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Ochratoxin A (OTA) is a toxic secondary metabolite that widely contaminates agro-products and poses a significant dietary risk to human health. Previously, a carboxypeptidase CP4 was characterized for OTA degradation in Lysobacter sp. CW239, but the degradation activity was much lower than its host strain CW239. In this study, an amidohydrolase ADH2 was screened for OTA hydrolysis in this strain. The result showed that 50 μg/L OTA was completely degraded by 1.0 μg/mL rADH2 within 5 min, indicating ultra-efficient activity. Meanwhile, the two hydrolases (i.e., CP4 and ADH2) in the strain CW239 showed the same degradation manner, which transformed the OTA to ochratoxin α (OTα) and l-β-phenylalanine. Gene mutants (Δcp4, Δadh2 and Δcp4-adh2) testing result showed that OTA was co-degraded by carboxypeptidase CP4 and amidohydrolase ADH2, and the two hydrolases are sole agents in strain CW239 for OTA degradation. Hereinto, the ADH2 was the overwhelming efficient hydrolase, and the two types of hydrolases co-degraded OTA in CW239 by synergistic effect. The results of this study are highly significant to ochratoxin A contamination control during agro-products production and postharvest., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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. IMPORTANCE Polycyclic 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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

Two Gram-negative bacteria, designated as strains LF1 T and HM2-2 T , were isolated from an artificial pond in a honey farm at Hoengseong-gun, Gangwon-do, Republic of Korea. The 16S rRNA sequence analysis results revealed that strain LF1 T belonged to the genus Lysobacter and had the highest sequence similarity to Lysobacter niastensis GH41-7 T (99.0 %), Lysobacter panacisoli CJ29 T (98.9 %), and Lysobacter prati SYSU H10001 T (98.2 %). Its growth occurred at 20-37 °C, at pH 5.0-12.0, and in the presence of 0-2% NaCl. The major fatty acids were iso-C 15 : 0 , iso-C 16 : 0 , and summed feature 9 (iso-C 17 : 1  ω 9 c and/or C 16 : 0 10-methyl). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G+C content was 67.5 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain LF1 T and species of the genus Lysobacter were 79.1-84.4% and 22.0-27.5 %, respectively. The 16S rRNA sequence analysis results revealed that strain HM2-2 T belonged to the genus Limnohabitans and was most closely related to Limnohabitans planktonicus II-D5 T (98.9 %), Limnohabitans radicicola JUR4 T (98.4%), and Limnohabitans parvus II-B4 T (98.4 %). Its growth occurred at 10-35 °C, at pH 5.0-11.0, and in the presence of 0-2% NaCl. The major fatty acids were C 16 : 0 and summed feature 3 (C 16 : 1  ω 7 c /C 16 : 1  ω 6 c ). The major polar lipid was phosphatidylethanolamine. The DNA G+C content was 59.9 mol%. The ANI and dDDH values between strain HM2-2 T and its closely related strains were 75.1-83.0% and 20.4-26.4 %, respectively. Phenotypic, genomic, and phylogenetic data revealed that strains LF1 T and HM2-2 T represent novel species in the genera Lysobacter and Limnohabitans , for which the names Lysobacter stagni sp. nov. and Limnohabitans lacus sp. nov. are proposed, respectively. The type strain of Lys. stagni is LF1 T (=KACC 23251 T =TBRC 17648 T ), and that of Lim. lacus is HM2-2 T (=KACC 23250 T =TBRC 17649 T ).

Diffusible signal factor (DSF) family signals represent a unique group of quorum sensing (QS) chemicals that modulate a wide range of behaviors for bacteria to adapt to different environments. However, whether DSF-mediated QS signaling acts as a public language to regulate the behavior of biocontrol and pathogenic bacteria remains unknown. In this study, we present groundbreaking evidence demonstrating that RpfF Xc1 or RpfF OH11 could be a conserved DSF-family signal synthase in Xanthomonas campestris or Lysobacter enzymogenes . Interestingly, we found that both RpfF OH11 and RpfF Xc1 have the ability to synthesize DSF and BDSF signaling molecules. DSF and BDSF positively regulate the biosynthesis of an antifungal factor (heat-stable antifungal factor, HSAF) in L. enzymogenes . Finally, we show that RpfF Xc1 and RpfF OH11 have similar functions in regulating HSAF production in L. enzymogenes , as well as the virulence, synthesis of virulence factors, biofilm formation, and extracellular polysaccharide production in X. campestris . These findings reveal a previously uncharacterized mechanism of DSF-mediated regulation in both biocontrol and pathogenic bacteria., Competing Interests: The author(s) declare no conflict of interest.

Effector proteins secreted by bacteria that infect mammalian and plant cells often subdue eukaryotic host cell defenses by simultaneously affecting multiple targets. However, instances when a bacterial effector injected in the competing bacteria sabotage more than a single target have not been reported. Here, we demonstrate that the effector protein, LtaE, translocated by the type IV secretion system from the soil bacterium Lysobacter enzymogenes into the competing bacterium, Pseudomonas protegens, affects several targets, thus disabling the antibacterial defenses of the competitor. One LtaE target is the transcription factor, LuxR1, that regulates biosynthesis of the antimicrobial compound, orfamide A. Another target is the sigma factor, PvdS, required for biosynthesis of another antimicrobial compound, pyoverdine. Deletion of the genes involved in orfamide A and pyoverdine biosynthesis disabled the antibacterial activity of P. protegens, whereas expression of LtaE in P. protegens resulted in the near-complete loss of the antibacterial activity against L. enzymogenes. Mechanistically, LtaE inhibits the assembly of the RNA polymerase complexes with each of these proteins. The ability of LtaE to bind to LuxR1 and PvdS homologs from several Pseudomonas species suggests that it can sabotage defenses of various competitors present in the soil or on plant matter. Our study thus reveals that the multi-target effectors have evolved to subdue cell defenses not only in eukaryotic hosts but also in bacterial competitors., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

In the present work, we characterized in detail strain CM-3-T8 T , which was isolated from the rhizosphere soil of strawberries in Beijing, China, in order to elucidate its taxonomic position. Cells of strain CM-3-T8 T were Gram-negative, non-spore-forming, aerobic, short rod. Growth occurred at 25-37 °C, pH 5.0-10.0, and in the presence of 0-8% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CM-3-T8 T formed a stable clade with Lysobacter soli DCY21 T and Lysobacter panacisoli CJ29 T , with the 16S rRNA gene sequence similarities of 98.91% and 98.50%. The average nucleotide identity and digital DNA-DNA hybridization values between strain SG-8  T and the two reference type strains listed above were 76.3%, 79.6%, and 34.3%, 27%, respectively. The DNA G + C content was 68.4% (mol/mol). The major cellular fatty acids were comprised of C15:0 iso (36.15%), C17:0 iso (8.40%), and C11:0 iso 3OH (8.28%). The major quinone system was ubiquinone Q-8. The major polar lipids were phosphatidylethanolamine (PE), phosphatidylethanolamine (PME), diphosphatidylglycerol (DPG), and aminophospholipid (APL). On the basis of phenotypic, genotypic, and phylogenetic evidence, strain CM-3-T8 T (= ACCC 61714  T  = JCM 34576  T ) represents a new species within the genus Lysobacter, for which the name Lysobacter changpingensis sp. nov. is proposed., (© 2023. The Author(s).)

A bacterial strain designated as UC was isolated from farmland soil. Strain UC T formed a pale yellow colony on nutrient agar. Cell morphology revealed it as the rod-shaped bacterium that stained Gram-negative. The 16S rRNA gene sequence analysis identified strain UC T as a member of the genus Lysobacter that showed high identity with L. soli DCY21 T (99.5%), L. panacisoli CJ29 T (98.7%), and L. tabacisoli C8-1 T (97.9%). It formed a distinct cluster with these strains in the neighbor-joining phylogenetic tree. A similar tree topology was observed in TYGS-based phylogenomic analysis. However, genome sequence analyses of strain UC T showed 87.7% average nucleotide identity and 34.7% digital DNA-DNA hybridization similarity with the phylogenetically closest species, L. soli DCY21 T . The similarity was much less with other closely related strains of the genus Lysobacter. The G + C content of strain UC T was 68.1%. Major cellular fatty acids observed were C 14:0 iso (13.4%), C 15:0 iso (13.6%), and C 15:0 anteiso (14.8%). Quinone Q-8 was the major respiratory ubiquinone. Predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol. Production of xanthomonadin pigment was observed. Based on phenotypic differences and phylogenomic analysis, strain UC T represents a novel species of the genus Lysobacter, for which the name Lysobacter arvi is proposed. The type strain of the novel species is UC T (= KCTC 92613 T  = JCM 23757 T  = MTCC 12824 T )., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Lysobacter harbors a plethora of cryptic biosynthetic gene clusters (BGCs), albeit only a limited number have been analyzed to date. In this study, we described the activation of a cryptic polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) gene cluster (lsh) in Lysobacter sp. DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp. S001. As a result of this methodology, we were able to isolate two novel linear lipopeptides, lysohexaenetides A (1) and B (2), from the recombinant strain S001-lsh. Furthermore, we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs. This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes, particularly in the absence of genetic manipulation tools., (Copyright © 2023 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Lysobacter is a genus of bacteria emerging as new biocontrol agents in agriculture. Although iron acquisition is essential for the bacteria, no siderophore has been identified from any Lysobacter . Here, we report the identification of the first siderophore, N 1, N 8-bis(2,3-dihydroxybenzoyl)spermidine (lysochelin), and its biosynthetic gene cluster from Lysobacter enzymogenes . Intriguingly, the deletion of the spermidine biosynthetic gene encoding arginine decarboxylase or SAM decarboxylase eliminated lysochelin and the antifungals, HSAF and its analogues, which are key to the disease control activity and to the survival of Lysobacter under oxidative stresses caused by excess iron. The production of lysochelin and the antifungals is greatly affected by iron concentration. Together, the results revealed a previously unrecognized system, in which L. enzymogenes produces a group of small molecules, lysochelin, spermidine, and HSAF and its analogues, that are affected by iron concentration and critical to the growth and survival of the biocontrol agent.

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 Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

To isolate β-galactosidase producing bacterial resources, a novel Gram-stain-negative, strictly aerobic bacterial strain designated as A6 T was obtained from a farmland soil sample. Cells of the strain were rod-shaped (0.4-0.7 μm × 1.8-2.2 μm) without flagella and motility. Strain A6 T grew optimally at 30 °C, pH 7.0 with 0% (w/v) NaCl. Based on phylogenetic analysis, strain A6 T clustered within the genus Lysobacter clade and branched with Lysobacter dokdonensis KCTC 12822 T (99.5%, 16S rRNA gene sequence similarity) and Lysobacter caseinilyticus KACC 19816 T (98.5%). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain A6 T and Lysobacter dokdonensis KCTC 12822 T were 82.7% and 26.2%, and the values for strain A6 T and KACC 19816 T were 81.4% and 23.8%, respectively. Iso-C 16:0 , iso-C 15:0 , summed feature 9 (C 17:1 iso ω9c and/or C 16:0 10-methyl) and summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c) were the major fatty acids, diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine were the major polar lipids, and ubiquinone 8 (Q-8) was the major ubiquinone. The genomic DNA G+C content was 67.2 mol%. Furthermore, under the condition of 30 °C, pH 7.0, 4% inoculation with 10.0 g L -1 lactose, the β-galactosidase activity produced by strain A6 T was highest, reaching 95.3 U mL -1 , indicating that this strain could be applied as a potential strain for β-galactosidase production. Strain A6 T represents a novel species of the genus Lysobacter, and Lysobacter lactosilyticus sp. nov. is proposed on the basis of phenotypic, genotypic, and chemotaxonomic analysis. The type strain is A6 T (=KCTC 82184 T =CGMCC 1.18582 T )., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A Gram-stain-negative, yellow-pigmented, motile, flagellated and rod-shaped bacterium, designated as 13A T , was isolated from a river sediment sample of Fuyang River in Hengshui City, Hebei Province, PR China. Strain 13A T grew at 10-37 °C (optimum, 30 °C), at pH 5.0-11.0 (optimum, pH 7.0) and at 0-7 % (w/v) NaCl concentration (optimum, 0 %). Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain 13A T belongs to the genus Lysobacter , and was most closely related to Lysobacter spongiicola DSM 21749 T (97.8 %), Lysobacter concretionis DSM 16239 T (97.5 %), Lysobacter daejeonensis GIM 1.690 T (97.3 %) and Lysobacter arseniciresistens CGMCC 1.10752 T (96.9 %). Meanwhile, the type species Lysobacter enzymogenes ATCC 29487 T was selected as a reference strain (95.2 %). The genomic size of strain 13A T was 3.0 Mb and the DNA G+C content was 69.0 %. The average nucleotide identity values between strain 13A T and each of the reference type strains L. spongiicola DSM 21749 T , L. concretionis DSM 16239 T , L. daejeonensis GIM 1.690 T , L. arseniciresistens CGMCC 1.10752 T and L. enzymogenes ATCC 29487 T were 75.9, 76.1, 77.7, 78.0 and 73.2 %, respectively. The digital DNA-DNA hybridization values between strain 13A T and each of the reference type strains were 21.7, 22.2, 21.9, 22.7 and 23.2 %, respectively. The average amino acid identity values between strain 13A T and each of the reference type strains were 72.5, 72.9, 72.3, 75.0 and 69.2 %, respectively. The major fatty acids were iso-C 15 : 0 , iso-C 16 : 0 and summed feature 9 (iso-C 17 : 1  ω 9 c and/or C 16 : 0 10-methyl). The sole respiratory quinone was identified as ubiquinone-8. The polar lipid profile contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid, an unidentified lipid, four unidentified phospholipids and two unidentified glycolipids. Based on the phenotypic, physiological, phylogenetic and chemotaxonomic data, strain 13A T represents a novel species of the genus Lysobacter , for which the name Lysobacter selenitireducens sp. nov. is proposed. The type strain is 13A T (=JCM 34786 T =GDMCC 1.2722 T ).

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A successful homologous expression system based on Lysobacter capsici VKM B-2533 T and the plasmid pBBR1-MCS5 was first developed for a promising bacteriolytic enzyme of this bacterium, β-lytic protease (Blp). In the expression strains, blp gene expression under the regulation of the GroEL(A) and T5 promoters increased by 247- and 667-fold, respectively, as compared with the wild-type strain. After the cultivation of the expression strains L. capsici P GroEL(A) - blp and L. capsici P T5 - blp , the Blp yield increased by 6.7- and 8.5-fold, respectively, with respect to the wild-type strain. The cultivation of the expression strain L. capsici P T5 - blp was successfully scaled up. Under fermentation conditions the yield of the enzyme increased by 1.6-fold. The developed homologous system was used to express the gene of the bacteriolytic serine protease (Serp) of L. capsici VKM B-2533 T . The expression of the serp gene in L. capsici P T5 - serp increased by 585-fold. The developed homologous system for the gene expression of bacteriolytic Lysobacter enzymes is potentially biotechnologically valuable, and is promising for creating highly efficient expression strains.

Covering: up to August 2021 Lysobacter 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.

Background: Most of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. The M23 family is further classified into two subfamilies, M23A and M23B. Proteases of the M23A subfamily are thought to lack the capacity for self-maturation by auto-processing of a propeptide, which has been a challenge in heterologous production and application research. In this study, we investigated the heterologous expression, in Bacillus subtilis, of the Lysobacter enzymogenes beta-lytic protease (BLP), a member of the M23A subfamily., Results: We found that B. subtilis can produce BLP in its active form. Two points were shown to be important for the production of BLP in B. subtilis. The first was that the extracellular proteases produced by the B. subtilis host are essential for BLP maturation. When the host strain was deficient in nine extracellular proteases, pro-BLP accumulated in the supernatant. This observation suggested that BLP lacks the capacity for self-maturation and that some protease from B. subtilis contributes to the cleavage of the propeptide of BLP. The second point was that the thiol-disulfide oxidoreductases BdbDC of the B. subtilis host are required for efficient secretory production of BLP. We infer that intramolecular disulfide bonds play an important role in the formation of the correct BLP conformation during secretion. We also achieved efficient protein engineering of BLP by utilizing the secretory expression system in B. subtilis. Saturation mutagenesis of Gln116 resulted in a Q116H mutant with enhanced staphylolytic activity. The minimum bactericidal concentration (MBC) of the wild-type BLP and the Q116H mutant against Staphylococcus aureus NCTC8325 was 0.75 μg/mL and 0.375 μg/mL, respectively, and the MBC against Staphylococcus aureus ATCC43300 was 6 μg/mL and 3 μg/mL, respectively., Conclusions: In this study, we succeeded in the secretory production of BLP in B. subtilis. To our knowledge, this work is the first report of the successful heterologous production of BLP in its active form, which opens up the possibility of industrial use of BLP. In addition, this study proposes a new strategy of using the extracellular proteases of B. subtilis for the maturation of heterologous proteins., (© 2021. The Author(s).)

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., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Two Gram-stain negative, yellow-pigmented, and mesophilic bacteria, designated strains R7 T and R19 T , were isolated from sandy and forest soil, South Korea, respectively. Both strains were non-motile rods showing catalase- and oxidase-positive activities. Both strains were shown to grow at 10-37°C and pH 6.0-9.0, and in the presence of 0-1.5% (w/v) NaCl. Strain R7 T contained iso-C 14:0 , iso-C 15:0 , iso-C 16:0 , and summed feature 9 (comprising C 16:0 10-methyl and/or iso-C 17:1 ω9c), whereas strain R19 T contained iso-C 11:0 3-OH, C 16:1 ω7c alcohol, iso-C 11:0 , iso-C 15:0 , iso-C 16:0 , and summed feature 9 (comprising C 16:0 10-methyl and/or iso-C 17:1 ω9c) as major cellular fatty acids (> 5%). Both strains contained ubiquin-one-8 as the sole isoprenoid quinone and phosphatidylglycerol, phosphatidylethanolamine, and an unidentified phospholipid as the major polar lipids. The DNA G + C contents of strains R7 T and R19 T calculated from their genomes were 66.9 mol% and 68.9 mol%, respectively. Strains R7 T and R19 T were most closely related to Lysobacter panacisoli C8-1 T and Lysobacter niabensis GH34-4 T with 98.7% and 97.8% 16S rRNA sequence similarities, respectively. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains R7 T and R19 T formed distinct phylogenetic lineages within the genus Lysobacter. Based on phenotypic, chemotaxonomic, and molecular features, strains R7 T and R19 T represent novel species of the genus Lysobacter, for which the names Lysobacter arenosi sp. nov. and Lysobacter solisilvae sp. nov. are proposed. The type strains of L. arenosi and L. solisilvae are R7 T (= KACC 21663 T = JCM 34257 T ) and R19 T (= KACC 21767 T = JCM 34258 T ), respectively., (© 2021. The Microbiological Society of Korea.)

Soil microbiome comprises numerous microbial species that continuously interact with each other. Among the modes of diverse interactions, cell-cell killing may play a key role in shaping the microbiome composition. Bacteria deploy various secretion systems to fend off other microorganisms and Type IV Secretion System (T4SS) in pathogenic bacteria was shown to function as a contact-dependent, inter-bacterial killing system only recently. The present study investigated the role played by T4SS in the killing behaviour of the soilborne biocontrol bacterium Lysobacter enzymogenes OH11. Results showed that L. enzymogenes OH11 genome encompasses genes encoding all the components of T4SS and effectors potentially involved in inter-bacterial killing system. Generation of knock-out mutants revealed that L. enzymogenes OH11 uses T4SS as the main contact-dependent weapon against other soilborne bacteria. The T4SS-mediated killing behaviour of L. enzymogenes OH11 decreased the antibacterial and antifungal activity of two Pseudomonas spp. but at the same time, protected carrot from infection by Pectobacterium carotovorum. Overall, this study showed for the first time the involvement of T4SS in the killing behaviour of L. enzymogenes and its impact on the multiple interactions occurring in the soil microbiome., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

This is a research comment on the ochratoxin A (OTA) degradation mechanism by Lysobacter sp. CW239 regarding the previous publication in Environmental Pollution (Wei et al., 2020). Three possible degradation mechanisms were discussed in the referred publication, but without definite evidences, it was not clear which one worked actually. Here, the gene cp4 deficient mutant CW239 Δcp4 was successfully constructed, and the carboxypeptidase CP4 role on OTA degradation in strain CW239 was validated in vivo. As a result, the mutant CW239 Δcp4 without gene cp4 showed less than 10% reduction of 24 hrs degradation ratio compared to wide-type strain CW239. After the gene cp4 complemented to CW239 Δcp4 , the complementary strain (+)cp4 recovered the degradation ability to wide-type. The validation result indicated that the third degradation mechanism (i.e., OTA is degraded by joint action of multiple enzymes in CW239) proposed previous (Wei et al., 2020) was correct route for the degradation strain. This commentary was significant to the following studies on the pollutant detoxify strains with similar degradation characters between identified enzyme and the host strain., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Hypeptin is a cyclodepsipeptide antibiotic produced by Lysobacter sp. K5869, isolated from an environmental sample by the iChip technology, dedicated to the cultivation of previously uncultured microorganisms. Hypeptin shares structural features with teixobactin and exhibits potent activity against a broad spectrum of gram-positive pathogens. Using comprehensive in vivo and in vitro analyses, we show that hypeptin blocks bacterial cell wall biosynthesis by binding to multiple undecaprenyl pyrophosphate-containing biosynthesis intermediates, forming a stoichiometric 2:1 complex. Resistance to hypeptin did not readily develop in vitro. Analysis of the hypeptin biosynthetic gene cluster (BGC) supported a model for the synthesis of the octapeptide. Within the BGC, two hydroxylases were identified and characterized, responsible for the stereoselective β-hydroxylation of four building blocks when bound to peptidyl carrier proteins. In vitro hydroxylation assays corroborate the biosynthetic hypothesis and lead to the proposal of a refined structure for hypeptin., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

In the soil gammaproteobacterium Lysobacter enzymogenes, a natural fungal predator, the response regulator PilR controls type IV pili (T4P)-mediated twitching motility as well as synthesis of the heat-stable antifungal factor (HSAF). Earlier we showed that PilR acts via the second messenger, c-di-GMP; however, the mechanism remained unknown. Here, we describe how PilR, c-di-GMP signalling, and HSAF synthesis are connected. We screened genes for putative diguanylate cyclases (c-di-GMP synthases) and found that PilR binds to the promoter region of lchD and down-regulates its transcription. The DNA-binding affinity of PilR, and therefore its repressor function, are enhanced by phosphorylation by its cognate histidine kinase, PilS. The lchD gene product is a diguanylate cyclase, and the decrease in LchD levels shifts the ratio of c-di-GMP-bound and c-di-GMP-free transcription factor Clp, a key activator of the HSAF biosynthesis operon expression. Furthermore, Clp directly interacts with LchD and enhances its diguanylate cyclase activity. Therefore, the PilS-PilR two-component system activates T4P-motility while simultaneously decreasing c-di-GMP levels and promoting HSAF production via the highly specific LchD-c-di-GMP-Clp pathway. Coordinated increase in motility and secretion of the "long-distance" antifungal weapon HSAF is expected to ensure safer grazing of L. enzymogenes on soil or plant surfaces, unimpeded by fungal competitors, or to facilitate bacterial preying on killed fungal cells. This study uncovered the mechanism of coregulated pili-based motility and production of an antifungal antibiotic in L. enzymogenes, showcased the expanded range of functions of the PilS-PilR system, and highlighted exquisite specificity in c-di-GMP-mediated circuits., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The chitin-assimilating gram-negative bacterium, Lysobacter sp. MK9-1, was isolated from soil and was the source of a glycoside hydrolase family 19-type chitinase (Chi19MK) gene that is 933-bp long and encodes a 311-residue protein. The deduced amino acid sequence of Chi19MK includes a signal peptide, an uncharacterized sequence, a carbohydrate-binding module family 12-type chitin binding domain, and a catalytic domain. The catalytic domain of Chi19MK is approximately 60% similar to those of ChiB from Burkholderia gladioli CHB101, chitinase N (ChiN) from Chitiniphilus shinanonensis SAY3 T , ChiF from Streptomyces coelicolor A3(2), Chi30 from Streptomyces olivaceoviridisis, ChiA from Streptomyces cyaneus SP-27, and ChiC from Streptomyces griseus HUT6037. Chi19MK lacking the signal and uncharacterized sequences (Chi19MKΔNTerm) was expressed in Escherichia coli Rosetta-gami B(DE3), resulting in significant chitinase activity in the soluble fraction. Purified Chi19MKΔNTerm hydrolyzed colloidal chitin and released disaccharide. Furthermore, Chi19MKΔNTerm inhibited hyphal extension in Trichoderma reesei and Schizophyllum commune. Based on quantitative antifungal activity assays, Chi19MKΔNTerm inhibits the growth of Trichoderma viride with an IC 50 value of 0.81 μM., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

A Gram-stain-negative, aerobic, non-motile, non-spore-forming light-yellow-coloured rod-shaped bacterial strain, designated YJ15 T , was isolated from soil at Bigeum island in Korea. Growth was observed at 10-37 °C (optimum, 28 °C), at pH 6.0-7.5 (optimum, pH 7.0) and in the absence of NaCl. Based on 16S rRNA gene sequence analysis, strain YJ15 T was closely related to 'Lysobacter tongrenensis' YS037 T (97.8%), Lysobacter pocheonensis Gsoil193 T (96.5%) and Lysobacter daecheongensis Dae08 T (95.8%) and phylogenetically grouped together with 'Lysobacter tongrenensis' YS037 T , Lysobacter dokdonensis DS-58 T and Lysobacter pocheonensis Gsoil 193 T . The DNA-DNA relatedness between strain YJ15 T and 'Lysobacter tongrenensis' KCTC 52206 T was 12% and the phylogenomic analysis based on the whole genome sequence demonstrated that strain YJ20 T formed a distinct phyletic line with Lysobacterlter dokdonensis DS-58 T showing average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of 76.3 and 21.3%, respectively. The predominant ubiquinone was identified as Q-8, and polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unidentified aminolipids. The major fatty acids were iso-C 17:1 ω9c, iso-C 15:0 , iso-C 16:0 and iso-C 17:0 . The genomic DNA G + C content was 68.2 mol %. On the basis of phenotypic, chemotaxonomic properties and phylogenetic analyses in this study, strain YJ15 T is considered to represent a novel species of the genus Lysobacter, for which the name Lysobacter telluris sp. nov. is proposed. The type strain is YJ15 T (= KACC 19552 T  = NBRC 113197 T ).

Strain HX-5-24 T was isolated from the sludge collected from the outlet of the biochemical treatment facility of an agricultural chemical plant in Maanshan city, Anhui province, PR China (118° 28' N, 31° 47' E). Cells were observed to be Gram-reaction-negative, rod-shaped, non-motile and aerobic. Strain HX-5-24 T shared 99.1% 16S rRNA gene sequence similarity with Lysobacter dokdonensis DS-58 T and less than 97% similarities with other type strains. The phylogenetic analysis based on 16S rRNA indicated that strain HX-5-24 T belonged to the genus Lysobacter and formed a subclade with L. dokdonensis DS-58 T . The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain HX-5-24 T and L. dokdonensis DS-58 T were 87.5% and 35.3%, respectively. The genomic DNA G + C content of the strain was 66.4%. The major fatty acids (> 5%) were iso-C 15:0 , anteiso-C 15:0 , iso-C 16:0 , C 16:0 and summed feature 9 (iso-C 17:1 ω9c and/or C 16:0 10-methyl). The predominant quinone was ubiquinone Q-8. The polar lipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phospholipids (PL). On the basis of phenotypic and phylogenetic evidences, strain HX-5-24 T is considered as a novel species in the genus Lysobacter, for which the name Lysobacter gilvus sp. nov. is proposed. The type strain is HX-5-24 T (= KCTC 72470 T  = CCTCC AB 2019228 T ).

Colletotrichum fructicola, is an important fungal pathogen that has been reported to cause pear (Pyrus) anthracnose in China, resulting in substantial economic losses due to severe defoliation and decreased fruit quality and yield. In the search for novel strategies to control pear anthracnose, Lysobacter strains have drawn a great deal of attention due to their high-level production of extracellular enzymes and bioactive metabolites. In the present study, we compared four Lysobacter strains including Lysobacter enzymogenes OH11, Lysobacter antibioticus OH13, Lysobacter gummosus OH17 and Lysobacter brunescens OH23 with respect to their characteristics and activity against pear anthracnose caused by C. fructicola. The results showed that the evaluated Lysobacter species presented various colony morphologies when cultured on different media and were proficient in producing protease, chitinase, cellulase and glucanase, with L. enzymogenes OH11 showing typical twitching motility. L. enzymogenes OH11 and L. gummosus OH17 showed potent activity against the tested fungi and oomycetes. L. gummosus OH17 produced HSAF (heat-stable antifungal factor) which was demonstrated to be a major antifungal factor in L. enzymogenes OH11 and C3. Furthermore, L. antibioticus OH13 and L. brunescens OH23 exhibited strong antibacterial activity, especially against Xanthomonas species. Cultures of L. enzymogenes OH11 protected pear against anthracnose caused by C. fructicola, and the in vivo results indicated that treatment with an L. enzymogenes OH11 culture could decrease the diameter of lesions in pears by 35 % and reduce the severity of rot symptoms compared to that observed in the control. In the present study, we systemically compared four Lysobacter strains and demonstrated that they have strong antagonistic activity against a range of pathogens, demonstrating their promise in the development of biological control agents., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

A novel proteobacterial strain designated SYSU H10001 T was isolated from a soil sample collected from plateau meadow in Hongyuan county, Sichuan province, south-western China. The taxonomic position of the strain was investigated using a polyphasic approach. On the basis of 16S rRNA gene sequence similarities and phylogenetic analysis, strain SYSU H10001 T was most closely related to Lysobacter soli KCTC 22011 T (98.6%, sequence similarity) and Lysobacter panacisoli JCM 19212 T (98.2%). The prediction result of secondary metabolites based on genome shown that the strain SYSU H10001 T contained 3 clusters of bacteriocins, 1 cluster of non-ribosomal peptide synthetase, 1 cluster of type 1 polyketide synthase and 1 cluster of arylpolyene. In addition, the major isoprenoid quinone was Q-8 and the major fatty acids were identified as iso-C 15:0 , iso-C 17:0 and Summed feature 9. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and three unidentified phospholipids. The genomic DNA G + C content of strain SYSU H10001 T was 66.5% (genome). On the basis of phenotypic, genotypic and phylogenetic data, strain SYSU H10001 T represents a novel species of the genus Lysobacter, for which the name Lysobacter prati sp. nov. is proposed. The type strain is SYSU H10001 T (= KCTC 72062 T  = CGMCC 1.16662 T ).

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.

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.

The bacterial flagellum is one of the best-studied surface-attached appendages in bacteria. Flagellarassembly in vivo is promoted by its own protein export apparatus, a type III secretion system (T3SS) in pathogenic bacteria. Lysobacter enzymogenes OH11 is a non-flagellated soil bacterium that utilizes type IV pilus (T4P)-driven twitching motility to prey upon nearby fungi for food. Interestingly, the strain OH11 encodes components homologous to the flagellar type III protein apparatus (FT3SS) on its genome, but it remains unknown whether this FT3SS-like system is functional. Here, we report that, despite the absence of flagella, the FT3SS homologous genes are responsible not only for the export of the heterologous flagellin in strain OH11 but also for twitching motility. Blocking the FT3SS-like system by in-frame deletion mutations in either flhB or fliI abolished the secretion of heterologous flagellin moleculesinto the culture medium, indicating that the FT3SS is functional in strain OH11. A deletion of flhA , flhB , fliI , or fliR inhibited T4P-driven twitching motility, whereas neither that of fliP nor fliQ did, suggesting that FlhA, FlhB, FliI, and FliR may obtain a novel function to modulate the twitching motility. The flagellar FliI ATPase was required for the secretion of the major pilus subunit, PilA, suggesting that FliI would have evolved to act as a PilB-like pilus ATPase. These observations lead to a plausible hypothesis that the non-flagellated L. enzymogenes OH11 could preserve FT3SS-like genes for acquiring a distinct function to regulate twitching motility associated with its predatory behavior.

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 coli fadD mutant JW1794. RpfB activates long-chain fatty acids (n-C 16:0 and n-C 18:0 ) 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 ( Le DSF3) 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. enzymogenes IMPORTANCE 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., (Copyright © 2020 Li et al.)

A bacterial strain isolated from a soil collected in Jeju Island, designated as 17J7-1 T , 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 17J7-1 T as a member of the genus Lysobacter with close sequence similarity with Lysobacter mobilis 9NM-14 T (97.4%), Lysobacter xinjiangensis RCML-52 T (97.0%), and Lysobacter humi FJY8 T (96.9%). The genomic DNA G + C content of the isolate was 67.9 mol%. DNA-DNA relatedness between strain 17J7-1 T 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 17J7-1 T and L. enzymogenes ATCC 29487 T , the type species of this genus, and several other close Lysobacter species were less than 77% and 22%, respectively. Major fatty acids were C 16:0 iso (29.8%), summed feature 9 (C 17:1 iso ω9c/C 16:0 10-methyl; 20.1%), and C 15:0 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 17J7-1 T is considered to represent a novel species in the genus Lysobacter, for which name Lysobacter terrigena sp. nov. is proposed. The type strain is 17J7-1 T (= KCTC 62217 T  = JCM 33057 T ).

Ochratoxin A (OTA) is a potent mycotoxin that frequently contaminates agro-products and threatens food safety. A highly efficient OTA degrading strain Lysobacter sp. CW239 was isolated, and the OTA degradation characteristics were investigated. A novel OTA degrading gene carboxypeptidase cp4 was successfully cloned and characterized from CW239. The heterologous recombinant was constructed by gene cp4 and expression vector pET-32a (+) and overexpressed by E. coli BL21 CodonPlus™ (DE3). The recombinant protein rCP4 was purified, and the OTA-degrading activity was evaluated. Although OTA was efficiently degraded by CW239 (24-h degradation ratio of 86.2%), the 24-h OTA degradation ratio for rCP4 was only 36.8% at fairly high concentration (0.25 mg/mL) protein. The degraded product was obtained by immune affinity column (IAC) and determined by mass spectrometry (MS), and the degraded product was the less toxic ochratoxin α (OTα). Based on the serial investigations of this study, OTA might be simultaneously co-degraded by CP4 and another unknown degrading agent in that degrading strain., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A Gram-negative, aerobic, motile by gliding, rod-shaped bacterium, strain 17J68-2 T , 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 FJY8 T (98.4% similarity), Lysobacter xinjiangensis RCML-52 T (98.3%), and Lysobacter mobilis 9NM-14 T (98.1%) as closest phylogenetic neighbors. Growth of strain 17J68-2 T 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 C 16:0 iso (39.4%), summed feature 3 (C 16:1 ω7c/C 16:1 ω6c) (6.6%), C 11:0 iso 3-OH (6.4%), C 15:0 iso (6.4%), and C 16:1 iso H (6.2%). The DNA-DNA relatedness between strain 17J68-2 T 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 17J68-2 T represents a novel species of the genus Lysobacter, for which the name Lysobacter segetis sp. nov. (=KCTC 62237 T  = JCM 33058 T ) is proposed.

Chitin amendment is an agricultural management strategy for controlling soil-borne plant disease. We previously reported an exponential decrease in chitin added to incubated upland soil. We herein investigated the transition of the bacterial community structure in chitin-degrading soil samples over time and the characteristics of chitinolytic bacteria in order to elucidate changes in the chitinolytic bacterial community structure during chitin degradation. The addition of chitin to soil immediately increased the population of bacteria in the genus Streptomyces, which is the main decomposer of chitin in soil environments. Lysobacter, Pseudoxanthomonas, Cellulosimicrobium, Streptosporangium, and Nonomuraea populations increased over time with decreases in that of Streptomyces. We isolated 104 strains of chitinolytic bacteria, among which six strains were classified as Lysobacter, from chitin-treated soils. These results suggested the involvement of Lysobacter as well as Streptomyces as chitin decomposers in the degradation of chitin added to soil. Lysobacter isolates required yeast extract or casamino acid for significant growth on minimal agar medium supplemented with glucose. Further nutritional analyses demonstrated that the six chitinolytic Lysobacter isolates required methionine (Met) to grow, but not cysteine or homocysteine, indicating Met auxotrophy. Met auxotrophy was also observed in two of the five type strains of Lysobacter spp. tested, and these Met auxotrophs used d-Met as well as l-Met. The addition of Met to incubated upland soil increased the population of Lysobacter. Met may be a factor increasing the population of Lysobacter in chitin-treated upland soil.

A Gram-stain negative, rod-shaped bacterial, catalase and oxidase positive strain (83-4 T ) that formed yellow colonies was isolated from human Meibomian gland secretions. Strain 83-4 T 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 83-4 T and the closely related type strain Lysobacter tolerans UM1 T . The estimated DNA-DNA hybridization value between strain 83-4 T and L. tolerans UM1 T was 41.6%. Diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol were the major polar lipids. Iso-C 15:0 , iso-C 11:0 3-OH, iso-C 11:0 and summed feature 9 (iso-C 17:1 ω9c) 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 83-4 T represents a novel species of the genus Lysobacter with the name of Lysobacter oculi sp. nov. The type strain is 83-4 T (= CGMCC 1.13464 T  = NRBC 113451 T ).

Objectives: Ubiquitous Gram-negative Lysobacter species are known to confer intrinsic antibiotic resistance and are being considered as new sources for novel anti-methicillin-resistant Staphylococcus aureus (MRSA) antibiotics. This study aimed to determine the intrinsic antibiotic resistance profiles of Lysobacter enzymogenes strain C3 (LeC3) and Lysobacter antibioticus strain ATCC29479 (LaATCC29479), and to in silico identify their intrinsic resistomes and compare with Xanthomonas campestris, a close relative and plant pathogen., Methods: The intrinsic resistant profiles of LeC3 and LaATCC29479 were determined by minimum inhibitory concentration (MIC) and disk diffusion assays. Resistance Gene Identifier (RGI) in the Comprehensive Antibiotic Resistance Database (CARD) was used to predict resistomes. Selected resistance genes were mutated and their roles in resistance to antibiotics were determined by spot dilution assays., Results: MIC and disk diffusion assays revealed that both LeC3 and LaATCC29479 exhibited high levels of multidrug resistance to 12 common antibiotics. Comparative resistomic analyses using the RGI revealed possible antibiotic resistance genes (ARGs) related to the antibiotic resistance profiles in LeC3 and LaATCC29479, and the core resistome of Lysobacter spp. Functional studies confirmed that three ARGs (bla, aac and sph) conferred antibiotic resistance in LeC3, and also in X. campestris when expressed in trans., Conclusion: The findings show that LeC3 and LaATCC29479 exhibited multidrug resistance at very high levels and the resistomes of Lysobacter strains were more abundant than those of X. campestris, which might provide novel targets for studies in the intrinsic antibiotic resistance of Lysobacter and other environmental bacterial species., (Copyright © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.)

Lysobacter enzymogenes, a member of Xanthomonadaceae, is a promising tool to control crop-destroying fungal pathogens. One of its key antifungal virulence factors is the type IV pili that are required for twitching motility. Transposon mutagenesis of L. enzymogenes revealed that the production of type IV pili required the presence of the Le2152 gene, which encodes an AlgC-type phosphomannomutase/phosphoglucomutase (PMM). However, in addition to the cytoplasmic PMM domain, the Le2152 gene product contains a ~200-aa N-terminal periplasmic domain that is anchored in the membrane by two transmembrane segments and belongs to the dCache superfamily of periplasmic sensor domains. Sequence analysis identified similar membrane-anchored PMMs, encoded in conserved coaBC-dut-algC gene clusters, in a variety of gammaproteobacteria, either as the sole PMM gene in the entire genome or in addition to the gene encoding the stand-alone enzymatic domain. Previously overlooked N-terminal periplasmic sensor domains were detected in the well-characterized PMMs of Pseudomonas aeruginosa and Xanthomonas campestris, albeit not in the enzymes from Pseudomonas fluorescens, Pseudomonas putida or Azotobacter vinelandii. It appears that after the initial cloning of the enzymatically active soluble part of P. aeruginosa AlgC in 1991, all subsequent studies utilized N-terminally truncated open reading frames. The N-terminal dCache sensor domain of AlgC is predicted to modulate the PMM activity of the cytoplasmic domain in response to as yet unidentified environmental signal(s). AlgC-like membrane-bound PMMs appear to comprise yet another environmental signalling system that regulates the production of type IV pili and potentially other systems in certain gammaproteobacteria., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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 P groEL 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.

A novel bacterial strain, designated KVB24 T , was isolated from sea-water of Busan Harbour in South Korea. Cells of strain KVB24 T were Gram-stain negative, aerobic, rod shaped and non-motile. Strain KVB24 T grew optimally at 25-28 °C and pH 6.5-7.0. Based on 16S rRNA gene sequence analysis, strain KVB24 T was shown to belong to the genus Lysobacter within the class Gammaproteobacteria and to be closely related to Lysobacter dokdonensis DS-58 T , Lysobacter hankyongensis KTce-2 T and Lysobacter niastensis GH41-7 T . DNA-DNA relatedness between strain KVB24 T and its current closest relative was below 70%. The predominant fatty acids of strain KVB24 T were iso-C 11:0 , iso-C 11:0 3-OH, iso-C 14:0 , iso-C 15:0 , anteiso-C 15:0 , iso-C 16:0 and summed feature 9 comprising (iso-C 17:1 ω9c and/or 10 methyl C 16:0 ); the prominent isoprenoid was Q-8 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The G + C content of genomic DNA from strain KVB24 T was determined to be 67.5 mol%. Based on the phenotypic, genotypic and chemotaxonomic analyses, strain KVB24 T represents a novel species of the genus Lysobacter, for which the name Lysobacter caseinilyticus sp. nov. is proposed. The type strain is KVB24 T (= KACC19816 T  = JCM32879 T ).

Lysobacter are ubiquitous in the environment but remain largely underexplored, although the bacteria are considered "peptide specialists". Here, we identified a new cyclic lipodepsipeptide, WBP-29479A1 ( 1 ), through genome mining of L. antibioticus ATCC 29479. 1 is biosynthesized by a large NRPS gene cluster, and its structure, including the six nonproteinogenic residues and 3-hydroxy fatty acid, was determined by extensive spectroscopic analyses and chemical derivatization. 1 exhibits potent anti-MRSA activity in a menaquinone-dependent manner.

Two bacterial strains, designated D10 T and U8 T , 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 D10 T showed high 16S rRNA gene sequence similarities with Lysobacter humi FJY8 T (99.0%), Lysobacter xinjiangensis RCML-52 T (98.9%) and Lysobacter mobilis 9NM-14 T (97.2%), whereas strain U8 T showed high sequence similarities to L. mobilis 9NM-14 T (97.9%), L. xinjiangensis RCML-52 T (97.8%), L. humi FJY8 T (97.5%) and Lysobacter bugurensis ZLD-29 T (97.1%). The 16S rRNA gene sequence similarity between D10 T and U8 T was 97.0%. Strain D10 T showed low DNA-DNA relatedness to U8 T (57.7 ± 3.4%), L. humi FJY8 T (48.8 ± 4.3%), L. xinjiangensis RCML-52 T (60.1 ± 2.4%) and L. mobilis 9NM-14 T (55.9 ± 1.9%). The level of DNA-DNA relatedness for strain U8 T with respect to D10 T , L. mobilis 9NM-14 T , L. xinjiangensis RCML-52 T , L. humi FJY8 T , and L. bugurensis ZLD-29 T 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-C 15:0 , iso-C 16:0 and summed feature 9 (iso-C 17:1 ω9c/C 16:0 10-methyl), and ubiquinone (Q-8) as the only isoprenoid quinone for both strains. The DNA G + C contents of the strains D10 T and U8 T were determined to be 70.2 mol% and 70.6 mol%. On the basis of phenotypic, genotypic, chemotaxonomic, and phylogenetic analysis, both strains D10 T and U8 T 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 D10 T (= KCTC 62111 T  = JCM 32364 T ) and the type strain of L. xanthus is U8 T (= KCTC 62112 T  = JCM 32365 T ).

Bacterial antibiotic resistance modulation by small signaling molecules is an emerging mechanism that has been increasingly reported in recent years. Several studies indicate that indole, an interkingdom signaling molecule, increases bacterial antibiotic resistance. However, the mechanism through which indole reduces antibiotic resistance is largely unknown. In this study, we demonstrated a novel mechanism for indole-mediated reversal of intrinsic antibiotic resistance in Lysobacter This reversal was facilitated by a novel BtuD-associated dual-function importer that can transfer both vitamin B 12 and antibiotics. Indole stimulated btuD overexpression and promoted efficient absorption of extracellular vitamin B 12 ; meanwhile, the weak selectivity of the importer caused cells to take up excessive doses of antibiotics that resulted in cell death. Consistently, btuD deletion and G48Y/K49D substitution led to marked reductions in the uptake of both antibiotics and vitamin B 12 This novel mechanism is common across multiple bacterial species, among which the Q-loop amino acid of BtuD proteins is Glu (E) instead of Gln (Q). Interestingly, the antibiotic resistance of Lysobacter spp. can be restored by another small quorum sensing signaling factor, 13-methyltetradecanoic acid, designated Le DSF, in response to bacterial population density. This work highlights the mechanisms underlying dynamic regulation of bacterial antibiotic resistance by small signaling molecules and suggests that the effectiveness of traditional antibiotics could be increased by coupling them with appropriate signaling molecules. IMPORTANCE Recently, signaling molecules were found to play a role in mediating antibiotic resistance. In this study, we demonstrated that i ndole r eversed the intrinsic a ntibiotic r esistance (IRAR) of multiple bacterial species by promoting the expression of a novel dual-function importer. In addition, population-dependent behavior induced by 13-methyltetradecanoic acid, a quorum sensing signal molecule designated Le DSF, was involved in the IRAR process. This study highlights the dynamic regulation of bacterial antibiotic resistance by small signaling molecules and provides direction for new therapeutic strategies using traditional antibiotics in combination with signaling molecules., (Copyright © 2019 Wang et al.)

The bacterial strains 4284/11T and 812/17 isolated from the respiratory tract of two royal pythons in 2011 and 2017, respectively were subjected to taxonomic characterization. The 16S rRNA gene sequences of the two strains were identical and showed highest sequence similarities to Lysobacter tolerans UM1 T (97.2%) and Luteimonas aestuarii DSM 19680 T (96.7 %). The two strains were identical in the sequences of the 16S-23S rRNA internal transcribed spacer (ITS) and partial groEL gene sequences and almost identical in genomic fingerprints. In the ITS sequence Ly. tolerans DSM 28473 T and in the groEL nucleotide sequence Luteimonas mephitis DSM 12574 T showed the highest similarity. In silico DDH analyses using genome sequence based ANIb and gANI similarity coefficients demonstrated that strain 4284/11 T represents a novel species and revealed Ly. tolerans UM1 T as the next relative (ANIb = 76.2 %, gANI = 78.0 %). Based on the topology of a core gene phylogeny strain 4284/11 T could be assigned to the genus Lysobacter. Chemotaxonomic characteristics including polyamine pattern, quinone system, polar lipid profile and fatty acid profile were in accordance with the characteristics of the genera Lysobacter and Luteimonas. Strains 4284/11 T and 812/17 could be differentiated from the type strains of the most closely related species by several physiological tests. In conclusion we are here proposing the novel species Lysobacter pythonis sp. nov. The type strain is 4284/11 T (= CCM 8829 T = CCUG 72164 T = LMG 30630 T ) and strain 812/17 (CCM 8830) is a second strain of this species., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Spermidine is a common polyamine compound produced in bacteria, but its roles remain poorly understood. The bacterial SpeD encodes an S-adenosylmethionine decarboxylase that participates in spermidine synthesis. Lysobacter enzymogenes is an efficient environmental predator of crop fungal pathogens by secreting an antifungal antibiotic HSAF (heat-stable antifungal factor), while Clp is a master transcription factor essential for the antifungal activity of L. enzymogenes. In this work, we observed that speD was a close genomic neighbor of the clp gene. This genomic arrangement also seems to occur in many other bacteria, but the underlying reason remains unclear. By using L. enzymogenes OH11 as a working model, we showed that SpeD was involved in spermidine production that was essential for the L. enzymogenes antifungal activity. Spermidine altered the bacterial growth capability and HSAF production, both of which critically contributed to the L. enzymogenes antifungal activity. We further found that spermidine in L. enzymogenes was able to play a crucial, yet indirect role in maintaining the Clp level in vivo, at least partially accounting for its role in the antifungal activity. Thus, our findings suggested that spermidine probably plays an uncharacterized role in maintaining the levels of the master transcription regulator Clp to optimize its role in antifungal activity in an agriculturally beneficial bacterium.

Background: Heat-stable antifungal factor (HSAF) is a newly identified broad-spectrum antifungal antibiotic from the biocontrol agent Lysobacter enzymogenes and is regarded as a potential biological pesticide, due to its novel mode of action. However, the production level of HSAF is quite low, and little research has reported on the fermentation process involved, representing huge obstacles for large-scale industrial production., Results: Medium capacity, culture temperature, and fermentation time were identified as the most significant factors affecting the production of HSAF and employed for further optimization through statistical methods. Based on the analysis of kinetic parameters at different temperatures, a novel two-stage temperature control strategy was developed to improve HSAF production, in which the temperature was increased to 32 °C during the first 12 h and then switched to 26 °C until the end of fermentation. Using this strategy, the maximum HSAF production reached 440.26 ± 16.14 mg L - 1 , increased by 9.93% than that of the best results from single-temperature fermentation. Moreover, the fermentation time was shortened from 58 h to 54 h, resulting in the enhancement of HSAF productivity (17.95%) and yield (9.93%)., Conclusions: This study provides a simple and efficient method for producing HSAF that could be feasibly applied to the industrial-scale production of HSAF.

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.

A Gram-negative, non-motile, aerobic, catalase-, and oxidasepositive bacterial strain, designated DCY117 T , was isolated from ginseng cultivated soil in Gochang-gun, Republic of Korea, and was characterized taxonomically using a multifaceted approach. 16S rRNA gene sequence analysis revealed that strain DCY117 T showed highest similarity to Lysobacter ruishenii CTN-1 T (95.3%). Phylogenetic analysis revealed that closely related relatives of strain DCY117 T were L. aestuarii S2-C T (95.1%), L. daejeonensis GH1-9 T (95.0%), and L. caeni BUT-8 T (94.9%). Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) were the major polar lipids of strain DCY117 T . The major isoprenoid quinone was Q-8. The major cellular fatty acids of strain DCY117 T were iso-C 15:0 , iso-C 16:0 , and summed feature 9 (comprising iso-C 17:1 ω9c and/or 10-methyl-C 16:0 ). Genomic DNA G + C content was 61.8 mol%. On the basis of our findings, strain DCY117 T is a novel species in the genus Lysobacter. We propose the name Lysobacter panacihumi sp. nov., and the type strain is DCY117 T (= KCTC 62019 T = JCM 32168 T ).