Immunological heterogeneity of archaebacterial protein synthesis elongation factors Tu (EF-Tu)

Abstract Polyclonal antibodies were raised against the EF-Tu of the archaebacterium Sulfolobus solfataricus and cross-reactivities of EF-Tus of other, phylogenetically disparate archaebacteria were determined using Western blotting and ELISA. The results demonstrate a high degree of heterogeneity of archaebacterial Tu factors with recognition by S. solfataricus EF-Tu antibodies ranging from 48% to 1.5% that observed with the homologous antigen. The immunochemical relatedness between the heterologous and the cognate ( Sulfolobus ) antigens correlates satisfactorily with similarities in 16 S rRNA sequences, there being no recognition of eubacterial and eukaryotic factors by the S. solfataricus EF-Tu antibodies.     

Unique antibiotic sensitivity of archaebacterial polypeptide elongation factors.

The antibiotic sensitivity of the archaebacterial factors catalyzing the binding of aminoacyl-tRNA to ribosomes (elongation factor Tu [EF-Tu] for eubacteria and elongation factor 1 [EF1] for eucaryotes) and the translocation of peptidyl-tRNA (elongation factor G [EF-G] for eubacteria and elongation factor 2 [EF2] for eucaryotes) was investigated by using two EF-Tu and EF1 [EF-Tu(EF1)]-targeted drugs, kirromycin and pulvomycin, and the EF-G and EF2 [EF-G(EF2)]-targeted drug fusidic acid. The interaction of the inhibitors with the target factors was monitored by using polyphenylalanine-synthesizing cell-free systems. A survey of methanogenic, halophilic, and sulfur-dependent archaebacteria showed that elongation factors of organisms belonging to the methanogenic-halophilic and sulfur-dependent branches of the "third kingdom" exhibit different antibiotic sensitivity spectra. Namely, the methanobacterial-halobacterial EF-Tu(EF1)-equivalent protein was found to be sensitive to pulvomycin but insensitive to kirromycin, whereas the methanobacterial-halobacterial EF-G(EF2)-equivalent protein was found to be sensitive to fusidic acid. By contrast, sulfur-dependent thermophiles were unaffected by all three antibiotics, with two exceptions; Thermococcus celer, whose EF-Tu(EF1)-equivalent factor was blocked by pulvomycin, and Thermoproteus tenax, whose EF-G(EF2)-equivalent factor was sensitive to fusidic acid. On the whole, the results revealed a remarkable intralineage heterogeneity of elongation factors not encountered within each of the two reference (eubacterial and eucaryotic) kingdoms.     

Nucleotide sequence of the gene coding for the elongation factor Tu from the extremely thermophilic eubacterium Thermotoga maritima

Abstract The gene coding for the elongation factor Tu (EF-Tu) of Thermatoga maritima was cloned and sequenced. The predicted amino acid sequence was compared with those of other eubacteria, an archaebacterium and two eukaryotes as well. The similarity values and the distance matrix tree show that Thermotoga is more closely related to the eubacteria than to the representatives of the other urkingdoms. Thermotoga maritima represents the deepest branching within the tree of EF-Tu sequences from all eubacteria studied so far.     

Phylogenetic depth ofThermotoga maritima inferred from analysis of thefus gene: Amino acid sequence of elongation factor G and organization of theThermotoga str operon

Summary The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacteriumThermotoga 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). TherpsG, fus, andtuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in thestr operon ofEscherichia 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 dendrogram, 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 betweenThermotoga and the other bacterial lineages.     

Archaebacterial elongation factor Tu insensitive to pulvomycin and kirromycin

A spermine-dependent, polyphenylalanine-synthesizing cell-free system having an optimum activity at 75-85 degrees C, has been developed from the extremely thermoacidophilic archaebacterium Caldariella acidophila. The C. acidophila system is totally insensitive to the EF-Tu targeted antibiotics pulvomycin (at 40 degrees C) and kirromycin (at 47-72 degrees C) contrary to control systems derived from both mesophilic (Escherichia coli) and thermoacidophilic (Bacillus acidocaldarius) eubacteria. The archaebacterial EF-Tu-equivalent factor is also immunologically unrelated to eubacterial EF-Tu and does not cross react with antibodies against Escherichia coli EF-Tu. The pulvomycin and kirromycin reactions thus provide new phyletic markers for archaebacterial ancestry.     

Evolution of HSP70 gene and its implications regarding relationships between archaebacteria,eubacteria, and eukaryotes

The 70-kDa heat-shock protein (HSP70) constitutes the most conserved protein present in all organisms that is known to date. Based on global alignment of HSP70 sequences from organisms representing all three domains, numerous sequence signatures that are specific for prokaryotic and eukaryotic homologs have been identified. HSP70s from the two archaebacterial species examined (viz., Halobacterium marismortui and Methanosarcina mazei) have been found to contain all eubacterial but no eukaryotic signature sequences. Based on several novel features of the HSP70 family of proteins (viz., presence of tandem repeats of a 9-amino-acid [a.a.] polypeptide sequence and structural similarity between the first and second quadrants of HSP70, homology of the N-terminal half of HSP70 to the bacterial MreB protein, presence of a conserved insert of 23–27 a.a. in all HSP70s except those from archaebacteria and gram-positive eubacteria) a model for the evolution of HSP70 gene from an early stage is proposed. The HSP70 homologs from archaebacteria and gram-positive bacteria lacking the insert in the N-terminal quadrants are indicated to be the ancestral form of the protein. Detailed phylogenetic analyses of HSP70 sequence data (viz., by bootstrap analyses, maximum parsimony, and maximum likelihood methods) provide evidence that archaebacteria are not monophyletic and show a close evolutionary linkage with the gram-positive eubacteria. These results do not support the traditional archaebacterial tree, where a close relationship between archaebacterial and eukaryotic homologs is observed. To explain the phylogenies based on HSP70 and other gene sequences, a model for the origin of eukaryotic cells involving fusion between archaebacteria and gram-negative eubacteria is proposed. Correspondence to: R. S. Gupta     

Molecular phylogenies based on ribosomal protein L11, L1, L10, and L12 sequences

Summary Available sequences that correspond to the E. coli ribosomal proteins L11, L1, L10, and L12 from eubacteria, archaebacteria, and eukaryotes have been aligned. The alignments were analyzed qualitatively for shared structural features and for conservation of deletions or insertions. The alignments were further subjected to quantitative phylogenetic analysis, and the amino acid identity between selected pairs of sequences was calculated. In general, eubacteria, archaebacteria, and eukaryotes each form coherent and well-resolved nonoverlapping phylogenetic domains. The degree of diversity of the four proteins between the three groups is not uniform. For L11, the eubacterial and archaebacterial proteins are very similar whereas the eukaryotic L11 is clearly less similar. In contrast, in the case of the L12 proteins and to a lesser extent the L10 proteins, the archaebacterial and eukaryotic proteins are similar whereas the eubacterial proteins are different. The eukaryotic L1 equivalent protein has yet to be identified. If the root of the universal tree is near or within the eubacterial domain, our ribosomal protein-based phylogenies indicate that archaebacteria are monophyletic. The eukaryotic lineage appears to originate either near or within the archaebacterial domain. Correspondence to: P. Dennis     

The binding site of ribosomal protein L10 in eubacteria and archaebacteria is conserved: reconstitution of chimeric 50S subunits.

It has been shown by electron microscopy that the selective removal of the stalk from 50S ribosomal subunits of two representative archaebacteria, namely Methanococcus vaniellii and Sulfolobus solfataricus, is accompanied by loss of the archaebacterial L10 and L12 proteins. The stalk was reformed if archaebacterial core particles were reconstituted with their corresponding split proteins. Next, structurally intact chimeric 50S subunits have been reconstituted in vitro by addition of Escherichia coli ribosomal proteins L10 and L7/L12 to 50S core particles from M vaniellii or S solfataricus, respectively. In the reverse experiment, using core particles from E coli and split proteins from M vaniellii, stalk-bearing 50S particles were also obtained. Analysis of the reconstituted 50S subunits by immunoblotting revealed that E coli L10 was incorporated into archaebacterial core particles in both presence or absence of E coli L7/L12. In contrast, incorporation of E coli L7/L12 into archaebacterial cores was only possible in the presence of E coli L10. Our results suggest that in archaebacteria - as in E coli - the stalk is formed by archaebacterial L12 proteins that bind to the ribosome via L10. The structural equivalence of eubacterial and archaebacterial L10 and L12 proteins has thus for the first time been established. The chimeric reconstitution experiments provide evidence that the domain of protein L10 that interacts with the ribosomal particle is highly conserved between eubacteria and archaebacteria.     

Evolutionary Relationship Between Translation Initiation Factor eIF-2γ and Selenocysteine-Specific Elongation Factor SELB: Change of Function in Translation Factors

Eubacterial and eukaryotic translation initiation systems have very little in common, and therefore the evolutionary events that gave rise to these two disparate systems are difficult to ascertain. One common feature is the presence of initiation, elongation, and release factors belonging to a large GTPase superfamily. One of these initiation factors, the γ subunit of initiation factor 2 (eIF-2γ), is found only in eukaryotes and archaebacteria. We have sequenced eIF-2γ gene fragments from representative diplomonads, parabasalia, and microsporidia and used these new sequences together with new archaebacterial homologues to examine the phylogenetic position of eIF-2γ within the GTPase superfamily. The archaebacterial and eukaryotic eIF-2γ proteins are found to be very closely related, and are in turn related to SELB, the selenocysteine-specific elongation factor from eubacteria. The overall topology of the GTPase tree further suggests that the eIF-2γ/SELB group may represent an ancient subfamily of GTPases that diverged prior to the last common ancestor of extant life. Received: 2 January 1998 / Accepted: 1 June 1998     

The structure and evolution of archaebacterial ribosomal RNAs

A cladistic analysis of 553 5S rRNA sequences has revealed a Ur-5S rRNA, the ancestor of all present-day 5S rRNA molecules. Previously stated characteristic differences between the eubacterial and eukaryotic molecules, namely, the length base-pairing schemes of helices D, can be used as a marker for the various archaebacterial branches. One model comprises Thermococcus, Thermoplasma, methanobacteria, and halobacteria; a second comprises the Sulfolobales; and a third is represented only by the single organism Octopus Spring species 1. A relaxed selection pressure on helix E with subsequent deletions is observed in Methanobacteriales, Methanococcales, and eubacteria. The secondary structures are supported by enzymatic digestion and chemical modification studies of the 5S rRNAs. Reconstitution of eubacterial 50S ribosomal subunits with 5S rRNA from Halobacterium and Thermoplasma has revealed 100% incorporation, while eukaryotic 5S rRNAs yielded a 50% incorporation. Relevant positions of the small-subunit rRNA are selected to answer the question of the monophyly of archaebacteria. Eight positions account for monophyly, eight for an ancestry of eubacteria with halophile methanogens and eukaryotes with eocytes (paraphyly of archaebacteria), and two for an ancestry of eubacteria with eocytes. A refinement of the neighborliness method of S. Sattath and A. Tversky resulted in a monophyly of archaebacteria when all positions are treated equally and in a paraphyly when tranversions are weighted twice over transitions.     

Sequence and identification of the nucleotide binding site for the elongation factor Tu from Thermus thermophilus HB8.

Two structural genes for the Thermus thermophilus elongation factor Tu (tuf) were identified by cross-hybridization with the tufA gene from E. coli. The sequence of one of these tuf genes, localized on a 6.6 kb Bam HI fragment, was determined and confirmed by partial protein sequencing of an authentic elongation factor Tu from T. thermophilus HB8. Expression of this tuf gene in E. coli minicells provided a low amount of immuno-precipitable thermophilic EF-Tu. Affinity labeling of the T. thermophilus EF-Tu and sequence comparison with homologous proteins from other organisms were used to identify the guanosine-nucleotide binding domain.     

Structure and evolution of the L11, L1, L10, and L12 equivalent ribosomal proteins in eubacteria, archaebacteria, and eucaryotes

The genes corresponding to the L11, L1, L10, and L12 equivalent ribosomal proteins (L11e, L1e, L10e, and L12e) of Escherichia coli have been cloned and sequenced from two widely divergent species of archaebacteria, Halobacterium cutirubrum and Sulfolobus solfataricus, and the L10 and four different L12 genes have been cloned and sequenced from the eucaryote Saccharomyces cerevisiae. Alignments between the deduced amino acid sequences of these proteins and to other available homologous proteins of eubacteria and eucaryotes have been made. The data suggest that the archaebacteria are a distinct coherent phylogenetic group. Alignment of the proline-rich L11e proteins reveals that the N-terminal region, believed to be responsible for interaction with release factor 1, is the most highly conserved region and that there is specific conservation of most of the proline residues, which may be important in maintaining the highly elongated structure of the molecule. Although L11 is the most highly methylated protein in the E. coli ribosome, the sites of methylation are not conserved in the archaebacterial L11e proteins. The L1e proteins of eubacteria and archaebacteria show two regions of very high similarity near the center and the carboxy termini of the proteins. The L10e proteins of all kingdoms are colinear and contain approximately three fourths of an L12e protein fused to their carboxy terminus, although much of this fusion has been lost in the truncated eubacterial protein. The archaebacterial and eucaryotic L12e proteins are colinear, whereas the eubacterial protein has suffered a rearrangement through what appear to be gene fusion events. Within the L12e derived region of the L10e proteins there exists a repeated module of 26 amino acids, present in two copies in eucaryotes, three in archaebacteria, and one in eubacteria. This modular sequence is apparently also present in the L12e proteins of all kingdoms and may play a role in L12e dimerization, L10e-L12e complex formation, and the function of the L10e-L12e complex in translation.     

Cross-genomic analysis of the translational systems of various organisms

We have characterized the genes encoding ribosomal proteins (r-proteins) as well as other translation-related factors of 15 eubacteria and four archaebacteria, and the genes for the mitochondrial r-proteins of Saccharomyces cerevisiae by using the complete genomic nucleotide sequence data of these organisms. In eubacteria, including two species of Mycoplasma, the operon structure of the r-protein genes is well conserved, while their relative orientation and chromosomal location are quite divergent. The operon structure of the r-protein genes in archaebacteria, on the other hand, is quite different from eubacteria and also among themselves. In addition, many archaebacterial r-proteins show similarity to rat cytoplasmic r-proteins. Nonetheless, characteristic features of several genes encoding proteins of functional importance are well conserved throughout the bacterial species including archaebacteria, as well as in S. cerevisiae. We searched for the genes encoding mitochondrial r-proteins in yeast by combining informatics and genetic experiments. Furthermore, we characterized some of the r-proteins genes by exchanging portions between Escherichia coli and S. cerevisiae and performed functional analysis of some of the genes from different evolutionary points of view. Our work may be extended towards phylogenetic analysis of organisms producing secondary metabolites of various sorts. Journal of Industrial Microbiology & Biotechnology (2001) 27, 163–169. Received 21 September 1999/ Accepted in revised form 22 September 2000     

Cloning of the HSP70 gene from Halobacterium marismortui: relatedness of archaebacterial HSP70 to its eubacterial homologs and a model for the evolution of the HSP70 gene.

Heat shock induces the synthesis of a set of proteins in Halobacterium marismortui whose molecular sizes correspond to the known major heat shock proteins. By using the polymerase chain reaction and degenerate oligonucleotide primers for conserved regions of the 70-kDa heat shock protein (HSP70) family, we have successfully cloned and sequenced a gene fragment containing the entire coding sequence for HSP70 from H. marismortui. HSP70 from H. marismortui shows between 44 and 47% amino acid identity with various eukaryotic HSP70s and between 51 and 58% identity with its eubacterial and archaebacterial homologs. On the basis of a comparison of all available HSP70 sequences, we have identified a number of unique sequence signatures in this protein family that provide a clear distinction between eukaryotic organisms and prokaryotic organisms (archaebacteria and eubacteria). The archaebacterial (viz., H. marismortui and Methanosarcina mazei) HSP70s have been found to contain all of the signature sequences characteristic of eubacteria (particularly the gram-positive bacteria), which suggests a close evolutionary relationship between these groups. In addition, detailed analyses of HSP70 sequences that we have carried out have revealed a number of additional novel features of the HSP70 protein family. These include (i) the presence of an insertion of about 25 to 27 amino acids in the N-terminal quadrants of all known eukaryotic and prokaryotic HSP70s except those from archaebacteria and the gram-positive group of bacteria, (ii) significant sequence similarity in HSP70 regions comprising its first and second quadrants from organisms lacking the above insertion, (iii) highly significant similarity between a protein, MreB, of Escherichia coli and the N-terminal half of HSP70s, (iv) significant sequence similarity between the N-terminal quadrant of HSP70 (from gram-positive bacteria and archaebacteria) and the m-type thioredoxin of plant chloroplasts. To account for these and other observations, a model for the evolution of HSP70 proteins involving gene duplication is proposed. The model proposes that HSP70 from archaebacteria (H. marismortui and M. mazei) and the gram-positive group of bacteria constitutes the ancestral form of the protein and that all other HSP70s (viz., other eubacteria as well as eukaryotes) containing the insert have evolved from this ancient protein.     

Distribution of folates and modified folates in extremely thermophilic bacteria.

Analyses were made of the structures and levels of folates and modified folates present in extremely thermophilic bacteria. These procedures involved the chemical analysis of products resulting from the oxidative cleavage of the 6-substituted, folatelike tetrahydropterins present in the cells. Air-oxidized cell extracts of extreme thermophiles from two members of the archaebacterial order Thermococcales, Thermococcus celer and Pyrococcus furiosus, contained only 7-methylpterin, indicating that these cells contain a modified folate with a methylated pterin. Cell extracts also contained 6-acetyl-7-methyl-7,8-dihydropterin, another product derived from the oxidative cleavage of a dimethylated folate, demonstrating that both the C-7 and C-9 carbons of the pterin were methylated. Extracts, however, contained neither p-aminobenzoylpolyglutamates nor methaniline, the oxidative cleavage products of folates and methanopterin, respectively, indicating that they contain a previously undescribed C1 carrier(s). On the basis of the level of the 7-methylpterin isolated, the levels of modified folate were 2 to 10 times higher than those typically found in mesophilic bacteria and 10 to 100 times less than the level of methanopterin found in the methanogenic bacteria. Oxidized cell extracts of Sulfolobus spp. of the archaebacterial order Sulfolobales contained only pterin, and, like members of the order Thermococcales, they contained neither-p-aminobenzoylpolyglutamates nor methaniline. Oxidized cell extracts of the extreme thermophiles Pyrobaculum sp. strain H10 and Pyrodictium occultum, from the archaebacterial orders Thermoproteales and Pyrodictiales, respectively, and Thermotoga maritima from the eubacterial order Thermotogales, contained pterin and p-aminobenzoylpolyglutamates, indicating that these cells contained unmodified folates. The levels of p-aminobenzoylpolyglutamates in these archaebacterial cell extracts indicate that the folates were present in the cells at levels 4 to 10 times higher than generally found in those mesophilic eubacteria which do not folates in energy metabolism. The levels and chain lengths of the of p-aminobenzoylpolyglutamates present in Thermotoga maritima were typical of those found in mesophilic eubacteria.     

Antitumor drugs inhibit the growth of halophilic archaebacteria

Permeability mutants of Escherichia coli have been used to prescreen antitumor drugs. However, most compounds active against eucaryotic proteins have no effect on isofunctional proteins of eubacteria. In contrast, we show that growth of halophilic archaebacteria, procaryotes as distantly related to eubacteria as to eucaryotes, is inhibited by several drugs known to interact with tubulin, actomyosin and DNA topoisomerase II of eucaryotes. Actually, different types of evidence indicate the presence of analogous proteins in halophilic archaebacteria: (a) a yeast actin probe hybridizes with DNA restriction digests of Halobacterium halobium; (b) antibodies against tubulin and actin from chicken react in a crude extract of H. halobium with polypeptides having Mr of 55,000 and 80,000, respectively; (c) the epipodophyllotoxin VP16, a eucaryotic DNA topoisomerase II inhibitor, induces DNA strand breaks with DNA-protein covalent linkage in H. halobium as in eucaryotes. Besides the evolutionary implications, these data indicate that halophilic archaebacteria can be used to prescreen antitumor drugs active on eucaryotic proteins.     

A genome phylogeny for mitochondria among alpha-proteobacteria and a predominantly eubacterial ancestry of yeast nuclear genes

Analyses of 55 individual and 31 concatenated protein data sets encoded in Reclinomonas americana and Marchantia polymorpha mitochondrial genomes revealed that current methods for constructing phylogenetic trees are insufficiently sensitive (or artifact-insensitive) to ascertain the sister of mitochondria among the current sample of eight alpha-proteobacterial genomes using mitochondrially-encoded proteins. However, Rhodospirillum rubrum came as close to mitochondria as any alpha-proteobacterium investigated. This prompted a search for methods to directly compare eukaryotic genomes to their prokaryotic counterparts to investigate the origin of the mitochondrion and its host from the standpoint of nuclear genes. We examined pairwise amino acid sequence identity in comparisons of 6,214 nuclear protein-coding genes from Saccharomyces cerevisiae to 177,117 proteins encoded in sequenced genomes from 45 eubacteria and 15 archaebacteria. The results reveal that approximately 75% of yeast genes having homologues among the present prokaryotic sample share greater amino acid sequence identity to eubacterial than to archaebacterial homologues. At high stringency comparisons, only the eubacterial component of the yeast genome is detectable. Our findings indicate that at the levels of overall amino acid sequence identity and gene content, yeast shares a sister-group relationship with eubacteria, not with archaebacteria, in contrast to the current phylogenetic paradigm based on ribosomal RNA. Among eubacteria and archaebacteria, proteobacterial and methanogen genomes, respectively, shared more similarity with the yeast genome than other prokaryotic genomes surveyed.     

Unusually strong immunological cross-reaction between elongation factor Tu of Escherichia coli and Bacillus subtilis

Polyclonal antibodies were prepared against the purified elongation factor Tu (EF-Tu) of Escherichia coli and Bacillus subtilis. Using the methods of Western blotting and microcomplement fixation the cross-reactivities of EF-Tu of 19 different prokaryotes were determined. The immunological distance were compared with the results of 16S rRNA oligonucleotide analysis. An unexpectedly high cross-reactivity was revealed between the EF-Tu of B. subtilis and the antiserum against the EF-Tu of E. coli. A comparison of the predicted amino acid sequences from the tuf-genes of E. coli and B. subtilis yielded two identical peptide fragments that are likely candidates for antibody binding sites.Abbreviations EF-Tu elongation factor Tu - GDP guanosine 5-diphosphate - GTP guanosine 5-triphosphate - MCF microcomplement fixation - T type strain     

Appearance of elongation factor Tu in the outer membrane of sucrose-dependent spectinomycin-resistant mutants of Escherichia coli

When sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were grown in the absence of sucrose, a new protein appeared in the membrane fraction insoluble in Triton X-100. The protein had a hydrophobic nature. However, unlike other outer membrane proteins the new protein was extracted with sodium dodecyl sarcosinate. The new protein was found to be identical with elongation factor Tu (EF-Tu), as judged from the electrophoretic mobility in three different gel systems, coprecipitation with the antiserum against EF-Tu, the profiles of peptide fragments produced with three different proteases and analyses of N-terminal and C-terminal amino acids. This membrane EF-Tu accounted for 5-10% of total cell EF-Tu. When spheroplasts were pretreated with trypsin, EF-Tu in the outer membrane disappeared. Incubation of cytosol EF-Tu with the outer membrane did not result in the binding of EF-Tu to the membrane. These results indicate that the appearance of EF-Tu in the outer membrane is not due to artificial binding during membrane preparation. It is suggested that the ribosomal alteration resulted in dislocation of the cytosol protein into the outer membrane.     

Molecular properties of elongation factor Tu fromStreptomyces aureofaciens andEscherichia coli

Some molecular properties of the elongation factor Tu of protein synthesis purified in an aggregated state from gram-positive Streptomyces aureofaciens were studied and compared with those of Tu from gram-negative Escherichia coli. Electrofocussing under reducing conditions showed that the molecule of EF-Tu from S. aureofaciens has an isoelectric point shifted more to the acidic side compared with EF-Tu from E. coli. A comparison of amino acid composition revealed minor differences in the content of several amino acids in the two factors and showed that EF-Tu from S. aureofaciens contains four half-cystines per molecule. Under denaturing conditions only two mercapto groups reacted with 5,5'-dithiobis(2-nitrobenzoic acid). Limited tryptic digestion of aggregated EF-Tu from S. aureofaciens yields six fragments: the four main fragments are of a similar size as those of the E. coli factor. All fragments detected after trypsin digestion of S. aureofaciens EF-Tu were immunologically cross-reactive with antibodies against E. coli EF-Tu. However, even after 2 h of the reaction there still remains a small part of streptomycete factor uncleaved, which documents high resistance of aggregated EF-Tu towards trypsin.