Production of the siderophore lysochelin in rich media through maltose-promoted high-density growth of Lysobacter sp. 3655.

Siderophores are produced by bacteria in iron-restricted conditions. However, we found maltose could induce the biosynthesis of the siderophore lysochelin in Lysobacter sp. 3655 in rich media that are not compatible with siderophore production. Maltose markedly promoted cell growth, with over 300% increase in cell density (OD600) when LB medium was added with maltose (LBM). While lysochelin was not detectable when OD600 in LBM was below 5.0, the siderophore was clearly produced when OD600 reached 7.5 and dramatically increased when OD600 was 15.0. Coincidently, the transcription of lysochelin biosynthesis genes was remarkably enhanced following the increase of OD600. Conversely, the iron concentration in the cell culture dropped to 1.2 µM when OD600 reached 15.0, which was 6-fold lower than that in the starting medium. Moreover, mutants of the maltose-utilizing genes (orf2677 and orf2678) or quorum-sensing related gene orf644 significantly lowered the lysochelin yield. Transcriptomics analysis showed that the iron-utilizing/up-taking genes were up-regulated under high cell density. Accordingly, the transcription of lysochelin biosynthetic genes and the yield of lysochelin were stimulated when the iron-utilizing/up-taking genes were deleted. Finally, lysochelin biosynthesis was positively regulated by a TetR regulator (ORF3043). The lysochelin yield in orf3043 mutant decreased to 50% of that in the wild type and then restored in the complementary strain. Together, this study revealed a previously unrecognized mechanism for lysochelin biosynthetic regulation, by which the siderophore could still be massively produced in Lysobacter even grown in a rich culture medium. This finding could find new applications in large-scale production of siderophores in bacteria.

Type IV secretion system effector sabotages multiple defense systems in a competing bacterium.

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.

Lysobacter stagni sp. nov. and Limnohabitans lacus sp. nov., isolated from a pond.

Two Gram-negative bacteria, designated as strains LF1T and HM2-2T, 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 LF1T belonged to the genus Lysobacter and had the highest sequence similarity to Lysobacter niastensis GH41-7T (99.0 %), Lysobacter panacisoli CJ29T (98.9 %), and Lysobacter prati SYSU H10001T (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-C15 : 0, iso-C16 : 0, and summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 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 LF1T 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 HM2-2T belonged to the genus Limnohabitans and was most closely related to Limnohabitans planktonicus II-D5T (98.9 %), Limnohabitans radicicola JUR4T (98.4%), and Limnohabitans parvus II-B4T (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 C16 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). The major polar lipid was phosphatidylethanolamine. The DNA G+C content was 59.9 mol%. The ANI and dDDH values between strain HM2-2T and its closely related strains were 75.1-83.0% and 20.4-26.4 %, respectively. Phenotypic, genomic, and phylogenetic data revealed that strains LF1T and HM2-2T 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 LF1T (=KACC 23251T=TBRC 17648T), and that of Lim. lacus is HM2-2T (=KACC 23250T=TBRC 17649T).

Characterization of soybean chitinase genes induced by rhizobacteria involved in the defense against Fusarium oxysporum.

Rhizobacteria are capable of inducing defense responses via the expression of pathogenesis-related proteins (PR-proteins) such as chitinases, and many studies have validated the functions of plant chitinases in defense responses. Soybean (Glycine max) is an economically important crop worldwide, but the functional validation of soybean chitinase in defense responses remains limited. In this study, genome-wide characterization of soybean chitinases was conducted, and the defense contribution of three chitinases (GmChi01, GmChi02, or GmChi16) was validated in Arabidopsis transgenic lines against the soil-borne pathogen Fusarium oxysporum. Compared to the Arabidopsis Col-0 and empty vector controls, the transgenic lines with GmChi02 or GmChi16 exhibited fewer chlorosis symptoms and wilting. While GmChi02 and GmChi16 enhanced defense to F. oxysporum, GmChi02 was the only one significantly induced by Burkholderia ambifaria. The observation indicated that plant chitinases may be induced by different rhizobacteria for defense responses. The survey of 37 soybean chitinase gene expressions in response to six rhizobacteria observed diverse inducibility, where only 10 genes were significantly upregulated by at least one rhizobacterium and 9 genes did not respond to any of the rhizobacteria. Motif analysis on soybean promoters further identified not only consensus but also rhizobacterium-specific transcription factor-binding sites for the inducible chitinase genes. Collectively, these results confirmed the involvement of GmChi02 and GmChi16 in defense enhancement and highlighted the diverse inducibility of 37 soybean chitinases encountering F. oxysporum and six rhizobacteria.

Unveiling the Role of Diffusible Signal Factor-Family Quorum Sensing Signals in Regulating Behavior of Xanthomonas and Lysobacter.

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 RpfFXc1 or RpfFOH11 could be a conserved DSF-family signal synthase in Xanthomonas campestris or Lysobacter enzymogenes. Interestingly, we found that both RpfFOH11 and RpfFXc1 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 RpfFXc1 and RpfFOH11 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.

Metabolites from Lysobacter gummosus YMF3.00690 Against Meloidogyne javanica.

The strains in Lysobacter spp. have the potential to control plant-parasitic nematodes. In our experiment, L. gummosus YMF3.00690 showed antagonistic effects against plant root-knot nematode. Nine metabolites were isolated and identified from cultures of L. gummosus YMF3.00690, of which compound 1 was identified as a new metabolite tetrahydro-4,4,6-trimethyl-6-[(tetrahydro-6,6-dimethyl-2-oxo-4(1H)-pyrimidinylidene) methyl]-2(1H)-pyrimidinone. The activity assay showed that two compounds, 5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (2) and 1H-pyrrole-2-carboxylic acid (3), had nematicidal activities against Meloidogyne javanica with mortalities of 69.93 and 90.54% at 400 ppm for 96 h, respectively. These two compounds were further tested for the inhibition activity of eggs hatching, and compound 3 showed a significant inhibition rate of 63.36% at 50 ppm for 48 h. In the chemotactic activity assay, three compounds (1 to 3) were found to have concentration-dependent chemotactic activity, of which compound 1 showed attractive activity. This experiment explored the active metabolites of L. gummosus YMF3.00690 against M. javanica and laid the foundation for biopesticide development.

Lysobacter gummosus 10.1.1, a Producer of Antimicrobial Agents.

This work investigated the antimicrobial potential of Lysobacter gummosus 10.1.1. The culture fluid of the strain was found to contain antimicrobial agents active against Staphylococcus aureus, Micrococcus luteus, and Bacillus cereus. L. gummosus was first shown to be capable of forming outer membrane vesicles, which have a bacteriolytic effect against not only Gram-positive bacteria but also against the Gram-negative pathogen Pseudomonas aeruginosa. Transcriptomic analysis revealed the genes of almost all known bacteriolytic enzymes of Lysobacter, as well as the genes of enzymes with putative bacteriolytic activity. Also identified were genes involved in the biosynthesis of a number of secondary metabolites for which antimicrobial activities are known. This research is indicative of the relevance of isolating and studying L. gummosus antimicrobial agents.

Iron ions regulate antifungal HSAF biosynthesis in Lysobacter enzymogenes by manipulating the DNA-binding affinity of the ferric uptake regulator (Fur).

Heat-stable antifungal factor (HSAF), produced by Lysobacter enzymogenes OH11, is regarded as a potential biological pesticide due to its broad-spectrum antifungal activity and novel mode of action. However, the current production of HSAF is low and cannot meet the requirements for large-scale production. Herein, we discovered that iron ions greatly promoted HSAF production, and the ferric uptake regulator (Fur) was involved in this regulatory process. Fur was also found to participate in the regulation of iron homeostasis in OH11 via the classic inhibition mechanism of Holo-Fur. Furthermore, Fur was collectively observed to directly bind to the promoter of the HSAF biosynthesis gene, and its DNA-binding affinity was attenuated by the addition of iron ions in vitro and in vivo. Its regulatory mechanism followed the uncommon inhibition mechanism of Apo-Fur. In summary, Fur exhibited a bidirectional regulatory mechanism in OH11. This study reveals a novel regulatory mechanism whereby Fur upregulates the biosynthesis of secondary metabolites. These findings contribute to the improvement of HSAF production and may guide its development into biological pesticides. IMPORTANCE HSAF possesses potent and broad antifungal activity with a novel mode of action. The HSAF yield is critical for fermentation production. In this study, iron ions were found to increase HSAF production, and the specific mechanism was elaborated. These results provide theoretical support for genetic transformation to improve HSAF yield, supporting its development into biological pesticides.

Characterization and Antioxidant Activity of Exopolysaccharides Produced by Lysobacter soyae sp. nov Isolated from the Root of Glycine max L.

Microbial exopolysaccharides (EPSs) have attracted attention from several fields due to their high industrial applicability. In the present study, rhizosphere strain CJ11T was isolated from the root of Glycine max L. in Goyang-si, Republic of Korea, and a novel exopolysaccharide was purified from the Lysobacter sp. CJ11T fermentation broth. The exopolysaccharide's average molecular weight was 0.93 × 105 Da. Its monosaccharide composition included 72.2% mannose, 17.2% glucose, 7.8% galactose, and 2.8% arabinose. Fourier-transform infrared spectroscopy identified the exopolysaccharide carbohydrate polymer functional groups, and the structural properties were investigated using nuclear magnetic resonance. In addition, a microstructure of lyophilized EPS was determined by scanning electron microscopy. Using thermogravimetric analysis, the degradation of the exopolysaccharide produced by strain CJ11T was determined to be 210 °C. The exopolysaccharide at a concentration of 4 mg/mL exhibited 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity of 73.47%. Phylogenetic analysis based on the 16S rRNA gene sequencing results revealed that strain CJ11T was a novel isolate for which the name Lysobacter soyae sp. nov is proposed.

A novel and high-efficient method for the preparation of heat-stable antifungal factor from Lysobacter enzymogenes by high-speed counter-current chromatography.

Heat-stable antifungal factor (HSAF) produced by the biocontrol bacterium Lysobacter enzymogenes shows considerable antifungal activity and has broad application potential in the agricultural and medical fields. There is a great demand for pure HSAF compounds in academic or industrial studies. However, an efficient preparation method that produces a high yield and high purity of HSAF is lacking, limiting the development of HSAF as a new drug. In the present study, high-speed counter-current chromatography (HSCCC) combined with column chromatography was successfully developed for the separation and preparation of HSAF from the crude extract of L. enzymogenes OH11. The crude extract was obtained by macroporous resin adsorption and desorption, and the main impurities were partly removed by ultraviolet light (254 nm) and gel filtration (Sephadex LH-20). In the HSCCC procedure, the selected suitable two-phase solvent system (n-hexane/ethyl acetate/methanol/water = 3:5:4:5, v/v, the lower phase added with 0.1% TFA) with a flow rate of 2.0 mL/min and a sample loading size of 100 mg was optimized for the separation. As a result, a total of 42 mg HSAF with a purity of 97.6% and recovery of 91.7% was yielded in one separation. The structure elucidation based on HR-TOF-MS, 1H and 13C NMR, and antifungal activities revealed that the isolated compound was unambiguously identified as HSAF. These results are helpful for separating and producing HSAF at an industrial scale, and they further demonstrate that HSCCC is a useful tool for isolating bioactive constituents from beneficial microorganisms.

Assembly of an active microbial consortium by engineering compatible combinations containing foreign and native biocontrol bacteria of kiwifruit.

Assembling functional bacterial biocontrol consortia is expected to expand the scope and efficiency of biocontrol agents. Generally, bacterial interspecies interactions lead to incompatibility events, as bacteria can produce antibacterial compounds and/or assemble contact-dependent killing (CDK) devices. Here, we aimed to assemble a bacterial consortium comprising Lysobacter enzymogenes OH11 and Bacillus safensis ZK-1 for the synergistic control of bacterial and fungal diseases of kiwifruit. ZK-1, a native kiwifruit biocontrol bacterium, is effective against Pseudomonas syringae pv. actinidiae (Psa) that causes bacterial kiwifruit canker, but has weak antifungal activity. OH11 is a foreign kiwifruit biocontrol agent with strong antifungal activity. While OH11 was unable to produce anti-Gram-negative metabolites, this strain could utilize type IV secretion system as an antibacterial CDK weapon. We first observed that OH11 could inhibit growth of ZK-1 by generating diffusible anti-Gram-positive antibiotic WAP-8294A2, whereas ZK-1 failed to generate diffusible antibacterial compound to inhibit growth of OH11. To disrupt this interspecies incompatibility, we generated a transgenic OH11-derived strain, OH11W, by deleting the WAP-8294A2 biosynthetic gene and found that OH11W did not kill ZK-1. We further observed that when OH11W and ZK-1 were co-inoculated on agar plates, no CDK effect was observed between them, whereas co-culture of OH11W or ZK-1 with Psa on agar plates resulted in Psa killing, suggesting L. enzymogenes and B. safensis assemble antibacterial CDK weapons against bacterial pathogens, and these CDK weapons did not affect the compatibility between OH11W and ZK-1. Based on these findings, we assembled an OH11W/ZK-1 dependent consortium that was shown to be functional in controlling bacterial canker and several representative fungal diseases of kiwifruit.

Genetic Basis and Expression Pattern Indicate the Biocontrol Potential and Soil Adaption of Lysobacter capsici CK09.

Lysobacter species have attracted increasing attention in recent years due to their capacities to produce diverse secondary metabolites against phytopathogens. In this research, we analyzed the genomic and transcriptomic patterns of Lysobacter capsici CK09. Our data showed that L. capsici CK09 harbored various contact-independent biocontrol traits, such as fungal cell wall lytic enzymes and HSAF/WAP-8294A2 biosynthesis, as well as several contact-dependent machineries, including type 2/4/6 secretion systems. Additionally, a variety of hydrolytic enzymes, particularly extracellular enzymes, were found in the L. capsici CK09 genome and predicted to improve its adaption in soil. Furthermore, several systems, including type 4 pili, type 3 secretion system and polysaccharide biosynthesis, can provide a selective advantage to L. capsici CK09, enabling the species to live on the surface in soil. The expression of these genes was then confirmed via transcriptomic analysis, indicating the activities of these genes. Collectively, our research provides a comprehensive understanding of the biocontrol potential and soil adaption of L. capsici CK09 and implies the potential of this strain for application in the future.

Lysohexaenetides A and B, linear lipopeptides from Lysobacter sp. DSM 3655 identified by heterologous expression in Streptomyces.

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.

Transcriptomic Analysis Followed by the Isolation of Extracellular Bacteriolytic Proteases from Lysobacter capsici VKM B-2533T.

The aim of the study was to search for, isolate and characterize new bacteriolytic enzymes that show promising potential for their use in medicine, agriculture and veterinary. Using a transcriptomic analysis, we annotated in Lysobacter capsici VKM B-2533T the genes of known bacteriolytic and antifungal enzymes, as well as of antibiotics, whose expression levels increased when cultivated on media conducive to the production of antimicrobial agents. The genes of the secreted putative bacteriolytic proteases were also annotated. Two new bacteriolytic proteases, Serp and Serp3, were isolated and characterized. The maximum bacteriolytic activities of Serp and Serp3 were exhibited at low ionic strength of 10 mM Tris-HCl, and high temperatures of, respectively, 80 °C and 70 °C. The pH optimum for Serp was 8.0; for Serp3, it was slightly acidic, at 6.0. Both enzymes hydrolyzed autoclaved cells of Micrococcus luteus Ac-2230T, Proteus vulgaris H-19, Pseudomonas aeruginosa and Staphylococcus aureus 209P. Serp also digested cells of Bacillus cereus 217. Both enzymes hydrolyzed casein and azofibrin. The newly discovered enzymes are promising for developing proteolytic antimicrobial drugs on their basis.

Lysobacter changpingensis sp. nov., a novel species of the genus Lysobacter isolated from a rhizosphere soil of strawberry in China.

In the present work, we characterized in detail strain CM-3-T8T, which was isolated from the rhizosphere soil of strawberries in Beijing, China, in order to elucidate its taxonomic position. Cells of strain CM-3-T8T 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-T8T formed a stable clade with Lysobacter soli DCY21T and Lysobacter panacisoli CJ29T, 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-T8T (= ACCC 61714 T = JCM 34576 T) represents a new species within the genus Lysobacter, for which the name Lysobacter changpingensis sp. nov. is proposed.

p-Aminobenzoic acid inhibits the growth of soybean pathogen Xanthomonas axonopodis pv. glycines by altering outer membrane integrity.

BACKGROUND: p-Aminobenzoic acid (pABA) is an environmentally friendly bioactive metabolite synthesized by Lysobacter antibioticus. This compound showed an unusual antifungal mode of action based on cytokinesis inhibition. However, the potential antibacterial properties of pABA remain unexplored. RESULTS: In this study, pABA showed antibacterial activity against Gram-negative bacteria. This metabolite inhibited growth (EC50 = 4.02 mM), and reduced swimming motility, extracellular protease activity, and biofilm formation in the soybean pathogen Xanthomonas axonopodis pv. glycines (Xag). Although pABA was previously reported to inhibit fungal cell division, no apparent effect was observed on Xag cell division genes. Instead, pABA reduced the expression of various membrane integrity-related genes, such as cirA, czcA, czcB, emrE, and tolC. Consistently, scanning electron microscopy observations revealed that pABA caused major alternations in Xag morphology and blocked the formation of bacterial consortiums. In addition, pABA reduced the content and profile of outer membrane proteins and lipopolysaccharides in Xag, which may explain the observed effects. Preventive and curative applications of 10 mM pABA reduced Xag symptoms in soybean plants by 52.1% and 75.2%, respectively. CONCLUSIONS: The antibacterial properties of pABA were studied for the first time, revealing new insights into its potential application for the management of bacterial pathogens. Although pABA was previously reported to show an antifungal mode of action based on cytokinesis inhibition, this compound inhibited Xag growth by altering the outer membrane's integrity. © 2023 Society of Chemical Industry.

Physiological and genomic analyses of cobalamin (vitamin B12)-auxotrophy of Lysobacter auxotrophicus sp. nov., a methionine-auxotrophic chitinolytic bacterium isolated from chitin-treated soil.

A novel bacterium, designated 5-21aT, isolated from chitin-treated upland soil, exhibits methionine (Met) auxotrophy and chitinolytic activity. A physiological experiment revealed the cobalamin (synonym, vitamin B12)(Cbl)-auxotrophic property of strain 5-21aT. The newly determined complete genomic sequence indicated that strain 5-21aT possesses only the putative gene for Cbl-dependent Met synthase (MetH) and lacks that for the Cbl-independent one (MetE), which implies the requirement of Cbl for Met-synthesis in strain 5-21aT. The set of genes for the upstream (corrin ring synthesis) pathway of Cbl synthesis is absent in the genome of strain 5-21aT, which explains the Cbl-auxotrophy of 5-21aT. This strain was characterized via a polyphasic approach to determine its taxonomic position. The nucleotide sequences of two copies of the 16S rRNA gene of strain 5-21aT indicated the highest similarities to Lysobacter soli DCY21T(99.8 and 99.9 %) and Lysobacter panacisoli CJ29T(98.7 and 98.8 %, respectively), whose Cbl-auxotrophic properties were revealed in this study. The principal respiratory quinone was Q-8. The predominant cellular fatty acids were iso-C15:0, iso-C16:0 and iso-C17:1 ω9c. The complete genome sequence of strain 5-21aT revealed that the genome size was 4 155 451 bp long and the G+C content was 67.87 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain 5-21aT and its most closely phylogenetic relative L. soli DCY21T were 88.8 and 36.5%, respectively. Based on genomic, chemotaxonomic, phenotypic and phylogenetic data, strain 5-21aT represents a novel species in the genus Lysobacter, for which the name Lyobacter auxotrophicus sp. nov. is proposed. The type strain is 5-21aT (=NBRC 115507T=LMG 32660T).

LexR Positively Regulates the LexABC Efflux Pump Involved in Self-Resistance to the Antimicrobial Di-N-Oxide Phenazine in Lysobacter antibioticus.

Myxin, a di-N-oxide phenazine isolated from the soil bacterium Lysobacter antibioticus, exhibits potent activity against various microorganisms and has the potential to be developed as an agrochemical. Antibiotic-producing microorganisms have developed self-resistance mechanisms to protect themselves from autotoxicity. Antibiotic efflux is vital for such protection. Recently, we identified a resistance-nodulation-division (RND) efflux pump, LexABC, involved in self-resistance against myxin in L. antibioticus. Expression of its genes, lexABC, was induced by myxin and was positively regulated by the LysR family transcriptional regulator LexR. The molecular mechanisms, however, have not been clear. Here, LexR was found to bind to the lexABC promoter region to directly regulate expression. Moreover, myxin enhanced this binding. Molecular docking and surface plasmon resonance analysis showed that myxin bound LexR with valine and lysine residues at positions 146 (V146) and 195 (K195), respectively. Furthermore, mutation of K195 in vivo led to downregulation of the gene lexA. These results indicated that LexR sensed and bound with myxin, thereby directly activating the expression of the LexABC efflux pump and increasing L. antibioticus resistance against myxin. IMPORTANCE Antibiotic-producing bacteria exhibit various sophisticated mechanisms for self-protection against their own secondary metabolites. RND efflux pumps that eliminate antibiotics from cells are ubiquitous in Gram-negative bacteria. Myxin is a heterocyclic N-oxide phenazine with potent antimicrobial and antitumor activities produced by the soil bacterium L. antibioticus. The RND pump LexABC contributes to the self-resistance of L. antibioticus against myxin. Herein, we report a mechanism involving the LysR family regulator LexR that binds to myxin and directly activates the LexABC pump. Further study on self-resistance mechanisms could help the investigation of strategies to deal with increasing bacterial antibiotic resistance and enable the discovery of novel natural products with resistance genes as selective markers.

Identification and Biosynthetic Study of the Siderophore Lysochelin in the Biocontrol Agent Lysobacter enzymogenes.

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, N1,N8-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.

Lysobacter enzymogenes prevents Phytophthora infection by inhibiting pathogen growth and eliciting plant immune responses.

The Phytophthora pathogen causes enormous damage to important agricultural plants. This group of filamentous pathogens is phylogenetically distant from fungi, making them difficult to control by most chemical fungicides. Lysobacter enzymogenes OH11 (OH11) is a biocontrol bacterium that secretes HSAF (Heat-Stable Antifungal Factor) as a broad-spectrum antifungal weapon. Here, we showed that OH11 could also control a variety of plant Phytophthora diseases caused by three major oomycetes (P. sojae, P. capsici and P. infestans). We provided abundant evidence to prove that OH11 protected host plants from Phytophthora pathogen infection by inhibiting mycelial growth, digesting cysts, suppressing cyst germination, and eliciting plant immune responses. Interestingly, the former two processes required the presence of HSAF, while the latter two did not. This suggested that L. enzymogenes could prevent Phytophthora infection via multiple previously unknown mechanisms. Therefore, this study showed that L. enzymogenes could serve as a promising alternative resource for promoting plant resistance to multiple Phytophthora pathogens.