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. Various other prosthecate bacteria: Rhodomicrobium vannielii 450 2.45 0 0 0 0 63.8b Prosthecomicrobium enhydrum 3.90 0 0 0 0 65.8c P. pneumaticum 3.58 0 0 0 0 69.4c Ancalomicrobium adetum 2.75 0 0 0 0 Caulobacter crescentus CB-2 2.10 0 0 0 0 67.0d aDeterminations reported by Mandel, Hirsch, and Conti (in press). " For determinations see reference 16. c For determinations see reference 27. d Determination by Mandel (personal communication). of DNA lost from the filters during incubation was by a 10-min incubation in 2 ml of 0.1 x SSC at each examined with the use of the unsheared, tritium-la- of the specified temperatures (20). beled test DNA species and found to be negligible. This is similar to previous results with Escherichia RESULTS coli DNA (8, 20). The thermal denaturation profiles were deter- The genetic relatedness among the various mined from the amount of tritium-labeled DNA prosthecate bacteria was determined from the eluted from the filter-bound DNA-DNA complexes extent to which the denatured DNA of these VOL. 110, 1972 DNA HOMOLOGIES IN BUDDING BACTERIA 259 organisms reannealed with the separated . strands of radiolabeled DNA from Hyphomi- 100-01 AO crobium EA-617 or H. neptunium LE-670 la (Table 2). With the use of such direct binding 0 )o 80. measurements, four of the six Hyphomi- E P4 crobium strains of group I were found to be .4 identical to strain EA-617. The extent of cross- reaction between the DNA of strain EA-617 0- 601 and strains MEV-533 and MEV-533gr was 68 0c and 70%, respectively. Physiological differ- C I ences between these subcultures and EA-617 *-. EA-617 40 0-0 NQ-521gr have also been observed (Hirsch, unpublished 0 4 data). U 1 O-ONQ*521 1 ,&-AWH -563 Other hyphomicrobia isolated from aquatic 0 0 4A-A MEV-533gr habitats (group II, Table 2) gave values ranging 20~ 0-0 MEV-533 0- M from 9 to 26% with strain EA-617. Lower A. +-+ NQ- 528 values ranging from 3 to 5% were obtained ^_ ~~~~~~~~~~~~~~~~I**-P.aeruginosa I I I I with DNA species from hyphomicrobia iso- 60 70 80 90 100 lated from soil (group III). Temperature [CO] The base compositions (moles per cent G + C) of the DNA species from these soil strains FIG. 1. Thermal stabilities of the duplexes formed were determined by Mandel, Hirsch, and between the DNA of Hyphomicrobium EA-617 and Conti (in press) to be lower than those of the that of closely related strains. Filters containing DNA from the various bacterial strains were incu- aquatic strains but similar to the amino acid- bated for 12 hr at 70 C in 0.2 ml of SSC containing 4 utilizing organisms of group IV. The amino gg of tritium-labeled DNA from strain EA-617 (522 acid-utilizing strains appeared to be only very counts per min per ug). The DNA-DNA duplexes distantly related to the aquatic hyphomicrobia were then disassociated by heating in 0.1 x SSC. of groups I and II, and they were not detect- ably related to the soil forms of group III. The largest degree of homology with DNA of H. neptunium LE-670 was obtained with the two DISCUSSION Hyphomonas strains, which had values of 28 The function of the cellular appendages of and 40%, respectively. these bacteria is still uncertain. Poindexter No base sequence homology was observed (25) has suggested that they act by retarding between the DNA species of prosthecate bac- the sedimentation rate of the organism, thus teria from the other genera studied and either helping to keep it at an optimal location in its one of the reference DNA species used. aquatic habitat. Pate and Ordal (23) have Thermal stability measurements of DNA- stressed the importance of an increase in sur- DNA or ribonucleic acid (RNA)-DNA com- face membranes which could enhance the plexes have been useful in determining phylo- ability of the bacterium to take up nutrients genetic relationships among the enterobacteria present in low concentrations. A correlation (2, 3, 20), Neisseria (13), and halobacteria (21). has also been observed between stalk forma- The mid-point of the thermal denaturation tion and the normal process of reproduction in profiles (Tm,i) of DNA-DNA duplexes between Caulobacter sp. (25), or between the formation Hyphomicrobium EA-617 DNA and the DNA of a hypha and the budding process in Rho- species of closely related strains was found to domicrobium (22) and hyphomicrobia (10, 14). be 78 C in 0.1 x SSC (Fig. 1). This is the ex- One of these possibilities, or some combination pected result for duplexes of this base compo- of them, could be assumed to apply in the case sition with a high degree of base pairing and of a particular prosthecate bacterium. What- indicates that the conditions used for rean- ever additional functions eventually may be nealing were optimal (12). The similarity of assigned to these organelles, it appears likely the various profiles supports the conclusion that survival of many different kinds of bac- made from direct binding measurements, that teria is enhanced by the formation and modifi- these DNA species show a high degree of base cation of such appendages. Thus, one might sequence homology to the DNA of strain EA- expect present-day prosthecate species to be of 617. The thermal stability profile obtained very diverse origin. with P. aeruginosa DNA is shown for compar- The results reported here indicate a wide ison. diversity in the base sequence of the DNA spe- 260 MOORE ANq]D HIRSCH J. BACTERIOL. cies from the various prosthecate bacteria. ACKNOWLEDGMENTS Even within the genus Hyphomicrobium, in We express our appreciation to N. Band and R. Costilow which common ancestry of the strains investi- (East Lansing) for the use of a French pressure cell, a Beckman scintillation counter, and a Beckman DU spectro- gated seems likely, only a low degree of base photometer. Continuous support by P. Gerhardt and many sequence homology exists. A barely observable helpful discussions with R. Brubacher (East Lansing) are level of relatedness, or none at all, was seen also gratefully acknowledged. between aquatic isolates or soil isolates and This work was supported by postdoctoral fellowship 1 F02 GM30669-01 from the National Institute of General those hyphomicrobia utilizing amino acids. A Medical Sciences to R. L. Moore, and by research grant no. computerized analysis of biochemical and GB-7403 from the National Science Foundation to P. morphological characteristics of these and Hirsch. other Hyphomicrobium strains has also sug- Agricultural Experiment Station article no. 5684. gested the existence of several discrete groups of strains (species) within the genus (Hirsch, manuscript in preparation). These groups LITERATURE CITED in the 1. Aristovskaya, T. V. 1961. The accumulation of iron ac- agreed well with the grouping suggested companying the decomposition of organomineral present study. complexes of humus substances by microorganisms. The usefulness of DNA base compositions in Dokl. Akad. Nauk SSSR 136:954-957. bacterial taxonomy has been discussed by 2. Brenner, D. J., and D. B. Cowie. 1968. Thermal stability of Escherichia coli-Salmonella typhimurium deoxy- Marmur et al. (18) and more recently by ribonucleic acid duplexes. J. Bacteriol. 95:2258-2262. Mandel (15). The arrangement of the Hypho- 3. Brenner, D. J., G. R. Fanning, K. E. Johnson, R. V. Ci- microbium strains into groups based upon tarella, and S. Falkow. 1969. Polynucleotide sequence DNA homology values produced groups of relationships among members of Enterobacteriaceae. Al- J. Bacteriol. 98:637-650. similar DNA base composition (Table 2). 4. Cohen-Bazire, G., W. R. Sistrom, and R. Y. Stanier. though the G + C contents of the various hy- 1957. Kinetic studies of pigment synthesis by non- phomicrobia used in this study ranged from sulfur purple bacteria. J. Cell. Comp. Physiol. 49:25- 59.2 to 66.8%, the maximum difference within 68. % G C. The DNA 5. Conti, S. F., and P. Hirsch. 1965. Biology of budding a single group is 2 moles + bacteria. m. Fine structure of Rhodomicrobium and base compositions of strains from the other Hyphomicrobium sp. J. Bacteriol. 89:503-512. genera of prosthecate bacteria are in the same 6. Denhardt, D. T. 1966. A membrane-filter technique for general range as those of the genus Hypho- the detection of complementary DNA. Biochem. Bio- (16, 27; Mandel, Hirsch, and Conti, phys. Res. Commun. 23:641-46. microbium 7. Duchow, E., and H. C. Douglas. 1949. Rhodomicrobium in press). vannielii, a new photoheterotrophic bacterium. J. In addition to similarities in DNA base Bacteriol. 58:409-416. composition, other resemblances also exist 8. Gillespie, D., and S. Spiegelman. 1965. A quantitative between and the genera of assay for DNA-RNA hybrids with DNA immobilized hyphomicrobia on a membrane. J. Mol. Biol. 12:829-842. prosthecate bacteria discussed here. R. vannie- 9. Hirsch, P. 1968. Biology of budding bacteria. IV. Epicel- 1ii, originally described by Duchow and lular deposition of iron by budding, aquatic bacteria. Douglas (7), is morphologically, structurally, Arch. Mikrobiol. 60:201-216. similar to certain Hyphomi- 10. Hirsch, P., and S. F. Conti. 1964. Biology of budding and biochemically bacteria. I. Enrichment, isolation and morphology of crobium strains (5). For this reason, Hyphomi- Hyphomicrobium spp. Arch. Mikrobiol. 48:339-357. crobium has been referred to as the colorless 11. Hirsch, P., and S. F. Conti. 1964. Biology of budding counterpart of R. vannielii (28). Despite these bacteria. II. Growth and nutrition of Hyphomicrobium no base sequence ho- spp. Arch. Mikrobiol. 48: 358-367. similarities, however, 12. Johnson, J. L., and E. J. Ordal. 1968. Deoxyribonucleic mology was discernible between the reference acid homology in bacterial taxonomy: effect of incu- DNA of two representative forms (EA-617 and bation temperature on reaction specificity. J. Bac- LE-670) and that of R. vannielii. There is still teriol. 95:893-900. some 13. Kingsbury, D. T., G. R. Fanning, K. E. Johnson, and D. the possibility, though, that homology J. Brenner. 1969. Thermal stability of interspecies might have been observed between the DNA of Neisseria DNA duplexes. J. Gen. Microbiol. 55:201- R. vaniellii and one of the strains of group II, 208. in which the DNA base compositions are more 14. Leifson, E. 1964. Hyphomicrobium neptunium sp. n. like that of R. vaniellii than those of the refer- Antonie van Leeuwenhoek J. Microbiol. Serol. 30:249- 251. ence strains used. 15. Mandel, M. 1969. New approaches to bacterial tax- These results indicate that the development onomy: perspective and prospects. Annu. Rev. Micro- of cellular appendages may have been a mul- biol. 23:239-274. event. Differences noted in size, fine 16. Mandel, M., E. R. Leadbetter, N. Pfennig, and H. G. tiple Truper. 1971. Deoxyribonucleic acid base composi- structure, and the mode of formation of these tions of phototrophic bacteria. Int. J. Syst. Bacteriol. appendages lend support to this conclusion (5, 21:222-230. 25, 27). 17. Marmur, J. 1961. A procedure for the isolation of deoxy- VOL. 110, 1972 DNA HOMOLOGIES IN BUDDING BACTERIA 261

ribonucleic acid from microorganisms. J. Mol. Biol. 3: 157-167. 208-218. 23. Pate, J. L., and E. J. Ordal. 1965. The fine structure of 18. Marmur, J., S. Falkow, and M. Mandel. 1963. New ap- two unusual stalked bacteria. J. Cell. Biol. 27:130- proaches to bacterial taxonomy. Annu. Rev. Micro- 133. biol. 17:329-372. 24. Pfennig, N. 1969. Rhodopseudomonas acidophila, sp.n., 19. Mevius, W., Jr. 1953. Beitrage zur Kenntnis von Hy- a new species of the budding purple nonsulfur bac- phomicrobium vulgare Stutzer et Harleb. Arch. Mik- teria. J. Bacteriol. 99:597-602. robiol. 19:1-29. 25. Poindexter, J. S. 1964. Biological properties and classifi- 20. Moore, R. L., and B. J. McCarthy. 1967. Comparative cation of the Caulobacter group. Bacteriol. Rev. 28: study of ribosomal ribonucleic acid cistrons in entero- 231-295. bacteria and myxobacteria. J. Bacteriol. 94:1066-1074. 26. Pongratz, E. 1957. D'une bacterie pediculee isolee d'un 21. Moore, R. L., and B. J. McCarthy. 1969. Base sequence pus de sinus. Schweiz. Z. Allg. Pathol. Bakteriol. 20: homology and renaturation studies of the deoxyribo- 593-608. nucleic acid of extremely halophilic bacteria. J. Bac- 27. Staley, J. T. 1968. Prosthecomicrobium and Ancalomi- teriol. 99:255-262. crobium: new prosthecate freshwater bacteria. J. Bac- 22. Murray, R. G. E., and H. C. Douglas. 1950. The repro- teriol. 95:1921-1942. ductive mechanism of Rhodomicrobium vannielii and 28. Van Niel, C. B. 1954. The chemoautotrophic and photo- the accompanying nuclear changes. J. Bacteriol. 59: synthetic bacteria. Annu. Rev. Microbiol. 8:105-132.