The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and responsible for cell separation at the end of cell division; lytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytB_E585A). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, this is, lytB mutants (either lytB or producing the inactive LytB_E585A) 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 LytB_E585A mutant formed biofilm more rapidly than lytB 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.

肺炎链球菌的N-乙酰氨基葡萄糖苷酶LytB参与鼻咽定植，并在细胞分裂结束时负责细胞分离。lytB突变体形成细胞的长链。本文报道了产生失活的LytB蛋白（LytB _E585A）的有缺陷的肺炎球菌突变体的构建和特性。结果表明，体外生物膜形成需要酶促活性的LytB ，即lytB突变体（lytB或产生失活的LytB _E585A即使生物膜基质中存在细胞外DNA，也无法形成大量的生物膜。添加少量（0.5–2.0μg/ ml）外源LytB或某些LytB构建体可将lytB突变体的生物膜形成能力恢复至野生型水平。LytB _E585A突变体比lytB更快形成生物膜LytB存在的突变体。这表明存在的突变蛋白可能通过与细胞外DNA形成复合物而发挥结构作用。LytB的链分散能力使子细胞得以分离，大概有助于微菌落的形成，并最终促进生物膜的形成。还讨论了LytB可能参与生物膜基质的细胞外多糖成分合成的作用。

重要性肺炎链球菌的生物膜形成是多基因性状，因为仅一个基因的突变可能仅导致生物膜产生的部分抑制。但是，在本研究中，提供了N-乙酰氨基葡萄糖苷LytB在生物膜形成中至关重要的证据。尽管存在细胞外DNA，但不足或有缺陷的lytB突变体仅形成浅生物膜。