Comparative genome analysis and genome-guided physiological analysis of Roseobacter litoralis

<p>Abstract</p> <p>Background</p> <p><it>Roseobacter litoralis </it>OCh149, the type species of the genus, and <it>Roseobacter denitrificans </it>OCh114 were the first described organisms of the <it>Roseobacter </it>clade, an ecologically important group of marine bacteria. Both species were isolated from seaweed and are able to perform aerobic anoxygenic photosynthesis.</p> <p>Results</p> <p>The genome of <it>R. litoralis </it>OCh149 contains one circular chromosome of 4,505,211 bp and three plasmids of 93,578 bp (pRLO149_94), 83,129 bp (pRLO149_83) and 63,532 bp (pRLO149_63). Of the 4537 genes predicted for <it>R. litoralis</it>, 1122 (24.7%) are not present in the genome of <it>R. denitrificans</it>. Many of the unique genes of <it>R. litoralis </it>are located in genomic islands and on plasmids. On pRLO149_83 several potential heavy metal resistance genes are encoded which are not present in the genome of <it>R. denitrificans</it>. The comparison of the heavy metal tolerance of the two organisms showed an increased zinc tolerance of <it>R. litoralis</it>. In contrast to <it>R. denitrificans</it>, the photosynthesis genes of <it>R. litoralis </it>are plasmid encoded. The activity of the photosynthetic apparatus was confirmed by respiration rate measurements, indicating a growth-phase dependent response to light. Comparative genomics with other members of the <it>Roseobacter </it>clade revealed several genomic regions that were only conserved in the two <it>Roseobacter </it>species. One of those regions encodes a variety of genes that might play a role in host association of the organisms. The catabolism of different carbon and nitrogen sources was predicted from the genome and combined with experimental data. In several cases, e.g. the degradation of some algal osmolytes and sugars, the genome-derived predictions of the metabolic pathways in <it>R. litoralis </it>differed from the phenotype.</p> <p>Conclusions</p> <p>The genomic differences between the two <it>Roseobacter </it>species are mainly due to lateral gene transfer and genomic rearrangements. Plasmid pRLO149_83 contains predominantly recently acquired genetic material whereas pRLO149_94 was probably translocated from the chromosome. Plasmid pRLO149_63 and one plasmid of <it>R. denitrifcans </it>(pTB2) seem to have a common ancestor and are important for cell envelope biosynthesis. Several new mechanisms of substrate degradation were indicated from the combination of experimental and genomic data. The photosynthetic activity of <it>R. litoralis </it>is probably regulated by nutrient availability.</p

Wild-type and mutant D-xylose isomerase from Actinoplanes missouriensis: metal-ion dissociation constants, kinetic parameters of deuterated and non-deuterated substrates and solvent-isotope effects.

The metal-ion dissociation constants (Mg2+, Mn2+) of wild-type and mutant D-xylose isomerases from Actinoplanes missouriensis have been determined by titrating the metal-ion-free enzymes with Mg2+ and Mn2+ respectively. Substitution of amino acids co-ordinated to metal-ion 1 (E181D, D245N) dramatically affects the dissociation constants, pH-activity profiles and apparent substrate binding. Mutagenesis of groups ligated to metal-ion 2 is less drastic except for that of Asp-255: a decrease in metal-ion affinity, a change in metal-ion preference and an improved apparent substrate binding (at pH values above the optimum), especially in the presence of Mn2+, are observed for the D255N enzyme. Similar effects, except for a slightly increased metal-ion affinity, are obtained by mutagenesis of the adjacent Glu-186 to Gln and the unconserved Ala-25 to Lys. Moreover, the striking acidic-pH shifts observed for the D255N and E186Q enzymes support the crucial role of the water molecule, Wa-690, Asp-255 and the adjacent Glu-186 in proton transfer from 2-OH to O-1 of the open and extended aldose substrate. Mutations of other important groups scarcely affect the metal-ion dissociation constants and pH-activity profiles, although pronounced effects on the kinetic parameters may be observed

Binding characteristics of Mn2+, Co2+ and Mg2+ ions with several D-xylose isomerases.

D-Xylose isomerases are metal-ion (Mn2+, Co2+, Mg2+)-requiring tetrameric enzymes. Both the stoichiometry and the binding constants have been determined by titrating the metal-ion-free enzymes from five organisms (Actinomycetaceae and more divergent bacteria) with the respective metal ions using the enzyme activity as indicator of active complex-formation. The following characteristics have been observed for each specific isomerase: (i) two essential metal ion sites (one structural and one catalytic) exist per subunit; (ii) the metal ion binding at one site does not affect the binding at the other site; (iii) of the four possible configurations E, aE, Eb and aEb, only the double-occupied enzyme is active; (iv) the metal ion activation is a time-dependent process; (v) the dissociation constants for both the structural and catalytic sites may be identical or may differ by one or higher orders of magnitude; (vi) metal ion binding is stronger in the order Mn2+ greater than Co2+ much greater than Mg2+; (vii) pronounced increases in Km values concomitant with decreasing equivalents of metal ion added are only observed in the presence of Mg2+ ions

Risk factors for delay in the diagnosis and treatment of tuberculosis at a referral hospital in Rwanda

SETTING: Kigali University Hospital, the main referral centre for TB in Rwanda. OBJECTIVE: To evaluate delays in the diagnosis and treatment of tuberculosis (TB) and associated risk factors. DESIGN: Prospective data collection of patients treated for pulmonary TB (PTB) or extra-pulmonary TB (EPTB) between June and September 2006. RESULTS: Of 104 patients with a mean age of 35 years (range 17-84) recruited into the study, 62% were HIV-positive. EPTB was diagnosed in 60 cases. The median total, health care and patient delays were respectively 57, 28 and 25 days. The health system delay before referral was significantly longer than the delay at our institution (18 vs. 6 days, P<0.0001). Risk factors for a longer health system delay at our institution were smear-negative PTB or EPTB (OR 5.12) and a trial of antibiotics (OR 2.96). The latter was also found to significantly prolong total delay (OR 2.85), as did rural residence (OR 4.86). No significant association was found between patient delay and age, sex, profession or health insurance status. CONCLUSION: Smear-negative PTB and EPTB were associated with longer health system delays. A trial of antibiotics significantly increased the health system delay. Its use, recommended by the World Health Organization in case of smear-negative TB and EPTB in developing countries, needs validation at the tertiary health care level