA from bacteria to yeast, there has been no evidence of classical bacterial conjugation to higher eukaryotes . Here, I present evidence of such transfer, using Escherichia coli, the RK2 plasmid system and Chinese hamster ovary CHO K1 cells.

Biochemistry, 2001 Dec 4, 40(48), 14547 - 56
Controlling the functionality of cytochrome c(1) redox potentials in the Rhodobacter capsulatus bc(1) complex through disulfide anchoring of a loop and a beta-branched amino acid near the heme-ligating methionine; Osyczka A et al.; The cytochrome c(1) subunit of the ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) contains a single heme group covalently attached to the polypeptide via thioether bonds of two conserved cysteine residues . In the photosynthetic bacterium Rhodobacter (Rba.) capsulatus, cytochrome c(1) contains two additional cysteines, C144 and C167 . Site-directed mutagenesis reveals a disulfide bond (rare in monoheme c-type cytochromes) anchoring C144 to C167, which is in the middle of an 18 amino acid loop that is present in some bacterial cytochromes c(1) but absent in higher organisms . Both single and double Cys to Ala substitutions drastically lower the +320 mV redox potential of the native form to below 0 mV, yielding nonfunctional cytochrome bc(1) . In sharp contrast to the native protein, mutant cytochrome c(1) binds carbon monoxide (CO) in the reduced form, indicating an opening of the heme environment that is correlated with the drop in potential . In revertants, loss of the disulfide bond is remediated uniquely by insertion of a beta-branched amino acid two residues away from the heme-ligating methionine 183, identifying the pattern betaXM, naturally common in many other high-potential cytochromes c . Despite the unrepaired disulfide bond, the betaXM revertants are no longer vulnerable to CO binding and restore function by raising the redox potential to +227 mV, which is remarkably close to the value of the betaXM containing but loop-free mitochondrial cytochrome c(1) . The disulfide anchored loop and betaXM motifs appear to be two independent but nonadditive strategies to control the integrity of the heme-binding pocket and raise cytochrome c midpoint potentials.

Biochemistry, 2001 Dec 4, 40(48), 14509 - 17
Crystal structure of ATP sulfurylase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila; Beynon JD et al.; In sulfur chemolithotrophic bacteria, the enzyme ATP sulfurylase functions to produce ATP and inorganic sulfate from APS and inorganic pyrophosphate, which is the final step in the biological oxidation of hydrogen sulfide to sulfate . The giant tubeworm, Riftia pachyptila, which lives near hydrothermal vents on the ocean floor, harbors a sulfur chemolithotroph as an endosymbiont in its trophosome tissue . This yet-to-be-named bacterium was found to contain high levels of ATP sulfurylase that may provide a substantial fraction of the organisms ATP . We present here, the crystal structure of ATP sulfurylase from this bacterium at 1.7 A resolution . As predicted from sequence homology, the enzyme folds into distinct N-terminal and catalytic domains, but lacks the APS kinase-like C-terminal domain that is present in fungal ATP sulfurylase . The enzyme crystallizes as a dimer with one subunit in the crystallographic asymmetric unit . Many buried solvent molecules mediate subunit contacts at the interface . Despite the high concentration of sulfate needed for crystallization, no ordered sulfate was observed in the sulfate-binding pocket . The structure reveals a mobile loop positioned over the active site . This loop is in a "closed" or "down" position in the reported crystal structures of fungal ATP sulfurylases, which contained bound substrates, but it is in an "open" or "up" position in the ligand-free Riftia symbiont enzyme . Thus, closure of the loop correlates with occupancy of the active site, although the loop itself does not interact directly with bound ligands . Rather, it appears to assist in the orientation of residues that do interact with active-site ligands . Amino acid differences between the mobile loops of the enzymes from sulfate assimilators and sulfur chemolithotrophs may account for the significant kinetic differences between the two classes of ATP sulfurylase.

Ugeskr Laeger, 2001 Nov 5, 163(45), 6287 - 8
{Kingella kingae osteomyelitis}; Wildt S et al.; A case of Kingella kingae osteomyelitis in a 1-year-old child is described . Kingella kingae osteoarticular infections in children and the difficulties of isolating this slow growing, fastidious bacterium are discussed.

J Biol Chem, 2002 Feb 15, 277(7), 4628 - 35 Epub 2001 Nov 26.
Properties and substrate specificity of RppA, a chalcone synthase-related polyketide synthase in Streptomyces griseus; Funa N et al.; RppA, a chalcone synthase-related polyketide synthase (type III polyketide synthase) in the bacterium Streptomyces griseus, catalyzes the formation of 1,3,6,8-tetrahydroxynaphthalene (THN) from five molecules of malonyl-CoA . The K(m) value for malonyl-CoA and the k(cat) value for THN synthesis were determined to be 0.93 +/- 0.1 microm and 0.77 +/- 0.04 min(-1), respectively . RppA accepted aliphatic acyl-CoAs with the carbon lengths from C(4) to C(8) as starter substrates and catalyzed sequential condensation of malonyl-CoA to yield alpha-pyrones and phloroglucinols . In addition, RppA yielded a hexaketide, 4-hydroxy-6-(2',4',6'-trioxotridecyl)-2-pyrone, from octanoyl-CoA and five molecules of malonyl-CoA, suggesting that the size of the active site cavity of RppA is larger than any other chalcone synthase-related enzymes found so far in plants and bacteria . RppA was also found to synthesize a C-methylated pyrone, 3,6-dimethyl-4-hydroxy-2-pyrone, by using acetoacetyl-CoA as the starter and methylmalonyl-CoA as an extender . Thus, the broad substrate specificity of RppA yields a wide variety of products.

Appl Environ Microbiol, 2001 Dec, 67(12), 5771 - 9
Sequence analysis of a 101-kilobase plasmid required for agar degradation by a Microscilla isolate; Zhong Z et al.; An agar-degrading marine bacterium identified as a Microscilla species was isolated from coastal California marine sediment . This organism harbored a single 101-kb circular DNA plasmid designated pSD15 . The complete nucleotide sequence of pSD15 was obtained, and sequence analysis indicated a number of genes putatively encoding a variety of enzymes involved in polysaccharide utilization . The most striking feature was the occurrence of five putative agarase genes . Loss of the plasmid, which occurred at a surprisingly high frequency, was associated with loss of agarase activity, supporting the sequence analysis results.

Appl Environ Microbiol, 2001 Dec, 67(12), 5568 - 80
Isolation and characterization of a novel As(V)-reducing bacterium: implications for arsenic mobilization and the genus Desulfitobacterium; Niggemyer A et al.; Dissimilatory arsenate-reducing bacteria have been implicated in the mobilization of arsenic from arsenic-enriched sediments . An As(V)-reducing bacterium, designated strain GBFH, was isolated from arsenic-contaminated sediments of Lake Coeur d'Alene, Idaho . Strain GBFH couples the oxidation of formate to the reduction of As(V) when formate is supplied as the sole carbon source and electron donor . Additionally, strain GBFH is capable of reducing As(V), Fe(III), Se(VI), Mn(IV) and a variety of oxidized sulfur species . 16S ribosomal DNA sequence comparisons reveal that strain GBFH is closely related to Desulfitobacterium hafniense DCB-2(T) and Desulfitobacterium frappieri PCP-1(T) . Comparative physiology demonstrates that D . hafniense and D . frappieri, known for reductively dechlorinating chlorophenols, are also capable of toxic metal or metalloid respiration . DNA-DNA hybridization and comparative physiological studies suggest that D . hafniense, D . frappieri, and strain GBFH should be united into one species . The isolation of an Fe(III)- and As(V)-reducing bacterium from Lake Coeur d'Alene suggests a mechanism for arsenic mobilization in these contaminated sediments while the discovery of metal or metalloid respiration in the genus Desulfitobacterium has implications for environments cocontaminated with arsenious and chlorophenolic compounds.

Appl Environ Microbiol, 2001 Dec, 67(12), 5403 - 9
Isolation from agricultural soil and characterization of a Sphingomonas sp . able to mineralize the phenylurea herbicide isoproturon; Sorensen SR et al.; A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil . Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp . within the alpha-subdivision of the proteobacteria . Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy . Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization . Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure . Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products . In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed . This report is the first characterization of a pure bacterial culture able to mineralize IPU.

Mol Microbiol, 2001 Nov, 42(3), 809 - 19
Light-induced carotenogenesis in Myxococcus xanthus: evidence that CarS acts as an anti-repressor of CarA; Whitworth DE et al.; In the bacterium Myxococcus xanthus, carotenoids are produced in response to illumination, as a result of expression of the crt carotenoid biosynthesis genes . The majority of crt genes are clustered in the crtEBDC operon, which is repressed in the dark by CarA . Genetic data suggest that, in the light, CarS is synthesized and achieves activation of the crtEBDC operon by removing the repressive action of CarA . As CarS contains no known DNA-binding motif, the relief of CarA-mediated repression was postulated to result from a direct interaction between these two proteins . Use of the yeast two-hybrid system demonstrated direct interaction between CarA and CarS . The two-hybrid system also implied that CarA and, possibly, CarS are capable of homodimerization . Direct evidence for CarS anti-repressor action was provided in vitro . A glutathione S-transferase (GST)-CarA protein fusion was shown to bind specifically to a palindromic operator sequence within the crtEBDC promoter . CarA was prevented from binding to its operator, and prebound CarA was removed by the addition of purified CarS . CarS is therefore an anti-repressor.

Protein Expr Purif, 2001 Dec, 23(3), 476 - 82
Cloning, expression, and purification of the uropathogenic Escherichia coli invasin DraD; Zalewska B et al.; In this study we presented a very efficient expression system, based on pET30LIC/Ek vector, for producing DraD invasin of the uropathogenic Escherichia coli and a one-step chromatography purification procedure for obtaining pure recombinant protein (DraD-C-His(6)) . This protein has a molecular weight of 14,818 and calculated pI of 6.6 . It contains a polyhistidine tag at the C-terminus (13 additional amino acids) that allowed single-step isolation by Ni affinity chromatography . Also, we obtained specific antibodies against DraD invasin to develop tools for characterizing the expression and biological function of this protein . The amount and quality of DraD-C-His(6) fusion protein purified from E . coli overexpression system seems to be fully appropriate for crystallographic studies (soluble form), and for establishing role of the protein in bacterium (cultured cell line interaction and in the internalization process) and for obtaining rabbit polyclonal antisera (insoluble form) .

Anal Chem, 2001 Nov 1, 73(21), 5334 - 8
Flow cytometry of Escherichia coli on microfluidic devices; McClain MA et al.; Flow cytometry of the bacterium Escherichia coli was demonstrated on a microfabricated fluidic device (microchip) . The channels were coated with poly(dimethylacrylamide) to prevent cell adhesion, and the cells were transported electrophoretically by applying potentials to the fluid reservoirs . The cells were electrophoretically focused at the channel cross and detected by coincident light scattering and fluorescence . The E . coli were labeled with a membrane-permeable nucleic acid stain (Syto15), a membrane-impermeable nucleic acid stain (propidium iodide), or a fluorescein-labeled antibody and counted at rates from 30 to 85 Hz . The observed labeling efficiencies for the dyes and antibody were greater than 94%.

Heart Dis, 1999 Sep-Oct, 1(4), 233 - 40
The role of infection in atherosclerosis and coronary artery disease: a new therapeutic target; Ismail A et al.; There is growing evidence that inflammatory processes may be involved in the development of atherosclerosis and its complications . Viral and bacterial pathogens have been implicated as possible causative factors in the pathogenesis of coronary artery disease (CAD) and restenosis after angioplasty . Antibiotic trials are now in progress to examine whether treatment of infection can prevent the complications of CAD . Atherosclerosis, the primary pathologic process in coronary artery disease (CAD), carotid artery disease, abdominal aortic aneurysm, and peripheral vascular disease, is no longer considered to be an obscure, slowly progressive, degenerative disease . Indeed, recent molecular studies on the atherosclerotic plaque have shown that the initiation, progression, and acute sequelae of atherosclerosis can be explained in part by a low-grade inflammatory process . Studies show that mediators of inflammation can be found at all stages of the life cycle of the atherosclerotic plaque . These include activated macrophages and lymphocytes, cytokines, growth factors, matrix degenerating proteinases, and tissue factor . It is hypothesized that risk factors such as hypertension, smoking, or elevated levels of low-density lipoprotein (LDL) cholesterol result in injury to the endothelial cell of the artery, and this injury initiates the inflammatory process . However, many patients with vascular disease do not have these established risk factors, and this observation has galvanized efforts to find new risk factors . Because inflammation is now considered to be an operative paradigm for atherosclerosis, it is not a major leap to the hypothesis that infectious agents, such as viral or bacterial, may play a role . Certainly this is not a new concept, and with the recent discovery that peptic ulcer disease, heretofore considered a disease of excess acid and reduced mucosal resistance, is caused by the ubiquitous bacterium Helicobacter pylori, interest in finding an infectious etiology for atherosclerosis has increased . Accordingly, the purpose of this discussion is to review in a historical manner the evidence that infectious agents-including herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Enterovirus (adenovirus, Coxsackie virus), Chlamydia pneumoniae, and H . pylori-may play a role in atherosclerosis and its manifestations, especially as they relate to CAD.

Zhonghua Kou Qiang Yi Xue Za Zhi, 2001 Jul, 36(4), 289 - 91
{Influence of surface roughness of pure titanium on accumulation of bacterium}; Li X et al.; OBJECTIVE: The aim of this investigation was to evaluate the influence of surface roughness of pure titanium on adhesion of bacterium . METHODS: A total of six edentulous volunteers with healthy oral mucosa participated in an in vivo study . Four kinds of pure Titanium testing pieces with different surfaces were fixed in the polished surface of upper complete dentures and the other in the tissue surface of the dentures . After 6-month wearing the dentures, the amount and species of bacterium adhered on pure Titanium were examined . RESULTS: (1) Individual difference had a significant influence on amount of bacterium adhered on pure Titanium, but had no relation to species of bacterium according to Gram's staining . (2) To the same patient, with the increase of roughness, bacterium adhered on samples improved in amount (P < 0.01), but remained the same in composition . Wrinkly samples collected more bacterium than plane samples and exhibited G- coccus beside G+ coccus . Comparing two samples with same roughness, the one with roughness on the tissue surface of the denture collected less bacterium than that on the polished surface (P < 0.01), and predominantly presented G- rod bacterium and coccus, which was completely different to that on the polished surface . CONCLUSION: From the perspective of benefit to periodontal tissue, plane surface should be adopted when framework of pure Titanium is made, and polishing of the prosthesis after being modified should be paid more attention, especially on the tissue surface.

Chin Med J (Engl), 1999 Oct, 112(10), 938 - 41
Etiology of trachoma: a great success in isolating and cultivating Chlamydia trachomatis; Wang Y; PURPOSE: To review the studies on the etiology of trachoma and to honor Professor Tang Feifan (Prof . FF Tang) . He is the first scientist who was successful in isolating and cultivating chlamydia trachomatis in 1946 . DATA SOURCES: The principal literatures are cited from Tang's papers published from the 1930s to the 1950s . Earlier literatures concerning early hypotheses for trachoma pathogenic agent are also studied . STUDY SELECTION AND DATA EXTRACTION: Only important and conclusive breakthroughs in Tang's papers are selected and extracted . RESULTS AND CONCLUSIONS: In the 1930s Tang was intending to repeat Noguchi's experiments in isolating bacterium granulosis from cases of trachoma in China and to employ bacterium granulosis isolated by Noguchi in 1928 for reproducing experimental trachomatous human clinical manifestations in China . Both experiments showed negative results . In the 1950s, before isolated chlamydia trachomtas, Tang and his colleagues had finished two fundamental studies: (1) the histological nature of trachoma, their relationship to etiological agent as well as to the host cells; and (2) the clinical manifestations and the morphological pictures of trachoma in monkeys . Eventually chlamydia trachomatis was isolated successfully and cultivated continuously.

J Bacteriol, 2001 Dec, 183(24), 7231 - 40
Different physiological roles of ATP- and PP(i)-dependent phosphofructokinase isoenzymes in the methylotrophic actinomycete Amycolatopsis methanolica; Alves AM et al.; Cells of the actinomycete Amycolatopsis methanolica grown on glucose possess only a single, exclusively PP(i)-dependent phosphofructokinase (PP(i)-PFK) (A . M . C . R . Alves, G . J . W . Euverink, H . J . Hektor, J . van der Vlag, W . Vrijbloed, D.H.A . Hondmann, J . Visser, and L . Dijkhuizen, J . Bacteriol . 176:6827-6835, 1994) . When this methylotrophic bacterium is grown on one-carbon (C(1)) compounds (e.g., methanol), an ATP-dependent phosphofructokinase (ATP-PFK) activity is specifically induced, completely replacing the PP(i)-PFK . The two A . methanolica PFK isoenzymes have very distinct functions, namely, in the metabolism of C(6) and C(1) carbon substrates . This is the first report providing biochemical evidence for the presence and physiological roles of PP(i)-PFK and ATP-PFK isoenzymes in a bacterium . The novel ATP-PFK enzyme was purified to homogeneity and characterized in detail at the biochemical and molecular levels . The A . methanolica ATP-PFK and PP(i)-PFK proteins possess a low level of amino acid sequence similarity (24%), clearly showing that the two proteins are not the result of a gene duplication event . PP(i)-PFK is closely related to other (putative) actinomycete PFK enzymes . Surprisingly, the A . methanolica ATP-PFK is most similar to ATP-PFK from the protozoon Trypanosoma brucei and PP(i)-PFK proteins from the bacteria Borrelia burgdorferi and Treponema pallidum, both spirochetes, very distinct from actinomycetes . The data thus suggest that A . methanolica obtained the ATP-PFK-encoding gene via a lateral gene transfer event.

Protein Sci, 2001 Dec, 10(12), 2577 - 86
Molecular design of Mycoplasma hominis Vaa adhesin; Boesen T et al.; The variable adherence-associated (Vaa) adhesin of the opportunistic human pathogen Mycoplasma hominis is a surface-exposed, membrane-associated protein involved in the attachment of the bacterium to host cells . The molecular masses of recombinant 1 and 2 cassette forms of the protein determined by a light-scattering (LS) method were 23.9 kD and 36.5 kD, respectively, and corresponded to their monomeric forms . Circular dichroism (CD) spectroscopy of the full-length forms indicated that the Vaa protein has an alpha-helical content of approximately 80% . Sequence analysis indicates the presence of coiled-coil domains in both the conserved N-terminal and antigenic variable C-terminal part of the Vaa adhesin . Experimental results obtained with recombinant proteins corresponding to the N- or C-terminal parts of the shortest one-cassette form of the protein were consistent with the hypothesis of two distinct coiled-coil regions . The one-cassette Vaa monomer appears to be an elongated protein with a axial shape ratio of 1:10 . Analysis of a two-cassette Vaa type reveals a similar axial shape ratio . The results are interpreted in terms of the topological organization of the Vaa protein indicating the localization of the adherence-mediating structure.

J Biol Inorg Chem, 2001 Oct, 6(8), 791 - 800
Structure refinement of the aldehyde oxidoreductase from Desulfovibrio gigas (MOP) at 1.28 A; Rebelo JM et al.; The sulfate-reducing bacterium aldehyde oxidoreductase from Desulfovibrio gigas (MOP) is a member of the xanthine oxidase family of enzymes . It has 907 residues on a single polypeptide chain, a molybdopterin cytosine dinucleotide (MCD) cofactor and two {2Fe-2S} iron-sulfur clusters . Synchrotron data to almost atomic resolution were collected for improved cryo-cooled crystals of this enzyme in the oxidized form . The cell constants of a=b=141.78 A and c=160.87 A are about 2% shorter than those of room temperature data, yielding 233,755 unique reflections in space group P6(1)22, at 1.28 A resolution . Throughout the entire refinement the full gradient least-squares method was used, leading to a final R factor of 14.5 and Rfree factor of 19.3 (4sigma cut-off) with "riding" H-atoms at their calculated positions . The model contains 8146 non-hydrogen atoms described by anisotropic displacement parameters with an observations/parameters ratio of 4.4 . It includes alternate conformations for 17 amino acid residues . At 1.28 A resolution, three Cl- and two Mg2+ ions from the crystallization solution were clearly identified . With the exception of one Cl- which is buried and 8 A distant from the Mo atom, the other ions are close to the molecular surface and may contribute to crystal packing . The overall structure has not changed in comparison to the lower resolution model apart from local corrections that included some loop adjustments and alternate side-chain conformations . Based on the estimated errors of bond distances obtained by blocked least-squares matrix inversion, a more detailed analysis of the three redox centres was possible . For the MCD cofactor, the resulting geometric parameters confirmed its reduction state as a tetrahydropterin . At the Mo centre, estimated corrections calculated for the Fourier ripples artefact are very small when compared to the experimental associated errors, supporting the suggestion that the fifth ligand is a water molecule rather than a hydroxide . Concerning the two iron-sulfur centres, asymmetry in the Fe-S distances as well as differences in the pattern of NH.S hydrogen-bonding interactions was observed, which influences the electron distribution upon reduction and causes non-equivalence of the individual Fe atoms in each cluster.

Mol Genet Genomics, 2001 Nov, 266(3), 406 - 16
Bacteriophage P2: recombination in the superinfection preprophage state and under replication control by phage P4; Bertani G et al.; Genetic crosses (mixed infection, lytic cycle) with bacteriophage P2 are known to give extremely low recombination frequencies, and these are unaffected by the recA status of the host bacterium . We now show the following: (1) the satellite bacteriophage P4, which interacts with P2 in a number of ways, but is quite different from it in terms of DNA replication and its control, is clearly dependent on the host recA+ function for recombination; (2) a chimeric phage (Lindqvist's P2/P4 Hy19), in which P2 replication early genes have been replaced by those of P4, recombines in a recA+-dependent manner; (3) immunity-sensitive P2 phages, in mixed infections of P2-immune bacteria, and hence blocked in their replication, recombine in a recA+-dependent manner; (4) an analysis of the distribution of exchanges based on a simple model confirms that in mixed infections of sensitive cells (where P2 is actively multiplying) recombinational exchanges tend to be statistically clustered in a segment of the chromosome containing the origin of replication, and also shows that, under conditions in which P2 DNA replication is blocked, the distribution of exchanges correlates well with the physical distances between markers on the P2 DNA.

J Bioenerg Biomembr, 2001 Aug, 33(4), 343 - 52
The succinate dehydrogenase from the thermohalophilic bacterium Rhodothermus marinus: redox-Bohr effect on heme bL; Fernandes AS et al.; The succinate dehydrogenase from the thermohalophilic bacterium Rhodothermus marinus is a member of the succinate:menaquinone oxidoreductases family . It is constituted by three subunits with apparent molecular masses of 70, 32, and 18 kDa . The optimum temperature for succinate dehydrogenase activity is 80 degrees C, higher than the optimum growth temperature of R . marinus, 65 degrees C . The enzyme shows a high affinity for both succinate (Km = 0.165 mM) and fumarate (Km = 0.10 mM) . It contains the canonical iron-sulfur centers S1, S2, and S3, as well as two B-type hemes . In contrast to other succinate dehydrogenases, the S3 center has an unusually high reduction potential of +130 mV and is present in two different conformations, one of which presents an unusual EPR signal with g values at 2.035, 2.009, and 2.001 . The apparent midpoint reduction potentials of the hemes, +75 and -65 mV at pH 7.5, are also higher than those reported for other enzymes . The heme with the lower potential (heme bL) presents a considerable dependence of the reduction potential with pH (redox-Bohr effect), having a pKa(OX) = 6.5 and a pKa(red) = 8.7 . This behavior is consistent with the proposal that in these enzymes menaquinone reduction occurs close to heme bL, near to the periplasmic side of the membrane, and involving dissipation of the proton transmembrane gradient.

Biomacromolecules, 2001 Fall, 2(3), 1061 - 5
Biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptobutyrate) as a sulfur analogue to poly(3-hydroxybutyrate) (PHB); Lutke-Eversloh T et al.; A hitherto unknown copolymer that contains sulfur in the backbone linking 3-hydroxybutyrate and 3-mercaptobutyrate by thioester linkages, poly(3HB-co-3MB), was synthesized by the polyhydroxyalkanoate-(PHA-) accumulating bacterium Ralstonia eutropha, when 3-mercaptobutyric acid was fed as the carbon source in addition to gluconate . The chemical structure of this novel polymer was confirmed by gas chromatography/mass spectrometry, infrared spectroscopy, 1H and 13C nuclear magnetic resonance spectroscopy, and elemental sulfur analysis . Different poly(3HB-co-3MB) samples were isolated and characterized, and the molar 3MB fraction contributed up to 72 mol% . Regarding the chemical structure of 3-mercaptobutyrate, which is analogous to 3-hydroxybutyrate, the incorporation of this novel polymer constituent represents a second example of 3-mercaptoalkanoates that are incorporated into biopolymers by the PHA synthase of R . eutropha.

Antimicrob Agents Chemother, 2001 Dec, 45(12), 3610 - 2
Chloramphenicol-sensitive Escherichia coli strain expressing the chloramphenicol acetyltransferase (cat) gene; Potrykus J et al.; An Escherichia coli strain (strain CM2555) bearing the chloramphenicol acetyltransferase (cat) gene was found to be sensitive to chloramphenicol . We demonstrate that the cat gene is efficiently expressed in strain CM2555 . Our results suggest that decreased levels of acetyl coenzyme A in cat-expressing CM2555 cells in the presence of chloramphenicol may cause the bacterium to be sensitive to this antibiotic.

Infect Immun, 2001 Dec, 69(12), 7820 - 31
Dynamic nature of host-pathogen interactions in Mycobacterium marinum granulomas; Bouley DM et al.; Mycobacterium marinum causes long-term subclinical granulomatous infection in immunocompetent leopard frogs (Rana pipiens) . These granulomas, organized collections of activated macrophages, share many morphological features with persistent human tuberculous infection . We examined organs of frogs with chronic M . marinum infection using transmission electron microscopy in conjunction with immunohistochemistry and acid phosphatase cytochemistry to better define the bacterium-host interplay during persistent infection . Bacteria were always found within macrophage phagosomes . These phagosomes were often fused to lysosomes, in sharp contrast to those formed during in vitro infection of J774 macrophage-like cells by M . marinum . The infected macrophages in frog granulomas showed various levels of activation, as evidenced by morphological changes, including epithelioid transformation, recent phagocytic events, phagolysosomal fusion, and disintegration of bacteria . Our results demonstrate that even long-term granulomas are dynamic environments with regard to the level of host cell activation and bacterial turnover and suggest a continuum between constantly replicating bacteria and phagocytic killing that maintains relatively constant bacterial numbers despite an established immune response . Infection with a mutant bacterial strain with a reduced capacity for intracellular replication shifted the balance, leading to a greatly reduced bacterial burden and inflammatory foci that differed from typical granulomas.

Infect Immun, 2001 Dec, 69(12), 7753 - 9
Chlamydia pneumoniae infects and multiplies in lymphocytes in vitro; Haranaga S et al.; The obligate intracellular pathogen Chlamydia (Chlamydophila) pneumoniae is known to be associated with some chronic inflammatory diseases, such as atherosclerosis . Interaction between C . pneumoniae and immune cells is important in the development of such diseases . However, susceptibility of immune cells, particularly lymphocytes, to C . pneumoniae infection has not been reported, even though lymphocytes play a pivotal role in the development of the diseases caused by this bacterium . In this regard, we examined the susceptibility of lymphocytes to C . pneumoniae infection in vitro . The results demonstrated that human peripheral blood lymphocytes as well as mouse spleen lymphocytes could be infected with C . pneumoniae . Furthermore, purified T lymphocytes as well as established T-lymphocyte cell line cells showed an obvious susceptibility to C . pneumoniae infection, indicating that T cells could be one of the host cells for this bacterial infection . These findings reveal a new infection site for C . pneumoniae, i.e., lymphocytes.

Infect Immun, 2001 Dec, 69(12), 7527 - 34
Role of RgpA, RgpB, and Kgp proteinases in virulence of Porphyromonas gingivalis W50 in a murine lesion model; O'Brien-Simpson NM et al.; Extracellular Arg-x- and Lys-x-specific cysteine proteinases are considered important virulence factors and pathogenic markers for Porphyromonas gingivalis, a bacterium implicated as a major etiological agent of chronic periodontitis . Three genes . rgpA, rgpB, and kgp, encode an Arg-x-specific proteinase and adhesins (RgpA), an Arg-x-specific proteinase (RgpB), and a Lys-x-specific proteinase and adhesins (Kgp), respectively . The contribution to pathogenicity of each of the proteinase genes of P . gingivalis W50 was investigated in a murine lesion model using isogenic mutants lacking RgpA, RgpB, and Kgp . Whole-cell Arg-x-specific proteolytic activity of both the RgpA(-) and RgpB(-) isogenic mutants was significantly reduced (3- to 4-fold) relative to that of the wild-type W50 . However, for the Kgp(-) isogenic mutant, whole-cell Arg-x activity was similar to that of the wild-type strain . Whole-cell Lys-x proteolytic activity of the RgpA(-) and RgpB(-) mutants was not significantly different from that of wild-type W50, whereas the Kgp(-) mutant was devoid of Lys-x whole-cell proteolytic activity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using proteinase-specific antibodies of cell sonicates of the wild-type and mutant strains showed that the proteinase catalytic domain of each of the mutants was not expressed . This analysis further showed that RgpB appeared as 72- and 80-kDa bands, and the catalytic domains of RgpA and Kgp appeared as processed 45-kDa and 48-kDa bands, respectively . In the murine lesion model, mice were challenged with three doses of each mutant and wild-type strain . At the lower dose (3.0 x 10(9) viable-cells), no lesions were recorded for each of the mutants, whereas wild-type W50 induced large ulcerative lesions . At a dose of 6.0 x 10(9) viable-cells, all the mice challenged with the wild-type strain died, whereas mice challenged with the RgpA(-) and RgpB(-) isogenic mutants did not die but developed lesions . Mice challenged with the Kgp(-) isogenic mutant at this dose did not develop lesions . At a 1.2 x 10(10) viable-cell dose, only 40% of mice challenged with the Kgp(-) mutant developed lesions, and these lesions were significantly smaller than lesions induced by the wild-type strain at the 3.0 x 10(9) viable-cell dose . All the mice challenged with the RgpA(-) mutant died at the 1.2 x 10(10) viable-cell dose, whereas only 20% died when challenged with the RgpB(-) mutant at this dose . Wild-type phenotype was restored to the RgpB(-) mutant by complementation with plasmid pNJR12::rgpB containing the rgpB gene . There was no difference between the pNJR12::rgpB-complemented RgpB(-) mutant and the wild-type W50 strain in whole-cell Arg-x activity, protein profile, or virulence in the murine lesion model . These results show that the three proteinases, RgpA, RgpB, and Kgp, all contributed to virulence of P . gingivalis W50 in the murine lesion model and that the order in which they contributed was Kgp >> RgpB > or = RgpA.

Am J Physiol Gastrointest Liver Physiol, 2001 Dec, 281(6), G1440 - 8
Helicobacter pylori cytotoxin VacA increases alkaline secretion in gastric epithelial cells; Debellis L et al.; Human infection by the bacterium Helicobacter pylori (Hp) may lead to severe gastric diseases by an ill-understood process involving several virulence factors . Among these, the cytotoxin VacA is associated with higher tissue damage . In this study, the isolated frog stomach model was used to characterize the acute effects of VacA on the gastric epithelium . Our results show that VacA partially inhibits gastric acid output by increasing HCO(3)(-) efflux . Experiments conducted with double-barrelled pH or Cl(-)-selective microelectrodes on surface epithelial gastric cells (SECs) and single gastric glands show that VacA does not impair the activity of the oxyntic cells but renders the apical membrane of SECs more permeable to HCO(3)(-) and Cl(-) . Inhibition of this permeation by 5-nitro-2-(3-phenylpropylamino) benzoic acid indicates that this may be due to the formation of anion-selective pores by the toxin . We suggest that VacA-dependent HCO(3)(-) efflux from SECs improves the environmental conditions (pH, CO(2) concentration) of the niche parasitized by Hp, that is the gastric surface . This may favor Hp persistence in the tissue and the secondary development of a chronic inflammation.

Biochemistry, 2001 Nov 20, 40(46), 13767 - 73
Blue light drives B-side electron transfer in bacterial photosynthetic reaction centers; Lin S et al.; The core of the photosynthetic reaction center from the purple non-sulfur bacterium Rhodobacter sphaeroides is a quasi-symmetric heterodimer, providing two potential pathways for transmembrane electron transfer . Past measurements have demonstrated that only one of the two pathways (the A-side) is used to any significant extent upon excitation with red or near-infrared light . Here, it is shown that excitation with blue light into the Soret band of the reaction center gives rise to electron transfer along the alternate or B-side pathway, resulting in a charge-separated state involving the anion of the B-side bacteriopheophytin . This electron transfer is much faster than normal A-side transfer, apparently occurring within a few hundred femtoseconds . At low temperatures, the B-side charge-separated state is stable for at least 1 ns, but at room temperature, the B-side bacteriopheophytin anion is short-lived, decaying within approximately 15 ps . One possible physiological role for B-side electron transfer is photoprotection, rapidly quenching higher excited states of the reaction center.

Dev Cell, 2001 Sep, 1(3), 317 - 8
Plagiarism and pathogenesis: common themes in actin remodeling; May RC et al.; The involvement of Nck in pedestal formation by EPEC highlights the similar strategies adopted by this bacterium and the Vaccinia virus to hijack the host cell's cytoskeleton.

Annu Rev Phytopathol, 2001, 39, 53 - 77
Apomictic, polyphagous root-knot nematodes: exceptionally successful and damaging biotrophic root pathogens; Trudgill DL et al.; Most apomictic root-knot nematodes (RKN; Meloidogyne spp.) have host ranges that encompass the majority of flowering plants, and M . incognita is possibly the world's most damaging crop pathogen . The ancestors, age, and origins of the polyphagous RKN are obscure, but there is increasing evidence that M . incognita, M . javanica, and M . arenaria are closely related, heterogeneous species with a recent, hybrid (reticulate) origin . If so, they must owe much of their current worldwide distributions to spread by agriculture . Host resistance appears to be generally durable in the field, but laboratory studies suggest that apomixis does not prevent evolution in response to selection by a parasitic bacterium (Pasteuria penetrans) and host resistance . Maintaining general fitness may be the evolutionary priority for most populations of polyphagous RKN, and a wide host range, important in the field but not in the laboratory, may be conserved by apomixis . Several factors may help confer a wide host range, including suppression of host resistance, perhaps as a consequence of the strength of the induced susceptible response . Resistance genes effective against RKN appear not to have resulted from coevolution . Rates of juvenile invasion and/or development are low in many wild and some crop plants, with the result that they are both poor hosts and sustain less damage . Overall, it is suggested that greater coordination, particularly of fundamental research, is required.

Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13619 - 24 Epub 2001 Nov 06.
Mechanism of ATP-driven electron transfer catalyzed by the benzene ring-reducing enzyme benzoyl-CoA reductase; Unciuleac M et al.; Benzoyl-CoA reductase (BCR) from the bacterium Thauera aromatica catalyzes the two-electron reduction of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene . In a process analogous to enzymatic nitrogen reduction, BCR couples the electron transfer to the aromatic ring to a stoichiometric hydrolysis of 2 ATP/2e(-) . Reduced but not oxidized BCR hydrolyzes ATP to ADP . In this work, purified BCR was shown to catalyze an isotope exchange from {(14)C}ADP to {(14)C}ATP, which was approximately 15% of the ATPase activity in the presence of equimolar amounts of ADP and ATP . In accordance, BCR (alpha beta gamma delta-composition) autophosphorylated its gamma-subunit when incubated with {gamma-(32)P}ATP . Formation of the enzyme-phosphate was independent of the redox state, whereas only dithionite-reduced BCR catalyzed a dephosphorylation associated with the ATPase activity . This finding suggests that the ATPase- and autophosphatase-partial activities of BCR exhibit identical redox dependencies . BCoA or the nonphysiological electron-accepting substrate hydroxylamine stimulated the redox-dependent effects; the rates of both the overall ATPase and the autophosphatase activities of reduced BCR were increased 6-fold . In contrast, BCoA and hydroxylamine had no effect on oxidized and phosphorylated BCR . The reactivity of the phosphoamino acid fits best with a phosphoamidate or acylphosphate linkage . The results obtained suggest a mechanism of ATP hydrolysis-driven electron transfer, which differs from that of nitrogenase by the transient formation of a phosphorylated enzyme.

Mol Biotechnol, 2001 Sep, 19(1), 1 - 12
Chromosomal editing in Escherichia coli . Vectors for DNA integration and excision; Balbas P et al.; Chromosomal editing constitutes the direct and specific modification of the genetic information present in the chromosome . In the bacterium Escherichia coli, strategies were originally developed for the production of specific proteins, the genotypic improvement of strains, and the analysis of regulation of gene expression . However, with the emerging field of metabolic engineering and genomics, efficient means of targeting specific genetic mutations into the chromosome are most useful . In this review, a summary of the systems currently available to generate insertions and deletions in the chromosome of E . coli are presented, as well as the current knowledge about the genetic mechanisms responsible for these processes.

Cell Microbiol, 2001 Nov, 3(11), 745 - 51
Talin, a host cell protein, interacts directly with the translocated intimin receptor, Tir, of enteropathogenic Escherichia coli, and is essential for pedestal formation; Cantarelli VV et al.; Enteropathogenic Escherichia coli (EPEC) is able to inject its own receptor, a transmembrane protein called translocated intimin receptor, Tir, into the host epithelial cell . The bacterium then uses an outer membrane protein, intimin, to bind to Tir and remains firmly attached to the host cell surface for the duration of the infection . The bacterium is also able to trigger the rearrangement of several host cell proteins, culminating with the formation of an actin-rich, pedestal-like structure beneath the EPEC adherence site . Although several cytoskeletal proteins are rearranged following EPEC infection, the exact role played by these proteins during pedestal formation remains unknown . We report here that talin, an integrin-binding protein, is recruited by EPEC and associates directly with Tir . By surface plasmon resonance (SPR), the predicted value for the dissociation constant (KD) for Tir-talin binding was 1.86 x 10(-7) M . We also demonstrate that microinjection of anti-talin antibodies into HeLa cells resulted in the complete inability to focus actin filaments beneath the attached bacterium . These findings demonstrate that talin is essential for EPEC-induced pedestal formation in infected cells.

Biochemistry, 2001 Nov 13, 40(45), 13690 - 8
Cytochromes c555 from the hyperthermophilic bacterium Aquifex aeolicus . 2 . Heterologous production of soluble cytochrome c555s and investigation of the role of methionine residues; Aubert C et al.; The cycB2 gene encoding the soluble cytochrome c555s from Aquifex aeolicus, an hyperthermophilic organism, has been cloned and expressed using Escherichia coli as the host organism . The cytochrome was successfully produced in the periplasm of an E . coli strain coexpressing the ccmABCDEFGH genes involved in the cytochrome c maturation process . Comparison of native and recombinant cytochrome c555s shows that both proteins are indistinguishable in terms of spectroscopic and physicochemical properties . Since two different methionine residues are present in the sequence stretch usually providing the sixth ligand to the heme iron, site-directed mutagenesis has been performed in order to identify the methionine serving as the axial ligand . Two single mutations were introduced, leading to the replacement of each methionine by a histidine residue . Characterization of both mutants, M78H and M84H cytochromes c555s, using biochemical and biophysical techniques has been carried out . The M84H mutant exhibits spectral features identical to those of native cytochrome . Its redox midpoint potential is decreased by 40 mV . By contrast, substitution of methionine 78 by a histidine residue strongly alters the structural and physicochemical properties of the molecule which exhibits characteristics of His/His iron coordination type rather than His/Met . These results allow us to identify methionine 78 as the sixth ligand of cytochrome c555s heme iron . Preliminary results on the thermostability of the native and mutant cytochromes c555 are also reported.

Biochemistry, 2001 Nov 13, 40(45), 13681 - 9
Cytochromes c555 from the hyperthermophilic bacterium Aquifex aeolicus (VF5) . 1 . Characterization of two highly homologous, soluble and membranous, cytochromes c555; Baymann F et al.; Two distinct class I (monoheme) c-type cytochromes from the hyperthermophilic bacterium Aquifex aeolicus were studied by biochemical and biophysical methods (i.e., optical and EPR spectroscopy, electrochemistry) . The sequences of these two heme proteins (encoded by the cycB1 and cycB2 genes) are close to identical (85% identity in the common part of the protein) apart from the presence of an N-terminal stretch of 62 amino acid residues present only in the cycB1 gene . A soluble cytochrome was purified and identified by N-terminal sequencing as the cycB2 gene product . It showed an alpha-peak at 555 nm, an E(m) value of +220 mV, and electron paramagnetic resonance parameters of gz = 2.89, gy = 2.287, and gx = 1.52 . A firmly membrane-bound cytochrome characterized by nearly identical properties was detected and attributed to the cycB1 gene product . The very high degree of homology of its N-terminal part to cytochrome c553 from Heliobacterium gestii strongly suggests it to be anchored to the membrane via N-terminally attached lipid molecules . The two heme proteins were named cytochrome c555s (soluble) and cytochrome c555m (membranous) . Electron paramagnetic resonance on partially ordered membrane multilayers suggests that the solvent-exposed heme domain of cytochrome c555m is flexible with respect to the membrane plane . Possible functional roles for both cytochromes are discussed.

Bioelectrochemistry, 2001 Nov, 54(2), 145 - 50
Electrochemical reactions of redox cofactors in Rhodobacter sphaeroides reaction center proteins in lipid films; Munge B et al.; Cyclic voltammetry of thin films made from the lipid dimyristoylphosphatidyl choline and reaction centers from the purple bacterium Rhodobacter sphaeroides on pyrolytic graphite electrodes in bromide-free pH 8 buffers at 4 degrees C revealed an oxidation peak at 0.98 V and a reduction peak at -0.17 V vs . NHE . No reverse CV peaks were found, suggesting chemical irreversibility . The reduction peak disappeared for reaction centers depleted of quinones, suggesting that the peak represents reduction of this cofactor . The oxidation peak showed a catalytic current increase in the presence of small amounts of ferrous cytochrome c, and decreased by 85% when illuminated by visible light, suggesting assignment to the primary donor (P) cofactor . While oxidized primary donor P(+) is destroyed upon electrochemical formation in the film, reaction of ferrous cyt c with P(+) suggests its persistence in the films on the microsecond time scale.

Appl Microbiol Biotechnol, 2001 Oct, 57(1-2), 212 - 5
Benzo{b}thiophene desulfurization by Gordonia rubropertinctus strain T08; Matsui T et al.; A benzothiophene-desulfurizing bacterium which has a novel desulfurization pathway was isolated and identified as Gordonia rubropertinctus strain T08 . Gas chromatography/mass spectroscopy analysis of the ethyl acetate extract of the culture broth detected benzothiophene sulfoxide, benzothiophene sulfone, benzo{e}{1,2}oxathiin S-oxide (BT-sultine), benzo{e}{1,2}oxathiin S,S-dioxide (BT-sultone), o-hydroxystyrene, and 2-coumaranone, but not 2-(2'-hydroxyphenyl)ethan-1-al, which has been reported to be a desulfurized product of mesophilic nocardioforms.

Nucleic Acids Res, 2001 Nov 1, 29(21), 4441 - 51
Replication intermediate analysis confirms that chromosomal replication origin initiates from an unusual intergenic region in Caulobacter crescentus; Brassinga AK et al.; The alpha-proteobacterium Caulobacter crescentus possesses a developmental cell cycle that restricts chromosome replication to a stalked cell type . The proposed C.crescentus chromosome replication origin (Cori) lies between hemE and RP001, an unusual intergenic region not previously associated with bacterial replication origins, although a similar genomic arrangement is also present at the putative replication origin in the related bacterium Rickettsia prowazekii . The cloned Cori supports autonomous plasmid replication selectively in the stalked cell type implying that replication of the entire chromosome also initiates between hemE and RP001 . To confirm this location, we applied the 2-D (N/N) agarose gel electrophoresis technique to resolve and identify chromosome replication intermediates throughout a 30 kb region spanning Cori . Replication initiation in Cori was uniquely characterized by an 'origin bubble and Y-arc' pattern and this observation was supported by simple replication fork 'Y-arc' patterns that characterized the regions flanking Cori . These replication forks originated bi-directionally from within Cori as determined by the fork direction assay . Therefore, chromosomal replication initiates from the unusual hemE/RP001 intergenic region that we propose represents a new class of replication origins.

Rev Esp Enferm Dig, 2001 Jul, 93(7), 471 - 80
Geographic differences and the role of cagA gene in gastroduodenal diseases associated with Helicobacter pylori infection; Valmaseda Perez T et al.; Helicobacter pylori (H . pylori) is the major causal agent of gastritis, peptic ulcer and gastric cancer . Several bacterium genes seem to be involved in the pathogenicity mechanism . One of them, the cagA gene, has been extensively studied and characterized . In this article we have carried out a study of characteristics and genetic variability of cagA gene in different geographic areas of the world . At the same time, we have summarized several studies that evaluate possible relation of cagA with gastroduodenal diseases associated by H . pylori infection . In our study we found that the presence of the cagA gene has been confirmed in more than 60% H . pylori strains distributed throughout the world . The prevalence of cagA genotype is of 65.4% in gastritis patients, 84.2% in patients with peptic ulcer and 86.5% in those with gastric cancer . It shows a high genetic variability of cagA associated with gastroduodenal diseases that could serve as a virulence marker in H . pylori infected subjects . However, the high prevalence of H . pylori cagA positive strains in some geographic areas does not confirm the strong association between cagA and virulence of strains as described in other countries . Nowadays, cagA gene is considered as a marker for the presence of cag pathogenicity island (cag-PAI) in H . pylori genoma . This region contains several genes that has been involved with the production of cytokines that results in an increased inflammation of host gastric mucosa, but its function is unknown . Probably, others bacterium factors, such as susceptibility host and environmental cofactors could influence in the risk of developing different gastroduodenal diseases associated with H . pylori infection.

Arch Microbiol, 2001 Oct, 176(4), 301 - 5
In vivo role of adenosine-5'-phosphosulfate reductase in the purple sulfur bacterium Allochromatium vinosum; Sanchez O et al.; Adenosine-5'-phosphosulfate (APS) reductase participates in the oxidation of sulfite to APS in Allochromatium vinosum . Oxidation of sulfite via the APS pathway yields ATP through substrate-level phosphorylation . An alternative enzyme for the oxidation of sulfite to sulfate, sulfite:acceptor oxidoreductase, has also been reported in Ach . vinosum . Oxidation of sulfite through this enzyme does not yield ATP . APS reductase is expressed constitutively in Ach . vinosum, suggesting that it performs an important role in this organism . However, studies carried out with batch cultures of an APS reductase mutant showed little or no differences in growth or in the rates of substrate oxidation when compared to the wild-type, therefore questioning the role of this enzyme . In an attempt to establish whether the ATP gain derived from APS-reductase-mediated oxidation of sulfite is relevant for energy-limited cultures, we compared growth of the wild-type SM50 and the APS-reductase-deficient mutant D3 when grown in continuous culture under different degrees of illumination . Little differences in the specific growth rates of the two strains were observed at light-limiting irradiances, suggesting that the ATP gained during sulfite oxidation through the APS reductase pathway does not constitute a significant energy input . However, at saturating irradiances, wild-type Ach . vinosum grew considerably faster than the mutant . Increasing the irradiance even further resulted in inhibition of the wild-type strain down to the level of the APS reductase mutant . The implications of these results are discussed.

Arch Microbiol, 2001 Oct, 176(4), 278 - 84
Light responses in the green sulfur bacterium Prosthecochloris aestuarii: changes in prosthecae length, ultrastructure, and antenna pigment composition; Guyoneaud R et al.; The morphology (mainly prosthecae length), ultrastructure, and antenna pigment composition of the green sulfur bacterium Prosthecochloris aestuarii changed when grown under different light intensities . At light intensities of 0.5 and 5 micromol quanta m(-2) s(-1), the cells had a star-like morphology . Prosthecae, the characteristic appendages of the genus Prosthecochloris, were 232 nm and 194 nm long, respectively . In contrast, when grown at 100 micromol quanta m(-2) s(-1), these appendages were shorter (98 nm) and the cells appeared more rod-shaped . Transmission electron microscopy revealed a significant decrease in the cell perimeter to area ratio and in the number of chlorosomes per linear microm of membrane as light intensity increased . In addition to these morphological and ultrastructural responses, Prosthecochloris aestuarii exhibited changes in its pigment composition as a function of light regime . Lower specific pigment content and synthesis rates were found in cultures grown at light intensities above 5 micromol quanta m(-2) s(-1) . A blue shift in the bacteriochlorophyll (BChl) c Q(y) absorption maximum of up to 17.5 nm was observed under saturating light conditions (100 micromol quanta m(-2) s(-1)) . This displacement was accompanied by changes in the composition of BChl c homologs and by a very low carotenoid content . The morphological, ultrastructural and functional changes exhibited by Prosthecochloris aestuarii revealed the strong light-response capacity of this bacterium to both high and low photon-flux densities.

J Clin Microbiol, 2001 Nov, 39(11), 3920 - 6
Helicobacter typhlonius sp . nov., a Novel Murine Urease-Negative Helicobacter Species; Franklin CL et al.; Over the past decade, several Helicobacter species have been isolated from rodents . With the advent of PCR for the diagnosis of infectious agents, it has become clear that several previously uncharacterized Helicobacter species also colonize rodents . In this report, we describe a novel urease-negative helicobacter, Helicobacter typhlonius sp . nov., which was isolated from colonies of laboratory mice independently by two laboratories . Infection of immunodeficient mice by this bacterium resulted in typhlocolitis similar to that observed with other helicobacter infections . H . typhlonius is genetically most closely related to H . hepaticus . Like H . hepaticus, it is a spiral bacterium with bipolar sheathed flagella . However, this novel species contains a large intervening sequence in its 16S rRNA gene and is biochemically distinct from H . hepaticus . Notably, H . typhlonius does not produce urease or H(2)S nor does it hydrolize indoxyl-acetate . Compared to other Helicobacter species that commonly colonize rodents, H . typhlonius was found to be less prevalent than H . hepaticus and H . rodentium but as prevalent as H . bilis . H . typhlonius joins a growing list of helicobacters that colonize mice and are capable of inducing enteric disease in various strains of immunodeficient mice.

Evolution Int J Org Evolution, 2001 Sep, 55(9), 1746 - 52
Epistasis between new mutations and genetic background and a test of genetic canalization; Elena SF et al.; The importance for fitness of epistatic interactions among mutations is poorly known, yet epistasis can exert important effects on the dynamics of evolving populations . We showed previously that epistatic interactions are common between pairs of random insertion mutations in the bacterium Escherichia coli . In this paper, we examine interactions between these mutations and other mutations by transducing each of twelve insertion mutations into two genetic backgrounds, one ancestral and the other having evolved in, and adapted to, a defined laboratory environment for 10,000 generations . To assess the effect of the mutation on fitness, we allowed each mutant to compete against its unmutated counterpart in that same environment . Overall, there was a strong positive correlation between the mutational effects on the two genetic backgrounds . Nonetheless, three of the twelve mutations had significantly different effects on the two backgrounds, indicating epistasis . There was no significant tendency for the mutations to be less harmful on the derived background . Thus, there is no evidence supporting the hypothesis that the derived bacteria had adapted, in part, by becoming buffered against the harmful effects of mutations.

J Econ Entomol, 2001 Oct, 94(5), 1031 - 6
Effect of selected insecticides on Homalodisca coagulata (Homoptera: Cicadellidae) and transmission of oleander leaf scorch in a greenhouse study; Bethke JA et al.; Homalodisca coagulata (Say) is a recent introduction to California . It is known to spread a strain of the bacterium Xylella fastidiosa Wells, Raju, Hung, Weisberg, Mandelco-Paul & Brenner that induces oleander leaf scorch disease in oleander, Nerium oleander L . Oleander leaf scorch is lethal to oleander and threatens to decimate one of the most important landscape shrubs in California . Towards developing a management strategy for H . coagulata-spread oleander leaf scorch, we documented the affects of selected insecticides on H . coagulata mortality, feeding behavior, and disease transmission in a greenhouse study . Oleanders treated with fenpropathrin, fenpropathrin + acephate, and imidacloprid caused significant mortality to caged H . coagulata within 4 h of exposure . Within 24 h, these pesticides caused nearly 100% mortality 3 wk after treatment . In other experiments, acetamiprid and fenpropathrin treatments reduced time spent feeding and total time on plants . H . coagulata on fenpropathrin-, acetamiprid-, and imidacloprid-treated oleander died in less than 13 min on average . Oleander leaf scorch transmission by H . coagulata was blocked by applications of foliar-applied acetamiprid, and soil-applied imidacloprid and thiamethoxam.

Proteomics, 2001 Apr, 1(4), 508 - 15
Construction of a Francisella tularensis two-dimensional electrophoresis protein database; Hernychova L et al.; We have started the construction of a two-dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia . The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database . We have analyzed the F . tularensis live vaccine strain by two-dimensional gel electrophoresis with immobilized pH 3-10 gradient in the first dimension and 9-16% gradient or tricine SDS-PAGE in the second dimension . In both cases about 2000 spots were detected . Furthermore, we compared the protein pattern of the nonvirulent F . tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted . The separated proteins are going to be identified by peptide mass fingerprinting . However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified . Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1498 - 505 Epub 2001 Oct 25.
Structure of cytochrome c2 from Rhodospirillum centenum; Camara-Artigas A et al.; Cytochrome c(2) from the purple photosynthetic bacterium Rhodospirillum centenum has been crystallized by the sitting-drop vapour-diffusion method . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 29.7, b = 59.9, c = 65.4 A, and diffract to a resolution limit of 1.7 A . The Fe-atom position was determined from its anomalous scattering contribution and a molecular-replacement solution was calculated . The correctness of the solution was confirmed by parallel isomorphous replacement studies . The resulting model has a type I cytochrome fold with two features, an extended alpha-helix and a surface-charge distribution, that are distinctive to this protein . The implications of these structural features for the ability of the cytochrome to serve as an electron carrier are discussed.

Appl Environ Microbiol, 2001 Nov, 67(11), 5315 - 20
Secondary intracellular symbiotic bacteria in aphids of the genus Yamatocallis (Homoptera: Aphididae: Drepanosiphinae); Fukatsu T; A novel secondary intracellular symbiotic bacterium from aphids of the genus Yamatocallis (subfamily Drepanosiphinae) was characterized by using molecular phylogenetic analysis, in situ hybridization, and diagnostic PCR detection . In the aphid tissues, this bacterium (tentatively designated YSMS {Yamatocallis secondary mycetocyte symbiont}) was found specifically in large cells surrounded by primary mycetocytes harboring Buchnera cells . Of nine drepanosiphine aphids examined, YSMS was detected in only two species of the same genus, Yamatocallis tokyoensis and Yamatocallis hirayamae . In natural populations of these aphids, YSMS was present in 100% of the individuals . Phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences demonstrated that YSMS of Y . tokyoensis and Y . hirayamae constitute a distinct and isolated clade in the gamma subdivision of the class Proteobacteria . No 16S rDNA sequences of secondary endosymbionts characterized so far from other aphids showed phylogenetic affinity to YSMS . Based on these results, I suggest that YSMS was acquired by an ancestor of the genus Yamatocallis and has been conserved throughout the evolution of the lineage . By using the nucleotide substitution rate for 16S rDNA of Buchnera spp., the time of acquisition of YSMS was estimated to be about 13 to 26 million years ago, in the Miocene epoch of the Tertiary period.

Biosens Bioelectron, 2001 Dec, 16(9-12), 667 - 74
Soil biosensor for the detection of PAH toxicity using an immobilized recombinant bacterium and a biosurfactant; Gu MB et al.; A biosensor for detecting the toxicity of polycylic aromatic hydrocarbons (PAHs) contaminated soil has been successfully constructed using an immobilized recombinant bioluminescent bacterium, GC2 (lac::luxCDABE), which constitutively produces bioluminescence . The biosurfactant, rhamnolipids, was used to extract a model PAH, phenanthrene, and was found to enhance the bioavailability of phenanthrene via an increase in its rate of mass transfer from sorbed soil to the aqueous phase . The monitoring of phenanthrene toxicity was achieved through the measurement of the decrease in bioluminescence when a sample extracted with the biosurfactant was injected into the minibioreactor . The concentrations of phenanthrene in the aqueous phase were found to correlate well with the corresponding toxicity data obtained by using this toxicity biosensor . In addition, it was also found that the addition of glass beads to the agar media enhanced the stability of the immobilized cells . This biosensor system using a biosurfactant may be applied as an in-situ biosensor to detect the toxicity of hydrophobic contaminants in soils and for performance evaluation of PAH degradation in soils.

Proteomics, 2001 May, 1(5), 683 - 90
Cell fingerprinting: an approach to classifying cells according to mass profiles of digests of protein extracts; Zhou X et al.; We present a statistical framework for classifying cells according to the set of peptide masses obtained by mass spectrometric analysis of digestions of whole cell protein extracts . The digest is separated by high performance liquid chromatography (HPLC) coupled directly to a mass spectrometer either by an electrospray interface or by collection to a matrix-assisted laser desorption/ionization target plate . Here, the mass to charge ratio, intensity, and HPLC retention time of the peptides are measured . We have used defined bacterial strains to test this approach . For each bacterium, this process is repeated for extracts obtained at different points in the growth curve in order to try and define an invariant set of signals that uniquely identify the bacterium . This paper presents algorithms for the creation of this cell fingerprint database and develops a Bayesian classification scheme for deciding whether or not an unknown bacterium has a match in the database . Our initial testing based on a limited data set of three bacteria indicates that our approach is feasible . Via a jack-knife test, our Bayesian classification scheme correctly identified the bacterium in 67.8% of the cases.

Panminerva Med, 2001 Dec, 43(4), 279 - 82
The journey from hepatitis to hepatocellular carcinoma . Bridging role of Helicobacter species; Fagoonee S et al.; Hepatocellular carcinoma (HCC) is a long-term consequence of chronic liver disease, whose aetiology could result from viral, environmental and hereditary causes . Viral infection, by itself, could only partially explain the pathogenesis of cirrhosis and HCC . A new aetiologic agent capable of inducing chronic active hepatitis and hepatocellular tumours was discovered: it is a bacterium belonging to the genus Helicobacter, and named H . hepaticus . Presence of sequences belonging to the 16S rRNA of Helicobacter species (spp.) has been demonstrated in liver of most patients with cirrhosis and HCC . H . pylori and related bacteria, such as H . hepaticus, produce toxins that kill hepatocyte by a granulating effect on liver cell lines . In vivo, such toxins might reach the liver through the portal tract, thereby causing hepatocellular damage . The recognition of Helicobacter spp . as a possible risk factor for cirrhosis and HCC might have a practical impact on the general population: the treatment of this infection is easy and far less expensive than liver transplantation or any long term treatment for the other risk factors of HCC . Any confirmation of the involvement of Helicobacter in liver disease would eventually come from the success of culturing the bacterium from liver tissues . Future research is needed to clarify the importance of Helicobacter spp . in respect to the other pathogens already known as causative agents of chronic inflammation of the liver and its long term sequelae, namely cirrhosis and HCC.

Protein Expr Purif, 2001 Nov, 23(2), 242 - 8
Expression in Escherichia coli of the thermostable inorganic pyrophosphatase from the Aquifex aeolicus and purification and characterization of the recombinant enzyme; Hoe HS et al.; The gene encoding the inorganic pyrophosphatase from a hyperthermophilic bacterium, Aquifex aeolicus (Aae), was amplified by PCR . Then, the gene was overexpressed in Escherichia coli using a pJR-based expression plasmid, pAIPD . The recombinant Aae pyrophosphatase was purified 16.2-fold with a 53.4% yield and a specific activity of 34 U/mg protein by a combination of heating (to denature E . coli proteins) and two steps of DEAE-Sephacel column chromatography (nonabsorbed enzyme at pH 7.3 and absorbed enzyme at pH 8.0) . This enzyme has an approximate molecular mass of 105,000 Da and consists of four subunits, each with a molecular mass of 24,500 Da . The enzyme shows the optimal activity in the pH range 7.5-8.0 . The enzyme was stable at 80-95 degrees C . A divalent cation was absolutely required for the enzyme activity, Mg(2+) being most effective .

Biosci Biotechnol Biochem, 2001 Sep, 65(9), 1949 - 56
Super-channel in bacteria: function and structure of a macromolecule import system mediated by a pit-dependent ABC transporter; Hashimoto W et al.; In a soil isolate, Sphingomonas sp . A1, the transport of a macromolecule (alginate: 27 kDa) is mediated by a pit-dependent ATP-binding cassette (ABC) transporter . The transporter is different from other ABC transporters so far analyzed in that its function is dependent on the pit, a mouth-like organ formed on the cell surface only when the cells are compelled to assimilate macromolecules, and in that it allows direct import of macromolecules into cells . The ABC transporter coupled with the pit, which functions as a funnel and/or concentrator of macromolecules to be imported, was designated as the "Super-channel", and in this review, we discuss the three-dimensional structure and specific function of the "Super-channel" for macromolecule import found for the first time in a bacterium.

J Mol Evol, 2001 Oct-Nov, 53(4-5), 555 - 95
Obcells as proto-organisms: membrane heredity, lithophosphorylation, and the origins of the genetic code, the first cells, and photosynthesis; Cavalier-Smith T; I attempt to sketch a unified picture of the origin of living organisms in their genetic, bioenergetic, and structural aspects . Only selection at a higher level than for individual selfish genes could power the cooperative macromolecular coevolution required for evolving the genetic code . The protein synthesis machinery is too complex to have evolved before membranes . Therefore a symbiosis of membranes, replicators, and catalysts probably mediated the origin of the code and the transition from a nucleic acid world of independent molecular replicators to a nucleic acid/protein/lipid world of reproducing organisms . Membranes initially functioned as supramolecular structures to which different replicators attached and were selected as a higher-level reproductive unit: the proto-organism . I discuss the roles of stereochemistry, gene divergence, codon capture, and selection in the code's origin . I argue that proteins were primarily structural not enzymatic and that the first biological membranes consisted of amphipathic peptidyl-tRNAs and prebiotic mixed lipids . The peptidyl-tRNAs functioned as genetically-specified lipid analogues with hydrophobic tails (ancestral signal peptides) and hydrophilic polynucleotide heads . Protoribosomes arose from two cooperating RNAs: peptidyl transferase (large subunit) and mRNA-binder (small subunit) . Early proteins had a second key role: coupling energy flow to the phosphorylation of gene and peptide precursors, probably by lithophosphorylation by membrane-anchored kinases scavenging geothermal polyphosphate stocks . These key evolutionary steps probably occurred on the outer surface of an 'inside out-cell' or obcell, which evolved an unambiguous hydrophobic code with four prebiotic amino acids and proline, and initiation by isoleucine anticodon CAU; early proteins and nucleozymes were all membrane-attached . To improve replication, translation, and lithophosphorylation, hydrophilic substrate-binding and catalytic domains were later added to signal peptides, yielding a ten-acid doublet code . A primitive proto-ecology of molecular scavenging, parasitism, and predation evolved among obcells . I propose a new theory for the origin of the first cell: fusion of two cup-shaped obcells, or hemicells, to make a protocell with double envelope, internal genome and ribosomes, protocytosol, and periplasm . Only then did water-soluble enzymes, amino acid biosynthesis, and intermediary metabolism evolve in a concentrated autocatalytic internal cytosolic soup, causing 12 new amino acid assignments, termination, and rapid freezing of the 22-acid code . Anticodons were recruited sequentially: GNN, CNN, INN, and *UNN . CO2 fixation, photoreduction, and lipid synthesis probably evolved in the protocell before photophosphorylation . Signal recognition particles, chaperones, compartmented proteases, and peptidoglycan arose prior to the last common ancestor of life, a complex autotrophic, anaerobic green bacterium.

Proc R Soc Lond B Biol Sci, 2001 Nov 7, 268(1482), 2245 - 51
Wolbachia-induced parthenogenesis in a genus of phytophagous mites; Weeks AR et al.; The vertically transmitted endosymbiotic bacterium Wolbachia modifies host reproduction in several ways in order to enhance its own spread . One such modification results in the induction of parthenogenesis, where males, which are unable to transmit Wolbachia, are not produced . Interestingly, parthenogenesis-inducing Wolbachia have only been found within haplodiploid insects and it is not known whether this exclusivity is the result of functional constraints of Wolbachia . Here we find a unique pattern of Wolbachia infection that is associated with parthenogenesis in six species within the phytophagous mite genus Bryobia . Through antibiotic treatment we show that, in two species, Bryobia praetiosa and an unidentified species, the Wolbachia infection is strictly associated with parthenogenesis . Microsatellite loci show the mechanism of parthenogenesis to be functionally apomictic and not gamete duplication, with progeny identical to their infected mother . Crossing experiments within B . praetiosa showed no evidence of sexual reproduction . These results are discussed with reference to the distribution of parthenogenesis-inducing Wolbachia and the diversification of the Bryobia genus.

Plant Cell Physiol, 2001 Oct, 42(10), 1112 - 8
Phytoene desaturase, CrtI, of the purple photosynthetic bacterium, Rubrivivax gelatinosus, produces both neurosporene and lycopene; Harada J et al.; Biosynthetic pathways for carotenoids in the purple photosynthetic bacterium, Rubrivivax gelatinosus, which synthesizes spirilloxanthin in addition to spheroidene and OH-spheroidene, were investigated by means of genetic manipulation . A phytoene desaturase gene (crtI) found in the photosynthesis gene cluster of this bacterium was expressed in an Escherichia coli strain that can produce phytoene . Both neurosporene and lycopene were synthesized in the recombinant, probably by three- and four-step desaturation reactions of CrtI . A mutant of RVI: gelatinosus lacking the crtI gene produced only phytoene, indicating that this organism had no other phytoene desaturases . When the crtI deletion mutant was complemented by the three-step phytoene desaturase of Rhodobacter capsulatus, spirilloxanthin and its precursors were not synthesized, although spheroidene and OH-spheroidene were accumulated . It was concluded that neurosporene and lycopene are produced by a single phytoene desaturase in RVI: gelatinosus resulting in the synthesis of spheroidene and spirilloxanthin, and that there are no pathways for spirilloxanthin synthesis via spheroidene.

Am J Physiol Gastrointest Liver Physiol, 2001 Nov, 281(5), G1140 - 50
Lactoferrin protects neonatal rats from gut-related systemic infection; Edde L et al.; Lactoferrin is a milk protein that reportedly protects infants from gut-related, systemic infection . Proof for this concept is limited and was addressed during in vivo and in vitro studies . Neonatal rats pretreated orally with recombinant human lactoferrin (rh-LF) had less bacteremia and lower disease severity scores (P < 0.001) after intestinal infection with Escherichia coli . Control animals had 1,000-fold more colony-forming units of E . coli per milliliter of blood than treated animals (P < 0.001) . Liver cultures from control animals had a twofold increase in bacterial counts compared with cultures from rh-LF-treated pups (P < 0.02) . Oral therapy with rh-LF + FeSO(4) did not alter the protective effect . In vitro studies confirmed that rh-LF interacted with the infecting bacterium and rat macrophages . An in vitro assay showed that rh-LF did not kill E . coli, but a combination of rh-LF + lysozyme was microbicidal . In vitro studies showed that rat macrophages released escalating amounts of nitric oxide and tumor necrosis factor-alpha when stimulated with increasing concentrations of rh-LF . The in vitro studies suggest that rh-LF may act with other "natural peptide antibiotics" or may prime macrophages to kill E . coli in vivo.

Arthritis Rheum, 2001 Oct, 44(10), 2392 - 401
The reprogrammed host: Chlamydia trachomatis-induced up-regulation of glycoprotein 130 cytokines, transcription factors, and antiapoptotic genes; Hess S et al.; OBJECTIVE: Infection with Chlamydia trachomatis is a known cause of sexually transmitted diseases, eye infections (including trachoma), and reactive arthritis (ReA) . Because the mechanisms of Chlamydia-induced changes leading to ReA are poorly defined, this study sought to identify the target genes involved at the molecular level . METHODS: Chlamydia-induced changes in host cells were investigated by combining a screening technique, which utilized complementary DNA arrays on C trachomatis-infected and mock-infected epithelial HeLa cells, with real-time reverse transcription-polymerase chain reaction or enzyme-linked immunosorbent assay of gene products . Some responses were additionally demonstrated on human primary chondrocytes and a human synovial fibroblast cell line, both of which served as model cells for ReA . RESULTS: Eighteen genes (of 1,176) were found to be up-regulated after 24 hours of infection with this obligate intracellular bacterium, among them the glycoprotein 130 family members IL-11 and LIF, the chemokine gene MIP2-alpha, the transcription factor genes EGR1, ETR101, FRA1, and c-jun, the apoptosis-related genes IEX-1L and MCL-1, adhesion molecule genes such as ICAM1, and various other functionally important genes . In the context of this rheumatic disease, the cytokines and transcription factors seem to be especially involved, since various connections to chondrocytes, synoviocytes, bone remodeling, joint pathology, and other rheumatic diseases have been demonstrated . CONCLUSION: Infection with C trachomatis seems to reprogram the host cells (independent of activation by lipopolysaccharide or other ultraviolet-resistant bacterial components) at various key positions that act as intra- or intercellular switches, suggesting that these changes and similar Chlamydia-induced functional alterations constitute an important basis of the pathogenic inflammatory potential of these cells in ReA . Our results suggest that this approach is generally useful for the broad analysis of host-pathogen interactions involving obligate intracellular bacteria, and for the identification of target genes for therapeutic intervention in this rheumatic disease.

Proc Natl Acad Sci U S A, 1990 Dec 1, 87(23), 9449 - 53
Reduced coenzyme F420: heterodisulfide oxidoreductase, a proton- translocating redox system in methanogenic bacteria; Deppenmeier U et al.; Washed everted vesicles of the methanogenic bacterium strain Go1 were found to couple the F420H2-dependent heterodisulfide reduction with the transfer of protons across the membrane into the lumen of the everted vesicles . The transmembrane electrochemical potential of protons thereby generated was shown to be competent in driving ATP synthesis from ADP + Pi, exhibiting a stoichiometry of 2 H+ translocated or 0.4 ATP synthesized per F420H2 oxidized . This enzyme system exhibits the phenomenon of coupling and uncoupling and represents a different kind of electron transport chain with the heterodisulfide of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate as terminal electron acceptor . The heterodisulfide and methane are formed in the methyl coenzyme M reductase reaction . The reducing equivalents are derived from reduced coenzyme F420, which represents an analogue of NADH + H+ in other respiratory chains . It is assumed that the proton-translocating oxidoreductase discovered in strain Go1 is of principal importance to all methanogenic bacteria not utilizing H2.

Proc Natl Acad Sci U S A, 1990 Jul, 87(13), 5168 - 72
Initial electron-transfer in the reaction center from Rhodobacter sphaeroides; Holzapfel W et al.; The initial electron transfer steps in the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides have been investigated by femtosecond time-resolved spectroscopy . The experimental data taken at various wavelengths demonstrate the existence of at least four intermediate states within the first nanosecond . The difference spectra of the intermediates and transient photodichroism data are fully consistent with a sequential four-step model of the primary electron transfer: Light absorption by the special pair P leads to the state P* . From the excited primary donor P*, the electron is transferred within 3.5 +/- 0.4 ps to the accessory bacteriochlorophyll B . State P+B- decays with a time constant of 0.9 +/- 0.3 ps passing the electron to the bacteriopheophytin H . Finally, the electron is transferred from H- to the quinone QA within 220 +/- 40 ps.

J Mol Biol, 2001 Oct 12, 313(1), 35 - 48
Central domain assembly: thermodynamics and kinetics of S6 and S18 binding to an S15-RNA complex; Recht MI et al.; The 30 S ribosomal subunit assembles in vitro through the hierarchical binding of 21 ribosomal proteins to 16 S rRNA . The central domain of 16 S rRNA becomes the platform of the 30 S subunit upon binding of ribosomal proteins S6, S8, S11, S15, S18 and S21 . The assembly of the platform is nucleated by binding of S15 to 16 S rRNA, followed by the cooperative binding of S6 and S18 . The prior binding of S6 and S18 is required for binding of S11 and S21 . We have studied the mechanism of the cooperative binding of S6 and S18 to the S15-rRNA complex by isothermal titration calorimetry and gel mobility shift assays with rRNA and proteins from the hyperthermophilic bacterium Aquifex aeolicus . S6 and S18 form a stable heterodimer in solution with an apparent dissociation constant of 8.7 nM at 40 degrees C . The S6:S18 heterodimer binds to the S15-rRNA complex with an equilibrium dissociation constant of 2.7 nM at 40 degrees C . Consistent with previous studies using rRNA and proteins from Escherichia coli, we observed no binding of S6 or S18 in the absence of the other protein or S15 . The presence of S15 increases the affinity of S6:S18 for the RNA by at least four orders of magnitude . The kinetics of S6:S18 binding to the S15-rRNA complex are slow, with an apparent bimolecular rate constant of 8.0 x 10(4) M(-1) s(-1) and an apparent unimolecular dissociation rate of 1.6 x 10(-4) s(-1) . These results, which are consistent with a model in which S6 and S18 bind as a heterodimer to the S15-rRNA complex, provide a mechanistic framework to describe the previously observed S15-mediated cooperative binding of S6 and S18 in the ordered assembly of a multi-protein ribonucleoprotein complex .

Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 750 - 6
Carotenoid accumulation in the psychrotrophic bacterium Arthrobacter agilis in response to thermal and salt stress; Fong NJ et al.; A psychrotrophic strain of Arthrobacter agilis, isolated from Antarctic sea ice, grows from 5 degrees C to 40 degrees C and in culture media containing 0-10% (w/v) NaCl . Maximum growth rate occurred at 30-35 degrees C with a drastic decline as the cultivation temperatures diverged . Adaptation to extremes of low temperature may be partially attributed to the production of the C-50 carotenoid bacterioruberin, and its glycosylated derivatives . Lowering of the cultivation temperature resulted in a concomitant increase in carotenoid production, which may contribute to membrane stabilisation at low temperature . Maximum biomass accumulation occurred at 5-30 degrees C with a tenfold reduction at 40 degrees C . Changes in growth rates were minimal in culture media containing 0-2% (w/v) NaCl at 10 degrees C while a gradual decrease in growth rates occurred at higher salinity . Biomass accumulation at different salinity followed a trend similar to that observed with different cultivation temperatures . Maximum biomass accumulation was observed in culture media containing 0-5% (w/v) NaCl with a tenfold reduction at 10% (w/v) NaCl . Carotenoid production also decreased as salinity increased.

J Med Microbiol, 2001 Oct, 50(10), 889 - 95
Reactivity of dog sera to whole-cell or recombinant antigens of Borrelia burgdorferi by ELISA and immunoblot analysis; Magnarelli LA et al.; Enzyme-linked immunosorbent assays (ELISAs) with separate preparations of 10 purified recombinant antigens of Borrelia burgdorferi sensu stricto were used to test sera from 36 dogs not vaccinated with whole cells of this agent and from five dogs vaccinated with whole-cell B . burgdorferi bacteria . All dogs lived in tick-infested areas of Connecticut and south-eastern New York state, USA . The non-vaccinated dogs had limb or joint disorder, lameness and fever during the period 1984-1991 and had antibodies to B . burgdorferi, as determined by a polyvalent ELISA with whole-cell antigen . In re-analyses of sera for total immunoglobulins in ELISAs with recombinant antigens, reactions were most frequently recorded when outer-surface protein (Osp) F, protein (p)35, p37, p39 and p-41G (a flagellin component) were tested separately . Western immunoblots of a subset of 16 sera, positive by ELISA with whole-cell antigen and representing a range of antibody titres (640-40960), verified immune responses to these or other lysed whole-cell antigens . Sera from vaccinated dogs contained antibodies to OspA, OspB, p22, p37 and p41-G . Therefore, serological reactions to OspF, p35 and p39 were the most important indicators of natural exposure to B . burgdorferi . Serum reactivities to these recombinant antigens in ELISAs can be used to help identify possible natural infections of canine borreliosis in dogs not vaccinated with whole-cell B . burgdorferi and to provide information on the geographic distribution of this bacterium.

Infect Immun, 2001 Nov, 69(11), 7121 - 9
Characterization of antiapoptotic activities of Chlamydia pneumoniae in human cells; Fischer SF et al.; Chlamydia pneumoniae is an obligate intracellular bacterium which frequently causes airway infection in humans and has been implicated in atherosclerosis . Here we show that infection with C . pneumoniae protects HeLa human epithelioid cells against apoptosis induced by external stimuli . In infected HeLa cells, apoptosis induced by staurosporine and CD95-death-receptor signaling was strongly reduced . Upon treatment with staurosporine, generation of effector caspase activity, processing of caspase-3 and caspase-9 and cytochrome c redistribution were all profoundly inhibited in cells infected with C . pneumoniae . Bacterial protein synthesis during early infection was required for this inhibition . Furthermore, cytochrome c-induced processing and activation of caspases were inhibited in cytosolic extracts from infected cells, suggesting that a C . pneumoniae-dependent antiapoptotic factor was generated in the cytosol upon infection . Infection with C . pneumoniae failed to induce significant NF-kappaB activation in HeLa cells, indicating that no NF-kappaB-dependent cellular factors were involved in the protection against apoptosis . These results show that C . pneumoniae is capable of interfering with the host cell's apoptotic apparatus at probably at least two steps in signal transduction and might explain the propensity of these bacteria to cause chronic infections in humans.

J Physiol Paris, 2001 Jan-Dec, 95(1-6), 461 - 7
mRNA expression of cytokines and chemokines in the normal gastric surface mucous epithelial cell line GSM06 during bacterial infection with Helicobacter felis; Debreceni A et al.; BACKGROUND AND AIM: A group of the proinflammatory and chemotactic cytokines (chemokines) has been considered as an important factor in the pathomechanism of different bacterial diseases, among them the common Helicobacter pylori infection . Experimental results obtained with gastric biopsy samples of H . pylori positive patients, and with H . pylori infected tumor originated gastric cell lines indicated that these cytokines have essential roles in the development and maintenance of the immune response and inflammation of the gastric mucosa during H . pylori infection . Although the mRNA expression was shown in these biopsy samples and cell lines, it is not yet proved that the normal gastric mucosal epithelial cells themselves express these cytokines . The establishment of a gastric surface mucous cell line with non-tumor origin (GSM06), and the usage of Helicobacter felis as a model of the classic H . pylori infection gave us the possibility to check this question . MATERIALS AND METHODS: in this study GSM06 cells were infected with different numbers (10(5), 10(6), 10(7), 10(8), 10(9) bacterium/ml medium) of H . felis for two different time periods (2, 4 h) . Cells treated with medium only were used as control . Then the mRNA expression of the following cytokines was measured by RT-PCR method in the GSM06 cells: proinflammatory cytokine IL1-beta, and chemokine RANTES, eotaxin, MCP-1, MIP1-alpha and MIP1-beta . RESULTS: we found that neither mRNA of the investigated cytokines was expressed constitutively, however the GSM06 cells expressed the mRNA of each cytokine during H . felis infection . CONCLUSION: our results prove that normal gastric surface mucous epithelial cells express immunologically active peptides during H . felis infection . We may suppose that the epithelial cells of the gastric mucosa contribute to the immune response and inflammation by expressing proinflammatory (IL1-beta) and chemotactic (RANTES, eotaxin, MCP-1, MIP1-alpha and beta) cytokines during H . pylori infection in human.

Arch Biochem Biophys, 2001 Oct 15, 394(2), 265 - 74
Sensitivity of Deinococcus radiodurans to gamma-irradiation: a novel approach by Fourier transform infrared spectroscopy; Melin AM et al.; Deinococcus radiodurans is a red-pigmented coccus known to be particularly resistant to both chemical and radiative agents . Fourier transform infrared (FT-IR) spectroscopy was used as a convenient and easy-to-run method to monitor damage induced in this bacterium by ionizing radiations . First, stationary-phase cultures were submitted to increasing doses of gamma-irradiation ((137)Cs source) . Beyond a threshold of 11 kGy, striking changes occurred in spectra of irradiated samples compared with unirradiated ones, especially in the 1750-900 cm(-1) region, which is spectroscopically assigned to amide I and II components, nucleotide bases, the phosphodiester backbone, and the sugar ring . Second, bacterial cultures were postirradiation reincubated . After a reincubation time of 15 h, the oxidative stress was in part overwhelmed, and the growth of D . radiodurans again occurred, although some biocellular components remained altered . Consequently, FT-IR analysis is an accurate means to rapidly visualize biomolecular changes undergone by cells both after gamma-irradiation and during the repair mechanism .

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1831 - 7
Xenophilus azovorans gen . nov., sp . nov., a soil bacterium that is able to degrade azo dyes of the Orange II type; Blumel S et al.; The taxonomy of strain KF46FT, which was isolated previously after an aerobic enrichment with the azo compound 1-(4'-carboxyphenylazo)-2-naphthol as the sole source of energy and carbon, was investigated by a polyphasic approach . The organism contained a quinone system with ubiquinone Q-8 and 2-hydroxyputrescine and putrescine as the major polyamines, suggesting that strain KF46FT belonged to the beta-subclass of the Proteobacteria . The polar lipid profile consisted mainly of phosphatidylethanolamine and minor amounts of phosphatidylglycerol and diphosphatidylglycerol . Sequencing of the 16S rRNA gene supported its placement in the family Comamonadaceae, but the sequence similarities to the most closely related species of the genera Hydrogenophaga, Acidovorax, Comamonas and Xylophilus were only in the range 95.0 to 96.1% . Different methods for the construction of phylogenetic trees showed the separate position of strain KF46FT 'between' the genera Hydrogenophaga, Variovorax, Comamonas and Xylophilus . Analysis of the fatty acids revealed an unusual profile, with the presence of 8:0 3-OH, 10:0 3-OH, 16:1 2-OH, 16:0 2-OH and 18:1 2-OH in addition to 17:0 cyclo, which is unique among the previously described genera of the family Comamonadaceae . Thus, a new taxon is proposed for strain KF46FT, with the name Xenophilus azovorans gen . nov., sp . nov.

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1715 - 22
Mycobacterium frederiksbergense sp . nov., a novel polycyclic aromatic hydrocarbon-degrading Mycobacterium species; Willumsen P et al.; A polycyclic aromatic hydrocarbon-degrading bacterium isolated from coal tar-contaminated soil in Denmark was characterized by a polyphasic approach . Phylogenetically and chemotaxonomically, it was related to members of the genus Mycobacterium . The isolate contains chemotaxonomic markers that are diagnostic for the genus Mycobacterium; i.e . the meso isomer of 2,6-diaminopimelic acid, arabinose and galactose as diagnostic whole-cell sugars, MK-9(H2) as the principal isoprenoid quinone, a mycolic acid pattern of alpha-mycolates, ketomycolates and wax-ester mycolates, unbranched saturated and unsaturated fatty acids plus a small amount of tuberculostearic acid and a significant amount of a C18:0 secondary alcohol . Based on the unique combination of chemical markers among mycobacteria, it is proposed that the isolate should be assigned to a new species, Mycobacterium frederiksbergense sp . nov . This novel species is phylogenetically closely related to Mycobacterium diernhoferi, Mycobacterium neoaurum and Mycobacterium hodleri . The type strain of M . frederiksbergense is strain FAn9T (= DSM 44346T = NRRL B-24126T).

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1699 - 701
Transfer of Pfennigia purpurea tindall 1999 (Amoebobacter purpureus Eichler and Pfennig 1988) to the genus Lamprocystis as Lamprocystis purpurea comb . nov; Imhoff JF; On the basis of its close phylogenetic relationship to Lamprocystis roseopersicina, the phototrophic purple sulfur bacterium originally described as Amoebobacter purpureus and recently transferred to Pfennigia purpurea is reclassified as Lamprocystis purpurea comb . nov . In addition, an emended description of the genus Lamprocystis is given.

Chin Med J (Engl), 1999 May, 112(5), 392 - 5
Development of a universal immunoenzyme quantitative assay for detecting amplified products of nucleic acid and its preliminary application in hepatitis C virus; Tong W et al.; OBJECTIVE: To develop a universal quantitative immunoenzyme assay (EIA) for detecting amplified products of nucleic acid and its application in hepatitis C virus (HCV) . METHODS: The appropriate cycle number of amplification was selected to stop polymerase chain reaction (PCR) before the "plateau stage" . At the same time, primers HCV (3) of the second PCR were modified with biotin so that the amplified products were labeled . The products were diluted and subsequently added to the streptavidin-coated wells, and the biotinylated products were captured, followed by denaturation of NaOH, and non-biotinylated strands were removed . Hybridization was performed by adding the specific probe labeled with fluorescein . Finally antifluorescein horse radish peroxidase (HRP) conjugates were added, after washing, 3, 3', 5, 5',-tetramethylbenzidine (TMB) was added to the wells and then measured on a microplate reader . RESULTS: EIA detection of amplified products of HCV showed that this assay was rapid, sensitive, specific and accurate . Correlation between the initial number of viral template and the EIA of amplified products was good . We also prospectively investigated the response to interferon in five patients with HCV coinfection . Results showed that this assay could be used as a guidance to the clinical therapy in directing the use of antiviral drugs . CONCLUSIONS: This assay could be widely used as a universal technique for the quantitative detection of amplified products of all nucleic acid (such as virus, bacterium) and other human genes (such as HLA B27), it has vast vistas.

Proc Natl Acad Sci U S A, 2001 Oct 23, 98(22), 12555 - 60 Epub 2001 Oct 09.
A newly discovered bacterium associated with parthenogenesis and a change in host selection behavior in parasitoid wasps; Zchori-Fein E et al.; The symbiotic bacterium Wolbachia pipientis has been considered unique in its ability to cause multiple reproductive anomalies in its arthropod hosts . Here we report that an undescribed bacterium is vertically transmitted and associated with thelytokous parthenogenetic reproduction in Encarsia, a genus of parasitoid wasps . Although Wolbachia was found in only one of seven parthenogenetic Encarsia populations examined, the "Encarsia bacterium" (EB) was found in the other six . Among seven sexually reproducing populations screened, EB was present in one, and none harbored Wolbachia . Antibiotic treatment did not induce male production in Encarsia pergandiella but changed the oviposition behavior of females . Cured females accepted one host type at the same rate as control females but parasitized significantly fewer of the other host type . Phylogenetic analysis based on the 16S rDNA gene sequence places the EB in a unique clade within the Cytophaga-Flexibacter-Bacteroid group and shows EB is unrelated to the Proteobacteria, where Wolbachia and most other insect symbionts are found . These results imply evolution of the induction of parthenogenesis in a lineage other than Wolbachia . Importantly, these results also suggest that EB may modify the behavior of its wasp carrier in a way that enhances its transmission.

J Immunol, 2001 Oct 15, 167(8), 4738 - 46
Identification of HLA-B27-restricted peptides from the Chlamydia trachomatis proteome with possible relevance to HLA-B27-associated diseases; Kuon W et al.; The association of HLA-B27 with ankylosing spondylitis and reactive arthritis is the strongest one known between an MHC class I Ag and a disease . We have searched the proteome of the bacterium Chlamydia trachomatis for HLA-B27 binding peptides that are stimulatory for CD8(+) cells both in a model of HLA-B27 transgenic mice and in patients . This was done by combining two biomathematical computer programs, the first of which predicts HLA-B27 peptide binding epitopes, and the second the probability of HLA-B27 peptide generation by the proteasome system . After preselection, immunodominant peptides were identified by Ag-specific flow cytometry . Using this approach we have identified for the first time nine peptides derived from different C . trachomatis proteins that are stimulatory for CD8(+) T cells . Eight of these nine murine-derived peptides were recognized by cytotoxic T cells . The same strategy was used to identify B27-restricted chlamydial peptides in three patients with reactive arthritis . Eleven peptides were found to be stimulatory for patient-derived CD8(+) T cells, of which eight overlapped those found in mice . Additionally, we applied the tetramer technology, showing that a B27/chlamydial peptide containing one of the chlamydial peptides stained CD8(+) T cells in patients with Chlamydia-induced arthritis . This comprehensive approach offers the possibility of clarifying the pathogenesis of B27-associated diseases.

J Biol Chem, 2002 Jan 4, 277(1), 127 - 34 Epub 2001 Oct 08.
Catabolism of pyrimidine nucleotides in the deep-sea tube worm Riftia pachyptila; Minic Z et al.; The present study describes the distribution and properties of enzymes of the catabolic pathway of pyrimidine nucleotides in Riftia pachyptila, a tubeworm living around deep-sea hydrothermal vents and known to be involved in a highly specialized symbiotic association with a bacterium . The catabolic enzymes, 5'-nucleotidase, uridine phosphorylase, and uracil reductase, are present in all tissues of the worm, whereas none of these enzymatic activities were found in the symbiotic bacteria . The 5'-nucleotidase activity was particularly high in the trophosome, the symbiont-harboring tissue . These results suggest that the production of nucleosides in the trophosome may represent an alternative source of carbon and nitrogen for R . pachyptila, because these nucleosides can be delivered to other parts of the worm . This process would complement the source of carbon and nitrogen from organic metabolites provided by the bacterial assimilatory pathways . The localization of the enzymes participating in catabolism, 5'-nucleotidase and uridine phosphorylase, and of the enzymes involved in the biosynthesis of pyrimidine nucleotides, aspartate transcarbamylase and dihydroorotase, shows a non-homogeneous distribution of these enzymes in the trophosome . The catabolic enzymes 5'-nucleotidase and uridine phosphorylase activities increase from the center of the trophosome to its periphery . In contrast, the anabolic enzymes aspartate transcarbamylase and dihydroorotase activities decrease from the center toward the periphery of the trophosome . We propose a general scheme of anatomical and physiological organization of the metabolic pathways of the pyrimidine nucleotides in R . pachyptila and its bacterial endosymbiont.

J Bacteriol, 2001 Nov, 183(21), 6159 - 68
Functional characterization of three GlnB homologs in the photosynthetic bacterium Rhodospirillum rubrum: roles in sensing ammonium and energy status; Zhang Y et al.; The GlnB (P(II)) protein, the product of glnB, has been characterized previously in the photosynthetic bacterium Rhodospirillum rubrum . Here we describe identification of two other P(II) homologs in this organism, GlnK and GlnJ . Although the sequences of these three homologs are very similar, the molecules have both distinct and overlapping functions in the cell . While GlnB is required for activation of NifA activity in R . rubrum, GlnK and GlnJ do not appear to be involved in this process . In contrast, either GlnB or GlnJ can serve as a critical element in regulation of the reversible ADP ribosylation of dinitrogenase reductase catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase-activating glycohydrolase (DRAG) regulatory system . Similarly, either GlnB or GlnJ is necessary for normal growth on a variety of minimal and rich media, and any of the proteins is sufficient for normal posttranslational regulation of glutamine synthetase . Surprisingly, in their regulation of the DRAT/DRAG system, GlnB and GlnJ appeared to be responsive not only to changes in nitrogen status but also to changes in energy status, revealing a new role for this family of regulators in central metabolic regulation.

Gene, 2001 Oct 3, 276(1-2), 57 - 72
Delineating relative homogeneous G+C domains in DNA sequences; Li W; The concept of homogeneity of G+C content is always relative and subjective . This point is emphasized and quantified in this paper using a simple example of one sequence segmented into two subsequences . Whether the sequence is homogeneous or not can be answered by whether the two-subsequence model describes the DNA sequence better than the one-sequence model . There are at least three equivalent ways of looking at the 1-to-2 segmentation: Jensen-Shannon divergence measure, log likelihood ratio test, and model selection using Bayesian information criterion . Once a criterion is chosen, a DNA sequence can be recursively segmented into multiple domains . We use one subjective criterion called segmentation strength based on the Bayesian information criterion . Whether or not a sequence is homogeneous and how many domains it has depend on this criterion . We compare six different genome sequences (yeast S . cerevisiae chromosome III and IV, bacterium M . pneumoniae, human major histocompatibility complex sequence, longest contigs in human chromosome 21 and 22) by recursive segmentations at different strength criteria . Results by recursive segmentation confirm that yeast chromosome IV is more homogeneous than yeast chromosome III, human chromosome 21 is more homogeneous than human chromosome 22, and bacterial genomes may not be homogeneous due to short segments with distinct base compositions . The recursive segmentation also provides a quantitative criterion for identifying isochores in human sequences . Some features of our recursive segmentation, such as the possibility of delineating domain borders accurately, are superior to those of the moving-window approach commonly used in such analyses.

Int J Biol Macromol, 2001 Oct 22, 29(3), 193 - 202
Structure of Acetobacter cellulose composites in the hydrated state; Astley OM et al.; The structure of composites produced by the bacterium Acetobacter xylinus have been studied in their natural, hydrated, state . Small-angle X-ray diffraction and environmental scanning electron microscopy has shown that the ribbons have a width of 500 A and contain smaller semi-crystalline cellulose microfibrils with an essentially rectangular cross-section of approximately 10 x 160 A(2) . Incubation of Acetobacter in xyloglucan or pectin results in no changes in the size of either the microfibrils or the ribbons . Changes in the cellulose crystals are seen upon dehydration of the material, resulting in either a reduction in crystal size or an increase in crystal disorder.

Peptides, 2001 Oct, 22(10), 1597 - 601
A two-component signal-transduction cascade in Carnobacterium piscicola LV17B: two signaling peptides and one sensor-transmitter; Kleerebezem M et al.; In the lactic acid bacterium Carnobacterium piscicola LV17B a peptide-pheromone dependent quorum-sensing mode is involved in the regulation of bacteriocin production . Bacteriocin CB2 was identified as an environmental signal that induces bacteriocin production . Here, we demonstrate that a second 24 amino acid peptide (CS) also induces bacteriocin production . Transcription activation of several carnobacteriocin operons is triggered by CB2 or CS via a two-component signal transduction system composed of CbnK and CbnR.

Front Biosci, 2001 Oct 01, 6, E104 - 18
Effects of H . pylori infection of gastric epithelial cells on cell cycle control; Shirin H et al.; Chronic infection of the gastric mucosa by the bacterium H . pylori results in an intense inflammatory response which can last for decades . An associated host response is a chronic hyperproliferative state, in which there is increased cell turnover and also increased apoptosis of the gastric epithelial cells . Recent studies have also demonstrated abnormalities in the expression of cell cycle control proteins . This review describes these events, emphasizing recent studies on the effects of H . pylori infection on cell cycle progression and the expression of cell cycle regulatory proteins . The systems that have been studied include in vivo studies in humans and in experimental animals, and in vitro studies in which gastric epithelial cells were co-cultivated with H . pylori . The earliest event following H . pylori's interaction with epithelial cells appears to be growth inhibition and apoptosis . The hyperproliferative response observed in the gastric mucosa is secondary to this initial insult and is associated with increased expression of cyclin D1, the cyclin dependent kinase inhibitor p16ink4a and of p53 and decreased expression of the cyclin dependent kinase inhibitor p27kip1 . Dysregulation of the hyperproliferative response may, ultimately, be responsible for the ability of H . pylori to enhance the development of gastric cancer.

J Periodontol, 2001 Sep, 72(9), 1172 - 7
Effects of whole cell sonicates of Treponema lecithinolyticum on osteoclast differentiation; Choi BK et al.; BACKGROUND: Alveolar bone destruction is a characteristic feature of periodontal diseases and multinucleated osteoclast cells derived from hemopoietic cells are responsible for bone resorption . Treponema lecithinolyticum is a novel oral spirochete isolated from the periodontal lesions . METHODS: The effect of whole cell sonicates on the osteoclast differentiation was examined in a co-culture system of hemopoietic mouse bone marrow cells and calvaria derived-osteoblastic cells to clarify the role of T . lecithinolyticum in the alveolar bone destruction associated with periodontal diseases . The differentiated osteoclasts were confirmed by tartrate-resistant acid phosphatase (TRAP) staining . RESULTS: Sonicates of this bacterium stimulated the osteoclast formation in the co-culture system in a dose-dependent manner . The sonicates-induced osteoclast formation was partially inhibited by the heat treatment of sonicates . Indomethacin, which is a prostaglandin inhibitor, decreased the osteoclast formation induced by the bacterial sonicates . CONCLUSIONS: These findings suggest that T . lecithinolyticum induces osteoclast differentiation by a prostaglandin E2-dependent mechanism and that heat-labile components may be involved in this process.

Nat Struct Biol, 2001 Oct, 8(10), 848 - 52
Crystal structure and kinetic analysis of beta-lactamase inhibitor protein-II in complex with TEM-1 beta-lactamase; Lim D et al.; The structure of the 28 kDa beta-lactamase inhibitor protein-II (BLIP-II) in complex with the TEM-1 beta-lactamase has been determined to 2.3 A resolution . BLIP-II is a secreted protein produced by the soil bacterium Streptomyces exfoliatus SMF19 and is able to bind and inhibit TEM-1 with subnanomolar affinity . BLIP-II is a seven-bladed beta-propeller with a unique blade motif consisting of only three antiparallel beta-strands . The overall fold is highly similar to the core structure of the human regulator of chromosome condensation (RCC1) . Although BLIP-II does not share the same fold with BLIP, the first beta-lactamase inhibitor protein for which structural data was available, a comparison of the two complexes reveals a number of similarities and provides further insights into key components of the TEM-1-BLIP and TEM-1-BLIP-II interfaces . Our preliminary results from gene knock-out studies and scanning electron microscopy also reveal a critical role of BLIP-II in sporulation.

Clin Lab Med, 2001 Sep, 21(3), 619 - 29, x
The emerging impact of genomics on the development of biological weapons . Threats and benefits posed by engineered extremophiles; Daly MJ; During the past decades, representatives of Archaea, Bacteria, and Protista have been found thriving in many newly discovered extremely hostile habitats, which hitherto were regarded as too harsh to harbor life . To illustrate how an extremophile could be targeted for development as a biowarfare agent, an example is presented describing current advances in engineering Deinococcus radiodurans . Using a generally applicable combination of conventional genetic engineering and genomic informatics, this extremely radiation-resistant and environmentally robust bacterium is being developed for biotechnology.

Appl Environ Microbiol, 2001 Oct, 67(10), 4922 - 5
Isolation of methyl parathion-degrading strain M6 and cloning of the methyl parathion hydrolase gene; Zhongli C et al.; A degradative bacterium, M6, was isolated and presumptively identified as Plesiomonas sp . strain M6 was able to hydrolyze methyl parathion to p-nitrophenol . A novel organophosphate hydrolase gene designated mpd was selected from its genomic library prepared by shotgun cloning . The nucleotide sequence of the mpd gene was determined . The gene could be effectively expressed in Escherichia coli.

Appl Environ Microbiol, 2001 Oct, 67(10), 4834 - 41
Isolation and characterization of intracellular protein inclusions produced by the entomopathogenic bacterium Photorhabdus luminescens; Bowen DJ et al.; Cells of the entomopathogenic bacterium Photorhabdus luminescens contain two types of morphologically distinct crystalline inclusion proteins . The larger rectangular inclusion (type 1) and a smaller bipyramid-shaped inclusion (type 2) were purified from cell lysates by differential centrifugation and isopycnic density gradient centrifugation . Both structures are composed of protein and are readily soluble at pH 11 and 4 in 1% sodium dodecyl sulfate (SDS) and in 8 M urea . Electrophoretic analysis reveals that each inclusion is composed of a single protein subunit with a molecular mass of 11,000 Da . The proteins differ in amino acid composition, protease digestion pattern, and immunological cross-reactivity . The protein inclusions are first visible in the cells at the time of late exponential growth . Western blot analyses showed that the proteins appeared in cells during mid- to late exponential growth . When at maximum size in stationary-phase cells, the proteins constitute 40% of the total cellular protein . The protein inclusions are not used during long-term starvation of the cells and were not toxic when injected into or fed to Galleria mellonella larvae.

Appl Environ Microbiol, 2001 Oct, 67(10), 4583 - 7
Reduction of technetium(VII) by Desulfovibrio fructosovorans is mediated by the nickel-iron hydrogenase; De Luca G et al.; Resting cells of the sulfate-reducing bacterium Desulfovibrio fructosovorans grown in the absence of sulfate had a very high Tc(VII)-reducing activity, which led to the formation of an insoluble black precipitate . The involvement of a periplasmic hydrogenase in Tc(VII) reduction was indicated (i) by the requirement for hydrogen as an electron donor, (ii) by the tolerance of this activity to oxygen, and (iii) by the inhibition of this activity by Cu(II) . Moreover, a mutant carrying a deletion in the nickel-iron hydrogenase operon showed a dramatic decrease in the rate of Tc(VII) reduction . The restoration of Tc(VII) reduction by complementation of this mutation with nickel-iron hydrogenase genes demonstrated the specific involvement of the periplasmic nickel-iron hydrogenase in the mechanism in vivo . The Tc(VII)-reducing activity was also observed with cell extracts in the presence of hydrogen . Under these conditions, Tc(VII) was reduced enzymatically to soluble Tc(V) or precipitated to an insoluble black precipitate, depending on the chemical nature of the buffer used . The purified nickel-iron hydrogenase performed Tc(VII) reduction and precipitation at high rates . These series of genetic and biochemical approaches demonstrated that the periplasmic nickel-iron hydrogenase of sulfate-reducing bacteria functions as a Tc(VII) reductase . The role of cytochrome c(3) in the mechanism is also discussed.

Appl Environ Microbiol, 2001 Oct, 67(10), 4573 - 82
A large gene cluster encoding several magnetosome proteins is conserved in different species of magnetotactic bacteria; Grunberg K et al.; In magnetotactic bacteria, a number of specific proteins are associated with the magnetosome membrane (MM) and may have a crucial role in magnetite biomineralization . We have cloned and sequenced the genes of several of these polypeptides in the magnetotactic bacterium Magnetospirillum gryphiswaldense that could be assigned to two different genomic regions . Except for mamA, none of these genes have been previously reported to be related to magnetosome formation . Homologous genes were found in the genome sequences of M . magnetotacticum and magnetic coccus strain MC-1 . The MM proteins identified display homology to tetratricopeptide repeat proteins (MamA), cation diffusion facilitators (MamB), and HtrA-like serine proteases (MamE) or bear no similarity to known proteins (MamC and MamD) . A major gene cluster containing several magnetosome genes (including mamA and mamB) was found to be conserved in all three of the strains investigated . The mamAB cluster also contains additional genes that have no known homologs in any nonmagnetic organism, suggesting a specific role in magnetosome formation.

Appl Environ Microbiol, 2001 Oct, 67(10), 4440 - 7
Effect of selenite on growth and protein synthesis in the phototrophic bacterium Rhodobacter sphaeroides; Bebien M et al.; The effect of selenite on the growth rate and protein synthesis has been investigated in Rhodobacter sphaeroides . This photosynthetic bacterium efficiently reduces selenite with intracellular accumulation under both dark aerobic and anaerobic photosynthetic conditions . Addition of 1 mM selenite under these two growth conditions does not affect the final cell density, although a marked slowdown in growth rate is observed under aerobic growth . The proteome analysis of selenite response by two-dimensional gel electrophoresis shows an enhanced synthesis of some chaperones, an elongation factor, and enzymes associated to oxidative stress . The induction of these antioxidant proteins confirms that the major toxic effect of selenite is the formation of reactive oxygen species during its metabolism . In addition, we show that one mutant unable to precipitate selenite, selected from a transposon library, is affected in the smoK gene . This encodes a constituent of a putative ABC transporter implicated in the uptake of polyols . This mutant is less sensitive to selenite and does not express stress proteins identified in the wild type in response to selenite . This suggests that the entry of selenite into the cytoplasm is mediated by a polyol transporter in R . sphaeroides.

Appl Environ Microbiol, 2001 Oct, 67(10), 4432 - 9
Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth; Strahl ED et al.; Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis . There was a linear relationship between FAM and colony counts of M . avium cells both from cultures and within protozoa . The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles . Starved, 7-day-old cultures of T . pyriformis transferred into fresh medium readily phagocytized M . avium, M . intracellulare, and M . scrofulaceum . Phagocytosis was rapid and reached a maximum in 30 min . M . avium, M . intracellulare, and M . scrofulaceum grew within T . pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30 degrees C . Intracellular M . avium numbers remained constant over a 25-day period of growth (by transfer) of T . pyriformis . Intracellular M . avium cells also survived protozoan encystment and germination . The growth and viability of T . pyriformis were not affected by mycobacterial infection . The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M . avium, M . intracellulare, and M . scrofulaceum.

J Bacteriol, 2001 Oct, 183(20), 6107 - 18
Cytochrome complex essential for photosynthetic oxidation of both thiosulfate and sulfide in Rhodovulum sulfidophilum; Appia-Ayme C et al.; Many photosynthetic bacteria use inorganic sulfur compounds as electron donors for carbon dioxide fixation . A thiosulfate-induced cytochrome c has been purified from the photosynthetic alpha-proteobacterium Rhodovulum sulfidophilum . This cytochrome c(551) is a heterodimer of a diheme 30-kDa SoxA subunit and a monoheme 15-kDa SoxX subunit . The cytochrome c(551) structural genes are part of an 11-gene sox locus . Sequence analysis suggests that the ligands to the heme iron in SoxX are a methionine and a histidine, while both SoxA hemes are predicted to have unusual cysteine-plus-histidine coordination . A soxA mutant strain is unable to grow photoautotrophically on or oxidize either thiosulfate or sulfide . Cytochrome c(551) is thus essential for the metabolism of both these sulfur species . Periplasmic extracts of wild-type R . sulfidophilum exhibit thiosulfate:cytochrome c oxidoreductase activity . However, such activity can only be measured for a soxA mutant strain if the periplasmic extract is supplemented with purified cytochrome c(551) . Gene clusters similar to the R . sulfidophilum sox locus can be found in the genome of a green sulfur bacterium and in phylogenetically diverse nonphotosynthetic autotrophs.

J Bacteriol, 2001 Oct, 183(20), 5991 - 6
RNA polymerase sigma factor that blocks morphological differentiation by Streptomyces coelicolor; Gehring AM et al.; The filamentous bacterium Streptomyces coelicolor undergoes a complicated process of morphological differentiation that begins with the formation of an aerial mycelium and culminates in sporulation . Genes required for the initiation of aerial mycelium formation have been termed bld (bald), describing the smooth, undifferentiated colonies of mutant strains . By using an insertional mutagenesis protocol that relies on in vitro transposition, we have isolated a bld mutant harboring an insertion in a previously uncharacterized gene, SCE59.12c, renamed here rsuA . The insertion mutant exhibited no measurable growth defect but failed to produce an aerial mycelium and showed a significant delay in the production of the polyketide antibiotic actinorhodin . The rsuA gene encodes an apparent anti-sigma factor and is located immediately downstream of SCE59.13c, renamed here sigU, whose product is inferred to be a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors . The absence of rsuA in a strain that contained sigU caused a block in development, and the overexpression of sigU in an otherwise wild-type strain caused a delay in aerial mycelium formation . However, a strain in which both rsuA and sigU had been deleted was able to undergo morphological differentiation normally . We conclude that the rsuA-encoded anti-sigma factor is responsible for antagonizing the function of the sigma factor encoded by sigU . We also conclude that the sigU-encoded sigma factor is not normally required for development but that its uncontrolled activity obstructs morphological differentiation at an early stage.

Biochemistry (Mosc), 2001 Aug, 66(8), 894 - 7
O-specific polysaccharide of the marine bacterium "Alteromonas marinoglutinosa" NCIMB 1770; Komandrova NA et al.; The O-specific polysaccharide of the marine bacterium "Alteromonas marinoglutinosa" NCIMB 1770 was obtained by mild acid degradation of the corresponding lipopolysaccharide and found to contain D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosamine residues in equimolar ratio . Based on methylation analysis, periodate oxidation, and 13C-NMR spectroscopy data of native and modified polysaccharides, the following structure of the trisaccharide repeating unit of the O-specific polysaccharide was established: {structure: see text}

Microb Pathog, 2001 Oct, 31(4), 173 - 84
Chlamydia trachomatis-induced apoptosis occurs in uninfected McCoy cells late in the developmental cycle and is regulated by the intracellular redox state; Schoier J et al.; Infections with the obligate intracellular bacterium Chlamydia trachomatis are characterized by avoidance of fusion between chlamydia-containing endosomes and lysosomes, bacterial persistence and development of post-infectious sequelae . In this report we show that C . trachomatis induces apoptosis in McCoy and HeLa cells . Apoptosis was monitored by three different techniques; enzyme-linked immunoassay (EIA) of fragmented nucleosomes, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and flow cytometry of propidium iodide-stained cells . Apoptosis occurred in uninfected cells, was induced late in the chlamydial developmental cycle, beyond 24 h post-infection and was dependent on bacterial protein synthesis . Apoptosis was not significantly increased in infected, inclusion-containing cells . Treatment of cells with the antioxidants ascorbic acid (10 microM) and alpha-tocopherol (10 microM) reduced the degree of apoptosis . These results suggest that host cells infected with C . trachomatis generate proapoptotic stimuli that induce apoptosis in uninfected, neighbouring cells and that the redox state of the cell is a regulator in chlamydia-induced apoptosis .

Health Soc Care Community, 1998 Mar, 6(2), 78 - 83
Helicobacter pylori: an assessment of public awareness and acceptance of screening; Stone MA et al.; Research studies involving population screening could be important in assessing short and long-term benefits of eradicating H . pylori . Compliance with screening is an important factor to be considered . Two population studies sought to assess public awareness of H . pylori, interest in screening for the bacterium, and the acceptability of two blood tests . One hundred adults were interviewed in a street survey in Market Harborough, Leicestershire . Although only 4-14% of those interviewed had heard of H . pylori, 73% expressed an interest in screening . In the second study, a population sample of 50 men and women aged 21-55 years, from a general practice in the same town, were offered screening . This sample was divided into two matched groups of 25; one group was offered a test involving venepuncture and the other group was offered a 'near-patient' finger prick test . To confirm results, this exercise was subsequently repeated with a further two groups of 25 subjects . There was an overall acceptance rate of 41% for the actual offer of screening . Overall, 28/50 subjects (56%) accepted venepuncture, but only 13/50 (26%) accepted the finger prick test.

Biofizika, 2001 Jul-Aug, 46(4), 625 - 30
{A viscosimetric study of thermally induced release of ds-DNA from Un phage}; Mdzinarashvili TD et al.; The thermally induced ejection of DNA from the head of the Un phage was studied by viscosimetry, pH was used as a variable external factor . The results obtained suggest that the infection of the bacterium by the phage occurs in the pH range 6-10 rather than in the acidic medium (pH 4-5) because the infection can take place only if the DNA is completely ejected from the phage head . It was shown that the ejection of DNA upon dilution of the initial concentration of the phage depends on the time required for the equilibrium of the phage suspension in the corresponding buffer solution to be established after which the measurements can be performed . In our experiments, this time interval was 24 h.

Science, 2001 Sep 14, 293(5537), 2093 - 8
Mechanisms of evolution in Rickettsia conorii and R . prowazekii; Ogata H et al.; Rickettsia conorii is an obligate intracellular bacterium that causes Mediterranean spotted fever in humans . We determined the 1,268,755-nucleotide complete genome sequence of R . conorii, containing 1374 open reading frames . This genome exhibits 804 of the 834 genes of the previously determined R . prowazekii genome plus 552 supplementary open reading frames and a 10-fold increase in the number of repetitive elements . Despite these differences, the two genomes exhibit a nearly perfect colinearity that allowed the clear identification of different stages of gene alterations with gene remnants and 37 genes split in 105 fragments, of which 59 are transcribed . A 38-kilobase sequence inversion was dated shortly after the divergence of the genus.

J Biol Chem, 2001 Dec 21, 276(51), 48183 - 8 Epub 2001 Sep 13.
A magnetosome-specific GTPase from the magnetic bacterium Magnetospirillum magneticum AMB-1; Okamura Y et al.; Magnetic bacteria produce intracellular vesicles that envelope single domain magnetite crystals . Although many proteins are present in this intracellular vesicle membrane, five are specific to this membrane . A 16-kDa protein, designated Mms16, is the most abundant of the magnetosome-specific proteins, and to establish its function we cloned and sequenced its gene from Magnetospirillum magneticum AMB-1 . This was achieved by determination of the N-terminal amino acid sequence of the protein following two dimensional polyacrylamide gel electrophoresis, and sequencing of the gene was performed by gene walking using anchored polymerase chain reaction . Mms16 contains a putative ATP/GTP binding motif (P-loop) . Recombinant Mms16 with a hemagglutinin tag, was expressed in Escherichia coli and purified . Recombinant Mms16 protein could bind GTP and showed GTPase activity . GTP was the preferred substrate for Mms16-catalyzed nucleotide triphosphate hydrolysis . These results suggest that a novel protein specifically localized on the magnetic particle membrane, Mms16, is a GTPase . Mms16 protein showed similar characteristics to small GTPases involved in the formation of intracellular vesicles . Furthermore, addition of the GTPase inhibitor AlF(4)- also inhibited magnetic particle synthesis, suggesting that GTPase is required for magnetic particles synthesis.

Antimicrob Agents Chemother, 2001 Oct, 45(10), 2871 - 6
Inhibition of trypsin-like cysteine proteinases (gingipains) from Porphyromonas gingivalis by tetracycline and its analogues; Imamura T et al.; Extracellular cysteine proteinases, referred to as gingipains, are considered important virulence factors for Porphyromonas gingivalis, a bacterium recognized as a major etiologic agent of chronic periodontitis . We investigated the effect of tetracycline and its analogues, doxycycline and minocycline, on the enzymatic activities of gingipains . Tetracyclines at 100 microM totally inhibited the amidolytic activity of arginine-specific gingipains (HRgpA and RgpB) . In contrast, inhibition of Kgp was less efficient and required a somewhat higher concentration of the antibiotic to achieve the same effect . Among tetracycline derivatives, the most potent gingipain inhibitor was doxycycline, followed by tetracycline and minocycline . RgpB was inhibited by doxycycline in an uncompetitive and reversible manner with a 50% inhibitory concentration of 3 microM . Significantly, inhibition was unaffected by calcium, excluding the chelating activity of tetracyclines as the mechanism of gingipain inactivation . In contrast, the inhibitory activities of the tetracyclines were reduced by cysteine, a reducing agent, suggesting an interference of the drug at the oxidative region with the catalytic system of the enzyme . Doxycycline, at 10 microM, significantly inhibited the RgpB-mediated production of vascular permeability-enhancing activity from human plasma, thus proving an effective inhibition of gingipain in vivo . These results indicate a new activity of tetracyclines as cysteine proteinase inhibitors and may explain the therapeutic efficiency of these antibiotics in the treatment of periodontitis.

Oral Microbiol Immunol, 2001 Oct, 16(5), 296 - 305
Soluble products from Eikenella corrodens stimulate oral epithelial cells to induce inflammatory mediators; Yumoto H et al.; In the inflammatory response elicited by bacterial colonization in periodontal pockets, pocket epithelial cells not only serve as a barrier to isolate the pocket microenvironment from external stimuli but also regulate the functions of neighboring cells including fibroblasts and inflammatory cells . To elucidate this mechanism, we characterized the effects of periodontopathic bacterium Eikenella corrodens 1073 components on the production of some inflammatory mediators in a human oral epithelial cell line (KB) . In enzyme-linked immunosorbent assay (ELISA), the E . corrodens supernatant induced interleukin-6 (IL-6), IL-8 and prostaglandin E2 but not interferon-gamma (IFN-gamma) production by KB cells . After incubation with E . corrodens supernatant, KB cells showed a marked increase in the levels of IL-6, IL-8 and PG G/H synthase (cyclooxygenase)-2, but not IFN-gamma, gene expression by reverse-transcriptase polymerase chain reaction . All these E . corrodens products responsible for production of these inflammatory mediators resisted freezing and boiling and were present in a 10-kDa filtrate . These results imply that these soluble small-molecular-mass products from E . corrodens stimulate various inflammatory mediator productions by human oral epithelial cells and may play a role in the initiation of periodontal inflammation and subsequently perpetuate the inflammatory response during chronic infection.

Plant Physiol, 2001 Sep, 127(1), 78 - 89
Srchi24, a chitinase homolog lacking an essential glutamic acid residue for hydrolytic activity, is induced during nodule development on Sesbania rostrata; Goormachtig S et al.; The interaction between the tropical legume Sesbania rostrata and the bacterium Azorhizobium caulinodans results in the formation of nodules on both stem and roots . Stem nodulation was used as a model system to isolate early markers by differential display . One of them, Srchi24 is a novel early nodulin whose transcript level increased already 4 h after inoculation . This enhancement depended on Nod factor-producing bacteria . Srchi24 transcript levels were induced also by exogenous cytokinins . In situ hybridization and immunolocalization experiments showed that Srchi24 transcripts and proteins were present in the outermost cortical cell layers of the developing nodules . Sequence analyses revealed that Srchi24 is similar to class III chitinases, but lacks an important catalytic glutamate residue . A fusion between a maltose-binding protein and Srchi24 had no detectable hydrolytic activity . A function in nodulation is proposed for the Srchi24 protein.

Infect Immun, 2001 Oct, 69(10), 6172 - 8
Transcriptional analysis of p30 major outer membrane multigene family of Ehrlichia canis in dogs, ticks, and cell culture at different temperatures; Unver A et al.; Ehrlichia canis, an obligatory intracellular bacterium of monocytes and macrophages, causes canine monocytic ehrlichiosis . E . canis immunodominant 30-kDa major outer membrane proteins are encoded by a polymorphic multigene family consisting of more than 20 paralogs . In the present study, we analyzed the mRNA expression of 14 paralogs in experimentally infected dogs and Rhipicephalus sanguineus ticks by reverse transcription-PCR using gene-specific primers followed by Southern blotting . Eleven out of 14 paralogs in E . canis were transcribed in increasing numbers and transcription levels, while the mRNA expression of the 3 remaining paralogs was not detected in blood monocytes of infected dogs during the 56-day postinoculation period . Three different groups of R . sanguineus ticks (adult males and females and nymphs) were separately infected with E . canis by feeding on the infected dogs . In these pools of acquisition-fed ticks as well as in the transmission-fed adult ticks, the transcript from only one paralog was detected, suggesting the predominant transcription of that paralog or the suppression of the remaining paralogs in ticks . Expression of the same paralog was higher whereas expression of the remaining paralogs was lower in E . canis cultivated in dog monocyte cell line DH82 at 25 degrees C than in E . canis cultivated at 37 degrees C . Analysis of differential expression of p30 multigenes in dogs, ticks, or monocyte cell cultures would help in understanding the role of these gene products in pathogenesis and E . canis transmission as well as in designing a rational vaccine candidate immunogenic against canine ehrlichiosis.

Trends Microbiol, 2001 Sep, 9(9), 405 - 7
Global analysis of a bacterial cell cycle: tracking down necessary functions and their regulators; Brun YV; New, post-genomic analyses are increasing the rate at which information about highly complex processes such as bacterial growth and development can be acquired . The recent use of DNA-microarray and proteomic analysis to study the differentiating bacterium Caulobacter crescentus has provided the first global view of the requirements of a bacterium as it progresses through its cell cycle . Potential regulators of cell cycle progression have been identified, and it has been suggested that proteolysis could have a global role in regulating the bacterial cell cycle.

Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 486 - 90
Mineralization and co-metabolic degradation of phenoxyalkanoic acid herbicides by a pure bacterial culture isolated from an aquifer; Mai P et al.; A mecoprop {(+/-)-2-(4-chloro-2-methylphenoxy)propionic acid; MCPP}-degrading bacterium identified as Stenotrophomonas maltophilia PM was isolated from a Danish aquifer . Besides mecoprop, the bacterium was also able to degrade MCPA {(4-chloro-2-methylphenoxy)acetic acid)}, MCPB {(4-chloro-2-methylphenoxy)butyric acid}, 4-CPA {(4-chlorophenoxy)acetic acid}, 2, 4-D {(2, 4-dichlorophenoxy)acetic acid}, 2, 4-DP {(+/-)-2-(2, 4-dichlorophenoxy)propionic acid} and 2, 4-DB {(2, 4-dichlorophenoxy)butyric acid} . The bacterium was able to grow using these individual phenoxyalkanoic acids as the sole source of carbon and energy . In addition, it was able to co-metabolically degrade the phenoxyalkanoic acid 2, 4, 5-T {(2, 4, 5-trichlorophenoxy)acetic acid)} in the presence of mecoprop . At high 2, 4, 5-T concentrations (100 and 52 mg/l), however, only partial degradation of both mecoprop and 2, 4, 5-T was obtained, thus indicating the production of toxic metabolites . Bacterial yields were highest when grown on the monochlorinated phenoxyalkanoic acids as compared to the dichlorinated analogues, an exception being growth on 4CPA, which resulted in the lowest yield at all . Using {ring-U-14C}-labeled herbicides it was shown that the lower yield on 2, 4-D than on mecoprop was accompanied by greater CO2 generation, thus indicating that less energy is available from the complete oxidation of the dichlorinated phenoxyalkanoic acids than the monochlorinated analogues.

Stroke, 2001 Sep, 32(9), 1973 - 6
Chlamydia pneumoniae in atherosclerotic middle cerebral artery; Virok D et al.; BACKGROUND AND PURPOSE: Atherosclerotic middle cerebral arteries are frequent sites of thrombosis, leading to stroke . Previous studies have suggested a role for Chlamydia pneumoniae in the pathogenesis of atherosclerosis . However, the presence of this pathogen in atherosclerotic middle cerebral arteries has heretofore not been documented . In the present study, we analyzed atheromatous plaques from middle cerebral arteries for the presence of C pneumoniae . METHODS: Atherosclerotic middle cerebral arteries from 15 cadavers who died of natural causes and corresponding nonatherosclerotic arteries from 4 otherwise healthy trauma victims were examined . Assays for C pneumoniae DNA were carried out by nested polymerase chain reaction (nPCR) specific for the C pneumoniae ompA gene . The presence of the bacterium was assessed by transmission electron microscopy . RESULTS: Five of the 15 atherosclerotic arterial samples and none of the control tissues were positive for C pneumoniae by nPCR . Particles similar in morphology and size to C pneumoniae elementary bodies were detected by transmission electron microscopy in 4 of the 5 nPCR-positive atherosclerotic samples . CONCLUSIONS: The demonstration of C pneumoniae in atherosclerotic middle cerebral arteries is consistent with the hypothesis that this bacterium is involved in acute and chronic cerebrovascular diseases.

Adv Space Res, 2000, 25(10), 2103 - 6
Effect of the space environment on the induction of DNA-repair related proteins and recovery from radiation damage; Kobayashi Y et al.; Recovery of bacterial cells from radiation damage and the effects of microgravity were examined in an STS-79 Shuttle/Mir Mission-4 experiment using the extremely radioresistant bacterium Deinococcus radiodurans . The cells were irradiated with gamma rays before the space flight and incubated on board the Space-Shuttle . The survival of the wild type cells incubated in space increased compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under microgravity . No difference was observed for the survival of radiosensitive mutant rec30 cells whether incubated in space or on the ground . The amount of DNA-repair related RecA protein induced under microgravity was similar to those of ground controls, however, induction of PprA protein, the product of a newly found gene related to the DNA repair mechanism of D . radiodurans, was enhanced under microgravity compared with ground controls.

Arch Microbiol, 1987, 147, 213 - 20
Association of a new type of gliding, filamentous, purple phototrophic bacterium inside bundles of Microcoleus chthonoplastes in hypersaline cyanobacterial mats; D'Amelio ED et al.; An unidentified filamentous purple bacterium, probably belonging to a new genus or even a new family, is found in close association with the filamentous, mat-forming cyanobacterium Microcoleus chthonoplastes in a hypersaline pond at Guerrero Negro, Baja California Sur, Mexico, and in Solar Lake, Sinai, Egypt . This organism is a gliding, segmented trichome, 0.8-0.9 micrometer wide . It contains intracytoplasmic stacked lamellae which are perpendicular and obliquely oriented to the cell wall, similar to those described for the purple sulfur bacteria Ectothiorhodospira . These bacteria are found inside the cyanobacterial bundle, enclosed by the cyanobacterial sheath . Detailed transmission electron microscopical analyses carried out in horizontal sections of the upper 1.5 mm of the cyanobacterial mat show this cyanobacterial-purple bacterial association at depths of 300-1200 micrometers, corresponding to the zone below that of maximal oxygenic photosynthesis . Sharp gradients of oxygen and sulfide are established during the day at this microzone in the two cyanobacterial mats studied . The close association, the distribution pattern of this association and preliminary physiological experiments suggest a co-metabolism of sulfur by the two-membered community . This probable new genus of purple bacteria may also grow photoheterotrophically using organic carbon excreted by the cyanobacterium . Since the chemical gradients in the entire photic zone fluctuate widely in a diurnal cycle, both types of metabolism probably take place . During the morning and afternoon, sulfide migrates up to the photic zone allowing photoautotrophic metabolism with sulfide as the electron donor . During the day the photic zone is highly oxygenated and the purple bacteria may either use oxidized species of sulfur such as elemental sulfur and thiosulfate in the photoautotrophic mode or grow photoheterotrophically using organic carbon excreted by M . chthonoplastes . The new type of filamentous purple sulfur bacteria is not available yet in pure culture, and its taxonomical position cannot be fully established . This organism is suggested to be a new type of gliding, filamentous, purple phototroph.

Acta Hortic, 1996 Dec, 440, 75 - 80
Measurement of Fe2+ ion by coulometry method at incubation of Thiobacillus ferrooxidans; Tsuda I et al.; Thiobacillus ferrooxidans is a chemoautotrophic bacterium that is capable of using Fe2+ oxidation by O2 as the sole source of energy for growth and CO2 fixation . The idea of the solar bacterial biomass farm by using of this bacterium is proposed . The incubation experiment of these bacteria was carried out, and the 9K culture medium as the standard medium for T . ferrooxidans was used . The measurement of Fe2+ in the growth stage was carried out as the first step of the experiments to clarify the possibility of this system . The items of measurement were Fe2+ ion density, pH of the medium, bacterium density and quantity of total organic carbon (TOC) . The density of Fe2+ ion in the medium was measured by coulometry method . This method has the following advantage, high accuracy (<1%), easy operation, short measurement time (a few minutes) and small sample quantity (about 0.1 ml) . The experimental results show that the Fe 2+ ion density is measured as same as the accuracy of pH measurement . At the final stage of the growth, the pH decreased due to the generation of the iron hydroxide (Fe(OH)3) . The bacterium density and TOC slightly increased after that Fe2+ runs short . This result shows that the CO2 fixation speed is slower than Fe2+ oxidation speed . It is shown by the experiment that the growth limit of T . ferrooxidans is caused by the disappearance of the Fe2+ ion . It may be possible that the bacterium density increases by the continuous supply of Fe2+ ion.

Photosynth Res, 1990, 24, 47 - 53
A reverse KREBS cycle in photosynthesis: consensus at last; Buchanan BB et al.; The Krebs cycle (citric acid or tricarboxylic acid cycle), the final common pathway in aerobic metabolism for the oxidation of carbohydrates, fatty acids and amino acids, is known to be irreversible . It liberates CO2 and generates NADH whose aerobic oxidation yields ATP but it does not operate in reverse as a biosynthetic pathway for CO2 assimilation . In 1966, our laboratory described a cyclic pathway for CO2 assimilation (Evans, Buchanan and Arnon 1966) that was unusual in two respects: (i) it provided the first instance of an obligate photoautotroph that assimilated CO2 by a pathway different from Calvin's reductive pentose phosphate cycle (Calvin 1962) and (ii) in its overall effect the new cycle was a reversal of the Krebs cycle . Named the 'reductive carboxylic acid cycle' (sometimes also called the reductive tricarboxylic acid cycle) the new cycle appeared to be the sole CO2 assimilation pathway in Chlorobium thiosulfatophilum (Evans et al . 1966) (now known as Chlorobium limicola forma thiosulfatophilum) . Chlorobium is a photosynthetic green sulfur bacterium that grows anaerobically in an inorganic medium with sulfide and thiosulfate as electron donors and CO2 as an obligatory carbon source . In the ensuing years, the new cycle was viewed with skepticism . Not only was it in conflict with the prevailing doctrine that the 'one important property .. . shared by all (our emphasis) autotrophic species is the assimilation of CO2 via the Calvin cycle' (McFadden 1973) but also some of its experimental underpinnings were challenged . It is only now that in the words of one of its early skeptics (Tabita 1988) 'a long and tortuous controversy' has ended with general acceptance of the reductive carboxylic acid cycle as a photosynthetic CO2 assimilation pathway distinct from the pentose cycle . (Henceforth, to minimize repetitiveness, the reductive pentose phosphate cycle will often be referred to as the pentose cycle and the reductive carboxylic acid cycle as the carboxylic acid cycle.) Aside from photosynthetic pathways which are the focus of this article, CO2 assimilation is also known to sustain autotrophic growth via the acetyl-CoA pathway (Wood et al . 1986) . Our aim here is to discuss (i) the findings that led our group to the discovery of the reductive carboxylic acid cycle, (ii) the nature and resolution of the controversy that followed, and (iii) the possible evolutionary implications of the cycle as an ancient mechanism for photosynthetic CO2 assimilation that preceded the pentose cycle and served as a precursor of the Krebs cycle in aerobic metabolism.

FEMS Microbiol Lett, 1985, 27, 227 - 32
Isotope effects associated with the anaerobic oxidation of sulfite and thiosulfate by the photosynthetic bacterium, Chromatium vinosum; Fry B et al.; The purple photosynthetic bacterium Chromatium vinosum, strain D, catalyzes several oxidations of reduced sulfur compounds under anaerobic conditions in the light: e.g., sulfide --> sulfur --> sulfate, sulfite --> sulfate, and thiosulfate --> sulfur + sulfate . Here it is shown that no sulfur isotope effect is associated with the last of these processes; isotopic compositions of the sulfur and sulfate produced can differ, however, if the sulfane and sulfonate positions within the thiosulfate have different isotopic compositions . In the second process, an observed change from an inverse to a normal isotope effect during oxidation of sulfite may indicate the operation of 2 enzymatic pathways . In contrast to heterotrophic anaerobic reduction of oxidized sulfur compounds, anaerobic oxidations of inorganic sulfur compounds by photosynthetic bacteria are characterized by relatively small isotope effects.

Chem Phys, 1995 May 15, 194(2-3), 245 - 58
Ultrafast energy transfer in light-harvesting chlorosomes from the green sulfur bacterium Chlorobium tepidum; Savikhin S et al.; Two independent pump-probe techniques were used to study the antenna energy transfer kinetics of intact chlorosomes from the green sulfur bacterium Chlorobium tepidum with femtosecond resolution . The isotropic kinetics revealed by one-color experiments in the BChl c antenna were inhomogeneous with respect to wavelength . Multiexponential analyses of the photobleaching/stimulated emission (PB/SE) decay profiles typically yielded (apart from a approximately 10 fs component that may stem from the initial coherent oscillation) components with lifetimes 1-2 ps and several tens of ps . The largest amplitudes for the latter component occur at 810 nm, the longest wavelength studied . Analyses of most two-color pump-probe profiles with the probe wavelength red-shifted from the pump wavelength yielded no PB/SE rise components . PB/SE components with approximately 1 ps risetime were found in 790 --> 810 and 790 --> 820 nm profiles, in which the probe wavelength is situated well into the BChl a absorption region . A 760 --> 740 nm uphill two-color experiment yielded a PB/SE component with 4-6 ps risetime . Broadband absorption difference spectra of chlorosomes excited at 720 nm (in the blue edge of the 746 nm BChl c Qy band) exhibit approximately 15 nm red-shifting of the PB/SE peak wavelength during the first several hundred fs . Analogous spectra excited at 760 nm (at the red edge) show little dynamic spectral shifting . Our results suggest that inhomogeneous broadening and spectral equilibration play a larger role in the early BChl c antenna kinetics in chlorosomes from C . tepidum than in those from C . aurantiacus, a system studied previously . As in C . aurantiacus, the initial one-color anisotropies r(0) for most BChl c wavelengths are close to 0.4 . The corresponding residual anisotropies r(infinity) are typically 0.19-0.25, which is much lower than found in C . aurantiacus (> or = 0.35); the transition moment organization is appreciably less collinear in the BChl c antenna of C . tepidum . However, the final one-color anisotropies at 789 and 801 nm are approximately 0 and 0.09 respectively, and the final anisotropy in time 780 --> 800 nm experiment is approximately -0.1 . These facts indicate that the BChI a transition moments themselves exhibit some order, and are directed at an angle > 54.7 degrees on the average from the BChl c moments . The one-color profiles exhibit coherent oscillations at most wavelengths, including 800 nm; Fourier analyses of these oscillations frequently yield components with frequencies 70-80 and 130-140 cm-1.

Can J Microbiol, 1985, 31, 675 - 80
Isolation and partial chemical analysis of firmly bound exopolysaccharide from adherent cells of a freshwater sediment bacterium; Platt RM et al.; Cells of a freshwater sediment bacterium produced firmly bound extracellular polymers in laboratory cultures which, at the ultrastructural level, resembled those produced by natural sediment bacterial populations . Production of the exopolymers during subculture was maintained by using as a source of inoculum the population of cells which adhered to each other and to the wall of the glass culture vessel . The exopolymers were selectively released from the cells by blending and centrifugation in the presence of EDTA . Evaluation of glucose-6-phosphate dehydrogenase activity and 2-keto-3-deoxyoctonate concentration indicated that only small amounts of intracellular and cell wall components were released from the cells during exopolymer removal . Chemical analysis of the isolated crude exopolymer material indicated that it contained protein, polysaccharide, and DNA . The treatment promoted the selective isolation of firmly bound polymers from the surface of adherent cells.

Adv Space Res, 1994 Oct, 14(10), 213 - 6
Effects of heavy ions on inactivation and DNA double strand breaks in Deinococcus radiodurans R1; Zimmermann H et al.; Inactivation and double strand break (dsb) induction after heavy ion irradiation were studied in stationary phase cells of the highly radiation resistant bacterium Deinococcus radiodurans R1 . There is evidence that the radiation sensitivity of this bacterium is nearly independent on energy in the range of up to 15 MeV/u for lighter ions (Ar) . The responses to dsb induction for charged particles show direct relationship between increasing radiation dose and residual intact DNA.

Photosynth Res, 1994 Jul, 41(1), 89 - 96
Redox effects on the bacteriochlorophyll a-containing Fenna-Matthews-Olson protein from Chlorobium tepidum; Zhou W et al.; The BChl a-containing Fenna-Matthews-Olson (FMO) protein from the green sulfur bacterium Chlorobium tepidum was purified and characterized . Fluorescence spectra indicate that efficient excited state quenching occurs at neutral or oxidizing redox potentials . The major fluorescence lifetime at room temperature is approximately 60 ps in samples that are in neutral or oxidizing conditions, and approximately 2 ns in samples where the strong reductant sodium dithionite has been added . A similar change is observed in pump-probe picosecond absorbance difference experiments, where the long life time component increases after dithionite addition . A 16 Gauss wide EPR signal with g factor = 2.005 is observed in samples without dithionite . This signal largely disappears upon addition of dithionite . Dithionite induces large reversible changes in the 77 K absorbance spectra of the purified FMO protein and in whole cells . These results indicate that the FMO protein contains redox active groups, which may be involved in the regulation of energy transfer . Room temperature circular dichroism and low temperature absorption spectra show that dithionite also induces conformational or structural changes of the FMO protein complex.

Environ Sci Technol, 1995 Oct, 29(10), 2535 - 40
Dissolution and reduction of magnetite by bacteria; Kostka JE et al.; Magnetite (Fe3O4) is an iron oxide of mixed oxidation state {Fe(II), Fe(III)} that contributes largely to geomagnetism and plays a significant role in diagenesis in marine and freshwater sediments . Magnetic data are the primary evidence for ocean floor spreading and accurate interpretation of the sedimentary magnetic record depends on an understanding of the conditions under which magnetite is stable . Though chemical reduction of magnetite by dissolved sulfide is well known, biological reduction has not been considered likely based upon thermodynamic considerations . This study shows that marine and freshwater strains of the bacterium Shewanella putrefaciens are capable of the rapid dissolution and reduction of magnetite, converting millimolar amounts to soluble Fe(II)in a few days at room temperature . Conditions under which magnetite reduction is optimal (pH 5-6, 22-37 degrees C) are consistent with an enzymatic process and not with simple chemical reduction . Magnetite reduction requires viable cells and cell contact, and it appears to be coupled to electron transport and growth . In a minimal medium with formate or lactate as the electron donor, more than 10 times the amount of magnetite was reduced over no carbon controls . These data suggest that magnetite reduction is coupled to carbon metabolism in S . putrefaciens . Bacterial reduction rates of magnetite are of the same order of magnitude as those estimated for reduction by sulfide . If such remobilization of magnetite occurs in nature, it could have a major impact on sediment magnetism and diagenesis.

J Phys Chem, 1996 Feb 29, 100(9), 3320 - 2
Ultrafast energy transfer in chlorosomes from the green photosynthetic bacterium Chloroflexus aurantiacus; Savikhin S et al.; Energy transfers between the bacteriochlorophyll c and a antennae in light-harvesting chlorosomes from the green bacterium Chloroflexes aurantiacus have been studied in two-color pump-probe experiments with improved sensitivity and wavelength versatility . The BChl c --> BChl a energy transfers are well simulated with biexponential kinetics, with lifetimes of 2-3 and 11 ps . They do not exhibit an appreciable subpicosecond component . In the context of a kinetic model for chlorosomes, these lifetimes suggest that both internal BChl c processes and the BChl c --> BChl a energy-transfer step contribute materially to the empirical rod-to-baseplate energy-transfer kinetics.

J Gen Microbiol, 1992 Aug, 138(8), 1759 - 66
Presence of methyl sterol and bacteriohopanepolyol in an outer-membrane preparation from Methylococcus capsulatus (Bath); Jahnke LL et al.; Cytoplasmic/intracytoplasmic and outer membrane preparations of Methylococcus capsulatus (Bath) were isolated by sucrose density gradient centrifugation of a total membrane fraction prepared by disruption using a French pressure cell . The cytoplasmic and/or intracytoplasmic membrane fraction consisted of two distinct bands, Ia and Ib (buoyant densities 1.16 and 1.8 g ml-1, respectively) that together contained 57% of the protein, 68% of the phospholipid, 73% of the ubiquinone and 89% of the CN-sensitive NADH oxidase activity . The only apparent difference between these two cytoplasmic bands was a much higher phospholipid content for Ia . The outer membrane fraction (buoyant density 1.23 - 1.24 g ml-1) contained 60% of the lipopolysaccharide-associated, beta-hydroxypalmitic acid, 74% of the methylsterol, and 66% of the bacteriohopanepolyol (BHP); phospholipid to methyl sterol or BHP ratios were 6:1 . Methanol dehydrogenase activity and a c-type cytochrome were also present in this outer membrane fraction . Phospholipase A activity was present in both the cytoplasmic membrane and outer membrane fractions . The unique distribution of cyclic triterpenes may reflect a specific role in conferring outer membrane stability in this methanotrophic bacterium.

Plant Physiol, 1992 Jul, 99(3), 919 - 24
Specific reduction of wheat storage proteins by thioredoxin h; Kobrehel K et al.; Gliadins and glutenins, the major storage proteins of wheat endosperm (Triticum durum, Desf . cv Monroe), were reduced in vitro by the NADP/thioredoxin system (NADPH, NADP-thioredoxin reductase and thioredoxin; in plants, the h type) from either the same source or the bacterium Escherichia coli . A more limited reduction of certain members of these protein groups was achieved with the reduced form of glutathione or glutaredoxin, a protein known to replace thioredoxin in certain bacterial and mammalian enzyme systems but not known to occur in higher plants . Endosperm extracts contained the enzymes necessary to reduce NADP by the oxidative pentose phosphate pathway (hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase) . The gliadins and glutenins were also reduced in vivo during germination--an event that accompanied their proteolytic breakdown . The results suggest that thioredoxin, reduced by NADPH generated via the oxidative pentose phosphate pathway, functions as a signal in germination to enhance metabolic processes such as the mobilization of storage proteins and, as found earlier, the activation of enzymes.

Orig Life Evol Biosph, 1998 Oct, 28(4-6), 515 - 21
Photosynthesis and the origin of life; Hartman H; The origin and evolution of photosynthesis is considered to be the key to the origin of life . This eliminates the need for a soup as the synthesis of the bioorganics are to come from the fixation of carbon dioxide and nitrogen . No soup then no RNA world or Protein world . Cyanobacteria have been formed by the horizontal transfer of green sulfur bacterial photoreaction center genes by means of a plasmid into a purple photosynthetic bacterium . The fixation of carbon dioxide is considered to have evolved from a reductive dicarboxylic acid cycle (Chloroflexus) which was then followed by a reductive tricarboxylic acid cycle (Chlorobium) and finally by the reductive pentose phosphate cycle (Calvin cycle) . The origin of life is considered to have occurred in a hot spring on the outgassing early earth . The first organisms were self-replicating iron-rich clays which fixed carbon dioxide into oxalic and other dicarboxylic acids . This system of replicating clays and their metabolic phenotype then evolved into the sulfide rich region of the hotspring acquiring the ability to fix nitrogen . Finally phosphate was incorporated into the evolving system which allowed the synthesis of nucleotides and phospholipids . If biosynthesis recapitulates biopoesis, then the synthesis of amino acids preceded the synthesis of the purine and pyrimidine bases . Furthermore the polymerization of the amino acid thioesters into polypeptides preceded the directed polymerization of amino acid esters by polynucleotides . Thus the origin and evolution of the genetic code is a late development and records the takeover of the clay by RNA.

Arch Microbiol, 1984 Jun, 138(2), 96 - 101
Chloroherpeton thalassium gen . nov . et spec . nov., a non-filamentous, flexing and gliding green sulfur bacterium; Gibson J et al.; A flexing and gliding green sulfur bacterium has been isolated from marine sources off the North East coast of the USA . Chloroherpeton thalassium is an obligate phototroph, and requires CO2 and S2 for growth; some organic acids can contribute to cell carbon, and N2 may be fixed . The cells contain typical chlorosomes, and gas vesicles may be present . Bacteriochlorophyll c is the main light harvesting pigment, and a small quantity of bacteriochlorophyll a is also present . Over 80% of the carotenoid is gamma-carotene . DNA base composition of the isolates ranges from 45.0-48.2 mol% G + C.

Plasmid, 2001 Jul, 46(1), 37 - 46
Development of small high-copy-number plasmid vectors for gene expression in Caulobacter crescentus; Umelo-Njaka E et al.; Caulobacter crescentus is a bacterium with a distinctive life cycle and so it is studied as a cell development model . In addition, we have adapted this bacterium for recombinant protein production and display based on the crystalline surface protein (S)-layer and its C-terminal secretion signal . We report here the development of small, high-copy-number plasmid vectors and methods for producing an obligate expression host . The vectors are based on a narrow-host-range colE1-replicon-based plasmid commonly used in Escherichia coli, to which was added the replication origin of the IncQ plasmid RSF1010 . C . crescentus strains were modified to enable plasmid replication by introduction of the RSF1010 repBAC genes at the recA locus . The small (4.0-4.5 kb) plasmids were in high copy numbers in both C . crescentus and E . coli and amenable to rapid methods for plasmid isolation and DNA sequencing . The method for introducing repBAC is suitable for other C . crescentus strains or any bacterium with an adequately homologous recA gene . Application of the vector for protein expression, based on the type I secretion system of the S-layer protein, when compared to constructs in broad-host-range plasmids, resulted in reduced time and steps required from clone construction to recombinant protein recovery and increased protein yield .

Cytobios, 2001, 106 Suppl 1, 99 - 117
Characterization of the agarase system of a multiple carbohydrate degrading marine bacterium; Whitehead LA et al.; A marine bacterium strain 2-40 (2-40) degraded numerous complex carbohydrates, such as agar, chitin and alginate . It may play an important role in altering carbon fluxes in marine environments . End-product analyses revealed that 2-40 synthesized an agarase system that consisted of at least three enzymes, beta-agarase I, beta-agarase II and alpha-agarase, which acted in concert to degrade polymeric agar to D-galactose and 3,6-anhydro-L-galactose . The agarase system was shown to be both cell envelope-associated and extracellular, with the relative concentrations depending on the growth phase . The principal depolymerase, a beta-agarase I, hydrolysed agar to both neoagarotetrose and neoagarobiose, as identified by thin layer chromatography . This agarase had a mass of 98 kD and a Pi of 4.3 . The agarase system was repressed by D-glucose and D-galactose and induced by agar, agarose, neoagarobiose, neoagarotetrose and neoagarohexose.

Mol Microbiol, 2001 Aug, 41(4), 787 - 800
Discovery of a haem uptake system in the soil bacterium Bradyrhizobium japonicum; Nienaber A et al.; In Bradyrhizobium japonicum, the nitrogen-fixing symbiont of soybeans, we have identified a haem uptake system, Hmu, that comprises a cluster of nine open reading frames . Predicted products of these genes include: HmuR, a TonB-dependent haem receptor in the outer membrane; HmuT, a periplasmic haem-binding protein; and HmuUV, an ABC transporter in the inner membrane . Furthermore, we identified homologues of ExbBD and TonB, that are required for energy transduction from the inner to the outer membrane . Mutant analysis and complementation tests indicated that HmuR and the ExbBD-TonB system, but not the HmuTUV transporter, are essential for haem uptake or haem acquisition from haemoglobin and leghaemoglobin . The TonB system seems to be specific for haem uptake as it is dispensable for siderophore uptake . Therefore, we propose the existence of a second TonB homologue functioning in the uptake of Fe-chelates . When tested on soybean host plants, hmuT-hmuR and exbD-tonB mutants exhibited wild-type symbiotic properties . Thus, haem uptake is not essential for symbiotic nitrogen fixation but it may enable B . japonicum to have access to alternative iron sources in its non-symbiotic state . Transcript analysis and expression studies with lacZ fusions showed that expression of hmuT and hmuR is induced under low iron supply . The same was observed in fur and irr mutant backgrounds although maximal induction levels were decreased . We conclude either that both regulators, Fur and Irr, independently mediate transcriptional control by iron or that a yet unknown iron regulatory system activates gene expression under iron deprivation . An A/T-rich cis-acting element, located in the promoter region of the divergently transcribed hmuTUV and hmuR genes, is possibly required for this type of iron control.

Eur J Biochem, 2001 Sep, 268(17), 4769 - 75
Characterization of the formyltransferase from Methylobacterium extorquens AM1; Pomper BK et al.; Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate-reducing archaea . Here we describe the purification and characterization of formylmethanofuran-tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylmethanofuran (formylMFR) and tetrahydromethanopterin (H4MPT) from N5-formylH4MPT and methanofuran (MFR) . Formyltransferase from M . extorquens AM1 showed activity with MFR and H4MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent Km for formylMFR = 50 microM; apparent Km for H4MPT = 30 microM) . The enzyme is encoded by the ffsA gene and exhibits a sequence identity of approximately 40% with Ftr from methanogenic and sulfate-reducing archaea . The 32-kDa Ftr protein from M . extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa . Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively . The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M . extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit . Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability . Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR . Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active-site cofactors molybdenum, molybdopterin and a {4Fe-4S} cluster . Cytochrome c was found to be spontaneously reduced by H4MPT . On the basis of this property, a novel assay for Ftr activity and MFR is described.

J Mol Biol, 2001 Aug 31, 311(5), 1037 - 48
Structure of the soluble domain of a membrane-anchored thioredoxin-like protein from Bradyrhizobium japonicum reveals unusual properties; Capitani G et al.; TlpA is an unusual thioredoxin-like protein present in the nitrogen-fixing soil bacterium Bradyrhizobium japonicum . A hydrophobic N-terminal transmembrane domain anchors it to the cytoplasmic membrane, whereby the hydrophilic thioredoxin domain becomes exposed to the periplasmic space . There, TlpA catalyses an essential reaction, probably a reduction, in the biogenesis of cytochrome aa(3) . The soluble thioredoxin domain (TlpA(sol)), devoid of the membrane anchor, was purified and crystallized . Oxidized TlpA(sol) crystallized as a non-covalent dimer in the space group P2(1)2(1)2(1) . The X-ray structure analysis was carried out by isomorphous replacement using a xenon derivative . This resulted in a high-resolution (1.6 A) three-dimensional structure that displayed all of the features of a classical thioredoxin fold . A number of peculiar structural details were uncovered: (i) Only one of the two active-site-cysteine sulphurs (Cys72, the one closer to the N terminus) is exposed on the surface, making it the likely nucleophile for the reduction of target proteins . (ii) TlpA(sol) possesses a unique structural disulphide bond, formed between Cys10 and Cys155, which connects an unprecedented N-terminal alpha helix with a beta sheet near the C terminus . (iii) An insertion of about 25 amino acid residues, not found in the thioredoxin prototype of Escherichia coli, contributes only marginally to the thioredoxin fold, but forms an extra, surface-exposed alpha helix . This region plus another surface-exposed stretch (-Ile-Gly-Arg-Ala-), which is absent even in the closest TlpA relatives, might be considered as specificity determinants for the recognition of target proteins in the periplasm . The TlpA(sol) structure paves the way towards unraveling important structure-function relationships by rational mutagenesis .

Biochimie, 2001 Aug, 83(8), 819 - 30
Hexuronyl C5-epimerases in alginate and glycosaminoglycan biosynthesis; Valla S et al.; The sugar residues in most polysaccharides are incorporated as their corresponding monomers during polymerization . Here we summarize the three known exceptions to this rule, involving the biosynthesis of alginate, and the glycosaminoglycans, heparin/heparan sulfate and dermatan sulfate . Alginate is synthesized by brown seaweeds and certain bacteria, while glycosaminoglycans are produced by most animal species . In all cases one of the incorporated sugar monomers are being C5-epimerized at the polymer level, from D-mannuronic acid to L-guluronic acid in alginate, and from D-glucuronic acid to L-iduronic acid in glycosaminoglycans . Alginate epimerization modulates the mechanical properties of seaweed tissues, whereas in bacteria it seems to serve a wide range of purposes . The conformational flexibility of iduronic acid units in glycosaminoglycans promotes apposition to, and thus functional interactions with a variety of proteins at cell surfaces and in the extracellular matrix . In the bacterium Azotobacter vinelandii the alginates are being epimerized at the cell surface or in the extracellular environment by a family of evolutionary strongly related modular type and Ca(2+)-dependent epimerases (AlgE1-7) . Each of these enzymes introduces a specific distribution pattern of guluronic acid residues along the polymer chains, explaining the wide structural variability observed in alginates isolated from nature . Glycosaminoglycans are synthesized in the Golgi system, through a series of reactions that include the C5-epimerization reaction along with extensive sulfation of the polymers . The single, Ca(2+)-independent, epimerase in heparin/heparan sulfate biosynthesis and the Ca(2+)-dependent dermatan sulfate epimerase(s) also generate variable epimerization patterns, depending on other polymer-modification reactions . The alginate and heparin epimerases appear unrelated at the amino acid sequence level, and have probably evolved through independent evolutionary pathways; however, hydrophobic cluster analysis indicates limited similarity . Seaweed alginates are widely used in industry, while heparin is well established in the clinic as an anticoagulant.

J Biol Chem, 2001 Nov 2, 276(44), 40864 - 72 Epub 2001 Aug 29.
Role of D-cysteine desulfhydrase in the adaptation of Escherichia coli to D-cysteine; Soutourina J et al.; D-cysteine, a powerful inhibitor of Escherichia coli growth, is decomposed in vitro into pyruvate, H2S, and NH3 by D-cysteine desulfhydrase . To assess the role of this reaction in the adaptation of the bacterium to growth on D-cysteine, the gene of the desulfhydrase was cloned . It corresponds to the open reading frame yedO at 43.03 min on the genetic map of E . coli . The amino acid sequence deduced from this gene is homologous to those of several 1-aminocyclopropane-carboxylate deaminases . However, the E . coli desulfhydrase does not use 1-aminocyclopropane-1-carboxylate as substrate . Various mutants in which the yedO gene was inactivated or overexpressed were constructed . They exhibited hypersensitivity or resistance, respectively, to the presence of d-cysteine in the culture medium . Growth protection against D-cysteine in minimal medium was conferred by the simultaneous addition of isoleucine, leucine, and valine . In agreement with this behavior, D-cysteine inhibited the activity of threonine deaminase, a key enzyme of the isoleucine, leucine, and valine pathway . Finally, in the presence of the intact yedO gene, E . coli growth was improved by addition of D-cysteine as the sole sulfur source . In agreement with a role of the desulfhydrase in sulfur metabolism, yedO expression was induced under conditions of sulfate limitation.

Curr Opin Investig Drugs, 2001 Jan, 2(1), 40 - 4
Towards the development of vaccines against Helicobacter pylori: status and issues; Del Giudice G; Helicobacter pylori is one of the most common bacteria worldwide . It infects more than 50% of the human population, and causes serious gastric diseases, such as chronic gastritis, peptic ulcer and, in some individuals, gastric cancer . The current treatment with antibiotics is not without drawbacks . The development of vaccines could help enormously in reducing the burden of H . pylori-associated diseases . A large body of evidence from experimental animal models has shown that vaccine-mediated prevention or vaccine-induced eradication of H . pylori is feasible . Several issues, however, are still unresolved, concerning the quality of the induced immune response necessary to achieve strong and sustained protective immunity in man . Several trials in humans are planned for the next few years . These studies will tell whether vaccination against H . pylori is feasible in man, and will offer the great opportunity to understand better the relationship between this ancient bacterium and humans.

Appl Environ Microbiol, 2001 Sep, 67(9), 4390 - 2
In vivo 23Na nuclear magnetic resonance study of maintenance of a sodium gradient in the ruminal bacterium Fibrobacter succinogenes S85; Schwaab V et al.; Sodium gradients (DeltapNa) were measured in resting cells of Fibrobacter succinogenes by in vivo 23Na nuclear magnetic resonance using Tm(DOTP)5- {thulium(III) 1,4,7,10-tetraazacyclododecane-N',N",N"'-tetramethylenephosphonate} as the shift reagent . This bacterium was able to maintain a DeltapNa of -55 to -40 mV for extracellular sodium concentrations ranging from 30 to 200 mM . Depletion of Na+ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).

Appl Environ Microbiol, 2001 Sep, 67(9), 3866 - 72
The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection; Grey BE et al.; The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system . The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil . To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection . The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease . The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined . When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R . solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants . This is the first report of R . solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.

Prog Nucleic Acid Res Mol Biol, 2001, 67, 35 - 63
CooA: a heme-containing regulatory protein that serves as a specific sensor of both carbon monoxide and redox state; Roberts GP et al.; CooA, the heme-containing carbon monoxide (CO) sensor from the bacterium Rhodospirillum rubrum, is a transcriptional factor that activates expression of certain genes in response to CO . As with other heme proteins, CooA is unable to bind CO when the Fe heme is oxidized, consistent with the fact that some of the regulated gene products are oxygen-labile . Upon reduction, there is an unusual switch of protein ligands to the six-coordinate heme and the reduced heme is able to bind CO . CO binding stabilizes a conformation of the dimeric protein that allows sequence-specific DNA binding, and transcription is activated through contacts between CooA and RNA polymerase . CooA is therefore a novel redox sensor as well as a specific CO sensor . CooA is a homolog of catabolite responsive protein (CRP), whose transcriptionally active conformation has been known for some time . The recent solution of the crystal structure of the CO-free (transcriptionally inactive) form of CooA has allowed insights into the mechanism by which both proteins respond to their specific small-molecule effectors.

Biochemistry, 2001 Sep 4, 40(35), 10562 - 9
Novel heme ligation in a c-type cytochrome involved in thiosulfate oxidation: EPR and MCD of SoxAX from Rhodovulum sulfidophilum; Cheesman MR et al.; The SoxAX complex of the bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that plays an essential role in photosynthetic thiosulfate and sulfide oxidation . The three heme sites of SoxAX have been analyzed using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism spectroscopies . Heme-3 in the ferric state is characterized by a Large g(max) EPR signal and has histidine and methionine axial heme iron ligands which are retained on reduction to the ferrous state . Hemes-1 and -2 both have thiolate plus nitrogenous ligand sets in the ferric state and give rise to rhombic EPR spectra . Heme-1, whose ligands derive from cysteinate and histidine residues, remains ferric in the presence of dithionite ion . Ferric heme-2 exists with a preparation-dependent mixture of two different ligand sets, one being cysteinate/histidine, the other an unidentified pair with a weaker crystal-field strength . Upon reduction of the SoxAX complex with dithionite, a change occurs in the ligands of heme-2 in which the thiolate is either protonated or replaced by an unidentified ligand . Sequence analysis places the histidine/methionine-coordinated heme in SoxX and the thiolate-liganded hemes in SoxA . SoxAX is the first naturally occurring c-type cytochrome in which a thiolate-coordinated heme has been identified.

Thromb Haemost, 2001 Aug, 86(2), 668 - 71
Serial determinations of platelet counts in mice by flow cytometry; Alugupalli KR et al.; Elucidation of the pathophysiological basis of platelet disorders in murine models requires a reliable method for the frequent determinations of platelet counts in individual mice . Here, we present a rapid, reproducible and accurate flow cytometric method for enumeration of platelets that involves fluorescent staining of platelets in whole blood with specific antibody and the addition of known numbers of fluorescent beads for standardization of the sample volume . Analysis of platelets obtained by tail bleeding indicated that this sampling procedure did not activate platelets, and that only five microliters of blood were required for platelet counting . Using this method, we followed platelet counts in mice infected with the relapsing fever spirochete Borrelia turicatae for 26 days, and found that this bacterium induces thrombocytopenia, a common manifestation of human relapsing fever . Therefore, this method can be used to follow the number and the activation state of circulating platelets from individual mice over extended periods of time and is applicable to a wide range of murine models of platelet disorders.

FEMS Microbiol Lett, 2001 Aug 21, 202(2), 251 - 6
Two new loci affecting cell division identified as suppressors of an ftsQ-null mutation in Streptomyces coelicolor A3(2); Bennett JA et al.; Mutations in fts genes partially or completely block both vegetative cell division and sporulation septation in the filamentous bacterium Streptomyces coelicolor A3(2) . Using a novel screen, we independently isolated two double-mutant strains, each containing a spontaneous suppressor mutation, which partially restores division to an ftsQ-null mutant . Genetic complementation experiments revealed that the suppressor mutations alone confer no observable defect in sporulation . The suppressor mutations were genetically mapped to regions of the chromosome, distinct from each other and the division and cell wall cluster containing ftsQ . Therefore, the genes identified by the suppressor mutations were named sqnA and sqnB (suppressor of ftsQ-null) and may be representatives of a novel class of genes involved in cell division or the regulation of cell division in this mycelial organism.

Heredity, 2001 Apr, 86(Pt 4), 497 - 505
Rickettsia associated with male-killing in a buprestid beetle; Lawson ET et al.; Many populations of the buprestid leaf-mining beetle, Brachys tessellatus, from central South Carolina, USA, show highly skewed sex ratios, ranging from 1.3 to 6.0 females per male . We have identified a Rickettsia bacterium that is associated with sex ratio distortion (SRD) and selective killing of male embryos in B . tessellatus . Molecular assays of infection by this bacterium are highly associated with SRD within families, and treatment with an antibiotic (tetracycline) increases the number of male eggs that hatch and develop . The 16S rDNA sequence indicates that this is a novel Rickettsia, most closely related to Rickettsia bellii (a tick-associated bacterium) and a pea-aphid Rickettsia . It is also related to a Rickettsial bacterium that causes male-killing in an unrelated ladybird beetle species . Low levels of parthenogenesis are also observed in this system (about 10% of females) and may be the result of selection due to male rarity, or a direct result of infection by the Rickettsia.

Genome Biol . 2001;2(7):REVIEWS1020 . Epub 2001 Jul 03.
Anatomy of a bacterial cell cycle; Amick JD et al.; Two recent reports describe mRNA and protein expression patterns in the bacterium Caulobacter crescentus . The combined use of DNA microarray and proteomic analyses provides a powerful new perspective for unraveling the global regulatory networks of this complex bacterium.

Braz J Med Biol Res, 2001 Sep, 34(9), 1105 - 13
Partial characterization of nif genes from the bacterium Azospirillum amazonense; Potrich DP et al.; Azospirillum amazonense revealed genomic organization patterns of the nitrogen fixation genes similar to those of the distantly related species A . brasilense . Our work suggests that A . brasilense nifHDK, nifENX, fixABC operons and nifA and glnB genes may be structurally homologous to the counterpart genes of A . amazonense . This is the first analysis revealing homology between A . brasilense nif genes and the A . amazonense genome . Sequence analysis of PCR amplification products revealed similarities between the amino acid sequences of the highly conserved nifD and glnB genes of A . amazonense and related genes of A . brasilense and other bacteria . However, the A . amazonense non-coding regions (the upstream activator sequence region and the region between the nifH and nifD genes) differed from related regions of A . brasilense even in nitrogenase structural genes which are highly conserved among diazotrophic bacteria . The feasibility of the 16S ribosomal RNA gene-based PCR system for specific detection of A . amazonense was shown . Our results indicate that the PCR primers for 16S rDNA defined in this article are highly specific to A . amazonense and can distinguish this species from A . brasilense.

J Bacteriol, 2001 Sep, 183(18), 5268 - 78
Two similar gene clusters coding for enzymes of a new type of aerobic 2-aminobenzoate (anthranilate) metabolism in the bacterium Azoarcus evansii; Schuhle K et al.; In the beta-proteobacterium Azoarcus evansii, the aerobic metabolism of 2-aminobenzoate (anthranilate), phenylacetate, and benzoate proceeds via three unprecedented pathways . The pathways have in common that all three substrates are initially activated to coenzyme A (CoA) thioesters and further processed in this form . The two initial steps of 2-aminobenzoate metabolism are catalyzed by a 2-aminobenzoate-CoA ligase forming 2-aminobenzoyl-CoA and by a 2-aminobenzoyl-CoA monooxygenase/reductase (ACMR) forming 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA . Eight genes possibly involved in this pathway, including the genes encoding 2-aminobenzoate-CoA ligase and ACMR, were detected, cloned, and sequenced . The sequence of the ACMR gene showed that this enzyme is an 87-kDa fusion protein of two flavoproteins, a monooxygenase (similar to salicylate monooxygenase) and a reductase (similar to old yellow enzyme) . Besides the genes for the initial two enzymes, genes for three enzymes of a beta-oxidation pathway were found . A substrate binding protein of an ABC transport system, a MarR-like regulator, and a putative translation inhibitor protein were also encoded by the gene cluster . The data suggest that, after monooxygenation/reduction of 2-aminobenzoyl-CoA, the nonaromatic CoA thioester intermediate is metabolized further by beta-oxidation . This implies that all subsequent intermediates are CoA thioesters and that the alicyclic carbon ring is not cleaved oxygenolytically . Surprisingly, the cluster of eight genes, which form an operon, is duplicated . The two copies differ only marginally within the coding regions but differ substantially in the respective intergenic regions . Both copies of the genes are coordinately expressed in cells grown aerobically on 2-aminobenzoate.

Biophys J, 2001 Sep, 81(3), 1613 - 23
Self diffusion and spectral modifications of a membrane protein, the Rubrivivax gelatinosus LH2 complex, incorporated into a monoolein cubic phase; Tsapis N et al.; The light-harvesting complex LH2 from a purple bacterium, Rubrivivax gelatinosus, has been incorporated into the Q230 cubic phase of monoolein . We measured the self-diffusion of LH2 in detergent solution and in the cubic phase by fluorescence recovery after photobleaching . We investigated also the absorption and fluorescence properties of this oligomeric membrane protein in the cubic phase, in comparison with its beta-octyl glucoside solution . In these experiments, native LH2 and LH2 labeled by a fluorescent marker were used . The results indicate that the inclusion of LH2 into the cubic phase induced modifications in the carotenoid and B800 binding sites . Despite these significant perturbations, the protein seems to keep an oligomeric structure . The relevance of these observations for the possible crystallization of this protein in the cubic phase is discussed.

Biophys J, 2001 Sep, 81(3), 1208 - 19
Photoreduction and reoxidation of the three iron-sulfur clusters of reaction centers of green sulfur bacteria; Setif P et al.; Iron-sulfur clusters are the terminal electron acceptors of the photosynthetic reaction centers of green sulfur bacteria and photosystem I . We have studied electron-transfer reactions involving these clusters in the green sulfur bacterium Chlorobium tepidum, using flash-absorption spectroscopic measurements . We show for the first time that three different clusters, named F(X), F(1), and F(2), can be photoreduced at room temperature during a series of consecutive flashes . The rates of electron escape to exogenous acceptors depend strongly upon the number of reduced clusters . When two or three clusters are reduced, the escape is biphasic, with the fastest phase being 12-14-fold faster than the slowest phase, which is similar to that observed after single reduction . This is explained by assuming that escape involves mostly the second reducible cluster . Evidence is thus provided for a functional asymmetry between the two terminal acceptors F(1) and F(2) . From multiple-flash experiments, it was possible to derive the intrinsic recombination rates between P840(+) and reduced iron-sulfur clusters: values of 7, 14, and 59 s(-1) were found after one, two and three electron reduction of the clusters, respectively . The implications of our results for the relative redox potentials of the three clusters are discussed.

Vet Microbiol, 2001 Oct 1, 82(4), 331 - 45
Lawsonia intracellularis: getting inside the pathogenesis of proliferative enteropathy; Smith DG et al.; Although proliferative enteropathy (PE) has been recognised for several decades, Lawsonia intracellularis, the aetiological agent, was identified formally in only 1995 . This organism is both highly fastidious and obligately intracellular bacterium, characteristics which have inevitably restricted investigations in all aspects of its biology . Despite these limitations, advances have been made in characterising and understanding L . intracellularis-host interaction both in vivo and in vitro . Based upon evidence provided by mainly pathological and histological investigations conducted to date, we review salient features of our current understanding of processes involved throughout the course of infection by this unique pathogen.

FEMS Microbiol Lett, 2001 Aug 7, 202(1), 109 - 14
Intraspecific diversity of Oenococcus oeni isolated during red wine-making in Japan; Sato H et al.; Using molecular and chemotaxonomic techniques, we studied the intraspecific diversity of Oenococcus oeni, a lactic acid bacterium isolated during red wine-making in Japan . The results confirmed high values of DNA-DNA relatedness and strong similarity among 16S rDNA sequences of the isolates with the O . oeni-type strain . Pulsed-field gel electrophoresis (PFGE) by NotI identified four patterns among the strains . Three different patterns of lactate dehydrogenase mobility were seen and there was a strong correlation between PFGE pattern and mobility . The present results suggest that the different strains of O . oeni comprise one species, and that variations in the genomic profiles of the different strains of O . oeni, including Japanese isolates are well correlated.

J Cardiothorac Vasc Anesth, 2001 Aug, 15(4), 451 - 4
Endotoxin-neutralizing capacity of serum from cardiac surgical patients; Bennett-Guerrero E et al.; OBJECTIVE: To determine if endotoxin core antibody (EndoCAb) from the serum of cardiac surgical patients neutralizes endotoxin in an ex vivo biologic assay . DESIGN: Prospective blinded cohort study . SETTING: Academic medical center . PARTICIPANTS: Patients (n = 203) undergoing cardiac surgery . INTERVENTIONS: Sera were obtained from patients preoperatively . MEASUREMENTS AND MAIN RESULTS: EndoCAb levels were determined by enzyme-linked immunosorbent assay . Sera were incubated for 15 minutes at 37 degrees C with varying concentrations of endotoxin from a clinically relevant bacterium (Escherichia coli serotype O18), then tested for the presence of endotoxin activity using the validated Limulus amebocyte lysate assay . Median (interquartile range) IgM and IgG EndoCAb levels were 118 median units (range, 31 to 259 median units) and 208 median units (range, 108 to 401 medium units) . Increasing levels of IgM EndoCAb were associated with increased neutralization of endotoxin (p < 0.0001) . Increasing levels of IgG EndoCAb were associated with increased neutralization of endotoxin (p < 0.0001) . An additive effect of IgM and IgG EndoCAb levels on endotoxin neutralization was observed without evidence of synergistic or plateau effects . EndoCAb levels did not completely predict serum neutralization capacity . CONCLUSION: Anti-EndoCAbs of both classes (IgM and IgG) were able to neutralize lipopolysaccharide from a clinically relevant bacterium in an ex vivo model . Neither Igm nor IgG appeared to be more capable of neutralization in this model . These antibodies did not completely predict neutralization capacity; other endogenous factors in human serum must be capable of lipopolysaccharide neutralization .

Mod Pathol, 2001 Aug, 14(8), 752 - 9
Identification of the target cells of Orientia tsutsugamushi in human cases of scrub typhus; Moron CG et al.; Orientia tsutsugamushi is the etiologic agent of scrub typhus, a chigger-borne zoonosis that is a highly prevalent, life-threatening illness of greatest public health importance in tropical Asia and the islands of the western Pacific Ocean . The target cell of this bacterium is poorly defined in humans . In this study, O . tsutsugamushi were identified by immunohistochemistry using a rabbit polyclonal antibody raised against O . tsutsugamushi Karp strain in paraffin-embedded archived autopsy tissues of three patients with clinical suspicion of scrub typhus who died during World War II and the Vietnam War . Rickettsiae were located in endothelial cells in all of the organs evaluated, namely heart, lung, brain, kidney, pancreas, and skin, and within cardiac muscle cells and in macrophages located in liver and spleen . Electron microscopy confirmed the location of rickettsiae in endothelium and cardiac myocytes.

Eur J Biochem, 2001 Aug, 268(16), 4375 - 83
Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp . KUC-1: purification, molecular characterization and expression; Oikawa T et al.; We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp . KUC-1, isolated from Antarctic seawater and purified the enzyme to homogeneity . The molecular mass of the enzyme was determined to be approximately 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer . The enzyme required NAD+ as a coenzyme, and catalyzed the oxidative deamination of L-valine, L-isoleucine, L-leucine and the reductive amination of alpha-ketoisovalerate, alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketocaproate . The reaction proceeds through an iso-ordered bi-bi mechanism . The enzyme was highly susceptible to heat treatment and the half-life at 45 degrees C was estimated to be 2.4 min . The kcat/Km (micro(-1).s(-1)) values for L-valine and NAD+ at 20 degrees C were 27.48 and 421.6, respectively . The enzyme showed pro-S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme . The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme . The enzyme contains 370 amino-acid residues, and is highly homologous with S . coelicolor ValDH (identity, 46.7%) and S . fradiae ValDH (43.1%) . Cytophaga sp . KUC-1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs . The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures.

Biochemistry, 2001 Aug 21, 40(33), 9799 - 809
Site-directed mutagenesis and X-ray crystallography of the PQQ-containing quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome c(L); Afolabi PR et al.; Two proteins specifically involved in methanol oxidation in the methylotrophic bacterium Methylobacterium extorquens have been modified by site-directed mutagenesis . Mutation of the proposed active site base (Asp303) to glutamate in methanol dehydrogenase (MDH) gave an active enzyme (D303E-MDH) with a greatly reduced affinity for substrate and with a lower activation energy . Results of kinetic and deuterium isotope studies showed that the essential mechanism in the mutant protein was unchanged, and that the step requiring activation by ammonia remained rate limiting . No spectrally detectable intermediates could be observed during the reaction . The X-ray structure, determined to 3 A resolution, of D303E-MDH showed that the position and coordination geometry of the Ca2+ ion in the active site was altered; the larger Glu303 side chain was coordinated to the Ca2+ ion and also hydrogen bonded to the O5 atom of pyrroloquinoline quinone (PQQ) . The properties and structure of the D303E-MDH are consistent with the previous proposal that the reaction in MDH is initiated by proton abstraction involving Asp303, and that the mechanism involves a direct hydride transfer reaction . Mutation of the two adjacent cysteine residues that make up the novel disulfide ring in the active site of MDH led to an inactive enzyme, confirming the essential role of this remarkable ring structure . Mutations of cytochrome c(L), which is the electron acceptor from MDH was used to identify Met109 as the sixth ligand to the heme.

Res Microbiol, 2001 Jul-Aug, 152(6), 539 - 49
Expression of horizontally transferred gene clusters: activation by promoter-generating mutations; Dabizzi S et al.; The occurrence of promoter-generating mutations allowing the transcription of heterologous genes has been studied in a system based on the plasmid-mediated conjugal transfer of histidine biosynthetic genes from a donor bacterium (Azospirillum brasilense) into a heterologous Escherichia coli mutant population lacking histidine biosynthetic ability and initially unable to recognize the transcriptional signal of the introgressed gene(s) . Under selective stressful conditions, His+ revertants accumulated in the E . coli His- culture . The number of His+ colonies was dependent on the time of incubation under selective conditions, the strength of selective pressure, and on the crowding of cells plated; moreover, it was independent of the physiological status of the cell (i.e . the growth phase) . Sequence analysis of plasmid DNA extracted from E . coli His+ revertants revealed that single base substitutions in the region upstream of the A . brasilense his operon resulted in an adjustment of the pre-existing sequence that was rendered similar to the E . coli -10 promoter sequence and transcriptable by the host RNA-polymerase . One particular transition (C --> T) was predominant in the His+ revertants . Data presented here indicated that the barriers to the expression of horizontally transferred heterologous genes or operons may be overcome in a short time scale and at high frequency, and supported the selfish operon model on the origin and evolution of gene clusters.

Folia Microbiol (Praha), 2001, 46(1), 33 - 5
Non-specific DNAases from the rumen bacterium Prevotella bryantii; Accetto T et al.; Extracellular non-specific nucleases were observed in some strains belonging to the ruminal species of the genus Prevotella, mostly P . brevis and P . bryantii . The nuclease from P . bryantii appeared to be extracellular; it mediates the degradation of the supercoiled plasmid DNA via an open circle intermediate . The cleavage is not site specific although a preference for certain cleavage sites does seem to exist . Our attempts to clone the wild-type P . bryantii B(1)4 nuclease in E . coli strain ER1992 that reports on the DNA damage sustained, were unsuccessful probably due to excessive intracellular nuclease activity that killed the cells bearing the gene for the nuclease . On the other hand, the nuclease from a related strain TCl-1, which has a less active enzyme of the same type, was successfully cloned.

Environ Toxicol, 2001, 16(4), 337 - 43
Degradation of the cyanobacterial hepatotoxin microcystin by a new bacterium isolated from a hypertrophic lake; Park HD et al.; A bacterium capable of degrading microcystins-RR, -YR, and -LR was isolated from a hypertrophic lake . The bacterium, designated Y2 and classified phenotypically as a member of the genus Sphingomonas, was shown to be distinct phylogenetically from any established species of Sphingomonas on the basis of 16S rDNA sequencing . The bacterium was tentatively identified as Sphingomonas by manual chemotaxonomy, but 16S rRNA sequencing analysis suggests that it is in fact a new species or even a new genus . When the Y2 bacterium was added to microcystins present in culture medium, the microcystins were degraded thoroughly in 4 days . The highest degradation rates of microcystins-RR and -LR were 13 and 5.4 mg L-1 day-1, respectively . The degradation rates were strongly dependent on temperature and the maximum rate was at 30 degrees C.

Angew Chem Int Ed Engl . 2001 Aug 3;40(15):2725.
Cover Picture; Merkx M et al.; The cover picture shows in the background the whole cell of a methanotrophic bacterium on which are superimposed components of methane monooxygenase (the structure of the hydroxylase component (top), one of the two four-helix bundles that house the catalytic diiron centers (left)) and a schematic diagram of the catalytic cycle by which the enzyme converts dioxygen and methane into methanol and water . More about this unusual enzyme system is reported by Lippard et al . on p . 2782 ff.

Infect Immun, 2001 Sep, 69(9), 5925 - 30
Recombinant Actinobacillus actinomycetemcomitans cytolethal distending toxin proteins are required to interact to inhibit human cell cycle progression and to stimulate human leukocyte cytokine synthesis; Akifusa S et al.; It has recently been discovered that Actinobacillus actinomycetemcomitans, an oral bacterium causing periodontitis, produces cytolethal distending toxin (CDT), a cell cycle-modulating toxin that has three protein subunits: CdtA, CdtB, and CdtC . In this study, we have cloned and expressed each toxin gene from A . actinomycetemcomitans in Escherichia coli and purified the recombinant Cdt proteins to homogeneity . Individual Cdt proteins failed to induce cell cycle arrest of the human epithelial cell line HEp-2 . The only combinations of toxin proteins causing cell cycle arrest were the presence of all three Cdt proteins and the combination of CdtB and CdtC . A similar experimental protocol was used to determine if recombinant Cdt proteins were able to induce human peripheral blood mononuclear cells (PBMCs) to produce cytokines . The individual Cdt proteins were able to induce the synthesis by PBMCs of interleukin-1beta (IL-1beta), IL-6, and IL-8 but not of tumor necrosis factor alpha, IL-12, or granulocyte-macrophage colony-stimulating factor, with CdtC being the most potent and CdtB being the least potent cytokine inducer . There was evidence of synergism between these Cdt proteins in the stimulation of cytokine production, most markedly with gamma interferon, which required the minimum interaction of CdtB and -C to stimulate production.

Infect Immun, 2001 Sep, 69(9), 5577 - 88
Exploitation of interleukin-8-induced neutrophil chemotaxis by the agent of human granulocytic ehrlichiosis; Akkoyunlu M et al.; The agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium with a tropism for neutrophils; however, the mechanisms of bacterial dissemination are not yet understood . Interleukin-8 (IL-8) is a chemokine that induces neutrophil migration to sites of infection for host defense against pathogens . We now show that HGE bacteria, and the HGE-44 protein, induce IL-8 secretion in a promyelocytic (HL-60) cell line that has been differentiated along the neutrophil lineage with retinoic acid and in neutrophils . Infected HL-60 cells also demonstrate upregulation of CXCR2, an IL-8 receptor, but not CXCR1 . Human neutrophils migrate towards Ehrlichia sp.-infected cells in a chemotaxis chamber assay, and this movement can be blocked with antibodies to IL-8 . Finally, immunocompetent and severe combined immunodeficient mice administered CXCR2 antisera, and CXCR2(-/-) mice that lack the human IL-8 receptor homologue, are much less susceptible to granulocytic ehrlichiosis than are control animals . These results demonstrate that HGE bacteria induce IL-8 production by host cells and, paradoxically, appear to exploit this chemokine to enhance infection.

Infect Immun, 2001 Sep, 69(9), 5423 - 9
Chlamydia pneumoniae expresses genes required for DNA replication but not cytokinesis during persistent infection of HEp-2 cells; Byrne GI et al.; Chlamydia pneumoniae causes community-acquired pneumonia and is associated with several chronic diseases, including asthma and atherosclerosis . The intracellular growth rate of C . pneumoniae slows dramatically during chronic infection, and such persistence leads to attenuated production of new elementary bodies, appearance of morphologically aberrant reticulate bodies, and altered expression of several chlamydial genes . We used an in vitro system to further characterize persistent C . pneumoniae infection, employing both ultrastructural and transcriptional activity measurements . HEp-2 cells were infected with C . pneumoniae (TW-183) at a multiplicity of infection of 3:1, and at 2 h postinfection gamma interferon (IFN-gamma) was added to the medium at 0.15 or 0.50 ng/ml . Treated and untreated cultures were harvested at several times postinfection . RNA was isolated and reverse transcribed, and reverse transcription (RT)-PCR analyses targeting primary transcripts from chlamydial rRNA operons as well as dnaA, polA, mutS, minD, ftsK, and ftsW mRNA were done . Some cultures were fixed and stained for electron microscopic analysis, and a real-time PCR assay was used to assess relative chlamydial chromosome accumulation under each culture condition . The latter assays showed that bacterial chromosome copies accumulated severalfold during IFN-gamma treatment of infected HEp-2 cells, although less accumulation was observed in cells treated with the higher dose . Electron microscopy demonstrated that high-dose IFN-gamma treatment elicited aberrant forms of the bacterium . RT-PCR showed that chlamydial primary rRNA transcripts were present in all IFN-gamma-treated and untreated cell cultures, indicating bacterial metabolic activity . Transcripts from dnaA, polA, mutS, and minD, all of which encode products for bacterial chromosome replication and partition, were expressed in IFN-gamma-treated and untreated cells . In contrast, ftsK and ftsW, encoding products for bacterial cell division, were expressed in untreated cells, but expression was attenuated in cells treated with low-dose IFN-gamma and absent in cells given the high dose of cytokine . Thus, the development of persistence included production of transcripts for DNA replication-related, but not cell division-related, genes . These results provide new insight regarding molecular activities that accompany persistence of C . pneumoniae, as well as suggesting requirements for reactivation from persistent to productive growth.

Infect Immun, 2001 Sep, 69(9), 5395 - 402
Roles of fructosyltransferase and levanase-sucrase of Actinomyces naeslundii in fructan and sucrose metabolism; Bergeron LJ et al.; The ability of Actinomyces naeslundii to convert sucrose to extracellular homopolymers of fructose and to catabolize these types of polymers is suspected to be a virulence trait that contributes to the initiation and progression of dental caries and periodontal diseases . Previously, we reported on the isolation and characterization of the gene, ftf, encoding the fructosyltransferase (FTF) of A . naeslundii WVU45 . Allelic exchange mutagenesis was used to inactivate ftf, revealing that FTF-deficient stains were completely devoid of the capacity to produce levan-type (beta2,6-linked) polysaccharides . A polyclonal antibody was raised to a histidine-tagged, purified A . naeslundii FTF, and the antibody was used to localize the enzyme in the supernatant fluid . A sensitive technique was developed to detect levan formation by proteins that had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the method was used to confirm that the levan-synthesizing activity of A . naeslundii existed predominantly in a cell-free form, that a small amount of the activity was cell associated, and that the ftf mutant was unable to produce levans . By using the nucleotide sequence of the levanase gene of a genospecies 2 A . naeslundii, formerly Actinomyces viscosus, a portion of a homologue of this gene (levJ) was amplified by PCR and inserted into a suicide vector, and the resulting construct was used to inactivate the levJ gene in the genospecies 1 strain WVU45 . A variety of physiologic and biochemical studies were performed on the wild-type and LevJ-deficient strains to demonstrate that (i) this enzyme was the dominant levanase and sucrase of A . naeslundii; (ii) that LevJ was inducible by growth in sucrose; (iii) that the LevJ activity was found predominantly (>90%) in a cell-associated form; and (iv) that there was a second, fructose-inducible fructan hydrolase activity produced by these strains . The data provide the first detailed molecular analysis of fructan production and catabolism in this abundant and important oral bacterium.

Infect Immun, 2001 Sep, 69(9), 5375 - 84
Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f; Kaplan JB et al.; The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP) . A . actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules . Serotype b has been reported to be the dominant serotype isolated from LJP patients . We determined the lipopolysaccharide O-PS structure from A . actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b . The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of L-rhamnose and 2-acetamido-2-deoxy-D-galactose (molar ratio, 2:1) with the structure -->2)-alpha-L-Rhap-(1-3)-2-O-(beta-D-GalpNAc)-alpha-L-Rhap-(1-->* O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A . actinomycetemcomitans serotypes . Strain CU1000 was mutagenized with transposon IS903phikan, and three mutants that were deficient in O-PS synthesis were isolated . All three transposon insertions mapped to a single 1-kb region on the chromosome . The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A . actinomycetemcomitans serotype b, c, and e O-PS antigens . The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A . actinomycetemcomitans serotypes . These data indicate that strain CU1000 should be assigned to a new A . actinomycetemcomitans serotype, designated serotype f . A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A . actinomycetemcomitans strains carrying the serotype f gene cluster . The finding of an A . actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.

Membr Cell Biol, 2001, 14(4), 463 - 74
UV-Induced destruction of light-harvesting complexes from purple bacterium Chromatium minutissimum; Solov'ev AA et al.; We studied UV-induced photodestruction of the native forms of bacteriochlorophyll a (Bchl a) from chromatophores and light harvesting complexes (LHC) of the sulphur photosynthetic bacterium Chromatium minutissimum . Irradiation of chromato- phores with 365-nm light (Soret band) or 280-nm light (absorption region of aromatic amino acids) led to the destruction of all long-wavelength forms of Bchl a . The quantum yields of photodestruction produced by the 280-nm light was higher than that produced by the 365-nm light . For the spectral forms of Bchl a absorbing at 850 nm and 890 nm, the difference was about one order of magnitude, and for the form absorbing at 800 nm the difference was almost two orders of magnitude . Similar UV sensitivity was observed for the Bchl a forms from isolated LHC . As a rule, the quantum yields of photodestruction induced by UV irradiation at 280 nm were about 100-1000 times higher (approximately 10(-3)-10(-4)) than that upon red light irradiation (approximately 10(-6)-10(-7)) . We found that irradiation of chromatophores at 280 nm resulted in a crosslink between the core and peripheral LHC.

Asian Pac J Allergy Immunol, 2001 Mar, 19(1), 59 - 62
The effects of exogenous interleukin-12 on the induction of immune response to Porphyromonas gingivalis in vivo in mice; Rochim A et al.; The effects of treatment with exogenous interleukin-12 (IL-12) on the induction of immune response to Porphyromonas gingivalis, a black pigmented periodontopathic oral bacterium in mice, were determined in the present study . An increased footpad swelling representing a delayed type hypersensitivity (DTH) response to P . gingivalis in IL-12-treated mice could be observed, although increasing doses of IL-12 did not produce cumulative effects on this cellular Immune response . Multiple injections with IL-12 also resulted in elevated serum IFN-gamma levels . Treatment with this cytokine the day before, on and after immunization with heat-killed P . gingivalis augmented the levels of serum antigen-specific IgG2a and IgG3 antibodies, but had obviously little or no effects on those of serum antigen-specific IgG1 and IgG2b antibodies . The results of this study suggest that treatment with exogenous IL-12 In P . gingivalis-immunized mice may enhance DTH response and Th1 cell-associated antibody production.

Environ Toxicol Chem, 2001 Aug, 20(8), 1824 - 30
Toxicity evaluation of natural and synthetic phenanthrenes in aquatic systems; DellaGreca M et al.; Seven natural 9,10-dihydrophenanthrenes were isolated from the common reed Juncus effusus by means of chromatographic processes and identified by spectroscopic means . Furthermore, mimics of natural isolated compounds were synthesized to try to evaluate the influence of functional groups on the dihydrophenanthrene skeleton . Syntheses of compounds were based on the cross-coupling of 1-(2-iodo-5-methoxy)phenyl-ethanol with variously substituted iodobenzenes by zerovalent nickel . All the chemicals were tested to evaluate their effects on freshwater organisms from different trophic levels . Toxicity tests were performed on reducers (the bacterium Escherichia coli); producers (the alga Raphidocelis subcapitata, previously known as Selenastrum capricornutum); and consumers including a rotifer (Brachionus calyciflorus), a cladoceran (Daphnia pulex), and an anostracan (Thamnocephalus platyurus) . Results suggested no one organism was uniquely sensitive to the chemicals tested . Toxicity depended on the kind and position of substituents on the aromatic skeleton.

Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1323 - 6
Actinomyces suimastitidis sp . nov., isolated from pig mastitis; Hoyles L et al.; An unusual Actinomyces-like bacterium originating from a pig with mastitis was subjected to a polyphasic taxonomic investigation . The morphological and biochemical characteristics of the organism were consistent with its preliminary assignment to the genus Actinomyces but it did not appear to correspond to any recognized species . PAGE analysis of whole-cell proteins confirmed the phenotypic distinctiveness of the bacterium and 16S rRNA gene sequence analysis demonstrated that it represents a hitherto unknown sub-line amongst a cluster of Actinomyces species which embraces Actinomyces canis, Actinomyces georgiae, Actinomyces hyovaginalis, Actinomyces meyeri, Actinomyces odontolyticus, Actinomyces radingae and Actinomyces turicensis . Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium isolated from pig mastitis be classified as Actinomyces suimastitidis sp . nov . The type strain of Actinomyces suimastitidis is CCUG 39279T (= CIP 106779T).

Arch Microbiol, 2001 Jun, 175(6), 462 - 5
Growth of the purple bacterium Rhodobacter capsulatus on the aromatic compound hippurate; Madigan MT et al.; The purple nonsulfur bacterium Rhodobacter capsulatus strain B10 grew phototrophically on the aromatic compound hippurate (N-benzoyl-L-glycine) and related benzoyl amino acids . Absorption spectra, extraction, and GC/MS analysis of culture supernatants showed that hippurate was stoichiometrically converted to benzoate and glycine, with the latter used as a carbon or nitrogen source for growth . This conclusion was supported by detection of the enzyme hippuricase in permeabilized intact cells . Chemotrophic growth on hippurate by Rba . capsulatus, either at full or reduced oxygen tensions, was not observed . The type strain of Rhodobacter sphaeroides as well as four strains of Rhodopseudomonas palustris also grew phototrophically on hippurate, while several other aromatic-degrading species of purple bacteria did not.

J Bacteriol, 2001 Sep, 183(17), 5180 - 6
Cytological evidence for association of the ends of the linear chromosome in Streptomyces coelicolor; Yang MC et al.; The chromosome of the filamentous bacterium Streptomyces coelicolor is linear, but the genetic map is circular . We present cytological evidence based on the use of fluorescence in situ hybridization showing that the ends of the chromosome frequently colocalize, in agreement with the idea that the ends are held together, effectively forming a circular chromosome . These observations provide a possible explanation for how a linear bacterial chromosome can exhibit a circular genetic map.

Trends Genet, 2001 Aug, 17(8), 431 - 3
Origin of an animal mitochondrial DNA polymerase subunit via lineage-specific acquisition of a glycyl-tRNA synthetase from bacteria of the Thermus-Deinococcus group; Wolf YI et al.; Phylogenetic tree analysis shows that the accessory subunit animal mitochondrial DNA polymerase emerges as a result of horizontal transfer of the gene encoding glycyl-tRNA synthetase from a bacterium of the Thermus-Deinococcus group into the animal nuclear genome . This acquisition by a distinct eukaryotic lineage of a gene encoding a mitochondrial protein from a nonmitochondrial bacterial source underscores the contribution of different types of horizontal transfer event to the evolution of eukaryotes.

EMBO J, 2001 Aug 1, 20(15), 3902 - 9
Mycobacterium tuberculosis hemoglobin N displays a protein tunnel suited for O2 diffusion to the heme; Milani M et al.; Macrophage-generated oxygen- and nitrogen-reactive species control the development of Mycobacterium tuberculosis infection in the host . Mycobacterium tuberculosis 'truncated hemoglobin' N (trHbN) has been related to nitric oxide (NO) detoxification, in response to macrophage nitrosative stress, during the bacterium latent infection stage . The three-dimensional structure of oxygenated trHbN, solved at 1.9 A resolution, displays the two-over-two alpha-helical sandwich fold recently characterized in two homologous truncated hemoglobins, featuring an extra N-terminal alpha-helix and homodimeric assembly . In the absence of a polar distal E7 residue, the O2 heme ligand is stabilized by two hydrogen bonds to TyrB10(33) . Strikingly, ligand diffusion to the heme in trHbN may occur via an apolar tunnel/cavity system extending for approximately 28 A through the protein matrix, connecting the heme distal cavity to two distinct protein surface sites . This unique structural feature appears to be conserved in several homologous truncated hemoglobins . It is proposed that in trHbN, heme Fe/O2 stereochemistry and the protein matrix tunnel may promote O2/NO chemistry in vivo, as a M.tuberculosis defense mechanism against macrophage nitrosative stress.

Chemosphere, 2001 Aug, 44(4), 519 - 28
Influence of n-alkanes and petroleum on fatty acid composition of a hydrocarbonoclastic bacterium: Marinobacter hydrocarbonoclasticus strain 617; Doumenq P et al.; This study concerns the effects of various long-chain n-alkanes, n-alkane mixtures and Arabian Light crude oil on the fatty acid (FA) composition of a sedimentary marine bacteria (Marinobacter hydrocarbonoclasticus strain 617), growing under aerobic conditions . The cultures with n-alkanes, as compared with soluble carbon sources, led to greater amounts of saturated and methyl branched FA (mainly belonging to a delta10 series) . We observed the appearance or increase of saturated and unsaturated FA with the same carbon chain length (CCL) as the n-alkane carbon source (maximum for n-alkane CCL corresponding to the 'range' of the de novo synthesized fatty acids) . We also observed a strong control of the oddness/evenness of the CCL of the FA by the oddness/evenness of the n-alkane . A n-alkane utilization index, (saturated + branched)/monounsaturated fatty acids (SAFA + BFA/MUFA) enabled discriminating between soluble carbon sources and hydrocarbons.

Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9883 - 8 Epub 2001 Jul 31.
Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid; Barnett MJ et al.; The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome . We report here the complete 1,354,226-nt sequence of pSymA . In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S . meliloti an advantage in its specialized niche.

Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9889 - 94 Epub 2001 Jul 31.
The complete sequence of the 1,683-kb pSymB megaplasmid from the N2-fixing endosymbiont Sinorhizobium meliloti; Finan TM et al.; Analysis of the 1,683,333-nt sequence of the pSymB megaplasmid from the symbiotic N(2)-fixing bacterium Sinorhizobium meliloti revealed that the replicon has a high gene density with a total of 1,570 protein-coding regions, with few insertion elements and regions duplicated elsewhere in the genome . The only copies of an essential arg-tRNA gene and the minCDE genes are located on pSymB . Almost 20% of the pSymB sequence carries genes encoding solute uptake systems, most of which were of the ATP-binding cassette family . Many previously unsuspected genes involved in polysaccharide biosynthesis were identified and these, together with the two known distinct exopolysaccharide synthesis gene clusters, show that 14% of the pSymB sequence is dedicated to polysaccharide synthesis . Other recognizable gene clusters include many involved in catabolic activities such as protocatechuate utilization and phosphonate degradation . The functions of these genes are consistent with the notion that pSymB plays a major role in the saprophytic competence of the bacteria in the soil environment.

Dig Dis Sci, 2001 Jul, 46(7), 1573 - 83
Helicobacter pylori infection and gastric function in patients with fundic atrophic gastritis; Tucci A et al.; In the present study we evaluated the relation among histology, H . pylori, IgG to H . pylori, gastric emptying, and acid secretion in 43 patients with fundic atrophic gastritis . On the basis of gastric acid secretion, patients were divided into three subgroups: patients with preserved acid secretion (Group 1), patients with hypochlorhydria (Group 2), and patients with achlorhydria (Group 3) . Fundic glandular atrophy was more severe in hypoachlorhydric patients than in those with preserved acid secretion (P < 0.05 vs Group 2, P < 0.005 vs Group 3) . H . pylori colonization was found in 94% of patients in Group 1, in 61% of patients in Group 2, and in only 8% of patients in Group 3 (P < 0.001 vs Group 1, P < 0.05 vs Group 2) . Conversely, serological positivity to H . pylori was high in all three subgroups of patients (100% in Group 1, 77% in Group 2, 92% in Group 3) . Gastric emptying was delayed in atrophic patients, particularly in those with hypoachlorhydria . Our data suggest that fundic atrophic gastritis represents a possible end stage of H . pylori infection, characterized by a progressive disappearance of the bacterium and a progressive deterioration of gastric functions.

Evolution Int J Org Evolution, 2001 Jun, 55(6), 1146 - 52
Temporal patterns of genetic variation for resistance and infectivity in a Daphnia-microparasite system; Little TJ et al.; Theoretical studies have indicated that the population genetics of host-parasite interactions may be highly dynamic . with parasites perpetually adapting to common host genotypes and hosts evolving resistance to common parasite genotypes . The present study examined temporal variation in resistance of hosts and infectivity of parasites within three populations of Daphnia magna infected with the sterilizing bacterium Pasteuria ramosa . Parasite isolates and host clones were collected in each of two years (1997, 1998) from one population; in two other populations, hosts were collected from both years, but parasites from only the first year . We then performed infection experiments (separately for each population) that exposed hosts to parasites from the same year or made combinations involving hosts and parasites from different years . In two populations, patterns were consistent with the evolution of host resistance: either infectivity or the speed with which parasites sterilized hosts declined from 1997 to 1998 . In another population, infectivity, virulence, and parasite spore production did not vary among host-year or parasite-year . For this population, we also detected strong within-population genetic variation for resistance . Thus, in this case, genetic variability for fitness-related traits apparently did not translate into evolutionary change . We discuss a number of reasons why genetic change may not occur as expected in parasite-host systems, including negative correlations between resistance and other traits, gene flow, or that the dynamic process itself may obscure the detection of gene frequency changes.

Appl Environ Microbiol, 2001 Aug, 67(8), 3767 - 70
Sinorhizobium meliloti cells require biotin and either cobalt or methionine for growth; Watson RJ et al.; Sinorhizobium meliloti is usually cultured in rich media containing yeast extract . It has been suggested that some components of yeast extract are also required for growth in minimal medium . We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation . By fractionation of yeast extract, two required growth factors were identified . Biotin was found to be absolutely required for growth, whereas previously the need for this vitamin was considered to be strain specific . All strains also required supplementation with cobalt or methionine, consistent with the requirement for a vitamin B(12)-dependent homocysteine methyltransferase for methionine biosynthesis.

J Biol Inorg Chem, 2001 Jun, 6(5-6), 601 - 7
Chlorite dismutase from Ideonella dechloratans; Stenklo K et al.; Chlorite dismutase has been purified from the chlorate-metabolizing bacterium Ideonella dechloratans . The purified enzyme is tetrameric, with a relative molecular mass of 25,000 for the subunit, and contains about 0.6 heme/subunit as isolated . Its catalytic properties are similar, but not identical, to those found for a similar enzyme purified earlier from the bacterium GR-1 . The heme group in Ideonella chlorite dismutase is readily reduced by dithionite, in contrast to the GR-1 enzyme, and redox titration gave a value of -21 mV for the midpoint potential at pH 7 . The heme group has been characterized by optical and EPR spectroscopy . It is high-spin ferric at neutral pH, with spectroscopic properties similar to those found for cytochrome c peroxidase . In the alkaline pH range, a low-spin compound is formed . A 22-residue N-terminal amino acid sequence has been determined and no homologue has been found in the protein sequence databases.

Mutat Res, 2001 Aug 8, 479(1-2), 63 - 70
Reduction of ENU-induced transversion mutations by the isoflavone genistein in Escherichia coli; Yang Y et al.; In studies of mutagenesis induced by the carcinogen N-ethyl-N-nitrosourea (ENU) in the bacterium Escherichia coli FX-11, it was observed that G:C to A:T transitions did not require the inducible umuDC gene products, while a portion of the A:T to G:C transitions and all transversion mutations were dependent on a functional umuC gene . This observation suggested that the different base substitutions may result from differential processing of specific DNA adducts produced by ENU . To further understand these processes, we have investigated the effect of the soybean isoflavone genistein on the production of ENU-induced mutations . This compound, in particular, has been shown to exhibit numerous effects including the inhibition of the growth or proliferation of a variety of cancers, inhibition of angiogenesis, inhibition of tyrosine protein kinases and anti-oxidant properties . In our experiments, tyrosine defective (TyrA(-)) E . coli were exposed to ENU and a portion of the ENU-treated cells were exposed to genistein . The results showed a three-fold reduction in the overall mutation frequency when cells were treated with genistein subsequent to ENU-exposure and this anti-mutagenic effect was dependent on the dose of genistein employed . However, only certain types of base substitution mutagenesis were affected . In particular, transversion mutations were reduced an average of about 8.5-fold, while transitions were not greatly affected . In addition, UV-mutagenesis was reduced about three-fold and induction of the SOS response (as monitored with a sulA-lacZ fusion) was decreased . These results suggest that genistein may interfere with expression of the SOS response, including the UmuC-mediated lesion bypass mechanism that is necessary for UV-mutagenesis and the generation of transversions by ENU in E . coli.

Biochim Biophys Acta, 2001 Jul 30, 1520(1), 63 - 70
In the facultative sulphate/nitrate reducer Desulfovibrio desulfuricans ATCC 27774, the nine-haem cytochrome c is part of a membrane-bound redox complex mainly expressed in sulphate-grown cells; Saraiva LM et al.; The bacterium Desulfovibrio desulfuricans ATCC 27774 belongs to the group of sulphate reducers also capable of utilising nitrate as its terminal electron acceptor for anaerobic growth . One of the complex multihaem proteins found in nitrate- or sulphate-grown cells of Desulfovibrio desulfuricans ATCC 27774 is the nine-haem cytochrome c . The present work shows that the gene encoding for Desulfovibrio desulfuricans ATCC 27774 nine-haem cytochrome c is part of an operon formed by the gene cluster 9hcA-D . Besides 9hcA, the gene encoding for the nine-haem cytochrome c, genes 9hcB to D encode for a protein containing four {4Fe-4S}(2+/1+) centres, for a dihaem transmembrane cytochrome b and for an unknown hydrophobic protein, respectively . The four proteins have a predicted topology that is in accordance with the formation of a membrane-bound redox complex . Furthermore, the transcriptional studies show that not only the expression of the 9HcA-D complex is dependent on the growth phase, but also is markedly increased in sulphate-grown cells.

Biochemistry, 2001 Jul 31, 40(30), 8738 - 48
Multiple cleavage activities of endonuclease V from Thermotoga maritima: recognition and strand nicking mechanism; Huang J et al.; Endonuclease V is a deoxyinosine 3'-endonuclease which initiates removal of inosine from damaged DNA . A thermostable endonuclease V from the hyperthermophilic bacterium Thermotoga maritima has been cloned and expressed in Escherichia coli . The DNA recognition and reaction mechanisms were probed with both double-stranded and single-stranded oligonucleotide substrates which contained inosine, abasic site (AP site), uracil, or mismatches . Gel mobility shift and kinetic analyses indicate that the enzyme remains bound to the cleaved inosine product . This slow product release may be required in vivo to ensure an orderly process of repairing deaminated DNA . When the enzyme is in excess, the primary nicked products experience a second nicking event on the complementary strand, leading to a double-stranded break . Cleavage at AP sites suggests that the enzyme may use a combination of base contacts and local distortion for recognition . The weak binding to uracil sites may preclude the enzyme from playing a significant role in repair of such sites, which may be occupied by uracil-specific DNA glycosylases . Analysis of cleavage patterns of all 12 natural mismatched base pairs suggests that purine bases are preferrentially cleaved, showing a general hierarchy of A = G > T > C . A model accounting for the recognition and strand nicking mechanism of endonuclease V is presented.

J Bacteriol, 2001 Aug, 183(16), 4860 - 5
Conserved promoter motif is required for cell cycle timing of dnaX transcription in Caulobacter; Keiler KC et al.; Cells use highly regulated transcriptional networks to control temporally regulated events . In the bacterium Caulobacter crescentus, many cellular processes are temporally regulated with respect to the cell cycle, and the genes required for these processes are expressed immediately before the products are needed . Genes encoding factors required for DNA replication, including dnaX, dnaA, dnaN, gyrB, and dnaK, are induced at the G(1)/S-phase transition . By analyzing mutations in the dnaX promoter, we identified a motif between the -10 and -35 regions that is required for proper timing of gene expression . This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors . Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor . Consistent with this hypothesis, Caulobacter extracts contain an activity that binds specifically to the RRF in vitro.

J Bacteriol, 2001 Aug, 183(16), 4702 - 8
Treponema pallidum 3-phosphoglycerate mutase is a heat-labile enzyme that may limit the maximum growth temperature for the spirochete; Benoit S et al.; In the causative agent of syphilis, Treponema pallidum, the gene encoding 3-phosphoglycerate mutase, gpm, is part of a six-gene operon (tro operon) that is regulated by the Mn-dependent repressor TroR . Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP in T . pallidum and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium . To study this, T . pallidum gpm was cloned, Gpm was purified from Escherichia coli, and antiserum against the recombinant protein was raised . Immunoblots indicated that Gpm was expressed in freshly extracted infective T . pallidum . Enzyme assays indicated that Gpm did not require Mn(2+) while 2,3-diphosphoglycerate (DPG) was required for maximum activity . Consistent with these observations, Mn did not copurify with Gpm . The purified Gpm was stable for more than 4 h at 25 degrees C, retained only 50% activity after incubation for 20 min at 34 degrees C or 10 min at 37 degrees C, and was completely inactive after 10 min at 42 degrees C . The temperature effect was attenuated when 1 mM DPG was added to the assay mixture . The recombinant Gpm from pSLB2 complemented E . coli strain PL225 (gpm) and restored growth on minimal glucose medium in a temperature-dependent manner . Increasing the temperature of cultures of E . coli PL225 harboring pSLB2 from 34 to 42 degrees C resulted in a 7- to 11-h period in which no growth occurred (compared to wild-type E . coli) . These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of T . pallidum.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8235 - 40
Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled; McGlynn P et al.; Replication forks formed at bacterial origins often encounter template roadblocks in the form of DNA adducts and frozen protein-DNA complexes, leading to replication-fork stalling and inactivation . Subsequent correction of the corrupting template lesion and origin-independent assembly of a new replisome therefore are required for survival of the bacterium . A number of models for replication-fork restart under these conditions posit that nascent strand regression at the stalled fork generates a Holliday junction that is a substrate for subsequent processing by recombination and repair enzymes . We show here that early replication intermediates containing replication forks stalled in vitro by the accumulation of excess positive supercoils could be cleaved by the Holliday junction resolvases RusA and RuvC . Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecG, confirming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent strand regression at stalled replication forks . Furthermore, RecG promoted Holliday junction formation when replication intermediates in which the replisome had been inactivated were negatively supercoiled, suggesting that under intracellular conditions, the action of RecG, or helicases with similar activities, is necessary for the catalysis of nascent strand regression.

Mol Cells, 2001 Jun 30, 11(3), 312 - 20
Presumptive mechanisms of peptic ulceration by Helicobacter pylori VacA involving mucoprotease and CagA; Choi KM et al.; Helicobacter pylori vacuolating toxin (VacA) appears to be unusually stable, not only against extreme pH conditions or high temperatures, but also against common organic solvents or detergents . Under acidic conditions, its activity was markedly increased in the manner of temperature-independent, suggesting a spontaneous activation . A similar finding was also observed under alkaline conditions, however, it should have an appropriate temperature . From these observations, the mechanisms of VacA activation were suggested to be so redundant that either the case of acidic or basic amino acid residues could be involved in the VacA activation . Separately, we also found that the VacA production by H . pylori was pH-dependent: Its production was increased at a low pH region with a broad range (1.0-5.0), and at a high pH region with a narrow range (8.0-9.0) . Astonishingly, a highly immunogenic CagA did not appear to be expressed under the acidic conditions . Its expression, however, was shown to be enhanced when the surrounding pH of this bacterium was raised . In contrast, mucoproteolytic activity in the H . pylori membrane was found to be increased at acidic conditions . Considering these observations, together with the stomach and duodenal pH of humans, two presumptive mechanisms of H . pylori VacA-associated ulceration may be deduced; namely, an acid- and an alkali-dependent type, involving mucoprotease and CagA, respectively.

Biochemistry, 2001 Jul 24, 40(29), 8523 - 30
Inactivation of C30A trimethylamine dehydrogenase by N-cyclopropyl-alpha-methylbenzylamine, 1-phenylcyclopropylamine, and phenylhydrazine; Mitchell DJ et al.; Trimethylamine dehydrogenase (TMADH) from the bacterium Methylophilus methylotrophus (sp . W(3)A(1)) and its C30A mutant were inactivated with three known inactivators of monoamine oxidase, namely, phenylhydrazine, N-cyclopropyl-alpha-methylbenzylamine, and 1-phenylcyclopropylamine . All three compounds irreversibly inactivated both the wild-type and C30A mutant enzymes, although phenylhydrazine was 10 times more potent than N-cyclopropyl-alpha-methylbenzylamine, which was much more potent than 1-phenylcyclopropylamine . The change in the UV--visible absorption spectra upon modification indicated that the flavin was modified . In the case of the C30A mutant, the absence of a covalent attachment of the flavin to the polypeptide has permitted LC-electrospray mass spectrometry of the reaction product to be undertaken, demonstrating new mass peaks corresponding to various chemically modified forms of the flavin cofactor . In the case of N-cyclopropyl-alpha-methylbenzylamine, masses corresponding to hydroxy-FMN and hydroxyriboflavin were detected . 1-Phenylcyclopropylamine inactivation of the C30A mutant produced three modified flavins, as evidenced by the electrospray mass spectrum: hydroxy-FMN, FMN plus C(6)H(5)COCH(2)CH(2), and hydroxy-FMN plus C(6)H(5)COCH(2)CH(2) . Phenylhydrazine inactivation of the C30A mutant gave at least seven different modified flavins: hydroxyriboflavin, hydroxy-FMN, two apparently isomeric compounds corresponding to hydroxy-FMN plus one phenyl group, two apparently isomeric compounds corresponding to FMN plus one phenyl group, and FMN plus two phenyl groups . Covalent flavin adduct formation appears to be the only modification because dialysis of the inactive enzyme followed by reconstitution with FMN restores the enzyme activity to that of a noninactivated control.

J Food Prot, 2001 Jul, 64(7), 1030 - 4
Nannocystis exedens: a potential biocompetitive agent against Aspergillus flavus and Aspergillus parasiticus; Taylor WJ et al.; This study examined the potential for controlling toxigenic Aspergillus flavus and Aspergillus parasiticus by biological means using a myxobacterium commonly found in soil . The ability of Nannocystis exedens to antagonize A . flavus ATCC 16875, A . flavus ATCC 26946, and A . parasiticus NRRL 3145 was discovered . Cultures of aflatoxigenic fungi were grown on 0.3% Trypticase peptone yeast extract agar for 14 days at 28 degrees C . When N . exedens was grown in close proximity with an aflatoxigenic mold, zones of inhibition (10 to 20 mm) developed between the bacterium and mold colony . A flattening of the mold colony on the sides nearest N . exedens and general stunting of growth of the mold colony were also observed . When N . exedens was added to the center of the cross-streak of a mold colony, lysis of the colony by the bacterium was observed after 24 h . Microscopic observations revealed that N . exedens grew on spores, germinating spores, hyphae, and sclerotia of the molds . These results indicate that N . exedens may be a potential biocontrol agent against A . flavus and A . parasiticus.

Extremophiles, 2001 Jun, 5(3), 161 - 8
Molecular cloning and characterization of thermostable DNA ligase from Aquifex pyrophilus, a hyperthermophilic bacterium; Lim JH et al.; A DNA ligase gene from the hyperthermophilic bacterium Aquifex pyrophilus (Ap) was cloned and sequenced . An open reading frame of 2,157 bp that codes for a 82-kDa protein showed 40%-60% homology with a series of NAD+-dependent DNA ligases from different organisms . The recombinant enzyme Ap DNA ligase expressed in Escherichia coli was purified to homogeneity and characterized . The activity of Ap DNA ligase gradually increased in proportion to the concentration of monovalent salt up to 200 mM NaCl, 150 mM KCl, 200 mM NH4Cl, and 350 mM potassium glutamate . The optimum temperature and pH of Ap DNA ligase were greater than 65 degrees C and 8.0-8.6, respectively, for nick-closing activity . More than 75% of the ligation activity was retained after incubation at 95 degrees C for 60 min, whereas the half-lives of Thermus aquaticus and Escherichia coli DNA ligases at 95 degrees C were < or =15 min and 5 min, respectively . Thermostable Ap DNA ligase was applied to repeat expansion detection (RED) and could be a useful enzyme in DNA diagnostics.

Science, 2001 Jul 13, 293(5528), 297 - 300
Molecular evolution of protein atomic composition; Baudouin-Cornu P et al.; Living organisms encounter various growth conditions in their habitats, raising the question of whether ecological fluctuations could alter biological macromolecules . The advent of complete genome sequences and the characterization of whole metabolic pathways allowed us to search for such ecological imprints . Significant correlations between atomic composition and metabolic function were found in sulfur- and carbon-assimilatory enzymes, which appear depleted in sulfur and carbon, respectively, in both the bacterium Escherichia coli and the eukaryote Saccharomyces cerevisiae . In addition to genetic instructions, genomic data thus also provide paleontological records of environmental nutrient availability and of metabolic costs.

FEMS Microbiol Ecol, 2001 Jul, 36(2-3), 131 - 137
Adaptation of Ruminococcus flavefaciens resulting in increased degradation of ryegrass cell walls; Saluzzi L et al.; This study investigated the long term adaptation of a ruminal bacterium to growth on four different plant cell wall substrates . No significant increase in degradation was detected for lucerne, barley straw or weeping lovegrass after 23 serial subcultures of the cellulolytic rumen bacterium Ruminococcus flavefaciens strain 17 on each of these substrates . Significantly increased substrate degradation by R . flavefaciens strain 17 was however observed after 23 subcultures on perennial ryegrass . The increase in dry matter solubilisation (from 24.3 to 39.5% in 24 h incubation and from 52.3 to 61% in 72 h) was at least partially due to an increase in solubilisation of xylose, glucose and arabinose . Enhanced growth of the adapted strains occurred on this substrate . Significant increases in xylanase and beta-xylosidase specific activities were detected but no effect was detected on xylanase profiles in zymogram analyses . Similar responses were observed for two cultures originally derived from single-colony re-isolates . The most likely explanation for the observed adaptation involves selection for mutations affecting the regulation of xylanolytic enzymes.

Mikrobiologiia, 2001 May-Jun, 70(3), 391 - 7
{Ecophysiological features of mat-forming bacteria Thioploca in bottom sediments of Frolikha Bay, northern Baikal}; Zemsiakaia TI et al.; A colorless sulfur bacterium of the genus Thioploca, which forms bacterial mats, was studied in the region of underwater thermal vents (Frolikha Bay, northern Baikal) . The organism occurs under microaerobic conditions in top sediment layers, and its biomass can amount to 65 mg of wet weight per 1 kg of silt . Individual filaments of the bacterium penetrate the anaerobic zone to the depth of 19 cm . Thioploca is distributed in a mosaic pattern over the bottom of the bay . Thioploca mats are typically found near vents that discharge low-temperature underground water . In the form of separate filaments, this bacterium is more widely distributed in the top sediment layer, particularly in sediments with a more active sulfate reduction . The bacteria from the deep-water and coastal areas of the bay have different morphology . Cells of Thioploca are able to accumulate nitrate, and the coefficient of nitrate accumulation in wet bacterial mass in relation to the near-bottom water is 1.3 x 10(4), suggesting a similarity of metabolism with seawater species . A more lightweight isotopic composition of nitrogen in cell mass as compared to that of representatives of zoobenthos also indicates an active metabolism of nitrogen, apparently, in the process of nitrogen respiration . Comparison of the composition of stable isotopes of carbon in the biomass of representatives of different trophic levels, including Thioploca, found at a depth of 105 m indicates its planktonic origin, whereas, in the deeper bay region, the biomass of Thioploca incorporates more of the light carbon originating from biogenic methane.

Infect Immun, 2001 Aug, 69(8), 5166 - 72
Role of gingipains in growth of Porphyromonas gingivalis in the presence of human serum albumin; Grenier D et al.; Porphyromonas gingivalis, a bacterium associated with active chronic periodontitis lesions, produces several proteolytic enzymes that are thought to be involved in host colonization, perturbation of the immune system, and tissue destruction . The aim of the present study was to investigate the contribution of Arg- and Lys-gingipains produced by P . gingivalis to its growth . Although all of the proteins studied were degraded by P . gingivalis, only human serum albumin and transferrin supported growth during serial transfers in a chemically defined medium (CDM) . Growth studies with site-directed gingipain-deficient mutants of P . gingivalis revealed that inactivation of both gingipains prevents growth, whereas inactivation of either Arg- or Lys-gingipain activity extended the doubling times to 33 or 13 h, respectively, compared to 9 h for the parent strain . Growth of the mutants and the parent strain was similar when the CDM was supplemented with a protein hydrolysate instead of human serum albumin . Incubation of resting P . gingivalis ATCC 33277 cells with fluorophore-labeled albumin indicated that the proteolytic fragments generated by the gingipains were internalized by the bacterial cells . Internalization of fluorophore-labeled albumin fragments was reduced or completely inhibited in the proteinase-deficient mutants . Interestingly, gingival crevicular fluid samples from diseased periodontal sites contained low-molecular-mass albumin fragments, whereas samples from healthy sites did not . The critical role of proteinases in the growth of P . gingivalis was further investigated using specific Arg- and Lys-gingipain inhibitors . Adding the inhibitors to CDM containing albumin revealed that leupeptin (Arg-gingipain A and B inhibitor) was more efficient at inhibiting growth than cathepsin B inhibitor II (Lys-gingipain inhibitor) . Our study suggests that Arg-gingipains and, to a lesser extent, Lys-gingipain play an important role in the growth of P . gingivalis in a defined medium containing a human protein as the sole carbon and nitrogen source.

Infect Immun, 2001 Aug, 69(8), 4874 - 83
Characterization of a stress-induced alternate sigma factor, RpoS, of Coxiella burnetii and its expression during the development cycle; Seshadri R et al.; Coxiella burnetii is an obligate intracellular bacterium that resides in an acidified phagolysosome and has a remarkable ability to persist in the extracellular environment . C . burnetii has evolved a developmental cycle that includes at least two morphologic forms, designated large cell variants (LCV) and small cell variants (SCV) . Based on differential protein expression, distinct ultrastructures, and different metabolic activities, we speculated that LCV and SCV are similar to typical logarithmic- and stationary-phase growth stages . We hypothesized that the alternate sigma factor, RpoS, a global regulator of genes expressed under stationary-phase, starvation, and stress conditions in many bacteria, regulates differential expression in life cycle variants of C . burnetii . To test this hypothesis, we cloned and characterized the major sigma factor, encoded by an rpoD homologue, and the stress response sigma factor, encoded by an rpoS homologue . The rpoS gene was cloned by complementation of an Escherichia coli rpoS null mutant containing an RpoS-dependent lacZ fusion (osmY::lacZ) . Expression of C . burnetii rpoS was regulated by growth phase in E . coli (induced upon entry into stationary phase) . A glutathione S-transferase-RpoS fusion protein was used to develop polyclonal antiserum against C . burnetii RpoS . Western blot analysis detected abundant RpoS in LCV but not in SCV . These results suggest that LCV and SCV are not comparable to logarithmic and stationary phases of growth and may represent a novel adaptation for survival in both the phagolysosome and the extracellular environment.

FEMS Microbiol Lett, 2001 Jul 10, 201(1), 9 - 14
What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function; Poteete AR; Recent studies have generated interest in the use of the homologous recombination system of bacteriophage lambda for genetic engineering . The system, called Red, consists primarily of three proteins: lambda exonuclease, which processively digests the 5'-ended strand of a dsDNA end; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme and inhibits its activities . These proteins induce a 'hyper-rec' state in Escherichia coli and other bacteria, in which recombination events between DNA species with as little as 40 bp of shared sequence occur at high frequency . Red-mediated recombination in the hyper-rec bacterium proceeds via a number of different pathways, and with the involvement of different sets of bacterial proteins, depending in part on the nature of the recombining DNA species . The role of high-frequency double-strand break repair/recombination in the life cycle of the lambdoid phages is discussed.

Oral Microbiol Immunol, 2001 Aug, 16(4), 229 - 34
Porphyromonas endodontalis binds, reduces and grows on human hemoglobin; Zerr M et al.; Porphyromonas endodontalis is a black-pigmented, obligate anaerobic rod-shaped bacterium implicated as playing a major role in endodontic infections . We have previously shown that P . endodontalis requires the porphyrin nucleus, preferably supplied as hemoglobin, as a growth supplement . The bacteria also actively transport free iron, although this activity does not support growth in the absence of a porphyrin source . The purpose of this study was to further investigate the binding and subsequent utilization of human hemoglobin by P . endodontalis . P . endodontalis binds hemoglobin and reduces the Fe(III) porphyrin, resulting in a steady accumulation of ferrous hemoglobin . Reduction of methemoglobin was similar to the extracellular reduction of nitrobluetetrazolium in the presence of oxidizable substrate . Turbidimetric and viable cell determinations showed that P . endodontalis grew when supplied only hemoglobin . Therefore, we conclude that hemoglobin appears to serve as a sole carbon and nitrogen source, and that these bacteria reduce extracellular compounds at the expense of oxidized substrates.

Oral Microbiol Immunol, 2001 Aug, 16(4), 212 - 7
Studies on the aminopeptidase activities of Porphyromonas gingivalis; Grenier D et al.; Porphyromonas gingivalis is an asaccharolytic bacterium that requires nitrogen substrates as carbon and energy sources . The aims of this study were to investigate the aminopeptidase activities of P . gingivalis and to evaluate the effect of aminopeptidase inhibitors on bacterial growth . Only arginine aminopeptidase and dipeptidyl aminopeptidase IV activities were detected . Experimental evidence was obtained suggesting that the Arg-gingipains of P . gingivalis can function as both an endopeptidase and an aminopeptidase . Firstly, the arginine aminopeptidase activity was found to be inhibited by leupeptin, a well-known inhibitor of Arg-gingipain activity . Secondly, a preparation of Arg-gingipain activity could hydrolyze the chromogenic substrate for arginine aminopeptidase . Lastly, a mutant of P . gingivalis constructed via gene disruption by use of suicide plasmids and deficient in both Arg-gingipain A and B was also devoid of arginine aminopeptidase activity . To investigate the key role of aminopeptidase activities in growth of P . gingivalis, aminopeptidase inhibitors were incorporated in the culture medium prior to inoculation . Bestatin and actinonin were the only ones to inhibit growth of P . gingivalis . Their mechanism of growth inhibition appears to be different but does not involve inhibition of the two major aminopeptidase activities (arginine aminopeptidase and dipeptidyl aminopeptidase IV).

Lett Appl Microbiol, 2001 Jul, 33(1), 56 - 60
Pleomorphism of the marine bacterium Teredinobacter turnirae; Ferreira GM et al.; AIMS: A morphology transition for the marine bacterium, Teredinobacter turnirae is reported . METHODS AND RESULTS: When grown in the rod-shaped morphology, the cells require high concentrations of NaCl (0.3 mol x l(-1)) and secrete extracellular protease and endoglucanase activity . When this bacterium is grown in a medium containing casein as a sole carbon and nitrogen source, a major change in morphology to a stable aggregated form is obtained . CONCLUSION: In the aggregated morphology, much higher protease production rates (170 Units x ml(-1) x d-1 for aggregates vs . 15 Units x ml(-1) x d(-1) for rods, for the same initial biomass) and negligible endoglucanase titres are obtained . In addition, the aggregated morphology does not require sodium chloride for growth . SIGNIFICANCE AND IMPACT OF THE STUDY: The phenomenon reported here describes a novel relationship between the cell morphology and the biochemical characteristics of the bacterium.

Biotech Histochem, 2001 Jan, 76(1), 31 - 4
A stain for demonstrating Helicobacter pylori in gastric biopsies; Young DG; Helicobacter pylori continues to be a significant health care problem . It is associated with a variety of stomach disorders such as gastritis, gastric ulcer disease, gastric carcinoma and B-cell gastric lymphoma . One common method diagnosing an infection by this bacterium is microscopic examination of routine processed gastric or duodenal biopsies . With this type of specimen, it is necessary to demonstrate visually the presence of H . pylori using an appropriate staining technique . This paper presents a simple staining technique for demonstrating H . pylori in gastric biopsy specimens using carbol fuchsin staining against a contrasting background of light green.

Biosci Biotechnol Biochem, 2001 May, 65(5), 1095 - 103
Purification and characterization of a cold-adapted isocitrate lyase and a malate synthase from Colwellia maris, a psychrophilic bacterium; Watanabe S et al.; Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state . The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa . MS was a dimeric enzyme composed of 76.3 kDa subunits . N-Terminal amino acid sequences of the ICL and MS were analyzed . Purified ICL had its maximum activity at 20 degrees C and was rapidly inactivated at the temperatures above 30 degrees C, but the optimum temperature for the activity of MS was 45 degrees C . NaCl was found to protect ICL from heat inactivation above 30 degrees C, but the salt did not stabilize MS . Effects of temperatures on the kinetic parameters of both the enzymes were examined . The Km for the substrate (isocitrate) of ICL was decreased with decreasing temperature . On the other hand, the Km for the substrate (glyoxylate) of MS was increased with decreasing temperature . The calculated value of free energy of activation of ICL was on the same level as that of MS.

J Biol Chem, 2001 Jun 15, 276(24), 10971 - 6
Biosynthetic controls on the 13C contents of organic components in the photoautotrophic bacterium Chloroflexus aurantiacus; van der Meer MT et al.; To assess the effects related to known and proposed biosynthetic pathways on the (13)C content of lipids and storage products of the photoautotrophic bacterium Chloroflexus aurantiacus, the isotopic compositions of bulk cell material, alkyl and isoprenoid lipids, and storage products such as glycogen and polyhydroxyalkanoic acids have been investigated . The bulk cell material was 13 per thousand depleted in (13)C relative to the dissolved inorganic carbon . Evidently, inorganic carbon fixation by the main carboxylating enzymes used by C . aurantiacus, which are assumed to use bicarbonate rather than CO(2), results in a relatively small carbon isotopic fractionation compared with CO(2) fixation by the Calvin cycle . Even carbon numbered fatty acids, odd carbon numbered fatty acids, and isoprenoid lipids were 14, 15, and 17-18 per thousand depleted in (13)C relative to the carbon source, respectively . Based on the (13)C contents of alkyl and isoprenoid lipids, a 40 per thousand difference in (13)C content between the carboxyl and methyl carbon from acetyl-coenzyme A has been calculated . Both sugars and polyhydroxyalkanoic acid were enriched in (13)C relative to the alkyl and isoprenoid lipids . To the best of our knowledge this is the first report in which the stable carbon isotopic composition of a large range of biosynthetic products in a photoautotrophic organism has been investigated and interpreted based on previously proposed inorganic carbon fixation and biosynthetic pathways . Our results indicate that compound-specific stable carbon isotope analysis may provide a rapid screening tool for carbon fixation pathways.

Biochem J, 2001 Jul 15, 357(Pt 2), 505 - 11
Determination of the chemical structure of the capsular polysaccharide of strain B33, a fast-growing soya bean-nodulating bacterium isolated from an arid region of China; Rodriguez-Carvajal MA et al.; We have determined the structure of a polysaccharide from strain B33, a fast-growing bacterium that forms nitrogen-fixing nodules with Asiatic and American soya bean cultivars . On the basis of monosaccharide analysis, methylation analysis, one-dimensional 1H- and 13C-NMR and two-dimensional NMR experiments, the structure was shown to consist of a polymer having the repeating unit -->6)-4-O-methyl-alpha-D-Glcp-(1-->4)-3-O-methyl-beta-D-GlcpA-(1--> (where GlcpA is glucopyranuronic acid and Glcp is glucopyranose) . Strain B33 produces a K-antigen polysaccharide repeating unit that does not have the structural motif sugar-Kdx {where Kdx is 3-deoxy-D-manno-2-octulosonic acid (Kdo) or a Kdo-related acid} proposed for different Sinorhizobium fredii strains, all of them being effective with Asiatic soya bean cultivars but unable to form nitrogen-fixing nodules with American soya bean cultivars . Instead, it resembles the K-antigen of S . fredii strain HH303 (rhamnose, galacturonic acid)n, which is also effective with both groups of soya bean cultivars . Only the capsular polysaccharide from strains B33 and HH303 have monosaccharide components that are also present in the surface polysaccharide of Bradyrhizobium elkanii strains, which consists of a 4-O-methyl-D-glucurono-L-rhamnan.

J Dent Res, 2001 May, 80(5), 1425 - 9
Preferential utilization of dipeptides by Porphyromonas gingivalis; Takahashi N et al.; Although Porphyromonas gingivalis is known to utilize peptides preferentially, instead of free amino acids, as the source of energy and cell material, there is only limited information on what sizes and kinds of peptide this bacterium preferentially utilizes . In this study, therefore, we tested aspartate or glutamate monopolymers consisting of from 2 to 100 amino acids as metabolic substrates for P . gingivalis . The washed cells of P . gingivalis consumed aspartylaspartate and glutamylglutamate, and produced large amounts of ammonia and organic acids such as propionate and butyrate, while the cells formed only small amounts of end-products from aspartate, glutamate, and other peptides longer than a dipeptide . P . gingivalis also metabolized valylvaline and leucylleucine and produced isobutyrate and isovalerate, respectively, only in the presence of aspartylaspartate or glutamylglutamate . This suggests a metabolic linkage between these dipeptides . These results clearly indicate that P . gingivalis utilizes dipeptides preferentially as its metabolic substrates.

Genome Res, 2001 Jul, 11(7), 1246 - 55
Phenotype microarrays for high-throughput phenotypic testing and assay of gene function; Bochner BR et al.; The bacterium Escherichia coli is used as a model cellular system to test and validate a new technology called Phenotype MicroArrays (PMs) . PM technology is a high-throughput technology for simultaneous testing of a large number of cellular phenotypes . It consists of preconfigured well arrays in which each well tests a different cellular phenotype and an automated instrument that continuously monitors and records the response of the cells in all wells of the arrays . For example, nearly 700 phenotypes of E . coli can be assayed by merely pipetting a cell suspension into seven microplate arrays . PMs can be used to directly assay the effects of genetic changes on cells, especially gene knock-outs . Here, we provide data on phenotypic analysis of six strains and show that we can detect expected phenotypes as well as, in some cases, unexpected phenotypes.

Environ Sci Technol, 2001 Jun 15, 35(12), 2482 - 90
Kinetic analysis of the bacterial reduction of goethite; Liu C et al.; The kinetics of dissimilatory reduction of goethite (alpha-FeOOH) was studied in batch cultures of a groundwater bacterium, Shewanella putrefaciens, strain CN32 in pH 7 bicarbonate buffer . The rate and extent of goethite reduction were measured as a function of electron acceptor (goethite) and donor (lactate) concentrations . Increasing goethite concentrations increased both the rate and extent of Fe(III) reduction when cell and lactate concentrations were held constant . However, constant initial reduction rates were observed after normalization to the Fe(II) sorption capacity of FeOOH, suggesting that the bacterial reduction rate was first orderwith respect to surface site concentration . Increasing the lactate concentration also increased the rate and extent of FeOOH reduction . Monod-type kinetic behaviorwas observed with respectto lactate concentration . Fe(II) sorption on FeOOH was well-described by the Langmuir sorption isotherm . However, the Fe(II) sorption capacities hyperbolically decreased with increasing FeOOH concentration (10-100 mM) implying aggregation, while the affinity constant between Fe(II) and goethite was constant (log K approximately equals 3) . Evaluation of the end states of the variable FeOOH and lactate experiments when iron reduction ceased indicated a consistent excess in reaction free energy of -22.7 kJ/mol . This value was remarkably close to the minimum value reported for bacteria to mediate a given reaction (-20 kJ/mol) . X-ray diffraction (XRD) and scanning electron microscopy (SEM) indicated that siderite (FeCO3) was the only biogenic Fe(II) solid formed upon FeOOH bioreduction . A kinetic biogeochemical model that incorporated Monod kinetics with respect to lactate concentration, first-order kinetics with respectto goethite surface concentration, a Gibbs free energy availability factor, the rates of Fe(II) sorption on goethite and siderite precipitation, and aqueous speciation reactions was applied to the experimental data . Using independently estimated parameters, the developed model successfully described bacterial goethite reduction with variable FeOOH and lactate concentrations.

Science, 2001 Jun 29, 292(5526), 2479 - 82
A mite species that consists entirely of haploid females; Weeks AR et al.; The dominance of the diploid state in higher organisms, with haploidy generally confined to the gametic phase, has led to the perception that diploidy is favored by selection . This view is highlighted by the fact that no known female organism within the Metazoa exists exclusively (or even for a prolonged period) in a haploid state . We used fluorescence microscopy and variation at nine microsatellite loci to show that the false spider mite, Brevipalpus phoenicis, consists of haploid female parthenogens . We show that this reproductive anomaly is caused by infection by an undescribed endosymbiotic bacterium, which results in feminization of haploid genetic males.

Evolution Int J Org Evolution, 2001 May, 55(5), 889 - 96
Evolution of thermal dependence of growth rate of Escherichia coli populations during 20,000 generations in a constant environment; Cooper VS et al.; Twelve experimental populations of the bacterium Escherichia coli evolved for 20,000 generations in a defined medium at 37 degrees C . We measured their maximum growth rates across a broad range of temperatures and at several evolutionary time points to quantify the extent to which they became thermal specialists with diminished performance at other temperatures . We also sought to determine whether antagonistic pleiotropy (genetic trade-offs) or mutation accumulation (drift decay) was primarily responsible for any thermal specialization . Populations showed consistent improvement in growth rate at moderate temperatures (27-39 degrees C), but tended to have decreased growth rate at both low (20 degrees C) and high (41-42 degrees C) temperatures . Most loss occurred early in the experiment, when adaptation was most rapid . This dynamic is predicted by antagonistic pleiotropy but not by mutation accumulation . Several populations evolved high mutation rates due to defects in their DNA repair, but they did not subsequently undergo a greater decrease in growth rate at thermal extremes than populations that retained low mutation rates, contrary to the acceleration of decay predicted by mutation accumulation . Antagonistic pleiotropy therefore is more likely to be responsible for the evolution of thermal specialization observed in maximum growth rate.

Appl Environ Microbiol, 2001 Jul, 67(7), 3299 - 303
True chemotaxis in oxygen gradients of the sulfur-oxidizing bacterium Thiovulum majus; Thar R et al.; Observations of free-swimming Thiovulum majus cells show that these bacteria exhibit a phobic response as well as true chemotaxis in oxygen gradients . Both phenomena of their chemotactic behavior are integrated into a single model of helical klinotaxis, which is demonstrated by computer simulations.

Environ Mol Mutagen, 2001, 37(4), 356 - 60
Estimation of the average burst size of Phix174 am3, cs70 for use in mutation assays with transgenic mice; Delongchamp RR et al.; In mutation assays using transgenic mice, with recoverable vectors such as PhiX174 am3, cs70, mutations originate from two sources: (1) in vivo mutations, that is, mutations that were fixed in the mouse, or (2) ex vivo mutations, that is, mutations that were fixed during recovery or plating . When a bacteriophage infects a bacterium, it multiplies and bursts the cell, releasing a number of phages referred to as the burst size . Our method for distinguishing between in vivo mutations and ex vivo mutations estimates the average number of bursts, the denominator of in vivo mutant frequencies, by dividing the total plaque-forming units (PFU) by the average number of phages in a burst . Herein, we outline a probability model relating observed plaque counts to the burst size and present the statistical method used to estimate the burst size . The average size of a single burst from nonrevertant phages was estimated in eight studies under the conditions of our mutation assay . The average burst size was stable across studies at 182.5 plaques per burst (standard error, 14.25) . The probability that a burst is a specific size was approximated by a negative binomial distribution, which implies a Poisson-Pascal distribution for the observed plaque counts . The observed plaque counts were adequately fit by this approximation . Environ . Mol . Mutagen . 37:356-360, 2001 Published 2001 Wiley-Liss, Inc.

J Ind Microbiol Biotechnol, 1999 Oct, 23(4-5), 425 - 435
Special cell surface structure, and novel macromolecule transport/depolymerization system of Sphingomonas sp A1; Momma K et al.; A bacterium isolated from soil as an alginate lyase producer shows characteristic morphological and taxonomical properties consistent with being classified in the genus Sphingomonas . The bacterium utilizes high molecular weight (HMW)-alginate for growth by depolymerization of the polymer with intracellular alginate lyases, which are generated from a common precursor protein through autoregulated post-translational modifications . Electron microscopic observations of the cell surface and of thin sections of cells grown on HMW-alginate revealed dynamic changes in both cell surface and membrane structures . The most remarkable change is recognized in the formation of mouth-like pits which open and close depending on the presence or absence of HMW-alginate . Enzymatic and genetic analyses of HMW-alginate incorporation processes confirmed the presence of a pit-dependent and macromolecule-specific ABC transporter system in cells of Sphingomonas species A1 . This is the first description of a bacterium with a pit on the cell surface and a pit-dependent endocytosic uptake system for macromolecules.

J Ind Microbiol Biotechnol, 1999 Oct, 23(4-5), 303 - 313
Induction of aromatic catabolic activity in Sphingomonas aromaticivorans strain F199; Romine MF et al.; Enzyme induction studies with Sphingomonas aromaticivorans F199 demonstrated that both toluene and naphthalene induced expression of both naphthalene and toluene catabolic enzymes . However, neither aromatic compound induced expression of all the enzymes required for complete mineralization of either naphthalene or toluene . Activity measurements in combination with gene sequence analyses indicate that growth on either aromatic substrate in the absence of the other is, therefore, sub-optimal and is predicted to lead to the build-up of metabolites due to imbalance in toluene or naphthalene catabolic enzyme activities . Growth on toluene may be further inhibited by the co-expression of two toluene catabolic pathways, as predicted from gene sequence analyses . One of these pathways may potentially result in the formation of a dead-end intermediate, possibly benzaldehyde . In contrast, either p-cresol or benzoate can support high levels of growth . Analyses of promoter region sequences on the F199 aromatic catabolic plasmid, pNL1, suggest that additional regulatory events are modulated through the interaction of BphR with Sigma54 type promoters and through the binding of a regulator upstream of p-cresol catabolic genes and xylM . We hypothesize that the unusual gene clustering in strain F199 is optimized for simultaneous degradation of multiple aromatic compound classes, possibly in response to the heterogeneous composition of aromatic structures in the fossil organic matter present in the deep Atlantic Coastal Plain sediments from which this bacterium was isolated.

Postgrad Med J, 2001 Jul, 77(909), 447 - 50
Quadruple therapy for symptomatic spontaneous duodenal ulcer disease; Bateson MC; AIM: To investigate Helicobacter pylori eradication in duodenal ulcer patients with a new regimen, lansoprazole 30 mg daily for one or four weeks plus twice daily tetracycline 500 mg, clarithromycin 250 mg, and metronidazole 400 mg . BACKGROUND: Spontaneous duodenal ulcer is regularly associated with H pylori, and permanent cure follows eradication of this bacterium . Numerous treatments have been proposed and none is ideal, possibly because of primary or acquired antibiotic resistance . Quadruple regimens with proton pump inhibitor therapy and three antibiotics offer promise as the most effective therapy . METHODS: From November 1995 all patients with spontaneous duodenal ulcer were offered quadruple therapy . A month after completion a carbon 14 urea breath test (UBT) was performed . Sensitivity of H pylori to the antibiotics used was tested in 1992-3, 1996, and 1999 . RESULTS: A total of 331 patients were treated; 313 attended for a UBT, of which 299 were negative (95.5%) . Of those patients who had an endoscopy with positive urease test immediately before treatment, 95/101 (94.0%) on lansoprazole for one week and 116/121 (95.8%) on lansoprazole for four weeks had a negative UBT . H pylori antibiotic sensitivity did not change . CONCLUSION: This regimen produced some of the best results yet seen and may be generally recommended as first line therapy.

Carbohydr Res, 2001 Jun 22, 333(1), 87 - 93
Structural analysis of a new glycosphingolipid from the lipopolysaccharide-lacking bacterium Sphingomonas adhaesiva; Kawahara K et al.; A new glycosphingolipid, GSL-4B, was isolated from Sphingomonas adhaesiva and found to share the ceramide moiety with GSL-1 and GSL-3 from Sphingomonas capsulata studied earlier {Kawahara, K.; Moll, H.; Knirel, Y . A.; Seydel, U.; Zahringer, U . Eur . J . Biochem . 2000, 267, 1837-1846} . It is heterogeneous with respect to the long-chain bases erythro-2-amino-1,3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1,3-diol, and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol which in GSL-4B are present in the ratios of 1.1:1.0:1.1, and all bearing amide-linked (S)-2-hydroxymyristic acid . Methylation analysis and MALDI-TOF-MS along with 1H and 13C NMR spectroscopy showed that the carbohydrate part of GSL-4B has the structure of alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-GlcpA-(1-->1)-Cer

Carbohydr Res, 2001 Jun 22, 333(1), 41 - 6
Structure of a colitose-containing O-specific polysaccharide of the marine bacterium Pseudoalteromonas tetraodonis IAM 14160(T); Muldoon J et al.; O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Pseudoalteromonas tetraodonis type strain IAM 14160(T) and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, 1H,(13)C HMQC and HMBC experiments . The polysaccharide was found to consist of hexasaccharide repeating units containing one residue each of D-Gal, D-GlcA, D-GalNAc and D-GlcNAc and two residues of 3,6-dideoxy-L-xylo-hexose (colitose, Col) and having the following structure:In common with the polysaccharides of some other bacteria, the polysaccharide studied contains a tetrasaccharide fragment alpha-Colp-(1-->2)-beta-D-Galp-(1-->3)-{alpha-Colp-(1-->4)}-beta-D-GlcpNAc, which is a colitose ('3-deoxy-L-fucose') analogue of the Lewis(b) blood group antigenic determinant.

Helicobacter, 2001 Jun, 6(2), 146 - 50
Correlation between Helicobacter pylori infection, gastric inflammation and serum homocysteine concentration; Leung WK et al.; BACKGROUND: Epidemiological studies have suggested a link between chronic Helicobacter pylori infection and ischemic heart disease but the underlying mechanism remains elusive . We hypothesized that H . pylori-associated chronic gastritis causes impairment of absorption of vitamin cofactors that are essential in the metabolism of homocysteine and results in hyperhomocysteinemia . MATERIALS AND METHODS: Forty-nine dyspeptic patients were studied . H . pylori infection was defined by rapid urease test and histology . Fasting serum homocysteine level, which was measured by a validated commercial fluorescence polarization immunoassay, was correlated with H . pylori infection statuses and gastric histology . H . pylori-infected patients were followed up for 24 weeks post eradication for changes in serum homocysteine concentration . RESULTS: Univariate analyses showed that serum homocysteine level correlated with increasing age (p <.001), male sex (p =.003) and smoking habit (p =.025) . There was no significant difference in serum homocysteine levels between H . pylori infected and uninfected subjects (median 10.5 vs . 10.2 micromol/l) . After successful eradication of the bacterium, there was no significant reduction in homocysteine level . Moreover, there was no correlation between homocysteine level and gastric histology including H . pylori density, activity and inflammation scores, presence of atrophy or intestinal metaplasia . CONCLUSIONS: The postulated link between H . pylori infection and ischemic heart disease, if it actually exists, is unlikely to be mediated through hyperhomocysteinemia.

Helicobacter, 2001 Jun, 6(2), 100 - 9
Probing 23S ribosomal RNA cleavage sites in coccoid Helicobacter pylori; Monstein HJ et al.; BACKGROUND: Previous studies have revealed that extensive nonrandom fragmentation of ribosomal RNA occurs during conversion of Helicobacter pylori to the coccoid form . The 16S rRNA fragmentation has been characterised in some detail . The aim of the present study was to define corresponding cleavage-sites in the 3'-half of the 23S rRNA molecule . MATERIALS AND METHODS: Northern blot analysis using 23S rRNA specific antisense riboprobes and a 5'-end-labelled oligonucleotide probe was used to analyse the 23S rRNA fragmentation pattern in coccoid H . pylori type strain CCUG 17874T and H . pylori 26695, for which the genome has been sequenced . A double-stranded cDNA-dependent (ds-cDNA) primer-extension analysis technique using 23S rRNA ds-cDNA and a primer targeting the vicinity of the peptidyl-transferase centre was used to determine cleavage sites at the nucleotide level . RESULTS: We report here the mapping of putative cleavage sites within domains IV and V, enclosing the peptidyl transferase centre, in the 3'-half of the 23S rRNA molecule . Three cleavage sites were located in domain IV . Two other cleavage sites were located in the peptidyl transferase centre, and one presumptive multiple-break site between helices 77 and 78 in domain V . The DNA motifs were different from the postulated A + U rich single-strand cleavage sites recognised by RNase E, which has been implicated in rRNA degradation in Escherichia coli . CONCLUSIONS: The present analysis suggests that a hitherto unknown mechanism is responsible for the nonrandom fragmentation of rRNA in coccoid H . pylori, which may have important consequences for the growth, and survival of the bacterium.

Helicobacter, 2001 Jun, 6(2), 93 - 9
Cell lysis is responsible for the appearance of extracellular urease in Helicobacter pylori; Marcus EA et al.; BACKGROUND: Helicobacter pylori is a neutralophilic bacterium that colonizes the acidic human gastric surface using the neutralizing capacity of a constitutively produced urease . Urease is present both in the cytoplasm and bound to the outside surface of the bacteria . The origin of the surface urease continues to be controversial . This study provides additional evidence that the origin of surface urease is cell lysis, not secretion . METHODS: H . Pylori was transformed with a plasmid encoding green fluorescent protein (GFP), a non-native cytoplasmic protein . Cultures supplemented with beta-cyclodextrin or horse serum were collected over various time periods and spun through a ficoll cushion to gently separate whole bacteria from released protein . The pellet and supernatant fractions were analyzed by fluorimetry, SDS-PAGE and Coomassie blue or Western analysis . RESULTS: GFP fluorescence and antigenic reactivity in the supernatant increased at each time point . GFP, the non-native cytoplasmic protein, and UreB, a native cytoplasmic protein, increased over time in the supernatant and both proteins were always present in the pellet fraction . UreI, an inner membrane protein, was only present in the pellet fraction . beta-galactosidase, a protein not found in H . pylori, was used as a negative control . CONCLUSIONS: Since it is unlikely that there is an intrinsic secretion system for GFP, a non-native protein, its increasing presence over time in the supernate fraction along with UreB, and retention of UreI in the pellet fraction implies that cell lysis accounts for the presence of urease on the surface of H . pylori.

Eur J Biochem, 2001 Jun, 268(12), 3577 - 86
2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans: characterization and comparison of both enzymes; Brock M et al.; In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle . The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase . The enzymes from both organisms were assayed with chemically synthesized threo-2-methylisocitrate; the erythro-diastereomer was not active . 2-Methylisocitrate lyase from E . coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation . The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources ( approximately 48 kDa) . 2-Methylisocitrate lyase from A . nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism . Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography . The enzymes show no measurable activity with isocitrate . Starting from 250 mM pyruvate, 150 mM succinate and 10 microM PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield . As revealed by chiral HPLC, the product consisted of a single enantiomer . This isomer is cleaved by 2-methylisocitrate lyases from A . nidulans and E . coli . The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E . coli . PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved.

Eur J Biochem, 2001 Jun, 268(12), 3375 - 82
Methionine oxidation and its effect on the stability of a reconstituted subunit of the light-harvesting complex from Rhodospirillum rubrum; Wang ZY et al.; An additional component in the purified core light-harvesting complex (LH1) from wild-type purple photosynthetic bacterium Rhodospirillum rubrum has been identified as an oxidized species of alpha-polypeptide by MALDI-TOF mass spectrometry . This component appears as a slightly earlier-eluting peak in the RP-HPLC chromatogram compared with the authentic alpha-polypeptide . The oxidation site has been determined to be the N-terminal methionine residue by high-resolution NMR spectroscopy, where the methionine is oxidized to methionine sulfoxide in a diastereoisomeric form . Interconversion between the oxidized and authentic alpha-polypeptides has been confirmed by selective oxidation and reduction . The oxidative modification of methionine is shown to have discernible effects on the ability to form B820 subunit with beta-polypeptide and bacteriochlorophyll a, and on the stability of the reconstituted B820 subunit . Both the ability and the stability for the samples using the oxidized alpha-polypeptide are moderately reduced, indicating that the oxidation-induced conformational change in the N-terminal domain of alpha-polypeptide may affect the pigment-binding environment through a long-range interaction . The MALDI-TOF mass results also reveal that the N-terminus of alpha-polypeptide is formylated and no phosphorylation has occurred in this polypeptide.

J Bacteriol, 2001 Jul, 183(14), 4305 - 16
Autotrophic CO(2) fixation by Chloroflexus aurantiacus: study of glyoxylate formation and assimilation via the 3-hydroxypropionate cycle; Herter S et al.; In the facultative autotrophic organism Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation . An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed . In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO(2) molecules are thereby fixed . Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product . Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells . Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor . Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions . So far, no clue as to the first step in glyoxylate assimilation has been obtained . One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine . However, such a pathway does not occur, as shown by labeling of whole cells with {1,2-(13)C(2)}glycine . Glycine carbon was incorporated only into glycine, serine, and compounds that contained C(1) units derived therefrom and not into other cell compounds.

FEBS Lett, 2001 Jun 15, 499(1-2), 45 - 9
A deeper investigation on carbohydrate cycling in Sinorhizobium meliloti; Gosselin I et al.; Recycling of triose-phosphate and pentose-phosphate was previously reported on glucose in Sinorhizobium meliloti, a polysaccharide-synthesizing bacterium, but the metabolic basis of such processes remained unclear . In this work, we have used (13)C-labelling strategies to demonstrate that carbohydrate cycling in this organism is independent of the gluconate bypass, the alternative pathway for glucose assimilation involving its periplasmic oxidation into gluconate . Furthermore, carbohydrate cycling in S . meliloti is also observed on fructose, making the situation in this bacterium significantly different from that depicted for alginate-synthesizing species.

Biochim Biophys Acta, 2001 Jul 2, 1506(1), 23 - 30
In vitro and in vivo electron transfer to the triheme cytochrome subunit bound to the photosynthetic reaction center complex in the purple bacterium Rhodovulum sulfidophilum; Yoshida M et al.; The cytochrome subunit bound to the photosynthetic reaction center (RC) complex in Rhodovulum sulfidophilum lacks one heme-binding motif (CXXCH) out of four motifs found in other purple bacteria resulting in the absence of the most distal heme from the RC-core complex (S . Masuda et al., J . Biol . Chem . 274 (1999) 10795) . Cytochrome c(2), which acts as the electron donor to the RC was purified, and its gene was cloned and sequenced . The redox midpoint potential of cytochrome c(2) was determined to be E(m)=357 mV . The photo-oxidation and re-reduction of purified cytochrome c(2) were observed in the presence of membrane preparations . Flash-induced photo-oxidation and re-reduction of the RC-bound cytochrome were also observed in intact cells . Despite the unusual nature of the RC-bound cytochrome subunit, the cyclic electron transfer system in Rdv . sulfidophilum was shown to be similar to those in other purple bacteria.

Phys Rev E Stat Nonlin Soft Matter Phys . 2001 Jun;63(6 Pt 1):061904 . Epub 2001 May 23.
Phase transition in a spatial Lotka-Volterra model; Szabo G et al.; Spatial evolution is investigated in a simulated system of nine competing and mutating bacterium strains, which mimics the biochemical war among bacteria capable of producing two different bacteriocins (toxins) at most . Random sequential dynamics on a square lattice is governed by very symmetrical transition rules for neighborhood invasions of sensitive strains by killers, killers by resistants, and resistants by sensitives . The community of the nine possible toxicity/resistance types undergoes a critical phase transition as the uniform transmutation rates between the types decreases below a critical value P(c) above that all the nine types of strains coexist with equal frequencies . Passing the critical mutation rate from above, the system collapses into one of three topologically identical (degenerated) states, each consisting of three strain types . Of the three possible final states each accrues with equal probability and all three maintain themselves in a self-organizing polydomain structure via cyclic invasions . Our Monte Carlo simulations support that this symmetry-breaking transition belongs to the universality class of the three-state Potts model.

Microbios, 2001, 105(412), 141 - 52
Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA; Kamimura K et al.; The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR . The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III . The RFLP patterns revealed that T . ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T . thiooxidans NB1-3, T . caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11 . The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T . ferrooxidans except for strain ATCC 13661 . The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T . ferrooxidans strains . The 16S rDNA sequence of T . ferrooxidans NASF-1 possessed an additional restriction site for Hha I . These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T . ferrooxidans by the method combining RFLP analysis with physiological analysis.

Am Surg, 2001 Jun, 67(6), 589 - 93
Chlamydia pneumoniae in atherosclerotic carotid artery plaques: high prevalence among heavy smokers; Dobrilovic N et al.; This study was designed to determine the prevalence of Chlamydia pneumoniae in carotid artery plaques . Although there have been numerous studies evaluating coronary plaques for this bacterium fewer studies have assessed noncoronary vasculature . In addition we wished to evaluate whether correlation exists between the presence of C . pneumoniae in carotid plaques and established risk factors for atherosclerosis . Sixty intact carotid artery plaques removed during surgery (carotid endarterectomy) were formalin-fixed and paraffin-embedded according to conventional techniques . These samples were evaluated by polymerase chain reaction analysis to detect presence of C . pneumoniae DNA . Results were tabulated and compared against established risk factors for atherosclerosis: diabetes, hypertension, hyperlipidemia, age, and smoking . Forty-two (70.0%) of the 60 plaques that were evaluated tested positive for the presence of C . pneumoniae DNA by polymerase chain reaction analysis . In the sample defined as being from heavy smokers (greater than 15-pack-year history) 33 (94.3%) of 35 plaques tested positive whereas two (5.7%) tested negative . This correlation demonstrated statistical significance (P = 1.36 x 10(-6), two-tailed Fisher exact test) . Presence of C . pneumoniae in carotid plaques demonstrated no statistically significant correlation with diabetes, hypertension, or hyperlipidemia . Age as a risk factor was examined but not statistically evaluated because of the narrow range within our patient sample . Analysis of the data reveals that C . pneumoniae is present in large numbers of atheromatous plaques as is consistent with emerging data . What is interesting though is that 33 (94.3%) of the 35 smokers had plaques that tested positive for the bacterium as opposed to only nine (36.0%) of the 25 nonsmokers . Identification of specific populations exhibiting a high prevalence of C . pneumoniae may serve to focus future studies . Ongoing investigation will seek to determine whether C . pneumoniae plays an active role in the pathogenesis of atherosclerosis.

Arch Microbiol, 2001 May, 175(5), 376 - 83
Isolation and characterization of a veratrol:corrinoid protein methyl transferase from Acetobacterium dehalogenans; Engelmann T et al.; From 3-methoxyphenol-grown cells of Acetobacterium dehalogenans, an inducible enzyme was purified that mediated the transfer of the methyl groups of veratrol (1,2-dimethoxybenzene) to a corrinoid protein enriched from the same cells . In this reaction, veratrol was converted via 2-methoxyphenol to 1,2-dihydroxybenzene . The veratrol:corrinoid protein methyl transferase, designated MTIver, had an apparent molecular mass of about 32 kDa . With respect to the N-terminal amino acid sequence and other characteristics, MTIver is different from the vanillate:corrinoid protein methyl transferase (MTIvan) isolated earlier from the same bacterium . For the methyl transfer from veratrol to tetrahydrofolate, two additional protein fractions were required, one of which contained a corrinoid protein . This protein was not identical with the corrinoid protein of the vanillate O-demethylase system . However, the latter corrinoid protein could also serve as methyl acceptor for the veratrol:corrinoid protein methyl transferase . MTIver catalyzed the demethylation of veratrol, 3,4-dimethoxybenzoate, 2-methoxyphenol, and 3-methoxyphenol . Vanillate (3-methoxy-4-hydroxybenzoate), 2-methoxybenzoate, or 4-methoxybenzoate could not serve as substrates.

Biochemistry, 2001 Jun 26, 40(25), 7542 - 8
Role of the ATP synthase alpha-subunit in conferring sensitivity to tentoxin; Tucker WC et al.; Tentoxin, produced by phytopathogenic fungi, selectively affects the function of the ATP synthase enzymes of certain sensitive plant species . Binding of tentoxin to a high affinity (K(i) approximately 10 nM) site on the chloroplast F(1) (CF(1)) strongly inhibits catalytic function, whereas binding to a second, lower affinity site (K(d) > 10 microM) leads to restoration and even stimulation of catalytic activity . Sensitivity to tentoxin has been shown to be due, in part, to the nature of the amino acid residue at position 83 on the catalytic beta subunit of CF(1) . An aspartate in this position is required, but is not sufficient, for tentoxin inhibition . By comparison with the solved structure of mitochondrial F(1) {Abrahams, J . P., Leslie, A . G . W., Lutter, R., and Walker, J . E . (1994) Nature 370, 621-628}, Asp83 is probably located at an interface between alpha and beta subunits on CF(1) where residues on the alpha subunit could also participate in tentoxin binding . A hybrid core F(1) enzyme assembled with beta and gamma subunits of the tentoxin-sensitive spinach CF(1), and an alpha subunit of the tentoxin-insensitive photosynthetic bacterium Rhodospirillum rubrum F(1) (RrF(1)), was stimulated but not inhibited by tentoxin {Tucker, W . C., Du, Z., Gromet-Elhanan, Z . and Richter, M . L . (2001) Eur . J . Biochem . 268, 2179-2186} . In this study, chimeric alpha subunits were prepared by introducing short segments of the spinach CF(1) alpha subunit from a poorly conserved region which is immediately adjacent to beta-Asp83 in the crystal structure, into equivalent positions in the RrF(1) alpha subunit using oligonucleotide-directed mutagenesis . Hybrid enzymes containing these chimeric alpha subunits had both the high affinity inhibitory tentoxin binding site and the lower affinity stimulatory site . Changing beta-Asp83 to leucine resulted in loss of both inhibition and stimulation by tentoxin in the chimeras . The results indicate that tentoxin inhibition requires additional alpha residues that are not present on the RrF(1) alpha subunit . A structural model of a putative inhibitory tentoxin binding pocket is presented.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 971 - 6
Taylorella asinigenitalis sp . nov., a bacterium isolated from the genital tract of male donkeys (Equus asinus); Jang SS et al.; Three bacterial isolates that were phenotypically indistinguishable from Taylorella equigenitalis were obtained from the urethral fossae of three male donkeys (Equus asinus), one located in the state of California and the other two in the state of Kentucky, USA . Based on results of pulsed-field gel electrophoresis, the isolate from California differed from the two Kentucky isolates, which were the same . Mares bred artificially (California) or naturally (Kentucky) did not show signs of disease, even though infection with the organism was established in those bred naturally . Mares and, uncharacteristically, all three jacks produced antibodies that reacted in the complement fixation test utilized to identify mares recently infected with T . equigenitalis . Sequence analysis of DNA encoding the 16S rRNA revealed that the gene sequences of these isolates were virtually identical to each other (>99.8% similarity), but different (97.6% similarity) from those of several confirmed isolates of T . equigenitalis . The 16S rDNA sequences of the latter were 100% identical . DNA-DNA hybridization studies revealed a mean hybridization level of 89% between the donkey isolate from California and the donkey isolate from Kentucky . On the other hand, the mean DNA-DNA hybridization level from the donkey isolates with DNA from a strain of T . equigenitalis was 23% . The DNA G+C composition was 37.8 mol% for the two donkey isolates, as well as the strain of T . equigenitalis used in the hybridization studies . These data support our opinion that micro-organisms isolated from the male donkeys are different from T . equigenitalis and it is proposed that they be considered a new species within the genus Taylorella and named Taylorella asinigenitalis sp . nov . The type strain is strain UCD-1T (= ATCC 700933T = LMG 19572T).

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 853 - 5
Actinomyces funkei sp . nov., isolated from human clinical specimens; Lawson PA et al.; Three strains of a previously undescribed Actinomyces-like bacterium were isolated from human clinical specimens . Phenotypic studies indicated that the strains were members of the genus Actinomyces and were presumptively identified as Actinomyces turicensis . Comparative 16S rRNA gene sequencing studies showed that although the bacterium is phylogenetically closely related to Actinomyces turicensis, it nevertheless constitutes a new sub-line within the genus Actinomyces . Based on phenotypic and molecular chemical and molecular genetic evidence, it is proposed that the unknown Actinomyces-like bacterium from human clinical specimens be classified as Actinomyces funkei sp . nov . The type strain of Actinomyces funkei is CCUG 42773T (= CIP 106713T).

Plasmid, 2001 May, 45(3), 184 - 99
Genetic organization of plasmid pXF51 from the plant pathogen Xylella fastidiosa; Marques MV et al.; The sequence of plasmid pXF51 from the plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, has been analyzed . This plasmid codes for 65 open reading frames (ORFs), organized into four main regions, containing genes related to replication, mobilization, and conjugative transfer . Twenty-five ORFs have no counterparts in the public sequence databases, and 7 are similar to conserved hypothetical proteins from other bacteria . A pXF51 incompatibility group has not been determined, as we could not find a typical replication origin . One cluster of conjugation-related genes (trb) seems to be incomplete in pXF51, and a copy of this sequence is found in the chromosome, suggesting it was generated by a duplication event . A second cluster (tra) contains all genes necessary for conjugation transfer to occur, showing a conserved organization with other conjugative plasmids . An identifiable origin of transfer similar to oriT from IncP plasmids is found adjacent to genes encoding two mobilization proteins . None of the ORFs with putative assigned function could be predicted as having a role in pathogenesis, except for a virulence-associated protein D homolog . These results indicate that even though pXF51 appears not to have a direct role in Xylella pathogenesis, it is a conjugative plasmid that could be important for lateral gene transfer in this bacterium . This property may be of great importance for future development of transformation techniques in X . fastidiosa.

Biotechnol Bioeng, 2001, 76(1), 11 - 6
Swimming characteristics of magnetic bacterium, Magnetospirillum sp . AMB-1, and implications as toxicity measurement; Seong S et al.; To develop a novel toxicity measurement system using the persistent swimming property of magnetic bacteria along an externally applied magnetic field, certain characteristics of Magnetospirillum sp . AMB-1 cells were examined, including their growth pattern, motility, magnetosensitivity, swimming speed, and cell length distribution . In addition, the effect of toxic compounds on the swimming speed was assessed relative to application as a toxicity sensor . With an inoculum of 1.0 x 10(8) cells/mL, the cells reached the stationary phase with a concentration of about 5 x 10(8) cells/mL after 20 h, under both aerobic and anaerobic conditions . The distribution of the cell length did not vary significantly during the growth period, and both aerobically and anaerobically growing cells showed a similar cell length distribution . Although the cells showed similar growth patterns under both conditions, the anaerobically grown cells exhibited higher motility and magnetosensitivity . Actively growing cells under anaerobic conditions had an average swimming speed of 49 microm/s with a standard deviation of 20 microm/s . When the anaerobically growing cells were exposed to various concentrations of toxic compounds, such as 1-propanol and acetone, the swimming speed decreased with an increased concentration of the toxic compound . Accordingly, the relationship between swimming speed and toxicity can be used as an effective quantitative toxicity measurement; furthermore, the relative sensitivity of the proposed system was comparable to Microtox, which is commercially available .

Curr Microbiol, 2001 May, 42(5), 323 - 9
Phosphorylation of LHI beta during membrane synthesis in the photosynthetic bacterium Rhodovulum sulfidophilum; Iustman LJ et al.; Cells of Rhv . sulfidophilum were grown under different conditions in the presence of 32P-phosphate and the corresponding H and L membrane fractions obtained and fractionated by SDS-PAGE . Both membranes showed almost identical polypeptide composition . The bacteriochlorophyll (Bchl) specific content in H was always lower that in L . As described before, oxygen did not regulate gene expression . Under high light, an almost two- to threefold decrease of the cellular specific Bchl content was observed . Pulse and chase experiments showed that transitions from aerobiosis to light-anaerobiosis did not quantitatively affect the Bchl content of the membranes, although a turnover of the 32P-phosphate and 35S-methionine was observed . LHI beta was the only polypeptidic subunit of the Bchl-binding polypeptides that was phosphorylated in vivo, and phosphotyrosine was the only phosphorylated amino acid detectable . The phosphorylated LHI beta was determined to be insoluble in the organic solvent mixture of (vol/vol) 1:1 chloroform-methanol containing ammonium acetate (0.1 m final concentration) . Treatment with a chaotropic agent such as Na2CO3 solubilized the phosphorylated LHI beta, indicating that part of this posttranslationally modified polypeptide was not inserted in a transmembrane position . These results were used to speculate about the regulatory properties of this posttranslational modification of LHI beta on membrane differentiation.

Parasitol Res, 2001 May, 87(5), 417 - 20
Molecular identification of Wolbachia from the filarial nematode Mansonella ozzardi; Casiraghi M et al.; Mansonella ozzardi, a filarial parasite of humans in Latin America, has been shown to harbour intracellular bacteria not yet identified . Here we show that these bacteria, like those of other filarial nematodes, belong to the genus Wolbachia (alpha 2 Proteobacteria; Rickettsiales) . Their unambiguous placement in the Wolbachia group was shown by 16S rDNA sequence analysis . However, the exact position of the Wolbachia from M . ozzardi relative to the other wolbachiae is not clear . Indeed, 16S rDNA sequence analysis places this bacterium at a deep branch in Wolbachia evolution . It is interesting that analysis of the 5S rDNA gene spacer of the nematode host also suggests that the genus Mansonella, together with the genus Loa, could represent a deep-branching lineage in filarial evolution.

Infect Immun, 2001 Jul, 69(7), 4639 - 46
Granulomatous skin lesions in moray eels caused by a novel Mycobacterium species related to Mycobacterium triplex; Herbst LH et al.; An outbreak of granulomatous dermatitis was investigated in a captive population of moray eels . The affected eels had florid skin nodules concentrated around the head and trunk . Histopathological examination revealed extensive granulomatous inflammation within the dermis and subcutaneous fascial plane between the fat and axial musculature . Acid-fast rods were detected within the smallest lesions, which were presumably the ones that had developed earliest . Eventually, after several months of incubation at room temperature, a very slowly growing acid-fast organism was isolated . Sequencing of the 16S rRNA gene identified it as a Mycobacterium species closely related (0.59% divergence) to M . triplex, an SAV mycobacterium . Intradermal inoculation of healthy green moray eels with this organism reliably reproduced the lesion . Experimentally induced granulomatous dermatitis appeared within 2 weeks of inoculation and slowly but progressively expanded during the 2 months of the experiment . Live organisms were recovered from these lesions at all time points, fulfilling Koch's postulates for this bacterium . In a retrospective study of tissues collected between 1993 and 1999 from five spontaneous disease cases, acid-fast rods were consistently found within lesions, and a nested PCR for the rRNA gene also demonstrated the presence of mycobacteria within affected tissues.

Mol Microbiol, 2001 May, 40(4), 1027 - 36
Identifying regulators of transcription in an obligate intracellular pathogen: a metal-dependent repressor in Chlamydia trachomatis; Wyllie S et al.; A prominent feature exhibited by Chlamydia trachomatis growing in an iron-limiting environment is a differential pattern of protein expression . In many bacteria, iron-responsive proteins are regulated at the level of transcription by a family of repressors resembling the Escherichia coli ferric uptake regulator (Fur) protein . Although the chlamydial genome sequencing project did not unveil an obvious Fur homologue, a detailed examination indicated five unassigned open reading frames (ORFs) that would encode products with limited sequence homology to Fur . In this report, each chlamydial ORF was engineered in E . coli, and recombinant proteins were examined for functional characteristics resembling Fur . A Fur-specific polyclonal antiserum revealed that the protein encoded by ORF CT296 shares antigenic cross-recognition . Moreover, this protein forms dimers in solution in a fashion analogous to E . coli Fur . Further studies confirmed that the product of ORF CT296 is able to (i) complement Fur activity in a mutant strain of E . coli; and (ii) specifically bind to a 19 bp consensus sequence found in promoters of iron-regulated genes in E . coli . We propose a designation of dcrA (divalent cation-dependent regulator A) for ORF CT296, which encodes a protein distantly related to E . coli Fur . DcrA represents the first repressor described for this obligate intracellular bacterium.

J Periodontol, 2001 May, 72(5), 641 - 50
Protease-active extracellular protein preparations from Porphyromonas gingivalis W83 induce N-cadherin proteolysis, loss of cell adhesion, and apoptosis in human epithelial cells; Chen Z et al.; BACKGROUND: The protease-induced cytotoxicity of P . gingivalis may partly result from alteration of the extracellular matrix and/or surface receptors that mediate interaction between the host cells and their matrix . While P . gingivalis-induced degradation of E-cadherin has been documented, there is no information on the effects of P . gingivalis proteases on other members of this family of cell adhesion proteins . METHODS: Human epithelial KB cells were exposed to protease-active extracellular protein preparations from isogenic mutants of P . gingivalis . Quantification of apoptosis was performed by visualization of nuclei stained with 4,6'-diamidino-2-phenylindole . Alteration of cell adhesion proteins was examined by immunoblotting of cell lysates using monoclonal antibodies to those proteins . RESULTS: Treated cells exhibited loss of cell adhesion properties with apoptotic cell death subsequently observed . These effects correlated with the different levels of cysteine-dependent proteolytic activities of the isogenic mutants tested . Cleavage of N-cadherin was observed in immunoblots of lysates from detached cells . There was a direct correlation between the kinetics of N-cadherin cleavage and loss of cell adhesion properties . Loss of cell adhesion, as well as N-cadherin cleavage, could be inhibited by preincubation of P . gingivalis protease active extracellular protein preparations with the cysteine protease inhibitor TLCK . In control experiments, the cleavage of N-cadherin was detected after treatment of KB cells with trypsin but not after cell dissociation by a non-enzymatic method . CONCLUSIONS: These results suggest that extracellular proteases from P . gingivalis can induce degradation of N-cadherin, which could have implications for the pathogenicity of this bacterium.

Curr Microbiol, 2001 Aug, 43(2), 83 - 8
Monitoring of bio-oxidation process of ferrous ion by using piezoelectric impedance analysis; Zhang J et al.; A new method of monitoring the bio-oxidation process of ferrous ion in the presence of Thiobacillus ferroxidans was proposed by piezoelectric impedance analysis . The time courses of the responses of impedance parameters for a quartz crystal in a culture system were simultaneously obtained and discussed . It was found that the frequency shift response originates mainly from the adsorption of bacterial metabolites on the surface of gold electrode . Experiments also examined the effect of culture temperature on the bio-oxidation process . Combined with the growth situation of the bacterium, an impedance response model reflecting the process was established . By fitting Delta f vs . time curves toward the proposed model, we obtained and discussed the bacterial growth parameters . The results showed that the proposed method could provide real time and multidimensional information to monitoring of the bio-oxidation process.

Altern Lab Anim, 2001 May-Jun, 29(3), 269 - 75
Cytotoxicity testing of wound-dressing materials; Sahlin H et al.; A method was developed for testing the cytotoxicity of various bandage-like wound dressings and gel wound dressings . In this method, the ability of human polymorphonuclear neutrophils (PMNs) to initiate a respiratory burst after exposure to the various wound dressings is used as a marker of cytotoxicity . Luminol-amplified chemiluminescence stimulated with opsonised zymosan or phorbol 12-myristate 13-acetate (PMA) is used to measure the degree of activation of the respiratory burst, i.e . the NADPH oxidase activity, after exposure to wound dressings . Opsonised zymosan (material from yeast cell walls) is a phagocytic stimulus that activates the NADPH oxidase by binding to FC-receptors and complement receptors, and functions as an artificial bacterium, whereas PMA activates the NADPH oxidase by direct activation of protein kinase C . NADPH oxidase activity was inhibited by several wound dressings . The down-regulation of the respiratory burst is detrimental to the bactericial effect of PMNs, and can be used as a marker for the cytotoxicity of wound dressing materials.

Mikrobiologiia, 2001 Mar-Apr, 70(2), 182 - 8
{Lithoautotrophic growth of the freshwater colorless sulfur bacterium Beggiatoa "leptomitiformis" D-402}; Patritskaia VIu et al.; The freshwater colorless sulfur bacterium Beggiatoa "leptomitiformis" D-402 was shown to be capable of lithoautotrophic growth in a batch culture under microaerobic conditions at O2 concentrations in the medium of no higher than 0.5 mg/l . The cell yield was maximum at a dissolved oxygen concentration of 0.15 mg/l . A high activity level of key enzymes of the Calvin cycle and of enzymes involved in dissimilatory oxidation of thiosulfate was recorded in the cells . The high rate of CO2 assimilation (112-139 nmol/(min mg protein)) and the cell yield (12 mg dry cells/mmol thiosulfate oxidized), 91-92% of which was accounted for by CO2 carbon, were close to those typical of autotrophic bacteria . Thiosulfate was oxidized almost completely to sulfate, and the fraction of elemental sulfur in the final products did not exceed 0.2-1.7% of the thiosulfate sulfur . The cell membrane fraction contained cytochromes (b + o) and two cytochromes c with M(r) of 23 and 26 kDa; the soluble fraction contained cytochrome c with M(r) of 12 kDa.

Int Arch Allergy Immunol, 2001 May, 125(1), 66 - 72
Helicobacter pylori infection affects eosinophilic cationic protein in the gastric juice of patients with idiopathic chronic urticaria; Ojetti V et al.; BACKGROUND: Helicobacter pylori, the main cause of gastritis and peptic ulcer, has been associated with idiopathic chronic urticaria (ICU), an immunological skin disorder of unknown origin . Eosinophilic cationic protein (ECP) is a cytotoxic molecule secreted by the activated eosinophils involved in the pathogenesis of ICU . We assessed serum/gastric juice ECP levels and gastric mucosal eosinophil infiltration in ICU patients infected or not with H . pylori and evaluated the modification after bacterium eradication . METHODS: 33 patients with ICU and 25 dyspeptic controls underwent upper gastrointestinal endoscopy for histological evaluation and assessment of H . pylori infection . One-week triple therapy was given to H . pylori-positive patients . Serum and gastric juice ECP levels, eosinophil infiltration from gastric mucosal sections and urticaria symptoms were evaluated in all patients at enrollment and 8 weeks after eradication . RESULTS: 19 of 33 (57%) ICU patients and 16 of 25 (64%) controls were found to be infected with H . pylori . Serum ECP was significantly higher in ICU patients compared to controls, regardless of infectious status . Gastric juice ECP and gastric eosinophil infiltration were significantly higher in infected ICU patients when compared both to uninfected ICU patients and controls . H . pylori eradication determined a significant decrease in gastric juice ECP and gastric eosinophil infiltration only in ICU patients . Moreover, a total or partial remission of urticaria symptoms was observed only in ICU patients in whom the bacterium was eradicated . CONCLUSIONS: Although H . pylori infection affects gastric juice ECP and eosinophil infiltration of ICU patients, the role of the bacterium in the pathogenesis of this skin disorder still remains uncertain .

Curr Opin Microbiol, 2001 Jun, 4(3), 237 - 45
Genomics and proteomics converge on Helicobacter pylori; Bjorkholm BM et al.; During the past year, a series of studies have provided new perspectives about genetic diversity in Helicobacter pylori . The results illustrate how the current revolution in genomics and proteomics is being used to understand how this organism co-evolves with its host . The approaches should have broad applications to other host-bacterium relationships.

FEMS Microbiol Ecol, 2001 Jun, 36(1), 43 - 50
An aphid-borne bacterium allied to the secondary symbionts of whitefly; Darby AC et al.; Bacterial 16S rDNA amplified by PCR from the pea aphid Acyrthosiphon pisum included a sequence with >98% similarity to secondary symbionts in the whitefly Bemisia tabaci . The 'pea aphid Bemisia-like bacterium' (PABS) and B . tabaci secondary symbionts are estimated to have diverged 17-34 million years ago, a time considerably more recent than the common ancestor of aphids and whitefly and suggestive of horizontal transmission of this bacterial lineage . PABS was scored in both the gut and ovaries of aphids by PCR and identified as a small rod by in situ hybridisation . PABS was not universal in pea aphids: 2/3 laboratory strains and 13/35 of field aphids were PABS-positive . It is suggested that the incidence of PABS in pea aphids is determined by the balance between loss (processes may include occasional failure of vertical transmission and selection against PABS-positive aphids) and horizontal transfer between insects.

Appl Environ Microbiol, 2001 Jun, 67(6), 2657 - 64
Secretion of active recombinant human tissue plasminogen activator derivatives in Escherichia coli; Manosroi J et al.; The DNA fragment coding for kringle 2 plus serine protease domains (K2S) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS . In the recombinant vector, pComb3H-K2S, the K2S gene was fused to gpIII of PhiM13 and linked to the OmpA signal sequence . The resulting gene, rK2S-gpIII, was inducibly expressed in Escherichia coli XL-1 Blue . The protein was presented on the phage particle . To stop the expression of gpIII, a stop codon between K2S and the gpIII gene was inserted by site-directed mutagenesis . This mutated vector, MpComb3H-K2S, was transformed in XL-1 Blue . After induction with IPTG (isopropyl-beta-D-thiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68% . The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation . The periplasmic form was isolated from whole cells by chloroform extraction . The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B) . Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/microg . No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form . The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes . This finding is an important technological advance in the development of large-scale, bacterium-based tPA production systems.

Appl Environ Microbiol, 2001 Jun, 67(6), 2538 - 44
Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation; Frigaard NU et al.; Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established . As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers . Growth of wild-type C . tepidum at 40 degrees C on agar plates could be completely inhibited by 100 microg of gentamicin ml(-1), 2 microg of erythromycin ml(-1), 30 microg of chloramphenicol ml(-1), or 1 microg of tetracycline ml(-1) or a combination of 300 microg of streptomycin ml(-1) and 150 microg of spectinomycin ml(-1) . Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h . Transformation frequencies on the order of 10(-7) were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10(-3) were observed with a streptomycin-spectinomycin marker . The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency . Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence . Linearized plasmids transformed at least an order of magnitude better than circular plasmids . This work forms a foundation for the systematic targeted inactivation of genes in C . tepidum, whose 2.15-Mb genome has recently been completely sequenced.

Appl Environ Microbiol, 2001 Jun, 67(6), 2476 - 83
Transposon mutagenesis in purple sulfur photosynthetic bacteria: identification of hypF, encoding a protein capable of processing {NiFe} hydrogenases in alpha, beta, and gamma subdivisions of the proteobacteria; Fodor B et al.; A random transposon-based mutagenesis system was optimized for the purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS . Screening for hydrogenase-deficient phenotypes resulted in the isolation of six independent mutants in a mini-Tn5 library . One of the mutations was in a gene showing high amino acid sequence similarity to HypF proteins in other organisms . Inactivation of hydrogen uptake activity in the hypF-deficient mutant resulted in a dramatic increase in the hydrogen evolution capacity of T . roseopersicina under nitrogen-fixing conditions . This mutant is therefore a promising candidate for use in practical biohydrogen-producing systems . The reconstructed hypF gene was able to complement the hypF-deficient mutant of T . roseopersicina BBS . Heterologous complementation experiments, using hypF mutant strains of T . roseopersicina, Escherichia coli, and Ralstonia eutropha and various hypF genes, were performed . They were successful in all of the cases tested, although for E . coli, the regulatory region of the foreign gene had to be replaced in order to achieve partial complementation . RT-PCR data suggested that HypF has no effect on the transcriptional regulation of the structural genes of hydrogenases in this organism.

J Nat Prod, 2001 May, 64(5), 664 - 7
New lactone-containing metabolites from a marine-derived bacterium of the genus Streptomyces; Cho KW et al.; Six novel metabolites containing a lactone moiety as a common structural feature, along with the previously described (-)-blastmycinolactol, have been isolated from the cultivation broth of a bacterium of the genus Streptomyces isolated from marine sediment . On the basis of the results of combined spectroscopic analysis, the structures of the new compounds have been determined as butenolides and 3-hydroxy-gamma-butyrolactones.

J Nat Prod, 2001 May, 64(5), 620 - 3
New methoxylated fatty acids from the Caribbean sponge Callyspongia fallax; Carballeira NM et al.; The saturated 2-methoxylated fatty acids 2-methoxytetradecanoic acid (1), 2-methoxypentadecanoic acid (2), and 2-methoxyoctadecanoic acid (3) as well as the Delta6 monoenoic methoxylated fatty acids (6Z)-2-methoxy-6-tetradecenoic acid (4), (6Z)-2-methoxy-6-pentadecenoic acid (5), and (6Z)-2-methoxy-13-methyl-6-tetradecenoic acid (7) were identified for the first time in nature in the phospholipids from the Caribbean sponge Callyspongia fallax . These findings expand the occurrence of 2-methoxylated fatty acids to C14-C15 chain lengths and establish new fatty acid biosynthetic possibilities for marine organisms . The novel methoxylated fatty acids could have originated from the phospholipids of a bacterium in symbiosis with the sponge.

South Med J, 2001 May, 94(5), 525 - 8
Cardiac tamponade as a manifestation of tuberculosis; Gladych E et al.; Tuberculosis has been increasing in incidence in recent years . Pericardial involvement and pericardial effusions are well-documented and may result in pericardial tamponade . Despite this, large pericardial effusions are uncommon, and manifestation as cardiac tamponade is rare . We report two cases of tuberculous pericarditis in which the initial feature was tamponade . Since the diagnosis of tuberculosis may be delayed due to the slow-growing nature of the bacterium, physicians need to be aware of this possibility and consider the use of modern diagnostic techniques that may permit an earlier diagnosis.

What Is Biotechnology?, What Is Listeria Monocytogenes?, What Is Nitrification?, What Is Water Purification?, What Is Cell Biology?, e, Microbes, o, Microbiology, e, Bacteriology, e, Microorganisms, c, Microorganism, s, Bacillus, i, Antimicrobial, c, Acinetobacter, c, MIC, e, Prokaryotes, o, Escherichia coli, a, Corynebacter, c, Listeriosis, n, S. cerevisiae, e, Bactericidal, i, Antimicrobial, a, Bactericidal, i, Escherichia coli, s, Escherichia coli, o, Yeasts, i, Aeromonades, r, Escherichia coli, i, Antimicrobials, o, Clostridia, c, Fermentations, i, Antimicrobial

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005