Honours applications close next Friday (3 June)

We still have space in the group for one more Honours student, with a variety of projects available to work on PacBio SMRT data for bacteria, yeast or cane toad de novo genome assembly. Please get in touch if you are interested. (Better still, fill in the lab application form, making it clear it’s for Honours, not PhD!) The deadline is Friday, 3rd of June. See the BABS website for more details.

Congratulations, Dr Joe Jenkins!

Congratulations to Joe Jenkins, who successfully defended his PhD on 12th May and was awarded his doctorate following his viva, with Professor Gary Bending as external examiner. It’s been a busy week for Joe, with the main paper from his PhD out this week too!

Biochar alters the soil microbiome and soil function: results of next generation amplicon sequencing across Europe

Jenkins JR, Viger M, Arnold EC, Harris ZM, Ventura M, Miglietta F, Girardin C, Edwards RJ, Rumpel C, Fornasier F, Zavalloni C, Tonon G, Alberti G & Taylor G (2016): Biochar alters the soil microbiome and soil function: results of next generation amplicon sequencing across Europe. GCB Bioenergy Adv. Access DOI: 10.1111/gcbb.12371

Abstract

Wide scale application of biochar to soil has been suggested as a mechanism to offset increases in CO2 emissions through the long-term sequestration of a carbon rich and inert substance to the soil, but the implications of this for soil diversity and function remain to be determined. Biochar is capable of inducing changes in soil bacterial communities, but the exact impacts of its application are poorly understood. Using three European sites (UK SRC, short rotation coppice, French grassland (FR) and Italian SRF, short rotation forestry (IT)) treated with identical biochar applications; we undertook 16S and ITS amplicon DNA sequencing. In addition, we carried out assessments of community change over time and N and P mobilisation in the UK.

Significant changes in bacterial and community structure occurred due to treatment, although the nature of the changes varied by site. STAMP differential abundance analysis showed enrichment of Gemmatimonadete and Acidobacteria in UK biochar plots one year after application, whilst control plots exhibited enriched Gemmataceae, Isosphaeraceae and Koribacteraceae. Increased mobility of ammonium and phosphates were also detected after one year, coupled with a shift from acid to alkaline phophomonoesterase activity, which may suggest an ecological and functional shift towards a more copiotrophic ecology. Italy also exhibited enrichments, in both the Proteobacteria (driven by an increase in the order Rhizobiales) and the Gemmatimonadetes. No significant change in the abundance of individual taxa were noted in FR, although a small significant change in unweighted UNIFRAC occurred, indicating variation in the identities of taxa present due to treatment. Fungal β diversity was affected by treatment in IT and FR, but was unaffected in UK samples. The effects of time and site were greater than that of biochar application in UK samples. Overall, this report gives a tantalising view of the soil microbiome at several sites across Europe, and suggests that although application of biochar has significant effects on microbial communities, these may be small compared with the highly variable soil microbiome that is found in different soils and changes with time.

ARC Linkage Success! - Elucidating the genetic basis of newly evolved metabolic functions in yeast

We are very happy to report a successful ARC Linkage Projects 2016 grant application:

LP160100610: Elucidating the genetic basis of newly evolved metabolic functions in yeast

Dr Richard Edwards; Professor Marc Wilkins; Associate Professor Mark Tanaka; Dr Paul Attfield; Dr Phillip Bell

This project intends to research how complex metabolic pathways originate and evolve. This project will use cutting edge genome sequencing and molecular techniques to elucidate the heritable genetic basis of Baker’s yeast, which has been the selectively evolved to use xylose as a sole carbon source: something vital for second generation biofuel production that wild yeast cannot do. This project will combine detailed molecular characterisation of highly adapted yeast strains with a novel “molecular palaeontology” approach to trace the evolutionary process and identify functionally significant loci under selection. Detailed characterisation of this trait will accelerate the development of future yeast strains and test fundamental evolutionary theories.

This will continue the work we have been doing on PacBio sequencing and yeast genomics in collaboration with our industrial partners, Microbiogen Pty Ltd.

There will be job and studentship opportunities associated with this grant, so watch this space! (Or get in touch!)

Parallel evolution in Streptococcus pneumoniae biofilms

Churton NWV, Misra RV, Howlin RP, Allan RN, Jefferies J, Faust SN, Gharbia SE, Edwards RJ, Clarke SC & Webb JS (2016): Parallel evolution in Streptococcus pneumoniae biofilms. Genome Biology and Evolution Adv. Access doi: 10.1093/gbe/evw072

Abstract

Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and non-invasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51-264 bp, one insertion resulting in a coding frameshift, and seven nonsense single nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae, and therefore may have important implications for colonization, carriage and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development.

Lorne #Genome2016 poster 132: PacBio sequencing and comparative genomics of three Saccharomyces cerevisiae strains

Richard J. Edwards, Åsa Pérez-Bercoff, Tonia L. Russell, Zhiliang Chen , Marc R. Wilkins, Paul V. Attfield & Philip J.L. Bell. F1000Research 2016, 5:172 (poster) (doi: 10.7490/f1000research.1111305.1).

Abstract

PacBio Single Molecule Real Time (SMRT™) sequencing is rapidly becoming the technology of choice for de novo whole genome sequencing. The long read lengths and random error of PacBio data make genome assembly considerably easier and more accurate than short read data. Here, we report on de novo genome sequencing and assembly of three Saccharomyces cerevisiae genomes using the PacBio RSII at the UNSW Ramaciotti Centre for Genomics. A haploid reference yeast genome strain, S288C, and two novel diploid strains were sequenced as part of a larger functional genomics project. For each strain, 20kb SMRT Bell library preps were performed and sequenced on two SMRT Cells using the P6-C4 chemistry with read lengths of up to 53.3 kb. Whole genome de novo assemblies are then generated through the PacBio SMRT Portal.

We are using the S288C data to explore performance in comparison to the published genome as a reference. An initial assembly of S288C yielded over 99.97% genome coverage at 99.99% accuracy on only 26 contigs, with 16/17 reference chromosomes (16 nuclear chromosomes plus mitochondrion) essentially returned as a single, complete contig. The long reads enable accurate reconstruction of tandemly repeated genes (except >900kb of rRNA repeats), transposition and chromosomal translocations. We are now using the S288C data to optimise the assembly process and derive assembly settings for the two novel diploid strains. To this end, we have developed a new pipeline for the comparative assessment of high quality whole genomes against a reference, which we are now adapting for the additional challenge of appropriately handling diploid data.

Quantitative Proteomics of the Infectious and Replicative Forms of Chlamydia trachomatis

Skipp PJS, Hughes C, McKenna T, Edwards R, Langridge J, Thomson NR & Clarke IN (2016): Quantitative Proteomics of the Infectious and Replicative Forms of Chlamydia trachomatis. PLoS ONE 11(2): e0149011. doi:10.1371/journal.pone.0149011

Abstract

The obligate intracellular developmental cycle of Chlamydia trachomatis presents significant challenges in defining its proteome. In this study we have applied quantitative proteomics to both the intracellular reticulate body (RB) and the extracellular elementary body (EB) from C. trachomatis. We used C. trachomatis L2 as a model chlamydial isolate for our study since it has a high infectivity:particle ratio and there is an excellent quality genome sequence. EBs and RBs (>99% pure) were quantified by chromosomal and plasmid copy number using PCR, from which the concentrations of chlamydial proteins per bacterial cell/genome were determined. RBs harvested at 15h post infection (PI) were purified by three successive rounds of gradient centrifugation. This is the earliest possible time to obtain purified RBs, free from host cell components in quantity, within the constraints of the technology. EBs were purified at 48h PI. We then used two-dimensional reverse phase UPLC to fractionate RB or EB peptides before mass spectroscopic analysis, providing absolute amount estimates of chlamydial proteins. The ability to express the data as molecules per cell gave ranking in both abundance and energy requirements for synthesis, allowing meaningful identification of rate-limiting components. The study assigned 562 proteins with high confidence and provided absolute estimates of protein concentration for 489 proteins. Interestingly, the data showed an increase in TTS capacity at 15h PI. Most of the enzymes involved in peptidoglycan biosynthesis were detected along with high levels of muramidase (in EBs) suggesting breakdown of peptidoglycan occurs in the non-dividing form of the microorganism. All the genome-encoded enzymes for glycolysis, pentose phosphate pathway and tricarboxylic acid cycle were identified and quantified; these data supported the observation that the EB is metabolically active. The availability of detailed, accurate quantitative proteomic data will be invaluable for investigations into gene regulation and function.