The photosynthetic alphaproteobacterium Rhodospirillum rubrum S1H is part of the Micro-Ecological Life Support System Alternative (MELiSSA) project that is aiming to develop a closed life support system for oxygen, water and food production to support human life in space in forthcoming long-term space exploration missions. In the present study, R. rubrum S1H was cultured in a rotating wall vessel (RWV), simulating partial microgravity conditions on Earth. The bacterium showed a significant response to cultivation in simulated microgravity at the transcriptomic, proteomic and metabolic levels. In simulated microgravity conditions three N-acyl-l-homoserine lactones (C10-HSL, C12-HSL and 3-OH-C14-HSL) were detected in concentrations that were twice those detected under normal gravity, while no differences in cell density was detected. In addition, R. rubrum cultivated in modelled microgravity showed higher pigmentation than the normal gravity control, without change in culture oxygenation. When compared to randomized microgravity cultivation using a random positioning machine, significant overlap for the top differentially expressed genes and proteins was observed. Cultivation in this new artificial environment of simulated microgravity showed new properties of this well-known bacterium, including its first, to our knowledge, complete quorum-sensing-related N-acylhomoserine lactone profile.
NADP-dependent glutamate dehydrogenase (NADP-GDH) is a key enzyme in the assimilation of alternative nitrogen nutrient sources through ammonium in fungi. In Aspergillus nidulans, NADP-GDH is encoded by gdhA. Several transcription factors are known to regulate gdhA expression, including AreA, the major transcription activator of nitrogen metabolic genes, and TamA, a co-activator of AreA. TamA also interacts with LeuB, the regulator of leucine biosynthesis. We have investigated the effects of leucine biosynthesis on gdhA regulation, and found that leucine regulates the levels of NADP-GDH activity and gdhA expression. We show, using mutants with perturbed levels of α-isopropylmalate (α-IPM), that this leucine biosynthesis intermediate affects gdhA regulation. Leucine regulation of gdhA requires a functional LeuB with an intact Zn(II)2Cys6 DNA-binding domain. By analysing the prevalence of putative LeuB DNA-binding sites in promoters of gdhA orthologues we predict broad conservation of leucine regulation of NADP-GDH expression within ascomycetes except in the fusaria and fission yeasts. Using promoter mutations in gdhA–lacZ reporter genes we identified two sites of action for LeuB within the A. nidulans gdhA promoter. These two sites lack sequence identity, with one site conforming to the predicted LeuB DNA-binding site consensus motif, whereas the second site is a novel regulatory sequence element conserved in Aspergillus gdhA promoters. These data suggest that LeuB regulates NADP-GDH expression in response to leucine levels, which may act as an important sensor of nitrogen availability.
Expression of DNA transfer (tra) genes of F-type conjugative plasmids is required for the assembly of a functional type IV secretion machinery and subsequent plasmid DNA transfer from donor to recipient cells. Transcription of tra genes depends on the activation of a single promoter, designated PY, by the plasmid encoded TraJ protein. We here determine plasmid specificity of TraJ proteins from various subgroups of F-like plasmids and find that plasmid R1 conjugation and PY promoter activation can be achieved only by its cognate activator and by TraJ of the Salmonella plasmid pSLT and not by F or R100 TraJ proteins. In addition, we characterize the PY promoter of plasmid R1. We show that TraJ binds to PY DNA in vivo and that H-NS acts as a silencer of the PY promoter. In the natural plasmid context, H-NS silences transfer gene expression and horizontal plasmid DNA transfer. In contrast to what was found for the F plasmid, lack of H-NS did not abolish the requirement for ArcA and TraJ to reach full tra gene expression and DNA transfer activity. We propose that, besides a passive de-silencing activity, both ArcA and TraJ play a direct role in synergistically stimulating tra operon transcription and subsequent DNA transfer.
Methylisocitrate lyase (MCL), a signature enzyme of the methylcitrate cycle, which cleaves methylisocitrate to pyruvate and succinate, is required for propionate metabolism, for secondary metabolite production and for virulence in bacteria and fungi. Here we investigate the role of the methylcitrate cycle by generating an mcl deletion mutant in the fungal biocontrol agent Trichoderma atroviride. Gene expression analysis shows that a basal expression of mcl is observed in all growth conditions tested. Phenotypic analysis of an mcl deletion mutant suggests the requirement of MCL in propionate resistance, growth, conidial pigmentation and germination, and abiotic stress tolerance. A plate confrontation assay did not show a difference between the WT and the Δmcl strain in antagonism towards Botrytis cinerea. However, the Δmcl strain displays reduced antagonism towards B. cinerea based on a secretion assay. Furthermore, an in vitro root colonization assay shows that the Δmcl strain had reduced ability to colonize Arabidopsis thaliana roots, which results in reduced induction of systemic resistance towards B. cinerea. These data show that MCL is important not only for growth and development in T. atroviride but also in antagonism, root colonization and induction of defence responses in plants.