Studies on the role of protein secretion systems in Pseudomonas fluorescens biofilm formation, and development of a tunable gene expression system for such analyses

Abstract

Plant growth-promoting rhizobacteria (PGPRs), such as Pseudomonas fluorescens, support plants via different mechanisms such as biocontrol (suppressing pathogenic organisms), phytostimulation (modulation of plant-hormone levels) or biofertilization (by improving nutrient supply) and thus are important for growth yields. Therefore, it is important to understand interactions of the bacteria and plant roots. Many PGPRs are able to form a biofilm on plant roots, so they live in an adherent cell community on the root surface. Different bacterial surface structures (e.g. pili or fimbriae) and secreted proteins (e.g. adhesins or enzymes) are involved in biofilm formation or plant growth promotion. Therefore, the first part of this study investigated the influence of selected secreted proteins or protein secretion systems on biofilm formation of P. fluorescens. Genes encoding translocated substrates or secretion system components were deleted to abolish their function. Two genes encoding type 1 secretion system (T1SS) components (lapA, encoding an adhesin and aprE, encoding a membrane fusion protein) were identified, whose deletion lead to weaker biofilm formation. Further analyses of the Apr T1SS revealed that its substrate AprA (an extracellular protease) was not directly involved in biofilm formation. The deletion of aprE most likely resulted in mistargeting AprA into the periplasm, which could influence biofilm formation. In the second part of the study, a new tightly controlled sugar-independent expression system was established for P. fluorescens A506. The gene PflA506_4486 was predicted to encode a putative anthranilic acid inducible regulator for the expression of an adjacent antABC operon for anthranilic acid degradation to catechol. PflA506_4486 and the putative promoter region for antABC expression were cloned into a shuttle vector system for Escherichia coli and Pseudomonas. gfp was put under control of this anthranilate-inducible promoter (PantA) system as reporter for the examination of promoter activity. In this study it was shown that the anthranilate-inducible promoter system is tightly controlled by inducer concentration and can be tuned to expression levels suitable for physiological analyses. Compared with a rhamnose-inducible promoter system, the PantA-system was advantageous in P. fluorescens A506. Moreover, this new system is working in P. fluorescens as well as in Pseudomonas putida and Pseudomonas aeruginosa, which are the most intensively studies pseudomonads.