Bacteria become highly tolerant to antibiotics when nutrients are limited. The inactivity of antibiotic targets caused by starvation-induced growth arrest is thought to be a key mechanism producing tolerance (1). Here we show that the antibiotic tolerance of nutrient-limited and biofilm Pseudomonas aeruginosa is mediated by active responses to starvation, rather than by the passive effects of growth arrest. The protective mechanism is controlled by the starvation-signaling stringent response (SR), and our experiments link SR–mediated tolerance to reduced levels of oxidant stress in bacterial cells. Furthermore, inactivating this protective mechanism sensitized biofilms by several orders of magnitude to four different classes of antibiotics, and markedly enhanced the efficacy of antibiotic treatment in experimental infections.

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knockins, as well as single nucleotide insertions, deletions and substitutions in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selection are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ~2 weeks.

The lack of new antibiotics is among the most critical challenges facing medicine. The problem is particularly acute for Gram-negative bacteria. A novel antibiotic strategy is to target bacterial nutrition and metabolism. The metal gallium can disrupt bacterial iron metabolism as gallium can be taken up by bacteria, and replace iron. Here we performed pre-clinical work and a phase 1 human trial to investigate the antibiotic activity of gallium in people with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa airway infections. We found that CF sputum was iron-limited, and that low micromolar concentrations of gallium inhibited P. aeruginosa growth in CF sputum. Ex vivo experiments indicated that gallium inhibited key iron-dependent enzymes, and increased bacterial sensitivity to oxidants. We also found that gallium resistance developed at low rates, its activity was synergistic with some antibiotics, and it did not affect P. aeruginosa killing by human macrophages. Finally, we tested parenteral gallium in murine lung infections, and in CF patients with chronic P. aeruginosa lung infections and found indications of safety and efficacy. These data represent a small step toward targeting iron metabolism, or other nutritional vulnerabilities of bacteria, to treat human infections.

Quorum-sensing (QS) systems allow organisms, such as the pathogen Pseudomonas aeruginosa, to link gene expression with their population density and the diffusion and flow characteristics of their environment. The leading hypotheses about QS systems' biological functions necessitate that QS-controlled gene expression be suppressed until a threshold culture density (the quorum) is reached. Despite a detailed understanding of QS in P. aeruginosa, known regulatory elements do not fully explain how the quorum threshold for gene activation is produced. Here we investigated the mechanism with a screening approach that used random gene activation. These experiments uncovered a regulator without close homologs in other species that produces the quorum expression threshold. Expression of this regulator (named QteE) reduces LasR protein stability without affecting LasR transcription or translation. QteE also independently reduces RhlR levels. Because QteE can block QS when signal levels are high, it could provide a mechanism for individual cells to exert autonomous control over their QS regulons. This unique regulator governs two central QS control points in P. aeruginosa and shapes the expression pattern thought fundamental to the biological functions of QS.cell to cell signaling | quorum sensing | LasR | gene regulation

In this review, we describe the outer membrane proteins of Pseudomonas aeruginosa and related strains from the Pseudomonas fluorescens rRNA homology group of the Pseudomonadaceae, with emphasis on the physiological function and biochemical characteristics of these proteins. The use of opr (for outer membrane protein) is proposed as the genetic designation for the P. aeruginosa outer membrane proteins and letters are assigned, in conjunction with this designation, to known outer membrane proteins. Proteins whose primary functions involve pore formation, transport of specific substrates, cell structure determination and membrane stabilization are discussed. The conservation of selected proteins in the above Pseudomonas species is also examined.