Collateral Toxicity Limits the Evolution of Bacterial Release Factor 2 toward Total Omnipotence.

When new genes evolve through modification of existing genes, there are often tradeoffs between the new and original functions, making gene duplication and amplification necessary to buffer deleterious effects on the original function. We have used experimental evolution of a bacterial strain lacking peptide release factor 1 (RF1) in order to study how peptide release factor 2 (RF2) evolves to compensate the loss of RF1. As expected, amplification of the RF2-encoding gene prfB to high copy number was a rapid initial response, followed by the appearance of mutations in RF2 and other components of the translation machinery. Characterization of the evolved RF2 variants by their effects on bacterial growth rate, reporter gene expression, and in vitro translation termination reveals a complex picture of reduced discrimination between the cognate and near-cognate stop codons and highlights a functional tradeoff that we term "collateral toxicity." We suggest that this type of tradeoff may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary tradeoffs between "old" and "new" functions of a gene, as it cannot be overcome by gene copy number changes. Further, we suggest a model for how RF2 autoregulation responds to alterations in the demand not only for RF2 activity but also for RF1 activity.

Structural and functional innovations in the real-time evolution of new (ßα)8 barrel enzymes.

New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. Here we used crystallography, NMR dynamics, kinetics, and mass spectrometry to explain the molecular innovations that arose during a previous real-time evolution experiment. In that experiment, the (ßα)8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bifunctional generalists. We found that selection affects enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bifunctionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead, we observed thresholds for each activity above which further improvements in catalytic efficiency had little if any effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with sufficiently high activities such that they no longer limit the growth of their host organism, and confirms the (ßα)8 barrel as an inherently evolvable protein scaffold.

Structural mechanism of AadA, a dual-specificity aminoglycoside adenylyltransferase from Salmonella enterica.

Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from Salmonella enterica is an aminoglycoside (3″)(9) adenylyltransferase that O-adenylates position 3″ of streptomycin and position 9 of spectinomycin. We previously reported the apo-AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of WT AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that, despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site and, in agreement with mutational data, forms critical interactions with fewer residues. Using structure-guided sequence analyses of ANT(3″)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the nonadenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin.

Experimental Determination and Prediction of the Fitness Effects of Random Point Mutations in the Biosynthetic Enzyme HisA.

The distribution of fitness effects of mutations is a factor of fundamental importance in evolutionary biology. We determined the distribution of fitness effects of 510 mutants that each carried between 1 and 10 mutations (synonymous and nonsynonymous) in the hisA gene, encoding an essential enzyme in the l-histidine biosynthesis pathway of Salmonella enterica. For the full set of mutants, the distribution was bimodal with many apparently neutral mutations and many lethal mutations. For a subset of 81 single, nonsynonymous mutants most mutations appeared neutral at high expression levels, whereas at low expression levels only a few mutations were neutral. Furthermore, we examined how the magnitude of the observed fitness effects was correlated to several measures of biophysical properties and phylogenetic conservation.We conclude that for HisA: (i) The effect of mutations can be masked by high expression levels, such that mutations that are deleterious to the function of the protein can still be neutral with regard to organism fitness if the protein is expressed at a sufficiently high level; (ii) the shape of the fitness distribution is dependent on the extent to which the protein is rate-limiting for growth; (iii) negative epistatic interactions, on an average, amplified the combined effect of nonsynonymous mutations; and (iv) no single sequence-based predictor could confidently predict the fitness effects of mutations in HisA, but a combination of multiple predictors could predict the effect with a SD of 0.04 resulting in 80% of the mutations predicted within 12% of their observed selection coefficients.

Duplication-Insertion Recombineering: a fast and scar-free method for efficient transfer of multiple mutations in bacteria.

We have developed a new λ Red recombineering methodology for generating transient selection markers that can be used to transfer mutations between bacterial strains of both Escherichia coli and Salmonella enterica. The method is fast, simple and allows for the construction of strains with several mutations without any unwanted sequence changes (scar-free). The method uses λ Red recombineering to generate a marker-held tandem duplication, termed Duplication-Insertion (Dup-In). The Dup-Ins can easily be transferred between strains by generalized transduction and are subsequently rapidly lost by homologous recombination between the two copies of the duplicated sequence, leaving no scar sequence or antibiotic resistance cassette behind. We demonstrate the utility of the method by generating several Dup-Ins in E. coli and S. enterica to transfer genetically linked mutations in both essential and non-essential genes. We have successfully used this methodology to re-construct mutants found after various types of selections, and to introduce foreign genes into the two species. Furthermore, recombineering with two overlapping fragments was as efficient as recombineering with the corresponding single large fragment, allowing more complicated constructions without the need for overlap extension PCR.

Compensating the Fitness Costs of Synonymous Mutations.

Synonymous mutations do not change the sequence of the polypeptide but they may still influence fitness. We investigated in Salmonella enterica how four synonymous mutations in the rpsT gene (encoding ribosomal protein S20) reduce fitness (i.e., growth rate) and the mechanisms by which this cost can be genetically compensated. The reduced growth rates of the synonymous mutants were correlated with reduced levels of the rpsT transcript and S20 protein. In an adaptive evolution experiment, these fitness impairments could be compensated by mutations that either caused up-regulation of S20 through increased gene dosage (due to duplications), increased transcription of the rpsT gene (due to an rpoD mutation or mutations in rpsT), or increased translation from the rpsT transcript (due to rpsT mutations). We suggest that the reduced levels of S20 in the synonymous mutants result in production of a defective subpopulation of 30S subunits lacking S20 that reduce protein synthesis and bacterial growth and that the compensatory mutations restore S20 levels and the number of functional ribosomes. Our results demonstrate how specific synonymous mutations can cause substantial fitness reductions and that many different types of intra- and extragenic compensatory mutations can efficiently restore fitness. Furthermore, this study highlights that also synonymous sites can be under strong selection, which may have implications for the use of dN/dS ratios as signature for selection.

Evolution of Antibiotic Resistance without Antibiotic Exposure.

Antibiotic use is the main driver in the emergence of antibiotic resistance. Another unexplored possibility is that resistance evolves coincidentally in response to other selective pressures. We show that selection in the absence of antibiotics can coselect for decreased susceptibility to several antibiotics. Thus, genetic adaptation of bacteria to natural environments may drive resistance evolution by generating a pool of resistance mutations that selection could act on to enrich resistant mutants when antibiotic exposure occurs.

Two-step Ligand Binding in a (ßα)8 Barrel Enzyme: SUBSTRATE-BOUND STRUCTURES SHED NEW LIGHT ON THE CATALYTIC CYCLE OF HisA.

HisA is a (ßα)8 barrel enzyme that catalyzes the Amadori rearrangement of N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) to N'-((5'-phosphoribulosyl) formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the histidine biosynthesis pathway, and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo-state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp-7 acts as the catalytic base, and Asp-176 acts as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen bond network including His-47, Asp-129, Thr-171, and Ser-202, all shown to be functionally important. The closed conformation structure is highly similar to the bifunctional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.

Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3'')(9) adenyltransferase.

Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3'')(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5 Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.

Minor fitness costs in an experimental model of horizontal gene transfer in bacteria.

Genes introduced by horizontal gene transfer (HGT) from other species constitute a significant portion of many bacterial genomes, and the evolutionary dynamics of HGTs are important for understanding the spread of antibiotic resistance and the emergence of new pathogenic strains of bacteria. The fitness effects of the transferred genes largely determine the fixation rates and the amount of neutral diversity of newly acquired genes in bacterial populations. Comparative analysis of bacterial genomes provides insight into what genes are commonly transferred, but direct experimental tests of the fitness constraints on HGT are scarce. Here, we address this paucity of experimental studies by introducing 98 random DNA fragments varying in size from 0.45 to 5 kb from Bacteroides, Proteus, and human intestinal phage into a defined position in the Salmonella chromosome and measuring the effects on fitness. Using highly sensitive competition assays, we found that eight inserts were deleterious with selection coefficients (s) ranging from ≈ -0.007 to -0.02 and 90 did not have significant fitness effects. When inducing transcription from a PBAD promoter located at one end of the insert, 16 transfers were deleterious and 82 were not significantly different from the control. In conclusion, a major fraction of the inserts had minor effects on fitness implying that extra DNA transferred by HGT, even though it does not confer an immediate selective advantage, could be maintained at selection-transfer balance and serve as raw material for the evolution of novel beneficial functions.

Activation of cryptic aminoglycoside resistance in Salmonella enterica.

Aminoglycoside resistance in bacteria can be acquired by several mechanisms, including drug modification, target alteration, reduced uptake and increased efflux. Here we demonstrate that increased resistance to the aminoglycosides streptomycin and spectinomycin in Salmonella enterica can be conferred by increased expression of an aminoglycoside adenyl transferase encoded by the cryptic, chromosomally located aadA gene. During growth in rich medium the wild-type strain was susceptible but mutations that impaired electron transport and conferred a small colony variant (SCV) phenotype or growth in glucose/glycerol minimal media resulted in activation of the aadA gene and aminoglycoside resistance. Expression of the aadA gene was positively regulated by the stringent response regulator guanosine penta/tetraphosphate ((p)ppGpp). SCV mutants carrying stop codon mutations in the hemA and ubiA genes showed a streptomycin pseudo-dependent phenotype, where growth was stimulated by streptomycin. Our data suggest that this phenotype is due to streptomycin-induced readthrough of the stop codons, a resulting increase in HemA/UbiA levels and improved electron transport and growth. Our results demonstrate that environmental and mutational activation of a cryptic resistance gene can confer clinically significant resistance and that a streptomycin-pseudo-dependent phenotype can be generated via a novel mechanism that does not involve the classical rpsL mutations.

Dup-In and DIRex: Techniques for Single-Step, Scar-Free Mutagenesis with Marker Recycling.

This chapter describes two related recombineering-based techniques: "Duplication Insertion" (Dup-In) and "Direct- and Inverted Repeat stimulated excision" (DIRex). Dup-In is used for transferring existing mutations between strains, and DIRex for generating almost any type of mutation. Both techniques use intermediate insertions with counter-selectable cassettes, flanked by directly repeated sequences that enable exact and spontaneous excision of the cassettes. These constructs can be transferred to other strains using generalized transductions, and the final intended mutation is obtained following selection for spontaneous loss of the counter-selectable cassette, which leaves only the intended mutation behind in the final strain. The techniques have been used in several strains of Escherichia coli and Salmonella enterica, and should be readily adaptable to other organisms where λ Red recombineering or similar methods are available.

Mutational Pathways and Trade-Offs Between HisA and TrpF Functions: Implications for Evolution via Gene Duplication and Divergence.

When a new activity evolves by changes in a pre-existing enzyme this is likely to reduce the original activity, generating a functional trade-off. The properties of this trade-off will affect the continued evolution of both functions. If the trade-off is strong, gene duplication and subsequent divergence would be favored whereas if the trade-off is weak a bi-functional enzyme could evolve that performs both functions. We previously showed that when a bi-functional HisA enzyme was evolved under selection for both HisA and TrpF functions, evolution mainly proceeded via duplication-divergence and specialization, implying that the trade-off is strong between these two functions. Here, we examined this hypothesis by identifying the mutational pathways (i.e., the mutational landscape) in the Salmonella enterica HisA enzyme that conferred a TrpF-like activity, and examining the trade-offs between the original and new activity. For the HisA enzyme there are many different paths toward the new TrpF function, each with its own unique trade-off. A total of 16 single mutations resulted in HisA enzyme variants that acquired TrpF activity and only three of them maintained HisA activity. Twelve mutants were evolved further toward increased TrpF activity and during evolution toward improved TrpF activity the original HisA activity was completely lost in all lineages. We propose that, aside from various relevant ecological factors, two main genetic factors influence whether evolution of a new function proceeds via duplication - divergence (specialization) or by evolution of a generalist: (i) the relative mutation supply of the two pathways and (ii) the shape of the trade-off curve between the native and new function.

Synonymous Mutations in rpsT Lead to Ribosomal Assembly Defects That Can Be Compensated by Mutations in fis and rpoA.

We previously described how four deleterious synonymous mutations in the Salmonella enterica rpsT gene (encoding ribosomal protein S20) result in low S20 levels that can be compensated by mutations that restore [S20]. Here, we have further studied the cause for the deleterious effects of S20 deficiency and found that the S20 mutants were also deficient in four other 30S proteins (S1, S2, S12, and S21), which is likely due to an assembly defect of the S20 deficient 30S subunits. We examined the compensatory effect by six additional mutations affecting the global regulator Fis and the C-terminal domain of the α subunit of RNA polymerase (encoded by rpoA). The fis and rpoA mutations restored the S20 levels, concomitantly restoring the assembly defect and the levels of S1, S2, S12, and S21. These results illustrate the complexity of compensatory evolution and how the negative effects of deleterious mutations can be suppressed by a multitude of mechanisms. Additionally, we found that the mutations in fis and rpoA caused reduced expression of other ribosomal components. Notably, some of the fis mutations and the rpoA mutation corrected the fitness of the rpsT mutants to wild-type levels, although expression of other ribosomal components was reduced compared to wild-type. This finding raises new questions regarding the relation between translation capacity and growth rate.

Selection for novel metabolic capabilities in Salmonella enterica.

Bacteria are known to display extensive metabolic diversity and many studies have shown that they can use an extensive repertoire of small molecules as carbon- and energy sources. However, it is less clear to what extent a bacterium can expand its existing metabolic capabilities by acquiring mutations that, for example, rewire its metabolic pathways. To investigate this capability and potential for evolution of novel phenotypes, we sampled large populations of mutagenized Salmonella enterica to select very rare mutants that can grow on minimal media containing 124 low molecular weight compounds as sole carbon sources. We found mutants growing on 18 of these novel carbon sources, and identified the causal mutations that allowed growth for four of them. Mutations that relieve physiological constraints or increase expression of existing pathways were found to be important contributors to the novel phenotypes. For the remaining 14 novel phenotypes, whole genome sequencing of independent mutants and genetic analysis suggested that these novel metabolic phenotypes result from a combination of multiple mutations. This work, by virtue of identifying the genetic and mechanistic basis for new metabolic capabilities, sheds light on the properties of adaptive landscapes underlying the evolution of novel phenotypes.

Genetic Adaptation to Growth Under Laboratory Conditions in Escherichia coli and Salmonella enterica.

Experimental evolution under controlled laboratory conditions is becoming increasingly important to address various evolutionary questions, including, for example, the dynamics and mechanisms of genetic adaptation to different growth and stress conditions. In such experiments, mutations typically appear that increase the fitness under the conditions tested (medium adaptation), but that are not necessarily of interest for the specific research question. Here, we have identified mutations that appeared during serial passage of E. coli and S. enterica in four different and commonly used laboratory media and measured the relative competitive fitness and maximum growth rate of 111 genetically re-constituted strains, carrying different single and multiple mutations. Little overlap was found between the mutations that were selected in the two species and the different media, implying that adaptation occurs via different genetic pathways. Furthermore, we show that commonly occurring adaptive mutations can generate undesired genetic variation in a population and reduce the accuracy of competition experiments. However, by introducing media adaptation mutations with large effects into the parental strain that was used for the evolution experiment, the variation (standard deviation) was decreased 10-fold, and it was possible to measure fitness differences between two competitors as small as |s| < 0.001.

Direct and Inverted Repeat stimulated excision (DIRex): Simple, single-step, and scar-free mutagenesis of bacterial genes.

The need for generating precisely designed mutations is common in genetics, biochemistry, and molecular biology. Here, I describe a new λ Red recombineering method (Direct and Inverted Repeat stimulated excision; DIRex) for fast and easy generation of single point mutations, small insertions or replacements as well as deletions of any size, in bacterial genes. The method does not leave any resistance marker or scar sequence and requires only one transformation to generate a semi-stable intermediate insertion mutant. Spontaneous excision of the intermediate efficiently and accurately generates the final mutant. In addition, the intermediate is transferable between strains by generalized transductions, enabling transfer of the mutation into multiple strains without repeating the recombineering step. Existing methods that can be used to accomplish similar results are either (i) more complicated to design, (ii) more limited in what mutation types can be made, or (iii) require expression of extrinsic factors in addition to λ Red. I demonstrate the utility of the method by generating several deletions, small insertions/replacements, and single nucleotide exchanges in Escherichia coli and Salmonella enterica. Furthermore, the design parameters that influence the excision frequency and the success rate of generating desired point mutations have been examined to determine design guidelines for optimal efficiency.

Evolution of new functions de novo and from preexisting genes.

How the enormous structural and functional diversity of new genes and proteins was generated (estimated to be 10(10)-10(12) different proteins in all organisms on earth [Choi I-G, Kim S-H. 2006. Evolution of protein structural classes and protein sequence families. Proc Natl Acad Sci 103: 14056-14061] is a central biological question that has a long and rich history. Extensive work during the last 80 years have shown that new genes that play important roles in lineage-specific phenotypes and adaptation can originate through a multitude of different mechanisms, including duplication, lateral gene transfer, gene fusion/fission, and de novo origination. In this review, we focus on two main processes as generators of new functions: evolution of new genes by duplication and divergence of pre-existing genes and de novo gene origination in which a whole protein-coding gene evolves from a noncoding sequence.

Real-time evolution of new genes by innovation, amplification, and divergence.

Gene duplications allow evolution of genes with new functions. Here, we describe the innovation-amplification-divergence (IAD) model in which the new function appears before duplication and functionally distinct new genes evolve under continuous selection. One example fitting this model is a preexisting parental gene in Salmonella enterica that has low levels of two distinct activities. This gene is amplified to a high copy number, and the amplified gene copies accumulate mutations that provide enzymatic specialization of different copies and faster growth. Selection maintains the initial amplification and beneficial mutant alleles but is relaxed for other less improved gene copies, allowing their loss. This rapid process, completed in fewer than 3000 generations, shows the efficacy of the IAD model and allows the study of gene evolution in real time.