Defense Date


Document Type


Degree Name

Doctor of Philosophy


Microbiology & Immunology

First Advisor

James D. Punch


The molecular nature and replicative behavior of R factor 222 was examined in Proteus mirabilis . In deoxyribonucleic acid (DNA} from R+ P. mirabilis , R factor 222 was identified by CsCl density gradient centrifugation as 2 satellite DNA bands at densities corresponding to 50 and 58 moles percent guanine plus cytosine (% GC) . Replication of the 50 and 58% GC components of R factor 222 in P. mirabilis was analyzed during growth in the presence and absence of chloramphenicol (CAM} and after shifting exponentialand stationary- phase cells to conditions which inhibit host protein or DNA synthesis . CAM reduced the cellular growth rate but increased the amount of both R factor components relative to host chromosomal DNA . However, the 58% GC component showed a larger proportionate increase. This was inferred to indicate reduced synthesis of an inhibitor that acts on both R factor components and an initiator required for replication of the 50% GC component . Replicative patterns observed after shifting exponential- and stationary-phase cel ls grown with or without CAM to minimal medium or CAM for one generation, or puromycin for 3 hr , corroborated this interpretation. After shifts of exponential- phase cells from either medium, replication of the 50% GC components parallel ed host replication, thus indicating a requirement for protein synthesis . Under these conditions , replication of the 58% GC replicon increased due to reduced inhibitor synthesis . R factor DNA content remained constant after shifting stationary- phase cells from drug-free medium, whereas increased replication of the 58% GC component occurred after identical shifts of CAM- grown cells of the same chronological age. This indicated that effective concentrations of the regulatory inhibitor were attained in the stationary-phase cells grown in drug-free medium . Similar responses were observed after shifts to 5 C or to medium containing streptomycin or tetracycline . Absence of replication of the 50% GC component after shifting to medium containing nalidixic acid or phenethyl alcohol and the hereditary persistence of this replicon during growth indicated that the 50% GC replicon was attached to the membrane . Thus , in P . mirabilis the three replicons of R factor 222 are regulated as follows : the composite R factor and transfer portion (RTF) replicons , represented by the 50% GC component, require protein synthesis and membrane attachment for replication and are negatively regulated by an inhibitor ; the 58% GC or resistance determinant s replicon exists cytoplasmically and is subject only to negative replicative control .

The unusually low hyper chromic shift and the abnormal buoyant density shifts in CsCl observed after thermal denaturation of R f actor DNA indicated an abnormal chemical composition . DNA from P. mirabilis harboring R factor 222 was examined chromatographically after enzymatic and chemical hydrolyses . Preliminary results indicated the presence of an unusual chemical component in R factor DNA which reacts positively to carbohydrate development and possesses chromatographic and spectrophotometric properties similar to 5-hydroxymethyl cytosine.


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