Erythromycin is the drug of choice for treatment of Mycoplasma pneumoniae infections due to its susceptibility to low levels of this antibiotic. After exposure of susceptible strains to erythromycin in vitro and in vivo, mutants resistant to erythromycin and other macrolides were isolated. Their phenotypes have been characterized, but the genetic basis for resistance has never been determined. We isolated two resistant mutants (M129-ER1 and M129-ER2) by growing M. pneumoniae M129 on agar containing different amounts of erythromycin. In broth dilution tests both strains displayed resistance to high levels of several macrolide-lincosamide-streptogramin B (MLS) antibiotics. In binding studies, ribosomes isolated from the resistant strains exhibited significantly lower affinity for Mycoplasma pneumoniae is a common cause of respiratory tract infections particularly in school-aged children and young adults. All levels of the respiratory tract are involved, and while the most characteristic manifestations are acute bronchitis and pneumonia (8, 11), serious complications can occur (5). The sensitivity of M. pneumoniae to erythromycin and many other macrolide antibiotics makes them the drugs of choice for chemotherapy (3, 33). Treatment with erythromycin results in relatively rapid alleviation of symptoms; however, viable M. pneumoniae can frequently be isolated from infected individuals for a prolonged period of time following therapy (6,41,45). Isolation of resistant strains from patients following treatment is not uncommon (26,28,(43)(44)(45), and erythromycinresistant mutants are readily derived by selection in vitro (27,43,44). Phenotypic studies have demonstrated that most of these strains simultaneously developed resistance to macrolide, lincosamide, and group B streptogramin (MLS) antibiotics (48). It was suspected that development of resistance to erythromycin contributed to the prolonged colonization of the respiratory tract following chemotherapy.As part of our efforts to understand what role, if any, development of antibiotic resistance may play in the course of M. pneumoniae infection, we are attempting to determine the molecular mechanisms by which resistance to erythromycin can arise. While the genetic basis for MLS resistance has been extensively studied for other bacteria, except for a rudimentary study of Ureaplasma urealyticum (30) it has not been explored in mycoplasmas. We show that MLS-type resistance in two isolates derived in vitro is correlated with A-to-G transitions at two conserved sites in the central loop in domain V of the 23S rRNA which are known to result in similar patterns of resistance in other organisms. The possible clinical implications of these mechanisms are discussed. MATERIALS AND METHODSMedia and growth conditions. M. pneumoniae M129-B16 (ATCC 33530) was grown in glass culture bottles or 96-well microtiter plates in modified Hayflick's medium (19) without penicillin. One-percent Bacto-Agar (Difco, Detroit, Mich.) was added for growth on solid medium. Antibiotics were obtained ...

We measured the kinetics of growth and mutagenesis of UV-irradiated DNA of phages S13 and A that were undergoing SOS repair; the kinetics strongly suggest that most of SOS mutagenesis arises from the deamination of cytosine in cyclobutane pyrimidine dimers, producing C -3 T transitions. This occurs because the SOS mechanism bypasses T'T dimers promptly, while bypass of cytosine-containing dimers is delayed long enough for deamination to occur. The mutations are thus primarily the product of a faithful mechanism of lesion bypass by a DNA polymerase and are not, as had been generally thought, the product of an error-prone mechanism. All of these observations are explained by the A-rule, which is that adenine nucleotides are inserted noninstructionally opposite DNA lesions.SOS repair and SOS mutagenesis were discovered nearly 40 years ago by Jean Weigle, who observed that preirradiation of the bacterial host increased survival of UV-irradiated phage A (1). When applied to phage DNA these SOS phenomena are now called Weigle reactivation and Weigle mutagenesis; their mechanism has thus been an intriguing puzzle for some time. We show here that the primary, but not the sole, mechanism of UV mutagenesis in phages S13 and A is simply the spontaneous deamination of cytosine in cyclobutane pyrimidine dimers. This deamination occurs during a deliberate, and arguably an unnecessary, delay in the bypass of the lesion by DNA polymerase; the delay is caused by cytosine-containing dimers, possibly because of the mispairing of the cytosine with adenine. SOS repair is induced by damage to DNA. In Escherichia coli, the damage results in an activated form of the RecA protein, which can mediate the cleavage of LexA, the repressor ofthe SOS regulon. The principal known components of the repair system are the products of the recA and the umuDC genes. The activated RecA protein is needed for cleavage of the UmuD protein to produce UmuD', the active C-terminal fragment (2-4). The RecA protein can also be activated by mutations designated recA(Prtc) to what is termed the protease constitutive state (5); with mutants such as recAl202(Prtc) and recAJ237(Prtc), which were used in the work described here, Weigle reactivation is achieved without irradiation of the host cell.It is generally thought that a high frequency of erroneous base pairing during DNA synthesis opposite the distorted lesion is responsible for the mutagenesis that accompanies SOS repair, and that idea has evoked the term error-prone repair. We propose, instead, that most, though certainly not all, ofthe mutations arise through accurate base pairing-i.e., an error-free bypass mechanism. This paradox will be resolved by the fact that mutations can arise by deamination of cytosine in cyclobutane dimers; if the uracils formed are read correctly during bypass of the dimers, adenines would be found in the newly synthesized complementary strand. If there is adequate time for deamination to occur, and that is the very central issue here, bypass of lesions will usually invo...

Repair of UV-irradiated bacteriophage in Escherichia coli by Weigle reactivation requires functional recA+ and umuD+C+ genes. When the cells were UV irradiated, the groE heat shock gene products, GroES and GroEL, were needed for at least 50% of the Weigle reactivation of the single-stranded DNA phage S13. Because of repression of the umuDC and recA genes, Weigle reactivation is normally blocked by the lexA3(Ind-) mutation (which creates a noncleavable LexA protein), but it was restored by a combination of a high-copynumber umuD+C+ plasmid and a UV dose that increases groE expression. Maximal reactivation was achieved by elevated amounts of the Umu proteins, which was accomplished in part by UV-induced expression of the groE genes. By increasing the number of copies of the umuD+C+ genes, up to 50% of the normal amount of reactivation of S13 was achieved in an unirradiated recA+ host.

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