In a comparative study the lipoquinones of some chemoorganotrophic, facultatively aerobic bacteria, and representative Acholeplasma, Mycoplasma, Spiroplasma, and Thermoplasma strains were investigated. The quinones were partly purified by preparative thin layer chromatography of lipid extracts, and characterized by their difference spectra (reduced minus oxidized) and Rf values. Respiring bacteria expectedly contained benzoquinones and/or naphthoquinones in micromolar concentrations whereas some aerotolerant, cytochrome-less, gram-positive bacteria were found to contain menaquinones in nanomolar concentrations, or even no quinones; only Streptococcus faecalis, an organism supposed to use a rudimentary, flavin-terminated respiratory chain system produced desmethyl menaquinone in amounts ranging between "high" and "low" quinone contents. Among the mycoplasmas investigated, only Thermoplasma acidophilum was found to be capable of synthesizing quinones (MK-7) in the micromolar order of magnitude indicating a respiratory electron transport system. The presence of energetically useful respiratory chain systems in Acholeplasma, Mycoplasma, and Spiroplasma is questioned since these organisms contain quinones (MK-4) in nanomolar concentrations, or no quinones, depending on the presence of exogeneous MK-6 in the growth medium. The possible metabolite role of menaquinones present in "low" amounts, as well as the role of NADH oxidase systems more or less tightly bound to the cytoplasmic membrane with the mycoplasmas deserves further investigation.

The lipoquinones and associated respiratory functions of 34 cultures representing 17 bacterial species hitherto assigned to the genus Actinobacillus or Pasteurella were investigated. Strains representing recognized Actino bacillus or Pasteurella species regularly contained desmethylmenaquinones and were capable of anaerobic electron transport with fumarate as a terminal acceptor when grown in oxygen-limited cultures. On aeration, the majority of the strains produced ubiquinones in addition to desmethylmenaquinones, with inter-and intraspecific variations. The use of lipoquinone patterns and features indicating fumarate respiration, such as stimulation of oxygen-limited growth by fumarate, in the classification and identification of Actinobacillus, Pasteurella, and phenotypically similar organisms is discussed.In the 8th edition of Bergey's Manual, four species of the genus Pasteurella Trevisan and two species of the genus Actinobacillus Brumpt are recognized, and, furthermore, some species incertae sedis are listed (7, 10). Several species of Pasteurella and Actinobacillus described more recently are waiting for recognition or rejection. On the basis of phenotypical data, both circumscription of the two traditional genera and their classification below the generic level are at present difficult to assess. Moreover, Pasteurella and Actinobacillus are closely related at least to some members of the conventional genus Haemophilus (10). Thus, additional classifying features of the organisms in question are desired.After a preliminary study on the composition of the respiratory chain system of some Actinobacillus and Pasteurella strains (R. Zabel for 20 min before pouring) was used for maintenance and purity checks. Proteose peptone medium (14) with proteose peptone no. 3 (Difco) was used throughout for mass cultivation; the peptone lots used were known to contain less than lo-'' mol of both benzo-and naphthoquinones per g of dry weight (R. Hollander and W. Mannheim, unpublished data). Disodium fumarate (Fluka AG., Buchs, Switzerland; sterilized by filtration) was supplemented to a final concentration of 50 mM where indicated. "Aerobic" cells were grown by shaking 250-ml batches in Fernbach flasks (no. 20511, Schott & Gen., Mainz, Germany), using a rotary shaker (model G-25, New Brunswick Scientific Co., New Brunswick, N.J.), the shaking frequency depending on the sensitivity of the cultivated organism to aeration. "Oxygen-limited" cells were harvested from Fernbach flasks filled to the neck (about 2,000 ml of medium) and incubated statically. Incubation temperature was 37°C except for P. piscicida, which was grown at 25°C. Late-exponential-phase cells were sedimented at 12,000 x g, 0 to 4"C, and washed twice with 0.1 M tris(hydroxymethy1)aminomethane-hydrochloride buffer, pH 7.2.Determination of protein. Protein was determined, after sonic disintegration of the cells, by a modified biuret method (12).Determination of NADH oxidase and NADHfumarate reductase activities. Oxidation of reduced p-nicotinamade adenine dinu...