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 cultur