Pseudomonas aeruginosa has emerged as an important opportunistic human pathogen that is often highly resistant to eradication strategies, mediated in part by the formation of multicellular aggregates. Cellular aggregates may occur attached to a surface (biofilm), at the air-liquid interface (pellicle), or as suspended aggregates. Compared to surface attached communities, knowledge about the regulatory processes involved in the formation of suspended cell aggregates is still limited. We have recently described the SiaA/D signal transduction module that regulates macroscopic cell aggregation during growth with, or in the presence of the surfactant SDS. Targets for SiaA/D mediated regulation include the Psl polysaccharide, the CdrAB two-partner secretion system and the CupA fimbriae. While the global regulators c-di-GMP and RsmA are known to inversely coordinate cell aggregation and regulate the expression of several adhesins, their potential impact on the expression of the cupA operon remains unknown. Here, we investigated the function of SiaA (a putative ser/thr phosphatase) and SiaD (a di-guanylate cyclase) in cupA1 expression using transcriptional reporter fusions and qRT-PCR. These studies revealed a novel interaction between the RsmA posttranscriptional regulatory system and SiaA/D mediated macroscopic aggregation. The RsmA/rsmY/Z system was found to affect macroscopic aggregate formation in the presence of surfactant by impacting the stability of the cupA1 mRNA transcript and we reveal that RsmA directly binds to the cupA1 leader sequence in vitro. We further identified that transcription of the RsmA antagonist rsmZ is controlled in a SiaA/D dependent manner during growth with SDS. Finally, we found that the siaD transcript is also under regulatory control of RsmA and that overproduction of RsmA or the deletion of siaD results in decreased cellular cyclic di-guanosine monophosphate (c-di-GMP) levels quantified by a transcriptional reporter, demonstrating that SiaA/D connects c-di-GMP and RsmA/rsmY/Z signaling to reciprocally regulate cell aggregation in response to environmental conditions.

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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...

The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen.

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