Differential Roles of the Pseudomonas aeruginosa PA14 rpoN Gene in Pathogenicity in Plants, Nematodes, Insects, and Mice

Abstract

We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor 54 from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14. A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen r . PA14 rpoN::Gen r synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14, rpoN::Gen r was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans. In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen r exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P. aeruginosa PA14 at later stages of infection. rpoN::Gen r was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model. These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P. aeruginosa pathogenicity in diverse hosts.In gram-negative bacteria, the alternate sigma factor 54 , working in concert with a transcriptional activator that belongs to the NtrC superfamily, activates a variety of genes that are regulated in response to external stimuli (1). For example, in various bacteria, 54 is required for expression of the enzymatic pathways responsible for nitrogen utilization, dicarboxylate transport, xylene degradation, and hydrogen utilization (6,32,39,41,61). 54 is also involved in the regulation of virulence-related factors in both plant and animal pathogens, including pilin, flagellin, and alginate synthesis in Pseudomonas aeruginosa (19,58,60); capsular expression in Klebsiella pneumoniae (3); and regulation of hrp gene expression and coronatine biosynthesis in Pseudomonas syringae (23,24).Our laboratory has developed a bacterial pathogenicity model that utilizes a clinical isolate of P. aeruginosa (strain UCBPP-PA14 [referred to here as PA14]) that elicits severe soft-rot-like symptoms and proliferates when infiltrated into Arabidopsis leaves (52), kills the larvae of the wax moth caterpillar Galleria mellonella (30), causes lethal sepsis in a mouse full-skin-thickness burn model (52), and kills the nematode Caenorhabditis elegans (40,56,57). Interestingly, there is significant overlap among the PA14 virulence factors required for pathogenesis in plants, nematodes, insects, and mice. For example, among 21 genes identified as being involved in pathogenesis by screening transposon-induced PA14 mutants in plants and nematodes, 18, 17, 19, and 21 of these genes were required for pathogenicity in Arabidopsis, nematodes, wax moths, and mice, respectively (30,40,53,57).In other studies...

Regulation of the Histidine Utilization (Hut) System in Bacteria

“…argues strongly that hutC and hutD form a single hutCD transcription unit (175). It is interesting that like the Hut systems from enteric bacteria (46,84), all of the hut operons of P. fluorescens (and perhaps other pseudomonads [52,64]) can be transcribed by the "housekeeping" RNA polymerase, which carries 70 as its promoter recognition subunit (175,176). However, the hutU-G operon of P. fluorescens can also be transcribed from a promoter that is recognized by the unusual RNA polymerase that carries 54 (also known as N ) as its promoter recognition subunit (175,176), as described in more detail in the section on regulation (see below).…”

Sticky Situations: Key Components That Control Bacterial Surface Attachment

“…For instance, the sigma factor RpoS is a positive regulator of psl gene expression in P. aeruginosa (66), yet inactivation of rpoS has been correlated with more-substantial biofilm biomass accumulation compared to that of the wild type (59). Similarly, P. aeruginosa rpoN mutants are capable of forming biofilms but are impaired in attachment while demonstrating in-creased production of sadB, which is essential for attachment (20,57,143,151). SadC has been demonstrated to contribute to attachment.…”

P. aeruginosa Biofilms in CF Infection

“…Nevertheless, several genes that have been identified as QS regulated, including rhlA, rpoS, sadA, and those in the denitrification pathway, have been shown to be important in P. aeruginosa biofilm development, maintenance, and dispersion [10,59,68,69]. A recent study of P. aeruginosa biofilms in the mouse thermal injury model suggested that QS was not important in the formation of biofilms in acute infection [70]; however, several previous studies have clearly demonstrated a role for QS in this model [71][72][73][74]. Further investigation is clearly needed to precisely define the role of QS in both in vitro and in vivo biofilm development.…”

“…Numerous models of infection, including plant, invertebrate, and animal models, have supported the premise that QS contributes to P. aeruginosa pathogenesis [71][72][73][74][93][94][95][96][97][98][99][100][101]. In animal models of lung pathogenesis, including the mouse model of acute pneumonia, the mouse model of chronic lung infection, and the rat chronic lung infection model, lasR, lasI, and rhlI have been demonstrated to be required for virulence [73,93,[102][103][104][105].…”