Ammonia acquisition in enteric bacteria: Physiological role of the ammonium/methylammonium transport B (AmtB) protein

Abstract

Homologues of the amtB gene of enteric bacteria exist in all three domains of life. Although their products are required for transport of the ammonium analogue methylammonium in washed cells, only in Saccharomyces cerevisiae have they been shown to be necessary for growth at low NH 4 ؉ concentrations. We now demonstrate that an amtB strain of Escherichia coli also grows slowly at low NH 4 ؉ concentrations in batch culture, but only at pH values below 7. In addition, we find that the growth defect of an S. cerevisiae triple-mutant strain lacking the function of three homologues of the ammonium͞methylammonium transport B (AmtB) protein [called methylammonium͞ammonium permeases (MEP)] that was observed at pH 6.1 is relieved at pH 7.1. These results provide direct evidence that AmtB participates in acquisition of NH 4 ؉ ͞NH 3 in bacteria as well as eucarya. Because NH 3 is the species limiting at low pH for a given total concentration of NH 4 ؉ ؉ NH 3 , results with both organisms indicate that AmtB͞MEP proteins function in acquisition of the uncharged form. We confirmed that accumulation of [ 14 C]methylammonium depends on its conversion to ␥-Nmethylglutamine, an energy-requiring reaction catalyzed by glutamine synthetase, and found that at pH 7, constitutive expression of AmtB did not relieve the growth defects of a mutant strain of Salmonella typhimurium that appears to require a high internal concentration of NH 4 ؉ ͞NH 3 . Hence, contrary to previous views, we propose that AmtB͞MEP proteins increase the rate of equilibration of the uncharged species, NH 3 , across the cytoplasmic membrane rather than actively transporting-that is, concentrating-the charged species, NH 4 ؉ .

Ammonium Toxicity in Bacteria

“…A possible explanation of this observation is an energyindependent transport by ammonium channels, which facilitates diffusion, rather than by permeases, which concentrate ammonium inside the cell by an energydependent transport mechanism. This kind of transport mechanism was first suggested by Soupene and coworkers for ammonium transporter AmtB from Salmonella typhimurium [32,33] and was further supported by the recently published structure of E. coli AmtB [17] as well as by transport data [14].…”

“…In ammonium-rich medium, the diffusion of NH 3 across the cytoplasmic membrane is sufficient to promote bacterial growth, and wild-type cells of E. coli [32], B. subtilis [10], and C. glutamicum [23,31] synthesize special ammonium transporters only in a situation of nitrogen limitation. As one reason for this strict regulation, prevention of a putative energy-wasting futile cycling of ammonium was discussed.…”

Control of acute, chronic, and constitutive hyperammonemia by wild-type and genetically engineered Lactobacillus plantarum in rodents

“…36 Especially when the pH is below 7.0, AmtB could participate to the acquisition of NH 4 ϩ / NH 3 and could be involved in ammonia consumption in the gut. [37][38][39] The mutant strain AmtB Ϫ did not show any effect on blood ammonia, survival rate, and fecal ammonia in our hepatitis-induced HA mouse model. A similar survival of this genetically engineered L. plantarum strain compared to the wild-type strain was observed into the gut, showing that the difference observed after treatment with L. plantarum AmtB Ϫ is not due to bacterial death or to a lower number of CFU.…”

The NtcA-activated amt1 gene encodes a permease required for uptake of low concentrations of ammonium in the cyanobacterium Synechococcus sp. PCC 7942 The GenBank accession number for the nucleotide sequence of the amt1 gene described in this paper is AJ311900.

“…This proposed role for the Amt1 protein is consistent with the observed regulation of the amt1 gene, whose expression is increased in the absence of added ammonium. The concentration of ammonia present in solutions of high ammonium concentration appears to be sufficient to permit entrance into the cell, by diffusion, of an amount of ammonia high enough to support cell growth (Soupene et al, 1998).…”