An effector strain has been constructed for use in the replacement therapy of dental caries. Recombinant DNA methods were used to make the Streptococcus mutans supercolonizing strain, JH1140, lactate dehydrogenase deficient by deleting virtually all of the ldh open reading frame (ORF). To compensate for the resulting metabolic imbalance, a supplemental alcohol dehydrogenase activity was introduced by substituting the adhB ORF from Zymomonas mobilis in place of the deleted ldh ORF. The resulting clone, BCS3-L1, was found to produce no detectable lactic acid during growth on a variety of carbon sources, and it produced significantly less total acid due to its increased production of ethanol and acetoin. BCS3-L1 was significantly less cariogenic than JH1140 in both gnotobiotic-and conventional-rodent models. It colonized the teeth of conventional rats as well as JH1140 in both aggressive-displacement and preemptive-colonization models. No gross or microscopic abnormalities of major organs were associated with oral colonization of rats with BCS3-L1 for 6 months. Acid-producing revertants of BCS3-L1 were not observed in samples taken from infected animals (reversion frequency, <10 ؊3 ) or by screening cultures grown in vitro, where no revertants were observed among 10 5 colonies examined on pH indicator medium. The reduced pathogenic potential of BCS3-L1, its strong colonization potential, and its genetic stability suggest that this strain is well suited to serve as an effector strain in the replacement therapy of dental caries in humans.

Streptococcus mutans JH1000 and its derivatives were previously shown (J. D. Hillman, K. P. Johnson, and B. I. Yaphe, Infect. Immun. 44:141–144, 1984) to produce a low-molecular-weight, broad-spectrum bacteriocin-like inhibitory substance (BLIS). The thermosensitive vector pTV1-OK harboring Tn917 was used to isolate a BLIS-deficient mutant, DM25, and the mutated gene was recovered by shotgun cloning inEscherichia coli. Sequence analysis of insert DNA adjacent to Tn917 led to the identification of four open reading frames including two (lanA and lanB) which have substantial homology to the Staphylococcus epidermidisstructural gene (epiA) and a modifying enzyme gene (epiB) for biosynthesis of the lantibiotic epidermin, respectively. Although the BLIS activity could not be recovered from broth cultures, high yields were obtained from a solid medium consisting of Todd-Hewitt broth containing 0.5% agarose that was stab inoculated with JH1140 (a spontaneous mutant of JH1000 that produces threefold-elevated amounts of activity). Agar could not substitute for agarose. Chloroform extraction of the spent medium produced a fraction which yielded two major bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The faster-migrating band was absent in chloroform extracts of the mutant, DM25. The amino acid sequence of this band was determined by Edman sequencing and mass spectroscopy. The results showed that it is a lantibiotic, which we have named mutacin 1140, and that the sequence corresponded to that deduced from thelanA sequence. We observed a number of similarities of mutacin 1140 to epidermin and an S. mutans lantibiotic, B-Ny266, but it appears to have significant differences in the positions of its thioether bridges. It also has other unique features with regard to its leader sequence and posttranslational modification. A proposed structure for mutacin 1140 is presented.

Unsorted, mixed waste office paper (MWOP) is an excellent substrate for conversion into fuel ethanol using a recombinant strain of Klebsiella oxytoca which ferments cellobiose and cellotriose to ethanol at near theoretical yields, eliminating the need for supplemental β‐glucosidase. This organism was tested with commercial fungal cellulase in optimized simultaneous saccharification and fermentation experiments (SSF) using MWOP as a substrate (pH 5–15.2, 35 °C). Similar rates and yields were obtained with dilute acid‐pulped (hydrolysis of hemicellulose) and water‐pulped MWOP on a dry weight basis although viscosity was reduced by the acid pretreatment. In simple batch fermentations, 40 g/L ethanol was produced after 48–72 h with 100 g/L MWOP and 1000 filter paper units (FPU) of cellulase/L, a yield of 550 L of ethanol/metric ton. Cellulase usage was further reduced by recycling SSF residues containing bound enzymes in multistage fermentations. This approach reduced the requirement for fungal cellulase while retaining rapid ethanol production and high ethanol yield. In our optimal design, broths containing an average of 39.6 g/L ethanol were produced in three successive stages with an average fermentation time of 80 h (567 FPU of fungal cellulase/L; 6.1 FPU/g of substrate). This represents a yield of 0.426 g of ethanol/g of substrate, 539 L/metric ton, 129 gal/U.S. ton. MWOP contains approximately 90% carbohydrate. Thus the combined efficiency for saccharification and fermentation to ethanol was 83.3% of the theoretical maximum.