Kolter and Losick (37
) noted that until recently, even though most microorganisms grow as biofilms and the physical structures of different biofilms are well characterized, biofilms had not been studied using molecular genetic approaches. A well-studied, genetically amenable oral bacterium, S. gordonii
Challis, was used in this study to identify genes that are required for biofilm formation on abiotic surfaces.
The observation that S. gordonii
Challis biofilm formation was enhanced in a minimal medium but not in a nutritionally rich environment indicates that sessile growth may represent a survival strategy in a nutritionally limited environment, as surface colonization provides advantages such as increased capture of nutrients that may be adsorbed to surfaces (64
). Starvation conditions have previously been reported to initiate adhesion as bacteria attempt to exploit a source of essential nutrients which may be in short supply in the surrounding environment (38
). Oral surfaces in vivo also represent a nutritionally limited environment, and biofilm formation may be required for survival, as bacteria depend on degraded salivary constituents as nutrients (40
Interspecies variation was observed in the ability of oral streptococci to form biofilm on abiotic surfaces, which may reflect differences in the mechanisms of colonization by different streptococcal species. For example, S. oralis
C104, which demonstrated poor biofilm-forming ability, may lack effective colonization factors for binding to abiotic surfaces but still participate in plaque formation by binding to initial colonizing cells of other species. Most human viridans streptococci participate in intrageneric coaggregation, the cell-to-cell adherence among genetically distinct streptococci, and these interactions may foster the primary colonization of the tooth surfaces (36
). Furthermore, an S. gordonii
DL1 mutant that did not coaggregate with its streptococcal partner S. oralis
C104 exhibited wild-type levels of coaggregation with actinomyces (8
The oral environment experiences significant fluctuations in O2
tension, pH, and carbohydrate content due to variations in microflora, diet, and oral hygiene habits. The primary indicators of the two major dental diseases, periodontal disease and caries, are a shift in dental plaque flora from initial gram-positive facultative aerobes to mainly gram-negative anaerobes (58
) and an increased acidogenicity due to bacterial metabolism of dietary sucrose (41
), respectively. Some of these environmental changes, namely, in pH, osmolarity, and carbohydrate content, were found to influence streptococcal biofilm formation in vitro. As nutritional (48
) and environmental (64
) signals play a role in biofilm development, these observations may be useful in attempts to identify the cellular factors and molecular mechanisms involved in streptococcal biofilm formation.
Bacteria sense a large number of environmental signals and process this information into specific transcriptional responses. In gram-positive bacteria, cell-density-dependent gene expression regulatory modes appear to follow a common theme, in which the signal molecule is a posttranslationally processed peptide that is secreted by an ATP-binding cassette exporter. This peptide pheromone accumulates extracellularly in proportion to the total number of cells, providing an index of population densities (15
), and functions as the input signal for the sensing component of a two-component signal transduction system (34
). Therefore, these bacterial autoinduction systems represent cell-to-cell communication, which is also referred to as quorum sensing.
In S. gordonii
, the sensing component of the two-component signal transduction system is ComD, an autophosphorylating histidine kinase. ComD is the receptor for the comC
-encoded competence-stimulating peptide, a 50-amino-acid peptide pheromone that induces competence in the bacterial population at a critical extracellular concentration (27
). The second component, the cognate response regulator ComE, becomes activated after receiving the phosphoryl group from ComD at an aspartate residue and binds to specific promoter regions of appropriate target genes, therefore acting as a transcriptional factor (45
One of the biofilm-defective mutants isolated had a transposon insertion within comD
of S. gordonii
, which encodes ComD (42
). To our knowledge, this is the first report that cell-to-cell signaling is involved in the biofilm formation of a gram-positive species on an abiotic surface and is consistent with a previous report that the differentiation and integrity of P. aeruginosa
biofilms are controlled by a specific quorum-sensing signal (12
). A P. aeruginosa
mutant defective in the production of N
-homoserine lactone, one of the acylhomoserine lactones that mediate quorum sensing, produced abnormal biofilms that were sensitive to the detergent biocide sodium dodecyl sulfate, indicating that a quorum-sensing signal is involved in biofilm differentiation and integrity (12
). A recent study implicated cell density signaling in activation of the recovery process of nitrogen-starved Nitrosomonas europaea
). Results from this study demonstrates that cell-to-cell signaling in biofilm formation may not be a characteristic restricted to P. aeruginosa
or gram-negative bacteria.
PBPs are responsible for the assembly, maintenance, and regulation of peptidoglycan peptide structures. Identification of biofilm-associated genes that are involved in peptidoglycan biosynthesis indicates the importance of cell envelope integrity to the biofilm phenotype in streptococci, as mutations in peptidoglycan biosynthesis genes may result in cells with morphologies that lack a rigid cell envelope component.
Disruptions in genes regulating peptidoglycan synthesis are also likely to affect their ability to respond to environmental changes such as extracellular osmolarity, which is important during sessile growth. Change in environmental osmolarity can elicit structural alterations by cellular remodeling. Cellular remodeling has been shown to accompany long-term osmoadaptation, whereas the P-type ATPases are involved in acute-phase osmoadaptation (65
). Identification of biofilm-associated genes that are involved in peptidoglycan biosynthesis suggest that osmoadaptation systems may play a role in biofilm formation.
One of the 18 biofilm-defective mutants (9F8) had two transposon insertions, one of which is the 5′ region of a salivary α-amylase-binding gene of S. gordonii
). This is the only adhesion-related gene of S. gordonii
found to have a potential role in biofilm formation. A previous study has shown that the mannose-sensitive hemagglutination pilus of Vibrio cholerae
, which is involved in biofilm formation on abiotic surfaces, is important for attachment but not pathogenicity (63
). The type IV pili of Pseudomonas aeruginosa
, which is required for biofilm formation on an abiotic surface, is also important for bacterial adhesion to eukaryotic cell surfaces and pathogenesis (47
). These observations suggest that there may be an overlap in the factors required for biofilm formation and those for bacterial adhesion and/or pathogenesis in vivo. As the most abundant enzyme in human saliva, α-amylase, binds with high affinity to oral streptococci (14
), one of the multiple amylase-binding proteins of S. gordonii
) may promote biofilm formation during early plaque formation on nutritionally poor, saliva-coated tooth surfaces. This potential role of α-amylase-binding protein in biofilm formation can be determined only when the two transpositions in the biofilm-defective mutant are separated and analyzed for biofilm formation.
Although flagella and/or motility are important for biofilm formation in motile bacteria, nonmotile bacteria can also form biofilms, indicating that other genes may be involved (47
). The successful isolation of biofilm-defective mutants clearly demonstrates the utility of the assay used. In addition, these mutants were used to identify genes that may be important in biofilm formation after initial adhesion. Nine of the 18 biofilm-defective mutants have disruptions in genetic loci that have no homology to genes in the databases. This polystyrene assay coupled with other assays such as flow cell and animal colonization studies will identify functions for some of these genes.
The relative proportion of the biofilm-deficient mutants isolated in this study (0.07%) was similar to the 0.08% obtained with Tn917
mutants of Staphylococcus epidermidis
were screened for biofilm deficiency (28
) but lower than the 0.3% obtained from Tn5
-based mutants of P. aeruginosa
). Results from this study, together with those of a previous report (57
), indicate that Tn916
appears to preferentially transpose into AT-rich regions of the bacterial chromosome. Therefore, the biofilm-associated genes identified in this study may not represent the full complement of the genes necessary for the sessile growth of S. gordonii
. Additional genes may be identified by insertion-duplication analysis or by using transposons from other gram-positive bacteria for mutagenesis.
In order to understand the processes involved in dental-plaque formation, different approaches, such as confocal scanning laser microscopy, which enables the study of biofilm communities without disturbance (9
), and genetic approaches, need to be utilized. Two-component signal transduction systems (2
) and other cell-to-cell signaling systems (18
) have already become novel targets in the design of new types of microbial anti-infective therapy. Putative biofilm-associated genes may identify other processes important in sessile growth and facilitate the development of therapeutic agents that target the biofilm phenotype and cell-to-cell signaling agents and subsequently prevent the formation or promote the detachment of biofilms. Biofilm-associated genes will provide insight into the unique process of biofilm formation and may facilitate the development of therapeutic agents and strategies to control biofilm-mediated infections.