Basal-Level Effects of (p)ppGpp in the Absence of Branched-Chain Amino Acids in Actinobacillus pleuropneumoniae

ABSTRACT The (p)ppGpp-mediated stringent response (SR) is a highly conserved regulatory mechanism in bacterial pathogens, enabling adaptation to adverse environments, and is linked to pathogenesis. Actinobacillus pleuropneumoniae can cause damage to the lungs of pigs, its only known natural host. Pig lungs are known to have a low concentration of free branched-chain amino acids (BCAAs) compared to the level in plasma. We had investigated the role for (p)ppGpp in viability and biofilm formation of A. pleuropneumoniae. Now, we sought to determine whether (p)ppGpp was a trigger signal for the SR in A. pleuropneumoniae in the absence of BCAAs. Combining transcriptome and phenotypic analyses of the wild type (WT) and an relA spoT double mutant [which does not produce (p)ppGpp], we found that (p)ppGpp could repress de novo purine biosynthesis and activate antioxidant pathways. There was a positive correlation between GTP and endogenous hydrogen peroxide content. Furthermore, the growth, viability, morphology, and virulence were altered by the inability to produce (p)ppGpp. Genes involved in the biosynthesis of BCAAs were constitutively upregulated, regardless of the existence of BCAAs, without accumulation of (p)ppGpp beyond a basal level. Collectively, our study shows that the absence of BCAAs was not a sufficient signal to trigger the SR in A. pleuropneumoniae. (p)ppGpp-mediated regulation in A. pleuropneumoniae is different from that described for the model organism Escherichia coli. Further work will establish whether the (p)ppGpp-dependent SR mechanism in A. pleuropneumoniae is conserved among other veterinary pathogens, especially those in the Pasteurellaceae family. IMPORTANCE (p)ppGpp is a key player in reprogramming transcriptomes to respond to nutritional challenges. Here, we present transcriptional and phenotypic differences of A. pleuropneumoniae grown in different chemically defined media in the absence of (p)ppGpp. We show that the deprivation of branched-chain amino acids (BCAAs) does not elicit a change in the basal-level (p)ppGpp, but this level is sufficient to regulate the expression of BCAA biosynthesis. The mechanism found in A. pleuropneumoniae is different from that of the model organism Escherichia coli but similar to that found in some Gram-positive bacteria. This study not only broadens the research scope of (p)ppGpp but also further validates the complexity and multiplicity of (p)ppGpp regulation in microorganisms that occupy different biological niches.

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The ilvGMEDA Operon Is Regulated by Transcription Attenuation in Vibrio alginolyticus ZJ-T

Applied and Environmental Microbiology ◽

10.1128/aem.00880-19 ◽

2019 ◽

Vol 85 (19) ◽

Author(s):

Yiqin Deng ◽

Xing Luo ◽

Mei Xie ◽

Philippe Bouloc ◽

Chang Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Branched Chain Amino Acids ◽

Vibrio Alginolyticus ◽

Gram Negative ◽

Content Type ◽

Wide Range ◽

Transcription Attenuation ◽

Branched Chain ◽

Fish And Shellfish

ABSTRACT Bacteria synthesize amino acids according to their availability in the environment or, in the case of pathogens, within the host. We explored the regulation of the biosynthesis of branched-chain amino acids (BCAAs) (l-leucine, l-valine, and l-isoleucine) in Vibrio alginolyticus, a marine fish and shellfish pathogen and an emerging opportunistic human pathogen. In this species, the ilvGMEDA operon encodes the main pathway for biosynthesis of BCAAs. Its upstream regulatory region shows no sequence similarity to the corresponding region in Escherichia coli or other Enterobacteriaceae, and yet we show that this operon is regulated by transcription attenuation. The translation of a BCAA-rich peptide encoded upstream of the structural genes provides an adaptive response similar to the E. coli canonical model. This study of a nonmodel Gram-negative organism highlights the mechanistic conservation of transcription attenuation despite the absence of primary sequence conservation. IMPORTANCE This study analyzes the regulation of the biosynthesis of branched-chain amino acids (leucine, valine, and isoleucine) in Vibrio alginolyticus, a marine bacterium that is pathogenic to fish and humans. The results highlight the conservation of the main regulatory mechanism with that of the enterobacterium Escherichia coli, suggesting that such a mechanism appeared early during the evolution of Gram-negative bacteria, allowing adaptation to a wide range of environments.

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YjeH Is a Novel Exporter of l-Methionine and Branched-Chain Amino Acids in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.02242-15 ◽

2015 ◽

Vol 81 (22) ◽

pp. 7753-7766 ◽

Cited By ~ 12

Author(s):

Qian Liu ◽

Yong Liang ◽

Yun Zhang ◽

Xiuling Shang ◽

Shuwen Liu ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Fluorescent Protein ◽

Branched Chain Amino Acids ◽

Transport Systems ◽

Content Type ◽

Overexpression Strain ◽

Branched Chain ◽

Amino Acid Efflux

ABSTRACTAmino acid efflux transport systems have important physiological functions and play vital roles in the fermentative production of amino acids. However, no methionine exporter has yet been identified inEscherichia coli. In this study, we identified a novel amino acid exporter, YjeH, inE. coli. TheyjeHoverexpression strain exhibited high tolerance to the structural analogues ofl-methionine and branched-chain amino acids, decreased intracellular amino acid levels, and enhanced export rates in the presence of a Met-Met, Leu-Leu, Ile-Ile, or Val-Val dipeptide, suggesting that YjeH functions as an exporter ofl-methionine and the three branched-chain amino acids. The export of the four amino acids in theyjeHoverexpression strain was competitively inhibited in relation to each other. The expression ofyjeHwas strongly induced by increasing cytoplasmic concentrations of substrate amino acids. Green fluorescent protein (GFP)-tagged YjeH was visualized by total internal reflection fluorescence microscopy to confirm the plasma membrane localization of YjeH. Phylogenetic analysis of transporters indicated that YjeH belongs to the amino acid efflux family of the amino acid/polyamine/organocation (APC) superfamily. Structural modeling revealed that YjeH has the typical “5 + 5” transmembrane α-helical segment (TMS) inverted-repeat fold of APC superfamily transporters, and its binding sites are strictly conserved. The enhanced capacity ofl-methionine export by the overexpression ofyjeHin anl-methionine-producing strain resulted in a 70% improvement in titer. This study supplements the transporter classification and provides a substantial basis for the application of the methionine exporter in metabolic engineering.

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Molecular Mechanisms Underlying the Positive Stringent Response of the Bacillus subtilis ilv-leu Operon, Involved in the Biosynthesis of Branched-Chain Amino Acids

Journal of Bacteriology ◽

10.1128/jb.00606-08 ◽

2008 ◽

Vol 190 (18) ◽

pp. 6134-6147 ◽

Cited By ~ 33

Author(s):

Shigeo Tojo ◽

Takenori Satomura ◽

Kanako Kumamoto ◽

Kazutake Hirooka ◽

Yasutaro Fujita

Keyword(s):

Amino Acids ◽

Bacillus Subtilis ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Stringent Response ◽

Branched Chain Amino Acids ◽

Base Substitution ◽

Branched Chain

ABSTRACT Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5′ end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

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[31] Transport of branched-chain amino acids in escherichia coli

Methods in Enzymology - Branched-Chain Amino Acids ◽

10.1016/s0076-6879(88)66033-2 ◽

1988 ◽

pp. 245-251 ◽

Cited By ~ 1

Author(s):

Tammy K. Antonucci ◽

Dale L. Oxender

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Branched Chain Amino Acids ◽

Branched Chain

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Mechanism of De Novo Branched-Chain Amino Acid Synthesis as an Alternative Electron Sink in Hypoxic Aspergillus nidulans Cells

Applied and Environmental Microbiology ◽

10.1128/aem.02135-09 ◽

2010 ◽

Vol 76 (5) ◽

pp. 1507-1515 ◽

Cited By ~ 36

Author(s):

Motoyuki Shimizu ◽

Tatsuya Fujii ◽

Shunsuke Masuo ◽

Naoki Takaya

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Aspergillus Nidulans ◽

De Novo ◽

Acid Synthesis ◽

Branched Chain Amino Acids ◽

Amino Acid Synthesis ◽

Lactate Dehydrogenase Activity ◽

Branched Chain Amino Acid ◽

Branched Chain

ABSTRACT Although branched-chain amino acids are synthesized as building blocks of proteins, we found that the fungus Aspergillus nidulans excretes them into the culture medium under hypoxia. The transcription of predicted genes for synthesizing branched-chain amino acids was upregulated by hypoxia. A knockout strain of the gene encoding the large subunit of acetohydroxy acid synthase (AHAS), which catalyzes the initial reaction of the synthesis, required branched-chain amino acids for growth and excreted very little of them. Pyruvate, a substrate for AHAS, increased the amount of hypoxic excretion in the wild-type strain. These results indicated that the fungus responds to hypoxia by synthesizing branched-chain amino acids via a de novo mechanism. We also found that the small subunit of AHAS regulated hypoxic branched-chain amino acid production as well as cellular AHAS activity. The AHAS knockout resulted in higher ratios of NADH/NAD+ and NADPH/NADP+ under hypoxia, indicating that the branched-chain amino acid synthesis contributed to NAD+ and NADP+ regeneration. The production of branched-chain amino acids and the hypoxic induction of involved genes were partly repressed in the presence of glucose, where cells produced ethanol and lactate and increased levels of lactate dehydrogenase activity. These indicated that hypoxic branched-chain amino acid synthesis is a unique alternative mechanism that functions in the absence of glucose-to-ethanol/lactate fermentation and oxygen respiration.

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