A Survey of Agrobacterium Strains Associated with Georgia Pecan Trees and an Immunological Study of the Bacterium

AN ABSTRACT OF THE THESIS OF

Hacene Bouzar for the degree of Master of Science in Botany & Plant Pathology presented on December 17, 1982

Title: A SURVEY OF AGROBACTERIUM STRAINS ASSOCIATED WITH GEORGIA

PECAN TREES AND AN IMMUNOLOGICAL STUDY OF THE BACTERIUM

Abstract approved: Redacted for Privacy Larry W. Moore

Crown gall was found in numerous pecan orchards in Georgia. In some instances, 60% of the trees were diseased. Galled trees were less vigorous than noninfected trees. Among the pathogenic

Agrobacterium strains isolated from 18 galled trees from six counties, biovar 1 strains predominated and most were sensitive to agrocin 84 in vitro. A representative biovar 1 strain from pecan was inhibited from infecting tomato seedlings by A. radiobacter K84. We would expect that biological control of crown gallin Georgia pecan orchards with strain K84 would be successful.

Because the taxonomy of the members of the Rhizobiaceae has not been resolved by classical and modern methods of microbial classification, a serological analysis of the 50S ribosomal subunits was made. Antisera raised against 50S ribosomal subunits of five different agrobacteria were used to arrange 31 Agrobacterium strains and 6 Rhizobium strains into 16 serovars. The immunological relationships of 50S subunits of Agrobacterium did not confirm either of the major taxonomic groupings; fast-growing rhizobiacould not be

immunologically differenciated from the agrobacteria andwere closely related to the biovar 3 strain CG64. The five antisera were specific

at the family level.

The antigenic structure of 50S ribosomal subunits of tumorigenic

strain C58 and rhizogenic strain A4 were compared to that of their avirulent derivatives (NT1 and A4R1, respectively) and to strain A323

(A NT1 that was transformed with the large nopaline plasmid from K84) using Ouchterlony doubly diffusion tests. A unique precipitin band was observed with50S subunits of the virulent plasmid-containing strains; this band whichrevealedthepresence of an additional immunogenwas absent from 50S subunits oftheplasmid-deficient mutants. This unique band observed in 50S subunits of the virulent strain C58 was also present in the transformant A323, but was absent from both strainsNT1 andK84. Therefore, it appears thatthe combination of plasmid and chromosome can induce formation of new 50S ribosomal subunits which have a major immunological difference from

50S subunits of cells of the same chromosomal background but lacking the plasmid. A SURVEY OF AGROBACTERIUM STRAINS ASSOCIATED WITH GEORGIA PECAN TREES AND AN IMMUNOLOGICAL STUDY OF THE BACTERIUM

Hacene Bouzar

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of Master of Science

Completed December 17, 1982

Commencement June, 1983 APPROVED: Redacted for Privacy Professor of Bot0VY and Plant Pathology in charge of'Plant Pathology Major

Redacted for Privacy Chairman of Department of Botany and Plant Pathology

Redacted for Privacy

Dean of Gradua School dr.-

Date thesis is presented December 17, 1982

Typed by WORD PROCESSING SPECIALISTS for Hacene Bouzar ACKNOWLEDGEMENTS

Thanks aregiven tothe Algerian Ministere de L'Enseignement

Superieur et dela Recherche Scientifique for its financial support of this study.

I wish to sincerely thank Dr. Larry W. Moore for his encouragement, counsel, and generosity in the giving of his time.

I would alsolike to thank Dr.Norman W.Schaad and Ruth C.

Donaldson for sharing their knowledge and research techniques during my stay at the Georgia Experiment Station.

To my dear friends,both farandnear, whocared enoughto provide a great dealof moral support, I humbly say thank you. I would also like to express my appreciation to all the kind people at the Agricultural Research Laboratory of Corvallis for their support, especially Carol Bender, Don Cooksey, Phil Garrett, John Kough, and

Dave Schisler.

Most importantly, my parents and family are deserving of special thanks. I am deeply grateful for their love, patience, encouragement,and assistance,not only during the course of this study, but throughout my life.

This thesis is dedicated to myyoung cousin and friend,

Farid Hafiz, who left us without being understood and without understanding our love. TABLE OF CONTENTS

Page

INTRODUCTION 1

CHAPTER 1: CROWN GALL OF PECAN: A SURVEY OF AGRO- BACTERIUM STRAINS AND POTENTIAL FOR BIOLOGICAL CONTROL IN GEORGIA

Summary 3 Introduction 4 Materials and Methods 5 Results and Discussion 8 References 12

CHAPTER 2: IMMUNOLOGICAL HETEROGENEITY OF 50S RIBOSOMAL SUBUNITS FROM AGROBACTERIUM STRAINS

Summary 17 Introduction Materials and Methods 22 Results 25 Discussion 28 References 32

CHAPTER 3: IMMUNOLOGICAL DIFFERENCES BETWEEN 50S RIBOSOMAL SUBUNITS FROM AGROBACTERIA AND THEIR PLASMID-CURED DERIVATIVES

Summary 44 Introduction 45 Materials and Methods 47 Results 48 Discussion 51 References 54

SUMMARY AND CONCLUSIONS 60

BIBLIOGRAPHY 63 LIST OF FIGURES

Figure Page

Chapter 2

2.1 Ouchterlony double diffusion patterns of ribosomes 35 from different strains of Agrobacterium and Rhizobium tested against antiserum raised to 50S ribosomal subunits of A. tumefaciens B6

2.2 Ouchterlony double diffusion patterns of ribosomes 36 from different strains of Agrobacterium tested against antiserum to 50S ribosomal subunits of A. tumefaciens M63/79

2.3 Ouchterlony double diffusion pattern of ribosomes 37 from different species of the Enterobacteriaceae and Rhizobiaceae families tested against antiserum to 70S ribosomes of Escherichia coli

Chapter 3

3.1 Ouchterlony double diffusion pattern showing the 56 serological relationships of the tumorigenic strain M63/79 with the rhizogenic strain A4 and, the A4 mutant, A4R1 which lacks a major antigenic determinant

3.2 Serological relationship of 50S subunits from the 57 virulent wild type C58, its plasmid-deficient mutant NT1, the transformant A323, and K84

3.3 Ouchterlony double diffusion pattern of phenol- 58 extracted antigens tested against antiserum to 50S ribosomal subunits of C58 LIST OF TABLES

Table Page

Chapter 1

1.1 Number of Agrobacterium strains isolated from 14 pecan galls on three selective media

1.2 Number of Agrobacterium strains pathogenic and 15 sensitive to agrocin 84 produced by A. radiobacter K84

1.3 Biological control of representative Agrobacter- 16 ium tumefaciens strains on tomato seedlings by A. radiobacter K84

Chapter 2

2.1 Source and origin of bacterial strains used as 38 antigens

2.2 Serovars developed in Ouchterlony double diffu- 41 sion tests from reactions of antisera to 50S subunits from five Agrobacterium strains to ribosomes from 31 agrobacteria, 6 rhizobia, and 8 miscellaneous species

2.3 Distribution of the 16 serovars within the 43 different serological groups revealed by each antiserum

Chapter 3

3.1 Agrobacterium strains used as test-antigens 59 CONTRIBUTIONS OF AUTHORS

Dr. Schaad provided laboratory space, equipment, and some consultation for much of this study, because the survey was made in Georgia. Dr. Schaad helped me make contacts with the pecan orchardists, Extension Personnel, and provided transportation for the surveys. A SURVEY OF AGROBACTERIUM STRAINS ASSOCIATED WITH GEORGIA PECAN TREES AND AN IMMUNOLOGICAL STUDY OF THE BACTERIUM

INTRODUCTION

Crowngall, a disease caused by Agrobacterium tumefaciens, affectsmanypecan trees grown in Georgia. Becausebiological control of thepathogenby thebacteriocinogenic A. radiobacter strain K84 was reported to be unsuccessful on peach trees in Mary- land,Tennessee,and South Carolina (Alconero,1980); there was a need to determine whether thestrainsinfecting pecantrees were physiologically different from those reported by Alconero and whether they would be sensitive to agrocin 84.

The classical means for the identification of bacteria (mor- phological,biochemical,cultural,and pathological characters) are time consuming. In addition, these characters can be variable, which has caused some confusion in the classification of the agrobacteria

(Kerr, Young,and Panagopoulos, 1978; Holmes and Roberts, 1981). A quicker andmoredefiniteidentificationprocedurefortheagro- bacteriawould be valuable. Because, serology has been used successfully to identifyother bacteria, and recently a higher specific antiserum was produced when ribosomes from the phytopathogenic bacterium Xanthomonas vesicatoria were used as inject antigens (Schaad, 1976), it seemed worthwhile to examine serology as a diagnostic tool for identification of Agrobacterium strains.

The major objectiveof this study was todetermine whether antisera raised against 50S ribosomal subunits of Agrobacterium would be specific and allow rapid identification of unknown agrobacteria. 2

Because oncogenicity of the agrobacteria is coded for by genes on a large plasmid,a second objective was to see whether the presence or absence of these extrachromosomal elements would code for any immunologically different cell products. 3

CHAPTER 1

CROWN GALL OF PECAN: A SURVEY OF AGOBACTERIUM STRAINS AND POTENTIAL FOR BIOLOGICAL CONTROL IN GEORGIA

HACENE BOUZAR

Department of Botany and Plant Pathology Oregon State University, Corvallis, Oregon 97331

LARRY W. MOORE

Department of Botany and Plant Pathology Oregon State University, Corvallis, Oregon 97331

and

NORMAN W. SCHAAD

Department of Plant Pathology, Georgia Experiment Station, University of Georgia, Experiment, Georgia 30212

SUMMARY

Crown gall was found in numerous pecan orchards in Georgia. In

some instances, 60% of the trees were diseased. Galled trees were

less vigorous than noninfected trees and often were stunted. Among

the pathogenic Agrobacterium strainsisolated from 18 galled trees

fromsix counties, biovar 1 strains predominatedand most were

sensitive to agrocin 84 in vitro. A representative biovar 1 strain from pecan was inhibited from infecting tomato seedlings by A. radio-

bacter strain K84. We would expect that biological control of crown

gallin Georgia pecan orchards with strain K84 would be successful.

Additionalputative antagonists for biocontrol of crowngall were

isolated from a potentially suppressive soil located in a young peach orchard. 4

INTRODUCTION

Pecan [Carya illinoinensis (Wangenh) C. Koch ] sales in Georgia

average morethan$50 millionannually, andaccount for about 50

percent of the pecan production in the U.S.A. This production comes

from approximately two million bearing trees, with the highest

concentration of trees in Southcentral Georgia (Livingston and Bruce,

1979) where this study was done.

Rand (1914) first reported the occurence of crown gall onpecan

trees grown in anursery in Mississippi andin Northern Florida.

Today, crowngall is a major concernto many pecan growers whose

trees are afflicted by large and numerous tumors at the base of the trunk and in the root system. This paper reports the incidence of crown gall in the pecan growing counties of Georgia, the predominant biovar of Agrobacterium tumefaciens isolated from infected trees, and the sensitivity of these strains to agrocin 84 produced in vitro by

A. radiobacter K84. Such characterization is important to determine whether crown gallon pecan could be controlled with the bacteriocinogenic A. radiobacter strain K84 (New and Kerr, 1972). 5

MATERIALS AND METHODS

Agrobacterium spp.wereisolated from gallsamples obtained mostly from trees of 40 to 60 year old pecan orchards.The incidence of diseased treesin these orchards ranged from 25% to 60%. The galled trees appearedless vigorous than nongalled trees and were often stunted. The trees were produced in Florida and Mississippi nurseries on unspecified pecanroot stocks, graftedwitheither

Moneymaker, Schley, Stuart, or Teshe scions.

Galls from 18 trees were collected from orchardsin Mitchell,

Dougherty, Calhoun, Tift,and Baker counties. One gall sample from

Jefferson County came from young pecan trees which had been grown from nursery stock that weredipped three yearsbefore in the

bacteriocinogenic A. radiobacterK84 beforebeingplanted; these

treated trees were planted in soil where old pecan treeshad been

killed prior to replanting, reportedly from crown gall. To isolate

for A. tumefaciens the gall exteriors were washedthoroughly, and one

gram of living tissue was ground with amortar and pestle for two

minutes in 10 ml of sterile distilled water. The suspension was left

standingfor 30 minutes and 0.1 ml of selected 10-fold serial

dilutions spread onto the selective media of Schroth,et al. (1965),

New and Kerr (1971), and D1M (Kado, C.I. and M.G. Heskett--

unpublished) selective media,as described by Moore,Anderson, and

Kado (1980). The inoculated plates were incubated for one week at

28C. Fifteen colonies (five from each medium) resembling Agrobacterium were selected at random from each gall sample. After purification, the identity of each suspected strain of Agrobacterium was determined by the appropriate biovar 1 and 2 diagnostic tests

(Moore, Anderson, and Kado, 1980).

Sensitivityof the characterized strains to agrocin 84was tested on mannitol-glutamate agar plates following Stonier's method

(1960) as modified by Cooksey and Moore (1980). A 10 Jul sample of

107 CFU/ml suspension of A. radiobacter K84 was spotted in the center oftheagar plates. After two days ofincubationat 28C, a108

CFU/ml suspension of the strain to be tested was sprayed over the plates. Growthinhibitionofthetest strain was recorded after three additional days of incubation.

Pathogenicity tests wereperformed onstemsof four-week-old seedlings of Datura stramonium and tomato (Lycopersicon esculentum

'Tampa')(Anderson and Moore,1979). Tumor formation was recorded two months after inoculation. Biocontrol by K84 of two representative A. tumefaciens strains from pecan was tested on wounded tomato stems. One strain was a biovar 2 agrocin-resistant strain GA003, and the other was a biovar 1 agrocin-sensitivestrain GA1010. A. tumefaciens strains B6 (Dr. R. Baker,Colorado State University,

Colorado) (biovar 1 andagrocin-resistant)and K24 (Dr. A. Kerr,

UniversityofAdelaide, South Australia) (biovar 2 and agrocin- sensitive) were included as reference strains. For each pathogenic

strain, stems of ten woundedtomatoseedlings wereeach wounded 7

longitudinally (4-5 mm long slit) with a sterile scalpel blade, anda

10,../ suspension of K84 (108CFU/ml) was applied with a micropipet to each wound. Twenty-four hours later, these inoculated wounds were challenged with a 10..,ufsuspension (107 CFU/ml)of a pathogenic strain.

Becausenot all pathogenic strains aresubjectto biological control by K84 (Alconero, 1980; Moore, 1977), a search was made for other bacterial antagonists that would inhibit strains resistant to agrocin 84. Potential antagonists wereisolated from the rhizo- spheres of healthy and galled peach andpecan trees located in

Brooks, Thomas,and Tift counties. Soil surrounding the roots was suspended in sterile distilled water, diluted serially, and 0.1 ml of the selected dilution was spread over duplicate plates of mannitol- glutamate agar medium. After two days of incubation at 28C, plates with separated colonies were over-sprayed with 108 CFU/ml suspensions of the agrocin-sensitive strain K24 or the agrocin-resistant strain

B6. The plates were incubated for an additional three to five days at 28C, and colonies producing compounds inhibitory to growth of both

K24 and B6 were recovered, purified,and tested as described above for K84. 8

RESULTS AND DISCUSSION

Nearly equalnumbers of biovar 1 and biovar 2 strains were isolated from all the Georgia samples. However, there was a variability in the selectivity of the various selective media.Of 81

Agrobacterium colonies selected from the three different media,39 were biovar 1 strains and most of these were isolated on Schroth et al.and D1M media (Table 1.1). In addition, three or four strains isolated earlier (N.W.Schaad) from pecan galls were identified as biovar 1. The correlation between 3-ketolactose production, biovar type,and growth on Schroth et al. or New-Kerr selective medium was high. No biovar 2 strains were isolated on Schroth et al. medium, and onlyfour biovar 1 strains were isolated on the New-Kerr medium. In this study, fewer strains were isolated on the D1M medium than on the other two media (Table 1.1).

Virulentstrains of both biovars wereisolated from the same gallin four of the 18 galls examined. Most pathogenic strains were sensitive in vitro to agrocin 84 (Table 1.2). Interestingly, eight biovar 1 and four biovar 2 strains were nonpathogenic on tomato and

Datura, but were sensitive to agrocin 84. Because agrocin sensitivity is coded for by genes on the Ti-plasmid (Engler et al.,

1975),the inability of these strains to infect tomato and Datura seedlings may be due to their host specificity (Anderson and Moore,

1979) or loss of uncogenicity genes, but not agrocin-sensitive genes from the Ti-plasmid. Strain GA003 also may be less virulent because 9

it infected fewer tomato seedlings (Table 1.3). About 73 percent of

the pathogens isolated from galled pecan trees in Georgiawere biovar

1, most of which were sensitive to agrocin 84(Table 1.2). Thus,

pathogenic biovar 1 strains predominated in this study. In contrast,

more biovar 2 than biovar 1 Agrobacterium strains were sensitive to

K84 in Australia (Kerr and Htay, 1974) and Oregon (Moore, 1977).

Infection of tomato plantsby agrocin-sensitive strain GA1010

isolated from pecangall wassuccessfully preventedin greenhouse

tests with K84 (Table 1.3). Despite the limited success of biologi-

cal control of crown gall of peaches by K84 in two other states in

the South (Alconero,1980), 94% of the pathogenic biovar 1 strains

from pecans in Georgia were agrocin-sensitive while only 29% (two out

of seven pathogenic strains)of theless prevalent biovar 2 were

agrocin-sensitive (Table 1.2). From the results of these greenhouse

tomato tests (Table 1.3) and because biological control of crown gall

is highly correlated with agrocin sensitivity of the pathogen (Kerr

and Htay, 1974),it appears that K84 would prevent the majority of

pathogenic agrobacteria from infecting pecans in Georgia. In addi-

tion, some pathogenic strains resistant to agrocin 84 in vitro have

been prevented from infecting certain hosts by K84 in field

experiments (Kerr and Htay, 1974; Moore,1977; Schroth and Moller,

1976).

K84 is used as a preventative and not a curative control agent; therefore, latent infections are not prevented (Moore, 1976). Latent 10 infections may have occurred on the Jefferson County pecans that were treatedwith K84, but subsequently developedcrown gall. This explanation seemsplausible sincethe pathogenic strainsisolated from this sample were sensitive to agrocin 84. Alternatively, K84 may not survive well on roots of pecan, or the K84 inoculum may have been adversely affected by other unknown factors following treatment of the young trees (Moore and Warren, 1979).

The inhibition of A.tumefaciensin vitro by other bacterial antagonists isolated from Georgia suggests that these new antagonists may provide an alternativecontrol forsome agrocin84 resistant pathogenicstrains. B6 (biovar 1 and agrocin-resistant)and K24

(biovar 2 and agrocin-sensitive) strains were inhibited in vitro by these new rhizosphere antagonists. The putative antagonists were isolated from a potentially suppressive soil located in a young peach orchardin Brooks County whereno galled trees were detected and included strains of actinomycetes, fluorescent pseudomonads, Bacillus spp., and agrobacteria. From these antagonists,two actinomycete strains produced large zones of inhibition against the two reference strains, and no resistant mutants were observed. Because little successhas been noted with other antibiotic producing antagonists

(Garrett, 1979; KerrandPanagopoulos, 1977), thesenewputative antagonists,as well asK84,should be tested under Georgia field conditions for control of crown gall on pecan trees. 11

ACKNOWLEDGEMENTS

We thank Dr. PaulF. Bertrand (Cooperative Extension Service,

University of Georgia, Tifton) for his help during the field trips;

Dr. Norman E. McGlohon (Head, Cooperative Extension Service,

University of Georgia, Athens); and Dr. Thomas C.Allen (Professor,

Department of Botany and Plant Pathology, Oregon State University,

Corvallis) for reviewing the manuscript. 12

REFERENCES

Alconero, R. 1980--Crown gall of peachesfromMaryland, South Carolina, and Tennesseeand problems with biological control. Plant Disease 64:835-838.

Anderson, A.R.,and L.W. Moore. 1979--Host specificity in the genus Agrobacterium. Phytopathology 69:320-323.

Cooksey, D.A., andL.W. Moore. 1980--Biologicalcontrolof crown gall with fungal and bacterial antagonists. Phytopathology 70:506-509.

Engler, G., M. Holsters, M. Van Montagu, J. Schell, J.P.Hernal- steens,and R. Schilperoort. 1975--Agrocin 84 sensitivity: a plasmid determined property in Agrobacterium tumefaciens. Mol. Gen. Genet. 138:345-349.

Garrett,C.M.E. 1979--Biologicalcontrolof crown gall in cherry rootstock propagation. Ann. Appl. Biol. 21:221-226.

Kerr, A., and K. Htay. 1974--Biological control ofcrown gall through bacteriocin production. Physiol. Plant Pathol. 4:37-44.

Kerr, A., andC.G. Panagopoulos. 1977--Biotypesof Agrobacterium radiobacter var. tumefaciens and their biological control. Phytopathol. Z. 90:172-179.

Livingston, R.L. and C.M. Bruce. 1979--Pecan in Georgia. Hortic. Bull.609, Coop. Ext. Serv., University of Georgia, College of Agriculture, Athens, Georgia. 32 pages.

Moore, L.W. 1976--Latentinfections andseasonal variability of crown gall development in seedlingsof three Prunus species. Phytopathology 66:1097-1101.

Moore, L.W. 1977--Prevention ofcrown gall on Prunusroots by bacterial antagonists. Phytopathology 67:139-144.

Moore, L.W. and G. Warren. 1979--Agrobacterium radiobacter strain 84 and biological control of crown gall. Annu. Rev. Phytopathol. 17:163-179.

Moore, L.W., A. Anderson, and C.I. Kado. 1980--Agrobacterium. pp17-25 in Schaad, N.W. ed. Laboratory Guide for Identification of Plant Pathogenic Bacteria. Am. Phytopathol. Soc., St. Paul, Minnesota. 13

New, P.B.,and A. Kerr. 1971--A selective medium for Agrobacterium radiobacter biotype 2. J. Appl. Bacteriol. 34:233-236.

New, P.B., and A.Kerr. 1972--Biological controlof crown gall: Field measurement and glasshouse experiments. J. Appl. Bacteriol. 35:279-287.

Rand, F.V. 1914--Some diseases of pecans. J. Agric. Res. 1:334-335.

Schroth, M.N.,J.P. Thompson, and D.C. Hildebrand. 1965--Isolation of Agrobacterium tumefaciens - A. radiobacter group from soil. Phytopathology 55:645-647.

Schroth, M.N.,and M.J. Moller. 1976--Crown gall controlled in the field with a nonpathogenic bacterium. Plant Dis. Rep. 60:275-278.

Stonier, T. 1960--Agrobacterium tumefaciens Conn. II: Production of an antibiotic substance. J. Bacteriol. 79:889-898. 14

TABLE 1.-1

Number of Agrobacterium strains isolated from pecan galls on three selective media

3-ketolactose Selective Medium

reactiona New - Kerr Schroth et al. 01M Total

4 24 11 39

38 0 4 42

There was a high correlation between the ability of a strainto oxidize lactose to3-ketolactose and the physiological-biochemical tests that distinguish biovar 1 strains. 15

TABLE 1.2

Number a of Agrobacterium strains pathogenic and sensitive to agrocin 84 produced by A. radiobacter K84

Pathogens Nonpathogens

. .Agrocin...... Agrocin

Biovar sensitive resistant sensitiveb resistant

1 15 1 8 18

2 2 5 4 32 aThese data include the four strains isolated earlier by N.W. Schaad. b0f 29 agrocin-sensitive strains, 12were nonpathogenic. 16

TABLE 1.3

Biological control of representative Agrobacterium tumefaciens strains on tomato seedlings by A. radiobacter K84

No. infected seedlings

Pathogenic strainsa

K84 86 GA003 K24 GA1010

Absent 10/10 4/10 10/10 9/10

Present 10/10 2/10 0/10 0/10 aB6 and GA003 are agrocin-resistant; AC24 and GA1010 are sensitive. Stem wounds were in9culated with 10° CFU/ml K84 then challenged 24 hours later with 10 CFU/ml of the pathogen. 17

CHAPTER 2

IMMUNOLOGICAL HETEROGENEITY OF 50S RIBOSOMAL SUBUNITS FROM AGROBACTERIUM STRAINS

SUMMARY

The taxonomy of both Agrobacterium and Rhizobium and their relationship in the familyRhizobiaceae is still an unsettled matter. Because the taxonomy of the Rhizobiaceae has not been resolved by classical and modern methods of microbial classification, a serological analysis of the 50S ribosomal subunits wasmade.

Antiseraraised against 50Sribosomal subunits of fivedifferent agrobacteria wereused to arrange31 Agrobacterium strainsand 6

Rhizobium strains into 16 serovars. The immunological relationships of 50Ssubunits of ribosomes from Agrobacteriumdid notconfirm either one of the major taxonomic groupings; the antigenic structure did not correlatewitheitherthetumorigenic state, the biovar affiliation, the host, or the area of origin. Fast-growing rhizobia couldnot beimmunologically differentiated from the agrobacteria, and rhizobia in general were immunologically more closely related to biovar 3 strain CG64. The five sera were specific at the family levelbecause none of them reacted with ribosomes of species from outside the Rhizobiaceae. 18

INTRODUCTION

The taxonomy ofthe genus Agrobacterium inBergey's manual

(Allen and Holding, 1974) is based primarily on phytopathogenic

characters. Those strains causing crown gall disease are placed in

the species A. tumefaciens, those causing hairy root in A.

rhizogenes,those causing cane gallon Rubus spp.in A.rubi, and

nonpathogens in A. radiobacter. The Approved List of Bacterial Names

(Skerman, McGowan, and Sneath, 1980) recognizes these four species of

Agrobacterium. However, these species are indistinguishable by

biochemical and physiologicalmeans. Andbecausethe genesfor

pathogenicity are plasmid-borne (Zaenen et al., 1974; Moore, Warren,

and Strobel, 1978), and the plasmids are readily transferred between

strains of Agrobacterium, many plant pathologists consider that this

classification does not accurately reflect the taxonomic relationship

of these organisms. Kerr, Young,and Panagopoulos(1978) proposed

that these speciesbe amalgamated into a single species A.radio-

bacter. Thisspecies wouldinclude threepathovars (pv) charac-

terizingdifferent pathogenic states: the saprophyticstate A. radiobacter pv radiobacter, the tumorigenic state A. radiobacter pv tumefaciens,and the rhizogenic state A. radiobacter pv rhizogenes.

In addition,this species would also be divided into three biovars

(Keane, Kerr, and New, 1970; White, 1972; Kersters et al., 1973; Kerr

and Panagopoulos, 1977). Thus strains of the same pathogenic state would be physiologically subdivided into biovar 1, 2, or 3. 19

Becausebiovar 2 strains hybridize at 20%or less with the

strains ofbiovar 1, Holmes and Roberts(1981) believedthatthe

taxonomy of Kerr etal. did not follow the International Rules of

Nomenclature. They proposed at least three species in the genus

Agrobacteriumwhich were based on phenotypic characters without

regard to phytopathogenic effect. That is, the taxon A. tumefaciens

corresponded to the biovar 1 group and was comprised predominantly of

3-ketolactose-positive strains; the taxon A. rhizogeneswas used for

the 3-ketolactose-negative strains corresponding to the biovar 2

group; and the taxon A. rubi was used for a heterogenous group which

included the biovar 3 group. Holmes and Roberts proposed that an

additional cluster of yellow-pigmented, 3-ketolactose-positive

strains of the agrobacteria and Chromobacterium lividum might

constitute a fourth species. This lattergroup is less closely

related to the other agrobacteria thanis Rhizobium meliloti which was closer to the A. rubi.

Thecloserelatedness of thefast-growingrhizobia withthe

agrobacteria has been known for some time because of DNA homology

studies (Heberlein, DeLey, and Tijtgat, 1967), comparison of ribosomal RNAcistrons (DeSmedt and DeLey, 1977), physiological studies (White,1972), andimmunologicalstudies(Graham, 1969 and

1971; Schroth et al., 1971; Abd-El-Rahim et al., 1975). Even with these reports, the taxonomy of both Agrobacterium abd Rhizobium, and their relationship in the Rhizobiaceae is still an unsettled 20

matter. This lack ofclarity is alsothe majorreason for the absence of quick and precise methods of identification, and the lack of such methods has prevented an in-depth study of the ecology of

Agrobacterium. This continuing need for a precise and fast method of identification prompted us to explore the use of antisera developed against ribosomal subunits as a method of identification.

Other phytopathogenic bacteria are presently identified by antisera raised primarily against external antigens(Schaad, 1979).

Unfortunately this is not the case for Agrobacterium, where most of the serological studies are contradictory. In some cases, serological specificity was reported at the species or biovar level

(Berquistand Elrod, 1948; Keane, Kerr,and New,1970;Schroth et

al., 1971; Sine, 1982), whereas in others cross-reactions and strain specificity werereported (Rikeret al., 1930;Coleman and Reid,

1945; Miller and Vruggink, 1981).

In 1962, Quash et al. first reportedthe immunogenicity of ribosomes. Later, Friedman, Olenick, and Hahn (1968) reported that

50Ssubunitswerespecific at the species level. Schaad (1974)

studied the immunogenicity of ribosomes from seven species of.

Enterobacteriaceae and observed a specific immunogenic determinant at

the subspecies level. In the first detailed study of the antigenic

structure of ribosomes of a plant pathogenic bacterium, Schaad(1976)

examined and compared, by Ouchterlony double diffusion,ribosomes of

25 isolates of Xanthomonas vesicatoria toribosomes of15other 21 closely and distantly related bacteria. He showed that the antigenic structure of ribosomes was similar at the subspecies level, but different at the specific and generic levels. X. vesicatoria strains grouped into three serotypes. This work demonstrated the usefulness of ribosomal immunology as a tool to distinguish species and subspecies of these bacteria.

The objectives of this research were to study the immunogenicity of the 50S ribosomal subunits of Agrobacterium, investigate whether their immunological relationship would support any of the taxonomical schemes, and determine whether the sera were specific enough to be used in the identification of unknown agrobacteria. For comparison purposes thesesera weretested against phenol-extracted antigens from whole cells. 22

MATERIALS AND METHODS

ANTISERUM PRODUCTION: Pre-immunesera werecollected from

marginal ear-veins of each animal beforebeginning the immunization

schedule. Highly purified 50S ribosomal subunitswere prepared after

Schaad's procedure (1974). Because isolation of 50S subunits is long

and complex, only a single New Zealand White rabbitwas immunized for

each of the following strains: three biovar 1 (B6, C58, and M63/79),

one biovar 2 (U11), and one biovar 3 (CG64). Antigen injections con-

sisted of an emulsion of equal proportion ofantigen suspension and

incomplete Freund's adjuvant. The equivalent of1.5 mg ribosomes

(Schaad, 1974) was first injected intraperitoneally. The three following injections were administered intramuscularly andten days apart, with increasing antigen concentrations of 2.5mg, 3.5 mg, and

4.5 mg. After the last injection the rabbits were bled twice at weekly intervals. For comparative purposes, antiserum to 70S ribosomes of E.coli was graciously provided by Dr. H. W. Schaup,

Department of Biochemistry and Biophysics, Oregon State University.

PREPARATION OF TEST-ANTIGENS: In contast to inject-antigens,

70S ribosomesand phenol-extracts were usedas test-antigens(e.g. antigens run in double diffusion tests). The strains used to provide testantigenswerechosen from differentbiovars andrepresented pathogens and nonpathogens isolated from different hosts and geographical areas (Table 2.1). Several other bacteria from different genera and families were also included. Crude 70S ribosomes were 23

prepared following Schaad's procedure(1974), and phenol-extracted

antigens from whole cells were obtained following the techniqueof

DeBoer, Copeman, and Vruggink (1979). Because the rate of cell

multiplicationin the early log-phase of four Agrobacterium strains

grown in medium 523 was very low,strains Ull,K47,CG64,and 6/6

were grownin YDP medium (yeast-extract 0.4%, glucose 2.0%, peptone

0.4%,and NH4SO4 0.5%). The rhizobia were grown on yeast-mannitol

medium (mannitol 1.0%, K2HPO4 0.1%, yeast-extract 0.05%, MgSO4.7H20

0.02%, NaC1 0.01%, and CaC12.2H20 0.003%) (Dr. D.O. Wilson, Depart- ment of Agronomy, Georgia Experiment Station, University of Georgia).

DETERMINATION OF SEROLOGICAL RELATEDNESS: The Ouchterlony

Double Diffusion test was used to determine the specificity of the

antisera and theserological variability of theorganisms. The medium for Ouchterlony plates was prepared with 7.5 g of agarose,

0.2 g of sodium azide, 8.5 g of sodium chloride, 2.0 g of magnesium

chloride, and 10 ml of 1% Trypan blue solution, mixed in 990 ml of distilled water. The medium was autoclaved and 10 mlaliquots were poured in 88-mm diameter plastic petri-dishes. A template was used to cut the wells (3.5 mm in diameter). The center well contained

10..44 of antiserum, and theouter wells contained 5 to 15a of test- antigen. Theplateswereallowed to incubate at 4C in a moist chamber for two to three days before readings were made.

BASIS FOR SEROVAR GROUPING: Immunological relationships were determined by comparing patterns of precipitin bands of every strain 24 run next to the homologous strain (e.g. the strain used to prepare the antiserum). If precipitin bands developed and fused with those of the homologous strain without spur formation (reaction of identity), the strain was considered to belong to the same serological group (Figure 2.2B). If a reaction of partial identity

(e.g. a precipitin band partially fused with a band of the homologous strain causing the formation of a spur),or ifa reaction of non- identity (e.g. the two bands crossed over each other forming a double spur), or if no reaction developed, the strain was considered to be of another serological group. The strains that fell into the latter categorywere compared to each other, and assigned to various serological groups following the type of reaction they developed when adjacent (Figure 2.1; Figure 2.2A). 25

RESULTS

Several precipitin bands were produced when ribosomes from Agro- bacterium strains were used as test-antigens against antisera to 50S ribosomal subunits (Figure 2.1 and 2.2). Each precipitinband constitutedofa group of lines that could notbe separated into individual lines. Therefore, the precipitin band was considered as a single reaction. Various degrees of cross reaction were observed when ribosomal preparations of 37 strains of the Rhizobiaceae were run against the five antisera (Table 2.2). Because ribosomes from the tumorigenic biovar 2 strain M9/79 failed to react with any of the sera, this strain could not be typed. None of the antisera reacted withribosomes of speciesfromoutsidethe Rhizobiaceae. In a reciprocal reaction, ribosomes from the agrobacteria and rhizobia did notreact with theantiserum raised against ribosomes of E.coli

(Figure 2.3), whereas ribosomes from members of the

Enterobacteriaceae and Pseudomonadaceae did react with the E.coli antiserum. Therefore, the antisera raised against our Rhizobiaceae strains were specific at the family level.

Different serological groups were observed for each of the five antisera (Table 2.3). Seven different serological groups were detected using the B6-antiserum, whereas only threeserological groups were recognizable with antiserum to C58. This difference in serological grouping suggests that the antigenic structures of the inject-antigens were sufficiently different to trigger production of 26

different antibodies by the rabbit immune system. Therefore, strains

from the same serological group that reacted differently to a given

antiserum were consideredto belong to a different serovar. For

example, althoughstrainsof serovar IVandV were serologically

grouped together usingantiseratoB6, C58,Ull, and CG64,they

reacted differentlyagainst theantiserum toM63/79. Because a

serovar is defined as an immunologically distinct group of strains,

the uniqueness of the antigenic structure of its members allows the

strains of the same serovar to react identically against any given

serum. On this basis,16 serovars of Agrobacterium and Rhizobium

strains were identified and designated by Roman numerals (Table 2.2).

A strain-specific immunogen was observed in the homologous

systems of strains B6, M63/79, and Ull. It appeared that the serovar

I strain B6 had all the antigenic determinants of serovar V strains

because antisera to strains C58 and CG64 (both strains were included

in serovar V) failed to show a strain-specific immunogen that would

separate serovar I from serovar V. In contrast, B6-antiserum

revealed the absence of the major band (e.g. the closest band to the

antigen well)in serovar V strains. Strain B6 seemed to have the

greatest number of antigenic determinants because it reacted strongly with all the sera. It is also possible that the immune system of the

particular rabbitinjected with B6-50S subunits was more reactive than the other rabbit systems. 27

Interestingly, the antigens from C58 and CG64 (serovar V) apparently did not trigger the same antibody responsein rabbits.

Antisera to these antigens reacted differently with members of eight serovars (II,III,IX, X, XI, XII, XV, and XVI) (Table 2.2), and the major difference was that antiserum to the biovar 3 strain CG64 had more antibodies that recognized immunogens from rhizobia strains than antiserum to C58. Differences in the antigenic structure of the two strains were suggested by the reaction of the C58 and CG64 antisera with heterologous strains,but were not revealed when C58 and CG64 were run in adjacent wells; the reason might have been the development of weak spurs that were too dilute to be detected by our technique.

In contrast to ribosomal antigens, the phenol-extracted antigens were strain-specific, thus -serological typing of the strains was not feasible. Out of the 37 strains of the Rhizobiaceae, phenol-extracts from B6, M63/79, and U11 reacted only with their homologous antiserum to produce a single strain-specific precipitin band. C58-antiserum developed a common precipitin band against phenol-extracts of C58 and the C58 avirulent mutant NT1. However, antiserum to the biovar 3 strain CG64 developed a reaction of identity between phenol-extracts of CG64 and the biovar 3 strain 6/6. 28

DISCUSSION

The Ouchterlony double diffusion technique allows identification ofantigens in a mixtureand canestablish relationships between antigens without complete knowledge of all the antigenic groups that may be present. This technique was used to arrange 31 Agrobacterium strains, of different pathogenic states and biovars, and 6 Rhizobium strains, of different species, into 16 serovars.

The immunological relationship of 50S subunits from

Agrobacteriumdid not confirmthe taxonomicschemes proposed by

Skerman et al. (1980) or Kerr et al. (1978). The antigenic structure of the 31 agrobacteria did not correlate with the speciation based on the pathogenic state. For example,strains M3/73 and GA003 of A. tumefaciens, N2/73 of A. rubi, K47 of A. rhizogenes, and K84 of A. radiobacterwere immunologically indistinguishable and thuswere includedin serovar XII. Although all five of these strainsare classified biovar 2,it was not possible to differentiate the other

26 strains on the basis of theirbiovar classification. For instance, serovar V had members from all three biovars and serovar VI had members from both biovar 1 and 2.

Area and host of origin did not correlate with the antigenic structure of the 50S subunits of Agrobacterium. Strains isolated from the same host species were grouped into different serovars, and strains of the same serovarwere isolatedfromdifferent hosts located in different areas. 29

The fast-growing rhizobia could not be immunologically differenciated from the agrobacteria, thus confirming the close relatedness of the two groups. Rhizobium meliloti YA 15 was a member of serovar V, R. phaseoli 127K12b and R. trifolii 16257a were included in serovar VI, whereas R.leguminosarum 128Al2 formed the distinct serovar XV. The two slow-growing species reacted poorly against all five sera, but shared more immunogens with the biovar 3 strain CG64. Holmes and Roberts (1981) reported a close relationship between R. melilotiand their A. rubi taxon,a taxon they used to symbolize the biovar 3 group. Our resultsalso showedthat 50S subunits from thesix rhizobia wereimmunologically more closely related to the biovar 3 strain CG64.

Contradictory reports about the classification of members of the

Rhizobiaceaefamily seem to bedueto the lackof standardized methodology and to the use of different culture collections. Because standardization is needed tofacilitate parallel comparisons and interrelate the data, a list of strains common to different laboratories should be used as a starting base for every comparative study of the Rhizobiaceae. The pathogenic strain TR2 was used in the presentstudy as well as in othertaxonomicstudies. TR2 was isolated from a cane gall, therefore traditionally classified as A. rubi and is typed in the biovar 2 group (Keane et al., 1970). How- ever, strain TR2 shows the closest similarity to the A. rubi group of

Holmes and Roberts (1981), which corresponds to the biovar 3 group. 30

Immunologically, 50S subunits of TR2 formed the distinct serovar IX because the major band (closest to the antigen well) that developed against B6-antiserum was unique. The contradictory results observed with TR2 reflects the superficial nature of our knowledge; standardization will help understand discrepancies, therefore adequate corrections will be made.

The combination of the five antisera demonstrated 16 different serovars when tested against 37 strains of the Rhizobiaceae family.

Therefore, in this study one serovar was demonstrated for approximately every twostrainstested. The differencesbetween antisera to C58 and CG64 point out that either serovar V was hetero- geneous or the immune response of the two rabbits differed; therefore, to avoid a loss in precision it is recommended to increase the number of strains used as antigens and the number of animals to be immunized per strain. However, based on our data, the use of sera from additional strains would probably reveal more differences among strains,evenofthesame serovar. Similarly,the use of test- antigens from more strains would probably increase the number of serovars; therefore, the immunological relationships of the species would be simultaneously more precise but also complex. However, if the needs are to detect unknown strains in mixed populations, the use of a single serum specific at the species level is required; this serum must contain antibodies that would react with immunogens common to members of the species. 31

From this serologicalstudy, it is evident that 50S ribosomal subunits from Agrobacterium strainsinduce quite different immuno- chemical reactions, suggesting that they differ in molecular structure. The sera were specific atthe family level, andthe

Ouchterlony doublediffusiontests of50Ssubunits oftheagro- bacteria and rhizobia appeared to be a good method for determining therelatedness of strains. However, the existenceof numerous serovars and therelative complexity ofextracting theribosomal

antigens casts doubt on this method for rapid identification of the agrobacteria, unlessa mixture of antisera proved effectiveas a

reagent to detect strains from different serovars.

Most of the antisera appeared to have a strain specific antigenic determinant, which agrees with the findings of Hochster and

Cole (1967) and Miller and Vruggink (1981). Therefore, the use of such antisera in ecological studies may prove useful for "tracking"

the movement and population dynamics of a particular strain on plant

roots, in soil, or water. 32

REFERENCES

Abd-El-Rehim, M.A., A.S.A. Ghaffar, K. Fawaz, and W.N. Gobtial. 1975--Serological and immuneolectrophoretical relationships between Rhizobiumand Agrobacteriumstrains. Egypt. J. Soil Sci. Specia issue 471-476. Alexandria University, Egypt.

Berquist, K.R.,and R.P. Elrod. 1948--The somatic antigens of the genus Agrobacterium. Proc. S.D. Acad. Sci. 27:104-111.

Coleman, M.F.,and J.J. Reid. 1945--A serological study of strains of Alcaligenes radiobacter and Phytomonas tumefaciens in the "M" and "S" phases. J. Bacteriol. 49:187-192.

DeBoer, S.H.,R.J.Copeman,and H.Vruggink. 1979--Serogroups of Erwinia carotovora potato strains determined with diffusible somatic antigens. Phytopathology 69:316-319.

DeSmedt J., and J. DeLey. 1977--Intra- and intergeneric similarities of Agrobacterium ribosomalribonucleic acid cistrons. Int. J. Syst. Bacteriol. 27:222-240.

Friedman, D.I., J.G. Olenick, and F.E. Hahn. 1968--A 50S ribosomal determinant: immunological studies correlating function and structure. J. Mol. Biol. 32:579-586.

Graham, P.H. 1969--Analytical serology of the Rhizobiaceae. In Analyticalserology of microorganisms. Kwapinski,J.B.G., eds. John Wiley and Sons. 2:353-378.

Graham, P.H. 1971--Serological studies with Agrobacterium radiobacter, A. tumefaciens, and Rhizobium strains. Arch. Mikrobiol. 78:70-75.

Heberlein, G.T., J.DeLey,and R. Tijtgat. 1967--Deoxyribonucleic acid homology and taxonomy of Agrobacterium, Rhizobium, and Chromobacterium. J. Bacteriol. 94:116-124.

Hochster, R.M., and S.E. Cole. 1967--Serological comparison between strains of Agrobacterium tumefaciens. Can. J. Microbial. 13:569- 572.

Holmes, B. and P. Roberts. 1981--The classification, identification, and nomenclature of agrobacteria. J. Appl. Bacteriol. 50:443-467. 33

Keane, P.J., A. Kerr, and P.B. New. 1970--Crown gall ofstone fruit. II. Identification and Nomenclature of Agrobacterium isolates. Aust. J. Biol. Sci. 23:585-595.

Kerr, A.,and C.G. Panagopoulos. 1977--Biotypes of A. radiobacter var. tumefaciens,and their biological control. Phytopathol. Z. 90:172-179.

Kerr, A., J.M. Young, and C.G. Panagopoulos. 1978--Genus II Agrobacterium. Conn. 1942. In Young,J.M.,D.W.Dye, J.F. Bradburg, C.G. Panagopoulos, and C.F. Robbs. A proposed nomenclature and classification forplantpathogenic bacteria. New Zealand J. of Agric. Res. 21:153-177.

Kersters, K., J. DeLey, P.H.A. Sneath, and M. Sakin. 1973--Numerical taxonomic analysis of Agrobacterium. J. Gen. Microbiol. 78:227-239.

Miller, N.J.,and H. Vruggink. 1981--An assessment of biochemical and serological tests for Agrobacterium radiobacter subsp. tumefaciens. Phytopathol. Z. 102:292-300.

Moore, L.W., G. Warren, and G. Strobel. 1978--Plasmid involvement in the hairy-root infection caused byAgrobacterium rhizogenes. Proc. IVth Int. Conf. Plant Path. Batt., Angers, France. 1:127- 131.

Quash, G., J.P. Dandeu, E. Barbu. et J. Panijel. 1962--Rechereches preliminaires sur les antigenes des ribosomes. Ann. Inst. Pasteur. 103:3-23.

Riker, A.J., W.M. Banfield, W.J. Wright, G.W. Keitt, and H.E. Sagen. 1930--Studies on infectious hairy root of nursery apple trees. J. Agric. Res. 41:507-540.

Schaad, N.W. 1974--Comparative immunology of ribosomes and disc gel electrophoresis of ribosomal proteins from erwiniae, pectobacteria, and othermembers of the familyEnterobacter- iaceae. Int. J. Syst. Bacteriol. 24:42-53.

Schaad, N.W. 1976--Immunological comparison and characterization of ribosomes of Xanthomonas vesicatoria. Phytopathology. 66:770-776.

Schaad, N.W., 1979--Serological identification of plant pathogenic bacteria. Ann. Rev. Phytopathol. 17:123-147. 34

Schroth,M.N., A. Weinhold,A.H.McCain, D.C. Hildebrand,and N. Ross. 1971--Biologyand control of Agrobacterium tumefaciens. Hilgardia. 40:537-552.

Skerman, V.B.D., V. McGowan, and P.H.A. Sneath. 1980--Approved lists of bacterial names. Int. J. Syst. Bacteriol. 225-420.

S. 1982--Serological studies on agrobacteria. Int. Work. on CrownGall (Agrobacterium tumefaciens),Wadenswil,Switzerland. 11 pages.

White, L.O. 1972--The taxonomy of the crown gall organism Agrobacterium tumefaciens and its relationship torhizobi.aand other agrobacteria. J. Gen. Microbiol. 72:565-574.

Zaenen, I., N. Van Larebeke, H. Teuchy, M. Van Montagu,and J. Schell. 1974--Supercoiled circular DNAincrown gallinducing Agrobacterium strains. J. Mol. Biol. 86:109-127. 35

Fig. 2.1-Ouchterlonydoublediffusion patterns of ribosomes from differentstrainsof Agrobacterium and Rhizobium tested against antiserum raised to 50S ribosomal subunits of A. tumefaciensB6. The bandspecific to B6 is the sharp bandclosest tothe B6 antigen well. Center wells contained antiserum to 50S subunits of strain B6. A. Outer wells contained ribosomes of B6 (1,4), AB2/73(2), G2/79 (3), GA001 (5), and K30 (6). B. Outer wells contained ribosomes of B6 (1), T20-73(2), M63/79 (3), RR5 (4), H27/79 (5), and C58 (6). C. Outer wells contained ribosomes of B6 (1,4), T20/73(2), RR5 (3), R. meliloti YA15 (5), and CG64 (6). D. Outer wells contained ribosomes of B6(1,4), TR2 (2), GA105 (3), S1/73 (5), and GA002 (6). 36

Fig. 2.2-Ouchterlonydouble diffusion patterns of ribosomes from different strains of Agrobacterium tested against antiserum to 50S ribosomal subunits of A. tumefaciens M63/79 (center wells). A. Reaction of identity between serovar XII strains. Outer wells contained ribosomes of M63/79 (1), GA003 (2), M3/73 (3), K84 (4), K47 (5), and N2/73 (6). B. Reaction ofidentity between strains of the same serological group. Outer wells contained ribosomes of M63/79 (1,6), T20/73 (2), B6 (3), RR5 (4), and H27/79 (5). 37

Fig. 2.3-Ouchterlony double diffusion pattern of ribosomes from different species of the Enterobacteriaceae and Rhizobiaceae families tested against antiserum to 70S ribosomes of Escherichia coli (center well). Outer wells contained ribosomes of E.coli (1,2,4), Erwinia carotovora (3), Agrobacterium tumefaciens15T and R. japonicum (6). TABLE 2.1 Source and origin of bacterial strains used as antigens

Host of Obtained Strain origin Location from

RHIZOBIACEAE Agrobacterium tumefaciens Biovar 1 B6 Apple Iowa R. Baker C58 Cherry New York R. Dickey G2/79 Cottonwood Oklahoma L. Moore G18/79 Poplar Oklahoma L. Moore GA001 Pecan Georgia N. Schaad GA002 Pecan Georgia N. Schaad GA015 Pecan Georgia H. Bouzar GA105 Pecan Georgia H. Bouzar H27/79 Rose Columbia, S.A. L. Moore K30 Peach Australia A. Kerr M63/79 Cottonwood Oklahoma L. Moore S1/73 Lippia Arizona L. Moore

Biovar 2 AB2/73 Lippia Arizona L. Moore B234 INAa California J. DeVay GA003 Pecan Georgia N. Schaad M3/73 Birch Oregon L. Moore M9/79 Cottonwood Oklahoma L. Moore U11 Willow Oregon L. Moore Biovar 3 6/6 Grapevine Hungary S. SUle

Ag63 Grapevine Greece C. Panagopoulos 8,6, Table 2.1--Page 2 of 3

CG48 Grapevine New York T. Burr CG 54 Grapevine New York T. Burr CG64 Grapevine New York T. Burr

Agrobacterium rubi Biovar 1 RR5 Raspberry Oregon L. Moore Biovar 2 N2/73 Raspberry Oregon L. Moore TR2 Raspberry Washington E. Nester

Agrobacterium rhizogenes Biovar 2 A4 INA California R. Durbin K47 INA Australia A. Kerr UCBPP 604 INA California M. Starr

Agrobacterium radiobacter Biovar 1 T20/73 Rose Oregon L. Moore Biovar 2 K84 Soil Australia A. Kerr

Rhizobium japonicum 61A101 INA Wisconsin R. Stewart Smithb

Rhizobium lupini 96B9 INA Wisconsin R. Stewart Smith

Rhizobium meliloti YA15 INA Oregon L. Barber

Rhizobium phaseoli 127K12b INA Wisconsin R. Stewart Smith

Rhizobium leguminosarum 128Al2 INA Wisconsin R. Stewart Smith TABLE 2.1 -- Page 3 of 3

Rhizobium trifolii 162S7a INA Wisconsin R. Stewart Smith

PSEUDOMONADACEAE Pseudomonas solanacearum 51 INA Wisconsin L. Sequeira

Pseudomonas syringae pv syringae B3 INA Georgia Chandler

Xanthomonas campestris pv campestris B24 INA Oregon L. Moore

ENTEROBACTERIACEAE Erwinia carotovora EVEIT. carotovora 105 INA Missouri R. Goodman

Escherichia coli CDC-01A INA Georgia Center for Disease control, Atlanta

Salmonella typhimurium E26 INA Georgia N. Schaad

BACILLACEAE Bacillus subtilis J42 INA Georgia N. Schaad

CORYNEFORM Corynebacterium michiganense 1 INA N. Carolina Eschandi aInformation Not Available bfrom the Nitragin Company TABLE 2.2 Serovars developed in Ouchterlony double diffusion tests from reaction of antisera to 50S subunitsfrom five Agrobacterium strains to ribosomes from 31 Agrobacterium strains, 6 Rhizobium, and 8 miscellaneous species.

Antiserum to

50S ribosomal subunits of Agrobacterium 705 ribosomes

Serovar Strains B6b C58 M63/79 Ull CG64 of E. coli

86 ++ ++ ++ ++ ++ ++ o ++ ++ + + ++ ++++ ++ 0

II 105 + ++ ++ + ++ ++ ++++ + + ++ o ++ ++ ND

III s1/73;GA002 + ++ ++ + ++ ++ o + ++ + ++++ o ++ ++ ND

IV M63/79 o ++ ++ ++ ++ ++ ++++++ + + ++ ++++ ++ 0 T20/73;RR5;C58 CG64;6/6;Ag63; H27/79;G18/79;YA15 o ++ ++ ++ ++ ++ o ++++ + + ++ ++++ ++ 0

VI B234;GA015;A4; UCBPP 604; 0 ++ ++ ++ ++ ++ 127K12b; 162S7a o ++++ + ++++ + ++ ++ 0

VII CG48 o ++ ++ ND ND ND o 0 ++ o 0 ++ o ++ ++ ND

VIII GA001;K30 + o ++ + ++ ++ o o ++ - ++++ o + +4 ND

IX TR2 - + ++ + ++ ++ o ++++ - ++++ o ++ ++ ND ++ ++ 0 ++++ + ++++ o ++ ++ ND X CG54 o + ++ Table 2.2- Page 2 of 2 XI Ull 0 0 ++ 0 4+++ 0 0 ++ ++ ++ ++ 0 + ++ XII K84;K47;M3/73; GA003;N2/73 0 0 ++ 0 ++++ 0 0 ++ + ++ ++ o + ++ NO A82/73 XIII 0 0 ++ + ++++ 0 o + ++ ++ ++ o + ++ ND xlv G2/79 0 0 ++ + ++++ 0 o ++ - + ++ 0 + ++ ND XV 128Al2 0 0 4+ 0 ++++ o o ++ 0 0 ++ + ++ ++ NO XVI 96139:61A101 0 0 ++ 0 + ++ 0 0 0 0 0 ++ o + ++ o

M9/79 0 0 0 0 0 0 o 0 0 0 0 0 0 0 o NO

P. solanacearum 51 o o o 0 0 0 0 0 0 0 0 0 0 0 0 157 syrinqae B3 + 0 0 0 0 0 0 0 0 0 000 0 X. campestris 0 0 + o o o 0 0 0 0 0 0 0 0 0 0 0 0 + E. carotovora 105 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ++ r".- soli COC-01A o o o 0 0 0 0 0 0 0 0 0 00 0 ++ 37 typhimurium E26 0 0 o 0 0 o 0 0 o o 0 o o o o ND IT subtilis J42 0 0 0 o 0 o o o 0 o o n o o o ND C. 77F7517ense 1 0 o o o o o o 0 0 o o o 0 o o 0

Symbols defined: ++ reaction of identity; += partial identity; - . non-identity; o = no reaction; ND = not determined

aCrude 70S ribosomes

b Each main column represents the reactions of each antiserum with 50S subunits from each listed strain; of which three bandswere analyzed. The first subcolumn (left) refers to the reaction of the band closestto the antigen well, the second subcolumn (middle) refers to the reaction of the intermediate band, and the third subcolumn (right) refers to the reaction ofthe band closest to the antiserum. TABLE 2.3

Distribution of the 16 serovars within the different serologicalgroups revealed by each antiserum

Antiserum to

B6 C58 M63/79 Ull CG64

7 serological 3 serological 5 serological 6 serological 4 serological groups: groups: groups: groups: groups: (I),(II,III),(IV, (I,IV,V,VII),(II, (IV) (I,II,V,VII, (XI),(III,VII,X, (I,IV,V),(VII,XV), V,VI,VII),(VII), III,VIII,IX,X, IX,X1,(III),(VI, XII,XIII),(VIII, (II,III,VI,IX,X), (IX),(X),(XI,XII, XIII,XIV),(XI,XII, VIII,XI,XII,XIII, IX),(I,II,IV,V), (VIII,XI,XII,XIII, XIII,XIV,XV,XVI) XV,XVI) XIV,XV),(XVI) (XIV),(VI,XV,XVI) XIV,XVI) 44

CHAPTER 3

IMMUNOLOGICAL DIFFERENCES BETWEEN 50S SUBUNITS FROM AGROBACTERIA AND THEIR PLASMID-CURED DERIVATIVES

SUMMARY

The antigenic structure of 50S ribosomal subunits oftumorigenic

Agrobacterium tumefaciens C58 and rhizogenic A. rhizogenes A4 strains were compared to that of their avirulentderivatives (NT1 and A4R1, respectively) and to strain A323 (a NT1 that was transformed with the large nopaline plasmid from K84) using Ouchterlonydouble diffusion test. A unique precipitin band was observed in the virulentplasmid- containingstrains; this band whichrevealedthe presenceof an additional immunogen wasabsent from 50S subunits ofthe plasmid- deficient mutants. This unique band observed in 50S subunits of the virulent strain C58 was also presentin the transformant A323, but was absent from both strainsNT1 and K84. From these observations, it appears that the combination of plasmid and chromosome caninduce formation of new 50S ribosomal subunits which have a major

immunological difference from 50Ssubunits of cells of the same

chromosomal background but lacking the plasmid. 45

INTRODUCTION

The ability of Agrobacterium to cause tumor development (Zaenen et al., 1974) androot proliferation (Moore,Warren,and Strobel,

1978) is plasmid borne. The bacterium cantransferor loseits plasmid, thus transferring or losing its virulence.

Plasmid-associated characteristics other than virulence have also been reported. For example Sonoki and Kado (1979) isolated two plasmid-mediated proteins which were associated with the outer membrane and periplasmicfraction of the cell of thewildtype tumorigenic strain C58 using slab gel electrophoretic methods. These proteins were absent from NT1, the avirulent derivative of C58. In otherinstances, using whole cells and sonic-extracts as inject- antigens, serological differences have been reported between virulent strains and their avirulent mutants (Schnathorst, DeVay, and Kosuge,

1964; Hochster and Cole, 1967; Sule, 1982). These authors reported plasmid-associated cell products, and cell products are either proteins or products of reactions catalyzed by proteins. Because protein synthesisis the function of the ribosome machinery and we reported heterogeneity inthe antigenic structure of 50S ribosomal subunits of various agrobacteria (see Chapter 2),the objective of thisstudy was to determine whether plasmids could influence the antigenic structure of the 50S subunit.

In the present study, antiseraraised against 50Sribosomal subunits were used to compare the antigenic structure of 50S subunits 46 from the tumorigenic C58 and rhizogenic A4 strains to that of their

avirulentderivatives (NT1 and A4R1, respectively) and to strain

A323,which is a NT1strain that wastransformed withthe large

defective Ti-plasmid from strain K84 that codes for catabolism of

nopaline. Schaad (1976) reported that important immunological

differences existed between the 30S and 50S subunits of Xanthomonas

vesicatoria ribosmes and Tischendorf,Stbffler, and Wittmann (as

citedby Wittmann, 1974) reportedthatthe30S and50S subunits, which constitute the 70S ribosome of Escherichia colicontained 55

differentproteins with only one protein common tobothsubunits.

Therefore, 70S ribosomes could be used as test-antigens to

immunologically analyze the 50S subunits usingantibodies raised

against 50S subunits. 47

MATERIALS AND METHODS

The Ouchterlony double diffusion technique was used to test the immunological properties of strains with different chromosomal backgrounds in thepresence or absenceof largeplasmids (Table

3.1). Two virulent vs avirulent systems were tested (C58 vs NT1 and

A4 vs A4R1) using antisera raised against highly purified 50S ribosomal subunits extracted from tumorigenic C58, B6, M63/79, Ull, and CG64 strains (refer to Chapter 2 for methods).

Because the extraction of the 50S subunits is long and relatively complex, crude 70S ribosomes were used as test-antigens in the double diffusion tests (see Chapter 2). Phenol-extracts of whole cells(DeBoer,Copeman, and Vruggink,1979) which we found tobe strain specific with the agrobacteria (see Chapter 2) were used to determine whether the C58 strain-specific band was present in both

NT1 and A323. 48

RESULTS

Antisera to 50S ribosomal subunits of all five virulent strains produced an additional precipitin band when ribosomes of the virulent wild type strains C58 and A4 were reactedin adjacent wells with their avirulent plasmid-deficient mutants, NT1 and A4R1, respectively

(Figure 3.1 and 3.2).

Interestingly,this unique band developed against 50S subunits of the rhizogenic strain A4 even though all five antisera were raised against 50S subunits from tumorigenic strains.

When M63/79-antiserum was reacted with 50S subunits of M63/79 a strain-specific precipitin bandwasobservedclose to the antigen well (see Chapter 2). In addition,a thick inner band of numerous lines formeda reaction ofidentity with a band producedby A4.

However, a spur developed between this thick inner band and the thin band producedby A4R1(Figure3.1), indicating the presence of an immunogenic componentin 50S subunits of A4 that is absent in 50S subunits of A4R1.

Ribosome preparations isolated from C58, NT1, A323, and K84 were also comparedimmunologically toeach other ina double diffusion testagainst antiserum-to 50S subunitsof C58(Figure 3.2). The antigenic structure of 50S subunits of the homologous wild type C58 and the transformant strain A323 was identical. Both C58 and A323 have the same C58-chromosomal background and carry a large molecular weight plasmid thatis cryptic (Table 3.1), and each produced three 49 different precipitin bands that met to form a reaction of identity.

However, NT1 (a pTi-cured C58), which also has the same C58- chromosomal background, lacks the major antigen which was immunochemically visualized by the presence of the outer precipitin band with both C58 and A323. The other two bands produced by the reaction of the C58-antiserum with 50S subunits of NT1 fused ina pattern of identity with the two inner bands of C58 and A323 (Figure

3.2).

When 50S subunits of K84 were testedin wells adjacent to the three strains with C58-chromosomal background, the onlycommon reaction was the inner most band (Figure 3.2). The lack of any other commonbandbetween K84 andA323, as compared to C58and A323, suggests that the unique precipitin band between C58 and A323 cannot be attributed to a specific product of the pAt-84b plasmid, which is present in both K84 and A323. Rather, it appears that the "plasmid- chromosome" complex is responsiblefor the additional immunogen present in the 50S subunits of the wild type C58 and the transformant

A323.

In contrast to ribosomal antigens, phenol-extracted antigens from whole cells of C58 gave only one band and this band was strain- specific(see Chapter2). Predictably,thisbandshowed identity with the phenol-extracted antigens of A323 and NT1, two other strains with C58-chromosomal background (Figure 3.3). No bands were observed with C58-antiserum and the phenol-extracted antigens of K84. These 50 patterns of identity suggest that the antigen extracted from phenol- treated cells is coded for by chromosomal genes because this antigen is specificonlyto Agrobacterium cells of identical chromosomal background and the presence, absence, or addition ofa plasmid from another strain does not affect it in any way.

On the basis of the results of the phenol-extracts and the 50S subunits antigens, we hypothesizedthatphenol-extractedantigens

(Figure 3.3) were responsible for the middle precipitin band common to 50S subunits of NT1, C58, and A323 (Figure 3.2). This hypothesis was tested by running 50S subunits of C58 next to phenol-extracts of

C58 cells,and the hypothesis proved correct because a reaction of identity developedbetweenthe intermediateband producedby50S subunits and the single band produced by phenol-extracts. 51

DISCUSSION

Striking immunological differences occurred when 50S subunits

from the virulent A4 and C58 strains werecompared to the 50S

subunits of their avirulent, plasmid-deficient mutants, A4R1 and NT1

respectively. The plasmid-deficient strains always lackedone unique

precipitin band that was common to the plasmid-bearing parent

strain. Thisdifference suggested thatthe additional immunogen

observed with the wild type parents was coded for by the plasmid.

Interestingly, the same antigenic structure was presentin both C58

andA323, two strains withthe same chromosomal background but different plasmids. Both plasmids are the nopaline-type and havea similar molecular weight,but their restriction enzyme digests are different (Sciaky,Montoya,and Chilton,1978). In addition,the pTi-058 is an oncogenic plasmidwhilethepAt-84bfrom A323 is considered a defective Ti-plasmid. Apparently, this additional unique immunogen is not specifically related toor itssynthesis coded for by either the pTi-058 nor the pAt-84b plasmid. Therefore, theadditional immunogenis morelikely the product ofa general response between the plasmid and the chromosome.

Because there was a unique immunogen associated with 50S subunits from the pTi- and pRi- plasmid-containing cells of the two systems studied (C58 vs NT1 and A4 vs A4R1), we would predict that an additional immunogen ofsimilar function would be produced by K84

(and other Agrobacterium strains that carry a plasmid). One 52 hypothesis being that this additional immunogen would help translate the messages coded by the plasmid; but being specific to the K84 cell machinery it would be immunologically different from the one present in cells with different chromosomalbackground (i.e. C58). This would explain why Robert and Kerr (1974) could not detect differences

in the reactions ofthe pathogens andtheir nonpathogenic parents when they tested antibodies raised against whole cellsand sonic- extracts from four avirulent biovar 1 strains that carried a biovar 2 oncogenic plasmid.

Because important immunological differences were observed between50S subunitsofcells ofidenticalchromosomal background

(the strains differed only in their plasmid constitution), this would suggest that the "plasmid- chromosome" complex may induce formation of new 50S subunits that have a major immunological difference from50S subunits ofplasmid-deficient cells. At thistime it isunknown whether theadditional immunogen is a closely bound protein ora

"true" constituant of the "new 50S subunits." Hypothetically, the

additional antigen appears necessry to the 50S subunit structure for

the ribosome to translate and express the information carried by the

plasmid.

Proteinsassociatedwiththe outer membrane and periplasmic

fraction of the cell were reported to be associated with the presence

of the pTi-plasmid (Sonokiand Kado, 1979) and 50S subunits of the

plasmid-deficient cell, tested in adjacent cells to 50S subunitsof 53

the plasmid-containing cell, were lacking one major antigenic structure. Therefore,another hypothesis might be that the unique immunogen present in the "new 50S subunit" is the bacterial equivalent of the signal recognition particle (SRP) discovered recently in eukaryotic cells by Walter and Blobel (as cited by Lewin,

1982) and described as a "third ribosomalsubunit" because of the combination of protein (6 polypeptides) and RNA (7S RNA)in the SRP and its role in mediating protein translocation.

The presence ofthe plasmid seemsto derepressgenes onthe chromosome which code for the synthesis of an unidentified structure on the 50S subunitthathypothetically would be needed forthe reading and expression ofthe informationencoded in theplasmid genome. More investigation is needed to determine the nature of the antigen, its role, and if it is a "true" element of the 50S subunit, a supernatant protein of the cytoplasm strongly bound to the ribosome, or the SRP equivalent in Agrobacterium. If the new antigenic structure is the SRP equivalent our results would indicate that the SRP is closely associated to the 50S subunit. 54

REFERENCES

Cooksey, D.A., andL.W. Moore. 1982--High frequency spontaneous mutations to agrocin 84 resistance in Agrobacterium tumefaciens and A. rhizogenes. Physiol. Plant Pathol. 20:129-135.

DeBoer,S.H., R.J. Copeman, andH. Vruggink. 1979--Serogroups of Erwiniacarotovorapotato strains determined withdiffusible somatic antigens. Phytopathology. 69:316-319.

Hochster, R.M., and S.E. Cole. 1967--Serological comparison between strains of Agrobacterium tumefaciens. Can. J. Microbiol. 13: 569 -572.

Lewin, R. 1982--Surprising discovery witha smallRNA. Science. 218:777-778.

Moore, L.W., G. Warren, and G. Strobel. 1978--Plasmid involvement in the hairy-root infectioncaused by Agrobacterium rhizogenes. Proc. IVth Int. Conf. Plant Path. Bact., Angers, France. 1:127- 131.

Robert, W.P.,and A. Kerr. 1974--Crown gall induction: serological reactions,isozyme patterns and sensitivity to mitomycin C and to bacteriocin, of pathogenic and non-pathogenic strains of Agrobacterium radiobacter. Physiol. Plant Pathol. 4:81-91.

Schaad, N.W. 1976--Immunological comparison and characterization of ribosomes of Xanthomonas vesicatoria. Phytopathology. 66:770- 776.

Schnathorst,W.C., J.E. DeVay,and T. Kosuge. 1964--Virulence in Agrobacterium tumefaciens in relationto glycineattenuation, host, temperature,bacteriophage,and antigenicity of pathogen and host. in Proc. Conf. Abnormal Growth Plants. DeVay, J.E., and E.E. Wilson, eds. Cancer -Research Coordination Committee, University of California, Berkeley. 30-32.

Sciaky, D., A.L. Montoya, and M. Chilton. 1978--Fingerprintsof AgrobacteriumTi plasmids. Plasmid. 1:238-253.

Sonoki, S., and C.I. Kado. 1978--Proteins conferred by the virulence - specifying plasmid of Agrobacterium tumefaciens C58. Cell Biology. Proc. Nat. Acad. Sci., U.S.A. 75 (8):3796-3800.

Sine,S. 1982--Serological studies on agrobacteria. Int. Work. on Crown Gall (Agrobacterium tumefaciens), Wgdenswil, Switzerland. 11 pages. 55

Wittmann, N.G. 1974--Purification and identification of Escherichia coli ribosomal proteins. in Ribosomes. Nomura, M., A. Tissieres, and P. Lengyel, eds. Cold Spring Harbor Laboratory, Cold Spring Harbor, Massachusetts. 93-114.

Zaenen, I., N. Van Larebeke, H. Teuchy, M. VanMontagu, and J. Schell. 1974--Supercoiled circular DNA in crown gallinducing Agrobacterium strains. J. Mol. Biol. 86-109-127. 56

Figure 3.1-Ouchterlony double diffusion pattern showing the serological relationships of the tumorigenic strain M63/79 with the rhizogenic strain A4 and, the A4 mutant, A4R1 which lacks a major antigenic determinant. The center well contained antiserum to 50S ribosomal subunits of M63/79; outerwells contained ribosomes of A4 (1,4,6), A4R1 (2,3), and M63/79 (5). 57

Figure 3.2-Serological relationships of 50S subunits from the virulent wild type C58, its plasmid-deficient mutant NT1, the transformant A323, and K84. The center well contained antiserum to 50S subunits of C58; outer wells contained ribosomes ofC58 (1,5), A323 (2,6), NT1 (3,8), and K84 (4,7). 58

co co 9o0

Figure 3.3-Ouchterlony double diffusion pattern of phenol-extracted antigenstested against antiserum to 50S ribosomal subunits of C58 (center well); outer wells contained phenol-extracts of C58 (1,2,4),its plasmid-deficient mutant NT1 (3), the transformant A323 (5), and K84 (6). TABLE 3.1 Agrobacterium strains used as test-antigens

ONCO- STRAIN SOURCE GENECITY PLASMIDS Designation Moleculag WeightxlO

A. tumefaciensa C58 R. Dickey, Cornell University, pTi-058 120f5 New York

NT1 Heat treated C58 that has lost its pTi-plasmid - E. Nester, Univ. of Washington, Seattle

A323 Transformation of the plasmid- pAt-84b 1241'4 cured C58 (NT1) by a plasmid from K84 - E. Nester, Uni- versity of Washington, Seattle

A. rhizogenesb A4 R. Durbin, University of Wis- three consin, Madison plasmids

A4R1 An Agrocin-resistant mutant of one A4 that has lost the largest plasmid and smallest plasmids

A. radiobactera K84 A. Kerr, University of Adelaide, pAt-84a 3012 Australia pAt-84b 12414

From Sciaky, Montoya, and Chilton (1978) From Cooksey and Moore (1982) 60

SUMMARY AND CONCLUSIONS

Pecanorchardsin Georgia were affected by the crown gall disease and the infected trees were less vigorous than noninfected ones. From 18 gall samples, 81 Agrobacterium strains were isolated and typed. Nearly equalnumbers of biovar 1 and biovar 2 strains were isolated;but, about 73 percent of the pathogens were biovar 1 strain, mostof which were sensitive to agrocin 84. Infection of tomato plants in greenhouse tests by a representative biovar 1 strain was successfully preventedwith K84. It appearsthat K84 would prevent the majority of pathogenic agrobacteria from infecting pecan, however more than one fourth of the strains were resistant to agrocin

84. Putative bacterialantagonists that produced a large zone of inhibition against the agrocin-resistant reference strain were isolated from a potentially suppressive soil. These new putative antagonists,as wellas K84, should be tested on pecan trees under

Georgia field conditions.

The lack of a quick and reliable method for the identification of Agrobacterium prompted us to study the possible use of ribosomal serology. Antisera to 50S subunits of five tumorigenic strains from the three different biovars allowed us to arrange 31 agrobacteria and

6 rhizobia into16 serovars. The antigenic structureof the 31 agrobacteria did not correlate with either, the speciation based on the pathogenic state, the biovar affiliation, the area, nor the host of origin. The fast-growing rhizobia could not be immunologically 61 differenciated from the agrobacteria and were closely related to the biovar 3 strain CG64. The heterogeneity observed among 50S subunits of the agrobacteria and their close relatedness with 50S subunits of the rhizobia cast doubt on this method for rapid identification of the agrobacteria. However, someantisera showedstrainspecific antigens, thus they may prove useful for "tracking" the movement and population dynamic of the specific strain in a natural environment.

Membrane proteins associated with the presence of the pTi-058 plasmid was reported (Sonokiand Kado,1979)and the heterogeneity observed among 50Ssubunits ofagrobacteria prompted ustostudy whether plasmidswould affect theantigenic structureof the50S subunit. Striking immunological differences were observed when 50S subunits from the rhizogenic A4 and the tumorigenic C58 strains were compared to 50S subunits of their avirulent plasmid-deficient mutants, A4R1 and NT1 respectively. The plasmid-deficient strains always lacked one unique precipitin band. Interestingly, the additional band present in C58 was also present in the transformant

A323, but absent from K84. This additional immunogen is not specifically associated to either the pTi-058 nor the pAt-84b plasmid, but is more likely the product of a general response between the plasmid and the chromosome. Hypothetically, the "plasmid- chromosome" complex induces the formation of "new 50S subunits" that have an additionalunique antigenic structure. However, itis not yet known whether this new structure is a "true" constituantof the 62

"new 50S subunit," a closely bound protein, or the signal recognition particle (SRP) which acts as a transcient third ribosomal subunit in linking protein translation with export from the cell. 63

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