RESUMO
Three symbiotic nitrogen-fixing bacteria (BD68T, BD66 and BD73) isolated from root nodules of Lotus tenuis in lowland soils of the Flooding Pampa (Argentina), previously classified as members of the Mesorhizobium genus, were characterized in this study. Phylogenetic analysis of their 16S rRNA gene sequences showed a close relationship to M. japonicum MAFF 303099T, M. erdmanii USDA 3471T, M. carmichaelinearum ICMP 18942T, M. opportunistum WSM 2975T and M. jarvisii ATCC 33699T, with sequence identities of 99.72%-100%. Multilocus sequence analysis of other housekeeping genes revealed that the three isolates belonged to a phylogenetically distinct clade within the genus Mesorhizobium. Strain BD68T was designated as the group representative and its genome was fully sequenced. The average nucleotide identity and in silico DNA-DNA hybridization comparisons between BD68T and the most related type strains showed values below the accepted threshold for species discrimination. Phenotypic and chemotaxonomic features were also studied. Based on these results, BD68T, BD66 and BD73 could be considered to represent a novel species of the genus Mesorhizobium, for which the name Mesorhizobium intechi sp. nov. is hereby proposed. The type strain of this species is BD68T (=CECT 9304T=LMG 30179T).
Assuntos
Lotus/microbiologia , Mesorhizobium/classificação , Filogenia , Nódulos Radiculares de Plantas/microbiologia , Argentina , DNA Bacteriano/genética , Ácidos Graxos/análise , Genes Bacterianos/genética , Genes Essenciais/genética , Genoma Bacteriano/genética , Mesorhizobium/química , Mesorhizobium/citologia , Mesorhizobium/fisiologia , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Microbiologia do SoloRESUMO
BACKGROUND: Arctic Mesorhizobium strain N33 was isolated from nodules of the legume Oxytropis arctobia in Canada's eastern Arctic. This symbiotic bacterium can grow at temperatures ranging from 0 to 30 °C, fix nitrogen at 10 °C, and is one of the best known cold-adapted rhizobia. Despite the economic potential of this bacterium for northern regions, the key molecular mechanisms of its cold adaptation remain poorly understood. RESULTS: Using a microarray printed with 5760 Arctic Mesorhizobium genomic clones, we performed a partial transcriptome analysis of strain N33 grown under eight different temperature conditions, including both sustained and transient cold treatments, compared with cells grown at room temperature. Cells treated under constant (4 and 10 °C) low temperatures expressed a prominent number of induced genes distinct from cells treated to short-term cold-exposure (<60 min), but exhibited an intermediate expression profile when exposed to a prolonged cold exposure (240 min). The most prominent up-regulated genes encode proteins involved in metabolite transport, transcription regulation, protein turnover, oxidoreductase activity, cryoprotection (mannitol, polyamines), fatty acid metabolism, and membrane fluidity. The main categories of genes affected in N33 during cold treatment are sugar transport and protein translocation, lipid biosynthesis, and NADH oxidoreductase (quinone) activity. Some genes were significantly down-regulated and classified in secretion, energy production and conversion, amino acid transport, cell motility, cell envelope and outer membrane biogenesis functions. This might suggest growth cessation or reduction, which is an important strategy to adjust cellular function and save energy under cold stress conditions. CONCLUSION: Our analysis revealed a complex series of changes associated with cold exposure adaptation and constant growth at low temperatures. Moreover, it highlighted some of the strategies and different physiological states that Mesorhizobium strain N33 has developed to adapt to the cold environment of the Canadian high Arctic and has revealed candidate genes potentially involved in cold adaptation.
Assuntos
Adaptação Fisiológica/genética , Temperatura Baixa , Perfilação da Expressão Gênica , Mesorhizobium/genética , Mesorhizobium/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Transporte Biológico/genética , Metabolismo dos Carboidratos/genética , Membrana Celular/metabolismo , Análise por Conglomerados , Reparo do DNA/genética , Replicação do DNA/genética , Metabolismo Energético/genética , Genômica , Metabolismo dos Lipídeos/genética , Mesorhizobium/citologia , Mesorhizobium/metabolismo , Chaperonas Moleculares/metabolismo , Anotação de Sequência Molecular , Dados de Sequência Molecular , Fixação de Nitrogênio/genética , Nucleotídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Recombinação Genética/genética , Ribossomos/genética , Ribossomos/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Estresse Fisiológico/genética , Simbiose/genética , Fatores de Transcrição/metabolismoRESUMO
Several molecular chaperones are known to be involved in bacteria stress response. To investigate the role of chaperone ClpB in rhizobia stress tolerance as well as in the rhizobia-plant symbiosis process, the clpB gene from a chickpea microsymbiont, strain Mesorhizobium ciceri LMS-1, was identified and a knockout mutant was obtained. The ClpB knockout mutant was tested to several abiotic stresses, showing that it was unable to grow after a heat shock and it was more sensitive to acid shock than the wild-type strain. A plant-growth assay performed to evaluate the symbiotic performance of the clpB mutant showed a higher proportion of ineffective root nodules obtained with the mutant than with the wild-type strain. Nodulation kinetics analysis showed a 6- to 8-day delay in nodule appearance in plants inoculated with the ΔclpB mutant. Analysis of nodC gene expression showed lower levels of transcript in the ΔclpB mutant strain. Analysis of histological sections of nodules formed by the clpB mutant showed that most of the nodules presented a low number of bacteroids. No differences in the root infection abilities of green fluorescent protein-tagged clpB mutant and wild-type strains were detected. To our knowledge, this is the first study that presents evidence of the involvement of the chaperone ClpB from rhizobia in the symbiotic nodulation process.
