Biotechnology for Crop Protection - American Chemical Society

Chapter

Phylogenetically

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 21, 2018 | https://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch029

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David A. Stahl College of Veterinary Medicine, University of Illinois, Urbana, IL 61801 The use of comparative ribosomalRNAsequencing for studies in microbial ecology and microbial evolution is discussed. A specific study, using the 16S ribosomal RNA to monitor the bovine rumen microbial community, is described. The response of this community to antibiotic addition was evaluated by hybridization of oligonucleotide probes to nucleic acid isolated from the rumen. The application of the ribosomal RNA based measure of microbial communities to general evaluations of community disruption, as might follow the release of genetically engineered microorganisms into the environment, is discussed. Current, concern over the pending and pilot release of genetically engineered microorganisms (GEMS) and viruses into the environment (1,2) is a reflection of our very limited understanding of natural microbial ecosystems. Recent advances in biotechnology have greatly expanded the repertoire of genetic constructions and so, the phenotypic variety available for commercial application. However, unlike other products, microorganisms are self-replicating. Their release must be treated as irreversible. This has served to fuel concern. Two opposing attitudes concerning "deliberate release*' prevail, or at least dominate attention. The conservative viewpoint would ask that, any novel organism (either the product of genetic engineering or more conventional genetic manipulations) be closely scrutinized (1). This position holds that we know very little about how microorganisms compete in nature, what constitutes a functionally stable microbial community and how buffered those communities are to insult... so it is essential to assess ecosystem effects. The alternative position claims that the products of recombinant technology differ little (if at all) from those arising from natural processes and those of more traditional genetic selections (2). This controversy has shown how poorly prepared the scientific community is to evaluate natural microbial communities and the impact of engineered microorganisms on these communities. Fortunately, the same technical revolution that 0097-6156/88/0379-0373$06.00/0 • 1988 American Chemical Society

Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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spawned genetically engineered microorganisms has also served to provide new tools for the evaluation of natural microbial communities. This chapter describes the application of some of these techniques to the characterization of the most poorly characterized of natural communities. The foundation of this work is the analysis of ribosomal RNAs (rRNAs) or their genes i n the environment.


Historical Impediments to the Study of Microbial Ecology There is l i t t l e disagreement that the greatest handicap i n the study of microbial ecology is characterizing the members of microbial communities. This is a two part problem. The first part is the prerequisite pure culture isolation of the various microorganisms making up a natural community. The puVe culture description is the foundation of microbiology and is an essential prelude to the complete description (biochemical, physiological and genetic) of a microorganism. Yet, the greater part of many natural populations cannot readily be grown using standard techniques (3). This problem has often been cited as the greatest blind spot i n microbial ecology and i n measures of microbial diversity. The second part of the problem of characterization is nomenclature. Although the necessity of cultivation is perhaps the better appreciated limitation i n studies of microbial ecology, the more fundamental limitation is taxonomy. The phenotypic characteristics of a microorganism frequently do not adequately define i t . Differences i n physiological attributes examined may belie an underlying genetic similarity. Alternatively, similar physiological and morphological attire may obscure genetic diversity ( 1 2 ) . There are many examples of each. Classification would therefore seem to be the blind spot i n studies of microbial ecology. The study of microbial ecology rests upon a correct and workable taxonomy. The later rests upon an understanding of microbial phylogeny. Microbial Phylogeny and the Use of Biopolymers as Historical Documents Historically, the classification of microorganisms has been driven by two considerations. These are the practical need for a useful determinative scheme for identification and, an ideal, that the classification reflect natural relationships. The drive toward a phylogejnetically correct taxonomy was early mired in technical and conceptual shortcomings. The ranking by morphology and physiology (e.g. photosynthetic, chemosynthetic and heterotrophic metabolisms) was ultimately shown to be arbitrary ( 4 ) . An added confusion concerned diversity within designated taxons. The "splitting" or "lumping" of classes of microorganisms often reflected the immediate needs (and resources) of the study, not a uniform classification. Insights into the natural relationships among microorganisms had to await the development of methods for studying their molecular architecture ( 5 ) . The genealogy of microorganisms is most clearly recorded i n their common biopolymers, the amino acid sequence of homologous proteins and the nucleotide sequence of homologous nucleic acids. When certain conditions are met (below), divergence of sequence can



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be related to evolutionary divergence of the organism. This can be inferred by several means. Methods commonly used include nucleic acid hybridization, antigenic relatedness, electron microscopy (e.g. of ribosomes), and sequencing. Among these, the least ambiguous is comparative sequencing. Unlike the other methods, sequencing generates a cumulative data collection that can be treated analytically. Thus, the recent revolution in nucleic acid sequencing technology has fostered comparative sequencing studies and is placing microbiology for the first time within an evolutionary framework. Comparative sequence analysis, most importantly of the ribosomal RNAs, has yielded the most complete understanding of microbial phylogeny (4,6). This, and a clear evolutionary perspective has served as the foundation for the explicit characterization of natural microbial communities (7,8,9,10). The ribosomal RNAs and the Universal Phylogeny The use of ribosomal RNAs to infer evolutionary relationships is now well documented (4). These ubiquitous biopolymers are ideally suited to studies of microbial phylogeny and to studies in microbial ecology for the following reasons. 1.

As integral elements of the protein-synthesizing machinery, they are functionally and evolutionarily homologous in a l l organisms. Thus they can be used to infer a universal phylogeny of extant life.

2.

They are highly conserved in both sequence, size and higher-order structure. Consequently, the rRNAs (or their genes) can be identified in natural samples by both size and sequence.

3.

The conservation of nucleotide sequence across the length of the molecules is variable. Some regions of sequence (and structure) are invariant. These can be used to identify these molecules in the environment and also for the sequence alignment necessary for calculations of similarity. The more variable regions are used to infer evolutionary distance and to provide signatures of identify for individual species or strains of microorganisms.

4.

The rRNAs provide ample sequence information for statistically significant comparisons.

5.

The rRNA genes appear not to be transferred among different microorganisms. The phylogeny of the molecules therefore reflects the phylogeny of the parent organisms.

There are three ribosomal RNAs in bacteria; 5S (ca 120 nucleotides), 16S (ca 1600 nucleotides), and 23S (ca 3,000 nucleotides). The eukaryotic versions of the 16S and 23s rRNAs are somewhat larger. In addition, most eukaryotes have a fourth species, the 5.8S rRNA, that is homologous with the 5' end of the 23S rRNA. For historical reasons, most early analyses used the 5S and 16S species in comparative studies. The smaller species were focused upon mostly because the then-available sequencing technology was not adequate for sequencing the larger 23S rRNA. However, recent developments in



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sequencing technology have now made a l l species equally accessible (11). Since the precision of comparative analysis grows with the amount information (sequence) analyzed, greater use of the 23S rRNA is anticipated for the study of both microbial evolution and microbial ecology. The first inclusive phylogeny (encompassing both eukaryotes and prokaryotes) was derived from the comparative sequencing of 16S rRNAs (6). This study was primarily the effort of Carl Woese and collaborators and has served as the foundation for the studies in microbial ecology discussed here. This earlier work relied upon the sequencing of oligonucleotides (RNA fragments from one to ca 20 nucleotides in length) derived from ribonuclease T^ digestion of the 16S rRNA. The collection (the catalogue) of oligonucleotide sequences comprises a partial, and discontinuous, sequence of the parent molecule. Similarity between organisms is inferred from the fraction of oligonucleotides common to both and from the limited application of signature sequences (6). The latter are relatively highly conserved oligonucleotides that define major lines of evolutionary descent. Although, much of the existing data collection of 16S rRNA sequence is in the form of oligonucleotide catalogues, the use of rRNA templated reverse transcriptase sequencing is rapidly expanding the collection of complete or nearly complete 16S rRNA sequences (11). Continuous sequences will soon replace the catalogue collection. It. was the analysis of the 16S rRNAs that first revealed the unique evolutionary position of the archaebacteria, and defined the primary evolutionary divisions of life on this planet (4,6). Figure 1 shows an unrooted phylogenetic tree based upon complete 16S rRNA sequences of representatives of the three primary lines of evolutionary descent; the archaebacteria, the eubacteria and the eukaryotes. Within these primary lines of descent other major lines of descent have been delineated. Within the eubacteria, for example, some ten major divisions are now recognized. It has been suggested that these be given a systematic rank equivalent to phylum (12). However, this chapter will not elaborate upon the emerging phylogenetic description of life on this planet. The reader is referred to a recent review for a dedicated treatment, of this subject (4,12). Microbial Ecology and Microbial Phylogeny: Common Ground Full appreciation of microbial evolution must come from a complete knowledge of extant microbial diversity. As well, full appreciation of microbial diversity can only come from an understanding of their evolution. Although these studies generally have been treated independently, this is mostly because there has been no unifying conceptual and technical framework. The ribosomal RNAs now provide the framework. They therefore offer the criteria to explicitly characterize any organism on this planet and to place it in a phylogenetic context. They can be used to characterize natural communities without the requirements of cultivation or previous pure-culture descriptions of the community members (7,10). Two general technical approaches to using the ribosomal RNAs for the characterization of natural microbial communities have so far been evaluated (8). The method first explored used the 5S rRNAs to



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ARCHAEBACTERIA Halobacterium volcanii

0.1fixedi per sequence position

Sutfolobus solfataricus Thermoproteus tenax

Methanospiriltum hungatei Methanobacterium formidcum Methanococcus vannfefff

Homosapiens

EUKARYOTES

EUBACTERIA

Figure 1. Universal phylogenetic tree based on comparative 16S rRNA sequencing showing the relationship of representatives of the three primary lines of evolutionary descent. Sequence differences through regions of unambiguous alignment: (about 950 nucleotides) were corrected for multiple nucleotide substitutions per site and these estimates of sequence divergence (mutations fixed per sequence position) used to infer the phylogenetic tree as previously described (26). (Reproduced with permission from Ref. 23. Copyright 1986 Cell Press.)



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c h a r a c t e r i z e n a t u r a l communities of l i m i t e d c o m p l e x i t y ( 9 , 1 3 ) . In t h i s approach 5S rRNAs are d i r e c t l y i s o l a t e d from a v a i l a b l e biomass, s e p a r a t e d on h i g h - r e s o l u t i o n a c r y l a m i d e g e l s , and sequenced. The number o f unique 5S rRNAs d e f i n e s p o p u l a t i o n c o m p l e x i t y . Each community member i s c h a r a c t e r i z e d by a unique sequence t h a t can be r e l a t e d t o a r e f e r e n c e c o l l e c t i o n o f 5S rRNAs from organisms i n pure culture. An i d e n t i c a l sequence i n the r e f e r e n c e c o l l e c t i o n i s not r e q u i r e d , s i n c e the r e f e r e n c e organism rRNA sequence most c l o s e l y r e l a t e d t o the e n v i r o n m e n t a l l y d e r i v e d sequence i s always known. Because t h i s approach i s dependent upon the a n a l y t i c a l f r a c t i o n a t i o n o f s i m i l a r m o l e c u l e s , i t can not e a s i l y r e s o l v e the m i c r o b i a l components o f v e r y complex communities (> 10-20 s p e c i e s ) . This i s the g r e a t e s t l i m i t a t i o n o f the a n a l y s i s . A l s o , the 5S rRNA i s r e l a t i v e l y s m a l l (ca 120 n u c l e o t i d e s ) and does not p r o v i d e enough i n f o r m a t i o n t o d i s c r i m i n a t e between v e r y c l o s e l y r e l a t e d organisms or f o r d e t e r m i n i n g meaningful e s t i m a t e s o f g r e a t e r p h y l o g e n e t i c d i s t a n c e s , as s e p a r a t e the v a r i o u s e u b a c t e r i a l p h y l a . The second approach i s not so l i m i t e d by p o p u l a t i o n c o m p l e x i t y or information content. Here 16S rRNA genes are "shotgun c l o n e d " u s i n g DNA p u r i f i e d from a v a i l a b l e biomass ( 7 , 8 ) . In t h i s c a s e , the o r i g i n a t i n g p o p u l a t i o n c o m p l e x i t y does not matter s i n c e the rRNA genes are c l o n a l l y i s o l a t e d from a recombinant l i b r a r y o f DNA d i r e c t l y d e r i v e d from the n a t u r a l m i c r o b i a l community. The d i f f e r e n t c l o n e s are s o r t e d and sequenced u s i n g r a p i d s e q u e n c i n g t e c h n i q u e s . The r e a d e r i s r e f e r r e d to p r e v i o u s r e v i e w a r t i c l e s on the e c o l o g i c a l use o f rRNAs f o r d e t a i l s o f the 5S rRNA based a n a l y s i s and the c l o n a l i s o l a t i o n o f rRNA genes from n a t u r a l samples ( 8 , 9 , 1 3 ) . The i n t e r p r e t a t i o n o f rRNAs i d e n t i f i e d i n the environment i s dependent upon the completeness o f the r e f e r e n c e c o l l e c t i o n o f sequences. A l t h o u g h t h e r e w i l l not soon be a r e f e r e n c e c o l l e c t i o n complete enough t o a s s i g n e x a c t i d e n t i t i e s , t h i s s h o u l d not be p e r c e i v e d as too g r e a t a l i m i t a t i o n o f t e c h n i q u e . The more immediate change t h i s new t e c h n o l o g y w i l l b r i n g i s i n m i n d s e t . Pure c u l t u r e i s o l a t i o n has so l o n g been a n e c e s s a r y p r e l u d e t o s t u d i e s i n m i c r o b i a l e c o l o g y t h a t t h i s has n e a r l y become an a c c e p t e d l i m i t a t i o n . Now the pure c u l t u r e r e p r e s e n t a t i o n o f a community can be compared t o e x p l i c i t measure o f community c o m p o s i t i o n ; rRNA sequences. This a l o n e s h o u l d g r e a t l y a l t e r how n a t u r a l systems are c h a r a c t e r i z e d . R e c o g n i t i o n o f a dominant community member by sequence but not by c u l t u r e s e r v e s as powerful i n c e n t i v e t o pursue the more e l u s i v e community member. A second change t h i s m o l e c u l a r approach b r i n g s t o s t u d i e s o f m i c r o b i a l e c o l o g y i s more d i f f i c u l t t o s u c c i n c t l y s t a t e . This d e r i v e s from u n d e r s t a n d i n g e v o l u t i o n a r y r e l a t i o n s h i p s and the v a l u e of a p h y l o g e n e t i c framework i n d e s i g n i n g s t u d i e s o f n a t u r a l systems. As s h o u l d become a p p a r e n t , a p h y l o g e n e t i c framework f o r c e s i n t e g r a t e d s t u d i e s o f n a t u r a l systems. The b a s i s f o r the p h y l o g e n e t i c framework (rRNA sequence) p r o v i d e s e x p l i c i t c h a r a c t e r i s t i c s ( s i g n a t u r e sequences) f o r m o n i t o r i n g m i c r o o r g a n i s m s i n any s e t t i n g . The same measure i s a p p l i e d t o e v e r y s y s t e m . F o r the f i r s t t i m e , m i c r o b i a l e c o l o g i s t s w o r k i n g w i t h d i f f e r e n t m i c r o b i a l systems can c r o s s - t a l k , u s i n g the r i b o s o m a l RNAs as a common tongue. To more c l e a r l y i l l u s t r a t e the c o u p l e between phylogeny and e c o l o g y , a s p e c i f i c s t u d y w i l l be d i s c u s s e d . T h i s i s p a r t o f an ongoing study o f r u m i n a l m i c r o b i a l ecology (14). a

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The Rumen as a Model Microbial Ecosystem The rumen is the modified stomach region of herbivores responsible for fiber digestion. (15,16,17). Having a volume of about 30 gallons in cattle, the rumen is an anaerobic environment harboring large numbers of obligately anaerobic bacteria and eukaryotes. Total microbial numbers average 10 to 10 cells/ml. These microbes are the principal agents of fiber breakdown, converting it to the protein and energy sources (primarily volatile fatty acids) used by the animal. The animal is therefore entirely dependent upon these microbial symbionts. We have elected to use this microbial ecosystem for developing some of the molecular techniques described in this chapter.


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Why the Rumen? There are a number of reasons for using the rumen microbial community as a model system. 1.

The rumen offers an abundant and complex microbial population. It is composed of representatives of a l l the primary kingdoms (archaebacteria, eubacteria, and eukaryotes), including anaerobic fungi and protozoa in addition to the better described bacteria.

2.

It is well characterized bacteriologically, and therefore offers a basis for judging the fidelity of these molecular descriptions. Although the resident bacteria are quite well described biochemically and physiologically, there has been relatively l i t t l e work on the ecological relationships between the different groups.

3.

It is quite analogous to other systems of anaerobic decomposition (e.g. sediments and sewage digesters), except that fatty acids and lowly substituted benzenoids are not completely degraded (18).

4.

It is a contained and easily sampled population. Exogenous microorganisms are easily introduced into the system and the system as a whole readily perturbed, for example, by altering diet, introducing exogenous organisms, or administering antibiotics.

The last point is of particular interest from the standpoint of assessing the risk of releasing genetically engineered microorganisms (GEMS) into the environment. One principal concern of GEM release is their impact on existing microbial communities. But there is l i t t l e understanding of what constitutes a stable microbial community. Measures of normal population variation and functional redundancy among natural community members are virtually absent in the literature. Yet these must be done to predict risk or to evaluate the ecosystem effects of GEM release. Community perturbation must be defined relative to normal variation in community composition. To more clearly establish these baseline criteria for risk assessment, the rumen stands as a very useful model.



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We have initiated a comparative sequencing survey of dominant rumen microbial flora (Montgomery, L . ; Flesher, B.; Stahl, D.A. Submitted to Int. J . Syst. Bacterid.). The goal is to define each organism by 16S rRNA sequence. There are two components to the sequence characterization, with current effort directed to the first. The first is the comparative sequence analysis of rumen microorganisms now in pure culture. This provides a molecular signature (16S sequence) for each and also validates or serves as a basis for restructuring the existing taxonomy. Regions of variable sequence are used to determine phylogenetic relationships and are also used as hybridization targets for synthetic oligonucleotides. The relative amount of hybridization of specific or general oligonucleotide probes to rRNA isolated from the environment offers a rapid and quantitative measure of organism abundance without need for cultivation. The second component of the characterization is the comparative sequencing of 16S rRNA genes cloned from DNA isolated from total rumen contents. The comparative sequencing of 16S rRNA genes clonally isolated from naturally available DNA offers the most unbiased measure of rumen microbial content. Yet, as already discussed, the clonal analysis of the population cannot stand alone. It must be related to the existing culture collection of rumen microflora and their corresponding 16S rRNA sequences. Although a reference collection large enough to adequately cover the microbial diversity of this planet is not attainable in the foreseeable future, the rumen bacterial community is possibly well enough circumscribed to be reasonably well-defined by both the pure culture and 16S rRNA sequence criteria. Use of rRNA-Targeted Hybridization Probes for Studies of Microbial Ecology This section will cover two aspects of the use of DNA oligonucleotides as hybridization probes to monitor environmental populations of microorganisms. The first will discuss the nuts-and-bolts of technique. The second is more conceptual and addresses the design of these probes within a phylogenetic framework. Overview of Methodology. An unbiased assessment of microbial makeup based upon nucleic acid hybridization necessitates an unbiased recovery of nucleic acid from the various organisms represented in the environment. This is not straightforward. Cells greatly vary in susceptibility to breakage by enzymatic, chemical and mechanical disruption. In general, however, mechanical disruption offers the most uniform breakage of different cell types. Although mechanical disruption cannot be used to recover high molecular weight DNA, it can be used to recover intact DNA and rRNA suitable for oligonucleotide probe hybridization. In our experience, disruption with glass beads in a reciprocating shaker offers excellent recovery of nucleic acid from a great, variety of organisms (Stahl, D.A., unpublished observations). Even the more recalcitrant types (e.g. Gram-positive cocci, mycobacteria) are efficiently broken (unpublished observations).



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For the rumen p e r t u r b a t i o n s t u d y , t o t a l n u c l e i c a c i d was r e c o v e r e d from u n f r a c t i o n a t e d rumen c o n t e n t s by m e c h a n i c a l d i s r u p t i o n ( M i n i - b e a d b e a t e r , B i o s p e c P r o d u c t s , B a r t l e s v i l l e , OK) w i t h g l a s s beads (150-200 m i c r o n s ) and p h e n o l . Phenol was p r e s e n t t o m i n i m i z e the d e g r a d a t i o n o f n u c l e i c a c i d s by n u c l e a s e s . F o l l o w i n g a d d i t i o n a l e x t r a c t i o n s w i t h p h e n o l / C H C l 3 , t o t a l n u c l e i c a c i d ( p r i m a r i l y rRNA) was r e c o v e r e d by e t h a n o l p r e c i p i t a t i o n , denatured w i t h 1.5% g l u t a r a l d e h y d e , and s p o t t e d on n y l o n membranes ( 1 9 ) . S i n c e t h e r e are u s u a l l y 10,000 t o 20,000 c o p i e s o f the r i b o s o m a l RNAs per c e l l , r R N A - t a r g e t e d h y b r i d i z a t i o n i s a much more s e n s i t i v e measure o f organism abundance than h y b r i d i z a t i o n to DNA sequences. DNA o l i g o n u c l e o t i d e probes were l a b e l e d w i t h 32p u s i n g p o l y n u c l e o t i d e k i n a s e and gamma-labeled ATP ( 1 9 ) . H y b r i d i z a t i o n c o n d i t i o n s were o p t i m i z e d f o r s e n s i t i v i t y and s p e c i f i c i t y r e l a t i v e t o t o t a l n u c l e i c a c i d i s o l a t e d from v a r i o u s o t h e r rumen and non-rumen microorganisms (19,20). For t h e s e s t u d i e s we have used r a d i o a c t i v e p r o b e s . T h i s at p r e s e n t remains the most s e n s i t i v e d e t e c t i o n system. N o n - r a d i o a c t i v e s i g n a l l i n g systems ( e . g . b i o t i n and a l k a l i n e phosphatase tagged o l i g o n u c l e o t i d e s ) have y e t t o demonstrate comparable o r b e t t e r sensitivity. T h i s s h o u l d change, but d i s c u s s i o n o f t h e s e a l t e r n a t i v e s i s beyond the scope o f t h i s c h a p t e r . For the rumen p e r t u r b a t i o n s t u d y , h y b r i d i z a t i o n o f the v a r i o u s o l i g o n u c l e o t i d e probes was measured by d e n s i t o m e t r y o f f i l m exposed to the n y l o n s u p p o r t membrane f o l l o w i n g h y b r i d i z a t i o n . F i l m response throughout the range o f exposures used was l i n e a r . F i g u r e 2 i s an example o f one h y b r i d i z a t i o n s e r i e s t a k e n from our rumen s t u d y . D e s i g n o f H y b r i d i z a t i o n Probes and t h e i r A p p l i c a t i o n to M o n i t o r i n g M i c r o b i a l Communities I t i s the c a p a c i t y t o d e f i n e rRNA t a r g e t e d probe s p e c i f i c i t y w i t h i n a p h y l o g e n e t i c framework t h a t l e n d s such v e r s a t i l i t y to these s t u d i e s . F i g u r e 3 d i s p l a y s a 16S rRNA f o l d e d i n t o a consensus secondary s t r u c t u r e w i t h p o s i t i o n s shaded a c c o r d i n g to r e l a t i v e e v o l u t i o n a r y c o n s e r v a t i o n among t h i r t y d i f f e r e n t e u b a c t e r i a l s p e c i e s . Darker s h a d i n g corresponds t o h i g h e r c o n s e r v a t i o n . As d i s p l a y e d by t h i s f i g u r e , the m o l e c u l e i s a patchwork o f r e l a t i v e e v o l u t i o n a r y conservation. Some p o s i t i o n s and l o c a l e s are i n v a r i a n t ; o t h e r s are common t o a g i v e n kingdom but v a r y between kingdoms; more v a r i a b l e r e g i o n s c o n t a i n s i g n a t u r e s f o r the v a r i o u s e u b a c t e r i a l p h y l a . The most v a r i a b l e domains can be used t o d i s c r i m i n a t e between s p e c i e s o r even s u b s p e c i e s o f b a c t e r i a . Thus the spectrum o f organisms addressed by a s i n g l e h y b r i d i z a t i o n probe v a r i e s a c c o r d i n g t o the r e g i o n o f the m o l e c u l e s e l e c t e d as the h y b r i d i z a t i o n t a r g e t . The net c a s t by a g i v e n h y b r i d i z a t i o n probe i s d e f i n e d by the needs o f the s t u d y . The c a s t i n g o f wide o r narrow n e t s i n s t u d i e s o f m i c r o b i a l e c o l o g y i s b e s t i l l u s t r a t e d by example. O l i g o n u c l e o t i d e probes o f "nested" s p e c i f i c i t y have been used by us to monitor p o p u l a t i o n s o f one o f the more i m p o r t a n t c e l l u l o l y t i c b a c t e r i a i n the rumen, Bacteroides succinogenes. P r i o r t o our c h a r a c t e r i z a t i o n o f v a r i o u s s t r a i n s o f t h i s rumen b a c t e r i u m , i t was viewed as a coherent s p e c i e s . Now, based upon the marked d i v e r g e n c e among the 16S rRNA sequences o f s t r a i n s o f t h i s " s p e c i e s " , i t i s now r e c o g n i z e d t o be made up o f a c o l l e c t i o n o f



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Figure 2. Representative hybridization of the runtinal probe (2A) and cecal probe (2B) to nucleic acid extracted from daily rumen fluid samples and spotted to a nylon membrane. The oligonucleotide probes listed were used in the complete study. The following are complementary to positions 207 226 in the Escherichia coli 16S rRNA numbering: Lachnospira multiparus -specific, CTTATACCACCGGAGTTTTTCA; Bacteroides succinogenes strains NR9- and DR7-specific (cecal probe) CCGCATCGATGAATCTTTCGT ; and B. succinogenes strains S85- and A3e-specific (rumjnal probe) CCATACCGATAAATCTCTAGT. The B. succinogenes signature probe AATCGGACGCAAGCTCATCCC is complementary to positions 225 245 in the E. coli numbering. Total 16S rRNA was estimated by hybridization to an oligonucleotide ACGGGCGGTGTGTRC complementary to a region (near position 1400, E. coli numbering) of virtually a l l 16S-like rRNAs so far characterized (17). Bound probe was quantitated by densitometry relative to reference standards after autoradiography with preflashed film exposed at -85o C. Arrows mark the addition to, arid removal from, the feed of the ionophore antibiotic monensin.


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Secondary Structure of eubacterial 16S Ribosomal RNA

F i g u r e 3 . P o s i t i o n a l n u c l e o t i d e c o n s e r v a t i o n i n 27 d i v e r s e e u b a c t e r i a l 16S r i b o s o m a l RNA sequences. Shading i n t e n s i t y increases i n proportion with conservation. Invariant positions w i t h i n t h i s sequence s e t are b l a c k . W i t h the e x c e p t i o n o f the u n i v e r s a l p r o b e , the o l i g o n u c l e o t i d e probes used i n t h i s s t u d y h y b r i d i z e w i t h i n the boxed r e g i o n o f the 16S rRNA sequence. (Reproduced w i t h p e r m i s s i o n from R e f . 10. C o p y r i g h t 1986 N a t u r e Publishing Co.)



384


g e n e t i c a l l y d i s t i n c t b a c t e r i a (Montgomery, L . ; F l e s h e r , B . ; S t a h l , D.A. Submitted to I n t . J . S y s t . B a c t e r i o l . ) . A l t h o u g h the assemblage o f s t r a i n s i s p h y l o g e n e t i c a l l y c o h e r e n t , t o the e x c l u s i o n o f o t h e r c h a r a c t e r i z e d b a c t e r i a , the p h y l o g e n e t i c depth among them (ca 90% s i m i l a r i t y ) i s g r e a t e r than t h a t which s e p a r a t e s P r o t e u s v u l g a r i s from E s c h e r i c h i a c o l i (ca 93% s i m i l a r i t y ) . The p h y l o g e n e t i c depth among r e p r e s e n t a t i v e s t r a i n s o f B . s u c c i n o g e n e s i s b e s t i l l u s t r a t e d by the p h y l o g e n e t i c t r e e ( f i g . 4) i n f e r r e d from 16S rRNA sequence s i m i l a r i t y . The depth s e p a r a t i n g the two major l i n e s o f descent w i t h B . succinogenes i s deep enough t o d i v i d e t h e c o l l e c t i o n i n t o d i f f e r e n t s p e c i e s o r g e n e r a , but t h e r e a r e , as y e t , no s a t i s f y i n g p h e n o t y p i c c r i t e r i a t o base such a d i v i s i o n upon; the v a r i o u s s t r a i n s are now d i s t i n g u i s h e d m a i n l y by v i t a m i n r e q u i r e m e n t s and v a r i a t i o n s i n morphology. As we c o n t i n u e t o b u i l d our r e f e r e n c e c o l l e c t i o n c r y p t i c d i v e r s i t y i s a common theme. A single strain cannot be assumed r e p r e s e n t a t i v e o f a s p e c i e s . These o b s e r v a t i o n s h i g h l i g h t the l i m i t a t i o n s o f t r a d i t i o n a l i d e n t i f i c a t i o n schemes f o r studies i n microbial ecology. To m o n i t o r p o p u l a t i o n s o f B . s u c c i n o g e n e s i n the rumen over t i m e , t h r e e d i f f e r e n t o l i g o n u c l e o t i d e probes were s y n t h e s i z e d . The t a r g e t r e g i o n s w i t h i n the 16S rRNA a r e i n d i c a t e d i n f i g u r e 3 . The " s i g n a t u r e " probe i s complementary t o the 16S rRNA o f a l l but one s t r a i n o f B . succinogenes (DR7, S85, HM2, REH9-1, and A3c but not NR9) so f a r c h a r a c t e r i z e d by c o m p a r a t i v e s e q u e n c i n g . S t r a i n NR9 d i f f e r s i n a s i n g l e A t o G t r a n s i t i o n . The o t h e r probes i d e n t i f y e i t h e r o f two n a t u r a l groups w i t h i n the l a r g e r assemblage. One group ( r u m i n a l type) i s r e p r e s e n t e d by rumen s t r a i n s S85 and A3c; the second group ( c e c a l type) i s r e p r e s e n t e d by s t r a i n s NR9 and DR7 i s o l a t e d from r a t and p i g c e c a , r e s p e c t i v e l y . These probes t h e r e f o r e are of nested s p e c i f i c i t y . A f o u r t h o l i g o n u c l e o t i d e probe i d e n t i f i e d L a c h n o s p i r a m u l t i p a r u s , a G r a m - p o s i t i v e p e c t i n o l y t i c rumen b a c t e r i u m . One a d d i t i o n a l o l i g o n u c l e o t i d e was used t o measure the t o t a l 16S rRNA c o n t e n t o f each n u c l e i c a c i d sample a p p l i e d t o n y l o n membranes. This probe i s complementary t o a sequence element p r e s e n t i n a l l s m a l l s u b u n i t r i b o s o m a l RNAs so f a r c h a r a c t e r i z e d ( e u k a r y o t i c and p r o k a r y o t i c ) ( 1 1 ) . H y b r i d i z a t i o n t o t h i s probe was used t o n o r m a l i z e h y b r i d i z a t i o n o f each s p e c i f i c p r o b e . Abundance i s e x p r e s s e d as the t a r g e t - g r o u p f r a c t i o n o f the t o t a l 16S rRNAs i n the sample. P e r t u r b a t i o n o f the Rumen M i c r o b i a l

E c o l o g y by Monensin

Monensin i s a sodium ionophore a n t i b i o t i c t h a t i s r o u t i n e l y i n c l u d e d i n c a t t l e f e e d l o t d i e t s t o improve feed u t i l i z a t i o n e f f i c i e n c y ( 2 1 ) . I n c r e a s e d p r o p i o n a t e p r o d u c t i o n , d e c r e a s e d methane p r o d u c t i o n and a p r o t e i n s p a r i n g e f f e c t have been suggested t o be i n p a r t r e s p o n s i b l e f o r improved feed c o n v e r s i o n ( 2 1 ) . Y e t , i t remains u n r e s o l v e d whether a l t e r a t i o n o f the rumen p o p u l a t i o n o r a l t e r a t i o n o f the p h y s i o l o g y o f a r e l a t i v e l y unchanged p o p u l a t i o n c o n t r i b u t e s most t o these e f f e c t s . . P o l y e t h e r a n t i b i o t i c s , such as monensin and l a s a l o c i d , a r e g e n e r a l l y c o n s i d e r e d t o be most a c t i v e a g a i n s t G r a m - p o s i t i v e o r g a n i s m s , such as L a c h n o s p i r a s p p . and Ruminococcus s p p . , whereas organisms w i t h Gram-negative c e l l w a l l s ( e . g . Selenomonas and B a c t e r o i d e s s p p . ) a r e c o n s i d e r e d t o be r e l a t i v e l y r e s i s t a n t ( 2 2 ) . In t h i s s t u d y we have used monensin p r i m a r i l y as a means t o p e r t u r b t h i s ecosystem i n o r d e r t o e v a l u a t e our methods f o r measuring



29. STAHL


385

Bacteroides fragilis

Flavobacterium heparinum

Bacteroides succinogenes S85

REH9-1

HM2

V* J 7

I

ruminal type

cecal type

Arthrobacter globiformis Mycobacterium flavescens Bacillus subtls

Pseudomonas testosteroni • Proteus vulgaris Escherichia coE

F i g u r e 4 . P h y l o g e n e t i c t r e e i n f e r r e d by comparative a n a l y s i s o f 16S rRNAs o f v a r i o u s s t r a i n s o f B a c t e r o i d e s succinogenes and t h e most c l o s e l y r e l a t e d e u b a c t e r i a l s p e c i e s (see F i g . 1 and R e f . 20 f o r d e s c r i p t i o n s o f method).



386


change i n community c o m p o s i t i o n . The e x p e r i m e n t a l a n i m a l was a r u m i n a l l y c a n n u l a t e d , n o n - l a c t a t i n g H o l s t e i n cow (ca 12 y r s ) fed a maintenance d i e t o f 1.8 Kg g r a i n mix and 1.8 Kg a l f a l f a hay a t 8 AM and 4 PM each day. The cow was adapted t o the d i e t f o r 20 days b e f o r e s a m p l i n g . The rumen c o n t e n t s were sampled d a i l y f o r 77 days a t 2 PM from the mid rumen r e g i o n . Monensin was added a t 35 mg per Kg o f feed from s a m p l i n g day 16 to day 6 2 , comparable to l e v e l s used i n feed l o t d i e t s . T o t a l n u c l e i c a c i d s were e x t r a c t e d from about 1 mL o f each d a i l y sample and h y b r i d i z e d to each o f the v a r i o u s o l i g o n u c l e o t i d e probes d e s c r i b e d above. The r e s u l t s o f t h i s s t u d y are i n f i g u r e 5. The two groups o f B . s u c c i n o g e n e s responded v e r y d i f f e r e n t l y t o a d d i t i o n o f the a n t i b i o t i c . The p r o p o r t i o n (as a percentage o f t o t a l 1 6 S - l i k e rRNAs) o f r u m i n a l - t y p e B . s u c c i n o g e n e s i n c r e a s e d about f i v e - f o l d ( r e l a t i v e t o the b a s e l i n e p e r i o d ) i m m e d i a t e l y a f t e r a d d i t i o n , whereas the p r o p o r t i o n o f c e c a l - t y p e B . s u c c i n o g e n e s was depressed. The r u m i n a l type remained e l e v a t e d ( a c c o u n t i n g t r a n s i e n t l y f o r about 1% o f t o t a l ribosome numbers) f o r s e v e r a l days b e f o r e d r o p p i n g , over the next two week p e r i o d , to below b a s e l i n e proportions. A t t h a t time the c e c a l type g a i n e d r e l a t i v e dominance, a l t h o u g h p r e s e n t i n much lower l e v e l s than the e a r l i e r peak o f the ruminal type. Over the next t h r e e week p e r i o d the r u m i n a l type p r e d o m i n a t e d , showing two c l e a r peaks o f s e v e r a l days d u r a t i o n e a c h . Upon w i t h d r a w a l o f monensin, the c e c a l types reached t h e i r h i g h e s t l e v e l s (0.4%), t r a n s i e n t l y e q u a l l i n g the p r o p o r t i o n o f the r u m i n a l - t y p e b e f o r e a g a i n d r o p p i n g t o e a r l i e r l e v e l s (

Biotechnology for Crop Protection - American Chemical Society

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