Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 3 de 3
Filter
Add more filters










Database
Language
Publication year range
1.
J Biotechnol ; 329: 80-91, 2021 Mar 10.
Article in English | MEDLINE | ID: mdl-33539896

ABSTRACT

The nitrogen-fixing α-proteobacterium Sinorhizobium meliloti genome codifies at least 50 response regulator (RR) proteins mediating different and, in many cases, unknown processes. RR-mutant library screening allowed us to identify genes potentially implicated in survival to acid conditions. actJ mutation resulted in a strain with reduced growth rate under mildly acidic conditions as well as a lower capacity to tolerate a sudden shift to lethal acidic conditions compared with the parental strain. Mutation of the downstream gene actK, which encodes for a histidine kinase, showed a similar phenotype in acidic environments suggesting a functional two-component system. Interestingly, even though nodulation kinetics, quantity, and macroscopic morphology of Medicago sativa nodules were not affected in actJ and actK mutants, ActK was required to express the wild-type nitrogen fixation phenotype and ActJK was necessary for full bacteroid development and nodule occupancy. The actJK regulatory system presented here provides insights into an evolutionary process in rhizobium adaptation to acidic environments and suggests that actJK-controlled functions are crucial for optimal symbiosis development.


Subject(s)
Sinorhizobium meliloti , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Medicago sativa/metabolism , Nitrogen Fixation , Sinorhizobium meliloti/genetics , Sinorhizobium meliloti/metabolism , Symbiosis/genetics
2.
Sci Rep ; 6: 29278, 2016 07 11.
Article in English | MEDLINE | ID: mdl-27404346

ABSTRACT

Abiotic stresses in general and extracellular acidity in particular disturb and limit nitrogen-fixing symbioses between rhizobia and their host legumes. Except for valuable molecular-biological studies on different rhizobia, no consolidated models have been formulated to describe the central physiologic changes that occur in acid-stressed bacteria. We present here an integrated analysis entailing the main cultural, metabolic, and molecular responses of the model bacterium Sinorhizobium meliloti growing under controlled acid stress in a chemostat. A stepwise extracellular acidification of the culture medium had indicated that S. meliloti stopped growing at ca. pH 6.0-6.1. Under such stress the rhizobia increased the O2 consumption per cell by more than 5-fold. This phenotype, together with an increase in the transcripts for several membrane cytochromes, entails a higher aerobic-respiration rate in the acid-stressed rhizobia. Multivariate analysis of global metabolome data served to unequivocally correlate specific-metabolite profiles with the extracellular pH, showing that at low pH the pentose-phosphate pathway exhibited increases in several transcripts, enzymes, and metabolites. Further analyses should be focused on the time course of the observed changes, its associated intracellular signaling, and on the comparison with the changes that operate during the sub lethal acid-adaptive response (ATR) in rhizobia.


Subject(s)
Cytochromes/metabolism , Fabaceae/microbiology , Hydrogen-Ion Concentration , Rhizobium/physiology , Sinorhizobium meliloti/physiology , Stress, Physiological/physiology , Acids/metabolism , Nitrogen Fixation , Oxygen Consumption , Pentose Phosphate Pathway , Soil , Symbiosis
3.
J Biotechnol ; 155(2): 147-55, 2011 Sep 10.
Article in English | MEDLINE | ID: mdl-21723338

ABSTRACT

RIVET (Recombination Based in vivo Expression Technology) is a powerful genetic tool originally conceived for the identification of genes induced in complex biological niches where conventional transcriptomics is difficult to use. With a broader application, genetic recombination-based technologies have also been used, in combination with regulatory proteins and specific transcriptional regulators, for the development of highly sensitive biosensor systems. RIVET systems generally comprise two modules: a promoter-trap cassette generating genomic transcriptional fusions to the tnpR gene encoding the Tn-γδ TnpR resolvase, and a reporter cassette carrying res-flanked selection markers that are excised upon expression of tnpR to produce an irreversible, inheritable phenotypic change. We report here the construction and validation of a new set of positive-selection RIVET systems that, upon induction of the promoter-trap module, generate the transcriptional activation of an antibiotic-resistant and a green-fluorescent phenotype. Two classes of promoter-trap tools were constructed to generate transcriptional fusions to tnpR: one based on the use of a narrow-host-range plasmid (pRIVET-I), integrative in several Gram-negative bacteria, and the other based on the use of a broad-host-range plasmid (pRIVET-R). The system was evaluated in the model soil bacterium Sinorhizobium meliloti, where a clear-cut phenotypic transition from Nm(R)-Gm(S)-GFP(-) to Nm(S)-Gm(R)-GFP(+) occurred upon expression of tnpR. A S. meliloti integrative RIVET library was constructed in pRIVET-I and, as expected, changes in the extracellular conditions (e.g., salt stress) triggered a significant increase in the appearance of Gm(R)-GFP(+) (excised) clones. The sacB-independent positive-selection RIVET systems here described provide suitable basic tools both for the construction of new recombination-based biosensors and for the search of bacterial markers induced when microorganisms colonize and invade complex environments and eukaryotic hosts.


Subject(s)
Biosensing Techniques/methods , Gene Expression Profiling/methods , Gene Expression Regulation, Bacterial/genetics , Recombinant Fusion Proteins/metabolism , Recombination, Genetic/genetics , Sinorhizobium meliloti/metabolism , Transcriptional Activation/genetics , Drug Resistance, Bacterial/genetics , Escherichia coli , Gene Library , Green Fluorescent Proteins , Plasmids/genetics , Promoter Regions, Genetic/genetics , Sinorhizobium meliloti/genetics , Transposon Resolvases/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...