The ComQXPA Quorum Sensing System May Play an Important Role in the Synthesis of Bacillomycin D in Bacillus Amyloliquefaciens Q-426

Background: Bacillus amyloliquefaciens Q-426 can secrete numerous cyclic lipopeptides that have antifungal and antitumor activities. ComQXPA is a common quorum sensing (QS) system in Bacillus species. Most B. amyloliquefaciens strains are encoding the QS gene cluster comQXPA, however, the biological function of the ComQXPA system in B. amyloliquefaciens has not been well studied. In this study, we identied the comQXPA gene locus and the chemical structure of ComX Q-426 in B. amyloliquefaciens Q-426, and explored the function of ComX Q-426 in regulating lipopeptide production. Results: We identied and analyzed the comQXPA locus in Q-426. The full length of the comQXPA gene cluster was 4,014 bp, including 912 bp of comQ, 165 bp of comX, 2292 bp of comP, and 645 bp of comA. The comQXPA locus belongs to group B, as comQ and comX overlap by only one base pair. ComX Q-426 consists of six amino acids (GGDWKY) that contain a modied tryptophan residue. The antifungal activity of Q426ΔcomX was signicantly affected, and almost no antifungal activity was observed, while the antifungal activity of strain Q426ΔcomX /comQX was restored to the same level as that of the wild-type strain. When the ComX Q-426 was added to the culture medium at a nal concentration of 8 μg/L at the early stage of the log-phase, the antifungal activity of the wild-type strain Q-426 was signicantly improved. Knocking out the comX gene did not affect the growth of the bacteria, however, the strain Q426ΔcomX lost its swimming ability, was unable to form colonies when spread on a solid surface, and could not form biolms on the interface between the gas and liquid medium. Conclusions: Disruption of the ComPA signaling pathway in the Q-426 strain resulted in

The ComQXPA QS system of B. subtilis is a typical QS system of Bacillus bacteria; the system controls the expression of nearly 200 genes, including both extracellular and intracellular factors [11]. This QS system consist of an isoprenyl transferase (ComQ), an autoinducer (ComX), a histidine kinase (ComP), and a response regulator (ComA). The autoinducer ComX is initially synthesized as a 47-73 residue propeptide (pre-ComX) and then processed and modi ed by ComQ. Extracellular accumulation of the modi ed ComX leads to phosphorylation of ComA, and then the phosphorylated ComA directly modulates the expression of various genes, including the srfA operon required for surfactin biosynthesis [12][13][14][15][16].
Several studies have indicated that the comQXPA gene cluster is widespread in Bacillus and that it exhibits a remarkable degree of intraspeci c diversity [14,15,17,18]. These studies have shown that the locus encoding the ComQXPA system is highly polymorphic, especially the coding regions for comQ, comX, and the 5´-end of comP, which are poorly conserved. However, the conservation of the genes followed the same order: ComA > ComP > ComQ > ComX. The genomes of many bacteria contain substantial fractions of overlapping co-directional gene pairs (resulting in overlapping reading frames), and gene overlaps are known to play an important role in their evolution and biological function [19,20]. Dogsa et al. performed comparative experiments with comQXPA-like gene arrangements in 2620 complete and 6970 draft prokaryotic genomes. Their results demonstrated that in addition to B. subtilis and close relatives, other bacteria species have comQXPA-like loci and characteristic gene-overlap patterns as in the comQXPA loci. Dogsa and co-workers divided the types of gene-overlaps into ve groups based on the number of overlaps in the reading frames and the number of amino acids (Fig. S1).
Group A exhibits no overlapping reading frames between genes of comQ, comX, comA, and comP; group B exhibits an overlapping reading frame between comQ and comX; group C exhibits a continuous overlapping reading frame between comQ, comX, and comP; group D exhibits an overlapping reading frame between comP and comA, and group E exhibits a continuous overlapping reading frame between comX, comP, and comA. The overlap types are dominated by an apparent mutation in the comQ stop codon that results in a 13-18 amino acid long C-terminal extension [19].
The ComX pheromone is a post-translationally modi ed oligopeptide. The modi cation is catalyzed by ComQ and is essential for the function of the pheromone. Previous studies have demonstrated that striking polymorphism occurs in the amino acid sequence of the ComX pheromone in certain Bacillus species, but each ComX pheromone possesses a tryptophan residue at either the 3rd or 4th residue from the C-terminus. Okada et al. noted that the tryptophan residue is modi ed with either a geranyl group or a farnesyl group at its γ-position, resulting in the formation of a tricyclic structure that includes a newlyformed proline-like ve-membered ring [17,[21][22][23][24][25][26][27][28].Thus far, the chemical structures of only seven ComX pheromones have been identi ed.
In previous studies, we reported that B. amyloliquefaciens Q-426 isolated from compost exhibits strong antifungal activity and produces various lipopeptides, including bacilomycin D, fengycin A, and fengycin B [29][30][31]. Whether the production of those lipopeptides is directly regulated by ComQXPA QS system and is a critical function for cell survival of B. amyloliquefaciens Q-426 remains unclear. Here, we identi ed and analyzed the comQXPA locus of B. amyloliquefaciens Q-426. Moreover, we identi ed the chemical structure of ComX Q−426 and investigated its function in regulating lipopeptide production and its biological characteristics. We found that the production of lipopeptide-like antifungal compounds is controlled by the ComPA signaling pathway in B. amyloliquefaciens Q-426, and we demonstrate that a lack of production of lipopeptides affects the swarming ability and bio lm formation of Q-426.

Results
Gene overlapping in the comQXPA locus of B. amyloliquefaciens Q-426 The entire comQXPA gene cluster was ampli ed from the chromosomal DNA of B. amyloliquefaciens Q-426. The full length of the comQXPA gene cluster was 4,014 bp, including 912 bp of comQ, 165 bp of comX, 2292 bp of comP, and 645 bp of comA. The chromosomal arrangement of the comQXPA locus in strain Q-426 is shown schematically in Fig. 1A.
In Bacillus, each gene in the comQXPA locus is differentially conserved and overlapped. Approximately half of the comQXPA loci exhibit no overlapping gene pairs belonging to group A; 31.6% of the loci contain overlapping gene pairs belonging to group B and only a few loci belonging to groups C, D and E [19]. The comQXPA locus in strain Q-426 belongs to group B, because only one base pair overlapped between comQ and comX.
We analyzed 40 comQXPA sequences from completely annotated Bacillus genomes that had at least one lipopeptide synthesis gene cluster, for example iturins, surfactins, and fengycins. Some of the sequences were from important commercial strains (B. velezensis QST713, B. velezensis FZB42). A phylogenetic tree was constructed based on the comQ gene sequences, as the comQ gene shows high diversity in the comQXPA locus. The gene overlapping types are marked in different colors in the Fig. 2. As shown in Fig. 2, most of the comQXPA loci belong to group B, and the number of overlapping base pairs varied from 1 to 49 base pair overlaps. There was no corresponding relationship between species and overlapping type, and the relationship between the number of overlaps and the function was unclear.
Many of B. amyloliquefaciens strains were registered as biological control agents and are commercially available because they can produce numerous antifungal metabolites with well-established activity in vitro such as bacillomycin, fengycin, and surfactin [32,33]. B. amyloliquefaciens is one member of the B. subtilis group that includes B. subtilis, B. licheniformis, B. pumilus, and B. amyloliquefaciens. However, these species were frequently misidenti ed for many years due to the lack of distinguishing phenotypes, and they are poorly resolved by 16S rRNA sequencing. In recent years, the taxonomy of the B. subtilis species group has been updated and clari ed based on their whole genome sequences. Some of the strains that were previously registered as B. subtilis or B. amyloliquefaciens are actually strains of B. velezensis [34,35]. For example, some important commercial strains, B. subtilis QST713 and B. amyloliquefaciens subsp. plantarum FZB42, were reidenti ed as B. velezensis QST713 and B. velezensis FZB42 based on phylogenetic analysis [36,37]. We also found that many strains belonging to the B. subtilis group were not consistent with the current taxonomy (Table S1). In this study, it was noticed that strain Q-426 was in the same clade with B. velezensis GB03, B. velezensis YJ11-1-4 and B. velezensis IT-45 (Fig. 2). Furthermore, strains in the same clade with Q-426 on the phylogenic tree reported in a previous paper [30] have been updated to B. velezensis. Therefore, we need to perform whole-genome sequencing of Q-426 in the future to ensure its taxonomic status.
Puri cation and characterization of the ComX pheromone of B. amyloliquefaciens Q-426 The QS pheromone, ComX, is a strain-speci c signaling oligopeptide that is modi ed from pro-ComX by the corresponding protein ComQ. The ComX pheromone has a unique modi ed tryptophan (W) residue with a geranyl group or a farnesyl group at the 3-position of its indole ring, resulting in the formation of a tricyclic structure. The structural variability of ComX also is related to the sequences of the peptide backbones [14,15,17,22,38]. As shown in Fig. 3A, conservation appears to be restricted to the Nterminus of the protein, whereas high diversity in the C-terminus marks divergence within the pheromoneforming region (in red letters). To date, the chemical structures of ComX pheromones have been identi ed for only a few Bacillus strains (Fig. 3A). No chemical structures have been identi ed for ComX from B. amyloliquefaciens.
To determine the chemical structure of ComX from B. amyloliquefaciens Q-426, we co-expressed ComQ Q426 and ComX Q426 in E. coli BL21(DE3) and puri ed the ComX Q426 by gradient reverse-phase HPLC

Deletion of comX decreased lipopeptide production in B. amyloliquefaciens Q-426
To investigate the effects of the quorum sensing system on lipopeptide production, comX was deleted in wild-type strain Q-426, and ComQX was overexpressed in Q426ΔcomX, resulting in the gene deletion strain Q426ΔcomX and overexpression strain Q426ΔcomX/comQX. The antifungal activity of Q426ΔcomX was signi cantly affected by the deletion, and almost no antifungal activity was observed ( Fig. 4A-a), while the antifungal activity of strain Q426ΔcomX /comQX was restored to the same level as that of the wild-type strain ( Fig. 4A-c, 4B-c). When the puri ed ComX Q−426 pheromone was added to the culture medium at a nal concentration of 8 µg/L at the early stage of the log-phase, the antifungal activity of the wild-type strain Q-426 was signi cantly improved ( Fig. 4A-d). Also, it was observed that the antifungal activity was restored by adding extra ComX Q−426 (at a nal concentration of 8 µg/L) during the culture of Q426ΔcomX. (Fig. 4A-e and 4B-e).
The previous results revealed that the Q-426 strain could produce a variety of cyclic lipopeptide compounds which were identi ed as C-14, C-15, C-16 bacillomycin D, C-15, C-16, C-17 fengycin A, and c-bacillomycin D, respectively. As shown in Fig. 4B, after the addition of the puri ed ComX Q−426 pheromone, the yield of lipopeptide production, especially, the yields of C-15 bacillomycin D (peak 1) and C-16 bacillomycin D (peak 2), were signi cantly increased ( Fig. 4B-d and 4B-e), while there were no obvious peaks at the retention time corresponding to C-15 bacillomycin D or C-16 bacillomycin D in strain Q426ΔcomX ( Fig. 4B-a). The above results showed that the lipopeptide synthesis is related to the quorum sensing system in strain Q-426. Collectively, these results suggested that ComX might positively affect lipopeptide synthesis, and deletion of comX contributed to the decrease of lipopeptide production.

Effects Of Comx De ciency On Morphology And Motility
The wild-type strains were able to grow normally on all ve media; the colony surface was rough and wrinkled; the edges were rough, and the color and size of the colonies changed according to the different media. For example, in FM medium, the colony color was dark yellow, and the folds were more obvious. In the MSgg and NB media, the colony color was white, the frill was lighter, and the edge was relatively neat ( Fig. 5A and 5C). The mutant strain only formed colonies at the initial inoculation site on all the tested media and could not expand to the periphery. Except for growth on FM medium, the colonies were smooth with neat edges (Fig. 5B and 5D). The swarming ability of strain Q426ΔcomX was signi cantly reduced. It was di cult to expand colonies on the surface of solid medium, because the biosynthesis of lipopeptides was downregulated. As shown in Fig. 6A, when cells were gown in FM medium with a reduced agar concentration, the whole plate (bottom, wild-type strain) was quickly covered with colonies. The mutant could not move within the agar plates at any concentration of agar, and there was no signi cant change in the colony size. We also investigated the effects of the comX gene deletion on bio lm formation. As shown in Fig. 6B, in both of FM and LB media, the mutant could not form bio lms, and the bacterial cells were deposited at the bottom of the wells. Figure 7 shows the growth curves of the wild-type strain and mutant strains. As shown from the growth curve, the lag-phase of the mutant was approximately 0-3 h and then the cell soon entered the log-phase, while the lag-phase of the wild-type strain was 0-5 h. However, the wild-type strain had a longer log-phase than the mutant, and the nal cell density of the wild-type strain was higher than that of the mutant. Therefore, the deletion of comX had a certain effect on the growth, but the effect was not signi cant.

Discussions
In this study, we identi ed and characterized the comQXPA gene locus of B. amyloliquefaciens Q-426 and found that the stop codon of comQ and the start codon of comX overlapped by one base pair. Furthermore, we demonstrated that most of the comQXPA loci from the Bacillus genus belong to group B in the variations of gene overlap. Disruption of the ComPA signaling pathway in the Q-426 strain resulted in signi cant effects on lipopeptide production, morphology, and motility.
Recent studies have demonstrated that the ComQXPA QS system not only exists in Bacillus-related species but also in other Gram-positive species such as Lysinbacillus, Geobacillus, and Anoxybacillus [19]. In Bacillus, the ComQXPA QS system exhibits striking polymorphism and is coupled to the bacterial pherotypes, which are de ned as groups of bacteria that are able to communicate through comX pheromones [14,15,17,38,39]. The evolutionary history of the comQXPA locus and the interrelation between the structural characteristics of the comQXPA locus and its ecological functions are still unclear. In this study, we analyzed 40 comQXPA sequences from completely annotated Bacillus genomes. Most of the comQXPA loci from the Bacillus genus belong to group B, in which the comQ and comX genes overlap, and the number of overlapping base pairs varied from 1 to 49 base pairs. From these results, although we cannot see any corresponding relation between species with overlapping type, the number of overlaps was the same within the strains located in the same clade. This suggests that overlapping reading frames in the comQXPA locus may result in more e cient or individual transcriptional control and may be associated with the evolution of speci c biological functions.
Bacillomycin D, one member of the iturinic lipopeptide family, has strong antifungal activity, especially against lamentous fungi. Iturinic lipopeptides are mainly produced by members of the B. subtilis group through non-ribosomal synthetases machinery, and they exhibit strong diversity within the B. subtilis species group; some lipopeptides are only produced by one species, whereas certain others can be produced by up to three species [40]. The chemical principles for the biosynthesis of iturinic lipopeptide are also largely characterized [41,42], but little is known about the regulatory mechanisms that control the expression of the iturinic lipopeptide. To date, the biosynthesis mechanisms and expression regulation mechanisms of lipopeptides except surfactin have not been extensively studied. Surfactin biosynthesis is regulated by the ComQXPA QS system in B. subtilis. Upon reaching a threshold concentration, ComX activates ComP autophosphorylation, and then ComP∼P transfers the phosphoryl group to ComA [25],which controls the expression of srfA encoding a very large protein complex for surfactin production that contributes to swarming motility and bio lm formation [13,35,38,[43][44][45][46][47][48]. Spacapan et al. also found that exoproteases required for bio lm formation are regulated by the ComQXPA system through ComX induction in B. subtilis [9]. In B. amyloliquefaciens FZB42, it has been shown that bacillomycin D is under direct transcriptional control of DegU, and DegQ and ComA also are required for the full transcriptional activation [49].
In our previous studies, we demonstrated that B. amyloliquefaciens Q-426 can produce seven lipopeptides (C-14, C-15, and C-16 bacillomycin D; C-15, C-16, and C-17 fengycin A; C-17 fengycin B), among which the levels of C-15 and C-16 bacillomycin D were higher than others [29,30,50]. To investigate whether the ComQXPA QS system is involved in regulating the lipopeptide synthesis in B. amyloliquefaciens Q-426, we disrupted the ComQXPA QS system through constructing the Q426ΔcomX mutant. Our results revealed that the ComQXPA QS system might be involved in regulating bacillomycin D production in B. amyloliquefaciens Q-426. Interestingly, knocking out the comX gene does not affect the growth of the bacteria, but the growth capacity of the Q426ΔcomX mutant in liquid medium was slightly lower than that of the wild-type strain. However, the Q426ΔcomX lost its swimming ability, was unable to form colonies when spread on a solid surface, and could not form bio lms on the interface between the gas and liquid medium. Unfortunately, we did not con rm the direct effects of the ComQXPA QS system or ComA on lipopeptide synthesis, and this is our immediate goal. It is widely known that lipopeptides exhibit strong antifungal activities, but their other physiological roles in Bacillus cells are still unclear. We speculate that the ComQXPA QS system of strain Q-426 modulates some physiological and biochemical properties by regulating the biosynthesis of lipopeptides. These lipopeptide compounds themselves may also be signal molecules, and each lipopeptide has its own speci c biological regulatory circuit. In a recent study, Xu and co-researchers pointed out that bacillomycin D is a signal that promotes bio lm development of B. velezensis SQR9 [51].
B. amyloliquefaciens Q-426 produces an oligopeptide pheromone, ComX Q−426 , that inducts its QS system. ComX Q−426 consists of six amino acids that contain a modi ed tryptophan residue located at the third position from the C-terminus, resulting in the formation of a tricyclic scaffold with a pyrrolidine ring (Fig. 3C). ComX pheromones from different Bacillus strains exhibit unique amino acid sequences and residues, indicating that each bacterium has its own oligopeptide language for activating QS signaling. The linear oligopeptide (GGDWKY) without modi cation of the tryptophan residue with farnesyl did not activate QS signaling in the Q426ΔcomX strain to produce lipopeptides such as bacillomycin D (Fig. S4). This result was in close agreement with other research in that the tricyclic scaffold of the tryptophan residues in the ComX pheromone is critical to activating kinase activity of the receptor protein ComP, but there is no effect on the recognition of the receptor with pheromone molecules [17,21,[23][24][25]. Further research should clarify how the sequence of amino acids of ComX Q−426 contributes to the speci c recognition of ComX Q−426 by the receptor protein ComQ Q−426 .
For Bacillus strains, it is now estimated that at least 5%-10% of the genome is devoted to the production of antimicrobial compounds [45,52]. These compounds are mainly antimicrobial cyclic peptides containing D-amino acids and hydrophobic residues. At present, studies of lipopeptides mainly focus on their physicochemical properties and biological activities, but further studies of their biological functions and the regulatory mechanisms of their biosynthesis are needed. The understanding of such biological functions and the molecular regulatory mechanism of biosynthesis will provide a theoretical basis for constructing a high-yielding lipopeptide-producing strain. This will help us to better understand the molecular mechanisms of secondary metabolism in Bacillus and its environmental adaptations.

Conclusion
In this study, we identi ed and characterized the comQXPA gene locus of B. amyloliquefaciens Q-426 and analysed 40 comQXPA sequences from completely annotated Bacillus genomes. Most of the comQXPA loci from the Bacillus genus belong to group B, in which the comQ and comX genes overlap and the number of overlapping base pairs varied from 1 to 49 base pair overlaps. We identi ed the chemical structure of ComX Q−426 that consists of six amino acids that contain a modi ed tryptophan residue located at the third position from the C-terminus, resulting in the formation of a tricyclic scaffold with a pyrrolidine ring. We found that strain Q426ΔcomX exhibited signi cantly reduced antifungal ability, almost no antifungal activity was observed, while the antifungal activity of strain Q426/comX could be restored to the same level as that of the wild-type strain. Moreover, the antifungal activity of B. amyloliquefaciens Q-426 was signi cantly improved by adding the ComX Q−426 pheromone to the culture medium. Interestingly, knocking out the comX gene did not affect bacterial growth. However, the Q-426 ΔcomX mutant lost swimming ability, was unable to form colonies when spread in on solid surface, and could not form bio lms on the interface between the gas and liquid medium. These funding suggest that the production of lipopeptides is controlled by the ComPA signalling pathway and relate to the swarming ability and bio lm formation in B. amyloliquefaciens Q-426.

Dna Manipulation Techniques
Oligonucleotide synthesis and DNA sequencing were performed by Sangon Biotech Co., Ltd. (Shanghai, China). The isolation and manipulation of recombinant DNA were carried out as described previously [53]. All enzymes were commercial preparations. Phusion DNA high-delity polymerase was purchased from NEB (Shanghai, China). B. amyloliquefaciens transformations were performed by arti cially inducing genetic competence [54].
Cloning and sequence analysis of QS-related genes ( comQXPA ) Genomic DNA of strain Q-426 was isolated from the bacterial culture using the Bacterial DNA Isolation Kit (Foregen, Beijing, China). To amplify the whole gene of the comQXPA locus of B. amyloliquefaciens Q-426, the upstream and downstream primers (com_F and com_R; the location is indicated in Fig. 1A in red) were designed from the degQ and luxR gene sequences, which are located upstream of comQ and downstream of comA, respectively. PCR ampli cation was employed in a 50 µL nal volume using the following program: at 95 °C for 5 min; 30 cycles of denaturation at 95 °C for 1 min, annealing at 58 °C for 1 min and extension at 72 °C for 1 min; nal extension at 72 °C for 10 min. PCR products were detected by 1% w/v agarose gel electrophoresis at 100 V for 30 min. The nucleotide sequences of ampli ed PCR fragments were determined by Sangon Biotech Co., Ltd. using an ABI DNA Sequencer (3730XL, USA) and then submitted to NCBI GenBank for BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST, GenBank accession no. MF579444).
The nucleotide sequence of the ampli ed PCR fragment was then compared with the corresponding gene clusters from strains with homologous genes from other Bacilli, which were obtained from the NCBI nucleotide/protein database. Alignments for the phylogenetic tree were made using ClustalX-1.81 software. Phylogenetic analyses were conducted using MEGA version 7.0.20 for Neighbor-Joining and ME analyses using the Kimura 2-parameter model. Measures of bootstrap support for internal branches were obtained from 1000 pseudoreplicates.

Deletion of the comX gene in B. amyloliquefaciens strain Q-426
The left anking (LF) region (85 bp), kan region (813 bp), and right anking (RF) region (80 bp) were ampli ed from the genomic DNA of Q-426 and pET-28-(a+) using the primer pairs of comX_F/comXI_R, kan_F/kan_R and comXП_F/comXП_R (Table 1, Fig. 1B), respectively. These three fragments were fused using overlap PCR in the order of LF, KAN, and RF. The resulting 1.0-kb comX deletion amplicon (1 µg) was directly transformed (25 µF, 600 Ω) into strain Q-426, and 53 colonies were obtained from LB plates containing Kanamycin (30 µg·mL − 1 ). Three colonies con rmed by PCR were cultivated to an OD 600 of 1.0 without Kan, and a 100-µL aliquot of a 10-fold dilution of the cultures (approximately 10 5 cells) was plated on LB-Kan medium. Mutants growing on LB-Kan were further con rmed by PCR and DNA sequencing. Mutants selected were retreated on LB-Kan plates for 20 times to con rm their stability. The ComQX expression vector was constructed according to a previously reported method [55]. Brie y, the gene comQX was ampli ed by the corresponding primers (ComQX_F/R, Table 2). Then, the fragment was inserted into pHY300PLK, a shuttle vector for E coli and B. subtilis (BioVector NTCC Inc., China), at the restriction enzyme sites BamHI/XbaI, resulting in the comQX expression vector named as pHY-comQX. The vectors pHY-comQX were rst electroporated into E. coli DH 5α to con rm and prepare an e cient expression vector, and then electroporated into Q426ΔcomX competent cells. The recombinant strains were selected by tetracycline-resistance (25 µg·mL − 1 ) and named Q-426ΔcomX /ComX. For growth curve determination, a fresh single colony of each tested strain was inoculated into 200 mL FM liquid medium in a 1-L ask with 1% inoculation and incubated at 30°C and 180 rpm. A certain amount of liquid culture was taken from the ask, and the OD 600 was measured every hour on the rst day and then measured every 4 or 8 h after reaching a stable period. The average value was measured three times for each group of data.

Pheromone Overproduction And Identi cation
The E. coli ComX-producing strain that co-expressed comX and comQ was grown overnight in completed M9 medium supplemented with a mixture of amino acids (Phe, Ser, His, Met, at 40 ug/mL; and Gln at 0.4 mg/mL), kanamycin (30 µg/mL), and ampicillin (50 µg/mL). At the stationary phase, 20 mL of the pre-culture was added to 1980 mL of the supplemented M9 medium to make a 2-L bacterial culture, which was then incubated at 37 °C and 110 rpm for 8 h. The expression of the comQX gene was induced with 0.1 mM IPTG at 37 °C and 110 rpm overnight. The culture broth was centrifuged for 10 min at 8000 × g. The supernatant was ltered through a 0.22 µm lter, and CH 3 CN was added to a nal concentration of 20%. The ltrate was loaded onto a column packed with HZ resin (Huazhen Tech, Shanghai, China) that was equilibrated with 10% CH 3 CN and 0.1 TFA. The column was eluted with a step gradient of CH 3 CN (20, 40, 50, 65, and 80% in 0.1% aqueous TFA). The 50% elute containing ComX was immediately neutralized with aqueous ammonia, concentrated with a rotary evaporator, and dried with a freeze dryer.
The dried extract was analyzed through HPLC (1260 In nity, Agilent Technologies, Santa Clara, CA, USA) equipped with a C18 column (Agilent Technologies, 5 µm × 4.6 × 150 mm) to detect the ComX pheromone. MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) was used to determine the mass and the amino acid sequences of the pheromone peptide present in the samples collected from HPLC.
Effects of the ComX pheromone and ComX gene deletion on lipopeptide production The wild-type, Q426ΔcomX, and Q426ΔcomX /ComX strains were streaked on LB agar plates and then inoculated into 100 mL of FM liquid culture medium. Q426ΔcomX and Q426ΔcomX /ComX strains were transferred to the FM liquid culture medium containing corresponding antibiotics in a 1% inoculation volume. The fermentation process was conducted at 180 rpm for 72-76 h at 30°C. Lipopeptide productivity and antifungal activity assays were evaluated according to a previous study [31].