Conjugative co-transfer of lactose and bacteriophage resistance plasmids from Streptococcus cremoris UC653

Abstract When conjugative transfer of lactose-fermenting ability (Lac) was observed between Streptococcus cremoris UC653 (donor) and S. lactis MG1363 Sm (recipient), 70% of the Lac⁺ transconjugants had acquired total resistance to phage 712 and propagated phage C2 at a lower efficiency and with a reduced plaque size. Plasmid analysis of transconjugants combined with curing experiments showed that the Lac and phage resistance markers were associated with plasmids of 26 and 50 MDa, respectively. Some transconjugants contained a large plasmid of either 77 or 83 MDa which coded for both Lac and phage resistance. The phage resistance mechanism did not act at the adsorption stage and was not affected by incubation at 37°C.     

Transposition studies of mini-Mu plasmids constructed from the chemically synthesized ends of bacteriophage Mu

We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage λ p_L promoter. Derepression of p_L leads to a high frequency of mini-Mu transposition (5.6 × 10⁻²) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.     

Identification of palindromic sequences recognized by restriction endonucleases, as based on the tabularized sequencing data for seven viral and plasmid DNAs

Computer search of DNA sequences for phages φX174, G4, M13 and fd, plasmids pBR322 and pA03, and virus SV40, was employed to prepare tables specifying the size classes and frequencies of DNA segments located between all possible tetra-, penta- and hexanucleotide palindromes. As described earlier (Fuchs et al., 1978), these tables permit identifying sequences recognized by most of the restriction endonucleases. The effect of sequencing errors on the accuracy of the present identification method is evaluated. Only four of the 224 listed sequences do not appear in any of the seven DNAs, leading to discussion (see Appendix) on the natural sequence distribution.     

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IneW plasmid R388 and comparison with the related IncN plasmid R46

Summary We cloned and sequenced a 402 by DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 by of oriT was sufficient for efficient mobilization. The first 86 by of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 by perfect inverted repeat. Deletion of the first 95 by reduced the frequency of transfer by a hundred-fold. The sequence between by 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 by segment (from by 176–236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between by 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, whch coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.     

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.     

真菌的老化和质粒

真菌老化的研究工作在过去的三十五年中已有了很大的进展,本文综述了几种典型真菌老化的生长特征,以及线粒体 DNA 的部分缺失和部分扩增与老化发生的相关性。并且着重强调了 plDNA 的产生与插入可能是造成老化的一个重要原因。plDNA 的产生与反录酶有关,plDNA 可以自我复制,并能在菌丝中转移。因此,它极相似于在其它植物中被发现的转座子。本文中还介绍了几种老化发生机制的理论,并列举了迄今为止已发现的真菌质粒。     

Large plasmids in an actinomycete

Abstract Using a modified procedure large indigenous plasmids were detected in cells of three strains belonging to a group of phenotypically similar actinomycetes isolated from the rhizoplane and root nodules of Alnus spp. M _r values of the plasmids were estimated to be about 80 · 10⁶ and 120 · 10⁶. The plasmid profiles of different strains of the group were found to be almost identical. This remarkable plasmid similarly is discussed in relation to the common source of isolation.     

Development of an intergeneric conjugal transfer system for rimocidin‐producing Streptomyces rimosus

Aims: To develop an intergeneric conjugation system for rimocidin‐producing Streptomyces rimosus. Methods and Results: High efficiencies of conjugation [10^?2–10^?3 transconjugants/recipient colony forming units (CFU)] were obtained when spores of S. rimosus were heat treated at 40°C for 10 min prior to mixing with E. coli ET12567(pUZ8002/pIJ8600) as donor. Mycelium from liquid grown cultures of S. rimosus could also be used as recipient instead of spores, with 24‐h cultures giving optimal results. TSA (Oxoid) medium containing 10 m mol l^?1 MgCl₂ was the preferred medium for conjugation. Southern hybridization was used to confirm that transconjugants of S. rimosus contained a single copy of pIJ8600 integrated at a unique chromosomal attachment site (attB). The transconjugants exhibited a high stability of plasmid integration and showed strong expression of green fluorescent protein when using pIJ8655 as the conjugative vector. Conclusion: Intergeneric conjugation between E. coli and S. rimosus was achieved at high efficiency using both spores and mycelium. Significance and Impact of the Study: The conjugation system developed provides a convenient gene expression system for S. rimosus R7 and will enable the genetic manipulation of the rimocidin gene cluster.     

Mechanisms of deletion formation in Escherichia coli plasmids. II. Deletions mediated by short direct repeats.

Summary A set of plasmids containing 42, 21 and 13 bp direct repeats was used to analyze the effect of repeat length on the frequencies of deletion formation and the structure of the deleted derivatives of different recombination-deficient Escherichia coli strains. Agarose gel electrophoresis of plasmid DNA demonstrated that the formation of deletions in these plasmids was associated with dimerization of plasmid DNA. Restriction analysis of the dimers showed that deletions at short direct repeats arose non-conservatively, that is, the formation of a deletion in one monomeric plasmid unit was not associated with a duplication in the other. Mutations in the recA, recF, recJ and recO genes had no marked effect on either the frequencies of deletion formation or the structure of dimers. In contrast, recB recC mutations greatly increased the frequencies of deletion formation, 6-fold for 42 bp, and 115-fold for 21 by direct repeats. Conversion of DNA replication to the rolling circle mode in a recB recC strain, resulting in the formation of double-stranded ends, is suggested as the stimulatory effector.