Predictable tuning of protein expression in bacteria.

We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expression level of any Escherichia coli gene by changing only a few bases. Measured protein levels for 91% of our designed sequences were within twofold of the desired target level.

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.

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.

Minimizing acetate formation from overflow metabolism in Escherichia coli: comparison of genetic engineering strategies to improve robustness toward sugar gradients in large-scale fermentation processes.

Introduction: Escherichia coli, a well characterized workhorse in biotechnology, has been used to produce many recombinant proteins and metabolites, but have a major drawback in its tendency to revert to overflow metabolism. This phenomenon occurs when excess sugar triggers the production of mainly acetate under aerobic conditions, a detrimental by-product that reduces carbon efficiency, increases cell maintenance, and ultimately inhibits growth. Although this can be prevented by controlled feeding of the sugar carbon source to limit its availability, gradients in commercial-scale bioreactors can still induce it in otherwise carbon-limited cells. While the underlying mechanisms have been extensively studied, these have mostly used non-limited cultures. In contrast, industrial production typically employs carbon-limited processes, which results in a substantially different cell physiology. Objective: The objective of this study was to evaluate and compare the efficiency of different metabolic engineering strategies with the aim to reduce overflow metabolism and increase the robustness of an industrial 2'-O-fucosyllactose producing strain under industrially relevant conditions. Methods: Three distinct metabolic engineering strategies were compared: i) alterations to pathways leading to and from acetate, ii) increased flux towards the tricarboxylic acid (TCA) cycle, and iii) reduced glucose uptake rate. The engineered strains were evaluated for growth, acetate formation, and product yield under non-limiting batch conditions, carbon limited fed-batch conditions, and after a glucose pulse in fed-batch mode. Results and Discussion: The findings demonstrated that blockage of the major acetate production pathways by deletion of the pta and poxB genes or increased carbon flux into the TCA cycle by overexpression of the gltA and deletion of the iclR genes, were efficient ways to reduce acetate accumulation. Surprisingly, a reduced glucose uptake rate did not reduce acetate formation despite it having previously been shown as a very effective strategy. Interestingly, overexpression of gltA was the most efficient way to reduce acetate accumulation in non-limited cultures, whereas disruption of the poxB and pta genes was more effective for carbon-limited cultures exposed to a sudden glucose shock. Strains from both strategies showed increased tolerance towards a glucose pulse during carbon-limited growth indicating feasible ways to engineer industrial E. coli strains with enhanced robustness.

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.

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.

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.

Repression of the lysogenic PR promoter in bacteriophage TP901-1 through binding of a CI-MOR complex to a composite OM-OR operator.

A functional genetic switch from the lactococcal bacteriophage TP901-1, deciding which of two divergently transcribing promoters becomes most active and allows this bi-stable decision to be inherited in future generations requires a DNA region of less than 1 kb. The fragment encodes two repressors, CI and MOR, transcribed from the PR and PL promoters respectively. CI can repress the transcription of the mor gene at three operator sites (OR, OL, and OD), leading to the immune state. Repression of the cI gene, leading to the lytic (anti-immune) state, requires interaction between CI and MOR by an unknown mechanism, but involving a CI:MOR complex. A consensus for putative MOR binding sites (OM sites), and a common topology of three OM sites adjacent to the OR motif was here identified in diverse phage switches that encode CI and MOR homologs, in a search for DNA sequences similar to the TP901-1 switch. The OR site and all putative OM sites are important for establishment of the anti-immune repression of PR, and a putative DNA binding motif in MOR is needed for establishment of the anti-immune state. Direct evidence for binding between CI and MOR is here shown by pull-down experiments, chemical crosslinking, and size exclusion chromatography. The results are consistent with two possible models for establishment of the anti-immune repression of cI expression at the PR promoter.

Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1.

The CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD1 and CTD2. Full-length CI is a hexameric protein, whereas a truncated version, CI∆58, forms dimers. We identify the dimerization region of CI∆58 as CTD1 and determine its secondary structure to be helical both within the context of CI∆58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CI∆58 interacts with the OL operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CI∆58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function.

The functional half-life of an mRNA depends on the ribosome spacing in an early coding region.

Bacterial mRNAs are translated by closely spaced ribosomes and degraded from the 5'-end, with half-lives of around 2 min at 37 °C in most cases. Ribosome-free or "naked" mRNA is known to be readily degraded, but the initial event that inactivates the mRNA functionally has not been fully described. Here, we characterize a determinant of the functional stability of an mRNA, which is located in the early coding region. Using literature values for the mRNA half-lives of variant lacZ mRNAs in Escherichia coli, we modeled how the ribosome spacing is affected by the translation rate of the individual codons. When comparing the ribosome spacing at various segments of the mRNA to its functional half-life, we found a clear correlation between the functional mRNA half-life and the ribosome spacing in the mRNA region approximately between codon 20 and codon 45. From this finding, we predicted that inserts of slowly translated codons before codon 20 or after codon 45 should shorten or prolong, respectively, the functional mRNA half-life by altering the ribosome density in the important region. These predictions were tested on eight new lacZ variants, and their experimentally determined mRNA half-lives all supported the model. We thus suggest that translation-rate-mediated differences in the spacing between ribosomes in this early coding region is a parameter that determines the mRNAs functional half-life. We present a model that is in accordance with many earlier observations and that allows a prediction of the functional half-life of a given mRNA sequence.

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.

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.

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.

Identification of DNA-binding sites for the activator involved in late transcription of the temperate lactococcal phage TP901-1.

Alt, encoded by the lactococcal phage TP901-1, is needed for late transcription. We identify Alt as a DNA-binding protein, and footprint analysis shows that Alt binds to a region containing four imperfect direct repeats (ALT boxes) located -76 to -32 relative to the P(late) transcriptional start site. The importance of the ALT boxes was confirmed by deletion of one or two ALT boxes and by introducing mutations in ALT boxes 1 and 4. Alt is proposed to act as a tetramer or higher multimer activating transcription of TP901-1 late genes by binding to the four ALT boxes, and bending of the DNA may be important for transcriptional activation of P(late). Furthermore, our results suggest that DNA replication may be required for late transcription in TP901-1. Additionally, we identify gp28 of the related lactococcal phage Tuc2009 as an activator and show that the activators required for late transcription in TP901-1 and Tuc2009 are interchangeable.