Publications

4748

In Australia, Stylosanthes little leaf (StLL) phytoplasma has been detected in Stylosanthes scabra Vogel, Arachis pintoi Krapov, Saccharum officinarum L., Carica papaya L., Medicago sativa L., and Solanum tuberosum L. The 16S rRNA gene sequence of StLL phytoplasma strains from S. scabra, C. papaya, S. officinarum and S. tuberosum were compared and share 99.93–100 % nucleotide sequence identity. Phylogenetic comparisons between the 16S rRNA genes of StLL phytoplasma and other ‘Candidatus Phytoplasma’ species indicate that StLL represents a distinct phytoplasma lineage. It shares its most recent known ancestry with ‘Ca. Phytoplasma luffae’ (16SrVIII-A), with which it has 97.17–97.25 % nucleotide identity. In silico RFLP analysis of the 16S rRNA amplicon using iPhyClassifier indicate that StLL phytoplasmas have a unique pattern (similarity coefficient below 0.85) that is most similar to that of ‘Ca. Phytoplasma luffae’. The unique in silico RFLP patterns were confirmed in vitro. Nucleotide sequences of genes that are more variable than the 16S rRNA gene, namely tuf (tu-elongation factor), secA (partial translocation gene), and the partial ribosomal protein (rp) gene operon (rps19-rpl22-rps3), produced phylogenetic trees with similar branching patterns to the 16S rRNA gene tree. Sequence comparisons between the StLL 16S rRNA spacer region confirmed previous reports of rrn interoperon sequence heterogeneity for StLL, where the spacer region of rrnB encodes a complete tRNA-Isoleucine gene and the rrnA spacer region does not. Together these results suggest that the Australian phytoplasma, StLL, is unique according to the International Organization for Mycoplasmology (IRPCM) recommendations. The novel taxon ‘Ca. Phytoplasma stylosanthis’ is proposed, with the most recent strain from a potato crop in Victoria, Australia, serving as the reference strain (deposited in the Victorian Plant Pathology Herbarium as VPRI 43683).

Microbiology is in a new age in which sequence databases are primary sources of information about many microbes. However, in-depth analysis of environmental genomes thus retrieved is essential to substantiate the new knowledge.

Significance The marine sedimentary subsurface is a vast and inhospitable ecosystem, often described as a place where microbes “race to their death,” as microbial cells are buried and available energy is severely diminished with increasing depth/age. By combining a variety of biogeochemical and molecular methods to describe the energetics and genetics of the bacteria specialized in anaerobic ammonium oxidation, we show that despite prolonged exposure to highly unfavorable conditions for tens of thousands of years, these bacteria exhibit remarkable net population growth when reaching their niche: the nitrate–ammonium transition zone. This common, yet understudied, geochemical transition zone represents an oasis in the sedimentary energetic desert, and the growth it supports is of major importance for the global nitrogen cycle.

Recent genome-resolved metagenomic analyses of microbial communities from diverse environments have led to the discovery of many novel lineages that significantly expand the phylogenetic breadth of Archaea. Here, we report the genomic characterization of a new archaeal family based on five metagenome-assembled genomes retrieved from acid mine drainage sediments. Phylogenomic analyses placed these uncultivated archaea at the root of the candidate phylum Parvarchaeota, which expand this lesser-known phylum into two family levels. Genes involved in environmental adaptation and carbohydrate and protein utilization were identified in the ultra-small genomes (estimated size 0.53–0.76 Mb), indicating a survival strategy in this harsh environment (low pH and high heavy metal content). The detection of genes with homology to sulfocyanin suggested a potential involvement in iron cycling. Nevertheless, the absence of the ability to synthesize amino acids and nucleotides implies that these archaea may acquire these biomolecules from the environment or other community members. Applying evolutionary history analysis to Parvarchaeota suggested that members of the two families could broaden their niches by acquiring the potentials of utilizing different substrates. This study expands our knowledge of the diversity, metabolic capacity, and evolutionary history of the Parvarchaeota.

Aims: In this study, the best sampling time and tissues for phytoplasma detection in twenty pear trees (cv. Deveci) infected by ‘Candidatus Phytoplasma pyri’, causal agent of pear decline disease, in Bursa province of Turkey were investigated.Methods and Results: Sampling was done throughout the year in leaf midribs, shoot and root tissues, where as the flower tissues were tested once a year in March and fruit tissues in September. All samples were analyzed by nested-PCR using P1/P7 and fU5/rU3 universal primer pairs. Nested PCR products were digested with RsaI and SspI restriction enzymes. The results revealed that the detection rate of ‘Ca. P. pyri’ in different plant tissues was greatly depending on the sample collection period. The fruit tissues, which were only sampled in September due to the ripening time of Deveci pear cultivar in Bursa, showed the highest detection rate of ‘Ca. P. pyri’ (100%) followed by flower tissues (75%). The average detection rate in root, shoot tissues and leaf midribs was found as 43.75, 39.58 and 16.25%, respectively. The present results showed that the best plant tissues for detecting ‘Ca. P. pyri’ in pear trees were fruit columella and flowers. The highest detection rate of this phytoplasma in root tissues was found from November to March, whereas it could be detected whole year around except summer months in shoot samples in Turkey.Conclusions: For 'Candidatus Phytoplasma pyri', detection, if there is no seasonal limitation for testing, the most suitable tissues are fruits and flowers. When it comes to testing throughout the year, the most suitable tissues were determined as the root, the phloem and cambium layer of the shoots and the leaves, respectively.Significance and Impact of the Study: This study on seasonal variations of ‘Candidatus Phytoplasma pyri’ in different pear tissues has been first time investigated in Turkey. This preliminary data provides important knowledge on molecular detection of Ca. P. pyri, causal agent of pear decline disease for further studies and sertification-quarantine programmes of pear trees in Turkey.

Two metagenome-assembled genomes (MAGs), obtained from laboratory-scale enhanced biological phosphorus removal bioreactors, were analyzed. The values of 16S rRNA gene sequence identity, average nucleotide identity, and average amino acid identity indicated that these genomes, designated as RT and SSD2, represented two novel species within the genus Thiothrix, ‘Candidatus Thiothrix moscowensis’ and ‘Candidatus Thiothrix singaporensis’. A complete set of genes for the tricarboxylic acid cycle and electron transport chain indicates a respiratory type of metabolism. A notable feature of RT and SSD2, as well as other Thiothrix species, is the presence of a flavin adenine dinucleotide (FAD)-dependent malate:quinone oxidoreductase instead of nicotinamide adenine dinucleotide (NAD)-dependent malate dehydrogenase. Both MAGs contained genes for CO2 assimilation through the Calvin–Benson–Bassam cycle; sulfide oxidation (sqr, fccAB), sulfur oxidation (rDsr complex), direct (soeABC) and indirect (aprBA, sat) sulfite oxidation, and the branched Sox pathway (SoxAXBYZ) of thiosulfate oxidation to sulfur and sulfate. All these features indicate a chemoorganoheterotrophic, chemolithoautotrophic, and chemolithoheterotrophic lifestyle. Both MAGs comprise genes for nitrate reductase and NO-reductase, while SSD2 also contains genes for nitrite reductase. The presence of polyphosphate kinase and exopolyphosphatase suggests that RT and SSD2 could accumulate and degrade polyhosphates during the oxic-anoxic growth cycle in the bioreactors, such as typical phosphate-accumulating microorganisms.

Microbial communities with the ability to convert long-chain fatty acids (LCFA) coupled to sulfate reduction can be important in the removal of these compounds from wastewater. In this work, an enrichment culture, able to oxidize the long-chain fatty acid palmitate (C16:0) coupled to sulfate reduction, was obtained from anaerobic granular sludge. Microscopic analysis of this culture, designated HP culture, revealed that it was mainly composed of one morphotype with a typical collar-like cell wall invagination, a distinct morphological feature of theDesulfomonilegenus. 16S rRNA gene amplicon and metagenome-assembled genome (MAG) indeed confirmed that the abundant phylotype in HP culture belong toDesulfomonilegenus [ca.92% 16S rRNA gene sequences closely related toDesulfomonilespp.; andca. 82% whole genome shotgun (WGS)]. Based on similar cell morphology and average nucleotide identity (ANI) (77%) between theDesulfomonilesp. in HP culture and the type strainDesulfomonile tiedjeistrain DCB-1T, we propose a novel species designated as “CandidatusDesulfomonile palmitatoxidans.” This bacterium shares 94.3 and 93.6% 16S rRNA gene identity withDesulfomonile limimarisstrain DCB-MTandD. tiedjeistrain DCB-1T, respectively. Based on sequence abundance ofDesulfomonile-morphotype in HP culture, its predominance in the microscopic observations, and presence of several genes coding for enzymes involved in LCFA degradation, the proposed species “Ca.Desulfomonile palmitatoxidans” most probably plays an important role in palmitate degradation in HP culture. Analysis of the growth of HP culture andD. tiedjeistrain DCB-1Twith short- (butyrate), medium- (caprylate) and long-chain fatty acids (palmitate, stearate, and oleate) showed that both cultures degraded all fatty acids coupled to sulfate reduction, except oleate that was only utilized by HP culture. In the absence of sulfate, neither HP culture, norD. tiedjeistrain DCB-1Tdegraded palmitate when incubated withMethanobacterium formicicumas a possible methanogenic syntrophic partner. UnlikeD. tiedjeistrain DCB-1T, “Ca.Desulfomonile palmitatoxidans” lacks reductive dehalogenase genes in its genome, and HP culture was not able to grow by organohalide respiration. An emended description of the genusDesulfomonileis proposed. Our study reveals an unrecognized LCFA degradation feature of theDesulfomonilegenus.

AbstractA recent survey in Germany revealed the wide presence of ‘Candidatus Phytoplasma ulmi’ in native elm stands. Accessions were studied for their genetic variability and phylogenetic relationship based on the conserved groEL and the variable imp gene. While the groEL sequences revealed a high intraspecific homology of more than 99%, the homology of the imp gene dropped to 71% between distantly related sequences. Twenty-nine groEL and 74 imp genotypes were distinguished based on polymorphic sites. Phylogenetic analysis of the groEL gene clustered all ‘Ca. P. ulmi’ strains and separated them from related phytoplasmas of the 16SrV group. The inferred phylogeny of the imp gene resulted in a different tree topology and separated the ‘Ca. P. ulmi’ genotypes into two clusters, one closely related to the flavescence dorée phytoplasma strain FD-D (16SrV-D), the other affiliated with the flavescence dorée phytoplasma strains FD-C and FD70 and the alder yellows phytoplasma (16SrV-C). In both phylograms, ‘Ca. P. ulmi’ genotypes from Scots elm trees formed a coherent cluster, while genotypes from European white elms and field elms grouped less strictly. The regional distribution pattern was congruent for some of the groEL and imp genotypes, but a strict linkage for all genotypes was not apparent.

AbstractA key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we isolate a member of the ubiquitous, yet-to-be-cultivated phylum ‘Candidatus Atribacteria’ (also known as OP9) that has an intracytoplasmic membrane apparently surrounding the nucleoid. The isolate, RT761, is a subsurface-derived anaerobic bacterium that appears to have three lipid membrane-like layers, as shown by cryo-electron tomography. Our observations are consistent with a classical gram-negative structure with an additional intracytoplasmic membrane. However, further studies are needed to provide conclusive evidence for this unique intracellular structure. The RT761 genome encodes proteins with features that might be related to the complex cellular structure, including: N-terminal extensions in proteins involved in important processes (such as cell-division protein FtsZ); one of the highest percentages of transmembrane proteins among gram-negative bacteria; and predicted Sec-secreted proteins with unique signal peptides. Physiologically, RT761 primarily produces hydrogen for electron disposal during sugar degradation, and co-cultivation with a hydrogen-scavenging methanogen improves growth. We propose RT761 as a new species, Atribacter laminatus gen. nov. sp. nov. and a new phylum, Atribacterota phy. nov.