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Journal of Bacteriology, August 2006, p. 5762-5774, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.00347-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Two Transsulfurylation Pathways in Klebsiella pneumoniae

Thomas A. Seiflein and Jeffrey G. Lawrence*

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Received 10 March 2006/ Accepted 7 June 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In most bacteria, inorganic sulfur is assimilated into cysteine, which provides sulfur for methionine biosynthesis via transsulfurylation. Here, cysteine is transferred to the terminal carbon of homoserine via its sulfhydryl group to form cystathionine, which is cleaved to yield homocysteine. In the enteric bacteria Escherichia coli and Salmonella enterica, these reactions are catalyzed by irreversible cystathionine-{gamma}-synthase and cystathionine-ß-lyase enzymes. Alternatively, yeast and some bacteria assimilate sulfur into homocysteine, which serves as a sulfhydryl group donor in the synthesis of cysteine by reverse transsulfurylation with a cystathionine-ß-synthase and cystathionine-{gamma}-lyase. Herein we report that the related enteric bacterium Klebsiella pneumoniae encodes genes for both transsulfurylation pathways; genetic and biochemical analyses show that they are coordinately regulated to prevent futile cycling. Klebsiella uses reverse transsulfurylation to recycle methionine to cysteine during periods of sulfate starvation. This methionine-to-cysteine (mtc) transsulfurylation pathway is activated by cysteine starvation via the CysB protein, by adenosyl-phosphosulfate starvation via the Cbl protein, and by methionine excess via the MetJ protein. While mtc mutants cannot use methionine as a sulfur source on solid medium, they will utilize methionine in liquid medium via a sulfide intermediate, suggesting that an additional nontranssulfurylation methionine-to-cysteine recycling pathway(s) operates under these conditions.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacterial physiology is a complex network of interwoven biochemical pathways involving numerous shared intermediates, disparately regulated enzymes, and keystone metabolites with important roles as substrates, products, regulatory effectors, or a combination of all three. When genomes gain and lose genes—as they do with startling frequency (20)—the addition of new enzymes and loss of others alter pools of metabolites, overall energy demands, drains on carbon and nitrogen pools, and the balance between transport and de novo biosynthesis. While comparative genomics provides a record of changes in the gene inventory, insight into how the cell responds to such changes is achieved by comparative genetic analyses, where metabolic differences among related organisms are correlated with changes both in their genetic and genomic makeup and in the deployment of their regulatory apparatuses.

Here we examine differences in sulfur amino acid metabolism among three well-studied enteric bacteria, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. E. coli and Salmonella serve as model systems for the physiology of sulfur assimilation. In these organisms, sulfate is reduced to sulfide and assimilated into acyl-activated serine to form cysteine (14); cysteine then serves as the sulfur group donor, either directly or indirectly, for the synthesis of all other sulfur-bearing molecules in the cell, including methionine. In methionine biosynthesis, cysteine is combined with acyl-activated homoserine to form an intermediate compound, LL-cystathionine (7). Cystathionine is cleaved to form homocysteine, pyruvate, and ammonia (Fig. 1), and homocysteine is subsequently methylated to form methionine. These two reactions—together termed transsulfurylation—are catalyzed by the cystathionine-{gamma}-synthase (MetB) and cystathionine-ß-lyase (MetC) enzymes. Transsulfurylation is an irreversible process, and neither E. coli nor S. enterica can use homocysteine as a sulfur group donor in the synthesis of cysteine.


Figure 1
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  		FIG. 1. Transsulfurylation pathways in K. pneumoniae W70. The MetBC enzymes catalyze the transsulfurylation of homoserine to form homocysteine, a precursor of methionine. The MtcBC enzymes catalyze reverse transsulfurylation of serine to form cysteine; both pathways employ cystathionine intermediates. SAH, S-adenosylhomocysteine; HS-CoA, coenzyme A.

 
Alternatively, yeast and some bacteria assimilate sulfide into acyl-activated homoserine to form homocysteine directly (31), which is used in the synthesis of both methionine and cysteine. In these cases, an L-allo-cystathionine intermediate is formed when homocysteine donates its sulfhydryl group to activated serine through a cystathionine-ß-synthase; cystathionine is cleaved by cystathionine-{gamma}-lyase to yield cysteine, {alpha}-keto-butyrate, and ammonia (Fig. 1). This process is termed reverse transsulfurylation. In both transsulfurylation pathways, a sulfhydryl group from the donor molecule (either cysteine or homocysteine) is transferred to an activated alcohol (either homoserine or serine) to form a cystathionine intermediate, which is cleaved on the opposite side of the central sulfur atom to complete sulfhydryl group transfer.

K. pneumoniae—formally designated Klebsiella aerogenes—is an enteric bacterium closely related to both E. coli and S. enterica. Studies of physiological processes in Klebsiella provide context for interpreting the physiology of the these other model organisms (2). We reported previously that, unlike E. coli and S. enterica, Klebsiella is able to use methionine as a sole source of sulfur (26). We suggested that Klebsiella may harbor two transsulfurylation pathways, one that is used in methionine biosynthesis (MetBC) and one that is used in methionine-to-cysteine recycling (MtcBC). Homocysteine would be created from methionine intracellularly during the use and recycling of the methyl group donor S-adenosylmethionine (SAM), thereby allowing methionine to serve as a sole source of sulfur for cell growth.

Here we report physical, genetic, and biochemical evidence for both transsulfurylation pathways in K. pneumoniae. The metB and metC genes encode the methionine biosynthetic enzymes cystathionine-{gamma}-synthase and cystathionine-ß-lyase, respectively. These genes are highly similar to their well-studied E. coli and S. enterica homologues and are repressed during methionine excess; loss of their function results in methionine auxotrophy. We have also characterized the mtcBC operon, which encodes cystathionine-ß-synthase and cystathionine-{gamma}-lyase, respectively. These proteins are expressed during periods of sulfur starvation, and the presence of cysteine or sulfate in the medium reduces their expression. While mtcBC mutants fail to use methionine as a sole sulfur source on solid medium, significant methionine-to-cysteine recycling activity is found in cells grown in liquid medium, suggesting the presence of at least one additional methionine-recycling pathway in Klebsiella; this pathway appears to proceed via a sulfide intermediate, indicating that it is metabolically distinct from the MtcBC-catalyzed pathway for methionine utilization. Partial dissection of the regulation of the metB, metC, and mtcBC loci provides a framework for understanding both the physiological significance of the pathways and the selective forces leading to retention or loss of the genes over evolutionary time.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and plasmids. The strains used in these studies (Table 1) were constructed from K. pneumoniae (formally designated K. aerogenes) W70 derivative KC2668 [hsdR suc+ hutC515(Con) dadA lac {Delta}bla-2]. Plasmid pTAS1 bears the lamB region from E. coli and renders Klebsiella sensitive to bacteriophage {lambda} (26); plasmid pMMK1 (13) is pTAS1 with the gene encoding the altered target specificity Tn10 transposase (12).


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  		TABLE 1. Bacterial strains and plasmids used in this study

 
Media and antibiotics. The rich medium used was LB; the minimal defined medium was E (34). Sulfur-free E medium (NSE) was created by substituting MgCl2 for MgSO4; glucose was used as a carbon source at 0.2%. Solid media were made by the addition of Bacto Agar (Difco) or agarose (Gibco BRL) to 1.2%. P1 buffer contained 5 mM CaCl2 and 10 mM MgSO4. Ampicillin was used at 200 µg/ml; kanamycin was used at 20 µg/ml; chloramphenicol was used at 50 µg/ml for selection of Tn10dCm transposition mutants and at 20 µg/ml otherwise; tetracycline was used at 10 µg/ml for selection of transductants and at 20 µg/ml otherwise; and zeomycin was used at 100 µg/ml. For growth curve determination, cells from a fresh overnight culture were rinsed and diluted 200-fold into NSE-glucose medium; growth curves were determined with 200-µl samples in a PowerwaveX 96-well spectrophotometer, where absorbance at 600 nm was measured every 5 min.

Genetic methods. Transposition-defective derivatives of Tn10-Tn10LK (kanamycin resistant), Tn10dTc (tetracycline resistant), and Tn10dCm (chloramphenicol resistant) were delivered into pTAS1- or pMMK1-bearing strains of K. pneumoniae with bacteriophage {lambda} delivery vectors {lambda}NK1205, {lambda}NK1323, and {lambda}NK1324 as previously described (13). All insertion mutations were transduced into their maternal parent strain following mutagenesis to ensure 100% linkage of the antibiotic resistance marker to the observed mutant phenotype; transduction was mediated by bacteriophage P1-vir as previously described (13).

Enzyme assays. Cells were grown to mid-log phase, concentrated by centrifugation, rinsed, and resuspended in 1/30 volume of 100 mM KxPO4 (pH 8.0). Cells were disrupted in a Parr cell bomb by two successive rounds of compression for 5 min under 1,000 lb/in2 N2. Cell debris was removed by centrifugation, and the protein-bearing supernatant was desalted on a PD-10 gel filtration column (Pharmacia), eluting in 10 mM KxPO4 (pH 8.0). Enzyme extracts were used immediately in assays for cystathionine-{gamma}-lyase activity (18), with final concentrations of 8.4 mM cystathionine (all four stereoisomers) as the substrate and detecting {alpha}-ketobutyrate as a product; assays were performed in triplicate. Specific activities were calculated as micromoles of {alpha}-keto-butyrate produced per minute per milligram of protein. Activities were calculated for the substrate only, the extract only, and the complete reaction mixture (for calculation of substrate-only activities, the protein concentration of the corresponding extract was used); values were normalized to no-substrate controls, leading to some insignificantly negative activities. Assays for ß-galactosidase activity were performed as previously described (17). Protein concentrations were determined by the Bradford assay (4).

Molecular methods. For the creation of chromosomal libraries, genomic DNA from Tn10dCm-bearing cells was partially digested with Sau3AI and ligated into pNEB193, which was digested with BamHI and treated with alkaline phosphatase. Plasmids were introduced into E. coli XL-2 Gold (Stratagene) selecting for ampicillin resistance. Chloramphenicol-resistant colonies were detected by replica printing to LB-chloramphenicol medium, and plasmids were prepared with QIAprep Spin Miniprep kits (QIAGEN). DNA sequences were determined on an ABI 3700 automated sequencer according to the manufacturer's instructions. The sequences across transposon insertion sites were determined from DNA fragments amplified by the PCR from DNA prepared from wild-type cells. Inverse PCR was performed as previously described (19).

For directed gene knockouts, genes conferring resistance to chloramphenicol (cat), kanamycin (aph), and zeomycin (ble) were amplified by PCR with primers whose 3' ends were complementary to the antibiotic resistance genes but whose 5' ends comprised ~45 bases identical to regions flanking the target gene to be replaced. Following amplification of the appropriate antibiotic resistance gene by PCR with these tailed primers, allelic replacement was performed by electroporation of the fragment into a host cell bearing plasmid pTP223 (24), which expresses the {lambda} red recombinase system. Replacement of the chromosomal gene with the antibiotic resistance gene was confirmed by (i) amplification of the target locus via PCR and determination of the nucleotide sequences of the join points between native chromosomal DNA and the introduced antibiotic resistance genes and (ii) linkage analysis of the antibiotic resistance gene to known neighboring loci via P1 cotransduction assays.

Nucleotide sequence accession numbers. The sequences determined in this study have been deposited in GenBank and assigned accession numbers DQ643993 to DQ64998.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the metB and metC loci. Transposon insertion mutations in the metB and metC loci were isolated as methionine auxotrophs as described previously (26). The metB locus was identified by its mutant phenotype (methionine auxotrophy corrected by cystathionine) and by linkage to the arg and rha loci (26). The metC locus was identified by its methionine auxotrophy being corrected by homocysteine but not by cystathionine (26). Chromosomal libraries were constructed from a strain bearing a metB4031::Tn10dCm or a metC4029::Tn10dCm insertion, chloramphenicol-resistant transformants were isolated, and the DNA sequences of appropriate plasmid inserts were determined as described above. A contiguous sequence was constructed for both the metB and metC regions from overlapping clones where appropriate (Fig. 2A and B).


Figure 2
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  		FIG. 2. Comparison of the metB and metC loci of K. pneumoniae and E. coli. Open reading frames in Klebsiella are named after their E. coli homologues; percent nucleotide and protein identities to homologous Klebsiella open reading frames are noted above each E. coli gene. The gray bars denote the extents of plasmid inserts. (A) The metJBL locus. The triangles denote the sites of Tn10 insertions in strains LD859, LD866, and LD834. (B) The metC locus. The triangles denote the sites of Tn10 insertions in strains LD850 and LD894. (C) Comparison of the metJB promoter region among K. pneumoniae (Kpn), E. coli (Eco), and S. enterica (Sen). MetJ binding sites lie between vertical lines; consensus –35 and –10 binding sites for four promoters are noted. (D) Comparison of the metC promoter region. Consensus MetJ binding sites are shown between vertical lines; sites in E. coli were inferred on the basis of footprinting (1, 11, 28) and the crystal structure of the MetJ::SAM::DNA ternary complex (29).

 
The Klebsiella metB locus encodes homologues of the E. coli metJ, metB, and metL genes (Fig. 2A); gene functions can be inferred by the high degree of similarity (proteins are 92 to 96% identical) to their E. coli homologues. The metB gene encodes a cystathionine-{gamma}-synthase (Fig. 1), and disruption of the metB gene by a Tn10dCm insertion at codon 222 results in methionine auxotrophy. The metL gene encodes an aspartate kinase which produces aspartyl-phosphate; this compound serves as a precursor in lysine biosynthesis, as well as a precursor in the synthesis of homoserine, an intermediate in methionine and threonine biosyntheses. No metB insertion results in lysine or threonine auxotrophy by polarity on the metL gene, suggesting that Klebsiella, like E. coli and Salmonella, may harbor multiple genes encoding aspartate kinases, each potentially differentially regulated by an amino acid synthesized from aspartate, i.e., methionine, threonine, or lysine.

The divergently transcribed metJ gene encodes a repressor of methionine biosynthetic genes; in E. coli and Salmonella, MetJ senses high levels of methionine by binding to SAM and effecting transcriptional repression. Good consensus MetJ binding sites are found upstream of the Klebsiella metJ gene and metBL operon (Fig. 2C) in appropriate positions to affect transcriptional repression of PmetB, PmetJ1, and PmetJ2 (Fig. 2C). In addition, good conservation of constitutive PmetJ3 is also seen upstream of the Klebsiella metJ gene. The conservation of the MetJ binding sites among E. coli, S. enterica, and Klebsiella suggests that a similar mode of regulation may be present in Klebsiella.

The Klebsiella metC genes encodes a protein that is 92% identical to its E. coli metC homolog (Fig. 2B). The metC gene encodes a cystathionine-ß-lyase (Fig. 1), and disruption by a Tn10 insertion at codon 77 or codon 280 results in methionine auxotrophy (Fig. 2B). Two MetJ binding sites (each matching seven of eight bases to the consensus sequence) are found upstream of the metC gene (Fig. 2D); the conservation of these sites in E. coli, Salmonella, and Klebsiella suggests that MetJ may repress transcription initiation at the Klebsiella metC gene during periods of methionine excess. Homologs of the E. coli yghB and exbB genes are found adjacent to the metC gene (Fig. 2B); in addition, a gene unannotated in E. coli, B3007 (gray gene in Fig. 2B), appears to be encoded upstream of the exbB gene since this region shows the excess of synonymous substitutions expected for a protein-coding region. These data support the hypothesis that this locus in Klebsiella is the bona fide metC gene and not merely a related sequence.

Regulation of methionine biosynthetic genes. Mutations in the metB and metC loci both result in methionine auxotrophy in Klebsiella (26), as do mutations in their E. coli and Salmonella homologues. If the primary role for the Klebsiella metBC gene products were in methionine biosynthesis, one would predict that these genes would be induced under periods of methionine starvation and be repressed during periods of methionine excess. In E. coli and Salmonella, this regulation is provided by the MetJ protein, which senses excess methionine via excess SAM and represses methionine biosynthetic genes (11). The presence of a metJ homologue in Klebsiella—and good consensus MetJ binding sites upstream of the metJ, metBL, and metC transcription units—suggests that the same regulatory paradigm may apply.

To test this hypothesis, we isolated 12 mutants with Tn10LK insertions in methionine biosynthesis genes; two fusions appeared to be in frame and showed regulated behavior (Table 2). Linkage analysis showed that one insertion affected the metA gene and one insertion affected the metB gene (creating a fusion at codon 381 of the MetB protein; Fig. 2A). The Tn10LK insertions result in MetA::LacZ and MetB::LacZ fusion proteins that are transcribed and translated from endogenous promoters and ribosome-binding signals. In both cases, expression of the gene fusions was decreased when the cells were provided with excess methionine (Table 2). When the cells were provided with the methionine biosynthetic intermediate homocysteine (Fig. 1) as a sole sulfur source, the metA and metB genes were expressed to ~100-fold greater levels (Table 2). To determine if the repression of the metA and metB genes was mediated by the MetJ protein, a metJ::ble mutation—whereby the entirety of the metJ coding sequence is replaced with a cassette expressing a protein conferring zeomycin resistance—was constructed by directed gene knockout (24). As expected, the MetA::LacZ and MetB::LacZ fusion proteins were expressed to high levels in the presence of methionine in a metJ::ble mutant background (Table 2). These results support the hypothesis that the MetBC transsulfurylation pathway is expressed only during periods of methionine starvation and serves to synthesize methionine from cysteine.


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  		TABLE 2. Expression of met::lacZ translational fusions

 
Isolation of mtc mutations. Unlike E. coli and Salmonella, Klebsiella can utilize methionine as a sole source of sulfur. On solid medium, auxotrophs with lesions in the cysPTWA or cysDN locus are completely corrected by cysteine or by methionine (26). We proposed (26) that methionine utilization could proceed by a reverse transsulfurylation pathway, one distinct from the metBC-encoded transsulfurylation pathway inferred from genetic analyses and characterized above. With cysDN::Tn10 insertion mutants as parents, we isolated 14 Tn10dCm, 6 Tn10dTc, and 4 Tn10LK insertion mutants that failed to grow on methionine on solid medium; the cysDN mutation served to eliminate growth on the residual sulfate that is seen in wild-type strains propagated on "sulfur-free" medium. All 24 mtc mutants failed to show significant growth on methionine as a sole sulfur source after 5 days of growth on solid medium but grew well on cysteine, sulfite, and sulfide (the cysDN mutation prevents growth on sulfate); most of the mtc mutations fell into a single linkage group (the exception, a cysE mutation, is described below).

Characterization of the mtcBC locus. A chromosomal library was constructed from strain LD859, bearing the mtc-4028::Tn10dCm mutation, and chloramphenicol-resistant transformants were isolated as described above. The nucleotide sequences were determined for the inserts of several clones to generate a 3.4-kb contiguous sequence (Fig. 3A); the sequence of the promoter region of the mtcBC operon was determined from a fragment generated by inverse PCR (19) with genomic DNA as the template. The mtc-4028::Tn10dCm insertion affects the mtcB gene, which encodes a likely cystathionine-ß-synthase. The predicted MtcB protein sequence is homologous to predicted cystathionine-ß-synthases from numerous bacteria and eukaryotes.


Figure 3
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  		FIG. 3. (A) mtcBC locus of K. pneumoniae. The triangles denotes the sites of Tn10 insertions in strains LD859, LD860, and LD872. The gray bars denote the extents of plasmid inserts or inverse PCR (iPCR) fragments. (B) Promoter region of the mtcBC operon. Possible ribosome binding and {sigma}70 binding sites are underlined. (C) Locations of mutations in the Klebsiella cysJIH (LD893), cysE (LD891), and cysK (LD892) loci.

 
The gene downstream of the mtcB gene, termed mtcC, encodes a likely cystathionine-{gamma}-lyase; the predicted MtcC protein sequence is homologous to predicted cystathionine-{gamma}-lyases from bacterial species and eukaryotes. These two proteins are sufficient to catalyze the reverse transsulfurylation pathway, allowing methionine to serve as a sulfur donor in the synthesis on methionine via SAM, S-adenosylhomocysteine, and homocysteine intermediates. Unlike the metB and metC genes, there are no good consensus MetJ binding sites between mtcBC operon and the upstream pheV gene, suggesting that the mtcBC promoter is not regulated directly by the MetJ repressor. However, there is a good consensus CysB binding site upstream of a potential mtcBC promoter (Fig. 3B).

The gene downstream of the mtcC gene encodes a likely LysR family transcriptional regulator; although these genes are convergently transcribed and their stop codons abut one another, there is no obvious rho-independent terminator for either transcript. The pheV gene, which encodes a tRNAPhe, is located upstream of the mtcBC operon. A pheV homologue is located at min 67 of the E. coli genetic map, 42 kb from the metC locus. Linkage analysis by P1 cotransduction shows that the mtcBC operon is ~55% linked to the metC gene in K. pneumoniae, indicating that it is situated at a corresponding location in the chromosome. However, no evidence of an mtcBC operon is found in the genome sequences of E. coli strain K-12 (3), O157:H7 (23), or CFT073 (36) or S. enterica serovar Typhimurium (16), Typhi (22), or Choleraesuis (6) at this or any other chromosomal location.

A nontranssulfurylation pathway allows methionine utilization in liquid medium. Although all mtcBC mutants fail to use methionine as a sole sulfur source on solid medium, they do utilize methionine as a sole sulfur source when grown in liquid medium (Fig. 4). The apparent diauxie of these growth curves is consistent with the utilization of methionine only after internal pools of cysteine and other sulfur sources have been exhausted; external methionine spares these resources, allowing cells to achieve a higher density. Three possible routes for the Klebsiella secondary pathway are possible. (i) A gaseous methanethiol intermediate (26) may be reduced directly to formaldehyde and sulfide; (ii) methanethiol may be oxidized to methanesulfonate, as has been proposed in Pseudomonas (10, 33), and then reduced to sulfide via a sulfite intermediate; or (iii) a third transsulfurylation pathway operates in liquid medium.


Figure 4
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  		FIG. 4. Growth of Klebsiella strains on sulfur-free minimal medium. Sole sulfur sources were added as indicated. Strains used were LD561 (wild type), LD870 (cbl), LD871 (cysB), LD872 (mtcB), LD873 (mtcB cbl), LD874 (mtcB cysIJ), LD876 (mtcB cysM), LD877 (mtcB cysK), and LD875 (mtcB cysM cysK). Triangles denote the point during methionine utilization when internal sulfur stores have been exhausted (see text). cysB strains grow significantly more slowly and consistently reach this point at a later time. Values indicate the doubling times of strains growing on methionine during exponential growth after this point. OD600, optical density at 600 nm.

 
To discriminate among these alternatives, mutants with changes in the cysJ (sulfite reductase), cysK (cysteine synthase I), and cysM (cysteine synthase II) genes were isolated. As shown in Fig. 4, the alternative pathway appears to proceed via a sulfide intermediate, as a cysM cysK double mutant (eliminating all cysteine synthase activity) eliminates methionine utilization in an mtcB background, while mtc mutants also lacking the CysIJ sulfite reductase are still able to grow on methionine in liquid medium. These data indicate that mtcBC-independent methionine utilization in liquid medium proceeds through a sulfide intermediate (Fig. 5) but not a sulfite intermediate, as proposed in Pseudomonas putida (33), also excluding the action of a third transsulfurylation pathway in Klebsiella. Importantly, this behavior facilitates the characterization of MtcBC functions, since mtcBC mutants do grow with methionine as a sole sulfur source in liquid medium.


Figure 5
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  		FIG. 5. Sulfur amino acid metabolism in K. pneumoniae. Steps for cysteine synthesis are shown with black arrows, methionine synthesis is shown with gray arrows, and methionine-recycling pathways are shown with open arrows. Regulatory interactions are shown with dashed lines.

 
Cysteine, but not sulfate, represses MtcC activity. To test the hypothesis that the mtcC gene encodes a cystathionine-{gamma}-lyase, we assayed for this activity in wild-type and mtcB and mtcC mutant strains. To avoid any residual activity potentially conferred by the MetB or MetC enzyme, assays were performed in both the wild-type and metB metC mutant backgrounds. While no cystathionine-{gamma}-lyase activity was observed in mtcC::Tn10dCm mutants (Fig. 6A), wild-type cells and nonpolar mtcB::Tn10dCm mutants (where the mtcC gene is expressed from the Tn10dCm promoter) showed cystathionine-{gamma}-lyase activity, indicating that the mtcC gene encodes the cystathionine-{gamma}-lyase; we infer that the mtcB gene encodes the cystathionine-ß-synthase. Cystathionine-{gamma}-lyase activity is not evident in wild-type cells grown in the presence of cysteine (Fig. 6B) or sulfate (Fig. 6B and C). These data suggest that cysteine is a preferred sulfur source and the mtcBC operon is induced only during periods of sulfur starvation.


Figure 6
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  		FIG. 6. Cystathionine-{gamma}-lyase activity in K. pneumoniae. Specific activities were calculated as micromoles of {alpha}-keto-butyrate produced per minute per milligram of protein. Strains assayed were LD862, LD863, LD864, LD860, LD887, LD888, LD561, LD826, and LD828.

 
Control of the mtcBC operon by the CysB protein. Since cysteine has a repressive effect on cystathionine-{gamma}-lyase activity in liquid medium (Fig. 6), we postulated that the CysB protein may regulate mtcBC expression; a potential CysB binding site is found in the mtcBC promoter region (Fig. 3B). The CysB protein detects sulfur starvation as accumulation of o-acetyl-serine (via its isomer N-acetyl-serine [15]), the product of the CysE protein. In the screen detailed above, a cysE mutation prevented growth on methionine on solid and liquid media and caused cysteine auxotrophy even when transduced into an otherwise wild-type background. The Tn10dCm element in strain LD891 is located 4 bp upstream of the translation initiation site of the cysE gene and likely results in cysteine auxotrophy because of polar effects.

We posited that the cysE mutant fails to express the mtcBC operon because the lack of N-acetyl-serine fails to activate CysB (Fig. 5). To test this hypothesis, we created a cysB null mutant by directed insertion of a chloramphenicol resistance gene (cat) into the cysB coding region, replacing the cysB gene with the cat gene and its promoter. The cysB mutation prevents utilization of methionine as a sole sulfur source on solid medium (Table 3). Methionine utilization is also impaired in liquid medium (Fig. 4); the cysB strain grows more slowly, exhausting internal sulfur stores at ~9 h (see no-sulfur and sulfate curves). These results indicate that the mtcBC operon is not expressed in the cysB::cat mutant background.


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  		TABLE 3. Growth of K. pneumoniae strains on solid medium with various sulfur sources

 
The mtcBC operon is regulated by the Cbl protein on solid medium. The E. coli Cbl (CysB-like) protein regulates many genes involved in the use of alternative sources of sulfur upon sulfate depletion (9, 32). It has been proposed that Cbl recognizes the sulfate reduction intermediate adenosyl-phosphosulfate (APS) as an effector, which inhibits its ability to activate transcription (5), and purified Cbl protein appears to bind APS (30). The phenotypes of Klebsiella cysDN and cysC mutants support the hypothesis that Cbl mediates gene expression; cysC mutants would accumulate APS and do not express mtcBC on solid medium, whereas cysTWA and cysDN mutants, affecting sulfate transport and APS synthesis, do. This behavior is consistent with APS acting as an anti-inducer via Cbl.

This hypothesis was tested by creating a cbl::aph mutation by directed gene knockout; linkage of this mutation to the nac locus (25) confirmed that we had eliminated the cbl orthologue. MtcBC function was assessed by testing the abilities of strains bearing cbl::aph and cysB::cat mutations to utilize methionine as a sole source of sulfur. While cysD mutants utilized methionine as a sole sulfur source on solid medium, neither a cysD cysB nor a cysD cbl double mutant could use methionine as a sole sulfur source on solid medium (Table 3). These data suggest that the Cbl protein may activate the mtcBC operon in response to the lack of APS in the cell, rather than repressing the mtc operon in the absence of APS. Consistent with this hypothesis, cbl mutations did not allow cysC mutants to utilize methionine as a sulfur source (Table 3).

Although cbl mutants fail to use methionine on solid medium, they do utilize methionine in liquid medium. The addition of a cbl mutation results in a short lag before growth on methionine at rates comparable to that of wild-type cells (Fig. 4). In addition, cbl mtcB mutants (whereby all cysteine is made via the alternative pathway) show no growth defect relative to cbl+ mtcB strains aside from the short lag following exhaustion of internal sulfur stores, indicating that expression of the alternative pathway is not affected by Cbl. The less dramatic phenotype of cbl mutations relative to cysB mutations (Table 3; Fig. 4) suggests that CysB does not regulate mtcBC expression solely through Cbl.

The MetJ protein affects MtcBC activity. To test if MetJ plays a role in methionine recycling, we examined the growth of wild-type and metJ mutant strains on various sulfur sources. On solid medium, cysD metJ double mutants failed to utilize methionine as a sulfur source whereas cysD mutants grew well, suggesting that MetJ activity is required for methionine utilization (Table 3). On liquid medium (Fig. 7A to F), the lack of the MetJ protein decreased methionine-recycling capabilities. This result may have two underlying causes. First, the lack of MetJ repression may cause constitutive expression of the metB and metC genes, causing a futile cycle to occur. Second, MetJ may be required for proper expression of the mtcBC operon or other genes involved in methionine utilization as a sulfur source. To separate these phenomena, we tested the effect of a metJ mutation in the metB and mtcC mutant backgrounds, thereby eliminating any deleterious effect of futile recycling (Fig. 7). In these strains, a defect in methionine-recycling abilities was still observed, suggesting a direct or indirect role of the MetJ protein in regulating mtcBC operon expression. We believe the effect is likely indirect, since there is no evidence of consensus MetJ binding sites upstream of the mtcBC promoter.


Figure 7
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  		FIG. 7. (A to F) Effect of a metJ mutation on methionine utilization. Strains were grown in sulfur-free minimal medium with the sulfur sources specified. Strains used were LD561 (wild type), LD883 (metB), LD882 (mtcC), LD884 (metJ), LD885 (metJ metB), and LD886 (metJ mtcC). (G to H) Effect of a metR mutation on methionine utilization. Strains were grown in sulfur-free minimal medium with the sulfur sources specified. Strains used were LD561 (wild type) and LD881 (metR). Triangles denote the point during methionine utilization when internal sulfur stores have been exhausted (see text). OD600, optical density at 600 nm.

 
The MetR protein plays no role in methionine utilization. The MetR protein up-regulates genes responsible for the conversion of homocysteine to methionine (metE and metH) upon the accumulation of excess homocysteine concentrations. Since this pathway is insensitive to the absolute concentration of either methionine or homocysteine, we did not expect this protein to regulate the mtcBC operon. We examined metR mutants for defects in methionine utilization and found none on either solid medium (data not shown) or liquid medium (Fig. 7G and H).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Roles of CysB and Cbl in mtcBC expression. MtcBC activity, and likely mtcBC expression, is eliminated in wild-type cells in the presence of excess cysteine—which would inhibit the CysE protein (Fig. 5)—or in cysE mutants (Table 3). The inability of cysE mutants to assimilate sulfide prevents methionine utilization via the proposed alternative pathway as well, resulting in complete lack of growth on methionine in liquid medium. The CysE protein produces O-acetyl-serine, both the substrate for sulfide assimilation and the effector for the CysB activator via its isomerization product N-acetyl-serine (15). Since MtcBC activity produces cysteine directly from homocysteine—without the use of a sulfide intermediate—we cannot attribute the lack of MtcBC action to a lack of sulfide incorporation. Rather, we suspect that expression of the mtcBC operon requires activation by CysB, which is unavailable in the cysE mutant because of the lack of N-acetyl-serine.

CysB may act either directly on the mtcBC operon or indirectly because CysB activates the cbl operon. There is a plausible CysB binding site upstream of a likely mtcBC promoter, but the sequence requirements of a CysB binding site are few and have not been readily distinguished from the sequences defining a Cbl binding site. To discriminate among these alternatives, both cysB and cbl null mutants were created by directed gene knockout. As expected, the cysB::cat mutant was unable to grow on methionine as a sole sulfur source on solid or liquid medium. However, cbl mutants showed a more modest defect in methionine utilization on solid medium than cysB mutants did (Table 3). Therefore, Cbl protein cannot be the sole activator of the mtcBC operon and cysB mutants likely have additional defects beyond their inability to transcribe the cbl gene. The CysB and Cbl proteins may work in tandem at the mtcBC promoter, or CysB may have additional indirect effects. The failure of cysDN cbl and cysC cbl mutants to utilize methionine on solid medium is consistent with the Cbl protein serving to activate the mtcBC operon, with APS serving as an anti-inducer. The likely accumulation of APS in cysCHIF mutants would prevent these strains from using methionine as a sulfur source, whereas its absence from cysDN mutants would allow Cbl to activate mtcBC.

But things are more complex in liquid medium, where the alternative pathway allows for growth on methionine. Because cysTWA and cysD mutants utilize methionine on solid medium while cysC, cysH, and cysIJ mutants do not, we had anticipated that the accumulation of APS in cysC mutants (residual sulfate is present in all solid media) would prevent MtcBC activity in both solid and liquid media. Although cystathionine-{gamma}-lyase activity was repressed by the presence of cysteine, the addition of sulfate had no direct effect for cells grown in liquid medium (Fig. 6B and C). Here, wild-type cells showed no cystathionine-{gamma}-lyase activity when grown on methionine plus sulfate, likely because cysteine was being synthesized to levels sufficiently high to inhibit CysE, lowering levels of acetyl-serine, thus preventing CysB activity. Yet sulfate had no effect on MtcC activity in liquid medium (Fig. 6), even though we expect that APS levels should be quite high, preventing Cbl from activating the mtcBC operon. In addition, cbl mutations resulted only in a slight lag period following exhaustion of internal sulfur stores before robust growth on methionine (Fig. 4). Therefore, either another protein besides Cbl activates the mtcBC operon in liquid medium or cysCHIJ mutants fail to grow on solid medium for regulatory reasons that are specific to solid medium.

Role of MetJ in modulating MtcBC function. We expect that the CysB and Cbl proteins would be involved in mtcBC expression if the operon is to be expressed during periods of sulfur starvation. However, we also expect that mtcBC operon expression may require methionine excess since homocysteine is a substrate. As detailed above, levels of methionine in the cells are sensed by the MetJ protein via concentrations of SAM. While it normally serves as a repressor of methionine biosynthetic genes (8, 27, 35, 37), it is possible that MetJ may act as an activator of the mtcBC operon. Such behavior is not unprecedented, as the CysB protein acts as both a transcriptional activator (15) and a transcriptional repressor (21). As expected, elimination of MetJ activity through directed gene knockout showed that this protein is involved in regulating mtcBC operon expression as well. The MetJ protein operates as a transcriptional repressor of met genes, occluding the promoter when bound to excess SAM. There were no MetJ binding sites detected upstream of the mtcBC operon, while such sites were readily found upstream of the metBL and metC transcriptional units (Fig. 2). These data suggest that the MetJ protein acts indirectly to affect mtcBC transcription or that its binding site is significantly different when acting as an activator at this site.

Evolution of transsulfurylation pathways in members of the family Enterobacteriaceae. The absence of homologues of the mtcBC genes from the genomes of E. coli and S. enterica could reflect one of two processes: gain of the mtcBC operon by the Klebsiella lineage after its divergence from the E. coli-Salmonella ancestor or their loss from this ancestor's genome following relaxation of selection for function. That is, either Klebsiella acquired the reverse transsulfurylation pathway as a novel trait or the ancestor of E. coli and Salmonella no longer found it useful. To address this question, we looked for homologues of these genes in the genomes of completely sequenced {gamma}-proteobacteria, including the Enterobacteriaceae (Fig. 8). The distribution of metB and metC genes suggests that the transsulfurylation pathway is ancestral to the Enterobacteriaceae, although it has been lost in some of the pathogenic lineages. Not only are these genes found throughout these {gamma}-proteobacteria, but—as suggested by their similarity to the Klebsiella genes (Fig. 8)—their phylogeny is also consistent with vertical inheritance from their common ancestor (phylogeny not shown). The only exception is that the metB genes in the Pasteurellaceae have been replaced by an orthologue from gram-positive bacteria (the closest relatives are found in the genera Geobacillus, Bacillus, Clostridium, and Listeria, indicated by G+ in Fig. 8).


Figure 8
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  		FIG. 8. Distribution of transsulfurylation genes among {gamma}-proteobacteria. The similarity of homologues to the Klebsiella genes is noted. The phylogeny of 16S rRNA genes was constructed by the maximum-likelihood method with PhyML. Genes were detected via BLAST. Thick lines denote lineages bearing mtcBC genes, while gray lines denote lineages with orthologous replacement. C{gamma}S, cystathionine-{gamma}-synthase; CßL, cystathionine-ß-lyase; CßS, cystathionine-ß-synthase; C{gamma}L, cystathionine-{gamma}-lyase; G+, homologues to genes found in gram-positive bacteria (see text); unk, unknown because a complete genome sequence was unavailable.

 
In contrast, the mtcBC genes for the reverse transsulfurylation pathway are only found in the genera Klebsiella, Photorhabdus, and Yersinia (Fig. 8A), where the genes also form an operon. Homologues are also found in Pantoea agglomerans, although the complete genome sequence of this organism is not available. The relationships among the mtcBC homologues (Fig. 8B) are consistent with their introduction into the ancestor of the family Enterobacteriaceae and subsequent loss in several lineages, including the ancestor of E. coli and S. enterica. While the gain of these genes in an ancestor and subsequent multiple losses in many lineages are not traditionally thought of as most parsimonious, we favor this scenario for two reasons. First, the relationships among these genes are consistent with vertical inheritance. Second, repeated loss is not unexpected when considering the genomes of pathogenic bacteria, where gene loss is common. Rather, multiple losses of the mtc operon, while the metBC genes are retained, suggest that the reverse transsulfurylation pathway plays a less important role in enteric bacterial physiology.

Summary. K. pneumoniae encodes two transsulfurylation pathways, one functioning in methionine biosynthesis and another functioning in methionine-to-cysteine recycling during sulfate starvation. This reverse transsulfurylation pathway is regulated by at least three proteins, including the CysB protein required for activation, the Cbl protein required for activation on solid medium, and the MetJ protein.


   ACKNOWLEDGMENTS
 
We thank L. Kapetanovich for technical assistance in the isolation of met::Tn10LK fusions, J. Floyd for assistance with mtc linkage analysis, M. M. Kolko for assistance with nucleotide sequencing, and E. A. Presley for helpful discussions.

This work was supported by grant GM62805 from the National Institutes of Health and by a grant from the David and Lucille Packard Foundation.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260. Phone: (412) 624-4204. Fax: (412) 624-4759. E-mail:jlawrenc{at}pitt.edu. Back


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 Discussion
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Journal of Bacteriology, August 2006, p. 5762-5774, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.00347-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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