Publications

3867

Few described archaeal, and fewer bacterial, lineages thrive at salt-saturating conditions, such as solar saltern crystallizers (salinity above 30%-w/v). They accumulate molar K+ cytoplasmic concentrations to maintain osmotic balance ("salt-in" strategy), and have proteins adaptively enriched in negatively charged, acidic amino acids. Here, we analyzed metagenomes and metagenome-assembled genomes (MAGs) from geothermally influenced hypersaline ecosystems with increasing chaotropicity in the Danakil Depression. Normalized abundances of universal single-copy genes confirmed that haloarchaea and Nanohaloarchaeota encompass 99% of microbial communities in the near life-limiting conditions of the Western-Canyon Lakes (WCLs). Danakil metagenome- and MAG-inferred proteomes, compared to those of freshwater, seawater and solar saltern ponds up to saturation (6-14-32% salinity), showed that WCL archaea encode the most acidic proteomes ever observed (median protein isoelectric points ≤4.4). We identified previously undescribed Halobacteria families as well as an Aenigmatarchaeota family and a bacterial phylum independently adapted to extreme halophily. Despite phylum-level diversity decreasing with increasing salinity-chaotropicity, and unlike in solar salterns, adapted archaea exceedingly diversified in Danakil ecosystems, challenging the notion of decreasing diversity under extreme conditions. Metabolic flexibility to utilize multiple energy and carbon resources generated by local hydrothermalism along feast-and-famine strategies seemingly shape microbial diversity in these ecosystems near life limits.

Citri Reticulatae Pericarpium, especially the pericarp of Citrus reticulata Blanco cv. Chachiensis (PCRC), is an important edible and medicinal ingredient for health and pharmacological properties. Citrus Huanglongbing, a devastating disease that currently threatens the citrus industry worldwide, is caused by a phloem-limited alpha-proteobacterium, “Candidatus Liberibacter asiaticus” (CLas). The industry of cultivar Chachiensis has been suffering from HLB. Although HLB affected the quality of citrus fruit, whether the quality of PCRC was affected by HLB remains unclear. In this study, we compared the metabolite profiles between HLB-affected and healthy PCRC from three sources: fresh, 6-month-old, and 9-year-old PCRC, through the untargeted LC–MS method. Compared to healthy controls, various types of bioactive compounds, mainly flavonoids, terpenoids, alkaloids, coumarins, polysaccharides, and phenolic acids, accumulated in HLB-affected PCRC, especially in the HLB-affected 9-year PCRC. In particular, isorhamnetin, isoliquiritigenin, luteolin 7-O-beta-D-glucoside, limonin, geniposide, pyrimidodiazepine, scoparone, chitobiose, m-coumaric acid, malonate, and pantothenic acid, which contributed to the pharmacological activity and health care effects of PCRC, were highly accumulated in HLB-affected 9-year-old PCRC compared to the healthy control. Multibioassay analyses revealed that HLB-affected 9-year-old PCRC had a higher content of total flavonoids and total polyphenols and exhibited similar antioxidant capacity as compared to healthy controls. The results of this study provided detailed information on the quality of HLB-affected PCRC.

Abstract Candidatus Liberibacter asiaticus (Las) is one of the causal agents of huanglongbing (HLB), the most devastating disease of citrus worldwide. Due to the intracellular lifestyle and significant genome reduction, culturing Las in vitro has proven to be extremely challenging. In this study, we optimized growth conditions and developed a semi-selective medium based on the results of nutritional and antibiotic screening assays. Using these optimized conditions, we were able to grow Las in the LG liquid medium with ca.100- to 1000-fold increase, which peaked after 4 to 6 weeks and were estimated to contain 106 to 107 cells/ml. The cultured Las bacteria remained in a dynamic state of growth for over 20 months and displayed limited growth in subcultures. The survival and growth of Las was confirmed by fluorescence in situ hybridization with Las-specific probes and expression of its metabolic genes. Growth of Las in the optimized medium relied on the presence of a helper bacterium, Stenotrophomonas maltophilia FLMAT-1 that is multi-drug resistant and dominant in the Las co-culture system. To recapitulate the disease, the co-cultured Las was inoculated back to citrus seedlings via psyllid feeding. Although the Las-positive rate of the fed psyllids and inoculated plants were relatively low, this is the first demonstration of partial fulfillment of Koch’s postulates with significant growth of Las in vitro and a successful inoculation of cultured Las back to psyllids and citrus plants that resulted in HLB symptoms. These results provide new insights into Las growth in vitro and a system for improvement towards axenic culture and anti-Las compound screening.

The recent emergence of anaplasmosis in camels has raised global interest in the pathogenicity and zoonotic potential of the pathogen causing it and the role of camels as reservoir hosts. In the United Arab Emirates (UAE), molecular studies and genetic characterization of camel-associated Anaplasma species are limited. This study aimed to characterize molecularly Anaplasmataceae strains circulating in dromedary camels in the UAE. Two hundred eighty-seven whole-blood samples collected from dromedary camels across regions of the Abu Dhabi Emirate were received between 2019 and 2023 at the Abu Dhabi Agriculture and Food Safety Authority (ADAFSA) veterinary laboratories for routine diagnosis of anaplasmosis. The animals were sampled based on field clinical observation by veterinarians and their tentative suspicion of blood parasite infection on the basis of similar clinical symptoms as those caused by blood parasites in ruminants. The samples were screened for Anaplasmataceae by PCR assay targeting the groEL gene. Anaplasmataceae strains were further characterized by sequencing and phylogenetic analysis of the groEL gene. Thirty-five samples (35/287 = 12.2%) tested positive for Anaplasmataceae spp. by PCR assay. Nine positive samples (9/35 = 25.7%) were sequenced using groEL gene primers. GenBank BLAST analysis revealed that all strains were 100% identical to the Candidatus A. camelii reference sequence available in the GenBank nucleotide database. Phylogenetic analysis further indicated that the sequences were close to each other and were located in one cluster with Candidatus A. camelii sequences detected in Saudi Arabia, Morocco, and the UAE. Pairwise alignment showed that the UAE sequences detected in this study were completely identical and shared 100% identity with Candidatus A. camelii from Morocco and Saudi Arabia and 99.5% identity with Candidatus A. camelii from the UAE. This study demonstrates the presence of Candidatus A. camelii in UAE dromedary camels. Further critical investigation of the clinical and economical significance of this pathogen in camels needs to be carried out.

Abstract Syndrome ‘basses richesses’ (SBR) disease in sugar beet caused by two phloem-limited pathogens, 'Candidatus Arsenophonus phytopathogenicus' and 'Candidatus Phytoplasma solani' is a fastspreading disease in Central Europe. The planthopper vector, Pentastiridius leporinus (Cixiidae), has recently expanded its host range to potato in Germany. However, the genetic diversity of 'Ca P. solani' in potato and possible association to SBR phytoplasma is unknown. In this study we charachterized ‘Ca. P. solani’ infecting sugar beet and potato plants in close distance fields in Southern Germany. Initially, the pathogen was detected in the potato tubers (18.7%) and sugar beet roots (60%) using TaqMan Real-Time PCR. Then, the sequences of 16S rRNA and other informative genes (stamp, vmp1, tuf and secY) were analysed in a number of infected potato and sugar beet plants. The phytoplasma strain infecting sugar beet in Southern Germany was classified into 16SrXII-P subgroup, a novel subgroup recently reported from sugar beet in Eastern Germany. While, the potato related strains were close to 16SrXII-A, which is a common subgroup for potato stolbur reported in Europe. The multilocus sequence analysis (MLSA) of non-ribosomal genes of the phytoplasma strains showed that the potato strain is clearly different from the sugar beet associated strain in this region. The presence and prevalence of 16SrXII-P in sugar beet in Southern and Eastern Germany suggests that this subgroup is dominant in sugar beet in Germany. In addition, this study elucidates for the first time, the genetic diversity of ‘Ca. P. solani’ strains in potato in Germany with a possible different source rather than sugar beet. Further investigation is required to investigate genetic variation of ‘Ca. P. solani’ in all sugar beet and potato-growing regions including weeds host in Central Europe to better understand the epidemiology of both sugar beet SBR and potato stolbur disease.

AbstractBackgroundCandidate Phyla Radiation (CPR) is a large monophyletic group thought to cover about 25% of bacterial diversity. Due to peculiar characteristics and unusual 16S rRNA gene structure, they are often under-represented or lost in 16S rRNA-based microbiota surveys. Among CPR, “CandidatusSaccharibacteria” is a phylum experimentally found to modulate the immune response and enriched in the oral microbiota of subjects suffering from several immune-mediated disorders, e.g. food allergies, as reported by us in a previous work. Due to the growing evidence of “Ca. Saccharibacteria”’s role in clinical settings and in order to unravel its role in host physiology and pathology, it is crucial to have a reliable method to detect and quantify this lineage.Methods and ResultsFour qPCR protocols for quantifying “Ca.Saccharibacteria” (one targeting the 23S rRNA gene and three the 16S) were selected from the literature among the few available. Efficiency and coverage of primer pairs used in these protocols were preliminary evaluated viain silicoanalyses on the “Ca.Saccharibacteria” known taxonomic variability, and then testedin vitroon the salivary DNA previously investigated by 16S metagenomics in the food allergy study.In silicoanalyses evidenced that the 23S qPCR protocol covered more “Ca. Saccharibacteria” variability compared to the 16S-based ones, and that the 16S metagenomics primers were the most comprehensive. qPCR experiments confirmed that 16S-based protocols strongly underestimated “Ca. Saccharibacteria” while the 23S protocol was the only one to yield results comparable to 16S metagenomics both in terms of correlation and absolute quantification. However, only 16S metagenomics evidenced an expansion of “Ca. Saccharibacteria” in allergic subjects compared to controls, while none of the four qPCR protocols detected it.ConclusionThese results underline the current limits in experimentally approaching “Ca. Saccharibacteria”. To obtain a more realistic picture of their abundance within bacterial communities, and to enable more efficient taxonomic resolution, it is essential to find novel experimental strategies. This is a necessary premise for more targeted and systematic functional studies to clarify the role of “Ca. Saccharibacteria” and, generally, CPR bacteria, in maintaining the health of the host.

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus , including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.

AbstractZinnia peruviana (Asteraceae) is an annual flowering plant cultivated in various gardens of Mysore, the south of Karnataka state, India. During 2021, phytoplasma‐like symptoms such as virescence and phyllody were observed on Zinnia in Mysore local gardens. A total of 10 symptomatic and five asymptomatic samples were collected, and the DNA was amplified by PCR using primers specific to 16S RNA and secA genes. The nucleotide sequence analysis showed that the 16S RNA and SecA gene sequences were most similar to those of ‘Ca. P. aurantifolia’, with identities greater than 99%. The phytoplasma sequence from this study shared a common clade with ‘Ca. P. aurantifolia’ when phylogenetic analysis was done. Similarly, the reference sequences for the 16S rRNA and SecA region confirmed the identity of the phytoplasma associated with the leaves of Zinnia. This is the new host record for ‘Ca. P. aurantifolia’ (16SrII‐D) phytoplasma associated with Z. peruviana from India.