Genetic and Biochemical Properties of Streptococcal NAD-glycohydrolase Inhibitor

Hisashi Kimoto,Yutaka Fujii,Satoko Hirano,Yoshifumi Yokota,Akira Taketo

doi:10.1074/jbc.m506879200

Hisashi Kimoto, Yutaka Fujii + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m506879200

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Abstract

The gene encoding streptolysin O (slo), a cytolysin of hemolytic streptococci, is transcribed polycistronically from the promoter of the preceding NAD-glycohydrolase (NADase) gene (nga). Between nga and slo, a putative open reading frame (orf1) is located whose function has been totally unknown. Present investigation demonstrated that the orf1 encodes a protein designated as streptococcal NADase inhibitor (SNI). From its nucleotide sequence, SNI was inferred to comprise 161 amino acid residues and the deduced molecular weight was 18,800. This protein was detectable only within cells. Coexpression of SNI was essential for production of streptococcal NADase, and NADase precursor existed as an inactive complex with SNI, in recombinant Escherichia coli. Monomeric NADase and SNI rapidly formed in vitro a stable heterodimer complex in the ratio 1:1, resulting in complete suppression of the hydrolase activity. Unlike other bacterial NADase inhibitors, SNI was thermostable. This protein, coexpressed and complexed with NADase, may protect the producer cocci from exhaustion of NAD.

Highlights

We demonstrated that slo is a member of an operon covering the upper-stream nusG and nga genes, from which transcription of slo proceeds polycistronically, and major transcript is produced by readthrough from nga promoter [16]
The data presented above demonstrate that this gene encodes a 18.8-kDa protein functioning as a streptococcal NADase inhibitor (SNI)
In contrast to failure of subcloning of the single nga gene in E. coli [16], coexpression of the gene with orf1 takes place steadily, in the heterologous host harboring the recombinant plasmid with ngaorf1 insert

Summary

EXPERIMENTAL PROCEDURES

Materials—Mutanolysin and lysozyme were purchased from Sigma. Ampicillin sodium and L-arabinose were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Preparation of TSKgel BioAssist Ni2ϩ-chelate Column—A TSKgel BioAssist chelate column (Tosoh Co., Tokyo, Japan), prewashed with 50 mM sodium phosphate buffer, pH 7.4, containing 0.01% sodium azide (Buffer A), was loaded with 4 ml of 0.1 M Ni2SO4 solution, washed with 10 ml of Buffer A containing 500 mM imidazole, and equilibrated with Buffer A containing 20 mM imidazole at room temperature, with a flow rate of 1 ml/min, using a Tosoh HPLC system equipped with a PEEK-type pump (Tosoh Co.) Purification of His Tag-fused NADase-Orf Complex—The BugBuster extract of E. coli TOP10 harboring recombinant plasmid pBAD/ His I (encoding His tag-fused NADase and intact Orf1) was dialyzed against 2 liters of Buffer A, at 4 °C overnight. 50 mM potassium phosphate buffer (pH 7.5) containing bovine serum albumin alone was used, instead of the incubated Orf solution

RESULTS

Whole cell extract treated with

NaSCN in

DISCUSSION