Metaproteomics reveals methyltransferases implicated in dichloromethane and glycine betaine fermentation by ‘Candidatus Formimonas warabiya’ strain DCMF

Holland SI, Vázquez-Campos X, Ertan H, Edwards RJ, Manefield MJ & Lee M (2022): Metaproteomics reveals methyltransferases implicated in dichloromethane and glycine betaine fermentation by ' Candidatus Formimonas warabiya' strain DCMF. Front Microbiol. 13:1035247. doi: 10.3389/fmicb.2022.1035247 [Front Microbiol.] [PubMed]

Dichloromethane (DCM; CH2Cl2) is a widespread pollutant with anthropogenic and natural sources. Anaerobic DCM-dechlorinating bacteria use the Wood–Ljungdahl pathway, yet dechlorination reaction mechanisms remain unclear and the enzyme(s) responsible for carbon-chlorine bond cleavage have not been definitively identified. Of the three bacterial taxa known to carry out anaerobic dechlorination of DCM, ‘Candidatus Formimonas warabiya’ strain DCMF is the only organism that can also ferment non-chlorinated substrates, including quaternary amines (i.e., choline and glycine betaine) and methanol. Strain DCMF is present within enrichment culture DFE, which was derived from an organochlorine-contaminated aquifer. We utilized the metabolic versatility of strain DCMF to carry out comparative metaproteomics of cultures grown with DCM or glycine betaine. This revealed differential abundance of numerous proteins, including a methyltransferase gene cluster (the mec cassette) that was significantly more abundant during DCM degradation, as well as highly conserved amongst anaerobic DCM-degrading bacteria. This lends strong support to its involvement in DCM dechlorination. A putative glycine betaine methyltransferase was also discovered, adding to the limited knowledge about the fate of this widespread osmolyte in anoxic subsurface environments. Furthermore, the metagenome of enrichment culture DFE was assembled, resulting in five high quality and two low quality draft metagenome-assembled genomes. Metaproteogenomic analysis did not reveal any genes or proteins for utilization of DCM or glycine betaine in the cohabiting bacteria, supporting the previously held idea that they persist via necromass utilization.

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.

Whole genome sequencing of a novel, dichloromethane-fermenting Peptococcaceae from an enrichment culture

Holland​ SI*, Edwards​ RJ*, Ertan H, Wong YK, Russell TL, Deshpande NP, Manefield M & Lee MJ​ (2019) Whole genome sequencing of a novel, dichloromethane-fermenting Peptococcaceae from an enrichment culture. PeerJ 7:e7775. DOI 10.7717/peerj.7775 [*Joint first authors]

Abstract

Bacteria capable of dechlorinating the toxic environmental contaminant dichloromethane (DCM, CH2Cl2) are of great interest for potential bioremediation applications. A novel, strictly anaerobic, DCM-fermenting bacterium, “DCMF”, was enriched from organochlorine-contaminated groundwater near Botany Bay, Australia. The enrichment culture was maintained in minimal, mineral salt medium amended with dichloromethane as the sole energy source. PacBio whole genome SMRTTM sequencing of DCMF allowed de novo, gap-free assembly despite the presence of cohabiting organisms in the culture. Illumina sequencing reads were utilised to correct minor indels. The single, circularised 6.44 Mb chromosome was annotated with the IMG pipeline and contains 5,773 predicted protein-coding genes. Based on 16S rRNA gene and predicted proteome phylogeny, the organism appears to be a novel member of the Peptococcaceae family. The DCMF genome is large in comparison to known DCM-fermenting bacteria and includes 96 predicted methylamine methyltransferases, which may provide clues to the basis of its DCM metabolism. Full annotation has been provided in a custom genome browser and search tool, in addition to multiple sequence alignments and phylogenetic trees for every predicted protein, available at http://www.slimsuite.unsw.edu.au/research/dcmf/.

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.

Honours and undergrad research opportunities

Honours

BABS are currently recruiting the next cohort of Honours students for Semester 1 2017. As usual, the EdwardsLab is looking to recruit enthusiastic students in two main areas:

1. Functional genomics using long-read PacBio sequencing. We are particularly keen to get a student to work on either (a) aspects of our ARC Linkage grant, investigating the evolution of a novel biochemical pathway in yeast, or (b) de novo whole genome sequencing of the cane toad. We also have a number of projects with bacteria for those with a keen interest in microbiology. In each case, the lab is collaborating with experts in the relevant organisms.

2. Applying biological sequence analysis and molecular evolution to study the molecular basis of protein-protein interactions. The main lab software, SLiMSuite has a number of improvements and developments that would benefit from some dedicated attention from a research student. We are also looking for someone who might want to help develop the lab servers.

More details of honours can be found on the BABS website, or please get in touch if you have questions about specific projects. Applications from non-UNSW students are also encouraged.

* BABS are also running an Honours information and networking night on 16^th September.*

Summer Vacation Research Scholarships

BABS is once again running its highly successful Summer Vacation Research Scholarship (SVRS) scheme and the EdwardsLab are looking to take on one or two students in the same areas as indicated above.

How to apply

We do not yet have a specific undergraduate application form but it is helpful if you can follow the PhD application process and just make it clear that you are interested in Honours or SVRS. As well as helping select between applicants, this form is also useful for me to make sure that students are assigned an appropriate project.

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.

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.

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.

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.