Na ⁺ -Stimulated Transport of l -Methionine in Brevibacterium linens CNRZ 918

Abstract

The transport of L-methionine by the gram-positive species Brevibacterium linens CNRZ 918 is described. The one transport system (Km = 55 ,uM) found is constitutive for L-methionine, stereospecific, and pH and temperature dependent. Entry of L-methionine into cells is controlled by the internal methionine pool. Competition studies indicate that L-methionine and aL-aminobutyric acid share a common carrier for their transport. Neither methionine derivatives substituted on the amino or carboxyl groups nor D-methionine was an inhibitor, whereas powerful inhibition was shown by L-cysteine, s-methyl-L-cysteine, DL-selenomethionine and DL-homocysteine. Sodium plays important and varied roles in L-methionine transport by B. linens CNRZ 918: (i) it stimulates transport without affecting the Km, (ii) it increases the specific activity (on a biomass basis) of the L-methionine transport system when present with methionine in the medium, suggesting a coinduction mechanism. L-Methionine transport requires an exogenous energy source, which may be succinic, lactic, acetic, or pyruvic acid but not glucose or sucrose. The fact that L-methionine transport was stimulated by potassium arsenate and to a lesser extent by potassium fluoride suggests that high-energy phosphorylated intermediates are not involved in the process. Monensin eliminates stimulation by sodium. Gramicidin and carbonyl cyanide-m-chlorophenylhydrazone act in the presence or absence of Na+. N-Ethylmaleimide, p-chloromercurobenzoate, valinomycin, sodium azide, and potassium cyanide have no or only a partial inhibitory effect. These results tend to indicate that the proton motive force reinforced by the Na+ gradient is involved in the mechanism of energy coupling of L-methionine transport by B. linens CNRZ 918. Thus, this transport is partially similar to the well-described systems in gram-negative bacteria, except for the role of sodium, which is very effective in B. linens, a species adapted to the high sodium levels of its niche.

Characterization of amino acid transport in the dairy strain Leuconostoc mesenteroides subsp. mesenteroides CNRZ 1273

“…Cells grown to mid-exponential phase were harvested by centrifugation at 4°C, washed twice with 50 mmol l )1 potassium phosphate, pH 6AE0, and suspended in the same buffer. For transport measurement, cells were energized for 2 min at 30°C with 20 mmol l )1 glucose; then transport was initiated by addition of a labelled amino acid, and at specific time intervals aliquots were removed to measure radioactivity (Ferchichi et al 1987;Gendrot et al 2000). The initial rate of transport was determined from the initial slopes of transport kinetic curves.…”

Transport and deamination of amino acids by a gram-positive, monensin-sensitive ruminal bacterium

“…Early work with Escherichia coli indicated that bacterial transport could be coupled to protons (15,16), but subsequent experiments showed that glutamate transport was stimulated by Na (11). It has since become apparent that Na gradients can serve as a driving force for transport in a variety of bacteria (10,18,27,30). The rumen is a Na-rich environment (approximately 90 mM), and recent work indicated that the ruminal bacteria Streptococcus bovis (24) and Peptostreptococcus strain C (7) used Na symport mechanisms to take up neutral amino acids and branched-chain amino acids, respectively.…”

“…Proton-motive force. When exponentially growing cultures were incubated with [14C]TPP+ and corrected for nonspecific binding (cells treated with 5 or 50 uM valinomycin, 10 uM TCS, or 1% toluene), Aqi was only -3 mV. Very little ['4C]benzoate was taken up, and the intracellular pH (6.67) was lower inside than outside (pH 6.9).…”