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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1993, p. 1310-13170099-2240/93/051310-08$02.00/0Copyright 1993, American Society for Microbiology
Correlative Association between Resident Plasmids and theHost Chromosome in a Diverse Agrobacterium
Soil PopulationHACENE BOUZAR,lt* DJAOUIDA OUADAH,1 ZOULIKHA KRIMI,' JEFFREY B. JONES,2
MAURIZIO TROVATO,3t ANNIK PETIT,3 AND YVES DESSAUX3
Institut d'Agronomie, Universite des Sciences et Technologies de Blida, 09000 Blida, Algeria'; Gulf CoastResearch and Education Center, University ofFlorida, Bradenton, FL 342032; and Institut des Sciences
Vegetales, Centre National de la Recherche Scientifique, F-91198 Gif-sur-Yvette, France3
Received 4 January 1993/Accepted 25 February 1993
Soil samples collected from a fallow field which had not been cultivated for 5 years harbored a populationofAgrobacterium spp. estimated at 3 x 107 CFU/g. Characterization of 72 strains selected from four differentisolation media showed the presence of biovar 1 (56%) and bv. 2 (44%) strains. Pathogenicity assays on fivedifferent test plants revealed a high proportion (33%) of tumorigenic strains in the resident population. Alltumorigenic strains belonged to bv. 1. Differentiation of the strains by restriction fragment length polymor-phism analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cellular proteins, and utilizationpatterns of 95 carbon substrates (Biolog GN microplate) revealed a diversified bv. 1 population, composed offive distinct chromosomal backgrounds (chr A, C, D, E, and F), and a homogeneous bv. 2 population (chr B).chr A, B, C, and D were detected at similar levels throughout the study site. According to opine metabolism,pathogenicity, and agrocin sensitivity, chrA strains carried a nopaline Ti plasmid (pTi), whereas chr C strainshad an octopine pTi. In addition, four of six nontumorigenic bv. 1 strains (two chr D, one chr E, and one chrF) had distinct and unusual opine catabolism patterns. chr B (bv. 2) strains were nonpathogenic andcatabolized nopaline. Although agrocin sensitivity is a pTi-borne trait, 14 chr B strains were sensitive to agrocin84, apparently harboring a defective nopaline pTi similar to pAtK84b. The other two chr B strains wereagrocin resistant. The present analysis of chromosomal and plasmid phenotypes suggests that in thisAgrobacterium soil population, there is a preferential association between the resident plasmids and theirbacterial host.
Agrobacterium species are gram-negative soil bacteriabelonging to the family Rhizobiaceae (29). This genus iscomposed of at least three basic chromosomal groups whichhave been allocated the rank of biovar (29). Each biovar isidentified by an array of biochemical and physiological tests(36). Some Agrobacterium strains can induce a neoplasticdisease, known as crown gall, on a large number of plantfamilies (15). This tumorigenic ability is borne on largeextrachromosomal DNA elements, called Ti plasmids (pTi),which are transferable by conjugation to nontumorigenicAgrobacterium strains (56). During the infection process, aspecific DNA fragment (the T-DNA) is transferred from thepTi to wounded plant cells and integrated into the nucleargenome (12). Transfer of T-DNA is directed by a set ofvirulence (vir) genes located in the nontransferred region ofthe pTi (54). The T-DNA carries both oncogenes, whichperturb plant cell division (1, 50), and genes coding for thesynthesis of low-molecular-weight compounds, termedopines, specifically metabolized by the tumor-inducing bac-terium for its growth (4, 44). Some opines also induceconjugal transfer of the pTi to nontumorigenic agrobacteria(46). However, different opines are synthesized in tumorsincited by different pTi, and induction of conjugation is
* Corresponding author.t Present address: Gulf Coast Research and Education Center,
University of Florida, 5007 60th St. E., Bradenton, FL 34203.Present address: Dipartimento di Genetica e Biologia Molecu-
lare, Universita di Roma "La Sapienza," 00185 Rome, Italy.
relatively specific. Octopine induces conjugation by strainscarrying an octopine-type pTi, whereas nopaline strains arenot induced to conjugate by octopine or even nopaline but byanother opine, agrocinopine A, which is also present intissue transformed by T-DNA of nopaline-type pTi. Thisselective opine induction contributes to the propagation ofpathogenicity genes and, consequently, plays a subtle role inthe ecology of this bacterium and the epidemiology of thedisease it causes (16).Crown gall infection may have significant detrimental
effects on the host plant, generating major economic losses(25, 37, 51). To evaluate the potential for crown gall epidem-ics and the possible efficacy of control measures such as theintroduction of antagonists (e.g., strain K84 ), moreinformation on the behavior of this bacterium and of its pTioutside the tumor environment is needed. At present, infor-mation on survival potential and genetic stability of tumori-genic agrobacteria in the soil environment is, at best, limited.This paucity of information is due in part to the raredetection of tumorigenic strains in the soil and on roots, eventhough nontumorigenic agrobacteria are commonly found (2,7, 10, 35, 52). These apparently low levels of tumorigenicbacteria in soil are in sharp contrast to the regular occur-rence of crown gall epidemics and the efficacious protectionagainst soil infections by the biocontrol agent K84 (5, 35).The need for a better understanding of the ecology of
agrobacteria and resident pathogenic plasmids led us toexamine the composition of an Agrobacterium soil popula-tion and the type of plasmids associated with the hostbacterium. This bacterial population was indigenous to a soil
Vol. 59, No. 5
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CORRELATIVE ASSOCIATION BETWEEN PLASMIDS AND CHROMOSOME 1311
which was fallow for 5 years after 2 years of stone-fruit treenursery production.(A portion of this work was presented at the Annual
Meeting of the American Phytopathological Society, St.Louis, Mo. .)
MATERIALS AND METHODS
Isolation and culture of bacteria. Bacteria were isolatedfrom soil samples collected at the experiment station of theInstitut Technique d'Arboriculture Fruitiere et de Viticul-ture located in the Mitidja plain (Algeria). Four samplingsites were randomly chosen from a plot (32 by 38 m).Samples for analysis were taken at a soil depth of 5 to 10 cm.Soil analysis revealed a loamy texture and an alkalinereaction (pH 8.5). From each sample, 1.0 g of soil wassuspended by shaking in 10 ml of sterile distilled water, andthe suspension was allowed to settle for 5 min. Tenfold serialdilutions were made, and a 100-pl aliquot of selected dilu-tions was spread onto duplicate plates containing the follow-ing media: 1A (selective for bv. 1) (9), 2E (selective for bv.2) (9), RS (selective for bv. 3) (48), and MG (nonselective)(24). Plates were incubated for 5 days at 27C before colonycounts were made. From each soil sample, 10 coloniesresemblingAgrobactenum spp. (36) were selected from eachmedium and purified by successive streakings on potatodextrose agar (PDA; Difco Laboratories, Detroit, Mich.)supplemented with 0.05% (wt/vol) calcium carbonate andKing's medium B (30). Gram-negative strains (20), nonfluo-rescent on King's medium B, were stored at 4C on PDA-CaCO3 slants and in sterile distilled water (36).
Bacterial identification. The biovar affiliation of each puta-tive Agrobacterium strain was determined by classical bio-chemical and physiological tests (36). To determine thetaxonomic structure of this Agrobactenum community be-low the biovar level, subpopulations were differentiated bymorphology of colonies growing on PDA-CaCO3 at 27C,profiles of cellular proteins separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE),and profiles of HindIII- and BamHI-digested genomic DNAfrom individual strains. In addition, strains representative ofeach subpopulation were compared by their ability to utilizeas a sole carbon source 95 substrates from the Biolog GNmicroplates (Biolog Inc., Hayward, Calif.); incubation, in-oculation, data acquisition, and analysis were done as de-scribed in the manufacturer's recommendations. Utilizationprofiles were compared by using a newly developed Agro-bacterium data base (6a) and subjected to cluster analysis byusing the Biolog MLCLUST program for phenotypic group-ing.
Cellular proteins were extracted from bacteria grown tothe stationary phase in 1.5 ml of AT minimal medium (45)supplemented with mannose (2.0 g/liter), ammonium sulfate(2.0 g/liter), and yeast extract (0.1 g/liter). One milliliter ofbacterial suspension was transferred to a microcentrifugetube, and cells were recovered by centrifugation for 2 min at13,500 x g. Bacteria were washed once with 0.9% NaCl andtreated with 100 ,ul of lysis buffer (31) for 5 min at 100C.Five microliters of lysate was electrophoretically separated(35 mA for 5 h) in a 12% acrylamide gel, and proteins werevisualized by using Coomassie blue staining.Genomic (total) DNA was extracted as described previ-
ously (18), endonuclease digested, and separated in an 0.8%agarose gel by using standard procedures (49). Total DNArestriction patterns were visualized by ethidium bromidestaining. When DNA hybridization was required, digested
genomic DNA was transferred to nylon membranes (Hy-bond N+; Amersham Co., Arlington Heights, Ill.) as de-scribed in the manufacturer's instructions. Chromosomalbackgrounds were compared by using a phosphomannoseisomerase (PMI) probe and a proline utilization (Put) probe.Both probes were cosmid clones selected from a genomicDNA library of the bv. 1 Agrobacterium strain C58. PMIconsisted of the labeled pCD1 clone