cleic acid (DNA) molecules, plasmids, as well as ... tion defect of certain dna- mutants (11). Two insertion ..... extrachromosomal plasmid DNA in strains that.
JOURNAL OF BACTERIOLOGY, Aug. 1975, P. 724-738 Copyright i 1975 American Society for Microbiology
Vol. 123, No. 2 Printed in U.S.A.
Prophage-Dependent Plasmid Integration in
Staphylococcus aureus M. D. SCHWESINGER
AND
R. P. NOVICK*
Department of Microbiology, The Public Health Research Institute of The City of New York, Inc., New York, New York 10016 Received for publication 17 April 1975
A study has been done of reversion to thermostability of thermosensitive, replication-defective (TSR) mutant penicillinase plasmids. All three of the expected classes of reversions were encountered: back mutation, suppression, and integration. The latter class was examined in some detail and it was found that the presence of the 11 prophage enhanced the frequency of reversion by integration some 103-fold. Prophage-dependent integration resulted in inactivation of plasmid-linked arsenate and arsenite resistance; these revertant strains gave rise to high frequency transducing lysates where the plasmid was restored upon transduction to its original TSR state including recovery of these resistances. The integrated plasmid-prophage complexes were stable at high temperatures (43 C) but slow growing and unstable at low (32 C); loss of either plasmid or prophage restored normal growth and stability. Sometimes restoration of the plasmid to its autonomous TSR state was observed and molecular studies showed that in most cases the plasmid was essentially the same size as before integration. In some cases an excision complex was recovered that was more than twice the size of the plasmid and could have been a plasmid-phage co-integrate. Integration also took place in the absence of the 11 prophage. These integrations retained all plasmid-linked resistances, were stable at all temperatures, and gave rise to low frequency transducing lysates in which the integrated state was retained upon transduction. On the basis of these results it is suggested that the prophage promotes integration at or near its attachment site.
gration, as exemplified by F, is sporadic, involves preferred sites, and is usually not mutagenic but may sometimes suppress the replication defect of certain dna- mutants (11). Two insertion sequences, recently identified in Escherichia coli by their ability to interrupt gene continuity by integration (22), are smaller than prophages (4 x 105 to 7 x 10O daltons) and as yet genetically unmarked. One insertion sequence recently identified in Staphylococcus aureus, carrying determinants of resistance to erythromycin and spectinomycin but seemingly unable to exist as an independent plasmid, can integrate in the absence of the host rec function (15, 27). Such insertion sequences appear to constitute a reservoir of endogenous integrate into one another, and a variety of genome-modifying potentiality. Moreover, prodifferent patterns have been observed. Thus, of phages and insertion sequences may integrate the prophages, some are integration-site- into plasmids (5, 22) and may be responsible specific (e.g., A), others may have several dis- for the integration of the latter into one another tinct integration sites (e.g., P2 [1]), and still or into the cell's chromosome or for the transothers may be able to integrate nearly anywhere location of particular markers between non(e.g., Mul) and in so doing interfere with or homologous replicons (4). modify local gene functions (26). Plasmid inteIn this paper we report the results of an Recent years have revealed an unsuspected complexity in the composition of cellular genomes which suggests their development as an assemblage of endosymbiotic partnerships (8). Thus, a bacterial cell may contain, in addition to its chromosome, any number of smaller independently replicating circular deoxyribonucleic acid (DNA) molecules, plasmids, as well as several integrated prophages and shorter insertion sequences (25). The existence and the behavior of such genetic elements are doubtless of overriding importance in the evolutionary plasticity of prokaryotic (and presumably, therefore, eukaryotic) genomes. A key feature of the interrelationships among these various genetic elements is their ability to
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PROPHAGE-DEPENDENT PLASMID INTEGRATION
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TABLE 1. Host strains investigation of plasmid integration in S. aureus which has revealed a hitherto unsuspected plasStock Derivation or mid-prophage relationship and so contributes to no. reference the description of prokaryotic replicon interacRN25 8325-34 (14) tions. RN25 lysogenized with 8325-3 (411) Our approach was based upon the earlier RN26 411 finding of Jacob et al. (6) that integration was a RN27 8325-3 (80a) (24) frequent consequence of selection for tempera- RN92 147 (pII147 -) (17) 147 (80) (pII147 -) RN92 lysogenized with ture-stable revertants of an F-lac plasmid ther- RN93 80 mosensitive for replication (TSR). Previous RN94 147 (80a) (pII147-) RN92 lysogenized with findings in this laboratory with TSR penicillin80a ase plasmids of S. aureus (L. Wyman, E. RN450 8325-4d (14) 8325-4 (411) RN450 lysogenized with Murphy, and R. Novick, unpublished results) RN451 411 have been that the majority of such revertants RN746 8325-4 (412) RN450 lysogenized with were plasmid-linked back mutants (or suppres012 sors) and a minority contained integrated plas- RN782 8325-4 (4147) RN450 lysogenized with 4147 mids. Of the latter, upon transduction to a new 8325-4 his-7recAl (28) host (outcrossing), the majority of transductant RN981 8325-4 (411) his-7 (28) plasmids in any one outcross remained stable RN1030 { recA (integrated) and only a minority were restored RN1617 147 (11) (pI147) RN92 lysogenized with 46~ ~ ~
