JOURNAL OF BACTERIOLOGY, Apr. 1972, p. 256-261 Vol. 110, No. 1 Copyright © 1972 American Society for Microbiology Printed in U.S.A. Deoxyribonucleic Acid Base Sequence Homologies of Some Budding and Prosthecate Bacterla RICHARD L. MOORE' AND PETER HIRSCH2 Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48823 Received for publication 21 December 1971 The genetic relatedness of a number of budding and prosthecate bacteria was determined by deoxyribonucleic acid (DNA) homology experiments of the di- rect binding type. Strains of Hyphomicrobium sp. isolated from aquatic habi- tats were found to have relatedness values ranging from 9 to 70% with strain "EA-617," a subculture of the Hyphomicrobium isolated by Mevius from river water. Strains obtained from soil enrichments had lower values with EA-617, ranging from 3 to 5%. Very little or no homology was detected between the amino acid-utilizing strain Hyphomicrobium neptunium and other Hyphomi- crobium strains, although significant homology was observed with the two Hyphomonas strains examined. No homology could be detected between pros- thecate bacteria of the genera Rhodomicrobium, Prosthecomicrobium, Ancal- omicrobium, or Caulobacter, and Hyphomicrobium strain EA-617 or H. nep- tunium LE-670. The grouping of Hyphomicrobium strains by their relatedness values agrees well with a grouping according to the base composition of their DNA species. It is concluded that bacteria possessing cellular extensions repre- sent a widely diverse group of organisms. Two genera of bacteria, Hyphomicrobium drum, P. pneumaticum, Ancalomicrobium adetum, and Rhodomicrobium, are listed under the and Caulobacter crescentus were obtained from J. T. family of Hyphomicrobiaceae in the seventh Staley (Seattle); Rhodomicrobium vannielii was re- edition of Bergey's Manual of Determinative ceived from H. C. Douglas (Seattle). Hyphomi- Bacteriology. Both genera are represented by a crobium sp. ZV-580 came from G. A. Zavarzin (Mos- cow), and strain KB-677 from T. Y. Kingma-Boltjes single species only. However, in recent years a (Amsterdam). Hyphomicrobium neptunium was a large number of new and related genera have gift of E. Leifson (Chicago), and the two Hypho- been described (1, 26, 27). Several new strains monas strains were obtained from E. Pongratz of hyphomicrobia and Rhodomicrobium sp. (Geneva). All other strains were selected from the have been isolated from various habitats (5, 9, culture collection of one of the authors (P.H.). 10, 24). The biochemical, morphological, and R. vannielii was grown as described by Duchow fine-structural characteristics of many of these and Douglas (7). The remaining cultures were incu- strains have been investigated in some detail bated in the various media listed (Table 1) at 30 C by Hirsch (manuscript in preparation). The on a gyratory shaker. Before entering the stationary present investigation has been undertaken to phase of growth, cells were harvested by centrifuga- tion (16,000 x g, 5 C, 20-40 min), washed twice with extend these studies through the examination 0.1 M ethylenediaminetetraacetate and 0.15 M NaCl, of genetic relatedness among selected isolates pH 8 (saline-EDTA), and stored frozen until used. of prosthecate bacteria by use of deoxyribonu- DNA preparation. DNA samples were obtained cleic acid (DNA) homology techniques. by the method of Marmur (17), with an additional incubation at 37 C for 1 hr with 100 gg of self-di- MATERIALS AND METHODS gested Pronase per ml (Calbiochem, Los Angeles, Bacteria and media. Prosthecomicrobium enhy- Calif.) after the digestion step with ribonuclease A (50 gg/ml; Worthington Biochemical Co., Freehold, I Present address: Division of Pathology, Faculty of Med- N.J.). icine, University of Calgary, Alberta, Canada. Before the addition of 1% sodium lauryl sulfate 2 Present address: Institut fiir Allgemeine Mikrobiologie (SLS) for lysis, Rhodomicrobium, A. adetum, and der Universitlit, 23 Kiel, West Germany. the Hyphomicrobium strains isolated from aquatic 256 VOL. 110, 1972 DNA HOMOLOGIES IN BUDDING BACTERIA 257 habitats (groups I and II, Table 2) required incuba- homology experiments. This yielded single strands tion with 100 Mg of lysozyme (Calbiochem) per ml for with a size of about 300,000 daltons. 1 hr at 37 C. DNA-DNA duplex formation. The various DNA Best DNA yields from the soil hyphomicrobia species were heat-denatured and immobilized on (group III, Table 2) were obtained by washing the nitrocellulose membrane filters (B-6 coarse, C. saline-EDTA from the cells with 0.01 M tris(hydrox- Schleicher and Schuell Co., Keene, N.H.) by the ymethyl)aminomethane-hydrochloride, pH 9, and method of Gillespie and Spiegelman (8). The suspending them in 0.15 M NaCl and 0.015 M sodium amount of DNA bound to the filters was determined citrate (SSC), pH 7.2, prior to treatment with lyso- from absorbancy measurements at 260 nm of the zyme. Afterwards, the cells were digested with DNA solutions before and after passage through the Pronase (100 jg/ml, 37 C, 8 hr) in the presence of filters. Filters with a diameter of 4.8 mm were 0.2% SLS. The concentration of SLS was then in- punched from larger filters and treated with Den- creased to 1%, and DNA was prepared from the hardt's preincubation mixture (6). These filters were lysate as described above. incubated for 12 hr at 70 C in 0.25 ml of SSC with Tritium-labeled DNA was prepared from refer- tritium-labeled DNA from Hyphomicrobium EA-617 ence strains Hyphomicrobium EA-617 and H. nep- (522 counts per min per Mg) or H. neptunium LE-670 tunium LE-670 after growth in media containing 10 (1,200 counts per min per ug). The ratio of tritium- Mg of 3H-adenine per ml (6.0 Ci/mmole) and 2 Mg of labeled DNA to filter-bound DNA was 5 to 1. After 3H-thymidine per ml (6.7 Ci/mmole; New England incubation, the filters were rinsed with SSC at 70 C, Corp., Boston, Mass.). These DNA species were di- dried, and counted in a Beckman LS-100 liquid scin- luted with the respective nonlabeled DNA species, tillation counter. The values given were corrected for heat-denatured in 0.01 x SSC, and sheared in a the background counts obtained with filters con- French pressure cell at 15,000 psi before use in DNA taining Pseudomonas aeruginosa DNA. The amount TABLE 1. Culture media used for the growth of test strains Organism Medium Reference Rhodomicrobium vannielii Rhodomicrobium medium with 0.2% (7) sodium lactate Prosthecomicrobium enhydrum 0.1% yeast extract, 0.1% glucose, 20 J. T. Staley, personal com- ml of modified Hutner's basal salts munication and (4) per liter Prosthecomicrobium pneumaticum 0.1% Casamino Acids, 0.1% glucose, J. T. Staley, personal com- 20 ml of modified Hutner's basal munication and (4) salts per liter, 10 ml of vitamin mixture per liter Anacalomicrobium adetum 0.2% mannitol, 0.01% peptone, 0.01% (4, 27) yeast extract, 0.025% (NH4) 2SO4, 20 ml of modified Hutner's basal salts per liter, 10 ml of vitamin mixture per liter Caulobacter crescentus 0.2% peptone, 0.1% yeast extract, (25) 0.02% MgSO4-7H2O, 0.2% glucose Amino acid-utilizing hyphomicrobia 0.2% Casitone, 0.1% yeast extract, (14) (group IV, Table 2) 0.1% MgCl2 Hyphomicrobia isolated from soil 0.5% methylamine hydrochloride in (11) (group III, Table 2) medium 337 with the amount of Na2HPO4 reduced to 1.13 gfliter, and one-fifth the amount of trace lement solution Hyphomicrobium sp. 0.5% methylamine hydrochloride in (11) strains NB-762 and medium 337 with 1.05 g of NaNO, NM-765 per liter in place of (NH4) ,SO, All other hyphomicrobia Medium 337 plus 0.5% methylamine (11) hydrochloride 258 MOORE AND HIRSCH J. BACTERIOL. TABLE 2. Duplex formation between the DNA species of two Hyphomicrobium strains and DNA species from various hyphomicrobia and other prosthecate bacteria Hyphomicrobium Hyphomicrobium strain EA-617 neptunium LE-670 Base corn- Organism DNA per 'H counts/ 'H counts/ filter (Mg) mn per Per centofmper (molesposition% DNgof Per c g of Per6n0 G+C) filter- EA67 filter- L-7 DNA DNA I. Hyphomicrobium strain EA-617 5.92 38.2 100 0.93 1 66.3a NQ-521 5.34 38.2 100 1.20 1 66.8 NQ-521gr 3.54 38.4 101 1.20 1 66.8 NQ-528 3.41 37.3 98 0 0 - WH-563 5.63 38.8 102 1.91 2 66.8 MEV-533 5.56 26.1 68 1.89 2 65.8 MEV-533 gr 5.31 26.8 70 0.66 1 66.3 II. Hyphomicrobium strain NB-762 3.51 9.97 26 0.85 1 64.3 NM-765 3.97 7.15 19 2.16 2 64.3 ZV-580 2.84 4.19 11 0 0 63.8 KB-677 3.28 3.93 10 0 0 63.8 MY-618 6.55 3.50 9 0 0 62.7 III. Hyphomicrobium strain B-522 3.46 1.01 3 0 0 60.2 C-523 6.03 1.74 5 0 0 60.2 D-524 3.65 1.81 5 0 0 60.2 E-525 1.75 1.04 3 0 0 60.2 G-527 7.43 1.13 3 0 0 60.2 H-526 3.74 1.23 3 0 0 60.2 K-529 2.26 1.23 3 0 0 61.2 L-530 3.95 1.47 4 0 0 59.2 IV. Hyphomicrobium neptunium LE-670 5.19 0.39 1 93.9 100 61.7 Hyphomonas polymorpha PR-727 3.19 0 0 37.8 40 61.2 PS-728 6.40 0 0 28.2 28 60.2 V.