Crowdsourcing biocuration: The Community Assessment of Community Annotation with Ontologies (CACAO).

Experimental data about gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5,000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a 10-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills.

Allosteric Effector ppGpp Potentiates the Inhibition of Transcript Initiation by DksA.

DksA and ppGpp are the central players in the stringent response and mediate a complete reprogramming of the transcriptome. A major component of the response is a reduction in ribosome synthesis, which is accomplished by the synergistic action of DksA and ppGpp bound to RNA polymerase (RNAP) inhibiting transcription of rRNAs. Here, we report the X-ray crystal structures of Escherichia coli RNAP in complex with DksA alone and with ppGpp. The structures show that DksA accesses the template strand at the active site and the downstream DNA binding site of RNAP simultaneously and reveal that binding of the allosteric effector ppGpp reshapes the RNAP-DksA complex. The structural data support a model for transcriptional inhibition in which ppGpp potentiates the destabilization of open complexes by DksA. This work establishes a structural basis for understanding the pleiotropic effects of DksA and ppGpp on transcriptional regulation in proteobacteria.

Circuitry Linking the Global Csr- and σE-Dependent Cell Envelope Stress Response Systems.

CsrA of Escherichia coli is an RNA-binding protein that globally regulates a wide variety of cellular processes and behaviors, including carbon metabolism, motility, biofilm formation, and the stringent response. CsrB and CsrC are small RNAs (sRNAs) that sequester CsrA, thereby preventing CsrA-mRNA interaction. RpoE (σE) is the extracytoplasmic stress response sigma factor of E. coli Previous RNA sequencing (RNA-seq) studies identified rpoE mRNA as a CsrA target. Here, we explored the regulation of rpoE by CsrA and found that CsrA represses rpoE translation. Gel mobility shift, footprint, and toeprint studies identified three CsrA binding sites in the rpoE leader transcript, one of which overlaps the rpoE Shine-Dalgarno (SD) sequence, while another overlaps the rpoE translation initiation codon. Coupled in vitro transcription-translation experiments showed that CsrA represses rpoE translation by binding to these sites. We further demonstrate that σE indirectly activates the transcription of csrB and csrC, leading to increased sequestration of CsrA, such that repression of rpoE by CsrA is reduced. We propose that the Csr system fine-tunes the σE-dependent cell envelope stress response. We also identified a 51-amino-acid coding sequence whose stop codon overlaps the rpoE start codon and demonstrate that rpoE is translationally coupled with this upstream open reading frame (ORF51). The loss of coupling reduces rpoE translation by more than 50%. Identification of a translationally coupled ORF upstream of rpoE suggests that this previously unannotated protein may participate in the cell envelope stress response. In keeping with existing nomenclature, we named ORF51 rseD, resulting in an operon arrangement of rseD-rpoE-rseA-rseB-rseC IMPORTANCE CsrA posttranscriptionally represses genes required for bacterial stress responses, including the stringent response, catabolite repression, and the RpoS (σS)-mediated general stress response. We show that CsrA represses the translation of rpoE, encoding the extracytoplasmic stress response sigma factor, and that σE indirectly activates the transcription of csrB and csrC, resulting in reciprocal regulation of these two global regulatory systems. These findings suggest that extracytoplasmic stress leads to derepression of rpoE translation by CsrA, and CsrA-mediated repression helps reset RpoE abundance to prestress levels once envelope damage is repaired. The discovery of an ORF, rseD, translationally coupled with rpoE adds further complexity to translational control of rpoE.

Themes and variations in gene regulation by extracytoplasmic function (ECF) sigma factors.

The ECF sigma family was identified 23 years ago as a distinct group of σ70-like factors. ECF sigma factors have since emerged as a major form of bacterial signal transduction that can be grouped into over 50 phylogenetically distinct subfamilies. Advances in our understanding of these sigma factors and the signaling pathways governing their activity have elucidated conserved features as well as aspects that have evolved over time. All ECF sigma factors are predicted to share a common streamlined domain structure and mode of promoter interaction. The activity of most ECF sigma factors is controlled by an anti-sigma factor. The nature of the anti-sigma factor and the activating signaling pathways appear to be conserved within ECF families, while considerable diversity exists between different families.

Appropriate Regulation of the σE-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli.

The alternative sigma factor σE is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of σE increases during entry into stationary phase, suggesting an important role for σE when nutrients are limiting. Elevated σE activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand σE-directed cell lysis and the role of σE in stationary phase, we investigated the effects of elevated σE activity in cultures grown for 10 days. We demonstrate that high σE activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced σE activity, due primarily to point mutations in the region of σE that binds the -35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High σE activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg2+ and buffering the growth medium or by deleting the σE-dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of σE activity. Our results demonstrate that appropriate regulation of σE activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability.IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The σE response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of σE is tightly regulated to match the production of σE regulon members to the needs of the cell. In E. coli, loss of σE results in lethality. Here we demonstrate that excessive σE activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent.

Teaching broader impacts of science with undergraduate research.

Science plays an important role in most aspects of society, and scientists face ethical decisions as a routine part of their work, but science education frequently omits or segregates content related to ethics and broader impacts of science. Undergraduate research experiences have the potential to bridge traditional divides in education and provide a holistic view of science. In practice, these experiences can be inconsistent and may not provide the optimal learning environment. We developed a course that combines seminar and independent research elements to support student learning during undergraduate research, makes ethical and societal impacts of science clear by relating them to the students' own research projects, and develops students' ethical decision-making skills. Here, we describe the course and provide resources for developing a similar course.

Cell-based assay to identify inhibitors of the Hfq-sRNA regulatory pathway.

Noncoding small RNAs (sRNAs) act in conjunction with the RNA chaperone Hfq to regulate gene expression in bacteria. Because Hfq is required for virulence in several bacterial pathogens, the Hfq-sRNA system is an attractive target for antibiotic development. A reporter strain in which the expression of yellow fluorescent protein (YFP) is controlled by Hfq-sRNA was engineered to identify inhibitors of this system. A reporter that is targeted by Hfq in conjunction with the RybB sRNA was used in a genetic screen to identify inhibitors from a library of cyclic peptides produced in Escherichia coli using split-intein circular ligation of peptides and proteins (SICLOPPS), an intein-based technology. One cyclic peptide identified in this screen, RI20, inhibited Hfq-mediated repression of gene expression in conjunction with both RybB and an unrelated sRNA, MicF. Gel mobility shift assays showed that RI20 inhibited binding of Hfq to RybB and MicF with similar Ki values. These data suggest that RI20 inhibits Hfq activity by blocking interactions with sRNAs and provide a paradigm for inhibiting virulence genes in Gram-negative pathogens.

Co-ordinated regulation of the extracytoplasmic stress factor, sigmaE, with other Escherichia coli sigma factors by (p)ppGpp and DksA may be achieved by specific regulation of individual holoenzymes.

The E. coli alternative sigma factor, σ(E) , transcribes genes required to maintain the cell envelope and is activated by conditions that destabilize the envelope. σ(E) is also activated during entry into stationary phase in the absence of envelope stress by the alarmone (p)ppGpp. (p)ppGpp controls a large regulatory network, reducing expression of σ(70) -dependent genes required for rapid growth and activating σ(70) -dependent and alternative sigma factor-dependent genes required for stress survival. The DksA protein often potentiates the effects of (p)ppGpp. Here we examine regulation of σ(E) by (p)ppGpp and DksA following starvation for nutrients. We find that (p)ppGpp is required for increased σ(E) activity under all conditions tested, but the requirement for DksA varies. DksA is required during amino acid starvation, but is dispensable during phosphate starvation. In contrast, regulation of σ(S) is (p)ppGpp- and DksA-dependent under all conditions tested, while negative regulation of σ(70) is DksA- but not (p)ppGpp-dependent during phosphate starvation, yet requires both factors during amino acid starvation. These findings suggest that the mechanism of transcriptional regulation by (p)ppGpp and/or DksA cannot yet be explained by a unifying model and is specific to individual promoters, individual holoenzymes, and specific starvation conditions.

Regulated proteolysis: control of the Escherichia coli σ(E)-dependent cell envelope stress response.

Over the past decade, regulatory proteolysis has emerged as a paradigm for transmembrane signal transduction in all organisms, from bacteria to humans. These conserved proteolytic pathways share a common design that involves the sequential proteolysis of a membrane-bound regulatory protein by two proteases. Proteolysis releases the regulator, which is inactive in its membrane-bound form, into the cytoplasm where it performs its cellular function. One of the best-characterized examples of signal transduction via regulatory proteolysis is the pathway governing the σ(E)-dependent cell envelope stress response in Escherichia coli. In unstressed cells, σ(E) is sequestered at the membrane by the transmembrane anti-sigma factor, RseA. Stresses that compromise the cell envelope and interfere with the proper folding of outer membrane proteins (OMPs) activate the proteolytic pathway. The C-terminal residues of unfolded OMPs bind to the inner membrane protease, DegS, to initiate the proteolytic cascade. DegS removes the periplasmic domain of RseA creating a substrate for the next protease in the pathway, RseP. RseP cleaves RseA in the periplasmic region in a process called regulated intramembrane proteolysis (RIP). The remaining fragment of RseA is released into the cytoplasm and fully degraded by the ATP-dependent protease, ClpXP, with the assistance of the adaptor protein, SspB, thereby freeing σ(E) to reprogram gene expression. A growing body of evidence indicates that the overall proteolytic framework that governs the σ(E) response is used to regulate similar anti-sigma factor/sigma factor pairs throughout the bacterial world and has been adapted to recognize a wide variety of signals and control systems as diverse as envelope stress responses, sporulation, virulence, and iron-siderophore uptake. In this chapter, we review the extensive physiological, biochemical, and structural studies on the σ(E) system that provide remarkable insights into the mechanistic underpinnings of this regulated proteolytic signal transduction pathway. These studies reveal design principles that are applicable to related proteases and regulatory proteolytic pathways in all domains of life.

sigE facilitates the adaptation of Bordetella bronchiseptica to stress conditions and lethal infection in immunocompromised mice.

BACKGROUND: The cell envelope of a bacterial pathogen can be damaged by harsh conditions in the environment outside a host and by immune factors during infection. Cell envelope stress responses preserve the integrity of this essential compartment and are often required for virulence. Bordetella species are important respiratory pathogens that possess a large number of putative transcription factors. However, no cell envelope stress responses have been described in these species. Among the putative Bordetella transcription factors are a number of genes belonging to the extracytoplasmic function (ECF) group of alternative sigma factors, some of which are known to mediate cell envelope stress responses in other bacteria. Here we investigate the role of one such gene, sigE, in stress survival and pathogenesis of Bordetella bronchiseptica. RESULTS: We demonstrate that sigE encodes a functional sigma factor that mediates a cell envelope stress response. Mutants of B. bronchiseptica strain RB50 lacking sigE are more sensitive to high temperature, ethanol, and perturbation of the envelope by SDS-EDTA and certain ß-lactam antibiotics. Using a series of immunocompromised mice deficient in different components of the innate and adaptive immune responses, we show that SigE plays an important role in evading the innate immune response during lethal infections of mice lacking B cells and T cells. SigE is not required, however, for colonization of the respiratory tract of immunocompetent mice. The sigE mutant is more efficiently phagocytosed and killed by peripheral blood polymorphonuclear leukocytes (PMNs) than RB50, and exhibits decreased cytotoxicity toward macrophages. These altered interactions with phagocytes could contribute to the defects observed during lethal infection. CONCLUSIONS: Much of the work on transcriptional regulation during infection in B. bronchiseptica has focused on the BvgAS two-component system. This study reveals that the SigE regulon also mediates a discrete subset of functions associated with virulence. SigE is the first cell envelope stress-sensing system to be described in the bordetellae. In addition to its role during lethal infection of mice deficient in adaptive immunity, our results indicate that SigE is likely to be important for survival in the face of stresses encountered in the environment between hosts.

Physiological response to membrane protein overexpression in E. coli.

Overexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially contributing to the difficulties encountered in IMP crystallization.

Promoter strength properties of the complete sigma E regulon of Escherichia coli and Salmonella enterica.

The sigma(E)-directed envelope stress response maintains outer membrane homeostasis and is an important virulence determinant upon host infection in Escherichia coli and related bacteria. sigma(E) is activated by at least two distinct mechanisms: accumulation of outer membrane porin precursors and an increase in the alarmone ppGpp upon transition to stationary phase. Expression of the sigma(E) regulon is driven from a suite of approximately 60 sigma(E)-dependent promoters. Using green fluorescent protein fusions to each of these promoters, we dissected promoter contributions to the output of the regulon under a variety of in vivo conditions. We found that the sigma(E) promoters exhibit a large dynamic range, with a few strong and many weak promoters. Interestingly, the strongest promoters control either transcriptional regulators or functions related to porin homeostasis, the very functions conserved among E. coli and its close relatives. We found that (i) the strength of most promoters is significantly affected by the presence of the upstream (-35 to -65) region of the promoter, which encompasses the UP element, a binding site for the C-terminal domain of the alpha-subunit of RNA polymerase; (ii) ppGpp generally activates sigma(E) promoters, and (iii) sigma(E) promoters are responsive to changing sigma(E) holoenzyme levels under physiological conditions, reinforcing the idea that the sigma(E) regulon is extremely dynamic, enabling cellular adaptation to a constantly changing environment.

Regulation by destruction: design of the sigmaE envelope stress response.

The signal transduction pathway governing the sigma(E)-dependent cell envelope stress response in Escherichia coli communicates information from the periplasm to sigma(E) in the cytoplasm via a regulated proteolytic cascade that results in the destruction of the membrane-bound antisigma factor, RseA, and the release of sigma(E) to direct transcription. Regulated proteolysis is used for signal transduction in all domains of life, and these pathways bear remarkable similarities in their architecture and the proteases involved. Work with the pathway governing the sigma(E) response has elucidated key design principles that ensure a rapid yet graded response that is buffered from inappropriate activation. Structural and biochemical studies of the proteases that mediate signal transduction reveal the molecular underpinnings enabling this design.

The extracytoplasmic stress factor, sigmaE, is required to maintain cell envelope integrity in Escherichia coli.

Extracytoplasmic function or ECF sigma factors are the most abundant class of alternative sigma factors in bacteria. Members of the rpoE subclass of ECF sigma factors are implicated in sensing stress in the cell envelope of Gram-negative bacteria and are required for virulence in many pathogens. The best-studied member of this family is rpoE from Escherichia coli, encoding the sigma(E) protein. sigma(E) has been well studied for its role in combating extracytoplasmic stress, and the members of its regulon have been largely defined. sigma(E) is required for viability of E. coli, yet none of the studies to date explain why sigma(E) is essential in seemingly unstressed cells. In this work we investigate the essential role of sigma(E) in E. coli by analyzing the phenotypes associated with loss of sigma(E) activity and isolating suppressors that allow cells to live in the absence of sigma(E). We demonstrate that when sigma(E) is inhibited, cell envelope stress increases and envelope integrity is lost. Many cells lyse and some develop blebs containing cytoplasmic material along their sides. To better understand the connection between transcription by sigma(E) and cell envelope integrity, we identified two multicopy suppressors of the essentiality of sigma(E), ptsN and yhbW. yhbW is a gene of unknown function, while ptsN is a member of the sigma(E) regulon. Overexpression of ptsN lowers the basal level of multiple envelope stress responses, but not that of a cytoplasmic stress response. Our results are consistent with a model in which overexpression of ptsN reduces stress in the cell envelope, thereby promoting survival in the absence of sigma(E).

The Rcs phosphorelay is a cell envelope stress response activated by peptidoglycan stress and contributes to intrinsic antibiotic resistance.

Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the beta-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to beta-lactam antibiotics.

ppGpp and DksA likely regulate the activity of the extracytoplasmic stress factor sigmaE in Escherichia coli by both direct and indirect mechanisms.

One of the major signalling pathways responsible for intercompartmental communication between the cell envelope and cytoplasm in Escherichia coli is mediated by the alternative sigma factor, sigmaE. sigmaE has been studied primarily for its role in response to the misfolding of outer membrane porins. This response is essentially reactionary; cells are stressed, porin folding is disrupted, and the response is activated. sigmaE can also be activated following starvation for a variety of nutrients by the alarmone ppGpp. This response is proactive, as sigmaE is activated in the absence of any obvious damage to the cell envelope sensed by the stress signalling pathway. Here we examine the mechanism of regulation of sigmaE by ppGpp. ppGpp has been proposed to activate at least two alternative sigma factors, sigmaN and sigmaS, indirectly by altering the competition for core RNA polymerase between the alternative sigma factors and the housekeeping sigma factor, sigma70. In vivo experiments with sigmaE are consistent with this model. However, ppGpp and its cofactor DksA can also activate transcription by EsigmaEin vitro, suggesting that the effects of ppGpp on sigmaE activity are both direct and indirect.

Growth phase-dependent regulation of the extracytoplasmic stress factor, sigmaE, by guanosine 3',5'-bispyrophosphate (ppGpp).

The sigma subunit of procaryotic RNA polymerases is responsible for specific promoter recognition and transcription initiation. In addition to the major sigma factor, sigma 70, in Escherichia coli, which directs most of the transcription in the cell, bacteria possess multiple, alternative sigma factors that direct RNA polymerase to distinct sets of promoters in response to environmental signals. By activating an alternative sigma factor, gene expression can be rapidly reprogrammed to meet the needs of the cell as the environment changes. Sigma factors are subject to multiple levels of regulation that control their levels and activities. The alternative sigma factor sigmaE in Escherichia coli is induced in response to extracytoplasmic stress. Here we demonstrate that sigmaE can also respond to signals other than extracytoplasmic stress. sigmaE activity increases in a growth phase-dependent manner as a culture enters stationary phase. The signaling pathway that activates sigmaE during entry into stationary phase is dependent upon the alarmone guanosine 3',5'-bispyrophosphate (ppGpp) and is distinct from the pathway that signals extracytoplasmic stress. ppGpp is the first cytoplasmic factor shown to control sigmaE activity, demonstrating that sigmaE can respond to internal signals as well as signals originating in the cell envelope. ppGpp is a general signal of starvation stress and is also required for activation of the sigmaS and sigma 54 alternative sigma factors upon entry into stationary phase, suggesting that this is a key mechanism by which alternative sigma factors can be activated in concert to provide a coordinated response to nutritional stress.

Proteolysis: Adaptor, adaptor, catch me a catch.

The ClpXP protease of bacteria can degrade a wide variety of proteins while maintaining remarkable substrate selectivity. New work in Escherichia coli implicates adaptor proteins in enhancing substrate selectivity and regulating the flow of substrates to cellular proteases.

Control of the alternative sigma factor sigmaE in Escherichia coli.

Signal transduction pathways that communicate information from the cell envelope to the cytoplasm of bacteria are crucial to maintain cell envelope homeostasis. In Escherichia coli, one of the key pathways that ensures the integrity of the cell envelope during stress and normal growth is controlled by the alternative sigma factor sigmaE. Recent studies have elucidated the signal transduction pathway that activates sigmaE in response to misfolded outer membrane porins. Unfolded porins trigger the degradation of the sigmaE-specific antisigma factor RseA by the sequential action of two inner membrane proteases, leading to release of sigmaE from RseA, and induction of the stress response. This mechanism of signal transduction, regulated intramembrane proteolysis, is used in transmembrane signaling pathways from bacteria to humans.