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Acid tolerance in legume root nodule bacteria and selecting for it

M. J. Dilworth, J. G. Howieson, W. G. Reeve, R. P. Tiwari and A. R. Glenn

Australian Journal of Experimental Agriculture 41(3) 435 - 446
Published: 2001


Bacteria face a variety of problems in trying to survive and grow in acidic environments. These include maintaining intracellular pH (pHi) in order to protect internal cell components, modifying or abandoning those external structures inevitably exposed to acidity, and resisting stresses whose interaction with pH may be the actual determinant of survival or growth rather than H+ toxicity per se.

An important aspect of acid resistance in Gram-negative bacteria (including the root nodule bacteria) is the adaptive acid tolerance response (ATR), whereby cells grown at moderately acid pH are much more resistant to being killed under strongly acidic conditions than are cells grown at neutral pH. Survival during pH shock is also markedly affected by the calcium concentration in the medium. The pH at which commercial legume inoculants are grown and supplied for inoculation into acid soils may therefore be of considerable importance for initial inoculant survival.

The mechanisms of resistance to acidity in root nodule bacteria have been investigated via 3 approaches: (i) creation of acid-sensitive mutants from acid-tolerant strains, and identification of the genes involved; (ii) random insertion of reporter genes to create mutants with pH-dependent reporter expression; and (iii) proteomics and identification of proteins regulated in response to acidity.

The results of the first approach, directed at genes essential for growth at acid pH, have identified a sensor–regulator gene pair (actS–actR), a copper-transporting ATPase (actP), and another gene involved in lipid metabolism (actA), inactivation of which results in sensitivity to heavy metals. While the ActS–ActR system is undoubtedly required for both acid tolerance and the ATR, it is also involved in global regulation of a wide range of cellular processes.

The second approach has allowed identification of a range of acid-responsive genes, which are not themselves critical to growth at low pH. One of these (phrR) is itself a regulator gene induced by a range of stresses including acid pH, but not controlled by the ActS–ActR system. Another, lpiA, responds specifically to acidity (not to other stresses) and may well be an antiporter related to nhaB, which is involved in Na+ transport in other bacteria.

The third approach indicates a number of proteins whose concentration changes with a switch from neutral to acidic growth pH; most of these seem to have no homologues in the protein databases, while the blocked N-terminal sequences of others have prevented identification.

It has been common experience that strains of root nodule bacteria selected for acid tolerance in the laboratory are not necessarily successful as inoculants in acid soils. In the light of the complex interactive effects on growth and survival of H+, Ca2+ and Cu2+ concentrations in our studies, this lack of correlation is no longer surprising. It remains to be seen whether it will be possible to improve the correlation between growth on laboratory media and performance in acid soils by determining which strains show an ATR, and by screening on media with defined ranges of concentration of some of these critical metal ions, perhaps approximating those to be expected in the soils in question.

© CSIRO 2001

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