Project description:Gluconacetobacter diazotrophicus has been the focus of several studies aiming to understand the mechanisms behind this endophytic diazotrophic bacterium. The present study is the first global analysis of the early transcriptional response of exponentially growing G. diazotrophicus to iron, an essential cofactor for many enzymes involved in various metabolic pathways. RNA-seq, targeted gene mutagenesis and computational motif discovery tools were used to define the G. diazotrophicus Fur regulon. The data analysis showed that genes encoding functions related to iron homeostasis, were significantly upregulated in response to iron limitation. Certain genes involved in the secondary metabolism were overexpressed under iron-limited conditions. In contrast, it was observed that the expression of genes involved in Fe-S cluster biosynthesis, flagellar biosynthesis and type IV secretion systems were downregulated in an iron-depleted culture medium. Our results support a model that control transcription in G. diazotrophicus by Fur function. The G. diazotrophicus Fur protein was able to complement an E. coli fur mutant. These results provide new insights into the effects of iron on the metabolism of G. diazotrophicus, as well as demonstrating the essentiality of this micronutrient for the main characteristic of plant growth promotion by G. diazotrophicus.
Project description:Heavy metal accumulation in agricultural areas is a global environmental problem that affects microorganisms and plants, with serious implications for human health. This study aimed to investigate the molecular responses of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus PAL5 to cobalt stress. We evaluated bacterial growth and cell viability under cobalt stress and performed comparative proteomic and reverse genetics analyses. Cobalt significantly inhibited bacterial growth but did not cause cell death. Proteomic analysis in the presence of 2.5 mM CoCl2, which caused approximately 50% growth inhibition, revealed the induction of pathways related to iron uptake, carbohydrate metabolism, amino acid metabolism, quality control, and efflux. Knockout mutants for genes involved in these pathways (∆tbdR, ∆zwf, ∆pdhB, ∆argH and ∆czcC) confirmed the essential role of the CzcC efflux system in cobalt tolerance. Cobalt stress triggers molecular responses in G. diazotrophicus PAL5, with efflux systems playing a crucial role in stress tolerance.
Project description:Investigation of whole genome gene expression level changes in a Gluconacetobacter xylinus NBRC 3288 delta-fnrG mutant, compared to the wild-type strain.
Project description:The biosynthesis of exopolysaccharides (EPSs) is essential for endophytic bacterial colonisation in plants bacause this exopolymer both protects bacterial cells against the defence and oxidative systems of plants and acts on the plant colonisation mechanism in Gluconacetobacter diazotrophicus. The pathway involved in the biosynthesis of bacterial EPS has not been fully elucidated, and several areas related to its molecular regulation mechanisms are still lacking. G. diazotrophicus relies heavily on EPS for survival indirectly by protecting plants from pathogen attack as well as for endophytic maintenance and adhesion in plant tissues. Here, we report that EPS from G. diazotrophicus strain Pal5 is a signal polymer that controls its own biosynthesis. EPS production depends on a bacterial tyrosine (BY) kinase (Wzc) that consists of a component that is able to phosphorylate a glycosyltranferase or to self-phosphorylate. EPS interacts with the extracellular domain of Wzc, which regulates kinase activity. In G. diazotrophicus strains that are deficient in EPS production, the Wzc is rendered inoperative by self-phosphorylation. The presence of EPS promotes the phosphorylation of a glycosyltransferase in the pathway, thus producing EPS. Wzc-mediated self-regulation is an attribute for the control of exopolysaccharide biosynthesis in G. diazotrophicus.
Project description:Plant growth-promoting (PGP) bacteria are important to the development of sustainable agricultural systems. PGP microbes that fix atmospheric nitrogen (diazotrophs) could minimize the application of industrially derived fertilizers and function as a biofertilizer. The bacterium Gluconacetobacter diazotrophicus is a nitrogen-fixing PGP microbe originally discovered in association with sugarcane plants, where it functions as an endophyte. It also forms endophyte associations with a range of other agriculturally relevant crop plants. G. diazotrophicus requires microaerobic conditions for diazotrophic growth. We generated a transposon library for G. diazotrophicus and cultured the library under various growth conditions and culture medium compositions to measure fitness defects associated with individual transposon inserts (transposon insertion sequencing [Tn-seq]). Using this library, we probed more than 3,200 genes and ascertained the importance of various genes for diazotrophic growth of this microaerobic endophyte. We also identified a set of essential genes. IMPORTANCE Our results demonstrate a succinct set of genes involved in diazotrophic growth for G. diazotrophicus, with a lower degree of redundancy than what is found in other model diazotrophs. The results will serve as a valuable resource for those interested in biological nitrogen fixation and will establish a baseline data set for plant free growth, which could complement future studies related to the endophyte relationship.
Project description:Seawater exposure to the gram negative marine bacterium Vibrio diazotrophicus induces a robust cellular response in sea urchin larvae that includes the migration of pigment cells to the gut epithelium, changes in cell behavior and altered gut morphology (Ho et al., 2016; PMID 27192936). To investigate the transcriptional underpinnings of this response, whole transcriptome sequencing was performed on mRNA isolated from larval samples collected at 0, 6, 12 and 24 hr of exposure to V. diazotrophicus. The morphological simplicity of the sea urchin larva provides a systems-level model for identifying biologically relevant transcriptional state changes in response to dysbiosis in the gut lumen.