Hybrid de novo Genome Assembly of Erwinia sp. E602 and Bioinformatic Analysis Characterized a New Plasmid-Borne lac Operon Under Positive Selection

Our previous study identified a new β-galactosidase in Erwinia sp. E602. To further understand the lactose metabolism in this strain, de novo genome assembly was conducted by using a strategy combining Illumina and PacBio sequencing technology. The whole genome of Erwinia sp. E602 includes a 4.8 Mb chromosome and a 326 kb large plasmid. A total of 4,739 genes, including 4,543 protein-coding genes, 25 rRNAs, 82 tRNAs and 7 other ncRNAs genes were annotated. The plasmid was the largest one characterized in genus Erwinia by far, and it contained a number of genes and pathways responsible for lactose metabolism and regulation. Moreover, a new plasmid-borne lac operon that lacked a typical β-galactoside transacetylase (lacA) gene was identified in the strain. Phylogenetic analysis showed that the genes lacY and lacZ in the operon were under positive selection, indicating the adaptation of lactose metabolism to the environment in Erwinia sp. E602. Our current study demonstrated that the hybrid de novo genome assembly using Illumina and PacBio sequencing technologies, as well as the metabolic pathway analysis, provided a useful strategy for better understanding of the evolution of undiscovered microbial species or strains.

Boosting Auto-induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives

Background: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. Objectives: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. Method: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7lac promoter-based expression). Results: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. Conclusion: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

The Selective Advantage of the lac Operon for Escherichia coli Is Conditional on Diet and Microbiota Composition

The lac operon is one of the best known gene regulatory circuits and constitutes a landmark example of how bacteria tune their metabolism to nutritional conditions. It is nearly ubiquitous in Escherichia coli strains justifying the use of its phenotype, the ability to consume lactose, for species identification. Lactose is the primary sugar found in milk, which is abundant in mammals during the first weeks of life. However, lactose is virtually non-existent after the weaning period, with humans being an exception as many consume dairy products throughout their lives. The absence of lactose during adulthood in most mammals and the rarity of lactose in the environment, means that the selective pressure for maintaining the lac operon could be weak for long periods of time. Despite the ability to metabolize lactose being a hallmark of E. coli’s success when colonizing its primary habitat, the mammalian intestine, the selective value of this trait remains unknown in this ecosystem during adulthood. Here we determine the competitive advantage conferred by the lac operon to a commensal strain of E. coli when colonizing the mouse gut. We find that its benefit, which can be as high as 11%, is contingent on the presence of lactose in the diet and on the presence of other microbiota members in the gut, but the operon is never deleterious. These results help explaining the pervasiveness of the lac operon in E. coli, but also its polymorphism, as lac-negative E. coli strains albeit rare can naturally occur in the gut.

Historical Contingency Causes Divergence in Adaptive Expression of the lac Operon

Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes.

Lac Operon Boolean Models: Dynamical Robustness and Alternative Improvements

In Veliz-Cuba and Stigler 2011, Boolean models were proposed for the lac operon in Escherichia coli capable of reproducing the operon being OFF, ON and bistable for three (low, medium and high) and two (low and high) parameters, representing the concentration ranges of lactose and glucose, respectively. Of these 6 possible combinations of parameters, 5 produce results that match with the biological experiments of Ozbudak et al., 2004. In the remaining one, the models predict the operon being OFF while biological experiments show a bistable behavior. In this paper, we first explore the robustness of two such models in the sense of how much its attractors change against any deterministic update schedule. We prove mathematically that, in cases where there is no bistability, all the dynamics in both models lack limit cycles while, when bistability appears, one model presents 30% of its dynamics with limit cycles while the other only 23%. Secondly, we propose two alternative improvements consisting of biologically supported modifications; one in which both models match with Ozbudak et al., 2004 in all 6 combinations of parameters and, the other one, where we increase the number of parameters to 9, matching in all these cases with the biological experiments of Ozbudak et al., 2004.

Modeling complex biological systems: Tackling the parameter curse through evolution

In this perspective paper we review a previously published evolutionary model of the lac-operon to argue and demonstrate the importance of using evolutionary methods to derive relevant parameters. We show that by doing so we can debug experimental and modeling artifacts.

Modelling Biological Systems: A New Algorithm for the Inference of Boolean Networks

Biological systems are commonly constituted by a high number of interacting agents. This great dimensionality hinders biological modelling due to the high computational cost. Therefore, new modelling methods are needed to reduce computation time while preserving the properties of the depicted systems. At this point, Boolean Networks have been revealed as a modelling tool with high expressiveness and reduced computing times. The aim of this work has been to introduce an automatic and coherent procedure to model systems through Boolean Networks. A synergy that harnesses the strengths of both approaches is obtained by combining an existing approach to managing information from biological pathways with the so-called Nested Canalising Boolean Functions (NCBF). In order to show the power of the developed method, two examples of an application with systems studied in the bibliography are provided: The epithelial-mesenchymal transition and the lac operon. Due to the fact that this method relies on directed graphs as a primary representation of the systems, its applications exceed life sciences into areas such as traffic management or machine learning, in which these graphs are the main expression of the systems handled.

The cost of evolved constitutive lac gene expression is usually, but not always, maintained during evolution of generalist populations

Beneficial mutations can become costly following an environmental change. Compensatory mutations can relieve these costs, while not affecting the selected function, so that the benefits are retained if the environment shifts back to be similar to the one in which the beneficial mutation was originally selected. Compensatory mutations have been extensively studied in the context of antibiotic resistance, responses to specific genetic perturbations and in the determination of interacting gene network components. Few studies have focused on the role of compensatory mutations during more general adaptation, especially as the result of selection in fluctuating environments where adaptations to different environment components may often involve tradeoffs. We examine if costs of a mutation in lacI, which deregulated expression of the lac operon in evolving populations of Escherichia coli bacteria, was compensated. This mutation occurred in multiple replicate populations selected in environments that fluctuated between growth on lactose, where the mutation was beneficial, and on glucose, where it was deleterious. We found that compensation for the cost of the lacI mutation was rare, but, when it did occur, it did not negatively affect the selected benefit. Compensation was not more likely to occur in a particular evolution environment. Compensation has the potential to remove pleiotropic costs of adaptation, but its rarity indicates that the circumstances to bring about the phenomenon may be peculiar to each individual or impeded by other selected mutations.