The terms prokaryote and eukaryote actually refer to the basic structure of the cell (karyos= Greek for kernel or nucleus). Prokaryotes (pro= primitive) have a cell struc- ture that appears simple: a single heterogeneous compartment containing nucleic acids, sites of protein synthesis, enzymes for metabolic reactions and myriad other cell components. By contrast, eukaryotic (eu= true) cells appear complex, containing a nucleus and many other separate membrane-bound compartments or organelles in which specialized conditions for specific sets of cellular reactions are created. This structural specialization of eukaryotic cells creates a very clear demarcation separating eukaryotes – whether an amoeba, a rice plant or a camel – from prokaryotes, which,
although they may carry out similar sets of metabolic reactions, show no obvious intracellular structural sophistication.
The taxonomic classification of eukaryotes continues to be based primarily on morphological differences, though molecular analyses are also increasingly applied in combination with morphological criteria. For prokaryotes, this is not really possible or useful. At school we may have been taught that bacteria were either rods, cocci or spirilla – an attempt to classify bacteria according to their shape – but this classification is almost meaningless. For example, application of antibiotics or simple mutations can cause bacteria of several species, which normally exist as cocci, to form rods instead (e.g. Lleoet al., 1990). Prokaryote classification has also been based on other criteria such as the ability to carry out photosynthesis or to fix N2but, as will be repeatedly emphasized, such classification based on a limited set of phenotypic properties can in fact obscure significant similarities or differences.
The classification of prokaryotes remains very difficult, but the application of molecular biology has made possible a prokaryote classification that is systematic, in other words that does indicate evolutionary relationships between groups. This change is reflected in the renaming of the key book used for the identification of bacteria, ‘Bergey’s Manual’. Formerly called Bergey’s Manual of Determinative Bacteriology, indicating only that the identity of a particular bacterial isolate could be determined, the 8th edition was renamedBergey’s Manual of Systematic Bacteriology, indicating that the classifications actually try to reflect true evolutionary relationships (Krieg and Holt, 1984). The history of prokaryote classification is reviewed in an article defending the concept of the Bacteria and Archaea as two separate domains of prokaryotes (Woese, 1998).
The molecular method that has had the greatest impact on bacterial classifica- tion is the use of nucleic acid sequence homologies (Woese, 1987). There are many advantages to the use of nucleic acid sequences as a tool for taxonomic analysis.
First, unlike characters such as metabolic capabilities, protein profiles, or even morphology, the structure of DNA of any organism remains constant throughout its life cycle (with very few exceptions). Thus, whetherMyxobacteriaare present in the soil as a loose colony of single cells or aggregated together forming a multicellular fruiting body, the sequence of their DNA, and the information that can be derived from it, remains unaltered (Shimkets and Woese, 1992).
Secondly, the DNA sequence does in fact change with time through the process of random mutations, but, with certain exceptions, this is time measured in billions of generations and is tempered by the process of natural selection. Thus, while changes may occur at equal rates in all regions of the DNA, organisms that bear dele- terious changes, such as those that lead to loss of an essential enzyme, will not survive.
The result is that perceived rates of change of nucleic acid sequence – changes that can be seen in surviving organisms – vary in different regions of the genome. This is very fortunate for molecular evolutionists because it means that, to them, the DNA sequence can be viewed as a series of molecular clocks all ticking at different rates.
Thus, in one region of the genome there might be sufficient differences to discriminate between two very closely related bacteria, whereas in another the rate of accumulation of nucleotide changes may be so slow that comparisons can be
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made between bacteria separated by hundreds of millions of years of evolution. For example, sequence differences in the genes encoding the respiratory-chain protein cytochromec have been useful in the reordering of one bacterial subdivision, the a-purple bacteria, but the changes are too great to allow cytochromecgenes to be useful in determining phylogenetic relationships between more widely dispersed groups of bacteria. For these purposes, the sequences of ribosomal RNA genes (5S, 16S or 23S) have proved most useful (Woese, 1987) (Fig. 2.1). This pervasive application of molecular tools to bacterial systematics is reflected in a spurt of hand- books to molecular prokaryotic taxonomy published in the mid 1990s (Goodfellow and O’Donnell, 1993; Priest and Austin, 1993; Towner and Cockayne, 1993;
Logan, 1994) and, above all, in the massive rise in sequence information available. At the end of 1999 there were more than 12,000 16S rRNA gene sequences from eubacterial species in the public nucleic acid databases and a further 600–700 from Archaea.
To return specifically to the subject of N2-fixing organisms, these developments in molecular phylogeny of prokaryotes have underscored the variety of prokaryotic taxa that contain N2-fixing species, including both Bacteria and Archaea. This begs the questions of whether all N2-fixing organisms are derived from a common ances- tor (one that pre-dates the divergence of archaebacteria and eubacteria), whether the capacity for N2-fixation has evolved independently several times, or whether genes encoding functions needed for N2-fixation have spread between organisms more recently by lateral gene transfer.
Fig. 2.1. Sequence divergence between (a) variable and (b) conserved regions of genes encoding 16S rRNA from several bacteria (differences between sequences are shaded).
Nitrogenase genes found among diazotrophic archaebacteria show strong similarity to genes found among eubacteria, and thus it is generally accepted that this system of N2-fixation (there is now one other system known – see section on other actinomycetes in this chapter) has evolved only once. A corollary of this is that N2-fixation capability must have been lost many times in descendants of this
‘common ancestor’ to account for its current sporadic phylogenetic distribution (Young, 2000).
When the genes encoding the two components of dinitrogenase,nifDandnifK, and their homologues are considered, the genes cluster according to the metal requirements of the nitrogenase (Mo, Vn, Fe; see Chapter 3), suggesting that the presumed gene duplication that led to subsequent evolution of the alternative nitrogenases occurred only once (Kessleret al., 1997). However, when Component 1 nitrogenase genes are considered (nifH,vnfH,anfH), they fall into four groups and there is no clear separation according to metal requirements; anfH genes cluster with group IInifHgenes, andvnfHgenes cluster with group InifHgenes (Kessler et al., 1997). Furthermore, a nifH sequence-based phylogeny shows only partial concordance with accepted 16S rRNA-based phylogenies, suggesting that there has been either some gene duplication or lateral gene transfer in the course of the evolution of nitrogenase genes (Young, 1996, 2000).