Microbiology Spectrum

ABSTRACT “ Candidatus Liberibacter solanacearum” (Lso) is a highly destructive plant pathogen within the alpha-proteobacteria group. Multiple Lso haplotypes occur worldwide, each uniquely associated with a specific psyllid vector. Haplotypes A and B, found in the United States, cause serious damage to solanaceous crops and are transmitted by Bactericera cockerelli , known as the potato psyllid in a circulative and persistent manner. The psyllid gut is the first organ encountered by Lso and may act as a barrier to its transmission; however, the immune response of the gut to Lso infection remains largely uncharacterized. In this study, we examined autophagic responses in the gut of potato psyllids at early, mid, and late infection stages. Based on gene expression analyses, microscopic observations, and Western blotting, we found no clear evidence of autophagy induction despite Lso infection. Nevertheless, during mid and late phases of LsoB infection, we observed downregulation of the mechanistic target of rapamycin and decreased accumulation of ATG8-II. Notably, inducing autophagy with rapamycin significantly reduced LsoA titers in the psyllid guts after a 5-day acquisition access period and lowered transmission efficiency, while LsoB remained unaffected. These findings suggest that modulating autophagy in the gut of potato psyllids could be a promising strategy to limit LsoA acquisition and transmission, while highlighting LsoA and LsoB possess distinct molecular regulatory mechanisms in the psyllid gut. IMPORTANCE Liberibacters are devastating plant pathogens transmitted by psyllids. Because these bacteria are fastidious, the study of the molecular mechanisms involved in plant infection and transmission is difficult. Here, we determined that inducing autophagy in the potato psyllid can affect the acquisition and transmission of Lso haplotype A but not haplotype B. Comparing the host and vector responses to different liberibacters can help identify their transmission and infection mechanisms and find targets to disrupt these processes.

ABSTRACT Psyllids (Psylloidea) are host-specific, phloem-feeding insects that are associated with the transmission of destructive plant diseases caused by the bacterial genus “ Candidatus Liberibacter”. While psyllid-liberibacter interactions have been studied in great detail, most research mainly focused on species associated with important crop diseases. Therefore, a more general understanding of the associations between these two groups of organisms remains limited. The present study investigated the occurrence of infection by “ Ca . Liberibacter” across undercharacterized psyllid lineages in Taiwan. Samples of 46 psyllid species, representing six of the seven known Psylloidea families from host plants spanning 22 families, were obtained. The presence of “ Ca . Liberibacter” in adult psyllid DNA samples was determined by PCRs targeting 16S rDNA of “ Ca . Liberibacter”. PCR tests amplifying mitochondrial 16S DNA of the psyllids were also carried out for quality confirmation. The assays detected “ Ca . Liberibacter” in five of the psyllid species tested, namely Calophya nigridorsalis , Homotoma radiata , Cacopsylla tobirae , Epipsylla albolineata , and Trioza quadrimaculata . BLASTn searches and phylogenetic analysis on near-full-length 16S rDNA sequences identified the strain detected in C. tobirae as “ Ca . Liberibacter europaeus” and showed that E. albolineata carried a strain closely related to a “ Ca . Liberibacter” strain found in Bhutan, while the remaining detected strains could potentially be novel “ Ca . Liberibacter” species. The findings from this study showed that a large diversity of “ Ca . Liberibacter” could exist among underexplored psyllid and plant species, opening new avenues for investigating the ecology of “ Ca . Liberibacter”. IMPORTANCE Bacteria of the genus “ Candidatus Liberibacter” can cause some of the most devastating plant diseases. Gaining a broader perspective on the diversity of associations between these bacteria and their psyllid vectors is crucial for both fundamental and applicative purposes. By taking advantage of the biological diversity in Taiwan, the present study conducted one of the broadest surveys on the presence of “ Ca . Liberibacter” in psyllids, in terms of the diversities of the psyllids examined. The data from this work indicated that previously unknown “ Ca . Liberibacter” genotypes and perhaps even novel species may be more prevalent among psyllid species than previously known. These findings highlight the potential importance of exploring psyllid-liberibacter associations from a broader ecological perspective.

ABSTRACT Although the high-altitude limit for microbial survival in the Earth–atmosphere system has remained a scientific curiosity and topic of study, the ecological significance of long-distance microbial dispersal in the atmosphere has been perceived to have marginal relevance. Here, we report the characterization of novel plant pathogenic species of Curtobacterium that were isolated from samples collected at altitudes ranging from 1.5 to 29 km above sea level. Whole genome-based phylogenies of three strains, paired with plant challenge assays, indicate that each is a previously unrecognized species and causes disease on beans comparable to Curtobacterium flaccumfaciens . Isolates from the stratosphere (strain L6-1) and agricultural millet (G77) were identified to be the same species and designated as Curtobacterium aetherium sp. nov. C. aetherium displays high levels of tolerance to desiccation and UV radiation, which are stresses that increase in intensity with altitude. Back trajectory air mass analysis implied that the phytopathogens may have had an intercontinental source, but regional origins in the continental US cannot be excluded. The environmentally robust phytopathogens we have documented in the upper atmosphere provide new perspective on the role that high-altitude transport may play in microbial dispersal, gene flow, and the epidemiology of aerially dispersed plant disease. IMPORTANCE Enormous quantities and varieties of microorganisms are continually aerosolized and transported in the atmosphere, yet there is a limited understanding of the consequences to downwind ecosystems. While studying bacteria that survive extreme conditions at altitudes up to 29 km in the atmosphere, we discovered new species that have the capacity to cause disease in agriculturally relevant bean varieties. The hardiest isolate we characterized from the stratosphere is a member of the same species as an isolate from an agricultural source, which we have designated Curtobacterium aetherium . C. aetherium is a phytopathogen capable of enduring high-altitude and long-distance atmospheric transport while also possessing the potential to opportunistically infect crops in deposition locations.

ABSTRACT Korea, being surrounded by the sea, provides a rich habitat for marine sponges, which have been a prolific source of bioactive natural products. Although a diverse array of structurally novel natural products has been isolated from Korean marine sponges, their biosynthetic origins remain largely unknown. To explore the biosynthetic potential of Korean marine sponges, we conducted metagenomic analyses of sponges inhabiting the East Sea of Korea. This analysis revealed a symbiotic association of Candidatus Entotheonella bacteria with Halichondria sponges. Here, we report a new chemically rich Entotheonella variant, which we named Ca . Entotheonella halido. Remarkably, this symbiont makes up 69% of the microbial community in the sponge Halichondira dokdoensis . Genome-resolved metagenomics enabled us to obtain a high-quality Ca . E. halido genome, which represents the largest (12 Mb) and highest quality among previously reported Entotheonella genomes. We also identified the biosynthetic gene cluster (BGC) of the known sponge-derived Halicylindramides from the Ca . E. halido genome, enabling us to determine their biosynthetic origin. This new symbiotic association expands the host diversity and biosynthetic potential of metabolically talented bacterial genus Ca . Entotheonella symbionts. IMPORTANCE Our study reports the discovery of a new bacterial symbiont Ca . Entotheonella halido associated with the Korean marine sponge Halichondria dokdoensis . Using genome-resolved metagenomics, we recovered a high-quality Ca . E. halido MAG (Metagenome-Assembled Genome), which represents the largest and most complete Ca . Entotheonella MAG reported to date. Pangenome and BGC network analyses revealed a remarkably high BGC diversity within the Ca . Entotheonella pangenome, with almost no overlapping BGCs between different MAGs. The cryptic and genetically unique BGCs present in the Ca . Entotheonella pangenome represents a promising source of new bioactive natural products.

ABSTRACT Huanglongbing (HLB) is a severe citrus disease in China caused by Candidatus Liberibacter asiaticus ( C Las). Since its initial identification, the pathogen has spread to 10 mainland provinces in China and caused devastating loss. Three distinct prophage types have been identified in C Las; however, their distribution and diversity in China remain inadequately understood. In this study, we collected 500 C Las samples from 10 provinces in China, employing three specific genomic loci to identify prophage types. Subsequently, Sanger sequencing was employed to analyze the genetic diversity of prophage within populations of C Las in China. In addition, the MaxEnt model optimized by the ENMeval software package, was used to predict the habitat suitability of populations of C Las and assess the potential impact of future climate change on its distribution in China. Our analysis revealed that type 2 prophage is the most prevalent, accounting for 55% in China. Among the 10 provinces tested, C Las populations in Yunnan and Sichuan demonstrated higher genetic diversity. Further analysis reveals that C Las populations harboring type 1 prophage remain relatively stable, whereas those carrying type 2 and type 3 prophages undergo population expansion. Furthermore, our predictive models indicate that the presently suitable habitat for C Las populations is concentrated in the southern and certain central regions of China, with an anticipated expansion under future climate change conditions. Presently, the center of populations of C Las characterized by favorable living conditions is situated in Zunyi City, Guizhou Province. Nevertheless, a projected trend indicates a shift toward the northeast, particularly targeting Tongren City in the foreseeable future. IMPORTANCE This study offers significant insights into the distribution and genetic diversity of three types of prophages associated with Candidatus Liberibacter asiaticus ( C Las) in China. Our predictions underscore the implications of climate change on the future distribution of C Las. These findings contribute to a better understanding of Huanglongbing management strategies and can facilitate the development of effective measures to control the spread of this devastating disease within the citrus industry.

ABSTRACT “ Candidatus Liberibacter asiaticus” (CLas), the causal agent of citrus Huanglongbing (HLB), is able to multiply to a high abundance in citrus fruit pith. However, little is known about the biological processes and phytochemical substances that are vital for CLas colonization and growth in fruit pith. In this study, CLas-infected fruit pith of three citrus cultivars (“Shatangju” mandarin, “Guanxi” pomelo, and “Shatian” pomelo) exhibiting different tolerance to CLas were collected and used for dual RNA-Seq and untargeted metabolome analysis. Comparative transcriptome analysis found that the activation of the CLas noncyclic TCA pathway and pathogenic-related effectors could contribute to the colonization and growth of CLas in fruit pith. The pre-established Type 2 prophage in the CLas genome and the induction of its CRISPR/ cas system could enhance the phage resistance of CLas and, in turn, facilitate CLas population growth in fruit pith. CLas infection caused the accumulation of amino acids that were correlated with tolerance to CLas. The accumulation of most sugars and organic acids in CLas-infected fruit pith, which could be due to the phloem blockage caused by CLas infection, was thought to be beneficial for CLas growth in localized phloem tissue. The higher levels of flavonoids and terpenoids in the fruit pith of CLas-tolerant cultivars, particularly those known for their antimicrobial properties, could hinder the growth of CLas. This study advances our understanding of CLas multiplication in fruit pith and offers novel insight into metabolites that could be responsible for tolerance to CLas or essential to CLas population growth. IMPORTANCE Citrus Huanglongbing (HLB, also called citrus greening disease) is a highly destructive disease currently threatening citrus production worldwide. HLB is caused by an unculturable bacterial pathogen, “ Candidatus Liberibacter asiaticus” (CLas). However, the mechanism of CLas colonization and growth in citrus hosts is poorly understood. In this study, we utilized the fruit pith tissue, which was able to maintain the CLas at a high abundance, as the materials for dual RNA-Seq and untargeted metabolome analysis, aiming to reveal the biological processes and phytochemical substances that are vital for CLas colonization and growth. We provided a genome-wide CLas transcriptome landscape in the fruit pith of three citrus cultivars with different tolerance and identified the important genes/pathways that contribute to CLas colonization and growth in the fruit pith. Metabolome profiling identified the key metabolites, which were mainly affected by CLas infection and influenced the population dynamic of CLas in fruit pith.

ABSTRACT Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense ( Ca . M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca . M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca . M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca . M. massiliense and possible transfer between hosts. IMPORTANCE Methanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca . M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated.

ABSTRACT We present here a comprehensive phylogenomic analysis of Acetobacteraceae , a vast group of alphaproteobacteria that has been widely studied for their economic importance. Our results indicate that the ancestor of Acetobacteraceae most likely was photosynthetic and evolved via a progressive transition from versatile photoferrotrophy to the incomplete oxidation of organic substrates defining acetous physiology. Vestigial signs of photosynthetic carotenoid metabolism are present in non-photosynthetic acetous taxa that have lost cytochrome oxidase, while their sister taxa retain such traits. The dominant terminal oxidase of acetous bacteria, the bo 3 ubiquinol oxidase, is derived from duplication and diversification of operons present in Acidocella taxa that have lost photosynthesis. We analyzed the bioenergetic traits that can compensate for the electron transfer function of photosynthetic reaction centers or constitute alternative pathways for the oxidoreduction of c -type cytochromes, such as iron oxidation. The latter pathway bypasses the deranged cytochrome bc 1 complex that is characteristically present in acidophilic taxa due to the loss of conserved ligands in both the Rieske iron-sulfur protein and cytochrome b subunit. The deranged or non-functional bc 1 complex may be retained for its structural role in stabilizing Complex I. The combination of our phylogenetic analysis with in-depth functional evaluations indicates that the order Acetobacterales needs to be emended to include three families: Acetobacteraceae sensu stricto , Roseomonadaceae fam. nov., and Acidocellaceae fam. nov. IMPORTANCE Acetobacteraceae are one of the best known and most extensively studied groups of bacteria, which nowadays encompasses a variety of taxa that are very different from the vinegar-producing species defining the family. Our paper presents the most detailed phylogeny of all current taxa classified as Acetobacteraceae , for which we propose a taxonomic revision. Several of such taxa inhabit some of the most extreme environments on the planet, from the deserts of Antarctica to the Sinai desert, as well as acidic niches in volcanic sites like the one we have been studying in Patagonia. Our work documents the progressive variation of the respiratory chain in early branching Acetobacteraceae into the different respiratory chains of acidophilic taxa such as Acidocella and acetous taxa such as Acetobacter . Remarkably, several genomes retain remnants of ancestral photosynthetic traits and functional bc 1 complexes. Thus, we propose that the common ancestor of Acetobacteraceae was photosynthetic.

ABSTRACT Autophagy plays an important role against pathogen infection in many organisms; however, little has been done with regard to vector-borne plant and animal pathogens, that sometimes replicate and cause deleterious effects in their vectors. Candidatus Liberibacter solanacearum (CLso) is a fastidious gram-negative phloem-restricted plant pathogen and vectored by the carrot psyllid, Bactericera trigonica . The plant disease caused by this bacterium is called carrot yellows and has recently gained much importance due to worldwide excessive economical losses. Here, we demonstrate that calcium ATPase, cytosolic calcium, and most importantly Beclin-1 have a role in regulating autophagy and its association with Liberibacter inside the psyllid. The presence of CLso generates reactive oxygen species and induces the expression of detoxification enzymes in the psyllid midguts, a main site for bacteria transmission. CLso also induces the expression of both sarco/endoplasmic reticulum Ca2+pump (SERCA) and 1,4,5-trisphosphate receptors (ITPR) in midguts, resulting in high levels of calcium in the cellular cytosol. Silencing these genes individually disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and subsequently on Liberibacter persistence and transmission. Inhibiting Beclin1-phosphorylation through different calcium-induced kinases altered the expression of autophagy and CLso titers and persistence. Based on our results obtained from the midgut, we suggest the existence of a direct correlation between cytosolic calcium levels, autophagy, and CLso persistence and transmission by the carrot psyllid. IMPORTANCE Plant diseases caused by vector-borne Liberibacter species are responsible for the most important economic losses in many agricultural sectors. Preventing these diseases relies mostly on chemical sprays against the insect vectors. Knowledge-based interference with the bacteria-vector interaction remains a promising approach as a sustainable solution. For unravelling how Liberibacter exploits molecular pathways in its insect vector for transmission, here, we show that the bacterium manipulates calcium levels on both sides of the endoplasmic reticulum membrane, resulting in manipulating autophagy. Silencing genes associated with these pathways disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and Liberibacter transmission. These results demonstrate major pathways that could be exploited for manipulating and controlling the disease transmission.

ABSTRACT Bacterial cells can vary greatly in size, from a few hundred nanometers to hundreds of micrometers in diameter. Filamentous cable bacteria also display substantial size differences, with filament diameters ranging from 0.4 to 8 µm. We analyzed the genomes of cable bacterium filaments from 11 coastal environments of which the resulting 23 new genomes represent 10 novel species-level clades of Candidatus Electrothrix and two clades that putatively represent novel genus-level diversity. Fluorescence in situ hybridization with a species-level probe showed that large-sized cable bacteria belong to a novel species with the proposed name Ca . Electrothrix gigas. Comparative genome analysis suggests genes that play a role in the construction or functioning of large cable bacteria cells: the genomes of Ca . Electrothrix gigas encode a novel actin-like protein as well as a species-specific gene cluster encoding four putative pilin proteins and a putative type II secretion platform protein, which are not present in other cable bacteria. The novel actin-like protein was also found in a number of other giant bacteria, suggesting there could be a genetic basis for large cell size. This actin-like protein (denoted big bacteria protein, Bbp) may have a function analogous to other actin proteins in cell structure or intracellular transport. We contend that Bbp may help overcome the challenges of diffusion limitation and/or morphological complexity presented by the large cells of Ca . Electrothrix gigas and other giant bacteria. IMPORTANCE In this study, we substantially expand the known diversity of marine cable bacteria and describe cable bacteria with a large diameter as a novel species with the proposed name Candidatus Electrothrix gigas. In the genomes of this species, we identified a gene that encodes a novel actin-like protein [denoted big bacteria protein (Bbp)]. The bbp gene was also found in a number of other giant bacteria, predominantly affiliated to Desulfobacterota and Gammaproteobacteria, indicating that there may be a genetic basis for large cell size. Thus far, mostly structural adaptations of giant bacteria, vacuoles, and other inclusions or organelles have been observed, which are employed to overcome nutrient diffusion limitation in their environment. In analogy to other actin proteins, Bbp could fulfill a structural role in the cell or potentially facilitate intracellular transport.