What It Takes to Be a Pseudomonas aeruginosa? The Core Genome of the Opportunistic Pathogen Updated

Pseudomonas aeruginosa is an opportunistic bacterial pathogen able to thrive in highly diverse ecological niches and to infect compromised patients. Its genome exhibits a mosaic structure composed of a core genome into which accessory genes are inserted en bloc at specific sites. The size and the content of the core genome are open for debate as their estimation depends on the set of genomes considered and the pipeline of gene detection and clustering. Here, we redefined the size and the content of the core genome of P. aeruginosa from fully re-analyzed genomes of 17 reference strains. After the optimization of gene detection and clustering parameters, the core genome was defined at 5,233 orthologs, which represented ~ 88% of the average genome. Extrapolation indicated that our panel was suitable to estimate the core genome that will remain constant even if new genomes are added. The core genome contained resistance determinants to the major antibiotic families as well as most metabolic, respiratory, and virulence genes. Although some virulence genes were accessory, they often related to conserved biological functions. Long-standing prophage elements were subjected to a genetic drift to eventually display a G+C content as higher as that of the core genome. This contrasts with the low G+C content of highly conserved ribosomal genes. The conservation of metabolic and respiratory genes could guarantee the ability of the species to thrive on a variety of carbon sources for energy in aerobiosis and anaerobiosis. Virtually all the strains, of environmental or clinical origin, have the complete toolkit to become resistant to the major antipseudomonal compounds and possess basic pathogenic mechanisms to infect humans. The knowledge of the genes shared by the majority of the P. aeruginosa isolates is a prerequisite for designing effective therapeutics to combat the wide variety of human infections.     

Gene Expression in Transformed Lymphocytes Reveals Variation in Endomembrane and HLA Pathways Modifying Cystic Fibrosis Pulmonary Phenotypes

Variation in cystic fibrosis (CF) phenotypes, including lung disease severity, age of onset of persistent Pseudomonas aeruginosa (P. aeruginosa) lung infection, and presence of meconium ileus (MI), has been partially explained by genome-wide association studies (GWASs). It is not expected that GWASs alone are sufficiently powered to uncover all heritable traits associated with CF phenotypic diversity. Therefore, we utilized gene expression association from lymphoblastoid cells lines from 754 p.Phe508del CF-affected homozygous individuals to identify genes and pathways. LPAR6, a G protein coupled receptor, associated with lung disease severity (false discovery rate q value = 0.0006). Additional pathway analyses, utilizing a stringent permutation-based approach, identified unique signals for all three phenotypes. Pathways associated with lung disease severity were annotated in three broad categories: (1) endomembrane function, containing p.Phe508del processing genes, providing evidence of the importance of p.Phe508del processing to explain lung phenotype variation; (2) HLA class I genes, extending previous GWAS findings in the HLA region; and (3) endoplasmic reticulum stress response genes. Expression pathways associated with lung disease were concordant for some endosome and HLA pathways, with pathways identified using GWAS associations from 1,978 CF-affected individuals. Pathways associated with age of onset of persistent P. aeruginosa infection were enriched for HLA class II genes, and those associated with MI were related to oxidative phosphorylation. Formal testing demonstrated that genes showing differential expression associated with lung disease severity were enriched for heritable genetic variation and expression quantitative traits. Gene expression provided a powerful tool to identify unrecognized heritable variation, complementing ongoing GWASs in this rare disease.     

The cost of multiple drug resistance in Pseudomonas aeruginosa

The spread of bacterial antibiotic resistance mutations is thought to be constrained by their pleiotropic fitness costs. Here we investigate the fitness costs of resistance in the context of the evolution of multiple drug resistance (MDR), by measuring the cost of acquiring streptomycin resistance mutations (StrepR) in independent strains of the bacterium Pseudomonas aeruginosa carrying different rifampicin resistance (RifR) mutations. In the absence of antibiotics, StrepR mutations are associated with similar fitness costs in different RifR genetic backgrounds. The cost of StrepR mutations is greater in a rifampicin‐sensitive (RifS) background, directly demonstrating antagonistic epistasis between resistance mutations. In the presence of rifampicin, StrepR mutations have contrasting effects in different RifR backgrounds: StrepR mutations have no detectable costs in some RifR backgrounds and massive fitness costs in others. Our results clearly demonstrate the importance of epistasis and genotype‐by‐environment interactions for the evolution of MDR.     

Development of Antibiotic Resistance during Simulated Treatment of Pseudomonas aeruginosa in Chemostats

During treatment of infections with antibiotics in critically ill patients in the intensive care resistance often develops. This study aims to establish whether under those conditions this resistance can develop de novo or that genetic exchange between bacteria is by necessity involved. Chemostat cultures of Pseudomonas aeruginosa were exposed to treatment regimes with ceftazidime and meropenem that simulated conditions expected in patient plasma. Development of antibiotic resistance was monitored and mutations in resistance genes were searched for by sequencing PCR products. Even at the highest concentrations that can be expected in patients, sufficient bacteria survived in clumps of filamentous cells to recover and grow out after 3 to 5 days. At the end of a 7 days simulated treatment, the minimal inhibitory concentration (MIC) had increased by a factor between 10 and 10,000 depending on the antibiotic and the treatment protocol. The fitness costs of resistance were minimal. In the resistant strains, only three mutations were observed in genes associated with beta-lactam resistance. The development of resistance often observed during patient treatment can be explained by de novo acquisition of resistance and genetic exchange of resistance genes is not by necessity involved. As far as conclusions based on an in vitro study using P. aeruginosa and only two antibiotics can be generalized, it seems that development of resistance can be minimized by treating with antibiotics in the highest concentration the patient can endure for the shortest time needed to eliminate the infection.     

Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates

The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is ‘open’, with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.     

Exome Sequencing of Phenotypic Extremes Identifies CAV2 and TMC6 as Interacting Modifiers of Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis

Discovery of rare or low frequency variants in exome or genome data that are associated with complex traits often will require use of very large sample sizes to achieve adequate statistical power. For a fixed sample size, sequencing of individuals sampled from the tails of a phenotype distribution (i.e., extreme phenotypes design) maximizes power and this approach was recently validated empirically with the discovery of variants in DCTN4 that influence the natural history of P. aeruginosa airway infection in persons with cystic fibrosis (CF; MIM219700). The increasing availability of large exome/genome sequence datasets that serve as proxies for population-based controls affords the opportunity to test an alternative, potentially more powerful and generalizable strategy, in which the frequency of rare variants in a single extreme phenotypic group is compared to a control group (i.e., extreme phenotype vs. control population design). As proof-of-principle, we applied this approach to search for variants associated with risk for age-of-onset of chronic P. aeruginosa airway infection among individuals with CF and identified variants in CAV2 and TMC6 that were significantly associated with group status. These results were validated using a large, prospective, longitudinal CF cohort and confirmed a significant association of a variant in CAV2 with increased age-of-onset of P. aeruginosa airway infection (hazard ratio = 0.48, 95% CI=[0.32, 0.88]) and variants in TMC6 with diminished age-of-onset of P. aeruginosa airway infection (HR = 5.4, 95% CI=[2.2, 13.5]) A strong interaction between CAV2 and TMC6 variants was observed (HR=12.1, 95% CI=[3.8, 39]) for children with the deleterious TMC6 variant and without the CAV2 protective variant. Neither gene showed a significant association using an extreme phenotypes design, and conditions for which the power of an extreme phenotype vs. control population design was greater than that for the extreme phenotypes design were explored.     

Genome Changes After Gene Duplication: Haploidy vs. Diploidy

Since genome size and the number of duplicate genes observed in genomes increase from haploid to diploid organisms, diploidy might provide more evolutionary probabilities through gene duplication. It is still unclear how diploidy promotes genomic evolution in detail. In this study, we explored the evolution of segmental gene duplication in haploid and diploid populations by analytical and simulation approaches. Results show that (1) under the double null recessive (DNR) selective model, given the same recombination rate, the evolutionary trajectories and consequences are very similar between the same-size gene-pool haploid vs. diploid populations; (2) recombination enlarges the probability of preservation of duplicate genes in either haploid or diploid large populations, and haplo-insufficiency reinforces this effect; and (3) the loss of duplicate genes at the ancestor locus is limited under recombination while under complete linkage the loss of duplicate genes is always random at the ancestor and newly duplicated loci. Therefore, we propose a model to explain the advantage of diploidy: diploidy might facilitate the increase of recombination rate, especially under sexual reproduction; more duplicate genes are preserved under more recombination by originalization (by which duplicate genes are preserved intact at a special quasi-mutation-selection balance under the DNR or haplo-insufficient selective model), so genome sizes and the number of duplicate genes in diploid organisms become larger. Additionally, it is suggested that small genomic rearrangements due to the random loss of duplicate genes might be limited under recombination.USUALLY genome size becomes larger from haploid to diploid organisms (Lynch and Conery 2003), and so does the number of duplicate genes observed in genomes (Zhang 2003). It is extensively hypothesized that diploidy might facilitate the preservation and accumulation of duplicate genes, but it is still unclear how diploidy supports the evolution of duplicate genes in detail. The superiority of diploidy is classically attributed to preventing expression of deleterious mutations (Crow and Kimura 1965), but it is also argued that the sheltering of deleterious mutations cannot adequately explain the advantages of diploidy (Perrot et al. 1991).Recombination is a common phenomenon in all three kingdoms of life, Bacteria, Eukarya, and Archaea. It has been reported that recombination influences the loss of duplicate genes (Zhang and Kishino 2004; Xue et al. 2010). In diploid organisms, if recombination between the ancestor locus and the newly duplicated locus is free, the rate of recombination is maximally 0.5, which is commonly observed especially when the two loci are located on different chromosomes. Although recombination should not be regarded as an exception in haploid organisms (Fraser et al. 2007), recombination events usually occur more frequently in diploid populations than they do in haploid populations. In other words, diploidy might facilitate the occurrence of recombination. The difference of recombination behaviors between haploid and diploid organisms is an obvious and important feature during genomic evolution.In our recent studies of genomic duplication, we proposed a new possible way of preserving and accumulating duplicate genes in genomes—originalization (Xue and Fu 2009a). As is well known, for a locus in an infinite diploid population, the frequencies of wild-type and degenerative alleles will move to an equilibrium under purifying selection and mutation, which is known as the mutation–selection balance. After genomic duplication, under two simple selective models, double null recessive (DNR, under which valid individuals require at least one active wild-type allele on the ancestor and newly duplicated loci) and haplo-insufficient (HI or partial dominant, under which valid individuals require at least two active wild-type alleles on both loci) models, a special equilibrium of allele frequencies at the ancestor and newly duplicated loci will be reached under recombination, in which the frequency of wild-type allele is kept high at both loci. Under the HI selective model this balance becomes so stable and flexible that the fixation of a degenerative allele at one of these two loci (or the balance being broken) becomes very difficult even in a modest population (Xue and Fu 2009a,b). However, if the two loci are tightly linked (recombination rate r = 0), this balance of allele frequencies does not appear. As r increases, the balance becomes more stable and the frequency of the wild-type allele at two loci becomes higher. High frequency of the wild-type allele at both loci means that duplicate genes are preserved intact in genomes, so this phenomenon was named originalization.Although many duplicate genes originated from genomic duplications in some species, such as yeast, maize, and fish (Li et al. 2005), those from segmental duplications are also very popular (Zhang et al. 2000; Leister 2004). In haploid populations, most duplication events are small segmental duplications. Therefore, to understand genomic evolution comprehensively, it is necessary to explore the evolution of segmental genomic duplication.Lynch et al. (2001) and Tanaka et al. (2009) have studied the evolution of segmental gene duplication in diploid populations theoretically. However, in this study, we further compared the evolution of segmental gene duplication in haploid vs. diploid populations by numerical and simulation approaches under the DNR and HI selective models. We observed that haploid and diploid populations with the same-size gene pool are very similar under the DNR model and the same recombination rate. Recombination enlarges the probability of preservation of duplicate genes in either haploid or diploid populations via originalization, and haplo-insufficiency reinforces this effect. The loss of duplicate genes at the ancestor locus might be limited under recombination, while under complete linkage, the loss of duplicate genes is random at the ancestor and newly duplicated loci. According to these results, we propose a model with which to explain the revolutionary genomic transition from haploidy to diploidy.     

Formation and Longevity of Chimeric and Duplicate Genes in Drosophila melanogaster

Historically, duplicate genes have been regarded as a major source of novel genetic material. However, recent work suggests that chimeric genes formed through the fusion of pieces of different genes may also contribute to the evolution of novel functions. To compare the contribution of chimeric and duplicate genes to genome evolution, we measured their prevalence and persistence within Drosophila melanogaster. We find that ~80.4 duplicates form per million years, but most are rapidly eliminated from the genome, leaving only 4.1% to be preserved by natural selection. Chimeras form at a comparatively modest rate of ~11.4 per million years but follow a similar pattern of decay, with ultimately only 1.4% of chimeras preserved. We propose two mechanisms of chimeric gene formation, which rely entirely on local, DNA-based mutations to explain the structure and placement of the youngest chimeric genes observed. One involves imprecise excision of an unpaired duplication during large-loop mismatch repair, while the other invokes a process akin to replication slippage to form a chimeric gene in a single event. Our results paint a dynamic picture of both chimeras and duplicate genes within the genome and suggest that chimeric genes contribute substantially to genomic novelty.     

Pleiotropic Mutations Are Subject to Strong Stabilizing Selection

The assumption that pleiotropic mutations are more deleterious than mutations with more restricted phenotypic effects is an important premise in models of evolution. However, empirical evidence supporting this assumption is limited. Here, we estimated the strength of stabilizing selection on mutations affecting gene expression in male Drosophila serrata. We estimated the mutational variance (V_M) and the standing genetic variance (V_G) from two well-matched panels of inbred lines: a panel of mutation accumulation (MA) lines derived from a single inbred ancestral line and a panel of inbred lines derived from an outbred population. For 855 gene-expression traits, we estimated the strength of stabilizing selection as s = V_M/V_G. Selection was observed to be relatively strong, with 17% of traits having s > 0.02, a magnitude typically associated with life-history traits. Randomly assigning expression traits to five-trait sets, we used factor analytic mixed modeling in the MA data set to identify covarying traits that shared pleiotropic mutations. By assigning traits to the same trait sets in the outbred line data set, we then estimated s for the combination of traits affected by pleiotropic mutation. For these pleiotropic combinations, the median s was three times greater than s acting on the individual component traits, and 46% of the pleiotropic trait combinations had s > 0.02. Although our analytical approach was biased toward detecting mutations with relatively large effects, likely overestimating the average strength of selection, our results provide widespread support for the prediction that stronger selection can act against mutations with pleiotropic effects.THE extent to which new mutations have pleiotropic effects on multiple traits, and ultimately on fitness is central to our understanding of the maintenance of genetic variation and the process of adaptation (Kondrashov and Turelli 1992; Otto 2004; Johnson and Barton 2005; Zhang and Hill 2005). Analyses of Fisher’s (1930) geometric model of adaptation have shown that a mutation with effects on many traits will have a reduced probability of contributing to adaptive evolution (Orr 2000; Welch and Waxman 2003; see also Haygood 2006). For a population close to its optimum under mutation–selection balance, a direct corollary of this is that selection must act more strongly against mutations with wider pleiotropic effects (Zhang 2012).Evidence for the strength of selection increasing with the number of traits that are pleiotropically affected by a mutation is limited. At a phenotypic level, nonlinear (stabilizing) selection is much stronger on combinations of metric traits than on each individual trait contributing to the combination (Blows and Brooks 2003; Walsh and Blows 2009). Given that genetic correlations among such traits are expected to be a consequence of pleiotropic alleles (Lande 1980), stronger selection on trait combinations is consistent with stronger selection on pleiotropic mutations that are likely to underlie the genetic covariance among such traits. There is some evidence that per-trait allelic effects might be greater for alleles with more widespread pleiotropic effects (Wagner et al. 2008; Wang et al. 2010); as mutations with larger phenotypic effects might be more effectively targeted by selection, this also suggests stronger selection against more pleiotropic mutation.Mutation accumulation (MA) breeding designs, in which the opportunity for selection is reduced, allowing new mutations to drift to fixation, provide an opportunity to characterize the strength of selection acting directly against new mutations. Rice and Townsend (2012) proposed an approach for determining the strength of selection acting against mutations at individual loci, combining information from QTL mapping and MA studies. This approach could conceivably be extended to associate the strength of selection with the number of traits a QTL affects. More typically, estimates of selection from MA designs are focused on traits, rather than alleles. Under the assumption that most mutations are deleterious, an assumption supported by MA studies (Halligan and Keightley 2009), the strength of selection acting on mutations affecting quantitative traits can be measured as the ratio of the mutational to the standing genetic variance, s = V_M/V_G, where s is the selection coefficient of the mutation in heterozygous form (Barton 1990; Houle et al. 1996). While estimating s in this way provides a framework for estimating selection on pleiotropic combinations of traits, we are not aware of any studies adopting this approach to directly estimate the strength of selection acting on mutations affecting multiple traits.Within an MA framework, Estes and Phillips (2006) manipulated the opportunity for selection, providing rare direct evidence of stronger selection against mutations with pleiotropic effects. In a DNA repair-deficient strain of Caenorhabditis elegans, Estes and Phillips (2006) observed lower mutational covariance among life-history components when selection was allowed (larger populations) than when the opportunity for selection was limited (small populations). Similarly, McGuigan et al. (2011) compared Drosophila serrata MA lines accumulating mutations in the presence or absence of sexual selection on males, reporting reduced covariance between two fitness components in the selection treatment. These studies reveal that selection can eliminate nonlethal alleles with pleiotropic effects, but whether traits other than life-history components exhibit similar evidence of selection against pleiotropic alleles remains unknown.In parallel to the quantitative genetic predictions that pleiotropic alleles will be under stronger selection, molecular genetic theory predicts that the rate of gene evolution will be negatively correlated with pleiotropy (Pal et al. 2006; Salathe et al. 2006). More highly pleiotropic genes, as identified through the extent of connectivity (the number of interactions) in protein–protein interaction networks (Jeong et al. 2001), or the number of gene ontology (GO) terms (Jovelin and Phillips 2009) are more likely to be essential (i.e., knockout mutations result in lethality), suggesting that selection is stronger against large-effect (knockout) mutations in more highly pleiotropic genes. However, the selection acting against small-effect, nonlethal mutations in pleiotropic genes is less clear (Pal et al. 2006). Several studies have found an association between gene pleiotropy indices, such GO annotation of the number of biological processes or tissue specificity of expression, and the rate of sequence evolution (e.g., Pal et al. 2001; Salathe et al. 2006; Jovelin and Phillips 2009; Su et al. 2010). These pleiotropy indices typically explain little of the variation in sequence evolutionary rates, and it remains unclear whether more highly pleiotropic mutations are typically under stronger selection (Pal et al. 2006; Salathe et al. 2006).Here, we estimate the selection coefficients acting against naturally occurring mutations affecting gene-expression traits in male D. serrata to quantitatively test if selection is stronger on mutations that affect multiple traits. Gene-expression phenotypes are uniquely positioned to enable detailed investigations of pleiotropy: there are many of them, they represent a broad coverage of biological function, they can be analyzed to quantify developmental pleiotropy in the same way as traits traditionally considered in quantitative genetics, and GO information can be used to index molecular genetic pleiotropy. We use multivariate mixed-model analyses of expression traits in a set of inbred lines from a mutation accumulation experiment to estimate the mutational variance in individual expression traits, and the pleiotropic mutational covariance among random sets of five expression traits. Using a second panel of inbred lines, derived from a natural, outbred, population, we estimate the standing genetic variance in the same individual traits and five-trait combinations. From these estimates of mutational and standing genetic variance, we calculate s for each of the individual traits and trait combinations to determine whether selection has typically been stronger on mutations with pleiotropic effects than on other mutations affecting each trait. We complement this quantitative genetic analysis of developmental pleiotropy with an analysis of molecular genetic pleiotropy (Paaby and Rockman 2013), determining whether the strength of selection acting on individual expression traits can be predicted from the number of biological functions that the gene annotates to in the GO database or to the range of tissues in which the gene is expressed.     

A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene

Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb) carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNA^Thr-associated attachment site. A corresponding integrated form of pHS87b at the tRNA^Thr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3’-end of the tRNA^Thr gene. The pHS87a plasmid (26.8 kb) displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa.     

Recent Origin of the Methacrylate Redox System in Geobacter sulfurreducens AM-1 through Horizontal Gene Transfer

The origin and evolution of novel biochemical functions remains one of the key questions in molecular evolution. We study recently emerged methacrylate reductase function that is thought to have emerged in the last century and reported in Geobacter sulfurreducens strain AM-1. We report the sequence and study the evolution of the operon coding for the flavin-containing methacrylate reductase (Mrd) and tetraheme cytochrome с (Mcc) in the genome of G. sulfurreducens AM-1. Different types of signal peptides in functionally interlinked proteins Mrd and Mcc suggest a possible complex mechanism of biogenesis for chromoproteids of the methacrylate redox system. The homologs of the Mrd and Mcc sequence found in δ-Proteobacteria and Deferribacteres are also organized into an operon and their phylogenetic distribution suggested that these two genes tend to be horizontally transferred together. Specifically, the mrd and mcc genes from G. sulfurreducens AM-1 are not monophyletic with any of the homologs found in other Geobacter genomes. The acquisition of methacrylate reductase function by G. sulfurreducens AM-1 appears linked to a horizontal gene transfer event. However, the new function of the products of mrd and mcc may have evolved either prior or subsequent to their acquisition by G. sulfurreducens AM-1.     

Relationship between gene duplicability and diversifiability in the topology of biochemical networks

Background

Selective gene duplicability, the extensive expansion of a small number of gene families, is universal. Quantitatively, the number of genes (P_(K)) with K duplicates in a genome decreases precipitously as K increases, and often follows a power law (P_(k)∝k^-α). Functional diversification, either neo- or sub-functionalization, is a major evolution route for duplicate genes.

Results

Using three lines of genomic datasets, we studied the relationship between gene duplicability and diversifiability in the topology of biochemical networks. First, we explored scenario where two pathways in the biochemical networks antagonize each other. Synthetic knockout of respective genes for the two pathways rescues the phenotypic defects of each individual knockout. We identified duplicate gene pairs with sufficient divergences that represent this antagonism relationship in the yeast S. cerevisiae. Such pairs overwhelmingly belong to large gene families, thus tend to have high duplicability. Second, we used distances between proteins of duplicate genes in the protein interaction network as a metric of their diversification. The higher a gene’s duplicate count, the further the proteins of this gene and its duplicates drift away from one another in the networks, which is especially true for genetically antagonizing duplicate genes. Third, we computed a sequence-homology-based clustering coefficient to quantify sequence diversifiability among duplicate genes – the lower the coefficient, the more the sequences have diverged. Duplicate count (K) of a gene is negatively correlated to the clustering coefficient of its duplicates, suggesting that gene duplicability is related to the extent of sequence divergence within the duplicate gene family.

Conclusion

Thus, a positive correlation exists between gene diversifiability and duplicability in the context of biochemical networks – an improvement of our understanding of gene duplicability.     

Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis

Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa’s ability to colonize the GI tract but does decrease P. aeruginosa’s cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.