Complete Genome Sequences of 13 Bacillus subtilis Soil Isolates for Studying Secondary Metabolite Diversity

Kiesewalter, Heiko T.; Lozano-Andrade, Carlos N.; Maróti, Gergely; Snyder, Dan; Cooper, Vaughn S.; Jørgensen, Tue Sparholt; Weber, Tilmann; Kovács, Ákos T.

doi:10.1128/mra.01406-19

Cited by 18 publications

(18 citation statements)

References 21 publications

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“…Our results show that for B. subtilis , surfactin production is not required for robust biofilm formation, which is in contradiction with many reports for surfactin requirement in various Bacilli [ 23 , 24 , 45 ]. In many of these reports however, the species or the strain examined also produce an iturin, bacillomycin, which is not the case for B. subtilis 3610 or the newly isolated B. subtilis strains [ 30 ]. Of note, Luo et al.…”

Section: Discussionmentioning

confidence: 99%

“…Soil samples were mixed with 0.9% saline solution, vortexed on a rotary shaker for 2 min, incubated at 80 °C for 25 min and serially diluted on LB medium solidified with 1.5% agar [ 29 ]. Highly structured colonies were targeted and isolation of B. subtilis strains was confirmed using 16S sequencing followed by whole genome [ 30 ]. These new isolates are all naturally competent allowing easy genetic modification using standard protocol for B. subtilis [ 31 ] .…”

Section: Methodsmentioning

confidence: 99%

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Surfactin production is not essential for pellicle and root-associated biofilm development of Bacillus subtilis

Thérien

Kiesewalter

Auria

et al. 2020

Biofilm

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Surfactin production is not essential for pellicle and root-associated biofilm development of Bacillus subtilis

Thérien

Kiesewalter

Auria

et al. 2020

Biofilm

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“…Soil samples were mixed with 0.9 % saline solution, vortexed on a rotary shaker for 2 min, incubated at 80 °C for 25 min and serially diluted on LB medium solidified with 1.5 % agar (53). Highly structured colonies were targeted and isolation of B. subtilis strains was confirmed using 16S sequencing followed by whole-genome sequencing of 13 selected strains (42) (43). The pan-genome pipeline Roary was applied to the Prokka annotations of the B. subtilis genomes to construct a pan-genome of genes having 95% similarity in 99% of the isolates (59,60).…”

Section: Methodsmentioning

confidence: 99%

“…sequences. Based on the antifungal screening results and the origin of the isolate, 13 B. subtilis strains were selected for genome sequencing, in addition to one B. licheniformis strain used as an outlier (42). The genomes were analyzed with antiSMASH (43) to obtain an overview of all predicted BGCs ( Fig.…”

Section: Prediction Of Biosynthetic Gene Cluster Potential Of the Isomentioning

confidence: 99%

Genomic and chemical diversity ofBacillus subtilissecondary metabolites against plant pathogenic fungi

Kiesewalter

Lozano-Andrade

Wibowo

et al. 2020

Preprint

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Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant-growth-promoting and biocontrol abilities. These secondary metabolites include non-ribosomal peptides (NRPs) with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates and therefore, a comparative overview of B. subtilis secondary metabolites is missing.In this study, we have isolated 23 B. subtilis strains from eleven sampling sites, compared the fungal inhibition profiles of wild types and their NRPs mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that non-ribosomal peptide production varied among B. subtilis strains co-isolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium sp., a combination of plipastatin and surfactin is required to hinder the growth of Botrytis cinerea. Detailed genomic analysis revealed that altered NRP production profiles in certain isolates is due to missing core genes, nonsense mutation, or potentially altered gene regulation.Our study combines microbiological antagonism assays with chemical NRPs detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis.IMPORTANCESecondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which non-ribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack production of certain secondary metabolites.

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“…Subsequently, B. velezensis AGVL-005 was found to contain many frame-shifted proteins; however, it was retained. A further 13 in-house genomes of B. subtilis and one of B. licheniformis(13) were included. This led to a final count of 310 genomes.…”

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Phylogenetic distribution of secondary metabolites in theBacillus subtilisspecies complex

Steinke

Mohite

Weber

et al. 2020

Preprint

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Microbes produce a plethora of secondary metabolites that although not essential for primary metabolism benefit them to survive in the environment, communicate, and influence differentiation. Biosynthetic gene clusters (BGCs) responsible for the production of these secondary metabolites are readily identifiable on the genome sequence of bacteria. Understanding the phylogeny and distribution of BGCs helps us to predict natural product synthesis ability of new isolates. Here, we examined the inter- and intraspecies patterns of absence/presence for all BGCs identified with antiSMASH 5.0 in 310 genomes from the B. subtilis group and assigned them to defined gene cluster families (GCFs). This allowed us to establish patterns in distribution for both known and unknown products. Further, we analyzed variations in the BGC structure of particular families encoding for natural products such as plipastatin, fengycin, iturin, mycosubtilin and bacillomycin. Our detailed analysis revealed multiple GCFs that are species or clade specific and few others that are scattered within or between species, which will guide exploration of the chemodiversity within the B. subtilis group. Uniquely, we discovered that partial deletion of BGCs and frameshift mutations in selected biosynthetic genes are conserved within phylogenetically related isolates, although isolated from around the globe. Our results highlight the importance of detailed analysis of BGCs and the remarkable phylogenetically conserved errodation of secondary metabolite biosynthetic potential in the B. subtilis group.

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Complete Genome Sequences of 13 Bacillus subtilis Soil Isolates for Studying Secondary Metabolite Diversity

Abstract: Bacillus subtilis is a plant-benefiting soil-dwelling Gram-positive bacterium with secondary metabolite production potential. Here, we report the complete genome sequences of 13 B. subtilis strains isolated from different soil samples in Germany and Denmark.

Cited by 18 publications

References 21 publications

Surfactin production is not essential for pellicle and root-associated biofilm development of Bacillus subtilis

Surfactin production is not essential for pellicle and root-associated biofilm development of Bacillus subtilis

Genomic and chemical diversity ofBacillus subtilissecondary metabolites against plant pathogenic fungi

Phylogenetic distribution of secondary metabolites in theBacillus subtilisspecies complex

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