Roxanna N. Andersen

Communication among Oral Bacteria

SUMMARY Human oral bacteria interact with their environment by attaching to surfaces and establishing mixed-species communities. As each bacterial cell attaches, it forms a new surface to which other cells can adhere. Adherence and community development are spatiotemporal; such order requires communication. The discovery of soluble signals, such as autoinducer-2, that may be exchanged within multispecies communities to convey information between organisms has emerged as a new research direction. Direct-contact signals, such as adhesins and receptors, that elicit changes in gene expression after cell-cell contact and biofilm growth are also an active research area. Considering that the majority of oral bacteria are organized in dense three-dimensional biofilms on teeth, confocal microscopy and fluorescently labeled probes provide valuable approaches for investigating the architecture of these organized communities in situ. Oral biofilms are readily accessible to microbiologists and are excellent model systems for studies of microbial communication. One attractive model system is a saliva-coated flowcell with oral bacterial biofilms growing on saliva as the sole nutrient source; an intergeneric mutualism is discussed. Several oral bacterial species are amenable to genetic manipulation for molecular characterization of communication both among bacteria and between bacteria and the host. A successful search for genes critical for mixed-species community organization will be accomplished only when it is conducted with mixed-species communities.

SPATIAL ORGANIZATION OF ORAL BACTERIA IN BIOFILMS

Publisher Summary This chapter describes the formation of a monospecies oral streptococcal biofilm that is established with only saliva as both the conditioning film and the growth medium. The chapter discusses confocal laser scanning microscopic images obtained of streptococcal biofilms formed in flowcells. The flowcell described in this chapter is especially useful for studying bacterial biofilms because it is easy to manipulate, is camera ready, and is disposable. It allows real time examination of biofilms by placing the flowcell directly on the microscope stage without interrupting the flow dynamics. Because the flow rate is slow, maintenance of the temperature in the flowcell while on the microscope stage is problematic but can be accomplished by passing the fluid through a heating/cooling device just prior to entering the flow cell. Flowcell is used to study the characteristics of colonization of several streptococci. The chapter investigates the accretion of various other oral bacteria onto the initial colonizing streptococci. The combination of fluorescent probes and the flowcell provides an excellent approach for investigating the spatial organization of oral bacterial biofilms.

Characterization of the Novel Fusobacterium nucleatum Plasmid pKH9 and Evidence of an Addiction System

ABSTRACT Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.

Determination of the Transcript Size and Start Site of the Putative sca Operon of Streptococcus gordonii ATCC 51656 (Formerly Strain PK488)

Streptococci are the primary early colonizers in the accretion of dental plaque. Studies have shown that they can adhere to the acquired pellicle of the tooth, to other oral genera, and to other species of oral streptococci9. The surface protein ScaA from Streptococcus gordonii ATCC 51656 has been characterized and is thought to mediate coaggregation of this organism with another early colonizer Actinomyces naeslundii ATCC 51655 (formerly strain PK606)4. ScaA appears as a 38-kDa protein in immunoblots of surface preparations of the wild type strain ATCC 51656, but the protein is absent or greatly reduced in strain PK1804, an isogenic mutant of strain ATCC 51656 which fails to coaggregate with strain ATCC 51655. Immunoblots of surface preparations from eleven other streptococcal coaggregation partners of ATCC 51655 showed the presence of the 38-kDa protein when probed with PK1804-absorbed anti-ATCC 51656 serum1.