The predatory soil bacterium Lysobacter reprograms quorum sensing system to regulate antifungal antibiotic production in a cyclic-di-GMP-independent manner.

Soil bacteria often harbour various toxins to against eukaryotic or prokaryotic. Diffusible signal factors (DSFs) represent a unique group of quorum sensing (QS) chemicals that modulate interspecies competition in bacteria that do not produce antibiotic-like molecules. However, the molecular mechanism by which DSF-mediated QS systems regulate antibiotic production for interspecies competition remains largely unknown in soil biocontrol bacteria. In this study, we find that the necessary QS system component protein RpfG from Lysobacter, in addition to being a cyclic dimeric GMP (c-di-GMP) phosphodiesterase (PDE), regulates the biosynthesis of an antifungal factor (heat-stable antifungal factor, HSAF), which does not appear to depend on the enzymatic activity. Interestingly, we show that RpfG interacts with three hybrid two-component system (HyTCS) proteins, HtsH1, HtsH2, and HtsH3, to regulate HSAF production in Lysobacter. In vitro studies show that each of these proteins interacted with RpfG, which reduced the PDE activity of RpfG. Finally, we show that the cytoplasmic proportions of these proteins depended on their phosphorylation activity and binding to the promoter controlling the genes implicated in HSAF synthesis. These findings reveal a previously uncharacterized mechanism of DSF signalling in antibiotic production in soil bacteria.

Outer Membrane Vesicle-Mediated Codelivery of the Antifungal HSAF Metabolites and Lytic Polysaccharide Monooxygenase in the Predatory Lysobacter enzymogenes.

Lysobacter are new biocontrol agents known for their prolific production of lytic enzymes and bioactive metabolites. L. enzymogenes is a predator of fungi and produces several structurally distinct antimicrobial compounds, such as the antifungal HSAF (heat stable antifungal factor) and analogs. The mechanism by which L. enzymogenes interacts with fungal prey is not well understood. Here, we found that the production of HSAF and analogs in L. enzymogenes OH11 was significantly induced in media supplemented with ground fungal mycelia or chitin. In the OH11 genome, we identified a gene (LeLPMO10A) that was annotated to encode a chitin-binding protein. The stimulation of HSAF and analogs by chitin was diminished when LeLPMO10A was deleted. We expressed the gene in E. coli and demonstrated that purified LeLPMO10A oxidatively cleaved chitin into oligomeric products, including 1,5 δ-lactones and aldonic acids. The results revealed that LeLPMO10A encodes a lytic polysaccharide monooxygenase, which has not been reported in Lysobacter. The metabolite analysis, antifungal assay, and proteomic analysis showed that the antifungal compounds and the chitin-cleaving LeLPMO10A are colocalized in outer membrane vesicles. The enzymatic products that resulted from in vitro LeLPMO10A-cleaved chitin also significantly induced HSAF and analogs in OH11. Scanning electron microscopic analysis indicated that spherical vesicles were formed outside of OH11 cells, and fewer OH11 cells were observed to attach to fungal hyphae when LeLPMO10A was deleted. Together, the study revealed a previously uncharacterized synergistic strategy utilized by the predatory Lysobacter during interaction with fungal prey.

Interspecies and Intraspecies Signals Synergistically Regulate Lysobacter enzymogenes Twitching Motility.

The twitching motility of bacteria is closely related to environmental adaptability and pathogenic behaviors. Lysobacter is a good genus in which to study twitching motility because of the complex social activities and distinct movement patterns of its members. Regardless, the mechanism that induces twitching motility is largely unknown. In this study, we found that the interspecies signal indole caused Lysobacter to have irregular, random twitching motility with significantly enhanced speed. Deletion of qseC or qseB from the two-component system for indole signaling perception resulted in the disappearance of rapid, random movements and significantly decreased twitching activity. Indole-induced, rapid, random twitching was achieved through upregulation of expression of gene cluster pilE1-pilY11-pilX1-pilW1-pilV1-fimT1 In addition, under conditions of extremely low bacterial density, individual Lysobacter cells grew and divided in a stable manner in situ without any movement. The intraspecies quorum-sensing signaling factor 13-methyltetradecanoic acid, designated L. enzymogenes diffusible signaling factor (LeDSF), was essential for Lysobacter to produce twitching motility through indirect regulation of gene clusters pilM-pilN-pilO-pilP-pilQ and pilS1-pilR-pilA-pilB-pilC These results demonstrate that the motility of Lysobacter is induced and regulated by indole and LeDSF, which reveals a novel theory for future studies of the mechanisms of bacterial twitching activities.IMPORTANCE The mechanism underlying bacterial twitching motility is an important research area because it is closely related to social and pathogenic behaviors. The mechanism mediating cell-to-cell perception of twitching motility is largely unknown. Using Lysobacter as a model, we found in this study that the interspecies signal indole caused Lysobacter to exhibit irregular, random twitching motility via activation of gene cluster pilE1-pilY11-pilX1-pilW1-pilV1-fimT1 In addition, population-dependent behavior induced by 13-methyltetradecanoic acid, a quorum-sensing signaling molecule designated LeDSF, was involved in twitching motility by indirectly regulating gene clusters pilM-pilN-pilO-pilP-pilQ and pilS1-pilR-pilA-pilB-pilC The results demonstrate that the twitching motility of Lysobacter is regulated by these two signaling molecules, offering novel clues for exploring the mechanisms of twitching motility and population-dependent behaviors of bacteria.

Effects of Lysobacter antibioticus HS124, an effective biocontrol agent against Fusarium graminearum, on crown rot disease and growth promotion of wheat.

Lysobacter antibioticus HS124 inhibited mycelial growth of Fusarium graminearum (74.66%) under the dual culture method. Microscopic investigation clearly showed that amendment with different concentrations (10%, 30%, and 50%) of HS124 bacterial culture filtrate on potato dextrose agar plates caused abnormal hyphal structures, including swelling and distortion. Its inhibition toward mycelial growth of F. graminearum was increased with increasing concentration of n-butanol crude extract of HS124. The highest inhibition (43.14%) was detected at a crude concentration of 10 mg/disc, whereas the lowest inhibition (21.57%) was observed at 2 mg/disc. Although mycelial growth of F. graminearum was promoted by volatile organic compounds (VOCs) produced by HS124 as compared with the control, these VOCs clearly decreased fungal pigmentation resulting in a reduction of fungal sporulation. Microscopic investigation revealed hyphal deformation of F. graminearum due to VOCs. These compounds also had a negative effect on spore germination of F. graminearum. In vivo evaluations demonstrated that HS124 inoculation of wheat plants reduced crown rot disease incidence by 73.70% as compared with the control. HS124 inoculation of wheat plants also promoted most of the growth characteristics compared with the control or fungicide-treated plants. Our results provide strong evidence that HS124 could control F. graminearum infections and promote growth of wheat plants as part of management strategies for crown rot disease.

Lysobacter helvus sp. nov. and Lysobacter xanthus sp. nov., isolated from Soil in South Korea.

Two bacterial strains, designated D10T and U8T, were isolated from soil samples from the Dong-angyeong cave and Geommeolle wharf sea-coast, Udo-Island, Jeju, South Korea. Both novel bacterial strains are yellow-pigmented, Gram-stain negative, motile by means of monotrichous flagella, short rod shaped and strictly aerobic. A phylogenetic tree was reconstructed based on their 16S rRNA gene sequences, which indicated that these two strains belong to the genus Lysobacter within the family Xanthomonadaceae. Strain D10T showed high 16S rRNA gene sequence similarities with Lysobacter humi FJY8T (99.0%), Lysobacter xinjiangensis RCML-52T (98.9%) and Lysobacter mobilis 9NM-14T (97.2%), whereas strain U8T showed high sequence similarities to L. mobilis 9NM-14T (97.9%), L. xinjiangensis RCML-52T (97.8%), L. humi FJY8T (97.5%) and Lysobacter bugurensis ZLD-29T (97.1%). The 16S rRNA gene sequence similarity between D10T and U8T was 97.0%. Strain D10T showed low DNA-DNA relatedness to U8T (57.7 ± 3.4%), L. humi FJY8T (48.8 ± 4.3%), L. xinjiangensis RCML-52T (60.1 ± 2.4%) and L. mobilis 9NM-14T (55.9 ± 1.9%). The level of DNA-DNA relatedness for strain U8T with respect to D10T, L. mobilis 9NM-14T, L. xinjiangensis RCML-52T, L. humi FJY8T, and L. bugurensis ZLD-29T was 55.5 ± 0.5%, 54.5 ± 2.1%, 58.1 ± 0.8%, and 51.9 ± 3.4%, respectively. The major polar lipids for both strains were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major cellular fatty acids for both strains were identified as iso-C15:0, iso-C16:0 and summed feature 9 (iso-C17:1 ω9c/C16:0 10-methyl), and ubiquinone (Q-8) as the only isoprenoid quinone for both strains. The DNA G + C contents of the strains D10T and U8T were determined to be 70.2 mol% and 70.6 mol%. On the basis of phenotypic, genotypic, chemotaxonomic, and phylogenetic analysis, both strains D10T and U8T represent a novel species in the genus Lysobacter, for which the names Lysobacter helvus sp. nov. and Lysobacter xanthus sp. nov. are proposed, respectively. The type strain of L. helvus is D10T (= KCTC 62111T = JCM 32364T) and the type strain of L. xanthus is U8T (= KCTC 62112T = JCM 32365T).

Optimization of culture conditions for promoting heat-stable antifungal factor production level in Lysobacter enzymogenes.

Heat-stable antifungal factor (HSAF), which was first isolated from Lysobacter enzymogenes, exhibits inhibitory activities against a wide range of pathogens; however, a low level of HSAF was obtained from L. enzymogenes cultured in 0.1 × tryptic soy broth (TSB), an amount that does not satisfy HSAF application in disease control. In this study, the optimization of media components and environmental conditions were examined for improving the production of HSAF from L. enzymogenes OH11. The one factor at a time method was used to screen optimal nitrogen and carbon sources and inorganic salt. Then the orthogonal matrix method was used to determine the optimal concentration of the media components and environmental factors. The results showed that the maximum level of HSAF (23361 mAU·s) was achieved when OH11 cultured in the media of 0.7% (w/v) soybean powder, 0.5% (w/v) glucose and 0.08% CaCl2 at 200 rpm at 30°C for 60 h, which is much higher than that cultured in 0.1 × TSB. This opens up the possibility of HSAF or L. enzymogenes utilization for biological control of plant disease.

Lysobacter pedocola sp. nov., a novel species isolated from Korean soil.

A Gram-negative, yellow-pigmented bacterial strain, designated IPC6T, was isolated from soil in an arid region of Goyang-si (Gyeonggi-do, South Korea). Cells were strictly aerobic, non-spore-forming, rod-shaped. The strain grew within a temperature range of 10-42°C (optimum, 30°C) and pH of 5.0-11.0 (optimum, pH 8.0) in the presence of 0-2% (w/v) NaCl. Phylogenetically, the novel strain was closely related to members of the Lysobacter genus based on 16S rRNA sequence similarity, and showed the highest sequence similarity to Lysobacter niastensis KACC 11588T (98.5%). The predominant fatty acids were iso-C15:0, iso-C16:0, and summed feature 9 (iso-C17:1ω9c), with Q-8 identified as the major ubiquinone. The polar lipid content included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unknown aminophospholipid, and an unidentified phospholipid. DNA-DNA hybridization results indicated that the strain IPC6T was distinct from Lysobacter niastensis KACC 11588T (37.9 ± 0.14%), Lysobacter panacisoli KACC 17502T (56.4 ± 0.13%), Lysobacter soli KCTC 22011T (8.1 ± 0.04%), Lysobacter gummosus KCTC 12132T (9.6 ± 0.03%), and Lysobacter cavernae KCTC 42875T (37.5 ± 0.14%), respectively. The DNA G + C content of the novel strain was 71.1 mol%. Based on the collective phenotypic, genotypic and chemotaxonomic data, the IPC6T strain is considered to represent a novel species in the genus Lysobacter, for which the name Lysobacter pedocola sp. nov. (= KCTC 42811T = JCM 31020T) is proposed.

ACC Deaminase from Lysobacter gummosus OH17 Can Promote Root Growth in Oryza sativa Nipponbare Plants.

Although Lysobacter species are a remarkable source of natural compounds with antibacterial and antifungal activities, the ability of these bacteria to produce plant growth promoters remains practically unknown. In this work, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) has been isolated from the secretions of Lysobacter gummosus OH17, indicating the presence of an ACC deaminase, which was shown to be encoded in the gene peg_1256. The recombinant enzyme could not only deaminate ACC to provide 2-oxobutanoic acid but also catalyzed the amination of the 2-oxobutanoic acid, demonstrating, for the first time, that ACC deaminases can produce ACC. After the treatment of rice Oryza sativa Nipponbare plants with OH17 ACC deaminase, the ethylene production levels were 44% higher in comparison with the control experiments, allowing significant improvements in root, 10%, and stem, 14%, growth.

Lysobacter spongiae sp. nov., isolated from spongin.

A Gram-negative, motile, aerobic and rod-shaped bacterial strain designated 119BY6-57T was isolated from spongin. The taxonomic position of the novel isolate was confirmed using the polyphasic approach. Strain 119BY6-57T grew well at 25-30°C on marine agar. On the basis of 16S rRNA gene sequence similarity, strain 119BY6-57T belongs to the family Xanthomonadaceae and is related to Lysobacter aestuarii S2-CT (99.8% sequence similarity), L. maris KMU-14T (97.5%), and L. daejeonensis GH1-9T (97.3%). Lower sequence similarities (97.0%) were found with all of the other recognized members of the genus Lysobacter. The G + C content of the genomic DNA was 69.9 mol%. The major respiratory quinone was Q-8 and the major fatty acids were C16:0 iso, C15:0 iso, summed feature 9 (comprising C17:1 iso ω9c and/or C16:0 10-methyl), summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c), and C11:0 iso 3-OH. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, three unidentified phospholipids, and an unidentified polar lipid. DNADNA relatedness values between strain 119BY6-57T and its closest phylogenetically neighbors were below 48.0 ± 2.1%. Based on genotypic and phenotypic characteristics, it is concluded that strain 119BY6-57T is a new member within the genus Lysobacter, for which the name Lysobacter spongiae sp. nov. is proposed. The type strain is 119BY6-57T (= KACC 19276T = LMG 30077T).

The impact of the omics era on the knowledge and use of Lysobacter species to control phytopathogenic micro-organisms.

Omics technologies have had a tremendous impact on underinvestigated genera of plant disease biocontrol agents such as Lysobacter. Strong evidence of the association between Lysobacter spp. and the rhizosphere has been obtained through culture-independent methods, which has also contributed towards highlighting the relationship between Lysobacter abundance and soil suppressiveness. It is conceivable that the role played by Lysobacter spp. in soil suppressiveness is related to their ability to produce an impressive array of lytic enzymes and antibiotics. Indeed, genomics has revealed that biocontrol Lysobacter strains share a vast number of genes involved in antagonism activities, and the molecular pathways underlying how Lysobacter spp. interact with the environment and other micro-organisms have been depicted through transcriptomic analysis. Furthermore, omics technologies shed light on the regulatory pathways governing cell motility and the biosynthesis of antibiotics. Overall, the results achieved so far through omics technologies confirm that the genus Lysobacter is a valuable source of novel biocontrol agents, paving the way for studies aimed at making their application in field conditions more reliable.

LesR is a novel upstream regulator that controls downstream Clp expression to modulate antibiotic HSAF biosynthesis and cell aggregation in Lysobacter enzymogenes OH11.

BACKGROUND: Heat-stable antifungal factor (HSAF) is a polycyclic tetramate macrolactam secondary metabolite that exhibits broad-spectrum inhibitory activities against filamentous fungal pathogens. The native yield of this chemical is low. It is also a great challenge to synthesize HSAF artificially, due to its complex structure. Understanding the regulatory mechanism underlying HSAF biosynthesis could provide genetic basis for engineering high HSAF-producing strain. The transcription factor Clp is a global regulator that controls bacterial pathogenicity and the expression of one hundred related genes in the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc). Diffusible signal factor (DSF) chemical signaling is the only well-characterized upstream regulatory pathway that involves downstream Clp regulation in Xcc. Such a regulatory hierarchy between DSF signaling and Clp is also conserved in the Gram-negative biological control agent Lysobacter enzymogenes, where the DSF signaling system controls antifungal antibiotic HSAF biosynthesis via Clp. RESULTS: Here, using LLysobacter enzymogenes OH11 as a working organism, we examined a novel upstream regulator, LesR, a LuxR solo that controls Clp expression to modulate HSAF biosynthesis as well as cell aggregation. We found that the overexpression of lesR in strain OH11 almost entirely shut down HSAF production and accelerated cell aggregation. These changed phenotypes could be rescued by the introduction of plasmid-borne clp in the lesR overexpression background. Consistent with findings, we further found that overexpression of lesR led to a decrease in the Clp level. CONCLUSIONS: These results collectively have shown that LesR could exert its function, i.e., HSAF biosynthesis, via downstream Clp. These findings were subsequently validated by a comparative transcriptome analysis, where the regulatory action of LesR was found to largely overlap with that of Clp. Therefore, in addition to the well-known DSF signaling system, the present study reveals that LesR functions as a new upstream regulatory factor of Clp in L. enzymogenes. The key factor was important for the production of HSAF. The strains with high HSAF yield can presumably be constructed by deletion of the negative regulators or overexpression of the positive regulators by genetic engineering.

Lysobacter zhanggongensis sp. nov. Isolated from a Pit Mud.

The Gram-stain-negative, rod-shaped and non-motile bacterial strain, designated ZGLJ7-1T, was isolated from a pit mud. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain ZGLJ7-1T was related to the genus Lysobacter and had the highest 16S rRNA gene sequence similarity with the type strain of Lysobacter arseniciresistens ZS79T (97.4%). The predominant cellular fatty acids were iso-C15:0, iso-C17:1ω9c, iso-C11:0 and iso-C11:03-OH. Strain ZGLJ7-1T had Q-8 as the predominant ubiquinone. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid, two unidentified aminolipids and two unidentified lipids. The genomic DNA G+C content of strain ZGLJ7-1T was 69.5 mol%. Strain ZGLJ7-1T shared DNA relatedness with 35% Lysobacter arseniciresistens CGMCC 1.10752T. Combined data from phenotypic, phylogenetic and DNA-DNA relatedness studies demonstrated that the strain ZGLJ7-1T is a representative of a novel species of the genus Lysobacter, for which we propose the name Lysobacter zhanggongensis sp. nov. (type strain ZGLJ7-1T = KACC 18547T = CGMCC 1.15404T).

ChpA Controls Twitching Motility and Broadly Affects Gene Expression in the Biological Control Agent Lysobacter enzymogenes.

Lysobacter enzymogenes (L. enzymogenes) is an agriculturally important Gram-negative bacterium that employs T4P (type IV pili)-driven twitching motility to exhibit its antifungal function. Yet, it is still unclear how this bacterium regulates its twitching motility. Here, by using strain OH11 as the working model organism, we showed that a hybrid two-component system ChpA acts as a positive regulator in controlling twitching motility in L. enzymogenes. ChpA is a hybrid TCS (two-component transduction system) contains 7 domains including those for auto-phosphorylation and phosphate group transfer, as well as a phosphate receiver (REC) domain. Mutation of chpA completely abolished the wild-type twitching motility, as evidenced by the absence of mobile cells at the margin of the mutant colonies. Further studies of domain-deletion and phenotypic characterization reveal that domains responsible for phosphorylation and phosphotransfer, but not the REC domain, were indispensable for ChpA in regulating twitching motility. Transcriptome analyses of the chpA knockout strain indicated that ChpA was extensively involved in controlling expression of a wide variety of genes (totaling 243). The products of these differentially expressed genes were involved in multiple physiological and biological functions in L. enzymogenes. Thus, we have not only identified a new regulator controlling twitching motility in L. enzymogenes, but also provided the first report demonstrating the broad impact of the conserved ChpA in gene regulation in Gram-negative bacteria.

Outer membrane vesicles of Lysobacter sp. XL1: biogenesis, functions, and applied prospects.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have been intensively investigated in recent times. Vesicle formation models have been proposed, some factors affecting the process were established, and important roles vesicles play in vital activities of their producing cells were determined. Studies of pathogenic bacterial vesicles contribute to understanding the causes of acute infection and developing drugs on their basis. Despite intensive research, issues associated with the understanding of vesicle biogenesis, the mechanisms of bacterium-bacterium and pathogen-host interactions with participation of vesicles, still remain unresolved. This review discusses some results obtained in the research into OMVs of Lysobacter sp. XL1 VKM B-1576. This bacterium secretes into the environment a spectrum of bacteriolytic enzymes that hydrolyze peptidoglycan of competing bacteria, thus leading to their lysis. One of these enzymes, lytic endopeptidase L5, has been shown not only to be secreted by means of vesicles but also to be involved in their formation. As part of vesicles, the antimicrobial potential of L5 enzyme has been found to be considerably expanded. Vesicles have been shown to have a therapeutic effect in respect of anthrax infection and staphylococcal sepsis modelled in mice. The scientific basis for constructing liposomal antimicrobial preparations from vesicle phospholipids and recombinant bacteriolytic enzyme L5 has been formed.

Polyphasic Characterization of Lysobacter maris sp. nov., a Bacterium Isolated from Seawater.

A strictly aerobic, Gram-negative, apricot-pigmented, non-motile, rod-shaped strain designated KMU-14(T) was isolated from seawater collected from the coastal zone of Yokji Island, Gyeongsangnam-do, Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the novel isolate was affiliated with the genus Lysobacter within the class Gammaproteobacteria and that it showed the highest sequence similarity (97.1 %) to Lysobacter concretionis Ko07(T). The hybridization value for DNA-DNA relatedness between the strains of KMU-14(T) and L. concretionis Ko07(T) was 34.8 %, which was lower than 70 %, the recommended delineation value for differentiation of species. The DNA G+C content of strain KMU-14(T) was 64.9 mol%. The major respiratory quinone was ubiquinone 8 (Q-8), and iso-C15:0, iso-C16:0, and 10-methyl C16:0 and/or iso-C17:1 ω9c were the major (>10 %) cellular fatty acids. A polar lipid profile was present consisting of diphosphatidylglycerol, phosphatidylethanolamine, an unidentified phosphoglycolipid, two unidentified aminophospholipids, and two unidentified phospholipids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel species for which the name Lysobacter maris sp. nov. is proposed. The type strain of L. maris sp. nov. is KMU-14(T) (=KCTC 42381(T) =NBRC 110750(T)).

Lysobacter chengduensis sp. nov. Isolated from the Air of Captive Ailuropoda melanoleuca Enclosures in Chengdu, China.

A novel bacterial strain, designated as CF21(T), was isolated from the air of Ailuropoda melanoleuca enclosures in China. Cells were gram-negative, aerobic, non-motile, and rod shaped. Strain CF21(T) grew at 10-40 °C (optimum 28-30 °C) and pH 6.0-9.0 (optimum pH 7.0-8.0) and in the presence of NaCl concentrations ranging from 0.0% (w/v) to 2.0 % (optimum 0.0-1.0%). 16SrRNA gene sequence analysis indicated that strain CF21(T) belonged to genus Lysobacter within class Gammaproteobacteria and was most closely related to Luteimonas dalianensi OB44-3(T) (95.8% similarity), Lysobacter ruishenii CTN-1(T) (95.1%), Lysobacter spongiicola KMM329(T) (94.8 %), and Lysobacter daejeonensis GH1-9T (94.6%). The genomic G+C DNA content was 68.72 mol%. Major cellular fatty acids of CF21(T) were iso-C16:0 (30.22%), iso-C15:0 (25.70%), and the sum of 10-methyl C16 : 0 and/or iso-C17 : 1ω9c (21.94%). The prominent isoprenoid quinone was ubiquinone 8 (Q-8). Primary polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unknown phospholipid. DNA sequence relatedness between strain CF21(T) and L. ruishenii CTN-1(T) was 56%, which was clearly below the 70% threshold for prokaryotic species delineation. These analyses indicated that CF21(T) is a novel member of genus Lysobacter, for which the name Lysobacter chengduensis sp. nov. is proposed. The type strain is CF21(T) (=CGMCC1.15145(T) = DSM 100306(T)).

Monitoring Lysobacter capsici AZ78 using strain specific qPCR reveals the importance of the formulation for its survival in vineyards.

Survival in the phyllosphere is a critical feature for biofungicides based on non-spore forming bacteria. Moreover, knowledge of their persistence on plants is important to design effective formulations and application techniques. With this scope, the aim of this work was to develop a specific method to monitor the fate in the environment of Lysobacter capsici AZ78, a biocontrol agent of Plasmopara viticola, and to evaluate the contribution of formulation in its persistence on grapevine leaves. A strain-specific primer pair derived from REP-PCR fingerprinting was used in quantitative PCR experiments to track the evolution of L. capsici AZ78 population in vineyards. The population reached between 5 and 6 log10 cells gram of leaf(-1) after application and decreased by more than 100 times in one week. Multiple regression analysis showed that unfavourable temperature was the main environmental factor correlating with the decrease of L. capsici AZ78 persistence on grapevine leaves. Importantly, the use of formulation additives protected L. capsici AZ78 against environmental factors and improved its persistence on the leaves by more than 10 times compared to nude cells. Formulation and the knowledge about the persistence of L. capsici AZ78 in vineyards will be useful to develop commercial biofungicides for foliar application.

Comparative genomics and metabolic profiling of the genus Lysobacter.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Stepwise flow diagram for the development of formulations of non spore-forming bacteria against foliar pathogens: The case of Lysobacter capsici AZ78.

The formulation is a significant step in biopesticide development and is an efficient way to obtain consistency in terms of biological control under field conditions. Nonetheless, there is still a lack of information regarding the processes needed to achieve efficient formulation of non spore-forming bacterial biological control agents. In response to this, we propose a flow diagram made up of six steps including selection of growth parameters, checking of minimum shelf life, selection of protective additives, checking that the additives have no adverse effects, validation of the additive mix under field conditions and choosing whether to use additives as co-formulants or tank mix additives. This diagram is intended to provide guidance and decision-making criteria for the formulation of non spore-forming bacterial biological control agents against foliar pathogens. The diagram was then validated by designing an efficient formulation for a Gram-negative bacterium, Lysobacter capsici AZ78, to control grapevine downy mildew caused by Plasmopara viticola. A harvest of 10(10)L. capsici AZ78cellsml(-1) was obtained in a bench top fermenter. The viability of cells decreased by only one order of magnitude after one year of storage at 4°C. The use of a combination of corn steep liquor, lignosulfonate, and polyethyleneglycol in the formulation improved the survival of L. capsici AZ78 cells living on grapevine leaves under field conditions by one order of magnitude. Furthermore, the use of these additives also guaranteed a reduction of 71% in P. viticola attacks. In conclusion, this work presents a straightforward stepwise flow diagram to help researchers develop formulations for biological control agents that are easy to prepare, stable, not phytotoxic and able to protect the microorganims under field conditions.

PilG is Involved in the Regulation of Twitching Motility and Antifungal Antibiotic Biosynthesis in the Biological Control Agent Lysobacter enzymogenes.

Lysobacter enzymogenes strain C3 is a gliding bacterium which produces the antifungal secondary metabolite heat-stable antifungal factor (HSAF) and type IV pilus (T4P) as important mechanisms in biological control activity against fungal pathogens. To date, the regulators that control HSAF biosynthesis and T4P-dependent twitching motility in L. enzymogenes are poorly explored. In the present study, we addressed the role of pilG in the regulation of these two traits in L. enzymogenes. PilG of L. enzymogenes was found to be a response regulator, commonly known as a component of a two-component transduction system. Mutation of pilG in strain C3 abolished its ability to display spreading colony phenotype and cell movement at the colony margin, which is indicative of twitching motility; hence, PilG positively regulates twitching motility in L. enzymogenes. Mutation of pilG also enhanced HSAF production and the transcription of its key biosynthetic gene hsaf pks/nrps, suggesting that PilG plays a negative regulatory role in HSAF biosynthesis. This finding represents the first demonstration of the regulator PilG having a role in secondary metabolite biosynthesis in bacteria. Collectively, our results suggest that key ecological functions (HSAF production and twitching motility) in L. enzymogenes strain C3 are regulated in opposite directions by the same regulatory protein, PilG.