Dataset Information

RNA-seq analysis in simulated microgravity unveils down-regulation of the beta-rhizobial siderophore phymabactin

ABSTRACT: Exploiting the symbiotic interaction between crops and nitrogen-fixing bacteria is a simple and ecological solution to promote plants growth in prospective extraterrestrial human outposts. In this study, we investigated the adaptation of the legume symbiont Paraburkholderia phymatum to simulated microgravity (s0-g) at the transcriptome level by performing an RNA-Seq analysis. The results revealed a drastic effect on gene expression, with roughly 23 % of P. phymatum genes being differentially regulated in s0-g. Among those, 951 genes were upregulated and 858 downregulated in the cells grown in s0-g compared to terrestrial gravity (1g). Genes involved in posttranslational modification, proteins turnover and chaperones production were upregulated in s0-g, while those involved in translation, ribosomal structure and biosynthesis, motility or inorganic ions transport were downregulated. Specifically, the whole phm gene cluster, previously predicted to be involved in the production of a hypothetical siderophore, phymabactin, was approximatively 20-fold downregulated in microgravity. Accordingly, a phm-gfp reporter strain showed less expression in s0-g and iron uptake was reduced in microgravity. By constructing a mutant strain (ΔphmJK) we confirmed that the phm gene cluster codes for the only siderophore secreted by P. phymatum. In fact, in contrast to P. phymatum wild-type, ΔphmJK did not produce any siderophores on chrome azurol S plates. These results not only provide a deeper understanding of the physiology of symbiotic organisms exposed to space-like conditions, but also increase our understanding of iron acquisition in rhizobia.

ORGANISM(S): Paraburkholderia phymatum STM815

PROVIDER: GSE255155 | GEO | 2024/04/10

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including Enterobactin (Ent). However, Ent is bound by the host protein Lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. In two experiments we treated A549 (lung cancer cell line) cells with Lcn2, Ent, and iron, alone and in combination. In experiment 1, biological duplicates of 4 conditions were used: PBS control, Lcn2, Lcn2+Ent, and Lcn2+Ent+iron. In experiment 2, 4 biological replicates of 4 conditions were used: PBS control, Ent, iron, and Ent+iron. Targets made from the samples were hybridized to Affymetrix Human Gene 1.0 ST arrays to measure transcript abundances. The RMA algorithm was used to estimate transcript levels. Replicate samples were exchangeable, so we fit one-way ANOVA models to log2-transformed data separately to each experiment, and tested for pairwise differences between groups in each experiment, as well as asking if the Ent vs. PBS differences were larger or smaller than the Ent+iron vs. iron differences (Ent by iron interactions). We report results for 29096 probe-sets that were not annotated as positive or negative controls on the array. A supplementary Excel workbook is provided that contains the estimated expression level, some probe-set annotation, and simple statistical analysis for each probe-set. It may be convenient for some users, however obtaining newer probe-set annotation may be advisable. A549 (ATCC CCL-185) cells, a human type II pneumocyte cell line, were cultured in F12K (Invitrogen, Carlsbad, CA) media supplemented with 10% fetal bovine serum (Invitrogen) and 1:100 penicillin streptomycin (Invitrogen). 24-well plates were seeded with A549 cells at a concentration of 35000 cells/well. After two days, cells were weaned from serum and antibiotics overnight. Cells were then stimulated overnight with the indicated combinations of 50 uM Ferric ammonium citrate (FAC) (Sigma, St. Louis, MO), 50 um Ent (Sigma or EMC Microcollections, Tubingen, Germany), and/or 25 uM lipocalin 2 (Lcn2) in F12K media lacking serum or antibiotics. Prior to incubation with cells, siderophore-Lcn2 complexes were prepared by sequential incubation at room temperature of FAC and Ent for 30 minutes followed by addition of Lcn2 and incubation for an additional 30 minutes. In experiment 1, 2 biological replicates of each of these 4 conditions were grown: PBS, 25 uM lipocalin 2 (Lcn2), a combination of 25 uM Lcn2 with 50 uM Enterobactin (Ent), and a combination of Lcn2, Ent and 50 uM Ferric Ammonium Citrate (FAC). In experiment 2, 4 biological replicates of each of these 4 conditions were grown: PBS, 50 uM Ent, 50 uM FAC, and a combination of 50 uM Ent and 50 uM FAC. Cells were harvested and RNA was extracted using an miRNeasy Mini Kit (Qiagen). Biotinylated cDNA targets were prepared according to the Ambion WT expression kit protocol from 150 ng total RNA. There are no batches within each experiment - the samples in each group are exchangeable. Targets were hybridized to Affymetrix Human Gene 1.0 ST arrays. Arrays were scanned and quantified by usual procedures, and transcript abundances for 29096 non-control probe-sets estimated by an RMA algorithm.

Dataset Information

RNA-seq analysis in simulated microgravity unveils down-regulation of the beta-rhizobial siderophore phymabactin

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure