INTERNATIONAL JOURNAL OF SYSTEMATICBACTERIOLOGY, July 1989, p. 304-308 Vol. 39. No. 3 0020-7713/89/030304-05$02.00/0 Copyright 0 1989, International Union of Microbiological Societies Lipoquinones in Members of the Family Pasteurellaceae R. M. KROPPENSTEDT’ AND W. MANNHEIM2* Deutsche Sammlung von Mikroorganismen, 0-3300 Braunschweig,’ and Institut fur Medizinische Mikrobiologie, Philipps- Universitdt, Pilgrimstein 2, 0-3550Marburg,2 Federal Republic. of Germany Selected members of the family Pasteurellaceae Pohll981 were investigated for their lipoquinone contents by using high-performance liquid chromatography. In addition to ubiquinones and demethylmenaquinones, menaquinones (MK-7 or MK-8 or both) were detected, mostly as minor naphthoquinone components, in several Actinobacillus, Pasteurella, and related species. Previous studies that relied on difference spectropho- tometry and thin-layer chromatographydid not identify menaquinone components in lipid extracts of members of the Pasteurellaceae. The view that this family can be differentiated from the Enterobacteriaceae and other fermenting gram-negative bacteria by a lack of menaquinones cannot be maintained. Although the situation seems to be more complex than previously recognized, the distribution patterns of lipoquinone structural types and their isoprenologs appear to remain a valuable chemotaxonomic tool for these bacteria. Isoprenoid quinones are essential mediators of phosphor- lone et al. (2) reported the presence of both menaquinones ylating electron transport in respiring membranes of many and demethylmenaquinones in Huemophilus ducreyi and procaryotes and eucaryotic organelles. They are associated questioned the inclusion of this species in the family Pas- with respiratory functions and are present in micromolar teurellaceae . amounts per gram of cellular protein, with considerable In this paper we present evidence that by performing variation depending on environmental conditions (9, 12, 14, HPLC analyses of samples containing at least 100 mg of 15, 19). As a rule, aerobic gram-negative chemoorgan- bacterial dry mass, menaquinones can be detected, mostly otrophic eubacteria and mitochondria contain ubiquinones as minor components, in addition to demethylmenaquinones as the sole respiratory quinones involved in aerobic metab- and ubiquinones, in selected strains representing well-estab- olism and nitrate respiration. Facultatively anaerobic and lished groups of the Pasteurellaceac. Moreover, our data fermentative gram-negative bacteria belonging to ribosomal contain additional information on the distribution patterns of ribonucleic acid superfamily I sensu De Ley (5) or to the quinone isoprenologs in this family. gamma group of the recently proposed class Proteobacteria (27) may be capable of producing respiratory naphthoqui- nones. The Flavobacterium-Cytophaga group and related MATERIALS AND METHODS taxa, gram-negative strictly anaerobic eubacteria, and aero- bic gram-positive eubacteria contain naphthoquinones as Microorganisms. Selected type or reference strains of sole respiratory quinones (3, 4). Menaquinones, methylm- species covering the whole range of phylogenetic diversity in enaquinones, and demethylmenaquinones may support the family Pasteurellaceae (Table 1) were grown from the phosphorylating electron transport to low-potential accep- lyophilized state on chocolate agar, cloned by subculturing tors (e.g., fumarate) (13, 16). Both the distribution of lipo- typical single colonies, and identified by conventional meth- quinone structural types and the length and degree of satu- ods (11). ration of their isoprenoid side chains are of chemotaxonomic Production of cell mass. Oxygen-limited cultures were interest (3, 4). grown in 500-ml batches of Proteose Peptone-glucose me- Previous studies on the respiratory quinones of members dium in 2-liter Fernbach flasks which were moderately of the family Pasteurellaceae Pohl 1979 (6-8, 23) were shaken (gyratory shaker, 80 rpm) at 36°C; cells were har- performed with lipid extracts of wet cell homogenates con- vested in the late exponential growth phase, washed in taining less than 10 mg of bacterial protein (17, 18) by using deionized water, and lyophilized. The medium consisted of difference spectrophotometry of reduced-minus-oxidized 2% (wt/vol) Proteose Peptone no. 3 (Difco Laboratories, ethanolic solutions and thin-layer chromatography (20). Detroit, Mich.), 0.5% (wt/vol) sodium chloride, 0.2% (wt/ These studies indicated that members of the family Pus- vol) D-( +)-glucose (separately sterilized), and 0.02 M sodium teurellaceae produce demethylmenaquinones as the sole hydrogen carbonate (sterilized by filtration); the final pH was respiratory naphthoquinones or demethylmenaquinones and 7.2. Anaerobic cultures were grown in 1-liter batches of ubiquinones. Several species that produced only menaqui- Proteose Peptone medium supplemented with 0.05 M diso- nones or only ubiquinones were excluded from the family dium fumarate (sterilized by filtration) instead of NaHCO, in Pasteurellaceae, and the absence of menaquinones in the Erlenmeyer flasks incubated statically. Before quinone anal- naphthoquinone fraction was used as a taxonomic marker to ysis dried cells were stored at -25°C for up to 2 months. separate this group from other gram-negative organisms Extraction, purification, and analysis of isoprenoid qui- belonging to the first and fifth ribosomal ribonucleic acid nones. Extraction, purification, and analysis of isoprenoid superfamilies (7, 22). More recently, however, the develop- quinones were performed as previously described (26). To ment of improved analytical methods, e.g., high-perfor- avoid confusion of quinone structural types that exhibited mance liquid chromatography (HPLC), have allowed more identical retention characteristics during HPLC, the individ- refined separation of quinone mixtures (3). Recently, Car- ual quinone peaks were further identified by their ultraviolet (UV) spectra. Each peak was scanned at its apex from 220 to 400 nm by using a diode array detector (model 1090/1040; * Corresponding author Hewlett-Packard Co., Palo Alto, Calif.). Each quinone ex- 304 VOL. 39, 1989 LIPOQUINONES IN THE PASTEURELLACEAE 305 TABLE 1. Lipoquinones of selected members of the Pasteurellaceue as determined by HPLC 96 of total quinones Terminal Species or group Strain" electron Menaquinones menaquinonesDemethyl- Ubiquinones acceptor MK-7 MK-8 DMK-7 DMK-8 4-6 Q-7 Q-8 Pasteurella multocidu NCTC 10322Th 02 23.6 30.9 45.5 Fumarate 1.0 60.0 39.0 Pasteurella gallinarum ATCC 13361T 02 3 .O 2.0 14.0 22.0 59.0 Actinobacillus lignieresii NCTC 4189T 02 19.0 2.0 74.0 5.0 Actinobacillus equuli NCTC 3365 02 28.0 72.0 Pasteurella pneumotropica bioty pe NCTC 8141T 02 97.9 2.3 Jawetz Fumarate 99.8 0.2 Pusteurella haemolyticu NCTC 9380T 02 22.4 17.9 59.7 Fumarate 57.1 18.9 24.0 Pasteurella haemolyticu biotype A NCTC 10610 02 11.0 23.0 66.0 Pasteurella haernolytica biotype T NCTC 10624 0, 11.0 21.0 68.0 Haemophilus paragallinurum NCTC 10926 Fumarate 18.0 10.0 72.0 Haemophilus injhenzae NCTC 8143T 02 99.0 1.0 Haemophilus parainjuenzae NCTC 7857T 02 100.0 Haemophilus parahaemolyticus NCTC 8457T 02 100.0 Haemophilus parasuis NCTC 4557T 02 4.0 17.0 69.0 10.0 Haemophilus uphrophilus NCTC 5886T 02 1.0 93 .O 6.0 Actinobacillus actinomycetem- FDC-Y4 02 96.0 4.0 comitans (HIM 829-8) Fumarate 97.6 2.4 Pasteurella aerogenes ATCC 27783T 02 13.1 8.1 78.8 Fumarate 24.2 52.2 23.6 Bisgaard taxon 14 HIM 913-2 02 8.8 26.5 64.7 Fumarate 12.0 76.5 11.5 a ATCC, American Type Culture Collection, Rockville, Md.; HIM, Culture Collection, Department of Medical Microbiology, University of Marburg, Marburg, Federal Republic of Germany; NCTC, National Collection of Type Cultures, Colindale, London, England: FDC, Forsyth Dental Center, Boston, Mass. ' T = type strain. hibited specific UV spectrum which could be used for ponents, in addition to demethylmenaquinones. These or- positive identification (Fig. 1 and 2). ganisms represented the genus Pasteurella sensu stricto Mutters et- al. 1985 (25), the genus Actinobacillus, the RESULTS AND DISCUSSION Pasteurella haernolytica complex (24), Haemophilus Of the 16 strains of members of the Pasteurellaceae paragallinarurn, Haernophilus parasuis, Haernophihs aph- studied, 12 produced menaquinones, mostly as minor com- rophilus, Pasteurella aerogenes, and Bisgaard provisional Pasteurel la haemolytica biotype A NCTC 10610 I Q-7 40- 30- DMK-7 1 20- MK-7 r 1 / 4 I I I 2 4 6 8 Time (min.1 FIG. 1. Separation of Pusteurella haemolytica NCTC 10610 quinones by HPLC. A 100-mg portion of lyophilized cells was extracted twice with 10 ml of chloroform-methanol (2:1, vol/vol) for 1h. A lO-~lportion (5%) of the reduced filtered extract was used for separation by HPLC. The following conditions were used: ODS column (125 by 4 mm [inside diameter]); particle size, 5 p,m (Hypersil); column temperature, 40°C; flow rate, 1 ml/min; eluant, acetonitrile-isopropanol (65:35). mAU, Milliabsorbance units. 4-7, ubiquinone 7 (2,3-dirnethoxy-S-methyl- 3-heptaprenyl-l,4-benzoquinone);MK-7, menaquinone 7 (2-methyl-3-heptaprenyl-l,4-naphthoquinone);DMK-7, demethylmenaquinone 7. 306 KROPPENSTEDT AND MANNHEIM INT. J. SYST.BACTERIOL. UV spectrum of MK-7 NCTC 18618 a 40i J 1 220 240 260 280 300 320 340 360 380 400 Wave 1 angth (nm) spectrum of DMK-7 NCTC 18618 1 . , . , . , . , . ,>., --, . .\, . , , 220 240 260 280 300 328 340 360 380 400 Wavalenath (nm) C 1 i.,...,...,...,..~,,...,...,...,...,..., 220 240 260 280 300 320 340 360 380 400 Wavelenath (nm) FIG. 2. UV spectra of the separated quinones of Pasteurella haernolytica NCTC 10610. (a) Scanned peak at 3.1 min (MK-7), showing the characteristic UV absorption spectrum of menaquinones. mAU, Milliabsorbance units. (b) Peak at 2.8 min (DMK-7), showing the characteristic UV absorption spectrum of demethylmenaquinones. (c) Peaks at 2.3 and 1.9 min (Q-7), showing the characteristic UV absorption spectrum of ubiquinones. taxon 14 (l),which has been incorporated in the family De Ley, and W. Mannheim, Abstr. XIV Int. Congr. Micro- Pasteurellaceae on the basis of deoxyribonucleic acid-ribo- biol., abstr. no. P.B4-8, 1986). Pasteurella pneumotropica soma1 ribonucleic acid hybridization results (U.