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Long non-coding RNAs (lncRNAs) are transcripts longer than 200 bp with low or no protein-coding ability, which play essential roles in various biological processes in plants. Tobacco is an ideal model plant for studying nicotine biosynthesis and metabolism, and there is little research on lncRNAs in this field. Therefore, how to take advantage of the mature tobacco system to profoundly investigate the lncRNAs involved in the nicotine pathway is intriguing. By exploiting 549 public RNA-Seq datasets of tobacco, 30,212 lncRNA candidates were identified, including 24,084 large intervening non-coding RNAs (lincRNAs), 5,778 natural antisense transcripts (NATs) and 350 intronic non-coding RNAs (incRNAs). Compared with protein-coding genes, lncRNAs have distinct properties in terms of exon number, sequence length, A/U content, and tissue-specific expression pattern. lincRNAs showed an asymmetric evolutionary pattern, with a higher proportion (68.71%) expressed from the Nicotiana sylvestris (S) subgenome. We predicted the potential cis/trans-regulatory effects on protein-coding genes. One hundred four lncRNAs were detected as precursors of 30 known microRNA (miRNA) family members, and 110 lncRNAs were expected to be the potential endogenous target mimics for 39 miRNAs. By combining the results of weighted gene co-expression network analysis with the differentially expressed gene analysis of topping RNA-seq data, we constructed a sub-network containing eight lncRNAs and 25 nicotine-related coding genes. We confirmed that the expression of seven lncRNAs could be affected by MeJA treatment and may be controlled by the transcription factor NtMYC2 using a quantitative PCR assay and gene editing. The results suggested that lncRNAs are involved in the nicotine pathway. Our findings further deepened the understanding of the features and functions of lncRNAs and provided new candidates for regulating nicotine biosynthesis in tobacco.

The advent of single-cell sequencing opened a new era in transcriptomic and genomic research. To understand cell composition using single-cell studies, a variety of cell markers have been widely used to label individual cell types. However, the specific database of cell markers for use by the plant research community remains very limited. To overcome this problem, we developed the Plant Cell Marker DataBase (PCMDB, http://www.tobaccodb.org/pcmdb/), which is based on a uniform annotation pipeline. By manually curating over 130 000 research publications, we collected a total of 81 117 cell marker genes of 263 cell types in 22 tissues across six plant species. Tissue- and cell-specific expression patterns can be visualized using multiple tools: eFP Browser, Bar, and UMAP/TSNE graph. The PCMDB also supports several analysis tools, including SCSA and SingleR, which allows for user annotation of cell types. To provide information about plant species currently unsupported in PCMDB, potential marker genes for other plant species can be searched based on homology with the supported species. PCMDB is a user-friendly hierarchical platform that contains five built-in search engines. We believe PCMDB will constitute a useful resource for researchers working on cell type annotation and the prediction of the biological function of individual cells.

Long non-coding RNAs (lncRNAs) regulate many biological processes in plants, including defense against pathogens and herbivores. Recently, many small ORFs embedded in lncRNAs have been identified to encode biologically functional peptides (small ORF-encoded peptides [SEPs]) in many species. However, it is unknown whether lncRNAs mediate defense against herbivore attack and whether there are novel functional SEPs for these lncRNAs. By sequencing Spodoptera litura-treated leaves at six time-points in Nicotiana tabacum, 22,436 lncRNAs were identified, of which 787 were differentially expressed. Using a comprehensive mass spectrometry (MS) pipeline, 302 novel SEPs derived from 115 tobacco lncRNAs were identified. Moreover, 61 SEPs showed differential expression after S. litura attack. Importantly, several of these peptides were characterized through 3D structure prediction, subcellular localization validation by laser confocal microscopy, and western blotting. Subsequent bioinformatic analysis revealed some specific chemical and physical properties of these novel SEPs, which probably represent the largest number of SEPs identified in plants to date. Our study not only identifies potential lncRNA regulators of plant response to herbivore attack but also serves as a valuable resource for the functional characterization of SEP-encoding lncRNAs.

Background Long noncoding RNAs (lncRNAs) are transcripts longer than 200bp with low or no protein-coding ability, which play essential roles in various biological processes in plants. Tobacco is an ideal model plant for studying nicotine biosynthesis and metabolism, and there is little research on lncRNAs in this field. Therefore, how to take advantage of the mature tobacco system to profoundly investigate the lncRNAs involved in the nicotine pathway is intriguing. Results By exploiting 549 public RNA-Seq datasets of tobacco, 30,212 lncRNA candidates were identified, including 24,084 large intervening noncoding RNAs (lincRNAs) and 5,778 natural antisense transcripts (NATs), and 350 intronic noncoding RNAs (incRNAs). Compared with protein-coding genes, lncRNAs have distinct properties in terms of exon number, sequence length, A/U content, and tissue-specific expression pattern. incRNAs showed an asymmetric evolutionary pattern, with a higher proportion (68.71%) expressed from N.sylvestris (S) subgenome. We predicted the potential cis/trans-regulatory effects on protein-coding genes. One hundred four lncRNAs were detected as precursors of 30 known miRNA family members, and 110 lncRNAs were expected to be the potential endogenous target mimic for 39 miRNAs. By combining the results of weighted gene co-expression network analysis with the differential expressed gene analysis of topping RNA-seq data, we constructed a sub-network containing eight lncRNAs and 25 nicotine-related coding genes. We confirmed that seven lncRNAs could be affected by MeJA treatment and transcription factor NtMYC2 using a quantitative PCR assay. Conclusions Nicotine pathway-related lncRNA candidates were predicted and investigated in our study. We verified that seven lncRNAs could be affected by MeJA treatment and the transcription factor NtMYC2. Results suggested that they are involved in the nicotine pathway. Our findings further deepened the understanding of the features and functions of lncRNAs and provided new candidates for regulating nicotine biosynthesis in tobacco.

Root-associated compartments, including the rhizosphere, rhizoplane, and endosphere, live with diverse microbial communities which profoundly affect plant growth and health. However, a systematic understanding of the microbiome assembly across the rhizosphere, rhizoplane, and endosphere under pathogen invasion remains elusive. Using 16S high-throughput sequencing, we studied how bacterial wilt disease affected the variation and assembly of the three continuous root-associated microbiomes of tobacco. The results indicated that microorganisms were gradually filtered from the rhizosphere to the endosphere. With the pathogen invasion, the rhizosphere, rhizoplane and endosphere microbiomes selected and recruited different beneficial bacterial taxa. Some recruited bacteria were also identified as keystone members in networks (i.e., Bosea in the endosphere). The microbiomes of endosphere and rhizoplane were more sensitive to plant disease than the rhizosphere microbiome. Still, response strategies of the rhizoplane and endosphere to disease were obviously different. Microbial networks of the rhizoplane became complex in diseased samples and genes involved in sporulation formation and cell cycle were enriched. However, microbial networks of the diseased endosphere were disrupted, and functional genes related to nitrogen utilization and chemotaxis were significantly increased, indicating the importance of nitrogen resources supply of plants for the endosphere microbiome under pathogen invasion. Our results provide novel insights for understanding the different responses of the root-associated microbiomes to plant disease.

Long non-coding RNAs (lncRNAs) regulate many biological processes in plants, including defense against pathogens and herbivores. Recently, many small ORFs embedded in lncRNAs have been identified to encode biologically functional peptides (small ORF-encoded peptides [SEPs]) in many species. However, it is unknown whether lncRNAs mediate defense against herbivore attack and whether there are novel functional SEPs for these lncRNAs. By sequencing

Root-knot nematode (RKN) disease is a destructive soil disease that affects crop health and causes huge losses in crop production. To explore the relationships between soil environments, rhizobacterial communities, and plant health, rhizosphere bacterial communities were analyzed using metagenomic sequencing in tobacco samples with different grades of RKN disease. The results showed that the community structure and function of the plant rhizosphere were significantly correlated to the RKN disease. RKN density and urease content were key factors affecting the rhizosphere bacterial community. Urease accelerated the catabolism of urea and led to the production of high concentrations of ammonia, which directly suppressed the development of RKNs or by improving the nutritional and growth status of microorganisms that were antagonistic to RKNs. Further experiments showed that the suppression role of ammonia should be attributed to the direct inhibition of NH

The root phenotypic traits have been considered as important factors in shaping the rhizosphere microbiome and regulating plant growth. However, the relationships between root phenotypic traits and the rhizosphere bacterial community remain unclear. We investigated two fields with different developing tobacco roots by a long-term positioning test in Hengshi. The well-developed root system (WDR) showed much more superiority in root phenotypic traits, including total root length, total projection area, surface area, and root tip number, than the underdeveloped root system. The specific root traits in WDR provided more ecological niches for the rhizosphere microorganisms, contributing to a more diverse microbial community and a more complex microbial network. The total root length and root tip number were the key factors shaping bacterial communities in the rhizosphere. In turn, the phyla Acidobacteria and Bacteroidetes might play vital roles in modifying root development and promoting plant growth according to their positive correlation with root phenotypic traits. Linking root phenotypic traits to the microbiome may enhance our understanding of rhizospheric interactions and their roles in developing rhizosphere ecosystems.

Extremely endangered wildlife can be effectively protected by ex situ conservation programs. Despite that, the effects of human activity on the health of captive populations are still unclear. Using reduced representation bisulfite sequencing and RNA-seq, we assessed epigenetic and gene transcriptional variations between different types of human intervention (“Custodial” and “Sanctuary” groups) and a “Wild” group of golden snub-nosed monkeys (Rhinopithecus roxellana), which is one of the most endangered primates worldwide. Consequently, we found striking genome-scale divergence of both DNA methylation and gene expression for the Custodial group, which agreed with stronger human interventions on the food supply and management of the group. KEGG pathway analyses indicated that the differentially expressed genes were enriched in a wide range of physiologically functional pathways, most of which are involved in immune and metabolic functions. In particular, a group of core genes in autophagy related pathways, e.g., ATP6V0A1 and PPM1D, were both differentially expressed and methylated. Our findings revealed that the ex situ conserved golden snub-nosed monkey exhibited significant epigenetic and transcriptional adaptation to human intervention, thereby may provide a foundation for future biomarker development that can be used to evaluate the unintended risks of ex situ conservation programs for endangered animal species.

Microbial degradation of organic compounds is an environmentally benign and energy efficient part in product processing. Fermentation of plant leaves involves enzymatic actions of many microorganisms. However, microbes and enzymes discovered from natural degradation communities were still limited by cultural methods. In this study, we used a metagenomics sequence-guided strategy to identify the microbes and enzymes involved in compound degradation and explore the potential synergy among community members in fermented tobacco leaves. The results showed that contents of protein, starch, pectin, lignin, and cellulose varied in fermented leaves from different growing sites. The different compound contents were closely related to taxonomic composition and functional profiles of foliar microbial communities. Microbial communities showed significant correlations with protein, lignin, and cellulose. Vital species for degradations of protein (Bacillus cereus and Terribacillus aidingensis), lignin (Klebsiella pneumoniae and Pantoea ananatis) and cellulose (Pseudomonas putida and Sphingomonas sp. Leaf20) were identified and relating hydrolytic enzymes were annotated. Further, twenty-two metagenome-assembled genomes (MAGs) were assembled from metagenomes and six potential cellulolytic genomes were used to reconstruct the cellulose-degrading process, revealing the potential metabolic cooperation related to cellulose degradation. Our work should deepen the understanding of microbial roles in plant fermentation and provide a new viewpoint for applying microbial consortia to convert plant organic components to small molecules.

Plant leaves are colonized by a remarkably diverse fungal microbiome, which contributes to host plant growth and health. However, responses of foliar fungal community to phytopathogen invasion and measures of the fungal community taken to resist or assist pathogens remain elusive. By utilizing high-throughput sequencing of internal transcribed spacer (ITS) amplicons, we studied the relationships between the foliar fungal community around the disease spot and the pathogen of brown spot disease. The pathogenic Alternaria was found to follow a dramatically decreased trend from the disease spot to its surrounding fungal communities, whose community structure also diverged substantially away from the disease spot community. With the increase of pathogenic Alternaria, diversity indexes, including Shannon, Pielou and Simpson, showed a trend of increasing first and then decreasing. Total network links and the average path distance exhibited strong negative and positive correlations with Alternaria, respectively. Five keystone members showed direct interactions with pathogenic Alternaria. Members of Botryosphaeria, Paraphoma and Plectosphaerella might act as key 'pathogen facilitators' to increase the severity and development of brown spot disease, while Pleospora and Ochrocladosporium might be important 'pathogen antagonists' to suppress the expansion of pathogenic Alternaria. Our study provides new insights in developing new strategies for leaf disease prediction or prevention.

Alicyclobacillus species inhabit diverse environments and have adapted to broad ranges of pH and temperature. However, their adaptive evolutions remain elusive, especially regarding the role of mobile genetic elements (MGEs). Here, we characterized the distributions and functions of MGEs in Alicyclobacillus species across five environments, including acid mine drainage (AMD), beverages, hot springs, sediments, and soils. Nine Alicyclobacillus strains were isolated from AMD and possessed larger genome sizes and more genes than those from other environments. Four AMD strains evolved to be mixotrophic and fell into distinctive clusters in phylogenetic tree. Four types of MGEs including genomic island (GI), insertion sequence (IS), prophage, and integrative and conjugative element (ICE) were widely distributed in Alicyclobacillus species. Further, AMD strains did not possess CRISPR-Cas systems, but had more GI, IS, and ICE, as well as more MGE-associated genes involved in the oxidation of iron and sulfide and the resistance of heavy metal and low temperature. These findings highlight the differences in phenotypes and genotypes between strains isolated from AMD and other environments, and the important role of MGEs in rapid environment niche expansions. This article is protected by copyright. All rights reserved.

The improper stacking of chromium (Cr) slag poses a great threat to the environment and human health. The toxicity of Cr in soil is not only related to its total amount, but also to its fractions. A simulated experiment was conducted in laboratory to assess the environmental risk of Cr fractions migration and distribution in red soil. The results showed the content of acid-soluble and reducible Cr significantly decreased (P &lt, 0.05) in top layer but increased in middle and substratum layers over time. This indicated that acid-soluble and reducible Cr migrated downward with time and the relative mobility of acid-soluble Cr (0.038 mg/kg&middot, d&middot, m) was higher than that of reducible Cr (0.028 mg/kg&middot, m). Furthermore, correlation analysis between microbial community and chromium fraction showed the relative abundance of Lysobacter, Flavihumibacter, Flavisolbacter, and Altererythrobacter was significantly (P &lt, 0.05) correlated with acid-soluble and reducible fractions. Thus, these microorganisms might be evaluators to assess the migration of acid-soluble and reducible fractions in red soil. In summary, this study provided a new comprehension on remediation of Cr-contaminated soil by monitoring the migration of acid-soluble and reducible fractions and the changes of related microbial groups.

Bacterial wilt, caused by Ralstonia solanacearum, occurs occasionally during tobacco planting and potentially brings huge economic losses in affected areas. Soil microbes in different management stages play important roles in influencing bacterial wilt incidence. Studies have focused on the impacts of species diversity and composition during cropping periods on disease morbidity; however, the effects of the soil bacterial biomass, species diversity, species succession, and population interactions on morbidity remain unclear during non-cropping periods. In this study, we explored the soil bacterial communities in the non-cropping winter fallow (WF) and cropping late growing (LG) periods under consecutive monoculture systems using 16S ribosomal RNA gene sequencing and qPCR and further analyzed their effects on tobacco bacterial wilt incidence. We found that the bacterial communities in the WF period were significantly different from those in the LG period based on detrended correspondence analysis and dissimilarity tests. Crop morbidity was significantly related to bacterial community structure and to the presence of some genera during WF and LG periods. These genera, such as Arthrobacter, Pseudomonas, Acidobacteria GP6, and Pasteuria, may be potential biological control agents for bacterial wilt. Further analysis indicated that low soil bacterial diversity during the WF period, decrease of bacterial interactions from the WF to LG periods, and low soil biomass during the LG period all have the potential to increase morbidity. In conclusion, an increase of soil bacterial diversity and control of some bacterial abundances in the WF period might be an effective approach in controlling bacterial wilt incidence.

Introducing exogenous species to an indigenous microbial community is an effective way to reveal the connections between metabolic processes, ecological function and microbial community structure. Herein, three different functional consortia (ferrous oxidizers, sulfur oxidizers and ferrous/sulfur oxidizers) were added to a natural leaching solution system derived from Zijin copper mine, China. The leaching experiment showed that the copper extraction rate of the community invaded by a sulfur-oxidizing consortium was 50.40% higher than that of the indigenous leachate at the endpoint of bioleaching. The variations of ferrous content, total iron, pH and redox potential in leachates also provided evidence that the community with exogenous sulfur oxidizers was more efficient. XRD analysis demonstrated that a proper addition of the sulfur-oxidizing consortium could eliminate sulfur passivation, promote production of chalcocite and enhance leaching. Furthermore, an exogenous ferrous-oxidizing consortium and a sulfur-oxidizing consortium greatly changed the community structure and microbial succession and promoted the cell growth rate during the bioleaching process, while ferrous/sulfur oxidizers showed no obvious effects on the indigenous community. Exogenous ferrous oxidizers, mainly L. ferriphilum, and sulfur oxidizers, mainly A. thiooxidans, successfully established and colonized in the indigenous community. However, only colonized A. thiooxidans, rather than L. ferriphilum, showed advantageous enhancement in the dissolution of chalcopyrite. Results indicated that exogenous sulfur oxidizer A. thiooxidans, which was scarce in the indigenous community, could easily colonize in the indigenous community, significantly change the community structure, sufficiently execute its function, and greatly enhance copper dissolution.

Continuous cropping has become the most common system in intensive, modern agricultural production; however, obstacles often appear in continuous cropping patterns after a few years of use. There have been several studies about the impacts of continuous cropping on soil microbial, but few about differences between soil experiencing continuous cropping obstacles and those where such obstacles had been resisted. Here, after ten or twenty years of continuous tobacco cropping, we collected soil samples investigating discrepancies in soil property and bacterial community between soils experiencing continuous cropping obstacles and soils where the obstacles were resisted providing insight into preventing and controlling continuous cropping obstacles. Results showed that soil organic matter (SOM), available phosphorus (AP), total nitrogen (TN), nitrate-N (NO3−-N), and bacterial diversity of samples where continuous cropping obstacles had been resisted were significantly higher than those where continuous cropping obstacles were present. Besides, SOM, AP, TN, and Ammonium-N (NH4+-N) considerably affected the bacterial community. Among all variables, NH4+-N explained the largest proportion of bacterial community variation. Molecular ecological networks were used to putatively identify keystone taxa, including Acidobacteria Gp1, Acidobacteria Gp2, Acidobacteria Gp16, and WPS-1_genera_incertae_sedis. Their relative abundance significantly changed between the two conditions. Overall, our results indicate that decreases in soil nutrient content and bacterial diversity, and significant changes in some keystone taxa abundances may be important factors leading to increased soil-borne diseases and reduced tobacco production potential or quality. Thus, during agricultural production, we could regulate the stability of the soil-crop-microbial ecological system via crop rotation, intercropping, or the use of specialized bio-fertilizers and soil conditioners to mitigate continuous cropping obstacles.

Bioleaching has been widely applied to recover sulfide minerals, however, little is known about the bioleaching behavior and mechanism of mixed oxide-sulfide copper ore with high-oxidative ratio. The leaching efficiency of artificial indigenous and exogenous microbial communities on a mixed oxide-sulfide copper ore with and without energy substrate was investigated. The results indicated that extra energy substrate was needed when bioleaching was applied to this kind of ore. The variation of leaching parameters caused by indigenous community was inferior to that caused by exogenous ones due to its lower activity. The leaching efficiency of sulfide part induced by indigenous strains was superior to that induced by exogenous ones, due to the stronger colonization ability of indigenous strains. It verified that both microbial activity and colonization ability were important properties affecting leaching efficiency. Sulfur oxidizers, which can oxidize sulfur or polysulfide formed on mineral surface and decrease leachate pH, played a more important role than ferrous oxidizers in bioleaching of high-oxidative oxide-sulfide ore.

Toxic ions accumulation in leachate is one of the limitations for bioleaching application. The effects of fluoride ions on microbial growth relevant to bioleaching have been extensively emphasized. However, the reasons that the microorganisms present different fluoride tolerance in bioleaching of different minerals have not yet been proposed. A fundamental exploration on the differential fluoride tolerance of sulfur- and ferrous iron-grown Acidithiobacillus ferrooxidans was performed. The results showed that the critical fluoride concentration for sulfur-grown A. ferrooxidans was less than 0.1 mM whenever fluoride was introduced. The critical fluoride concentration for ferrous iron-grown A. ferrooxidans was 1.0 mM when fluoride was introduced prior to the lag phase, but it was higher than 10.0 mM when fluoride was introduced at the log phase. The ferric ions generated from ferrous oxidation could significantly decrease the concentration of HF and finally reduce the toxicity of fluoride to A. ferrooxidans. The changes of concentration of metal ions with strong complex ability to fluoride ions, resulted in an illusion of high fluorine-resistant strain in industrial bioleaching heap. Finally, the feasibility of fluoride removal by ferric ion was put forward. This work has meaningful implications for the industrial bioleaching of high-fluorine containing uranium ore.

Six artificial communities with different function or biodiversity were reconstructed by six typical bioleaching species for chalcopyrite leaching. Absence of sulfur oxidizers in communities significantly reduced copper extraction rates, and low diversity communities also exhibited slightly poor bioleaching performances. The variations of pH, redox potential, ferrous and copper ions indicated that the community with both sulfur oxidizers and high diversity showed fast adaptation to the environment and rapid dissolution of chalcopyrite. Integrated analysis of mineralogical and microbial parameters demonstrated that functional types of microorganisms made more contributions in mediating chalcopyrite dissolution than microbial diversity. Further correlation analysis between microbial types and chalcopyrite dissolution performances showed that sulfur oxidizers, especially Acidithiobacillus caldus, could greatly accelerate chalcopyrite dissolution by regulating solution physicochemical factors, such as redox potential and pH. This study provided a theoretical basis for improving bioleaching efficiency by balancing microbial functional types and biodiversity.

The interaction between microorganisms and minerals was a hot topic to reveal the transformation of key elements that affecting bioleaching efficiency. Three typical low-grade copper ores, the main copper-bearing components of which were primary sulfide, secondary sulfide and high-oxidative sulfide copper, were obtained from Dexing, Zijinshan and Luanshya copper mine, respectively. Meanwhile, six typical microorganisms were isolated from each of the three habitats, and assembled as communities based on their origins. Cross bioleaching was carried out under identical conditions. The leaching parameters showed that native strains played excellent roles in their corresponding ore bioleaching process, and community structure was greatly determined by mineral composition, indicating that domestication for longitudinal adaption was an effective way to improve microbial leaching performance. Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans promoted copper release by shifting redox potential and pH of the leachate, respectively, indicating that microbial population regulation was another effective way to improve bioleaching efficiency.

Members of the genus Acidithiobacillus, which can adapt to extremely high concentrations of heavy metals, are universally found at acid mine drainage (AMD) sites. Here, we performed a comparative genomic analysis of 37 strains within the genus Acidithiobacillus to answer the untouched questions as to the mechanisms and the evolutionary history of metal resistance genes in Acidithiobacillus spp. The results showed that the evolutionary history of metal resistance genes in Acidithiobacillus spp. involved a combination of gene gains and losses, horizontal gene transfer (HGT), and gene duplication. Phylogenetic analyses revealed that metal resistance genes in Acidithiobacillus spp. were acquired by early HGT events from species that shared habitats with Acidithiobacillus spp., such as Acidihalobacter, Thiobacillus, Acidiferrobacter, and Thiomonas species. Multicopper oxidase genes involved in copper detoxification were lost in iron-oxidizing Acidithiobacillus ferridurans, Acidithiobacillus ferrivorans, and Acidithiobacillus ferrooxidans and were replaced by rusticyanin genes during evolution. In addition, widespread purifying selection and the predicted high expression levels emphasized the indispensable roles of metal resistance genes in the ability of Acidithiobacillus spp. to adapt to harsh environments. Altogether, the results suggested that Acidithiobacillus spp. recruited and consolidated additional novel functionalities during the adaption to challenging environments via HGT, gene duplication, and purifying selection. This study sheds light on the distribution, organization, functionality, and complex evolutionary history of metal resistance genes in Acidithiobacillus spp.IMPORTANCE Horizontal gene transfer (HGT), natural selection, and gene duplication are three main engines that drive the adaptive evolution of microbial genomes. Previous studies indicated that HGT was a main adaptive mechanism in acidophiles to cope with heavy-metal-rich environments. However, evidences of HGT in Acidithiobacillus species in response to challenging metal-rich environments and the mechanisms addressing how metal resistance genes originated and evolved in Acidithiobacillus are still lacking. The findings of this study revealed a fascinating phenomenon of putative cross-phylum HGT, suggesting that Acidithiobacillus spp. recruited and consolidated additional novel functionalities during the adaption to challenging environments via HGT, gene duplication, and purifying selection. Altogether, the insights gained in this study have improved our understanding of the metal resistance strategies of Acidithiobacillus spp.

Additional file 1. Additional figures and table.

Members of the genus Acidithiobacillus, which can adapt to extremely high concentrations of heavy metals, are universally found at acid mine drainage (AMD) sites. Here, we performed a comparative genomic analysis of 37 strains within the genus Acidithiobacillus to answer the untouched questions as to the mechanisms and the evolutionary history of metal resistance genes in Acidithiobacillus spp. The results showed that the evolutionary history of metal resistance genes in Acidithiobacillus spp. involved a combination of gene gains and losses, horizontal gene transfer (HGT), and gene duplication. Phylogenetic analyses revealed that metal resistance genes in Acidithiobacillus spp. were acquired by early HGT events from species that shared habitats with Acidithiobacillus spp., such as Acidihalobacter, Thiobacillus, Acidiferrobacter, and Thiomonas species. Multicopper oxidase genes involved in copper detoxification were lost in iron-oxidizing Acidithiobacillus ferridurans, Acidithiobacillus ferrivorans, and Acidithiobacillus ferrooxidans and were replaced by rusticyanin genes during evolution. In addition, widespread purifying selection and the predicted high expression levels emphasized the indispensable roles of metal resistance genes in the ability of Acidithiobacillus spp. to adapt to harsh environments. Altogether, the results suggested that Acidithiobacillus spp. recruited and consolidated additional novel functionalities during the adaption to challenging environments via HGT, gene duplication, and purifying selection. This study sheds light on the distribution, organization, functionality, and complex evolutionary history of metal resistance genes in Acidithiobacillus spp. IMPORTANCE Horizontal gene transfer (HGT), natural selection, and gene duplication are three main engines that drive the adaptive evolution of microbial genomes. Previous studies indicated that HGT was a main adaptive mechanism in acidophiles to cope with heavy-metal-rich environments. However, evidences of HGT in Acidithiobacillus species in response to challenging metal-rich environments and the mechanisms addressing how metal resistance genes originated and evolved in Acidithiobacillus are still lacking. The findings of this study revealed a fascinating phenomenon of putative cross-phylum HGT, suggesting that Acidithiobacillus spp. recruited and consolidated additional novel functionalities during the adaption to challenging environments via HGT, gene duplication, and purifying selection. Altogether, the insights gained in this study have improved our understanding of the metal resistance strategies of Acidithiobacillus spp.

Introducing exogenous species to an indigenous microbial community is an effective way to reveal the connections between metabolic processes, ecological function and microbial community structure. Herein, three different functional consortia (ferrous oxidizers, sulfur oxidizers and ferrous/sulfur oxidizers) were added to a natural leaching solution system derived from Zijin copper mine, China. The leaching experiment showed that the copper extraction rate of the community invaded by a sulfur-oxidizing consortium was 50.40% higher than that of the indigenous leachate at the endpoint of bioleaching. The variations of ferrous content, total iron, pH and redox potential in leachates also provided evidence that the community with exogenous sulfur oxidizers was more efficient. XRD analysis demonstrated that a proper addition of the sulfur-oxidizing consortium could eliminate sulfur passivation, promote production of chalcocite and enhance leaching. Furthermore, an exogenous ferrous-oxidizing consortium and a sulfur-oxidizing consortium greatly changed the community structure and microbial succession and promoted the cell growth rate during the bioleaching process, while ferrous/sulfur oxidizers showed no obvious effects on the indigenous community. Exogenous ferrous oxidizers, mainly

Acidithiobacillus ferrivorans is an acidophile that often occurs in low temperature acid mine drainage, e.g., that located at high altitude. Being able to inhabit the extreme environment, the bacterium must possess strategies to copy with the survival stress. Nonetheless, information on the strategies is in demand. Here, genomic and transcriptomic assays were performed to illuminate the adaptation mechanisms of an A. ferrivorans strain YL15, to the alpine acid mine drainage environment in Yulong copper mine in southwest China. Genomic analysis revealed that strain has a gene repertoire for metal-resistance, e.g., genes coding for the mer operon and a variety of transporters/efflux proteins, and for low pH adaptation, such as genes for hopanoid-synthesis and the sodium:proton antiporter. Genes for various DNA repair enzymes and synthesis of UV-absorbing mycosporine-like amino acids precursor indicated hypothetical UV radiation-resistance mechanisms in strain YL15. In addition, it has two types of the acquired immune system-type III-B and type I-F CRISPR/Cas modules against invasion of foreign genetic elements. RNA-seq based analysis uncovered that strain YL15 uses a set of mechanisms to adapt to low temperature. Genes involved in protein synthesis, transmembrane transport, energy metabolism and chemotaxis showed increased levels of RNA transcripts. Furthermore, a bacterioferritin Dps gene had higher RNA transcript counts at 6°C, possibly implicated in protecting DNA against oxidative stress at low temperature. The study represents the first to comprehensively unveil the adaptation mechanisms of an acidophilic bacterium to the acid mine drainage in alpine regions.

The use of green manures in agriculture can provide nutrients, affect soil microbial communities, and be a more sustainable management practice. The activities of soil microbes can effect crop growth, but the extent of this effect on yield remains unclear. We investigated soil bacterial communities and soil properties under four different green manure fertilization regimes (Vicia villosa, common vetch, milk vetch, and radish) and determined the effects of these regimes on maize growth. Milk vetch showed the greatest potential for improving crop productivity and increased maize yield by 31.3 %. This change might be related to changes in soil microbes and soil properties. The entire soil bacterial community and physicochemical properties differed significantly among treatments, and there were significant correlations between soil bacteria, soil properties, and maize yield. In particular, abundance of the phyla Acidobacteria and Verrucomicrobia was positively correlated with maize yield, while Proteobacteria and Chloroflexi were negatively correlated with yield. These data suggest that the variation of maize yield was related to differences in soil bacteria. The results also indicate that soil pH, alkali solution nitrogen, and available potassium were the key environmental factors shaping soil bacterial communities and determining maize yields. Both soil properties and soil microbes might be useful as indicators of soil quality and potential crop yield.

Acidophilic microorganisms involved in uranium bioleaching are usually suppressed by dissolved fluoride ions, eventually leading to reduced leaching efficiency. However, little is known about the regulation mechanisms of microbial resistance to fluoride. In this study, the resistance of Acidithiobacillus ferrooxidans ATCC 23270 to fluoride was investigated by detecting bacterial growth fluctuations and ferrous or sulfur oxidation. To explore the regulation mechanism, a whole genome microarray was used to profile the genome-wide expression. The fluoride tolerance of A. ferrooxidans cultured in the presence of FeSO4 was better than that cultured with the S0 substrate. The differentially expressed gene categories closely related to fluoride tolerance included those involved in energy metabolism, cellular processes, protein synthesis, transport, the cell envelope, and binding proteins. This study highlights that the cellular ferrous oxidation ability was enhanced at the lower fluoride concentrations. An overview of the cellular regulation mechanisms of extremophiles to fluoride resistance is discussed.