Project description:This research focuses on the design, manufacturing and validation of a new Agrobacterium tumefaciens C58 whole-genome tiling microarray platform for novel RNA transcript discovery. A whole-genome tiling microarray allows both annotated genes as well as previously unknown RNA transcripts to be detected and quantified at once. The Agrobacterium tumefaciens C58 genome is re-acquired with next-generation sequencing and then used to design the tilinlg microarray with the thermodynamic analysis program Picky. Validations are performed by subjecting Agrobacterium tumefaciens C58 under various growth conditions and then using the tling microarrays to verify expected gene expression patterns.
Project description:Comparative genomic hybridizations obtained with an original Agrobacterium tumefaciens strain C58 genome-based micro-array were used to detect the presence or absence of genes homologous to those of strain C58 in 25 agrobacterial strains. These strains included six other members of genomovar G8, one to three strains for each of the nine other A. tumefaciens genomovars and one for A. larrymoorei, a sister species of the A. tumefaciens complex. An original probabilistic method was used to segment C58 replicon sequences into regions, that are absent or present in tested strains, allowing us to detect the presence of homologues of C58 coding sequences (CDSs) in tested strains.
Project description:The genome of Agrobacterium tumefaciens C58 harbors three genes encoding small proteins (<50 aa) with a domain of unknown function DUF1127 (ATU_RS08170, ATU_RS08175, ATU_RS21905). To investigate their function we created a triple-deletion mutant lacking those genes. Total RNA was isolated from A. tumefaciens wild type and from the triple mutant (∆∆∆) at optical densities (OD) of 0.5 and 1.5, and after 24 h of growth (30 °C in YEB medium at 180 rpm) and sequenced on an Illumina platform. For data analysis, 672 small regulatory RNA (sRNA) genes annotated by Wilms et al. (1), Lee et al. (2) and Möller et al. (3) were added to the genome. At OD 0.5 a total of 76 genes showed significantly altered expression in ΔΔΔ. The number increased to 896 and 2,575 genes at OD 1.5 and after 24 h, respectively. 15 genes were upregulated in all three growth phases in ΔΔΔ. At ODs 0.5 and 1.5 numerous genes associated with glycine/serine synthesis were upregulated in the mutant. Additionally, phosphorus starvation and denitrification genes were highly induced at OD 1.5, whereas almost all sugar ABC-transporter genes were downregulated. Overall, the results demonstrate a great impact of DUF1127 proteins on the transcriptome of A. tumefaciens. 1. Wilms I, Overlöper A, Nowrousian M, Sharma CM, Narberhaus F. 2012. Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens. RNA Biol 9:446–57. 2. Lee K, Huang X, Yang C, Lee D, Ho V, Nobuta K, Fan JB, Wang K. 2013. A genome-wide survey of highly expressed non-coding RNAs and biological validation of selected candidates in Agrobacterium tumefaciens. PLoS One 8:e70720. 3. Möller P, Overlöper A, Förstner KU, Wen TN, Sharma CM, Lai EM, Narberhaus F. 2014. Profound impact of Hfq on nutrient acquisition, metabolism and motility in the plant pathogen Agrobacterium tumefaciens. PLoS One 9:e110427.
Project description:The plant pathogen Agrobacterium tumefaciens attaches to and forms biofilms on both biotic and abiotic surfaces. The transition between free-living, planktonic A. tumefaciens and multicellular biofilms is regulated by several well-defined environmental and nutritional inputs, including pH, oxygen tension, and phosphate concentration. In many bacterial species limiting iron levels inhibit attachment and biofilm formation. We demonstrate that A. tumefaciens biofilm formation is reduced under limiting iron conditions. Treatment of A. tumefaciens cultures with EDDHA, an iron-specific extracellular chelator, inhibited both planktonic growth rate and adherent biomass. These effects were reversed upon addition of exogenous ferrous iron. This reduced biofilm formation effect is independent of the known iron-responsive regulators Irr and RirA. Transcriptome analysis comparing gene expression under iron-replete versus iron-deficient conditions identified hundreds of genes that are differentially regulated. Downregulated genes suggest an iron sparing response.
