Publications

4467

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. 

ABSTRACT The complete genome, 173,958 bp in size, of “ Candidatus Carsonella ruddii” DC-OKEB1 , an obligate bacterial endosymbiont of the Asian citrus psyllid Diaphorina citri , was determined. The genome sequence provides valuable information for comparative and evolutionary aspects of the intimate insect–microbe mutualism.

ABSTRACT The complete genome, 226,287 bps in size, of “ Candidatus Nardonella sp.” Pin-AIST, an obligatory bacterial endosymbiont of the black hard weevil Pachyrhynchus infernalis , was sequenced. The extremely reduced endosymbiont genome is specialized for tyrosine synthesis, which contributes to the hardness of the beetle’s exoskeleton.

ABSTRACT We report the draft genomes of five Clostridium isolates from soil and agricultural by-products, four of which are proposed as Candidatus species. Members of the genus Clostridium are of significant industrial interest, and the availability of their genome sequences facilitates the understanding and exploration of their functional potential.