Competitive Fitness Advantage Research Articles

Enterococcus faecalis Sex Pheromone cCF10 Enhances Conjugative Plasmid Transfer In Vivo.

ABSTRACTCell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency plasmid transfer in vitro in Enterococcus faecalis. To examine the role of pheromone signaling in vivo, we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro, no extracellular complementation occurred in vivo. This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo. In the absence of CF+ recipients, a low level of transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum.

Prophage Rs551 and Its Repressor Gene orf14 Reduce Virulence and Increase Competitive Fitness of Its Ralstonia solanacearum Carrier Strain UW551.

We previously characterized a filamentous lysogenic bacteriophage, ϕRs551, isolated directly from the race 3 biovar 2 phylotype IIB sequevar 1 strain UW551 of Ralstonia solanacearum grown under normal culture conditions. The genome of ϕRs551 was identified with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum, indicating evolutionary and biological importance, and ORF14 of ϕRs551 was annotated as a putative type-2 repressor. In this study, we determined the effect of the prophage and its ORF14 on the virulence and competitive fitness of its carrier strain UW551 by deleting the orf14 gene only (the UW551 orf14 mutant), and nine of the prophage’s 14 genes including orf14 and six out of seven structural genes (the UW551 prophage mutant), respectively, from the genome of UW551. The two mutants were increased in extracellular polysaccharide production, twitching motility, expression of targeted virulence and virulence regulatory genes (pilT, egl, pehC, hrPB, and phcA), and virulence, suggesting that the virulence of UW551 was negatively regulated by ϕRs551, at least partially through ORF14. Interestingly, we found that the wt ϕRs551-carrying strain UW551 of R. solanacearum significantly outcompeted the wt strain RUN302 which lacks the prophage in tomato plants co-inoculated with the two strains. When each of the two mutant strains was co-inoculated with RUN302, however, the mutants were significantly out-competed by RUN302 for the same colonization site. Our results suggest that ecologically, ϕRs551 may play an important role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment, which in turn prolongs the symbiotic relationship between the phage ϕRs551 and the R. solanacearum strain UW551. Our study is the first toward a better understanding of the co-existence between a lysogenic phage and its carrier plant pathogenic bacterial strain by determining the effect of the prophage Rs551 and its repressor on the virulence and competitive fitness of its carrier strain UW551 of R. solanacearum.

Clonal Evolution During Stress Hematopoiesis

Hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations with age resulting in a heterogeneous cell population, with each HSPC possessing its own unique set of private mutations. HSPCs that acquire somatic mutations that confer a competitive fitness advantage relative to their normal counterparts may clonally expand. Indeed, several groups have documented the presence of clonal hematopoiesis in healthy individuals. Although originally thought to be limited to older individuals, a recent study using an ultra-sensitive sequencing technique showed that expanded hematopoietic clones are detectable in the majority of healthy 50-60-year-old individuals. With some notable exceptions, the same genes that are commonly mutated in clonal hematopoiesis also are somatically mutated in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). However, only a small fraction of individuals with clonal hematopoiesis subsequently develop a myeloid malignancy and the size of the mutant clone in clonal hematopoiesis can remain stable for years without disease progression. These observations raise several questions that will be addressed in this presentation. What drives expansion of hematopoietic clones? What role do external hematopoietic stressors, such as exposure to chemotherapy, play in the development of clonal hematopoiesis? Why do so few people with clonal hematopoiesis develop a myeloid malignancy, and are there certain mutations that confer a higher risk of transformation? Disclosures No relevant conflicts of interest to declare.

Rapid expansion of preexisting nonleukemic hematopoietic clones frequently follows induction therapy for de novo AML

AbstractThere is interest in using leukemia-gene panels and next-generation sequencing to assess acute myelogenous leukemia (AML) response to induction chemotherapy. Studies have shown that patients with AML in morphologic remission may continue to have clonal hematopoiesis with populations closely related to the founding AML clone and that this confers an increased risk of relapse. However, it remains unknown how induction chemotherapy influences the clonal evolution of a patient's nonleukemic hematopoietic population. Here, we report that 5 of 15 patients with genetic clearance of their founding AML clone after induction chemotherapy had a concomitant expansion of a hematopoietic population unrelated to the initial AML. These populations frequently harbored somatic mutations in genes recurrently mutated in AML or myelodysplastic syndromes and were detectable at very low frequencies at the time of AML diagnosis. These results suggest that nonleukemic hematopoietic stem and progenitor cells, harboring specific aging-acquired mutations, may have a competitive fitness advantage after induction chemotherapy, expand, and persist long after the completion of chemotherapy. Although the clinical importance of these “rising” clones remains to be determined, it will be important to distinguish them from leukemia-related populations when assessing for molecular responses to induction chemotherapy.

Regulated Formation of lncRNA-DNA Hybrids Enables Faster Transcriptional Induction and Environmental Adaptation

Long non-coding (lnc)RNAs, once thought to merely represent noise from imprecise transcription initiation, have now emerged as major regulatory entities in all eukaryotes. In contrast to the rapidly expanding identification of individual lncRNAs, mechanistic characterization has lagged behind. Here we provide evidence that the GAL lncRNAs in the budding yeast S. cerevisiae promote transcriptional induction in trans by formation of lncRNA-DNA hybrids or R-loops. The evolutionarily conserved RNA helicase Dbp2 regulates formation of these R-loops as genomic deletion or nuclear depletion results in accumulation of these structures across the GAL cluster gene promoters and coding regions. Enhanced transcriptional induction is manifested by lncRNA-dependent displacement of the Cyc8 co-repressor and subsequent gene looping, suggesting that these lncRNAs promote induction by altering chromatin architecture. Moreover, the GAL lncRNAs confer a competitive fitness advantage to yeast cells because expression of these non-coding molecules correlates with faster adaptation in response to an environmental switch.

Non-Malignant Oligoclonal Hematopoiesis Commonly Follows Cytoreductive Chemotherapy in Adult De Novo AML Patients

Our group (Welch, Cell 2012) previously showed that hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations with age. This produces a genetically heterogeneous HSPC population with each HSPC possessing its own unique set of mutations. Later work from our group (Xie, Nature Medicine 2014) and others (Genovese, Jaiswal, NEJM 2014) demonstrated that some these mutations may provide HSPCs with a fitness advantage, allowing them to clonally expand over time in healthy individuals. We recently published data (Wong, Nature 2015) suggesting that cytotoxic therapy can select for HSPC clones with TP53 mutations, resulting in their clonal expansion and contributing to the subsequent development of therapy-related AML/MDS. From these data, we hypothesized that the intensive cytoreductive chemotherapy used to treat AML poses a significant selection pressure on a patient's non-malignant HSPC population, favoring HSPCs with specific somatic mutations and potentially resulting in oligoclonal hematopoiesis even after elimination of the founding AML clone.To test this hypothesis, we performed enhanced exome sequencing on cryopreserved bone marrow cells from 25 adult de novo AML patients (who received a "7+3" regimen for induction of remission) at time of their initial diagnosis, at first morphologic remission (~day 30), and at long-term follow up (at first relapse or during a prolonged first remission) (Klco, JAMA, in press). In 15 patients, we observed genetic clearance of the AML founding clone at the time of first morphologic remission (defined as all AML founding clone mutations declining to a variant allele frequency (VAF) < 2.5%). Surprisingly, in 5 of the 15 patients exhibiting clearance of their AML founding clone, we observed a concomitant expansion of a non-malignant clonal population during cytoreductive therapy, resulting in long-lived clonal hematopoiesis. Somatic mutations harbored by these expanding hematopoietic clones were validated with a high-coverage PCR-based sequencing approach. In contrast to the studies highlighting clonal hematopoiesis in individuals unexposed to chemotherapy, patients with evidence of persistent clonal hematopoiesis after cytoreductive therapy (median age = 52.2 years) were similar in age to patients without such evidence (median age = 54.1 years).The majority of these "rising clones" harbored somatic mutations in genes frequently mutated in AML such as DNMT3A, TET2 and TP53. Using next-generation sequencing and droplet digital PCR, we determined that in all of the patients with an expanding non-malignant clone, the clone was, in fact, present in the initial AML diagnosis sample at very low VAFs (0.007-0.75%). These populations rapidly expanded with chemotherapy, comprising 13-57% of the total hematopoietic population upon its completion. In all 4 of cases with sample availability, these clones remained at an expanded level a year or more after initial chemotherapy exposure. These results suggest that certain non-malignant HSPCs, having previously acquired specific aging-related somatic mutations, may gain a competitive fitness advantage after cytoreductive therapy, expand, and persist long after the completion of chemotherapy.Two of the five patients with clonal non-leukemic hematopoiesis post-chemotherapy relapsed. In both patients, the relapsed AML clone evolved from the original AML founding clone and did not involve the non-malignant clonal population, which also persisted at relapse. Both patients re-achieved morphologic remission with salvage therapy. A post-salvage therapy bone marrow sample was available in one of the cases. Interestingly, it showed that the patient's non-malignant clonal population expanded even further with salvage therapy, eventually comprising almost 80% of the total bone marrow cells.These results show that non-malignant oligoclonal hematopoiesis is common in AML patients after cytoreductive chemotherapy, with non-malignant HSPCs carrying certain somatic mutations often gaining a fitness advantage and expanding. The long-term clinical consequences of oligoclonal hematopoiesis after cytoreductive chemotherapy are unknown but are likely to be different from oligoclonal hematopoiesis developing in healthy elderly individuals. Additional studies will be required to define the mechanisms by which certain HSPCs gain a fitness advantage after cytoreductive chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Expanding the RpoS/σS-network by RNA sequencing and identification of σS-controlled small RNAs in Salmonella.

The RpoS/σS sigma subunit of RNA polymerase (RNAP) controls a global adaptive response that allows many Gram-negative bacteria to survive starvation and various stresses. σS also contributes to biofilm formation and virulence of the food-borne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). In this study, we used directional RNA-sequencing and complementary assays to explore the σS-dependent transcriptome of S. Typhimurium during late stationary phase in rich medium. This study confirms the large regulatory scope of σS and provides insights into the physiological functions of σS in Salmonella. Extensive regulation by σS of genes involved in metabolism and membrane composition, and down-regulation of the respiratory chain functions, were important features of the σS effects on gene transcription that might confer fitness advantages to bacterial cells and/or populations under starving conditions. As an example, we show that arginine catabolism confers a competitive fitness advantage in stationary phase. This study also provides a firm basis for future studies to address molecular mechanisms of indirect regulation of gene expression by σS. Importantly, the σS-controlled downstream network includes small RNAs that might endow σS with post-transcriptional regulatory functions. Of these, four (RyhB-1/RyhB-2, SdsR, SraL) were known to be controlled by σS and deletion of the sdsR locus had a competitive fitness cost in stationary phase. The σS-dependent control of seven additional sRNAs was confirmed in Northern experiments. These findings will inspire future studies to investigate molecular mechanisms and the physiological impact of post-transcriptional regulation by σS.

Research highlights

With a changing climate, increasing need for food security, growing threats to marine and freshwater fisheries, an impending antibiotic crisis, invading species, and continued risks of emerging human and agricultural diseases, the need for creative and forward-thinking solutions is critical. The application of evolutionary theory to these everyday problems is proving to be a reliable and powerful tool, without which our ability to predict the responses of populations to change, both human-mediated and otherwise, is not possible. Evolutionary Applications is the only journal specializing specifically in publishing papers that make contributions to fundamental questions in evolutionary biology using study systems that are of practical or applied importance in topics including, but not limited to: agriculture, aquaculture, biomedicine, biotechnology, climate change, conservation biology, disease biology, forestry, invasion biology, and fisheries and wildlife management. As such, one of our main goals is to promote interest of applied evolution to a diverse audience including evolutionary biologists, ecologists, biomedical researchers, environmental consultants, and biologists within industry, government, and health care. Toward this end, this new series of research highlights will offer brief synopses of new work with direct relevance to readers of Evolutionary Applications from across other journals with the aim of exploring the breadth of potential applications of evolutionary theory from across fields and disciplines. This past year was full of noteworthy evolutionary applications, including a strong focus on the use of combination therapy to slow or stop the spread of drug resistance. In particular, the application of evolutionary theory to resistance of cancers was a hot issue, with a special issue of Evolutionary Applications (volume 6, issue 1), a Nature Reviews Cancer piece about the importance of life-history trade-offs to our understanding of cancer progression (Aktipis et al. 2013), a Nature review article on genomic instability of cancer (Burrell et al. 2013), a Jacques Monod conference on ‘Ecological and evolutionary perspectives in cancer,’ and the 2nd International Biannual Evolution and Cancer Conference. This blossoming interest is uncovering the roles of evolutionary processes in shaping the development, spread, and virulence of cancers, but is also highlighting the current gap in our ability to translate evolutionary theory into successful treatment. An elegant example of the great potential of this approach is the recent work by Shi and coauthors identifying two core resistance pathways in metastatic melanomas (Shi et al. 2014). The researchers found that drug resistance of these cancers occurred most often via two distinct ‘drug escape pathways,’ explaining 70% and 22% of resistance among disease-progressive tissues. These data act as strong justification for the use of combination treatment to target both BRAF inhibitor resistance pathways simultaneously to block these common routes of drug escape. Similar approaches are being applied to tackle the current antibiotic crisis, as has been reviewed in a recent Nature Reviews Genetics article (Palmer and Kishony 2013). The authors point out the importance of using optimal combinations of antibiotics, ideally chosen based on negative cross-resistance and antagonistic interactions among resistance mechanisms. However, a recent paper by Pena-Miller and colleagues, combining experimental and genomic techniques with mathematical modeling, highlights that caution must be employed when employing combination treatments, as the use of synergistic antibiotics can in fact increase pathogen load under suboptimal treatment durations or subinhibitory drug concentrations (Pena-Miller et al. 2013). A similar warning has now been raised for combination treatment of malaria due to the discovery of high association among resistance alleles in multiple regions of China (Ding et al. 2013). Furthermore, a clear understanding of epistasis among resistance mutations is key to predicting the spread of multidrug-resistant bacteria, as has been nicely demonstrated by Borrell et al. (2013). They found that combinations of drug resistance mutations in Mycobacterium smegmatis showing a competitive fitness advantage in vitro were also the most frequently observed combinations found in multidrug-resistant clinical isolates of human tuberculosis in South Africa. The application of evolutionary theory to development of combined treatments is certainly not limited to human disease. Experimental evolution of the green chlorophyte, Chlamydomonas reinhardtii, was used by Lagator and coauthors to demonstrate the utility of combined herbicides for slowing the evolution of resistance in agricultural settings (Lagator et al. 2013). However, their data come with a similar word of warning: using these combinations at low doses can select for both increased rates of resistance and a more general resistance mechanism relative to single herbicide treatment. This small subsample of recent research published in Evolutionary Applications and other high-profile journals on combination treatment emphasizes the power of applying evolutionary theory to treatment design, both in terms of creating more ‘evolution-proof’ combinations and in predicting potential negative consequences of this approach. We look forward to future research on the topic and continued monitoring of treatments designed in the light of evolutionary theory.

Evolution in a test tube: rise of the Wrinkly Spreaders

Understanding evolutionary mechanisms is fundamental to a balanced biological education, yet practical demonstrations are rarely considered. In this paper we describe a bacterial liquid microcosm which can be used to demonstrate aspects of evolution, namely adaptive radiation, niche colonisation and competitive fitness. In microcosms inoculated with Pseudomonas fluorescens SBW25, evolved mutants such as the Wrinkly Spreader (WS) rapidly arise to form biofilms covering the air–liquid (A–L) interface. WS are readily isolated due to a distinctive colony morphology and reach ∼30% of the population within five days. When re‐inoculated into static microcosms, WS preferentially colonises the A–L interface by producing a biofilm, demonstrating a niche preference distinct from the ancestral SBW25 which grows throughout the liquid column. This ability provides the WS with a ∼2.5× competitive fitness advantage over the non‐biofilm forming ancestral SBW25. However, WS and SBW25 have similar fitness in shaken microcosms where biofilms cannot form. These practical demonstrations of WS evolution, suitable for secondary or tertiary‐level classes, can be linked with a literature‐based review of the underlying molecular biology of the WS phenotype to provide a true exemplar of the modern evolutionary synthesis, the current paradigm in evolutionary biology.

Loss of Expression of cspC , a Cold Shock Family Gene, Confers a Gain of Fitness in Escherichia coli K-12 Strains

The CspA family of cold shock genes in Escherichia coli K-12 includes nine paralogs, cspA to cspI. Some of them have been implicated in cold stress adaptation. Screening for mutations among common laboratory E. coli strains showed a high degree of genetic diversity in cspC but not in cspA and cspE. This diversity in cspC was due to a wide spectrum of variations including insertions of IS elements, deletion, and point mutation. Northern analysis of these mutants showed loss of cspC expression in all but one case. Further analysis of the loss-of-function cspC mutants showed that they have a fitness advantage in broth culture after 24 h over their isogenic wild-type derivatives. Conversely, introduction of mutated cspC alleles conferred a competitive fitness advantage to AB1157, a commonly used laboratory strain. This provides the evidence that loss of cspC expression is both necessary and sufficient to confer a gain of fitness as seen in broth culture over 24 h. Together, these results ascribe a novel role in cellular growth at 37 degrees C for CspC, a member of the cold shock domain-containing protein family.

Competitive growth advantage of nontoxigenic mutants in the stationary phase in archival cultures of pathogenic Vibrio cholerae strains.

Spontaneous nontoxigenic mutants of highly pathogenic Vibrio cholerae O1 strains accumulate in large numbers during long-term storage of the cultures in agar stabs. In these mutants, production of the transcriptional regulator ToxR was reduced due to the presence of a mutation in the ribosome-binding site immediately upstream of the toxR open reading frame. Consequently, the ToxR-dependent virulence regulon was turned off, with concomitant reduction in the expression of cholera toxin and toxin-coregulated pilus. An intriguing feature of these mutants is that they have a competitive fitness advantage when grown in competition with the parent strains in stationary-phase cocultures which is independent of RpoS, the only locus known to be primarily associated with acquisition of a growth advantage phenotype in bacteria.

The growth advantage in stationary-phase phenotype conferred by rpoS mutations is dependent on the pH and nutrient environment.

Escherichia coli cells that are aged in batch culture display an increased fitness referred to as the growth advantage in stationary phase, or GASP, phenotype. A common early adaptation to this culture environment is a mutant rpoS allele, such as rpoS819, that results in attenuated RpoS activity. However, it is important to note that during long-term batch culture, environmental conditions are in flux. To date, most studies of the GASP phenotype have focused on identifying alleles that render an advantage in a specific environment, Luria-Bertani broth (LB) batch culture. To determine what role environmental conditions play in rendering relative fitness advantages to E. coli cells carrying either the wild-type or rpoS819 alleles, we performed competitions under a variety of culture conditions in which either the available nutrients, the pH, or both were manipulated. In LB medium, we found that while the rpoS819 allele confers a strong competitive fitness advantage at basic pH, it confers a reduced advantage under neutral conditions, and it is disadvantageous under acidic conditions. Similar results were found using other media. rpoS819 conferred its greatest advantage in basic minimal medium in which either glucose or Casamino Acids were the sole source of carbon and energy. In acidic medium supplemented with either Casamino Acids or glucose, the wild-type allele conferred a slight advantage. In addition, populations were dynamic under all pH conditions tested, with neither the wild-type nor mutant rpoS alleles sweeping a culture. We also found that the strength of the fitness advantage gained during a 10-day incubation is pH dependent.

SOS-induced DNA polymerases enhance long-term survival and evolutionary fitness.

Escherichia coli encodes three SOS-induced DNA polymerases: pol II, pol IV, and pol V. We show here that each of these polymerases confers a competitive fitness advantage during the stationary phase of the bacterial life cycle, in the absence of external DNA-damaging agents known to induce the SOS response. When grown individually, wild-type and SOS pol mutants exhibit indistinguishable temporal growth and death patterns. In contrast, when grown in competition with wild-type E. coli, mutants lacking one or more SOS polymerase suffer a severe reduction in fitness. These mutants also fail to express the "growth advantage in stationary phase" phenotype as do wild-type strains, instead expressing two additional new types of "growth advantage in stationary phase" phenotype. These polymerases contribute to survival by providing essential functions to ensure replication of the chromosome and by generating genetic diversity.