Comparative Study of Bacterial SPOR Domains Identifies Functionally Important Differences in Glycan Binding Affinity

• Published in: Journal of bacteriology 2022-09, Vol.204 (9), p.e0025222
• Main Authors: Yahashiri, Atsushi, Kaus, Gabriela M, Popham, David L, Houtman, Jon C D, Weiss, David S
• Format: Article
• Language: English
• Subjects: Affinity; Amidohydrolases - metabolism; Bacteria - metabolism; Bacteriology; Binding; Calorimetry; Cell division; Cell walls; Comparative studies; E coli; Editor's Pick; Escherichia coli; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; Glycan; Microbial Genetics; Penicillin; Peptides; Peptidoglycan - metabolism; Peptidoglycans; Polysaccharides; Protein Binding; Proteins; Research Article; Spotlight Selection; Titration; Titration calorimetry
• ISSN: 0021-9193; 1098-5530; 1098-5530
• DOI: 10.1128/jb.00252-22

Bacterial SPOR domains target proteins to the divisome by binding septal peptidoglycan (PG) at sites where cell wall amidases have removed stem peptides. These PG structures are referred to as denuded glycans. Although all characterized SPOR domains bind denuded glycans, whether there are differences in affinity is not known. Here, we use isothermal titration calorimetry (ITC) to determine the relative PG glycan binding affinity ( K d ) of four Escherichia coli SPOR domains and one Cytophaga hutchinsonii SPOR domain. We found that the K d values ranged from approximately 1 μM for E. coli DamX SPOR and C. hutchinsonii CHU2221 SPOR to about 10 μM for E. coli FtsN SPOR . To investigate whether these differences in PG binding affinity are important for SPOR domain protein function, we constructed and characterized a set of DamX and FtsN "swap" proteins. As expected, all SPOR domain swap proteins localized to the division site, and, in the case of FtsN, all of the heterologous SPOR domains supported cell division. However, for DamX, only the high-affinity SPOR domain from CHU2221 supported normal function in cell division. In summary, different SPOR domains bind denuded PG glycans with different affinities, which appears to be important for the functions of some SPOR domain proteins (e.g., DamX) but not for the functions of others (e.g., FtsN). IMPORTANCE SPOR domain proteins are prominent components of the cell division apparatus in a wide variety of bacteria. The primary function of SPOR domains is targeting proteins to the division site, which they accomplish by binding to septal peptidoglycan. However, whether SPOR domains have any functions beyond septal targeting is unknown. Here, we show that SPOR domains vary in their PG binding affinities and that, at least in the case of the E. coli cell division protein DamX, having a high-affinity SPOR domain contributes to proper function.