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ABSTRACTResistance to macrolides in pneumococci is generally mediated by methylation of 23S rRNA via erm(B) methylase which can confer a macrolide (M)-, lincosamide (L)-, and streptogramin B (SB)-resistant (MLSB) phenotype or by drug efflux via mef(A) which confers resistance to 14- and 15-membered macrolides only. We studied 20 strains with unusual ML or MSBphenotypes which did not harbor erm(B) ormef(A). The strains had been isolated from patients in Eastern Europe and North America from 1992 to 1998. These isolates were found to contain mutations in genes for either 23S rRNA or ribosomal proteins. Three strains from the United States with an ML phenotype, each representing a different clone, were characterized as having an A2059G (Escherichia colinumbering) change in three of the four 23S rRNA alleles. Susceptibility to macrolides and lincosamides decreased as the number of alleles in isogenic strains containing A2059G increased. Sixteen MSBstrains from Eastern Europe were found to contain a 3-amino-acid substitution (69GTG71to TPS) in a highly conserved region of the ribosomal protein L4 (63KPWRQKGTGRAR74). These strains formed several distinct clonal types. The single MSBstrain from Canada contained a 6-amino-acid L4 insertion (69GTGREKGTGRAR), which impacted growth rate and also conferred a 500-fold increase in MIC on the ketolide telithromycin. These macrolide resistance mechanisms from clinical isolates are similar to those recently described for laboratory-derived mutants.

Urea has been shown to be an obligate intermediate in and the penultimate product of the catabolism of pyrimidine-ring nitrogen in Rhodosporidium toruloides (Rhodotorula). One of a series of mutants selected for its inability to utilize uracil as a sole source of nitrogen was unable to utilize urea also. The mutant accumulated urea and failed to form 14CO2 during supplementation with [2-14C]uracil. Radioautograms from the resulting cell extracts and media failed to reveal expected intermediates. Cell-free extracts of the mutant were shown to lack urease activity. Revertants of the mutant were essentially wild type in all tested attributes. Elements of the reductive pathway for pyrimidine catabolism are present in Rhodosporidium (O. A. Milstein and M. L. Bekker, J. Bacteriol. 127: 1-6, 1976), but is has not been determined whether this pathway is involved with production of urea.

Rough colonial morphology and bacteriocin production, two properties which may be associated with the cariogenicity of Streptococcus mutans, were transformed into several strain GS-5 mutants defective in each respective property. Transformation was determined by observing the frequency of cotransfer of these properties with different reference markers. The rough colonial transformants were identical to the parental GS-5 strain with respect to ability to synthesize water-insoluble glucans and undergo in vitro sucrose-dependent colonization of glass surfaces. Alterations in the growth medium and the concentration of the initial cell inoculum resulted in an approximate 10-fold increase in the frequency of transformation of strain GS-5 compared to previous observations.

The antibiotic cerulenin differentially inhibited the production of glucosyltransferase activity by Streptococcus mutans GS5. Cerulenin preferentially inhibited [14C]acetate incorporation into cellular lipids but did not affect protein synthesis or ribonucleic acid synthesis in the same manner. No significant intracellular accumulation of glucosyltransferase activity could be demonstrated in cultures treated with cerulenin. On the other hand, another inhibitor of lipid synthesis, sodium chlorophenoxyisobutyrate, did not differentially inhibit glucosyltransferase expression. In addition, the role of a metal-requiring protease in the production of glucosyltransferase activity was suggested by the observation that the chelator quinacrine differentially inhibited the production of the enzyme.

Both dextransucrase and mutansynthetase activities have been purified from the culture fluids of Streptococcus mutans GS-5 (serotype c). Although homogeneous dextransucrase preparations normally synthesize little insoluble glucan, essentially all of the glucan synthesized by this enzyme in the presence of 1.5 M (NH4)2SO4 was water insoluble. Linkage analysis of the insoluble glucans indicated that the presence of NH4+ increased the portion of alpha-1,3-glucose linkages relative to alpha-1,6-glucose units in the product. Chromatofocusing of aggregated glucosyltransferase fractions synthesizing predominantly insoluble glucan yielded primarily dextransucrase activity separable from relatively low levels of mutansynthetase activity. The latter enzyme was detected only in 18-h assays and synthesized primer-dependent insoluble glucan, which was decreased in the presence of NH4+. In the absence of primer dextran T10, the addition of dextransucrase also stimulated insoluble glucan synthesis by mutansynthetase. Dextransucrase and mutansynthetase appear to be distinct enzymes, since the latter possesses a higher molecular weight (155,000 compared to 140,000), a much lower isoelectric point, and did not cross-react with antibody directed against dextransucrase. These results are discussed relative to the mechanism of insoluble glucan synthesis by S. mutans serotype c strains.

The Streptococcus mutansGS5 glucosyltransferase activities (both water-soluble and -insoluble glucan-synthesizing fractions) were inhibited by purified lipoteichoic acid. In vitro sucrose-dependent colonization of smooth surfaces by strain GS5 was also markedly reduced in the presence of the amphipathic molecules. The inhibition of soluble glucan synthesis by lipoteichoic acid appeared to be competitive with respect to both sucrose and primer dextran T10. These inhibitory effects were dependent on the presence of the fatty acid components of lipoteichoic acid since deacylated lipoteichoic acids did not inhibit glucosyltransferase activity. However, the deacylated molecules did interact with the enzymes since deacylated lipoteichoic acid partially protected the enzyme activity against heat inactivation and also induced the formation of high-molecular-weight enzyme complexes from the soluble glucan-synthesizing fraction. The presence of teichoic acid in high-molecular-weight aggregates of glucosyltransferase isolated from the culture fluids of strain GS5 was suggested by the detection of polyglycerophosphate in these fractions. In addition to strain GS5, two other organisms containing polyglycerophosphate teichoic acids, Lactobacillus caseiand Lactobacillus fermentum, were demonstrated to bind glucosyltransferase activity. These results are discussed relative to the potential role of teichoic acid-glucosyltransferase interactions in enzyme binding to the cell surface of S. mutansand the formation of high-molecular-weight enzyme aggregates in the culture fluids of the organism.