David K. Summers

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.

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 sub‐population 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.

Timing, self-control and a sense of direction are the secrets of multicopy plasmid stability

Multicopy plasmids of Escherichia coli are distributed randomly at cell division and, as long as copy number remains high, plasmid‐free cells arise only rarely. Copy number variation is minimized by plasmid‐encoded control circuits, and the limited data available suggest that deviations are corrected efficiently under most circumstances. However, plasmid multimers confuse control circuits, leading to copy number depression. To make matters worse, multimers out‐replicate monomers and accumulate clonally within the culture, creating a subpopulation of cells with a significantly increased rate of plasmid loss. Multimers of natural multicopy plasmids, such as ColE1, are resolved to monomers by a site‐specific recombination system (Xer‐cer ) whose activity is limited to intramolecular recombination. Recombination requires the heterodimeric XerCD recombinase plus two accessory proteins (ArgR and PepA), which activate recombination and prevent intermolecular events. Evidence is accumulating that Xer‐cer recombination is relatively slow, and there is a risk that cells might divide before multimer resolution is complete. The Rcd transcript encoded within cer may solve this problem by preventing the division of multimer‐containing cells. Working in concert, the triumvirate of copy number control, multimer resolution and cell division control achieve an extremely high fidelity of plasmid maintenance.

Indole signalling contributes to the stable maintenance of Escherichia coli multicopy plasmids.

The efficient transmission of multicopy plasmids to daughter cells at division requires that a high copy number is maintained. Plasmid multimers depress copy number, thereby causing instability. Various mechanisms exist to counter multimerization and thus ensure stable maintenance. One well‐studied example is the multimer resolution system of the Escherichia coli plasmid ColE1 which carries a recombination site (cer) at which multimers are resolved to monomers by the XerCD recombinase. A promoter within cer initiates synthesis of a short transcript (Rcd) in multimer‐containing cells. The Rcd checkpoint hypothesis proposes that Rcd delays cell division until multimer resolution is complete. We have identified tryptophanase (which catabolizes tryptophan to pyruvate and indole) as an Rcd binding protein. Furthermore, the stabilization of multicopy plasmids by Rcd is shown to be tryptophanase dependent, and a tryptophanase‐deficient strain is resistant to growth inhibition by Rcd overexpression. Rcd increases the affinity of tryptophanase for its substrate tryptophan which causes increased indole production by cells in low‐density cultures. Thus Rcd‐mediated stabilization of multicopy plasmids is dependent upon indole acting as a signalling molecule. This is an novel role for this molecule which previously has been implicated in quorum sensing‐like processes at high cell density.

Bacterial Metabolite Indole Modulates Incretin Secretion from Intestinal Enteroendocrine L Cells

Summary It has long been speculated that metabolites, produced by gut microbiota, influence host metabolism in health and diseases. Here, we reveal that indole, a metabolite produced from the dissimilation of tryptophan, is able to modulate the secretion of glucagon-like peptide-1 (GLP-1) from immortalized and primary mouse colonic L cells. Indole increased GLP-1 release during short exposures, but it reduced secretion over longer periods. These effects were attributed to the ability of indole to affect two key molecular mechanisms in L cells. On the one hand, indole inhibited voltage-gated K+ channels, increased the temporal width of action potentials fired by L cells, and led to enhanced Ca2+ entry, thereby acutely stimulating GLP-1 secretion. On the other hand, indole slowed ATP production by blocking NADH dehydrogenase, thus leading to a prolonged reduction of GLP-1 secretion. Our results identify indole as a signaling molecule by which gut microbiota communicate with L cells and influence host metabolism.

Indole Transport across Escherichia coli Membranes

Indole has many, diverse roles in bacterial signaling. It regulates the transition from exponential to stationary phase, it is involved in the control of plasmid stability, and it influences biofilm formation, virulence, and stress responses (including antibiotic resistance). Its role is not restricted to bacteria, and recently it has been shown to include mutually beneficial signaling between enteric bacteria and their mammalian hosts. In many respects indole behaves like the signaling component of a quorum-sensing system. Indole synthesized within the producer bacterium is exported into the surroundings where its accumulation is detected by sensitive cells. A view often repeated in the literature is that in Escherichia coli the AcrEF-TolC and Mtr transporter proteins are involved in the export and import, respectively, of indole. However, the evidence for their involvement is indirect, and it has been known for a long time that indole can pass directly through a lipid bilayer. We have combined in vivo and in vitro approaches to examine the relative importance of protein-mediated transport and direct passage across the E. coli membrane. We conclude that the movement of indole across the E. coli membrane under normal physiological conditions is independent of AcrEF-TolC and Mtr. Furthermore, direct observation of individual liposomes shows that indole can rapidly cross an E. coli lipid membrane without the aid of any proteinaceous transporter. These observations not only enhance our understanding of indole signaling in bacteria but also provide a simple explanation for the ability of indole to signal between biological kingdoms.

Importance of the Genetic Diversity within the Mycobacterium tuberculosis Complex for the Development of Novel Antibiotics and Diagnostic Tests of Drug Resistance

ABSTRACT Despite being genetically monomorphic, the limited genetic diversity within the Mycobacterium tuberculosis complex (MTBC) has practical consequences for molecular methods for drug susceptibility testing and for the use of current antibiotics and those in clinical trials. It renders some representatives of MTBC intrinsically resistant against one or multiple antibiotics and affects the spectrum and consequences of resistance mutations selected for during treatment. Moreover, neutral or silent changes within genes responsible for drug resistance can cause false-positive results with hybridization-based assays, which have been recently introduced to replace slower phenotypic methods. We discuss the consequences of these findings and propose concrete steps to rigorously assess the genetic diversity of MTBC to support ongoing clinical trials.

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 Red, a transcript encoded within cer and expressed in multimer‐containing cells, is also required. The appearance of Red correlates with the inhibition of division of multimer‐containing cells, presumably allowing time for the conversion of multimers to monomers by site‐specific recombination.

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.

Studies of single-chain antibody expression in quiescent Escherichia coli.

ABSTRACT Quiescent Escherichia coli cells are generated by overexpressing the Rcd transcript in an hns-205 mutant host. The resulting nongrowing, metabolically active cells were used here to express a single-chain antibody fragment (scFv) in shake flask and fermentor cultures. The expression system is based on two plasmids; one carries the product gene expressed from λPL under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from λPR. Shifting the culture from 30 to 42°C induces Rcd expression and product expression simultaneously. Our scFv carried a PelB leader, and 90% of the protein was secreted into the culture supernatant. In a batch culture, the supernatant concentration of scFv in the quiescent-cell culture (optical density at 600 nm [OD600] of 3.5) was 37 mg liter−1, compared to a maximum of 13 mg liter−1 in the control culture (final OD600 of 20). In a fed-batch fermentor culture, quiescent cells were held at an OD600 of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg liter−1. A control culture with a similar feed reached an OD600 of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg liter−1. Quiescent cells at an OD600 of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg liter−1 was achieved in 8 h. The rate of extracellular accumulation was 10-fold greater in the quiescent culture than in the control culture. This study demonstrates that it is possible to establish high-density quiescent E. coli cultures that are capable of efficient synthesis, folding, and export of proteins.