RESUMO
Contamination of aquifers by a combination of vanadate [V(V)] and nitrate (NO3-) is widespread nowadays. Although bioremediation of V(V)- and nitrate-contaminated environments is possible, only a limited number of functional species have been identified to date. The present study demonstrates the effectiveness of V(V) reduction and denitrification by a denitrifying bacterium Acidovorax sp. strain BoFeN1. The V(V) removal efficiency was 76.5 ± 5.41 % during 120 h incubation, with complete removal of NO3- within 48 h. Inhibitor experiments confirmed the involvement of electron transport substances and denitrifying enzymes in the bioreduction of V(V) and NO3-. Cyt c and riboflavin were important for extracellular V(V) reduction, with quinone and EPS more significant for NO3- removal. Intracellular reductive compounds including glutathione and NADH directly reduce V(V) and NO3-. Reverse transcription quantitative PCR confirmed the important roles of nirK and napA genes in regulating V(V) reduction and denitrification. Bioaugmentation by strain BoFeN1 increased V(V) and NO3- removal efficiency by 55.3 % ± 2.78 % and 42.1 % ± 1.04 % for samples from a contaminated aquifer. This study proposes new microbial resources for the bioremediation of V(V) and NO3-contaminated aquifers, and contributes to our understanding of coupled vanadium, nitrogen, and carbon biogeochemical processes.
Assuntos
Biodegradação Ambiental , Comamonadaceae , Desnitrificação , Nitratos , Oxirredução , Vanadatos , Comamonadaceae/metabolismo , Comamonadaceae/genética , Vanadatos/metabolismo , Nitratos/metabolismo , Poluentes Químicos da Água/metabolismo , Água Subterrânea/microbiologiaRESUMO
The cold-adapted bacterium Variovorax sp. PAMC28711 possesses two distinct glycoside hydrolase (GH) families of trehalase, GH15 and GH37. While numerous studies have explored bacterial trehalase, the presence of two different trehalase genes within a single strain has not been reported until now. Interestingly, despite both GH37 and GH15 trehalases serving the same purpose of degrading trehalose, but do not share the sequence similarity. The substrate specificity assay confirmed that Vtre37 and Vtre15 displayed hydrolytic activity on α, α-trehalose. The key catalytic sites were identified as D280 and E469 in Vtre37 and E389 and E554 in Vtre15 through site-directed mutation and confirmed these two enzymes belong to trehalase. In addition, Vtre37 exhibited a relatively high level of enzyme activity of 1306.33 (±53.091) µmolmg-1, whereas Vtre15 showed enzyme activity of 408.39 (±12.503) µmolmg-1. Moreover, Vtre37 performed admirably showing resistance to ethanol (10 %), with high stable at acidic pH range. Furthermore, both prediction and experimental results indicate that validoxylamine A showed a potent inhibitory activity against Vtre37 trehalase with a Ki value of 16.85 nM. Therefore, we postulate that Vtre37 could be utilized as an ethanol enhancer and designed for screening inhibitors related to the trehalose degradation pathway. Additionally, we believe that characterizing these bacterial trehalase contributes to a better understanding of trehalose metabolism and its biological importance in bacteria.
Assuntos
Temperatura Baixa , Comamonadaceae , Trealase , Trealase/metabolismo , Trealase/genética , Trealase/química , Especificidade por Substrato , Comamonadaceae/enzimologia , Comamonadaceae/genética , Domínio Catalítico , Trealose/metabolismo , Trealose/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Sequência de Aminoácidos , Estabilidade Enzimática , Adaptação FisiológicaRESUMO
A Gram-stain-negative, rod-shaped, non-motile, catalase-positive, denitrifying bacterium, designated strain Y-1T, was isolated from an aeration tank of a sewage treatment plant in China and characterized using polyphasic taxonomic approaches. Strain Y-1T could grow at 10-37 °C (optimum 25 °C), at pH 5.0-10.0 (optimum 7.0) and in the presence of 0-3.0% (w/v) NaCl (optimum 0.5%). The phylogenetic tree based on the 16S rRNA gene sequences revealed that strain Y-1T was a member of genus Diaphorobacter, and showed the highest sequence similarities with Diaphorobacter oryzae RF3T (97.50%), Diaphorobacter nitroreducens NA10BT (97.38%) and Diaphorobacter aerolatus 8604S-37T (96.56%). In terms of carbon source utilization and enzyme activities, strain Y-1T was significantly different from its similar strains. The major respiratory quinone was Q-8, and the main polar lipid was phosphatidylethanolamine. Comparative genomic analysis of strain Y-1T and other Diaphorobacter species was conducted to explore the mechanisms underlying the differences among these strains. Strain Y-1T encoded 3957 genes, consisting of 3813 protein-coding genes and 144 RNA coding genes, and encoded 652 enzymes with 31 unique enzymes compared with other related species. The DNA G + C content was 69.95 mol%. Strain Y-1T exhibited 41.71% DNA-DNA relatedness and 95% ANIb with the most related type strains.On the basis of the evidence presented from polyphasic analysis, strain Y-1T was suggested as a novel species within the genus Diaphorobacter, for which the name Diaphorobacter limosus sp. nov. is proposed, with the type strain Y-1T (= KCTC 92852T = CCTCC AB 2023032T).
Assuntos
Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano , Filogenia , RNA Ribossômico 16S , Esgotos , Esgotos/microbiologia , RNA Ribossômico 16S/genética , DNA Bacteriano/genética , China , Genoma Bacteriano , Ácidos Graxos/química , Comamonadaceae/genética , Comamonadaceae/classificação , Comamonadaceae/isolamento & purificação , Análise de Sequência de DNA , Hibridização de Ácido NucleicoRESUMO
The main metabolic product of the pyridinecarboxamide insecticide flonicamid, N-(4-trifluoromethylnicotinyl)glycinamide (TFNG-AM), has been shown to have very high mobility in soil, leading to its accumulation in the environment. Catabolic pathways of flonicamid have been widely reported, but few studies have focused on the metabolism of TFNG-AM. Here, the rapid transformation of TFNG-AM and production of the corresponding acid product N-(4-trifluoromethylnicotinoyl) glycine (TFNG) by the plant growth-promoting bacterium Variovorax boronicumulans CGMCC 4969 were investigated. With TFNG-AM at an initial concentration of 0.86 mmol/L, 90.70 % was transformed by V. boronicumulans CGMCC 4969 resting cells within 20 d, with a degradation half-life of 4.82 d. A novel amidase that potentially mediated this transformation process, called AmiD, was identified by bioinformatic analyses. The gene encoding amiD was cloned and expressed recombinantly in Escherichia coli, and the enzyme AmiD was characterized. Key amino acid residue Val154, which is associated with the catalytic activity and substrate specificity of signature family amidases, was identified for the first time by homology modeling, structural alignment, and site-directed mutagenesis analyses. When compared to wild-type recombinant AmiD, the mutant AmiD V154G demonstrated a 3.08-fold increase in activity toward TFNG-AM. The activity of AmiD V154G was greatly increased toward aromatic L-phenylalanine amides, heterocyclic TFNG-AM and IAM, and aliphatic asparagine, whereas it was dramatically lowered toward benzamide, phenylacetamide, nicotinamide, acetamide, acrylamide, and hexanamid. Quantitative PCR analysis revealed that AmiD may be a substrate-inducible enzyme in V. boronicumulans CGMCC 4969. The mechanism of transcriptional regulation of AmiD by a member of the AraC family of regulators encoded upstream of the amiD gene was preliminarily investigated. This study deepens our understanding of the mechanisms of metabolism of toxic amides in the environment, providing new ideas for microbial bioremediation.
Assuntos
Amidoidrolases , Biodegradação Ambiental , Comamonadaceae , Inseticidas , Niacinamida/análogos & derivados , Inseticidas/metabolismo , Comamonadaceae/metabolismo , Comamonadaceae/genética , Amidoidrolases/metabolismo , Amidoidrolases/genética , Ácidos Nicotínicos/metabolismoRESUMO
Pteris vittata (P. vittata), an arsenic (As) hyperaccumulator commonly used in the phytoremediation of As-contaminated soils, contains root-associated bacteria (RAB) including those that colonize the root rhizosphere and endosphere, which can adapt to As contamination and improve plant health. As(III)-oxidizing RAB can convert the more toxic arsenite (As(III)) to less toxic arsenate (As(V)) under As-rich conditions, which may promote plant survial. Previous studies have shown that microbial As(III) oxidation occurs in the rhizospheres and endospheres of P. vittata. However, knowledge of RAB of P. vittata responsible for As(III) oxidation remained limited. In this study, members of the Comamonadaceae family were identified as putative As(III) oxidizers, and the core microbiome associated with P. vittata roots using DNA-stable isotope probing (SIP), amplicon sequencing and metagenomic analysis. Metagenomic binning revealed that metagenome assembled genomes (MAGs) associated with Comamonadaceae contained several functional genes related to carbon fixation, arsenic resistance, plant growth promotion and bacterial colonization. As(III) oxidation and plant growth promotion may be key features of RAB in promoting P. vittata growth. These results extend the current knowledge of the diversity of As(III)-oxidizing RAB and provide new insights into improving the efficiency of arsenic phytoremediation.
Assuntos
Arsenitos , Biodegradação Ambiental , Comamonadaceae , Oxirredução , Raízes de Plantas , Pteris , Microbiologia do Solo , Poluentes do Solo , Raízes de Plantas/microbiologia , Raízes de Plantas/metabolismo , Arsenitos/metabolismo , Poluentes do Solo/metabolismo , Pteris/metabolismo , Comamonadaceae/metabolismo , Comamonadaceae/genética , Rizosfera , Arsênio/metabolismoRESUMO
Acidovorax citrulli is the main pathogen causing bacterial fruit blotch, which seriously threatens the global watermelon industry. At present, rapid, sensitive, and low-cost detection methods are urgently needed. The established CRISPR/LbCas12a visual detection method can specifically detect A. citrulli and does not cross-react with other pathogenic bacteria such as Erwinia tracheiphila, Pseudomonas syringae, and Xanthomonas campestris. The sensitivity of this method for genomic DNA detection is as low as 0.7 copies/µL, which is higher than conventional PCR and real-time PCR. In addition, this method only takes 2.5 h from DNA extraction to quantitative detection and does not require complex operation and sample treatment. Additionally, the technique was applied to test real watermelon seed samples for A. citrulli, and the results were contrasted with those of real-time fluorescence quantitative PCR and conventional PCR. The high sensitivity and specificity have broad application prospects in the rapid detection of bacterial fruit blotch bacterial pathogens of watermelon.IMPORTANCEBacterial fruit blotch, Acidovorax citrulli, is an important seed-borne bacterial disease of watermelon, melon, and other cucurbits. The lack of rapid, sensitive, and reliable pathogen detection methods has hampered research on fruit spot disease prevention and control. Here, we demonstrate the CRISPR/Cas12a system to analyze aspects of the specificity and sensitivity of A. citrulli and to test actual watermelon seed samples. The results showed that the CRISPR/Cas12a-based free-amplification method for detecting bacterial fruit blotch pathogens of watermelons was specific for A. citrulli target genes and 100-fold more sensitive than conventional PCR with quantitative real-time PCR. This method provides a new technical tool for the detection of A. citrulli.
Assuntos
Citrullus , Comamonadaceae , Citrullus/genética , Citrullus/microbiologia , Frutas/microbiologia , Doenças das Plantas/microbiologia , Comamonadaceae/genética , DNARESUMO
In recent years, extremophilic microorganisms have been employed as producers of the microbial bioplastics polyhydroxyalkanoates (PHA), which are of great biotechnological value. Nevertheless, cold-loving or psychrophilic (cryophilic) bacteria have been neglected in this regard. Here, we present an investigation of the Arctic glacier-derived PHA producer Acidovorax sp. A1169. Biolog GEN III Microplates were used as a screening tool to identify the most suitable carbon substrate concerning PHA synthesis. The strain produced homopolymer poly(3-hydroxybutyrate) (PHB) most efficiently (2 g/L) at a temperature of 15 °C when supplied with fructose or mannitol as carbon sources with a substantial decrease of PHB biosynthesis at 17.5 °C. The PHB yield did not increase considerably or even decreased when carbon source concentration exceeded 10 g/L hinting that the strain is oligotrophic in nature. The strain was also capable of introducing 3-hydroxyvalerate (3HV) into the polymer structure, which is known to improve PHA thermoplastic properties. This is the first investigation providing insight into a PHA biosynthesis process by means of a true psychrophile, offering guidelines on polar-region bacteria cultivation, production of PHA and also on the methodology for genetic engineering of psychrophiles.
Assuntos
Comamonadaceae , Poli-Hidroxialcanoatos , Temperatura , Engenharia Genética , Carbono , Comamonadaceae/genéticaRESUMO
Acidovorax citrulli is a seed-borne bacterial pathogen that causes bacterial fruit blotch in cucurbits and severely affects the production of cucumbers and watermelons globally. In this study, we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP) on the growth, metabolism, and virulence of A. citrulli. Bacterial population was not affected by DEHP exposure; moreover, significant changes were not observed in lipid peroxidation, membrane permeability, and nucleic acid leakage. However, palmitoleic acid content was increased in the cell membrane of DEHP-exposed A. citrulli. Further, DEHP exposure increased the activity of TCA cycle-related enzymes, including α-ketoglutarate dehydrogenase and succinyl-CoA synthetase, along with increase in the content of glutamate, succinate, fumarate, and malate in TCA cycle. Additionally, total 270 genes were differentially expressed by the treatment, of which 28 genes were upregulated and 242 genes, including those related to translation, flagellum-dependent cell motility, and flagellum assembly, were downregulated. Regarding virulence traits, swimming activity was decreased in DEHP-exposed A. citrulli; however, biofilm formation was not affected in in vitro assay. Moreover, relative expression of pathogenicity genes, including hrpX and hrpG, were decreased in DEHP-exposed A. citrulli compared to that of unexposed A. citrulli. Therefore, these results suggest that DEHP accumulation in soil could potentially influence the metabolism and virulence traits of A. citrulli.
Assuntos
Comamonadaceae , Dietilexilftalato , Virulência , Comamonadaceae/genéticaRESUMO
In recent years Acidovorax avenae subsp. avenae was identified as a major cause of bacterial etiolation and decline (BED) in turfgrasses and has become a growing economical concern for the turfgrass industry. The symptoms of BED resemble those of "bakanae," or foolish seedling disease, of rice (Oryzae sativa), in which the gibberellins produced by the infecting fungus, Fusarium fujikuroi, contribute to the symptom development. Additionally, an operon coding for the enzymes necessary for bacterial gibberellin production was recently characterized in plant-pathogenic bacteria belonging to the γ-proteobacteria. We therefore investigated whether this gibberellin operon might be present in A. avenae subsp. avenae. A homolog of the operon has been identified in two turfgrass-infecting A. avenae subsp. avenae phylogenetic groups but not in closely related phylogenetic groups or strains infecting other plants. Moreover, even within these two phylogenetic groups, the operon presence is not uniform. For that reason, the functionality of the operon was examined in one strain of each turfgrass-infecting phylogenetic group (A. avenae subsp. avenae strains KL3 and MD5). All nine operon genes were functionally characterized through heterologous expression in Escherichia coli and enzymatic activities were analyzed by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. All enzymes were functional in both investigated strains, thus demonstrating the ability of phytopathogenic ß-proteobacteria to produce biologically active GA4. This additional gibberellin produced by A. avenae subsp. avenae could disrupt phytohormonal balance and be a leading factor contributing to the pathogenicity on turf grasses. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Assuntos
Comamonadaceae , Giberelinas , Filogenia , Poaceae , Comamonadaceae/genética , PlantasRESUMO
A non-canonical biosynthetic pathway furnishing the first natural brexane-type bishomosesquiterpene (chlororaphen, C17 H28 ) was elucidated in the γ-proteobacterium Pseudomonas chlororaphis O6. A combination of genome mining, pathway cloning, in vitro enzyme assays, and NMR spectroscopy revealed a three-step pathway initiated by C10 methylation of farnesyl pyrophosphate (FPP, C15 ) along with cyclization and ring contraction to furnish monocyclic γ-presodorifen pyrophosphate (γ-PSPP, C16 ). Subsequent C-methylation of γ-PSPP by a second C-methyltransferase furnishes the monocyclic α-prechlororaphen pyrophosphate (α-PCPP, C17 ), serving as the substrate for the terpene synthase. The same biosynthetic pathway was characterized in the ß-proteobacterium Variovorax boronicumulans PHE5-4, demonstrating that non-canonical homosesquiterpene biosynthesis is more widespread in the bacterial domain than previously anticipated.
Assuntos
Comamonadaceae , Pseudomonas chlororaphis , Metilação , Difosfatos , Comamonadaceae/genéticaRESUMO
Bacterial fruit blotch (BFB) is a disease of cucurbit plants caused by Acidovorax citrulli. Although A. citrulli has great destructive potential, the molecular mechanisms of pathogenicity of A. citrulli are not clear, particularly with regard to its type III secreted effectors. In this study, we characterized the type III secreted effector protein, AopV, from A. citrulli strain Aac5. We show that AopV significantly inhibits reactive oxygen species and the expression of PTI marker genes, and helps the growth of Pseudomonas syringae D36E in Nicotiana benthamiana. In addition, we found that the aromatic dehydratase ADT6 from watermelon was a target of AopV. AopV interacts with ADT6 in vivo and in vitro. Subcellular localization indicated ADT6 and AopV were co-located at the cell membrane. Together, our results reveal that AopV suppresses plant immunity and targets ADT6 in the cell membrane. These findings provide an new characterization of the molecular interaction of A. citrulli effector protein AopV with host cells.
Assuntos
Citrullus , Comamonadaceae , Citrullus/genética , Citrullus/microbiologia , Comamonadaceae/genética , Hidroliases , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Espécies Reativas de OxigênioRESUMO
BACKGROUND: Folic acid (FA) is a synthetic vitamin (B9) and the oxidized form of a metabolic cofactor that is essential for life. Although the biosynthetic mechanisms of FA are established, its environmental degradation mechanism has not been fully elucidated. The present study aimed to identify bacteria in soil that degrade FA and the mechanisms involved. RESULTS: We isolated the soil bacterium Variovorax sp. F1 from sampled weed rhizospheres in a grassland and investigated its FA degradation mechanism. Cultured Variovorax sp. F1 rapidly degraded FA to pteroic acid (PA), indicating that FA hydrolysis to PA and glutamate. We cloned the carboxypeptidase G (CPG) gene and found widely distributed paralogs within the Variovorax genus. Recombinant CPG preferred FA and deaminofolic acid as substrates, indicating its involvement in FA degradation by Variovorax. Prolonged culture of Variovorax sp. F1 resulted in decreased rates of deaminofolic acid (DFA) and deaminopteroic acid (DPA) accumulation. This indicated that the deamination reaction also comprised a route of FA degradation. We also identified an F1 gene that was orthologous to the pterin deaminase gene (Arad3529) of Agrobacterium radiobacter. The encoded protein deaminated FA and PA to DFA and DPA, which was consistent with the deamination activity of FA and PA in bacterial cell-free extracts. CONCLUSION: We discovered that the two enzymes required for FA degradation pathways in isolates of Variovorax sp. F1 comprise CPG and pterin deaminase, and that DFA and PA are intermediates in the generation of DPA.
Assuntos
Comamonadaceae , Ácido Fólico , Aminoidrolases , Comamonadaceae/genética , Ácido Fólico/metabolismo , Glutamatos/metabolismo , Redes e Vias Metabólicas/genética , Solo , Vitaminas , gama-Glutamil Hidrolase/genética , gama-Glutamil Hidrolase/metabolismoRESUMO
Many genera closest to the family Comamonadaceae have not been classified into any family; moreover, some of them are not monophyletic groups beyond the genus level. To resolve the taxonomic uncertainty of the closest-to-Comamonadaceae (CTC) group, we performed 16S rRNA gene- and genome-based phylogenetic analyses combined with genome relatedness indices and phenotypic traits comparison. Phylogenies based on the 16S rRNA gene and genome sequences demonstrated that the CTC group formed a coherent and robust monophyletic lineage and was sister to the family Comamonadaceae, thereby proposing the CTC group as a novel family, Sphaerotilaceae fam. nov. The resolved genus- and species-level taxonomic relationships of this new family were then validated by the phylogenomic reconstruction and comparisons of genome relatedness indices including digital DNA-DNA hybridization and average nucleotide identity (ANI) as well as comprehensive phenotypic analysis for type strains. Finally, we reclassified all misidentified genera and species, resulting in 19 new combinations, and proposed Sphaerotilaceae-specific thresholds of ANI and average amino acid identity for genus delineation. Collectively, this study has established a sound taxonomic framework of the novel family Sphaerotilaceae and will help guide future taxonomic efforts and prevent the propagation of taxonomic errors.
Assuntos
Comamonadaceae , RNA Ribossômico 16S/genética , Comamonadaceae/genética , Filogenia , DNA Bacteriano/genética , Análise de Sequência de DNA , Hibridização de Ácido Nucleico , Técnicas de Tipagem Bacteriana , Ácidos Graxos/análiseRESUMO
Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is a devastating watermelon disease that severely impacts the global watermelon industry. Like other Gram-negative bacteria, the type three secretion system (T3SS) is the main pathogenicity factor of A. citrulli. The T3SS apparatus gene hrpE codes for the Hrp pilus and serves as a conduit to secret effector proteins into host cells. In this study, we found that the deletion of hrpE in A. citrulli results in the loss of pathogenicity on hosts and the hypersensitive response on non-hosts. In addition, the A. citrulli hrpE mutant showed a reduction in in vitro growth, in planta colonization, swimming and twitching motility, and displayed increases in biofilm formation ability compared to the wild type. However, when HrpE was transiently expressed in hosts, the defense responses, including reactive oxygen species bursts, callose deposition, and expression of defense-related genes, were activated. Thus, the A. Citrulli growth in HrpE-pretreated hosts was suppressed. These results indicated that HrpE is essential for A. citrulli virulence but can also be used by hosts to help resist A. citrulli. Our findings provide a better understanding of the T3SS pathogenesis in A. citrulli, thus providing a molecular basis for biopesticide development, and facilitating the effective control of BFB.
Assuntos
Citrullus , Comamonadaceae , Citrullus/genética , Citrullus/microbiologia , Comamonadaceae/genética , Imunidade , Virulência/genéticaRESUMO
Bacteria that inhabit soils and the leaves of trees partially mitigate the release of the abundant volatile organic compound, isoprene (2-methyl-1,3-butadiene). While the initial steps of isoprene metabolism were identified in Rhodococcus sp. AD45 two decades ago, the isoprene metabolic pathway still remains largely undefined. Limited understanding of the functions of isoG, isoJ and aldH and uncertainty in the route of isoprene-derived carbon into central metabolism have hindered our understanding of isoprene metabolism. These previously uncharacterised iso genes are essential in Variovorax sp. WS11, determined by targeted mutagenesis. Using combined 'omics-based approaches, we propose the complete isoprene metabolic pathway. Isoprene is converted to propionyl-CoA, which is assimilated by the chromosomally encoded methylmalonyl-CoA pathway, requiring biotin and vitamin B12, with the plasmid-encoded methylcitrate pathway potentially providing robustness against limitations in these vitamins. Key components of this pathway were induced by both isoprene and its initial oxidation product, epoxyisoprene, the principal inducer of isoprene metabolism in both Variovorax sp. WS11 and Rhodococcus sp. AD45. Analysis of the genomes of distinct isoprene-degrading bacteria indicated that all of the genetic components of the methylcitrate and methylmalonyl-CoA pathways are not always present in isoprene degraders, although incorporation of isoprene-derived carbon via propionyl-CoA and acetyl-CoA is universally indicated.
Assuntos
Comamonadaceae , Rhodococcus , Hemiterpenos/metabolismo , Butadienos/metabolismo , Rhodococcus/genética , Rhodococcus/metabolismo , Comamonadaceae/genética , Comamonadaceae/metabolismo , Carbono/metabolismoRESUMO
Since the role of biobased plastics increases every year, the search for alternatives to petrol-based polymers is very important. Variovorax paradoxus TBEA6 is able to grow with 3,3'-thiodipropionic acid (TDP) as sole source for carbon and energy. TDP can be used as a precursor substrate for the synthesis of polythioesters (PTE). To increase the feasibility of PTE synthesis, a good understanding of the degradation pathway of TDP in V. paradoxus TBEA6 is essential. Therefore, two putative 3-hydroxyisobutyryl-CoA hydrolases (VPARA_03110 & VPARA_05510) and two putative 3-hydroxypropionate dehydrogenases (VPARA_41140 & VPARA_54550) were investigated in this study. The deletion mutant V. paradoxus ∆VPARA_05510 showed a TDP-negative phenotype during growth experiments. The ability to grow with TDP as sole carbon source was successfully restored by complementation. Supernatant analysis revealed that the deletion mutant did not metabolize TDP or 3MP anymore. A specific enzyme activity up to 0.032 U/mg for the purified 3-hydroxyisobutyryl-CoA hydrolase VPARA_05510 was determined. A shift in the proteins (VPARA_54550) melting temperature of 6 °C with 2000 µM 3HP in comparison to protein without ligand was observed during thermal shift assays with the putative 3-hydroxypropionate dehydrogenase.
Assuntos
Comamonadaceae , Alcaligenes , Carbono/metabolismo , Comamonadaceae/genética , Proteínas de Ligação a DNA/metabolismo , PropionatosRESUMO
Rhodoferax sp. PAMC 29310 was isolated from a surface marine sediment of the East Siberian Sea, Arctic. Whole-genome sequencing of the strain Rhodoferax sp. PAMC 29310 was achieved using PacBio RS II and Illumina platform. The resulting complete genome comprised of 4,593,249 base pairs (G + C content of 58.0%) with a single chromosome, 4546 protein-coding genes, 57 tRNAs and 6 rRNA operons. A complete set of genes encoding the enzymes of glycolysis and citric acid cycle were identified. No genes encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and nitrogenase reductase (nif) were present indicating that strain PAMC 29310 is not capable of fixing of carbon and nitrogen. PAMC 29310 genome contains genes for dissimilatory and assimilatory nitrate reduction. Gene encoding choline dehydrogenase enzyme which functions at the first step in the synthesis of betaine, one of the most effective osmoprotectants, was detected. In particular, among the genomes of the genus Rhodoferax strains, gene encoding nitrite reductase (nirK), which reduces nitrite to nitric oxide and tetA gene encoding tetracycline resistance protein involved in the resistance to tetracycline were identified only in the genome of Rhodoferax sp. PAMC 29310. As the first genome from the strain which was isolated from marine sediment in the genus Rhodoferax, investigation of physiological characteristics based on the complete genome sequences will help understand the adaptation of Rhodoferax sp. PAMC 29310 in the marine sediment.
Assuntos
Comamonadaceae , Sedimentos Geológicos , Composição de Bases , Comamonadaceae/genética , DNA Bacteriano/genética , Genoma Bacteriano , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Schistosomiasis is a neglected tropical parasitic and chronic disease affecting hundreds of millions of people. Adult schistosomes reside in the blood stream of the definitive mammalian host. These helminth parasites possess two epithelial surfaces, the tegument and the gastrodermis, both of which interact with the host during immune evasion and in nutrient uptake. METHODS: Female ARC Swiss mice (4-6 weeks old) were infected percutaneously with Schistosoma japonicum cercariae freshly shed from Oncomelania hupensis quadrasi snails (Philippines strain). Fluorescent in situ hybridisation (FISH) was performed by using fresh adult S. japonicum perfused from those infected mice. Adult S. japonicum worms were processed to isolate the tegument from the carcass containing the gastrodermis; blood and bile were collected individually from infected and uninfected mice. Total DNA extracted from all those samples were used for microbiome profiling. RESULTS: FISH and microbiome profiling showed the presence of bacterial populations on two epithelial surfaces of adult worms, suggesting they were distinct not only from the host blood but also from each other. Whereas microbial diversity was reduced overall in the parasite epithelial tissues when compared with that of host blood, specific bacterial taxa, including Anoxybacillus and Escherichia, were elevated on the tegument. Minimal differences were evident in the microbiome of host blood during an active infection, compared with that of control uninfected blood. However, sampling of bile from infected animals identified some differences compared with controls, including elevated levels of Limnohabitans, Clostridium and Curvibacter. CONCLUSIONS: Using FISH and microbial profiling, we were able to demonstrate, for the first time, that bacteria are presented on the epithelial surfaces of adult schistosomes. These schistosome surface-associated bacteria, which are distinct from the host blood microenvironment, should be considered as a new and important component of the host-schistosome interaction. The importance of individual bacterial species in relation to schistosome parasitism needs further elucidation.
Assuntos
Sangue/microbiologia , Epitélio/microbiologia , Microbiota/genética , Schistosoma japonicum/microbiologia , Esquistossomose Japônica/sangue , Animais , Anoxybacillus/genética , Bile/microbiologia , Cercárias , Clostridium/genética , Comamonadaceae/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Modelos Animais de Doenças , Escherichia coli/genética , Feminino , Hibridização in Situ Fluorescente/métodos , Masculino , Camundongos , RNA Ribossômico 16S/genética , Schistosoma japonicum/isolamento & purificação , Esquistossomose Japônica/parasitologia , Caramujos/parasitologiaRESUMO
Caldimonas manganoxidans is a Gram-negative, thermophilic, bioplastic-producing bacterium that is a promising strain to overcome the drawbacks of existing bioplastic manufacturing methods. However, genetic manipulation of this species has not previously been studied. Here, we developed an optimized electrotransformation protocol for C. manganoxidans by screening conditions, including the bacterial growth phase, electroporation buffer, pulse strength, and recovery time. The optimized transformation protocol obtained (3.1 ± 0.78) × 108 colony-forming units/µg DNA of plasmid pBBR1MCS-2. High transformation efficiency was observed when using plasmid DNA isolated from C. manganoxidans. The DNA methylases of Escherichia coli did not affect the transformation efficiency of C. manganoxidans. The electrotransformation technique proposed here will be beneficial for the genetic manipulation of thermophilic Caldimonas species.
Assuntos
Comamonadaceae/genética , Eletroporação/métodos , Transformação Bacteriana/genética , DNA Bacteriano/genética , Plasmídeos/genéticaRESUMO
Beta-proteobacteria belonging to the genus Acidovorax have been described from various environments. Many strains can interact with a range of hosts, including humans and plants, forming neutral, beneficial or detrimental associations. In the frame of this study, we investigated the genomic properties of 52 bacterial strains of the genus Acidovorax, isolated from healthy roots of Lotus japonicus, with the intent of identifying traits important for effective plant-growth promotion. Based on single-strain inoculation bioassays with L. japonicus, performed in a gnotobiotic system, we distinguished seven robust plant-growth promoting strains from strains with no significant effects on plant-growth. We showed that the genomes of the two groups differed prominently in protein families linked to sensing and transport of organic acids, production of phytohormones, as well as resistance and production of compounds with antimicrobial properties. In a second step, we compared the genomes of the tested isolates with those of plant pathogens and free-living strains of the genus Acidovorax sourced from public repositories. Our pan-genomics comparison revealed features correlated with commensal and pathogenic lifestyle. We showed that commensals and pathogens differ mostly in their ability to use plant-derived lipids and in the type of secretion-systems being present. Most free-living Acidovorax strains did not harbour any secretion-systems. Overall, our data indicate that Acidovorax strains undergo extensive adaptations to their particular lifestyle by horizontal uptake of novel genetic information and loss of unnecessary genes.