JOURNAL OF BACTERIOLOGY, Sept. 1999, p. 5833–5837 0021-9193/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 181, No. 18
Pathways of Assimilative Sulfur Metabolism in Pseudomonas putida PAUL VERMEIJ
AND
MICHAEL A. KERTESZ*
Institute of Microbiology, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zu ¨rich, Switzerland Received 17 May 1999/Accepted 12 July 1999
Cysteine and methionine biosynthesis was studied in Pseudomonas 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 ␥-synthase and cystathionine -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 ␥-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 ␥-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. evisiae and P. aeruginosa use homocysteine both as the direct precursor for methionine biosynthesis and for the conversion to cysteine via the reverse transsulfuration pathway. They also contain the normal transsulfuration pathway, allowing them to grow equally well on either cysteine or methionine as the sole sulfur source. Recently, it was demonstrated that Pseudomonas syringae synthesizes methionine via the transsulfuration pathway (1), although the acylated homoserine intermediate appears to be O-acetyl-L-homoserine (1) rather then O-succinyl-L-homoserine, as is the case in E. coli (15) and P. aeruginosa (8). This pathway followed by P. syringae most closely resembles the pathway in Neurospora crassa (16) and is distinct from the one in P. aeruginosa. In this paper, we report that Pseudomonas putida synthesizes both cysteine and homocysteine primarily by direct sulfhydrylation and that, although the organism grows well with methionine as the sulfur source, it does not contain the reverse transsulfuration pathway.
In most microorganisms, the major route for the biosynthesis of cysteine is the sulfate assimilation pathway. This process has been best characterized for enteric bacteria (15) where it involves uptake and activation of inorganic sulfate followed by stepwise reduction to sulfide. The sulfide is then condensed with O-acetyl-L-serine, catalyzed by O-acetyl-L-serine sulfhydrylase (CysK/CysM), to yield cysteine (15). In enteric bacteria, this process is controlled by the CysB protein, a LysR-type transcriptional activator, which is required for the expression of the corresponding genes, grouped together as the cys regulon (15). Recently, it has been demonstrated that the genes involved in the uptake and utilization of taurine (2-aminoethanesulfonate) and other alkanesulfonates in Escherichia coli and of methanesulfonate and aromatic sulfate esters in Pseudomonas aeruginosa are also members of this regulon (14, 23, 24). In enteric bacteria, methionine biosynthesis is not a part of the cys regulon, although this pathway, the transsulfuration pathway, begins with cysteine. In this pathway, cysteine displaces the succinyl moiety of O-succinyl-L-homoserine to yield cystathionine, catalyzed by cystathionine ␥-synthase (MetB). This is then followed by a straightforward -elimination that converts cystathionine to homocysteine, pyruvate, and ammonia, catalyzed by cystathionine -lyase. Homocysteine is subsequently methylated to methionine by either metE or metH gene products. By contrast, yeasts, Rhizobium spp., Leptospira spp., P. aeruginosa, and all gram-positive bacteria examined (Bacillus, Brevibacterium, Corynebacterium, and Arthrobacter spp.) use a direct sulfhydrylation for methionine biosynthesis involving the immediate transfer of sulfide onto an O-acyl-Lhomoserine to yield homocysteine, catalyzed by homocysteine synthase (MET25/MetZ) (1, 2, 8, 20, 21). Saccharomyces cer-
MATERIALS AND METHODS P. putida S-313 (29) and P. aeruginosa PAO1 (11) were cultivated in a sulfurfree succinate-salts medium, as previously described (13), at 30 and 37°C, respectively. For the cultivation of P. aeruginosa, all the naturally occurring amino acids (40 g 䡠 ml⫺1), except methionine and cysteine, were added to the medium. E. coli MC4100 (3) was grown in a sulfur-free M63 medium (25) at 37°C. Sulfur sources were added as described in the text (250 M). Growth curves were determined in microtiter plates with 150 l of culture by using a SPECTRAmax Plus microtiter plate reader with SOFTmax PRO software (Molecular Devices). Overnight cultures were washed and diluted 100-fold in sulfur-free succinatesalts medium and pipetted (75 l) into the wells of a microtiter plate. Subsequently, 75 l of minimal medium containing a 500 M concentration of the appropriate sulfur source was added to each well. The plate was prewarmed at 30°C for 20 min and placed in the SPECTRAmax Plus. The optical density at 600 nm was measured every 5 min. Before every measurement, the plate was shaken for a period of 30 s to ensure aerobic growth conditions. Cell extracts for enzyme assays were prepared from cells grown in 5-liter Erlenmeyer flasks containing 500 ml of medium, harvested in the mid-exponential growth phase as described by Hummerjohann et al. (12), and stored at ⫺20°C in the presence of 20% glycerol until further use. Cystathionine -lyase was measured by the method of Uren (22) and was always tested immediately, since the enzyme activity was lost upon freezing. Cystathionine ␥-lyase was assayed as 2-oxobutanoic acid formation
* Corresponding author. Mailing address: Mikrobiologisches Institut, ETH-Zentrum/LFV, CH-8092 Zu ¨rich, Switzerland. Phone: 41 1 632 33 57. Fax: 41 1 632 11 48. E-mail:
[email protected]. 5833
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TABLE 1. O-Acetyl-L-serine sulfhydrylase and O-succinyl-L-homoserine sulfhydrylase activities in cell extracts of P. aeruginosa PAO1, E. coli MC4100, and P. putida S-313 during growth with various sulfur sourcesa Sp act (nmol/min/mg of protein) of the following enzyme in the indicated cell extract O-Acetyl-L-serine sulfhydrylase (no. 1)
Sulfur source
Sulfate Cysteine Methionine Taurine Pentanesulfonate Toluenesulfonate
O-Succinyl-L-homoserine sulfhydrylase (no. 5)
MC4100
S-313
PAO1
MC4100
S-313
PAO1
1,390 160 7,320 3,470 1,430
73.6 65.1 70.0 53.9 75.4 62.5
66.7 50.1 53.2 42.7 64.3
0.0 0.0 0.0 0.0 0.0
31.6 65.8 56.0 72.6 69.2 91.5
33.7 89.4 77.1 76.6 78.0
a Enzyme activities were determined as described in Materials and Methods and represent three independent experiments. Numbers in parentheses after the enzyme names correspond with the enzyme numbers in Fig. 1 and 3.
carries homologues of the E. coli cysK (64% amino acid identity) and cysM (66% amino acid identity) genes on its chromosome, encoding O-acetyl-L-serine sulfhydrylases A and B, respectively. O-Succinyl-L-homoserine sulfhydrylase activity was also found at similar levels in both pseudomonads but not in E. coli, as expected. The direct sulfhydrylation to homocysteine has been previously characterized in P. aeruginosa (8), and the homocysteine synthase activity was found to be encoded by the metZ gene. A mutation in this gene could be complemented with the E. coli metB gene (8), encoding cystathionine ␥-synthase, an enzyme which normally uses O-succinyl-L-homoserine and cysteine for cystathionine synthesis. This suggests the presence of a cysteine pool independent of the reverse transsulfuration pathway in P. aeruginosa, which is consistent with our finding that this species can synthesize cysteine directly from sulfide and O-acetylserine and not only via homocysteine. To test the importance of the transsulfuration pathway for P. putida and P. aeruginosa, we tested the cell extracts for cystathionine -lyase activity (Table 2), which cleaves cystathionine into homocysteine and serine, and for cystathionine ␥-synthase activity (data not shown). The activities of these enzymes in P. putida and P. aeruginosa were low compared to those observed in E. coli. In the pseudomonads, they were up to 10-fold higher during growth with cysteine, whereas in E. coli, the enzyme activity was always high, except when methionine was used as the sulfur source. Earlier work on the metZ gene had already shown that the transsulfuration pathway in P. aeruginosa is probably not very active (8), as is the case in yeast. Together, these results suggest that P. putida S-313 and P. aeruginosa PAO1 utilize the sulfhydrylation of O-acetyl-Lserine for the synthesis of cysteine and the direct sulfhydryla-
from L-homoserine or as cysteine formation from cystathionine (17). O-AcetylL-serine sulfhydrylase (cysteine synthase) activity was assayed according to Nagasawa and Yamada (18). The amount of cysteine formed was determined by using the ninhydrin reaction (9). O-Succinyl-L-homoserine sulfhydrylase (homocysteine synthase) activity was tested by the same method described for cysteine synthase with O-succinyl-L-homoserine as the substrate, with the exception that the homocysteine formed was quantified by using the nitroprusside reaction (28). Cystathionine ␥-synthase was assayed in the same reaction mixture as that used for cysteine synthase with 1 mM (final concentration) L-cysteine. The disappearance of L-cysteine was measured by using the ninhydrin reaction (9). Methionine ␥-lyase was measured by the method of Esaki and Soda (7). Taurine desulfonation was followed by assaying the 2-oxoglutarate-dependent dioxygenase as previously described (6), with the exception that 5,5⬘-dithio-bis-(2-nitrobenzoate) (DTNB; 50 M) was added to the assay mixture and sulfite release was measured continuously at 412 nm. The two-component alkanesulfonate mono-oxygenase activity (SsuED/MsuED) was tested routinely with DTNB as previously described (5). The alkanesulfonatase assays were incubated on an Eppendorf Thermomixer (30°C; 450 rpm). Total protein in cell extracts was determined with the Bio-Rad protein reagent, using bovine serum albumin as the standard.
RESULTS AND DISCUSSION Direct sulfhydrylation reactions for cysteine and homocysteine biosynthesis in P. putida and P. aeruginosa. The ability of P. aeruginosa PAO1 and P. putida S-313 to transfer sulfide directly onto acylserine or acylhomoserine acceptors was tested in extracts of cells cultivated with a variety of different sulfur sources. Both organisms exhibited significant cysteine synthase activity (Table 1), a feature which had previously been thought to be absent in P. aeruginosa (8). Although the cysteine synthase activities were much lower (20- to 50-fold) than those measured for E. coli, they were high enough to lead to sufficient cysteine production to support the growth rates observed (approximately 60 nmol/min/mg of protein). Here, it is perhaps interesting to note that analysis of the data emerging from the Pseudomonas Genome Project (19) revealed that P. aeruginosa
TABLE 2. Cystathionine -lyase, cystathionine ␥-lyase, and methionine ␥-lyase activities in cell extracts of P. aeruginosa PAO1, E. coli MC4100, and P. putida S-313 during growth with various sulfur sourcesa Sp act (nmol/min/mg of protein) of the following enzyme in the indicated cell extract Sulfur source
Sulfate Cysteine Methionine Taurine Pentanesulfonate Toluenesulfonate
Cystathionine -lyase (no. 3)
Cystathionine ␥-lyase (no. 8)
Methionine ␥-lyase (no. 9)
MC4100
S-313
PAO1
MC4100
S-313
PAO1
MC4100
S-313
PAO1
38.07 33.92 5.24 28.23 38.73
0.55 2.13 0.48 0.45 1.87 1.36
0.41 4.08 0.04 0.30 1.14
0.36 0.37 0.34 0.36 0.30
0.29 0.13 0.07 0.00 0.10 0.06
2.21 3.07 5.38 3.15 3.11
0.23 0.05 1.79 0.06 0.08
0.10 0.04 0.44 0.05 0.17 0.15
0.08 0.76 0.97 0.08 0.17
a Enzyme activities were determined as described in Materials and Methods and represent three independent experiments. Numbers in parentheses after the enzyme names correspond with enzyme numbers in Fig. 1 and 3.
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FIG. 1. Pathways of cysteine and methionine biosynthesis in P. aeruginosa and E. coli. Enzymes: 1, O-acetyl-L-serine sulfhydrylase; 2, cystathionine ␥-synthase; 3, cystathionine -lyase; 4, methionine synthase; 5, O-succinyl-L-homoserine sulfhydrylase; 6, S-adenosylmethionine synthase–methyltransferases–S-adenosylhomocysteine hydrolase pathway; 7, cystathionine -synthase; 8, cystathionine ␥-lyase. The dotted arrow indicates the putative utilization of methionine by E. coli via inorganic sulfate.
tion pathway for the synthesis of methionine. Both pathways are expressed simultaneously. Under most growth conditions, cysteine is not converted to methionine via cystathionine and homocysteine. However, when cysteine is supplied as the sole sulfur source, cystathionine -lyase and cystathionine ␥-synthase are expressed, allowing a direct cysteine-to-methionine conversion to occur. Although direct sulfhydrylation has now been found in a growing number of microorganisms (2, 4, 8, 20), the reverse transsulfuration pathway (homocysteine to cysteine) is still found only in P. aeruginosa and S. cerevisiae. We were therefore interested in how P. putida S-313 converts methionine to cysteine during growth with the former as the sole sulfur source. Methionine-to-cysteine conversion in P. putida. When methionine is provided as the sole sulfur source for bacterial growth, two main metabolic pathways are known that allow conversion of this compound to cysteine. The methionine may be desulfurized to yield an inorganic sulfur moiety which enters
the cysteine biosynthetic pathway via the normal sulfate assimilation route. Alternatively, the sulfur may be retained on the carbon skeleton and the methionine converted to cysteine via demethylation to homocysteine and subsequent reverse transsulfuration via cystathionine (Fig. 1). Enteric bacteria, such as E. coli, which use the former pathway, grow poorly with methionine as the sulfur source. In the presence of selenate, growth with methionine is completely halted. This has been taken as evidence that methionine utilization in E. coli proceeds by desulfurization of the methionine molecule to yield inorganic sulfate, since selenate is a known inhibitor of sulfate uptake and ATP sulfurylase in bacteria and therefore acts as an inhibitor of the sulfate assimilation pathway. In P. aeruginosa, by contrast, growth is rapid with methionine and is not inhibited by selenate (10), supporting the presence of a direct transsulfuration pathway. Like P. aeruginosa, P. putida grows equally well with methionine as with other sulfur sources (Fig. 2). Growth with me-
FIG. 2. Growth of P. putida S-313 on various sulfur sources in the absence or presence of 1 mM selenate. Growth was measured in microtiter plates in a SPECTRAmax instrument, as described in Materials and Methods. O.D.600, optical density at 600 nm.
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TABLE 3. Alkanesulfonate desulfonation activities and inhibition of alkanesulfonatase activity by selenate in cell extracts of P. aeruginosa PAO1, E. coli MC4100, and P. putida S-313 during growth with various sulfur sourcesa Sp act (pmol/min/mg of protein) of the following enzyme in the indicated cell extract in the presence or absence of selenate Sulfur source
Sulfate Cysteine Methionine Pentanesulfonate
Methanesulfonate sulfonatase (no. 10) MC4100
S-313
Pentanesulfonate sulfonatase (no. 10)
PAO1
MC4100
S-313
PAO1
⫺Se
⫺Se
⫹Se
⫺Se
⫹Se
⫺Se
⫹Se
⫺Se
⫹Se
⫺Se
⫹Se
0.0 0.0 0.0 0.0
5.3 4.3 83.0 22.6
0.0 0.0 35.9 10.8
0.0 14.0 51.2 137.8
0.0 5.7 24.1 72.5
0.0 0.0 0.0 41.6
0.0 0.0 0.0 24.2
0.0 27.1 29.3 188.4
0.0 16.4 13.9 102.4
0.0 14.0 34.1 330.8
0.0 5.7 16.5 180.1
a Enzyme activities were determined as described in Materials and Methods and represent six independent experiments. Numbers in parentheses after the enzyme names correspond with enzyme numbers in Fig. 3. ⫹Se, enzyme activity measured in the presence of 1 mM selenate; ⫺Se, enzyme activity measured in the absence of selenate.
thionine was slowed but not halted in the presence of 1 mM selenate, a concentration which stopped growth with sulfate completely (Fig. 2). We therefore initially postulated that P. putida also contains the reverse transsulfuration pathway, allowing a direct methionine-to-cysteine conversion. To confirm the presence of the reverse transsulfuration pathway in P. putida, we measured the key enzyme of this pathway, cystathionine ␥-lyase (Table 2). High levels of this enzyme were found in P. aeruginosa during growth with all sulfur sources tested, but no activity was found in cell extracts of P. putida or E. coli. This ruled out the possibility of a direct methionine-to-cysteine conversion in P. putida. However, during growth with methionine, we found that methionine lyase activity was approximately 10-fold upregulated in P. putida (Table 2), and we therefore propose that under these conditions methionine is converted into methanethiol, 2-oxobutyrate, and ammonia by the methionine ␥-lyase. Subsequently, methanethiol can then be converted, via an unknown pathway, to methanesulfonate, which is then desulfonated to yield sulfite. A mini Tn5 trans-
poson mutant of P. putida S-313, SN34, which was isolated for its inability to grow with sulfonates as the sulfur source (27), also grew very slowly with methionine, confirming an interconnection between alkanesulfonate and methionine utilization in this species. Complementation of this mutant with the P. putida ssuED genes, which encode a reduced flavin mononucleotidedependent alkanesulfonatase (26), restored growth with both methanesulfonate and methionine to wild-type levels. P. aeruginosa contains a second sulfonatase operon (msuEDC) which is involved in methanesulfonate desulfonation (14), and though it is not yet clear whether P. putida also contains this second operon, our results suggest that in P. putida methanesulfonate desulfonation is catalyzed primarily by the ssuED gene products. Conversion of methionine to cysteine via methanesulfonate and sulfite is not expected to be inhibited by selenate, since this compound affects only the upper pathway of sulfate assimilation. Unexpectedly, growth with methanesulfonate was completely halted in the presence of 1 mM selenate (Fig. 2), and so
FIG. 3. Proposed pathways for the biosynthesis of cysteine and methionine in P. putida S-313. The conversion of methanethiol to methanesulfonate is still hypothetical. Dotted arrows indicate that the transsulfuration pathway is not very active in P. putida S-313. Enzymes: 1, O-acetyl-L-serine sulfhydrylase; 2, cystathionine ␥-synthase; 3, cystathionine -lyase; 4, methionine synthase; 5, O-succinyl-L-homoserine sulfhydrylase; 9, methionine ␥-lyase; 10, methanesulfonatase (SsuED/MsuED). APS, adenosine-5⬘-phosphosulfate; PAPS, 3⬘-phosphoadenosine-5⬘-phosphosulfate.
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the inhibitory effect of selenate on the broad-substrate-range alkanesulfonate sulfonatase SsuED was tested in cell extracts with pentanesulfonate or methanesulfonate as the substrate (Table 3). Selenate led to approximately 50% inhibition of this enzyme in extracts of P. putida and P. aeruginosa. In E. coli, the same effect was seen for pentanesulfonate cleavage (methanesulfonate is not a substrate of the E. coli enzyme), and this was confirmed with the purified E. coli SsuED enzyme. This level of inhibition is consistent with the reduction in the P. putida growth rate with methionine that was found when selenate was added (Fig. 2). The observed complete inhibition by selenate of the growth of P. putida with methanesulfonate (Fig. 2) is therefore probably due to inhibition of methanesulfonate uptake. As a comparison, we also tested 2-oxoglutarate-dependent taurine dioxygenase (TauD) with and without 1 mM selenate. All three strains used in this study exhibited low TauD activity when grown with sulfate and cysteine and high TauD activity when grown with other sulfur sources, as previously reported (24), and the activity was not affected by selenate at all (data not shown). In this study, we have demonstrated that P. putida and P. aeruginosa are capable of methionine biosynthesis through either transsulfuration or direct sulfhydrylation, although the direct sulfhydrylation pathway is strongly favored. In both organisms, the transsulfuration pathway was found to be poorly active and cysteine is normally not converted to methionine, except when the organism is grown with cysteine as the sole sulfur source. The pathways found for the synthesis of methionine and cysteine in P. putida S-313 (Fig. 3) resemble those of P. aeruginosa. However, compared to the latter organism, P. putida lacks the reverse transsulfuration pathway, raising the question of whether P. aeruginosa has acquired this pathway during evolution or P. putida has lost it. ACKNOWLEDGMENTS This work was supported by the Swiss Federal Office for Education and Sciences (grant no. BBW 97.0190) as part of the EC program SUITE (ENV4-CT98-0723). The purified SsuD and SsuE proteins from E. coli were a kind gift from E. Eichhorn. REFERENCES 1. Andersen, G. L., G. A. Beattie, and S. E. Lindow. 1998. Molecular characterization and sequence of a methionine biosynthetic locus from Pseudomonas syringae. J. Bacteriol. 180:4497–4507. 2. Belfaiza, J., A. Martel, D. Margarita, and I. Saint Girons. 1998. Direct sulfhydrylation for methionine biosynthesis in Leptospira meyeri. J. Bacteriol. 180:250–255. 3. Casadaban, M. J. 1976. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J. Mol. Biol. 104:541–555. 4. Cherest, H., and Y. Surdin-Kerjan. 1992. Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway. Genetics 130:51–58. 5. Eichhorn, E., J. R. van der Ploeg, and T. Leisinger. Characterization of a two component alkanesulfonate monooxygenase from Escherichia coli. J. Biol. Chem., in press.
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