Bacillus subtilis 5'-nucleotidases with various functions and substrate specificities

Abstract

Background: In Escherichia coli, nagD, yrfG, yjjG, yieH, yigL, surE, and yfbR encode 5'-nucleotidases that hydrolyze the phosphate group of 5'-nucleotides. In Bacillus subtilis, genes encoding 5'-nucleotidase have remained to be identified.

Results: We found that B. subtilis ycsE, araL, yutF, ysaA, and yqeG show suggestive similarities to nagD. Here, we expressed them in E. coli to purify the respective His₆-tagged proteins. YcsE exhibited significant 5'-nucleotidase activity with a broader specificity, whereas the other four enzymes had rather weak but suggestive activities with various capacities and substrate specificities. In contrast, B. subtilis yktC shares high similarity with E. coli suhB encoding an inositol monophosphatase. YktC exhibited inositol monophosphatase activity as well as 5'-nucleotidase activity preferential for GMP and IMP. The ycsE, yktC, and yqeG genes are induced by oxidative stress and were dispensable, although yqeG was required to maintain normal growth on solid medium. In the presence of diamide, only mutants lacking yktC exhibited enhanced growth defects, whereas the other mutants without ycsE or yqeG did not.

Conclusions: Accordingly, in B. subtilis, at least YcsE and YktC acted as major 5'-nucleotidases and the four minor enzymes might function when the intracellular concentrations of substrates are sufficiently high. In addition, YktC is involved in resistance to oxidative stress caused by diamide, while YqeG is necessary for normal colony formation on solid medium.

Keywords: 5′-nucleotidase; Bacillus subtilis; Haloacid dehalogenase superfamily; Inositol monophosphatase; Inositol phosphate; Nucleoside/nucleotide metabolism; Oxidative stress; Phosphatase; Protein motif.

Fig. 1

Similarities among the amino acid sequences of the proteins encoded by E. coli nagD homologs in the B. subtilis genome. The amino acid sequences of the proteins encoded by B. subtilis araL, yutF, yqeG, ysaA, ycsE, gapB, ftsA, and hprP, and E. coli nagD were analyzed using CLUSTALW (default fast/approximate settings [http://www.genome.jp/tools/clustalw/]) to generate the phylogenic tree

Fig. 2

Phosphatase activities of B. subtilis proteins homologous to E. coli NagD. Each purified protein (0.25 mg/ml, indicated on the right) was incubated with 2 mM (a) or 10 mM (b) of various substrates for 12 h and assayed for phosphatase activity as described in Methods. All presented data are the mean values of three independent experiments ± SD

Fig. 3

Growth curves of B. subtilis strains. Strains 168 (a), NON01 (b ΔycsE), NON02 (c ΔyktC), NON03 (d ΔycsE ΔyktC), NON05 (e and f, ΔyqeG Pspac-yqeG), and NON06 (g and h, ΔycsE ΔyktC ΔyqeG Pspac-yqeG) were inoculated into liquid medium and their growth was monitored. At the times indicated by the arrowheads, diamide was added to final concentrations of 0 mM (open circle), 1 mM (solid square), and 4 mM (solid triangle). Strains NON05 and NON06 were grown in the presence (e and g) and absence (f and h) of 1 mM IPTG. All experiments were repeated more than three times and similar results were observed

Fig. 4

Colony formation by B. subtilis strains. Suspensions of strains 168, NON05, and NON06 were streaked on LB plates with (right) or without (left) 1 mM IPTG and incubated overnight at 37 °C. All experiments were repeated more than three times and similar results were observed