Burkholderia cepacia complex (Bcc) species are opportunistic pathogens in patients with cystic fibrosis (CF), which are able to cause lethal infections. The Bcc are inherently resistant to most classes of antibiotics, which makes successful treatment problematic. Lipid A (also known as endotoxin), the hydrophobic anchor of lipopolysaccaride (LPS), is the bio-active component of LPS. One of several unique characteristics of the lipid A of the Bcc, is the permanent attachment of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A molecule. Also, the genes involved in L-Ara4N biosynthesis are necessary for viability in B. cenocepacia. Here we present research on lipid A biosynthesis, modi cation, and transport in the Bcc and highlight promising antimicrobial targets. The synthetic antibiotic CHIR-090 is an inhibitor of LpxC, an enzyme involved in the lipid A biosynthetic pathway. I investigated the activity of CHIR-090 against the Bcc and found that sensitivity to this antibiotic was both species- and strain-specific. CHIR-090 displayed MICs between 0.1 and 12.5 μg/ml against a panel of B. multivorans, the most prevalent Burkholderia species in CF. The species- and strain-specific sensitivity towards CHIR-090 was further explored and a strong correlation was found between the presence of a unique open reading frame, named LpxC2, in resistant species. To address the problem of multiple drug-resistance of the Bcc, we investigated the activity of the pyridoxal 50-phosphate (PLP)-dependent enzyme inhibitor cycloserine (CS) against the Bcc. CS is used as a second line of defense against M. tuberculosis. The activity of the D-enantiomer of CS (DCS) against the Bcc was tested and displayed MICs between 2 and 128 μg/ml and acted bactericidal towards the Bcc. Additionally, DCS inhibition of recombinant ArnB from B. cenocepacia J2315, a PLP-dependent enzyme necessary for viability in the Bcc, was studied. ArnB was inhibited reversibly by DCS. ArnB was further explored as a promising drug-target in the Bcc, but only CS has been identified as an inhibitor so far. In this thesis it was attempted to find the reason why is L-Ara4N modification of lipid A necessary for viability in B. cenocepacia. Therefore, two proteins were characterised, which are involved in lipid A transport: LptA, the periplasmic lipid A binding protein, and LptB, the cytoplasmic ATP-ase. LptA was found to be able to bind both modified and unmodified lipid A in vitro and therefore is not L-Ara4N specific. Furthermore, LptA could bind deep-rough-, rough-, and smooth- LPS, similar to that described for Escherichia coli LptA. The kinetic parameters of LptB were determined in vitro (kcat = 5.71 min-1 and KM = 0.88 mM), and were comparable to E. coli LptB. The ATP-ase activity of LptB was not influenced by the presence of any forms of LPS (modified or non-modified). Therefore, we concluded that both B. cenocepacia J2315 LptA and LptB are not L-Ara4N specific.

To show how predaceous fish and dragonflies affect benthic community structure, we conducted enclosure experiments in the littoral zone of Bays Mountain Lake, Tennessee. A 'natural' benthic assemblage was subjected to all combinations of 2 densities of 3 predator treatments - small sunfish, 0 or 4/m2; large dragonfly larvae, 0 or 15/m2; and dragonfly eggs at 2 densities, 90 or 900/m2. Treatments were assigned randomly in each of 6 spatiotemporal blocks. Net effects of predation over 4 mo show that small sunfish had 'strong' effects (>50% reduction of densities) on triclads, large daphnid cladocerans, and snails, and 'moderate' effects (50% > reduction > 25%) on small dragonflies and ostracods; all these effects were statistically significant, except the one on ostracods. Large dragonflies had moderate non-significant effects on triclads and ostracods. There were no significant increases in prey density associated with fish predation; but chydorid cladocerans and midge larvae showed 'weak' non-significant increases that might be caused by 'indirect effects' of fish predation on invertebrate predators or grazers. There were only 2 significant interaction terms indicative of 'higher-order interactions': Fish X Dragonfly on the large daphnid Simocephalus, and Fish X Egg Density on snails, which were associated with relatively low probabilities. In both cases, the net effect of dragonflies was to reduce prey densities more when fish were present.