Two Homologous EF-G Proteins from Pseudomonas aeruginosa Exhibit Distinct Functions

Abstract

Genes encoding two proteins corresponding to elongation factor G (EF-G) were cloned from Pseudomonas aeruginosa. The proteins encoded by these genes are both members of the EFG I subfamily. The gene encoding one of the forms of EF-G is located in the str operon and the resulting protein is referred to as EF-G1A while the gene encoding the other form of EF-G is located in another part of the genome and the resulting protein is referred to as EF-G1B. These proteins were expressed and purified to 98% homogeneity. Sequence analysis indicated the two proteins are 90/84% similar/identical. In other organisms containing multiple forms of EF-G a lower degree of similarity is seen. When assayed in a poly(U)-directed poly-phenylalanine translation system, EF-G1B was 75-fold more active than EF-G1A. EF-G1A pre-incubate with ribosomes in the presence of the ribosome recycling factor (RRF) decreased polymerization of poly-phenylalanine upon addition of EF-G1B in poly(U)-directed translation suggesting a role for EF-G1A in uncoupling of the ribosome into its constituent subunits. Both forms of P. aeruginosa EF-G were active in ribosome dependent GTPase activity. The kinetic parameters (K M) for the interaction of EF-G1A and EF-G1B with GTP were 85 and 70 μM, respectively. However, EF-G1B exhibited a 5-fold greater turnover number (observed k cat) for the hydrolysis of GTP than EF-G1A; 0.2 s-1 vs. 0.04 s-1. These values resulted in specificity constants (k cat obs/K M) for EF-G1A and EF-G1B of 0.5 x 103 s-1 M-1 and 3.0 x 103 s-1 M-1, respectively. The antibiotic fusidic acid (FA) completely inhibited poly(U)-dependent protein synthesis containing P. aeruginosa EF-G1B, but the same protein synthesis system containing EF-G1A was not affected. Likewise, the activity of EF-G1B in ribosome dependent GTPase assays was completely inhibited by FA, while the activity of EF-G1A was not affected.

Discovery and Analysis of Natural-Product Compounds Inhibiting Protein Synthesis in Pseudomonas aeruginosa

“…During the earlier work, we also tested fusidic acid as a control antibiotic against the E. coli A/T system; however, only low levels of inhibition were observed as fusidic acid was titrated at levels up to 1 mM (data not shown). Assuming similar results would be observed in the P. aeruginosa A/T system, we were surprised to find that fusidic acid inhibited protein synthesis in this system with an IC 50 near 10 M. There are two forms of EF-G (the target of fusidic acid) present in P. aeruginosa but only one (EF-G1B) functions in the elongation phase of protein synthesis (25). EF-G1B was the form of EF-G used in the P. aeruginosa A/T assay.…”

“…Reactions were performed at 37°C for 30 min, and the assays were stopped by the addition of 150 l of 50 mM EDTA. The amount of GTPase activity was determined by measuring the amount of P i liberated using a colorimetric GTPase assay kit (Novus Biologicals) according to the manufacturer's directions (25). The final concentrations of compounds in the assays were 132 M.…”

Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients

“…Most mutations were found in domains 2 and 6 of the enzyme, which are responsible for the maintenance of the open complex and for RNA binding and catalysis, respectively (36)(37)(38). The fusA1 and fusA2 genes code for the highly homologous elongation factors EF-G1a and EF-G1b, which mediate translocation along the mRNA during translation (14,39,40). Twenty-two of 52 amino acid substitutions in EF-G1a and 11 of 24 amino acid substitutions in EF-G1b resided in the catalytically active GTP binding domain (40).…”

Global reprogramming of virulence and antibiotic resistance inPseudomonas aeruginosaby a single nucleotide polymorphism in the elongation factor-encoding gene,fusA1

“…FusA1 encodes one of the two paralogous elongation factor G (EF-G) proteins in P. aeruginosa, and plays a pivotal role in protein synthesis and ribosomal recycling (Palmer et al, 2013). The amino acid sequence of the two paralogues (denoted fusA1 (PA4266) and fusA2 (PA2071)) is highly conserved, with a shared identity of 84%.…”

“…The amino acid sequence of the two paralogues (denoted fusA1 (PA4266) and fusA2 (PA2071)) is highly conserved, with a shared identity of 84%. EF-G1B, encoded by fusA2, is thought to have greater involvement in elongation and polypeptide synthesis (Palmer et al, 2013). By contrast, EF-G1A (encoded by fusA1)…”