Properties and reactivity of myoglobin reconstituted with chemically modified protohemin complexes.

The synthetic complexes protohemin-6(7)-L-arginyl-L-alanine (HM-RA) and protohemin-6(7)-L-histidine methyl ester (HM-H) were prepared by condensation of suitably protected Arg-Ala or His residues with protohemin IX. HM-RA and HM-H were used for reconstitution of apomyoglobin from horse heart, yielding the Mb-RA and Mb-H derivatives, respectively, of the protein. The spectral, binding and catalytic properties of Mb-RA and Mb-H are significantly different from those of Mb. As shown by MM and MD calculations, these differences are determined by some local structural changes around the heme which are generated by increased mobility of a key peptide segment (Phe43-Lys47), containing the residue (Lys45) that in native Mb interacts with one of the porphyrin carboxylate groups. In the reconstituted Mbs this carboxylate group is bound to the Arg-Ala or His residue and is no longer available for electrostatic interaction with Lys45. The mobility of the peptide segment near the active site allows the distal histidine to come to a closer contact with the heme, and in fact Mb-RA and Mb-H exist as an equilibrium between a high-spin form and a major low-spin, six-coordinated form containing a bis-imidazole ligated heme. The two forms are clearly distinguishable in the NMR spectra, that also show that each of them consists of a mixture of the two most stable isomers resulting from cofactor reconstitution, as also anticipated by MM and MD calculations. Exogenous ligands such as cyanide, azide, or hydrogen peroxide can displace the bound distal histidine, but their affinity is reduced. On the other hand, mobilization of the peptide chain around the heme in the reconstituted Mbs increases the accessibility of large donor molecules at the heme periphery, with respect to native Mb, where a rigid backbone limits access to the distal pocket. The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and electron transfer of phenolic substrates in peroxidase-type oxidations catalyzed by the reconstituted proteins in the presence of hydrogen peroxide.

Phylogenetic depth of the bacterial genera Aquifex and Thermotoga inferred from analysis of ribosomal protein, elongation factor, and RNA polymerase subunit sequences.

The phylogenetic placement of the Aquifex and Thermotoga lineages has been inferred from (i) the concatenated ribosomal proteins S10, L3, L4, L23, L2, S19, L22, and S3 encoded in the S10 operon (833 aa positions); (ii) the joint sequences of the elongation factors Tu(1alpha) and G(2) coded by the str operon tuf and fus genes (733 aa positions); and (iii) the joint RNA polymerase beta- and beta'-type subunits encoded in the rpoBC operon (1130 aa positions). Phylogenies of r-protein and EF sequences support with moderate (r-proteins) to high statistical confidence (EFs) the placement of the two hyperthermophiles at the base of the bacterial clade in agreement with phylogenies of rRNA sequences. In the more robust EF-based phylogenies, the branching of Aquifex and Thermotoga below the successive bacterial lineages is given at bootstrap proportions of 82% (maximum likelihood; ML) and 85% (maximum parsimony; MP), in contrast to the trees inferred from the separate EF-Tu(1alpha) and EF-G(2) data sets, which lack both resolution and statistical robustness. In the EF analysis MP outperforms ML in discriminating (at the 0.05 level) trees having A. pyrophilus and T. maritima as the most basal lineages from competing alternatives that have (i) mesophiles, or the Thermus genus, as the deepest bacterial radiation and (ii) a monophyletic A. pyrophilus-T. maritima cluster situated at the base of the bacterial clade. RNAP-based phylogenies are equivocal with respect to the Aquifex and Thermotoga placements. The two hyperthermophiles fall basal to all other bacterial phyla when potential artifacts contributed by the compositionally biased and fast-evolving Mycoplasma genitalium and Mycoplasma pneumoniae sequences are eschewed. However, the branching order of the phyla is tenuously supported in ML trees inferred by the exhaustive search method and is unresolved in ML trees inferred by the quartet puzzling algorithm. A rooting of the RNA polymerase-subunit tree at the mycoplasma level seen in both the MP trees and the ML trees reconstructed with suboptimal amino acid substitution models is not supported by the EF-based phylogenies which robustly affiliate mycoplasmas with low-G+C gram-positives and, most probably, reflects a "long branch attraction" artifact.

The archaea monophyly issue: A phylogeny of translational elongation factor G(2) sequences inferred from an optimized selection of alignment positions.

A global alignment of EF-G(2) sequences was corrected by reference to protein structure. The selection of characters eligible for construction of phylogenetic trees was optimized by searching for regions arising from the artifactual matching of sequence segments unique to different phylogenetic domains. The spurious matchings were identified by comparing all sections of the global alignment with a comprehensive inventory of significant binary alignments obtained by BLAST probing of the DNA and protein databases with representative EF-G(2) sequences. In three discrete alignment blocks (one in domain II and two in domain IV), the alignment of the bacterial sequences with those of Archaea-Eucarya was not retrieved by database probing with EF-G(2) sequences, and no EF-G homologue of the EF-2 sequence segments was detected by using partial EF-G(2) sequences as probes in BLAST/FASTA searches. The two domain IV regions (one of which comprises the ADP-ribosylatable site of EF-2) are almost certainly due to the artifactual alignment of insertion segments that are unique to Bacteria and to Archaea-Eucarya. Phylogenetic trees have been constructed from the global alignment after deselecting positions encompassing the unretrieved, spuriously aligned regions, as well as positions arising from misalignment of the G' and G" subdomain insertion segments flanking the "fifth" consensus motif of the G domain (AE varsson, 1995). The results show inconsistencies between trees inferred by alternative methods and alternative (DNA and protein) data sets with regard to Archaea being a monophyletic or paraphyletic grouping. Both maximum-likelihood and maximum-parsimony methods do not allow discrimination (by log-likelihood difference and difference in number of inferred substitutions) between the conflicting (monophyletic vs. paraphyletic Archaea) topologies. No specific EF-2 insertions (or terminal accretions) supporting a crenarchaeal-eucaryal clade are detectable in the new EF-G(2) sequence alignment.

Discontinuous occurrence of the hsp70 (dnaK) gene among Archaea and sequence features of HSP70 suggest a novel outlook on phylogenies inferred from this protein.

Occurrence of the hsp70 (dnaK) gene was investigated in various members of the domain Archaea comprising both euryarchaeotes and crenarchaeotes and in the hyperthermophilic bacteria Aquifex pyrophilus and Thermotoga maritima representing the deepest offshoots in phylogenetic trees of bacterial 16S rRNA sequences. The gene was not detected in 8 of 10 archaea examined but was found in A. pyrophilus and T. maritima, from which it was cloned and sequenced. Comparative analyses of the HSP70 amino acid sequences encoded in these genes, and others in the databases, showed that (i) in accordance with the vicinities seen in rRNA-based trees, the proteins from A. pyrophilus and T. maritima form a thermophilic cluster with that from the green nonsulfur bacterium Thermomicrobium roseum and are unrelated to their counterparts from gram-positive bacteria, proteobacteria/mitochondria, chlamydiae/spirochetes, deinococci, and cyanobacteria/chloroplasts; (ii) the T. maritima HSP70 clusters with the homologues from the archaea Methanobacterium thermoautotrophicum and Thermoplasma acidophilum, in contrast to the postulated unique kinship between archaea and gram-positive bacteria; and (iii) there are exceptions to the reported association between an insert in HSP70 and gram negativity, or vice versa, absence of insert and gram positivity. Notably, the HSP70 from T. maritima lacks the insert, although T. maritima is phylogenetically unrelated to the gram-positive bacteria. These results, along with the absence of hsp70 (dnaK) in various archaea and its presence in others, suggest that (i) different taxa retained either one or the other of two hsp70 (dnaK) versions (with or without insert), regardless of phylogenetic position; and (ii) archaea are aboriginally devoid of hsp70 (dnaK), and those that have it must have received it from phylogenetically diverse bacteria via lateral gene transfer events that did not involve replacement of an endogenous hsp70 (dnaK) gene.

Manipulation of the tuf gene provides clues to the localization of sequence element(s) involved in the thermal stability of Thermotoga maritima elongation factor Tu.

Truncated versions of the tuf gene for elongation factor Tu (EF-Tu; 400 aa) from the hyperthermophilic bacterium Thermotoga maritima have been produced by progressive 3'-->5' trimming. The truncated genes have been expressed in Escherichia coli and the thermal stability of the gene products has been assayed by monitoring their GDP-binding capacity after preheating the cell-free extracts at various temperatures (65-95 degrees C). One of the truncated proteins, corresponding to the nucleotide-binding domain (G domain; aa 1-200) appears to be only slightly less stable than the full-length EF-Tu. Replacement of the first 90 N-terminal residues of both the full-length Thermotoga EF-Tu and the isolated G domain with the corresponding sequence of the mesophilic bacterium E. coli, drastically destabilizes both the complete and the truncated protein, indicating that sequence element(s) that are crucial for the attainment of a thermally stable conformation of the Thermotoga EF-Tu lie well within the initial portion of the G domain between residues 1 and 90. The relevant residues defy identification, however, as no amino acid preferences, or exclusive sequence element(s), appear to distinguish the N-terminal region of the thermophilic proteins from those of mesophilic counterparts. It is suggested that the thermal stability of Thermotoga EF-Tu is critically dependent upon unique tertiary structural interactions involving certain N-terminal residues of the molecule.

Arrangement and nucleotide sequence of the gene (fus) encoding elongation factor G (EF-G) from the hyperthermophilic bacterium Aquifex pyrophilus: phylogenetic depth of hyperthermophilic bacteria inferred from analysis of the EF-G/fus sequences.

The gene fus (for EF-G) of the hyperthermophilic bacterium Aquifex pyrophilus was cloned and sequenced. Unlike the other bacteria, which display the streptomycin-operon arrangement of EF genes (5'-rps12-rps7-fus-tuf-3'), the Aquifex fus gene (700 codons) is not preceded by the two small ribosomal subunit genes although it is still followed by a tuf gene (for EF-Tu). The opposite strand upstream from the EF-G coding locus revealed an open reading frame (ORF) encoding a polypeptide having 52.5% identity with an E. coli protein (the pdxJ gene product) involved in pyridoxine condensation. The Aquifex EF-G was aligned with available homologs representative of Deinococci, high G+C Gram positives, Proteobacteria, cyanobacteria, and several Archaea. Outgroup-rooted phylogenies were constructed from both the amino acid and the DNA sequences using first and second codon positions in the alignments except sites containing synonymous changes. Both datasets and alternative tree-making methods gave a consistent topology, with Aquifex and Thermotoga maritima (a hyperthermophile) as the first and the second deepest offshoots, respectively. However, the robustness of the inferred phylogenies is not impressive. The branching of Aquifex more deeply than Thermotoga and the branching of Thermotoga more deeply than the other taxa examined are given at bootstrap values between 65 and 70% in the fus-based phylogenies, while the EF-G(2)-based phylogenies do not provide a statistically significant level of support (< or = 50% bootstrap confirmation) for the emergence of Thermotoga between Aquifex and the successive offshoot (Thermus genus). At present, therefore, the placement of Aquifex at the root of the bacterial tree, albeit reproducible, can be asserted only with reservation, while the emergence of Thermotoga between the Aquificales and the Deinococci remains (statistically) indeterminate.

Chromosomal organization and nucleotide sequence of the genes for elongation factors EF-1 alpha and EF-2 and ribosomal proteins S7 and S10 of the hyperthermophilic archaeum Desulfurococcus mobilis.

The Desulfurococcus mobilis genes fus (encoding EF-2) and tuf (for EF-1 alpha) were cloned and sequenced together with genes for ribosomal proteins S10 (rps10) and S7 (rps7). Unlike Methanococcus, which displays the bacterial-like fus and tuf gene context 5'-rps12-rps7-fus-tuf-3', and similar to Sulfolobus and Pyrococcus, the Desulfurococcus fus gene (734 codons) has a distinct chromosomal location. Moreover, tuf (441 codons) is the promoter-proximal unit of a three-gene cluster comprising the genes rps10 (98 codons) and tRNA(Ser); the arrangement of the cluster is 5'-tuf-91 bp spacer -rps10-138 bp spacer -tRNA(Ser)-3' and the tuf gene is preceded by a canonical archaeal promoter. The D. mobilis gene rps7 (198 codons) is located further upstream from tuf (535 bp 'silent' intergenic spacing) and no rps12 homolog occurs in its immediate vicinity. Also, judging from putative promoter and transcription termination sequences, rps7 appears to be separately transcribed. Analysis of the predicted fus and tuf gene products revealed the three consensus motifs characteristic of GTP-binding proteins, and the fus-encoded EF-2 protein also displayed the consensus sequence required for ADP-ribosylation by Diphtheria toxin. Both EF sequences were definitely crenarchaeal by comparison with available homologs from other Archaea. Outgroup-rooted phylogenies derived from the sequences of ribosomal proteins S10 and S7 yielded the Sulfolobus-Desulfurococcus association at a high bootstrap confidence level.

Phylogenetic depth of S10 and spc operons: cloning and sequencing of a ribosomal protein gene cluster from the extremely thermophilic bacterium Thermotoga maritima.

A segment of Thermotoga maritima DNA spanning 6,613 bp downstream from the gene tuf for elongation factor Tu was sequenced by use of a chromosome walking strategy. The sequenced region comprised a string of 14 tightly linked open reading frames (ORFs) starting 50 bp downstream from tuf. The first 11 ORFs were identified as homologs of ribosomal protein genes rps10, rpl3, rpl4, rpl23, rpl2, rps19, rpl22, rps3, rpl16, rpl29, and rps17 (which in Escherichia coli constitute the S10 operon, in that order); the last three ORFs were homologous to genes rpl14, rpl24, and rpl5 (which in E. coli constitute the three promoter-proximal genes of the spectinomycin operon). The 14-gene string was preceded by putative -35 and -10 promoter sequences situated 5' to gene rps10, within the 50-bp spacing between genes tuf and rps10; the same region exhibited a potential transcription termination signal for the upstream gene cluster (having tuf as the last gene) but displayed also the potential for formation of a hairpin loop hindering the terminator; this suggests that transcription of rps10 and downstream genes may start farther upstream. The similar organization of the sequenced rp genes in the deepest-branching bacterial phyla (T. maritima) and among Archaea has been interpreted as indicating that the S10-spc gene arrangement existed in the (last) common ancestor. The phylogenetic depth of the Thermotoga lineage was probed by use of r proteins as marker molecules: in all except one case (S3), Proteobacteria or the gram-positive bacteria, and not the genus Thermotoga, were the deepest-branching lineage; in only two cases, however, was the inferred branching order substantiated by bootstrap analysis.

Evolution of translational elongation factor (EF) sequences: reliability of global phylogenies inferred from EF-1 alpha(Tu) and EF-2(G) proteins.

The EF-2 coding genes of the Archaea Pyrococcus woesei and Desulfurococcus mobilis were cloned and sequenced. Global phylogenies were inferred by alternative tree-making methods from available EF-2(G) sequence data and contrasted with phylogenies constructed from the more conserved but shorter EF-1 alpha(Tu) sequences. Both the monophyly (sensu Hennig) of Archaea and their subdivision into the kingdoms Crenarchaeota and Euryarchaeota are consistently inferred by analysis of EF-2(G) sequences, usually at a high bootstrap confidence level. In contrast, EF-1 alpha(Tu) phylogenies tend to be inconsistent with one another and show low bootstrap confidence levels. While evolutionary distance and DNA maximum parsimony analyses of EF-1 alpha(Tu) sequences do show archaeal monophyly, protein parsimony and DNA maximum-likelihood analyses of these data do not. In no case, however, do any of the tree topologies inferred from EF-1 alpha(Tu) sequence analyses receive significant bootstrap support.

The chromosomal location of genes for elongation factor Tu and ribosomal protein S10 in the cyanobacterium Spirulina platensis provides clues to the ancestral organization of the str and S10 operons in prokaryotes.

The structural gene (rps10) encoding ribosomal protein S10 of the cyanobacterium Spirulina platensis has been localized both on chromosomal DNA and the previously characterized recombinant plasmid pSp7 harbouring the 3'-terminal portion of the gene for elongation factor G (fus) and the gene for elongation factor Tu (tuf). Alignment of the predicted S10 sequence of S. platensis with the homologous sequences from cyanelles, bacteria, archaea and eukarya showed that the cyanobacterial S10 shares a high degree of sequence homology (74% amino acid identity) with the cyanellar protein. Unlike the situation in Escherichia coli, the rps10 gene of S. plantensis is unlinked to the S10 operon genes, being adjacent to the str operon genes. Since a similar organization could be observed in cyanelles of Cyanophora paradoxa and in all archaea so far analysed, this probably represents the ancestral state.

Cloning and sequencing of the gene encoding glutamine synthetase I from the archaeum Pyrococcus woesei: anomalous phylogenies inferred from analysis of archaeal and bacterial glutamine synthetase I sequences.

The gene glnA encoding glutamine synthetase I (GSI) from the archaeum Pyrococcus woesei was cloned and sequenced with the Sulfolobus solfataricus glnA gene as the probe. An operon reading frame of 448 amino acids was identified within a DNA segment of 1,528 bp. The encoded protein was 49% identical with the GSI of Methanococcus voltae and exhibited conserved regions characteristic of the GSI family. The P. woesei GSI was aligned with available homologs from other archaea (S. solfataricus, M. voltae) and with representative sequences from cyanobacteria, proteobacteria, and gram-positive bacteria. Phylogenetic trees were constructed from both the amino acid and the nucleotide sequence alignments. In accordance with the sequence similarities, archaeal and bacterial sequences did not segregate on a phylogeny. On the basis of sequence signatures, the GSI trees could be subdivided into two ensembles. One encompassed the GSI of cyanobacteria and proteobacteria, but also that of the high-G + C gram-positive bacterium Streptomyces coelicolor (all of which are regulated by the reversible adenylylation of the enzyme subunits); the other embraced the GSI of the three archaea as well as that of the low-G + C gram-positive bacteria (Clostridium acetobutilycum, Bacillus subtilis) and Thermotoga maritima (none of which are regulated by subunit adenylylation). The GSIs of the Thermotoga and the Bacillus-Clostridium lineages shared a direct common ancestor with that of P. woesei and the methanogens and were unrelated to their homologs from cyanobacteria, proteobacteria, and S. coelicolor. The possibility is presented that the GSI gene arose among the archaea and was then laterally transferred from some early methanogen to a Thermotoga-like organism. However, the relationship of the cyanobacterial-proteobacterial GSIs to the Thermotoga GSI and the GSI of low-G+C gram-positive bacteria remains unexplained.

Early evolutionary relationships among known life forms inferred from elongation factor EF-2/EF-G sequences: phylogenetic coherence and structure of the archaeal domain.

Phylogenies were inferred from both the gene and the protein sequences of the translational elongation factor termed EF-2 (for Archaea and Eukarya) and EF-G (for Bacteria). All treeing methods used (distance-matrix, maximum likelihood, and parsimony), including evolutionary parsimony, support the archaeal tree and disprove the "eocyte tree" (i.e., the polyphyly and paraphyly of the Archaea). Distance-matrix trees derived from both the amino acid and the DNA sequence alignments (first and second codon positions) showed the Archaea to be a monophyletic-holophyletic grouping whose deepest bifurcation divides a Sulfolobus branch from a branch comprising Methanococcus, Halobacterium, and Thermoplasma. Bootstrapped distance-matrix treeing confirmed the monophyly-holophyly of Archaea in 100% of the samples and supported the bifurcation of Archaea into a Sulfolobus branch and a methanogen-halophile branch in 97% of the samples. Similar phylogenies were inferred by maximum likelihood and by maximum (protein and DNA) parsimony. DNA parsimony trees essentially identical to those inferred from first and second codon positions were derived from alternative DNA data sets comprising either the first or the second position of each codon. Bootstrapped DNA parsimony supported the monophyly-holophyly of Archaea in 100% of the bootstrap samples and confirmed the division of Archaea into a Sulfolobus branch and a methanogen-halophile branch in 93% of the bootstrap samples. Distance-matrix and maximum likelihood treeing under the constraint that branch lengths must be consistent with a molecular clock placed the root of the universal tree between the Bacteria and the bifurcation of Archaea and Eukarya. The results support the division of Archaea into the kingdoms Crenarchaeota (corresponding to the Sulfolobus branch and Euryarchaeota). This division was not confirmed by evolutionary parsimony, which identified Halobacterium rather than Sulfolobus as the deepest offspring within the Archaea.

The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli.

The structural gene (glnA) encoding the glutamine synthetase (GS) of the extremely thermophilic eubacterium Thermotoga maritima has been cloned on a 6.0 kb HindIII DNA fragment. Sequencing of the region containing the glnA gene (1444 bp) showed an ORF encoding a polypeptide (439 residues) with an estimated mass of 50,088 Da, which shared significant homology with the GSI sequences of other Bacteria (Escherichia coli, Bacillus subtilis) and Archaea (Pyrococcus woesei, Sulfolobus solfataricus). The T. maritima glnA gene was expressed in E. coli, as shown by the ability to complement a glnA lesion in the glutamine-auxotrophic strain ET8051. The recombinant GS has been partially characterized with respect to the temperature dependence of enzyme activity, molecular mass and mode of regulation. The molecular mass of the Thermotoga GS (590,000 Da), estimated by gel filtration, was compatible with a dodecameric composition for the holoenzyme, as expected for a glutamine synthetase of the GSI type. Comparison of the amino acid sequence of T. maritima GS with those from thermophilic and mesophilic micro-organisms failed to detect any obvious features directly related to thermal stability.

Nucleotide sequence of a DNA region comprising the gene for elongation factor 1 alpha (EF-1 alpha) from the ultrathermophilic archaeote Pyrococcus woesei: phylogenetic implications.

The gene encoding elongation factor 1 alpha (EF-1 alpha, 1290 bp) of the ultrathermophilic, sulfur-reducing archaeote Pyrococcus woesei was localized within a Bg/II fragment of chromosomal DNA. Sequence analysis showed that the EF-1 alpha gene is the upstream unit of a three-gene cluster comprising the genes for ribosomal protein S10 (306 bp) and transfer RNAser (GGA). The three genes follow each other immediately in the order EF-1 alpha.S10.tRNA(ser) after a putative promoter located 55 bp upstream of the EF-1 alpha gene. Alignment of the derived EF-1 alpha sequence with the corresponding sequences from Eukarya, Bacteria/organelles, and with available archaeal sequences (Sulfolobus, Thermococcus, Methanococcus, Halobacterium) showed that Pyrococcus EF-1 alpha is highly homologous (89% identity) to Thermococcus celer EF-1 alpha, both being strikingly more similar to eukaryotic EF-1 alpha than to bacterial EF-Tu. Unrooted dendrograms computed from aligned sequences by distance matrix and DNA parsimony methods, including evolutionary parsimony, showed the Archaea to be a monophyletic-holophyletic cluster closer to Eukarya than to Bacteria. Both distance matrix and DNA parsimony--although not evolutionary parsimony--support the partition of the known archaeal lineages between the kingdoms Crenarchaeota and Euryarchaeota, and the affiliation of the Pyrococcus-Thermococcus lineage to the Euryarchaeota, of which it is the most primitive offspring. A closer relation of Pyrococcus to Euryarchaeota than to Crenarchaeota was also inferred from sequence analysis of S10 ribosomal proteins.

Phylogenetic depth of Thermotoga maritima inferred from analysis of the fus gene: amino acid sequence of elongation factor G and organization of the Thermotoga str operon.

The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacterium Thermotoga maritima was identified and sequenced. The EF-G coding sequence (2046 bp) was found to lie in an operon-like structure between the ribosomal protein S7 gene (rpsG) and the elongation factor Tu (EF-Tu) gene (tuf). The rpsG, fus, and tuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in the str operon of Escherichia coli. The derived amino acid sequence of the EF-G protein (682 residues) was aligned with the homologous sequences of other eubacteria, eukaryotes (hamster), and archaebacteria (Methanococcus vannielii). Unrooted phylogenetic dendrograms, obtained both from the amino acid and the nucleotide sequence alignments, using a variety of methods, lend further support to the notion that the (present) root of the (eu)bacterial tree lies between Thermotoga and the other bacterial lineages.

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: phylogenetic implications.

The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%-49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.

Organization and nucleotide sequence of the genes for ribosomal protein S2 and elongation factor Ts in Spirulina platensis.

A 6.5 kb region from the genome of the cyanobacterium Spirulina platensis was cloned using as a probe the Escherichia coli gene for ribosomal protein S2. Sequence analysis revealed, in this region, the presence of the gene for ribosomal protein S2 and part of the gene for the elongation factor Ts (EF-Ts). The arrangement rpsB-spacer-tsf resembles that reported for E. coli. The deduced amino acid sequences of the platensis S2 and EF-Ts show significant homology with the E. coli counterparts.

Phylogenetic conservation of antigenic determinants in archaebacterial elongation factors (Tu proteins).

By using affinity chromatography methods, we have purified elongation factor Tu (EF-Tu) proteins from a host of archaebacteria covering all known divisions in the archaebacterial tree except halophiles, and from such distantly related eubacteria as Thermotoga maritima and Escherichia coli. Polyclonal antibodies were raised against the Tu proteins of Sulfolobus solfataricus, Thermoproteus tenax, Thermococcus celer, Pyrococcus wosei, Archaeoglobus fulgidus, Methanococcus thermolitotrophicus, Thermoplasma acidophilum, and Thermotoga and used to probe the immunochemical relatedness of elongation factors both within and across kingdom boundaries. A selection of the results, presented here, indicates that (i) every archaebacterial EF-Tu is closer (immunochemically) to every other archaebacterial EF-Tu than to the functionally analogous proteins of eubacteria and eukaryotes, with only one possible exception concerning the recognition of eukaryotic (EF-1 alpha) factors by Thermococcus EF-Tu antibodies, and (ii) within the archaebacteria there appears to be a correlation between EF-Tu immunochemical similarities and the phylogenetic relatedness of the organisms inferred from other (sequence) criteria. On the whole, immunochemical similarity data argue against the proposal that the archaebacterial taxon should be split and redistributed between two superkingdoms.

Altered methionyl-tRNA synthetase in a Spirulina platensis mutant resistant to ethionine.

Compared with the parental strain, a Spirulina platensis mutant that is resistant to ethionine incorporated methionine into protein at a reduced rate, whereas ethionine incorporation was practically nil. The methionyl-tRNA synthetase present in crude extracts from the resistant strain showed a reduced affinity for methionine and ethionine.