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Spelling mutations and evolutionary advantages

Different people sometimes spell the same word differently - organisation versus organization, or analogue versus analog. In such words, despite the variation in the strings of letters, the meaning conveyed by the alternatives remains the same. Similarly, DNA codes carrying instructions for creating a protein can sometimes be 'spelt' differently, although they specify the exact same sequence of amino acids to create that protein. Until recently, most biologists believed that such 'synonymous' DNA codes had very weak effects on the evolution of organisms. However, a new study by an international team of scientists, including those from the National Centre for Biological Sciences (NCBS), Bangalore, shows that a different set of DNA codes specifying the same product can have major effects on the survival and evolution of living beings.

The code of life - composed of triplet codons of the four DNA alphabets A, T, G and C - is quite redundant. For example, the amino acid Alanine is specified by no less than four alternative triplet codes (GCT, GCC, GCA and GCG), or codons. This redundancy is at the root of what molecular biologists term 'synonymous mutations', where a change in the DNA sequence of a gene does not change the sequence of the protein it codes for. Mutations resulting in changes to protein sequences are expected to cause disruptions in function, and are hence likely to affect an organism's abilities or fitness. Contrary to this, synonymous mutations have been generally ignored in this context. Deepa Agashe at NCBS and her team of collaborators have reinforced a growing body of evidence that synonymous variants of a gene affect an organism's fitness. Moreover, they have now shown that single highly beneficial synonymous mutations can allow organisms to rapidly evolve and adapt to their environment.

Working on the bacterium Methylobacterium extorquens, the research group created several synonymous variants of a gene called fae. This gene codes for a metabolic enzyme essential for survival and growth in an environment where the only source of carbon comes from methanol or methylamine. Under such conditions, bacteria undergo strong selection for retaining the fae gene function. M. extorquens, like many other organisms, exhibits a trait known as 'codon bias'. This means that in M. extorquens, the usage of alternative codons in genes is not uniform - while some codons are extensively used, others are relatively rare. The normal or 'wild type' fae gene is composed of a mixture of such frequently used and rarely used codons. The gene variants generated by the research group had different levels of disruptions to the natural codon usage patterns seen in the normal version of fae. When grown in conditions with methanol being the sole carbon source, every one of the bacterial populations carrying synonymous fae variants performed poorly when compared to the population carrying the normal fae gene. This effect was attributed to very low levels of FAE enzyme production in all the variants, though the exact reason for this was unclear. These results provided proof that synonymous mutations could have a more profound effect on survival than previously expected.

However, when bacterial populations carrying these synonymous variants of fae were grown over long periods of time with methanol as the only carbon source - that is, under strong selection- an interesting phenomenon emerged. Within 100 to 200 generations, these bacterial populations began to regain fitness through additional mutations to the gene variants. Many of these mutations were again synonymous and occurred at single points within the gene. All of these mutations occurred in the N-terminal region of fae, and seemed to affect some intrinsic properties of the mRNA transcripts. The mutants had small but consistently higher levels of fae transcripts, enzyme levels and enzyme activity than their ancestors, which led to large beneficial effects on their survival and growth. However, proposed hypotheses on mRNA secondary structure and internal ribosome pause sites fail to explain the beneficial effects of these mutations, and the molecular mechanisms of this process are still unclear. Furthermore, these mutations have recurred in many independent replicate populations- in one case, 100% of the replicates carried the same mutation. This study also provides a rare example of multiple, highly parallel cases of bacterial adaptation driven by synonymous point mutations.

"Adaptation is sometimes considered akin to climbing a hill, to reach a 'fitness peak'. Beneficial mutations help you climb higher and faster, but deleterious mutations push you down towards a valley. Generally, synonymous mutations are thought to be irrelevant for this process. However, we found multiple cases when synonymous mutations allowed bacteria to rapidly climb different fitness peaks. What is also surprising is that this was highly repeatable at the nucleotide level for specific gene variants. You can think of this process with an analogy to climbers - different climbers who start independently from the bottom of a hill are using the exact same strategy to reach the top!", says Deepa Agashe, the lead author of the publication detailing these findings.

Studies like the one described here are critical in understanding the genetic basis of adaptation. Understanding adaptation, in turn, is the key to comprehending evolution and for predicting future dynamics of populations. For example, altering codon composition in the poliovirus genome can create an attenuated form of the virus, and is proposed as a strategy - 'death by a thousand cuts' - to generate vaccines since the virus would require too many mutations to regain fitness. The current study demonstrates that single point synonymous mutations could easily reverse the deleterious attenuating effects of a large number of other synonymous mutations, showing that synonymous mutations do not make a virus "evolution-proof". Until now, synonymous mutations and gene variants were considered relatively unimportant for studies on adaptation due to a lack of information about their effects on organism fitness. This study reinforces the view that these mutations can no longer be ignored as irrelevant in the processes of adaptation and evolution.

The work described in this article has been published in a paper titled 'Large-effect beneficial synonymous mutations mediate rapid and parallel adaptation in a bacterium' in Feb 2016 in the journal Molecular Biology and Evolution and can be accessed here.