Cells are complex systems continuously exposed to changing, dynamic environments. Understanding how cells respond and adapt is of great interest not only from a fundamental viewpoint but also for the development of solutions for current challenges in medical, industrial and environmental research fields. In this thesis, the bacterial community member Candidatus Accumulibacter phosphatis (hereafter referred to as Accumulibacter) from the functional group of Polyphosphate Accumulating Organisms (PAOs) was selected as study object due to their key-role in phosphorus removal at wastewater treatment processes and their adaptive metabolic strategies to thrive in fluctuating environments. These microorganisms are enriched in Enhanced Biological Phosphorus removal (EBPR) systems where they experience cyclic absence and presence of external electron acceptors (here, anaerobic and aerobic conditions, respectively). These fluctuations together with non-continuous availability of nutrients lead to intricate metabolic strategies. While the overall metabolic traits of these bacteria are well described, the non-availability of isolates has led to controversial hypotheses on which metabolic pathways are used - structure, when are these pathways active - function, and what mechanisms control the operation of these pathways - regulation. These hypotheses were further analysed and discussed in this dissertation. While the bacterial community member Accumulibacter was the cellular system example studied here, this doctoral dissertation explored and combined systems biology methods to improve the mechanistic understanding of cells as structured metabolic systems with functional and regulatory frameworks, which respond and adapt to changing external conditions (environments). The developed and applied approaches are not only specific to Accumulibacter and can be applied to other cell systems and communities exposed to changing environments.@en

Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation of fermentation products is more cost-effective. Aiming to find a key piece for the production of (poly)-3-hydroxybutyrate (PHB) as a fermentation product, here we characterize an acetoacetyl-CoA reductase, isolated from a Candidatus Accumulibacter phosphatis-enriched mixed culture, showing a (kcatNADH/KMNADH)/(kcatNADPH/KMNADPH)>500. Further kinetic analyses indicate that, at physiological concentrations, this enzyme clearly prefers NADH, presenting the strongest NADH preference so far observed among the acetoacetyl-CoA reductases. Structural and kinetic analyses indicate that residues between E37 and P41 have an important role for the observed NADH preference. Moreover, an operon was assembled combining the phaCA genes from Cupriavidus necator and the gene encoding for this NADH-preferring acetoacetyl-CoA reductase. Escherichia coli cells expressing that assembled operon showed continuous accumulation of PHB under oxygen limiting conditions and PHB titer increased when decreasing the specific oxygen consumption rate. Taken together, these results show that it is possible to generate PHB as a fermentation product in E. coli, opening opportunities for further protein/metabolic engineering strategies envisioning a more efficient anaerobic production of PHB.@en

Metaproteomics has emerged as one of the most promising approaches for determining the composition and metabolic functions of complete microbial communities. Conventional metaproteomics approaches rely on the construction of protein sequence databases and efficient peptide-spectrum-matching algorithms, an approach that is intrinsically biased towards the content of the constructed sequence database. Here, we introduce a highly efficient, database-independent de novo metaproteomics approach and systematically evaluate its quantitative performance using synthetic and natural microbial communities comprising dozens of taxonomic families. Our work demonstrates that the de novo sequencing approach can vastly expand many metaproteomics applications by enabling rapid quantitative profiling and by capturing unsequenced community members that otherwise remain inaccessible for further interpretation. Kleikamp et al., describe a novel de novo metaproteomics pipeline (NovoBridge) that enables rapid community profiling without the need for constructing protein sequence databases.@en

Natural habitats of microorganisms are dynamic environments with non-continuous supply of carbon and energy sources, in which intermediate storage of substrates can increase competitiveness. Plasticicumulans acidivorans are polyhydroxybutyrate (PHB) accumulating bacteria enriched from activated sludge using carbon feast-famine cycles as selective pressure. Despite growing slowly, P. acidivorans outcompetes other bacteria by quickly taking up acetate and storing it intracellularly as PHB to later use it for growth. As soon as acetate is depleted, these bacteria immediately ‘switch’ their metabolism from PHB production to consumption entailing a very interesting regulatory challenge as parallel activity could lead to significant losses (futile cycling). While the stoichiometry for both feast and famine phases has been extensively described in literature, the switch regulation is not yet fully understood. To elucidate the responsible regulatory processes, an enrichment of P. acidivorans was studied using targeted intracellular metabolite analysis over time, with emphasis on the feast to famine switch. In combination with extracellular rates, the measured intracellular metabolite pools are used to design a labelling experiment to obtain actual intracellular fluxes (dynamic 13C flux analysis). Here the challenge is to create an isotopically non-stationary state (usually mediated by changing the substrate’s isotopic composition) to study the metabolic response in the transition from presence-to-absence of substrate.In this way, we aim to unravel the responsible regulatory mechanism governing the metabolic switch from storage-to-consumption and use this knowledge not only to understand its ecological relevance, but to also propose novel metabolic strategies for microbial cell factory design.@en