OxyR-Dependent Transcription Response of Sinorhizobium meliloti to Oxidative Stress.

Reactive oxygen species such as peroxides play an important role in plant development, cell wall maturation, and defense responses. During nodulation with the host plant Medicago sativa, Sinorhizobium meliloti cells are exposed to H2O2 in infection threads and developing nodules (R. Santos, D. Hérouart, S. Sigaud, D. Touati, and A. Puppo, Mol Plant Microbe Interact 14:86-89, 2001, https://doi.org/10.1094/MPMI.2001.14.1.86). S. meliloti cells likely also experience oxidative stress, from both internal and external sources, during life in the soil. Here, we present microarray transcription data for S. meliloti wild-type cells compared to a mutant deficient in the key oxidative regulatory protein OxyR, each in response to H2O2 treatment. Several alternative sigma factor genes are upregulated in the response to H2O2; the stress sigma gene rpoE2 shows OxyR-dependent induction by H2O2, while rpoH1 expression is induced by H2O2 irrespective of the oxyR genotype. The activity of the RpoE2 sigma factor in turn causes increased expression of two more sigma factor genes, rpoE5 and rpoH2 Strains with deletions of rpoH1 showed improved survival in H2O2 as well as increased levels of oxyR and total catalase expression. These results imply that ΔrpoH1 strains are primed to deal with oxidative stress. This work presents a global view of S. meliloti gene expression changes, and of regulation of those changes, in response to H2O2IMPORTANCE Like all aerobic organisms, the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti experiences oxidative stress throughout its complex life cycle. This report describes the global transcriptional changes that S. meliloti makes in response to H2O2 and the roles of the OxyR transcriptional regulator and the RpoH1 sigma factor in regulating those changes. By understanding the complex regulatory response of S. meliloti to oxidative stress, we may further understand the role that reactive oxygen species play as both stressors and potential signals during symbiosis.

Preparation of Low-Input and Ligation-Free ChIP-seq Libraries Using Template-Switching Technology.

Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low-abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP-seq workflow that can be applied to small cell numbers, including a robust single-tube and ligation-free method for preparation of sequencing libraries from sub-nanogram amounts of ChIP DNA. An example ChIP protocol is first presented, resulting in selective enrichment of DNA-binding proteins and cross-linked DNA fragments immobilized on beads via an antibody bridge. This is followed by a protocol for fast and easy cross-linking reversal and DNA recovery. Finally, we describe a fast, ligation-free library preparation protocol, featuring DNA SMART technology, resulting in samples ready for Illumina sequencing. © 2016 by John Wiley & Sons, Inc.

Exopolysaccharides from Sinorhizobium meliloti can protect against H2O2-dependent damage.

Sinorhizobium meliloti requires exopolysaccharides in order to form a successful nitrogen-fixing symbiosis with Medicago species. Additionally, during early stages of symbiosis, S. meliloti is presented with an oxidative burst that must be overcome. Levels of production of the exopolysaccharides succinoglycan (EPS-I) and galactoglucan (EPS-II) were found to correlate positively with survival in hydrogen peroxide (H2O2). H2O2 damage is dependent on the presence of iron and is mitigated when EPS-I and EPS-II mutants are cocultured with cells expressing either exopolysaccharide. Purified EPS-I is able to decrease in vitro levels of H2O2, and this activity is specific to the symbiotically active low-molecular-weight form of EPS-I. This suggests a potential protective function of exopolysaccharides against H2O2 during early symbiosis.

Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response.

Horizontal gene transfer contributes to the evolution of bacterial species. Mobile genetic elements play an important role in horizontal gene transfer, and characterization of the regulation of these elements should provide insight into conditions that influence bacterial evolution. We characterized a mobile genetic element, ICEBs1, in the Gram-positive bacterium Bacillus subtilis and found that it is a functional integrative and conjugative element (ICE) capable of transferring to Bacillus and Listeria species. We identified two conditions that promote ICEBs1 transfer: conditions that induce the global DNA damage response and crowding by potential recipients that lack ICEBs1. Transfer of ICEBs1 into cells that already contain the element is inhibited by an intercellular signaling peptide encoded by ICEBs1. The dual regulation of ICEBs1 allows for passive propagation in the host cell until either the potential mating partners lacking ICEBs1 are present or the host cell is in distress.