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.

Dynamic Control of 4-Hydroxyisoleucine Biosynthesis by Modified l-Isoleucine Biosensor in Recombinant Corynebacterium glutamicum.

4-Hydroxyisoleucine (4-HIL), a promising drug for treating diabetes, can be synthesized from the self-produced l-isoleucine (Ile) by expressing the Ile dioxygenase gene ido in Corynebacterium glutamicum. However, the requirement of three substrates, Ile, α-ketoglutarate (α-KG), and O2, makes such de novo biosynthesis difficult to be fulfilled effectively under static engineering conditions. In this study, dynamic control of 4-HIL biosynthesis by the Ile biosensor Lrp-PbrnFE was researched. The native PbrnFE promoter of natural Ile biosensor was still weak even under Ile induction. Through tetA dual genetic selection, several modified stronger PbrnFEN promoters were obtained from the synthetic library of the Ile biosensor. Dynamic regulation of ido expression by modified Ile biosensors increased the 4-HIL titer from 24.7 mM to 28.9-74.4 mM. The best strain ST04 produced even a little more 4-HIL than the static strain SN02 overexpressing ido by the strong PtacM promoter (69.7 mM). Further dynamic modulation of α-KG supply in ST04 by expressing different PbrnFEN-controlled odhI decreased the 4-HIL production but increased the l-glutamate or Ile accumulation. However, synergistic modulation of α-KG supply and O2 supply in ST04 by different combinations of PbrnFEN-odhI and PbrnFEN-vgb improved the 4-HIL production significantly, and the highest titer (135.3 mM) was obtained in ST17 strain regulating all the three genes by PbrnFE7. This titer was higher than those of all the static metabolic engineered C. glutamicum strains ever constructed. Therefore, dynamic regulation by modified Ile biosensor is a predominant strategy for enhancing 4-HIL de novo biosynthesis in C. glutamicum.

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.

Transcriptional regulation of a leucine-responsive regulatory protein for directly controlling lincomycin biosynthesis in Streptomyces lincolnensis.

Leucine-responsive regulatory proteins (Lrps) are a family of transcription factors involved in diverse biological processes in bacteria. So far, molecular mechanism of Lrps for regulating antibiotics biosynthesis in actinomycetes remains largely unexplored. This study, for the first time in Streptomyces lincolnensis, identified an Lrp (named as SLCG_Lrp) associated with lincomycin production. SLCG_Lrp was validated to be a positive regulator for lincomycin biosynthesis by directly stimulating transcription of two structural genes (lmbA and lmbV), three resistance genes (lmrA, lmrB and lmrC), and a regulatory gene (lmbU) within the lincomycin biosynthetic gene (lin) cluster. SLCG_Lrp was transcriptionally self-inhibited and triggered the expression of its adjacent gene SLCG_3127 encoding a LysE superfamily protein. Further, the binding site of SLCG_Lrp in the intergenic region of SLCG_3127 and SLCG_Lrp was precisely identified. Inactivation of SLCG_3127 in S. lincolnensis resulted in yield improvement of lincomycin, which was caused by intracellular accumulation of proline and cysteine. Arginine and phenylalanine were identified as specific regulatory ligands, respectively, to reduce and promote DNA-binding affinity of SLCG_Lrp. We further found that SLCG_Lrp was directly repressed by SLCG_2919, the first identified transcription factor outside lin cluster for lincomycin production. Therefore, our findings revealed SLCG_Lrp-mediated transcriptional regulation of lincomycin biosynthesis. This study extends the understanding of molecular mechanisms underlying lincomycin biosynthetic regulation.

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 Leucine-Responsive Regulatory Protein Lrp Participates in Virulence Regulation Downstream of Small RNA ArcZ in Erwinia amylovora.

Erwinia amylovora causes the devastating fire blight disease of apple and pear trees. During systemic infection of host trees, pathogen cells must rapidly respond to changes in their environment as they move through different host tissues that present distinct challenges and sources of nutrition. Growing evidence indicates that small RNAs (sRNAs) play an important role in disease progression as posttranscriptional regulators. The sRNA ArcZ positively regulates the motility phenotype and transcription of flagellar genes in E. amylovora Ea1189 yet is a direct repressor of translation of the flagellar master regulator, FlhD. We utilized transposon mutagenesis to conduct a forward genetic screen and identified suppressor mutations that increase motility in the Ea1189ΔarcZ mutant background. This enabled us to determine that the mechanism of transcriptional activation of the flhDC mRNA by ArcZ is mediated by the leucine-responsive regulatory protein, Lrp. We show that Lrp contributes to expression of virulence and several virulence-associated traits, including production of the exopolysaccharide amylovoran, levansucrase activity, and biofilm formation. We further show that Lrp is regulated posttranscriptionally by ArcZ through destabilization of lrp mRNA. Thus, ArcZ regulation of FlhDC directly and indirectly through Lrp forms an incoherent feed-forward loop that regulates levansucrase activity and motility as outputs. This work identifies Lrp as a novel participant in virulence regulation in E. amylovora and places it in the context of a virulence-associated regulatory network.IMPORTANCE Fire blight disease continues to plague the commercial production of apples and pears despite more than a century of research into disease epidemiology and disease control. The causative agent of fire blight, Erwinia amylovora coordinates turning on or off specific virulence-associated traits at the appropriate time during disease development. The development of novel control strategies requires an in-depth understanding of E. amylovora regulatory mechanisms, including regulatory control of virulence-associated traits. This study investigates how the small RNA ArcZ regulates motility at the transcriptional level and identifies the transcription factor Lrp as a novel participant in the regulation of several virulence-associated traits. We report that ArcZ and Lrp together affect key virulence-associated traits through integration of transcriptional and posttranscriptional mechanisms. Further understanding of the topology of virulence regulatory networks can uncover weak points that can subsequently be exploited to control E. amylovora.

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.

Engineering of leucine-responsive regulatory protein improves spiramycin and bitespiramycin biosynthesis.

BACKGROUND: Bitespiramycin (BT) is produced by recombinant spiramycin (SP) producing strain Streptomyces spiramyceticus harboring a heterologous 4″-O-isovaleryltransferase gene (ist). Exogenous L-Leucine (L-Leu) could improve the production of BT. The orf2 gene found from the genomic sequence of S. spiramyceticus encodes a leucine-responsive regulatory protein (Lrp) family regulator named as SSP_Lrp. The functions of SSP_Lrp and L-Leu involved in the biosynthesis of spiramycin (SP) and BT were investigated in S. spiramyceticus. RESULTS: SSP_Lrp was a global regulator directly affecting the expression of three positive regulatory genes, bsm23, bsm42 and acyB2, in SP or BT biosynthesis. Inactivation of SSP_Lrp gene in S. spiramyceticus 1941 caused minor increase of SP production. However, SP production of the ΔSSP_Lrp-SP strain containing an SSP_Lrp deficient of putative L-Leu binding domain was higher than that of S. spiramyceticus 1941 (476.2 ± 3.1 µg/L versus 313.3 ± 25.2 µg/L, respectively), especially SP III increased remarkably. The yield of BT in ΔSSP_Lrp-BT strain was more than twice than that in 1941-BT. The fact that intracellular concentrations of branched-chain amino acids (BCAAs) decreased markedly in the ΔSSP_Lrp-SP demonstrated increasing catabolism of BCAAs provided more precursors for SP biosynthesis. Comparative analysis of transcriptome profiles of the ΔSSP_Lrp-SP and S. spiramyceticus 1941 found 12 genes with obvious differences in expression, including 6 up-regulated genes and 6 down-regulated genes. The up-regulated genes are related to PKS gene for SP biosynthesis, isoprenoid biosynthesis, a Sigma24 family factor, the metabolism of aspartic acid, pyruvate and acyl-CoA; and the down-regulated genes are associated with ribosomal proteins, an AcrR family regulator, and biosynthesis of terpenoid, glutamate and glutamine. CONCLUSION: SSP_Lrp in S. spiramyceticus was a negative regulator involved in the SP and BT biosynthesis. The deletion of SSP_Lrp putative L-Leu binding domain was advantageous for production of BT and SP, especially their III components.

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.