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.conjugation ͉ horizontal gene transfer ͉ quorum sensing ͉ peptide signaling ͉ DNA microarrays

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 (H 2 O 2 ). H 2 O 2 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 H 2 O 2 , 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 H 2 O 2 during early symbiosis.

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 , cells are exposed to HO 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). cells likely also experience oxidative stress, from both internal and external sources, during life in the soil. Here, we present microarray transcription data for wild-type cells compared to a mutant deficient in the key oxidative regulatory protein OxyR, each in response to HO treatment. Several alternative sigma factor genes are upregulated in the response to HO; the stress sigma gene shows OxyR-dependent induction by HO, while expression is induced by HO irrespective of the genotype. The activity of the RpoE2 sigma factor in turn causes increased expression of two more sigma factor genes, and Strains with deletions of showed improved survival in HO as well as increased levels of and total catalase expression. These results imply that Δ strains are primed to deal with oxidative stress. This work presents a global view of gene expression changes, and of regulation of those changes, in response to HO Like all aerobic organisms, the symbiotic nitrogen-fixing bacterium experiences oxidative stress throughout its complex life cycle. This report describes the global transcriptional changes that makes in response to HO and the roles of the OxyR transcriptional regulator and the RpoH1 sigma factor in regulating those changes. By understanding the complex regulatory response of to oxidative stress, we may further understand the role that reactive oxygen species play as both stressors and potential signals during symbiosis.

Next-generation sequencing is empowering a deeper understanding of biology by enabling RNA expression analysis over the entire transcriptome with high sensitivity and a wide dynamic range. One powerful application within this field is stranded RNA sequencing (RNA-seq), which is necessary to distinguish overlapping genes and to conduct comprehensive annotation and quantification of long non-coding RNAs. Commonly used methods for generating strand-specific RNA-seq libraries are often complicated by protocols that require several rounds of enzymatic treatments and clean-up steps, making them time-intensive, insensitive, and unsuitable for processing several samples simultaneously. An additional challenge in the generation of RNA-seq libraries from total RNA involves the high amount of ribosomal RNA (rRNA) in the starting material. This unit presents streamlined workflows for generating strand-specific RNA-seq libraries from 10 ng to 1 µg total RNA, representing a minimum of 1000 cells, in less than 7 hr with minimal carryover rRNA. These methods allow scientists to evaluate the expression of all transcripts, including non-polyadenylated long non-coding RNAs, even in limited biological samples. Combination of the RNase H-based RiboGone rRNA removal system and SMARTer Stranded RNA-seq technology enables depletion of over 95% of rRNA from mammalian samples, and direct production of Illumina-ready libraries that maintain strand-of-origin information. An alternate method for low input of highly degraded samples is also presented. © 2016 by John Wiley & Sons, Inc.

RNA sequencing (RNA-seq) is a powerful method for analyzing cell state, with minimal bias, and has broad applications within the biological sciences. However, transcriptome analysis of seemingly homogenous cell populations may in fact overlook significant heterogeneity that can be uncovered at the single-cell level. The ultra-low amount of RNA contained in a single cell requires extraordinarily sensitive and reproducible transcriptome analysis methods. As next-generation sequencing (NGS) technologies mature, transcriptome profiling by RNA-seq is increasingly being used to decipher the molecular signature of individual cells. This unit describes an ultra-sensitive and reproducible protocol to generate cDNA and sequencing libraries directly from single cells or RNA inputs ranging from 10 pg to 10 ng. Important considerations for working with minute RNA inputs are given. © 2016 by John Wiley & Sons, Inc.