Publications

4364

Jujube witches’ broom (JWB) disease, caused by Candidatus Phytoplasma ziziphi (Ca. P. ziziphi), severely threatens the production of Chinese jujube (Ziziphus jujuba Mill.). Emerging evidence highlights the critical role of phytoplasma-secreted effectors in pathogenesis, though few have been functionally characterized. Here, we identified a Sec-dependent effector, JWB790, from Ca. P. ziziphi, which was shown to suppress plant immunity. Through transient expression assays in Nicotiana benthamiana, pathogen inoculation assays, the generation of transgenic Arabidopsis thaliana plants, and RNA-seq-based transcriptomic profiling, we systematically investigated the virulence function of JWB790. Our findings revealed that JWB790 is highly expressed in JWB-infected tissues. The transient expression of JWB790 in N. benthamiana suppressed BAX-induced cell death and H2O2 accumulation. Furthermore, the stable overexpression of JWB790 in A. thaliana compromised disease resistance, accompanied by reduced H2O2 accumulation and callose deposition triggered by flg22. Additionally, the RNA-seq analysis of JWB790 transgenic Arabidopsis plants indicated that the overexpression of JWB790 altered the expression of biotic stress-related genes. In summary, JWB790 is a virulence factor that suppresses plant immunity and promotes pathogen proliferation. These results advance our understanding of Ca. P. ziziphi pathogenesis.

IntroductionEndosymbiotic bacteria show diverse strategies to manipulate host reproduction for their survival in nature. The diversity of symbionts infecting hematophagous insects and their impact on host ecology could be crucial for developing effective control measures of disease-transmitting vectors. Sand flies are a group of small insects, with some species serving as vectors for leishmaniasis, bartonellosis, and arboviral diseases. Sergentomyia squamirostris is the only known species of sand flies found on the main islands of Japan. Although no medical implications exist for S. squamirostris, we conducted whole-genome sequencing to explore its potential relevance.MethodsWe conducted whole-genome sequencing and de novo assembly of S. squamirostris using genomic DNA isolated from a single field-collected female insect sample. During this attempt, we incidentally obtained closed genomes of two new bacteria, wSSQ and RiSSQ, belonging to Wolbachia and Candidatus Tisiphia, respectively. We then investigated infection rates of both bacteria in two natural populations of S. squamirostris in Japan.ResultsPhylogenetic analysis indicated that wSSQ and RiSSQ belonged to Wolbachia and Ca. Tisiphia, respectively. Ca. Tisiphia is also known as “Torix Rickettsia,” which is considered a large group of endosymbionts of invertebrates with no known pathogenicity to humans and animals. NGS read depths for both wSSQ and RiSSQ genomes were found to be high coverages, indicating that these bacteria are S. squamirostris endosymbionts. The infection rates of wSSQ and RiSSQ in the wild population of S. squamirostris varied in the two different localities in Japan, and co-infection with both bacteria was commonly seen. wSSQ was detected in both sexes of S. squamirostris, whereas RiSSQ was detected only in female sand flies.ConclusionCa. Tisiphia has recently been recognized as an underexplored endosymbiont with a widespread presence in invertebrates, including disease vectors. RiSSQ represents the first complete genomic information resource of Ca. Tisiphia infecting sand flies. Further research is needed to understand potential interactions between its host and other endosymbionts, as well as to explore the potential implications of disease control in the future.

A strictly anaerobic bacterial strain, designated as AMB_02T, was isolated from a propionate enrichment culture obtained from a high-ammonia biogas digester. The cells were anaerobic and coccoid (0.5 µm), often appearing as diplococci or in a short chain of three to four cells. Growth was observed between 20 and 45 °C (optimum at 37–39 °C), with an initial pH of 6.5–9.0 (optimum pH 8.0–8.5), and the species tolerated up to 0.8 M NH4Cl and 0.5 M NaCl. The major cellular fatty acids were C16 : 0 (31.6%), C14 : 0 (14.6%) and C18 : 0 (13.3%). AMB_02T grew with formate, carbohydrates and aa, including asparagine, histidine, tryptone and tryptophan. Acetate was the major product formed. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain AMB_02T was most closely related to the species Citroniella saccharovorans (92.5%). The genome of strain AMB_02T was 2.5 Mb in length with a G+C content of 34.8 mol%, and 2,354 protein-coding genes were predicted. Furthermore, genes coding for the reductive glycine pathway potentially used for formate metabolism were identified. Comparative genomic analysis of AMB_02T revealed the closest similarity to C. saccharovorans [21.2% digital DNA–DNA hybridization (dDDH) and 77.4% average nt identity (ANI)] and to Parvimonas micra (24.4% dDDH and 76.9% ANI). Based on the phenotypic characteristics and phylogenetic analyses, AMB_02T is regarded as a novel genus, Microaceticoccus, within the family Peptoniphilaceae for which the species name Microaceticoccus formicicus is proposed. The type strain is AMB_02T=DSM 110248T=JCM 39108T.

The genus Pseudalkalibacillus, created by the reclassification of specific deep-branching Alkalihalobacillus species, exhibits polyphyletic branching. The Genome Taxonomy Database (GTDB) also assigns Pseudalkalibacillus species into two families and three genera. To clarify the evolutionary relationships and classification of Pseudalkalibacillus species, we report detailed investigations using phylogenomic and molecular signature-based approaches. In phylogenomic trees, Pseudalkalibacillus species are distributed within two family-level lineages. One of these clades, containing the type species of Pseudalkalibacillus (viz. Pseudalkalibacillus decolorationis), represents the genus Pseudalkalibacillus, groups within the family Fictibacillaceae. Ten novel conserved signature indels (CSIs) identified in this study are specific for this clade, providing a robust means for the differentiation of the emended genus Pseudalkalibacillus. The remaining Pseudalkalibacillus species form a separate family-level clade, designated as f_HBI72195 in the GTDB. Within this clade, all species except Pseudalkalibacillus caeni form a robust clade designated as Pseudalkalibacillus clade −2 in our work and g_Anaerobacillus_A in the GTDB. We have also identified 15 novel CSIs specific to this clade. As the Pseudalkalibacillus clade −2 is distinct from Pseudalkalibacillus, we propose transferring species from this clade into a new genus, Guptibacillus gen. nov. The species P. caeni branches distinctly from other Pseudalkalibacillus species, and the GTDB considers it a novel genus (g_Bacillus_BR). Six newly identified CSIs are specific to this species, and we are proposing the transfer of this species into a new genus, Exobacillus gen. nov. Two additional identified CSIs are shared by members of the novel family-level taxon (f_HBI72195) comprising the proposed genera Guptibacillus and Exobacillus, for which we are proposing the name Guptibacillaceae fam. nov. Lastly, the results presented here also show that ‘Pseudalkalibacillus hemicentroti’ and ‘Pseudalkalibacillus macyae’ are later heterotypic synonyms of Guptibacillus hwajinpoensis. These changes, which reliably depict the evolutionary relationships among Pseudalkalibacillus species, should be helpful in future studies of these organisms.

AbstractBackgroundRickettsiaare obligate intracellular gram-negative bacteria of the class Alphaproteobacteria and are vector-borne species that cause diseases in humans and animals. NewRickettsiaspecies have been involved as human pathogens; however, the pathogenic potential of other species is unknown.CandidatusRickettsia colombiensis is a new species in the spotted fever group of unknown pathogenicity that is phylogenetically related to other pathogenic species.ObjetiveTo evaluate the pathogenic potential ofCa. R. colombiensis in Syrian hamsters (Mesocricetus auratus) and analyzed its cross-reactivity against other SFG rickettsia species in human and Syrian hamster sera.MethodsCa. R. colombiensis was isolated fromAmblyomma dissimileusing a shell vial. Subsequently, five male hamsters were inoculated intraperitoneally (IP) and five intradermally (ID) with 1 × 106 Vero cells infected withCa. R. colombiensis. One control animal was used in each group of the study. The health status of the animals was assessed daily, and necropsies were performed on days 5, 10, 15, and 16 DPI. Serum samples for indirect immunofluorescence and tissues were processed for qPCR and immunohistochemistry.ResultsAll animals remained healthy during the trial and showed no changes in their physiological parameters. No Rickettsia DNA was detected in any of the tissues. Animals infected withCa. R. colombiensis showed IgG antibody titers ranging from 1:64 to 1:1024. The control animals were negative. Regarding human sera, 56% (84/150) had IgG antibodies against Ca. R. colombiensis antigen. Sera with titers equal to or higher than 1:64 were 100% seroreactive.ConclusionsCa. R. colombiensis caused a subclinical infection in hamsters, suggesting the possibility of infecting other mammals. However, the clinical, pathological, and molecular findings are inconclusive in establishing thatCa.R. colombiensis is a pathogenic species. Regarding cross-reactivity, it is possible to serologically diagnose Rickettsial infection usingCa. R. colombiensis as antigens.Author summaryRickettsiosis is a human and animal disease caused by the bacterial Rickettsia species, which are mainly transmitted by arthropods. The clinical spectrum of rickettsiosis ranges from asymptomatic infection to severe and fatal diseases, such as Rocky Mountain spotted fever caused by Rickettsia rickettsii. In recent years, the number of species belonging to the genus Rickettsia has increased, in part due to the progress in the detection and identification of these agents by the application of modern molecular techniques. Much of this newRickettsiaspecies, known as Candidatus, has been involved as animal and human pathogens; however, for other Candidatus Rickettsia species, the ability to cause diseases is unknown.CandidatusRickettsia colombiensis is a new species phylogenetically related to pathogenic species. Here, we evaluated, for the first time, the pathogenic potential of this microorganism in a hamster animal model and analyzed its cross-reactivity against other members of the Rickettsia genus in human and hamster sera to determine the possibility of usingCandidatusRickettsia colombiensis as antigens to diagnose rickettsiosis in regions where immunologic reagents are scarce and high biosafety level laboratories do not exist. In this report, we demonstrate thatCandidatusRickettsia colombiensis causes an inapparent infection in hamsters and cross-reactivity between other members of the Rickettsia genus.

Psyllids (Hemiptera: Psylloidea) are the main vectors of various phloem-limited plant pathogens, including ‘Candidatus Liberibacter’ species. ‘Candidatus Liberibacter solanacearum’ (CLso) has been associated with various plant disorders and economic losses in plants from the Solanaceae and Apiaceae families. Recently, it has been reported in Europe, primarily linked to carrots and celery. This situation presents a significant threat, prompting the need for a survey to assess the presence of the bacterium and its potential vectors. Plant and psyllid samples were collected from potato (Solanum tuberosum), carrot (Daucus carota) and other wild weed species in commercial fields and urban areas over two consecutive years (2022 and 2023). DNA was extracted from the samples, followed by conventional PCR and the sequencing of positive samples. The psyllid species Bactericera nigricornis was the dominant species in potato fields, while Bactericera trigonica was the most abundant in carrot fields, followed by Heterotrioza chenopodii, ranking as the second most abundant species in both cases. CLso-positive samples were found in D. carota, B. trigonica, H. chenopodii, B. nigricornis, and Trioza urticae. The sequencing results suggest the detected haplotypes are D and U. These findings raise concerns about the potential spread of CLso and the associated risk of significant economic losses.

ABSTRACT Although the high-altitude limit for microbial survival in the Earth–atmosphere system has remained a scientific curiosity and topic of study, the ecological significance of long-distance microbial dispersal in the atmosphere has been perceived to have marginal relevance. Here, we report the characterization of novel plant pathogenic species of Curtobacterium that were isolated from samples collected at altitudes ranging from 1.5 to 29 km above sea level. Whole genome-based phylogenies of three strains, paired with plant challenge assays, indicate that each is a previously unrecognized species and causes disease on beans comparable to Curtobacterium flaccumfaciens . Isolates from the stratosphere (strain L6-1) and agricultural millet (G77) were identified to be the same species and designated as Curtobacterium aetherium sp. nov. C. aetherium displays high levels of tolerance to desiccation and UV radiation, which are stresses that increase in intensity with altitude. Back trajectory air mass analysis implied that the phytopathogens may have had an intercontinental source, but regional origins in the continental US cannot be excluded. The environmentally robust phytopathogens we have documented in the upper atmosphere provide new perspective on the role that high-altitude transport may play in microbial dispersal, gene flow, and the epidemiology of aerially dispersed plant disease. IMPORTANCE Enormous quantities and varieties of microorganisms are continually aerosolized and transported in the atmosphere, yet there is a limited understanding of the consequences to downwind ecosystems. While studying bacteria that survive extreme conditions at altitudes up to 29 km in the atmosphere, we discovered new species that have the capacity to cause disease in agriculturally relevant bean varieties. The hardiest isolate we characterized from the stratosphere is a member of the same species as an isolate from an agricultural source, which we have designated Curtobacterium aetherium . C. aetherium is a phytopathogen capable of enduring high-altitude and long-distance atmospheric transport while also possessing the potential to opportunistically infect crops in deposition locations.

Abstract Numerous bacteria in the human gut microbiome remain unknown and/or have yet to be cultured. While collections of human gut bacteria have been published, few strains are accessible to the scientific community. We have therefore created a publicly available collection of bacterial strains isolated from the human gut. The Human intestinal Bacteria Collection (HiBC) (https://www.hibc.rwth-aachen.de) contains 340 strains representing 198 species within 29 families and 7 phyla, of which 29 previously unknown species are taxonomically described and named. These included two butyrate-producing species of Faecalibacterium and new dominant species associated with health and inflammatory bowel disease, Ruminococcoides intestinale and Blautia intestinihominis, respectively. Plasmids were prolific within the HiBC isolates, with almost half (46%) of strains containing plasmids, with a maximum of six within a strain. This included a broadly occurring plasmid (pBAC) that exists in three diverse forms across Bacteroidales species. Megaplasmids were identified within two strains, the pMMCAT megaplasmid is globally present within multiple Bacteroidales species. This collection of easily searchable and publicly available gut bacterial isolates will facilitate functional studies of the gut microbiome.

Abstract The genus “Candidatus Kouleothrix” (Eikelboom type 1851) is the major filamentous bacterium responsible for activated sludge bulking in Japanese activated sludge plants, where it is present in many in unusually high abundances. Global surveys have shown this genus embraces several species, although the contribution each of these makes to bulking is not known. This study followed their population dynamics in a full-scale bulking sludge plant in Japan over a 12-month period, which required the development of species-specific FISH probes, used to measure their relative abundances. Six of the 9 recognised species of “Ca. Kouleothrix” were detected there, two of which (midas_s_3423 and midas_s_35412) were at consistently high abundances and responsible for increases in the sludge volume index (SVI). Their abundances were also statistically correlated with their filament lengths, which showed higher correlations with SVI increases than did species abundances. This study examined the impacts of both operational parameters and other communities on the abundances of these two species over the 12-month period and showed marked interspecies differences in responses to both. Together these findings stress the importance in studies of this kind to identify populations to species level to reveal possible important ecological differences not seen at the genus level.