Cent Guanine Research Articles

Purification of DNA from the intracellular symbiotes of Sitophilus oryzae and Sitophilus zeamais (Coleoptera: Curculionidae)

DNAs from the endosymbiotes of two species of grain weevils, Sitophilus oryzae and S. zeamais, were separated from host-weevil DNA and purified by cesium chloride gradient centrifugation. Endosymbiote DNA was identified by hybridization with a probe of the gene encoding the 16S rRNA of Escherichia coli. Estimations of the CsCl buoyant densities of symbiote DNAs of S. oryzae and S. zeamais were similar ( ϱ = 1.712 and 1.714, respectively) corresponding to a mole per cent guanine + cytosine (GC%) of ≈52–55%. The GC%s of host-weevil DNAs were ≈33%.

Guanine plus cytosine content of the DNA ofCoccidioides immitis

The mole per cent guanine plus cytosine was determined for DNA extracted from Coccidioides immitis to add information pertinent to the taxonomy of this zoopathogenic fungus. Four strains of C. immitis, three in the mycelial phase, one in the spherule/endospore phase yielded guanine plus cytosine in the narrow range of 49.41 to 49.61 mole percent. These values are lower than those for ascomycetes that mimic the arthroconidial form of C. immitis, higher than those for ascomycetous yeasts, but closer to those of 'transition zone' or basidiomycetous yeasts.

β-Haemolysis and pigment production by the oral bacterium Streptococcus mutans

The prevalence of β-haemolysis and yellow pigmentation among 89 isolates of Streptococcus mutans from human dental plaque and 59 Strep. mutans isolates from positive cultures of human dental root canals were investigated. β-Haemolytic strains were more frequent among the root-canal isolates (25.4 per cent) than among the dental plaque isolates (12.4 per cent). The distribution of pigment production among β-haemolytic strains (22 pigmented strains out of 26 strains) and among non-haemolytic strains (5 pigmented strains out of 122 strains) showed a relationship between haemolysin and pigment production. β-Haemolytic, pigmented strains (biotype c and e and 35–39 per cent guanine plus cytosine) were common in plaque and were the dominant type of Strep. mutans in some plaques. As β-haemolysis by Strep. mutans was only demonstrated after strict anaerobic growth, whereas pigment production was demonstrated after growth under less anaerobic conditions (candle jar) on sucrose-containing agar plates, the observation of pigment may facilitate a presumptive identification of β-haemolytic strains of Strep. mutans.

Streptococcus mutans isolates identified by biochemical tests and DNA base contents

Fifty-five strains of Streptococcus mutans were isolated from dental plaque in a clinical survey of the presence of this species and others resembling it. The species identifications were based on biochemical tests; determination of DNA base contents (mol per cent G + C) of 43 isolates confirmed the identifications. The results showed that Strep. mutans, as currently defined, can be easily and reliably identified by biochemical tests, and that DNA of clinical isolates have the expected 36 to 38 mol per cent guanine plus cytosine. Strep. mutans was clearly the predominant Strep. mutans-like streptococcus in the population sampled.

Identification of named cultures of Bacillus calidolactis as Bacillus stearothermophilus.

A study was made of 23 cultures of Bacillus calidolactis. By using cultural, biochemical, and per cent guanine plus cytosine methods, the cultures were found to be B. stearothermophilus.

Taxonomy of Aerobic Marine Eubacteria

Two hundred and eighteen strains of nonfermentative marine bacteria were submitted to an extensive morphological, physiological, and nutritional characterization. All the strains were gram-negative, straight or curved rods which were motile by means of polar or peritrichous flagella. A wide variety of organic substrates served as sole sources of carbon and energy. The strains differed extensively in their nutritional versatility, being able to utilize from 11 to 85 carbon compounds. Some strains had an extracellular amylase, gelatinase, lipase, or chitinase and were able to utilize n-hexadecane and to denitrify. None of the strains had a yellow, cell-associated pigment or a constitutive arginine dihydrolase system, nor were they able to hydrolyze cellulose or agar. The results of the physiological and nutritional characterization were submitted to a numerical analysis which clustered the strains into 22 groups on the basis of phenotypic similarities. The majority of these groups were separable by a large number of unrelated phenotypic traits. Analysis of the moles per cent guanine plus cytosine (GC) content in the deoxyribonucleic acid of representative strains indicated that the peritrichously flagellated groups had a GC content of 53.7 to 67.8 moles%; polarly flagellated strains had a GC content of 30.5 to 64.7 moles%. The peritrichously flagellated groups were assigned to the genus Alcaligenes. The polarly flagellated groups, which had a GC content of 43.2 to 48.0 moles%, were placed into a newly created genus, Alteromonas; groups which had a GC content of 57.8 to 64.7 moles% were placed into the genus Pseudomonas; and the remaining groups were left unassigned. Twelve groups were given the following designations: Alteromonas communis, A. vaga, A. macleodii, A. marinopraesens, Pseudomonas doudoroffi, P. marina, P. nautica, Alcaligenes pacificus, A. cupidus, A. venustus, and A. aestus. The problems of assigning species of aerobic marine bacteria to genera are discussed.

Taxonomic Consideration of Microbacterium lacticum, Microbacterium flavum, and Microbacterium thermosphactum

Classification of the microbacteria in a separate genus, Microbacterium Orla-Jensen, has been rejected by some authors. In this study three strains of Microbacterium lacticum Orla-Jensen, the type species of the genus, one strain of Microbacterium flavum Orla-Jensen, and two strains of Microbacterium thermosphactum McLean and Sulzbacher were examined with the hope that the somewhat precarious taxonomic position of these species might be clarified. Included in this examination were: The determination of deoxyribonucleic acid (DNA) base composition by the thermal denaturation technique, the development of protein and esterase profiles by polyacrylamide gel electrophoresis, and selected enzyme assays associated with the Embden-Meyerhof, hexosemono-phosphate, and tricarboxylic acid pathways. The DNA base ratio studies showed M. thermosphactum to have a per cent guanine plus cytosine content of 36 compared with 58 for M. flavum and 64 for M. lacticum. The enzyme assays also indicated heterogeneity. All possessed enzymes associated with the Embden-Meyerhof and the hexosemonophosphate pathways, but M. thermosphactum alone failed to yield enzymatic evidence for an operational tricarboxylic acid cycle. Also, only M. flavum showed evidence of a glyoxylate shunt. Protein profiles and esterase patterns were also sufficiently different to suggest a separation of M. thermosphactum from M. lacticum and M. flavum. These studies furnish additional evidence that M. flavum be placed in the genus Corynebacterium Lehmann and Neumann 1896. M. thermosphactum is an unusual organism which does not appear to be assignable to any of the presently recognized families of bacteria. The placement of M. lacticum in the genus Corynebacterium Lehmann and Neumann 1896 would render Microbacterium Orla-Jensen 1919 a later, subjective synonym of Corynebacterium. M. lacticum does possess characteristics not unlike those of the plant pathogenic corynebacteria.

Base ratio determination using unpurified DNA

A technique is described for extracting DNA and measuring the mole per cent guanine plus cytosine of DNA from animals, bacteria, and plants. Cells were disrupted by lysis or homogenization, and in the case of animals and plants, total nucleic acids were then extracted. The extract, or in the case of bacteria the total lysate, was subjected to the combined effects of ribonuclease and alkaline hydrolysis to remove RNA. DNA was acid precipitated, collected, washed, and hydrolyzed in 0.1 M HCl at 100°. This quantitatively liberated guanine and adenine from the impure DNA, and the bases were separated by ion-exchange chromatography on Dowex 50. The mole fractions of guanine and adenine (and therefore mole per cent guanine plus cytosine) were then calculated from simple spectrophotometric determinations on the eluted peaks. The technique is of superior precision to those in present use, does not require purified and highly polymerized DNA, and should be capable of automation with commercially available liquid chromatographs.

Characteristics of S organism isolated from Methanobacillus omelianskii.

Previous work showed that Methanobacillus omelianskii was a mixed culture of an ethanol-oxidizing organism called S organism and a hydrogen-utilizing methane bacterium, strain MOH. S organism grows poorly on ethanol unless a hydrogen-utilizing methanogenic bacterium is included to utilize the H(2) produced during growth. Further studies have shown that, among many substrates tested, only ethanol, n-propanol, n-butanol, isobutanol, n-pentanol, acetaldehyde, oxalacetate, and pyruvate are fermented by S organism, either alone or in combination with Methanobacterium ruminantium. It grew better in pure culture with pyruvate than with alcohols. H(2) gas phase inhibited growth on pyruvate as well as on alcohol. When grown alone on pyruvate, S organism produced mainly acetate, ethanol, and CO(2), in addition to a small amount of H(2). When combined with M. ruminantium, no H(2) and very little ethanol were produced and acetate production was increased. When M. ruminantium was present, electrons from pyruvate oxidation by S organism were channeled almost entirely to H(2) and hence to methane formation rather than ethanol. Also, S organism utilized more pyruvate when grown with M. ruminantium. Attempts to obtain better growth of S organism on ethanol by addition of many possible electron acceptors were unsuccessful. It grew best between 32 and 45 C, had a per cent guanine plus cytosine content of deoxyribonucleic acid bases of 47.27 +/- 0.1, contained no cytochrome, and could be grown on a defined medium with pyruvate as the energy and carbon source and with (NH(4))(2)SO(4) as the main nitrogen source. These and other results suggest that S organism belongs in a new genus, but assignment of a definite taxonomic status should await isolation and characterization of more strains.

Deoxyribonucleic acid of Anaplasma marginale.

Deoxyribonucleic acid from isolated marginal bodies and calf erythrocytes infected with Anaplasma marginale is found to be double stranded and to contain 51 moles per cent guanine plus cytosine.

Positive and negative control of R-factor replication in Proteus mirabilis.

Replication of the 50 and 58 moles per cent guanine plus cytosine (%GC) components of R factor 222 in Proteus mirabilis during growth in the presence and absence of chloramphenicol and after shifting exponential- and stationary-phase cells to conditions which inhibit host protein or deoxyribonucleic acid (DNA) synthesis was examined. Chloramphenicol reduced the growth rate but increased the amount of both R-factor components; the 58% GC component showed a larger proportionate increase. This was inferred to indicate reduced synthesis of an inhibitor that acts on both R-factor components and an initiator for replication of the 50% GC component. Replicative patterns observed after shifting exponential- and stationary-phase cells grown with or without chloramphenicol to minimal medium or chloramphenicol for one generation, or puromycin for 3 hr, corroborated this interpretation. After shifts of exponential cells from either medium, replication of the 50% GC components paralleled host replication, thus indicating a requirement for protein synthesis; replication of the 58% GC component increased due to reduced inhibitor synthesis. R-factor DNA remained constant after shifting stationary cells from drug-free medium, thus indicating that the cells contained effective concentrations of the regulatory inhibitor, whereas increased replication of the 58% GC component occurred after identical shifts of chloramphenicol-grown cells of the same chronological age. Similar responses were observed after shifts to 5 C or to medium containing streptomycin or tetracycline. Absence of replication of the 50% GC component after shifting to medium containing nalidixic acid or phenethanol and its hereditary persistence during growth indicated that the 50% GC replicon was attached to the membrane. Thus, in P. mirabilis the three replicons of R factor 222 are regulated as follows: The composite and transfer portion (RTF) replicons represented by the 50% GC component require protein synthesis and membrane attachment and are negatively regulated by an inhibitor; the 58% GC or resistance-determinants replicon exists cytoplasmically and is subject only to negative control.

Characterization of Deoxyribonucleic Acid of Entamoeba histolytica by Cesium Chloride Density Centrifugation

Buoyant density of deoxyribonucleic acid (DNA) from Entamoeba histolytica was determined in cesium chloride gradients in the analytical ultracentrifuge. The DNA was extracted from fresh or lyophilized cells in a solution containing 8 M urea. It was purified by absorption on and elution from hydroxylapatite, and on a cesium chloride gradient in the preparative ultracentrifuge. DNA from axenically grown amebae contained a single component and it banded in the region of 1.69 g/ml. DNA prepared from 13 strains of amebae grown with penicillin-inhibited bacterial cells also banded in this region. Some of the latter preparations contained a second component of buoyant density 1.708 g/ml which is attributed to the food bacteria, Bacteroides symbiosus. Buoyant density of amebal DNA ranged from 1.6881 to 1.6913 g/ml corresponding to guanine plus cytosine contents of 27.5 to 30.8 mole per cent. Buoyant density is not a sensitive parameter for making interstrain distinctions among strains of E. histolytica, but it appears to offer promise in an area where little else has succeeded. A number of statistically significant interstrain differences were encountered. Thus low density DNA from NRS or HK9 strains was distinguished from DNA of intermediate density from DKB or 200 strains and from the high density DNA of the F22 strain at probability levels to 0.001. Guanine plus cytosine contents calculated from thermal hyperchromicity (Tin) were lower than those calculated from buoyant density data. The DNA from Bacteroides symbiosus was calculated to contain 48.2 mole per cent guanine plus cytosine, using buoyant density data. Knowledge concerning the base composition of DNA has been useful in taxonomic studies on bacteria. The less extensive work with protozoa was reviewed by Mandel (1967). Recently Adam, Blewett, and Flamm (1969) encountered differences in base composition among DNAs from various Acanthamoeba. The purpose of this investigation was to probe for evidence of different base composition among DNAs from various strains of Entamoeba histolytica. We were particularly interested in comparing the group of high-temperature strains which grow in vitro between 33 and 39 C with the group of low-temperature strains which grow at temperatures of 25, or less, to about 37 C. The chemical method for determining DNA base composition by separation and quantitation of the hydrolyzed bases requires more purified DNA than we could afford for this work. Most of our data were obtained by the buoyant density method which requires only about 1 I/g of DNA for a single centrifuge run. This method has the added capability of resolving DNAs of different densities which may be present in a single preparation. Thus we were able to survey DNA not only from four Received for publication 13 April 1971. axenically grown strains, but also from a larger number of strains grown with penicillin-inhibited cells of the associate organism, Bacteroides symbiosus. Many of the latter strains have not yet been placed in axenic culture. MATERIALS AND METHODS Amebae were grown in axenic culture by the method of Diamond (1968), or with penicillininhibited cells of Bacteroides symbiosus by the method of Reeves and Ward (1965). The hightemperature strains of E. histolytica were grown at 36 to 37 C, the others at 25 to 31 C. Cultures were harvested soon after population peaks had been reached. Cells were collected by centrifugation and washed 3 times with a solution containing 0.11 M NaCl, 0.02 M KC1, 1 mM potassium phosphate (pH 7.4), 0.5 mM MgCl2, and 0.1 mM CaCI2. Centrifugally packed cell volumes were noted and cell counts were made in a Fuchs-Rosenthal counting chamber. The final wash fluid was withdrawn as completely as possible from the packed cells and the cell pellet was quickly frozen in an acetone-dry ice bath. The frozen cells were lyophilized, sealed in glass under vacuum, and stored at -20 C until used. Source of the axenic cultures has been reported by Diamond (1968), and of the monoxenic cultures by Reeves et al. (1967), except for the N strain which is from South Africa and was obtained from Dr. Shirley Madison.

Serological Investigations of Planococcus Strains

Boháček et al. have proposed that the flagellated, gram-positive cocci be included in the genus Planococcus Migula, particularly on the basis of their per cent guanine plus cytosine content. A serological examination of these organisms (10 strains) by the author revealed no antigenic relationship to staphylococci or micrococci, thus substantiating the conclusion of Boháček et al. that they do not belong to the genus Micrococcus. Antigenically, the group of strains was rather heterogeneous.

Comparison of the morphology and deoxyribonucleic acid composition of 27 strains of nitrifying bacteria.

The gross morphology, fine structure, and per cent guanine plus cytosine (GC) composition of deoxyribonucleic acid of 27 strains of nitrifying bacteria were compared. Based on morphological differences, the ammonia-oxidizing bacteria were separated into four genera. Nitrosomonas species and Nitrosocystis species formed one homogenous group, and Nitrosolobus species and Nitrosospira species formed a second homogenous group in respect to their deoxyribonucleic acid GC compositions. Similarly, the nitrite-oxidizing bacteria were separated into three genera based on their morphology. The members of two of these nitrite-oxidizing genera, Nitrobacter and Nitrococcus, had similar GC compositions, but Nitrospina gracilis had a significantly lower GC composition than the members of the other two genera.

A Comparison of Genome Sizes and Thermal-Denaturation-Derived Base Composition of DNAs from Several Members of Entamoeba (Histolytica Group)

The DNA base composition and genome size of several strains of Entamoeba that form mature 4-nucleated cysts (histolytica group) was compared. These 2 measurements of genetic potential were used to differentiate species. Significant differences in genome size were observed for E. histolytica, E. histolytica-like amebae, and E. moshkovskii. Significant differences in DNA base composition were found in strains of amebae thought to be of the same species. These data clearly indicate the existence of more than one genospecies for classical E. histolytica, E. histolytica-like amebae, and E. invadens. Members of the genus Entamoeba can be readily divided into four major morphological groups on the basis of the existence or absence of a cyst stage and by the number of nuclei in the mature cyst. One group is characterized by having uninucleated cysts, another has 4-nucleated cysts, a third has 8-nucleated cysts, and one is without any known cyst stage. Levine (1961) conveniently designates these groups as bovis, histolytica, coli, and gingivalis, respectively. Within each of these groups separation into species is especially difficult. Currently, the classification is based primarily on morphology, host specificity, and pathogenicity. This system is inadequate. There are few consistent morphological differences among members of a group other than size. Host specificity is not a particularly reliable character, and pathogenicity is a highly variable property. Additional criteria for classification must be sought. In recent years, advances in our knowledge of the biology of the Entamoeba, gained primarily through study of monoxenically and axenically cultivated organisms, gives promise that the antigenic, biochemical, metabolic, and physiological properties of the amebae can be used to develop more meaningful systems of classification as has been done for bacteria. We have been characterizing entamoebal DNA and using specific differences as a means of distinguishing members of the histolytica group, or those Entamoeba forming 4-nucleated Received for publication 1 December 1970. cysts. In our previous study (Gelderman et al., 1971) we demonstrated that three members of the group, classical Entamloeba histolytica, E. histolytica-like ameba, and E. moshkovskii were sufficiently different with respect to genome size, DNA reassociation kinetics, mole per cent guanine plus cytosine (%GC), and DNA to DNA homologies to be considered distinct species. In that work only one strain of each type was studied. In this study, the results of two parameters of genetic potential (genome size and %GC) are reported for several strains of the histolytica group. GENOSPECIES AND TAXONOMY The ideal taxonomic classification must consider the widest possible range of relatedness such as morphology, host specificity, metabolism, growth requirements, immunological characteristics, specific isozymes, and DNA composition. Many of the parameters change with differing environmental conditions, thus requiring a different part of the DNA to be expressed. However, genome size and %GC of a cell is a biological constant and any significant change in the nucleotide composition of the DNA of an organism with 107 to 108 nucleotide pairs would be lethal to the cell. The DNA base ratio has been extensively studied in bacteria and used as a taxonomic tool (Mandel, 1969). The %GC of the DNA from all bacteria studied varied from 27 to 72%(Shapiro, 1968). The range of the DNA base ratios from the whole family of Enterobacteriaceae varied by only 20% (Shapiro, 1968). The

Taxonomy of marine bacteria: the genus Beneckea.

One-hundred-and-forty-five isolates of marine origin were submitted to an extensive physiological, nutritional, and morphological characterization. All strains were gram-negative, facultatively anaerobic, straight or curved rods which were motile by means of flagella. Glucose was fermented with the production of acid but no gas. Sodium but no organic growth factors were required. None of the strains were able to denitrify or fix molecular nitrogen. The results of nutritional and physiological tests were submitted to a numerical analysis. On the basis of phenotypic similarity, nine groups were established. These groups could be distinguished from one another by multiple, unrelated, phenotypic traits. Six groups which had deoxyribonucleic acid (DNA) containing 45 to 48 moles per cent guanine plus cytosine (GC) were assigned to a redefined genus Beneckea. All of the strains in this genus, when grown in liquid medium, had a single, polar flagellum. When grown on a solid medium, many strains had peritrichous flagella. Two groups were similar to previously described species and were designated B. alginolytica and B. natriegens. The remaining four groups were designated B. campbellii, B. neptuna, B. nereida, and B. pelagia. An additional group of phenotypically similar strains having the properties of the genus Beneckea was not included in the numerical analysis. These strains were readily separable from species of this genus and were designated B. parahaemolytica. Of the remaining groups, one was identified as Photobacterium fischeri. The other group (B-2) which had about 41 moles% GC content in its DNA could not be placed into existing genera.

Growth and morphological characteristics of a species of Flexibacter.

1. An organism belonging to the genus Flexibacter (Soriano emend. Lewin) has been isolated from soil and designated FS-1. 2. Flexibacter FS-1 is a Gram negative flexuous, gliding, multinucleate filament 0.7 μm in diameter. The moles per cent guanine plus cystosine is 48.7%. The organism possesses orange carotenoid-like pigments but no photosynthetic pigments. 3. The length of the cells was shown to be a function of the growth temperature and composition of the medium, and can range from 10–400 μm long. Cross-walls are rarely seen along the length of the long filaments. The filamentous cells fragment extensively when the culture enters the stationary phase of growth. 4. Starch, protein, peptides, or a limited variety of mono-and disaccharides can serve as the sole source of carbon and energy when the cells are grown at 25°C or 30° C. However, cells grown at 35° C on a glucose+salts medium require a supplement of yeast extract. Single amino acids or mixtures of such can provide nitrogen but cannot replace the carbon and energy sources. None of the carboxylic acids tested supported growth.

Deoxyribonucleic acid base composition of Rothia dentocariosa as determined by thermal denaturation.

The moles per cent guanine plus cytosine (GC) of 10 filamentous strains of Rothia dentocariosa ranged from 65.4 to 69.7. Major differences were not observed in the base composition of a filamentous form (69.7 moles% GC) and its coccal variant (68.0 moles% GC).

Lysis of blue-green algae by myxobacter.

Enrichment from local fishponds led to the isolation of a bacterium capable of lysing many species of unicellular and filamentous blue-green algae, as well as certain bacteria. The isolate is an aflagellate, motile rod which moves in a gliding, flexuous manner; the organism is capable of digesting starch and agar, but not cellulose and gelatin. Its deoxyribonucleic acid base pair composition (per cent guanine plus cytosine approximately 70) shows a close resemblance to that of the fruiting myxobacteria. Algae in lawns on agar plates were lysed rapidly by the myxobacter, but only limited and slow lysis occurred in liquid media, and no lysis took place when liquid cultures were shaken. No diffusible lytic factors would be demonstrated. Continuous observation of the lytic process under a phase-contrast microscope suggested that a close contact between the polar tip of the myxobacter and the alga is necessary for lysis. The lytic action is limited to the vegetative cells of the algae, whereas heterocysts are not affected. The gas vacuoles of the algal host are the only remnant visible after completion of digestion by the myxobacter.

Deoxyribonucleic Acid Heterogeneity Between Human and Murine Strains of Chlamydia trachomatis

We compared the polynucleotide sequence relationships of three strains of Chlamydia trachomatis of human origin (MRC-1/G, TW-3, and Lgv), one of murine origin (MoPn), and the MN strain of C. psittaci. The four strains of C. trachomatis have the same base ratio, about 42.5 moles per cent guanine plus cytosine, which is significantly higher than the base ratio of MN (39.5). Single strands of deoxyribonucleic acid (DNA) fragments of MRC-1/G reassociated with immobilized DNA of TW-3 and Lgv almost as well as with the homologous DNA. The duplexes produced in these reactions were about equally thermostable. On the other hand, reassociations between MRC-1/G and MoPn involved 60 or 30% of the DNA, depending on the stringency of the conditions for reassociation, and the duplexes were thermolabile. MoPn reassociated only to a very small degree with MN. We also compared glucose catabolism of MRC-1/G, MoPn, and MN under several sets of conditions. These tests failed to reveal any qualitative phenotypic differences among the three strains. It can be concluded that, judging by polynucleotide sequence, the three human strains of C. trachomatis are closely related but appreciably different from a murine strain.