Involvement of the adc Operon and Manganese Homeostasis in Streptococcus gordonii Biofilm Formation

Oral streptococci (viridans streptococci) initiate the formation of oral biofilms known as dental plaque and are ubiquitous primary colonizers that are the predominant cultivable bacteria found in supragingival plaque (45). Viridans streptococci are also frequent etiologic agents of bacterial endocarditis (21, 64) and are becoming significant opportunistic pathogens and a major cause of bacteremia in immunocompromised patients, accounting for 40% of infections in neutropenic patients (25, 32). Fully developed, surface-attached oral biofilms are highly structured, with distinct architectural and physiochemical properties commonly observed with other biofilm communities (12). Sessile bacteria found in biofilms represent a distinct mode of growth that differs from planktonic cells, characterized by differences in gene expression and cellular physiology and an increased resistance to antibiotics. The multistep process of biofilm formation has just begun to be understood (12). This complex biofilm developmental process is initiated by the attachment of the colonizing bacteria to a surface. Subsequent accumulation and growth of attached bacteria result in microcolonies that increase in size and eventually appear as towering pillar- and mushroom-shaped biofilms. The growth of this complex, multilayered, cellular matrix-embedded community and its eventual dispersal and spread require the coordinated expression of an array of genes (57, 59). These include general housekeeping genes and some global regulatory genes, which have been identified by using genetic analyses of biofilm-defective phenotypes, DNA microarrays, and proteomic analysis (4, 38, 46, 61, 63, 68, 69). Thus, the transition between planktonic and sessile phenotypes does not appear to arise from the expression of unique genes but probably requires a differential pattern of gene expression as the bacteria enter a distinct physiological state. The same attached bacteria can revert to planktonic cells by switching off this coordinated gene expression (59). Assays that measure the ability of bacteria to form adherent biofilms on abiotic surfaces by staining attached bacterial biofilm with crystal violet or safarinin have been used extensively to screen for biofilm-defective mutants. Specific genes involved in single-species biofilms have been identified in a variety of bacteria, including Escherichia coli (24, 52, 54), Staphylococcus aureus (13), Staphylococcus epidermidis (43), Streptococcus gordonii (42), Streptococcus mutans (68, 71), Pseudomonas aeruginosa (48), Pseudomonas fluorescens (49), Salmonella enterica (44), and Vibrio cholerae (67). Identifying and analyzing biofilm-defective mutants resulted in the identification of a number of biofilm-associated genetic loci. These include genetic competence (39, 40, 42), quorum sensing and signaling (4, 39, 40, 42), exopolysaccharide synthesis (27, 70), and specific bacterial surface proteins (22, 36, 52, 55, 67). A number of transcriptional regulators that are stress inducible and growth phase dependent have been found to modulate bacterial biofilm formation. Biofilm formation of S. epidermidis was found to require the expression of RsbU, a positive regulator of σB, an alternative sigma factor that modulates biofilm formation during environmental challenges by altering the expression of the polysaccharide intercellular adhesin (33). A transcriptional repressor in S. epidermidis encoded by icaR is involved in both the environmental regulation of the ica operon and biofilm formation (10). The E. coli OmpR/EnvZ two-component regulatory system modulates biofilm formation in response to changes in osmolarity through a complex regulatory network that controls expression of curli (53), whereas the Cpx two-component signal transduction system is involved in the attachment of stationary-phase E. coli cells to hydrophobic abiotic surfaces (18, 50). PvrR, a putative sensor regulator homolog, modulates antibiotic resistance and biofilm formation of P. aeruginosa during phase variation (19). Crc, the global carbon metabolism regulator in P. aeruginosa (47), and CcpA, the catabolite control protein in S. mutans (68), are necessary during biofilm formation on abiotic surfaces. CytR, a transcriptional repressor that inhibits polysaccharide synthesis in V. cholerae, also modulates biofilm development (26). A ToxR homolog that regulates its own synthesis in Vibrio anguillarum serotype 01 has been implicated in biofilm formation on glass surfaces (66). The availability of microbial genome sequences has enabled investigators to mutate global transcriptional regulators identified in silico and to identify several biofilm-associated genetic loci among these regulators in the oral pathogen S. mutans (4, 38-40, 68). Taken together, these studies demonstrated that certain transcriptional regulators play a crucial role in biofilm formation by regulating the expression of biofilm-related genes and reinforced the concept that coordinated gene expression may modulate the transition between the planktonic and sessile phenotypes. A mutant generated by transcriptional fusion with a reporter gene would represent an important advantage in the study of genes involved in biofilm formation, as it is an important tool for facilitating studies of gene regulation. In order to investigate factors that may regulate the expression of biofilm-associated genes, the gram-positive transposon Tn917, containing the easily detectable promoterless E. coli lacZ reporter gene, was used to generate insertion-mediated transcriptional fusions in S. gordonii and the mutants generated were screened to identify genes associated with biofilm formation. An in vitro biofilm formation assay (42) was used to screen for biofilm-defective mutants. The use of Tn917-lac (Eryr, Gal+) (14) enabled the detection of the levels of gene expression under various environmental conditions. The aims of this study were to identify a biofilm-associated gene(s) in S. gordonii and to determine the influence of environmental factors on the expression of the gene(s) identified.