Transmembrane Amino Acid Flux in Bacterial Cells

Milner, Jocelyn L.; Vink, Bernadette; Wood, Janet M.

doi:10.3109/07388558709044151

Cited by 26 publications

(13 citation statements)

References 234 publications

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“…5A). Using a diffusion rate constant of 0.09 l mg (dry weight) Ϫ1 min Ϫ1 (18,24), the calculated export rates for L-Val are Ϫ0.1 nmol min Ϫ1 mg (dry weight) Ϫ1 with the deletion mutant and 4.5 nmol min Ϫ1 mg (dry weight) Ϫ1 with the same strain but overexpressing brnFE (Table 2). In addition, with the wild-type strain carrying pJC1brnFE, the L-Val export rate proved to be 3.3 nmol min Ϫ1 mg (dry weight) Ϫ1 .…”

Section: Mg (Dry Weight)mentioning

confidence: 99%

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Export of l -Isoleucine from Corynebacterium glutamicum : a Two-Gene-Encoded Member of a New Translocator Family

et al. 2002

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Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes L-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for L-isoleucine export, we have used transposon mutagenesis to isolate mutants of C. glutamicum sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices. brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports L-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of L-leucine and L-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in ␣-proteobacteria but not in other prokaryotes analyzed.

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Section: Mg (Dry Weight)mentioning

confidence: 99%

“…Calculations.Observed efflux rates of branched-chain amino acids are due to diffusion, active export, and active import (44). Therefore, the carrier-mediated export rate is calculated as: Ϫ1 min Ϫ1 (18,24). V in is the rate of uptake of the branched-chain amino acids.…”

Section: Methodsmentioning

confidence: 99%

Export of l -Isoleucine from Corynebacterium glutamicum : a Two-Gene-Encoded Member of a New Translocator Family

et al. 2002

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“…The generally accepted hypothesis for glutamate secretion under biotin limitation V"leak model") is based on the observation that lack of biotin decreases fatty acid synthesis, causing alterations in the plasma membrane, i.e., decreasing the lipid content and changing the phospholipid and fatty acid composition (45). Thus, the plasma membrane is postulated to become permeable to glutamate, leading to secretion -by passive diffusion (4,9,21,22,30,(39)(40)(41). The internal concentration of glutamate then decreases to such an extent that the feedback inhibition of glutamate synthesis is abolished.…”

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confidence: 99%

Membrane alteration is necessary but not sufficient for effective glutamate secretion in Corynebacterium glutamicum

1990

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We showed recently that secretion of glutamate in biotin-limited cells of Corynebacterium glutamicum is mediated by carrier systems in the plasma membrane (C. Hoischen -and R. Kramer, Arch. Microbiol. 151:342-347, 1989). In view of the generally accepted hypothesis that glutamate efflux is directly caused by alterations of the membrane, it was necessary to examine the kind of correlation between changes in lipid content and composition of the bacterial membrane and glutamate secretion activity. Two new experimental approaches were'used. (i) Changes in lipid content and composition were analyzed in glutamate-producing cells which were forced to switch to nonproducers by addition of biotin in a short-term fermentation. (ii) The time courses of both the fatty acid or phospholipid composition and the efflux activity were analyzed within the first minutes of the switch from high to low secretion activity. The following results were obtained. (i) The time course of the change i,n fatty acid or phospholipid content and composition was not related to the change in secretion behavior. (ii) There was no specific fatty acid or phospholipid compound which regulated glutamate efflux. (iii) High efflux activity could only be induced when the total lipid content of the membrane was reduced.

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“…There is apparently no preferred export of D-or L-Lys. Translocation of charged amino acids through lipid bilayers requires active export (18,23), and we have demonstrated that L-Lys export with C. glutamicum is fully dependent on the lysine exporter LysE (40). We hypothesize here that D-Lys is also exported actively and that LysE might possibly translocate D-Lys, too.…”

Section: Resultsmentioning

confidence: 96%

“…This occurs by (i) passive diffusion, (ii) active export catalyzed by the threonine exporter ThrE (34), and (iii) further active export by unknown carriers. Due to its related structure, Ser is also expected to be exported partly by diffusion (18,23), and this physical process, of course, relates to both enantiomers. Since ThrE also utilizes L-Ser as a substrate, it would be interesting to test the consequences of ThrE deletion for Ser accumulation.…”

Section: Discussionmentioning

confidence: 99%

Corynebacterium glutamicum as a Host for Synthesis and Export of d -Amino Acids

et al. 2011

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A number of D-amino acids occur in nature, and there is growing interest in their function and metabolism, as well as in their production and use. Here we use the well-established L-amino-acid-producing bacterium Corynebacterium glutamicum to study whether D-amino acid synthesis is possible and whether mechanisms for the export of these amino acids exist. In contrast to Escherichia coli, C. glutamicum tolerates D-amino acids added extracellularly. Expression of argR (encoding the broad-substrate-specific racemase of Pseudomonas taetrolens) with its signal sequence deleted results in cytosolic localization of ArgR in C. glutamicum. The isolated enzyme has the highest activity with lysine (100%) but also exhibits activity with serine (2%). Upon overexpression of argR in an L-arginine, L-ornithine, or L-lysine producer, equimolar mixtures of the D-and L-enantiomers accumulated extracellularly. Unexpectedly, argR overexpression in an L-serine producer resulted in extracellular accumulation of a surplus of D-serine (81 mM D-serine and 37 mM L-serine) at intracellular concentrations of 125 mM D-serine plus 125 mM L-serine. This points to a nonlimiting ArgR activity for intracellular serine racemization and to the existence of a specific export carrier for D-serine. Export of D-lysine relies fully on the presence of lysE, encoding the exporter for L-lysine, which is apparently promiscuous with respect to the chirality of lysine. These data show that D-amino acids can also be produced with C. glutamicum and that in special cases, due to specific carriers, even a preferential extracellular accumulation of this enantiomer is possible.Corynebacterium glutamicum is well known for its extraordinary L-amino acid production properties. Its most prominent feature is probably its capacity to produce L-glutamate, 1.8 million tons of which are currently produced per year and used as a sodium salt to be added to food (24). Another amino acid made with C. glutamicum is L-lysine, which is required for animal nutrition. Indeed, over the years, C. glutamicum strains have been developed for the production of almost all the L-amino acids with commercial potential. For instance, our own group has contributed to the development of C. glutamicum strains producing L-isoleucine (7), L-valine (30), L-threonine (5), and L-serine (37).Obviously, given the success of amino acid production by C. glutamicum and other bacteria, and the fact that sugar is a renewable substrate, bacterial production of further compounds has great appeal. Processes to enable the production of succinate (28), lactate (29), cadaverine (14), putrescine (32), 2-oxoisovalerate (19), and isobutanol (35) by C. glutamicum have been described. D-Amino acids constitute another interesting group of products, although their direct biotechnological production by bacteria has not yet been investigated. A significant reason is probably that it is unclear whether the cell can tolerate the coexistence of D-and L-amino acids. In fact, the growth of Currently, D-amino acids are produced ...

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Transmembrane Amino Acid Flux in Bacterial Cells

Cited by 26 publications

References 234 publications

Export of l -Isoleucine from Corynebacterium glutamicum : a Two-Gene-Encoded Member of a New Translocator Family

Export of l -Isoleucine from Corynebacterium glutamicum : a Two-Gene-Encoded Member of a New Translocator Family

Membrane alteration is necessary but not sufficient for effective glutamate secretion in Corynebacterium glutamicum

Corynebacterium glutamicum as a Host for Synthesis and Export of d -Amino Acids

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