Genetic analysis of the cell division protein FtsI (PBP3): amino acid substitutions that impair septal localization of FtsI and recruitment of FtsN.
FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of several proteins that localize to the septal ring. FtsI comprises a small cytoplasmic domain, a transmembrane helix, a noncatalytic domain of unknown function, and a catalytic (transpeptidase) domain. The last two domains reside in the periplasm. We used PCR to randomly mutagenize ftsI, ligated the products into a green fluorescent protein fusion vector, and screened approximately 7,500 transformants for gfp-ftsI alleles that failed to complement an ftsI null mutant. Western blotting and penicillin-binding assays were then used to weed out proteins that were unstable, failed to insert into the cytoplasmic membrane, or were defective in catalysis. The remaining candidates were tested for septal localization and ability to recruit another division protein, FtsN, to the septal ring. Mutant proteins severely defective in localization to the septal ring all had lesions in one of three amino acids-R23, L39, or Q46-that are in or near the transmembrane helix and implicate this region of FtsI in septal localization. Mutant FtsI proteins defective in recruitment of FtsN all had lesions in one of eight residues in the noncatalytic domain. The most interesting of these mutants had lesions at G57, S61, L62, or R210. Although separated by approximately 150 residues in the primary sequence, these amino acids are close together in the folded protein and might constitute a site of FtsI-FtsN interaction.
Wissel
Mark C
MC
Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA.
Weiss
David S
DS
D019943:Amino Acid Substitution; D000818:Animals; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005816:Genetic Complementation Test; D006358:Hot Temperature; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D010406:Penicillins / Q000378:metabolism; D010458:Peptidyl Transferases / Q000737:chemistry / Q000235:genetics*; D011817:Rabbits
D029968:Escherichia coli Proteins; C098711:FtsN protein, E coli; D008565:Membrane Proteins; D010406:Penicillins; D010458:Peptidyl Transferases