Bacillus subtilis synthesizes pyridoxal 5'-phosphate, the active form of vitamin B₆, by a poorly characterized pathway involving the yaaD and yaaE genes . The pdxS [yaaD] mutant was confirmedto be a strict B₆ auxotroph, but the pdxT [yaaE] mutant turnedout to be a conditional auxotroph depending on the availabilityof ammonium in the growth medium . The PdxS and PdxT proteinscopurified during affinity chromatography and apparently forma complex that has glutaminase activity . PdxS and PdxT appearto encode the synthase and glutaminase subunits, respectively,of a glutamine amidotransferase of as-yet-unknown specificityessential for B₆ biosynthesis.

 
Pyridoxal 5'-phosphate [PLP], the biologically active form ofvitamin B₆, is an essential cofactor for numerous metabolic enzymes [17] . Two pathways of de novo PLP synthesis are known.The PdxA/PdxJ pathway, comprising six dedicated steps, has beenextensively characterized in Escherichia coli [9, 15] . Genesencoding similar enzymes can be found in the genomes of manyother gram-negative bacteria . A second pathway of PLP synthesis,PDX1/PDX2, has been recently discovered in fungi through identificationof two proteins [PDX1/SNZ/SOR1/PYROA and PDX2/SNO] that arerequired for PLP synthesis [2, 10, 11, 27, 31] . Genes codingfor similar proteins are highly conserved in plants, sponges,plasmodia, archaea, and many bacteria [12, 25, 36, 37].

The adjacent Bacillus subtilis genes, yaaD and yaaE, homologousto PDX1 and PDX2, respectively, have recently been shown tobe required for PLP biosynthesis [33] . We present evidence herethat YaaD [renamed PdxS] and YaaE [renamed PdxT] form a glutamineamidotransferase of as-yet-unknown specificity required forthe pathway of de novo PLP synthesis.

 
To create a deletion mutation within the pdxS gene, pBB1181 [Fig . 1] was cut with AatII and HindIII and religated after filling in the ends . The actual deletion endpoint in the resulting plasmid, pBB1185 [Fig . 1], as determined by sequencing, turnedout to be beyond the HindIII site; the sequence removed was 606 bp long and did not alter the reading frame of pdxS . pBB1218, a truncated version of pBB1185 [Fig . 1], was used to replacethe wild-type pdxS gene of B . subtilis strain SMY with the pdxS allele, resulting in strain BB2253 . Methods for plasmid isolation, agarose and polyacrylamide gel electrophoresis, use of restriction and DNA modification enzymes, DNA ligation, PCR, and electroporation of E . coli JM107 cells were as described by Sambrook et al. [35] . Growth of B . subtilis cells, transformation by plasmidDNA, and procedures for gene replacement were as described previously[4, 5].

 
Strain BB2253 [pdxS] lost the ability to grow in minimal medium, even if the medium was enriched with amino acids; growth was fully restored by addition of 0.2 µM pyridoxal [PL] . Thisresult is consistent with the phenotype of the pdxS insertionmutant [33] and the documented role in PLP biosynthesis of fungalpdxS homologs [2, 10, 27, 31] . Pyridoxine [PN] was 1,000-foldless effective in restoration of BB2253 growth, indicating thatB . subtilis cells utilize exogenous PN for PLP biosynthesisweakly or not at all [growth in the presence of PN may be dueto PL contamination or to oxidation of PN to PL] . Pyridoxaminewas several times less effective than PL [probably due to thespecificity of the pyridoxal kinase required for phosphorylationof all simple B₆ vitamers [45] or the corresponding transportsystem].

 
A deletion-insertion mutation within the pdxT gene was created by replacing the 135-bp SphI-EcoRI fragment of pBB1213 [Fig. 1] with a 1.4-kb SphI-EcoRI ble cassette determining resistanceto phleomycin, excised from pJPM136 [5] . pBB1217 was introducedinto B . subtilis SMY, and strain BB2256 [pdxT::ble] was isolatedas described previously [5].

In contrast to strain BB2253 [pdxS], strain BB2256 [pdxT] behavedin minimal medium as a conditional PL auxotroph depending on ammonium availability . Virtually no growth was observed in glucose-glutamateor glucose-1 mM NH₄Cl medium, but growth at almost the wild-typerate was observed in the presence of 60 to 120 mM NH₄Cl [Fig.2]; intermediate growth was observed in glucose-0.2% [37.4 mM]NH₄Cl medium . Growth of strain BB2256 was restored to the wild-typerate by addition of PL . Similarly, less severe effects of fungalPDX2 gene defects on PLP synthesis, compared with what is seenwith PDX1 defects, have been described previously [11, 31, 40].In fact, the B₆ dependency of the Aspergillus nidulans PYROB[similar to PDX2; GenBank accession number AAK50016] mutantwas also rescued by a high concentration of ammonium [1] . Thepartial B₆ auxotrophy of pdxT mutants and the poor capacityof B . subtilis cells to utilize PN explain the ability of 50µM PN to relieve the growth defect of the pdxT mutantbut not that of the pdxS mutant [33].

 
A Bacillus circulans mutant defective in the yaaE [pdxT] genehas been described recently . Though the function of the gene was not determined, a conditional growth defect dependent on ammonium availability was also observed for this mutant [41]. The authors of this earlier study tried to relieve the growth defect by adding PN but failed in accord with the very poorability of B . subtilis cells to utilize PN.

A strain [BB2254] carrying a deletion of parts of both pdxS and pdxT was constructed as described above by using pBB1190, which lacks the 898-bp fragment of pBB1181 starting at the AatIIsite [Fig . 1] . BB2254 had the same strict PL auxotrophy as a single pdxS null mutant . No pseudorevertants of such mutants have been found . Because both pdxS and pdxST deletions cause a strict requirement for B₆ vitamers, we conclude that only one pathway of de novo PLP synthesis, which cannot be easily bypassed due to mutations in other genes, operates in B . subtilis.

B . subtilis mutants requiring PN, PL, or pyridoxamine for growth have been isolated previously [30] but have not been characterizedgenetically . The B₆ requirement of most such mutants was partiallyor completely alleviated by isoleucine or isoleucine plus lowconcentrations of PN [30] . In our work, no effect on the growthof the pdxS or pdxT mutants was observed when isoleucine [500µg/ml], with or without a suboptimal amount of PN, wasadded [data not shown], suggesting that our mutants are differentfrom the ones isolated previously.

The fact that PL is utilized preferentially and PN only very poorly correlates well with an apparent lack in B . subtilisof the E . coli pdxH-like gene and the corresponding pyridoxine 5'-phosphate oxidase activity [29, 30] required for conversionof PN to PLP . No genes likely to encode pyridoxine 5'-phosphateoxidase can be found in most bacteria harboring the pdxS- andpdxT-like genes [data not shown] . It is likely that all suchbacteria will prove to depend on PL if their de novo pathwayof PLP synthesis becomes inactivated . In accord with this conclusion,several lactic acid bacteria whose genomes lack pdxH and thegenes for de novo PLP synthesis were shown to use PL preferentially[38].

 
PdxT and other PDX2-like proteins are similar to some glutamine amidotransferases [47], consistent with the observation that in some fungi and bacteria the nitrogen group of PLP originates in glutamine [42] . In E . coli and some other bacteria, the nitrogengroup of PLP comes from glutamate [42] through the SerC-catalyzedtransamination step [21]; in B . subtilis, SerC aminotransferaseis not involved in B₆ synthesis [34].

The sequence of pdxT suggests that this gene is unlikely to encode a full glutamine amidotransferase by itself . It is morelikely to encode a glutaminase subunit of a heteromultimericglutamine amidotransferase responsible for hydrolysis of glutamineto glutamate and ammonia and for channeling the latter to thecorresponding synthase subunit . The location of pdxS adjacentto pdxT makes pdxS the most likely candidate to encode the synthase subunit of this glutamine amidotransferase of a novel, as-yet-unknown specificity . The phenotypes of pdxS and pdxT null mutants [strict and conditional requirements for B₆ vitamers, respectively] are entirely consistent with this suggestion as [i] most glutamine amidotransferases are able to utilize ammonium, though withlow efficiency, as a substitute for glutamine, [ii] the glutaminase subunit is completely dispensable when ammonium is the substrate,and [iii] inactivation of glutaminase subunits of other glutamine amidotransferases often leads to conditional, ammonium-dependent phenotypes of corresponding mutants [46].

In the absence of precise knowledge of the reaction catalyzedby the putative PdxST complex, we tested a known partial activityof glutamine amidotransferases, the glutaminase reaction which frequently occurs in the absence of other substrates [46] . A His₆-tagged version of B . subtilis PdxS [the predicted molecularmass of the unmodified protein is 31.6 kDa] was overproducedin E . coli LMG194 [ara] cells [14] containing pBB1261 [Fig.1] after addition of 0.2% L-arabinose to Luria broth culturesat an A₆₀₀ of 0.25 to 0.4 and incubation for 4 h . The cellswere pelleted, washed in 50 mM Tris-Cl [pH 8.0]-5% glycerol,and disrupted by sonication in 20 mM Tris-Cl [pH 7.9]-500 mMNaCl-5 mM imidazole-5% glycerol-1 mM phenylmethylsulfonyl fluoride-0.1%Nonidet P-40 . The supernatant was clarified by low-speed centrifugation,and PdxS was purified to virtual homogeneity on a Ni²⁺ affinitycolumn [His•Bind resin; Novagen] as described by the manufacturer,using 485 mM imidazole for elution [Fig . 3, lane 1] . A His₆-tagged version of B . subtilis PdxT [the predicted molecular mass of the unmodified protein is 21.4 kDa] was purified from E . coli [pBB1256] cells in a similar manner using 385 mM imidazole for elution [Fig . 3, lane 3] . The modified proteins, when expressedin B . subtilis cells, were active and could complement nullmutations in the corresponding chromosomal genes [data not shown].

 
Glutaminase activity was detected in a coupled reaction withbovine glutamate dehydrogenase as the increase in optical densityat 363 nm due to reduction of 3-acetylpyridine adenine dinucleotide[APAD], which accompanies conversion of glutamine-derived glutamateto 2-ketoglutarate . Samples were assayed at room temperaturein 1 ml of 50 mM Tris-Cl [pH 8.2]-10 mM glutamine-0.6 mM APADcontaining 1 U of bovine glutamate dehydrogenase [Sigma] . Theuse of APAD, an analog of NAD, shifts the unfavorable equilibriumof the glutamate dehydrogenase reaction [44] . The data were corrected for very low reduction of APAD in reactions lackingPdx proteins . Protein concentration was determined using a Bio-Rad protein assay with bovine serum albumin as a standard.

Neither PdxT [Fig . 4, curve 1] nor PdxS [data not shown] alonehad glutaminase activity when glutamine was provided as the only substrate . When PdxS and PdxT were both present, significant glutaminase activity was observed . Higher glutaminase activity was obtained when the ratio of PdxS to PdxT was increased [Fig. 4, curves 2 and 3].

 
Glutaminase subunits of some other glutamine amidotransferasesare also activated by the presence of the corresponding synthasesubunits [18, 46] . This result strongly confirms our hypothesisthat PdxS and PdxT are components of the same enzyme . Additionally,we conclude that the conditional growth phenotype of the pdxT-likemutants reflects the ammonium-dependent activity of PdxS inthe absence of its cognate glutaminase subunit.

 
An E . coli strain containing pBB1252 [Fig . 1] and overproducingthe wild-type version of the B . subtilis PdxS protein and aHis₆-tagged version of PdxT was used for purification of thePdxT-His₆ protein by Ni²⁺ affinity chromatography . Wild-typePdxS, which by itself had no affinity for the Ni²⁺ affinitycolumn, copurified with PdxT-His₆ [though not in the stoichiometricamount], indicating that the two proteins form a complex thatdoes not completely dissociate even under the high salt conditions[0.5 M NaCl, 0.4 M imidazole] used for purification [Fig . 3, lane 2] . This result is consistent with the postulated interaction of yeast PDX1 and PDX2 homologs, as deduced from experiments with two-hybrid systems [28] . No protein copurified with PdxT-His₆if part of the pdxS gene was deleted from pBB1252, as in pBB1256[Fig . 1 and 3, lane 3].

 
All gram-positive bacteria have either the PdxST pathway orno PLP synthetic pathway; most other bacteria have the PdxA/PdxJpathway [data not shown; published genome sequence data weresearched by using the National Center for Biotechnology InformationBlast server available at http://www.ncbi.nlm.nih.gov/BLAST;preliminary sequence data were searched at http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgiand http://www.sanger.ac.uk/DataSearch] . Some lactic acid bacteriaand Clostridium spp . are known to be B₆ auxotrophs [6, 19, 26,38], consistent with the absence of the genes of de novo PLPsynthesis in their genomes . No single bacterial genome has genescoding for all components of both pathways . Some bacteria withthe PdxST pathway have a PdxA homolog; it is likely that theseorphan PdxA-like proteins, as well as additional PdxA paralogsin some gram-negative bacteria [data not shown], have a dehydrogenaseactivity unrelated to PLP synthesis . The pdxS- and pdxT-likegenes are linked in all bacterial genomes except that of Mycobacteriumleprae [8].

In Saccharomyces cerevisiae, a glucose-derived five-carbon-containing compound and a triose are utilized for PLP synthesis [13, 48].No other components of the PDX1/PDX2 pathway, other than glutamine,are known, and it is unclear whether all steps of the PDX1/PDX2pathway are common in different organisms . In our work, we demonstratedthat the PdxST complex is likely to catalyze the correspondingglutamine amidotransferase reaction though neither the additionalsubstrate[s] nor the products of the reaction have been identified.PdxS may have a phosphate-binding site [12], implicating a phosphorylatedcompound as a substrate; another possible role for this siteis binding of flavin mononucleotide [FMN; the IPR003009 motifavailable at www.ebi.ac.uk/interpro].The latter designationmay be misleading, however . Many enzymes containing the IPR003009motif are not known to bind FMN; instead, they have pent[ul]ose5-phosphate derivatives as their substrates . Since FMN containsa ribityl-5-phosphate group, we suggest that the IPR003009 motifreflects binding of a five-carbon unit phosphorylated at position5 . In that case, one of the PdxST substrates, in addition to glutamine, is a phosphorylated five-carbon carbohydrate derivative, as has been suggested by tracer experiments for yeast [13]. Guanylylation of a protein that turned out to be PdxS by extracts of B . subtilis cells has been reported [24] . The physiologicalrole of this modification remains unknown.

In yeast grown anaerobically, glutamine does not serve as B₆ precursor [16] . It is possible that under these conditions theyeast PdxS-like SNZ protein uses ammonium as its substrate.Interestingly, the genomes of several bacteria, such as Fusobacteriumnucleatum, Corynebacterium efficiens, Clostridium botulinum,and Treponema denticola, include pdxS-like genes but no pdxT-likegene [data not shown] . PdxS proteins of these organisms mayoperate as ammonium-dependent synthases of the PLP pathway,may use other types of glutaminase subunits, or may not synthesizePLP in vivo . The tight B₆ requirement of the pdxT null mutantin the absence of ammonium indicates that no other B . subtilisglutaminase can substitute for PdxT.

1-Deoxy-D-xylulose-5-phosphate, whose formation is catalyzedby the dxs gene product [22, 39], is required for PLP synthesisin E . coli [7, 20] . Most bacteria that have pdxS- and pdxT-likegenes have dxs orthologs, but no orthologs of dxs can be foundin the genomes of archaea, Staphylococcus aureus, or Streptococcuspneumoniae, all of which apparently utilize the PdxST pathway[data not shown] . Moreover, the B . subtilis conditional dxsmutant was not impaired in B₆ synthesis [32] . Additionally, the five-carbon-containing compound utilized for PLP synthesis in yeast is derived from glucose as an intact unit [13]; in contrast, 1-deoxy-D-xylulose-5-phosphate originates from condensationof pyruvate and glyceraldehyde 3-phosphate [9, 15] . Therefore,Dxs-produced 1-deoxy-D-xylulose-5-phosphate is an unlikely intermediatein the PdxST-dependent pathway of PLP synthesis . Though an alternativeroute of 1-deoxy-D-xylulose-5-phosphate synthesis was describedin E . coli, its physiological role is unknown [43].

Exogenous 2'-hydroxypyridoxine can be converted to PLP in yeast[49] . It is possible that 2'-hydroxypyridoxine 5'-phosphateis an immediate precursor of PLP in B . subtilis . If so, theputative five-carbon intermediate of the PdxST pathway shouldhave a hydroxyl group at its 1 position.

 
The crystal structure of the B . subtilis PdxT [YaaE] protein was determined recently [J . A . Bauer, E . M . Bennett, T . P . Begley, and S . E . Ealick, J . Biol . Chem., in press].

 
I am grateful to A . L . Sonenshein and J . Smith for many helpful discussions and for careful reading of the manuscript.

This work was supported by a grant from the National Science Foundation [MCB-0110651] to B.R.B.

 
* Mailing address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111 . Phone: [617] 636-3618 . Fax: [617] 636-0337 . E-mail: bbelit02@granite.tufts.edu.

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