Biochemical Characterization of the C4-Dicarboxylate Transporter DctA from Bacillus subtilis

Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C₄-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C₄-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter Glt_Ph from Pyrococcus horikoshii.The DctA family is one of several diverse families of secondary transporters that catalyze the uptake of C₄-dicarboxylates from the Krebs cycle in bacteria (16, 27). In Escherichia coli, DctA mediates the uptake of succinate, fumurate, and malate under aerobic conditions; genomic disruption of dctA in E. coli prevents growth with malate or fumarate as the sole carbon source, and the mutant grows poorly on succinate (5). Similarly, a dctA knockout mutant of Bacillus subtilis cannot grow with succinate or fumarate as the sole carbon source (1). DctA plays a major role in the symbiotic relationship between nitrogen-fixing rhizobia (43) and root nodule-forming plants (30, 37, 38). Transport assays with Sinorhizobium meliloti cells showed previously that in addition to succinate, malate, and fumarate, orotate is transported and that a range of other substrates such as succinamic acid and succinamide may be transported, because they inhibit the transport of orotate (42). In Corynebacterium glutamicum, malate transport by DctA is inhibited by α-ketoglutarate, oxaloacetate, and glyoxylate, indicating that these compounds may be substrates also (41).DctA transporters belong to a large family of secondary transporters (the DAACS [dicarboxylate/amino acid:cation symporter] family), which also comprises well-characterized glutamate/aspartate transporters and neutral amino acid transporters (32, 33). While DctA-type dicarboxylate transporters are found only in bacteria, glutamate/aspartate transporters of the DAACS family are found both in prokaryotes (e.g., GltT in Bacillus stearothermophilus, GltP in E. coli, and Glt_Ph in Pyrococcus horikoshii [2, 7, 34]) and in higher eukarya, where they play a pivotal role in the reuptake of the excitatory neurotransmitter glutamate from the synaptic cleft (4). Neutral amino acid (alanine, serine, and threonine) transporters are found in mammals (see, e.g., references 36 and 44) as well as bacteria (17).Secondary transporters of the DAACS family use (electro)chemical gradients of cations across the membrane to drive transport. The type of cotransported ions varies among family members: eukaryotic glutamate transporters couple the transport of glutamate to the symport of one proton and three sodium ions and the antiport of one potassium ion (24, 45). Bacterial and archaeal glutamate transporters utilize either sodium ions or protons for symport (2) and are independent of potassium ions (28, 31). The bacterial and mammalian neutral amino acid transporters are sodium ion coupled. Glutamate/aspartate transporters and bacterial serine/threonine transporters (SstTs) are electrogenic, but mammalian neutral amino acid transporters are obligate electroneutral amino acid antiporters (44).Insight into the structure-function relationships of the DAACS family members has greatly increased since crystal structures of the P. horikoshii aspartate transporter Glt_Ph have been determined (2, 29, 40). The protein consists of eight membrane-spanning helices and two reentrant regions (helical hairpins HP1 and HP2) (40). The C-terminal part of the protein (helices 7 and 8 and HP1 and HP2) is most strongly conserved with respect to other family members and binds the substrate and cotransported ions, with HP1 and HP2 functioning as lids that allow alternating access to the substrate- and ion-binding sites from either side of the membrane (3, 29). Glt_Ph forms a homotrimeric complex in which each protomer functions independently of the other subunits (11, 12, 18, 19, 23). The fold and oligomeric state are likely to be conserved throughout the family.Whereas the transport mechanisms of bacterial glutamate and neutral amino acid transporters of the DAACS family have been studied extensively in vitro, the C₄-dicarboxylate transporters of the DAACS family (DctA proteins) have been studied using whole cells only. To fully characterize these transporters, in vitro activity assays using either membrane vesicles or proteoliposomes containing purified protein are necessary. In such assays, the internal and external buffer compositions can be controlled, thus allowing manipulation of the chemical ion gradients and the electrical potential across the membrane. Here, we present the first biochemical characterization of a DctA family member in membrane vesicles. We have studied the DctA homologue from B. subtilis, which is annotated as DctP (1) but which we propose to rename DctA to reflect the homology to other DctA proteins. B. subtilis DctA (DctA_Bs) has 30 to 32% sequence identity to the aspartate transporter Glt_Ph and human excitatory amino acid transporter (EAAT) family members, over 40% sequence identity to the characterized bacterial glutamate transporters from E. coli and B. stearothermophilus, and 41 and 56% identity to DctA homologues from C. glutamicum and E. coli, respectively. We determined the substrate specificity of DctA_Bs, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns.     

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

DNA helicases have important roles in genome maintenance. The RecD helicase has been well studied as a component of the heterotrimeric RecBCD helicase-nuclease enzyme important for double-strand break repair in Escherichia coli. Interestingly, many bacteria lack RecBC and instead contain a RecD2 helicase, which is not known to function as part of a larger complex. Depending on the organism studied, RecD2 has been shown to provide resistance to a broad range of DNA-damaging agents while also contributing to mismatch repair (MMR). Here we investigated the importance of Bacillus subtilis RecD2 helicase to genome integrity. We show that deletion of recD2 confers a modest increase in the spontaneous mutation rate and that the mutational signature in ΔrecD2 cells is not consistent with an MMR defect, indicating a new function for RecD2 in B. subtilis. To further characterize the role of RecD2, we tested the deletion strain for sensitivity to DNA-damaging agents. We found that loss of RecD2 in B. subtilis sensitized cells to several DNA-damaging agents that can block or impair replication fork movement. Measurement of replication fork progression in vivo showed that forks collapse more frequently in ΔrecD2 cells, supporting the hypothesis that RecD2 is important for normal replication fork progression. Biochemical characterization of B. subtilis RecD2 showed that it is a 5′-3′ helicase and that it directly binds single-stranded DNA binding protein. Together, our results highlight novel roles for RecD2 in DNA replication which help to maintain replication fork integrity during normal growth and when forks encounter DNA damage.     

Genetic and Biochemical Characterization of Mutants of Bacillus subtilis Defective in Succinate Dehydrogenase

Eleven succinate-accumulating mutants of Bacillus subtilis have been mapped by transformation and transduction crosses and characterized with respect to activities of citric acid cycle enzymes. These mutants could be divided into three genetic groups. Nine of the mutants were found to map between argA and leu in the citF locus. A second group was located between lys-1 and trpC2 and the third group could not be located on the B. subtilis chromosome in extensive transduction crosses. All of the citF mutants lack detectable succinate dehydrogenase activity, whereas both of the other groups show a reduced level of this enzyme. In addition, most of the mutants in the citF locus lack cytochrome a, whereas the level of this cytochrome is normal in the other two groups. A procedure has been devised for the solubilization of the succinate dehydrogenase from the membrane of B. subtilis with the non-ionic detergent Brij 58. Some properties of the soluble and bound forms of succinate dehydrogenase are described.     

Characterization of Bacillus subtilis recombinational pathways

Recombination in Bacillus subtilis requires the products of numerous rec loci. To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele. On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups. Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mutations, while group beta comprises the addA and addB mutations. Group gamma consists of the recH and recP mutations. These results suggest that B. subtilis has multiple pathways for genetic recombination and that the products of the genes within the alpha, beta, and gamma epistatic groups are involved in these alternative recombination pathways. The RecA protein is required in all three pathways of intermolecular recombination.     

Characterization of recombination-deficient mutants of Bacillus subtilis

An isogenic set of "prophage-free," DNA repair-proficient and -deficient strains of Bacillus subtilis were characterized phenotypically. The mutant strains were provisionally classified into four categories on the basis of their sensitivity to DNA-damaging agents, their ability to release phage after lysogenization followed by damage to chromosomal DNA, and their impairment in genetic exchange. The properties of double Rec- mutants showed that recF and addA belong to different epistatic groups, whereas recF, recL, and recH fall into the same group. More than one pathway for genetic exchange might be operative in B. subtilis.     

嗜热脂肪芽孢杆菌DNA解链蛋白1的纯化及性质

以嗜热脂肪芽孢杆菌为材料,通过ＰｏｌｙｍｉｎＰ沉淀,硫酸铵分级及Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ,ＤＥＡＥ纤维素,磷酸纤维素,ＦＰＬＣ ＭｏｎｏＱ,ＦＰＬＣ Ｓｕｐｅｒｏｓｅ１２等柱层析,得到部分纯化的ＤＮＡ解链蛋白１。ＢｓｔＨ１具有依赖ＤＮＡ和Ｍｇ＾２＋的ＡＴＰ酶活力,不同类型的核酸对ＢｓｔＨ１的ＡＴＰ酶活力的促进作用不同。     

Properties of regeneration mutants of Bacillus subtilis

Abstract Higher regeneration mutants were isolated from Bacillus subtilis . Protoplasts from two out of four mutants regenerated at a 100% frequency on a semi-synthetic hypertonic medium. They conferred less autolytic productivity, and a revertant regained the parental levels of regeneration frequency and autolytic activities. This mutation ( rgn -1) expressed the other pleiotropic properties, i.e., nonmotility, phage PBS1 resistance and different cell morphology.     

Characterization of recF suppressors in Bacillus subtilis

A recF mutation renders Bacillus subtilis cells very sensitive to DNA-damaging agents. Such a recF defect is partially suppressed either by the presence of the recA73 mutation or by the presence of a plasmid-borne, heterologous, single-stranded DNA-binding (ssb) protein gene. Plasmids carrying ssb genes also suppressed the recR and recL defects. Our results suggest that suppression occurs by increasing recombinational repair. The effect of the suppressors may be at the level of induction of the SOS response.     

Overexpression and Biochemical Characterization of a Thermostable Phytase from Bacillus subtilis US417 in Pichia pastoris

The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50–65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100 % of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.     

重组磷脂酰丝氨酸合成酶的分离纯化及酶学性质研究

利对重组枯草芽孢杆菌(pBES-pss)表达的磷脂酰丝氨酸合成酶进行分离纯化及酶学性质研究.pBES-pss发酵后的粗酶液经硫酸铵盐析、中空纤维膜除盐浓缩、SP-Sepharose HP离子交换层析和Sephadex G-75凝胶层析,基本获得电泳纯的重组磷脂酰丝氨酸合成酶,比活力可达13.62 U/mg,分子量约为53 kD.酶学性质研究表明,该酶催化卵磷脂水解反应的最适pH8.0,最适温度为35℃.稳定性研究表明:该酶在pH 6.5～9.5 区间和低于45℃温度下稳定.表面活性剂及金属离子对该酶水解活性的影响结果表明,SDS、Tween20、Tween80对该酶有抑制作用,Triton X-100对该酶有增强作用;Mg2+、Zn2+、K+对该酶有抑制作用,Ca2+、Mn2+和EDTA对该酶有增强作用.     

Biochemical Characterization of Adeno-Associated Virus Rep68 DNA Helicase and ATPase Activities

The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45°C, and an optimal divalent-cation concentration of 5 mM MgCl₂. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The K_m values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 μM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3′-to-5′ direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5′ or 3′ tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3′ tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.     

A Nucleotide-dependent and HRDC Domain-dependent Structural Transition in DNA-bound RecQ Helicase

The allosteric communication between the ATP- and DNA-binding sites of RecQ helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded DNA (ssDNA) and, in turn, the restructuring of multistranded DNA substrates during genome maintenance processes. In this study, we used the tryptophan fluorescence signal of Escherichia coli RecQ helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. Rapid kinetic experiments revealed that ssDNA binding occurs in a two-step mechanism in which the initial binding step is followed by a structural transition of the DNA-bound helicase. We found that the nucleotide state of RecQ greatly influences the kinetics of the detected structural transition, which leads to a high affinity DNA-clamped state in the presence of the nucleotide analog ADP-AlF₄. The DNA binding mechanism is largely independent of ssDNA length, indicating the independent binding of RecQ molecules to ssDNA and the lack of significant DNA end effects. The structural transition of DNA-bound RecQ was not detected when the ssDNA binding capability of the helicase-RNase D C-terminal domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multistranded DNA processing by RecQ helicases.     

Biochemical Characterization of AtRECQ3 Reveals Significant Differences Relative to Other RecQ Helicases

Members of the conserved RecQ helicase family are important for the preservation of genomic stability. Multiple RecQ homologs within one organism raise the question of functional specialization. Whereas five different homologs are present in humans, the model plant Arabidopsis (Arabidopsis thaliana) carries seven RecQ homologs in its genome. We performed biochemical analysis of AtRECQ3, expanded upon a previous analysis of AtRECQ2, and compared their properties. Both proteins differ in their domain composition. Our analysis demonstrates that they are 3′ to 5′ helicases with similar activities on partial duplex DNA. However, they promote different outcomes with synthetic DNA structures that mimic Holliday junctions or a replication fork. AtRECQ2 catalyzes Holliday junction branch migration and replication fork regression, while AtRECQ3 cannot act on intact Holliday junctions. The observed reaction of AtRECQ3 on the replication fork is in line with unwinding the lagging strand. On nicked Holliday junctions, which have not been intensively studied with RecQ helicases before, AtRECQ3, but not AtRECQ2, shows a clear preference for one unwinding mechanism. In addition, AtRECQ3 is much more efficient at catalyzing DNA strand annealing. Thus, AtRECQ2 and AtRECQ3 are likely to perform different tasks in the cell, and AtRECQ3 differs in its biochemical properties from all other eukaryotic RECQ helicases characterized so far.RecQ helicases are important players in the maintenance of genomic stability in prokaryotes and eukaryotes (for reviews covering several aspects of RecQ helicases, see Bachrati and Hickson, 2003; Hickson, 2003; Opresko et al., 2004; Sharma et al., 2006; Brosh and Bohr, 2007; Hanada and Hickson, 2007; Bachrati and Hickson, 2008). Interestingly, organisms contain different numbers of RecQ homologs. In humans, mutations in three of the five RecQ helicases have been linked to distinct, cancer-associated genetic diseases. Consequently, the human RecQ helicases have been intensively studied. All RecQ helicases share a characteristic helicase domain, due to which they are classified as RecQ helicases. In addition, a RecQ C-terminal (RQCt) and/or a Helicase and RNAseD C-terminal (HRDC) domain can be identified in some RecQ helicases. The possible functions of the latter in protein stability and DNA and protein binding are summarized, for example, in a recent review (Chu and Hickson, 2009). From the enzymatic point of view, most RecQ helicases both unwind DNA and promote the opposite reaction, called strand annealing. In addition, some RecQ homologs combine these activities to catalyze branch migration.One of the central questions regarding RecQ helicases is the extent to which the homologs have unique or overlapping functions (Ellis et al., 2008). This question originally raised for the human RecQ helicases is of equal importance for other organisms, such as plants. Analysis of plant RecQ homologs could reveal species- or kingdom-specific tasks, and such comparisons could shed light on evolutionary questions, such as how selection pressures induce multiple homologs within a single species.In the dicotyledonous model plant Arabidopsis (Arabidopsis thaliana), seven RecQ helicase homologs have been identified (Hartung et al., 2000; Hartung and Puchta, 2006). A biochemical comparative analysis may provide information about the molecular basis of their cellular functions, which are likely to be different. As has been shown, sequence homology does not necessarily imply functional homology (Hartung et al., 2007).Previously, we were able to characterize basic biochemical properties of the helicase RECQ2 of the model plant Arabidopsis (Kobbe et al., 2008). In this study, we chose to study AtRECQ3 as a RecQ helicase that differs in the domain structure from AtRECQ2, lacking the HRDC domain and the winged helix subdomain of the RQCt domain (Fig. 1A). AtRECQ3 has the same domain structure as human RECQ5β. AtRECQ3 was expressed in Escherichia coli, purified, and biochemically characterized. The specificity of AtRECQ2 and AtRECQ3 was compared with several substrates, extending the analyzed spectrum of substrates for AtRECQ2 and for RecQ helicases in general. We identified several differences in the enzymatic properties of AtRECQ2 and AtRECQ3, suggesting that these two enzymes might play different roles in vivo.Open in a separate windowFigure 1.Domain structure, purification, and directionality of AtRECQ2 and AtRECQ3. A, Schematic drawing of the domain structure of AtRECQ2 and AtRECQ3. In the helicase domain, the different helicase motifs (0, I, Ia, II, III, IV, V, and VI) are depicted in dark gray. B, 10% SDS-PAGE analysis stained with colloidal Coomassie Brilliant Blue of representative purifications of AtRECQ2-K117M (lane 1, 0.5 pmol), AtRECQ2 (lane 2, 0.8 pmol), AtRECQ3-K64M (lane 3, 0.9 pmol), and AtRECQ3 (lane 4, 1.25 pmol). The proteins were overexpressed in E. coli and purified by nickel-immobilized metal ion affinity chromatography and Calmodulin affinity chromatography. The gray arrow indicates AtRECQ2/-K117M with a predicted molecular mass of 85.5 kD, and the black arrow indicates AtRECQ3/-K64M with a predicted molecular mass of 86 kD. C, AtRECQ2 (8 nm), AtRECQ3 (8 nm), AtRECQ3-K64M (8 nm), and AtRECQ2-K117M (5 nm) were incubated with 150 pm of the indicated substrates for 30 min at 37°C. Error bars indicate sd of the mean of three independent experiments.     

Isolation and Characterization of Rifampin-Resistant and Streptolydigin-Resistant Mutants of Bacillus subtilis with Altered Sporulation Properties

Mutants of Bacillus subtilis with altered deoxyribonucleic-dependent ribonucleic acid polymerase activity have been isolated and characterized. These mutants, selected as strains resistant to rifampin or streptolydigin, demonstrate drug-resistant in vitro ribonucleic acid synthesis. Sporeforming ability and support of phage infection are altered in many of the mutants. Mutations to rifampin and streptolydigin resistance have been located on the B. subtilis chromosome and ordered relative to the markers cysA14 and str.     

Genetic Characterization of Recombination-Deficient Mutants of Bacillus subtilis

Recombination-deficient (rec), radiation-sensitive mutations in Bacillus subtilis are grouped in at least seven distinct loci. Map positions are determined for six of these loci.     

Biochemical characterization of ribosome assembly GTPase RbgA in Bacillus subtilis

The ribosome biogenesis GTPase A protein RbgA is involved in the assembly of the large ribosomal subunit in Bacillus subtilis, and homologs of RbgA are implicated in the biogenesis of mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA give rise to a large ribosomal subunit that is immature and migrates at 45 S in sucrose density gradients. Here, we report a detailed biochemical analysis of RbgA and its interaction with the ribosome. We found that RbgA, like most other GTPases, exhibits a very slow k(cat) (14 h(-1)) and has a high K(m) (90 μM). Homology modeling of the RbgA switch I region using the K-loop GTPase MnmE as a template suggested that RbgA requires K(+) ions for GTPase activity, which was confirmed experimentally. Interaction with 50 S subunits, but not 45 S intermediates, increased GTPase activity by ～55-fold. Stable association with 50 S subunits and 45 S intermediates was nucleotide-dependent, and GDP did not support strong interaction with either of the subunits. GTP and guanosine 5'-(β,γ-imido)triphosphate (GMPPNP) were sufficient to promote association with the 45 S intermediate, whereas only GMPPNP was able to support binding to the 50 S subunit, presumably due to the stimulation of GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the ribosome.     

Characterization of aspartate kinase III of Bacillus subtilis

A search in the Bacillus subtilis genome sequence found that the gene designated yclM encode(s) a protein showing significant identity in amino acid sequence to aspartate kinases. When yclM was introduced into Escherichia coli cells deficient in all three aspartate kinase genes, production of a protein with molecular size 50 kDa, which was similar to the value deduced from the nucleotide sequence of the gene, was observed. Expectedly, the protein purified to homogeneity had aspartate kinase activity. The enzyme was significantly inhibited by simultaneous addition of both threonine and lysine, which is a typical feature of aspartate kinase III of B. subtilis. The enzyme was very unstable in 10 mM tris-HCl (pH 7.5) buffer, but was stabilized by addition of 500 mM ammonium sulfate. Although all the aspartate kinases so far investigated are oligomeric enzymes, this aspartate kinase was suggested to be a monomer.