APPLIED MICROBIAL AND CELL PHYSIOLOGY Analysis of the alginate O-acetylation machinery in Pseudomonas aeruginosa Wankuson Chanasit 1 & Zennia Jean C. Gonzaga 2 & Bernd H. A. Rehm 2 Received: 6 November 2019 /Revised: 6 November 2019 /Accepted: 8 December 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract O-acetylation of alginate produced by the opportunistic human pathogen Pseudomonas aeruginosa significantly contributes to its pathogenesis. Three proteins, AlgI, AlgJ and AlgF have been implicated to form a complex and act together with AlgX for O- acetylation of alginate. AlgI was proposed to transfer the acetyl group across the cytoplasmic membrane, while periplasmic AlgJ was hypothesised to transfer the acetyl group to AlgX that acetylates alginate. To elucidate the proposed O-acetylation multiprotein complex, isogenic knockout mutants of algI, algJ and algF genes were generated in the constitutively alginate overproducing P. aeruginosa PDO300 to enable mutual stability studies. All knockout mutants were O-acetylation negative and complementation with the respective genes in cis or trans restored O-acetylation of alginate. Interestingly, only the AlgF deletion impaired alginate production suggesting a link to the alginate polymerisation/secretion multiprotein complex. Mutual stability experiments indicated that AlgI and AlgF interact independent of AlgJ as well as impact on stability of the alginate polymerisation/secretion multiprotein complex. Deletion of AlgJ did not destabilise AlgX and vice versa. When the alginate polymerase, Alg8, was absent, then AlgI and AlgF stability was strongly impaired supporting a link of the O-acetylation machinery with alginate polymerisation. Pull-down experiments suggested that AlgI interacts with AlgJ, while AlgF interacts with AlgJ and AlgI. Overall, these results suggested that AlgI-AlgJ-AlgF form a multiprotein complex linked via Alg8 to the envelope-spanning alginate polymerisation/secretion multiprotein complex to mediate O-acetylation of nascent alginate. Here, we provide the first insight on how the O-acetylation machinery is associated with alginate production. Keywords Pseudomonas aeruginosa . Alginate . O-acetylation . AlgI . AlgJ . AlgF Introduction Pseudomonas aeruginosa is an opportunistic pathogen caus- ing acute and chronic infections in humans. Patients with cys- tic fibrosis (CF) are particularly susceptible to P. aeruginosa chronic infections that are associated with morbidity and mor- tality (Leid et al. 2005; Lyczak et al. 2002; Moradali et al. 2017b). Clinical isolates of P. aeruginosa obtained from CF patients with chronic pulmonary infections typically comprise a highly mucoid strain that overproduces the extracellular polysaccharide, alginate (May et al. 1991). Alginate produc- tion contributes to clogging of the patient’s lung and protects the bacteria from host immune responses and antibiotic treat- ments (Baltimore and Mitchell 1980; Hay et al. 2014; Simpson et al. 1988, 1989; Song et al. 2003; Waters and Smyth 2015). Hence, once P. aeruginosa has converted to its mucoid alginate overproducing phenotype, eradication of P. aeruginosa from infected patients becomes nearly impossi- ble (Moradali et al. 2017b). Alginate is composed of β-1,4-linked β-D-mannuronic acid and its C-5 epimer α-L-guluronic acid. Twelve proteins are re- quired for alginate polymerisation, modification and secretion and are located in a single operon (AlgD, Alg8, Alg44, AlgK, AlgE, AlgG, AlgX, AlgL, AlgI, AlgJ, AlgF and AlgA) (Rehm Wankuson Chanasit and Zennia Jean C. Gonzaga contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-019-10310-6) contains supplementary material, which is available to authorized users. * Bernd H. A. Rehm b.rehm@griffith.edu.au 1 Department of Biology, Faculty of Science, Thaksin University, Pa Phayom, Patthalung 93210, Thailand 2 Centre for Cell Factories and Biopolymers, Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Brisbane, QLD 4111, Australia Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-019-10310-6