Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter P LtetO-1 allows the regulation of gene expression over an up to 5000-fold range with anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.

/pdf/independent-and-tight-regulation-of-transcriptional-units-in-2ea2uolqxl.pdf

Independent and Tight Regulation of Transcriptional Units in Escherichia Coli Via the LacR/O, the TetR/O and AraC/I1-I2 Regulatory Elements

SUMMARY An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3′-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can “sense” and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.

Replication and control of circular bacterial plasmids.

Antisense RNA control is now recognized as an efficient and specific means of regulating gene expression at the posttranscriptional level. Almost all naturally occurring cases have been found in prokaryotes, often in their accessory genetic elements. Several antisense RNA systems are now well-understood, and these display a spectrum of mechanisms of action, binding pathways, and kinetics. This review summarizes antisense RNA control in prokaryotes, emphasizing the biology of the systems involved.

Antisense RNA Control in Bacteria, Phages, and Plasmids

An increasing number of small RNAs (sRNAs) have been shown to regulate critical pathways in prokaryotes and eukaryotes. In bacteria, regulation by trans-encoded sRNAs is predominantly found in the coordination of intricate stress responses. The mechanisms by which sRNAs modulate expression of its targets are diverse. In common to most is the possibility that interference with the translation of mRNA targets may also alter the abundance of functional sRNAs. Aiming to understand the unique role played by sRNAs in gene regulation, we studied examples from two distinct classes of bacterial sRNAs in Escherichia coli using a quantitative approach combining experiment and theory. Our results demonstrate that sRNA provides a novel mode of gene regulation, with characteristics distinct from those of protein-mediated gene regulation. These include a threshold-linear response with a tunable threshold, a robust noise resistance characteristic, and a built-in capability for hierarchical cross-talk. Knowledge of these special features of sRNA-mediated regulation may be crucial toward understanding the subtle functions that sRNAs can play in coordinating various stress-relief pathways. Our results may also help guide the design of synthetic genetic circuits that have properties difficult to attain with protein regulators alone.

/pdf/quantitative-characteristics-of-gene-regulation-by-small-rna-47rduiconw.pdf

Quantitative characteristics of gene regulation by small RNA.

The development and maturation of E. coli biofilms in flow-chambers was investigated. We found that the presence of transfer constitutive IncF plasmids induced biofilm development forming structures resembling those reported for Pseudomonas aeruginosa. The development occurred in a step-wise process: (i). attachment of cells to the substratum, (ii). clonal growth and microcolony formation, and (iii). differentiation into expanding structures rising 70-100 microm into the water phase. The first two steps were the same in the plasmid-carrying and plasmid-free strains, whereas the third step only occurred in conjugation pilus proficient plasmid-carrying strains. The final shapes of the expanding structures in the mature biofilm seem to be determined by the pilus configuration, as various mutants affected in the processing and activity of the transfer pili displayed differently structured biofilms. We further provide evidence that flagella, type 1 fimbriae, curli and Ag43 are all dispensable for the observed biofilm maturation. In addition, our results indicate that cell-to-cell signalling mediated by autoinducer 2 (AI-2) is not required for differentiation of E. coli within a biofilm community. We suggest on the basis of these results that E. coli K-12 biofilm development and maturation is dependent on cell-cell adhesion factors, which may act as inducers of self-assembly processes that result in differently structured biofilms depending on the adhesive properties on the cell surface.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2003.03490.x

Development and maturation of Escherichia coli K-12 biofilms.

New cloning vectors for integration into the λ attachment site attB of the Escherichia coli chromosome

The phage-plasmid hybrid phasyl can replicate as a phage in the presence of a filamentous phage of Escherichia coli (M13, fl, fd). The extragenic region of phasyl shows homology with the plus and the minus origins of filamentous phages. Insertion of a Cmr fragment into the plus origin or of a Kmr fragment into the minus origin resulted in a reduced transduction frequency, while insertion into other parts of the extragenic region did not. This suggests that phagelike replication of phasyl is mediated by an origin that coincides with the two homologous elements in the extragenic region. Autonomous replication of phasyl occurs from a second origin (oriA) that is located between positions 297 and 636. This fragment mediates replication if the Arp protein is supplied in trans. Arp is the only phage-encoded protein and is essential for plasmidlike replication. No sequence homology to other known origins was found. Phasyl derivatives with either one of the two origins inactivated can be rescued via the alternative replication mode, suggesting that the two replication pathways are independent.

Characterization of a phage-plasmid hybrid (phasyl) with two independent origins of replication isolated from escherichia coli

Characterization ofa Phage-Plasmid Hybrid (Phasyl) withTwo Independent Origins ofReplication Isolated from Escherichia coli

The termination of transcription in the dnaA gene of E. coli was analyzed using transcriptional fusions to the galactokinase gene, S1 nuclease mapping and quantification of translation products by Western blots. The majority of transcripts originating from dnaA promoters terminated at several positions within a 200 by region inside the dnaA reading frame.

Transcription termination in the dnaA gene.

Phasyl is the smallest naturally occurring replicon found so far in Escherichia coli. It encodes a protein which is essential for autonomous replication (Arp). The transcriptional start of the arp gene was mapped. A strong antisense promoter was found in close proximity to the arp promoter. The inactivation of this promoter led in cis to a strong increase of the transcription of the arp gene and to the inactivation of autonomous replication of phasyl. The product of the antisense promoter is an 83 nt RNA molecule, which is not translated. The antisense RNA led in trans to the inhibition of the translation of the arp mRNA, presumably mediated by the formation of an RNA-RNA hybrid in which the Shine-Dalgarno sequence of the arp transcript is sequestered. The expression of the arp gene is thus controlled by two negatively acting mechanisms: it is subject to a transcriptional control in cis exerted by the antisense promoter and to a translational control in trans mediated by the antisense RNA. Inactivation of one mechanism of control cannot be compensated by the remaining one.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1991.tb07857.x

Ludger Diederich

Papers

New cloning vectors for integration into the λ attachment site attB of the Escherichia coli chromosome

Characterization of a phage-plasmid hybrid (phasyl) with two independent origins of replication isolated from escherichia coli

Characterization ofa Phage-Plasmid Hybrid (Phasyl) withTwo Independent Origins ofReplication Isolated from Escherichia coli

Transcription termination in the dnaA gene.

Expression of the replication protein Arp of phasyl shows dual regulation by an antisense promoter.