Publications

4874

Abstract The evolution of phototrophy and carbon fixation in the phylum Chloroflexota remains incompletely understood, largely due to the scarcity of cultured mesophilic representatives. Here, we report the isolation and genomic characterization of a novel filamentous anoxygenic phototrophic bacterium from a microbial mat in the mesothermal Lake Umkhey (Southern Siberia). Although the original culture was lost, its genome was recovered via metagenome-assembled genome reconstruction from environmental samples, enabling a detailed phylogenomic and metabolic analysis. The genome of umkhey_bin13 (proposed as ‘Candidatus Viridifilum namsaraevi’ gen. nov., sp. nov.) encodes a complete 3-hydroxypropionate bicycle for autotrophic CO₂ fixation, a type II sulfide:quinone oxidoreductase, but lacks nitrogenase and chemolithotrophic markers. Comparative genomics across 18 Chloroflexota genomes reveals that this new lineage occupies an intermediate phylogenetic position, with distinct gene content for sulfur, nitrogen, and hydrogen metabolism, and highlights a complex evolutionary history within the group. Our findings provide new insights into the evolutionary transitions of photosynthetic and carbon-fixation pathways, and underscore the value of integrating cultivation-independent genomics with classical microbiology. The proposed novel genus and species contribute to a revised framework of Chloroflexota evolution and offer a genomic reference for studying metabolic adaptation in alkaline, sulfide-influenced microbial mats.

‘ Candidatus Liberibacter asiaticus’ ( C Las) is a phloem-limited bacterium that causes Huanglongbing (HLB), the devastating citrus greening disease that has led to billions of dollars in losses throughout global citrus production. There is very little epidemiological data available regarding fine-scale molecular variability of C Las populations in different regions of the world or their distribution across various geographical landscapes. Here, we present an assessment of C Las genetic diversity using a hypervariable variable number tandem repeat (VNTR) marker (AGACACA) at the CLIBASIA_01645 locus of 344 genomic sequence data obtained from GenBank from five regions of the world (India-177, China-113, Saudi Arabia-37, Bhutan-10, and Nepal-7). The findings revealed high evolutionary plasticity and non-random distribution of TRN within four distinct classes, significantly deviating from a uniform distribution ( p  < 0.05). The analyzed populations were stratified into four distinct classes: Class I (TRN ≤ 5), Class II (TRN > 5 ≤ 10), Class III (TRN > 10 ≤ 15), and Class IV (TRN > 15). The predominant type of subpopulation for all field samples was intermediate variants, with the highest frequencies observed for TRN6 (25%) and TRN7 (17.15%), possibly indicating genomic stability and/or adaptive advantages of specific tandem repeats. On the other hand, lower frequency rare variants (e.g., TRN17, TRN18, and TRN20 at 0.29%) represent some form of continued low frequency micro evolutional replication slippage and corresponding absence of some individual TRs indicates evidence of local purging forces. Crucially, the Indian C Las populations exhibited significantly greater densities of molecular diversity than populations from China, Saudi, Bhutan, and Nepal examined in this study. The distinct TRN signature profiles of these populations’ subpopulations exhibit strong geographic correlation with ecological regions, reinforcing the CLIBASIA_01645 locus as a strong marker for epidemiological surveillance for tracking global strain movement to develop target-specific HLB management programs.

In Germany, maize (Zea mays L.) is cultivated on approximately 2.4 Mha, making it one of the most widely grown arable crops alongside wheat and barley (German Federal Statistical Office, 2025). Although maize has previously been identified as a host of Candidatus Phytoplasma solani (PHYPSO, 16SrXII-A), particularly in relation to maize redness (MR) disease detected in southeastern Europe (Jović et al., 2009), its role as a host of ‘Candidatus Arsenophonus phytopathogenicus’ (ARSEPH) has not been reported previously. The planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) is the major vector for transmitting both pathogens in many crops in Germany (Rinklef et al., 2024). MR symptoms include patchy premature drying, reddish discoloration of leaves and midribs, poorly filled or aborted ears, and malformed kernels. In August and September 2025, maize plants showing MR-like symptoms were observed in different fields in Otzberg 49°48'25.3"N, 8°52'28.8"E (South Hesse), Bellheim 49°10′19.9N, 8°15′50.8E, Bockenheim 49°36'18.6"N, 8°11'30. E"O, Ibersheim 49°43′36.5″N, 8°24′34.6″E (Rhineland-Palatinate) and Ettling 48°48'50.0"N, 11°40'17.2"E (Baden-Württemberg). Samples from roots (25), leaves (10), ears (14) and kernels (9) of 14 selected symptomatic plants were collected for molecular analysis. DNA was extracted using a CTAB-based protocol. ARSEPH was detected by qPCR targeting the manA gene according to Zübert and Kube (2021). Positive results were obtained from ears of six plants from Ibersheim and Ettling, leaves of three plants from Bockenheim and Otzberg, kernels of two plants from Bellheim, and roots of one plant from Bockenheim. Four ARSEPH-positive samples (two ears and two kernels) were further analyzed using the Fra4/Fra5 PCR assay (Zreik et al. 1998). Sanger sequencing of the resulting amplicons followed by BLAST analysis confirmed all four sequences as ‘Candidatus Arsenophonus phytopathogenicus’ with 100% identity to reference sequence PP400342. The sequences have been deposited in GenBank under accession numbers PZ623476, PZ623478, PZ623483, and PZ626926. Furthermore, all samples were screened for PHYPSO using the qPCR assay described by Behrmann et al. (2022) with primers KL464/KL465. Seven ear samples from Ibersheim yielded positive qPCR signals. To further validate these results, DNA from qPCR-positive samples was subjected to nested PCR targeting the 16S rRNA gene using primer pairs P1/P7 (Schneider et al., 1995) and R16F2n/R16R2 (Gundersen and Lee, 1996), followed by Sanger sequencing and BLAST analysis against the NCBI GenBank database. However, repeated sequencing did not provide sufficient sequence identity to the reference strain STOL11 (GenBank accession number AF248959) to confirm the qPCR-positive samples as PHYPSO. To our knowledge, this is the first report of ARSEPH in maize in Germany. Its detection in maize extends the known host range of this phloem-limited bacterium. The high ARSEPH loads in ears and kernels detected in reproductive tissues are consistent with preferential accumulation in sink organs during grain filling. Whether maize contributes to pathogen persistence and transmission within the Pentastiridius leporinus or other potential vectors’ pathosystem remains to be investigated.

SUMMARY The ubiquitin proteasome system (UPS) and autophagy cooperatively orchestrate plant immune homeostasis, while diverse phytopathogens deploy secreted effectors to disrupt their crosstalk for successful colonization. Proteaphagy, the autophagic turnover of intact 26S proteasome holoenzymes, is a conserved stress response co‐opted by Pseudomonas syringae effector HopM1 to cripple host proteolysis. However, how the Huanglongbing (HLB) pathogen ‘ Candidatus Liberibacter asiaticus’ ( C Las) simultaneously manipulates UPS and autophagy to subvert citrus immunity remains largely uncharacterized. Here, we functionally characterize the secreted C Las effector SDE5640, which exacerbates HLB susceptibility in sweet orange ( Citrus sinensis ). SDE5640 directly binds the 19S proteasome chaperone CsPSMD10, whose homodimerization via Cys163/Cys190 disulfide bonds is required for immune suppression. CsPSMD10 overexpression enhances citrus susceptibility to C Las, whereas the dimerization‐deficient CsPSMD10‐C190S mutant confers robust HLB resistance. Mechanistically, CsPSMD10 targets CsATG8 isoforms for UPS‐dependent degradation to restrain autophagic flux; SDE5640 competitively occupies the Ank2/Ank3 domains of CsPSMD10 to disrupt CsPSMD10‐CsATG8c complexes, thereby stabilizing CsATG8c and triggering autophagy dependent on CsPSMD10. Moreover, SDE5640‐induced autophagy drives selective autophagic degradation of the 20S proteasome catalytic subunit CsPBA1, which ultimately disrupts cellular proteasome activity and attenuates salicylic acid‐mediated citrus immunity. Collectively, our findings identify a novel SDE5640‐CsPSMD10‐CsATG8c‐CsPBA1 regulatory cascade, illustrating how a single C Las effector synchronously rewires the UPS‐autophagy crosstalk to facilitate pathogen proliferation.

The family Gallionellaceae includes neutrophilic, aerobic, iron-oxidizing bacteria, many of which remain uncultured and known only from metagenomic data. In this study, two high-quality Gallionellaceae genomes were assembled from a ferruginous spring in the Voronezh region, Russia. Phylogenetic analysis based on 120 concatenated proteins, ANI (average nucleotide identity), and AAI (average amino acid identity) indicates that BG-057 belongs to the genus Gallionella and BG-364 to the genus 39-52-133. We propose the names “Candidatus Gallionella aquifuscii” sp. nov. for BG-057 and “Candidatus Dubininella ochracea” gen. nov., sp. nov. for BG-364. Both genomes encode genes for the Calvin-Benson-Bassham (CBB) cycle. Both are potentially capable of aerobic respiration due to the presence of electron transfer chain components. Both genomes encode the iron oxidase Cyc2, and BG-057 also encodes MtoA. BG-057 is potentially capable of lithotrophic growth in the presence of H2 and H2S. BG-364 is potentially capable of growth via Mn (II) oxidation; arsenite oxidation; the direct oxidation of thiosulfate; H2S, elemental sulfur, and sulfite oxidation. BG-364 encodes denitrification genes (narGHI, nirS, norBC). BG-057 possesses systems for the biosynthesis and export of siderophores, while BG-364 lacks these systems but retains Ton-dependent receptors, suggesting the use of exogenous siderophores.

‘ Candidatus Sarcina troglodytae’ (‘ Ca . S. troglodytae’), a proposed bacterial species in the family Clostridiaceae , has been epidemiologically associated with a lethal disease in sanctuary chimpanzees in Sierra Leone, epizootic neurologic and gastroenteric syndrome (ENGS). As sarcinae are known to be difficult to culture, a lack of viable in vitro growth conditions has limited further characterization of this bacterium. Such studies are critical to elucidating the relationship between ‘ Ca . S. troglodytae’ and ENGS. Here, we isolate a Gram-stain-positive, endospore-forming coccus and anaerobic bacterial strain, designated JB3 T , from brain tissue of a western chimpanzee ( Pan troglodytes verus ) using a reinforced clostridial medium. We describe the growth of strain JB3 T at a pH range of 6 to 9 (optimum 6) at 37 °C in an atmosphere composed of 5% H 2 , 5% CO 2 and 90% N 2 and demonstrate endospore production under alkaline conditions, suggesting the possibility of an environmental reservoir. We identify ethanol as the primary non-gaseous fermentation byproduct which we hypothesize to have relevance for ENGS. Antibiotic susceptibility testing shows susceptibility to common antimicrobial agents with notable resistance to ceftriaxone. By establishing in vitro conditions for the viable culture, propagation and storage of ‘ Ca . S. troglodytae’, this study lays the groundwork for future infection studies to test hypotheses about pathogenic mechanisms and prevention/treatment measures for ENGS.

Abstract Antimicrobial peptides (AMPs) are widely used in disease-resistant breeding, and their application in citrus has recently been reported. However, the molecular mechanism by which AMPs inhibit the Huanglongbing (HLB) remains unclear. This study aimed to clarify the inhibitory mechanism of Hinnavin I (an AMP) against HLB-associated pathogens. We first demonstrated that Hinnavin I exhibits antibacterial activity against several tested Gram-negative bacterial strains. Using a transgenic citrus hairy root system, the heterologous expression of Hinnavin I was found to significantly reduce Candidatus Liberibacter asiaticus (CLas) colonization. Transcriptomic and phytohormone analyses further showed that constitutive Hinnavin I expression was associated with altered defense-related gene expression and enhanced SA-related responses in citrus hairy roots. Multiple interaction assays identified the CLas outer membrane lipoprotein Omlp as a direct interaction partner of Hinnavin I both in vitro and in vivo.

Abstract Purpose The objective of this study was to characterize Bradyrhizobium strains currently recommended as inoculants for Arachis pintoi in Brazil using comparative genomic and symbiotic analyses. In addition, this study aimed to expand current knowledge on the diversity, evolution, and bacterial symbiotic traits of Arachis -associated Bradyrhizobium while providing robust taxonomic evidence to support the description of a novel Bradyrhizobium species isolated from root nodules of Arachis . Methods Phylogenetic reconstruction was performed based on the 16S rRNA gene and by phylogenomics using 116 single- copy core gene sequences. Average Nucleotide Identity (ANI) analyses were conducted to evaluate the genomic relatedness of strains BR 1432ᵀ, BR 1433 and BR 14999 to 1,301 reference Bradyrhizobium genomes, including all available type strain strains. The results were validated using digital DNA–DNA hybridization (dDDH). Additional analyses included phylogenetic reconstruction of symbiotic genes ( nodC and nifH ) and, assessment of the denitrification pathway, and phenotypic characterization. Results Phylogenetic analyses based on 16S rRNA gene sequences and whole-genome data indicated that strains BR 1432ᵀ and BR 14999 form a distinct lineage within the genus Bradyrhizobium , clearly separated from BR 1433, which clustered with Bradyrhizobium brasilense . ANI and dDDH values between BR 1432ᵀ and BR 14999 and their closest related type strains, including B. zhanjiangense LMG 29279ᵀ, were below the species delineation thresholds of 96% and 70%, respectively, supporting the proposal of a novel species. In addition, the publicly available RefSeq genomes Bradyrhizobium sp. DN5 and Rc2d were found to be closely related to BR 1432ᵀ. Phylogenetic analyses of the nodC and nifH genes revealed distinct evolutionary patterns, while genomic analyses showed that the proposed novel species harbors a complete denitrification pathway, unlike strain BR 1433. Furthermore, phenotypic characterization revealed differences in carbon source utilization and enzymatic activities. Conclusion Based on genomic, phylogenetic, and phenotypic evidence, strain BR 1433 was identified as a member of B. brasilense , whereas strains BR 1432ᵀ (= UCCC 277ᵀ = LMG 34120ᵀ = CCP577ᵀ), BR 14999, DN5 and Rc2d represent a novel species within the genus Bradyrhizobium , for which the name Bradyrhizobium boddeyi sp. nov. is proposed.