The complete mitochondrial genome of the New Zealand parasitic roundworm Teladorsagia circumcincta (Trichostrongyloidea: Haemonchidae) field strain NZ_Teci_NP

The complete mitochondrial genome of the New Zealand parasitic nematod

Tackling hypotheticals in helminth genomes

Advancements in genome sequencing have led to the rapid accumulation of uncharacterized ‘hypothetical proteins’ in the public databases. Here we provide a community perspective and some best-practice approaches for the accurate functional annotation of uncharacterized genomic sequences

Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut microbiome data sets. Comparison with the human microbiome revealed rumen-specific enrichment for genes encoding de novo synthesis of vitamin B12, ongoing evolution by gene loss and potential vertical inheritance of the rumen microbiome based on underrepresentation of markers of environmental stress. We estimate that our Hungate genome resource represents ?75% of the genus-level bacterial and archaeal taxa present in the rumen.publishersversionPeer reviewe

Genome sequencing of rumen bacteria involved in lignocellulose digestion : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in the Institute of Molecular Biosciences at Massey University, Palmerston North, New Zealand

Determining the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals. In order to learn more about lignocellulose degradation in pasture-grazed dairy cows under NZ conditions, twenty representative strains from five major phylotype clusters (Butyrivibrio fibrisolvens/hungatei cluster 383, Pseudobutyrivibrio xylanivorans clusters 247 and 245, Selenomonas ruminantium cluster 212, and Lachnospiraceae cluster 121), cultivated directly from the fibre-adherent rumen microbial fraction of dairy cows were selected. Genotypic and phenotypic analysis of these strains led to identification of Butyrivibrio sp. MB2003 that adheres to and efficiently degrades the plant fibre. The 3.3 Mb MB2003 genome was sequenced and annotated and found to consist of four replicons: a chromosome (7 contigs, in 1 super scaffold), a chromid (Bhu II), a megaplasmid (pNP144) and a small plasmid (pNP6). A novel feature of the MB2003 genome is the presence of a chromid (Bhu II) which is now the smallest chromid reported for all bacteria. The MB2003 polysaccharide-degrading enzymes, surface structures and predicted strategy for attachment to, and degradation of, complex polysaccharides was found to be comparable to that of the fibrolytic bacterium Butyrivibrio proteoclasticus B316. Both MB2003 and B316 are non-motile, despite the presence of flagellar gene clusters, and utilise a range of insoluble plant polysaccharides, but not cellulose. Xylan is the preferred insoluble substrate of MB2003 and its genome encodes a large repertoire of enzymes predicted to metabolise this complex polysaccharide. The MB2003 draft genome produced in this work is the first opportunity to conduct comparative analysis of two rumen bacteria belonging to the same genus. Although both MB2003 and B316 have similar phenotypic characteristics and occupy the same habitat, the genome of MB2003 is much smaller and contains fewer extracellular polysaccharide degrading enzymes. From this comparison it can be concluded that MB2003 is a secondary hemicellulose degrader, offering an alternate view of the genes required for a xylanolytic lifestyle in the rumen, and posing an interesting question about the purpose of the wider range of polysaccharide degrading enzymes found in B316

Comparative genomics of Butyrivibrio and Pseudobutyrivibrio from the rumen : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Palmerston North, New Zealand

Determining the role of rumen microbes in plant polysaccharide breakdown is fundamental to understanding digestion, and maximising productivity, in ruminant animals. Rumen bacterial species belonging to the genera Butyrivibrio and Pseudobutyrivibrio are important degraders of plant hemicellulose, an abundant heterogeneous, branched polymer, involved in crosslinking cellulose microfibrils to lignin. To investigate their genes required for hemicellulose degradation, the genomes of 40 Butyrivibrio and 6 Pseudobutyrivibrio strains isolated from the plant-adherent microbiome of New Zealand bovine ruminants, were sequenced, and their CAZyme-encoding genes compared. Within the Butyrivibrio and Pseudobutyrivibrio pan-genomes, respectively, there were a total of 4,421 and 441 glycoside hydrolases, as well as 1,283 and 122 carbohydrate esterases with predicted activities involved in the degradation of the insoluble plant polysaccharides such as xylan and pectin. To examine species differences, the genes of the previously characterised bacterium B. proteoclasticus B316 were compared in detail with those from the newly sequenced B. hungatei MB2003. B316 was found to encode a much more developed polysaccharide-degrading repertoire and it was thus hypothesised that B316 would out-compete MB2003 when grown in co-culture on the insoluble hemicellulose substrate, xylan. To test this hypothesis, the two strains were grown on xylan and pectin, either alone in mono-cultures, or in direct competition in a co-culture. The results showed that MB2003 had little ability to utilise xylan or pectin alone, but was capable of significant growth when co-cultured with B316. This indicates a commensalistic interaction between these species, in which B316 initiates the primary attack on the insoluble substrate, while MB2003 has a secondary role, competing for the released soluble sugars. This work provides the first systematic phenotypic, comparative genomic and functional analysis of ruminal Butyrivibrio and Pseudobutyrivibrio species, which not only defines their conserved features involved in hemicellulose degradation, but is also beginning to differentiate their unique gene complements and growth characteristics that separate them as discrete species

Characterization of the complete mitochondrial genome of the New Zealand parasitic blowfly Calliphora vicina (Insecta: Diptera: Calliphoridae)

In this study, the complete mitochondrial (mt) genome of the New Zealand parasitic blowfly Calliphora vicina (blue bottle blowfly) field strain NZ_CalVic_NP was generated using next-generation sequencing technology and annotated. The 16,518 bp mt genome consists of 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and a 1689 bp non-coding region, similar to the two other available C. vicina and most metazoan mt genomes. Phylogenetic analysis showed that C. vicina NZ_CalVic_NP forms a monophyletic cluster with the remaining three Calliphorinae species. The complete mt genome sequence of C. vicina NZ_CalVic_NP is a resource to facilitate future species- and strain-level identification research and investigations into the evolutionary provenance within the Calliphoridae

The complete mitochondrial genome of the New Zealand parasitic blowfly Lucilia sericata (Insecta: Diptera: Calliphoridae)

In this study, the complete mitochondrial (mt) genome of the New Zealand parasitic blowfly Lucilia sericata (green bottle blowfly) field strain NZ_LucSer_NP was generated using next-generation sequencing technology. The length of complete the mt genome is 15,938 bp, with 39.4% A, 13.0% C, 9.3% G, and 38.2% T nucleotide distribution. The complete mt genome consists of 13 protein-coding genes (PCGs), two ribosomal RNAs, 22 transfer RNAs, and a 1124 bp non-coding region, similar to most metazoan mt genomes. Phylogenetic analysis showed that L. sericata NZ_LucSer_NP forms a monophyletic cluster with the remaining six Lucilia species and the Calliphoridae are polyphyletic. This study provides the first complete mt genome sequence for a L. sericata blowfly species derived from New Zealand to facilitate species identification and phylogenetic analysis

Teladorsagia circumcincta 1,6-Bisphosphate Aldolase: Molecular and Biochemical Characterisation, Structure Analysis and Recognition by Immune Hosts

A 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO-1) was cloned and expressed in Escherichia coli. Recombinant TciALDO-1 was purified, and its kinetic properties determined. The predicted protein consisted of 365 amino acids, and was present as a single band of about 44 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TciALDO-1 with homologues from other helminths showed the greatest similarity (93%) to the aldolases of Haemonchus contortus and Dictyocaulus viviparus, 82–86% similarity to the other nematode sequences, and 68–71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified, and were completely conserved in other homologues. At 30 °C, the optimum pH for TciALDO-1 activity was pH 7.5, the Vmax was 432 ± 23 nmol × min−1 × mg−1 protein, and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 ± 0.01 µM (mean ± SEM, n = 3). Recombinant TciALDO-1 was recognized by antibodies in both serum and saliva from field-immune sheep in ELISA, however, that was not the case with nematode-naïve sheep. Teladorsagia circumcincta fructose 1,6-bisphosphate aldolase appears to have potential as a vaccine candidate to control this common sheep parasite