Expression by Bacteria of Nodulation Genesfrom Rhizobium ... Gram-negative, rod-shaped bacteria fromthe

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  • APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1989, p. 1426-1434 Vol. 55, No. 6 0099-2240/89/061426-09$02.00/0 Copyright © 1989, American Society for Microbiology

    Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii

    B. D. W. JARVIS,* L. J. H. WARD, AND E. A. SLADE

    Department of Microbiology and Genetics, Massey University, Palinelston Nor-th, Newv Zealaknd

    Received 13 December 1988/Accepted 28 February 1989

    Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr5l4a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosa- rum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.

    The genus Rhizobiuim is "characteristically able to invade the root hairs of temperate zone and some tropical zone leguminous plants (family Leguminosae) and incite the pro- duction of root nodules wherein the bacteria occur as intracellular symbionts. . . which are normally involved in fixing atmospheric nitrogen" (25). Thus, nodulation is re- garded as the characteristic function of Rhizobilam species and nitrogen fixation as a normal but not essential conse- quence of nodulation. This definition of the genus Rhizobinm is unsatisfactory because it usually depends on the presence of a Sym plasmid which can be lost in strains maintained on laboratory media (25). It would be preferable to define rhizobia in terms of DNA-DNA or rRNA-DNA homology to accepted reference bacteria.

    Primary isolation of Rhizobium strains is usually from nodulated legumes (31, 38, 47, 50, 51). We believe that this has tended to obscure relationships between rhizobia and other soil bacteria. Strains isolated from nodules on red (Trifoliium pratense) and white (Trifoliium repens) clover are all classified as Rhizobium leguminosariitm biovar trifolii (25), but they exhibit considerable genotypic (6, 21) and phenotypic (51, 52) diversity. Jarvis et al. (21) and Crow et al. (6) used recognized inoculum strains as sources of reference DNA and found values for optimal DNA homol- ogy and divergence in related sequences consistent with the presence of subspecies or perhaps multiple species by ac- cepted criteria (2, 23). Young and co-workers (51, 52) used enzyme polymorphism to show the existence of a number of genetically distinct lineages among R. legiuminosarumn bio- vars viceae, trifolii, and phaseoli in soil. They further showed that distinct lineages could carry any of several different host-range determinants. They postulated that soil might also contain bacteria which were electrophoretically indistinguishable from rhizobia but did not contain a Sym plasmid and were therefore unrecognizable as rhizobia by conventional methods (51).

    * Corresponding author.

    Numerous reports have shown that Sym plasmids can be transferred between Rhizobiium strains in the laboratory. Some have involved mobilization by cointegrate formation (27, 40), others self-transmissible Sym plasmids (10, 19, 24, 28). It has also been shown that nodule formation but not nitrogen fixation can be expressed on legume roots by Sym plasmids in Agrobacteriium species (19, 27, 32), Pseiudomo- nas aeruiginosa, and Ligniobacter species (33). The effects of Sym plasmid transfer to Rhizobiium sp. have

    been studied in relation to nodulation specificity (10, 12, 19, 24, 28), nitrogen fixation (19, 24, 28), competitiveness (4), and symbiotic efficiency (7, 45). Several aspects of Sym plasmid expression in R. legliminosarimn are partly depen- dent on chromosomal factors contributed by the recipient bacteria. These include rhizosphere competitiveness, nodu- lation competitiveness (4), the onset of nitrogen fixation, the carbon cost of nitrogen fixation, and the host plant biomass resulting from the symbiosis (45).

    In view of these considerations, it was decided to examine soil for the presence of free-living Rhizobilln-related bacte- ria unable to nodulate white clover and to examine the expression of symbiotic genes from R. leguminosarum bio- var trijolii in these bacteria.

    MATERIALS AND METHODS Bacterial strains and plasmids. The bacteria and plasmids

    used in this study are listed in Table 1. Isolation of soil bacteria. Soil cores 70 to 100 mm long were

    collected, placed in plastic bags, and stored at 4°C until they were cultured. Samples were diluted in 0.05% tryptone broth and immediately placed on yeast-mannitol-glucose (YMG) agar (47) or on soil extract (SE) agar (34). Cultures were incubated for 5 to 7 days at 28°C. Isolated colonies were restreaked on YMG or SE agar and reincubated. The OR strains were gram-negative rod-shaped bacteria from these plates. The NR strains were gram-negative rod-shaped bac- teria from Rhizobiium sp.-like colonies grown on YMG agar. Cultures were maintained on YMG at 4°C or as cell suspen-


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    TABLE 1. Bacteria and plasmids used in this study

    Bacterial strains and plasmids Characteristics" Source or reference

    Rlhizobilon legiuminosairim biovar trifolii

    ANU843 ICMP2668 (NZP582)


    NZP561 PN100 PN 165

    Escherichia c oli ATCC 9637

    HB101 RR1 PN200 PN291 PN362 PN600 PN298

    Soil bacteria NR41

    NR64 OR168

    Spontaneous antibiotic- resistant dervatives

    BJ1 BJ2 BJ3 BJ4

    Transconjugant bacteria SJ20 to SJ25 SJ1 to SJ6 SJ30, SJ32, SJ33 SJ40, SJ41 SJ43, SJ44

    Plasmids pBR328 R68.45 pRK290 pRK2013 pRtrSl4a


    pPN1 pLAFR1 pPN26

    Nod' Fix' on white and subterranean clovers Nod' Fix'


    Nod' Fix' Nod+ Fix+ Str-1 Rif-1 Sym plasmid-cured derivative of NZP561

    Source of nonrhizobial DNA for colony hybrid- izations and dot blots

    pro leiu thi lacY Str-1 recA r m pro lel thi lacY Str-1 r- m- HB101(pPN1) RR1(pRt572) HB101 (pRK2013) HB101(pPN26) HB101(R68.45)

    Gram-negative rod from a Rhizobiltin-like colony on YMG agar

    As for NR41 Gram-negative rod

    NR41Rif` NR64 Rif OR168 Rif PN165 Rift

    Rif Kmr Rif' Kmr Rif' Kmr Rif' Kmr Rif' Kans

    Ap' Tc' Cm' Km" Tc' Cb' IncP Derivative of RK2 Tcr Km" Nod' symbiotic plasmid from R. legium7inosaruim

    biovar trifolii NZP514 R. legiuminosarulm biovar trifolii ANU 843 7.2-kb EcoRl niod fragment cloned in pBR328

    pRtrSl4a: :R68.45 Acos derivative of pRK290 pLAFR1 cosmid containing niod region of PN100

    41 DSIR culture collection (Palmerston North, New

    Zealand) isolated from white clover inoculated with ICMP2153 (NZP560)

    DSIR culture collection 41 DSIR culture collection Spontaneous mutant of NZP514 (41) 35

    Massey University culture collection (Palmerston North, New Zealand)

    40 1 35 37, 43 D. B. Scott D. B. Scott D. B. Scott

    This work

    This work This work

    This work This work This work This work

    This work (BJ1 x This work (BJ2 x This work (BJ3 x This work (BJ3 x This work (BJ3 x

    PN200) PN200) PN200 nodule 1) PN200 nodule 2) PN200 nodule 2)

    1 16 8 8 45

    37, 42, 43

    41 14 42

    " Abbreviations: Ap, ampicillin; Cm, chloramphenicol; Km, kanamycin; Rif. rifampin: Tc, tetracycline.

    sions in TY broth (0.5% tryptone, 0.3% yeast extract, 0.13%Y CaCl, * 6H,O) containing 50% glycerol at -20°C.

    Nodulation tests. Nodulation was tested on sterile white clover seedlings (T. repens cv. Grasslands Huia) growing on Thornton agar with trace elements (17) in the enclosed culture method described by Vincent (47). In initial screen- ing experiments, two seedlings were inoculated with each soil bacterium and seedlings inoculated with R. legiumlinosa- ru(m biovar trifjoii ICMP2668, TAl, or ATCC 10004 and Escherichia coli W were used as positive and negative controls, respectively. In transconjugation experiments, three seedlings were used for each transconjugant or transconjugation mixture and for each donor and recipient strain. Strain ICMP2668 was used as a positive control. All tubes were inserted in wooden blocks and grown under

    controlled environment conditions at 22°C under artificial light (180 W/m2) with a 12-h photoperiod. Plants were examined for nodule number, shoot length, and color at weekly intervals.

    Isolation of bacteria from nodules. For isolation of bacteria from nodules, the methods described by Vincent (47) were used. DNA preparation. Genomic DNA was isolated by a mod-

    ification (40) of the me