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The mechanism by which L-nicotine is taken up by bacteria that are able to grow on it is unknown. Nicotine degradation by Arthrobacter nicotinovorans, a Gram-positive soil bacterium, is linked to the presence of the catabolic megaplasmid pAO1. L-[sup.14]C]Nicotine uptake assays with A. nicotinovorans showed transport of nicotine across the cell membrane to be energy-independent and saturable with a [K.sub.m] of 6.2 [+ or -] 0.1 [micro]M and a [V.sub.max] of 0.70 [+ or -] 0.08 [micro]mol [min.sup.-1] [(mg protein).sup.-1]. This is in accord with a mechanism of facilitated diffusion, driven by the nicotine concentration gradient. Nicotine uptake was coupled to its intracellular degradation, and an A. nicotinovorans strain unable to degrade nicotine ([pAO1.sup.-]) showed no nicotine import. However, when the nicotine dehydrogenase genes were expressed in this strain, import of L-[[sup.14]C]nicotine took place. A. nicetinovorans [pAO1.sup.-] and Escherichia ceil were also unable to import 6-hydroxy-L-nicotine, but expression of the 6-hydroxy-L-nicotine oxidase gene allowed both bacteria to take up this compound. L-Nicotine uptake was inhibited by D-nicotine, 6-hydroxy-L-nicotine and 2-amino-L-nicotine, which may indicate transport of these nicotine derivatives by a common permease. Attempts to correlate nicotine uptake with pAO1 genes possessing similarity to amino acid transporters failed. In contrast to the situation at the blood-brain barrier, nicotine transport across the cell membrane by these bacteria was not by passive diffusion or active transport but by facilitated diffusion.

The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [[sup.14]C]nicotine was added to the growth medium the bacteria exported the [sup.14]C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (COOP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [[sup.14]C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR: cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, [gamma]-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate [gamma]-N-methylaminobutyrate.

Sealed cariogenic bacteria are deprived from dietary carbohydrate, but could be provided with nutrients by pulpal fluids, with adaptive strain-specific activities being possible. We investigated survival and metabolic activity of the cariogenic bacteria Streptococcus sobrinus, Actinomyces naeslundii and Lactobacillus rhamnosus in different carbohydrate-limited media without carbon source (CLM), or containing glucose (CLM-G), albumin (CLM-A), or α1-acid glycoprotein (CLM-AGP) in vitro. Bacterial metabolite concentrations (lactate, pyruvate, oxaloacetate, citrate, acetate, formate, ethanol, acetoin) after 20 and 4 hours incubation, and bacterial numbers (CFU) after 24 hours incubation were analyzed using multivariate-analysis-of-variance (MANOVA). The medium (p = 0.02/MANOVA), strain and incubation-time (both p < 0.001) had significant impact on metabolite concentrations. Bacteria secreted mainly lactate (80.3 µg/106 bacteria S. sobrinus) and acetate (54.5 µg/106 bacteria A. naeslundii). Nearly all metabolites were produced in higher concentrations in S. sobrinus than in A. naeslundii or L. rhamnosus (p < 0.05/HSD). Metabolite concentration was significantly higher in CLM-G than in other media for most metabolites (p < 0.05). L. rhamnosus showed significantly lower survival than S. sobrinus and A. naeslundii (p < 0.05/HSD) regardless of the media, while S. sobrinus and A. naeslundii showed medium-specific survival. Survival of carbon starvation was strain- and medium-specific. Sustained organic acid production was found for all strains and media. [ABSTRACT FROM AUTHOR]