INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The enteric bacteria Escherichia coli and Salmonella enterica serve as model systems for the physiology and regulation of numerous metabolic processes in bacteria, including sulfur assimilation. Sulfur is a constituent of several indispensable biomolecules, including cysteine, methionine, thiamine, biotin, lipoic acid, and coenzyme A; in these contexts, sulfur is used in its fully reduced state (S²). In many environments, sulfur is found primarily in the oxidized state of sulfate (SO₄²) and must be reduced to sulfide before assimilation into organic material. While the reduction of sulfate to sulfide follows a common pathway in all organisms studied to date, the assimilation of sulfide itself into an organic molecule may take one of two routes. In most bacteria, including enteric bacteria, sulfide is incorporated into activated forms of serine to form cysteine (18); cysteine then serves as the sulfur group donor, either directly or indirectly, for the synthesis of all other sulfur-bearing molecules in the cell (Fig. 1A). Alternatively, yeast and some bacteria assimilate sulfide primarily into activated homoserine to form homocysteine, an intermediate in methionine biosynthesis (30); homocysteine is used for biosynthesis of cysteine, methionine, and other sulfur-bearing molecules (Fig. 1B). Some organisms, such as Pseudomonas putida, can assimilate sulfide into either compound (31).

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Pathways for sulfur assimilation. (A) Sulfur assimilation in enteric bacteria E. coli and S. enterica. Genes whose products contribute to each step are noted. (B) Sulfur assimilation in yeast Saccharomyces cerevisiae. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine.

In E. coli and S. enterica, auxotrophs corrected by the addition of sulfur-bearing amino acids fall neatly into two classes (Fig. 1A). Mutants defective in sulfate reduction or sulfide assimilation are corrected by the addition of cysteine to the growth medium; by definition the associated mutations affect cys genes and their defects are not corrected by the addition of methionine (18). Mutants defective in the synthesis of methionine (affecting met genes) are corrected by methionine but not by cysteine (9). One unusual class of mutants with leaky point mutations (termed cym) which are corrected by the addition of either cysteine or methionine has been reported in S. enterica (14, 15, 29); these mutations mapped to various cysteine biosynthetic genes. Their leaky behavior sustained very slow growth rates when the cell was spared the need to use cysteine to synthesize methionine (which comprises 40% of the reduced sulfur requirement for the cell), which then provided just enough cysteine to satisfy other requirements.

The metabolism of E. coli and S. enterica are often used as paradigms in unraveling the physiology of diverse and unrelated organisms, wherein detection of enzyme homologues in these species implies their implementation in specific metabolic pathways. The validity of these inferences relies heavily on a satisfactory understanding of these processes within E. coli. The enteric bacterium Klebsiella aerogenes is related to E. coli and S. enterica and therefore serves as an outside reference taxon for understanding the evolution of the metabolic capabilities in these organisms (2). As reported below, Klebsiella expresses at least two pathways that allow the sulfur atom of methionine to be recycled back into cysteine. Although present in many enteric bacteria, this capability appears to have been lost from the E. coli/Salmonella lineage. Hence, the cysteine and methionine biosynthetic pathways of Klebsiella are far more intertwined than suspected, being only two portions of a complex series of metabolic cycles. Initial genetic and biochemical characterization of these pathways is presented, and their implication in the evolution of enteric bacteria is discussed.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains and plasmids. Strains used in these studies (Table 1) were constructed from K. aerogenes W70 derivative KC2668 (hsdR suc⁺ hutC515(Con) dadA lac bla-2), kindly provided by R. Bender. Plasmid pTAS1 bears the lamB region from E. coli and renders Klebsiella sensitive to bacteriophage . Plasmid pTAS1 was constructed from pTROY11 (6) by cleavage with HindIII and BamHI, treatment with T4 DNA polymerase to create blunt ends, and ligation with T4 DNA ligase according to the manufacturer's instructions; this treatment removed a portion of the tetracycline resistance gene and eliminated the appearance of spontaneous tetracycline-resistant colonies. Plasmid pMMK1 (17) is pTAS1 with the gene encoding the altered-target specificity (ATS) Tn10 transposase from plasmid pNK2881 (16) inserted into the EcoRI site.

Media and antibiotics. The rich medium used was Luria-Bertani (LB) medium; minimal defined medium was E (32). Sulfur-free E medium (NSE) was created by substituting MgCl₂ for MgSO₄; glucose was used as a carbon source. Solid medium was made by the addition of Bacto agar (Difco) to 1.2%, agarose (Gibco-BRL) to 1.3%, Gelrite (Schweizer Hall) to 1.3%, or Phytagel (Sigma) to 1.3%. P1 buffer contained 5 mM CaCl₂ and 10 mM MgSO₄. Ampicillin was used at 200 µg/ml; kanamycin was used at 20 µg/ml; tetracycline was used at 10 µg/ml for selection of transductants and at 20 µg/ml otherwise; chloramphenicol was used at 50 µg/ml for selection of Tn10dCm transposition mutants and at 20 µg/ml otherwise. For growth curves, 5 ml of NSE-0.2% glucose was inoculated with 50 µl of a fresh, overnight culture that had been washed and resuspended in an equal volume of NSE.

Genetic methods. Transposition-defective derivatives of Tn10 Tn10dKn (kanamycin resistance), Tn10dTc (tetracycline resistance), and Tn10dCm (chloramphenicol resistance) were delivered into pTAS1-bearing strains of K. aerogenes using bacteriophage  delivery vectors NK1316, NK1323, and NK1324 as described by Kolko et al. (17). Transduction was mediated by bacteriophage P1 vir as described by Kolko et al. (17).

Enzyme assays. Cells were prepared by growth to mid-log phase, concentration by centrifugation, and resuspension in 1/10 volume of 100 mM K_xPO₄, pH 8.0. Cells were lysed by sonication in a 30-ml Corex tube using eight 10-s pulses on ice. Debris was removed by centrifugation, and the protein-bearing supernatant was desalted on a PD-10 gel filtration column (Pharmacia) and eluted in 10 mM K_xPO₄, pH 8.0. Enzyme extracts were used immediately in assays for cystathionine--lyase (26) or methionine--lyase (8) as described; assays were performed in triplicate. Assays for cystathionine--lyase activity used 50 mM homoserine, 8.4 mM cystathionine (all four stereoisomers), and 2.1 mM LL-cystathionine (all are final concentrations) as substrates. In all assays, -ketobutyrate was detected as a product. Protein concentration was determined by a Bradford assay (4).

Cloning and sequencing. Chromosomal DNA was prepared from Tn10dCm-bearing cells and partially digested with the Sau3AI restriction endonuclease. DNA was size fractionated on agarose gels; high-molecular-weight fragments were purified using the JetSorb kit (Promega) and ligated into pNEB193 prepared by BamHI digestion and phosphatase treatment. Ligations were introduced into XL2-Gold-Kn competent cells (Stratagene) according to the manufacturer's instructions, and transformants were selected on ampicillin-containing media. Chloramphenicol-resistant colonies were isolated by replica printing. Positive clones were identified by DNA sequencing using an ABI 310 sequencer and primers directing replication out of Tn10dCm. Homology to E. coli genes was inferred from BLAST analysis (1).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Klebsiella cym mutants are corrected by cysteine or by methionine. To isolate Klebsiella mutants defective for biosynthesis of sulfur-bearing amino acids, insertion mutagenesis was performed with transposition-defective derivatives of Tn10 (Tn10dTc, Tn10dCm, and Tn10dKn). Mutants were isolated on LB plates bearing the appropriate antibiotic and screened for auxotrophs by replica printing to minimal media and to minimal media containing both cysteine and methionine; a total of 130 independent mutants were isolated in four different mutagenic screens. Mutants were sorted into nutritional groups by determining which of the two amino acids suppressed their growth defect. As noted above, auxotrophs of E. coli and S. enterica corrected by the addition of sulfur-bearing amino acids fall into two classes: those corrected by the addition of cysteine and those corrected by the addition of methionine. In contrast, auxotrophs of K. aerogenes corrected by sulfur-bearing amino acids defined three distinct classes (Table 2). In addition to the expected cys and met gene classes, up to 40% of insertion mutations (corrected by the combination of cysteine and methionine in the original screen) were corrected by the addition of either amino acid to the growth medium (Table 2). We termed this class of mutations cym (corrected by cysteine or methionine).

Klebsiella cym mutations confer gain-of-function phenotypes. Klebsiella cym mutations are not leaky cys mutations, like those described for S. enterica (14, 15, 29). Rather, cym mutations confer two gain-of-function phenotypes that are inconsistent with leaky cys mutations. First, cys cym double mutants are corrected by methionine (Table 2). Therefore, the cym mutation is not a leaky cys mutation since it allows tight cys mutants to utilize methionine as a sulfur source; the cym mutation uncovered a metabolic activity that was masked in the cys mutant on this growth medium, i.e., growth on methionine as the sole sulfur source. Second, cym mutations confer selenate resistance (Table 2). Selenate (SeO₄²) is an analogue of sulfate that is toxic when metabolized into selenide (subsequent assimilation into selenocysteine results in tRNA^Ser mischarged with selenocysteine). In E. coli, wild-type cells are sensitive to selenate unless they are growing in the presence of excess cysteine, which prevents CysB activation (10, 11) of genes for sulfate and selenate transport (23), reduction, and assimilation. In wild-type Klebsiella, the presence of methionine does not mitigate the toxic effects of selenate (Table 2). In contrast, Klebsiella cym mutants grow on methionine as the sole sulfur source even in the presence of selenate (Table 2). As discussed below, selenate resistance likely results from the lack of selenate transport or activation in cym mutants.

Genetic mapping and identification of cys, cym, and met loci. To identify the cym loci and to infer their mode of action, mutations conferring cys, cym, and met nutritional phenotypes were sorted into 11 groups by transductional analysis and nutritional profiling (Table 3, Fig. 2). The likely identities of these groups were assigned by linkage to loci which flank the corresponding genes in E. coli and S. enterica and/or by distinctive growth and nutritional phenotypes.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Linkage groups for cys, cym, and met genes in K. aerogenes. Corresponding genes on the E. coli chromosome are noted on the inner circle. Gene identifications were made by linkage analysis and/or DNA sequence. Dotted lines denote linkage groups that have not yet been cloned from Klebsiella. Linkages were calculated on the basis of cotransduction frequencies using bacteriophage P1 vir. Tn10dCm insertions in strains LD823 and LD831 are noted between cysC and srl and between cysJ and fuc, respectively.

Klebsiella uses methionine as a sole sulfur source in liquid medium. The behavior of cym mutants on solid media suggests that methionine can be employed as a sole sulfur source by K. aerogenes. Since agar media contain trace amounts of sulfate and usable alkane sulfates, this hypothesis was tested by growing wild-type cells and cys, met, and cym mutants on a variety of sulfur sources in sulfur-free liquid media (Fig. 3). Growth of wild-type K. aerogenes when cysteine was provided as the sole source of sulfur was similar to that with methionine as the sole source; no significant growth was detected on unsupplemented NSE media, attesting to the low sulfur source content of this medium (Fig. 3A). In addition, cym mutants grew well on methionine as a sole sulfur source in liquid media (Fig. 3D and E). Surprisingly, cys mutants, corrected only by cysteine on solid medium, were readily corrected by methionine in liquid medium (Fig. 3C and F to I), although both cysH and cysB mutants showed a substantial lag before utilizing methionine that is not seen when cysteine is used as a sulfur source.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   Growth of K. aerogenes strains on sulfur sources. (A) Wild-type strain LD561; (B) LD827 (cysIJ4009::Tn10dTc); (C) LD828 (cysC4022::Tn10dCm); (D) LD822 (met-4004::Tn10dTc); (E) LD825 (cysT4071::Tn10dCm [cymX]); (F) LD830 (cysB4020::Tn10dTc); (G) LD824 (cysQ4007::Tn10dTc); (H) LD826 (cysDN4070::Tn10dTc [cymY]); (I) LD829 (cysH4005::Tn10dTc).

Methionine utilization does not require assimilation of sulfite or sulfide. It is possible that methionine is degraded and that free sulfide, or an oxidized form of sulfur, is released. Alternatively, methionine may be converted to cysteine via a cystathionine intermediate, similar to the transsulfurylation pathway of yeasts and some bacteria (Fig. 1). To test the hypothesis that methionine utilization proceeds through a sulfite intermediate, a cymY (i.e., cysDN) cysIJ double mutant was created (LD841) by P1 transduction. This mutant fails to use sulfite as a sulfur source but does use methionine, implying that sulfite is not a requisite intermediate in methionine utilization. Since mutants lacking cysteine synthase activity were not isolated (likely because Klebsiella bears two cysteine synthases homologous to the E. coli CysM and CysK enzymes), we examined the growth of a cysB mutant in liquid media (Fig. 4). Lacking the requisite positive activator, cysB mutants cannot assimilate any form of inorganic sulfur on solid media and are cysteine auxotrophs (Table 3). However, they do assimilate alternative sulfur sources in liquid media after a substantial lag period; the growth rate after the lag phase is comparable to the growth rate of wild-type cells (serial dilution and plating experiments verify that this apparent lag is not attributable to contamination of the culture or reversion of the cysB mutation).

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Growth curves of LD830 (cysB::Tn10dTc) in NSE-glucose medium. Compounds serving as sole sources of sulfur are noted. The doubling time of cells grown on cysteine is 76.7 min. Between points A and B, the doubling times of cells grown on homocysteine and methionine are 583 and 775 min, respectively. Between points A and C, the doubling times of cells grown on sulfate, sulfite, and sulfide are 7,180, 1,530, and 1,410 min, respectively. OD600, optical density at 600 nm.

Klebsiella expresses a cystathionine--lyase. The conversion of homocysteine to cysteine by transsulfurylation proceeds in fungi via an L-allo-cystathionine intermediate, followed by its subsequent cleavage to yield cysteine, -ketobutyrate, and ammonia (Fig. 1B). To test for cystathionine--lyase activity in Klebsiella, enzyme extracts were prepared from wild-type K. aerogenes grown on sulfate, cysteine, methionine, or cysteine plus methionine as the sulfur source (Fig. 5A). Cystathionine--lyase activity was detected in Klebsiella, but it was present only in cells grown with methionine as the sole sulfur source; sulfate or cysteine in the growth medium, regardless of the presence of methionine, eliminated this activity. Cystathionine--lyase activity was detected using either homoserine or cystathionine as the substrate. When assaying for cystathionine utilization, we employed a metB metC double mutant, eliminating any contribution of the associated methionine-biosynthetic enzymes, which could act on cystathionine as a substrate. Although the mixture of all four stereoisomers of cystathionine served as a substrate, the LL-cystathionine stereoisomer alone appears to be unsuitable (Fig. 5B). We attribute the residual activity seen on LL-cystathionine to contaminants in the LL-cystathionine preparation, which is reported to be 90% pure; alternatively, cystathionine--lyase could act on more than one stereoisomer of cystathionine. Therefore, we conclude that an inducible cystathionine--lyase activity allows K. aerogenes to utilize methionine as a sole sulfur source in the absence of cysteine or sulfate. We should note that although -ketobutyrate was effectively detected as a product of cystathionine cleavage, cysteine was not reproducibly detected. This result may reflect difficulties with the assay or may be due to intact cysteine not being released as a product. Although cystathionine--lyase has been reported to use the L-allo-cystathionine isomer in other organisms, from these data we can only conclude that LL-cystathionine is not the substrate. Assays of E. coli and S. enterica protein extracts failed to detect cystathionine--lyase activity, even when cells were grown (as well as they could be, see below) on methionine as the sole potential source of sulfur.

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Assays for cystathionine--lyase in K. aerogenes LD561 grown in different sulfur sources. Specific activities were calculated as increases in absorbance at 320 nm per minute per milligram of protein. Activities were calculated for substrate only, extract only, and complete reaction mixtures (for calculation of substrate-only activities, the protein concentration of the corresponding extract was used). Values are mean activities for four assays. (A) Strain LD561. (B) LD856.

Evidence for a second pathway for methionine recycling. Although the data presented above provide compelling evidence for a pathway converting homocysteine to cysteine in K. aerogenes, these data do not preclude the presence of an additional pathway for methionine recycling. Since methionine serves as a poor sulfur source under anaerobic conditions (Fig. 6), we reasoned that a second pathway, proceeding via methanethiol (CH₃SH) and methanesulfonate (CH₃SO₃) intermediates, could contribute to aerobic methionine recycling. Production of methanethiol via 3-methylthiopropionate uses the oxygen-dependent enzyme aci-reductone oxidase (CO forming) (33, 34). Methanethiol would be oxidized to methanesulfonate, and the sulfur would be recovered as sulfite, through the action of an oxygen-dependent alkane sulfonatase (5, 13), which has been described for Klebsiella (7).

View larger version (28K): [in this window] [in a new window]   FIG. 6.   Growth of wild-type K. aerogenes (LD561) on different sulfur sources under aerobic and anaerobic conditions. The residual growth observed in media without added sulfur can be attributed to the use of internal sulfur stores and the scavenging of residual sulfate. The addition of methionine extends the period of stored- and scavenged-sulfur use under anaerobic growth conditions. After this point (arrow), methionine utilization results in slower growth than that under aerobic conditions.

Klebsiella does not express a methionine--lyase. Methanethiol can be produced from methionine in two ways. First, Klebsiella is known to use an aci-reductone oxidase to produce 3-methylthiopropionate from an intermediate generated during the recycling of methylthioadenosine via methylthioribose (33, 34); methanethiol is formed upon 3-methylthiopropionate degradation. Alternatively, a methionine--lyase may produce methanethiol and -ketobutyrate directly. We assayed for methionine--lyase activity and found no significant activity in cells grown with methionine as the sole sulfur source, or under any other growth condition. We conclude that either our strains of K. aerogenes do not encode a methionine--lyase or we have failed to induce its production. Since Klebsiella is known to produce 3-methylthiopropionate from methylthioadenosine, we favor this route as the likely source of methanethiol in Klebsiella.

The ability to recycle methionine as the sulfur source was lost from the E. coli/Salmonella lineage. To determine if strains of E. coli or S. enterica could recycle methionine into cysteine, we tested both standard laboratory strains of each species (K-12 and LT2, respectively), as well as a number of isolates from representative collections of natural isolates (the ECOR [28] and SARB [3] collections, respectively), for their ability to use methionine as the sole source of sulfur; representative data are shown in Fig. 7. No strain of E. coli or S. enterica, either a laboratory strain or a natural isolate, could degrade methionine as the sole source of sulfur; the initial increase in cell density on methionine-grown cells can be attributed to the utilization of cytoplasmic stores of sulfur, which are rapidly exhausted. Long-term growth experiments show that strains of E. coli and Salmonella never reach a higher cell density, so it is unlikely that a cryptic pathway has remained uninduced, as in Klebsiella cysB mutants (Fig. 4). In addition, no cystathionine--lyase activity was detected in enzyme extracts of E. coli or Salmonella cells.

View larger version (32K): [in this window] [in a new window]   FIG. 7.   Growth of enteric bacteria on sulfur sources. (A) Klebsiella aerogenes LD561; (B) Klebsiella pneumoniae M5aL; (C) Escherichia vulneris LD126; (D) Enterobacter cloacae LD118; (E) Serratia marcescens LD137; (F) E. fergusonii LD130; (G) E. coli ECOR-1; (H) E. coli ECOR-16; (I) E. coli ECOR-47; (J) S. enterica SARB-3; (K) S. enterica SARB-9; (L) S. enterica SARB-19. The residual growth observed in media without added sulfur can be attributed to the use of internal sulfur stores and scavenging of residual sulfate. The addition of methionine extends the period of stored- and scavenged-sulfur use for strains of E. coli and S. enterica. OD600, optical density at 600 nm.

View larger version (31K): [in this window] [in a new window]   FIG. 8.   Distribution of methionine recycling among enteric bacteria. Open bars, organisms that fail to use methionine as a sulfur source; solid bars, organisms that use methionine as the sole sulfur in liquid media. Growth curves for representative strains are shown in Fig. 7. The dendrogram is based on sequences of the gapA, ompA, and trpR loci, as presented in reference 21. Despite their nomenclature, other strains of Escherichia are not necessarily closely related to E. coli (21) and were classified as Escherichia based on their ability to degrade lactose.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Assimilation of sulfur in Klebsiella is similar to the E. coli model. Nutritional analysis of Klebsiella mutants shows that sulfur is assimilated into cysteine, as in E. coli and S. enterica (green pathway in Fig. 9), and not into homocysteine, as in Saccharomyces (Fig. 1). That is, all mutants failing to reduce sulfate are corrected by sulfide and cysteine and no mutants corrected only by methionine are not corrected by sulfide. These data, as well as the preliminary correspondence between the E. coli and Klebsiella genes for sulfur assimilation, justify our use of the E. coli models for sulfate reduction, cysteine biosynthesis, and methionine biosynthesis. Yet, in E. coli and S. enterica, methionine cannot serve as the sole sulfur source, and these organisms appear to contain a single transsulfurylation pathway, allowing conversion of cysteine to homocysteine (and then to methionine; blue pathway in Fig. 9) but not of homocysteine to cysteine. Therefore, a comprehensive model for sulfur-bearing amino acid metabolism in Klebsiella requires additional metabolic pathways not found in E. coli or Salmonella.

View larger version (27K): [in this window] [in a new window]   FIG. 9.   Model for methionine recycling pathways in K. aerogenes. The formal cysteine-biosynthetic pathway is shown in green, with genes identified in Klebsiella noted. Similarly, the methionine-biosynthetic pathway is shown in blue. The inferred pathway of homocysteine recycling via the transsulfurylation pathway is shown in red, and a possible pathway for methionine recycling via methanethiol is shown in magenta; both these pathways await rigorous biochemical confirmation. Regulatory interactions inferred from studies of E. coli and S. enterica are shown in gray, and regulatory interactions inferred from this work are shown in yellow. Ellipses, regulatory proteins. Arrows with open heads direct compounds serving as effectors to their cognate proteins. Dotted lines, proteins, steps, and interactions without experimental evidence for Klebsiella. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; CoA, coenzyme A.

Evidence for the transsulfurylation pathway of methionine utilization in Klebsiella. K. aerogenes, like yeasts (30) and some Archaea (35), appears to have both transsulfurylation pathways, allowing either cysteine or methionine to serve as the sole sulfur source (red pathway in Fig. 9). Evidence for this pathway comes from four sources. (i) Klebsiella can use methionine as the sole source of sulfur, although a cym mutation is required to allow its use on solid medium. (ii) A cysIJ cym double mutant still uses methionine as the sole source of sulfur, meaning sulfite is not a requisite intermediate. (iii) A cysB mutant utilizes methionine and homocysteine as sulfur sources during a period when sulfide, sulfite, and thiosulfate cannot be used. These data suggest that sulfide assimilation is not required for methionine utilization but are not conclusive. (iv) Cystathionine--lyase activity has been detected in Klebsiella cells. This activity is induced by methionine and is repressed by cysteine.

Evidence for the methanethiol pathway. The transsulfurylation pathway is not sufficient to explain all phenotypes associated with Klebsiella's growth on methionine as the sole source of sulfur. We propose that a second pathway is employed, entailing the utilization of 3-methylthiopropionate. This compound is produced by the enzyme aci-reductone oxidase, which has been described for Klebsiella (33, 34) and which acts on an intermediate in the recycling of methylthioadenosine, a compound produced during spermidine synthesis. The utilization of 3-methylthiopropionate would proceed via methanethiol (CH₃SH) and methanesulfonate (CH₃SO₃) intermediates; methanesulfonate utilization has been reported for Klebsiella (7). Three pieces of evidence suggest, albeit only indirectly, that a second pathway allows for methionine utilization in Klebsiella. (i) Methionine utilization is partially impaired under anaerobic growth conditions (Fig. 6). While the transsulfurylation pathway has no requirement for molecular oxygen, the methanethiol pathway requires molecular oxygen both in the production of 3-methylthiopropionate and in the utilization of alkane sulfonates (magenta pathway in Fig. 9). (ii) Klebsiella mtc mutants, which have defects in methionine utilization, are uniformly leaky, suggesting that any one mutant cannot eliminate methionine recycling activity entirely. (ii) Klebsiella cysB mutants grown on methionine show two distinct growth phases (Fig. 4), consistent with immediate deployment of an ineffective pathway for methionine recycling and the late deployment of a more effective means. These data are clearly speculative, and confirmation that two pathways operate for methionine recycling in Klebsiella and the nature of these pathways await the further characterization of mtc mutants.

Regulation of methionine utilization involves the CysB protein. CysB acts as a positive activator to allow transcription of genes involved in sulfate assimilation in enteric bacteria (11, 12). This is accomplished by sensing high levels of o-acetylserine (via the isomer N-acetylserine), the form of activated serine into which sulfide is assimilated (green pathway in Fig. 9). Two lines of evidence suggest that CysB is involved, either directly or indirectly, in activating the transsulfurylation pathway of methionine utilization. First, activity of cystathionine--lyase is repressed by the presence of cysteine in the media. Cysteine inhibits the activity of the CysE serine transacetylase (19, 20), thereby decreasing the production of N-acetylserine and preventing transcription activation by CysB. Second, cysB mutants fail to use methionine as a sulfur source in liquid media until after a very long lag (Fig. 4), whereas wild-type cells use it immediately (compare Fig. 3A with 3I and 4). Since defects in sulfur reduction and assimilation (also affected by a cysB mutation) are not required by the transsulfurylation pathway (although they are required for the methanethiol pathway), these data suggest that CysB activity is required for the transsulfurylation pathway and that the repression of cystathionine--lyase activity is the result of CysB-mediated transcriptional control (yellow interactions in Fig. 9). This regulation is not unexpected if sulfate or cysteine is preferred as a sulfur source over methionine. Activation of methionine recycling would require both the absence of sulfate (lowering APS levels; see below) and the absence of cysteine.

APS as a signal molecule and the origin of the cym phenotype. Although a requirement for CysB activation may prevent methionine utilization when cysteine is abundant, CysB does not respond directly to the concentration of sulfate in the environment. Naturally, CysB activates transcription of the sulfur assimilation genes when cysteine concentration is low, independent of the concentration of sulfate. Therefore, sulfate concentrations must be sensed if methionine, or any other alternative sulfur source, is to be used by Klebsiella when sulfate concentrations are low and sulfate cannot be assimilated efficiently.

Failure to use methionine on solid media. Although both wild-type Klebsiella and cys mutants use methionine as a sulfur source in liquid media quite effectively (Fig. 3), they cannot use methionine as a sulfur source on solid medium. We don't believe that agar possesses a compound that inhibits this activity since (i) this inhibition is also seen on agarose-, Phytagel-, and Gelrite-based media and (ii) agar does not inhibit methionine utilization when the bacteria are grown embedded within an agar matrix (data not shown). Moreover, the high oxygen tension experienced on plates does not prevent methionine utilization, as plates incubated anaerobically show the same effect. In fact, oxygen assists in methionine utilization (both in liquid for all cells and on plates for cym mutants), likely by allowing production of 3-methylthiopropionate and subsequent employment of the methanethiol pathway (magenta pathway in Fig. 9).

Why two pathways? While both the transsulfurylation pathway and the methanethiol pathway serve to recycle methionine into cysteine under laboratory conditions, they likely serve different purposes in natural environments. The repression of cystathionine--lyase by CysB implies that sulfate is a preferred sulfur source and that other compounds are used only under sulfate-limiting conditions. A similar behavior is seen in the regulation of alkane sulfatase activity in Klebsiella (24, 25). Methionine can be recovered from proteins, and we envision that it is recycled primarily through the transsulfurylation pathway, which is specific for the transfer of sulfhydryl groups from homocysteine to serine to form cysteine.