scholarly journals Role of N,N-Dimethylglycine and Its Catabolism to Sarcosine in Chromohalobacter salexigens DSM 3043

2020 ◽  
Vol 86 (17) ◽  
Author(s):  
Ting Yang ◽  
Ya-Hui Shao ◽  
Li-Zhong Guo ◽  
Xiang-Lin Meng ◽  
Hao Yu ◽  
...  
Keyword(s):  
Glycine Betaine ◽  
Synthetic Medium ◽  
Physiological Role ◽  
Intermolecular Electron Transfer ◽  
Coenzyme Specificity ◽  
Content Type ◽  
Cell Extracts ◽  
N Source ◽  
Chromohalobacter Salexigens

ABSTRACT Chromohalobacter salexigens DSM 3043 can grow on N,N-dimethylglycine (DMG) as the sole C, N, and energy source and utilize sarcosine as the sole N source under aerobic conditions. However, little is known about the genes and enzymes involved in the conversion of DMG to sarcosine in this strain. In the present study, gene disruption and complementation assays indicated that the csal_0990, csal_0991, csal_0992, and csal_0993 genes are responsible for DMG degradation to sarcosine. The csal_0990 gene heterologously expressed in Escherichia coli was proven to encode an unusual DMG dehydrogenase (DMGDH). The enzyme, existing as a monomer of 79 kDa with a noncovalently bound flavin adenine dinucleotide, utilized both DMG and sarcosine as substrates and exhibited dual coenzyme specificity, preferring NAD+ to NADP+. The optimum pH and temperature of enzyme activity were determined to be 7.0 and 60°C, respectively. Kinetic parameters of the enzyme toward its substrates were determined accordingly. Under high-salinity conditions, the presence of DMG inhibited growth of the wild type and induced the production and accumulation of trehalose and glucosylglycerate intracellularly. Moreover, exogenous addition of DMG significantly improved the growth rates of the four DMG– mutants (Δcsal_0990, Δcsal_0991, Δcsal_0992, and Δcsal_0993) incubated at 37°C in S-M63 synthetic medium with sarcosine as the sole N source. 13C nuclear magnetic resonance (13C-NMR) experiments revealed that not only ectoine, glutamate, and N-acetyl-2,4-diaminobutyrate but also glycine betaine (GB), DMG, sarcosine, trehalose, and glucosylglycerate are accumulated intracellularly in the four mutants. IMPORTANCE Although N,N-dimethylglycine (DMG) dehydrogenase (DMGDH) activity was detected in cell extracts of microorganisms, the genes encoding microbial DMGDHs have not been determined until now. In addition, to our knowledge, the physiological role of DMG in moderate halophiles has never been investigated. In this study, we identified the genes involved in DMG degradation to sarcosine, characterized an unusual DMGDH, and investigated the role of DMG in Chromohalobacter salexigens DSM 3043 and its mutants. Our results suggested that the conversion of DMG to sarcosine is accompanied by intramolecular delivery of electrons in DMGDH and intermolecular electron transfer between DMGDH and other electron acceptors. Moreover, an unidentified methyltransferase catalyzing the production of glycine betaine (GB) from DMG but sharing no homology with the reported sarcosine DMG methyltransferases was predicted to be present in the cells. The results of this study expand our understanding of the physiological role of DMG and its catabolism to sarcosine in C. salexigens.

scholarly journals Phosphoserine Phosphatase Is Required for Serine and One-Carbon Unit Synthesis in Hydrogenobacter thermophilus

2017 ◽  
Vol 199 (21) ◽  
Author(s):  
Keugtae Kim ◽  
Yoko Chiba ◽  
Azusa Kobayashi ◽  
Hiroyuki Arai ◽  
Masaharu Ishii
Keyword(s):  
Physiological Role ◽  
Tca Cycle ◽  
Content Type ◽  
Phosphoserine Phosphatase ◽  
Genes Encoding ◽  
Hydrogenobacter Thermophilus ◽  
Glycine Cleavage ◽  
Major Donor ◽  
Serine Biosynthesis

ABSTRACT Hydrogenobacter thermophilus is an obligate chemolithoautotrophic bacterium of the phylum Aquificae and is capable of fixing carbon dioxide through the reductive tricarboxylic acid (TCA) cycle. The recent discovery of two novel-type phosphoserine phosphatases (PSPs) in H. thermophilus suggests the presence of a phosphorylated serine biosynthesis pathway; however, the physiological role of these novel-type metal-independent PSPs (iPSPs) in H. thermophilus has not been confirmed. In the present study, a mutant strain with a deletion of pspA, the catalytic subunit of iPSPs, was constructed and characterized. The generated mutant was a serine auxotroph, suggesting that the novel-type PSPs and phosphorylated serine synthesis pathway are essential for serine anabolism in H. thermophilus. As an autotrophic medium supplemented with glycine did not support the growth of the mutant, the reversible enzyme serine hydroxymethyltransferase does not appear to synthesize serine from glycine and may therefore generate glycine and 5,10-CH2-tetrahydrofolate (5,10-CH2-THF) from serine. This speculation is supported by the lack of glycine cleavage activity, which is needed to generate 5,10-CH2-THF, in H. thermophilus. Determining the mechanism of 5,10-CH2-THF synthesis is important for understanding the fundamental anabolic pathways of organisms, because 5,10-CH2-THF is a major one-carbon donor that is used for the synthesis of various essential compounds, including nucleic and amino acids. The findings from the present experiments using a pspA deletion mutant have confirmed the physiological role of iPSPs as serine producers and show that serine is a major donor of one-carbon units in H. thermophilus. IMPORTANCE Serine biosynthesis and catabolism pathways are intimately related to the metabolism of 5,10-CH2-THF, a one-carbon donor that is utilized for the biosynthesis of various essential compounds. For this reason, determining the mechanism of serine synthesis is important for understanding the fundamental anabolic pathways of microorganisms. In the present study, we experimentally confirmed that a novel phosphoserine phosphatase in the obligate chemolithoautotrophic bacterium Hydrogenobacter thermophilus is essential for serine biosynthesis. This finding indicates that serine is synthesized from an intermediate of gluconeogenesis in H. thermophilus. In addition, because glycine cleavage system activity and genes encoding an enzyme capable of producing 5,10-CH2-THF were not detected, serine appears to be the major one-carbon donor to tetrahydrofolate (THF) in H. thermophilus.

scholarly journals Importance of Bacillithiol in the Oxidative Stress Response of Staphylococcus aureus

2013 ◽  
Vol 82 (1) ◽  
pp. 316-332 ◽  
Author(s):  
Ana C. Posada ◽  
Stacey L. Kolar ◽  
Renata G. Dusi ◽  
Patrice Francois ◽  
Alexandra A. Roberts ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Physiological Role ◽  
Content Type ◽  
Chromatography Analysis ◽  
Expression Of Genes ◽  
Fold Reduction ◽  
Fosfomycin Resistance ◽  
Microarray Analyses

ABSTRACTInStaphylococcus aureus, the low-molecular-weight thiol called bacillithiol (BSH), together with cognateS-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH inS. aureus, we constructed mutants with the deletion ofbshA(sa1291), which encodes the glycosyltransferase that catalyzes the first step of BSH biosynthesis, andfosB(sa2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in severalS. aureusstrains, including clinical isolates. Mutation offosBorbshAcaused a 16- to 60-fold reduction in fosfomycin resistance in theseS. aureusstrains. High-pressure liquid chromatography analysis, which quantified thiol extracts, revealed some variability in the amounts of BSH present acrossS. aureusstrains. Deletion offosBled to a decrease in BSH levels. ThefosBandbshAmutants of strain COL and a USA300 isolate, upon further characterization, were found to be sensitive to H2O2and exhibited decreased NADPH levels compared with those in the isogenic parents. Microarray analyses of COL and the isogenicbshAmutant revealed increased expression of genes involved in staphyloxanthin synthesis in thebshAmutant relative to that in COL under thiol stress conditions. However, thebshAmutant of COL demonstrated decreased survival compared to that of the parent in human whole-blood survival assays; likewise, the naturally BSH-deficient strain SH1000 survived less well than its BSH-producing isogenic counterpart. Thus, the survival ofS. aureusunder oxidative stress is facilitated by BSH, possibly via a FosB-mediated mechanism, independently of its capability to produce staphyloxanthin.

scholarly journals Diverse Energy-Conserving Pathways in Clostridium difficile: Growth in the Absence of Amino Acid Stickland Acceptors and the Role of the Wood-Ljungdahl Pathway

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Simonida Gencic ◽  
David A. Grahame
Keyword(s):  
Amino Acid ◽  
Clostridium Difficile ◽  
Central Component ◽  
Content Increase ◽  
Efficient System ◽  
Content Type ◽  
Atp Production ◽  
Cell Extracts ◽  
Complete Set

ABSTRACT Clostridium difficile is the leading cause of hospital-acquired antibiotic-associated diarrhea and is the only widespread human pathogen that contains a complete set of genes encoding the Wood-Ljungdahl pathway (WLP). In acetogenic bacteria, synthesis of acetate from 2 CO2 molecules by the WLP functions as a terminal electron accepting pathway; however, C. difficile contains various other reductive pathways, including a heavy reliance on Stickland reactions, which questions the role of the WLP in this bacterium. In rich medium containing high levels of electron acceptor substrates, only trace levels of key WLP enzymes were found; therefore, conditions were developed to adapt C. difficile to grow in the absence of amino acid Stickland acceptors. Growth conditions were identified that produce the highest levels of WLP activity, determined by Western blot analyses of the central component acetyl coenzyme A synthase (AcsB) and assays of other WLP enzymes. Fermentation substrate and product analyses, enzyme assays of cell extracts, and characterization of a ΔacsB mutant demonstrated that the WLP functions to dispose of metabolically generated reducing equivalents. While WLP activity in C. difficile does not reach the levels seen in classical acetogens, coupling of the WLP to butyrate formation provides a highly efficient system for regeneration of NAD+ “acetobutyrogenesis,” requiring only low flux through the pathways to support efficient ATP production from glucose oxidation. Additional insights redefine the amino acid requirements in C. difficile, explore the relationship of the WLP to toxin production, and provide a rationale for colocalization of genes involved in glycine synthesis and cleavage within the WLP operon. IMPORTANCE Clostridium difficile is an anaerobic, multidrug-resistant, toxin-producing pathogen with major health impacts worldwide. It is the only widespread pathogen harboring a complete set of Wood-Ljungdahl pathway (WLP) genes; however, the role of the WLP in C. difficile is poorly understood. In other anaerobic bacteria and archaea, the WLP can operate in one direction to convert CO2 to acetic acid for biosynthesis or in either direction for energy conservation. Here, conditions are defined in which WLP levels in C. difficile increase markedly, functioning to support metabolism of carbohydrates. Amino acid nutritional requirements were better defined, with new insight into how the WLP and butyrate pathways act in concert, contributing significantly to energy metabolism by a mechanism that may have broad physiological significance within the group of nonclassical acetogens.

scholarly journals Thermoprotection of Bacillus subtilis by Exogenously Provided Glycine Betaine and Structurally Related Compatible Solutes: Involvement of Opu Transporters

2004 ◽  
Vol 186 (6) ◽  
pp. 1683-1693 ◽  
Author(s):  
Gudrun Holtmann ◽  
Erhard Bremer
Keyword(s):  
Bacillus Subtilis ◽  
Glycine Betaine ◽  
Compatible Solutes ◽  
Physiological Role ◽  
Growth Conditions ◽  
Transport Systems ◽  
Moderate Increase ◽  
Low Concentrations ◽  
In Cells

ABSTRACT Bacillus subtilis possesses five osmotically regulated transporters (Opu) for the uptake of various compatible solutes for osmoprotective purposes. We have now found that compatible solutes also function as thermoprotectants for B. subtilis. Low concentrations of glycine betaine enhanced the growth of the B. subtilis wild-type strain JH642 at its maximal growth temperature (52°C) but did not allow an extension of the upper growth limit. A similar enhancement in the growth of B. subtilis was also observed by the addition of several other compatible solutes that are structurally related to glycine betaine or by the addition of proline. Each of these compatible solutes was taken up under heat stress by the cell through the same Opu transporters that are used for their acquisition under osmostress conditions. Northern blot analysis revealed a moderate increase in transcription of the structural genes for each of the Opu transport systems in cells that were propagated at 52°C. In contrast, the uptake level of radiolabeled glycine betaine was very low under high-temperature growth conditions but nevertheless allowed the buildup of an intracellular glycine betaine pool comparable to that found in cells grown at 37°C in the absence of salt stress. Although exogenously added glutamate has only a limited osmoprotective potential for B. subtilis, it was found to be a very effective thermoprotectant. Collectively, our data demonstrate thermoprotection by a variety of compatible solutes in B. subtilis, thus ascribing a new physiological function for this class of compounds in this microorganism and broadening the physiological role of the known osmoprotectant uptake systems (Opu).

scholarly journals Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Chunhua Zhao ◽  
Zhao Lin ◽  
Hongjun Dong ◽  
Yanping Zhang ◽  
Yin Li
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Kinase ◽  
Physiological Role ◽  
Glucose Consumption ◽  
General Strategy ◽  
Anaerobic Conditions ◽  
Content Type ◽  
Intracellular Atp ◽  
Atp Levels

ABSTRACT Pyruvate kinase is one of the three rate-limiting glycolytic enzymes that catalyze the last step of glycolysis, conversion of phosphoenolpyruvate (PEP) into pyruvate, which is associated with ATP generation. Two isozymes of pyruvate kinase, PykF and PykA, are identified in Escherichia coli. PykF is considered important, whereas PykA has a less-defined role. Prior studies inactivated the pykA gene to increase the level of its substrate, PEP, and thereby increased the yield of end products derived from PEP. We were surprised when we found a pykA::Tn5 mutant in a screen for increased yield of an end product derived from pyruvate (n-butanol), suggesting that the role of PykA needs to be reexamined. We show that the pykA mutant exhibited elevated intracellular ATP levels, biomass concentrations, glucose consumption, and n-butanol production. We also discovered that the pykA mutant expresses higher levels of a presumed pyruvate transporter, YhjX, permitting the mutant to recapture and metabolize excreted pyruvate. Furthermore, we demonstrated that the nucleotide diphosphate kinase activity of PykA leads to negative regulation of the intracellular ATP levels. Taking the data together, we propose that inactivation of pykA can be considered a general strategy to enhance the production of pyruvate-derived metabolites under anaerobic conditions. IMPORTANCE This study showed that knocking out pykA significantly increased the intracellular ATP level and thus significantly increased the levels of glucose consumption, biomass formation, and pyruvate-derived product formation under anaerobic conditions. pykA was considered to be encoding a dispensable pyruvate kinase; here we show that pykA negatively regulates the anaerobic glycolysis rate through regulating the energy distribution. Thus, knocking out pykA can be used as a general strategy to increase the level of pyruvate-derived fermentative products.

scholarly journals Role of Glutathione in Buffering Excess Intracellular Copper in Streptococcus pyogenes

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Louisa J. Stewart ◽  
Cheryl-lynn Y. Ong ◽  
May M. Zhang ◽  
Stephan Brouwer ◽  
Liam McIntyre ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Metal Binding ◽  
Metal Ion ◽  
Physiological Role ◽  
Metal Availability ◽  
Content Type ◽  
Bacterial Physiology ◽  
Group A

ABSTRACT Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]), this system is encoded by the copYAZ operon. This study demonstrates that although the site of GAS infection represents a Cu-rich environment, inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde-3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of noncognate metal-binding sites by Cu. Surprisingly, the onset of these effects is delayed by ∼4 h even though expression of copZ is upregulated immediately upon exposure to Cu. Further biochemical investigations show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. These results indicate that GSH buffers excess intracellular Cu when the transcriptionally responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance and allows bacteria to maintain metabolism even in the presence of an excess of this metal ion. IMPORTANCE The control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it has been established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and has long been assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this physiological role of glutathione beyond measuring growth of glutathione-deficient mutants in the presence of Cu. Our work with group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.

scholarly journals Physiological Role of Acyl Coenzyme A Synthetase Homologs in Lipid Metabolism in Neurospora crassa

2013 ◽  
Vol 12 (9) ◽  
pp. 1244-1257 ◽  
Author(s):  
Christine M. Roche ◽  
Harvey W. Blanch ◽  
Douglas S. Clark ◽  
N. Louise Glass
Keyword(s):  
Neurospora Crassa ◽  
Metabolic Pathways ◽  
Lipid Membrane ◽  
Coenzyme A ◽  
De Novo ◽  
Physiological Role ◽  
Functional Redundancy ◽  
Content Type ◽  
Acyl Coenzyme A

ABSTRACTAcyl coenzyme A (CoA) synthetase (ACS) enzymes catalyze the activation of free fatty acids (FAs) to CoA esters by a two-step thioesterification reaction. Activated FAs participate in a variety of anabolic and catabolic lipid metabolic pathways, includingde novocomplex lipid biosynthesis, FA β-oxidation, and lipid membrane remodeling. Analysis of the genome sequence of the filamentous fungusNeurospora crassaidentified seven putative fatty ACSs (ACS-1 through ACS-7). ACS-3 was found to be the major activator for exogenous FAs for anabolic lipid metabolic pathways, and consistent with this finding, ACS-3 localized to the endoplasmic reticulum, plasma membrane, and septa. Double-mutant analyses confirmed partial functional redundancy of ACS-2 and ACS-3. ACS-5 was determined to function in siderophore biosynthesis, indicating alternative functions for ACS enzymes in addition to fatty acid metabolism. TheN. crassaACSs involved in activation of FAs for catabolism were not specifically defined, presumably due to functional redundancy of several of ACSs for catabolism of exogenous FAs.

scholarly journals Occurrence of Choline and Glycine Betaine Uptake and Metabolism in the Family Rhizobiaceae and Their Roles in Osmoprotection

1999 ◽  
Vol 65 (5) ◽  
pp. 2072-2077 ◽  
Author(s):  
Eric Boncompagni ◽  
Magne Østerås ◽  
Marie-Christine Poggi ◽  
Daniel le Rudulier
Keyword(s):  
Glycine Betaine ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Physiological Role ◽  
Betaine Aldehyde Dehydrogenase ◽  
Choline Uptake ◽  
Rhizobium Tropici ◽  
Mesorhizobium Loti ◽  
The Family

ABSTRACT The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium,Mesorhizobium, Agrobacterium, andBradyrhizobium reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of Rhizobium tropici,Sinorhizobium meliloti, Sinorhizobium fredii,Rhizobium galegae, Agrobacterium tumefaciens,Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such asRhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum. Except for B. japonicum, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R. leguminosarum and S. meliloti or, more surprisingly, reduced in R. tropici, S. fredii, and M. loti. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception ofB. japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M. huakuii and B. japonicum. The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.

scholarly journals The Extracellular Wall-Bound β-N-Acetylglucosaminidase from Lactobacillus casei Is Involved in the Metabolism of the Human Milk Oligosaccharide Lacto-N-Triose

2015 ◽  
Vol 82 (2) ◽  
pp. 570-577 ◽  
Author(s):  
Gonzalo N. Bidart ◽  
Jesús Rodríguez-Díaz ◽  
María J. Yebra
Keyword(s):  
Carbon Source ◽  
Human Milk ◽  
Lactobacillus Casei ◽  
Physiological Role ◽  
Metabolic Adaptation ◽  
Knockout Mutant ◽  
Content Type ◽  
Whole Cells

ABSTRACTHuman milk oligosaccharides (HMOs) are considered to play a key role in establishing and maintaining the infant gut microbiota. Lacto-N-triose forms part of both type 1 and type 2 HMOs and also of the glycan moieties of glycoproteins. Upstream of the previously characterized gene cluster involved in lacto-N-biose and galacto-N-biose metabolism fromLactobacillus caseiBL23, there are two genes,bnaGandmanA, encoding a β-N-acetylglucosaminidase precursor and a mannose-6-phosphate isomerase, respectively. In this work, we show thatL. caseiis able to grow in the presence of lacto-N-triose as a carbon source. Inactivation ofbnaGabolished the growth ofL. caseion this oligosaccharide, demonstrating that BnaG is involved in its metabolism. Interestingly, whole cells of abnaGmutant were totally devoid of β-N-acetylglucosaminidase activity, suggesting that BnaG is an extracellular wall-attached enzyme. In addition to hydrolyzing lacto-N-triose intoN-acetylglucosamine and lactose, the purified BnaG enzyme also catalyzed the hydrolysis of 3′-N-acetylglucosaminyl-mannose and 3′-N-acetylgalactosaminyl-galactose.L. caseican be cultured in the presence of 3′-N-acetylglucosaminyl-mannose as a carbon source, but, curiously, thebnaGmutant strain was not impaired in its utilization. These results indicate that the assimilation of 3′-N-acetylglucosaminyl-mannose is independent of BnaG. Enzyme activity and growth analysis with amanA-knockout mutant showed that ManA is involved in the utilization of the mannose moiety of 3′-N-acetylglucosaminyl-mannose. Here we describe the physiological role of a β-N-acetylglucosaminidase in lactobacilli, and it supports the metabolic adaptation ofL. caseito theN-acetylglucosaminide-rich gut niche.

scholarly journals AnethA-ethR-Deficient Mycobacterium bovis BCG Mutant Displays Increased Adherence to Mammalian Cells and Greater PersistenceIn Vivo, Which Correlate with Altered Mycolic Acid Composition

2014 ◽  
Vol 82 (5) ◽  
pp. 1850-1859 ◽  
Author(s):  
Michelle Lay Teng Ang ◽  
Zarina Zainul Rahim Siti ◽  
Guanghou Shui ◽  
Petronela Dianišková ◽  
Jan Madacki ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Bovis ◽  
Mammalian Cells ◽  
Physiological Role ◽  
Multidrug Resistant ◽  
Mycolic Acid ◽  
Content Type ◽  
Antitubercular Drugs

ABSTRACTTuberculosis remains a major worldwide epidemic because of its sole etiological agent,Mycobacterium tuberculosis. Ethionamide (ETH) is one of the major antitubercular drugs used to treat infections with multidrug-resistantM. tuberculosisstrains. ETH is a prodrug that requires activation within the mycobacterial cell; its bioactivation involves theethA-ethRlocus, which encodes the monooxygenase EthA, while EthR is a transcriptional regulator that binds to the intergenic promoter region of theethA-ethRlocus. While most studies have focused on the role of EthA-EthR in ETH bioactivation, its physiological role in mycobacteria has remained elusive, although a role in bacterial cell detoxification has been proposed. Moreover, the importance of EthA-EthRin vivohas never been reported on. Here we constructed and characterized an EthA-EthR-deficient mutant ofMycobacterium bovisBCG. Our results indicate that absence of theethA-ethRlocus led to greater persistence ofM. bovisBCG in the mouse model of mycobacterial infection, which correlated with greater adherence to mammalian cells. Furthermore, analysis of cell wall lipid composition by thin-layer chromatography and mass spectrometry revealed differences between theethA-ethRKO mutant and the parental strain in the relative amounts of α- and keto-mycolates. Therefore, we propose here thatM. bovisBCGethA-ethRis involved in the cell wall-bound mycolate profile, which impacts mycobacterial adherence properties andin vivopersistence. This study thus provides some experimental clues to the possible physiological role ofethA-ethRand proposes that this locus is a novel factor involved in the modulation of mycobacterial virulence.

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