Open Access

Miriam Sand

• This thesis describes the adaptation of Acinetobacter species to dry environments with the soil bacterium A. baylyi and the opportunistic hospital pathogen A. baumanii in its focus. The adaptation of A. baylyi and A. baumannii to osmotic stress was investigated. Compatible solutes that were uptaken from the environment or synthesized de novo to cope with the loss of water at high salinity were identified. The corresponding transporters and enzymes involved were characzerized. In addition, the desiccation resistance of A. baumannii was analyzed to elucidate its survival in hospital environments. The usage of compatible solutes during desiccation stress was analyzed and proteins that were produced were identified. The availability of water is essential for bacterial life and if environmental conditions are awkward, bacteria have to cope with high salinitiy to prevent loss of water. In this thesis it was shown that A. baylyi synthesizes glutamate and mannitol de novo as compatible solutes in response to osmotic stress to balance the osmotic potential. The pathway for mannitol biosynthesis from Fructose-6-Phosphate (F-6-P) via Mannitol-1-Phosphate (Mtl-1-P) was elucidated and the isolation and characterization of a novel type of biofunctional enzyme was described. Interestingly, the unique bifunctional enzyme MtlD, acting as dehydrogenase and phosphatase, mediates both steps of the mannitol biosynthesis pathway. This enzyme catalyzes the reduction of F-6-P to Mtl-1-P with NADPH as reducing equivalent. The dehydrogenase activity of MtlD was salt dependent and the phosphatase activity was dependent on Mg2+ as cofactor. Phylogenetic analyses revealed that MtlD is broadly distributed among other Acinetobacter strains but not in other phylogenetic tribes. In this thesis it is also described that, besides de novo synthesis of compatible solutes, A. baylyi takes up glycine betaine (GB) or its precursor choline by different transport systems and uses this solutes as osmoprotectants. The uptake of GB occurs via a secondary transporter (ACIAD3460) of the BCCT family. Choline is taken up as precursor and oxidized to GB by two dehydrogenases. The uptake and use of choline as GB precursor involves two transporters, whose genes are encoded in the bet cluster (BetT1, BetT2), two dehydrogenases (BetA, BetB) and a regulatory protein (BetI). Both transporters differ from each other in structure and function: BetT1 is osmo-independent and active independently of osmotic stress. BetT2 contains - in contrast to BetT1 - a long C-terminal domain for osmo-sensing and its activity highly increases in the presence of high osmolarity. The oxidation of choline occurs independently of the osmolarity of the medium but in the absence of salt stress, GB is exported. In contrast, in the presence of high salinity, GB is accumulated in the cytoplasm to balance the osmotic potential in order to prevent loss of water. The regulation of both transporters, the uptake of choline independently of the osmolarity and the export of GB under isoosmotic conditions are regulated by the transcriptional regulator BetI. A. baumannii ATCC 19606 was also shown to cope with high salinity. Analogously to A. baylyi, A. baumannii ATCC19606 synthesizes glutamate and mannitol de novo in response to osmotic stress. The genes for the synthesis of these compatible solutes are identical to those found in A. baylyi. This suggests that the solute biosynthesis pathways of A. baumannii and A. baylyi are identical. A. baumannii was also able to take up GB and choline in response to osmotic stress and growth at high salinity was restored upon addition of GB and its precursor choline. The bet cluster was also present in the genome A. baumannii and also contains the two different choline transporters BetT1 and BetT2. Our suggestion that choline or GB or the utilization of phosphatidylcholine as carbon source led to an increase in the survival under desiccation stress was not confirmed. However, 2D analysis of proteins produced during desiccation stress in A. baumannii led to elevated amounts of proteins implicated in biofilm formation, regulation, cell morphology and general stress response, such as Hsp60 or superoxide dismutase, both might play a role in general stress protection.
• Die Gattung Acinetobacter umfasst eine Reihe von Bakterien, die alle sehr eng miteinander verwandt sind und sich allgemein durch ihre hohe Anpassungsfähigkeit an sich ändernde Umweltbedingungen auszeichnen. Durch ihre hohe metabolische Versatilität sind diese Organismen zudem in der Lage, sich optimal auf die Nahrungsverfügbarkeit in ihre Umwelt einzustellen. Die wohl am bekanntesten Vertreter sind hierbei das Bodenbakterium A. baylyi und der opportunistisch humanpathogene Krankenhauskeim A. baumannii. Vor allem A. baumnannii rückt immer mehr in den Fokus des Interesses, da dieser weltweit zunehmend zu nosokomialen Infektionen führt. Zudem weist dieser Pathogen steigende Antibiotikaresistenzen - und mittlerweile auch Multiresistenzen - auf und zählt daher laut IDSA (Infectious Diseases Society of America) mittlerweile zu den sechs wichtigsten Krankheitserregern weltweit. Problematisch ist hierbei auch, dass A. baumannii durch seine hohe Anpassungsfähigkeit in der Lage ist, auch bei ungünstigen Bedingungen im Krankenhaus zu überleben. So ist dieser Organismus fähig, durch seine hohe Austrocknungsresistenz auch ohne Wasser monatelang auf Oberflächen im Krankenhaus zu überdauern. Diese Persistenz führt dazu, dass dieser Erreger vor allem durch das Krankenhauspersonal an Patieten weiter übertragen wird und immer wieder neue Infektionen auslöst...