25The looming antibiotic crisis and the critical role of biofilms in chronic infections call for novel and tailor-26 made anti-infective strategies. We previously characterised the PP2C-like phosphatase SiaA and the di-27 guanylate cyclase SiaD control the formation of macroscopic, suspended biofilms of P. aeruginosa cells in 28 response to surfactant stress. We now demonstrate that this regulation is also important for cellular 29 aggregation in response to carbon availability and provide compelling evidence that the SiaABCD pathway 30 functions trough a partner switch mechanism. Our study reveals that SiaA and SiaB represent a threonine-31 specific protein phosphatase/kinase switch regulating cellular aggregation by balancing the 32 phosphorylation status of SiaC. From these data, we hypothesize that fine-tuning cellular aggregation 33 through SiaABCD constitutes a general strategy used by P. aeruginosa to adapt to various environmental 34 conditions and that this pathway represents a novel and promising target for the development of anti-35 infective drugs against this aggressive opportunistic pathogen. 36 specific phosphatases are usually not required to inactivate the response over time. In contrast, 61 phosphorylation of a serine or threonine residue are much more stable and, thus, require the additional 62 presence of a phosphatase to facilitate reversible regulation 47,48 . 63Pseudomonas aeruginosa, an opportunistic human pathogen of critical concern, is often highly resistant 64 to antimicrobial therapies. It is an ubiquitous organism that can thrive in multiple environmental niches 65 and genetic evidence indicates that infections usually arise from environmental sources [49][50][51][52] . Various 66 regulatory pathways affect its biofilm formation and virulence traits, many of which are based on protein 67 phosphorylation including, but not limited to, the GacS/GacA system, the threonine phosphorylation 68 pathway (TPP) and the Wsp, Yfie or HptB pathways 24,53-58 . While P. aeruginosa does not generally infect 69 healthy humans, it is a serious threat in hospital environments, particularly for individuals with large burn 70 wounds or chronic diseases such as obstructive pulmonary disease (COPD) and the genetic disorder cystic 71 fibrosis (CF) 29,[59][60][61][62][63] . In addition to surface attached biofilms, suspended biofilms of P. aeruginosa (i.e. 72 cellular aggregates) have been characterised first for growth under laboratory conditions 28,64 . Notably, 73 such cellular aggregates are also regularly found in chronic infections in vivo 26,27,[65][66][67] . Moreover, 74 suspended biofilms greatly influence the development, structure and function of their surface-attached 75 counterparts and have thus been suggested to play an important role in niche colonisation and the chronic 76 manifestation of infections 68,69 . 77We previously demonstrated that SiaA and the di-guanylate cyclase (DGC) SiaD regulate the formation of 78 large, macroscopic, DNA-containing suspended biofilms in response to the to...

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