Long-term effects of inducible mutagenic DNA repair on relative fitness and phenotypic diversification in Pseudomonas cichorii 302959

Mutagenic DNA repair (MDR) employs low-fidelity DNA polymerases capable of replicating past DNA lesions resulting from exposure to high-energy ultraviolet radiation (UVR). MDR confers UVR tolerance and activation initiates a transient mutator phenotype that may provide opportunities for adaptation. To investigate the potential role of MDR in adaptation, we have propagated parallel lineages of the highly mutable epiphytic plant pathogen Pseudomonas cichorii 302959 with daily UVR activation (UVR lineages) for ~500 generations. Here we examine those lineages through the measurement of relative fitness and observation of distinct colony morphotypes that emerged. Isolates and population samples from UVR lineages displayed gains in fitness relative to the ancestor despite increased rates of inducible mutation to rifampicin resistance. Regular activation of MDR resulted in the maintenance of genetic diversity within UVR lineages, including the reproducible diversification and coexistence of “round” and “fuzzy” colony morphotypes. These results suggest that inducible mutability may present a reasonable strategy for adaptive evolution in stressful environments by contributing to gains in relative fitness and diversification.     

Mutagenic DNA repair potential in Pseudomonas spp., and characterization of the rulABPc operon from the highly mutable strain Pseudomonas cichorii 302959

We assessed the tolerance to ultraviolet B (UVB; 290-320 nm) radiation and UVB-induced mutability in 28 Pseudomonas spp. and four Burkholderia cepacia strains. The UVB survival of 23 (72%) of the strains was elevated (>46% survival following irradiation with a 2250 J m-2 dose), and 17 (53%) strains were defined as mutable by UVB. A mutagenic DNA repair determinant was cloned and characterized from the highly mutable strain P. cichorii 302959 and shown by sequence analysis to be an allele of rulAB, a mutagenic DNA repair determinant previously characterized from Pseudomonas syringae. Phylogenetic analyses of RulA- and RulB-related sequences indicated that the sequences identified in environmental bacteria shared a common ancestor with UmuDC-like sequences from enteric bacteria but were considerably diverged. The dynamics of UVB-induced mutability to nalidixic acid resistance (NalR) and rifampicin resistance (RifR) were studied in replicate populations of P. cichorii 302959 subjected to a daily UVB dose of 2250 J m-2 for 14 consecutive days. While there was an initial spike in the frequency of NalR and RifR mutants recovered on Days 1 and 2 of two separate experiments, the frequencies were sharply reduced and then fluctuated throughout the duration of both experiments. These experimental results are intriguing because they point to the possibility that P. cichorii possesses additional mechanisms to curtail the induction of spontaneous mutants following repeated episodes of UVB irradiation.     

Divergent evolution during an experimental adaptive radiation

How repeatable a process is evolution? Comparative studies of multicellular eukaryotes and experimental studies with unicellular prokaryotes document the repeated evolution of adaptive phenotypes during similar adaptive radiations, suggesting that the outcome of adaptive radiation is broadly reproducible. The goal of this study was to test this hypothesis by using phenotypic traits to infer the genetic basis of adaptation to simple carbon-limited environments in an extensive adaptive radiation. We used a clone of the bacterium Pseudomonas fluorescens to found two sets of experimental lines. The first set of lines was allowed to adapt to one of 23 novel environments for 1100 generations while the second set of lines was allowed to accumulate mutations by drift for 2000 generations. All lines were then assayed in the 95 environments provided by Biolog microplates to determine the phenotypic consequences of selection and drift. Replicate selection lines propagated in a common environment evolved similar adaptive components of their phenotype but showed extensive variation in non-adaptive phenotypic traits. This variation in non-adaptive phenotypic traits primarily resulted from the ascendance of different beneficial mutations in different lines. We argue that these results reconcile experimental and comparative approaches to studying adaptation by demonstrating that the convergent phenotypic evolution that occurs during adaptive radiation may be associated with radically different sets of beneficial mutations.     

Fitness evolution and the rise of mutator alleles in experimental Escherichia coli populations

We studied the evolution of high mutation rates and the evolution of fitness in three experimental populations of Escherichia coli adapting to a glucose-limited environment. We identified the mutations responsible for the high mutation rates and show that their rate of substitution in all three populations was too rapid to be accounted for simply by genetic drift. In two of the populations, large gains in fitness relative to the ancestor occurred as the mutator alleles rose to fixation, strongly supporting the conclusion that mutator alleles fixed by hitchhiking with beneficial mutations at other loci. In one population, no significant gain in fitness relative to the ancestor occurred in the population as a whole while the mutator allele rose to fixation, but a substantial and significant gain in fitness occurred in the mutator subpopulation as the mutator neared fixation. The spread of the mutator allele from rarity to fixation took >1000 generations in each population. We show that simultaneous adaptive gains in both the mutator and wild-type subpopulations (clonal interference) retarded the mutator fixation in at least one of the populations. We found little evidence that the evolution of high mutation rates accelerated adaptation in these populations.     

Bypass of genetic constraints during mutator evolution to antibiotic resistance

Genetic constraints can block many mutational pathways to optimal genotypes in real fitness landscapes, yet the extent to which this can limit evolution remains to be determined. Interestingly, mutator bacteria elevate only specific types of mutations, and therefore could be very sensitive to genetic constraints. Testing this possibility is not only clinically relevant, but can also inform about the general impact of genetic constraints in adaptation. Here, we evolved 576 populations of two mutator and one wild-type Escherichia coli to doubling concentrations of the antibiotic cefotaxime. All strains carried TEM-1, a β-lactamase enzyme well known by its low availability of mutational pathways. Crucially, one of the mutators does not elevate any of the relevant first-step mutations known to improve cefatoximase activity. Despite this, both mutators displayed a similar ability to evolve more than 1000-fold resistance. Initial adaptation proceeded in parallel through general multi-drug resistance mechanisms. High-level resistance, in contrast, was achieved through divergent paths; with the a priori inferior mutator exploiting alternative mutational pathways in PBP3, the target of the antibiotic. These results have implications for mutator management in clinical infections and, more generally, illustrate that limits to natural selection in real organisms are alleviated by the existence of multiple loci contributing to fitness.     

The evolution of low mutation rates in experimental mutator populations of Saccharomyces cerevisiae

Mutation is the source of both beneficial adaptive variation and deleterious genetic load, fueling the opposing selective forces than shape mutation rate evolution. This dichotomy is well illustrated by the evolution of the mutator phenotype, a genome-wide 10- to 100-fold increase in mutation rate. This phenotype has often been observed in clonally expanding populations exposed to novel or frequently changing conditions. Although studies of both experimental and natural populations have shed light on the evolutionary forces that lead to the spread of the mutator allele through a population, significant gaps in our understanding of mutator evolution remain. Here we use an experimental evolution approach to investigate the conditions required for the evolution of a reduction in mutation rate and the mechanisms by which populations tolerate the accumulation of deleterious mutations. We find that after ～6,700 generations, four out of eight experimental mutator lines had evolved a decreased mutation rate. We provide evidence that the accumulation of deleterious mutations leads to selection for reduced mutation rate clones in populations of mutators. Finally, we test the long-term consequences of the mutator phenotype, finding that mutator lines follow different evolutionary trajectories, some of which lead to drug resistance.     

Respiratory energy transduction by membrane fragments of the phytopathogenic bacteria Pseudomonas cichorii and Pseudomonas aptata

The energy transduction by respiratory membranes from the fluorescent phytopathogenic bacteria Pseudomonas cichorii and Pseudomonas aptata has been examined. Both species have shown to perform ATP synthesis linked to oxidation of NADH with P/2e^- ratios ranging between 0.25 and 0.42. This phosphorylation activity is largely insensitive to antimycin A (10^-6 M) and KCN (5·10^-6 M) in membranes from P. aptata, a strain deficient in c type complement (Zannoni 1982). In contrast, the phosphorylation efficiency is partially lowered by antimycin A and KCN in P. cichorii a strain containing a branched respiratory chain (Zannoni 1982). Oxidation of NADH by ubiquinone-1 (UQ-1) in antimycin A-treated membranes from these two pseudomonads is not coupled to ATP generation. This finding indicates that both strains contain a nonenergy conserving membrane-bound NADH dehydrogenase.The location of the sites of energy conservation was investigated by respiratory-induced quenching of the fluorescence of atebrine. This approach has confirmed the P/2e^--ratios measurements along with indication of a energy conserving step at the UQ/cyt. b levels of both bacterial strains. This study has also shown that the cytochrome c oxidase activity by P. cichorii is linked to a proton gradient generation which in turn drives ATP synthesis (P/2e^-=0.1). Previous data indicated that a high-potential cytochrome of b type (cyt. b380, Em_7.0=+380 mV) is involved in the cytochrome c oxidase activity of P. cichorii (Zannoni 1982). The possibility that this bacterial strain is endowed with a terminal b type oxidase operating with a proton pump mechanism is therefore suggested.     

Cytochrome b type oxidases in the respiratory chains of the phytopathogenic fluorescent bacteria Pseudomonas cichorii and Pseudomonas aptata

Membrane fragments from the phytopathogenic bacteria Pseudomonas cichorii and Pseudomonas aptata have been examined. A branched respiratory chain is operative in P. cichorii whereas a linear electron transport system characterizes the related bacterium P. aptata. Both species contain several b type cytochromes resolved by redox titration analysis, but no a type components may be detected. In contrast, only P. cichorii is endowed with c type cytochromes and hence with cytochrome c oxidase activity. Among the b type cytochromes, two high-potential components, with Em_7.0 at +250 mV and +380 mV, have been kinetically characterized and tentatively associated with cyanideresistant and cytochrome c oxidase activities, respectively. Cytochrome b-250 should correspond to the spectrally detectable cytochrome o whereas cytochrome b-380 is functionally similar to cytochrome b-410 described in Rhodopseudomonas capsulata. This conclusion seems to blur previous reported data on other obligate aerobes in which cytochrome o has been generally associated with cytochrome c oxidase and also suggests that a more accurate reconsideration of the actual physiological role of cyt. o in bacterial respiration is necessary. Furthermore the question arises whether cyt. b-410 like oxidases, i. e. high-potential b's similar to cyt. b-410 of R. capsulata, may be widely distributed among aerobes rather than restricted to facultative photosynthetic prokaryotes.     

The evolution of Pseudomonas syringae host specificity and type III effector repertoires

The discovery 45 years ago that many Pseudomonas syringae pathovars elicit the hypersensitive response in plant species other than their hosts fostered the use of these bacteria as experimental models. However, the basis for host specificity and the corresponding resistance of nonhosts remain unclear. Pseudomonas syringae is now known to inject into the host cytoplasm, via the type III secretion system, effector proteins that suppress basal innate immunity, but may be recognized by cognate resistance (R) proteins in a second level of defence. The identification and manipulation of complete repertoires of type III effectors have revealed the highly polymorphic nature of effector repertoires and their potential to limit the host range. However, the maintenance of compatible effector repertoires may be driven by adaptations to life in a given plant species involving many factors. Tools are now available to test several hypotheses for the nature and evolution of P. syringae host specificity and nonhost resistance.     

The repeatability of adaptive radiation during long-term experimental evolution of Escherichia coli in a multiple nutrient environment

Adaptive radiations occur when a species diversifies into different ecological specialists due to competition for resources and trade-offs associated with the specialization. The evolutionary outcome of an instance of adaptive radiation cannot generally be predicted because chance (stochastic events) and necessity (deterministic events) contribute to the evolution of diversity. With increasing contributions of chance, the degree of parallelism among different instances of adaptive radiations and the predictability of an outcome will decrease. To assess the relative contributions of chance and necessity during adaptive radiation, we performed a selection experiment by evolving twelve independent microcosms of Escherichia coli for 1000 generations in an environment that contained two distinct resources. Specialization to either of these resources involves strong trade-offs in the ability to use the other resource. After selection, we measured three phenotypic traits: 1) fitness, 2) mean colony size, and 3) colony size diversity. We used fitness relative to the ancestor as a measure of adaptation to the selective environment; changes in colony size as a measure of the evolution of new resource specialists because colony size has been shown to correlate with resource specialization; and colony size diversity as a measure of the evolved ecological diversity. Resource competition led to the rapid evolution of phenotypic diversity within microcosms. Measurements of fitness, colony size, and colony size diversity within and among microcosms showed that the repeatability of adaptive radiation was high, despite the evolution of genetic variation within microcosms. Consistent with the observation of parallel evolution, we show that the relative contributions of chance are far smaller and less important than effects due to adaptation for the traits investigated. The two-resource environment imposed similar selection pressures in independent populations and promoted parallel phenotypic adaptive radiations in all independently evolved microcosms.     

Insertion and replication of the Pseudomonas aeruginosa mutator phage D3112

D3112 is a temperate bacteriophage of P. aeruginosa with heterogeneous sequences at one extremity of the virion DNA molecule. Infection of strain PAOl with phage D3112 results in a 40- to 65-fold increase in the frequency of ami mutants resistant to fluoroacetamide. Nine ami::D3112 prophages have been mapped to distinct sites within the ami locus by Southern blotting experiments with a cloned ami+ probe. All prophages have the same restriction map as the D3112 genome extracted from phage particles. The position of D3112 insertions correlates with the phenotype and reversion behavior of the ami mutants. Induction of D3112cts prophages results in amplification of internal prophage segments as discrete restriction fragments before the terminal viral fragments are visible as sharp hybridizing species. This indicates that D3112 replication is accompanied by recombination of prophage termini to numerous sites in the bacterial genome. Chromosomal junction fragments of an ami::D3112cts prophage are maintained through most of the replication cycle but are cleaved shortly before cell lysis, apparently by the viral encapsidation system.     

Actinomycins inhibit the production of the siderophore pyoverdines in the plant pathogen Pseudomonas cichorii SPC9018

ABSTRACT

Pyoverdines, a group of peptide siderophores produced by Pseudomonas species, function not only in iron acquisition, but also in their virulence in hosts. Thus, chemical inhibition of pyoverdine production may be an effective strategy to control Pseudomonas virulence. In the plant pathogen Pseudomonas cichorii SPC9018 (SPC9018), pyoverdine production is required for virulence on eggplant. We screened microbial culture extracts in a pyoverdine-production inhibition assay of SPC9018 and found Streptomyces sp. RM-32 as a candidate-producer. We isolated two active compounds from RM-32 cultures, and elucidated their structures to be actinomycins X₂ and D. Actinomycins X₂ and D inhibited pyoverdine production by SPC9018 with IC₅₀ values of 17.6 and 29.6 μM, respectively. Furthermore, pyoverdine production in other Pseudomonas bacteria, such as the mushroom pathogen P. tolaasii, was inhibited by the actinomycins. Therefore, these actinomycins may be useful as chemical tools to examine pyoverdine functions and as seed compounds for anti-Pseudomonas virulence agents.     

Generation of mutator mutants during carcinogenesis

Mutations are rare in normal cells. In contrast, multiple mutations are characteristic in most tumors. Previously we proposed a "mutator phenotype" hypothesis to explain how pre-cancer cells may acquire large number of mutations during carcinogenesis. Here we extend the "mutator phenotype" hypothesis considering recently discovered biochemical activities whose aberrant expression may result in genome-wide random mutations. The scope of this article is to emphasize that simple random point mutations can drive carcinogenesis and highlight new emerging pathways that generate these mutations. We focus specifically on random point mutations generated by replication errors, oxidative base damage, covalent base modifications by enzymes, and spontaneously generated abasic sites as a source of mutator mutants.     

Acquisition of endonuclease specificity during evolution of L1 retrotransposon

L1 is the most proliferative autonomous retroelement that comprises about 20% of mammalian genomes. Why L1s have proliferated so extensively in mammalian genomes is an important yet unsolved question. L1 copies are amplified via retrotransposition, in which the DNA cleavage specificity by the L1-encoded endonuclease (EN) primarily dictates sites of insertion. Whereas mammalian L1s show target preference for 5'-TTAAAA-3', other L1-like elements exhibit various degrees of target specificity. To gain insights on diversification of the EN specificity during L1 evolution, ENs of zebrafish L1 elements were analyzed here. We revealed that they form 3 discrete clades, M, F, and Tx1, which is in stark contrast to a single L1 clade in mammalian species. Interestingly, zebrafish clade M elements cluster as a sister group of mammalian L1s and show target-site preference for 5'-TTAAAA-3'. In contrast, elements of the clade F, the immediate outgroup of the clade M, show little specificity. We identified certain clade-specific amino acid residues in EN, many of which are located in the cleft that recognizes the substrate, suggesting that these amino acid alterations have generated 2 types of ENs with different substrate specificities. The distribution pattern of the 3 clades suggests a possibility that the acquisition of target specificity by the L1 ENs improved the L1 fitness under the circumstances in mammalian hosts.     

Genetic requirements and mutational specificity of the Escherichia coli SOS mutator activity.

To better understand the mechanisms of SOS mutagenesis in the bacterium Escherichia coli, we have undertaken a genetic analysis of the SOS mutator activity. The SOS mutator activity results from constitutive expression of the SOS system in strains carrying a constitutively activated RecA protein (RecA730). We show that the SOS mutator activity is not enhanced in strains containing deficiencies in the uvrABC nucleotide excision-repair system or the xth and nfo base excision-repair systems. Further, recA730-induced errors are shown to be corrected by the MutHLS-dependent mismatch-repair system as efficiently as the corresponding errors in the rec+ background. These results suggest that the SOS mutator activity does not reflect mutagenesis at so-called cryptic lesions but instead represents an amplification of normally occurring DNA polymerase errors. Analysis of the base-pair-substitution mutations induced by recA730 in a mismatch repair-deficient background shows that both transition and transversion errors are amplified, although the effect is much larger for transversions than for transitions. Analysis of the mutator effect in various dnaE strains, including dnaE antimutators, as well as in proofreading-deficient dnaQ (mutD) strains suggests that in recA730 strains, two types of replication errors occur in parallel: (i) normal replication errors that are subject to both exonucleolytic proofreading and dnaE antimutator effects and (ii) recA730-specific errors that are not susceptible to either proofreading or dnaE antimutator effects. The combined data are consistent with a model suggesting that in recA730 cells error-prone replication complexes are assembled at sites where DNA polymerization is temporarily stalled, most likely when a normal polymerase insertion error has created a poorly extendable terminal mismatch. The modified complex forces extension of the mismatch largely at the exclusion of proofreading and polymerase dissociation pathways. SOS mutagenesis targeted at replication-blocking DNA lesions likely proceeds in the same manner.     

Mutational specificity of a conditional Escherichia coli mutator, mutD5

Summary MutD5, a conditional mutator in Escherichia coli, causes the stimulation of mutation frequencies 50 to 100 fold in minimal medium. In rich medium mutation frequencies are further increased 50 to 100 fold. We show here that all possible base-pair mutations are increased in a mutD5 strain grown in rich medium. A:TG:C transitions as well as A:TC:G, A:TT:A aud G:CC:G transversions are stimulated. Transitions occur more frequently than transversions. MutD5 also increases the reversion frequencies of three trpA frameshift mutations by causing base-pair additions, and, possibly, base-pair deletions.     

Induction of apoptotic cell death leads to the development of bacterial rot caused by Pseudomonas cichorii

Pseudomonas cichorii is the major causal agent of bacterial rot of lettuce. Collapse and browning symptoms were observed in lettuce leaf tissue from 15 to 24 h after inoculation (HAI) with P. cichorii; superoxide anion generation was detected at 1 to 6 HAI; and cell death was induced at 6 HAI, reaching a maximum at approximately 9 and 12 HAI. Heterochromatin condensation and DNA laddering also were observed within 3 HAI. Pharmacological studies showed that induction of cell death and DNA laddering was closely associated with de novo protein synthesis, protein kinase, intracellular reactive oxygen species, DNase, serine protease, and caspase III-like protease. Moreover, chemicals, which inhibited the induction of cell death and DNA laddering, also suppressed the development of disease symptoms. These results suggest that apoptotic cell death might be closely associated with the development of bacterial rot caused by P. cichorii.     

alpha-Santonin 1,2-reductase and its role in the formation of dihydrosantonin and lumisantonin by Pseudomonas cichorii S

1,2-Dihydrosantonin is the first stable product in the degradative pathway of alpha-santonin by Pseudomonas cichorii S. Its formation is catalyzed by an oxidoreductase, which is NADH or NADPH dependent and has an apparent Km value of 66.66 microM for santonin and 44.33 microM for NADH. The enzyme activity is stable at pH 6.0, 7.0, and 8.0, and is not affected by EDTA and divalent metal ions. It is postulated that the enzymic reduction of santonin occurs via formation of a transient zwitterionic intermediate, which undergoes nonenzymatic 1,4-sigmatropic rearrangement to yield lumisantonin during the solvent extraction process. Lumisantonin is, thus, not a true metabolic intermediate but an artifact.