We are interested in the root microbiome of the fast-growing Eastern cottonwood tree, Populus deltoides. There is a large bank of bacterial isolates from P. deltoides, and there are 44 draft genomes of bacterial endophyte and rhizosphere isolates. As a first step in efforts to understand the roles of bacterial communication and plant-bacterial signaling in P. deltoides, we focused on the prevalence of acyl-homoserine lactone (AHL) quorum-sensing-signal production and reception in members of the P. deltoides microbiome. We screened 129 bacterial isolates for AHL production using a broad-spectrum bioassay that responds to many but not all AHLs, and we queried the available genome sequences of microbiome isolates for homologs of AHL synthase and receptor genes. AHL signal production was detected in 40% of 129 strains tested. Positive isolates included members of the Alpha-, Beta-, and Gammaproteobacteria. Members of the luxI family of AHL synthases were identified in 18 of 39 proteobacterial genomes, including genomes of some isolates that tested negative in the bioassay. Members of the luxR family of transcription factors, which includes AHL-responsive factors, were more abundant than luxI homologs. There were 72 in the 39 proteobacterial genomes. Some of the luxR homologs appear to be members of a subfamily of LuxRs that respond to as-yet-unknown plant signals rather than bacterial AHLs. Apparently, there is a substantial capacity for AHL cell-to-cell communication in proteobacteria of the P. deltoides microbiota, and there are also Proteobacteria with LuxR homologs of the type hypothesized to respond to plant signals or cues.

The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorumsensing signal p-coumaroyl-homoserine lactone (pC-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by pC-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His 6 -RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although pC-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is pC-HSL-RpaR-activated antisense transcription of rpaR. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon.Many bacteria control subsets of genes in a cell densitydependent manner. This coordinated group behavior is known as quorum sensing and response. More than 100 species of Proteobacteria contain acyl-homoserine lactone (acyl-HSL) quorum-sensing (QS) circuits (12, 45). Acyl-HSLs can diffuse into and out of cells, and once a threshold concentration is reached, acyl-HSLs bind specific transcriptional regulators that control target genes. A variety of genes are controlled by QS depending on the bacterial species, including protease genes, conjugal transfer genes, antibiotic synthesis genes, and bioluminescence genes (12, 45). Many QS-regulated gene products are "public goods," exoproducts that can be shared by all of the individuals in a group.Two types of genes are involved in most acyl-HSL-type QS systems: luxI-and luxR-type genes. LuxI proteins are QS signal synthases that catalyze amide bond formation between an acyl group on an appropriate side chain donor (most often acyl-acyl carrier protein) and S-adenosylmethionine (SAM) resulting in the final acyl-HSL product (22,26,34,35). For fatty acyl-HSLs, signal specificity is conferred by the length (4 to 18 carbons) and side chain modifications of the fatty acyl group. LuxR homologs are homodimeric transcription factors, with each monomer consisting of two domains: an N-terminal acyl-HSL binding domain and a C-terminal DNA-binding domain that contains a helix-turn-helix motif (5,6,13,50). Genes controlled by LuxR homologs often have specific inverted repeat DNA sequences in their promoter regions. These elements are known as lux box-like sequences. The lux box is a 20-bp palindromic sequence centered at bp Ϫ42.5 from the transcription start of the Vibrio fischeri lux operon, which encodes...

High-throughput sequencing of cDNA prepared from RNA, an approach known as RNA-seq, is coming into increasing use as a method for transcriptome analysis. Despite its many advantages, widespread adoption of the technique has been hampered by a lack of easy-to-use, integrated, open-source tools for analyzing the nucleotide sequence data that are generated. Here we describe Xpression, an integrated tool for processing prokaryotic RNA-seq data. The tool is easy to use and is fully automated. It performs all essential processing tasks, including nucleotide sequence extraction, alignment, quantification, normalization, and visualization. Importantly, Xpression processes multiplexed and strand-specific nucleotide sequence data. It extracts and trims specific sequences from files and separately quantifies sense and antisense reads in the final results. Outputs from the tool can also be conveniently used in downstream analysis. In this paper, we show the utility of Xpression to process strand-specific RNA-seq data to identify genes regulated by CouR, a transcription factor that controls p-coumarate degradation by the bacterium Rhodopseudomonas palustris. RNA-seq is a recently developed technique for global analysis of mRNA transcripts that involves the use of high-throughput sequencing technology (18). It has a number of advantages over traditional microarray-based technologies, including improved sensitivity, increased dynamic range, and lower cost. As a result, it is becoming the preferred tool for gene expression studies. Despite many advantages, widespread adoption of RNA-seq is impeded by a lack of easy-to-use, integrated, open-source tools for processing of the nucleotide sequence data that are generated as the output of the technique. Millions of raw sequence reads are generated for each RNA-seq experiment, making it impossible to process the sequencing data without bioinformatic tools.