De Novo Emergence of Peptides That Confer Antibiotic Resistance

Abstract

The origin of novel genes and beneficial functions is of fundamental interest in evolutionary biology. New genes can originate from different mechanisms, including horizontal gene transfer, duplication-divergence, and de novo from noncoding DNA sequences. Comparative genomics has generated strong evidence for de novo emergence of genes in various organisms, but experimental demonstration of this process has been limited to localized randomization in preexisting structural scaffolds. This bypasses the basic requirement of de novo gene emergence, i.e., lack of an ancestral gene. We constructed highly diverse plasmid libraries encoding randomly generated open reading frames and expressed them in Escherichia coli to identify short peptides that could confer a beneficial and selectable phenotype in vivo (in a living cell). Selections on antibiotic-containing agar plates resulted in the identification of three peptides that increased aminoglycoside resistance up to 48-fold. Combining genetic and functional analyses, we show that the peptides are highly hydrophobic, and by inserting into the membrane, they reduce membrane potential, decrease aminoglycoside uptake, and thereby confer high-level resistance. This study demonstrates that randomized DNA sequences can encode peptides that confer selective benefits and illustrates how expression of random sequences could spark the origination of new genes. In addition, our results also show that this question can be addressed experimentally by expression of highly diverse sequence libraries and subsequent selection for specific functions, such as resistance to toxic compounds, the ability to rescue auxotrophic/temperature-sensitive mutants, and growth on normally nonused carbon sources, allowing the exploration of many different phenotypes. IMPORTANCE De novo gene origination from nonfunctional DNA sequences was long assumed to be implausible. However, recent studies have shown that large fractions of genomic noncoding DNA are transcribed and translated, potentially generating new genes. Experimental validation of this process so far has been limited to comparative genomics, in vitro selections, or partial randomizations. Here, we describe selection of novel peptides in vivo using fully random synthetic expression libraries. The peptides confer aminoglycoside resistance by inserting into the bacterial membrane and thereby partly reducing membrane potential and decreasing drug uptake. Our results show that beneficial peptides can be selected from random sequence pools in vivo and support the idea that expression of noncoding sequences could spark the origination of new genes.

De novo emergence of adaptive membrane proteins from thymine-rich genomic sequences

“…We expect that this proportion might rise above the 10% observed in this study (Fig. 3), a fraction that already far exceeds observations based on random sequences 17,18,55,80 , if emerging sequences were screened across additional conditions and wider ranges of expression levels. Hence, while emerging sequences show no evidence of encoding a useful protein product in the present state of the organism, they have the potential to do so in the future.…”

“…Beyond yeast, putative de novo genes with TM domains have also been characterized [51][52][53][54] . Furthermore, evidence suggests that the fitness-enhancing capacities of small TM proteins might extend to bacteria as well as to mouse 18,[55][56][57] . Finally, unannotated TM sequences may also be pervasively translated in bacteria, insects and mammals [58][59][60] .…”

Random peptides rich in small and disorder-promoting amino acids are less likely to be harmful

“…Neme et al's (2017) experiment was suitable for this purpose because it used a large library of peptides with diverse properties, and selected under relatively permissive conditions, simply for relative growth rates in the presence of peptide expression. In contrast, a study design such as that of Knopp et al (2019), who selected random peptides that rescue viability in the presence of antibiotics, is less suitable for our purposes because so few peptides, including harm-avoiding peptides, are viable. Neme et al's (Neme, et al 2017) study was also convenient because all peptides were the same length -65 amino acids with 50 amino acids of random sequenceallowing us to neglect length in our analysis.…”

A novel type of colistin resistance genes selected from random sequence space

“…by accidental expression of normally non-functional sequences that could confer a beneficial effect. Comparative genomics has provided many convincing examples of the de novo emergence of genes across distant organisms [ 9 – 13 ], but experimental demonstration of this process is limited [ 14 – 16 ]. Initial studies have shown that resistance genes can be isolated from random sequences, however these de novo genes likely have limited clinical significance due high associated fitness costs and their inferiority to naturally occurring resistance determinants [ 16 – 18 ].…”

“…Comparative genomics has provided many convincing examples of the de novo emergence of genes across distant organisms [ 9 – 13 ], but experimental demonstration of this process is limited [ 14 – 16 ]. Initial studies have shown that resistance genes can be isolated from random sequences, however these de novo genes likely have limited clinical significance due high associated fitness costs and their inferiority to naturally occurring resistance determinants [ 16 – 18 ]. It remains unclear if and how de novo gene evolution contributes to the evolution of antibiotic resistance genes, but conceivably experimental selection of de novo resistance genes may allow the identification of possible resistance pathways that either have not yet emerged, or, more importantly from a medical perspective, are present but yet undetected in natural bacterial isolates.…”