Regulation of the PQQ-dependent quinoprotein enzyme systems in Pseudomonas aeruginosa
Zusammenfassung der Projektergebnisse
Pseudomonas aeruginosa expresses two pyrroloquinoline quinone (PQQ) dependent enzymes: quinoprotein ethanol dehydrogenase (QEDH) and quinoprotein glucose dehydrogenase (QGDH). Regulation of PQQ cofactor and QEDH biosynthesis had been studied in our laboratory. In this project we were interested in the regulation of QGDH biosynthesis and the identification of regulatory proteins involved in controlling both enzyme systems. QGDH was found to be encoded by the gcd-gene, which was co-transcribed with gene oprB2, coding for a putative glucose sensitive porin. The highest transcriptional activity of the oprB2,gcd-operon was found on fructose as substrate, rather than glucose. The same was found for the pqq-operon. A mutant with a defective gcd gene showed no QGDH activity and exhibited an extended lag phase on glucose or fructose. Mutants with inactivated regulatory genes which control ethanol utilization showed normal expression of QEDH activity and enzymes for PQQ biosynthesis after growth on glucose. In addition to the known response regulator AgmR and the two component regulatory system ExaDE, four additional regulatory proteins have been identified to be involved in controlling transcription of the ethanol oxidation system in P. aeruginosa. Two putative sensor kinases and one response regulator were found, all of which show significant similarity to the two component flhSR system in Paracoccus denitrificans controlling methanol and formaldehyde metabolism. In addition an iron and NAD+ dependent alcohol dehydrogenase Fe-ADH is involved in regulating ethanol oxidation in P. aeruginosa.Inactivation of one of these genes resulted in extremely extended lag-phases on ethanol or no growth at all. Using reporter gene constructs of all identified regulatory genes in the different mutants allowed to establish the hierarchy of the complex regulatory network. Of the 3 sensor kinases and 3 response regulators identified thus far, only the exaDE system forms a corresponding kinase/regulator pair. So, two additional sensor kinases and two response regulators, which are hidden among the numerous regulatory genes annotaded in the genome of P. aeruginosa, remain to be identified. All three identified response regulators are members of the luxR family. The three sensor kinases are localised in the cytoplasm and therefore recognise cytoplasmic signals. This is in accord with the observation that several mutants with defects in genes coding for ethanol or acetate metabolizing enzymes showed impaired regulation of the ethanol oxidation system. So far the results do not allow to pinpoint a metabolite which might serve as internal signal for one of the sensor kinases.
