Publications

4372

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.

Grapevine (Vitis vinifera L.) is one of the most important horticultural crops in Iran, with >200,000 ha of cultivated area. Recently, outbreaks of the grapevine yellows Bois noir that is associated with phytoplasma strains related to ‘Candidatus Phytoplasma solani’ were recorded in several Iranian regions. This has resulted in severe economic losses. We carried out a survey in 2015, followed by collection of leaf samples from symptomatic grapevines and weeds. Because no information is available on the molecular epidemiology of ‘Ca. P. solani’ in Iran, multiple gene analyses were carried out here according to molecular characterization of the tuf and vmp1 genes. From the molecular characterization, all of the samples (i.e., grapevines, weeds) were infected with tuf b type. Detailed molecular characterization of the vmp1 gene (i.e., PCR–restriction fragment length polymorphism, sequence analysis) defined five molecular types: V1, V4, V10, V15, and V20. The abundance of Convolvulus arvensis in vineyards and detection of the same ‘Ca. P. solani’ molecular types in grapevines and weeds suggest that C. arvensis has a major role in Bois noir epidemiology of Iranian vineyards. Therefore, control strategies should be developed to manage these host plants to reduce inoculum sources of the phytoplasma in vineyards.

Citrus huanglongbing (HLB) is one of the most destructive diseases affecting citrus plants. ‘Candidatus Liberibacter asiaticus’, an uncultivated α-proteobacteria, is the most widely spread causal agent of HLB and is transmitted by the Asian citrus psyllid Diaphorina citri. ‘Ca. L. asiaticus’ attachment to the psyllid midgut is believed to be critical to further infect other organs, including the salivary gland. In this study, the type IVc tight adherence (Tad) pilus locus encoded by ‘Ca. L. asiaticus’ was characterized. The Tad loci are conserved among members of Rhizobiaceae, including ‘Ca. L. asiaticus’ and Agrobacterium spp. Ectopic expression of the ‘Ca. L. asiaticus’ cpaF gene, an ATPase essential for the biogenesis and secretion of the Tad pilus, restored the adherence phenotype in cpaF mutant of A. tumefaciens, indicating CpaF of ‘Ca. L. asiaticus’ was functional and critical for bacterial adherence mediated by Tad pilus. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that ‘Ca. L. asiaticus’ Tad pilus-encoding genes and ‘Ca. L. asiaticus’ pilin gene flp3 were upregulated in psyllids compared with in planta. A bacterial one-hybrid assay showed that ‘Ca. L. asiaticus’ VisN and VisR, members of the LuxR transcriptional factor family, were bound to the flp3 promoter. VisNR regulate flp3. Negative regulation of the flp3 promoter by both VisN and VisR was demonstrated using a shuttle strategy, with analysis of the phenotypes and immunoblotting together with quantification of the expression of the flp3 promoter fused to the β-galactosidase reporter gene. Comparative expression analysis confirmed that ‘Ca. L. asiaticus’ visNR was less expressed in the psyllid than in the plant host. Further, motility and biofilm phenotypes of the visNR mutant of A. tumefaciens were fully complemented by expressing ‘Ca. L. asiaticus’ visNR together. The physical interaction between VisN and VisR was confirmed by pull-down and stability assays. The interaction of the flp3 promoter with VisR was verified by electrophoretic mobility shift assay. Taken together, the results revealed the contribution of the Tad pilus apparatus in the colonization of the insect vector by ‘Ca. L. asiaticus’ and shed light on the involvement of VisNR in regulation of the Tad locus.

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.

The amino acid‐binding receptors, a component of ABC transporters, have evolved to cater to different specificities and functions. Of particular interest are cystine‐binding receptors, which have shown broad specificity. In the present study, a putative periplasmic cystine‐binding protein from Candidatus Liberibacter asiaticus (CLasTcyA) was characterized. Analysis of the CLasTcyA sequence and crystal structures in the ligand‐bound state revealed novel features of CLasTcyA in comparison to related proteins. One of the unique features found in CLasTcyA structure was the positioning of the C‐terminal extended loop of one chain very close to the substrate‐binding site of the adjacent monomer in the asymmetric unit. The presence of a disulphide bond, unique to Candidatus Liberibacter family, holds the C‐terminal extended loop in position. Analysis of the substrate‐binding pocket of CLasTcyA suggested a broad specificity and a completely different orientation of the bound substrates in comparison to related protein structures. The open conformation for one of the two chains of the asymmetric unit in the Arg‐bound structure revealed a limited open state (18.4°) for CLasTcyA as compared to open state of other related proteins (~ 60°). The strong interaction between Asp126 on helix‐α5 of small domain and Arg82 (bigger domain) restricts the degree of opening in ligand‐free open state. The dissociation constant of 1.26 μm by SPR and 3.7 μm by MST exhibited low affinity for the cystine. This is the first structural characterization of an l‐cystine ABC transporter from plant pathogen and our results suggest that CLasTcyA may have evolved to cater to its specific needs for its survival in the host.