The Escherichia coli tauD gene is required for the utilization of taurine (2-aminoethanesulfonic acid) as a sulfur source and is expressed only under conditions of sulfate starvation. The sequence relatedness of the TauD protein to the ␣-ketoglutarate-dependent 2,4-dichlorophenoxyacetate dioxygenase of Alcaligenes eutrophus suggested that TauD is an ␣-ketoglutarate-dependent dioxygenase catalyzing the oxygenolytic release of sulfite from taurine (van der Ploeg, J. R., Weiss, M. A., Saller, E., Nashimoto, H., Saito, N., Kertesz, M. A., and Leisinger, T. (1996) J. Bacteriol. 178, 5438 -5446). TauD was overexpressed in E. coli to ϳ70% of the total soluble protein and purified to apparent homogeneity by a simple two-step procedure. The apparent M r of 81,000 of the native protein and the subunit M r of 37,400 were consistent with a homodimeric structure. The pure enzyme converted taurine to sulfite and aminoacetaldehyde, which was identified by high pressure liquid chromatography after enzymatic conversion to ethanolamine. The reaction also consumed equimolar amounts of oxygen and ␣-ketoglutarate; ferrous iron was absolutely required for activity; and ascorbate stimulated the reaction. The properties and amino acid sequence of this enzyme thus define it as a new member of the ␣-ketoglutarate-dependent dioxygenase family. The pure enzyme showed maximal activity at pH 6.9 and retained activity on storage at ؊20°C for several weeks. Taurine (K m ‫؍‬ 55 M) was the preferred substrate, but pentanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, and 1,3-dioxo-2-isoindolineethanesulfonic acid were also desulfonated at significant rates. Among the cosubstrates tested, only ␣-ketoglutarate (K m ‫؍‬ 11 M) supported significant dioxygenase activity.In the absence of sulfate, Escherichia coli can utilize aliphatic sulfonates as sulfur sources for growth. Sulfonates known to provide sulfur include ethanesulfonate, butanesulfonate, L-cysteate, isethionate (2-hydroxyethanesulfonate), and taurine (2-aminoethanesulfonate) (1, 2). None of these sulfonates served as sulfur source under anaerobic conditions, nor could they be utilized as a source of carbon and energy or of carbon, energy, and sulfur under either aerobic or anaerobic conditions (1). The mechanisms of sulfur assimilation from aliphatic sulfonates are unknown, but it has been shown that sulfonate/sulfur enters the assimilatory sulfate reduction pathway at the stage of sulfite (3).Recently, we have identified the tauABCD gene cluster, located at 8.5 min on the E. coli chromosome, which is specifically involved in the utilization of taurine as a sulfur source (2). Disruption of tauB, tauC, or tauD resulted in the loss of the ability to utilize taurine as a source of sulfur, but did not affect the utilization of a range of other aliphatic sulfonates as sulfur sources. The tau genes were only expressed during growth in the absence of sulfate or cysteine (2). The amino acid sequences of TauABC exhibit similarity to components of ABC-type transport systems (4). TauA has a p...

The Escherichia coli ssuEADCB gene cluster is required for the utilization of alkanesulfonates as sulfur sources, and is expressed under conditions of sulfate or cysteine starvation. The SsuD and SsuE proteins were overexpressed and characterized. SsuE was purified to homogeneity as an N-terminal histidine-tagged fusion protein. Native SsuE was a homodimeric enzyme of M r 58,400, which catalyzed an NAD(P)H-dependent reduction of FMN, but it was also able to reduce FAD or riboflavin. The SsuD protein was purified to >98% purity using cation exchange, anion exchange, and hydrophobic interaction chromatography. The pure enzyme catalyzed the conversion of pentanesulfonic acid to sulfite and pentaldehyde and was able to desulfonate a wide range of sulfonated substrates including C-2 to C-10 unsubstituted linear alkanesulfonates, substituted ethanesulfonic acids and sulfonated buffers. SsuD catalysis was absolutely dependent on FMNH 2 and oxygen, and was maximal for SsuE/SsuD molar ratios of 2.1 to 4.2 in 10 mM Tris-HCl, pH 9.1. Native SsuD was a homotetrameric enzyme of M r 181,000. These results demonstrate that SsuD is a broad range FMNH 2 -dependent monooxygenase catalyzing the oxygenolytic conversion of alkanesulfonates to sulfite and the corresponding aldehydes. SsuE is the FMN reducing enzyme providing SsuD with FMNH 2 .In Escherichia coli, sulfate starvation causes increased synthesis of several proteins involved in scavenging sulfur from alternative sulfur sources (1). Among these proteins are the tauABCD-encoded proteins required for uptake and desulfonation of taurine (2-aminoethanesulfonic acid) (2, 3) and the proteins SsuE and SsuD of the ssuEADCB gene cluster. We have shown that the ssuEADCB gene cluster, located at 21.4 min on the E. coli chromosome, is specifically involved in the utilization of alkanesulfonates as a source of sulfur for growth (4). Deletion of ssuEADCB resulted in the loss of the ability to utilize alkanesulfonates as a sulfur source but did not affect the utilization of taurine for this purpose. The amino acid sequences of SsuABC exhibit similarity to components of ABCtype transport systems (4, 5). SsuA has a putative signal sequence, indicating that it functions as a periplasmic binding protein, and the sequences of SsuB and SsuC are significantly similar to those of ATP-binding proteins and membrane components, respectively, of members of the ABC transporter superfamily. It thus appears that the proteins encoded by ssuABC constitute an uptake system for alkanesulfonates.The ssuD gene product shows 25% sequence identity to a characterized nitrilotriacetate two-component monooxygenase of Chelatobacter heintzii (6) and to the pristinamycin II A synthase subunit A of Streptomyces pristinaespiralis (7), suggesting that SsuD is involved in the oxygenolytic release of sulfite from alkanesulfonates. Here we report the purification of the SsuD and SsuE proteins, describe their biochemical properties, and demonstrate that SsuD is a monooxygenase that catalyzes the desulfonation of alkanesulf...

Orange I1 azoreductase [NAD(P)H : l-(4'-sulfophenylazo)-2-naphthol oxidoreductase], an enzyme catalyzing the reductive cleavage of the azo bridge of Orange I1 and related dyes, was purified to electrophoretic homogeneity from Pseudomonas species, strain KF46. This organism utilized carboxy-Orange I1 [I -(4'-carboxypheny1azo)-2-naphthol] but not Orange I1 as the sole source of carbon, energy, and nitrogen. Orange I1 azoreductase was induced 80-fold by both Orange I1 and carboxy-Orange 11. With two successive runs of affinity chromatography using two chromatographic media with different triazinyl dyes as ligands, the enzyme was purified 120-fold with 43% yield. The purified enzyme is a monomer with a molecular weight of 30000. Its K , values were 1.5 pM for both Orange I1 and carboxy-Orange 11, 5 pM for NADPH, and 180 pM for NADH. A survey of the efficiency of various Orange dyes as substrates for Orange I1 azoreductase showed that: (a) a hydroxy group in the 2-position of the naphthol ring is required; (b) charged groups in proximity to the azo group hinder the reaction; (c) a second polar substituent on the dye molecule impedes the reaction; (d) electronwithdrawing groups on the phenyl ring accelerate the reaction.

Genes whose expression is regulated by sulfate starvation in Escherichia coli were identified by generating random translational lacZ fusions in the chromosome with the placMu9 system. Nine lacZ fusion strains which expressed ␤-galactosidase after growth under sulfate starvation conditions but not after growth in the presence of sulfate were found. These included two strains with insertions in the dmsA and rhsD genes, respectively, and seven strains in which the insertions were located within a 1.8-kb region downstream of hemB at 8.5 minutes on the E. coli chromosome. Analysis of the nucleotide sequence of this region indicated the presence of four open reading frames designated tauABCD. Disruption of these genes resulted in the loss of the ability to utilize taurine (2-aminoethanesulfonate) as a source of sulfur but did not affect the utilization of a range of other aliphatic sulfonates as sulfur sources. The TauA protein contained a putative signal peptide for transport into the periplasm; the TauB and TauC proteins showed sequence similarity to ATP-binding proteins and membrane proteins, respectively, of ABC-type transport systems; and the TauD protein was related in sequence to a dichlorophenoxyacetic acid dioxygenase. We therefore suggest that the proteins encoded by tauABC constitute an uptake system for taurine and that the product of tauD is involved in the oxygenolytic release of sulfite from taurine. The transcription initiation site was detected 26 to 27 bp upstream of the translational start site of tauA. Expression of the tauD gene was dependent on CysB, the transcriptional activator of the cysteine regulon.

Methylobacterium sp. strain CM4, an aerobic methylotrophic ␣-proteobacterium, is able to grow with chloromethane as a carbon and energy source. Mutants of this strain that still grew with methanol, methylamine, or formate, but were unable to grow with chloromethane, were previously obtained by miniTn5 mutagenesis. The transposon insertion sites in six of these mutants mapped to two distinct DNA fragments. The sequences of these fragments, which extended over more than 17 kb, were determined. Sequence analysis, mutant properties, and measurements of enzyme activity in cell-free extracts allowed the definition of a multistep pathway for the conversion of chloromethane to formate. The methyl group of chloromethane is first transferred by the protein CmuA (cmu: chloromethane utilization) to a corrinoid protein, from where it is transferred to H 4 folate by CmuB. Both CmuA and CmuB display sequence similarity to methyltransferases of methanogenic archaea. In its C-terminal part, CmuA is also very similar to corrinoid-binding proteins, indicating that it is a bifunctional protein consisting of two domains that are expressed as separate polypeptides in methyl transfer systems of methanogens. The methyl group derived from chloromethane is then processed by means of pterinelinked intermediates to formate by a pathway that appears to be distinct from those already described in Methylobacterium. Remarkable features of this pathway for the catabolism of chloromethane thus include the involvement of a corrinoiddependent methyltransferase system for dehalogenation in an aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from chloromethane into central metabolism.

The growth properties of an Escherichia coli strain carrying a chromosomal deletion of the ssuEADCB genes (formerly designated ycbPONME) indicated that the products of this gene cluster are required for the utilization of sulfur from aliphatic sulfonates. Sequence similarity searches indicated that the proteins encoded by ssuA, ssuB, and ssuC are likely to constitute an ABC type transport system, whereas ssuD and ssuE encode an FMNH 2 -dependent monooxygenase and an NAD(P)Hdependent FMN reductase, respectively (Eichhorn, E., van der Ploeg, J. R., and Leisinger, T. (1999) J. Biol. Chem. 274, 26639 -26646). Synthesis of ␤-galactosidase from a transcriptional chromosomal ssuE-lacZ fusion was repressed by sulfate or cystine and depended on the presence of a functional cbl gene, which encodes a LysRtype transcriptional regulator. Electrophoretic mobility shift assays with the ssu promoter region and measurements of ␤-galactosidase from plasmid-encoded ssuElacZ fusions showed that full expression of the ssu operon required the presence of a Cbl-binding site upstream of the ؊35 region. CysB, the LysR transcriptional regulator for the cys genes, was not required for expression of a chromosomal ssuE-lacZ fusion although the ssu promoter region contained three CysB-binding sites. Integration host factor could also occupy three binding sites in the ssu promoter region but had no influence on expression of a chromosomal ssuE-lacZ fusion.

Six loci coding for arginine biosynthetic enzymes in Pseudomonas aeruginosa strain PAO were identified by enzyme assay: argA (N-acetylglutamate synthase), argB (N-acetylglutamate 5-phosphotransferase), argC (N-acetylglutamate 5-semialdehyde dehydrogenase), argF (anabolic ornithine carbamoyl-transferase), argG (argininosuccinate synthetase), and argH (argininosuccinase). One-step mutants which had a requirement for arginine and uracil were defective in carbamoylphosphate synthase, specified by a locus designated car. To map these mutations we used the sex factor FP2 in an improved interrupted mating technique as well as the generalized transducing phages F116L and G101. We confirmed earlier studies, and found no clustering of arg and car loci. However, argA, argH, and argB were mapped on a short chromosome segment (approx. 3 min long), and argF and argG were cotransducible, but not contiguous. N-Acetylglutamate synthase, the enzyme which replenishes the cycle of acetylated intermediates in ornithine synthesis of Pseudomonas, appears to be essential for arginine synthesis since argA mutants showed no growth on unsupplemented minimal medium.

Five anaerobic bacteria were tested for their abilities to transform tetrachloromethane so that information about enzymes involved in reductive dehalogenations of polychloromethanes could be obtained. Cultures of the sulfate reducer Desulfobactenium autotrophicum transformed some 80 ,uM tetrachloromethane to trichloromethane and a small amount of dichloromethane in 18 days under conditions of heterotrophic growth. The acetogens Acetobacterium woodii and Clostridium thermoaceticum in fructose-salts and glucose-salts media, respectively, degraded some 80 ,M tetrachloromethane completely within 3 days. Trichloromethane accumulated as a transient intermediate, but the only chlorinated methanes recovered at the end of the incubation were 8 ,uM dichloromethane and traces of chloromethane. Desulfobacter hydrogenophilus and an autotrophic, nitrate-reducing bacterium were unable to transform tetrachloromethane. Reduction of chlorinated methanes was thus observed only in the organisms with the acetyl-coenzyme A pathway. Experiments with ['4C]tetrachloromethane were done to determine the fate of this compound in the acetogen A. woodii. Radioactivity in an 11-day heterotrophic culture was largely (67%) recovered in C02, acetate, pyruvate, and cell material. In experiments with cell suspensions to which [14C]tetrachloromethane was added, 14Co2 appeared within 20 s as the major transformation product. A. woodii thus catalyzes reductive dechlorinations and transforms tetrachloromethane to CO2 by a series of unknown reactions. * Corresponding author. MATERIALS AND METHODS Materials. [U-'4C]acetate (2.07 TBq/mol), [2-'4C]acetate (1.96 TBq/mol), and ['4C]tetrachloromethane (2.3 TBq/mol;