Problems of multi-species organisms: endosymbionts to holobionts

The organism is one of the fundamental concepts of biology and has been at the center of many discussions about biological individuality, yet what exactly it is can be confusing. The definition that we find generally useful is that an organism is a unit in which all the subunits have evolved to be highly cooperative, with very little conflict. We focus on how often organisms evolve from two or more formerly independent organisms. Two canonical transitions of this type—replicators clustered in cells and endosymbiotic organelles within host cells—demonstrate the reality of this kind of evolutionary transition and suggest conditions that can favor it. These conditions include co-transmission of the partners across generations and rules that strongly regulate and limit conflict, such as a fair meiosis. Recently, much attention has been given to associations of animals with microbes involved in their nutrition. These range from tight endosymbiotic associations like those between aphids and Buchnera bacteria, to the complex communities in animal intestines. Here, starting with a reflection about identity through time (which we call “Theseus’s fish”), we consider the distinctions between these kinds of animal–bacteria interactions and describe the criteria by which a few can be considered jointly organismal but most cannot

Chance and necessity in the genome evolution of endosymbiotic bacteria of insects

[EN] An open question in evolutionary biology is how does the selection¿drift balance determine the fates of biological interactions. We searched for signatures of selection and drift in genomes of five endosymbiotic bacterial groups known to evolve under strong genetic drift. Although most genes in endosymbiotic bacteria showed evidence of relaxed purifying selection, many genes in these bacteria exhibited stronger selective constraints than their orthologs in free-living bacterial relatives. Remarkably, most of these highly constrained genes had no role in the host¿symbiont interactions but were involved in either buffering the deleterious consequences of drift or other host-unrelated functions, suggesting that they have either acquired new roles or their role became more central in endosymbiotic bacteria. Experimental evolution of Escherichia coli under strong genetic drift revealed remarkable similarities in the mutational spectrum, genome reduction patterns and gene losses to endosymbiotic bacteria of insects. Interestingly, the transcriptome of the experimentally evolved lines showed a generalized deregulation of the genome that affected genes encoding proteins involved in mutational buffering, regulation and amino acid biosynthesis, patterns identical to those found in endosymbiotic bacteria. Our results indicate that drift has shaped endosymbiotic associations through a change in the functional landscape of bacterial genes and that the host had only a small role in such a shiftThis work was supported by Science Foundation Ireland (12/IP/1637) and grants from the Spanish Ministerio de Economia y Competitividad (MINECO-FEDER; BFU2012-36346 and BFU2015-66073-P) to MAF. DAP and CT were supported by Juan de la Cierva fellowships from MINECO (references: JCI-2011-11089 and JCA-2012-14056, respectively). DAP is supported by funds from the University of Nevada, Reno, NV, USA.Sabater-Muñoz, B.; Toft, C.; Alvarez-Ponce, D.; Fares Riaño, MA. (2017). Chance and necessity in the genome evolution of endosymbiotic bacteria of insects. The ISME Journal. 11(6):1291-1304. https://doi.org/10.1038/ismej.2017.18S12911304116Aguilar-Rodriguez J, Sabater-Munoz B, Montagud-Martinez R, Berlanga V, Alvarez-Ponce D, Wagner A et al. (2016). The molecular chaperone DnaK is a source of mutational robustness. Genome Biol Evol 8: 2979–2991.Alvarez-Ponce D, Sabater-Munoz B, Toft C, Ruiz-Gonzalez MX, Fares MA . (2016). Essentiality is a strong determinant of protein rates of evolution during mutation accumulation experiments in Escherichia coli. Genome Biol Evol 8: 2914–2927.Anders S, Huber W . (2010). Differential expression analysis for sequence count data. Genome Biol 11: R106.Archibald J . (2014) One Plus One Equals One: Symbiosis and the Evolution of Complex Life. Oxford University Press: Oxford, UK.Aussel L, Loiseau L, Hajj Chehade M, Pocachard B, Fontecave M, Pierrel F et al. (2014). ubiJ, a new gene required for aerobic growth and proliferation in macrophage, is involved in coenzyme Q biosynthesis in Escherichia coli and Salmonella enterica serovar Typhimurium. J Bacteriol 196: 70–79.Baumann P, Baumann L, Clark MA . (1996). Levels of Buchnera aphidicola chaperonin groEL during growth of the aphid Schizaphis graminum. Curr Microbiol 32: 7.Benjamini Y, Yekutieli Y . (2005). False discovery rate controlling confidence intervals for selected parameters. J Am Stat Assoc 100: 10.Bennett GM, Moran NA . (2015). Heritable symbiosis: the advantages and perils of an evolutionary rabbit hole. Proc Natl Acad Sci USA 112: 10169–10176.Bermingham J, Rabatel A, Calevro F, Vinuelas J, Febvay G, Charles H et al. (2009). Impact of host developmental age on the transcriptome of the symbiotic bacterium Buchnera aphidicola in the pea aphid (Acyrthosiphon pisum. Appl Environ Microbiol 75: 7294–7297.Bogumil D, Dagan T . (2010). Chaperonin-dependent accelerated substitution rates in prokaryotes. Genome Biol Evol 2: 602–608.Carbon S, Ireland A, Mungall CJ, Shu S, Marshall B, Lewis S et al. (2009). AmiGO: online access to ontology and annotation data. Bioinformatics 25: 288–289.Chen Z, Wang Y, Li Y, Li Y, Fu N, Ye J et al. (2012). Esre: a novel essential non-coding RNA in Escherichia coli. FEBS Lett 586: 1195–1200.Clark JW, Hossain S, Burnside CA, Kambhampati S . (2001). Coevolution between a cockroach and its bacterial endosymbiont: a biogeographical perspective. Proc Biol Sci 268: 393–398.Dale C, Wang B, Moran N, Ochman H . (2003). Loss of DNA recombinational repair enzymes in the initial stages of genome degeneration. Mol Biol Evol 20: 1188–1194.Deatherage DE, Barrick JE . (2014). Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol Biol 1151: 165–188.Douglas AE . (2003). The nutritional physiology of aphids. Adv Insect Physiol 31: 68.Fares MA, Barrio E, Sabater-Munoz B, Moya A . (2002a). The evolution of the heat-shock protein GroEL from Buchnera, the primary endosymbiont of aphids, is governed by positive selection. Mol Biol Evol 19: 1162–1170.Fares MA, Ruiz-Gonzalez MX, Moya A, Elena SF, Barrio E . (2002b). Endosymbiotic bacteria: groEL buffers against deleterious mutations. Nature 417: 398.Gancedo C, Flores CL, Gancedo JM . (2016). The expanding landscape of moonlighting proteins in yeasts. Microbiol Mol Biol Rev 80: 765–777.Gerardo NM, Altincicek B, Anselme C, Atamian H, Barribeau SM, de Vos M et al. (2010). Immunity and other defenses in pea aphids, Acyrthosiphon pisum. Genome Biol 11: R21.Gomez-Valero L, Latorre A, Silva FJ . (2004). The evolutionary fate of nonfunctional DNA in the bacterial endosymbiont Buchnera aphidicola. Mol Biol Evol 21: 2172–2181.Gomez-Valero L, Silva FJ, Christophe Simon J, Latorre A . (2007). Genome reduction of the aphid endosymbiont Buchnera aphidicola in a recent evolutionary time scale. Gene 389: 87–95.Gonzalez-Domenech CM, Belda E, Patino-Navarrete R, Moya A, Pereto J, Latorre A . (2012). Metabolic stasis in an ancient symbiosis: genome-scale metabolic networks from two Blattabacterium cuenoti strains, primary endosymbionts of cockroaches. BMC Microbiol 12 (Suppl 1): S5.Hansen AK, Moran NA . (2011). Aphid genome expression reveals host-symbiont cooperation in the production of amino acids. Proc Natl Acad Sci USA 108: 2849–2854.Hansen AK, Moran NA . (2014). The impact of microbial symbionts on host plant utilization by herbivorous insects. Mol Ecol 23: 1473–1496.Henderson B, Fares MA, Lund PA . (2013). Chaperonin 60: a paradoxical, evolutionarily conserved protein family with multiple moonlighting functions. Biol Rev Camb Philos Soc 88: 955–987.Humphreys NJ, Douglas AE . (1997). Partitioning of symbiotic bacteria between generations of an insect: a quantitative study of a Buchnera sp. in the pea aphid (Acyrthosiphon pisum reared at different temperatures. Appl Environ Microbiol 63: 3294–3296.International Aphid Genomics Consortium. (2010). Genome sequence of the pea aphid Acyrthosiphon pisum. PLoS Biol 8: e1000313.Kadibalban AS, Bogumil D, Landan G, Dagan T . (2016). DnaK-dependent accelerated evolutionary rate in prokaryotes. Genome Biol Evol 8: 1590–1599.Katoh K, Standley DM . (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30: 772–780.Kelkar YD, Ochman H . (2013). Genome reduction promotes increase in protein functional complexity in bacteria. Genetics 193: 303–307.Koga R, Meng XY, Tsuchida T, Fukatsu T . (2012). Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte-embryo interface. Proc Natl Acad Sci USA 109: E1230–E1237.Kuo CH, Moran NA, Ochman H . (2009). The consequences of genetic drift for bacterial genome complexity. Genome Res 19: 1450–1454.Kuo CH, Ochman H . (2009). Deletional bias across the three domains of life. Genome Biol Evol 1: 145–152.Law R, Lewis DH . (1983). Biotic environments and the maintenance of sex-some evidence from mutualistic symbioses. Biol J Linnean Soc 20: 28.Liu XD, Xie L, Wei Y, Zhou X, Jia B, Liu J et al. (2014). Abiotic stress resistance, a novel moonlighting function of ribosomal protein RPL44 in the halophilic fungus Aspergillus glaucus. Appl Environ Microbiol 80: 4294–4300.Lohse M, Bolger AM, Nagel A, Fernie AR, Lunn JE, Stitt M et al. (2012). RobiNA: a user-friendly, integrated software solution for RNA-Seq-based transcriptomics. Nucleic Acids Res 40: W622–W627.Macdonald SJ, Lin GG, Russell CW, Thomas GH, Douglas AE . (2012). The central role of the host cell in symbiotic nitrogen metabolism. Proc Biol Sci 279: 2965–2973.McClure R, Balasubramanian D, Sun Y, Bobrovskyy M, Sumby P, Genco CA et al. (2013). Computational analysis of bacterial RNA-Seq data. Nucleic Acids Res 41: e140.McCutcheon JP, Moran NA . (2012). Extreme genome reduction in symbiotic bacteria. Nat Rev Microbiol 10: 13–26.McFall-Ngai M, Hadfield MG, Bosch TC, Carey HV, Domazet-Loso T, Douglas AE et al. (2013). Animals in a bacterial world, a new imperative for the life sciences. Proc Natl Acad Sci USA 110: 3229–3236.Mira A, Ochman H, Moran NA . (2001). Deletional bias and the evolution of bacterial genomes. Trends Genet 17: 589–596.Moran NA . (1996). Accelerated evolution and Muller's rachet in endosymbiotic bacteria. Proc Natl Acad Sci USA 93: 2873–2878.Moran NA, Dunbar HE, Wilcox JL . (2005). Regulation of transcription in a reduced bacterial genome: nutrient-provisioning genes of the obligate symbiont Buchnera aphidicola. J Bacteriol 187: 4229–4237.Moran NA, McCutcheon JP, Nakabachi A . (2008). Genomics and evolution of heritable bacterial symbionts. Annu Rev Genet 42: 165–190.Moran NA, McLaughlin HJ, Sorek R . (2009). The dynamics and time scale of ongoing genomic erosion in symbiotic bacteria. Science 323: 379–382.Nakabachi A, Ishida K, Hongoh Y, Ohkuma M, Miyagishima SY . (2014). Aphid gene of bacterial origin encodes a protein transported to an obligate endosymbiont. Curr Biol 24: R640–R641.Nilsson AI, Koskiniemi S, Eriksson S, Kugelberg E, Hinton JC, Andersson DI . (2005). Bacterial genome size reduction by experimental evolution. Proc Natl Acad Sci USA 102: 12112–12116.Patino-Navarrete R, Moya A, Latorre A, Pereto J . (2013). Comparative genomics of Blattabacterium cuenoti: the frozen legacy of an ancient endosymbiont genome. Genome Biol Evol 5: 351–361.Pettersson ME, Berg OG . (2007). Muller's ratchet in symbiont populations. Genetica 130: 199–211.Price DR, Feng H, Baker JD, Bavan S, Luetje CW, Wilson AC . (2014). Aphid amino acid transporter regulates glutamine supply to intracellular bacterial symbionts. Proc Natl Acad Sci USA 111: 320–325.Reyes-Prieto M, Vargas-Chavez C, Latorre A, Moya A . (2015). SymbioGenomesDB: a database for the integration and access to knowledge on host-symbiont relationships. Database 2015: bav109 (1–8).Robinson MD, McCarthy DJ, Smyth GK . (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26: 139–140.Sabater-Muñoz B, Prats-Escriche M, Montagud-Martinez R, Lopez-Cerdan A, Toft C, Aguilar-Rodriguez J et al. (2015). Fitness trade-offs determine the role of the molecular chaperonin groel in buffering mutations. Mol Biol Evol 32: 2681–2693.Schlicker A, Domingues FS, Rahnenfuhrer J, Lengauer T . (2006). A new measure for functional similarity of gene products based on Gene Ontology. BMC Bioinformatics 7: 302.Shigenobu S, Watanabe H, Hattori M, Sakaki Y, Ishikawa H . (2000). Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407: 81–86.Supek F, Bosnjak M, Skunca N, Smuc T . (2011). REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS ONE 6: e21800.Tamas I, Klasson L, Canback B, Naslund AK, Eriksson AS, Wernegreen JJ et al. (2002). 50 million years of genomic stasis in endosymbiotic bacteria. Science 296: 2376–2379.Toft C, Fares MA . (2008). The evolution of the flagellar assembly pathway in endosymbiotic bacterial genomes. Mol Biol Evol 25: 2069–2076.van Ham RC, Kamerbeek J, Palacios C, Rausell C, Abascal F, Bastolla U et al. (2003). Reductive genome evolution in Buchnera aphidicola. Proc Natl Acad Sci USA 100: 581–586.Wernegreen JJ . (2002). Genome evolution in bacterial endosymbionts of insects. Nat Rev Genet 3: 850–861.Wernegreen JJ . (2011). Reduced selective constraint in endosymbionts: elevation in radical amino acid replacements occurs genome-wide. PLoS One 6: e28905.Williams TA, Fares MA . (2010). The effect of chaperonin buffering on protein evolution. Genome Biol Evol 2: 609–619.Yang Z . (2007). PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol 24: 1586–1591

Amino Acid Usage Is Asymmetrically Biased in AT- and GC-Rich Microbial Genomes.

INTRODUCTION: Genomic base composition ranges from less than 25% AT to more than 85% AT in prokaryotes. Since only a small fraction of prokaryotic genomes is not protein coding even a minor change in genomic base composition will induce profound protein changes. We examined how amino acid and codon frequencies were distributed in over 2000 microbial genomes and how these distributions were affected by base compositional changes. In addition, we wanted to know how genome-wide amino acid usage was biased in the different genomes and how changes to base composition and mutations affected this bias. To carry this out, we used a Generalized Additive Mixed-effects Model (GAMM) to explore non-linear associations and strong data dependences in closely related microbes; principal component analysis (PCA) was used to examine genomic amino acid- and codon frequencies, while the concept of relative entropy was used to analyze genomic mutation rates. RESULTS: We found that genomic amino acid frequencies carried a stronger phylogenetic signal than codon frequencies, but that this signal was weak compared to that of genomic %AT. Further, in contrast to codon usage bias (CUB), amino acid usage bias (AAUB) was differently distributed in AT- and GC-rich genomes in the sense that AT-rich genomes did not prefer specific amino acids over others to the same extent as GC-rich genomes. AAUB was also associated with relative entropy; genomes with low AAUB contained more random mutations as a consequence of relaxed purifying selection than genomes with higher AAUB. CONCLUSION: Genomic base composition has a substantial effect on both amino acid- and codon frequencies in bacterial genomes. While phylogeny influenced amino acid usage more in GC-rich genomes, AT-content was driving amino acid usage in AT-rich genomes. We found the GAMM model to be an excellent tool to analyze the genomic data used in this study

Pathogenic Bacillus anthracis in the progressive gene losses and gains in adaptive evolution

Background: Sequence mutations represent a driving force of adaptive evolution in bacterial pathogens. It is especially evident in reductive genome evolution where bacteria underwent lifestyles shifting from a free-living to a strictly intracellular or host-depending life. It resulted in loss of function mutations and/or the acquisition of virulence gene clusters. Bacillus anthracis shares a common soil bacterial ancestor with its closely related bacillus species but is the only obligate, causative agent of inhalation anthrax within the genus Bacillus. The anthrax-causing Bacillus anthracis experienced the similar lifestyle changes. We thus hypothesized that the bacterial pathogen would follow a compatible evolution path. Results: In this study, a cluster-based evolution scheme was devised to analyze genes that are gained by or lost from B. anthracis. The study detected gene losses/gains at two separate evolutionary stages. The stage I is when B. anthracis and its sister species within the Bacillus cereus group diverged from other species in genus Bacillus. The stage II is when B. anthracis differentiated from its two closest relatives: B. cereus and B. thuringiensis. Many genes gained at these stages are homologues of known pathogenic factors such those for internalin, B. anthracis-specific toxins and large groups of surface proteins and lipoproteins. Conclusion: The analysis presented here allowed us to portray a progressive evolutionary process during the lifestyle shift of B. anthracis, thus providing new insights into how B. anthracis had evolved and bore a promise of finding drug and vaccine targets for this strategically important pathogen

Evolutionary Instability of Symbiotic Function in Bradyrhizobium japonicum

Bacterial mutualists are often acquired from the environment by eukaryotic hosts. However, both theory and empirical work suggest that this bacterial lifestyle is evolutionarily unstable. Bacterial evolution outside of the host is predicted to favor traits that promote an independent lifestyle in the environment at a cost to symbiotic function. Consistent with these predictions, environmentally-acquired bacterial mutualists often lose symbiotic function over evolutionary time. Here, we investigate the evolutionary erosion of symbiotic traits in Bradyrhizobium japonicum, a nodulating root symbiont of legumes. Building on a previous published phylogeny we infer loss events of nodulation capability in a natural population of Bradyrhizobium, potentially driven by mutation or deletion of symbiosis loci. Subsequently, we experimentally evolved representative strains from the symbiont population under host-free in vitro conditions to examine potential drivers of these loss events. Among Bradyrhizobium genotypes that evolved significant increases in fitness in vitro, two exhibited reduced symbiotic quality, but no experimentally evolved strain lost nodulation capability or evolved any fixed changes at six sequenced loci. Our results are consistent with trade-offs between symbiotic quality and fitness in a host free environment. However, the drivers of loss-of-nodulation events in natural Bradyrhizobium populations remain unknown

Structure and dynamics of the operon map of Buchnera aphidicola sp. strain APS

<p>Abstract</p> <p>Background</p> <p>Gene expression regulation is still poorly documented in bacteria with highly reduced genomes. Understanding the evolution and mechanisms underlying the regulation of gene transcription in <it>Buchnera aphidicola</it>, the primary endosymbiont of aphids, is expected both to enhance our understanding of this nutritionally based association and to provide an intriguing case-study of the evolution of gene expression regulation in a reduced bacterial genome.</p> <p>Results</p> <p>A Bayesian predictor was defined to infer the <it>B. aphidicola </it>transcription units, which were further validated using transcriptomic data and RT-PCR experiments. The characteristics of <it>B. aphidicola </it>predicted transcription units (TUs) were analyzed in order to evaluate the impact of operon map organization on the regulation of gene transcription.</p> <p>On average, <it>B. aphidicola </it>TUs contain more genes than those of <it>E. coli</it>. The global layout of <it>B. aphidicola </it>operon map was mainly shaped by the big reduction and the rearrangements events, which occurred at the early stage of the symbiosis. Our analysis suggests that this operon map may evolve further only by small reorganizations around the frontiers of <it>B. aphidicola </it>TUs, through promoter and/or terminator sequence modifications and/or by pseudogenization events. We also found that the need for specific transcription regulation exerts some pressure on gene conservation, but not on gene assembling in the operon map in <it>Buchnera</it>. Our analysis of the TUs spacing pointed out that a selection pressure is maintained on the length of the intergenic regions between divergent adjacent gene pairs.</p> <p>Conclusions</p> <p><it>B. aphidicola </it>can seemingly only evolve towards a more polycistronic operon map. This implies that gene transcription regulation is probably subject to weak selection pressure in <it>Buchnera </it>conserving operons composed of genes with unrelated functions.</p

Reduced Selective Constraint in Endosymbionts: Elevation in Radical Amino Acid Replacements Occurs Genome-Wide

As predicted by the nearly neutral model of evolution, numerous studies have shown that reduced Ne accelerates the accumulation of slightly deleterious changes under genetic drift. While such studies have mostly focused on eukaryotes, bacteria also offer excellent models to explore the effects of Ne. Most notably, the genomes of host-dependent bacteria with small Ne show signatures of genetic drift, including elevated Ka/Ks. Here, I explore the utility of an alternative measure of selective constraint: the per-site rate of radical and conservative amino acid substitutions (Dr/Dc). I test the hypothesis that purifying selection against radical amino acid changes is less effective in two insect endosymbiont groups (Blochmannia of ants and Buchnera of aphids), compared to related gamma-Proteobacteria. Genome comparisons demonstrate a significant elevation in Dr/Dc in endosymbionts that affects the majority (66–79%) of shared orthologs examined. The elevation of Dr/Dc in endosymbionts affects all functional categories examined. Simulations indicate that Dr/Dc estimates are sensitive to codon frequencies and mutational parameters; however, estimation biases occur in the opposite direction as the patterns observed in genome comparisons, thereby making the inference of elevated Dr/Dc more conservative. Increased Dr/Dc and other signatures of genome degradation in endosymbionts are consistent with strong effects of genetic drift in their small populations, as well as linkage to selected sites in these asexual bacteria. While relaxed selection against radical substitutions may contribute, genome-wide processes such as genetic drift and linkage best explain the pervasive elevation in Dr/Dc across diverse functional categories that include basic cellular processes. Although the current study focuses on a few bacterial lineages, it suggests Dr/Dc is a useful gauge of selective constraint and may provide a valuable alternative to Ka/Ks when high sequence divergences preclude estimates of Ks. Broader application of Dr/Dc will benefit from approaches less prone to estimation biases

Selection-Driven Gene Loss in Bacteria

Gene loss by deletion is a common evolutionary process in bacteria, as exemplified by bacteria with small genomes that have evolved from bacteria with larger genomes by reductive processes. The driving force(s) for genome reduction remains unclear, and here we examined the hypothesis that gene loss is selected because carriage of superfluous genes confers a fitness cost to the bacterium. In the bacterium Salmonella enterica, we measured deletion rates at 11 chromosomal positions and the fitness effects of several spontaneous deletions. Deletion rates varied over 200-fold between different regions with the replication terminus region showing the highest rates. Approximately 25% of the examined deletions caused an increase in fitness under one or several growth conditions, and after serial passage of wild-type bacteria in rich medium for 1,000 generations we observed fixation of deletions that substantially increased bacterial fitness when reconstructed in a non-evolved bacterium. These results suggest that selection could be a significant driver of gene loss and reductive genome evolution