Effective use of a horizontally-transferred pathway for dichloromethane catabolism requires post–transfer refinement

When microbes acquire new abilities through horizontal gene transfer, the genes and pathways must function under conditions with which they did not coevolve. If newly-acquired genes burden the host, their utility will depend on further evolutionary refinement of the recombinant strain. We used laboratory evolution to recapitulate this process of transfer and refinement, demonstrating that effective use of an introduced dichloromethane degradation pathway required one of several mutations to the bacterial host that are predicted to increase chloride efflux. We then used this knowledge to identify parallel, beneficial mutations that independently evolved in two natural dichloromethane-degrading strains. Finally, we constructed a synthetic mobile genetic element carrying both the degradation pathway and a chloride exporter, which preempted the adaptive process and directly enabled effective dichloromethane degradation across diverse Methylobacterium environmental isolates. Our results demonstrate the importance of post–transfer refinement in horizontal gene transfer, with potential applications in bioremediation and synthetic biology. DOI: http://dx.doi.org/10.7554/eLife.04279.00

Phylogeny poorly predicts the utility of a challenging horizontally transferred gene in Methylobacterium strains

Horizontal gene transfer plays a crucial role in microbial evolution. While much is known about the mechanisms that determine whether physical DNA can be transferred into a new host, the factors determining the utility of the transferred genes are less clear. We have explored this issue using dichloromethane consumption in Methylobacterium strains. Methylobacterium extorquens DM4 expresses a dichloromethane dehalogenase (DcmA) that has been acquired through horizontal gene transfer and allows the strain to grow on dichloromethane as the sole carbon and energy source. We transferred the dcmA gene into six Methylobacterium strains that include both close and distant evolutionary relatives. The transconjugants varied in their ability to grow on dichloromethane, but their fitness on dichloromethane did not correlate with the phylogeny of the parental strains or with any single tested physiological factor. This work highlights an important limiting factor in horizontal gene transfer, namely, the capacity of the recipient strain to accommodate the stress and metabolic disruption resulting from the acquisition of a new enzyme or pathway. Understanding these limitations may help to rationalize historical examples of horizontal transfer and aid deliberate genetic transfers in biotechnology for metabolic engineering

Effective use of a horizontally-transferred pathway for dichloromethane catabolism requires post-transfer refinement. eLife 2014

Abstract When microbes acquire new abilities through horizontal gene transfer, the genes and pathways must function under conditions with which they did not coevolve. If newly-acquired genes burden the host, their utility will depend on further evolutionary refinement of the recombinant strain. We used laboratory evolution to recapitulate this process of transfer and refinement, demonstrating that effective use of an introduced dichloromethane degradation pathway required one of several mutations to the bacterial host that are predicted to increase chloride efflux. We then used this knowledge to identify parallel, beneficial mutations that independently evolved in two natural dichloromethane-degrading strains. Finally, we constructed a synthetic mobile genetic element carrying both the degradation pathway and a chloride exporter, which preempted the adaptive process and directly enabled effective dichloromethane degradation across diverse Methylobacterium environmental isolates. Our results demonstrate the importance of post-transfer refinement in horizontal gene transfer, with potential applications in bioremediation and synthetic biology

Effective use of a horizontally-transferred pathway for dichloromethane catabolism requires post-transfer refinement

When microbes acquire new abilities through horizontal gene transfer, the genes and pathways must function under conditions with which they did not coevolve. If newly-acquired genes burden the host, effective use will depend on further evolutionary refinement of the recombinant strain. We used laboratory evolution to recapitulate this process of transfer and refinement, demonstrating that effective use of an introduced dichloromethane degradation pathway required one of several mutations to the bacterial host that are predicted to increase chloride efflux. We then used this knowledge to identify parallel, beneficial mutations that independently evolved in two natural dichloromethane-degrading strains. Finally, we constructed a synthetic mobile genetic element carrying both the degradation pathway and a chloride exporter, which preempted the adaptive process and directly enabled effective dichloromethane degradation across diverse Methylobacterium environmental isolates. Our results demonstrate the importance of post-transfer refinement in horizontal gene transfer, with potential applications in bioremediation and synthetic biology

Diversity and abundance of solitary and primitively eusocial bees in an urban centre: a case study from Northampton (England)

The apparent reduction of solitary and primitively eusocial bees populations has remained a huge concern over the past few decades and urbanisation is considered as one of the factors affecting bees at different scales depending on bee guild. As urbanisation is increasing globally it necessitates more research to understand the complex community dynamics of solitary and primitively eusocial bees in urban settings. We investigated the urban core of a British town for diversity and abundance of solitary bees using standardized methods, and compared the results with nearby meadows and nature reserves. The study recorded 48 species within the town, about 22 % of the total species and 58 % of the genera of solitary bees in the United Kingdom. Furthermore we found the urban core to be more diverse and abundant in solitary and primitively eusocial bees compared to the meadows and nature re-serves. Of particular note was an urban record of the nationally rare Red Data Book species Coelioxys quadridentata and its host Anthophora quadrimaculata. This research demonstrates that urban settings can contribute significantly to the conservation of solitary and primitively eusocial bees in Britain

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

Metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neither enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity. Keywords: Lignin, Protocatechuate, Experimental evolutio

Transfer of a Catabolic Pathway for Chloromethane in Methylobacterium Strains Highlights Different Limitations for Growth with Chloromethane or with Dichloromethane

Chloromethane (CM) is an ozone-depleting gas, produced predominantly from natural sources, that provides an important carbon source for microbes capable of consuming it. CM catabolism has been difficult to study owing to the challenging genetics of its native microbial hosts. Since the pathways for CM catabolism show evidence of horizontal gene transfer, we reproduced this transfer process in the laboratory to generate new CM-catabolizing strains in tractable hosts. We demonstrate that six putative accessory genes improve CM catabolism, though heterologous expression of only one of the six is strictly necessary for growth on CM. In contrast to growth of Methylobacterium strains with the closely related compound dichloromethane (DCM), we find that chloride export does not limit growth on CM and, in general that the ability of a strain to grow on DCM is uncorrelated with its ability to grow on CM. This heterologous expression system allows us to investigate the components required for effective CM catabolism and the factors that limit effective catabolism after horizontal transfer

Data from: Controlled measurement and comparative analysis of cellular components in E. coli reveals broad regulatory changes in response to glucose starvation

How do bacteria regulate their cellular physiology in response to starvation? Here, we present a detailed characterization of Escherichia coli growth and starvation over a time-course lasting two weeks. We have measured multiple cellular components, including RNA and proteins at deep genomic coverage, as well as lipid modifications and flux through central metabolism. Our study focuses on the physiological response of E. coli in stationary phase as a result of being starved for glucose, not on the genetic adaptation of E. coli to utilize alternative nutrients. In our analysis, we have taken advantage of the temporal correlations within and among RNA and protein abundances to identify systematic trends in gene regulation. Specifically, we have developed a general computational strategy for classifying expression-profile time courses into distinct categories in an unbiased manner. We have also developed, from dynamic models of gene expression, a framework to characterize protein degradation patterns based on the observed temporal relationships between mRNA and protein abundances. By comparing and contrasting our transcriptomic and proteomic data, we have identified several broad physiological trends in the E. coli starvation response. Strikingly, mRNAs are widely down-regulated in response to glucose starvation, presumably as a strategy for reducing new protein synthesis. By contrast, protein abundances display more varied responses. The abundances of many proteins involved in energy-intensive processes mirror the corresponding mRNA profiles while proteins involved in nutrient metabolism remain abundant even though their corresponding mRNAs are down-regulated

Controlled Measurement and Comparative Analysis of Cellular Components in E. coli Reveals Broad Regulatory Changes in Response to Glucose Starvation.

How do bacteria regulate their cellular physiology in response to starvation? Here, we present a detailed characterization of Escherichia coli growth and starvation over a time-course lasting two weeks. We have measured multiple cellular components, including RNA and proteins at deep genomic coverage, as well as lipid modifications and flux through central metabolism. Our study focuses on the physiological response of E. coli in stationary phase as a result of being starved for glucose, not on the genetic adaptation of E. coli to utilize alternative nutrients. In our analysis, we have taken advantage of the temporal correlations within and among RNA and protein abundances to identify systematic trends in gene regulation. Specifically, we have developed a general computational strategy for classifying expression-profile time courses into distinct categories in an unbiased manner. We have also developed, from dynamic models of gene expression, a framework to characterize protein degradation patterns based on the observed temporal relationships between mRNA and protein abundances. By comparing and contrasting our transcriptomic and proteomic data, we have identified several broad physiological trends in the E. coli starvation response. Strikingly, mRNAs are widely down-regulated in response to glucose starvation, presumably as a strategy for reducing new protein synthesis. By contrast, protein abundances display more varied responses. The abundances of many proteins involved in energy-intensive processes mirror the corresponding mRNA profiles while proteins involved in nutrient metabolism remain abundant even though their corresponding mRNAs are down-regulated