Embrane protein YuaF from B. subtilis is a member of the NfeD-like clan with a potential role in maintaining membrane integrity during conditions of cellular stress [18]. We constructed a POR 8 web pathway of the rapid response phase, which incorporated gene expression and protein-protein interaction data (Fig. 3). It appears that the genes involved belong to a 2-component system (TCS). The TCS that is activated in response to fusaricidin includes modules involved in 
cellular membrane dynamics, as well as phosphorylation and dephosphorylation events associated with detoxification. By the string 25033180 analysis of pairwise combinations of TCS and kinases, we found that YbdK-YbdJ, KinA-Spo0F, KinB-Spo0F, and KinC were closely correlated with the rapid-response phase (5 min post treatment; see Fig. 2). The analysis also revealed that YdjPQ and YuaFGI were close to KapB, and KapB anchored with KinB, indicating that fusaricidins acted on the 2 TCS, KinB-SpoF and YbdK-YbdJ. In the next step, YbdKYbdJ was activated by fusaricidin, and transcription of the genes downstream of this operon (yvlA, yvlB, ymcC, pksA, and yeaA) was significantly altered. The second TCS, KinB-SpoOF, was alsoinduced by the fusaricidin 
treatment, changing the transcription of some of the downstream genes (yvlA, yvlB, ymcC, and pksA) involved in cell membrane dynamics. Furthermore, we found alterations in the other genes, although the precise biological implication of this remains unclear. It is possible that some of these genes modulate bacterial aggregation and/or growth.Effect of Fusaricidins on Carbon and Nitrogen MetabolismFusaricidin exposure for 20 and 170 min led to the induction of 194 genes by at least 3-fold, and many of these genes are members of known antibiotic-responsive stimulons (Table S1). A NHS-Biotin site prominent feature was that a high proportion of these genes are regulated by SigA, which encodes the primary s factor of RNA polymerase and is essential for cell growth. This result is in agreement with other studies on antibiotic treatment in B. subtilis, as there are some genes known to be induced by different antibiotics, such as yvgN, ywiE, pyrB, and purC. Although some characterized enzymes were present, including phosphoribosylglycinamide synthetase, many of the other genes encode proteins with no known function. The dlt operon, including dltA, is involved in the D-alanine esterification of lipoteichoic and wall teichoic acids, which increases bacterialMechanisms of Fusaricidins to Bacillus subtilisTable 2. Gene groups with E ,0.05 at 5, 20, and 170 min.Time point of fermentation: 5 min Gene groups SigW CcpA-negative SigK SigE AbrB-negative AbrB-positive GerE-negative FNR-positive SigBt value15.46 7.12 6.50 4.99 4.94 4.80 4.64 4.47 28.E value,10E-15 1.50E-09 1.12E-07 8.38E-04 1.09E-03 2.20E-03 4.83E-03 1.08E-02 9.25E-Mean 0.978 0.376 0.390 0.263 0.374 0.592 0.847 0.871 20.ORFs 63 120 93 148 61 21 10 9Time point of fermentation: 20 min Gene groups CcpA-negative SigK AbrB-positive StrCon-negative SigE SigH StrCon-positive PyrR-negative CtsR-negative PurR-negative SigBt value7.09 6.34 5.86 5.48 5.22 24.37 24.62 24.86 25.93 28.76 225.E value 1.86E-09 3.19E-07 6.43E-06 5.91E-05 2.49E-04 1.71E-02 5.32E-03 1.63E-03 4.21E-06 ,1.0E-15 ,1.0E-Mean 0.737 0.750 1.380 0.672 0.537 20.587 20.414 21.547 21.579 1.314 22.ORFs 120 93 21 92 148 32 59 9 12 32Time point of fermentation: 170 min Gene groups SigD Fur-negative SigA Ccp-negative CodY-negative StrCon-negative SinR-negative lolR-negative.Embrane protein YuaF from B. subtilis is a member of the NfeD-like clan with a potential role in maintaining membrane integrity during conditions of cellular stress [18]. We constructed a pathway of the rapid response phase, which incorporated gene expression and protein-protein interaction data (Fig. 3). It appears that the genes involved belong to a 2-component system (TCS). The TCS that is activated in response to fusaricidin includes modules involved in cellular membrane dynamics, as well as phosphorylation and dephosphorylation events associated with detoxification. By the string 25033180 analysis of pairwise combinations of TCS and kinases, we found that YbdK-YbdJ, KinA-Spo0F, KinB-Spo0F, and KinC were closely correlated with the rapid-response phase (5 min post treatment; see Fig. 2). The analysis also revealed that YdjPQ and YuaFGI were close to KapB, and KapB anchored with KinB, indicating that fusaricidins acted on the 2 TCS, KinB-SpoF and YbdK-YbdJ. In the next step, YbdKYbdJ was activated by fusaricidin, and transcription of the genes downstream of this operon (yvlA, yvlB, ymcC, pksA, and yeaA) was significantly altered. The second TCS, KinB-SpoOF, was alsoinduced by the fusaricidin treatment, changing the transcription of some of the downstream genes (yvlA, yvlB, ymcC, and pksA) involved in cell membrane dynamics. Furthermore, we found alterations in the other genes, although the precise biological implication of this remains unclear. It is possible that some of these genes modulate bacterial aggregation and/or growth.Effect of Fusaricidins on Carbon and Nitrogen MetabolismFusaricidin exposure for 20 and 170 min led to the induction of 194 genes by at least 3-fold, and many of these genes are members of known antibiotic-responsive stimulons (Table S1). A prominent feature was that a high proportion of these genes are regulated by SigA, which encodes the primary s factor of RNA polymerase and is essential for cell growth. This result is in agreement with other studies on antibiotic treatment in B. subtilis, as there are some genes known to be induced by different antibiotics, such as yvgN, ywiE, pyrB, and purC. Although some characterized enzymes were present, including phosphoribosylglycinamide synthetase, many of the other genes encode proteins with no known function. The dlt operon, including dltA, is involved in the D-alanine esterification of lipoteichoic and wall teichoic acids, which increases bacterialMechanisms of Fusaricidins to Bacillus subtilisTable 2. Gene groups with E ,0.05 at 5, 20, and 170 min.Time point of fermentation: 5 min Gene groups SigW CcpA-negative SigK SigE AbrB-negative AbrB-positive GerE-negative FNR-positive SigBt value15.46 7.12 6.50 4.99 4.94 4.80 4.64 4.47 28.E value,10E-15 1.50E-09 1.12E-07 8.38E-04 1.09E-03 2.20E-03 4.83E-03 1.08E-02 9.25E-Mean 0.978 0.376 0.390 0.263 0.374 0.592 0.847 0.871 20.ORFs 63 120 93 148 61 21 10 9Time point of fermentation: 20 min Gene groups CcpA-negative SigK AbrB-positive StrCon-negative SigE SigH StrCon-positive PyrR-negative CtsR-negative PurR-negative SigBt value7.09 6.34 5.86 5.48 5.22 24.37 24.62 24.86 25.93 28.76 225.E value 1.86E-09 3.19E-07 6.43E-06 5.91E-05 2.49E-04 1.71E-02 5.32E-03 1.63E-03 4.21E-06 ,1.0E-15 ,1.0E-Mean 0.737 0.750 1.380 0.672 0.537 20.587 20.414 21.547 21.579 1.314 22.ORFs 120 93 21 92 148 32 59 9 12 32Time point of fermentation: 170 min Gene groups SigD Fur-negative SigA Ccp-negative CodY-negative StrCon-negative SinR-negative lolR-negative.