Transposition of DNA fragments flanked by two inverted Tn1 sequences: translocation of the plasmid RP4::Tn1 region harboring the Tcr marker.

We have demonstrated the possibility of transposition of the plasmid RP4::Tn1 fragment (21.2 kb) carrying the tetracycline resistance (Tcr) gene and flanked by two Tn1 copies. The new transposon, designated Tn1756, bears lethal genes that kill host cells. Therefore, its transposition can only be revealed in the presence of lethality-compensating helper regions of the plasmid RP4. Thus, RP4::Tn1 consists of two transposons, Tn1755 (Tn1-Kmr-Tn1) and Tn1756 (Tn1-Tcr-Tn1), sharing the Tn1 sequences. Both of these transposons are capable of recA-independent translocation to other plasmids. Therefore, transposition of DNA fragments flanked by two inverted Tn1 sequences does not depend on Tn1 orientation.

[New class of RP4 plasmid mutations inducing a mucoid-type of Escherichia coli K-12 cell growth]. / Novyi klass mutatsii plazmidy RP4, indutsiruiushchikh mukoidnyi kharakter rosta kletok Escherichia coli K-12.

A new class of mutations is described which induce mucoid growth of Escherichia coli K-12. Unlike classical capR and capS mutations, the mucoid phenotype of colonies of the cap forms obtained is determined by mutations in genomes of thermosensitive plasmids pEG1 and RP1-6Repts12, derivates of the RP1 Inc P1 Ap Tc Km factor and accompanied by a complete or partial loss of the thermosensitive character of maintenance. The morphological character induced by plasmids is not associated with changes in the sensitivity of bacteria to UV irradiation and is determined by superproduction of capsular polysaccharide differing in the chemical structure from colanic acid, a common capsular polysaccharide of E. coli K-12.

Transposition of a DNA fragment flanked by two inverted Tn1 sequences.

The 32 Md fragment (derived from plasmid RP4::Tn1) carrying the Kmr gene and flanked by two inverted Tn1 elements is capable of recA-independent translocation to other plasmids. We designated this new transposon Tn1755. In various crosses, frequencies of Tn1755 transposition to plasmids Co1B-R3, R15 and F'Co1VBtrp varied from 2.5 to 90% of the frequencies of Tn1 transposition. Tn1755 can integrate into various sites of the recipient plasmids. We failed to observe transposition of another RP4::Tn1 fragment flanked by two opposingly oriented Tn1 transposons and harboring the Tcr gene. Presumably, to form a new transposable structure, other features must also be of importance.

Isolation of transposon TnA from plasmid RP4 carrying two copies of this element.

Employing heteroduplex and restriction analyses, two inverted copies of a 3.2.10(6) dalton transposable sequence, TnA, were found in RP4::TnA, a spontaneously arisen derivative of the plasmid RP4. Integration of the second copy of TnA causes loss of the conjugative properties of RP4. Both TnA sequences in RP4::TnA were localized and found to have opposite orientations. The DNA fragment corresponding to the individual transposon TnA was isolated after the endonuclease S1 digestion of RP4::TnA molecules annealed under conditions favoring intramolecular renaturation. The attempts to transform the cells of Escherichia coli QD5003, HB101[pCRI] and JC7623 with the isolated transposon were unsuccessful.

[Effect of rifampicin on the synthesis of bacterial RNA polymerase mRNA by means of hybrid plasmids]. / Issledovanie vliianiia rifampitsina na skorost' sinteza mRNK bakterial'noi RNK-polimerazy s pomoshch'iu gibridnykh plazmid.

We studied the rate of synthesis of beta- and beta'-subunits of DNA-dependent RNA polymerase and the rate of beta-polypeptide mRNA synthesis in rifampicin-treated bacteria. The antibiotic doses used did not significantly inhibit the total RNA and protein synthesis in rifampicin-sensitive bacteria. For RNA-DNA hybridization experiments a pOD162 plasmid was constructed carrying a fragment of the rpoB gene and no other chromosome DNA regions. It is found that low doses of rifampicin cause an absolute and differential increase in the rate of synthesis of the specific mRNA for the beta-subunit, suggesting a stimulation of the corresponding gene transcription. However the absolute transcription stimulation does not fully correlate with the relative acceleration of beta-mRNA and the corresponding polypeptide synthesis. The stimulating effect of rifampicin on the beta-polypeptide synthesis was demonstrated also in a coupled system of transcription and translation directed by lambda rifd 47 DNA. The possible mechanisms of the rifampicin action are discussed.

The effect of rifampicin upon the transcription of RNA polymerase beta-gene in Escherichia coli.

We studied the rate of synthesis of beta-and beta'-subunits of DNA-dependent RNA polymerase and the rate of beta-polypeptide mRNA synthesis in rifampicin-treated bacteria. The chosen antibiotic doses did not significantly inhibit the total RNA and protein synthesis in rifampicin-sensitive bacteria. For RNA-DNA hybridization experiments a pOD162 plasmid was constructed carrying a fragment of the rpoB gene and no other chromosome DNA regions. It was found that low doses of rifampicin cause an absolute and a relative increase in the rate of synthesis of the specific mRNA for the beta-subunit, suggesting a stimulation of the corresponding gene transcription and excluding the possibility of a less pronounced inhibition of the rpoB gene expression compared to that of most other genes. However the relative acceleration of transcription is substantially higher than the absolute one. The stimulating effect of rifampicin on the beta-polypeptide synthesis is also demonstrated in a coupled system of transcription and translation directed by lambda rifd47 DNA. The possible mechanisms of the rifampicin action are discussed.