INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1986, p. 323-332 Vol. 36, No. 2 0020-7713/86/020323-10$02.OO/O Copyright 0 1986, International Union of Microbiological Societies

Inter- and Intrageneric Similarities of Ribosomal Ribonucleic Acid Cistrons of the R. ROSSAU,’ A. VAN LANDSCHOOT,? W. MANNHEIM,2 AND J. DE LEY1* Laboratorium voor Microbiologie en microbiele Genetica, Rijksuniversiteit, B-9000 Gent, Belgium’ and Institut fur Bakteriologie, Zentrum fur Hygiene und Medizinische Mikrobiologie, Universitat, 0-3550MarburglLahn, Federal Republic of Germany2

Filter-fixed deoxyribonucleic acid preparations from organisms of the Neisseriaceae and from two unpigmented, psychrophilic, misnamed “Achromobacter” strains were hybridized with labeled ribosomal ribonucleic acids (rRNAs) from Acinetobacter calcoaceticus ATCC 2305ST (T = type strain), Alteromonas macleodii ATCC 2712@, B, NCTC 10322T, Actinobacillus lignieresii NCTC 4189T, ATCC 17802T, Pseudomonas fluorescens MMCA 40T, Deleya aquamarina NCMB 557, Marinomonas vaga ATCC 27119T,Xanthomonas campestris NCPPB 52gT,Alcaligenes denitrijicans ATCC 15173T, Pseudomonas solanacearum NCPPB 32ST, Pseudomonas acidovorans ATCC 15668T,Alcaligenes faecalis NCIB 8156T,Janthinobacterium lividum NCTC 979fiT, violaceum NCTC 9757T, Acetobacter aceti NCIB 8621T,Agrobacterium tumefaciens ICPB TTIII, and Rhizobium meliloti NZP 4009. The results revealed that the present members of the Neisseriuceae are genetically very heterogeneous. This family contains at least five unrelated groups. One group is located in rRNA superfamily I11 (containing the Pseudomonas acidovorans complex, Alcaligenes, Janthinobacterium, etc.) and includes the “true neisseriae,” kingae, and Kingella denitri3cans. The present emended family Neisseriaceae should be limited to these organisms. Its closest genetic relative is Chrornobacterium. Another group, formed by Acinetobacter, Moraxella, Branhamella, the “false neisseriae,” and the misnamed achromobacters, should be removed from the Neisseriaceae; it is somewhat related to organisms belonging to rRNA superfamily I1 (containing the Pseudomonasfluorescens complex, Marinomonas, etc.). Kingella indologenes does not belong to either of these groups and is situated between rRNA superfamily I (containing the , Pasteurellaceae, etc.) and rRNA superfamily 11. Moraxella urethralis and Moraxella anatipestifer are not at all related to other members of the genus Moraxella.

In The Prokaryotes (9) and in Bergey’s Manual of System- positive that are parasites of the mucosal mem- atic Bacteriology (6) the family Neisseriaceae was divided branes of human beings and warm-blooded animals. In into the following four genera: Acinetobacter, Kingella, contrast to Moraxella, most species form acids Moraxella, and Neisseria. Branhamella was included in from sugars. Because of their coccoid micromorphology, Moraxella as a subgenus. In The Prokaryotes (9), the previ- Branhamella catarrhalis and the false neisseriae were orig- ously so-called “false neisseriae” (Neisseria ovis, Neisseria inally thought to be “true neisseriae.” However, transfor- caviae, and Neisseria cuniculi) were also described as mation studies (5, 12) revealed that these bacteria are more members of this subgenus of Moraxella. In Bergey’s Man- closely related to Moraxella. As a result, Moraxella was ual, however, Vedros (44) cautiously maintained these three incorporated into the family Neisseriaceae (22). Henriksen species in the genus Neisseria as species incertae sedis. and Bevre (21) initially placed in the genus Moraxella urethralis was considered a temporary member of Moraxella, but later gave genus status to this group of the genus until the exact taxonomic position of this species is bacteria (23). Snell and Lapage (38) added two other species elucidated. Moraxella anatipestifer is not considered to be a to Kingella on phenotypic grounds. true member of the Neisseriaceae. For many years a vast array of classical and genetic The members of the Neisseriaceae have been described as approaches has been used to unravel the of the gram-negative rods or cocci which are often arranged in Neisseriaceae. The current taxonomy of this family owes a pairs, are aerobic, and are nonmotile. Acinetobacter differs lot to the extensive investigations by Henriksen and Bevre greatly from the other members of the Neisseriaceae in and colleagues, who used mainly genetic transformation habitat, growth characteristics, and various other pheno- and, later, other techniques, such as gas chromatography of typic features. It is the only oxidase-negative genus of the cellular fatty acids. Nevertheless, the relationships within family. For these reasons, Henriksen (20) suggested a new and among the genera remained obscure. DNA-DNA hy- family for these bacteria. Van Landschoot and De Ley bridization experiments performed by Kingsbury (3 l), (submitted for publication) studied the taxonomy of the Johnson et al. (27), Hoke and Vedros (24), Riou et al. (36) genus Acinetobacter by using deoxyribonucleic acid (DNA)- and Sugimoto et al. (39) revealed significant homologies only ribosomal ribonucleic acid (rRNA) hybridization. This genus within the genera Neisseria and Acinetohacter and among was found to be rather heterogeneous and quite separate the “classical moraxellae” (Moraxella lacunata, Moraxella from all other genera. nonliquefaciens , and ). Some homology The genera Moraxella and Neisseria both contain oxidase- among these Moraxella species was also found by pulse- labeled ribonucleic acid-DNA hybridization experiments (4); * Corresponding author. however, only low to negligible values were obtained in t Present address: Industriele Hogeschool van het Rijk C.T.L., hybridizations with other Moraxella species and species of B-9000 Gent, Belgium. Acinetobacter, Neisseria, and Kingella. Within the Neis-

323 324 ROSSAU ET AL. INT. J. SYST.BACTERIOL.

TABLE 1. Organisms used, strain numbers, guanine-plus-cytosine contents, and growth media Guanine-plus- Growth conditions“ Sequence Strain no. as cytosine no.a Organism used for DNA isolation receivedb content Culture Medium Supplement(s1 (mol %)‘ type 1 Moraxella lacunata subsp. lacunata ATCC 17967T 43.9 FF PP 2 Moraxella lacunata subsp. liquefuciens ATCC 17952 45.5 RF HIA 3 Moraxella nonliquefaciens ATCC 19975T 42.5 FF PP 4 Moraxella bovis CCUG 2133T 42.5‘ RF HIA 5 Moraxella osloensis CCUG 350T 43.5‘ RF HIA 6 Moraxella osloensis H19116-51 43.5‘ RF HIA 7 Moraxella osloensis H18522-51 44.3s RF HIA 8 Moraxella phenylpyruvica NCTC 10526T 42.9 FF PP 9 Moraxella urethralis ATCC 17960T 47.7 RF HIA 10 Moraxelta urethralis NCTC 11008 47.4 FF TS 11 Moraxella anatipestifer ATCC 1184ST 34.9 FF PP Y 12 Branhamella catarrhalis ATCC 25238T 41.0 FF PP Y,H 13 Branhamella catarrhalis NCTC 4103 44.6 FF PP Y,H 14 “Achrornobacter” sp. NCMB 98 44.1 RF HIA 15 “Achrornobacter” sp. NCMB 307 46.3 RF HIA 16 Neisseria ovisg NCTC 11227T 45.9 FF PP 17 Neisseria caviaeg ATCC 14659T 46.9 FF Mh 18 Neisseria cuniculig ATCC 1468gT 46.1 FF Mh 19 NCTC 8375T 53.2h FF PP 20 NCTC 10025T 52.8 FF PP 21 Neisseria lactarnica NCTC 10617T 53 .O FF TS 22 Neisseria jlavescens ATCC 13120T 49.6 RF HIA 23 CIP 5951T 51.6 FF PP Y 24 ATCC 10555 49.6h RF HIA 25 Neisseria animalis NCTC 10212T 51.3 FF PP 26 Neisseria denitrificans ATCC 14686T 56.2 FF PP 27 subsp, elongata NCTC 10660T 53.1 FF Mh 28 Neisseria elongata subsp. glycolytica NCTC 11050T 54.8 FF Mh 29 Kingella kingae NCTC 10529T 48.2 FF sc 30 Kingella kingae NCTC 10746 47.4 FF sc 31 Kingella denitrificans NCTC 10995T 53.5 FF sc 32 Kingella denitrificans NCTC 10997 55.3 FF sc 33 Kingella indologenes NCTC 10717T 48.2 FF PP 34 Kingella indologenes NCTC 10883 48.7‘ FF PP

a Sequence numbers are not strain numbers. ATCC, American Type Culture Collection, Rockville, Md.; CCUG, Culture Collection of the University of Goteborg, Goteborg, Sweden; CIP, Collection de 1’Institut Pasteur, Paris, France; NCMB National Collection of Marine Bacteria, Torry Research Station, Aberdeen, United Kingdom; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom. Guanine-plus-cytosine contents were determined in our laboratories unless indicated otherwise. FF, Shake cultures in Fernbach flasks containing 500-ml volumes of liquid media; RF, aerobic cultures in Roux flasks containing agar media; B, Bovine serum (final concentration, 2%, vol/vol); H, Horse serum (final concentration, 2%, vol/vol); HIA, heart infusion agar (Difco Laboratories); Mh, Mueller-Hinton broth (BBL Microbiology Systems); PP, proteose peptone medium (45); TS, tryptic soy broth (Difco); Sc, Schaedler broth (BioMCrieux); Y, Filter-sterilized yeast extract (final concentration, OS%, wthol; Oxoid Ltd.). ‘ Data from reference 7. Data from reference 13. False neisseriae. Data from reference 24. ’ Data from reference 38. seriaceae genetic transformation is of practical value for and Branhamella catarrhalis by using a competition hybrid- identification at the species level (28, 29), and, when strep- ization method. Unfortunately, limited taxonomic conclu- tomycin resistance is used as a marker, it appears to be a sions were possible because the study included only three very powerful taxonomic tool which is capable of tracing representative strains of the Neisseriaceae. affinities even beyond the range of most other methods (5). In this work we studied the intergeneric relationships Although some marginal affinities between Acinetobacter within the Neisseriaceae by using the DNA-rRNA hybrid- and Moraxella and between Kingelta kingae and Neisseria ization method of De Ley and De Smedt (15). Because this elongata have been reported (9), no transforming ability method quite easily detects distant relationships between between the other genera and some species of Moraxella and bacteria, it frequently has resolved taxonomic problems Kingella was observed in tests with competent strains. It when other classical and even genetic methods have failed, seems that the overall DNA or messenger ribonucleic acid as was the case in the Azotobacteraceae (17) and Pseudomo- sequences of these bacteria differ too much to elucidate the nas (18). Based on the results of this method, an extensive taxonomy of the entire family. However, there is some detailed rRNA cistron similarity dendrogram covering the evidence that the rRNA cistron similarities are useful. In- gram-negative bacteria is being constructed. Within this deed, Johnson et al. (27) demonstrated homology between dendrogram at least four large groups are obvious; each one Acinetobacter rRNA and DNA from Moraxella osloensis has been called an rRNA superfamily (14). In this study VOL.36, 1986 rRNA CISTRONS OF THE NEISSERIACEAE 325

TABLE 2. TmC,,and rRNA binding values for hybrids between DNAs from species of the Neisseriaceae and labeled rRNAs from the type strain of Acinetobacter and from several other type and representative strains of rRNA superfamilies I, 11, and IV Source of rRNA superfamily I

DNA from strain: Acinetobacter Alteromonas Escherichia Pasteurelln Actinohmillus calcoaceticus macleodii coli multocidu lignieresii A TCC 230ST ATCC 27126T B NCTC 10322’ NCTC 4189T % of % of 5% of % of % of Tmc P) rRNA Tr’ic‘3) rRNA Tmce) rRNA T”,,,, rRNA Tnl(t’) rRNA (OC) binding (OC) binding rC) binding (”‘) binding (”‘) binding Moraxella lacunata 70.8 0.15 64.7 0.11 ATCC 17967T Moraxella lacunata 70.4 0.14 65.0 0.09 subsp. liquefaciens ATCC 17952 Moraxella 71.0 0.21 66.9 0.11 nonliquefaciens ATCC 1997ST Moraxella bovis CCUG 71.1 0.18 2133T Moraxella osloensis 71.1 0.18 CCUG 350T Moraxella osloensis 72.2 0.22 H19116-51 Moraxella osloensis 72.1 0.21 64.3 0.09 66.6 0.16 H18522-51 Moraxella phenylpyruvica 70.3 0.19 NCTC 10526T Moraxella urethralis 61.5 0.17 ATCC 17960T Moraxella urethralis 59.2 0.14 NCTC 11008 Moraxella anatipestifer 55.1 0.09 58.1 0.07 ATCC 1184ST Branhamella catarrhalis 73.8 0.20 66.0 0.10 ATCC 2523gT Branhamella catarrhalis 71.7 0.20 NCTC 4103 Neisseria ovis NCTC 70.3 0.18 65.8 0.10 11227T Neisseria caviae ATCC 70.8 0.16 14659T Neisseria cuniculi ATCC 69.9 0.20 14688T Neisseria gonorrhoeae 62.5 0.12 NCTC 8375T Neisseria meningitidis NCTC 10025T 60.8 0.14 NCTC 10617T “Achromobacter” sp. 70.6 0.18 strain NCMB 98 “Achromobacter” sp. 71.6 0.17 strain NCMB 307 Kingella denitr$cans 61.0 0.13 NCTC 1099ST Kingella indologenes 65.1 0.11 NCTC 10717T Kingella indologenes 64.2 0.11 NCTC 10883 Continued on following page

DNAs from members of several typical species of the IV). In this way (i) the different members of the Neis- Neisseriaceae, Moraxella anatipestifer, and two misnamed seriaceae could be grouped, (ii) the relationships among psychrophilic achromobacters were hybridized with labeled these groups could be detected, and (iii) the taxonomic rRNAs, from either Acinetobacter spp. or from species position of each group compared with the other gram- belonging to the four main superfamilies (superfamilies I to negative bacteria could be determined. 326 ROSSAU ET AL. INT. J. SYST.BACTERIOL.

TABLE 2-Continued labeled rRNA" rRNA superfamily I1 rRNA superfamily IV Pseudomonas Deleya Marinomonas Xanthomonas Rhizobium Agrobacterium fluorexens aquamarina vaga campestris meliloti tumefaciens MMCA 40T NCMB 557' ATCC 27119T NCPPB 528' NZP 4009 ICPB TTlll % of % of % of % of % of % of Tm(e) rRNA Tm(r) rRNA Tmcp) rRNA Tmcp) rRNA Tmce' rRNA Tm(r) rRNA ("') binding ("'I binding ("') binding ("') binding ("'I binding ("') binding 65.9 0.08 67.1 0.08 63 .O 0.10

68.4 0.14 64.4 0.12

66.3 0.17

67.4 0.11 56.3 0.10

68.5 0.12

57.5 0.06

66.4 0.18 64.0

60.3 0.18

62.9 0.14

66.5 0.14 62.9 0.15 64.1 0.10

65.2 0.10 57.5 0.08

a ICPB, International Collection of Phytopathogenic Bacteria, Department of Bacteriology, University of California, Davis: MMCA, Medical Microbiology Culture Collection, Institute of Medical Microbiology, Aarhus, Denmark; NCPPB, National Collection of Plant , Plant Pathology Laboratory, Harpenden, United Kingdom; NZP, Applied Biochemistry Division Department of Scientific and Industrial Research, Palmerston North, New Zealand. For other abbreviations, see Table 1, footnote b. Unpublished data from our laboratories.

MATERIALS AND METHODS used are either type strains or representative strains that have been used in other taxonomic studies, thus allowing easy Organisms used. The strains and growth media which we comparison of the results. used are listed in Table 1. We used various temperatures (30 Isolation of DNA. DNA was prepsred by the method of to 36°C) and incubation times (1to 6 days). Most of the strains Marmur (32). Sometimes the sodium perchlorate step was TABLE 3. T,,+,, and rRNA binding values forhybrids between DNAs from species of the Neisseriaceaeand labeled rRNAsfrom Acinetobacterand from several other typestrains of rRNA superfamilies I, 111, and IV Source of labeled rRNA"

rRNA superfamily111 rRNA superfamilyIV

Acinetobacter Alcaligenes Pseudomonas PseudomonasAlcaligenes Janthinobac- Chrornobae- Rhizobium Acetobacter Vibrioparahue- rnolvficusATCC calcoaceticus denitriJicans solanacearum acidovorans fnecalis terium lividum terium meliloti NZP DNA from strain: acykEFIB17802T(rRNA su- ATCC 23055T ATCC 15173TNCPPB 325' ATCC 15668T NCIB8156T NCTC9796T ceu~,~~TC 4009 perfamily I)

% of % of 5% of % of Tmre, 5% of 96 of % of % of Trnce, rRNA TTcr, rRNA Tm~e) ,.RNA Tm~c, rRNA TtnCc, ("C) (oc) rRNA Tm(e) rRNA Trnf,) rRNA Tmfe, rRNA T,,,,P,("0 (OC) binding ( binding ("'I binding ("') binding binding ("') binding ("') binding ("') binding

~~ ~~~~~ ~~ Neisseriagonorr- 67.3 0.16 69.1 0.07 hoeaeNCTC 8375iT Neisseriarneningiti- 61.6 0.21 67.3 0.18 68.6 0.23 72.4 0.18 59.7 0.10 dis NCTC 10025T Neisseria lactamica 60.4 0.29 67.0 0.15 70.1 0.06 66.9 0.18 NCTC 10617T Neisseriarnucosa 61.6 0.17 67.2 0.15 66.8 0.15 CIP 5951T Neisseriasubfrava 68.8 0.14 69.6 0.19 67.7 0.14 ATCC 10555 Neisseria JIavescens 72.6 0.15 ATCC 13120T Neisseriaanimalis 65.7 0.19 NCTC 10212T Neisseriadenitrifi- 61.6 0.14 68.1 0.14 72.1 0.13 56.8 0.09 cans ATCC 14686T Neisseria elongata 59.2 0.13 65.0 0.14 subsp. elongata NCTC 10660T Neisseria elongata 65.7 0.16 subsp. glycolytica NCTC 11050T Kingella kingae 58.9 0.22 66.4 0.13 67.8 0.08 65.1 0.16 66.7 61.9 NCTC 10529T Kingella kingae 61.4 0.18 68.7 0.17 71.8 0.17 NCTC 10746 Kingella denitrifi- 61.6 0.18 66.3 0.16 65.5 0.18 cans NCTC 10995T Kingella denitriji- 61.9 0.18 69.8 0.20 72.2 0.19 cans NCTC 10997 Kingella indologenes 63.2 0.17 61.6 0.11 61.8 0.05 NCTC 10717T Kingella indologenes 62.9 0.12 64.2 0.12 NCTC 10883 Moraxella nonlique- 62.1 0.14 61.6 0.08 faciens ATCC 19975T Moraxella osloensis 63.5 0.10 CCUG 350T Moraxella lacunata 62.0 0.07 subsp. lacunata ATCC 17967T Moraxella anatipes- 54.0 0.07 tifer ATCC 1184ST

a NCIB,National Collectionof IndustrialBacteria, Torry Research Station, Aberdeen, United Kingdom.For other abbreviations,see Table 1, footnoteb, and Table 2, footnotea. 328 ROSSAU ET AL. INT. J. SYST.BACTERIOL. omitted, and 50 pg of proteinase K (E. Merck AG, Kingella indologenes, is probably not closely related to Darmstadt Federal Republic of Germany) per ml was added other Kingella species. Undoubtedly, the isolated position of instead immediately before lysis in 1% sodium dodecyl Moraxella anatipestfer excluded this organism from all sulfate at 60°C. other genetic groups of the Neisseriaceae and from Fixation of single-stranded DNA. The procedure of Gillis superfamilies I, 11, and 111. In order to reveal the taxonomic and De Ley (19) was used to fix single-stranded DNA. relationships of the strains in this low-T,(,, area, more Determination of filter-fixed DNA. The chemical procedure accurate positioning was obtained by hybridizing the DNAs of Richards (35) was used to determine filter-fixed DNA. of these organisms with labeled rRNAs from various refer- DNA base composition. Average guanine-plus-cytosine ence strains of taxa belonging to other superfamilies (Tables contents were determined by the thermal denaturation 2 and 3). method (16). DNA-rRNA hybridization. Hybridizations were performed DISCUSSION as described by De Ley and De Smedt (15). The following We detected considerable genetic heterogeneity within the labeled rRNAs were used: Acinetobacter calcoaceticus Neisseriaceae as defined in Bergey’s Manual (6). Table 4 ATCC 230ST (T = type strain) [3H]rRNA (specific activity, shows the average TmCejvalues of the different genetic groups 21 x lo3 cpm/p,g of rRNA); Alteromonas macleodii ATCC found within the Neisseriaceae compared with each 27126T [14C]rRNA (6 x lo3 cpm/pg); Escherichia coli B superfamily. The species included in each group are also [14C]rRNA (7 x lo3 cpm/pgj; Pasteurella multocida NCTC shown in Table 4. 10322T [3H]rRNA (30 x lo3 cpm/pg); Actinobacillus As mentioned above, Moraxella, Branhamella, and the lignieresii NCTC 4189T [3H]rRNA (24 X lo3cpm/pg); Vibrio false neisseriae have their highest levels of homology with parahaemolyticus ATCC 17802T [14C]rRNA (6 X lo3 Acinetobacter. Although the average Tm(,) values of cpm/pg); Pseudomonas fEuorescens MMCA 40T [14C]rRNA Branhamella catarrhalis (72.8”C) and Moraxella osloensis (6 X lo3 cpm/Fg); Deleya aquamarina NCMB 557 [14C]r- (71.8”C) were somewhat higher than those of the other RNA (7 X lo3 cpm/pg); Marinomonas vaga ATCC 27119T Moraxella species (70.6”C), there seems to be enough evi- [3H]rRNA (7 x lo3 cpm/pg); Xanthomonas campestris dence in the literature to consider these organisms members NCPPB 52ST [14C]rRNA (5 X lo3 cpm/pg); Alcaligenes of one genetic group, which branches from the genus Acine- denitrifcans ATCC 15173T [14C]rRNA (9 X lo3 cpm/pg); tobacter at a Tm,e)level of 71.2 5 1.0”C (Table 4). Baumann Pseudomonas solanacearum NCPPB 3ZT [3H]rRNA (92 X et al. (2,3) have pointed out that the “oxidase-positive group lo3 cpm/kg); Pseudomonas acidovorans ATCC 15668T of moraxellae” is distinct from the “oxidase-negative [3H]rRNA (63 x lo3 cpm/pg); Alcaligenes faecalis NCIB group” (i.e., Acinetobacter). Pulse-labeled ribonucleic acid- 8156T [14C]rRNA (11 X lo3 cpm/pg); Janthinobacterium DNA hybridization results (4) allowed the same conclusion. fividum NCTC 9796T [3H]rRNA (8 X lo3 cpm/pg); Chromo- These results and DNA-DNA hybridization results (27) also bacterium violaceum NCTC 9757T [3H]rRNA (34 x lo3 suggest that our genetic group 1 (Table 4) might be rather cpmlpg); Acetobacter aceti NCIB 8621T [14C]rRNA(9 X lo3 heterogeneous. Indeed, the level of the relatedness between cpm/pg); Agrobacterium tumefaciens ICPB TTIII [3H]r- Acinetobacter and the Moraxella cluster is not high and is of RNA (33 X lo3 cpm/pg); and Rhizohium meliloti NZP 4009 the same order as the level of relatedness between the [3H]rRNA (24 x lo3 cpm/pg). Enterobacteriaceae and the Vibrionaceae (18). Strains NCMB 98 and NCMB 307 were received as RESULTS Achromobacter sp.; these organisms were reported as Mor- The main parameters of the DNA-rRNA hybrids are axella-like in the 1985 National Collection of Marine Bacte- shown in Tables 2 and 3. The temperature at which 50% of a ria Catalogue (33) and belong to a group of unpigmented hybrid was denatured [T,,,)], which was the most significant psychrophilic oxidase-positive rods which Thornley (42) parameter (19), was a measure of the base sequence similar- assigned to the genus Acinetobacter. Bgvre et al. (8) sug- ity between rRNA cistrons. The higher the Tm(,)value of a gested that both strain NCMB 98 and strain NCMB 307 hybrid, the higher the level of relatedness between the belong to Moraxella or to Acinetobacter or are part of an hybridized organisms. The percentage of rRNA binding was intermediate taxon between Moraxella and Acinetobacter. the total amount of rRNA that remained bound per 100 kg of Wax patterns indicated a closer relationship to Moraxella filter-fixed DNA after ribonuclease treatment. than to Acinetobacter (10). Later, it was shown by transfor- Figure 1 shows an rRNA cistron similarity map, which mation that both of the strains, together with 107 other was the result of hybridizations between various DNAs and psychrophilic strains, form a genetic group which exhibits no Acinetobacter calcoaceticus ATCC 2305ST rRNA. Three genetic affinity to Moraxella or Acinetobacter (30). Our different homology areas could be differentiated in the hybridization experiments (Table 2) indicated that these Neisseriaceae. The upper area contained Acinetobacter misnamed bbAchromobacter”strains do not belong to the strains (Van Landschoot and De Ley, submitted). Immedi- genus Acinetobacter but seem to cluster with Moraxella. ately below this area, between Tm(ejvalues of 69.9 and However, this does not necessarily mean that they are 733°C and rRNA binding values of 0.14 and 0.2296, there moraxellae; the possibility that they are as far removed from was a second area, which included strains of Moraxella Moraxella as they are from Acinetobacter, as suggested (except Moraxella urethralis and Moraxella anatipestifer), previously (8), cannot be ruled out. Acinetobacter, Morax- Branhamella, the false neisseriae, and misnamed ella, Branhamella, the false neisseriae, and both misnamed “Achromobacter” sp. strains NCMB 98 and NCMB 307. “Achromobacter” strains constitute a cluster which is sep- Below a Tm(e)value of 67.5”C strains belonging to the arated from rRNA superfamily I1 at an average T,+) value of different rRNA superfamilies were distributed all over the 67.2”C (calculated from the data in Table 4). The average map. True neisseriae, Kingella spp. strains, and Moraxella Tm(e) value with Xanthomonas spp. is 64.5”C; the average urethralis were found in an area encompassing superfamily Tm(.o)values with superfamilies I, 111, and IV are 65.7, 61.8, I11 organisms (i.e., Pseudomonas solanacearum, Pseu- and 58.6“C, respectively (calculated from the data in Table domonas acidovorans, and Bordetella sp.). However, 4). VOL. 36, 1986 rRNA CISTRONS OF THE NEISSERIACEAE 329

Branhamella Moraxella false neisserlae

2

XaFt.

M. urethralis 1 Rhiz.

11. M. anatipestifer I I I 0.10 0.20 0.30 %rRNA binding FIG. 1. Similarity map of hybrids made with 3H-labeled rRNA from Acinetobacter calcoaceticus ATCC 23055T. T,Hc,,is plotted versus percentage of rRNA binding. The definitions of both of these parameters are given in the text. The sequence numbers refer to the strains listed in Table 1. The values for Acinetohacter and species not belonging to the Neisseriaceae were taken from unpublished data from our laboratory. Symbols: 0,members of rRNA superfamily I; x , members of rRNA superfamily 11; A,members of rRNA superfamily 111; V, members of rRNA superfamily IV. Abbreviations: A. halo., Alteromonas haloplunktis; A. macl., Alteromonas macleodii; Aer., ; Bord., Bordetella bronchiseptica; E.,Escherichia; K., Kingella; M., Moraxella; Mar. corn., Marinomonas communis; Mar. vaga, Marinomonas vaga; Past., Pasteurella multocidu and Pasteurella bettii; P. acid, Pseudomonas acidovorans; P. fluor., Pseudomonus fluorescens; P. sol., Pseudomonas solanacearum; Rhiz., Rhizobium trifolii; Vibrio, Vihrio parahaemolyticus; Xant., Xanthomonas campestris.

The above mentioned cluster is not at all related to a bacterium. It is quite possible that Neisseria and Kingella second large cluster within the Neisseriaceae which is branch off together to form one cluster. This has been formed by the true neisseriae and two Kingella species, suggested for Kingella kingae on the basis of transformation Kingella kingae and Kingella denitrijicans. The highest studies (9) and fatty acid analyses (26). average Tm(e)value (72.2 f 0.3"C) (Table 4) was obtained The level of relatedness between the Neisseria-Kingella with rRNA from Chromobacterium violaceum, a member of cluster and Chromobacterium is on the same order as the rRNA superfamily 111. Since all of the species of true level of relatedness between Acinetobacter and Moraxella . neisseriae studied do have some DNA-DNA homology with The third Kingella species is Kingella indologenes. Two one another (24, 36), they can be regarded as one genetic strains were examined. One of these (strain NCTC 10717) group. Therefore, the Tm(e)values of the type strains of was originally isolated and described by van Bijsterveld (43); Neisseria meningitidis, , and Neisseria the other (strain NCTC 10883) was isolated and described by denitrijicans (Table 3) are representative of the whole group. Sutton et al. (41). These two strains are quite similar, as We can safely state that Neisseria branches from shown by Bflvre et al. (8). According to Snell and Lapage Chromobacterium at an average Tm(e)value of 72.4"C (Table (38), they belong phenotypically to the genus Kingella. 4). Only the following two Kingella species seem to be However, our results strongly suggest that, genetically, related to the true neisseriae: the type species, Kingella Kingella indologenes is not a member of the genus Kingella. kingae, and Kingella denitrijicans (formerly referred to as It belongs neither to the Neisseria-Kingella cluster nor to the TM1 group of Hollis et al. [25]). Both of these Kingella rRNA superfamily I11 (Fig. 1 and Table 4). This conclusion species have Tm(e)values (approximately 72°C) (Table 4) is partly supported by the lack of transforming ability similar to those of the true neisseriae against Chromo- between this species and members of Kingella or Neisseria 330 ROSSAU ET AL. INT. J. SYST.BACTERIOL.

TABLE 4. Average T,,*(')values of the hybrids between DNAs from members of different genetic groups in the Neisseriaceae and labeled rRNAs from Acinetobucter and from other taxa in rRNA superfamilies I to IV" T,,,((>,value ("C) with the following sources of labeled rRNA:

Genetic Source of DNA rRNA rRNA rRNA superfamily I11 r~~~ Species included' group Acinetobacter super- super- Xanthomonas Others super- family I family I1 taxa, Chrornobacteriurn family IV

1 Acinetobacter 74.5-79.5 65.8 67.3 64.7 61.7 ND" 57.6 Acinetobacter calcoaceticus Moraxella and 71.2 65.6 67.1 64.2 62.1 62.6 59.7 Moraxella iacunata, Branhamella Moraxella bovis, Moraxella nonliquefaciens, Moruxella osloensis, Moraxella False neisseriae phenylpyruvica, Neisseria o vis , Neisse ria cunicul i, Neisse ria ca via e , Branhamella catarrhalis "Achromobacter" 71.1 ND ND ND ND ND ND SP. 2 Kingella 63.1 64.7 64.7 64.7 62.5 ND 57.5' Kingella indologenes indologenes 3 True neisseriae 60.8 61.7 61.6 ND 67.5 72.4 8.3 Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactarnica, Neisseria, subjava, Kingelld 61 .O 61.5 ND NC 67.0 72.0 ND Neisseria javescens, Neisseria rnucosa, Neisseria animalis, Neisseria denitriJicans, Neisseria elongata, Kingella kingae, Kingella denitrificans 4 Moraxella 60.4 ND ND ND ND ND ND Moraxella urethralis urethralis 5 Moraxella 55.1" 58.1" ND ND 54.0' ND 57.5' Moraxella anatipestifer anatipestifer

~~

a This table summaries the results shown in Tables 2 and 3. The data for Acinetobacter were obtained mainly from work done by Van Landschoot and De Ley (manuscript in preparation). The higher the Tmcc)value, the closer the relationship. The species in each genetic group are also indicated. ' See Table 3. ' Only species used in this study are listed. ND, Not determined. Values obtained from only one hybridization. To calculate the average T,,,(,, values, Kingella kingae and Kingella denitriJcans were considered one group, as proposed by Snell and Lapage (38), although no genetic evidence for this is available.

(9). The highest average Tm(elvalues were obtained with cally very far removed from Moraxella, and (ii) is not a rRNAs from species belonging to superfamilies I and I1 member of rRNA superfamily I or 11. The exact taxonomic (Table 4); however, these values do not allow inclusion in position of Moraxella urethralis remains to be determined. either one of these superfamilies. Distinct relationships with Some organisms causing septicemia in ducks are known as other well-known genera have not been found yet. In our Moraxella anatipestifer or Pasteurella anatipestifer. Morax- opinion, Kingella indologenes is taxonomically best situated ella anatipestifer is the valid name (36), although in Bergey's somewhere between rRNA superfamilies I and 11. Manual (11) these bacteria are classified as species incertae Morphologically and phenotypically, Mnraxella urethralis sedis under Pasteurella. Our hybridization results with DNA resembles some Moraxella species, but it can be differenti- from the type strain clearly showed that Moraxella ated from them by its distinct fatty acid composition, which, anatipestifer is related neither to Moraxella nor to Pasteu- however, seems to fit within the overall fatty acid pattern of rella (Table 2). This is in agreement with previous observa- the Neisseriaceae (26). According to Sugimoto et al. (40), tions (1, 34, 39). The very low Tm(e)values also rule out a there is a very close fatty acid similarity among Moraxella possible relationship with other genera belonging to rRNA urethralis, Branhamella catarrhalis, and Neisseria ovis. superfamilies I to IV. At present we cannot propose a However, genetic affinities between Moraxella urethralis definite genus. Moraxella anatipestifer might be related to and other genera have never been found (27, 39). We used the Flavobacteriurn-Cytophagagroup (W. Mannheim et al. , two strains of Moraxella urethralis, strain ATCC 17960T unpublished data). (cited on the Approved Lists [37] as the type strain) and Figure 2 shows the taxonomic positions of the different strain NCTC 11008 (Table 1). The DNA-rRNA hybrids of groups of the Neisseriaceae relative to other gram-negative these strains with Acinetobacter spp. strains gave Tm(e) bacteria; it demonstrates the extreme genetic heterogeneity values of 61.5 and 59.2"C, respectively (Table 2). These low in this family. The Neisseria-Kingella cluster and the Acine- Tm(e)values indicate that Moraxella urethralis (i) is geneti- tobacter-Moraxella cluster are as far removed from each VOL. 36, 1986 rRNA CISTRONS OF THE NEZSSERZACEAE 331

80 -Y C hromobac ter ium Wa8 E I- Moraxellg - 6r an hamella I false neisseriae 70 NCM 6 98,307 other rRNA super- r RNA superfamily II family IU organisms & rRNA superfamlly I

rRNA superfamily IP yestifer]

FIG. 2. Part of the rRNA cistron similarity dendrogram showing the Chromobacterium and Acinetobacter rRNA branches only. The levels of similarity are expressed as TmC,,values. The different groups within the Neisseriaceae are shown as boxed names. The taxonomic position of Moraxella urethralis is not yet clear. Solid and open bars indicate the TmC,,areas of the strains within the different taxa. The arrows indicate the roots of the superfamilies and taxa. other as Alcaligenes is from the Pseudomonas fluorescens 6. B~vre,K. 1984. Family VIII. The Neisseriuceae PrCvot 1933, complex. Thus, it is not surprising at all that most taxonomic 119AL,p. 288-308. In N. R. Krieg and J. G. Holt (ed.), Bergey’s methods failed to detect significant levels of relatedness manual of systematic bacteriology, vol. 1. The Williams & between species of the former two groups. In any case, Wilkins Co., Baltimore. genetically a single family Neisseriaceae including species of 7. B~vre,K., M. Fiandt, and W. Szybalski. 1969. DNA base composition of Neisseria, Moraxella, and Acinetobacter, as both groups cannot be maintained. Our results suggest determined by measurement of buoyant density in CsCl gradi- strongly that the family Neisseriaceae should now be limited ents. Can. J. Microbiol. 15335-338. to the true neisseriae (e.g., Neisseria gonorrhoeae and other 8. B~vre,K., J. E. Fuglesang, S. D. Henriksen, S. P. Lapage, H. Neisseria species), Kingella kingae, and Kingella Lautrop, and J. J. S. Snell. 1974. Studies on a collection of denitrijicans. Acinetohacter, Moraxella, Branhamella, the gram-negative bacterial strains showing resemblance to false neisseriae (e.g., Neisseria ovis and other Neisseria moraxellae: examination by conventional bacteriological meth- species), and the misnamed psychrophilic achromobacters ods. Int. J. Syst. Bacteriol. 24:438446. are members of at least one other family, which is still to be 9. B~vre,K., and N. Hagen. 1981. The family Neisseriaceue: named. rod-shaped species of the genera Moraxella, Acinetobacter, Kingella, and Neisseria, and the Branhamella group of cocci, p. 1506-1529. In M. P. Starr, H. Stolp, H. G. Triiper, A. Balows, ACKNOWLEDGMENTS and H. G. Schlegel (ed.), The prokaryotes, vol. 2. Springer- J.D.L. is indebted to the National Fonds voor Wetenschappelijk Verlag, Berlin. 10. Bryn, K., E. Jantzen, and K. B~vre.1977. Occurrence and Onderzoek and the Fonds voor Geneeskundig Wetenschappelijk patterns of waxes in Neisseriuceae. J. Gen. Microbiol. Onderzoek for several research and personnel grants. This 102:3343. investigation was supported in part by a grant from the Deutsche 11. Carter, G. R. 1984. Pasteurella 1887, 94AL, Forschungs-Gemeinschaft to W.M. R.R. is indebted to the Instituut Genus I. Trevisan Nom. cons. Opin. 13, Jud. Comm. 1954, 153, p. 552-557. In tot Aanmoediging van het Wetenschappelijk Onderzoek in N. R. Krieg and J. G. 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