produces an acid-inducible form of ADC, encoded by adiA
, that contributes to the survival of this organism in extremely acidic environments (4
). Mutants of E
that do not possess this enzyme fail to demonstrate arginine-dependent resistance to pH 2.5. Inducible amino acid decarboxylase systems typically possess, along with the amino acid decarboxylase, an antiporter that imports an extracellular amino acid substrate in exchange for the intracellular decarboxylation product. This exchange is needed to constantly replenish intracellular substrate and rid the cell of product. Prior to this report, the identity of the arginine:agmatine antiporter was unknown.
Twenty six Tn10 insertion mutants specifically defective in arginine-dependent acid resistance were found to have mutations located within or upstream of adiC (yjdE), a gene predicted to encode an antiporter. Mutants lacking AdiC (YjdE) possessed normal levels of ADC activity, indicating that the gene does not regulate adiA expression or function. However, the mutants failed to convert extracellular arginine to agmatine, clearly supporting a role for AdiC as the requisite arginine:agmatine antiporter.
Northern blot analysis indicated that adiC is induced by growth under acidic conditions, but transcription appears to be independent of the adiAY promoter. Three transcripts were observed in the adi region, namely, (i) adiAY (minor), (ii) adiA (major), and (iii) adiC. These transcripts appear to result from two promoters, one before adiA and one preceding adiC. The finding that six of the Tn10 insertions eliminating AdiC activity occurred upstream of the adiC open reading frame but not within adiY is consistent with adiC having an independent promoter (Fig. ).
The data presented also revealed that maximal activity of the ADC-antiporter system in whole cells occurs at pH 2.5. At this pH, other transporters of arginine likely do not function, as evidenced by the failure of adiA and adiC mutants to remove arginine from the extracellular medium. Although adiC expression is induced by low pH, it is not known whether AdiC antiporter activity is directly under pH control or is constitutively active but used only at a pH where intracellular ADC is active. In either case, we predict that the arginine-dependent acid resistance system, to work efficiently at an external pH of 2.5, will maintain intracellular pH around 5, the optimum pH for inducible ADC. Proton consumption would be maximal in this pH range for this system.
locus also includes the gene adiY
located between adiA
. Earlier reports indicated that AdiY, a member of the XylS/AraC family of transcriptional regulators, was a positive regulator of adiA
). In that study, overexpressing AdiY increased adiA
transcription. However, in our screen for mutants defective in arginine-dependent acid resistance, no adiY
mutants emerged. The targeted deletion of adiY
also failed to alter arginine-dependent acid resistance under the conditions tested (data not shown). Another regulator controlling adiA
is CysB (4
). CysB mutants are clearly defective in arginine-dependent acid resistance. Thus, as is the case for the glutamate decarboxylase (gadA/BC
)-dependent acid resistance system, there may be multiple, and perhaps redundant, regulators for adiA
. The AraC-like regulator GadX, for example, is needed to activate the gadA/BC
genes only when cells are grown in complex media, not in minimal salts media (12
). AdiY may fill an analogous role as a conditional regulator of the arginine-dependent system.
In sum, the inducible arginine:agmatine antiporter required for arginine-dependent acid resistance has been identified. This discovery will allow direct comparison between the arginine:agmatine antiporter and the putative glutamate:GABA antiporter employed by the glutamate-dependent acid resistance system. In addition, questions of pH control, exchange rates, and membrane configurations can now be addressed.