Citrobacter, and Providencial

Transcription

1 JOURNAL OF BACTERIOLOGY, Apr. 1973, p Copyright American Society for Microbiology Vol. 114, No. 1 Printed in U.SA. Fatty Acid Compositions of Paracolons: Arizona, Citrobacter, and Providencial NEAL A. MACHTIGER' AND WILLIAM M. O'LEARY Department of Microbiology, Cornell University Medical College, New York, New York Received for publication 15 November 1972 The fatty acid compositions of stationary-phase cultures of Arizona arizonae, Citrobacter freundii, Providencia alcalifaciens, Providencia stuartii, and Providencia sp. were studied. The major fatty acids of A. arizonae, C. freundii, and Providencia were 16:0, 16: 1, 17: cyclopropane, and 19: cyclopropane. The fatty acid compositions of the two strains of A. arizonae examined were similar to each other, but the three strains of C. freundii differed from one another in their fatty acid compositions. In both A. arizonae and C. freundii, the relative quantities of saturated, unsaturated, and cyclopropane fatty acids were similar to those which have been found in stationary-phase cultures of other members of the Enterobacteriaceae. The three strains of Providencia also differed from one another in their fatty acid compositions. In all three strains, the total quantity of unsaturated fatty acids was larger and that of the cyclopropane fatty acids was smaller than those found in stationary-phase cultures of other enteric bacteria. Interest in the lipids of the Enterobacteriaceae has grown in recent years, and the lipid compositions of several members of this family have been the subject of many investigations. However, detailed information about the lipid chemistry and physiology is available for only a few species within this family, which have been one of the most useful tools in biochemical research in recent years. The paracolons are among those enteric bacteria about whose lipid biochemistry virtually nothing is known. The position of the paracolons within the family Enterobacteriaceae has been poorly defined for many years. Recently, efforts have been made to clarify the relationship of these organisms to the other members of the family (3-5). As virtually nothing is known about the chemical composition of the paracolons, and in light of this proposed clarification of the relationship of these organisms to the other genera within the Enterobacteriaceae, we undertook an investigation of the fatty acid compositions of strains of Arizona arizonae, Citrobacter freundii, Providencia alcalifaciens, P. stuartii, and Providencia sp. 'This work was part of the thesis of N.A.M. which has been accepted by the faculty of Cornell University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 'Present address: Department of Bacteriology, University of California, Los Angeles, Calif MATERIALS AND METHODS Bacteria. The organisms used for these investigations were acquired from a number of sources, as we desired to study the fatty acid compositions of several different isolates of each organism. A. arizonae 13314, C. freundii 8090, and Prouidencia sp were from the American Type Culture Collection; M. J. Pickett (University of California, Los Angeles) supplied A. arizonae UCLA and C. freundii 17; F. G. Jarvis (Idaho State University) supplied C. freundii 10053; and W. J. Martin (National Center for Disease Control) supplied P. alcalifaciens 3300 and P. stuartii 247. The biochemical characteristics of these strains were the subject of an earlier communication (13). Bacterial cultivation and harvest. All of the organisms studied were grown in chemically defined media. Cultures of A. arizonae were grown in the medium of Davis and Mingioli (1) containing 0.75% glucose as carbon and energy source. Cultures of C. freundii were grown in the medium of Jarvis et al. supplemented with glutamate, pantothenate, and NaCl (8), and containing 0.75% glucose. Cultures of Providencia were grown in the defined medium for these organisms developed in this laboratory (13), containing 0.75% glucose. Cultures of 1 liter of medium in baffled 2-liter Erlenmeyer flasks (Bellco Glass Inc., Vineland, N.J.) were incubated at 37 C in a New Brunswick model G-25 shaking incubator operating at 250 oscillations per min. Stationary-phase cells were harvested by centrifugation at 8,200 x g for 20 min in a Lourdes model A-2 centrifuge maintained at 5 C, washed once 80

2 VOL. 114, 1973 FATTY ACID COMPOSITIONS OF PARACOLONS with distilled water, and recentrifuged. The cell pastes were then lyophilized and weighed. Growth was measured turbidimetrically with a Bausch & Lomb Spectronic-20 colorimeter, at a wavelength of 600 nm. Extraction and characterization of total celular fatty acids. The total cellular fatty acids were extracted from dry cells as described by Dunnick and O'Leary (2). The fatty acids were methylated by the method of Metcalfe and Schmitz (14) and analyzed by gas-liquid chromatography. Gas-liquid chromatography was performed by use of a Perkin Elmer Model 801 dual column gas chromatograph, equipped with dual flame ionization detectors and a Varian model G 2000 recorder, and connected to a Varian model 475 digital electronic integrator. Analyses were run on columns of diethylene glycol succinate (DEGS) and butylene diol succinate (BDS). Stainless-steel columns (1/8 inch by 6 ft) packed with either DEGS (15% liquid phase (wt/wt] on Chromsorb W-NAW 80/100 mesh) or BDS (10% liquid phase (wt/wt] on Chromsorb W-HMDS 80/100 mesh) were purchased from Applied Science Co., Stste- College, Pa. The instrument was operated in the linear temperature programming mode over the range of 150 to 220 C, at an 8 C/min rise in temperature. The injection port temperature was 270 C, the detector temperature was 210 C, and the flow rate of the He carrier gas was 20 cc/min. All analyses were run on both columns, and the retention times of the bacterial fatty acids on each column were compared with the retention times of fatty acid methylester standards, including the use of plots of the log of retention time versus carbon number for each homologous series of fatty acids. Chemicals and standards. All organic reagents and solvents were obtained commercially and were of the highest quality available. All solvents were redistilled before use. The cyclopropane and,b-hydroxy fatty acid methyl ester standards were purchased from Analabs Inc., North Haven, Conn. All other fatty acid standards were purchased from Applied Scienco Co. RESULTS The conditions employed for gas-liquid chromatography resulted in the resolution of all major classes of fatty acids which have been reported by other investigators to be present in bacteria. The results of the analyses of bacterial fatty acid composition employing two different types of columns (BDS and DEGS) were quite similar. The percentage compositions of the total cellular fatty acids reported here are the means of duplicate determinations on each of the two types of columns. A. arizonae. The fatty acid compositions of A. arizonae strains and UCLA are summarized in Table 1. The major fatty acids of this organism were 16: 0, 17: cy, 19: cy, and 14:0. There were smaller quantities of 16: 1 and 18: 1, the unsaturated fatty acids amounting to TABLE 1. Fatty acids of two Arizona arizonae strains Fatty acida Percent fatty acid of each chain length Strain Strain UCLA 10:0 Q.07 Trace 12: : 1 Trace Trace 13:0 13:cy : : : :cy : : :0 Trace Trace 17: cy : : : : cy Total saturated acids Total unsaturated acids Total cyclopropane acids a Number preceding colon indicates number of carbons; number after colon designates degree of unsaturation; cyclopropane acids are represented by the symbol cy. no more than 10% of the total cellular fatty acids. On the other hand, the cyclopropane fatty acids amounted to more than one-third of the cellular fatty acids. This observation of a larger total quantity of cyclopropane than unsaturated fatty acids is typical of the organisms in the family Enterobacteriaceae, when cells have been harvested in stationary phase (2, 9, 10, 19). The compositions of the cellular fatty acids of the two strains of A. arizonae studied are represented graphically in Fig. 1. Analysis of the amounts of 14: 0, 16: 0, 16: 1, 17: cy, 18: 1, and 19: cy reveals that the differences between the quantities of these fatty acids found in the two strains were not statistically significant (P > 0.05 in each case). C. freundii. At the beginning of these investigations, the fatty acids of two strains of C. freundii were extracted and analyzed. The results of these analyses, summarized in Table 2, indicated that the two strains, 8090 and 10053, differed in their fatty acid compositions. We thought it possible that one of the two strains might be atypical in its fatty acid composition, and hence added an additional strain of C. freundii, strain 17, to our investiga- 81

3 82 MACHTIGER AND O'LEARY J. BACTERIOL. 4U 30 U MUCLA 20 la nv U1--. m Anokm 12:0 14:0 16:0 18:0 19:0 12:1 14:1 16:1 18:4 15:Cy 17:Cy: FIG. 1. Comparison of fatty acid compositions of Arizona arizonae strains and UCLA. tions. The results of the analysis of the fatty acids of this strain are also summarized in Table 2. It is apparent from these data that the three strains of C. freundii differed quantitatively in their fatty acid compositions. These differences, most easily appreciated from Fig. 2, are evident both in the total amounts of saturated, unsaturated, and cyclopropane fatty acids found in these strains, and in the quantities of individual fatty acids in the extracts. Providencia. The fatty acid compositions of P. alcalifaciens 3300, P. stuartii 247, and Providencia sp are summarized in Table 3. The major fatty acids of P. alcalifaciens were 16: 1, 16:0 (present in nearly equal quantities), 19: cy, and 14:0. The total quantity of unsaturated fatty acids was nearly twice the total of cyclopropane fatty acids in this organism, although the cells had been harvested in stationary phase. The most notable feature of the fatty acid composition of P. stuartii was the large quantity of 16:0 (44%) found. This represents the largest amount of any single compound found in the fatty acids of the paracolons we studied. This is also the only organism studied in which 18: 1 was the predominant monoenoic fatty acid and in which there was more than a very small quantity of 17: cy. The fatty acid composition of Providencia sp was also determined. The fatty acids h1 of stationary-phase cells of Providencia sp contained a larger quantity of unsaturated than cyclopropane fatty acids. The major fatty acids of this organism were 16:0, 16:1, 19:cy. From the foregoing description of the three strains of Providencia, it is apparent that these organisms differed markedly in their fatty acid compositions. These differences, as can be seen in Fig. 3, were in both the quantities of individual fatty acids as well as in the total amounts of saturated, unsaturated, and cyclopropane fatty acids. DISCUSSION The fatty acids of A. arizonae, C. freundii, P. alcalifaciens, P. stuartii, and Providencia sp. were characterized in this study. Since it is well known that bacterial lipid composition may vary with the conditions under which cells are cultivated (10, 17), the growth conditions employed here were carefully standardized. The methods used for fatty acid analysis were also carefully controlled so that any comparisons of the fatty acid compositions of the organisms studied would be based on actual differences in the fatty acids of these organisms. The major fatty acids of A. arizonae and C. freundii have generally been found among the cellular fatty acids of the Enterobacteriaceae (2, 7, 9, 15, 16). In all of the strains studied here, the principal saturated fatty acid was

4 VOL. 114, 1973 palmitic acid and the principal monoenoic acid was hexadecenoic acid. In A. arizonae, the principal cyclopropane fatty acid was 17: cy, whereas in C. freundii the 19 carbon acid TABLE 2. FATTY ACID COMPOSITIONS OF PARACOLONS Fatty acids of three Citrobacter freundii strains Fatty acida Percent fatty acid of each chain length Strain Strain Strain : : :1 - trace :0-13:cy : : : trace : cy : : : :cy : : : : cy Total saturated acids Total unsaturated acids Total cyclopropane acids a Number preceding colon indicates number of carbons; number after colon designates degree of unsaturation; cyclopropane acids are represented by the symbol cy r predominated. The two strains of A. arizonae, and UCLA, did not differ from each other in their fatty acid compositions. There was a larger TABLE 3. Fatty acids of Providencia Percent fatty acid of each chain length Fatty acida P. P Provialcali- dencia faciens 247 sp :0 trace trace trace 12: :1 trace 0.12 trace 13:0 _ : cy trace trace trace 14: : : : cy : , 16: : : cy : : :0 trace - trace 19: cy Total saturated acids Total unsaturated acids Total cyclopropane acids a Number preceding colon indicates number of carbons; number after colon designates degree of unsaturation; cyclopropane -acids are represented by symbol cy. * fiit~~~00 asb1 M- 1-12:0 14:0 16:0 18:0 19:0 12:1 14:1 16:1 18:1 15:cy 17:cy 19:cy FIG. 2. Comparison of fatty acid compositions of Citrobacter freundii strains 8090, 10053, and 17.

5 84 MACHTIGER AND O'LEARY J. BACTERIOL *33W 0247 U S LI :0 14:0 16:0 FIG. 3. Comparison of fatty Providencia sp total quantity of cyclopropane than unsaturated fatty acids in A. arizonae, consistent with the fact that the cultures were harvested in stationary phase. The principal fatty acids of C. freundii were 16:0, 16: 1, and 19: cy, but different quantities of these compounds were found in the three strains studied. Palmitic acid was the fatty acid found in largest quantity in strains 8090 and 17, whereas the predominant fatty acid in strain was 19: cy. Unsaturated fatty acids comprised only about 11% of the total fatty acids of strain 8090; in the other strains, the amount of unsaturated fatty acids was considerably larger. Finally, the ratio of the total quantity of cyclopropane to unsaturated fatty acids in strain 8090 was 3.4, whereas in the other strains this ratio was much smaller (1.8 for and 1.9 for 17). Our analyses of several strains of C. freundii thus indicate a heterogeneity of cellular fatty acid composition within this species. This finding parallels those of Sacks et al. (18), who found a variability of cellular protein composition among different strains of C. freundii, as determined by polyacrylamide gel electrophoresis. It appears that heterogeniety of cellular chemical composition is characteristic of the species C. freundii. 18:0 19:0oo 12:1 14:1 16:1 18:1 15:cy 17:cy 19:cy acid compositions of Providencia acalifaciens 3300, P. stuartii 247, and The strains of Providencia that we studied included representatives of the two presently recognized species within this genus (4) as well as a strain whose biochemical characteristics did not correspond to those of either of these two species (13). We found that these three organisms differed from one another in several features of their fatty acid compositions. The principal monoenoic fatty acid in P. alcalifaciens and in Providencia sp. was 16:1, whereas 18:1 predominated in P. stuartii. Although 19: cy was the major cyclopropane fatty acid in each of these organisms, a significant quantity of 17: cy was found only in the fatty acids of P. stuartii. In all three organisms, the principal saturated fatty acid was 16: 0. Perhaps the most striking feature of the fatty acid composition of Providencia is the large total quantity of unsaturated fatty acids found in cells of stationary-phase cultures of these organisms. In the strains of A. arizonae and C. freundii that we examined, and in other members of the Enterobacteriaceae whose fatty acids have been characterized in other laboratories, the quantity of cyclopropane fatty acids has been found to exceed that of monoenoic fatty acids. In only one Providencia organism, P. stuartii, have we found this to be the case, and in this instance the ratio of the total

6 VOL. 114, 1973 FATTY ACID COMPOSITIONS OF PARACOLONS quantity of cyclopropane to unsaturated fatty, acids was 1.1. In the other two organisms, there was a marked excess of unsaturated over cyclopropane fatty acids. These findings suggest that the relationship between growth of Providencia and the conversion of unsaturated to cyclopropane fatty acids warrants further study, since it appears that the changes in fatty acid compositions which take place in organisms of this genus during the course of the growth cycle occur at a different rate than in other enteric bacteria. Hydroxy fatty acids were not present in detectable quantities in the cells of the paracolons which we studied. This class of fatty acids are components of the cellular lipopolysaccharide of many gram-negative bacteria and are intermediates in the biosynthesis of monoenoic fatty acids in bacteria (6, 11, 17). The study of the fatty acids specifically associated with the lipopolysaccharides of paracolons was beyond the scope of our investigations. Such studies require the use of rigorous extraction and degradative procedures designed specifically for the release and recovery of the fatty acids from lipopolysaccharides (15, 17). Although hydroxy fatty acids have been reported by a number of other workers to be present in the cellular fatty acids of gram-negative bacteria released by the alkaline hydrolytic extraction employed in our investigations (6, 15, 17), they have not been found in all such organisms whose fatty acids have been studied after such extraction (2, 10, 20). The cultures we studied were harvested in stationary phase, when the rate of monoenoic fatty acid biosynthesis would be below that of log phase, and the cellular content of hydroxy fatty acid intermediates would have declined to below the limit of detection by the techniques used. ACKNOWLEDGMENTS We thank F. G. Jarvis and M. J. Pickett for their gifts of bacterial cultures. We also thank Maija Skangalis and Anna Smith for their able technical assistance. This work was supported by Public Health Service grants AI and lt1-ai from the National Institute for Allergy and Infectious Diseases. LITERATURE CITED 1. Davis, B. D., and E. S. Mingioli Mutants of Escherichia coli requiring methionine or vitamin B,2. J. Bacteriol. 60: Dunnick, J. K., and W. M. O'Leary Correlation of bacterial lipid composition with antibiotic resistance. J. Bacteriol. 101: Edwards, P. R., and W. H. Ewing Identification of Enterobacteriaceae. Burgess Publishing Co., Minneapolis. 4. Ewing, W. H Differentiation of Enterobacteriaceae by biochemical reactions. National Center for Disease Control Publications, Atlanta, Ga. 5. Ewing, W. H., and P. R. Edwards The principal divisions and groups of Enterobacteriaceae and their differentiation. Int. Bull. Bacteriol. Nomen. Taxon. 10: Goldfine, H Comparative aspects of bacterial lipids. Advan. Microbial Physiol. 8: Gray, G. M The cyclopropane ring fatty acids of Salmonella typhimurium. Biochim. Biophys. Acta 65: Jarvis, F. G., M. T. Mesenko, and K. E. Tibbs Production of Vi antigen on a chemically defined medium by a coliform bacterium. J. Bacteriol. 80: Kaneshiro, T., and A. G. Marr Cis-9, 10- methylene hexadecanoic acid from the phospholipids of Escherichia coli. J. Biol. Chem. 236: Kates, M Bacterial lipids. Advan. Lipid Res. 2: Kates, M Biosynthesis of lipids in microorganisms. Annu. Rev. Microbiol. 20: Letters, R Phospholipids of yeast. Biochim. Biophys. Acta 116: Machtiger, N. A., and W. M. O'Leary The nutritional requirements of Arizona, Citrobacter, and Providencia. J. Bacteriol. 108: Metcalfe, L. D., and A. A. Schmitz The rapid preparation of fatty acid esters for gas chromatography. Anal. Chem. 13: Nesbitt, J. A., and W. J. Lennarz Comparison of lipids and lipopolysaccharide from the bacillary and L-forms of Proteus P18. J. Bacteriol. 89: O'Leary, W. M S-adenosylmethionine in the biosynthesis of bacterial fatty acids. J. Bacteriol. 84: O'Leary, W. M The chemistry and metabolism of microbial lipids. World Publishing Co., Cleveland. 18. Sacks, T. G., M. N. Hass, and S. Razin Polyacrylamide-gel electrophoresis of cell proteins of Enterobacteriaceae. Israel J. Med. Sci. 5: Sud, K. J., and D. S. Feingold Mechanism of polymyxin resistance in Proteus mirabilis. J. Bacteriol. 104: White, D. C Lipid composition of the electron 85 transport membrane of Haemophilus parainfluenza. J. Bacteriol. 96: