Acidinduced Genes Identified with Fusions

Mutants with genes transcriptionally activated at low pH have been obtained by using insertions carrying a promoterless gusA gene. The sequences surrounding the insertions have allowed identification of a number of them, including a putative regulator (phrR; Reeve et al. 1998), a membrane protein (IpiA), ABC transporters, kdpBC (for K+ transport), a peptide synthetase, and fixNO (for cytochrome oxidase ebb3). These insertions do not destroy acid-tolerance, though the functional products may be required to mount an effective ATR.

8.1. phrR gene. This gene is adjacent to the essential act A gene. Its sequence suggests that it encodes a regulator protein; it has an H-T-H motif for DNA-binding, but thus far we do not know what it regulates. It is induced 5-fold by low pH, but its responses to other stresses (copper, zinc, ethanol, hydrogen peroxide) indicate that it functions in a range of stress response systems. However, not all stresses induce its expression - it does not respond to high temperature, high sucrose concentration, phosphate starvation or in stationary phase. Its expression is not dependent on an intact actSR system, and we do not know how it is controlled. Its inactivation results in no loss of symbiotic effectiveness in nodulation tests with Medicago murex, nor is there any impairment of acid tolerance (Reeve et al. 1998).

8.2. IpiA gene. This gene is the most strongly induced by low pH of any of those identified using gusA fusions. Unlike phrR, its induction is specific to low pH - none of the other stresses to which phrR responds results in induction. It nodulates M. murex normally and effectively, and staining with X-glc indicates that it is strongly expressed in the nodules. Increasing concentrations of calcium markedly affect the pH at which induction occurs, the calcium preventing induction until a lower pH is applied. Its induction, like that of phrR, does not depend on an intact actSR system.

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