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1.
Nat Commun ; 12(1): 3829, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158504

RESUMO

While beneficial plant-microbe interactions are common in nature, direct evidence for the evolution of bacterial mutualism is scarce. Here we use experimental evolution to causally show that initially plant-antagonistic Pseudomonas protegens bacteria evolve into mutualists in the rhizosphere of Arabidopsis thaliana within six plant growth cycles (6 months). This evolutionary transition is accompanied with increased mutualist fitness via two mechanisms: (i) improved competitiveness for root exudates and (ii) enhanced tolerance to the plant-secreted antimicrobial scopoletin whose production is regulated by transcription factor MYB72. Crucially, these mutualistic adaptations are coupled with reduced phytotoxicity, enhanced transcription of MYB72 in roots, and a positive effect on plant growth. Genetically, mutualism is associated with diverse mutations in the GacS/GacA two-component regulator system, which confers high fitness benefits only in the presence of plants. Together, our results show that rhizosphere bacteria can rapidly evolve along the parasitism-mutualism continuum at an agriculturally relevant evolutionary timescale.


Assuntos
Arabidopsis/genética , Raízes de Plantas/genética , Pseudomonas/genética , Rizosfera , Simbiose/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Aptidão Genética , Interações Hospedeiro-Patógeno/genética , Mutação , Fenótipo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Pseudomonas/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
BMC Genomics ; 22(1): 464, 2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34157973

RESUMO

BACKGROUND: Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn2+. So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn2+. We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment. RESULTS: Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn2+ oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn2+ oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity. CONCLUSIONS: The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water.


Assuntos
Alcaloides , Burkholderiales/enzimologia , Manganês , Oxirredutases , Pseudomonas/enzimologia , Burkholderiales/genética , Genoma Bacteriano , Leptothrix , Oxirredução , Oxirredutases/metabolismo , Pseudomonas/genética
3.
Methods Mol Biol ; 2290: 187-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009591

RESUMO

Polymerase chain reaction (PCR) is a popular molecular tool for detection of bacteria. PCR allows millions of copies of a target segment of DNA to be produced. The DNA is extracted from overnight grown cultures of pure bacterial isolates using either the organo-solvent method or a commercial DNA extraction kit. The quality and purity of the DNA is determined by performing gel electrophoresis on 0.8% agarose gel. The DNA is amplified by performing PCR assay. Bands of approximately 1.5 kb in size are obtained from the amplified products of DNA. The PCR products run on 1.5% agarose gel are visualized with UV light and imaged by gel documentation system. This chapter outlines the protocol for isolation and amplification of DNA from cellulolytic bacteria. Cellulolytic bacteria are considered a potential source of cellulases for pretreatment of crop residues during biogas production. PCR is considered a very powerful, sensitive, specific, fast, and reliable tool in molecular detection and diagnostics.


Assuntos
Biocombustíveis/microbiologia , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Bacillus/genética , Bactérias/classificação , Bactérias/genética , Cellulomonas/genética , Clostridium/genética , DNA Bacteriano/genética , Eletroforese/métodos , Pseudomonas/genética , Rhodothermus/genética
4.
Environ Monit Assess ; 193(5): 294, 2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33893564

RESUMO

Aquatic environments are hotspots for the spread of antibiotic-resistant bacteria and genes due to pollution caused mainly by anthropogenic activities. The aim of this study was to evaluate the impact of wastewater effluents, informal settlements, hospital, and veterinary clinic discharges on the occurrence, antibiotic resistance profile and virulence signatures of Aeromonas spp. and Pseudomonas spp. isolated from surface water and wastewater. High counts of Aeromonas spp. (2.5 (± 0.8) - 3.3 (± 0.4) log10 CFU mL-1) and Pseudomonas spp. (0.6 (± 1.0) - 1.8 (± 1.0) log10 CFU mL-1) were obtained. Polymerase chain reaction (PCR) and MALDI-TOF characterization identified four species of Aeromonas and five of Pseudomonas. The isolates displayed resistance to 3 or more antibiotics (71% of Aeromonas and 94% of Pseudomonas). Aeromonas spp. showed significant association with the antibiotic meropenem (χ2 = 3.993, P < 0.05). The virulence gene aer in Aeromonas was found to be positively associated with the antibiotic resistance gene blaOXA (χ2 = 6.657, P < 0.05) and the antibiotic ceftazidime (χ2 = 7.537, P < 0.05). Aeromonas recovered from both wastewater and surface water displayed high resistance to ampicillin and had higher multiple antibiotic resistance (MAR) indices close to the hospital. Pseudomonas isolates on the other hand exhibited low resistance to carbapenems but very high resistance to the third-generation cephalosporins and cefixime. The results showed that some of the Pseudomonas spp. and Aeromonas spp. isolates were extended-spectrum ß-lactamase producing bacteria. In conclusion, the strong association between virulence genes and antibiotic resistance in the isolates shows the potential health risk to communities through direct and indirect exposure to the water.


Assuntos
Aeromonas , Aeromonas/genética , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Monitoramento Ambiental , Testes de Sensibilidade Microbiana , Pseudomonas/genética , Virulência , Águas Residuárias , Água
5.
Curr Microbiol ; 78(5): 1871-1881, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33830318

RESUMO

Prometryne is a widely used herbicide in China to control annual grasses and broadleaf weeds. However, the stability of prometryne makes it difficult to be degraded, which poses a threat to human health. This study presents a bacterial strain isolated from soil samples with a prometryne application history, designated strain DY-1. Strain DY-1, identified as Pseudomonas sp., is capable of utilizing prometryne as a sole carbon source for growth and degrading 100% of prometryne within 48 h from an initial concentration of 50 mg L-1. To further optimize the degradation of prometryne, the prometryne concentration, temperature, pH, and salt concentration were examined. The optimal conditions for degradation of prometryne by strain DY-1 were an initial prometryne concentration of 50 mg L-1, 30 °C, pH 7-8, and NaCl concentration of 200 mg L-1. The same strain also degraded other s-triazine herbicides, including simetryne, ametryne, desmetryne, and metribuzin, under the same conditions. The biodegradation pathway of prometryne was established by isolating sulfoxide prometryne as the first metabolite and by the identification of sulfone prometryne and 2-hydroxy prometryne by liquid chromatography-mass spectrometry (LC-MS/MS). The results illustrated that strain DY-1 achieved the removal of prometryne by gradually oxidizing and hydrolyzing the methylthio groups. A bioremediation trial with contaminated soil and pot experiments showed that after treating the prometryne-contaminated soil with strain DY-1, the content of prometryne was significantly reduced (P < 0.05). This study provides an efficient bacterial strain and approach that could be potentially useful for detoxification and bioremediation of prometryne analogs.


Assuntos
Herbicidas , Poluentes do Solo , Biodegradação Ambiental , China , Cromatografia Líquida , Prometrina , Pseudomonas/genética , Solo , Microbiologia do Solo , Espectrometria de Massas em Tandem
6.
Mol Genet Genomics ; 296(4): 893-903, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33909166

RESUMO

Whole-genome sequence of Pseudomonas sp. Kongs-67 retrieved from Kongsfjorden, an Arctic fjord, has been investigated to understand the molecular machinery required for microbial association and survival in a polar fjord. The genome size of Kongs-67 was 4.5 Mb and was found to be closely related to the Antarctic P. pelagia strain CL-AP6. This genome encodes for chemotaxis response regulator proteins (CheABB1RR2VWYZ), chemoreceptors (methyl-accepting chemotaxis proteins), and flagellar system proteins (FliCDEFGOPMN, FlhABF, FlgBCDEFGHIJKL, and MotAB proteins) vital in cellular interactions in the dynamic fjord environment. A high proportion of genes were assigned to biofilm formation (pgaABCD operon) and signal transduction protein categories (EnvZ/OmpR, CpxA/CpxR, PhoR/PhoB, PhoQ) indicating that the biofilm formation in Kongs-67 could be tightly regulated in response to the availability of signalling-metabolites. The genome of Kongs-67 encoded for HemBCD, CbiA, CobABNSTOQCDP, and BtuBFR proteins involved in cobalamin biosynthesis and transport along with proteins for siderophore-mediated iron channelling (PchR, Fur protein, FpvA); crucial in a microbial association. The genomes of Arctic strain Kongs-67 and Antarctic strain CL-AP6 were similar which is indicative of retainment of the core genes in the polar Pseudomonas strains that could be vital in conferring evolutionary adaptation for its survival in a polar fjord. Thus, our study contributes to the knowledge on the genetics of a polar Pseudomonas member exhibiting biosynthetic potentials and suggest Pseudomonas sp. Kongs-67 as a suitable candidate for the investigation of functional aspects of molecular adaptations in the polar marine environment.


Assuntos
Clima Frio , Viabilidade Microbiana/genética , Pseudomonas , Adaptação Biológica/genética , Organismos Aquáticos/genética , Regiões Árticas , Proteínas de Bactérias/genética , Estuários , Genoma Bacteriano/genética , Óperon , Pseudomonas/classificação , Pseudomonas/genética , Análise de Sequência de DNA
7.
Science ; 371(6533): 1033-1037, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674490

RESUMO

Microbial production of antibiotics is common, but our understanding of their roles in the environment is limited. In this study, we explore long-standing observations that microbes increase the production of redox-active antibiotics under phosphorus limitation. The availability of phosphorus, a nutrient required by all life on Earth and essential for agriculture, can be controlled by adsorption to and release from iron minerals by means of redox cycling. Using phenazine antibiotic production by pseudomonads as a case study, we show that phenazines are regulated by phosphorus, solubilize phosphorus through reductive dissolution of iron oxides in the lab and field, and increase phosphorus-limited microbial growth. Phenazines are just one of many examples of phosphorus-regulated antibiotics. Our work suggests a widespread but previously unappreciated role for redox-active antibiotics in phosphorus acquisition and cycling.


Assuntos
Antibacterianos/biossíntese , Fenazinas/metabolismo , Fósforo/metabolismo , Pseudomonas/metabolismo , Técnicas de Cultura Celular por Lotes , Disponibilidade Biológica , Oxirredução , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento
8.
Curr Microbiol ; 78(4): 1670-1677, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33660045

RESUMO

The genomes of two Pseudomonas strains, IzPS23T and IzPS32dT isolated from soil samples of Izu Oshima were compared to Pseudomonas type strains. Whole-genome sequence analysis revealed both belong to the Pseudomonas fluorescens lineage. The average nucleotide identity values of the whole-genome sequences of IzPS23T and IzPS32dT compared with other type strains showed high correlations with Pseudomonas kribbensis (93.1%) and Pseudomonas glycinae (93.5%), respectively. Genome-to-genome distances between the whole-genome sequences of IzPS23T and IzPS32dT showed correlations with Pseudomonas kribbensis (51.0%) and Pseudomonas glycinae (53.2%), respectively. Genotypic and phenotypic analysis indicated the two strains were novel species, and were named Pseudomonas allokribbensis (IzPS23T = CECT 9961T, = LMG 31525T) and Pseudomonas gozinkensis (IzPS32dT = CECT 9962T, = LMG 31526T), respectively.


Assuntos
Ácidos Graxos , Pseudomonas , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ilhas , Japão , Hibridização de Ácido Nucleico , Filogenia , Pseudomonas/genética , RNA Ribossômico 16S , Análise de Sequência de DNA
9.
Arch Microbiol ; 203(5): 2735-2742, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33646340

RESUMO

Genomic islands, defined as large clusters of genes mobilized through horizontal gene transfer, have a profound impact on evolution of prokaryotes. Recently, we developed a new program, IslandCafe, for identifying such large localized structures in bacterial genomes. A unique attribute of IslandCafe is its ability to decipher mosaic structures within genomic islands. Mosaic genomic islands have generated immense interest due to novel traits that have been attributed to such islands. To provide the Pseudomonas research community a catalogue of mosaic islands in Pseudomonas spp., we applied IslandCafe to decipher genomic islands in 224 completely sequenced genomes of Pseudomonas spp. We also performed comparative genomic analysis using BLAST to infer potential sources of distinct segments within genomic islands. Of the total 4271 genomic islands identified in Pseudomonas spp., 1036 were found to be mosaic. We also identified drug-resistant and pathogenic genomic islands and their potential donors. Our analysis provides a useful resource for Pseudomonas research community to further examine and interrogate mosaic islands in the genomes of interest and understand their role in the emergence and evolution of novel traits.


Assuntos
Genoma Bacteriano/genética , Ilhas Genômicas/genética , Pseudomonas/genética , Biologia Computacional , Transferência Genética Horizontal , Genômica , Software
10.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557119

RESUMO

Coumarins are well known secondary metabolites widely found in various plants. However, the degradation of these compounds in the environment has not been studied in detail, and, especially, the initial stages of the catabolic pathways of coumarins are not fully understood. A soil isolate Pseudomonas mandelii 7HK4 is able to degrade 7-hydroxycoumarin (umbelliferone) via the formation of 3-(2,4-dihydroxyphenyl)propionic acid, but the enzymes catalyzing the α-pyrone ring transformations have not been characterized. To elucidate an upper pathway of the catabolism of 7-hydroxycoumarin, 7-hydroxycoumarin-inducible genes hcdD, hcdE, hcdF, and hcdG were identified by RT-qPCR analysis. The DNA fragment encoding a putative alcohol dehydrogenase HcdE was cloned, and the recombinant protein catalyzed the NADPH-dependent reduction of 7-hydroxycoumarin both in vivo and in vitro. The reaction product was isolated and characterized as a 7-hydroxy-3,4-dihydrocoumarin based on HPLC-MS and NMR analyses. In addition, the HcdE was active towards 6,7-dihydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin and coumarin. Thus, in contrast to the well-known fact that the ene-reductases usually participate in the reduction of the double bond, an alcohol dehydrogenase catalyzing such reaction has been identified, and, for P. mandelii 7HK4, 7-hydroxycoumarin degradation via a 7-hydroxy-3,4-dihydrocoumarin pathway has been proposed.


Assuntos
Álcool Desidrogenase/metabolismo , Biodegradação Ambiental , Pseudomonas/metabolismo , Umbeliferonas/metabolismo , Álcool Desidrogenase/genética , Catálise , Cumarínicos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genoma Bacteriano , Estrutura Molecular , Família Multigênica , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Filogenia , Pseudomonas/classificação , Pseudomonas/enzimologia , Pseudomonas/genética , Reação em Cadeia da Polimerase em Tempo Real , Umbeliferonas/química
11.
J Environ Manage ; 284: 112030, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33529882

RESUMO

This study prioritizes the biodegradation potential of novel bacterial consortia formulated from cow dung samples towards low-density polyethylene (LDPE) and polypropylene (PP) in comparison with our previous studies. Ten possible consortia were formulated using 10 selected isolates with >10% weight reduction of LDPE and PP, these were pre-treated under UV for 1 h, and their biodegradation potential was studied for 160 days. The isolates present in prioritized consortia were characterized by standard microbiology and 16SrRNA gene sequencing methods. Out of 10 bacterial consortia formulated, potential consortium-CB3 showed greater percentage degradation (weight reduction) of 64.25 ± 2% and 63.00 ± 2% towards LDPE and PP films, respectively (p < 0.05) at 37 °C compared to other consortia. Significant structural variations due to the formation of bacterial biofilm were observed in CB3 treated LDPE and PP films. The three bacteria-IS1, IS2, and IS3-that constituted CB3 were found to be novel strains and designated to be Enterobacter sp nov. bt DSCE01, Enterobacter cloacae nov. bt DSCE02, and Pseudomonas aeruginosa nov. bt DSCE-CD03, respectively. This novel consortium can be scaled up for enhanced degradation of plastic polymers and probably design cost-effective bio-digester for industrial applications using CB3 as potential inoculum.


Assuntos
Polietileno , Polipropilenos , Animais , Biodegradação Ambiental , Bovinos , Enterobacter , Feminino , Pseudomonas/genética
12.
Appl Microbiol Biotechnol ; 105(4): 1693-1708, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33527148

RESUMO

The heat-stable peptidase AprX, secreted by psychrotolerant Pseudomonas species in raw milk, is a major cause of destabilization and premature spoilage of ultra-high temperature (UHT) milk and milk products. To enable rapid detection and quantification of seven frequent and proteolytic Pseudomonas species (P. proteolytica, P. gessardii, P. lactis, P. fluorescens, P. protegens, P. lundensis, and P. fragi) in raw milk, we developed two triplex qPCR assays taking into account species-dependent differences in AprX activity. Besides five species-specific hydrolysis probes, targeting the aprX gene, a universal rpoB probe was included in the assay to determine the total Pseudomonas counts. For all six probes, linear regression lines between Cq value and target DNA concentration were obtained in singleplex as well as in multiplex approaches, yielding R2 values of > 0.975 and amplification efficiencies of 85-97%. Moreover, high specificity was determined using genomic DNA of 75 Pseudomonas strains, assigned to 57 species, and 40 other bacterial species as templates in the qPCR. Quantification of the target species and total Pseudomonas counts resulted in linear detection ranges of approx. 103-107 cfu/ml, which correspond well to common Pseudomonas counts in raw milk. Application of the assay using 60 raw milk samples from different dairies showed good agreement of total Pseudomonas counts calculated by qPCR with cell counts derived from cultivation. Furthermore, a remarkably high variability regarding the species composition was observed for each milk sample, whereby P. lundensis and P. proteolytica/P. gessardii were the predominant species detected. KEY POINTS: • Multiplex qPCR for quantification of seven proteolytic Pseudomonas species and total Pseudomonas counts in raw milk • High specificity and sensitivity via hydrolysis probes against aprX and rpoB • Rapid method to determine Pseudomonas contamination in raw milk and predict spoilage potential.


Assuntos
Leite , Pseudomonas , Animais , Temperatura Alta , Leite/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Pseudomonas/genética , Pseudomonas/metabolismo
13.
Appl Environ Microbiol ; 87(9)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33579686

RESUMO

Although enzyme-encoding genes involved in the degradation of carbaryl have been reported in Pseudomonas sp. strain XWY-1, no regulator has been identified yet. In the mcbABCDEF cluster responsible for the upstream pathway of carbaryl degradation (from carbaryl to salicylate), the mcbA gene is constitutively expressed, while mcbBCDEF is induced by 1-naphthol, the hydrolysis product of carbaryl by McbA. In this study, we identified McbG, a transcriptional activator of the mcbBCDEF cluster. McbG is a 315-amino-acid protein with a molecular mass of 35.7 kDa. It belongs to the LysR family of transcriptional regulators and shows 28.48% identity to the pentachlorophenol (PCP) degradation transcriptional activation protein PcpR from Sphingobium chlorophenolicum ATCC 39723. Gene disruption and complementation studies reveal that mcbG is essential for transcription of the mcbBCDEF cluster in response to 1-naphthol in strain XWY-1. The results of the electrophoretic mobility shift assay (EMSA) and DNase I footprinting show that McbG binds to the 25-bp motif in the mcbBCDEF promoter area. The palindromic sequence TATCGATA within the motif is essential for McbG binding. The binding site is located between the -10 box and the transcription start site. In addition, McbG can repress its own transcription. The EMSA results show that a 25-bp motif in the mcbG promoter area plays an important role in McbG binding to the promoter of mcbG This study reveals the regulatory mechanism for the upstream pathway of carbaryl degradation in strain XWY-1. The identification of McbG increases the variety of regulatory models within the LysR family of transcriptional regulators.IMPORTANCE Pseudomonas sp. strain XWY-1 is a carbaryl-degrading strain that utilizes carbaryl as the sole carbon and energy source for growth. The functional genes involved in the degradation of carbaryl have already been reported. However, the regulatory mechanism has not been investigated yet. Previous studies demonstrated that the mcbA gene, responsible for hydrolysis of carbaryl to 1-naphthol, is constitutively expressed in strain XWY-1. In this study, we identified a LysR-type transcriptional regulator, McbG, which activates the mcbBCDEF gene cluster responsible for the degradation of 1-naphthol to salicylate and represses its own transcription. The DNA binding site of McbG in the mcbBCDEF promoter area contains a palindromic sequence, which affects the binding of McbG to DNA. These findings enhance our understanding of the mechanism of microbial degradation of carbaryl.


Assuntos
Proteínas de Bactérias/genética , Carbaril/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , Fatores de Transcrição/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Família Multigênica , Fatores de Transcrição/metabolismo
14.
Chemosphere ; 274: 129819, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33582538

RESUMO

Heavy metal and metalloid toxicity in agricultural land needs special attention for crop production essential to feed increasing population globally. Plant growth-promoting rhizobacteria (PGPR) are native biological agents that have tremendous potential to augment crop production in contaminated fields. This study involves selection and identification (through 16S rRNA gene sequence and FAME analysis) of a potent Pseudomonas sp. (strain K32) isolated from a metal-contaminated rice rhizosphere, aimed to its application for sustainable agriculture. Apart from multi-heavy metal(loid) resistance (Cd2+, Pb2+ and As3+ upto 4000, 3800, 3700 µg/ml respectively) along with remarkable Cd bioaccumulation potential (∼90%), this strain showed IAA production, nitrogen-fixation and phosphate solubilization under Cd stress. This bioaccumulation efficiency coupled with PGP traits resulted in the significant enhancement of rice seedling growth under Cd stress. This positive impact of K32 strain was clearly manifested in morphological and biochemical improvements under Cd stress including successful root colonization with rice roots. Cd uptake was also reduced significantly in seedlings in presence of K32 strain. Together with all mentioned properties, K32 showed bio-control potential against plant pathogenic fungi viz. Aspergillus flavus, Aspergillus parasiticus, Paecilomyces sp., Cladosporium herbarum, Rhizopus stolonifer and Alternaria alternata which establish K32 strain a key player in effective bioremediation of agricultural fields. Biocontrol potential was found to be the result of enzymatic activities viz. chitinase, ß-1,3-glucanase and protease which were estimated as 8.17 ± 0.44, 4.38 ± 0.35 and 7.72 ± 0.28 U/mg protein respectively.


Assuntos
Metais Pesados , Oryza , Poluentes do Solo , Alternaria , Aspergillus , Cádmio/toxicidade , Cladosporium , Metais Pesados/toxicidade , Raízes de Plantas , Pseudomonas/genética , RNA Ribossômico 16S , Rhizopus , Rizosfera , Plântula , Microbiologia do Solo , Poluentes do Solo/toxicidade
15.
Appl Environ Microbiol ; 87(9)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33608298

RESUMO

Biosurfactant production is a common trait in leaf surface-colonizing bacteria that has been associated with increased survival and movement on leaves. At the same time, the ability to degrade aliphatics is common in biosurfactant-producing leaf colonizers. Pseudomonads are common leaf colonizers and have been recognized for their ability to produce biosurfactants and degrade aliphatic compounds. In this study, we investigated the role of biosurfactants in four non-plant-pathogenic Pseudomonas strains by performing a series of experiments to characterize their surfactant properties and their role during leaf colonization and diesel degradation. The biosurfactants produced were identified using mass spectrometry. Two strains produced viscosin-like biosurfactants, and the other two produced massetolide A-like biosurfactants, which aligned with the phylogenetic relatedness between the strains. To further investigate the role of surfactant production, random Tn5 transposon mutagenesis was performed to generate knockout mutants. The knockout mutants were compared to their respective wild types with regard to their ability to colonize gnotobiotic Arabidopsis thaliana and to degrade diesel or dodecane. It was not possible to detect negative effects during plant colonization in direct competition or individual colonization experiments. When grown on diesel, knockout mutants grew significantly slower than their respective wild types. When grown on dodecane, knockout mutants were less impacted than during growth on diesel. By adding isolated wild-type biosurfactants, it was possible to complement the growth of the knockout mutants.IMPORTANCE Many leaf-colonizing bacteria produce surfactants and are able to degrade aliphatic compounds; however, whether surfactant production provides a competitive advantage during leaf colonization is unclear. Furthermore, it is unclear if leaf colonizers take advantage of the aliphatic compounds that constitute the leaf cuticle and cuticular waxes. Here, we tested the effect of surfactant production on leaf colonization, and we demonstrate that the lack of surfactant production decreases the ability to degrade aliphatic compounds. This indicates that leaf surface-dwelling, surfactant-producing bacteria contribute to degradation of environmental hydrocarbons and may be able to utilize leaf surface waxes. This has implications for plant-microbe interactions and future studies.


Assuntos
Arabidopsis/microbiologia , Gasolina , Folhas de Planta/microbiologia , Pseudomonas/metabolismo , Tensoativos/metabolismo , Alcanos/metabolismo , Biodegradação Ambiental , Mutagênese , Filogenia , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , RNA Ribossômico 16S , Tensoativos/química
16.
Appl Environ Microbiol ; 87(9)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33608299

RESUMO

Gram-negative bacteria employ secretion systems to translocate proteinaceous effectors from the cytoplasm to the extracellular milieu, thus interacting with the surrounding environment or microniche. It is known that bacteria can benefit from the type VI secretion system (T6SS) by transporting ions to combat reactive oxygen species (ROS). Here, we report that T6SS activities conferred tolerance to nicotine-induced oxidative stress in Pseudomonas sp. strain JY-Q, a highly active nicotine degradation strain isolated from tobacco waste extract. AA098_13375 was identified to encode a dual-functional effector with antimicrobial and anti-ROS activities. Wild-type strain JY-Q grew better than the AA098_13375 deletion mutant in nicotine-containing medium by antagonizing increased intracellular ROS levels. It was, therefore, tentatively designated TseN (type VI secretion system effector for nicotine tolerance), homologs of which were observed to be broadly ubiquitous in Pseudomonas species. TseN was identified as a Tse6-like bacteriostatic toxin via monitoring intracellular NAD+ TseN presented potential antagonism against ROS to fine tune the heavy traffic of nicotine metabolism in strain JY-Q. It is feasible that the dynamic tuning of NAD+ driven by TseN could satisfy demands from nicotine degradation with less cytotoxicity. In this scenario, T6SS involves a fascinating accommodation cascade that prompts constitutive biotransformation of N-heterocyclic aromatics by improving bacterial robustness/growth. In summary, the T6SS in JY-Q mediated resistance to oxidative stress and promoted bacterial fitness via a contact-independent growth competitive advantage, in addition to the well-studied T6SS-dependent antimicrobial activities.IMPORTANCE Mixtures of various pollutants and the coexistence of numerous species of organisms are usually found in adverse environments. Concerning biodegradation of nitrogen-heterocyclic contaminants, the scientific community has commonly focused on screening functional enzymes that transform pollutants into intermediates of attenuated toxicity or for primary metabolism. Here, we identified dual roles of the T6SS effector TseN in Pseudomonas sp. strain JY-Q, which is capable of degrading nicotine. The T6SS in strain JY-Q is able to deliver TseN to kill competitors and provide a growth advantage by a contact-independent pattern. TseN could monitor the intracellular NAD+ level by its hydrolase activity, causing cytotoxicity in competitive rivals but metabolic homeostasis on JY-Q. Moreover, JY-Q could be protected from TseN toxicity by the immunity protein TsiN. In conclusion, we found that TseN with cytotoxicity to bacterial competitors facilitated the nicotine tolerance of JY-Q. We therefore reveal a working model between T6SS and nicotine metabolism. This finding indicates that multiple diversified weapons have been evolved by bacteria for their growth and robustness.


Assuntos
Proteínas de Bactérias/metabolismo , Nicotina/metabolismo , Pseudomonas/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Proteínas de Bactérias/genética , Biodegradação Ambiental , Homeostase , Família Multigênica , Pseudomonas/genética , Espécies Reativas de Oxigênio/metabolismo , Sistemas de Secreção Tipo VI/genética
17.
Curr Microbiol ; 78(4): 1227-1237, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33625570

RESUMO

Selection and dissemination of resistant bacteria and antibiotic resistance genes (ARGs) require a deeper understanding since antibiotics are permanently released to the environment. The objective of this paper was to evaluate the phenotypic resistance of 499 isolates of Pseudomonas spp. from urban water sources, and the prevalence of 20 ARGs within those isolates. Resistance to penicillins, cephalosporins, carbapenems, quinolones, macrolides, and tetracyclines was mainly observed in the hospital effluent, municipal wastewater and river water downstream the city. Resistant strains were frequently identified as P. aeruginosa and P. putida. P. aeruginosa isolates were mostly resistant to cefepime, ceftazidime, imipenem, and gentamycin, while P. putida strains were especially resistant to piperacillin-tazobactam. ARGs such as blaTEM-1, blaSHV-1, blaPER-1, blaAmpC, blaVIM-1, PstS, qnrA, qnrB, ermB, tetA, tetB and tetC have been detected. The blaAmpC gene was found in P. aeruginosa, while blaTEM-1 and blaPER-1 genes were found in P. putida. Class 1 integron integrase gene was found in 6.81% of the Pseudomonas isolates.


Assuntos
Pseudomonas , Ciclo Hidrológico , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Integrons/genética , Testes de Sensibilidade Microbiana , Pseudomonas/genética , Pseudomonas aeruginosa/genética , Águas Residuárias
18.
ACS Chem Biol ; 16(3): 501-509, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33595276

RESUMO

Cell-to-cell communication via chemical signals is an essential mechanism that pathogenic bacteria use to coordinate group behaviors and promote virulence. The Pseudomonas virulence factor (pvf) gene cluster is distributed in more than 500 strains of proteobacteria including both plant and human pathogens. The pvf cluster has been implicated in the production of signaling molecules important for virulence; however, the regulatory impact of these signaling molecules on virulence had not been elucidated. Using the insect pathogen Pseudomonas entomophila L48 as a model, we demonstrated that pvf-encoded biosynthetic enzymes produce PVF autoinducers that regulate the expression of pvf genes and a gene encoding the toxin monalysin via quorum sensing. In addition, PVF autoinducers regulate the expression of nearly 200 secreted and membrane proteins, including toxins, motility proteins, and components of the type VI secretion system, which play key roles in bacterial virulence, colonization, and competition with other microbes. Deletion of pvf also altered the secondary metabolome. Six major compounds upregulated by PVF autoinducers were isolated and structurally characterized, including three insecticidal 3-indolyl oxazoles, the labradorins, and three antimicrobial pyrrolizidine alkaloids, the pyreudiones. The signaling properties of PVF autoinducers and their wide-ranging regulatory effects indicate multifaceted roles of PVF in controlling cell physiology and promoting virulence. The broad genome distribution of pvf suggests that PVF-mediated signaling is relevant to many bacteria of agricultural and biomedical significance.


Assuntos
Proteínas de Bactérias/metabolismo , Pseudomonas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Extratos Celulares/química , Regulação Bacteriana da Expressão Gênica , Oxazóis/química , Pseudomonas/genética , Percepção de Quorum , Metabolismo Secundário , Transdução de Sinais , Virulência , Fatores de Virulência/genética
19.
World J Microbiol Biotechnol ; 37(4): 55, 2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33615389

RESUMO

Biosurfactants are environment compatible surface-active biomolecules with multifunctional properties which can be utilized in various industries. In this study a biosurfactant producing novel plant growth promoting isolate Pseudomonas guariconensis LE3 from the rhizosphere of Lycopersicon esculentum is presented as biostimulant and biocontrol agent. Biosurfactant extracted from culture was characterized to be mixture of various mono- and di-rhamnolipids with antagonistic activity against Macrophomina phaseolina, causal agent of charcoal rot in diverse crops. Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR) analysis confirmed the rhamnolipid nature of biosurfactant. PCR analysis established the presence of genes involved in synthesis of antibiotics diacetylphloroglucinol, phenazine 1-carboxylic acid and pyocyanin, and lytic enzymes chitinase and endoglucanase suggesting biocontrol potential of the isolate. Plant growth promoting activities shown by LE3 were phosphate solubilization and production of siderophores, indole acetic acid (IAA), ammonia and 1-aminocyclopropane-1-carboxylate deaminase (ACCD). To assemble all the characteristics of LE3 various bioformuations were developed. Amendment of biosurfactant in bioformulation of LE3 cells improved the shelf life. Biosurfactant amended formulation of LE3 cells was most effective in biocontrol of charcoal rot disease of sunflower and growth promotion in field conditions. The root adhered soil mass of plantlets inoculated with LE3 plus biosurfactant was significantly higher over control. Biosurfactant amended formulation of LE3 cells caused maximum yield enhancement (80.80%) and biocontrol activity (75.45%), indicating that addition of biosurfactant improves the plant-bacterial interaction and soil properties leading to better control of disease and overall improvement of plant health and yield.


Assuntos
Agentes de Controle Biológico/farmacologia , Helianthus/microbiologia , Desenvolvimento Vegetal , Doenças das Plantas/microbiologia , Pseudomonas/fisiologia , Tensoativos/metabolismo , Antifúngicos , Ascomicetos/efeitos dos fármacos , Agentes de Controle Biológico/química , Carbono-Carbono Liases , Linhagem Celular , Celulase , Quitinases , Helianthus/crescimento & desenvolvimento , Ácidos Indolacéticos , Lycopersicon esculentum/microbiologia , Fenazinas , Filogenia , Doenças das Plantas/prevenção & controle , Raízes de Plantas/microbiologia , Pseudomonas/genética , Piocianina , RNA Ribossômico 16S/genética , Rizosfera , Sideróforos/biossíntese , Microbiologia do Solo
20.
J Glob Antimicrob Resist ; 24: 395-397, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33577996

RESUMO

OBJECTIVES: Pseudomonas is a Gram-negative bacterial genus with numerous member species. In this study, using whole-genome sequencing, we characterized a novel Pseudomonas sp. strain TUM18999, isolated as a pathogen from a human patient. METHODS: The TUM18999 strain was isolated from a patient's burn wound. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method. The whole-genome sequence was obtained using Miseq and MinION, and we conducted phylogenetic analysis based on single nucleotide polymorphisms of the core genome. RESULTS: Antimicrobial susceptibility testing revealed a high ceftazidime MIC (32 mg/L). Moreover, carbapenemase production was confirmed using the modified carbapenem inactivation method. We found that the complete genome of TUM18999 was 6,826,062 bp long, with 6175 coding sequences (CDS) and a DNA G+C content (non-plasmid) of 66.4 mol%. Consistent with the high similarities with the 16S rRNA sequences of P. otitidis MCC10330 (98.6%) and P. alcaligenes NBRC 14159 (99.2%), similarities (<90%) were also observed with the gyrB genes of both strains. The average nucleotide identities for P. alcaligenes NBRC 14159 and P. otitidis MCC10330 were also <90%. The core-genome single nucleotide polymorphism phylogenetic tree indicated that the TUM18999 strain was most closely related to P. otitidis MCC10330. In addition, the TUM18999 strain carried the novel gene, species-specific subclass B3 metallo-ß-lactamase (MBL), and its similarities with P. alcaligenes metallo-ß-lactamase-1 (PAM-1) and P. otitidis metallo-ß-lactamase-1 (POM-1) were 90.24% and 73.14%, respectively. CONCLUSION: We characterized the complete whole genome sequence of the novel Pseudomonas sp. TUM18999 carrying the novel gene species-specific subclass B3 MBL.


Assuntos
Queimaduras , Genoma Bacteriano , Pseudomonas , Queimaduras/microbiologia , Humanos , Japão , Filogenia , Pseudomonas/genética , RNA Ribossômico 16S/genética
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