Escherichia coli Δglk ΔmanZ ΔptsG glucose strains that lack the glucose phosphotransferase system (PTS) and the mannose PTS as well as glucokinase have been widely used by researchers studying the PTS. In this study we show that both fast- and slow-growing spontaneous glucose revertants can be readily obtained from Δglk ΔmanZ ΔptsG glucose strains. All of the fast-growing revertants either altered the N-acetylglucosamine PTS or caused its overproduction by inactivating the NagC repressor protein, which regulates the N-acetylglucosamine PTS, and these revertants could utilize either glucose or N-acetylglucosamine as a sole carbon source. When a ΔnagE deletion, which abolishes the N-acetylglucosamine PTS, was introduced into the Δglk ΔmanZ ΔptsG glucose strains, fast-growing revertants could no longer be isolated. Based on our results and other studies, it is clear that the N-acetylglucosamine PTS is the most easily adaptable PTS for transporting and phosphorylating glucose, other than the glucose PTS and mannose PTS, which are the primary glucose transport systems. While the slow-growing glucoserevertants were not characterized, they were likely mutations that other researchers have observed before and affect other PTSs or sugar kinases.
The isolation and characterization of 42 unique nonfunctional missense mutants in the bacterial cytosolic β-galactosidase and catechol 2,3-dioxygenase enzymes allowed us to examine some of the basic general trends regarding protein structure and function. A total of 6 out of the 42, or 14.29% of the missense mutants were in α-helices, 17 out of the 42, or 40.48%, of the missense mutants were in β-sheets and 19 out of the 42, or 45.24% of the missense mutants were in unstructured coil, turn or loop regions. While α-helices and β-sheets are undeniably important in protein structure, our results clearly indicate that the unstructured regions are just as important. A total of 21 out of the 42, or 50.00% of the missense mutants caused either amino acids located on the surface of the protein to shift from hydrophilic to hydrophobic or buried amino acids to shift from hydrophobic to hydrophilic and resulted in drastic changes in hydropathy that would not be preferable. There was generally good consensus amongst the widely used algorithms, Chou-Fasman, GOR, Qian-Sejnowski, JPred, PSIPRED, Porter and SPIDER, in their ability to predict the presence of the secondary structures that were affected by the missense mutants and most of the algorithms predicted that the majority of the 42 inactive missense mutants would impact the α-helical and β-sheet secondary structures or the unstructured coil, turn or loop regions that they altered.
While most missense suppressors have very narrow specificities and only suppress the allele against which they were isolated, the sumA missense suppressor from Salmonella enterica serovar Typhimurium is a promiscuous or broad-acting missense suppressor that suppresses numerous missense mutants. The sumA missense suppressor was identified as a glyV tRNA Gly3(GAU/C) missense suppressor that can recognize GAU or GAC aspartic acid codons and insert a glycine amino acid instead of aspartic acid. In addition to rescuing missense mutants caused by glycine to aspartic acid changes as expected, sumA could also rescue a number of other missense mutants as well by changing a neighboring (contacting) aspartic acid to glycine, which compensated for the other amino acid change. Thus the ability of sumA to rescue numerous missense mutants was due in part to the large number of glycine codons in genes that can be mutated to an aspartic acid codon and in part to the general tolerability and/or preference for glycine amino acids in proteins. Because the glyV tRNA Gly3(GAU/C) missense suppressor has also been extensively characterized in Escherichia coli as the mutA mutator, we demonstrated that all gain-of-function mutants isolated in a glyV tRNA Gly3(GAU/C) missense suppressor are transferable to a wild-type background and thus the increased mutation rates, which occur in glyV tRNA Gly3(GAU/C) missense suppressors, are not due to the suppression of these mutants.
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