Hans Joachim Burkardt

Plasmid replication functions

SummaryR6-5 is a low copy number, conjugative, FII incompatibility group plasmid that has a molecular length of 102 kb and that specifies resistance against several antibiotics (chloramphenicol, fusidic acid, kanamycin, streptomycin and sulphonamide) and mercury salts. By means of in vitro cloning procedures, mini plasmids have been generated that contain a DNA segment from the essential region of R6-5 that is only 2.6 kb in length. This DNA segment, which consists of two PstI fragments that are adjacent in the parent plasmid, carries all genes and sequences required for the regulated replication and incompatibility properties of R6-5, including its origin of replication, OriV, an essential function that has been designated RepA, and the copy control function, Cop. Three different polypeptides, having monomer molecular weights of 23,000, 10,000 and 9,500 daltons, are synthesized in detectable quantities by minicells carrying pBR322 hybrid plasmids that contain DNA segments from the R6-5 essential region. A spontaneous deletion derivative of a pBR322 hybrid plasmid that carries the R6-5 origin of replication was isolated. Heteroduplex analysis of this derivative plasmid indicates that the deleted DNA segment carries the R6-5 replication origin and that its termini consist of short inverted repeat sequences.

Morphological and Molecular Characterization of Several Actinophages Isolated from Soil Which Lyse Streptomyces cattleya or S. venezuelae

Several lytic and lysogenic actinophages were isolated from soil samples infected with Streptomyces cattleya and S. venezuelae. The morphologies and some biological properties of the phages, and the physico-chemical characteristics of their DNAs, were compared. Electron micrographs indicated that all the phage heads were of an icosahedral form, but head size and length of the tail varied. Two of the phages had a broad host range; the other isolates could lyse only a limited number of species. The molecular sizes of the phage DNAs were between 32.2 and 98.5 kb as estimated by electron microscopy and restriction enzyme analysis. The same study also indicated that one of the DNA species contained cohesive ends. The G + C content of the DNAs ranged between 45.1 and 74.2 mol % as estimated from melting studies. Sedimentation velocity experiments implied that several of the phage DNAs were probably heavily glycosylated or methylated. These modifications might explain the partial or slow digestion of some of the DNAs by several of the 23 restriction enzymes tested. Protoplasts of the appropriate Streptomyces strains could be efficiently transfected with phage DNA in the presence of 25% (w/v) polyethylene glycol (mol. wt 6000).

Repetition of tetracycline resistance determinant genes on R plasmid pRSD1 in Escherichia coli.

SummaryThe 30 megadalton (Mdal)-conjgaative, fi- plasmid pRSD1 determines inducible tetracycline resistance (Tc) in Escherichia coli. As shown by restriction analysis, a 3.5 Mdal-EcoRI fragment of pRSD1 spliced into the small plasmid pRSD2124 comprises the entire Tc determinant (tet) region. A restriction map of pRSD1 is presented which includes the location of the tet region and of an “underwound” loop not related to Tc (Burkardt et al., 1978). Selective amplification of tet genes is demonstrated by three lines of evidence. (i) The resistance level of cell harbouring pRSD1 increases approximately tenfold by induction with 10μg/ml of tetracycline. Further groth in the presence of 100 μg/ml of the drug (“tet-racycline stress”) selects for cells with even higher resistance levels (about 300 μg/ml) in rec+ cells. In a recA strain, a smaller proportion of cells attains these high resistance levels suggesting the involvement of host recombination. (ii) Electron micrographs of pRSD1-DNA isolated from tetracycline-stressed cells reveal a heterogeneous population of circular DNA molecules ranging between 1.7 and 21.6 μm. The distribution of contour lengths shows a discrete pattern ascribed to the presence of autonomous single-and multiple-copy Tc determinants and to intact plasmids containing zero to six tet regions in tandem repeats. (iii) This interpretation is supported by heteroduplex and restriction analyses which demonstrate the presence of multiple copies of the 3.5 Mdal-element encompassing the tet region in pRSD1 molecules selected by tetracycline stress. It has been concluded that gene amplification leading to tandem repetition of the tet region ensues in pRSD1. Such plasmids confer increased tetracycline resistance and can, therefore, be selected by high doses of the drug.

Properties of two conjugative plasmids mediating tetracycline resistance, raffinose catabolism and hydrogen sulfide production in Escherichia coli

SummaryThe tetracycline resistance (Tc) and raffi nose-hydrogen sulfide (Raf-Hys) characters of Escherichia coli D1021 are located on two compatible, conjugative fi− plasmids named pRSDI and pRSD2, respectively. These were transferred together or separately to the isogenic background of E. coli K12 and isolated as transconjugants. Plasmid molecules isolated as covalently closed circular DNA have been analysed by buoyant density centrifugation, sucrose gradient sedimentation and electron microscopy. pRSDI (Tc) showed a buoyant density of 1.716 g/cm3 (56% GC), the open circular (OC) form had a sedimentation coefficient of 37s. If grown without tetracycline prior to isolation the contour length of most pRSDI molecules was 14.6 (±0.1) μm (30.3 × 106 daltons) with a minor species of 13.9 (± 0.1) μm owing to a small deletion. Pronounced length variation of pRSDI by growth in the presence of tetracycline owing to gene amplification is subject of a subsequent paper. In contrast, pRSD2 (Raf-Hys) molecules are stable under all conditions tested. The buoyant density was 1.713 g/cm3 (53% GC), the sedimentation coefficient of OGDNA was 58s and the contour length was 40.2 ± 0.6 μm (83 × 106 daltons). During exponential growth one copy of pRSD2 per chromosome was found indicating stringent control of plasmid replication. At the onset of stationary growth the copy number rose from one to four per host chromosome indicating relaxation of the replication control. The level of plasmid-coded α-galactosidase increases with copy numbers and has been used to test the gene dosage.

Cloning of the entire region for nitrogen fixation from Klebsiella pneumoniae on a multicopy plasmid vehicle in Escherichia coli

SummaryTwo deletion mutants pAP1 (MW 82 Mdals) and pAP2 (MW 64 Mdals) were isolated by P1 transduction of the plasmid pRD1 (MW 101 (Mdals). These plasmid mutants still carry the his-nif region of K. pneumoniae. They are selftransmissible and mediate resistance to ampicillin, kanamycin and tetracycline. Comparing the HindIII maps of pRD1, pAP1 and pAP2 showed that pAP1 was derived from pRD1 by an 8 μm deletion and pAP2 by two deletions — the same 8 μm deletion and a further 9 μm deletion. The plasmids pAP1 and pAP2 helped us to locate the his-nif region of pRD1 on 3 adjacent HindIII fragments (number 5, 4 and 3 according to gelelectrophoresis). The molecular weights of these fragments were 8.2, 10 and 15 Mdals. These 3 fragments were cloned separately on the multicopy plasmid vehicle pWL625 giving rise to the hybrid plasmids pWK1 (pWL625+HindIII fragment 4), pWK2 (pWL625+HindIII fragment 5). None of these hybrid plasmids conferred nitrogen fixation capacity on E. coli C cells. By combining HindIII fragment 4 and 3 in the same alignment as in pRD1 and cloning them together on pWL625 the hybrid plasmid pWK120 (pLW625+HindIII fragments 4 and 3) was found to carry the entire nif region. An E. coli C strain harbouring the plasmid pWK120 grew on nitrogen free medium and reduced acetylene. The plasmid pWK 120 had a contourlength of 17 μm, a buoyant density of 1.715 g/ml and a copy number up to 65.

ISR1: an insertion element isolated from the soil bacterium Rhizobium lupini.

The insertion element ISR1 was isolated from the soil bacterium R. lupini. In this strain, ISR1 shows a very strong affinity to plasmid RP4. It causes RP4 mutations at the strikingly high frequency of 10(-2) to 10(-1), either by the integration itself or by generating deletions. In E. coli, ISR1 seems to be inactive. No evidence could be obtained for a promoter site on ISR1 or for an ISR1-encoded protein. Our results indicate, however, an ISR1-specific termination signal for either transcription or translation.

Spontaneous degradation of pRD1 DNA into unique size classes is recA dependent

SummaryThe his and nif genes of the Pl type plasmid pRD1 were lost at a high frequency in a recA+ but not in a recA-Escherichia coli host during growth in a non-selective medium. 92% of the His- Nif- segregants after 6 subcultures retained the genetic markers of the precursor plasmid RP4, while the remainder lost all of the pRD1 markers with the concomintant loss of ccc-DNA. Plasmids purified from the His- Nif- segregants resembled RP4 in the physical and genetic properties examined.The contour length of pRD1 DNA molecules from a recA- strain was 49 μm corresponding to a molecular weight of 101 Mdals and the buoyant density was 1.715 g/cm3. In contrast, the contour lengths of plasmid molecules isolated from a recA- host carrying pRD1 fell into 3 size classes of 49, 19 and 2 μm corresponding to molecular weights of 101, 39 and 4 Mdals respectively and two DNA species of buoyant density 1.715 and 1.719 g/cm3 were observed.

Fertility inhibition in Rhizobium lupini by the resistance plasmid RP4

SummaryR plasmid RP4 inhibits the fertility of R. lupini. An RP4 carrying R. lupini donor strain is no longer capable of transferring chromosomal genes. After loss of RP4 the R. lupini fertility reappears. Plasmid RP4 spontaneously mutates at high frequency in R. lupini. RP4 mutants which do not inhibit fertility were isolated. These mutants were always transfer-defective, too. It is postulated that the genetic information for fertility inhibition in R. lupini is a substantial part of the transfer unit of the RP4 plasmid.

Transposon mutagenesis of the gene encoding the bacteriophage P1 restriction endonuclease: Co-linearity of the gene and gene product

Abstract The gene encoding the restriction activity of the restriction-modification enzyme EcoPI has been delineated by transposon mutagenesis. Insertion of Tn1 or Tn5 into this non-essential gene has been used to identify the gene product, gpres, as a polypeptide with an apparent molecular weight of 110,000. Minicells infected by P1 res:: Tn1 or P1 res:: Tn5 phages synthesize truncated gpres molecules. Correlation of the size of these polypeptides with the site of transposon insertion, determined by heteroduplex analyses using DNA from P1res::Tn1 and from a λ/P1 hybrid phage containing the EcoRI fragment 2 of the P1 genome (λ-P1.2) has demonstrated the direction of transcription of the res gene. The DNA sequence encoding gpres contains two EcoRI restriction sites whereas the modification activity of EcoPI is completely encoded by EcoRI fragment 2 of the P1 genome and is functional in λ-P1.2-infected cells. Infection of minicells with phage carrying Tn1 DNA leads to the synthesis of Tn1-encoded β-lactamase and transposase. The results obtained suggest that transposition should occur at a higher than normal frequency immediately following infection of cells with transposon-carrying phages.

Adenine + Thymine Content of Different Genes Located on the Broad Host Range Plasmid RP4

The genetic map of plasmid RP4 was correlated with its adenine+thymine (AT) map. For this purpose, RP4 DNA was digested with one or both of the restriction enzymes EcoRI and HindIII and the resulting linear RP4 molecules and fragments were partially denatured, examined in the electron microscope and their AT maps were determined using a computer program. From these AT maps the EcoRI and HindIII restriction sites were located on the AT map of RP4. Since the positions of these restriction sites on the genetic map of RP4 are known, the maps could be compared. They revealed a high AT content for the Tn1 transposon and the kanamycin resistance gene. The tra-1 region is also distinguished by a sharply defined AT-rich region, whereas tra-2 and the tetracycline resistance gene have an AT content which is not distinctly different from the average AT content of RP4.