Purine nucleotides are essential precursors for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and the biosynthesis of several amino acids and vitamins such as riboflavin. GTP is the immediate precursor for riboflavin biosynthesis, and its formation through the purine pathway is subject to several regulatory mechanisms in different steps. Extracellular purines repress the transcription of most genes required for de novo ATP and GTP synthesis. Additionally, three enzymes of the pathway, phosphoribosyl pyrophosphate (PRPP) amidotransferase, adenylosuccinate synthetase, and IMP dehydrogenase, are subject to feedback inhibition by their end products. Here we report the characterization and manipulation of the committed step in the purine pathway of the riboflavin overproducer Ashbya gossypii. We report that phosphoribosylamine biosynthesis in A. gossypii is negatively regulated at the transcriptional level by extracellular adenine. Furthermore, we show that ATP and GTP exert a strong inhibitory effect on the PRPP amidotransferase from A. gossypii. We constitutively overexpressed the AgADE4 gene encoding PRPP amidotransferase in A. gossypii, thereby abolishing the adenine-mediated transcriptional repression. In addition, we replaced the corresponding residues (aspartic acid 310 , lysine 333 , and alanine 417 ) that have been described to be important for PRPP amidotransferase feedback inhibition in other organisms by site-directed mutagenesis. With these manipulations, we managed to enhance metabolic flow through the purine pathway and to increase the production of riboflavin in the triple mutant strain 10-fold (228 mg/liter).

Selective interaction of mutans streptococci with benign bacteria could present an opportunity for their removal from the mouth without disruption of other oral flora. This study was conducted to find probiotic lactobacilli that could specifically co-aggregate in vitro with mutans streptococci, but not with other plaque commensals. A search of 624 lactobacilli among a large culture library revealed 6 strains, all classifiable as L. paracasei or L. rhamnosus, which met this criterion. Such novel, specific co-aggregation, however, was not a general characteristic of these species or the genus Lactobacillus. The co-aggregation by these specific lactobacilli was characterized as heat treatment (autoclaving)- and protease-resistant, lectin-independent, not inactivated by sugar substitutes, operational over a wide pH range, unaffected by whole saliva, but calcium-dependent. It is thus seen to present a potential strategy for in vivo alteration of plaque biofilm and caries.

A gene (bos) coding for bacterioopsin (BO), the apoprotein of bacteriorhodopsin was assembled from chemically synthesized oligonucleotides by a new method of repeated rounds of insertion mutagenesis. The gene sequence was designed for convenient manipulation in future protein engineering experiments. In-frame fusion of bos to the lacZ454 gene allowed high-yield production in Escherichia coli of a fl-Ga1454/BO fusion protein, deposited as intracellular inclusion bodies. These were enriched by virtue of their insolubility in 0.5% Triton X-100 and cleaved in aqueous suspension with IgA protease at a specific site provided at the P-Ga1454/BO boundary. Pure BO could be obtained from the mixture of water-insoluble cleavage products by selective extraction into organic solvent. The yield was in the range 30-50 mg pure protein/l culture medium, depending on individual preparation. This material could be used for reconstitution of fully functional bacteriorhodopsin. Taken together, the procedure constitutes a practical basis for the production of genetically engineered bacteriorhodopsins.

Lactobacillus paracasei DSMZ16671, even if heat-killed, sensitively co-aggregates mutans streptococci specifically. Mutans streptococci are strongly implicated in caries induction in humans and rodents. We hypothesized: (1) that S. mutans recoveries from rats' teeth in vivo will decrease, with an associated decrease in caries, if these lactobacilli are fed to rats in an established caries model; and (2) that toxicological assays of these lactobacilli will show them to be benign. Four groups of specific-pathogen-free rats were formed: S. mutans 10449S-inoculated/16671-supplemented diet; un-inoculated/16671-supplemented diet; S. mutans 10449S-inoculated/placebo diet; and un-inoculated/placebo diet. Standard tests of toxicity and mutagenicity of heat-killed DSMZ16671 were performed. S. mutans recoveries were significantly reduced both in mid-experiment and at termination, as were caries lesion scores for the rats inoculated by S. mutans and fed the DSMZ16671 supplement, by comparison with controls. Neither toxicity nor mutagenicity of DSMZ16671 was detected. Use of heat-killed DSMZ16671 is efficacious in rats and appears safe.