Running title: LytB is required for pneumococcal biofilm formation

29 p.-5 fig.+ 3 fig. supl.The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and responsible for cell separation at the end of cell division; ΔlytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytBE585A). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, this is, lytB mutants (either ΔlytB or producing the inactive LytBE585A) are incapable of forming substantial biofilms, even although extracellular DNA is present in the biofilm matrix. Adding small amounts (0.5-2.0 μg/ml) of exogenous LytB or some LytB constructs restored the biofilm-forming capacity of lytB mutants to wild-type levels. The LytBE585A mutant formed biofilm more rapidly than ΔlytB mutants in the presence of LytB. This suggests that the mutant protein that was present acted in a structural role, likely through the formation of complexes with extracellular DNA. The chain-dispersing capacity of LytB allowed the separation of daughter cells, presumably facilitating the formation of microcolonies and, finally, of biofilms. A role for the possible involvement of LytB in the synthesis of the extracellular polysaccharide component of the biofilm matrix is also discussed.IMPORTANCE It had been accepted that biofilm formation in S. pneumoniae is a multigenic trait because the mutation of just one gene might lead only to a partial inhibition of biofilm production. In the present study, however, evidence that the N-acetylglucosaminidasa LytB is crucial in biofilm formation is provided. Although extracellular DNA was present, either deficient or defective lytB mutants only formed shallow biofilms.This research was supported by grant SAF2017-88664-R from the Spanish Ministerio de Economía, Industria y Competitividad (MEICOM). The Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES) is an initiative of the Instituto de Salud Carlos III (ISCIII).Peer reviewe