Table 1.
Bacteria and plasmids used in this study.
Table 2.
Primers used in this study.
Fig 1.
Comparison of the deduced amino acid sequence of B. subtilis YutF with characterized members of a type IIA subfamily of HADSF.
The conserved residues involved in catalysis are shown in red, and the residues required for coordinating the Mg ion in the active site are underlined. Approximate areas of the four conserved motifs (I-IV) are shaded in yellow. The conserved residues from the cap domain C2 that can act as a substrate specificity loop (SSL) are shaded in green. Similar (‘.’ and ‘:’) and identical (‘*’) amino acids are indicated. The following protein sequences were used (GenBank accession numbers are indicated in parentheses): YutF_Bs, putative hydrolase from B. subtilis (NP_391109.1); UmpH_Eco, UMP phosphatase from E. coli (NP_415201.1); and AraL_Bs, sugar phosphatase from B. subtilis (NP_390755.1). The multiple sequence alignment was generated using the CLUSTALW program [34].
Fig 2.
Comparison of the 3D structures of UmpH and YutF.
Ribbon diagram representations of the 3D structures of UmpH (PDB id: 2c4n) and YutF (PDB id: 3pdw) (in the center) and magnified views of the substrate specificity loop (SSL) and the core domain configurations (on the top and bottom, respectively). The core domain and SSL residues are shown in yellow, and their regions are highlighted by black boxes. The identities of conserved residues involved in catalysis are indicated. This figure was prepared using 3D-Mol Viewer (a component of Vector NTI Advance 10 software, https://www.invitrogen.com/).
Table 3.
pNPPase activity in strains with various levels of yutF expression.
Table 4.
Study of the substrate specificity of recombinant Ht-YutF.
Table 5.
Kinetic parameters of recombinant Ht-YutF.
Fig 3.
Schematic representation of the B. subtilis 168 yutDEF region in the constructed strains.
(A). Left: The B. subtilis 168 yutD-yutE-yutF region (top) and chromosomal transcription fusions of the yutF region to a promoterless lacZ in derivatives of B. subtilis 168, strains BsA1, BsA2, BsA3, BsB1, BsB2 and BsB3 (bottom). The yutD-yutE-yutF region fragments fused to a promoterless lacZ are denoted by thick black lines. Promoters (P and Pspac) and rho-independent transcription terminators are indicated. Right: specific β-galactosidase activity (Miller units, MU) of crude cell extracts from the indicated strains. The values are the means ± standard errors of at least three independent experiments. (B). The yutDEF region in pMUTIN2-yutF-containing strains. The deleted fragments in the yutDEF promoter region and in the yutF coding region in BsΔPMTNyutF and BsMTNΔyutF, respectively, are indicated by Δ.
Table 6.
The influence of YutF production on yutF expression in strains with pMUTIN2-borne transcriptional fusions.
Fig 4.
Effect of inorganic phosphate and IPTG on the induction of β-galactosidase in BsMTNyutF.
β-galactosidase activity in BsMTNyutF during cultivation in glucose phosphate-free minimal medium without IPTG or KH2PO4 (circles), 1 mM IPTG without KH2PO4 (triangles), 1 mM KH2PO4 without IPTG (diamonds), and 1 mM IPTG and 1 mM KH2PO4 (squares) was measured as described in Materials and methods. The results are expressed as the means ± standard errors of at least three independent experiments.