Abstract
ABSTRACTSpores of Bacillus species have novel properties, which allow them to lie dormant for years and then germinate under favourable conditions. In the current work, the role of a key metabolic integrator, coenzyme A (CoA), in redox regulation of growing cells and during spore formation in Bacillus megaterium and Bacillus subtilis is studied. Exposing these growing cells to oxidising agents or carbon deprivation resulted in extensive covalent protein modification by CoA (termed protein CoAlation), through disulphide bond formation between the CoA thiol group and a protein cysteine. Significant protein CoAlation was observed during sporulation of B. megaterium, and increased largely in parallel with loss of metabolism in spores. Mass spectrometric analysis identified four CoAlated proteins in B. subtilis spores as well as one CoAlated protein in growing B. megaterium cells. All five of these proteins have been identified as moderately abundant in spores. Based on these findings and published studies, protein CoAlation might be involved in facilitating establishment of spores’ metabolic dormancy, and/or protecting sensitive sulfhydryl groups of spore enzymes.
Highlights
Bacterial spores of Firmicute species are likely nature's most resilient life forms, and are extremely resistant to desiccation, pressure, chemicals, heat, cold, and radiation, and can survive in unfavourable conditions for long periods (Paul 2019; Setlow 2014)
T Dormant bacterial spores are “preloaded” with low energy molecules such as NAD+ and IP AMP, key compounds needed to generate ATP and NADH (Loshon and Setlow 1993; R Setlow and Setlow 1977a). Another of these compounds is coenzyme A (CoA-SH, SC hereinafter referred to as CoA), a key metabolic integrator in all living organisms, not in an acylated form in spores but mostly disulphide bonded with protein cysteine residues
Analysis of the sporulation E efficiency by phase-contrast microscopy revealed that levels of phase-bright spores in cells N were:
Summary
Bacterial spores of Firmicute species are likely nature's most resilient life forms, and are extremely resistant to desiccation, pressure, chemicals, heat, cold, and radiation, and can survive in unfavourable conditions for long periods (Paul 2019; Setlow 2014). IGTo identify the heavily CoAlated proteins in wild-type spores, and to locate the sites of RCoAlation, three slices corresponding to the location of anti-CoA immunoreactive bands on O the Western blot (slice 1 (~12-22 kDa); slice 2 (~44-55 kDa); and slice 3 (~30-44 kDa)) were excised from the stained gel (Figure 3B) and subjected to digestion, treatment with Nudix to remove the 3-phosphoAMP and enrichment with an IMAC column before LC-MS/MS analysis (Figure 4A) This analysis identified CoAlated peptides from 4 proteins - YneT, the H1 homolog of the thiol-specific peroxidase AhpC-H1, alcohol dehydrogenase AdhB and phosphopentomutase Drm (Figure 4B and Table 1). YneT is of particular interest as its molecular weight is in the range of the multiple CoAlated spore proteins at much lower levels in spores that lack most coat proteins
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