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1.
PLoS One ; 15(7): e0235508, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614917

RESUMO

This study examined the influence of bioaugmentation on metal concentrations (aluminum, cadmium, chromium, cobalt, copper, iron, lead, manganese, molybdenum, nickel and zinc) in anaerobically digested sewage sludge. To improve the digestion efficiency, bioaugmentation with a mixture of wild-living Archaea and Bacteria (MAB) from Yellowstone National Park, USA, was used. The total concentration of all metals was higher in the digestate than in the feedstock. During anaerobic digestion, the percent increase in the concentration of most of metals was slightly higher in the bioaugmented runs than in the un-augmented runs, but these differences were not statistically significant. However, the percent increase in cadmium and cobalt concentration was significantly higher in the bioaugmented runs than in the un-augmented runs. At MAB doses of 9 and 13% v/v, cadmium concentration in the digestate was 211 and 308% higher than in the feedstock, respectively, and cobalt concentration was 138 and 165%, respectively. Bioaugmentation increased over 4 times the percentage of Pseudomonas sp. in the biomass that are able to efficiently accumulate metals by both extracellular adsorption and intracellular uptake. Biogas production was not affected by the increased metal concentrations. In conclusion, bioaugmentation increased the concentration of metals in dry sludge, which means that it could potentially have negative effects on the environment.


Assuntos
Metais/metabolismo , Esgotos/química , Adsorção , Anaerobiose , Archaea/genética , Archaea/crescimento & desenvolvimento , Archaea/metabolismo , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Biomassa , Cádmio/análise , Cádmio/metabolismo , Cobalto/análise , Cobalto/metabolismo , Metais/química , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/metabolismo , Esgotos/microbiologia , Eliminação de Resíduos Líquidos
2.
Ecotoxicol Environ Saf ; 200: 110767, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32470679

RESUMO

The occurrence and dissemination of antibiotic resistant genes (ARGs) that are associated with clinical pathogens and the evaluation of associated risks are still under-investigated in developing countries under tropical conditions. In this context, cultivable and molecular approaches were performed to assess the dissemination of bacteria and the antibiotic resistance genes in aquatic environment in Kinshasa, Democratic Republic of the Congo. Cultivable approach quantified ß-lactam, carbapenem resistant, and total Escherichia coli and Enterobacteriaceae in river sediments and surface waters that receive raw hospital effluents. The molecular approach utilized Quantitative Polymerase Chain Reaction (qPCR) to quantify the total bacteria and the richness of relevant bacteria (Escherichia coli, Enterococcus, and Pseudomonas), and antibiotic resistance genes (ARGs: blaOXA-48, blaCTX-M, blaIMP, blaTEM) in sediment samples. Statistical analysis were employed to highlight the significance of hospital contribution and seasonal variation of bacteria and ARGs into aquatic ecosystems in suburban municipalities of Kinshasa, Democratic Republic of the Congo. The contribution of hospitals to antibiotic resistance proliferation is higher in the dry season than during the wet season (p < 0.05). Hospital similarly contributed Escherichia coli, Enterococcus, and Pseudomonas and ARGs significantly to the sediments in both seasons (p < 0.05). The organic matter content correlated positively with E. coli (r = 0.50, p < 0.05). The total bacterial load correlated with Enterococcus, and Pseudomonas (0.49 < r < 0.69, p < 0.05). Each ARG correlated with the total bacterial load or at least one relevant bacteria (0.41 < r < 0.81, p < 0.05). Our findings confirm that hospital wastewaters contributed significantly to antibiotic resistance profile and the significance of this contribution increased in the dry season. Moreover, our analysis highlights this risk from untreated hospital wastewaters in developing countries, which presents a great threat to public health.


Assuntos
Resistência Microbiana a Medicamentos/genética , Genes Bacterianos/efeitos dos fármacos , Hospitais , Rios/microbiologia , Águas Residuárias/microbiologia , Antibacterianos/análise , Antibacterianos/farmacologia , Cidades , República Democrática do Congo , Ecossistema , Enterococcus/efeitos dos fármacos , Enterococcus/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Pseudomonas/efeitos dos fármacos , Pseudomonas/genética , Rios/química , Clima Tropical , Águas Residuárias/química
3.
PLoS One ; 15(4): e0232115, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32339192

RESUMO

Crop diseases are responsible for considerable yield losses worldwide and particularly in sub-Saharan Africa. To implement efficient disease control measures, detection of the pathogens and understanding pathogen spatio-temporal dynamics is crucial and requires the use of molecular detection tools, especially to distinguish different pathogens causing more or less similar symptoms. We report here the design a new molecular diagnostic tool able to simultaneously detect five bacterial taxa causing important diseases on rice in Africa: (1) Pseudomonas fuscovaginae, (2) Xanthomonas oryzae, (3) Burkholderia glumae and Burkholderia gladioli, (4) Sphingomonas and (5) Pantoea species. This new detection tool consists of a multiplex PCR, which is cost effective and easily applicable. Validation of the method is presented through its application on a global collection of bacterial strains. Moreover, sensitivity assessment for the detection of all five bacteria is reported to be at 0.5 ng DNA by µl. As a proof of concept, we applied the new molecular detection method to a set of 256 rice leaves collected from 16 fields in two irrigated areas in western Burkina Faso. Our results show high levels of Sphingomonas spp. (up to 100% of tested samples in one field), with significant variation in the incidence between the two sampled sites. Xanthomonas oryzae incidence levels were mostly congruent with bacterial leaf streak (BLS) and bacterial leaf blight (BLB) symptom observations in the field. Low levels of Pantoea spp. were found while none of the 256 analysed samples was positive for Burkholderia or Pseudomonas fuscovaginae. Finally, many samples (up to 37.5% in one studied field) were positive for more than one bacterium (co-infection). Documenting co-infection levels are important because of their drastic consequences on epidemiology, evolution of pathogen populations and yield losses. The newly designed multiplex PCR for multiple bacterial pathogens of rice is a significant improvement for disease monitoring in the field, thus contributing to efficient disease control and food safety.


Assuntos
Burkholderia/genética , Coinfecção/diagnóstico , DNA Bacteriano/análise , Reação em Cadeia da Polimerase Multiplex/métodos , Oryza/microbiologia , Doenças das Plantas/microbiologia , Pseudomonas/genética , Xanthomonas/genética , Burkholderia/isolamento & purificação , Burkholderia/patogenicidade , Burkina Faso/epidemiologia , Coinfecção/epidemiologia , Coinfecção/genética , DNA Bacteriano/genética , Incidência , Pseudomonas/isolamento & purificação , Pseudomonas/patogenicidade , Xanthomonas/isolamento & purificação , Xanthomonas/patogenicidade
4.
Nat Commun ; 11(1): 1370, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170080

RESUMO

Multidrug resistance (MDR) represents a global threat to health. Here, we used whole genome sequencing to characterise Pseudomonas aeruginosa MDR clinical isolates from a hospital in Thailand. Using long-read sequence data we obtained complete sequences of two closely related megaplasmids (>420 kb) carrying large arrays of antibiotic resistance genes located in discrete, complex and dynamic resistance regions, and revealing evidence of extensive duplication and recombination events. A comprehensive pangenomic and phylogenomic analysis indicates that: 1) these large plasmids comprise an emerging family present in different members of the Pseudomonas genus, and associated with multiple sources (geographical, clinical or environmental); 2) the megaplasmids encode diverse niche-adaptive accessory traits, including multidrug resistance; 3) the accessory genome of the megaplasmid family is highly flexible and diverse. The history of the megaplasmid family, inferred from our analysis of the available database, suggests that members carrying multiple resistance genes date back to at least the 1970s.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos/genética , Plasmídeos/genética , Pseudomonas/genética , Antibacterianos/farmacologia , DNA Bacteriano/genética , Evolução Molecular , Genômica , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Plasmídeos/classificação , Plasmídeos/isolamento & purificação , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Tailândia , Sequenciamento Completo do Genoma
5.
J Mol Biol ; 432(7): 2232-2252, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32084414

RESUMO

Protein fibrillation is traditionally associated with misfolding, loss of functional phenotype, and gain of toxicity in neurodegenerative diseases. However, many organisms exploit fibrils in the form of functional amyloids (FA), as seen in bacteria, such as E. coli, Salmonella, Bacillus, and Pseudomonas. Here, we provide structural information and mechanistic data for fibrillation of the smallest amyloidogenic truncation unit along with the full-length version (FL) of the major amyloid protein FapC from Pseudomonas, predicted to consist of three ß-hairpin-forming imperfect repeats separated by disordered regions. Using a series of truncation mutants, we establish that the putative loops (linkers) increase the rate of aggregation. The minimal aggregation unit consisting of a single repeat with flanking disordered regions (R3C) aggregates in a pathway dominated by secondary nucleation, in contrast to the primary nucleation favored by full-length (FL) FapC. SAXS on FapC FL, R3C, and remaining truncation constructs resolves two major coexisting species in the fibrillation process, namely pre-fibrillar loosely aggregated monomers, and cylindrical, elliptical cross-section fibrils. Solid-state NMR spectra identified rigid parts of the FapC fibril. We assigned Cα-Cß chemical shifts, indicative of a predominant ß-sheet topology with some α-helix or loop chemical shifts. Our work emphasizes the complex nature of FapC fibrillation. In addition, we are able to deduce the importance of non-repeat regions (i.e., predicted loops), which enhance the amyloid protein aggregation and their influence on the polymorphism of the fibril architecture.


Assuntos
Amiloide/química , Proteínas Amiloidogênicas/metabolismo , Proteínas de Bactérias/metabolismo , Agregados Proteicos , Pseudomonas/metabolismo , Sequência de Aminoácidos , Proteínas Amiloidogênicas/genética , Proteínas de Bactérias/genética , Mutação , Pseudomonas/genética
6.
PLoS One ; 15(1): e0227927, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31986172

RESUMO

Pseudomonas brassicacearum GS20 is an antagonistic strain of bacteria recently isolated from the rhizosphere of Codonopsis pilosula. No validated reference gene has been indentified from P. brassicacearum to use in the normalization of real-time quantitative reverse transcription-PCR data. Therefore, in this study, nine candidate reference genes (recA, gyrA, rpoD, proC, gmk, rho, 16S, ftsz, and secA) were assessed at different growth phases and under various growth conditions. The expression stability of these candidate genes was evaluated using BestKeeper, NormFinder and GeNorm. In general, the results showed rho, rpoD and gyrA were the most suitable reference genes for P. brassicacearum GS20. The relative expression of iron-regulated gene (fhu) was normalized to verify the reliability of the proposed reference genes under iron-replete and iron-limited conditions. The trend in relative expression was consistent with the change in siderophore production under different iron conditions. This study presents reliable reference genes for transcriptional studies in P. brassicacearum GS20 under the chosen experimental conditions.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Pseudomonas/genética , Perfilação da Expressão Gênica/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Padrões de Referência , Rizosfera
7.
J Med Microbiol ; 69(3): 347-360, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31976855

RESUMO

Pseudomonas brassicacearum is one of over fifty species of bacteria classified into the P. fluorescens group. Generally considered a harmless commensal, these bacteria are studied for their plant-growth promotion (PGP) and biocontrol characteristics. Intriguingly, P. brassicacearum is closely related to P. corrugata, which is classified as an opportunistic phytopathogen. Twenty-one P. brassicacearum genomes have been sequenced to date. In the current review, genomes of P. brassicacearum and strains from the P. corrugata clade were mined for regions associated with PGP, biocontrol and pathogenicity. We discovered that 'beneficial' bacteria and those classified as plant pathogens have many genes in common; thus, only a fine line separates beneficial/harmless commensals from those capable of causing disease in plants. The genotype and physiological state of the plant, the presence of biotic/abiotic stressors, and the ability of bacteria to manipulate the plant immune system collectively contribute to how the bacterial-plant interaction plays out. Because production of extracellular metabolites is energetically costly, these compounds are expected to impart a fitness advantage to the producer. P. brassicacearum is able to reduce the threat of nematode predation through release of metabolites involved in biocontrol. Moreover this bacterium has the unique ability to form biofilms on the head of Caenorhabditis elegans, as a second mechanism of predator avoidance. Rhizobacteria, plants, fungi, and microfaunal predators have occupied a shared niche for millions of years and, in many ways, they function as a single organism. Accordingly, it is essential that we appreciate the dynamic interplay among these members of the community.


Assuntos
Biofilmes/crescimento & desenvolvimento , Caenorhabditis elegans/microbiologia , Genoma Bacteriano/genética , Doenças das Plantas/microbiologia , Plantas/microbiologia , Pseudomonas/fisiologia , Animais , Lipopeptídeos/genética , Controle Biológico de Vetores , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , Transdução de Sinais , Simbiose
8.
J Agric Food Chem ; 68(3): 826-837, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31895558

RESUMO

A total of five strains of nicosulfuron-degrading bacteria were isolated from a continuously cultivated microbial consortium using culturomics. Among them, a novel Pseudomonas strain, LAM1902, with the highest degradation efficiency was investigated in detail. The characteristics of nicosulfuron-degradation by LAM1902 were investigated and optimized by response surface analysis. Furthermore, non-targeted metabolomic analysis of extracellular and intracellular biodegradation of nicosulfuron by LAM1902 was carried out by liquid chromatography/mass spectroscopy (LC-MS) and gas chromatography-time-of-flight/mass spectroscopy (GC-TOF/MS). It was found that nicosulfuron was degraded by LAM1902 mainly via breaking the sulfonylurea bridge, and this degradation might be attributed to oxalate accumulation. The results of GC-TOF/MS also showed that the intracellular degradation of nicosulfuron did not occur. However, nicosulfuron exerted a significant influence on the metabolism of inositol phosphate, pyrimidine, arginine/proline, glyoxylate, and dicarboxylate metabolism and streptomycin biosynthesis. The changes of myo-inositol, trehalose, and 3-aminoisobutanoic acid were proposed as a mechanism of self-protection against nicosulfuron stress.


Assuntos
Herbicidas/metabolismo , Pseudomonas/metabolismo , Piridinas/metabolismo , Compostos de Sulfonilureia/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Herbicidas/química , Concentração de Íons de Hidrogênio , Metabolômica , Filogenia , Pseudomonas/classificação , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Piridinas/química , Compostos de Sulfonilureia/química
9.
Artigo em Inglês | MEDLINE | ID: mdl-31913782

RESUMO

A stain of Pseudomonas sp. Lphe-2, which could degrade phenanthrene as the main carbon and energy source, was isolated from the aerobic sludge of a coking plant. Then its biodegradation characteristics, whole genome sequence and biodegradation pathway were examined. The Lphe-2 strain exhibited broad-spectrum degradation activities for various polycyclic aromatic hydrocarbons (PAHs), including naphthalene (NAP), phenanthrene (PHE), and pyrene (PYR). Under the optimal conditions, the degradation efficiency of phenanthrene (100 mg/L) is 92.76% on the 7th day, and 2-carboxybenzaldehyde and 1-hydroxy-2-naphthoic acid are the major metabolites found in phenanthrene metabolism. Genomic analysis of Pseudomonas sp. Lphe-2 showed that a total of 3879 genes from the Lphe-2 strain were annotated based on the COG classification, and the genomic information was annotated to 185 metabolic pathways. Based on the intermediate metabolites detected by Gas Chromatography-Mass Spectrometer (GC-MS) and all potential phenanthrene-degrading genes identified by BLAST search, a phenanthrene biodegradation pathway of Lphe-2 strain was proposed. These results suggested that Lphe-2 strain has a good prospect in the bioremediation of PAHs pollution.


Assuntos
Genes Bacterianos , Fenantrenos/análise , Pseudomonas/metabolismo , Esgotos/microbiologia , Poluentes do Solo/análise , Biodegradação Ambiental , Cromatografia Gasosa-Espectrometria de Massas , Genômica , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Naftalenos/análise , Naftalenos/metabolismo , Fenantrenos/metabolismo , Pseudomonas/genética , Pirenos/análise , Pirenos/metabolismo , Poluentes do Solo/metabolismo
10.
Nucleic Acids Res ; 48(5): 2388-2400, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31925438

RESUMO

Tight and coordinate regulation of virulence determinants is essential for bacterial biology and involves dynamic shaping of transcriptional regulatory networks during evolution. The horizontally transferred two-partner secretion system ExlB-ExlA is instrumental in the virulence of different Pseudomonas species, ranging from soil- and plant-dwelling biocontrol agents to the major human pathogen Pseudomonas aeruginosa. Here, we identify a Cro/CI-like repressor, named ErfA, which together with Vfr, a CRP-like activator, controls exlBA expression in P. aeruginosa. The characterization of ErfA regulon across P. aeruginosa subfamilies revealed a second conserved target, the ergAB operon, with functions unrelated to virulence. To gain insights into this functional dichotomy, we defined the pan-regulon of ErfA in several Pseudomonas species and found ergAB as the sole conserved target of ErfA. The analysis of 446 exlBA promoter sequences from all exlBA+ genomes revealed a wide variety of regulatory sequences, as ErfA- and Vfr-binding sites were found to have evolved specifically in P. aeruginosa and nearly each species carries different regulatory sequences for this operon. We propose that the emergence of different regulatory cis-elements in the promoters of horizontally transferred genes is an example of plasticity of regulatory networks evolving to provide an adapted response in each individual niche.


Assuntos
Toxinas Bacterianas/metabolismo , Fatores de Transcrição/metabolismo , Células A549 , Proteínas de Bactérias/metabolismo , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Humanos , Óperon/genética , Regiões Promotoras Genéticas , Ligação Proteica , Pseudomonas/genética , Pseudomonas/patogenicidade , Proteínas Repressoras/metabolismo , Especificidade da Espécie , Virulência
11.
J Appl Microbiol ; 128(6): 1720-1734, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31957222

RESUMO

AIMS: This study investigated the adaptation strategies of Pseudomonas protegens to hyperosmotic growth environments (HGE). METHODS AND RESULTS: The current study combined transcriptomics and proteomics to provide an overview of the molecular mechanism of P. protegens adaptation to HGE. The results revealed that HGE exerted prominent impact on the synthesis of proteins involved in multiple cellular functions. Comparison of the differentially accumulated proteins at both transcriptomics and proteomics levels indicated the existence of complex post-transcriptional modification during P. protegens hyperosmotic adaptation. CONCLUSIONS: During HGE adaptation, the cells form a complex self-protection mechanism. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the current study can help researchers to gain insights regarding the mechanism of P. protegens adaptation to HGE. These findings provide information for the application of stress methods and facilitate its broad commercial utilization.


Assuntos
Aclimatação , Pressão Osmótica , Pseudomonas/fisiologia , Aclimatação/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Proteômica , Pseudomonas/genética , Pseudomonas/metabolismo
12.
FEMS Microbiol Ecol ; 96(2)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31930390

RESUMO

The Pseudomonas genus, which includes environmental and pathogenic species, is known to present antibiotic resistances, and can receive resistance genes from multi-resistant enteric bacteria released into the environment via faecal rejects. This study was aimed to investigate the resistome of Pseudomonas populations that have been in contact with these faecal bacteria. Thus, faecal discharges originating from human or cattle were sampled (from 12 points and two sampling campaigns) and 41 Pseudomonas species identified (316 isolates studied). The resistance phenotype to 25 antibiotics was determined in all isolates, and we propose a specific antibiotic resistance pattern for 14 species (from 2 to 9 resistances). None showed resistance to aminoglycosides, tetracycline, or polymyxins. Four species carried a very low number of resistances, with none to ß-lactams. Interestingly, we observed the absence of the transcriptional activator soxR gene in these four species. No plasmid transfer was highlighted by conjugation assays, and a few class 1 but no class 2 integrons were detected in strains that may have received resistance genes from Enterobacteria. These results imply that the contribution of the Pseudomonas genus to the resistome of an ecosystem first depends on the structure of the Pseudomonas populations, as they may have very different resistance profiles.


Assuntos
Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Pseudomonas/efeitos dos fármacos , Microbiologia da Água , Animais , Bovinos , Ecossistema , Fezes , Humanos , Integrons/genética , Plasmídeos , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Tetraciclina/farmacologia
13.
Microbiol Res ; 231: 126356, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31722286

RESUMO

In Rhizobium-legume symbiosis, the nodule is the most frequently studied compartment, where the endophytic/symbiotic microbiota demands critical investigation for development of specific inocula. We identified the bacterial diversity within root nodules of mung bean from different growing areas of Pakistan using Illumina sequencing of 16S rRNA gene. We observed specific OTUs related to specific site where Bradyrhizobium was found to be the dominant genus comprising of 82-94% of total rhizobia in nodules with very minor fraction of sequences from other rhizobia at three sites. In contrast, Ensifer (Sinorhizobium) was single dominant genus comprising 99.9% of total rhizobial sequences at site four. Among non-rhizobial sequences, the genus Acinetobacter was abundant (7-18% of total sequences), particularly in Bradyrhizobium-dominated nodule samples. Rhizobia and non-rhizobial PGPR isolated from nodule samples include Ensifer, Bradyrhizobium, Acinetobacter, Microbacterium and Pseudomonas strains. Co-inoculation of multi-trait PGPR Acinetobacter sp. VrB1 with either of the two rhizobia in field exhibited more positive effect on nodulation and plant growth than single-strain inoculation which favors the use of Acinetobacter as an essential component for development of mung bean inoculum. Furthermore, site-specific dominance of rhizobia and non-rhizobia revealed in this study may contribute towards decision making for development and application of specific inocula in different habitats.


Assuntos
Rhizobiaceae , Nódulos Radiculares de Plantas/microbiologia , Vigna/microbiologia , Acinetobacter/genética , Acinetobacter/isolamento & purificação , Bradyrhizobium/genética , Bradyrhizobium/isolamento & purificação , DNA Bacteriano/genética , Ecossistema , Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica , Microbiota/genética , Paquistão , Filogenia , Pseudomonas/genética , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S , Rhizobiaceae/classificação , Rhizobiaceae/genética , Sinorhizobium/genética , Sinorhizobium/isolamento & purificação
14.
J Appl Microbiol ; 128(2): 528-543, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31606926

RESUMO

AIMS: The characterization of bacterial communities diversity on four local plum cultivars in two phenological stages using culture-dependent and culture-independent methods and screening among culturable plum community for indigenous bacteria active against phytopathogens. METHODS AND RESULTS: The bacterial communities associated with leaves and fruits of four local Serbian plum cultivars (Pozegaca, Ranka, Cacanska Lepotica and Cacanska Rodna) were investigated in two phenological stages during early (May) and late (July) fruit maturation. Metagenomic approach revealed Methylobacterium, Sphingomonas and Hymenobacter as dominant genera. The most frequently isolated representatives with cultivable approach were pseudomonads with Pseudomonas syringae and Pseudomonas graminis, the most likely resident species of plum community. Antagonistic Bacillus thuringiensis R3/3 isolate from plum phyllosphere had ability to produce exoenzymes, reduce the growth of phytopathogenic bacteria in co-culture environment and show quorum quenching activity. CONCLUSIONS: Plum cultivar and growth season contribute to the structure of the bacterial community associated with plum. Plum phyllosphere is good source of antagonists effective against phytopathogens. SIGNIFICANCE AND IMPACT OF STUDY: Knowledge of bacterial communities on plum will have an impact on studies related to phyllosphere ecology and biocontrol. The indigenous antagonistic isolate, B. thuringiensis R3/3, from plum could be further investigated for its potential use in biological control of plum diseases.


Assuntos
Antibiose , Bacillus thuringiensis/isolamento & purificação , Bacillus thuringiensis/fisiologia , Doenças das Plantas/microbiologia , Prunus domestica/microbiologia , Bacillus thuringiensis/classificação , Bacillus thuringiensis/genética , Folhas de Planta/microbiologia , Pseudomonas/classificação , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Pseudomonas/fisiologia
15.
Nat Microbiol ; 5(2): 314-330, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31844298

RESUMO

Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling native regulation can turn on nitrogenase expression, even in the presence of nitrogenous fertilizer and low oxygen, but continuous nitrogenase production confers an energy burden. Here, we engineer inducible nitrogenase activity in two cereal endophytes (Azorhizobium caulinodans ORS571 and Rhizobium sp. IRBG74) and the well-characterized plant epiphyte Pseudomonas protegens Pf-5, a maize seed inoculant. For each organism, different strategies were taken to eliminate ammonium repression and place nitrogenase expression under the control of agriculturally relevant signals, including root exudates, biocontrol agents and phytohormones. We demonstrate that R. sp. IRBG74 can be engineered to result in nitrogenase activity under free-living conditions by transferring a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca. For P. protegens Pf-5, the transfer of an inducible cluster from Pseudomonas stutzeri and Azotobacter vinelandii yields ammonium tolerance and higher oxygen tolerance of nitrogenase activity than that from K. oxytoca. Collectively, the data from the transfer of 12 nif gene clusters between 15 diverse species (including Escherichia coli and 12 rhizobia) help identify the barriers that must be overcome to engineer a bacterium to deliver a high nitrogen flux to a cereal crop.


Assuntos
Grão Comestível/metabolismo , Grão Comestível/microbiologia , Fixação de Nitrogênio , Azorhizobium caulinodans/genética , Azorhizobium caulinodans/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Engenharia Metabólica , Família Multigênica , Fixação de Nitrogênio/genética , Nitrogenase/genética , Nitrogenase/metabolismo , Nodulação/genética , Pseudomonas/genética , Pseudomonas/metabolismo , Rhizobium/genética , Rhizobium/metabolismo , Simbiose/genética
16.
J Biotechnol ; 307: 182-192, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31697976

RESUMO

The emergence of antibiotic resistant bacterial strains demands the development of new antimicrobial agents. In the last decades, bacteriocins have gained significant interest due to their potential application as biopreservatives in the food industry and as therapeutic agents in medicine. Recent studies project the use of these antimicrobials in agriculture as biocontrol agents. The characterization of bacteriocins and their genetic regulation, however, have been scarcely studied in plant-associated bacteria. In this report, an in-silico and proteomic analysis was performed to identify the bacteriocins produced by Pseudomonas fluorescens SF4c. More than one functional bacteriocin was detected in this strain (S-type bacteriocins and phage-tail-like bacteriocins [tailocins]). It is known that the regulator PrtR represses bacteriocin production in P. aeruginosa under normal condition. However, the mechanism for tailocin regulation remains unknown in plant-associated pseudomonads. In this work, an orthologue of the prtR of P. aeruginosa was identified in the SF4c-tailocin cluster and a prtR null mutant constructed. The expression and production of tailocins was abolished in this mutant; thus evidencing that, unlike P. aeruginosa, PrtR is a positive regulator of tailocins expression in P. fluorescens.


Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Regiões Promotoras Genéticas/genética , Proteômica , Pseudomonas/metabolismo , Bacteriocinas/genética , Plantas/microbiologia , Pseudomonas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo
17.
Genome Biol Evol ; 11(12): 3529-3533, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31800028

RESUMO

Many of the soil-dwelling Pseudomonas species are known to produce secondary metabolite compounds, which can have antagonistic activity against other microorganisms, including important plant pathogens. It is thus of importance to isolate new strains of Pseudomonas and discover novel or rare gene clusters encoding bioactive products. In an effort to accomplish this, we have isolated a bioactive Pseudomonas strain DTU12.1 from leaf-covered soil in Denmark. Following genome sequencing with Illumina and Oxford Nanopore technologies, we generated a complete genome sequence with the length of 5,943,629 base pairs. The DTU12.1 strain contained a complete gene cluster for a rare thioquinolobactin siderophore, which was previously described as possessing bioactivity against oomycetes and several fungal species. We placed the DTU12.1 strain within Pseudomonas gessardii subgroup of fluorescent pseudomonads, where it formed a distinct clade with other Pseudomonas strains, most of which also contained a complete thioquinolobactin gene cluster. Only two other Pseudomonas strains were found to contain the gene cluster, though they were present in a different phylogenetic clade and were missing a transcriptional regulator of the whole cluster. We show that having the complete genome sequence and establishing phylogenetic relationships with other strains can enable us to start evaluating the distribution and evolutionary origins of secondary metabolite clusters.


Assuntos
Vias Biossintéticas , Pseudomonas/genética , Pseudomonas/metabolismo , Quinolinas/metabolismo , Metabolômica , Filogenia , Pseudomonas/classificação , Pseudomonas/enzimologia , Microbiologia do Solo , Sequenciamento Completo do Genoma
18.
Fish Shellfish Immunol ; 95: 481-490, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31698069

RESUMO

The interactions between host and pathogen is exceedingly complex, which involves alterations at multiple molecular layers. However, research to simultaneously monitor the alterations of transcriptome and proteome between a bacterial pathogen and aquatic animal host through integrated dual RNA-seq and dual iTRAQ of tissue during infection is currently lacking. The important role of a diguanylate cyclase gene (L321_RS15240) in pathogenicity of Pseudomonas plecoglossicida against Epinephelus coioides was suggested by previous dual RNA-seq of our lab. Then L321_RS15240-RNAi strains of P. plecoglossicida were constructed with pCM130/tac, and the mutant with the best silencing effect was selected for follow-up study. The RNAi of L321_RS15240 resulted in a significant decrease in bacterial virulence of P. plecoglossicida. The E. coioides spleens infected by wild type strain or L321_RS15240-RNAi strain of P. plecoglossicida were subjected to dual RNA-seq and dual iTRAQ, respectively. The results showed that: RNAi of L321_RS15240 led to 1)alterations of host transcriptome associated with complement and coagulation cascades, ribosome, arginine and proline metabolism, and oxidative phosphorylation; 2)high expression of host proteins which related to phagosome and metabolism responses (metabolism of glutathione, amino sugar and nucleotide sugar); 3)the highly differentially expression of host lncRNAs and miRNAs. The differentially expressed proteins and mRNAs of pathogen were different after infection, but the functions of these proteins and mRNAs were mainly related to metabolism and virulence. This study provides a new insight to comprehensively understand the gene functions of pathogens and hosts at multiple molecular layers during in vivo infection.


Assuntos
Bass/microbiologia , Proteínas de Escherichia coli/genética , Interações Hospedeiro-Patógeno , Fósforo-Oxigênio Liases/genética , Infecções por Pseudomonas/veterinária , Pseudomonas/enzimologia , Pseudomonas/genética , Animais , Proteínas de Escherichia coli/imunologia , Doenças dos Peixes/microbiologia , Perfilação da Expressão Gênica , Imunidade Inata , Fósforo-Oxigênio Liases/imunologia , Infecções por Pseudomonas/imunologia , Interferência de RNA , RNA-Seq , Transcriptoma , Virulência
19.
Molecules ; 24(22)2019 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-31744250

RESUMO

Pseudomonas species are metabolically robust, with capacity to produce secondary metabolites including cyclic lipopeptides (CLPs). Herein we conducted a chemical analysis of a crude CLP extract from the cocoyam rhizosphere-derived biocontrol strain Pseudomonas sp. COW3. We performed in silico analyses on its whole genome, and conducted in vitro antagonistic assay using the strain and purified CLPs. Via LC-MS and NMR, we elucidated the structures of four novel members of the bananamide group, named bananamides D-G. Besides variability in fatty acid length, bananamides D-G differ from previously described bananamides A-C and MD-0066 by the presence of a serine and aspartic acid at position 6 and 2, respectively. In addition, bananamide G has valine instead of isoleucine at position 8. Kendrick mass defect (KMD) allowed the assignment of molecular formulae to bananamides D and E. We unraveled a non-ribosomal peptide synthetase cluster banA, banB and banC which encodes the novel bananamide derivatives. Furthermore, COW3 displayed antagonistic activity and mycophagy against Pythium myriotylum, while it mainly showed mycophagy on Pyricularia oryzae. Purified bananamides D-G inhibited the growth of P. myriotylum and P. oryzae and caused hyphal distortion. Our study shows the complementarity of chemical analyses and genome mining in the discovery and elucidation of novel CLPs. In addition, structurally diverse bananamides differ in their antimicrobial activity.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Pseudomonas/metabolismo , Cromatografia Líquida de Alta Pressão , Genes Bacterianos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Filogenia , Pseudomonas/classificação , Pseudomonas/genética , Pythium/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
J Basic Microbiol ; 59(12): 1229-1237, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31642093

RESUMO

Arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria inhabit the plant rhizosphere. Both functional groups can influence plant community structures, and interactions between them can vary from being synergistic to antagonistic. HCN-producing Pseudomonas protegens CHA0 is a plant growth-promoting rhizobacterium. P. protegens CHA0 has been shown to weakly attach to AMF hyphae. Here, we analyze the effect of P. protegens CHA0 on the viability of intraradical AMF hyphae. Using pot experiments, we have grown mycorrhizal and nonmycorrhizal Sorghum vulgare var. M35 with P. protegens CHA0 or HCN- mutant P. protegens CHA77, which did not produce HCN. Mycorrhizal and nonmycorrhizal Sorghum grown without CHA0 or CHA77 served as the control. While metabolically active AMF was not detected in mycorrhizal plants grown with HCN+ CHA0, the percentage of root colonization of metabolically active AMF in plants grown with HCN- CHA77 was lower than in the control. Root phosphorus was highest in mycorrhizal plants grown with HCN+ CHA0, but root Fe was higher in plants grown with the bacterial strains. Our results indicate that HCN-producing P. protegens can affect the viability of intraradical AMF.


Assuntos
Glomeromycota/fisiologia , Cianeto de Hidrogênio/metabolismo , Interações Microbianas , Micorrizas/fisiologia , Reguladores de Crescimento de Planta/metabolismo , Pseudomonas/metabolismo , Sorghum/microbiologia , Biomassa , Nutrientes/metabolismo , Reguladores de Crescimento de Planta/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Pseudomonas/genética , Rizosfera , Microbiologia do Solo , Sorghum/metabolismo
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