ExsB, an unusually highly phosphorylated protein required for the stable attachment of the exosporium of Bacillus anthracis.
The outermost layer of the Bacillus anthracis spore, the exosporium, is composed of a paracrystalline basal layer and an external hair-like nap. The nap is formed from a single collagen-like glycoprotein, while the basal layer contains many different proteins, including a 186-amino acid protein called ExsB. In this study, we discovered that ExsB is unusually highly phosphorylated, with at least 14 of its 19 threonine residues modified. The phosphorylated threonines are included in seven contiguous approximately 12-residue imperfect repeats, which presumably contain kinase recognition sequences. We demonstrated that a B. anthracis DeltaexsB mutant unable to synthesize ExsB produced spores with an exosporium that was readily sloughed, indicating that ExsB was required for stable exosporium attachment. This unstable exosporium also lacked the enzyme alanine racemase, which is normally tightly associated with the exosporium. Additionally, purified DeltaexsB spores lacking a visible exosporium were devoid of most exosporium proteins but, surprisingly, retained the putative exosporium proteins BxpC and CotB-1. Finally, we showed that transcription of the exsB gene occurred only during the late stages of sporulation, and we used an active and phosphorylated ExsB-EGFP fusion protein to monitor ExsB localization to wild-type and DeltabxpB mutant exosporia.
McPherson
Sylvia A
SA
Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Li
Mei
M
Kearney
John F
JF
Turnbough
Charles L
CL
D000411:Alanine Racemase / Q000032:analysis; D000595:Amino Acid Sequence; D001408:Bacillus anthracis / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D002473:Cell Wall / Q000737:chemistry* / Q000378:metabolism*; D017353:Gene Deletion; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013171:Spores, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013912:Threonine; D000411:Alanine Racemase