INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1986, p. 241-245 Vol. 36, No. 2 0020-7713/86/020241-05$02.00/0 Copyright 0 1986, International Union of Microbiological Societies

Deoxyribonucleic Acid Homologies of Hyphomicrobium spp. Hyphomonas spp. and Other Hyphal, Budding MINER GEBERS, BEATE MARTENS, UTA WEHMEYER, AND PETER HIRSCH* Institut fur Allgemeine Mikrobiologie, Universitat Kiel, 0-2300 Kiel, Federal Republic of Germany

The levels of genetic relatedness of 19 Hyphomicrobium strains which utilize one-carbon compounds were determined by deoxyribonucleic acid (DNA)-DNA hybridization in solution under optimal conditions (S1 nuclease technique). Most of these hyphomicrobia fell into four groups with high levels of relatedness (level of homology within each group, 86 to 110%). These groups were only distantly related to each other (levels of homology between groups, 1 to 9%). Three additional groups of C1-utilizing hyphomicrobia were represented by only one strain each. In addition, the levels of DNA-DNA homology of four Hyphomonas and 11 Hyphomicrobium-Hyphomonas-likeisolates were determined. Seven of these isolates formed three groups containing two or three strains each; the level of homology within each group was 94 to 120%. These groups of peptide-utilizing strains were related to Hyphomonas spp. at a DNA-DNA homology level of 13 to 43 % ;thus, they represented new species of Hyphomonas. Four of the isolates had less than 10% DNA homology with either Hyphomicrobium reference strains or Hyphomonas spp. Strain B-1408 was distinguished by its DNA base composition of 46.19 mol% guanine plus cytosine, which is 14 mol% below the average base composition of Hyphomicrobium spp. or Hyphomonas spp. The levels of genetic relatedness of other hyphal, budding bacteria, such as Pedomicrobium spp., F, Rhodomicrobium vannielii, and genus T, to Hyphomicrobium sp. strain MC-750 were too low to be evaluated by DNA-DNA hybridization techniques.

The genus Hyphomicrobium was described in 1898 as the when they were compared with Hyphomonas polymorpha first of a group of hyphal, budding bacteria (35). Later, PS-728T (T = type strain), Hyphomonas neptunium morphologically similar organisms belonging to the genera LE-670T, or Hyphomonas jannaschiana VP-1382, which Rhodomicrobium (3, Hyphomonas (29), and Pedo- were previously shown to be genetically related to other microbium (1, 2) were discovered. While Rhodomicrobium Hyphomonas species (9). spp. were clearly distinguished by their ability to produce Additionally, we determined the genetic relatedness of 19 photosynthetic pigments (5, 23, 28), the remaining three C1-utilizing Hyphomicrobium strains, which split into seven genera could not be differentiated with certainty by morpho- groups, corresponding to previous results (10, 21, 26). logical and physiological properties (6, 28). However, The DNAs of representative strains of Pedomicrobium Pedomicrobium spp. could be distinguished from other spp., Rhodomicrobium vannielii, and two new genera, pre- budding and hyphal bacteria by means of deoxyribonucleic liminarily designated genera F and T (lo), were hybridized acid (DNA) base composition and nucleotide distribution (7, with Hyphomicrobium sp. strain MC-750 DNA to confirm lo), genome size (19), and DNA-DNA homology (8, 9). The that they are distinct from the C1-utilizing hyphomicrobia. differentiation of Hyphomicrobium spp. from Hyphomonas spp. by means of morphology, DNA base composition (9, MATERIALS AND METHODS 10,21), nucleotide distribution (lo), and genome size (19,27) Strains and cultivation. All of the bacterial strains which we was still afflicted with uncertainty. However, DNA-DNA used were supplied by the culture collection of the Institut fur hybridization experiments (9, 26) performed with 20 Allgemeine Mikrobiologie, Kiel, Federal Republic of Ger- Hyphomicrobium strains and 3 strains of Hyphomonas spp. many (Tables 1 and 2). Strains SCH-1427, SCH-1495, and revealed 2% homology, at most. Ribosomal ribonucleic SCH-1497 were isolated from brackish water by H. acid-DNA hybridization between Hyphomicrobium strain Schlesner, Kiel. Strains SCH-1427 and SCH-1495 were B-522 and three Hyphomonas strains detected high thermal grown in medium 387+1/4 ASW (10). Strains SCH-1497 and denaturation (T,) values, which indicated distant related- B-1408 (strain B-1408 was isolated from brackish water by W. ness between these representatives of the two genera (25). Bockelmann, Kiel) were grown in PYGV medium (33) Unfortunately, the first description of Hyphomicrobium supplemented with 250 ml of artificial seawater per liter (20) vulgare (35) did not provide information about the utilization and 50 ml of Tris(hydroxymethy1)aminomethane (10) per of one-carbon compounds by this organism. On the other liter. Strain P-1258 was isolated from freshwater by J. S. hand, most of the isolates presently named Hyphomicrobium Poindexter, New York, N.Y., and was cultivated in PSM specialize on these compounds (11, 14, 16). Therefore, in medium (6). All of these strains were grown aerobically in the practice the term Hyphomicrobium has been applied to dark at 30°C. The origins and growth conditions of all of the Cl-utilizing strains, while Hyphomonas is used for amino other strains studied have been published previously (6, 10). acid utilizers (12, 28, 29). Since this way of distinguishing DNA preparation. The methods used for cell wall disinte- these genera has not been officially confirmed, we chose to gration, DNA extraction, and purification have been de- rely on DNA-DNA homology to determine the taxonomic scribed previously for most strains (8, 10). Strains positions of 11 Hyphomicrobium-Hyphomonas-likeisolates SCH-1427, SCH-1495, SCH-1497, and B-1408 were lysed by and two new Hyphomonas species (36). Seven of these enzyme treatment E (lo), while strain P-1258 was disinte- strains had levels of DNA homology of between 13 and 43% grated mechanically by the cell mill A procedure (10). All DNAs were sheared to an average double-stranded fragment * Corresponding author. M, of 420,000 to 450,000 (8).

24 1 242 GEBERS ET AL. INT.J. SYST.BACTERIOL.

TABLE 1. Levels of DNA-DNA homology of Hyphomicrobium spp. and other hyphal, budding bacteria Source of unlabeled DNA' DNAbase % Homology with labeled DNA from strain?

Hyphomicrobium spp. MC-750 27500 61.38' 100 4 3 2 0 A MEV -533gr 27488 64.69' 24 103 4 B EA-617 63.46' 20 110 2 B WH-563 63.09' 19 92 4 B NQ-521gr 27483 64.11' 18 100 9 5 0 B ZV -622 64.8d 8 100 0 C ZV -620 64.3d 100 C ZV -580 61.77' 14 91 5 C KB -677 27498 62.41' 12 3 4 4 0 D MC-651 27497 62.91' 11 D CO-582 27492 60.54' 11 1 2 87 0 E CO-559 60.02' 10 98 E CO-558 27491 59.78' 7 91 E 1-551 27489 59.40" 5 1 1 86 0 E F-550 59.91' 5 106 E H-526 27485 59.53' 5 88 E B-522 27484 59.34' 4 0 2 100 0 E T-854 57.88' 7 1 1 0 F Wi -926 59.29' 3 1 4 0 G New isolate SW-815 60.23' 5 0 1 1 101 New isolate SW-814 59.11' 5 100 New isolate SW -808 55.15' 3 1 0 Pedomicrobium ferrugineum P- 1196 33116 65.7 5 P-1225 33122 649 5 S - 1290T 33119 65.v 4 Pedomicrobium manganicum E- 1129T 33121T 65.6 5 Pedomicrobium-like ST-1306 65.00' 5 WD-1355 62.80' 5 869 64.41' 4 868 64.72' 3 Genus F SCH-1315 61.15' 3 Rhodomicrobium vannilii 117gT 17100T 62. 8g 2 DSM 162T Genus T 1300 62.82' 1 ST-1307 61.91' 1

~ a IFAM, Institut fur Allgemeine Mikrobiologie, Kiel, Federal Republic of Germany; DSM, Deutsche Sammlung von Mikroorganisrnen, Gottingen, Federal Republic of Germany; ATCC, American Type Culture Collection, Rockville, Md. Mean of at least two reactions corrected for the background values obtained with the self-reassociation controls. The self-reassociation values obtained by hybridization with unrelated E. coli K-12 DNA were as follows: strain MC-750,5.9%; strain NQ-521gr, 1.1%; strain ZV-622,0.7%; strain B-522, 1.2%; strain SW- 814, 1.3%. Data obtained by T, determinations (10). Data derived from buoyant density determinations (21). Data obtained by T, determinations (9). Data obtained by T, determinations (7). Data derived from buoyant density determinations (23).

DNA base composition. At least six T, profiles of each SCH-1416, and SCH-1417 was done by using the method of DNA species investigated were recorded at 260 nm with a Selin et al. (32) and Na1251(specific activity, 603 MBq/pg of Gilford model 250 spectrophotometer as described previ- iodine; Amersham Buchler). ously (7). From the T, values of these curves the molar DNA-DNA reassociation experiments. DNA-DN A reas- fractions of the DNA bases (guanine-plus-cytosine [G + C] sociation experiments were carried out under optimal con- contents) were calculated by using the following equation ditions in solution as described previously (8, 9). Unreacted (22): G+C content = (T, in 0.1X SSC/50.2) - 0.990. (IX DNA fragments were digested with S1 nuclease (4). Reas- SSC is 0.15 M NaCl plus 0.015 M sodium citrate.) sociated DNA strands were precipitated with 5.5% (wt/vol) DNA labeling. In vivo labeling of Hyphomicrobium sp. trichloroacetic acid. The hybridization conditions used are strain MC-750 DNA was achieved by adding (per milliliter of shown in Table 3. Nonspecific binding levels and amounts of exponentially growing culture) 118 kBq each of [methyl- available DNA were determined by using 150 pg of Esche- 3H]thymidine and [8-3H]adenine (specific activities, 684.5 richia coli K-12 DNA and the labeled DNAs. Radioactivity GBq/mmol and 925 GBq/mmol, respectively; Amersham was expressed as the percentage of available input (Tables 1 Buchler, Braunschweig, Federal Republic of Germany). and 2). All reactions were performed at least three times. Extracted, sheared DNA was further purified by Tritium-labeled hybrids were counted with a Packard Tri- hydroxyapatite column chromatography (3). In vitro iodina- Carb model 3390/544 liquid scintillation counter; iodine tion of the DNAs of strains NQ-521gr, B-522, ZV-622, decay values were determined with a Berthold model LE-670T, PS-728T, SW-814, SW-815, SCH-1325T, VP-1382, MAG315 gamma counter. VOL. 36, 1986 DNA HOMOLOGIES OF HYPHAL, BUDDING BACTERIA 243

TABLE 2. Levels of DNA-DNA homology of Hyphomonas spp. and other hyphal, budding bacteria Source of unlabeled DNA" % Homologv with labeled DNA from strainf

-V..'rV"...V.. strain (mol% G+C)b PS-728T LE-670' SCH-1416 VP-1382 SW-815 SW-814 SCH-1417 SCH-1325T MC-750

Hyphomonas PS-72gT 33881T 60.07" 100 10 10 3 1 0 i 1 polymorpha (28,21)' PR-727 33880 60.05" (Ill)" (40,20)e 2 Hyphomonas LE-670T 15444" 60.40" 6 100 27 2 4 0 4 1 neptunium (23)" New isolate SCH-1416 58.w 15 26 100 6 3 2 2 New isolate SCH-1497 58.94 13 27 94 1 3 0 3 Hyphomonas VP-1382 33882 6o.B" (29)" (15)" 1 100 25 3 2 jannaschiana New isolate SW-815 60.29 2 6 3 43 100 101 10 5 5 New isolate SW-814 59.11f 100 5 New isolate SX-821 59.66 2 7 7 38 96 98 6 7 New isolate SCH-1417 60.05" 0 9 2 20 13 100 3 New isolate SCH-1495 58.77 0 9 3 13 9 120 4 Hyphomonas SCH-1325T 33879T 58.98" (6)" (3)" 1 9 3 5 100 2 oceanitis New isolate SCH-1427 60.30 0 5 3 6 3 7 15 New isolate B-1408 46.19 0 3 2 0 0 0 0 New isolate P-1258 58.53 0 5 1 3 2 0 1 New isolate SW-808 55.15" 0 2 0 0 0 0 0 0 3 2.1 1.5 0.8 1.0 0.9 1.3 1.2 0.7 5.9

a IFAM, Institut fur Allgemeine Mikrobiologie, Kiel, Federal Republic of Germany; ATCC, American Type Culture Collection, Rockville, Md. Mean of at least six T, determinations calculated by using the following equation (22): G+C content = (T, in 0.1~SSCbO.2) - 0.99. Mean of at least two reactions corrected for the background value obtained with the self-reassociation controls. The self-reassociation values obtained by hybridization with unrelated E. coli K-12 DNA were as follows: strain PS-728', 2.1%; strain LE-670T, 1.5%; strain SCH-1416, 0.8%; strain VP-1382, 1.0%; strain SW-815, 0.9%; strain SW-814, 1.3%; strain SCH-1417, 1.2%; strain SCH-1325', 0.7%; strain MC-750, 5.9%. Data from reference 9. The homology values in parentheses are included for comparison. Data in parentheses from references 9 and 26. Data from reference 10.

RESULTS AND DISCUSSION 15, 21). These strains were 86 to 106% homologous to strain B-522 and were distantly related (4 to 11%) to strain MC-750. The C1-utilizing Hyphomicrobium strains which we stud- Both group D strains, strain KB-677, which was isolated ied showed 0 to 24 or 86 to 110% DNA-DNA homology to from sewage (18), and strain MC-651, which was isolated each other (Table 1). The hybridization experiments re- from soil (21), were about as closely related to strain MC-750 vealed that there were four groups of highly related strains, as group E hyphomicrobia were, but neither isolate had a which were interconnected at only low levels of homology. significant level of homology to strain B-522 or strain Group E consisted of soil hyphomicrobia which had been ZV-622. Therefore, they represent a separate group. isolated in the presence or absence of carbon monoxide (13, The strains of group C, which were isolated from swamp

TABLE 3. Conditions used for DNA-DNA hybridization reactions

Source of Sp act for Amt Of Ratio of Amt of sp act after hybridization labeled DNA labeled DNA Hybridization Incubation S1 Incubation Filter (CDm,Up of labeledDNA R~$~~elabeling (cpm/Fg Per of DNA) hybridizatibn to unlabeled temp ("C) time (h) nuclease temp ("C) type (strain) reaction (ugl DNA (U)

~ _. MC-750 3H 11,100 (13,400) 11,100 (13,400) 0.25 or 0.28 1:600 or 69 16 1.250" 51 GFIC~ (0.23) 1536 (1:652) NQ-521gr 12'1 6.71 X lo6 1.15 X lo6 0.10 1: 1,500 70 20 500' 56 BA83d B-522 1251 1.45 x lo6 1.45 x lo6 0.07 1:2,143 68 20 500' 54 BA83d ZV-622 1251 4.08 X lo6 1.33 X lo6 0.10 1: 1,500 69 20 500' 55 BA83" SW-814 1251 4.51 x lo6 1.35 X lo6 0.10 1:1,500 68 20 500' 54 BA83" PS-72gT 1251 5.2 x lo6 lo6 0.10 1:1,500 69 20 500" 55 BA83d LE-670T 1251 5.2 X lo6 lo6 0.10 1: 1,500 69 20 500' 55 BA83d SCH-1416 '*'I 7.3 x lo6 lo6 0.10 1:1,500 68 20 500' 54 BA83" VP-1382 1251 5.3 x lo6 lo6 0.10 1: 1,500 69 20 500' 55 BA83d SW-815 1251 6.0 X lo6 lo6 0.10 1: 1,500 69 20 500' 55 BA83d SCH-1417 1251 4.2 X lo6 lo6 0.10 1:1,500 69 20 500' 55 BA83" SCH-1325T 1251 5.4 x lo6 lo6 0.10 1:1,500 68 20 500' 54 BA83"

(I Obtained from Miles Laboratories, Inc., Elkhart, Ind. Obtained from Whatman, Maidstone, Kent, United Kingdom. Obatined from Sigma Chemie, Taufkirchen, Federal Republic of Germany. Obtained from Schleicher & Schuell, Dassel, Federal Republic of Germany. 244 GEBERS ET AL. INT. J. SYST.BACTERIOL. soil by G. A. Zavarzin (personal communication), were Although technical differences between the experiments almost identical to each other. They were only distantly (e.g., membrane filter technique versus S1 nuclease tech- related to strain MC-750 (strain ZV-580, 14%) and to the nique; DNA fragment M, of 600,000 versus DNA fragment other reference strains. The exposed surface antigens were M, of 420,000 to 450,000) reduced the comparability of the serologically identical (30). These three strains may be results, a fixed limit for generic relatedness of 20 or 25% descendants of one original isolate (37). DNA-DNA homology does not seem to be applicable to The group B strains were isolated from brackish water (13, these bacteria. Until stronger evidence calls for a change, we 17). They were homologous to representative strain NQ- recommend keeping strain LE-670T as Hyphomonas 521gr. This was not surprising, since strains NQ-521gr and neptunium. This was confirmed by levels of homology of 13 EA-617 were subcultures of the original strain B of Mevius to 27% between new isolates SCH-1416 and SCH-1497 and (24) and were maintained over a period of 16 years by both Hyphomonas type strains (strains PS-728 and LE-670). different investigators. Strains SCH-1416 and SCH-1497 were 94% homologous to Obviously, these cultures did not suffer dramatic changes each other and thus may represent a new Hyphomonas in their DNA base sequences during subculturing, but the species. serological relationship of strains NQ-521gr and MEV-533gr Strains SW-815 and SX-821 were 43 and 38% homologous, was completely lost (30). However, previously published respectively, to Hyphomonas jannaschiana VP-1382 and data on the levels of DNA-DNA homology between strain were completely homologous to each other and to strain EA-617 and strain NQ-521gr (101%) and between strain SW-814. These three strains, which were isolated from EA-617 and strain MEV-533s (70%) differed by 30%; these brackish water (13), were only 5% homologous to values were calculated from filter hybridization data by Hyphomicrobium sp. strain MC-750 (Table 1); they may Moore and Hirsch (26). constitute an additional new species of Hyphomonas. Strains MC-750, Wi-926, and T-854 were not sufficiently Homologous strains SCH-1417 and SCH-1495 shared 20 homologous to any other hyphomicrobia to be included in and 13% DNA homology, respectively, with Hyphomonas one of the four groups. jannaschiana VP-1382, as well as 13 and 9% DNA homol- Supported by the results of previous studies on DNA base ogy, respectively, with strain SW-815 and 9% DNA homol- composition (21) and nucleotide distribution (lo), on DNA- ogy with Hyphomonas neptunium LE-670T. Although these DNA homology (26), and on ribosomal RNA-DNA related- homology values were below the arbitrary limit of 20 or 25% ness (25), our results suggest that the C1 utilizing for generic relatedness (31,34), we suggest that both of these Hyphomicrobium strains should be separated according to strains should be added to the genus Hyphomonas. the group designations (groups A through G) (Table 1). The levels of DNA homology between Hyphomonas Numerical studies (P. Hirsch and R. R. Colwell, oceanitis SCH-1325T and other Hyphomonas strains were unpublished data) revealed that strain MC-750 is the less than 10% (Table 2). Nevertheless, we suggest keeping hyphothetical median organism of 84 Hyphomicrobium this strain as a Hyphomonas species, because its morphol- strains and is the strain that is most similar to the original ogy, physiology, and lysing behavior of the cell envelope description of Hyphomicrobium vulgare (35). If levels of suggest some relatedness to Hyphomonas (36). DNA-DNA homology of 20 or 25% were used as the minimal Strain SCH-1427 was 15% homologous to Hyphomonas values for relatedness on the generic level (31, 34), none of oceanitis SCH-1325T and less than 8% homologous to all our groups B through G could be classified as other reference DNAs; thus, its taxonomic position remains Hyphomicrobium spp. Furthermore, most of the presently unclear. known C1-utilizing hyphomicrobia would have to be de- Red-pigmented strain B-1408 was clearly different from all scribed as members of new genera. However, with our other strains studied on the basis of its DNA base composi- still-limited knowledge of the relatedness of hyphal and tion (46.19 mol% G+C) (Table 2). Such a low G+C content budding bacteria and with more than 80 intensely investi- is quite unusual for the hyphal, budding bacteria which we gated strains still awaiting proper classification, we suggest studied; on@ orange-red-pigmented strain SCH-1415, which some caution in applying generic limits to hyphal, budding was investigated previously (lo), had a similar value (46.34 bacteria by using definitions employed for different bacterial mol% G+C). Most likely, both of these strains belong to a groups. From a practical point of view, we recommend new genus of pigmented, hyphal, budding bacteria. designating groups A through E (Table 1) as Unlike all Hyphomonas strains which we studied, the cell Hyphomicrobium species, to recognize their distant related- envelopes of strains P-1258 and SW-808 resisted enzymatic ness to this genus. Eventually further changes in their and detergent treatments, so the cells had to be disrupted by taxonomic rank (species designations) should await collec- grinding in a cell mill (10). Strain SW-808 was not homolo- tion of more comprehensive data. The proper classification gous to either the C1-utilizinghyphomicrobia (Table 1) or the of groups F and G will require further investigation. Hyphomonas reference strains (Table 2). The levels of DNA-DNA homology of Hyphomonas spp. Reciprocal hybridization reactions between the reference and some hew isolates (Table 2) which grew on media strains of the C1-utilizing hyphomicrobia and Hyphomonas containing peptides confronted us with the same questions spp. revealed different levels of DNA-DNA homology for concerning taxonomic ranking as discussed above. Re- each pair of strains depending on which genome carried the cently, Hyphomonas neptunium LE-670T was transferred radioactive label (Table 1). These deviations correlated to from the genus Hyphomicrobium to Hyphomonas (28) be- some extent with the different sizes of the genomes (19), so cause of its morphological and physiological characteristics that, if the smaller genome was labeled, it yielded high (9, 12, 28, 29) and levels of DNA-DNA homology (9, 26). homology values and vice versa. The reciprocal reactions Data from these previous homology studies were included between strain NQ-52lgr and strain ZV-622 gave almost for comparison in Table 2. The present hybridization exper- identical results (8 and 9% homology); since the genome size iments revealed 6 and 10% levels of homology between of strain ZV-580 was similar to that of strain NQ-521gr (19), DNAs of Hyphomonas polymorpha and Hyphomonas we assume that the genome size of strain ZV-622 also falls neptunium; the previous results were between 20 and 40%. into this range. VOL. 36, 1986 DNA HOMOLOGIES OF HYPHAL, BUDDING BACTERIA 245

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