SynopsisThe CD and absorption (OD) spectra of single-stranded nucleic acids in complex with the helix-destabilizing protein of either bacteriophage T4 (GP32) or bacteriophage fd (GP5) show similar and unusual features for all polynucleotides investigated. The change in the CD spectra between 310 and 240 nm is in all cases characterized by a considerable decrease in the positive band, while the negative band (if present) remains relatively intense. These changes are different from those due to temperature or solvent denaturation and, moreover, cannot be induced by the binding of simple oligopeptides. Absorption measurements show that all polynucleotides remain hypochromic in the complex. Both CD and OD spectra point to a specific and probably similar conformation in complex for all polynucleotides with substantial interactions between the bases. The spectral properties are almost temperature independent (0-40°C). Therefore, we conclude that the conformation must be regular and rigid. To investigate the relation between these optical properties and the specific polynucleotide structure, CD and OD spectra were calculated for an adenine hexamer over a wide range of the conformational parameters. It appears that the calculated CD intensity is not very sensitive to an increase in the axial increment and that many different conformations can give rise to more or less similar CD spectra. However, simulation of the very nonconservative experimental CD spectrum of the poly(rA)-GP32 complex requires that the conformation satisfies two criteria: (1) a considerable tilt of the bases (< -10") in combination with (2) a small rotation per base (=ZOO) andor a position of the bases close to the helix axis (dx = 0 A). Such conformations can also explain the observed hyperchromism upon binding of GP32 to poly(rA)/(dA). Very similar structural characteristics also account for the optical properties of the complexes with GP5. These are discussed as an alternative to the structure suggested by Alma-Zeestraten for poly(dA) in the complex [N. C. M. Alma-Zeestraten (1982) Doctoral thesis Catholic University, Nijmegen, The Netherlands]. The secondary structure proposed in this work can be reconciled with the overall dimensions of the complex, assuming that the polynucleotide helix is further organized in a superhelix.

We divided the asaccharolytic, black-pigmented Bacteroides strains into two groups on the basis of deoxyribonucleic acid (DNA) base ratios, DNA hybridization (Sl nuclease method) results, and direct hemagglutination. One homologous group of strains, which included the type strain of Bacteroides asaccharolyticus and had guanine-plus-cytosine contents of 52 to 54 mol%, contained only nonoral isolates. Another DNA homology group contained all of the strains from periodontal pockets and some nonoral isolates. These strains appeared to belong to the recently described new species Bacteroides gingivalis Coykendall et al. B. gingivalis strains had guanine-plus-cytosine contents of 48 to 50 mol%, showed high DNA homology values, and shared hemagglutinating activity. No DNA homology was observed between the two groups. Using a different method of analysis for DNA homology (Sl nuclease method), we confirmed the conclusion of Coykendall et al., who separated these two groups into different species, B. asaccharolyticus and B. gingivalis. Two strains from infected root canals could not be placed in either of these two species. On the basis of the DNA homology results, all asaccharolytic strains were distinguished clearly from the saccharolytic, black-pigmented Bacteroides strains, which at present are classified in the species Bacteroides melaninogenicus.The asaccharolytic, black-pigmented strains originally identified as members of Bacteroides melaninogenicus have been reclassified as members of Bacteroides asaccharolyticus (6). Recently, Coykendall et al. (2) proposed separating these strains into the following two different species: B. asaccharolyticus and a new species, Bacteroides gingivalis. B. asaccharolyticus strains can be isolated from many different kinds of infection (5), whereas high numbers of B. gingivalis are isolated from periodontal pockets (19, 22). The genetic heterogeneity within the group of asaccharolytic, black-pigmented Bacteroides strains is shown by the deoxyribonucleic acid (DNA) base contents of these organisms; one group has low guanine-plus-cytosine (G+C) values, and another has high G+C values (17, 24). In a preliminary report (25), we stated that these two groups showed no DNA-DNA hybridization. These groups were also heterogeneous with respect to polypeptide content (23), electrophoretic mobility of the enzyme malate dehydrogenase (17), fatty acid composition, and isoprenoid quinone composition (16). Differences between oral and nonoral asaccharolytic, black-pigmented Bacteroides strains were demonstrated by direct hemagglutination (20), by morphological and immunochemical methods (ll), by antigenic studies (13, 15), and by the production of phenylacetic acid (9). In this paper we present the results of studies on DNA hybridization and G+C contents and hemagglutination by asaccharolytic, black-pigmented Bacteroides strains. Our results confirm the proposal by Coykendall et al. of a new species, B. gingivalis, which is different from B. asaccharolyticus (2). MATERIALS AND METHODS Bacterial st...

The rotation diffusion coefficient of a complex of GP32, the single stranded DNA binding protein of the bacteriophage T4, with a single stranded DNA fragment with about 270 bases was determined to obtain further information on the flexibility of this particle. The rotation diffusion of these molecules is used as a sensitive measure of the flexibility of different DNA protein complexes. Using the theory of Hagerman and Zimm (Biopolymers 20, 1481 (1981)) and assuming a bending persistence length of about 35 nanometer it can be shown that the axial increment for GP32 complexes with single stranded DNA is close to 0.5 nm per base. The value for the bending persistence length is in agreement with values found for much larger DNA protein complexes using light scattering experiments. This value for the persistence length also implies that the complex is thin. The radius is estimated to be around 1.7 nm, which shows a moderate degree of hydration. With this set of parameters we can describe all the hydrodynamic experiments on GP32 complexes from 76 to more than 7000 bases obtained using electric birefringence, quasi-elastic light scattering and sedimentation experiments performed in our group over the last few years.

Saccharolytic, black‐pigmented Bacteroides strains, which at present belong to the species Bacteroides melaninogenicus were classified on the basis of deoxyribonucleic acid (DNA) base ratios and DNA hybridization studies. These strains were divided into several DNA homology groups, which showed no or low mutual DNA homology. A DNA homology group with a percentage guanine plus cytosine (G + C) of 42–43% was formed by three strains of Bact. melaninogenicus subsp. melaninogenicus; the type strain of this subspecies, strain ATCC 25845, had about 60% DNA homology with this group. Strain ATCC 15930, which has been assigned to this subspecies, had a percentage G + C of 47% and showed no DNA homology with the former group. All strains of Bact. melaninogenicus subsp. intermedius had a percentage G + C of 39–45%. A DNA homology group was formed by eight strains of this subspecies. The type strain of Bact. melaninogenicus subsp. intermedius, ATCC 25611, showed relatively low DNA homology with this main DNA homology group. A strain of Bact. melaninogenicus subsp. intermedius serotype C1 showed no DNA homology with the other strains tested. Furthermore two strains labelled ‘Bact. melaninogenicus subsp. levii’ were found to form a distinct DNA homology group. On the basis of the DNA homology results, the strains, which at present are classified in the species Bact. melaninogenicus, were clearly distinguished from strains of Bact. asaccharolyticus and Bact. gingivalis, and also from strains of related non‐pigmented Bacteroides species.

Binding of the single-stranded DNA-binding protein (SSB) of Escherichia coli to single-stranded (ss) polynucleotides produces characteristic changes in the absorbance (OD) and circular dichroism (CD) spectra of the polynucleotides. By use of these techniques, complexes of SSB protein and poly(rA) were shown to display two of the binding modes reported by Lohman and Overman [Lohman, T.M., & Overman, L. (1985) J. Biol. Chem. 260, 3594-3603]. The circular dichroism spectra of the "low salt" (10 mM NaCl) and "high salt" (greater than 50 mM NaCl) binding mode are similar in shape, but not in intensity. SSB binding to poly(rA) yields a complexed CD spectrum that shares several characteristics with the spectra obtained for the binding of AdDBP, GP32, and gene V protein to poly(rA). We therefore propose that the local structure of the SSB-poly(rA) complex is comparable to the structures proposed for the complexes of these three-stranded DNA-binding proteins with DNA (and RNA) and independent of the SSB-binding mode. Electric field induced birefringence experiments were used to show that the projected base-base distance of the complex is about 0.23 nm, in agreement with electron microscopy results. Nevertheless, the local distance between the successive bases in the complex will be quite large, due to the coiling of the DNA around the SSB tetramer, thus partly explaining the observed CD changes induced upon complexation with single-stranded DNA and RNA.