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Australian typographer Harry Pears continues to explore ancient type forms while maintaining his consultancy business Typeface Research Pty. Ltd., of Lake Cathie, Australia. Born in Quirindi, Australia, Harry has had a long career in printing and graphic arts and has been the guiding force behind the creation of the Lindisfarne Nova family. Lindisfarne Nova Incised and Lindisfarne Runes are wonderful illustrative companions to the Lindisfarne Nova text fonts.In a unique partnership, Harry develops the concepts, and calligrapher Margaret Layson brings the designs to life. They both then work on the digital incarnation in a true collaboration.
In a process termed quorum sensing (QS), the opportunistic bacterial pathogen ______________________ uses diffusible signaling molecules to regulate the expression of numerous secreted factors or public goods that are shared within the population. But not all cells respond to QS signals. These social cheaters typically harbor a mutation in the QS receptor gene 1____ and exploit the public goods produced by cooperators. Here we show that non-social adaptation under growth conditions that require QS-dependent public goods increases tolerance to cheating and defers a tragedy of the commons. The underlying mutation is in the transcriptional repressor gene 2____. This mutation has no effect on public goods expression but instead increases individual fitness by derepressing growth-limiting intracellular metabolism. Even though 3____ mutant populations remain susceptible to invasion by isogenic 4_________ cheaters, they bear a lower cheater load than do wild-type populations, and they are completely resistant to invasion by 5____ cheaters with functional 6____. Mutations in 7____ also sustain growth near wild-type levels when paired with certain partial loss-of-function 8____ mutations. Targeted sequencing of multiple evolved isolates revealed that mutations in 9____ arise before mutations in 10____, and rapidly sweep through the population. Our results indicate that a QS-favoring environment can lead to adaptations in non-social, intracellular traits that increase the fitness of cooperating individuals and thereby contribute to population-wide maintenance of QS and associated cooperative behaviors.
Strains were initially grown overnight in MOPS-buffered LB liquid culture, and then diluted to an OD600 of 0.02 in fresh CAA medium. Expression cultures were harvested at OD600 values of 0.5 and 1.5, corresponding to exponential and early stationary phases in this medium, respectively. Total RNA was isolated and complementary DNA synthesized as previously described (Schuster and Greenberg, 2007; Schuster, 2011). Quantitative reverse-transcriptase PCR was carried out according to established protocols (Schuster and Greenberg, 2007; Schuster, 2011) using an Applied Biosystems 7300 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). Identical amounts of complementary DNA were used as template. Transcript levels were quantified using the relative standard curve method.
In our previous 11________ evolution studies, we cultured the PAO1 wild-type strain in caseinate medium for 20 days, subculturing into fresh medium each day (Sandoz et al., 2007; Wilder et al., 2011). We used two phenotypic screens as a proxy for QS proficiency, namely (1) protease production on skim-milk agar plates and (2) growth on minimal agar plates with adenosine as the sole carbon source. Negative results in both phenotypic screens correspond to mutations in the gene coding for the primary QS regulator, 12____, thereby conferring a cheater phenotype.
To confirm that QS-controlled extracellular proteolysis is solely responsible for the growth deficiency of the pleiotropic 13____ mutant in caseinate medium, we cultured the 14____ single mutant in the presence of purified elastase. Addition of elastase restored 15____ mutant fitness, expressed as Malthusian growth parameter, 16_ (Lenski et al., 1991), to a level indistinguishable from wild-type and significantly above that of the 17____ mutant without elastase (Figure 1).
To correlate the absolute fitness of each strain with its exoprotease activity, we quantified caseinolysis of culture supernatants using a FITC-casein assay (Twining, 1984; Wilder et al., 2011). This method is more precise than the qualitative skim-milk plate assay used previously (Sandoz et al., 2007). In order to uncouple exoprotease activity from its effect on growth, we replaced caseinate in our growth medium with CAA, a C-source that does not require QS-dependent proteolysis. All strains harboring the 18_____ allele showed intermediate levels of extracellular caseinolysis at half the levels of the wild-type 19____ alleles and roughly three times higher than the 20____ alleles (Figure 3b). Strains containing 21____ mutations did not show elevated caseinolysis compared with the wild-type. These results confirm that 22_____ is a partial loss-of-function mutation, and further show that 23____ has no effect on QS-dependent exoprotease production, consistent with its role in regulating intracellular dipeptide metabolism.
First, we sought to ensure that selection for 24____ mutants in the original 25________ evolution experiments was not just a general feature of prolonged growth but was tied to the specific growth medium. We therefore initiated defined co-cultures of the 26____ mutant and the wild-type in different growth media, at a mutant frequency of 0.01. We used a complex medium (MOPS-buffered LB) and M9 minimal medium with essentially fully hydrolyzed casein (CAA) as the sole C-source. The 27____ mutant did not enrich in either LB+MOPS or CAA media, confirming that adaptive mutation of 28____ is linked to the cooperative media we employed (Figure 5a). To confirm that the increased absolute fitness of a 29____ mutant can be attributed in part to increased uptake and metabolism of dipeptides, we also employed M9-minimal medium with the dipeptide GlyGlu as the sole carbon source (Kiely et al., 2008). The 30____ mutant exhibited a high degree of relative fitness in this medium very similar to the 31_____ mutant in caseinate medium (Figure 5a and first column of Figure 5b), indicating that dipeptide uptake and metabolism is indeed a target of selection in our cooperative growth environment.
An increase in the absolute fitness of 32_____________ during proteolytic growth was realized through a loss-of-function mutation in the transcriptional repressor PsdR that, in turn, increases intracellular dipeptide transport and processing. This adaptation suggests that QS-dependent extracellular proteolysis is not growth-rate limiting during 33________ evolution, at least not exclusively. Presumably, proteolysis is only limiting during an initial lag period at the beginning of each growth cycle in caseinate medium. Abundant protease secretion during this period may lead to an excess in proteolytic break-down products that await uptake and processing later in growth. Here, 34____ mutants would benefit. This effective separation in cooperative and non-cooperative selective targets during QS-dependent 35________ evolution of 36_____________ is illustrated in Figure 7. The 37____ mutation proportionally increased the growth rates of cooperators and cheaters in co-culture, because the 38_________ mutant showed the same relative fitness in 39____ mutant co-culture as did the 40____ mutant in wild-type co-culture (Figure 5b). This general impact on growth is nevertheless sufficient to explain its cooperation-stabilizing effect during 41________ evolution: mixed cooperator/cheater populations deficient in PsdR reach saturation faster than those with functional PsdR and are consequently more robust to cycles of dilution and regrowth.
More broadly, cycles of non-social, genetic adaptation and cheating are unlikely to maintain cooperative behavior in the long term as environmental adaptation is expected to eventually reach an optimum. Non-social adaptation through mutation likely works in concert with other mechanisms that stabilize cooperative behavior, and may be particularly beneficial early in the evolution of cooperative behavior. The generally high phenotypic plasticity of present-day microbes with unpredictable life histories would appear sufficient for coping with most changes in their natural environment. In microbes, other stabilizing mechanisms include positive assortment of cooperating individuals through, for example, colonial growth (Fletcher and Doebeli, 2009), the linkage of cooperative behaviors with other essential traits through pleiotropy (Foster et al., 2004) and metabolically prudent regulation of public goods such that their production is only initiated if the limiting nutrient is not also a building block of the good (Xavier et al., 2011; Mellbye and Schuster, 2014). It seems that pleiotropic control of extracellular proteolysis and subsequent intracellular metabolism via QS would be a reasonable strategy to curtail cheating, and a recent investigation using a similar 42________ evolution system has provided support for this notion (Dandekar et al., 2012). QS cheaters that do not contribute to proteolysis would be punished with reduced nutrient uptake and processing. We found that 43____ indeed controls 44____ transcription in rich medium (Schuster et al., 2003), but not in the minimal medium used in this study. Of course, the relative fitness advantage of 45____ mutant cheaters is consistent with the latter. Even if pleiotropic control played a role here, our results suggest that QS regulation of 46____ or related genes would be subject to strong counterselection whenever dipeptide uptake and processing was growth-rate limiting. 5376163bf9
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