Comparative genomics and metabolic profiling of the genus Lysobacter
- Irene de Bruijn1, 2Email author,
- Xu Cheng2,
- Victor de Jager1,
- Ruth Gómez Expósito1, 2,
- Jeramie Watrous3,
- Nrupali Patel4,
- Joeke Postma5,
- Pieter C. Dorrestein3,
- Donald Kobayashi†4 and
- Jos M. Raaijmakers†1
© de Bruijn et al. 2015
Received: 18 May 2015
Accepted: 3 November 2015
Published: 23 November 2015
Lysobacter species are Gram-negative bacteria widely distributed in soil, plant and freshwater habitats. Lysobacter owes its name to the lytic effects on other microorganisms. To better understand their ecology and interactions with other (micro)organisms, five Lysobacter strains representing the four species L. enzymogenes, L. capsici, L. gummosus and L. antibioticus were subjected to genomics and metabolomics analyses.
Comparative genomics revealed a diverse genome content among the Lysobacter species with a core genome of 2,891 and a pangenome of 10,028 coding sequences. Genes encoding type I, II, III, IV, V secretion systems and type IV pili were highly conserved in all five genomes, whereas type VI secretion systems were only found in L. enzymogenes and L. gummosus. Genes encoding components of the flagellar apparatus were absent in the two sequenced L. antibioticus strains. The genomes contained a large number of genes encoding extracellular enzymes including chitinases, glucanases and peptidases. Various nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) gene clusters encoding putative bioactive metabolites were identified but only few of these clusters were shared between the different species. Metabolic profiling by imaging mass spectrometry complemented, in part, the in silico genome analyses and allowed visualisation of the spatial distribution patterns of several secondary metabolites produced by or induced in Lysobacter species during interactions with the soil-borne fungus Rhizoctonia solani.
Our work shows that mining the genomes of Lysobacter species in combination with metabolic profiling provides novel insights into the genomic and metabolic potential of this widely distributed but understudied and versatile bacterial genus.
KeywordsLysobacter Microbial interactions Comparative genomics Mass spectrometry imaging Nonribosomal peptide synthesis
Lysobacter species are Gram-negative bacteria commonly found in diverse ecosystems, including soil, rhizosphere and freshwater habitats [1, 2]. Lysobacter cells are thin, gliding and mucilaginous on agar medium . The genus Lysobacter belongs to the family Xanthomonadaceae and was first described in 1978 by Christensen and Cook (Christensen and Cook 1978) and initially included four species that were isolated from Canadian soil or lake water: L. antibioticus, L. brunescens, L. enzymogenes, and L. gummosus. Prior to the description of Lysobacter as a separate genus, isolates were often confused with the myxobacteria Polyangium and Sorangium  or misidentified as Xanthomonas and Stenotrophomonas. In the last decade, many more species have been identified, with the majority of isolates coming from soils of Asian origin . Approximately 25 species of Lysobacter are now described (International Journal of Systematic and Evolutionary Microbiology, IJSEM) but only two incomplete genome sequences, consisting of multiple contigs [4, 5], are publically available.
Lysobacter is named after its lytic effects on many other (micro)organisms including fungi, oomycetes, nematodes, unicellular algae, Gram-negative and Gram-positive bacteria . Originally, isolations were performed by enrichment with chitin or ground mushrooms, followed by plating on agar media containing yeast or bacterial cells as a nutrient source . Their broad-spectrum lytic activity has been attributed to the production of extracellular enzymes like proteases and endopeptidases [6–9], glucanases [10, 11], lipases [12–14], chitinases [15, 16], secondary metabolites [17, 18] and other yet unknown bioactive compounds. Several metabolites produced by Lysobacter species, mostly L. enzymogenes, have attracted considerable interest for their activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) . These metabolites include tripropeptins, lysobactin (also known as katanosin B) and WAP-8294A [18–22]. Antifungal metabolites produced by Lysobacter species are the maltophilins and derivatives thereof, including dihydromaltophillin and xanthobaccin A [18, 23–25]. Other compounds discovered for Lysobacter include the antibacterial cephabacins [26, 27], lactivicin and myxin [1, 18]. Recently, a 2,5-diketopiperazine (cyclo(L-Pro-L-Tyr)) was identified in L. capsici AZ78 with strong activity against the oomycete plant pathogens Phytophthora infestans and Plasmopara viticola . The majority of these compounds are encoded by nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) genes . For the antimicrobial activities of Lysobacter species other than L. enzymogenes, however, only few corresponding metabolites and their biosynthetic genes are known and many more bioactive compounds remain to be discovered. Hence, Lysobacter species are considered as a neglected resource for novel antibiotics .
To begin to unravel the genomic diversity and metabolic potential of the Lysobacter genus, we sequenced and closed the genomes of five Lysobacter strains representing four species: L. enzymogenes, L. capsici, L. gummosus and L. antibioticus. Comparative genomics was performed to refine the phylogenetic delineation of the Lysobacter genus and to identify the core and pan genomes. We then focused specifically on common and unique genes potentially involved in the secretion, regulation and biosynthesis of known and unknown bioactive compounds. Metabolic fingerprinting was performed by dry droplet colony mass spectrometry and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging  to complement the in silico genome analyses and to elucidate the spatial distribution patterns of these bioactive compounds in interactions between Lysobacter species and Rhizoctonia solani, an economically important fungal pathogen of multiple crops including wheat, rice and sugar beet.
Strains and culture conditions
Lysobacter strains were grown on R2A (Merck) medium at 20–25 °C, unless specified otherwise. All pre-cultures were performed in Luria-Burti (LB) or Tryptic Soy Broth (TSB) and incubated for 2–3 days at 25 °C. All fungi and oomycetes were pre-cultured on potato dextrose agar (PDA; Difco) at 20–25 °C. Xanthomonas campestris pv. campestris and Pectobacterium atrosepticum were pre-cultured in LB medium.
Isolation of genomic DNA
Strains L. antibioticus 55, L. capsici 76 and L. gummosus 3.2.11 were cultured in TSB medium for 3 days at 25 °C, the cells were washed three times with 0.9 % NaCl and subsequently genomic DNA was extracted using the Master Pure kit from Epicentre according to manufacturer’s protocol, excluding the beat beating step.
Genomic DNA sequencing
Genomic DNA of strains L. antibioticus 55, L. capsici 76 and L. gummosus 3.2.11 were sequenced using the Pacific Biosciences (PacBio) RS sequencing platform. From the genomic DNA, 20 Kb insert size libraries were prepared and size selected using Blue Pippin™ and sequenced using C3 in combination with P5 polymerase chemistry for 2 SMRT cells per genome with 180 min or longer movie time and stage start. The 20-kb continuous-long-read (CLR) data were de novo assembled using the PacBio hierarchical genome assembly process (HGAP3)/Quiver software package version 2.2.0 . Annotation was performed by RAST  and CloVR-Microbe . Further annotation was performed by InterProScan , KEGG  and the NR database. The genome sequences of L. enzymogenes C3 and L. antibioticus ATCC29479 were obtained from Kobayshi (personal communication) and L. antibioticus 13–6 and L. capsici AZ78 were downloaded from NCBI.
Whole genome phylogeny was performed using Gegenees . This program fragments the genome sequences in 200 bp fragment using overlap of 100 bp and performs all-against-all BLASTn comparison. From the obtained matrix a phylogenetic tree was generated. Phylogenetic analyses were also performed by concatenated alignments of eight core housekeeping genes: cys, dnaX, gly, recA, recN, rpoB, rpoD, uvrC and separately, the 16S rRNA gene. Prior to alignment of the concatenated sequence, the sequence of each housekeeping gene was aligned separately and trimmed where necessary. The trees were generated using the best fitted model for Maximum Likelihood with 1000 bootstrap repetitions in MEGA . The seven Lysobacter genomes were compared to each other using an all-against-all protein BLASTp  of all proteins with a minimum length of 20 residues followed by orthology prediction using orthAgogue with at least 40 % overlap and an inflation value of 1.5 . The Markov cluster (MCL) algorithm  was used to extract the orthologous groups. The resulting orthologous groups were used to deduce the core-, variable- and pan-genome of the corresponding dataset. Core- and pan-genome size evolution graphs were generated by calculating the median core- and pan-genome size for each number of combinations in the given species set. The standard deviation is calculated for the number of species involved and plotted with the median core- or pan-genome size. Core- and pan-genome plots are also calculated based on the CDS number ordering of the genomes with the largest genome first. Syntheny analyses were performed using Progressive MAUVE . Gene clusters encoding bioactive compounds were identified using BLASTp analysis of reference gene clusters, using E-value cut-off of 10−5 and a identity >60 % and a identity of >70 % in case of NRPS/PKS biosysnthesis clusters. NRPS/PKS gene clusters were also identified by AntiSmash analysis , subsequent substrate selection prediction by PKS/NPRS prediction , NRPSpredictor2  and phylogenetic analyses. For the phylogenetic analyses, alignments of the adenylation and condensation domains were were made with CLUSTALX (version 1.81). Trees were inferred by Neighbour Joining in CLUSTALX using 1000 bootstrap replicates.
Glycoside hydrolase (GH) domain analysis of the CDSs encoding chitinases and glucanases was performed by first extracting the GH domain by PFAM analysis  of CDSs of reference strains described in the CAZy database . The PFAM database did not contain the glycoside hydrolase GH23 domain, but the reference CDSs indicated as GH23 family in the CAZy database gave a significant hit with “SLT” domain. Therefore, those CDSs that contained a significant hit with “SLT” domain were considered as containing a GH23 domain. The GH domain sequences were used for BLASTp analysis against the Lysobacter genome sequences with and E-value cut-off of 10−5. Blast hits were subjected to PFAM analysis and only those that contained a significant hit with a GH domain were considered as CDSs containing a GH with chitinase of glucanase activity.
To identify CDSs encoding peptidases, the protein sequences of all five strains were blasted against the MEROPS database with default E-value cut-off of 10−4 . Subsequently, the CDSs were grouped based on peptidase family and number of CDSs per family counted.
The cells from pre-cultures were washed 3x with 0.9 % NaCl and the cell density was adjusted to OD600 of 1. Subsequently, 2–5 μl droplets were spotted on different media containing 1.5-2 % agar. For chitinase activity, R2A and 1/10th strength TSB agar plates were used containing 0.2 % colloidal chitin prepared form crab shell chitin (Sigma) according to . After incubation for 3–7 days at 25 °C, clearing zones surrounding the colonies could be observed. For glucanase activity, R2A medium containing 0.5 % laminarin was used. After growth for 3 days at 25 °C, the colonies were removed by washing with water and the medium was stained with 1 % congo red. After destaining, coloration of the medium was determined. R2A with and without laminarin was included as controls. For lipase activity, the strains were inoculated on R2A medium supplemented with 0.01 % (w/w) CaCl2 and 1 % (w/v) Tween 80 and after 3–5 days of incubation at 25 °C, plates were investigated for opaque halos surrounding the colonies, which indicates lipase activity . For protease activity, a medium was prepared containing 15 g/l skimmed milk powder, 4 g/l blood agar base, 0.5 g/l yeast extract. After incubation for 3–7 days at 25 °C, clearing zones surrounding the colonies could be observed.
Antimicrobial activity against fungal/oomycete pathogens was determined by spotting suspension of washed cells (see above) on R2A, TSA, PDA, 1/5th strength PDA or 1/10th strength TSA medium at the border of the plate and inoculating a plug of the fungus or oomycete in the middle. After incubation for 3–7 days at 25 °C, hyphal growth inhibition could be determined. Fungal isolates were grown in potato dextrose agar (PDA, Oxoid) or vegetable juice agar plates (vegetable juice (V8) solified with 1.5 % agar) until spore production at 25 °C. To enhance sporulation, Cercospora and Stemphylium were grown on V8 agar plates under 16 h photoperiod, and spores of Verticillium and Aspergillus were collected from mycelium and streaked on PDA plates. Fungal spores were collected as described in Trifonova et. al. (2009) with slight modifications. In brief, spores were released from the mycelium by adding 10 ml of 0.9 % NaCl and scratching the surface with a sterile spatula, collected, 10-fold diluted and added to the culture media (PDA, 1/5 PDA and R2A cooled at 48-55 °C at a final concentration of 5 % (v/v). Four 5 μl droplets of each bacterial suspension were spotted at the edges of 90 mm spore inoculated plates. Non spore-inoculated plates were used as control. Plates were incubated at 25 °C for one week and subsequently inhibitory halos were monitored. Antimicrobial activity against bacterial pathogens was determined by preparing R2A, PDA, TSA and 1/5th strength PDA plates with an overlay of 1 % water agar cooled down to 50 °C to which washed cells of a culture of the bacterial pathogens was added. Subsequently, 2–5 μl washed cells from the Lysobacter strains were spotted on top of the plates. After incubation for 3–7 days at 25 °C, clearing zones surrounding the colonies could be observed.
Viability staining of R. solani AG2-2IIIB was performed comparable to  by inoculating small mycelial plugs (2 mm) in 1 ml 1/5 PDB in 24-wells plate (Nunc). The plates were incubated for 4 days at 25 °C, non-shaking. Lysobacter strains were grown in R2B for four days at 25 °C, 200 rpm and the cultures were centrifuged for 20 min at maximum speed and the supernatant was collected and filter-sterilised by 0.2 μm (Whatman) The mycelium of R. solani was washed twice with 0.9 % NaCl and 1 ml of Lysobacter supernatant was added. The samples were incubated for 1, 2, 4, or 6 h. For each timepoint, 100 uM 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) + 60 uM menadione was added and after 1 h incubation 200 ul of each sample was transferred into 96-well plate (Nunc) and measured the absorbance at 490 nm.
Dried droplet mass spectrometry
Lysobacter strains, L. antibioticus L08, L. antibioticus ATCC29479, L. capsici L14, L. gummosus L15, L. enzymogenes C3 and L. enzymogenes DCA were grown in 5 ml LB broth overnight at 25 °C. Five μl of a bacterial suspension with an optical density of 1.0 at 600 nm was spotted on R2A agar plates and incubated at 25 °C. After two days of incubation, a plug of 2-mm diameter of the bacterial colony was placed into 100 ul methanol and the sample was vortexed and centrifuged briefly. The supernatant was collected and 0.7 μl of supernatant was mixed 1:1 with saturated Universal MALDI matrix (1:1 mixture of 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxy-cinnamic Acid, Sigma-Aldrich) and 1.0 μl was deposited onto the MALDI target and allowed to dry. The sample plates were subjected to MALDI-TOF mass spectrometry (Microflex from Bruker Daltonics, Billerica, MA, USA) for MS acquisition, and were run in positive reflector mode with a mass range of 0–4 kDa. The data of two technical replicates (two MALDI-TOF runs) of two biological replicates per strain was analysed using FlexAnalysis 3.3 (Bruker Daltonics) and ClinProTool 3.0 software (Bruker Daltonics).
Cultures were prepared as described above and 5 μl cell suspensions were spot-inoculated on thin R2A agar plates (1–1.5 mm thick; 10 ml in petridish with 9 cm diameter). After 2 days of incubation at 25 °C, regions of agar containing Lysobacter colony and surrounding area were cut and placed on top of a MALDI MSP 96 anchor plate (Bruker Daltonics, Billerica, MA, USA). A photograph was taken and a layer of Universal MALDI matrix (1:1 mixture of 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxy-cinnamic Acid, Sigma-Aldrich) was applied to the sample using a 53 μm sieve. Samples were dried at 37 °C for a minimum of 6 h until they were completely dry and adhered to the MALDI plate. The sample plates were subjected to MALDI-TOF mass spectrometry (Autoflex from Bruker Daltonics, Billerica, MA, USA) for IMS acquisition, and were run in positive linear mode, with 500 μm laser intervals in XY and a mass range of 0–4 kDa. Data of two biological replicates per strain was analysed. To study the interaction with R. solani: after 2 days of incubation of the Lysobacter strains on thin agar plates, a mycelial plug of 2-mm diameter of R.solani was placed next to Lysobacter colony and incubated for another 2 days at 25 °C. The mycelial plug was removed and the agar region containing both the Lysobacter spp. and R. solani were cut and placed on top of a MALDI MSP 96 anchor plate and further processed as described above. Data of one sample per interaction was obtained. The data was analysed using FlexImaging 3.0 software (Bruker Daltonics).
Results and discussion
Lysobacter strains used for comparative genomics
Isolation source with references
accession number and reference
L. ant ATCC29479
L. antibioticus ATCC29479
soil Central Experimental Farm, Ontario, Canada 
CP013141; this study
L. ant 76
L. antibioticus 76
CP011129; this study
L. ant 13-6
L. antibioticus 13–6
rhizosphere Chinese cabbage 
L. cap 55
L. capsici 55
CP011130; this study
L. cap AZ78
L. capsici AZ78
tobacco rhizosphere 
L. enz C3
L. enzymogenes C3
Kentucky bluegrass leave, Nebraska 
CP013140; this study
L. gum 3.2.11
L. gummosus 3.2.11
CP011131; this study
Assembly statisitics and features of the Lysobacter genome sequences
L. ant ATCC29479
L. ant 76
L. ant 13-6 a
L. cap 55
L. cap AZ78 a
L. gum 3.2.11
L. enz C3
Genome size (Mb)
Number of Reads
11 524 134
7 512 266
N50 Read Length
Mean Read Length
Coding DNA sequences (CDSs)
# hypothetical proteins
Gene/gene clusters encoding bioactive compounds
Selection of genes or gene clusters putatively encoding bioactive metabolites by Lysobacter species. The geneID numbers are indicated and for a gene cluster the first gene and the last gene of the cluster is indicated separated by a hyphen
L. ant ATCC29479
L. ant 76
L. cap 55
L. gum 3.2.11
L. enz C3
Number of genes encoding other putative antimicrobial compounds
# unknown NRPS clusters
Many of the bioactive compounds that are produced by Lysobacter spp. are encoded by NRPS/PKS genes . Several of the known NRPS/PKS gene clusters are present in either one or more of the Lysobacter genomes, including those encoding dihydromaltophilin, lysobactin and WAP-8294A biosynthesis (Table 3). The dihydromaltophilin biosynthesis cluster is present in L. capsici, L. gummosus and L. enzymogenes, but not in the two L. antibioticus strains. Recently, a partial PKS gene cluster was identified in L. capsici AZ78 , which had 85 % identity with the predicted proteins encoding Heat-Stable Antifungal Factor (HSAF) in L. enzymogenes C3 (LEC3_3215) and 100 % and 91 % identity to the predicted proteins that are HSAF-encoding homologs in our L. capsici 55 (LA55_2005) and L. gummosus 3.2.11 (LG3211_3209), respectively. The lysobactin biosynthesis cluster is present only in L. gummosus 3.2.11. The WAP-8294A biosynthesis cluster is present in L. capsici 76 and L. enzymogenes C3. The two L. antibioticus strains have an NRPS gene cluster which seems partly similar to the WAP-8294A gene cluster of L. enzymogenes OH11 , but these gene clusters most likely encode a novel compound based on in silico structure prediction [42, 58] (Additional file 1: Figure S7). Subsequent Liquid Chromatography- tandem Mass Spectrometry (LC-MS/MS) and Nuclear Magnetic Resonance (NMR) analyses will be required for further structural identification of the metabolites, as well as random or site-directed mutagenesis to identify the corresponding genes. Multiple attempts and approaches to generate mutants, however, have not been successful for the differerent Lysobacter species, except for L. enzymogenes [15, 59]. Cloning and heterologous expression of several of the identified gene clusters will be an alternative strategy to explore in future experiments to resolve the structures and functions of the encoded metabolites.
Siderophores play an important role in iron acquisition and in competition with other microorganisms. Many siderophores are encoded by NRPS gene clusters. In the Lysobacter genomes, however, gene clusters encoding pyoverdin, pyochelin, pseudomonin, yersiniabactin, enterobactin, aerobactin, achromobactin, vibroferrin, staphyloferrin, rhizobactin and Xss were absent or only partially present (Additional file 3: Table S12). Even though iron availability did have an effect on antibacterial activity, L. capsici PG4 did not produce siderophores on CAS medium , indicating that our Lysobacter strains potentially do not possess the siderophores commonly found in other bacterial genera. However, all our strains, except L. antibioticus ATCC29479, exhibit iron-chelating capacity on CAS medium (Fig. 3).
Besides the NRPS/PKS gene clusters encoding known antibiotics, several unknown NRPS gene clusters were identified in the Lysobacter genomes. These gene clusters could be a new resource for novel antibiotics (Additional file 3: Table S13) but in-depth chemical and functional analyses are required to further investigate this.
Gene/gene clusters involved in bacterial movement
In Lysobacter species, gliding motility is a common form of movement while flagellar motility is not. No flagellar genes were identified in the genomes of either L. antibioticus strain (Additional file 3: Table S14). However, components of the flagellar apparatus were found in L. enzymogenes C3; the 22 genes coding for a flagellar apparatus (including basal body, hook and regulatory genes) are also conserved in the genomes of L. capsici 55 and L. gummosus 3.2.11 (Additional file 3: Table S14). The flagellar gene clusters in the genomes of L. enzymogenes C3 and L. gummosus 3.2.11, but not of L. capsici 55, contained an additional gene encoding a transcription factor TCP domain protein downstream of the Sigma-70 regulatory factor gene. The presence of flagellar biosynthesis genes that appear non-functional within bacterial genomes has been observed previously  and is suggestive of evolved mechanisms for evasion of host detection as flagellar filament proteins serve as antigenic triggers of host immune responses [62, 63].
The type IV pilus genes were found in 5 different clusters in all five Lysobacter genomes and the genetic organization is mostly conserved (Additional file 3: Table S14). L. capsici 55, L. gummosus 3.2.11 and L. enzymogenes C3 contain duplicate regions of cluster IV consisting of pilE,Y1,X,W,V and pre-pilin genes.
Gene/gene clusters involved in regulation and secretion
We observed that the cell-free supernatant of the Lysobacter strains could inhibit viability of R. solani hyphae (Additional file 1: Figure S8), indicating that several bioactive compounds are secreted. L. antibioticus 76 supernant does not reduce cell viability, indicating that its bioactive compounds are cell-bound or not produced in liquid medium. Multiple secretion systems have been identified in Gram-negative bacteria , and most are represented in the Lysobacter genomes. All strains contain a number of genes associated with Type I (ABC transport) and Type V (autotransport) systems (data not shown). All strains investigated here also contain full complements of genes encoding Type II secretion (T2S) systems involving both the general secretory pathway (GSP) and the twin-arginine translocation (TAT) pathway (Additional file 2: Table S15). GSP consists of one gene cluster consisting of the genes gspD-N, and yajC and secA,B,D,E,F, and Y genes dispersed throughout the genome. The TAT pathway genes, consisting of tatA/E, tatB, tatC and an RDD family protein predicted as a transmembrane protein associated with transport, were organized in an operon structure. Absent from the gene cluster is the exonuclease gene tatD, which is not required for pathway function . However, two tatD gene homologues were located distally from the TAT gene cluster within the genomes of all strains.
Taken together, the presence of T3S, T4S, T6S, the flagellar components and type IV pilus suggests that Lysobacter species can establish pathogenic interactions with microbial eukaryotic hosts like plant pathogenic fungi (Kobayashi, personal communication).
Live colony MALDI imaging
A compound with a similar m/z value to lysobactin (m/z 1276) is only found in L. gummosus 3.2.11 and detected inside and surrounding the colony indicating that this compound is secreted into the medium. Multiple peaks were detected with slightly different m/z values (Fig. 6, Additional file 1: Figure S9-S10). It is well-known that the adenylation domains in the NRPS proteins encoding for these types of compounds can have relaxed substrate selectivity , leading to the biosynthesis of structural analogues. For L. capsici 55 and L. enzymogenes C3, a compound with a m/z value similar to WAP-8294A2 (m/z 1562) is present (Additional file 1: Figure S9-S10), but hardly detected with MALDI-IMS (data not shown). The putative structural analogue with m/z 1586 was readily detected inside the colonies of L. capsici 55 and L. enzymogenes C3 (Fig. 6).
Collectively, the metabolomics data obtained by MALDI matched well with the gene clusters identified in the genomes of these five strains (Table 3). For both L. antibioticus strains, a gene cluster partly similar to WAP-8294A2 was identified, but with 11 instead of 12 NRPS modules (Additional file 1: Figure S7). The mass spectra of these strains show peaks with a different m/z value (m/z 1508), which potentially could correspond to the 11 module gene cluster. This unknown compound was only detected inside the colonies of L. antibioticus.
Strikingly, in L. capsici 55 these compounds were not detected anymore in interaction with R. solani. Instead, a group of compounds with m/z values ranging from 1135–1181 was detected (Fig. 7). The compound with a mass of m/z 1150 was detected at high concentrations in the inhibition zone, diffusing even underneath the R. solani mycelial mat. This suggests that this compound is involved in the R. solani inhibition. R. solani by itself did not produce compounds with masses of m/z 1135–1181 (Additional file 1: Figure S11), suggesting that the production of these compounds by L. capsici 55 is induced upon interaction with the fungus. We identified an NRPS/PKS gene cluster that is unique to L. capsici 55 (Additional file 1: Figure S12), but based on the number of NRPS modules this gene cluster is most likely not encoding these compounds.
Conclusions Collectively, these results showed that microbial interactions can induce or suppress the expression of cryptic gene clusters and can be used as a tool to trigger cryptic bioactive metabolites [76, 77]. Several other Lysobacter metabolites are also interesting for further analysis, including those with m/z ranging from 1720–1840. Also several novel NRPS gene clusters identified in the genome sequences (Additional file 2: Table S13) are worth to look into for their ecological functions. In conclusion, mining the genomes of Lysobacter species in combination with metabolic profiling provides novel insights into the genomic and metabolic potential of this widely distributed, versatile but understudied bacterial genus.
Availability of supporting data
The genome sequences are available at the National Center for Biotechnology Information (NCBI) and the accession numbers are mentioned in Table 1. The mass spectrometry imaging data is available on http://hdl.handle.net/10411/20585. All other supporting data are included as additional files.
This manuscript is publication number 5958 of Netherlands Institute of Ecology (NIOO-KNAW).
This project was funded by the Dutch Technology Foundation (STW).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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