CERTAINDATACOI'ITAINEDINTHISDOCUMENTMAYBEDIFFICULTTOREAD

IN MICROFICHEPRODUCTS"

DOE/ER/14029--2

Grant # DE92 014590PROGRF.SS REPORT June 1989-June 1991

We have realized many of the goals of our proposed research in a timely fashion.For the sake of this review, we are restating here, verbatim, the objectives stated in ouroriginal proposal:

1. To characterize, at a structural level, the differences between the lipopolysaccharidesof a representative number of strains from different Rhizobium species to determinewhich features of LPS structure are species-specific and might, therefore, bedeterminants of host sPecificity.

2. Determine the effect(s) of nod gene induction on the structure of Rhizobiumlipopolysaccharides and determine whether synthesis of a modified LPS molecule or anew surface glycoconjugate is initiated by nod gene induction.

3. Develop a non-chemical means for rapidly screening large numbers of bacterial 'strains in order to determine which glycoconjugate structural features are conservedbetween strains of the same species.

4. Provide the necessary structural information which, when coupled with developmentsin the rapidly expanding field of Rhizobium genetics, should lead to a clearunderstanding of the role of Rhizobium surface glycoconjugates in host/symbiontinteractions.

Over the past two years, we have succeeded in isolating and purifyinglipopolysact_haridesfrom the cell surfaces of several bacteria from the species R. trifolii, R.leguminosarum and R. meliloti. We have determined that there are some very strikingsimilarities in the fatty acyl components of these species and the other Rhizobial species since

, they ali contain a variety of 3-hydroxy fatty acids as well as the usual long-chain fatty acid,27-hydroxyoctacosanic acid (Appendix I). A reprint of our article describing a lipid Acomponent containing this is also appended (Appendix 2). More ideas on our general

' perspective of the structure and function of gram negative bacterial outer membranes ininteraction with eukaryotic systems appear in Appendix 3.

A novel core tetrasaccharide we reported on in the Journal of Biological Chemistry(Hollingsworth, Carlson, Garcia and Gage, 1989, 264, 9294-9299)has been found in both R.trifolii, R. phaseoli and R. leguminosarum strains. However, we have ascertained that theactual quantities produced do depend on the exact growth phase of the bacteria and alsodepend on environmental factors. The bacterial cell surface has emerged as a complex,dynamic mosaic of carbohydrate structures which must be carefully defined to adocumentable metabolic state in defined environmental conditions. We now know that,

several different types of lipopolysaccharides are synthesized at once by cells in a givenculture. The major question that arises is whether the different LPS types appear on the cellor whether these variations reflect population differences. Using immunofluorescencemicroscopy, we have documented that the latter is often the case. We have documented that

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different LPS types are made and can be resolved by SDS polyacrylamide gel electrophoresis, and hydrophobic, gel filtration chromatography (Figure 1) and by regular gel filtration

chromatography. We have also demonstrated that the relative proportions of these LPS typescan be altered by simply adding flavones which aet as nod gene inducers (Figures 2 and 3).Similar experiments using alterations in pH, oxygen concentration or high sueeinate (whichinduces bacteroid formation) have already been initiated and are a fairly advanced stage. Wehav,e selectively hydrolyzed the lipopolysaecharide of R. leguminosarum to yield an "O-antigen" fraction, a tetrasaceharide and a trisaccharide (Figure 4). We have found that theexact quantifies of these components are altered dramatically by altering environmentalfactors such as pH, oxygen tension carbon source or host factor concentrations. (Figures 4and 5.) We synthesize,polymer antigens incorporating the trisaccharide or tetrasaeeharidesby making allyl glycosides of the components and performing a copolymerization withacrylamide to give structures _1and 2 respectively. The O-antigen fragments from cellsgrown under normal conditions (pH 7 in BIII medium) were separated into two distinctfragments and were also integrated separately into polymer matrices. Antibodies to thesefragments were raised in rabbits and then used to study the spatial and population distributionof surface antigens of bacteria grown under different environmental conditions (see colorpanels at the end of this report). In these panels plates on the left are phase-contrast light'microscopy pictures and plates on the fight are fluorescence pictures of the same field usfnga secondary labelling method fluorescence labelled goat anti-rabbit antibody. Plate A1 showsa phase-contrast picture of cells grown in Bergensens medium (liquid) labelled with an antiO-antigen fragment antibody. A1 shows the san_,lefield using fluorescence. Note that thisepitope is not aece._sibleon these cells except for at the very tips. We think that this iscaused by blocking by the capsule. This might explain the phenomenon of "polarattachment" of bacteria to root hairs. The antibody used in Plate B was raised to thetrisaccharide copolymer !. Note again the polar localization of this antigen in thefluorescence field (B2) indicated by the arrow. We expect to have anti-capsule antibody in 3weeks in order to address the question of encapsulation. When cells are grown in thepresence of succinate, the tetrasaeeharide antigen 2 becomes the dominant antigen (comparedto normal cells) as shown in Figure 4. Note how the ant;body to the tetrasaccharide reaet_with ali of the cells in the field (Plates C1 and C2) in this case. The trisaccharide is now

" minor (Figure 4) and there is weak labelling by this antibody (DI, D2). The anti-O-antigenantibody reacts quite well with ali of the cells (El and E2). We have isolated a very highmolecular glucan similar to the small, cytoplasmic, cyclic/3-1,2-glucans from cells grown :insuccinate. These small glucans have been the subject of the excellent work of the Kennedyand, now, Miller groups and we keep looking forward for the point of convergence withtheir work and ours. In cells grown under low oxygen, the major event is the loss ofcapsule. Both O-antigen epitopes are now available for binding (F1, F2 and G1, G2) as wellas the trisaccharide (K1, K2). The O-antigen is now the dominant antigen. Note that ali ofthe cells fluoresce strongly. Compare this with the situation in the Al, A2 and Bl, B2 pairswhere the cells do not fluoresce because they are completely covered by capsule. When cellsare grown at pH 4.5 the proportion of O-antigen diminishes considerably (Figure 5) and thetrisaccharide disappears. In accordance with this, there was low reaction to cells grownunder these conditions with the anti-trisaccharide antibody (J1,J2). The O-antigen epitopeswere located only on the bacteroid forms!!! (Hl amdH2.) Bacteroides are induced simply by

, lowering pH and are easily distinguished from vegetative cells by their larger size in plate

2-

al11 •

(Hl). Note the intense fluorescence due to the O-antigen antibody binding in H2 to the Y-shaped bacteroides. The tetrasaecharide is uniformly distributed (II, 12). Our work on theeffects of flavone is also very advanced and difficult to cover here inside of page limitations.We have also engaged on double and triple labeling experiments and are beginning ourelectron microscopy work using immunogold conjugates.

It appears that this tetrasaccharide and a trisaeehaxide that we reported on earlier areobligatory for proper nodule and infection thread development (Carlson, Gareia, Noel,Hollingsworth (1989) Carbohydr. Res. 195 101-110). We have determined that this peculiarproperty of switching surface antigens in response to environmental influences is animportant aspect of the symbiosis. Using the R. meliloti system, we and collaborators havedemonstrated that there are at least three loci which regulate the surface presented by thebacterium (Appendices 4 and 5) by controlling biosynthesis of LPS types. We are carryingout parallel work in R. trifolii ANU843.

Much has been said recently about the roles of certain excreted, sulphated,lipopolysaccharides in the nodulation process. We have stated and still maintain that suchmolecules are probably localized on the bacterial cell surface. This has proven to be aminority opinion. We do not disagree with the nod RM1 model, but our very strong roots instructural and mechanistic chemistry and our understanding of membrane phenomena raisesseveral points which have to be addressed and answers to which, we feel, will advance thefield and lead to a hasty crystallization of ideas. We are indeed excited that a carbohydratemolecule may be at the heart of the entire recognition process. We have recentlydemonstrated that there are, indeed, several sulphated, lipid,linked carbohydrates on the cellsurface R. meliloti (manuscript under review). We have addressed the possibility of nodRM1 precursors surface bound by synthesizing an immunogen with the terminal nod RM1residue. This will be used to raise antibodies for addressing this question_t,_._t 0) •

We now have a very good picture of the surface chemistry of R. leguminosarum 300compared to R. trifolii ANU843. We have isolated and purified representative cell surfacecarbohydrate components from each strain. Some of these have been fully characterized andsome only partially. We have succeeded in integrating these purified oligomer into largepolymers to make synthetic antigens. Over the next few months, we will expand thisapproach to a few more representative stains and, using the antibodies so generated, begin

" screening of a large number of common wild types.Some interesting and important carbohydrate chemistry has developed from our work,

'- In this course of developing chemical syntheses for the 3-hydroxy fatty acids found inRhizobial strains, we developed a means of synthesizing the valuable intermediate (S)-3, 4-dihydroxybutanoic acid from maltose, starch or maltodextrins. In addition, we havedeveloped a general method for activating the anomeric position of sialic acids and theirliposaccharides for further conjunction to complex matrices for antibody production. Wehave also developed a sensitive method for sequencing carbohydrates by mass spectrometry_hich we have perfected on test peptides (Appendix VI) as a sensitivity enhancementbenchmark and as a contribution to the D.O.E.-funded high field MS facility. Ourcarbohydrate work will be submitted soon.

Our analyses of mutants lacking the ability to carry out certain aspects of the infectionprocess continue to show a clear correlation between phenotype and surface chemistry. Thisis true of the R. leguminosarum Vf39 system (Figure 6 and 7) where we have found that theinability of certain mutants to be released from infection threads is due to LPS defects. The

3

4

. pH 7.0, normal aeration. Succinate _s q low oxygenBergensen's medium, carbon source tension.

LPS isolated with phenol/

, water and chromatographed

on SEPHAROSE 4B. SEPH 4B

ii :::{ l,!2:-giluCan _I/ S

/. _

%

BIOGEL P2

N iiii!!!iii ...

f I" T'" trisac - tri

charide trisaccharidetetrasac -

. .charide tetra

O- an t±gen tet ra s ac char id e

O-antigenO-antigen

NOTE INCREASE IN THE RELA-

AMOUNT OF TETRASACCHARIDE.

Figure 4 Gel Filtration profile of lipopolysaccharide isolates from Rhizobiumleguminosarum 300. The shaded peaks were hydrolyzed with dilute acid andthe carbohydrates further separated on Biogel P2 gel filtrationchromatography. The plots are off total carbohydrate versus fraction number.Positions of elution for O-antigen tetrasaccharide and trisaccharide antigens are

, indicated. 5

i pH 4.5 Succinate/ low pH, low phenol layer from

oxygen, narigenin, succinate/ low pH,' low oxygen, narigenin

SEPH 4B SE

i<

]......................... \H+/ H20 H+/ H20 H+/ N20

Biogel P2 Biogel P2 Biogel P2

". ' accharide t_'isaccharide tetrasaccharide

tetrasacch :etrasaccharide 'O-antigen

*-antigen O-antigen

Figure 5 Gel Filtration profile of lipopolysaccharide i_lates from Rhizobiumleguminosarum 300. The shaded peaks were hydrolyzed with dilute acid andthe carbohydrates further separated on Biogel P2 gel filtrationchromatography. The plots are off total carbohydrate versus fraction number.Positions of elution for O-antigen tetrasaccharide and trisaccharide antigens are

" indicated.

6

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lipopolysaccharic e

2) BIOGEB P2 , '

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trisaccharide ri- tri-

tetrasaccharide tetra- tetra-O-ant.

O-antigen O-ant.

FIGURES 6, 7 Gel Filtration profile of'lipopolysaccharide isolates from Rhizobiumleguminosarum VF-39 and nodulation defective mutants. The shaded peakswere hydrolyzed with dilute acid and the carbohydrates further separated onBiogel P2 gel filtration chromatography. The plots are off total carbohydrateversus fraction number. Positions of elution for O-antigen tetrasaccharide andtrisaccharide antigens are indicated.

q

t

R _- leguminosarum VF39-86A

SEPHAROSE 4B

Peak 1 is an all peaks are new several oligomers

O-antigen fragment are present in the

the other peaks are major peak.

being characterized.

Figure 7

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R. leguminosarum VF39-32

SEPHAROSE 4B

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Fig 7 (continued)

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