Characterization of the glucose ABC transporter of Pseudomonas aeruginosa

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Adewoye, Lateef O.
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'Pseudomonas aeruginosa' is a multiple antibiotic-resistant opportunistic pathogen which is responsible for serious infections in individuals whose immune status is compromised, especially in patients with cystic fibrosis, pneumonia, and AIDS. A major factor contributing to the antibiotic resistance of this organism is the porin-dependent low permeability of its outer membrane. Interestingly, one of these porins, the high-affinity glucose transport OprB porin, which is only induced during growth in low glucose concentration, is expressed in the human host upon infection. This suggests that the OprB porin or the OprB-associated transport system might play a role in the pathogenesis of 'P. aeruginosa' infections. This thesis describes the characterization and the identification of components of the high-affinity glucose transport system in 'P. aeruginosa'. In an attempt to determine whether the specificity of this system is well conserved throughout various members of the genus 'Pseudomonas', homologs of the OprB porin were purified and their structural and functional properties were examined by a combination of liposome swelling assays and circular dichroism spectropolarimetry. It was demonstrated that the high-affinity glucose transport system is primarily specific for glucose and well conserved in the genus 'Pseudomonas', but the outer membrane porin component may differ in channe architecture and specificity for other carbohydrates. Homologs of the 'oprB' gene were also identified, by PCR, in 'P. putida, P. chlororaphis', and 'P. fluorescens'. The promoter region of the 'P. aeruginosa oprB' gene was determined by primer extension and subsequently cloned into a promoterless 'lacZ' translational fusion vector to monitor gene expression under various growth conditions. Temperature, pH, salicylate, osmolarity and carbohydrate source were found to influence ' oprB' gene expression which in most cases reflected the pattern of whole-cell uptake of [U-14C]glucose by cells grown under similar conditions. The glucose transport genes seemed to be negatively regulated by salicylic acid, differentially regulated by pH and are specifically responsive to exogenous glucose induction. Furthermore two open reading frames (ORFs), ' gltK' and 'orfX', Banking the 'oprB' gene, were mutagenized and mobilized into 'P. aeruginosa' to replace the wild-type copies by homologous recombination. Analysis of the [U-14C]glucose uptake activity of the mutants showed that ' gltK', wile 'orfX', is essential for optimum glucose uptake. The 'gltK' gene expressed a ~29 KDa protein in ' E. coli'. Sequence analysis showed that 'gltK' encodes a putative cytoplasmic membrane-associated polypeptide which exhibits strong homology to several bacterial carbohydrate transport ATPases suggesting that the putative GltK is likely the inner membrane component of the 'P. aeruginosa' glucose ABC transporter. Moreover a genomic locus consisting of three ORFs, designated 'orfBCD', which affects the glucose ABC transporter as well as the fructose phosphoenolpyruvate-dependent fructose phosphotransferase system. was identified. However, none of the three ORFs has homology to any known bacterial genes directly involved in carbohydrate transport and regulation.