ThessuLocus Plays a Key Role in Organosulfur Metabolism inPseudomonas putidaS-313

Abstract

Pseudomonas putida S-313 can utilize a broad range of aromatic sulfonates as sulfur sources for growth in sulfate-free minimal medium. The sulfonates are cleaved monooxygenolytically to yield the corresponding phenols. miniTn5 mutants of strain S-313 which were no longer able to desulfurize arylsulfonates were isolated and were found to carry transposon insertions in the ssuEADCBF operon, which contained genes for an ATPbinding cassette-type transporter (ssuABC), a two-component reduced flavin mononucleotide-dependent monooxygenase (ssuED) closely related to the Escherichia coli alkanesulfonatase, and a protein related to clostridial molybdopterin-binding proteins (ssuF). These mutants were also deficient in growth with a variety of other organosulfur sources, including aromatic and aliphatic sulfate esters, methionine, and aliphatic sulfonates other than the natural sulfonates taurine and cysteate. This pleiotropic phenotype was complemented by the ssu operon, confirming its key role in organosulfur metabolism in this species. Further complementation analysis revealed that the ssuF gene product was required for growth with all of the tested substrates except methionine and that the oxygenase encoded by ssuD was required for growth with sulfonates or methionine. The flavin reductase SsuE was not required for growth with aliphatic sulfonates or methionine but was needed for growth with arylsulfonates, suggesting that an alternative isozyme exists for the former compounds that is not active in transformation of the latter substrates. Aryl sulfate ester utilization was catalyzed by an arylsulfotransferase, and not by an arylsulfatase as in the related species Pseudomonas aeruginosa.The sulfur content of aerobic soils is made up almost entirely of sulfonates and sulfate esters of undefined structure. Inorganic sulfate, by contrast, is comparatively poorly represented and constitutes less than 5% of the sulfur in these environments (2). In order to meet their sulfur requirements, soil bacteria must therefore be able to mobilize this organically bound sulfur and assimilate it into cell material. Utilization of the naturally occurring alkanesulfonates taurine, isethionate, and cysteate as sulfur sources is widespread in soil isolates (24, 38), although complete degradation of these compounds as carbon and energy sources appears to be limited to a few species. The ability to hydrolyze alkyl or aromatic sulfate esters is also common in bacteria from soil and water environments. Arylsulfatase is a stable soil enzyme and has been used as a marker for biological activity in soils (37). Alkyl sulfatase enzymes are also very common and were found in 15% of isolates obtained nonselectively from a noncontaminated environment (52).In contrast to the naturally occurring alkanesulfonates and aliphatic and aromatic sulfate esters, aromatic sulfonates are regarded as xenobiotic compounds (20) and are produced industrially as surfactants, dyestuffs, and cement additives. These compounds are mineralized as a carbon source by a number ...

Crystal Structures of NADH:FMN Oxidoreductase (EmoB) at Different Stages of Catalysis

“…The phosphoribityl group of this first FMN molecule is in the relatively shallow pocket formed by the two loops between ␤1 and ␣1 and between ␤5 and ␣5, and the edge of its isoalloxazine ring is surrounded by parts of the loop connecting ␤3 and ␣3 and the loop connecting ␤4 and ␣4. In detail, the phosphate group of the first FMN molecule is within hydrogen bond distance of the side chains of Ser 11 , Ser 16 , and Thr 18 and the backbone nitrogen atoms of Thr 17 and Thr 18 , which together constitute the hydrophilic bottom of the pocket (Fig. 4).…”

“…Recently, more FMNH 2 -dependent monooxygenases have been discovered, including pristinamycin IIA synthase of Streptomyces pristinaespiralis (16), two monooxygenases involved in desulfurization of dibenzothiophene in Rhodococcus sp. strain IGTS8 (17), organosulfur monooxygenases (SsuD) in Pseudomonas putida S-313 (18), and EDTA monooxygenase in Mesorhizobium sp. bacterium BNC1 (6).…”

RNA-Seq Analysis of Sulfur-Deprived Chlamydomonas Cells Reveals Aspects of Acclimation Critical for Cell Survival

“…While no TAUD homologs have been identified in plants or animals, the putative TAUD proteins of Chlamydomonas have significant similarity to bacterial and fungal TAUD/TfdA family proteins (;19 to 35% identity), as shown in Supplemental Figure 2 online. Moreover, the Escherichia coli TauD, the Saccharomyces cerevisiae YLL057c, and the Pseudomonas syringae AtsK a-ketoglutarate dioxygenases can efficiently metabolize various sulfonates in addition to taurine (Eichhorn et al, 1997;Hogan et al, 1999;Kahnert et al, 2000). Similarly, the putative TAUD proteins of Chlamydomonas might use alkanesulfonates other than taurine.…”

Saccharin as a sole source of carbon and energy for Sphingomonas xenophaga SKN

“…An initial attack on the unsubstituted carbon atoms of the saccharin ring (Fig. 1) would lead to very complex intermediates, and, as with an initial monooxygenase of the type known in assimilative desulfonation (see Kahnert et al 2000), would probably require at least three oxygenases in the pathway.…”