The leucine-responsive regulatory proteins/feast-famine regulatory proteins: an ancient and complex class of transcriptional regulators in bacteria and archaea.

Since the discovery of the Escherichia coli leucine-responsive regulatory protein (Lrp) almost 50 years ago, hundreds of Lrp homologs have been discovered, occurring in 45% of sequenced bacteria and almost all sequenced archaea. Lrp-like proteins are often referred to as the feast/famine regulatory proteins (FFRPs), reflecting their common regulatory roles. Acting as either global or local transcriptional regulators, FFRPs detect the environmental nutritional status by sensing small effector molecules (usually amino acids) and regulate the expression of genes involved in metabolism, virulence, motility, nutrient transport, stress tolerance, and antibiotic resistance to implement appropriate behaviors for the specific ecological niche of each organism. Despite FFRPs' complexity, a significant role in gene regulation, and prevalence throughout prokaryotes, the last comprehensive review on this family of proteins was published about a decade ago. In this review, we integrate recent notable findings regarding E. coli Lrp and other FFRPs across bacteria and archaea with previous observations to synthesize a more complete view on the mechanistic details and biological roles of this ancient class of transcription factors.

A Shift to Human Body Temperature (37°C) Rapidly Reprograms Multiple Adaptive Responses in Escherichia coli That Would Facilitate Niche Survival and Colonization.

One of the first environmental cues sensed by a microbe as it enters a human host is an upshift in temperature to 37°C. In this dynamic time point analysis, we demonstrate that this environmental transition rapidly signals a multitude of gene expression changes in Escherichia coli. Bacteria grown at 23°C under aerobic conditions were shifted to 37°C, and mRNA expression was measured at time points after the shift to 37°C (t = 0.5, 1, and 4 h). The first hour is characterized by a transient shift to anaerobic respiration strategies and stress responses, particularly acid resistance, indicating that temperature serves as a sentinel cue to predict and prepare for various niches within the host. The temperature effects on a subset of stress response genes were shown to be mediated by RpoS and directly correlated with RpoS, DsrA, and RprA levels, and increased acid resistance was observed that was dependent on 23°C growth and RpoS. By 4 h, gene expression shifted to aerobic respiration pathways and decreased stress responses, coupled with increases in genes associated with biosynthesis (amino acid and nucleotides), iron uptake, and host defense. ompT, a gene that confers resistance to antimicrobial peptides, was highly thermoregulated, with a pattern conserved in enteropathogenic and uropathogenic E. coli strains. An immediate decrease in curli gene expression concomitant with an increase in flagellar gene expression implicates temperature in this developmental decision. Together, our studies demonstrate that temperature signals a reprogramming of gene expression immediately upon an upshift that may predict, prepare, and benefit the survival of the bacterium within the host. IMPORTANCE As one of the first cues sensed by the microbe upon entry into a human host, understanding how bacteria like E. coli modulate gene expression in response to temperature improves our understanding of how bacteria immediately initiate responses beneficial for survival and colonization. For pathogens, understanding the various pathways of thermal regulation could yield valuable targets for anti-infective chemotherapeutic drugs or disinfection measures. In addition, our data provide a dynamic examination of the RpoS stress response, providing genome-wide support for how temperature impacts RpoS through changes in RpoS stability and modulation by small regulatory RNAs.

Two-Tier Screening Platform for Directed Evolution of Aminoacyl-tRNA Synthetases with Enhanced Stop Codon Suppression Efficiency.

Genetic code expansion through amber stop codon suppression provides a powerful tool for introducing non-proteinogenic functionalities into proteins for a broad range of applications. However, ribosomal incorporation of noncanonical amino acids (ncAAs) by means of engineered aminoacyl-tRNA synthetases (aaRSs) often proceeds with significantly reduced efficiency compared to sense codon translation. Here, we report the implementation of a versatile platform for the development of engineered aaRSs with enhanced efficiency in mediating ncAA incorporation by amber stop codon suppression. This system integrates a white/blue colony screen with a plate-based colorimetric assay, thereby combining high-throughput capabilities with reliable and quantitative measurement of aaRS-dependent ncAA incorporation efficiency. This two-tier functional screening system was successfully applied to obtain a pyrrolysyl-tRNA synthetase (PylRS) variant (CrtK-RS(4.1)) with significantly improved efficiency (+250-370 %) for mediating the incorporation of Nϵ -crotonyl-lysine and other lysine analogues of relevance for the study of protein post-translational modifications into a target protein. Interestingly, the beneficial mutations accumulated by CrtK-RS(4.1) were found to localize within the noncatalytic N-terminal domain of the enzyme and could be transferred to another PylRS variant, improving the ability of the variant to incorporate its corresponding ncAA substrate. This work introduces an efficient platform for the improvement of aaRSs that could be readily extended to other members of this enzyme family and/or other target ncAAs.

Escherichia coli Leucine-Responsive Regulatory Protein Bridges DNA In Vivo and Tunably Dissociates in the Presence of Exogenous Leucine.

Feast-famine response proteins are a widely conserved class of global regulators in prokaryotes, the most highly studied of which is the Escherichia coli leucine-responsive regulatory protein (Lrp). Lrp senses the environmental nutrition status and subsequently regulates up to one-third of the genes in E. coli, either directly or indirectly. Lrp exists predominantly as octamers and hexadecamers (16mers), where leucine is believed to shift the equilibrium toward the octameric state. In this study, we analyzed the effects of three oligomerization state mutants of Lrp in terms of their ability to bind to DNA and regulate gene expression in response to exogenous leucine. We find that oligomerization beyond dimers is required for Lrp's regulatory activity and that, contrary to previous speculation, exogenous leucine modulates Lrp activity at its target promoters exclusively by inhibiting Lrp binding to DNA. We also show evidence that Lrp binding bridges DNA over length scales of multiple kilobases, revealing a new range of mechanisms for Lrp-mediated transcriptional regulation. IMPORTANCE Leucine-responsive regulatory protein (Lrp) is one of the most impactful regulators in E. coli and other bacteria. Lrp senses nutrient conditions and responds by controlling strategies for virulence, cellular motility, and nutrient acquisition. Despite its importance and being evolutionarily highly conserved across bacteria and archaea, several mysteries remain regarding Lrp, including how it actually responds to leucine to change its regulation of targets. Previous studies have led to the hypothesis that Lrp switches between two states, an octamer (8 Lrp molecules together) and a hexadecamer (16 Lrp molecules together), upon exposure to leucine; these are referred to as different oligomerization states. Here, we show that contrary to previous expectations, it is Lrp's propensity to bind DNA, rather than its oligomerization state, that is directly affected by leucine in the cell's environment. Our new understanding of Lrp activity will aid in identifying and disrupting pathways used by bacteria to cause disease.

A Thermosensitive, Phase-Variable Epigenetic Switch: pap Revisited.

It has been more than a decade since the last comprehensive review of the phase-variable uropathogen-associated pyelonephritis-associated pilus (pap) genetic switch. Since then, important data have come to light, including additional factors that regulate pap expression, better characterization of H-NS regulation, the structure of the Lrp octamer in complex with pap regulatory DNA, the temperature-insensitive phenotype of a mutant lacking the acetyltransferase RimJ, evidence that key components of the regulatory machinery are acetylated, and new insights into the role of DNA binding by key regulators in shaping both the physical structure and regulatory state of the papI and papBA promoters. This review revisits pap, integrating these newer observations with older ones to produce a new model for the concerted behavior of this virulence-regulatory region.

Genome-wide identification of H-NS-controlled, temperature-regulated genes in Escherichia coli K-12.

DNA microarrays demonstrate that H-NS controls 69% of the temperature regulated genes in Escherichia coli K-12. H-NS is shown to be a common regulator of multiple iron and other nutrient acquisition systems preferentially expressed at 37 degrees C and of general stress response, biofilm formation, and cold shock genes highly expressed at 23 degrees C.

Metabolic Cycles in Yeast Share Features Conserved among Circadian Rhythms.

Cell-autonomous circadian rhythms allow organisms to temporally orchestrate their internal state to anticipate and/or resonate with the external environment. Although ∼24-hr periodicity is observed across aerobic eukaryotes, the central mechanism has been hard to dissect because few simple models exist, and known clock proteins are not conserved across phylogenetic kingdoms. In contrast, contributions to circadian rhythmicity made by a handful of post-translational mechanisms, such as phosphorylation of clock proteins by casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3), appear conserved among phyla. These kinases have many other essential cellular functions and are better conserved in their contribution to timekeeping than any of the clock proteins they phosphorylate. Rhythmic oscillations in cellular redox state are another universal feature of circadian timekeeping, e.g., over-oxidation cycles of abundant peroxiredoxin proteins. Here, we use comparative chronobiology to distinguish fundamental clock mechanisms from species and/or tissue-specific adaptations and thereby identify features shared between circadian rhythms in mammalian cells and non-circadian temperature-compensated respiratory oscillations in budding yeast. We find that both types of oscillations are coupled with the cell division cycle, exhibit period determination by CK1 and GSK3, and have peroxiredoxin over-oxidation cycles. We also explore how peroxiredoxins contribute to YROs. Our data point to common mechanisms underlying both YROs and circadian rhythms and suggest two interpretations: either certain biochemical systems are simply permissive for cellular oscillations (with frequencies from hours to days) or this commonality arose via divergence from an ancestral cellular clock.

Low temperature (23 degrees C) increases expression of biofilm-, cold-shock- and RpoS-dependent genes in Escherichia coli K-12.

Temperature serves as a cue to regulate gene expression in Escherichia coli and other bacteria. Using DNA microarrays, we identified 297 genes whose expression is increased at 23 degrees C compared to 37 degrees C in E. coli K-12. Of these genes, 122 are RpoS-controlled, confirming genome-wide the model that low temperature serves as a primary cue to trigger the general stress response. Several genes expressed at 23 degrees C overlap with the cold-shock response, suggesting that strategies used to adapt to sudden shifts in temperature also mediate long-term growth at 23 degrees C. Another category of genes more highly expressed at 23 degrees C are associated with biofilm development, implicating temperature as an important cue influencing this developmental pathway. In a candidate set of genes tested, the biofilm genes (adrA, bolA, mlrA, nhaR, csgA, yceP/bssS) and cold-shock genes (otsA, yceP/bssS) were found to be RpoS- and DsrA-dependent for their transcription at 23 degrees C. In contrast, transcription of three genes (ycgZ, dps and ymgB) was either partially or fully independent of these regulators, signifying there is an alternative thermoregulatory mechanism(s) that increases gene expression at 23 degrees C. Increased expression at 23 degrees C compared to 37 degrees C is retained in various media tested for most of the genes, supporting the relative importance of this cue in adaptation to changing environments. Both the RpoS-dependent gene otsA and the RpoS-independent gene ymgB demonstrated increased expression levels within 1 h after a shift from 37 to 23 degrees C, indicating a rapid response to this environmental cue. Despite changes in gene expression for many RpoS-dependent genes, experiments assessing growth rate at 23 degrees C and viability at 4 degrees C did not demonstrate significant impairment in rpoS : : Tn10 or dsrA : : cat mutant strains in comparison to the wild-type strain. Biofilm formation was favoured at low temperature and is moderately impaired in both the rpoS : : Tn10 and dsrA : : cat mutants at 23 degrees C, suggesting genes controlled by these regulators play a role necessary for optimal biofilm formation at 23 degrees C. Taken together, our data demonstrate that a large number of genes are increased in expression at 23 degrees C to globally respond to this environmental change and that at least two thermoregulatory pathways are involved in co-ordinating this response - the RpoS/DsrA pathway and an alternative thermoregulatory pathway, independent of these regulators.

Human body temperature (37degrees C) increases the expression of iron, carbohydrate, and amino acid utilization genes in Escherichia coli K-12.

Using DNA microarrays, we identified 126 genes in Escherichia coli K-12 whose expression is increased at human body temperature (37 degrees C) compared to growth at 23 degrees C. Genes involved in the uptake and utilization of amino acids, carbohydrates, and iron dominated the list, supporting a model in which temperature serves as a host cue to increase expression of bacterial genes needed for growth. Using quantitative real-time PCR, we investigated the thermoregulatory response for representative genes in each of these three categories (hisJ, cysP, srlE, garP, fes, and cirA), along with the fimbrial gene papB. Increased expression at 37 degrees C compared to 23 degrees C was retained in both exponential and stationary phases for all of the genes and in most of the various media tested, supporting the relative importance of this cue in adapting to changing environments. Because iron acquisition is important for both growth and virulence, we analyzed the regulation of the iron utilization genes cirA and fes and found that growth in iron-depleted medium abrogated the thermoregulatory effect, with high-level expression at both temperatures, contrasting with papB thermoregulation, which was not greatly altered by limiting iron levels. A positive role for the environmental regulator H-NS was found for fes, cirA, hisJ, and srlE transcription, whereas it had a primarily negative effect on cysP and garP expression. Together, these studies indicate that temperature is a broadly used cue for regulating gene expression in E. coli and that H-NS regulates iron, carbohydrate, and amino acid utilization gene expression.

The N-acetyltransferase RimJ responds to environmental stimuli to repress pap fimbrial transcription in Escherichia coli.

In uropathogenic Escherichia coli, P pili (Pap) facilitate binding to host epithelial cells and subsequent colonization. Whereas P pili can be produced at 37 degrees C, the expression of these fimbriae is suppressed at 23 degrees C. Previously, insertion mutations in rimJ, a gene encoding the N-terminal acetyltransferase of ribosomal protein S5, were shown to disrupt this thermoregulatory response, allowing papBA transcription at low temperature. In this study, we created an in-frame deletion of rimJ. This deletion relieved the repressive effects not only of low temperature but also of rich (Luria-Bertani [LB]) medium and glucose on papBA transcription, indicating that RimJ modulates papBA transcription in response to multiple environmental stimuli. papI transcription was also shown to be regulated by RimJ. papBA transcription is also controlled by a phase variation mechanism. We demonstrated that the regulators necessary to establish a phase ON state--PapI, PapB, Dam, Lrp, and cyclic AMP-CAP-are still required for papBA transcription in a rimJ mutant strain. rimJ mutations increase the rate at which bacteria transition into the phase ON state, indicating that RimJ inhibits the phase OFF-->ON transition. A DeltarimJ hns651 mutant is viable on LB medium but not on minimal medium. This synthetic lethality, along with transcriptional analyses, indicates that RimJ and H-NS work through separate pathways to control papBA transcription. Mutations in rimJ do not greatly influence the transcription of the fan, daa, or fim operon, suggesting that RimJ may be a pap-specific regulator. Overexpression of rimJ under conditions repressive for papBA transcription complements the DeltarimJ mutation but has little effect on transcription under activating conditions, indicating that the ability of RimJ to regulate transcription is environmentally controlled.