RESUMEN
Actively dividing cells perform robust and accurate DNA replication during fluctuating nutrient availability, yet factors that prevent disruption of replication remain largely unknown. Here we report that DksA, a nutrient-responsive transcription factor, ensures replication completion in Escherichia coli by removing transcription roadblocks. In the absence of DksA, replication is rapidly arrested upon amino acid starvation. This arrest requires active transcription and is alleviated by RNA polymerase mutants that compensate for DksA activity. This replication arrest occurs independently of exogenous DNA damage, yet it induces the DNA-damage response and recruits the main recombination protein RecA. This function of DksA is independent of its transcription initiation activity but requires its less-studied transcription elongation activity. Finally, GreA/B elongation factors also prevent replication arrest during nutrient stress. We conclude that transcription elongation factors alleviate fundamental conflicts between replication and transcription, thereby protecting replication fork progression and DNA integrity.
Asunto(s)
Replicación del ADN , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Transcripción Genética , Aminoácidos/metabolismo , Daño del ADN , Reparación del ADN , Guanosina Pentafosfato/metabolismoRESUMEN
Horizontal gene transfer by conjugation plays a major role in bacterial evolution, allowing the acquisition of new traits, such as virulence and resistance to antibacterial agents. With the increased antibiotic resistance in bacterial pathogens, a better understanding of how bacteria modulate conjugation under changing environments and the genetic factors involved is needed. Despite the evolutionary advantages conjugation may confer, the process can be quite stressful for the donor cell. Here, we characterize the ability of TraR, encoded on the episomal F' plasmid, to upregulate the σ(E) extracytoplasmic stress pathway in Escherichia coli. TraR, a DksA homolog, modulates transcription initiation through the secondary channel of RNA polymerase. We show here that TraR activates transcription directly; however, unlike DksA, it does so without using ppGpp as a cofactor. TraR expression can stimulate the σ(E) extracytoplasmic stress response independently of the DegS/RseA signal transduction cascade. In the absence of TraR, bacteria carrying conjugative plasmids become more susceptible to external stress. We propose that TraR increases the concentrations of periplasmic chaperones and proteases by directly activating the transcription of σ(E)-dependent promoters; this increased protein folding capacity may prepare the bacterium to endure the periplasmic stress of sex pilus biosynthesis during mating.
Asunto(s)
Conjugación Genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Factor sigma/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Operón , Regiones Promotoras Genéticas , Factor sigma/metabolismo , Estrés Fisiológico , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
An engineered reversal of the ß-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6-10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned ß-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (bktB) and thioesterase (ydiI) in combination with previously established core ß-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C6-C10 carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes. Using this approach, the expression of the Pseudomonas putida alkane monooxygenase system, encoded by alkBGT, in combination with all ß-oxidation reversal enzymes resulted in the production of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid. Following identification and characterization of potential alcohol and aldehyde dehydrogenases, chnD and chnE from Acinetobacter sp. strain SE19 were expressed in conjunction with alkBGT to demonstrate the synthesis of the C6-C10 dicarboxylic acids, adipic acid, suberic acid, and sebacic acid. The potential of a ß-oxidation cycle with ω-oxidation termination pathways was further demonstrated through the production of greater than 0.8 g/L C6-C10 ω-hydroxyacids or about 0.5 g/L dicarboxylic acids of the same chain lengths from glycerol (an unrelated carbon source) using minimal media.
Asunto(s)
Ácidos Carboxílicos/metabolismo , Ingeniería Metabólica , Pseudomonas putida/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Oxidación-Reducción , Pseudomonas putida/genética , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismoRESUMEN
We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the -oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541554, 2012, http://dx.doi.org/10.1021/sb3000782).While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a -oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled deltaffabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the ß-oxidation reversal in E. coli.
Asunto(s)
Enoil-ACP Reductasa (NADH)/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Acilcoenzima A/metabolismo , Catálisis , Enoil-ACP Reductasa (NADH)/química , Enoil-ACP Reductasa (NADH)/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo II/genética , Acido Graso Sintasa Tipo II/metabolismo , Ácidos Grasos/metabolismo , Cinética , NAD/metabolismo , Oxidación-ReducciónRESUMEN
At specific times during bacterial growth, the transcription factor DksA and the unusual nucleotide regulator ppGpp work synergistically to inhibit some Escherichia coli promoters (e.g. rRNA promoters) and to stimulate others (e.g. promoters for amino-acid synthesis and transport). However, the mechanism of DksA action remains uncertain, in part because DksA does not function like conventional transcription factors. To gain insights into DksA function, we identified mutations in dksA that bypassed the requirement for ppGpp by selecting for growth of cells lacking ppGpp on minimal medium without amino acids. We show here that two substitutions in DksA, L15F and N88I, result in higher DksA activity both in vivo and in vitro, primarily by increasing the apparent affinity of DksA for RNA polymerase (RNAP). The mutant DksA proteins suggest potential roles for ppGpp in DksA function, identify potential surfaces on DksA crucial for RNAP binding, and provide tools for future studies to elucidate the mechanism of DksA action.
Asunto(s)
Sustitución de Aminoácidos/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Transcripción Genética , Aminoácidos/metabolismo , Proteínas Portadoras/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Guanosina Tetrafosfato/metabolismo , Semivida , Holoenzimas/metabolismo , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutación/genética , Regiones Promotoras Genéticas/genética , Activación Transcripcional/genética , Operón de ARNr/genéticaRESUMEN
BACKGROUND: Due to its abundance and low-price, glycerol has become an attractive carbon source for the industrial production of value-added fuels and chemicals. This work reports the engineering of E. coli for the efficient conversion of glycerol into L-lactic acid (L-lactate). RESULTS: Escherichia coli strains have previously been metabolically engineered for the microaerobic production of D-lactic acid from glycerol in defined media by disrupting genes that minimize the synthesis of succinate, acetate, and ethanol, and also overexpressing the respiratory route of glycerol dissimilation (GlpK/GlpD). Here, further rounds of rationale design were performed on these strains for the homofermentative production of L-lactate, not normally produced in E. coli. Specifically, L-lactate production was enabled by: 1), replacing the native D-lactate specific dehydrogenase with Streptococcus bovis L-lactate dehydrogenase (L-LDH), 2) blocking the methylglyoxal bypass pathways to avoid the synthesis of a racemic mixture of D- and L-lactate and prevent the accumulation of toxic intermediate, methylglyoxal, and 3) the native aerobic L-lactate dehydrogenase was blocked to prevent the undesired utilization of L-lactate. The engineered strain produced 50 g/L of L-lactate from 56 g/L of crude glycerol at a yield 93% of the theoretical maximum and with high optical (99.9%) and chemical (97%) purity. CONCLUSIONS: This study demonstrates the efficient conversion of glycerol to L-lactate, a microbial process that had not been reported in the literature prior to our work. The engineered biocatalysts produced L-lactate from crude glycerol in defined minimal salts medium at high chemical and optical purity.
Asunto(s)
Escherichia coli/metabolismo , Glicerol/metabolismo , Ácido Láctico/biosíntesis , Ingeniería Metabólica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Glicerol Quinasa/genética , Glicerol Quinasa/metabolismo , Glicerolfosfato Deshidrogenasa/genética , Glicerolfosfato Deshidrogenasa/metabolismo , Cinética , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Piruvaldehído/metabolismo , Estereoisomerismo , Streptococcus bovis/enzimología , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/metabolismoRESUMEN
Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Epigénesis Genética , Escherichia coli/metabolismo , Retroalimentación Fisiológica , Genes de Cambio , Operón Lac/genética , Fenotipo , Multimerización de Proteína , Procesos EstocásticosRESUMEN
Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Aminoácidos , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , ARN Bacteriano/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Glycerol has become an ideal feedstock for the microbial production of bio-based chemicals due to its abundance, low cost, and high degree of reduction. We have previously reported the pathways and mechanisms for the utilization of glycerol by Escherichia coli in minimal salts medium under microaerobic conditions. Here we capitalize on such results to engineer E. coli for the production of value-added succinate from glycerol. Through metabolic engineering of E. coli metabolism, succinate production was greatly elevated by (1) blocking pathways for the synthesis of competing by-products lactate, ethanol, and acetate and (2) expressing Lactococcus lactis pyruvate carboxylase to drive the generation of succinate from the pyruvate node (as opposed to that of phosphoenolpyruvate). As such, these metabolic engineering strategies coupled cell growth to succinate production because the synthesis of succinate remained as the primary route of NAD+ regeneration. This feature enabled the operation of the succinate pathway in the absence of selective pressure (e.g. antibiotics). Our biocatalysts demonstrated a maximum specific productivity of approximately 400 mg succinate/g cell/h and a yield of 0.69 g succinate/g glycerol, on par with the use of glucose as a feedstock.
Asunto(s)
Escherichia coli/fisiología , Mejoramiento Genético/métodos , Glicerol/metabolismo , Lactobacillus/fisiología , Piruvato Carboxilasa/metabolismo , Ácido Succínico/metabolismo , Piruvato Carboxilasa/genética , Proteínas Recombinantes/metabolismoRESUMEN
The use of plasmonic nanoparticle complexes for biomedical applications such as imaging, gene therapy, and cancer treatment is a rapidly emerging field expected to significantly improve conventional medical practices. In contrast, the use of these types of nanoparticles to noninvasively trigger biochemical pathways has been largely unexplored. Here we report the light-induced activation of the thermophilic enzyme Aeropyrum pernix glucokinase, a key enzyme for the decomposition of glucose via the glycolysis pathway, increasing its rate of reaction 60% with light by conjugating the enzyme onto Au nanorods. The observed increase in enzyme activity corresponded to a local temperature increase within a calcium alginate encapsulate of ~20 °C when compared to the bulk medium maintained at standard, nonthermophilic temperatures. The encapsulated nanocomplexes were reusable and stable for several days, making them potentially useful in industrial applications. This approach could significantly improve how biochemical pathways are controlled for in vitro and, quite possibly, in vivo use.
Asunto(s)
Proteínas Arqueales/química , Glucoquinasa/química , Oro/química , Nanopartículas del Metal/química , Proteínas Arqueales/efectos de la radiación , Catálisis , Activación Enzimática/efectos de la radiación , Glucoquinasa/efectos de la radiación , Oro/efectos de la radiación , Luz , Ensayo de Materiales , Nanopartículas del Metal/efectos de la radiaciónRESUMEN
While we have recently constructed a functional reversal of the ß-oxidation cycle as a platform for the production of fuels and chemicals by engineering global regulators and eliminating native fermentative pathways, the system-level approach used makes it difficult to determine which of the many deregulated enzymes are responsible for product synthesis. This, in turn, limits efforts to fine-tune the synthesis of specific products and prevents the transfer of the engineered pathway to other organisms. In the work reported here, we overcome the aforementioned limitations by using a synthetic biology approach to construct and functionally characterize a reversal of the ß-oxidation cycle. This was achieved through the in vitro kinetic characterization of each functional unit of the core and termination pathways, followed by their in vivo assembly and functional characterization. With this approach, the four functional units of the core pathway, thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase, and acyl-CoA dehydrogenase/trans-enoyl-CoA reductase, were purified and kinetically characterized in vitro. When these four functional units were assembled in vivo in combination with thioesterases as the termination pathway, the synthesis of a variety of 4-C carboxylic acids from a one-turn functional reversal of the ß-oxidation cycle was realized. The individual expression and modular construction of these well-defined core components exerted the majority of control over product formation, with only highly selective termination pathways resulting in shifts in product formation. Further control over product synthesis was demonstrated by overexpressing a long-chain thiolase that enables the operation of multiple turns of the reversal of the ß-oxidation cycle and hence the synthesis of longer-chain carboxylic acids. The well-defined and self-contained nature of each functional unit makes the engineered reversal of the ß-oxidation cycle "chassis neutral" and hence transferrable to the host of choice for efficient fuel or chemical production.
Asunto(s)
Ácidos Carboxílicos/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasas/genética , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Acil-CoA Deshidrogenasa/genética , Acil-CoA Deshidrogenasa/metabolismo , Biocombustibles/microbiología , Enoil-CoA Hidratasa/genética , Enoil-CoA Hidratasa/metabolismo , Escherichia coli/genética , Ingeniería Genética/métodos , Cinética , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Biología Sintética/métodos , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismoRESUMEN
Maintenance of genomic stability is critical for all cells. Homologous recombination (HR) pathways promote genome stability using evolutionarily conserved proteins such as RecA, SSB, and RecQ, the Escherichia coli homologue of five human proteins at least three of which suppress genome instability and cancer. A previous report indicated that RecQ promotes the net accumulation in cells of intermolecular HR intermediates (IRIs), a net effect opposite that of the yeast and two human RecQ homologues. Here we extend those conclusions. We demonstrate that cells that lack both UvrD, an inhibitor of RecA-mediated strand exchange, and RecG, a DNA helicase implicated in IRI resolution, are inviable. We show that the uvrD recG cells die a "death-by-recombination" in which IRIs accumulate blocking chromosome segregation. First, their death requires RecA HR protein. Second, the death is accompanied by cytogenetically visible failure to segregate chromosomes. Third, FISH analyses show that the unsegregated chromosomes have completed replication, supporting the hypothesis that unresolved IRIs prevented the segregation. Fourth, we show that RecQ and induction of the SOS response are required for the accumulation of replicated, unsegregated chromosomes and death, as are RecF, RecO, and RecJ. ExoI exonuclease and MutL mismatch-repair protein are partially required. This set of genes is similar but not identical to those that promote death-by-recombination of DeltauvrD Deltaruv cells. The data support models in which RecQ promotes the net accumulation in cells of IRIs and RecG promotes resolution of IRIs that form via pathways not wholly identical to those that produce the IRIs resolved by RuvABC. This implies that RecG resolves intermediates other than or in addition to standard Holliday junctions resolved by RuvABC. The role of RecQ in net accumulation of IRIs may be shared by one or more of its human homologues.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RecQ Helicasas/metabolismo , Recombinación Genética , ADN Helicasas/genética , Proteínas de Escherichia coli/genética , MutaciónRESUMEN
The RecQ-helicase family is widespread, is highly conserved, and includes human orthologs that suppress genomic instability and cancer. In vivo, some RecQ homologs promote reduction of steady-state levels of bimolecular recombination intermediates (BRIs), which block chromosome segregation if not resolved. We find that, in vivo, E. coli RecQ can promote the opposite: the net accumulation of BRIs. We report that cells lacking Ruv and UvrD BRI-resolution and -prevention proteins die and display failed chromosome segregation attributable to accumulation of BRIs. Death and segregation failure require RecA and RecF strand exchange proteins. FISH data show that replication is completed during chromosome-segregation failure/death of ruv uvrD recA(Ts) cells. Surprisingly, RecQ (and RecJ) promotes this death. The data imply that RecQ promotes the net accumulation of BRIs in vivo, indicating a second paradigm for the in vivo effect of RecQ-like proteins. The E. coli RecQ paradigm may provide a useful model for some human RecQ homologs.