Publications

4670

Huanglongbing, a destructive citrus disease of global importance that is also present in Saudi Arabia, is associated with Candidatus Liberibacter asiaticus (CLas) and remains a major threat to citrus production. Although previous studies have documented sequence variation and prophage polymorphism in CLas, broader comparisons of prophage-associated gene content remain limited. In particular, comparative orthogroup analysis of prophage gene-content conservation across geographically structured CLas populations has rarely been explored. In this study, we analyzed 42 CLas prophage genomes from Saudi Arabia and other geographic regions using a comparative orthogroup framework. OrthoFinder assigned 99.1% of predicted proteins (1825 of 1841) to 64 orthogroups, with only 16 genes remaining unassigned. A small number of rare orthogroups restricted to only a few genomes were identified, and no orthogroup was detected in all genomes. Presence–absence analyses supported a predominantly conserved prophage gene repertoire together with a small accessory component, while also indicating that apparent absences should be interpreted in light of mixed assembly status and prophage-region completeness. Saudi Arabian genomes were distributed within the broader global framework and exhibited generally similar gene-content profiles rather than a deeply separated lineage. Functional interpretation of representative orthogroups identified conserved prophage-associated genes related to replication, helicase activity, and phage packaging, whereas variable orthogroups were primarily associated with hypothetical or accessory prophage-related functions. Overall, these results are consistent with a model in which CLas prophage diversification is associated more with sequence-level variation and localized structural differences than with extensive gain or loss of prophage genes. These findings further refine current understanding of CLas genome evolution and highlight conserved prophage-associated targets that may support molecular diagnostics and epidemiological surveillance.

Chokecherry (Prunus virginiana L.) is a native shrub that is widely found across North America and is a host of ‘Candidatus Phytoplasma pruni’ associated with X-disease of stone fruits. This pathogen is of major concern to U.S. cherry and peach growers, with the most recent and ongoing outbreak in the Pacific Northwest causing major economic harm. To investigate the role of chokecherry as a potential reservoir and source of orchard-associated strains, we surveyed wild chokecherry trees across 12 U.S. states, and compared the strains present to those in commercially cultivated Prunus species. Approximately 47% of chokecherry sampled tested positive for a ‘Ca. P. pruni’-related strain and phylogenetic analysis revealed high strain diversity, including six distinct clades and four newly identified lineages. In contrast, ‘Ca. P. pruni’ strains isolated from cultivated cherry and peach trees exhibited lower diversity and were infected by known disease outbreak-associated strains such as NWX. These data suggest limited transmission between wild and cultivated hosts in the western U.S., with patterns indicating spillover from orchards into nearby chokecherry populations rather than the reverse. However, in states east of the Rocky Mountains, chokecherry likely functions as a reservoir for orchard infections.

ABSTRACT The Sphaerotilus-Leptothrix group of bacteria includes one of the first described microorganisms, Leptothrix ochracea , an uncultured type strain, plus isolates of Leptothrix and Sphaerotilus . This group is unified by the ability to form sheaths and oxidize metals, although L. ochracea exhibits obvious ecological, morphological, and functional differences from the rest of Sphaerotilus-Leptothrix . Recently, there have been calls to combine the group into one genus, Sphaerotilus ; however, these studies lacked adequate genomic representation of L. ochracea . Here, we present a comprehensive comparative genomic analysis of the Sphaerotilus-Leptothrix group, including expanded representation of L. ochracea , a closely related novel species, Leptothrix toolikensis , and two new isolates ( Leptothrix mechoopdaensis ). Analysis of 38 genomes resolves three phylogenetic and functional groups: the ochracea -type Leptothrix (Group 1), the mobilis -type Leptothrix (Group 2), and Sphaerotilus (Group 3). Group 1 genomes form a separate genus based on average nucleotide identity and alignment fraction. The genomes clearly diverge from the rest of Sphaerotilus-Leptothrix in phylogeny, size, and metabolic potential. Group 1 genomes are much smaller (2.59–3.04 Mb) than those of Groups 2 (4.55–6.06 Mb) and 3 (3.94–5.07 Mb), while encoding more metal oxidases and fewer carbohydrate-active enzymes. Group 2 clusters with Group 3 phylogenetically and is similar in organic carbon metabolisms but maintains more metal oxidation genes. Group 2 members lack homogeneity in phenotype and genotype, suggesting that additional isolates and genomes are needed for confident classification. However, Group 1 genomes ( L. ochracea and L. toolikensis ) show clear divergence, precluding their inclusion in Sphaerotilus and supporting the retention of the genus Leptothrix . IMPORTANCE Researchers have long noted differences in metal oxidation, morphology, and ecology among Sphaerotilus-Leptothrix , but longstanding confusion over phylogeny and genus boundaries led to inconsistent taxonomic classification between the two genera. This confusion stems from previous work that used isolates that are unavailable or lost distinguishing traits in culture, and from limited genomic data. Furthermore, the Leptothrix type strain L. ochracea has never been isolated. This study provides molecular evidence that substantiates calls to reassign some Leptothrix members to the genus Sphaerotilus but adds to an emerging body of evidence that Group 1 L. ochracea and now L. toolikensis represent a functionally distinct lineage. While genomic similarity metrics left taxonomic divisions unclear, integrating metabolic potential with phylogeny resolved genus boundaries based on clear functional groupings. This polyphasic approach for delineating genera clarifies longstanding taxonomic confusion and refines our understanding of functional diversity both across and within Sphaerotilus-Leptothrix lineages.

GroE is a chaperonin folding system consisting of GroEL (Cpn60, a 60 kDa chaperonin), and the smaller co-chaperonin GroES (Cpn10). Many ‘client’ proteins require GroE to fold properly, including several that are essential for cell viability. GroE is found in nearly all bacteria and eukaryotes. Mollicutes are the only micro-organisms that lack GroE in almost all cases. Only two clades of Mollicutes have retained the ancestral GroE system, or perhaps reacquired one; these exceptions include the family Acholeplasmataceae , consisting of the genera Acholeplasma and ‘ Candidatus Phytoplasma’. The role of GroEL in these unique Mollicutes is a source of speculation, given how many non-canonical ‘moonlighting’ roles have been ascribed to this protein. GroEL has been suggested to play a role in pathogenesis in plant and animal pathogenic Mollicutes by binding to host cells and facilitating invasion. However, in one further layer of exception, the phytopathogenic taxon ‘ Candidatus Phytoplasma pruni’ (ribosomal group 16SrIII) was reported in 2012 to lack a GroE system. This study confirms the lack of a functional GroE system in 16SrIII by providing two new, high-quality, non-fragmented genome assemblies, as well as a thorough survey of other 16SrIII genomes for genes encoding GroEL/GroES, including those that may not resemble phytoplasma GroEL (i.e. acquired by horizontal gene transfer, HGT). We discuss the implications of a clearly phytopathogenic, invasive group of Mollicutes that nevertheless lacks GroE, in light of the presumed role of GroEL for this species. We determined that multiple genomes of 16SrIII contain short, non-functional groEL pseudogenes, while most of the reported genomes lack any semblance of a GroE system. Examination of the new assemblies allowed us to rule out HGT as a means of GroE acquisition.

Soils are critical microbial habitats that support terrestrial ecosystem functioning and harbor numerous uncultured and functionally uncharacterized microbial groups. The black soil region in northeast China is a key agricultural ecosystem globally, yet the classification and functional understanding of its crucial microbial groups remain underexplored. In this study, we identified three high-completeness metagenome-assembled genomes (MAGs) from the Global Mollisols Genomic Atlas (GMGA). Phylogenetic and comparative genomic analyses identified these genomes as representing a novel evolutionary branch within the genus Flavobacterium, classified under the phylum Bacteroidota. Their novel taxonomic position is further supported by average nucleotide identity (ANI) and average amino acid identity (AAI) thresholds, demonstrating significant divergence from all known reference genomes. Functional annotation indicated that this species possesses strong plant polysaccharide degradation potential and a chemoheterotrophic lifestyle, together with environmental stress tolerance and a specialized nitrogen metabolic network adapted to agricultural inputs, thereby conferring a metabolic advantage in black soil environments characterized by high organic matter input and marked seasonal fluctuations. In addition, global distribution analysis showed that this lineage is widely distributed across diverse ecosystems and is significantly enriched in soil habitats, particularly in environments with fluctuating carbon sources and high organic matter inputs. The new species is most abundant in temperate soils, with the northeast black soil region of China emerging as a key hotspot. Based on these findings, and because no pure culture is currently available, we propose Candidatus Flavobacterium genomatis based on genome-resolved metagenomic evidence and in alignment with the International Code of Nomenclature of Prokaryotes rules for uncultivated prokaryotes. Our results expand the known species diversity of the genus Flavobacterium and suggest potential ecological roles of uncultured black-soil microbes in carbon and nitrogen cycling, including possible involvement in N2O reduction under suitable environmental conditions.

“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 JB3T, from brain tissue of a western chimpanzee (Pan troglodytes verus) using a reinforced clostridial medium. We describe the growth of strain JB3T at a pH range of 6 to 9 (optimum 6) at 37°C in an atmosphere composed of 5% H2, 5% CO2, and 90% N2 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.