Plasmid resistance system

More recently additional systems have been developed that rely on other DBDs. Golemis and co-workers developed a system that uses the DBD from the bacteriophage X repressor cl protein [25]. Interestingly, the plasmid encoding the cl fusion protein carries the zeoR gene, which makes both yeast and E. coli containing the plasmids resistant to zeocin. Also, a system has been developed that uses the DBD from the human estrogen receptor [26]. 

Many microbes that thrive in environments contaminated with copper contain plasmid-borne copper-resistance systems. Such plasmid-borne... 

Wang, L., Chen, S., Xiao, X., Huang, X., You, D., Zhou, X., Deng, Z. (2006). arsRBOCT arsenic resistance system encoded by linear plasmid pHZ227 in Streptomyces sp. strain FR-008. Appl. Environ. Microbiol. 72 3738 2. 

Arsenic resistance is not the only toxic heavy metal ion resistance system found in bacteria. Bacteria have known plasmid and chromosomal genes for resistances to Ag+, As02, ASO4 -, Cd ", Co ", CrO/-, Cu ", Hg +, Ni +, Pb ", Sb ",... 

Resistance to the toxic effects of mercury is quite commonly found in bacteria (Silver and Walderhaug 1992 Misra 1992). Since most bacteria are rarely exposed to toxic levels of mercury, the resistance mechanism is inducible and is frequently found on plasmids and/or transposons. Many of the multi-antibiotic resistance plasmids that are frequently found in clinical collections have determinants of mercury resistance as well. Furthermore, mercury resistance is a consistent component of the chromosomal resistance determinant of MRS A (methicillin-resistant Staphylococcus aureus), a current clinical problem and one with no apparent connection to the use of mercurials. If the mercury resistance system is present, the expression of the detoxifying activities is tightly regulated and turned on only when needed. The MerR regulatory protein turns on mRNA synthesis by a positive activator mechanism (for primary references, see Silver and Walderhaug 1992 Misra 1992). 

We have reviewed the best-known prokaryotic plasmid-based resistance systems from the view of metal ion homeostasis. Plasmid-based systems are, with few exceptions, inducible and are regulated by activators (mercury and copper) or repressors (cadmium) in different cases. The metal-binding motifs used by these systems are frequently localized (e.g., Cys-Xaa-Xaa-Cys, Glu-His-His, and His-Xaa-His), but occasionally coordinating cysteine residues on different subunits interact (as in the MerR regulatory protein and mercuric reductase enzyme). 

Brown NL, Rouch DA, Lee BTO (1992) Copper resistance systems in bacteria. Plasmid 27 41-51... 

Resistance to a range of antibiotics is of increasing concern in clinical practice since the genes are often carried on transmissible plasmids. There are different types of mechanism that confer resistance, inclnding enzymatic covalent modification of the antibiotic, effective efflnx systems, and indnction of a cellnlar enzyme that is resistant to the antibiotic. Examples of these are used as illustration. 

In microbes without a permeability barrier, or when the barrier fails, a mechanism must be in place to export metals from the cytoplasm. These active transport systems involve energy-dependent, membrane-bound efflux pumps that can be encoded by either chromosomal- or plasmid-borne genes. Active transport is the most well-studied metal resistance mechanism. Some of these include the ars operon for exporting arsenic from E. coli, the cad system for exporting cadmium from Staphylococcus aureus, and the cop operon for removing excess copper from Enterococcus hiraeP i9A0... 

Chracteristics of B. m terium s plasmid system have been summarized by Carlton (3p. The plasmids are typical in that they band as covalently closed circular DNA in ethidium bromide-cesium chloride gradients and they are resistant to irreversible alkaline denaturation (17). However, B. megaterium plasmids are atypical in that they exist in approximately 10 size classes and as many copies per cell ( ). In fact, for the smaller plasmids there are hundreds of copies per cell so that plasmid DNA may represent up to 40% of the total extractable DNA (31). This is unusual since for most plasmids there are usually no more than a few copies per cell. Also, hybridization studies suggest that there is extensive homology between three B. megaterium plasmids of different sizes and between these plasmids and the chromosomal DNA (31,33). Carlton (31) concludes that the most likely explanation of the origin of B. megaterium plasmids is that they are molecular hybrids between one or more plasmid elements and various portions of the chromosomal DNA. 

B. Overproduction (A) of PABA is one of the resistance mechanisms of sulfonamides. Changes in the synthesis of DNA gyrases (B) is a well-described mechanism for quinolone resistance. Plasmid-mediated resistance (C) does not occur with quinolones. An active efflux system for transport of drug out of the cell has been described for quinolone resistance, but it is not plasmid mediated. Inhibition of structural blocks (D) in bacterial cell wall synthesis is a basic mechanism of action of p-lactam antibiotics. Inhibition of folic acid synthesis (E) by blocking different steps is the basic mechanism of action of sulfonamides. 

Clones with ampicillin resistance but without kanamycin resistance are examined with the CloneChecker System. By this antibiotic selection, the undesired vector heterodimer is removed. If both of the clones are false, additional clones are analyzed using the same protocol. When appropriate clones are obtained for almost all of the genes, they are inoculated from the ampicillin plates to prepare plasmids in a 96-well format by using the Wizard SV 96 Plasmid DNA Purification System see Note 6). 

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