Resistance to lincomycin and clindamycin in the clinical isolateEnterococcus faecium HM1025 is due to a ribosomal methylase encoded by an ermAM-like gene and the plasmid-mediated inactivation of these antibiotics. We have cloned and determined the nucleotide sequence of the gene responsible for the inactivation of lincosamides, linB. This gene encodes a 267-amino-acid lincosamide nucleotidyltransferase. The enzyme catalyzes 3-(5′-adenylation) (the adenylation of the hydroxyl group in position 3 of the molecules) of lincomycin and clindamycin. Expression oflinB was observed in both Escherichia coli andStaphylococcus aureus. The deduced amino acid sequence of the enzyme did not display any significant homology with staphylococcal nucleotidyltransferases encoded by linA andlinA′ genes. Sequences homologous to linB were found in 14 other clinical isolates of E. faecium, indicating the spread of the resistance trait in this species.

The development and use of a new assay system for the simultaneous determination of identity, purity, and concentration of sample components from combinatorial libraries produced by parallel synthesis are described. The system makes use of high-performance liquid chromatography with UV/vis photodiode array (PDA), evaporative light scattering (ELSD), chemiluminescent nitrogen (CLND), and time-of-flight mass spectrometer (TOFMS) detectors (HPLC-PDA-ELSD-CLND-TOFMS). Although these detectors have previously been utilized separately for the analysis of combinatorial chemistry libraries, the use of TOFMS along with CLND provides a synergistic combination enabling target and side-product structures to be identified and their concentrations and purities determined in a single experiment from a solution containing microgram levels of material. The CLND was found to give a linear response based on the number of moles of nitrogen present. Therefore, if the number of nitrogens per molecule is known, the concentration of each nitrogen-containing sample component may be determined utilizing an unrelated co-injected standard. A molecular formula for an impurity may often be calculated from the exact mass determined by the TOFMS and knowledge of the chemistry involved. Thus, if the sample components contain nitrogen, the concentration of every identified HPLC peak may be determined even in the absence of primary standards. This combination of detectors enabled the characterization of both target compounds and byproducts in combinatorial libraries, allowing the optimization of library synthetic procedures. This system was also used to survey the quality of libraries, enabling the selection of the best libraries for screening. This method also facilitated the characterization of samples from combinatorial libraries found as hits in high-throughput screening to establish the potency of the leads based on their actual concentration. In addition, concentrations and potencies of impurities were determined after identification of their structures, utilizing exact mass data, determination of charge states, and knowledge of the synthetic chemistry.

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An accumulated lincomycin intermediate in UC8292, a lincomycin nonproducing strain of Streptomyces lincolnensis, has been isolated and purified by employing an assay system based on complementation of UC11066, another lincomycin nonproducing strain of S. lincolnensis. The structure of the purified intermediate is shown to be 3-propylidene-z1 1-pyrroline-5-carboxylic acid, or 1, 2, 3, 6-tetradehydro-propylproline by mass spectrometry and NMRspectroscopic studies. Based on the structure of this newly found intermediate, a biosynthetic pathway for propylproline is proposed as tyrosine-»L-3-hydroxytyrosine (Dopa) -^-> -^->3-propylidene-zl 1-pyrroline-5-carboxylic acid -åº 3-propyl-zl 2-pyrroline-5-carboxylic acid ->>propylproline. 1773It has been shown previously that Streptomyces lincolnensis strain UC8292 (originally designated NTG-3), a lincomycin nonproducer, is incapable of synthesizing lincomycin cosynthetic factor (LCF), a deazariboflavin1'2^Lacking this deazariboflavin, UC 8292 cannot synthesize propylproline and consequently lincomycin. In this paper, we report that a previously unidentified lincomycin intermediate in UC 8292 has been isolated and purified by employing an assay system based on complementation of S. lincolnensis strain UC 1 1066 (originally designated IIc-2-3-3)3), another lincomycin nonproducer. Materials and MethodsBacterial Strains Two mutants of S. lincolnensis blocked in the biosynthesis of lincomycin were used in this study, UC 8292, a riboflavin auxotroph which does not produce LCFnecessary for the biosynthesis of lincomycin1'2), and UC1 1066, a mutant which does not produce propylproline, an intermediate in the lincomycin biosynthetic pathway. The latter strain was obtained from a derivative of S. lincolnensis strain UC5124 by transposon mutagenesis with the Streptomycesfradiae transposon Tn45603\Fermentation Media and Conditions The media and fermentation conditions for shake flask fermentations were described previously2*. Twenty liter fermentations were carried out in the same media at 29°C with an agitation speed of 700 rpm and an aeration rate of 20liters per minute. Both flasks and tanks were harvested at 4 days. Assay for Lincomycin Biosynthetic Intermediates (LBI)In an effort to find a nonproducing strain of S. lincolnensis capable of complementing UC8292, several other strains, including UC1 1066, were tested in cross-feeding experiments. Fermentations of all strains were carried out in 500ml shake flasks and supernatants and mycelia mixed in all combinations at 96 hours. Mixed test flasks were incubated for an additional 24 hours and assayed for lincomycin activity. Addition ofUC 8292 supernatant to mycelia of UC1 1066 also resulted in lincomycin production indicating that the genetic lesion in UC11066 precedes the block in UC8292. Based on these results, S. lincolnensis