We present the genomic sequence of Legionella pneumophila, the bacterial agent of Legionnaires' disease, a potentially fatal pneumonia acquired from aerosolized contaminated fresh water. The genome includes a 45-kilobase pair element that can exist in chromosomal and episomal forms, selective expansions of important gene families, genes for unexpected metabolic pathways, and previously unknown candidate virulence determinants. We highlight the genes that may account for Legionella's ability to survive in protozoa, mammalian macrophages, and inhospitable environmental niches and that may define new therapeutic targets.

We describe a comprehensive quantitative measure of the splicing impact of a complete set of RNA 6-mer sequences by deep sequencing successfully spliced transcripts. All 4096 6-mers were substituted at five positions within two different internal exons in a 3-exon minigene, and millions of successfully spliced transcripts were sequenced after transfection of human cells. The results allowed the assignment of a relative splicing strength score to each mutant molecule. The effect of 6-mers on splicing often depended on their location; much of this context effect could be ascribed to the creation of different overlapping sequences at each site. Taking these overlaps into account, the splicing effect of each 6-mer could be quantified, and 6-mers could be designated as enhancers (ESEseqs) and silencers (ESSseqs), with an ESRseq score indicating their strength. Some 6-mers exhibited positional bias relative to the two splice sites. The distribution and conservation of these ESRseqs in and around human exons supported their classification. Predicted RNA secondary structure effects were also seen: Effective enhancers, silencers and 39 splice sites tend to be single stranded, and effective 59 splice sites tend to be double stranded. 6-mers that may form positive or negative synergy with another were also identified. Chromatin structure may also influence the splicing enhancement observed, as a good correspondence was found between splicing performance and the predicted nucleosome occupancy scores of 6-mers. This approach may prove of general use in defining nucleic acid regulatory motifs, substitute for functional SELEX in most cases, and provide insights about splicing mechanisms.[Supplemental material is available for this article.]The transfer of genetic information from DNA to protein in living things is accomplished with accuracy, precision, and fidelity. These qualities characterize pre-mRNA splicing as much as transcription and translation (Fox-Walsh and Hertel 2009). The accurate identification of splice sites in long metazoan transcripts depends not only on the splice-site sequences that are substrates for the splicing reaction, but also on short RNA stretches known as exonic and intronic splicing enhancers (ESEs and ISEs) and silencers (ESSs and ISSs). These so-called splicing regulatory motifs are manifold and,

During host injury, Pseudomonas aeruginosa can be cued to express a lethal phenotype within the intestinal tract reservoir-a hostile, nutrient scarce environment depleted of inorganic phosphate. Here we determined if phosphate depletion activates a lethal phenotype in P. aeruginosa during intestinal colonization. To test this, we allowed Caenorhabditis elegans to feed on lawns of P. aeruginosa PAO1 grown on high and low phosphate media. Phosphate depletion caused PAO1 to kill 60% of nematodes whereas no worms died on high phosphate media. Unexpectedly, intense redness was observed in digestive tubes of worms before death. Using a combination of transcriptome analyses, mutants, and reporter constructs, we identified 3 global virulence systems that were involved in the ''red death'' response of P. aeruginosa during phosphate depletion; they included phosphate signaling (PhoB), the MvfR-PQS pathway of quorum sensing, and the pyoverdin iron acquisition system. Activation of all 3 systems was required to form a red colored PQS؉Fe 3؉ complex which conferred a lethal phenotype in this model. When pyoverdin production was inhibited in P. aeruginosa by providing excess iron, red death was attenuated in C. elegans and mortality was decreased in mice intestinally inoculated with P. aeruginosa. Introduction of the red colored PQS؉Fe 3؉ complex into the digestive tube of C. elegans or mouse intestine caused mortality associated with epithelial disruption and apoptosis. In summary, red death in C. elegans reveals a triangulated response between PhoB, MvfR-PQS, and pyoverdin in response to phosphate depletion that activates a lethal phenotype in P. aeruginosa.pyoverdin ͉ P. aeruginosa transcriptome ͉ mice ͉ phosphate depletion ͉ PQS/Fe3ϩ/rhamnolipid complex

Legionella pneumophila is the causative agent of the severe and potentially fatal pneumonia Legionnaires' disease. L. pneumophila is able to replicate within macrophages and protozoa by establishing a replicative compartment in a process that requires the Icm/Dot type IVB secretion system. The signals and regulatory pathways required for Legionella infection and intracellular replication are poorly understood. Mutation of the rpoS gene, which encodes S , does not affect growth in rich medium but severely decreases L. pneumophila intracellular multiplication within protozoan hosts. To gain insight into the intracellular multiplication defect of an rpoS mutant, we examined its pattern of gene expression during exponential and postexponential growth. We found that S affects distinct groups of genes that contribute to Legionella intracellular multiplication. We demonstrate that rpoS mutants have a functional Icm/Dot system yet are defective for the expression of many genes encoding Icm/Dot-translocated substrates. We also show that S affects the transcription of the cpxR and pmrA genes, which encode two-component response regulators that directly affect the transcription of Icm/Dot substrates. Our characterization of the L. pneumophila small RNA csrB homologs, rsmY and rsmZ, introduces a link between S and the posttranscriptional regulator CsrA. We analyzed the network of S -controlled genes by mutational analysis of transcriptional regulators affected by S . One of these, encoding the L. pneumophila arginine repressor homolog gene, argR, is required for maximal intracellular growth in amoebae. These data show that S is a key regulator of multiple pathways required for L. pneumophila intracellular multiplication.Legionella pneumophila is a gram-negative opportunistic human pathogen that causes the severe and potentially fatal pneumonia Legionnaires' disease (30,47,67,83). L. pneumophila's ability to replicate within human alveolar macrophages is essential for its capacity to cause disease (44-46). Transmission of L. pneumophila to the human lung occurs as a result of the inhalation of aerosolized contaminated water droplets (74), often from exposure to showers or whirlpool baths (96). Legionella species are ubiquitous in most naturally occurring and man-made aquatic systems, where the organism replicates within a variety of unicellular protozoan hosts (28,38,96). It has been suggested that the interaction of Legionella species with environmental protozoa has selected for the bacterium's evolutionary adaptation to intracellular life in mammalian cells (99).Intracellular multiplication of L. pneumophila requires a series of ordered events that disrupt normal endocytic trafficking in both macrophage and protozoan host cells. These include preventing phagolysosome fusion and the acidification of the Legionella-containing vacuole (LCV), followed by the acquisition of membrane material derived from the Golgi and endoplasmic reticulum compartments of the host (71,85,93,95). These events are dependent upon the Icm/Dot type ...

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a singlemolecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotidebased polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time singlemolecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.single-molecule sequencing | nanopore | DNA sequencing by synthesis | polymer-tagged nucleotides | chip array T he importance of DNA sequencing has increased dramatically from its inception four decades ago. It is recognized as a crucial technology for most areas of biology and medicine and as the underpinning for the new paradigm of personalized and precision medicine. Information on individuals' genomes and epigenomes can help reveal their propensity for disease, clinical prognosis, and response to therapeutics, but routine application of genome sequencing in medicine will require comprehensive data delivered in a timely and cost-effective manner (1). Although 35 years of technological advances have improved sequence throughput and have reduced costs exponentially, genome analysis still takes several days and thousands of dollars to complete (1, 2). To realize the potential of personalized medicine fully, the speed and cost of sequencing must be brought down another order of magnitude while increasing sequencing accuracy and read length. Singlemolecule approaches are thought to be essential to meet these requirements and offer the additional benefit of eliminating amplification bias (3, 4). Although optical methods for singlemolecule sequencing have been achieved and commercialize...