The sanguis streptococci are primary colonizers of the tooth surface and thus form the foundation for the complex multiple species biofilm known as dental plaque. In addition, these bacteria can colonize native and prosthetic heart valves and are a common cause of endocarditis. Little is known about the molecular mechanisms governing multiple or single species biofilm development within this group of organisms. Using an in vitro assay for biofilm formation, we determined that (i) Streptococcus parasanguis FW213 can form biofilms on inert surfaces such as polystyrene and (ii) environmental and nutritional factors, such as glucose, affect S. parasanguis biofilm formation. Several isogenic mutants of FW213 were tested in the biofilm assay. Strains containing mutations in fap1, a gene encoding a protein required for assembly of fimbriae, were deficient in biofilm formation. Mutants defective in recA, PepO endopeptidase activity, or the production of a fimbriaeassociated protein, FimA, were still capable of biofilm formation. Phase-contrast microscopy was used to follow biofilm development by wild-type and fap1 mutant strains on plastic coverslips over time. Wild-type FW213 attached to the surface, formed aggregates of cells, and eventually formed a dense layer of cells that included microcolonies. In contrast, few fap1 mutant cells were observed attached to the surface, and no cell aggregates or microcolonies were formed. These results suggest that the long peritrichous fimbriae of FW213 are critical for the formation of biofilms on solid surfaces.

NUT1, a gene homologous to the major nitrogen regulatory genes nit-2 of Neurospora crassa and areA of Aspergillus nidulans, was isolated from the rice blast fungus, Magnaporthe grisea. NUT1 encodes a protein of 956 amino acid residues and, like nit-2 and areA, has a single putative zinc finger DNA-binding domain. Functional equivalence of NUT1 to areA was demonstrated by introducing the NUT1 gene by DNA-mediated transformation into an areA loss-of-function mutant of A. nidulans. The introduced NUT1 gene fully complemented the areA null mutation, restoring to the mutant the ability to utilize a variety of nitrogen sources. In addition, the sensitivity of Aspergillus NUT1 transformants to ammonium repression of extracellular protease activity was comparable to that of wild-type A. nidulans. Thus, NUT1 and areA encode functionally equivalent gene products that activate expression of nitrogen-regulated genes. A one-step disruption strategy was used to generate nut1- mutants of M. grisea by transforming a rice-infecting strain with a disruption vector in which a gene for hygromycin B phosphotransferase (Hyg) replaced the zinc-finger DNA-binding motif of NUT1. Of 31 hygromycin B (hyg-B)-resistant transformants shown by Southern hybridization to contain a disrupted NUT1 gene (nut1 : : Hyg), 26 resulted from single-copy replacement events at the NUT1 locus. Although nut1- transformants of M. grisea failed to grown on a variety of nitrogen sources, glutamate, proline and alanine could still be utilized. This contrasts with A. nidulans where disruption of the zinc-finger region of areA prevents utilization of nitrogen sources other than ammonium and glutamine. The role of NUT1 and regulation of nitrogen metabolism in the disease process was evaluated by pathogenicity assays. The infection efficiency of nut1- transformants on susceptible rice plants was similar to that of the parental strain, although lesions were reduced in size. These studies demonstrate that the M. grisea NUT1 gene activates expression of nitrogen-regulated genes but is dispensable for pathogenicity.

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The study of how bacteria respond to and obtain divalent metal ions provides insight into the regulation of virulence factors in the host environment. Regulation of metal permease operons in gram-positive bacteria may involve the binding of metal-responsive repressors to palindromic domains in their control regions. The Streptococcus parasanguis fimA operon, which encodes an ATP-binding cassette (ABC) transporter system with sequence homology to the LraI family of metal transporters, possesses a palindromic regulatory region with high homology to that of the Streptococcus gordonii ScaR binding domain. Mapping of the promoter and regulatory regions of fimA and the divergently transcribed pepO gene, which encodes a zinc metalloendopeptidase, indicated that their promoter and regulatory elements overlap. fimA had one transcriptional start site, whereas pepO had three. Analysis of truncated versions of the pepO promoter suggested that all three transcriptional start sites are functional. Analysis of promoter activity under various environmental conditions indicated that the fimA operon promoter and the pepO promoter are not coordinately regulated. Streptococcus parasanguis, along with other members of the mitis group of oral streptococci, are among some of the most successful colonizers of the human body. These commensal organisms in the oral cavity have the ability to attach, colonize, and thrive in an environment of continual flux of pH, temperature, mechanical stress, and nutrient availability. Introduction of these oral organisms into the bloodstream of individuals with predisposing heart valve damage can result in endocarditis, a life-threatening illness (3). Nutrients, in particular divalent metal ions, are often sequestered by the host in such a way that the colonizing bacteria must actively gain access to these resources in order to survive in the host environment. Iron in the form of ferric and ferrous compounds is essential for the growth and survival of gram-negative bacterial pathogens, and the ability to acquire these nutrients is considered a virulence trait (8,34). In gram-positive organisms, such as the streptococci, the role of divalent metals in virulence is less welldefined. Evidence indicates that the lipoprotein receptor-associated antigen I (LraI) family of polypeptides found in a variety of streptococci (4,7,22,23,35) and of which FimA of S. parasanguis FW213 is a member forms part of a new family of solute-binding receptors of ABC metal ion transporters. Previous studies have shown that Streptococcus gordonii (24) and Streptococcus pneumoniae (9) transporters mediate uptake of Mn 2ϩ , while recent work indicates that the Streptococcus pyogenes LraI polypeptide binds Zn 2ϩ , Cu 2ϩ , and Fe 3ϩ (21). S. parasanguis FimA is a major virulence factor associated with endocarditis, and it has been suggested that FimA functions in the development of the infection by facilitating adherence to fibrin (5). Other members of the LraI family, including PsaA of S. pneumoniae and SloC of Streptococcus mutans,...