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Biomedical subjects

Paul B Rainey

Publications and source records attributed to Paul B Rainey.

At least 19 recordsLinked to original sources
PubMed2006

The impact of phages on interspecific competition in experimental populations of bacteria.

BACKGROUND: Phages are thought to play a crucial role in the maintenance of diversity in natural bacterial communities. Theory suggests that phages impose density dependent regulation on bacterial populations, preventing competitive dominants from excluding less competitive species. To test this, we constructed experimental communities containing two bacterial species (Pseudomonas fluorescens and Pseudomonas aeruginosa) and their phage parasites. Communities were propagated at two environmental temperatures that reversed the outcome of competition in the absence of phage. RESULTS: The evenness of coexistence was enhanced in the presence of a phage infecting the superior competitor and in the presence of phage infecting both competitors. This occurred because phage altered the balance of competitive interactions through reductions in density of the superior competitor, allowing concomitant increases in density of the weaker competitor. However, even coexistence was not equally stable at the two environmental temperatures. CONCLUSION: Phage can alter competitive interactions between bacterial species in a way that is consistent with the maintenance of coexistence. However, the stability of coexistence is likely to depend upon the nature of the constituent bacteria-bacteriophage interactions and environmental conditions.

Ecosystem↗
PubMed2006

Unraveling adaptive evolution: how a single point mutation affects the protein coregulation network.

Understanding the mechanisms of evolution requires identification of the molecular basis of the multiple (pleiotropic) effects of specific adaptive mutations. We have characterized the pleiotropic effects on protein levels of an adaptive single-base pair substitution in the coding sequence of a signaling pathway gene in the bacterium Pseudomonas fluorescens SBW25. We find 52 proteomic changes, corresponding to 46 identified proteins. None of these proteins is required for the adaptive phenotype. Instead, many are found within specific metabolic pathways associated with fitness-reducing (that is, antagonistic) effects of the mutation. The affected proteins fall within a single coregulatory network. The mutation 'rewires' this network by drawing particular proteins into tighter coregulating relationships. Although these changes are specific to the mutation studied, the quantitatively altered proteins are also affected in a coordinated way in other examples of evolution to the same niche.

Adaptation, Physiological↗
PubMed2006

Adaptive divergence in experimental populations of Pseudomonas fluorescens. II. Role of the GGDEF regulator WspR in evolution and development of the wrinkly spreader phenotype.

Wrinkly spreader (WS) genotypes evolve repeatedly in model Pseudomonas populations undergoing adaptive radiation. Previous work identified genes contributing to the evolutionary success of WS. Here we scrutinize the GGDEF response regulator protein WspR and show that it is both necessary and sufficient for WS. Activation of WspR occurs by phosphorylation and different levels of activation generate phenotypic differences among WS genotypes. Five alleles of wspR, each encoding a protein with a single amino acid substitution, were generated by mutagenesis. Two alleles are constitutively active and cause the ancestral genotype to develop a WS phenotype; the phenotypic effects are allele specific and independent of phosphorylation. Three alleles contain changes in the GGDEF domain and when overexpressed in WS cause reversion to the ancestral phenotype. Ability to mimic this effect by overexpression of a liberated N-terminal domain shows that in WS, regulatory components upstream of WspR are overactive. To connect changes at the nucleotide level with fitness, the effects of variant alleles were examined in both structured and unstructured environments: alleles had adaptive and deleterious effects with trade-offs evident across environments. Despite the proclivity of mutations within wspR to generate WS, sequence analysis of wspR from 53 independently obtained WS showed no evidence of sequence change in this gene.

Alleles↗
PubMed2006

The histidine utilization (hut) genes of Pseudomonas fluorescens SBW25 are active on plant surfaces, but are not required for competitive colonization of sugar beet seedlings.

The ability to monitor the spatial and temporal distribution of signals in complex environments is necessary for an understanding of the function of bacteria in the wild. To this end, an existing recombinase-based transcriptional reporter strategy (recombinase-based in vivo expression technology, RIVET) has been extended and applied to the plant-colonizing bacterium Pseudomonas fluorescens SBW25. Central to the project was a rhizosphere-inducible locus, rhi14, which functional analyses show is hutT, a histidine-inducible gene that is required for histidine utilization. A transcriptional fusion between hutT and a promoterless site-specific recombinase (tnpR(mut168)) results in excision of a chromosomally integrated tetracycline-resistance cassette in a histidine-dependent manner. The dose- and time-responsiveness of the promoterless recombinase to histidine closely mirrored the histidine responsiveness of an identical hutT fusion to promoterless lacZ. To demonstrate the effectiveness of the strategy, the activity of hutT was monitored on sugar beet seedlings. Low levels of transcriptional activity were detected in the phyllosphere, rhizosphere and in plant extract, but not in vermiculite devoid of seedlings. The histidine concentration in the rhizosphere was estimated to be 0.6 microg ml(-1). The ecological significance of the hut locus was examined by competing a hutT deletion mutant against the wild-type during colonization of sugar beet seedlings. No impact on competitive fitness was detected, suggesting that the ability to utilize plant-derived histidine is not essential for bacterial colonization.

Bacterial Proteins↗
PubMed2005

The effect of a bacteriophage on diversification of the opportunistic bacterial pathogen, Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that colonizes the lungs of cystic fibrosis (CF) patients. CF lungs often contain a diverse range of P. aeruginosa phenotypes, some of which are likely to contribute to the persistence of infection, yet the causes of diversity are unclear. While the ecological heterogeneity of the lung environment and therapeutic regimes are probable factors, a role for parasitic bacteriophage cannot be ruled out. Parasites have been implicated as a key ecological variable driving the evolution of diversity in host populations. PP7 drove cycles of morphological diversification in host populations of P. aeruginosa due to the de novo evolution of small-rough colony variants that coexisted with large diffuse colony morph bacteria. In the absence of phage, bacteria only displayed the large diffuse colony morphology of the wild-type. Further assays revealed there to be two distinct types of resistant bacteria; these had very different ecological phenotypes, yet each carried a cost of resistance.

Colony Count, Microbial↗
PubMed2005

The PIN-domain toxin-antitoxin array in mycobacteria.

PIN-domains (homologues of the pilT N-terminal domain) are small protein domains of approximately 140 amino acids. They are found in a diverse range of organisms and recent evidence from bioinformatics, biochemistry, structural biology and microbiology suggest that the majority of the prokaryotic PIN-domain proteins are the toxic components of toxin-antitoxin (TA) operons. Several microorganisms have a large cohort of these operons. For example, the genome of Mycobacterium tuberculosis encodes 48 PIN-domain proteins, of which 38 are thought to be involved in TA interactions. This large array of PIN-domain TA operons raises questions as to their evolutionary origin and contemporary functional significance. We suggest that the evolutionary origin of genes encoding mycobacterial PIN-domain TA operons is linked to the mobile gene pool, but that TA operons can become resident within the chromosome of host cells from where they might be recruited to fulfil a variety of roles associated with retardation of cell growth and persistence in stressful environments.

Adenosine Triphosphatases↗
PubMed2005

The Pseudomonas fluorescens SBW25 wrinkly spreader biofilm requires attachment factor, cellulose fibre and LPS interactions to maintain strength and integrity.

The wrinkly spreader (WS) isolate of Pseudomonas fluorescens SBW25 forms a substantial biofilm at the air-liquid interface. The biofilm is composed of an extracellular partially acetylated cellulose-fibre matrix, and previous mutagenesis of WS with mini-Tn5 had identified both the regulatory and cellulose-biosynthetic operons. One uncharacterized WS mutant, WS-5, still expressed cellulose but produced very weak biofilms. In this work, the mini-Tn5 insertion site in WS-5 has been identified as being immediately upstream of the tol-pal operon. Like Tol-Pal mutants of other Gram-negative bacteria, WS-5 showed a "leaky-membrane" phenotype, including the serendipitous ability to utilize sucrose, increased uptake of the hydrophilic dye propidium iodide, and the loss of lipopolysaccharide (LPS) expression. WS-5 cells were altered in relative hydrophobicity, and showed poorer recruitment and maintenance in the biofilm than WS. The WS-5 biofilm was also less sensitive to chemical interference during development. However, growth rate, cellulose expression and attachment were not significantly different between WS and WS-5. Finally, WS-5 biofilms could be partially complemented with WS-4, a biofilm- and attachment-deficient mutant that expressed LPS, resulting in a mixed biofilm with significantly increased strength. These findings show that a major component of the WS air-liquid biofilm strength results from the interactions between LPS and the cellulose matrix of the biofilm--and that in the WS biofilm, cellulose fibres, attachment factor and LPS are required for biofilm development, strength and integrity.

Bacterial Adhesion↗
PubMed2005

Genetic characterization of Pseudomonas fluorescens SBW25 rsp gene expression in the phytosphere and in vitro.

The plant-colonizing Pseudomonas fluorescens strain SBW25 harbors a gene cluster (rsp) whose products show similarity to type III protein secretion systems found in plant and animal pathogens. Here we report a detailed analysis of the expression and regulation of the P. fluorescens rsp pathway, both in the phytosphere and in vitro. A combination of chromosomally integrated transcriptional reporter fusions, overexpressed regulatory genes, and specific mutants reveal that promoters controlling expression of rsp are actively transcribed in the plant rhizosphere but not (with the exception of the rspC promoter) in the phyllosphere. In synthetic medium, regulatory (rspL and rspR) and structural (rspU, plus the putative effector ropE) genes are poorly expressed; the rspC promoter is subject to an additional level of regulatory control. Ectopic expression of regulatory genes in wild-type and mutant backgrounds showed that RspR controls transcription of the alternate sigma factor, rspL, and that RspL controls expression of gene clusters encoding structural genes. Mutation of rspV did not affect RspR-mediated expression of rspU. A search for additional regulators revealed two candidates--one with a role in the conversion of alanine to pyruvate--suggesting that expression of rsp is partly dependent upon the metabolic status of the cell. Mutations in rsp regulators resulted in a significant reduction in competitive colonization of the root tips of sugar beet seedlings but also caused a marked increase in the lag phase of laboratory-grown cultures, indicating that rsp regulatory genes play a more significant general role in the function of P. fluorescens SBW25 than previously appreciated.

Arabidopsis↗
PubMed2005

Unraveling the secret lives of bacteria: use of in vivo expression technology and differential fluorescence induction promoter traps as tools for exploring niche-specific gene expression.

A major challenge for microbiologists is to elucidate the strategies deployed by microorganisms to adapt to and thrive in highly complex and dynamic environments. In vitro studies, including those monitoring genomewide changes, have proven their value, but they can, at best, mimic only a subset of the ensemble of abiotic and biotic stimuli that microorganisms experience in their natural habitats. The widely used gene-to-phenotype approach involves the identification of altered niche-related phenotypes on the basis of gene inactivation. However, many traits contributing to ecological performance that, upon inactivation, result in only subtle or difficult to score phenotypic changes are likely to be overlooked by this otherwise powerful approach. Based on the premise that many, if not most, of the corresponding genes will be induced or upregulated in the environment under study, ecologically significant genes can alternatively be traced using the promoter trap techniques differential fluorescence induction and in vivo expression technology (IVET). The potential and limitations are discussed for the different IVET selection strategies and system-specific variants thereof. Based on a compendium of genes that have emerged from these promoter-trapping studies, several functional groups have been distinguished, and their physiological relevance is illustrated with follow-up studies of selected genes. In addition to confirming results from largely complementary approaches such as signature-tagged mutagenesis, some unexpected parallels as well as distinguishing features of microbial phenotypic acclimation in diverse environmental niches have surfaced. On the other hand, by the identification of a large proportion of genes with unknown function, these promoter-trapping studies underscore how little we know about the secret lives of bacteria and other microorganisms.

Adaptation, Physiological↗
PubMed2004

The indigenous Pseudomonas plasmid pQBR103 encodes plant-inducible genes, including three putative helicases.

Plasmid pQBR103 ( approximately 400 kb) is representative of many self-transmissible, mercury resistant plasmids observed in the Pseudomonas community colonising the phytosphere of sugar beet. A promoter trapping strategy (IVET) was employed to identify pQBR103 genes showing elevated levels of expression on plant surfaces. Thirty-seven different plant-inducible gene fusions were isolated that were silent in laboratory media, but active in the plant environment. Three of the fusions were to DNA sequences whose protein products show significant homology to DNA-unwinding helicases. The three helicase-like genes, designated helA, helB and helC, are restricted to a defined group of related Pseudomonas plasmids. They are induced in both the root and shoot environments of sugar beet seedlings. Sequence analysis of the three plasmid-encoded helicase-like genes shows that they are phylogenetically distinct and likely to have independent evolutionary histories. The helA gene is predicted to encode a protein of 1121 amino acids, containing conserved domains found in the ultraviolet (UV) resistance helicase, UvrD. A helA knockout mutant was constructed and no phenotypic changes were found with plasmid-conferred UV resistance or plasmid conjugation. The other 34 fusions are unique with no homologues in the public gene databases, including the Pseudomonas genomes. These data demonstrate the presence of plant responsive genes in plasmid DNA comprising a component of the genomes of plant-associated bacteria.

Adaptation, Physiological↗
PubMed2004

Ecological constraints on diversification in a model adaptive radiation.

Taxonomic diversification commonly occurs through adaptive radiation, the rapid evolution of a single lineage into a range of genotypes or species each adapted to a different ecological niche. Radiation size (measured as the number of new types) varies widely between phylogenetically distinct taxa and between replicate radiations within a single taxon where the ecological opportunities available seem to be identical. Here we show how variation in energy input (productivity) and environmental disturbance combine to determine the extent of diversification in a single radiating lineage of Pseudomonas fluorescens adapting to laboratory conditions. Diversity peaked at intermediate rates of both productivity and disturbance and declined towards the extremes in a manner reminiscent of well-known ecological patterns. The mechanism responsible for the decrease in diversity arises from pleiotropic fitness costs associated with niche specialization, the effects of which are modulated by gradients of productivity and disturbance. Our results indicate that ecological gradients may constrain the size of adaptive radiations, even in the presence of the strong diversifying selection associated with ecological opportunity, by decoupling evolutionary diversification from ecological coexistence.

Animals↗
PubMed2004

The evolution of a pleiotropic fitness tradeoff in Pseudomonas fluorescens.

The evolution of ecological specialization is expected to carry a cost, due to either antagonistic pleiotropy or mutation accumulation. In general, it has been difficult to distinguish between these two possibilities. Here, we demonstrate that the experimental evolution of niche-specialist genotypes of the bacterium Pseudomonas fluorescens that colonize the air-broth interface of spatially structured microcosms is accompanied by pleiotropic fitness costs in terms of reduced carbon catabolism. Prolonged selection in spatially structured microcosms caused the cost of specialization to decline without loss of the benefits associated with specialization. The decline in the cost of specialization can be explained by either compensatory adaptation within specialist lineages or clonal competition among specialist lineages. These results provide a possible explanation of conflicting accounts for the cost of specialization.

Adaptation, Physiological↗
PubMed2004

Global analysis of predicted proteomes: functional adaptation of physical properties.

The physical characteristics of proteins are fundamentally important in organismal function. We used the complete predicted proteomes of >100 organisms spanning the three domains of life to investigate the comparative biology and evolution of proteomes. Theoretical 2D gels were constructed with axes of protein mass and charge (pI) and converted to density estimates comparable across all types and sizes of proteome. We asked whether we could detect general patterns of proteome conservation and variation. The overall pattern of theoretical 2D gels was strongly conserved across all life forms. Nevertheless, coevolved replicons from the same organism (different chromosomes or plasmid and host chromosomes) encode proteomes more similar to each other than those from different organisms. Furthermore, there was disparity between the membrane and nonmembrane subproteomes within organisms (proteins of membrane proteomes are on the average more basic and heavier) and their variation across organisms, suggesting that membrane proteomes evolve most rapidly. Experimentally, a significant positive relationship independent of phylogeny was found between the predicted proteome and Biolog profile, a measure associated with the ecological niche. Finally, we show that, for the smallest and most alkaline proteomes, there is a negative relationship between proteome size and basicity. This relationship is not adequately explained by AT bias at the DNA sequence level. Together, these data provide evidence of functional adaptation in the properties of complete proteomes.

Animals↗
PubMed2004

The effect of spatial heterogeneity and parasites on the evolution of host diversity.

Both spatial heterogeneity and exploiters (parasites and predators) have been implicated as key ecological factors driving population diversification. However, it is unclear how these factors interact. We addressed this question using the common plant-colonizing bacterium Pseudomonas fluorescens, which has been shown to diversify rapidly into spatial niche-specialist genotypes when propagated in laboratory microcosms. Replicate populations were evolved in spatially homogeneous and heterogeneous environments (shaken and static microcosms, respectively) with and without viral parasites (bacteriophage) for approximately 60 bacterial generations. Consistent with previous findings, exploiters reduced diversity in heterogeneous environments by relaxing the intensity of resource competition. By contrast, exploiters increased diversity in homogeneous environments where there was little diversification through resource competition. Competition experiments revealed this increase in diversity to be the result of fitness trade-offs between exploiter resistance and competitive ability. In both environments, exploiters increased allopatric diversity, presumably as a result of divergent selection for resistance between populations. Phage increased total diversity in homogeneous environments, but had no net effect in heterogeneous environments. Such interactions between key ecological variables need to be considered when addressing diversification and coexistence in future studies.

Animals↗
PubMed2004

Evolution of bacterial diversity and the origins of modularity.

A characteristic feature of all organisms is modular organisation: the tendency for groups of genes to interact in such a way as to limit the extent of pleiotropic effects among characters belonging to different functional complexes. While the implications of modularity for the evolution of variability have been much discussed the evolutionary origins remain obscure. Here we develop a model, with special reference to signal transduction cascades of bacteria, which predicts that in the face of ecological opportunity and lateral gene transfer, selection will favour modular genome architectures because such architectures minimise the pleiotropic effects associated with accommodation of potentially beneficial foreign DNA.

Base Composition↗
PubMed2004

Big questions, small worlds: microbial model systems in ecology.

Although many biologists have embraced microbial model systems as tools to address genetic and physiological questions, the explicit use of microbial communities as model systems in ecology has traditionally been more restricted. Here, we highlight recent studies that use laboratory-based microbial model systems to address ecological questions. Such studies have significantly advanced our understanding of processes that have proven difficult to study in field systems, including the genetic and biochemical underpinnings of traits involved in ecological interactions, and the ecological differences driving evolutionary change. It is the simplicity of microbial model systems that makes them such powerful tools for the study of ecology. Such simplicity enables the high degrees of experimental control and replication that are necessary to address many questions that are inaccessible through field observation or experimentation.

Journal Article↗
PubMed2004

Functional and phylogenetic analysis of a plant-inducible oligoribonuclease (orn) gene from an indigenous Pseudomonas plasmid.

Application of a promoter-trapping strategy to identify plant-inducible genes carried on an indigenous Pseudomonas plasmid, pQBR103, revealed the presence of a putative oligoribonuclease (orn) gene that encodes a highly conserved 3' to 5' exoribonuclease specific for small oligoribonucleotides. The deduced amino acid sequence of the plasmid-derived orn (orn(pl)) showed three conserved motifs characteristic of Orn from both prokaryotes and eukaryotes. Deletion of orn(pl) generated no observable phenotype, but inactivation of the chromosomal copy caused slow growth in Pseudomonas putida KT2440. This defect was fully restored by complementation with orn from Escherichia coli (orn(E.coli)). Plasmid-derived orn(pl) was capable of partially complementing the P. putida orn mutant, demonstrating functionality of orn(pl). Phylogenetic analysis showed that plasmid-encoded Orn was distinct from Orn encoded by the chromosome of proteobacteria. A survey of orn(pl) from related Pseudomonas plasmids showed a sporadic distribution but no sequence diversity. These data suggest that the orn(pl) was acquired by pQBR103 in a single gene-transfer event: the donor is unknown, but is unlikely to be a member of the Proteobacteria.

Amino Acid Sequence↗