Positive and Negative Control of R-Factor Replication in Proteus mirabilis

Abstract

Replication of the 50 and 58 moles per cent guanine plus cytosine (%GC) components of R factor 222 in Proteus mirabilis during growth in the presence and absence of chloramphenicol and after shifting exponential- and stationary-phase cells to conditions which inhibit host protein or deoxyribonucleic acid (DNA) synthesis was examined. Chloramphenicol reduced the growth rate but increased the amount of both R-factor components; 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 for replication of the 50% GC component. Replicative patterns observed after shifting exponential- and stationary-phase cells grown with or without chloramphenicol to minimal medium or chloramphenicol for one generation, or puromycin for 3 hr, corroborated this interpretation. After shifts of exponential cells from either medium, replication of the 50% GC components paralleled host replication, thus indicating a requirement for protein synthesis; replication of the 58% GC component increased due to reduced inhibitor synthesis. R-factor DNA remained constant after shifting stationary cells from drug-free medium, thus indicating that the cells contained effective concentrations of the regulatory inhibitor, whereas increased replication of the 58% GC component occurred after identical shifts of chloramphenicol-grown cells of the same chronological age. 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 phenethanol and its hereditary persistence 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 and transfer portion (RTF) replicons represented by the 50% GC component require protein synthesis and membrane attachment and are negatively regulated by an inhibitor; the 58% GC or resistance-determinants replicon exists cytoplasmically and is subject only to negative control.

Molecular Nature of R-Factor Deoxyribonucleic Acid Isolated from Salmonella typhimurium Minicells

“…In E. coli and S. marcesens, R-factor replication is under stringent (11) control and is coordinated with host cell division, and R-factor DNA makes up about 4% of the total extractable DNA (6,21). In contrast, when R factors are present in Proteus, R-factor DNA may constitute up to 50% of the total extractable DNA (22). In addition, the numbers of copies of the three molecular species of R-100 in Proteus depend on both the stage of the bacterial growth cycle and the presence or absence of antibiotics in the culture (22,26).…”

Alternate Forms of the Resistance Factor R1 in Proteus mirabilis

“…& Based on a molecular weight value of 2.8 x 10' for the P. mirabilis chromosome (15) and 65 x 10' for the 1.711 g/cm' satellite component (4). although here a greater proportion of drug-sensitive cells is encountered.…”

“…This form of Ri is stable in Proteus and replicates under stringent control. Based on a molecular weight of 65 x 106 for the composite structure of Ri (4), and 2.8 x 10' for the Proteus chromosome (15), then the resistance factor is present to the extent of one to two copies per chromosome in both logand stationary-phase cells ( Tables 1 and 2).…”

“…The amount of relaxed replication of R factors in stationary-phase Proteus has been observed to be a function of whether or not cells are grown in medium containing drugs (15,17). According to Rownd and Mickel, cells grown in drug-free medium lack the large R-factor molecules described above, with most of the R DNA being in the form of separate RTF and r determinants.…”

Effect of Growth Phase of Host Bacteria on Synthesis of R Factor‐Mediated Penicillinase

“…One of the possible interpretations regarding this phenomenon is that the transcriptions of amp and cml genes are affected differently by the age of the growth, and that the effectors may be metabolites which accumulate or diminish according to the advancement of the host growth, although we presently do not have any convincing evidence for such factors. Punch and Kopecko [7] suggested the existence of a negative control over replication of the resistance determinants which is detached from the transfer portion (RTF) of the R factor and located cytoplasmically in P. mirabilis. A possibility that amp and cml genes of the R factors replicate under a different control mechanism in P. mirabilis cannot be excluded.…”