Coaggregation by the freshwater bacterium Sphingomonas natatoria alters dual-species biofilm formation

Coaggregation is hypothesized to enhance freshwater biofilm development. To investigate this hypothesis, the ability of the coaggregating bacterium Sphingomonas natatoria to form single- and dual-species biofilms was studied and compared to that of a naturally occurring spontaneous coaggregation-deficient variant. Attachment assays using metabolically inactive cells were performed using epifluorescence and confocal laser scanning microscopy. Under static and flowing conditions, coaggregating S. natatoria 2.1gfp cells adhered to glass surfaces to form diaphanous single-species biofilms. When glass surfaces were precoated with coaggregation partner Micrococcus luteus 2.13 cells, S. natatoria 2.1gfp cells formed densely packed dual-species biofilms. The addition of 80 mM galactosamine, which reverses coaggregation, mildly reduced adhesion to glass but inhibited the interaction and attachment to glass-surface-attached M. luteus 2.13 cells. As opposed to wild-type coaggregating cells, coaggregation-deficient S. natatoria 2.1COGgfp variant cells were retarded in colonizing glass and did not interact with glass-surface-attached M. luteus 2.13 cells. To determine if coaggregation enhances biofilm growth and expansion, viable coaggregating S. natatoria 2.1gfp cells or the coaggregation-deficient variant S. natatoria 2.1COGgfp cells were coinoculated in flow cells with viable M. luteus 2.13 cells and allowed to grow together for 96 h. Coaggregating S. natatoria 2.1gfp cells outcompeted M. luteus 2.13 cells, and 96-h biofilms were composed predominantly of S. natatoria 2.1gfp cells. Conversely, when coaggregation-deficient S. natatoria 2.1COGgfp cells were coinoculated with M. luteus 2.13 cells, the 96-h biofilm contained few coaggregation-deficient S. natatoria 2.1 cells. Thus, coaggregation promotes biofilm integration by facilitating attachment to partner species and likely contributes to the expansion of coaggregating S. natatoria 2.1 populations in dual-species biofilms through competitive interactions.