We have sequenced, annotated, and analyzed the genome of Ca. Pandoraea novymonadis, a recently described bacterial endosymbiont of the trypanosomatid Novymonas esmeraldas. When compared with genomes of its free-living relatives, it has all the hallmarks of the endosymbionts' genomes, such as significantly reduced size, extensive gene loss, low GC content, numerous gene rearrangements, and low codon usage bias. In addition, Ca. P. novymonadis lacks mobile elements, has a strikingly low number of pseudogenes, and almost all genes are single copied. This suggests that it already passed the intensive period of host adaptation, which still can be observed in the genome of Polynucleobacter necessarius, a certainly recent endosymbiont. Phylogenetically, Ca. P. novymonadis is more related to P. necessarius, an intracytoplasmic bacterium of free-living ciliates, than to Ca. Kinetoplastibacterium spp., the only other known endosymbionts of trypanosomatid flagellates. As judged by the extent of the overall genome reduction and the loss of particular metabolic abilities correlating with the increasing dependence of the symbiont on its host, Ca. P. novymonadis occupies an intermediate position P. necessarius and Ca. Kinetoplastibacterium spp. We conclude that the relationships between Ca. P. novymonadis and N. esmeraldas are well-established, although not as fine-tuned as in the case of Strigomonadinae and their endosymbionts.

• Klíčová slova
• Pandoraea, Trypanosomatidae, bacterial endosymbiont, metabolism, phylogenomics,
• Publikační typ
• časopisecké články MeSH

Five isolates, designated TA2, TA4, TA25(T), KOx(T) and NS15(T) were isolated in previous studies by enrichment in mineral medium with potassium oxalate as the sole carbon source and were characterized using a polyphasic approach. The isolates were Gram-reaction-negative, aerobic, non-spore-forming rods. Phylogenetic analyses based on 16S rRNA and DNA gyrase B subunit (gyrB) gene sequences confirmed that the isolates belonged to the genus Pandoraea and were most closely related to Pandoraea sputorum and Pandoraea pnomenusa (97.2-99.7 % 16S rRNA gene sequence similarity). The isolates could be differentiated from their closest relatives on the basis of several phenotypic characteristics. The major cellular fatty acid profiles of the isolates comprised C₁₆:₀, C₁₈:₁ω7c, C₁₇:₀ cyclo and summed feature 3 (C₁₆:₁ω7c and/or iso-C₁₅:₀ 2-OH). On the basis of DNA-DNA hybridization studies and phylogenetic analyses, the isolates represent three novel species within the genus Pandoraea, for which the names Pandoraea oxalativorans sp. nov. (TA25(T) = NBRC 106091(T) = CCM 7677(T) = DSM 23570(T)), Pandoraea faecigallinarum sp. nov. (KOx(T) = NBRC 106092(T) = CCM 2766(T) = DSM 23572(T)) and Pandoraea vervacti sp. nov. (NS15(T) = NBRC 106088(T) = CCM 7667(T) = DSM 23571(T)) are proposed.

• MeSH
• aerobióza MeSH
• Burkholderiaceae klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• DNA bakterií chemie   genetika   MeSH
• DNA gyráza genetika   MeSH
• fylogeneze MeSH
• hybridizace nukleových kyselin MeSH
• kultivační média chemie   MeSH
• mastné kyseliny analýza   MeSH
• molekulární sekvence - údaje MeSH
• oxaláty metabolismus   MeSH
• ribozomální DNA chemie   genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• shluková analýza MeSH
• techniky typizace bakterií MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• DNA gyráza MeSH
• kultivační média MeSH
• mastné kyseliny MeSH
• oxaláty MeSH
• ribozomální DNA MeSH
• RNA ribozomální 16S MeSH

The closest relative of human pathogen Leishmania, the trypanosomatid Novymonas esmeraldas, harbors a bacterial endosymbiont "Candidatus Pandoraea novymonadis." Based on genomic data, we performed a detailed characterization of the metabolic interactions of both partners. While in many respects the metabolism of N. esmeraldas resembles that of other Leishmaniinae, the endosymbiont provides the trypanosomatid with heme, essential amino acids, purines, some coenzymes, and vitamins. In return, N. esmeraldas shares with the bacterium several nonessential amino acids and phospholipids. Moreover, it complements its carbohydrate metabolism and urea cycle with enzymes missing from the "Ca. Pandoraea novymonadis" genome. The removal of the endosymbiont from N. esmeraldas results in a significant reduction of the overall translation rate, reduced expression of genes involved in lipid metabolism and mitochondrial respiratory activity, and downregulation of several aminoacyl-tRNA synthetases, enzymes involved in the synthesis of some amino acids, as well as proteins associated with autophagy. At the same time, the genes responsible for protection against reactive oxygen species and DNA repair become significantly upregulated in the aposymbiotic strain of this trypanosomatid. By knocking out a component of its flagellum, we turned N. esmeraldas into a new model trypanosomatid that is amenable to genetic manipulation using both conventional and CRISPR-Cas9-mediated approaches. IMPORTANCENovymonas esmeraldas is a parasitic flagellate of the family Trypanosomatidae representing the closest insect-restricted relative of the human pathogen Leishmania. It bears symbiotic bacteria in its cytoplasm, the relationship with which has been established relatively recently and independently from other known endosymbioses in protists. Here, using the genome analysis and comparison of transcriptomic profiles of N. esmeraldas with and without the endosymbionts, we describe a uniquely complex cooperation between both partners on the biochemical level. We demonstrate that the removal of bacteria leads to a decelerated growth of N. esmeraldas, substantial suppression of many metabolic pathways, and increased oxidative stress. Our success with the genetic transformation of this flagellate makes it a new model trypanosomatid species that can be used for the dissection of mechanisms underlying the symbiotic relationships between protists and bacteria.

• Klíčová slova
• Leishmaniinae, Trypanosomatidae, bacterial endosymbiont, genomics, metabolism,
• MeSH
• Bacteria klasifikace   genetika   metabolismus   MeSH
• fylogeneze MeSH
• genom bakteriální * MeSH
• genomika MeSH
• symbióza genetika   MeSH
• Trypanosoma klasifikace   metabolismus   mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The stability of endosymbiotic associations between eukaryotes and bacteria depends on a reliable mechanism ensuring vertical inheritance of the latter. Here, we demonstrate that a host-encoded protein, located at the interface between the endoplasmic reticulum of the trypanosomatid Novymonas esmeraldas and its endosymbiotic bacterium Ca. Pandoraea novymonadis, regulates such a process. This protein, named TMP18e, is a product of duplication and neo-functionalization of the ubiquitous transmembrane protein 18 (TMEM18). Its expression level is increased at the proliferative stage of the host life cycle correlating with the confinement of bacteria to the nuclear vicinity. This is important for the proper segregation of bacteria into the daughter host cells as evidenced from the TMP18e ablation, which disrupts the nucleus-endosymbiont association and leads to greater variability of bacterial cell numbers, including an elevated proportion of aposymbiotic cells. Thus, we conclude that TMP18e is necessary for the reliable vertical inheritance of endosymbionts.

• Klíčová slova
• Leishmaniinae, Pandoraea, TMEM18, Trypanosomatidae, endosymbiosis,
• MeSH
• Bacteria MeSH
• Eukaryota MeSH
• symbióza fyziologie   MeSH
• Trypanosomatina * mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

High soil pH and excess CaCO3 are major contributors to calcareous soil limitations on crops' access to essential nutrients, especially phosphorus (P) and micronutrients, which in turn impact pulses yields and growth. The purpose of this study was to determine the effect of bio sulfur granules (BSG) on the growth of black gram and the availability of nutrients in calcareous vertisols deficient in sulfur. BSG was developed by using sulfur-oxidizing bacteria (SOB) and elemental sulfur (ES) through an incubation study. Developed BSG was tested in a pot and field conditions to evaluate their effectiveness on black gram growth and yield. In the incubation study, soil treated with Methylobacterium thiocyanatum VRI7-A4 and ES (40 kg S/ha) significantly decreased pH and increased available S (SO42-) in calcareous soils. After 40 days of incubation, the solubility of P, Fe, and Zn was greatly increased by the addition of ES @ 40 kg S/ ha in combination with M. thiocyanatum VRI7-A4 or Pandoraea thiooxydans ATSB16. Black gram in S-deficient calcareous soil was improved by the application of BSG (ES @ 40 kg S/ ha with M. thiocyanatum VRI7-A7) in terms of root and shoot lengths, nodule number, plant biomass, pod yield, and biological yield as compared to control. The same treatment greatly increased plant nutrient intake as well as the concentrations of P, Fe, and Zn in the soil. The results showed that the addition of BSG granules (ES @ 40 kg S/ha + M. thiocyanatum VRI7-A4) to calcareous vertisol deficient in S enhanced the nutrient solubility through S oxidation. The developed bio sulfur granules may be added to the fertilizer schedule of the pulses growers to get improved crop growth and yield of black gram in calcareous soil.

• Klíčová slova
• Methylobacterium thiocyanatum, Pandoraea thiooxydans, Bio sulfur granules, Black gram, Calcareous soil, Elemental sulfur, Nutrient availability,
• Publikační typ
• časopisecké články MeSH

Parasitic trypanosomatids diverged from free-living kinetoplastid ancestors several hundred million years ago. These parasites are relatively well known, due in part to several unusual cell biological and molecular traits and in part to the significance of a few - pathogenic Leishmania and Trypanosoma species - as aetiological agents of serious neglected tropical diseases. However, the majority of trypanosomatid biodiversity is represented by osmotrophic monoxenous parasites of insects. In two lineages, novymonads and strigomonads, osmotrophic lifestyles are supported by cytoplasmic endosymbionts, providing hosts with macromolecular precursors and vitamins. Here we discuss the two independent origins of endosymbiosis within trypanosomatids and subsequently different evolutionary trajectories that see entrainment vs tolerance of symbiont cell divisions cycles within those of the host. With the potential to inform on the transition to obligate parasitism in the trypanosomatids, interest in the biology and ecology of free-living, phagotrophic kinetoplastids is beginning to enjoy a renaissance. Thus, we take the opportunity to additionally consider the wider relevance of endosymbiosis during kinetoplastid evolution, including the indulged lifestyle and reductive evolution of basal kinetoplastid Perkinsela.

• Klíčová slova
• Angomonas deanei, Candidatus Kinetoplastibacterium, Kentomonas, Novymonas esmeraldas, Pandoraea, cytostome,
• MeSH
• biodiverzita MeSH
• biologická evoluce * MeSH
• genom protozoální MeSH
• Kinetoplastida genetika   MeSH
• Leishmania genetika   fyziologie   MeSH
• molekulární evoluce MeSH
• symbióza * MeSH
• Trypanosoma genetika   fyziologie   MeSH
• Trypanosomatina genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

UNLABELLED: We describe a novel symbiotic association between a kinetoplastid protist, Novymonas esmeraldas gen. nov., sp. nov., and an intracytoplasmic bacterium, "Candidatus Pandoraea novymonadis" sp. nov., discovered as a result of a broad-scale survey of insect trypanosomatid biodiversity in Ecuador. We characterize this association by describing the morphology of both organisms, as well as their interactions, and by establishing their phylogenetic affinities. Importantly, neither partner is closely related to other known organisms previously implicated in eukaryote-bacterial symbiosis. This symbiotic association seems to be relatively recent, as the host does not exert a stringent control over the number of bacteria harbored in its cytoplasm. We argue that this unique relationship may represent a suitable model for studying the initial stages of establishment of endosymbiosis between a single-cellular eukaryote and a prokaryote. Based on phylogenetic analyses, Novymonas could be considered a proxy for the insect-only ancestor of the dixenous genus Leishmania and shed light on the origin of the two-host life cycle within the subfamily Leishmaniinae. IMPORTANCE: The parasitic trypanosomatid protist Novymonas esmeraldas gen. nov., sp. nov. entered into endosymbiosis with the bacterium "Ca. Pandoraea novymonadis" sp. nov. This novel and rather unstable interaction shows several signs of relatively recent establishment, qualifying it as a potentially unique transient stage in the increasingly complex range of eukaryotic-prokaryotic relationships.

• MeSH
• Burkholderiaceae klasifikace   cytologie   izolace a purifikace   fyziologie   MeSH
• fylogeneze MeSH
• symbióza * MeSH
• Trypanosomatina klasifikace   cytologie   genetika   mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Ekvádor MeSH

Using the established commercial system Sherlock (MIDI, Inc.), cellular fatty acid methyl ester analysis for differentiation among Burkholderia cepacia complex species was proven. The identification key based on the diagnostic fatty acids is able to discern phenotypically related Ralstonia pickettii and Pandoraea spp. and further distinguish Burkholderia pyrrocinia, Burkholderia ambifaria, and Burkholderia vietnamiensis.

• MeSH
• Burkholderia cepacia komplex chemie   klasifikace   MeSH
• druhová specificita MeSH
• fenotyp MeSH
• infekce bakteriemi rodu Burkholderia mikrobiologie   MeSH
• lidé MeSH
• mastné kyseliny analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• mastné kyseliny MeSH

Bacteria were identified associated with biodegradation of aromatic pollutants biphenyl, benzoate, and naphthalene in a long-term polychlorinated biphenyl- and polyaromatic hydrocarbon-contaminated soil. In order to avoid biases of culture-based approaches, stable isotope probing was applied in combination with sequence analysis of 16 S rRNA gene pyrotags amplified from (13)C-enriched DNA fractions. Special attention was paid to pyrosequencing data analysis in order to eliminate the errors caused by either generation of amplicons (random errors caused by DNA polymerase, formation of chimeric sequences) or sequencing itself. Therefore, sample DNA was amplified, sequenced, and analyzed along with the DNA of a mock community constructed out of 8 bacterial strains. This warranted that appropriate tools and parameters were chosen for sequence data processing. (13)C-labeled metagenomes isolated after the incubation of soil samples with all three studied aromatics were largely dominated by Proteobacteria, namely sequences clustering with the genera Rhodanobacter Burkholderia, Pandoraea, Dyella as well as some Rudaea- and Skermanella-related ones. Pseudomonads were mostly labeled by (13)C from naphthalene and benzoate. The results of this study show that many biphenyl/benzoate-assimilating bacteria derive carbon also from naphthalene, pointing out broader biodegradation abilities of some soil microbiota. The results also demonstrate that, in addition to traditionally isolated genera of degradative bacteria, yet-to-be cultured bacteria are important players in bioremediation. Overall, the study contributes to our understanding of biodegradation processes in contaminated soil. At the same time our results show the importance of sequencing and analyzing a mock community in order to more correctly process and analyze sequence data.

• MeSH
• Bacteria genetika   izolace a purifikace   metabolismus   MeSH
• benzoáty metabolismus   MeSH
• bifenylové sloučeniny metabolismus   MeSH
• biodegradace MeSH
• DNA bakterií metabolismus   MeSH
• fylogeneze MeSH
• izotopové značení MeSH
• izotopy uhlíku MeSH
• látky znečišťující půdu analýza   MeSH
• naftaleny metabolismus   MeSH
• půdní mikrobiologie * MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzoáty MeSH
• bifenylové sloučeniny MeSH
• biphenyl MeSH Prohlížeč
• DNA bakterií MeSH
• izotopy uhlíku MeSH
• látky znečišťující půdu MeSH
• naftaleny MeSH
• naphthalene MeSH Prohlížeč