Construction and characterization of yciGFE mutants in Escherichia coli



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Texas A&M University


Escherichia coli has served as a model organism for studies in molecular genetics and physiology since the 1960s. Yet the function of 20% of the 4288 known and predicted proteins in E. coli is still not known. Almost two-thirds of these proteins have homologs in other microorganisms, but their function(s) is not known in any organism. One such protein is YciG. YciG was chosen as the focus of this study because, intriguingly, an ortholog of YciG is found in the genome of the fungus Neurospora crassa. The gene encoding YciG is predicted to be in an operon with two other genes, yciF and yciE. Genes in the same operon often encode proteins with related functions, so the study was extended to include YciF and YciE. To determine the function of these proteins, in-frame deletion alleles were constructed and strains lacking one or more of the three proteins were tested for mutant phenotypes. Expression of the yciGFE operon is induced by several stresses and is regulated by RpoS, which controls the general stress response in E. coli. Therefore, we tested the ability of the mutant strains to survive environmental stresses. Our results revealed that YciG was important for stationary-phase resistance to thermal stress, oxidative stress and, in particular, acid stress. Both RpoS-dependent and RpoS-independent acid resistance mechanisms are found in E. coli. YciG was shown to be required for RpoS-independent acid resistance, but further experiments are needed to determine whether YciG also is required for RpoS-dependent acid resistance. YciG was not required for normal exponential growth of E. coli, as mutants lacking YciG had the same growth rate as the wild-type parent. No mutant phenotypes have been found yet for mutants lacking YciF or YciE. yciE deletion mutants showed the same growth rate and the same level of acid resistance as wild-type cells. The acid resistance of yciF mutants has not yet been tested, and strains lacking YciE and/or YciF need to be assayed for their ability to survive stresses other than acid stress.