RESUMO
Soil salinity is one of the major plant growth and yield-limiting constraints in arid and semi-arid regions of the world. In addition to the oxidative damage, increasing salt stress is associated with elevated cellular ethylene levels due to the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) in large amounts. The objective of the current study was to elucidate the inoculation effect of an ACC deaminase (ACCD)-producing phytobeneficial strain Achromobacter sp. FB-14 on rice plants to alleviate the salinity effects by upregulation of the stress-responsive CIPK genes. The strain FB-14 was isolated by using nutrient agar medium at 855 mM NaCl concentration and it was taxonomically identified as Achromobacter sp. with more than 99% 16S rRNA gene sequence similarity with many Achromobacter species. The strain FB-14 demonstrated substantial in vitro potential for ACCD activity, synthesis of indole compounds, and phosphate solubilization up to 100 mM NaCl concentration in the culture medium. The gene corresponding to ACCD activity (acdS) was amplified and sequenced in order to confirm the inherent enzyme activity of the strain at a molecular level. The rifampicin-resistant derivative of strain FB-14 was recovered from the rice rhizosphere on antibiotic medium up to 21 days of sowing. Moreover, the strain FB-14 was inoculated on rice plants under salinity and it not only enhanced the growth of rice plants in terms of root and shoot length, and fresh and dry weight, but also upregulated the expression of stress-responsive CIPK genes (OsCIPK03, OsCIPK12, and OsCIPK15) according to the results of qRT-PCR analysis. To the best of our knowledge, this is the first report deciphering the role of plant-beneficial Achromobacter strain relieving the rice plants from salt stress by promoting the growth and enhancing the expression of stress-responsive CIPK genes.
Assuntos
Achromobacter/enzimologia , Carbono-Carbono Liases/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/microbiologia , Proteínas Serina-Treonina Quinases/genética , Estresse Salino/genética , Achromobacter/genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , RNA Ribossômico 16S/genética , Solo/química , Microbiologia do Solo , Regulação para CimaRESUMO
OBJECTIVES: The aim of this study was to characterise OXA-258 variants and other features that may contribute to carbapenem resistance in Achromobacter ruhlandii. METHODS: Kinetic parameters for purified OXA-258a and OXA-258b were determined measuring the rate of hydrolysis of a representative group of antimicrobial agents. Whole-genome shotgun sequencing was performed on A. ruhlandii 38 (producing OXA-258a) and A. ruhlandii 319 (producing OXA-258b), and in silico analysis of antimicrobial resistance determinants was conducted. Substrates of the AxyABM efflux pump were investigated by inhibition assays using phenylalanine-arginine ß-naphthylamide (PAßN). Outer membrane protein profiles were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Kinetic measurements of purified OXA-258 variants displayed an overall weak catalytic efficiency toward ß-lactams. A detectable hydrolysis of imipenem was observed. In silico genomic analysis confirmed the presence of 32 and 35 putative efflux pump-encoding genes in A. ruhlandii strains 38 and 319, respectively. Complete sequences for AxyABM and AxyXY efflux pumps, previously described in Achromobacter xylosoxidans, were detected. Decreases in the MICs for chloramphenicol, nalidixic acid and trimethoprim/sulfamethoxazole were observed in the presence of the inhibitor PAßN, suggesting that these antibiotics are substrates of AxyABM. AxyXY-encoding genes of A. ruhlandii 38 and A. ruhlandii 319 displayed 99% identity. No differences were observed in the outer membrane protein profiles. CONCLUSIONS: The contribution of OXA-258 enzymes to the final ß-lactam resistance profile may be secondary. Further studies on other putative resistance markers identified in the whole-genome analysis should be conducted to understand the carbapenem resistance observed in A. ruhlandii.
Assuntos
Achromobacter/enzimologia , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Sequenciamento Completo do Genoma/métodos , Resistência beta-Lactâmica , beta-Lactamases/genética , Achromobacter/genética , Antibacterianos/química , Proteínas de Bactérias/genética , Cloranfenicol/química , Cloranfenicol/farmacologia , Simulação por Computador , Variação Genética , Hidrólise , Imipenem/química , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Ácido Nalidíxico/química , Ácido Nalidíxico/farmacologia , Combinação Trimetoprima e Sulfametoxazol/química , Combinação Trimetoprima e Sulfametoxazol/farmacologiaRESUMO
The behavior of three isolates retrieved from different cellulolytic consortia, Bacillus sp. AR03, Paenibacillus sp. AR247 and Achromobacter sp. AR476-2, were examined individually and as co-cultures in order to evaluate their ability to produce extracellular cellulases and xylanases. Utilizing a peptone-based medium supplemented with carboxymethyl cellulose (CMC), an increase estimation of 1.30 and 1.50 times was obtained by the co-culture containing the strains AR03 and AR247, with respect to enzyme titles registered by their individual cultivation. On the contrary, the extracellular enzymatic production decreased during the co-cultivation of strain AR03 with the non-cellulolytic Achromobacter sp. AR476-2. The synergistic behavior observed through the combined cultivation of the strains AR03 and AR247 might be a consequence of the consumption by Paenibacillus sp. AR247 of the products of the CMC hydrolysis (i.e., cellobiose and/or cello-oligosaccharides), which were mostly generated by the cellulase producer Bacillus sp. AR03. The effect observed could be driven by the requirement to fulfill the nutritional supply from both strains on the substrate evaluated. These results would contribute to a better description of the degradation of the cellulose fraction of the plant cell walls in nature, expected to an efficient utilization of renewable sources.
Assuntos
Achromobacter/enzimologia , Bacillus/enzimologia , Celulase/metabolismo , Técnicas de Cocultura/métodos , Xilosidases/metabolismo , Achromobacter/crescimento & desenvolvimento , Achromobacter/metabolismo , Bacillus/crescimento & desenvolvimento , Bacillus/metabolismo , Carboximetilcelulose Sódica/metabolismo , Celobiose/metabolismo , Celulose/metabolismo , Microbiologia Industrial/métodosRESUMO
The accurate species identification of Achromobacter isolates is difficult and the clinical isolates of this genus are mostly referred as A. xylosoxidans. Here, we report new OXA variants in 2 isolates identified as A. insuavis (A114, A79) and 1 isolate identified as A. dolens (A336). These results suggest that different bla OXA genes are ubiquitous in the different species of Achromobacter spp. The role of the other species of Achromobacter in clinical samples needs to be reevaluated, and the proper identification is absolutely necessary to understand the epidemiology of this genus.
Assuntos
Achromobacter denitrificans/enzimologia , Achromobacter/enzimologia , Proteínas de Bactérias/genética , Fibrose Cística/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , beta-Lactamases/genética , Achromobacter/efeitos dos fármacos , Achromobacter/genética , Achromobacter/isolamento & purificação , Achromobacter denitrificans/efeitos dos fármacos , Achromobacter denitrificans/genética , Achromobacter denitrificans/isolamento & purificação , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Humanos , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
A new blaOXA-258 gene is described as a species-specific taxonomic marker for Achromobacter ruhlandii isolates (all recovered from cystic fibrosis patients). Even though OXA-258 differs from OXA-114 variants, isolates could be misidentified as A. xiloxosidans by the amplification of an inner fragment from the OXA-coding gene. A robust identification of A. ruhlandii can be achieved by sequencing this single OXA gene, as well as by a more laborious recently proposed multilocus sequence-typing (MLST) scheme.