- Research article
- Open Access
Comparative genomics and metabolic profiling of the genus Lysobacter
BMC Genomicsvolume 16, Article number: 991 (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.
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
L. antibioticus strain 76, L. capsici strain 55 and L. gummosus strain 3.2.11 (Table 1) were selected for PacBio genome sequencing of size-selected 20-kb-insert libraries. Subsequent de novo assembly resulted in closed genome sequences. Comparative genomics of these three genomes including two genomes provided by D. Kobayashi and the two recently deposited, partial Lysobacter genome sequences [4, 5], showed that the genome sizes ranged from 5.8–6.4 Mb with a GC content of 66–70 % (Table 2). The number of Coding DNA Sequences (CDSs) in the closed genomes ranged from 5,146 in L. antibioticus 76 to 5,686 in L. capsici 55 (Table 2). Whole genome phylogeny showed that the Lysobacter genomes were distinct from the Stenotrophomonas and Xanthomonas genomes (Fig. 1). The three L. antibioticus and the two L. capsici strains clustered closely together and more distant from the other Lysobacter species (Fig. 1). Phylogenetic analysis based on multi-locus sequence analysis (MLSA) and 16S rDNA showed similar results (Additional file 1: Figure S1). The L. antibioticus 13–6 genome sequence was not included for further analyses, since no predicted protein sequences were available .
All-against-all BLASTp analyses and subsequent orthology clustering of the predicted protein sequences of all five strains and L. capsici AZ78  resulted in a pangenome of 10,028 orthologous groups. The core genome consisted of 2,891 CDSs, representing 54 % of the CDSs of each strain (Fig. 2, Additional file 1: Figure S2, Additional file 2: Table S1). This number is larger than the core genome of Xanthomonads (±30 % of the total genome ) but comparable to the highly diverse Pseudomonas fluorescens clade (±50 %, ). Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation , the core genome consists, for a large part, of CDSs for enzymes, transporters and transcription factors (Additional file 1: Figure S3). For the two L. antibioticus strains 76 and ATCC29479 and the two L. capsici strains, approximately 90 % and 85 % of the CDSs were shared between the strains, indicating that within these species there is a high level of similarity. However, genome alignment of L. antibioticus ATCC29479 with L. antibioticus 76 showed many rearrangements between the two genomes (Additional file 1: Figure S4). CDSs unique for each strain ranged from 7.5 % for L. antibioticus 76 to 22.4 % for L. enzymogenes C3 (Additional file 2: Table S2). Approximately 13 % and 9 % of the CDSs were unique for the L. antibioticus and L. capsici species, respectively (Additional file 2: Table S3). Several of these unique CDSs gene clusters will be discussed in further detail below, focussing primarily on the gene clusters from the strains/species with closed genome sequences.
Gene/gene clusters encoding bioactive compounds
The Lysobacter strains had a different activity spectrum against fungi, oomycetes and bacteria (Fig. 3; Additional file 3: Table S4), suggesting that they harbour or express different or multiple sets of genes coding for antimicrobial compounds. Lysobacter owes its name to the lytic activity against bacteria, fungi and algae. This activity has been partially attributed to the production of lytic enzymes such as chitinases, endopeptidases and proteases . All Lysobacter strains showed chitinase activity (Fig. 3) and 1,3-β-glucanase activity (data not shown) and their genomes contained multiple CDSs encoding glycoside hydrolase domains conferring to chinase and glucanase activity (Additional file 3: Table S5-S8) according to the Carbohydrate-Active enZYmes (CAZy) database . Almost all strains showed extracellular protease activity, except the L. gummosus strain under the condition tested (Additional file 2: Table S4) and 5.7-7.2 % of the total CDSs were predicted to encode peptidases with the majority belonging to the serine proteases followed by the metalloproteases (Additional file 1: Figure S5; Additional file 3: Table S9). Several of these peptidases are secreted in outer membrane vesicles, which are proposed to deliver the lytic enzymes at high concentrations at the site of interaction with other microbes [8, 9, 53–55]. Several peptidases were shown to have potential biofilm degrading activity in L. gummosus DSMZ6980 . Only the α-lytic protease (accession number KF738068) had a high identity (99 %) in our strain L. gummosus 3.2.11 (Additional file 3: Table S10), indicating that this enzyme might be specific for the L. gummosus lineage.
In literature, several antibiotics and their gene clusters have been described for Lysobacter species (Additional file 3: Table S11). In our strains, several phenazine-related genes were predicted only for the L. antibioticus strains (Table 3). The organization of these putative phenazine biosynthesis genes is different from those in Pseudomonas aeruginosa (Additional file 1: Figure S6) and in other bacterial genera .
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
Comparable to other Xanthomonads, the production of several bioactive compounds in Lysobacter have been shown to be regulated by intercellular signalling or quorum sensing mediated by the diffusible signal factor (DSF)-dependent system and Clp (cyclic AMP receptor (CRP)-like protein) regulator [59, 64–69]. Most components of the DSF-dependent system are present in the Lysobacter genomes, although some components had a lower identity to the DSF system described in Stenotrophomonas (Fig. 4). In L. capsici 55, the rpfC and rpfF genes were absent (Fig. 4). The Clp protein was present in all genomes with 99 % identity (Fig. 4), indicating that the Clp regulator is highly conserved among Lysobacter species.
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.
The genetic organization of the core genes encoding for Type III secretion (T3S) are highly conserved in all five Lysobacter strains (Fig. 5a). Conspicuously absent within the T3S pathways for all Lysobacter strains is a gene encoding the T3S secretin, a protein necessary for pore formation and transport across the outer membrane and essential for T3S apparatus function [72, 73]. No effector-encoding genes (related to those characterized in animal and plant pathogens) were identified. However, the presence of hypothetical proteins embedded in the core T3S genes suggests their putative effector function. All five strains possess only two secretin homologues, those belonging to T2S and Type IV Pilus (Additional file 3: Table S14). Also the virD4 - virB1-11 required for Type IV secretion (T4S) are present in conserved genetic organization in all Lysobacter genomes with virD4 located distally from the virB1-11 gene complex; only the order of virB8 and virB9 in L. gummosus 3.2.11 was reversed. There are two copies of virB5 (designated virB5a and b) within the virB gene cluster, as well as multiple predicted virB5-6 gene pairs distributed throughout the genomes of all three strains (Fig. 5b). In contrast to the T3S and T4S pathways, genes encoding for T6S were found only in L. enzymogenes strain C3 and L. gummosus strain 3.2.11 and with different organization (Fig. 5C). L. enzymogenes harbors two distinct Type VI secretion (T6S) pathways encoded by genes located in separate clusters, but only one cluster consisting of 13 genes was identified in L. gummosus 3.2.11. The lack of conservation suggests that T6S represents a more recent acquisition among and within Lysobacter species.
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
Using MALDI-Imaging Mass Spectrometry (MALDI-IMS), the spatial distribution of extracellular metabolites produced by Lysobacter colonies was investigated. Several peaks ranging from 0–2000 mass-to-charge ratios (m/z) were detected (Fig. 6). Similar masses were detected in methanol extracts of plate cultures (Additional file 1: Figure S9-S10; Additional file 4). Most peaks with m/z 0–350 were considered as background since they were present in the matrix and R2A medium control (Additional file 1: Figure S10). The two L. antibioticus strains appear to produce similar compounds (Fig. 6). The other strains show different metabolic profiles, although L. capsici 55 and L. enzymogenes C3 have specific peaks in common (Fig. 6, Additional file 1: Figure S9-S10). In the metabolic profile of the clp mutant of L. enzymogenes C3, designated mutant strain DCA, most of the compounds produced by wild type strain C3 were absent (Additional file 1: Figure S9-10), confirming that the Clp transcription factor is indeed required for the production of several of these extracellular compounds. The m/z values described previously [21, 22, 74] for the dihydromaltophilin, lysobactin, and WAP-8294A2 were 513, 1276, 1562, respectively. The mass spectra show that a compound with an m/z value similar to dihydromaltophilin (m/z 513) is present in L. capsici 55, L. gummosus 3.2.11 and L. enzymogenes C3 (Additional file 1: Figure S10). This compound was secreted by L. enzymogenes C3, but was mostly retained in the colony of L. capsici 55 (Fig. 6). A putative structural analogue of dihydromaltophilin with m/z 510 was detected at high intensity in L. capsici 55 (Fig. 6).
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.
All strains inhibited hyphal growth of the soil-borne, plant pathogenic fungus R. solani (Fig. 3). In interaction with R. solani, we observed that the dihydromaltophilin-like compound (m/z 513) was mainly secreted by L. gummosus 3.2.11 and L. enzymogenes C3 and diffused radially from the bacterial colony (Fig. 7). Since the diffusion area of this compound does not match well with the inhibition zone, it is doubtful if this compound is the main contributor to hyphal growth inhibition of R. solani. The lysobactin-like compound (m/z 1276) was closely surrounding the colony and is diffusing radially in the medium (Fig. 7). Since its distribution matches better with the hyphal inhibition zone, this compound could be a major contributor to the antifungal activity of L. capsici 55. For L. antibioticus 76, the unknown compound with m/z 1508 stays localised within the colony and most likely is not responsible for the growth inhibition (Fig 7). The compounds with m/z 1562–1586 similar to WAP-8294A and derivatives thereof were detected at high levels within and closely surrounding the colony of L. enzymogenes C3.
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.
Christensen P, Cook FD. Lysobacter, a new genus of nonfruiting, gliding bacteria with a high base ratio. Int J Syst Bacteriol. 1978;28(3):367–93.
Hayward AC, Fegan N, Fegan M, Stirling GR. Stenotrophomonas and Lysobacter: ubiquitous plant-associated gamma-proteobacteria of developing significance in applied microbiology. J Appl Microbiol. 2010;108(3):756–70.
Reichenbach H. The genus Lysobacter. In: Balows A, Trüper HG, Dworkin M, Harder W, Schleifer K-H, editors. The Prokaryotes, second edition A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, vol. IV. New York: Springer; 1992. p. 3256–75.
Puopolo G, Sonego P, Engelen K, Pertot I. Draft genome sequence of Lysobacter capsici AZ78, a bacterium antagonistic to plant-pathogenic oomycetes. Genome Announc. 2014;2(2):e00325–00314.
Zhou L, Li M, Yang J, Wei L, Ji G. Draft genome sequence of antagonistic agent Lysobacter antibioticus 13–6. Genome Announc. 2014; 2(5).
Ahmed K, Chohnan S, Ohashi H, Hirata T, Masaki T, Sakiyama F. Purification, bacteriolytic activity, and specificity of beta-lytic protease from Lysobacter sp. IB-9374. J Biosci Bioeng. 2003;95(1):27–34.
Bone R, Frank D, Kettner CA, Agard DA. Structural analysis of specificity: alpha-lytic protease complexes with analogues of reaction intermediates. Biochemistry. 1989;28(19):7600–9.
Lapteva YS, Zolova OE, Shlyapnikov MG, Tsfasman IM, Muranova TA, Stepnaya OA, et al. Cloning and expression analysis of genes encoding lytic endopeptidases L1 and L5 from Lysobacter sp. strain XL1. Appl Environ Microbiol. 2012;78(19):7082–9.
Vasilyeva NV, Tsfasman IM, Suzina NE, Stepnaya OA, Kulaev IS. Secretion of bacteriolytic endopeptidase L5 of Lysobacter sp. XL1 into the medium by means of outer membrane vesicles. FEBS Journal. 2008;275(15):3827–35.
Palumbo JD, Sullivan RF, Kobayashi DY. Molecular characterization and expression in Escherichia coli of three β-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7. J Bacteriol. 2003;185(15):4362–70.
Palumbo JD, Yuen GY, Jochum CC, Tatum K, Kobayashi DY. Mutagenesis of β-1,3-glucanase genes in Lysobacter enzymogenes strain C3 results in reduced biological control activity toward Bipolaris leaf spot of tall fescue and Pythium damping-off of sugar beet. Phytopathology. 2005;95(6):701–7.
Folman LB, Postma J, Van Veen JA. Characterisation of Lysobacter enzymogenes (Christensen and Cook 1978 strain 3.1 T8, a powerful antagonist of fungal diseases of cucumber. Microbiol Res. 2003;158(2):107–15.
Ko HS, Jin RD, Krishnan HB, Lee SB, Kim KY. Biocontrol ability of Lysobacter antibioticus HS124 against Phytophthora blight is mediated by the production of 4-hydroxyphenylacetic acid and several lytic enzymes. Curr Microbiol. 2009;59(6):608–15.
Kobayashi DY, El-Barrad NEH. Selection of bacterial antagonists using enrichment cultures for the control of summer patch disease in Kentucky bluegrass. Curr Microbiol. 1996;32(2):106–10.
Qian G, Wang Y, Qian D, Fan J, Hu B, Liu F. Selection of available suicide vectors for gene mutagenesis using chiA (a chitinase encoding gene) as a new reporter and primary functional analysis of chiA in Lysobacter enzymogenes strain OH11. World J Microbiol Biotechnol. 2012;28(2):549–57.
Zhang Z, Yuen GY. The role of chitinase production by Stenotrophomonas maltophilia strain C3 in biological control of Bipolaris sorokiniana. Phytopathology. 2000;90(4):384–9.
Pidot SJ, Coyne S, Kloss F, Hertweck C. Antibiotics from neglected bacterial sources. Int J Med Microbiol. 2014;304(1):14–22.
Xie Y, Wright S, Shen Y, Du L. Bioactive natural products from Lysobacter. Nat Prod Rep. 2012;29(11):1277–87.
Hashizume H, Hirosawa S, Sawa R, Muraoka Y, Ikeda D, Naganawa H, et al. Tripropeptins, novel antimicrobial agents produced by Lysobacter sp. II. Structure elucidation. J Antibiotics. 2004;57(1):52–8.
Hashizume H, Igarashi M, Sawa R, Adachi H, Nishimura Y, Akamatsu Y. A new type of tripropeptin with anteiso-branched chain fatty acid from Lysobacter sp. BMK333-48 F3. J Antibiot. 2008;61(9):577–82.
Hou J, Robbel L, Marahiel MA. Identification and characterization of the lysobactin biosynthetic gene cluster reveals mechanistic insights into an unusual termination module architecture. Chem Biol. 2011;18(5):655–64.
Zhang W, Li Y, Qian G, Wang Y, Chen H, Li YZ, et al. Identification and characterization of the anti-methicillin-resistant Staphylococcus aureus WAP-8294A2 biosynthetic gene cluster from Lysobacter enzymogenes OH11. Antimicrob Agents Chemother. 2011;55(12):5581–9.
Islam MT, Hashidoko Y, Deora A, Ito T, Tahara S. Suppression of damping-off disease in host plants by the rhizoplane bacterium Lysobacter sp. strain SB-K88 is linked to plant colonization and antibiosis against soilborne peronosporomycetes. Appl Environ Microbiol. 2005;71(7):3786–96.
Lou L, Qian G, Xie Y, Hang J, Chen H, Zaleta-Rivera K, et al. Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes. J Am Chem Soc. 2011;133(4):643–5.
Nakayama T, Homma Y, Hashidoko Y, Mizutani J, Tahara S. Possible role of xanthobaccins produced by Stenotrophomonas sp. strain SB-K88 in suppression of sugar beet damping-off disease. Appl Environ Microbiol. 1999;65(10):4334–9.
Ono H, Nozaki Y, Katayama N, Okazaki H. Cephabacins, new cephem antibiotics of bacterial origin. I. Discovery and taxonomy of the producing organisms and fermentation. J Antibiot. 1984;37(12):1528–35.
Sohn YS, Nam DH, Ryu DDY. Biosynthetic pathway of cephabacins in Lysobacter lactamgenus: Molecular and biochemical characterization of the upstream region of the gene clusters for engineering of novel antibiotics. Metab Eng. 2001;3(4):380–92.
Puopolo G, Cimmino A, Palmieri MC, Giovannini O, Evidente A, Pertot I. Lysobacter capsici AZ78 produces cyclo(l-Pro-l-Tyr), a 2,5-diketopiperazine with toxic activity against sporangia of Phytophthora infestans and Plasmopara viticola. J Appl Microbiol. 2014;117(4):1168–80.
Cornett DS, Reyzer ML, Chaurand P, Caprioli RM. MALDI imaging mass spectrometry: molecular snapshots of biochemical systems. Nat Methods. 2007;4(10):828–33.
Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods. 2013;10(6):563–9.
Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, et al. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res. 2014;42(Database issue):D206–214.
Angiuoli SV, Matalka M, Gussman A, Galens K, Vangala M, Riley DR, et al. CloVR: a virtual machine for automated and portable sequence analysis from the desktop using cloud computing. BMC Bioinformatics. 2011;12:356.
Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014;30(9):1236–40.
Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.
Agren J, Sundstrom A, Hafstrom T, Segerman B. Gegenees: fragmented alignment of multiple genomes for determining phylogenomic distances and genetic signatures unique for specified target groups. PLoS One. 2012;7(6), e39107.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403–10.
Ekseth OK, Kuiper M, Mironov V. OrthAgogue: an agile tool for the rapid prediction of orthology relations. Bioinformatics. 2014;30(5):734–6.
Enright AJ, Van Dongen S, Ouzounis CA. An efficient algorithm for large-scale detection of protein families. Nucleic Acids Res. 2002;30(7):1575–84.
Darling AE, Mau B, Perna NT. ProgressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One. 2010;5(6), e11147.
Blin K, Medema MH, Kazempour D, Fischbach MA, Breitling R, Takano E, Weber T. antiSMASH 2.0--a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res. 2013;41(Web Server issue):W204-212.
Bachmann BO, Ravel J. Methods for in silico prediction of microbial polyketide and nonribosomal peptide biosynthetic pathways from DNA sequence data. Methods Enzymol. 2009;458:181–217.
Rottig M, Medema MH, Blin K, Weber T, Rausch C, Kohlbacher O: NRPSpredictor2 - a web server for predicting NRPS adenylation domain specificity. Nucleic Acids Res. 2011;39(Web Server issue):W362-367.
Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, et al. Pfam: the protein families database. Nucleic Acids Res. 2014;42(1):D222–230.
Henrissat B, Davies G. Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol. 1997;7(5):637–44.
Rawlings ND, Waller M, Barrett AJ, Bateman A. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 2014;42(1):D503–509.
Hsu SC, Lockwood JL. Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. Appl Microbiol. 1975;29(3):422–6.
Sierra G. A simple method for the detection of lipolytic activity of micro-organisms and some observations on the influence of the contact between cells and fatty substrates. Antonie Van Leeuwenhoek. 1957;23(1):15–22.
Patel N, Oudemans PV, Hillman BI, Kobayashi DY. Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica. Antonie Van Leeuwenhoek. 2013;103(6):1271–80.
Darrasse A, Carrere S, Barbe V, Boureau T, Arrieta-Ortiz M, Bonneau S, et al. Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads. BMC Genomics. 2013;14(1):761.
Loper JE, Hassan KA, Mavrodi DV, Davis EW, Lim CK, Shaffer BT, et al. Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions. Plos Genetics. 2012;8(7):e1002784.
Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 2014;42(1):D490–495.
Tsfasman IM, Sitkin BV, Lysanskaya VY, Stepnaya OA, Kulaev IS. Substrate specificity and some physicochemical properties of autolytic enzymes of the bacterium Lysobacter sp. XL 1. Biochemistry (Moscow). 2007;72(7):760–5.
Vasilyeva NV, Tsfasman IM, Kudryakova IV, Suzina NE, Shishkova NA, Kulaev IS, et al. The role of membrane vesicles in secretion of Lysobacter sp. bacteriolytic enzymes. J Mol Microbiol Biotechnol. 2013;23(1–2):142–51.
Vasilyeva NV, Tsfasman IM, Suzina NE, Stepnaya OA, Kulaev IS. Outer membrane vesicles of Lysobacter sp. Dokl Biochem Biophys. 2009;426(1):139–42.
Gokcen A, Vilcinskas A, Wiesner J. Biofilm-degrading enzymes from Lysobacter gummosus. Virulence. 2014;5(3):378–87.
Mavrodi DV, Peever TL, Mavrodi OV, Parejko JA, Raaijmakers JM, Lemanceau P, et al. Diversity and evolution of the phenazine biosynthesis pathway. Appl Environ Microbiol. 2010;76(3):866–79.
Rausch C, Weber T, Kohlbacher O, Wohlleben W, Huson DH. Specificity prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using transductive support vector machines (TSVMs). Nucleic Acids Res. 2005;33(18):5799–808.
Kobayashi DY, Reedy RM, Palumbo JD, Zhou JM, Yuen GY. A clp gene homologue belonging to the Crp gene family globally regulates lytic enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes strain C3. Appl Environ Microbiol. 2005;71(1):261–9.
Puopolo G, Raio A, Zoina A. Identification and characterization of Lysobacter capsici strain PG4: a new plant health-promoting rhizobacterium. J Plant Pathol. 2010;92:157–64.
Rasko DA, Ravel J, Okstad OA, Helgason E, Cer RZ, Jiang L, et al. The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1. Nucleic Acids Res. 2004;32(3):977–88.
Ren T, Zamboni DS, Roy CR, Dietrich WF, Vance RE. Flagellin-deficient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity. PLoS Pathog. 2006;2(3), e18.
Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature. 2001;410(6832):1099–103.
Deng Y, Wu J, Tao F, Zhang LH. Listening to a new language: DSF-based quorum sensing in Gram-negative bacteria. Chem Rev. 2011;111(1):160–73.
Han Y, Wang Y, Tombosa S, Wright S, Huffman J, Yuen G, et al. Identification of a small molecule signaling factor that regulates the biosynthesis of the antifungal polycyclic tetramate macrolactam HSAF in Lysobacter enzymogenes. Appl Microbiol Biotechnol. 2015;99(2):801–11.
He YW, Zhang LH. Quorum sensing and virulence regulation in Xanthomonas campestris. FEMS Microbiol Rev. 2008;32(5):842–57.
Qian G, Wang Y, Liu Y, Xu F, He YW, Du L, et al. Lysobacter enzymogenes uses two distinct cell-cell signaling systems for differential regulation of secondary-metabolite biosynthesis and colony morphology. Appl Environ Microbiol. 2013;79(21):6604–16.
Wang Y, Zhao Y, Zhang J, Shen Y, Su Z, Xu G, et al. Transcriptomic analysis reveals new regulatory roles of Clp signaling in secondary metabolite biosynthesis and surface motility in Lysobacter enzymogenes OH11. Appl Microbiol Biotechnol. 2014;98(21):9009–20.
Wright SJ. Regulation of secondary metabolism in Lysobacter enzymogenes: studies of intercellular and intracellular signaling. University of Nebraska: Lincoln; 2013.
Tseng T-T, Tyler B, Setubal JC. Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology. BMC Microbiol. 2009;9(1):1–9.
Wexler M, Sargent F, Jack RL, Stanley NR, Bogsch EG, Robinson C, et al. TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in sec-independent protein export. J Biol Chem. 2000;275(22):16717–22.
Ghosh P. Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev. 2004;68(4):771–95.
Linderoth NA, Simon MN, Russel M. The filamentous phage pIV multimer visualized by scanning transmission electron microscopy. Science. 1997;278(5343):1635–8.
Yu F, Zaleta-Rivera K, Zhu X, Huffman J, Millet JC, Harris SD, et al. Structure and biosynthesis of heat-stable antifungal factor (HSAF), a broad-spectrum antimycotic with a novel mode of action. Antimicrob Agents Chemother. 2007;51(1):64–72.
Lautru S, Challis GL. Substrate recognition by nonribosomal peptide synthetase multi-enzymes. Microbiology. 2004;150(Pt 6):1629–36.
Garbeva P, Silby MW, Raaijmakers JM, Levy SB, Boer W. Transcriptional and antagonistic responses of Pseudomonas fluorescens Pf0-1 to phylogenetically different bacterial competitors. ISME J. 2011;5(6):973–85.
Traxler MF, Watrous JD, Alexandrov T, Dorrestein PC, Kolter R. Interspecies interactions stimulate diversification of the Streptomyces coelicolor secreted metabolome. MBio. 2013;4(4).
Postma J, Scheper RWA, Schilder MT. Effect of successive cauliflower plantings and Rhizoctonia solani AG 2–1 inoculations on disease suppressiveness of a suppressive and a conducive soil. Soil Biol Biochem. 2010;42:804–12.
Postma J, Schilder MT, van Hoof RA. Indigenous populations of three closely related Lysobacter spp. in agricultural soils using Real-Time PCR. Microb Ecol. 2011;62(4):948–58.
Postma J, Nijhuis EH, Yassin AF. Genotypic and phenotypic variation among Lysobacter capsici strains isolated from Rhizoctonia suppressive soils. Syst Appl Microbiol. 2010;33(4):232–5.
Giesler LJ, Yuen GY. Evaluation of Stenotrophomonas maltophilia strain C3 for biocontrol of brown patch disease. Crop Protection. 1998;17:509–13.
Postma J, Schilder MT, Bloem J, van Leeuwen-Haagsma WK. Soil suppressiveness and functional diversity of the soil microflora in organic farming systems. Soil Biol Biochem. 2008;40(9):2394–406.
This manuscript is publication number 5958 of Netherlands Institute of Ecology (NIOO-KNAW).
This project was funded by the Dutch Technology Foundation (STW).
All authors declare no competing interests.
IdB carried out the comparative genomics, genome mining, enzymatic activity testing, mass spectrometry data analysis and drafted the manuscript. XC carried out the mass spectrometry analyses with help of JW and PCD. VdJ participated in the sequence assembly, annotation and pan-core genome analysis. RG participated in activity screening of the Lysobacter strains. NP and DK provided genome sequences of L. enzymogenes and L. antibioticus ATCC29479 and performed analyses on secretion systems and motility associated gene clusters. JP and JRM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Donald Kobayashi and Jos M. Raaijmakers contributed equally to this work.
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