Influence of salt concentration on the cellular fatty acid ... - ScienceDirect
~) INSTITUTPASTEUPJELSEVIER Paris 1998
Res. Microbiol. 1998, 149, 6 7 5 - 6 7 9
Influence of salt concentration on the cellular fatty acid composition of the moderately halophilic bacterium Halomonas salina M.J. Valderrama t*), M. Monteoliva-Sanchez (**), E. Quesada and A. Ramos-Cormenzana Department of Microbiology, Facul~ of Pharmacy, Universi~ of Granada, Granada (Spain)
SUMMARY
The cellular fatty acid composition of Halomonas salina, a moderately halophilic bacterium grown at different salt concentrations, is reported. Fatty acids C16:0 and C18:1 were major components and significant amounts of C16:1, C18:0 and cyc-C19:0 were also detected. The results showed clear chemotaxonomic relationships with recognized members of the genus Halomonas. The salt concentration greatly influenced the fatty acid composition, suggesting activation of cyciopropane synthetase at high levels of salt, since increases in cyclopropane fatty acids with decreases in monounsaturated fatty acids were observed as the salt concentration in the medium rose.
Key-words: Fatty acid, Halomonas safina, Salt concentration.
INTRODUCTION Halophiles, which constitute a wide group of organisms including protozoa, algae and bacteria, are characterized by their requirement for a high concentration of inorganic salts, mostly sodium chloride, to grow and survive. They inhabit saline environments where several conditions of osmolaxity and ionic strength challenge the stability of lipid bilayers and the structure of proteins, forcing those organisms to develop specialized molecules and physiological mechanisms to cope with this environmental stress. Among these adaptational properties, the changes in fatty acid com-
position may play an important role in regulating the membrane lipid phase (Russell, 1989). On the other hand, the cellular fatty acid composition has been shown to be a useful chemotaxonomic marker (Minnikin et al., 1984; Jantzen and Bryn, 1985; Komagata and Suzuki, 1987; Welch, 1991 ). The genus Deleva was constructed by Baumann et al. (1983) to include five species isolated from marine habitats that were slight halophiles specifically requiring added NaCI for growth. In 1984, Quesada et al. described the first moderately halophilic species in this genus, Del~3"a halophila, isolated from hypersaline soil. Deleva salina is a moderately halophile species of the
Submitted June 17, 1998, accepted August 1 I, 1998. (*) New address: Department of Microbiology, Faculty of Biology, Universidad Complutense de Madrid, Madrid. (**) Corresponding address: M. Monteoliva-Sanchez, Departamento de Microbiologia, Facuitad de Farmacia, Campus Universitario de Cartuja, 18071 Granada (Spain).
676
M.J. VALDERRAMA ET AL.
genus Deleya (Valderrama et al., 1991) with a strong eurihaline character, as it is able to grow between 2.5 and 25 % (wt/vol) salts, x. ith optimal growth at 5% (wt/vol) salts and 32°C. In 1996, Donson and Franzmann, by dete~nafion of 16S r ~ A sequences, proposed ~at members of the genus Deleya should be placed in the genus Halomonas. This paper presents the cellular fatty acid composition of Halomonas salina (previously Deleya salina) together with the modifications observed in that composition as a consequence of exposure to environments of differing salt concentrations.
MATERIALS AND METHODS Organisms and growth conditions The type strain of H. salina (strain F8-10=ATCC 49509), a moderately halophilic species, was used in this study. This species was isolated from a hypersaline soil found in Aiicante, Spain, and the description and reclassification have been previously reported (Valderrama et al., 1991 ; Mellado et al., 1996 ~Donson and Franzmann, 1996). Cells were grown in the MH complex medium described by Quesada et ai. (1983). Its composition is as follows: proteose-peptone no 3 (Difco), 5 g/l, yeast extract (Difco), 10 g~; and glucose, 1 g/I, supplemented with a balanced mixture of sea salts (Rodriguez-Valem et al., 1981)to give a final concentration of 2.5, 7,5 and 20% (w/v). Cultures were incubated at 32°C in a rotato~ shaker and harvested in early-exponential, exponential and stationary phase of growth by cold cen~fugation at 1 5 , ~ g tbr 15 min. Cells were washed twice with cold saline solution at the appropriate salt concentration, and lyophilized.
RESULTS The results of the analysis of fatty acid methyl esters from H. salina are given in table I. Under optimal culture conditions (7.5 %, w/v, salts), the major fatty acid components of cells harvested in the stationary phase of growth were C l6:0 and C lS:l, together with significant proportions of C 18:0; significant amounts of C 16: l and cyc-Cl9:0 were also detected. Only very small amounts of branched acids appeared, and acids of short chain length, those of greater than 20 carbons and hydroxy acids were not detected. The fatty acid composition of H. salina was altered when it was grown in media containing different salt concentrations (table 1; fig. I). For each phase of growth, increases in the proportion of cyclopropane (cyc-Cl7:0 and cyc-Cl9:0) and saturated fatty acids (Cl6:0 and C18:0) could be detected as the salt concentration of the growth medium rose. In contrast, the relative amounts of the mono-unsaturated C 16: l and C 18: l fatty acids decreased. In order to study whether the phase of growth influenced the fatty acid composition, cells in the early exponential, exponential and stationary phases were analysed (table I; fig. 2). Increased relative proportions of saturated fatty acids (C 16:0+C 18:0) and decreased respective unsaturated acids were observed in old cultures when the cells were grown at 7.5% and 20% (w/v) salts. The opposite occurred in the presence of 2.5 % salts.
DISCUSSION Fatty acid analysis Fatty acid methyl esters (FAME) were prepared from lyophilized cells (50 rag) following methods described by Monteoliva-Sanchez and Ramos-Cormenzana (1987). The FAMEs were examined by gas-liquid chromatography in a Carlo Erba Fractoyap 2 2 ~ instrument equipped with a flame ionization detector. A 2-m glass column packed with 15 % diethylene glycol succinate coated on 100/120 mesh G a s - ~ m P was used. Standards of FAMEs were obtained from Applied Science Lab., Pennsylvania (USA).
As with the taxonomic characterization of halophilic eubacteria, examination of the fatty acid profiles of the cells and the relationships to salt tolerance has lagged somewhat behind other developments. This study is the first report on the fatty acid composition of H. salina and has revealed that it is significantly affected by different salinities in the growth medium. The fatty acid profile of H. salina cultured in 7.5 % (w/v) salts is similar to that reported for
F A T T Y A C I D C O M P O S I T I O N OF H A L O M O N A S S A L I N A
677
T a b l e | . Fatty acid composition o f H. salina as a function o f phase of g r o w t h and salt concentration.
2.5 E
Fatty acid
EE
C12:0 C13:0 Ci4:0 CI5:0 CI6:0 C17:0 CI8:0 C19:0 C20:0 C!6:1 CI8:1 br-Cl4:0 br-Cl5:0 br-Cl6:0 br-Cl7:0 c y c - C 17:0 cyc-Cl9:0
--T 0.2 31.4 . 2.5 0.2 1.6 19.1 36.3 0.2 0.4 . 1.7 1.9 4.3
S
T T ~ 1.3 32.1 .
Salt concentration (% w/v) 7.5 EE E S
T 0.4 T i.7 26.8 . 1.9 ~ 0.9 22.6 38.4 ~ 0.7
. 2.7 T 0.5 15.9 42.9 ~ 1.5 .
. 0.8 0.6 1.3
. -0.2 0.9 38.7
.
. 0.2 T 2.3 41. I T 8.4 T 0.4 14.2 27.9 T T
0.3 ~ T 15.2 34.9 0.3 T .
.
0.3 3.1 2.9
. 0.6 4.9 3.8
.
. 1.2 0.7 3.2
. -T 1.4 38.0 T 14.9 . . 1.2 2.1 32.0 ~ 2.2 . T 0.5 7.6
20 E
EE
T 0.1 T 0.3 41.5 -8.6
T T T 0.8 42.0 0.5 10.1 T T 1.8 5.4 10.2 26.8 22.3 . . . . ~ T
~ T 39.8 0.3 8.8 . 2.1 10.4 27.6 0.3 T . T 6.9 3.6
.
2.4 3.6 10.6
T 4.0 5.2
Values are expressed as percentage of the total and are means of three determinations. EE = early exponential ; E = exponential" S = stationary ; dash = not detected" T = trace content of less than 0. 1% ; br = branched acid; cyc = cyclopropane acid.
_o F-
GROWTHPHASE
30
S
z
A 0
SALT CONCENTRATION
EXPONENTIAL
t
i 2.s~,
20 STAIlOflARY
EARLY
10
7.596
+
i
20%
--"C)-- I
EXPONENTIAL i t ...............................................................
u~
.....
I
I
I
2.5
7.5
20
i
EE
E
S
SALT CONCENTRATION (%) GROWTH PHASE
Fig. I. Effect of salt c o n c e n t r a t i o n of the ratio of monounsaturated ( C I 6 : 1 + C i 8 : 1 ) and cyclopropane fatty acids (cyc-Cl7:0+cyc-Cl9:0) of H. salina at different phases of growth (early exponential, exponential and stationary).
Fig. 2. Effect of phase of growth (EE, early ext,oncnt i a l ; E, e x p o n e n t i a l ; S, .,,tationary) on the ratio of monounsaturated (C16: i+C18: !) and saturated fat~.y acids (C16:0+C!8:0) of H. salina grown at different salt concentrations (%, w/v).
other moderately halophilic Gram-negative rods
et al., 1 9 8 8 ; F r a n z m a n n a n d T i n d a l l , 1 9 9 0 ; R u s -
( H a n n a et al., 1 9 8 4 ; M o n t e o l i v a - S a n c h e z a n d Ramos-Cormenzana, 1987; Monteoliva-Sanchez
sell, 1993). The major components are C16:0 and C18:1, and include significants a m o u n t s of
678
M.J. VALDERRAMA ET AL.
C18:0 and C16:1 (table I). Our results are in close agreement with the chemotaxonomic study carried out by Franzmann and Tindall (1990) on members of the family Halomonadaceae, i.e. validly described species of the genus Halomollt'lS.
Differences were found, however, in e.g. cyclopropane fatty acids cyc-Cl7:0 and cycCl9:0 composition. H. salina contained lower proportions of these (table l) than reported for another species of the genus Halomona,~ (previously designated as Deleya) by Franzmann and Tindall (1990). Our results more closely resembled the composition found for D. halophila, now H, halophila, by Monteoliva-Sanchez et al. (1988). Differences in compositional fatty acids have been reported as a consequence of the distinct culture media used (Drucker, 198 l). With respect to the influence of the phase of growth on the cellular fatty acid composition in H. salina, the results obtained when the organisms were grown at three different saline concentrations are shown in figure 2. At 7.5 and 20% (w/v) salts, the relative proportions of saturated and monounsaturated fatty acids change with the growth stages" an increase in saturated (CI6:0+C18:0) and a decrease in unsaturated (CI6:1 +C18:1) was observed with the age of the cultures. Younger cells of Pseudomonas halosaccharolytica also contained more C 16 and C 18 monoenoic acids (Monteoliva-Sanchez et al,, 1993), but in this microorganism, higher amounts of cyc-Cl9:0 were found in cells of the late logarithmic and stationary stages. However, no significant changes in the fatty acid composition as a consequence of phase of growth were r e p o r t e d in Vibrio c o s t i c o l a (Hanna et al., 1984), now designated as Salinivibrio costicola by Mellado et al. (1996), or Halomonas halophila (Monteoliva-Sanchez et al., 1988). The predominant alterations in fatty acid composition of H. salina, in response to variable salt concentrations in the culture medium, are shown in figure I. For each phase of growth studied, increasing the salt c o n c e n t r a t i o n resulted in a decrease in the proportion of mono-
unsaturated fatty acids, with a concomitant increase in cyclopropane fatty acids; the relative proportion of saturated fatty acids was also increased as a consequence of raising the salt concentration. There are few comparable date available in the literature concerning moderate halophiles. H. halophila (Monteoliva-Sanchez et al., 1988) and Pseudomonas halossaccharolytica (Monteoliva-Sanchez et al., 1993) showed similar adaptational fatty acid patterns. However, S. costicola did not show a significant degree of cyclization of fatty acids when cultured at a high salt concentration (Hanna et aL, 1984), since cyclopropanoic acids were not detected by the authors in that study. This particular microorganism showed a slightly different fatty acid response: total monounsaturated acids were highest at the optimal copcentrations, whereas total saturated fatty acids showed the opposite effect. Our results are more closely related to those reported for H. halophila by Monteoliva-Sanchez et al. (1988), which on the other hand, agree with the taxonomic relationship between this species and H. salina. The results presented here on the modification of fatty acid of the H. salina are in accord with the hypothesis concerning the activation or induction of cyclopropane synthetase when there is a high salinity in the medium and the bacteria show increases in compatible solutes (Monteoliva-Sanchez et al., 1993). The physical properties of acyl lipids containing cyclopropane chains (e.g., cyc-17:0) can be considered to be intermediate between the corresponding saturated fatty acid (C17:0) and the unsaturated fatty acid from which it was derived (C16:1). Therefore, an increase in cyclopropane fatty acid content at the expense of unsaturated fatty acid would tend to decrease membrane fluidity (Russell, 1993). In this sense, the increase in the proportions of cyclopropane tatty acids could contribute to preserving the membrane integrity of some halophilic bacteria, since they could prevent the formation of abnormal structures (Harwood and Russell, 1984; Russell and Kogut, 1985; Quin, 1986). Further studies are necessary in which fatty acid composition and membrane properties will be compared in cultures grown over a range of salinities.
FATTY ACID COMPOSITION OF HALOMONAS SAL|NA
Acknowledgements This investigation was partially supported by grants to the Grupo de lnvestigaci6n CVI 0190 from Junta de Andalucia, Spain.
Influence de la concentration saline sur la composition en acides gras cellulaires de Halomonas salina, bact6rie mod6r6ment halophile Les acides gras cellulaires de H a l o m o n a s salina, une bact6rie m o d 6 r f m e n t h a l o p h i l e , c u l t i v d c "~ diverses concentrations salines, ont 6t6 6tudi6s. Les acides gras en C i 6 : 0 et CI8:1 sont des composants majoritaires; des quantit6s significatives d'acides en C16:1, C18:8 et c y c - C l 9 : 0 ont 6t6. dfcel6es. Lcs r6sultats illustrent nettement les relations chimiot a x o n o m i q u e s avec des repr6sentants c o n n u s du genre Halomonas. La concentration saline influence amplement la composition en acides gras, ce qui sugg6re une activation de la cyclopropane-synth6tase ?a des concentrations salines 61evfes: une augmentation des acides gras h cyclopropane et une diminution des a c i d e s m o n o - i n s a t u r 6 s sont o b s e r v 6 e s Iors de l'616vation de la concentration saline dans le milieu. Mots-clds : Acide gras, Halomonas Concentration saline.
salina,
References Baumann, L., Bowditch, R.D. & Baumann, P. (1983), Description of Deleya gen. nov. created to accommodate the marine species Ah:aligenes aestus, A. pac~ficus, A. cupidus, A. venustus and Pseudomonas marina, hit. J. Syst. Bacteriol., 33, 793-802. Donson, S.J. & Franzmann, P.D. (1996), Unification of the genera Deleya (Baumann et al. 1983), Italomonas (Vreeland et al. 1980), and Halovibrio (Fendrich 1988) and the species Paracnccus halodenitrificans (Robinson and Gibtxms 1952) into a single genus, Halomonas. and placement of the genus Zvmohacter in the Family thdontonadaceae, htt. .L Sv~t. Bactetqol.. 46, 550-558. Drucker, D.B. ( 1981), Microbiological applications of gas chromatography. Cambridge Universi~,y Press, Cambridge. Franzmann, P.D. & Tindall, B.J. (1990), A chemotaxonomic study of members of the family Halomonathtceae. Syst. Appl. Microbiol., 13, ! 42- ! 47. ttanna, K., Bengis-Garber, C., Kushner, D.J., Kogut, M. & Kales, M. (1984), The effect of salt concentration on the phospholipid and fatty acid composition of the moderate halophile Vibrio cnsticola. Can. J. Microbiol., 30, 669-675.
679
Harwood, J.L. & Russell, N.J. (1984), Lipids in plants and microbes. George Arun and Unwin, London. Jantzen, E. & Bryn, K. (1985), Whole-cell and lipopolysaccharide fatty acids and sugars of Gram-negative bacteria, #z "Chemical Methods in Bacterial Systematic" (D.E. Goodfellow & D.E. Minnikin) (pp. 145171). Academic Press, London. Komagata, K. & Suzuki, K. (1987), Lipid and cell wall analysis in bacterial systematics, in "Methods in Microbiology "~ (R.R. Colweil & R. Grigorova) (pp. 161-207). Academic Press, London. Mellado, E., Moore, E.R.B., Nieto, J.J. & Ventosa, A. (1996), Analysis of 16S rRNA gene sequences of Vibrio costicola strains: description of Salinivibrio costicola gen. nov., comb. nov. Int. J. Syst. Bacteriol.. 46, 8 ! 7-82 !. Minnikin, D.F., O'Donnell, A.G., Goodfellow, M., Alderson, G., Ath',.dye,M., Schaal, A. & Parlett, J. (1984), An integrated procedure tor the extraction of bacteri',d ixoprenoids and polar lipids. J. MicrobioL Meth., 2, 233-241. Monteoliva-Sanchez, M. & Ramos-Cormenzana, A. (1987), Cellular fatty acid composition in moderately halophilic Gram-negative rods. J. Appl. Bacteriol., 62, 361-366. Monteoliva-Sanchez, M., Ferrer, M.R., Ramos-Cormenzana, A., Quesada, E. & Monteoliva, M. (1988), Cellular fatty acid composition of Deleya haiophila: effect of growth, temperature and salt concentration. J. Gen. MicrobioL, 134, i 99-203. Monteoliva-Sanchez, M., Ramos-Cormenzana, A. & Russell, N.J. ( ! 993), The effect of salinity and solutes on the biosynthesis of cyt:lopropane fatty acids in Pseudomonas halosaccarolytic:l. J. Gen. MicrobioL, 139, 1877- ! 884. Quesada, E., Ventosa, A., Rodriguez-Valera, F., Mejias, L. & Ranlos-Cormenzana, A. (1983), Numerical taxonomy of moderately halophilic Gram-negative bacteria from hypers',dine soils. J. Gen. Microbiol., 129, 2649-2657. Quesada, E., Ventosa, A., Rodriguez-Valera, F., Mejias, L. & Ramos-Cormenzana, A. (1984), Deleya halophila, a new species of moderately halophilic bacteria, h~t. Z Syst. Bacteriol., 34, 287-292. Quin, P.J. (1986), Models of haloadaptation in bacterial membranes. FEMS Microbiol. Rev., 39, 87-94. Rodriguez-Valera, F., Ruiz-Berraquero, F. & Ramos-Cormenzana, A. (1981), Characteristic of the heterotrophic bacterial populations in hyperaline environments of differen t ,~alt concentrations. Microbial Ecology, 7, 235-243. Russell, N.J. & Kogut, M. (1985), ltaioadaptalion: salt sensing and cell-envelope changes. Microhiol. Sci.. 2, 345-350. Russell, N.J. (1989), Adaptative modifications in membrane t)t" halotolerant and halophilic microorganisms. .!. Bioenerg. Binmcm.. 2 !, 93- ! 13. Russell, N. (1993), Lipids of halophilic and halotolcrant Microorganisms, in "'The Biology of Halophilic Bacteria" (R.H. Vreeland & L.I. ltochstcin) (pp. 163210). CRC Pres:~, Boca Raton. Valdcrrama, M.J., Quesada, E., Bcjar, V., Vcntosa, A., Gutierrez. M.C., Ruiz-Berraqucro, !:. & Ramos-Cormenzana, A. (1991), Dch, va salina sp. nov., a moderately halophilic Gram-negative bacterium. Int. J. Svst. Bacterinl., 41,377-384. Welch~ D. (1991), Applications of cellular fatty acid analysis. Clin. Microbhd. Rev., 4, 422-438.
Res. Microbiol. 1998, 149, 6 7 5 - 6 7 9
Influence of salt concentration on the cellular fatty acid composition of the moderately halophilic bacterium Halomonas salina M.J. Valderrama t*), M. Monteoliva-Sanchez (**), E. Quesada and A. Ramos-Cormenzana Department of Microbiology, Facul~ of Pharmacy, Universi~ of Granada, Granada (Spain)
SUMMARY
The cellular fatty acid composition of Halomonas salina, a moderately halophilic bacterium grown at different salt concentrations, is reported. Fatty acids C16:0 and C18:1 were major components and significant amounts of C16:1, C18:0 and cyc-C19:0 were also detected. The results showed clear chemotaxonomic relationships with recognized members of the genus Halomonas. The salt concentration greatly influenced the fatty acid composition, suggesting activation of cyciopropane synthetase at high levels of salt, since increases in cyclopropane fatty acids with decreases in monounsaturated fatty acids were observed as the salt concentration in the medium rose.
Key-words: Fatty acid, Halomonas safina, Salt concentration.
INTRODUCTION Halophiles, which constitute a wide group of organisms including protozoa, algae and bacteria, are characterized by their requirement for a high concentration of inorganic salts, mostly sodium chloride, to grow and survive. They inhabit saline environments where several conditions of osmolaxity and ionic strength challenge the stability of lipid bilayers and the structure of proteins, forcing those organisms to develop specialized molecules and physiological mechanisms to cope with this environmental stress. Among these adaptational properties, the changes in fatty acid com-
position may play an important role in regulating the membrane lipid phase (Russell, 1989). On the other hand, the cellular fatty acid composition has been shown to be a useful chemotaxonomic marker (Minnikin et al., 1984; Jantzen and Bryn, 1985; Komagata and Suzuki, 1987; Welch, 1991 ). The genus Deleva was constructed by Baumann et al. (1983) to include five species isolated from marine habitats that were slight halophiles specifically requiring added NaCI for growth. In 1984, Quesada et al. described the first moderately halophilic species in this genus, Del~3"a halophila, isolated from hypersaline soil. Deleva salina is a moderately halophile species of the
Submitted June 17, 1998, accepted August 1 I, 1998. (*) New address: Department of Microbiology, Faculty of Biology, Universidad Complutense de Madrid, Madrid. (**) Corresponding address: M. Monteoliva-Sanchez, Departamento de Microbiologia, Facuitad de Farmacia, Campus Universitario de Cartuja, 18071 Granada (Spain).
676
M.J. VALDERRAMA ET AL.
genus Deleya (Valderrama et al., 1991) with a strong eurihaline character, as it is able to grow between 2.5 and 25 % (wt/vol) salts, x. ith optimal growth at 5% (wt/vol) salts and 32°C. In 1996, Donson and Franzmann, by dete~nafion of 16S r ~ A sequences, proposed ~at members of the genus Deleya should be placed in the genus Halomonas. This paper presents the cellular fatty acid composition of Halomonas salina (previously Deleya salina) together with the modifications observed in that composition as a consequence of exposure to environments of differing salt concentrations.
MATERIALS AND METHODS Organisms and growth conditions The type strain of H. salina (strain F8-10=ATCC 49509), a moderately halophilic species, was used in this study. This species was isolated from a hypersaline soil found in Aiicante, Spain, and the description and reclassification have been previously reported (Valderrama et al., 1991 ; Mellado et al., 1996 ~Donson and Franzmann, 1996). Cells were grown in the MH complex medium described by Quesada et ai. (1983). Its composition is as follows: proteose-peptone no 3 (Difco), 5 g/l, yeast extract (Difco), 10 g~; and glucose, 1 g/I, supplemented with a balanced mixture of sea salts (Rodriguez-Valem et al., 1981)to give a final concentration of 2.5, 7,5 and 20% (w/v). Cultures were incubated at 32°C in a rotato~ shaker and harvested in early-exponential, exponential and stationary phase of growth by cold cen~fugation at 1 5 , ~ g tbr 15 min. Cells were washed twice with cold saline solution at the appropriate salt concentration, and lyophilized.
RESULTS The results of the analysis of fatty acid methyl esters from H. salina are given in table I. Under optimal culture conditions (7.5 %, w/v, salts), the major fatty acid components of cells harvested in the stationary phase of growth were C l6:0 and C lS:l, together with significant proportions of C 18:0; significant amounts of C 16: l and cyc-Cl9:0 were also detected. Only very small amounts of branched acids appeared, and acids of short chain length, those of greater than 20 carbons and hydroxy acids were not detected. The fatty acid composition of H. salina was altered when it was grown in media containing different salt concentrations (table 1; fig. I). For each phase of growth, increases in the proportion of cyclopropane (cyc-Cl7:0 and cyc-Cl9:0) and saturated fatty acids (Cl6:0 and C18:0) could be detected as the salt concentration of the growth medium rose. In contrast, the relative amounts of the mono-unsaturated C 16: l and C 18: l fatty acids decreased. In order to study whether the phase of growth influenced the fatty acid composition, cells in the early exponential, exponential and stationary phases were analysed (table I; fig. 2). Increased relative proportions of saturated fatty acids (C 16:0+C 18:0) and decreased respective unsaturated acids were observed in old cultures when the cells were grown at 7.5% and 20% (w/v) salts. The opposite occurred in the presence of 2.5 % salts.
DISCUSSION Fatty acid analysis Fatty acid methyl esters (FAME) were prepared from lyophilized cells (50 rag) following methods described by Monteoliva-Sanchez and Ramos-Cormenzana (1987). The FAMEs were examined by gas-liquid chromatography in a Carlo Erba Fractoyap 2 2 ~ instrument equipped with a flame ionization detector. A 2-m glass column packed with 15 % diethylene glycol succinate coated on 100/120 mesh G a s - ~ m P was used. Standards of FAMEs were obtained from Applied Science Lab., Pennsylvania (USA).
As with the taxonomic characterization of halophilic eubacteria, examination of the fatty acid profiles of the cells and the relationships to salt tolerance has lagged somewhat behind other developments. This study is the first report on the fatty acid composition of H. salina and has revealed that it is significantly affected by different salinities in the growth medium. The fatty acid profile of H. salina cultured in 7.5 % (w/v) salts is similar to that reported for
F A T T Y A C I D C O M P O S I T I O N OF H A L O M O N A S S A L I N A
677
T a b l e | . Fatty acid composition o f H. salina as a function o f phase of g r o w t h and salt concentration.
2.5 E
Fatty acid
EE
C12:0 C13:0 Ci4:0 CI5:0 CI6:0 C17:0 CI8:0 C19:0 C20:0 C!6:1 CI8:1 br-Cl4:0 br-Cl5:0 br-Cl6:0 br-Cl7:0 c y c - C 17:0 cyc-Cl9:0
--T 0.2 31.4 . 2.5 0.2 1.6 19.1 36.3 0.2 0.4 . 1.7 1.9 4.3
S
T T ~ 1.3 32.1 .
Salt concentration (% w/v) 7.5 EE E S
T 0.4 T i.7 26.8 . 1.9 ~ 0.9 22.6 38.4 ~ 0.7
. 2.7 T 0.5 15.9 42.9 ~ 1.5 .
. 0.8 0.6 1.3
. -0.2 0.9 38.7
.
. 0.2 T 2.3 41. I T 8.4 T 0.4 14.2 27.9 T T
0.3 ~ T 15.2 34.9 0.3 T .
.
0.3 3.1 2.9
. 0.6 4.9 3.8
.
. 1.2 0.7 3.2
. -T 1.4 38.0 T 14.9 . . 1.2 2.1 32.0 ~ 2.2 . T 0.5 7.6
20 E
EE
T 0.1 T 0.3 41.5 -8.6
T T T 0.8 42.0 0.5 10.1 T T 1.8 5.4 10.2 26.8 22.3 . . . . ~ T
~ T 39.8 0.3 8.8 . 2.1 10.4 27.6 0.3 T . T 6.9 3.6
.
2.4 3.6 10.6
T 4.0 5.2
Values are expressed as percentage of the total and are means of three determinations. EE = early exponential ; E = exponential" S = stationary ; dash = not detected" T = trace content of less than 0. 1% ; br = branched acid; cyc = cyclopropane acid.
_o F-
GROWTHPHASE
30
S
z
A 0
SALT CONCENTRATION
EXPONENTIAL
t
i 2.s~,
20 STAIlOflARY
EARLY
10
7.596
+
i
20%
--"C)-- I
EXPONENTIAL i t ...............................................................
u~
.....
I
I
I
2.5
7.5
20
i
EE
E
S
SALT CONCENTRATION (%) GROWTH PHASE
Fig. I. Effect of salt c o n c e n t r a t i o n of the ratio of monounsaturated ( C I 6 : 1 + C i 8 : 1 ) and cyclopropane fatty acids (cyc-Cl7:0+cyc-Cl9:0) of H. salina at different phases of growth (early exponential, exponential and stationary).
Fig. 2. Effect of phase of growth (EE, early ext,oncnt i a l ; E, e x p o n e n t i a l ; S, .,,tationary) on the ratio of monounsaturated (C16: i+C18: !) and saturated fat~.y acids (C16:0+C!8:0) of H. salina grown at different salt concentrations (%, w/v).
other moderately halophilic Gram-negative rods
et al., 1 9 8 8 ; F r a n z m a n n a n d T i n d a l l , 1 9 9 0 ; R u s -
( H a n n a et al., 1 9 8 4 ; M o n t e o l i v a - S a n c h e z a n d Ramos-Cormenzana, 1987; Monteoliva-Sanchez
sell, 1993). The major components are C16:0 and C18:1, and include significants a m o u n t s of
678
M.J. VALDERRAMA ET AL.
C18:0 and C16:1 (table I). Our results are in close agreement with the chemotaxonomic study carried out by Franzmann and Tindall (1990) on members of the family Halomonadaceae, i.e. validly described species of the genus Halomollt'lS.
Differences were found, however, in e.g. cyclopropane fatty acids cyc-Cl7:0 and cycCl9:0 composition. H. salina contained lower proportions of these (table l) than reported for another species of the genus Halomona,~ (previously designated as Deleya) by Franzmann and Tindall (1990). Our results more closely resembled the composition found for D. halophila, now H, halophila, by Monteoliva-Sanchez et al. (1988). Differences in compositional fatty acids have been reported as a consequence of the distinct culture media used (Drucker, 198 l). With respect to the influence of the phase of growth on the cellular fatty acid composition in H. salina, the results obtained when the organisms were grown at three different saline concentrations are shown in figure 2. At 7.5 and 20% (w/v) salts, the relative proportions of saturated and monounsaturated fatty acids change with the growth stages" an increase in saturated (CI6:0+C18:0) and a decrease in unsaturated (CI6:1 +C18:1) was observed with the age of the cultures. Younger cells of Pseudomonas halosaccharolytica also contained more C 16 and C 18 monoenoic acids (Monteoliva-Sanchez et al,, 1993), but in this microorganism, higher amounts of cyc-Cl9:0 were found in cells of the late logarithmic and stationary stages. However, no significant changes in the fatty acid composition as a consequence of phase of growth were r e p o r t e d in Vibrio c o s t i c o l a (Hanna et al., 1984), now designated as Salinivibrio costicola by Mellado et al. (1996), or Halomonas halophila (Monteoliva-Sanchez et al., 1988). The predominant alterations in fatty acid composition of H. salina, in response to variable salt concentrations in the culture medium, are shown in figure I. For each phase of growth studied, increasing the salt c o n c e n t r a t i o n resulted in a decrease in the proportion of mono-
unsaturated fatty acids, with a concomitant increase in cyclopropane fatty acids; the relative proportion of saturated fatty acids was also increased as a consequence of raising the salt concentration. There are few comparable date available in the literature concerning moderate halophiles. H. halophila (Monteoliva-Sanchez et al., 1988) and Pseudomonas halossaccharolytica (Monteoliva-Sanchez et al., 1993) showed similar adaptational fatty acid patterns. However, S. costicola did not show a significant degree of cyclization of fatty acids when cultured at a high salt concentration (Hanna et aL, 1984), since cyclopropanoic acids were not detected by the authors in that study. This particular microorganism showed a slightly different fatty acid response: total monounsaturated acids were highest at the optimal copcentrations, whereas total saturated fatty acids showed the opposite effect. Our results are more closely related to those reported for H. halophila by Monteoliva-Sanchez et al. (1988), which on the other hand, agree with the taxonomic relationship between this species and H. salina. The results presented here on the modification of fatty acid of the H. salina are in accord with the hypothesis concerning the activation or induction of cyclopropane synthetase when there is a high salinity in the medium and the bacteria show increases in compatible solutes (Monteoliva-Sanchez et al., 1993). The physical properties of acyl lipids containing cyclopropane chains (e.g., cyc-17:0) can be considered to be intermediate between the corresponding saturated fatty acid (C17:0) and the unsaturated fatty acid from which it was derived (C16:1). Therefore, an increase in cyclopropane fatty acid content at the expense of unsaturated fatty acid would tend to decrease membrane fluidity (Russell, 1993). In this sense, the increase in the proportions of cyclopropane tatty acids could contribute to preserving the membrane integrity of some halophilic bacteria, since they could prevent the formation of abnormal structures (Harwood and Russell, 1984; Russell and Kogut, 1985; Quin, 1986). Further studies are necessary in which fatty acid composition and membrane properties will be compared in cultures grown over a range of salinities.
FATTY ACID COMPOSITION OF HALOMONAS SAL|NA
Acknowledgements This investigation was partially supported by grants to the Grupo de lnvestigaci6n CVI 0190 from Junta de Andalucia, Spain.
Influence de la concentration saline sur la composition en acides gras cellulaires de Halomonas salina, bact6rie mod6r6ment halophile Les acides gras cellulaires de H a l o m o n a s salina, une bact6rie m o d 6 r f m e n t h a l o p h i l e , c u l t i v d c "~ diverses concentrations salines, ont 6t6 6tudi6s. Les acides gras en C i 6 : 0 et CI8:1 sont des composants majoritaires; des quantit6s significatives d'acides en C16:1, C18:8 et c y c - C l 9 : 0 ont 6t6. dfcel6es. Lcs r6sultats illustrent nettement les relations chimiot a x o n o m i q u e s avec des repr6sentants c o n n u s du genre Halomonas. La concentration saline influence amplement la composition en acides gras, ce qui sugg6re une activation de la cyclopropane-synth6tase ?a des concentrations salines 61evfes: une augmentation des acides gras h cyclopropane et une diminution des a c i d e s m o n o - i n s a t u r 6 s sont o b s e r v 6 e s Iors de l'616vation de la concentration saline dans le milieu. Mots-clds : Acide gras, Halomonas Concentration saline.
salina,
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