Browsing by Author "Moreira, Leonilde M."
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- Absence of functional TolC protein causes increased stress response gene expression in Sinorhizobium melilotiPublication . Santos, Mario R.; Cosme, Ana M.; Becker, Jorg D.; Medeiros, Joao M C.; Mata, Marcia F.; Moreira, Leonilde M.The TolC protein from Sinorhizobium meliloti has previously been demonstrated to be required for establishing successful biological nitrogen fixation symbiosis with Medicago sativa. It is also needed in protein and exopolysaccharide secretion and for protection against osmotic and oxidative stresses. Here, the transcriptional profile of free-living S. meliloti 1021 tolC mutant is described as a step toward understanding its role in the physiology of the cell.
- The regulator LdhR and the d-lactate dehydrogenase LdhA of Burkholderia multivorans play a role in carbon overflow and in planktonic cellular aggregates formationPublication . Silva, Inês N.; Ramires, Marcelo J.; Azevedo, Lisa A.; Guerreiro, Ana R.; Tavares, Andreia C.; Becker, Jörg D.; Moreira, Leonilde M.LysR-type transcriptional regulators (LTTR) are the most commonly found regulators in Burkholderia cepacia complex, comprising opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. Despite LTTRs being global regulators of pathogenicity in several bacteria, few have been characterized in Burkholderia Here, we showed that gene ldhR of B. multivorans encoding a LTTR is co-transcribed with ldhA encoding a d-lactate dehydrogenase, and evaluate their implication in virulence traits like exopolysaccharide (EPS) synthesis and biofilm formation. Comparison of wild-type (WT) and its isogenic ΔldhR mutant grown in medium with 2% d-glucose revealed a negative impact on EPS biosynthesis and on cells' viability in the presence of LdhR. Loss of viability in WT cells was caused by intracellular acidification as consequence of cumulative organic acids secretion including d-lactate, this last one absent from the ΔldhR mutant supernatant. Furthermore, LdhR is implicated in the formation of planktonic cellular aggregates. WT cell aggregates reached 1000 μm after 24 hours in liquid cultures; in contrast to ΔldhR mutant aggregates that never grew more than 60 μm. Overexpression of d-lactate dehydrogenase LdhA in the ΔldhR mutant partially restored formed aggregates size, suggesting a role for fermentation inside aggregates. Similar results were obtained for surface-attached biofilms, with WT cells producing more biofilm. A systematic evaluation of planktonic aggregates in Burkholderia CF clinical isolates showed aggregates in 40 out of 74. As CF patients' lung environment is microaerophilic and bacteria are found as free aggregates/biofilms, LdhR and LdhA might have central roles in adaptation to this environment.IMPORTANCE Cystic fibrosis patients often suffer from chronic respiratory infections caused by several microorganisms. Among them are the Burkholderia cepacia complex bacteria which cause progressive deterioration of lung function and, in some patients, might develop into fatal necrotizing pneumoniae with bacteremia, known as "cepacia syndrome". Burkholderia pathogenesis is multifactorial since they express several virulence factors, form biofilms, and are highly resistant to antimicrobial compounds, making their eradication from the CF patients' airways very difficult. As Burkholderia is commonly found in the CF lungs in the form of cell aggregates and biofilms, the need to investigate the mechanisms of cellular aggregation is obvious. In this study we demonstrate the importance of a d-lactate dehydrogenase and a regulator, in regulating carbon overflow, cellular aggregates and surface-attached biofilm formation. This not only enhances our understanding of Burkholderia pathogenesis, but can also lead to the development of drugs against these proteins to circumvent biofilm formation.
- The Sinorhizobium meliloti EmrR Regulator Is Required for Efficient Colonization of Medicago sativa Root NodulesPublication . Santos, Mário R.; Marques, Andreia T.; Becker, Jörg D.; Moreira, Leonilde M.The nitrogen-fixing bacterium Sinorhizobium meliloti must adapt to diverse conditions encountered during its symbiosis with leguminous plants. We characterized a new symbiotically relevant gene, emrR (SMc03169), whose product belongs to the TetR family of repressors and is divergently transcribed from emrAB genes encoding a putative major facilitator superfamily-type efflux pump. An emrR deletion mutant produced more succinoglycan, displayed increased cell-wall permeability, and exhibited higher tolerance to heat shock. It also showed lower tolerance to acidic conditions, a reduced production of siderophores, and lower motility and biofilm formation. The simultaneous deletion of emrA and emrR genes restored the mentioned traits to the wild-type phenotype, except for survival under heat shock, which was lower than that displayed by the wild-type strain. Furthermore, the ΔemrR mutant as well as the double ΔemrAR mutant was impaired in symbiosis with Medicago sativa; it formed fewer nodules and competed poorly with the wild-type strain for nodule colonization. Expression profiling of the ΔemrR mutant showed decreased expression of genes involved in Nod-factor and rhizobactin biosynthesis and in stress responses. Expression of genes directing the biosynthesis of succinoglycan and other polysaccharides were increased. EmrR may therefore be involved in a regulatory network targeting membrane and cell wall modifications in preparation for colonization of root hairs during symbiosis.