Aspartic Acid Transport in <i>Mycobacterium smegmatis</i>

Yabu, Kunihiko

doi:10.1111/j.1348-0421.1971.tb00603.x

Cited by 9 publications

(8 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In order to calculate the permeability from the kinetic data, we assumed that the nutrients are actively transported across the cytoplasmic membrane (see Materials and Methods). Such an assumption is justified, since energy-coupled systems of amino acid transport have been found in M. phlei (21,33,34) and since the uptake kinetics of amino acids and sugars suggested the existence of active transport systems in M. smegmatis, M phlei, and M. tuberculosis (1,23,40,44), as well as in Nocardia asteroides, a species related to the mycobacteria (3). The Km of the overall transport of glycerol and glucose ranged from 200 to 1,000 ,uM, very high values which suggest a rather low permeability of the cell wall.…”

Section: Resultsmentioning

confidence: 99%

Permeability barrier to hydrophilic solutes in Mycobacterium chelonei

1990

View full text Add to dashboard Cite

In order to define the permeability barrier to hydrophilic molecules in mycobacteria, we used as a model a smooth, P-lactamase-producing strain of Mycobacterium chelonei. The rates of hydrolysis of eight cephalosporins by intact and sonicated cells were measured, and the permeability coefficient (P) was calculated from these rates by the method of Zimmermann and Rosselet (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368-372, 1977). P ranged from (0.9 ± 0.3) x 10-8 (benzothienylcephalosporin) to (10 ± 3.3) x 10-8 cm/s (cephaloridine); i.e., the P values were lower than those reported for Pseudomonas aeruginosa and Escherichia coli by 1 and 3 orders of magnitude, respectively. The permeability barrier was shown to reduce drastically the stream of drug molecules entering the cell, allowing the rather low level of P-lactamase (0.1 U/mg of protein with penicillin G) to decrease radically the concentration of the drug at the target; this explains the poor in vitro activities of the Plactams against M. chelonei. We also estimated P for small, hydrophilic molecules (glucose, glycerol, glycine, leucine), by studying their uptake kinetics. The values found, ranging from 15 x 10-8 to 490 x 10-8 cm/s, were consistent again with a very low permeability of M. chelonei cell wall. The permeation of cephalosporins was not very dependent on the hydrophobicity of the molecules or on the temperature, suggesting a hydrophilic pathway of penetration for these molecules.It is commonly believed that the mycobacterial cell wall constitutes an efficient permeability barrier, and several properties of mycobacteria, such as acid fastness, the slow rates of growth, and the natural resistance to a wide range of antibiotics, are often thought to be at least partially related to the poor penetration of solutes across the cell wall (11,35,37). It is also well known that the mycobacterial cell wall is extremely rich in lipids, which, principally in the form of very-long-chain fatty acids (mycolic acids), account for 30 to 60% of the weight of the wall (24, 35). Such a thick layer of lipidic material obviously acts as a barrier to penetration of hydrophilic solutes. However, mycobacteria must take up nutrients, which are mostly hydrophilic molecules, from the outside medium. Currently little is known about the mechanism of entry of these molecules through the mycobacterial cell wall.There are some suggestive and qualitative pieces of evidence on the role of the mycobacterial cell wall as a permeation barrier. For example, the sensitivity to antibiotics is increased when the cell wall is altered by growth in the presence of a detergent, such as Tween 80 (16), by the removal of cell wall material through spheroplasting (10), or by mutations that make the cell surface rough (10). In species that produce P-lactamase presumably located inside the cell wall but outside the cytoplasmic membrane, such as Mycobacterium smegmatis, M. phlei, and M. fortuitum, the enzyme is cryptic, suggesting that the cell wall slows down the permeation ...

show abstract

Section: Resultsmentioning

confidence: 99%

Permeability barrier to hydrophilic solutes in Mycobacterium chelonei

1990

View full text Add to dashboard Cite

show abstract

“…L-arginine transport by the fast-growing species M. phlei has been measured as part of a larger study of the energetics of amino acid transport (45,54). From our data, we estimate initial uptake rates of L-arginine in the range of only 0.014 nmol/mg/min, which is 10-fold lower than those measured in E. coli (48), P. aeruginosa (52), and Clostridium (50).…”

Section: Discussionmentioning

confidence: 40%

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

Seth

Connell

2000

J Bacteriol

View full text Add to dashboard Cite

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 from Mycobacterium bovis BCG and the effects of its deletion on L-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to the rocE 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 14 C-labeled substrates. Greatly reduced uptake of L-arginine and ␥-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-[ 14 C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenous L-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.The mycobacteria are distinguishable from most other organisms on the basis of their low permeability. Cell wall structural analysis (41) and permeability studies (18, 28) have provided an increased understanding of this characteristic. However, few of the mycobacterial transporters mediating the uptake of nutrients have been characterized at the genetic, molecular, or biochemical levels. There are few recent studies of nutrient transport by mycobacteria (9; for a review, see reference 18), and, in particular, there are no studies of the role of nutrient transport in the intracellular survival of mycobacteria. The nature and availability of carbon, nitrogen, and energy sources within the macrophage can best be studied with genetic mutants altered in intermediary metabolism and transport. This study presents a genetic approach to the transport of L-arginine in mycobacteria.Many microorganisms use L-arginine as a source of energy and/or nitrogen, and the pathways of L-arginine biosynthesis and utilization are well understood in some cases. At least five pathways of L-arginine catabolism have been identified in microorganisms, listed here by the first enzyme to act upon the substrate: arginase, arginine deiminase, arginine decarboxylase, arginine succinyl transferase, and arginine oxidase. L-Arginine metabolism has not previously been ...

show abstract

“…It was shown early in seminal papers by Yabu that D-amino acids are taken up rapidly by mycobacteria while the Lforms are transported at a much lower rate (Yabu, 1967(Yabu, , 1970(Yabu, , 1971. These results can be attributed to the specificity of the inner-membrane transporters for the natural form of amino acids.…”

Section: Transporters Of Amino Acidsmentioning

confidence: 62%

Nutrient acquisition by mycobacteria

Niederweis

2008

Microbiology

126

View full text Add to dashboard Cite

The growth and nutritional requirements of mycobacteria have been intensively studied since the discovery of Mycobacterium tuberculosis more than a century ago. However, the identity of many transporters for essential nutrients of M. tuberculosis and other mycobacteria is still unknown despite a wealth of genomic data and the availability of sophisticated genetic tools. Recently, considerable progress has been made in recognizing that two lipid permeability barriers have to be overcome in order for a nutrient molecule to reach the cytoplasm of mycobacteria. Uptake processes are discussed by comparing M. tuberculosis with Mycobacterium smegmatis. For example, M. tuberculosis has only five recognizable carbohydrate transporters in the inner membrane, while M. smegmatis has 28 such transporters at its disposal. The specificities of inner-membrane transporters for sulfate, phosphate and some amino acids have been determined. Outer-membrane channel proteins in both organisms are thought to contribute to nutrient uptake. In particular, the Msp porins have been shown to be required for uptake of carbohydrates, amino acids and phosphate by M. smegmatis. The set of porins also appears to be different for M. tuberculosis and M. smegmatis. These differences likely reflect the lifestyles of these mycobacteria and the availability of nutrients in their natural habitats: the soil and the human body. The comprehensive identification and the biochemical and structural characterization of the nutrient transporters of M. tuberculosis will not only promote our understanding of the physiology of this important human pathogen, but might also be exploited to improve tuberculosis chemotherapy. IntroductionThe growth and nutritional requirements of mycobacteria have been intensively studied since the discovery of Mycobacterium tuberculosis (Koch, 1882). These studies have resulted in an overwhelming body of literature on the physiology of mycobacterial metabolism in the years before the dawn of molecular biology (Edson, 1951;Ramakrishnan et al., 1972;Ratledge, 1982). For example, the utilization of many solutes such as carbohydrates, alcohols, carboxy acids, fatty acids and amino acids by mycobacteria was examined by measuring oxygen consumption (Edson, 1951). It is striking that the identity of many transporters for essential nutrients of M. tuberculosis and other mycobacteria is still unknown despite considerable progress in the development of genetic methods for mycobacteria (Kana & Mizrahi, 2004;Machowski et al., 2005). Recently, in particular carbon metabolism of mycobacteria has attracted renewed interest since the observation that M. tuberculosis relies on the glyoxylate cycle for survival in mice (McKinney et al., 2000; MunozElias & McKinney, 2005). This indicates that M. tuberculosis uses lipids as the main carbon source during infection. On the other hand, genes that encode a putative disaccharide transporter were found to be essential for M. tuberculosis during the first week of infection (Sassetti & Rubin, 2003), indicating...

show abstract

Aspartic Acid Transport in Mycobacterium smegmatis

Cited by 9 publications

References 17 publications

Permeability barrier to hydrophilic solutes in Mycobacterium chelonei

Permeability barrier to hydrophilic solutes in Mycobacterium chelonei

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

Nutrient acquisition by mycobacteria

Contact Info

Product

Resources

About