TABLE 3.
Grouping of eight mercury resistance plasmids based on replicon, broad-host-range Inc group PCR, and restriction-hybridization typing data
| Plasmid | Groupa | Size (kb)b | Detection of Inc group, marker gene or plasmidc
|
Positive resistance markersf | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| IncP
|
IncN
|
IncW
|
IncQd
|
IncA/C
|
merRT
|
pEC10
|
|||||||||||
| Replicon typing | PCR-hybridization | Replicon typing | PCR-hybridization | Replicon typing | PCR-hybridization | Replicon typing | PCR-hybridization | Hybridization with probe | Replicon typing | PCR-hybridizatione | PCR-hybridization | Hybridization with probe | PCR-hybridization | ||||
| pEC10 | I | 45 | − | − | − | − | − | − | − | − | − | − | − | + | + | + | Hg Cu Sm Cm tcbCD |
| pEC8 | II | 46 | − | − | − | − | − | −g | − | − | − | − | − | w | + | + | Hg Cu Sm Cm |
| pF7 | II | 45 | − | −h | − | − | − | −i | − | − | − | − | − | − | + | − | Hg Cu Cmj |
| pP4 | II | 45 | − | − | − | − | − | −g | − | − | − | − | − | + | + | + | Hg Cu |
| pP2 | III | 42 | − | − | − | −k | − | −g | − | − | − | − | − | + | + | + | Hg Cu Sml |
| pF17 | III | 42 | − | − | − | − | − | −i | − | − | − | − | − | + | + | w | Hg Cu Sm Cmj |
| pP11 | IV | 49 | − | − | − | − | − | −g | − | − | − | − | − | + | w | + | Hg Cu Sml Cm As |
| pF10 | V | 40 | − | − | − | − | − | −i | − | − | − | − | − | + | + | + | Hg Cu Cmj |
Group based on PstI-generated restriction patterns and determined directly and after reaction with probes pEC10 and pP11.
Molecular size, as estimated from restriction digests.
Replicon typing was performed with Couturier probes (5). The PCR-hybridization analyses of Inc groups (7) were performed with the following systems: for IncP (α and β), trfA, oriT, korA, and traG; for IncN, repB, kikA, and oriT; for IncW, oriV, oriT, and trwA; for IncQ, repB, oriV, and oriT; and for IncA/C, rep (16). The PCR-hybridization analysis of merRT was performed with the merRTΔP system (consensus Tn501, Tn21, and pMJ100 mer genes), as described by Bruce et al. (3). The PCR-hybridization analysis of pEC10 was performed with the system developed in this study. The probes used for hybridization analysis were oriV (for IncA/C) and the whole-plasmid and PCR-generated pEC10-specific probe (for pEC10). +, positive signal; w, weak signal; −, no signal.
The IncQ oriV system generated nonspecific, nonhybridizing products of about 300 bp with all plasmids.
A weak, nonhybridizing band was detected with all plasmids.
In this study we tested resistance to Co, Ni, Cu, Zn, Ag, As, Cr, Cd, Hg, ampicillin, tetracycline, streptomycin (Sm), trimethoprim, chloramphenicol (Cm), gentamicin, kanamycin, nalidixic acid, erythromycin, lincomycin, and catabolic probes.
The IncW trwA system gave different nonspecific products with pEC8 and with pP11, pP4, and pP2.
The IncP traG system generated a nonspecific product with pF7.
The IncW oriV system generated a nonspecific, nonhybridizing product with pF7, pF10, and pF17.
Resistance to chloramphenicol was difficult to determine because of the high level of intrinsic resistance of the host.
The IncN oriT system generated a nonspecific product with pP2.
Resistance to streptomycin was difficult to determine because of the high level of intrinsic resistance of the host.