Extremophilic red algae as models for understanding adaptation to hostile environments and the evolution of eukaryotic life on the early earth.

Extremophiles have always garnered great interest because of their exotic lifestyles and ability to thrive at the physical limits of life. In hot springs environments, the Cyanidiophyceae red algae are the only photosynthetic eukaryotes able to live under extremely low pH (0-5) and relatively high temperature (35ºC to 63ºC). These extremophiles live as biofilms in the springs, inhabit acid soils near the hot springs, and form endolithic populations in the surrounding rocks. Cyanidiophyceae represent a remarkable source of knowledge about the evolution of extremophilic lifestyles and their genomes encode specialized enzymes that have applied uses. Here we review the evolutionary origin, taxonomy, genome biology, industrial applications, and use of Cyanidiophyceae as genetic models. Currently, Cyanidiophyceae comprise a single order (Cyanidiales), three families, four genera, and nine species, including the well-known Cyanidioschyzon merolae and Galdieria sulphuraria. These algae have small, gene-rich genomes that are analogous to those of prokaryotes they live and compete with. There are few spliceosomal introns and evidence exists for horizontal gene transfer as a driver of local adaptation to gain access to external fixed carbon and to extrude toxic metals. Cyanidiophyceae offer a variety of commercial opportunities such as phytoremediation to detoxify contaminated soils or waters and exploitation of their mixotrophic lifestyles to support the efficient production of bioproducts such as phycocyanin and floridosides. In terms of exobiology, Cyanidiophyceae are an ideal model system for understanding the evolutionary effects of foreign gene acquisition and the interactions between different organisms inhabiting the same harsh environment on the early Earth. Finally, we describe ongoing research with C. merolae genetics and summarize the unique insights they offer to the understanding of algal biology and evolution.

Endosymbiotic ratchet accelerates divergence after organelle origin: The Paulinella model for plastid evolution: The Paulinella model for plastid evolution.

We hypothesize that as one of the most consequential events in evolution, primary endosymbiosis accelerates lineage divergence, a process we refer to as the endosymbiotic ratchet. Our proposal is supported by recent work on the photosynthetic amoeba, Paulinella, that underwent primary plastid endosymbiosis about 124 Mya. This amoeba model allows us to explore the early impacts of photosynthetic organelle (plastid) origin on the host lineage. The current data point to a central role for effective population size (Ne ) in accelerating divergence post-endosymbiosis due to limits to dispersal and reproductive isolation that reduce Ne , leading to local adaptation. We posit that isolated populations exploit different strategies and behaviors and assort themselves in non-overlapping niches to minimize competition during the early, rapid evolutionary phase of organelle integration. The endosymbiotic ratchet provides a general framework for interpreting post-endosymbiosis lineage evolution that is driven by disruptive selection and demographic and population shifts. Also see the video abstract here: https://youtu.be/gYXrFM6Zz6Q.

Comparative genomics of Ascetosporea gives new insight into the evolutionary basis for animal parasitism in Rhizaria.

BACKGROUND: Ascetosporea (Endomyxa, Rhizaria) is a group of unicellular parasites infecting aquatic invertebrates. They are increasingly being recognized as widespread and important in marine environments, causing large annual losses in invertebrate aquaculture. Despite their importance, little molecular data of Ascetosporea exist, with only two genome assemblies published to date. Accordingly, the evolutionary origin of these parasites is unclear, including their phylogenetic position and the genomic adaptations that accompanied the transition from a free-living lifestyle to parasitism. Here, we sequenced and assembled three new ascetosporean genomes, as well as the genome of a closely related amphizoic species, to investigate the phylogeny, origin, and genomic adaptations to parasitism in Ascetosporea. RESULTS: Using a phylogenomic approach, we confirm the monophyly of Ascetosporea and show that Paramyxida group with Mikrocytida, with Haplosporida being sister to both groups. We report that the genomes of these parasites are relatively small (12-36 Mb) and gene-sparse (~ 2300-5200 genes), while containing surprisingly high amounts of non-coding sequence (~ 70-90% of the genomes). Performing gene-tree aware ancestral reconstruction of gene families, we demonstrate extensive gene losses at the origin of parasitism in Ascetosporea, primarily of metabolic functions, and little gene gain except on terminal branches. Finally, we highlight some functional gene classes that have undergone expansions during evolution of the group. CONCLUSIONS: We present important new genomic information from a lineage of enigmatic but important parasites of invertebrates and illuminate some of the genomic innovations accompanying the evolutionary transition to parasitism in this lineage. Our results and data provide a genetic basis for the development of control measures against these parasites.

Comparison of wild-type KT2440 and genome-reduced EM42 Pseudomonas putida strains for muconate production from aromatic compounds and glucose.

Pseudomonas putida KT2440 is a robust, aromatic catabolic bacterium that has been widely engineered to convert bio-based and waste-based feedstocks to target products. Towards industrial domestication of P. putida KT2440, rational genome reduction has been previously conducted, resulting in P. putida strain EM42, which exhibited characteristics that could be advantageous for production strains. Here, we compared P. putida KT2440- and EM42-derived strains for cis,cis-muconic acid production from an aromatic compound, p-coumarate, and in separate strains, from glucose. To our surprise, the EM42-derived strains did not outperform the KT2440-derived strains in muconate production from either substrate. In bioreactor cultivations, KT2440- and EM42-derived strains produced muconate from p-coumarate at titers of 45 g/L and 37 g/L, respectively, and from glucose at 20 g/L and 13 g/L, respectively. To provide additional insights about the differences in the parent strains, we analyzed growth profiles of KT2440 and EM42 on aromatic compounds as the sole carbon and energy sources. In general, the EM42 strain exhibited reduced growth rates but shorter growth lags than KT2440. We also observed that EM42-derived strains resulted in higher growth rates on glucose compared to KT2440-derived strains, but only at the lowest glucose concentrations tested. Transcriptomics revealed that genome reduction in EM42 had global effects on transcript levels and showed that the EM42-derived strains that produce muconate from glucose exhibit reduced modulation of gene expression in response to changes in glucose concentrations. Overall, our results highlight that additional studies are warranted to understand the effects of genome reduction on microbial metabolism and physiology, especially when intended for use in production strains.

A genome-reduced Corynebacterium glutamicum derivative discloses a hidden pathway relevant for 1,2-propanediol production.

BACKGROUND: 1,2-propanediol (1,2-PDO) is widely used in the cosmetic, food, and drug industries with a worldwide consumption of over 1.5 million metric tons per year. Although efforts have been made to engineer microbial hosts such as Corynebacterium glutamicum to produce 1,2-PDO from renewable resources, the performance of such strains is still improvable to be competitive with existing petrochemical production routes. RESULTS: In this study, we enabled 1,2-PDO production in the genome-reduced strain C. glutamicum PC2 by introducing previously described modifications. The resulting strain showed reduced product formation but secreted 50 ± 1 mM D-lactate as byproduct. C. glutamicum PC2 lacks the D-lactate dehydrogenase which pointed to a yet unknown pathway relevant for 1,2-PDO production. Further analysis indicated that in C. glutamicum methylglyoxal, the precursor for 1,2-PDO synthesis, is detoxified with the antioxidant native mycothiol (MSH) by a glyoxalase-like system to lactoylmycothiol and converted to D-lactate which is rerouted into the central carbon metabolism at the level of pyruvate. Metabolomics of cell extracts of the empty vector-carrying wildtype, a 1,2-PDO producer and its derivative with inactive D-lactate dehydrogenase identified major mass peaks characteristic for lactoylmycothiol and its precursors MSH and glucosaminyl-myo-inositol, whereas the respective mass peaks were absent in a production strain with inactivated MSH synthesis. Deletion of mshA, encoding MSH synthase, in the 1,2-PDO producing strain C. glutamicum ΔhdpAΔldh(pEKEx3-mgsA-yqhD-gldA) improved the product yield by 56% to 0.53 ± 0.01 mM1,2-PDO mMglucose-1 which is the highest value for C. glutamicum reported so far. CONCLUSIONS: Genome reduced-strains are a useful basis to unravel metabolic constraints for strain engineering and disclosed in this study the pathway to detoxify methylglyoxal which represents a precursor for 1,2-PDO production. Subsequent inactivation of the competing pathway significantly improved the 1,2-PDO yield.

Combinatorial metabolic engineering of Streptomyces sp. CB03234-S for the enhanced production of anthraquinone-fused enediyne tiancimycins.

BACKGROUND: Anthraquinone-fused enediynes (AFEs) are excellent payloads for antibody-drug conjugates (ADCs). The yields of AFEs in the original bacterial hosts are extremely low. Multiple traditional methods had been adopted to enhance the production of the AFEs. Despite these efforts, the production titers of these compounds are still low, presenting a practical challenge for their development. Tiancimycins (TNMs) are a class of AFEs produced by Streptomyces sp. CB03234. One of their salient features is that they exhibit rapid and complete cell killing ability against various cancer cell lines. RESULTS: In this study, a combinatorial metabolic engineering strategy guided by the CB03234-S genome and transcriptome was employed to improve the titers of TNMs. First, re-sequencing of CB03234-S (Ribosome engineered mutant strains) genome revealed the deletion of a 583-kb DNA fragment, accounting for about 7.5% of its genome. Second, by individual or combined inactivation of seven potential precursor competitive biosynthetic gene clusters (BGCs) in CB03234-S, a double-BGC inactivation mutant, S1009, was identified with an improved TNMs titer of 28.2 ± 0.8 mg/L. Third, overexpression of five essential biosynthetic genes, including two post-modification genes, and three self-resistance auxiliary genes, was also conducted, through which we discovered that mutants carrying the core genes, tnmE or tnmE10, exhibited enhanced TNMs production. The average TNMs yield reached 43.5 ± 2.4 mg/L in a 30-L fermenter, representing an approximately 360% increase over CB03234-S and the highest titer among all AFEs to date. Moreover, the resulting mutant produced TNM-W, a unique TNM derivative with a double bond instead of a common ethylene oxide moiety. Preliminary studies suggested that TNM-W was probably converted from TNM-A by both TnmE and TnmE10. CONCLUSIONS: Based on the genome and transcriptome analyses, we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a high-yield strain of TNMs based on CB03234-S. Our study establishes a solid basis for the clinical development of AFE-based ADCs.

Toward a high-quality pan-genome landscape of Bacillus subtilis by removal of confounding strains.

Pan-genome analysis is widely used to study the evolution and genetic diversity of species, particularly in bacteria. However, the impact of strain selection on the outcome of pan-genome analysis is poorly understood. Furthermore, a standard protocol to ensure high-quality pan-genome results is lacking. In this study, we carried out a series of pan-genome analyses of different strain sets of Bacillus subtilis to understand the impact of various strains on the performance and output quality of pan-genome analyses. Consequently, we found that the results obtained by pan-genome analyses of B. subtilis can be influenced by the inclusion of incorrectly classified Bacillus subspecies strains, phylogenetically distinct strains, engineered genome-reduced strains, chimeric strains, strains with a large number of unique genes or a large proportion of pseudogenes, and multiple clonal strains. Since the presence of these confounding strains can seriously affect the quality and true landscape of the pan-genome, we should remove these deviations in the process of pan-genome analyses. Our study provides new insights into the removal of biases from confounding strains in pan-genome analyses at the beginning of data processing, which enables the achievement of a closer representation of a high-quality pan-genome landscape of B. subtilis that better reflects the performance and credibility of the B. subtilis pan-genome. This procedure could be added as an important quality control step in pan-genome analyses for improving the efficiency of analyses, and ultimately contributing to a better understanding of genome function, evolution and genome-reduction strategies for B. subtilis in the future.

Bacterial genome reduction for optimal chassis of synthetic biology: a review.

Bacteria with streamlined genomes, that harbor full functional genes for essential metabolic networks, are able to synthesize the desired products more effectively and thus have advantages as production platforms in industrial applications. To obtain streamlined chassis genomes, a large amount of effort has been made to reduce existing bacterial genomes. This work falls into two categories: rational and random reduction. The identification of essential gene sets and the emergence of various genome-deletion techniques have greatly promoted genome reduction in many bacteria over the past few decades. Some of the constructed genomes possessed desirable properties for industrial applications, such as: increased genome stability, transformation capacity, cell growth, and biomaterial productivity. The decreased growth and perturbations in physiological phenotype of some genome-reduced strains may limit their applications as optimized cell factories. This review presents an assessment of the advancements made to date in bacterial genome reduction to construct optimal chassis for synthetic biology, including: the identification of essential gene sets, the genome-deletion techniques, the properties and industrial applications of artificially streamlined genomes, the obstacles encountered in constructing reduced genomes, and the future perspectives.

Opacimonas viscosa gen. nov., sp. nov., a novel bacterium isolated from phycosphere.

A novel strain of a member of the family Alteromonadaceae was isolated from the phycosphere of a diatom and designated as LMIT007T. LMIT007T could form milk-white, opaque, circular and smooth colonies on 2216E marine agar. LMIT007T cells were around 1.0-1.8 µm long, 0.8-1.8 µm wide, round or oval shaped and had polar flagella but were non-motile. Optimum conditions for growth were 25 °C, pH 7.0 and 6 % (w/v) NaCl. The results of 16S rRNA gene-based analysis indicated that LMIT007T had the highest similarity with the type strains Aestuaribacter halophilus JC2043T (95.95 %), Alteromonas lipolytica JW12T (95.60 %) and Alteromonas halophila KCTC 22164T (94.21 %). Furthermore, the results of phylogenetic analysis based on 16S rRNA gene sequences and of phylogenomic analysis indicated that LMIT007T could be clustered into the family Alteromonadaceae but formed a separate branch. The genome size of the strain was 2.95 Mbp and the DNA G+C content was 41.6 %. The average nucleotide identity (ANI) values of orthologous genes between LMIT007T and species of other closely related genera within the family Alteromonadaceae ranged from 66.9 to 69.2 %, and the average amino acid identity (AAI) values ranged from 60.0 to 65.7 %. The main respiratory quinone was ubiquinone-8. The major fatty acids were summed feature 3 (C16 : 1ω7c / C16 : 1ω6c) and C16 : 0. The polar lipid profile contain phosphatidylethanolamine, phosphatidylglycerol, aminolipid, two phospholipid and an unknown polar lipid. On the basis of the results of the polyphasic analysis, strain LMIT007T is suggested to represent a novel genus and species within the family Alteromonadaceae, for which the name Opacimonas viscosa gen. nov., sp. nov. is proposed. The type strain is LMIT007T (=MCCC 1K08161T=KCTC 92597T).

Multiple origins of obligate nematode and insect symbionts by a clade of bacteria closely related to plant pathogens.

Obligate symbioses involving intracellular bacteria have transformed eukaryotic life, from providing aerobic respiration and photosynthesis to enabling colonization of previously inaccessible niches, such as feeding on xylem and phloem, and surviving in deep-sea hydrothermal vents. A major challenge in the study of obligate symbioses is to understand how they arise. Because the best studied obligate symbioses are ancient, it is especially challenging to identify early or intermediate stages. Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium, a well-studied nematode parasite of Drosophila flies. We have found that Haoronymphium and its sister species harbor a maternally inherited intracellular bacterial symbiont. We never find the symbiont in nematode-free flies, and virtually all nematodes in the field and the laboratory are infected. Treating nematodes with antibiotics causes a severe reduction in fly infection success. The association is recent, as more distantly related insect-parasitic tylenchid nematodes do not host these endosymbionts. We also report that the Howardula nematode symbiont is a member of a widespread monophyletic group of invertebrate host-associated microbes that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, and that is sister to Pectobacterium, a lineage of plant pathogenic bacteria. Comparative genomic analysis of this group, which we name Candidatus Symbiopectobacterium, shows signatures of genome erosion characteristic of early stages of symbiosis, with the Howardula symbiont's genome containing over a thousand predicted pseudogenes, comprising a third of its genome.

Gene loss, pseudogenization, and independent genome reduction in non-photosynthetic species of Cryptomonas (Cryptophyceae) revealed by comparative nucleomorph genomics.

BACKGROUND: Cryptophytes are ecologically important algae of interest to evolutionary cell biologists because of the convoluted history of their plastids and nucleomorphs, which are derived from red algal secondary endosymbionts. To better understand the evolution of the cryptophyte nucleomorph, we sequenced nucleomorph genomes from two photosynthetic and two non-photosynthetic species in the genus Cryptomonas. We performed a comparative analysis of these four genomes and the previously published genome of the non-photosynthetic species Cryptomonas paramecium CCAP977/2a. RESULTS: All five nucleomorph genomes are similar in terms of their general architecture, gene content, and gene order and, in the non-photosynthetic strains, loss of photosynthesis-related genes. Interestingly, in terms of size and coding capacity, the nucleomorph genome of the non-photosynthetic species Cryptomonas sp. CCAC1634B is much more similar to that of the photosynthetic C. curvata species than to the non-photosynthetic species C. paramecium. CONCLUSIONS: Our results reveal fine-scale nucleomorph genome variation between distantly related congeneric taxa containing photosynthetic and non-photosynthetic species, including recent pseudogene formation, and provide a first glimpse into the possible impacts of the loss of photosynthesis on nucleomorph genome coding capacity and structure in independently evolved colorless strains.

Exploring the Potential of a Genome-Reduced Escherichia coli Strain for Plasmid DNA Production.

The global demand for nucleic acid-based vaccines, including plasmid DNA (pDNA) and mRNA vaccines, needs efficient production platforms. However, conventional hosts for plasmid production have encountered challenges related to sequence integrity due to the presence of insertion sequences (ISs). In this study, we explored the potential of a genome-reduced Escherichia coli as a host for pDNA production. This strain had been constructed by removing approximately 23% of the genome which were unessential genes, including the genomic unstable elements. Moreover, the strain exhibits an elevated level of NADPH, a coenzyme known to increase plasmid production according to a mathematical model. We hypothesized that the combination of genome reduction and the abundance of NADPH would significantly enhance pDNA production capabilities. Remarkably, our results confirmed a three-fold increase in pDNA production compared to the widely employed DH5α strain. Furthermore, the genome-reduced strain exhibited heightened sensitivity to various antibiotics, bolstering its potential for large scale industrial pDNA production. These findings suggest the genome-reduced E. coli as an exciting candidate for revolutionizing the pDNA industry, offering unprecedented efficiency and productivity.

Simplification of Ribosomes in Bacteria with Tiny Genomes.

The ribosome is an essential cellular machine performing protein biosynthesis. Its structure and composition are highly conserved in all species. However, some bacteria have been reported to have an incomplete set of ribosomal proteins. We have analyzed ribosomal protein composition in 214 small bacterial genomes (<1 Mb) and found that although the ribosome composition is fairly stable, some ribosomal proteins may be absent, especially in bacteria with dramatically reduced genomes. The protein composition of the large subunit is less conserved than that of the small subunit. We have identified the set of frequently lost ribosomal proteins and demonstrated that they tend to be positioned on the ribosome surface and have fewer contacts to other ribosome components. Moreover, some proteins are lost in an evolutionary correlated manner. The reduction of ribosomal RNA is also common, with deletions mostly occurring in free loops. Finally, the loss of the anti-Shine-Dalgarno sequence is associated with the loss of a higher number of ribosomal proteins.

Enhanced Mutation Rate, Relaxed Selection, and the "Domino Effect" are associated with Gene Loss in Blattabacterium, A Cockroach Endosymbiont.

Intracellular endosymbionts have reduced genomes that progressively lose genes at a timescale of tens of million years. We previously reported that gene loss rate is linked to mutation rate in Blattabacterium, however, the mechanisms causing gene loss are not yet fully understood. Here, we carried out comparative genomic analyses on the complete genome sequences of a representative set of 67 Blattabacterium strains, with sizes ranging between 511 and 645 kb. We found that 200 of the 566 analyzed protein-coding genes were lost in at least one lineage of Blattabacterium, with the most extreme case being one gene that was lost independently in 24 lineages. We found evidence for three mechanisms influencing gene loss in Blattabacterium. First, gene loss rates were found to increase exponentially with the accumulation of substitutions. Second, genes involved in vitamin and amino acid metabolism experienced relaxed selection in Cryptocercus and Mastotermes, possibly triggered by their vertically inherited gut symbionts. Third, we found evidence of epistatic interactions among genes leading to a "domino effect" of gene loss within pathways. Our results highlight the complexity of the process of genome erosion in an endosymbiont.

A highly divergent Wolbachia with a tiny genome in an insect-parasitic tylenchid nematode.

Wolbachia symbionts are the most successful host-associated microbes on the planet, infecting arthropods and nematodes. Their role in nematodes is particularly enigmatic, with filarial nematode species either 100% infected and dependent on symbionts for reproduction and development, or not at all infected. We have discovered a highly divergent strain of Wolbachia in an insect-parasitic tylenchid nematode, Howardula sp., in a nematode clade that has not previously been known to harbour Wolbachia. While this nematode is 100% infected with Wolbachia, we did not detect it in related species. We sequenced the Howardula symbiont (wHow) genome and found that it is highly reduced, comprising only 550 kilobase pairs of DNA, approximately 35% smaller than the smallest Wolbachia nematode symbiont genomes. The wHow genome is a subset of all other Wolbachia genomes and has not acquired any new genetic information. While it has lost many genes, including genes involved in cell wall synthesis and cell division, it has retained the entire haem biosynthesis pathway, suggesting that haem supplementation is critical. wHow provides key insights into our understanding of what are the lower limits of Wolbachia cells, as well as the role of Wolbachia symbionts in the biology and convergent evolution of diverse parasitic nematodes.

Communities of Niche-optimized Strains (CoNoS) - Design and creation of stable, genome-reduced co-cultures.

Current bioprocesses for production of value-added compounds are mainly based on pure cultures that are composed of rationally engineered strains of model organisms with versatile metabolic capacities. However, in the comparably well-defined environment of a bioreactor, metabolic flexibility provided by various highly abundant biosynthetic enzymes is much less required and results in suboptimal use of carbon and energy sources for compound production. In nature, non-model organisms have frequently evolved in communities where genome-reduced, auxotrophic strains cross-feed each other, suggesting that there must be a significant advantage compared to growth without cooperation. To prove this, we started to create and study synthetic communities of niche-optimized strains (CoNoS) that consists of two strains of the same species Corynebacterium glutamicum that are mutually dependent on one amino acid. We used both the wild-type and the genome-reduced C1* chassis for introducing selected amino acid auxotrophies, each based on complete deletion of all required biosynthetic genes. The best candidate strains were used to establish several stably growing CoNoS that were further characterized and optimized by metabolic modelling, microfluidic experiments and rational metabolic engineering to improve amino acid production and exchange. Finally, the engineered CoNoS consisting of an l-leucine and l-arginine auxotroph showed a specific growth rate equivalent to 83% of the wild type in monoculture, making it the fastest co-culture of two auxotrophic C. glutamicum strains to date. Overall, our results are a first promising step towards establishing improved biobased production of value-added compounds using the CoNoS approach.

Biobased PET from lignin using an engineered cis, cis-muconate-producing Pseudomonas putida strain with superior robustness, energy and redox properties.

Polyethylene terephthalate (PET), the most common synthetic polyester today, is largely produced from fossil resources, contributing to global warming. Consequently, sustainable sources must be developed to meet the increasing demand for this useful polymer. Here, we demonstrate a cascaded value chain that provides green PET from lignin, the world's most underutilized renewable, via fermentative production of cis, cis-muconate (MA) from lignin-based aromatics as a central step. Catechol, industrially the most relevant but apparently also a highly toxic lignin-related aromatic, strongly inhibited MA-producing Pseudomonas putida MA-1. Assessed by 13C metabolic flux analysis, the microbe substantially redirected its carbon core fluxes, resulting in enhanced NADPH supply for stress defense but causing additional ATP costs. The reconstruction of MA production in a genome-reduced P. putida chassis yielded novel producers with superior pathway fluxes and enhanced robustness to catechol and a wide range of other aromatics. Using the advanced producer P. putida MA-10 catechol, MA could be produced in a fed-batch process from catechol (plus glucose as additional growth substrate) up to an attractive titer of 74 g L-1 and a space-time-yield of 1.4 g L-1 h-1. In terms of co-consumed sugar, the further streamlined strain MA-11 achieved the highest yield of 1.4 mol MA (mol glucose)-1, providing a striking economic advantage. Following fermentative production, bio-based MA was purified and used to chemically synthetize the PET monomer terephthalic acid and the comonomer diethylene glycol terephthalic acid through five steps, which finally enabled the first green PET from lignin.

Phylogenomics and plastome evolution of a Brazilian mycoheterotrophic orchid, Pogoniopsis schenckii.

PREMISE: Pogoniopsis likely represents an independent photosynthesis loss in orchids. We use phylogenomic data to better identify the phylogenetic placement of this fully mycoheterotrophic taxon, and investigate its molecular evolution. METHODS: We performed likelihood analysis of plastid and mitochondrial phylogenomic data to localize the position of Pogoniopsis schenckii in orchid phylogeny, and investigated the evolution of its plastid genome. RESULTS: All analyses place Pogoniopsis in subfamily Epidendroideae, with strongest support from mitochondrial data, which also place it near tribe Sobralieae with moderately strong support. Extreme rate elevation in Pogoniopsis plastid genes broadly depresses branch support; in contrast, mitochondrial genes are only mildly rate elevated and display very modest and localized reductions in bootstrap support. Despite considerable genome reduction, including loss of photosynthesis genes and multiple translation apparatus genes, gene order in Pogoniopsis plastomes is identical to related autotrophs, apart from moderately shifted inverted repeat (IR) boundaries. All cis-spliced introns have been lost in retained genes. Two plastid genes (accD, rpl2) show significant strengthening of purifying selection. A retained plastid tRNA gene (trnE-UUC) of Pogoniopsis lacks an anticodon; we predict that it no longer functions in translation but retains a secondary role in heme biosynthesis. CONCLUSIONS: Slowly evolving mitochondrial genes clarify the placement of Pogoniopsis in orchid phylogeny, a strong contrast with analysis of rate-elevated plastome data. We documented the effects of the novel loss of photosynthesis: for example, despite massive gene loss, its plastome is fully colinear with other orchids, and it displays only moderate shifts in selective pressure in retained genes.

Novel genetic code and record-setting AT-richness in the highly reduced plastid genome of the holoparasitic plant Balanophora.

Plastid genomes (plastomes) vary enormously in size and gene content among the many lineages of nonphotosynthetic plants, but key lineages remain unexplored. We therefore investigated plastome sequence and expression in the holoparasitic and morphologically bizarre Balanophoraceae. The two Balanophora plastomes examined are remarkable, exhibiting features rarely if ever seen before in plastomes or in any other genomes. At 15.5 kb in size and with only 19 genes, they are among the most reduced plastomes known. They have no tRNA genes for protein synthesis, a trait found in only three other plastid lineages, and thus Balanophora plastids must import all tRNAs needed for translation. Balanophora plastomes are exceptionally compact, with numerous overlapping genes, highly reduced spacers, loss of all cis-spliced introns, and shrunken protein genes. With A+T contents of 87.8% and 88.4%, the Balanophora genomes are the most AT-rich genomes known save for a single mitochondrial genome that is merely bloated with AT-rich spacer DNA. Most plastid protein genes in Balanophora consist of ≥90% AT, with several between 95% and 98% AT, resulting in the most biased codon usage in any genome described to date. A potential consequence of its radical compositional evolution is the novel genetic code used by Balanophora plastids, in which TAG has been reassigned from stop to tryptophan. Despite its many exceptional properties, the Balanophora plastome must be functional because all examined genes are transcribed, its only intron is correctly trans-spliced, and its protein genes, although highly divergent, are evolving under various degrees of selective constraint.

DELEAT: gene essentiality prediction and deletion design for bacterial genome reduction.

BACKGROUND: The study of gene essentiality is fundamental to understand the basic principles of life, as well as for applications in many fields. In recent decades, dozens of sets of essential genes have been determined using different experimental and bioinformatics approaches, and this information has been useful for genome reduction of model organisms. Multiple in silico strategies have been developed to predict gene essentiality, but no optimal algorithm or set of gene features has been found yet, especially for non-model organisms with incomplete functional annotation. RESULTS: We have developed DELEAT v0.1 (DELetion design by Essentiality Analysis Tool), an easy-to-use bioinformatic tool which integrates an in silico gene essentiality classifier in a pipeline allowing automatic design of large-scale deletions in any bacterial genome. The essentiality classifier consists of a novel logistic regression model based on only six gene features which are not dependent on experimental data or functional annotation. As a proof of concept, we have applied this pipeline to the determination of dispensable regions in the genome of Bartonella quintana str. Toulouse. In this already reduced genome, 35 possible deletions have been delimited, spanning 29% of the genome. CONCLUSIONS: Built on in silico gene essentiality predictions, we have developed an analysis pipeline which assists researchers throughout multiple stages of bacterial genome reduction projects, and created a novel classifier which is simple, fast, and universally applicable to any bacterial organism with a GenBank annotation file.