Hyper-susceptibility of a fusidic acid-resistant mutant of Salmonella to different classes of antibiotics

Fusidic acid resistance (Fus(R)) in Salmonella enterica serovar Typhimurium is caused by mutations in fusA, encoding elongation factor G (EF-G). Pleiotropic phenotypes are observed in Fus(R) mutants. Thus, the fusA1 allele (EF-G P413L) is associated with slow growth rate, reduced ppGpp and RpoS levels, reduced heme levels, and increased sensitivity to oxidative stress. The fusA1-15 allele, (EF-G P413L and T423I) derived from fusA1 in a selection for growth rate compensation, is partially compensated in each of these phenotypic defects but maintains its resistance to fusidic acid. We show here that the fusA1 allele is associated with sensitivity to ultraviolet light and increased susceptibility to the inhibitory action of several unrelated antibiotic classes (beta-lactam, fluoroquinolone, aminoglycoside, rifampicin, and chloramphenicol). The fusA1-15 allele, in contrast, is less susceptible to UV and to other antibiotics than fusA1. The hyper-susceptibility to multiple antibiotics associated with fusA1 and fusA1-15 is revealed in a novel growth competition assay at sub-MIC concentrations, but not in a standard MIC assay.     

Biological cost and compensatory evolution in fusidic acid-resistant Staphylococcus aureus

Fusidic acid resistance resulting from mutations in elongation factor G (EF-G) of Staphylococcus aureus is associated with fitness costs during growth in vivo and in vitro. In both environments, these costs can be partly or fully compensated by the acquisition of secondary intragenic mutations. Among clinical isolates of S. aureus, fusidic acid-resistant strains have been identified that carry multiple mutations in EF-G at positions similar to those shown experimentally to cause resistance and fitness compensation. This observation suggests that fitness-compensatory mutations may be an important aspect of the evolution of antibiotic resistance in the clinical environment, and may contribute to a stabilization of the resistant bacteria present in a bacterial population.     

In vivo selection of conditional-lethal mutations in the gene encoding elongation factor G of Escherichia coli.

The ribosome translocation step that occurs during protein synthesis is a highly conserved, essential activity of all cells. The precise movement of one codon that occurs following peptide bond formation is regulated by elongation factor G (EF-G) in eubacteria or elongation factor 2 (EF-2) in eukaryotes. To begin to understand molecular interactions that regulate this process, a genetic selection was developed with the aim of obtaining conditional-lethal alleles of the gene (fusA) that encodes EF-G in Escherichia coli. The genetic selection depends on the observation that resistant strains arose spontaneously in the presence of sublethal concentrations of the antibiotic kanamycin. Replica plating was performed to obtain mutant isolates from this collection that were restrictive for growth at 42 degrees C. Two tightly temperature-sensitive strains were characterized in detail and shown to harbor single-site missense mutations within fusA. The fusA100 mutant encoded a glycine-to-aspartic acid change at codon 502. The fusA101 allele encoded a glutamine-to-proline alteration at position 495. Induction kinetics of beta-galactosidase activity suggested that both mutations resulted in slower elongation rates in vivo. These missense mutations were very near a small group of conserved amino acid residues (positions 483 to 493) that occur in EF-G and EF-2 but not EF-Tu. It is concluded that these sequences encode a specific domain that is essential for efficient translocase function.     

Mutations in the G-domain of elongation factor G from Thermus thermophilus affect both its interaction with GTP and fusidic acid

Two hypersensitive and two resistant variants of elongation factor-G (EF-G) toward fusidic acid are studied in comparison with the wild type factor. All mutated proteins are active in a cell-free translation system and ribosome-dependent GTP hydrolysis. The EF-G variants with the Thr-84-->Ala or Asp-109-->Lys mutations bring about a strong resistance of EF-G to the antibiotic, whereas the EF-Gs with substitutions Gly-16-->Val or Glu-119-->Lys are the first examples of fusidic acid-hypersensitive factors. A correlation between fusidic acid resistance of EF-G mutants and their affinity to GTP are revealed in this study, although their interactions with GDP are not changed. Thus, fusidic acid-hypersensitive mutants have the high affinity to an uncleavable GTP analog, but the association of resistant mutants with GTP is decreased. The effects of either fusidic acid-sensitive or resistant mutations can be explained by the conformational changes in the EF-G molecule, which influence its GTP-binding center. The results presented in this paper indicate that fusidic acid-sensitive mutant factors have a conformation favorable for GTP binding and subsequent interaction with the ribosomes.     

The function of conserved amino acid residues adjacent to the effector domain in elongation factor G

Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino acid positions necessary for EF-G functions, a series of mutations were constructed in the EF-G structural gene (fusA) of Escherichia coli, specifically at positions flanking the effector domain. A mutated allele was isolated in which the wild-type sequence from codons 29 to 47 ("EFG2947") was replaced with a sequence encoding 28 amino acids from ribosomal protein S7. This mutated gene was unable to complement a fusAts strain when supplied in trans at the nonpermissive temperature. In vitro biochemical analysis demonstrated that nucleotide crosslinking was unaffected in EFG2947, while ribosome binding appeared to be completely abolished. A series of point mutations created within this region, encoding L30A, Y32A, H37A, and K38A were shown to give rise to fully functional proteins, suggesting that side chains of these individual residues are not essential for EF-G function. A sixth mutant, E41A, was found to inefficiently rescue growth in a fusAts background, and was also unable to bind ribosomes normally in vitro. In contrast E41Q could restore growth at the nonpermissive temperature. These results can be explained within the context of a three-dimensional model for the effector region of EF-G. This model indicates that the effector domain contains a negative potential field that may be important for ribosome binding.     

Mutations in the <Emphasis Type="Italic">fusA</Emphasis> Gene Encoding Elongation Factor G in the Coryneform Bacterium Lead to Increased Lysine Production

Resistance to fusidic acid in Corynebacterium glutamicum and Brevibacterium flavum is associated with mutations in the fusA gene, which encodes the elongation factor G (EF-G). Two to ten percent of fusidic acid-resistant clones were shown to produce more lysine than parent strains. Sequencing of the fusA gene in clones with a high level of lysine production made it possible to find two mutations in the gene at position 1383—С1383G and С1383А. These mutations cause amino acid replacement at position 461 in the protein EF-G, namely, histidine is substituted by glutamine (H461Q). The mutation С1383G was introduced in the chromosomal copy of the fusA gene in C. glutamicum and B. flavum strains by homologous recombination. All clones containing the mutant variant of the fusA gene produced 10% more lysine than the parent strains.     

Molecular basis of fusB-mediated resistance to fusidic acid in Staphylococcus aureus

The primary mechanism of fusidic acid resistance in clinical strains of Staphylococcus aureus involves acquisition of the fusB determinant. The genetic elements(s) responsible are incompletely defined, and the mechanism of resistance is unknown. Here we report the cloning, sequencing and overexpression of a single gene (fusB) from plasmid pUB101 capable of conferring resistance to fusidic acid in S. aureus. The fusB gene is located on a transposon-like element and encodes a small (25 kDa), cytoplasmic protein for which homologues exist in a number of clinically important and environmental Gram-positive bacterial species. Bioinformatic analysis of regions immediately upstream of fusB suggested that expression of resistance is regulated by translational attenuation, which was confirmed through use of reporter fusions. FusB was overexpressed in Escherichia coli as a polyhistidine-tagged fusion product, and the purified protein shown to protect an in vitro staphylococcal translation system from inhibition by fusidic acid in a specific and dose-dependent fashion. Purified FusB bound staphylococcal EF-G, the target of fusidic acid. The protein provided no protection from inhibition by fusidic acid when added to an in vitro E. coli translation system, consistent both with the observed failure of FusB to bind E. coli EF-G, and its inability to confer resistance in E. coli.     

Susceptibility to and resistance determinants of fusidic acid in Staphylococcus aureus isolated from Chinese children with skin and soft tissue infections

This study aims to determine the resistance rates and determinants of fusidic acid among Staphylococcus aureus isolates collected from Chinese pediatric patients with skin and soft tissue infections (SSTIs). Between 2008 and 2009, a total of 186 clinical S. aureus isolates were collected from the pediatric patients with SSTIs, abscess (44.6%) was the most common SSTI in children 0-16 years old. Four clinical isolates (4/186, 2.2%) were resistant to fusidic acid. Two of these isolates were methicillin-resistant S. aureus (MRSA) that carry the fusC gene. The other two isolates were methicillin-sensitive S. aureus (MSSA) that carry the fusB gene. In the two fusB-positive clinical isolates, the fusB gene was located in a transposon-like element that has 99% identity with a pUB101 fragment from S. aureus. The four fusidic acid-resistant clinical isolates were ST1-MRSA-SCCmecV-t127, ST93-MRSA-SCCmecIII-t202, ST680-MSSA-t5415, and ST680-MSSA-t377. The fusidic acid resistance rate of S. aureus isolated from Chinese pediatric patients with SSTIs was low, and the genes fusB and fusC were the main resistance determinants. The transposon-like element that contains the fusB gene might participate in the transmission of fusidic acid resistance genes. This is the first report regarding the emergence of fusidic acid-resistant clinical S. aureus isolates in mainland China.     

Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus

Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by secondary mutations. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that a significantly slower tRNA translocation and ribosome recycling, plus increased peptidyl-tRNA drop-off, are the causes for fitness defects of the primary FA-resistant mutant F88L. The double mutant F88L/M16I is three to four times faster than F88L in both reactions and showed no tRNA drop-off, explaining its fitness compensatory phenotype. The M16I mutation alone showed hypersensitivity to FA, higher activity, and somewhat increased affinity to GTP. The crystal structures demonstrate that Phe-88 in switch II is a key residue for FA locking and also for triggering interdomain movements in EF-G essential for its function, explaining functional deficiencies in F88L. The mutation M16I loosens the hydrophobic core in the G domain and affects domain I to domain II contact, resulting in improved activity both in the wild-type and F88L background. Thus, FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome.     

Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium

Most chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium, the nucleotide substitution AAA42 --> AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eighty-one independent lineages of the low-fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be > or = 10-7 per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin-sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single-nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.     

Genetic complexity of fusidic acid-resistant small colony variants (SCV) in Staphylococcus aureus

FusE mutants are fusidic acid-resistant small colony variants (SCVs) of Staphylococcus aureus that can be selected with aminoglycosides. All FusE SCVs have mutations in rplF, encoding ribosomal protein L6. However, individual FusE mutants including some with the same mutation in rplF display auxotrophy for either hemin or menadione, suggesting that additional mutations are involved. Here we show that FusE SCVs can be divided into three genetic sub-groups and that some carry an additional mutation, in one of the genes required for hemin biosynthesis, or in one of the genes required for menadione biosynthesis. Reversion analysis and genome sequencing support the hypothesis that these combinations of mutations in the rplF, hem, and/or men genes can account for the SCV and auxotrophic phenotypes of FusE mutants.     

Alterations in the peptidyltransferase and decoding domains of ribosomal RNA suppress mutations in the elongation factor G gene

The translocation stage of protein synthesis is a highly conserved process in all cells. Although the components necessary for translocation have been delineated, the mechanism of this activity has not been well defined. Ribosome movement on template mRNA must allow for displacement of tRNA-mRNA complexes from the ribosomal A to P sites and P to E sites, while ensuring rigid maintenance of the correct reading frame. In Escherichia coli, translocation of the ribosome is promoted by elongation factor G (EF-G). To examine the role of EF-G and rRNA in translocation we have characterized mutations in rRNA genes that can suppress a temperature-sensitive (ts) allele of fusA, the gene in E. coli that encodes EF-G. This analysis was performed using the ts E. coli strain PEM100, which contains a point mutation within fusA. The ts phenotype of PEM100 can be suppressed by either of two mutations in the decoding region of the 16S rRNA when present in combination with a mutation at position 2058 in the peptidyltransferase domain of the 23S rRNA. Communication between these ribosomal domains is essential for coordinating the events of the elongation cycle. We propose a model in which EF-G promotes translocation by modulating this communication, thereby increasing the efficiency of this fundamental process.     

Isolation and characterization of fusidic acid-resistant, sporulation-defective mutants of Bacillus subtilis.

Fusidic acid-resistant, sporulation-defective mutants were isolated from Bacillus subtilis 168 thy trp. About two-thirds of the fusidic acid-resistant (fusr) mutants were defective in sporulation ability and fell into three classes with respect to sporulation character. The representative mutants FUS426 and FUS429 were characterized in detail. FUS426 [fusr spo (Ts)], a temperature-sensitive sporulation mutant, grew well at 30 and 42 degrees C but did not sporulate at 42 degrees C. FUS429 [fusr spo (Con)], conditional sporulation mutant, grew and sporulated normally in the absence of fusidic acid, but its sporulation and growth rates decreased in the presence of fusidic acid, depending on the concentration of the drug. Although electron microscopic observation showed that both mutants were blocked at stage I of sporulation, the physiological analyses indicate that these mutants belong to the SpoOB class. Both mutants formed a thickened cell wall as compared with that of the parental strain. Genetic and in vitro protein synthesis analyses led to the conclusion that the sporulation-defective character of mutants FUS426 and FUS429 resulted from an alteration in elongation factor G caused by a single lesion in the fus locus. The possible role of elongation factor G in sporulation is discussed.     

Structure of a mutant EF-G reveals domain III and possibly the fusidic acid binding site

The crystal structure of Thermus thermophilus elongation factor G (EF-G) carrying the point mutation His573Ala was determined at a resolution of 2.8 A. The mutant has a more closed structure than that previously reported for wild-type EF-G. This is obtained by a 10 degrees rigid rotation of domains III, IV and V with regard to domains I and II. This rotation results in a displacement of the tip of domain IV by approximately 9 A. The structure of domain III is now fully visible and reveals the double split beta-alpha-beta motif also observed for EF-G domain V and for several ribosomal proteins. A large number of fusidic acid resistant mutations found in domain III have now been possible to locate. Possible locations for the effector loop and a possible binding site for fusidic acid are discussed in relation to some of the fusidic acid resistant mutations.     

A central interdomain protein joint in elongation factor G regulates antibiotic sensitivity, GTP hydrolysis, and ribosome translocation

The antibiotic fusidic acid potently inhibits bacterial translation (and cellular growth) by lodging between domains I and III of elongation factor G (EF-G) and preventing release of EF-G from the ribosome. We examined the functions of key amino acid residues near the active site of EF-G that interact with fusidic acid and regulate hydrolysis of GTP. Alanine mutants of these residues spontaneously hydrolyzed GTP in solution, bypassing the normal activating role of the ribosome. A conserved phenylalanine in the switch II element of EF-G was important for suppressing GTP hydrolysis in solution and critical for catalyzing translocation of the ribosome along mRNA. These experimental results reveal the multipurpose roles of an interdomain joint in the heart of an essential translation factor that can both promote and inhibit bacterial translation.     

Effects of sarA inactivation on the intrinsic multidrug resistance mechanism of Staphylococcus aureus

The sarA locus of Staphylococccus aureus regulates the synthesis of over 100 genes on the S. aureus chromosome. We now report the effects of sarA inactivation on intrinsic multidrug resistance expression by S. aureus. In a strain-dependent fashion, sarA::kan mutants of three unrelated strains of S. aureus demonstrated significantly increased susceptibility to five or more of the following substances: the antibiotics ciprofloxacin, fusidic acid, and vancomycin; the DNA-intercalating agent ethidium; and four common household cleaner formulations. In addition, all three sarA::kan mutants demonstrated significantly increased accumulation of ciprofloxacin and one sarA::kan mutant demonstrated increased ethidium accumulation. Our data therefore indicate that sarA plays a role in the intrinsic multidrug resistance mechanism expressed by S. aureus, in part by regulating drug accumulation.     

Genetic lesions involved in temperature sensitivity of the src gene products of four Rous sarcoma virus mutants.

The src genes of four Rous sarcoma virus (RSV) mutants temperature-sensitive (ts) for cell transformation were analyzed. The mutant src genes were cloned into a replication-competent RSV expression vector, and the contribution of individual mutations to the ts phenotype was assessed by in vitro recombination with wild-type src sequences. Three of the mutants, which were derived from the Schmidt-Ruppin strain of RSV, each encoded two mutations within the conserved kinase domain. In all three cases, one of the two mutations was an identical valine to methionine change at amino acid position 461. Virus encoding recombinant src genes containing each of these mutations alone were not ts for transformation, demonstrating that two mutations are required for temperature sensitivity. The sequence of the src gene of the Bryan high-titer strain of RSV was determined and compared with that of the fourth ts mutant which was derived from it, again revealing two lesions in the kinase domain of the mutant.