Publications

3734

Nitrobacterstrain NHB1 is a nitrite-oxidising bacterium previously co-enriched with the neutrophilic ammonia-oxidising bacteriumNitrosospiraAHB1, a consortium that nitrifies in acidic conditions in co-culture. Here we characterise the growth of the isolateNitrobacterstrain NHB1 as a function of pH and nitrite (NO2-) concentration, and its influence on the activity of acidophilic soil ammonia-oxidising archaea (AOA). NHB1 is acidotolerant and grows optimally at pH 6.0 (range 5.0 - 7.5) at initial NO2-concentrations of 500 μM. However, the optimum decreases to pH 5.0 at lower initial NO2-concentrations typically found in soil, with detectable growth down to pH 3.5. NHB1 has a comparatively high affinity for NO2-with an apparent-half-saturation constant (54 μM) one order of magnitude lower than its closest relative, the neutrophilic strainNitrobacter hamburgensisX14. In co-culture, NHB1 enhances the growth of acidophilic AOA. Specifically,Nitrosotalea devaniterraeNd1 andNitrosotalea sinensisNd2 are sensitive to NO2--derived compounds and only oxidise ~200-300 μM ammonia (NH3) in batch cultures. However, in co-culture with NHB1, pH ranges were lowered by ~0.5 pH units and both strains could oxidise up to 3 mM NH3, only limited by buffering capacity. NHB1 possesses a cyanase facilitating reciprocal cross-feeding via generating cyanate-derived NH3and utilising AOA-derived NO2-. Removal of NO2-is likely crucial for the growth of nitrifiers in acidic soils and this study highlights the importance of considering substrate and metabolic product concentrations when characterising physiology. Genome analysis reveals NHB1 is distinct from validated species and the name "Nitrobacter laanbroekii" is proposed.

Onion (Allium cepa L.) is the most produced vegetable after tomato worldwide and is grown on about 15,000 ha in Germany. In Lampertheim, Hesse in southwest Germany (49°40'02.3"N, 8°26'00.0"E) bulbs of the cultivar ‘Red Baron F1’ were harvested in September 2023 in an apparently healthy state. Four months later some of the onions showed rotting symptoms, which could not be assigned to a known storage disease. At first, the bulbs became glassy, later they showed soft rot. They originated from a field located in a growing region severely affected by “Syndrome Basses Richesses” (SBR). ‘Candidatus Arsenophonus phytopathogenicus’ as well as ‘Candidatus Phytoplasma solani’ are associated with this disease in sugar beet (Gatineau et al. 2002). Moreover, ‘Ca. A. phytopathogenicus’ was recently reported in association of bacterial wilt and yellowing in potato (Behrmann et al. 2023). Both phloem-restricted bacteria are vectored by the polyphagous planthopper Pentastiridius leporinus (Therhaag et al. 2024), which is highly abundant in this region. To examine, if the unknown symptoms in onion might be related to the presence of these pathogens, DNA of 69 bulbs showing a different degree of softening were analyzed. The samples were tested for the presence of ‘Ca. Phytoplasma solani’ in a TaqMan assay (Behrmann et al. 2022). All showed negative results. To demonstrate the presence of ‘Ca. A. phytopathogenicus’, universal and genus-specific primers for the amplification of 16S rDNA and a real-time qPCR assay amplifying an hsp20 fragment were employed (Christensen et al. 2004, Zübert and Kube 2021). Two bulbs of the five positive samples were in an apparently healthy state, the other three showed light to moderate softening symptoms. The 16S rDNA fragments from two samples were sequenced on both strands and aligned. Both fragments were homologous. One fragment of 1474 bp fragment showing 100% homology to the 16S rDNA from SBR (accession no. AY057392) was submitted to GenBank (accession no. PP400342). Other taxa of ‘Ca. Arsenophonus’ showed 16S rDNA homologies of less than 99.3 %. To corroborate the finding onion samples were subjected to PCR reactions employing genus-specific primers for the conserved tufB, secY and manA gene, which had been derived from multiple alignments of ‘Ca. A. spp’ sequence submissions (Sela et al. 1989, Lee et al. 2010). The tufB, secY and manA primers amplified fragments of about 980 bp, 640 bp and 930 bp, respectively, from all previously positive samples. Samples which had been tested negative for ‘Ca. P. phytopathogenicus’ remained negative. Fragments from two accessions were sequenced and the sequences from both isolates were 100 % identical. A BLAST search of the partial tufB gene (acc. no. PP950434) showed 98.57 % sequence identity to a yet unnamed Arsenophonus endosymbiont (acc. no. OZ026540) and 91.85 to 91.83 % to ‘Ca. A. nasoniae’ and ‘Ca. A. apicola’, respectively. A similar result was obtained for the partial secY sequence (acc. no. PP950433). The manA sequence (acc. no. PP942231) was identical to a partial sequence of ‘Ca. A. phytopathogenicus’ strain HN (acc. no. OK335757) and 97.42 % to ‘Ca. A. nasoniae and about 87 % to related Arsenophonus species. The finding of ‘Ca. A. phytopathogenicus’ in onion is novel and might indicate an expanding host range of vector and pathogen in the regional crop rotation. As a correlation between the pathogen and the soft rot symptom is unclear at present, further investigations are needed.

Abstract Background The Atribacterota are widely distributed in the subsurface biosphere. Recently, the first Atribacterota isolate was described and the number of Atribacterota genome sequences retrieved from environmental samples has increased significantly; however, their diversity, physiology, ecology, and evolution remain poorly understood. Results We report the isolation of the second member of Atribacterota, Thermatribacter velox gen. nov., sp. nov., within a new family Thermatribacteraceae fam. nov., and the short-term laboratory cultivation of a member of the JS1 lineage, Phoenicimicrobium oleiphilum HX-OS.bin.34TS, both from a terrestrial oil reservoir. Physiological and metatranscriptomics analyses showed that Thermatribacter velox B11T and Phoenicimicrobium oleiphilum HX-OS.bin.34TS ferment sugars and n-alkanes, respectively, producing H2, CO2, and acetate as common products. Comparative genomics showed that all members of the Atribacterota lack a complete Wood-Ljungdahl Pathway (WLP), but that the Reductive Glycine Pathway (RGP) is widespread, indicating that the RGP, rather than WLP, is a central hub in Atribacterota metabolism. Ancestral character state reconstructions and phylogenetic analyses showed that key genes encoding the RGP (fdhA, fhs, folD, glyA, gcvT, gcvPAB, pdhD) and other central functions were gained independently in the two classes, Atribacteria (OP9) and Phoenicimicrobiia (JS1), after which they were inherited vertically; these genes included fumarate-adding enzymes (faeA; Phoenicimicrobiia only), the CODH/ACS complex (acsABCDE), and diverse hydrogenases (NiFe group 3b, 4b and FeFe group A3, C). Finally, we present genome-resolved community metabolic models showing the central roles of Atribacteria (OP9) and Phoenicimicrobiia (JS1) in acetate- and hydrocarbon-rich environments. Conclusion Our findings expand the knowledge of the diversity, physiology, ecology, and evolution of the phylum Atribacterota. This study is a starting point for promoting more incisive studies of their syntrophic biology and may guide the rational design of strategies to cultivate them in the laboratory.

An anaerobic, mesophilic, syntrophic, archaeon strain MK-D1T, was isolated as a pure co-culture with Methanogenium sp. strain MK-MG from deep-sea methane seep sediment. This organism is, to our knowledge, the first cultured representative of ‘Asgard’ archaea, an archaeal group closely related to eukaryotes. Here, we describe the detailed physiology and phylogeny of MK-D1T and propose Promethearchaeum syntrophicum gen. nov., sp. nov. to accommodate this strain. Cells were non-motile, small cocci, approximately 300–750 nm in diameter and produced membrane vesicles, chains of blebs and membrane-based protrusions. MK-D1T grew at 4–30 °C with optimum growth at 20 °C. The strain grew chemoorganotrophically with amino acids, peptides and yeast extract with obligate dependence on syntrophy with H2-/formate-utilizing organisms. MK-D1T showed the fastest growth and highest maximum cell yield when grown with yeast extract as the substrate: approximately 3 months to full growth, reaching up to 6.7×106 16S rRNA gene copies ml−1. MK-D1T had a circular 4.32 Mb chromosome with a DNA G+C content of 31.1 mol%. The results of phylogenetic analyses of the 16S rRNA gene and conserved marker proteins indicated that the strain is affiliated with ‘Asgard’ archaea and more specifically DHVC1/DSAG/MBG-B and ‘Lokiarchaeota’/‘Lokiarchaeia’. On the basis of the results of 16S rRNA gene sequence analysis, the most closely related isolated relatives were Infirmifilum lucidum 3507LTT (76.09 %) and Methanothermobacter tenebrarum RMAST (77.45 %) and the closest relative in enrichment culture was Candidatus ‘Lokiarchaeum ossiferum’ (95.39 %). The type strain of the type species is MK-D1T (JCM 39240T and JAMSTEC no. 115508). We propose the associated family, order, class, phylum, and kingdom as Promethearchaeaceae fam. nov., Promethearchaeales ord. nov., Promethearchaeia class. nov., Promethearchaeota phyl. nov., and Promethearchaeati regn. nov., respectively. These are in accordance with ICNP Rules 8 and 22 for nomenclature, Rule 30(3)(b) for validation and maintenance of the type strain, and Rule 31a for description as a member of an unambiguous syntrophic association.

Cottony ash psyllid (CAP, Psyllopsis discrepans) is an important, invasive insect pest of ash trees in North America, where it has established populations and is the host of a newly identified strain of ‘ Candidatus Liberibacter solanacearum’. However, not much is known about the diversity of its introduced population. In this study, a CAP mitochondrial genome (mitogenome) sequence was obtained from a collection in Saskatoon, Saskatchewan, Canada. The CAP mitogenome is a circular DNA of 18,824 bp, encoding 13 protein-coding genes, 21 transfer RNA genes, and two ribosomal RNA genes. BLAST search using the CAP mitogenome as a query against the GenBank sequence database showed the mitogenome of Euphyllura phillyreae (15,202 bp) was the most similar (query coverage = 77%; percentage identity = 78.90%). The CAP mitogenome is significantly different from other known psyllid mitogenomes with the presence of a 4,357-bp control region. The mitogenome sequence will further the genomic understanding of CAP. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

A novel slightly halophilic, aerobic, and Gram-stain-negative strain, designated as CH-27T, was isolated during a bacterial resource investigation of intertidal sediment collected from Xiaoshi Island in Weihai, PR China. Cells of strain CH-27T were rod-shaped with widths of 0.3–0.6 µm and lengths of 2.0–11.0 µm. Strain CH-27T grew optimally at 37 °C, pH 7.0 and with 2.0 % (w/v) NaCl. Catalase activity was weakly positive and oxidase activity was positive. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CH-27T was most related to Marinihelvus fidelis KCTC 92639T (93.6 %), followed by Wenzhouxiangella marina MCCC 1K00261T (92.0 %). Based on genome comparisons between strain CH-27T and M. fidelis KCTC 92639T, the average amino acid identity was 63.6 % and the percentage of conserved proteins was 48.3 %. The major cellular fatty acid of strain CH-27T (≥10 %) was iso-C15 : 0 and the sole respiratory quinone was quinone-8. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, and aminophospholipid. The DNA G+C content was 62.7 mol%. Based on comprehensive analysis of its phylogenetic, physiological, biochemical, and chemotaxonomic characteristics, strain CH-27T represents a novel species in a novel genus, for which the name Elongatibacter sediminis gen. nov., sp.nov. is proposed. The type strain is CH-27T (=MCCC 1H00480T=KCTC 8011T).

The unculturable bacterium ‘ Candidatus Liberibacter solanacearum’ (CLso) is responsible for a growing number of emerging crop diseases. However, we know little about the diversity and ecology of CLso and its psyllid vectors outside of agricultural systems, which limits our ability to manage crop disease and understand the impacts this pathogen may have on wild plants in natural ecosystems. In North America, CLso is transmitted to crops by the native potato psyllid ( Bactericera cockerelli). However, the geographic and host plant range of the potato psyllid and CLso beyond the borders of agriculture are not well understood. A recent study of historic herbarium specimens revealed that a unique haplotype of CLso was present infecting populations of the native perennial Solanum umbelliferum in California decades before CLso was first detected in crops. We hypothesized that this haplotype and other potentially novel CLso variants are still present in S. umbelliferum populations. To test this, we surveyed populations of S. umbelliferum in Southern California for CLso and potato psyllid vectors. We found multiple haplotypes of CLso and the potato psyllid associated with these populations, with none of these genetic variants having been previously reported in California crops. These results suggest that CLso and its psyllid vectors are much more widespread and diverse in North American natural plant communities than suggested by data collected solely from crops and weeds in agricultural fields. Further characterization of these apparently asymptomatic haplotypes will facilitate comparison with disease-causing variants and provide insights into the continued emergence and spread of CLso.