Publications

4478

ABSTRACT The Coxiellaceae bacterial family, within the order Legionellales, is defined by a collection of poorly characterized obligate intracellular bacteria. The zoonotic pathogen and causative agent of human Q fever, Coxiella burnetii , represents the best-characterized member of this family. Coxiellaceae establish replicative niches within diverse host cells and rely on their host for survival, making them challenging to isolate and cultivate within a laboratory setting. Here, we describe a new genus within the Coxiellaceae family that has been previously shown to infect economically significant freshwater crayfish. Using culture-independent long-read metagenomics, we reconstructed the complete genome of this novel organism and demonstrate that the species previously referred to as Candidatus Coxiella cheraxi represents a novel genus within this family, herein denoted Candidatus Paracoxiella cheracis . Interestingly, we demonstrate that Candidatus P. cheracis encodes a complete, putatively functional Dot/Icm type 4 secretion system that likely mediates the intracellular success of this pathogen. In silico analysis defined a unique repertoire of Dot/Icm effector proteins and highlighted homologs of several important C. burnetii effectors, including a homolog of CpeB that was demonstrated to be a Dot/Icm substrate in C. burnetii . IMPORTANCE Using long-read sequencing technology, we have uncovered the full genome sequence of Candidatus Paracoxiella cheracis , a pathogen of economic importance in aquaculture. Analysis of this sequence has revealed new insights into this novel member of the Coxiellaceae family, demonstrating that it represents a new genus within this poorly characterized family of intracellular organisms. Importantly, the genome sequence reveals invaluable information that will support diagnostics and potentially both preventative and treatment strategies within crayfish breeding facilities. Candidatus P. cheracis also represents a new member of Dot/Icm pathogens that rely on this system to establish an intracellular niche. Candidatus P. cheracis possesses a unique cohort of putative Dot/Icm substrates that constitute a collection of new eukaryotic cell biology-manipulating effector proteins.

Phytoplasmas (genus ‘Candidatus Phytoplasma’) encompass a group of uncultivated bacteria affecting numerous plant species and causing significant damage in agriculture worldwide. They have a dual parasitic cycle, including colonization of both plant phloem and insect cells. Their genomes are small, diverse, repetitive, prone to rearrangements and harbour transposon-like elements known as potential mobile units (PMUs). In the Euro-Mediterranean region, ‘Ca. P. solani’ is an important species due to its broad range of plant hosts and insect vectors. To provide insights into the genomic diversity of this species, particularly the repertoire of putative effectors and PMUs, this study conducted genome sequencing and analyses of two ‘Ca. P. solani’ strains originating from different plants and transmitted by different insects. Based on de novo assembly, we obtained 19 contigs totalling 656 141 bp for strain STOL and 28 contigs totalling 707 036 bp for strain ST19. The prevalence of repetitive sequences and PMUs contributed to the fragmentation of these draft assemblies. The annotation identified 28 and 26 genes that encode putative secreted proteins in these two strains, respectively, including several homologues of previously characterized phytoplasma effectors. Our comparative analyses further identified species- and strain-specific genes. Frequently, genes that encode putative secreted proteins and effectors were found within PMU-like regions in both genomes. Moreover, strain STOL showed characteristics of a more reduced genome, having fewer PMU-like repetitive elements and genome rearrangements, while strain ST19 exhibited a higher level of sequence divergence in its PMU genes. The high levels of genomic diversity among ‘Ca. P. solani’ strains suggested rapid evolution of this species, which may contribute to its wide host range and adaptability potential. This study provides novel data on the diversification of ‘Ca. P. solani’ genomes. These results provide a foundation for future functional studies of putative effectors and their interactions with host targets, which could facilitate deciphering the pathogenicity strategies of this successful and versatile pathogen.

A Gram-stain-negative, aerobic, rod-shaped bacterial strain, SYSU D60014T, was isolated from a sandy soil sample collected from the Gurbantunggut Desert in Xinjiang, PR China. Colonies of SYSU D60014T were pink-coloured, crystalline, irregular edge with a rough surface. Phylogenetic analyses based on both the 16S rRNA gene and whole-genome sequences assigned strain SYSU D60014T belonged to the family Rhodospirillaceae, formed a distinct lineage and showed 92.5–91.0% similarity to closely related strains. Digital DNA–DNA hybridization, average nucleotide identity and average amino acid identity values between strain SYSU D60014T and members of its related species were 17.2–19.6%, 70.8–74.4% and 48.9–64.5%, respectively. The complete genome of strain SYSU D60014T was 5,100,926 bp with a DNA G+C content of 65.1%. Cells were oxidase-positive and catalase-negative. The strain could grow at 28–40 °C (optimum, 37 °C), pH 5.0–8.0 (optimum, pH 7.0), and in the presence of up to 3% NaCl (optimum, 1–1.5%, w/v) on R2A. The predominant menaquinone was Q-10. The main polar lipids were phosphatidylethanolamine, phosphatidylmethyl ethanolamine, diphosphatidylglycerol, unidentified aminophospholipid, unidentified aminolipid, unidentified phospholipid, four unidentified polar lipids and two unidentified phosphoglycolipids. Major cellular fatty acids were C19 : 0 cyclo ω8c, summed feature 8 (C18 : 1  ω7c and/or C18 : 1  ω6c) and C16 : 0. Based on the results obtained through genotypic and phenotypic analyses, we propose that strain SYSU D60014T represents a novel species and genus within the family Rhodospirillaceae, for which we propose the name Virgifigura deserti gen. nov., sp. nov. (type strain SYSU D60014T=NBRC 112951T=CPCC 101030T).

Background Hyaluronan synthases (HASs) are ubiquitous in living organisms, and the hyaluronic acid (HA) synthesized by them are important to their body and well used in medicine, cosmetics and other fields. HAS from deep-sea creatures has not yet been explored before. The study aims to analyse the characteristics and enzyme kinetics of a novel hyaluronan synthase derived from the symbiont “Candidatus Mycoplasma liparidae” found in deep-sea snailfish (snHAS). Methodology snHAS was over-expressed using His 6 as tag in the study. The sequence alignment was conducted by Cluster W and then the phylogenetic analyse of HASs was performed by Mega 6.0 to investigate the position of snHAS during evolution. Km and Vmax were detected to study the enzyme kinetics of snHAS wildtype and its mutant. The molecular weight of HA was evaluated by high performance gel permeation chromatography (HPGPC). The cardiolipin was added to investigate whether it had a promoting effect on the snHAS. Results The length of snHAS was 933 bp with an open reading frame (ORF) of 310 amino acids. Unlike other repoted HASs, snHAS had no transmembrane region and was not classified into the currently known Class I or Class II. snHAS could synthesize hyaluronan with lower molecular weights using the substrates of uridine-diphosphate—N-acetylglucosamine (UDP-GlcNAc) and uridine-diphosphate—glucuronic acid (UDP-GlcA) in vitro. The Km values of snHAS were 258 ± 45 µM and 39 ± 5 µM for UDP-GlcNAc and UDP-GlcA, respectively, much lower than those from mice (Km for UDP-GlcA: 55 ± 5 µM; Km for UDP-GlcNAc: 870 ± 60 µM). The kcat/Km values of snHAS were 163.5 s−1 mM−1 and 8.08 s−1 mM−1 for UDP-GlcA and UDP-GlcNAc, respectively. Furthermore, the activity of snHAS was independent of cardiolipin. Conclusions snHAS was a novel HAS based on the characteristics of the animo acid sequence, which could produce low molecular weight of HA with high efficiency. This provides a molecular basis for the biosynthesis of low molecular weight of HA.

In Graneros, O’Higgins Region, Chile, the mallow psyllid (Russelliana solanicola Tuthill, 1959) from Malva nicaeensis L. was identified as a potential vector of ‘Candidatus Phytoplasma pruni’. Over an 8-month period, 2089 specimens of a species of Psylloidea, including immatures and adults, were captured. We only selected the adults used for transmission trials in Catharanthus roseus (L.) G. Don (periwinkle) plants. By nested PCR, using primer pairs for phytoplasma detection in 16S rRNA and IdpA genes, 7 out of 113 (6.2%) periwinkle plants used in transmission trials were found to be infected by phytoplasmas. Insects that fed on these plants also tested positive for the same phytoplasmas. Periwinkle plants never showed virescence and phyllody, as commonly occurs with phytoplasma 16SrIII-J infection due to the effector SAP54. In this case, using primer pairs for the SAP54 gene, an amplification product was never obtained. Virtual restriction fragment length polymorphism (RFLP) analysis of F2nR2 fragments indicated that the phytoplasma, found in both periwinkle plants and insects used in transmission trials, belongs to the 16SrIII-J ribosomal subgroup. The COI gene of the psyllids samples was amplified and sequenced, showing a similarity ranging from 84.84% to 85.02% with R. solanicola from Solanum tuberosum L. The mitochondrial genome of the psyllid was also sequenced, revealing a 14,835 bp circular DNA molecule with 37 genes. The mallow psyllid transmitted the phytoplasma 16SrIII-J to periwinkle plants. The molecular identification of the insect does not match the morphological one, indicating that the mallow psyllid may constitute a cryptic species within the polyphagous R. solanicola species. This is the first report of a psyllid as a vector of the phytoplasma 16SrIII-J.

A Gram-positive, aerobic, non-motile coccoid bacterium, designated strain ZF39T, was isolated from a farm in Quandian Village, Zhengzhou City, Henan Province, China, and subjected to taxonomic characterization. Optimal growth occurred at 32 ℃, pH 7.0 and 0–1% NaCl (w/v). Mannose and glucose were identified as the sugar components in the whole-cell hydrolysates. The strain’s peptidoglycan type was classified as A3γ (ll-Dpm-Gly), and MK-9(H4) was identified as the predominant respiratory quinone. Aminoglycolipid, aminophospholipid, glycolipids, phosphatidylglycerol and phospholipid were detected as the major polar lipids. The predominant cellular fatty acids were anteiso-C15 : 0 (48.7%) and iso-C15 : 0 (20.2%). Phylogenetic analysis based on 16S rRNA gene sequences placed strain ZF39T within the family Propionibacteriaceae. The strain formed a distinct genetic lineage adjacent to the genus Desertihabitans, with 16S rRNA gene sequence similarities of 94.70% to Desertihabitans aurantiacus CPCC 204711T and 94.56% to Desertihabitans brevis KCTC 49116T. The genomic G+C content was 67.7 mol%. Genome comparisons revealed that the average nucleotide identity values between strain ZF39T and D. aurantiacus CPCC 204711T, D. brevis KCTC 49116T, Granulicoccus phenolivorans DSM 17626T were 72.50%, 71.98% and 72.36%, respectively. Moreover, phylogenetic analysis and phenotypic characterization supported the classification of strain ZF39T as a novel species of a new genus within the family Propionibacteriaceae. The name Ammonicoccus fulvus gen. nov., sp. nov. is proposed, with ZF39T (=CGMCC 1.60047T=KCTC 59250T=MCCC 1K08851T) as the type strain.

Abstract Uncultured UBA5794 actinobacteria are frequently found in marine and inland water environments by using metagenomic approaches. However, knowledge about these actinobacteria is limited, hindering their isolation and cultivation, and they are always confused with “Candidatus Actinomarinales” based on 16S rRNA gene classification. Here, to conduct genomic characterization of them, we obtained three high-quality UBA5794 metagenome-assembled genomes (MAGs) from a hydrothermal sediment on the Carlsberg Ridge (CR) and retrieved 131 high-quality UBA5794 genomes from public datasets. Phylogenomic analysis confirms UBA5794 as an independent order within the class Acidimicrobiia. Genome-based metabolic predictions reveal that flexible metabolism and diversified energy acquisition, as well as heavy metal(loid) detoxification capacity, are crucial for the ability of UBA5794 to thrive in diverse environments. Moreover, there is separation between sponge-associated and free-living UBA5794 groups in phylogeny and functional potential, which can be attributed to the symbiotic nature of the sponge-associated group and the extensive horizontal gene transfer (HGT) events observed in these bacteria. Ancestral state reconstruction suggests that the UBA5794 clade may have originated from a free-living environment and then some members gradually migrated to the sponge host. Overall, our study sheds light on the ecological adaptation and evolutionary history of the ubiquitous but poorly understood UBA5794 actinobacteria.

‘Candidatus Phytoplasma prunorum’ has been associated with severe disease in Prunus spp., which are commodities of economic importance in the USA. The introduction and establishment of ‘Ca. P. prunorum’ in the USA could result in huge economic losses, thus creating a need for validated diagnostic tools, which are the cornerstone of successful surveillance, quarantine, and eradication measures. Whole-genome comparisons led to the identification of a diagnostic marker gene specific to ‘Ca. P. prunorum’ (PE639). The PE639 assay was duplexed with an 18S rDNA plant internal control and compared to modified 23S (phytoplasmas) and imp (‘Ca. P. mali’) assays. The PE639 assay produced congruent results to 23S and imp assays for all metrics, demonstrating high linearity, repeatability, intermediate precision, and reproducibility. The limit of detection was comparable for all assays tested, and all demonstrated 100% analytical specificity, selectivity, and diagnostic specificity for their respective target species. Assays metrics were consistent across two platforms, the ABI QuantStudio™ 5 and Bio-Rad CFX96™ OPUS. A synthetic gBlocks™ control was designed and validated to work with all assays, as well as conventional PCR assays targeting 16S rDNA and tuf genes. These validated assays and synthetic control represent beneficial tools that support efforts to protect USA agriculture and facilitate safe trade.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an invasive citrus pest that vectors the phloem-dwelling bacterium, Candidatus Liberibacter asiaticus (CLas), which is a putative causal agent of citrus greening disease known as Huanglongbing (HLB). In Florida, the D. citri–CLas pathosystem complex is at present considered the greatest threat the citrus industry must manage. Brassinosteroids (Brs), a relatively new class of plant hormones, are approved for commercial use in Florida citrus and are believed to delay HLB progression. Individual protective covers (IPCs), a type of psyllid exclusion mesh bags, are increasingly being adopted to efficiently protect newly planted citrus trees from HLB infection. However, IPCs typically must be removed after 2–3 years due to tree growth. With the aim of prolonging tree health after IPC removal, we are investigating the efficacy Brs in protecting and/or preventing trees from ACP and CLas infection once they are left exposed. Upon IPC removal from trees covered for 3 years, monthly Br treatments started in trees from three mandarin cultivars (Early Pride, SugarBelle and Tango) on two different rootstocks, sour orange and US-942. The IPCs effectively excluded psyllids, prevented CLas infection, and clearly improved most of the horticultural traits measured. Although this is an on going experiment, we have already found that treatment with Brs reduced CLas bacterial titer by 10-fold on average two months after IPC removal. The long-term efficacy of the treatments still needs to be assessed and will be discussed.