Characterization of the amp genes involved in the regulation of beta-lactamase expression in Pseudomonas aeruginosa

Antibiotic resistance, production of alginate and virulence factors, and altered host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection. Failure of antibiotic therapy has been attributed to the emergence of P. aeruginosa strains that produce β-lactamase constitutively. In Enterobacteriaceae, β-lactamase induction involves four genes with known functions: ampC, ampR, ampD, and ampG, encoding the enzyme, transcriptional regulator, amidase and permease, respectively. In addition to all these amp genes, P. aeruginosa possesses two ampG paralogs, designated ampG and ampP. In this study, P. aeruginosa ampC, ampR, ampG and ampP were analyzed. Inactivation of ampC in the prototypic PAO1 failed to abolish the β-lactamase activity leading to the discovery of P. aeruginosa oxacillinase PoxB. Cloning and expression of poxB in Escherichia coli confers β-lactam resistance. Both AmpC and PoxB contribute to P. aeruginosa resistance against a wide spectrum of β-lactam antibiotics. The expression of PoxB and AmpC is regulated by a LysR-type transcriptional regulator AmpR that up-regulates AmpC but down-regulates PoxB activities. Analyses of P. aeruginosa ampR mutant demonstrate that AmpR is a global regulator that modulates the expressions of Las and Rhl quorum sensing (QS) systems, and the production of pyocyanin, LasA protease and LasB elastase. Introduction of the ampR mutation into an alginate-producing strain reveals the presence of a complex co-regulatory network between antibiotic resistance, QS alginate and other virulence factor production. Using phoA and lacZ protein fusion analyses, AmpR, AmpG and AmpP were localized to the inner membrane with one, 16 and 10 transmembrane helices, respectively. AmpR has a cytoplasmic DNA-binding and a periplasmic substrate binding domains. AmpG and AmpP are essential for the maximal expression of β-lactamase. Analysis of the murein breakdown products suggests that AmpG exports UDP-N-acetylmuramyl-L-alanine-γ-D-glutamate-meso-diaminopimelic acid-D-alanine-D-alanine (UDP-MurNAc-pentapeptide), the corepressor of AmpR, whereas AmpP imports N-acetylglucosaminyl-beta-1,4-anhydro-N-acetylmuramic acid-Ala-γ-D-Glu-meso-diaminopimelic acid (GlcNAc-anhMurNAc-tripeptide) and GlcNAc-anhMurNAc-pentapeptide, the co-inducers of AmpR. This study reveals a complex interaction between the Amp proteins and murein breakdown products involved in P. aeruginosa β-lactamase induction. In summary, this dissertation takes us a little closer to understanding the P. aeruginosa complex co-regulatory mechanism in the development of β-lactam resistance and establishment of chronic infection. ^

Multi-Scaled Grassland-Woody Plant Dynamics in the Heterogeneous Marl Prairies of the Southern Everglades

The Everglades freshwater marl prairie is a dynamic and spatially heterogeneous landscape, containing thousands of tree islands nested within a marsh matrix. Spatial processes underlie population and community dynamics across the mosaic, especially the balance between woody and graminoid components, and landscape patterns reflect interactions among multiple biotic and abiotic drivers. To better understand these complex, multi-scaled relationships we employed a three-tiered hierarchical design to investigate the effects of seed source, hydrology, and more indirectly fire on the establishment of new woody recruits in the marsh, and to assess current tree island patterning across the landscape. Our analyses were conducted at the ground level at two scales, which we term the micro- and meso-scapes, and results were related to remotely detected tree island distributions assessed in the broader landscape, that is, the macro-scape. Seed source and hydrologic effects on recruitment in the micro- and meso-scapes were analyzed via logistic regression, and spatial aggregation in the macro-scape was evaluated using a grid-based univariate O-ring function. Results varied among regions and scales but several general trends were observed. The patterning of adult populations was the strongest driver of recruitment in the micro- and meso-scape prairies, with recruits frequently aggregating around adults or tree islands. However in the macro-scape biologically associated (second order) aggregation was rare, suggesting that emergent woody patches are heavily controlled by underlying physical and environmental factors such as topography, hydrology, and fire.