Critical analysis of polycyclic tetramate macrolactam biosynthetic gene cluster phylogeny and functional diversity.

Polycyclic tetramate macrolactams (PTMs) are bioactive natural products commonly associated with certain actinobacterial and proteobacterial lineages. These molecules have been the subject of numerous structure-activity investigations since the 1970s. New members continue to be pursued in wild and engineered bacterial strains, and advances in PTM biosynthesis suggest their outwardly simplistic biosynthetic gene clusters (BGCs) belie unexpected product complexity. To address the origins of this complexity and understand its influence on PTM discovery, we engaged in a combination of bioinformatics to systematically classify PTM BGCs and PTM-targeted metabolomics to compare the products of select BGC types. By comparing groups of producers and BGC mutants, we exposed knowledge gaps that complicate bioinformatics-driven product predictions. In sum, we provide new insights into the evolution of PTM BGCs while systematically accounting for the PTMs discovered thus far. The combined computational and metabologenomic findings presented here should prove useful for guiding future discovery.IMPORTANCEPolycyclic tetramate macrolactam (PTM) pathways are frequently found within the genomes of biotechnologically important bacteria, including Streptomyces and Lysobacter spp. Their molecular products are typically bioactive, having substantial agricultural and therapeutic interest. Leveraging bacterial genomics for the discovery of new related molecules is thus desirable, but drawing accurate structural predictions from bioinformatics alone remains challenging. This difficulty stems from a combination of previously underappreciated biosynthetic complexity and remaining knowledge gaps, compounded by a stream of yet-uncharacterized PTM biosynthetic loci gleaned from recently sequenced bacterial genomes. We engaged in the following study to create a useful framework for cataloging historic PTM clusters, identifying new cluster variations, and tracing evolutionary paths for these molecules. Our data suggest new PTM chemistry remains discoverable in nature. However, our metabolomic and mutational analyses emphasize the practical limitations of genomics-based discovery by exposing hidden complexity.

Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

The posttranscriptional regulator CsrA affects multidrug resistance and biocontrol activity in Lysobacter enzymogenes.

AIMS: The posttranscriptional regulator CsrA regulates many cellular processes, including stress responses in diverse bacteria. However, the role of CsrA in multidrug resistance (MDR) and biocontrol activity in Lysobacter enzymogenes strain C3 (LeC3) remains unknown. METHODS AND RESULTS: In this study, we demonstrated that deletion of the csrA gene resulted in the initial slow growth of LeC3 and reduced its resistance to multiple antibiotics, including nalidixic acid (NAL), rifampicin (RIF), kanamycin (Km), and nitrofurantoin (NIT). Loss of the csrA gene also reduced its ability in inhibiting hypha growth of Sclerotium sclerotiorum and influenced its extracellular cellulase and protease activities. Two putative small noncoding regulatory RNAs (sRNAs), referred to as csrB and csrC, were also revealed in the genome of LeC3. Double deletion of csrB and csrC in LeC3 led to increased resistance to NAL, RIF, Km, and NIT. However, no difference was observed between LeC3 and the csrB/csrC double mutant in their suppression of S. sclerotiorum hypha growth and production of extracellular enzymes. CONCLUSION: These results suggest that CsrA in LeC3 not only conferred its intrinsic MDR, but also contributed to its biocontrol activity.

The Transcription Regulator GntR/HutC Regulates Biofilm Formation, Motility and Stress Tolerance in Lysobacter capsici X2-3.

Lysobacter capsici X2-3, a plant growth-promoting rhizobacteria (PGPR), was isolated from wheat rhizosphere and has inhibitory effects against a wide range of pathogens. One important characteristic of L. capsici is its ability to produce diverse antibiotics and lytic enzymes. The GntR family of transcription factors is a common transcription factor superfamily in bacteria that has fundamental roles in bacterial metabolism regulation. However, the GntR family transcription factor in Lysobacter has not been identified. In this study, to obtain an understanding of the GntR/HutC gene function in L. capsici X2-3, a random Tn5-insertion mutant library of X2-3 was constructed to select genes showing pleiotropic effects on phenotype. We identified a Tn5 mutant with an insertion in LC4356 that showed reduced biofilm levels, and sequence analysis indicated that the inserted gene encodes a GntR/HutC family transcription regulator. Furthermore, the LC4356 mutant showed reduced extracellular polysaccharide (EPS) production, diminished twitching motility and decreased survival under UV radiation and high-temperature. The RT‒qPCR results indicated that the pentose phosphate pathway-related genes G6PDH, 6PGL and PGDH were upregulated in the LC4356 mutant. Thus, since L. capsici is an efficient biocontrol agent for crop protection, our findings provide fundamental insights into GntR/HutC and will be worthwhile to improve PGPR biocontrol efficacy.

Biosynthesis, regulation, and engineering of natural products from Lysobacter.

Covering: up to August 2021Lysobacter is a genus of Gram-negative bacteria that was classified in 1987. Several Lysobacter species are emerging as new biocontrol agents for crop protection in agriculture. Lysobacter are prolific producers of new bioactive natural products that are largely underexplored. So far, several classes of structurally interesting and biologically active natural products have been isolated from Lysobacter. This article reviews the progress in Lysobacter natural product research over the past ten years, including molecular mechanisms for biosynthesis, regulation and mode of action, genome mining of cryptic biosynthetic gene clusters, and metabolic engineering using synthetic biology tools.

An intrinsic mechanism for coordinated production of the contact-dependent and contact-independent weapon systems in a soil bacterium.

Soil bacteria possess multiple weapons to fend off microbial competitors. Currently, we poorly understand the factors guiding bacterial decisions about weapon systems deployment. In this study, we investigated how such decisions are made by the soil bacterium Lysobacter enzymogenes, used in antifungal plant protection. We found that weapons production is guided by environmental cues. In rich media, which likely mimic environments crowded with other microbes, L. enzymogenes produces a contact-dependent weapon, type six secretion system (T6SS). In nutrient-poor media, likely dominated by filamentous oomycetes and fungi, L. enzymogenes synthesizes and secretes a heat-stable antifungal factor (HSAF), a contact-independent weapon. Surprisingly, the T6SS inner tube protein Hcp is accumulated intracellularly even in nutrient-poor media, when the T6SS is not assembled. We found that Hcp interacts with the transcription factor Clp required for activating HSAF biosynthesis operon expression. Hcp protects Clp from binding to c-di-GMP, an intracellular second messenger inhibiting DNA binding. The increased concentration of c-di-GMP-free Clp thus leads to higher gene expression and HSAF production. Therefore, when the contact-dependent weapon, T6SS, is not in use, accumulation of one of its structural components, Hcp, serves as a signal to enhance production of the contact-independent weapon, HSAF. The uncovered environment-dependent and auto-regulatory mechanisms shed light on the processes governing deployment of various weapon systems in environmental bacteria.

Enhancing proteolytic activity of Lysobacter enzymogenes using cold atmospheric plasma.

Cold atmospheric plasma (CAP) is being used recently as a modern technique for microbial random mutagenesis. In the present study, CAP was used to induce mutagenesis in L. enzymogenes which is the bacteria known for producing proteolytic enzymes especially lysyl endopeptidase (Lys C). Enhanced proteolytic activity was the main criteria to select mutant strains. Therefore, the cell suspension of L. enzymogenes strain (ATCC 29487), was exposed to CAP for 30, 45, 90, and 150 s. The proteolytic activity of mutant strains was screened initially by radial caseinolytic assay and then by Ansons method in different phases of bacterial growth in the selected mutants. The purification process of Lysyl endopeptidase as the target enzyme was optimized and for enlightening molecular aspect of CAP mutagenesis, the sequences of the upstream and coding regions of lys C gene from 10 selected mutant strains were determined. The bacterial survival assessment showed that the more CAP treatment time, the less survival rate, however, in all exposure times, a number of survived mutants showed enhanced proteolytic activity. Among 38 out of 100 examined mutants which showed higher proteolytic activity than that of wild type, the M1-30 s mutant exhibited the highest increment to 1.94 fold. The SDS-PAGE analysis showed expected size of purified Lys C from M1-30 s. The Lys C gene from M14-150 s mutant strain (1.4-fold increment) harbored three point mutations which can be effective in enhancing protease activity. In conclusion, the results highlighted the role of CAP for strain improvement process to obtain industrial strains.

Antifungal weapons of Lysobacter, a mighty biocontrol agent.

Bacteria interact with fungi in a variety of ways to inhibit fungal growth, while the underlying mechanisms remain only partially characterized. The plant-beneficial Bacillus and Pseudomonas species are well-known antifungal biocontrol agents, whereas Lysobacter are far less studied. Members of Lysobacter are easy to grow in fermenters and are safe to humans, animals and plants. These environmentally ubiquitous bacteria use a diverse arsenal of weapons to prey on other microorganisms, including fungi and oomycetes. The small molecular toxins secreted by Lysobacter represent long-range weapons effective against filamentous fungi. The secreted hydrolytic enzymes act as intermediate-range weapons against non-filamentous fungi. The contact-dependent killing devices are proposed to work as short-range weapons. We describe here the structure, biosynthetic pathway, action mode and applications of one of the best-characterized long-range weapons, the heat-stable antifungal factor (HSAF) produced by Lysobacter enzymogenes. We discuss how the flagellar type III secretion system has evolved into an enzyme secretion machine for the intermediate-range antifungal weapons. We highlight an intricate mechanism coordinating the production of the long-range weapon, HSAF and the proposed contact-dependent killing device, type VI secretion system. We also overview the regulatory mechanisms of HSAF production involving specific transcription factors and the bacterial second messenger c-di-GMP.

A non-flagellated, predatory soil bacterium reprograms a chemosensory system to control antifungal antibiotic production via cyclic di-GMP signalling.

Lysobacter enzymogenes is a non-flagellated, soil proteobacterium that secretes a diffusible antibiotic known as heat-stable antifungal factor (HSAF) to kill nearby fungi for food. The genome of the model strain OH11 encodes a homologous Wsp system, which is generally deployed by flagellated bacteria to achieve flagella-dependent outputs via a c-di-GMP-FleQ complex, in which c-di-GMP is a ubiquitous dinucleotide second messenger and FleQ is a transcription factor (TF). Here, we show that the Wsp system in the non-flagellated OH11 participates in a unique c-di-GMP-dependent signalling pathway and forms a WspR-CdgL binary complex to alter HSAF production, in which WspR and CdgL act as a c-di-GMP diguanylate cyclase (DGC) and a non-TF binding protein respectively. We found that the phosphorylation of WspR activates its DGC activity and enhances c-di-GMP production while inhibiting HSAF biosynthesis. The phosphorylation of WspR also plays a key role in weakening WspR-CdgL binding and HSAF generation. Interestingly, c-di-GMP binding to CdgL did not seem to induce the disassociation of the WspR-CdgL complex. These observations, along with our earlier findings, lead us to propose a model in which L. enzymogenes re-programs the Wsp system via c-di-GMP signalling to regulate HSAF biosynthesis for the benefit of ecological adaptation.

An Antifungal Polycyclic Tetramate Macrolactam, Heat-Stable Antifungal Factor (HSAF), Is a Novel Oxidative Stress Modulator in Lysobacter enzymogenes.

Polycyclic tetramate macrolactams (PoTeMs) are a fast-growing family of antibiotic natural products found in phylogenetically diverse microorganisms. Surprisingly, none of the PoTeMs have been investigated for potential physiological functions in their producers. Here, we used heat-stable antifungal factor (HSAF), an antifungal PoTeM from Lysobacter enzymogenes, as a model to show that PoTeMs form complexes with iron ions, with an association constant (Ka ) of 2.71 × 106 M-1 The in vivo and in vitro data showed formation of 2:1 and 3:1 complexes between HSAF and iron ions, which were confirmed by molecular mechanical and quantum mechanical calculations. HSAF protected DNA from degradation in high concentrations of iron and H2O2 or under UV radiation. HSAF mutants of L. enzymogenes barely survived under oxidative stress and exhibited markedly increased production of reactive oxygen species (ROS). Exogenous addition of HSAF into the mutants significantly prevented ROS production and restored normal growth in the mutants under the oxidative stress. The results reveal that the function of HSAF is to protect the producer microorganism from oxidative damage rather than as an iron-acquisition siderophore. The characteristic structure of PoTeMs, a 2,4-pyrrolidinedione-embedded macrolactam, may represent a new iron-chelating scaffold of microbial metabolites. The study demonstrated a previously unrecognized strategy for microorganisms to modulate oxidative damage to the cells.IMPORTANCE PoTeMs are a family of structurally distinct metabolites that have been found in a large number of bacteria. Although PoTeMs exhibit diverse therapeutic properties, the physiological function of PoTeMs in the producer microorganisms had not been investigated. HSAF from Lysobacter enzymogenes is an antifungal PoTeM that has been subjected to extensive studies for mechanisms of biosynthesis, regulation, and antifungal activity. Using HSAF as a model system, we here showed that the characteristic structure of PoTeMs, a 2,4-pyrrolidinedione-embedded macrolactam, may represent a new iron-chelating scaffold of microbial metabolites. In L. enzymogenes, HSAF functions as a small-molecule modulator for oxidative damage caused by iron, H2O2, and UV light. Together, the study demonstrated a previously unrecognized strategy for microorganisms to modulate oxidative damage to the cells. HSAF represents the first member of the fast-growing PoTeM family of microbial metabolites whose potential biological function has been studied.

Effect of culture medium optimization on the secondary metabolites activity of Lysobacter antibioticus 13-6.

Fermentation products of Lysobacter antibioticus 13-6 have antagonistic activity against devastating phytopathogenic bacerium Xanthomonas oryzae pv. oryzicola. The production of Lysobacter antibioticus 13-6 secondary metabolites was increased by optimizing the fermentation medium; using a single-factor screening test, Plackett-Burman Design, and Box-Behnken Design. The medium's final formulation for active secondary metabolites high-yield included peptone 5 g/L, glucose 4.73 g/L, MgSO4·7H2O 2.33 g/L, and K2HPO4 2.21 g/L. We compared phenazine-1-carboxylic acid (PCA) contents of L. antibioticus 13-6 in the initial and optimized mediums through HPLC. It was found PCA contents of the optimized medium are two folds more than in the initial medium. We also detected the relative expression of five phenazine genes of L. antibioticus 13-6 via RT-qPCR, and it was found that genes: phzB, C, S, and NO1 have more significant expression compared with the initial medium, while gene phzD has found just significant. Further, we revealed that the optimal fermentation conditions for secondary metabolites were: fermentation time 60 hours, shaking speed 160 rpm, inoculum size 3%, and the initial pH = 7.0. In the end, it was determined that the antimicrobial activity and quality of L. antibioticus 13-6 secondary metabolites were increased by about 41.75% and 2-times, respectively, after the optimization of the fermentation medium.

Two Functional Fatty Acyl Coenzyme A Ligases Affect Free Fatty Acid Metabolism To Block Biosynthesis of an Antifungal Antibiotic in Lysobacter enzymogenes.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia colifadD mutant JW1794. RpfB activates long-chain fatty acids (n-C16:0 and n-C18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2 In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenesIMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

Lysobacter terrigena sp. nov., isolated from a Korean soil sample.

A bacterial strain isolated from a soil collected in Jeju Island, designated as 17J7-1T, was Gram-negative, rod-shaped, yellow colored, and motile by gliding. This strain was able to grow at temperature range from 10 to 42 °C, pH 7-9, and tolerated up to 1% NaCl. Analysis of 16S rRNA sequence identified strain 17J7-1T as a member of the genus Lysobacter with close sequence similarity with Lysobacter mobilis 9NM-14T (97.4%), Lysobacter xinjiangensis RCML-52T (97.0%), and Lysobacter humi FJY8T (96.9%). The genomic DNA G + C content of the isolate was 67.9 mol%. DNA-DNA relatedness between strain 17J7-1T and L. mobilis, L. humi, and L. xinjiangensis were 42.3%, 39.5%, and 35.8%, respectively, clearly showing that the isolate is distinct from its closest phylogenetic neighbors in the genus Lysobacter. Average nucleotide identity (ANI) and digital DNA-DNAhybridization (dDDH) values between strain 17J7-1T and L. enzymogenes ATCC 29487T, the type species of this genus, and several other close Lysobacter species were less than 77% and 22%, respectively. Major fatty acids were C16:0 iso (29.8%), summed feature 9 (C17:1 iso ω9c/C16:0 10-methyl; 20.1%), and C15:0 iso (17.7%). The predominant respiratory quinone was ubiquinone Q-8 and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. In the light of the polyphasic evidence accumulated in this study, strain 17J7-1T is considered to represent a novel species in the genus Lysobacter, for which name Lysobacter terrigena sp. nov. is proposed. The type strain is 17J7-1T (= KCTC 62217T = JCM 33057T).

Lysobacter enzymogenes LE16 autolysates have potential as biocontrol agents-Lysobacter sp. autolysates as biofungicide.

AIMS: Biological techniques can manage plant diseases safely and in environmentally friendly ways, but their efficacy needs improvement. It is of the utmost importance to search for powerful microbes for the effective control of plant diseases. METHODS AND RESULTS: Unheated self-digestive solutions (SDS) that were heated at 100°C for 30 min(H-SDS) or stored for 12 months at room temperature (S-SDS) were prepared from Lysobacter enzymogenes LE16 broth culture to study their potential as biocontrol agents. This bacterium produced protease, phosphatase, lysozyme and siderophores in pure culture as well as 12 secondary metabolites including novel antibiotics lysobactin, WAP-8294A2 and mupirocin determined based on the antiSMASH 5.0.0 blast database. A poison plate assay revealed the antagonistic activities of SDS, H-SDS and S-SDS against an animal pathogenic bacterium Staphylococcus aureus, a phytopathogenic bacterium Pseudomonas syringae pv. tabaci, and numerous plant pathogenic fungi and oomycetes, including Colletotrichum gloeosporioides, Penicillium italicum, Alternaria alternate, Rhizoctonia solani, Didymella bryoniae, Sclerotinia sclerotiorum, Phytophthora nicotianae and Phytophthora capsici. The greenhouse experiment showed that SDS was highly effective in controlling pepper blight disease, which is caused by P. capsici. Compared with only pathogen inoculation, the application of SDS to the soil in preventive or curative treatments significantly reduced the disease incidence and index with relatively high control efficacy of 86·2-93·1%. CONCLUSIONS: SDS enriched lytic enzymes, siderophores and antibiotics, has a wide antimicrobial spectrum, and shows potential as a new, safe and effective biocontrol agent against plant diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: Autolysates of the new biocontrol bacterium L. enzymogenes LE16 demonstrated the potential for industrial production and commercial use as a promising biocontrol agent in agriculture.

Lysobacter oculi sp. nov., isolated from human Meibomian gland secretions.

A Gram-stain negative, rod-shaped bacterial, catalase and oxidase positive strain (83-4T) that formed yellow colonies was isolated from human Meibomian gland secretions. Strain 83-4T belongs to the genus Lysobacter according to phylogenetic analysis based on 16S rRNA gene sequences. The DNA G+C content was 67.1 mol%. The circular genome was 2.6 Mb, which contained 2431 protein-coding sequences, 75 pseudogenes, 46 tRNAs, 3 rRNAs and 4 ncRNAs. A bacteriocin cluster and aryl polyene cluster were also found in the genome. The average nucleotide identity value was 79.6% between isolate 83-4T and the closely related type strain Lysobacter tolerans UM1T. The estimated DNA-DNA hybridization value between strain 83-4T and L. tolerans UM1T was 41.6%. Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol were the major polar lipids. Iso-C15:0, iso-C11:0 3-OH, iso-C11:0 and summed feature 9 (iso-C17:1ω9c) were the major fatty acids. Ubiquinone (Q-8) was the only respiratory quinone. Therefore, based on the data of phylogenetic analysis, chemotaxonomical and biochemical analyses, it is concluded that strain 83-4T represents a novel species of the genus Lysobacter with the name of Lysobacter oculi sp. nov. The type strain is 83-4T (= CGMCC 1.13464T = NRBC 113451T).

Lysobacter segetis sp. nov., Isolated from Soil.

A Gram-negative, aerobic, motile by gliding, rod-shaped bacterium, strain 17J68-2T, was isolated from a soil sample taken from Jeju Island, Republic of Korea. The isolate displayed high 16S rRNA gene sequence similarity to the members of the genus Lysobacter in the family Lysobacteraceae, with Lysobacter humi FJY8T (98.4% similarity), Lysobacter xinjiangensis RCML-52T (98.3%), and Lysobacter mobilis 9NM-14T (98.1%) as closest phylogenetic neighbors. Growth of strain 17J68-2T occurred at 15-42 °C, pH 7-8, and in the presence of 0-1.0% NaCl. Draft genome was 2.94 Mb in size with G+C content of 70.5 mol%. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine. Ubiquinone Q-8 was the predominant respiratory quinone and the major fatty acids were C16:0 iso (39.4%), summed feature 3 (C16:1ω7c/C16:1ω6c) (6.6%), C11:0 iso 3-OH (6.4%), C15:0 iso (6.4%), and C16:1 iso H (6.2%). The DNA-DNA relatedness between strain 17J68-2T and L. humi, L. xinjiangensis, and L. mobilis were 39.9, 39.4, and 25.3%, respectively. From these results, it is concluded that the novel isolate possesses sufficient characteristics to differentiate it from the most closely affiliated Lysobacter species, and strain 17J68-2T represents a novel species of the genus Lysobacter, for which the name Lysobacter segetis sp. nov. (=KCTC 62237T = JCM 33058T) is proposed.

Knockout of Diguanylate Cyclase Genes in Lysobacter enzymogenes to Improve Production of Antifungal Factor and Increase Its Application in Seed Coating.

Heat-stable antifungal factor (HSAF) is a broad-spectrum antifungal antibiotic produced by the biological control agent, Lysobacter enzymogenes. In our earlier works, we have applied HSAF to effectively control wheat and pear fungal disease. However, a major bottleneck in its practical application is the low HSAF production level; therefore, boosting its production is essential for its wide application. In the past, we find that c-di-GMP, a universal bacterial second messenger, is inhibitory to HSAF production. In this work, we further identified eight active diguanylate cyclases (DGCs) responsible for c-di-GMP synthesis in Lysobacter enzymogenes via both bioinformatics and genetic analyses. We generated a strain lacking seven active DGC genes and found that this DGC-modified strain, OH11LC, produced a higher HSAF amount in a c-di-GMP concentration-dependent manner. Subsequently, by employing OH11LC as the host fermentation strain, we could even produce a much higher HSAF amount (> 200-fold). After improving the HSAF production, we further developed a technique of seed coating method with HSAF, which turned out to be effective in fighting against the maize seed-borne filamentous pathogen, Pythium gramineacola. Overall, via combining strain modification and fermentation optimization, we demonstrated a good example of translating fundamental knowledge of bacterial c-di-GMP signaling into biological control application in which we relieved the inhibitory effect of c-di-GMP on HSAF biosynthesis by deleting a bunch of potentially active L. enzymogenes DGC genes to improve HSAF yield and to expand its usage in antifungal seed coating.

Signaling specificity in the c-di-GMP-dependent network regulating antibiotic synthesis in Lysobacter.

Enzymes controlling intracellular second messengers in bacteria, such as c-di-GMP, often affect some but not other targets. How such specificity is achieved is understood only partially. Here, we present a novel mechanism that enables specific c-di-GMP-dependent inhibition of the antifungal antibiotic production. Expression of the biosynthesis operon for Heat-Stable Antifungal Factor, HSAF, in Lysobacter enzymogenes occurs when the transcription activator Clp binds to two upstream sites. At high c-di-GMP levels, Clp binding to the lower-affinity site is compromised, which is sufficient to decrease gene expression. We identified a weak c-di-GMP phosphodiesterase, LchP, that plays a disproportionately high role in HSAF synthesis due to its ability to bind Clp. Further, Clp binding stimulates phosphodiesterase activity of LchP. An observation of a signaling complex formed by a c-di-GMP phosphodiesterase and a c-di-GMP-binding transcription factor lends support to the emerging paradigm that such signaling complexes are common in bacteria, and that bacteria and eukaryotes employ similar solutions to the specificity problem in second messenger-based signaling systems.

LeTetR Positively Regulates 3-Hydroxylation of the Antifungal HSAF and Its Analogs in Lysobacter enzymogenes OH11.

The biocontrol agent Lysobacter enzymogenes OH11 produces several structurally distinct antibiotic compounds, including the antifungal HSAF (Heat Stable Antifungal Factor) and alteramides, along with their 3-dehydroxyl precursors (3-deOH). We previously showed that the 3-hydroxylation is the final step of the biosynthesis and is also a key structural moiety for the antifungal activity. However, the procedure through which OH11 regulates the 3-hydroxylation is still not clear. In OH11, the gene orf3232 was predicted to encode a TetR regulator (LeTetR) with unknown function. Here, we deleted orf3232 and found that the LeTetR mutant produced very little HSAF and alteramides, while the 3-deOH compounds were not significantly affected. The production of HSAF and alteramides was restored in orf3232-complemented mutant. qRT-PCR showed that the deletion of orf3232 impaired the transcription of a putative fatty acid hydroxylase gene, orf2195, but did not directly affect the expression of the HSAF biosynthetic gene cluster (hsaf). When an enzyme extract from E. coli expressing the fatty acid hydroxylase gene, hsaf-orf7, was added to the LeTetR mutant, the production of HSAF and alteramides increased by 13-14 fold. This study revealed a rare function of the TetR family regulator, which positively controls the final step of the antifungal biosynthesis and thus controls the antifungal activity of the biocontrol agent.

A novel heterologous expression strategy for the quorum-quenching enzyme MomL in Lysobacter enzymogenes to the inhibit pathogenicity of Pectobacterium.

Quorum-quenching (QQ) enzymes can block the quorum-sensing (QS) system and prevent the expression of QS-controlled pathogenic factors in bacteria. However, the low expression levels of QQ proteins in the original host bacteria have affected their widespread application. In this study, we heterologously expressed momL, encoding a QQ enzyme with high activity, in Lysobacter enzymogenes. A "yellow-to-white" selection marker and the high-constitutive-expression promoter PgroEL were used in this novel heterologous expression system. In addition, we optimized the spacer between the SD sequence and the initiator to improve the efficiency of the expression system by 1.54-fold. The engineered strain LeMomL degraded the AHL molecule and the virulence factors of Pectobacterium carotovorum subsp. carotovora (Pcc). Additionally, LeMomL significantly decreased the disease caused by Pcc in Chinese cabbages and carrot root tissues. In conclusion, this novel and facile L. enzymogenes expression strategy has good prospects and is an ideal approach for foreign protein expression.