Arginine Repressor Research Articles

Symmetry of a partially-ligated state maintained by dynamics in a negatively cooperative system

Symmetry was a key concept underlying the MWC model for allostery advanced in 1965 by Monod, Wyman, and Changeux. The reciprocal interactions of symmetrically-arranged identical subunits were proposed to stabilize multimeric assemblies together with the free energy from bound ligands that progressively favor a monomer-like state. Structural symmetry of subunits was assumed to be maintained in the partially-ligated states, even if ligand placement itself is not symmetric. Partially-ligated states can be populated sufficiently for experimental study only in negatively cooperative systems, which were not considered in the MWC model. The work reported here uses 1H, 13C, 15N, and 19F NMR to evaluate the structural symmetry of the hexameric arginine repressor of E. coli, a negatively cooperative system, with a single bound L-arginine ligand. The analysis indicates that the singly-ligated hexamer maintains structural symmetry as probed by these four NMR nuclei. The results are consistent with earlier molecular dynamics simulations suggesting that the global dynamics of the singly-ligated assembly are harnessed to maintain structural symmetry. The results extend MWC symmetry concepts to this negatively cooperative system, and indicate a role for global dynamics in allostery.

Metabolic reprogramming in the food-borne pathogen Listeria monocytogenes as a critical defence against acid stress.

The ability to sense and respond effectively to acidic stress is important for microorganisms to survive and proliferate in fluctuating environments. As specific metabolic activities can serve to buffer the cytoplasmic pH, microorganisms rewire their metabolism to favour these reactions and thereby mitigate acid stress. The orally acquired pathogen Listeria monocytogenes exploits alternative metabolic activities to overcome the acidic stress encountered in the human stomach or food products. In this minireview, we discuss the metabolic processes in L. monocytogenes that mitigate acid stress, with an emphasis on the proton-depleting reactions, including glutamate decarboxylation, arginine/agmatine deimination, and fermentative acetoin production. We also summarize the recent findings on regulatory mechanisms that control the expression of genes that are responsible for these metabolic activities, including the general stress response regulator SigB, arginine repressor ArgR, and the recently discovered RofA-like transcriptional regulatory GadR. We further discuss the importance of this metabolic reprogramming in the context of food products and within the host. Finally, we highlight some outstanding challenges in the field, including an understanding of acid-sensing mechanisms, the role of intraspecies heterogeneity in acid resistance, and how a fundamental understanding of acid stress response can be exploited for food formulation to improve food safety and reduce food waste.

Metabolic engineering of Thermoanaerobacterium aotearoense strain SCUT27 for biofuels production from sucrose and molasses

BackgroundSucrose-rich sugarcane trash surpasses 28 million tons globally per year. Effective biorefinery systems could convert these biomasses to bioproducts, such as bioethanol from sugarcane sucrose in Brazil. Thermophilic microbes for biofuels have attracted great attention due to their higher fermentation temperature and wide substrate spectrum. However, few thermophiles using sucrose or molasses for biofuels production was reported. Thermoanaerobacterium aotearoense SCUT27 has been considered as an efficient ethanol producer, but it cannot directly utilize sucrose. In this study, various sucrose metabolic pathways were introduced and analyzed in Thermoanaerobaterium.ResultsThe sucrose-6-phosphate hydrolase (scrB), which was from a screened strain Thermoanaerobacterium thermosaccharolyticum G3-1 was overexpressed in T. aotearoense SCUT27 and endowed this strain with the ability to utilize sucrose. In addition, overexpression of the sucrose-specific PTS system (scrA) from Clostridium acetobutylicum accelerated the sucrose transport. To strengthen the alcohols production and substrates metabolism, the redox-sensing transcriptional repressor (rex) in T. aotearoense was further knocked out. Moreover, with the gene arginine repressor (argR) deleted, the ethanologenic mutant P8S10 showed great inhibitors-tolerance and finally accumulated ~ 34 g/L ethanol (a yield of 0.39 g/g sugars) from pretreated cane molasses in 5 L tank by fed-batch fermentation. When introducing butanol synthetic pathway, 3.22 g/L butanol was produced by P8SB4 with a yield of 0.44 g alcohols/g sugars at 50℃. This study demonstrated the potential application of T. aotearoense SCUT27 for ethanol and butanol production from low cost cane molasses.ConclusionsOur work provided strategies for sucrose utilization in thermophiles and improved biofuels production as well as stress tolerances of T. aotearoense SCUT27, demonstrating the potential application of the strain for cost-effective biofuels production from sucrose-based feedstocks.

Engineering Thermoanaerobacterium aotearoense SCUT27 with the deficiency of a hypothetic protein regulated by ArgR1864 for enhanced ethanol production from lignocellulosic hydrolysates

Arginine repressor (ArgR1864) is a multifunctional regulator in Thermoanaerobacterium aotearoense SCUT27 (SCUT27). For better understanding its function in SCUT27, the transcriptome of SCUT27/ΔargR1864 under xylose was further investigated. 2014 (V518_2014) denoted as a hypothetic protein gene was mined, which played important roles on xylose metabolism and carbon metabolic flux distribution in SCUT27. In 2014 deletion mutant, no lactic acid could be produced along with enhanced ethanol formation. Same changes were also observed in SCUT27/ΔargR1864. ArgR1864 is the direct regulator of 2014 identified by electrophoretic mobility shift assays (EMSA). When 2014 was complemented in SCUT27/ΔargR1864, the fermentation traits of SCUT27/ΔargR1864/2014 were returned to wild-type levels, suggesting that the improved xylose utilization and ethanol formation of SCUT27/ΔargR1864 were attributed to the low expression of 2014. When cultured under sorghum stalk, corn cob and wheat straw hydrolysates, SCUT27/Δ2014 also showed the excellent capability for sugar utilization and ethanol production with the consumption rate of xylose and glucose increased by 20%–37.93 and 64.29%–190.91% separately, the ethanol production improved by 326.61%–547.89% and ethanol yield enhanced by 216.67%–337.50%. Thus, 2014, as a hypothetic protein gene, could be used as the new genetic reference to predict functional genes to engineer the microorganism for biofuel production from lignocellulosic hydrolysates.

AhrC Negatively Regulates Streptococcus mutans Arginine Biosynthesis.

ABSTRACTStreptococcus mutans is a primary cariogenic pathogen in humans. Arginine metabolism is required for bacterial growth. In S. mutans, however, the involvement of transcription factors in regulating arginine metabolism is unclear. The purpose of this study was to investigate the function and mechanism of ArgR family transcription factors in S. mutans. Here, we identified an ArgR (arginine repressor) family transcription factor named AhrC, which negatively regulates arginine biosynthesis and biofilm formation in S. mutans. The ahrC in-frame deletion strain exhibited slow growth and significantly increased intracellular arginine content. The strain overexpressing ahrC showed reduced intracellular arginine content, decreased biofilm biomass, reduced production of water-insoluble exopolysaccharides (EPS), and different biofilm structures. Furthermore, global gene expression profiles revealed differential expression levels of 233 genes in the ahrC-deficient strain, among which genes related to arginine biosynthesis (argJ, argB, argC, argD, argF, argG, argH) were significantly upregulated. In the ahrC overexpression strain, there are 89 differentially expressed genes, mostly related to arginine biosynthesis. The conserved DNA patterns bound by AhrC were identified by electrophoretic mobility shift assay (EMSA) and DNase I footprinting. In addition, the analysis of β-galactosidase activity showed that AhrC acted as a negative regulator. Taken together, our findings suggest that AhrC is an important transcription factor that regulates arginine biosynthesis gene expression and biofilm formation in S. mutans. These findings add new aspects to the complexity of regulating the expression of genes involved in arginine biosynthesis and biofilm formation in S. mutans.IMPORTANCE Arginine metabolism is essential for bacterial growth. The regulation of intracellular arginine metabolism in Streptococcus mutans, one of the major pathogens of dental caries, is unclear. In this study, we found that the transcription factor AhrC can directly and negatively regulate the expression of N-acetyl-gamma-glutamyl-phosphate reductase (argC), thus regulating arginine biosynthesis in S. mutans. In addition, the ahrC overexpression strain exhibited a significant decrease in biofilm and water-insoluble extracellular polysaccharides (EPS). This study adds new support to our understanding of the regulation of intracellular arginine metabolism in S. mutans.

The Arginine Repressor ArgR2 Controls Conjugated Linoleic Acid Biosynthesis by Activating the cla Operon in Lactiplantibacillus plantarum.

ABSTRACTCLA (conjugated linoleic acid) has attracted substantial attention due to its physiological functions, including regulating immunity, reducing obesity, and contributing to cancer suppression. In Lactiplantibacillus plantarum, CLA oleate hydratase (CLA-HY), CLA short-chain dehydrogenase (CLA-DH), and CLA acetoacetate decarboxylase (CLA-DC) catalyze the biotransformation of linoleic acid (LA) to CLA. However, the underlying transcriptional regulation mechanism of this pathway remains largely unknown. In this study, the potential transcriptional regulators that might bind to the cla promoter of L. plantarum AR195 were investigated by DNA pulldown. Interestingly, ArgR2, the transcriptional regulator of arginine metabolism, was identified as a potential regulator involved in the regulation of CLA biotransformation. Electrophoretic mobility shift assay (EMSA) and molecular interaction results demonstrated the specific binding of ArgR2 to the regulatory region of the cla operon. The knockout of argR2 led to the downregulation of cla-dh and cla-dc by 91% and 34%, respectively, resulting in a decline in the CLA yield by 14%. A segmental EMSA revealed that ArgR2 bound to three distinct sites in the cla regulatory region, and these binding sites were highly conserved and rich in AT. The regulatory mechanism of ArgR2 on CLA biosynthesis further expanded our knowledge of the regulatory mechanism of CLA biosynthesis in L. plantarum and laid the theoretical foundation for the production and application of CLA.IMPORTANCE CLA (conjugated linoleic acid) has received extensive attention owing to its important physiological functions. CLA from natural sources is far from meeting people’s demands. Lactic acid bacteria can efficiently synthesize cis-9,trans-11-CLA and trans-10,cis-12-CLA, which possess physiological activities. However, little is known about the regulatory mechanism. In this study, we identified that the biosynthesis of CLA in L. plantarum AR195 was transcriptionally regulated by the arginine biosynthesis regulatory protein ArgR2. The regulation mechanism of ArgR2 on CLA biosynthesis lays a theoretical foundation for the regulation of CLA synthesis and industrial production.

Abundance of integrons in halophilic bacteria.

Integrons are genetic platforms used for expressing open reading frames (ORFs) arranged in gene cassettes. Excision and integration of gene cassettes are controlled by their associated integron integrase (IntI). Using IntegronFinder software, we analyzed all complete halophilic genomes available in the HaloDom database, along with selected partial halophilic genomes. We identified 18 new complete bacterial integrons and 46 clusters of attC sites lacking a neighboring integron integrase (CALINs). Different classes of insertion sequences (ISs) were also identified within and near integrons and CALINs, with an abundance of IS1182 elements and different ISs that can presumably mobilize adjacent genomic structures. Different promoters of intI genes (PintI) showed nearby binding sites for arginine repressor (ArgR), raising the possibility that IntI expression and recombination activity are regulated by these proteins. Our findings revealed the existence of new integrons in halophilic bacteria with possible adaptive roles.

HYPER-SOLUBILIZING TRICALCIUM PHOSPHATE MUTANTS OF Klebsiella sp. GMD08

Klebsiella sp. GMD08 is one of the bacteria that has the capability to dissolve insoluble inorganic phosphate into soluble phosphate ion through their organic acid production. Transposon is a genetic element agent usually used to generate mutant through mutagenesis. Thus it can be used to identify the genetic functions involved in those phosphate solubilizing mechanisms. This research was conducted to identify the genes of Klebsiella sp. GMD08 involved in phosphate solubilization through sequence detection obtained from a hyper-solubilizing phosphate mutant library. Mutation was conducted by inserting mini-Tn5 transposon hosted in Escherichia coli S17-1/λpir [pBSL202] into Klebsiella sp. GMD08 chromosome by the filter mating conjugation method. Trans conjugant mutant candidates were then qualitatively and quantitatively analyzed for their solubilizing ability to dissolve tricalcium phosphate [Ca3(PO4)2] using pikovskaya medium. The organic acid characteristics of transconjugant mutants were detected using High-performance liquid chromatography (HPLC). Meanwhile, suspected genes involved in phosphate solubilizing were detected using the sequencing method obtained from the transposon insertion result. Nucleotide Basic Local Alignment Search Tool (nucleotide BLAST) was used to identify the nucleotide base sequence similarity with the database. The results showed that PB116 and PB122 were the two main transconjugant mutants obtained from transposon mutagenesis which had higher tricalcium phosphate dissolving ability. Gluconic acid was the main organic acid produced by Klebsiella sp. GMD08 phosphate solubilizing mechanism. Moreover, arginine repressor (ArgR) and malate dehydrogenase gene (mdh) coding gene were involved in Klebsiella sp. GMD08 phosphate solubilizing mechanism.

A new coumarin compound DCH combats methicillin-resistant Staphylococcus aureus biofilm by targeting arginine repressor.

Staphylococcus aureus infection is difficult to eradicate because of biofilm formation and antibiotic resistance. The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection necessitates the development of a new agent against bacterial biofilms. We report a new coumarin compound, termed DCH, that effectively combats MRSA in vitro and in vivo and exhibits potent antibiofilm activity without detectable resistance. Cellular proteome analysis suggests that the molecular mechanism of action of DCH involves the arginine catabolic pathway. Using molecular docking and binding affinity assays of DCH, and comparison of the properties of wild-type and ArgR-deficient MRSA strains, we demonstrate that the arginine repressor ArgR, an essential regulator of the arginine catabolic pathway, is the target of DCH. These findings indicate that DCH is a promising lead compound and validate bacterial ArgR as a potential target in the development of new drugs against MRSA biofilms.

Conserved Dynamic Mechanism of Allosteric Response to L-arg in Divergent Bacterial Arginine Repressors.

Hexameric arginine repressor, ArgR, is the feedback regulator of bacterial L-arginine regulons, and sensor of L-arg that controls transcription of genes for its synthesis and catabolism. Although ArgR function, as well as its secondary, tertiary, and quaternary structures, is essentially the same in E. coli and B. subtilis, the two proteins differ significantly in sequence, including residues implicated in the response to L-arg. Molecular dynamics simulations are used here to evaluate the behavior of intact B. subtilis ArgR with and without L-arg, and are compared with prior MD results for a domain fragment of E. coli ArgR. Relative to its crystal structure, B. subtilis ArgR in absence of L-arg undergoes a large-scale rotational shift of its trimeric subassemblies that is very similar to that observed in the E. coli protein, but the residues driving rotation have distinct secondary and tertiary structural locations, and a key residue that drives rotation in E. coli is missing in B. subtilis. The similarity of trimer rotation despite different driving residues suggests that a rotational shift between trimers is integral to ArgR function. This conclusion is supported by phylogenetic analysis of distant ArgR homologs reported here that indicates at least three major groups characterized by distinct sequence motifs but predicted to undergo a common rotational transition. The dynamic consequences of L-arg binding for transcriptional activation of intact ArgR are evaluated here for the first time in two-microsecond simulations of B. subtilis ArgR. L-arg binding to intact B. subtilis ArgR causes a significant further shift in the angle of rotation between trimers that causes the N-terminal DNA-binding domains lose their interactions with the C-terminal domains, and is likely the first step toward adopting DNA-binding-competent conformations. The results aid interpretation of crystal structures of ArgR and ArgR-DNA complexes.

Engineering Thermoanaerobacterium aotearoense SCUT27 with argR knockout for enhanced ethanol production from lignocellulosic hydrolysates

Although Thermoanaerobacterium aotearoense SCUT27 (SCUT27) could co-utilize glucose and xylose, the presence of glucose still repressed xylose catabolism. Arginine repressors (ArgRs) were involved in several key metabolic pathways and might be the global regulator. In SCUT27, three genes (V518_0585; V518_1870; V518_1864) were annotated as argR and only the deficiency of argR1864 could greatly improve the co-utilization of glucose and xylose, due to the enhanced activity of xylose isomerase, xylulokinase and the higher energy level. The metabolic flux of SCUT27/ΔargR1864 indicated that new carbon distribution had been re-established and the ethanol yield had increased by 82.95%, strains growth and acetate yield improved by ~35.91% without detectable lactate for the poor activity of lactate dehydrogenase. The improved concentration of ATP and NAD(H) in SCUT27/ΔargR1864 provided more energy to respond the stress, which enabled the mutant the better cell viability to utilize lignocellulosic hydrolysates for enhanced ethanol formation.

A single P115Q mutation modulates specificity in the Corynebacterium pseudotuberculosis arginine repressor

The arginine repressor (ArgR) regulates the expression of genes involved in arginine biosynthesis. Upon attaining a threshold concentration of arginine in the cytoplasm, the trimeric C-terminal domain of ArgR binds three arginines in a shallow surface cleft and subsequently hexamerizes forming a dimer of trimers containing six Arg co-repressor molecules which are buried at the subunit interfaces. The N-terminal domains of this complex bind to the DNA promoter thereby interrupting the transcription of the genes related to Arg biosynthesis.The crystal structures of the wild type and mutant Pro115Gln ArgR from Corynebacterium pseudotuberculosis determined at 1.7 Å demonstrate that a single amino acid substitution switches co-repressor specificity from Tyr to Arg.Molecular dynamics simulations indicate that the first step, i.e., the binding of the co-repressor, occurs in the trimeric state and that Pro115Gln ArgR preferentially binds Arg. It was also shown that, in Pro115 ArgR hexamers, the concomitant binding of sodium ions shifts selectivity to Tyr.Structural data combined with phylogenetic analyses of ArgR from C. pseudotuberculosis suggest that substitutions in the binding pocket at position 115 may alter its specificity for amino acids and that the length of the protein interdomain linker can provide further functional flexibility. These results support the existence of alternative ArgR regulatory mechanisms in this pathogenic bacterium.

Systematic metabolic pathway modification to boost L-ornithine supply for bacitracin production in Bacillus licheniformis DW2.

Bacitracin is a cyclic dodecyl peptide antibiotic that is an effective bacteriocide against Gram-positive and some Gram-negative bacteria. Bacitracin has been widely used as an antibacterial feed additive for livestock since it is not absorbed easily by the intestine and is easily excreted. Precursor availability has been proven to be one of the core factors for bacitracin production by many previous studies. In this study, we focused on enhancing the supply of the precursor amino acid L-ornithine to enhance bacitracin production by Bacillus licheniformis DW2 through systematic metabolic pathway modification. Several genes encoding rate-limiting enzymes for L-ornithine biosynthesis were episomally overexpressed, including argB, rocF, ppnk1, and ppnk2. The results showed that the overexpression of ppnK1 was the most effective for both L-ornithine and bacitracin biosynthesis. Secondly, the competitive branch pathways for L-ornithine biosynthesis were blocked, and the repressor was also deleted to boost L-ornithine biosynthesis. The results suggested that the deletion of genes proB and proJ to prevent proline biosynthesis and the disruption of the gene encoding the arginine repressor ArgR could enhance the intracellular concentration of L-ornithine by 49% and 2.1 times respectively, and the bacitracin production also increased accordingly by 6.6% and 11.9% respectively. Finally, several most effective efforts were combined to construct the optimal strain DW2ΔproBΔproJΔargR::ppnk1. In the optimal strain, the NADPH availability was improved and the expression levels of several essential genes for L-ornithine biosynthesis were upregulated, resulting in the enhancement of both L-ornithine and bacitracin production by 71.4% and 16.5% respectively. The final bacitracin production titer was 950 U/mL, which reached the level for industrial production.

Competitive Repression of the artPIQM Operon for Arginine and Ornithine Transport by Arginine Repressor and Leucine-Responsive Regulatory Protein in Escherichia coli.

Two out of the three major uptake systems for arginine in Escherichia coli are encoded by the artJ-artPIQM gene cluster. ArtJ is the high-affinity periplasmic arginine-specific binding protein (ArgBP-I), whereas artI encodes the arginine and ornithine periplasmic binding protein (AO). Both ArtJ and ArtI are supposed to combine with the inner membrane-associated ArtQMP2 transport complex of the ATP-binding cassette-type (ABC). Transcription of artJ is repressed by arginine repressor (ArgR) and the artPIQM operon is regulated by the transcriptional regulators ArgR and Leucine-responsive regulatory protein (Lrp). Whereas repression by ArgR requires arginine as corepressor, repression of PartP by Lrp is partially counteracted by leucine, its major effector molecule. We demonstrate that binding of dimeric Lrp to the artP control region generates four complexes with a distinct migration velocity, and that leucine has an effect on both global binding affinity and cooperativity in the binding. We identify the binding sites for Lrp in the artP control region, reveal interferences in the binding of ArgR and Lrp in vitro and demonstrate that the two transcription factors act as competitive repressors in vivo, each one being a more potent regulator in the absence of the other. This competitive behavior may be explained by the partial steric overlap of their respective binding sites. Furthermore, we demonstrate ArgR binding to an unusual position in the control region of the lrp gene, downstream of the transcription initiation site. From this unusual position for an ArgR-specific operator, ArgR has little direct effect on lrp expression, but interferes with the negative leucine-sensitive autoregulation exerted by Lrp. Direct arginine and ArgR-dependent repression of lrp could be observed with a 25-bp deletion mutant, in which the ArgR binding site was artificially moved to a position immediately downstream of the lrp transcription initiation site. This finding is reminiscent of a previous observation made for the carAB operon encoding carbamoylphosphate synthase, where ArgR bound in overlap with the downstream promoter P2 does not block transcription initiated 67 bp upstream at the P1 promoter, and further supports the hypothesis that ArgR does not act as an efficient roadblock.

Metabolic engineering of Corynebacterium glutamicum for improved L-arginine synthesis by enhancing NADPH supply.

Corynebacterium glutamicum SNK 118 was metabolically engineered with improved L-arginine titer. Considering the crucial role of NADPH level in L-arginine production, pntAB (membrane-bound transhydrogenase) and ppnK (NAD+ kinase) were co-expressed to increase the intracellular NADPH pool. Expression of pntAB exhibited significant effects on NADPH supply and L-arginine synthesis. Furthermore, argR and farR, encoding arginine repressor ArgR and transcriptional regulator FarR, respectively, were removed from the genome of C. glutamicum. The competitive branch pathway gene ldh was also deleted. Eventually, an engineered C. glutamicum JML07 was obtained for L-arginine production. Fed-batch fermentation in 5-L bioreactor employing strain JML07 allowed production of 67.01gL-1L-arginine with productivity of 0.89gL-1h-1 and yield of 0.35gg-1 glucose. This study provides a productive L-arginine fermentation strain and an effective cofactor manipulating strategy for promoting the biosynthesis of NADPH-dependent metabolites.

Systematic pathway engineering of Corynebacterium glutamicum S9114 for l-ornithine production

Backgroundl-Ornithine is a non-protein amino acid with extensive applications in medicine and the food industry. Currently, l-ornithine production is based on microbial fermentation, and few microbes are used for producing l-ornithine owing to unsatisfactory production titer.ResultsIn this study, Corynebacterium glutamicum S9114, a high glutamate-producing strain, was developed for l-ornithine production by pathway engineering. First, argF was deleted to block l-ornithine to citrulline conversion. To improve l-ornithine production, ncgl1221 encoding glutamate transporter, argR encoding arginine repressor, and putP encoding proline transporter were disrupted. This base strain was further engineered by attenuating oxoglutarate dehydrogenase to increase l-ornithine production. Plasmid-based overexpression of argCJBD operon and lysine/arginine transport protein LysE was tested to strengthen l-ornithine synthesis and transportation. This resulted in efficient l-ornithine production at a titer of 18.4 g/L.ConclusionThese results demonstrate the potential of Corynebacterium glutamicum S9114 for efficient l-ornithine production and provide new targets for strain development.

Shotgun proteomic analysis of Anaeroglobus geminatus

ABSTRACTObjectives: Anaeroglobus geminatus is a gram-negative species belonging to the family of Veillonellaceae and close associate to genus Megasphaera. The role of the species within dysbiotic microbial communities is not yet clear. However, this putative pathogen has been identified in a number of anaerobic infections, including periodontal diseases. The aim of this proteomic study was to analyse the global protein expression of A. geminatus.Methods: The whole proteome of A. geminatus CCUG 44773 strain was analysed by shotgun proteomics, using Orbitrap Fusion mass spectrometer.Results: A total of 1164 A. geminatus proteins were identified by this analysis, with an FDR of 2.2 % and 0.19 %, at peptide and protein level, respectively. Functional analysis of the identified proteins was performed using the Uniprot knowledge-base. Among those, 100 proteins were predicated to be transferase, 47 were hydrolases, and 41 were ligases, based on computational analysed functions. The top-scored pathways were “amino-acid biosynthesis” and “cofactor biosynthesis”, and each of these pathways included 20 proteins, such as arginine repressor and aspartokinase.Conclusion: According to our knowledge, this is the first study that gives an overview of the global proteome of A. geminatus and hopefully will help to unravel its role in anaerobic infections.

Listeria monocytogenes 10403S Arginine Repressor ArgR Finely Tunes Arginine Metabolism Regulation under Acidic Conditions.

Listeria monocytogenes is able to colonize human and animal intestinal tracts and to subsequently cross the intestinal barrier, causing systemic infection. For successful establishment of infection, L. monocytogenes must survive the low pH environment of the stomach. L. monocytogenes encodes a functional ArgR, a transcriptional regulator belonging to the ArgR/AhrC arginine repressor family. We aimed at clarifying the specific functions of ArgR in arginine metabolism regulation, and more importantly, in acid tolerance of L. monocytogenes. We showed that ArgR in the presence of 10 mM arginine represses transcription and expression of the argGH and argCJBDF operons, indicating that L. monocytogenes ArgR plays the classical role of ArgR/AhrC family proteins in feedback inhibition of the arginine biosynthetic pathway. Notably, transcription and expression of arcA (encoding arginine deiminase) and sigB (encoding an alternative sigma factor B) were also markedly repressed by ArgR when bacteria were exposed to pH 5.5 in the absence of arginine. However, addition of arginine enabled ArgR to derepress the transcription and expression of these two genes. Electrophoretic mobility shift assays showed that ArgR binds to the putative ARG boxes in the promoter regions of argC, argG, arcA, and sigB. Reporter gene analysis with gfp under control of the argG promoter demonstrated that ArgR was able to activate the argG promoter. Unexpectedly, deletion of argR significantly increased bacterial survival in BHI medium adjusted to pH 3.5 with lactic acid. We conclude that this phenomenon is due to activation of arcA and sigB. Collectively, our results show that L. monocytogenes ArgR finely tunes arginine metabolism through negative transcriptional regulation of the arginine biosynthetic operons and of the catabolic arcA gene in an arginine-independent manner during lactic acid-induced acid stress. ArgR also appears to activate catabolism as well as sigB transcription by anti-repression in an arginine-dependent way.

Tyrosine binding and promiscuity in the arginine repressor from the pathogenic bacterium Corynebacterium pseudotuberculosis

The arginine repressor (ArgR) regulates arginine biosynthesis in a number of microorganisms and consists of two domains interlinked by a short peptide; the N-terminal domain is involved in DNA binding and the C-terminal domain binds arginine and forms a hexamer made-up of a dimer of trimers. The crystal structure of the C-terminal domain of ArgR from the pathogenic Corynebacterium pseudotuberculosis determined at 1.9 Å resolution contains a tightly bound tyrosine at the arginine-binding site indicating hitherto unobserved promiscuity. Structural analysis of the binding pocket displays clear molecular adaptations to accommodate tyrosine binding suggesting the possible existence of an alternative regulatory process in this pathogenic bacterium.

Structural Analysis and Insights into the Oligomeric State of an Arginine-Dependent Transcriptional Regulator from Bacillus halodurans

The arginine repressor (ArgR) is an arginine-dependent transcription factor that regulates the expression of genes encoding proteins involved in the arginine biosynthesis and catabolic pathways. ArgR is a functional homolog of the arginine-dependent repressor/activator AhrC from Bacillus subtilis, and belongs to the ArgR/AhrC family of transcriptional regulators. In this research, we determined the structure of the ArgR (Bh2777) from Bacillus halodurans at 2.41 Å resolution by X-ray crystallography. The ArgR from B. halodurans appeared to be a trimer in a size exclusion column and in the crystal structure. However, it formed a hexamer in the presence of L-arginine in multi-angle light scattering (MALS) studies, indicating the oligomerization state was dependent on the presence of L-arginine. The trimeric structure showed that the C-terminal domains form the core, which was made by inter-subunit interactions mainly through hydrophobic contacts, while the N-terminal domains containing a winged helix-turn-helix DNA binding motif were arranged around the periphery. The arrangement of trimeric structure in the B. halodurans ArgR was different from those of other ArgR homologs previously reported. We finally showed that the B. halodurans ArgR has an arginine-dependent DNA binding property by an electrophoretic mobility shift assay.