Margit Pedersen

Evolution of Escherichia coli to 42°C and Subsequent Genetic Engineering Reveals Adaptive Mechanisms and Novel Mutations

Adaptive laboratory evolution (ALE) has emerged as a valuable method by which to investigate microbial adaptation to a desired environment. Here, we performed ALE to 42 °C of ten parallel populations of Escherichia coli K-12 MG1655 grown in glucose minimal media. Tightly controlled experimental conditions allowed selection based on exponential-phase growth rate, yielding strains that uniformly converged toward a similar phenotype along distinct genetic paths. Adapted strains possessed as few as 6 and as many as 55 mutations, and of the 144 genes that mutated in total, 14 arose independently across two or more strains. This mutational recurrence pointed to the key genetic targets underlying the evolved fitness increase. Genome engineering was used to introduce the novel ALE-acquired alleles in random combinations into the ancestral strain, and competition between these engineered strains reaffirmed the impact of the key mutations on the growth rate at 42 °C. Interestingly, most of the identified key gene targets differed significantly from those found in similar temperature adaptation studies, highlighting the sensitivity of genetic evolution to experimental conditions and ancestral genotype. Additionally, transcriptomic analysis of the ancestral and evolved strains revealed a general trend for restoration of the global expression state back toward preheat stressed levels. This restorative effect was previously documented following evolution to metabolic perturbations, and thus may represent a general feature of ALE experiments. The widespread evolved expression shifts were enabled by a comparatively scant number of regulatory mutations, providing a net fitness benefit but causing suboptimal expression levels for certain genes, such as those governing flagellar formation, which then became targets for additional ameliorating mutations. Overall, the results of this study provide insight into the adaptation process and yield lessons important for the future implementation of ALE as a tool for scientific research and engineering.

Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects.

Homologous recombination of single-stranded oligonucleotides is a highly efficient process for introducing precise mutations into the genome of E. coli and other organisms when mismatch repair (MMR) is disabled. This can result in the rapid accumulation of off-target mutations that can mask desired phenotypes, especially when selections need to be employed following the generation of combinatorial libraries. While the use of inducible mutator phenotypes or other MMR evasion tactics have proven useful, reported methods either require non-mobile genetic modifications or costly oligonucleotides that also result in reduced efficiencies of replacement. Therefore a new system was developed, Transient Mutator Multiplex Automated Genome Engineering (TM-MAGE), that solves problems encountered in other methods for oligonucleotide-mediated recombination. TM-MAGE enables nearly equivalent efficiencies of allelic replacement to the use of strains with fully disabled MMR and with an approximately 12- to 33-fold lower off-target mutation rate. Furthermore, growth temperatures are not restricted and a version of the plasmid can be readily removed by sucrose counterselection. TM-MAGE was used to combinatorially reconstruct mutations found in evolved salt-tolerant strains, enabling the identification of causative mutations and isolation of strains with up to 75% increases in growth rate and greatly reduced lag times in 0.6 M NaCl.

An Activator of Transcription Regulates Phage TP901-1 Late Gene Expression

ABSTRACT A promoter active in the late phase of the lytic cycle of lactococcal bacteriophage TP901-1 has been identified. The promoter is tightly regulated and requires the product of the phage TP901-1orf29 for activity. A deletion analysis of the late promoter region showed that a fragment as small as 99 bp contains both the promoter and the region necessary for activation by ORF29. The transcriptional start site of the promoter was identified by primer extension to position 13073 on the TP901-1 genome, thus located 87 bp downstream of orf29 in a 580-bp intergenic region between orf29 and orf30. Furthermore, the region located −85 to −61 bp upstream of the start site was shown to be necessary for promoter activity. During infection, the transcript arising from the late promoter is fully induced at 40 min postinfection, and our results suggest that a certain level of ORF29 must be reached in order to activate transcription of the promoter. Several lactococcal bacteriophages encode ORF29 homologous proteins, indicating that late transcription may be controlled by a similar mechanism in these phages. With the identification of this novel regulator, our results suggest that within the P335 group of lactococcal phages at least two regulatory systems controlling transcription in the late stage of infection exist.

The role of MOR and the CI operator sites on the genetic switch of the temperate bacteriophage TP901-1.

A genetic switch controls whether the temperate bacteriophage TP901-1 will enter a lytic or a lysogenic life cycle after infection of its host, Lactococcus lactis. We studied this bistable switch encoded in a small DNA fragment of 979 bp by fusing it to a reporter gene on a low-copy-number plasmid. The cloned DNA fragment contained the two divergently oriented promoters, P(R) and P(L), transcribing the lysogenic and lytic gene clusters; the two promoter-proximal genes, cI and mor; and the three CI operator sites, O(R), O(L) and O(D). We show that mor encodes a protein and that this protein in concert with CI is required for the bistability. Furthermore, interaction of CI at O(R) represses transcription from the lysogenic promoter, P(R). Thus, CI regulates its own transcription. Interaction of CI at O(L) represses transcription from the lytic promoter, P(L). The presence of only O(L) (absence of O(R) and O(D)) is enough to maintain a bistable system. The distantly located operator site, O(D), functions as a helper site by increasing binding of CI at O(R) and O(L). In the immune state, O(D) increases repression of the lytic promoter, P(L). Our results strongly support the model that a hexameric form of CI binds cooperatively to the three operator sites in the immune state forming a CI-DNA loop structure. Finally, we show that in the anti-immune state, repression of the lysogenic promoter is independent of the known CI operator sites but requires the presence of both CI and MOR.

Structure and crystallinity of water dispersible photoactive nanoparticles for organic solar cells

Water based inks would be a strong advantage for large scale production of organic photovoltaic devices. Formation of water dispersible nanoparticles produced by the Landfester method is a promising route to achieve such inks. We provide new insights into the key ink properties of poly(3-hexylthiophene) (P3HT) and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) nanoparticles such as the internal structure and crystallinity of the dispersed nanoparticles and the previously unreported drastic changes that occur when the inks are cast into a film. We observe through transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS) that the nanoparticles in dispersion are spherical with the nanodomains of P3HT being partly crystalline. When wet processed and dried into films, the nanoparticles lose their spherical shape and become flattened into oblate shapes with a large aspect ratio. Most particles are observed to have a diameter 13 times of the particle height. After casting into a film, the crystal domains adopt a preferred orientation with the majority of the nanocrystals (68%) with face-on orientation to the substrate. We propose that low substrate surface energy is responsible for particle deformation and texturing.

Identification of Quaternary Structure and Functional Domains of the CI Repressor from Bacteriophage TP901-1

The bacteriophage-encoded repressor protein plays a key role in determining the life cycle of a temperate phage following infection of a sensitive host. The repressor protein CI, which is encoded by the temperate lactococcal phage TP901-1, represses transcription from both the lytic promoter P(L) and the lysogenic promoter P(R) by binding to multiple operator sites on the DNA. In this study, we used a small bistable genetic switch element from phage TP901-1 to study the effect of cI deletions in vivo and showed that 43 amino acids could be removed from the C-terminal end of CI without destroying the ability of CI to repress transcription from the P(L) or the bistable switch properties. We showed that a helix-turn-helix motif located in the N-terminal part of CI is involved in DNA binding by introducing specific point mutations. Purification of CI and truncated forms of CI followed by analytical gel filtration and chemical cross-linking demonstrated that the C-terminal end of CI was required for oligomerization and that CI may exist as a hexamer in solution. Furthermore, expression and purification of the C-terminal part of CI (amino acids 92-180) showed that this part of the protein contained all the amino acids required to form an oligomer with an apparent molecular weight corresponding to a hexamer. We found that the C-terminal end of CI was required for de-repression of the P(L) following SOS induction, suggesting that the hexameric form of CI is needed for this or that this part of the protein is involved in the interaction with host proteins. By using small-angle X-ray scattering, we show for the first time the overall solution structure of a full-length wild-type bacteriophage repressor at low resolution revealing that the TP901-1 repressor forms a flat oligomer, most probably a trimer of dimers.

Characterization of the CI Repressor Protein Encoded by the Temperate Lactococcal Phage TP901-1

The gene regulatory mechanism determining the developmental pathway of the temperate bacteriophage TP901-1 is regulated by two phage-encoded proteins, CI and MOR. Functional domains of the CI repressor were investigated by introducing linkers of 15 bp at various positions in cI and by limited proteolysis of purified CI protein. We show that insertions of five amino acids at positions in the N-terminal half of CI resulted in mutant proteins that could no longer repress transcription from the lytic promoter, P(L). We confirmed that the N-terminal domain of CI contains the DNA binding site, and we showed that this part of the protein is tightly folded, whereas the central part and the C-terminal part of CI seem to contain more flexible structures. Furthermore, insertions at several different positions in the central part of the CI protein reduced the cooperative binding of CI to the operator sites and possibly altered the interaction with MOR.

Binding of the N-terminal domain of the lactococcal bacteriophage TP901-1 CI repressor to its target DNA: a crystallography, small angle scattering, and nuclear magnetic resonance study.

In most temperate bacteriophages, regulation of the choice of lysogenic or lytic life cycle is controlled by a CI repressor protein. Inhibition of transcription is dependent on a helix-turn-helix motif, often located in the N-terminal domain (NTD), which binds to specific DNA sequences (operator sites). Here the crystal structure of the NTD of the CI repressor from phage TP901-1 has been determined at 1.6 Å resolution, and at 2.6 Å resolution in complex with a 9 bp double-stranded DNA fragment that constitutes a half-site of the OL operator. This N-terminal construct, comprising residues 2-74 of the CI repressor, is monomeric in solution as shown by nuclear magnetic resonance (NMR), small angle X-ray scattering, and gel filtration and is monomeric in the crystal structures. The binding interface between the NTD and the half-site in the crystal is very similar to the interface that can be mapped by NMR in solution with a full palindromic site. The interactions seen in the complexes (in the crystal and in solution) explain the observed affinity for the OR site that is lower than that for the OL site and the specificity for the recognized DNA sequence in comparison to that for other repressors. Compared with many well-studied phage repressor systems, the NTD from TP901-1 CI has a longer extended scaffolding helix that, interestingly, is strongly conserved in putative repressors of Gram-positive pathogens. On the basis of sequence comparisons, we suggest that these bacteria also possess repressor/antirepressor systems similar to that found in phage TP901-1.

Modeling of the Genetic Switch of Bacteriophage TP901-1: A Heteromer of CI and MOR Ensures Robust Bistability

The lytic-lysogenic switch of the temperate lactococcal phage TP901-1 is fundamentally different from that of phage lambda. In phage TP901-1, the lytic promoter P(L) is repressed by CI, whereas repression of the lysogenic promoter P(R) requires the presence of both of the antagonistic regulator proteins, MOR and CI. We model the central part of the switch and compare the two cases for P(R) repression: the one where the two regulators interact only on the DNA and the other where the two regulators form a heteromer complex in the cytoplasm prior to DNA binding. The models are analyzed for bistability, and the predicted promoter repression folds are compared to experimental data. We conclude that the experimental data are best reproduced the latter case, where a heteromer complex forms in solution. We further find that CI sequestration by the formation of MOR:CI complexes in cytoplasm makes the genetic switch robust.

Key Players in the Genetic Switch of Bacteriophage TP901-1

After infection of a sensitive host temperate phages may enter either a lytic or a lysogenic pathway leading to new phage assembly or silencing as a prophage, respectively. The decision about which pathway to enter is centered in the genetic switch of the phage. In this work, we explore the bistable genetic switch of bacteriophage TP901-1 through experiments and statistical mechanical modeling. We examine the activity of the lysogenic promoter P(R) at different concentrations of the phage repressor, CI, and compare the effect of CI on P(R) in the presence or absence of the phage-encoded MOR protein expressed from the lytic promoter P(L). We find that the presence of large amounts of MOR prevents repression of the P(R) promoter, verifying that MOR works as an antirepressor. We compare our experimental data with simulations based on previous mathematical formulations of this switch. Good agreement between data and simulations verify the model of CI repression of P(R). By including MOR in the simulations, we are able to discard a model that assumes that CI and MOR do not interact before binding together at the DNA to repress P(R). The second model of Pr repression assumes the formation of a CI:MOR complex in the cytoplasm. We suggest that a CI:MOR complex may exist in different forms that either prevent or invoke P(R) repression, introducing a new twist on mixed feedback systems.