Evolution of multipartite mitochondrial genomes in the booklice of the genus Liposcelis (Psocoptera)

Background

The genus Liposcelis (Psocoptera: Troctomorpha) has more than 120 species with a worldwide distribution and they pose a risk for global food security. The organization of mitochondrial (mt) genomes varies between the two species of booklice investigated in the genus Liposcelis. Liposcelis decolor has its mt genes on a single chromosome, like most other insects; L. bostrychophila, however, has a multipartite mt genome with genes on two chromosomes.

Results

To understand how multipartite mt genome organization evolved in the genus Liposcelis, we sequenced the mt genomes of L. entomophila and L. paeta in this study. We found that these two species of booklice also have multipartite mt genomes, like L. bostrychophila, with the mt genes we identified on two chromosomes. Numerous pseudo mt genes and non-coding regions were found in the mt genomes of these two booklice, and account for 30% and 10% respectively of the entire length we sequenced. In L. bostrychophila, the mt genes are distributed approximately equally between the two chromosomes. In L. entomophila and L. paeta, however, one mt chromosome has most of the genes we identified whereas the other chromosome has largely pseudogenes and non-coding regions. L. entomophila and L. paeta differ substantially from each other and from L. bostrychophila in gene content and gene arrangement in their mt chromosomes.

Conclusions

Our results indicate unusually fast evolution in mt genome organization in the booklice of the genus Liposcelis, and reveal different patterns of mt genome fragmentation among L. bostrychophila, L. entomophila and L. paeta.

Electronic supplementary material

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A unique tRNA gene family and a novel,highly expressed ORF in the mitochondrial genome of the silver-lip pearl oyster, Pinctada maxima (Bivalvia: Pteriidae)

Characteristics of mitochondrial (mt) DNA such as gene content and arrangement, as well as mt tRNA secondary structure, are frequently used in comparative genomic analyses because they provide valuable phylogenetic information. However, most analyses do not characterize the relationship of tRNA genes from the same mt genome and, in some cases, analyses overlook possible novel open reading frames (ORFs) when the 13 expected protein-coding genes are already annotated. In this study, we describe the sequence and characterization of the complete mt genome of the silver-lip pearl oyster, Pinctada maxima. The 16,994-bp mt genome contains the same 13 protein-coding genes (PCGs) and two ribosomal RNA genes typical of metazoans. The gene arrangement, however, is completely distinct from that of all other available bivalve mt genomes, and a unique tRNA gene family is observed in this genome. The unique tRNA gene family includes two trnS^− AGY and trnQ genes, a trnM isomerism, but it lacks trnS^− CUN. We also report the first clear evidence of alloacceptor tRNA gene recruitment (trnP → trnS^− AGY) in mollusks. In addition, a novel ORF (orfUR1) expressed at high levels is present in the mt genome of this pearl oyster. This gene contains a conserved domain, “Oxidored_q1_N”, which is a member of Complex I and thus may play an important role in key biological functions. Because orfUR1 has a very similar nucleotide composition and codon bias to that of other genes in this genome, we hypothesize that this gene may have been moved to the mt genome via gene transfer from the nuclear genome at an early stage of speciation of P. maxima, or it may have evolved as a result of gene duplication, followed by rapid sequence divergence. Lastly, a 319-bp region was identified as the possible control region (CR) even though it does not correspond to the longest non-coding region in the genome. Unlike other studies of mt genomes, this study compares the evolutionary patterns of all available bivalve mt tRNA and atp8 genes.     

Complete Mitochondrial Genome of Haplorchis taichui and Comparative Analysis with Other Trematodes

Mitochondrial genomes have been extensively studied for phylogenetic purposes and to investigate intra- and interspecific genetic variations. In recent years, numerous groups have undertaken sequencing of platyhelminth mitochondrial genomes. Haplorchis taichui (family Heterophyidae) is a trematode that infects humans and animals mainly in Asia, including the Mekong River basin. We sequenced and determined the organization of the complete mitochondrial genome of H. taichui. The mitochondrial genome is 15,130 bp long, containing 12 protein-coding genes, 2 ribosomal RNAs (rRNAs, a small and a large subunit), and 22 transfer RNAs (tRNAs). Like other trematodes, it does not encode the atp8 gene. All genes are transcribed from the same strand. The ATG initiation codon is used for 9 protein-coding genes, and GTG for the remaining 3 (nad1, nad4, and nad5). The mitochondrial genome of H. taichui has a single long non-coding region between trnE and trnG. H. taichui has evolved as being more closely related to Opisthorchiidae than other trematode groups with maximal support in the phylogenetic analysis. Our results could provide a resource for the comparative mitochondrial genome analysis of trematodes, and may yield genetic markers for molecular epidemiological investigations into intestinal flukes.     

The complete mitochondrial genome of Macrobrachium nipponense

The complete mitochondrial (mt) genome sequence plays an important role in the accurate determination of phylogenetic relationships among metazoans. Herein, we determined the complete mt genome sequence, structure and organization of Macrobrachium nipponense (M. nipponense) (GenBank ID: NC_015073.1) and compared it to that of Macrobrachium lanchesteri (M. lanchesteri) and Macrobrachium rosenbergii (M. rosenbergii). The 15,806 base pair (bp) M. nipponense mt genome, which is comprised of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs), is slightly larger than that of M. lanchesteri (15,694 bp, GenBank ID: NC_012217.1) and M. rosenbergii (15,772 bp, GenBank ID: NC_006880.1). The M. nipponense genome contains a high AT content (66.0%), which is a common feature among metazoan mt genomes. Compared with M. lanchesteri and M. rosenbergii, we found a peculiar non-coding region of 950 bp with a microsatellite-like (TA)₆ element and many hairpin structures. The 13 PCGs are comprised of a total of 3707 codons, excluding incomplete termination codons, and the most frequently used amino acid is Leu (16.0%). The predicted start codons in the M. nipponense mt genome include ATG, ATC and ATA. Seven PCGs use TAA as a stop codon, whereas two use TAG, three use T and only one uses TA. Twenty-three of the genes are encoded on the L strand, and ND1, ND4, ND5, ND4L, 12S rRNA, 16S rRNA, tRNA^His, tRNA^Pro, tRNA^Phe, tRNA^Val, tRNA^Gln, tRNA^Cys, tRNA^Tyr and a tRNA^Leu are encoded on the H strand. The two rRNAs of M. nipponense and M. rosenbergii are encoded on the H strand, whereas the M. lanchesteri rRNAs are encoded on the L stand.     

Accelerated evolution of the mitochondrial genome in an alloplasmic line of durum wheat

Background

Wheat is an excellent plant species for nuclear mitochondrial interaction studies due to availability of large collection of alloplasmic lines. These lines exhibit different vegetative and physiological properties than their parents. To investigate the level of sequence changes introduced into the mitochondrial genome under the alloplasmic condition, three mitochondrial genomes of the Triticum-Aegilops species were sequenced: 1) durum alloplasmic line with the Ae. longissima cytoplasm that carries the T. turgidum nucleus designated as (lo) durum, 2) the cytoplasmic donor line, and 3) the nuclear donor line.

Results

The mitochondrial genome of the T. turgidum was 451,678 bp in length with high structural and nucleotide identity to the previously characterized T. aestivum genome. The assembled mitochondrial genome of the (lo) durum and the Ae. longissima were 431,959 bp and 399,005 bp in size, respectively. The high sequence coverage for all three genomes allowed analysis of heteroplasmy within each genome. The mitochondrial genome structure in the alloplasmic line was genetically distant from both maternal and paternal genomes. The alloplasmic durum and the Ae. longissima carry the same versions of atp6, nad6, rps19-p, cob and cox2 exon 2 which are different from the T. turgidum parent. Evidence of paternal leakage was also observed by analyzing nad9 and orf359 among all three lines. Nucleotide search identified a number of open reading frames, of which 27 were specific to the (lo) durum line.

Conclusions

Several heteroplasmic regions were observed within genes and intergenic regions of the mitochondrial genomes of all three lines. The number of rearrangements and nucleotide changes in the mitochondrial genome of the alloplasmic line that have occurred in less than half a century was significant considering the high sequence conservation between the T. turgidum and the T. aestivum that diverged from each other 10,000 years ago. We showed that the changes in genes were not limited to paternal leakage but were sufficiently significant to suggest that other mechanisms, such as recombination and mutation, were responsible. The newly formed ORFs, differences in gene sequences and copy numbers, heteroplasmy, and substoichiometric changes show the potential of the alloplasmic condition to accelerate evolution towards forming new mitochondrial genomes.

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RPB2 sequences reveal a close phylogenetic relationship between tetraploid Hordelymus and diploid Hordeum species in Triticeae (Poaceae)

The origin of Hordelymus genome has been debated for years, and no consensus conclusion was reached. In this study, we sequenced and analyzed the RPB2 (RNA polymerase subunit II) gene from Hordelymus europaeus (L.) Harz, and its potential diploid ancestor species those were suggested in previous studies. The focus of this study was to examine the phylogenetic relationship of Hordelymus genomes with its potential donor Hordeum, Psathyrostachys, and Taeniatherum species. Two distinguishable copies of sequences were obtained from H. europaeus. The obvious difference between the two copies of sequences is a 24 bp indel (insertion/deletion). Phylogenetic analysis showed a strong affinity between Hordeum genome and Hordelymus with 85% bootstrap support. These results suggested that one genome in tetraploid H. europaeus closely related to the genome in Hordeum species. Another genome in H. europaeus is sister to the genomes in Triticeae species examined here, which corresponds well with the recently published EF-G data. No obvious relationship was found between Hordelymus and either Ta genome donor, Taeniatherum caput-medusae or Ns genome donor, Psathyrostachys juncea. Our data does not support the presence of Ta and Ns genome in H. europaeus, and further confirms that H. europaeus is allopolyploid.     

Comparison of complete mitochondrial genomes of pine wilt nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus (Nematoda: Aphelenchoidea) and development of a molecular tool for species identification

We determined the complete mitochondrial genome sequences for Bursaphelenchus mucronatus, one species of pinewood nematode. The genome is a circular-DNA molecule of 14,583 bp (195 bp smaller than its congener Bursaphelenchus xylophilus) and contains 12 protein-coding genes (lacking atp8), 22 tRNA genes, and 2 rRNA genes encoded in the same direction, consistent with most other nematodes. Based on sequence comparison of mtDNA genomes, we developed a PCR-based molecular assay to differentiate B. xylophilus (highly pathogenic) and B. mucronatus (relatively less virulent) using species-specific primers. The molecular identification system employs multiplex-PCR and is very effective and reliable for discriminating these Bursaphelenchus species, which are economically important, but difficult to distinguish based on morphology. The comparison of the mitochondrial genomes and molecular identification system of the two species of Bursaphelenchus spp. should provide a rich source of genetic information to support the effective control and management (quarantine) of the pine wilt disease caused by pinewood nematodes.     

Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum

Background

Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis–associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent yearsIaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome.

Results

Altogether, 8.59-9.05 Mb of Cryptosporidium sequences in 45–767 assembled contigs were obtained from the four specimens, representing 94.36-99.47% coverage of the expected genome. These genomes had complete synteny in gene organization and 96.86-97.0% and 99.72-99.83% nucleotide sequence similarities to the published genomes of C. parvum and C. hominis, respectively. Several major insertions and deletions were seen between C. hominis and C. parvum genomes, involving mostly members of multicopy gene families near telomeres. The four C. hominis genomes were highly similar to each other and divergent from the reference IaA25R3 genome in some highly polymorphic regions. Major sequence differences among the four specimens sequenced in this study were in the 5′ and 3′ ends of chromosome 6 and the gp60 region, largely the result of genetic recombination.

Conclusions

The sequence similarity among specimens of the two dominant outbreak subtypes and genetic recombination in chromosome 6, especially around the putative virulence determinant gp60 region, suggest that genetic recombination plays a potential role in the emergence of hyper-transmissible C. hominis subtypes. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of MEDLE family secreted proteins and insulinase-like proteases indicate that telomeric gene duplications could potentially contribute to host expansion in C. parvum.

Electronic supplementary material

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Genome sequence of B. amyloliquefaciens type strain DSM7(T) reveals differences to plant-associated B. amyloliquefaciens FZB42

The complete genome sequence of Bacillus amyloliquefaciens type strain DSM7^T is presented. A comparative analysis between the genome sequences of the plant associated strain FZB42 (Chen et al., 2007) with the genome of B. amyloliquefaciens DSM7^T revealed obvious differences in the variable part of the genomes, whilst the core genomes were found to be very similar. The strains FZB42 and DSM7^T have in common 3345 genes (CDS) in their core genomes; whilst 547 and 344 CDS were found to be unique in DSM7^T and FZB42, respectively. The core genome shared by both strains exhibited 97.89% identity on amino acid level. The number of genes representing the core genome of the strains FZB42, DSM7^T, and Bacillus subtilis DSM10^T was calculated as being 3098 and their identity was 92.25%. The 3,980,199 bp genome of DSM7^T contains numerous genomic islands (GI) detected by different methods. Many of them were located in vicinity of tRNA, glnA, and glmS gene copies. In contrast to FZB42, but similar to B. subtilis DSM10^T, the GI were enriched in prophage sequences and often harbored transposases, integrases and recombinases. Compared to FZB42, B. amyloliquefaciens DSM7^T possessed a reduced potential to non-ribosomally synthesize secondary metabolites with antibacterial and/or antifungal action. B. amyloliquefaciens DSM7^T did not produce the polyketides difficidin and macrolactin and was impaired in its ability to produce lipopeptides other than surfactin. Differences established within the variable part of the genomes, justify our proposal to discriminate the plant-associated ecotype represented by FZB42 from the group of type strain related B. amyloliquefaciens soil bacteria.     

Genome based analysis of type-I polyketide synthase and nonribosomal peptide synthetase gene clusters in seven strains of five representative Nocardia species

Background

Actinobacteria of the genus Nocardia usually live in soil or water and play saprophytic roles, but they also opportunistically infect the respiratory system, skin, and other organs of humans and animals. Primarily because of the clinical importance of the strains, some Nocardia genomes have been sequenced, and genome sequences have accumulated. Genome sizes of Nocardia strains are similar to those of Streptomyces strains, the producers of most antibiotics. In the present work, we compared secondary metabolite biosynthesis gene clusters of type-I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) among genomes of representative Nocardia species/strains based on domain organization and amino acid sequence homology.

Results

Draft genome sequences of Nocardia asteroides NBRC 15531^T, Nocardia otitidiscaviarum IFM 11049, Nocardia brasiliensis NBRC 14402^T, and N. brasiliensis IFM 10847 were read and compared with published complete genome sequences of Nocardia farcinica IFM 10152, Nocardia cyriacigeorgica GUH-2, and N. brasiliensis HUJEG-1. Genome sizes are as follows: N. farcinica, 6.0 Mb; N. cyriacigeorgica, 6.2 Mb; N. asteroides, 7.0 Mb; N. otitidiscaviarum, 7.8 Mb; and N. brasiliensis, 8.9 - 9.4 Mb. Predicted numbers of PKS-I, NRPS, and PKS-I/NRPS hybrid clusters ranged between 4–11, 7–13, and 1–6, respectively, depending on strains, and tended to increase with increasing genome size. Domain and module structures of representative or unique clusters are discussed in the text.

Conclusion

We conclude the following: 1) genomes of Nocardia strains carry as many PKS-I and NRPS gene clusters as those of Streptomyces strains, 2) the number of PKS-I and NRPS gene clusters in Nocardia strains varies substantially depending on species, and N. brasiliensis strains carry the largest numbers of clusters among the species studied, 3) the seven Nocardia strains studied in the present work have seven common PKS-I and/or NRPS clusters, some of whose products are yet to be studied, and 4) different N. brasiliensis strains have some different gene clusters of PKS-I/NRPS, although the rest of the clusters are common within the N. brasiliensis strains. Genome sequencing suggested that Nocardia strains are highly promising resources in the search of novel secondary metabolites.

Electronic supplementary material

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Predicting relatedness of bacterial genomes using the chaperonin-60 universal target (cpn60 UT): application to Thermoanaerobacter species

D.R. Zeigler determined that the sequence identity of bacterial genomes can be predicted accurately using the sequence identities of a corresponding set of genes that meet certain criteria [32]. This three-gene model for comparing bacterial genome pairs requires the determination of the sequence identities for recN, thdF, and rpoA. This involves the generation of approximately 4.2 kb of genomic DNA sequence from each organism to be compared, and also normally requires that oligonucleotide primers be designed for amplification and sequencing based on the sequences of closely related organisms. However, we have developed an analogous mathematical model for predicting the sequence identity of whole genomes based on the sequence identity of the 542-567 base pair chaperonin-60 universal target (cpn60 UT). The cpn60 UT is accessible in nearly all bacterial genomes with a single set of universal primers, and its length is such that it can be completely sequenced in one pair of overlapping sequencing reads via di-deoxy sequencing. These mathematical models were applied to a set of Thermoanaerobacter isolates from a wood chip compost pile and it was shown that both the one-gene cpn60 UT-based model and the three-gene model based on recN, rpoA, and thdF predicted that these isolates could be classified as Thermoanaerobacter thermohydrosulfuricus. Furthermore, it was found that the genomic prediction model using cpn60 UT gave similar results to whole-genome sequence alignments over a broad range of taxa, suggesting that this method may have general utility for screening isolates and predicting their taxonomic affiliations.     

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous,multidrug resistant,biofilm producing isolate from India

Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniaespecies from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1,blaSHV-28, aac(6’)1b-cr,catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1,aac(6’)-Ib, aac(3)-IId,sul1,2, blaTEM-1A,1B,blaOXA-9, blaCTX-M-15,blaSHV-11, cmlA1, erm(B),mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniaestrains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13 was found inserted.     

SL2-like spliced leader RNAs in the basal nematode Prionchulus punctatus: New insight into the evolution of nematode SL2 RNAs

Spliced-leader (SL) trans-splicing has been found in all molecularly characterized nematode species to date, and it is likely to be a nematode synapomorphy. Most information regarding SL trans-splicing has come from the study of nematodes from a single monophyletic group, the Rhabditida, all of which employ SL RNAs that are identical to, or variants of, the SL1 RNA first characterized in Caenorhabditis elegans. In contrast, the more distantly related Trichinella spiralis, belonging to the subclass Dorylaimia, utilizes a distinct set of SL RNAs that display considerable sequence diversity. To investigate whether this is true of other members of the Dorylaimia, we have characterized SL RNAs from Prionchulus punctatus. Surprisingly, this revealed the presence of a set of SLs that show clear sequence similarity to the SL2 family of spliced leaders, which have previously only been found within the rhabditine group (which includes C. elegans). Expression of one of the P. punctatus SL RNAs in C. elegans reveals that it can compete specifically with the endogenous C. elegans SL2 spliced leaders, being spliced to the pre-mRNAs derived from downstream genes in operons, but does not compete with the SL1 spliced leaders. This discovery raises the possibility that SL2-like spliced leaders were present in the last common ancestor of the nematode phylum.     

Genome Wide Search for Biomarkers to Diagnose Yersinia Infections

Bacterial identification on the basis of the highly conserved 16S rRNA (rrs) gene is limited by its presence in multiple copies and a very high level of similarity among them. The need is to look for other genes with unique characteristics to be used as biomarkers. Fifty-one sequenced genomes belonging to 10 different Yersinia species were used for searching genes common to all the genomes. Out of 304 common genes, 34 genes of sizes varying from 0.11 to 4.42 kb, were selected and subjected to in silico digestion with 10 different Restriction endonucleases (RE) (4–6 base cutters). Yersinia species have 6–7 copies of rrs per genome, which are difficult to distinguish by multiple sequence alignments or their RE digestion patterns. However, certain unique combinations of other common gene sequences—carB, fadJ, gluM, gltX, ileS, malE, nusA, ribD, and rlmL and their RE digestion patterns can be used as markers for identifying 21 strains belonging to 10 Yersinia species: Y. aldovae, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. pestis, Y. pseudotuberculosis, Y. rohdei, Y. ruckeri, and Y. similis. This approach can be applied for rapid diagnostic applications.

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The complete mitochondrial genome of Arctic Calanus hyperboreus (Copepoda,Calanoida) reveals characteristic patterns in calanoid mitochondrial genome

Copepoda is the most diverse and abundant group of crustaceans, but its phylogenetic relationships are ambiguous. Mitochondrial (mt) genomes are useful for studying evolutionary history, but only six complete Copepoda mt genomes have been made available and these have extremely rearranged genome structures. This study determined the mt genome of Calanus hyperboreus, making it the first reported Arctic copepod mt genome and the first complete mt genome of a calanoid copepod. The mt genome of C. hyperboreus is 17,910 bp in length and it contains the entire set of 37 mt genes, including 13 protein-coding genes, 2 rRNAs, and 22 tRNAs. It has a very unusual gene structure, including the longest control region reported for a crustacean, a large tRNA gene cluster, and reversed GC skews in 11 out of 13 protein-coding genes (84.6%). Despite the unusual features, comparing this genome to published copepod genomes revealed retained pan-crustacean features, as well as a conserved calanoid-specific pattern. Our data provide a foundation for exploring the calanoid pattern and the mechanisms of mt gene rearrangement in the evolutionary history of the copepod mt genome.