Oligonucleotide library assisted sequence mining reveals promoter sequences with distinct temporal expression dynamics for applications in <i>Curvibacter</i> sp. AEP1-3

The <i>β-proteobacterial</i> species <i>Curvibacter</i> sp. AEP1-3 is a model organism for the study of symbiotic interactions as it is the most abundant colonizer of <i>Hydra vulgaris</i>. Yet, genetic tools for <i>Curvibacter</i> are still in their infancy; few promoters have been characterized so far. Here, we employ an oligonucleotide-based strategy to develop novel expression systems <i>Curvibacter</i>. Potential promoters were systematically mined from the genome <i>in silico</i>. The sequences were cloned as a mixed library into a mCherry reporter vector and positive candidates were selected by Flow Cytometry to be further analysed through plate reader measurements. From 500 candidate sequences, 25 were identified as active promoters of varying expression strength levels. Plate reader measurements revealed unique activity profiles for these sequences across growth phases. The expression levels of these promoters ranged over two orders of magnitudes and showed distinct temporal expression dynamics over the growth phases: while three sequences showed higher expression levels in the exponential phase, we found 12 sequences saturating expression during stationary phase and 10 that showed little discrimination between growth phases. From our library, promoters of the genes <i>dnaK, rpsL</i> and an acyl-homoserine-lactone (AHL) synthase stood out as the most interesting candidates fit for a variety of applications. We identified enriched transcription factor binding motifs among the sorted 33 sequences and genes encoding for homologs of these transcription factors in close proximity to the identified motifs. In this work, we show the value of employing comprehensive high-throughput strategies to establish expression systems for novel model organisms.