Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp

1 Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada V6T 1Z3 2 Department of Microbiology, Montana State University, Bozeman, MT 59717, USA 3 Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY 13902, USA 4 Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717, USA Correspondence Michael J. Franklin umbfm{at}montana.edu Bacteria undergo a variety of physiological changes following a switch from planktonic growth to surface-associated biofilm growth. Here, it is shown that biofilm development of a marine isolate, Pseudoalteromonas sp. 1398, results in global changes in its cytosolic and extracellular proteomes. Calcium influences these proteome responses, and affects the amount of surface-associated biomass and extracellular matrix material produced by Pseudoalteromonas sp. 1398. Four extracellular proteins, characterized by N-terminal sequencing, showed increased abundances, while one protein, flagellin, showed reduced abundance at higher [Ca 2+ ]. Immunoblotting and transmission-electron-microscopy analysis confirmed that higher [Ca 2+ ] and surface-associated growth results in the repression of flagella production. Two-dimensional gel electrophoresis (2DGE) studies combined with cluster analysis of global proteome responses demonstrated that Ca 2+ had a greater regulatory influence on Pseudoalteromonas sp. growing in biofilms than on planktonic cultures. Approximately 22 % of the total cytosolic proteins resolved by 2DGE had differing abundances in response to a switch from planktonic growth to surface-associated growth when the cells were cultivated in 1 mM Ca 2+ . At higher [Ca 2+ ] this number increased to 38 %. Fifteen cellular proteins that were differentially expressed in response to biofilm growth and/or Ca 2+ were analysed by N-terminal sequencing and/or MS/MS. These proteins were identified as factors involved in cellular metabolic functions, putative proteases and transport proteins, although there were several proteins that had not been previously characterized. These results indicate that Ca 2+ causes global changes in matrix material, as well as in cellular and extracellular protein profiles of Pseudoalteromonas sp. 1398. These changes are more pronounced when the bacterium grows in biofilms than when it grows in planktonic culture. Abbreviations: 2DGE, two-dimensional gel electrophoresis; MS/MS, tandem mass spectrometry; SCLM, scanning