Résumé des résultats (Abstract)
(Anglais)
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Desulfurization of aromatic sulfonates was studied in the sewage sludge isolate Pseudomonas pulido S-313. Using transposon mutagenesis, three gene loci were found to be involved in this process, the as!. ssu and als-sft gene clusters. Deletion analysis was used to defme the minimum set of genes required for growth with aromatic sulfonates in this species. The FMNH2-dependent monooxygenase SsuD was found to be responsible for the desulfonation reaction, and to require reduced FMN supplied by the NADH:FMN oxidoreductase SsuE. SsuD also cleaved aliphatic sulfonates -for desulfurization of arylsulfonates a further reductase-ferredoxin pair was required, encoded by the asfAB genes. Uptake of aromatic sulfonates into the cell was catalyzed by an ABC-type transporter, composed of the AtsB membrane protein and the AtsC ATP-binding component, together with either AsiC or AtsR as the periplasmic binding protein. In addition, a small protein related to molybdopterin-binding proteins, SsuF, was required for growth with arylsulfonates, but its exact role is not yet defmed.
The monooxygenase SsuD and the FMN reductase SsuE have been overexpressed and purified. SsuD desulfonates a broad range of aliphatic sulfonates in vitro, including methanesulfonate and taurine, which are not substrates for the E. coli SsuD enzyme.
Overexpression of the additional proteins required for desulfurization of arylsulfonates was hampered by their extreme insolubility. Conditions were optimized for their production by co-expression with three chaperones, GroES, GroEL and trigger factor. In parallel to these experiments, a 'desulfonation cassette' was constructed, carrying all the genes determined to be for aromatic desulfonation, under the control of the lac promoter on a broad host-range plasmid.
The transcriptional start sites for asjR and asiA were determined. Expression of the asfABC genes is repressed in the presence of sulfate by the CysB protein, and by the product of the asjR gene. Depression in the presence of aromatic sulfonates involves AsfR and an unidentified inducer protein that is propose to interact with the region immediately upstream of the asjR gene. An additional LysR-type regulator (SftR) was identified in the als-sft cluster, and shown to be required for expression of sulfate ester utilization genes in this strain and related species. sftR mutants of P. pulido S-313 showed significantly reduced survival in mesocosms constructed with three different Danish soils (agricultural, grassland and forest soils), either in soil alone or in an Arabidopsis rhizosphere in each soil. The results suggest that sulfate esters play an important role in bacterial nutrition in soil, and that correct expression of the SftR-regulated sulfatases is crucial for bacterial soil survival.
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