Publications (27)198.48 Total impact
-
Article: A novel pair of inducible expression vectors for use in Methylobacterium extorquens.
[show abstract] [hide abstract]
ABSTRACT: BACKGROUND: Due to the ever increasing use of diverse microbial taxa in basic research and industrial settings, there is a growing need for genetic tools to alter the physiology of these organisms. In particular, there is a dearth of inducible expression systems available for bacteria outside commonly used ¿-proteobacteria, such as Escherichia coli or Pseudomonas species. To this end, we have sought to develop a pair of inducible expression vectors for use in the ¿-proteobacterium Methylobacterium extorquens, a model methylotroph. FINDINGS: We found that the P_R promoter from rhizobial phage 16-3 was active in M. extorquens and engineeredthe promoter to be inducible by either p-isopropyl benzoate (cumate) or anhydrotetracycline. These hybridpromoters P_R/cmtO and P_R/tetO were found to have high levels of expression in M. extorquens with a regulatory range of 10-fold and 30-fold, respectively. Compared to an existing cumate-inducible (10-fold range), high-level expression system for M. extorquens, P_R/cmtO and P_R/tetO have 33% of the maximal activity but were able to repress gene expression 3 and 8-fold greater, respectively. Both promoters were observed to exhibit homogeneous, titratable activation dynamics rather than on-off, switch-like behavior. The utility of these promoters was further demonstrated by complementing loss of function of ftfL - essential for growth on methanol - where we show P_R/tetO is capable of not only fully complementing function but also producing a conditional null phenotype. These promoters have been incorporated into a broad-host-range backbone allowing for potential use in a variety of bacterial hosts. CONCLUSIONS: We have developed two novel expression systems for use in M. extorquens. The expression range of these vectors should allow for increased ability to explore cellular physiology in M. extorquens. Further, the P_R/tetO promoter is capable of producing conditional null phenotypes, previously unattainable in M. extorquens. As both expression systems rely on the use of membrane permeable inducers, we suspect these expression vectors will be useful for ectopic gene expression in numerous proteobacteria.BMC Research Notes 05/2013; 6(1):183. -
Article: Synchronous Waves of Failed Soft Sweeps in the Laboratory: Remarkably Rampant Clonal Interference of Alleles at a Single Locus.
[show abstract] [hide abstract]
ABSTRACT: It has increasingly been recognized that adapting populations of microbes contain not one, but many lineages continually arising and competing at once. This process, termed clonal interference, alters the rate and dynamics of adaptation, and biases winning mutations toward those with the largest selective effect. Here we uncovered a dramatic example of clonal interference between multiple, similar mutations occurring at the same locus within replicate populations of Methylobacterium extorquens AM1. Because these mutational events involved the transposition of an insertion sequence into a narrow window of a single gene, they were both readily detectable at low frequencies and could be distinguished due to differences in insertion sites. This allowed us to detect up to 17 beneficial alleles of this type coexisting in a single population. Despite conferring a large selective benefit, the majority of these alleles rose and then fell in frequency due to other lineages emerging that were more fit. By comparing allele frequency dynamics to the trajectories of fitness gains by these populations, we estimated the fitness values of the genotypes that contained these mutations. Collectively across all populations, these alleles arose upon backgrounds with a wide range of fitness values. Within any single population, however, multiple alleles tended to rise and fall synchronously during a single wave of multiple genotypes with nearly identical fitness values. These results suggest that alleles of large benefit arose repeatedly in failed "soft sweeps" during narrow windows of adaptation due to the combined effects of epistasis and clonal interference.Genetics 01/2013; · 4.01 Impact Factor -
Article: Development of an Optimized Medium, Strain and High-Throughput Culturing Methods for Methylobacterium extorquens.
[show abstract] [hide abstract]
ABSTRACT: Methylobacterium extorquens strains are the best-studied methylotrophic model system, and their metabolism of single carbon compounds has been studied for over 50 years. Here we develop a new system for high-throughput batch culture of M. extorquens in microtiter plates by jointly optimizing the properties of the organism, the growth media and the culturing system. After removing cellulose synthase genes in M. extorquens strains AM1 and PA1 to prevent biofilm formation, we found that currently available lab automation equipment, integrated and managed by open source software, makes possible reliable estimates of the exponential growth rate. Using this system, we developed an optimized growth medium for M. extorquens using response surface methodologies. We found that media that used EDTA as a metal chelator inhibited growth and led to inconsistent culture conditions. In contrast, the new medium we developed with a PIPES buffer and metals chelated by citrate allowed for fast and more consistent growth rates. This new MethylobacteriumPIPES ('MP') medium was also robust to large deviations in its component ingredients which avoided batch effects from experiments that used media prepared at different times. MP medium allows for faster and more consistent growth than other media used for M. extorquens.PLoS ONE 01/2013; 8(4):e62957. · 4.09 Impact Factor -
Article: Complete genome sequences of six strains of the genus Methylobacterium.
[show abstract] [hide abstract]
ABSTRACT: The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.Journal of bacteriology 09/2012; 194(17):4746-8. · 3.94 Impact Factor -
Article: OASIS: an automated program for global investigation of bacterial and archaeal insertion sequences.
[show abstract] [hide abstract]
ABSTRACT: Insertion sequences (ISs) are simple transposable elements present in most bacterial and archaeal genomes and play an important role in genomic evolution. The recent expansion of sequenced genomes offers the opportunity to study ISs comprehensively, but this requires efficient and accurate tools for IS annotation. We have developed an open-source program called OASIS, or Optimized Annotation System for Insertion Sequences, which automatically annotates ISs within sequenced genomes. OASIS annotations of 1737 bacterial and archaeal genomes offered an unprecedented opportunity to examine IS evolution. At a broad scale, we found that most IS families are quite widespread; however, they are not present randomly across taxa. This may indicate differential loss, barriers to exchange and/or insufficient time to equilibrate across clades. The number of ISs increases with genome length, but there is both tremendous variation and no increase in IS density for genomes >2 Mb. At the finer scale of recently diverged genomes, the proportion of shared IS content falls sharply, suggesting loss and/or emergence of barriers to successful cross-infection occurs rapidly. Surprisingly, even after controlling for 16S rRNA sequence divergence, the same ISs were more likely to be shared between genomes labeled as the same species rather than as different species.Nucleic Acids Research 08/2012; · 8.03 Impact Factor -
Article: Epistasis from functional dependence of fitness on underlying traits.
[show abstract] [hide abstract]
ABSTRACT: Epistasis between mutations in two genes is thought to reflect an interdependence of their functions. While sometimes epistasis is predictable using mechanistic models, its roots seem, in general, hidden in the complex architecture of biological networks. Here, we ask how epistasis can be quantified based on the mathematical dependence of a system-level trait (e.g. fitness) on lower-level traits (e.g. molecular or cellular properties). We first focus on a model in which fitness is the difference between a benefit and a cost trait, both pleiotropically affected by mutations. We show that despite its simplicity, this model can be used to analytically predict certain properties of the ensuing distribution of epistasis, such as a global negative bias, resulting in antagonism between beneficial mutations, and synergism between deleterious ones. We next extend these ideas to derive a general expression for epistasis given an arbitrary functional dependence of fitness on other traits. This expression demonstrates how epistasis relative to fitness can emerge despite the absence of epistasis relative to lower level traits, leading to a formalization of the concept of independence between biological processes. Our results suggest that epistasis may be largely shaped by the pervasiveness of pleiotropic effects and modular organization in biological networks.Proceedings of the Royal Society B: Biological Sciences 08/2012; 279(1745):4156-64. · 5.41 Impact Factor -
Article: Repeated, selection-driven genome reduction of accessory genes in experimental populations.
[show abstract] [hide abstract]
ABSTRACT: Genome reduction has been observed in many bacterial lineages that have adapted to specialized environments. The extreme genome degradation seen for obligate pathogens and symbionts appears to be dominated by genetic drift. In contrast, for free-living organisms with reduced genomes, the dominant force is proposed to be direct selection for smaller, streamlined genomes. Most variation in gene content for these free-living species is of "accessory" genes, which are commonly gained as large chromosomal islands that are adaptive for specialized traits such as pathogenicity. It is generally unclear, however, whether the process of accessory gene loss is largely driven by drift or selection. Here we demonstrate that selection for gene loss, and not a shortened genome, per se, drove massive, rapid reduction of accessory genes. In just 1,500 generations of experimental evolution, 80% of populations of Methylobacterium extorquens AM1 experienced nearly parallel deletions removing up to 10% of the genome from a megaplasmid present in this strain. The absence of these deletion events in a mutation accumulation experiment suggested that selection, rather than drift, has dominated the process. Reconstructing these deletions confirmed that they were beneficial in their selective regimes, but led to decreased performance in alternative environments. These results indicate that selection can be crucial in eliminating unnecessary genes during the early stages of adaptation to a specialized environment.PLoS Genetics 05/2012; 8(5):e1002651. · 8.69 Impact Factor -
Article: Optimization of gene expression through divergent mutational paths.
[show abstract] [hide abstract]
ABSTRACT: Adaptation under similar selective pressure often leads to comparable phenotypes. A longstanding question is whether such phenotypic repeatability entails similar (parallelism) or different genotypic changes (convergence). To better understand this, we characterized mutations that optimized expression of a plasmid-borne metabolic pathway during laboratory evolution of a bacterium. Expressing these pathway genes was essential for growth but came with substantial costs. Starting from overexpression, replicate populations founded by this bacterium all evolved to reduce expression. Despite this phenotypic repetitiveness, the underlying mutational spectrum was highly diverse. Analysis of these plasmid mutations identified three distinct means to modulate gene expression: (1) reducing the gene copy number, (2) lowering transcript stability, and (3) integration of the pathway-bearing plasmid into the host genome. Our study revealed diverse molecular changes beneath convergence to a simple phenotype. This complex genotype-phenotype mapping presents a challenge to inferring genetic evolution based solely on phenotypic changes.Cell reports. 02/2012; 1(2):133-40. -
Article: FREQ-Seq: A Rapid, Cost-Effective, Sequencing-Based Method to Determine Allele Frequencies Directly from Mixed Populations.
[show abstract] [hide abstract]
ABSTRACT: Understanding evolutionary dynamics within microbial populations requires the ability to accurately follow allele frequencies through time. Here we present a rapid, cost-effective method (FREQ-Seq) that leverages Illumina next-generation sequencing for localized, quantitative allele frequency detection. Analogous to RNA-Seq, FREQ-Seq relies upon counts from the >10(5) reads generated per locus per time-point to determine allele frequencies. Loci of interest are directly amplified from a mixed population via two rounds of PCR using inexpensive, user-designed oligonucleotides and a bar-coded bridging primer system that can be regenerated in-house. The resulting bar-coded PCR products contain the adapters needed for Illumina sequencing, eliminating further library preparation. We demonstrate the utility of FREQ-Seq by determining the order and dynamics of beneficial alleles that arose as a microbial population, founded with an engineered strain of Methylobacterium, evolved to grow on methanol. Quantifying allele frequencies with minimal bias down to 1% abundance allowed effective analysis of SNPs, small in-dels and insertions of transposable elements. Our data reveal large-scale clonal interference during the early stages of adaptation and illustrate the utility of FREQ-Seq as a cost-effective tool for tracking allele frequencies in populations.PLoS ONE 01/2012; 7(10):e47959. · 4.09 Impact Factor -
Article: Regulatory revolution: evolving the "anti-LacI" repressor.
[show abstract] [hide abstract]
ABSTRACT: Much of adaptation is based upon changes in gene expression, but the emergence of new regulatory logic has not been observed directly. Now, Poelwijk et al. report evolving the lac repressor (LacI) to reverse its regulatory logic, resulting in an "anti-LacI" that represses transcription when bound to its "inducer."Cell 08/2011; 146(3):350-2. · 32.40 Impact Factor -
Article: Diminishing returns epistasis among beneficial mutations decelerates adaptation.
[show abstract] [hide abstract]
ABSTRACT: Epistasis has substantial impacts on evolution, in particular, the rate of adaptation. We generated combinations of beneficial mutations that arose in a lineage during rapid adaptation of a bacterium whose growth depended on a newly introduced metabolic pathway. The proportional selective benefit for three of the four loci consistently decreased when they were introduced onto more fit backgrounds. These three alleles all reduced morphological defects caused by expression of the foreign pathway. A simple theoretical model segregating the apparent contribution of individual alleles to benefits and costs effectively predicted the interactions between them. These results provide the first evidence that patterns of epistasis may differ for within- and between-gene interactions during adaptation and that diminishing returns epistasis contributes to the consistent observation of decelerating fitness gains during adaptation.Science 06/2011; 332(6034):1190-2. · 31.20 Impact Factor -
Article: Introduction to focus issue: genetic interactions.
[show abstract] [hide abstract]
ABSTRACT: The perturbation of a gene in an organism's genome often causes changes in the organism's observable properties or phenotypes. It is not obvious a priori whether the simultaneous perturbation of two genes produces a phenotypic change that is easily predictable from the changes caused by individual perturbations. In fact, this is often not the case: the nonlinearity and interdependence between genetic variants in determining phenotypes, also known as epistasis, is a prevalent phenomenon in biological systems. This focus issue presents recent developments in the study of epistasis and genetic interactions, emphasizing the broad implications of this phenomenon in evolutionary biology, functional genomics, and human diseases.Chaos (Woodbury, N.Y.) 06/2010; 20(2):026101. · 1.80 Impact Factor -
Article: Fast growth increases the selective advantage of a mutation arising recurrently during evolution under metal limitation.
[show abstract] [hide abstract]
ABSTRACT: Understanding the evolution of biological systems requires untangling the molecular mechanisms that connect genetic and environmental variations to their physiological consequences. Metal limitation across many environments, ranging from pathogens in the human body to phytoplankton in the oceans, imposes strong selection for improved metal acquisition systems. In this study, we uncovered the genetic and physiological basis of adaptation to metal limitation using experimental populations of Methylobacterium extorquens AM1 evolved in metal-deficient growth media. We identified a transposition mutation arising recurrently in 30 of 32 independent populations that utilized methanol as a carbon source, but not in any of the 8 that utilized only succinate. These parallel insertion events increased expression of a novel transporter system that enhanced cobalt uptake. Such ability ensured the production of vitamin B(12), a cobalt-containing cofactor, to sustain two vitamin B(12)-dependent enzymatic reactions essential to methanol, but not succinate, metabolism. Interestingly, this mutation provided higher selective advantages under genetic backgrounds or incubation temperatures that permit faster growth, indicating growth-rate-dependent epistatic and genotype-by-environment interactions. Our results link beneficial mutations emerging in a metal-limiting environment to their physiological basis in carbon metabolism, suggest that certain molecular features may promote the emergence of parallel mutations, and indicate that the selective advantages of some mutations depend generically upon changes in growth rate that can stem from either genetic or environmental influences.PLoS Genetics 09/2009; 5(9):e1000652. · 8.69 Impact Factor -
Article: Asymmetric, bimodal trade-offs during adaptation of Methylobacterium to distinct growth substrates.
[show abstract] [hide abstract]
ABSTRACT: Trade-offs between selected and nonselected environments are often assumed to exist during adaptation. This phenomenon is prevalent in microbial metabolism, where many organisms have come to specialize on a narrow breadth of substrates. One well-studied example is methylotrophic bacteria that can use single-carbon (C(1)) compounds as their sole source of carbon and energy, but generally use few, if any, multi-C compounds. Here, we use adaptation of experimental populations of the model methylotroph, Methylobacterium extorquens AM1, to C(1) (methanol) or multi-C (succinate) compounds to investigate specialization and trade-offs between these two metabolic lifestyles. We found a general trend toward trade-offs during adaptation to succinate, but this was neither universal nor showed a quantitative relationship with the extent of adaptation. After 1500 generations, succinate-evolved strains had a remarkably bimodal distribution of fitness values on methanol: either an improvement comparable to the strains adapted on methanol or the complete loss of the ability to grow on C(1) compounds. In contrast, adaptation to methanol resulted in no such trade-offs. Based on the substantial, asymmetric loss of C(1) growth during growth on succinate, we suggest that the long-term maintenance of C(1) metabolism across the genus Methylobacterium requires relatively frequent use of C(1) compounds to prevent rapid loss.Evolution 07/2009; 63(11):2816-30. · 5.15 Impact Factor -
Article: Microbiology. Getting in touch with your friends.
Science 06/2009; 324(5931):1150-1. · 31.20 Impact Factor -
Article: Methylobacterium genome sequences: a reference blueprint to investigate microbial metabolism of C1 compounds from natural and industrial sources.
[show abstract] [hide abstract]
ABSTRACT: Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name "island integration determinant" (iid). These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.PLoS ONE 02/2009; 4(5):e5584. · 4.09 Impact Factor -
Article: Development of a broad-host-range sacB-based vector for unmarked allelic exchange.
[show abstract] [hide abstract]
ABSTRACT: Although genome sequences are available for an ever-increasing number of bacterial species, the availability of facile genetic tools for physiological analysis have generally lagged substantially behind traditional genetic models. Here I describe the development of an improved, broad-host-range "in-out" allelic exchange vector, pCM433, which permits the generation of clean, marker-free genetic manipulations. Wild-type and mutant alleles were reciprocally exchanged at three loci in Methylobacterium extorquens AM1 in order to demonstrate the utility of pCM433. The broad-host-range vector for marker-free allelic exchange described here, pCM433, has the advantages of a high copy, general Escherichia coli replicon for easy cloning, an IncP oriT enabling conjugal transfer, an extensive set of restriction sites in its polylinker, three antibiotic markers, and sacB (encoding levansucrase) for negative selection upon sucrose plates. These traits should permit pCM433 to be broadly applied across many bacterial taxa for marker-free allelic exchange, which is particularly important if multiple manipulations or more subtle genetic manipulations such as point mutations are desired.BMC Research Notes 02/2008; 1:1. -
Article: Analysis of gene islands involved in methanopterin-linked C1 transfer reactions reveals new functions and provides evolutionary insights.
[show abstract] [hide abstract]
ABSTRACT: In this study, the occurrence and chromosomal clustering of genes encoding C(1) transfer reactions linked to tetrahydromethanopterin (H(4)MPT) were analyzed in a variety of proteobacteria and in representatives of the Planctomycetes via genomic analysis or via partial sequencing by cosmid walking. Although a tendency for clustering was found common for the genes of interest, significant variations in gene order and the degree of clustering were uncovered both between and within different groups of Proteobacteria and between Proteobacteria and Planctomycetes. Phylogenetic analyses suggested that the evolution of genes encoding H(4)MPT-linked reactions in Proteobacteria involved lateral transfers within Proteobacteria and possibly between Proteobacteria and other phyla. Gene cluster comparisons revealed a number of novel genes potentially involved in the C(1) transfer reactions, and these were analyzed by mutation and expression analyses. Four genes, a homolog of pabB, and three genes conserved between methanogenic Archaea and Bacteria possessing H(4)MPT-linked functions, orfY, orf1, and afpA were shown to be involved in formaldehyde oxidation/detoxification, as judged by specific mutant phenotypes. In particular, pabB contributes to the biosynthesis of para-aminobenzoic acid, a precursor of both tetrahydrofolate and H(4)MPT, and afpA apparently encodes a novel dihydromethanopterin reductase, based on mutant complementation experiments.Journal of Bacteriology 08/2005; 187(13):4607-14. · 3.83 Impact Factor -
Article: Flux analysis uncovers key role of functional redundancy in formaldehyde metabolism.
[show abstract] [hide abstract]
ABSTRACT: Genome-scale analysis of predicted metabolic pathways has revealed the common occurrence of apparent redundancy for specific functional units, or metabolic modules. In many cases, mutation analysis does not resolve function, and instead, direct experimental analysis of metabolic flux under changing conditions is necessary. In order to use genome sequences to build models of cellular function, it is important to define function for such apparently redundant systems. Here we describe direct flux measurements to determine the role of redundancy in three modules involved in formaldehyde assimilation and dissimilation in a bacterium growing on methanol. A combination of deuterium and (14)C labeling was used to measure the flux through each of the branches of metabolism for growth on methanol during transitions into and out of methylotrophy. The cells were found to differentially partition formaldehyde among the three modules depending on the flux of methanol into the cell. A dynamic mathematical model demonstrated that the kinetic constants of the enzymes involved are sufficient to account for this phenomenon. We demonstrate the role of redundancy in formaldehyde metabolism and have uncovered a new paradigm for coping with toxic, high-flux metabolic intermediates: a dynamic, interconnected metabolic loop.PLoS Biology 03/2005; 3(2):e16. · 11.45 Impact Factor -
Article: The enigmatic planctomycetes may hold a key to the origins of methanogenesis and methylotrophy.
[show abstract] [hide abstract]
ABSTRACT: Methanogenesis and methane oxidation are the major biological processes affecting the global cycling of the powerful greenhouse gas methane. To carry out the two alternative bioconversions, Nature has cleverly recycled key reactions for the C1 transfers between the oxidation levels of formaldehyde and formate, and these involve analogous enzyme systems and common specialized cofactors, methanopterin and methanofuran. Until recently, the distribution of these functions has been limited to methanogenic archaea and methylotrophic proteobacteria, and their evolutionary history remained obscure. Single interdomain lateral transfer of the respective genes has been suggested to play a role. Here we show that genes for C1 transfer reactions linked to methanopterin and methanofuran are also present in diverse representatives of the enigmatic bacterial clade, the Planctomycetes. Phylogenetic analysis places the planctomycete sequences as distantly from their archaeal counterparts as from their proteobacterial counterparts, suggesting novel scenarios for the evolution of the C1 transfer functions in both methanogens and methylotrophs. This finding suggests a possible role for Planctomycetes in the evolution of the methane cycle on Earth.Molecular Biology and Evolution 08/2004; 21(7):1234-41. · 5.55 Impact Factor
Top co-authors
Top Journals
- Journal of Bacteriology (5)
- PLoS ONE (3)
- Science (2)
- PLoS Genetics (2)
- BMC Research Notes (2)
Institutions
-
2008–2013
-
Harvard University
- Department of Organismic and Evolutionary Biology
Boston, MA, USA
-
-
2010
-
Boston University
- Department of Biology
Boston, MA, USA
-
-
2009
-
Université de Strasbourg
Strasbourg, Alsace, France
-
-
2002–2004
-
University of Washington Seattle
- Department of Microbiology
Seattle, WA, USA
-
