Five independent DNA microarray experiments were used to study the gene expression profile of a 5-day Bacillus subtilis air-liquid interface biofilm relative to planktonic cells. Both wild-type B. subtilis and its sporulation mutant (DeltaspollGB::erm) were investigated to discern the important biofilm genes (in the presence and absence of sporulation). The microarray results indicated that suspension cells were encountering anaerobic conditions, and the air-liquid interface biofilm was metabolically active, For the statistically significant differential expression (P < 0.05), there were 342 genes induced and 248 genes repressed in the wild-type biofilm, whereas 371 genes were induced and 128 genes were repressed in the sporulation mutant biofilm. The microarray results were confirmed with RNA dot blotting. A small portion of cells (1.5%) in the wild-type biofilm formed spores and sporulation genes were highly expressed. In the biofilm formed by the sporulation mutant, competence genes (comGA, srfAA, srfAB, srfAD, and comS) were induced which indicate a role for quorum sensing (bacterial gene expression controlled by sensing their population) in biofilms. There were 53 genes consistently induced in the biofilms of both the wild-type strain and its spollGB mutant-those genes have functions for transport, metabolism, antibiotic production-and 26 genes with unknown functions. Besides the large number of genes with known functions induced in the biofilm (121 genes in the wild-type biofilm and 185 genes in the sporulation mutant biofilm), some genes with unknown functions were also induced (221 genes in the wild-type biofilm and 186 genes in the sporulation mutant biofilm), such as the yve operon which appears to be involved in polysaccharide synthesis and the ybc operon which inhibits the growth of competitors for nutrients. A knockout mutant of yveR was constructed, and the mutant showed major defects in biofilm maintenance. Both the wild-type strain and its sporulation mutant formed normal biofilms, suggesting complete sporulation is not necessary for biofilm formation. The expression profiles of these two strains share more repressed genes than induced genes, suggesting that the biofilm cells repress similar pathways in response to starvation and high cell density. (C) 2004 Wiley Periodicals, Inc.