AbstractThe bacterium Candidatus Liberibacter asiaticus (CLas) causes citrus Huanglongbing disease. Our understanding of the pathogenicity and biology of this microorganism remains limited because CLas has not yet been cultivated in artificial media. Its genome is relatively small and encodes approximately 1136 proteins, of which 415 have unknown functions. Here, we use a high-throughput yeast-two-hybrid (Y2H) screen to identify interactions between CLas proteins, thus providing insights into their potential functions. We identify 4245 interactions between 542 proteins, after screening 916 bait and 936 prey proteins. The false positive rate of the Y2H assay is estimated to be 2.9%. Pull-down assays for nine protein-protein interactions (PPIs) likely involved in flagellar function support the robustness of the Y2H results. The average number of PPIs per node in the CLas interactome is 15.6, which is higher than the numbers previously reported for interactomes of free-living bacteria, suggesting that CLas genome reduction has been accompanied by increased protein multi-functionality. We propose potential functions for 171 uncharacterized proteins, based on the PPI results, guilt-by-association analyses, and comparison with data from other bacterial species. We identify 40 hub-node proteins, including quinone oxidoreductase and LysR, which are known to protect other bacteria against oxidative stress and might be important for CLas survival in the phloem. We expect our PPI database to facilitate research on CLas biology and pathogenicity mechanisms.
AbstractPhotosynthesis is a fundamental biogeochemical process, thought to be restricted to a few bacterial and eukaryotic phyla. However, understanding the origin and evolution of phototrophic organisms can be impeded and biased by the difficulties of cultivation. Here, we analyzed metagenomic datasets and found potential photosynthetic abilities encoded in the genomes of uncultivated bacteria within the phylum Myxococcota. A putative photosynthesis gene cluster encoding a type-II reaction center appears in at least six Myxococcota families from three classes, suggesting vertical inheritance of these genes from an early common ancestor, with multiple independent losses in other lineages. Analysis of metatranscriptomic datasets indicate that the putative myxococcotal photosynthesis genes are actively expressed in various natural environments. Furthermore, heterologous expression of myxococcotal pigment biosynthesis genes in a purple bacterium supports that the genes can drive photosynthetic processes. Given that predatory abilities are thought to be widespread across Myxococcota, our results suggest the intriguing possibility of a chimeric lifestyle (combining predatory and photosynthetic abilities) in members of this phylum.
AbstractAnaerobic methanotrophic archaea (ANME) carry out anaerobic oxidation of methane, thus playing a crucial role in the methane cycle. Previous genomic evidence indicates that multi-heme c-type cytochromes (MHCs) may facilitate the extracellular electron transfer (EET) from ANME to different electron sinks. Here, we provide experimental evidence supporting cytochrome-mediated EET for the reduction of metals and electrodes by ‘Candidatus Methanoperedens nitroreducens’, an ANME acclimated to nitrate reduction. Ferrous iron-targeted fluorescent assays, metatranscriptomics, and single-cell imaging suggest that ‘Ca. M. nitroreducens’ uses surface-localized redox-active cytochromes for metal reduction. Electrochemical and Raman spectroscopic analyses also support the involvement of c-type cytochrome-mediated EET for electrode reduction. Furthermore, several genes encoding menaquinone cytochrome type-c oxidoreductases and extracellular MHCs are differentially expressed when different electron acceptors are used.
AbstractTaurine-respiring gut bacteria produce H2S with ambivalent impact on host health. We report the isolation and ecophysiological characterization of a taurine-respiring mouse gut bacterium. Taurinivorans muris strain LT0009 represents a new widespread species that differs from the human gut sulfidogen Bilophila wadsworthia in its sulfur metabolism pathways and host distribution. T. muris specializes in taurine respiration in vivo, seemingly unaffected by mouse diet and genotype, but is dependent on other bacteria for release of taurine from bile acids. Colonization of T. muris in gnotobiotic mice increased deconjugation of taurine-conjugated bile acids and transcriptional activity of a sulfur metabolism gene-encoding prophage in other commensals, and slightly decreased the abundance of Salmonella enterica, which showed reduced expression of galactonate catabolism genes. Re-analysis of metagenome data from a previous study further suggested that T. muris can contribute to protection against pathogens by the commensal mouse gut microbiota. Together, we show the realized physiological niche of a key murine gut sulfidogen and its interactions with selected gut microbiota members.
Symbiotic relationships are ubiquitous throughout the world’s oceans, yet for many marine organisms, including those in the high latitudes, little is understood about symbiotic associations and functional relationships. From a recently determined genome sequence of a filter-feeding basket star from Argentina, Gorgonocephalus chilensis, we discovered a novel Mycoplasma species with a 796Kb genome (CheckM completeness of 97.9%, G+C content = 30.1%). Similar to other Mycoplasma spp. within Mycoplasmatota, genomic analysis of the novel organism revealed reduced metabolic pathways including incomplete biosynthetic pathways, suggesting an obligate association with their basket star host. Results of 16S rRNA and multi-locus phylogenetic analyses revealed that this organism belonged to a recently characterized non-free-living lineage of Mycoplasma spp. specifically associated with marine invertebrate animals. Thus, the name “Candidatus Mycoplasma mahonii” is proposed for this novel species. Based on 16S rRNA PCR-screening, we found that Ca. M. mahonii also occurs in Gorgonocephalus eucnemis from the Northwest Pacific and other Gorgonocephalus chilensis from Argentinian waters. The level of sequence conservation within Ca. M. mahonii is considerable between widely disparate high-latitude Gorgonocephalus species, suggesting that oceanic dispersal of this microbe may be greater than excepted.
Bamboo is used for making structures, furniture, handicrafts, and ropes, as well as a source of food in the Philippines. One of the emerging diseases of bamboo in the country is the bamboo witches’ broom (BWB), which has been occasionally noted in three genera of bamboo – including Dendrocalamus, Gigantochloa, and Schizostachyum from various provinces in the Philippines (Ilocos Norte, Laguna, Batangas, Quezon, Agusan del Sur, Bukidnon, and South Cotabato) since the 1990s. However, studies and information about BWB in the country remain lacking and largely unexplored. In this study, we report a similar disease affecting Dendrocalamus and Gigantochloa bamboo species from Bohol and Davao and – for the first time – in Dendrocalamus merrillianus ("bayog") and Bambusa spinosa ("kawayan-tinik") from Isabela and Nueva Vizcaya. As a result of the surveys conducted from 1999–2019, the disease is now identified in six species across four genera of bamboo – namely, Gigantochloa spp. (G. levis and G. atter), Dendrocalamus spp. (D. asper and D. merrillianus), Schizostachyum lumampao, and Bambusa spinosa recorded in 11 provinces in the Philippines. The BWB symptoms include clustering of leaves forming a rosette-like structure, leaf proliferation, excessive limb formation from a single node, and shortening of internodes. Nested PCR using the universal primers P1/P7 and R16MF2/R1 targeting the phytoplasma 16S ribosomal RNA gene revealed positive amplification in five symptomatic BWB samples from Isabela, Philippines. Subsequent sequencing (~ 1.3kbp) and phylogenetic analysis using the representative BWB isolates from Isabela revealed > 98.65% genetic similarity and clustering to Candidatus Phytoplasma luffae, which belongs to the 16SrVIII group (Loofah Witches’ Broom Group). This paper determined the distribution of BWB in different species of bamboo in the Philippines, as well as the association of 'Ca. Phytoplasma luffae'-related strain (16SrVIII) to BWB.
Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus plants caused by the obligate and phloem-limiting bacterium Candidatus Liberibacter asiaticus (Las). Reliable detection methods are important for successful control of the disease. This study was aimed to develop a rapid and user-friendly on-site detection system for Las using the TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) assay. The Las-specific on-site detection system could be completed within one hour by simple DNA extraction coupled with a portable POCKIT device, which can perform PCR amplification and automatically provide qualitative results derived from fluorescence signals. The sensitivity of the TaqMan probe-iiPCR assay could be as low as single copy of Las, comparable to a real-time PCR method. Further testing of the field citrus samples showed 100% agreement between the TaqMan probe-iiPCR assay and the real-time PCR method, and the on-site detection system also demonstrated a great performance of Las detection. With high specificity and sensitivity, the on-site detection system developed in this study becomes a simple, rapid and powerful tool for detecting Las in fields.
AbstractThree hundred and forty-four tick samples were collected from vegetation at Taksin Maharat National Park, Tak province, northwestern Thailand. They were morphologically identified and molecularly confirmed by 16S rRNA and COI genes as Dermacentor laothaiensis (n = 105), D. steini (n = 139), and D. auratus (n = 100). These ticks were examined for the spotted fever group rickettsiae (SFGRs) using PCR and DNA sequencing of six genes; 17-kDa, gltA, 16S rRNA, ompA, ompB, and sca4. Of these ticks, 6.10% (21/344) gave positive results for the presence of SFGRs. Phylogenetic analyses of the SFGRs clearly indicated that a novel genotype assigned as Candidatus Rickettsia takensis was detected in D. laothaiensis (19/105) and at lesser frequency in D. steini (1/139). Furthermore, Candidatus Rickettsia laoensis was also found at a low frequency in D. auratus (1/100), the first record in Thailand. Although, the pathogenicities of these SFGRs remain unknown, our findings suggest potential risks of SFGRs being transmitted via ticks near the border between Thailand and Myanmar, a gateway of daily migrations of local people and visitors both legal and illegal.
‘Candidatus Liberibacter solanacearum’ (Lso) is a bacterial pathogen infecting several crops and causing damaging diseases. Several Lso haplotypes have been identified. Among the seven haplotypes present in North America, LsoA and LsoB are transmitted by the potato psyllid, Bactericera cockerelli (Šulc), in a circulative and persistent manner. The gut, which is the first organ pathogen encounters, could be a barrier for Lso transmission. However, the molecular interactions between Lso and the psyllid vector at the gut interface remain largely unknown. In this study, we investigated the global transcriptional responses of the adult psyllid gut upon infection with two Lso haplotypes (LsoA and LsoB) using Illumina sequencing. The results showed that each haplotype triggers a unique transcriptional response, with most of the distinct genes elicited by the highly virulent LsoB. The differentially expressed genes were mainly associated with digestion and metabolism, stress response, immunity, detoxification as well as cell proliferation and epithelium renewal. Importantly, distinct immune pathways were triggered by LsoA and LsoB in the gut of the potato psyllid. The information in this study will provide an understanding of the molecular basis of the interactions between the potato psyllid gut and Lso, which may lead to the discovery of novel molecular targets for the control of these pathogens.
AbstractFour pathogenic bacterial species of the genus ‘Candidatus Liberibacter’, transmitted by psyllid vectors, have been associated with serious diseases affecting economically important crops of Rutaceae, Apiaceae and Solanaceae families. The most severe disease of citrus plants, huanglongbing (HLB), is associated with ‘Ca. Liberibacter asiaticus’ (CaLas), ‘Ca. Liberibacter americanus’ (CaLam) and ‘Ca. Liberibacter africanus’ (CaLaf), while ‘Ca. Liberibacter solanacearum’ (CaLsol) is associated with zebra chip disease in potatoes and vegetative disorders in apiaceous plants. Since these bacteria remain non-culturable and their symptoms are non-specific, their detection and identification are done by molecular methods, mainly based on PCR protocols. In this study, a new quantitative real-time PCR protocol based on TaqMan probe, which can also be performed in a conventional PCR version, has been developed to detect the four known phytopathogenic species of the genus Liberibacter. The new protocol has been validated according to European Plant Protection Organization (EPPO) guidelines and is able to detect CaLas, CaLam, CaLaf and CaLsol in both plants and vectors, not only using purified DNA but also using crude extracts of potato and citrus or psyllids. A comparative analysis with other previously described qPCR protocols revealed that this new one developed in this study is more specific and equally or more sensitive. Thus, other genus-specific qPCR protocols have important drawbacks regarding the lack of specificity, while with the new protocol there was no cross-reactions in 250 samples from 24 different plant and insect species from eight different geographical origins. Therefore, it can be used as a rapid and time-saving screening test, as it allows simultaneous detection of all plant pathogenic species of ‘Ca. Liberibacter’ in a one-step assay.