Exploring the molecular mechanisms of Lrp/AsnC-type transcription regulator DecR, an L-cysteine-responsive feast/famine regulatory protein.

The Lrp/AsnC family of transcriptional regulators is commonly found in prokaryotes and is associated with the regulation of amino acid metabolism. However, it remains unclear how the L-cysteine-responsive Lrp/AsnC family regulator perceives and responds to L-cysteine. Here, we try to elucidate the molecular mechanism of the L-cysteine-responsive transcriptional regulator. Through 5'RACE and EMSA, we discovered a 15 bp incompletely complementary pair palindromic sequence essential for DecR binding, which differed slightly from the binding sequence of other Lrp/AsnC transcription regulators. Using alanine scanning, we identified the L-cysteine binding site on DecR and found that different Lrp/AsnC regulators adjust their binding pocket's side-chain residues to accommodate their specific effector. MD simulations were then conducted to explore how ligand binding influences the allosteric behavior of the protein. PCA and in silico docking revealed that ligand binding induced perturbations in the linker region, triggering conformational alterations and leading to the relocalization of the DNA-binding domains, enabling the embedding of the DNA-binding region of DecR into the DNA molecule, thereby enhancing DNA-binding affinity. Our findings can broaden the understanding of the recognition and regulatory mechanisms of the Lrp/AsnC-type transcription factors, providing a theoretical basis for further investigating the molecular mechanisms of other transcription factors.

The replication enhancer crtS depends on transcription factor Lrp for modulating binding of initiator RctB to ori2 of Vibrio cholerae.

Replication of Vibrio cholerae chromosome 2 (Chr2) initiates when the Chr1 locus, crtS (Chr2 replication triggering site) duplicates. The site binds the Chr2 initiator, RctB, and the binding increases when crtS is complexed with the transcription factor, Lrp. How Lrp increases the RctB binding and how RctB is subsequently activated for initiation by the crtS-Lrp complex remain unclear. Here we show that Lrp bends crtS DNA and possibly contacts RctB, acts that commonly promote DNA-protein interactions. To understand how the crtS-Lrp complex enhances replication, we isolated Tn-insertion and point mutants of RctB, selecting for retention of initiator activity without crtS. Nearly all mutants (42/44) still responded to crtS for enhancing replication, exclusively in an Lrp-dependent manner. The results suggest that the Lrp-crtS controls either an essential function or more than one function of RctB. Indeed, crtS modulates two kinds of RctB binding to the origin of Chr2, ori2, both of which we find to be Lrp-dependent. Some point mutants of RctB that are optimally modulated for ori2 binding without crtS still remained responsive to crtS and Lrp for replication enhancement. We infer that crtS-Lrp functions as a unit, which has an overarching role, beyond controlling initiator binding to ori2.

The Lrp transcriptional factor of an entomopathogenic bacterium, Xenorhabdus hominickii, activates non-ribosomal peptide synthetases to suppress insect immunity.

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. The infective juveniles deliver the symbiotic bacteria to the hemocoel of target insects, in which the bacteria proliferate and help the development of the host nematode. The successful parasitism of the nematode-bacterial complex depends on host immunosuppression by the bacteria via their secondary metabolites. Leucine-responsive regulatory protein (Lrp) is a global bacterial transcriptional factor that plays a crucial role in parasitism. However, its regulatory targets to suppress insect immunity are not clearly understood. This study investigated the bacterial genes regulated by Lrp and the subsequent production of secondary metabolites in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage of the bacteria in a target insect, Spodoptera exigua. A preliminary in silico screening indicated that 3.7% genes among 4075 predicted genes encoded in X. hominickii had the Lrp-response element on their promoters, including two non-ribosomal peptide synthetases (NRPSs). Eight NRPS (NRPS1-NRPS8) genes were predicted in the bacterial genome, in which six NRPS (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites and the NRPS expression levels. The immunosuppressive activities of X. hominickii were dependent on the Lrp expression level. The metabolites produced by Lrp expression included the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (=cPF) was produced by the bacteria at high Lrp expression and inhibited the phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays a crucial role in insect immunosuppression by modulating NRPS expression.

Molecular mechanisms of regulation by a ß-alanine-responsive Lrp-type transcription factor from Acidianus hospitalis.

The leucine-responsive regulatory protein (Lrp) family of transcriptional regulators is widespread among prokaryotes and especially well-represented in archaea. It harbors members with diverse functional mechanisms and physiological roles, often linked to the regulation of amino acid metabolism. BarR is an Lrp-type regulator that is conserved in thermoacidophilic Thermoprotei belonging to the order Sulfolobales and is responsive to the non-proteinogenic amino acid ß-alanine. In this work, we unravel molecular mechanisms of the Acidianus hospitalis BarR homolog, Ah-BarR. Using a heterologous reporter gene system in Escherichia coli, we demonstrate that Ah-BarR is a dual-function transcription regulator that is capable of repressing transcription of its own gene and activating transcription of an aminotransferase gene, which is divergently transcribed from a common intergenic region. Atomic force microscopy (AFM) visualization reveals a conformation in which the intergenic region appears wrapped around an octameric Ah-BarR protein. ß-alanine causes small conformational changes without affecting the oligomeric state of the protein, resulting in a relief of regulation while the regulator remains bound to the DNA. This regulatory and ligand response is different from the orthologous regulators in Sulfolobus acidocaldarius and Sulfurisphaera tokodaii, which is possibly explained by a distinct binding site organization and/or by the presence of an additional C-terminal tail in Ah-BarR. By performing site-directed mutagenesis, this tail is shown to be involved in ligand-binding response.

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.

The leucine-responsive regulatory protein SCAB_Lrp modulates thaxtomin biosynthesis, pathogenicity, and morphological development in Streptomyces scabies.

Streptomyces scabies is the best-characterized plant-pathogenic streptomycete, which is a special species among the large genus Streptomyces. The pathogenicity of S. scabies relies on the production of the secondary metabolite thaxtomin A. Little is known about the molecular mechanisms underlying the regulation of thaxtomin biosynthesis in S. scabies beyond the pathway-specific activator TxtR and the cellulose utilization repressor CebR. The leucine-responsive regulatory protein (Lrp) family modulates secondary metabolism in nonpathogenic streptomycetes. However, the regulatory relationship between the Lrp and pathogenic streptomycetes remains unknown. In this study, we demonstrated that SCAB_Lrp (SCAB_77931) from S. scabies significantly affects thaxtomin biosynthesis, pathogenicity, and morphological development. SCAB_Lrp deletion resulted in a dramatic decline in thaxtomin A production and a low-virulence phenotype of S. scabies. An in-depth dissection of the regulatory mechanism of SCAB_Lrp revealed that it positively regulates the transcription of the thaxtomin biosynthetic gene cluster by directly binding to the promoter of the cluster-situated regulator gene txtR. SCAB_Lrp also controls the morphological development of S. scabies by directly activating the transcription of amfC, whiB, and ssgB. SCAB_Lrp directly controls the transcription of its own gene by binding a specific sequence (5'-GGACAGTCGCCGTGCTACG-3'). Moreover, phenylalanine and methionine have been characterized as SCAB_Lrp effectors by strengthening the binding affinity and complex status between SCAB_Lrp and DNA. Our findings characterize a multifunctional regulatory protein, SCAB_Lrp, that controls secondary metabolism, pathogenicity, and sporulation in S. scabies and provide new insights into the complex regulatory network that modulates thaxtomin phytotoxins in pathogenic Streptomyces.

Co-expressing Leucine Responsive Regulatory protein (Lrp) enhances recombinant L-Asparaginase-II production in Escherichia coli.

Over expression of recombinant proteins triggers a cellular stress response (CSR) that down-regulates numerous genes that have a key role in sustaining expression. Instead of trying to individually up-regulate these genes we hypothesized that a superior strategy would be to modulate the expression of global regulators that control the expression of many such downstream genes. Transcriptomic profiling of post induction cultures expressing recombinant asparaginase in Escherichia coli showed the down-regulation of several critical genes many of which were under the control of the global regulator lrp which is known to have a significant impact on both amino acid metabolism and protein translation. Therefore, to ameliorate the deleterious effects of the CSR we decided to supplement the activity of lrp using plasmid-based co-expression. We observed that the test culture containing an additional plasmid expressing lrp under the arabinose promoter gave a 50% higher yield of recombinant L-Asparaginase after 32 h in batch culture compared to the control, which had only one plasmid expressing the recombinant protein. This approach helped us design a better performing strain, which could sustain expression rates for a significantly longer time period. This work illustrates that modifying the expression of regulatory genes could serve as a better strategy to prevent the reprogramming of the cellular machinery which is the hallmark of the CSR and help in the design better hosts for recombinant protein expression.

Leucine-Responsive Regulatory Protein in Acetic Acid Bacteria Is Stable and Functions at a Wide Range of Intracellular pH Levels.

Acetic acid bacteria grow while producing acetic acid, resulting in acidification of the culture. Limited reports elucidate the effect of changes in intracellular pH on transcriptional factors. In the present study, the intracellular pH of Komagataeibacter europaeus was monitored with a pH-sensitive green fluorescent protein, showing that the intracellular pH decreased from 6.3 to 4.7 accompanied by acetic acid production during cell growth. The leucine-responsive regulatory protein of K. europaeus (KeLrp) was used as a model to examine pH-dependent effects, and its properties were compared with those of the Escherichia coli ortholog (EcLrp) at different pH levels. The DNA-binding activities of EcLrp and KeLrp with the target DNA (Ec-ilvI and Ke-ilvI) were examined by gel mobility shift assays under various pH conditions. EcLrp showed the highest affinity with the target at pH 8.0 (Kd [dissociation constant], 0.7 µM), decreasing to a minimum of 3.4 µM at pH 4.0. Conversely, KeLrp did not show significant differences in binding affinity between pH 4 and 7 (Kd, 1.0 to 1.5 µM), and the highest affinity was at pH 5.0 (Kd, 1.0 µM). Circular dichroism spectroscopy revealed that the α-helical content of KeLrp was the highest at pH 5.0 (49%) and was almost unchanged while being maintained at >45% over a range of pH levels examined, while that of EcLrp decreased from its maximum (49% at pH 7.0) to its minimum (36% at pH 4.0). These data indicate that KeLrp is stable and functions over a wide range of intracellular pH levels. IMPORTANCE Lrp is a highly conserved transcriptional regulator found in bacteria and archaea and regulates transcriptions of various genes. The intracellular pH of acetic acid bacteria (AAB) changes accompanied by acetic acid production during cell growth. The Lrp of AAB K. europaeus (KeLrp) was structurally stable over a wide range of pH and maintained DNA-binding activity even at low pH compared with Lrp from E. coli living in a neutral environment. An in vitro experiment showed DNA-binding activity of KeLrp to the target varied with changes in pH. In AAB, change of the intracellular pH during a cell growth would be an important trigger in controlling the activity of Lrp in vivo.

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.

Automated Rational Strain Construction Based on High-Throughput Conjugation.

Molecular cloning is the core of synthetic biology, as it comprises the assembly of DNA and its expression in target hosts. At present, however, cloning is most often a manual, time-consuming, and repetitive process that highly benefits from automation. The automation of a complete rational cloning procedure, i.e., from DNA creation to expression in the target host, involves the integration of different operations and machines. Examples of such workflows are sparse, especially when the design is rational (i.e., the DNA sequence design is fixed and not based on randomized libraries) and the target host is less genetically tractable (e.g., not sensitive to heat-shock transformation). In this study, an automated workflow for the rational construction of plasmids and their subsequent conjugative transfer into the biotechnological platform organism Corynebacterium glutamicum is presented. The whole workflow is accompanied by a custom-made software tool. As an application example, a rationally designed library of transcription factor-biosensors based on the regulator Lrp was constructed and characterized. A sensor with an improved dynamic range was obtained, and insights from the screening provided evidence for a dual regulator function of C. glutamicum Lrp.

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.

A MARTX Toxin rtxA Gene Is Controlled by Host Environmental Signals through a CRP-Coordinated Regulatory Network in Vibrio vulnificus.

A multifunctional autoprocessing repeats-in-toxin (MARTX) toxin plays an essential role in the virulence of many pathogens, including a fulminating human pathogen Vibrio vulnificus H-NS and HlyU repress and derepress expression of the MARTX toxin gene rtxA in V. vulnificus, respectively. However, little is known about other regulatory proteins and environmental signals involved in rtxA regulation. In this study, we found that a leucine-responsive regulatory protein (Lrp) activates rtxA by binding directly and specifically to the rtxA promoter, P rtxA Phased hypersensitivity resulting from DNase I cleavage of the P rtxA regulatory region suggests that Lrp probably induces DNA bending in P rtxA Lrp activates P rtxA independently of H-NS and HlyU, and leucine inhibits Lrp binding to P rtxA and reduces the Lrp-mediated activation. Furthermore, a cyclic AMP receptor protein (CRP) represses P rtxA , and exogenous glucose relieves the CRP-mediated repression. Biochemical and mutational analyses demonstrated that CRP binds directly and specifically to the upstream region of P rtxA , which presumably alters the DNA conformation in P rtxA and thus represses rtxA Moreover, CRP represses expression of lrp and hlyU by binding directly to their upstream regions, forming coherent feed-forward loops with Lrp and HlyU. In conclusion, expression of rtxA is controlled by a regulatory network comprising CRP, Lrp, H-NS, and HlyU in response to changes in host environmental signals such as leucine and glucose. This collaborative regulation enables the elaborate expression of rtxA, thereby enhancing the fitness and pathogenesis of V. vulnificus during the course of infection.IMPORTANCE A MARTX toxin, RtxA, is an essential virulence factor of many pathogens, including Vibrio species. H-NS and HlyU repress and derepress, respectively, rtxA expression of a life-threatening pathogen, Vibrio vulnificus We found that Lrp directly activates rtxA independently of H-NS and HlyU, and leucine inhibits the Lrp-mediated activation of rtxA Furthermore, we demonstrated that CRP represses rtxA but derepresses in the presence of exogenous glucose. CRP represses rtxA not only directly by binding to upstream of rtxA but also indirectly by repressing lrp and hlyU This is the first report of a regulatory network comprising CRP, Lrp, H-NS, and HlyU, which coordinates the rtxA expression in response to environmental signals such as leucine and glucose during infection. This elaborate regulatory network will enhance the fitness of V. vulnificus and contribute to its successful infection within the host.

Cystine import is a valuable but risky process whose hazards Escherichia coli minimizes by inducing a cysteine exporter.

The structure of free cysteine makes it vulnerable to oxidation by molecular oxygen; consequently, organisms that live in oxic habitats have acquired the ability to import cystine as a sulfur source. We show that cystine imported into Escherichia coli can transfer disulfide bonds to cytoplasmic proteins. To minimize this problem, the imported cystine is rapidly reduced. However, this conversion of cystine to cysteine precludes product inhibition of the importer, so cystine import continues into cells that are already sated with cysteine. The burgeoning cysteine pool is itself hazardous, as cysteine promotes the formation of reactive oxygen species, triggers sulfide production and competitively inhibits a key enzyme in the isoleucine biosynthetic pathway. The Lrp transcription factor senses the excess cysteine and induces AlaE, an export protein that pumps cysteine back out of the cell until transcriptional controls succeed in lowering the amount of the importer. While it lasts, the overall phenomenon roughly doubles the NADPH demand of the cell. It comprises another example of the incompatibility of the reduced cytoplasms of microbes with the oxic world in which they dwell. It also reveals one natural source of cytoplasmic disulfide stress and sheds light on a role for broad-spectrum amino acid exporters.

The Transcriptional Regulator Lrp Contributes to Toxin Expression, Sporulation, and Swimming Motility in Clostridium difficile.

Clostridium difficile is a Gram-positive, spore-forming bacterium, and major cause of nosocomial diarrhea. Related studies have identified numerous factors that influence virulence traits such as the production of the two primary toxins, toxin A (TcdA) and toxin B (TcdB), as well as sporulation, motility, and biofilm formation. However, multiple putative transcriptional regulators are reportedly encoded in the genome, and additional factors are likely involved in virulence regulation. Although the leucine-responsive regulatory protein (Lrp) has been studied extensively in Gram-negative bacteria, little is known about its function in Gram-positive bacteria, although homologs have been identified in the genome. This study revealed that disruption of the lone lrp homolog in C. difficile decelerated growth under nutrient-limiting conditions, increased TcdA and TcdB production. Lrp was also found to negatively regulate sporulation while positively regulate swimming motility in strain R20291, but not in strain 630. The C. difficile Lrp appeared to function through transcriptional repression or activation. In addition, the lrp mutant was relatively virulent in a mouse model of infection. The results of this study collectively demonstrated that Lrp has broad regulatory function in C. difficile toxin expression, sporulation, motility, and pathogenesis.

The ridA gene of E. coli is indirectly regulated by BglG through the transcriptional regulator Lrp in stationary phase.

Regulators encoded by the beta-glucoside (bgl) operon of Escherichia coli are known to influence the expression of downstream target genes that confer a fitness advantage in stationary phase. We have examined the role of bglG in the regulation of ridA that encodes an enamine/imine deaminase essential for the elimination of reactive intermediates generated during the catabolism of amino acids such as serine. We report here that ridA is positively regulated by leucine responsive regulatory protein (Lrp) and leucine antagonizes the activation by Lrp. We also show that Lrp itself is under the indirect regulation of BglG, which brings about the overexpression of ridA in Bgl+ strains during stationary phase. Loss of ridA function in a Bgl+ background results in a significant growth retardation in serine-containing media compared to that in a Bgl- background. We propose that overexpression of ridA in Bgl+ background during stationary phase is physiologically relevant to eliminate toxic metabolites generated by the catabolism of serine-containing peptides as a result of elevated levels of their uptake.

Escherichia coli Lrp Regulates One-Third of the Genome via Direct, Cooperative, and Indirect Routes.

The global regulator Lrp plays a crucial role in regulating metabolism, virulence, and motility in response to environmental conditions. Lrp has previously been shown to activate or repress approximately 10% of the genes in Escherichia coli However, the full spectrum of targets, and how Lrp acts to regulate them, have stymied earlier study. We have combined matched chromatin-immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) under nine physiological conditions to comprehensively map the binding and regulatory activity of Lrp as it directs responses to nutrient abundance. In addition to identifying hundreds of novel Lrp targets, we observe two new global trends, as follows: first, that Lrp will often bind to promoters in a poised position under conditions when it has no regulatory activity to enable combinatorial interactions with other regulators, and second, that nutrient levels induce a global shift in the equilibrium between less-sequence-specific and more-sequence-specific DNA binding. The overall regulatory behavior of Lrp, which as we now show extends to 38% of E. coli genes directly or indirectly under at least one condition, thus arises from the interaction between changes in Lrp binding specificity and cooperative action with other regulators.IMPORTANCE To survive, bacteria such as E. coli must rapidly respond to changing environmental conditions, including nutrient levels. A decrease in nutrient availability causes bacteria to stop rapid replication and enter stationary phase, where they perform limited to no cell division. The E. coli global regulatory protein Lrp has been previously implicated in modulating the expression of genes particularly important at this transition from rapid to slowed growth. Here, we monitor Lrp's DNA binding locations and effect on gene expression under three different nutrient conditions across three growth stages. We find that Lrp's role is even broader than previously suspected and that it appears to interact with many other bacterial regulators to perform its function in a condition-specific manner.

Leucine responsive regulatory protein is involved in methionine metabolism and polyamine homeostasis in acetic acid bacterium Komagataeibacter europaeus.

The leucine responsive regulatory protein (Lrp) is a global transcription factor that regulates the expression of genes involved in amino acid metabolism. To identify metabolic pathways and related genes under the control of Lrp in the acetic acid bacterium Komagataeibacter europaeus, the Kelrp null mutant (KGMA7110), which requires supplementation of all 20 amino acids for normal growth, was cultivated in minimal media containing or lacking particular amino acids. The results confirmed that KGMA7110 was auxotrophic for methionine and its catabolites S-adenosylmethionine (SAM) and spermidine (SPD). Quantitative reverse-transcription PCR analysis revealed lower metK (SAM synthetase) and mdtI (SPD efflux pump) expression in KGMA7110 than in wild-type KGMA0119. By contrast, these genes were significantly up-regulated in the Kelrp mutant lacking the putative C-terminal ligand-sensing domain (KGMA7203), indicating abnormal regulation of target genes by the KeLrp variant in KGMA7203. KGMA7110 (0.69±0.27 µM) and KGMA7203 (4.90±0.61 µM) excreted lower and higher quantities of SPD, respectively, than KGMA0119 (2.28±0.26 µM). This was attributed to imbalanced carbon flow caused by Kelrp disruption that respectively attenuated and stimulated metK and mdtI expression. These findings indicate that KeLrp plays a key role in SAM biosynthesis and intracellular polyamine homeostasis in K. europaeus.

Lrp, a global regulator, regulates the virulence of Vibrio vulnificus.

BACKGROUND: An attenuated mutant (designated NY303) of Vibrio vulnificus, which causes serious wound infection and septicemia in humans, was isolated fortuitously from a clinical strain YJ016. This mutant was defective in cytotoxicity, migration on soft agar and virulence in the mouse. The purpose of this study was to map the mutation in this attenuated mutant and further explore how the gene thus identified is involved in virulence. METHODS: The whole genome sequence of mutant NY303 determined by next-generation sequencing was compared with that of strain YJ016 to map the mutations. By isolating and characterizing the specific gene-knockout mutants, the gene associated with the phenotype of mutant NY303 was identified. This gene encodes a global regulator, Lrp. A mutant, YH01, deficient in Lrp was isolated and examined in vitro, in vivo and ex vivo to find the affected virulence mechanisms. The target genes of Lrp were further identified by comparing the transcriptomes, which were determined by RNA-seq, of strain YJ016 and mutant YH01. The promoters bound by Lrp were identified by genome footprinting-sequencing, and those related with virulence were further examined by electrophoretic mobility shift assay. RESULTS: A mutation in lrp was shown to be associated with the reduced cytotoxicity, chemotaxis and virulence of mutant NY303. Mutant YH01 exhibited a phenotype resembling that of mutant NY303, and was defective in colonization in the mouse and growth in mouse serum, but not the antiphagocytosis ability. 596 and 95 genes were down- and up-regulated, respectively, in mutant YH01. Many of the genes involved in secretion of the MARTX cytotoxin, chemotaxis and iron-acquisition were down-regulated in mutant YH01. The lrp gene, which was shown to be negatively autoregulated, and 7 down-regulated virulence-associated genes were bound by Lrp in their promoters. A 14-bp consensus sequence, mkCrTTkwAyTsTG, putatively recognized by Lrp was identified in the promoters of these genes. CONCLUSIONS: Lrp is a global regulator involved in regulation of cytotoxicity, chemotaxis and iron-acquisition in V. vulnificus. Down-regulation of many of the genes associated with these properties may be responsible, at least partly, for loss of virulence in mutant NY303.

Characterization of an Lrp/AsnC family regulator SCO3361, controlling actinorhodin production and morphological development in Streptomyces coelicolor.

Lrp/AsnC family regulators have been found in many bacteria as crucial regulators controlling diverse cellular processes. By genomic alignment, we found that SCO3361, an Lrp/AsnC family protein from Streptomyces coelicolor, shared the highest similarity to the SACE_Lrp from Saccharopolyspora erythraea. Deletion of SCO3361 led to dramatic reduction in actinorhodin (Act) production and delay in aerial mycelium formation and sporulation on solid media. Dissection of the mechanism underlying the function of SCO3361 in Act production revealed that it altered the transcription of the cluster-situated regulator gene actII-ORF4 by directly binding to its promoter. SCO3361 was an auto-regulator and simultaneously activated the transcription of its adjacent divergently transcribed gene SCO3362. SCO3361 affected aerial hyphae formation and sporulation of S. coelicolor by activating the expression of amfC, whiB, and ssgB. Phenylalanine and cysteine were identified as the effector molecules of SCO3361, with phenylalanine reducing the binding affinity, whereas cysteine increasing it. Moreover, interactional regulation between SCO3361 and SACE_Lrp was discovered for binding to each other's target gene promoter in this work. Our findings indicate that SCO3361 functions as a pleiotropic regulator controlling secondary metabolism and morphological development in S. coelicolor.

Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli.

The alaE gene in Escherichia coli encodes an l-alanine exporter that catalyzes the active export of l-alanine using proton electrochemical potential. In our previous study, alaE expression was shown to increase in the presence of l-alanyl-l-alanine (Ala-Ala). In this study, the global regulator leucine-responsive regulatory protein (Lrp) was identified as an activator of the alaE gene. A promoter less ß-galactosidase gene was fused to an alaE upstream region (240 nucleotides). Cells that were lacZ-deficient and harbored this reporter plasmid showed significant induction of ß-galactosidase activity (approximately 17-fold) in the presence of 6 mM l-alanine, l-leucine, and Ala-Ala. However, a reporter plasmid possessing a smaller alaE upstream region (180 nucleotides) yielded transformants with strikingly low enzyme activity under the same conditions. In contrast, lrp-deficient cells showed almost no ß-galactosidase induction, indicating that Lrp positively regulates alaE expression. We next performed an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay using purified hexahistidine-tagged Lrp (Lrp-His). Consequently, we found that Lrp-His binds to the alaE upstream region spanning nucleotide -161 to -83 with a physiologically relevant affinity (apparent KD, 288.7 ± 83.8 nM). Furthermore, the binding affinity of Lrp-His toward its cis-element was increased by l-alanine and l-leucine, but not by Ala-Ala and d-alanine. Based on these results, we concluded that the gene expression of the alaE is regulated by Lrp in response to intracellular levels of l-alanine, which eventually leads to intracellular homeostasis of l-alanine concentrations.