Identification of the initiation codon for the atpB gene in Chlamydomonas chloroplasts excludes translation of a precursor form of the beta subunit of the ATP synthase

The chloroplast atpB gene of Chlamydomonas reinhardtii, which encodes the beta subunit of the ATP synthase, contains three in-frame ATGs that are candidate translation initiation codons. An earlier study revealed that the N terminus of the assembled beta subunit maps at the +2 position with respect to the second in-frame methionine codon (Fiedler et al. 1995). Using chloroplast transformation, we have examined the possibility that either of the two additional in-frame ATG codons is competent for translation initiation. We provide evidence that translation of atpB is initiated exclusively at the second ATG codon. We conclude that the beta subunit is not synthesized with an N-terminal leader before its assembly into a functional ATP synthase complex.     

Molecular characterization of two point mutants in the chloroplast atpB gene of the green alga Chlamydomonas reinhardtii defective in assembly of the ATP synthase complex

Two point mutants of Chlamydomonas reinhardtii, previously found by recombination and complementation analysis to map in the chloroplast atpB gene encoding the beta subunit of the CF1/CF0 ATP synthase, are here shown to be missense alterations near the 5' end of that gene. One mutant (ac-u-c-2-9) has a change at amino acid position 47 of the beta subunit from leucine (CTA) to arginine (CGA). In the second mutant (ac-u-c-2-29), the codon AAA (lysine) is changed to AAC (asparagine) at position 154. Spontaneous revertants of each mutant were isolated that restore the original wild type base pair. Northern analysis of total RNA and in vivo pulse labeling followed by immunoprecipitation reveals that both mutant atpB genes are transcribed and translated normally. However, immunoblots show that the amount of beta subunit associated with mutant thylakoids is only approximately 3% of that seen in wild type and that the CF1 alpha and gamma subunits are missing entirely. The disruption of ATP synthase complex assembly in these mutants is much more severe than in Escherichia coli beta subunit gene point mutants, which retain significant amounts of alpha and beta subunits on their membranes (Noumi, T., Oka, N., Kanazawa, H., and Futai, M. (1986) J. Biol. Chem. 261, 7070-7075). These results support the hypothesis that there are differences in assembly of the ATP synthase between E. coli and chloroplasts. In particular they indicate that beta must be present for assembly of the alpha and gamma subunits of CF1 onto chloroplast membranes.     

Factors affecting codon bias in the mitochondrial genomes of the streptophyte Mesostigma viride and the chlorophyte Chlamydomonas reinhardtii

Mitochondrial genomes typically show genome-wide patterns of synonymous codon usage bias. In animals and land plants, mutation appears more dominant than selection in shaping this bias, while in green algae the relative importance of these factors is not well studied. Based on our analysis of mitochondrial DNA sequence from the green algae Mesostigma viride (NIES-296) and Chlamydomonas reinhardtii (CC-277) and a closely related relative of each, we conclude that both mutation and selection are important in shaping synonymous codon usage bias in their mitochondrial genomes, with selection being more dominant. The possible confounding influence of mutational context dependence on our analyses is discussed.     

Analysis of the codon usage of the cephamycin C producer Nocardia lactamdurans

Abstract The G + C content in a sequenced region of 27 kb of the Nocardia lactamdurans genome is 70.4 and 70.6% in the 14 characterized ORFs, showing an extreme average G + C content (94.9%) in the third codon position. The codon usage parameters of the N. lactamdurans genes studied are closely related and depart weakly from the values of other species of the genus Nocardia . The homologies and differences in the codon usage between N. lactamdurans and Streptomyces sp. or other high-G + C Gram-positive genera are analysed.     

Comparative analysis of codon usage patterns in chloroplast genomes of the Asteraceae family

Codon usage bias (CUB) is an important evolutionary feature in a genome and has been widely documented from prokaryotes to eukaryotes. However, the significance of CUB in the Asteraceae family has not been well understood, with no Asteraceae species having been analyzed for this characteristic. Here, we use bioinformatics approaches to comparatively analyze the general patterns and influencing factors of CUB in five Asteraceae chloroplast (cp) genomes. The results indicated that the five genomes had similar codon usage patterns, showing a strong bias towards a high representation of NNA and NNT codons. Neutrality analysis showed that these cp genomes had a narrow GC distribution and no significant correlation was observed between GC₁₂ and GC₃. Parity Rule 2 (PR2) plot analysis revealed that purines were used more frequently than pyrimidines. Effective number of codons (ENc)-plot analysis showed that most genes followed the parabolic line of trajectory, but several genes with low ENc values lying below the expected curve were also observed. Furthermore, correspondence analysis of relative synonymous codon usage (RSCU) yielded a first axis that explained only a partial amount of variation of codon usage. These findings suggested that both natural selection and mutational bias contributed to codon bias, while selection was the major force to shape the codon usage in these Asteraceae cp genomes. Our study, which is the first to investigate codon usage patterns in Asteraceae plastomes, will provide helpful information about codon distribution and variation in these species, and also shed light on the genetic and evolutionary mechanisms of codon biology within this family.     

Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast

Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5'- and 3'-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.     

The typically mitochondrial DNA-encoded ATP6 subunit of the F1F0-ATPase is encoded by a nuclear gene in Chlamydomonas reinhardtii.

The atp6 gene, encoding the ATP6 subunit of F(1)F(0)-ATP synthase, has thus far been found only as an mtDNA-encoded gene. However, atp6 is absent from mtDNAs of some species, including that of Chlamydomonas reinhardtii. Analysis of C. reinhardtii expressed sequence tags revealed three overlapping sequences that encoded a protein with similarity to ATP6 proteins. PCR and 5'- and 3'-RACE were used to obtain the complete cDNA and genomic sequences of C. reinhardtii atp6. The atp6 gene exhibited characteristics of a nucleus-encoded gene: Southern hybridization signals consistent with nuclear localization, the presence of introns, and a codon usage and a polyadenylation signal typical of nuclear genes. The corresponding ATP6 protein was confirmed as a subunit of the mitochondrial F(1)F(0)-ATP synthase from C. reinhardtii by N-terminal sequencing. The predicted ATP6 polypeptide has a 107-amino acid cleavable mitochondrial targeting sequence. The mean hydrophobicity of the protein is decreased in those transmembrane regions that are predicted not to participate directly in proton translocation or in intersubunit contacts with the multimeric ring of c subunits. This is the first example of a mitochondrial protein with more than two transmembrane stretches, directly involved in proton translocation, that is nucleus-encoded.     

Analysis of five presumptive protein-coding sequences clustered between the primosome genes, 41 and 61, of bacteriophages T4, T2, and T6.

In bacteriophage T4, there is a strong tendency for genes that encode interacting proteins to be clustered on the chromosome. There is 1.6 kb of DNA between the DNA helicase (gene 41) and the DNA primase (gene 61) genes of this virus. The DNA sequence of this region suggests that it contains five genes, designated as open reading frames (ORFs) 61.1 to 61.5, predicted to encode proteins ranging in size from 5.94 to 22.88 kDa. Are these ORFs actually genes? As one test, we compared the DNA sequence of this region in bacteriophages T2, T4, and T6 and found that ORFs 61.1, 61.3, 61.4, and 61.5 are highly conserved among the three closely related viruses. In contrast, ORF 61.2 is conserved between phages T4 and T6 yet is absent from phage T2, where it is replaced by another ORF, T2 ORF 61.2, which is not found in the T4 and T6 genomes. As a second, independent test for coding sequences, we calculated the codon base position preferences for all ORFs in this region that could encode proteins that contain at least 30 amino acids. Both the T4/T6 and T2 versions of ORF 61.2, as well as the other ORFs, have codon base position preferences that are indistinguishable from those of known T4 genes (coefficients of 0.81 to 0.94); the six other possible ORFs of at least 90 bp in this region are ruled out as genes by this test (coefficients less than zero). Thus, both evolutionary conservation and codon usage patterns lead us to conclude that ORFs 61.1 to 61.5 represent important protein-coding sequences for this family of bacteriophages. Because they are located between the genes that encode the two interacting proteins of the T4 primosome (DNA helicase plus DNA primase), one or more may function in DNA replication by modulating primosome function.     

Complete chloroplast genome sequences of Praxelis (Eupatorium catarium Veldkamp), an important invasive species

Praxelis (Eupatorium catarium Veldkamp) is a new hazardous invasive plant species that has caused serious economic losses and environmental damage in the Northern hemisphere tropical and subtropical regions. Although previous studies focused on detecting the biological characteristics of this plant to prevent its expansion, little effort has been made to understand the impact of Praxelis on the ecosystem in an evolutionary process. The genetic information of Praxelis is required for further phylogenetic identification and evolutionary studies. Here, we report the complete Praxelis chloroplast (cp) genome sequence. The Praxelis chloroplast genome is 151,410 bp in length including a small single-copy region (18,547 bp) and a large single-copy region (85,311 bp) separated by a pair of inverted repeats (IRs; 23,776 bp). The genome contains 85 unique and 18 duplicated genes in the IR region. The gene content and organization are similar to other Asteraceae tribe cp genomes. We also analyzed the whole cp genome sequence, repeat structure, codon usage, contraction of the IR and gene structure/organization features between native and invasive Asteraceae plants, in order to understand the evolution of organelle genomes between native and invasive Asteraceae. Comparative analysis identified the 14 markers containing greater than 2% parsimony-informative characters, indicating that they are potential informative markers for barcoding and phylogenetic analysis. Moreover, a sister relationship between Praxelis and seven other species in Asteraceae was found based on phylogenetic analysis of 28 protein-coding sequences. Complete cp genome information is useful for plant phylogenetic and evolutionary studies within this invasive species and also within the Asteraceae family.     

Molecular characterization influencing metal resistance in the Cupriavidus/Ralstonia genomes

Our environment is stressed with a load of heavy and toxic metals. Microbes, abundant in our environment, are found to adapt well to this metal-stressed condition. A comparative study among five Cupriavidus/Ralstonia genomes can offer a better perception of their evolutionary mechanisms to adapt to these conditions. We have studied codon usage among 1051 genes common to all these organisms and identified 15 optimal codons frequently used in highly expressed genes present within 1051 genes. We found the core genes of Cupriavidus metallidurans CH34 have a different optimal codon choice for arginine, glycine and alanine in comparison with the other four bacteria. We also found that the synonymous codon usage bias within these 1051 core genes is highly correlated with their gene expression. This supports that translational selection drives synonymous codon usage in the core genes of these genomes. Synonymous codon usage is highly conserved in the core genes of these five genomes. The only exception among them is C. metallidurans CH34. This genomewide shift in synonymous codon choice in C. metallidurans CH34 may have taken place due to the insertion of new genes in its genomes facilitating them to survive in heavy metal containing environment and the co-evolution of the other genes in its genome to achieve a balance in gene expression. Structural studies indicated the presence of a longer N-terminal region containing a copper-binding domain in the cupC proteins of C. metallidurans CH3 that helps it to attain higher binding efficacy with copper in comparison with its orthologs.     

Predicting Statistical Properties of Open Reading Frames in Bacterial Genomes

An analytical model based on the statistical properties of Open Reading Frames (ORFs) of eubacterial genomes such as codon composition and sequence length of all reading frames was developed. This new model predicts the average length, maximum length as well as the length distribution of the ORFs of 70 species with GC contents varying between 21% and 74%. Furthermore, the number of annotated genes is predicted with high accordance. However, the ORF length distribution in the five alternative reading frames shows interesting deviations from the predicted distribution. In particular, long ORFs appear more often than expected statistically. The unexpected depletion of stop codons in these alternative open reading frames cannot completely be explained by a biased codon usage in the +1 frame. While it is unknown if the stop codon depletion has a biological function, it could be due to a protein coding capacity of alternative ORFs exerting a selection pressure which prevents the fixation of stop codon mutations. The comparison of the analytical model with bacterial genomes, therefore, leads to a hypothesis suggesting novel gene candidates which can now be investigated in subsequent wet lab experiments.     

The Complete Chloroplast Genome Sequence of the Medicinal Plant Salvia miltiorrhiza

Salvia miltiorrhiza is an important medicinal plant with great economic and medicinal value. The complete chloroplast (cp) genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family, is reported here. The genome is 151,328 bp in length and exhibits a typical quadripartite structure of the large (LSC, 82,695 bp) and small (SSC, 17,555 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,539 bp). It contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs and four rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Four forward, three inverted and seven tandem repeats were detected in the Salvia miltiorrhiza cp genome. Simple sequence repeat (SSR) analysis among the 30 asterid cp genomes revealed that most SSRs are AT-rich, which contribute to the overall AT richness of these cp genomes. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the cp genomes. Entire cp genome comparison of Salvia miltiorrhiza and three other Lamiales cp genomes showed a high degree of sequence similarity and a relatively high divergence of intergenic spacers. Sequence divergence analysis discovered the ten most divergent and ten most conserved genes as well as their length variation, which will be helpful for phylogenetic studies in asterids. Our analysis also supports that both regional and functional constraints affect gene sequence evolution. Further, phylogenetic analysis demonstrated a sister relationship between Salvia miltiorrhiza and Sesamum indicum. The complete cp genome sequence of Salvia miltiorrhiza reported in this paper will facilitate population, phylogenetic and cp genetic engineering studies of this medicinal plant.