General Stress Protein CTC from Bacillus subtilis Specifically Binds to Ribosomal 5S RNA

Two recombinant proteins of the CTC family were prepared: the general stress protein CTC from Bacillus subtilis and its homolog from Aquifex aeolicus. The general stress protein CTC from B. subtilis forms a specific complex with 5S rRNA and its stable fragment of 60 nucleotides, which contains internal loop E. The ribosomal protein TL5 from Thermus thermophilus, which binds with high affinity to 5S rRNA in the loop E region, was also shown to replace the CTC protein from B. subtilis in its complexes with 5S rRNA and its fragment. The findings suggest that the protein CTC from B. subtilis binds to the same site on 5S rRNA as the protein TL5. The protein CTC from A. aeolicus, which is 50 amino acid residues shorter from the N-terminus than the proteins TL5 from T. thermophilus and CTC from B. subtilis, does not interact with 5S rRNA.     

Polypurine.polypyrimidine sequences in complete bacterial genomes: preference for polypurines in protein-coding regions

The genomes of Methanococcus jannaschii, Mycoplasma genitalium, Haemophilus influenzae, Archaeoglobus fulgidus, Helicobacter pylori, Treponema pallidum, Borrelia burgdorferri, Rickettsia prowazekeii, Mycobacterium tuberculosis, Methanobacterium thermoautotrophicum, Synechocystis sp. PCC6803, Bacillus subtilis, Chlamydia trachomatis, Pyrococcus horikoshii, Aquifex aeolicus, Mycoplasma pneumoniae and Escherichia coli have been analysed for the presence of polypurine.polypyrimidine tracts, in order to understand their distribution in these genomes. We observed a variation in abundance of such sequences in these bacteria, with the archaeal genomes forming a high-abundance group and the canonical eubacteria forming a low-abundance group. The genomes of M. tuberculosis and A. aeolicus are unique among the organisms analysed here in the abnormal underrepresentation and overrepresentation of polypurine.polypyrimidine, respectively. We also observe a strand bias, i.e., a preferential occurrence of polypurines in coding strands. It varies widely among the bacteria, from the very high bias in M. jannaschii to the slightly inverse bias in the parasitic genomes of T. pallidum and C. trachomatis. The extent of strand bias, however, cannot be explained on the basis of the GC-content of the genome, use of all-purine codons or an excess in the amino acids that are encoded by such codons. The probable causes and effects of this phenomenon are discussed.     

Gene expression level shapes the amino acid usages in Prochlorococcus marinus MED4

Prochlorococcus species are the first example of free-living bacteria with reduced genome. Codon and amino acid usages bias of Prochlorococcus marinus MED4 was investigated using all protein coding genes having length greater than or equal to 100 amino acids. Correspondence analysis on relative synonymous codon usage (RSCU) values shows that there is no such influence of translational selection in shaping the codon usage variation among the genes in this organism. However, amino acid usages were markedly different between the highly and lowly expressed genes in this organism and in particular, GC rich amino acids were found to occur significantly higher in highly expressed genes than the lowly expressed genes. Comparative analysis of the homologous genes of Synechococcus sp. WH8102 and Prochlorococcus marinus MED4 shows that amino acids conservation in highly expressed genes is significantly higher than lowly expressed genes. Based on our results we concluded that conservation of GC rich amino acids in the highly expressed genes to its ancestor is the major source of variation in amino acid usages in the organism.     

On the organizational dynamics of the genetic code

The organization of the canonical genetic code needs to be thoroughly illuminated. Here we reorder the four nucleotides-adenine, thymine, guanine and cytosine-according to their emergence in evolution, and apply the organizational rules to devising an algebraic representation for the canonical genetic code. Under a framework of the devised code, we quantify codon and amino acid usages from a large collection of 917 prokaryotic genome sequences, and associate the usages with its intrinsic structure and classification schemes as well as amino acid physicochemical properties. Our results show that the algebraic representation of the code is structurally equivalent to a content-centric organization of the code and that codon and amino acid usages under different classification schemes were correlated closely with GC content, implying a set of rules governing composition dynamics across a wide variety of prokaryotic genome sequences. These results also indicate that codons and amino acids are not randomly allocated in the code, where the six-fold degenerate codons and their amino acids have important balancing roles for error minimization. Therefore, the content-centric code is of great usefulness in deciphering its hitherto unknown regularities as well as the dynamics of nucleotide, codon, and amino acid compositions.     

Expression and characterization of the terminal heme synthetic enzymes from the hyperthermophile Aquifex aeolicus

The terminal two heme biosynthetic pathway enzymes, protoporphyrinogen oxidase and ferrochelatase, of the hyperthermophilic bacterium Aquifex aeolicus have been expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Ferrochelatase and protoporphyrinogen oxidase of this organism are both monomeric, as was found for the corresponding enzymes of Bacillus subtilis. However, unlike the B. subtilis proteins, both A. aeolicus enzymes are membrane-associated. Both proteins have temperature optima over 60 degrees C. This is the first demonstration of functional heme biosynthetic enzymes in an extreme thermophilic bacterium.     

Does the Genetic Code Have A Eukaryotic Origin?

In the RNA world, RNA is assumed to be the dominant macromolecule performing most, if not all, core "house-keeping" functions. The ribo-cell hypothesis suggests that the genetic code and the translation machinery may both be born of the RNA world, and the introduction of DNA to ribo-cells may take over the informational role of RNA gradually, such as a mature set of genetic code and mechanism enabling stable inheritance of sequence and its variation. In this context, we modeled the genetic code in two content variables-GC and purine contents-of protein-coding sequences and measured the purine content sensitivities for each codon when the sensitivity (% usage) is plotted as a function of GC content variation. The analysis leads to a new pattern-the symmetric pattern-where the sensitivity of purine content variation shows diagonally symmetry in the codon table more significantly in the two GC content invariable quarters in addition to the two existing patterns where the table is divided into either four GC content sensitivity quarters or two amino acid diversity halves. The most insensitive codon sets are GUN (valine) and CAN (CAR for asparagine and CAY for aspartic acid) and the most biased amino acid is valine (always over-estimated) followed by alanine (always under-estimated). The unique position of valine and its codons suggests its key roles in the final recruitment of the complete codon set of the canonical table. The distinct choice may only be attributable to sequence signatures or signals of splice sites for spliceosomal introns shared by all extant eukaryotes.     

Nucleotide sequence of a cellulase gene of Bacillus subtilis

The nucleotide sequence of an endolytic cellulase gene of Bacillus subtilis was determined and compared with the NH2-terminal amino acid sequence of the purified enzyme. The mature protein appeared to be extended by a signal sequence of 36 amino acids. The putative AUG initiation codon was preceded by a sigma 43-type promoter of B. subtilis and an AAGGAGG sequence, typical of procaryotic ribosomal binding sites. Partial homology of amino acid sequences was found between B. subtilis cellulase and an alkalophilic Bacillus cellulase.     

A content-centric organization of the genetic code

The codon table for the canonical genetic code can be rearranged in such a way that the code is divided into four quarters and two halves according to the variability of their GC and purine contents, respectively. For prokaryotic genomes, when the genomic GC content increases, their amino acid contents tend to be restricted to the GC-rich quarter and the purine-content insensitive half, where all codons are fourfold degenerate and relatively mutation-tolerant. Conversely, when the genomic GC content decreases, most of the codons retract to the AUrich quarter and the purine-content sensitive half; most of the codons not only remain encoding physicochemically diversified amino acids but also vary when transversion （between purine and pyrimidine） happens. Amino acids with sixfolddegenerate codons are distributed into all four quarters and across the two halves; their fourfold-degenerate codons are all partitioned into the purine-insensitive half in favorite of robustness against mutations. The features manifested in the rearranged codon table explain most of the intrinsic relationship between protein coding sequences （the informational content） and amino acid compositions （the functional content）. The renovated codon table is useful in predicting abundant amino acids and positioning the amino acids with related or distinct physicochemical properties.     

Analysis of synonymous codon usage in <Emphasis Type="Italic">Zea mays</Emphasis>

It is important and meaningful to understand the codon usage pattern and the factors that shape codon usage of maize. In this study, trends in synonymous codon usage in maize have been firstly examined through the multivariate statistical analysis on 7402 cDNA sequences. The results showed that the genes positions on the primary axis were strongly negatively correlated with GC3s, GC content of individual gene and gene expression level assessed by the codon adaptation index (CAI) values, which indicated that nucleotide composition and gene expression level were the main factors in shaping the codon usage of maize, and the variation in codon usage among genes may be due to mutational bias at the DNA level and natural selection acting at the level of mRNA translation. At the same time, CDS length and the hydrophobicity of each protein were, respectively, significantly correlated with the genes locations on the primary axis, GC3s and CAI values. We infer that genes length and the hydrophobicity of the encoded protein may play minor role in shaping codon usage bias. Additional 28 codons ending with a G or C base have been defined as “optimal codons”, which may provide useful information for maize gene-transformation and gene prediction.     

Across bacterial phyla, distantly-related genomes with similar genomic GC content have similar patterns of amino acid usage

The GC content of bacterial genomes ranges from 16% to 75% and wide ranges of genomic GC content are observed within many bacterial phyla, including both gram negative and gram positive phyla. Thus, divergent genomic GC content has evolved repeatedly in widely separated bacterial taxa. Since genomic GC content influences codon usage, we examined codon usage patterns and predicted protein amino acid content as a function of genomic GC content within eight different phyla or classes of bacteria. We found that similar patterns of codon usage and protein amino acid content have evolved independently in all eight groups of bacteria. For example, in each group, use of amino acids encoded by GC-rich codons increased by approximately 1% for each 10% increase in genomic GC content, while the use of amino acids encoded by AT-rich codons decreased by a similar amount. This consistency within every phylum and class studied led us to conclude that GC content appears to be the primary determinant of the codon and amino acid usage patterns observed in bacterial genomes. These results also indicate that selection for translational efficiency of highly expressed genes is constrained by the genomic parameters associated with the GC content of the host genome.     

Cloning,analysis, and expression of the gene for inorganic pyrophosphatase of Aquifex pyrophilus and properties of the enzyme

The gene encoding Aquifex pyrophilus (Apy) pyrophosphatase was cloned and sequenced. The deduced amino acid sequence of Apy pyrophosphatase showed a 94.2% homology to Aquifex aeolicus (Aae) pyrophosphatase. The gene exhibits a difference in the codon usage at the third position from Aae pyrophosphatase. The gene was expressed under the control of a tac promoter in E. coli. The recombinant Apy pyrophosphatase was purified 18.7-fold with a 52.8% yield and a specific activity of 26.2 U mg(-1) protein. The native enzyme has a homotetramer of 177 amino acids. The enzyme shows optimal activity in pH 7.5. The optimum temperature was approximately 70 degrees C. A divalent cation was absolutely required for the enzyme activity; Mg2+ was the most effective.     

Translation in Bacillus subtilis: roles and trends of initiation and termination, insights from a genome analysis.

We analysed the Bacillus subtilis protein coding sequences termini, and compared it to other genomes. The analysis focused on signals, com-positional biases of nucleotides, oligonucleotides, codons and amino acids and mRNA secondary structure. AUG is the preferred start codon in all genomes, independent of their G+C content, and seems to induce less stable mRNA structures. However, it is not conserved between homologous genes neither is it preferred in highly expressed genes. In B.subtilis the ribosome binding site is very strong. We found that downstream boxes do not seem to exist either in Escherichia coli or in B.subtilis. UAA stop codon usage is correlated with the G+C content and is strongly selected in highly expressed genes. We found less stable mRNA structures at both termini, which we related to mRNA-ribosome and mRNA-release-factor interactions. This pattern seems to impose a peculiar A-rich nucleotide and codon usage bias in these regions. Finally the analysis of all proteins from B.subtilis revealed a similar amino acid bias near both termini of proteins consisting of over-representation of hydrophilic residues. This bias near the stop codon is partially release-factor specific.     

Complete nucleotide sequence of a cryptic plasmid from the ruminal bacterium Selenomonas ruminantium HD4 and identification of two predicted open reading frames.

A cryptic plasmid (pSR1) isolated from Selenomonas ruminantium HD4 was previously cloned into the HindIII site of pBR322 and a restriction map was constructed using HindIII, ClaI, BamHI, and PvuII (S. A. Martin and R. G. Dean, Appl. Environ. Microbiol. 55(12), 3035-3038, 1989). Analysis of the nucleotide sequence of pSR1 revealed two major open reading frames (ORFs) located in the minus strand at different frames. Analysis of ORF-1 revealed that it has 325 amino acids with a predicted MW of 36,588, and ORF-2 has 379 amino acids with a predicted MW of 42,651. The ORF-1 amino acids showed 30 to 32% sequence homology to the hypothetical protein YtqA in Bacillus subtilis and another hypothetical protein in the thermophilic bacterium Aquifex aeolicus. ORF-2 showed limited homology (23%) to the hypothetical protein ICFG in the photosynthetic cyanobacteria Synechocystis PCC6803.     

Changes in body temperature pattern in vertebrates do not influence the codon usages of alpha-globin genes

Codon usages are known to vary among vertebrates chiefly due to variations in isochore structure. Under the assumption that marked differences exist in isochore structure between warm-blooded and cold-blooded animals, the variations among vertebrates were previously attributed to an adaptation to homeothermy. However, based on data from a turtle species and a crocodile (Archosauromorpha), it was recently proposed that the common ancestors of mammals, birds and extent reptiles already had the "warm-blooded" isochore structure. We determined the nucleotide sequences of alpha-globin genes from two species of heterotherms, cuckoo (Cuculus canorus) and bat (Pipistrellus abramus), and three species of snakes (Lepidosauromorpha), Naja kaouthia from a tropical terrestrial habitat, Elaphe climacophora from a temperate terrestrial habitat, and Hydrophis melanocephalus from a tropical marine habitat. Our purposes were to assess the influence of differential body temperature patterns on codon usage and GC content at the third position of a codon (GC3), and to test the hypothesis concerning the phylogenetic position at which GC contents had increased in vertebrates. The results of principal component analysis (PCA) using the present data and data for other taxa from GenBank indicate that the primary difference in codon usage in globin genes among amniotes and other vertebrates lies in GC3. The codon usages (and GC3) in alpha-globin genes from two heterotherms and three snakes are similar to those in alpha-globin genes from warm-blooded vertebrates. These results refute the influence of body temperature pattern upon codon usages (and GC3) in alpha-globin genes, and support the hypothesis that the increase in GC content in the genome occurred in the common ancestor of amniotes.     

The relationship between synonymous codon usage and protein structure in Escherichia coli and Homo sapiens

The role of silent position in the codon on the protein structure is an interesting and yet unclear problem. In this paper, 563 Homo sapiens genes and 417 Escherichia coli genes coding for proteins with four different folding types have been analyzed using variance analysis, a multivariate analysis method newly used in codon usage analysis, to find the correlation between amino acid composition, synonymous codon, and protein structure in different organisms. It has been found that in E. coli, both amino acid compositions in differently folded proteins and synonymous codon usage in different gene classes coding for differently folded proteins are significantly different. It was also found that only amino acid composition is different in different protein classes in H. sapiens. There is no universal correlation between synonymous codon usage and protein structure in these two different organisms. Further analysis has shown that GC content on the second codon position can distinguish coding genes for different folded proteins in both organisms.     

A simple model based on mutation and selection explains trends in codon and amino-acid usage and GC composition within and across genomes

Background  

Correlations between genome composition (in terms of GC content) and usage of particular codons and amino acids have been widely reported, but poorly explained. We show here that a simple model of processes acting at the nucleotide level explains codon usage across a large sample of species (311 bacteria, 28 archaea and 257 eukaryotes). The model quantitatively predicts responses (slope and intercept of the regression line on genome GC content) of individual codons and amino acids to genome composition.