INTRODUCTION

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Sulfonates are chemically stable compounds which are common xenobiotics released into the environment (22) but which are also natural products that contribute significantly to the global biogeochemical sulfur cycle. They constitute a large proportion of the sulfur found in aerobic soils and together with sulfate esters make up >95% of the sulfur content of soil environments (2). Sulfonates have also been found to accumulate to levels of 20 to 40% of the total organic sulfur in near-surface marine sediments (46). Methanesulfonic acid is the main biogenic sulfur component in the atmosphere, being generated by photooxidation of dimethyl sulfide released by marine ecosystems and subsequently returned to terrestrial environments in precipitation. Bacteria which can utilize methanesulfonate and other naturally occurring alkanesulfonates, such as cysteate, taurine, or isethionate, as sources of sulfur for growth can readily be isolated from soil environments (25, 40), even under nonselective conditions, and several strains that can use these compounds as carbon sources have also been reported (16, 43, 44).

With the exception of taurine (2-aminoethanesulfonate), the detailed enzymology of desulfonation of alkanesulfonates within the sulfur cycle has not yet been reported. In Escherichia coli, taurine is desulfonated to aminoacetaldehyde by the -ketoglutarate-dependent taurine dioxygenase encoded by the tauD gene (10). Taurine sulfur is released by taurine dioxygenase as sulfite (10). Sulfite also appears to be the direct desulfurization product of cysteate and isethionate in E. coli, since utilization of these compounds by E. coli required an intact sulfite reductase enzyme (45). Synthesis of TauD is repressed in the presence of sulfate or cysteine in the growth medium and is dependent on two very similar LysR-type transcriptional activators, CysB and Cbl (48).

For Pseudomonas aeruginosa, physiological data suggest that release of alkanesulfonate sulfur is also catalyzed by an oxygenase (21), although the enzyme responsible for the reaction has not yet been identified or characterized. As in E. coli, desulfonation activity in whole cells is repressed in the presence of cysteine or sulfate in the growth medium. In pseudomonads thiocyanate also leads to repression of desulfonation (in contrast, E. coli is unable to grow with thiocyanate as a sulfur source [24]). This pattern of regulation corresponds with that observed for a set of 10 proteins in P. aeruginosa whose synthesis is controlled by the sulfur supply to the cell, being synthesized only in the absence of sulfate, cysteine, or thiocyanate (17). These proteins, called sulfate starvation-induced proteins (SSI proteins), have been found in several gram-positive and gram-negative species (24) and have been studied in detail in E. coli, where they constitute a subset of the cysteine regulon products (35, 48, 49).

In this study we have carried out a more detailed investigation of the PA13 protein, one of the SSI proteins of P. aeruginosa. The gene encoding this protein was identified, and the putative operon in which it is located was sequenced. Overexpression studies showed that the encoded proteins are involved in methanesulfonate metabolism and constitute a novel reduced flavin mononucleotide (FMNH₂)-dependent desulfonating sulfonatase system.

	MATERIALS AND METHODS

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Chemicals, bacterial strains, and growth conditions. All chemicals used as sulfur sources were of the highest quality available and were obtained from Fluka (Buchs, Switzerland). Oligonucleotides were obtained from Microsynth (Balgach, Switzerland). Bacterial strains, plasmids, and phages used in this study are listed in Table 1. All P. aeruginosa strains were grown at 37°C either in Luria-Bertani medium (1) or in MMAA medium, which was a succinate-salts medium supplemented with all of the proteinogenic amino acids except methionine and cysteine, as previously described (3). Sulfur sources were added as described in Results, to a final concentration of 250 to 500 µM. Antibiotics were added at the following concentrations (micrograms per milliliter): ampicillin, 100 for E. coli; tetracycline, 25 for E. coli and 125 for P. aeruginosa; gentamicin, 15 for E. coli and 200 for P. aeruginosa; streptomycin and carbenicillin, 500 for P. aeruginosa; and kanamycin, 25 for E. coli. Sulfur-limited solid media were prepared by addition of 0.6% SeaPlaque agarose (FMC BioProducts). 5-Bromo-4-chloro-3-indolylgalactoside (X-Gal) (80 µg/ml) was added when necessary. Growth in liquid cultures was monitored as the optical density at 650 nm (OD₆₅₀), and cultures used for enzyme assays were harvested in the mid-exponential phase (OD₆₅₀ = 0.5 to 0.7).

Enzyme assays. Arylsulfatase was assayed by using 4-nitrocatecholsulfate as the substrate as described previously (3). Catechol-2,3-dioxygenase was quantified by measuring the A₃₇₅ in a whole-cell continuous assay (19). NADH-dependent flavin mononucleotide (FMN) reductase activity was measured as the disappearance of NADH at 340 nm, in a reaction mixture (1 ml) consisting of 100 µM FMN, 100 µM NADH, and 50 mM Tris-HCl, pH 7.5. The reaction was started by addition of a suitable amount of enzyme, and the mixture was incubated at 25°C. No electron acceptor other than oxygen was provided, and so initial, linear reduction rates were determined, before oxygen became rate limiting. Sulfonate desulfonation was assayed in a reaction mixture (0.5 ml) consisting of 5 mM substrate, 100 µM FMN, 5 mM NADH, and 50 mM Tris-HCl (pH 7.5), and the reaction was started by addition of cell extract (ca 0.5 mg of protein). Sulfite release was quantified at 430 nm after a suitable time by diluting samples into stop buffer [125 µg of 5,5'-dithio-bis(2-nitrobenzoate) per ml, 62 mM EDTA]. Desulfonation assay mixtures were shaken at 37°C (180 rpm). -Ketoglutarate-dependent taurine dioxygenase activity was measured as previously described (10). Oxygen consumption was measured with a Clark oxygen electrode (Rank Bros, Bottisham, United Kingdom). Formaldehyde was determined by chemical derivatization, and formaldehyde dehydrogenase was measured in an NAD-dependent assay (31). Protein was measured by the method of Bradford (4), with bovine serum albumin as the standard.

DNA manipulations. For plasmid isolation, restriction enzyme digestion, and transformation, published procedures were used (1). Where required, DNA fragments were isolated from agarose gels with GeneClean (Bio101, La Jolla, Calif.) or Qiaquick (Qiagen, Basel, Switzerland) spin columns. PCR was carried out in a Trio Block (BioMetra, Göttingen, Germany). Standard reaction mixtures consisted of 50 pmol of primers, 200 nmol of deoxynucleoside triphosphates, 0.2 U of Taq DNA polymerase (Fermentas, Vilnius, Lithuania), and 1 to 100 ng of template in a final volume of 50 µl. Dimethyl sulfoxide (10%, vol/vol) was routinely added, and for cloning purposes the Taq polymerase was supplemented with 1 U of Vent DNA polymerase (New England Biolabs, Bad Schwalbach, Germany). When required, PCR-derived fragments were sequenced to confirm that no point mutations had occurred. Southern analysis was carried out by using digoxigenin-labelled probes which had been labelled either by random-primed labelling (1) or by using PCR of a suitable DNA fragment to incorporate digoxigenin-11-dUTP. Hybridization on nylon membranes (Hybond; Amersham) was carried out at 68°C in 500 mM sodium phosphate (pH 7.2)-7% sodium dodecyl sulfate (SDS) for 24 to 48 h, and the membranes were washed at 65°C with 50 mM sodium phosphate (pH 7.2)-1% SDS twice for 30 min each.

Isolation of the msuEDC genes and construction of insertion and deletion mutants. The cosmid pME4006, which contained the msuEDC operon, was identified in a gene bank of P. aeruginosa PAO1 constructed in the pLAFR3 cosmid vector (50) by PCR with primers PA13F1 and PA13R1. A 7.5-kb EcoRV fragment of pME4006 was subcloned into pBluescript, yielding plasmid pME4095. The xylE reporter gene was inserted into the NcoI site in the msuD gene as a 2.2-kb SmaI fragment from plasmid pX1918GT. Allele replacement was performed by subcloning into the ColE1 suicide vector pME3087 (29) to yield plasmid pME4088, followed by conjugative transfer into P. aeruginosa PAO1 by plate mating. Recombinants were sought by selection for a Gm^r Tc^s phenotype. Correct allele replacement in strain SLF3 was confirmed by PCR and Southern analysis. An identical strategy was used to construct strain SLF2, which had the xylE-Gm^r cassette inserted in the reverse orientation.

Overexpression of MsuE, MsuD, and MsuC. For overexpression and partial characterization of the MsuE, MsuD, and MsuC proteins, pET-24b-based plasmids were used. To make pME4503, the 5' end of the msuE gene was amplified by PCR with primers SlfA-N and 960377. The resulting fragment was digested with NdeI and DrdI. This fragment was then ligated with NdeI- and XhoI-digested pET-24b and the 2,940-bp DrdI/XhoI fragment of pME4095 containing the rest of the msuE gene and the msuD and msuC genes, yielding plasmid pME4503.

Sequence analysis. Nucleotide and protein sequences were analyzed with the University of Wisconsin Genetics Computer Group package version 9.1 and compared to the GenEMBL database and the SWISS-PROT database, respectively.

Nucleotide sequence accession number. The nucleotide sequence of the msuEDC operon has been deposited in the GenBank database under accession no. AF026067.

	RESULTS

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Cloning and sequence analysis of the msuEDC genes. When it is cultivated in the absence of sulfate, cysteine, or thiocyanate, P. aeruginosa synthesizes a set of additional SSI proteins (17). The reported N-terminal amino acid sequences of these SSI proteins yielded little information about their function in the cell, and it was speculated that they were involved in desulfurization of organosulfur compounds, similarly to the SSI proteins of E. coli (10, 49). One of the reported P. aeruginosa SSI proteins, PA13, was further investigated. With the reported N-terminal sequence as a probe, a DNA fragment encoding protein PA13 was identified in a P. aeruginosa cosmid library (50), carried on cosmid pME4006. Southern analysis showed that the PA13 N-terminal region was encoded on a 7.2-kb EcoRV fragment of pME4006. This fragment was therefore subcloned into pBluescript to give plasmid pME4095, and the nucleotide sequence of a 5,000-bp region centered on PA13 was determined. Analysis of the sequence revealed the presence of three open reading frames, arranged in a putative operon structure, which have been designated msuE, msuD, and msuC, for methanesulfonate sulfur utilization (Fig. 1). All three msu reading frames were preceded by acceptable ribosome binding sites, and their codon usage corresponded to that reported previously for P. aeruginosa (51). The overall G+C content of the coding region was 70.3%, which is within the range reported for this species (51).

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Genetic organization of the msu locus of P. aeruginosa. The position of the msuD::xylE fusion is shown, as are selected restriction sites: (B, BamHI; D, DrdI; E, EcoRI; K, KpnI; N, EcoNI; Nc, NcoI; S, SphI; Sa, SauI; V, EcoRV; and X, XhoI). The DNA fragments in several plasmids described in the text are shown at the bottom.

View larger version (139K): [in this window] [in a new window]   FIG. 2.   Sequence comparison of FMNH2-dependent monooxygenases. Sequence alignment was done with the program CLUSTALW. Amino acid identities (*) and similarities (:) are shown. The proteins are B. subtilis (Bs) SsuD (OrfM) (accession no. L16808), E. coli (Ec) SsuD (Ssi6) (accession no. P80645), P. putida (Pp) SsuD (accession no. AF075709), P. aeruginosa (Pa) SsuD (preliminary results from the Pseudomonas sequencing project [34]), P. aeruginosa MsuD (accession no. AF026067), Streptomyces pristinaespiralis pristinamycin synthase A (SnaA) (accession no. P54991), R. erythropolis DBT oxide monooxygenase (DszA [SoxA]) (accession no. P54995), and Chelatobacter heintzii nitrilotriacetate monooxygenase (NtaA) (accession no. P54989).

Chromosomal location of the msuEDC genes. The msuEDC genes were localized on the chromosome of P. aeruginosa PAO by hybridization of a digoxigenin-labelled SphI/KpnI fragment (msuED) (Fig. 1) to genomic DNA which had been digested with SpeI or DpnI. The probe hybridized strongly to chromosomal SpeI fragment J and DpnI fragment B (37), corresponding to a map position of 46.5 to 49.5 min on the chromosomal map (15). To date, the only genes in this region that have been characterized are related to pyoverdine synthesis and transport. A second, weak signal was also detected on SpeI fragment C, corresponding to a map position of 32 to 37 min. The presence of this second signal suggests that a second copy of closely related genes exists on the chromosome of P. aeruginosa, and this second copy may well correspond to the ssuD gene mentioned above.

Analysis of mutants with mutations in the msuEDC region. In order to investigate the function of the msu genes, we constructed strain SLF3, in which the msuD gene was interrupted by a cassette carrying a promoterless xylE reporter gene and a gentamicin resistance gene. The gentamicin resistance gene in this cassette is flanked by transcriptional termination signals, so a polar effect on expression of msuC was also expected. Since SSI protein PA13 was not synthesized at all in the presence of inorganic sulfate, we anticipated that strain SLF3 would be deficient in utilization of some alternative sulfur source. Unexpectedly, the mutant was able to grow with any of over 20 sulfur sources tested, including sulfate, thiocyanate, sulfonates, sulfate esters, sulfamates, sulfur amino acids, and glucosinolates. However, the fact that the msu genes hybridized to the chromosome at two sites (see above) suggested a gene duplication, and so the lack of an observable phenotype for this mutant was perhaps not surprising.

Overexpression of the MsuEDC proteins and characterization of enzyme activities. To examine the enzyme activities encoded by the msuEDC genes, the entire operon was inserted into the expression vector pET-24b, placing the msu genes under the control of a T7 promoter. After overexpression of the operon, two strongly overexpressed proteins corresponding to MsuE and MsuD were observed, together with a weakly expressed band corresponding to MsuC (Fig. 3). The weak expression of msuC observed with plasmid pME4503 (Fig. 3, lane 2) was probably due to the GTG start codon of msuC being poorly recognized in E. coli, since expression of msuC alone under T7 control gave high levels of the corresponding protein (Fig. 3, lane 3).

View larger version (73K): [in this window] [in a new window]   FIG. 3.   Overexpression of the Msu proteins. The Msu proteins were overexpressed in E. coli BL21(DE3) containing various pET24b-derived constructs, as described in Materials and Methods. Total cell extracts were separated by SDS-polyacrylamide gel electrophoresis (12% gel), and the proteins were visualized with Coomassie blue. The positions of the MsuE, MsuD, and MsuC proteins are indicated. Lanes: M, molecular weight marker; 1, BL21(pET24b); 2, BL21(pME4503); 3, BL21(pME4529). Approximately 10 µg of protein was loaded per lane.

Transcriptional regulation of msuEDC. The msuD::xylE transcriptional fusion in strain SLF3 was used to investigate the pattern of expression of the msuD gene during growth with several different sulfur sources (Table 5). Catechol oxygenase (XylE) specific activity was measured in whole cells harvested in the mid-exponential growth phase and compared with the specific activity of the well-characterized, sulfate-regulated enzyme arylsulfatase (3). The specific activities of both enzymes were much higher during growth with organosulfur sources such as sulfonates or methionine than with sulfate as the sole sulfur source. After growth with a mixture of pentanesulfonate and sulfate, enzyme activity was low, confirming that regulation was mediated by repression by sulfate or a related metabolite (see below). This corresponds well with the qualitative behavior previously reported for protein PA13 on two-dimensional electropherograms (17). In the presence of cysteine, expression of msuD and atsA was not completely repressed, perhaps due to partial oxidation to cystine, which acts as a derepressing growth substrate in this species (17). When the xylE-Gm^r cassette was inserted in the reverse orientation (strain SLF2), no catechol oxygenase activity was observed (data not shown).

Effect of cys mutations. Several genetic systems involved in sulfur metabolism are regulated by metabolites involved in the cysteine biosynthetic pathway. Thus, cysteine biosynthesis in E. coli is positively regulated by O-acetylserine and is negatively controlled by cysteine, sulfide, and thiosulfate (27). In P. aeruginosa, repression of arylsulfatase synthesis under sulfur-replete conditions has been shown to be under the control of at least two effectors, probably sulfite and sulfide (17). To investigate the dependence of msuD expression on cysteine biosynthetic intermediates, the msuD::xylE allele was transduced into cysI and cysN genetic backgrounds (strains AX18 and AC309, respectively) to give strains SLF4 and SLF5. The cells were grown under derepressing conditions (methionine or pentanesulfonate [100 µM] as a sulfur source), and the negative effect of sulfate, sulfite, or sulfide (500 µM) on arylsulfatase and catechol oxygenase synthesis was tested (Table 6). msuD expression was negatively regulated in response to all three sulfur-containing compounds tested, in both the cysI and cysN backgrounds, in contrast to the case for arylsulfatase, expression of which was not directly affected by sulfate. It therefore appears that sulfate, sulfite, and sulfide or cysteine play independent roles in the negative regulation of the msu genes and that multiple negative regulatory inputs are involved.

Distribution of the msu genes. Analysis of the gene sequence of msuD revealed that it was closely related to the ssuD genes of B. subtilis and E. coli. A closely related gene is also present in Pseudomonas putida (accession no. AF075709), and the desulfonation function that these genes encode therefore seems to be widespread. To test this, a pair of oligonucleotides was designed from the regions of the msuD sequence which were most similar to the homologous sequences from E. coli and B. subtilis, and these primers were used to carry out PCR with chromosomal DNAs from a variety of bacterial species (Fig. 4). A band corresponding to the msuD gene was observed in a range of species, including enteric bacteria, a variety of pseudomonads, and a Bacillus species. Indeed, in a number of species, two bands were observed, consistent with the observation above that the msuD gene maps to two positions on the P. aeruginosa genome (in Fig. 4 the single band obtained with P. aeruginosa was shown to be two superimposed bands by digestion of the PCR product with NcoI or PvuI, both of which cut within the msuD gene). The specificity of the PCR was confirmed by Southern hybridization, which demonstrated that all of the bands shown hybridized with the msuD gene (not shown). No PCR signal was observed for anaerobic bacteria such as Clostridium or Methanobacterium or for rhizobial species such as Bradyrhizobium or Rhizobium. This is not surprising, since the gene products encode an oxygenative sulfonatase and these species live in environments containing low or no oxygen. However, two of the species tested (Salmonella typhimurium and Stenotrophomonas maltophilia) cannot grow with alkanesulfonates as a sulfur source (Fig. 4), although PCR analysis showed a band corresponding to the msuD gene. In S. typhimurium the gene may have become cryptic during evolution, whereas S. maltophilia is by nature a cysteine/methionine auxotroph.

View larger version (60K): [in this window] [in a new window]   FIG. 4.   Distribution of the msuD gene. PCR was done with genomic DNA of the indicated species, using primers orfM500 and orfM970rev, and the PCR products were separated on a 2% agarose gel. Growth of the various species in succinate-minimal medium with taurine or methane- or pentanesulfonate (Me/Pn sulfonate) is indicated. a, S. maltophilia will grow only with cysteine or methionine as a sulfur source.

	DISCUSSION

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Alkanesulfonates are ubiquitous in nature, but very few enzyme systems for their metabolism have yet been characterized. In this report, we have shown that the msuD and msuE genes encode an FMNH₂-dependent sulfonatase with specificity for methanesulfonate and the NADH-dependent FMN reductase required to supply reduced FMN to the sulfonatase, respectively. A third gene, msuC, is involved in the desulfonation reaction but is not an essential component. Methanesulfonate is a natural oxidation product of dimethyl sulfide; it is the main biogenic organic sulfur compound in the atmosphere and is present in significant quantities in rainwater. The regulatory pattern exhibited by the msu genes (Tables 4 and 5) indicates that they have evolved to allow bacteria to use this compound as a source of sulfur under conditions where more favored sulfur sources (sulfate, sulfide, or cysteine) are not available.

The expected product of methanesulfonate desulfonation is formaldehyde (20) (cf. previous work with other alkanesulfonates [44] and with taurine [10]). Although this compound was not observed in the present study, it may have been metabolized too quickly for determination, since control experiments showed that formaldehyde disappeared rapidly when added to the E. coli cell extracts used. In vivo, P. aeruginosa does not release an aldehyde as the desulfonation product of alkanesulfonates but rather releases its oxidation product, the corresponding carboxylic acid (21). It therefore seemed possible that MsuC might be involved in oxidation of formaldehyde produced in the desulfonation reaction. This reaction could involve either a direct dehydrogenation or an FMNH₂-dependent oxygenation reaction (MsuC shows a high level of similarity to DszC, which catalyzes the FMNH₂-dependent monooxygenolytic oxidation of DBT). However, overexpressed MsuC protein did not show formaldehyde dehydrogenase activity, nor was any formaldehyde oxidation observed when reduced FMN was provided by addition of overexpressed MsuE protein. The role of MsuC is therefore still unclear and is being further investigated in our laboratory.

The biochemistry of methanesulfonate metabolism has previously been studied with a methylotrophic soil isolate, Methylosulfonomonas methylovora M2 (16, 20), and with two marine methylotrophs, strain TR3 and Marinosulfonomonas methylotropha PSCH4 (16, 43). Those studies differed from the present one in that the strains investigated were isolated by enrichment for their ability to utilize methanesulfonate as a source of carbon and energy, whereas the P. aeruginosa strain used in this study is a standard laboratory strain and can utilize methanesulfonate only as a sulfur source. The key enzyme responsible for methanesulfonate metabolism in methylotrophs is an NADH-dependent monooxygenase (MSAMO) which requires FAD and Fe²⁺ and cleaves the substrate into formaldehyde and sulfite. The MSAMO enzyme system consists of three components, one of which has been characterized as a [2Fe-2S] ferredoxin (13). No further information is yet available on the remaining components, and a molecular-level comparison with the Msu system is therefore not yet possible. The reconstituted MSAMO enzyme is highly specific for short-chain (C₁ to C₃) sulfonates as substrates and therefore displays a specificity similar to that of the MsuED system (Table 4). The narrow substrate specificity of MsuED contrasts with the broad substrate tolerance that has been reported for other enzymes that are involved in the sulfur cycle, e.g., the desulfurization of aromatic sulfonates as sulfur source by P. putida S-313 (23, 53) or the arylsulfatase of P. aeruginosa (3, 9). In this sense the MsuED methanesulfonic acid desulfurization system appears to be unusual, and it could be argued that the msu genes may have been recruited from the carbon cycle to exploit methanesulfonic acid as a sulfur source. However, given the similarity between the msu genes and the more widely distributed ssu genes (see below), this seems unlikely. Indeed, since E. coli K-12 possesses the ssu genes yet does not grow with methanesulfonate (our unpublished results), it is more probable that msuD represents a mutant form of ssuD which has adapted its substrate specificity to allow utilization of the common, sulfur-containing methanesulfonate.

Sulfonate utilization seems to be a more important part of sulfur metabolism for bacteria than has previously been recognized. Homologues of the msu genes are present in the majority of strains selected for testing from our strain collection (Fig. 4), and several of these bacteria even contain several different desulfonation systems, with partially redundant substrate specificities. In E. coli, desulfonation of alkanesulfonates is catalyzed by both TauD and SsuD (10), and a desulfonation-negative phenotype was seen only in a double mutant (unpublished data). In P. aeruginosa, our mapping studies revealed the presence of an msuD homologue, and this has been confirmed by preliminary data from the Pseudomonas genome sequencing project, which has demonstrated the presence in this species of an ssuD gene very similar to those of B. subtilis and E. coli (Fig. 2). A comparison of the SsuD and MsuD proteins with other FMNH₂-dependent monooxygenases (Fig. 2) shows a conserved domain (residues 89 to 135 of MsuD) which may form part of the active site of the enzyme. However, this domain does not contain any known protein motifs (as determined with PROSITE), and further clarification of the enzyme mechanism will require detailed biochemical characterization of these proteins.

In enteric bacteria, sulfate assimilation is controlled by the CysB protein, a LysR-type transcriptional activator which is required for expression of the genes of the cys regulon. Recent work has shown that the cys regulon includes not only the genes for sulfate activation and reduction but also those for uptake and desulfonation of taurine (tauABCD) (49). Expression of the latter genes, however, is dependent not only on CysB but also on the Cbl protein, a second regulator of the LysR family that is closely related to CysB (18, 48). The cysB gene has recently also been identified in P. aeruginosa, and cysB mutants of this species were found to be auxotrophic for cysteine, as expected. Our results show that CysB is also required for utilization of several organosulfur sources, including sulfonates and sulfate esters, and a cysB mutant of P. aeruginosa (strain PAO-CB) could not grow with sulfate but retained the ability to grow with sulfite or sulfide as a sulfur source. This suggests that the genes encoding sulfite reductase and O-succinylhomoserine sulfhydrylase are not part of the cys regulon in this species. Growth with methionine as a sulfur source (via the reverse transsulfuration pathway [12]) was not affected by the cysB mutation, and it is likely that this pathway is separately regulated. CysB is also important in regulating alginate biosynthesis in this species (6), since it acts as an activator in expression of the algD gene, but the physiological significance of this is not yet fully understood. Sequencing of the P. aeruginosa genome is still in progress, but from preliminary analysis there appears to be only one copy of cysB, and there is no evidence for an additional cbl gene.

Recent work has shown that alkanesulfonates not only can be desulfonated by aerobic microorganisms but also can be utilized by a variety of anaerobic bacteria, either as a source of sulfur (5, 7) or to satisfy energy requirements for growth as electron donors or electron sinks (8, 28, 32). The presence of duplicate sulfonate-assimilatory pathways in aerobic bacteria, and of a variety of desulfonation pathways in anaerobes, serves to underline the importance of natural sulfonates in the bacterial life cycle.