Plasmid RA1 Research Articles

Nucleotide sequence of class D tetracycline resistance genes from Salmonella ordonez.

Plasmid pIP173, isolated from Salmonella ordonez strain BM2000, confers resistance to tetracycline and a number of other antibiotics. We determined the nucleotide sequence of the pIP173 tetR repressor and tetA resistance genes. The pIP173 tetR gene is essentially identical to the class D tetR gene from plasmid RA1. The pIP173 tet genes are flanked by directly repeated copies of the insertion sequence IS26. Interestingly, the 3' end of the tetR gene, encoding the C-terminal 16 amino acids of the TetR protein, extends into the flanking IS26 sequence. The relationships between the class A, B, C, and D TetA sequences parallel the relationships between the corresponding TetR sequences; class D is more closely related to class B than to either class A or C. Overall, the four TetA sequences show 38% identity and 57% similarity.

Conjugation system of IncC plasmid RA1, and the interaction of RA1 pili with specific RNA phage C-1

RA1 was the only IncC plasmid that was slightly temperature-sensitive for replication and transfer. At 30°C, RA1 determined constitutive synthesis of conjugative pili and yet was transfer-repressed. Attachment of shaft-adsorbing RNA phage C-1 virions prevented the probable retraction of pili under heat stimulus (55°C). Electron microscopy showed single adsorbed virions at pilus bases where they were thought to have stopped retraction.

Frequency of tetracycline resistance determinant classes among lactose-fermenting coliforms.

Using colony hybridization techniques and DNA probes derived from four distinct tetracycline resistance determinants, we have examined the frequency of these determinants among 225 lactose-fermenting coliforms isolated from fecal samples of both humans and animals. The class B, or Tn10-type determinant, occurred most frequently at 73.3%, followed by class A (on RP1) at 21.7%, and class C (on pSC101) at 8%; 3.5% of isolates harbored two of these determinants. Hybridization to class D, carried by plasmid RA1, was not found among any of the isolates. One isolate failed to hybridize to any of the probes and represents a fifth class of determinant. No dramatic differences were observed in the frequencies of these determinants among four populations examined: hospital, urban, rural, and laboratory. At low stringency conditions of hybridization we were able to demonstrate cross-hybridization of determinant A with class C DNA and limited reaction with class B DNA, but no reaction with class D DNA.

Tetracycline resistance determinants from groups A to D vary in their ability to confer decreased accumulation of tetracycline derivatives by Escherichia coli.

The ability of four genetically distinct plasmid-located tetracycline resistance determinants (TetA, B, C and D) to confer decreased accumulation of tetracycline and some of its analogues by Escherichia coli K12 was examined. Accumulation of oxytetracycline, tetracycline, demethylchlorotetracycline, 6-demethyl-6-deoxy-5-hydroxy-6-methylene-tetracycline, chlorotetracycline, doxycycline and 6-demethyl-6-deoxytetracycline was examined by fluorescence spectroscopy. The determinants varied in their ability to promote decreased accumulation of tetracyclines, defined as an R+/R- fluorescence ratio of less than 0.85. Plasmid pIP7 (TetA) caused reduced accumulation of only oxytetracycline, tetracycline and chlorotetracycline, but plasmid pDU301 (TetB) promoted reduced accumulation of all the compounds tested except 6-demethyl-6-deoxytetracycline. The TetC determinant of pBR322 caused decreased uptake of five derivatives, but not doxycycline or 6-demethyl-6-deoxytetracycline. Plasmid RA1 (TetD) encoded reduced accumulation of oxytetracycline, tetracycline, 6-demethyl-6-deoxy-5-hydroxy-6-methylenetetracycline and chlorotetracycline. In general, the resistance determinants were more efficient in promoting decreased accumulation of hydrophilic tetracyclines. These accumulation studies provide a satisfactory method for the phenotypic identification of Tet resistance determinants.

Characterization of Conjugative R Plasmids Belonging to the New Incompatibility Group IncU

Five conjugative plasmids governing different antibiotic resistance patterns were identified in wild strains of enteric bacteria isolated in Czechoslovakia and the G.D.R. between 1976 and 1979. They have been characterized as members of the new incompatibility group IncU (reference plasmid RA3 from Japan). The molecular sizes of the IncU plasmids ranged between 18 and 37 megadaltons; their restriction fragment patterns indicated them to be distinct types.

Heterogeneity of tetracycline resistance determinants

We have found that tetracycline resistance on different naturally occurring bacterial plasmids is encoded by more than one genetic determinant. Using restriction enzyme analyses and DNA-DNA hybridization to specific 32P-labeled genetic probes, we can define at least four genetically distinct tetracycline resistance determiants: Class A (the determinant on prototype plasmid RP1), Class B (that on R222), and Class C (that on plasmid pSC101). At least one other determinant, encoded by plasmid RA1, belongs to none of these three groups and has been designated Class D. These genetic classes confirm phenotypic differences in expression of resistance to tetracycline and tetracycline analogs encoded by the different plasmids.