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(1α) and G(2) coded by the str operon tuf and fus genes (733 aa positions); and (iii) the joint RNA polymerase β- and β′-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(1α) 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. Received: 22 September 1999 / Accepted: 11 January 2000     

Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction.

Taking advantage of sequence conservation of portions of the alpha, beta, and beta' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the beta subunit and the 5' portion of the beta' subunit from a library of cloned murine Chlamydia trachomatis DNA. Similar attempts to recover the alpha subunit were unsuccessful. Sequence analysis demonstrated that the beta subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the beta' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli. The C. trachomatis beta subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit. Although this polyclonal antibody specifically immunoprecipitated the beta subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the beta subunit appeared early in infection.     

Evolved RNA Secondary Structure and the Rooting of the Universal Tree of Life

The origin and diversification of RNA secondary structure were traced using cladistic methods. Structural components were coded as polarized and ordered multi-state characters, following a model of character state transformation outlined by considerations in statistical mechanics. Several classes of functional RNA were analyzed, including ribosomal RNA (rRNA). Considerable phylogenetic signal was present in their secondary structure. The intrinsically rooted phylogenies reconstructed from evolved RNA structure depicted those derived from nucleic acid sequence at all taxonomical levels, and grouped organisms in concordance with traditional classification, especially in the archaeal and eukaryal domains. Natural selection appears therefore to operate early in the information flow that originates in sequence and ends in an adapted phenotype. When examining the hierarchical classification of the living world, phylogenetic analysis of secondary structure of the small and large rRNA subunits reconstructed a universal tree of life that branched in three monophyletic groups corresponding to Eucarya, Archaea, and Bacteria, and was rooted in the eukaryotic branch. Ribosomal characters involved in the translational cycle could be easily traced and showed that transfer RNA (tRNA) binding domains in the large rRNA subunit evolved concurrently with the rest of the rRNA molecule. Results suggest it is equally parsimonious to consider that ancestral unicellular eukaryotes or prokaryotes gave rise to all extant life forms and provide a rare insight into the early evolution of nucleic acid and protein biosynthesis. Received: 13 September 2000 / Accepted: 27 August 2001     

The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the beta beta' subunits of RNA polymerase following a nutritional shiftup of Escherichia coli.

The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the beta and beta' subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT-). In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the beta and beta' subunits of RNA polymerase increased with little or no lag. In contrast, in the spoT- strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup. An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and beta and beta' subunits occurred concurrently with the transient increase in ppGpp. In addition, the DNA-dependent synthesis in vitro of the beta and beta' subunits of RNA polymerase was inhibited by physiological levels of ppGpp. Because of the timing and magnitude of the changes in ppGpp levels in the spoT- strain versus the timing when the new rates of stable RNA, ribosomal protein, and beta and beta' subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes.     

Mutation slowing down the degradation of the beta beta'-subunits of Escherichia coli RNA-polymerase

The rpoC1 ts mutation affecting the RNA polymerase beta' subunit accelerates synthesis of RNA polymerase beta beta' subunits at 42 degrees C, while the surplus amount of subunits degrades in an hour's time. In a Ts strain with two RNA polymerase mutations, rpoC1 and rpoB251, we obtained a ts+ reversion designated opr24 which slows down degradation of surplus beta beta' subunits. The slowing down of degradation and the resulting accumulation of beta beta' subunits does not affect the kinetics of beta beta' subunit synthesis after the transfer to 42 degrees C. The effects of the opr24 are allele non-specific. The mutation also slows down degradation of beta' subunit and the amber fragment of beta subunit in the strain with subunit amber mutation rpoB22. Besides, the opr24 mutation reduces proteolysis of anomalous proteins containing canavanine. The opr24 mutation has been mapped between 17 and 21 minutes on the Escherichia coli map.     

Evidence for the early divergence of tryptophanyl- and tyrosyl-tRNA synthetases

Each amino acid is attached to its cognate tRNA by a distinct aminoacyl-tRNA synthetase (aaRS). The conventional evolutionary view is that the modern complement of synthetases existed prior to the divergence of eubacteria and eukaryotes. Thus comparisons of prokaryotic and eukaryotic aminoacyl-tRNA synthetases of the same type (charging specificity) should show greater sequence similarities than comparisons between synthetases of different types—and this is almost always so. However, a recent study [Ribas de Pouplana L, Furgier M, Quinn CL, Schimmel P (1996) Proc Natl Acad Sci USA 93:166–170] suggested that tryptophanyl- (TrpRS) and tyrosyl-tRNA (TyrRS) synthetases of the Eucarya (eukaryotes) are more similar to each other than either is to counterparts in the Bacteria (eubacteria). Here, we reexamine the evolutionary relationships of TyrRS and TrpRS using a broader range of taxa, including new sequence data from the Archaea (archaebacteria) as well as species of Eucarya and Bacteria. Our results differ from those of Ribas de Pouplana et al.: All phylogenetic methods support the separate monophyly of TrpRS and TyrRS. We attribute this result to the inclusion of the archaeal data which might serve to reduce long branch effects possibly associated with eukaryotic TrpRS and TyrRS sequences. Furthermore, reciprocally rooted phylogenies of TrpRS and TyrRS sequences confirm the closer evolutionary relationship of Archaea to eukaryotes by placing the root of the universal tree in the Bacteria. Received: 7 December 1996 / Accepted: 11 February 1997     

Molecular analysis of RNA polymerase alpha subunit gene from Streptomyces coelicolor A3(2).

The rpoA gene, encoding the alpha subunit of RNA polymerase, was cloned from Streptomyces coelicolor A3(2). It is preceded by rpsK and followed by rplQ, encoding ribosomal proteins S11 and L17, respectively, similar to the gene order in Bacillus subtilis. The rpoA gene specifies a protein of 339 amino acids with deduced molecular mass of 36,510 Da, exhibiting 64.3 and 70.7% similarity over its entire length to Escherichia coli and B. subtilis alpha subunits, respectively. Using T7 expression system, we overexpressed the S. coelicolor alpha protein in E. coli. A small fraction of this protein was found to be assembled into E. coli RNA polymerase. Antibody against S. coelicolor alpha protein crossreacted with that of B. subtilis more than with the E. coli alpha subunit. The ability of recombinant alpha protein to assemble beta and beta' subunits into core enzyme in vitro was examined by measuring the core enzyme activity. Maximal reconstitution was obtained at alpha2:beta+beta' ratio of 1:2.3, indicating that the recombinant alpha protein is fully functional for subunit assembly. Similar results were also obtained for natural alpha protein. Limited proteolysis with endoproteinase Glu-C revealed that S. coelicolor alpha contains a tightly folded N-terminal domain and the C-terminal region is more protease-sensitive than that of E. coli alpha.     

The Rooting of the Universal Tree of Life Is Not Reliable

Several composite universal trees connected by an ancestral gene duplication have been used to root the universal tree of life. In all cases, this root turned out to be in the eubacterial branch. However, the validity of results obtained from comparative sequence analysis has recently been questioned, in particular, in the case of ancient phylogenies. For example, it has been shown that several eukaryotic groups are misplaced in ribosomal RNA or elongation factor trees because of unequal rates of evolution and mutational saturation. Furthermore, the addition of new sequences to data sets has often turned apparently reasonable phylogenies into confused ones. We have thus revisited all composite protein trees that have been used to root the universal tree of life up to now (elongation factors, ATPases, tRNA synthetases, carbamoyl phosphate synthetases, signal recognition particle proteins) with updated data sets. In general, the two prokaryotic domains were not monophyletic with several aberrant groupings at different levels of the tree. Furthermore, the respective phylogenies contradicted each others, so that various ad hoc scenarios (paralogy or lateral gene transfer) must be proposed in order to obtain the traditional Archaebacteria–Eukaryota sisterhood. More importantly, all of the markers are heavily saturated with respect to amino acid substitutions. As phylogenies inferred from saturated data sets are extremely sensitive to differences in evolutionary rates, present phylogenies used to root the universal tree of life could be biased by the phenomenon of long branch attraction. Since the eubacterial branch was always the longest one, the eubacterial rooting could be explained by an attraction between this branch and the long branch of the outgroup. Finally, we suggested that an eukaryotic rooting could be a more fruitful working hypothesis, as it provides, for example, a simple explanation to the high genetic similarity of Archaebacteria and Eubacteria inferred from complete genome analysis.     

Necessity of polyamines for maximum in vivo synthesis of beta beta' subunits of RNA polymerase

The possibility that polyamines can stimulate the in vivo synthesis of beta beta' subunits of RNA polymerase has been examined through the use of a polyamine-requiring mutant of Escherichia coli. Results from autoradiographic estimation and activity measurement of RNA polymerase in cell extracts prepared from polyamine starved and unstarved bacteria showed that polyamines stimulate the synthesis of beta beta' subunits of RNA polymerase 2- to 3.6-fold.     

Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents.

The quaternary structures of Escherichia coli DNA-dependent RNA polymerase holenzyme (alpha 2 beta beta' sigma) and core enzyme (alpha 2 beta beta') have been investigated by chemical cross-linking with a cleavable bifunctional reagent, methyl 4-mercaptobutyrimidate, and noncleavable reagents, dimethyl suberimidate and N,N'-(1,4-phenylene)bismaleimide. A model of the subunit organization deduced from cross-linked subunit neighbors identified by dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the large beta and beta' subunits constitute the backbone of both core and holoenzyme, while sigma and two alpha subunits interact with this structure along the contact domain of beta and beta' subunits. In holoenzyme, sigma subunit is in the vicinity of at least one alpha subunit. The two alpha subunits are close to each other in holoenzyme, core enzyme, and the isolated alpha 2 beta complex. Cross-linking of the "premature" core and holoenzyme intermediates in the in vitro reconstitution of active enzyme from isolated subunits suggests that these species are composed of subunit complexes of molecular weight lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzyme-promoter recognition as well as the mechanism of subunit assembly of the enzyme.     

Evidence that sigma factors are components of chloroplast RNA polymerase.

Plastid genes are transcribed by DNA-dependent RNA polymerase(s), which have been incompletely characterized and have been examined in a limited number of species. Plastid genomes contain rpoA, rpoB, rpoC1, and rpoC2 coding for alpha, beta, beta', and beta" RNA polymerase subunits that are homologous to the alpha, beta, and beta' subunits that constitute the core moiety of RNA polymerase in bacteria. However, genes with homology to sigma subunits in bacteria have not been found in plastid genomes. An antibody directed against the principal sigma subunit of RNA polymerase from the cyanobacterium Anabaena sp. PCC 7120 was used to probe western blots of purified chloroplast RNA polymerase from maize, rice, Chlamydomonas reinhardtii, and Cyanidium caldarium. Chloroplast RNA polymerase from maize and rice contained an immunoreactive 64-kD protein. Chloroplast RNA polymerase from C. reinhardtii contained immunoreactive 100- and 82-kD proteins, and chloroplast RNA polymerase from C. caldarium contained an immunoreactive 32-kD protein. The elution profile of enzyme activity of both algal chloroplast RNA polymerases coeluted from DEAE with the respective immunoreactive proteins, indicating that they are components of the enzyme. These results provide immunological evidence for sigma-like factors in chloroplast RNA polymerase in higher plants and algae.     

The Vertebrate 7S K RNA Separates Hagfish (Myxine glutinosa) and Lamprey (Lampetra fluviatilis)

7S RNA sequences from the hagfish (Myxiniformes) and lamprey (Petromyzontiformes) were cloned and analyzed. In both species, 7S L RNA (also designated SRP RNA, since it represents the RNA constituent of the signal recognition particle) was clearly detectable. The sequence similarity between the two species was 86%, compared with about 75% similarity between either of these species and mammals. 7S K RNA was also cloned from the lamprey. The similarity between the 7S K RNA of the lamprey and that of mammals was 68%. Interestingly, several interspersed elements were found with nearly 100% similarity compared with mammals. In contrast to the lamprey, no 7S K RNA-related sequences were detectable among hagfish RNA, neither in northern blots nor with the PCR assay. In view of the significant conservation between the 7S K RNA of lamprey and that of mammals (human), this unexpected result clearly separates lamprey and hagfish. In addition, the lack of detectable 7S K RNA sequences in an outgroup, such as amphioxus, indicates that these results do not reflect an autapomorphy of hagfish. Therefore, our data provide additional support to the notion of a sister group relationship between Petromyzontiformes and gnathostomous vertebrates to the exclusion of Myxiniformes. Received: 24 September 1999 / Accepted: 9 February 2000