Universiẗat, Diss., 2006--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2005.

Der Abbau der PBS unter Stickstoffmangel ist in Cyanobakterien ein ubiquitärer Mechanismus. Essenziell für den Abbau ist das Protein NblA. Das in Nostoc sp. PCC 7120 gut untersuchte ~7 kDa große Protein interagiert als Homodimer mit den PBS und dem Chaperon ClpC. Soweit bekannt kodieren alle Cyanobakterien ein essenzielles NblA-Protein. Der Modellorganismus Synechocystis sp. PCC 6803, dagegen kodiert mit NblA1 und NblA2 sogar zwei für den PBS-Abbau entscheidende Proteine. In dieser Arbeit konnte erstmalig durch in vitro und in vivo Interaktionsstudien mithilfe von pull down-Versuchen und FRET gezeigt werden, dass NblA1 und NblA2 als biologisch aktive Form ein Heterodimer bilden. In vitro-pulldown-Versuche zeigten für Synechocystis eine Interaktion des Heterodimers mit den PBS und ClpC. Durch die Ausbildung dieses ternären Komplexes markiert NblA1/NblA2 die PBS für den Abbau durch eine Clp-Protease mit ClpC als Chaperonpartner. In Photobionten gibt es eine Reihe von clp-Genen, so besitzen Cyanobakterien vier proteolytische clp Untereinheiten. Aus diesen bilden sich gemischte Heptamere aus je zwei Clp-Untereinheiten. Zwei lösliche, im Cytoplasma vorkommende Proteasen wurden bis jetzt zweifelsfrei identifiziert, eine dritte, an der Thylakoidmembran assoziierte Protease wird außerdem vermutet. Diese putative Protease, vermutlich aus dem Chaperon ClpC und den proteolytischen Untereinheiten ClpP1 und ClpR aufgebaut, wurde heterolog exprimiert. Mittels Koreinigung konnte ein funktioneller proteolytischer Kern gereinigt werden, der zusammen mit dem Chaperon ClpC eine aktive Clp-Protease bildet. Durch Größenausschlusschromatografie und Untersuchungen zur Proteaseaktivität konnte diese dann erstmalig charakterisiert werden. Des Weiteren zeigten in vitro-Degradationsversuche mit der aktiven Protease zweifelsfrei, dass das NblA1/NblA2-Heterodimer durch ClpC-ClpP1/ClpR abgebaut wird. Der Abbau von NblA1/NblA2 kann demnach auch ohne ein Substrat durch die Protease erfolgen. / The degradation of pycobilisomes under nitrogen deficiency, which is visible as a color change from blue green to yellow green, is an ubiquitary mechanism. Essential for the degradation is the small NblA protein. The well characterized NblA protein from Nostoc sp. PCC 7120 builds as biological active form a homodimer and interacts with the phycobilisomes and ClpC. However, the model organism Synechocystis sp. PCC 6803 possess two nblA genes, nblA1 and nblA2, which are both essential for phycobilisome degradation. In this work it could be shown by interaction studies and Förster resonance energy transfer that NblA1 and NblA2 builds as biological active form a heterodimer. Furthermore it could be shown that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease. Clp proteases of Cyanobacteria consist, besides the chaperones ClpC and ClpX of three different proteolytic subunits (ClpP1, 2, 3) and the ClpP variant ClpR which lacks the catalytic triad typical of Serine-type proteases. In Synechococcus 7942 two Clp proteases could be identified by gel filtration and immunoblot analyses, which provided evidence that each protease consists of a unique proteolytic core comprised of two separate Clp subunits, one core consisting of ClpP1 and ClpP2, and the other of ClpP3 and ClpR, in which subunits ClpP1/ClpP2 interact with ClpX and the ClpP3/ClpR protease with ClpC. In addition to these two soluble proteases, a third, membrane associated proteolytic complex is assumed, consisting of ClpP1 and ClpR. In Synechocystis 6803, homologs of the Clp machinery could be found by BLAST analyzes. This putative ClpP1/ClpR protease could be characterized in vitro by size-exclusion chromatography and degradation assays. Furthermore it could be shown a degradation of the NblA1/NblA2 heterodimer by the protease.

Cytosolic protein degradation is crucial for cellular homeostasis as it orchestrates protein turnover by destruction of misfolded, unstable and abnormal proteins. This process has two main stages: (i) an ATP-dependent stage mediated by unfoldases and proteases, and (ii) an ATP-independent stage mediated by various peptidases. The ATP dependent proteases recognise target proteins and cleave them into smaller peptides. These enzymes comprise the ATPase-family-associated-with-various-cellular-activities domain that is important for unfolding target proteins. Subsequently, unfolded proteins enter a barrel-shaped proteolytic chamber, an architecture conserved throughout prokaryotes, archea and eukaryotes, where the peptide bond is hydrolysed in an ATP-independent manner. The smaller peptides released are broken down by ATP-independent peptidases into free amino acids recycled into the cellular pool. In prokaryotes, major cellular protein degradation functions are performed by Clp and Lon proteases. Earlier studies in our laboratory have shown the role of an ATP-independent peptidase, AminopeptidaseN, in sodium salicylate (NaSal) induced growth inhibition. NaSal belongs to the family of Non-Steroidal Anti-Inflammatory Drugs and its acetyl ester, Aspirin, is a very widely used analgesic. It is produced by plants as a defence response and is known to cause different effects, including xenobiotic stress i. e. stress mediated by compounds which are not naturally produced or expected to be present in the organism in bacteria. In bacteria, salicylate modulates outer membrane proteins, virulence factors, and reduces motility. In addition, NaSal is able induce “phenotypic antibiotic resistance” by binding to MarR and de-repressing the mar operon. NaSal promotes the dissociation of MarR from the promoter site leading to transcription of MarA, a transcription factor that induces several genes that encode the AcrAB-TolC pump which effluxes multiple antibiotics from the cell. The present study investigates the possible roles of ATP-dependent proteases, Lon and Clp, during growth reduction of E. coli induced with high (2-3 mM) amounts of NaSal. The growth of the Lon mutant (lon), but not clpP, was found to be greatly reduced with high doses of NaSal in the media. Our hypothesis was that the lack of Lon led to the accumulation of high amounts of substrate proteins, which led to its greater sensitivity with high doses of NaSal. To identify the substrate protein/s and to better understand the mechanism of action, single and double mutants (with lon) of E. coli lacking several prominent Lon substrates, i.e. MarA, RcsA, StpA, SulA and UmuD, were generated and screened for rescue of growth with 2-3 mM NaSal. MarA, a transcription factor, was identified to be important during NaSal-induced growth reduction. It modulates outer membrane proteins and induces the AcrAB-TolC pump that increases the efflux of antibiotics from the cell. Also, RT-PCR analysis revealed that the levels of marA and its targets, acrA and acrB, were higher in the lon strain suggesting that the MarA protein levels were stabilised the cell in the absence of Lon. Further studies using approriate strains demonstrated that one of the effectors of MarA, i.e. the AcrAB-TolC efflux pump, was not involved in the NaSal-induced growth inhibition of lon. Therefore, in presence of higher doses of NaSal, MarA is upregulated due to de-repression of the operon. The levels of MarA are regulated by Lon via degradation but in the absence of Lon, MarA levels are stabilised and lead to upregulation of MarA and its target genes like AcrAB-TolC. This study identifies higher amounts of MarA to be responsible for NaSal-induced growth inhibition of lon. Subsequently, experiments were conducted to demonstrate the role of MarA and its targets in antibiotic resistance with low dose (0.5 mM) NaSal that does not affect growth. This low dose of NaSal was able to upregulate marA and its target genes, acrA, acrB and tolC. Quantification of antibiotic resistance further revealed an induction in resistance by 0.5 mM NaSal in a MarA- and AcrB-dependent fashion. Studies using atomic force microscopy demonstrated that ciprofloxacin-induced cell elongation was lower in lon due to higher levels of MarA. Therefore, low dose of NaSal is capable of upregulating MarA and inducing antibiotic resistance but does not affect cell growth. This part of the study addresses the roles of Lon protease, its substrate MarA and MarA-induced targets, e.g. AcrB, during NaSal-mediated growth reduction and antibiotic resistance. The factors contributing to antibiotic resistance in bacteria are an important area of study for the global public health care system. Antibiotic resistance can be acquired by transmittance of genetic material, accumulation of antibiotic resistant mutations in the target molecule or can be induced by certain compounds present in the environment like NaSal. For rapid identification of compounds that may behave in a similar fashion as NaSal, a 96-well plate based screen was developed utilising the growth inhibition feature of the lon strain. The compounds were selected on the basis of their structural (phenolic compounds) and functional (Non-Steroidal Anti Inflammatory Drugs or NSAIDs) similarity to NaSal. Through this screen, four compounds were identified that lowered the growth of lon more than that of wild type strain and may be important in inducing phenotypic antibiotic resistance: Acetaminophen (anti-pyretic), Ibuprofen (NSAID), and two phenolic uncouplers, Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-dinitrophenol (2,4-DNP). Notably, another compound Phenylbutazone (NSAID), which is used to lower inflammation in animals, did not reduce the growth of E. coli. RNA expression analysis revealed that these four compounds, but not phenylbutazone, induced the expression of marA and its target gene involved in antibiotic efflux, acrB. Furthermore, dose dependent and comparative studies with Nasal demonstrated differential effects of these four compounds in inducing antibiotic resistance with respect to ciprofloxacin, tetracycline and nalidixic acid. The two uncouplers were much more effective in inducing antibiotic resistance at lower doses than the NSAIDs. As NSAIDs are clinically important compounds, the study suggests that it would be desirable to screen them for induction of antibiotic resistance. The approach elucidated in this study has the potential to identify additional compounds present in the environment that may contribute to antibiotic resistance in bacteria. Overall, this study delineates the roles of Lon protease and its substrate, MarA, during NaSal-mediated responses, involving antibiotic resistance and/or growth reduction in E. coli. In addition, four other compounds were identified that could induce phenotypic antibiotic resistance in E. coli in a MarA-dependent manner. These observations may have implications in the adaptation of bacteria under different environmental conditions.