Serological techniques were used to assess the relationships among the following species: Actinobacillus equuli, Actinobacillus lignieresii, Actinobacillus pleuropneumoniae, "Actinobacillus seminis," Actinobacillus suis, Actinobacillus actinomycetemcomitans Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus segnis, Haemophilus parainjluenzae, Haemophilus injluenzae, Haemophilus parahaemolyticus, Haemophilus aegyptius, and Gardnerella vaginalis. Deoxyribonucleic acid-deoxyribonucleic acid hybridization analyses of A . actinomycetemcomitans, H . segnis, H. aphrophilus, and H . paraphrophilus showed that these four species have measurable deoxyribonucleic acid relatedness of 28% or greater, consistent with their placement in a single genus. These bacterial species also demonstrated common antigens by immunodiffusion. In view of these genetic and serological relationships and previous inclusion of X-and V-factor-independent bacterial species in the genus Haemophilus, reassignment of A . actinomycetemcomitans to the genus as Haemophilus actinomycetemcomitans comb. nov. is proposed. ~~ ~The morphological and cultural characteristics of Actinobacillus actinomycetemcomitans were first described by Klinger (12) in 1912. The initial isolates of this gram-negative facultative anaerobe were from actinomycotic lesions and were given the name "Bacterium actinomycetemcomitans." The name of this organism was subsequently changed twice, first by Lieske (13) in 1921 to "Bacterium comitans" and then by Topley and Wilson (31) in 1929 to Actinobacillus actinomycetemcomitans.The first detailed description of the biochemical and serological characteristics of this species was published by King and Tatum (ll), who compared A . actinomycetemcomitans with Haemophilus aphrophilus and found these species to be closely related. More recently, a number of studies have delineated the phenotypic (1, 6, 8, 10, 19, 26, 29), serological (24, 27, 33, 34) MATERIALS AND METHODSBacterial strains and culture conditions. The bacterial strains used in the production of rabbit antisera are shown in Table 1. Strains of Actinobacillus and Haemophilus species were grown to late logarithmic or early stationary phase in National Institutes of Health thioglycolate liquid medium * Corresponding author.(Difco Laboratories, Detroit, Mich.). X-and V-factor-requiring strains were grown on chocolate agar for 72 h. All strains were grown at 37°C in an anaerobic chamber (Coy Manufacturing Co., Ann Arbor, Mich.) containing 85% N2, 10% Hz, and 5% COz. The bacteria grown in liquid media were harvested by centrifugation and washed three times in phosphate-buffered saline (pH 7.2). The strains grown on agar plates were harvested with a platinum loop and washed three times in phosphate-buffered saline.Antiserum production. Whole bacterial cells to be used as immunogens were suspended in sterile saline to a concentration of 10 mg (wet weight) per ml. Eight-week-old female New Zealand white rabbits weighing 4 kg received intravenous injections of 1.0 ml of ba...
DNA was purified from 16 strains of Fusobacterium nucleatum and from five strains representing other Fusobacterium species. The relationships among fusobacteria were examined by DNA-DNA hybridization and by determining the guanine plus cytosine content of the DNA. F. nucleatum was found to comprise a heterogenous group of organisms related to Fusobacterium periodonticum and Fusobacterium simiae, but unrelated to any of the other species of Fusobacterium tested.
Isolates of Fusobacterium that differ from type strains of various fusobacterial species with respect to DNA sequence, cellular fatty acid composition, and biochemical activity, were obtained from periodontitis lesions in a patient with insulin-dependent diabetes mellitus. These isolates have the following distinguishing characteristics: 28% guanine + cytosine content; 40% or less DNA homology with type strains of representative fusobacterial species; cell size, 0.5 - 1 X 4 -100 microns; absence of motility; ability to ferment glucose, fructose, and galactose, but not 25 other carbohydrates; ability to produce indole; ability to hydrolyze hippurate but not esculin; sensitivity to bile; ability to produce little or no gas; ability to utilize threonine but not lactate. We propose that the organisms be classified as a distinct species of Fusobacterium to be named Fusobacterium periodonticum. The type strain of this new species has been deposited with the American Type Culture Collection under the designation ATCC 33693.
In order to assess the relationships among strains of the genera Actinobacillus and Haemophilus, DNAs from 50 strains of these genera were isolated and purified. The guanine plus cytosine (G + C) content of DNAs from strains of Haemophilus segnis and Haemophilus parainfluenzae were determined by thermal denaturation. DNA-DNA homologies were measured using labelled probes from one strain representing Haemophilus segnis (strain ATCC 10977), and two strains representing Haemophilus parainfluenzae (strains ATCC 9796 and ATCC 7901). Strains isolated as H. segnis had a G + C content of 39.0 to 42.9% and were 49-92% homologous with the ATCC 10977 DNA probe. All of the strains freshly isolated as H. parainfluenzae were 70-81% homologous with the ATCC 9796 DNA probe and had a G + C content of 34.9 to 38.3%. Strain ATCC 7901 was 11% homologous with the ATCC 9796 DNA probe, had a G + C content of 42.4%, and was 65-78% homologous to DNA from strains identified as Haemophilus aphrophilus and Haemophilus paraphrophilus. From these results we conclude that strain ATCC 7901 is a mislabelled strain of H. paraphrophilus. The results of multiple DNA-DNA hybridizations indicated that separate species designations were appropriate for H. segnis, H. parainfluenzae, Actinobacillus actinomycetemcomitans ("Haemophilus actinomycetemcomitans"), and H. aphrophilus. H. aphrophilus and H. paraphrophilus were closely related organisms and did not fulfill the generally accepted criteria for designation as separate species.
Deoxyribonucleic acids (DNAs) were isolated and purified from 20 strains of Actinobacillus actinomycetemcornitans, 6 strains of Haemophilus aphrophilus, 2 strains of Haemophilus paraphrophilus, 2 strains of Haemophilus pleuropneumoniae, 2 strains of Haemophilus paraphrohaemolyticus, 2 strains of Haemophilus injhenzae, and 1 strain each of Actinobacillus lignieresii, Actinobacillus suis, Haemophilus aegyptius, Haemophilus parainfluenzae, and Haemophilus parahaemolyticus. The guanine-plus-cytosine contents of the DNAs were determined, and they agreed closely with previous estimates. DNA-DNA hybridization analyses revealed that all of the strains identified as A . actinomycetemcomitans were at least 69% homologous to DNA probes from two A . actinomycetemcomitans strains (strains NCTC 9710T [T = type strain] and Y4). The H . aphrophilus and H . paraphrophilus strains were homologous to the two A . actinomyceterncomitans probes at levels of 25 to 37%. The DNAs of all A . actinomycetemcomitans strains were homologous to a DNA probe from H . aphrophilus strain NCTC 5906 at levels of 30 to 39%. The two strains of H . paraphrophilus tested were homologous to the H . aphrophilus probe at levels of 73 and 77%, indicating a very close relationship between these groups of organisms. H. parainfluenzae strain ATCC 9796 DNA seemed to be homologous to the H . aphrophilus and A . actinomycetemcomitans probes at low but significant levels (12 to 16%). All of the other strains of haemophili and actinobacilli tested gave 10% or less homology with the three test probes.
The ability of certain phages to successfully infect a restricting host at a high multiplicity of infection is known as cooperative infection or cooperation. We have examined the ability of unmodified Ti (Ti * 0) to participate in cooperative infection in cells possessing the P1 restriction system. We have found that cooperation is dependent upon protein synthesis during the first few minutes after phage infection. However, we have been unable to attribute the necessary protein to a known Ti cistron. Degradation of the restricted Ti genome is
When unmodified phage Ti infects restricting host cells at high multiplicities of infection, there is an increase in recombination frequency in all regions of the Ti map compared to the level of recombination in standard crosses when short distances are examined. The enhancement of recombination frequency is not uniform for all regions but is greatest for markers near the center of the map and not so great for markers near the ends. Crosses between markers at the extremities of the map show that there is no increase in recombination frequency under restriction conditions. An examination of phage Ti he'terozygotes suggests that an increase of ends created by the process of P1 restriction increases recombination. When Ti crosses are done in the absence of host restriction, recombination defects in the host have no effect on phage recombination and we conclude that phage Ti codes for its own recombination genes. Host recombination functions are also dispensable for the recombination occurring during infection ofrestricting host cells by unmodified phage at high multiplicities of infection.
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