Metagenomic Sequencing of Multiple Soil Horizons and Sites in Close Vicinity Revealed Novel Secondary Metabolite Diversity

Mantri, Shrikant; Negri, Timo; Sales-Ortells, Helena; Angelov, Angel; Peter, Silke; Neidhardt, Harald; Oelmann, Yvonne; Ziemert, Nadine

doi:10.1128/msystems.01018-21

Cited by 28 publications

(25 citation statements)

References 64 publications

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“…Furthermore, apart from the few metagenomic projects whose MAGs we incorporated in the first part of our analysis, there are multiple such projects publicly available, some of which have been the focus of NP studies 59 . Although the reconstruction of genomes from metagenomes remains a challenge 60 and the assembly will often miss BGCs 61 , which has indirectly prevented their comparison to the cultured bacteria in the current project, metagenomics is proving a promising source of information on NPs and their producers 7,37,48,58,59 , as made apparent in the present investigation. We expect the effect of this field on NP research to become more evident in the following years.…”

Section: Discussionmentioning

confidence: 80%

Compendium of secondary metabolite biosynthetic diversity encoded in bacterial genomes

Gavriilidou

Kautsar

Zaburannyi

et al. 2021

Preprint

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Bacterial secondary metabolites have been studied for decades for their usefulness as drugs, such as antibiotics. However, the identification of new structures has been decelerating, in part due to rediscovery of known compounds. Meanwhile, multi-resistant pathogens continue to emerge, urging the need for new antibiotics. It is unclear how much chemical diversity exists in Nature and whether discovery efforts should be focused on established antibiotic producers or rather on understudied taxa. Here, we surveyed around 170,000 bacterial genomes as well as several thousands of Metagenome Assembled Genomes (MAGs) for their diversity in Biosynthetic Gene Clusters (BGCs) known to encode the biosynthetic machinery for producing secondary metabolites. We used two distinct algorithms to provide a global overview of the biosynthetic diversity present in the sequenced part of the bacterial kingdom. Our results indicate that only 3% of genomic potential for natural products has been experimentally discovered. We connect the emergence of most biosynthetic diversity in evolutionary history close to the taxonomic rank of genus. Despite enormous differences in potential among taxa, we identify Streptomyces as by far the most biosynthetically diverse based on currently available data. Simultaneously, our analysis highlights multiple promising high-producing taxas that have thus far escaped investigation.

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Section: Discussionmentioning

confidence: 80%

Compendium of secondary metabolite biosynthetic diversity encoded in bacterial genomes

Gavriilidou

Kautsar

Zaburannyi

et al. 2021

Preprint

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“…It is uncertain if the V4V6 primers were under amplifying or V4V5 primers over amplifying planctomycetes . Mantri et al compared 16S amplicon sequencing and shotgun sequencing of DNA from four forest soils and found that the planctomycetes were overrepresented in the amplicons sequences by several orders of magnitude (Mantri et al, 2021), suggesting that some primers can over amplify planctomycetes . However this study used V3V4 amplicons and so cannot be directly compared to ours.…”

Section: Resultsmentioning

confidence: 99%

A comparison of hard and soft direct methods for DNA extraction from soil

Hill

Dextraze

Kroetsch

et al. 2022

Preprint

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Nucleic acid extraction is the first step in molecular biology studies of soil bacterial communities. The most common used soil DNA extraction method is the direct, hard extraction Mobio method, which uses bead beating to lyse bacteria. In this study we compared the Mobio method with a soft, enzymatic lysis extraction method. Next generation sequencing (Illumina and Pyrosequencing) of amplicons generated from four 16S primer pairs and DNA from 12 soils and 3 composts was used to compare the two extraction methods. Four bacterial orders, the delta proteobacterial Desulfuromonadales and gamma proteobacterial Pseudomonadales, Enterobacteriales, and Alteromonadales were more common in amplicons from soft extracted DNA, sometimes by two orders of magnitude. These groups can be a significant fraction of the bacterial population. For example the Pseudomonadales made up to 16 % and Enterobacteriales 10% of amplicons from Soft extracted DNA. The JG30-KF-CM45 order was under extracted by the enzymatic lysis extraction method. Results differed more by primer choice than extraction method and the phylogenetic resolution of differences between extraction methods changed with primer choice. Given how often Mobio extraction is used, these proteobacterial orders are probably under-represented in the studies of soil bacteria that use nucleic acid methods. Further improvements in soil DNA extraction are needed. Amplicons sequencing studies should use a range of different primers to confirm the phylogenetic resolution of their results.

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“…The A horizon of the soil type cambisol was sampled from the Schönbuch Forest nature reserve (close to Tübingen, Germany) in November 2016. Different metagenomic HMW DNA isolation protocols were applied for subsequent Nanopore and Illumina sequencing of the DNA as described in detail in our previous study (Mantri et al 2021). The HMW DNA used for fosmid library generation was isolated following two methods of our previous study, which are summarized in the following: a small part of the library (approx.…”

Section: Soil Sampling Isolation and Sequencing Of Metagenomic Hmw Dnamentioning

confidence: 99%

“…The lasso peptide BGC 44.1 that was detected on hybrid assembled contigs (Online Resource 3) derived from the metagenome sequencing data of our previous study (Mantri et al 2021) was used as a reference sequence to design the specific primers Cl_44_SalI_fw GTC GAC CCT CCG TCG CAG AGC TGT AT (SalI recognition site underlined) and Cl_44_SacI_rv GAG CTC AAG ATG TTC CTG ACC TGC GG (SacI recognition site underlined). Using this primer pair as well as the Q5 high-fidelity DNA polymerase kit (NEB), the lasso peptide-specific genes A, B1, B2, and C of BGC 44.1 were amplified via PCR.…”

Section: Amplification and Cloning Of A Lasso Peptide Bgc From Metage...mentioning

confidence: 99%

“…In this study in particular, the metagenomic DNA needed to be of sufficient quality for the downstream applications we used such as long-and short-read sequencing, fosmid library generation and direct amplification of complete BGCs via PCR. We used our optimized metagenomic DNA isolation protocol (Mantri et al 2021) to isolate high-quality HMW metagenomic DNA from a soil sample of the Schönbuch forest (Germany). Analysis of the DNA on a gel revealed an intense band migrating above the lambda DNA/HindIII 23 kb band with almost no smear below, which confirmed that the DNA was of a high molecular weight and showed minimal shearing (Fig.…”

Section: Isolation Of High-quality Hmw Metagenomic Dna From Soilmentioning

confidence: 99%

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A rapid and efficient strategy to identify and recover biosynthetic gene clusters from soil metagenomes

Negri

Mantri

Angelov

et al. 2022

Appl Microbiol Biotechnol

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Culture-independent metagenomic approaches offer a promising solution to the discovery of therapeutically relevant compounds such as antibiotics by enabling access to the hidden biosynthetic potential of microorganisms. These strategies, however, often entail laborious, multi-step, and time-consuming procedures to recover the biosynthetic gene clusters (BGCs) from soil metagenomes for subsequent heterologous expression. Here, we developed an efficient method we called single Nanopore read cluster mining (SNRCM), which enables the fast recovery of complete BGCs from a soil metagenome using long- and short-read sequencing. A metagenomic fosmid library of 83,700 clones was generated and sequenced using Nanopore as well as Illumina technologies. Hybrid assembled contigs of the sequenced fosmid library were subsequently analyzed to identify BGCs encoding secondary metabolites. Using SNRCM, we aligned the identified BGCs directly to Nanopore long-reads and were able to detect complete BGCs on single fosmids. This enabled us to select for and recover BGCs of interest for subsequent heterologous expression attempts. Additionally, the sequencing data of the fosmid library and its corresponding metagenomic DNA enabled us to assemble and recover a large nonribosomal peptide synthetase (NRPS) BGC from three different fosmids of our library and to directly amplify and recover a complete lasso peptide BGC from the high-quality metagenomic DNA. Overall, the strategies presented here provide a useful tool for accelerating and facilitating the identification and production of potentially interesting bioactive compounds from soil metagenomes. Key points • An efficient approach for the recovery of BGCs from soil metagenomes was developed to facilitate natural product discovery. • A fosmid library was constructed from soil metagenomic HMW DNA and sequenced via Illumina and Nanopore. • Nanopore long-reads enabled the direct identification and recovery of complete BGCs on single fosmids.

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Metagenomic Sequencing of Multiple Soil Horizons and Sites in Close Vicinity Revealed Novel Secondary Metabolite Diversity

Cited by 28 publications

References 64 publications

Compendium of secondary metabolite biosynthetic diversity encoded in bacterial genomes

Compendium of secondary metabolite biosynthetic diversity encoded in bacterial genomes

A comparison of hard and soft direct methods for DNA extraction from soil

A rapid and efficient strategy to identify and recover biosynthetic gene clusters from soil metagenomes

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