We report the construction of a series of Escherichia-Pseudomonas broad-host-range expression vectors utilizing the P BAD promoter and the araC regulator for routine cloning, conditional expression, and analysis of tightly controlled and/or toxic genes in pseudomonads.Gene cloning, disruption, deletion, complementation analysis, and allelic exchange are central to prokaryotic molecular genetics. In Pseudomonas aeruginosa, Schweizer and colleagues developed the pUCP family of general-purpose vectors for cloning and gene expression (24, 29) based on the well-characterized pUC18/19 vectors (32) and the cryptic mini-plasmid pRO1614 (19). Other promoters are also in routine use, such as the tac (4, 6), T7 (28), and araBAD promoter-based (8, 11) vectors for regulated expression in Escherichia coli and many other bacterial species (e.g., see references 2, 18, and 25). In E. coli, AraC represses the araBAD promoter (P BAD ) and the expression of a cloned gene is induced by the addition of L-arabinose. Pseudomonas researchers have used the inducible properties of the araC regulator and the P BAD promoter cassette for the controlled gene expression by integrating the araC-P BAD -specific transcription fusion into the chromosome by using a suicide vector or an integration-proficient vector (1,3,13,17,30,31). In the present study, we modified the existing EscherichiaPseudomonas shuttle vectors pUCP20T, -26, -28T, and -30T by replacing the lac promoter with the araC-P BAD cassette to allow conditional expression in pseudomonads and other bacteria, e.g., Burkholderia spp.Construction and features of pHERD vectors. Functional genetic analysis requires vectors capable of conditional expression. The P BAD promoter has been used for gene expression extensively in E. coli and some in P. aeruginosa and Burkholderia spp. (12,27,31). We first constructed three shuttle vectors, pHERD20T, -28T, and -30T (Fig. 1), based on EscherichiaPseudomonas shuttle vectors pUCP20T, pUCP28T, and pUCP30T (29) and the commercial expression vector pBAD/ Thio-TOPO (Invitrogen). The 368-bp fragment of the pUCP vectors spanning two restriction sites, AflII and EcoRI, was replaced with the araC-P BAD fragment (1.3 kb), produced via PCR with pBAD/Thio-TOPO as the template and primers pBAD-F and pBAD-R (Table 1). The PCR product was purified and directly digested with AflII and EcoRI, and the two fragments were ligated into the pUCP vectors, creating pHERD20T (Fig. 1). The EcoRI/AflII regions of these vectors were sequenced to confirm that no mutations were introduced during the cloning pro-

Determining bacterial gene expression during infection is fundamental to understand pathogenesis. In this study, we used dual RNA-seq to simultaneously measure P. aeruginosa and the murine host’s gene expression and response to respiratory infection. Bacterial genes encoding products involved in metabolism and virulence were differentially expressed during infection and the type III and VI secretion systems were highly expressed in vivo. Strikingly, heme acquisition, ferric-enterobactin transport, and pyoverdine biosynthesis genes were found to be significantly up-regulated during infection. In the mouse, we profiled the acute immune response to P. aeruginosa and identified the pro-inflammatory cytokines involved in acute response to the bacterium in the lung. Additionally, we also identified numerous host iron sequestration systems upregulated during infection. Overall, this work sheds light on how P. aeruginosa triggers a pro-inflammatory response and competes for iron with the host during infection, as iron is one of the central elements for which both pathogen and host fight during acute pneumonia.

Mucoidy, or overproduction of the exopolysaccharide known as alginate, in Pseudomonas aeruginosa is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of P. aeruginosa have a wild-type (wt) mucA. Here, we describe a loss-of-function mutation in kinB that causes overproduction of alginate in the wt mucA strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of kinB was correlated with high expression at the alginate-related promoters P algU and P algD . Deletion of alternative factor RpoN ( 54 ) or the response regulator AlgB in kinB mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the kinB algB double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when kinB was inactivated. The inactivation of the DegS-like protease AlgW in the kinB mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the kinB mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required algB and rpoN in the kinB mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt mucA strain PAO1.

Summary Pseudomonas aeruginosa, a Gram‐negative bacterium, is a significant opportunistic pathogen associated with skin and soft tissue infections, nosocomial pneumonia and sepsis. In addition, it can chronically colonize the lungs of cystic fibrosis (CF) patients. Overproduction of the exopolysaccharide called alginate provides P. aeruginosa with a selective advantage and facilitates survival in the CF lung. The in vitro phenotype of alginate overproduction observed on solid culture media is referred to as mucoid. Expression of the alginate machinery and biosynthetic enzymes are controlled by the extracytoplasmic sigma factor, σ22 (AlgU/T). The key negative regulator of both σ22 activity and the mucoid phenotype is the cognate anti‐sigma factor MucA. MucA sequesters σ22 to the inner membrane inhibiting the sigma factor's transcriptional activity. The well‐studied mechanism for transition to the mucoid phenotype is mutation of mucA, leading to loss of MucA function and therefore activation of σ22. Recently, regulated intramembrane proteolysis (RIP) has been recognized as a mechanism whereby proteolysis of the anti‐sigma factor MucA leads to active σ22 allowing P. aeruginosa to respond to environmental stress conditions by overproduction of alginate. The goal of this review is to illuminate the pathways leading to RIP that have been identified and proposed.

f Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor ( 22 ; AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN ( 54 ). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy.

Pseudomonas aeruginosa is a ubiquitous microorganism and the most common Gram-negative bacterium associated with nosocomial pneumonia, which is a leading cause of mortality among critically ill patients. Although many virulence factors have been identified in this pathogen, little is known about the bacterial components required to initiate infection in the host. Here, we identified a unique trimethyl lysine posttranslational modification of elongation factor Tu as a previously unrecognized bacterial ligand involved in early host colonization by P. aeruginosa. This modification is carried out by a novel methyltransferase, here named elongation factor Tu-modifying enzyme, resulting in a motif that is a structural mimic of the phosphorylcholine present in platelet-activating factor. This novel motif mediates bacterial attachment to airway respiratory cells through platelet-activating factor receptor and is a major virulence factor, expression of which is a prerequisite to pneumonia in a murine model of respiratory infection.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that requires iron for virulence. Iron homeostasis is maintained in part by the PrrF1 and PrrF2 small RNAs (sRNAs), which block the expression of iron-containing proteins under iron-depleted conditions. The PrrF sRNAs also promote the production of the Pseudomonas quinolone signal (PQS), a quorum sensing molecule that activates the expression of several virulence genes. The tandem arrangement of the prrF genes allows for expression of a third sRNA, PrrH, which is predicted to regulate gene expression through its unique sequence derived from the prrF1-prrF2 intergenic (IG) sequence (the PrrH IG sequence). Previous studies showed that the prrF locus is required for acute lung infection. However, the individual functions of the PrrF and PrrH sRNAs were not determined. Here, we describe a system for differentiating PrrF and PrrH functions by deleting the PrrH IG sequence [prrF(ΔH IG )]. Our analyses of this construct indicate that the PrrF sRNAs, but not PrrH, are required for acute lung infection by P. aeruginosa. Moreover, we show that the virulence defect of the ΔprrF1-prrF2 mutant is due to decreased bacterial burden during acute lung infection. In vivo analysis of gene expression in lung homogenates shows that PrrF-mediated regulation of genes for iron-containing proteins is disrupted in the ΔprrF1-prrF2 mutant during infection, while the expression of genes that mediate PrrF-regulated PQS production are not affected by prrF deletion in vivo. Combined, these studies demonstrate that regulation of iron utilization plays a critical role in P. aeruginosa's ability to survive during infection.KEYWORDS Pseudomonas aeruginosa, sRNA, PrrF, PrrH, iron regulation, PQS, small RNA P seudomonas aeruginosa is a Gram-negative bacterium that causes life-threatening infections in a variety of patient populations, including acute blood and lung infections in hospitalized patients and chronic lung infections in individuals with cystic fibrosis (CF) (1-4). Iron is an essential nutrient for virulence in P. aeruginosa (5-9), but it is sequestered by mammalian host proteins such as lactoferrin and transferrin (10). To overcome this barrier to infection, P. aeruginosa secretes two siderophores, pyoverdine and pyochelin, which scavenge ferric iron (Fe 3ϩ ) from host proteins and are required for acute infections (5,(7)(8)(9). P. aeruginosa also obtains iron from host heme using outer membrane heme transporters and a cytosolic HemO heme oxygenase that degrades heme (11,12). Ferrous iron (Fe 2ϩ ) can also be obtained through the Feo system in microaerobic environments, such as those found within biofilms and the CF lung (13-15).

After primary infection, varicella-zoster virus (VZV) establishes latency in neurons of the dorsal root and trigeminal ganglia. Many questions concerning the mechanism of VZV pathogenesis remain unanswered, due in part to the strict host tropism and inconsistent availability of human tissue obtained from autopsies and abortions. The recent development of induced pluripotent stem (iPS) cells provides great potential for the study of many diseases. We previously generated human iPS cells from skin fibroblasts by introducing four reprogramming genes with non-integrating adenovirus. In this study, we developed a novel protocol to generate sensory neurons from iPS cells. Human iPS cells were exposed to small molecule inhibitors for 10 days, which efficiently converted pluripotent cells into neural progenitor cells (NPCs). The NPCs were then exposed for two weeks to growth factors required for their conversion to sensory neurons. The iPS cell-derived sensory neurons were characterized by immunocytochemistry, flow cytometry, RT-qPCR, and electrophysiology. After differentiation, approximately 80% of the total cell population expressed the neuron-specific protein, βIII-tubulin. Importantly, 15% of the total cell population co-expressed the markers Brn3a and peripherin, indicating that these cells are sensory neurons. These sensory neurons could be infected by both VZV and herpes simplex virus (HSV), a related alphaherpesvirus. Since limited neuronal populations are capable of supporting the entire VZV and HSV life cycles, our iPS-derived sensory neuron model may prove useful for studying alphaherpesvirus latency and reactivation.