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Pleiotropic hubs drive bacterial surface competition through parallel changes in colony composition and expansion [1]

['Jordi Van Gestel', 'Department Of Evolutionary Biology', 'Environmental Studies', 'University Of Zürich', 'Zürich', 'Swiss Institute Of Bioinformatics', 'Lausanne', 'Department Of Environmental Systems Science', 'Eth Zürich', 'Department Of Environmental Microbiology']

Date: 2023-10

Mutations with pleiotropic effects on both colony expansion and composition drive adaptation

To determine what drives the evolution of rapid colony expansion, we focused our analysis on the Bacilli species that showed the largest increase in colony size in our evolution experiment (Fig 1C). This is B. subtilis subsp. spizizenii ATCC 6633 (from here onward referred to as B. subtilis) [37]. From the 4 replicate B. subtilis populations in our evolution experiment, 2 replicates increased in size the most, producing colonies with a 2.5-fold larger radius than the ancestor [37]. We concentrated most of our analysis on these 2 replicates and will refer to them as lineage 1 and 2. For comparison, we also examined the other replicate populations (lineage 3 and 4) as well as populations from Bacillus cereus ATCC 10987 (from here onward referred to as B. cereus), which showed the second to largest increase in colony size in our evolution experiment [37].

We systematically genotyped and phenotyped populations in lineage 1 and 2. As a starting point, we first randomly isolated 2 or 3 clones from each of our weekly archived populations from the evolution experiment. We sequenced each clone and examined colony growth dynamics (S2 Fig) by cultivating individual clones for a week and imaging their colonies daily (Methods in S5 Text). Clones showed distinct mutations (S3 Table). Some mutations were present in all clones isolated after their initial appearance. These mutations fixed in the evolved populations and are most relevant for our analysis. Other mutations did not fix and were only observed transiently. Since genotypes and growth dynamics were identical for most clones isolated from the same week (R2 = 0.99, P < 10−16; S2 Fig), we decided to examine the colony composition for 1 clone per week only. We did so for the first 6 or 7 weeks of our evolution experiment, where the increase in colony size was most pronounced. For each clone, we collected cells from the colony edge and center and counted the number of filamentous cells, single cells, sporulating cells, and spores, using flow cytometry (see Fig 1B and Methods in S5 Text for details). For weeks in which large changes in colony composition occurred (clones isolated from week 3 and 5 for lineage 1, and clones isolated from week 1, 2, and 6 for lineage 2; Fig 2), we also acquired transcriptomic data by collecting cells from the colony edge and/or center after 1, 2, 4, or 7 days of colony growth and performing RNA-seq (see S4 Data). Fig 2 gives an overview of the mutations that arose in lineage 1 and 2 and their effect on colony growth and composition.

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TIFF original image Download: Fig 2. Evolution of surface expansion. Changes in colony expansion (A, B), genetic makeup (C, D), and colony composition (E, F) for clones isolated from evolved populations in the first weeks of the evolution experiment in lineage 1 (left) and 2 (right). (A and B) Colony expansion. Grey lines show colony outlines from day 1 (black outline) to day 7 (light grey outline), superimposing replicate colonies. Graph shows changes in colony radius in time (grey polygon, distribution in colony radius across replicates; black dot, mean; n = 3–16 colonies). Source data can be found in S1 Data. (C and D) Genetic mutations detected in clones isolated from evolved populations in lineage 1 (C) and 2 (D). Fixed mutations were observed in all clones isolated in subsequent weeks and are shown in black and nonfixed mutations were not observed in (all) clones isolated from subsequent weeks and are shown in white. For detailed overview of mutations in all isolated clones, see S3 Table. Source data can be found in S3 Data. (E and F) Colony composition at both colony edge (upper) and center (lower): filamentous cells (green), vegetative cells (blue), sporulating cells (purple), spores (red) (see figure legend). Source data can be found in S2 Data. https://doi.org/10.1371/journal.pbio.3002338.g002

In both ancestral and evolved colonies, we observed strong changes in colony composition during colony growth (Fig 2), consistent with previous studies on B. subtilis colony development [33,34]. We expect that these temporal changes in colony composition result from changes in resource gradients to which cells are exposed inside the colony (see Fig 1A and Model in S1 Text). As cells consume resources during colony growth, resource gradients emerge from the colony edge to the center [22,34], where there are more resources at the edge than in the center. This results in a relatively high fraction of spores in the center (Fig 2). Compared to previous studies [33,34], we grew colonies on a relatively poor growth medium (S1 Table) and for a long period of time (1 week), which increases the impact of resource depletion on colony development. At the colony edge, cells can however escape resource depletion by expanding outwards (S1 Text). Changes in colony composition are therefore the product of both resource consumption and colony expansion (S1 Text).

Also, over evolutionary time, we observed strong changes in colony composition, where sporulation is almost completely lost at the colony edge in both lineage 1 and 2 (Fig 2). Correspondingly, we observed a strong reduction in the expression of spp genes, encoding for small spore proteins [40,41], between the ancestor and evolved colonies for both lineage 1 (log 2 FC = −4.63, P<10−10; S3A Fig) and 2 (log 2 FC = −5.3, P<10−7; S3A Fig). The reduction in sporulation in lineage 1 and 2 was associated with mutations in 3 global regulators: RicT (lineage 1), RNAse Y (lineage 2), and LexA (lineage 2). The mutations affecting RicT and LexA were loss-of-function mutations (see Sections 1 and 3 in S2 Text and S5, S6, S8, and S9 Figs), while the mutation affecting RNase Y occurred upstream in the promoter region and lowered Rnase Y expression (see Section 2 in S2 Text and S5 and S7 Figs).

Each of the 3 affected global regulators have been linked to sporulation before [42–45]. RicT is part of the Y-complex, which—together with RicA and RicF—is thought to be important for regulating Rnase Y [46]. In doing so, RicT forms a stable association with Rnase Y [47]. The mutation affecting RicT in lineage 1 might thus impair sporulation through a similar mechanism than the mutation affecting Rnase Y in lineage 2. Rnase Y is one of the major endoribonucleases underlying both mRNA maturation and degradation in B. subtilis [48–50]. It was previously observed that knockouts of either ricT or rny, the genes encoding RicT and RNAse Y, cause severe sporulation defects and lower the spore frequency >100-fold [42–45] (see also S4 Fig)—which is consistent with the near loss of sporulation at the colony edge in both lineage 1 and 2 (Fig 2). It is unclear how RicT and RNAse Y exert their effect, but some evidence suggests that RicT might interfere with the phosphorylation cascade of Spo0A [43,51–55]. In lineage 2, we observed an additional mutation in LexA that led to the constitutive expression of the SOS response (Section 3 in S2 Text) [56,57]. The SOS response reduces sporulation (S4 Fig) through the Sda-dependent inhibition of Spo0A activity [58,59] and thus further lowers the sporulation rate (Fig 2F). Thus, in both lineage 1 and 2, we find mutations that directly or indirectly lower the sporulation rate, consistent with the findings in our previous study [37]. Mutants with lower sporulation rates outcompete the ancestor during colony growth, because of their higher effective growth rates (see Model in S1 Text).

Considering that the observed mutations targeted global regulators, they are expected to have many pleiotropic effects, some of which could be maladaptive. For example, RicT is important for mRNA maturation. Accordingly, we observed that our ricT mutant is defective in polycistronic mRNA cleavage, which might have potential negative side effects (Section 1 in S2 Text and S6 Fig). In the case of Rnase Y, it was previously shown that depletion of Rnase Y substantially increases the average mRNA half-life [48,60], which affects gene expression [49,61,62], impedes growth [45] (S4 Fig), and can even result in massive cell lysis [60]. In our experiments, we observed that the reduced rny expression resulted in a near loss of sporulation at the colony edge but simultaneously increased the fraction of spores in the colony center. Paradoxically, the increased fraction of spores in the colony center was not associated with a larger fraction of sporulating cells. In fact, after 3 days of colony growth, there were hardly any sporulating cells in the center (Fig 2F), while the fraction of spores peaked at approximately 80% to 90%. This suggests that the depletion of RNAse Y also resulted in cell lysis in our experiment, which led to the enrichment of spores in the colony center. Since we only transferred cells from the colony edge, cell lysis in the center would not affect colony propagation.

Pleiotropic effects can also be adaptive. This is perhaps most evident in lineage 2, where the lexA knockout mutant not only altered colony composition but also strongly affected colony expansion, causing an approximately 50% increase in colony radius (Fig 2B). Many bacterial species induce filamentation when activating the SOS response, which is broadly referred to as SOS filamentation [57,63]. In B. subtilis, SOS filamentation is mediated by YneA, which is suppressed by LexA [64]. Correspondingly, we observed that the lexA knockout mutation caused strong filamentation (Fig 2F), which enhanced colony expansion (see Section 3 in S2 Text and S9 Fig for more details). A single mutation can thus both reduce sporulation rates and increase colony expansion rates. Both phenotypes are adaptive in surface competition (S1 Text and S1B Fig).

Furthermore, the genetic mutations affecting RicT and RNAse Y have adaptive pleiotropic effects on surface expansion. It was previously shown that depletion of ricT and rny expression results in reduced expression of the eps operon [43,65], which underlies the production of extracellular polysaccharide (EPS). Consistently, in both lineage 1 and 2, we found significantly reduced eps expression in the evolved colonies compared to the ancestor (lineage 1: log 2 FC = −2.4, P<10−8; lineage 2: log 2 FC = −1.8, P<10−4; S3B Fig). EPS production mediates adhesion between cells and can thereby limit cells from expanding over a surface [49,66,67]. By reducing EPS expression, our ricT and rny mutants could therefore promote surface expansion. Indeed, beside the mutations in ricT and rny in lineage 1 and 2, we found several independent mutations in the eps operon across our replicate populations (epsD, epsF, epsI, epsK mutants) (S3 Table), each of which resulted in increased colony size (Section 4 in S2 Text, Figs 2 and S10). Since mutations in the eps operon enhance colony expansion, they allow cells to escape resource depletion, thereby lowering the fraction of sporulating cells at the colony edge (see S1 Text and S10 Fig). The impact of these mutations on sporulation is however small compared to that of the sporulation-inhibiting mutations in ricT and rny (Fig 2). Thus, like the lexA mutant, the ricT and rny mutants also have adaptive pleiotropic effects on both sporulation and colony expansion (Fig 2).

Besides the mutations affecting RicT, RNAse Y, and LexA, we also observed mutations with minimal or no effect on either colony size or composition (Fig 2), as detailed in Section 4 in S2 Text. These mutations might solely increase the cell division rate (see Section 4 in S2 Text and S11 Fig), with no observable consequence for both colony size and composition (see S1 Text), or they may have no phenotypic effect at all (e.g., synonymous amino acid substitutions; Fig 2). Previous studies have shown that bacterial surface expansion can result in low effective population sizes, which could promote the fixation of neutral or even maladaptive mutations [68].

Finally, for comparison, we also analyzed the evolved populations of B. cereus (S4 Table), which yielded highly similar results. B. cereus populations showed the second to largest increase in colony size during our evolution experiment [37]. Similar to our observations in B. subtilis, they harbored mutations that had pleiotropic effects on both sporulation and colony expansion (S4 Table). For example, as observed in our previous study [37], in one of the B. cereus replicate populations, there is a mutation in Spo0A, which reduces sporulation. Spo0A, however, also affects EPS production [69]. Thus, similar to the mutations affecting RNase Y and RicT in B. subtilis, the mutation in Spo0A is expected to have adaptive pleiotropic effects on both sporulation and EPS production [70–73]. In another replicate population of B. cereus (S4 Table and S12 Fig), we found 2 independent mutations affecting sporulation (in spoVG) and EPS production (in epsF). The sporulation mutant mainly affects the colony composition (S1 Text), while the EPS mutant strongly improves colony expansion (see both S1 Text and S12 Fig). Altogether, the results in B. cereus corroborate our findings in B. subtilis.

In summary, we find that bacterial surface competition favors mutations in global regulators, like RicT, Rnase Y, LexA, and Spo0A [37], with comparable pleiotropic effects on both colony size and composition: lowering the rate of sporulation and facilitating expansion by either reducing EPS production or causing filamentation. These global regulators function as pleiotropic hubs. They explain why even in our short evolution experiment we observed rapid adaptive changes in both colony size and composition (Fig 2).

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002338

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