Quorum sensing allows bacteria to sense and respond to changes in population density. Acyl-homoserine lactones serve as quorumsensing signals for many Proteobacteria, and acyl-homoserine lactone signaling is known to control cooperative activities. Quorum-controlled activities vary from one species to another. Quorum-sensing controls a constellation of genes in the opportunistic pathogen Pseudomonas aeruginosa, which thrives in a number of habitats ranging from soil and water to animal hosts. We hypothesized that there would be significant variation in quorumsensing regulons among strains of P. aeruginosa isolated from different habitats and that differences in the quorum-sensing regulons might reveal insights about the ecology of P. aeruginosa. As a test of our hypothesis we used RNA-seq to identify quorumcontrolled genes in seven P. aeruginosa isolates of diverse origins. Although our approach certainly overlooks some quorum-sensingregulated genes we found a shared set of genes, i.e., a core quorum-controlled gene set, and we identified distinct, strain-variable sets of quorum-controlled genes, i.e., accessory genes. Some quorumcontrolled genes in some strains were not present in the genomes of other strains. We detected a correlation between traits encoded by some genes in the strain-variable subsets of the quorum regulons and the ecology of the isolates. These findings indicate a role for quorum sensing in extension of the range of habitats in which a species can thrive. This study also provides a framework for understanding the molecular mechanisms by which quorum-sensing systems operate, the evolutionary pressures by which they are maintained, and their importance in disparate ecological contexts.bacterial communication | systems biology | transcription control B acteria use quorum-sensing signals to communicate with each other and control gene expression in a cell density-dependent manner. Many species of Proteobacteria use diffusible acyl-homoserine lactones (AHLs) as quorum-sensing signals. AHLs are produced by signal synthase enzymes and are detected by signalspecific transcriptional regulators. AHL quorum-sensing circuits regulate a wide spectrum of phenotypes in a diverse array of α-, β-, and γ-Proteobacteria (1). Interspecies differences in quorum regulons often are a reflection of the diverse habitats that bacteria occupy, and quorum-controlled phenotypes often play a crucial role in niche persistence. The classic example is quorum control of luminescence in Vibrio fischeri, which allows this bacterium to discriminate between its free-living, low-populationdensity seawater habitat and its high-density symbiotic habitats, the light organs of certain fish and squid (2, 3). It is well established that there are species-specific differences in quorum regulons, but there is little information regarding the possibility of intraspecies strain-specific differences. We hypothesized that, particularly for versatile species that occupy diverse niches, there might be a shared core of quorum-controlled genes an...

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.