Publications

4368

ABSTRACT Candidatus Altiarchaea are widespread across aquatic subsurface ecosystems and possess a highly conserved core genome, yet adaptations of this core genome to different biotic and abiotic factors based on gene expression remain unknown. Here, we investigated the metatranscriptome of two Ca. Altiarchaeum populations that thrive in two substantially different subsurface ecosystems. In Crystal Geyser, a high‐CO 2 groundwater system in the USA, Ca. Altiarchaeum crystalense co‐occurs with the symbiont Ca. Huberiarchaeum crystalense, while in the Muehlbacher sulfidic spring in Germany, an artesian spring high in sulfide concentration, Ca. A . hamiconexum is heavily infected with viruses. We here mapped metatranscriptome reads against their genomes to analyse the in situ expression profile of their core genomes. Out of 537 shared gene clusters, 331 were functionally annotated and 130 differed significantly in expression between the two sites. Main differences were related to genes involved in cell defence like CRISPR‐Cas, virus defence, replication, transcription and energy and carbon metabolism. Our results demonstrate that altiarchaeal populations in the subsurface are likely adapted to their environment while influenced by other biological entities that tamper with their core metabolism. We consequently posit that viruses and symbiotic interactions can be major energy sinks for organisms in the deep biosphere.

Abstract Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm), and Candidatus Mycoplasma turicensis (CMt) are the major feline hemotropic Mycoplasma (FHM) species identified in cats worldwide. Data concerning FHM in Egypt is limited; therefore, the current study aimed to gain further insights into disease epidemiology by investigating FHM molecular prevalence, risk factors, and hemogram abnormalities in 246 Egyptian cats (shelter-housed and client-owned) during 2022–2024. Additionally, 16 S rRNA gene-based maximum-likelihood (ML) phylogenetic analysis was performed for all CMt-positive samples for the first time in Egypt. FHM was detected in 16.3% (n = 40) of cats and typed as CMhm, Mhf, and CMt in 15.4% (n = 38), 3.6% (n = 9), and 2.4% (n = 6) of cats, respectively. Single CMhm and Mhf infections were detected in 12.6% (n = 31) and 0.8% (n = 2) of cats, respectively. Dual (Mhf and CMhm) and triple (Mhf, CMhm, and CMt) infections were found in 0.4% (n = 1) and 2.4% (n = 6) of cats, respectively. CMhm was significantly associated with single infections rather than coinfections compared to other FHM species. Male sex and anemia were identified as predictors of FHM PCR positivity. FHM-infected cats had significantly lower hematocrit %, RBC counts, and hemoglobin concentrations than non-infected ones. Egyptian CMt sequences showed 97.6–100% nucleotide identity with each other. Egyptian and reference CMt strains represented nine nucleotide sequence types clustered into three well-supported clades on the ML tree without clear geographic distinction. The data generated in this study, conducted in Egypt, is crucial for enhancing our understanding of disease epidemiology and implementing effective preventive measures.

A Gram-stain-negative, non-motile, rod-shaped and imazamox-resistant bacterium, designated as H34T, was isolated from soil obtained from a farmland. Strain H34T grew at pH 6.0–9.0 (optimum, 7.0), temperature 15–40 °C (optimum, 30 °C) and salinity of 0–3.0% NaCl (optimum 0.5% NaCl, w/v). Phylogenetic analysis of the 16S rRNA gene and genome sequences showed that strain H34T was affiliated to the family Chitinophagaceae but formed a distinct phyletic branch. The values of 16S rRNA gene sequence similarity, average nt identity (ANI), digital DNA–DNA hybridization (dDDH) and average aa identity (AAI) between strain H34T and closely related type strains of the genera Flavisolibacter, Paraflavisolibacter, Paracnuella, Flaviaesturariibacter and Cnuella were 90.9–92.6%, 68.4–72.1%, 18.7–20.5% and 60.5–65.9%, respectively. The genome size was 7.01 Mb with a G+C content of 56.5 mol%. The major fatty acids (≥10%) were iso-C15 : 0, iso-C15 : 1 G and C16 : 1  ω5c. The only isoprenoid quinone was menaquinone-7. The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified aminophosphoglycolipid, two unidentified phospholipids, three unidentified aminolipids and four unidentified lipids. The distinct phylogenetic relationships; low values of 16S rRNA gene sequence similarity, AAI, ANI and dDDH; and the differences in phenotypic and genotypic characteristics support that strain H34T represents a novel species in a novel genus in the family Chitinophagaceae, for which the name Huijunlia imazamoxiresistens gen. nov., sp. nov. is proposed (type strain H34T=MCCC 1K09117T=KCTC 102287T).

Abstract Psyllids are major vectors of plant diseases, including Candidatus Liberibacter solanacearum (CLso), the bacterial agent associated with “zebra chip” disease in potatoes and “carrot yellows” disease in carrot. Despite their agricultural significance, there is limited knowledge on the genome structure and genetic diversity of psyllids. In this study, we provide chromosome-level genome assemblies for three psyllid species known to transmit CLso: Dyspersa apicalis (carrot psyllid), Dyspersa pallida, and Trioza urticae (nettle psyllid). As D. apicalis is recognized as the primary vector of CLso by carrot growers in Northern Europe, we also resequenced populations of this species from Finland, Norway, and Austria. Genome assemblies were constructed using PacBio HiFi and Hi–C sequencing data, yielding genome sizes of 594.01 Mbp for D. apicalis; 587.80 Mbp for D. pallida; and 655.58 Mbp for T. urticae. Over 90% of sequences anchored into 13 pseudo-chromosomes per species. D. apicalis and D. pallida assemblies exhibited high completeness, capturing over 92% of conserved Hemiptera single-copy orthologs. Furthermore, we identified sequences of the primary psyllid symbiont, Candidatus Carsonella ruddii, in all three species. Gene annotations were produced for each assembly: 17,932 unique protein-coding genes were predicted for D. apicalis; 18,292 for D. pallida; and 16,007 for T. urticae. We observed significant expansions in gene families, particularly those linked to potential insecticide detoxification, within the Dyspersa lineage. Resequencing also revealed the existence of multiple subpopulations of D. apicalis across Europe. These high-quality genome resources will support future research on genome evolution, insect–plant–pest interactions, and disease management strategies.

ABSTRACT Huanglongbing (HLB) is a bacterial disease that affects citrus trees and is considered the most severe citrus disease in the world. The three HLB-associated ‘Ca.Liberibacter’ species harbor prophage regions which have been described to play critical roles in their evolution and biology. In this study, we assembled and characterized the accessory genome of the first circularde novo‘CandidatusLiberibacter asiaticus’ (CLas) assembly (V1R1) from Réunion, one of the sparse areas worldwide hosting CLas and ‘Ca.Liberibacter africanus’ (CLaf). This 1,272 Mb-long whole-genome harbored 1,129 coding sequences and two complete prophages, including a 37,934 bp-long Type 1 prophage, frequently present in CLas genomes, and a 40,501 bp-long undescribed CLas prophage designated as P-V1R1-5. Comparative genomic approaches suggested that P-V1R1-5 have all the genetic elements to produce new viral particles and revealed that it likely belongs to a new CLas Type 5 prophage. While being reported for the first time in a CLas strain, P-V1R1-5-like prophages were previously identified in CLaf genomes, making it the first evidence of shared prophage-like sequence among HLB-associated ‘Ca, Liberibacter’ species. Using PCR amplifications targeting P-V1R1-5, we also showed that 85.7% of the tested CLas strains from Réunion and all tested CLaf strains from Madagascar and Réunion harbored a Type 5-like prophage. The identification of CLas Type 5-like prophages not only expanded our knowledge of CLas genomic diversity in Réunion, but also provided new insights into the role of horizontally transferred elements in the evolution of the sympatric HLB-associated bacteria. IMPORTANCE Huanglongbing (HLB) is the most severe citrus disease worldwide. The disease is associated with three ‘CandidatusLiberibacter’ species harboring prophage regions that have been described to play critical roles in their biology. In this study, we assembled and characterized the accessory genome of the first circularde novo‘Ca.Liberibacter asiaticus’ (CLas) assembly from Réunion, one of the very few areas in the world where both of CLas and ‘Ca.Liberibacter africanus’ (CLaf) coexist. Comparative genomic approaches demonstrated that this genome harbored two complete prophages, including a new CLas Type 5 prophage that was previously identified in CLaf but was reported for the first time in a CLas strain. This first evidence of shared prophage-like sequences among HLB-associated species expands our knowledge of CLas genomic diversity, but also provides new insights into the role of the accessory genome in the evolution of these bacteria.

Abstract Immunity is generally considered critical for plant health against pathogen infection. Citrus Huanglongbing (HLB) caused by the phloem colonizing bacterial pathogenCandidatusLiberibacter asiaticus (CLas) was suggested to be a pathogen triggered chronic immune disease. However, the genetic evidence and mechanistic understanding for such a disease model is lacking. Here, we show CLas triggers phloem cell death, reactive oxygen species (ROS) production and callose deposition in photosynthesis tissues, but little or none in non-photosynthesis tissues of citrus. We further demonstrate that CLas triggers ROS production in chloroplasts. Overexpression offlavodoxin(Fld), an electron shuttle, in chloroplasts reduced ROS production, cell death and HLB symptoms, but not phloem callose deposition induced by CLas. Knockout, silencing, and overexpression of phloem callose synthase genesCsCalS7a,CsCalS7b, orCsCalS7cdemonstrated that phloem callose deposition also caused phloem cell death. Trunk injection with 2-deoxy-d-glucose, a callose deposition inhibitor, reduced fruit drop and increased fruit yield of HLB symptomatic trees in field trials. Using tomato-Ca. L. psyllaurous (Lpsy) as a model, knockout ofEds1andPad4but notRbohB,Bik1andSobir1abolished ROS production, phloem callose deposition, cell death and disease damages caused by Lpsy. This study provides genetic evidence forCa. Liberibacter-triggered immune disease and reveals that tuning plant immune responses convertsCa. Liberibacter into a benign endophyte, providing a promising strategy for precision breeding to enhance resistance/tolerance against HLB.

A Gram-positive, oval-shaped and non-motile strain, designated MMS21-DH1MA12T, was isolated from riverside soil and subjected to polyphasic taxonomic characterization. Strain MMS21-DH1MA12T grew optimally in tryptic soy agar, forming yellow-coloured, convex and smooth colonies. The optimal growth conditions of the strain were 30 °C, pH 7 and absence of NaCl. Strain MMS21-DH1MA12T shared the highest 16S rRNA gene sequence similarity with Agrococcus sediminis NS18T (97.3%), and then with Diaminobutyricimonas aerilata 6408 J-67T (97.1%) and Mycetocola miduiensis CGMCC 1.11101 T (96.9%), all belonging to the family Microbacteriaceae. Strain MMS21-DH1MA12T had a genome size of 2.4 Mbp and G+C content of 62.6%. The digital DNA–DNA hybridization (dDDH), orthologous average nucleotide identity (orthoANI) and average amino acid identity (AAI) values between strain MMS21-DH1MA12T and related type strains of the genera within the family Microbacteriaceae were no higher than 22.2%, 71.54% and 60.19%, respectively. The dDDH, orthoANI and AAI values of MMS21-DH1MA12T with related taxa were no higher than 22.2%, 71.54% and 61.96%, thus showing a distant relationship of the strain with other members of Microbacteriaceae. It was also notable that the species sharing the highest similarities were different for all three indices. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. The main cellular fatty acids were anteiso-C15 : 1, antesio-C17 : 1 and iso-C15 : 0. The predominant menaquinones were MK-10 and MK-11. The combination of physiological and biochemical properties, as well as chemotaxonomic features, clearly separated strain MMS21-DH1MA12T from related taxa of Microbacteriaceae. It is, thus, evident that MMS21-DH1MA12T represents a novel species in a novel genus, for which the name Humidisolicoccus flavus gen. nov., sp. nov. is proposed (type strain=MMS21-DH1MA12T = KCTC 49751T = LMG 32525T=JCM 37009T).

‘Candidatus Phytoplasma mali’ is associated with apple proliferation, a devastating disease in fruit production. Using genome analysis, a gene encoding a hemolysin-like protein was identified. It was postulated that this protein could be an effector. However, the function of this protein is unknown. It is shown that the hemolysin-like protein binds to a GTP binding protein, Toc33, of Arabidopsis thaliana in yeast two-hybrid analysis and that the Toc33-binding domain is located in the C-terminus of the domain of unknown function (DUF21) of the protein. The biochemical studies reveal that the protein can hydrolyze phosphate of purine and pyrimidine nucleotides. Transgenic Nicotiana benthamiana plants expressing the protein show no discernible change in phenotype. Phytoplasma have a much-reduced genome, lacking important genes for catabolic pathways or nucleotide production; therefore, the hemolysin-like protein plays a role in the uptake of plant nucleotides from their host and hydrolyzes these nucleotides for energy and their own biosynthesis.

The order Sphingomonadales strains are globally distributed in various biomes and are renowned for their biodegradable and biosynthesis capabilities. At present, it consists of 4 families and 49 genera making it the third largest order within the class Alphaproteobacteria. However, their taxonomy remains complex, especially due to polyphyly in the family Sphingomonadaceae. In this study, we collected 429 Sphingomonadales type strain genomes, reconstructed robust phylogenomic relationships, and proposed delineation thresholds at the genus and family levels based on average amino acid identities (AAI) and evolutionary distances (ED). Based on the maximum-likelihood and Bayesian phylogenomic trees reconstructed by two molecular sets determined by orthologous sequence identity and the Genome Taxonomy Database, the consensus degree values were all higher than 90%, revealing that those phylogenomic trees had similar topological structures. By confirming monophyletic taxa and determining stable nodes, we reclassified the order Sphingomonadales into thirteen families including nine novel ones. AAI calculations indicated that the average intra-family AAI values ranged from 0.62 to 0.84, while inter-family ones were 0.51 to 0.60. ED summaries demonstrated that the average and median intra-family ED values were 0.16 to 0.57, and inter-family ones ranged from 0.50 to 1.22. Comparisons of AAI and ED values calculated by using genomic and phylogenetic analyses supported that those 13 families were significantly separated with p values < 2.2×10−16. Thus, it was speculated that the AAI and ED thresholds for distinguishing different families were <0.6 and >0.5, respectively. Additionally, we reclassified 163 species into new genera with their phylogenetic topologies, according to the previous genus AAI and ED boundaries of 0.7 and 0.4. Our study is the first genomic-based study of the order Sphingomonadales and will promote further insights into the evolution of this order.