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Evolution recovers the fitness of Acinetobacter baylyi strains with large deletions through mutations in deletion-specific targets and global post-transcriptional regulators [1]

['Isaac Gifford', 'Department Of Molecular Biosciences', 'Center For Systems', 'Synthetic Biology', 'The University Of Texas At Austin', 'Austin', 'Texas', 'United States Of America', 'Gabriel A. Suárez', 'Jeffrey E. Barrick']

Date: 2024-10

Organelles and endosymbionts have naturally evolved dramatically reduced genome sizes compared to their free-living ancestors. Synthetic biologists have purposefully engineered streamlined microbial genomes to create more efficient cellular chassis and define the minimal components of cellular life. During natural or engineered genome streamlining, deletion of many non-essential genes in combination often reduces bacterial fitness for idiosyncratic or unknown reasons. We investigated how and to what extent laboratory evolution could overcome these defects in six variants of the transposon-free Acinetobacter baylyi strain ADP1-ISx that each had a deletion of a different 22- to 42-kilobase region and two strains with larger deletions of 70 and 293 kilobases. We evolved replicate populations of ADP1-ISx and each deletion strain for ~300 generations in a chemically defined minimal medium or a complex medium and sequenced the genomes of endpoint clonal isolates. Fitness increased in all cases that were examined except for two ancestors that each failed to improve in one of the two environments. Mutations affecting nine protein-coding genes and two small RNAs were significantly associated with one of the two environments or with certain deletion ancestors. The global post-transcriptional regulators rnd (ribonuclease D), csrA (RNA-binding carbon storage regulator), and hfq (RNA-binding protein and chaperone) were frequently mutated across all strains, though the incidence and effects of these mutations on gene function and bacterial fitness varied with the ancestral deletion and evolution environment. Mutations in this regulatory network likely compensate for how an earlier deletion of a transposon in the ADP1-ISx ancestor of all the deletion strains restored csrA function. More generally, our results demonstrate that fitness lost during genome streamlining can usually be regained rapidly through laboratory evolution and that recovery tends to occur through a combination of deletion-specific compensation and global regulatory adjustments.

Genome streamlining reduces the complexity of organisms by eliminating large, non-essential portions of their genomes. This process occurs naturally in endosymbiont lineages and can be engineered to create microbial chassis that operate more efficiently and predictably. However, genome reduction often compromises the fitness of an organism when genes and combinations of genes are deleted that, while not essential, are advantageous. In this study, we used laboratory evolution to improve the fitness of a collection of Acinetobacter baylyi strains with large engineered deletions. In most cases, we found that spontaneous mutations could recover fitness lost due to deletions spanning many genes in these strains. These beneficial mutations were sometimes general, occurring in multiple strains and environments regardless of what genes were deleted, or specific, observed solely or more often in one environment or in strains with certain deletions. Mutations affecting proteins and small RNAs involved in post-transcriptional regulation of gene expression were especially common. Thus, recovering fitness often involves a combination of mutations that adjust global regulatory networks and compensate for lost gene functions. More broadly, our findings validate using laboratory evolution as a strategy for improving the fitness of reduced-genome strains created for biotechnology applications.

Funding: This work was supported by Welch Foundation grant F-1979 to J.E.B, National Science Foundation grant CBET-1554179 to J.E.B., National Science Foundation grant MCB-2123996 to J.E.B., a subcontract from the NSF BEACON Center for the Study of Evolution grant DBI-0939454 to J.E.B., and a UT Austin College of Natural Sciences Spark grant to J.E.B. The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Previously, we engineered strains of the metabolically versatile and naturally competent bacterium Acinetobacter baylyi ADP1-ISx with deletions of large genome segments that each encoded ≥17 nonessential genes [ 23 ]. In this study, we evolved replicate populations of eight reduced-genome strains of Acinetobacter baylyi ADP1-ISx in complex and defined medium laboratory environments. Deletions in these strains were constructed without examining the functions or expression levels of the genes being removed, which resulted in substantial fitness defects in some cases. Nevertheless, evolution restored fitness, at least partially, in most instances. Whole-genome sequencing revealed examples of both deletion-specific and shared genetic targets that recurrently evolved mutations in different populations. The most widespread mutations affected genes in a post-transcriptional global regulatory network. They appear to recapitulate or phenocopy a transposon insertion in the csrA gene in A. baylyi ADP1 that was removed during construction of the transposon-free ADP1-ISx progenitor strain. However, even in these cases, the effects of mutations within this network on gene function varied with the deletion strain and environment in which they evolved. Overall, our results validate laboratory evolution as a strategy for improving the fitness of reduced-genome strains, and they illuminate a path forward for engineering a minimal-genome A. baylyi strain.

The potential benefits of genome streamlining, however, may come at a cost. Removing what have been called "quasi-essential" genes [ 9 ] can result in fitness defects due to disrupting metabolic networks [ 18 ], reducing stress tolerance [ 8 , 14 ], or perturbing global gene expression [ 18 ], among other possibilities. Deletion of multiple, large genomic regions can also remove genes that individually have little impact on a cell’s function but when removed together produce synthetic lethality [ 9 , 19 ] or are more deleterious than expected due to "synthetic-sick" interactions [ 14 ]. Because of our incomplete understanding of the consequences of deleting genes in top-down streamlined strains, laboratory evolution has been used as a means to recover lost fitness [ 14 , 18 ]. This strategy often works for single-gene deletions: they acquire mutations that compensate for the lost function [ 20 , 21 ]. Less is known about whether and when strains with many genes deleted at once can recover wild-type fitness through laboratory evolution. Short evolution experiments were able to partially restore the fitness of hypermutator strains of Salmonella typhimurium after they spontaneously accumulated mutations that included deletions of tens to hundreds of kilobases [ 22 ]. Different outcomes have been reported, however, after evolving strains of E. coli [ 18 ] and Bacillus subtilis [ 14 ] with cumulative engineered deletions comprising ~20–30% of their genomes. The E. coli strain wholly recovered fitness lost in minimal medium, primarily through rewiring metabolic and regulatory networks [ 18 ]. The B. subtilis strain exhibited more limited fitness recovery [ 14 ]. In light of these results, questions still remain about the impact of large deletions on an organism’s fitness and how and whether laboratory evolution can recover lost fitness after this type of genome reduction.

It has been argued that streamlined genomes provide two major benefits to synthetic biology: removing genes that are detrimental in an environment of interest and reducing the complexity of cellular networks [ 4 – 7 ]. Most genomes encode pathways that are unnecessary in laboratory, bioreactor, or host environments; and these additional genes can negatively impact a desired phenotype. For example, genomes have been engineered to remove genes that cause autolysis [ 11 ] or block DNA uptake [ 12 ] to improve cell survival and competence, respectively. Other deletions, such as removing transposons, can also lower mutation rates [ 12 – 14 ]. Streamlining can be especially beneficial for metabolic engineering, when extraneous pathways redirect resources away from a desired product [ 7 ], as has been shown by deleting genes to increase antibiotic production in Streptomyces avermitilis [ 15 ] and recombinant protein production in Pseudomonas putida [ 16 ] and Lactococcus lactis [ 17 ]. Removing genes eliminates their interactions with remaining genes, simplifying modeling of metabolic and regulatory networks, especially when genes of unknown function are deleted. Reducing cellular complexity in this way should make engineering more predictable [ 7 ].

Genome streamlining is a process of genome reduction in which unnecessary or detrimental genes are lost or removed, resulting in a simpler genome. Gene loss occurs naturally in many microorganisms, including symbionts [ 1 ] and pathogens [ 2 ], that live in stable, nutrient-rich environments where genes encoding ancillary metabolic pathways are unnecessary. However, little is known about the detailed dynamics of natural streamlining, as most of our understanding of genome reduction comes from comparing the very different genomes of endosymbionts and their free-living relatives [ 3 ]. Two types of approaches have been used to experimentally study genome streamlining [ 4 – 7 ]. Top-down approaches are similar to natural processes in that they start with a free-living organism and sequentially delete non-essential pieces of its genome. This procedure has achieved various degrees of genome reduction in many bacterial and fungal species, including deleting up to 38.9% of the Escherichia coli genome [ 8 ]. The other approach, bottom-up design, assembles the DNA for a minimal chromosome de novo and then inserts it into a recipient cell to produce a synthetic organism. This procedure has been used to create Mycoplasma mycoides strains with extremely reduced genomes [ 9 ] and Saccharomyces cerevisiae strains with up to six and a half synthetic chromosomes [ 10 ].

Results

Multiple-gene deletion strains exhibit fitness defects Previously, we created Acinetobacter baylyi strain ADP1-ISx [13] by deleting all transposable elements from the genome of the laboratory strain ADP1 [24]. Then, we examined possibilities for additional genome streamlining by attempting to construct 55 multiple-gene deletion (MGD) strains from ADP1-ISx [23]. Each of the 18 MGD strains that were successfully constructed has a single contiguous stretch of nonessential genes removed. For this study, we selected six of these MGD strains (MGD4, MGD6, MGD9, MGD12, MGD15, and MGD17) with deletions ranging in size from 21.8 to 41.7 kb that removed from 17 to 47 genes (Fig 1A). We also examined a retained genome region (RGR) strain with a partially successful deletion, leading to loss of only six genes (RGR7), as an additional wild-type-like control, and two multiple-segment deletion (MSD) strains (MSD1 and MSD2) that were created by combining or expanding deletions in the original MGD strains. Strain MSD1 had the most-reduced genome, with a total of 293.5 kb and 268 genes removed, including a ribosomal RNA operon. MSD2 has a deletion of 69.8 kb that expands upon one of the original MGD segments (MGD11). It removes 67 genes. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Some reduced-genome variants of A. baylyi ADP1-ISx have fitness defects. (A) A. baylyi ADP1-ISx chromosome showing the locations and sizes of regions removed in six multiple-gene deletion (MGD) strains, one retained-genomic region (RGR) strain, and two multiple-segment deletion (MSD) strains used as ancestors for the evolution experiment. The numbers of protein-coding, tRNA, and rRNA genes deleted in each strain are indicated. (B, C) Fitness of ADP1-ISx and the six MGD strains relative to a GFP-expressing variant of ADP1-ISx. Filled bars are means. Error bars are 95% confidence intervals. Starred values are significantly different from ADP-ISx (Benjamini-Hochberg adjusted Welch’s t-tests). https://doi.org/10.1371/journal.pgen.1011306.g001 To examine how the large deletions affected bacterial fitness, we constructed a GFP-expressing variant of ADP1-ISx. Then, we performed co-culture competition assays comparing this reference strain to each MGD strain and the ADP1-ISx progenitor in a complex medium (LB) (Fig 1B) and a defined minimal succinate medium (MS) (Fig 1C). The six MGD ancestor strains had fitness defects of 17.0% on average and up to 49.3% relative to ADP1-ISx in LB. The fitness defects were 10.0% on average and as high as 39.9% in MS. Four MGD ancestors (MGD4, MGD6, MGD9, and MGD12) had significantly lower fitness than ADP1-ISx in LB, and three of these four (MGD4, MGD6, and MGD9) also had significantly lower fitness than ADP1-ISx in MS (Benjamini-Hochberg adjusted p < 0.05, Welch’s t-tests, see Methods). Two MGD ancestors (MGD4 and MGD6) exhibited significantly different fitness defects in the two different culture media (adjusted p < 0.05). For the MGD4 ancestor, the fitness defect was larger in MS by 18.3%, while the fitness defect of the MGD6 ancestor was 32.0% greater in LB.

Multiple-gene deletion strains recover fitness during laboratory evolution We evolved replicate populations of each of the six MGD, one RGR, and two MSD strains plus ADP1-ISx controls for 30 days (~300-generation) by performing 1000-fold serial-dilution transfers in either LB or MS. We began the experiment with six replicates of each ancestor strain containing a deletion in each environment with additional replicates of the ADP1-ISx controls. At the conclusion of the evolution experiment, we plated each population and picked a single large colony for further characterization. To understand how fitness changed during the evolution experiment, we compared the results of co-culture competition assays pitting each of the six endpoint MGD clonal isolates against the GFP-expressing ADP1-ISx reference strain (Fig 2). The ADP1-ISx progenitor already had relatively high fitness in LB. Its six LB-evolved endpoint clones that we tested exhibited very little change in fitness in this environment (+0.1% mean ± 3.3% standard deviation), and the change due to evolution was not significant for any one clone (adjusted p > 0.05). The six MS-evolved ADP1-ISx clones whose fitness we assayed gained fitness overall (+13.4 ± 13.6%), but the difference in any one clone was, again, not statistically significant (adjusted p > 0.05). The evolved MGD strains exhibited wider variation in their fitness changes. Overall, they exhibited fitness increases of 7.9% on average and up to 25.1% in LB and 18.5% on average and up to 38.0% in MS. At least three of the six evolved clones derived from the same MGD ancestor had significant fitness increases (adjusted p > 0.05) for three MGD strains in LB (MGD4, MGD6, and MGD12) and for two MGD strains in MS (MGD6 and MGD9). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Evolution of fitness in the evolution experiment. The inset in the upper right shows the design of the evolution experiment and how final endpoint clonal isolates from each replicate population were selected for characterization. The other panels show the fitness determined for each of six evolved endpoint isolates (numbered 1–6) and their respective ancestor (Anc) relative to a GFP-expressing variant of ADP1-ISx. Points are the results of individual replicates of co-culture fitness assays. Horizontal solid lines are the average fitness of the respective ancestral strain. Horizontal dashed lines at a value of one are the relative fitness of the ADP1-ISx progenitor because this reference comparison was used to normalize all relative fitness measurements. Filled bars are means. Error bars are 95% confidence intervals. Starred values are significantly different from ADP1-ISx (Benjamini-Hochberg adjusted Welch’s t-tests). https://doi.org/10.1371/journal.pgen.1011306.g002 We expected that, in general, MGD strains that had the lowest initial fitness would gain the most fitness during the evolution experiment. Often this was the case, but there were also exceptions (Fig 3). In LB, most MGD strains followed the expected trend, but MGD9 was an outlier. Despite initially having the third-largest fitness defect in LB relative to ADP1-ISx from its deletion, there was a negligible change in the fitness of its six LB-evolved descendant strains (−1.1 ± 4.7%). In MS, strains that evolved from MGD4, the ancestor with the greatest initial fitness defect of 39.9% in this medium, did not exhibit any increase in fitness (−2.0 ± 8.9%). MGD12 was an outlier in the opposite direction in MS. The ancestor strain had a 19.2% higher fitness than ADP1-ISx in this medium due to mutations that occurred during its construction (see below), yet the further increase in fitness of its six MS-evolved endpoint clones was also among the highest of all MGD strains (+33.5 ± 14.2%). Overall, these results show evolution was successful at improving the fitness of most MGD strains in most environments, though there were notable exceptions for two strains, each in a specific environment. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Fitness of evolved clones versus their ancestors. The relative fitness of each evolved endpoint clone that was characterized is plotted against the relative fitness of its ancestor. Fitness values measured for independently evolved clones from different replicate populations of the evolution experiment (small gray points) are summarized as means (larger colored points) with error bars for the standard deviations of the clone values. Points above the reference line with a slope of one represent improvements during evolution. https://doi.org/10.1371/journal.pgen.1011306.g003

Rates of genome evolution were similar for all deletions and in both environments We sequenced the genomes of the MGD, RGR, and MSD ancestor strains and endpoint clones from independent replicate populations to understand how they evolved. Some evolved clones shared mutations that arose during strain construction or evolved in the cultures from which colonies were picked to begin each replicate population (Fig 4). Multiple ancestral strains and/or cultures had pre-existing mutations in the same genomic region upstream of ACIAD2521, which encodes a putative divalent metal transporter. The MGD4 and MGD15 strains each had different mutations in this region, and the exact same mutations appear to have also evolved in subpopulations of cells in the initial MGD17 culture and in one culture of ADP1-ISx that was used to initiate six specific replicate populations (numbered 7–12). These and other mutations nearby in the same intergenic region were observed in other endpoint clones evolved in LB, suggesting that they both arise at high rates and are beneficial in this environment. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Mutated genes associated with certain ancestors and environments. Each symbol on the grids represents a mutation affecting the labeled gene, intergenic region (if two gene names are separated by a slash), any one of a set of labeled genes (if gene names are separated by commas), or a multigene region (if gene names are separated by a dash). Triangles are used for larger mutations that include the corresponding genes as well as their surrounding regions. Unfilled symbols are used for pre-existing mutations that fixed in the ancestor strain or were present in cultures used to start the evolution experiment. They were not included in the association analysis. Only genes or gene groups affected by mutations in three or more evolved isolates are shown. The bottommost lines of the grid show genes that were mutated only in the context of that one specific ancestor strain. A full list of evolved mutations is included in S4 Table. Genes or gene sets in which the rate of mutations differed depending on the culture environment are indicated by highlighting horizontal guidelines using thick (enriched) and thin (depleted) lines colored by the medium condition. Mutations with rates that differed depending on the ancestor strain within each environment are highlighted in color, with a different color used for each gene or gene set. In both cases the statistical significance of these associations was tested by using binomial regression models (Benjamini-Hochberg adjusted p < 0.05, likelihood ratio tests, see Methods). https://doi.org/10.1371/journal.pgen.1011306.g004 All other pre-existing mutations were restricted to one ancestor strain and its derivatives. The MGD12 ancestor had two such mutations, one in murB and one in rpoD. RpoD is the major housekeeping sigma factor (σ70), and this mutation was an in-frame deletion of one CAG unit from a (CAG) 3 repeat that is likely a mutational hotspot. Interestingly, this mutation appears to have reverted in all the MGD12 clones that evolved in LB, an outcome that is consistent with how this deletion ancestor had lower fitness in LB but unexpectedly exhibited 19.2% higher fitness in MS (Fig 1C), though this difference in fitness did not reach statistical significance in our measurements (adjusted p = 0.23). The engineered deletion in MGD12 overlapped the promoter region of murB, which encodes an enzyme involved in peptidoglycan biosynthesis that is essential in A. baylyi ADP1 [23,25]. The MGD12 ancestor and all strains that evolved from it shared a synonymous substitution in the third codon of this gene that may restore its expression. The same nonsynonymous mutation in ACIAD1813, a putative transporter that may be related to phenol metabolism, was present in the MSD2 ancestor and all its descendants. RGR7 and its evolved clones shared a base substitution in the intergenic region upstream of ACIAD1420, a putative carbonate dehydratase. Finally, the same intergenic base substitution upstream of rpoB, which encodes the β subunit of RNA polymerase, was observed in one of the two sequenced MSD1 ancestors and two of the nine clones that evolved from it, suggesting that it was present in a subpopulation of cells in the founding culture. Not counting these pre-existing mutations, at least one mutation and as many as nine mutations occurred in the lineages leading to the 140 sequenced endpoint clones from the evolution experiment. There were 1.86 mutations, on average, in the 73 LB-evolved clones and 1.36 mutations in the 67 MS-evolved clones. The difference in the rate at which mutations accumulated in the two environments was significant (p = 0.022, likelihood-ratio test comparing Poisson regression models), whereas there was not a significant effect of the ancestor strain on the rate (p = 0.99) or an ancestor by environment effect (p = 0.70). This held true even when comparing the very similar ADP1-ISx and RGR7 ancestors as one group against all MGD and MSD ancestors together (p = 0.92 for ancestor group effect and p = 0.96 for ancestor group by environment effect). In summary there were typically one or two mutations in each endpoint clone from the evolution experiment, with more on average in the LB-evolved clones.

Mutations in specific genes are associated with certain environments and deletions We next examined whether mutations affecting certain genes were associated with specific ancestral deletions or culture environments (Fig 4). We first tested for environment specificity by determining whether we could reject the hypothesis that the occurrence of mutations in a gene or set of genes was the same for endpoint clones that evolved in LB and MS. Then, within each environment, we determined whether we could reject the hypothesis that the rates of mutations in a gene or gene set were equal in clones that evolved from different ancestors. In each case we determined whether the environment or ancestor effects were significant by comparing binomial regression models fit to the occurrence of mutations in each gene or gene set (Benjamini-Hochberg adjusted p < 0.05, likelihood ratio tests, see Methods). We found that mutations in seven genes or sets of related genes were more likely in LB than in MS (Fig 4). One of these was the region upstream of the ACIAD2521 divalent metal transporter gene that had pre-existing mutations in many ancestral strains (see previous section), even though these instances were not included in the association analysis. Mutations in this target were also unevenly distributed across ancestor strains. Similarly, mutations in ACIAD0085, galU, and pgi—three genes grouped together because they are part of the same extracellular polysaccharide (EPS) biosynthesis operon—and a deletion of a 49-kb prophage region that extends from ACIAD2132 to ACIADtRNAVal_46 were both significantly more common in LB. Mutations in the three EPS biosynthesis genes were significantly more likely to be mutated in clones that evolved from certain ancestors in both environments. In LB, these mutations were concentrated in strains that evolved from ADP1-ISx, RGR7, MGD15 and MGD17. In MS, most were in MGD4 and MGD9 and MGD17. The prophage deletions only occurred in LB and were mostly found in clones that evolved from the MGD15 and MGD17 ancestors, though the overall ancestor-specific association was only marginally significant after adjusting for multiple testing in this case (adjusted p = 0.057). The four remaining targets that were significantly more likely to be mutated in LB-evolved clones were also significantly, and solely, associated with specific ancestors in this environment (Fig 4). In all seven MGD12 clones evolved in LB, mutations in rpoD reverted the in-frame deletion in a CAG trinucleotide repeat present in this ancestor strain (see previous section). All six LB-evolved MSD2 clones had nonsynonymous base substitutions in the pyridoxal phosphate-dependent aminotransferase ACIAD2087. Five of six LB-evolved MGD6 strains had base substitutions within or near a predicted homolog of the AbsR28 small RNA of unknown function first experimentally detected in Acinetobacter baumanii [26]. Four of six MSD1 endpoint clones evolved in LB had mutations in genes encoding two components of a zinc ABC transporter found in the same operon (znuB and znuC). One of these mutations is a single-base deletion that results in a frameshift in znuC, which suggests that loss of this transporter’s function is beneficial in the MSD1 deletion background. Ribonuclease D (rnd) was the most commonly mutated gene in the entire evolution experiment. It was also the only gene that was significantly more likely to be mutated in MS than in LB (Fig 4). In MS, 46 of 67 endpoint clones (68.7%) had an rnd mutation, including at least one clone derived from every ancestor. Mutations in rnd were also common in LB, just less so: 19 of 73 clones (26.0%) evolved in this environment had an rnd mutation. These widespread mutations in rnd were also significantly associated with certain ancestors in both environments. Many rnd mutations were nonsense mutations or small insertions or deletions (indels) that are expected to completely inactivate the function of this gene (see next section). While not significantly associated with either culture medium, mutations in the carbon storage regulatory protein csrA were associated with certain ancestors in MS, particularly MGD9 descendants that evolved in this environment. Mutations in csrA were either single-base substitutions or in-frame indels. They probably alter the function of this gene versus resulting in complete loss of function. A transposon that truncates the CsrA protein in A. baylyi relative to other Acinetobacter species was deleted during the creation of ADP1-ISx [16]. (S2 Data File). It is possible that the csrA mutations we observe have similar effects on its function to this transposon insertion. Mutations in glucose-6-phosphate isomerase (pgi) that include frameshifting indels were significantly associated with certain ancestors in LB, mostly ADP1-ISx and RGR7. LB-evolved mutations between the convergently oriented recD and ACIAD0400 genes were also significantly biased between ancestors, with three of five MSD1 isolates containing mutations in this region, which was also mutated in one ADP1-ISx-MS clone and one MGD4-LB clone. Four of these mutations are within and the two others would completely delete a computationally predicted small RNA of unknown function [27]. Mutations in genes involved in natural competence (comC, comN, pilB, pilD, pilF, and pilR) were significantly associated with the MGD12 ancestor in MS. All seven of the mutations observed in these genes are insertions or deletion and all but one of them introduces a frameshift in the affected gene.

Loss-of-function mutations in RNAse D were widespread There were 65 total mutations in RNase D (rnd) across all evolved clones, which allowed us to examine their effects on gene function with more granularity. These mutations include nonsynonymous mutations, which may preserve some level of rnd function, and nonsense mutations, small indels, and large deletions, most of which are likely to result in complete loss of rnd function (Fig 5A). As noted above, mutations in rnd were significantly more common in MS-evolved strains than LB-evolved strains. Mutations in rnd in each environment were also heavily biased in different ways: the odds that an rnd mutation in MS was of a type that is likely to result in complete loss of function versus a nonsynonymous change was 8.0 times that in LB (p = 0.00068, Fisher’s exact test). Some individual strains showed noticeable deviations from this general trend. For example, MGD9 was the only ancestor to have fewer rnd mutations in MS-evolved clones, and the spectrum was also reversed relative to other ancestors such that most of the rnd mutations observed in its LB-evolved clones were putative loss of function mutations. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Spectra of RNase D mutations vary with ancestor and environment. Wider bars are used for types of mutations that are likely to result in complete loss of gene function. https://doi.org/10.1371/journal.pgen.1011306.g005

Effects of deleting rnd on fitness vary with ancestor and environment To directly study the impact of RNAse D knockout on the different MGD strains, we deleted the rnd gene in the unevolved ancestor of each MGD strain and ADP1-ISx. Then, we used co-culture competition assays to measure the fitness effects of rnd deletion in both LB and MS environments (Fig 6). Overall, across all strain backgrounds, deletion of rnd increased fitness by 7.4%, on average, in MS-evolved strains, and this difference was significant (F 1,76 = 0.37, p = 0.016). In LB, it increased fitness by 1.2%, on average, which was not significant (F 1,76 = 6.1, p = 0.54). The higher fitness benefit in MS is in accordance with the observation of more mutations, including more loss-of-function mutations, in clones that evolved in MS (Fig 5). PPT PowerPoint slide

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TIFF original image Download: Fig 6. Effects of deleting RNase D in different ancestors on fitness in each environment. Each panel shows the fitness of an RNAse D deletion strain (Δrnd) and its ancestor (Ancestor) relative to GFP-expressing ADP1-ISx. Points are the results of individual replicates of co-culture fitness assays. Horizontal solid lines are the average fitness of the respective ancestral strain measured in these competitions. Horizontal dashed lines at a value of one are the relative fitness of the ADP1-ISx progenitor determined in a prior set of experiments, which was used as a reference value for normalizing all measurements in these assays. Error bars are 95% confidence intervals. Starred comparisons indicate a significant difference between the fitness values measured for an ancestor and its Δrnd mutant (Benjamini-Hochberg adjusted p < 0.05, t-tests). https://doi.org/10.1371/journal.pgen.1011306.g006 Most strains did not individually show a significant change in fitness in either medium after deleting rnd (Benjamini-Hochberg adjusted p > 0.05, t-tests) (Fig 6). The exceptions were MGD12, in which fitness decreased by 19.6% in LB (adjusted p = 0.0011); and MGD6, in which fitness increased by 33.8% in MS (adjusted p = 0.0070). Mutations in rnd were rare in MGD12 strains evolved in LB (Fig 5), which makes sense given the deleterious effects of deleting rnd in MGD12 in this environment. Similarly, there were no rnd mutations in LB-evolved MGD17 strains, which is in agreement with the apparent, though not statistically significant, reduction in fitness upon deleting rnd in MGD17. Overall, these results suggest that deleting RNase D is beneficial in many but not all combinations of ancestors and environments, in agreement with how putative knockout mutations are widespread, particularly in MS-evolved clones, but other types of mutations commonly evolved in certain strains.

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