INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY, Apr. 1975, p. 102-107 Vol. 25, No. 2 Copyright 0 1975 International Association of Microbiological Societies Printed in U.S.A.

Characterization of Hemophilic and Related by Their Respiratory Quinones and Cytochromes

R. HOLLANDER AND W. MANNHEIM Hygiene-lnstitut der Philipps- Universitat, MurburglLahn, Federal Republic of Germany Nineteen strains of gram-negative organisms representing 10 species, Eikenella corrodens, and the so-called Haemophilus vaginalis were investigated for cytochromes and respiratory quinones. Phenotypical variation of cytochrome and quinone contents was checked using cells grown with oxygen and fumarate as alternative electron acceptors. The cytochromes were detected by difference spectra of briefly sonicated cells at room temperature, and the quinones were determined in lipid extracts by difference spectrophotometry and partly by thin-layer chromatography. In contrast to other conventional groups of chemo-organotrophic, gram-negative bacteria that have been found to be highly homogeneous with respect to the respiratory chain components, the organisms investigated here may be divided into three categories by their respiratory quinones, namely (i) bacteria containing desmethylmenaquinone as sole respira - tory quinone-H. influenzae, H. aegyptius, H. paraphrophilus, H. parahue- molyticus, H. parainfluenzae (in part), (ii) bacteria containing both ubiquinone and desmethylmenaquinone-H. haemoglo binophilus, H. parasuis, H. paragal- linarum, H. parainfluenzae (in part), and (iii) bacteria containing only ubiquinone-H. piscium, so-called H. uaginalis, and E. corrodens. The data presented may contribute to further characterization, independent of growth factor requirements, of the taxonomic entities investigated. In the absence of information on deoxyribonucleic acid relatedness between the organisms under study, a provisional reclassification of Haemophilus and related taxa on the basis of respiratory quinones is discussed.

Requirements for protoheme (or protopor- ing to their respiratory quinones, there are phyrin IX) and/or nicotinamide adenine dinu- several different groups among the so-called cleotide (NAD) as growth factors have been haemophilic bacteria. These findings may con- used as the essential criteria for classification of tribute ’to a new definition of Haemophilus and the chemo-organotrophic, aerobic, nonmotile, of contiguous groups of organisms. gram-negative bacteria of the genus Haemophilus (19). However, there are a num- MATERIALS AND METHODS ber of examples that heme and/or NAD-requir- Microorganisms. The bacterial cultures used are ing bacteria may develop from unrelated paren- listed in Table 1. Before use, collection cultures were tal forms, e.g., Micrococcus pyogenes (12), examined for microscopic appearance, Gram reaction, (4),and Bacteroides ruminicola colonial morphology, growth factor requirements, and (7). Therefore, the lack of enzymes for the biochemical activities. Media and cultivation. Proteose peptone medium synthesis of heme or pyridine nucleotides is not (27) was the complex medium base used for all itself an adequate basis for the classification of organisms except H. pisciurn, which was cultivated in haemophiles. fish peptone medium (medium no. 69, ATCC cata- Besides growth factor and complex substrate logue, 1972), and “.H.uuginulis,” which was grown in requirements, a common feature of all liver broth (sliced,bovine liver was overlaid with two Haemophilus species hitherto described ap- volumes of meat broth [beef extract, 0.8%;peptone. pears to be vertebrate parasitism. If they were a 1.0%; Nacl, 0.3%; Na,HPO,, 0.2% wt/volJ and auto- phylogenetically homogeneous group, one claved at 121 C for 15 min. (Beef extract and peptone would have to expect a high degree of pheno- were from Merck, Darmstadt, Germany.) The media were sterilized by filtration and supplemented before typic similarity of the group members in many use with NAD (5.0 pglml), hemin (hemin chloride other respects. [Fluka, Buchs, Switzerland] dissolved in triethanol We investigated the question of such similar- amine; 0.5 pg/ml), and, where indicated, with diso- ity in the composition of the respiratory chain. dium fumarate (Fluka; 0.8%wt/vol), from 100-times- The data presented here indicate that, accord- concentrated stock solutions kept at 4 C. 102 VOL. 25, 1975 HEMOPHILIC AND RELATED BACTERIA 103

TABLE1. Bacterial cultures investigated calculated by the equations of Kr6ger et al. (14) using the difference extinction coefficients A21280-289 = -9.6, Source of At?$&,O = -1.4, A(t2250-255= 2.1 and Aty2t-255= 20.2 Species Strain supplylcollec- (mM-’ x cm-I). tion no. a When spectrophotometrically pure naphthoqui- Haemophilus RAMC 18 Bensted NCTC 4560 none extracts were obtained, the presence of even influenzae small quantities of Q was excluded by thin-layer H. influenzae HIM 412-7 chromatography on kieselgel (Merck, Darmstadt, H. influenzae 641 Pittman NCTC 8467 Germany). H. parainfluenzae HIM 412-6 Ethylmethylketone (15% vol/vol) in hexane was H. parainfluenzae HIM 170-1 used as the solvent system (9). The amount of H.parainfluenzae 1 Fleming NCTC 4101 contaminating Q was then determined by the method H.parainfluenzae 429 Pittman ATCC 7901 of Lester and Ramasarma (171, sensitive to 1.3 nmol of H. parainfluenzae Bossy No. 7 Leidy HIM 449-8 H. paragallinarum 6 Love11 NCTC 3438 Q. (5) Determination of cytochromes. Cytochromes H. haemoglobino- XI11 Friedberger NCTC 1659 were determined by difference spectrophotometry of philus (canis) turbid suspensions of morphologically intact bacteria H. parasuis (5) 1374 Shope NCTC 4557 (that had been sonically treated briefly to insure H. aegyptius 180-a Pittman ATCC 11116 uncoupling), using a Diffuse Reflectance Accessory of H. aegyptius Leidy HIM 450-4 the EPS-3T spectrophotometer. The characteristic H.piscium Snieszko ATCC 10801 spectral properties of cytochromes of aas, a,, d(a,), b, “H.vaginalis” Dukes 594 ATCC 14018 H.paraphrophilus Funagli NCTC 10557 and c types were recognized by the difference spec- H.parahaemolyti- 536 Pittman NCTC 8479. trum of Na,S,O,-reduced minus H,O,-oxidized Sam- cus (5) ples of a given bacterial suspension recorded from 390 H.paraphrohae- L 1 Zinnemann NCTC 10670 to 650 nm. molyticus Further spectral properties of cytochrome oxidases Eikenella 333154-55 Henriksen NCTC 10596 and, in particular, of cytochrome o were detected by corrodens the difference spectrum Na,S,O,-red/CO minus NCTC, National Collection of Type Cultures, London, Na,S,O,-red. The a-maxima of cytochrome pigments Great Britain; ATCC, The American Type Culture Collec- in difference spectra (redox) and the corresponding tion, Rockville, Maryland; HIM, Collection of the Hygiene- minima in carbon monoxide spectra were used for Institut, Marburg, Germany. their identification and quantitative assay (cf. 2, 21). Since in most instances molar extinction coeffi- Aerobic cultures were grown in Fernbach flasks (no. cients of bacterial cytochromes in situ are not known, 20511 Schott & Gen., Mainz, Germany) containing the amount of cytochrome was only specified as 400 ml of medium and aerated by shaking in a rotatory absorption difference per gram of bacterial protein shaker (model G-25, New Brunswick Scientific Co.) and centimeter of optical pathway, corrected for the at 200 rpm. Oxygen-limited cultures were grown sta- amplification effect of the diffuse reflectance equip- tically in Fernbach flasks filled with 2,000 ml of me- ment. The amplification factor was determined from dium containing fumarate as an additional supple- the absorption spectrum of a known amount of ment. The incubation temperature was 37 C except cytochrome c (Fluka, Buchs, Switzerland) added to for H.pisciurn, which was incubated at 20 C. Cells of the reference cuvette and recorded over the red minus the early stationary phase were harvested at 20,000 x red base line with each bacterial suspension. g, 0 to 4 C, and washed twice in 0.1 M tridhydroxy- methyl\aminomethFe-hydrochloridebuffer, pH 7.2. RESULTS Determination of protein. Protein was determined The scope of the present investigation was in sonically treated bacterial suspensions by the classification of haemophilic bacteria according biuret method using KCN (22). to phylogenetic variations in composition of the Extraction and determination of quinones. Qui- nones were extracted by the method of Kriiger and respiratory chain. Since, however, the pheno- Daddk (13) and determined by their characteristic typical dynamics of bacterial respiratory chain absorption spectra in ethanolic solution, using a systems have to be taken into consideration, the recording spectrophotometer (model EPS-3T, Hitachi bacteria were grown with two alternative elec- Ltd., Tokyo). The absorption differences of the re- tron acceptors, oxygen or fumarate, as de- duced minus oxidized quinones at 230 to 300 nm were scribed above. Fumarate may serve as a termi- recorded (13, 18). The quinone spectra were plotted nal acceptor in anaerobic electron transfer in by the methods of White (25) and Kriiger and some gram-negative bacteria (cf. 14, 26). With Klingenberg (15). organisms unable to reduce fumarate, however, The amounts of ubiquinone (Q) and desmethyl- menaquinone (DMK) were calculated using the dif- only oxygen-limited growth could be expected using the unaerated complex culture medium. ference extinction AZ280-289 = -8.8 mM-I x cm-1 (14) and = 26.7 mM-I x cm-1 (25). The cytochromes and respiratory quinones of If both Q and DMK were present in the lipid cells harvested in the early stationary phase are extract, the amounts of the individual quinones were shown in Table 2. As indicated by the table, 104 HOLLANDER AND MANNHEIM INT.J. SYST.BACTERIOL. cytochromes of the b and/or c type, as well as geneity with respect to the terpenoid quinones is cytochrome 0, are common to all organisms unusual, as other generic groups of bacteria are examined. In addition, cytochromes a and/or d highly invariable in this respect. As a rule, were detected in some strains. The amount of chemo-organotrophic gram-negative Eubacteri- cytochromes synthesized may vary considerably ales and that utilize organic among different strains and among derivatives compounds only by oxidation with oxygen or of a given strain according to culture conditions, nitrates as electron acceptors contain Q as the For example, strains of the species H. only quinone component of the respiratory influenzae and H. aeg-yptius (except strain 641 chain system (6, 24; E. Callies, manuscript in Pittman) produced only a few percent of the preparation). maximum amounts of cytochromes found in H. In contrast, many facultatively aerobic parainfluenzae cultures. gram-negative bacteria which produce acid Table 2 further indicates that the bacteria from carbohydrates in the absence of oxygen or investigated contain either Q or DMK, or both, nitrate are able to produce two different respira- respiratory chain components in varying pro- tory quinones, namely, Q in aerobic respiration, portions. Strains producing both Q and DMK and a naphthoquinone, predominantly mena- (H. parainflvenzae strains HIM 412-6 and 1 quinone (MK), in anaerobic respiration with Fleming, H. haemoglo binophilus, H. parasuis, fumarate as electron acceptor. This holds true H. paragallinarum) show the tendency to re- for the (14, 23, 24; R. Jediss, duce Q in favor of DMK when cultivated M.D. thesis, Philipps University, Marburg, W. anaerobically in the presence of fumarate. Fur- Germany, 1973), Yersinia, Vibrio, and Aero- thermore, in most of the strains producing moms (23; R. Zabel, M.D. thesis, Philipps DMK (H. influenzae, H. parainfluenzae strains University, ‘ Marburg, W. Germany, 1974), HIM 170-1, and Bossy No. 7 Leidy, H. aegyp- whereqs other groups of facultatively or strictly tius, H. paraphrophilus, H. parahaemolyticus, anaerobic gram-negative bacteria may contain H. paraphrohuemolyticus) the growth yields of only MK. oxygen-limited cultures are increased by fuma- MK is also the respiratory quinone of aerobic rate whereas growth of strains containing only Q and facultatively anaerobic gram-positive bac- (H. piscium, “H. vaginalis,” Eikenella teria (cf. 6, 11, 16). Among the fermentative corrodens) remains unaffected. , the leading Pasteurella and H. parainfluenzae strain 429 Pittman could species produce both Q and not be cultivated anaerobically; however, when DMK (R. Zabel, Ph.D. thesis), a quinone first grown aerobically in the presence of fumarate, detected in Streptococcus faecalis (3) and in a this organism contained, in addition to Q, re- strain of Haemophilus parainfluenzae (18). spectively small amounts of a redox active DMK may be accumulated as a precursor or quinone that has not yet been identified but is by-product in MK biosynthesis (23, 24). considered to be a naphthoquinone derivative The organisms hitherto ascribed to Hae- similar to DMK on the basis of spectral proper- mophilus may be subdivided, according to their ties and R, values. respiratory quinones, into (i) bacteria that con- tain DMK alone, (ii) bacteria containing both Q and DMK, and (iii) bacteria containing only Q. DISCUSSION This statement leads to the supposition that the The data presented suggest that, in addition genus Haemophilus in its present composition to aerobic respiration, the majority of the orga- is probably not a natural group, and even some nisms investigated possess a DMK-mediated of the conventional Haemophilus species ap- anaerobic fumarate respiration pathway, pear to be heterogeneous. However, we do not whereas other Haemophilus strains that have rule out that, with organisms in which the abil- been found to produce only Q appear to be ity of synthesizing several respiratory chain metabolically related to nonfermentative components varies so greatly, variation in bio- groups of bacteria. synthesis of the respiratory quinones might be The taxonomic significance of these findings possible as well. Therefore, the following tenta- should be considered in relation to the variabil- tive reclassification of Haemophilus and related ity of respiratory chain components in well bacteria, on the basis of the respiratory established groups in order to evaluate this type quinones, deserves further confirmation, in of criteria, rarely used to date in bacterial particular, by investigation of DNA relatedness. classification. As for the distribution of cyto- Genus Haemophilus sensu strictiori (s.s.). chromes, the bacterial strains examined here The type species of Haemophilus, H. influen- are qualitatively similar. However, their hetero- zae, produces DMK as sole respiratory quinone. TABLE2. C-ytochrornes and quinones of bacteria from earlv stationary cultures

Quinones (pmollp of Effect of Characteristic absorption (nmP (AOD x cm I x g of protein I) Growth protein) fumarate on Strain and collection no. condition anaerobic ab d 0 b b+c C Q DMK growth yield H. influenzae NCTC 1‘ - 555(0.48 1 552-560(0.82) - 1.06 4560 2d - - - - 1.44 + H. influenzae HIM 1 - 555( 1.2) 559(6.56) - 1.7 412-7 2 - - - - 1.5 + H. influenzae NCTC 1 - 555( 1.O) 559(2.5) - 2.9 8467 2 63%2.46) 554(46.0) - - 3.7 + H.parainfluenzae HIM 1 635(2.72) 557(4.5) 556(11.3) - 1.04 170-1 2 63X32.1) 559(2.2) 558(30.9 ) - 1.43 + H.parainfluenzae HIM 1 636( 12.3) 555(102.01 - - 3.04 449 -8 2 635(5.22) 555(24.4) - 2.8 + H. aegyptks ATCC 1 - 560(0.6) 560(2.96) - 3.35 11116 2 634(5.48) 558(20.6 1 550-560( 10.9) - 9.4 + H. aegyptius HIM 450-4 1 - 556(1 .O) 559(0.81) - 3.7 2 634(3.28) 560(6.2) 552-562( 10.5) - 2.0 + H. parap hrophil us 1 635(3.42 ) 554(4.02) 558(29.4) - 0.46 NCTC 10557 2 - 554(24.1 ) 557( 10.2) - 0.96 + H.parahaemolyticus 1 634(2.97) 557(3.96 560(27.6) - 2.19 NCTC 8479 2 - 554(21.1 ) 552-558(47.2) - 2.6 + H.paraphrohaemol.vti- 1 a - 7.6 cus NCTC 10670 2 - 555(20.5) 559(20.6) - 1.7 + H. parainfluenrae HIM 1 634(5.12) 556( 19.4) 553-560(43.8) <0.1 1.52 412-6 2 - 558 (9.8) 553-560( 102.3) - 1.61 + H. parainfluenrae NCT( 1 635(3.0) 556(9.4) 558(9.5 1 0.46 0.22 4101 2 - 554(92.8) - 0.21 0.29 + H. haemoglobinophilus 1 635(0.861 557(2.14 559(3.42) 1.21 1.2 NCTC 1659 2 635(1.9) 560(5.83) 559(8.6) 0.98 2.3 + H.parasuis NCTC 4557 1 634(7.35) 557(0.7) 560(19.1) 1.49 - 2 634(6.51 557(22.4) 558( 103.4 0.32 0.84 + H. paragallinarum 1 632 (4.95 557(2.0) 552-560( 15.8) 2.9 0.4 NCTC 3438 2 - 558(6.2) 552-560( 13.7) 2.2 0.55 + H.parainfluenzae ATCC 1 634 (24.3 555(3.4) 559(47.74 1.09 ? 7901 2 - 559(4.48 - 550-560( 5.6) 1.3 ? - H.piscium ATCC 10801 1 - 559(3.06) 558( 13.7) 0.3 - 2 - 560( 1.8) 559(2.36) 0.13 - - “H.uaginalis” ATCC 1 - - 559(2.04) <0.1 - 14018 2 - 560( 1.65) 559(0.33) <0.1 - - Eikenella corrodens 1 a a a 0.7 - NCTC 10596 2 a a <0.1 - -

a Cytochrome pigments were estimated using the a-maxima in their difference spectra (reduced minus oxidized) except cytochrome o, which was estimated from its characteristic absorption minimum between 555 and 560 nm in carbon monoxide spectrum (difference spectrum reduced + CO minus reduced). Symbols: - not indicated; e, not examined: +, increase in growth yield in the presence of fumarate; ?, not identified. Cytochrome. 1 = Cells from aerated, early stationary cultures in proteose peptone glucose medium. 2 = Cells from oxygen-limited, early stationary cultures in proteose peptone glucose medium supplemented with fumarate (0.8% wt/vol). 106 HOLLANDER AND MANNHEIM INT. J. SYST.BACTERIOL. Besides H. influenzae, the H. aegyptius, H. ACKNOWLEDGMENT paraphrophilus, H. parahaemolyticus, and H. We are indebted to K. S. Zinnemann, Leeds, for encour- paraphrohaemolyticus strains investigated, as agement and helpful criticism. well as two H. parainfluenzae strains (HIM REPRINT REQUESTS 170-1 and Bossy no. 7 Leidy), exhibit the same Address reprint requests to: Dr. W. Mannheim or Dr. R. property. Moreover, if V and X factor require- Hollgnder, Hygiene-Institut, Philipps University, 355 Mar- ments are not considered as taxonomic charac- burgbahn, D- Pilgrimstein 2, West Germany. ters, Haemophilus aphrophilus and Pasteurella pneumotropica, which both contain only DMK LITERATURE CITED (30), will fit into this group, which then could be 1. Amies, C. R., and M. Garabedian. 1963. The bacteriology redefined as follows: small, nonmotile, coccoid of human vaginitis. Can. J. Public Health 5450. 2. Bartsch, R. G. 1968. Bacterial cytochromes. Annu. Rev. to thread-forming, gram-negative rods that are Microbiol. 22:181-200. obligate vertebrate parasites, contain DMK as 3. Baum, R. H., and M. I. Dolin. 1963. Isolation of-a new sole respiratory quinone, and require highly naphthoquinone from Streptococcus faecalis 10 C 1. J. complex media. Species differentiation within Biol. Chem. 238:4109-4111. 4. Beljanski, M. 1955. Isolement des mutants d’Escherichia this genus Haemophilus sensu strictiori ad- coli streptomycinrbsistants de’pourvus d’enzymes re- heres to most of the traditional criteria. Fer- spiratoires. Action de l’hbmine sur la formation de ces mentation of carbohydrates is typical but not enzymes chez le mutant H,. C. R. Acad. Sci. Paris an obligatory character of this genus, since 240:374-377. nonfermenting strains of H. aegyptius do occur 5. Biberstein, E. L., and K. Zinnemann. 1971. Report (1966-1970) of the Subcommittee on the of (e. g., strain 180a Pittman). Haemophilus to the International Committee on No- Fermentative bacteria to be eliminated menclature of Bacteria. Int. J. Syst. Bacteriol. from Haemophilus S.S. The organisms pro- 21:133-134. 6. Bishop, D. W., K. P. Pandya, and H. K. King. 1962. ducing both DMK and Q, H. paragallinarum, Ubiquinone and Vitamin K in bacteria. Biochem. J. H. haemoglobinophilus, H. parasuis, and H. 83:606-614. parainfluenzae (strains 1 Fleming and HIM 7. Caldwell, D. R., D. C. White, M. P. Bryant, and R. N. 412-6), should be placed close to Actinobacillus Doetsch. 1965. Specificity of the heme requirement for growth of Bacteroides ruminicola. J. Bacteriol. lignieresii, Actinobacillus equuli, Pasteurella 90: 1645-1654. huemolytica, and (cf. R. 8. Criswell, B. S., J. H. Marston, W. A. Stenback, S. H. Zebel, M.D. thesis). Black, and H. L. Gardner. 1971. Haemophilus oaginalis The position of H. parainfluenzae strain 429 594, a Gram-negative organism? Can. J. Microbiol. 17:865-869. Pittman will remain uncertain until the nature 9. Dunphy, P. J., P. G. Phillips, and A. F. Brodie. 1971. of its second quinone is elucidated. Separation and identification of menaquinones from Nonfermentative bacteria to be eliminated microorganisms. J. Lipid Res. 12~442-449. from Haemophilus S.S. Those “haemophilic” 10. Jackson, F. L., and Y. E. Goodman. 1972. Transfer of the facultatively anaerobic organism Bacteroides corrodens bacteria that contain Q as sole respiratory Eiken to a new genus Eikenella. Int. J. Syst. Bacteriol. quinone, namely H. piscium and “H. uaginal- 22: 73-77. is,” are probably not a homogeneous group of 11. Jacobsen, B. K., and H. Dam. 1960. Vitamin K in organisms; they should be transferred to al- bacteria. Biochim. Biophys. Acta 40:211-216. 12. Jensen, J., and E. Thofern. 1953. Chlorhlmin (Ferropor- ready existing or new taxa of gram-negative phyrinchlorid) als Bakterienwuchsstoff. 11. Z. Natur- bacteria. Eikenella corrodens (Bacteroides cor- forsch. 8b:59?-603. rodens) (cf. 10) may be related to some of these 13. Kriiger, A., and V. DadAk. 1969. On the role of quinones organisms because of similar growth require- in bacterial electron transport. The respiratory system of Bacillus megaterium. Eur. J. Biochem. 11:328-340. ments and Q content. 14. Kriiger, A., V. Dadhk, M. Klingenberg, and F. Diemer. “Haemophilus uaginalis” Gardner and Dukes 1971. On the role of quinones in bacterial electron 1955, originally described as a gram-negative transport. Differential roles of ubiquinone and mena- organism, was removed from the genus Hae- quinone in Proteus rettgeri. Eur. J. Biochem. 21~322-333. mophilus by the majority of the more recent in- 15. Kroger, A., and M. Klingenberg. 1970. Quinones and vestigators mainly because its Gram reaction nicotinamide nucleotides associated with electron depends on the growth medium used (1, 20, 28, transfer. Vitam. Horm. 28533-574. and others). The data presented here, however, 16. Lester, R. L., and F. L. Crane. 1959. The natural occurrence of coenzyme Q and related compounds. J. although eliminatipg strain Dukes 594 from Biol. Chem. 234:2169-2173. Haemophilus, do not support the view that 17. Lester, R. L., and T. Ramasarma. 1959. Chromatography “H. uaginulis” could be related to gram-posi- of the coenzyme Q family of compounds on silicone- tive taxa; the presence of Q rather confirms the impregnated paper. J. Biol. Chem. 234:672-676. 18. Lester, R. L., D. C. White, and S. L. Smith. 1964. The statement of Criswell et al. (8) that strain 594 is 2-desmethyl vitamin K,’s. A new group of naphthoqui- a true gram-negative organism since gram-posi- nones isolated from Haemophilus parainfluenzae. Bio- tive bacteria do not form Q as far as is known. chemistry 3:949-954. VOL.25, 1975 HEMOPHILIC AND RELATED BACTERIA 107

19. Pittman, M. 1957. Genus IV: Haemophilus, p. 406-413. 24. Whistance, G. R., and D. R. Threlfall. 1968. Effect of In R. S. Breed, R. G. E. Murray, and N. R. Smith (ed.), anaerobiosis on the concentrations of demethyl- Bergey’s manual of determinative bacteriology, 7th ed. menaquinone, menaquinone and ubiquinone in Esche- William & Wilkins Co., Baltimore. richia freundii, and Aeromonas 20. Reyn, A., A. Birch-Anderson, and S. P. Lapage. 1966. An punctata. Biochem. J. 108:505-507. electron microscope study of thin sections of Haemo- 25. White, D. C. 1965. The function of 2-desmethyl vitamin phifus uaginulis (tiardner and Dukes) and some pos- Kzin the electron transport system of Haemophilus sibly related species. Can. J. Microbiol. 12:1125-1136. parainfluenzae. J. Biol. Chem. 240:1387-1394. 21. Smith, L. 1968. The respiratory chain system in bacteria, 26. White, D. C. 1966. The obligatory involvement of the p. 55-122. In T. P. Singer (ed.), Biological oxidations. electron transport system in the catabolic metabolism Interscience Publishers, New York. of Haemophilus parainfluenzae. Antonie van Leeuwen- 22. Szarkowska, L., and M. Klingenberg. 1963. On the role of hoek J. Microbiol. Serol. 32:139-158. ubiquinone in mitochondria. Spectrophotometric and 27. White, D. C., and L. Smith. 1962. Hematin enzymes of chemical measurements of its tedox reaction. Biochem. Haernophilus parainfluenzae. J. Biol. Chem. 2. 338 674-697. 237: 1332-1336. 23. Whistance, G. R., J. F. Dillon, and D. R. Threlfall. 1969. 28. Zinnemann, K., and G. C. Turner. 1963. The taxonomic The nature, intergeneric distribution and biosynthesis position of ‘Haemophilus uaginalis’ (Corynebacterium of isoprenoid quinones and phenols in Gram-negative uaginale). J. Pathol. Bacteriol. 85:213-219. bacteria. Biochem. J. 111:461-472.