Global Regulator Lrp Research Articles

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.

Pleiotropic Clostridioides difficile Cyclophilin PpiB Controls Cysteine-Tolerance, Toxin Production, the Central Metabolism and Multiple Stress Responses.

The Gram-positive pathogen Clostridioides difficile is the main bacterial agent of nosocomial antibiotic associated diarrhea. Bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) are well established modulators of virulence that influence the outcome of human pathologies during infections. Here, we present the first interactomic network of the sole cyclophilin-type PPIase of C. difficile (CdPpiB) and show that it has diverse interaction partners including major enzymes of the amino acid-dependent energy (LdhA, EtfAB, Had, Acd) and the glucose-derived (Fba, GapA, Pfo, Pyk, Pyc) central metabolism. Proteins of the general (UspA), oxidative (Rbr1,2,3, Dsr), alkaline (YloU, YphY) and cold shock (CspB) response were found bound to CdPpiB. The transcriptional (Lrp), translational (InfC, RFF) and folding (GroS, DnaK) control proteins were also found attached. For a crucial enzyme of cysteine metabolism, O-acetylserine sulfhydrylase (CysK), the global transcription regulator Lrp and the flagellar subunit FliC, these interactions were independently confirmed using a bacterial two hybrid system. The active site residues F50, F109, and F110 of CdPpiB were shown to be important for the interaction with the residue P87 of Lrp. CysK activity after heat denaturation was restored by interaction with CdPpiB. In accordance, tolerance toward cell wall stress caused by the exposure to amoxicillin was reduced. In the absence of CdPpiB, C. difficile was more susceptible toward L-cysteine. At the same time, the cysteine-mediated suppression of toxin production ceased resulting in higher cytotoxicity. In summary, the cyclophilin-type PPIase of C. difficile (CdPpiB) coordinates major cellular processes via its interaction with major regulators of transcription, translation, protein folding, stress response and the central metabolism.

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.

The Global Regulators Lrp, LeuO, and HexA Control Secondary Metabolism in Entomopathogenic Bacteria.

Photorhabdus luminescens TTO1 and Xenorhabdus nematophila HGB081 are insect pathogenic bacteria and producers of various structurally diverse bioactive natural products. In these entomopathogenic bacteria we investigated the role of the global regulators Lrp, LeuO, and HexA in the production of natural products. Lrp is a general activator of natural product biosynthesis in X. nematophila and for most compounds in TTO1. Microarray analysis confirmed these results in X. nematophila and enabled the identification of additional biosynthesis gene clusters (BGC) regulated by Lrp. Moreover, when promoters of two X. nematophila BGC were analyzed, transcriptional activation by Lrp was observed. In contrast, LeuO in X. nematophila and P. luminescens has both repressing and activating features, depending on the natural product examined. Furthermore, heterologous overexpression of leuO from X. nematophila in the closely related Xenorhabdus szentirmaii resulted in overproduction of several natural products including novel compounds. The presented findings could be of importance for establishing a tool for overproduction of secondary metabolites and subsequent identification of novel compounds.

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 β-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 β-galactosidase activity (approximately 17-fold) in the presence of 6mM 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 β-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±83.8nM). 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.

Enhancing the carbon flux and NADPH supply to increase L-isoleucine production in Corynebacterium glutamicum

Our previous work has shown that L-isoleucine production in Corynebacterium glutamicum IWJ001 could be increased by overexpressing ilvA1 encoding a feedback-resistant threonine dehydratase, ilvBN1 encoding a feedback-resistant acetohydroxy acid synthase, lrp encoding the global regulator Lrp, brnFE encoding the two-component export system BrnFE, or ppnk1 encoding NAD kinase. The main purpose of this study is to further increase the L-isoleucine production in C. glutamicum IWJ001 by overexpressing the above genes in various combinations. Several C. glutamicum strains IWJ001/pDXW-8-ppnk1-lrp-brnFE, IWJ001/pDXW-8-ilvBN1-ilvA1-lrp-brnFE, IWJ001/pDXW-8-ilvBN1-ilvA1-ppnk1, and IWJ001/pDXW-8-ppnk1-ilvBN1-ilvA1-lrp-brnFE were constructed, and L-isoleucine production and activities of several key enzymes in these strains were analyzed. Compared with the control strain IWJ001/pDXW-8, L-isoleucine production increased in all of the four strains. IWJ001/pDXW-8-ilvBN1-ilvA1-ppnk1 showed the highest L-isoleucine production and produced 32.3 g/L L-isoleucine in 72 h fed batch fermentation. The results indicate that L-isoleucine production in C. glutamicum could be increased by enhancing the carbon flux and NADPH supply in the biosynthetic pathway.

Increasingl-isoleucine production inCorynebacterium glutamicumby overexpressing global regulator Lrp and two-component export system BrnFE

To increase the l-isoleucine production in Corynebacterium glutamicum by overexpressing the global regulator Lrp and the two-component export system BrnFE. The brnFE operon and the lrp gene were cloned into the shuttle vector pDXW-8 individually or in combination. The constructed plasmids were transformed into an l-isoleucine-producing strain C. glutamicum JHI3-156, and the l-isoleucine production in these different strains was analysed and compared. More l-isoleucine was produced when only Lrp was expressed than when only BrnFE was expressed. Significant increase in l-isoleucine production was observed when Lrp and BrnFE were expressed in combination. Compared to the control strain, l-isoleucine production in JHI3-156/pDXW-8-lrp-brnFE increased 63% in flask cultivation, and the specific yield of l-isoleucine increased 72% in fed-batch fermentation. Both Lrp and BrnFE are important to enhance the l-isoleucine production in C. glutamicum. The results provide useful information to enhance l-isoleucine or other branched-chain amino acid production in C. glutamicum.

Rational Design ofEscherichia coliforl-Isoleucine Production

Metabolic engineering of Escherichia coli was performed to construct a 100% rationally engineered strain capable of overproducing L-isoleucine, an important branched-chain amino acid. The thrABC (encoding L-threonine biosynthetic enzymes), ilvA (encoding feedback-resistant threonine dehydratase), ilvIH (encoding feedback-resistant acetohydroxy acid synthase III), and ygaZH (encoding branched-chain amino acid exporter) genes were amplified by plasmid-based overexpression. The ilvCED (encoding L-isoleucine biosynthetic enzymes) and lrp (encoding global regulator Lrp) genes were also amplified by chromosomal promoter replacement in order to further increase the flux toward L-isoleucine. The final engineered E. coli strain was able to produce 9.46 g/L of L-isoleucine with a yield of 0.14 g/g of glucose by fed-batch culture. The overall design principles described here for the production of highly regulated product should be useful in designing strains for the production of other similar bioproducts.

Regulating the regulator: MicF RNA controls expression of the global regulator Lrp

Studies on the regulatory RNA MicF in Enterobacteriaceae reveal a pivotal role in gene regulation. Multiple target gene mRNAs were identified and, importantly, MicF RNA regulates the expression of the global regulatory gene lrp (Holmqvist et al., 2012; Corcoran et al., 2012). Thus MicF RNA is a central factor in a regulatory network that regulates bacterial cell physiology.

Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA

MicF is a textbook example of a small regulatory RNA (sRNA) that acts on a trans-encoded target mRNA through imperfect base pairing. Discovery of MicF as a post-transcriptional repressor of the major Escherichia coli porin OmpF established the paradigm for a meanwhile common mechanism of translational inhibition, through antisense sequestration of a ribosome binding site. However, whether MicF regulates additional genes has remained unknown for almost three decades. Here, we have harnessed the new superfolder variant of GFP for reporter-gene fusions to validate newly predicted targets of MicF in Salmonella. We show that the conserved 5' end of MicF acts by seed pairing to repress the mRNAs of global transcriptional regulator Lrp, and periplasmic protein YahO, while a second targeting region is also required to regulate the mRNA of the lipid A-modifying enzyme LpxR. Interestingly, MicF targets lpxR at both the ribosome binding site and deep within the coding sequence. MicF binding in the coding sequence of lpxR decreases mRNA stability through exacerbating the use of a native RNase E site proximal to the short MicF-lpxR duplex. Altogether, this study assigns the classic MicF sRNA to the growing class of Hfq-associated regulators that use diverse mechanisms to impact multiple loci.

A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp

Roughly 10% of all genes in Escherichia coli are controlled by the global transcription factor Lrp, which responds to nutrient availability. Bioinformatically, we identified lrp as one of several putative targets for the sRNA MicF, which is transcriptionally downregulated by Lrp. Deleting micF results in higher Lrp levels, while overexpression of MicF inhibits Lrp synthesis. This effect is by antisense; mutations in the predicted interaction region relieve MicF-dependent repression of Lrp synthesis, and regulation is restored by compensatory mutations. In vitro, MicF sterically interferes with initiation complex formation and inhibits lrp mRNA translation. In vivo, MicF indirectly activates genes in the Lrp regulon by repressing Lrp, and causes severely impaired growth in minimal medium, a phenotype characteristic of lrp deletion strains. The double negative feedback between MicF and Lrp may promote a switch for adequate Lrp-dependent adaptation to nutrient availability. Lrp adds to the growing list of transcription factors that are targeted by sRNAs, thus indicating that perhaps the majority of all bacterial genes may be directly or indirectly controlled by sRNAs.

The TonB3 System in the Human Pathogen Vibrio vulnificus Is under the Control of the Global Regulators Lrp and Cyclic AMP Receptor Protein

TonB systems transduce the proton motive force of the cytoplasmic membrane to energize substrate transport through a specific TonB-dependent transporter across the outer membrane. Vibrio vulnificus, an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels, possesses three TonB systems. While the TonB1 and TonB2 systems are iron regulated, the TonB3 system is induced when the bacterium grows in human serum. In this work we have determined the essential roles of the leucine-responsive protein (Lrp) and cyclic AMP (cAMP) receptor protein (CRP) in the transcriptional activation of this system. Whereas Lrp shows at least four very distinctive DNA binding regions spread out from position -59 to -509, cAMP-CRP binds exclusively in a region centered at position -122.5 from the start point of the transcription. Our results suggest that both proteins bind simultaneously to the region closer to the RNA polymerase binding site. Importantly, we report that the TonB3 system is induced not only by serum but also during growth in minimal medium with glycerol as the sole carbon source and low concentrations of Casamino Acids. In addition to catabolite repression by glucose, l-leucine acts by inhibiting the binding of Lrp to the promoter region, hence preventing transcription of the TonB3 operon. Thus, this TonB system is under the direct control of two global regulators that can integrate different environmental signals (i.e., glucose starvation and the transition between "feast" and "famine"). These results shed light on new mechanisms of regulation for a TonB system that could be widespread in other organisms.

Pervasive post‐transcriptional control of genes involved in amino acid metabolism by the Hfq‐dependent GcvB small RNA

GcvB is one of the most highly conserved Hfq-associated small RNAs in Gram-negative bacteria and was previously reported to repress several ABC transporters for amino acids. To determine the full extent of GcvB-mediated regulation in Salmonella, we combined a genome-wide experimental approach with biocomputational target prediction. Comparative pulse expression of wild-type versus mutant sRNA variants revealed that GcvB governs a large post-transcriptional regulon, impacting ~1% of all Salmonella genes via its conserved G/U-rich domain R1. Complementary predictions of C/A-rich binding sites in mRNAs and gfp reporter fusion experiments increased the number of validated GcvB targets to more than 20, and doubled the number of regulated amino acid transporters. Unlike the previously described targeting via the single R1 domain, GcvB represses the glycine transporter CycA by exceptionally redundant base-pairing. This novel ability of GcvB is focused upon the one target that could feedback-regulate the glycine-responsive synthesis of GcvB. Several newly discovered mRNA targets involved in amino acid metabolism, including the global regulator Lrp, question the previous assumption that GcvB simply acts to limit unnecessary amino acid uptake. Rather, GcvB rewires primary transcriptional control circuits and seems to act as a distinct regulatory node in amino acid metabolism.

Identification of ArgP and Lrp as transcriptional regulators of lysP, the gene encoding the specific lysine permease of Escherichia coli.

Expression of lysP, which encodes the lysine-specific transporter LysP in Escherichia coli, is regulated by the concentration of exogenous available lysine. In this study, the LysR-type transcriptional regulator ArgP was identified as the activator of lysP expression. At lysine concentrations higher than 25 μM, lysP expression was shut off and phenocopied an argP deletion mutant. Purified ArgP-His(6) bound to the lysP promoter/control region at a sequence containing a conserved T-N(11)-A motif. Its affinity increased in the presence of lysine but not in the presence of the other known coeffector, arginine. In vivo data suggest that lysine-loaded ArgP and arginine-loaded ArgP compete at the lysP promoter. We propose that lysine-loaded ArgP prevents lysP transcription at the promoter clearance step, as described for the lysine-dependent regulation of argO (R. S. Laishram and J. Gowrishankar, Genes Dev. 21:1258-1272, 2007). The global regulator Lrp also bound to the lysP promoter/control region. An lrp mutant exhibited reduced lysP expression in the absence of external lysine. These results indicate that ArgP is a major regulator of lysP expression but that Lrp modulates lysP transcription under lysine-limiting conditions.

The CRISPR/Cas Immune System Is an Operon Regulated by LeuO, H-NS, and Leucine-Responsive Regulatory Protein in Salmonella enterica Serovar Typhi

Prokaryotes have developed multiple strategies to survive phage attack and invasive DNA. Recently, a novel genetic program denominated the CRISPR/Cas system was demonstrated to have a role in these biological processes providing genetic immunity. This defense mechanism is widespread in the Archaea and Bacteria, suggesting an ancient origin. In the last few years, progress has been made regarding the functionality of the CRISPR/Cas system; however, many basic aspects of the system remain unknown. For instance, there are few studies about the conditions and regulators involved in its transcriptional control. In this work, we analyzed the transcriptional organization of the CRISPR/Cas system as well as the positive and negative regulators involved in its genetic expression in Salmonella enterica serovar Typhi. The results obtained show that in S. Typhi the CRISPR/Cas system is a LeuO-dependent operon silenced by the global regulator LRP, in addition to the previously known nucleoid-associated protein H-NS; both LRP and H-NS bind upstream and downstream of the transcriptional start site of casA. In this study, relevant nucleotides of the casA regulatory region that mediate its LeuO transcriptional activation were identified. Interestingly, specific growth conditions (N-minimal medium) were found for the LeuO-independent expression of the CRISPR/Cas system in S. Typhi. Thus, our work provides evidence that there are multiple modulators involved in the genetic expression of this immune system in S. Typhi IMSS-1.

Escherichia coli W as a new platform strain for the enhanced production of L‐Valine by systems metabolic engineering

A less frequently employed Escherichia coli strain W, yet possessing useful metabolic characteristics such as less acetic acid production and high L-valine tolerance, was metabolically engineered for the production of L-valine. The ilvA gene was deleted to make more pyruvate, a key precursor for L-valine, available for enhanced L-valine biosynthesis. The lacI gene was deleted to allow constitutive expression of genes under the tac or trc promoter. The ilvBN(mut) genes encoding feedback-resistant acetohydroxy acid synthase (AHAS) I and the L-valine biosynthetic ilvCED genes encoding acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and branched chain amino acid aminotransferase, respectively, were amplified by plasmid-based overexpression. The global regulator Lrp and L-valine exporter YgaZH were also amplified by plasmid-based overexpression. The engineered E. coli W (ΔlacI ΔilvA) strain overexpressing the ilvBN(mut) , ilvCED, ygaZH, and lrp genes was able to produce an impressively high concentration of 60.7 g/L L-valine by fed-batch culture in 29.5 h, resulting in a high volumetric productivity of 2.06 g/L/h. The most notable finding is that there was no other byproduct produced during L-valine production. The results obtained in this study suggest that E. coli W can be a good alternative to Corynebacterium glutamicum and E. coli K-12, which have so far been the most efficient L-valine producer. Furthermore, it is expected that various bioproducts including other amino acids might be more efficiently produced by this revisited platform strain of E. coli.

Competitive activation of the Escherichia coli argO gene coding for an arginine exporter by the transcriptional regulators Lrp and ArgP

In vivo and in vitro analyses indicate that transcription of the argO gene coding for an arginine exporter is regulated by the global transcriptional regulator Lrp, an effect that went by unnoticed in previous genome-scale screenings of the Lrp regulatory network in Escherichia coli. Lrp activates the argO promoter fourfold; exogenous leucine antagonizes, but does not completely eliminate this effect. Activation by Lrp interferes with the previously demonstrated activation of the argO promoter by ArgP. This interference results from the mutual inhibitory binding of the two activators to overlapping targets. As a consequence, each regulator acts more potently in the absence of the other. Dimeric Lrp binds cooperatively to at least three regularly spaced semi-palindromic binding sites. Leucine reduces complex formation approximately twofold but concomitantly enhances the cooperativity of the binding. Footprinting data suggest a severe Lrp-induced deformation of the argO control region. Combined, the effector modulated activation of argO transcription by ArgP and Lrp must ensure an adapted and fine-tuned synthesis of the transporter in response to environmental conditions. The repertoire of bacterial transcription regulation mechanisms is vast, but the competitive activation of a single promoter by two activator proteins as described here appears to be rare.

Xenorhabdus nematophila lrhAIs Necessary for Motility, Lipase Activity, Toxin Expression, and Virulence inManduca sextaInsects

The gram-negative insect pathogen Xenorhabdus nematophila possesses potential virulence factors including an assortment of toxins, degradative enzymes, and regulators of these compounds. Here, we describe the lysR-like homolog A (lrhA) gene, a gene required by X. nematophila for full virulence in Manduca sexta insects. In several other gram-negative bacteria, LrhA homologs are transcriptional regulators involved in the expression (typically repression) of virulence factors. Based on phenotypic and genetic evidence, we report that X. nematophila LrhA has a positive effect on transcription and expression of certain potential virulence factors, including a toxin subunit-encoding gene, xptD1. Furthermore, an lrhA mutant lacks in vitro lipase activity and has reduced swimming motility compared to its wild-type parent. Quantitative PCR revealed that transcript levels of flagellar genes, a lipase gene, and xptD1 were significantly lower in the lrhA mutant than in the wild type. In addition, lrhA itself is positively regulated by the global regulator Lrp. This work establishes a role for LrhA as a vital component of a regulatory hierarchy necessary for X. nematophila pathogenesis and expression of surface-localized and secreted factors. Future research is aimed at identifying and characterizing virulence factors within the LrhA regulon.

Metabolic engineering of Escherichia coli for the production of l -valine based on transcriptome analysis and in silico gene knockout simulation

The L-valine production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement based on transcriptome analysis and gene knockout simulation of the in silico genome-scale metabolic network. Feedback inhibition of acetohydroxy acid synthase isoenzyme III by L-valine was removed by site-directed mutagenesis, and the native promoter containing the transcriptional attenuator leader regions of the ilvGMEDA and ilvBN operon was replaced with the tac promoter. The ilvA, leuA, and panB genes were deleted to make more precursors available for L-valine biosynthesis. This engineered Val strain harboring a plasmid overexpressing the ilvBN genes produced 1.31 g/liter L-valine. Comparative transcriptome profiling was performed during batch fermentation of the engineered and control strains. Among the down-regulated genes, the lrp and ygaZH genes, which encode a global regulator Lrp and L-valine exporter, respectively, were overexpressed. Amplification of the lrp, ygaZH, and lrp-ygaZH genes led to the enhanced production of L-valine by 21.6%, 47.1%, and 113%, respectively. Further improvement was achieved by using in silico gene knockout simulation, which identified the aceF, mdh, and pfkA genes as knockout targets. The VAMF strain (Val DeltaaceF Deltamdh DeltapfkA) overexpressing the ilvBN, ilvCED, ygaZH, and lrp genes was able to produce 7.55 g/liter L-valine from 20 g/liter glucose in batch culture, resulting in a high yield of 0.378 g of L-valine per gram of glucose. These results suggest that an industrially competitive strain can be efficiently developed by metabolic engineering based on combined rational modification, transcriptome profiling, and systems-level in silico analysis.

The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdus nematophila

Xenorhabdus nematophila is a Gram-negative bacterium that leads both pathogenic and mutualistic lifestyles. In this study, we examine the role of Lrp, the leucine-responsive regulatory protein, in regulating both of these lifestyles. lrp mutants have attenuated virulence towards Manduca sexta insects and are defective in suppression of both cellular and humoral insect immunity. In addition, an lrp mutant is deficient in initiating colonization of and growth within mutualistic host nematodes. Furthermore, nematodes reared on lrp mutant lawns exhibit decreased overall numbers of nematode progeny. To our knowledge, this is the first demonstration of virulence attenuation associated with an lrp mutation in any bacterium, as well as the first report of a factor involved in both X. nematophila symbioses. Protein profiles of wild-type and mutant cells indicate that Lrp is a global regulator of expression in X. nematophila, affecting approximately 65% of 290 proteins. We show that Lrp binds to the promoter regions of genes known to be involved in basic metabolism, mutualism and pathogenesis, demonstrating that the regulation of at least some host interaction factors is likely direct. Finally, we demonstrate that Lrp influences aspects of X. nematophila phenotypic variation, a spontaneous process that occurs during prolonged growth in stationary phase.