Transcriptional mapping studies of the IncB minireplicon pMU720 demonstrated the existence of a long RNA molecule, RNA II, whose 5' portion is complementary to the product of the incompatibility gene RNA I. By using gene fusion and transcriptional fusion plasmids, it was shown that RNA I regulated the expression of the RNA II gene product and that it did so primarily at the level of translation. The target of RNA I was mapped to lie within a 216-base region of RNA II containing the sequence complementary to RNA I. Introduction of the target for RNA I in trans increased the copy number of an IncB minireplicon, indicating that RNA I and RNA II form the basis of the copy number control system of IncB plasmids.

The isolation and characterization of minireplicons corresponding to group B and I-complex plasmids are reported. The molecular structures of the small RNAs that may play a major role in the replication control and incompatibility reactions of the plasmids are compared. A mutant plasmid with changed copy number and incompatibility specificity is described. This mutant had a single-base-pair substitution in the DNA region that codes for the small RNA.Plasmid replication in bacteria is, in some cases, primarily controlled by a small RNA molecule whose sequence is complementary to a part of an RNA molecule essential for replication (rep) (5,6,16,17,30,(35)(36)(37). The rep RNA and the small countertranscript RNA are transcribed from complementary strands of the DNA double helix. The small RNAs act in trans to inhibit or control replication. In the case of ColEl, it is postulated that the rep RNA acts as a primer for the initiation of plasmid replication and that the interaction with the countertranscript RNA interferes with the processing of the primer (16,20,33,34,36). In the case of FII plasmids, although the function of the rep mRNA remains unclear, it has been shown that the countertranscript RNA interferes with its translation (18).Because they act in trans, these countertranscript RNAs also affect the replication of other plasmid molecules showing the same specificity of replication control. In this way, these RNAs appear to play a major role in defining the incompatibility reaction of these particular plasmids.The group B plasmids and plasmids previously grouped together in the I complex exhibit some cross-reactivity in their incompatibility phenotypes (7). Studies of a minireplicon derived from a group B plasmid have shown that a small RNA is synthesized from a region of DNA which specifies essential replication functions (4). More recent work has shown that this small RNA is a countertranscript RNA and that it interferes with the elongation, stability, or translation of the rep mRNA with which it shows complementarity (this paper; J. Praszkier, P. Bird, S. Nikoletti, and J. Pittard, manuscript in preparation).In this paper, we describe the isolation and characterization of minireplicons corresponding to group B, I1, and IY plasmids and compare the molecular structures of the small RNAs which appear to play a major role in both replication control and incompatibility reactions of these plasmids. We also describe a mutant plasmid which exhibited changes in both copy number and incompatibility specificity. This plasmid had a single-base-pair substitution in the region of DNA coding for the small RNA.

No abstract

Trimethoprim (Tp) resistant Gram negative bacteria were isolated from humans and pigs. The bacterial hosts were characterized by their resistance pattern and biotype. The presence of transferable Tp plasmids was demonstrated in 86% of 59 porcine isolates and 37% of 49 human isolates. The Tp R-plasmids carried a diversity of resistance determinants such as Tc, Cm, Sp, Sm and Su. Incompatibility tests distinguished two major groups, Inc FIV and Inc N. Thirty of 99 Tp R-plasmids isolated from humans were grouped as Inc FIV and eight as Inc N. The results of molecular weight determination of Tp R-plasmids performed by agarose gel electrophoresis were consistent with the existence of two groups--larger R-plasmids (76 to 104 Md) belonging to Inc FIV and lower molecular weight R-plasmids (25 to 35 Md) belonging to Inc N. Results from this study indicate that the Tp R-plasmids isolated in Perth have evolved independently from those described in Europe and the United Kingdom. There is also evidence for their local spread between Escherichia, Klebsiella, Enterobacter, Citrobacter and Acinetobacter from man and animals.