A UV-sensitive derivative was obtained from Streptococcus sanguis Challis. The organism could be transformed with a number of small streptococcal plasmids at frequencies equal to, or 1 logarithm below, the transformation frequencies for the parent organism. However, transformation with chromosomal DNA was greatly impaired in the UV-sensitive derivative.Streptococcus sanguis Challis is a transformable organism useful for the study of streptococcal gene expression. Before the initiation of a series of gene-cloning experiments, it seemed desirable to obtain a recombination-deficient derivative to minimize the occurrence of classical host recombination events (1). A series of recombination-deficient mutants of S. sanguis Challis has been described by Raina and Macrina (5). These derivatives were obtained after screening for sensitivity to methyl methanesulfonate (MMS), which occurred in 4% of 7,000 mutagenized colonies, followed by screening for UV sensitivity (five colonies) and further selection for inability to be transformed with chromosomal DNA (one colony, called cipA9). However, in both cipA9 and its cipA-11 derivative obtained by those workers, transformation was severely impaired when both chromosomal DNA and DNA obtained from plasmids in the 5to 7megadalton range was used. This last impairment would reduce the usefulness of the derivatives in gene-cloning experiments.We report here the isolation of an S. sanguis derivative which appears to be recombination deficient by standard criteria (1, 7) but is capable of transformation with the streptococcal plasmids pVA1 and pVA736 (7.3 and 5.0 megadaltons, respectively), as well as with a Streptococcus-Escherichia coli shuttle vector, pVA856 (9.2 megadaltons). The existence of such a strain suggests that the missing recombination function in our derivative affects chromosomal but not plasmid-mediated recombination events. The plasmids were provided by F. Macrina, Virginia Commonwealth University, Richmond (3, 5).S. sanguis 685 was mutagenized with 100-,ug/ml N-methyl-N'-nitro-N-nitrosoguanidine as described by Yagi and Clewell (7), and the survivors were washed and suspended in Todd-Hewitt broth containing 2% glucose and 0.1% cysteine. S. sanguis 685, which was provided by F. Macrina, is a derivative of the Challis strain V288 and contains the pVA380-1 helper plasmid (6). After replica plating onto the same medium (minus cysteine) containing 2% agar, 24-h-old colonies were picked and replica plated onto two plates, one of which was irradiated for 40 s with UV light (160 ergs/cm2 per s), and examined for viability at 48 h. Of 7,080 colonies tested, 1 colony, UV-48, was found to be UV sensitive by this criterion.When DNA from a rifampinor penicillin-resistant derivative of S. sanguis 685 was used to transform the parent and * Corresponding author. NOTESon August 1, 2020 by guest http://iai.asm.org/ Downloaded from

Tolerant strains of Streptococcus faecium had higher levels of muramidase 2 and lower levels of trypsinactivable muramidase 1 than did susceptible strains. Susceptible strains lysed faster than did tolerant strains in buffer and at some antibiotic concentrations. The addition of Triton X-100 produced equal lysis rates for susceptible and tolerant cultures.We recently reported that Streptococcus faecium ATCC 9790 was tolerant (T) of the lethal action of penicillin G as well as of that of bacitracin, vancomycin, and D-cyclOserine (8). Single-cell-colony isolates of ATCC 9790 exhibited an approximate 5% loss of tolerance. A lineage analysis over many transfers indicated that the change from tolerance to susceptibility was reversible and that susceptible (S) derivatives became tolerant at the same 5% frequency (8). The T and S cultures studied differed from each other in the extent of survival after exposure to penicillin G or to several other inhibitors of cell wall synthesis and also in the rate of penicillin-induced lysis (8). At concentrations that produced detectable lysis of S cultures, penicillin induced lysis of T cultures at a barely detectable rate. The availability of strains with a high rate of change between tolerant and susceptible states permitted an analysis of the physiological basis for tolerance. S and T derivatives recloned three times were used in this study.Cultures were grown in Todd-Hewitt medium supplemented with 0.15% glucose, and antibiotics were added in the exponential phase of growth when the cultures contained about 108 cells per ml. All glassware was acid washed. For autolysis assay cells were harvested as described previously (14) by filtration on 0.65-p.m-pore membrane filters (Millipore Corp., Bedford, Mass.), washed twice with about 5 ml (each time) of ice-cold distilled water, and suspended in 0.3 M potassium phosphate buffer (pH 6.8) (12). Autolysis rates (h-') were determined from the pseudo first-order reaction rate as described previously (14). Table 1 shows the rates of lysis of exponential cultures exposed to various antibiotics. In the case of penicillin G or D-cycloserine, S cultures lysed faster than did T cultures at antibiotic concentrations below the MIC but not at or above the MIC. For bacitracin, S cultures lysed faster than did T cultures above the MIC. Finally, for vancomycin, rates of lysis were not very different for S and T cultures examined both above and below the MIC. The data shown in Table 1 indicate that in terms of cell lysis, T strains were only relatively tolerant, since high enough concentrations of each antibiotic resulted in lysis. For any one antibiotic, lysis was not necessarily initiated near the MIC, which was determined at a much lower inoculum and after 24 h of incubation. We have shown previously that the MICs of each of the above-mentioned antibiotics for S and T derivatives are essentially indistinguishable but that the MBCs are 16-to 100-fold higher for T derivatives than for S derivatives (8).* Corresponding author. t Present address: CE...

Lineage studies of single‐colony isolates from the ATCC9790 strain of Streptococcus faecium are consistent with a reversible high‐frequency variation between a penicillin‐tolerant and a penicillin‐sensitive state. Tolerant and sensitive derivatives have been partially characterized.