Antarctic desert soil bacteria exhibit high novel natural product potential, evaluated through long-read genome sequencing and comparative genomics

The third of our collaborative “controlled bacterial metagenome” de novo whole genome assembly projects was published in Environmental Microbiology in November. This was a fun collaboration with the Ferrari lab at UNSW trying to maximise bang for buck to sequence some complete bacterial genomes using PacBio sequencing to identify biosynthetic gene clusters. As with a previous paper, we used pooled genomic DNA sequencing and were able to assemble complete genomes (and plasmids) of the 13/17 species that had sufficient depth of coverage. Coolest of all (if you excuse the pun), these were bugs from an Antarctic expedition! Head over to the Ferrari lab website to find out more about their research.

Benaud N, Edwards RJ, Amos TG, D’Agostino PM, Gutiérrez-Cháveza C, Montgomery K, Nicetic I & Ferrari BC (2020). Antarctic desert soil bacteria exhibit high novel natural product potential, evaluated through long-read genome sequencing and comparative genomics. Environmental Microbiology. https://doi.org/10.1111/1462-2920.15300

Abstract

Actinobacteria and Proteobacteria are important producers of bioactive natural products (NP), and these phyla dominate in the arid soils of Antarctica, where metabolic adaptations influence survival under harsh conditions. Biosynthetic gene clusters (BGCs) which encode NPs, are typically long and repetitious high G + C regions difficult to sequence with short‐read technologies. We sequenced 17 Antarctic soil bacteria from multi‐genome libraries, employing the long‐read PacBio platform, to optimize capture of BGCs and to facilitate a comprehensive analysis of their NP capacity. We report 13 complete bacterial genomes of high quality and contiguity, representing 10 different cold‐adapted genera including novel species. Antarctic BGCs exhibited low similarity to known compound BGCs (av. 31%), with an abundance of terpene, non‐ribosomal peptide and polyketide‐encoding clusters. Comparative genome analysis was used to map BGC variation between closely related strains from geographically distant environments. Results showed the greatest biosynthetic differences to be in a psychrotolerant Streptomyces strain, as well as a rare Actinobacteria genus, Kribbella, while two other Streptomyces spp. were surprisingly similar to known genomes. Streptomyces and Kribbella BGCs were predicted to encode antitumour, antifungal, antibacterial and biosurfactant‐like compounds, and the synthesis of NPs with antibacterial, antifungal and surfactant properties was confirmed through bioactivity assays.

Complete genome sequences of pooled genomic DNA from 10 marine bacteria using PacBio long-read sequencing

Song W, Thomas T & Edwards RJ (2019) Complete genome sequences of pooled genomic DNA from 10 marine bacteria using PacBio long-read sequencing. Marine Genomics 48:100687. DOI: 10.1016/j.margen.2019.05.002

Abstract

Background

High-quality, completed genomes are important to understand the functions of marine bacteria. PacBio sequencing technology provides a powerful way to obtain high-quality completed genomes. However individual library production is currently still costly, limiting the utility of the PacBio system for high-throughput genomics. Here we investigate how to generate high-quality genomes from pooled marine bacterial genomes.

Results

Pooled genomic DNA from 10 marine bacteria were subjected to a single library production and sequenced with eight SMRT cells on the PacBio RS II sequencing platform. In total, 7.35 Gbp of long-read data was generated, which is equivalent to an approximate 168× average coverage for the input genomes. Genome assembly showed that eight genomes with average nucleotide identities (ANI) lower than 91.4% can be assembled with high-quality and completion using standard assembly algorithms (e.g. HGAP or Canu). A reference-based reads phasing step was developed and incorporated to assemble the complete genomes of the remaining two marine bacteria that had an ANI > 97% and whose initial assemblies were highly fragmented.

Conclusions

Ten complete high-quality genomes of marine bacteria were generated. The findings and developments made here, including the reference-based read phasing approach for the assembly of highly similar genomes, can be used in the future to design strategies to sequence pooled genomes using long-read sequencing.

Peter Santosa (SVRS Student)

Peter is a 3rd year Advance Science student who worked in the lab in January-February 2017 on the Summer Vacation Research Scholarship (SVRS). Peter was working on a bacterial sequencing project in collaboration with Mike Manefield at UNSW. We have successfully used PacBio sequencing to fully and contiguously assemble the genome of a new bacterial strain from a mixed culture. Peter’s project was analysing assembled contigs from other organisms in the culture.

Peter is undertaking a double major of molecular and cell biology and microbiology at UNSW.

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.

iOES Poster: Using metagenomics to assess soil microbial diversity under future climate scenarios

Joseph Jenkins, Prof. Gail Taylor, Dr. Rich Edwards.

1st International Environmental Omics Synthesis (iEOS) Conference, Cardiff, UK (Sept 11-13, 2013). Poster CE-3.

Abstract:

Soil microbes are responsible for the function of biogeochemical cycles, which are essential to maintain soil quality. Anthropogenic climate change is resulting in variation of the soil habitat, through alteration of a multitude of soil variables. In particular, prevalence of drought and use of geoengineering methods to sequester carbon (such as biochar) are expected to increase.

To test the implications of drought and biochar amendment in soil habitats, soil samples were collected from two long term experimental sites, and shotgun metagenomic sequencing undertaken to determine changes in soil microbial diversity. Furthermore, analysis of the method itself will be undertaken to determine potential issues with the technique, and to improve the methodology for future studies.

Future work will attempt to compare results of shotgun and amplicon metagenomic methods, and sequencing of additional biochar treated samples from a range of European sites undertaken. This will provide a means to compare changes in microbial diversity after biochar incorporation under a variety of field conditions, providing insight into its likely effects for soil microbial ecology.

1st International Environmental Omics Synthesis (iEOS) Conference

The lab has a couple of posters at the 1st International Environmental Omics Synthesis (iEOS) Conference conference in Cardiff this week (Sept 11-13), so come and say hello to Joe and/or Alex if you are in Cardiff.

Poster EcO-15:

UNDERSTANDING PLANT ADAPTATION TO THE CHANGING ENVIRONMENT USING NEXT GENERATION RNA TRANSCRIPTOME SEQUENCING

Alex Watson-Lazowski, Yunan Lin, Jennifer DeWoody, Richard Edwards and Gail Taylor. Centre for Biological Sciences, University of Southampton.

Plant adaptation to elevated atmospheric carbon dioxide (CO2) is of great interest, as the concentration of this gas in the atmosphere has risen by more than 30% to 388 μmol mol-1 since the industrial revolution. On average there has been a rise of 3 ppm per year. Plant fossil samples suggest that atmospheric CO2 may be acting as a selective agent driving evolution, but limited evidence is available to support this idea for plants subjected to future predicted concentrations. Studying evolutionary responses to this aspect of environmental change is difficult, but here we use a CO2 spring site where plants have been exposed for multiple generations to concentrations of CO2 predicted for 2050. From this, detailed phenotyping data was collected, including data for stomatal patterning. Considerable evidence exists to show that stomatal numbers have declined across geological time and that this is linked to CO2 concentration, but few CO2-sensitive stomatal patterning genes have ever been identified. When grown under elevated CO2 concentrations P. lanceolata (the narrow leaf plantain), seeds collected from the spring site showed a counter-intuitive change in stomatal index and density. Here, in this non- model plant we have investigated the gene expression changes underlying this stomatal patterning response to elevated CO2.

RNA-Seq allows for in depth analysis of plant species with no previous information required, enabling rapid evaluation of any of novel plant acclimations and adaptions. Using this approach we have identified a set of novel genes for stomatal patterning in high CO2 and confirmed previously observed acclimation responses.

RNA-Seq refers to the use of high-throughput deep-sequencing technologies to sequence cDNA in order to get information about the transcriptome of a given biological sample.

Poster CE-3:

USING METAGENOMICS TO ASSESS SOIL MICROBIAL DIVERSITY UNDER FUTURE CLIMATE SCENARIOS

Joseph Jenkins, Prof. Gail Taylor, Dr. Rich Edwards. University of Southampton, ExpeER, EuroChar.

Soil microbes are responsible for the function of biogeochemical cycles, which are essential to maintain soil quality. Anthropogenic climate change is resulting in variation of the soil habitat, through alteration of a multitude of soil variables. In particular, prevalence of drought and use of geoengineering methods to sequester carbon (such as biochar) are expected to increase.

To test the implications of drought and biochar amendment in soil habitats, soil samples were collected from two long term experimental sites, and shotgun metagenomic sequencing undertaken to determine changes in soil microbial diversity. Furthermore, analysis of the method itself will be undertaken to determine potential issues with the technique, and to improve the methodology for future studies.

Future work will attempt to compare results of shotgun and amplicon metagenomic methods, and sequencing of additional biochar treated samples from a range of European sites undertaken. This will provide a means to compare changes in microbial diversity after biochar incorporation under a variety of field conditions, providing insight into its likely effects for soil microbial ecology.