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Asset Tag: Industrial biotechnology

April 21, 2020

Genomic analyses of two Alteromonas stellipolaris strains reveal traits with potential biotechnological applications.

The Alteromonas stellipolaris strains PQQ-42 and PQQ-44, previously isolated from a fish hatchery, have been selected on the basis of their strong quorum quenching (QQ) activity, as well as their ability to reduce Vibrio-induced mortality on the coral Oculina patagonica. In this study, the genome sequences of both strains were determined and analyzed in order to identify the mechanism responsible for QQ activity. Both PQQ-42 and PQQ-44 were found to degrade a wide range of N-acylhomoserine lactone (AHL) QS signals, possibly due to the presence of an aac gene which encodes an AHL amidohydrolase. In addition, the different colony morphologies exhibited by the strains could be related to the differences observed in genes encoding cell wall biosynthesis and exopolysaccharide (EPS) production. The PQQ-42 strain produces more EPS (0.36?g?l-1) than the PQQ-44 strain (0.15?g?l-1), whose chemical compositions also differ. Remarkably, PQQ-44 EPS contains large amounts of fucose, a sugar used in high-value biotechnological applications. Furthermore, the genome of strain PQQ-42 contained a large non-ribosomal peptide synthase (NRPS) cluster with a previously unknown genetic structure. The synthesis of enzymes and other bioactive compounds were also identified, indicating that PQQ-42 and PQQ-44 could have biotechnological applications.

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April 21, 2020

In-Depth Genomic and Phenotypic Characterization of the Antarctic Psychrotolerant Strain Pseudomonas sp. MPC6 Reveals Unique Metabolic Features, Plasticity, and Biotechnological Potential.

We obtained the complete genome sequence of the psychrotolerant extremophile Pseudomonas sp. MPC6, a natural Polyhydroxyalkanoates (PHAs) producing bacterium able to rapidly grow at low temperatures. Genomic and phenotypic analyses allowed us to situate this isolate inside the Pseudomonas fluorescens phylogroup of pseudomonads as well as to reveal its metabolic versatility and plasticity. The isolate possesses the gene machinery for metabolizing a variety of toxic aromatic compounds such as toluene, phenol, chloroaromatics, and TNT. In addition, it can use both C6- and C5-carbon sugars like xylose and arabinose as carbon substrates, an uncommon feature for bacteria of this genus. Furthermore, Pseudomonas sp. MPC6 exhibits a high-copy number of genes encoding for enzymes involved in oxidative and cold-stress response that allows it to cope with high concentrations of heavy metals (As, Cd, Cu) and low temperatures, a finding that was further validated experimentally. We then assessed the growth performance of MPC6 on glycerol using a temperature range from 0 to 45°C, the latter temperature corresponding to the limit at which this Antarctic isolate was no longer able to propagate. On the other hand, the MPC6 genome comprised considerably less virulence and drug resistance factors as compared to pathogenic Pseudomonas strains, thus supporting its safety. Unexpectedly, we found five PHA synthases within the genome of MPC6, one of which clustered separately from the other four. This PHA synthase shared only 40% sequence identity at the amino acid level against the only PHA polymerase described for Pseudomonas (63-1 strain) able to produce copolymers of short- and medium-chain length PHAs. Batch cultures for PHA synthesis in Pseudomonas sp. MPC6 using sugars, decanoate, ethylene glycol, and organic acids as carbon substrates result in biopolymers with different monomer compositions. This indicates that the PHA synthases play a critical role in defining not only the final chemical structure of the biosynthesized PHA, but also the employed biosynthetic pathways. Based on the results obtained, we conclude that Pseudomonas sp. MPC6 can be exploited as a bioremediator and biopolymer factory, as well as a model strain to unveil molecular mechanisms behind adaptation to cold and extreme environments.

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April 21, 2020

Comparative Genomics of Thiohalobacter thiocyanaticus HRh1T and Guyparkeria sp. SCN-R1, Halophilic Chemolithoautotrophic Sulfur-Oxidizing Gammaproteobacteria Capable of Using Thiocyanate as Energy Source.

The genomes of Thiohalobacter thiocyanaticus and Guyparkeria (formerly known as Halothiobacillus) sp. SCN-R1, two gammaproteobacterial halophilic sulfur-oxidizing bacteria (SOB) capable of thiocyanate oxidation via the “cyanate pathway”, have been analyzed with a particular focus on their thiocyanate-oxidizing potential and sulfur oxidation pathways. Both genomes encode homologs of the enzyme thiocyanate dehydrogenase (TcDH) that oxidizes thiocyanate via the “cyanate pathway” in members of the haloalkaliphilic SOB of the genus Thioalkalivibrio. However, despite the presence of conservative motives indicative of TcDH, the putative TcDH of the halophilic SOB have a low overall amino acid similarity to the Thioalkalivibrio enzyme, and also the surrounding genes in the TcDH locus were different. In particular, an alternative copper transport system Cus is present instead of Cop and a putative zero-valent sulfur acceptor protein gene appears just before TcDH. Moreover, in contrast to the thiocyanate-oxidizing Thioalkalivibrio species, both genomes of the halophilic SOB contained a gene encoding the enzyme cyanate hydratase. The sulfur-oxidizing pathway in the genome of Thiohalobacter includes a Fcc type of sulfide dehydrogenase, a rDsr complex/AprAB/Sat for oxidation of zero-valent sulfur to sulfate, and an incomplete Sox pathway, lacking SoxCD. The sulfur oxidation pathway reconstructed from the genome of Guyparkeria sp. SCN-R1 was more similar to that of members of the Thiomicrospira-Hydrogenovibrio group, including a Fcc type of sulfide dehydrogenase and a complete Sox complex. One of the outstanding properties of Thiohalobacter is the presence of a Na+-dependent ATP synthase, which is rarely found in aerobic Prokaryotes.Overall, the results showed that, despite an obvious difference in the general sulfur-oxidation pathways, halophilic and haloalkaliphilic SOB belonging to different genera within the Gammaproteobacteria developed a similar unique thiocyanate-degrading mechanism based on the direct oxidative attack on the sulfane atom of thiocyanate.

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April 21, 2020

Study of the whole genome, methylome and transcriptome of Cordyceps militaris.

The complete genome of Cordyceps militaris was sequenced using single-molecule real-time (SMRT) sequencing technology at a coverage over 300×. The genome size was 32.57?Mb, and 14 contigs ranging from 0.35 to 4.58?Mb with an N50 of 2.86?Mb were assembled, including 4 contigs with telomeric sequences on both ends and an additional 8 contigs with telomeric sequences on either the 5′ or 3′ end. A methylome database of the genome was constructed using SMRT and m4C and m6A methylated nucleotides, and many unknown modification types were identified. The major m6A methylation motif is GA and GGAG, and the major m4C methylation motif is GC or CG/GC. In the C. militaris genome DNA, there were four types of methylated nucleotides that we confirmed using high-resolution LCMS-IT-TOF. Using PacBio Iso-Seq, a total of 31,133 complete cDNA sequences were obtained in the fruiting body. The conserved domains of the nontranscribed regions of the genome include TATA boxes, which are the initial regions of genome replication. There were 406 structural variants between the HN and CM01 strains, and there were 1,114 structural variants between the HN and ATCC strains.

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April 21, 2020

Genome Features and Secondary Metabolites Biosynthetic Potential of the Class Ktedonobacteria.

The prevalence of antibiotic resistance and the decrease in novel antibiotic discovery in recent years necessitates the identification of potentially novel microbial resources to produce natural products. Ktedonobacteria, a class of deeply branched bacterial lineage in the ancient phylum Chloroflexi, are ubiquitous in terrestrial environments and characterized by their large genome size and complex life cycle. These characteristics indicate Ktedonobacteria as a potential active producer of bioactive compounds. In this study, we observed the existence of a putative “megaplasmid,” multiple copies of ribosomal RNA operons, and high ratio of hypothetical proteins with unknown functions in the class Ktedonobacteria. Furthermore, a total of 104 antiSMASH-predicted putative biosynthetic gene clusters (BGCs) for secondary metabolites with high novelty and diversity were identified in nine Ktedonobacteria genomes. Our investigation of domain composition and organization of the non-ribosomal peptide synthetase and polyketide synthase BGCs further supports the concept that class Ktedonobacteria may produce compounds structurally different from known natural products. Furthermore, screening of bioactive compounds from representative Ktedonobacteria strains resulted in the identification of broad antimicrobial activities against both Gram-positive and Gram-negative tested bacterial strains. Based on these findings, we propose the ancient, ubiquitous, and spore-forming Ktedonobacteria as a versatile and promising microbial resource for natural product discovery.

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April 21, 2020

Biodegradation of naphthalene, BTEX, and aliphatic hydrocarbons by Paraburkholderia aromaticivorans BN5 isolated from petroleum-contaminated soil.

To isolate bacteria responsible for the biodegradation of naphthalene, BTEX (benzene, toluene, ethylbenzene, and o-, m-, and p-xylene), and aliphatic hydrocarbons in petroleum-contaminated soil, three enrichment cultures were established using soil extract as the medium supplemented with naphthalene, BTEX, or n-hexadecane. Community analyses showed that Paraburkholderia species were predominant in naphthalene and BTEX, but relatively minor in n-hexadecane. Paraburkholderia aromaticivorans BN5 was able to degrade naphthalene and all BTEX compounds, but not n-hexadecane. The genome of strain BN5 harbors genes encoding 29 monooxygenases including two alkane 1-monooxygenases and 54 dioxygenases, indicating that strain BN5 has versatile metabolic capabilities, for diverse organic compounds: the ability of strain BN5 to degrade short chain aliphatic hydrocarbons was verified experimentally. The biodegradation pathways of naphthalene and BTEX compounds were bioinformatically predicted and verified experimentally through the analysis of their metabolic intermediates. Some genomic features including the encoding of the biodegradation genes on a plasmid and the low sequence homologies of biodegradation-related genes suggest that biodegradation potentials of strain BN5 may have been acquired via horizontal gene transfers and/or gene duplication, resulting in enhanced ecological fitness by enabling strain BN5 to degrade all compounds including naphthalene, BTEX, and short aliphatic hydrocarbons in contaminated soil.

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April 21, 2020

Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph “Candidatus Methylacidiphilum kamchatkense” strain Kam1 and comparison with its closest relatives.

The candidate genus “Methylacidiphilum” comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the “Ca. Methylacidiphilum” and the sister genus “Ca. Methylacidimicrobium” represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms.Here we present the closed genome of “Ca. Methylacidiphilum kamchatkense” strain Kam1 and a comparison with the genomes of its two closest relatives “Ca. Methylacidiphilum fumariolicum” strain SolV and “Ca. Methylacidiphilum infernorum” strain V4. The genome consists of a single 2,2 Mbp chromosome with 2119 predicted protein coding sequences. Genome analysis showed that the majority of the genes connected with metabolic traits described for one member of “Ca. Methylacidiphilum” is conserved between all three genomes. All three strains encode class I CRISPR-cas systems. The average nucleotide identity between “Ca. M. kamchatkense” strain Kam1 and strains SolV and V4 is =95% showing that they should be regarded as separate species. Whole genome comparison revealed a high degree of synteny between the genomes of strains Kam1 and SolV. In contrast, comparison of the genomes of strains Kam1 and V4 revealed a number of rearrangements. There are large differences in the numbers of transposable elements found in the genomes of the three strains with 12, 37 and 80 transposable elements in the genomes of strains Kam1, V4 and SolV respectively. Genomic rearrangements and the activity of transposable elements explain much of the genomic differences between strains. For example, a type 1h uptake hydrogenase is conserved between strains Kam1 and SolV but seems to have been lost from strain V4 due to genomic rearrangements.Comparing three closed genomes of “Ca. Methylacidiphilum” spp. has given new insights into the evolution of these organisms and revealed large differences in numbers of transposable elements between strains, the activity of these explains much of the genomic differences between strains.

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April 21, 2020

Denitrifying Bacteria Active in Woodchip Bioreactors at Low-Temperature Conditions.

Woodchip bioreactor technology removes nitrate from agricultural subsurface drainage by using denitrifying microorganisms. Although woodchip bioreactors have demonstrated success in many field locations, low water temperature can significantly limit bioreactor efficiency and performance. To improve bioreactor performance, it is important to identify the microbes responsible for nitrate removal at low temperature conditions. Therefore, in this study, we identified and characterized denitrifiers active at low-temperature conditions by using culture-independent and -dependent approaches. By comparative 16S rRNA (gene) analysis and culture isolation technique, Pseudomonas spp., Polaromonas spp., and Cellulomonas spp. were identified as being important bacteria responsible for denitrification in woodchip bioreactor microcosms at relatively low temperature conditions (15°C). Genome analysis of Cellulomonas sp. strain WB94 confirmed the presence of nitrite reductase gene nirK. Transcription levels of this nirK were significantly higher in the denitrifying microcosms than in the non-denitrifying microcosms. Strain WB94 was also capable of degrading cellulose and other complex polysaccharides. Taken together, our results suggest that Cellulomonas sp. denitrifiers could degrade woodchips to provide carbon source and electron donors to themselves and other denitrifiers in woodchip bioreactors at low-temperature conditions. By inoculating these denitrifiers (i.e., bioaugmentation), it might be possible to increase the nitrate removal rate of woodchip bioreactors at low-temperature conditions.

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April 21, 2020

Hybrid sequencing of the Gynostemma pentaphyllum transcriptome provides new insights into gypenoside biosynthesis.

Gypenosides are a group of triterpene saponins from Gynostemma pentaphyllum that are the same as or very similar to ginsenosides from the Panax species. Several enzymes involved in ginsenoside biosynthesis have been characterized, which provide important clues for elucidating the gypenoside biosynthetic pathway. We suppose that gypenosides and ginsenosides may have a similar biosynthetic mechanism and that the corresponding enzymes in the two pathways may have considerable similarity in their sequences. To further understand gypenoside biosynthesis, we sequenced the G. pentaphyllum transcriptome with a hybrid sequencing-based strategy and then determined the candidate genes involved in this pathway using phylogenetic tree construction and gene expression analysis.Following the PacBio standard analysis pipeline, 66,046 polished consensus sequences were obtained, while Illumina data were assembled into 140,601 unigenes with Trinity software. Then, these output sequences from the two analytical routes were merged. After removing redundant data with CD-HIT software, a total of 140,157 final unigenes were obtained. After functional annotation, five 2,3-oxidosqualene cyclase genes, 145 cytochrome P450 genes and 254 UDP-glycosyltransferase genes were selected for the screening of genes involved in gypenoside biosynthesis. Using phylogenetic analysis, several genes were divided into the same subfamilies or closely related evolutionary branches with characterized enzymes involved in ginsenoside biosynthesis. Using real-time PCR technology, their expression patterns were investigated in different tissues and at different times after methyl jasmonate induction. Since the genes in the same biosynthetic pathway are generally coexpressed, we speculated that GpOSC1, GpCYP89, and GpUGT35 were the leading candidates for gypenoside biosynthesis. In addition, six GpWRKYs and one GpbHLH might play a possible role in regulating gypenoside biosynthesis.We developed a hybrid sequencing strategy to obtain longer length transcriptomes with increased accuracy, which will greatly contribute to downstream gene screening and characterization, thus improving our ability to elucidate secondary metabolite biosynthetic pathways. With this strategy, we found several candidate genes that may be involved in gypenoside biosynthesis, which laid an important foundation for the elucidation of this biosynthetic pathway, thus greatly contributing to further research in metabolic regulation, synthetic biology and molecular breeding in this species.

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April 21, 2020

Adaptations of Alteromonas sp. 76-1 to Polysaccharide Degradation: A CAZyme Plasmid for Ulvan Degradation and Two Alginolytic Systems.

Studying the physiology and genomics of cultured hydrolytic bacteria is a valuable approach to decipher the biogeochemical cycling of marine polysaccharides, major nutrients derived from phytoplankton and macroalgae. We herein describe the profound potential of Alteromonas sp. 76-1, isolated from alginate-enriched seawater at the Patagonian continental shelf, to degrade the algal polysaccharides alginate and ulvan. Phylogenetic analyses indicated that strain 76-1 might represent a novel species, distinguished from its closest relative (Alteromonas naphthalenivorans) by adaptations to their contrasting habitats (productive open ocean vs. coastal sediments). Ecological distinction of 76-1 was particularly manifested in the abundance of carbohydrate-active enzymes (CAZymes), consistent with its isolation from alginate-enriched seawater and elevated abundance of a related OTU in the original microcosm. Strain 76-1 encodes multiple alginate lyases from families PL6, PL7, PL17, and PL18 largely contained in two polysaccharide utilization loci (PUL), which may facilitate the utilization of different alginate structures in nature. Notably, ulvan degradation relates to a 126 Kb plasmid dedicated to polysaccharide utilization, encoding several PL24 and PL25 ulvan lyases and monomer-processing genes. This extensive and versatile CAZyme repertoire allowed substantial growth on polysaccharides, showing comparable doubling times with alginate (2 h) and ulvan (3 h) in relation to glucose (3 h). The finding of homologous ulvanolytic systems in distantly related Alteromonas spp. suggests CAZyme plasmids as effective vehicles for PUL transfer that mediate niche gain. Overall, the demonstrated CAZyme repertoire substantiates the role of Alteromonas in marine polysaccharide degradation and how PUL exchange influences the ecophysiology of this ubiquitous marine taxon.

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April 21, 2020

Closing the Yield Gap for Cannabis: A Meta-Analysis of Factors Determining Cannabis Yield.

Until recently, the commercial production of Cannabis sativa was restricted to varieties that yielded high-quality fiber while producing low levels of the psychoactive cannabinoid tetrahydrocannabinol (THC). In the last few years, a number of jurisdictions have legalized the production of medical and/or recreational cannabis with higher levels of THC, and other jurisdictions seem poised to follow suit. Consequently, demand for industrial-scale production of high yield cannabis with consistent cannabinoid profiles is expected to increase. In this paper we highlight that currently, projected annual production of cannabis is based largely on facility size, not yield per square meter. This meta-analysis of cannabis yields reported in scientific literature aimed to identify the main factors contributing to cannabis yield per plant, per square meter, and per W of lighting electricity. In line with previous research we found that variety, plant density, light intensity and fertilization influence cannabis yield and cannabinoid content; we also identified pot size, light type and duration of the flowering period as predictors of yield and THC accumulation. We provide insight into the critical role of light intensity, quality, and photoperiod in determining cannabis yields, with particular focus on the potential for light-emitting diodes (LEDs) to improve growth and reduce energy requirements. We propose that the vast amount of genomics data currently available for cannabis can be used to better understand the effect of genotype on yield. Finally, we describe diversification that is likely to emerge in cannabis growing systems and examine the potential role of plant-growth promoting rhizobacteria (PGPR) for growth promotion, regulation of cannabinoid biosynthesis, and biocontrol.

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April 21, 2020

A survey of transcriptome complexity using PacBio single-molecule real-time analysis combined with Illumina RNA sequencing for a better understanding of ricinoleic acid biosynthesis in Ricinus communis.

Ricinus communis is a highly economically valuable oil crop plant from the spurge family, Euphorbiaceae. However, the available reference genomes are incomplete and to date studies on ricinoleic acid biosynthesis at the transcriptional level are limited.In this study, we combined PacBio single-molecule long read isoform and Illumina RNA sequencing to identify the alternative splicing (AS) events, novel isoforms, fusion genes, long non-coding RNAs (lncRNAs) and alternative polyadenylation (APA) sites to unveil the transcriptomic complexity of castor beans and identify critical genes related to ricinoleic acid biosynthesis. Here, we identified 11,285 AS-variants distributed in 21,448 novel genes and detected 520 fusion genes, 320 lncRNAs and 9511 (APA-sites). Furthermore, a total of 6067, 5983 and 4058 differentially expressed genes between developing beans of the R. communis lines 349 and 1115 with extremely different oil content were identified at 7, 14 and 21?days after flowering, respectively. Specifically, 14, 18 and 11 DEGs were annotated encoding key enzymes related to ricinoleic acid biosynthesis reflecting the higher castor oil content of 1115 compared than 349. Quantitative real-time RT-PCR further validated fifteen of these DEGs at three-time points.Our results significantly improved the existed gene models of R. communis, and a putative model of key genes was built to show the differences between strains 349 and 1115, illustrating the molecular mechanism of castor oil biosynthesis. A multi-transcriptome database and candidate genes were provided to further improve the level of ricinoleic acid in transgenic crops.

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April 21, 2020

Complete genome sequence of the Sulfodiicoccus acidiphilus strain HS-1T, the first crenarchaeon that lacks polB3, isolated from an acidic hot spring in Ohwaku-dani, Hakone, Japan.

Sulfodiicoccus acidiphilus HS-1T is the type species of the genus Sulfodiicoccus, a thermoacidophilic archaeon belonging to the order Sulfolobales (class Thermoprotei; phylum Crenarchaeota). While S. acidiphilus HS-1T shares many common physiological and phenotypic features with other Sulfolobales species, the similarities in their 16S rRNA gene sequences are less than 89%. In order to know the genomic features of S. acidiphilus HS-1T in the order Sulfolobales, we determined and characterized the genome of this strain.The circular genome of S. acidiphilus HS-1T is comprised of 2353,189 bp with a G+C content of 51.15 mol%. A total of 2459 genes were predicted, including 2411 protein coding and 48 RNA genes. The notable genomic features of S. acidiphilus HS-1T in Sulfolobales species are the absence of genes for polB3 and the autotrophic carbon fixation pathway, and the distribution pattern of essential genes and sequences related to genomic replication initiation. These insights contribute to an understanding of archaeal genomic diversity and evolution.

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April 21, 2020

Whole Genome Analysis of Lactobacillus plantarum Strains Isolated From Kimchi and Determination of Probiotic Properties to Treat Mucosal Infections by Candida albicans and Gardnerella vaginalis.

Three Lactobacillus plantarum strains ATG-K2, ATG-K6, and ATG-K8 were isolated from Kimchi, a Korean traditional fermented food, and their probiotic potentials were examined. All three strains were free of antibiotic resistance, hemolysis, and biogenic amine production and therefore assumed to be safe, as supported by whole genome analyses. These strains demonstrated several basic probiotic functions including a wide range of antibacterial activity, bile salt hydrolase activity, hydrogen peroxide production, and heat resistance at 70°C for 60 s. Further studies of antimicrobial activities against Candida albicans and Gardnerella vaginalis revealed growth inhibitory effects from culture supernatants, coaggregation effects, and killing effects of the three probiotic strains, with better efficacy toward C. albicans. In vitro treatment of bacterial lysates of the probiotic strains to the RAW264.7 murine macrophage cell line resulted in innate immunity enhancement via IL-6 and TNF-a production without lipopolysaccharide (LPS) treatment and anti-inflammatory effects via significantly increased production of IL-10 when co-treated with LPS. However, the degree of probiotic effect was different for each strain as the highest TNF-a and the lowest IL-10 production by the RAW264.7 cell were observed in the K8 lysate treated group compared to the K2 and K6 lysate treated groups, which may be related to genomic differences such as chromosome size (K2: 3,034,884 bp, K6: 3,205,672 bp, K8: 3,221,272 bp), plasmid numbers (K2: 3, K6 and K8: 1), or total gene numbers (K2: 3,114, K6: 3,178, K8: 3,186). Although more correlative inspections to connect genomic information and biological functions are needed, genomic analyses of the three strains revealed distinct genomic compositions of each strain. Also, this finding suggests genome level analysis may be required to accurately identify microorganisms. Nevertheless, L. plantarum ATG-K2, ATG-K6, and ATG-K8 demonstrated their potential as probiotics for mucosal health improvement in both microbial and immunological contexts.

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April 21, 2020

Pandoravirus Celtis Illustrates the Microevolution Processes at Work in the Giant Pandoraviridae Genomes.

With genomes of up to 2.7 Mb propagated in µm-long oblong particles and initially predicted to encode more than 2000 proteins, members of the Pandoraviridae family display the most extreme features of the known viral world. The mere existence of such giant viruses raises fundamental questions about their origin and the processes governing their evolution. A previous analysis of six newly available isolates, independently confirmed by a study including three others, established that the Pandoraviridae pan-genome is open, meaning that each new strain exhibits protein-coding genes not previously identified in other family members. With an average increment of about 60 proteins, the gene repertoire shows no sign of reaching a limit and remains largely coding for proteins without recognizable homologs in other viruses or cells (ORFans). To explain these results, we proposed that most new protein-coding genes were created de novo, from pre-existing non-coding regions of the G+C rich pandoravirus genomes. The comparison of the gene content of a new isolate, pandoravirus celtis, closely related (96% identical genome) to the previously described p. quercus is now used to test this hypothesis by studying genomic changes in a microevolution range. Our results confirm that the differences between these two similar gene contents mostly consist of protein-coding genes without known homologs, with statistical signatures close to that of intergenic regions. These newborn proteins are under slight negative selection, perhaps to maintain stable folds and prevent protein aggregation pending the eventual emergence of fitness-increasing functions. Our study also unraveled several insertion events mediated by a transposase of the hAT family, 3 copies of which are found in p. celtis and are presumably active. Members of the Pandoraviridae are presently the first viruses known to encode this type of transposase.

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