Establishment and application of quantitative detection of Bacillus velezensis HMB26553, a biocontrol agent against cotton damping-off caused by Rhizoctonia.

A highly sensitive quantitative PCR (qPCR) method was developed for detection and quantification of Bacillus velezensis HMB26553 in cotton rhizosphere. The study aimed to develop a quantitative detection method for the strain HMB26553, and explore the relationship between its colonization of the cotton rhizosphere and its control effect. The whole genome sequence of strain HMB26553 was obtained by genome sequencing and a unique specific sequence pB-gene0026 on plasmid plaBV2 was identified by using high-throughput alignment against NCBI. Plasmid plaBV2 could be stably genetically inherited. Based on this sequence, specific primers for amplifying 106 bp and a minor groove binder (MGB) TaqMan probe for enhancing sensitivity were designed. The copy number of plaBV2 in strain HMB26553, which was 2, was confirmed by internal reference primers and the MGB TaqMan probe based on housekeeping gene gyrB. The established detection technique based on these primers and probes had high specificity and sensitivity compared to traditional plate counting method, with a detection limit of 1.5 copy genome. Using this method, the study discovered a likely correlation between the quantity of colonization in cotton rhizosphere and efficacy against cotton damping-off caused by Rhizoctonia after seed soaking and irrigation with strain HMB26553. Thus, this method provides scientific support for the rational application of strain HMB26553 in the future.

Complete genome sequence of Bacillus velezensis B31, a potential biocontrol agent with ability of tolerance to fusaric acid and antagonism against Fusarium oxysporum.

Bacillus velezensis B31 is tolerant to fusaric acid, exhibits antagonism against Fusarium oxysporum, and has an excellent control effect on tomato fusarium wilt. Here, we present the complete genome sequence of B31, which contains 4,056,755 bp DNA with a G + C ratio of 46.39%. The genome has 3,838 protein-coding genes.

Arabinose Plays an Important Role in Regulating the Growth and Sporulation of Bacillus subtilis NCD-2.

A microbial fungicide developed from Bacillus subtilis NCD-2 has been registered for suppressing verticillium wilt in crops in China. Spores are the main ingredient of this fungicide and play a crucial role in suppressing plant disease. Therefore, increasing the number of spores of strain NCD-2 during fermentation is important for reducing the cost of the fungicide. In this study, five kinds of carbon sources were found to promote the metabolism of strain NCD-2 revealed via Biolog Phenotype MicroArray (PM) technology. L-arabinose showed the strongest ability to promote the growth and sporulation of strain NCD-2. L-arabinose increased the bacterial concentration and the sporulation efficiency of strain NCD-2 by 2.04 times and 1.99 times compared with D-glucose, respectively. Moreover, L-arabinose significantly decreased the autolysis of strain NCD-2. Genes associated with arabinose metabolism, sporulation, spore resistance to heat, and spore coat formation were significantly up-regulated, and genes associated with sporulation-delaying protein were significantly down-regulated under L-arabinose treatment. The deletion of msmX, which is involved in arabinose transport in the Bacillus genus, decreased growth and sporulation by 53.71% and 86.46% compared with wild-type strain NCD-2, respectively. Complementing the mutant strain by importing an intact msmX gene restored the strain's growth and sporulation.

Characterization of a Bacillus subtilis S-16 Thiazole-Synthesis-Related Gene thiS Knockout and Antimicrobial Activity Analysis.

Bacillus subtilis S-16 isolated from sunflower-rhizosphere soil is an effective biocontrol agent for preventing soilborne diseases in plants. Previous research revealed that the volatile organic compounds (VOCs) produced by the S-16 strain have strong inhibitory effects on Sclerotinia sclerotiorum. The identification of the VOCs of S-16 using gas chromatography-tandem mass spectrometry (GC-MS/MS) revealed 35 compounds. Technical-grade formulations of four of these compounds were chosen for further study: 2-pentadecanone, 6,10,14-trimethyl-2-octanone, 2-methyl benzothiazole (2-MBTH), and heptadecane. The major constituent, 2-MBTH, plays an important role in the antifungal activity of the VOCs of S-16 against the growth of Sclerotinia sclerotiorum. The purpose of this study was to determine the impact of the thiS gene's deletion on the 2-MBTH production and to conduct an antimicrobial activity analysis of the Bacillus subtilis S-16. The thiazole-biosynthesis gene was deleted via homologous recombination, after which the contents of 2-MBTH in the wild-type and mutant S-16 strains were analyzed using GC-MS. The antifungal effects of the VOCs were determined using a dual-culture technique. The morphological characteristics of the Sclerotinia sclerotiorum mycelia were examined via scanning-electron microscopy (SEM). Additionally, the lesion areas on the sunflower leaves with and without treatment with the VOCs from the wild-type and mutant strains were measured to explore the effects of the VOCs on the virulence of the Sclerotinia sclerotiorum. Moreover, the effects of the VOCs on the sclerotial production were assessed. We showed that the mutant strain produced less 2-MBTH. The ability of the VOCs produced by the mutant strain to inhibit the growth of the mycelia was also reduced. The SEM observation showed that the VOCs released by the mutant strain also caused more flaccid and gapped hyphae in the Sclerotinia sclerotiorum. The Sclerotinia sclerotiorum treated by the VOCs produced by the mutant strains caused more damage to the leaves than that treated by the VOCs produced by the wild type and the mutant-strain-produced VOCs inhibited sclerotia formation less. The production of 2-MBTH and its antimicrobial activities were adversely affected to varying degrees by the deletion of thiS.

Screening Biocontrol Agents for Cash Crop Fusarium Wilt Based on Fusaric Acid Tolerance and Antagonistic Activity against Fusarium oxysporum.

Fusarium wilt, caused by Fusarium oxysporum, is one of the most notorious diseases of cash crops. The use of microbial fungicides is an effective measure for controlling Fusarium wilt, and the genus Bacillus is an important resource for the development of microbial fungicides. Fusaric acid (FA) produced by F. oxysporum can inhibit the growth of Bacillus, thus affecting the control efficacy of microbial fungicides. Therefore, screening FA-tolerant biocontrol Bacillus may help to improve the biocontrol effect on Fusarium wilt. In this study, a method for screening biocontrol agents against Fusarium wilt was established based on tolerance to FA and antagonism against F. oxysporum. Three promising biocontrol bacteria, named B31, F68, and 30833, were obtained to successfully control tomato, watermelon, and cucumber Fusarium wilt. Strains B31, F68, and 30833 were identified as B. velezensis by phylogenetic analysis of the 16S rDNA, gyrB, rpoB, and rpoC gene sequences. Coculture assays revealed that strains B31, F68, and 30833 showed increased tolerance to F. oxysporum and its metabolites compared with B. velezensis strain FZB42. Further experiments confirmed that 10 µg/mL FA completely inhibited the growth of strain FZB42, while strains B31, F68, and 30833 maintained normal growth at 20 µg/mL FA and partial growth at 40 µg/mL FA. Compared with strain FZB42, strains B31, F68, and 30833 exhibited significantly greater tolerance to FA.

Functional Analyses of the Bacillus velezensis HMB26553 Genome Provide Evidence That Its Genes Are Potentially Related to the Promotion of Plant Growth and Prevention of Cotton Rhizoctonia Damping-Off.

Bacillus spp. is one kind of the important representative biocontrol agents against plant diseases and promoting plant growth. In this study, the whole genomic sequence of bacterial strain HMB26553 was obtained. A phylogenetic tree based on the genome and ANI (average nucleotide identity), as well as dDDH (digital DNA-DNA hybridization), was constructed, and strain HMB26553 was identified as Bacillus velezensis. Fourteen biosynthetic gene clusters responsible for secondary metabolite were predicted via anti-SMASH, and six secondary metabolites were identified by UHPLC-QTOF-MS/MS (ultra-high-performance liquid chromatography coupled to quadrupole-time-of-flight tandem mass spectrometry). When the phytopathogen Rhizoctonia solani was treated with B. velezensis HMB26553, the mycelial structure changed, ROS (reactive oxygen species) accumulated, and the mitochondrial membrane potential decreased. Characteristics of strain HMB26553 were predicted and confirmed by genomic information and experiments, such as producing IAA, siderophore, extracellular enzymes and biofilm, as well as moving and promoting cotton growth. All these results suggested the mechanisms by which B. velezensis HMB26553 inhibits pathogen growth and promotes cotton growth, which likely provided the potential biocontrol agent to control cotton Rhizoctonia damping-off.

Colonization Ability of Bacillus subtilis NCD-2 in Different Crops and Its Effect on Rhizosphere Microorganisms.

Bacillus subtilis strain NCD-2 is a promising biocontrol agent for soil-borne plant diseases and shows potential for promoting the growth of some crops. The purposes of this study were to analyze the colonization ability of strain NCD-2 in different crops and reveal the plant growth promotion mechanism of strain NCD-2 by rhizosphere microbiome analysis. qRT-PCR was used to determine the populations of strain NCD-2, and microbial communities' structures were analyzed through amplicon sequencing after application of strain NCD-2. Results demonstrated that strain NCD-2 had a good growth promotion effect on tomato, eggplant and pepper, and it was the most abundant in eggplant rhizosphere soil. There were significantly differences in the types of beneficial microorganisms recruited for different crops after application of strain NCD-2. PICRUSt analysis showed that the relative abundances of functional genes for amino acid transport and metabolism, coenzyme transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism, and defense mechanisms were enriched in the rhizospheres of pepper and eggplant more than in the rhizospheres of cotton, tomato and maize after application of strain NCD-2. In summary, the colonization ability of strain NCD-2 for five plants was different. There were differences in microbial communities' structure in rhizosphere of different plants after application of strain NCD-2. Based on the results obtained in this study, it was concluded that the growth promoting ability of strain NCD-2 were correlated with its colonization quantity and the microbial species it recruited.

Effect of incorporation of broccoli residues into soil on occurrence of verticillium wilt of spring-sowing-cotton and on rhizosphere microbial communities structure and function.

Cotton verticillium wilt (CVW) represented a typical plant soil-borne disease and resulted in widespread economic losses in cotton production. However, the effect of broccoli residues (BR) on verticillium wilt of spring-sowing-cotton was not clear. We investigated the effects of BR on CVW, microbial communities structure and function in rhizosphere of two cotton cultivars with different CVW resistance using amplicon sequencing methods. Results showed that control effects of BR on CVW of susceptible cultivar (cv. EJ-1) and resistant cultivar (cv. J863) were 58.49% and 85.96%, and the populations of V. dahliae decreased by 14.31% and 34.19%, respectively. The bacterial diversity indices significantly increased in BR treatment, while fungal diversity indices significantly decreased. In terms of microbial community composition, the abilities to recruit bacteria and fungi were enhanced in BR treatment, including RB41, Gemmatimonas, Pontibacter, Streptomyces, Blastococcus, Massilia, Bacillus, and Gibberella, Plectosphaerella, Neocosmospora, Aspergillus and Preussia. However, the relative abundances of Sphingomonas, Nocardioides, Haliangium, Lysobacter, Penicillium, Mortierella and Chaetomidium were opposite tendency between cultivars in BR treatment. According to PICRUSt analysis, functional profiles prediction showed that significant shifts in metabolic functions impacting KEGG pathways of BR treatment were related to metabolism and biosynthesis. FUNGuild analysis indicated that BR treatment altered the relative abundances of fungal trophic modes. The results of this study demonstrated that BR treatment decreased the populations of V. dahliae in soil, increased bacterial diversity, decreased fungal diversity, changed the microbial community structure and function, and increased the abundances of beneficial microorganisms.

Insights into complex infection by two Pectobacterium species causing potato blackleg and soft rot.

Pectobacterium spp. are causative agents of blackleg and soft rot of potato. However, little is known about the relationship between the pathogenicity of mixed infections of different Pectobacterium spp. at different temperatures. In this study, two pectinolytic strains of Pectobacterium spp. were isolated from the same potato plant with typical symptoms of blackleg and identified as P. brasiliense and P. carotovorum by multilocus sequence analysis (MLSA), whole-genome phylogenetic tree construction, average nucleotide identity (ANI) analysis and digital DNA-DNA hybridization (dDDH). Plant cell wall degrading enzyme, including pectinases, cellulases and proteases, as the most important virulence factors, as well as pathogenicity toward potato tuber, were compared between the strains P. brasiliense BL-2 and P. carotovorum BL-4 at 28 â„ƒ. The results showed that P. carotovorum had higher cell wall-degrading enzyme activities and brought more severe disease symptoms to potato tubers than P. brasiliense. Moreover, the pathogenicity of P. carotovorum and P. brasiliense increased with increasing temperature (20, 25, 28, 32 â„ƒ). The pathogenicity was more severe when P. carotovorum strain BL-4 was co-inoculated with P. brasiliense strain BL-2, especially when the former exhibited an advantage in bacterial number at the initial time. The results of this study provide new insight for understanding the pathogenicity caused by mixed infections with different species of Pectobacterium spp., and they may provide some guidance for controlling potato blackleg and soft rot.

iTRAQ-based proteomic analysis of Bacillus subtilis strain NCD-2 regulated by PhoPR two-component system: A comparative analysis with transcriptomics revealed the regulation for fengycin production by branched chain amino acids.

The PhoPR two-component system (TCS) is a signal transduction pathway to regulate the phosphate starvation response in Bacillus subtilis and regulated fengycin production in strain NCD-2 under low phosphate condition. The purpose of this study was to characterize the proteome level responses in the phoP-null mutant (MP) and the phoR-null mutant (MR), and to integrate the proteomics with the transcriptomic data obtained previously. The metabolic pathway for fengycin was predicted based on omics analysis as well as molecular genetics assay. Results showed the proteins and genes associated with biosynthesis of branched chain amino acids (BCAAs) were regulated by PhoPR TCS, and liquid chromatography mass spectrometry (LC-MS) analysis also confirmed that the production of BCAAs was down-regulated in the MP and MR mutants, when compared to wild-type strain NCD-2. Protein network analysis showed that the BCAA metabolism was linked to the biosynthesis of lipopeptides. The MP and MR strains decreased the fengycin production when cultured in modified Landy medium supplied with 0.42 mM phosphate, however, the fengycin production could be restored when the glutamic acid was replaced with BCAAs that were added to modified Landy medium. The lpdV gene, which is responsible for the BCAA degradation process, was deleted in strain NCD-2. Compared with the wild-type strain, the lpdV mutant produced significantly less fengycin in the medium supplied with BCAAs. Considered together, the results of this study indicate that the PhoPR TCS regulates fengycin production by affecting BCAA biosynthesis.

First Report of Verticillium Wilt of Watermelon Caused by Verticillium dahliae in China.

Watermelon (Citrullus lanatus T.) is one of the most important economic crops in China. Soil-borne diseases are becoming more and more serious with longer growing seasons and continuous cropping of watermelon in greenhouses. In May 2020, symptoms were observed on plants in greenhouses located at Xingtai, Hebei province of China and included wilted leaves, chlorosis and plant death. Among the 26 greenhouses examined, symptomatic plants were observed in 17 greenhouses. The incidences of infected plants ranged from 1% to 35%, and caused an average 10% yield loss. Symptoms began on lower part of the plants and progressed upward to the vines and leaves. At the early stage of infection, the edge of watermelon leaves changed from green to yellow, and became soft. As the disease progressed, infected leaves wilted and desicated. The vascular tissue of the stem exhibited a uniform brown discoloration that often extended throughout the vine. To identify the causal agent, small pieces approximate 3.0×3.0 mm size of infected stem tissues were collected and sterilized with 0.5% sodium hypochlorite solution for 1 min, rinsed three times with sterile water and transferred onto potato dextrose agar (PDA) medium amended with 100 µg·mL-1 of chloramphenicol. The plates were incubated at 25°C for 3 days in the dark and fungal isolates were purified using the single-spore isolation method. A total of 22 fungal isolates with identical colony morphology were collected from diseased plants. The color of the fungal colonies on PDA medium was creamy-white with an abundance of mycelia that darken after 5 days growth due to the formation of microsclerotia. Fungal colonies consisted of fine, hyaline hyphae with verticillate conidiophores producing hyaline, ellipsoidal to oval conidia with an average size of 5.12×3.41 µm (n=50). The morphological characters of the fungal isolates were identical to those of Verticillium dahliae Kleb. described by Hawksworth and Talboys (Hawksworth, D. and Talboys, P, 1970). Pathogenicity tests were performed by soaking 30 watermelon seedlings with wounded root tips in the fungal conidial suspension (1x107 conidium/mL) for 30 min (Ma, et al, 2004). The same number of non-inoculated watermelon seedlings was used as a control. All plants were kept in a greenhouse at 25°C and 90%-95% relative humidity. Seven days post-inoculation (dpi), leaves of treated plants began to show symptoms of wilt. At 10-dpi, lower leaves wilted and dry and by 15-dpi, whole plants were dead. Pathogenicity tests were repeated three times with consistent results. The pathogen was re-isolated from the diseased plants and displayed identical morphological characteristics to the original isolates. To further identity the pathogens, the ribosomal DNA Internal Transcribed Spacer (rDNA-ITS) region was amplified by PCR (White et al., 1990; Liu et al., 1999; Bellemain et al.. 2010). The amplicon was sequenced and showed 99%-100% identity to the ITS region of the V. dahliae reference strains deposited in the NCBI database (MK093977.1, MK287620.1, MT348570.1 and LC549667.1, respectively). Based on morphological and ITS sequence information, the fungal pathogen was identified as V. dahliae. V. dahliae is an economically important pathogen with a wide host range worldwide. The discovery of Verticillium wilt on watermelons indicates that there might be a risk of Verticillium wilt when watermelons are planted in subsequent crops of the host plants of the disease, such as cotton or eggplant. To our knowledge, this is the first report of V. dahliae causing Verticillium wilt of watermelon in China. Financed: the Special Fund for Agro-scientific Research in the Public Interest, China (201503109) References: Hawksworth, D. and Talboys, P. 1970. Description of Pathogenic Fungi and Bacteria, CMI, Surrey. Ma, P., et al. 2004. A New Inoculation Method for Verticillium Wilt on Cotton and Its Application in Evaluating Pathogenesis and Host Resistance. Acta Phytopathologica Sinica, 34(6): 536-541. White, T. J., et al. 1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. PCR protocols: a guide to methods and applications, 18(1), 315-322. Bellemain, E., et al. 2010. ITS as an Environmental DNA Barcode for Fungi: an in Silico Approach Reveals Potential PCR Biases. BMC microbiology, 10(1), 1-9. Liu, Y. J., et al. 1999. Phylogenetic Relationships Among Ascomycetes: Evidence from an RNA Polymerse II SubunitMol. Biol. Evol. 16:1799-1808.

Genome mining and UHPLC-QTOF-MS/MS to identify the potential antimicrobial compounds and determine the specificity of biosynthetic gene clusters in Bacillus subtilis NCD-2.

BACKGROUND: Bacillus subtilis strain NCD-2 is an excellent biocontrol agent against plant soil-borne diseases and shows broad-spectrum antifungal activities. This study aimed to explore some secondary metabolite biosynthetic gene clusters and related antimicrobial compounds in strain NCD-2. An integrative approach combining genome mining and structural identification technologies using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-MS/MS), was adopted to interpret the chemical origins of metabolites with significant biological activities. RESULTS: Genome mining revealed nine gene clusters encoding secondary metabolites with predicted functions, including fengycin, surfactin, bacillaene, subtilosin, bacillibactin, bacilysin and three unknown products. Fengycin, surfactin, bacillaene and bacillibactin were successfully detected from the fermentation broth of strain NCD-2 by UHPLC-QTOF-MS/MS. The biosynthetic gene clusters of bacillaene, subtilosin, bacillibactin, and bacilysin showed 100% amino acid sequence identities with those in B. velezensis strain FZB42, whereas the identities of the surfactin and fengycin gene clusters were only 83 and 92%, respectively. Further comparison revealed that strain NCD-2 had lost the fenC and fenD genes in the fengycin biosynthetic operon. The biosynthetic enzyme-related gene srfAB for surfactin was divided into two parts. Bioinformatics analysis suggested that FenE in strain NCD-2 had a similar function to FenE and FenC in strain FZB42, and that FenA in strain NCD-2 had a similar function to FenA and FenD in strain FZB42. Five different kinds of fengycins, with 26 homologs, and surfactin, with 4 homologs, were detected from strain NCD-2. To the best of our knowledge, this is the first report of a non-typical gene cluster related to fengycin synthesis. CONCLUSIONS: Our study revealed a number of gene clusters encoding antimicrobial compounds in the genome of strain NCD-2, including a fengycin synthetic gene cluster that might be unique by using genome mining and UHPLC-QTOF-MS/MS. The production of fengycin, surfactin, bacillaene and bacillibactin might explain the biological activities of strain NCD-2.

Using a phenotype microarray and transcriptome analysis to elucidate multi-drug resistance regulated by the PhoR/PhoP two-component system in Bacillus subtilis strain NCD-2.

The PhoRP two-component system (TCS), one of the most important signaling pathways in Bacillus subtilis, regulates cell physiological reactions mainly under phosphate starvation conditions. The mechanism by which PhoRP TCS regulates resistance towards antibiotics in B. subtilis strain NCD-2 was investigated in this study. Using phenotype microarray (PM) technology, the susceptibility of B. subtilis to 240 antimicrobial compounds was compared among the wild-type strain NCD-2, the phoR-null mutant (MR), and the phoP-null mutant (MP). Compared with the wild type, the MR mutant was more resistant to 13 antibiotics with different functions, and the MP mutant was more resistant to 14 antibiotics, of which 8 were 30S/50S ribosome-targeted. To investigate the molecular mechanisms involved in changing the level of antibiotic resistance, transcriptional analysis was performed to compare the differentially expressed genes among the wild-type strain and the MR and MP mutants. Compared with the wild-type strain, 294 genes were differentially expressed in the MR mutant, including 97 up-regulated genes and 197 down-regulated genes. Most of the differently expressed genes were associated with carbohydrate mechanism, amino acid mechanism, ABC-transporters and phosphotransferase systems. A total of 212 genes were differentially expressed in the MP mutant, including 10 up-regulated genes and 202 down-regulated genes, and most were associated with ribosome synthesis, amino acid metabolism, carbohydrate metabolism and ABC-transporters. The khtSTU operon (encoding the K+ efflux pump) that was up-regulated in the MP mutant was deleted by in-frame deletion in the MP mutant. The phoP and khtSTU operon double mutant MPK showed decreased antibiotic resistance to doxycycline, chlortetracycline, spiramycin, puromycin, and paromomycin when compared with the MP mutant. Thus, the results indicated that the khtSTU operon was responsible for the PhoP-mediated multiple antibiotic resistance.

Qualitative and Quantitative Analyses of the Colonization Characteristics of Bacillus subtilis Strain NCD-2 on Cotton Root.

Bacillus subtilis strain NCD-2 is an excellent biocontrol agent against plant soil-borne diseases. With the purpose of understanding the colonization characteristics of strain NCD-2, firstly, a constitutive expression promoter was cloned from strain NCD-2 and was used to construct GFP-labeled strain NCD-2. The GFP-labeled strain NCD-2 showed strong green fluorescence under planktonic cells and biofilm formation. The colonization characteristics of strain NCD-2 on different parts of cotton root were qualitatively observed by confocal laser scanning microscopy (CLSM). Results showed that strain NCD-2 mainly colonized on the zone of differentiation and elongation. Rhizosphere populations of B. subtilis strain NCD-2 on different cotton root were quantitatively evaluated by traditional plating count and quantitative PCR (qPCR) analysis in both autoclaved soil and non-autoclaved soil, respectively. Results showed that both traditional plating count and qPCR analysis showed similar trend for colonization characteristics of strain NCD-2. The greatest strain NCD-2 populations were in the root tip, at 9.19 × 107 CFU g-1 root and 6.75 × 107 CFU g-1 root as estimated by qPCR in non-autoclaved and autoclaved soil, respectively. This study provides a clearer understanding of the interactions between biocontrol agent and plant, as well as with the indigenous microorganisms in the soil.

Heat-Stable Antifungal Factor (HSAF) Biosynthesis in Lysobacter enzymogenes Is Controlled by the Interplay of Two Transcription Factors and a Diffusible Molecule.

Lysobacter enzymogenes is a Gram-negative, environmentally ubiquitous bacterium that produces a secondary metabolite, called heat-stable antifungal factor (HSAF), as an antifungal factor against plant and animal fungal pathogens. 4-Hydroxybenzoic acid (4-HBA) is a newly identified diffusible factor that regulates HSAF synthesis via L. enzymogenes LysR (LysRLe), an LysR-type transcription factor (TF). Here, to identify additional TFs within the 4-HBA regulatory pathway that control HSAF production, we reanalyzed the LenB2-based transcriptomic data, in which LenB2 is the enzyme responsible for 4-HBA production. This survey led to identification of three TFs (Le4806, Le4969, and Le3904). Of them, LarR (Le4806), a member of the MarR family proteins, was identified as a new TF that participated in the 4-HBA-dependent regulation of HSAF production. Our data show the following: (i) that LarR is a downstream component of the 4-HBA regulatory pathway controlling the HSAF level, while LysRLe is the receptor of 4-HBA; (ii) that 4-HBA and LysRLe have opposite regulatory effects on larR transcription whereby larR transcript is negatively modulated by 4-HBA while LysRLe, in contrast, exerts positive transcriptional regulation by directly binding to the larR promoter without being affected by 4-HBA in vitro; (iii) that LarR, similar to LysRLe, can bind to the promoter of the HSAF biosynthetic gene operon, leading to positive regulation of HSAF production; and (iv) that LarR and LysRLe cannot interact and instead control HSAF biosynthesis independently. These results outline a previously uncharacterized mechanism by which biosynthesis of the antibiotic HSAF in L. enzymogenes is modulated by the interplay of 4-HBA, a diffusible molecule, and two different TFs.IMPORTANCE Bacteria use diverse chemical signaling molecules to regulate a wide range of physiological and cellular processes. 4-HBA is an "old" chemical molecule that is produced by diverse bacterial species, but its regulatory function and working mechanism remain largely unknown. We previously found that 4-HBA in L. enzymogenes could serve as a diffusible factor regulating HSAF synthesis via LysRLe Here, we further identified LarR, an MarR family protein, as a second TF that participates in the 4-HBA-dependent regulation of HSAF biosynthesis. Our results dissected how LarR acts as a protein linker to connect 4-HBA and HSAF synthesis, whereby LarR also has cross talk with LysRLe Thus, our findings not only provide fundamental insight regarding how a diffusible molecule (4-HBA) adopts two different types of TFs for coordinating HSAF biosynthesis but also show the use of applied microbiology to increase the yield of the antibiotic HSAF by modification of the 4-HBA regulatory pathway in L. enzymogenes.

SERS Assay for Copper(II) Ions Based on Dual Hot-Spot Model Coupling with MarR Protein: New Cu2+-Specific Biorecognition Element.

We have developed a rapid and ultrasensitive surface-enhanced Raman scattering (SERS) assay for Cu2+ detection using the multiple antibiotic resistance regulator (MarR) as specific bridging molecules in a SERS hot-spot model. In the assay, Cu2+ induces formation of MarR tetramers, which provide Au nanoparticle (NP)-AuNP bridges, resulting in the formation of SERS hot spots. 4-Mercaptobenzoic acid (4-MBA) was used as a Raman reporter. The addition of Cu2+ increased the Raman intensity of 4-MBA. Use of a dual hot-spot signal-amplification strategy based on AuNP-AgNP heterodimers combined through antigen-antibody reactions increased the sensitivity of the sensing platform by 50-fold. The proposed method gave a linear response for Cu2+ detection in the range of 0.5-1000 nM, with a detection limit of 0.18 nM, which is 5 orders of magnitude lower than the U.S. Environmental Protection Agency limit for Cu2+ in drinking water (20 µM). In addition, all analyses can be completed in less than 15 min. The high sensitivity, high specificity, and rapid detection capacity of the SERS assay therefore provide a combined advantage over current assays.

Multipath colourimetric assay for copper(II) ions utilizing MarR functionalized gold nanoparticles.

We use the multiple antibiotic resistance regulator (MarR), as a highly selective biorecognition elements in a multipath colourimetric sensing strategy for the fast detection of Cu2+ in water samples. The colourimetric assay is based on the aggregation of MarR-coated gold nanoparticles in the presence of Cu2+ ions, which induces a red-to-purple colour change of the solution. The colour variation in the gold nanoparticle aggregation process can be used for qualitative and quantitative detection of Cu2+ by the naked eye, and with UV-vis and smartphone-based approaches. The three analysis techniques used in the multipath colourimetric assay complement each other and provide greater flexibility for differing requirements and conditions, making the assay highly applicable for Cu2+ detection. Under optimal conditions, the Cu2+ concentration was quantified in less than 5 min with limits of detection for the naked eye, UV-vis and smartphone-based approaches of 1 µM, 405 nM and 61 nM, respectively. Moreover, the sensing system exhibited excellent selectivity and practical application for Cu2+ detection in real water samples. Thus, our strategy has great potential for application in on-site monitoring of Cu2+, and the unique response of MarR towards copper ions may provide a new approach to Cu2+ sensing.

Lysobacter PilR, the Regulator of Type IV Pilus Synthesis, Controls Antifungal Antibiotic Production via a Cyclic di-GMP Pathway.

Lysobacter enzymogenes is a ubiquitous soil gammaproteobacterium that produces a broad-spectrum antifungal antibiotic, known as heat-stable antifungal factor (HSAF). To increase HSAF production for use against fungal crop diseases, it is important to understand how HSAF synthesis is regulated. To gain insights into transcriptional regulation of the HSAF synthesis gene cluster, we generated a library with deletion mutations in the genes predicted to encode response regulators of the two-component signaling systems in L. enzymogenes strain OH11. By quantifying HSAF production levels in the 45 constructed mutants, we identified two strains that produced significantly smaller amounts of HSAF. One of the mutations affected a gene encoding a conserved bacterial response regulator, PilR, which is commonly associated with type IV pilus synthesis. We determined that L. enzymogenes PilR regulates pilus synthesis and twitching motility via a traditional pathway, by binding to the pilA promoter and upregulating pilA expression. Regulation of HSAF production by PilR was found to be independent of pilus formation. We discovered that the pilR mutant contained significantly higher intracellular levels of the second messenger cyclic di-GMP (c-di-GMP) and that this was the inhibitory signal for HSAF production. Therefore, the type IV pilus regulator PilR in L. enzymogenes activates twitching motility while downregulating antibiotic HSAF production by increasing intracellular c-di-GMP levels. This study identifies a new role of a common pilus regulator in proteobacteria and provides guidance for increasing antifungal antibiotic production in L. enzymogenesIMPORTANCE PilR is a widespread response regulator of the two-component system known for regulating type IV pilus synthesis in proteobacteria. Here we report that, in the soil bacterium Lysobacter enzymogenes, PilR regulates pilus synthesis and twitching motility, as expected. Unexpectedly, PilR was also found to control intracellular levels of the second messenger c-di-GMP, which in turn inhibits production of the antifungal antibiotic HSAF. The coordinated production of type IV pili and antifungal antibiotics has not been observed previously.

4-Hydroxybenzoic acid is a diffusible factor that connects metabolic shikimate pathway to the biosynthesis of a unique antifungal metabolite in Lysobacter enzymogenes.

Heat-stable antifungal factor (HSAF) produced by Lysobacter enzymogenes is a potential lead compound for developing new antibiotics. Yet, how L. enzymogenes regulates the HSAF biosynthesis remains largely unknown. Here, we show that 4-hydroxybenzoic acid (4-HBA) serves as a diffusible factor for regulating HSAF biosynthesis. The biosynthesis of 4-HBA involved an oxygenase, LenB2, and mutation of lenB2 almost completely abolished 4-HBA production, leading to significantly impaired HSAF production. Introduction of a heterologous gene coding for 4-HBA biosynthetic enzyme into the lenB2 mutant restored the production of 4-HBA and HSAF to their corresponding wild-type levels. Exogenous addition of 0.5-1 µM 4-HBA was sufficient to restore HSAF production in the lenB2 mutant. Furthermore, the shikimate pathway was found to regulate the biosynthesis of HSAF via 4-HBA. Finally, we identified a LysR-family transcription factor (LysRLe ) with activity directed to HSAF production. LysRLe could bind to the HSAF promoter and, as a result, regulates expression of HSAF biosynthesis genes. The 4-HBA could bind to LysRLe and appeared to partly enhance formation of the LysRLe -DNA complex. Collectively, our findings suggest that L. enzymogenes produces 4-HBA to serve as an adaptor molecule to link the shikimate pathway to the biosynthesis of a unique antifungal metabolite (HSAF).

Transcriptomic analysis reveals new regulatory roles of Clp signaling in secondary metabolite biosynthesis and surface motility in Lysobacter enzymogenes OH11.

Lysobacter enzymogenes is a bacterial biological control agent emerging as a new source of antibiotic metabolites, such as heat-stable antifungal factor (HSAF) and the antibacterial factor WAP-8294A2. The regulatory mechanism(s) for antibiotic metabolite biosynthesis remains largely unknown in L. enzymogenes. Clp, a cyclic adenosine monophosphate (cAMP)-receptor-like protein, is shown to function as a global regulator in modulating biocontrol-associated traits in L. enzymogenes. However, the genetic basis of Clp signaling remains unclear. Here, we utilized transcriptome/microarray analysis to determine the Clp regulon in L. enzymogenes. We showed that Clp is a global regulator in gene expression, as the transcription of 775 genes belonging to 19 functional groups was differentially controlled by Clp signaling. Analysis of the Clp regulon detected previously characterized Clp-modulated functions as well as novel loci. These include novel loci involved in antibiotic metabolite biosynthesis and surface motility in L. enzymogenes. We further showed experimentally that Clp signaling played a positive role in regulating the biosynthesis of HSAF and WAP-8294A2, as well as surface motility which is a type-IV-pilus-dependent trait. The regulation by Clp signaling of antibiotic (HSAF and WAP-8294A2) biosynthesis and surface motility was found to be independent. Importantly, we identified a factor Lysobacter acetyltransferase (Lat), a homologue of histone acetyltransferase Hpa2, which was regulated by Clp and involved in HSAF biosynthesis, but not associated with WAP-8294A2 production and surface motility. Overall, our study provided new insights into the regulatory role and molecular mechanism of Clp signaling in L. enzymogenes.