Acid-Soluble Nucleotides in an Asporogenous

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JOURNAL OF BACTERIOLOGY, Mar. 1972, p. 1175-1180 Copyright 0 1972 American Society for Microbiology Vol. 109, No. 3 Printed in U.S.A. Acid-Soluble Nucleotides in an Asporogenous Mutant of Bacillus subtilis C.

1 JOURNAL OF BACTERIOLOGY, Mar. 1972, p Copyright American Society for Microbiology Vol. 109, No. 3 Printed in U.S.A. Acid-Soluble Nucleotides in an Asporogenous Mutant of Bacillus subtilis C. T. CHOW' AND I. TAKAHASHI Department of Biology, McMaster University, Hamilton, Ontario, Canada Received for publication 15 December 1971 An asporogenous mutant of Bacillus subtilis Sp-H12-3, which is considered to have a block at stage 0, showed general growth characteristics similar to those of sporulating cultures. However, a sudden increase in the total amount of acid-soluble nucleotides observed at t2 in sporulating bacteria was completely absent in this mutant. In sporulating cells, a marked increase in two nucleotides which were identified to be uridine diphosphate (UDP)-galactose and UDP-N-acetylglucosamine was noted, whereas UDP-glucose appeared to be accumulated in the mutant cells at t2. No unusual nucleotides were found in the strains of B. subtilis examined. The possible role of these UDP derivatives in early stages of sporulation in B. subtilis is discussed. It is generally believed that genes for bacterial sporulation are nonfunctional during vegetative growth but are induced or derepressed at the onset of sporulation. A number of models have been proposed to explain how the sporulation genes become functional. However, since none of the early physiological or biochemical events has been unequivocally shown to be involved in the process of sporulation, it is not possible to test these models experimentally. Thus far, only later events such as the synthesis of peptidoglycan for cortex formation, the synthesis of coat proteins, and the appearance of enzymes for dipicolinic acid synthesis are considered to be specifically related to sporulation. To investigate regulation of sporulation, a number of asporogenous (spo-) mutants of Bacillus subtilis have been isolated and analyzed by transduction and transformation in our laboratory (14, 15). One of these mutants, Sp-H12-3, is morphologically a stage 0 mutant which is blocked at a stage before the chromatin becomes an axial filament and is unable to produce "sporulation-related products" such as antibiotics, proteinases, etc. (15). Since the mutant has been shown to be a deletion mutant (15, 16), the ambiguity due to appearance of sporogenous (spo+) revertant cells is not possible. For these reasons, this mutant was used in investigations of early physiological and biochemical events which are inti- mately related to the initiation of sporulation. In the present study, general growth characteristics and changes in intracellular nucleotides of Sp-H12-3 were compared with those of sporulating cultures. Initially, the investigation on nucleotides was carried out to detect compounds which might be acting as inducing or derepressing agents for spore genes. Although no such compounds were found, a marked difference in uridine diphosphate (UDP) derivatives between Sp-H12-3 and spo+ strains was observed. MATERIALS AND METHODS Bacterial cultures. Strains of B. subtilis used were: A26 (spo+, ura), SB19E (spo+, prototrophic), Sp-H12-3 (spo-, phe), and Sp-N2-2 (spo-, ser). Broth cultures were obtained as described previously (16), except that uracil was omitted from the medium for growing cells of A26. Frequencies of sporulation in the broth cultures after 24 hr were 0.85 to 0.95 for A26 and SB19E, <10-' for Sp-H12-3, and 10-7 to 10-' for Sp-N2-2. Strains Sp-H12-3 and Sp-N2-2 were previously found to be a stage 0 mutant and a stage II mutant, respectively (15). Different stages of sporulation were designated as follows: to, the end of log phase; tn (t1, t2, etc.), n hr after to. Extraction of acid-soluble nucleotides. The method described by Leitzmanii and Bernlohr (8) was used with slight modifications to obtain acidsoluble nucleotides. Nucleotides were extracted from 10 g (wet weight) of cells with 150 ml of cold 5% trichloroacetic acid by shaking at 4 C for 15 min. The treated cells were collected by centrifugation and I Present address: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada. ground cells were suspended in 50 ml of cold 5% triground with an equal amount of quartz sand. The 1175

2 1176 CHOW AND TAKAHASHI J. BACTERIOL. chloroacetic acid, shaken for 15 min, and centrifuged. The supernatant fluid was combined with the trichloroacetic acid extract obtained earlier, and the ph was adjusted to 5.5 with KOH. To this solution, 5 g of acid-washed Norit A was added, and the mixture was stirred for 2 hr at 4 C to adsorb nucleotides. Norit A with bound nucleotides was separated by centrifugation, washed twice with 50 ml of 0.05 M sodium acetate buffer (ph 4.5) and once with distilled water, and eluted five times with 20 ml of cold 50% ethanol containing 1% NH4OH. The eluates were filtered and evaporated to 10 ml with a Rotovapor rotary vacuum evaporator at 35 C. Cromatography of nucleotides. Acid-soluble nucleotides were separated by column chromatography with Dowex-1 (X-8, 200 to 400 mesh, Bio-Rad Laboratories), formate form, by the procedure described by Leitzmann and Bernlohr (8). After the chromatography, formic acid was removed from nucleotide samples by vacuum evaporation at 35 C. To protect nucleotides from the drop in ph caused by evaporation, five to seven drops of 1 N KOH were added to neutralize formic acid. When the samples were reduced to about 0.2 of the original volume, 10 to 15 ml of distilled water was added, and the samples were again evaporated to a small volume. Major nucleotide peaks which were tentatively identified from their 275 nm/260 nm ratio were further purified by Dowex-1 chromatography with ammonium formate buffer as eluant (7). Ultraviolet absorption spectra of the final products were determined with a Gilford spectrophotometer over the range of 210 to 300 nm at ph values of 2, 7, and 11. The identity of purified nucleotides was further confirmed by paper chromatography according to Smith and Mills (12). Other analytical methods. The sugar moiety was separated from UDP derivatives by the technique of Cabib et al. (4). Samples were hydrolyzed with 0.1 N H2SO4 at 100 C for 10 min. Hydrolysates were neutralized in 0.3 N Ba(OH)2, and free UDP was precipitated by the addition of an equal volume of 5% ZnSO4 and 0.3 N Ba(OH)2. The precipitate was removed by centrifugation, and the supematant fluid was used for the determination of sugars. Acetylglucosamine was identified by the technique of Aminoff et al. (1); galactose and glucose were identified by the technique of Ashwell (2). Paper chromatography of sugar was carried out according to Smith (13). The phosphorus content of nucleotides was determined by the technique of Chen et al. (5) Ċellular nucleic acids were extracted and separated by the method of Schmidt and Thannhauser modified by Siminovitch and Graham (11). The amount of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) was determined by diphenylamine and orcinol reactions, respectively (3, 9). RESULTS Growth characteristics. The growth curve and changes in ph and pyruvate in cultures of A26 (spo+) and Sp-H12-3 are shown in Fig. 1. In both A26 and Sp-H12-3, the ph started to decrease in the middle of the log phase and reached a minimal value at to. Thereafter, the ph value began to increase steadily. Pyruvate which was accumulated during the log phase was completely utilized at t2 in both cultures. After 24 hr, 85 to 95% of A26 cells were converted to refractile spores, and, as a consequence, the optical density as measured by a Klett colorimeter increased considerably. In Sp-H12-3, on the other hand, a marked decrease in optical density due to cell lysis was observed after 24 hr. The growth curve and changes in ph and pyruvate concentration in another spo+ strain SB19E and a stage II mutant Sp-N2-2 were very similar to those of Sp-H12-3 and A26 illustrated in Fig. 1. Acid-soluble nucleotides. Nucleotides were extracted from cells of A26 or Sp-H12-3 harvested at log, t0, t1, t2 and t3. The nucleotide samples, after concentration, were adsorbed on a Dowex-1 column and eluted with a gradient of formic acid by the technique of Hurlbert et al. (7). Typical elution profiles of nucleotides extracted from cells at stages log and t2 are shown in Fig. 2 and 3, respectively. Ultraviolet (UV)-absorbing materials were identified by absorption spectra determined in the wavelength range of 210 to 300 nm and also by paper chromatography (12). Most UV-absorbing peaks contained 400F o0 I,P %X 0.4-D7 cn A ot~ ~ ~ ~~~~ TIME (HOUR) FIG. 1. Growth curve and changes in ph and pyruvate. Cells were grown in Schaeffer's sporulation medium (14) in a water-bath shaker at 37 C. The ph values and pyruvate concentration were determined according to Nagata and Halvorson (10). Solid lines, A26; broken lines, Sp-H12-3.

3 VOL. 109, 1972 common nucleotides except for three peaks eluted at the tube numbers 118 to 123, 126 to 130, and 133 to 138. They were designated as X,, X2, and X3 and are shown in Fig. 2 (log phase) and 3 (t2). The amounts of these compounds extracted at other stages were very small. Ultraviolet absorption spectra of these three compounds indicated that they might be derivatives of uridine. The chromatographic behavior of the unknown nucleotides on a Dowex-1 column suggested that nucleotide Xl, X,2 and X3 might be UDP-N-acetylglucosamine, UDP-galactose, and UDP-glucose, respectively. The R (adenosine) values obtained by paper chromatography for nucleo- ASPOROGENOUS MUTANT OF B. SUBTILIS 1177 tides X1 (0.55), X2 (0.42), and X3 (0.44) were very close to those of UDP-N-acetylglucosamine (0.56), UDP-galactose (0.41), and UDP-glucose (0.44). The sugar moieties in the three nucleotides were separated and identified by the techniques of Aminoff et al. (1) and Ashwell (2). It was found that the absorption spectrum of the sugar extracted from nucleotide Xl, after treatment with dimethylaminobenzaldehyde, was identical with that of N- acetylglucosamine. The sugars liberated from nucleotides X2 and X3 were identified as galactose and glucose from their characteristic color developed with the cystein-sulfuric acid reagent. The result of phosphorus determina ~~~~~ TUBE FIG. 2. Elution profile of acid-soluble nucleotides extracted from cells in the log phase. Solid line, A26; broken line, Sp-H I IIa. NO L ~~~I.27~~~~ TUBE NO FIG. 3. Elution profile of acid-soluble nucleotides extracted from cells at t2. Solid line, A26; broken line, Sp-H12-3.

4 CHOW AND TAKAHASHI J. BACTERIOL tion together with the above data on hexoses creased considerably between t, and t3. This revealed the ratio of hexose:phosphorus: tendency was also evident in another spo+ culuridine to be 1:2: 1 (Table 1). From the above ture SB19E and in a stage II mutant Sp-N2-2. results, nucleotides, X1, X2, and X3 were iden- The increase in acid-soluble nucleotides was tified as UDP-N-acetylglucosamine, UDP-ga- completely absent in Sp-H12-3. No marked lactose, and UDP-glucose, respectively. changes were noted in nucleoside di- and tri- Quantitative determination of acid-sol- phosphates in any strains studied. uble nucleotides. The amounts of nucleotides In A26, the concentration of diphosphodetermined from their absorbancy at ph 1 (7) pyridine nucleotide (DPN) which is a cofactor are shown in Table 2. The total amount of nu- for many enzymatic reactions became very low cleotides in sporulating A26 cells at to was at t1 but started to increase thereafter. At t3, only one-half of that in the log phase but in- the level of DPN was even higher than that of TABLE 1. Uridine, phosphorus, and hexose contents in nucleotides X1, X2, and X3 Components Nucleotide XXa x2a X3a X3b Concn (MM)I Ratio Concn(jiM) Ratio Concn (gm) Ratio Concn (imm) Ratio Uridine Total Pi Labile Pi Hexose a Nucleotide extracted from A26 cells. b Nucleotide extracted from Sp H12-3 cells. TABLE 2. Acid-soluble nucleotides in A26 and Sp -H12-3 Amt of nucleotides (Mmoles) per 10 g of cells Nucleotidea A26 Sp H12-3 log to ti t2 t3 log t0 t t2 t AMP ADP ATP GMP GDP GTP CMP CDP CTP UMP UDP UTP DPN UDPAG UDP-Gal UDPG Totals a AMP = adenosine monophosphate; ADP = adenosine diphosphate; ATP = adenosine triphosphate; GMP = guanosine monophosphate; GDP = guanosine diphosphate; GTP = guanosine triphosphate; CMP = cytidine monophosphate; CDP = cytidine diphosphate; CTP = cytidine triphosphate; DPN = diphosphopyridine nucleotide; UDPAG = UDP-N-acetylglucosamine; UDP-Gal = UDP-galactose; UDPG = UDPglucose.

VOL. 109, 1972 ASPOROGENOUS MUTANT OF B. SUBTILIS 1179 the log phase. In contrast, this nucleotide in Sp-H12-3 gradually decreased to a very low level during the presporulation period.

5 VOL. 109, 1972 ASPOROGENOUS MUTANT OF B. SUBTILIS 1179 the log phase. In contrast, this nucleotide in Sp-H12-3 gradually decreased to a very low level during the presporulation period. The amounts of the UDP derivatives were about the same in both Sp-H12-3 and A26 up to the stage t1. At t2, a sudden increase in UDP-N-acetylglucosamine and UDP-galactose occurred in A26, but no detectable amount of these two compounds was present in Sp-H12-3. On the other hand, UDP-glucose which might be a precursor of UDP-galactose was accumulated in Sp-H12-3 at t2, Changes in the total amount of acid-soluble nucleotides together with those of cellular nucleic acids were shown in Fig. 4. As expected, the amount of DNA remained almost constant in both A26 and Sp-H12-3. The decrease in RNA in A26 would probably reflect the increased ribonuclease activity and could account for the increase in acid-soluble nucleotides. This would suggest that the observed increase in acid-soluble nucleotides may be due to the degradation of RNA rather than to de novo synthesis of nucleotides. An analogous situation has been found in B. licheniformis by Leitzmann and Bernlohr (8). In Sp-H12-3, on the other hand, there was no appreciable change in either RNA or acid-soluble nucleotides throughout the experiment. DISCUSSION Many Bacillus species accumulate organic acids such as pyruvate and acetate in broth cultures during the log phase, causing the ph to become lower. The accumulated organic acids are utilized during early stages of sporulation, and ph values increase gradually. The same changes were observed with B. subtilis strain A26 (spo+) in the present study. However, the observation that these changes in a stage 0 mutant, Sp-H12-3, were indistinguishable from those of A26 raised the question as to whether they are directly related to the process of sporulation. Similarly, no qualitative and quantitative differences in acid-soluble nucleotides were detectable between A26 and Sp-H12-3 up to the stage t,. In addition, unusual nucleotides such as 3', 5'-cyclic adenosine monophosphate or similar compounds were not detected in either culture. Data accumulated thus far, therefore, reveal no marked difference in general physiology between the two strains during the log phase and at t,. An increase in acid-soluble nucleotides has been observed at early stages of sporulation in B. licheniformis by Leitzmann and Bernlohr (8). It appears that the increased ribonuclease activity and larger nucleotide pool in presporulation stages are common characteristics of sporulating cells. It has been observed in the present study that the size of the nucleotide pool increased markedly at t2 and t3 in sporulating cultures and in a stage II mutant (sp- N2-2). In Sp-H12-3, on the other hand, the amount of acid-soluble nucleotides remained at about the same level during the presporulating period. Furthermore, the increase in UDP-N-acetylglucosamine and UDP-galactose found at t2 in A26 was also absent in Sp-H12-3. Since UDPglucose, which may be a precursor of UDP-galactose, is accumulated in Sp-H12-3 at t2, a mutation borne by this strain is probably preventing the conversion of UDP-glucose to other UDP derivatives. Although care was taken to avoid the degradation of nucleic acids and nucleotides during extraction, the possibility exists that some of the changes in nucleotides observed here may not reflect the real situation in the cells. Nevertheless, it is interesting to note that UDP-galactose-4-epimerase (EC ), one of the enzymes involved in the synthesis of UDP-galactose, was Z E C-) C.) -J -) z 1oo- 50H -o ' ---o 7_ O W, 12 lp t l log t0 ti t2 t3 TIME (HOUR) FIG. 4. Changes in nucleic acids and acid-soluble nucleotides in strains A26 and Sp-H12-3 at early stages of sporulation. Nucleic acids were extracted from I g (wet weight) of cells at the stages indicated, and the amounts of DNA (-) and RNA (0) were determined. The total amounts of acid-soluble nucleotides are shown by (x). The values of nucleic acids are the average of three determinations, and those of nucleotides are the average of two determinations. Solid lines, A26; broken lines, Sp-H j z

1180 CHOW AND TAKAHASHI J. BACTERIOL. found to remain at a very low level in Sp-H12-3, whereas spo+ cultures show a marked increase in the enzyme activity at t2 (Chow and Takahashi, unpublished data).

6 1180 CHOW AND TAKAHASHI J. BACTERIOL. found to remain at a very low level in Sp-H12-3, whereas spo+ cultures show a marked increase in the enzyme activity at t2 (Chow and Takahashi, unpublished data). Of course, it is possible that this mutant has physiological or biochemical blocks other than UDP-galactose- 4-epimerase because of its large deletion (15, 16). Other enzymes concerning the synthesis of UDP derivatives are being investigated in our laboratory. Recently, Hitchins and Slepecky (6) proposed a hypothesis that bacterial sporulation may be a modified prokaryotic cell division and that the morphological changes that occur during sporulation are principally the result of changes in the relative timing and quantity of membrane and cell wall materials that are synthesized. It may not be fortuitous that at t2, when septum formation takes place, the amount of UDP-galactose and UDP-N-acetylglucosamine suddenly increases in wild-type strains and a stage II mutant (Sp-N2-2) but not in Sp-H12-3. Unlike Sp-N2-2 which has a block at a stage later than septum formation, Sp-H12-3 shows no morphological changes related to sporulation. Although the present data are not sufficient to determine the role of UDP-galactose and UDP-N-acetylglucosamine in sporulation, a reasonable interpretation of the morphological data is that these compounds could be involved in the synthesis of membrane or cell wall materials. Alternately, the accumulated UDP-derivatives may interfere with enzymes concerning the synthesis of membrane or cell wall materials, thus creating an unbalanced situation. In either case, the UDP-derivatives would have an important role in early stages of sporulation. ACKNOWLEDGMENT This investigation was supported by grant no. A-1996 from the National Research Council of Canada. LITERATURE CITED 1. Aminoff, D., W. T. J. Morgan, and W. M. Watkins Studies in immunochemistry. II. The action of dilute alkali on the N-acetylhexosamines and the specific blood-group mucoids. Biochem. J. 51: Ashwell, G Colorimetric analysis of sugars, p In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York. 3. Burton, K A study of the conditions and mechanism of the diphenylamaine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62: Cabib, E., L. F. Leloir, and C. E. Cardini Uridine diphosphate acetylglucosamine. J. Biol. Chem. 203: Chen, P. S., T. Y. Toribara, and H. Warner Microdetermination of phosphorus. Anal. Chem. 28: Hitchins, A. D., and R. A. Slepecky Bacterial sporulation as a modified procaryotic cell division. Nature (London) 223: Hurlbert, R. B., H. Schwitz, A. F. Brumm, and V. R. Potter Nucleotide metabolism. II. Chromatographic separation of acid soluble nucleotides. J. Biol. Chem. 209: Leitzmann, D., and R. W. Bernlohr Changes in the nucleotide pool of Bacillus licheniformis during sporulation. J. Bacteriol. 89: Mejbaum, W Uber die Bestimmung kleiner Pentosemengen insbesondere in Derivaten der Adenylsaiure. Hoppe-Seyler's Z. Physiol. Chem. 258: Nakata, H. M., and H. 0. Halvorson Biochemical changes occurring during growth and sporulation of Bacillus cereus. J. Bacteriol. 80: Siminovitch, L., and A. F. Graham Synthesis of nucleic acid in Escherichia coli. Can. J. Microbiol. 1: Smith, E. E. B., and G. T. Mills Uridine nucleotide compounds of liver. Biochim. Biophys. Acta 13: Smith, I Chromatographic and electrophoretic techniques, 3rd ed. Interscience Publishers, Inc., New York. 14. Takahashi, I Transduction of sporogenesis in Bacillus subtilis. J. Bacteriol. 89: Takahashi, I Genes controlling sporulation in Bacillus subtilis, p In L. L. Campbell (ed.), Spores IV. American Society for Microbiology, Bethesda, Md. 16. Yamagishi, H., and I. Takahashi Genetic transcription in asporogenous mutants of Bacillus subtilis. Biochim. Biophys. Acta 155:

NUCLEOSIDES AND NUCLEOTIDES OF THE COLD ACID-SOLUBLE PORTION OF THE BLOOD OF STEERS 1

NUCLEOSIDES AND NUCLEOTIDES OF THE COLD ACID-SOLUBLE PORTION OF THE BLOOD OF STEERS 1 Robert C. Smith and Connie M. Stricker Auburn University 2, Auburn, Alabama 36830 SUMMARY The ultraviolet absorption