Saturating mutagenesis of an essential gene: a majority of the Neisseria gonorrhoeae major outer membrane porin (PorB) is mutable.
The major outer membrane porin (PorB) of Neisseria gonorrhoeae is an essential protein that mediates ion exchange between the organism and its environment and also plays multiple roles in human host pathogenesis. To facilitate structure-function studies of porin's multiple roles, we performed saturating mutagenesis at the porB locus and used deep sequencing to identify essential versus mutable residues. Random mutations in porB were generated in a plasmid vector, and mutant gene pools were transformed into N. gonorrhoeae to select for alleles that maintained bacterial viability. Deep sequencing of the input plasmid pools and the output N. gonorrhoeae genomic DNA pools identified mutations present in each, and the mutations in both pools were compared to determine which changes could be tolerated by the organism. We examined the mutability of 328 amino acids in the mature PorB protein and found that 308 of them were likely to be mutable and that 20 amino acids were likely to be nonmutable. A subset of these predictions was validated experimentally. This approach to identifying essential amino acids in a protein of interest introduces an additional application for next-generation sequencing technology and provides a template for future studies of both porin and other essential bacterial genes.
Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
Adrienne
A
Chen
H Steven
HS
Seifert
D000595:Amino Acid Sequence; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009344:Neisseria gonorrhoeae / Q000235:genetics / Q000378:metabolism*; D018272:Porins / Q000235:genetics / Q000378:metabolism*
D018272:Porins; C057699:porin protein, Neisseria