Environmentally Friendly Degradation and Detoxification of Rifampicin by a Bacterial Laccase and Hydrogen Peroxide.

Antibiotics are micropollutants accumulating in our rivers and wastewaters, potentially leading to bacterial antibiotic resistance, a worldwide problem to which there is no current solution. Here, we have developed an environmentally friendly two-step process to transform the antibiotic rifampicin (RIF) into non-antimicrobial compounds. The process involves an enzymatic oxidation step by the bacterial CotA-laccase and a hydrogen peroxide bleaching step. NMR identified rifampicin quinone as the main product of the enzymatic oxidation. Growth of Escherichia coli strains in the presence of final degradation products (FP) and minimum inhibitory concentration (MIC) measurements confirmed that FP are non-anti-microbial compounds, and bioassays suggest that FP is not toxic to eukaryotic organisms. Moreover, competitive fitness assays between susceptible and RIF-resistant bacteria show that susceptible bacteria is strongly favoured in the presence of FP. Our results show that we have developed a robust and environmentally friendly process to effectively remediate rifampicin from antibiotic contaminated environments.

Microbes are potential key players in the evolution of life histories and aging in Caenorhabditis elegans.

Microbes can have profound effects on host fitness and health and the appearance of late-onset diseases. Host-microbe interactions thus represent a major environmental context for healthy aging of the host and might also mediate trade-offs between life-history traits in the evolution of host senescence. Here, we have used the nematode Caenorhabditis elegans to study how host-microbe interactions may modulate the evolution of life histories and aging. We first characterized the effects of two non-pathogenic and one pathogenic Escherichia coli strains, together with the pathogenic Serratia marcescens DB11 strain, on population growth rates and survival of C. elegans from five different genetic backgrounds. We then focused on an outbred C. elegans population, to understand if microbe-specific effects on the reproductive schedule and in traits such as developmental rate and survival were also expressed in the presence of males and standing genetic variation, which could be relevant for the evolution of C. elegans and other nematode species in nature. Our results show that host-microbe interactions have a substantial host-genotype-dependent impact on the reproductive aging and survival of the nematode host. Although both pathogenic bacteria reduced host survival in comparison with benign strains, they differed in how they affected other host traits. Host fertility and population growth rate were affected by S. marcescens DB11 only during early adulthood, whereas this occurred at later ages with the pathogenic E. coli IAI1. In both cases, these effects were largely dependent on the host genotypes. Given such microbe-specific genotypic differences in host life history, we predict that the evolution of reproductive schedules and senescence might be critically contingent on host-microbe interactions in nature.

Partial Selfing Can Reduce Genetic Loads While Maintaining Diversity During Experimental Evolution.

Partial selfing, whereby self- and cross- fertilization occur in populations at intermediate frequencies, is generally thought to be evolutionarily unstable. Yet, it is found in natural populations. This could be explained if populations with partial selfing are able to reduce genetic loads and the possibility for inbreeding depression while keeping genetic diversity that may be important for future adaptation. To address this hypothesis, we compare the experimental evolution of Caenorhabditis elegans populations under partial selfing, exclusive selfing or predominant outcrossing, while they adapt to osmotically challenging conditions. We find that the ancestral genetic load, as measured by the risk of extinction upon inbreeding by selfing, is maintained as long as outcrossing is the main reproductive mode, but becomes reduced otherwise. Analysis of genome-wide single-nucleotide polymorphisms (SNPs) during experimental evolution and among the inbred lines that survived enforced inbreeding indicates that populations with predominant outcrossing or partial selfing maintained more genetic diversity than expected with neutrality or purifying selection. We discuss the conditions under which this could be explained by the presence of recessive deleterious alleles and/or overdominant loci. Taken together, our observations suggest that populations evolving under partial selfing can gain some of the benefits of eliminating unlinked deleterious recessive alleles and also the benefits of maintaining genetic diversity at partially dominant or overdominant loci that become associated due to variance of inbreeding levels.

Slower environmental change hinders adaptation from standing genetic variation.

Evolutionary responses to environmental change depend on the time available for adaptation before environmental degradation leads to extinction. Explicit tests of this relationship are limited to microbes where adaptation usually depends on the sequential fixation of de novo mutations, excluding standing variation for genotype-by-environment fitness interactions that should be key for most natural species. For natural species evolving from standing genetic variation, adaptation at slower rates of environmental change may be impeded since the best genotypes at the most extreme environments can be lost during evolution due to genetic drift or founder effects. To address this hypothesis, we perform experimental evolution with self-fertilizing populations of the nematode Caenorhabditis elegans and develop an inference model to describe natural selection on extant genotypes under environmental change. Under a sudden environmental change, we find that selection rapidly increases the frequency of genotypes with high fitness in the most extreme environment. In contrast, under a gradual environmental change selection first favors genotypes that are worse at the most extreme environment. We demonstrate with a second set of evolution experiments that, as a consequence of slower environmental change and thus longer periods to reach the most extreme environments, genetic drift and founder effects can lead to the loss of the most beneficial genotypes. We further find that maintenance of standing genetic variation can retard the fixation of the best genotypes in the most extreme environment because of interference between them. Taken together, these results show that slower environmental change can hamper adaptation from standing genetic variation and they support theoretical models indicating that standing variation for genotype-by-environment fitness interactions critically alters the pace and outcome of adaptation under environmental change.

Reproductive assurance drives transitions to self-fertilization in experimental Caenorhabditis elegans.

BACKGROUND: Evolutionary transitions from outcrossing between individuals to selfing are partly responsible for the great diversity of animal and plant reproduction systems. The hypothesis of 'reproductive assurance' suggests that transitions to selfing occur because selfers that are able to reproduce on their own ensure the persistence of populations in environments where mates or pollination agents are unavailable. Here we test this hypothesis by performing experimental evolution in Caenorhabditis elegans. RESULTS: We show that self-compatible hermaphrodites provide reproductive assurance to a male-female population facing a novel environment where outcrossing is limiting. Invasions of hermaphrodites in male-female populations, and subsequent experimental evolution in the novel environment, led to successful transitions to selfing and adaptation. Adaptation was not due to the loss of males during transitions, as shown by evolution experiments in exclusively hermaphroditic populations and in male-hermaphrodite populations. Instead, adaptation was due to the displacement of females by hermaphrodites. Genotyping of single-nucleotide polymorphisms further indicated that the observed evolution of selfing rates was not due to selection of standing genetic diversity. Finally, numerical modelling and evolution experiments in male-female populations demonstrate that the improvement of male fitness components may diminish the opportunity for reproductive assurance. CONCLUSIONS: Our findings support the hypothesis that reproductive assurance can drive the transition from outcrossing to selfing, and further suggest that the success of transitions to selfing hinges on adaptation of obligate outcrossing populations to the environment where outcrossing was once a limiting factor.

The role of hermaphrodites in the experimental evolution of increased outcrossing rates in Caenorhabditis elegans.

BACKGROUND: Why most organisms reproduce via outcrossing rather than selfing is a central question in evolutionary biology. It has long ago been suggested that outcrossing is favoured when it facilitates adaptation to novel environments. We have previously shown that the experimental evolution of increased outcrossing rates in populations of the male-hermaphrodite nematode Caenorhabditis elegans were correlated with the experimental evolution of increased male fitness. However, it is unknown whether outcrossing led to adaptation, and if so, which fitness components can explain the observed increase in outcrossing rates. RESULTS: Using experimental evolution in six populations with initially low standing levels of genetic diversity, we show with head-to-head competition assays that population-wide fitness improved during 100 generations. Since outcrossing rates increased during the same period, this result demonstrates that outcrossing is adaptive. We also show that there was little evolution of hermaphrodite fitness under conditions of selfing or under conditions of outcrossing with unrelated tester males. We nonetheless find a positive genetic correlation between hermaphrodite self-fitness and population-wide fitness, and a negative genetic correlation between hermaphrodite mating success and population-wide fitness. These results suggest that the several hermaphrodite traits measured are fitness components. Tradeoffs expressed in hermaphrodites, particularly noticed between self-fitness and mating success, may in turn explain their lack of change during experimental evolution. CONCLUSIONS: Our findings indicate that outcrossing facilitates adaptation to novel environments. They further indicate that the experimental evolution of increased outcrossing rates depended little on hermaphrodites because of fitness tradeoffs between selfing and outcrossing. Instead, the evolution of increased outcrossing rates appears to have resulted from unhindered selection on males.

Experimental determination of invasive fitness in Caenorhabditis elegans.

Estimation of fitness is a key step in experimental evolution studies. However, no established methods currently exist to specifically estimate how successful new alleles are in invading populations. The main reason is that most assays do not accurately reflect the randomness associated with the first stages of the invasion, when invaders are rare and extinctions are frequent. In this protocol, I describe how such experiments can be done in an effective way. By using the nematode model, Caenorhabditis elegans, a large number of invasion experiments are set up, whereby invading individuals carrying a visual marker are introduced into populations in very low numbers. The number of invaders counted in consecutive generations, together with the number of extinctions, is then used in the context of individual-based computer simulations to provide likelihood (Lk) estimates for fitness. This protocol can take up to five generations of experimental invasions and a few hours of computer processing time.

An experimental test on the probability of extinction of new genetic variants.

In 1927, J.B.S. Haldane reasoned that the probability of fixation of new beneficial alleles is twice their fitness effect. This result, later generalized by M. Kimura, has since become the cornerstone of modern population genetics. There is no experimental test of Haldane's insight that new beneficial alleles are lost with high probability. Here we demonstrate that extinction rates decrease with increasing initial numbers of beneficial alleles, as expected, by performing invasion experiments with inbred lines of the nematode Caenorhabditis elegans. We further show that the extinction rates of deleterious alleles are higher than those of beneficial alleles, also as expected. Interestingly, we also find that for these inbred lines, when at intermediate frequencies, the fate of invaders might not result in their ultimate fixation or loss but on their maintenance. Our study confirms the key results from classical population genetics and highlights that the nature of adaptation can be complex.

The opportunity for balancing selection in experimental populations of Caenorhabditis elegans.

The role of balancing selection in maintaining diversity during the evolution of sexual populations to novel environments is poorly understood. To address this issue, we studied the impact of two mating systems, androdioecy and dioecy, on genotype distributions during the experimental evolution of Caenorhabditis elegans. We analyzed the temporal trajectories of 334 single nucleotide polymorphisms, covering 1/3 of the genome, and found extensive allele frequency changes and little loss of heterozygosities after 100 generations. As modeled with numerical simulations, SNP differentiation was consistent with genetic drift and average fitness effects of 2%, assuming that selection acted independently at each locus. Remarkably, inbreeding by self-fertilization was of little consequence to SNP differentiation. Modeling selection on deleterious recessive alleles suggests that the initial evolutionary dynamics can be explained by associative overdominance, but not the later stages because much lower heterozygosities would be maintained during experimental evolution. By contrast, models with selection on true overdominant loci can explain the heterozygote excess observed at all periods, particularly when negative epistasis or independent fitness effects were considered. Overall, these findings indicate that selection at single loci, including purging of recessive alleles, underlies most of the genetic differentiation accomplished during the experiment. Nonetheless, they also imply that maintenance of genetic diversity may in large part be due to balancing selection at multiple loci.

Evolution of outcrossing in experimental populations of Caenorhabditis elegans.

Caenorhabditis elegans can reproduce exclusively by self-fertilization. Yet, males can be maintained in laboratory populations, a phenomenon that continues to puzzle biologists. In this study we evaluated the role of males in facilitating adaptation to novel environments. For this, we contrasted the evolution of a fitness component exclusive to outcrossing in experimental populations of different mating systems. We introgressed a modifier of outcrossing into a hybrid population derived from several wild isolates to transform the wild-type androdioecious mating system into a dioecious mating system. By genotyping 375 single-nucleotide polymorphisms we show that the two populations had similar standing genetic diversity available for adaptation, despite the occurrence of selection during their derivation. We then performed replicated experimental evolution under the two mating systems from starting conditions of either high or low levels of diversity, under defined environmental conditions of discrete non-overlapping generations, constant density at high population sizes (N = 10(4)), no obvious spatial structure and abundant food resources. During 100 generations measurements of sex ratios and male competitive performance showed: 1) adaptation to the novel environment; 2) directional selection on male frequency under androdioecy; 3) optimal outcrossing rates of 0.5 under androdioecy; 4) the existence of initial inbreeding depression; and finally 5) that the strength of directional selection on male competitive performance does not depend on male frequencies. Taken together, these results suggest that androdioecious males are maintained at intermediate frequencies because outcrossing is adaptive.

Genotyping with Sequenom.

Often in evolutionary genetics research, one needs to analyze polymorphisms in populations for which cost-efficient high-throughput arrays are nonexistent, either because the species is not a model organism or because the populations have been subjected to such specific conditions that their base variation is almost unique. In this situation, custom-made genotyping assays are required. Sequenom's MassARRAY(®) genotyping platform is a powerful and flexible method for assaying up to a few thousand markers and up to thousands of individuals. It is based on distinguishing allele-specific primer extension products by mass spectrometry (MALDI-TOF). Most stages of the experimental protocol reflect adaptations of established PCR protocols to multiplexing, which allows the simultaneous amplification and detection of multiple markers per reaction.

Genome diversity in the genera Fructobacillus, Leuconostoc and Weissella determined by physical and genetic mapping.

Pulsed-field gel electrophoresis analysis of chromosomal single and double restriction profiles of 17 strains belonging to three genera of 'Leuconostocaceae' was done, resulting in physical and genetic maps for three Fructobacillus, six Leuconostoc and four Weissella strains. AscI, I-CeuI, NotI and SfiI restriction enzymes were used together with Southern hybridization of selected probes to provide an assessment of genomic organization in different species. Estimated genome sizes varied from 1408 kb to 1547 kb in Fructobacillus, from 1644 kb to 2133 kb in Leuconostoc and from 1371 kb to 2197 kb in Weissella. Other genomic characteristics of interest were analysed, such as oriC and terC localization and rrn operon organization. The latter seems markedly different in Weissella, in both number and disposition in the chromosome. Comparisons of intra- and intergeneric features are discussed in the light of chromosome rearrangements and genomic evolution.

Experimental evolution reveals natural selection on standing genetic variation.

Evolution depends on genetic variation generated by mutation or recombination from standing genetic variation. In sexual organisms, little is known about the molecular population genetics of adaptation and reverse evolution. We carry out 50 generations of experimental reverse evolution in populations of Drosophila melanogaster, previously differentiated by forward evolution, and follow changes in the frequency of SNPs in both arms of the third chromosome. We characterize the effects of sampling finite population sizes and natural selection at the genotype level. We demonstrate that selection has occurred at several loci and further that there is no general loss or gain of allele diversity. We also observe that despite the complete convergence to ancestral levels of adaptation, allele frequencies only show partial return.

Genome organization in Oenococcus oeni strains studied by comparison of physical and genetic maps.

The genomic organization of nine strains of Oenococcus oeni belonging to two previously suggested divergent groups was examined by a top-down approach, including analysis of isolated genes and construction of physical and genetic maps. Genomic sequence data from Oenococcus oeni strain PSU-1 were also examined by a bottom-up approach, using sequence data accessible from the U.S. Joint Genome Institute (Walnut Creek, CA, USA), which enabled the confirmation of gene location and the assessment of transcription direction. A comparison of the genomic maps revealed that O. oeni is a homogeneous species and supported the existence of two different genomic groups, although in a phase of divergence much too early for the recognition of subspecies. The genomic organization of O. oeni is characterized by an unusual conserved distribution of the two rrn operons, located at least 500 kb apart from the putative chromosome replication origin. Differential degrees of conservation are observed in O. oeni chromosomes, the neighboring region of the replication terminus being the most conserved one. Since most of the structural polymorphisms can be correlated to the presence of transposase genes and sites of prophage integration, the occurrence of macrodiversity events, such as insertions-deletions, duplications, or inversions of larger genomic regions, can most likely be ruled out in O. oeni evolution.

Congruence of evolutionary relationships inside the Leuconostoc-Oenococcus-Weissella clade assessed by phylogenetic analysis of the 16S rRNA gene, dnaA, gyrB, rpoC and dnaK.

The phylogenetic structure of the Leuconostoc-Oenococcus-Weissella clade was evaluated by comparison of 16S rRNA gene, dnaA, gyrB, rpoC and dnaK sequence analysis. Phylogenies obtained with the different genes were in overall good agreement and a well-supported, almost fully resolved phylogenetic tree was obtained when the combined data were analysed in a Bayesian approach. A rapid basal diversification of the three genera is suggested. Evolutionary rates of the 16S rRNA gene in these genera seem to be different and specifically related to the evolution of this group, revealing the importance of this sequence in the constitution of the present taxonomy, but preventing its straightforward use in phylogenetic inference.

Rickettsiae phylogeny: a multigenic approach.

The development of molecular taxonomic methods has provided a large amount of data in the reorganization of Rickettsiae taxonomy. Nevertheless, phylogenetic relationships among some groups and species delimitation remain unclear. To clarify rickettsial phylogeny, a multigenic approach was used for the first time for the genus Rickettsia, based on simultaneous analyses of eight loci: atpA, recA, virB4, dnaA, dnaK, rrl-rrf internal transcribed spacer, ompA and gltA. Concatenation of different nucleotide sequences resulted in an improvement in phylogenetic resolution when compared to single gene data. This multigenic approach has enabled the differentiation of many groups, including the spotted fever group which includes a great number of closely related species. The reliability of some previously recognized groups was evaluated.

Leuconostoc pseudoficulneum sp. nov., isolated from a ripe fig.

Six strains of lactic acid bacteria (LAB) were isolated from a ripe fig. These strains constituted a highly homogeneous, but distinct, cluster that was separate from other LAB species in a polyphasic approach including dot-blot DNA-DNA hybridization, SDS-PAGE whole-cell protein profiling, carbohydrate fermentation ability, growth characteristics, enzymic profiling, pulsed-field gel electrophoresis macrorestriction analysis and RFLPs. Phylogenetic analysis based on 16S rRNA gene sequencing positioned a representative strain, LC51(T), in a distinct line of descent within the recently described clade comprising Leuconostoc ficulneum, Leuconostoc fructosum and Leuconostoc durionis; L. ficulneum was its closest neighbour (98 % sequence similarity). DNA-DNA hybridization values and chemotaxonomic and biochemical characteristics, including enzymic profiles detected with API ZYM microtubes, confirmed that this group of strains is distinct from L. ficulneum and represents a novel species within the genus Leuconostoc. Taking into account the common origin and phylogenetic proximity, the name Leuconostoc pseudoficulneum sp. nov. is proposed. Strain LC51(T) (=DSM 15468(T) = CECT 5759(T)) is the type strain; the DNA G + C content of this strain is 44.5 mol%.

Physical and genetic map of the Weissella paramesenteroides DSMZ 20288T chromosome and characterization of different rrn operons by ITS analysis.

The Weissella paramesenteroides DSMZ 20288T chromosome was analysed by pulsed-field gel electrophoresis, enabling the construction of a physical and genetic map. A total of 21 recognition sites of the restriction enzymes AscI, I-CeuI, NotI and SfiI were mapped on the chromosome, which was found to be circular with an estimated size of 2026 kb. This is believed to constitute the first study into the genomic organization of a strain of this genus, addressing the localization of important chromosomal regions such as oriC and terC. A total of 23 genetic markers were mapped, including eight rrn operons that were precisely assigned in 37 % of the W. paramesenteroides chromosome. The transcription direction of rrn loci was determined and three different rrn clusters were recognized regarding the presence/absence of tRNA genes in ITS regions.