Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

Abstract

Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database. The biosynthetic route to the nucleotide dUMP, the precursor of dTTP, as shown in Fig. 1, differs between organisms and sets it apart from the rest of nucleotide metabolism (1). This is in agreement with the general belief that uracil was the original DNA base-pairing partner for adenine and that the biosynthesis of dTTP from dUMP arose later in evolution (2). The deoxyribonucleotides dGTP, dATP, and dCTP are all produced in a pathway that goes through the multisubstrate enzyme ribonucleotide reductase. This enzyme is highly regulated in order to maintain the balance of deoxyribonucleotide pools (3-5). dUTP is also formed by ribonucleotide reductase, but because of the toxicity of this nucleotide (6, 7), all organisms express an enzyme with dUTPase activity that hydrolyzes dUTP to dUMP (8).The direct formation of dUMP via ribonucleotide reductase (Fig. 1) is not an efficient process, as the ribonucleotide reductase has a reduced affinity for UDP compared to the affinities of three other canonical ribonucleotides (9-12). Instead, most of the dUMP arises from deamination of cytosine deoxyribonucleotides (13-15). Enteric bacteria synthesize an enzyme with dCTP deaminase activity that converts dCTP into dUTP (EC 3.5.4.13), which accounts for 70 to 80% of the dUMP production (15). The dCTP deaminase is a homotrimeric enzyme structurally related to the trimeric dUTPase and part of the dCTP deaminase/dUTPase superfamily (16-18). The genomes of many Gram-positive bacteria and all eukaryotes encode a dCMP deaminase (EC 3.5.4.12) that shares no resemblance to the enteric dCTP de...

The Bacillus subtilis dCMP deaminase ComEB acts as a dynamic polar localization factor for ComGA within the competence machinery

“…We show that ComEB Geo (from the related organism Geobacillus ) has dCMP deaminase (DCD) activity, important for nucleotide metabolism, also shown for ComEB of B. halodurans (Oehlenschlaeger et al, ). Our findings indicate an oligomeric structure for ComEB Geo , based on the sigmoidal curve of enzyme kinetics, as was found for ComEB of B. halodurans (Oehlenschlaeger et al, ).…”

“…So far, it has not been known whether the enzymatic activity of ComEB plays a role in the formation of the competence complex or the uptake of DNA during natural transformation. In B. halodurans strain 125, the homolog of B. subtilis ComEB carries out a 2′‐dCMP deaminase activity (Oehlenschlaeger et al, ), catalyzing the deamination of dCMP to 2′‐deoxyuridylic monophosphate acid (dUMP). We found that the B. subtilis protein remained insoluble during purification, but were able to purify ComEB of Geobacillus thermodenitrificans, heterologously expressed in E. coli BL21(DE3) cells via an N‐terminal 6 x Histidin tag .…”

“…The function of ComEB, encoded in the middle of the comE operon, is not known, while the product of the homologous gene of Bacillus halodurans carried out 2′‐deoxycytidylate monophosphate (dCMP) deaminase (dcd) activity in vitro (Oehlenschlaeger et al, ). To our knowledge, no cellular function in the context of B. subtilis competence was annotated for this protein so far.…”

“…An in‐frame deletion removing 44% of the protein did not yield any phenotype, indicating that the protein is dispensable for transformation, but the authors could not exclude that a domain relevant for transformation was still encoded (Inamine & Dubnau, ). Several Bacillus species, including B. halodurans , Bacillus cereus and Bacillus megaterium, encode a bifunctional dCTPase DCD:DUT, in addition to the comEB gene, to generate dUMP directly from dCMP (Oehlenschlaeger et al, ). The enzyme is able to deaminate dCTP and hydrolyze dUTP to form dUMP, which then serves as a general precursor for dTTP in nucleotide metabolism (Jensen, Dandanell, Hove‐Jensen, & WillemoËs, ; Oehlenschlaeger et al, ).…”

“…Several Bacillus species, including B. halodurans , Bacillus cereus and Bacillus megaterium, encode a bifunctional dCTPase DCD:DUT, in addition to the comEB gene, to generate dUMP directly from dCMP (Oehlenschlaeger et al, ). The enzyme is able to deaminate dCTP and hydrolyze dUTP to form dUMP, which then serves as a general precursor for dTTP in nucleotide metabolism (Jensen, Dandanell, Hove‐Jensen, & WillemoËs, ; Oehlenschlaeger et al, ). In B. subtilis, dUMP can be produced by a pathway involving ribonucleotide reductase (Reichard, ) and two dUTPases (YosS, YncF), in which dUTP is finally hydrolyzed to dUMP (Garcia‐Nafria, Timm, Harrison, Turkenburg, & Wilson, ; Johansson, Neuhard, Willemoes, & Larsen, ).…”