RESUMO
A link between the T3SS and inhibition of swimming motility by the transcriptional regulator TtsI in Mesorhizobium japonicum MAFF303099 has been previously reported. Here, we show that mutants in T3SS components display impaired biofilm formation capacity, indicating that a functional T3SS, or at least pili formation, is required for this process. As a first approach to the cdiG regulation network in this bacterium, we started a study of the second messenger cdiG by overexpressing or by deleting some genes encoding cdiG metabolizing enzymes. Overexpression of two putative PDEs as well as deletion of various DGCs led to reduced biofilm formation on glass tubes. Mutation of dgc9509 also affected negatively the nodulation and symbiosis efficiency on Lotus plants, which can be related to the observed reduction in adhesion to plant roots. Results from transcriptional nopX- and ttsI-promoter-lacZ fusions suggested that cdiG negatively regulates T3SS expression in M. japonicum MAFF303099.
Assuntos
Mesorhizobium , Simbiose , Mesorhizobium/genética , Membrana Celular , BiofilmesRESUMO
The specific interaction between rhizobia and legume roots leads to the development of a highly regulated process called nodulation, by which the atmospheric nitrogen is converted into an assimilable plant nutrient. This capacity is the basis for the use of bacterial inoculants for field crop cultivation. Legume plants have acquired tools that allow the entry of compatible bacteria. Likewise, plants can impose sanctions against the maintenance of nodules occupied by rhizobia with low nitrogen-fixing capacity. At the same time, bacteria must overcome different obstacles posed first by the environment and then by the legume. The present review describes the mechanisms involved in the regulation of the entire legume-rhizobium symbiotic process and the strategies and tools of bacteria for reaching the nitrogen-fixing state inside the nodule. Also, we revised different approaches to improve the nodulation process for a better crop yield.
Assuntos
Fabaceae , Rhizobium , Agricultura , Fixação de Nitrogênio , Simbiose , VerdurasRESUMO
Mesorhizobium loti MAFF303099 is a rhizobial strain that nodulates Lotus spp. A M. loti MAFF303099 mutant strain affected in the tatC gene was generated. This strain presented an altered protein secretion level to the culture supernatant and also a higher sensitivity to SDS. Its nodulation phenotype on Lotus showed the induction of small and colorless nodules, and in a larger number than those induced by the wild-type strain. In addition, these nodules presented defects in the degree of occupation by rhizobia. Two Rieske Fe/S proteins, encoded by the mll2707 and mlr0970 genes, were predicted as potential Tat substrates in M. loti MAFF303099. The transcriptional expression of mll2707 and mlr0970 genes was analyzed under different oxygen growth conditions. The mll2707 gene was expressed constitutively, while the expression of the mlr0970 gene was only detected under anaerobic and microaerophilic in vitro conditions. Both genes were down-regulated in the tatC mutant strain. mll2707 and mlr0970 mRNAs from the wild-type strain were detected in nodules. Using a translational reporter peptide fusion, we found that the Mll2707 protein was only detectable in the wild-type strain. On the other hand, although Mlr0970 protein was detected in wild-type and tatC mutant strains, its association with the membrane was favored in the wild-type strain. The tatC and the mll2707 mutant strains were affected in the cytochrome c oxidase activity. These results confirm that Mll2707 is required for cytochrome c-dependent respiration and that Tat functionality is required for the correct activity of Mll2707. The mll2707 mutant strain showed a nodulation phenotype similar to the tatC mutant strain, although it presented only a slight difference in comparison with wild-type strain in terms of nodule occupation. No defective phenotype was observed in the nodulation with the mlr0970 mutant strain. These results indicate that, of the two Rieske Fe/S proteins coded by M. loti MAFF303099, only Mll2707 expression is required for the induction of effective nodules, and that the functionality of the Tat system is necessary not only for the correct function of this protein, but also for some other protein required in an earlier stage of the nodulation process.
RESUMO
To better understand how the composition of bacterial communities changes in response to different environmental conditions, we examined the influence of increasing phenol load on the distribution of the protein-coding functional gene of the largest subunit of phenol hydroxylase (LmPH) and of the 16S rRNA gene in lab-scale activated sludge reactors. LmPH diversity was assessed initially from a total of 124 clone sequences retrieved from two reactors exposed to a low (0.25 g L(-1)) and a high (2.5 g L(-1)) phenol concentration. The quantitative changes in the concentration of the eight detected genotypes accompanied changes in the phenol degradation rates, indicating a community structure-function relationship. Nonmetric dimensional analysis showed that LmPH genotypes and the denaturing gradient gel electrophoresis banding patterns clustered together by phenol concentration, rather than by reactor identity. Seven isolates, representing cultivated strains of each of the observed LmPH genotypes, exhibited a rather narrow range of physiological diversity, in terms of the growth rate and the kinetic parameters of the phenol-degrading activity. We suggest that lab-scale reactors support many ecological niches, which allow the maintenance of a high diversity of ecotypes through varying concentrations of phenol, but the ability of particular strains to become dominant members of the community under the different environmental conditions cannot be predicted easily solely from their phenol-degrading properties.
Assuntos
Bactérias/enzimologia , Reatores Biológicos , Oxigenases de Função Mista/genética , Fenol/metabolismo , Esgotos/microbiologia , Sequência de Aminoácidos , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , DNA Bacteriano/genética , Genótipo , Dados de Sequência Molecular , Consumo de Oxigênio , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
We describe a quantitative analysis of the genetic diversity of phenol-degrading potential in bacterial communities from laboratory-scale activated sludge. Genomic DNA extracted from activated sludge from two sequential batch reactors fed with synthetic sewage plus phenol was amplified using conserved primers for the major subunit of the phenol hydroxylase (LmPH) gene and used to generate clone libraries. Following phylogenetic analysis, 59 sequences containing a 470-bp fragment clustered into six distinct subgroups with a genetic distance of 8%, most likely representing ecologically relevant variants of the enzyme. Seven sets of primers were designed to target the six clusters and used to obtain quantitative information on the dynamics of LmPH gene diversity using real-time PCR assays throughout 9 months of bioreactors operation. Total LmPH gene copy number remained approximately steady in phenol-amended and control reactors. However, a significant increase in phenol-degrading activity in the phenol-amended sludge was accompanied by a parallel increase in LmPH gene diversity, suggesting that phenol degradation in the activated sludge depends on the combined activity of a number of redundant species.