Serological Studies on Chloridazon-Degrading Bacteria

System. Appl. Mi crobi al. 4, 459-469 (1983)

Institut fiir Mikr obiologie, Univcrsirat H ohenheim, 7000 Stuttgart 70, Federa l Republic of Germany " Institut fiir Tierhygiene, Universita t H ohenheim, 7000 Stuttgart 70, Federal Republ ic of Germany

Serological Studies on Chloridazon-Degrading Bacteria GERLINDE LAYH, REINHARD BOHM ", JORGEN EBERSPACHER, and FRAN Z LINGENS

Received July 7, 1983

Summary Agglut ination tests and immunofluorescence tests with antisera against four stra ins of chloridazo n-degrading bacteria revealed the sero logical un iformity of a gro up of 22 chlor idazon-d egrading bacteria l stra ins. No serological relationship could be found betwe en chloridazon-degrading bacteria and representa tives of other Gram -negative bacteria. This was demon strated by agglutination tests, including testing of the ant iserum against Acinetobacter calcoaceticus, and by immunofluorescence tests, includ ing testing of the sera against Pseudom onas and Acinetobacter strains. T he tests were perform ed with 31 represent ati ves of different Gra m-negative bacteria, and with 22 strains of chloridazon-d egrading bacteri a as antige ns. Differences in the extent of agglutination reactions and ant ibod y titres among chlori dazon -degrad ing bacterial strai ns togeth er with cross-adso rpt ion xperiments, suggest a rough classification of chlori dazo n-degrad ing bacteria into two subg roups. On th e basis of immunofluoresce nce dat a, a linkage-map was wo rked out to represent sero logical relationships in the group of chloridazon -degra ding str ains.

Key words: Chloridazon-degrading bacteria - Degradation of aromatic compounds - Agglutination test - Immunofluorescence - Enzyme immunoassay Serological classification

Introduction Chlorida zon is the active ingredient of th e widel y used herb icide Pyrarnin". An enric hment technique, using soi l from different locations fro m all over the world, Abbreviatio ns: chloridazo n = 5-amino-4-chloro-2-phenyl-3(2H) -pyridazinone ant ipyrin = I-ph enyl-2,3-dimeth yl-pyrazolone pyrarnidon (aminopyrine) = I -phen yl-2,3-dimethyl-4-dim eth ylaminopyrazolon e

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G.Layh, RiBohm, j.Eberspacher, and F.Lingens

led to the isolation of 22 bacterial strains with the ability to utilize chloridazon or the structurally related analgesics antipyrin and pyramidon (aminopyrine) as the sole source of carbon (Eberspiicher and Lingens, 1981). Chloridazon-degrading bacteria are Gram-negative, non-motile, non-pigmented, and non-spore - forming aerobic cocci or coccobacilli of 1-2 {lm length and 0.7-1 {lm width. They occur singly, in pairs or short chains and the G + C-content of the 22 strains ranges between 65 and 68.5 mol %. The bacteria are highly specialized in nutrition with optimal growth on chloridazon or antipyrin and good growth on phenylalanine and phenylpropionare. The complex media usually applied in bacteriology do not support growth, and sugars, alcohols, acids or other simple carbon sources are not - or only very poorly utilized. In this paper serological studies on chloridazon-degrading bacteria are described. Immunological techniques are used to establish whether chloridazon-degrading bacteria are serologically related to other Gram-negative soil bacteria and whether the different strains are a serologically uniform group of bacteria.

Material and Methods Bacterial strains

Strains J1 and J2 of chloridazon-degrading bacteria were enriched in Denmark (Engvild and Jensen, 1969), all other strains were isolated at the Institut fur Mikrobiologie at Hohenheim. Strains A., All' A1 2 , A13 and A14 were isolated from soil of Germany (Blecher et al., 1978). Strain E from soil of Ecuador, South America tFrohner et al., 1970), strains K2 , K3 and K, from soil of Kenya, Africa (Lingens et al., 1977). Strains K2AP- and K3APare spontaneous mutants of strain K2 and K" respectively, having lost the ability to grow on antipyrin (Kreis et al., 1981). Strain L was isolated from soil of Limburgerhof, Germany, and strain R from Rothschwaige, Germany iFrohner et al., 1970), strain M ll from soil of Bad Wimpfen, Germany (Blecher et al., 1979), strains M 13 and M 1 5 from soil of Bad Wimpfen and Hohenheim, Germany (Blecher et al., 1981), strain N from soil of Nebraska, USA (Buck et al., 1979), and strains Z" Z., Z, and Z8from soil of Hohenheim, Germany (Blecher, 1980). Strain C2 was isolated from soil of Cardwell, Australia. The following representatives of Gram-negative bacteria were used: Acinetobacter calcoaceticus ATCC 23055, nine strains (12-1, 12-2, 16-2,22-1,22-4,26-1,27-1,28-1,28-2) of Acinetobacter calcoaceticus with the ability to utilize phenylalanine as sale carbon source isolated from soil by U.Tittmann, Achromobacter parvulus ATCC 4335, Alcaligenes faecalis ATCC 15557 and ATCC 8759, Caulobacter CI-13, Escherichia coli K 12, two Flavobacterium sp. P 16/1 and C 14/1, Pseudomonas acidovorans ATCC 15005, four Pseudomonas aeruginosa (ATCC 17934, ATCC 17933, NCTC 8506, NCTC 8203), Pseudomonas fluorescens ATCC 29574, Pseudomonas lemoignei ATCC 17989, Pseudomonas putida ATCC 23973, Pseudomonas sp. ATCC 13263, three Pseudomonas stutzeri (NCTC 10450, NCTC 10473, ATCC 19154), Pseudomonas testosteroni ATCC 17454 and Salmonella typhimurium 7-73. Acinetobacter calcoaceticus ATCC 23055 was obtained from the Deutsche Sammlung fur Mikroorganismen (DSM), all other ATCC-strains were directly obtained from the American Type Culture Collection, and were regularly transferred at the Institut fur Mikrobiologie, Hohenheim. All NCTC-strains were obtained from the National Collection of Type Cultures, and were regularly transferred at the Instirut fur Tierhygiene, Hohenheim.

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461

Media and growth conditions

Chloridazon-degrading bacteria were cultured in a mineral salts medium which containedper litre: 0.3 g KH 2PO"0.7 gNa 2HPO, . 12H 20,0.3 g (NH,)H 2PO"0.7 g (NH,)2HPO" 0.1 g (NH,),SO" 0.05 g CaCl 2 . 6H 20, 0.25 g MgSO, . 7H 20, 0.5 mg H 3B03 , 0.04 mg CuSO,' 5H 20, 0.1 mg K], 0.2 mg FeCI3 • 6H 20, 0.4 mg MnSO, . 4H 20, 0.4 mg ZnS04' 7H 20, 0.2 mg (NH')2Mo0 3 , 0.1 mg biotin, 0.03 mg vitamin B12 • As carbon source antipyrin (1.0 gil) was added, except for strains N, K2AP- and K3AP- which were grown on chloridazon (0.8 gil). All other bacteria were cultivated in a medium consisting of dehydrated nutrient broth (10 gil), Difco, Detroit, Michigan, USA, yeast extract (5 gil) and NaCI (5 gil). All bacterial strains were grown in 21 Erlenmeyer flasks at 30 DC on a rotary shaker at 120 rpm. Chloridazon-degrading bacteria were harvested after a growth period of three days, all other bacteria after 12 h. Preparations of antisera

Bacteria cultured as described above were inactivated by a 30 min treatment with formaldehyde (10% in water; w/v). After plating a sample of the bacterial suspension on either antipyrin or on nutrient agar plates no growth was observed. Bacteria were washed 5 times with a physiological NaCI solution (0.15 M). A bacterial suspension with 2 X 10" cells/ml was prepared in 0.15 M NaCI solution. This suspension was injected on alternate days into a rabbit's ear vein in the following amounts: 0.8, 1.0, 1.6, 1.6, 2.0, 2.0 and 2.0 ml (Chase, 1967). Sixteen days after the first injection the narcotized animals were bled by cardiac puncture. Clotted blood was centrifuged and the serum was filtered through a membrane filter (pore width 0.45 ,urn) yielding about 50 ml of antiserum per animal. Agglutination tests

Bacteria from agar slants were suspended in 0.15 M NaCI solution. The bacterial suspension with 2 x 10" cells/rnl (SId) was mixed with 5 III of the antiserum on a slide and agglutination was examined by light microscopy. For the determination of the antibodytitre 20,u1 of the bacterial suspension were mixed with 20ld of the antiserum in the well of a microtest-plate and then shaken for 10 min. The mixture was incubated at 30 DC for 30 min and then at 18 DC for 12 h. The antiserum was applied in serial dilutions from 1: 2 to 1: 1024. The titre is the reciprocal of the highest dilution where an agglutination reaction occurs. Enzyme immunoassay

Preparation of antigen and j erformance of the test was according to Bohm and Britzius (1982). A bacterial suspension with 2 x 10" cellsjml was prepared in sterile water. The suspension was ultrasonicated for 60 min with a Branson sonifier. Larger particles were allowed to settle for 12 h at 4 DC, thereafter 2/3 of the supernatant fluid was removed and used as antigen. The supernatant was diluted with sterile water to an absorption of 60% at 560 nm. Binding of the antigen on the surface of the wells was achieved by evaporating 80% of the fluid at 70 DC in an incubator. The residual solution was sucked off, the microtest-plates were washed twice with water and thereafter dried at 70 DC for 24 h. For the determination of the antibody-titre all wells of the microtest-plate were filled with 100,ul of a sterile salt solution (0.15 M NaCl, 0.03 M KCl, 0.0015 M KH 2PO, and 0.008 M Na 2HPO, in water). Antiserum (100 Ill, previously diluted 1: 10) was placed into the first row of the plate. After mixing, 100,ul were transferred from the first into the second row and so on, thus prod .cing serial dilutions of the serum from 1:20 to 1:40960. Serum of a non-immunized rabbit served as a control. The microtest-plate was sealed with a foil and incubated for 3 h at 37°C. The antisera were removed and the plates were rinsed four times for three minutes with a 0.05% solution of Tween 20 in water. Bound IgG-molecules were detected by incubating the test-plate at 37 DC for 1 h after the addition of 100 pI of peroxidase-labelled protein A. The commercially available product

462

G.Layh, R.B6hm, j.Eberspacher, and F.Lingens

from Sigma, Munich, was diluted 1: 800 prior to application. After incubation the supernatant was sucked off and the microtest-plate washed again four times for three minutes with Tween 20 (0.05% in water). Peroxidase-activity was assayed by adding 100,u1 of H 2 0 2 (0.05% in a buffer solution consisting of 0.008 M NaH 2PO'b 0.001 M Na 2HPO.! and 0.001 M EDT A) and 100 ,Ill of 5-aminosalicylic acid (0.1% in the buffer, mentioned above) into each well of the microtest-plate. For 30 min the plate, previously sealed with a foil, was shaken at room temperature and after another 12 h at the same temperature extinction at 492 nm was read with a Microelisa-minireader (MR 590) from Dynatech. Cross-adsorption experiments

The antisera were adsorbed on the cell surface of the bacteria by suspending pelleted bacteria in 1 ml of antiserum. The pellet was obtained by centrifuging 5 ml of a bacterial suspension (2 x 109 cells/ml) for 15 min at 10000 x g in the Eppendorf centrifuge No. 5414. The mixture of bacteria and serum was shaken for 10 min at room temperature, followed by an incubation of 30 min at 30 °C and a subsequent incubation of 12 h at 4 "C. The incubation mixture was centrifuged for 15 min at 10000 x g in an Eppendorf centrifuge and the supernatant was removed for the determination of the antibody titre with the agglutination test. Preparation of conjugated fluorescein-isothiocyanate labelled antibodies

Pure IgG was isolated from the immunesera by affinity chromatography with Protein-ASepharose, Pharmacia A.S., Sweden, according to Goding (1976). The conjugation of the immunoglobulins with fluorescein-isothiocyanate and the purification of the conjugates were performed according to Coons et al. (1941) with modifications as described by Bohm and Strauch (1973). Immunofluorescence test

Bacteria suspended in 0.15 M NaCl (cell density 2 x 108 czIlsjml) were dropped on slides, dried in air and fixed by heat. Conjugate (50 ,Ill) was then place on the slide and incubated in a moist chamber at 37°C for 30 min. The optimal use-dilution of every conjugate was determined in preliminary experiments. The slides were first rinsed with water and then washed twice for 10 min with 0.02 M sodium phosphate buffer, pH 8. The slides were dried, a drop of phosphate buffered glycerol (pH 7.2) was added and covered with a cover slip. Controls included unstained smears and smears stained with conjugates from the serum of a non-immunized rabbit. Specificity of each conjugate against the homologous strain was controlled by incubating the smears first with unlabelled and then with labelled antiserum from the same rabbit. The smears were examined under a fluorescence microscope, Typ Universal from Zeiss, Oberkochen, Germany, in the dark field with a 6CO fold magnification. Two persons independ ntly estimated fluorescence intensity in a procedure, based on a comparison between the fluorescence of the reference strain and the other bacterial smears, stained under the same conditions (Conway de Macario et aI., 1981; Fluck et aI., 1977). The results were recorded in a 5 step scheme as follows: a = no fluorescence; 1 = trace fluorescence, single bacterial cells cannot be seen, only coloured spots; 2 = diffusive fluorescence, the shape of the bacteria cannot be seen; 3 = fluorescence not as bright as at 4, but the bacterial shape is already marked by a bright green margin; 4 = bright fluorescence as with the positive control.

Results

Agglutination tests Antisera against four representative strains of chloridazon-degrading bacteria were prepared by injecting formalin-treated bacteria into rabbits. Strain E is the

Serology of Chloridazon-Degrading Bacteria

463

type strain and is deposited in the German collection of micro-organisms under DSM 1986. Strain N was chosen due to the results of metabolic studies (Buck et al. 1979) which showed that this strain was the only one to degrade phenylalanine without a lag-phase and did not grow on antipyrin. Strains K2 and M n were selected in the course of this serological study; M ll because it reacted differently than all other strains, and K2 because it was supposed to be a typical representative of a group of serologically related strains. Table 1. Results of the agglutination test with chloridazon-degrading bacteria, performed as described in Material and Methods Antigen (strain)

strain E

Rabbit-antiserum against strain M ll strain N strain K2

Acinetobacter calcoaceticus 22-4

A" Au, A", AI" M 13 ,

+

M", Z" Z" Z, Zs E

+ +

Mu L,R

J" J2' N

K" K3AP~, K, K" K,AP~ A' 2

+ +

+ + +

+

+

+

+ + + + +

+ +

+

The results of the agglutination test (Table 1) show cross-reactions for all strains except A12 with at least one type of antiserum. A serum sample taken from the rabbit before immunization as a control did not react with any strain of the chloridazon-degrading bacteria. The antiserum against the soil bacterium Acinetobacter calcoaceticus 22-4, prepared for further control experiments showed negative reactions with all strains of chloridazon-degrading bacteria. Thirtyone different strains of Gram-negative bacteria (10 Acinetobacter calcoaceticus strains, Achromobacter paruulus, 2 Alcaligenes faecalis strains, Caulobacter sp., Escherichia coli, 2 Flavobacterium sp ., Pseudomonas acidovorans, 4 Pseudomonas aeruginosa and 3 Pseudomonas stutzeri strains, Pseudomonas f/uorescens, Pseudomonas lemoignei, Pseudomonas putida, Pseudomonas sp., Pseudomonas testosteroni and Salmonella typhimurium) were examined with the antisera, mentioned above. As representatives of Gram-negative bacteria predominantly members of Pseudomonas and Acinetobacter were chosen, since chloridazon-degrading bacteria exhibit a certain degree of relationship to these genera. Not a single positive reaction was observed with antisera against chloridazon-degrading bacteria. Antiserum against Acinetobacter calcoaceticus 22-4 with a titre of 128, when tested with the homologous antigen, showed positive reactions only with the Acinetobacter calcoaceticus strains 22-4 and 26-1. For the antibody-titres of the four sera with their homologous antigens the following values were found: 512 for anti-E and 16 for anti-Kg, anti-Mj. and anti-No

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Owing to its high titre only the antiserum against strain E was applied for the determination of antibody-titres with other strains of chloridazon-degrading bacteria (Table 2). Sixteen strains with positive reactions in the agglutination test were examined. Table 2. Antibody-titres of chloridazon-degrading bacteria tested with antiserum against strain E in the agglutination test Strains

Titre (reciprocal of dilution)

E

512 255 128 64 32 8

A., Z5

Z.

Al 3 , Al " M l 3 , M l 5

A wZ7 Z8

K., K3AP~, K5 , L, R Al 2 , JD J2' K2 , K2AP-, N, M l1

4

o

Enzyme immunoassay When tested with their homologous antigens the four sera against chloridazondegrading bacteria showed a maximum extinction of 1.7 at 492 nm, if the antisera were applied in dilutions from 1: 20 to 1: 320. The next dilution step (1: 640) caused a decrease in extinction. Extinction values for the 1: 320 diluted antisera with their homologous and heterologous antigens are presented in Table 3. Table 3. Extinction at 492 nm in the enzyme immunoassay (see Material and Methods) with 1: 320 diluted antisera after reaction with homologous or heterologous antigens. Relative extinction in % in parentheses Antigen (strain) E K2 M l1 N

Rabbit-antiserum against strain K2 Mu

E 1.7 1.3 0.4 1.3

(100) (76) (23) (76)

1.1 (65) 1.7 (100) 0.4 (23) 1.3 (76)

0.6 (35) 0.5 (29) 1.7 (100) 0.7 (41)

N 1.2 (71) 1.2 (71) 0.5 (29) 1.7 (100)

Cross-adsorption experiments The antiserum against strain E, the only one with a reasonable high titre of 512 was adsorbed on ten representatives of chloridazon-degrading bacteria, having shown an agglutination reaction with this serum. By means of the agglutination test with each of these strains the titre was determined in the supernatant of the adsorption mixture (Table 4).

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Table 4. Cross adsorption experiment: antibody titre in the agglutination test after adsorption of antiserum against strain E Antigen (strain)

E A.

Z5 Z. Z7 Zs

K3,K5 , L, R

Rabbit-antiserum against strain E adsorbed with chloridazon-degrading bacteria (strain) K, Z, E L R K5 A. Z5 Z. Z7 16 0 0 0 0 0 0

64 0 0 0 0 0 0

32 0 0 0 0 0 0

64 0 0 0 0 0 0

64 0 0 0 0 0 0

64 8 32 4 0 0 0

256 256 64 16 16 0 0

256 255 32 32 32 0 0

256 256 8 32 32 0 0

256 256 32 32 32 0 0

Table 5. Results of the immunofluorescence test. Fluorescence intensity from 0 (no fluorescence) to 4 (brigth fluorescence). The values are averages of two independent experiments, each recorded by two observers Antigen (strain)

Fluorescence intensity of conjugate from rabbit-antiserum against strain K2 strain M" strain N strain E

As

4 3 3.5 3.5 4 3 4 3 3 3 3 3 3 3.5 3

All

AI 2 AI 3 Au

C2

E

JI J2

K2

K 2AP-

K3

K,AP-

K5 L

Mll M13

M I5 N R

Z5 Z. Z7 Zs

2

3.5 3 3.5 3 4 4 4 3

3 2 3 2.5 1.5 2

4 2 2.5 4 4

3 3

2.5 2 4 2 2 2 2 3 2.5 2 3

3.5 3.5 3.5 3 3 1.5 3 4 4 3 2.5 2.5 2 2 3 4 3 3.5 4 2.5 3 3 3 2.5

4 3 3.5 3.5 4 2.5 4 4

4 3 3 3 3

3 3 2 3

3 4 3 4 4

4 4

Immunofluorescence tests

Fluorescein labelled antibodies from the antisera against the chloridazon-degrading bacteria E, K2, M n , N against Acinetobacter calcoaceticus 22-4, Pseudomonas

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G. Layh, R. Bohrn,

J.Eberspacher, and F. Lingens

aeruglnosa NCTC 8203, Pseudomonas aeruginosa NCTC 8506, Pseud omonas stutzeri NCTC 10450 and Pseudomonas stutzeri NCTC 10473 were tested with chlorid azon-d egrading bacteria and with further 31 Gram-negative bacteria (listed in material and methods). Th e results in Table 5 indicate cross-reactions of chloridazon-degrading bacteri a with the conjugates from the four different stra ins. Due to the serological relati ons between the strains fluorescence intensities were found to vary from 1.5 to 4 dependin g on the antigen-antibody reaction. Th ese differences were utilized for a serological classification of chloridazon-degrading bacteria (see discussion ). No cross-reaction of the ot her 31 representatives of Gram-negative bacteria is observed with the conjugate from antiseru m against strain E.With conjugates against K2, M u and N ten Acinetobac ter calcoaceticus strains were tested and found to be negative. Again no cross-reaction takes place when chloridazon-degrading bacteri a are allowed to react with conjugates from anti sera against Acinetobacter calcoaceticus 22-4 and Pseudomonas aeruginosa NCTC 8203. Conjugates from ant isera against four strains of Pseudomonas (Pseudomonas aeruginosa NCTC 8203, Pseudomonas aeruginosa NCT C 8506, Pseudomonas stutze ri NCTC 10450 and Pseudomonas stut zeri NCT C 10473) were tested with th e corresponding Pseudom onas strains and with 4 representatives of the chlorid azon-d egrading bacteria (E, K2 , MIl and N). Pseudomonas conjugates only reacted wit h th eir homologous antigens and neith er with the other Pseudomonas strai ns nor wit h chloridazon-degrading bacteria. Conjugates fro m chloridazon-degrading bacteria did not reac t with any Pseudomonas strai n. Discussion By agglutination tests and with the aid of the immunofluorescence technique the different strains of chloridazon-d egrading soil bacteria were found to represent one gro up of immunologically related organisms. Repre sentatives of other Gram-negative bacteria did not show any immun ological reaction with chloridazo n-degrading bacteria. Th ese results are the basis for a rapid and specific demonstration of chloridazo n-degrading bacteria in natura l hab itats like soil samples with mixed popul ation s. Studies to develop a serological identific ation method are under progress. We hope, that this meth od will help to look for chloridazon -degrading bacteria in soil never treated with the herbicide, or to follow the process for the enrichment of these microorganisms. Although chloridazon-degrading bacteria are a serologically uniform group of organisms, differences are observed in the extent of immun e reactions between cert ain members of this group. In the agglutination test nin e strai ns (1\;, All> Ala, Au , MIa, M 15, Z5' Z6, Z7) reacted with every type of the four antisera, th ree strains (E, MIl, Zg) with three sera, five strai ns OJ> J2' L, N, R) with two sera , five strains (K2 , K2AP-, Ka, KaAP-, K5 ) with only one type of ant iserum and strain Al2 did not show any agglutination reaction . Correspondingly high titres with the antiserum against strain E were only found for str ains, which were positive with at least thr ee types of antisera. In the cross-adsorption experiment with the antiseru m against strain E (T able 4) again only the member s with high titres are able to block their mutual cross-reaction , the adsorption of strains with low titres, however, could not significantly reduce the reaction of the "well-agglutinating" group .

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467

The same titres for the four sera with the homologous antigens were determined in the enzyme immunoassay. This situation is in contrast to the results of the agglutination test. With the enzyme immunoassay the extent of antigen-antibody reaction is measured, whereas in the agglutination process the primary bindingreaction is followed by cross-linking of the antigen-antibody complexes to form macromolecular structures. Cross-linking may be inhibited by electrostatic or steric effects and steric hindrance in the reaction of bacterial cells as antigens has been reported to be caused by mucilage on the cell surface (Kauffmann, 1946). In accordance with this view a correlation exists between agglutination reaction and colony type of the four strains: strain E with good agglutination reaction and high titre has rough colonies, strains K2, M u and N with low titres have smooth colonies. Other strains with smooth colonies are also found to react poorly in the agglutination test and the well-agglutinating strains have rough colonies. The results of the immunofluorescence test were used for a classification of the chloridazon-degrading bacteria in serological sub-groups. Euclidian distances for each strain to all other strains were computed with the aid of immunofluorescence intensity values of Table 5. As a graphical tool to present the data-material a linkage-map according to Ohmayer et al., 1980, is shown in Figure 1. The following clusters can be discerned: strains K2, K2AP- form one cluster, strains Ks, KsAP-, K5 a second one, strains Au, L, MIS, M I5, R the third, strains 11' 12' N the fourth and strains At" AI2, AIs, Al4 , Z5' Zs, Zi the fifth cluster. Strain Zs is found in an intermediate position, strain C2, E and MIl in marginal positions. Clustering obtained by processing immunofluorescence data shows similarities to the situation of the agglutination test and the enzyme immunoassay. Thus strain M u was found to occupy a marginal position in the enzyme immunoassay (Table 3)

Fig. 1. Linkage map according to Ohmayer et al., 1980, constructed on the basis of Euclidian distances (data not shown), calculated from the immunofl orescence values of Table 5. Similarity levels are presented by unbroken lines for distances < 0.6 and by broken lines for distances of 0.6-0.8.

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G.Layh, R.B6hm, ].Eberspiicher, and F.Lingens

as well as in the linkage-map. All strains of the first, second and third cluster show poor agglutination reactions and the colony type is smooth, and strains of the fourth and fifth cluster, with the exception of Al2 , are found in the group with good agglutination reactions. The marginal position of strain E results from the exceptionally high immunofluorescence values of this strain with all antisera, on the other hand strain C2 is marginal in reacting poorly with every serum. Studies on the chemical nature of immunodeterminant groups shall verify the sub-group classification, presented here. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (DFG). We thank veterinary surgeon Eberhard llonicke for performing the immunization procedure. We are grateful to Dr. Herbert Seiler, Siiddeutsche Versuchs- und Forschungsanstalt fiir Milchwirtschaft, Weihenstephan, for linkage-map calculation by processing immunofluorescence data.

References Blecher, H., Blecher, R., Muller, R., Lingens, F.: Isolierung und Charakterisierung Antipyrin-abbauender Bakterien. Z. Naturforsch. 33c, 120-123 (1978) Blecher, R., Koch, V., Ballhause, B., Lingens, F.: MikrobieIIer und abiotischer Abbau des Triazinon-Herbizids Metamitron. Z. Pflanzenkr. Pflanzensch. 86, 93-102 (1979) Blecher, H.: MikrobieIIer Abbau von Amidopyrin und Phenazon. Dissertation, Tiibingen 1980 Blecher, H., Blecher, R., Wegst, W., Eberspdcher, [; Lingens, F.: Bacterial Degradation of Aminopyrine. Xenobiotica 11, 749-754 (1981) Bohm, R., Strauch, D.: Ein fluoreszenz-serologischer Mikrokulturnachweis von Bacillus anthracis auf Membranfiltern als SporenschneIInachweis. Wien. tierarztl. Mschr. 60, 327-332 (1973) Bohm, R., Britzius, E.: Eine einfache Mikromethode zur Bestimmung des Gehaltes von Kaninchenimmunserum an spezifischem IgG gegen bakterielle somatische Antigene mit Hilfe der Enzyrn-Immunotechnik. ZbI. Vet.-Med. B 29, 242-247 (1982) Buck, R., Eberspdcher, f., Lingens, F.: Abbau und Biosynthese von L-Phenylalanin in Chloridazon-abbauenden Bakterien. Hoppe-Seyler's Z. physioi. Chern. 360, 957-969 (1979) Chase, W.: Methods in Immunology and Immunchemistry, p. 197-224. New York-London, Academic Press 1967 Conway, E. de Macario, Wolin, M. l-, Macario, A. f. L.: Immunology of Archaebacteria that Produce Methane Gas. Science 214, 74-75 (1981) Coons, A.H., Creech, H.]., [ones, R.N.: Immunological properties of an antibody containing a fluorescent group. Proc. Soc. expo BioI. (N. Y.) 47,200-202 (1941) Eberspdcber, f., Lingens, F.: Microbial degradation of the herbicide chloridazon, p. 271285 in: Microbial degradation of xenobiotics and recalcitrant compounds (T.Leisinger, A.M.Cook, Ri Iliitter, ].Nuesch, eds.). London-New York-Toronto-Sydney-San Francisco, Academic Press 1981 Enguild, K. c., jensen, H. L.: Microbial decomposition of the herbicide pyrazon. Soil BioI. Biochem. 1,295-300 (1969) Fluck, R., Bohm, R., Strauch, D.: Fluoreszenzserologische Untersuchungen von Kreuzreaktionen zwischen Sporen von Bacillus antbracis und Sporen anderer aerober Sporenbildner. ZbI. Vet.-Med. B 24, 497-507 (1977)

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Frbhner, c., Oltmanns, 0 ., Lingens, F.: Isolierung und Charakrerisierung Pyrazon-abbauender Bakterien. Arch. Mik robi oJ. 74, 82-89 (1970) Goding, ]. W.: Conjugation of ant ibodies with fluorochromes: Modificati ons to the standard methods. ]. Immunol. Meth. 13, 215-2 26 (1976) Kauffmann, F.: The serology of th e coli group. ]. Immunol. 57, 71- 100 (1946) Kreis, M., Eberspdcher, ]., Lingens, F.: Detection and char acterization of plasmids in chlor idazon and antipyrin degrading bacteria. Zbl. Bakt. H yg., I. Abr. Ori g. C 2, 45-60 (1981) Lingens, F., Blecher, R. , Blecher, H., Koch, U. : Isolierung Chloridaz on-abbauender Bodenbakterien aus ostafrikanischen Bodenproben (Kenia). Z. Pflanzenkr. Pflanzensch. 84, 684-690 (1977) Ohmayer, G., Precht, M ., Seiler, 1-1., Busse, M . : Linkage-map and th eir Relat ions to Linkage Cluster Procedures. Zbl. Bakt. , II.Abt. 135, 22-3 7 (1980) Prof. Dr. F.Lingens, Institut fiir Mikrobiologie, Universit ar Hoh cnheim, Garbenstr. 30, 0-7000 Stuttgart 70