STREPTOCOCCAL L FORMS

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1 STREPTOCOCCAL L FORMS II. CHEMICAL COMPOSITION' CHARLES PANOS, S. S. BARKULIS, AND J. A. HAYASHI Department of Biological Chemistry, University of Illinois College of Medicine, Chicago, Illinois Received for publication June 5, 1959 Suspension of a streptococcal L form in hypotonic solutions is accompanied by loss of viability and structural disintegration (Panos and Barkulis, 1959). Consequently, a variable amount of water soluble material is lost in employing hypotonic solutions for washing procedures and results in only a residue portion remaining. Analyses of such residues in terms of the chemical constituents of the intact L form may, therefore, lead to erroneous interpretations and conclusions. A paper from this laboratory has described the preservation of viability and structural integrity of an L form derived from a nontypable group A 3-hemolytic streptococcus suspended in 0.3 M hypertonic sucrose (Panos and Barkulis, 1959). This report describes experiments designed to measure the loss of nucleic acids from a streptococcal L form when distilled water is employed in lieu of a protective solute during washing procedures. The first chemical analyses of various components from this osmotically protected L form are also presented and the results compared to the parent streptococcus. Part of these results have been reported in preliminary form (Panos et al., 1959). MATERIALS AND METHODS Growth of the organisms. The preparation of the liquid medium, conditions for growth and examination, and characteristics of the streptococcal AED L form have been described (Panos and Barkulis, 1959). The parent streptococcus was cultured in the same medium except that sodium chloride and penicillin were omitted. The dry weight yield of the parent streptococcus from 100 ml of broth after 24 hr incubation was from 90 to 107 mg and 23 to 27 mg when cultured in the presence of 1.5 and 0.1 per cent glucose, re- ' This investigation was supported by a grant from the Institute of Arthritis and Infectious Disease of the U. S. Public Health Service, no. E spectively. Under similar conditions the yield of the L form was from 15 to 23 mg and 8 to 14 mg, respectively, after 36 hr incubation. For chemical investigations, large cultures of the L form and streptococcus (0.8 to 1.0 L) grown in low or high glucose (0.1 or 1.5 per cent) concentrations were harvested at 6000 rpm in the Servall SS-1 centrifuge, washed twice, and resuspended in a third portion of either 0.3 M sucrose or distilled water. Following shell freezing, the cellular mass was lyophilized and stored at room temperature in a desiccator over P205 and NaOH. L form cultures were harvested after 36 hr and streptococci after 24 hr incubation at 35 C. L form content of lyophilized L formsucrose preparations was determined by weight difference and nitrogen content. Chemical methods. Hexosamine, expressed as glucosamine, was determined by the method of Dische and Borenfreund (1950) and Roseman and Daffner's modification (1956) of the Boas method. Prior to analyses of L form-sucrose preparations for rhamnose (Dische and Shettles, 1948), sucrose was removed by extensive washing with 0.3 M phosphate buffer (Panos and Barkulis, 1959) to prevent charring during the determination. As suggested by Dische (1955), the heating time during this analysis was extended from 3 to 10 min in order to destroy interfering chromogens. The nitrogen content of dried cells and hydrolyzates was determined by micro-kjeldahl or Nesslerization procedures. Dried cells (15 to 25 mg) were extracted for ultraviolet absorbing material by repeated agitation and centrifugation with 3 ml portions of 5 per cent trichloroacetic acid at 4 C. Cold trichloroacetic acid extraction was discontinued after removal of all ultraviolet absorbing material as determined with a Beckman spectrophotometer set at 260 m,u. Discoloration due to sucrose from L form-sucrose preparations during ribonucleic acid analyses was avoided by multiple 863

2 864 PANOS, BARKULIS, AND HAYASHI [VOL. 78 extractions with 10-ml quantities of 5 per cent trichloroacetic acid for 24 hr in the cold (4 C). Extracts for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) determinations were obtained, following centrifugation, by extracting the remaining cellular residue with 5 per cent trichloroacetic acid in a boiling water bath for 30 min and the volume adjusted to 2 ml. For DNA analyses, a modification of the method by Burton (1955) was employed. To 0.5 ml of the extract were added 0.2 ml of 0.07 M acetaldehyde and 4 ml of diphenylamine reagent, prepared by dissolving 7.5 g of this reagent into 500 ml of glacial acetic acid plus 7.5 ml concentrated sulfuric acid, and the blue color allowed to develop at 25 to 30 C for at least 12 hr. The modified procedure of Drury (1948) was employed for RNA analyses. To 0.5 ml or less of the extract were added 0.3 ml of a freshly prepared 10 per cent orcinol solution (95 per cent ethanol) and distilled water to 3 ml. To this were added 3 ml of iron reagent prepared by dissolving g Fe(NH4)(SO4)2-12H20 into 1 L of concentrated HCI, the tubes capped with glass marbles, and the green color developed by immersing into a boiling water bath for 45 min. Color intensities were read with a spectrophotometer set at 600 m,u for DNA and 660 ma for RNA analyses. Commercially available DNA (California Corporation for Biochemical Research) and RNA (Nutritional Biochemicals Corp.) heated in 5 per cent trichloroacetic acid, as described above, served as the standards. Hydrolysis and chromatographic analyses. For rhamnose and hexosamine determinations, samples were hydrolyzed with 2 N HCI in sealed tubes at 108 C for 3 hr and the acid removed under reduced pressure as previously described (Hayashi and Barkulis, 1959). Hydrolyzed preparations were dried and stored in vacuo over P205 and NaOH. The dried hydrolyzates were dissolved in distilled water, samples deposited onto 0.8 by 8.5 cm Dowex 50-X8 (H+ form) columns, and rhamnose and hexosamine eluted with distilled water and 2 N HCl, respectively. The hydrolysis of samples (25 mg) for amino acid analyses was performed in 100 ml of 1 N HCI for 36 hr as previously described (Hayashi and Barkulis, 1959). Humin from L form-sucrose preparations was removed by filtration and all samples were dissolved in ph 2.2, 0.2 N sodium citrate buffer prior to chromatographic analyses. Samples were analyzed by ion exchange chromatography on 0.9 by 150 cm columns of Dowex- 50 by the method of Moore and Stein as described by Tepper and Wilson (1958). Identification of each peak was accomplished by comparing its position with similar elution patterns previously confirmed by paper chromatography. RESULTS C Polysaccharide components. The glucosamine and rhamnose content of the L form appears in table 1. Rhamnose determinations were done either on hydrolyzates obtained as described under Materials and Methods or samples analyzed directly without prior hydrolysis. Both procedures gave comparable results. By comparison with various group A streptococci, the glucosamine and rhamnose content of the L form was appreciably lower. The rhamnose content of the L form washed with 0.3 M sucrose and 0.3 M phosphate buffer, however, was always found to be higher than that observed with water washed, lyophilized preparations. The glucosamine content remained the same. Total amino acid composition. Table 2 lists the results of the amino acid analyses of the streptococcus and its L form grown in 0.1 per cent glucose. Prior to lyophilization, the streptococcus was washed with and suspended in distilled water while hypertonic 0.3 M sucrose replaced distilled water for the L form. Each organism was hydrolyzed, without further extraction, to prevent TABLE 1 Rhamnose and glucosamine content of the osmotically protected and water washed streptococcal AED L form Organism Washing Dry Weight Procedure Glucos- Rhamamine nose % % Group A streptococci... Water AED L form... Water < AED L form... Sucrose- <0.4t 0.3 phosphate* * See text under Materials and Methods. t From column chromatographic analyses data, see table 2.

3 1959] STREPTOCOCCAL L FORMS 865 TABLE 2 Amino acid composition of the streptococcus and its intact L form Strepto- L Form Amino' Amino~~Acid Acid COCCUS Amino Amino Acid* Acid * jimoles,umoles Aspartic Threonine Serine Muramic t Proline Glutamic Glycine Alanine Valine Methionine Isoleucine Leucine Glucosamine Tyrosine Phenylalanine Ammonia Ornithine Lysine Histidine Arginine Per cent total nitrogen recovered Per cent nucleic acid nitrogen Total per cent nitrogen accounted for * Per 5.10 mg dry weight of cells. t Leucine equivalents. disintegration and loss of intracellular components of the L form and to allow for a more accurate comparison of their amino acid composition. The brownish discoloration remaining after humin removal from L form-sucrose hydrolyzates did not affect the chromatographic resolution or ninhydrin analyses of the various fractions. The percentage of protein nitrogen actually lost during analyses is small. As is shown in table 3, the streptococcus and its L form contain and per cent, respectively, of total nucleic acids. Since the average nitrogen content of the nucleotides is approximately 20 per cent, the total nitrogen content accounted for is from 103 to 108 per cent when the nucleic acid nitrogen is considered. Both quantitative and qualitative differences in amino acid content were found between streptococcal and L form hydrolyzates (table 2). In addition to finding the same predominating amino acids in both organisms, the L form had an equal or higher concentration of almost all of the other amino acids present as compared with the coccal form. A notable difference between these organisms was the absence of muramic acid and ornithine, the surprisingly high content of serine and leucine, and the lower aspartic acid, alanine, and lysine content found in the L form. Chemical constituents of the osmotically protected and water washed L form. Table 3 tabulates the percentages of the various chemical constituents of the L form and its parent streptococcus following the washing procedure indicated. TABLE 3 Comparison of some of the chemical constituents of the osmotically protected and water washed Lform Per Cent with 1.5% Glucose Per Cent with 0.1% Glucose Organism Washing Extract- Procedure Extractable RDTtlal / ultra- N RNA DNA R/D TotNA ultra- N RNA DNA violet ratiot NAt material ioletr NA material Streptococcus... Water L Form... Water L Form M Sucrose * From cold 5 per cent trichloroacetic acid extracts measured at 260 mg. See text for calculations. t R/D ratio = ribonucleic acid-deoxyribonucleic acid ratio. t NA = nucleic acid.

4 866 PANOS, BARKULIS, AND HAYASHI[ [VOL. 78 The relative percentage of extractable ultraviolet absorbing material present was calculated from the following equation and assumptions. Corrections have been made for the solvent absorbence. (A.) * (MW) g/l where Am = molar absorbancy = 10 X 103; for uridylic acid (Pabst Circular OR-10, 1956). As = absorbancy. MW = molecular weight; average of 340 for nucleotides. The nucleic acids used as standards were of the highest purity commercially available. Rhamnose simultaneously extracted while preparing nucleic acid containing extracts from streptococcal cells was found to interfere with the RNA analysis. Therefore, a standard RNA curve containing a predetermined amount of rhamnose equal to that found in such extracts was employed to compensate for the resulting increase in color intensity. However, since the error encountered is negligible when optical densities above are avoided, the internal rhamnose standard was omitted with aliquots for analyses indicating a low RNA content. Although the extractable ultraviolet absorbing material and nitrogen content did not vary markedly with the various washing procedures employed, approximately 40 per cent of the total nucleic acid content of the L form was lost during water washing treatment. No significant difference was found in the total nucleic acid content of the coccus and its intact L form washed with 0.3 M sucrose. The ribonucleic to deoxyribonucleic acid ratio is relatively constant following washing with an osmotically protective solution (0.3 M sucrose) and is similar with that of the parent streptococcus. DISCUSSION The osmotically protected L form was reexamined for the presence of C polysaccharide components and compared with water washed preparations (table 1). More rhamnose was found with water washed preparations than had been previously reported. However, the results obtained are in general agreement with those of Sharp et al. (1957). Although a higher rhamnose content was always found in the osmotically protected L form, the total percentage still remained so low as to exclude this carbohydrate as a major structural component. The cell walls of type 14, group A streptococci have been found to contain muramic acid, an amino sugar associated primarily with cell wall structure, as well as significant amounts of rhamnose and glucosamine (Hayashi and Barkulis, 1959). The failure to observe these carbohydrates in this group A B-hemolytic streptococcal L form reconfirms the absence of a bacterial cell wall similar to that of the parent organism. A comparison of the amino acid composition of the osmotically protected L form and its parent streptococcus revealed major differences (table 2). Barkulis and Jones (1957) have shown that the streptococcal cell walls of group A streptococci comprise approximately 25 per cent of the dry weight of the intact streptococcal cell. The reason for the presence of a comparatively high content of serine and leucine in the L form is at present obscure and cannot be accounted for by dry weight differences due to loss of the streptococcal cell wall. Since uridine diphosphate type compounds have been implicated as precursors for continual cell wall synthesis of the bacterial cell (Park, 1952; Park and Strominger, 1957), the extractable ultraviolet absorbing content of the L form and parent streptococcus was estimated. Although a greater amount of absorbing material was always found in the streptococcus, it was of interest to observe that appreciably more ultraviolet absorbing material accumulated within the L form and streptococcus when a low (0.1 per cent) rather than a high glucose concentration was employed (table 3). A chemical investigation of such fractions may provide an insight into the type of cell wall precursors necessary for cell wall synthesis to continue. As is documented in table 3, failure to consider osmotic fragility, by employing distilled water as a washing procedure, results in the complete disintegration of the L form and loss of as much as 35 to 40 per cent of the total nucleic acid content. This illustrates the ease with which the nucleic acids are removed from within the L form and, again, emphasizes the need for employing protective washing procedures for chemical analyses to be meaningful. The results of the analyses of the osmotically protected L form agree closely with those published for the intact

5 1959] STREPTOCOCCAL L FORMS 867 streptococcus. Over 11 per cent of the L form is composed of nucleic acids, of which ribonucleic acid (80 per cent or more) predominates. The ribonucleic acid to deoxyribonucleic acid ratio of the 0.3 M sucrose washed L form as compared to the parent streptococcus agrees favorably with that reported by Mitchell and Moyle (1950) for the gram-positive bacteria (4.1). SUMMARY These results illustrate the necessity for employing osmotically protective washing procedures in order for chemical analyses to be meaningful. Washing the L form with distilled water results in complete cellular disintegration and loss of as much as 40 per cent of the total nucleic acid content. A high content of cold trichloroacetic acid extractable ultraviolet absorbing material was consistently found in the streptococcus and its L form cultured at a low glucose concentration. Ornithine and the major cell wall carbohydrates (glucosamine, rhamnose, and muramic acid) were absent in the L form. The same predominating amino acids as well as a surprisingly high content of serine and leucine were found in the L form as compared with the parent streptococcus. REFERENCES BARKULIS, S. S. AND JONES, M. F Studies of streptococcal cell walls. I. Isolation, chemical composition and preparation of M protein. J. Bacteriol., 74, BURTON, K The relationship between the synthesis of deoxyribonucleic acid and the synthesis of protein in the multiplication of bacteriophage T2. Biochem. J., 61, DISCHE, Z Methods of biochemical analysis, Vol. 2, pp Interscience Publishers, Inc., New York. DISCHE, Z. AND BORENFREUND, E A spectrophotometric method for the microdetermination of hexosamines. J. Biol. Chem., 184, DISCHE, Z. AND SHETTLES, L. B A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J. Biol. Chem., 175, DRURY, H. F Identification and estimation of pentoses in the presence of glucose. Arch. Biochem., 19, HAYASHI, J. A. AND BARKULIS, S. S Studies of streptococcal cell walls. III. The amino acids of the trypsin-treated cell wall. J. Bacteriol., 77, MITCHELL, P. AND MOYLE, J Occurrence of a phosphoric ester in certain bacteria: Its relation to gram staining and penicillin sensitivity. Nature, 166, Pabst Circular OR Ultraviolet absorption spectra of 5'-ribonucleotides. Pabst Brewing Company, Milwaukee, Wisconsin. PANOS, C. AND BARKULIS, S. S Streptococcal L forms. I. The effect of osmotic change on viability. J Bacteriol.,78, PANOS, C., BARKULIS, S. S., AND HAYASHI, J. A Viability and structural integrity of a streptococcal L form. Bacteriol. Proc., 1959, 126. PARK, J. T Uridine-5'-pyrophosphate derivatives. I. Isolation from Staphylococcus aureus. J. Biol. Chem., 194, PARK, J. T. AND STROMINGER, J. L Mode of action of penicillin. Biochemical basis for the mechanism of action of penicillin and for its selective toxicity. Science, 125, ROSEMAN, S. AND DAFFNER, I Colorimetric method for determination of glucosamine and galactosamine. Anal. Chem., 28, SHARP, J. T., HIJMANS, W., AND DIENES, L Examination of the L forms of group A streptococci for the group-specific polysaccharide and M protein. J. Exptl. Med., 105, TEPPER, B. S. AND WILSON, J. B A comparison of the amino acid composition and nucleic acid content of strains of Brucella abortus. J. Infectious Diseases, 103,