Kathleen O'Connor 2003 6334

Applied and Environmental Microbiology, October 2003, p . 6334-6336, Vol . 69, No . 10

The High Salt Requirement of the Moderate Halophile Chromohalobacter salexigens DSM3043 Can Be Met Not Only by NaCl but by Other Ions

Kathleen O'Connor and Laszlo N . Csonka^*

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392

Received 3 June 2003/ Accepted 25 July 2003

ABSTRACT

The growth rate of Chromohalobacter salexigens DSM 3043 can be stimulated in media containing 0.3 M NaCl by a 0.7 M concentration of other salts of Na⁺, K⁺, Rb⁺, or NH₄⁺, Cl^-, Br^-, NO₃^-, or SO₄^2- ions . To our knowledge, growth rate stimulation by a general high ion concentration has not been reported for any organism previously .

INTRODUCTION

The halophilic gram-negative bacterium Chromohalobacter salexigens (1) has been reported to require at least 0.5 M NaCl for growth (2) . In this study, we carried out a more comprehensive characterization of the ion requirements of this organism and made the unexpected finding that while this organism requires moderate concentrations of Na⁺ and Cl^- ions, its growth rate was stimulated by a number of other salts, indicating that C . salexigens requires a combination of NaCl and high ionic strength for optimal growth .

Characterization of the cation requirements of C . salexigens DSM 3043.

For growth rate studies, C . salexigens DSM 3043 was cultured aerobically at 37°C in a modified form of M63 medium (3), consisting of 0.10 M KH₂PO₄, 0.075 M KOH, and 0.015 M (NH₄)₂SO₄, supplemented with MgSO₄ and FeSO₄ · 7H₂O, whose original concentrations in M63 were increased to 8.0 and 0.1 mM, respectively, as suggested by Martin et al . (9) . The Na⁺ requirement of C . salexigens was determined in media containing 2.0 M Cl^- salts, made up of various combinations of NaCl and KCl (Fig . 1) . The strain was unable to grow with 0.03 M Na⁺ plus 1.97 M K⁺ but was able to do so with 0.23 M Na⁺ plus 1.77 M K⁺ . Thus, although strain DSM 3043 needs Na⁺, its requirement for this cation can be reduced below the 0.5 M concentration suggested previously (2) if the total monovalent-ion concentration is maintained at 2 M with KCl . The growth rate was 35 to 65% higher in the presence of 1.77 M Na⁺ plus 0.23 M K⁺ than in the presence of 0.23 M Na⁺ plus 1.77 M K⁺, indicating that high concentrations of Na⁺ are more beneficial than high concentrations of K⁺ . As has been observed previously (2), glycine betaine was stimulatory at all Na⁺ and K⁺ concentrations, with the exception of 0.03 M Na⁺ (which was inadequate to support growth) .

FIG . 1 . Effect of Na⁺ on the growth rate of C . salexigens DSM 3043 in the presence of a constant 2.0 M concentration of Cl^- salts . The growth rate of the strain was determined in modified M63 medium (M63-5x Mg-Fe; see text) supplemented with 10 mM glucose, the indicated concentrations of Na⁺ (supplied as NaCl), and KCl, added at concentrations such that the sum of the NaCl and KCl was constant at 2.0 M . When used, glycine betaine was added at 1 mM . Cells were first grown to saturation in liquid Luria-Bertani medium (4) with 1 M NaCl, subcultured at a 1:100 dilution into M63-5x Mg-Fe-10 mM glucose-2.0 M NaCl, and grown to saturation . The cells were subcultured at a 1:50 dilution into M63-5x Mg-Fe-10 mM glucose containing various combinations of NaCl and KCl, and the growth rates were determined from the increases in cell density, measured as light scattering at 600 nm (A₆₀₀), as a function of time . The final density in all cultures containing

0.23 M Na⁺ was

2 x 10⁹ cells/ml (A₆₀₀ = 1.2 to 1.5).

To address whether the optimal growth of C . salexigens seen in the presence of high concentrations of Na⁺ is dependent specifically on this cation, we determined the organism's growth rate in media containing 0.3 M NaCl and higher concentrations of other salts or glucose (Fig . 2) . The strain could not grow rapidly with 0.3 M NaCl in the absence of additional salts . However, augmentation of this medium with 0.7 M NaCl, NaBr, NaNO₃, Na₂SO₄, KCl, RbCl, or NH₄Cl resulted in a marked stimulation of growth . Glucose at a concentration of 1.1 M (osmotically equivalent to 0.7 M NaCl) did not support growth, indicating that the growth stimulation seen with the salts was not due to high osmolality alone . Thus, the results presented in Fig . 1 and 2 suggest that in addition to 0.2 to 0.3 M Na⁺ and/or Cl^- ions, for optimum growth, C . salexigens has a requirement for a high ion concentration, which can be satisfied by a 0.7 M concentration of a number of ionic solutes, including the cations Na⁺, K⁺, Rb⁺, and NH₄⁺and the anions Cl^-, Br^-, NO₃^-, and SO₄^2- .

FIG . 2 . Effect of various salts on the growth rate of C . salexigens DSM 3043 . Cells were grown as described in the legend to Fig . 1, except that they were subcultured from Luria broth with 1 M NaCl into M63-5x Mg-Fe-10 mM glucose-1.0 M NaCl . Growth rates were determined after a second subculture into M63-5x Mg-Fe-10 mM glucose-0.3 M NaCl-0.7 M salt or 1.1 M glucose . The final density of all cultures was

2 x 10⁹ cells/ml (A₆₀₀ = 1.2 to 1.5), except for those grown in 0.3 M NaCl or 0.3 M NaCl-1.1 M glucose (in which case there was no growth).

	C . salexigens DSM 3043 requires Cl^- ions.

We investigated whether C . salexigens DSM 3043 has a specific requirement for Cl^- in experiments in which the strain was grown in the presence of various combinations of NaCl and NaNO₃ . Figure 3 shows that the organism was not able to grow in media containing 0.1 M Cl^- plus 1.0 M NaNO₃ but was able to grow at a rate of 5 generations/day in the presence of 0.3 M Cl^- plus 0.7 M NaNO₃ . The growth rate was increased to 10 and 11 generations/day in medium containing 1 M Cl^- in the absence and presence of glycine betaine, respectively . The lack of growth at a Cl^- concentration of 0.1 M in the presence of 1.0 M NO₃^- could not be attributed to inhibitory effects of the latter anion, not only because 0.7 M NaNO₃ was stimulatory in the presence of 0.3 M NaCl (Fig . 2) but also because the organism could grow in medium containing 1.6 M NaNO₃ and 0.4 M NaCl at rates of 3.1 and 4.2 generations/day in the absence and presence of glycine betaine, respectively (Fig . 3) . These results show that C . salexigens DSM 3043 has a Cl^- ion requirement of >0.1 M . We found that SO₄^2- could not substitute for Cl^- ions (data not shown), but we have not investigated whether the requirement for Cl^- ions could be met by Br^- or I^- as entire sources of anions .

FIG . 3 . C . salexigens requires Cl^- ions for growth . The cells were grown as described in the legend to Fig . 2 . Growth rates were determined after a second subculture into M63-5x Mg-Fe-10 mM glucose containing the indicated concentrations of Cl^- and NO₃^-, added as Na⁺ salts . The final density of all cultures was

2 x 10⁹ cells/ml (A₆₀₀ = 1.2 to 1.5), except for those containing

0.1 M NaCl (in which case there was no growth).

	Conclusions.

The major new observation we made is that C . salexigens DSM 3043 does not need high concentrations of NaCl . Provided that the medium contained 0.2 to 0.3 M concentrations of Na⁺ and Cl^- ions, the growth rate of this organism was enhanced by a number of salts of other ions, such as K⁺, Rb⁺, NH₄⁺, Br^-, NO₃^-, and SO₄^2- . Thus, C . salexigens DSM 3043 seems to grow optimally in a highly ionic environment, and not necessarily in the presence of high concentrations of NaCl alone . Vreeland and Martin reported that the moderate halophile Halomonas elongata 1H9 has a specific requirement for Na⁺ which cannot be met by other cations, including K⁺, Li⁺, Mg²⁺, or NH₄⁺ added as Cl^- salts (13) . Thus, the response of C . salexigens DSM 3043 to high concentrations of ions is very different from those of other H . elongata strains . To our knowledge, this is the first time that growth stimulation by nonspecific high ion concentrations has been reported for any organism . However, a generalized high ion concentration is not sufficient for C . salexigens DSM 3043; in addition, this organism requires moderate concentrations of Na⁺, which may be used to drive Na⁺ gradient-dependent processes (5, 6, 8, 12) .

In addition to requiring Na⁺ for growth, C . salexigens DSM 3043 needs Cl^- ions at a concentration of >0.1 M, and NO₃^- cannot be used in place of Cl^- ions . This observation points to a second important difference between C . salexigens DSM 3043 and H . elongata 1H9, because unlike C . salexigens DSM 3043, the latter organism was able to use NO₃^- instead of Cl^- (13) . It has been reported that Halobacillus halophilus has a requirement for Cl^- ions (10) . However, that organism was able to adapt to use NO₃^- instead of Cl^- (10), unlike C . salexigens, for which NO₃^- could not replace Cl^- (Fig . 3) . Like most other eubacteria, C . salexigens excludes Cl^- from the cytoplasm and accumulates the zwitterionic organic compounds ectoine, hydroxyectoine, and glycine betaine as compatible solutes (7, 11); therefore, the biochemical function of Cl^- in C . salexigens needs to be elucidated .

ACKNOWLEDGMENTS

This work was supported by the National Science Foundation Life in Extreme Environments program (grant MCB-9978253) .

FOOTNOTES

* Corresponding author . Mailing address: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392 . Phone: (765) 494-4969 . Fax: (765) 496-1496 . E-mail: lcsonka{at}bilbo.bio.purdue.edu .

REFERENCES

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