Frontiers in Microbiology

Introduction Citrus huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is a globally devastating plant disease characterized by severe disruption of host physiology. Although alterations in plant hormone homeostasis, including salicylic acid and abscisic acid (ABA), are hallmarks of HLB, the contribution of plant-associated microbiota to these hormonal changes remains poorly understood. Here, we investigated whether ABA depletion in HLB-affected citrus is associated with microbial metabolism. Methods ABA and its metabolites were quantified in HLB-affected citrus tissues by liquid chromatography–mass spectrometry. The capacity of CLas strain Ishi-1 and Flavobacterium spp. strains CGRL1 and CGRL2 to metabolize ABA was assessed using in vitro culture assays. The effects of exogenous ABA on bacterial growth were also evaluated. Results ABA concentrations were significantly decreased in symptomatic HLB-affected tissues, concomitant with the accumulation of the ABA catabolite dehydrovomifoliol. Furthermore, CLas strain Ishi-1 and Flavobacterium strains CGRL1 and CGRL2 degraded ABA and produced dehydrovomifoliol, whereas no such activity was detected in non-inoculated controls. Notably, exogenous ABA promoted bacterial growth, suggesting that ABA may serve as a metabolic substrate or growth-enhancing factor for these bacteria. Discussion These findings identify microbial ABA metabolism as a potential driver of hormone depletion in HLB-affected citrus. Our results further suggest that CLas and its associated bacteria may actively reshape host hormone homeostasis through ABA metabolism, providing a mechanistic link between pathogen-associated microbial communities and hormonal imbalance during HLB development.

Terrestrial mud volcanoes are surface geological features where fluidized sediments and gasses from the subsurface are discharged along a fracture network providing a window into the deep biosphere. Although mud volcanoes constitute an important source of methane emission from natural environments, their microbial communities responsible for methane cycling remain poorly characterized. Using a metagenomics approach, we investigated the taxonomic composition and metabolic potential of microbial communities in three active mud volcanoes in the Kerch-Taman mud volcanic province. Despite the volcanoes’ close proximity their microbial communities strongly differ. In the Kmv1 and Kmv2 volcanoes surface horizons mostly harbored organotrophic microbial communities, while the relative abundance of anaerobic methanotrophic archaea (ANME) increased with depth. The deep horizons (1.5 m) of Kmv1 were dominated by Ca. Methanoperedenaceae that lacked nitrate reductase and could couple methane oxidation to the reduction of metal oxides, while the abundance of sulfate-reducing bacteria was low. Consistently, with higher sulfate content, the deep horizon in Kmv2 was dominated by Ca. Methanoperedenaceae, ANME-2a/2b clade, sulfate-reducing Desulfobacterota and sulfur-oxidizing Gammaproteobacteria . No clear depth distribution of taxa was observed in the Kmv3 volcano where microorganisms of the methane and sulfur cycles, namely, methanogens, ANME-3 clade, methanotrophic bacteria, and sulfate reducers were simultaneously detected. A high-quality genome of a member of the archaeal candidate phylum EX4484-52 within the DPANN lineage was assembled from metagenomes. This archaeon, named Candidatus Lutivulcanarchaeum fermentans , has complete glycolytic pathway and ATP generation mechanisms, but lacked the biosynthetic pathways for many key cellular compounds, indicating a parasitic or symbiotic lifestyle.

Ocean warming, disease, and pollution have contributed to global declines in coral abundances and diversity. In the Caribbean, corals previously dominated reefs, providing an architectural framework for diverse ecological habitats, but have significantly declined due to infectious disease and anthropogenic climate change. Key species like the coral Acropora cervicornis are critically endangered, prompting researchers to focus on scientific endeavors to identify factors influencing coral disease resistance and resilience. We previously showed that disease susceptibility, growth rates, and bleaching risk were all associated with the abundance of a single bacterial parasite, Candidatus Aquirickettsia rohweri which proliferates in vivo under nutrient enrichment. Yet how nutrients influence parasite physiology in vivo remains unknown. Here, we analyzed parasite gene expression from a disease-susceptible A. cervicornis genotype exposed to ambient or nutrient enrichment conditions. Electron microscopy showed that Ca. A. rohweri was abundant in coral tissue and densely packed in mucocytes prior to nutrient enrichment. Under ambient conditions, the parasite upregulated genes involved in translation, protein maintenance, and cell envelope integrity, consistent with a conserve-and-maintain strategy. Nutrient enrichment induced expression of genes associated with central metabolism, nutrient import, stress response, host interaction, and two-component systems. Together, these results indicate that nutrient enrichment activates a growth-and-exploitation strategy, likely exacerbating parasitic pressure on A. cervicornis .

The Clostridium estertheticum complex (CEC) consists of closely related bacterial species that are mostly isolated from meat processing environment. Genome mining studies have recently established CEC as a source of class I bacteriocins. However, up until now, class II bacteriocins have not been reported from CEC. In the present study, we determined the presence of class II bacteriocin biosynthetic clusters in 33 CEC genomes through genome mining followed by bacteriocin production through heterologous expression in Escherichia coli . Six biosynthetic gene clusters belonging to class IIa ( n  = 1), IIb ( n  = 2), IId ( n  = 2) and an undefined class containing three precursor peptides were identified in six different CEC strains. Using molecular biology, we developed dedicated expression vectors for the class IIa bacteriocin, clesteriocin A. Its precursor peptide, CleA, and protease, CleB150, were initially expressed in insoluble form in E. coli . Through a systematic analysis of suitable solubility enhancing protein tags, both CleA and CleB150 were expressed in significant amounts of soluble fractions using tandem tags derived from Small Ubiquitin-like Modifier, superfolder Green fluorescent protein, Maltose binding protein and N-utilization substance. Mature clesteriocin A was obtained following in vitro maturation reactions between the tagged CleA and CleB150. The novel bacteriocin displayed antimicrobial activity against Listeria monocytogenes , which was mediated by the mptC gene. By combining genome mining and molecular biology, we have shown CEC is a source of novel class II bacteriocins that have potential for application as food biopreservatives.

“ Candidatus Liberibacter asiaticus” (CLas) is an uncultivable α -proteobacterium causing the most destructive and currently incurable citrus disease, Huanglongbing (HLB). The transcription factors (TFs) of CLas are involved in various biological processes. However, the functions of most TFs remain unverified. BolA is reported to be an important transcriptional regulator related to bacterial growth and virulence. Here, the role of BolA in CLas was investigated using gene deletion and complementation assays in the heterologous host Sinorhizobium meliloti ( Sme ). The results showed that BolA CLas and BolA Sme are similar in sequence and transcriptional regulation. BolA positively regulates biofilm formation—evidenced by the significant downregulation of a key gene ( cyaA ) in the mutant (Δ BolA Sme ), without affecting bacterial growth. The upregulation of 16 differentially expressed genes (DEGs) related to flagellar assembly indicated that BolA negatively regulates CLas motility. BolA deletion also led to the downregulation of ABC transporters (15 DEGs) and lipid metabolism genes (13 DEGs), correlating with reduced stress tolerance. Furthermore, BolA CLas is involved in modulating heme metabolism, as well as protein export, folding, sorting, and degradation. Finally, in vivo screening identified two compounds as BolA inhibitors, which significantly reduced CLas titer in infected periwinkle leaves. Taken together, this study constitutes a relevant step toward the understanding of CLas virulence by demonstrating that BolA is a key TF involved in biofilm formation, stress response, motility, and bacterial physiology, thereby presenting a potential target for disease control.

Polyphosphate-accumulating organisms (PAOs) play a crucial role in enhanced biological phosphorus removal (EBPR) processes. In addition to biosynthesis, they rely on phosphate for energy generation. However, Candidatus Accumulibacter, a model PAO, has been shown to adapt to low phosphate conditions by switching to a glycogen-accumulating metabolism (GAM), with variable success across genus members and experiments. This study aimed to explore the metabolic shift of several Accumulibacter species subjected to low-phosphate concentration in different operating conditions using metatranscriptomics analysis. Furthermore, the study enabled a comparison of the transcriptomic profiles of Accumulibacter with those of Propionivibrio , a glycogen-accumulating organism typically found in EBPR plants. Two sequencing batch reactors were operated with different carbon sources to enrich for different populations of Accumulibacter . After decreasing the influent phosphate concentration, carbon removal performance was maintained while anaerobic phosphate release dropped dramatically, suggesting a shift from a phosphate-accumulating to a glycogen-accumulating metabolism. Analysis of metatranscriptomics data indicated that Accumulibacter regalis (type I) and Propionivibrio aalborgensis remained the most abundant species after the phosphate decrease in the reactor with acetate-propionate and allylthiourea, while Accumulibacter delftensis (type I) and Accumulibacter phosphatis (type II) remained active in the reactor with acetate-glucose and no allylthiourea. Transcription of the genes from the ethylmalonyl-CoA pathway involved in the production of propionyl-CoA and regulation of the anaerobic redox balance was enhanced under low-phosphate conditions, especially for type I Accumulibacter . Conversely, the transcription of the methylmalonyl-CoA pathway was enhanced under low-phosphate conditions in Propionivibrio and type II Accumulibacter .

IntroductionEndosymbiotic bacteria show diverse strategies to manipulate host reproduction for their survival in nature. The diversity of symbionts infecting hematophagous insects and their impact on host ecology could be crucial for developing effective control measures of disease-transmitting vectors. Sand flies are a group of small insects, with some species serving as vectors for leishmaniasis, bartonellosis, and arboviral diseases. Sergentomyia squamirostris is the only known species of sand flies found on the main islands of Japan. Although no medical implications exist for S. squamirostris, we conducted whole-genome sequencing to explore its potential relevance.MethodsWe conducted whole-genome sequencing and de novo assembly of S. squamirostris using genomic DNA isolated from a single field-collected female insect sample. During this attempt, we incidentally obtained closed genomes of two new bacteria, wSSQ and RiSSQ, belonging to Wolbachia and Candidatus Tisiphia, respectively. We then investigated infection rates of both bacteria in two natural populations of S. squamirostris in Japan.ResultsPhylogenetic analysis indicated that wSSQ and RiSSQ belonged to Wolbachia and Ca. Tisiphia, respectively. Ca. Tisiphia is also known as “Torix Rickettsia,” which is considered a large group of endosymbionts of invertebrates with no known pathogenicity to humans and animals. NGS read depths for both wSSQ and RiSSQ genomes were found to be high coverages, indicating that these bacteria are S. squamirostris endosymbionts. The infection rates of wSSQ and RiSSQ in the wild population of S. squamirostris varied in the two different localities in Japan, and co-infection with both bacteria was commonly seen. wSSQ was detected in both sexes of S. squamirostris, whereas RiSSQ was detected only in female sand flies.ConclusionCa. Tisiphia has recently been recognized as an underexplored endosymbiont with a widespread presence in invertebrates, including disease vectors. RiSSQ represents the first complete genomic information resource of Ca. Tisiphia infecting sand flies. Further research is needed to understand potential interactions between its host and other endosymbionts, as well as to explore the potential implications of disease control in the future.

Marine heterotrophic bacteria in coastal waters respond to the influx of carbon from natural and anthropogenic sources. We identified two nearly identical, (99.9% average nucleotide identity; 100% amino acid identity; same DNA G + C content of 52.3 mol%) high-quality (≥99% CheckM completeness and ≤ 1.3% contamination) draft metagenome-assembled genomes (MAGs; SJ0813 and SJ0972) from seawater microbiomes of a southern island of Singapore that is in a protected marine park. The MAGs were only assigned to the Cellvibrionaceae family according to Genome Taxonomy Database. Overall genome related indices to Pseudomaricurvus alkylphenolicus KU41GT as the closest phylogenetic relative revealed no more than 70.45% average nucleotide identity (ANIcutoff < 95%), below the 50% percentage of conserved proteins (POCPcutoff = 43.54%) for genera cutoff and low digital DNA–DNA hybridization values (DDH = 20.6 and 20.8%). The major respiratory quinone is predicted to be ubiquinone-9 from the annotation of 3-demethylubiquinone-9 3-methyltransferase (ubiG, K00568) involved in the last step of the ubiquinone biosynthesis pathway (M00117), which differed from the ubiquinone-8 utilized by known members of Cellvibrionaceae. Both MAGs contained a complete pathway for dissimilatory nitrate reduction to ammonia, which increases bioavailability of nitrogen in seawater. An identical choline dehydrogenase found in both MAGs have a low amino-acid identity (≤64.47%) compared to existing GMC family oxidoreductases, expanding on the diversity of this family of enzymes. The MAGs meet nearly all the minimum requirements but lack a 16S rRNA gene of sufficient length required for the proposed novel genus and species under SeqCode. Nevertheless, phylogenetic trees based on core-genome and RpoB as an alternative phylogenetic marker are congruent with the taxon standing as a monophyletic clade to other taxa of the order Cellvibrionales. Taken together, the MAGs (SJ0813 and SJ0972) represent an uncultured, undescribed genus and species in which we tentatively propose the name Candidatus Pelagadaptatus aseana gen. nov., sp. nov. and strain SJ0813TS (=BAABNI000000000.1TS) as type sequence. Phylogenetic inference from core-genome and RpoB phylogenetic trees placed Umboniibacter marinipuniceus KMM 3891T outside Cellvibrionaceae. We, therefore, propose the transfer of the genus Umboniibacter from the family Cellvibrionaceae to a new family Umboniibacteraceae according to the International Code of Nomenclature of Prokaryotes.

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.