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Genome sequence data provide a framework for predicting potential microbial activities; however, the proteome content of the cell dictates its response to its environment. Microbiology is witnessing a major initiative to elucidate the nature of the proteome of large numbers of species. The tool driving the proteomic revolution is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. During the analysis process, proteins are ionized and separated on the basis of their mass-to-charge ratios, which results in a characteristic mass-spectral profile. Because of the dynamic nature of the cell and the large number of external parameters that could influence its mass-spectral profile, considerable work was needed initially to optimize sample analysis and obtain consistent and reproducible results. For many anaerobes that grow poorly or are nonreactive in most diagnostic systems, proteome analysis is likely to have a major impact on microbial diagnosis and the delineation of centers of diversity associated with infections.

There is now increasing evidence that surface-associated enzymes, previously considered to be involved in intermediary metabolism or virulence, play a role in physiological reactions such as signal transduction, transport systems, and metabolic processes. Herein we report the molecular aspects of two such enzymes, the cysteine proteinase gingivain and NAD-dependent glutamate dehydrogenase of Porphyromonas gingivalis. The gdh gene comprises an open reading frame of 1,335 base pairs that encodes a 49,000-M(r) protein of 445 amino acids. The gdh gene showed high homology (78.3%) with that of Clostridium symbiosum. Optimal codons accounted for 35.9% of the total codon usage, indicating high expression of this enzyme. These data are currently being used to carry out targeted mutagenesis, which was established here for gingivain. Conditions for targeted mutagenesis within the histidine domain of the catalytic site of gingivain using Tn 4351 was successfully achieved. Consequently, the catalytic functions, such as gingivain's capacity to hydrolyze the synthetic substrate alpha-benzoyl-arginine-4-nitroanilide, were disrupted.

The periodontal pocket provides a unique structural site for studies on host/bacterial interactions. The pocket is colonized by a complex but characteristic anaerobic bacterial flora. Many new taxa have been described that have now been supported by comparative rRNA sequence analysis. Within recent years, several molecular approaches have been used to describe both cultivable and noncultivable species, and new genotypes have been reported. Microbial activity rather than the mere presence of a microorganism at a site must be a major factor in the process of disease development. Information on metabolic activities of species and identification of substrates are therefore essential to elucidate the complex interactions that are likely to occur in vivo. We have been using a variety of analytical procedures such as 13C substrate-enrichment nuclear magnetic resonance, 14C isotopic labeling experiments, enzyme assays, impedance measurements, and various chromatographic and electrophoretic procedures to study key species of this predominantly asaccharolytic flora. Results have so far indicated a major role for cationic and anionic acids as sources of energy, but the mechanisms of substrate processing may differ significantly between species. In this ecosystem, crevicular fluid is the likely source of nutrients for species. Components of this fluid appear to have a role in the selection of species in subgingival sites.

Over the last decade, biochemical and chemical analyses have been used widely to study the intrageneric structure of Bacteroidaceae. New chromogenic substrates (e.g., naphthylamide-linked compounds) and fluorogenic substrates (e.g., 4-methylumbelliferyl or 7-amido-4-methyl-coumarin compounds) can be used to identify certain species within a few hours under aerobic conditions. Clarification of the taxonomy of many oral anaerobic species that are now considered important putative periodontal pathogens has permitted the development of panels of both polyclonal and monoclonal antibodies for their detection. Similarly, both DNA and RNA gene probes, derived through nucleic acid sequence analysis, have been constructed for several species; many such probes are now commercially available. In the long term, the application of these techniques will lead to a better understanding of the distribution, transmission, and ecological and clinical importance of many species that have hitherto remained poorly characterized.

The growth response of Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola to peptides (supplied as trypticase) and amino acids (supplied as casamino acids) was measured over 24 hours by monitoring changes in alternating-current conductivity at 37 degrees C. All species utilized peptides preferentially over amino acids. These results are consistent with those obtained previously with conventional growth-response experiments. The duration of the linear growth response to trypticase of P. gingivalis was 9.7 hours, whereas that of both F. nucleatum and T. denticola was > 24 hours. By contrast, there was more uniformity in the utilization of amino acids from the casamino acid mixture, which previously has been shown to be a poor growth substrate. Furthermore, subtle differences in growth patterns, such as the ability of F. nucleatum to metabolize its storage glycopolymers before utilizing amino acids, were clearly evident. The present method provides an excellent means of studying bacterial growth kinetics and delineating bacterial/substrate specificities of both synthetic and natural substrates.

Recent taxonomic changes among Bacteroides and related species has led to a clearer insight into the site specificity of these taxa. Two recently described centers of variation within the genus Bacteroides sensu stricto and Prevotella intermedia are reported. However, considerable taxonomic problems still remain unresolved within this large and complex group of microorganisms. These bacteria are metabolically diverse, and this is reflected in their colonization sites. In the rumen, gastrointestinal tract, or the oral cavity, specific members of this group contribute significantly to the initial attack on both simple and complex carbohydrates, and by doing so, obtain carbon and energy for their survival. Depletion of carbohydrates from the milieu results in a gradual change in microbial activity to proteolysis. In some sites, such as the periodontal pocket, potent proteinases produced by species such as Porphyromonas gingivalis may facilitate degradation of available proteins to their constituent peptides and amino acids. These products are not only likely to affect the growth of these species, but must profoundly affect the nutrient network of this ecosystem.