Date

1-4-2016 12:00 AM

Major

Microbiology; Genetics

Department

Microbiology

College

College of Agriculture and Life Sciences

Project Advisor

Gregory Phillips

Project Advisor's Department

Veterinary Diagnostic & Production Animal Medicine

Description

Complementation tests are fundamental for understanding gene function. Characterization of essential bacterial genes poses a challenge, however, because a functional copy of the gene must be provided in a haploid organism. We have developed a new visual genetic system to conduct complementation tests that characterize the essential Escherichia coli ffh gene, which encodes a subunit of the signal recognition particle (SRP). The Ffh protein contains a methionine-rich carboxy-terminal domain (M domain) important for binding the hydrophobic signal sequences of nascent membrane proteins. To assess the importance of these methionine residues, we created multiple alleles where each methionine of the M domain was converted to tryptophan, valine, leucine, phenylalanine, tyrosine or isoleucine. We also constructed an E. coli mutant (AC101) where an ffh deletion was complemented by an unstable plasmid encoding wild type ffh, along with a purple chromoprotein. To test the system, AC101 was transformed with plasmids carrying the different ffh alleles. Complementation was easily confirmed by observing white colonies, indicating loss of the unstable plasmid. Using this system we found that only valine could substitute for the methionine residues. This genetic system should be amenable to isolate and characterize mutant alleles of many other essential bacterial genes.

File Format

application/pdf

Included in

Microbiology Commons

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Apr 1st, 12:00 AM

Development of a visual complementation system: application to the essential ffh gene of Escherichia coli

Complementation tests are fundamental for understanding gene function. Characterization of essential bacterial genes poses a challenge, however, because a functional copy of the gene must be provided in a haploid organism. We have developed a new visual genetic system to conduct complementation tests that characterize the essential Escherichia coli ffh gene, which encodes a subunit of the signal recognition particle (SRP). The Ffh protein contains a methionine-rich carboxy-terminal domain (M domain) important for binding the hydrophobic signal sequences of nascent membrane proteins. To assess the importance of these methionine residues, we created multiple alleles where each methionine of the M domain was converted to tryptophan, valine, leucine, phenylalanine, tyrosine or isoleucine. We also constructed an E. coli mutant (AC101) where an ffh deletion was complemented by an unstable plasmid encoding wild type ffh, along with a purple chromoprotein. To test the system, AC101 was transformed with plasmids carrying the different ffh alleles. Complementation was easily confirmed by observing white colonies, indicating loss of the unstable plasmid. Using this system we found that only valine could substitute for the methionine residues. This genetic system should be amenable to isolate and characterize mutant alleles of many other essential bacterial genes.