Добірка наукової літератури з теми "Au-tolerant bacterial genome and function"

Pseudomonas simiae WCS417 is a root-colonizing bacterium with well-established plant-beneficial effects. Upon colonization of Arabidopsis roots, WCS417 evades local root immune responses while triggering an induced systemic resistance (ISR) in the leaves. The early onset of ISR in roots shows similarities with the iron deficiency response, as both responses are associated with the production and secretion of coumarins. Coumarins can mobilize iron from the soil environment and have a selective antimicrobial activity that impacts microbiome assembly in the rhizosphere. Being highly coumarin-tolerant, WCS417 induces the secretion of these phenolic compounds, likely to improve its own niche establishment, while providing growth and immunity benefits for the host in return. To investigate the possible signaling function of coumarins in the mutualistic Arabidopsis-WCS417 interaction, we analyzed the transcriptome of WCS417 growing in root exudates of coumarin-producing Arabidopsis Col-0 and the coumarin-biosynthesis mutant f6′h1. We found that coumarins in F6′H1-dependent root exudates significantly affected the expression of 439 bacterial genes (8% of the bacterial genome). Of those, genes with functions related to transport and metabolism of carbohydrates, amino acids, and nucleotides were induced, whereas genes with functions related to cell motility, the bacterial mobilome, and energy production and conversion were repressed. Strikingly, most genes related to flagellar biosynthesis were down-regulated by F6′H1-dependent root exudates and we found that application of selected coumarins reduces bacterial motility. These findings suggest that coumarins’ function in the rhizosphere as semiochemicals in the communication between the roots and WCS417. Collectively, our results provide important novel leads for future functional analysis of molecular processes in the establishment of plant-mutualist interactions.

Citrus huanglongbing (HLB), caused by phloem-limited ‘Candidatus Liberibacter’ bacteria, is a destructive disease threatening the worldwide citrus industry. The mechanisms of pathogenesis are poorly understood and no efficient strategy is available to control HLB. Here, we used a comparative genomics screen to identify candidate microbe-associated molecular patterns (MAMPs) from ‘Ca. Liberibacter’ spp. We identified the core genome from multiple ‘Ca. Liberibacter’ pathogens, and searched for core genes with signatures of positive selection. We hypothesized that genes encoding putative MAMPs would evolve to reduce recognition by the plant immune system, while retaining their essential functions. To efficiently screen candidate MAMP peptides, we established a high-throughput microtiter plate-based screening assay, particularly for citrus, that measured reactive oxygen species (ROS) production, which is a common immune response in plants. We found that two peptides could elicit ROS production in Arabidopsis and Nicotiana benthamiana. One of these peptides elicited ROS production and defense gene expression in HLB-tolerant citrus genotypes, and induced MAMP-triggered immunity against the bacterial pathogen Pseudomonas syringae. Our findings identify MAMPs that boost immunity in citrus and could help prevent or reduce HLB infection.

Bacteria employ secondary metabolism to combat competitors, and xenobiotic metabolism to survive their chemical environment. This project has aimed to introduce a bacterial collection enabling comprehensive comparative investigations of those functions. The collection comprises 120 strains (Proteobacteria, Actinobacteria and Firmicutes), and was compiled on the basis of the broad taxonomic range of isolates and their postulated biosynthetic and/or xenobiotic detoxification capabilities. The utility of the collection was demonstrated in two ways: first, by performing 5144 co-cultures, recording inhibition between isolates and employing bioinformatics to predict biosynthetic gene clusters in sequenced genomes of species; second, by screening for xenobiotic sensitivity of isolates against 2-benzoxazolinone and 2-aminophenol. The co-culture medium of Bacillus siamensis D9 and Lysinibacillus sphaericus DSM 28T was further analysed for possible antimicrobial compounds, using liquid chromatography-mass spectrometry (LC-MS), and guided by computational predictions and the literature. Finally, LC-MS analysis demonstrated N-acetylation of 3,4-dichloroaniline (a toxic pesticide residue of concern) by the actinobacterium Tsukamurella paurometabola DSM 20162T which is highly tolerant of the xenobiotic. Microbial collections enable "pipeline" comparative screening of strains: on the one hand, bacterial co-culture is a promising approach for antibiotic discovery; on the other hand, bioremediation is effective in combating pollution, but requires knowledge of microbial xenobiotic metabolism. The presented outcomes are anticipated to pave the way for studies that may identify bacterial strains and/or metabolites of merit in biotechnological applications.

AbstractForest soil microbiomes have crucial roles in carbon storage, biogeochemical cycling and rhizosphere processes. Wildfire season length, and the frequency and size of severe fires have increased owing to climate change. Fires affect ecosystem recovery and modify soil microbiomes and microbially mediated biogeochemical processes. To study wildfire-dependent changes in soil microbiomes, we characterized functional shifts in the soil microbiota (bacteria, fungi and viruses) across burn severity gradients (low, moderate and high severity) 1 yr post fire in coniferous forests in Colorado and Wyoming, USA. We found severity-dependent increases of Actinobacteria encoding genes for heat resistance, fast growth, and pyrogenic carbon utilization that might enhance post-fire survival. We report that increased burn severity led to the loss of ectomycorrhizal fungi and less tolerant microbial taxa. Viruses remained active in post-fire soils and probably influenced carbon cycling and biogeochemistry via turnover of biomass and ecosystem-relevant auxiliary metabolic genes. Our genome-resolved analyses link post-fire soil microbial taxonomy to functions and reveal the complexity of post-fire soil microbiome activity.

AbstractThe biogeochemical cycling of gold has been proposed from studies focusing on gold particle morphology, surface textures and associated bacteria living on the surface of gold particles. Additionally, it has been suggested that metabolically active bacteria on particles catalyse gold dissolution and gold re-precipitation processes, i.e. fluid–bacterial–mineral interaction within microenvironments surrounding particles. Therefore, the isolation and characterisation of viable bacteria from gold particles can be used as a model to improve the understanding of bacterial–gold interactions. In this study, classical microbiology methods were used to isolate a gold-tolerant bacterium (Acinetobacter sp. SK-43) directly from gold particles. The genome of this isolate contained diverse (laterally acquired) heavy-metal resistance genes and stress tolerance genes, suggesting that gene expression would confer resistance to a wide range of potentially toxic metals that could occur in the surrounding microenvironment. The presence of these genes, along with genes for nutrient cycling under nutrient-limited conditions highlights the genomic capacity of how Acinetobacter sp. SK-43 could survive on gold particles and remain viable. Laboratory experiments demonstrated that this isolate could grow in the presence of soluble gold up to 20 μM (AuCl3) and that >50% of soluble gold was reduced upon exposure. Collectively, these results suggest that Acinetobacter sp. SK-43 (and presumably similar bacteria) could survive the cytotoxic effects of soluble Au from particles undergoing dissolution. This study provides comprehensive insight on the possible bacterial contributions to gold biogeochemical cycling in natural environments.

ABSTRACT The hemiascomycete yeast Dekkera bruxellensis, also known as Brettanomyces bruxellensis, is a major cause of wine spoilage worldwide. Wines infected with D. bruxellensis develop distinctive, unpleasant aromas due to volatile phenols produced by this species, which is highly ethanol tolerant and facultatively anaerobic. Despite its importance, however, D. bruxellensis has been poorly genetically characterized until now. We performed genome survey sequencing of a wine strain of D. bruxellensis to obtain 0.4× coverage of the genome. We identified approximately 3,000 genes, whose products averaged 49% amino acid identity to their Saccharomyces cerevisiae orthologs, with similar intron contents. Maximum likelihood phylogenetic analyses suggest that the relationship between D. bruxellensis, S. cerevisiae, and Candida albicans is close to a trichotomy. The estimated rate of chromosomal rearrangement in D. bruxellensis is slower than that calculated for C. albicans, while its rate of amino acid evolution is somewhat higher. The proteome of D. bruxellensis is enriched for transporters and genes involved in nitrogen and lipid metabolism, among other functions, which may reflect adaptations to its low-nutrient, high-ethanol niche. We also identified an adenyl deaminase gene that has high similarity to a gene in bacteria of the Burkholderia cepacia species complex and appears to be the result of horizontal gene transfer. These data provide a resource for further analyses of the population genetics and evolution of D. bruxellensis and of the genetic bases of its physiological capabilities.

Background Cellulolytic, hemicellulolytic, and amylolytic (CHA) enzyme-producing halophiles are understudied. The recently defined taxon Iocasia fonsfrigidae consists of one well-described anaerobic bacterial strain: NS-1T. Prior to characterization of strain NS-1T, an isolate designated Halocella sp. SP3-1 was isolated and its genome was published. Based on physiological and genetic comparisons, it was suggested that Halocella sp. SP3-1 may be another isolate of I. fronsfrigidae. Despite being geographic variants of the same species, data indicate that strain SP3-1 exhibits genetic, genomic, and physiological characteristics that distinguish it from strain NS-1T. In this study, we examine the halophilic and alkaliphilic nature of strain SP3-1 and the genetic substrates underlying phenotypic differences between strains SP3-1 and NS-1T with focus on sugar metabolism and CHA enzyme expression. Methods Standard methods in anaerobic cell culture were used to grow strains SP3-1 as well as other comparator species. Morphological characterization was done via electron microscopy and Schaeffer-Fulton staining. Data for sequence comparisons (e.g., 16S rRNA) were retrieved via BLAST and EzBioCloud. Alignments and phylogenetic trees were generated via CLUTAL_X and neighbor joining functions in MEGA (version 11). Genomes were assembled/annotated via the Prokka annotation pipeline. Clusters of Orthologous Groups (COGs) were defined by eegNOG 4.5. DNA-DNA hybridization calculations were performed by the ANI Calculator web service. Results Cells of strain SP3-1 are rods. SP3-1 cells grow at NaCl concentrations of 5-30% (w/v). Optimal growth occurs at 37 °C, pH 8.0, and 20% NaCl (w/v). Although phylogenetic analysis based on 16S rRNA gene indicates that strain SP3-1 belongs to the genus Iocasia with 99.58% average nucleotide sequence identity to Iocasia fonsfrigida NS-1T, strain SP3-1 is uniquely an extreme haloalkaliphile. Moreover, strain SP3-1 ferments D-glucose to acetate, butyrate, carbon dioxide, hydrogen, ethanol, and butanol and will grow on L-arabinose, D-fructose, D-galactose, D-glucose, D-mannose, D-raffinose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, xylan and phosphoric acid swollen cellulose (PASC). D-rhamnose, alginate, and lignin do not serve as suitable culture substrates for strain SP3-1. Thus, the carbon utilization profile of strain SP3-1 differs from that of I. fronsfrigidae strain NS-1T. Differences between these two strains are also noted in their lipid composition. Genomic data reveal key differences between the genetic profiles of strain SP3-1 and NS-1T that likely account for differences in morphology, sugar metabolism, and CHA-enzyme potential. Important to this study, I. fonsfrigidae SP3-1 produces and extracellularly secretes CHA enzymes at different levels and composition than type strain NS-1T. The high salt tolerance and pH range of SP3-1 makes it an ideal candidate for salt and pH tolerant enzyme discovery.

AbstractCulture-independent analysis with high-throughput sequencing has been widely used to characterize bacterial communities. However, signals derived from non-viable bacteria and non-cell DNA may inhibit its characterization. Here, we present a method for viable bacteria-targeted single-cell genome sequencing, called PMA-SAG-gel, to obtain comprehensive whole-genome sequences of surviving uncultured bacteria from microbial communities. PMA-SAG-gel uses gel matrixes that enable sequential enzymatic reactions for cell lysis and genome amplification of viable single cells from the microbial communities. PMA-SAG-gel removed the single-amplified genomes (SAGs) derived from dead bacteria and enabled selective sequencing of viable bacteria in the model samples of Escherichia coli and Bacillus subtilis. Next, we demonstrated the recovery of near-complete SAGs of eight oxygen-tolerant bacteria, including Bacteroides spp. and Phocaeicola spp., from 1331 human feces SAGs. We found the presence of two different strains in each species and identified their specific genes to investigate the metabolic functions. The survival profile of an entire population at the strain level will provide the information for understanding the characteristics of the surviving bacteria under the specific environments or sample processing and insights for quality assessment of live bacterial products or fecal microbiota transplantation and for understanding the effect of antimicrobial treatments.

ABSTRACT A bacterial consortium was enriched from gold particles that ‘experienced’ ca. 80 years of biotransformation within waste-rock piles (Australia). This bacterial consortium was exposed to 10 µM AuCl3 to obtain Au-tolerant bacteria. From these isolates, Serratia sp. and Stenotrophomonas sp. were the most Au-tolerant and reduced soluble Au as pure gold nanoparticles, indicating that passive mineralisation is a mechanism for mediating the toxic effect of soluble Au produced during particle dissolution. Genome-wide analysis demonstrated that these isolates also possessed various genes that could provide cellular defence enabling survival under heavy-metal stressed condition by mediating the toxicity of heavy metals through active efflux/reduction. Diverse metal-resistant genes or genes clusters (cop, cus, czc, zntand ars) were detected, which could confer resistance to soluble Au. Comparative genome analysis revealed that the majority of detected heavy-metal resistant genes were similar (i.e. orthologous) to those genes of Cupriavidus metallidurans CH34. The detection of heavy-metal resistance, nutrient cycling and biofilm formation genes (pgaABCD, bsmAandhmpS) may have indirect yet important roles when dealing with soluble Au during particle dissolution. In conclusion, the physiological and genomic results suggest that bacteria living on gold particles would likely use various genes to ensure survival during Au-biogeochemical cycling.

Widespread antimicrobial resistance encourages repurposing/refining of non-antimicrobial drugs for antimicrobial indications. Gallium nitrate (GaNt), an FDA-approved medication for cancer-related hypercalcemia, recently showed good activity against several clinically significant bacteria. However, the mechanism of GaNt antibacterial action is still poorly understood. In the present work, resistant and tolerant mutants of Escherichia coli were sought via multiple rounds of killing by GaNt. Multi-round-enrichment yielded no resistant mutant; whole-genome sequencing of one representative GaNt-tolerant mutant uncovered mutations in three genes (evgS, arpA, kdpD) potentially linked to protection from GaNt-mediated killing. Subsequent genetic analysis ruled out a role for arpA and kdpD, but two gain-of-function mutations in evgS conferred tolerance. The evgS mutation-mediated GaNt tolerance depended on EvgS to EvgA phosphor-transfer; EvgA-mediated up-regulation of GadE. YdeO, and SarfA also contributed to tolerance, the latter two likely through their regulation of GadE. GaNt-mediated killing of wild-type cells correlated with increased intracellular ROS accumulation that was abolished by the evgS-tolerant mutation. Moreover, GaNt-mediated killing was mitigated by dimethyl sulfoxide, and the evgS-tolerant mutation upregulated genes encoding enzymes involved in ROS detoxification and in the glyoxylate shunt of the TCA cycle. Collectively, these findings indicate that GaNt kills bacteria through elevation of ROS; gain-of-function mutations in evgS confer tolerance by constitutively activating the EvgA-YdeO/GadE cascade of acid-resistance pathways and by preventing GaNt-stimulated ROS accumulation by upregulating ROS detoxification and shifting TCA cycle carbon flux. The striking lethal activity of GaNt suggests that clinical use of the agent may not quickly lead to resistance.

In natural environments, gold is dissolved (oxidized), dispersed, and reconcentrated (reduced and aggregated). Collectively, these processes constitute the biogeochemical cycle of gold, which can be catalysed by bacteria, and have been interpreted from the characterisation of gold particles that have undergone (bio)transformation. Therefore, the primary focus of this study is to explore the diversity and function of bacteria residing on gold particles to understand their influence on particle structure and chemistry and hence gold biogeochemical cycling. In doing so, both culture-independent (direct amplification of bacterial 16S rDNA gene from particles) and dependent (enrichment of viable bacteria from particles) techniques were applied to get a comprehensive overview of the composition of microbial communities on gold particles. Diverse bacterial species belonging to different phyla were detected on gold particles, which was consistent with previous studies. The culture-independent approach provided a record of both past and present bacterial existence on gold particles, whereas, the culture-dependent approach enriched viable bacteria living on gold particles at the time of sampling. The detection and enrichment of bacteria from gold particles indicated that on Earth’s surficial environment gold particles could provide a solid substrate for bacterial colonisation. The biochemical activity of these bacteria, along with other environmental conditions, create microenvironments on gold particle surfaces (i.e. polymorphic layers) which promote particle biotransformation. The microstructures (porous textures and pure gold nanoparticles) on gold particle surfaces were interpreted as “products” of past biogeochemical processes (gold dissolution and precipitation). By analysing gold particle structure, chemistry and secondary gold concentration in the soil, gold biogeochemical cycling was estimated to be 1.60 × 10-9 M year-1 and was attributed, in part, to the viable bacteria living on the surface of those particles. The dissolution of particles during biogeochemical transformation can produce toxic soluble Au complexes thereby creating an “extreme” microenvironment. In this study, Au-tolerant bacteria were enriched (using a soluble Au concentration equivalent to the kinetic estimate) and were taxonomically diverse. This provides in-vitro evidence that gold biogeochemical cycling could act as a selective pressure on bacteria living on particles undergoing biotransformation. As a result, Au-tolerant bacteria are selected over time. The physiology, genomic, and functional characterisation of Au-tolerant bacteria demonstrated that these bacteria possess the ability to reduce cytotoxic soluble Au to gold nanoparticles (i.e. gold biomineralisation), harbour various types of genes (heavy-metal resistance, general-stress tolerance, and metabolic genes), and employ multiple mechanisms to mediate the toxicity of soluble Au. Therefore, Autolerant bacteria can survive gold biogeochemical cycling as well as catalyse particle biotransformation. Additionally, this present study also demonstrated that heavy-metal contamination (derived from anthropogenic activities) alters the natural gold biogeochemical cycling and modifies the dynamics of microbe-gold interactions. Mercury contamination in soils can directly influence gold particle structure by “erasing” evidence of past biogeochemical processes that increase the gold purity on the outer surface of particles. Conversely, mercury and other heavy metals could selectively enrich heavy-metal resistant bacteria on gold particles. Bacteria with these functional capabilities could amplify gold biogeochemical cycling. Overall, the thesis highlights the diversity and function of bacteria occurring on the surface of gold particles and how these bacteria could contribute to particle transformation. Moreover, this study provides greater insight on bacteria-gold interactions and expands our understanding of gold biogeochemistry in natural to engineered systems.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2020

Genetic engineering plays a key role in plant functional research and genetic improvement. A novel and powerful gene editing technique, CRISPR/Cas9, which was developed from a type II bacterial immune system, opened up a new era in precision genetic engineering in plants. This technique is based on a non-permanent transgene system and is starting to be adopted for precise gene editing in major cereal crops. It offers tremendous potential to accelerate crop improvement in a way that potentially reduces or eliminates the cumbersome and expensive regulatory processes associated with traditional transgenic crops. This chapter describes the advance of gene editing applied to sorghum, a drought tolerant C4 crop, and a successful strategy of CRISPR/Cas9 mediated gene family editing to improve sorghum digestibility and protein quality. It also discusses future prospects of CRISPR/Cas9 gene editing for sorghum genetic improvement.