Publications

4358

The occurrence of ‘Candidatus Liberibacter’ spp. and ‘Ca. Phytoplasma’ spp. associated with blotchy mottle symptoms poses challenges to huanglongbing (HLB) diagnosis using molecular techniques. The ability to detect multiple targets simultaneously and specifically is a key aspect met by quantitative PCR (qPCR). A set of primers and hydrolysis probes useful in either single or multiplex reactions for the detection and quantification of HLB-associated bacteria were developed. Sequences from conserved genes of the ribosomal proteins for Liberibacter and phytoplasma circumvent the lack of specificity and cross-reactivity problems related to 16Sr DNA gene amplification, allowing precise and specific detection of HLB-associated bacteria in citrus and in the Liberibacter vector, Diaphorina citri. The triplex reaction exhibited high quality and precision as a robust tool for quantifying ‘Ca. L. asiaticus’ (CLas), ‘Ca. L. americanus’ (CLam), and 16Sr IX phytoplasma. Triplex qPCR showed consistent results and comparable sensitivity to the ribonuclease reductase test, although quantification cycle (Cq) values were higher when compared with 16SrDNA qPCR. Detection tests using field samples indicate that the qPCR triplex can identify HLB-associated bacteria in samples with varying levels of symptoms, ranging from typical to asymptomatic. Assessment of field samples from growers indicated more than 78.6% had Cq lower than 35.0, below the cutoff established for qPCR reactions used in this work. qPCR triplex is a safe, specific, and sufficiently sensitive technique for detecting CLas, CLam, and 16Sr IX phytoplasma simultaneously, in both citrus and D. citri samples. Its application is of importance in assisting growers in making decisions for HLB management.

Opinion 131 addresses a Request for an Opinion asking the Judicial Commission to conserve the genus name Proteus Hauser 1885 (Approved Lists 1980) over its earlier homonym, the protozoan genus name Proteus Müller 1786. The Judicial Commission agrees that the later homonym is illegitimate and that the replacement of the prokaryotic name Proteus would be undesirable. It is also concluded that Proteus Müller 1786 is an objectively invalid name under the International Code of Zoological Nomenclature. Judicial Opinions 9, 12 and 130 serve as precedents for the conservation of Proteus Hauser 1885 (Approved Lists 1980) over Proteus Müller 1786. This action is taken here and makes the prokaryotic name Proteus legitimate.

The anammox bacteria “Candidatus Kuenenia stuttgartiensis” (Ca. Kuenenia) are able to gain energy by combining ammonium and nitrite to produce nitrogen gas, which is an ecologically and technically significant activity process. In this reaction, nitric oxide serves as a recognized intermediate in the reduction of nitrite, which is subsequently combined with ammonium to produce hydrazine. However, the enzyme that converts nitrite to nitric oxide remains elusive. In this study, we investigated the nitrite-reducing activity in “Ca. Kuenenia stuttgartiensis” strain CSTR1 to identify candidates for such an enzyme. An optimized in vitro assay was established to measure nitrite-reducing activities, with which we followed the activity in protein fractions obtained from various fractionation methods. Separation of the cell extract of strain CSTR1 with size exclusion chromatography yielded active fractions corresponding to a molecular size range of 150–200 kDa. Several proteins coeluted with the nitrite-reducing activity, including the hydroxylamine dehydrogenase HOX, an NADP-dependent isopropanol dehydrogenase (Adh), an electron-transfer 4Fe-4S subunit protein (Fcp), and a nitric oxide detoxifying flavorubredoxin (NorVW). However, further separation of the cell extract with anion exchange chromatography, resulted in much lower activity yields, and activities were distributed among several fractions. In addition, fractionation of cell extracts using ultracentrifugation and ultrafiltration linked the activity to HOX, but could not exclude the involvement of other proteins in the activity. Overall, our results suggest that the molecular mechanism for nitrite reduction in “Ca. Kuenenia” strains is more complex than that currently described in the literature. Nitrite reduction appears to be strongly associated with HOX but may additionally require the participation of other proteins.

A novel, strictly aerobic, non-motile, and pink-pigmented bacterium, designated 7Alg 153T, was isolated from the Pacific green alga Cladophora stimpsonii. Strain 7Alg 153T was able to grow at 4–32 °C in the presence of 1.5–4% NaCl and hydrolyze L-tyrosine, gelatin, aesculin, Tweens 20, 40, and 80 and urea, as well as produce catalase, oxidase, and nitrate reductase. The novel strain 7Alg 153T showed the highest similarity of 96.75% with Pseudaestuariivita rosea H15T, followed by Thalassobius litorarius MME-075T (96.60%), Thalassobius mangrovi GS-10T (96.53%), Tritonibacter litoralis SM1979T (96.45%), and Marivita cryptomonadis CL-SK44T (96.38%), indicating that it belongs to the family Roseobacteraceae, the order Rhodobacteales, the class Alphaproteobacteria, and the phylum Pseudomonadota. The respiratory ubiquinone was Q-10. The main polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, two unidentified aminolipids, and one unidentified lipid. The predominant cellular fatty acids (>5%) were C18:1 ω7c, C16:0, C18:0, and 11-methyl C18:1 ω7c. The 7Alg 153T genome is composed of a single circular chromosome of 3,786,800 bp and two circular plasmids of 53,157 bp and 37,459 bp, respectively. Pan-genome analysis showed that the 7Alg 153T genome contains 33 genus-specific clusters spanning 92 genes. The COG20-annotated singletons were more often related to signal transduction mechanisms, cell membrane biogenesis, transcription, and transport, and the metabolism of amino acids. The complete photosynthetic gene cluster (PGC) for aerobic anoxygenic photosynthesis (AAP) was found on a 53 kb plasmid. Based on the phylogenetic evidence and phenotypic and chemotaxonomic characteristics, the novel isolate represents a novel genus and species within the family Roseobacteraceae, for which the name Algirhabdus cladophorae gen. nov., sp. nov. is proposed. The type strain is 7Alg 153T (=KCTC 72606T = KMM 6494T).

Huanglongbing (HLB) is a devastating disease of citrus plants caused by the non-culturable phloem-inhabiting bacterium Candidatus Liberibacter ssp., being Ca. Liberibacter asiaticus (CLas) the most aggressive species. CLas is vectored by the psyllid Diaphorina citri and introduced into sieve cells, establishing a successful infection in all Citrus species. Partial or complete resistance has been documented in the distant relatives Murraya paniculata and Bergera koenigii, respectively, providing excellent systems to investigate the molecular basis of HLB-resistance. It has been shown previously that the first weeks after bacterial release into the phloem are critical for the establishment of the bacterium. In this study, a thorough transcriptomic analysis of young flushes exposed to CLas-positive and negative psyllids has been performed in Citrus × sinensis, as well as in the aforementioned resistant species, along the first eight weeks after exposure. Our results indicate that the resistant species do not deploy a classical immunity response upon CLas recognition. Instead, transcriptome changes are scarce and only a few genes are differentially expressed when flushes exposed to CLas-positive and negative psyllid are compared. Functional analysis suggests that primary metabolism and other basic cellular functions could be rewired in the resistant species to limit infection. Transcriptomes of young flushes of the three species are very different, supporting the existence of distinct biochemical niches for the bacterium. These findings suggest that both intrinsic metabolic inadequacies to CLas survival, as well as inducible reprogramming of physiological functions upon CLas recognition, could orchestrate together restriction of bacterial multiplication in these resistant hosts.

Fungi engage in diverse symbiotic relationships with phototrophs. Lichens, symbiotic complexes involving fungi and either cyanobacteria, green algae, or both, have fungi forming the external layer and much of the interior. We found an erect thallus resembling a lichen yet with an unexpected thallus structure composed of interwoven cyanobacterial filaments with numerous fungal hyphae inserted within individual cyanobacterial sheaths, contrasting with typical lichen structure. Phylogenetics identified the fungus as a previously undescribed species, Serendipita cyanobacteriicola , closely related to endophytes, and the cyanobacterium belongs to the family Coleofasciculaceae, representing a genus and species not yet classified, Symbiothallus taiwanensis . These thalli exhibit nitrogen-fixing activity similar to mosses but lower than cyanolichens. Both symbiotic partners are distinct from known lichen-forming symbionts, uncovering a phylogenetically and morphologically unprecedented thallus-forming fungus-cyanobacterium symbiosis. We propose the name “phyllosymbia” for these thalli to underscore their unique symbiotic nature and leaf-like appearance. This finding marks a previously unknown instance of fungi solely residing within structures generated by cyanobacteria.

Two Gram-stain-negative, facultative anaerobic, rod-shaped and non-gliding bacteria, designated as HL-DH10T and HL-DH14, were isolated from the halophyte Suaeda japonica in a mudflat, Republic of Korea. Based on the results of 16S rRNA gene pairwise analysis, the two isolates were the members of the family Flavobacteriaceae, and Aestuariibaculum suncheonense SC17T was the most closely related to strains HL-DH10T and HL-DH14 with 96.3% and 95.4% sequence similarity, respectively. The average nt identity and digital DNA–DNA hybridization values between strains HL-DH10T and HL-DH14 and other related species were all less than 79.2% and 21.9%, respectively. The genomic DNA G+C contents of strains HL-DH10T and HL-DH14 were 32.0% and 31.5%, respectively. Cells of these strains showed optimal growth at 25 °C, pH 6.5–7.0 and 2.5–4.0% (w/v) sea salts. The major respiratory quinone was menaquinone-6. The major cellular fatty acids were iso-C15:0 (14.0–16.0%), iso-C15:1 G (10.0–12.0%), iso-C17:0 3-OH (12.4–13.9%), iso-C15:0 3-OH (11.8–14.9%) and anteiso-C15:0 (9.4–10.6%). The polar lipids consisted of phosphatidylethanolamine, an unidentified aminophospholipid, two to three unidentified aminolipids and three unidentified lipids. The comprehensive phylogenetic, genomic, phenotypic and chemotaxonomic results indicate that strains HL-DH10T and HL-DH14 are considered to represent a novel genus of Flavobacteriaceae. Hence, we propose the novel genus Thalassobellus suaedae gen. nov., sp. nov. The type strain is HL-DH10T (=KCCM 90512T=JCM 36598T).

In the domain Bacteria, one of the largest, most diverse and environmentally ubiquitous phylogenetic groups, Candidatus Patescibacteria (also known as candidate phyla radiation/CPR), remains poorly characterized, leaving a major knowledge gap in microbial ecology. We recently discovered a novel cross-domain symbiosis between Ca. Patescibacteria and Archaea in highly purified enrichment cultures and proposed Candidatus taxa for the characterized species, including Ca. Minisyncoccus archaeophilus and the corresponding family Ca. Minisyncoccaceae. In this study, we report the isolation of this bacterium, designated strain PMX.108T, in a two-strain co-culture with a host archaeon, Methanospirillum hungatei strain DSM 864T (JF-1T), and hereby describe it as the first representative species of Ca. Patescibacteria. Strain PMX.108T was isolated from mesophilic methanogenic sludge in an anaerobic laboratory-scale bioreactor treating synthetic purified terephthalate- and dimethyl terephthalate-manufacturing wastewater. The strain could not grow axenically and is obligately anaerobic and parasitic, strictly depending on M. hungatei as a host. The genome was comparatively large (1.54 Mbp) compared to other members of the clade, lacked some genes involved in the biosynthesis pathway and encoded type IV pili-related genes associated with the parasitic lifestyle of ultrasmall microbes. The G+C content of the genomic DNA was 36.6 mol%. Here, we report the phenotypic and genomic properties of strain PMX.108T; we propose Minisyncoccus archaeiphilus gen. nov., sp. nov. to accommodate this strain. The type strain of the species is PMX.108T (=JCM 39522T). We also propose the associated family, order, class and phylum as Minisyncoccaceae fam. nov. Minisyncoccales nov., Minisyncoccia class. nov. and Minisyncoccota phyl. nov. within the bacterial kingdom Bacillati.