To Eubacterial and Eucaryotic Systems
Before 1977, two classified groups of living organisms existed -- the procaryotes (eubacteria) and the eucaryotes. However, due to accumulation of ribosomal ribonucleic acid (rRNA) sequence data, a problem arose with this classification. A third group seemed to exist as a phylogenetic group on the basis of their 16S rRNA sequence (31). Thus, in 1977, C.R. Woese and his colleagues proposed a third, distinct group known as the archaebacteria that was based on homologies in the 16S rRNA (39). Today, the kingdom Archaea is recognized and well established but is still being developed. To elaborate, when considering general phenotypes, archaebacteria resemble ordinary bacteria. On the basis of molecular homology, however, they are as different as eucaryotes are from eubacteria. Finally, the archaebacteria arose approximately 3.5 x 109 years ago independently of the other two kingdoms (10), thus, they provide a third perspective of the processes of cellular evolution.
The archaebacteria are a very diverse group but have one characteristic in common; they all live in extreme environments -- i.e. high salt concentrations, low pH, or high temperature. They include anaerobes, aerobes, autotrophs, heterotrophs, thermophiles, acidophiles, and photosynthetics. The archaebacteria group consists of the methanogens, the extreme halophiles, and the thermoacidophiles.
The methanogens are fastidious anaerobes whose metabolism involves the use of hydrogen (H2) to reduce carbon dioxide (CO2) to methane (CH4). They are the most diverse group of archaebacteria as they can be extremely thermophilic, moderately thermophilic, or mesophilic and, unlike other archaebacteria, are found in a wide variety of extreme environments. Morphologically, methanogens can exist as cocci, rods, or spirillum. The extreme halophiles grow in areas near 45oC, neutral pH, and close to the saturation point of NaCl (16 to 26% wt/vol). They are usually aerobic chemoorganotrophs, but a few can grow anaerobically in the presence of nitrates. Their morphology consists of a rod or a disk shape. The thermoacidophiles are aerobic and grow at low pH (2-3.5) and high temperatures (70 to 90oC). They have chemolithotropic metabolism, or the capability of sulfur metabolism via the oxidation of sulfur to produce sulfuric acid. Because of this, they are also called sulfur-dependent archaebacteria. Finally, they have only been found to have cocciod morphology.
Taking the overall characteristics of archaebacterial, they contain several eucaryotic features as well as many eubacterial attributes. The archaebacteria exhibit a procaryotic cell on the basis of structure and cellular organization but possess features previously only seen in eucaryotes. Archaebacterial and eucaryotic cells have no general phenotypical characteristics but do possess certain molecular similarities. Some distinguishing characteristics of archaebacteria include their rRNA, tRNA, and r-protein sequences and structures, intervening sequences present in tRNA, rRNA, and possibly protein-encoding genes, and an RNA polymerase as complex as the eucaryotic counterpart.
The first consideration of the comparison of archaebacteria to eubacteria and eucaryotes will be genome characteristics, gene organization, and extrachromosomal DNA. To begin, the genome of the archaebacteria resembles the range of typical procaryotic genomes in size. For example, the methanogen genome possess typical eubacteria morphologies and is somewhat smaller than the genome of E. coli. It is circular, double stranded DNA, and approximately 1.9 Mbp in length (~45% the size of the E. coli genome) (23). On the level of gene organization, methanogenic gene organization and expression resembles bacterial patterns. For example, several genes are found in closely linked clusters, designated as operons. In addition, through Southern analysis experiments, the genes of archaebacteria are located throughout the genome as found in bacterial genomes and have few repetitive DNA sequences (31). Therefore, the genomes of archaebacteria appear to be unique sequences. In gene arrangement, both monocistronic and polycistronic genes seem to be possible (10). In the halophilic and sulfur-dependent Archaea, introns are present in some rRNA and tRNA (17). However, introns have yet to be discovered in methanogens. The halophilic genome contains families of mobile, repeated sequences due to a large number of transposable elements that resemble transposable elements found in eubacteria (35). A final characteristic of archaebacterial genomes and gene organization is that the methanogenic genome DNA has open reading frames separated by regions of high A+T content which are approximately 200 bp long with an A+T content close to 80% (6).
Extrachromosomal DNA is common in archaebacteria. In methanogens, for example, cryptic plasmids have been found (40). However, halophiles are found almost always to contain "satellite DNA" (24). Extreme halophiles show a high degree of genetic instability due to the presence of this extrachromosomal DNA material that include a large amount of insertion elements called ISHs (20). ISHs are responsible for high rates of phenotypic variability and promote rearrangements in the genome by inserting upstream or directly into genes, disrupting their expression (30). Therefore, their structure and function are similar to transposons. Now that genomic characteristics and organization has been reviewed, the DNA in relation to content and properties will be discussed.
An important distinction within the archaebacteria is the diversity of the G+C content, which can range from 28 to 68 mol%. In extreme halobacteria, the G+C content is always above 60% (20), and can be as high as 68% (11). In methanogens, the G+C content varies from 27.5 to 61% (3). The G+C content can even vary greatly within a genius. In one genius of methanogens, the mesophiles range from 32.7 to 40.7% G+C compared to 49.7% G+C for the one thermophile (3). However, the intergenic regions of methanogens are less G+C rich than the average value for the genome (Figure 1) (8).
Figure 1. G+C content of the intergenic regions with comparison to the genomic value of three different methanogenic archaebacteria
The archaebacteria contain unique proteins for the packaging of the DNA due to their existence in extreme environments. Histone-like proteins have been isolated that resemble the eucaryotic histones in the ability to protect small DNA fragments from nuclease digestion (9). Although some sequence homology exists with the eucaryotic histones 2A and 3, more homology exists with the eubacterial DNA-binding proteins HU-1 and HU-2 (9). These small, basic DNA proteins of archaebacteria contribute to the architecture of the genome and have been called HMf and HMt (each from different organisms). They have consensus of greater than 80% to each other and greater than 30% consensus to eucaryal core histones (32). HMf and HMt bind to DNA to form a nucleosome-like structure. The DNA molecule is wrapped in a positive toroidal supercoil (26). This is unlike eucaryal nucleosomes where the DNA is negatively supercoiled. Hyperthermophilic methanogens have an unusual topoisomerase called reverse gyrase. Together with DNA binding proteins, such as HMf, the genomes of these hyperthermophiles are protected from heat denaturation (27). Reverse gyrase is a type I topoisomerase, with requires both divalent cations and ATP to produce the positive, superhilical turns in DNA (28).
Transcription in archaebacteria contains several distinct and unique factors. The first one is the archaebacterial DNA-dependent RNA polymerase. A major difference between archaebacteria and eubacteria exists in the composition of their respective DNA-dependent RNA polymerase. Archaebacteria have only one type of DNA-dependent RNA polymerase. It is a multisubunit complex that is more complex than the a2bb´s eubacteria enzyme and simpler than the RNA polymerases type I, II, and III of eucaryotes (12). The Archaeal RNA polymerase contains 8-10 different polypeptide subunits, thus resembling the eucaryotic enzyme (8). Within the archaebacteria, small differences exist between the groups on their RNA polymerase structure. The sulfur-dependent thermophilic RNA polymerase contains a few more low-molecular weight subunits than do the halophilic and methanogenic RNA polymerases (4). The RNA polymerases of methanogens identify the translation initiation site by a Shine-Delgarno type of interaction (4). For halophilic and sulfur-dependent thermoacidophilic RNA polymerases, this is not the case since some of their mRNA's lack a 5' leader region. Reported in both eucaryotic and eubacterial RNA polymerase characteristics, the Methanococcus RNA polymerase exhibits preferred non-specific binding to the free ends of DNA and nicks (34). Another way in which methanogen RNA polymerase resembles bacterial RNA polymerase is that both of them bind directly to promoter-containing DNA sequences (35), whereas eucaryotic RNA polymerases require transcription factors for specific promoter recognition.
The second way in which transcription differs in the archaebacteria is the recognition signals used for the initiation of transcription. Both sequence and structure in a promoter region play an important role as signals for the initiation of transcription in strongly expressed genes of archaebacteria. The RNA polymerase binds to DNA to cover a stretch from -32 to +18 bp on the promoters of archaebacteria (34). From this initial discovery, a general promoter region has been accepted for archaebacteria that is shown not to start at the same sites typical to eubacterial start sites. Because of this difference in the eubacterial -35 sequence and the spacing of promoter regions, the discrimination between archaebacterial stable promoters and eubacterial promoters can clearly be seen. As stated by Michael Thomm (9), "The discovery of archaebacteria as a second procaryotic line of descent and the structural similarity of their RNA polymerases with the eucaryotic transcription enzyme poses the question on the structure of archaebacterial promoters" (pg. 151).
Two consensus boxes were found to exist in RNA genes which are transcriptional signals in archaebacteria. This was found by the analysis of an intergenic region between transcriptional units of rRNA and tRNA genes and RNA polymerase binding and footprinting analysis in front of structural genes (1). For rRNA and tRNA genes, the RNA polymerase binding site was located 31 (±1) bp and 32 (±1) bp, respectively, upstream from the start site of transcription (34). From the location of this binding area and subsequent sequencing, the consensus of TTTA[T/A]ATA was found located at the -25 region of archaebacterial promoters for stable RNA genes (34) and was designated as part of the box A region (figure 2A). Only the sequence TTTA[T/A]ATA (8 bp) of box A is protected by RNA polymerase (34). Therefore, this octanucleotide is very important in the box A motif. In addition, this sequence is conserved between all representatives of archaebacteria (expect for halophiles, whose consensus is CTTA[T/A]GTA) (16). However, the box A sequence from the -30 to -40 region is much less conserved. In comparison, eucaryotic promoters for RNA polymerase B, the DNA sequence consensus is 5' TATA[T/A]A[T/A] 3' (the TATA box) that exists 25 bp upstream from the state site of transcription (9). Homology exists in both similarity in sequence and the location of the box A octanucleotide with the eucaryotic TATA box (which proceeds protein encoding genes transcribed by RNA polymerase B) (figure 2B).
In addition to the box A motif, an additional homologous sequence was found 25 bp downstream of box A (31). This short 4 bp sequence was designated as box B and is now known as the site of transcriptional initiation. The box B motif, ATGC, is the same between the different groups of archaebacteria. Therefore, the two conserved sequences involved in the binding of RNA polymerase are box A, located 20-40 bp upstream of stable RNA genes, and box B, located at the start of transcription (figure 3). Now that the general archaebacterial promoter for RNA genes has been considered, let's briefly discuss them for protein-encoding genes.
Box A sequences are very well conserved for RNA genes, but are less clear when considering protein-encoding genes since few have been sequenced. Consensus sequences for transcriptional activation were found both upstream and downstream of the transcriptional start sites for protein-encoding genes that correspond to the stable RNA genes (1). Of the few
In addition to DNA sequence, the structure of DNA in front of the transcriptional unit influences the units function in both eucaryotes and eubacteria. For example, E. coli promoters usually are proceeded by regularly spaced oligonucleotide sequences that lead to DNA binding. In addition to the homology of promoter sequences, a bending of DNA in front of the transcriptional start sites was found along with a common DNA structure at the end of the 5' transcript (1). Therefore, both sequence and structure of DNA play a role in the initiation of transcription for protein-encoding genes of archaebacteria.
A way in which transcription in archaebacteria resembles eubacteria is the termination of transcription. A termination site has been identified that contains a G+C-rich inverted repeat followed by an A+T-rich region (25), thus resembling the termination sites of eubacteria. In methanogen genes, transcriptional termination conforms to one of two models -- 1) An inverted repeat sequence found after the termination unit forms a stem-loop structure in the transcript; and, 2) An oligo-T sequence immediately following the gene (8). However, the oligo-T sequences are found only in hyperthermophiles (14), suggesting that the stem-loop structures in transcripts are not stable as a termination structure in cells growing at greater than 80oC.
The third and final major difference of transcription in archaebacteria is the modification of RNA nucleotides. mRNA in archaebacteria, like eubacteria, is not capped and have poly-A+ tails averaging only 12 bp in length (7). For tRNAs, patterns of the modification for 16S rRNA have shown that nothing is in common between the archaebacteria and eubacteria even though the same location of each 16S rRNA sequence is modified (39). Long inverted-repeat sequences are found around the 16S and 23S genes in all the major archaebacteria groups (10). These long inverted-repeat sequences along with other conserved sequences are used for rRNA maturation and assembly (4). The next topic of discussion will be translation and the machinery involved.
The comparative studies of the archaebacterial ribosome and its 16S rRNA sequence initially identified and separated the archaebacteria into a distinct phylogenetic group. To do the comparison of archaebacteria to eubacteria and eucaryotes, the machinery of translation, i.e. ribosomes, elongation factors, and tRNAs, will be discussed, compared, and contrasted to the machinery of eubacteria and eucaryotes. In this sense, the full distinction of the archaebacteria will be pointed out.
The Archaeal ribosomes are an intermediate size between eubacterial and eucaryotic ribosomes (2). The ribosome's amino acid sequence shows more homology with eucaryotes than eubacteria (4). However, the ribosomal proteins of archaebacteria are unusually acidic (15), which is normal considering the intracellular high concentration of ions found in archaebacteria (especially the halophiles). The archaebacterial ribosome resembles the eubacterial ribosome in size, 70S, and rRNA content, 23S, 16S, and 5S (4). The ribosome also dissociates into 30S and 50S subunits (16). Even though Archaea ribosomes contain rRNAs that are bacterial-sized, they have additional r-proteins that are related to r-proteins only previously found in eucaryal ribosomes (2, 36), which explains the amino acid sequence homology with eucaryotes. For example, the ribosomal 'A' protein in archaebacteria has a high degree of homology with the eucaryotic 'A' protein and little homology with the eubacterial L12 equivalent protein (22). Still, some of the Archaeal r-proteins show homology to the S9 and S15 eubacterial r-proteins with no homologies to the equivalent eucaryotic r-proteins (16).
The gene order for rRNA in archaebacteria, like eucaryotes, is 5'-16S-23S-5S-3' (29). The sulfur-dependent thermoacidophiles show the conserved gene order, but the 5S gene is not connected with the 16S and 23S genes. For example, the distance between the 16S and 23S rRNA ranges from 57 base pairs to greater than 2 kilobases, and between 23S and 5S, the distance ranges from less than 1 kilobases up to 11 kilobases (10). The methanogen rRNA-encoding genes, showing the 16S-23S-5S order, are transcribed into one primary transcript and processed to give rise to mature rRNAs (31). rRNA genes are found with long, almost perfect inverted-repeat sequences around the 16S and 23S genes (10), which, as described earlier, are used for this maturation and assembly.
The rRNAs are transcribed in a gene cluster, forming a single 7S RNA - tRNAser - 16S rRNA - tRNAala - 23S rRNA - 5S rRNA transcript. The 7S RNA molecule is present in large amounts in all Archaeal species investigated so far (21). The function of the 7S RNA molecule is still unknown. Examination of the primary sequence and the secondary structure shows a similarity to the 7S RNA component of the eucaryal signal-recognition particle (21). Finally, The secondary structure of the 5S rRNA resembles the bacterial 5S rRNA in three segments -- the 'molecular stalk,' the 'turned helix,' and the 'common arm base.' However, an additional region exists called the 'procaryotic loop' that neither bacteria nor eucaryotes possess (39).
Two highly conserved elongation factors are required for translation in eubacteria and eucaryotes. The archaebacteria elongation factors show homologies to these. Methanogens have elongation factors that are related to eucaryal elongation factors EF-2 and EF-1a (2). The archaebacterial aEF-2 translocation factor, like the eucaryotic counterpart, has a post-translationally modified histidinyl residue, diphthamide (19). aEF-1a, the archaebacterial aminoacyl-tRNA binding factor, resembles the eubacterial and eucaryotic factors in both molecular weight and the ability to bind GDP and GTP (19). In vitro, the archaebacterial aEF-1a and the eucaryotic EF-1a are interchangeable.
For tRNAs, the general secondary structures are similar among the three kingdoms, but many structural details are unique to the archaebacteria. Also, the modifications found in eubacterial tRNAs are very rarely found in their archaebacterial counterparts (39). tRNA genes examined for a number of archaebacterial species have all shown that none encode for the 3' CCA termini that is normally found on all mature tRNAs (37,38). In addition, the archaebacteria tRNA does not possess the common arm sequence of GTYCG (13,39), which had previously been accepted as a universal feature of tRNAs. The arm possesses sequences of GYYCG, or GUYCG (39). tRNAs from thermophilic methanogens indicated that they have more base pairs than mesophilic methanogens and employ more G+C pairs (14). Introns for tRNA genes in methanogens have not been reported even though they have been found in the tRNAs for halophilic and sulfur-dependent thermophilic Archaea (17). Finally, the codons of GTG and TTG are the most frequently used translation initiation codons (5), compared to the AUG codon for eubacteria and eucaryotes.
The archaebacteria are a very diverse group that offers a third perspective in gene expression and regulation. Although not much information is available on the subject, regulated genes from archaebacteria have already been cloned and regulatory studies can thus begin. Because the archaebacteria have much in common with the eubacteria and eucaryotes but yet are unique, a deeper insight into the genesis of microorganisms may be gained. Although substantial progress has been made in understanding bacterial diversity, many existing question are still unanswered.
As of yet, no system of transformation is available for the introduction of recombinant DNA molecules in archaebacteria. This has really hindered the analysis of gene structure, function, and regulation. Such transformation systems, when available, will facilitate the unraveling of the mysteries and complexities of the Archaeal kingdom, and thus give us that additional perspective on the solution of early cellular evolution.
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