Publications

4378

‘Candidatus Liberibacter’ spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. ‘Ca. L. asiaticus’, causal agent of Huanglongbing or citrus “greening,” and ‘Ca. L. solanacearum’, causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters, and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step, and highly reproducible protocols for axenic culture, transformation, and targeted gene knockouts of L. crescens are described. In the course of developing these protocols, we found that L. crescens is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

Cable bacteria of the family Desulfobulbaceae form centimeter-long filaments comprising thousands of cells. They occur worldwide in the surface of aquatic sediments, where they connect sulfide oxidation with oxygen or nitrate reduction via long-distance electron transport. In the absence of pure cultures, we used single-filament genomics and metagenomics to retrieve draft genomes of 3 marine Candidatus Electrothrix and 1 freshwater Ca. Electronema species. These genomes contain >50% unknown genes but still share their core genomic makeup with sulfate-reducing and sulfur-disproportionating Desulfobulbaceae, with few core genes lost and 212 unique genes (from 197 gene families) conserved among cable bacteria. Last common ancestor analysis indicates gene divergence and lateral gene transfer as equally important origins of these unique genes. With support from metaproteomics of a Ca. Electronema enrichment, the genomes suggest that cable bacteria oxidize sulfide by reversing the canonical sulfate reduction pathway and fix CO 2 using the Wood–Ljungdahl pathway. Cable bacteria show limited organotrophic potential, may assimilate smaller organic acids and alcohols, fix N 2 , and synthesize polyphosphates and polyglucose as storage compounds; several of these traits were confirmed by cell-level experimental analyses. We propose a model for electron flow from sulfide to oxygen that involves periplasmic cytochromes, yet-unidentified conductive periplasmic fibers, and periplasmic oxygen reduction. This model proposes that an active cable bacterium gains energy in the anodic, sulfide-oxidizing cells, whereas cells in the oxic zone flare off electrons through intense cathodic oxygen respiration without energy conservation; this peculiar form of multicellularity seems unparalleled in the microbial world.

AbstractTheCampylobacterotais a novel phylum created from the reclassification of the proteobacterial classEpsilonproteobacteriaand orderDesulfurellales(Deltaproteobacteria). The phylum is organized according to phylogenies based on a concatenated alignment of 120 conserved protein marker sequences and the 16S rRNA gene. Like any phylogenetic inference, this road map is a hypothesis based on the data available at the time of writing and may be subject to change as new species are described, or other gene sequences considered. This road map incorporates relative evolutionary divergence to normalize subordinate ranks. It, therefore, includes a number of changes at the rank of order, family, and genus, which would otherwise not be affected by the merging of the former lineages.

Abstract Me.tha.no.hal'ar.chae.um N.L. neut. n. methanum methane; N.L. pref. methano ‐ pertaining to methane; Gr. masc. n. hals, halos salt; N.L. neut. n. archaeum (pertaining to archaios, ‐e, ‐on) ancient; N.L. neut. n. Methanohalarchaeum a salt‐loving archaeon forming methane. The candidate genus Ca . Methanohalarchaeum includes obligately anaerobic, extremely halophilic, neutrophilic, and moderately thermophilic methanogens with small coccoid cells producing methane by reduction of C 1 ‐methylated compounds using either H 2 or formate as electron donors. Amino acids or acetate is required as C‐source for assimilation. Core membrane lipids are represented by diphytanyl glycerol tetra‐ and diethers. Cells accumulate potassium as a compatible solute. They inhabit hypersaline lakes or salterns with neutral pH. The genus contains a single candidate species Ca . M. thermophilum, which is the type species. It is a member of the class Methanonatronarchaeia within a newly proposed phylum Halobacteriota (formerly part of the phylum Euryarchaeota ). DNA G + C content (mol%) : 35.4 (genome of the type strain). Type species : Ca . Methanohalarchaeum thermophilum Sorokin, Merkel, Abbas, Makarova, Rijpstra et al. 2018, 2206. Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the genus Candidatus Methanohalarchaeum is: preferred name (not correct name) (last update, February 2025) * . LPSN classification: Archaea / Methanobacteriati / Methanobacteriota / Methanonatronarchaeia / Methanonatronarchaeales / Methanonatronarchaeaceae / Candidatus Methanohalarchaeum The genus Candidatus Methanohalarchaeum can also be recovered in the Genome Taxonomy Database (GTDB) as g__Methanohalarchaeum (version v220) ** . GTDB classification: d__Archaea / p__Halobacteriota / c__Methanonatronarchaeia / o__Methanonatronarchaeales / f__Methanonatronarchaeaceae / g__Methanohalarchaeum * Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776

Abstract Sci.sci.o.nel'la. N.L. fem. dim. n. Sciscionella arbitrary name formed from the acronym of the South China Sea Institute of Oceanology, SCISCIO, where taxonomic studies on this taxon were performed. The genus Sciscionella is classified as a member of the family Pseudonocardiaceae , order Pseudonocardiales , and in the class Actinobacteria . The member of this genus has a 16S rRNA sequence identity of less than 94% with the other members of the family Pseudonocardiaceae . In the phylogenetic tree based on 16S rRNA gene sequences, the single member of this genus, Sciscionella marina , forms a distant clade, closely related to the genera Haloechinothrix, Thermocrispum , and Yuhushiella . Cells are characterized with cell‐wall chemotype IV and phospholipid pattern type III sensu Lechevalier et al. 1977. The predominant respiratory quinone is MK‐9(H 4 ), and the fatty acid composition is dominated by saturated branched‐chain fatty acids, but not mycolic acids. Phenotypically, strains are aerobic and stain Gram‐positive. They could grow at 10–37°C, pH 6.0–8.0, and in the presence of up to 13% NaCl (w/v). The genus comprises one species, Sciscionella marina (the type species of the genus). DNA G + C content (mol%) : 68 (WGS). Type species : Sciscionella marina Tian, Zhi, Qiu, Zhang, Tang, Xu et al. 2009, 225 VP . Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the genus Sciscionella is: correct name (last update, February 2025) * . LPSN classification: Bacteria / Bacillati / Actinomycetota / Actinomycetes / Pseudonocardiales / Pseudonocardiaceae / Sciscionella The genus Sciscionella can also be recovered in the Genome Taxonomy Database (GTDB) as g__Sciscionella (version v220) ** . GTDB classification: d__Bacteria / p__Actinomycetota / c__Actinomycetes / o__Mycobacteriales / f__Pseudonocardiaceae / g__Sciscionella * Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776

Citrus greening disease, also known as huanglongbing (HLB), is the most devastating disease of Citrus worldwide. This incurable disease is caused primarily by the bacterium Candidatus Liberibacter asiaticus and spread by feeding of the Asian Citrus Psyllid, Diaphorina citri . Ca . L. asiaticus cannot be cultured; its growth is restricted to citrus phloem and the psyllid insect. Management of infected trees includes use of broad-spectrum antibiotics, which have disadvantages. Recent work has sought to identify small molecules that inhibit Ca . L. asiaticus transcription regulators, based on a premise that at least some regulators control expression of genes necessary for virulence. We describe a synthetic, high-throughput screening system to identify compounds that inhibit activity of Ca . L. asiaticus transcription activators LdtR, RpoH, and VisNR. Our system uses the closely related model bacterium, Sinorhizobium meliloti , as a heterologous host for expression of a Ca . L. asiaticus transcription activator, the activity of which is detected through expression of an enhanced green fluorescent protein (EGFP) gene fused to a target promoter. We used this system to screen more than 120,000 compounds for compounds that inhibited regulator activity, but not growth. Our screen identified several dozen compounds that inhibit regulator activity in our assay. This work shows that, in addition to providing a means of characterizing Ca . L. asiaticus regulators, an S. meliloti host can be used for preliminary identification of candidate inhibitory molecules.

Candidatus Liberibacter solanacerum (CLso), transmitted by Bactericera trigonica in a persistent and propagative mode causes carrot yellows disease, inflicting hefty economic losses. Understanding the process of transmission of CLso by psyllids is fundamental to devise sustainable management strategies. Persistent transmission involves critical steps of adhesion, cell invasion, and replication before passage through the midgut barrier. This study uses a transcriptomic approach for the identification of differentially expressed genes with CLso infection in the midguts, adults, and nymphs of B. trigonica and their putative involvement in CLso transmission. Several genes related to focal adhesion and cellular invasion were upregulated after CLso infection. Interestingly, genes involved with proper functionality of the endoplasmic reticulum (ER) were upregulated in CLso infected samples. Notably, genes from the endoplasmic reticulum associated degradation (ERAD) and the unfolded protein response (UPR) pathway were overexpressed after CLso infection. Marker genes of the ERAD and UPR pathways were also upregulated in Diaphorina citri when infected with Candidatus Liberibacter asiaticus (CLas). Upregulation of the ERAD and UPR pathways indicate induction of ER stress by CLso/CLas in their psyllid vector. The role of ER in bacteria–host interactions is well-documented; however, the ER role following pathogenesis of CLso/CLas is unknown and requires further functional validation.

Abstract The actinobacterial acI lineage is among the most successful and ubiquitous freshwater bacterioplankton found on all continents, often representing more than half of all microbial cells in the lacustrine environment and constituting multiple ecotypes. However, stably growing pure cultures of the acI lineage have not been established despite various cultivation efforts based on ecological and genomic studies on the lineage, which is in contrast to the ocean from which abundant microorganisms such as Prochlorococcus, Pelagibacter, and Nitrosopumilus have been isolated. Here, we report the first two pure cultures of the acI lineage successfully maintained by supplementing the growth media with catalase. Catalase was critical for stabilizing the growth of acI strains irrespective of the genomic presence of the catalase-peroxidase (katG) gene. The two strains, representing two novel species, displayed differential phenotypes and distinct preferences for reduced sulfurs and carbohydrates, some of which were difficult to predict based on genomic information. Our results suggest that culture of previously uncultured freshwater bacteria can be facilitated by a simple catalase-supplement method and indicate that genome-based metabolic prediction can be complemented by physiological analyses.

Abstract Human activities such as deforestation, urbanization, and environmental pollution lead to a reduction in the spatial boundary between wild animals, domestic animals and humans. These activities increase the risk for the emergence of pathogens from the sylvatic cycle in the population of domestic animals and humans. Foxes are recognized as potential reservoirs for a number of bacterial pathogens of medical and public health concern. The aim of the present study was to investigate the prevalence and spatial distribution of bacterial tick-borne pathogens from the Anaplasmataceae family, Borrelia burgdorferi sensu lato (s.l.), Rickettsia spp., Coxiella burnetii, Francisella tularensis, Bartonella spp., in the red fox population from Serbia and to discuss the obtained results from the epidemiological point of view. Legally hunted red foxes (Vulpes vulpes) from 14 localities in Serbia were included in the study and spleen samples from 129 animals were tested with conventional PCR assays for the presence of bacterial tick-borne pathogens. DNA of Candidatus Neoehrlichia sp. (FU98), Borrelia burgdorferi sensu stricto, Borrelia lusitaniae, and Borrelia garinii was detected in 6 (4.7%), 1 (0.8%), 2 (1.6%) and 1 (0.8%) animals, respectively. Co-infection by Candidatus Neoehrlichia sp. (FU98) and B. garinii was detected in one animal. All samples were negative for other tested bacterial tick-borne pathogens. The results of the present study indicate the potential role of foxes in natural cycles of Candidatus Neoehrlichia sp. (FU98) and causative agents of Lyme borreliosis in the investigated areas. Further research is required to elucidate the role of foxes in the epidemiology of these and other tick-borne zoonotic pathogens in the Republic of Serbia.