Zebrafish (Danio rerio) have a number of strengths as a host model for infection, including genetic tractability, a vertebrate immune system similar to that of mammals, ease and scale of laboratory handling, which allows analysis with reasonable throughput, and transparency, which facilitates visualization of the infection. With these advantages in mind, we examined whether zebrafish could be used to study Pseudomonas aeruginosa pathogenesis and found that infection of zebrafish embryos with live P. aeruginosa (PA14 or PAO1) by microinjection results in embryonic death, unlike infection with Escherichia coli or heat-killed P. aeruginosa, which has no effect. Similar to studies with mice, P. aeruginosa mutants deficient in type three secretion (pscD) or quorum sensing (lasR and mvfR) are attenuated in zebrafish embryos infected at 50 h postfertilization (hpf), a developmental stage when both macrophages and neutrophils are present. In contrast, embryos infected at 28 hpf, when only macrophages are initially present, succumb to lethal challenge with far fewer P. aeruginosa cells than those required for embryos infected at 50 hpf, are susceptible to infection with lasR and pscD deletion mutants, and are moderately resistant to infection with an mvfR mutant. Finally, we show that we can control the outcome of infection through the use of morpholinos, which allow us to shift immune cell numbers, or small molecules (antibiotics), which rescue embryos from lethal challenge. Thus, zebrafish are a novel host model that is well suited for studying the interactions among individual pathogenic functions of P. aeruginosa, the role of individual components of host immune defense, and small-molecule modulators of infection.Pseudomonas aeruginosa, one of the most common causes of nosocomial infections in the United States, typically infects injured, burned, and immunocompromised patients and is the primary cause of mortality among cystic fibrosis patients. It is a ubiquitous, gram-negative bacterium adapted to a variety of niches, including water and soil in associations with other eukaryotic organisms. A number of evolutionarily divergent model hosts have been used to examine P. aeruginosa pathogenesis, including amoebae, plants, nematodes, insects, and rodents (25, 36, 37). While much has been learned about P. aeruginosa pathogenesis from these models, each model has different strengths and weaknesses. Invertebrate model hosts such as Caenorhabditis elegans offer greater genetic tractability than rodent models. Moreover, the size and life cycle of organisms like C. elegans enable experiments such as comprehensive genetic screens that require large numbers of animals, in contrast to rodent models, where such studies are often simply unfeasible due to cost and space requirements. The drawback to modeling human infections in invertebrate hosts is the dissimilarity between vertebrate and invertebrate immune responses. Invertebrate model hosts such as Drosophila melanogaster and C. elegans do not possess adaptive immunity, a true ...

ABSTRACT

The multisubunit RNA polymerase (RNAP) in bacteria consists of a catalytically active core enzyme (␣ 2␤␤ ) complexed with a factor that is required for promoter-specific transcription initiation. During early elongation the stability of interactions between and core decreases, in part because of the nascent RNA-mediated destabilization of an interaction between region 4 of and the flap domain of the ␤-subunit (␤-flap). The nascent RNA-mediated destabilization of the region 4/␤-flap interaction is required for the bacteriophage Q antiterminator protein ( Q) to engage the RNAP holoenzyme. Here, we provide an explanation for this requirement by showing that Q establishes direct contact with the ␤-flap during the engagement process, thus competing with 70 region 4 for access to the ␤-flap. We also show that Q's affinity for the ␤-flap is calibrated to ensure that Q activity is restricted to the late promoter PR . Specifically, we find that strengthening the Q/␤-flap interaction allows Q to bypass the requirement for specific cis-acting sequence elements, a Q-DNA binding site and a RNAP pause-inducing element, that normally ensure Q is recruited exclusively to transcription complexes associated with P R . Our findings demonstrate that the ␤-flap can serve as a direct target for regulators of elongation.sigma ͉ transcription elongation T he bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytically active core enzyme (␣ 2 ␤␤Ј ) in complex with a factor, which confers on the core enzyme the ability to initiate promoter-specific transcription (reviewed in ref. 1). In the context of the RNAP holoenzyme, 70 (the primary factor in Escherichia coli) contacts the conserved Ϫ10 and Ϫ35 promoter elements. All primary factors share four regions of conserved sequence (regions 1-4) (2). Regions 2 and 4 contain DNA-binding domains responsible for recognition of the promoter Ϫ10 element and Ϫ35 element, respectively. Holoenzyme formation depends on an interaction between 70 region 2 and a coiled-coil motif in the ␤Ј-subunit (the ␤Ј coiled coil) (3, 4); this interaction is also required for 70 to make functional contact with the promoter Ϫ10 element (5). Interaction between 70 region 4 and the flap domain of the ␤-subunit (the ␤-flap), although not required for holoenzyme formation, is required for contact with the promoter Ϫ35 element (6). In particular, the 70 region 4/␤-flap interaction properly positions 70 region 4 with respect to 70 region 2, thus enabling 70 region 4 and 70 region 2 to simultaneously contact promoter elements separated by Ϸ17 bp (6).During early elongation the stability of interactions between 70 and core RNAP decreases (reviewed in ref. 7), in part because of the nascent RNA-mediated destabilization of the 70 region 4/␤-flap interaction (8). Specifically, 70 region 4, when bound to the ␤-flap, is positioned within the path of the nascent RNA as it first emerges from the RNA exit channel (the channel through which the nascent RNA is extruded during transcription elongation) (9, 10) at a nascent RN...