Multiple Origins and Replication Proteins Influence Biological Properties of β-Lactamase-Producing Plasmids from Neisseria gonorrhoeae

Abstract

The ␤-lactamase-producing Asia-type plasmid pJD4 of Neisseria gonorrhoeae is a 7.4-kb, broad-host-range plasmid. It is part of a family of plasmids which are structurally related yet vary in size, found in both N. gonorrhoeae and Haemophilus ducreyi. Branch-point analysis by electron microscopy indicates that pJD4 carries three clustered but distinguishable origins of replication, which we named ori1, ori2, and ori3. Although pJD4 belongs to incompatibility (Inc) group W, it also carries a silent IncFII determinant which is expressed when ori2 and ori3 are absent. The Africa-type plasmid pJD5, a naturally occurring deletion derivative of pJD4, carries only ori1, belongs to the IncFII group, and, in contrast to pJD4, requires DNA polymerase I (Pol I) for replication. Plasmids constructed from pJD4 which lack ori1 but carry ori2 and ori3 do not require Pol I and are incompatible with IncW plasmids, suggesting that the ori2 or ori3 region contains the IncW determinant. We have cloned a replication initiation protein (RepB) that is necessary for ori2 and ori3 to function. This Rep protein is distinct from RepA, which is necessary for ori1. Thus, pJD4 is unique because it is the smallest plasmid characterized containing three origins of replication and two unique Rep proteins.Plasmids of gram-negative bacteria containing three origins of replication are rare (1, 9, 23); to date, only three such naturally occurring plasmids have been described. The first plasmid discovered, F (94.5 kb), contains remnants of three independent replication regions, RepFIC, RepFIA, and RepFIB (3,8). A 9-kb mini-F plasmid, containing the complete RepF1A region, includes seven genes encoding proteins involved in the replication and maintenance of the plasmid, including a single replication initiation protein (Rep) and two origins, oriV and oriS (8). Related plasmids lacking the RepF1A region require RepF1B, a less stable replicon (27). The RepF1C replicon was shown to have been rendered nonfunctional on the F plasmid by the natural insertion of the transposon Tn1000 (25,40). This replicon, in members of the F plasmid family, resembles the replication region of the plasmid R1 (25). The IncN plasmid pCU1 contains three origins of replication (oriB, oriS, and oriV), all located on a 2-kb DNA fragment, which are driven by a single Rep protein (1, 38). The third and best-characterized plasmid with three origins (␣, ␤, and ␥) is the 38-kb plasmid RK6. This plasmid also carries genes for two replication proteins, pir (encoding the protein for origins ␣ and ␥) and bis (Bis for origin ␤) (31). These origins and genes are located on a 4-kb DNA fragment (18, 31).It appears that the penicillinase-producing plasmids of N. gonorrhoeae contain different and multiple origins of replication as well. These plasmids belong to a family of plasmids that are genetically related (33). Since the isolation of the first penicillinase-producing isolate in 1976, their worldwide spread and prevalence (i.e., over 50 to 80% of all gonococci isolated in some countries) h...

Variation in Salmonella enterica Serovar Typhi IncHI1 Plasmids during the Global Spread of Resistant Typhoid Fever

“…These replication (Rep) proteins relate to incompatibility, and so this is a possible way in which different plasmid cores (10), or possibly different accessory gene linkage groups, could compete for replication space in the bacterial host. Expression of different Rep proteins, as seen with Neisseria gonorrhoeae (22), may explain the occasional reports of plasmids of different incompatibility groups in serovar Typhi (20). Three possible DNA-binding proteins (HCM1.58c, HCM1.175, and HCM1.286c) clustered within the core genes, as did the plasmid partition proteins parA (HCM1.86) and a putative parB encoding CDS (HCM1.87), with a series of 54-bp repeats upstream that could act as binding sites for the partition proteins.…”

Two Internal Origins of Replication in Streptomyces Linear Plasmid pFRL1

“…Only one origin of replication (i.e., ColEI) functioned, and the other was inhibited. Two or three origins have been discovered in some naturally occurring plasmids, e.g., pIP501 carrying a rollingcircle and a theta replicon (8,29), pTB19 containing two rolling-circle replicons (18,26), and pJD4 containing three origins of replication (27). As in artificially constructed plasmids, only one origin functioned in the naturally occurring plasmids.…”

Three functional replication origins of the linear and artificially circularized plasmid SCP1 of Streptomyces coelicolor

“…Replication with two or three origins in artificially constructed or naturally occurring plasmids in other bacteria has been known for a long time. For example, some artificially constructed plasmids, pSC101/ColEI and Rsc11/ColEI (Cabello et al, 1976;Boidol et al, 1977), contain two distinct replicons; some naturally occurring plasmids, such as pIP501 and pJD4, carry a rolling circle and a h replicon (Evans & Macrina, 1983;Pujol et al, 1998), and three origins of replication (Pagotto & Dillon, 2001), respectively. In these plasmids, only one replication origin functions and the others are inhibited (e.g.…”