Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose-Derived Sugars.

Currently, microbial conversion of lignocellulose-derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E.

Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification.

Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH).

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria.

The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function.

DksA guards elongating RNA polymerase against ribosome-stalling-induced arrest.

In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs.

Rho and NusG suppress pervasive antisense transcription in Escherichia coli.

Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes.

Complex physiology and compound stress responses during fermentation of alkali-pretreated corn stover hydrolysate by an Escherichia coli ethanologen.

The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E.

The RPB2 flap loop of human RNA polymerase II is dispensable for transcription initiation and elongation.

The flap domain of multisubunit RNA polymerases (RNAPs), also called the wall, forms one side of the RNA exit channel. In bacterial RNAP, the mobile part of the flap is called the flap tip and makes essential contacts with initiation and elongation factors. Cocrystal structures suggest that the orthologous part of eukaryotic RNAPII, called the flap loop, contacts transcription factor IIB (TFIIB), but the function of the flap loop has not been assessed.

Context-dependent function of "GATA switch" sites in vivo.

Master transcriptional regulators of development often function through dispersed cis elements at endogenous target genes. While cis-elements are routinely studied in transfection and transgenic reporter assays, it is challenging to ascertain how they function in vivo. To address this problem in the context of the locus encoding the critical hematopoietic transcription factor Gata2, we engineered mice lacking a cluster of GATA motifs 2.

A single cis element maintains repression of the key developmental regulator Gata2.

In development, lineage-restricted transcription factors simultaneously promote differentiation while repressing alternative fates. Molecular dissection of this process has been challenging as transcription factor loci are regulated by many trans-acting factors functioning through dispersed cis elements. It is not understood whether these elements function collectively to confer transcriptional regulation, or individually to control specific aspects of activation or repression, such as initiation versus maintenance.

Context-dependent GATA factor function: combinatorial requirements for transcriptional control in hematopoietic and endothelial cells.

GATA factors are fundamental components of developmentally important transcriptional networks. By contrast to common mechanisms in which transacting factors function directly at promoters, the hematopoietic GATA factors GATA-1 and GATA-2 often assemble dispersed complexes over broad chromosomal regions. For example, GATA-1 and GATA-2 occupy five conserved regions over approximately 100 kb of the Gata2 locus in the transcriptionally repressed and active states, respectively, in erythroid cells.

Patient-controlled analgesia.

One of the most common methods for providing postoperative analgesia is via patient-controlled analgesia (PCA). Although the typical approach is to administer opioids via a programmable infusion pump, other drugs and other modes of administration are available. This article reviews the history and practice of many aspects of PCA and provides extensive guidelines for the practice of PCA-administered opioids.

Molecular determinants of NOTCH4 transcription in vascular endothelium.

The process whereby the primitive vascular network develops into the mature vasculature, known as angiogenic vascular remodeling, is controlled by the Notch signaling pathway. Of the two mammalian Notch receptors expressed in vascular endothelium, Notch1 is broadly expressed in diverse cell types, whereas Notch4 is preferentially expressed in endothelial cells. As mechanisms that confer Notch4 expression were unknown, we investigated how NOTCH4 transcription is regulated in human endothelial cells and in transgenic mice.

Measurement of protein-DNA interactions in vivo by chromatin immunoprecipitation.

Elucidating mechanisms controlling nuclear processes requires an understanding of the nucleoprotein structure of genes at endogenous chromosomal loci. Traditional approaches to measuring protein-DNA interactions in vitro have often failed to provide insights into physiological mechanisms. Given that most transcription factors interact with simple DNA sequence motifs, which are abundantly distributed throughout a genome, it is essential to pinpoint the small subset of sites bound by factors in vivo.

Highly restricted localization of RNA polymerase II within a locus control region of a tissue-specific chromatin domain.

RNA polymerase II (Pol II) can associate with regulatory elements far from promoters. For the murine beta-globin locus, Pol II binds the beta-globin locus control region (LCR) far upstream of the beta-globin promoters, independent of recruitment to and activation of the betamajor promoter. We describe here an analysis of where Pol II resides within the LCR, how it is recruited to the LCR, and the functional consequences of recruitment.

GATA-1-dependent transcriptional repression of GATA-2 via disruption of positive autoregulation and domain-wide chromatin remodeling.

Interplay among GATA transcription factors is an important determinant of cell fate during hematopoiesis. Although GATA-2 regulates hematopoietic stem cell function, mechanisms controlling GATA-2 expression are undefined. Of particular interest is the repression of GATA-2, because sustained GATA-2 expression in hematopoietic stem and progenitor cells alters hematopoiesis.

Histone deacetylase-dependent establishment and maintenance of broad low-level histone acetylation within a tissue-specific chromatin domain.

The murine beta-globin locus in adult erythroid cells is characterized by a broad pattern of erythroid-specific histone acetylation. The embryonic beta-globin genes Ey and betaH1 are located in a approximately 30 kb central subdomain characterized by low-level histone acetylation, while the fetal/adult genes betamajor and betaminor and the upstream locus control region reside in hyperacetylated chromatin. Histone deacetylase (HDAC) inhibitors induce H4 acetylation at the Ey promoter [Forsberg, E.

Hematopoietic-specific activators establish an overlapping pattern of histone acetylation and methylation within a mammalian chromatin domain.

Posttranslational modification of histones through acetylation, methylation, and phosphorylation is a common mode of regulating chromatin structure and, therefore, diverse nuclear processes. One such modification, methylated histone H3 at lysine-4 (H3-meK4), colocalizes with hyperacetylated histones H3 and H4 in mammalian chromatin. Whereas activators directly recruit acetyltransferases, the process whereby H3-meK4 is established is unknown.

Cooperative activities of hematopoietic regulators recruit RNA polymerase II to a tissue-specific chromatin domain.

The hematopoietic transcription factor GATA-1 regulates erythropoiesis and beta-globin expression. Although consensus GATA-1 binding sites exist throughout the murine beta-globin locus, we found that GATA-1 discriminates among these sites in vivo. Conditional expression of GATA-1 in GATA-1-null cells recapitulated the occupancy pattern.