PubMed Health⌕ Search

Biomedical subjects

D K Summers

Publications and source records attributed to D K Summers.

14 recordsLinked to original sources
PubMed1999

The quiescent-cell expression system for protein synthesis in Escherichia coli.

The quiescent-cell expression system is a radical alternative to conventional fermentation for protein overproduction in Escherichia coli. It is dependent on the controlled overexpression of a small RNA called Rcd in hns mutant strains to generate nongrowing, quiescent cells which are not nutrient limited. Quiescent cells no longer produce biomass and have their metabolic resources channelled toward the expression of plasmid-based genes. The biosynthetic capacity of the system is demonstrated by its ability to express chloramphenicol acetyltransferase to more than 40% of total cell protein. Quiescent cells may provide an ideal environment for the expression of toxic as well as benign proteins.

Bacterial Proteins↗
PubMed1998

Nucleoprotein architecture and ColE1 dimer resolution: a hypothesis.

Dimers of plasmid ColE1 are converted to monomers by site-specific recombination, a process that requires 240 bp of DNA (cer) and four host-encoded proteins (XerC, XerD, ArgR and PepA). Here, we propose structures for nucleoprotein complexes involved in cer-Xer recombination based upon existing knowledge of the structures of component proteins and computational analyses of protein structure and DNA curvature. We propose that, in the nucleoprotein complex at a single cer site, a PepA hexamer acts as an adaptor, connecting the heterodimeric recombinase (XerCD) to an ArgR hexamer. This provides a protein core around which the cer site wraps, its exact path being defined by strong sequence-specific interactions with ArgR and XerCD, weak interactions with PepA and sequence-dependent flexibility of cer. The initial association of single-site complexes (pairing) is proposed to occur via an ArgR-PepA interaction. Pairing between sites in a plasmid dimer is stabilized by DNA supercoiling and is followed by a structural isomerization to form a recombination-proficient synaptic complex. We propose that paired structures formed between sites in trans are too short-lived to permit synaptic complex formation. There is thus an energetic barrier to inappropriate recombination reactions. Our proposals are consistent with a wide range of experimental observations.

Acetyltransferases↗
PubMed1994

The origins and consequences of genetic instability in prokaryotes.

The causes of genetic change include errors in replication and repair, transposition and recombination. Together, these processes generate a reservoir of genetic variants from which selection will amplify any changes which increase host fitness. Recombination, transposition and slippage during replication may all result in large-scale DNA rearrangements. Homologous and illegitimate recombination generate deletions, inversions, duplications and fusions. Intramolecular transposition can cause deletion or inversion of DNA sequences adjacent to the mobile element, while intermolecular movement may lead to fusion of donor and target replicons when transposition is replicative. Of crucial importance is how genetic variation affects the fitness of the host cell. For a neutral variant the probability of eventual fixation is small, but changes which increase host cell fitness pose a more serious problem because these cells will eventually dominate the culture. The most effective way to combat the proliferation of variant plasmid cloning vectors is to block the processes by which they arise. Cloning vectors should be "stress-tested" and DNA sequence analysis programs can be used to screen for sequence repetition which should be removed, as far as possible, from both vectors and cloned sequences. It is also important that vectors place modest metabolic demands on their hosts in order to minimise the potential increase in fitness associated with changes in plasmid structure.

Biotechnology↗
PubMed1993

ColE1 multimer formation triggers inhibition of Escherichia coli cell division.

Multimer formation and consequent copy number depression are acknowledged causes of multicopy plasmid instability. Multimer resolution sites (among which ColE1 cer is best-characterized) have been identified in a variety of plasmids. They participate in the conversion of multimers to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. We show that multimer resolution alone is insufficient to ensure stable maintenance of ColE1-like plasmids in a recombination-proficient host. The expression of Rcd, a transcript encoded within cer and expressed in multimer-containing cells, is also required. The appearance of Rcd correlates with the inhibition of division of multimer-containing cells, presumably allowing time for the conversion of multimers to monomers by site-specific recombination.

Bacterial Proteins↗
PubMed1993

Multicopy plasmid instability: the dimer catastrophe hypothesis.

Multimer formation reduces plasmid copy number and is an established cause of segregational instability. Nevertheless, it is difficult to rationalize observations that low levels of dimers can cause severe instability, if we assume they are distributed evenly in cell populations. We report here that dimer distribution is in fact heterogeneous in recombination-proficient strains. Most cells in the population contain only monomers; dimers are confined to a small subpopulation from which plasmid-free daughters arise at high frequency. In a rec+ culture where 4% of pBR322 molecules are dimers, more than half are in dimer-only cells. We show that this situation is inevitable because dimers replicate at twice the rate of monomers. Runaway multimerization is avoided because dimer-containing cells grow more slowly than their monomer-containing counterparts. A computer simulation is used to show how dimers proliferate after formation by homologous recombination. The equilibrium concentration of dimers is proportional to the inter-plasmid recombination rate and is essentially independent of the rate at which homologous recombination converts dimers to monomers.

Cell Division↗
PubMed1991

The kinetics of plasmid loss.

Instability of bacterial cloning vectors can present a serious problem when direct selection for plasmid-encoded phenotypes is undesirable, ineffective or impractical. Antibiotic selection may provide a satisfactory solution in enclosed fermentors but not where recombinant organisms are part of complex microbial consortia after release into the outside environment. In the past decade there has been significant progress towards understanding the causes of plasmid loss and the lessons learned from these studies can be used in the design of a new generation of stable vectors.

Kinetics↗
PubMed1989

Derivatives of ColE1 cer show altered topological specificity in site-specific recombination.

ColE1 contains a 250-bp sequence (cer) which is required in cis for the conversion of plasmid dimers to monomers. Recombination between cer and parB (a dimer resolution site from plasmid CloDF13) occurs in vivo at low frequency. The properties of the resulting hybrid sites have been studied. The type I hybrid closely resembles wild-type cer. It supports intramolecular recombination and requires the products of the chromosomal xerA, xerB and xerC genes together with the 250-bp site. In contrast, the type II hybrid (although differing from type I by only 2 bp) functions independently of the topological relationship of the participating sites, supporting both inter- and intramolecular recombination. Furthermore, recombination between type II sites is independent of the products of the xerA and xerB genes and requires a site of less than 50 bp.

Bacteriocin Plasmids↗
PubMed1988

Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site.

Plasmid ColE1 specifies a recombination site (cer) which participates in the conversion of plasmid dimers to monomers. The uncontrolled accumulation of dimers (and higher oligomeric forms) would otherwise lead to plasmid instability. Exonuclease III-generated deletions have been used to define the left-hand boundary of the cer site. Deletions which have lost up to 60 bp adjacent to the boundary no longer mediate the conversion of plasmid dimers to monomers, but still recombine with a wild-type site. Although this boundary region is essential for dimer resolution, its DNA sequence is poorly conserved among multimer resolution sites in related plasmids. We present evidence that its function is to influence the three-dimensional organization of the site and suggest that it may be required for the formation of a condensed nucleoprotein complex.

Chromosome Deletion↗
PubMed1984

Multimerization of high copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability.

Although the natural multicopy plasmid CoIE1 is maintained stably under most growth conditions, plasmid cloning vectors related to it are relatively unstable, being lost at frequencies of 10(-2)-10(-5) per cell per generation. Evidence suggests that CoIE1 and related plasmids are partitioned randomly at cell division and that plasmid stability is correlated inversely with plasmid multimerization; factors or conditions that reduce multimerization increase stability. Cells containing plasmid multimers segregate plasmid-free cells because the multimers are maintained at lower copy numbers than monomers, as predicted by origin-counting models for copy number control. CoIE1 is stable because it encodes a determinant, cer, that is necessary for recA-, recF-, and recE-independent recombination events that efficiently convert any multimers to monomers. We have localized monomerizing and stability determinants of CoIE1 to within a 0.38 kb region that, when cloned into plasmid vectors, greatly increases their stability.

Ampicillin↗
PubMed1983

Plasmid replication in a temperature-sensitive chromosome replication mutant of Staphylococcus aureus.

Replication of the antibiotic resistance plasmids pI258, pT10501 and pC221 has been investigated in a mutant of Staphylococcus aureus NCTC 8325, which is temperature-sensitive for the initiation of chromosome replication. Replication of pI258 stopped rapidly at the nonpermissive temperature, whilst replication of pT10501 and pC221 continued (although at a lower rate than in the wild-type). It is proposed that the product of the mutant gene may be required directly for pI258 replication, but not for replication of pT10501 or pC221.

Chromosomes, Bacterial↗
PubMed1982

Characterization of a temperature-sensitive DNA replication mutant of Staphylococcus aureus.

A temperature-sensitive DNA replication mutant of Staphylococcus aureus NCTC 8325 has been isolated and characterized. After transfer to the non-permissive-temperature (42 degrees C), DNA synthesis continued for 30 min and the mean DNA content increased by 56%. The amount of residual DNA synthesis was not reduced when the non-permissive temperature was raised, nor when chloramphenicol was added at the time of the temperature shift. During incubation at 42 degrees C, mutant bacteria accumulated the capacity to synthesize DNA after return to the permissive temperature (30 degrees C) in the presence of chloramphenicol. This capacity was lost when chloramphenicol was present at 42 degrees C. The properties of the mutant are consistent with a defect in the initiation of DNA replication at 42 degrees C.

Chloramphenicol↗