The acquired enamel pellicle is an organic film covering the surfaces of teeth. When this film was first discovered, it was thought to be of embryologic origin. Only in the middle of this century did it become clear that it was acquired after tooth eruption. Initially, the small amounts of material that could be obtained have virtually limited the investigation of pellicle proteins to amino acid analysis. Nevertheless, this technique revealed that the pellicle is mainly proteinaceous and is formed by selective adsorption of salivary proteins on tooth enamel. Later, immunologic techniques allowed for the identification of many salivary and fewer non-salivary proteins as constituents of pellicle. However, to this date, isolation and direct biochemical characterization of in vivo pellicle protein have not been possible, because only a few micrograms can be obtained from a single donor. Therefore, the composition and structure of the acquired enamel pellicle are still essentially unknown. Information on the functions of pellicle has been obtained mainly from in vitro experiments carried out with saliva-coated hydroxyapatite and enamel discs. It was found that pellicle protects enamel by reducing demineralization upon acid challenge. Improved pellicle harvesting procedures and analysis by state-of-the-art proteomics with mass spectroscopy approaches promise to make major inroads into the characterization of enamel pellicle.

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In natural environments heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present at a concentration of a few micrograms per liter or even less. Under such conditions no significant growth would be expected if cells utilized only one of the available carbon compounds, as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon compounds simultaneously. Whereas the kinetics of single-substrate and diauxic growth are well understood, little is known about how microbial growth rates depend on the concentrations of several simultaneously utilized carbon sources. In this study this question was answered for carbon-limited chemostat growth of Escherichia coli fed with mixtures of up to six sugars; the sugars used were glucose, galactose, maltose, ribose, arabinose, and fructose. Independent of the mixture composition and dilution rate tested, E. coli utilized all sugars simultaneously. Compared with growth with a single sugar at a particular growth rate, the steady-state concentrations were consistently lower during simultaneous utilization of mixtures of sugars. The steady-state concentrations of particular sugars depended approximately linearly on their contributions to the total carbon consumption rate of the culture. Our experimental data demonstrate that the simultaneous utilization of mixtures of carbon sources enables heterotrophic microbes to grow relatively fast even in the presence of low environmental substrate concentrations. We propose that the observed reductions in the steady-state concentrations of individual carbon sources during simultaneous utilization of mixtures of carbon sources by heterotrophic microorganisms reflect a general kinetic principle.

Histatins are a group of small cationic peptides in human saliva which are well known for their antibacterial and antifungal activities. In a previous study we demonstrated that histatin 5 kills both blastoconidia and germ tubes of Candida albicans in a time-and concentration-dependent manner at 37°C, whereas no killing was detected at 4°C. This indicated that killing activity depends on cellular energy. To test histatin 5 killing activity at lower cellular ATP levels at 37°C, respiratory mutants, or so-called petite mutants, of C. albicans were prepared. These mutants are deficient in respiration due to mutations in mitochondrial DNA. Mutants were initially identified by their small colony size and were further characterized with respect to colony morphology, growth characteristics, respiratory activity, and cytochrome spectra. The killing activity of histatin 5 at the highest concentration was only 28 to 30% against respiratory mutants, whereas 98% of the wild-type cells were killed. Furthermore, histatin 5 killing activity was also tested on wild-type cells in the presence of the respiratory inhibitor sodium azide or, alternatively, the uncoupler carbonyl cyanide m-chlorophenylhydrazone. In both cases histatin 5 killing activity was significantly reduced. Additionally, supernatants and pellets of cells incubated with histatin 5 in the presence or absence of inhibitors of mitochondrial ATP synthesis were analyzed by sodium dodecyl sulfate gel electrophoresis. It was observed that wild-type cells accumulated large amounts of histatin 5, while wild-type cells treated with inhibitors or petite mutants did not accumulate significant amounts of the peptide. These data showed first that cellular accumulation of histatin 5 is necessary for killing activity and second that accumulation of histatin 5 depends on the availability of cellular energy. Therefore, mitochondrial ATP synthesis is required for effective killing activity of histatin 5.

Several investigations have shown that during growth in carbon-limited chemostats the simultaneous utilisation of carbon substrates which usually provoke diauxie under batch conditions, i.e., 'mixed substrate growth,' is probably the rule under ecologically relevant growth conditions. In contrast, the models presently available for the description of the kinetics of microbial growth are all based on the use of single substrates. Systematic studies in chemostat culture have shown that steady-state residual concentrations of individual compounds were consistently lower during mixed substrate growth than during growth with the single substrates. This effect is clearly demonstrated for the case of Escherichia coli growing with mixtures of glucose plus galactose. The data presented indicate that the extent of reduction of steady-state residual substrate concentration is dependent on the proportions of the substrates in the mixture, the nature of substrates mixed and the regulation pattern of enzymes involved in their breakdown. If this behaviour can be shown to be typical for growth under environmental conditions, it may provide an explanation why microbes still grow relatively fast at the low substrate concentrations encountered in nature.

Most bacterial pathways for the degradation of aromatic compounds involve introduction of two hydroxyl groups either ortho or para to each other. Ring fission then occurs at the bond adjacent to one of the hydroxyl groups. In contrast, 2-aminophenol is cleaved to 2-aminomuconic acid semialdehyde in the nitrobenzenedegrading strain Pseudomonas pseudoalcaligenes JS45. To examine the relationship between this enzyme and other dioxygenases, 2-aminophenol 1,6-dioxygenase has been purified by ethanol precipitation, gel filtration, and ion exchange chromatography. The molecular mass determined by gel filtration was 140,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two subunits of 35,000 and 39,000 Da, which suggested an ␣ 2 ␤ 2 subunit structure. Studies with inhibitors indicated that ferrous iron was the sole cofactor. The K m values for 2-aminophenol and oxygen were 4.2 and 710 M, respectively. The enzyme catalyzed the oxidation of catechol, 6-amino-m-cresol, 2-amino-m-cresol, and 2-amino-4-chlorophenol. 3-Hydroxyanthranilate, protocatechuate, gentisate, and 3-and 4-methylcatechol were not substrates. The substrate range and the subunit structure are unique among those of the known ring cleavage dioxygenases.Ring cleavage is a key reaction in microbial degradation of aromatic compounds. In aerobic bacteria, it is usually catalyzed by dioxygenases (17). Typically, substrates of ring cleavage dioxygenases feature an aromatic ring substituted with two hydroxyl groups oriented either ortho or para to each other (26). Indeed, nearly all bacterial catabolic pathways transform aromatic substrates to catechol or gentisate and their derivatives, which subsequently undergo ring cleavage (10).Recently, we reported the discovery of 2-aminophenol 1,6-dioxygenase, a new ring cleavage enzyme that catalyzes the direct conversion of 2-aminophenol to 2-aminomuconic acid semialdehyde. The enzyme is induced during the growth of Pseudomonas pseudoalcaligenes JS45 on nitrobenzene. The degradation pathway (Fig. 1A) starts with reduction of nitrobenzene to hydroxylaminobenzene followed by rearrangement to 2-aminophenol (19).Two enzymes have been reported to catalyze the ring cleavage of 2-aminophenol at very low rates. Catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 catalyzes extradiol ring fission of 2-aminophenol at a rate that is 0.1% that of the intradiol cleavage of catechol (27). 2-Aminophenol was oxidized by catechol 2,3-dioxygenase from P. arvilla at a rate less than 0.01% of the rate with catechol (14, 22). These low turnover rates indicate that 2-aminophenol is not a physiological substrate of catechol 1,2-or 2,3-dioxygenase. To our knowledge, this is the first report of an enzyme that cleaves 2-aminophenol as its primary substrate.We have purified and characterized 2-aminophenol 1,6-dioxygenase from P. pseudoalcaligenes JS45 to determine how it is related to previously known ring cleavage enzymes. The tertiary structure, substrate range, and cofactor requirement were compared with that of other ri...