In vitro and in vivo oxidation of methionine residues in small, acid-soluble spore proteins from Bacillus species

Abstract

Methionine residues in alpha/beta-type small, acid-soluble spore proteins (SASP) of Bacillus species were readily oxidized to methionine sulfoxide in vitro by t-butyl hydroperoxide (tBHP) or hydrogen peroxide (H2O2). These oxidized alpha/beta-type SASP no longer bound to DNA effectively, but DNA binding protected alpha/beta-type SASP against methionine oxidation by peroxides in vitro. Incubation of an oxidized alpha/beta-type SASP with peptidyl methionine sulfoxide reductase (MsrA), which can reduce methionine sulfoxide residues back to methionine, restored the alpha/beta-type SASP's ability to bind to DNA. Both tBHP and H2O2 caused some oxidation of the two methionine residues of an alpha/beta-type SASP (SspC) in spores of Bacillus subtilis, although one methionine which is highly conserved in alpha/beta-type SASP was only oxidized to a small degree. However, much more methionine sulfoxide was generated by peroxide treatment of spores carrying a mutant form of SspC which has a lower affinity for DNA. MsrA activity was present in wild-type B. subtilis spores. However, msrA mutant spores were no more sensitive to H2O2 than were wild-type spores. The major mechanism operating for dealing with oxidative damage to alpha/beta-type SASP in spores is DNA binding, which protects the protein's methionine residues from oxidation both in vitro and in vivo. This may be important in vivo since alpha/beta-type SASP containing oxidized methionine residues no longer bind DNA well and alpha/beta-type SASP-DNA binding is essential for long-term spore survival.

FIG. 1

Analysis of tryptic peptides from untreated (A) and tBHP-oxidized (B) SspC. SspC and SspC oxidized with tBHP for 3 h were digested with trypsin, and the products were resolved by HPLC as described in Materials and Methods. Because tBHP was removed by dialysis, the actual exposure to oxidant was longer than 3 h. The SspC peptides identified are as follows: 1, A47 to K57; 2 and 2*, L62 to H72; 3, L29 to R46; 4 and 4*, S9 to K28; and 5, S9 to R46. Peptides 2*, 4*, and 5 contain methionine sulfoxide residues. Peptide 5 results from additional oxidation at lysine 28. The identity of this peptide was established by performing amino acid analysis and mass spectrometry and determining resistance to trypsin cleavage. No peptides were eluted prior to 10 min.

FIG. 2

Ability of untreated and tBHP-oxidized α/β-type SASP to provide DNase protection to plasmid pUC19. Purified SspC and SASP-C were treated with tBHP for 3 h, dialyzed, complexed with linearized plasmid pCU19 DNA, and DNase treated as described in Materials and Methods. DNA was isolated and analyzed by agarose gel electrophoresis. The arrows labeled a and b indicate the positions of 2.3- and 0.56-kb DNA size markers, respectively. Samples run in the various lanes are as follows: lane 1, SspC without DNase treatment; lane 2, no α/β-type SASP; lane 3, untreated SspC; lane 4, tBHP-treated SspC; lane 5, untreated SASP-C; and lane 6, tBHP-treated SASP-C. SspC contained ∼3 and ∼21% methionine sulfoxide at M27 and M67, respectively. Oxidized SspC contained ∼79 and ∼86% methionine sulfoxide at M27 and M67, respectively. SASP-C and oxidized SASP-C contained ∼8 and ∼88% methionine sulfoxide at M28, respectively. Oxidized SspC and SASP-C also showed ∼8 and ∼17% oxidation at lysines 28 and 29, respectively. The bands at ∼2.7 kb in lanes 3 and 6 are probably microdrops of the incubation mixtures which escaped exposure to DNase.