Site-directed mutagenesis of transcriptional regulator sinR decreases biofilm formation and increases MK-7 synthesis in BS168
Previous studies found that biofilm formation was beneficial for the synthesis of MK-7 in B. subtilis [4, 33], we were interested in examining which gene play important role in affecting both biofilm formation and MK synthesis. A comprehensive mutant library of B. subtilis was generated, the biofilm formation ability and MK-7 synthesis of all mutants were tested (Fig. 1). Our initial screening indicated that deletion of sinR gene resulted in an obviously increased biofilm phenotype, the biofilm biomass increased by 2.8-fold compared to the wild type (Fig. 1A, B). Furthermore, the concentration of MK-7 increased by 2.6-fold after 6 days cultivation (Fig. 1C). Therefore, sinR gene was chosen for the further study.
Although sinR knocking out (KO-sinR) increased the concentration of MK-7, the yield was far less than the maximum yield reported [34]. The reason maybe that complete deleted sinR led to a large number of spore and wrinkle forming (Fig. 2A), which slow down the MK-7 biosynthesis. Therefore, it might be a better way to reduce spore forming to further promote MK-7 production.
Aromatic residues Glu97, Tyr101, Trp104 and Arg105 in SinR (Fig. 3B), were replaced with Lys34, Leu38, Lys41 and Ser42 in SinI to examine the different effects on B. subtilis. Fig.2 showed the different biofilm morphology, biofilm biomass and MK-7 production of seven different strains (BS168, E97K, Y101L, W104K, R105S, SinRquad and KO-SinR) after 6 days incubation. B. subtilis 168 wild type strain made the biofilm look smooth with a few wrinkles, the OD570nm value and the MK-7 production were 0.46±0.04 and 10±0.84 mg/L, respectively. In contrast, visible rough and dry biofilm could be found for KO-sinR strain, the OD570nm value and the MK-7 production were 1.28±0.05 and 26 ±0.78 mg/L. In addition, it was found that E97K, Y101L, W104K and R105S formed more wrinkles than that of BS168 but less wrinkles than that of KO-SinR. The OD570nm value of E97K, Y101L, W104K and R105S increased slightly compared to BS168, which lower than that of KO-SinR. However, the MK-7 production of four mutants especially E97K increased obviously, 61.02±0.84 mg/L, 2.35- fold that of KO-SinR. When the four sites were mutated simultaneously, we obtained SinRquad strain. Different from KO-SinR, SinRquad formed more wrinkly but smooth biofilm. Although the highest biofilm biomass was obtained by KO-SinR, the maximum MK-7 value (102.56±2.84 mg/L) was obtained by SinRquad, which was ten times of that by BS168, indicated that the increase in MK-7 production was not induced by biomass growth.
Illumina HiSeq mRNA sequencing and functional classification of unigenes
In order to explore how SinRquad affects biofilm formation and MK-7 production of B. subtilis, the Illumina RNAseq method was used to link the genes in related metabolic pathways when site-directed mutagenesis of transcriptional regulator sinR. Fig. 4A shows the probability density distribution of expression of all genes. According to the venn diagram, 4063 (LOS) and 3768 (HOS) genes were identified (Fig. 4B). Among them, 3740 genes were co-expressed. Meanwhile, 323 and 28 genes were solely expressed with SinRquad and BS168 strains, respectively, but most of them encoded some hypothetical proteins or the same proteins with other co-expressed genes. In order to further study the gene expression profile of 3740 coexisting genes, the differences of gene expression with different strains were compared. In this case, 1875 DEGs were found with significant differences (Fig. 4C). In these genes, 958 genes were up-regulated and 917 genes were down-regulated. According to the volcano-plots (Fig. 4D), the expression of differential genes can also be observed intuitively.
For understanding their functional information and biological function, these DEGs were annotated into the GO database and mapped to different pathways (q≤0.05). As shown in Fig. 5A, most of DEGs belong to the category of biological process, cellular component and molecular function. More concretely, “membrane”, “cell”, “carbohydrate metabolic process” “sporulation” and “hydrolase activity” were included in the above three categories. Further KEGG analysis showed the most significant 20 different enrichment pathways (Fig. 5B). Specifically, the 20 enrichment pathways included ABC transporters, flagellar assembly, bacterial chemotaxis, ubiquinone and other terpenoid-quinone biosynthesis, two-component system, oxidative phosphorylation, and glycolysis/gluconeogenesis and so on. The pathway of ABC transporters and phosphotransferase system were associated with the state of cell membrane [35]. These DEGs involved in significant enrich pathway may be related to site-directed mutagenesis of sinR and MK-7 synthesis.
Differential gene expression related to QS system and biofilm
B. subtilis uses ComX and CSP to regulate the competence and sporulation processes. The expression of QS system and biofilm related genes of BS168 and SinRquad are shown in Fig. 6A and Table S2. With respect to the CSP QS system, after a series of phosphorylation, the increase of CSP concentration in cellular promotes phosphor-Spo0A levels which can induce biofilm formation. Spo0A activates biofilm formation by increasing production of SinI, a small protein antagonist of the biofilm master repressor SinR [36]. SinR directly represses two matrix component operons, epsA-O and tapA-sipW-tasA, which were encoded by epsE, tapA and tasA [37-38]. Two additional small antagonists of SinR, SlrA and SlrR, also play a role in the regulation of biofilm formation by interacting with SinR [39].
It can be seen from Fig.6B, with regard to the CSP QS system of SinRquad, the expression level of spo0A, abrB and sinI was basically unchanged, while sinR decreased significantly compared to that of BS168, with over 88.63% reduction. The expression of slrR were up-regulated by 1.93-fold. Besides, the expression of tapA, tasA and epsE were up-regulated by 9.79-, 0.95-, 4.42-fold, respectively. The upregulation of tapA, tasA and epsE implied that sinR downregulation led to the promotion of extracellular matrix and thus increase biofilm biomass.
With regard to the ComX QS system, the expression of comX, comA and comS were unchanged, indicated that the expression of ComX QS system and ComX regulated biofilm genes were unaffected by site-directed mutagenesis of SinR.
Numerous previous studies have reported that SinR was a positive effector of motility and cell separation, which was not conducive to the formation of biofilm [40]. Therefore, the expression of swrA and hag gene was investigated. As we know, swrA encodes proteins for the hook-basal body of flagella, chemotaxis proteins and the flagellum-associated sigma factor σD [41]. While the hag gene, a late-flagellar gene, encoded the flagellar filament structural protein flagellin, which is responsible for swimming motility [42]. Fig. 6B showed that the relative transcription of swrA gene, has no changes when site-directed mutagenesis of SinR. However, the expression of hag gene was downregulated by 75.65%. Therefore, site-directed mutagenesis of E97K, Y101L, W104K and R105S of sinR hindered the expression of late-flagellar gene.
The atpA gene involved in ATP synthesis and appD gene encodes oligopeptide ABC transporter (ATP-binding protein) [43] were up-regulated by 3.40-, 4.07-fold, respectively. The upregulation in expression of atpA and appD promoted ATP synthesis and membrane transport. In view of ComX is a 10-amino acid peptide and CSP is an oligopeptide autoinducer [44], the up-regulation of appD implied the transport of CSP might be improved by site-directed mutagenesis of transcriptional regulator SinR.
In summary, SinRquad decreased the transcriptional level of sinR, promoted the synthesis of extracellular polymeric substances, inhibited the swimming motility of late-flagellar and thus promoted the biofilm biomass. In addition, intracellular ATP synthesis and membrane transport were improved.
Differential gene expression related to MK-7 biosynthesis
The biosynthesis pathway of MK-7 in B. subtilis was presented in Fig. 7, which could be categorized into four modules, namely the glycerol metabolism pathway (Module I), the methylerythritol phosphate (MEP) pathway (Module II), the shikimate (SA) pathway (Module III), and the MK-7 pathway (Module IV) [15].
Previous studies compared the effect of four carbon sources (i.e., soluble starch, sucrose, glucose and glycerol) on the MK-7 synthesis and the growth of B. subtilis natto, and found that the presence of glycerol in the media resulted in higher MK-7 production [10, 45]. Therefore, the first step in the biosynthesis pathway was the uptake of glycerol, which is catalyzed in an energy-independent manner by a membrane channel protein, the glycerol facilitator (GlpF) [46]. The main pathway of glycerol dissimilation involves a glycerol kinase (GlpK) that phosphorylates glycerol to glycerol-3-phosphate (Gly-3P), and a Gly-3P dehydrogenase (GlpD) that oxidizes Gly-3P to dihydroxyacetone phosphate (DHAP), an intermediate in glycolysis. In the glycerol metabolism pathway, the expression of glpF, glpk, and glpD were all up-regulated by 1.21-, 2.20- and 1.11-fold. In addition, methylglyoxal synthase (MgsA) and glycerol-1-phosphate dehydrogenase (AraM), which catalyzes the conversion of DHAP to methylglyoxal (MG) and the reduction of DHAP to glycerol-1-phosphate (G1P), were down-regulated by 97% and 46%, respectively. The result indicated that SinRquad could increase the consumption of substrate glycerol and weaken the other two branch pathways of DHAP, allowing DHAP to flow into glycolysis as much as possible.
Module II provided isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) for the biosynthesis of isoprenoids, which is the precursor of side chain of MK-7. However, the studies on the key enzymes in the MEP pathway in B. subtilis were rare. Fig.7 showed that most of the enzymes in Module II were up-regulated for SinRquad strain. As the first rate-limiting enzyme in MEP pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxs) which catalyze the reaction of pyruvate and glyceraldehyde 3- phosphate to form 1-deoxy-D-xylose-5-phosphate (DXP), was up-regulated by 0.91-fold. The result was consistent with previous study which found overexpressing dxs could increase the yield of isoprene by 40% over that of the wild-type B. subtilis [47]. In addition, Yqfp, which catalyzed the synthesis of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), was up-regulated by 1.95- fold. Especially, YacM and Yqid, which catalyzed the reaction of MEP and cytosine triphosphate (CTP) to form 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME), and polymerized IPP to Farnesyl pyrophosphate (FPP), were up-regulated by 2.26-fold and 2.06-fold, respectively.
Module III provided chorismate (CHA), the precursor of main chain of MK-7. Meanwhile, it is essential in cellular metabolism for providing precursors for the biosynthesis of three aromatic amino acids such as tyrosine (Tyr), phenylalanine (Phe) and tryptophan (Trp) [48]. The four enzymes of AroA, B, C, D can catalyze D-erythrose 4-phosphate and phosphoenolpyruvate to form shikimate. Then, the shikimate will be converted to chorismate by AroK, E, F. Fig.7 showed that aroA, B, C, D, K, E and F were all up-regulated by 1.47-, 0.75-, 1.64-, 3.77-, 8.30-, 1.08-, 0.14-fold. While AroH, TrpE, PabA and PabB which involved in the synthesis of three aromatic amino acids, the encoded genes were down-regulated by 41%, 29%, 21% and 62%, respectively. The result indicated that AroA, D, K played the most important role in CHA synthesis. It was consistent with previous study which showed that simultaneous overexpression of aroA and aroK in B. subtilis resulted in 2- fold increase in MK-7 compared with that in strain BS168 [34].
Module IV was the last pathway for MK-7 synthesis. Seven enzymes (MenF, D, H, C, E, B, I) for the synthesis of 1,4- Dihydroxy-2-naphthoyl-CoA (DHNA-CoA) in the MK-7 pathway were detected with different expression changes (Supplementary table 2). Five genes were up-regulated especially menD, whose expression level was high-regulated by 3.87-fold. Finally, menA and menG, which could combine the isoprene side chain and naphthoquinone ring and then catalyzed the methylation to form MK-7, were both up-regulated by 2.03- and 0.72- fold for SinRquad strain. It is consistent with our previous studies that overexpression of the menG/ubiE in Elizabethkingia meningoseptica also enhanced the MK content by 1.41-fold [25]. These results provided a new idea for further understanding the effect of SinRquad on the biosynthesis of MK-7 at the transcriptional level.
Differential gene expression related to cell membrane
MK-7 is a component of bacterial cell membranes and plays an important role in electron transfer. Schematic electron flow mediated by MK in B. subtilis is illustrated in Fig. 8A. Respiration occurs in the cell membrane of Gram-positive bacteria. Electron donors, with the help of an enzyme, transfer two electrons to MK and cytochrome c. MK and cytochrome c, with the help of another enzyme, in turn transfer these two electrons to an electron acceptor oxygen to form water. In this study, we found the expression level of ctaC-G operator and qcrA-C operator which encode the cytochromes [49] were upregulated in SinRquard compared with that in BS168 (Fig. 8B). These results showed that SinRquard could significantly promote the synthesis of MK-7 and cytochromes, deliver more electrons and promote respiration of B. subtilis. Previous study found that in the process of oxalate to formate, and then convert to CO2, there was electron formed [50]. Therefore, we tested the expression level of oxalate-decarboxylase (OxdC), which was used to catalyze the conversion of oxalate to formate and CO2 with the help of Mn and O2. However, the expression level of oxdC decreased obviously, with 45% reduction. In addition, the expression level of fdhD and yrhE, which encoded formate dehydratase and used to oxidized formate to CO2 and electrons, was down-regulated by 60% and 94%, indicating that this process donated less electrons for SinRquard. Therefore, we speculate that there may be other processes that can donate large amounts of electrons. It has been reported that NADH is the most important electron donor which donates the electrons under action of NADH dehydrogenase and transfers electrons to the electron transport system (ETM) and pumps protons out of the cell [51]. The process of producing electrons is shown in Fig. 8C. Consequently, the expression level of NADH dehydrogenases (i.e. ndH, bdhA, sdhA-C, idH and glpD) was tested. As shown in Fig. 8B, the expression level of most NADH dehydrogenases was upregulated. Especially, sdhA-C and glpD, increased 1.01-, 3.93-, 1.87-, 1.11- fold, respectively.