Membrane Proteomes and Ion Transporters in Bacillus anthracis and Bacillus subtilis Dormant and Germinating Spores

Abstract

Bacterial endospores produced by Bacillus and Clostridium species can remain dormant and highly resistant to environmental insults for long periods, but they can also rapidly germinate in response to a nutrient-rich environment. Multiple proteins involved in sensing and responding to nutrient germinants, initiating solute and water transport, and accomplishing spore wall degradation are associated with the membrane surrounding the spore core. In order to more fully catalog proteins that may be involved in spore germination, as well as to identify protein changes taking place during germination, unbiased proteomic analyses of membrane preparations isolated from dormant and germinated spores of Bacillus anthracis and Bacillus subtilis were undertaken. Membrane-associated proteins were fractionated by SDS-PAGE, gel slices were trypsin digested, and extracted peptides were fractionated by liquid chromatography and analyzed by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. More than 500 proteins were identified from each preparation. Bioinformatic methods were used to characterize proteins with regard to membrane association, cellular function, and conservation across species. Numerous proteins not previously known to be spore associated, 6 in B. subtilis and 68 in B. anthracis, were identified. Relative quantitation based on spectral counting indicated that the majority of spore membrane proteins decrease in abundance during the first 20 min of germination. The spore membranes contained several proteins thought to be involved in the transport of metal ions, a process that plays a major role in spore formation and germination. Analyses of mutant strains lacking these transport proteins implicated YloB in the accumulation of calcium within the developing forespore.IMPORTANCE Bacterial endospores can remain dormant and highly resistant to environmental insults for long periods but can also rapidly germinate in response to a nutrient-rich environment. The persistence and subsequent germination of spores contribute to their colonization of new environments and to the spread of certain diseases. Proteins of Bacillus subtilis and Bacillus anthracis were identified that are associated with the spore membrane, a position that can allow them to contribute to germination. A set of identified proteins that are predicted to carry out ion transport were examined for their contributions to spore formation, stability, and germination. Greater knowledge of spore formation and germination can contribute to the development of better decontamination strategies.

Keywords: Bacillus anthracis; germination; membrane; proteome; spore; subtilis.

FIG 1

Predicted membrane-spanning domains of B. anthracis and B. subtilis spore membrane proteins. Predictions of membrane association mechanisms were made for proteins identified in membrane fractions as described in Materials and Methods. Proteins were further classified based upon their predicted number of membrane-spanning helices.

FIG 2

Mutant strains lacking YloB produce many phase-dark spores. B. subtilis strains were grown and sporulated in 2×SG medium, and spores were purified by water washing. Phase-contrast microscopy revealed that approximately 50% of the spores produced by all strains containing yloB deletion mutations were phase dark. Scale bars, 5 μm.

FIG 3

Ca²⁺ content of forespore and mother cell in sporulating cells. Strains were grown with shaking in 2×SG medium at 37°C and OD₆₀₀ was measured. Samples were removed at T₆ or T₈ and treated with lysozyme and several rounds of centrifugation to separate forespore and mother cell compartments. The samples were extracted with HCl and Ca²⁺ was quantified using atomic emission spectroscopy. Values give the averages from three assays, and error bars indicate standard deviations.

FIG 4

Ion contents of purified phase-bright spores. Purified spores were extracted with HCl, and the amounts of various ions were quantified using atomic emission spectroscopy. Values are averages from three to nine assays, depending on the strain, and are expressed as percentages of the values found in PS832 (wild type) spores prepared on the same day. The strains examined were DPVB693 (ΔyloB), DPVB706 (Δ3), DPVB715 (ΔyugS::mls), DPVB716 (ΔchaA::spec), and DPVB722 (Δ5ΔyloB). Error bars indicate standard errors. *, significant difference from PS832 (P ≤ 0.05).

FIG 5

Germination of purified phase-bright spores. Purified spores of B. subtilis wild-type and mutant strains were heat activated and stimulated to germinate by the addition of 10 mM l-alanine. Values are averages from three assays, and error bars indicate the standard deviations. For DPVB693 (ΔyloB) all points after 2 min and for DPVB722 (Δ5ΔyloB) all points are significantly different from those of PS832 (wild type) and DPVB706 (Δ3) (P ≤ 0.05).

FIG 6

Release of ions by germinating spores. Spores were germinated as described in Materials and Methods. Samples were collected at the indicated times and centrifuged briefly to pellet spores. Ions in the germination exudate were quantified using atomic emission spectroscopy. Values are averages from three assays, and error bars indicate standard deviations. *, points significantly different from those of PS832 (wild type) and DPVB693 (ΔyloB) (P ≤ 0.05).