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

The growth rate of Chromohalobacter salexigens DSM 3043 canbe stimulated in media containing 0.3 M NaCl by a 0.7 M concentrationof other salts of Na⁺, K⁺, Rb⁺, or NH₄⁺, Cl^-, Br^-, NO₃^-, orSO₄^2- ions. To our knowledge, growth rate stimulation by a generalhigh ion concentration has not been reported for any organismpreviously.

INTRODUCTION

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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 characterizationof the ion requirements of this organism and made the unexpectedfinding that while this organism requires moderate concentrationsof Na⁺ and Cl^- ions, its growth rate was stimulated by a numberof other salts, indicating that C. salexigens requires a combinationof NaCl and high ionic strength for optimal growth.

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

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Characterization of the cation...

For growth rate studies, C. salexigens DSM 3043 was culturedaerobically 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 originalconcentrations 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). Thestrain was unable to grow with 0.03 M Na⁺ plus 1.97 M K⁺ butwas able to do so with 0.23 M Na⁺ plus 1.77 M K⁺. Thus, althoughstrain DSM 3043 needs Na⁺, its requirement for this cation canbe reduced below the 0.5 M concentration suggested previously(2) if the total monovalent-ion concentration is maintainedat 2 M with KCl. The growth rate was 35 to 65% higher in thepresence of 1.77 M Na⁺ plus 0.23 M K⁺ than in the presence of0.23 M Na⁺ plus 1.77 M K⁺, indicating that high concentrationsof Na⁺ are more beneficial than high concentrations of K⁺. Ashas been observed previously (2), glycine betaine was stimulatoryat all Na⁺ and K⁺ concentrations, with the exception of 0.03M Na⁺ (which was inadequate to support growth).

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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 seenin the presence of high concentrations of Na⁺ is dependent specificallyon this cation, we determined the organism's growth rate inmedia containing 0.3 M NaCl and higher concentrations of othersalts or glucose (Fig. 2). The strain could not grow rapidlywith 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 equivalentto 0.7 M NaCl) did not support growth, indicating that the growthstimulation seen with the salts was not due to high osmolalityalone. Thus, the results presented in Fig. 1 and 2 suggest thatin addition to 0.2 to 0.3 M Na⁺ and/or Cl^- ions, for optimumgrowth, C. salexigens has a requirement for a high ion concentration,which can be satisfied by a 0.7 M concentration of a numberof ionic solutes, including the cations Na⁺, K⁺, Rb⁺, and NH₄⁺and the anions Cl^-, Br^-, NO₃^-, and SO₄^2-.

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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.

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C. salexigens DSM 3043...

We investigated whether C. salexigens DSM 3043 has a specificrequirement for Cl^- in experiments in which the strain was grownin the presence of various combinations of NaCl and NaNO₃. Figure3 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 inmedium containing 1 M Cl^- in the absence and presence of glycinebetaine, respectively. The lack of growth at a Cl^- concentrationof $<=$ 0.1 M in the presence of 1.0 M NO₃^- could not be attributedto inhibitory effects of the latter anion, not only because0.7 M NaNO₃ was stimulatory in the presence of 0.3 M NaCl (Fig.2) but also because the organism could grow in medium containing1.6 M NaNO₃ and 0.4 M NaCl at rates of 3.1 and 4.2 generations/dayin the absence and presence of glycine betaine, respectively(Fig. 3). These results show that C. salexigens DSM 3043 hasa Cl^- ion requirement of >0.1 M. We found that SO₄^2- couldnot substitute for Cl^- ions (data not shown), but we have notinvestigated whether the requirement for Cl^- ions could be metby Br^- or I^- as entire sources of anions.

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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.

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Conclusions.

The major new observation we made is that C. salexigens DSM3043 does not need high concentrations of NaCl. Provided thatthe medium contained 0.2 to 0.3 M concentrations of Na⁺ andCl^- ions, the growth rate of this organism was enhanced by anumber of salts of other ions, such as K⁺, Rb⁺, NH₄⁺, Br^-, NO₃^-,and SO₄^2-. Thus, C. salexigens DSM 3043 seems to grow optimallyin a highly ionic environment, and not necessarily in the presenceof high concentrations of NaCl alone. Vreeland and Martin reportedthat the moderate halophile Halomonas elongata 1H9 has a specificrequirement for Na⁺ which cannot be met by other cations, includingK⁺, Li⁺, Mg²⁺, or NH₄⁺ added as Cl^- salts (13). Thus, the responseof C. salexigens DSM 3043 to high concentrations of ions isvery different from those of other H. elongata strains. To ourknowledge, this is the first time that growth stimulation bynonspecific high ion concentrations has been reported for anyorganism. However, a generalized high ion concentration is notsufficient for C. salexigens DSM 3043; in addition, this organismrequires moderate concentrations of Na⁺, which may be used todrive Na⁺ gradient-dependent processes (5, 6, 8, 12).

In addition to requiring Na⁺ for growth, C. salexigens DSM 3043needs Cl^- ions at a concentration of >0.1 M, and NO₃^- cannotbe used in place of Cl^- ions. This observation points to a secondimportant difference between C. salexigens DSM 3043 and H. elongata1H9, because unlike C. salexigens DSM 3043, the latter organismwas able to use NO₃^- instead of Cl^- (13). It has been reportedthat Halobacillus halophilus has a requirement for Cl^- ions(10). However, that organism was able to adapt to use NO₃^- insteadof Cl^- (10), unlike C. salexigens, for which NO₃^- could notreplace Cl^- (Fig. 3). Like most other eubacteria, C. salexigensexcludes Cl^- from the cytoplasm and accumulates the zwitterionicorganic compounds ectoine, hydroxyectoine, and glycine betaineas compatible solutes (7, 11); therefore, the biochemical functionof Cl^- in C. salexigens needs to be elucidated.

ACKNOWLEDGMENTS

This work was supported by the National Science Foundation Lifein 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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