Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis

Abstract

The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B. subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B. anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B. anthracis genome for potential coat proteins. Our analysis suggests that the B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B. anthracis detection methods and understand the ecological role of the coat.

FIG. 1.

B. subtilis coat proteins resolved by 1D standard SDS-15% PAGE. The gel was stained with Coomassie blue G-250. The indicated bands were prepared for MALDI MS and MS/MS mapping as described in Materials and Methods. Protein identifications correspond to those described in Table 1.

FIG. 2.

Preliminary master gel of B. subtilis coat preparations and MALDI analysis. (A) pH 3 to 10 gel of B. subtilis coat preparations. Sample was solubilized in 10% LDS buffer. The solution was diluted in an excess of standard solubilization buffer containing 2% C8φ and 1% TX-100 and resolved by IEF. The second dimension was carried out on an SDS-11% polyacrylamide gel and silver stained (56). Spots identified by peptide mass fingerprinting are circled and numbered, and the identifications are presented in Table 2. (B) MALDI-MS spectrum for spot 1.

FIG. 3.

Improved 2D separation. One hundred eighty microliters of a spore pellet was solubilized in 100 μl of 1× SDS buffer after being boiled for 10 min. Ninety microliters of this solution was added to 810 μl of 1.1× 2% C8φ-1% TX-100 in standard rehydration buffer and incubated at 30°C for 1 h before rehydration. Four hundred fifty microliters each of this solution was run on a pH 3 to 10 (A) and a pH 4 to 7 (B) IEF strip. Frequent changes of filter paper wicks during IEF and focusing up to 120 kV · h until the dye ran out completely at the acidic end greatly improved separation. The second dimension is as in Fig. 2.

FIG. 4.

B. anthracis coat proteins resolved by 1D standard SDS-15% PAGE. The samples were solubilized with SDS, and the resulting gel was stained with Coomassie blue G-250. Proteins were identified by MALDI-TOF MS, and the identifications are presented in Table 4.