scholarly journals Contribution of YthA, a PspC Family Transcriptional Regulator ofLactococcus lactisF44 Acid Tolerance and Nisin Yield: a Transcriptomic Approach

2018 ◽  
Vol 84 (6) ◽  
Author(s):  
Hao Wu ◽  
Jingui Liu ◽  
Sen Miao ◽  
Yue Zhao ◽  
Hongji Zhu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Survival Rate ◽  
Transcriptome Analysis ◽  
Acid Stress ◽  
Acid Resistance ◽  
Transcriptional Regulator ◽  
Regulation Mechanism ◽  
Content Type ◽  
Regulation Mechanisms ◽  
Nisin Yield

ABSTRACTTo overcome the adverse impacts of environmental stresses during growth, different adaptive regulation mechanisms can be activated inLactococcus lactis. In this study, the transcription levels of eight transcriptional regulators ofL. lactissubsp.lactisF44 under acid stress were analyzed using quantitative reverse transcription-PCR. Eight gene-overexpressing strains were then constructed to examine their influences on acid-resistant capability. OverexpressingythA, a PspC family transcriptional regulator, increased the survival rate by 3.2-fold compared to the control at the lethal pH 3.0 acid shock. Moreover, the nisin yield was increased by 45.50%. TheythA-overexpressing strain FythA appeared to have higher intracellular pH stability and nisin-resistant ability. Subsequently, transcriptome analysis revealed that the vast majority of genes associated with amino acid biosynthesis, including arginine, serine, phenylalanine, and tyrosine, were predominantly upregulated in FythA. Arginine biosynthesis (argGandargH), arginine deiminase pathway, and polar amino acid transport (ysfEandysfF) were proposed to be the main regulation mechanisms of YthA. Furthermore, the transcription of genes associated with pyrimidine and exopolysaccharide biosynthesis were upregulated. The transcriptional levels ofnisIPRKFEGgenes were substantially higher in FythA, which directly contributed to the yield and resistance of nisin. Three potential DNA-binding sequences were predicted by computer analysis using the upstream regions of genes with prominent changes. This study showed that YthA could increase acid resistance and nisin yield and revealed a putative regulation mechanism of YthA.IMPORTANCENisin, produced byLactococcus lactissubsp.lactis, is widely used as a safe food preservative. Acid stress becomes the primary restrictive factor of cell growth and nisin yield. In this research, we found that the transcriptional regulator YthA was conducive to enhancing the acid resistance ofL. lactisF44. OverexpressingythAcould significantly improve the survival rate and nisin yield. The stability of intracellular pH and nisin resistance were also increased. Transcriptome analysis showed that nisin immunity and the biosynthesis of some amino acids, pyrimidine, and exopolysaccharides were enhanced in the engineered strain. This study elucidates the regulation mechanism of YthA and provides a novel strategy for constructing robust industrialL. lactisstrains.

scholarly journals D-Methionine and D-Phenylalanine Improve Lactococcus lactis F44 Acid Resistance and Nisin Yield by Governing Cell Wall Remodeling

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Hao Wu ◽  
Ershu Xue ◽  
Ning Zhi ◽  
Qianqian Song ◽  
Kairen Tian ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Lactococcus Lactis ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Acid Resistance ◽  
Cell Wall Synthesis ◽  
Nisin Production ◽  
Content Type ◽  
Nisin Yield

ABSTRACT Lactococcus lactis encounters various environmental challenges, especially acid stress, during its growth. The cell wall can maintain the integrity and shape of the cell under environmental stress, and d-amino acids play an important role in cell wall synthesis. Here, by analyzing the effects of 19 different d-amino acids on the physiology of L. lactis F44, we found that exogenously supplied d-methionine and d-phenylalanine increased the nisin yield by 93.22% and 101.29%, respectively, as well as significantly increasing the acid resistance of L. lactis F44. The composition of the cell wall in L. lactis F44 with exogenously supplied d-Met or d-Phe was further investigated via a vancomycin fluorescence experiment and a liquid chromatography-mass spectrometry assay, which demonstrated that d-Met could be incorporated into the fifth position of peptidoglycan (PG) muropeptides and d-Phe could be added to the fourth and fifth positions. Moreover, overexpression of the PG synthesis gene murF further enhanced the levels of d-Met and d-Phe involved in PG and increased the survival rate under acid stress and the nisin yield of the strain. This study reveals that the exogenous supply of d-Met or d-Phe can change the composition of the cell wall and influence acid tolerance as well as nisin yield in L. lactis. IMPORTANCE As d-amino acids play an important role in cell wall synthesis, we analyzed the effects of 19 different d-amino acids on L. lactis F44, demonstrating that d-Met and d-Phe can participate in peptidoglycan (PG) synthesis and improve the acid resistance and nisin yield of this strain. murF overexpression further increased the levels of d-Met and d-Phe incorporated into PG and contributed to the acid resistance of the strain. These findings suggest that d-Met and d-Phe can be incorporated into PG to improve the acid resistance and nisin yield of L. lactis, and this study provides new ideas for the enhancement of nisin production.

scholarly journals Acid Stress-Mediated Metabolic Shift in Lactobacillus sanfranciscensis LSCE1

2011 ◽  
Vol 77 (8) ◽  
pp. 2656-2666 ◽  
Author(s):  
Diana I. Serrazanetti ◽  
Maurice Ndagijimana ◽  
Sylvain L. Sado-Kamdem ◽  
Aldo Corsetti ◽  
Rudi F. Vogel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Solid Phase ◽  
Acid Stress ◽  
Redox Balance ◽  
Gas Chromatography Mass Spectrometry ◽  
Metabolic Shift ◽  
Content Type ◽  
Lactobacillus Sanfranciscensis ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACTLactobacillus sanfranciscensisLSCE1 was selected as a target organism originating from recurrently refreshed sourdough to study the metabolic rerouting associated with the acid stress exposure during sourdough fermentation. In particular, the acid stress induced a metabolic shift toward overproduction of 3-methylbutanoic and 2-methylbutanoic acids accompanied by reduced sugar consumption and primary carbohydrate metabolite production. The fate of labeled leucine, the role of different nutrients and precursors, and the expression of the genes involved in branched-chain amino acid (BCAA) catabolism were evaluated at pH 3.6 and 5.8. The novel application of the program XCMS to the solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) data allowed accurate separation and quantification of 2-methylbutanoic and 3-methylbutanoic acids, generally reported as a cumulative datum. The metabolites coming from BCAA catabolism increased up to seven times under acid stress. The gene expression analysis confirmed that some genes associated with BCAA catabolism were overexpressed under acid conditions. The experiment with labeled leucine showed that 2-methylbutanoic acid originated also from leucine. While the overproduction of 3-methylbutanoic acid under acid stress can be attributed to the need to maintain redox balance, the rationale for the production of 2-methylbutanoic acid from leucine can be found in a newly proposed biosynthesis pathway leading to 2-methylbutanoic acid and 3 mol of ATP per mol of leucine. Leucine catabolism to 3-methylbutanoic and 2-methylbutanoic acids suggests that the switch from sugar to amino acid catabolism supports growth inL. sanfranciscensisin restricted environments such as sourdough characterized by acid stress and recurrent carbon starvation.

scholarly journals Molecular Design of a Signaling System Influences Noise in Protein Abundance under Acid Stress in Different Gammaproteobacteria

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Sophie Brameyer ◽  
Elisabeth Hoyer ◽  
Sebastian Bibinger ◽  
Korinna Burdack ◽  
Jürgen Lassak ◽  
...  
Keyword(s):  
Copy Number ◽  
Molecular Design ◽  
Ph Sensor ◽  
Acid Stress ◽  
Acid Resistance ◽  
Signaling System ◽  
Content Type ◽  
Signaling Systems ◽  
E Coli ◽  
Cad System

ABSTRACT Bacteria have evolved different signaling systems to sense and adapt to acid stress. One of these systems, the CadABC system, responds to a combination of low pH and lysine availability. In Escherichia coli, the two signals are sensed by the pH sensor and transcription activator CadC and the cosensor LysP, a lysine-specific transporter. Activated CadC promotes the transcription of the cadBA operon, which codes for the lysine decarboxylase CadA and the lysine/cadaverine antiporter CadB. The copy number of CadC is controlled translationally. Using a bioinformatics approach, we identified the presence of CadC with ribosomal stalling motifs together with LysP in species of the Enterobacteriaceae family. In contrast, we identified CadC without stalling motifs in species of the Vibrionaceae family, and the LysP cosensor is missing. Therefore, we compared the outputs of the Cad system in single cells of the distantly related organisms E. coli and Vibrio campbellii using fluorescently tagged CadB as the reporter. We observed a heterogeneous output in E. coli, and all the V. campbellii cells produced CadB. The copy number of the pH sensor CadC in E. coli was extremely low (≤4 molecules per cell), but it was 10-fold higher in V. campbellii. An increase in the CadC copy number in E. coli correlated with a decrease in heterogeneous behavior. This study demonstrated how small changes in the design of a signaling system allow a homogeneous output and, thus, adaptation of Vibrio species that rely on the CadABC system as the only acid resistance system. IMPORTANCE Acid resistance is an important property for bacteria, such as Escherichia coli, to survive acidic environments like the human gastrointestinal tract. E. coli possesses both passive and inducible acid resistance systems to counteract acidic environments. Thus, E. coli evolved sophisticated signaling systems to sense and appropriately respond to environmental acidic stress by regulating the activity of its three inducible acid resistance systems. One of these systems is the Cad system, which is induced only under moderate acidic stress in a lysine-rich environment by the pH-responsive transcriptional regulator CadC. The significance of our research lies in identifying the molecular design of the Cad systems in different proteobacteria and their target expression noise at the single-cell level during acid stress conditions.

scholarly journals Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of theWHI2Gene Identified through Inverse Metabolic Engineering

2016 ◽  
Vol 82 (7) ◽  
pp. 2156-2166 ◽  
Author(s):  
Yingying Chen ◽  
Lisa Stabryla ◽  
Na Wei
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Metabolic Engineering ◽  
Acid Stress ◽  
Acid Resistance ◽  
Biofuel Production ◽  
Gene Target ◽  
Content Type ◽  
Inverse Metabolic Engineering ◽  
Acetic Acid Resistance

ABSTRACTDevelopment of acetic acid-resistantSaccharomyces cerevisiaeis important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target,WHI2(encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance inS. cerevisiae. Overexpression ofWHI2significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. TheWHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression ofWHI2gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 inS. cerevisiae. Meanwhile, thewhi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response inS. cerevisiae. Additionally, overexpression ofWHI2and of a cognate phosphatase gene,PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production.

scholarly journals Comparative Genome-Wide Transcriptome Analysis of Brucella suis and Brucella microti Under Acid Stress at pH 4.5: Cold Shock Protein CspA and Dps Are Associated With Acid Resistance of B. microti

2021 ◽  
Vol 12 ◽  
Author(s):  
Jorge A. de la Garza-García ◽  
Safia Ouahrani-Bettache ◽  
Sébastien Lyonnais ◽  
Erika Ornelas-Eusebio ◽  
Luca Freddi ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Protein Misfolding ◽  
Cold Shock ◽  
Stress Protein ◽  
Acid Stress ◽  
Acid Resistance ◽  
Cold Shock Protein ◽  
Physiological Adaptation ◽  
Energy Conditions ◽  
Brucella Suis

Brucellae are facultative intracellular coccobacilli causing brucellosis, one of the most widespread bacterial zoonosis affecting wildlife animals, livestock and humans. The genus Brucella comprises classical and atypical species, such as Brucella suis and Brucella microti, respectively. The latter is characterized by increased metabolic activity, fast growth rates, and extreme acid resistance at pH 2.5, suggesting an advantage for environmental survival. In addition, B. microti is more acid-tolerant than B. suis at the intermediate pH of 4.5. This acid-resistant phenotype of B. microti may have major implications for fitness in soil, food products and macrophages. Our study focused on the identification and characterization of acid resistance determinants of B. suis and B. microti in Gerhardt’s minimal medium at pH 4.5 and 7.0 for 20 min and 2 h by comparative RNA-Seq-based transcriptome analysis, validated by RT-qPCR. Results yielded a common core response in both species with a total of 150 differentially expressed genes, and acidic pH-dependent genes regulated specifically in each species. The identified core response mechanisms comprise proton neutralization or extrusion from the cytosol, participating in maintaining physiological intracellular pH values. Differential expression of 441 genes revealed species-specific mechanisms in B. microti with rapid physiological adaptation to acid stress, anticipating potential damage to cellular components and critical energy conditions. Acid stress-induced genes encoding cold shock protein CspA, pseudogene in B. suis, and stress protein Dps were associated with survival of B. microti at pH 4.5. B. suis response with 284 specifically regulated genes suggested increased acid stress-mediated protein misfolding or damaging, triggering the set-up of repair strategies countering the consequences rather than the origin of acid stress and leading to subsequent loss of viability. In conclusion, our work supports the hypothesis that increased acid stress resistance of B. microti is based on selective pressure for the maintenance of functionality of critical genes, and on specific differential gene expression, resulting in rapid adaptation.

scholarly journals Nuclear Localization of Haa1, Which Is Linked to Its Phosphorylation Status, Mediates Lactic Acid Tolerance in Saccharomyces cerevisiae

2014 ◽  
Vol 80 (11) ◽  
pp. 3488-3495 ◽  
Author(s):  
Minetaka Sugiyama ◽  
Shin-Pei Akase ◽  
Ryota Nakanishi ◽  
Hitoshi Horie ◽  
Yoshinobu Kaneko ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Subcellular Localization ◽  
Target Genes ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Acid Resistance ◽  
Nuclear Accumulation ◽  
Content Type ◽  
Adaptation Response

ABSTRACTImprovement of the lactic acid resistance of the yeastSaccharomyces cerevisiaeis important for the application of the yeast in industrial production of lactic acid from renewable resources. However, we still do not know the precise mechanisms of the lactic acid adaptation response in yeast and, consequently, lack effective approaches for improving its lactic acid tolerance. To enhance our understanding of the adaptation response, we screened forS. cerevisiaegenes that confer enhanced lactic acid resistance when present in multiple copies and identified the transcriptional factor Haa1 as conferring resistance to toxic levels of lactic acid when overexpressed. The enhanced tolerance probably results from increased expression of its target genes. When cells that expressed Haa1 only from the endogenous promoter were exposed to lactic acid stress, the main subcellular localization of Haa1 changed from the cytoplasm to the nucleus within 5 min. This nuclear accumulation induced upregulation of the Haa1 target genesYGP1,GPG1, andSPI1, while the degree of Haa1 phosphorylation observed under lactic acid-free conditions decreased. Disruption of the exportin geneMSN5led to accumulation of Haa1 in the nucleus even when no lactic acid was present. Since Msn5 was reported to interact with Haa1 and preferentially exports phosphorylated cargo proteins, our results suggest that regulation of the subcellular localization of Haa1, together with alteration of its phosphorylation status, mediates the adaptation to lactic acid stress in yeast.

scholarly journals Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Amanda He ◽  
Stephanie R. Penix ◽  
Preston J. Basting ◽  
Jessie M. Griffith ◽  
Kaitlin E. Creamer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Acid Stress ◽  
Acid Decarboxylase ◽  
Gamma Aminobutyric Acid ◽  
Knock Out ◽  
Content Type ◽  
Insertions And Deletions ◽  
Acid Ph ◽  
K 12

ABSTRACT Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932–1941, 2015, https://doi.org/10.1128/AEM.03494-14 ). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS5 insertion or IS-mediated deletion in cadC, while population B11 had a point mutation affecting the arginine activator adiY. The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR, yhiM, and Gad). Other strains showed downregulation of H2 consumption mediated by hydrogenases (hya and hyb) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes (dmsABC, frdABCD, hybABO, nikABCDE, and nrfAC). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism. IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain.

scholarly journals PccD Regulates Branched-Chain Amino Acid Degradation and Exerts a Negative Effect on Erythromycin Production inSaccharopolyspora erythraea

2018 ◽  
Vol 84 (8) ◽  
pp. e00049-18 ◽  
Author(s):  
Zhen Xu ◽  
Yong Liu ◽  
Bang-Ce Ye
Keyword(s):  
Growth Rate ◽  
Amino Acid ◽  
Transcriptional Regulator ◽  
Fermentation Medium ◽  
Industrial Fermentation ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Negative Effect ◽  
Branched Chain ◽  
Erythromycin Production

ABSTRACTBranched-chain amino acid (BCAA) degradation is a major source of propionyl coenzyme A (propionyl-CoA), a key precursor of erythromycin biosynthesis inSaccharopolyspora erythraea. In this study, we found that thebkdoperon, responsible for BCAA degradation, was regulated directly by PccD, a transcriptional regulator of propionyl-CoA carboxylase genes. The transcriptional level of thebkdoperon was upregulated 5-fold in apccDgene deletion strain (ΔpccDstrain) and decreased 3-fold in apccDoverexpression strain (WT/pIB-pccD), demonstrating that PccD was a negative transcriptional regulator of the operon. The deletion ofpccDsignificantly improved the ΔpccDstrain's growth rate, whereaspccDoverexpression repressed WT/pIB-pccDgrowth rate, in basic Evans medium with 30 mM valine as the sole carbon and nitrogen source. The deletion ofgdhA1and the BcdhE1 gene (genes in thebkdoperon) resulted in lower growth rates of ΔgdhA1and ΔBcdhE1 strains, respectively, on 30 mM valine, further suggesting that thebkdoperon is involved in BCAA degradation. Bothbkdoverexpression (WT/pIB-bkd) andpccDinactivation (ΔpccDstrain) improve erythromycin production (38% and 64%, respectively), whereas the erythromycin production of strain WT/pIB-pccDwas decreased by 48%. Lastly, we explored the applications of engineeringpccDandbkdin an industrial high-erythromycin-producing strain.pccDdeletion in industrial strainS. erythraeaE3 (E3pccD) improved erythromycin production by 20%, and the overexpression ofbkdin E3ΔpccD(E3ΔpccD/pIB-bkd) increased erythromycin production by 39% compared withS. erythraeaE3 in an industrial fermentation medium. Addition of 30 mM valine to industrial fermentation medium further improved the erythromycin production by 23%, a 72% increase from the initial strainS. erythraeaE3.IMPORTANCEWe describe abkdoperon involved in BCAA degradation inS. erythraea. The genes of the operon are repressed by a TetR regulator, PccD. The results demonstrated that PccD controlled the supply of precursors for biosynthesis of erythromycin via regulating the BCAA degradation and propionyl-CoA assimilation and exerted a negative effect on erythromycin production. The findings reveal a regulatory mechanism in feeder pathways and provide new strategies for designing metabolic engineering to increase erythromycin yield.

scholarly journals Escherichia coli Glutamate- and Arginine-Dependent Acid Resistance Systems Increase Internal pH and Reverse Transmembrane Potential

2004 ◽  
Vol 186 (18) ◽  
pp. 6032-6041 ◽  
Author(s):  
Hope Richard ◽  
John W. Foster
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Transmembrane Potential ◽  
Acid Stress ◽  
Acid Resistance ◽  
Physiological Parameter ◽  
Resistance System ◽  
Internal Ph ◽  
System 2 ◽  
Acidic Nature

ABSTRACT Due to the acidic nature of the stomach, enteric organisms must withstand extreme acid stress for colonization and pathogenesis. Escherichia coli contains several acid resistance systems that protect cells to pH 2. One acid resistance system, acid resistance system 2 (AR2), requires extracellular glutamate, while another (AR3) requires extracellular arginine. Little is known about how these systems protect cells from acid stress. AR2 and AR3 are thought to consume intracellular protons through amino acid decarboxylation. Antiport mechanisms then exchange decarboxylation products for new amino acid substrates. This form of proton consumption could maintain an internal pH (pHi) conducive to cell survival. The model was tested by estimating the pHi and transmembrane potential (ΔΨ) of cells acid stressed at pH 2.5. During acid challenge, glutamate- and arginine-dependent systems elevated pHi from 3.6 to 4.2 and 4.7, respectively. However, when pHi was manipulated to 4.0 in the presence or absence of glutamate, only cultures challenged in the presence of glutamate survived, indicating that a physiological parameter aside from pHi was also important. Measurements of ΔΨ indicated that amino acid-dependent acid resistance systems help convert membrane potential from an inside negative to inside positive charge, an established acidophile strategy used to survive extreme acidic environments. Thus, reversing ΔΨ may be a more important acid resistance strategy than maintaining a specific pHi value.

scholarly journals Role of Branched-Chain Amino Acid Transport in Bacillus subtilis CodY Activity

2015 ◽  
Vol 197 (8) ◽  
pp. 1330-1338 ◽  
Author(s):  
Boris R. Belitsky
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Growth Medium ◽  
Amino Acid Transport ◽  
Transcriptional Regulator ◽  
Acid Transport ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACTCodY is a branched-chain amino acid-responsive transcriptional regulator that controls the expression of several dozen transcription units inBacillus subtilis. The presence of isoleucine, valine, and leucine in the growth medium is essential for achieving high activity of CodY and for efficient regulation of the target genes. We identified three permeases—BcaP, BraB, and BrnQ—that are responsible for the bulk of isoleucine and valine uptake and are also involved in leucine uptake. At least one more permease is capable of efficient leucine uptake, as well as low-affinity transport of isoleucine and valine. The lack of the first three permeases strongly reduced activity of CodY in an amino acid-containing growth medium. BcaP appears to be the most efficient isoleucine and valine permease responsible for their utilization as nitrogen sources. The previously described strong CodY-mediated repression of BcaP provides a mechanism for fine-tuning CodY activity by reducing the availability of amino acids and for delaying the utilization of isoleucine and valine as nitrogen and carbon sources under conditions of nutrient excess.IMPORTANCEBacillus subtilisCodY is a global transcriptional regulator that is activated by branched-chain amino acids (BCAA). Since the level of BCAA achieved by intracellular synthesis is insufficient to fully activate CodY, transport of BCAA from the environment is critical for CodY activation, but the permeases needed for such activation have not been previously identified. This study identifies three such permeases, reports their amino acid transport specificity, and reveals their impact on CodY activation.

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