279
Immunology Today, vol. 3, No. 10, 7982
The immunology of methanogens" a new development in microbial biotechnology Alberto J. L. Macario and Everly Conway de Macario Laboratory Medicine Institute, Division of Laboratories and Research, New York State Department of Health, Albany, NY 12201, U.S.A. 77ze immunology of melhanogens is an ir, portant area of research into biotechnology-bioengineering involving lhese bacteria. Here, recent findings regarding lhe antzgenie characteristics qf methanogens are discussed, emphasizing the present availability of antibody probes and immunol%dcal methods for their ider~tifieation, classijicalion, and slructural analylis.
In the last tew y e a r s there h a s b e e n a r e - e v a l u a t i o n of t h e g r o u p of p r o k a r y o t e s k n o w n as t h e A r c h a e b a c t e r i a I 4. A r c h a e b a c t e r i a are q u i t e h e t e r o g e n e o u s , b u t t h e y have c h a r a c t e r i s t i c s t h a t u n i f y t h e m w i t h i n a single biological d o m a i n a n d s e p a r a t e t h e m from Eub a c t e r i a a n d E u k a r y o t e s . For all living o r g a n i s m s , therefore, t h r e e e v o l u t i o n a r y lines of d e s c e n t (kingd o m s ) have b e e n p r o p o s e d : E u k a r y o t e s , E u b a c t e r i a a n d the n e w l y defined A r c h a e b a c t e r i a . T h e m o l e c u l a r biology s u p p o r t i n g this p r o p o s a P 4 a n d its implic a t i o n s for o u r u n d e r s t a n d i n g of the e v o l u t i o n of proa n d e u - k a r y o t e s , have b e e n extensively d i s c u s s e d ~ ').
Methanogens: key elements in the forthcoming m i c r o b i a l revolution T h e m i c r o b i a l revolution p r e d i c t e d for thc 1980s will be c e n t r e d a r o u n d t h e intensive a n d varied use of m i c r o b e s for c o p i n g with w o r l d w i d e p r o b l e m s , especially t h o s e s t e m i n g from e n e r g y a n d food s h o r t a g e s a n d e n v i r o n m e n t a l p o l l u t i o n 1°-~3. T h e anaerobic Archaebacteria, notably those producing m e t h a n e (the m e t h a n o g e n s ) , are e x p e c t e d to have a vital role in t h e s e d e v e l o p m e n t s ~°qs. M e t h a n o g e n s are p l e i o m n r p h i c : s m a l l a n d large cocci, rods of various lengths, a n d spirilla have b e e n f o u n d 2,s,ls,19. T h e y also s h o w a n a r r a y of 16S r R N A c o d o n catalogs ~-4 a n d cell-wall c o m p o s i t i o n s s,~9. Yet m u c h of their genealogy, ecology a n d c o n t r i b u t i o n to t h e e n v i r o n m e n t r e m a i n s u n k n o w n 3,4,7,s, a n d until recently k n o w l e d g e of their a n t i g e n i c i t y a n d i m m u n o genici{y w a s poor 2° 22 But s t u d y of t h e i m m u n o l o g y of m e t h a n o g e n s h a s n o w b e g u n a n d in this article we c o n s i d e r the w a y s in w h i c h t h a t k n o w l e d g e will cont r i b u t e to t h e u s e of m e t h a n o g e n s .
Antibody probes for methanogens R e c e n t l y , w i t h M. J. W o l i n of t h e E n v i r o m n e n t a l Health Institute, we have b e g u n to p r e p a r e standardized antibody probes and calibrate immunoc h e m i c a l m e t h o d s for the a n a l y s i s of t h e s e microo r g a n i s m s , t a k i n g a d v a n t a g e of h y b r i d o m a technology 23,24 to a p p l y m o n o c l o n a l a n t i b o d i e s , p e r h a p s for t h e first time, to s y s t e m a t i c bacteriology 25 3o.
A n t i b o d i e s were raised, in r a b b i t s a n d mice, to all t h e m e t h a n o g e n s available in p u r e c u l t u r e z2,25,2G. A c o m p r e h e n s i v e p a n e l of s t a n d a r d i z e d rabbit a n t i s e r a to 22 m e t h a n o g e n s - p r a c t i c a l l y all the e x t a n t species - w a s a s s e m b l e d ( T a b l e I). I m m u n o f l u o r e s c e n c e a n d immunoenzymatic techniques and other immunological m e t h o d s were d e v e l o p e d in parallel 25-$°. TABLE I. Panel of antisera to methanogensa Antiserum numberb
l 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Immunizing bacterium c species
Methanobrevibactersmithii Melhanobaderiumfi.micicum Melhanosarcina bar,~eri Melhanobacleriumbryanlii Methanobacteriumbryanlii Melhanorarcinaharkeri Melhanospirillum hungalei Methanobrevibacterruminantium Methanobrevibacterarbor~phi/us Methanobrevibacter~mithii "MethanobacleriumY~e~moauiotr@hicum Melhanobacleffum thermoautolr@hicum Melhanococcusvannielii Methanococcusvogtae Melhanogeniummarisnigri Melhanosarcinaharkeri Methanogeniumcariaci Methanococcusmazei Methanosarcinabar/teri Methanosarcina Methanobrevibacterarborz/)hitus Methanobrevibaclerarboriphilus
strain PS MF MS Moll MoHG R 1M3 JFI M1 Dttl ALI GC 1 AH SB PS v JR1 ITI 227 JRI c MC6 W TM1 AZ DC
a This panel includes all antisera prepared and characterized up to .July 1981. New antisera which are being raised to other methanogens, will be numbered continuing the present series as they become available. b Antisera were numbered in the order of their preparation. Thus, antiserum No. 1 was obtained first, and antiserum No. 2 was obtained from a rabbit immunized after the donor of No. I antiserum, and so on. Moreover, each antiserum was defined by the rabbit letter and number, bleeding serial number and date, and vial letter t0r the serum bank records 2C'. c Complete infbrmation and relevant references regarding the immunizing microorganism can be found in references 2, 25, and 26. ~ElsevicrBiomedicalPress1982 0167~919/82/00000000/$t.00
280 Each antiserum was titrated against the immunizing bacterium and from each titration curve three dilutions were selected as probes for analysis 26,2v. T h e T probe (T from titer) is the antiserum titer, i.e. the highest dilution at which an a n t i g e n - a n t i b o d y reaction is still discernible. The S probe (S from screening) is the highest dilution at which the antiserum gives a maximal reaction, i.e. the last dilution of the titration curve's plateau. It was used in our initial surveys to screen methanogens for cross-reactions. The R probe (R from relatedness) is the optimal dilution for disclosing antigenic relatedness among methanogens. It is eightfold more concentrated than the S probe and higher antiserum concentrations do nut reveal relationships that have not been shown by it. Thus, the R probe is the most convenient tbr disclosing antigenic similarities among methanogens and for finding and counting these bacteria in samples from landfills, digesters, sludges, marine and lake sediments, etc., as described in following sections. T h e specificity spectrum of the three probes for the 22 rabbit antisera was determined. The T probe was found to be monospecific for the immunizing homologous bacterium 26. The S probe from some antisera cross reacted with a variable n u m b e r of methanogens. These cross-reactions were strong with strains of the same species of the immunizing bacterium and weak with methanogens of different species 25-2v. lnterfamily cross-reactions were not observed. Because of these cross-reaction characteristics, the S probe was choscn as a rapid means of assigning a new isolate to a particular species and family before the application of more definitive but cumbersome procedures (e.g. rRNA sequencingl-5).
The antigenic fingerprint of methanogens By conducting a multiple assay using the S probe and all the antisera the pattern of reactivity or antigenic fingerprint of each methanogen is revealed (some examples are shown in T a b l e II). Similar fingerprints can also be obtained with the R probe (unpublished results). T h e fingerprint itself is the degree to which a bacterium reacts with the probes, either positively or negatively. If the reaction is positive, the intensity is scored from 1 to 4, as measured by indirect immunofluorescence under standardized conditions >,2c'. Therefore, an antigenic fingerprint is a combination of the digits 0 (no reaction or negative), i, 2, 3 and 4. As a result of antigenic fingerprinting a n u m b e r of cross-reactions were discovered. W h e n all the fingerprints were arranged to conform the classification of methanogens 2 and the strains listed accordingly, two m a i n features emerged25: t h e r e were no crossreactions between members of different families, but there were four clusters of antigcnically related strains. These clusters coincided with families defined on the basis of comparative r R N A sequencing ~.
Immunology 7?)day, vof 3, No. It), 1982
Monoclonal antibodies to methanogens Once the cross-reactive clusters were identified, representative species from each cluster were selected for the preparation of monoclonal antibodies. In the process of generating hybridomas, the need to measure anti-methanogen antibodies in small, dilute samples from master- and cloning-plate microcultures emerged. Since no reliable technique was available, a slide immunoenzymatic assay, which proved to be sensitive and reproducible, was developed 26. Its outstanding features are the absence of background and of false-positive a n d false-negative results. With this method at hand, it was possible to form many hybrid cell lines producing monoclonal antibodies to several methanngens >,30. T h e following lines are now available: 16 that react with Methanospiri[lum hungatei JF1, 17 with Melflanococcu.r vannielii SB, 9 with Methanococcus ~oltae PSv, 7 with Methanohrevibacler arbor@hilus DH1, 6 with Melhanobacterium formicicum MF, 27 with Methanobacterium lhermoaulolr@hicum AH and 14 with Methanobaclerium thermoautotrophicum GC1. A systematic analysis of methanogens using monoclonal antibodies has.just begun, but it is already clear that it will provide insights into the surface structure of these unique microorganisms. Specific markers for strains, species, and genera, and perhaps for higher taxonomic groups are likely to be found. O t h e r applications of the monoclonal antibodies in research and biotechnology involving methanogens will be discussed below.
Structural analysis of methanogens by using monoclonal antibody probes Characterization of surface markers and elucidation of the cell-wall structure of methanogens can now he achieved quickly with monoclonal antibodies. These refined probes complement polyclonal antisera and standard immunochemical 2
Immunology Today, vol. 3, No. 10, 7982
The immunology of methanogens" a new development in microbial biotechnology Alberto J. L. Macario and Everly Conway de Macario Laboratory Medicine Institute, Division of Laboratories and Research, New York State Department of Health, Albany, NY 12201, U.S.A. 77ze immunology of melhanogens is an ir, portant area of research into biotechnology-bioengineering involving lhese bacteria. Here, recent findings regarding lhe antzgenie characteristics qf methanogens are discussed, emphasizing the present availability of antibody probes and immunol%dcal methods for their ider~tifieation, classijicalion, and slructural analylis.
In the last tew y e a r s there h a s b e e n a r e - e v a l u a t i o n of t h e g r o u p of p r o k a r y o t e s k n o w n as t h e A r c h a e b a c t e r i a I 4. A r c h a e b a c t e r i a are q u i t e h e t e r o g e n e o u s , b u t t h e y have c h a r a c t e r i s t i c s t h a t u n i f y t h e m w i t h i n a single biological d o m a i n a n d s e p a r a t e t h e m from Eub a c t e r i a a n d E u k a r y o t e s . For all living o r g a n i s m s , therefore, t h r e e e v o l u t i o n a r y lines of d e s c e n t (kingd o m s ) have b e e n p r o p o s e d : E u k a r y o t e s , E u b a c t e r i a a n d the n e w l y defined A r c h a e b a c t e r i a . T h e m o l e c u l a r biology s u p p o r t i n g this p r o p o s a P 4 a n d its implic a t i o n s for o u r u n d e r s t a n d i n g of the e v o l u t i o n of proa n d e u - k a r y o t e s , have b e e n extensively d i s c u s s e d ~ ').
Methanogens: key elements in the forthcoming m i c r o b i a l revolution T h e m i c r o b i a l revolution p r e d i c t e d for thc 1980s will be c e n t r e d a r o u n d t h e intensive a n d varied use of m i c r o b e s for c o p i n g with w o r l d w i d e p r o b l e m s , especially t h o s e s t e m i n g from e n e r g y a n d food s h o r t a g e s a n d e n v i r o n m e n t a l p o l l u t i o n 1°-~3. T h e anaerobic Archaebacteria, notably those producing m e t h a n e (the m e t h a n o g e n s ) , are e x p e c t e d to have a vital role in t h e s e d e v e l o p m e n t s ~°qs. M e t h a n o g e n s are p l e i o m n r p h i c : s m a l l a n d large cocci, rods of various lengths, a n d spirilla have b e e n f o u n d 2,s,ls,19. T h e y also s h o w a n a r r a y of 16S r R N A c o d o n catalogs ~-4 a n d cell-wall c o m p o s i t i o n s s,~9. Yet m u c h of their genealogy, ecology a n d c o n t r i b u t i o n to t h e e n v i r o n m e n t r e m a i n s u n k n o w n 3,4,7,s, a n d until recently k n o w l e d g e of their a n t i g e n i c i t y a n d i m m u n o genici{y w a s poor 2° 22 But s t u d y of t h e i m m u n o l o g y of m e t h a n o g e n s h a s n o w b e g u n a n d in this article we c o n s i d e r the w a y s in w h i c h t h a t k n o w l e d g e will cont r i b u t e to t h e u s e of m e t h a n o g e n s .
Antibody probes for methanogens R e c e n t l y , w i t h M. J. W o l i n of t h e E n v i r o m n e n t a l Health Institute, we have b e g u n to p r e p a r e standardized antibody probes and calibrate immunoc h e m i c a l m e t h o d s for the a n a l y s i s of t h e s e microo r g a n i s m s , t a k i n g a d v a n t a g e of h y b r i d o m a technology 23,24 to a p p l y m o n o c l o n a l a n t i b o d i e s , p e r h a p s for t h e first time, to s y s t e m a t i c bacteriology 25 3o.
A n t i b o d i e s were raised, in r a b b i t s a n d mice, to all t h e m e t h a n o g e n s available in p u r e c u l t u r e z2,25,2G. A c o m p r e h e n s i v e p a n e l of s t a n d a r d i z e d rabbit a n t i s e r a to 22 m e t h a n o g e n s - p r a c t i c a l l y all the e x t a n t species - w a s a s s e m b l e d ( T a b l e I). I m m u n o f l u o r e s c e n c e a n d immunoenzymatic techniques and other immunological m e t h o d s were d e v e l o p e d in parallel 25-$°. TABLE I. Panel of antisera to methanogensa Antiserum numberb
l 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Immunizing bacterium c species
Methanobrevibactersmithii Melhanobaderiumfi.micicum Melhanosarcina bar,~eri Melhanobacleriumbryanlii Methanobacteriumbryanlii Melhanorarcinaharkeri Melhanospirillum hungalei Methanobrevibacterruminantium Methanobrevibacterarbor~phi/us Methanobrevibacter~mithii "MethanobacleriumY~e~moauiotr@hicum Melhanobacleffum thermoautolr@hicum Melhanococcusvannielii Methanococcusvogtae Melhanogeniummarisnigri Melhanosarcinaharkeri Methanogeniumcariaci Methanococcusmazei Methanosarcinabar/teri Methanosarcina Methanobrevibacterarborz/)hitus Methanobrevibaclerarboriphilus
strain PS MF MS Moll MoHG R 1M3 JFI M1 Dttl ALI GC 1 AH SB PS v JR1 ITI 227 JRI c MC6 W TM1 AZ DC
a This panel includes all antisera prepared and characterized up to .July 1981. New antisera which are being raised to other methanogens, will be numbered continuing the present series as they become available. b Antisera were numbered in the order of their preparation. Thus, antiserum No. 1 was obtained first, and antiserum No. 2 was obtained from a rabbit immunized after the donor of No. I antiserum, and so on. Moreover, each antiserum was defined by the rabbit letter and number, bleeding serial number and date, and vial letter t0r the serum bank records 2C'. c Complete infbrmation and relevant references regarding the immunizing microorganism can be found in references 2, 25, and 26. ~ElsevicrBiomedicalPress1982 0167~919/82/00000000/$t.00
280 Each antiserum was titrated against the immunizing bacterium and from each titration curve three dilutions were selected as probes for analysis 26,2v. T h e T probe (T from titer) is the antiserum titer, i.e. the highest dilution at which an a n t i g e n - a n t i b o d y reaction is still discernible. The S probe (S from screening) is the highest dilution at which the antiserum gives a maximal reaction, i.e. the last dilution of the titration curve's plateau. It was used in our initial surveys to screen methanogens for cross-reactions. The R probe (R from relatedness) is the optimal dilution for disclosing antigenic relatedness among methanogens. It is eightfold more concentrated than the S probe and higher antiserum concentrations do nut reveal relationships that have not been shown by it. Thus, the R probe is the most convenient tbr disclosing antigenic similarities among methanogens and for finding and counting these bacteria in samples from landfills, digesters, sludges, marine and lake sediments, etc., as described in following sections. T h e specificity spectrum of the three probes for the 22 rabbit antisera was determined. The T probe was found to be monospecific for the immunizing homologous bacterium 26. The S probe from some antisera cross reacted with a variable n u m b e r of methanogens. These cross-reactions were strong with strains of the same species of the immunizing bacterium and weak with methanogens of different species 25-2v. lnterfamily cross-reactions were not observed. Because of these cross-reaction characteristics, the S probe was choscn as a rapid means of assigning a new isolate to a particular species and family before the application of more definitive but cumbersome procedures (e.g. rRNA sequencingl-5).
The antigenic fingerprint of methanogens By conducting a multiple assay using the S probe and all the antisera the pattern of reactivity or antigenic fingerprint of each methanogen is revealed (some examples are shown in T a b l e II). Similar fingerprints can also be obtained with the R probe (unpublished results). T h e fingerprint itself is the degree to which a bacterium reacts with the probes, either positively or negatively. If the reaction is positive, the intensity is scored from 1 to 4, as measured by indirect immunofluorescence under standardized conditions >,2c'. Therefore, an antigenic fingerprint is a combination of the digits 0 (no reaction or negative), i, 2, 3 and 4. As a result of antigenic fingerprinting a n u m b e r of cross-reactions were discovered. W h e n all the fingerprints were arranged to conform the classification of methanogens 2 and the strains listed accordingly, two m a i n features emerged25: t h e r e were no crossreactions between members of different families, but there were four clusters of antigcnically related strains. These clusters coincided with families defined on the basis of comparative r R N A sequencing ~.
Immunology 7?)day, vof 3, No. It), 1982
Monoclonal antibodies to methanogens Once the cross-reactive clusters were identified, representative species from each cluster were selected for the preparation of monoclonal antibodies. In the process of generating hybridomas, the need to measure anti-methanogen antibodies in small, dilute samples from master- and cloning-plate microcultures emerged. Since no reliable technique was available, a slide immunoenzymatic assay, which proved to be sensitive and reproducible, was developed 26. Its outstanding features are the absence of background and of false-positive a n d false-negative results. With this method at hand, it was possible to form many hybrid cell lines producing monoclonal antibodies to several methanngens >,30. T h e following lines are now available: 16 that react with Methanospiri[lum hungatei JF1, 17 with Melflanococcu.r vannielii SB, 9 with Methanococcus ~oltae PSv, 7 with Methanohrevibacler arbor@hilus DH1, 6 with Melhanobacterium formicicum MF, 27 with Methanobacterium lhermoaulolr@hicum AH and 14 with Methanobaclerium thermoautotrophicum GC1. A systematic analysis of methanogens using monoclonal antibodies has.just begun, but it is already clear that it will provide insights into the surface structure of these unique microorganisms. Specific markers for strains, species, and genera, and perhaps for higher taxonomic groups are likely to be found. O t h e r applications of the monoclonal antibodies in research and biotechnology involving methanogens will be discussed below.
Structural analysis of methanogens by using monoclonal antibody probes Characterization of surface markers and elucidation of the cell-wall structure of methanogens can now he achieved quickly with monoclonal antibodies. These refined probes complement polyclonal antisera and standard immunochemical 2
