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Purpose: The purpose of this study was to examine a potential increased cognitive processing bottleneck within Parkinson disease (PD) by extending a previous overlapping task methodology. Additionally, this study extends previous overlapping task methodology in PD to examine the influence of modality (vocal vs. manual) on response delays in overlapping tasks in PD., Method: This study used the psychological refractory period (PRP) paradigm (overlapping-task paradigm) to study processing limitations as participants complete two tasks that increasingly overlap in time. Three levels of temporal overlap of tasks were utilized to vary cognitive demands on manual and vocal response time tasks. Ten participants with PD (PwPD) and 12 participants without PD were included in this study., Results: Participants with PD demonstrated response time delays across temporal overlap conditions (likely indicating motor deficits) along with a larger increase in response delays in the most overlapped, cognitively taxing condition (likely indicating longer central processing bottleneck). Additionally, modality did not influence response times differently in overlapping task conditions or within participant groups., Conclusion: An extension of previous overlapping task methodologies within a complex task was successful in demonstrating an increased central processing deficit across manual and vocal response delays in PD, regardless of modality of response.

Qsp1 is a secreted quorum sensing peptide required for virulence of the fungal meningitis pathogen Cryptococcus neoformans. Qsp1 functions to control cell wall integrity in vegetatively growing cells and also functions in mating. Rather than acting on a cell surface receptor, Qsp1 is imported to act intracellularly via the predicted oligopeptide transporter Opt1. Here, we identify a transcription factor network as a target of Qsp1. Using whole-genome chromatin immunoprecipitation, we find Qsp1 controls the genomic associations of three transcription factors to genes whose outputs are regulated by Qsp1. One of these transcription factors, Cqs2, is also required for the action of Qsp1 during mating, indicating that it might be a shared proximal target of Qsp1. Consistent with this hypothesis, deletion of CQS2 impacts the binding of other network transcription factors specifically to Qsp1-regulated genes. These genetic and genomic studies illuminate mechanisms by which an imported peptide acts to modulate eukaryotic gene expression., Competing Interests: The authors have declared that no competing interests exist.

Persisters are a sub-population of genetically sensitive bacteria that survive antibiotic treatment by entering a dormant state. The emergence of persisters from dormancy after antibiotic withdrawal leads to recurrent infection. Indole is an aromatic molecule with diverse signalling roles, including a role in persister formation. Here we demonstrate that indole stimulates the formation of Escherichia coli persisters against quinolone antibiotics which target the GyrA subunit of DNA gyrase. However, indole has no effect on the formation of E. coli persisters against an aminocoumarin, novobiocin, which targets the GyrB subunit of DNA gyrase. Two modes of indole signalling have been described: persistent and pulse. The latter refers to the brief but intense elevation of intracellular indole during stationary phase entry. We show that the stimulation of quinolone persisters is due to indole pulse, rather than persistent, signalling. In silico docking of indole on DNA gyrase predicts that indole docks perfectly to the ATP binding site of the GyrB subunit. We propose that the inhibition of indole production offers a potential route to enhance the activity of quinolones against E. coli persisters.

Indole is a signalling molecule produced by many bacterial species and involved in intraspecies, interspecies, and interkingdom signalling. Despite the increasing volume of research published in this area, many aspects of indole signalling remain enigmatic. There is disagreement over the mechanism of indole import and export and no clearly defined target through which its effects are exerted. Progress is hindered further by the confused and sometimes contradictory body of indole research literature. We explore the reasons behind this lack of consistency and speculate whether the discovery of a new, pulse mode of indole signalling, together with a move away from the idea of a conventional protein target, might help to overcome these problems and enable the field to move forward., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bacterial cells are critically dependent upon pH regulation. Here we demonstrate that indole plays a critical role in the regulation of the cytoplasmic pH of Escherichia coli. Indole is an aromatic molecule with diverse signalling roles. Two modes of indole signalling have been described: persistent and pulse signalling. The latter is illustrated by the brief but intense elevation of intracellular indole during stationary phase entry. We show that under conditions permitting indole production, cells maintain their cytoplasmic pH at 7.2. In contrast, under conditions where no indole is produced, the cytoplasmic pH is near 7.8. We demonstrate that pH regulation results from pulse, rather than persistent, indole signalling. Furthermore, we illustrate that the relevant property of indole in this context is its ability to conduct protons across the cytoplasmic membrane. Additionally, we show that the effect of the indole pulse that occurs normally during stationary phase entry in rich medium remains as a "memory" to maintain the cytoplasmic pH until entry into the next stationary phase. The indole-mediated reduction in cytoplasmic pH may explain why indole provides E. coli with a degree of protection against stresses, including some bactericidal antibiotics.

The authors show that quiescent (Q-Cell) Escherichia coli cultures can maintain metabolic activity in the absence of growth for up to 24 h, leading to four times greater specific productivity of a model metabolite, 3-hydroxybutyrate (3HB), than a control. Q-cells can be created by using the proton ionophore indole to halt cell division of an hns mutant strain. This uncouples metabolism from cell growth and allows for more efficient use of carbon feedstocks because less metabolic effort is diverted to surplus biomass production. However, the reason for the increased productivity of cells in the quiescent state was previously unknown. In this study, proteome expression patterns between wild-type and Q-cell cultures show that Q-cells overexpress stress response proteins, which prime them to tolerate the metabolic imbalances incurred through indole addition. Metabolomic data reveal the accumulation of acetyl-coenzyme A and phosphoenolpyruvate: excellent starting points for high-value chemical production. We demonstrate the exploitation of these accumulated metabolites by engineering a simple pathway for 3HB production from acetyl-coenzyme A. Quiescent cultures produced half the cell biomass of control cultures lacking indole, but were still able to produce 39.4 g L -1 of 3HB compared to 18.6 g L -1 in the control. Q-cells therefore have great potential as a platform technology for the efficient production of a wide range of commodity and high value chemicals., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Indole is a bicyclic signaling molecule with effects on both eukaryotic and prokaryotic cells. The majority of studies of indole action have been performed with bacteria cultured under aerobic conditions and little information is available about its effects under anaerobic conditions. Here the effect of the indole on anaerobic metabolism of Escherichia coli WDHL was studied. Indole in the range 0.5-8mM was added to the culture medium and cell growth, hydrogen and metabolite production were compared to cultures lacking indole. Results showed that while 8mM indole abolished growth completely, 4mM indole had a partial bacteriostatic effect and the maximum optical density of the culture decreased by 44% compared to the control cultures. In addition, 4mM indole had an important effect on anaerobic metabolism. Hydrogen production increased from 650±115 to 1137±343mL H 2 /L, and hydrogen yield increased from 0.45±0.1 to 0.94±0.34mol H 2 /mol glucose, compared to the control culture. Carbon flux was also affected and the composition of the final by-products changed. Lactate (41mM) was the main metabolite in the control cultures, whereas ethanol (56.2mM) and acetate (41.2mM) were the main metabolites in the cultures with 2mM indole. We conclude that the supplementation of E. coli cultures with exogenous indole is a simple and novel strategy to improve the production of hydrogen as well as other metabolites such as ethanol used as biofuels., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C. neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence., (Copyright © 2016 Elsevier Inc. All rights reserved.)

An inherent problem with bacterial cell factories used to produce recombinant proteins or metabolites is that resources are channeled into unwanted biomass as well as product. Over several years, attempts have been made to increase efficiency by unlinking biomass and product generation. One example was the quiescent cell (Q-Cell) expression system that generated non-growing but metabolically active Escherichia coli by over-expressing a regulatory RNA (Rcd) in a defined genetic background. Although effective at increasing the efficiency with which resources are converted to product, the technical complexity of the Rcd-based Q-Cell system limited its use. We describe here an alternative method for generating Q-Cells by the direct addition of indole, or related indole derivatives, to the culture medium of an E. coli strain carrying defined mutations in the hns gene. This simple and effective approach is shown to be functional in both shake-flask and fermenter culture. The cells remain metabolically active and analysis of their performance in the fermenter suggests that they may be particularly suitable for the production of cellular metabolites., (Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 Ca(2+) 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., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Indole has diverse signalling roles, including modulation of biofilm formation, virulence and stress responses. Changes are induced by indole concentrations of 0.5-1.0 mM, similar to those found in the supernatant of Escherichia coli stationary phase culture. Here we describe an alternative mode of indole signalling that promotes the survival of E. coli cells during long-term stationary phase. A mutant that has lost the ability to produce indole demonstrates reduced survival under these conditions. Significantly, the addition of 1 mM indole to the culture supernatant is insufficient to restore long-term survival to the mutant. We provide evidence that the pertinent signal in this case is not 1 mM indole in the culture supernatant but a transient pulse of intra-cellular indole at the transition from exponential growth to stationary phase. During this pulse the cell-associated indole reaches a maximum of approximately 60 mM. We argue that this is sufficient to inhibit growth and division by an ionophore-based mechanism and causes the cells to enter stationary phase before resources are exhausted. The unused resources are used to repair and maintain cells during the extended period of starvation.

The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter(-1)), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter(-1)) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer.

Indole is an important biological signalling molecule produced by many Gram positive and Gram negative bacterial species, including Escherichia coli. Here we study the effect of indole on the electrical properties of lipid membranes. Using electrophysiology, we show that two indole molecules act cooperatively to transport charge across the hydrophobic core of the lipid membrane. To enhance charge transport, induced by indole across the lipid membrane, we use an indole derivative, 4 fluoro-indole. We demonstrate parallels between charge transport through artificial lipid membranes and the function of complex eukaryotic membrane systems by showing that physiological indole concentrations increase the rate of mitochondrial oxygen consumption. Our data provide a biophysical explanation for how indole may link the metabolism of bacterial and eukaryotic cells., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Indole is a bacterial signalling molecule that blocks E. coli cell division at concentrations of 3-5 mM. We have shown that indole is a proton ionophore and that this activity is key to the inhibition of division. By reducing the electrochemical potential across the cytoplasmic membrane of E. coli, indole deactivates MinCD oscillation and prevents formation of the FtsZ ring that is a prerequisite for division. This is the first example of a natural ionophore regulating a key biological process. Our findings have implications for our understanding of membrane biology, bacterial cell cycle control and potentially for the design of antibiotics that target the cell membrane.

Since its publication in 1998, the genome sequence of the Mycobacterium tuberculosis H37Rv laboratory strain has acted as the cornerstone for the study of tuberculosis. In this review we address some of the practical aspects that have come to light relating to the use of H37Rv throughout the past decade which are of relevance for the ongoing genomic and laboratory studies of this pathogen. These include errors in the genome reference sequence and its annotation, as well as the recently detected variation amongst isolates of H37Rv from different laboratories., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the absence of active partitioning, strict control of plasmid copy number is required to minimise the possibility of plasmid loss at bacterial cell division. An important cause of multicopy plasmid instability is the formation of plasmid dimers by recombination and their subsequent proliferation by over-replication in a process known as the dimer catastrophe. This leads to the formation of dimer-only cells in which plasmid copy number is substantially lower than in cells containing only monomers, and which have a greatly increased probability of plasmid loss at division. The accumulation of dimers triggers the synthesis of the regulatory small RNA, Rcd, which stimulates tryptophanase and increases the production of indole. This, in turn, inhibits Escherichia coli cell division. The Rcd checkpoint hypothesis proposes that delaying cell division allows time for the relatively slow conversion of plasmid dimers to monomers by Xer-cer site-specific recombination. In the present work we have re-evaluated this hypothesis and concluded that a cell division block is insufficient to prevent the dimer catastrophe. Plasmid replication must also be inhibited. In vivo experiments have shown that indole, when added exogenously to a broth culture of E. coli does indeed stop plasmid replication as well as cell division. We have also shown that indole inhibits the activity of DNA gyrase in vitro and propose that this is the mechanism by which plasmid replication is blocked. The simultaneous effects of upon growth, cell division and DNA replication in E. coli suggest that indole acts as a true cell cycle regulator., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In this study, we have constructed a stochastic simulation of the replication and distribution of the bacterial multicopy plasmid ColE1 in a population of exponentially growing cells. It is assumed that ColE1 is randomly distributed between daughter cells at division such that copy number is a critical determinant of plasmid loss. High copy number is threatened by plasmid dimers, which arises initially by homologous recombination and accumulate by replication in a process known as the 'dimer catastrophe'. Summers et al. (1993) modelled this process and demonstrated that the accumulation of dimers is limited by the metabolic load that they exert on their hosts. ColE1 also encodes the cer site, at which host-encoded proteins act to convert dimers to monomers by site-specific recombination. The cer site also encodes a regulatory RNA, Rcd, whose synthesis from plasmid dimers triggers a checkpoint that delays cell division, presumably allowing sufficient time for dimer resolution. Here we have developed the original dimer catastrophe model by incorporating copy number variance with a stochastic model of plasmid replication. We demonstrate that the Rcd checkpoint is necessary when the rate of dimer resolution is slow. Our results indicate that dimers over-replicate compared to monomers, suggesting a mechanism for their increased metabolic load. We find that the effect of dimers on plasmid stability is significantly less severe than suggested by the original model. Consequently, we propose that the primary role of dimer resolution and the Rcd checkpoint is to reduce the metabolic burden imposed by the plasmid in a recombinogenic host, rather than to ensure plasmid stability., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

PA-824 is a promising drug candidate for the treatment of tuberculosis (TB). It is in phase II clinical trials as part of the first newly designed regimen containing multiple novel antituberculosis drugs (PA-824 in combination with moxifloxacin and pyrazinamide). However, given that the genes involved in resistance against PA-824 are not fully conserved in the Mycobacterium tuberculosis complex (MTBC), this regimen might not be equally effective against different MTBC genotypes. To investigate this question, we sequenced two PA-824 resistance genes (fgd1 [Rv0407] and ddn [Rv3547]) in 65 MTBC strains representing major phylogenetic lineages. The MICs of representative strains were determined using the modified proportion method in the Bactec MGIT 960 system. Our analysis revealed single-nucleotide polymorphisms in both genes that were specific either for several genotypes or for individual strains, yet none of these mutations significantly affected the PA-824 MICs (≤ 0.25 μg/ml). These results were supported by in silico modeling of the mutations identified in Fgd1. In contrast, "Mycobacterium canettii" strains displayed a higher MIC of 8 μg/ml. In conclusion, we found a large genetic diversity in PA-824 resistance genes that did not lead to elevated PA-824 MICs. In contrast, M. canettii strains had MICs that were above the plasma concentrations of PA-824 documented so far in clinical trials. As M. canettii is also intrinsically resistant against pyrazinamide, new regimens containing PA-824 and pyrazinamide might not be effective in treating M. canettii infections. This finding has implications for the design of multiple ongoing clinical trials.

Sequence analyses of 74 strains that encompassed major phylogenetic lineages of the Mycobacterium tuberculosis complex revealed 10 polymorphisms in mshA (Rv0486) and four polymorphisms in inhA (Rv1484) that were not responsible for isoniazid or prothionamide resistance. Instead, some of these mutations were phylogenetically informative. This genetic diversity must be taken into consideration for drug development and for the design of molecular tests for drug resistance.

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.

Background: Rahnella is a widely distributed genus belonging to the Enterobacteriaceae and frequently present on vegetables. Although Rahnella has interesting agro-economical and industrial properties and several strains possess antibiotic resistances and toxin genes which might spread within microbial communities, little is known about plasmids of this genus. Thus, we isolated a number of Rahnella strains and investigated their complements of small plasmids., Results: In total 53 strains were investigated and 11 plasmids observed. Seven belonged to the ColE1 family; one was ColE2-like and three shared homology to rolling circle plasmids. One of them belonged to the pC194/pUB110 family and two showed similarity to poorly characterised plasmid groups. The G+C content of two rolling circle plasmids deviated considerably from that of Rahnella, indicating that their usual hosts might belong to other genera. Most ColE1-like plasmids formed a subgroup within the ColE1 family that seems to be fairly specific for Rahnella. Intriguingly, the multimer resolution sites of all ColE1-like plasmids had the same orientation with respect to the origin of replication. This arrangement might be necessary to prevent inappropriate synthesis of a small regulatory RNA that regulates cell division. Although the ColE1-like plasmids did not possess any mobilisation system, they shared large parts with high sequence identity in coding and non-coding regions. In addition, highly homologous regions of plasmids isolated from Rahnella and the chromosomes of Erwinia tasmaniensis and Photorhabdus luminescens could be identified., Conclusions: For the genus Rahnella we observed plasmid-containing isolates at a frequency of 19%, which is in the average range for Enterobacteriaceae. These plasmids belonged to different groups with members of the ColE1-family most frequently found. Regions of striking sequence homology of plasmids and bacterial chromosomes highlight the importance of plasmids for lateral gene transfer (including chromosomal sequences) to distinct genera.

Escherichia coli plasmid ColE1 lacks active partitioning, and copies are distributed randomly to daughter cells at division. The plasmid is maintained stably in the bacterial population as long as its copy number remains high. The accumulation of plasmid dimers and higher multimers depresses copy number, and is an important cause of multicopy plasmid instability. ColE1 dimers are restored to the monomeric state by site-specific recombination, which requires the host-encoded proteins XerCD, ArgR and PepA acting at the plasmid cer site. In addition, a 70 nt RNA expressed from the cer site of plasmid dimers delays the division of dimer-containing cells. Here, we report that the global regulator FIS binds to cer in a sequence-specific manner, close to the Rcd promoter (P(cer)). FIS is not required for plasmid dimer resolution, but is essential for repression of P(cer) in plasmid monomers. Repression also requires the XerCD recombinase, but not ArgR or PepA. We propose a model for monomer-dimer control of P(cer) in which the promoter is repressed in plasmid monomers by the concerted action of FIS and XerCD. Rcd transcription is triggered in plasmid dimers by the lifting of XerCD-mediated repression in the synaptic complex.

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.

The secretory production of recombinant proteins by the Gram-negative bacterium Escherichia coli has several advantages over intracellular production as inclusion bodies. In most cases, targeting protein to the periplasmic space or to the culture medium facilitates downstream processing, folding, and in vivo stability, enabling the production of soluble and biologically active proteins at a reduced process cost. This review presents several strategies that can be used for recombinant protein secretion in E. coli and discusses their advantages and limitations depending on the characteristics of the target protein to be produced.

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 lambdaP(L) under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from lambdaP(R). Shifting the culture from 30 to 42 degrees 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 [OD(600)] of 3.5) was 37 mg x liter(-1), compared to a maximum of 13 mg x liter(-1) in the control culture (final OD(600) of 20). In a fed-batch fermentor culture, quiescent cells were held at an OD(600) of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg x liter(-1). A control culture with a similar feed reached an OD(600) of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg x liter(-1). Quiescent cells at an OD(600) of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg x 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.

XerCD-mediated recombination at cer converts multimers of plasmid ColE1 to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. In addition to XerCD, recombination requires the accessory factors ArgR and PepA. The promoter P(cer), located centrally within cer, is also required for stable plasmid maintenance. P(cer) is active in plasmid multimers and directs transcription of a short RNA, Rcd, which appears to inhibit cell division. It has been proposed that Rcd is part of a checkpoint which ensures that multimer resolution is complete before the cell divides. This study has shown that ArgR does not act as a transcriptional repressor of P(cer) in plasmid monomers. P(cer) is unusual in that the -35 and -10 hexamers are separated by only 15 bp and this study has demonstrated that increasing this to a more conventional spacing results in elevated activity. An increase to 17 bp resulted in a 10- to 20-fold increase in activity, while smaller effects were seen when the spacer was increased to 16 bp or 18 bp. These observations are consistent with the hypothesis that P(cer) activation involves realignment of the -35 and -10 sequences within a recombinational synaptic complex. This predicts that a 17 bp spacer promoter derivative should be down-regulated by plasmid multimerization, and this is confirmed experimentally.

Multimer formation is an important cause of instability for many multicopy plasmids. Plasmid CoIE1 is maintained stably because multimers are converted to monomers by Xer-mediated site-specific recombination at the cer site. However, multimer resolution is not the whole story; inactivation of a promoter (Pcer) within cer causes plasmid instability even though recombination is unaffected. The promoter directs the synthesis of a short transcript (Rcd) which is proposed to delay the division of multimer-containing cells. Mapping of the 5' terminus of Rcd confirms that transcription initiates from Pcer. The 3' terminus shows considerable heterogeneity, consistent with a primary transcript of 95 nt being degraded via intermediates of 79 and 70 nt. Secondary structure predictions for Rcd are presented. Of four mutations which abolish Rcd-mediated growth inhibition, one reduces the activity of Pcer while the other three map to the rcd coding sequence and reduce the steady-state level of the transcript. RNA folding analysis suggests that these three mutant transcripts adopt a common secondary structure in which the major stem-loop differs from that of wild-type Rcd. A survey of 24 cer-like multimer resolution sites revealed six which contain Pcer-like sequences. The putative transcripts from these sites have similar predicted secondary structures to Rcd and contain a highly conserved 15 base sequence. To test the hypothesis that Rcd acts as an anti-sense RNA, interacting with its target gene(s) through the 15 nt sequence, we used DNA hybridization and sequence analysis to find matches to this sequence in the Escherichia coli chromosome. Our failure to find plausible anti-sense targets has led to the suggestion that Rcd may interact directly with a protein target.

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.

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.

Sequence comparisons have shown that part of pBR322 and other pSC101 derivatives contain a remnant resolvase gene closely related to that of plasmid mini-F. This observation illuminates part of the history of these plasmids.

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.

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.

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.

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.

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.

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.

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.

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.

Overall the 1976 Festival of American Folklife was a highly successful affair both from a cultural and environmental health standpoint. Although a number of health-related problems arose on a day-to-day basis, close surveillance and prompt action on the part of the onsite sanitarian made it possible to secure early resolution of them with no ill effects among either visitors or the Festival staff. Logistically, planning and conducting of the comprehensive Smithsonian Institute (SI) food operations involving 50 different ethnic groups was an accomplishment worthy of note. Even more important, however, was the fact that during the entire time the Government Services, Inc. and SI food service operations were in progress, there was not a single case of foodborne illness associated with foods and beverages served to an estimated 500,000 persons onsite. This was in large measure due to the excellent cooperative assistance furnished by health agency personnel in the District of Columbia and the surrounding counties in Maryland and Virginia. These personnel inspected the many food establishments within their jurisdictions which served as sources for foods served at the Festival. All individuals and groups assisting directly or indirectly with this aspect of the Festival may justly be proud of the achievement.

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.