Post-translational modifications on protein VII are important during the early stages of adenovirus infection.

Due to the importance of post-translational modification (PTM) in cellular function, viruses have evolved to both take advantage of and be susceptible to such modification. Adenovirus encodes a multifunctional protein called protein VII, which is packaged with the viral genome in the core of virions and disrupts host chromatin during infection. Protein VII has several PTMs whose addition contributes to the subnuclear localization of protein VII. Here, we used mutant viruses that abrogate or mimic these PTMs on protein VII to interrogate their impact on protein VII function during adenovirus infection. We discovered that acetylation of the lysine in positions 2 or 3 (K2 or K3) is deleterious during early infection as mutation to alanine led to greater intake of protein VII to the nucleus and enhanced early gene expression. Furthermore, we determined that protein VII is acetylated at alternative residues late during infection which may compensate for the mutated sites. Lastly, due to the role of the early viral protein E1A in viral gene activation, we investigated the interaction between protein VII and E1A and demonstrated that protein VII interacts with E1A through a chromatin-mediated interaction. Together, these results emphasize that the complexity of virus-host interactions is intimately tied to post-translational modification.

Regulators of rDNA array morphology in fission yeast.

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.

Differential roles of positive and negative supercoiling in organizing the E. coli genome.

This study aims to explore whether and how positive and negative supercoiling contribute to the three-dimensional (3D) organization of the bacterial genome. We used recently published Escherichia coli GapR ChIP-seq and TopoI ChIP-seq (also called EcTopoI-seq) data, which marks positive and negative supercoiling sites, respectively, to study how supercoiling correlates with the spatial contact maps obtained from chromosome conformation capture sequencing (Hi-C and 5C). We find that supercoiled chromosomal loci have overall higher Hi-C contact frequencies than sites that are not supercoiled. Surprisingly, positive supercoiling corresponds to higher spatial contact than negative supercoiling. Additionally, positive, but not negative, supercoiling could be identified from Hi-C data with high accuracy. We further find that the majority of positive and negative supercoils coincide with highly active transcription units, with a minor group likely associated with replication and other genomic processes. Our results show that both positive and negative supercoiling enhance spatial contact, with positive supercoiling playing a larger role in bringing genomic loci closer in space. Based on our results, we propose new physical models of how the E. coli chromosome is organized by positive and negative supercoils.

Adenovirus protein VII binds the A-box of HMGB1 to repress interferon responses.

Viruses hijack host proteins to promote infection and dampen host defenses. Adenovirus encodes the multifunctional protein VII that serves both to compact viral genomes inside the virion and disrupt host chromatin. Protein VII binds the abundant nuclear protein high mobility group box 1 (HMGB1) and sequesters HMGB1 in chromatin. HMGB1 is an abundant host nuclear protein that can also be released from infected cells as an alarmin to amplify inflammatory responses. By sequestering HMGB1, protein VII prevents its release, thus inhibiting downstream inflammatory signaling. However, the consequences of this chromatin sequestration on host transcription are unknown. Here, we employ bacterial two-hybrid interaction assays and human cell culture to interrogate the mechanism of the protein VII-HMGB1 interaction. HMGB1 contains two DNA binding domains, the A- and B-boxes, that bend DNA to promote transcription factor binding while the C-terminal tail regulates this interaction. We demonstrate that protein VII interacts directly with the A-box of HMGB1, an interaction that is inhibited by the HMGB1 C-terminal tail. By cellular fractionation, we show that protein VII renders A-box containing constructs insoluble, thereby acting to prevent their release from cells. This sequestration is not dependent on HMGB1's ability to bind DNA but does require post-translational modifications on protein VII. Importantly, we demonstrate that protein VII inhibits expression of interferon ß, in an HMGB1-dependent manner, but does not affect transcription of downstream interferon-stimulated genes. Together, our results demonstrate that protein VII specifically harnesses HMGB1 through its A-box domain to depress the innate immune response and promote infection.

Tailoring a Text Messaging and Fotonovela Program to Increase Patient Engagement in Colorectal Cancer Screening in a Large Urban Community Clinic Population: Quality Improvement Project.

BACKGROUND: Appropriate annual screenings for colorectal cancer (CRC) are an essential preventive measure for the second-leading cause of cancer-related death in the United States. Studies have shown that CRC screening rates are influenced by various social determinants of health (SDOH) factors, including race, ethnicity, and geography. According to 2018 national data, participation in screening is lowest among Hispanic or Latinx individuals (56.1%). At an urban Federally Qualified Health Center, a quality improvement project was conducted to evaluate a texting program with a motivational fotonovela-a short narrative comic. Fotonovelas have previously been used in programs to improve knowledge of cervical cancer and human papillomavirus, vaccinations, and treatments for depression. OBJECTIVE: This study aimed to encourage compliance with fecal immunochemical test (FIT) screening. Patient engagement involved a texting program with fotonovelas informed by behavior change techniques. This study sought to understand the qualitative characteristics of patient motivation, intention, and barriers to completing their screening. METHODS: A total of 5241 English-speaking or Spanish-speaking Federally Qualified Health Center patients aged 50 to 75 years were randomized to either intervention (a 4-week tailored 2-way texting program with a fotonovela comic) or usual care (an SMS text message reminder and patient navigator phone call). The texting vendor used a proprietary algorithm to categorize patients in the intervention group into SDOH bands based on their home addresses (high impact=high social needs and low impact=low social needs). Over 4 weeks, patients were texted questions about receiving and returning their FIT, what barriers they may be experiencing, and their thoughts about the fotonovela. RESULTS: The SDOH index analysis showed that most of the patient population was in the SDOH band categories of high impact (555/2597, 21.37%) and very high impact (1416/2597, 54.52%). Patients sent 1969 total responses to the texting system. Thematic analysis identified 3 major themes in these responses: messages as a reminder, where patients reported that they were motivated to return the FIT and had already done so or would do so as soon as possible; increasing patients' understanding of screening importance, where patients expressed an increased knowledge about the purpose and importance of the FIT; and expressing barriers, where patients shared reasons for not completing the FIT. CONCLUSIONS: The texting program and fotonovela engaged a subset of patients in each SDOH band, including the high and very high impact bands. Creating culturally tailored messages can encourage patient engagement for accepting the content of the messaging, confirming intentions to complete their FIT, and sharing insights about barriers to behavior change. To better support all patients across the continuum of care with CRC screening, it is important to continue to develop and assess strategies that engage patients who did not return their home-mailed FIT.

Adenovirus protein VII binds the A-box of HMGB1 to repress interferon responses.

Viruses hijack host proteins to promote infection and dampen host defenses. Adenovirus encodes the multifunctional protein VII that serves both to compact viral genomes inside the virion and disrupt host chromatin. Protein VII binds the abundant nuclear protein high mobility group box 1 (HMGB1) and sequesters HMGB1 in chromatin. HMGB1 is an abundant host nuclear protein that can also be released from infected cells as an alarmin to amplify inflammatory responses. By sequestering HMGB1, protein VII prevents its release, thus inhibiting downstream inflammatory signaling. However, the consequences of this chromatin sequestration on host transcription are unknown. Here, we employ bacterial two-hybrid interaction assays and human cell biological systems to interrogate the mechanism of the protein VII-HMGB1 interaction. HMGB1 contains two DNA binding domains, the A- and B-boxes, that bend DNA to promote transcription factor binding while the C-terminal tail regulates this interaction. We demonstrate that protein VII interacts directly with the A-box of HMGB1, an interaction that is inhibited by the HMGB1 C-terminal tail. By cellular fractionation, we show that protein VII renders A-box containing constructs insoluble, thereby acting to prevent their release from cells. This sequestration is not dependent on HMGB1's ability to bind DNA but does require post-translational modifications on protein VII. Importantly, we demonstrate that protein VII inhibits expression of interferon ß, in an HMGB1- dependent manner, but does not affect transcription of downstream interferon- stimulated genes. Together, our results demonstrate that protein VII specifically harnesses HMGB1 through its A-box domain to depress the innate immune response and promote infection.

Using Text Messages and Fotonovelas to Increase Return of Home-Mailed Colorectal Cancer Screening Tests: Mixed Methods Evaluation.

BACKGROUND: Colorectal cancer (CRC) is currently the second leading cause of cancer-related deaths in the United States; however, it is mostly preventable with appropriate screening and is often treatable when detected at early stages. Many patients enrolled in an urban Federally Qualified Health Center (FQHC) clinic were found to be past due for CRC screening. OBJECTIVE: This study described a quality improvement (QI) project to improve CRC screening rates. This project used bidirectional texting with fotonovela comics and natural language understanding (NLU) to encourage patients to mail fecal immunochemical test (FIT) kits back to the FQHC. METHODS: The FQHC mailed FIT kits to 11,000 unscreened patients in July 2021. Consistent with the usual care, all patients received 2 text messages and a patient navigator call within the first month of mailing. As part of a QI project, 5241 patients who did not return their FIT kit within 3 months, aged 50-75 years, and spoke either English or Spanish were randomized to either usual care (no further intervention) or intervention (4-week texting campaign with a fotonovela comic and remailing kits if requested) groups. The fotonovela was developed to address known barriers to CRC screening. The texting campaign used NLU to respond to patients' texts. A mixed methods evaluation used data from SMS text messages and electronic medical records to understand the impact of the QI project on CRC screening rates. Open-ended text messages were analyzed for themes, and interviews were completed with a convenience sample of patients to understand barriers to screening and impact of the fotonovela. RESULTS: Of the 2597 participants, 1026 (39.5%) in the intervention group engaged with bidirectional texting. Participating in bidirectional texting was related to language preference (χ22=11.0; P=.004) and age group (χ22=19.0; P<.001). Of the 1026 participants who engaged bidirectionally, 318 (31%) clicked on the fotonovela. Furthermore, 54% (32/59) of the patients clicked on the fotonovela and responded that they loved it, and 36% (21/59) of patients responded that they liked it. The intervention group was more likely to get screened (487/2597, 18.75%) than those in usual care (308/2644, 11.65%; P<.001), and this pattern held, regardless of demographic subgroup (sex, age, screening history, preferred language, and payer type). Interview data (n=16) indicated that the text messages, navigator calls, and fotonovelas were well received and not unduly invasive. Interviewees noted several important barriers to CRC screening and offered suggestions for reducing barriers and increasing screening. CONCLUSIONS: Texting using NLU and fotonovela is valuable in increasing CRC screening as observed by the FIT return rate for patients in the intervention group. There were patterns in which patients did not engage bidirectionally; future work should investigate how to ensure that populations are not left out of screening campaigns.

High-resolution, genome-wide mapping of positive supercoiling in chromosomes.

Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to Escherichia coli and Saccharomyces cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently oriented genes, consistent with the 'twin-domain' model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin-binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

Uncovering the basis of protein-protein interaction specificity with a combinatorially complete library.

Protein-protein interaction specificity is often encoded at the primary sequence level. However, the contributions of individual residues to specificity are usually poorly understood and often obscured by mutational robustness, sequence degeneracy, and epistasis. Using bacterial toxin-antitoxin systems as a model, we screened a combinatorially complete library of antitoxin variants at three key positions against two toxins. This library enabled us to measure the effect of individual substitutions on specificity in hundreds of genetic backgrounds. These distributions allow inferences about the general nature of interface residues in promoting specificity. We find that positive and negative contributions to specificity are neither inherently coupled nor mutually exclusive. Further, a wild-type antitoxin appears optimized for specificity as no substitutions improve discrimination between cognate and non-cognate partners. By comparing crystal structures of paralogous complexes, we provide a rationale for our observations. Collectively, this work provides a generalizable approach to understanding the logic of molecular recognition.

A CRISPR Interference System for Efficient and Rapid Gene Knockdown in Caulobacter crescentus.

CRISPR interference (CRISPRi) is a powerful new tool used in different organisms that provides a fast, specific, and reliable way to knock down gene expression. Caulobacter crescentus is a well-studied model bacterium, and although a variety of genetic tools have been developed, it currently takes several weeks to delete or deplete individual genes, which significantly limits genetic studies. Here, we optimized a CRISPRi approach to specifically downregulate the expression of genes in C. crescentus Although the Streptococcus pyogenes CRISPRi system commonly used in other organisms does not work efficiently in Caulobacter, we demonstrate that a catalytically dead version of Cas9 (dCas9) derived from the type II CRISPR3 module of Streptococcus thermophilus or from Streptococcus pasteurianus can each be effectively used in Caulobacter We show that these CRISPRi systems can be used to rapidly and inducibly deplete ctrA or gcrA, two essential well-studied genes in Caulobacter, in either asynchronous or synchronized populations of cells. Additionally, we demonstrate the ability to multiplex CRISPRi-based gene knockdowns, opening new possibilities for systematic genetic interaction studies in CaulobacterIMPORTANCECaulobacter crescentus is a major model organism for understanding cell cycle regulation and cellular asymmetry. The current genetic tools for deleting or silencing the expression of individual genes, particularly those essential for viability, are time-consuming and labor-intensive, which limits global genetic studies. Here, we optimized CRISPR interference (CRISPRi) for use in Caulobacter Using Streptococcus thermophilus CRISPR3 or Streptococcus pasteurianus CRISPR systems, we show that the coexpression of a catalytically dead form of Cas9 (dCas9) with a single guide RNA (sgRNA) containing a seed region that targets the promoter region of a gene of interest efficiently downregulates the expression of the targeted gene. We also demonstrate that multiple sgRNAs can be produced in parallel to enable the facile silencing of multiple genes, opening the door to systematic genetic interaction studies. In sum, our work now provides a rapid, specific, and powerful new tool for silencing gene expression in C. crescentus and possibly other alphaproteobacteria.

A Bacterial Chromosome Structuring Protein Binds Overtwisted DNA to Stimulate Type II Topoisomerases and Enable DNA Replication.

When DNA is unwound during replication, it becomes overtwisted and forms positive supercoils in front of the translocating DNA polymerase. Unless removed or dissipated, this superhelical tension can impede replication elongation. Topoisomerases, including gyrase and topoisomerase IV in bacteria, are required to relax positive supercoils ahead of DNA polymerase but may not be sufficient for replication. Here, we find that GapR, a chromosome structuring protein in Caulobacter crescentus, is required to complete DNA replication. GapR associates in vivo with positively supercoiled chromosomal DNA, and our biochemical and structural studies demonstrate that GapR forms a dimer-of-dimers that fully encircles overtwisted DNA. Further, we show that GapR stimulates gyrase and topo IV to relax positive supercoils, thereby enabling DNA replication. Analogous chromosome structuring proteins that locate to the overtwisted DNA in front of replication forks may be present in other organisms, similarly helping to recruit and stimulate topoisomerases during DNA replication.

Navigating Limited and Uncertain Access to Subsidized Housing After Prison.

An emerging literature has documented the challenges that formerly incarcerated individuals face in securing stable housing. Given the increasingly unaffordable rental market, rental subsidies represent an important and understudied source of stable housing for this population. The existing literature has described substantial discretion and a varied policy landscape that determine former prisoners' access to housing subsidies, or subsidized housing spaces that are leased to members of their social and family networks. Less is known about how former prisoners themselves interpret and navigate this limited and uncertain access to subsidized housing. Drawing on data from repeated qualitative interviews with 44 former prisoners, we describe the creative and often labor-intensive strategies that participants employed to navigate discretion and better position themselves for subsidized housing that was in high demand, but also largely out of reach. Our findings also illustrate the potential costs associated with these strategies for both participants and members of their social and family networks.

Evolutionary Convergence of Pathway-Specific Enzyme Expression Stoichiometry.

Coexpression of proteins in response to pathway-inducing signals is the founding paradigm of gene regulation. However, it remains unexplored whether the relative abundance of co-regulated proteins requires precise tuning. Here, we present large-scale analyses of protein stoichiometry and corresponding regulatory strategies for 21 pathways and 67-224 operons in divergent bacteria separated by 0.6-2 billion years. Using end-enriched RNA-sequencing (Rend-seq) with single-nucleotide resolution, we found that many bacterial gene clusters encoding conserved pathways have undergone massive divergence in transcript abundance and architectures via remodeling of internal promoters and terminators. Remarkably, these evolutionary changes are compensated post-transcriptionally to maintain preferred stoichiometry of protein synthesis rates. Even more strikingly, in eukaryotic budding yeast, functionally analogous proteins that arose independently from bacterial counterparts also evolved to convergent in-pathway expression. The broad requirement for exact protein stoichiometries despite regulatory divergence provides an unexpected principle for building biological pathways both in nature and for synthetic activities.

The Effects of Length of Residence and Exposure to Violence on Perceptions of Neighborhood Safety in an Urban Sample.

Perceptions of neighborhood safety shape the well-being of individuals and communities, affecting neighborhood walkability, associated physical activity behaviors, and health conditions. However, less is known about the factors that determine perceptions of safety. One factor that may affect perceptions of neighborhood safety is the length of time someone has lived in their neighborhood. We use a representative, adult sample of urban low-income residents from the 2015 New Haven Health Survey (n = 1189) to investigate the associations between length of residence (new residents of < 1 year in neighborhood versus longer-term residents of 1 or more years in neighborhood) and perceptions of neighborhood safety (whether feeling unsafe to walk at night). We then examine the potential moderating effect of exposure to neighborhood violence on these associations. We find that the association between length of residence and perceived safety differs by exposure to neighborhood violence. Among those unexposed to neighborhood violence, longer-term neighborhood residents were more likely to feel unsafe compared to new residents (OR = 2.03, 95% CI 1.19, 3.45). Additionally, the effect of exposure to violence on feelings of safety was larger for new residents (OR = 9.10, 95% CI 2.72, 30.44) compared to longer-term residents (OR = 1.88, 95% CI 1.28, 2.77). Our findings suggest that length of residence may have implications for feelings of safety, and that experiences of violence may uniquely contribute to feelings of unsafety among new residents. These findings hold implications for interventions and policy efforts aimed at neighborhood safety improvements through community development, housing, or city urban planning initiatives, particularly for new neighborhood residents or those who experience neighborhood violence.

"That wasn't really a place to worry about diabetes": Housing access and diabetes self-management among low-income adults.

Lack of affordable housing access represents a significant and growing problem for low-income households in the United States and these housing challenges may present barriers to the management of chronic diseases such as type 2 diabetes. In this qualitative study, we examined how both housing challenges and housing resources shaped diabetes self-management behaviors. We conducted semi-structured interviews with 40 low-income residents of one US city, New Haven, Connecticut, who had a diagnosis of type 2 diabetes and either resided in or qualified for subsidized housing. We purposively constructed our sample to include a range of housing experiences (subsidized, unsubsidized, homeless) and treatment regimes. We analyzed the data using grounded theory techniques of inductive coding and memo writing. Our findings suggest multiple ways that housing access can affect diabetes self-management with implications for blood glucose levels and future complications. Specifically, we describe the ways that housing access affected participants' ability to: 1) prioritize their diabetes care, 2) establish and maintain diabetes routines, and 3) afford diabetes-related expenses. Together, our findings show how housing challenges increased the cost of adherence to diabetes management regimes such that inadequately housed individuals had to both invest and sacrifice more for the same outcomes. Our findings suggest that improved affordable housing access may represent an opportunity to improve outcomes and reduce socioeconomic disparities among those living with type 2 diabetes.

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins.

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common.

MicL, a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein.

In enteric bacteria, the transcription factor σ(E) maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σ(E)-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σ(E) activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σ(E) regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σ(E) to repress the synthesis of all abundant outer membrane proteins in response to stress.

Stress-induced remodeling of the bacterial proteome.

Microorganisms live in fluctuating environments, requiring stress response pathways to resist environmental insults and stress. These pathways dynamically monitor cellular status, and mediate adaptive changes by remodeling the proteome, largely accomplished by remodeling transcriptional networks and protein degradation. The complementarity of fast, specific proteolytic degradation and slower, broad transcriptomic changes gives cells the mechanistic repertoire to dynamically adjust cellular processes and optimize response behavior. Together, this enables cells to minimize the 'cost' of the response while maximizing the ability to survive environmental stress. Here we highlight recent progress in our understanding of transcriptional networks and proteolysis that illustrates the design principles used by bacteria to generate the complex behaviors required to resist stress.

Dual molecular signals mediate the bacterial response to outer-membrane stress.

In Gram-negative bacteria, outer-membrane integrity is essential for survival and is monitored by the σ(E) stress-response system, which initiates damage-repair pathways. One activating signal is unassembled outer-membrane proteins. Using biochemical and genetic experiments in Escherichia coli, we found that off-pathway intermediates in lipopolysaccharide transport and assembly provided an additional required signal. These distinct signals, arising from disruptions in the transport and assembly of the major outer-membrane components, jointly determined the rate of proteolytic destruction of a negative regulator of the σ(E) transcription factor, thereby modulating the expression of stress-response genes. This dual-signal system permits a rapid response to dysfunction in outer-membrane biogenesis, while buffering responses to transient fluctuations in individual components, and may represent a broad strategy for bacteria to monitor their interface with the environment.

Signal integration by DegS and RseB governs the σ E-mediated envelope stress response in Escherichia coli.

In Escherichia coli, the σ(E) transcription factor monitors and maintains outer membrane (OM) integrity by activating genes required for assembly of its two key components, outer membrane proteins (OMPs) and lipopolysaccharide (LPS) and by transcribing small RNAs to down-regulate excess unassembled OMPs. σ(E) activity is governed by the rate of degradation of its membrane-spanning anti-σ factor, RseA. Importantly, the DegS protease can initiate RseA cleavage only when activated by binding to unassembled OMPs. The prevalent paradigm has been that the σ(E) response is controlled by the amount of activated DegS. Here we demonstrate that inactivation of a second negative regulator, the periplasmic protein RseB, is also required for σ(E) induction in vivo. Moreover, OMPs, previously known only to activate DegS, also generate a signal to antagonize RseB inhibition. This signal may be lipid related, as RseB is structurally similar to proteins that bind lipids. We propose that the use of an AND gate enables σ(E) to sense and integrate multivariate signals from the envelope.