Synonymous edits in the Escherichia coli genome have substantial and condition-dependent effects on fitness.

CRISPR-Cas-based genome editing is widely used in bacteria at scales ranging from construction of individual mutants to massively parallel libraries. This procedure relies on guide RNA-directed cleavage of the genome followed by repair with a template that introduces a desired mutation along with synonymous "immunizing" mutations to prevent re-cleavage of the genome after editing. Because the immunizing mutations do not change the protein sequence, they are often assumed to be neutral. However, synonymous mutations can change mRNA structures in ways that alter levels of the encoded proteins. We have tested the assumption that immunizing mutations are neutral by constructing a library of over 50,000 edits that consist of only synonymous mutations in Escherichia coli. Thousands of edits had substantial effects on fitness during growth of E. coli on acetate, a poor carbon source that is toxic at high concentrations. The percentage of high-impact edits varied considerably between genes and at different positions within genes. We reconstructed clones with high-impact edits and found that 69% indeed had significant effects on growth in acetate. Interestingly, fewer edits affected fitness during growth in glucose, a preferred carbon source, suggesting that changes in protein expression caused by synonymous mutations may be most important when an organism encounters challenging conditions. Finally, we showed that synonymous edits can have widespread effects; a synonymous edit at the 5' end of ptsI altered expression of hundreds of genes. Our results suggest that the synonymous immunizing edits introduced during CRISPR-Cas-based genome editing should not be assumed to be innocuous.

How to Recruit a Promiscuous Enzyme to Serve a New Function.

Promiscuous enzymes can be recruited to serve new functions when a genetic or environmental change makes catalysis of a novel reaction important for fitness or even survival. Subsequently, gene duplication and divergence can lead to evolution of an efficient and specialized new enzyme. Every organism likely has thousands of promiscuous enzyme activities that provide a vast reservoir of catalytic potential. However, much of this potential may not be accessible. We compiled kinetic parameters for promiscuous reactions catalyzed by 108 enzymes. The median value of kcat/KM is a very modest 31 M-1 s-1. Based upon the fluxes through metabolic pathways in E. coli, we estimate that many, if not most, promiscuous activities are too inefficient to impact fitness. However, mutations can elevate the level of an insufficient promiscuous activity by increasing enzyme expression, improving kcat/KM, or altering concentrations of the promiscuous and native substrates and allosteric regulators. Particularly in large bacterial populations, stochastic mutations may provide a viable pathway for recruitment of even inefficient promiscuous activities.

Amplicon Remodeling and Genomic Mutations Drive Population Dynamics after Segmental Amplification.

New enzymes often evolve by duplication and divergence of genes encoding enzymes with promiscuous activities that have become important in the face of environmental opportunities or challenges. Amplifications that increase the copy number of the gene under selection commonly amplify many surrounding genes. Extra copies of these coamplified genes must be removed, either during or after evolution of a new enzyme. Here we report that amplicon remodeling can begin even before mutations occur in the gene under selection. Amplicon remodeling and mutations elsewhere in the genome that indirectly increase fitness result in complex population dynamics, leading to emergence of clones that have improved fitness by different mechanisms. In this work, one of the two most successful clones had undergone two episodes of amplicon remodeling, leaving only four coamplified genes surrounding the gene under selection. Amplicon remodeling in the other clone resulted in removal of 111 genes from the genome, an acceptable solution under these selection conditions, but one that would certainly impair fitness under other environmental conditions.

Hidden resources in the Escherichia coli genome restore PLP synthesis and robust growth after deletion of the essential gene pdxB.

PdxB (erythronate 4-phosphate dehydrogenase) is expected to be required for synthesis of the essential cofactor pyridoxal 5'-phosphate (PLP) in Escherichia coli Surprisingly, incubation of the ∆pdxB strain in medium containing glucose as a sole carbon source for 10 d resulted in visible turbidity, suggesting that PLP is being produced by some alternative pathway. Continued evolution of parallel lineages for 110 to 150 generations produced several strains that grow robustly in glucose. We identified a 4-step bypass pathway patched together from promiscuous enzymes that restores PLP synthesis in strain JK1. None of the mutations in JK1 occurs in a gene encoding an enzyme in the new pathway. Two mutations indirectly enhance the ability of SerA (3-phosphoglycerate dehydrogenase) to perform a new function in the bypass pathway. Another disrupts a gene encoding a PLP phosphatase, thus preserving PLP levels. These results demonstrate that a functional pathway can be patched together from promiscuous enzymes in the proteome, even without mutations in the genes encoding those enzymes.

Synonymous mutations make dramatic contributions to fitness when growth is limited by a weak-link enzyme.

Synonymous mutations do not alter the specified amino acid but may alter the structure or function of an mRNA in ways that impact fitness. There are few examples in the literature, however, in which the effects of synonymous mutations on microbial growth rates have been measured, and even fewer for which the underlying mechanism is understood. We evolved four populations of a strain of Salmonella enterica in which a promiscuous enzyme has been recruited to replace an essential enzyme. A previously identified point mutation increases the enzyme's ability to catalyze the newly needed reaction (required for arginine biosynthesis) but decreases its ability to catalyze its native reaction (required for proline biosynthesis). The poor performance of this enzyme limits growth rate on glucose. After 260 generations, we identified two synonymous mutations in the first six codons of the gene encoding the weak-link enzyme that increase growth rate by 41 and 67%. We introduced all possible synonymous mutations into the first six codons and found substantial effects on growth rate; one doubles growth rate, and another completely abolishes growth. Computational analyses suggest that these mutations affect either the stability of a stem-loop structure that sequesters the start codon or the accessibility of the region between the Shine-Dalgarno sequence and the start codon. Thus, these mutations would be predicted to affect translational efficiency and thereby indirectly affect mRNA stability because translating ribosomes protect mRNA from degradation. Experimental data support these hypotheses. We conclude that the effects of the synonymous mutations are due to a combination of effects on mRNA stability and translation efficiency that alter levels of the weak-link enzyme. These findings suggest that synonymous mutations can have profound effects on fitness under strong selection and that their importance in evolution may be under-appreciated.

The physical basis and practical consequences of biological promiscuity.

Proteins interact with metabolites, nucleic acids, and other proteins to orchestrate the myriad catalytic, structural and regulatory functions that support life from the simplest microbes to the most complex multicellular organisms. These molecular interactions are often exquisitely specific, but never perfectly so. Adventitious "promiscuous" interactions are ubiquitous due to the thousands of macromolecules and small molecules crowded together in cells. Such interactions may perturb protein function at the molecular level, but as long as they do not compromise organismal fitness, they will not be removed by natural selection. Although promiscuous interactions are physiologically irrelevant, they are important because they can provide a vast reservoir of potential functions that can provide the starting point for evolution of new functions, both in nature and in the laboratory.

An evolutionary biochemist's perspective on promiscuity.

Evolutionary biochemists define enzyme promiscuity as the ability to catalyze secondary reactions that are physiologically irrelevant, either because they are too inefficient to affect fitness or because the enzyme never encounters the substrate. Promiscuous activities are common because evolution of a perfectly specific active site is both difficult and unnecessary; natural selection ceases when the performance of a protein is 'good enough' that it no longer affects fitness. Although promiscuous functions are accidental and physiologically irrelevant, they are of great importance because they provide opportunities for the evolution of new functions in nature and in the laboratory, as well as targets for therapeutic drugs and tools for a wide range of technological applications.

A Synonymous Mutation Upstream of the Gene Encoding a Weak-Link Enzyme Causes an Ultrasensitive Response in Growth Rate.

UNLABELLED: When microbes are faced with an environmental challenge or opportunity, preexisting enzymes with promiscuous secondary activities can be recruited to provide newly important functions. Mutations that increase the efficiency of a new activity often compromise the original activity, resulting in an inefficient bifunctional enzyme. We have investigated the mechanisms by which growth of Escherichia coli can be improved when fitness is limited by such an enzyme, E383A ProA (ProA*). ProA* can serve the functions of both ProA (required for synthesis of proline) and ArgC (required for synthesis of arginine), albeit poorly. We identified four genetic changes that improve the growth rate by up to 6.2-fold. Two point mutations in the promoter of the proBA* operon increase expression of the entire operon. Massive amplification of a genomic segment around the proBA* operon also increases expression of the entire operon. Finally, a synonymous point mutation in the coding region of proB creates a new promoter for proA* This synonymous mutation increases the level of ProA* by 2-fold but increases the growth rate by 5-fold, an ultrasensitive response likely arising from competition between two substrates for the active site of the inefficient bifunctional ProA*. IMPORTANCE: The high-impact synonymous mutation we discovered in proB is remarkable for two reasons. First, most polar effects documented in the literature are detrimental. This finding demonstrates that polar effect mutations can have strongly beneficial effects, especially when an organism is facing a difficult environmental challenge for which it is poorly adapted. Furthermore, the consequence of the synonymous mutation in proB is a 2-fold increase in the level of ProA* but a disproportionately large 5.1-fold increase in growth rate. While ultrasensitive responses are often found in signaling networks and genetic circuits, an ultrasensitive response to an adaptive mutation has not been previously reported.

Differential effects of a mutation on the normal and promiscuous activities of orthologs: implications for natural and directed evolution.

Neutral drift occurring over millions or billions of years results in substantial sequence divergence among enzymes that catalyze the same reaction. Although natural selection maintains the primary activity of orthologous enzymes, there is, by definition, no selective pressure to maintain physiologically irrelevant promiscuous activities. Thus, the levels and the evolvabilities of promiscuous activities may vary among orthologous enzymes. Consistent with this expectation, we have found that the levels of a promiscuous activity in nine gamma-glutamyl phosphate reductase (ProA) orthologs vary by about 50-fold. Remarkably, a single amino acid change from Glu to Ala near the active site appeared to be critical for improvement of the promiscuous activity in every ortholog. The effects of this change varied dramatically. The improvement in the promiscuous activity varied from 50- to 770-fold, and, importantly, was not correlated with the initial level of the promiscuous activity. The decrease in the original activity varied from 190- to 2,100-fold. These results suggest that evolution of a novel enzyme may be possible in some microbes, but not in others. Further, these results underscore the importance of using multiple orthologs as starting points for directed evolution of novel enzyme activities.

Reactivity landscape of pyruvate under simulated hydrothermal vent conditions.

Pyruvate is an important "hub" metabolite that is a precursor for amino acids, sugars, cofactors, and lipids in extant metabolic networks. Pyruvate has been produced under simulated hydrothermal vent conditions from alkyl thiols and carbon monoxide in the presence of transition metal sulfides at 250 °C [Cody GD et al. (2000) Science 289(5483):1337-1340], so it is plausible that pyruvate was formed in hydrothermal systems on the early earth. We report here that pyruvate reacts readily in the presence of transition metal sulfide minerals under simulated hydrothermal vent fluids at more moderate temperatures (25-110 °C) that are more conducive to survival of biogenic molecules. We found that pyruvate partitions among five reaction pathways at rates that depend upon the nature of the mineral present; the concentrations of H2S, H2, and NH4Cl; and the temperature. In most cases, high yields of one or two primary products are found due to preferential acceleration of certain pathways. Reactions observed include reduction of ketones to alcohols and aldol condensation, both reactions that are common in extant metabolic networks. We also observed reductive amination to form alanine and reduction to form propionic acid. Amino acids and fatty acids formed by analogous processes may have been important components of a protometabolic network that allowed the emergence of life.

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol.

Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN- and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s(-1)) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB.

Inhibitory cross-talk upon introduction of a new metabolic pathway into an existing metabolic network.

Evolution or engineering of novel metabolic pathways can endow microbes with new abilities to degrade anthropogenic pollutants or synthesize valuable chemicals. Most studies of the evolution of new pathways have focused on the origins and quality of function of the enzymes involved. However, there is an additional layer of complexity that has received less attention. Introduction of a novel pathway into an existing metabolic network can result in inhibitory cross-talk due to adventitious interactions between metabolites and macromolecules that have not previously encountered one another. Here, we report a thorough examination of inhibitory cross-talk between a novel metabolic pathway for synthesis of pyridoxal 5'-phosphate and the existing metabolic network of Escherichia coli. We demonstrate multiple problematic interactions, including (i) interference by metabolites in the novel pathway with metabolic processes in the existing network, (ii) interference by metabolites in the existing network with the function of the novel pathway, and (iii) diversion of metabolites from the novel pathway by promiscuous activities of enzymes in the existing metabolic network. Identification of the mechanisms of inhibitory cross-talk can reveal the types of adaptations that must occur to enhance the performance of a novel metabolic pathway as well as the fitness of the microbial host. These findings have important implications for evolutionary studies of the emergence of novel pathways in nature as well as genetic engineering of microbes for "green" manufacturing processes.

A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum.

Pentachlorophenol (PCP) hydroxylase, the first enzyme in the pathway for degradation of PCP in Sphingobium chlorophenolicum, is an unusually slow flavin-dependent monooxygenase (k(cat) = 0.02 s⁻¹) that converts PCP to a highly reactive product, tetrachlorobenzoquinone (TCBQ). Using stopped-flow spectroscopy, we have shown that the steps up to and including formation of TCBQ are rapid (5-30 s⁻¹). Before products can be released from the active site, the strongly oxidizing TCBQ abstracts an electron from a donor at the active site, possibly a cysteine residue, resulting in an off-pathway diradical state that only slowly reverts to an intermediate capable of completing the catalytic cycle. TCBQ reductase, the second enzyme in the PCP degradation pathway, rescues this nonproductive complex via two fast sequential one-electron transfers. These studies demonstrate how adoption of an ancestral catalytic strategy for conversion of a substrate with different steric and electronic properties can lead to subtle yet (literally) radical changes in enzymatic reaction mechanisms.

CodaChrome: a tool for the visualization of proteome conservation across all fully sequenced bacterial genomes.

BACKGROUND: The relationships between bacterial genomes are complicated by rampant horizontal gene transfer, varied selection pressures, acquisition of new genes, loss of genes, and divergence of genes, even in closely related lineages. As more and more bacterial genomes are sequenced, organizing and interpreting the incredible amount of relational information that connects them becomes increasingly difficult. RESULTS: We have developed CodaChrome (http://www.sourceforge.com/p/codachrome), a one-versus-all proteome comparison tool that allows the user to visually investigate the relationship between a bacterial proteome of interest and the proteomes encoded by every other bacterial genome recorded in GenBank in a massive interactive heat map. This tool has allowed us to rapidly identify the most highly conserved proteins encoded in the bacterial pan-genome, fast-clock genes useful for subtyping of bacterial species, the evolutionary history of an indel in the Sphingobium lineage, and an example of horizontal gene transfer from a member of the genus Enterococcus to a recent ancestor of Helicobacter pylori. CONCLUSION: CodaChrome is a user-friendly and powerful tool for simultaneously visualizing relationships between thousands of proteomes.

A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica.

BACKGROUND: Recently developed methods for genome editing in bacteria take advantage of the introduction of double-strand breaks by I-SceI in a mutation cassette to select for cells in which homologous recombination has healed the break and introduced a desired mutation. This elegantly designed method did not work well in our hands for most genes. RESULTS: We corrected a mutation in the gene encoding I-SceI that compromised the function of a previously used Red helper plasmid. Further, we found that transcription extending into the mutation cassette interferes with cleavage by I-SceI. Addition of two transcription terminators upstream of the cleavage site dramatically increases the efficiency of genome editing. We also developed an improved method for modification of essential genes. Inclusion of a segment of the essential gene consisting of synonymous codons restores an open reading frame when the mutation cassette is integrated into the genome and decreases the frequency of recombination events that fail to incorporate the desired mutation. The optimized protocol takes only 5 days and has been 100% successful for over 100 genomic modifications in our hands. CONCLUSIONS: The method we describe here is reliable and versatile, enabling various types of genome editing in Escherichia coli and Salmonella enterica by straightforward modifications of the mutation cassette. We provide detailed descriptions of the methods as well as designs for insertions, deletions, and introduction of point mutations.

An evolutionary perspective on protein moonlighting.

Moonlighting proteins serve one or more novel functions in addition to their canonical roles. Moonlighting functions arise when an adventitious interaction between a protein and a new partner improves fitness of the organism. Selective pressure for improvement in the new function can result in two alternative outcomes. The gene encoding the newly bifunctional protein may duplicate and diverge so as to encode two proteins, each of which serves only one function. Alternatively, genetic changes that minimize adaptive conflict between the two functions and/or improve control over the time and place at which each function is served can lead to a moonlighting protein. Importantly, genetic changes that enhance a moonlighting function can occur in the gene encoding the moonlighting protein itself, in a gene that affects the structure of its new partner or in a gene encoding a transcription factor that controls expression of either partner. The evolutionary history of each moonlighting protein is complex, depending on the stochastic occurrence of genetic changes such as gene duplication and point mutations, and the effects of those changes on fitness. Population effects, particularly loss of promising individuals due to random genetic drift, also play a role in the emergence of a moonlighting protein. The ultimate outcome is not necessarily the 'optimal' solution to the problem of serving two functions, but may be 'good enough' so that fitness becomes limited by some other function.

Moonlighting is mainstream: paradigm adjustment required.

Moonlighting--the performance of more than one function by a single protein--is becoming recognized as a common phenomenon with important implications for systems biology and human health. The different functions of a moonlighting protein may use different regions of the protein structure, or alternative structures that occur due to post-translational modifications and/or differences in binding partners. Often the different functions of moonlighting proteins are used at different times or in different places. The existence of moonlighting functions complicates efforts to understand metabolic and regulatory networks, as well as physiological and pathological processes in organisms. Because moonlighting functions can play important roles in disease processes, an improved understanding of moonlighting proteins will provide new opportunities for pharmacological manipulations that specifically target a function involved in pathology while sparing physiologically important functions.

CRISPR-Cas9-assisted genome editing in E. coli elevates the frequency of unintended mutations.

Cas-assisted lambda Red recombineering techniques have rapidly become a mainstay of bacterial genome editing. Such techniques have been used to construct both individual mutants and massive libraries to assess the effects of genomic changes. We have found that a commonly used Cas9-assisted editing method results in unintended mutations elsewhere in the genome in 26% of edited clones. The unintended mutations are frequently found over 200 kb from the intended edit site and even over 10 kb from potential off-target sites. We attribute the high frequency of unintended mutations to error-prone polymerases expressed in response to dsDNA breaks introduced at the edit site. Most unintended mutations occur in regulatory or coding regions and thus may have phenotypic effects. Our findings highlight the risks associated with genome editing techniques involving dsDNA breaks in E. coli and likely other bacteria and emphasize the importance of sequencing the genomes of edited cells to ensure the absence of unintended mutations.

The orphan protein bis-γ-glutamylcystine reductase joins the pyridine nucleotide disulfide reductase family.

Facile DNA sequencing became possible decades after many enzymes had been purified and characterized. Consequently, there are still "orphan" enyzmes for which activities are known but for which encoding genes have not been identified. Identification of the genes encoding orphan enzymes is important because it allows correct annotation of genes of unknown function or with misassigned function. Bis-γ-glutamylcystine reductase (GCR) is an orphan protein that was purified in 1988. This enzyme catalyzes the reduction of bis-γ-glutamylcystine. γ-Glutamylcysteine is the major low-molecular weight thiol in halobacteria. We purified GCR from Halobacterium sp. NRC-1 and identified the sequence of 23 tryptic peptides by nano-liquid chromatography electrospray ionization tandem mass spectrometry. These peptides cover 62% of the protein predicted to be encoded by a gene in Halobacterium sp. NRC-1 that is annotated as mercuric reductase. GCR and mercuric reductase activities were assayed using enzyme that was expressed in Escherichia coli and refolded from inclusion bodies. The enzyme had robust GCR activity but no mercuric reductase activity. The genomes of most, but not all, halobacteria for which whole genome sequences are available have close homologues of GCR, suggesting that there is more to be learned about the low-molecular weight thiols used in halobacteria.

Toward a systems biology perspective on enzyme evolution.

Large superfamilies of enzymes derived from a common progenitor have emerged by duplication and divergence of genes encoding metabolic enzymes. Division of the functions of early generalist enzymes enhanced catalytic power and control over metabolic fluxes. Later, novel enzymes evolved from inefficient secondary activities in specialized enzymes. Enzymes operate in the context of complex metabolic and regulatory networks. The potential for evolution of a new enzyme depends upon the collection of enzymes in a microbe, the topology of the metabolic network, the environmental conditions, and the net effect of trade-offs between the original and novel activities of the enzyme.