SpoIID-mediated peptidoglycan degradation is required throughout engulfment during Bacillus subtilis sporulation.
SpoIID is a membrane-anchored enzyme that degrades peptidoglycan and is essential for engulfment and sporulation in Bacillus subtilis. SpoIID is targeted to the sporulation septum, where it interacts with two other proteins required for engulfment: SpoIIP and SpoIIM. We changed conserved amino acids in SpoIID to alanine to determine whether there was a correlation between the effect of each substitution on the in vivo and in vitro activities of SpoIID. We identified one amino acid substitution, E88A, that eliminated peptidoglycan degradation activity and one, D210A, that reduced it, as well as two substitutions that destabilized the protein in B. subtilis (R106A and K203A). Using these mutants, we show that the peptidoglycan degradation activity of SpoIID is required for the first step of engulfment (septal thinning), as well as throughout membrane migration, and we show that SpoIID levels are substantially above the minimum required for engulfment. The inactive mutant E88A shows increased septal localization compared to the wild type, suggesting that the degradation cycle of the SpoIID/SpoIIP complex is accompanied by the activity-dependent release of SpoIID from the complex and subsequent rebinding. This mutant is also capable of moving SpoIIP across the sporulation septum, suggesting that SpoIID binding, but not peptidoglycan degradation activity, is needed for relocalization of SpoIIP. Finally, the mutant with reduced activity (D210A) causes uneven engulfment and time-lapse microscopy indicates that the fastest-moving membrane arm has greater concentrations of SpoIIP than the slower-moving arm, demonstrating a correlation between SpoIIP protein levels and the rate of membrane migration.
Gutierrez
Jennifer
J
Division of Biological Sciences, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0377, USA.
Smith
Rachelle
R
Pogliano
Kit
K
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D049452:Green Fluorescent Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010457:Peptidoglycan / Q000378:metabolism*; D021381:Protein Transport; D013171:Spores, Bacterial / Q000502:physiology*
D001426:Bacterial Proteins; D010457:Peptidoglycan; C030069:spore-specific proteins, Bacillus; D049452:Green Fluorescent Proteins
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