Cheating by Exploitation of Developmental Prestalk Patterning in Dictyostelium discoideum

The cooperative developmental system of the social amoeba Dictyostelium discoideum is susceptible to exploitation by cheaters—strains that make more than their fair share of spores in chimerae. Laboratory screens in Dictyostelium have shown that the genetic potential for facultative cheating is high, and field surveys have shown that cheaters are abundant in nature, but the cheating mechanisms are largely unknown. Here we describe cheater C (chtC), a strong facultative cheater mutant that cheats by affecting prestalk differentiation. The chtC gene is developmentally regulated and its mRNA becomes stalk-enriched at the end of development. chtC mutants are defective in maintaining the prestalk cell fate as some of their prestalk cells transdifferentiate into prespore cells, but that defect does not affect gross developmental morphology or sporulation efficiency. In chimerae between wild-type and chtC mutant cells, the wild-type cells preferentially give rise to prestalk cells, and the chtC mutants increase their representation in the spore mass. Mixing chtC mutants with other cell-type proportioning mutants revealed that the cheating is directly related to the prestalk-differentiation propensity of the victim. These findings illustrate that a cheater can victimize cooperative strains by exploiting an established developmental pathway

Bacterial Adaptation through Loss of Function

The metabolic capabilities and regulatory networks of bacteria have been optimized by evolution in response to selective pressures present in each species' native ecological niche. In a new environment, however, the same bacteria may grow poorly due to regulatory constraints or biochemical deficiencies. Adaptation to such conditions can proceed through the acquisition of new cellular functionality due to gain of function mutations or via modulation of cellular networks. Using selection experiments on transposon-mutagenized libraries of bacteria, we illustrate that even under conditions of extreme nutrient limitation, substantial adaptation can be achieved solely through loss of function mutations, which rewire the metabolism of the cell without gain of enzymatic or sensory function. A systematic analysis of similar experiments under more than 100 conditions reveals that adaptive loss of function mutations exist for many environmental challenges. Drawing on a wealth of examples from published articles, we detail the range of mechanisms through which loss-of-function mutations can generate such beneficial regulatory changes, without the need for rare, specific mutations to fine-tune enzymatic activities or network connections. The high rate at which loss-of-function mutations occur suggests that null mutations play an underappreciated role in the early stages of adaption of bacterial populations to new environments

Systematic identification of molecular mediators of interspecies sensing in a community of two frequently coinfecting bacterial pathogens.

Bacteria typically exist in dynamic, multispecies communities where polymicrobial interactions influence fitness. Elucidating the molecular mechanisms underlying these interactions is critical for understanding and modulating bacterial behavior in natural environments. While bacterial responses to foreign species are frequently characterized at the molecular and phenotypic level, the exogenous molecules that elicit these responses are understudied. Here, we outline a systematic strategy based on transcriptomics combined with genetic and biochemical screens of promoter-reporters to identify the molecules from one species that are sensed by another. We utilized this method to study interactions between the pathogens Pseudomonas aeruginosa and Staphylococcus aureus that are frequently found in coinfections. We discovered that P. aeruginosa senses diverse staphylococcal exoproducts including the metallophore staphylopine (StP), intermediate metabolites citrate and acetoin, and multiple molecules that modulate its iron starvation response. We observed that StP inhibits biofilm formation and that P. aeruginosa can utilize citrate and acetoin for growth, revealing that these interactions have both antagonistic and beneficial effects. Due to the unbiased nature of our approach, we also identified on a genome scale the genes in S. aureus that affect production of each sensed exoproduct, providing possible targets to modify multispecies community dynamics. Further, a combination of these identified S. aureus products recapitulated a majority of the transcriptional response of P. aeruginosa to S. aureus supernatant, validating our screening strategy. Cystic fibrosis (CF) clinical isolates of both S. aureus and P. aeruginosa also showed varying degrees of induction or responses, respectively, which suggests that these interactions are widespread among pathogenic strains. Our screening approach thus identified multiple S. aureus secreted molecules that are sensed by P. aeruginosa and affect its physiology, demonstrating the efficacy of this approach, and yielding new insight into the molecular basis of interactions between these two species

Multifactorial Competition and Resistance in a Two-Species Bacterial System

<div><p>Microorganisms exist almost exclusively in interactive multispecies communities, but genetic determinants of the fitness of interacting bacteria, and accessible adaptive pathways, remain uncharacterized. Here, using a two-species system, we studied the antagonism of <i>Pseudomonas aeruginosa</i> against <i>Escherichia coli</i>. Our unbiased genome-scale approach enabled us to identify multiple factors that explained the entire antagonism observed. We discovered both forms of ecological competition–sequestration of iron led to exploitative competition, while phenazine exposure engendered interference competition. We used laboratory evolution to discover adaptive evolutionary trajectories in our system. In the presence of <i>P</i>. <i>aeruginosa</i> toxins, <i>E</i>. <i>coli</i> populations showed parallel molecular evolution and adaptive convergence at the gene-level. The multiple resistance pathways discovered provide novel insights into mechanisms of toxin entry and activity. Our study reveals the molecular complexity of a simple two-species interaction, an important first-step in the application of systems biology to detailed molecular dissection of interactions within native microbiomes.</p></div

Energy, economic and environmental performance assessment of a grid-tied rooftop system in different cities of India based on 3E analysis

India is very rich in solar energy, with a total of 3000 sunshine hours annually in most places. The installation of on-grid rooftop electricity-generation photovoltaic (PV) systems is currently undergoing substantial growth and extension as an alternate source of energy that contributes to Indian buildings. This paper analyses the viability of mounting solar PV plants in distinct cities of India in various locations with different climate conditions such as Delhi, Bhopal, Udaipur, Ahmadabad, Thiruvananthapuram, Pune and Madurai. The technical feasibility of installing a 100-kWp system is evaluated using PVsyst software under local climatic conditions. The performance ratio is between 70% and 80%, with a capacity utilization factor of 19–21% and estimated energy output of 170 MWh annually at all sites. The system produces 400–500 kWh of energy daily at a per-unit cost of INR 6–7 (Indian rupees) in all locations. The lifespan of the system is ~25–30 years, reducing about 150–170 tons of carbon-dioxide emission to the atmosphere every year. The payback period of the system is ~5–6 years, which defines its feasibility. This information would encourage organizations and individuals to install such PV plants on the rooftops of buildings to use solar electricity for meeting the energy demands of the country

<i>Pseudomonas</i> siderophores and phenazines reduce fitness of <i>E</i>. <i>coli</i> in direct competition with <i>P</i>. <i>aeruginosa</i>.

<p><i>E</i>. <i>coli</i> cells were grown in direct competition with <i>P</i>. <i>aeruginosa</i> WT and mutant strains, and the fitness of both species was measured over a period of 20 hours. All mutant data were significantly different from WT (<i>q</i> < 0.001) as determined by a one-sided Mann-Whitney <i>U</i> test followed by the Benjamini-Hochberg procedure for multiple testing correction. The fitness of the mutant strains in competition with <i>E</i>. <i>coli</i> was similar to that of WT <i>P</i>. <i>aeruginosa</i>, supporting the idea that the siderophores and phenazines actively decrease <i>E</i>. <i>coli</i> fitness, rather than increasing <i>P</i>. <i>aeruginosa</i> fitness under our conditions.</p

The PQS pathway response in <i>P</i>. <i>aeruginosa</i> is important for <i>E</i>. <i>coli</i> growth inhibition.

<p><i>E</i>. <i>coli</i> cells were grown in the presence of 20% or 50% (v/v) spent media from either WT <i>P</i>. <i>aeruginosa</i> or various PQS pathway mutants, and the cell density was determined before and after 16 hours of growth. Data are the means from at least 5 replicates. Error bars represent standard deviation. All mutant and control data shown were significantly different from WT (<i>q</i> < 0.005) as determined by a one-sided Mann-Whitney <i>U</i> test followed by the Benjamini-Hochberg procedure for multiple testing correction (this correction included the mutant strains showed in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005715#pgen.1005715.g003" target="_blank">3B</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005715#pgen.1005715.g007" target="_blank">7A</a>).</p

Iron-limitation by <i>P</i>. <i>aeruginosa</i> siderophores inhibits <i>E</i>. <i>coli</i> growth.

<p><i>E</i>. <i>coli</i> cells were grown in the presence of <i>P</i>. <i>aeruginosa</i> spent media and the cell density was determined before and after 16 hours of growth. Data are the means from at least 5 replicates. Error bars represent standard deviation. <b>(A)</b> <i>E</i>. <i>coli</i> cells were grown in the presence of 50% (v/v) <i>P</i>. <i>aeruginosa</i> spent media supplemented with increasing levels of ferric citrate. Data from each concentration were significantly different from the preceding concentration (<i>q</i> < 0.005) as determined by a one-sided Mann-Whitney <i>U</i> test followed by the Benjamini-Hochberg procedure for multiple testing correction. <b>(B)</b> <i>E</i>. <i>coli</i> cells were grown in the presence of 20% or 50% (v/v) spent media from various <i>P</i>. <i>aeruginosa</i> wild-type and mutant strains. All mutant and control data shown (except for Δ<i>pchE</i> at 20%) were significantly different from WT (<i>q</i> < 0.005) as determined by a one-sided Mann-Whitney <i>U</i> test followed by the Benjamini-Hochberg procedure for multiple testing correction (this correction included the mutant strains showed in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005715#pgen.1005715.g005" target="_blank">5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005715#pgen.1005715.g007" target="_blank">7A</a>). Spent media from the Δ<i>pvdJ</i> and the Δ<i>pvdJ</i> Δ<i>pchE</i> mutants supported more growth than the media salts base control, likely due to the presence of unused nutrients or <i>P</i>. <i>aeruginosa</i> signaling molecules in the spent media.</p

<i>P</i>. <i>aeruginosa</i> secreted phenazines inhibit <i>E</i>. <i>coli</i> growth.

<p><i>E</i>. <i>coli</i> cells were grown in the presence of <i>P</i>. <i>aeruginosa</i> spent media or pyocyanin, and the cell density was determined before and after 16 hours of growth. Data are the means from at least 5 replicates. Error bars represent standard deviation. <b>(A)</b> <i>E</i>. <i>coli</i> cells were grown in the presence of 20% or 50% (v/v) spent media from <i>P</i>. <i>aeruginosa</i> wild-type and phenazine mutant strains. All mutant and control data shown were significantly different from WT (<i>q</i> < 0.005) as determined by a one-sided Mann-Whitney <i>U</i> test followed by the Benjamini-Hochberg procedure for multiple testing correction (this correction included the mutant strains showed in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005715#pgen.1005715.g003" target="_blank">3B</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005715#pgen.1005715.g005" target="_blank">5</a>). <b>(B)</b> <i>E</i>. <i>coli</i> cells were grown in the presence of increasing concentrations of pyocyanin. The 50μM and 100μM data were significantly different from the immediately lower concentration (25μM and 50μM, respectively) as determined by a one-sided Mann-Whitney <i>U</i> test followed by the Benjamini-Hochberg procedure for multiple testing correction (<i>q</i> < 0.01), but the 25μM data was not significantly different from the 0μM data (<i>q</i> > 0.5).</p

<i>P</i>. <i>aeruginosa</i> spent media upregulates the <i>soxS</i>-response in <i>E</i>. <i>coli</i>.

<p><i>E</i>. <i>coli</i> cells were grown in the presence of 45% (v/v) WT <i>P</i>. <i>aeruginosa</i> spent media supplemented with 100μM ferric citrate, and the transcriptional response was measured compared to unexposed cells. The genes were ordered by their fold-induction, and divided into 10 equal sized bins, represented in the 10 columns. The range of the log₁₀(fold-change) is shown on the top left. The global change in gene expression was analyzed using the ‘transcription factor regulon’ module in iPAGE [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005715#pgen.1005715.ref022" target="_blank">22</a>], which identifies the transcription factor regulons enriched and depleted across the bins, as shown in the heat map. The colors show the significance of the enrichment, with red representing the negative of log₁₀ of the over-representation <i>p</i>-values, and blue representing the log₁₀ of the under-representation <i>p</i>-values.</p