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The evolution of new genes and functions is considered to be a major contributor to biological diversity in organisms. Through de novo origination, “duplication and divergence”, and horizontal gene transfer, organisms can acquire new genetic material that can evolve to perform novel functions. In this thesis, we investigate how functional trade-offs, “gene duplication and amplification”, and neutral divergence contribute to the emergence of a new function from a preexisting gene.

 In Paper i, we investigated the ability of Salmonella enterica to compensate for the loss of peptide release factor 1 (RFI) and the potential of peptide release factor 2 (RF2) to gain a new function to replace RFI. The amplification of RF2 and accumulated mutations within RF2 were the main evolutionary routes by which the fitness cost was restored. However, further characterization of the evolved RF2 showed a toxic effect to the cell due to the termination on tryptophan codon (UGG). This evolutionary trade-off - which we named “collateral toxicity” - might present a serious barrier for evolving an efficient RF2 to replace RF1.

In Paper ii, we determined whether we could evolve a generalist enzyme with two functions (HisA + TrpF) from the specialist enzyme HisA, which can only synthesize histidine. In a previous study, we showed that HisA evolved a TrpF activity through strong trade-off trajectories. Here, we developed a selection scheme in which we constantly selected for keeping the original function (HisA), while intermittently selecting for the new function (TrpF). Our results showed that all evolved lineages shared the same “stepping stone” mutations in the hisA gene, which enabled them to grow well in the absence of both histidine and tryptophan. Additional accumulated mutations in the hisA gene gave the strains an increased ability to grow without both amino acids, indicating that the HisA enzyme evolved to be an efficient generalist.  

In Paper iii, we explored how differences between diverged orthologs influence evolvability. We generated artificial orthologs using a random mutagenesis approach. First, we screened for orthologs with a lower HisA activity and then selected for orthologs with a higher HisA activity; these steps were repeated in alternating rounds. We then tested the ability of each ortholog to evolve  TrpF activity. As expected, the orthologs showed varying abilities to evolve the new function. In particular, orthologs with higher HisA activity levels showed both a higher potential to evolve the new function and a higher TrpF activity when they acquired the new function. 

The divergence of orthologous genes is usually attributed to the slow and steady accumulation of neutral or nearly neutral mutations. It is known that present-day orthologous genes have a common ancestor and still perform the same function with about the same performance. Natural orthologs differ from each other not only in sequence, but also in physical properties such as their tolerance of mutations and their potential to evolve new functions. However, we currently have a poor understanding of how mutations that accumulate during the sequence divergence of orthologous genes affect their evolvability.

In this study, we generated a library of laboratory-evolved orthologous genes for studying how mutations and combinations of mutations (deleterious and compensatory mutations) affect evolvability. We simulated what could happen during the divergence of orthologous genes where fixation of a deleterious mutation is followed by a compensatory mutation. We have subjected one of histidine biosynthetic genes hisA from Salmonella enterica to alternating rounds of weak selection (by random mutagenesis through error-prone PCR subsequent screens for partial loss of hisA function) followed by strong purifying selection (another round of random mutagenesis and subsequent selection for restored hisA function).

The diverging lineages were tested for their ability to evolve TrpF activity and were compared with the WT hisA using a fluctuation test. Our results confirmed that orthologous enzymes had differing abilities to evolve the new function. The HisA orthologs with restored function had evolved adaptive genotypes with higher TrpF activity. We suggest that these orthologs may have a higher stability, which is known to increase evolutionary potential by increasing the tolerance for the otherwise destabilizing mutations that confer the new function.

A mutation that introduces a new enzymatic function can be accompanied by a trade-off between the original function and the new function. We previously tested the evolutionary trajectories of Salmonella enterica HisA enzyme (catalyze the fourth step in histidine biosynthesis) towards TrpF activity (catalyze the third step in tryptophan biosynthesis). The majority of the mutations that introduced TrpF activity led to a complete loss of detectable HisA activity. In the rare hisA mutants that kept HisA activity, HisA activity was lost when selecting for mutations that improved TrpF activity. However, some natural HisA orthologs had dual (HisA and TrpF) activities, which has evolved from a specialist HisA.

The present study was designed to investigate whether Salmonella enterica HisA can evolve into a generalist (HisA+TrpF) enzyme, despite the strong trade-off between the original function and the new function. Therefore, we allowed trpF-deleted Salmonella enterica with or without a mutator phenotype to evolve in a low concentration of tryptophan. This allowed it to grow for a few generations in relaxed selection, which was followed by several days of strong selection (tryptophan depletion). In this setup, it was never lethal to not have TrpF activity, but it would be lethal to lose HisA activity.

These cycled lineages circumvented the tryptophan starvation by loss of function in the tryptophan biosynthesis pathway or by accumulating mutations in hisA gene. The accumulated mutations introduced bifunctionality to the hisA alleles, which was more common in the lineages with the mutator phenotype. These alleles became increasingly efficient in performing both functions when evolved further using the same conditions. Our results indicate that it is possible to evolve an efficient generalist HisA when we use a selection condition that is more likely to occur in nature (a fluctuating environment).

When new genes evolve through modification of existing genes, there are often trade-offs 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 highlight a functional trade-off that we term “collateral toxicity”. We suggest that this type of trade-off may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary trade-offs 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 not only to alterations in the demand for RF2 activity, but also for RF1 activity.

The World Society for Virology (WSV) was founded and incorporated as a nonprofit organization in the United States in 2017. WSV seeks to strengthen and support both virological research and virologists who conduct research of viruses that affect humans, other animals, plants, and other organisms. One of the objectives of WSV is to connect virologists worldwide and support collaboration. Fulfilling this objective, virologists from fourteen countries in North America, Europe, Africa, Asia, and the Middle East met on 25-27th August 2019 in Stockholm, Sweden at the Karolinska University Hospital for the first Committee Meeting of WSV. This meeting included compelling keynote and honorary speeches and a series of 18 scientific talks were given encompassing a diverse array of subjects within virology. Followed by the scientific session, a business session was held where multiple aspects and next steps of the society were discussed and charted out.

It is known that there is an intraindividual amplitude variation in motor electroneurography when the same person is examined at different times. This variation affects the evaluation the status of the patient. The aim of this study was to investigate if the intraindividual amplitude variation decreased by photographing the electrode position, that later is used in the follow-up study. Twenty test persons were examined by four laboratory scientists. The nerves that were examined were median, ulnar, peroneal and tibial nerve. At the first examination the laboratory scientists used method guidelines and took photographs of the electrode position. The photographs were then used in the follow-up. The results showed that there was an indication of decreased of the intraindividual amplitude variation when photographic documentation was used instead of method guidelines.

Background: Most severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals are asymptomatic or only exhibit mild disease. In about 10% of cases, the infection leads to hypoxemic pneumonia, although it is much more rare in children. Objective: We evaluated 31 young patients aged 0.5 to 19 years who had preexisting inborn errors of immunity (IEI) but lacked a molecular diagnosis and were later diagnosed with coronavirus disease 2019 (COVID-19) complications. Methods: Genetic evaluation by whole-exome sequencing was performed in all patients. SARS-CoV-2-specific antibodies, autoantibodies against type I IFN (IFN-I), and inflammatory factors in plasma were measured. We also reviewed COVID-19 disease severity/outcome in reported IEI patients. Results: A potential genetic cause of the IEI was identified in 28 patients (90.3%), including mutations that may affect IFN signaling, T- and B-cell function, the inflammasome, and the complement system. From tested patients 65.5% had detectable virus-specific antibodies, and 6.8% had autoantibodies neutralizing IFN-I. Five patients (16.1%) fulfilled the diagnostic criteria of multisystem inflammatory syndrome in children. Eleven patients (35.4%) died of COVID-19 complications. All together, at least 381 IEI children with COVID-19 have been reported in the literature to date. Although many patients with asymptomatic or mild disease may not have been reported, severe presentation of COVID-19 was observed in 23.6% of the published cases, and the mortality rate was 8.7%. Conclusions: Young patients with preexisting IEI may have higher mortality than children without IEI when infected with SARS-CoV-2. Elucidating the genetic basis of IEI patients with severe/critical COVID-19 may help to develop better strategies for prevention and treatment of severe COVID-19 disease and complications in pediatric patients.

Background Inborn errors of immunity (IEI) and autoantibodies to type I interferons (IFNs) underlie critical COVID-19 pneumonia in at least 15% of the patients, while the causes of multisystem inflammatory syndrome in children (MIS-C) remain elusive. Objectives To detect causal genetic variants in very rare cases with concomitant critical COVID-19 pneumonia and MIS-C. Methods Whole exome sequencing was performed, and the impact of candidate gene variants was investigated. Plasma levels of cytokines, specific antibodies against the virus, and autoantibodies against type I IFNs were also measured. Results We report a 3-year-old child who died on day 56 of SARS-CoV-2 infection with an unusual clinical presentation, combining both critical COVID-19 pneumonia and MIS-C. We identified a large, homozygous loss-of-function deletion in IFNAR1, underlying autosomal recessive IFNAR1 deficiency. Conclusions Our findings confirm that impaired type I IFN immunity can underlie critical COVID-19 pneumonia, while suggesting that it can also unexpectedly underlie concomitant MIS-C. Our report further raises the possibility that inherited or acquired dysregulation of type I IFN immunity might contribute to MIS-C in other patients.