The gossip paradox: Why do bacteria share genes? - Related Documents




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923701.0000The gossip paradox: Why do bacteria share genes? Bacteria, in contrast to eukaryotic cells, contain two types of genes: chromosomal genes that are fixed to the cell, and plasmids, smaller loops of DNA capable of being passed from one cell to another. The sharing of plasmid genes between individual bacteria and between bacterial lineages has contributed vastly to bacterial evolution, allowing specialized traits to 'jump ship' between one lineage or species and the next. The benefits of this generosity from the point of view of both recipient cell and plasmid are generally understood: plasmids receive new hosts and ride out selective sweeps across the population, recipient cells gain new traits (such as antibiotic resistance). Explaining this behavior from the point of view of donor cells is substantially more difficult. Donor cells pay a fitness cost in order to share plasmids, and run the risk of sharing advantageous genes with their competition and rendering their own lineage redundant, while seemingly receiving no benefit in return. Using both compartment based models and agent based simulations we demonstrate that 'secretive' genes which restrict horizontal gene transfer are favored over a wide range of models and parameter values, even when sharing carries no direct cost. 'Generous' chromosomal genes which are more permissive of plasmid transfer are found to have neutral fitness at best, and are generally disfavored by selection. Our findings lead to a peculiar paradox: given the obvious benefits of keeping secrets, why do bacteria share information so freely?202235603365
938610.9998Bacteriophages limit the existence conditions for conjugative plasmids. Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. IMPORTANCE: Conjugative plasmids are infectious loops of DNA capable of transmitting DNA between bacterial cells and between species. Because plasmids often carry extra genes that allow bacteria to live in otherwise-inhospitable environments, their dynamics are central to understanding bacterial adaptive evolution. The plasmid-bacterium interaction has typically been studied in isolation, but in natural bacterial communities, bacteriophages, viruses that infect bacteria, are ubiquitous. Using experiments, mathematical models, and computer simulations we show that bacteriophages drive plasmid dynamics through their ecological and evolutionary effects on bacteria and ultimately limit the conditions allowing plasmid existence. These results advance our understanding of bacterial adaptation and show that bacteriophages could be used to select against plasmids carrying undesirable traits, such as antibiotic resistance.201526037122
929420.9998Plasmid persistence: costs, benefits, and the plasmid paradox. Plasmids are extrachromosomal DNA elements that can be found throughout bacteria, as well as in other domains of life. Nonetheless, the evolutionary processes underlying the persistence of plasmids are incompletely understood. Bacterial plasmids may encode genes for traits that are sometimes beneficial to their hosts, such as antimicrobial resistance, virulence, heavy metal tolerance, and the catabolism of unique nutrient sources. In the absence of selection for these traits, however, plasmids generally impose a fitness cost on their hosts. As such, plasmid persistence presents a conundrum: models predict that costly plasmids will be lost over time or that beneficial plasmid genes will be integrated into the host genome. However, laboratory and comparative studies have shown that plasmids can persist for long periods, even in the absence of positive selection. Several hypotheses have been proposed to explain plasmid persistence, including host-plasmid co-adaptation, plasmid hitchhiking, cross-ecotype transfer, and high plasmid transfer rates, but there is no clear evidence that any one model adequately resolves the plasmid paradox.201829562144
928430.9997The population and evolutionary dynamics of homologous gene recombination in bacterial populations. In bacteria, recombination is a rare event, not a part of the reproductive process. Nevertheless, recombination -- broadly defined to include the acquisition of genes from external sources, i.e., horizontal gene transfer (HGT) -- plays a central role as a source of variation for adaptive evolution in many species of bacteria. Much of niche expansion, resistance to antibiotics and other environmental stresses, virulence, and other characteristics that make bacteria interesting and problematic, is achieved through the expression of genes and genetic elements obtained from other populations of bacteria of the same and different species, as well as from eukaryotes and archaea. While recombination of homologous genes among members of the same species has played a central role in the development of the genetics and molecular biology of bacteria, the contribution of homologous gene recombination (HGR) to bacterial evolution is not at all clear. Also, not so clear are the selective pressures responsible for the evolution and maintenance of transformation, the only bacteria-encoded form of HGR. Using a semi-stochastic simulation of mutation, recombination, and selection within bacterial populations and competition between populations, we explore (1) the contribution of HGR to the rate of adaptive evolution in these populations and (2) the conditions under which HGR will provide a bacterial population a selective advantage over non-recombining or more slowly recombining populations. The results of our simulation indicate that, under broad conditions: (1) HGR occurring at rates in the range anticipated for bacteria like Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, and Bacillus subtilis will accelerate the rate at which a population adapts to environmental conditions; (2) once established in a population, selection for this capacity to increase rates of adaptive evolution can maintain bacteria-encoded mechanisms of recombination and prevent invasion of non-recombining populations, even when recombination engenders a modest fitness cost; and (3) because of the density- and frequency-dependent nature of HGR in bacteria, this capacity to increase rates of adaptive evolution is not sufficient as a selective force to provide a recombining population a selective advantage when it is rare. Under realistic conditions, homologous gene recombination will increase the rate of adaptive evolution in bacterial populations and, once established, selection for higher rates of evolution will promote the maintenance of bacteria-encoded mechanisms for HGR. On the other hand, increasing rates of adaptive evolution by HGR is unlikely to be the sole or even a dominant selective pressure responsible for the original evolution of transformation.200919680442
971640.9997Fitness effects of plasmids shape the structure of bacteria-plasmid interaction networks. Antimicrobial resistance (AMR) genes are often carried on broad host range plasmids, and the spread of AMR within microbial communities will therefore depend on the structure of bacteria–plasmid networks. Empirical and theoretical studies of ecological interaction networks suggest that network structure differs between communities that are predominantly mutualistic versus antagonistic, with the former showing more generalized interactions (i.e., species interact with many others to a similar extent). This suggests that mutualistic bacteria–plasmid networks—where antibiotics are present and plasmids carry AMR genes—will be more generalized than antagonistic interactions, where plasmids do not confer benefits to their hosts. We first develop a simple theory to explain this link: fitness benefits of harboring a mutualistic symbiont promote the spread of the symbiont to other species. We find support for this theory using an experimental bacteria–symbiont (plasmid) community, where the same plasmid can be mutualistic or antagonistic depending on the presence of antibiotics. This short-term and parsimonious mechanism complements a longer-term mechanism (coevolution and stability) explaining the link between mutualistic and antagonistic interactions and network structure.202235613058
931250.9997Why There Are No Essential Genes on Plasmids. Mobile genetic elements such as plasmids are important for the evolution of prokaryotes. It has been suggested that there are differences between functions coded for by mobile genes and those in the "core" genome and that these differences can be seen between plasmids and chromosomes. In particular, it has been suggested that essential genes, such as those involved in the formation of structural proteins or in basic metabolic functions, are rarely located on plasmids. We model competition between genotypically varying bacteria within a single population to investigate whether selection favors a chromosomal location for essential genes. We find that in general, chromosomal locations for essential genes are indeed favored. This is because the inheritance of chromosomes is more stable than that for plasmids. We define the "degradation" rate as the rate at which chance genetic processes, for example, mutation, deletion, or translocation, render essential genes nonfunctioning. The only way in which plasmids can be a location for functioning essential genes is if chromosomal genes degrade faster than plasmid genes. If the two degradation rates are equal, or if plasmid genes degrade faster than chromosomal genes, functioning essential genes will be found only on chromosomes.201525540453
929660.9997Genome plasticity: insertion sequence elements, transposons and integrons, and DNA rearrangement. Living organisms are defined by the genes they possess. Control of expression of this gene set, both temporally and in response to the environment, determines whether an organism can survive changing conditions and can compete for the resources it needs to reproduce. Bacteria are no exception; changes to the genome will, in general, threaten the ability of the microbe to survive, but acquisition of new genes may enhance its chances of survival by allowing growth in a previously hostile environment. For example, acquisition of an antibiotic resistance gene by a bacterial pathogen can permit it to thrive in the presence of an antibiotic that would otherwise kill it; this may compromise clinical treatments. Many forces, chemical and genetic, can alter the genetic content of DNA by locally changing its nucleotide sequence. Notable for genetic change in bacteria are transposable elements and site-specific recombination systems such as integrons. Many of the former can mobilize genes from one replicon to another, including chromosome-plasmid translocation, thus establishing conditions for interspecies gene transfer. Balancing this, transposition activity can result in loss or rearrangement of DNA sequences. This chapter discusses bacterial DNA transfer systems, transposable elements and integrons, and the contributions each makes towards the evolution of bacterial genomes, particularly in relation to bacterial pathogenesis. It highlights the variety of phylogenetically distinct transposable elements, the variety of transposition mechanisms, and some of the implications of rearranging DNA, and addresses the effects of genetic change on the fitness of the microbe.200415148416
928570.9997Bacterial genetic exchange in nature. Most bacteria are haploid organisms containing only one copy of each gene per cell for most of the growth cycle. This means that the chance for correcting random mutations in bacterial genes would depend entirely on the complementarity inherent in DNA structures, unless homologous DNA sequences can be imported from outside the cell. Bacteria, like all living organisms have evolved at least one autonomous mechanism, conjugation, for exchanging portions of genetic materials between two related cells. The ecological benefits of conjugation include the expansion of metabolic versatility and resistance to hazardous environmental conditions. Natural bacterial genetic exchange also occurs through virus infections (transduction) and through the uptake of extracellular DNA (transformation). The origin and ecological benefits of transduction and transformation are difficult to assess because they are driven by factors external to the affected cell. Bacterial genetic exchange has implications for the evolution of phenotypes that are either beneficial to humans, such as biodegradation of toxic xenobiotic chemicals, or that are detrimental, such as the evolution of pathogenesis and the spread of antibiotic resistance. Understanding natural bacterial genetic exchange mechanisms is also relevant to the assessment of dispersal risks associated with genetically engineered bacteria and recombinant genes in the environment.19958533067
934780.9997Multilevel populations and the evolution of antibiotic resistance through horizontal gene transfer. Horizontal gene transfer (HGT) can create diversity in the genetic repertoire of a lineage. Successful gene transfer likely occurs more frequently between more closely related organisms, leading to the formation of higher-level exchange groups that in some respects are comparable to single-species populations. Genes that appear fixed in a single species can be replaced through distant homologs or iso-functional analogs acquired through HGT. These genes may originate from other species or they may be acquired by an individual strain from the species pan-genome. Because of their similarity to alleles in a population, we label these gene variants that are exchanged between related species as homeoalleles. In a case study, we show that biased gene transfer plays an important role in the evolution of aminoacyl-tRNA synthetases (aaRS). Many microorganisms make use of these genes against naturally occurring antibiotics. We suggest that the resistance against naturally occurring antibiotics is the likely driving force behind the frequent switching between divergent aaRS types and the reason for the maintenance of these homeoalleles in higher-level exchange groups. Resistance to naturally occurring antibiotics may lead to the maintenance of different types of aminoacyl-tRNA synthetases in Bacteria through gene transfer.201121521245
969190.9997Defining pathogenic bacterial species in the genomic era. Actual definitions of bacterial species are limited due to the current criteria of definition and the use of restrictive genetic tools. The 16S ribosomal RNA sequence, for example, has been widely used as a marker for phylogenetic analyses; however, its use often leads to misleading species definitions. According to the first genetic studies, removing a certain number of genes from pathogenic bacteria removes their capacity to infect hosts. However, more recent studies have demonstrated that the specialization of bacteria in eukaryotic cells is associated with massive gene loss, especially for allopatric endosymbionts that have been isolated for a long time in an intracellular niche. Indeed, sympatric free-living bacteria often have bigger genomes and exhibit greater resistance and plasticity and constitute species complexes rather than true species. Specialists, such as pathogenic bacteria, escape these bacterial complexes and colonize a niche, thereby gaining a species name. Their specialization allows them to become allopatric, and their gene losses eventually favor reductive genome evolution. A pathogenic species is characterized by a gene repertoire that is defined not only by genes that are present but also by those that are lacking. It is likely that current bacterial pathogens will disappear soon and be replaced by new ones that will emerge from bacterial complexes that are already in contact with humans.201021687765
9688100.9997Indirect Selection against Antibiotic Resistance via Specialized Plasmid-Dependent Bacteriophages. Antibiotic resistance genes of important Gram-negative bacterial pathogens are residing in mobile genetic elements such as conjugative plasmids. These elements rapidly disperse between cells when antibiotics are present and hence our continuous use of antimicrobials selects for elements that often harbor multiple resistance genes. Plasmid-dependent (or male-specific or, in some cases, pilus-dependent) bacteriophages are bacterial viruses that infect specifically bacteria that carry certain plasmids. The introduction of these specialized phages into a plasmid-abundant bacterial community has many beneficial effects from an anthropocentric viewpoint: the majority of the plasmids are lost while the remaining plasmids acquire mutations that make them untransferable between pathogens. Recently, bacteriophage-based therapies have become a more acceptable choice to treat multi-resistant bacterial infections. Accordingly, there is a possibility to utilize these specialized phages, which are not dependent on any particular pathogenic species or strain but rather on the resistance-providing elements, in order to improve or enlengthen the lifespan of conventional antibiotic approaches. Here, we take a snapshot of the current knowledge of plasmid-dependent bacteriophages.202133572937
9342110.9997Natural transformation in Gram-negative bacteria thriving in extreme environments: from genes and genomes to proteins, structures and regulation. Extremophilic prokaryotes live under harsh environmental conditions which require far-reaching cellular adaptations. The acquisition of novel genetic information via natural transformation plays an important role in bacterial adaptation. This mode of DNA transfer permits the transfer of genetic information between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal gene transfer (HGT), significantly contributes to genome plasticity over evolutionary history and is a driving force for the spread of fitness-enhancing functions including virulence genes and antibiotic resistances. In particular, HGT has played an important role for adaptation of bacteria to extreme environments. Here, we present a survey of the natural transformation systems in bacteria that live under extreme conditions: the thermophile Thermus thermophilus and two desiccation-resistant members of the genus Acinetobacter such as Acinetobacter baylyi and Acinetobacter baumannii. The latter is an opportunistic pathogen and has become a world-wide threat in health-care institutions. We highlight conserved and unique features of the DNA transporter in Thermus and Acinetobacter and present tentative models of both systems. The structure and function of both DNA transporter are described and the mechanism of DNA uptake is discussed.202134542714
9282120.9997Could DNA uptake be a side effect of bacterial adhesion and twitching motility? DNA acquisition promotes the spread of resistance to antibiotics and virulence among bacteria. It is also linked to several natural phenomena including recombination, genome dynamics, adaptation and speciation. Horizontal DNA transfer between bacteria occurs via conjugation, transduction or competence for natural transformation by DNA uptake. Among these, competence is the only mechanism of transformation initiated and entirely controlled by the chromosome of the recipient bacteria. While the molecular mechanisms allowing the uptake of extracellular DNA are increasingly characterized, the function of competence for natural transformation by DNA uptake, the selective advantage maintaining it and the reasons why bacteria take up DNA in the first place are still debated. In this synthesis, I review some of the literature and discuss the four hypotheses on how and why do bacteria take up DNA. I argue that DNA uptake by bacteria is an accidental by-product of bacterial adhesion and twitching motility. Adhesion and motility are generally increased in stressful conditions, which may explain why bacteria increase DNA uptake in these conditions. In addition to its fundamental scientific relevance, the new hypothesis suggested here has significant clinical implications and finds further support from the fact that antibiotics sometimes fail to eliminate the targeted bacterium while inevitably causing stress to others. The widespread misuse of antibiotics may thus not only be selecting for resistant strains, but may also be causing bacteria to take up more DNA with the consequent increase in the chances of acquiring drug resistance and virulence-a scenario in full concordance with the previously reported induction of competence genes by antibiotics in Streptococcus pneumoniae and Legionella pneumophila.201323381940
9283130.9997Vibrio cholerae: Measuring Natural Transformation Frequency. Many bacteria can become naturally competent to take up extracellular DNA across their outer and inner membranes by a dedicated competence apparatus. Whereas some studies show that the DNA delivered to the cytoplasm may be used for genome repair or for nutrition, it can also be recombined onto the chromosome by homologous recombination: a process called natural transformation. Along with conjugation and transduction, natural transformation represents a mechanism for horizontal transfer of genetic material, e.g., antibiotic resistance genes, which can confer new beneficial characteristics onto the recipient bacteria. Described here are protocols for quantifying the frequency of transformation for the human pathogen Vibrio cholerae, one of several Vibrio species recently shown to be capable of natural transformation.201425367272
9387140.9997Indirect Fitness Benefits Enable the Spread of Host Genes Promoting Costly Transfer of Beneficial Plasmids. Bacterial genes that confer crucial phenotypes, such as antibiotic resistance, can spread horizontally by residing on mobile genetic elements (MGEs). Although many mobile genes provide strong benefits to their hosts, the fitness consequences of the process of transfer itself are less clear. In previous studies, transfer has been interpreted as a parasitic trait of the MGEs because of its costs to the host but also as a trait benefiting host populations through the sharing of a common gene pool. Here, we show that costly donation is an altruistic act when it spreads beneficial MGEs favoured when it increases the inclusive fitness of donor ability alleles. We show mathematically that donor ability can be selected when relatedness at the locus modulating transfer is sufficiently high between donor and recipients, ensuring high frequency of transfer between cells sharing donor alleles. We further experimentally demonstrate that either population structure or discrimination in transfer can increase relatedness to a level selecting for chromosomal transfer alleles. Both mechanisms are likely to occur in natural environments. The simple process of strong dilution can create sufficient population structure to select for donor ability. Another mechanism observed in natural isolates, discrimination in transfer, can emerge through coselection of transfer and discrimination alleles. Our work shows that horizontal gene transfer in bacteria can be promoted by bacterial hosts themselves and not only by MGEs. In the longer term, the success of cells bearing beneficial MGEs combined with biased transfer leads to an association between high donor ability, discrimination, and mobile beneficial genes. However, in conditions that do not select for altruism, host bacteria promoting transfer are outcompeted by hosts with lower transfer rate, an aspect that could be relevant in the fight against the spread of antibiotic resistance.201627270455
9493150.9997Regulatory integration of horizontally-transferred genes in bacteria. Horizontal transfer of genetic material is a fact of microbial life and bacteria can obtain new DNA sequences through the processes of conjugation, transduction and transformation. This offers the bacterium the possibility of evolving rapidly by importing new genes that code for new traits that may assist in environmental adaptation. Research in this area has focused in particular on the role of horizontal transfer in the dissemination through bacterial populations of genes for resistance to antimicrobial agents, including antibiotics. It is becoming clear that many other phenotypic characteristics have been acquired through horizontal routes and that these include traits contributing to pathogenesis and symbiosis. An important corollary to the acquisition of new genes is the problem of how best to integrate them in the existing gene regulatory circuits of the recipient so that fitness is not compromised initially and can be enhanced in the future through optimal expression of the new genes.200919273337
9286160.9997Bacterial sex in dental plaque. Genes are transferred between bacteria in dental plaque by transduction, conjugation, and transformation. Membrane vesicles can also provide a mechanism for horizontal gene transfer. DNA transfer is considered bacterial sex, but the transfer is not parallel to processes that we associate with sex in higher organisms. Several examples of bacterial gene transfer in the oral cavity are given in this review. How frequently this occurs in dental plaque is not clear, but evidence suggests that it affects a number of the major genera present. It has been estimated that new sequences in genomes established through horizontal gene transfer can constitute up to 30% of bacterial genomes. Gene transfer can be both inter- and intrageneric, and it can also affect transient organisms. The transferred DNA can be integrated or recombined in the recipient's chromosome or remain as an extrachromosomal inheritable element. This can make dental plaque a reservoir for antimicrobial resistance genes. The ability to transfer DNA is important for bacteria, making them better adapted to the harsh environment of the human mouth, and promoting their survival, virulence, and pathogenicity.201323741559
9346170.9997Horizontal gene transfer in prokaryotes: quantification and classification. Comparative analysis of bacterial, archaeal, and eukaryotic genomes indicates that a significant fraction of the genes in the prokaryotic genomes have been subject to horizontal transfer. In some cases, the amount and source of horizontal gene transfer can be linked to an organism's lifestyle. For example, bacterial hyperthermophiles seem to have exchanged genes with archaea to a greater extent than other bacteria, whereas transfer of certain classes of eukaryotic genes is most common in parasitic and symbiotic bacteria. Horizontal transfer events can be classified into distinct categories of acquisition of new genes, acquisition of paralogs of existing genes, and xenologous gene displacement whereby a gene is displaced by a horizontally transferred ortholog from another lineage (xenolog). Each of these types of horizontal gene transfer is common among prokaryotes, but their relative contributions differ in different lineages. The fixation and long-term persistence of horizontally transferred genes suggests that they confer a selective advantage on the recipient organism. In most cases, the nature of this advantage remains unclear, but detailed examination of several cases of acquisition of eukaryotic genes by bacteria seems to reveal the evolutionary forces involved. Examples include isoleucyl-tRNA synthetases whose acquisition from eukaryotes by several bacteria is linked to antibiotic resistance, ATP/ADP translocases acquired by intracellular parasitic bacteria, Chlamydia and Rickettsia, apparently from plants, and proteases that may be implicated in chlamydial pathogenesis.200111544372
9280180.9997Evolutionary Changes after Translational Challenges Imposed by Horizontal Gene Transfer. Genes acquired by horizontal gene transfer (HGT) may provide the recipient organism with potentially new functions, but proper expression level and integration of the transferred genes in the novel environment are not granted. Notably, transferred genes can differ from the receiving genome in codon usage preferences, leading to impaired translation and reduced functionality. Here, we characterize the genomic and proteomic changes undergone during experimental evolution of Escherichia coli after HGT of three synonymous versions, presenting very different codon usage preference, of an antibiotic resistance gene. The experimental evolution was conducted with and without the corresponding antibiotic and the mutational patterns and proteomic profiles after 1,000 generations largely depend on the experimental growth conditions (e.g., mutations in antibiotic off-target genes), and on the synonymous gene version transferred (e.g., mutations in genes responsive to translational stress). The transfer of an exogenous gene extensively modifies the whole proteome, and these proteomic changes are different for the different version of the transferred gene. Additionally, we identified conspicuous changes in global regulators and in intermediate metabolism, confirmed the evolutionary ratchet generated by mutations in DNA repair genes and highlighted the plasticity of bacterial genomes accumulating large and occasionally transient duplications. Our results support a central role of HGT in fuelling evolution as a powerful mechanism promoting rapid, often dramatic genotypic and phenotypic changes. The profound reshaping of the pre-existing geno/phenotype allows the recipient bacteria to explore new ways of functioning, far beyond the mere acquisition of a novel function.201930753446
9612190.9997Using experimental evolution to explore natural patterns between bacterial motility and resistance to bacteriophages. Resistance of bacteria to phages may be gained by alteration of surface proteins to which phages bind, a mechanism that is likely to be costly as these molecules typically have critical functions such as movement or nutrient uptake. To address this potential trade-off, we combine a systematic study of natural bacteria and phage populations with an experimental evolution approach. We compare motility, growth rate and susceptibility to local phages for 80 bacteria isolated from horse chestnut leaves and, contrary to expectation, find no negative association between resistance to phages and bacterial motility or growth rate. However, because correlational patterns (and their absence) are open to numerous interpretations, we test for any causal association between resistance to phages and bacterial motility using experimental evolution of a subset of bacteria in both the presence and absence of naturally associated phages. Again, we find no clear link between the acquisition of resistance and bacterial motility, suggesting that for these natural bacterial populations, phage-mediated selection is unlikely to shape bacterial motility, a key fitness trait for many bacteria in the phyllosphere. The agreement between the observed natural pattern and the experimental evolution results presented here demonstrates the power of this combined approach for testing evolutionary trade-offs.201121509046