Joakim Näsvall

Real-Time Evolution of New Genes by Innovation, Amplification, and Divergence

Duplicate Gene, New Tricks The fact that functionally new genes appear is clear, but the evolutionary process that allows for a new gain of function is not well understood. Näsvall et al. (p. 384) present the innovation-amplification-divergence model which suggests that after gene duplication, ancestral function is maintained but that the duplicate copies can gain new function that is selected for through the accumulation of mutations or changes in expression. Experimental selection on Salmonella enterica allowed an ancestral gene to evolve new enzymatic function in fewer than 3000 generations. Experimental validation of how new genes with divergent functions can evolve within a few thousand generations is described. 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.

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.

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.

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.

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.

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

Abstract 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.

Evolution of Antibiotic Resistance without Antibiotic Exposure

ABSTRACT 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 (βα)8 Barrel Enzyme: SUBSTRATE-BOUND STRUCTURES SHED NEW LIGHT ON THE CATALYTIC CYCLE OF HisA.

Background: HisA catalyzes a ring-opening isomerization reaction in histidine biosynthesis. Results: Catalytic residues and conformational changes upon substrate binding are clarified by structures, kinetics, and mutagenesis. Conclusion: Closing of active site loops in HisA brings the substrate into a product-like conformation before catalysis. Significance: This exemplifies coupled conformational changes in a (βα)8 barrel enzyme and its substrate and clarifies the mechanistic cycle of HisA. HisA is a (βα)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.

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

Significance New proteins can evolve by duplication of the genes that encode them, followed by specialization of the different copies. However, how the growth rate of an organism is coupled to the changes in a protein’s structure and function occurring during this process is not known. Here we show at atomic resolution how selection for the growth of a bacterium led to the evolution of HisA proteins with either a new function or two functions (old and new). We found that a distinct protein conformation is responsible for each function, and that a better enzyme leads to faster growth only up to a certain threshold. This study provides insight into how evolution works, from atomic to whole-organism levels. 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 (βα)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 (βα)8 barrel as an inherently evolvable protein scaffold.

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.