Genome sequences of three Bartonella schoenbuchensis strains from Czechia.

Bartonella schoenbuchensis causes bacteremia in ruminants and is transmitted by deer keds. Here, we report the complete genome sequences of three B. schoenbuchensis strains (L2, L19, and L24) recently isolated from deer keds (Lipoptena fortisetosa) in Czechia.

Phenotype-driven assessment of the ancestral trajectory of sulfur biooxidation in the thermoacidophilic archaea Sulfolobaceae.

Certain members of the family Sulfolobaceae represent the only archaea known to oxidize elemental sulfur, and their evolutionary history provides a framework to understand the development of chemolithotrophic growth by sulfur oxidation. Here, we evaluate the sulfur oxidation phenotype of Sulfolobaceae species and leverage comparative genomic and transcriptomic analysis to identify the key genes linked to sulfur oxidation. Metabolic engineering of the obligate heterotroph Sulfolobus acidocaldarius revealed that the known cytoplasmic components of sulfur oxidation alone are not sufficient to drive prolific sulfur oxidation. Imaging analysis showed that Sulfolobaceae species maintain proximity to the sulfur surface but do not necessarily contact the substrate directly. This indicates that a soluble form of sulfur must be transported to initiate cytoplasmic sulfur oxidation. Conservation patterns and transcriptomic response implicate an extracellular tetrathionate hydrolase and putative thiosulfate transporter in a newly proposed mechanism of sulfur acquisition in the Sulfolobaceae.IMPORTANCESulfur is one of the most abundant elements on earth (2.9% by mass), so it makes sense that the earliest biology found a way to use sulfur to create and sustain life. However, beyond evolutionary significance, sulfur and the molecules it comprises have important technological significance, not only in chemicals such as sulfuric acid and in pyritic ores containing critical metals but also as a waste product from oil and gas production. The thermoacidophilic Sulfolobaceae are unique among the archaea as sulfur oxidizers. The trajectory for how sulfur biooxidation arose and evolved can be traced using experimental and bioinformatic analyses of the available genomic data set. Such analysis can also inform the process by which extracellular sulfur is acquired and transported by thermoacidophilic archaea, a phenomenon that is critical to these microorganisms but has yet to be elucidated.

Complete genome sequence of a Marinococcus sp. PL1-022 isolated from the pink hypersaline Pearse Lakes, Rottnest Island, Western Australia.

Marinococcus sp. PL1-022 was isolated from Pearse Lakes, Western Australia. The sequenced genome consists of a chromosome (3,140,198 bp; 48.2% GC) and two plasmids (58,083 bp and 19,399 bp; 41.4 and 50.7% GC-content, respectively). Isolation of Marinococcus sp. PL1-022 adds to the increasing repertoire of culturable extremophiles.

Genome drafts of Pseudogracilibacillus spp. from soil and sediment of Mount Makiling, a dormant volcano in the Philippines.

We present the draft whole-genome sequences of Pseudogracilibacillus spp. isolated from the soils and sediments of Sipit Creek located at Mount Makiling, a dormant volcano in the southern part of Luzon Island, Philippines. This Pseudogracilibacillus spp. genome report extends the body of knowledge on a lesser-known genus of Bacillaceae.

Anti-diabetic effect of dicaffeoylquinic acids is associated with the modulation of gut microbiota and bile acid metabolism.

INTRODUCTION: The human gut microbiome plays a pivotal role in health and disease, notably through its interaction with bile acids (BAs). BAs, synthesized in the liver, undergo transformation by the gut microbiota upon excretion into the intestine, thus influencing host metabolism. However, the potential mechanisms of dicaffeoylquinic acids (DiCQAs) from Ilex kudingcha how to modulate lipid metabolism and inflammation via gut microbiota remain unclear. OBJECTIVES AND METHODS: The objectives of the present study were to investigate the regulating effects of DiCQAs on diabetes and the potential mechanisms of action. Two mice models were utilized to investigate the anti-diabetic effects of DiCQAs. Additionally, analysis of gut microbiota structure and functions was conducted concurrently with the examination of DiCQAs' impact on gut microbiota carrying the bile salt hydrolase (BSH) gene, as well as on the enterohepatic circulation of BAs and related signaling pathways. RESULTS: Our findings demonstrated that DiCQAs alleviated diabetic symptoms by modulating gut microbiota carrying the BSH gene. This modulation enhanced intestinal barrier integrity, increased enterohepatic circulation of conjugated BAs, and inhibited the farnesoid X receptor-fibroblast growth factor 15 (FGF15) signaling axis in the ileum. Consequently, the protein expression of hepatic FGFR4 fibroblast growth factor receptor 4 (FGFR4) decreased, accompanied by heightened BA synthesis, reduced hepatic BA stasis, and lowered levels of hepatic and plasma cholesterol. Furthermore, DiCQAs upregulated glucolipid metabolism-related proteins in the liver and muscle, including v-akt murine thymoma viral oncogene homolog (AKT)/glycogen synthase kinase 3-beta (GSK3ß) and AMP-activated protein kinase (AMPK), thereby ameliorating hyperglycemia and mitigating inflammation through the down-regulation of the MAPK signaling pathway in the diabetic group. CONCLUSION: Our study elucidated the anti-diabetic effects and mechanism of DiCQAs from I. kudingcha, highlighting the potential of targeting gut microbiota, particularly Acetatifactor sp011959105 and Acetatifactor muris carrying the BSH gene, as a therapeutic strategy to attenuate FXR-FGF15 signaling and ameliorate diabetes.

Genomic representativeness and chimerism in large collections of SAGs and MAGs of marine prokaryoplankton.

BACKGROUND: Single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) are the predominant sources of information about the coding potential of uncultured microbial lineages, but their strengths and limitations remain poorly understood. Here, we performed a direct comparison of two previously published collections of thousands of SAGs and MAGs obtained from the same, global environment. RESULTS: We found that SAGs were less prone to chimerism and more accurately reflected the relative abundance and the pangenome content of microbial lineages inhabiting the epipelagic of the tropical and subtropical ocean, as compared to MAGs. SAGs were also better suited to link genome information with taxa discovered through 16S rRNA amplicon analyses. Meanwhile, MAGs had the advantage of more readily recovering genomes of rare lineages. CONCLUSIONS: Our analyses revealed the relative strengths and weaknesses of the two most commonly used genome recovery approaches in environmental microbiology. These considerations, as well as the need for better tools for genome quality assessment, should be taken into account when designing studies and interpreting data that involve SAGs or MAGs. Video Abstract.

Plethora of new marsupial genomes informs our knowledge of marsupial MHC class II.

The major histocompatibility complex plays a vital role in the vertebrate immune system due to its role in infection, disease and autoimmunity, or recognition of "self". The marsupial MHC class II genes show divergence from eutherian MHC class II genes and are a unique taxon of therian mammals that give birth to altricial and immunologically naïve young providing an opportune study system for investigating evolution of the immune system. Additionally, the MHC in marsupials has been implicated in disease associations, including susceptibility to Chlamydia pecorum infection in koalas. Due to the complexity of the gene family, automated annotation is not possible so here we manually annotate 384 class II MHC genes in 29 marsupial species. We find losses of key components of the marsupial MHC repertoire in the Dasyuromorphia order and the Pseudochiridae family. We perform PGLS analysis to show the gene losses we find are true gene losses and not artefacts of unresolved genome assembly. We investigate the associations between the number of loci and life history traits, including lifespan and reproductive output in lineages of marsupials and hypothesise that gene loss may be linked to the energetic cost and trade-offs associated with pregnancy and reproduction. We found support for litter size being a significant predictor of the number of DBA and DBB loci, indicating a trade-off between the energetic requirements of immunity and reproduction. Additionally, we highlight the increased susceptibility of Dasyuridae species to neoplasia and a potential link to MHC gene loss. Finally, these annotations provide a valuable resource to the immunogenetics research community to move forward and further investigate diversity in MHC genes in marsupials.

Assembly and comparative analysis of the complete mitochondrial genome of Vaccinium carlesii Dunn.

Vaccinium L. is an important fruit tree with nutritional, medicinal, and ornamental values. However, the mitochondrial (mt) genome of Vaccinium L. remains largely unexplored. Vaccinium carlesii Dunn is an endemic wild resource in China, which is crucial for blueberry breeding. The V. carlesii mt genomes were sequenced using Illumina and Nanopore, which total length was 636,904 bp with 37 protein coding genes, 20 tRNA genes, and three rRNA genes. We found four pairs of long repeat fragments homologous recombination mediated the generation of substructures in the V. carlesii mt genome. We predicted 383 RNA editing sites, all converting cytosine (C) to uracil (U). According to the phylogenetic analysis, V. carlesii and V. macrocarpon of the Ericaceae exhibited the closest genetic relationship. This study provides a theoretical basis for understanding the evolution of higher plants, species classification and identification, and will also be useful for further utilization of Vaccinium germplasm resources.

The smallest angiosperm genomes may be the price for effective traps of bladderworts.

BACKGROUND: Species of the carnivorous family Lentibulariaceae exhibit the smallest genomes in flowering plants. We explored the hypothesis that their minute genomes result from the unique mitochondrial cytochrome c oxidase (COX) mutation. The mutation may boost mitochondrial efficiency, which is especially useful for suction-bladder traps of Utricularia, but also increase DNA-damaging reactive oxygen species, leading to genome shrinkage through deletion-biased DNA repair. We aimed to explore this mutation's impact on genome size, providing insights into genetic mutation roles in plant genome evolution under environmental pressures. METHODS: We compiled and measured genome and mean chromosome sizes for 127 and 67 species, respectively, representing all three genera (Genlisea, Pinguicula, and Utricularia) of Lentibulariaceae. We also isolated and analyzed COX sequences to detect the mutation. Through phylogenetic regressions and Ornstein-Uhlenbeck models of trait evolution, we assessed the impact of the COX mutation on the genome and chromosome sizes across the family. RESULTS: Our findings reveal significant correlations between the COX mutations and smaller genome and chromosome sizes. Specifically, species carrying the ancestral COX sequence exhibited larger genomes and chromosomes than those with the mutation. This evidence supports the notion that the COX mutation contributes to genome downsizing, with statistical analyses confirming a directional evolution towards smaller genomes in species harboring these mutations. CONCLUSIONS: Our study confirms that the COX mutation in Lentibulariaceae is associated with genome downsizing, likely driven by increased reactive oxygen species production and subsequent DNA damage requiring deletion-biased repair mechanisms. While boosting mitochondrial energy output, this genetic mutation compromises genome integrity and may potentially affect recombination rates, illustrating a complex trade-off between evolutionary advantages and disadvantages. Our results highlight the intricate processes by which genetic mutations and environmental pressures shape genome size evolution in carnivorous plants.

Label-free-based proteomics analysis reveals differential proteins of sheep, goat and cow milk.

Regarding the limited information on species protein differences between sheep, goat, and cow milk, the differentially expressed proteins in sheep, goat, and cow milk and their functional differences are analyzed using label-free proteomics technology to identify potential biomarkers. 770 proteins and 2914 peptide segments were identified. The statistical analysis showed significant differences in the relative abundances of the 74 proteins among the sheep, goat, and cow milk. CSN3 and LALBA can be used as potential biomarkers for goat milk, XDH can be used as potential biomarkers for cow milk, and CTSB and BPIFB1 can be used as potential biomarkers for sheep milk. The functional analysis using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes showed that these significantly different proteins were enriched by different pathways including thyroid hormone synthesis and glycerol phospholipid metabolism. The data revealed differences in the amounts and physiological functions of the milk proteins of different species, which may provide an important basis for research on the nutritional composition of dairy products and adulteration identification technology.

Divergent histopathological and molecular patterns in chemically induced interstitial cystitis/bladder pain syndrome rat models.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a complex chronic pain disorder with an elusive etiology and nonspecific symptoms. Although numerous animal models with phenotypes similar to human disease have been established, no available regimen can consistently alleviate clinical symptoms. This dilemma led us to question whether current animal models adequately represent IC/BPS. We compared four commonly used IC/BPS rat models to determine their diverse histopathological and molecular patterns. Female rats were given single treatments with hydrochloric acid (HCL), acetic acid (AA), protamine sulfate plus lipopolysaccharide (PS + LPS), or cyclophosphamide (CYP) to induce IC/BPS. Bladder sections were stained for histopathologic evaluation, and mRNA expression profiles were examined using next-generation sequencing and gene set analyses. Mast cell counts were significantly higher in the HCL and AA groups than in the PS + LPS, CYP, and control groups, but only the AA group showed significant collagen accumulation. The models differed substantially in terms of their gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our observations suggest that none of these rat models fully reflects the complexity of IC/BPS. We recommend that future studies apply and compare multiple models simultaneously to fully replicate the complicated features of IC/BPS.

Antimicrobial resistance and its risks evaluation in wetlands on the Qinghai-Tibetan Plateau.

Amidst the global antimicrobial resistance (AMR) crisis, antibiotic resistance has permeated even the most remote environments. To understand the dissemination and evolution of AMR in minimally impacted ecosystems, the resistome and mobilome of wetlands across the Qinghai-Tibetan Plateau and its marginal regions were scrutinized using metagenomic sequencing techniques. The composition of wetland microbiomes exhibits significant variability, with dominant phyla including Proteobacteria, Actinobacteria, Bacteroidetes, and Verrucomicrobia. Notably, a substantial abundance of Antibiotic Resistance Genes (ARGs) and Mobile Genetic Elements (MGEs) was detected, encompassing 17 ARG types, 132 ARG subtypes, and 5 types of MGEs (Insertion Sequences, Insertions Sequences, Genomic Islands, Transposons, and Integrative Conjugative Elements). No significant variance was observed in the prevalence of resistome and mobilome across different wetland types (i.e., the Yellow River, other rivers, lakes, and marshes) (R=-0.5882, P=0.607). The co-occurrence of 74 ARG subtypes and 22 MGEs was identified, underscoring the pivotal role of MGEs in shaping ARG pools within the Qinghai-Tibetan Plateau wetlands. Metagenomic binning and analysis of assembled genomes (MAGs) revealed that 93 out of 206 MAGs harbored ARGs (45.15 %). Predominantly, Burkholderiales, Pseudomonadales, and Enterobacterales were identified as the primary hosts of these ARGs, many of which represent novel species. Notably, a substantial proportion of ARG-carrying MAGs also contained MGEs, reaffirming the significance of MGEs in AMR dissemination. Furthermore, utilizing the arg_ranker framework for risk assessment unveiled severe contamination of high-risk ARGs across most plateau wetlands. Moreover, some prevalent human pathogens were identified as potential hosts for these high-risk ARGs, posing substantial transmission risks. This study aims to investigate the prevalence of resistome and mobilome in wetlands, along with evaluating the risk posed by high-risk ARGs. Such insights are crucial for informing environmental protection strategies and facilitating the management of water resources on the Qinghai-Tibetan Plateau.

Draft genome of Bacillus subtilis strain YB955, prophage-cured derivative of strain 168.

We report the genome sequence of Bacillus subtilis strain YB955, a prophage-cured strain used as a model in DNA repair, bacterial physiology, and mutagenesis studies. The assembled and annotated draft genome contains 4,031 coding genes, 5 rRNAs, and 73 tRNAs. Compared to 168, YB955 has a 134,402 bp deletion.

Whole-genome sequences of Helicobacter pylori isolated from patients with high risk of gastric cancer in the Andes of Nariño, Colombia.

We sequenced the complete genomes of 16 Helicobacter pylori isolates obtained from patients residing in Nariño, Colombia. These isolates were collected from patients presenting various gastric lesions classified according to the Correa cascade classification. The genomic characterization of these isolates provides valuable insights into the structure, composition, and pathogenicity.

Extremely halophilic brine community manipulation shows higher robustness of microbiomes inhabiting human-driven solar saltern than naturally driven lake.

Hypersaline ecosystems display taxonomically similar assemblages with low diversities and highly dense accompanying viromes. The ecological implications of viral infection on natural microbial populations remain poorly understood, especially at finer scales of diversity. Here, we sought to investigate the influence of changes in environmental physicochemical conditions and viral predation pressure by autochthonous and allochthonous viruses on host dynamics. For this purpose, we transplanted two microbiomes coming from distant hypersaline systems (solar salterns of Es Trenc in Spain and the thalassohaline lake of Aran-Bidgol lake in Iran), by exchanging the cellular fractions with the sterile-filtered accompanying brines with and without the free extracellular virus fraction. The midterm exposure (1 month) of the microbiomes to the new conditions showed that at the supraspecific taxonomic range, the assemblies from the solar saltern brine more strongly resisted the environmental changes and viral predation than that of the lake. The metagenome-assembled genomes (MAGs) analysis revealed an intraspecific transition at the ecotype level, mainly driven by changes in viral predation pressure, by both autochthonous and allochthonous viruses. IMPORTANCE: Viruses greatly influence succession and diversification of their hosts, yet the effects of viral infection on the ecological dynamics of natural microbial populations remain poorly understood, especially at finer scales of diversity. By manipulating the viral predation pressure by autochthonous and allochthonous viruses, we uncovered potential phage-host interaction, and their important role in structuring the prokaryote community at an ecotype level.

Diverse and unconventional methanogens, methanotrophs, and methylotrophs in metagenome-assembled genomes from subsurface sediments of the Slate River floodplain, Crested Butte, CO, USA.

We use metagenome-assembled genomes (MAGs) to understand single-carbon (C1) compound-cycling-particularly methane-cycling-microorganisms in montane riparian floodplain sediments. We generated 1,233 MAGs (>50% completeness and <10% contamination) from 50- to 150-cm depth below the sediment surface capturing the transition between oxic, unsaturated sediments and anoxic, saturated sediments in the Slate River (SR) floodplain (Crested Butte, CO, USA). We recovered genomes of putative methanogens, methanotrophs, and methylotrophs (n = 57). Methanogens, found only in deep, anoxic depths at SR, originate from three different clades (Methanoregulaceae, Methanotrichaceae, and Methanomassiliicoccales), each with a different methanogenesis pathway; putative methanotrophic MAGs originate from within the Archaea (Candidatus Methanoperedens) in anoxic depths and uncultured bacteria (Ca. Binatia) in oxic depths. Genomes for canonical aerobic methanotrophs were not recovered. Ca. Methanoperedens were exceptionally abundant (~1,400× coverage, >50% abundance in the MAG library) in one sample that also contained aceticlastic methanogens, indicating a potential C1/methane-cycling hotspot. Ca. Methylomirabilis MAGs from SR encode pathways for methylotrophy but do not harbor methane monooxygenase or nitrogen reduction genes. Comparative genomic analysis supports that one clade within the Ca. Methylomirabilis genus is not methanotrophic. The genetic potential for methylotrophy was widespread, with over 10% and 19% of SR MAGs encoding a methanol dehydrogenase or substrate-specific methyltransferase, respectively. MAGs from uncultured Thermoplasmata archaea in the Ca. Gimiplasmatales (UBA10834) contain pathways that may allow for anaerobic methylotrophic acetogenesis. Overall, MAGs from SR floodplain sediments reveal a potential for methane production and consumption in the system and a robust potential for methylotrophy.IMPORTANCEThe cycling of carbon by microorganisms in subsurface environments is of particular relevance in the face of global climate change. Riparian floodplain sediments contain high organic carbon that can be degraded into C1 compounds such as methane, methanol, and methylamines, the fate of which depends on the microbial metabolisms present as well as the hydrological conditions and availability of oxygen. In the present study, we generated over 1,000 MAGs from subsurface sediments from a montane river floodplain and recovered genomes for microorganisms that are capable of producing and consuming methane and other C1 compounds, highlighting a robust potential for C1 cycling in subsurface sediments both with and without oxygen. Archaea from the Ca. Methanoperedens genus were exceptionally abundant in one sample, indicating a potential C1/methane-cycling hotspot in the Slate River floodplain system.

Metagenomics analysis yields assembled genomes from prokaryotic anaerobes with polymer-degrading potential.

Anaerobic microbial communities are often highly degradative, such as those found in the herbivore rumen and large-scale anaerobic digesters. Since the microbial communities in these systems degrade recalcitrant organic polymers, we hypothesize that some microbes in anaerobic environments may be involved in man-made plastic association, deformation, or even breakdown. While efforts have been put toward characterizing microbial communities, many microbes remain unidentified until they can be sufficiently cultivated to generate enough genetic material to assemble high-quality metagenome assemblies and reference genomes. In this study, microbial consortia from goat fecal pellets and anaerobic digester sludge were cultivated for over 6 weeks to assemble metagenomes from novel anaerobic taxa with potential degradative activity. To select for microbes with potential plastic-degrading abilities, plastic strips were included in culture, though the presence of plastic did not appear to enrich for particularly degradative consortia, yet it did select for novel species that otherwise may not have been characterized. Whole-genome shotgun sequencing enabled assembly of 72 prokaryotic metagenome-assembled genomes (MAGs) with >90% completion, <5% contamination, and an N50 >10,000 bp; 17 of these MAGs are classified as novel species given their lack of similarity to publicly available genomes and MAGs. These 72 MAGs vary in predicted carbohydrate-degrading abilities, with genes predicted to encode fewer than 10 or up to nearly 400 carbohydrate-active enzymes. Overall, this enrichment strategy enables characterization of less abundant MAGs in a community, and the MAGs identified here can be further mined to advance understanding of degradative anaerobic microbial consortia.

Photostasis and photosynthetic adaptation to polar life.

Photostasis is the light-dependent maintenance of energy balance associated with cellular homeostasis in photoautotrophs. We review evidence that illustrates how photosynthetic adaptation in polar photoautrophs such as aquatic green algae, cyanobacteria, boreal conifers as well as terrestrial angiosperms exhibit an astonishing plasticity in structure and function of the photosynthetic apparatus. This plasticity contributes to the maintenance of photostasis, which is essential for the long-term survival in the seemingly inhospitable Antarctic and Arctic habitats. However, evidence indicates that polar photoautrophic species exhibit different functional solutions for the maintenance of photostasis. We suggest that this reflects, in part, the genetic diversity symbolized by inherent genetic redundancy characteristic of polar photoautotrophs which enhances their survival in a thermodynamically challenging environment.

Lacticaseibacillus rhamnosus Strains for Alleviation of Irritable Bowel Disease and Chronic Fatigue Syndrome.

Lacticaseibacillus rhamnosus is applied as a probiotic to alleviate various metabolic, gastrointestinal, and psychological symptoms and diseases, and its probiotic effectiveness is strain-specific. In this study, we obtained 21 strains of Ls. rhamnosus, and their genomes were sequenced. We defined the pan- and core-genomes of Ls. rhamnosus. Phenotypes such as the assimilation of carbohydrates and antibiotic resistance were experimentally characterized and associated with genome annotations. Nine strains were selected and tested for growth rates, tolerance to acidity/alkalinity and bile acids, the production of short-chain fatty acids, and competition with pathogenic microbes. Strains WL11 and WL17 were targeted as potential probiotics and were applied in mouse model tests for the alleviation of chronic fatigue syndrome (CFS) and irritable bowel syndrome (IBS). The results showed that WL11 and WL17 effectively alleviated slow body weight gain, anxiety, poor memory, and cognitive impairment in CFS mouse models. They also reduced the expression of pro-inflammatory factors, such as TNF-α and IL-6, and alleviated intestinal peristalsis, visceral hypersensitivity, and anxiety-like behavior in IBS mouse models. This study reports new Ls. rhamnosus strain resources and their effect on alleviation of both IBS and CFS symptoms with mouse models; the probiotic functions of those strains in human patients remain to be further tested.

Prevalence and diversity of TAL effector-like proteins in fungal endosymbiotic Mycetohabitans spp.

Endofungal Mycetohabitans (formerly Burkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus, Rhizopus microsporus. The one known secreted effector family from Mycetohabitans consists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenic Xanthomonas and Ralstonia spp. to activate host genes that promote disease. These 'Burkholderia TAL-like (Btl)' proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of two Mycetohabitans strains revealed that the single Btl in one Mycetohabitans endofungorum strain enhances fungal membrane stress tolerance, while others in a Mycetohabitans rhizoxinica strain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nine Mycetohabitans spp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal-bacterial metagenomes. We show that btl genes are present across diverse Mycetohabitans strains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested that Mycetohabitans might contain more species than previously recognized. Within our data set, Btl proteins were more conserved across M. rhizoxinica strains than across M. endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial-fungal symbioses in myriad ways.