MOLECULAR GENET/CS OF

Molecular genetics of Azorhizobium phenotypic and genotypic studies on tropical leading to the characterization of

M. Gillis 1, and iL Garcia 2 and B. Dreyfus 3

dy (10): it appeared to occupy a separate a homogeneous phenon, distinct from the 1. Introduction position in rRNA superfamily IV. other two, although a bit closes to the lat­ ter. The genus Rhizobium (which means ety­ The aim of our work was to determine the mologically that which lives in the roots') exact taxonomie structure and status of the The fast growing root-nodulating strains was created in 1889 (7) for those stem-nodulating strains and to from tropical Acacia and Leucaena spe­ which nodulate the roots of leguminous reveal their closest relatives by a polypha­ cies belong together with the Sesbania plants, wherein these bacteria live as endo­ sic approach, involving different modem root-nodulating strains in the Rhizobium symbionts. methods allowing differentiation on diffe­ cluster in which we distinguish 4 subphe­ rent taxonomie levels. The methods used na. Further studies with more tropical rhi­ were: numerical of phenotypic zobia (unpublished results from K. Since then, their classification has been features, comparison of the SDS gelelec­ Kersters and B. Dreyfus) confirm and changed considerably from a system based trophoretic wholecell protein patterns, to­ even extend this heterogeneity since more mainiy on plant cross inoculation groups tal DNA-DNA hybridizations, strains were found in the 4 subphena but (8,13) to a scheme based on results invol­ DNA-rRNA hybridizations and determi­ also new subphena were detected. The ving large parts if not total bacterial ge­ nation of their % (G+C). Bradyrhizobium cluster contains 2 sub­ nomes (2, 9, 10). In the first edition of Bergey's Manual of Systematic Bacterio­ phena. The features differentiating the The results lead to the proposai of a new stem-nodulating strains (Azorhizobium) logy, Jordan (14) distinguishes within the genus for the Sesbania root- and stem- no­ from Rhizobium and Bradyrhizobium were rhizobia two genera, Rhizobium and Bra­ dulating bacteria Azorhizobium; this new dyrhizobium with respectively 3 and 1 deterrnined. The most striking presence of genus contains one Azorhizobium one lateral flagellum when grown in liquid species. The comparison of ribosomal caulinodans. RRNA'S by cataloguing (18) or DNA­ medium, their colony morphology, their rRNA hybridizations (3, 6, 10) has proven generation time, the lack of sugar assimi­ to be an excellent tool to study intergene­ n. Phenotypic n~ults lation (except glucose). Moreover the Ses­ rie and even more remote relationships of bania stem-nodulating bacteria con fix N2 bacteria. DNA-rRNA hybridizations have A total of 20 strains isolated from stem in culture under microaerobic conditions been used to unravel in more detail the nodules of S. rostrata were compared by and grow at the expense of this fixed N2 inter- and intrageneric relationships with methods of numerical taxonomy with 9 (4). This seems to be (together with the and within Rhizobium and Bradyrhizo­ fast growing rhizobia strains isolated from stem-nodulating capacity) an important bium (10). Both genera belong in rRNA root nodules of different Sesbania species, discriminative character between Azorhi­ superfamily IV, but are further removed with 20 other strains of the genus Rhizo­ zobium, Rhizobium and Bradyrhizobium, from each other than they are from other bium and with 17 strains of the genes Bra­ although sorne strains belonging to Rhizo­ genera in this rRNA superfamily. Rhizo­ dyrhizobium. The latter groups contained bium have been described as showing bium is more closely related to Agrobacte­ both own isolates from different Acacia sorne degree of ex-planta nitrogen-fixing rium; Bradyrhizobium to Rhodopseudo­ species and from Leucaena leucocephala ability (1, 15). When compared with the monas palustris and Nitrobacter. R. loti from Senegal. We compared 221 charac­ stem-nodulating Sesbania strains the ni­ occupies a separate position and the taxo­ ters including 151 sole carbon sources. trogenase activity and oxygen tolerance nomie position of R. fredii and rhizobia The results of the complete linkage cluster are the highest for the latter strains (30 isolated from Galega species has been de­ analysis (Figure 1) reveal three clusters nmol of C2H2 produced per mg of protein termined. One stemnodulating Sesbania corresponding to Rhizobium, Bradyrhizo­ per min, and an oxygen tolerance up to 9 strain was preliminary included in this stu- bium and Azorhizobium, which constitutes nmol).

( 1) Labol'atorium 1'001' Microbiologie, Rijksuniversiteit Gent, B-9000, Belgium. (2) Laboratoire de Microbiologie, ORSrOM, Université de Provence, 13331, Marseille, France. (3) Laboratoire de Biologie des Sols, ORSrOM, Dakar, Senegal and Laboratol'ium l'(}OI' Genetica, Rijksunil'el'siteit Gent, B-9000 Gent, Belgium.

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The results are shown in a Tm(e) dendro­ 0050 0100 0.150 0.200 gram (Figure 2), representing part of the lM) B81 rRNA superfamily IV. OlO Olt ORSill ORS 107 ORS il' ORS t05 The most useful and significant parameter ORS "~ ORStoe of a DNA:rRNA hybrid is its Tm(e)-vaIUe QIilSlIl0 0115 tOI (3, 6, 10, 17), because this parameter is a ORS "2 f------RHIZOBIUM------. B" measure of the base sequence similarity ORS .10 ­ORS 001 between rRNA cistrons and has a decisive ~ ~ __r+---"------, ORS'" taxonomic significance. Our results show ORSto2 ORS 103 0115101 that the Sesbania stem-nodulating bacteria 00503 00..2 constitute a separate rRNA subbranch on ORS~I 0.522 the Rhodopseudomonas palustris-Brady­ :::: ==Jt-....;4:.- ---' ORS ~57 rhizobium rRNA branch. Four repre­ OllS .S) OllSS" sentative strains constitute a very narrow ORS HI OllS'" 0llS_ cluster (Tm(e) from 80.8 to 81.6 C) be­ 0llS ..' 0llS SM 0llS!IM AZORHIZOBIUM CAULlNOOANS -----, longs also on this subbranch, and this ge­ OllS ". OllSm nus is thus the closest relative of the 0llS MS OllSHl Sesbania stem-nodulating strains. OllSHJ OllS'" 0llS S70 I:=~ ORS 511 . These strains cannot be included in Xan­ Olt! 5" OllS 102 thobacter because a difference in Tm(e) of 1 ORS 101 ORS 6001 CI ,.. -.,1-t--1 4 C can indeed reflect an intergeneric rela­ ORS 407 OAS 101 tionship, provided that sufficient phenoty­ ORS 101 ORS lOt pic arguments are available to differentiate 00500) BRADYRHIZOBIUM------l frlIlG 2 IIYAII4 both genera. Since this condition was in­ liT. WJI 1 USD'" 1)5 deed fulfilled we consequently proposed a 1 usa.. ,. OllS lOI GJ separate genus rank for the Sesbania stem­ liTA ".

1 nodulating strains. At the moment of this conclusion only 2 species were described Fig. J. Dendrogram showing the results ofthe complete linkage cluster analysis ofphenotypic features. in (16) and we made our dif­ ferentiating table [Table 4 in (5)] accor­ All strains from the Azorhizobium cluster probes available in our research group. ding to the available data. Recently (Il), a formed effective stem and root nodules. The results (10) located ORS 22 and ORS third species X. agilis has been described Among the Sesbania root-nodulating Rhi­ 52 in the Rhizobium-Agrobacterium rRNA and X. flavus was emended (12). The type zobium strains, 2 strains formed pseudono­ complex in rRNA superfamily IV. Strains strain of X. agilis has since then also been dules on the stems of Sesbania rostrata. hybridized with the [3 H]-rRNA probe Two strains (ORS 609 and ORS 611), iso­ ORS 571 was located at the bifurcation from strain ORS 571, it has a Tm(e) value lated from root nodules of S. cannabina level (70.5 C) of the Bradyrhizobium-Rho­ of 77 C, indicating that it is also a member and S. grandiflora were capable to form dopseudomonas palustris rRNA branch of the Xanthobacter rRNA cluster. When effective nodules on both roots and stems, and the Beijerinckia rRNA branch. Theo­ the phenotypic results of the revised genus but did not fix N2 in culture. Both strains retically the other taxa from this Tm(e) le­ were not included in the phenotypic analy­ Xanthobacter were compared with these sis but our genotypic results indicated that vel can be more closely related to strain of the Sesbania stem-nodulating cluster, they were authentic rhizobia. ORS 571: Methylobacterium, Xanthobac­ we still found enough features to differen­ ter, Rhodopseudomonas acidophila and tiate both genera. The revised differentia­ Rhodopseudomonas viridis. In order to un­ ting table will be published elsewhere (M. ill. DNA:rRNA Gillis, l.L. Garcia and B. Dreyfus, manu­ ravel these relationships, we prepared a n.~ults script in preparation). hybridization eH]-labelled rRNA probe from strain ORS 571 and hybridized it with DNA's Initially, we hybridized DNA from repre­ Strains ORS 609 and ORS 611 have Tm(e) from other members of the stem-nodula­ sentative strains belonging to the Rhizo­ values of 79.7 C versus the rRNA probe bium phenon (ORS 52 and ORS 22) and ting Sesbania phenon and with DNA's from R. meli/ori NZP 4009 showing that the Sesbania stem-nodulating phenon from the above mentioned possibly related they are members of the Rhizobium-Agro­ (ORS 571) with 3 appropriate rRNA bacteria. bacterium rRNA complex.

- 20 - MOLECULAR GENET/CS OF AZORHIZOBIUM

N their closest relative, from which they U Ü ~ ~If) are phenotypically sufficiently diffe­ :::J ~ ~ ~z rent to deserve a separate generic rank. :::J :::J CD CD- O:::J --J

We thank A. Bianchi from the Laboratoire d'Ecologie et Biochimie microbienne du Fig. 2. Simplified Tm(e) cislron simiLCIritv dendrogram ofpari ofl'RNA supeifamil\" IV. Milieu marin, Centre National de la Re­ cherche Scientifique, Marseille, France, search is necessary to unravel their re­ for the computer analysis. We are indebted IV. Comparative sels gel­ lationships. to 1. De Ley, K. Kersters and M. Van electrophoresis of whole-eell '" 2.We propose a new genus and new Montagu for support and encouragement. proteins and DNA-DNA species for the stem-nodulating Sesba­ nia strains because the DNA:rRNA hybridiultions hybridization results show clearly that This investigation was partly supported by these strains constitute a separate grant N TSD-081-F from the European rRNA subbranch and do not belong in Economic Community and by grant Four strains of the stem-nodulating Sesba­ Rhizobiul11 nor in Brad\'rhizobium. 600/83 from the North Atlantic Treaty Or­ nia strains have almost identical protein Xa/1fhobaeter (3 species) appears Lo be ganisation. electrophoregrams (Figure 3) indicating that they constitute indeed a very homoge­ neous cluster. High percentages of total ORS 609 ] DNA:DNA binding (95%) were indeed ORS 6" RHIZOBIUM sp. found between representative strains of ORS 51 the Sesbania stem-nodulating bacteria ORS 591 ] showing that these strains belong genoty­ ORS 592 AZORHIZOBIUM CAUUNODANS ORS 590 pically in one species. ORS S71 T T LMG 7043 JXANTHOBACTER AUTOTROPHICUS V. Conclusions LMG 7044 LMG 7045T

'" l.Tropical rhizobia are heterogeneous Fig. 3. Nort/wli:ed SDS-polracn1al1lide gel eLecrrophorelic pa/lerns offour A:orhizobiwn and more phenotypic and genotypic re- cau!inoda/ls slmins. lhree Rhizobium sp. a/ld three Xa/llhobaclCl' sll'aim.

- 21 - MOLECULAR GENET/CS OF AZORHIZOBIUM

ribosomal ribonuc1eic acid cistrons. Int. J. 13. Jordan, DoC. and D.N. Allen. 1974. Fa­ References Syst. Bacteriol. 27: 222-240. mily III. Rhirobiaceae Conn 1938, p. 261-267. ln R.E. Buchanan & N.E. Gibbons (ed.), eight 1. Bender, Gregory L., Jacek Plazinski, and 7. Frank, B. 1989. Ueber die Pilzsymbiose edition of Bergeys manu al of determinative Barry G. Rolfe, 1986. Asymbiotic acetylene der Leguminosen. Ber. Deut. Bot. Ges. 7: 332­ bacteriology. The Williams & Wilkins Co., reduction by fast-growing cowpea Rhizobium 346. Baltimore. strain with nitrogenase structural genes located on symbiotic plasmid. App!. Environ. Micro­ 8. Fred, E., I.L. Baldwin, and E. McCoy. 14. Jordan, DoC. 1984. Family III. Rhizobia­ bio!. 51: 86S-S71. 1983. Root nodule bacteria and leguminous ceae Conn 1935, p. 234-244. ln N.R. Krieg plants. University of Wisconsin Studies in and J.G. Holt (ed.), Bergey's manual of syste­ 2. Crow, VoL., B.D.W. Jarvis, and R.M. Science, Number 5. University of Wisconsin matie bacteriology, vol. 1. The Williams & Greenwood. 19S1. Deoxyribonuc1eic acid ho­ Press, Madison. Wilkins Co., Baltimore. mologies among acid-producing strains of 15. Urban, James E., Lawrence C. Davis Rhizobium. lnt. J. Syst. Bacteriol. 31: 152-172. 9. Hollis, A.B., W.E. Kloos and G.H. Elkan. and Susan J. Brown. 1986. Rhizobium trifolii 1981. DNA:Dl'iA hybridization studies of Rhi­ 0403 is capable of growth in the absence of 3. De Ley, J., W. Mannheim, P. Segers, A. zobium japonicum and related Rhizobiacaea. combined nitrogen. Appl. Environ. Microbiol. Lievens, M. Denijn, M. Vanhoucke and M. 1. Gen. Microbiol. 123: 215-222. Gillis. 1987. Ribosomal ribonuc1eie acid cis­ 52: 1060-1067. tron similarities and taxonomie neighborhood 10. Jarvis, B.D.W., M. Gillis, and J. De Ley. of Brucella and CDC group Vd. 19H7. lnt. J. 16. Wiegel, J.K.W., and H.G. Schlegel. 1986. Intra- and intergeneric similarities be­ Syst. Bacteriol. 37: 35-42. 1984. Genus Xanthobacter Wiegel, Wilke, tween ribosomal ribonuc1eic acid cistrons of Baumgarten, Opitz and Schlegel. p. 325-333. Rhizobium and Bradvrhizobium species and 4. Dreyfus, B.L., C. Elmerich, and Y.R. ln KR. Krieg and J.G. Holt (ed.). Bergeys sorne related bacteria. Int. 1. Syst. Bacteriol. Dommergues. 1983. Free-living Rhizobium manual of systematic bacteriology, vol. 1. The 36: 129-138. strains able to grow on l'i2 as the sole nitrogen Williams & Wilkins Co., Baltimore. source. Appl. Environ. Mierobiol. 45: 711­ 713. 11. Jenni, B., and M. Aragno. 1987. Xantho­ 17. Willems, A., M. Gillis, K. Kersters, L. bacter agilis sp. nov., a motile, dinitrogen­ Van Den Broecke, and J. De Ley. 19H7. 5. Dreyfus. B.JoL. Garcia, and Mo Gillis. fixing hydrogen-oxydizing bacterium. System. Transfer of Xanthomonas ampelina Panago­ 1988. Characterization of Arorhizobium cauli­ App!. Microbiol. 9: 254-257. poulos 1969 to a new genus, Xvlophilus gen. nodans gen. nov .. sp. nov., a stem-nodulation nov., as Xvlophilus ampelinus (Panagopoulos nitrogen-fixing bacterium isolated from Sesba­ 12. Jenni, B., and M. Aragno, and J.K.W. 1969) comb. nov. lnt. J. Syst. Bacteriol. 37: nia rostrata. Int. J. Syst. Bacteriol. 38: S9-98. Wiegel. 1987. Numerical analysis and Dl'iA­ 422-430. DNA hybridization studies on Xanthobacter 6. De Smedt, J. and J. De Ley. 1977. Intra­ and emendation of Xanthobacter flavus. Sys­ 18. Woese, C.R. 1987. Bacterial evolution. and intergeneric similarities of Agrobacterium tem. Appl. Microbiol. 9: 247-253. Microbio!. Rev. 51: 221-271.

- 22- Gillis M., Garcia Jean-Louis, Dreyfus Bernard. Molecular genetics of Azorhizobium phenotypic and genotypic studies on tropical rhizobia leading to the characterization of Azorhizobium caulinodans. In : Sesbania rostrata. Wageningen (NLD), Dakar : CTA, ORSTOM, 1989, p. 19-22.

Sesbania rostrata : Congrès International, 1989, Dakar