UTILIZATION OF CARBOHYDRATES AND METABOLIC INTERMEDIATES

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1 UTILIZATION OF CARBOHYDRATES AND METABOLIC INTERMEDIATES BY PILIATED AND NONPILIATED BACTERIA J. A. WOHLHIETER, C. C. BRINTON, JR., AND L. S. BARON Division of Communicable Disease and Immunology, Walter Reed Army Institute of Research, Washington, D.C., and Department of Microbiology, University of Pittsbutrgh School of Medicine, Pittsburgh, Pennsylvania Received for publication March 28, 1962 ABSTRACT WOHLHIETER, J. A. (Walter Reed Army Institute of Research, Washington, D.C.), C. C. BRINTON, JR., AND L. S. BARON. Utilization of carbohydrates and metabolic intermediates by piliated and nonpiliated bacteria. J. Bacteriol. 84: The rate of aerobic and anaerobic metabolism of piliated and nonpiliated bacteria was studied in an effort to determine whether the presence of these surface structures altered the metabolic activity of bacteria. Respiration was measured manometrically when various piliated and nonpiliated strains metabolized glucose and several Krebs-cycle intermediates. Spontaneously produced nonpiliated variants of piliated cells were examined, but no difference in the rate of either aerobic or anaerobic metabolism was observed. Piliated and nonpiliated recombinants of a mating of Escherichia coli and Salmonella typhosa had different metabolic rates than the parent strains, but it was shown that these differences were not related to piliation. Clones of piliated and nonpiliated cells isolated from other crosses showed no difference between their respiration rate and that of either parent. Piliated and nonpiliated strains could mutate to higher rates of substrate utilization with no concomitant change in piliation. Since respiration and piliation can mutate independently and segregate independently in genetic crosses, we therefore conclude that there is no direct physiological relationship between them. Pili are thin, fragile appendages of bacteria, about 7 A in diameter, with varying lengths up to several microns, which grow out from the bacterial surface (Brinton, Buzzell, and Lauffer, 1954). These hairlike appendages are composed entirely of protein and are readily purified (Brinton and Stone, 1961). These protein structures 416 confer additional immunological properties on the cell. The exact function of pili is as yet rather obscure. They can, however, serve as organs of attachment, probably because of their hydrophobic surface properties. Recent studies by Brinton and Baron (196) and Brinton et al. (1961) have demonstrated that the piliation character can be transferred genetically from piliated (P+) to nonpiliated (P-) strains of the Enterobacteriaceae by recombination. Maccacaro and Dettori (1959) have investigated the utilization of carbohydrates and Krebs-cycle intermediates by P+ and P- hybrids independently isolated from a mating between Escherichia coli Pt and P- strains. Significant differences in 2 consumption and CO2 production with various substrates were correlated by them with differences in piliation, and interpreted as an indication of a relationship between piliation and metabolism. It is the purpose of this paper to investigate the ability of various P+ and P- strains to utilize some carbohydrates and Krebs-cycle intermediates in order to confirm or deny a biological function of piliation in this respect. MATERIALS AND METHODS Bacterial strains. A number of P+ and P strains were examined; the presence or absence of pili was determined by electron microscopy (Brinton and Baron, 196). B-L(E) P+ and a P- strain isolated as a one-step P+ to P- variation of B-L(E) P+ have been described in detail (Brinton, 1959). Bam Pt and a Pr form isolated from it as a one-step P+ to P variation are similar to B-L(E) P+ and P- in that the P+ form has a smaller denser colony and produces the P- form spontaneously at a relatively high rate. The recombinant strains of an intergeneric cross between W1895 Hfr P+ and Salmonella typhosa 643L+ FP, S. typhosa diploid X3D, and its haploid segregants Downloaded from on December 6, 218 by guest

2 VOL. 84, 1962 METABOLISM OF PILIATED BACTERIA 417 Organism Escherichia coli Salmonella typhosa S. typhosa S. typhosa S. typhosa S. typhimurium S. typhimurium TABLE 1. Identification of bacterial strains employed Strain B-L (E) P+ B-L (E) P- Bam P+ Bam P- K-12 W1895 K K-12 2U K-12 2U P+ K-12 2U P+ 643L+ X3D X3T X3P R7 R36 Mating polarity X3T and X3P have been described by Brinton and Baron (196). Salmonella typhimurium hybrid R7 P+ and R36 P- were independent isolates derived from an in vivo cross as reported by Schneider, Formal, and Baron (1961). The strains employed throughout this study are listed in Table 1. Media. Penassay broth (Difco) and meat extract agar (MEA) were employed for the cultivation of the strains. The minimal medium used in certain experiments has been described previously (Baron, Carey, and Spilman, 1959). Chemicals. The carbohydrates used were obtained from Pfanstiehl Laboratories, Inc., and the chemicals from the sources listed: malic acid and sodium succinate, Fisher Scientific Co; fumaric acid, disodium salt, California Corporation for Biochemical Research. Mlanometric experiments. Warburg experiments vere performed at 37 C using standard techniques described by Umbreit, Burris, and Stauffer (1957). The concentrated cell suspensions were prepared by harvesting the growth from MEA plates which had been incubated at 37 C for 18 hr. The cells were harvested from the plates and suspended in.7 M phosphate buffer (ph 7.3). The cell suspensions were centrifuged at 4, X g for 1 min. The pellets were washed and resuspended in buffer three times. After the final centrifugation the concentration of the bacterial suspension was adjusted with a Klett colorimeter to a reading of 6, which corresponds to approximately 11 cells/ml. The substrate concentrion used in the Warburg vessels was 2% for Pili Source F- + C. C. Brinton, Jr. F- - B-L (E) P+ F- + C. C. Brinton, Jr. F- - Bam P+ Hf r + J. Lederberg F+ + J. Lederberg F- - F. Jacob F X 2U F- + W1895 X 2U F- - W1895 X S. typhosa 643 F- + W1895 X 643L+ F- - Segregant from X3D F- + Segregant from X3D F- + W1895 X S. typhimurium 9Sr-2 F- - W1895 X S. typhimurium 9Sr-2 the sugars and.3% for the Krebs-cycle intermediates. The Krebs-cycle intermediates were adjusted to ph 7. before use. RESULTS The most clear-cut method of demonstrating that piliation plays some role in the metabolism of a given substrate is to investigate possible differences in respiration between the P+ and the P- forms of the same strain. Ideally, the respiration of a piliated strain should be compared with that of a spontaneously produced, nonpiliated, one-step variant which is identical to the original in all other characteristics except those associated with piliation itself (hemagglutination, autoagglutination, antigenicity, electrophoretic mobility, and colonial morphology). Two strains which approach this ideal are E. coli B-L(E) P+ and P- and Bam P+ and P-. B-L(E) P+ spontaneously produces P- forms which are similar to the parent in all other known characteristics except piliation and growth rate, which, depending on the temperature, can be either faster or slower than the P+ parent. Bam P+ spontaneously produces P- variants with the same growth rate as the P+ parent. The respiratory measurements were made on concentrated bacterial suspensions in which little or no growth occurs; therefore, any difference in growth rate between P+ and P- forms would have no effect on the results. It can be seen (Fig. 1A) that no significant difference was observed in the oxidative glycolysis of cell suspensions of P+ and P- variants of Downloaded from on December 6, 218 by guest

3 418 WOHLHIETER, BRINTON, AND BARON J. BACTERIOL. I12 r A 1 Lu E 8 cn z o 6 _ o 4 Lu 3 ocr- 3 L o 2 t 1 TIME (MIN.) BL (E) P + - BL (E) P BL (E) P + - BL (E) P TIME (MIN.) FIG. 1. Aerobic (A) and anaerobic (B) glycolysis of P+ and P- strains of Escherichia coli B-L(E). 12 w 1 z 8 N 6 4 a) FUMARAT E 2 I I I BL (E) P + *-- BL (E) P- b) 12 MALATE 1 _ B-L(E). The results of the anaerobic breakdown of glucose by P+ and P cells are shown in Fig. 1B, and here again the rate is similar for both. In Fig. 2A, B, and C, the results of the oxidation of fumarate, malate, and succinate by P+ and P- cell suspensions of B-L(E) are shown. No significant difference is observed in the utilization of these substrates. The curves obtained for Bam P+ and P- are similar to those shown, indicating no difference between the P+ and P variants of this strain. Fumarate, malate, and succinate were chosen because Maccacaro and Dettori (1959) found that the greatest difference between P+ and P- cells could be demonstrated with these substrates. These authors also reported that P+ cells are more active than P cells in the oxidative breakdown of glucose, but that nonpiliated cells were more active under anaerobic conditions. However, our experiments with the piliated and nonpiliated single-step variants of B-L(E) and Bam do not confirm these results. Two independently isolated hybrids from an in vivo cross between W1895 P+ and S. typhimurium 9Sr-2 P-, one stably piliated (R-7 P+) and the other nonpiliated (R-36 P; Schneider et al., 1961), were compared with respect to 2 consumption using glucose, fumarate, malate, and succinate. No significant difference was observed between R-7 P+ and R-36 P- on any of these substrates. A third group of piliated and nonpiliated bacteria was investigated. Measurements were made of the rate of utilization for glucose, malate, fumarate, and succinate by the male l TIME (MN) c) 12 SUCCINATE FIG. 2. Comparison of the utilization of fumarate (a), malate (b), and succinate (c) by P* and P- strains of Escherichia coli B-L(E). Downloaded from on December 6, 218 by guest

4 VOL. 84, 1962 METABOLISM OF PILIATED BACTERIA 419 TABLE 2. Utilization of various substrates by piliated and nonpiliated strains Bacterial strains Substrates (liters 2 /hr) Glucose Fumarate Malate Succinate W1895 P+ (stable) L+ P X3D P+ (un stable, diploid) X3T P X3P+ (stable) (W1895 Hfr Cavalli) and female (S. typhosa 643 L+) parents of a cross, an unstable partial diploid zygote (X3OD), and two stable haploid segregants from this zygote (X3OT and X3P; Baron, Spilman, and Carey, 196). The rates of 2 consumption of these strains with the various substrates are shown in Table 2. In an intergeneric cross one might expect the two parents to have different rates of utilization and the various independently isolated hybrids to have different rates of utilization. A hybrid might be like either parent or use a given substrate at an intermediate or higher rate. Table 2 shows that this is the case, depending upon the substrate. Both parents and all three hybrids use glucose at the same rate. For the other substrates tested (malate, fumarate, and succinate) there is a marked difference between the two parents; the W1895 male parent was able to utilize these substrates at about the same rate as glucose, and the 643L+ female parent used them at about onesixth the glucose rate. The unstable piliated diploid X3D had about the same rate as the female parent, as did the stable nonpiliated segregant X3T, demonstrating once more the lack of correlation between piliation and substrate utilization. The X3P stable piliated segregant, however, had a rate intermediate to that of the parents. It may be noted that had X3P (P+, intermediate rate of utilization) been compared only with X3T (P, low rate of utilization), one might mistakenly assume that nonpiliated cells had an impaired ability to utilize malate, fumarate, and succinate. One hypothesis which could account for the lower rate of utilization of 643L+ and the hybrids would be a more inefficient permeation mechanism. To test this possibility, attempts were made to force in substrates at higher concentrations. These experiments led to large variations in utilization rates which seemed to depend on the manner in which the cells were grown. It was found, however, that even if all conditions were maintained constant, large variations in utilization rate occurred. This variation suggested that random, spontaneous mutations to a higher utilization rate were occurring, causing some of the cultures to become biochemically mixed. When washed-cell suspensions were spread on minimal media containing the appropriate substrates as the sole carbon source, two types of colonies arose: one type consisted of minute colonies, and the other type, large mutant clones which could utilize the substrate at a higher rate. These mutants occurred at a high, but variable frequency of 1-5 to 1-4 depending upon the history of the particular culture. The mutant clones which appear are probably resistant to an inhibitory effect produced by the substrate. In Table 3 the rates of utilization for mutants of X3P, selected from fumarate plates, and for 643L+, selected from succinate and malate plates, are shown. It should be noted that mutation to higher rate of utilization for any one intermediate also permits the other intermediates tested to be utilized at a higher rate. Maccacaro and Hayes (1961) have recently isolated a strain of K , which resembles Brinton's strains B-L(E) and Bam in being a piliated form with small dense colonies which spontaneously and irreversibly produce a nonpiliated form with larger, more translucent colonies. These authors state that no metabolic difference exists between these TABLE 3. Utilization of substrates by mutant strains* Bacterial strains Substrates (uliters 2/hr) Glucose Fumarate Malate Succinate X3P X3P fum X3P fum i 643 L i 643 L+ suc l 643 L+ mal * Abbreviations: fum, fumarate; suc, succinate; mal, malate; +, large mutant clones; -, small background clones. Downloaded from on December 6, 218 by guest

5 42 WOHLHIETER, BRINTON, AND BARON J. BACTERIOL. two forms, in complete agreement with our experiments using B-L(E) and Bam. However, they interpret this lack of correlation between piliation and metabolic activity as being a particular property of the P+ to P type of irreversible variation, as distinguished from their "phenotypically piliated" to "phenotypically non-piliated" type of reversible variation which showed a metabolic difference. In one attempt, involving ten serial broth transfers and agar platings of each culture, we were unable to isolate a small colony variant of our strain of K In our hands, was a stably piliated strain even after ten subcultures on well-dried nutrient agar. The hemagglutinating ability of a few of the colonies was reduced but, upon restreaking, these usually regained their strong hemagglutinability. A well-piliated culture of showed no difference in aerobic or anaerobic metabolism of glucose, malate, fumarate, or succinate when compared with a culture of a stable nonpiliated strain, K12 2U. Furthermore, the piliated recombinants isolated from a mating between these two strains had the same metabolic activity as the parents. DISCUSSION It is clear from the results of respiration measurements on piliated cells and their spontaneously produced nonpiliated variants [B-L(E) P+ to B-L(E)P-, Bam, P+ to Bam P-, and X3D P+ to X3T PI- that a loss of piliation need not necessarily lead to an alteration in respiration. It is also clear that a change in respiration can occur without any change in piliation (X3D P+ to X3P P+, X3P P+ fum- to X3P P+ fum+). We may, therefore, conclude that piliation and respiration are completely independent of each other for the strains and substrates we have tested. All the observed respiratory differences can be explained by assuming that respiration is a property genetically and physiologically independent from piliation. In a cross, the two properties can segregate independently and they can also mutate independently. The results of Maccacaro and Dettori (1959), claiming a correlation of piliation with respiration, are contradictory to what we have found. Their conclusions are mainly based on the assumption that, when independently isolated recombinants are identical with respect to known markers, they are most probably identical with respect to all other characteristics. All of their recombinants acquired from the male parent threonine, leucine, maltose, and Ti phage-resistance markers; most acquired arabinose as well, and some acquired lactose in addition (Maccacaro, Colombo, and DiNardo, 1959). The genetic distance from Ti resistance to maltose on the K-12 chromosome is about one-third the length of the entire chromosome. Therefore, the male parent has transmitted a large piece of genetic material into the female including many markers not scored, which could have appeared in the recombinants. The results of Mlaccacaro and Dettori (1959) which seem to show some correlation between piliation and respiration in independently isolated recombinants could be interpreted to mean that there is genetic linkage between respiration and piliation. Linkage, however, is not a demonstration of a direct physiological relationship. If, as we suppose, piliation and respiration are independent properties, then Maccacaro and Dettori's "phenotypically non-piliated" cultures obtained by serial transfer on agar from a piliated culture should respire exactly like the piliated culture, unless respiration characteristics also change on serial agar cultivation. This could explain why their "phenotypically non-piliated" cultures respire like piliated cultures under aerobic conditions. During anaerobic glycolysis these authors found that "phenotypically nonpiliated" cultures behaved like "genotypically non-piliated" cultures, rather than like piliated cultures. This is the only instance in which they examined a less piliated culture directly derived from a more piliated culture and found a respiratory difference. During the 13 transfers through which Maccacaro and Dettori's strain was passed, the respiratory properties could easily have changed independently from piliation, since we have shown that mutation to a different rate of utilization can occur on solid media independent of piliation. ACKNOWLEDGMENTS We would like to acknowledge the technical assistance of Frank Kozcot, Jr., and Peter Gemski, Jr. Dr. Brinton's part in this research was supported by grant E-3242 and Senior Research Downloaded from on December 6, 218 by guest

6 VOL,. 8, 1962 METABOLISM OF PILIATED BACTERIA 421 Fellowship SF-47 of the National Institutes of Health, U.S. Public Health Service. LITERATURE CITED BARON, L. S., W. F. CAREY, AND W. M. SPILMAN Genetic recombination between Escherichia coli and Salmonella typhinurium Proc. Natl. Acad. Sci. U.S. 45: BARON,L. S., W. M. SPILMAN, AND W. F. CAREY Diploid heterozygous hybrids from matings between Escherichia coli and Salmonella typhosa. J. Exptl. Med. 112: BRINTON, C. C., JR Non-flagellar appendages of bacteria. Nature 183: BRINTON, C. C., JR., AND L. S. BARON Transfer of piliation from Escherichia coli to Salmonella typhosa by genetic recombination. Biochim. et Biophys. Acta 42: BRINTON, C. C., JR., A. BUZZELL, AND M. A. LAUFFER Electrophoresis and phage susceptibility studies on a filament producing variant of the B bacterium. Biochim. et Biophys. Acta 15: BRINTON, C. C., JR., AND P. GEMSKI, JR Transduction of piliation in Escherichia coli by phage P 1. Bacteriol. Proc., p. 8. BRINTON, C. C., JR., P. GEMSKI, JR., S. FALKOW, AND L. S. BARON Location of the piliation factor on the chromosome of Escherichia coli. Biochem. Biophys. Research Communs. 5: BRINTON, C. C., JR., AND M. J. STONE Chemical composition of bacterial pili-a fibrous protein. Bacteriol. Proc., p. 96. MACCACARO, G. A., C. COLOMBO, AND A. DINARDO Studi sulle fimbrie batteriche. I. lo studio genetico delle fimbrie. Giorn. microbiol. 7:1-2. MACCACARO, G. A., AND R. D)ETTORI Studi sulle fimbrie batteriche. IV. Metabolismo ossidativo e fermentativo in cellule fimbriate e efimbriate. Giorn. microbiol. 7: MACCACARO, G. A., AND W. HAYES The genetics of fimbriation in Escherichia coli. Genet. Research 2: SCHNEIDER, H., S. B. FORMAL, AND L. S. BARON Experimental genetic recombination in vivo between Escherichia coli and Salmonella typhimur-um. J. Exptl. Med. 114: UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF- FER Manometric techniques; a manual describing methods applicable to the study of tissue metabolism. Burgess Publishing Co., Minneapolis. Downloaded from on December 6, 218 by guest