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Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

Clostridioides difficile (Cdiff) infection (CDI) continues to be the leading threat of nosocomial deaths worldwide and a major burden on health-care systems. Broad-spectrum antibiotics eradicate the normal gut microbiome, killing protective commensal bacteria and increasing CDI recurrence. In contrast, Fidaxomicin (Fdx) is a narrow-spectrum antibiotic that inhibits Cdiff growth without affecting crucial gut microbes. However, the basis of the narrow-spectrum activity of Fdx on its target, RNA polymerase (RNAP), in Cdiff has been enigmatic. Recently, Cao et al. (Nature, doi: 10.1038/s41586-022-04545-z) combined transgenic RNAP design and synthesis with cryo-electron microscopy (cryo-EM) to identify a key determinant of Fdx inhibition of Cdiff RNAP. This finding was further corroborated by biochemical, bioinformatics, and genetic analysis. This microreview describes implications of this work for lineage-specific antibiotic design and new directions toward understanding transcription and regulation in Cdiff and other bacterial pathogens.

ABSTRACT Expression of virulence genes in pathogenic Escherichia coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-silence initiation by displacing H-NS from promoters, but how H-NS inhibition of elongation is overcome is not understood. In uropathogenic E. coli (UPEC), elongation regulator RfaH aids expression of some H-NS-silenced pathogenicity operons (e.g., hlyCABD encoding hemolysin). RfaH associates with elongation complexes (ECs) via direct contacts to a transiently exposed, nontemplate DNA strand sequence called operon polarity suppressor (ops). RfaH–ops interactions establish long-lived RfaH–EC contacts that allow RfaH to recruit ribosomes to the nascent mRNA and to suppress transcriptional pausing and termination. Using ChIP-seq, we mapped the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ70 in the UPEC strain CFT073. We identify eight RfaH-activated operons, all of which were bound by H-NS and StpA. Four are new additions to the RfaH regulon. Deletion of RfaH caused premature termination, whereas deletion of H-NS and StpA allowed elongation without RfaH. Thus, RfaH is an elongation counter-silencer of H-NS. Consistent with elongation counter-silencing, deletion of StpA alone decreased the effect of RfaH. StpA increases DNA bridging, which inhibits transcript elongation via topological constraints on RNAP. Residual RfaH effect when both H-NS and StpA were deleted was attributable to targeting of RfaH-regulated operons by a minor H-NS paralog, Hfp. These operons have evolved higher levels of H-NS–binding features, explaining minor-paralog targeting. IMPORTANCE Bacterial pathogens adapt to hosts and host defenses by reprogramming gene expression, including by H-NS counter-silencing. Counter-silencing turns on transcription initiation when regulators bind to promoters and rearrange repressive H-NS nucleoprotein filaments that ordinarily block transcription. The specialized NusG paralog RfaH also reprograms virulence genes but regulates transcription elongation. To understand how elongation regulators might affect genes silenced by H-NS, we mapped H-NS, StpA (an H-NS paralog), RfaH, σ70, and RNA polymerase (RNAP) locations on DNA in the uropathogenic E. coli strain CFT073. Although H-NS–StpA filaments bind only 18% of the CFT073 genome, all loci at which RfaH binds RNAP are also bound by H-NS–StpA and are silenced when RfaH is absent. Thus, RfaH represents a distinct class of counter-silencer that acts on elongating RNAP to enable transcription through repressive nucleoprotein filaments. Our findings define a new mechanism of elongation counter-silencing and explain how RfaH functions as a virulence regulator.

Summary: Gene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the structures, arrangements of DNA-binding domains (DBDs), and positions of DBD–DNA contacts in linear and bridged filaments are uncertain. To characterize the H-NS DBD contacts that silence transcription by RNA polymerase, we combined ·OH footprinting, molecular dynamics, statistical modeling, and DBD mapping using a chemical nuclease (Fe2+-EDTA) tethered to the DBDs (TEN-map). We find that H-NS DBDs contact DNA at indistinguishable locations in bridged or linear filaments and that the DBDs vary in orientation and position with ∼10-bp average spacing. Our results support a hemi-sequestration model of linear-to-bridged H-NS switching. Linear filaments able to inhibit only transcription initiation switch to bridged filaments able to inhibit both initiation and elongation using the same irregularly spaced DNA contacts.

In the canonical bacterial transcription, both nascent transcript and polymerase dissociate from template DNA. By employing multi-color single-molecule fluorescence imaging, here the authors show that RNA polymerases remain bound to DNA after the transcript release.

ABSTRACT Zymomonas mobilis is an ethanologenic alphaproteobacterium with promise for the industrial conversion of renewable plant biomass into fuels and chemical bioproducts. Limited functional annotation of the Z. mobilis genome is a current barrier to both fundamental studies of Z. mobilis and its development as a synthetic biology chassis. To gain insight, we collected sample-matched multiomics data, including RNA sequencing (RNA-seq), transcription start site (TSS) sequencing (TSS-seq), termination sequencing (term-seq), ribosome profiling, and label-free shotgun proteomic mass spectrometry, across different growth conditions and used these data to improve annotation and assign functional sites in the Z. mobilis genome. Proteomics and ribosome profiling informed revisions of protein-coding genes, which included 44 start codon changes and 42 added proteins. We developed statistical methods for annotating transcript 5′ and 3′ ends, enabling the identification of 3,940 TSSs and their corresponding promoters and 2,091 transcription termination sites, which were distinguished from RNA processing sites by the lack of an adjacent RNA 5′ end. Our results revealed that Z. mobilis σA −35 and −10 promoter elements closely resemble canonical Escherichia coli −35 and −10 elements, with one notable exception: the Z. mobilis −10 element lacks the highly conserved −7 thymine observed in E. coli and other previously characterized σA promoters. The σA promoters of another alphaproteobacterium, Caulobacter crescentus, similarly lack the conservation of −7 thymine in their −10 elements. Our results anchor the development of Z. mobilis as a platform for synthetic biology and establish strategies for empirical genome annotation that can complement purely computational methods. IMPORTANCE Efforts to rationally engineer synthetic pathways in Zymomonas mobilis are impeded by a lack of knowledge and tools for predictable and quantitative programming of gene regulation at the transcriptional, posttranscriptional, and posttranslational levels. With the detailed functional characterization of the Z. mobilis genome presented in this work, we provide crucial knowledge for the development of synthetic genetic parts tailored to Z. mobilis. This information is vital as researchers continue to develop Z. mobilis for synthetic biology applications. Our methods and statistical analyses also provide ways to rapidly advance the understanding of poorly characterized bacteria via empirical data that enable the experimental validation of sequence-based prediction for genome characterization and annotation.

Plant-derived fuels and chemicals from renewable biomass have significant potential to replace reliance on petroleum and improve global carbon balance. However, plant biomass contains significant fractions of oligosaccharides that are not usable natively by many industrial microorganisms, including Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis. Even after chemical or enzymatic hydrolysis, some carbohydrate remains as non-metabolizable oligosaccharides (e.g., cellobiose or longer cellulose-derived oligomers), thus reducing the efficiency of conversion to useful products. To begin to address this problem for Z. mobilis, we engineered a strain (Z. mobilis GH3) that expresses a glycosyl hydrolase (GH) with β-glucosidase activity from a related α-proteobacterial species, Caulobacter crescentus, and subjected it to an adaptation in cellobiose medium. Growth on cellobiose was achieved after a prolonged lag phase in cellobiose medium that induced changes in gene expression and cell composition, including increased expression and extracellular release of GH. These changes were reversible upon growth in glucose-containing medium, meaning they did not result from genetic mutation but could be retained upon transfer of cells to fresh cellobiose medium. After adaptation to cellobiose, our GH-expressing strain was able to convert about 50% of cellobiose to glucose within 24 h and use it for growth and ethanol production. Alternatively, pre-growth of Z. mobilis GH3 in sucrose medium enabled immediate growth on cellobiose. Proteomic analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid release or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are that Z. mobilis can be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural response of Z. mobilis to sucrose that promotes sucrase production.

Utilization of both C5 and C6 sugars to produce biofuels and bioproducts is a key goal for the development of integrated lignocellulosic biorefineries. Previously we found that although engineered Zymomonas mobilis 2032 was able to ferment glucose to ethanol when fermenting highly concentrated hydrolyzates such as 9% glucan-loading AFEX-pretreated corn stover hydrolyzate (9% ACSH), xylose conversion after glucose depletion was greatly impaired. We hypothesized that impaired xylose conversion was caused by lignocellulose-derived inhibitors (LDIs) in hydrolyzates. To investigate the effects of LDIs on the cellular physiology of Z. mobilis during fermentation of hydrolyzates, including impacts on xylose utilization, we generated synthetic hydrolyzates (SynHs) that contained nutrients and LDIs at concentrations found in 9% ACSH. Comparative fermentations of Z. mobilis 2032 using SynH with or without LDIs were performed, and samples were collected for end product, transcriptomic, metabolomic, and proteomic analyses. Several LDI-specific effects were observed at various timepoints during fermentation including upregulation of sulfur assimilation and cysteine biosynthesis, upregulation of RND family efflux pump systems (ZMO0282-0285) and ZMO1429-1432, downregulation of a Type I secretion system (ZMO0252-0255), depletion of reduced glutathione, and intracellular accumulation of mannose-1P and mannose-6P. Furthermore, when grown in SynH containing LDIs, Z. mobilis 2032 only metabolized ∼50% of xylose, compared to ∼80% in SynH without LDIs, recapitulating the poor xylose utilization observed in 9% ACSH. Our metabolomic data suggest that the overall flux of xylose metabolism is reduced in the presence of LDIs. However, the expression of most genes involved in glucose and xylose assimilation was not affected by LDIs, nor did we observe blocks in glucose and xylose metabolic pathways. Accumulations of intracellular xylitol and xylonic acid was observed in both SynH with and without LDIs, which decreased overall xylose-to-ethanol conversion efficiency. Our results suggest that xylose metabolism in Z. mobilis 2032 may not be able to support the cellular demands of LDI mitigation and detoxification during fermentation of highly concentrated lignocellulosic hydrolyzates with elevated levels of LDIs. Together, our findings identify several cellular responses to LDIs and possible causes of impaired xylose conversion that will enable future strain engineering of Z. mobilis.

Metabolic engineering of the biofuel-producing Zymomonas mobilis is necessary if we are to unlock the metabolic potential present in this non-model microbe. Manipulation of such organisms can be challenging because of the limited genetic tools for iterative genome modification. Here, we have developed an efficient method for generating markerless genomic deletions or additions in Z. mobilis. This is a two-step process that involves homologous recombination of an engineered suicide plasmid bearing Z. mobilis targeting sequences and a subsequent recombination event that leads to loss of the suicide plasmid and a genome modification. A key feature of this strategy is that GFP expressed from the suicide plasmid allows easy identification of cells that have lost the plasmid by using a fluorescence activated cell sorter. Using this method, we demonstrated deletion of the gene encoding lactate dehydrogenase (ldh) and the operon for cellulose synthase (bcsABC). In addition, by modifying the plasmid design, we demonstrated targeted insertion of the crtIBE operon encoding a neurosporene biosynthetic pathway into the Z. mobilis genome without addition of any antibiotic resistance genes. We propose this approach will provide an efficient and flexible platform for improved genetic engineering of Z. mobilis.

Abstract Background Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight Z. mobilis strains have been completely sequenced and found to contain 2–8 native plasmids. However, systematic verification of predicted Z. mobilis plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain. Results In this study, we determined the complete chromosome and plasmid sequences of ZM4 and its engineered xylose-utilizing derivatives 2032 and 8b. Compared to previously published and revised ZM4 chromosome sequences, the ZM4 chromosome sequence reported here contains 65 nucleotide sequence variations as well as a 2400-bp insertion. Four plasmids were identified in all three strains, with 150 plasmid genes predicted in strain ZM4 and 2032, and 153 plasmid genes predicted in strain 8b due to the insertion of heterologous DNA for expanded substrate utilization. Plasmid genes were then annotated using Blast2GO, InterProScan, and systems biology data analyses, and most genes were found to have apparent orthologs in other organisms or identifiable conserved domains. To verify plasmid gene prediction, RNA-Seq was used to map transcripts and also compare relative gene expression under various growth conditions, including anaerobic and aerobic conditions, or growth in different concentrations of biomass hydrolysates. Overall, plasmid genes were more responsive to varying hydrolysate concentrations than to oxygen availability. Additionally, our results indicated that although all plasmids were present in low copy number (about 1–2 per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion. Conclusions The complete genome of ZM4 and two xylose-utilizing derivatives is reported in this study, with an emphasis on identifying and characterizing plasmid genes. Plasmid gene annotation, validation, expression levels at growth conditions of interest, and contribution to host fitness are reported for the first time.

Riboswitches are RNA-based regulatory elements, which regulate downstream gene expression by binding of small molecular weight ligands. Here the authors demonstrate the molecular mechanism of a transcriptional riboswitch that integrates changes in transcription rates, metabolite concentration, and kinetic on- and off-rates of ligand binding.

Abstract Background Gamma valerolactone (GVL) treatment of lignocellulosic bomass is a promising technology for degradation of biomass for biofuel production; however, GVL is toxic to fermentative microbes. Using a combination of chemical genomics with the yeast (Saccharomyces cerevisiae) deletion collection to identify sensitive and resistant mutants, and chemical proteomics to monitor protein abundance in the presence of GVL, we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast. Results Chemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate cinnamic acid and its derivatives to vinyl forms, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain. Conclusions We found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol accumulation in aerobically grown cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates.

RNA polymerases (RNAPs) contain a conserved ‘secondary channel’ which binds regulatory factors that modulate transcription initiation. In Escherichia coli, the secondary channel factors (SCFs) GreB and DksA both repress ribosomal RNA (rRNA) transcription, but SCF loading and repression mechanisms are unclear. We observed in vitro fluorescently labeled GreB molecules binding to single RNAPs and initiation of individual transcripts from an rRNA promoter. GreB arrived and departed from promoters only in complex with RNAP. GreB did not alter initial RNAP-promoter binding but instead blocked a step after conformational rearrangement of the initial RNAP-promoter complex. Strikingly, GreB-RNAP complexes never initiated at an rRNA promoter; only RNAP molecules arriving at the promoter without bound GreB produced transcript. The data reveal that a model SCF functions by a ‘delayed inhibition’ mechanism and suggest that rRNA promoters are inhibited by GreB/DksA because their short-lived RNAP complexes do not allow sufficient time for SCFs to dissociate.

Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts γ-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against Mycobacterium tuberculosis RNAP in vitro, but clinical use of Fdx is limited to treating Clostridium difficile intestinal infections due to poor absorption. To identify the structural determinants of Fdx binding to RNAP, we determined the 3.4 Å cryo-electron microscopy structure of a complete M. tuberculosis RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin, explaining its strong effect on M. tuberculosis. Additional structures define conformational states of M. tuberculosis RNAP between the free apo-holoenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on the Fdx binding pocket.

CarD is an essential mycobacterial protein that binds the RNA polymerase (RNAP) and affects the transcriptional profile of Mycobacterium smegmatis and Mycobacterium tuberculosis [6]. We predicted that CarD was directly regulating RNAP function but our prior experiments had not determined at what stage of transcription CarD was functioning and at which genes CarD interacted with the RNAP. To begin to address these open questions, we performed chromatin immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. The distribution of RNAP subunits β and σA were also profiled. We expected that RNAP β would be present throughout transcribed regions and RNAP σA would be predominantly enriched at promoters based on work in Escherichia coli [3], however this had yet to be determined in mycobacteria. The ChIP-seq analyses revealed that CarD was never present on the genome in the absence of RNAP, was primarily associated with promoter regions, and was highly correlated with the distribution of RNAP σA. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. Here we describe in detail the data from the ChIP-seq experiments associated with the study published by Srivastava and colleagues in the Proceedings of the National Academy of Science in 2013 [5] as well as discuss the findings from this dataset in relation to both CarD and mycobacterial transcription as a whole. The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE48164).

In bacteria, the regulation of gene expression by cis-acting transcriptional riboswitches located in the 5'-untranslated regions of messenger RNA requires the temporal synchronization of RNA synthesis and ligand binding-dependent conformational refolding. Ligand binding to the aptamer domain of the riboswitch induces premature termination of the mRNA synthesis of ligand-associated genes due to the coupled formation of 3'-structural elements acting as terminators. To date, there has been no high resolution structural description of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA element. Here, we show that for the guanine-sensing xpt-pbuX riboswitch from Bacillus subtilis, the conformation of the full-length transcripts is static: it exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand. We show that only the combined matching of transcription rates and ligand binding enables transcription intermediates to undergo ligand-dependent conformational refolding.

[This corrects the article DOI: 10.1371/journal.pgen.1006372.].

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.

Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders E. coli more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene ref. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to E. coli in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens Neisseria gonorrhoeae and Staphylococcus aureus serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence.

Transcription control is the foundation of gene regulation. Whereas a cell is fully equipped for this task, viruses often depend on the host to supply tools for their transcription program. Over the course of evolution and adaptation, viruses have found diverse ways to optimally exploit cellular host processes such as transcription to their own benefit. Just as cells are increasingly understood to employ nascent RNAs in transcription regulation, recent discoveries are revealing how viruses use nascent RNAs to benefit their own gene expression. In this review, we first outline the two different transcription programs used by viruses, i.e., transcription (DNA-dependent) and RNA-dependent RNA synthesis. Subsequently, we use the distinct stages (initiation, elongation, termination) to describe the latest insights into nascent RNA-mediated regulation in the context of each relevant stage.

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the −10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the −10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.

Bacterial H-NS forms nucleoprotein filaments that spread on DNA and bridge distant DNA sites. H-NS filaments co-localize with sites of Rho-dependent termination in Escherichia coli, but their direct effects on transcriptional pausing and termination are untested. In this study, we report that bridged H-NS filaments strongly increase pausing by E. coli RNA polymerase at a subset of pause sites with high potential for backtracking. Bridged but not linear H-NS filaments promoted Rho-dependent termination by increasing pause dwell times and the kinetic window for Rho action. By observing single H-NS filaments and elongating RNA polymerase molecules using atomic force microscopy, we established that bridged filaments surround paused complexes. Our results favor a model in which H-NS-constrained changes in DNA supercoiling driven by transcription promote pausing at backtracking-susceptible sites. Our findings provide a mechanistic rationale for H-NS stimulation of Rho-dependent termination in horizontally transferred genes and during pervasive antisense and noncoding transcription in bacteria.

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). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts.

ABSTRACT Intrinsic terminators, which encode GC-rich RNA hairpins followed immediately by a 7-to-9-nucleotide (nt) U-rich “U-tract,” play principal roles of punctuating and regulating transcription in most bacteria. However, canonical intrinsic terminators with strong U-tracts are underrepresented in some bacterial lineages, notably mycobacteria, leading to proposals that their RNA polymerases stop at noncanonical intrinsic terminators encoding various RNA structures lacking U-tracts. We generated recombinant forms of mycobacterial RNA polymerase and its major elongation factors NusA and NusG to characterize mycobacterial intrinsic termination. Using in vitro transcription assays devoid of possible mycobacterial contaminants, we established that mycobacterial RNA polymerase terminates more efficiently than Escherichia coli RNA polymerase at canonical terminators with imperfect U-tracts but does not terminate at putative terminators lacking U-tracts even in the presence of mycobacterial NusA and NusG. However, mycobacterial NusG exhibits a novel termination-stimulating activity that may allow intrinsic terminators with suboptimal U-tracts to function efficiently. IMPORTANCE Bacteria rely on transcription termination to define and regulate units of gene expression. In most bacteria, precise termination and much regulation by attenuation are accomplished by intrinsic terminators that encode GC-rich hairpins and U-tracts necessary to disrupt stable transcription elongation complexes. Thus, the apparent dearth of canonical intrinsic terminators with recognizable U-tracts in mycobacteria is of significant interest both because noncanonical intrinsic terminators could reveal novel routes to destabilize transcription complexes and because accurate understanding of termination is crucial for strategies to combat mycobacterial diseases and for computational bioinformatics generally. Our finding that mycobacterial RNA polymerase requires U-tracts for intrinsic termination, which can be aided by NusG, will guide future study of mycobacterial transcription and aid improvement of predictive algorithms to annotate bacterial genome sequences.

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.

FNR is a well-studied global regulator of anaerobiosis, which is widely conserved across bacteria. Despite the importance of FNR and anaerobiosis in microbial lifestyles, the factors that influence its function on a genome-wide scale are poorly understood. Here, we report a functional genomic analysis of FNR action. We find that FNR occupancy at many target sites is strongly influenced by nucleoid-associated proteins (NAPs) that restrict access to many FNR binding sites. At a genome-wide level, only a subset of predicted FNR binding sites were bound under anaerobic fermentative conditions and many appeared to be masked by the NAPs H-NS, IHF and Fis. Similar assays in cells lacking H-NS and its paralog StpA showed increased FNR occupancy at sites bound by H-NS in WT strains, indicating that large regions of the genome are not readily accessible for FNR binding. Genome accessibility may also explain our finding that genome-wide FNR occupancy did not correlate with the match to consensus at binding sites, suggesting that significant variation in ChIP signal was attributable to cross-linking or immunoprecipitation efficiency rather than differences in binding affinities for FNR sites. Correlation of FNR ChIP-seq peaks with transcriptomic data showed that less than half of the FNR-regulated operons could be attributed to direct FNR binding. Conversely, FNR bound some promoters without regulating expression presumably requiring changes in activity of condition-specific transcription factors. Such combinatorial regulation may allow Escherichia coli to respond rapidly to environmental changes and confer an ecological advantage in the anaerobic but nutrient-fluctuating environment of the mammalian gut.

Chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) has been successfully used for genome-wide profiling of transcription factor binding sites, histone modifications, and nucleosome occupancy in many model organisms and humans. Because the compact genomes of prokaryotes harbor many binding sites separated by only few base pairs, applications of ChIP-Seq in this domain have not reached their full potential. Applications in prokaryotic genomes are further hampered by the fact that well studied data analysis methods for ChIP-Seq do not result in a resolution required for deciphering the locations of nearby binding events. We generated single-end tag (SET) and paired-end tag (PET) ChIP-Seq data for σ⁷⁰ factor in Escherichia coli (E. coli). Direct comparison of these datasets revealed that although PET assay enables higher resolution identification of binding events, standard ChIP-Seq analysis methods are not equipped to utilize PET-specific features of the data. To address this problem, we developed dPeak as a high resolution binding site identification (deconvolution) algorithm. dPeak implements a probabilistic model that accurately describes ChIP-Seq data generation process for both the SET and PET assays. For SET data, dPeak outperforms or performs comparably to the state-of-the-art high-resolution ChIP-Seq peak deconvolution algorithms such as PICS, GPS, and GEM. When coupled with PET data, dPeak significantly outperforms SET-based analysis with any of the current state-of-the-art methods. Experimental validations of a subset of dPeak predictions from σ⁷⁰ PET ChIP-Seq data indicate that dPeak can estimate locations of binding events with as high as 2 to 21 bp resolution. Applications of dPeak to σ⁷⁰ ChIP-Seq data in E. coli under aerobic and anaerobic conditions reveal closely located promoters that are differentially occupied and further illustrate the importance of high resolution analysis of ChIP-Seq data.

Despite the importance of maintaining redox homeostasis for cellular viability, how cells control redox balance globally is poorly understood. Here we provide new mechanistic insight into how the balance between reduced and oxidized electron carriers is regulated at the level of gene expression by mapping the regulon of the response regulator ArcA from Escherichia coli, which responds to the quinone/quinol redox couple via its membrane-bound sensor kinase, ArcB. Our genome-wide analysis reveals that ArcA reprograms metabolism under anaerobic conditions such that carbon oxidation pathways that recycle redox carriers via respiration are transcriptionally repressed by ArcA. We propose that this strategy favors use of catabolic pathways that recycle redox carriers via fermentation akin to lactate production in mammalian cells. Unexpectedly, bioinformatic analysis of the sequences bound by ArcA in ChIP-seq revealed that most ArcA binding sites contain additional direct repeat elements beyond the two required for binding an ArcA dimer. DNase I footprinting assays suggest that non-canonical arrangements of cis-regulatory modules dictate both the length and concentration-sensitive occupancy of DNA sites. We propose that this plasticity in ArcA binding site architecture provides both an efficient means of encoding binding sites for ArcA, σ(70)-RNAP and perhaps other transcription factors within the same narrow sequence space and an effective mechanism for global control of carbon metabolism to maintain redox homeostasis.

The Escherichia coli transcription system is the best characterized from a biochemical and genetic point of view and has served as a model system. Nevertheless, a molecular understanding of the details of E. coli transcription and its regulation, and therefore its full exploitation as a model system, has been hampered by the absence of high-resolution structural information on E. coli RNA polymerase (RNAP). We use a combination of approaches, including high-resolution X-ray crystallography, ab initio structural prediction, homology modeling, and single-particle cryo-electron microscopy, to generate complete atomic models of E. coli core RNAP and an E. coli RNAP ternary elongation complex. The detailed and comprehensive structural descriptions can be used to help interpret previous biochemical and genetic data in a new light and provide a structural framework for designing experiments to understand the function of the E. coli lineage-specific insertions and their role in the E. coli transcription program.