Limits...
Large genomic differences between the morphologically indistinguishable diplomonads Spironucleus barkhanus and Spironucleus salmonicida.

Roxström-Lindquist K, Jerlström-Hultqvist J, Jørgensen A, Troell K, Svärd SG, Andersson JO - BMC Genomics (2010)

Bottom Line: Here we address the tempo and mode of such changes within diplomonads, flagellated protists with two nuclei found in oxygen-poor environments.Sequence variations were found between individual S. barkhanus ESTs for many, but not all, protein coding genes.Conversely, no allelic variation was found in a previous genome survey of S. salmonicida.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.

ABSTRACT

Background: Microbial eukaryotes show large variations in genome structure and content between lineages, indicating extensive flexibility over evolutionary timescales. Here we address the tempo and mode of such changes within diplomonads, flagellated protists with two nuclei found in oxygen-poor environments. Approximately 5,000 expressed sequence tag (EST) sequences were generated from the fish commensal Spironucleus barkhanus and compared to sequences from the morphologically indistinguishable fish parasite Spironucleus salmonicida, and other diplomonads. The ESTs were complemented with sequence variation studies in selected genes and genome size determinations.

Results: Many genes detected in S. barkhanus and S. salmonicida are absent in the human parasite Giardia intestinalis, the most intensively studied diplomonad. For example, these fish diplomonads show an extended metabolic repertoire and are able to incorporate selenocysteine into proteins. The codon usage is altered in S. barkhanus compared to S. salmonicida. Sequence variations were found between individual S. barkhanus ESTs for many, but not all, protein coding genes. Conversely, no allelic variation was found in a previous genome survey of S. salmonicida. This difference was confirmed by sequencing of genomic DNA. Up to five alleles were identified for the cloned S. barkhanus genes, and at least nineteen highly expressed S. barkhanus genes are represented by more than four alleles in the EST dataset. This could be explained by the presence of a non-clonal S. barkhanus population in the culture, by a ploidy above four, or by duplications of parts of the genome. Indeed, genome size estimations using flow cytometry indicated similar haploid genome sizes in S. salmonicida and G. intestinalis (approximately 12 Mb), whereas the S. barkhanus genome is larger (approximately 18 Mb).

Conclusions: This study indicates extensive divergent genome evolution within diplomonads. Genomic traits such as codon usage, frequency of allelic sequence variation, and genome size have changed considerably between S. barkhanus and S. salmonicida. These observations suggest that large genomic differences may accumulate in morphologically indistinguishable eukaryotic microbes.

Show MeSH
Comparison of codon usage in S. barkhanus and S. salmonicida. (A) The effective number of codons (Nc') plotted against the G+C content in four-fold degenerate positions in the gene (GC3s) for 682 unique S. barkhanus genes. (B) Correspondence analysis of the relative synonymous codon usage (RSCU) of the 682 unique S. barkhanus genes. (C) GC3s of aligned regions of 233 pairs of putative S. barkhanus and S. salmonicida orthologs. (D) The difference of GC3s of aligned regions of putative orthologs plotted against the non-synonymous divergence values (dN). The color coding indicates the number of times the gene was sampled within the S. barkhanus EST data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2874811&req=5

Figure 2: Comparison of codon usage in S. barkhanus and S. salmonicida. (A) The effective number of codons (Nc') plotted against the G+C content in four-fold degenerate positions in the gene (GC3s) for 682 unique S. barkhanus genes. (B) Correspondence analysis of the relative synonymous codon usage (RSCU) of the 682 unique S. barkhanus genes. (C) GC3s of aligned regions of 233 pairs of putative S. barkhanus and S. salmonicida orthologs. (D) The difference of GC3s of aligned regions of putative orthologs plotted against the non-synonymous divergence values (dN). The color coding indicates the number of times the gene was sampled within the S. barkhanus EST data.

Mentions: S. barkhanus has a relatively G+C-poor genome; the average G+C-content of the EST sequences is 41% and GC3s values below 40% for most genes (Figure 2A), suggesting a general mutational bias towards A+T. We classify genes as "highly expressed" and "weakly expressed" if they have been found more than 20 times or less than 3 times, respectively, among the clones from the cDNA library. The number of occurrences of a specific gene within a non-normalized cDNA library is expected to be roughly correlated with the amount of mRNA in the cells harvested for the library preparation. Specific codons are preferred in highly expressed S. barkhanus genes, as previously found in S. salmonicida [14] (data not shown). The codon usage was explored in more detail by plotting the effective number of codons (Enc') [41] against the GC3s values (Figure 2A) and by correspondence analyses on the relative synonymous codon usage (Figure 2B). The S. barkhanus GC3s values are weakly correlated with expression levels (Figure 2A), whereas the correspondence analysis clearly separates highly expressed genes from weakly expressed genes (Figure 2B). This is in agreement with the observation that the preferred codons are a mixture of G+C rich and G+C poor codons [14]. Overall, the S. barkhanus codon usage is similar to S. salmonicida, except that the variation in GC3s values appear less extreme in S. barkhanus [14] (Figure 2A).


Large genomic differences between the morphologically indistinguishable diplomonads Spironucleus barkhanus and Spironucleus salmonicida.

Roxström-Lindquist K, Jerlström-Hultqvist J, Jørgensen A, Troell K, Svärd SG, Andersson JO - BMC Genomics (2010)

Comparison of codon usage in S. barkhanus and S. salmonicida. (A) The effective number of codons (Nc') plotted against the G+C content in four-fold degenerate positions in the gene (GC3s) for 682 unique S. barkhanus genes. (B) Correspondence analysis of the relative synonymous codon usage (RSCU) of the 682 unique S. barkhanus genes. (C) GC3s of aligned regions of 233 pairs of putative S. barkhanus and S. salmonicida orthologs. (D) The difference of GC3s of aligned regions of putative orthologs plotted against the non-synonymous divergence values (dN). The color coding indicates the number of times the gene was sampled within the S. barkhanus EST data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874811&req=5

Figure 2: Comparison of codon usage in S. barkhanus and S. salmonicida. (A) The effective number of codons (Nc') plotted against the G+C content in four-fold degenerate positions in the gene (GC3s) for 682 unique S. barkhanus genes. (B) Correspondence analysis of the relative synonymous codon usage (RSCU) of the 682 unique S. barkhanus genes. (C) GC3s of aligned regions of 233 pairs of putative S. barkhanus and S. salmonicida orthologs. (D) The difference of GC3s of aligned regions of putative orthologs plotted against the non-synonymous divergence values (dN). The color coding indicates the number of times the gene was sampled within the S. barkhanus EST data.
Mentions: S. barkhanus has a relatively G+C-poor genome; the average G+C-content of the EST sequences is 41% and GC3s values below 40% for most genes (Figure 2A), suggesting a general mutational bias towards A+T. We classify genes as "highly expressed" and "weakly expressed" if they have been found more than 20 times or less than 3 times, respectively, among the clones from the cDNA library. The number of occurrences of a specific gene within a non-normalized cDNA library is expected to be roughly correlated with the amount of mRNA in the cells harvested for the library preparation. Specific codons are preferred in highly expressed S. barkhanus genes, as previously found in S. salmonicida [14] (data not shown). The codon usage was explored in more detail by plotting the effective number of codons (Enc') [41] against the GC3s values (Figure 2A) and by correspondence analyses on the relative synonymous codon usage (Figure 2B). The S. barkhanus GC3s values are weakly correlated with expression levels (Figure 2A), whereas the correspondence analysis clearly separates highly expressed genes from weakly expressed genes (Figure 2B). This is in agreement with the observation that the preferred codons are a mixture of G+C rich and G+C poor codons [14]. Overall, the S. barkhanus codon usage is similar to S. salmonicida, except that the variation in GC3s values appear less extreme in S. barkhanus [14] (Figure 2A).

Bottom Line: Here we address the tempo and mode of such changes within diplomonads, flagellated protists with two nuclei found in oxygen-poor environments.Sequence variations were found between individual S. barkhanus ESTs for many, but not all, protein coding genes.Conversely, no allelic variation was found in a previous genome survey of S. salmonicida.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.

ABSTRACT

Background: Microbial eukaryotes show large variations in genome structure and content between lineages, indicating extensive flexibility over evolutionary timescales. Here we address the tempo and mode of such changes within diplomonads, flagellated protists with two nuclei found in oxygen-poor environments. Approximately 5,000 expressed sequence tag (EST) sequences were generated from the fish commensal Spironucleus barkhanus and compared to sequences from the morphologically indistinguishable fish parasite Spironucleus salmonicida, and other diplomonads. The ESTs were complemented with sequence variation studies in selected genes and genome size determinations.

Results: Many genes detected in S. barkhanus and S. salmonicida are absent in the human parasite Giardia intestinalis, the most intensively studied diplomonad. For example, these fish diplomonads show an extended metabolic repertoire and are able to incorporate selenocysteine into proteins. The codon usage is altered in S. barkhanus compared to S. salmonicida. Sequence variations were found between individual S. barkhanus ESTs for many, but not all, protein coding genes. Conversely, no allelic variation was found in a previous genome survey of S. salmonicida. This difference was confirmed by sequencing of genomic DNA. Up to five alleles were identified for the cloned S. barkhanus genes, and at least nineteen highly expressed S. barkhanus genes are represented by more than four alleles in the EST dataset. This could be explained by the presence of a non-clonal S. barkhanus population in the culture, by a ploidy above four, or by duplications of parts of the genome. Indeed, genome size estimations using flow cytometry indicated similar haploid genome sizes in S. salmonicida and G. intestinalis (approximately 12 Mb), whereas the S. barkhanus genome is larger (approximately 18 Mb).

Conclusions: This study indicates extensive divergent genome evolution within diplomonads. Genomic traits such as codon usage, frequency of allelic sequence variation, and genome size have changed considerably between S. barkhanus and S. salmonicida. These observations suggest that large genomic differences may accumulate in morphologically indistinguishable eukaryotic microbes.

Show MeSH