Resistance to hydrogen peroxide stress in sinorhizobium meliloti [electronic resource]

In nitrogen limiting conditions, leguminous plants can form a symbiosis with nitrogen-fixing bacteria called rhizobia. When this symbiosis is mature, rhizobia reduce, or fix, molecular dinitrogen to ammonia that can be incorporated into plant biomass. In exchange for fixed nitrogen products, legumes provide their rhizobia with the proper, low-oxygen environment for nitrogen fixation by generating a root nodule, as well as provide photosynthate for energy. In order to establish a productive symbiosis, both plant and bacterial partners must produce, and respond to, chemical signals. In the model Medicago truncatula -- Sinorhizobium meliloti symbiosis, the signal exchange begins with flavonoids, like luteolin, produced by the plant, initiating transcription of nod genes in the bacteria. The proteins encoded by these genes produce Nod Factor, a specifically modified lipochitooligosaccharide that induces both morphological and transcriptional changes in the plant root that lead to nodule formation. In addition to Nod Factor, S. meliloti must also produce exopolysaccharides (EPSs) for effective symbiosis formation. The function of EPSs in symbiosis is unknown, however, EPSs in other bacteria can protect against various stresses. S. meliloti are exposed to reactive oxygen species (ROS) in both free-living and symbiotic states. I investigated the role of S. meliloti EPSs in protection against the ROS hydrogen peroxide (H2O2). I found that the levels of EPSs correlate with survival in H2O2, and that purified EPSs can act as antioxidants and decrease H2O2 concentrations in vitro. To identify genes involved in protection against H2O2, I performed whole-genome transcriptional analysis of H2O2 treated and untreated S. meliloti. I found that in higher concentrations of H2O2 EPS biosynthesis genes are upreguated, confirming the role of EPSs in H2O2 protection. Additionally, genes differentially regulated by H2O2 are similarly regulated in response to other stresses, including heat shock and phosphate limitation, as well as nodulation. I also identified genes differentially regulated in response to H2O2 in a mutant lacking a functional oxyR. OxyR is a transcriptional regulator and sensor of the redox status in the cell. Comparing the transcriptional changes in the oxyR- mutant to wild type, I was able to generate a candidate list of OxyR-dependent genes. This work highlights the large transcriptional changes induced by oxidative stress in S. meliloti. It also shows conservation of responses to oxidative stress across bacterial species, and implies that some of the S. meliloti response to other conditions, including nodulation, may be due to oxidative stress. I found that the expression of four alternative sigma factor genes (rpoH1, rpoH2, rpoE2, and rpoE5) was induced by H2O2. To identify their roles in H2O2 response, I utilized deletion mutants of these sigma factors and tested for sensitivity to H2O2. Interestingly, deletion of rpoH1 improves survival in H2O2. This deletion also has higher levels of oxidative stress (as seen using a transcriptional reporter to oxyR, that encodes a redox sensor) and higher total catalase activity prior to addition of H2O2. These results indicate that RpoH1 plays a role in maintaining low levels of oxidative stress in S. meliloti under typical laboratory growth conditions. In total, this work shows that there are varied physiological and transcriptional responses to H2O2 stress in S. meliloti. It presents a role for the symbiotically important EPSs, and presents evidence that the transcriptional response to oxidative stress is part of the transcriptional changes induced by nodulation.

Keywords: Biology.
Advisor: Long Sharon R. primary advisor. Thesis advisor
Grossman Arthur (Arthur R.) advisor. Thesis advisor
Mudgett Mary Beth
Theriot Julie advisor. Thesis advisor