Regulatory and Enterotoxin Gene Expression and Enterotoxins Production in Staphylococcus aureus FRI913 Cultures Exposed to a Rotating Magnetic Field and trans-Anethole

The study aimed to examine the influence of a rotating magnetic field (RMF) of two different frequencies (5 and 50 Hz) on the expression of regulatory (agrA, hld, rot) and staphylococcal enterotoxin (SE—sea, sec, sel) genes as well as the production of SEs (SEA, SEC, SEL) by the Staphylococcus aureus FRI913 strain cultured on a medium supplemented with a subinhibitory concentration of trans-anethole (TA). Furthermore, a theoretical model of interactions between the bacterial medium and bacterial cells exposed to RMF was proposed. Gene expression and SEs production were measured using quantitative real-time PCR and ELISA techniques, respectively. Based on the obtained results, it was found that there were no significant differences in the expression of regulatory and SE genes in bacteria simultaneously cultured on a medium supplemented with TA and exposed to RMF at the same time in comparison to the control (unexposed to TA and RMF). In contrast, when the bacteria were cultured on a medium supplemented with TA but were not exposed to RMF or when they were exposed to RMF of 50 Hz (but not to TA), a significant increase in agrA and sea transcripts as compared to the unexposed control was found. Moreover, the decreased level of sec transcripts in bacteria cultured without TA but exposed to RMF of 50 Hz was also revealed. In turn, a significant increase in SEA and decrease in SEC and SEL production was observed in bacteria cultured on a medium supplemented with TA and simultaneously exposed to RMFs. It can be concluded, that depending on SE and regulatory genes expression as well as production of SEs, the effect exerted by the RMF and TA may be positive (i.e., manifests as the increase in SEs and/or regulatory gene expression of SEs production) or negative (i.e., manifests as the reduction in both aforementioned features) or none.

Magnetic fields (MFs) can be divided into two main types: direct-current magnetic field (DCMF) and alternating current magnetic field (ACMF). DCMF does not vary in time, or it changes very slowly. This type of MF does not have a frequency (the MF vector is constant in time and space). Static MF (SMF) is an example of DCMF [25][26][27][28]. In contrast to DCMF, ACMF varies in frequency. Pulsating MF that changes as a sine-wave with time at each point in space is an example of ACMF [29][30][31][32][33]. A superposition of two or more ACMFs of identical frequency but spatially displaced in phase with respect to one another causes rotation of the MF (RMF). RMF changes over time and can be characterized by its frequency [34].
The idea of gene expression, investigated in S. aureus under RMF and TA influence, originated from previous research performed by Fijałkowski et al. [35] and Qiu et al. [36]. The studies showed that RMF [35] and fennel essential oil (FEO), which contains a high level of TA [36], decreased the expression of genes encoding selected SEs. However, the studies on the influence of simultaneous exposure of S. aureus to RMF and TA at the same time on changes in SEs genes expression have not been published so far. Hence, the study aimed to investigate the influence of the RMF of two different frequencies (5 and 50 Hz) on the regulatory (agrA, hld, rot), as well as SE (sea, sec, sel) gene expression in S. aureus FRI913, cultured on a medium supplemented with a subinhibitory concentration of TA. Additional attention was paid to a possible effect of RMF and/or TA on the production of SEs (SEA, SEC, SEL). Moreover, a theoretical model explaining possible interactions between the RMF-activated bacterial medium and bacterial cells was proposed.

Evaluation of Antibacterial Activity of TA
Based on the agar dilution method, it was observed that TA showed antibacterial activity against the S. aureus FRI913 strain. The obtained minimum inhibitory concentration (MIC 100 ) and subinhibitory concentration (MIC 50 ) values of TA were 49.4 ± 0.0 mg/mL and 24.7 ± 0.0 mg/mL, respectively. It was also confirmed that an addition of 1% (v/v) Tween 80 in the MHA medium did not influence the viability of S. aureus FRI913 strain.

Selection of Housekeeping Gene
Among the tested housekeeping genes, the r-value of 16S rRNA (0.930) was the closest to 1. Thus, this gene appeared to be the most stable gene among all the analyzed genes (16S rRNA, rpoB, gyrB, recA, rho, pta, rplD, and tpo). Detailed statistical data of all housekeeping genes are listed in Supplementary Materials, Table S1.

Effect of RMF and TA on SEs and Regulatory Gene Expression
The S. aureus reference strain was screened for changes in the expression of sea, sec, sel genes exposed to RMF and/or TA. Detailed data on the quantification of the analyzed genes are listed in Supplementary Materials- Table S2.
As shown in Figure 1a, transcription of the sea gene did not differ statistically significantly between RMF of 5 Hz and unexposed control settings. In contrast, a significant increase (p < 0.001) in the sea gene expression (approx. 8-folds) was found due to exposure to the RMF of 50 Hz. Similarly, a significant increase (p < 0.001) in gene expression of sea, compared to the control (medium A unexposed to RMFs), was observed when bacteria were incubated on a medium with TA (approx. 4-folds). Nevertheless, the presence of RMF of 50 Hz and TA contributed to a significant decrease (p < 0.001) in the transcript level of sea. It was also found that the exposure to RMF (both frequencies) decreased the expression level of sec and sel genes (Figure 1b,c). However, in this case, only exposure to the RMF of 50 Hz resulted in significant changes (p < 0.05) in gene expression of sec in comparison to the control. uted to a significant decrease (p < 0.001) in the transcript level of agrA. The agrA gene was the only regulatory gene among those analyzed that showed significant differences in expression compared to the control (medium A unexposed to the RMFs). However, the level of upregulation was low and did not correlate with the expression of the dependent sec gene.
It was also observed that 1% Tween 80 (medium B) had no effect on the expression of all tested SE genes, excluding agrA and sea genes' exposure to RMF of 50 Hz. , and sel (c) and regulatory: agrA (d), hld (e), rot (f) genes in S. aureus FRI913 grown on Mueller-Hinton agar non-supplemented with trans-anethole (A), supplemented with 1% (v/v) Tween 80 (B), and supplemented with 1% (v/v) Tween 80 and subinhibitory concentration of trans-anethole (C), exposed to rotating magnetic fields (RMFs: RMF I: 5 Hz, RMF II: 50 Hz) and unexposed (without RMF). Control-bacteria cultured on a medium non-supplemented with trans-anethole (A) and unexposed to RMF. Data are shown as the mean ± standard deviation. * p < 0.05; *** p < 0.001.

Effect of RMF and TA on SEs Production
Bacteria grown on medium A and exposed to the RMF (5 and 50 Hz) was found to produce a similar amount of SEA (6.3 ± 0.7-7.6 ± 0.8 ng/mL). In accordance with the level of the transcript (Figure 2a), a significantly higher (p < 0.001) concentration of SEA (52.4 ± 2.5 ng/mL) was found in the S. aureus incubated on medium C (but not exposed to RMFs). Further exposure to RMF led to a decrease in SEA production to 26.8 ± 1.2 ng/mL for 5 Hz RMF treatment and 19.5 ± 0.6 ng/mL for 50 Hz RMF treatment (Figure 2a). hld (e), rot (f) genes in S. aureus FRI913 grown on Mueller-Hinton agar non-supplemented with trans-anethole (A), supplemented with 1% (v/v) Tween 80 (B), and supplemented with 1% (v/v) Tween 80 and subinhibitory concentration of trans-anethole (C), exposed to rotating magnetic fields (RMFs: RMF I: 5 Hz, RMF II: 50 Hz) and unexposed (without RMF). Control-bacteria cultured on a medium non-supplemented with trans-anethole (A) and unexposed to RMF. Data are shown as the mean ± standard deviation. * p < 0.05; *** p < 0.001.
The influence of the RMF and/or TA on the expression of regulatory genes (agrA, hld, rot) (Figure 1d-f) was also investigated. Detailed data on the quantification of the analyzed genes are listed in Supplementary Materials, Table S2. In our study, after 24 h of incubation, we observed a significant increase (p < 0.001) in the transcript level of the agrA gene in culture on TA-supplemented medium or culture exposed to the RMF of 50 Hz. Similar to the expression of the sea gene, the presence of RMF of 50 Hz and TA contributed to a significant decrease (p < 0.001) in the transcript level of agrA. The agrA gene was the only regulatory gene among those analyzed that showed significant differences in expression compared to the control (medium A unexposed to the RMFs). However, the level of upregulation was low and did not correlate with the expression of the dependent sec gene.
It was also observed that 1% Tween 80 (medium B) had no effect on the expression of all tested SE genes, excluding agrA and sea genes' exposure to RMF of 50 Hz.

Effect of RMF and TA on SEs Production
Bacteria grown on medium A and exposed to the RMF (5 and 50 Hz) was found to produce a similar amount of SEA (6.3 ± 0.7-7.6 ± 0.8 ng/mL). In accordance with the level of the transcript (Figure 2a), a significantly higher (p < 0.001) concentration of SEA (52.4 ± 2.5 ng/mL) was found in the S. aureus incubated on medium C (but not exposed to RMFs). Further exposure to RMF led to a decrease in SEA production to 26.8 ± 1.2 ng/mL for 5 Hz RMF treatment and 19.5 ± 0.6 ng/mL for 50 Hz RMF treatment (Figure 2a).
SEC production compared to the unexposed control.
In the used model, after 24 h of incubation, the production of SEA was mRNA dependent and regulated at the level of transcription in culture unexposed to the RMF of 5 Hz. Similar relationships between protein production and mRNA expression level were observed in the case of SEC without the use of RMFs. Expression at the level of the SEL protein was not regulated after this time at the level of transcription (Supplementary Materials, Table S3). , and SEL (c) production in S. aureus FRI913 grown on Mueller-Hinton agar non-supplemented with trans-anethole (A), supplemented with 1% (v/v) Tween 80 (B), and supplemented with 1% (v/v) Tween 80 and subinhibitory concentration of trans-anethole (C), exposed to rotating magnetic fields (RMFs: RMF I: 5 Hz, RMF II: 50 Hz) and unexposed (without RMF). Control-bacteria cultured on a medium non-supplemented with trans-anethole (A) and unexposed to RMFs. Data are shown as the mean ± standard deviation. *** p < 0.001.

Discussion
The results of basic microbiological studies presented in this article were an extension of the previous studies in this area presented in the cited reference works [34,35,[37][38][39][40]. The cited experimental studies were focused on the influence of the interaction between RMF and antibiotics, as well as the analysis of bacterial colony growth dynamics, the metabolic activity of cells, as well as genetic activity of selected bacterial species exposed to RMF. Nevertheless, a combined effect of TA and RMF on regulatory and SEs gene expression as well as on the concentration of selected SEs in S. aureus FRI913 strain has not been published so far. Moreover, our study is also the first to show the effect of TA on the above-mentioned analyzed parameters.
It is widely recognized that the effect of the magnetic field is to a major extent related to its frequency and intensity [34,37,[41][42][43][44] because these two factors determine the , and supplemented with 1% (v/v) Tween 80 and subinhibitory concentration of trans-anethole (C), exposed to rotating magnetic fields (RMFs: RMF I: 5 Hz, RMF II: 50 Hz) and unexposed (without RMF). Control-bacteria cultured on a medium non-supplemented with trans-anethole (A) and unexposed to RMFs. Data are shown as the mean ± standard deviation. *** p < 0.001. A significant effect of the investigated conditions was also observed in SEC concentrations ( Figure 2b). The highest inhibition effect was observed in S. aureus incubated on medium A and exposed to the RMF of 50 Hz. In this condition, no production of SEC was noticed. Bacteria incubated on medium C and unexposed to RMFs produced 52.4 ± 2.5 ng/mL of SEC. This concentration was approximately fourteen times lower than that of the control (medium A unexposed to RMFs; 737.8 ± 43.2 ng/mL). Moreover, the presence of both RMF and TA contributed to even a higher reduction in the SEC production: 26.8 ± 1.2 ng/mL and 19.5 ± 0.6 ng/mL, respectively, for RMFs of 5 and 50 Hz.
Moreover, when the bacteria were cultured on a medium supplemented with TA but were not exposed to RMF (52.4 ± 2.5 ng/mL) or when they were exposed to RMF of 5 Hz (26.8 ± 1.2 ng/mL) or 50 Hz (19.5 ± 0.6 ng/mL) and TA, a significant decrease (p < 0.001) in SEL production as compared to the unexposed control (76.0 ± 11.8 ng/mL) was found.
It was also observed that 1% Tween 80 (medium B) did not affect the production of all tested SEs, excluding SEC. There was a statistically significant reduction (p < 0.001) in SEC production compared to the unexposed control.
In the used model, after 24 h of incubation, the production of SEA was mRNA dependent and regulated at the level of transcription in culture unexposed to the RMF of 5 Hz. Similar relationships between protein production and mRNA expression level were observed in the case of SEC without the use of RMFs. Expression at the level of the SEL protein was not regulated after this time at the level of transcription (Supplementary Materials, Table S3).

Discussion
The results of basic microbiological studies presented in this article were an extension of the previous studies in this area presented in the cited reference works [34,35,[37][38][39][40]. The cited experimental studies were focused on the influence of the interaction between RMF and antibiotics, as well as the analysis of bacterial colony growth dynamics, the metabolic activity of cells, as well as genetic activity of selected bacterial species exposed to RMF. Nevertheless, a combined effect of TA and RMF on regulatory and SEs gene expression as well as on the concentration of selected SEs in S. aureus FRI913 strain has not been published so far. Moreover, our study is also the first to show the effect of TA on the above-mentioned analyzed parameters.
It is widely recognized that the effect of the magnetic field is to a major extent related to its frequency and intensity [34,37,[41][42][43][44] because these two factors determine the physical characteristics of the magnetic signal [45,46]. Therefore, a possible influence of these variables was also analyzed with regard to observed changes in the expression of regulatory genes (agrA, hld, rot) and genes encoding staphylococcal enterotoxins (SEs: sea, sec, sel), as well as the concentration of produced SEs. In the case of the RMF set-up used in the current study, the frequency of AC determines MF intensity as well as the physical characteristics of the magnetic wave shape. Therefore, the AC frequencies of 5 Hz (the lowest current frequency that can be used in the setup) and 50 Hz (the highest current frequency that can be used in the setup) at which the RMF was generated allowed obtaining MF of different parameters. At 5 Hz, the amplitude of the RMF was characterized by a longer period between maximal/minimal magnetic induction (B) states (100 ms) with B average 6.16 mT as compared to the RMF generated at 50 Hz (10 ms) with B average 6.41 mT (Figure 3a,b). It should also be noted, that the RMF was axially symmetric ( Figure 3c) and magnetic field lines rotated in a horizontal direction, with the rotation frequency equal to the frequency of the electric current, as can be seen in the simulations: 5 Hz: https://www.youtube.com/watch?v=sTpGK0bRJt8 (accessed on 1 January 2020) and 50 Hz: https://www.youtube.com/watch?v=_uLUocd1c9k (accessed on 1 January 2020). physical characteristics of the magnetic signal [45,46]. Therefore, a possible influence of these variables was also analyzed with regard to observed changes in the expression of regulatory genes (agrA, hld, rot) and genes encoding staphylococcal enterotoxins (SEs: sea, sec, sel), as well as the concentration of produced SEs. In the case of the RMF set-up used in the current study, the frequency of AC determines MF intensity as well as the physical characteristics of the magnetic wave shape. Therefore, the AC frequencies of 5 Hz (the lowest current frequency that can be used in the setup) and 50 Hz (the highest current frequency that can be used in the setup) at which the RMF was generated allowed obtaining MF of different parameters. At 5 Hz, the amplitude of the RMF was characterized by a longer period between maximal/minimal magnetic induction (B) states (100 ms) with Baverage 6.16 mT as compared to the RMF generated at 50 Hz (10 ms) with Baverage 6.41 mT (Figure 3a,b). It should also be noted, that the RMF was axially symmetric ( Figure 3c) and magnetic field lines rotated in a horizontal direction, with the rotation frequency equal to the frequency of the electric current, as can be seen in the simulations: 5 Hz: https://www.youtube.com/watch?v=sTpGK0bRJt8 (accessed on 1 January 2020) and 50 Hz: https://www.youtube.com/watch?v=_uLUocd1c9k (accessed on 1 January 2020). In our study, the influence of active factors such as RMF and TA on the expression level of regulatory (agrA, hld, rot) and SE (sea, sec, sel) genes' expression as well as SE (SEA SEC, SEL) production was investigated. The expression of genes encoding S. aureus virulence factors can be regulated by several systems [47]. The primary metabolism gene In our study, the influence of active factors such as RMF and TA on the expression level of regulatory (agrA, hld, rot) and SE (sea, sec, sel) genes' expression as well as SE (SEA SEC, SEL) production was investigated. The expression of genes encoding S. aureus virulence factors can be regulated by several systems [47]. The primary metabolism gene expression depends on many factors, such as pH, atmosphere, cell growth phase, and density of bacterial populations [10].
In our experimental study, RMF and TA were shown to be important additional factors controlling bacterial metabolism. Significant differences were observed depending on three main quantities, i.e., the induction value of RMF, TA content, and RMF exposure time of a sample.
Relationships between individual regulatory systems are still unexplained but they certainly determine the ability of microorganisms to adapt to various environmental conditions and also have a decisive impact on the intensity of the infection process [10].
The results presented in the current study suggest that the level of gene expression and SEs production depends on different frequencies of the RMF associated with TA. The relative normalized numerical statistical data indicated a significant increase in agrA (p < 0.001) gene expression observed due to exposure to the RMF of 50 Hz in comparison to the control (medium non-supplemented with TA and unexposed to RMFs). In addition, obtained results showed that the frequency of RMF did not affect rot and hld genes. Moreover, a significant increase and decrease, respectively, in sea (p < 0.001) and sec (p < 0.05) gene expression under this condition were observed. This fact revealed that only by the application of the RMF of 50 Hz can sea gene expression and production of SEA be controlled.
According to available data, there are several reports presenting the effects of MFs on gene expression in bacteria. Potenza et al. [48] proved that SMF (300 mT) induced cell proliferation and changes in Escherichia coli gene expression. Moreover, Giorgi et al. [49] observed that mini-Tn10 and Tn5 transposition decreased under sinusoidal MF and increased under pulsed-square MF. Finally, Fijałkowski et al. [35] reported a lower expression level of sea, seb, sec, sed, and see genes under RMF influence compared to non-exposure samples. Interestingly, these authors observed lower expressions of the sea gene compared to our data. These results may coincide due to the incubation conditions. In the current study, we used S. aureus cultured on MHA, whereas, in the mentioned study, broth culture was performed. According to our observations, due to the occurrence of an ouzo effect of TA in combination with MHB and difficulty in reading MIC by the microdilution method, we decided to immobilize TA in a solid medium (MHA). Nevertheless, the observed sec gene expression level was similar in both studies.
In the current study, an increase in the transcript level of the agrA gene in S. aureus exposed to TA was observed. It is the only regulatory gene among the ones tested which showed significantly different expression levels in comparison to the control (medium nonsupplemented with TA and unexposed to RMFs). The level of upregulation observed was high and could correlate with the expression of the sea gene as well as SEA production. What is more, there was no effect of TA on the sec expression gene. However, SEC production was lower (p < 0.001) than in the control. As stated in the literature, the expression of selected SEs could be influenced by subinhibitory concentrations of many natural compounds. For instance, Azizkhani et al. [50] revealed lower relative expression of sea, sec, see, and agrA in S. aureus ATCC 29213 cultured with a subinhibitory concentration of Zataria mulitflora Boiss. EO after 72 h in comparison to the control (without EO). Interestingly, data were obtained by Qiu et al. [36]. According to these studies, FEO (containing 88.91% of TA) in MHB reduced relative expression levels of hla, sea, seb, tst, and agrA, as well as the production of SEA, SEB, toxic shock syndrome toxin (TSST), and α-hemolysin. Results of the current study allow concluding that in addition to culture conditions, the presence of other compounds in FEO can also affect the expression/production level of the above-mentioned virulence factors. The influence of the EO solvent (dimethyl sulfoxide) used by these authors cannot be excluded here, either. Moreover, similar results were observed in other studies where the production of selected exotoxins, such as α-toxin, SEA, SEB, and TSST, in S. aureus was decreased by perilla [51], peppermint [52] EOs, or even thymol [53].
The above-mentioned results could be associated with the S. aureus Agr system involved in the regulation of SEs expression, which response to staphylococcal cell population density is known as quorum sensing [54]. The sea and sec genes products are well characterized and are well known for their superantigenic activity and ability to evoke emesis [55]. However, little is known about the sel gene, which is quite often clustered with the sec gene on a pathogenicity island SaPI in S. aureus [56,57]. However, the obtained results demonstrated a significant decrease in the sea gene expression and SEC production in the reference strain under TA and RMF (50 Hz) influence, but further research is needed to understand this mechanism.
It can be assumed that the impact of RMF on a microbial sample corresponds to the impact of this field on the free surface of the medium and on bacterial cells. It should be emphasized, that previously published studies concerning changes in the metabolism of microorganisms exposed to RMF in the context of the final effect and not the state of the medium with organic substance (OS) on the free surface [34,35,37,38]. Hence, this observation implies that either bacteria were sensitive to RMF or OS in the medium had magnetic properties, and the interaction between MF and the medium affected microorganisms on the surface of the medium.
In the theoretical model, we assumed, that both OS on the free surface of the medium and bacteria cultured on the medium were sensitive to RMF as well as to other possible external physical factors. For this purpose, we identified a microbial sample as a threephase system containing bacterial colonies and a multicomponent bacterial medium which were combined and simultaneously separated by a third phase called the pseudo surface phase (PSP). Additionally, the medium was physically defined as a solid and flexible substrate containing a mass of OS in the form of small irregularly shaped particles (POS). During the preparation of the bacterial medium, the OS particles were randomly dispersed throughout the volume, as well as in a thin layer, near the free surface of the medium as well as at different depths in such a way that the free surface of some POS entered the free surface formed by the continuous phase of the medium. Furthermore, it was assumed that the pseudo surface phase consisted of the free surface of the medium, the outer osmotic cell wall (OOPM), and the inner thin elastic cytoplasmic membrane (IECM) of the bacterial cell [58]. The PSP geometric shape and physical parameters depend on the shape and physical properties of the surrounding phases, respectively. The sigma phase (Σ-phase) exists in multiphase systems and participates in mass and heat transfer between phases [59]. The free surface in a real three-phase system is characterized by a complex geometry, and in the theoretical model, it can be treated as a regularly undulating free surface. RMF, medium free surface area, and OS mass flow are the main quantities affecting microorganisms on the medium surface. The physical nature of the free surface of the medium has the greatest influence on the growth of bacterial colonies. Thus, an increase or decrease in bacterial colony counts depends on the free surface with OS with activity controlled by available external factors. An example presenting one of such interactions is manifested by the fact that RMF significantly affects the physical state of the interfacial surface between the fluid and the solid phase in the technical process of manufacturing crystals from a liquid metal alloy [60].
In the first stage, we analyzed the free surface activity of the medium exposed to RMF. This type of analysis is of significant practical importance in microbiological processes, which are initially activated on such surfaces no matter whether the medium is controllably prepared or constitutes a free surface of any physical state. It should be kept in mind that in technical processes using RMF, magnetic fluids (medium) with a defined set of physical parameters of the electromagnetic field were used [36,37]. MF interacts with a multicomponent or dispersed system when volumes of these systems contain free electric charges, magnetic particles with their associated magnetizing effect, and perform an ability to accumulate and dissipate radiant magnetic energy [61][62][63]. We assumed that the magnetic susceptibility of OS can be generally defined, similar to any physical substance, by essential electromagnetic parameters, such as electrical conductivity and magnetic susceptibility [64]. In order to formulate the theoretical model which includes interaction between a medium and a bacterial colony, both exposed to RMF, we made specific assumptions concerning the geometric configuration as well as the mechanical and electromagnetic properties of the free surface of the medium. It was assumed that RMF influences OS if the investigated medium is adequately similar to magnetic fluid with defined magnetic properties. Furthermore, based on our assumptions, the experimental OS in our study contained chemical elements sensitive to MF, and hence, small, isolated POS with constant magnetic permeability should be treated as isolated magnetic particles of organic substance (MPOS). Moreover, we also assumed, that that the MPOS on a surface with a thin layer of the medium is influenced by RMF.
In the theoretical model, we assumed that the free surface of the medium has cylindricalshaped craters filled with MPOS, through which the outflow of OS is supplemented by a diffusive inflow of this substance from the interior of the medium. These craters are continually refilled with OS at a rate that depends on the depth of POS hiding under the free surface of the medium and on the rate of the mass diffusion process. This phenomenon repeats itself until the organic substance is exhausted or the bacterial colony is destroyed. In a microvolume of randomly dispersed craters filled with MPOS with their own certain electromagnetic parameters, MF induction lines penetrate OS, i.e., diffuse through the substance into the surface layer to a depth determined by the MF diffusion coefficient or magnetic kinematic viscosity coefficient depending on the electrical conductivity of OS, magnetic susceptibility of OS and RMF frequency [61,65,66]. Finally, MPOS of an equal number of poles (magnetic charges) of opposite polarity appears in the craters [67].
RMF orientates MPOS and forms magnetic dipoles. A collection of dipoles sends out a packet of induction lines and the volume of fluid in the crater becomes magnetized. These dipoles generate their inductive heterogeneous spin of MF inside OS whose magnetic force can move MPOS in a spin motion opposite to the direction of the external induction vector of RMF. MFs and especially RMFs, force a cylindrical volume of molten material into a uniform rotational motion with an azimuthal driving force proportional to the magnitude of RMF induction and frequency, electrical conductivity, and the diameter of the cylindrical volume of molten fluid [68]. However, the sample spins more slowly than the frequency of MF due to the viscosity of the surrounding fluid and the inertia of the cylindrical volume [65,69]. Hence, the magnetic dipoles of MPOS in a vortex motion can carry from the free surface of the medium an attached thin layer of mass of the medium inertial phase, an antimicrobial agent as well as other components present in the medium. Nevertheless, it can also be assumed that in the crater, a complex system of forces generated by various physical phenomena (collision, friction, oscillation, inertia) acts on their MPOS spin motion, and especially in the presence of small volume craters, the phenomena of non-magnetic nature can be neglected.
In the initial phase of the metabolic process, bacterial enzymes and RMF act on the local free surface area of small craters with POS organic substance. Surface tension in this area is then lowered and this area becomes an accessible source of OS. In view of the difference in organic substance concentration between the open free surface area of the medium as well as the bacterial cell, there is an outflow of an organic substance into the OOPM and thus through the IECM into the cell. As a result, the vital activity of bacteria increases, and the interaction of external RMF and induced internal RMF with magnetic dipoles of MPOS hidden in the layer under the free surface of the medium can decrease the surface tension of the medium-free surface partially interrupted by active craters. In turn, such a phenomenon contributes to an increase in the number of open OS-mass craters on this surface and an increase in OS influx into bacterial cells. Therefore, as a result of the RMF activity, MPOS moving through PSP reach OOPM of the bacterial cell, where, after penetrating IECM, they are absorbed by the cell. At a given point in the process, the metabolic activity of the bacteria increases, the surface organic substance content decreases, and the diffusive influx of OS from deeper layers of the medium to the layer below the free surface increases. MPOS can attach to TA molecules introduced into the medium. Therefore, the simultaneous presence of bacterial toxins induces counter-current movement of these chemicals. MPOS and TA molecules leave the crater and diffusely penetrate PSP to enter the pore target wall and then the bacterial cell. In the opposite direction, using the same type of movement, some of the toxins leave the bacterial cell and penetrate into some of the craters on the free surface contaminating the medium. Some of the toxins may remain in the cell, affecting the vital function of bacteria. These phenomena are dynamic processes. The biological resistance of the medium surface can be weakened by an introduction of a selective chemical substance into the layer under the free surface of the medium that lowers the surface tension. Ideally, this substance should also exhibit antimicrobial properties to some extent.
The introduction of chemically crafted small magnetic particles into the medium can modify the surface layer [70,71]. Such modification can be made by introducing inorganic Fe nanoparticles, Fe 3 O 2 , Fe 3 O 4 [72][73][74], or magnetic surface-modified particles dispersed in organic polymers to form a magnetic composite [75]. Bacteria can also be magnetically modified to form magnetotactic bacteria by the addition of immunomagnetic nano-and microparticles to the suspension with bacterial cells [46,[49][50][51][52][53]. Microorganisms can be biomineralized via the adsorption of magnetic fluids (aqueous electrolytes with ions Ni 2+ , Cu 2+ , Cd 2+ , or Ag 2+ as adsorbents, or can synthesize intracellular ferromagnetic crystalline with nanocrystals of magnetite (Fe 3 O 4 ) or greigite (Fe 3 S 4 ) [76][77][78][79][80][81][82]. Accumulation of a large number of nanocrystals of ferromagnetic metals in OOPM of the cell modifies its surface. In this case, one would expect a change in the OOPM of the bacterial cell, and the result would be either a change in mass and heat transport or in the elasticity of the bacterial cell membrane, which, like any physical membrane, is sensitive to external force actions of RMF. In this context, magnetotactic bacteria in RMF behave similarly to a magnetic dipole in suspension [50,51]. RMF and MPOS mass are sources that activate biological microparticles enclosed by the inner elastic membrane of the bacterial cell, where they are randomly distributed. The magnetic induction vector flux energetically activates MPOS present in OOPM pores and penetrates the interior of the bacterial cell.
Under the influence of MF, negatively charged biological microparticles and their own electromagnetic parameters randomly dispersed in the bacterial cell, form a material rotating "chain" oriented in the direction of MF lines. A similar phenomenon of chain formation occurs for magnetic particles Fe, Fe 3 O 2 , Fe 3 O 4 under the influence of RMF [29,81]. An electrically conductive "pathway" is formed and consists of a chain of biological microparticles, IECM and OOPM, as well as craters on the free surface of a MPOS-filled medium. An interaction between RMF and an electrically conductive "pathway" generates a microcurrent in the MPOS flow and the action of RMF on the microcurrent results in an elementary volume micro electromagnetic force that depends on the physical electromagnetic parameters of OS, frequency of the external RMF mechanically acting on the crater, OOPM, and IECM of the bacterial cell. It compresses the bacterial cell and elongates it in the direction of the MF induction vector, and with a high modulus of this force, the chain of molecules, as well as the bacterial cell, can get ruptured. Hence, like any elastic membrane, bacterial cell membranes can retain their original shape or get deformed under normal surface force. The mechanical properties of bacterial cells are described by Young's modulus and bending rigidity. If the stiffness of a bacterial membrane can be determined by the tensile elasticity and the stress/strain curve in the linear region, the bacterial cells will deform, whereas the removal of the load causes the membrane to return to its pre-load state [82]. At low-frequency RMF, an elementary volumetric electromagnetic microgrid is generated. It deforms the inner thin elastic membrane and generates slight oscillatory chain movement of particles in the cell, narrowing the pore on OOPM and the width of channels on the free surface of the medium, which in turn affects the resultant rate of the MPOS movement from the medium into the bacterial cell. When RMF frequency increases, the stress-strain curve is no longer linear. This results in micro-permanent dynamic deformation of the inner thin elastic membrane of the bacterial cell [83]. The highly increased intensity of RMF and simultaneous low elasticity of the inner thin elastic membrane contribute to the deformation which may irreversibly destroy the cell. Therefore, it can be expected that a sudden jump in high field stress will result in a mechanical rupture of the bacterial cells.

Reference Strain, Culture Condition, and Preparation of Staphylococcal Suspension
(Merck Life Science, Poznan, Poland) and incubated at 37 • C overnight. After incubation, the bacterial culture was centrifuged (5000× g for 10 min), washed three times using phosphate-buffered saline (PBS, pH 7.4), and adjusted to 1.5 × 10 8 CFU/mL density. The obtained suspension was used in all further experiments.

Minimum Inhibitory Concentration (MIC) Assay
The MIC of TA (99.0% purity; Merck Life Science, Poznan, Poland) was determined using the agar dilution method in compliance with the Clinical and Laboratory Standards Institute guidelines [85] with a few modifications, as described in our previous study [86]. Briefly, Mueller-Hinton agar (MHA; Merck Life Science, Poznan, Poland) was mixed with Tween 80 (1%, v/v; Merck Life Science, Poznan, Poland) and selected TA concentrations (0.31-10%, v/v). Next, the staphylococcal suspension was transferred onto prepared media (10 4 CFU per spot; 32 spots per plate) and incubated for 18 h at 37 • C.
MIC 100 of TA was evaluated after incubation and recognized as the lowest concentration of TA which completely inhibited the visible growth of S. aureus FRI913 on the MHA. The MIC 50 value (which is required to inhibit the growth of 50% staphylococcal cells) was calculated proportionally to the MIC 100 value. MHA supplemented with 1% (v/v) Tween 80 was used as a positive growth control for S. aureus FRI913. Concurrently, a sterility control was performed. The experiment was conducted in duplicates. The results were expressed in mg/mL.
The following culture media were prepared: MHA (medium A), MHA supplemented with 1% (v/v) Tween 80 (medium B), and MHA supplemented with 1% (v/v) Tween 80, and MIC 50 of TA (medium C). The media were used in all further stages of the experiment.

Rotating Magnetic Field Generator
The design of the RMF generator was based on a three-phase, four-pole stator with an internal diameter of 16 cm and a height of 20 cm, consisting of twelve groups of three coil sets (Figure 4). The frequency of the alternating current (AC) provided to the RMF generator was regulated using a Unidrive M200 inverter (Control Techniques, Nidec Industrial Automation, Poznan, Poland). The temperature in the RMF reactor chamber was controlled using a water thermostat (KISS K6, Huber, Germany) equipped with a set of temperature probes (accuracy range ± 1.0 • C). The distribution of magnetic induction (B) in the reactor chamber was performed at an initial voltage of AC of 100 V and AC frequencies of 5 Hz and 50 Hz using the Ansys Maxwell simulation software ver. 19.1 (Ansys, Inc., Canonsburg, PA, USA) and using a teslameter (SMS-102, Asonik, Tuczno, Poland).

Exposure of S. aureus FRI913 to RMF
In the first stage of the experiment, the staphylococcal suspension (100 μL) was introduced on A-C media, spread using a sterile spreader, and incubated for 12 h at 37 °C in the water bath (unexposed to RMFs) or in RMF generators at two different RMF frequencies: RMF I: 5 Hz, RMF II: 50 Hz. The arrangement and location of the Petri dishes in the RMF reactor chamber are graphically presented in Figure 5.
In the second stage of the experiment (after 12 h of incubation), media A-C incu-

Exposure of S. aureus FRI913 to RMF
In the first stage of the experiment, the staphylococcal suspension (100 µL) was introduced on A-C media, spread using a sterile spreader, and incubated for 12 h at 37 • C in the water bath (unexposed to RMFs) or in RMF generators at two different RMF frequencies: RMF I: 5 Hz, RMF II: 50 Hz. The arrangement and location of the Petri dishes in the RMF reactor chamber are graphically presented in Figure 5.

Exposure of S. aureus FRI913 to RMF
In the first stage of the experiment, the staphylococcal suspension (100 μL) was introduced on A-C media, spread using a sterile spreader, and incubated for 12 h at 37 °C in the water bath (unexposed to RMFs) or in RMF generators at two different RMF frequencies: RMF I: 5 Hz, RMF II: 50 Hz. The arrangement and location of the Petri dishes in the RMF reactor chamber are graphically presented in Figure 5.
In the second stage of the experiment (after 12 h of incubation), media A-C incubated in a water bath as well as exposed to RMFs were transferred to the incubator, and the cultures were incubated for the next 12 h (37 °C). In the RMF generator, a water bath as well as incubator, the same temperature (37 °C) and relative humidity RH (60%) were maintained throughout the entire experiment. Bacteria cultured on medium A and unexposed to RMFs were used as the control. A schematic diagram of the experimental setup is graphically presented in Supplementary Materials, Figure S1.  In the second stage of the experiment (after 12 h of incubation), media A-C incubated in a water bath as well as exposed to RMFs were transferred to the incubator, and the cultures were incubated for the next 12 h (37 • C). In the RMF generator, a water bath as well as incubator, the same temperature (37 • C) and relative humidity RH (60%) were maintained throughout the entire experiment. Bacteria cultured on medium A and unexposed to RMFs were used as the control. A schematic diagram of the experimental setup is graphically presented in Supplementary Materials, Figure S1.

RNA Isolation
After 24 h of incubation on A-C media unexposed and exposed to RMFs, bacteria were harvested, adjusted to 1.2 × 10 9 CFU/mL, transferred to 1.5 mL tubes, and washed three times using PBS. To ensure uniform exposure to the RMF, the bacteria were harvested from the zone 1.5 cm from the edges of the plate and 3 cm from the center, marked with a circle in Figure 5. The supernatant after the first washing was preserved for the ELISA test. Then, 400 µL of acid guanidinium thiocyanate-phenol-chloroform-based reagent (A&A Biotechnology, Gdansk, Poland) was added to each bacterial pellet and frozen at −20 • C to prevent RNA degradation. The RNA isolation procedure was carried out on the next day. After thawing, 50 mg of glass beads (diameter 150-212 µm; Merck, St. Louis, MO, USA) was added to the samples. Three cycles of beating (2 min each), with 1 min incubation on ice within cycles, were carried out in the Tissue Lyser LT (Qiagen, Hilden, Germany) at 4 • C. Total RNA was isolated with the RNA Isolation Mini Kit (A&A Biotechnology, Gdansk, Poland) in accordance with the manufacturer's instruction. Next, RNA was dissolved in 50 µL of RNase-free water (A&A Biotechnology, Gdansk, Poland). The RNA concentration was measured using a spectrophotometer (DeNovix DS-11 FX, Wilmington, DE, USA), and the purity of the isolated RNA was assessed spectrophotometrically (A 260 nm/A 280 nm). Contaminated DNA was removed using RNase-free DNase I (Merck, St. Louis, MO, USA) digestion for 1 h at 37 • C. The integrity of RNA was evaluated by electrophoresis on 1% (w/v) agarose gel. RT-PCR was performed using SuperScript™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). For cDNA synthesis, 100 ng of RNA was mixed with 1 µL of 50 µM Random Hexamers and 1 µL 10 mM dNTP Mix, heated to 65 • C for 5 min, and incubated on ice for 1 min. In the next step, 4 µL of 5XFirst-Strand Buffer, 1 µL of 0.1 dithiothreitols, 1 µL of RNaseOUT™ Recombinant RNase Inhibitor, and 1 µL of SuperScript™ III RT (Invitrogen, Carlsbad, CA, USA) were added and incubated at 50 • C for 60 min. The reaction was inactivated by heating at 70 • C for 15 min.  Table S4), 10 µL of SsoFast EvaGreen Supermix, and water up to 20 µL. Reaction mixtures were initially incubated for 30 s at 95 • C, followed by 35 cycles at 95 • C for 10 s, and 15 s at 62 • C. The specificity of PCR was evaluated by a melt curve analysis at a temperature ranging from 95 to 58 • C performed for each reaction. Residual DNA contamination was checked in each RNA sample by running no-RT controls. All samples were analyzed in triplicate.
The housekeeping gene was selected from 8 candidate genes, including rpoB, 16S rRNA, gyrB, recA, rho, pta, rplD, and tpo based on its expression stability [87]. The BestKeeper software (https://www.gene-quantification.de/bestkeeper.html, accessed on 5 January 2022) was used to assess the variability of housekeeping genes. As a positive control of the reaction, S. aureus FRI913 DNA was used. PCR efficiencies determined for all tested primer pairs were above 96%. Relative transcript levels were calculated according to results obtained by Pfaffl [88]. Data analysis was carried out using the Bio-Rad CFX Manager (Bio-Rad, Herkules, CA, USA) software.

Samples Preparation
After 24 h of incubation on A-C media unexposed and exposed to RMFs, 1 mg of the bacterial biomass was collected from each plate (to ensure uniform exposure to the RMF, the bacteria were harvested from the zone 1.5 cm from the edges of the plate and 3 cm from the center, marked with a circle in Figure 5) and mechanically lysed by homogenization with 0.5 mg of glass beads in 1 mL of supernatant after first washing the pellets, in the Tissue Lyser LT (Qiagen, Hilden, Germany). After centrifugation (2000× g, 5 min at 4 • C) supernatants were preincubated with 20% normal rabbit serum overnight at 4 • C (in order to bind protein A) and diluted 4 times in PBS supplemented with 0.1% (v/v) Tween 20 (Merck, St. Louis, MO, USA).

Sandwich ELISA Preparation
Sandwich ELISA was performed as described by Lis et al. [89]. Rabbit polyclonal anti-SEA and anti-SEC antibodies were purchased from Acris Antibodies (Herford, Germany). Rabbit polyclonal IgG antibodies against SEL were prepared by immunizing 3-month-old white male New Zealand rabbits. Recombinant proteins for immunization were obtained with a technique previously described by Podkowik et al. [84]. Briefly, the sel gene was cloned into the pET-22b plasmid vector and expressed in E. coli rosetta competent cells The ELISA was carried out in 96-well microtiter plates. Each well was coated with 50 µL of antibody (2 µg/mL) specific for the tested SEs in 0.05 M carbonate-bicarbonate buffer (pH 9.6; Merck, St. Louis, MO, USA) at room temperature overnight. A standard curve was prepared by using recombinant proteins: SEC (rSEC), SEL (rSEL) [84], and SEA (rSEA), purchased from Merck (St. Louis, MO, USA). Antibody-coated wells were blocked with 250 µL/well of PBS/0.1% bovine serum albumin for 1 h at 25 • C. Next, 100 µL/well of samples or standards were added and incubated at 25 • C for 2 h. After washing, biotinylated antibodies were added to each well and incubated at 37 • C for 1 h. Biotinylated antibody was detected with the conjugate of HRP-streptavidin (Merck, St. Louis, MO, USA). 3,3 ,5,5 -tetramethylbenzidine (TMB, Merck, St. Louis, MO, USA) was used as a substrate for HRP. The absorbance at 405 nm was read with a microplate reader (Spark Tecan, Männedorf, Switzerland).
In order to determine SE concentrations, a 4-parameter logistic curve was prepared using a known concentration of rSEC, rSEL, and SEA. Data were analyzed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA).

Statistics
The statistical significance of the results was determined using a one-way ANOVA test. Spearman's rank correlation coefficient was used to detect relations between SE mRNA expression and SE protein production. The following values: * p < 0.05, ** p < 0.01, *** p < 0.001 were considered statistically significant. Statistical analyses were performed using Statistica version 12 (StatSoft Inc., Cracow, Poland) and GraphPad Prism 5.02 (GraphPad Software Inc., San Diego, CA, USA).

Conclusions
The obtained results allowed us to conclude that there were no significant differences in the expression of regulatory and SE genes in bacteria simultaneously cultured on a medium supplemented with TA and exposed to RMF at the same time in comparison to the control (unexposed to TA and RMF). In contrast, when the bacteria were cultured on a medium supplemented with TA but were not exposed to RMF or when they were exposed to RMF of 50 Hz (but not to TA), a significant increase in agrA and sea transcripts as compared to the unexposed control was found. Moreover, the decreased level of sec transcripts in bacteria cultured without TA but exposed to RMF of 50 Hz was also revealed. In turn, a significant increase in SEA and decrease in SEC and SEL production was observed in bacteria cultured on a medium supplemented with TA and simultaneously exposed to RMFs. It can be concluded, that depending on SE and regulatory genes' expression, as well as production of SEs, the effect exerted by the RMF and TA may be positive (i.e., manifests as the increase in SE and/or regulatory gene expression of SEs production) or negative (i.e., manifests as the reduction in both aforementioned features) or none. The experimental results suggest that the above type of variables could be usefully, e.g., in the control of S. aureus gene expression, which in turn can have an enormous impact in the food industry (e.g., food safety and quality). However, the use of specific parameters and factors to determine the optimal conditions of such a process still require further development.


MsmR1, a global transcription factor, regulates polymyxin synthesis and carbohydrate metabolism in Paenibacillus polymyxa SC2

The multiple-sugar metabolism regulator (MsmR), a transcription factor belonging to the AraC/XylS family, participates in polysaccharide metabolism and virulence. However, the transcriptional regulatory mechanisms of MsmR1 in Paenibacillus polymyxa remain unclear. In this study, knocking out msmR1 was found to reduce polymyxin synthesis by the SC2-M1 strain. Chromatin immunoprecipitation assay with sequencing (ChIP-seq) revealed that most enriched pathway was that of carbohydrate metabolism. Additionally, electromobility shift assays (EMSA) confirmed the direct interaction between MsmR1 and the promoter regions of oppC3, sucA, sdr3, pepF, yycN, PPSC2_23180, pppL, and ydfp. MsmR1 stimulates polymyxin biosynthesis by directly binding to the promoter regions of oppC3 and sdr3, while also directly regulating sucA and influencing the citrate cycle (TCA cycle). In addition, MsmR1 directly activates pepF and was beneficial for spore and biofilm formation. These results indicated that MsmR1 could regulate carbohydrate and amino acid metabolism, and indirectly affect biological processes such as polymyxin synthesis, biofilm formation, and motility. Moreover, MsmR1 could be autoregulated. Hence, this study expand the current knowledge of MsmR1 and will be beneficial for the application of P. polymyxa SC2 in the biological control against the certain pathogens in pepper.

Carbohydrates exist in various forms, including monosaccharides, disaccharides, and polysaccharides. Energy released from carbohydrate catabolism is required for metabolic activity and growth (Warda et al., 2016). Pathways of carbohydrate catabolism involve complex networks that regulate gene expression. In bacteria, carbohydrate utilization is regulated by transcription factors (TFs) regulating the expression of genes which encodes transporters and enzymes (Khoroshkin et al., 2016). Efficient utilization of carbohydrates by bacteria is determined by various utilization systems and carbohydrate transporters (Warda et al., 2016).
The MsmR of the AraC/XylS family is involved in polysaccharide metabolism (Russell et al., 1992). In Streptococcus mutants, MsmR positively regulates an operon for the uptake and catabolism of carbon sources (Russell et al., 1992). In Streptococcus pyogenes, MsmR positively regulates fibronectin-and collagenbinding T-antigen (FCT) region genes, including nra, which inhibit the expression of crucial virulence factors (Kreikemeyer et al., 2007). Meanwhile, the transcriptional regulatory mechanisms of MsmR1 in P. polymyxa remain unclear.
Paenibacillus polymyxa SC2 was isolated from the rhizosphere of pepper plants in Guizhou province, China (Ma et al., 2011). Paenibacillus polymyxa SC2-M1 is spontaneously mutated from P. polymyxa SC2 during strain cultivation, making it suitable for molecular manipulations (Hou et al., 2016). A previous study reported that the msmR1 gene is upregulated by 2.17-fold during the interaction between P. polymyxa SC2 and pepper , indicating that msmR1 may play an important role in this interaction. In the present study, we aimed to explore the regulatory mechanism of the transcription factor MsmR1 in polymyxin synthesis and related biological processes in P. polymyxa SC2. To this end, chromatin immunoprecipitation with sequencing (ChIP-seq) was performed and the results were verified by electrophoretic mobility shift assay (EMSA). The oppC3, sucA, sdr3, pepF, yycN, PPSC2_23180, pppL, and ydfp genes were found to be directly regulated by the global regulator MsmR1; the functions of these genes were further explored to delineate the regulatory mechanism of MsmR1. The results of this study provide a theoretical basis for the improved application of P. polymyxa SC2 in the biological control against the certain pathogens.
Competent cells of E. coli were prepared using the transformation and storage solution (TSS) method and transformed by the heat pulse method (Chung et al., 1989). Competent cells were prepared and P. polymyxa SC2-M1 were electrotransformed as previously described (Hou et al., 2016).

Plasmid and strain construction
To construct knockout SC2-M1 strain mutants, 650-1,000 bp of homologous arm-flanking target genes were cloned from genomic DNA using PCR. The cat gene, encoding the chloramphenicol resistance cassette, was cloned from pDG1661 (Guérout-Fleury et al., 1996) using PCR to replace the target knockout genes. Two homologous arms and cat cassettes were fused by fusion PCR (Cha-Aim et al., 2012), and the fusion DNA fragments were ligated into the thermosensitive elimination plasmid pRN5101 to construct knockout vectors. The plasmid was demethylated by being further transformed into Trans 110. The demethylated knockout vectors were transferred into the SC2-M1 strain. Double-crossover recombinants were detected as previously reported (Hou et al., 2016), and the knockout strains were confirmed by PCR using the corresponding primers (Supplementary Table S2). For in situ complementation of the knockout genes, 650-1,000 bp of homologous arms fused with the corresponding complete target genes were cloned by PCR from genomic DNA (not involved in the resistance cassette), and ligated into pRN5101 to construct the complement vectors. Demethylated complementary vectors were introduced into the mutant strains. Double-crossover recombinants were detected as described above, and the complemented strains were confirmed by PCR using the corresponding primers (Supplementary Table S2). For ChIP-seq analysis, we obtained a Flag-tagged strain using homologous recombination. The Flag-tag was added in the front of termination codon of msmR1 and a chloramphenicol resistance cassette was also added after the Flag tag using overlapping PCR. The homologous arms (650-1,000 bp) were cloned by PCR amplification, and ligated to pRN5101 to construct the plasmid pRN5101-msmR1-Flag. Demethylated pRN5101-msmR1-Flag were introduced into SC2-M1 and double-crossover recombinants were detected as described above, designated as strain SC2-M1-Flag. For overexpression of target genes, fragments carrying complete target genes were cloned from genomic DNA, fused with the promoter fragments, and ligated into pHY300PLK between BglII and SalI sites by ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech, Nanjing, China) for overexpression in SC2-M1. To evaluate the promoter activity of target DNA fragments, the fragments were cloned from genomic DNA, and the promoterless green fluorescent protein (gfp) fragment from pGFP4412 was cloned (Li et al., 2019). The two fragments were fused using PCR and ligated into pHY300PLK between BglII and SalI sites as described above, thus generating the pHY300PLK-promoter-gfp plasmid.

Chromatin immunoprecipitation assay with sequencing (ChIP-seq)
The SC2-M1 and SC2-M1-Flag strains were cultured in 50 ml of LB medium at 37°C with shaking at 180 rpm for 10 h, inoculated with an initial OD 600 of 0.8 in 50 ml of LB broth by 2% (v/v), and cultured at 37°C with shaking at 180 rpm for 7 h. The two strains were then fixed with 1% formaldehyde for 10 min at 28°C and the reaction was stopped with 125 mM glycine for 5 min. The cells of the two strains were washed thrice with phosphate buffer solution (PBS) at 8000 rpm for 5 min at 4°C and stored at-80°C. ChIP was performed by Wuhan IGENEBOOK Biotechnology Co. Ltd., China. The manufacturer's instructions of Illumina TruSeq ChIP Sample Prep Set A were followed to construct sequencing libraries using chromatin immunoprecipitated DNA which was then sequenced on an Illumina Xten using the PE 150 method . The obtained reads were filtered by Trimmomatic (v 0.30). Clean reads were then mapped to the P. polymyxa SC2 genome using Bwa (v 0.7.15), which allowed for up to two mismatches (Pjanic, 2017). MsmR1 peaks were produced and visualized using MACS software and Integrative Genomics Viewer (IGV, v 2.3.91), respectively (Zhang et al., 2008;Thorvaldsdóttir et al., 2013). To understand the potential roles of genes, Kyoto Encyclopedia of Genes and Genomes (KEGG) and multiple EM for motif elicitation (MEME) were used for pathway enrichment analysis and motif analysis, respectively Wang et al., 2022).

Quantitative real-time PCR
SteadyPure Universal RNA Extraction Kit (Accurate Biology, Hunan, China) was used to purify the total RNA from the target bacteria. cDNA was obtained from 1 μg of total RNA using the Evo M-MLV RT Kit with gDNA clean for qPCR II (Accurate Biology, Hunan, China). qRT-PCR analysis was performed on a Roter-Gene Q PCR system using the SYBR Green Pro Taq HS qPCR Kit (Accurate Biology, Hunan, China). The relative expression of target genes was calculated using the 2 −ΔΔCt method . The housekeeping gene glyceraldehyde-3phosphate dehydrogenase (gapdh) was used as an internal control.

Purification of MsmR1 protein and electromobility shift assays
An 828 bp fragment (275 amino acids) of msmR1 was cloned from the genomic DNA of strain SC2-M1 using primers MsmR1F and MsmR1R (Supplementary Table S2). The fragments of msmR1 and plasmid pET-28b(+) were simultaneously cleaved with XhoI and SalI, purified, ligated using T4 DNA ligase, and transferred into E. coli DH5α to obtain pET28b(+)-msmR1.
To overexpress the MsmR1 protein, the plasmid pET28b(+)-msmR1 was transferred into E. coli BL21. The transformants were Frontiers in Microbiology 04 frontiersin.org cultured in kanamycin-resistant LB broth at 37°C with shaking at 180 rpm for 10 h. Next, the cells were inoculated into 50 ml of kanamycin-resistant LB broth at 2% (v/v) and cultured at 37°C with shaking at 180 rpm for approximately 1.2 h until the OD 600 reached 0.5-0.6. Then the cells were induced by 0.4 mM IPTG at 16°C for 20 h, harvested, washed, resuspended in PBS, and sonicated on ice. MsmR1-His6 protein was purified using BeyoGold™ His-tag Purification Resin (Beyotime, China) following the manufacturer's instructions. The purified protein was added to a final concentration of 10% (v/v) glycerol and stored at-80°C. EMSA was performed using a chemiluminescent EMSA kit (Beyotime, China; Zhang et al., 2021). The concentration of labeled probes and competitive probes were about 0.4 and 4 μM, respectively. The gradient concentration of protein were set up for the EMSA experiment.

Detection of the fluorescence intensity of promotors
Promoter reporter plasmids were transformed into E. coli DH5α cells. The strains were inoculated in LB plate containing 20 μg/ml tetracycline and fluorescence was observed with an OLYMPUS fluorescence microscope at 1000× (Li et al., 2019). The fluorescence intensity of the promoters was detected at different time points as previously reported (Li et al., 2019).

Antibacterial activity analysis
An Oxford cup assay was used to evaluate polymyxin production by the SC2-M1 strain, as previously described . Bacteria were inoculated in liquid LB medium and cultured at 37°C with shaking at 180 rpm for 10 h. Bacteria were then inoculated with an initial OD 600 of 0.8 in 50 ml fermentation medium (sucrose 40 g/l, (NH 4 ) 2 SO 4 8 g/l, CaCO 3 5 g/l, KH 2 PO 4 0.2 g/l, MgSO 4 0.2 g/l, NaCl 0.2 g/l) at 2% (v/v) and cultured at 37°C with shaking at 180 rpm for 48 h.

Motility assay
A motility assay was performed as previously reported (Guo et al., 2019). Bacteria were cultured on LB medium for approximately 12 h, after which the OD 600 was adjusted to 0.6. Diluted cell solutions (2 μl) were added to the center of the plate containing 0.5% glucose, 0.22% NaCl, 0.13% yeast extract, 0.45% tryptone, and 0.5% agar.

Growth curve assay
Bacteria were cultured overnight in liquid LB medium and adjusted to an OD 600 of 0.8. Diluted strain solutions were inoculated at 2% in 50 ml of fresh LB medium. The bacteria were cultured at 37°C with shaking at 180 rpm. The OD 600 and number of live bacterial cells were measured every 3 h. A total of 100 μl of the strain solution was serially diluted with 900 μl of sterile water; 100 μl of the dilution was then spread on an LB plate. The plates were subsequently incubated for 24 h at 37°C.
The growth curves of bacteria on the carbon sources were determined using a basal medium. Bacteria were cultured overnight in LB medium. The cultures were centrifuged, washed thrice with basal medium, and resuspended to inoculate 50 ml of fresh basal medium with 1% sole carbon source; the original OD 600 value was approximately 0.1. Cultures were incubated at 37°C with shaking at 180 rpm, and growth curves were determined according to OD 600 every 3 h.

Statistical analysis
For data analysis, the analysis of variance (ANOVA) and Least Significant Difference (LSD) test (p ≤ 0.05) were performed using statistical software SPSS (v 19.0, SPSS Inc., Chicago, IL, USA; Wang et al., 2020).

Results
Overview of the transcription factor MsmR1 from Paenibacillus polymyxa SC2 The MsmR1 of strain SC2, which is classified as an AraC/XylS family transcriptional regulator, contains two domains: an N-terminal heterologous dimerization domain and a C-terminal DNA-binding domain ( Figure 1A). The C-terminus of MsmR1 shows high similarities to that of AraC family members: RhaR, AraC, UreR, MelR, MsmR, XylS, and Toxt. Seven α-helix were identified in the C-terminal domain of MsmR1, which combined to form two helix-turn-helix (HTH) motifs ( Figure 1A).
AraC family TFs are often auto-regulated (Sun et al., 2016). Hence, the promoter activity of msmR1 was evaluated using the pHY300PLK-msmR1-gfp plasmid. This plasmid was found to possess promoter activity in E. coli DH5α cells. In contrast, the control (pHY300PLK-gfp) showed no fluorescence (Supplementary Figure S2A). The pHY300PLK-msmR1-gfp plasmid was introduced into the strains SC2-M1 and SC2-M1-ΔmsmR1 which was a msmR1 knock out strain obtained via homologous recombination (Supplementary Figure S2B). The fluorescence intensity of the strain SC2-M1-ΔmsmR1 was significantly higher than that of the SC2-M1 strain ( Figure 1B), revealing that MsmR1 could auto-regulate its transcription. EMSA was further performed to determine whether MsmR1 is capable of direct auto-regulation. MsmR1 was found to specifically bind to its own promoter resulting in a retarded band ( Figure 1C), indicating that MsmR1 auto-regulates its expression. Frontiers in Microbiology 06 frontiersin.org SC2-M1 strain; the diameter of the inhibitory zone was decreased by 9.0% ( Figure 1D; Supplementary Figure S2D). The qRT-PCR results revealed that the polymyxin synthetase genes were downregulated in the knockout strain compared with SC2-M1 ( Figure 1E). In SC2-M1-pmsmR1, the inhibitory zone diameter was increased by 10.03% compared with SC2-M1-p ( Figure 1D; Supplementary Figure S2D). The results were consistent with the qRT-PCR results which presented a significant increase in expression of polymyxin synthetase genes in SC2-M1-pmsmR1 when compared with that in SC2-M1-p ( Figure 1F). Given that AraC family members are involved in carbohydrate metabolism (Cortés-Avalos et al., 2021), the growth curves of the SC2-M1 and SC2-M1-ΔmsmR1 strains were determined using LB and basal media with sole carbon sources. Although phenotypic differences were noted between the two strains using glucose, xylose, galactose, fructose, and lactose, the differences were not significant (Supplementary Figure S2E).

Identification of MsmR1 binding genes
To determine the target genes directly regulated by MsmR1 in P. polymyxa SC2, ChIP-seq experiments were performed using C-terminal FLAG-tagged msmR1 in SC2-M1. The qRT-PCR results demonstrated that the expression of msmR1 was high during the early stage and low during the decline phase (Supplementary Figure S3A). Based on the qRT-PCR results and the growth curve, a culture time of 7 h (mid-logarithmic phase) was selected for subsequent ChIP-seq analysis (Supplementary Figure S3).
Approximately 3.83-4.86 million reads in the ChIP-seq results were derived from the six samples, which were mapped to the P. polymyxa SC2 genome (Supplementary Table S3). Overall, 649 peaks were identified between the two strains, SC2-M1 and SC2-M1-Flag. Among these, 96.15% were located in the coding sequence (CDS) and 3.85% were located in the intergenic regions. To categorize the functions of MsmR1 target genes, KEGG enrichment analysis was performed. The target genes were enriched in various functions, including cellular processes (1.56%), environmental information processing (6.25%), genetic information processing (10.94%), and metabolism (81.25%; Supplementary Table S4). In the metabolism category, carbohydrate metabolism was the most enriched pathway, while amino acid metabolism, metabolism of cofactors and vitamins, lipid metabolism, energy metabolism, and nucleotide metabolism were also significantly enriched ( Figure 2). Hence, MsmR1 primarily regulates basal metabolism in SC2 cells. According to the predicted results of MEME, we selected the fragments related to primary metabolism for further verification (Supplementary Table S5).
MsmR1 positively regulates polymyxin synthesis by directly binding to the oppC3 and sdr3 promoters As described previously, carbohydrate and amino acid could affect the biosynthesis of polymyxin (Yu et al., 2018). The oppC3 gene encodes a peptide ABC transporter together with the OppB protein forming a transmembrane channel that can transport and release peptides and nickel into cells (Ge et al., 2018;Slamti and Lereclus, 2019). The gene sdr3 whose predicted product belongs to the short-chain dehydrogenase/reductase (SDR) family is important for carbohydrate, amino acid, lipid metabolism, and redox sensor mechanisms (Kavanagh et al., 2008). oppC3 and sdr3 affected the growth state of strain SC2-M1 in LB medium ( Figures 4A,B). Therefore, we determined the expression levels of primary metabolism-related genes, such as those involved in carbohydrate, amino acid, and fatty acid metabolism (Supplementary Table S2), and all of these genes were downregulated in the mutant strains compared with SC2-M1 ( Figures 4C,D). The growth state of SC2-M1-ΔoppC3 and SC2-M1-Δsdr3 in basal medium with the sole carbon source supported their roles in carbohydrate metabolism (Supplementary Figures S4, S5). In the present study, polymyxin synthesis was significantly reduced in the knockout strains SC2-M1-ΔoppC3 and SC2-M1-Δsdr3 compared with SC2-M1, and was recovered in the complement/overexpression strain ( Figures 4E,F). Moreover, the qRT-PCR results confirmed that polymyxin synthetase genes were downregulated in the knockout strain compared with SC2-M1 ( Figures 4C,D). The oppC3 and sdr3 genes indirectly influence polymyxin synthesis by reducing the rate of primary metabolism and nutrient uptake, and further reduce precursors and energy for polymyxin synthesis.

MsmR1 positively regulates the TCA cycle by directly binding to the sucA promoter
The sucA gene encodes 2-oxoglutarate dehydrogenase (E1), part of the 2-oxoglutarate dehydrogenase complex (Gayán et al., 2019). In the TCA cycle, α-ketoglutarate is converted to succinate, which requires the catalytic action of the 2-oxoglutarate Frontiers in Microbiology 07 frontiersin.org dehydrogenase complex (sucAB) and succinate dehydrogenase (sucCD; Walshaw et al., 1997). To assess the function of sucA in SC2, a knockout mutant strain SC2-M1-ΔsucA was generated. The colony morphology on LB plates appeared flat and transparent following sucA knockout, whereas the original morphology was recovered in the complemented strain SC2-M1-ΔsucA/sucA ( Figure 5A). The growth curve in LB medium was determined to evaluate the growth status, and showed that growth of the mutant strain SC2-M1-ΔsucA was markedly reduced compared to SC2-M1 and SC2-M1-ΔsucA/sucA ( Figure 5B). Simultaneously, live bacterial counts were determined every 3 h; those of SC2-M1-ΔsucA were greatly reduced compared with those of SC2-M1 and SC2-M1-ΔsucA/sucA, which was consistent with the OD 600 results ( Figure 5C). Moreover, expression of the related gene sucB was markedly downregulated, while other genes involved in the TCA cycle, such as sucC, sucD, citC, lpdV, and pdhC (Supplementary Table S2) were markedly upregulated ( Figure 5D). Due to disruption of the TCA cycle, bacteria may metabolize carbohydrate sources through the pentose phosphate pathway and glyoxylate cycle. Therefore, the energy decreased and the bacterial mass was further reduced.
During carbon source (xylose, arabinose, glucose, galactose, fructose, mannose, sucrose, maltose, trehalose, lactose and raffinose) utilization, the SC2-M1-ΔsucA strain presented a longer lag phase compared with the SC2-M1 strain. For the utilization of xylose, galactose, fructose, lactose, and raffinose, SC2-M1-ΔsucA presented a markedly lower OD 600 value during the stationary phase compared with SC2-M1 (Supplementary Figure S6). Hence, sucA have essential roles in carbon source utilization. KEGG enrichment analysis of MsmR1 binding DNA according to ChIP-seq data. The fragments obtained from ChIP-seq were analyzed by KEGG, and were enriched in cellular process, environmental information process, genetic information process, and metabolism.
Frontiers in Microbiology 08 frontiersin.org MsmR1 regulates motility, biofilm, and spore synthesis, as well as other processes The pepF gene encodes an oligoendopeptidase F. In Bacillus subtilis, the pepF homolog phrA can regulate spore formation (Kanamaru et al., 2002), and in Aeromonas hydrophila, pepF can regulate biofilm formation . qRT-PCR results revealed that pepF regulated spore and biofilm formation. In other words, the relative expressions of genes related to biofilm and spore formation were downregulated ( Figure 6A). The relative expressions of these genes were also downregulated in strain SC2-M1-ΔmsmR1 compared with SC2-M1 (Supplementary Figure S7). Overall, MsmR1 could positively regulate spore and biofilm formation.
Chemotaxis enables microbes to sense environmental signals and move to more favorable environments, which is important for bacterial colonization (Ud-Din and Roujeinikova, 2017). We found that motility was lost following sdr3 deletion and was increased in the SC2-M1-psdr3 strain compared with that in the SC2-M1-p strain ( Figure 6B). Moreover, the relative expression levels of genes encoding chemotaxis and flagellum (Supplementary Table S2) were downregulated in the SC2-M1-Δsdr3 strain compared with those in the SC2-M1 strain ( Figure 6C). Taken together, these results indicate that MsmR1 may positively regulate cell motility by directly binding to the sdr3 promoter.
The yycN gene encodes GNAT family acetyltransferase. The ydfp gene acts as a DoxX family protein, combined with SodA and SseA, forming a membrane-associated oxidoreductase complex (Nambi et al., 2015). The pppL gene encodes a PPM family protein phosphatase, which participates in environmental stresses (Shi et al., 1998). Hence, MsmR1 in P. polymyxa SC2 may also be involved in other biological processes.

Discussion
The purpose of this study was to elucidate the transcriptional regulatory mechanism of MsmR1 in P. polymyxa SC2. Sequence analysis revealed that MsmR1 comprises seven α-helices forming two HTH motifs at the C-terminal domain, which is the DNA binding domain, as well as a sensing or oligomerization domain of approximately 140 amino acids at the N-terminal domain.
MsmR1 is a typical AraC/XylS family transcriptional regulator (Egan, 2002), which can act as activators, repressors, or both, and are involved in various biological processes ranging from metabolism to stress response and virulence (Gahlot et al., 2021). In this study, a ChIP-seq experiment combined with EMSA was performed, the regulatory network of the global regulator MsmR1 was proposed (Figure 7), and the role of MsmR1 in P. polymyxa was described.
MsmR1 directly and positively regulates the expression of oppC3, sucA, sdr3, pepF, yycN, PPSC2_23180, pppL, and ydfp. In particular, MsmR1 directly regulates sucA and influences the TCA cycle. OppC3, an ABC transporter, transports extracellular oligopeptides and oligosaccharides into cells, affecting primary  Microbiology  11 frontiersin.org metabolism and polymyxin synthesis. Meanwhile, as a member of the short-chain dehydrogenases/reductases (SDRs) family, Sdr3 can influence fatty acid, carbohydrate, amino acid metabolism, and motility, which in turn reduces energy and precursors for polymyxin synthesis. PepF affects spore and biofilm formation. Due to the fact that spontaneous mutant strain SC2-M1 lost the ability to form endospores and biofilm (Hou et al., 2016), we could not detect the physiological or biochemical characteristics of endospores and biofilm in the test strains. However, since the genes were not successfully knocked out, the functions of PPSC2_23180, ydfp, pppL, and yycN required further investigation. Nevertheless, the results of this study revealed the mechanism through which MsmR1 regulates polymyxin synthesis, carbohydrate metabolism, and other biological processes. We obtained 649 peaks by ChIP-seq. KEGG enrichment analysis of these sequences revealed that the genes were involved in various functions, including cellular processes, environmental information processing, genetic information processing, and metabolism. Among these, the proportion of genes enriched in metabolism was the highest, reaching 81.25%, of which carbohydrate metabolism was the most enriched, followed by amino acid metabolism. These results support previous findings indicating that MsmR is primarily involved in polysaccharide metabolism (Russell et al., 1992). In addition, only 3.85% of the identified fragments were located in intergenic regions, whereas 96.15% were located in coding regions. This may be due to differences in the DNA-binding properties of MsmR1 in vivo and in vitro. Meanwhile, in B. subtilis, 643 binding sites for AbrB and 411 binding sites for Abh were identified, and approximately half of these binding sites were located in the gene coding region. Although half of all binding sites are located in intergenic regions, this does not affect transcription, even with strong binding intensities (Chumsakul et al., 2011). RutR is a regulator of pyrimidine catabolism in E. coli, and possesses 20 binding sites primarily bound to the gene coding regions with minimal effects on transcription levels (Shimada et al., 2008). Hence, MsmR1 may be a transcriptional regulator with binding site preferences that are similar to those of RutR.

Frontiers in
Additionally, EMSA experiments revealed that MsmR1 could also bind its own promoter, however, this was not observed in the ChIP-seq results. In fact, most genes from the ChIP-seq results were not found to bind to MsmR1. There are several possible reasons for this. First, strains in the middle of the logarithmic growth phase were selected for ChIP-seq experiments; MsmR1 may interact weakly with these genes, and some binding sites may not have been detected (Galagan et al., 2013). Second, other cofactors may be required for MsmR1 to bind target genes. Transcription factors are Frontiers in Microbiology 12 frontiersin.org typically coordinated by two or more transcription factors (Zhang et al., 2021). Third, adding a Flag tag to the C-terminus of MsmR1 with a cat resistance gene in the genome of SC2-M1 may interfere with the DNA binding ability and result in false-positive or -negative results. ChIP results reported for Salmonella enterica did not reveal previously described OmpR-binding targets but revealed that binding to some OmpR-regulated genes was affected by the C-terminal Flag tag (Perkins et al., 2013;Zhang et al., 2021). Finally, the EMSA conditions may influence the binding process. In the EMSA experiment, some retarded bands were relatively clear at low or high protein concentrations, while other DNA-MsmR1 binding processes presented clearer retarded bands only at high protein concentrations and smeared bands at low concentrations. This might be due to the different binding abilities of MsmR1 to DNA or the stability of the MsmR1-DNA complex in vitro during electrophoresis. As reported previously, a weak interaction occurs between the regulator VjbR and the virB (PvirB) promoter; however, no retarded signal was observed in the EMSA experiment, indicating that VjbR and PvirB cannot withstand the electrophoretic conditions of EMSA (Arocena et al., 2010). Therefore, in ChIP-seq, some DNA may have bound weakly to MsmR1 causing it to not be detected during EMSA electrophoresis. MsmR is a regulator of polysaccharide metabolism, hence the growth curve of the msmR1 knockout strain was determined. The results revealed no significant change in the LB medium or minimal medium with different carbon sources (Supplementary Figure S2E). However, its role in carbohydrate metabolism cannot be ruled out, as most of the identified target genes are related to carbon source metabolism. We hypothesized that this may be due to the functional redundancy of MsmR1 homologs in the SC2 strain. For genome sequence analysis, three genes were annotated as msmR (msmR5, msmR7, and msmR9), and 44 AraC family transcription factors were identified in the genome of P. polymyxa SC2, most of which were related to carbon source metabolism, such as arabinose metabolism and L-rhamnose metabolism (Supplementary Table S6). This possibility was verified using qRT-PCR, which revealed that msmR5, msmR7, and msmR9 were all upregulated after msmR1 knockout (Supplementary Figures S8A-C). Since msmR5 was the most upregulated, we then assessed the impact of knocking out msmR5. The results showed no significant difference in SC2-M1, single mutant of msmR5 (SC2-M1-ΔmsmR5) and double mutant Proposed regulatory network of MsmR1 in P. polymyxa SC2. MsmR1 plays a regulatory role in the TCA cycle (sucA), amino acid metabolism, polymyxin synthesis (oppC3, sdr3), motility (sdr3), and biofilm and spore formation (pepF). Blue arrows: activation; bar-ended lines: repression; green arrows: metabolism process.
Frontiers in Microbiology 13 frontiersin.org (SC2-M1-ΔmsmR1/msmR5; Supplementary Figure S8D). In Pseudomonas aeruginosa PAO1161, PA3027 is an AraC-type transcriptional regulator with no effect on growth or carbon source utilization due to the functional redundancy of glpD homologs (Kotecka et al., 2021). Moreover, in Ustilaginoidea virens, due to the functional redundancy of other cutinase genes, cutinase activity was not affected after knockout of the cutinase G-box-binding protein UvCGBP1 . As a result of gene functional redundancy, growth status and carbon source utilization could not be considered the main markers of the msmR1 knockout mutant. AraC family transcription factors often form symmetric dimers and bind to palindromic DNA sequences (Kato et al., 2005;Sun et al., 2016). A 5-nt inverted repeat was identified by binding site analysis of the AraC family transcription factor SAV742, separated by 10 or 12 nt (GCCGA-n10/n12-TCGGC). Mutating the probe revealed that the 5-nt inverted repeat was essential for SAV742 binding (Sun et al., 2016). In E. coli, the TF RhaS could bind to the promoter region of rhaI 1 : ATCTTTCCCTGGTTGCC (Egan and Schleif, 1994). In Pseudomonas putida, Xyls could bind to two 15 bp direct repeats (TGCA-N6-GGNTA; Domínguez-Cuevas et al., 2008). In Vibrio cholerae, Toxt binds to a "toxbox" (yrTTTTwwTwAww). In the tcpA, ctxAB, and tcpI-2 promoters, two "toxboxes" are direct repeats, while in the acfD/acfA, tagA, and tcpI-1 promoters, two "toxboxes" are inverted repeats (Weber and Klose, 2011). In Streptomyces griseus, the AdpA regulon consensus binding sequence is 5'-TGGCSNGWWY-3′, serving as inverted repeats at both ends, separated by 13-14 bp loops (Yamazaki et al., 2004). In contrast, Streptococcus pyogenes possesses two identical MsmR-binding motifs, ACTGTGACCATAA and GAATAGCTATTC, without palindromic sequences or inverted repeats (Nakata et al., 2005). In a study on the binding sequences (emm, mrp, arp, enn, and scpA) of Mga in Streptococcus pyogenes, a 45-bp binding site was identified by DNase I protection experiments, containing three conserved nucleotide regions and 29 nucleotides, separated by several unrelated nucleotides (Almengor and McIver, 2004). This consensus sequence failed to identify the location of the Mga-binding sites in the mga-promoter (McIver et al., 1999), suggesting that Mga can bind with different sequences (Almengor and McIver, 2004). In a study on the DNA-binding site of MsmR1 in the SC2 strain, two motifs were preliminary identified, while no palindromic sequences or inverted repeats were found. At the same time, no similar motifs were identified with those of other homologue MsmR1 proteins.
Oligopeptide permease (Opp) is primarily involved in importing short-chain peptides, such as dipeptide and tripeptide, from the extracellular environment (Solomon et al., 2003;Ge et al., 2018). The Opp system play an important role in the uptake of limiting nutrients like nickel, proline and betaine, especially under starvation and stress conditions (Alloing et al., 2006;Ge et al., 2018). Additionally, the Opp system plays a crucial role in various processes controlled by signal peptides, including sporulation, virulence, and binding (Slamti and Lereclus, 2019). In fact, OppC is required for peptide uptake but not for cell viability (Solomon et al., 2003). In this study, construction of an oppC3-knockout mutant revealed that oppC3 also has an important role in amino acid metabolism. Simultaneously, the expression levels of genes related to nickel and sporulation were significantly downregulated following oppC3 knockout. Polymyxin synthesis requires various amino acids as precursors. Owing to the downregulation of amino acid metabolism-related genes, precursor substances for polymyxin biosynthesis were reduced, further inhibiting the synthesis of polymyxin. In addition, oppC3 may play an important role in oligosaccharide uptake by detecting the utilization of a sole carbon source. More specifically, oppC3 plays a crucial role in the uptake of lactose and sucrose, but not maltose and trehalose. Meanwhile, with raffinose, a trisaccharide composed of glucose, galactose, and fructose, strains with oppC3 knockout did not exhibit significant growth, suggesting that oppC3 is critical for raffinose uptake. Hence, given that the role of oppC in the uptake of oligosaccharides has not yet been reported, the findings of this study report an important novel role of oppC3 in the uptake of oligosaccharides.
During the interaction between P. polymyxa SC2 and pepper, the biosynthetic genes of polymyxin and fusaricidin were upregulated, which induced systemic resistance in pepper and enhanced its resistance to certain pathogens. Simultaneously, the upregulation of genes related to chemotaxis, motility, and biofilm formation is beneficial for the colonization of strains in the rhizosphere of plants . MsmR1, a global transcriptional regulator, regulates biological processes, including polymyxin synthesis, carbohydrate metabolism, biofilm formation, chemotaxis, and motility, which are beneficial for the interaction between P. polymyxa SC2 and pepper. The findings of this study provide a theoretical basis for polymyxin production and the application of strain P. polymyxa SC2.

Conclusion
In the study, we investigated the transcriptional regulatory mechanism of MsmR1 in P. polymyxa SC2. MsmR1, a AraC/ XylS family transcriptional regulator, is involved in polymyxin synthesis. In particular, MsmR1 was found to be highly associated with carbohydrate metabolism pathways. Indeed, MsmR1 affects polymyxin synthesis by directly regulating oppC3 and sdr3, affecting basal metabolism and precursors for polymyxin synthesis, while also directly regulating sucA and influencing the TCA cycle. In addition, MsmR1 could influence spore and biofilm formation and other biological processes. These results provide a theoretical basis for the application of P. polymyxa SC2 in the biological control of various pathogenic bacteria in pepper.

Data availability statement
The data presented in the study are deposited in the NCBI Sequence Read Archive (SRA) repository, accession number PRJNA885964.

Author contributions
DZ performed the laboratory work, analyzed the data, and wrote the manuscript. HL, YC, and ST analyzed the data. CW, BD, and YD provided insights on the manuscript. YD and BD supported the study. All authors contributed to the article and approved the submitted version.

Funding
This work was funded by the National Natural Science Foundation of China (31770115), Key Research and Development Program of Shandong Province (2021CXGC010804), and Tai-Shan Scholar Program from the Shandong Provincial Government.