Department of Biochemistry and Microbiology, Rutgers University Cook College, New Brunswick, NJ 08901, USA
Correspondence
palleroni{at}aesop.rutgers.edu
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ABSTRACT |
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Overview |
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In any case, the popularity of molecular taxonomic studies supplemented by the construction of so-called phylogenetic trees has contributed to the displacement of most of the pre-molecular experimental approaches developed during a long period that goes from the last decades of the 19th century to the 1960s, a span during which they enjoyed various degrees of success in determinative bacteriology.
Aside from their historical value, these systems have lost practically all their interest in the eyes of modern taxonomists. Those who are interested in discussions on the respective merits of the early classification systems are referred to the comprehensive articles by Kluyver & van Niel (1936) and by van Niel (1946)
, which place them in their correct perspective. I have taken from these articles many of the elements presented in the next section.
It is the purpose of this review to give just a glimpse of these early systems of classification, trying to emphasize the state of affairs at the time when a radical transformation in approaches took place in prokaryote taxonomy. The sequence of events that I shall outline clearly indicates that a sudden change in taxonomic methodologies occurred from the moment of publication of work performed on the genus Pseudomonas.
The impact of this work was manifested in two steps, the first of which was the demonstration of the advantage of an extensive phenotypic characterization of strains with emphasis on a nutritional analysis, of particular importance in the characterization of heterotrophic organisms. More importantly, the second step, a few years later, was the demonstration of the genomic complexity of Pseudomonas as shown by significant rRNA sequence differences among the species then assigned to the genus, and the possibility of defining RNA homology groups. The findings clearly showed the power of rRNA similarities in defining relationships above the species level, which until that moment had been elusive in prokaryote taxonomy.
Consequently, true to the subject that has occupied the largest portion of my scientific career, I dare here to divide the 20th century with respect to prokaryote taxonomy into two periods, which (in private and for the sake of simplification) I often call BP and AP (for Before and After Pseudomonas, respectively).
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The first half of the 20th century |
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With the publication of his bacterial classification system, Ferdinand Cohn is considered to be the father of bacterial taxonomy (Cohn, 1872). His system was based on the shape and general appearance of the bacterial cells, a choice that had its roots in the usefulness of morphology in the classification of higher organisms, but Cohn clearly realized the limitation of this approach for defining bacterial genera, and cautiously referred to the various morphological groups as form genera, without attaching to them any phylogenetic significance. We owe also to Cohn the important concept that bacteria are a special group of micro-organisms, in spite of which quite a few bacterial names still carry the ending mycetes, a reminder of the now discarded hypothesis that bacteria are fission fungi (schizomycetes). Such is the sticky property of nomenclature.
Cohn's morphological principle was universally accepted at the time, and most taxonomists following him used it in different variations as the basis of their own systems of classification, occasionally adding new form genera to the inventory (Lehmann & Neumann, 1896, 1927
; Migula, 1900
; Pringsheim, 1923
; Janke, 1924
; Prévot, 1933
). However, at the beginning of the 20th century, Orla-Jensen proposed using physiological characteristics as the basic criterion for the definition of the main lines of bacterial systematics (Orla-Jensen, 1909
). It seems clear that this alternative had its roots in the demonstration of the formidable metabolic diversity of prokaryotes, mainly in the laboratories of Beijerinck and Winogradsky, whose extraordinary contributions opened the vast horizon of bacterial metabolic diversity (Beijerinck, 1921
; Winogradsky, 1949
).
Anyhow, neither the physiological criterion nor the morphological principle gave a satisfactory answer to additional conflicting alternatives. Thus if morphology is the primary criterion, the main lines of the classification often include organisms of similar physiological properties, and vice versa, in a physiological system there will be organisms of similar morphology in each of the main lines. In other words, evolution seems to have taken its course in parallel in each of the independent primary lines of each system of classification. Behind the scene, in each case, is the unproven assumption of absence of genetic exchange among members of the various lines of descent.
The system proposed by Orla-Jensen, with its evolutionary implications, introduced an additional paradox. After the discovery of chemoautotrophy by Winogradsky, Orla-Jensen favoured the idea that organisms endowed with these properties were the most primitive, since they were able to grow under the simple environmental conditions of a primitive world. Notwithstanding the originality of this approach, however, Orla-Jensen failed to take into account the possibility that there may be an inverse relationship between the chemical complexity of the medium and the biochemical sophistication required to grow under such conditions.
Orla-Jensen's classification included many interesting points, but eventually morphology was favoured by most workers as the primary classification criterion. In fact, it was accepted by most European bacteriologists, who followed the system of Lehmann and Neumann, first proposed in 1896 and updated in 1927, and it was the basis of the classification scheme developed by Kluyver and van Niel in the 1930s (Kluyver & van Niel, 1936). In this system, morphological characters include the shape and size of the cells, type of motility, presence of flagella, their number and type of insertion, mode of reproduction, occurrence of endospores or gonidia, and various structural peculiarities. Amongst the physiological properties the most important are the behaviour towards temperature, oxygen and osmotic pressure, and various characteristics of the anabolic and catabolic reactions. Among the latter, a judicious selection was recommended; for example, the reactions providing energy were placed at a level of importance above hydrolytic reactions. Pathogenicity was considered of doubtful value, and differentiation of genera and even of species on its basis was objectionable as a taxonomic criterion.
In the meantime, America was on the way to adopting its own bacterial classification system. This started in 1923 with the publication of the first edition of Bergey's Manual of Determinative Bacteriology (Bergey et al., 1923). Its successive editions were closely followed by all American workers, thereby acquiring a taste of provincialism that could not escape Kluyver and van Niel's objections, expressed in sharply critical terms. Their analysis of Bergey's system is detailed order by order, and many errors are indicated as a consequence of the use of morphological, physiological, utilitarian, cultural and pathogenic properties in the most arbitrary way, with utter disregard for mutual relationships between natural groups' (Kluyver & van Niel, 1936
).
Unfortunately, the commercial success of the Bergey's Manual editions contributed to a disregard of substantial criticisms formulated mainly by European microbiologists, particularly those by Rahn (1929, 1937
) and others, which were left aside instead of being incorporated as useful suggestions in new revisions of the system (see Kluyver & van Niel, 1936
, for references). That was unfortunate, since the Manual represented the first formal cooperation in the history of bacterial taxonomy (Kluyver & van Niel, 1936
).
As mentioned before, Kluyver and van Niel were among those for whom morphology is the first and most reliable guide of taxonomic systems but, in designing their own classification system, they kept in mind Pringsheim's warnings that (a) the proposed relationships remain conjectural and (b) a number of links may have been lost in the course of evolution (Pringsheim, 1923). Accepting the fact that the scanty morphological variety in bacteria was the cause of the unsatisfactory state of taxonomy, physiological properties could be wisely added by the taxonomist when necessary, but, far from accepting this alternative, Kluyver and van Niel showed a most determined obstinacy by remaining truthful to morphology as the leading principle, and even going to the extreme of considering that physiology is nothing but the expression of submicroscopic morphology. For them, physiological principles were acceptable in classification as long as they could be subordinated to morphology.
With time, van Niel abandoned the idea that morphology has more than a restricted phylogenetic significance and his previous optimistic attitude did not permeate a communication at one of the Cold Spring Harbor Symposia (van Niel, 1946), where he seemed inclined to draw empirical keys for bacteria based on colour, shape, physiology, disease production, nutrition and other easily determinable characteristics to help in identification. Other bacteriologists shared van Niel's pessimistic attitude about the possibility of designing a definitive natural classification of bacteria. However, there was also a reaction against the tendency to use semantic arguments as substitute for original new approaches, which were badly needed. None of the proposed systems was wholly convincing, and the arbitrary nature of the principles on which they were based made them the target for constant revisions. In fact, it was inevitable that the basis of a true natural classification of bacteria would remain unsteady inasmuch as the course of phylogeny will always remain unknown (Kluyver & van Niel, 1936
). In the words of Bruce White, the present call is not for newer, more ingenious, more pretentious, systems of classification, but for patient and incisive investigation (White, 1937
), a criticism fully supported by van Niel.
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The situation of bacterial taxonomy in the 1960s: the Pseudomonas story |
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The Pseudomonas story has been told many times, and the reader interested in the details is referred to the citations included in other reports (Palleroni, 1984, 1993
), but following the opinion of T. S. Eliot, who said that the maddening thing is that we only have one thing to say, but must keep on finding new forms of saying it, a brief repetition may not be out of place here.
A project was launched in the mid-1960s mainly by my response to Roger Stanier's insistence to look for some improvement in the chaotic state of the definition and classification of the species of the important genus Pseudomonas, some of which had been the subjects of intense biochemical research in the Department of Bacteriology of the University of California at Berkeley for a number of years.
During the initial stage of this project, strains of different species were subjected to many phenotypic tests, the most important of which was the extensive nutritional screening that had been suggested as a taxonomic tool by den Dooren de Jong many years before (den Dooren de Jong, 1926). The results of this study were summarized in a subdivision of the collection into a number of species and species groups defined on purely phenotypic grounds (Stanier et al., 1966
). The studies were later supplemented with detailed analysis of some of the species groups, such as the mallei-pseudomallei species (Redfearn et al., 1966
), the diminuta-vesicularis complex (Ballard et al., 1968
), the stutzeri group (Palleroni et al., 1970
) and the cepacia group (Ballard et al., 1970
), studies on Pseudomonas solanacearum (Palleroni & Doudoroff, 1971
) and description of the related species Pseudomonas pickettii (Ralston et al., 1973
), the fluorescens group (Barrett et al., 1986
; Champion et al., 1980
; Johnson & Palleroni, 1989
; Palleroni et al., 1972
), the alcaligenes group (Ralston-Barrett et al., 1976
) and the hydrogenomonad species (Ralston et al., 1972
). The interest manifested by plant pathologists in these studies resulted in some of the early interesting papers on the phytopathogens assigned to the genus (Misaghi & Grogan, 1969
; Sands et al., 1970
; Pecknold & Grogan, 1973
).
DNADNA hybridization experiments on the above groups were performed with DNA immobilized on membranes, following a methodology developed for other groups of organisms (Johnson & Ordal, 1968), and the results were a general confirmation of the phenotypic classification. However, the wide range of DNA homology values suggested a high degree of genomic heterogeneity among the species assigned to the genus, and this required the exploration of an area that was quite unfamiliar to most bacterial taxonomists.
While these experiments were in progress, Roger Stanier decided to leave the country to accept a position in the Institut Pasteur in Paris, and in my discussions with Mike Doudoroff I manifested the need for further investigation by concentrating our attention on conservative regions of the genome. A few years before, work in two laboratories (Doi & Igarashi, 1965; Dubnau et al., 1965
) had demonstrated that the genes of rRNA met this criterion. The demonstration of the conservative nature of rRNA genes had been focused on the properties of strains of Bacillus, and a further confirmation came from experiments that included strains of several bacterial genera (Pace & Campbell, 1971a
, b
). These two interesting contributions were not, however, aimed at the solution of taxonomic problems, but rather at the demonstration of residual rRNA similarities in distantly related organisms, which, by the way, could be readily differentiated by following classical methods of taxonomic analysis. Organisms as distant as Escherichia coli and Bacillus stearothermophilus still preserved detectable levels of sequence similarity in these genes (Pace & Campbell, 1971b
). In contrast, the application of the rRNA hybridization technique to Pseudomonas species was of a more uncertain nature, since all the organisms that had been assigned to this genus shared many basic morphological and physiological properties, and nothing seemed to guarantee a priori any degree of success. However, the idea of using it to resolve the genomic complexity that seems to underline the properties of Pseudomonas species in the DNADNA hybridization experiments appeared to be particularly attractive.
We adapted to the rRNADNA hybridization experiments the competition method that we had used for our DNADNA experiments, and the results were strikingly clear. Pseudomonas, as classically defined, became subdivided into five well-defined rRNA homology groups which deserved at least five independent generic designations. The main conclusions were already available by the end of 1970, and were incorporated by Roger Stanier in the text of his presentation at a Congress of General Microbiology that took place in Mexico in 1971 (Stanier, 1971). He was pleasantly surprised when he heard the success of the line followed by us and of course, the tone of his comments at the congress differed significantly from that of his publication in collaboration with van Niel a few years before. Interestingly enough, Stanier said, it was van Niel (1946)
who first clearly pointed out the shortcomings of the deductive phylogenetic approach to bacterial classification. Today, there are few bacteriologists who still believe in its utility. This does not necessarily mean, however, that evolutionary considerations should have no place in bacterial taxonomy. And he goes on referring to the possibility of defining phylogenetic relationships by experiments involving rRNA. The actual results of the rRNADNA hybridization experiments were published later (Palleroni et al., 1973
).
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Further developments |
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Phenotypic studies
The long phenotypic report (Stanier et al., 1966) was so well received that soon it reached the level of a citation classic. In little more than a decade, the number of citations reached a figure well above 1000, which is rather uncommon for a paper on a taxonomic subject. Its success as a descriptive tool was in part due to its applicability to the study of other Gram-negative organisms and even selected Gram-positive groups (Hagedorn & Holt, 1975
). The importance of the contribution was acknowledged by many other workers, and it was cited even recently as a good example of a biodiversity study (Spiers et al., 2000
).
As expected, the methodology of phenotypic studies, which are very labour demanding, particularly with respect to nutritional screenings, has been simplified by means of the use of commercial kits. The simplification did not necessarily represent an improvement. Our nutritional studies included direct observation of growth on plates, and in many cases they benefited from the expertise of Mike Doudoroff in carbohydrate metabolism, the vast knowledge of Roger Stanier in the degradation of aromatic compounds, and Ed Adelberg's familiarity with the degradation of amino acids by E. coli. Frequently, the results of the nutritional tests were supplemented with observations on probable pathways to be included in more detailed studies. It is obvious, however, that these conditions are no longer found in most cases. The nutritional properties are reported as results obtained with commercial kits designed to determine the utilization of a long list of compounds, supplemented by a number of enzymic reactions. They are presented without further discussion on their meaning, and are usually accepted without discussion by editors and reviewers alike.
Studies on rRNA
Here the situation is just the opposite. With some modifications, the hybridization method was used for more extensive taxonomic studies of the pseudomonads (De Vos et al., 1985, 1989
; De Vos & De Ley, 1983
), but nowadays studies of rRNA similarities are no longer practised with the cumbersome hybridization methods, but with the more direct and informative methodology of sequencing. Starting with the sequences of oligonucleotides (Fox et al., 1977
) and the determination of signature oligonucleotides' (Woese et al., 1985
), the total 16S rRNA sequence after PCR amplification is the procedure of choice.
The paper describing our rRNA experiments (Palleroni et al., 1973) included, among the most striking findings, the fact that some of the rRNA groups of pseudomonads were closer to species of other genera (Escherichia, Xanthomonas) than to members of other Pseudomonas groups. At that time, the surprising suggestion that some species of Pseudomonas appeared to be closer to the enteric bacteria than to other species of the same genus would have been flatly rejected by most bacteriologists. I am quite convinced that the new findings not only helped substantially to unravel the heterogeneity of a single bacterial genus but, in addition, they contributed to inspiring spectacular developments in other laboratories by the use of hybridization techniques or of more direct refinements, such as oligonucleotide similarity determinations.
The advent of rRNA sequence comparisons (going from the crude rRNADNA hybridization methods to the actual 16S gene sequencing) had some unfortunate consequences, because soon they became the main if not the only criterion for the estimation of phylogenetic relationships, and later for the development of methods purported to avoid culture isolation procedures in studies of prokaryote diversity. The unjustified climate of optimism had its roots in extending the relationships of single macromolecules to those of the respective host cells. This, added to the impossibility of inferring the physiological properties of organisms on the basis of such information, provoked animated discussions (Gest, 1999a, b
; Meyer et al., 1986
; Palleroni, 1994
, 1997
; Postgate, 1995
), in spite of which the taxonomic literature was enriched by proposals of many new taxa chiefly on the basis of 16S rRNA sequence considerations.
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Impact of the Pseudomonas studies |
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rRNA methodology has been remarkably influential (Woese, 1987) and 16S rDNA sequencing soon confirmed the basic groupings defined by Palleroni et al. (1973)
. It is only fair to mention here that before this time very important taxonomic research had been done on the basis of other cell components as subjects of chemotaxonomic or immunological methods of analysis. These approaches, however, did not have the general applicability of the nucleic acid studies and their phylogenetic implications were not always clear.
Of the valuable pieces of research that followed our work, but done on a much larger collection of strains, the work of De Ley and his group mentioned above is outstanding. The results were essentially comparable with those of the Berkeley group. The Pseudomonas work example was followed after a short interval by the beginning of an era of methodological euphoria so that the methods proposed in recent years represent a magnificent display of ingenuity. Making use of the enormous amount of information contained in some macromolecules, mainly nucleic acids and proteins, which was added to a large volume of results of very valuable studies already available on the cellular lipids and the components of cell walls and outer membranes, the phylogenetic relationships among prokaryote groups could be traced. The list of procedures for these special studies on Pseudomonas is so extensive that no detailed description will be included here.
I am sure that up to this point I have been taxing the reader who has had the tolerance of reading my previous reviews on Pseudomonas. I guess, however, that in this communication some sense of temporal perspective has been added that allows us to see that, in the decade of the 1960s, a change of mood in the participants occurred as a consequence of having envisioned for the first time the possibility of achieving phylogenetic classifications of bacteria. In fact, one of the consequences of prior methodologies was evident in the fact that successive editions of Bergey's Manuals were unable to define taxonomic hierarchies above the genus level.
The term pseudomonad still applies to all the organisms that have a group of phenotypic properties common to strains of the five rRNA homology groups and described in the early paper of Stanier et al. (1966), and therefore it makes little sense to try to single out true (Busse et al., 1989
) or genuine (Amann et al., 1996
) members within the group. In practice, the identification of a new isolate as a Pseudomonas strain may not necessarily require the use of methods beyond the capability of the laboratory facilities, and the worker may consider the use of the excellent enzymic approach developed by Jensen and his collaborators based on the peculiarities of the biosynthesis of aromatic amino acids (Byng et al., 1983
) or the cellular fatty acid analysis procedures aptly described by Oyaizu & Komagata (1983)
and Stead (1992)
.
The Pseudomonas team at Berkeley became dispersed at a moment when the obvious continuation would have been to pursue further studies on bacterial phylogeny. Seen in retrospect, the Pseudomonas example was just the tip of an iceberg whose real dimensions are still being evaluated. In his obituary notice of Mike Doudoroff, Stanier considered the projections of the work so significant that it will certainly remain one of the great landmarks of bacterial taxonomy (Stanier, 1975). The circumstances had prevented him from participating in the final phase of the work, but he liked to think of it as the culmination of our most ambitious and rewarding taxonomic enterprise (Stanier, 1980
).
As to the future of taxonomic studies on the genus Pseudomonas, there is no doubt that 16S rDNA sequencing (and the sequencing of other highly conserved genes; see, for example, Yamamoto et al., 2000) will continue to play an important role, but for such studies to be biologically meaningful they need to reflect the underlying genetic structure of the strains and populations being studied. Until recently most microbiologists assumed that populations were strictly clonal and under such an assumption the phylogeny of a single gene was taken to reflect the phylogeny of a set of strains if not a species. Genome sequencing and rigorous population genetic studies have shown this need not be true. Lateral gene transfer is far more pervasive than once thought (Ochman et al., 2000
) and for populations undergoing even limited recombination a phylogeny based on 16S rDNA may reflect little more than the phylogeny of the 16S rDNA gene.
The solution of this problem and therefore, the problem of meaningfully naming Pseudomonas, is not straightforward. At the very least it requires a much better understanding of the genetic structure of Pseudomonas populations. The limited evidence so far indicates that populations of Pseudomonas do exchange DNA (Haubold & Rainey, 1996; Stover et al., 2000
; Lomholt et al., 2001
), but despite this they are defined by clear genetic and phenotypic groupings. Interestingly, the groups defined by one of these studies (Haubold & Rainey, 1996
) corresponded precisely to the fundamental divisions originally described by Stanier et al. (1966
). The challenge for the future is not just to define and name the biologically meaningful groups within the genus Pseudomonas, but also to explain why, in the face of lateral gene transfer, distinct genetic groupings are maintained.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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