Expression of UGA-containing Mycoplasma genes in Bacillus subtilis

Abstract

We used Bacillus subtilis to express UGA-containing Mycoplasma genes encoding the P30 adhesin (one UGA) of Mycoplasma pneumoniae and methionine sulfoxide reductase (two UGAs) of Mycoplasma genitalium. Due to natural UGA suppression, these Mycoplasma genes were expressed as full-length protein products, but at relatively low efficiency, in recombinant wild-type Bacillus. The B. subtilis-expressed Mycoplasma proteins appeared as single bands and not as multiple bands compared to expression in recombinant Escherichia coli. Bacillus mutants carrying mutations in the structural gene (prfB) for release factor 2 markedly enhanced the level of readthrough of UGA-containing Mycoplasma genes.

FIG. 1

Expression of M. pneumoniae gene p30 in B. subtilis and E. coli. B. subtilis strains BS-28 (control) and BS-P30 (p30) were cultivated in LB medium and induced with 1 mM IPTG. E. coli strain INVαF′ carrying the 4P3 plasmid (UGA-corrected p30) was grown in LB medium under anaerobic conditions for the expression of P30 from the nirB promoter. Sonicated protein extracts (120 μg) were fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting with P30 antibodies. (A) Immunoblot analysis of UGA readthrough of p30 gene in wild-type B. subtilis PY22. Lanes: 1, BS-28 with no IPTG; 2, BS-28 with 1 mM IPTG; 3, BS-P30 with no IPTG; 4, BS-P30 with 1 mM IPTG. (B) Immunoblot analysis of multiple P30-related proteins from the expression of the UGA-corrected p30 gene in E. coli. Lanes: 1, M. pneumoniae; 2, E. coli (4P3, UGA corrected); 3, E. coli (pDG28-30, not UGA corrected).

FIG. 2

Comparison of UGA readthrough of Mycoplasma genes p30 and msr among the different prfB mutant strains of B. subtilis. The B. subtilis strains were cultivated in LB medium with 0.5% glucose and induced with IPTG (1 mM). (A) Immunoblot analysis of the P30 protein induction. Lanes: 1, BS5-P30 (prfB5); 2, BS3-P30 (prfB3); 3, BS2-P30 (prfB2); 4, BS4-P30 (prfB4). (B) Immunoblot analysis of MSR protein induction. Lanes: 1, BS5-MSR (prfB5) uninduced; 2, BS5-MSR (prfB5); 3, BS2-MSR (prfB2); 4, BS4-MSR (pfrB4); 5, BS3-MSR (prfB3); 6, BS-MSR (wild type); 7, BS-MSR (wild type) uninduced.