Novel aspects of benzoate and crotonate metabolism by the strictly anaerobic bacterium Syntrophus aciditrophicus strain SB.

Although enzyme activities and metabolites detected in S. aciditrophicus indicated that benzoate was degraded by the pathway found in Rhodopseudomonas palustris, proteomic data detected the gene products homologous to that found in Thauera aromatica. Such observation supports the hypothesis of two routes for benzoate degradation exist in S. aciditrophicus, one involved in benzoate degradation to acetate and CO2 and the other involved in cyclohexane carboxylate formation from benzoate.S. aciditrophicus was shown to ferment benzoate to acetate and cyclohexane carboxylate via a dismutation process where reducing equivalents produced during benzoate oxidation to acetate and CO2 are used to reduce benzoate to cyclohexane carboxylate. The ability to ferment benzoate suggests that benzoate could serve as an electron acceptor if a suitable electron donor was present. To test whether benzoate can be respired, S. aciditrophicus was grown with crotonate and benzoate. Benzoate was stoichiometrically reduced to cyclohexane carboxylate while crotonate was oxidized to acetate. Cultures grown with [ring-13C]-benzoate and unlabeled crotonate formed ring-labeled 13C-cyclohexane carboxylate. No [13C]-labeled acetate was detected. The molar growth yield was 22.7 +/- 2.1 g (dry wt) cell per mol of crotonate compared to 14.0 +/- 0.1g per mol of crotonate when crotonate was used as a sole substrate. Furthermore, benzoate fermentation occurred only if traces amounts of crotonate were present.The metabolism of crotonate in the strictly anaerobic bacteria Syntrophus aciditrophicus was studied. S. aciditrophicus produced 1.4 +/- 0.24 moles of acetate and 0.16 +/- 0.02 moles of cyclohexane carboxylate per mole of crotonate degraded. [U- 13C] Crotonate was metabolized to [1, 2-13C] acetate and [1, 2, 3, 4, 5, 7-13C] cyclohexane carboxylate. Cultures grown with unlabeled crotonate and [13C] sodium bicarbonate formed [6-13C] cyclohexane carboxylate. Cyclohex-1-ene carboxylate, benzoate, pimelate, glutarate, 3-hydroxybutyrate, and acetoacetate were detected as intermediates. These are the same intermediates as that detected during syntrophic or fermentative benzoate metabolism by S. aciditrophicus . When S. aciditrophicus was grown with [1, 2- 13C] acetate and unlabeled crotonate, the m/z-15 of TMS-derivatized 3-hydroxybutyrate, acetoacetate, and glutarate each increased by +0, +2, and +4 mass units, and the m/z-15 of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, cyclohexane carboxylate, and 2-hydroxycyclohexane carboxylate each increased by +0, +2, +4 and +6 mass units. The data are consistent with a pathway for cyclohexane carboxylate formation involving the condensation of two-carbon units derived from crotonate degradation with CO2 addition, rather than the use of the intact four-carbon skeleton of crotonate.Fluorobenzoates and hydroxybenzoates were tested as substrates for S. aciditrophicus in order to detect potential intermediates of interest. The utilization of 3-fluorobenzoate allowed the detection of a metabolite, which had a mass ion increase of 2 units greater than the parent compound, or 3 or 4 units greater than the parent compound when deuterated water was used. These results were consistent with the formation of a fluorinated diene intermediate. The transient accumulation of benzoate when 2-hydroxybenzoate was the substrate showed that hydroxylation of the ring was not required for ring reduction. The metabolites detected with fluoro- and hydroxy-benzoates are consistent with the hypothesis that benzoyl-CoA reduction involves a two-electron reduction forming a diene intermediate, rather than a four- or six-electron reduction.Phototrophic and denitrifying bacteria couple the hydrolysis of two ATP molecules to reduce benzoyl-CoA to cyclohex-1,5-diene carboxyl-CoA. The use of such an energy intensive reaction by fermentative bacteria such as S. aciditrophicus has been questioned since it is not clear how net ATP synthesis would occur. Rather, a four- or six-electron reduction which thermodynamic calculations indicate is exergonic under standard conditions, or hydroxylation of the ring prior to its reduction have been proposed.Proteomic analysis of S. aciditrophicus grown with crotonate or crotonate and benzoate allowed the identification of gene products involved in benzoate metabolism. Two benzoyl-CoA ligases and a possible novel benzoyl-CoA reductase, a tungsten/molybdenum-containing aldehyde ferredoxin oxidoreductase associated with heterodisulfide reductase components similar to the benzoate-induced proteins found in G. metallireducens were identified. Cyclohex-1,5-diene carboxyl-CoA hydratase and the enzymes needed to form 3-hydroxypimelyl-CoA from the diene were also detected. The detection of subunits of ATP synthase, cytoplasmic and periplasmic formate dehydrogenases, a sodium-translocating glutaconyl-CoA, and sodium-driven membrane-bound NADH:ferredoxin oxidoreductase indicates that S. aciditrophicus has the potential to create and use both sodium and proton gradients. ATP synthesis from acetyl-CoA appears to occur by an archaeal-like acetyl-CoA synthetase (ADP-forming) rather than the typical bacterial phosphotransacetylaseacetate kinase system

L’accès différencié des ménages marocains aux soins : quels facteurs dominent ?

La compréhension du recours aux soins passe par l’analyse parallèle des déterminants de l’offre et de la demande de soins. Cet article a pour objectif d’analyser les déterminants de la demande de soins au Maroc. Sur un échantillon composé de 7.062 ménages représentant 36.000 individus, les résultats montrent que le niveau du revenu du ménage, la durée de la maladie et l’âge du patient influencent le recours aux soins modernes. Par ailleurs, l’affiliation à un régime de couverture médicale influence la décision des ménages, permettant notamment aux plus pauvres d’entre eux de recourir à des soins privés auxquels ils n’auraient pas recouru autrement

Molecular diagnosis of cutaneous leishmaniasis and identification of the causative Leishmania species in Morocco by using three PCR-based assays

Background: The diagnosis of cutaneous leishmaniasis (CL) might be difficult, in particular in endemic areas where different species of Leishmania can cause lesions of very similar appearance and where other skin diseases with similar clinical symptoms occur. Even today, the parasitological diagnosis of CL remains the gold standard and it is based on the direct identification of amastigotes in microscopy smears and/or culture of promastigotes from infected tissues. Although these techniques are highly specific, they are not sensitive enough. The objective of this study is to contribute to improving the diagnosis of CL and the identification of Leishmania species in Morocco by comparing three PCR-based assays applied directly on dermal samples. Methods: A total of 58 patients presenting with cutaneous lesions suggestive of CL were sampled for parasitological diagnosis by direct examination (DE), culture in NNN medium, two kinetoplast DNA (kDNA) PCRs (Lmj4/Uni21 and 13A/13B primers) and one rRNA gene internal transcribed spacer 1 (ITS1) PCR (LITSR/L5.8S primers). The techniques were statistically analyzed and compared. Results: According to our consensus positive, 44 out of 58 samples were true positives. The 13A/13B-PCR and ITS1-PCR showed the highest sensitivities (100%). Parasite microscopy and culture detected 43% and 29% of the true positives, respectively, while culture and microscopy together improved sensitivity to 52%. PCRs 13A/13B and ITS1 were associated to four and one false positives, respectively, while the other assays were 100% specific. Furthermore, the ITS1-PCR-RFLP assay clearly identified the Leishmania species for all the true positives (44/44), whereas Lmj4/Uni21-PCR identified 35/44 samples. The comparison between the Leishmania molecular characterizations and the expected species according to the national data from the Ministry of Health indicate 7 discrepant results. Conclusions: The PCR-based assays tested on our samples increased the speed and sensitivity of the diagnosis of CL compared to the conventional techniques. Furthermore, we showed that we can not base the species identification on the national data from the Ministry of Health. Finally, we suggest the use of PCR-ITS1-RFLP for diagnosis and simultaneous identification of the species in the Moroccan epidemiological context, but also in similar areas of the Mediterranean Basin.This study was supported by the University of Granada, Spain (CICODE program), the Faculty of Medicine and Pharmacy of Casablanca (Morocco), and the PHC Volubilis program

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation.

UnlabelledSyntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase.ImportanceBacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA