The ssu locus plays a key role in organosulfur metabolism in Pseudomonas putida S-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 ATP-binding 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.

FIG. 1

Map of the ssu region of the P. putida chromosome. The positions of the miniTn5Km insertions in strains SN34, PW2, PW7, and PW10 are shown, as are selected restriction sites: Bp, Bpn1102I; Bs, BsrG1; C, ClaI; E, EcoRI; Ea, Eam11051; H, HindIII; K, KpnI; S, SapI; St, StuI. X, XhoI; Xb, XbaI. Several plasmids described in the text are shown, and the location of the lac promoter in the vector is indicated with a solid triangle.

FIG. 2

Two-dimensional electropherograms of total cell protein from P. putida S-313. Cells were grown in succinate minimal medium with sulfur for growth provided as inorganic sulfate (A) or toluenesulfonate (B). Proteins that are upregulated during growth in the absence of sulfate are circled, and the SsuF protein is indicated.

FIG. 3

Growth of P. putida PW7 (A) and P. putida PW7(pME4578) (B) in succinate minimal medium with different sulfur sources. Growth curves were measured in a SPECTRAmax Plus microtiter plate reader, as described in Materials and Methods. Sulfur sources: ■, sulfate; ○, hexylsulfate; ●, nitrocatecholsulfate; ▴, pentanesulfonate; ▵, benzenesulfonate; ∗, methionine. —, no sulfur.

FIG. 4

Nucleotide sequence of the intergenic region between the P. putida lsfA and ssuE genes. The stop and start codons of lsfA and ssuE, respectively, are shown; the ribosome binding site of ssuE is underlined; and a putative rho-independent terminator structure downstream of lsfA is indicated with arrows. The −10 and −35 sequences of a putative ς⁷⁰-dependent promoter are boxed. Plasmids pME4424 and pME4443 begin at the points indicated.

FIG. 5

Growth of derivatives of P. putida SN34 in succinate minimal medium with different sulfur sources. Growth curves were measured in a SPECTRAmax Plus microtiter plate reader, as described in Materials and Methods. (A) SN34(pME4423); (B) SN34(pME4431); (C) SN34(pME4433). Sulfur sources: ■, sulfate; ○, hexylsulfate; ●, nitrocatecholsulfate; ▴, pentanesulfonate; ▵, benzenesulfonate; ∗, methionine. —, no sulfur.

FIG. 6

Genetic organization of related ssu and msu operons. The enzymes encoded in each gene cluster are putative oxygenases (■), NADH-dependent FMN reductases (), and the components of ABC-type transporters: periplasmic solute-binding proteins (), ATP-binding proteins (), and permease proteins (). No definite function is known for the products of the ssuF and msuC genes (reference and this paper). The genes are from E. coli (46, 47), B. subtilis (45), P. putida (reference and this paper), P. aeruginosa (; Pseudomonas Genome Project [http://www.pseudomonas.com/), and the unfinished chromosomes of Yersinia pestis and K. pneumoniae (Yersinia pestis Sequencing Group, Sanger Centre [ftp://ftp.Sanger.ac.uk/pub/pathogens/yp/]; Klebsiella pneumoniae Sequencing Group, University of Washington Genome Center [http://genome.wustl.edu/gsc/Projects/bacterial/klebsiella/klebsiella.shtml]).