Pseudomonas putida S‐313 is able to desulphonate a broad range of aromatic sulphonates to provide sulphur for growth by monooxygenolytic cleavage to yield the corresponding phenol. After miniTn5 transposon mutagenesis of this strain, 11 mutants were isolated that were no longer able to utilize benzenesulphonate as a sulphur source. Three of these mutants were defective in the utilization of all aromatic sulphonates tested, but they grew normally with other sulphur sources. These strains contained independent insertions in the novel 4.2 kb asfRABC gene cluster, encoding a putative reductase (AsfA), a ferredoxin (AsfB), a putative periplasmic binding protein (AsfC), which was localized to the periplasm using alkaline phosphatase fusions, and a divergently oriented fourth gene, asfR, that encoded a LysR‐type regulator protein. A further mutant was interrupted in the ssu locus, which includes the gene for a putative desulphonative monooxygenase. Transformation of Pseudomonas aeruginosa with the asfRAB genes was sufficient to allow arylsulphonate utilization by this species, which does not normally use these compounds, suggesting that the AsfAB proteins may constitute an arylsulphonate‐specific electron transport system that interacts with a less specific oxygenase. Expression of the asfABC genes in P. putida was induced by benzenesulphonate or toluenesulphonate, and it was repressed in the presence of sulphate in the growth medium. AsfR was a negative regulator of asfABC expression, and toluenesulphonate induced expression of these genes indirectly by reducing the expression of the asfR gene.
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 ...
The class 1 major outer membrane protein of Neisseria meningitidis is a serious candidate for a meningococcal vaccine. To facilitate studies on the function of this protein, mutants were isolated that lacked this protein or the structurally related class 3 protein. These mutants were obtained by using the antibody-dependent bactericidal action of the complement system. The class 1 protein-deficient strain grew normally in vitro, whereas growth of the class 3 protein-deficient strain was slightly retarded. The class 3 protein-deficient strain displayed increased resistance to the antibiotics tetracycline and cefsulodin, which is consistent with the proposed role of the protein as a pore-forming protein. The class 1 protein was purified to homogeneity from the class 3 protein-deficient strain. Lipid bilayer experiments revealed that this protein also formed pores. The class 1 protein pores were cation selective.
Cysteine and methionine biosynthesis was studied inPseudomonas putida S-313 and Pseudomonas aeruginosa PAO1. Both these organisms used direct sulfhydrylation of O-succinylhomoserine for the synthesis of methionine but also contained substantial levels of O-acetylserine sulfhydrylase (cysteine synthase) activity. The enzymes of the transsulfuration pathway (cystathionine γ-synthase and cystathionine β-lyase) were expressed at low levels in both pseudomonads but were strongly upregulated during growth with cysteine as the sole sulfur source. In P. aeruginosa, the reverse transsulfuration pathway between homocysteine and cysteine, with cystathionine as the intermediate, allows P. aeruginosa to grow rapidly with methionine as the sole sulfur source. P. putida S-313 also grew well with methionine as the sulfur source, but no cystathionine γ-lyase, the key enzyme of the reverse transsulfuration pathway, was found in this species. In the absence of the reverse transsulfuration pathway, P. putida desulfurized methionine by the conversion of methionine to methanethiol, catalyzed by methionine γ-lyase, which was upregulated under these conditions. A transposon mutant of P. putida that was defective in the alkanesulfonatase locus (ssuD) was unable to grow with either methanesulfonate or methionine as the sulfur source. We therefore propose that in P. putida methionine is converted to methanethiol and then oxidized to methanesulfonate. The sulfonate is then desulfonated by alkanesulfonatase to release sulfite for reassimilation into cysteine.
Methanobacterium thermoautotrophicum ⌬H was grown in a fed-batch fermentor and in a chemostat under a variety of 80% hydrogen-20% CO 2 gassing regimes. During growth or after the establishment of steady-state conditions, the cells were analyzed for the content of adenylylated coenzyme F 420 (factor F 390 -A) and other methanogenic cofactors. In addition, cells collected from the chemostat were measured for methyl coenzyme M reductase isoenzyme (MCR I and MCR II) content as well as for specific activities of coenzyme F 420 -dependent and H 2 -dependent methylenetetrahydromethanopterin dehydrogenase (F 420 -MDH and H 2 -MDH, respectively), total (viologen-reducing) and coenzyme F 420 -reducing hydrogenase (FRH), factor F 390 synthetase, and factor F 390 hydrolase. The experiments were performed to investigate how the intracellular F 390 concentrations changed with the growth conditions used and how the variations were related to changes in levels of enzymes that are known to be differentially expressed. The levels of factor F 390 varied in a way that is consistently understood from the biochemical mechanisms underlying its synthesis and degradation. Moreover, a remarkable correlation was observed between expression levels of MCR I and II, F 420 -MDH, and H 2 -MDH and the cellular contents of the factor. These results suggest that factor F 390 is a reporter compound for hydrogen limitation and may act as a response regulator of methanogenic metabolism.
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