Transcriptional regulation of the mannan utilization genes in the alkaliphilic Bacillus sp. N16-5

Transcriptional regulation of the mannan utilization genes in the alkaliphilic Bacillus sp. N16-5 Abstract Bacillus sp. N16-5 is an alkaliphile with a great ability to utilize mannan. Its mannan utilization gene cluster has been identified in a previous study. The ManR protein encoded by the cluster was predicted to be a LacI family regulator, and the transcription level of the mannan utilization gene cluster was upregulated after the manR gene was deleted, indicating that ManR is the repressor of this cluster. The transcription of the related genes was downregulated when manH, encoding the extracellular substrate-binding domain of the manno-oligosaccharide transporter, was deleted. Furthermore, isothermal titration calorimetry revealed that mannotetraose and mannopentose are ligands of ManR. These results all corroborate the hypothesis that the mannan utilization gene cluster is repressed by the transcription regulator ManR, and that the repression is removed when it binds to manno-oligosaccharides, which are generated by mannan degradation and transported into the cell by a specific transporter. mannan, manno-oligosaccharide, transcription regulation, transcription repressor, LacI family INTRODUCTION Bacillus sp. N16-5 is an alkaliphile isolated from the sediments of a soda lake; it has a broad substrate spectrum and exhibits an excellent ability to use many polysaccharides, such as xylan, mannan and pectin (Ma et al.2004; Song, Xue and Ma 2013). A 16.6 kb gene cluster involved in mannan utilization has been identified in this strain in a previous study (Song, Xue and Ma 2013). This gene cluster encodes seven enzymes involved in mannan metabolism, as well as a manno-oligosaccharide ABC transporter, a transcription regulator, and a protein with unknown function. The transcription of the cluster is dramatically induced by locust bean gum but cannot be induced by mannose (Song, Xue and Ma 2013). The β-mannanase encoded by manA has been characterized as an extracellular endo-acting enzyme with high activity (Ma et al.2004). The proteins encoded by manH, manI and manJ are thought to compose an ABC transporter complex. ManH is the encoding gene of the extracellular substrate-binding domain that was found to bind manno-oligosaccharides and present them to the transmembrane channel, while manI and manJ are encoding genes of two transmembrane subunits (Song et al.2016). This is particularly notable since the transport of manno-oligosaccharides was found to be important in mannan utilization by Bacillus sp. N16-5 (Song et al.2016). Mannans are the major components of hemicellulose and widely distributed in plants (Chauhan et al.2012). They are linear or branched polysaccharides composed of sugars such as d-mannose, d-glucose and d-galactose (Moreira and Filho 2008). A large number of enzymes that participate in mannan degradation have been identified in bacteria and investigated extensively (Chauhan et al.2012; Malgas, van Dyk and Pletschke 2015), but few studies have been conducted on the regulation of mannan utilization in bacteria. A glucomannan utilization operon with an internal regulator gene (gmuR) was identified in Bacillus subtilis, and its expression was found to be induced by degraded glucomannan (Sadaie et al.2008). However, the details of this operon's regulatory mechanism were not completely elucidated. Polysaccharides are an important carbon source for many bacteria. Because they are difficult to transport into the cell, bacteria developed efficient mechanisms to sense and respond to their presence in the environment (Newcomb, Chen and Wu 2007). A two-component system was found to regulate the xylan utilization of Cellvibrio japonicus and Geobacillus stearothermophilus (Shulami et al.2007; Emami et al.2009). Negative regulation by repressors was also found in the polysaccharide utilization operons of some bacteria. For example G. stearothermophilus uses the arabinose-responsive AraR repressor to control the regulation of its arabinan utilization system (Shulami et al.2011). The identification of the mannan utilization gene cluster of Bacillus sp. N16-5 gives us a chance to study the regulatory mechanism of its mannan utilization ability. In this study, the oligosaccharide transport was found to contribute to the induction of mannan utilization genes, and the function of the regulatory protein ManR was also studied. Study of the regulation of mannan use by Bacillus sp. N16-5 will deepen our understanding of the polysaccharide utilization mechanisms of bacteria. MATERIALS AND METHODS Plasmids, strains and chemicals The plasmids pMD18-T (Takara, Dalian, China) and pET-28a (+) (Novagen, Madison, WI, USA) were used as vectors for cloning and expression, respectively. The Bacillus–Escherichia coli shuttle vector pNNB194 (Connelly, Young and Sloma 2004) was used for gene deletions in Bacillus sp. N16-5. Escherichia coli DH5α and BL21(DE3) were used as hosts for gene cloning and expression, respectively. Bacillus sp. N16-5 (CGMCC No.0369) was cultivated in Horikoshi-II medium (Horikoshi 1971) at 37°C and in aerobic conditions. Protoplast regeneration was carried on SA5 medium (Gao, Xue and Ma 2011), and NCM medium (Ito and Nagane 2001) was used for the construction of deletion mutants. The primers used in this study are listed in Table 1. Mannobiose, mannotriose, mannotetraose and mannopentose from Megazyme (Wicklow, Ireland) and locust bean gum from Sigma (Sigma Chemical Co., St Louis, MO, USA) were used. All the chemicals were commercially available and of analytical grade. Table 1. Oligonucleotides used in this study. Name  Sequence  Description  R1  5΄-GGGGAATTCAGATGTTTCAATAGGCGCT-3΄  Cloning of manR upstream region, deletion of manR  R2  5΄-CTTAGTTGCGGAACAATGTGACGAACATGCATGCGGTAAAGATGAA-3΄  Cloning of manR upstream region, deletion of manR  R3  5΄-TTCATCTTTACCGCATGCATGTTCGTCACATTGTTCCGCAACTAAG-3΄  Cloning of manR downstream region, deletion of manR  R4  5΄-CATGGATCCGCTCTAGCGGTAAGTGACAC-3΄  Cloning of manR downstream region, deletion of manR  manR-f  5΄-ATCGGATCCATGAGGGACATTGCTGACAAA-3΄  manR cloning and expression  manR-r  5΄-GCCGAGCTCCTTTACCGCATGCATGTTTTA-3΄  manR cloning and expression  Name  Sequence  Description  R1  5΄-GGGGAATTCAGATGTTTCAATAGGCGCT-3΄  Cloning of manR upstream region, deletion of manR  R2  5΄-CTTAGTTGCGGAACAATGTGACGAACATGCATGCGGTAAAGATGAA-3΄  Cloning of manR upstream region, deletion of manR  R3  5΄-TTCATCTTTACCGCATGCATGTTCGTCACATTGTTCCGCAACTAAG-3΄  Cloning of manR downstream region, deletion of manR  R4  5΄-CATGGATCCGCTCTAGCGGTAAGTGACAC-3΄  Cloning of manR downstream region, deletion of manR  manR-f  5΄-ATCGGATCCATGAGGGACATTGCTGACAAA-3΄  manR cloning and expression  manR-r  5΄-GCCGAGCTCCTTTACCGCATGCATGTTTTA-3΄  manR cloning and expression  View Large Construction of plasmids and deletion mutants The manH deletion mutant ΔmanH was constructed in our previous study (Song et al.2016). The ΔmanR (manR deletion) strain used in this study was constructed using the same methods as described in that publication. The primers used to amplify the upstream and downstream homologous arms are listed in Table 1. Isolation of RNA and real-time quantitative PCR The cells were collected for RNA extraction when cultured to the mid-log phase. The culture was centrifuged at 10 000 × g for 2 min and the pellets were rapidly frozen in liquid nitrogen and ground using a mortar and pestle. Then the total RNA was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). PrimeScript RT reagent kit and SYBR® Premix Ex Taq (Takara, Dalian, China) were used for the RNA reverse-transcription and the real-time quantitative PCR (RT-qPCR) reaction. Mastercycler® ep realplex thermal cycler (Eppendorf, Hamburg, Germany) was used for the RT-qPCR reaction. Three biological replicates were performed and the 16S rDNA was chosen as the housekeeping gene. Cloning, expression and purification of ManR The gene encoding ManR was amplified using the primers manR-f and manR-r, and the genomic DNA of Bacillus sp. N16-5 was used as a template. Purified PCR product was digested by SacI and BamHI to be inserted into pET-28a(+). The resulting recombinant plasmid was transferred into E. coli DH5α for cloning, and after confirmation by sequencing, introduced into E. coli BL21(DE3) for expression. The expression strain was cultivated in Luria-Bertani (LB) medium containing 50 μg ml−1 kanamycin at 37°C. When the OD600 of the culture reached 1.0, 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added, and then the cells were induced overnight under 30°C. The cells were harvested by centrifugation at 8000 × g for 5 min, re-suspended in 20 mM Tris-HCl (pH 8.0), and disrupted by ultra-sonication. After centrifugation at 12 000 × g for 15 min and passing through a 0.22-μm filter, the sample was applied to a Ni-agarose resin column (Cwbiotech, Beijing, China) for purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the purified ManR, and a BCA protein assay kit (Solarbio, Beijing, China) with bovine serum albumin as protein standard was used to measure the concentration of the purified protein. Microcalorimetric titration assay An isothermal titration calorimeter (iTC200, MicroCal, Northampton, MA, USA) was used to measure the binding between ManR and the putative ligands. The protein and the ligand were both dissolved in 20 mM Tris-HCl buffer (pH 8.0) with 200 mM NaCl in it, and the titration temperature was set to 30°C. The concentrations of the protein and ligand were 20 and 200 μM, respectively. Aliquots (5 μl) of the ligand solution were injected from a 100-μl rotating stirrer-syringe. The titration curve was fitted by analysis software provided together with the MicroCal iTC200, and the values of the enthalpy (ΔHa [J mol−1]) and binding constant (Ka [M−1]) were generated by the same software. Determination of cell growth and concentration of reducing sugars The strains were firstly cultivated under aerobic conditions at 37°C in tubes with 5 ml Horikoshi-II medium for 12 h (Horikoshi 1971). Then 1 ml of the culture was inoculated into 250-ml shake flasks with 50 ml fermentation medium. The fermentation medium contained 5 g peptone (Oxoid), 1 g K2HPO4·3H2O, 0.2 g Mg2SO4·7H2O, 0.1 g yeast extract (Oxoid) and 5 g carbon source per liter, and 10% (v/v) of a 10% Na2CO3 solution was aseptically added into the medium after separate sterilization. The growth curve was charted according to the optical density at 600 nm (OD600) of the culture, and the OD600 was measured every 3 h on a spectrophotometer (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA) with a microplate containing 200 μl culture in each well. The dinitrosalicylic acid (DNS) method (Miller 1959) was used to measure the concentration of reducing sugars in the cultures. RESULTS AND DISCUSSION Sequence analysis of ManR The structure of the mannan utilization gene cluster is shown in Fig. 1A. The ManR protein (334 amino acids [aa]) encoded by the gene cluster was predicted to be a transcription regulator. In fact, it is a homologue of the lactose-inducible lac operon transcriptional repressor (LacI) of E. coli (360 aa) with 24% identities and 46% positives. It has the typical domain structure of LacI family proteins. The C-terminal end of ManR contains a sugar-binding domain (aa 58–324) and the N-terminal end contains a helix–turn–helix motif which usually binds to DNA (aa 2–52) (Fig. 1B). When the C-terminal domain of the LacI family proteins binds its ligand, the protein undergoes a structural change and separates from the DNA-binding site to which it is normally bound (Weickert and Adhya 1992). The sequence analysis of ManR suggested that this protein probably also works as a repressor of the mannan utilization gene cluster. Figure 1. View largeDownload slide Schematic drawing of the mannan utilization gene cluster (A) and the domain structure of ManR of Bacillus sp. N16-5 (B). ManA encodes endo-1,4-β-mannosidase; manH, manI and manJ encode components of a manno-oligosaccharide ABC transporter; manB, manC, manD, manE, manF and manG encode glycosidases; manK encodes a protein of unknown function. Figure 1. View largeDownload slide Schematic drawing of the mannan utilization gene cluster (A) and the domain structure of ManR of Bacillus sp. N16-5 (B). ManA encodes endo-1,4-β-mannosidase; manH, manI and manJ encode components of a manno-oligosaccharide ABC transporter; manB, manC, manD, manE, manF and manG encode glycosidases; manK encodes a protein of unknown function. Transcriptional levels of mannan utilization genes in a ΔmanR knockout mutant The transcriptional levels of manA, B, C, J and H in mid-log-phase cells were compared between the ΔmanR and the WT (wild type) strain using RT-qPCR. Glucose, xylose and locust bean gum were each used as carbon sources. When manR was deleted, the transcription of all the tested genes was upregulated regardless of whether the strain was cultivated on glucose (Fig. 2A) or xylose (Fig. 2B), proving that ManR acts as a repressor. By contrast, when locust bean gum was used as a carbon source, the deletion of manR did not lead to a significant change of gene transcription (Fig. 2C). This is probably because, in the WT strain, ManR can bind to the ligand when mannan is used as the carbon source, and the repression of the gene cluster is relieved, leading to transcriptional levels similar to those in the manR deletion mutant. Figure 2. View largeDownload slide The relative transcription levels of genes in the mannan utilization gene cluster in ΔmanR and WT when the strains were cultivated on glucose (A), xylose (B) and locust bean gum (C). Figure 2. View largeDownload slide The relative transcription levels of genes in the mannan utilization gene cluster in ΔmanR and WT when the strains were cultivated on glucose (A), xylose (B) and locust bean gum (C). Transcriptional levels of mannan utilization genes in the ΔmanH knockout mutant In our previous study, ManH was identified as the extracellular substrate-binding domain of a manno-oligosaccharide ABC transporter, and its absence was found to lead to decreased growth on mannan (Song et al.2016). Here, the transcriptional levels of manA, B, C, J and R between the ΔmanH and WT strains grown on locust bean gum were compared. It was found that the deletion of manH dramatically depressed the transcriptional levels of all the investigated genes (Fig. 3), suggesting that manno-oligosaccharide transport is involved in the transcription regulation of the mannan-utilization gene cluster. There have been a few reports about ABC transporters related to hemicellulose utilization in bacteria. XynEF from Geobacillus stearothermophilus and BxlEFG from Streptomyces thermoviolaceus were reported as transporters for xylo-oligosaccharides (Tsujibo et al.2004; Shulami et al.2007). AraNPQ from Bacillus subtilis and AraEFG from Geobacillus stearothermophilus were reported as transporters for arabino-oligosaccharides (Ferreira and Sa-Nogueira 2010; Shulami et al.2011). CpMnBP1 found in Caldanaerobius polysaccharolyticus was identified as a manno-oligosaccharide transporter (Chekan et al.2014). However, it is the first time it has been found that the deletion of the oligosaccharide ABC transporter suppresses the transcription of genes related to hemicellulose utilization. Figure 3. View largeDownload slide The relative transcription levels of manA, B, C, J and R in ΔmanH and WT at mid-logarithmic growth phase when the strains were grown on locust bean gum. Figure 3. View largeDownload slide The relative transcription levels of manA, B, C, J and R in ΔmanH and WT at mid-logarithmic growth phase when the strains were grown on locust bean gum. Substrate specificity of ManR In order to identify the ligands of ManR, its encoding gene manR was cloned from Bacillus sp. N16-5, heterologously expressed, and purified. The binding ability of recombinant ManR (rManR) to mannose and manno-oligosaccharides was measured using isothermal titration calorimetry. It was found that ManR had an affinity to mannotetraose and mannopentose under the experimental conditions (Fig. 4), but no obvious binding curve was observed for mannose, mannobiose, mannotriose or galactosyl mannotriose (data not shown). The dissociation constant (Kd) for the binding of mannotetraose and mannopentose to rManR is 13.8 ± 0.7 μM and 15.6 ± 0.5 μM, respectively. It indicated that mannotetraose and mannopentose act as the ligands of ManR, and that is the reason why the deletion of ManH led to the downregulation of the mannan utilization gene cluster. Figure 4. View largeDownload slide Isothermal calorimetric titration curves of ManR with mannotetraose (A) and mannopentose (B). Figure 4. View largeDownload slide Isothermal calorimetric titration curves of ManR with mannotetraose (A) and mannopentose (B). Production of reducing sugars during mannan utilization by Bacillus sp. N16-5 strains ΔmanR, ΔmanH and WT ΔmanR and WT had similar growth curves when the strains were inoculated into the medium containing locust bean gum (Fig. S1 in the online supplementary material), but the deletion of manR affected the production of reducing sugars. As shown in Fig. 5, for ΔmanR, the concentration of reducing sugars in the culture began to rapidly increase from 3 h and reached its peak at 9 h. By contrast, both the speed of concentration increase and the highest concentration of reducing sugars were lower in the WT culture. This was probably because the expression of the mannan utilization genes in the WT can be induced only after the repression by ManR is relieved. As this induction process takes some time, so the production of reducing sugars by the WT strain is slower, resulting in a more even balance between production and consumption. After 15 h, the concentration of reducing sugars was equal, because most of the generated reducing sugars were finally transported and metabolized by both ΔmanR and WT. Figure 5. View largeDownload slide Concentration of the reducing sugars in the culture when ΔmanR, ΔmanH and WT were grown on locust bean gum. Figure 5. View largeDownload slide Concentration of the reducing sugars in the culture when ΔmanR, ΔmanH and WT were grown on locust bean gum. Conversely, the increase of reducing sugars was dramatically delayed in the ΔmanH culture (Fig. 5). Since the expression of mannan utilization genes was suppressed by ManH deletion, the degradation of mannan was expected to be slower than in the WT. Nevertheless, the mannan was slowly hydrolyzed, most likely by enzymes expressed at a background level, so that the concentration of reducing sugars also reached a relatively high level after 18 h. However, since ManH was absent, the oligosaccharides generated in the culture of ΔmanH could not be transported efficiently, so that the concentration of reducing sugars remained high until the end of the fermentation. Regulatory mechanisms of mannan use of Bacillus sp. N16-5 Based on the above results, we propose the following hypothesis regarding the regulation of mannan use of the strain. When mannans such as locust bean gum are present outside the cells, a small amount is degraded into manno-oligosaccharides by ManA, whose gene is expressed at background level. The generated oligosaccharides are transported into the cell by the manno-oligosaccharide transporter ManHIJ. Oligosaccharides of suitable length such as mannotetraose and mannopentose bind to ManR, which releases its operator sequence, inducing the rapid transcription of the mannan utilization gene cluster. Finally, the enzymes and transporters related to mannan utilization are expressed in large amounts, which enables the strain to utilize the mannans efficiently. The expression of the polysaccharide utilization genes of bacteria is usually induced only when the substrate is present. As polymers, hemicelluloses cannot be transported into bacteria directly, so the bacteria should exhibit the ability to sense them. Cellvibrio japonicus was found to develop a two-component system to sense the decorated xylans and arabinoxylo-oligosaccharides directly and induce the expression of the related genes (Emami et al.2009). The polysaccharide degradative enzymes can also be regulated through soluble sugars generated by their own action (Newcomb, Chen and Wu 2007). The celC operon of Clostridium thermocellum encoding CelC and LicA, which act on β-1,3-glucan, can be induced by laminaribiose, which is the partial hydrolysis product of β-1,3-glucan (Newcomb, Chen and Wu 2007). Similarly, the expression of the mannan utilization genes can be induced even though mannans cannot be directly transported into the cells of Bacillus sp. N16-5. The regulation is achieved through the products of its partial degradation, manno-oligosaccharides. This regulation strategy enables the cells to sense and use the substrate in the environment accurately and rapidly. We analyzed additional gene clusters containing ManR homologues and found that they have a number of similar structural characters (Fig. S2 in the online supplementary material). All the investigated gene clusters encode CUT1 family transporters, which specifically transport di- or oligosaccharides (Schneider 2001). All the gene clusters also contain genes related to mannan utilization or polysaccharide degradation. This observation suggests that the regulatory mechanism of the mannan utilization cluster found in Bacillus sp. N16-5, which is based on oligosaccharide transport, is likely to exist in many other bacteria. CONCLUSIONS In this study, oligosaccharide transport was found to contribute to the induction of mannan utilization genes in Bacillus sp. N16-5. A novel regulatory protein, ManR, which represses the transcription of the mannan utilization gene cluster and uses manno-oligosaccharides as ligands was identified. Study of the regulation of mannan use by Bacillus sp. N16-5 will deepen our understanding of the polysaccharide utilization mechanisms of bacteria. SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. FUNDING This work was supported by the National Key R&D Program of China (2017YFD0400304) and the National Natural Science Foundation of China (No. 31400031). Conflict of interest. None declared. REFERENCES Chauhan PS, Puri N, Sharma P et al.   Mannanases: microbial sources, production, properties and potential biotechnological applications. Appl Microbiol Biotechnol  2012; 93: 1817– 30. Google Scholar CrossRef Search ADS PubMed  Chekan JR, Kwon IH, Agarwal V et al.   Structural and biochemical basis for mannan utilization by Caldanaerobius polysaccharolyticus strain ATCC BAA-17. J Biol Chem  2014; 289: 34965– 77. Google Scholar CrossRef Search ADS PubMed  Connelly MB, Young GM, Sloma A. Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis. J Bacteriol  2004; 186: 4159– 67. Google Scholar CrossRef Search ADS PubMed  Emami K, Topakas E, Nagy T et al.   Regulation of the xylan-degrading apparatus of Cellvibrio japonicus by a novel two-component system. J Biol Chem  2009; 284: 1086– 96. Google Scholar CrossRef Search ADS PubMed  Ferreira MJ, Sa-Nogueira I. 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N16-5. PLoS One  2013; 8: e54090. Google Scholar CrossRef Search ADS PubMed  Tsujibo H, Kosaka M, Ikenishi S et al.   Molecular characterization of a high-affinity xylobiose transporter of Streptomyces thermoviolaceus OPC-520 and its transcriptional regulation. J Bacteriol  2004; 186: 1029– 37. Google Scholar CrossRef Search ADS PubMed  Weickert MJ, Adhya S. A family of bacterial regulators homologous to Gal and Lac repressors. J Biol Chem  1992; 267: 15869– 74. Google Scholar PubMed  © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Microbiology Letters Oxford University Press

Transcriptional regulation of the mannan utilization genes in the alkaliphilic Bacillus sp. N16-5

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Abstract

Abstract Bacillus sp. N16-5 is an alkaliphile with a great ability to utilize mannan. Its mannan utilization gene cluster has been identified in a previous study. The ManR protein encoded by the cluster was predicted to be a LacI family regulator, and the transcription level of the mannan utilization gene cluster was upregulated after the manR gene was deleted, indicating that ManR is the repressor of this cluster. The transcription of the related genes was downregulated when manH, encoding the extracellular substrate-binding domain of the manno-oligosaccharide transporter, was deleted. Furthermore, isothermal titration calorimetry revealed that mannotetraose and mannopentose are ligands of ManR. These results all corroborate the hypothesis that the mannan utilization gene cluster is repressed by the transcription regulator ManR, and that the repression is removed when it binds to manno-oligosaccharides, which are generated by mannan degradation and transported into the cell by a specific transporter. mannan, manno-oligosaccharide, transcription regulation, transcription repressor, LacI family INTRODUCTION Bacillus sp. N16-5 is an alkaliphile isolated from the sediments of a soda lake; it has a broad substrate spectrum and exhibits an excellent ability to use many polysaccharides, such as xylan, mannan and pectin (Ma et al.2004; Song, Xue and Ma 2013). A 16.6 kb gene cluster involved in mannan utilization has been identified in this strain in a previous study (Song, Xue and Ma 2013). This gene cluster encodes seven enzymes involved in mannan metabolism, as well as a manno-oligosaccharide ABC transporter, a transcription regulator, and a protein with unknown function. The transcription of the cluster is dramatically induced by locust bean gum but cannot be induced by mannose (Song, Xue and Ma 2013). The β-mannanase encoded by manA has been characterized as an extracellular endo-acting enzyme with high activity (Ma et al.2004). The proteins encoded by manH, manI and manJ are thought to compose an ABC transporter complex. ManH is the encoding gene of the extracellular substrate-binding domain that was found to bind manno-oligosaccharides and present them to the transmembrane channel, while manI and manJ are encoding genes of two transmembrane subunits (Song et al.2016). This is particularly notable since the transport of manno-oligosaccharides was found to be important in mannan utilization by Bacillus sp. N16-5 (Song et al.2016). Mannans are the major components of hemicellulose and widely distributed in plants (Chauhan et al.2012). They are linear or branched polysaccharides composed of sugars such as d-mannose, d-glucose and d-galactose (Moreira and Filho 2008). A large number of enzymes that participate in mannan degradation have been identified in bacteria and investigated extensively (Chauhan et al.2012; Malgas, van Dyk and Pletschke 2015), but few studies have been conducted on the regulation of mannan utilization in bacteria. A glucomannan utilization operon with an internal regulator gene (gmuR) was identified in Bacillus subtilis, and its expression was found to be induced by degraded glucomannan (Sadaie et al.2008). However, the details of this operon's regulatory mechanism were not completely elucidated. Polysaccharides are an important carbon source for many bacteria. Because they are difficult to transport into the cell, bacteria developed efficient mechanisms to sense and respond to their presence in the environment (Newcomb, Chen and Wu 2007). A two-component system was found to regulate the xylan utilization of Cellvibrio japonicus and Geobacillus stearothermophilus (Shulami et al.2007; Emami et al.2009). Negative regulation by repressors was also found in the polysaccharide utilization operons of some bacteria. For example G. stearothermophilus uses the arabinose-responsive AraR repressor to control the regulation of its arabinan utilization system (Shulami et al.2011). The identification of the mannan utilization gene cluster of Bacillus sp. N16-5 gives us a chance to study the regulatory mechanism of its mannan utilization ability. In this study, the oligosaccharide transport was found to contribute to the induction of mannan utilization genes, and the function of the regulatory protein ManR was also studied. Study of the regulation of mannan use by Bacillus sp. N16-5 will deepen our understanding of the polysaccharide utilization mechanisms of bacteria. MATERIALS AND METHODS Plasmids, strains and chemicals The plasmids pMD18-T (Takara, Dalian, China) and pET-28a (+) (Novagen, Madison, WI, USA) were used as vectors for cloning and expression, respectively. The Bacillus–Escherichia coli shuttle vector pNNB194 (Connelly, Young and Sloma 2004) was used for gene deletions in Bacillus sp. N16-5. Escherichia coli DH5α and BL21(DE3) were used as hosts for gene cloning and expression, respectively. Bacillus sp. N16-5 (CGMCC No.0369) was cultivated in Horikoshi-II medium (Horikoshi 1971) at 37°C and in aerobic conditions. Protoplast regeneration was carried on SA5 medium (Gao, Xue and Ma 2011), and NCM medium (Ito and Nagane 2001) was used for the construction of deletion mutants. The primers used in this study are listed in Table 1. Mannobiose, mannotriose, mannotetraose and mannopentose from Megazyme (Wicklow, Ireland) and locust bean gum from Sigma (Sigma Chemical Co., St Louis, MO, USA) were used. All the chemicals were commercially available and of analytical grade. Table 1. Oligonucleotides used in this study. Name  Sequence  Description  R1  5΄-GGGGAATTCAGATGTTTCAATAGGCGCT-3΄  Cloning of manR upstream region, deletion of manR  R2  5΄-CTTAGTTGCGGAACAATGTGACGAACATGCATGCGGTAAAGATGAA-3΄  Cloning of manR upstream region, deletion of manR  R3  5΄-TTCATCTTTACCGCATGCATGTTCGTCACATTGTTCCGCAACTAAG-3΄  Cloning of manR downstream region, deletion of manR  R4  5΄-CATGGATCCGCTCTAGCGGTAAGTGACAC-3΄  Cloning of manR downstream region, deletion of manR  manR-f  5΄-ATCGGATCCATGAGGGACATTGCTGACAAA-3΄  manR cloning and expression  manR-r  5΄-GCCGAGCTCCTTTACCGCATGCATGTTTTA-3΄  manR cloning and expression  Name  Sequence  Description  R1  5΄-GGGGAATTCAGATGTTTCAATAGGCGCT-3΄  Cloning of manR upstream region, deletion of manR  R2  5΄-CTTAGTTGCGGAACAATGTGACGAACATGCATGCGGTAAAGATGAA-3΄  Cloning of manR upstream region, deletion of manR  R3  5΄-TTCATCTTTACCGCATGCATGTTCGTCACATTGTTCCGCAACTAAG-3΄  Cloning of manR downstream region, deletion of manR  R4  5΄-CATGGATCCGCTCTAGCGGTAAGTGACAC-3΄  Cloning of manR downstream region, deletion of manR  manR-f  5΄-ATCGGATCCATGAGGGACATTGCTGACAAA-3΄  manR cloning and expression  manR-r  5΄-GCCGAGCTCCTTTACCGCATGCATGTTTTA-3΄  manR cloning and expression  View Large Construction of plasmids and deletion mutants The manH deletion mutant ΔmanH was constructed in our previous study (Song et al.2016). The ΔmanR (manR deletion) strain used in this study was constructed using the same methods as described in that publication. The primers used to amplify the upstream and downstream homologous arms are listed in Table 1. Isolation of RNA and real-time quantitative PCR The cells were collected for RNA extraction when cultured to the mid-log phase. The culture was centrifuged at 10 000 × g for 2 min and the pellets were rapidly frozen in liquid nitrogen and ground using a mortar and pestle. Then the total RNA was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). PrimeScript RT reagent kit and SYBR® Premix Ex Taq (Takara, Dalian, China) were used for the RNA reverse-transcription and the real-time quantitative PCR (RT-qPCR) reaction. Mastercycler® ep realplex thermal cycler (Eppendorf, Hamburg, Germany) was used for the RT-qPCR reaction. Three biological replicates were performed and the 16S rDNA was chosen as the housekeeping gene. Cloning, expression and purification of ManR The gene encoding ManR was amplified using the primers manR-f and manR-r, and the genomic DNA of Bacillus sp. N16-5 was used as a template. Purified PCR product was digested by SacI and BamHI to be inserted into pET-28a(+). The resulting recombinant plasmid was transferred into E. coli DH5α for cloning, and after confirmation by sequencing, introduced into E. coli BL21(DE3) for expression. The expression strain was cultivated in Luria-Bertani (LB) medium containing 50 μg ml−1 kanamycin at 37°C. When the OD600 of the culture reached 1.0, 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added, and then the cells were induced overnight under 30°C. The cells were harvested by centrifugation at 8000 × g for 5 min, re-suspended in 20 mM Tris-HCl (pH 8.0), and disrupted by ultra-sonication. After centrifugation at 12 000 × g for 15 min and passing through a 0.22-μm filter, the sample was applied to a Ni-agarose resin column (Cwbiotech, Beijing, China) for purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the purified ManR, and a BCA protein assay kit (Solarbio, Beijing, China) with bovine serum albumin as protein standard was used to measure the concentration of the purified protein. Microcalorimetric titration assay An isothermal titration calorimeter (iTC200, MicroCal, Northampton, MA, USA) was used to measure the binding between ManR and the putative ligands. The protein and the ligand were both dissolved in 20 mM Tris-HCl buffer (pH 8.0) with 200 mM NaCl in it, and the titration temperature was set to 30°C. The concentrations of the protein and ligand were 20 and 200 μM, respectively. Aliquots (5 μl) of the ligand solution were injected from a 100-μl rotating stirrer-syringe. The titration curve was fitted by analysis software provided together with the MicroCal iTC200, and the values of the enthalpy (ΔHa [J mol−1]) and binding constant (Ka [M−1]) were generated by the same software. Determination of cell growth and concentration of reducing sugars The strains were firstly cultivated under aerobic conditions at 37°C in tubes with 5 ml Horikoshi-II medium for 12 h (Horikoshi 1971). Then 1 ml of the culture was inoculated into 250-ml shake flasks with 50 ml fermentation medium. The fermentation medium contained 5 g peptone (Oxoid), 1 g K2HPO4·3H2O, 0.2 g Mg2SO4·7H2O, 0.1 g yeast extract (Oxoid) and 5 g carbon source per liter, and 10% (v/v) of a 10% Na2CO3 solution was aseptically added into the medium after separate sterilization. The growth curve was charted according to the optical density at 600 nm (OD600) of the culture, and the OD600 was measured every 3 h on a spectrophotometer (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA) with a microplate containing 200 μl culture in each well. The dinitrosalicylic acid (DNS) method (Miller 1959) was used to measure the concentration of reducing sugars in the cultures. RESULTS AND DISCUSSION Sequence analysis of ManR The structure of the mannan utilization gene cluster is shown in Fig. 1A. The ManR protein (334 amino acids [aa]) encoded by the gene cluster was predicted to be a transcription regulator. In fact, it is a homologue of the lactose-inducible lac operon transcriptional repressor (LacI) of E. coli (360 aa) with 24% identities and 46% positives. It has the typical domain structure of LacI family proteins. The C-terminal end of ManR contains a sugar-binding domain (aa 58–324) and the N-terminal end contains a helix–turn–helix motif which usually binds to DNA (aa 2–52) (Fig. 1B). When the C-terminal domain of the LacI family proteins binds its ligand, the protein undergoes a structural change and separates from the DNA-binding site to which it is normally bound (Weickert and Adhya 1992). The sequence analysis of ManR suggested that this protein probably also works as a repressor of the mannan utilization gene cluster. Figure 1. View largeDownload slide Schematic drawing of the mannan utilization gene cluster (A) and the domain structure of ManR of Bacillus sp. N16-5 (B). ManA encodes endo-1,4-β-mannosidase; manH, manI and manJ encode components of a manno-oligosaccharide ABC transporter; manB, manC, manD, manE, manF and manG encode glycosidases; manK encodes a protein of unknown function. Figure 1. View largeDownload slide Schematic drawing of the mannan utilization gene cluster (A) and the domain structure of ManR of Bacillus sp. N16-5 (B). ManA encodes endo-1,4-β-mannosidase; manH, manI and manJ encode components of a manno-oligosaccharide ABC transporter; manB, manC, manD, manE, manF and manG encode glycosidases; manK encodes a protein of unknown function. Transcriptional levels of mannan utilization genes in a ΔmanR knockout mutant The transcriptional levels of manA, B, C, J and H in mid-log-phase cells were compared between the ΔmanR and the WT (wild type) strain using RT-qPCR. Glucose, xylose and locust bean gum were each used as carbon sources. When manR was deleted, the transcription of all the tested genes was upregulated regardless of whether the strain was cultivated on glucose (Fig. 2A) or xylose (Fig. 2B), proving that ManR acts as a repressor. By contrast, when locust bean gum was used as a carbon source, the deletion of manR did not lead to a significant change of gene transcription (Fig. 2C). This is probably because, in the WT strain, ManR can bind to the ligand when mannan is used as the carbon source, and the repression of the gene cluster is relieved, leading to transcriptional levels similar to those in the manR deletion mutant. Figure 2. View largeDownload slide The relative transcription levels of genes in the mannan utilization gene cluster in ΔmanR and WT when the strains were cultivated on glucose (A), xylose (B) and locust bean gum (C). Figure 2. View largeDownload slide The relative transcription levels of genes in the mannan utilization gene cluster in ΔmanR and WT when the strains were cultivated on glucose (A), xylose (B) and locust bean gum (C). Transcriptional levels of mannan utilization genes in the ΔmanH knockout mutant In our previous study, ManH was identified as the extracellular substrate-binding domain of a manno-oligosaccharide ABC transporter, and its absence was found to lead to decreased growth on mannan (Song et al.2016). Here, the transcriptional levels of manA, B, C, J and R between the ΔmanH and WT strains grown on locust bean gum were compared. It was found that the deletion of manH dramatically depressed the transcriptional levels of all the investigated genes (Fig. 3), suggesting that manno-oligosaccharide transport is involved in the transcription regulation of the mannan-utilization gene cluster. There have been a few reports about ABC transporters related to hemicellulose utilization in bacteria. XynEF from Geobacillus stearothermophilus and BxlEFG from Streptomyces thermoviolaceus were reported as transporters for xylo-oligosaccharides (Tsujibo et al.2004; Shulami et al.2007). AraNPQ from Bacillus subtilis and AraEFG from Geobacillus stearothermophilus were reported as transporters for arabino-oligosaccharides (Ferreira and Sa-Nogueira 2010; Shulami et al.2011). CpMnBP1 found in Caldanaerobius polysaccharolyticus was identified as a manno-oligosaccharide transporter (Chekan et al.2014). However, it is the first time it has been found that the deletion of the oligosaccharide ABC transporter suppresses the transcription of genes related to hemicellulose utilization. Figure 3. View largeDownload slide The relative transcription levels of manA, B, C, J and R in ΔmanH and WT at mid-logarithmic growth phase when the strains were grown on locust bean gum. Figure 3. View largeDownload slide The relative transcription levels of manA, B, C, J and R in ΔmanH and WT at mid-logarithmic growth phase when the strains were grown on locust bean gum. Substrate specificity of ManR In order to identify the ligands of ManR, its encoding gene manR was cloned from Bacillus sp. N16-5, heterologously expressed, and purified. The binding ability of recombinant ManR (rManR) to mannose and manno-oligosaccharides was measured using isothermal titration calorimetry. It was found that ManR had an affinity to mannotetraose and mannopentose under the experimental conditions (Fig. 4), but no obvious binding curve was observed for mannose, mannobiose, mannotriose or galactosyl mannotriose (data not shown). The dissociation constant (Kd) for the binding of mannotetraose and mannopentose to rManR is 13.8 ± 0.7 μM and 15.6 ± 0.5 μM, respectively. It indicated that mannotetraose and mannopentose act as the ligands of ManR, and that is the reason why the deletion of ManH led to the downregulation of the mannan utilization gene cluster. Figure 4. View largeDownload slide Isothermal calorimetric titration curves of ManR with mannotetraose (A) and mannopentose (B). Figure 4. View largeDownload slide Isothermal calorimetric titration curves of ManR with mannotetraose (A) and mannopentose (B). Production of reducing sugars during mannan utilization by Bacillus sp. N16-5 strains ΔmanR, ΔmanH and WT ΔmanR and WT had similar growth curves when the strains were inoculated into the medium containing locust bean gum (Fig. S1 in the online supplementary material), but the deletion of manR affected the production of reducing sugars. As shown in Fig. 5, for ΔmanR, the concentration of reducing sugars in the culture began to rapidly increase from 3 h and reached its peak at 9 h. By contrast, both the speed of concentration increase and the highest concentration of reducing sugars were lower in the WT culture. This was probably because the expression of the mannan utilization genes in the WT can be induced only after the repression by ManR is relieved. As this induction process takes some time, so the production of reducing sugars by the WT strain is slower, resulting in a more even balance between production and consumption. After 15 h, the concentration of reducing sugars was equal, because most of the generated reducing sugars were finally transported and metabolized by both ΔmanR and WT. Figure 5. View largeDownload slide Concentration of the reducing sugars in the culture when ΔmanR, ΔmanH and WT were grown on locust bean gum. Figure 5. View largeDownload slide Concentration of the reducing sugars in the culture when ΔmanR, ΔmanH and WT were grown on locust bean gum. Conversely, the increase of reducing sugars was dramatically delayed in the ΔmanH culture (Fig. 5). Since the expression of mannan utilization genes was suppressed by ManH deletion, the degradation of mannan was expected to be slower than in the WT. Nevertheless, the mannan was slowly hydrolyzed, most likely by enzymes expressed at a background level, so that the concentration of reducing sugars also reached a relatively high level after 18 h. However, since ManH was absent, the oligosaccharides generated in the culture of ΔmanH could not be transported efficiently, so that the concentration of reducing sugars remained high until the end of the fermentation. Regulatory mechanisms of mannan use of Bacillus sp. N16-5 Based on the above results, we propose the following hypothesis regarding the regulation of mannan use of the strain. When mannans such as locust bean gum are present outside the cells, a small amount is degraded into manno-oligosaccharides by ManA, whose gene is expressed at background level. The generated oligosaccharides are transported into the cell by the manno-oligosaccharide transporter ManHIJ. Oligosaccharides of suitable length such as mannotetraose and mannopentose bind to ManR, which releases its operator sequence, inducing the rapid transcription of the mannan utilization gene cluster. Finally, the enzymes and transporters related to mannan utilization are expressed in large amounts, which enables the strain to utilize the mannans efficiently. The expression of the polysaccharide utilization genes of bacteria is usually induced only when the substrate is present. As polymers, hemicelluloses cannot be transported into bacteria directly, so the bacteria should exhibit the ability to sense them. Cellvibrio japonicus was found to develop a two-component system to sense the decorated xylans and arabinoxylo-oligosaccharides directly and induce the expression of the related genes (Emami et al.2009). The polysaccharide degradative enzymes can also be regulated through soluble sugars generated by their own action (Newcomb, Chen and Wu 2007). The celC operon of Clostridium thermocellum encoding CelC and LicA, which act on β-1,3-glucan, can be induced by laminaribiose, which is the partial hydrolysis product of β-1,3-glucan (Newcomb, Chen and Wu 2007). Similarly, the expression of the mannan utilization genes can be induced even though mannans cannot be directly transported into the cells of Bacillus sp. N16-5. The regulation is achieved through the products of its partial degradation, manno-oligosaccharides. This regulation strategy enables the cells to sense and use the substrate in the environment accurately and rapidly. We analyzed additional gene clusters containing ManR homologues and found that they have a number of similar structural characters (Fig. S2 in the online supplementary material). All the investigated gene clusters encode CUT1 family transporters, which specifically transport di- or oligosaccharides (Schneider 2001). All the gene clusters also contain genes related to mannan utilization or polysaccharide degradation. This observation suggests that the regulatory mechanism of the mannan utilization cluster found in Bacillus sp. N16-5, which is based on oligosaccharide transport, is likely to exist in many other bacteria. CONCLUSIONS In this study, oligosaccharide transport was found to contribute to the induction of mannan utilization genes in Bacillus sp. N16-5. A novel regulatory protein, ManR, which represses the transcription of the mannan utilization gene cluster and uses manno-oligosaccharides as ligands was identified. Study of the regulation of mannan use by Bacillus sp. N16-5 will deepen our understanding of the polysaccharide utilization mechanisms of bacteria. SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. FUNDING This work was supported by the National Key R&D Program of China (2017YFD0400304) and the National Natural Science Foundation of China (No. 31400031). Conflict of interest. None declared. REFERENCES Chauhan PS, Puri N, Sharma P et al.   Mannanases: microbial sources, production, properties and potential biotechnological applications. Appl Microbiol Biotechnol  2012; 93: 1817– 30. Google Scholar CrossRef Search ADS PubMed  Chekan JR, Kwon IH, Agarwal V et al.   Structural and biochemical basis for mannan utilization by Caldanaerobius polysaccharolyticus strain ATCC BAA-17. J Biol Chem  2014; 289: 34965– 77. Google Scholar CrossRef Search ADS PubMed  Connelly MB, Young GM, Sloma A. Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis. J Bacteriol  2004; 186: 4159– 67. Google Scholar CrossRef Search ADS PubMed  Emami K, Topakas E, Nagy T et al.   Regulation of the xylan-degrading apparatus of Cellvibrio japonicus by a novel two-component system. J Biol Chem  2009; 284: 1086– 96. Google Scholar CrossRef Search ADS PubMed  Ferreira MJ, Sa-Nogueira I. 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FEMS Microbiology LettersOxford University Press

Published: Feb 1, 2018

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