Amino Acid Transport and Metabolism in Mycobacteria: Cloning, Interruption, and Characterization of an L-Arginine/gamma -Aminobutyric Acid Permease in Mycobacterium bovis BCG

Department of Microbiology and Molecular Genetics and New Jersey Medical School National Tuberculosis Center, UMDNJ/New Jersey Medical School, Newark, New Jersey 17103, USA.
Journal of Bacteriology (Impact Factor: 2.81). 03/2000; 182(4). DOI: 10.1128/JB.182.4.919-927.2000
Source: PubMed Central


Genes encoding l-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism
within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter fromMycobacterium bovis BCG and the effects of its deletion onl-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to
therocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene
interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis
of these strains was conducted using 14C-labeled substrates. Greatly reduced uptake of l-arginine and γ-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain
compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in
a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow),l-[14C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenousl-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type
cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium. l-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease
mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.

Download full-text


Available from: Nancy Connell,
11 Reads
  • Source
    • "The arginine transporter is the only inner-membrane amino acid transporter from mycobacteria characterized genetically and by transport experiments [133]. It was found that the Rv0522 gene product from Mycobacterium bovis BCG is an L-arginine and also a -aminobutyric acid permease [133]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Worldwide, tuberculosis is the leading cause of morbidity and mortality due to a single bacterial pathogen, Mycobacterium tuberculosis (Mtb). The increasing prevalence of this disease, the emergence of multi-, extensively, and totally drug-resistant strains, complicated by co-infection with the human immunodeficiency virus, and the length of tuberculosis chemotherapy have led to an urgent and continued need for the development of new and more effective antitubercular drugs. Within this context, the L-histidine biosynthetic pathway, which converts 5-phosphoribosyl 1-pyrophosphate to L-histidine in ten enzymatic steps, has been reported as a promising target of antimicrobial agents. This pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants but is absent in mammals, making these enzymes highly attractive targets for the drug design of new antimycobacterial compounds with selective toxicity. Moreover, the biosynthesis of L-histidine has been described as essential for Mtb growth in vitro. Accordingly, a comprehensive overview of Mycobacterium tuberculosis histidine pathway enzymes as attractive targets for the development of new antimycobacterial agents is provided, mainly summarizing the previously reported inhibition data for Mtb or orthologous proteins.
    Current topics in medicinal chemistry 10/2013; 13(22). DOI:10.2174/15680266113136660203 · 3.40 Impact Factor
  • Source
    • "Amino acid biosynthesis in mycobacteria has been a focus for creating auxotrophic knockouts that could serve as vaccine candidates (Guleria et al., 1996; Smith et al., 2001; Pavelka et al., 2003). However, the transport and catabolism of amino acids and their relevance in mycobacterial physiology and virulence have received little attention (Seth and Connell, 2000; Niederweis, 2008). Amino acids are mainly used as building blocks in protein biosynthesis, but under certain circumstances they can serve as alternative nitrogen or carbon sources, exhibit protective effects against oxidative and osmotic agents (Csonka, 1989; Krishnan et al., 2008a,b; Takagi, 2008; Szabados and Savoure, 2009) and can be important in virulence (Nakajima et al., 2008; Crawford et al., 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Genes with a role in proline metabolism are strongly expressed when mycobacterial cells are exposed to nutrient starvation and hypoxia. Here we show that proline metabolism in mycobacteria is mediated by the monofunctional enzymes Δ(1) -pyrroline-5-carboxylate dehydrogenase (PruA) and proline dehydrogenase (PruB). Proline metabolism was controlled by a unique membrane-associated DNA-binding protein PruC. Under hypoxia, addition of proline led to higher biomass production than in the absence of proline despite excess carbon and nitrogen. To identify the mechanism responsible for this enhanced growth, microarray analysis of wild-type Mycobacterium smegmatis versus pruC mutant was performed. Expression of the DNA repair machinery and glyoxalases was increased in the pruC mutant. Glyoxalases are proposed to degrade methylglyoxal, a toxic metabolite produced by various bacteria due to an imbalance in intermediary metabolism, suggesting the pruC mutant was under methylglyoxal stress. Consistent with this notion, pruB and pruC mutants were hypersensitive to methylglyoxal. Δ(1) -pyrroline-5-carboxylate is reported to react with methylglyoxal to form non-toxic 2-acetyl-1-pyrroline, thus providing a link between proline metabolism and methylglyoxal detoxification. In support of this mechanism, we show that proline metabolism protects mycobacterial cells from methylglyoxal toxicity and that functional proline dehydrogenase, but not Δ(1) -pyrroline-5-carboxylate dehydrogenase, is essential for this protective effect.
    Molecular Microbiology 04/2012; 84(4):664-81. DOI:10.1111/j.1365-2958.2012.08053.x · 4.42 Impact Factor
  • Source
    • "In this study, we found that branched chain amino-acid ABC transporter substrate-binding protein (RPA3297) and ABC transporter, periplasmic amino acid binding protein aapJ-1 (aapJ-1) are constituents of ATP-requiring ABC transporters responsible for amino acid uptake and efflux. Amino acids such as glutamate, aspartate and histidine are all substrates for periplasmic binding protein-dependent transport systems [47], [48]. An extracellular solute-binding protein family 1 (RPA4404), was also upregulated in transketolase-overexpressing strains. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Rhodopseudomonas palustris (R. palustris) is a purple non-sulfur anoxygenic phototrophic bacterium that belongs to the class of proteobacteria. It is capable of absorbing atmospheric carbon dioxide and converting it to biomass via the process of photosynthesis and the Calvin-Benson-Bassham (CBB) cycle. Transketolase is a key enzyme involved in the CBB cycle. Here, we reveal the functions of transketolase isoforms I and II in R. palustris using a systems biology approach. By measuring growth ability, we found that transketolase could enhance the autotrophic growth and biomass production of R. palustris. Microarray and real-time quantitative PCR revealed that transketolase isoforms I and II were involved in different carbon metabolic pathways. In addition, immunogold staining demonstrated that the two transketolase isoforms had different spatial localizations: transketolase I was primarily associated with the intracytoplasmic membrane (ICM) but transketolase II was mostly distributed in the cytoplasm. Comparative proteomic analysis and network construction of transketolase over-expression and negative control (NC) strains revealed that protein folding, transcriptional regulation, amino acid transport and CBB cycle-associated carbon metabolism were enriched in the transketolase I over-expressed strain. In contrast, ATP synthesis, carbohydrate transport, glycolysis-associated carbon metabolism and CBB cycle-associated carbon metabolism were enriched in the transketolase II over-expressed strain. Furthermore, ATP synthesis assays showed a significant increase in ATP synthesis in the transketolase II over-expressed strain. A PEPCK activity assay showed that PEPCK activity was higher in transketolase over-expressed strains than in the negative control strain. Taken together, our results indicate that the two isoforms of transketolase in R. palustris could affect photoautotrophic growth through both common and divergent metabolic mechanisms.
    PLoS ONE 12/2011; 6(12):e28329. DOI:10.1371/journal.pone.0028329 · 3.23 Impact Factor
Show more