程海舰1,2, 彭琦2, 张杰2, 宋福平1,2
1.东北农业大学生命科学学院, 黑龙江 哈尔滨 150030;
2.中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京 100193
收稿日期：2017-04-13；修回日期：2017-05-15；网络出版日期：2017-05-25
基金项目：国家自然科学基金（31530095）
*通信作者：宋福平, Tel:+86-10-62896634;E-mail:fpsong@ippcaas.cn
摘要：[目的]通过综合分析苏云金芽胞杆菌（Bacillus thuringiensis）HD73菌株Sigma54缺失突变体的转录组数据和蜡样芽胞杆菌（Bacillus cereus）ATCC 14579菌株CcpA缺失突变体的转录组数据，并进行启动子与CcpA蛋白的体外结合验证，明确Bt HD73菌株中Sigma54和CcpA共同调控的基因，丰富了对微生物的代谢调控网络的认识。[方法]以转录组测序结果为基础，通过基因同源性的比对在Bt HD73菌株中寻找受Sigma54和CcpA共同调控的基因，在这些基因中找到具有cre序列的启动子，通过凝胶阻滞验证这些启动子与CcpA蛋白的结合。[结果]Bt HD73菌株中有31个基因受Sigma54和CcpA共同调控，其中14个基因的启动子序列包含cre序列，这些启动子都可以与CcpA蛋白发生体外结合。[结论]Bt HD73菌株中有14个基因直接受CcpA的调控，同时其转录受Sigma54的控制。
关键词： 苏云金芽胞杆菌 Sigma54 CcpA 转录调控
Identification of genes regulated by Sigma54 and CcpA in Bacillus thuringiensis
Haijian Cheng1,2, Qi Peng2, Jie Zhang2, Fuping Song1,2
1.College of Life Sciences, Northeast Agricultural University, Harbin 150030, Heilongjiang Province, China;
2.State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Received 13 April 2017; Revised 15 May 2017; Published online 25 May 2017
*Corresponding author: Fuping Song, Tel:+86-10-62896634;E-mail:fpsong@ippcaas.cn
Supported by the National Natural Science Foundation of China (31530095)
Abstract: [Objective]We analyzed the Sigma54 mutant regulon data of Bacillus thuringiensis (Bt) HD73 strain and CcpA mutant regulon data of Bacillus cereus ATCC 14579 strain, and verified the binding of promoter and CcpA protein, to determine Sigma54 and CcpA co-regulated genes in Bt HD73 and enrich the understanding of microbial metabolic regulation network.[Methods]Based on the results of transcriptome sequencing, genes regulated by Sigma54 and CcpA were found via the comparison of homology gene in Bt HD73 strain. cre sequence of promoter was found by blast and verified the binding with CcpA protein by electrophoresis mobility shift assays.[Results]Thirty-one genes were co-regulated by Sigma54 and CcpA in Bt HD73 strain. The fourteen promoters of these genes contained the cre sequence, which could bind to the CcpA protein in vitro.[Conclusion]Fourteen genes were directly regulated by CcpA in Bt HD73, while their transcription was controlled by Sigma 54. This finding provides clues to the study of sporulation and crystalformation through the point of view the metabolic regulation, and enriches the Bt metabolic regulation network.
Key words: Bacillus thuringiensis Sigma54 CcpA transcriptional regulation
CcpA (catabolite control protein A)是LacI-GalR家族的蛋白，在革兰氏阳性菌中主要调控碳源分解代谢(carbon catabolite repression/carbon catabolite activation，CCR/CCA)[1-2]，如变异链球菌(Streptococcus mutans)的乙酸盐代谢[3]、金黄色葡萄球菌(Staphylococcus aureus)的谷氨酸盐代谢[4]、发酵乳杆菌(Lactobacillus fermentum)的木糖和葡萄糖代谢[5]等。CcpA是多效的调控因子，调控细菌的许多重要生理功能，如细菌与噬菌体的互作[6]、几丁质酶的活性[7]、生物膜的形成[8]、环境适应性[9]、毒力基因的表达[10]、芽胞形成[11]等。CcpA通过与启动子上的保守cre (catabolite responsive element)序列结合而发挥调控功能[12]，不同细菌中的cre位点表现出多样性，如枯草芽胞杆菌(Bacillus subtilis)的保守cre序列为TGWAANCGNTNWCA[13]，猪链球菌(Streptococcus suis)有2个保守的cre序列(WWGAAARCGYTTTCWW和TTTTYHWDH HWWTTTY)[14]，丙酮丁醇梭杆菌(Clostridium acetobutylicum)的cre序列表现出高度的灵活性，6个碱基的重复序列中间是多变的碱基(TGTAAA-Yx-TTTACA)[15]。
Sigma因子是RNA聚合酶的亚基，它通过识别目标基因的启动子，并与RNA聚合酶核心酶结合从而起始转录。根据功能不同分为Sigma70型和Sigma54型，区别在于Sigma70识别启动子的–35/–10区，Sigma54识别启动子的–24/–12区，同时需要增强子结合蛋白(enhance binding protein, EBPs)的激活才能起始转录[16]。不同细菌中Sigma54因子控制的基因参与多种生理功能，如碳氮源代谢[17-18]、生物膜形成[19]、环境适应能力[20-21]、毒力作用[22-24]等。通过转录组测序发现更多Sigma54控制的基因，大肠杆菌(Escherichia coli)有30个依赖于Sigma54的操纵子，其中一半以上参与氮源代谢[25]；霍乱弧菌(Vibrio Cholerae)有82个操纵子由Sigma54控制[26]；单核细胞增多性李斯特菌(Listeria Monocytogenes)有77个基因的转录由Sigma54控制，其中大多数与碳源代谢相关[27]；本实验室前期对苏云金芽胞杆菌(Bacillus thuringiensis，简称Bt)的Sigma54转录组分析发现47个基因的转录由Sigma54控制，大多数参与碳氮源的代谢[28]，包括γ-氨基丁酸代谢[29-30]、肌氨酸代谢[31]和赖氨酸代谢[32]等。目前关于Bt中Sigma54控制基因与CcpA调控基因间的关系仍不清楚，对于碳源代谢调控网络的认识仍不够。
Bt是革兰氏阳性细菌，在形成芽胞的同时能产生对多种农林害虫具有特异杀虫活性的伴胞晶体(主要成分是杀虫晶体蛋白，即Cry蛋白)，因其具有环境友好、生物安全等特点，在害虫的生物防治领域获得了广泛的应用[33]。而从代谢调控的角度对Bt芽胞和晶体蛋白形成研究的报道极少。转录组分析表明，Sigma54和CcpA都调控许多与芽胞形成相关基因的表达[28, 34]，并且Bt HD73菌株中Sigma54的缺失影响晶体蛋白的产量(待发表)，但作用机制不清楚。本文通过综合分析蜡样芽胞杆菌属遗传距离最近的两种细菌Bt HD73菌株Sigma54缺失突变体的转录组数据[28]和蜡样芽胞杆菌(Bacillus cereus) ATCC 14579菌株CcpA缺失突变体的转录组数据[34] (两个菌株的基因组相似性90%以上)，并进行启动子与CcpA蛋白的体外结合验证，明确Bt HD73菌株中共同受Sigma54和CcpA调控的基因，为从代谢调控的角度研究芽胞和晶体形成提供线索，并丰富对Bt的代谢调控网络的认识。
1 材料和方法 1.1 材料
1.1.1 菌株、质粒和培养基: 所用菌株和质粒见表 1。大肠杆菌(Escherichia coli，E. coli)和Bt的培养使用LB培养基(Tryptone 1.0%，Yeast extract 0.5%，NaCl 1.0%，pH 7.2)。Bt在30 ℃、220 r/min条件下培养；E. coli在37 ℃、220 r/min条件下培养，氨苄青霉素终浓度为100 μg/mL。
表 1. 菌株与质粒 Table 1. Strains and plasmids
| Strains and plasmids | Characterization | Resource |
| Strains | ||
| E. coli TG1 | Δ(lac-proAB) supE thi hsd-5 (F' traD36 proA+ proB+ lacIq lacZΔM15), general purpose cloning host | This lab |
| BL21(DE3) | E. coli B, F-, dcm, ompT hsdS(rB-mB-), gal, λ(DE3) | [35] |
| BL21(pETccpA) | BL21 (DE3) strain containing plasmid pETccpA | This study |
| BLpET | BL21 strain carrying pET21b | This lab |
| HD73 | B. thuringiensis subsp. kurstaki carrying the cry1Ac gene | This lab |
| Plasmids | ||
| pET21b | Expressional vector, Ampr, 5.4 kb | This lab |
| pETccpA | pET21b containing ccpA gene, Ampr | This study |
表选项
1.1.2 主要仪器和材料: 限制性内切酶、PrimeSTAR? HS DNA聚合酶和T4 DNA连接酶均购自宝生物工程(大连)有限公司；Taq DNA聚合酶购自北京博迈德科技发展有限公司；质粒提取、DNA回收和PCR产物纯化试剂盒购自Axygen公司。镍亲和层析柱填料(chelating sepharose fast flow)购自GE公司。poly(dI:dC)[Poly(ethylene glycol) di-(4-hydroxyphenyl)diphenylphosphine]购自Sigma公司。Gel Shift Assay Systems购自Promega公司。其他生化试剂和抗生素均为进口或国产生化或分析纯级试剂。
1.1.3 引物合成及序列测定: 根据Bt HD73基因组[36]序列设计引物，引物合成由生工生物工程公司北京合成部完成，序列测定由北京六合华大基因科技股份有限公司完成，引物名称及序列见表 2。
表 2. 引物序列 Table 2. Primers used in this study
| Primer name | Sequence (5'→3') |
| ccpA-F | CGGGATCCGATGAACGTAACAATCTATG |
| ccpA-R | ACGCGTCGACTTACTTCGTTGAATCTCTAAA |
| P0460-F | CAAAGAATTGTGCGATAA |
| P0460-R | GGTTTCCCCTCCACACC |
| P0493-F | GCTGGTACTATTATTTCAA |
| P0493-R | AAAATTCCCTCTCTCTTGC |
| P0737-F | CGTAGTAGGTGCCATCTTT |
| P0737-R | TCTCTTTTCTCCTCATTTCA |
| P1239-F | GAAGATGGATCATATGGAGATG |
| P1239-R | GAAATTCTCCCCCTTTGCA |
| P1416-F | CTCTGATAATTTAGAAAAGTG |
| P1416-R | CATGCTCATCGCTACTGTTG |
| P1464-F | GCATAATGAAACGTTTACAAAGG |
| P1464-R | GTCCTTCACCTCGCTCTG |
| P1489-F | TATTCCGTTATATTGTTG |
| P1489-R | TTGTTTAAACCTCCCAATG |
| P2061-F | GTTATGTATGAATGATAGGAG |
| P2061-R | ATTGTATGGTCAATTAACTTTGC |
| P4468-F | GCGTTAGGAATTTCCGTTCGAA |
| P4468-R | ATTTTGTAATCAACCCTTTCCGTCG |
| P4900-F | GCAAGTAATACAAGCGGATAAG |
| P4900-R | CGTCATTCCCCTTTCTTCTTC |
| P5854-F | TACTGAAATTCAGCCATGGTT |
| P5854-R | TTTGGCCACCTCTTTCGATTT |
| P0668-F | GAAATCGGGATAATCATTTAGGA |
| P0668-R | TATAGCTCACCTCTTTAATTG |
| P4302-F | TCTTTATTCCGCCATATTGGG |
| P4302-R | TTTCATTACCCCTTCTATAAGT |
| P5768-F | GTGGTTCCATTCCTCTACATA |
| P5768-R | TTTTTCCTCTCCTATTCTTC |
| PcwlX-F | TTGCAGAGGTTGAAAGAAGCTGTGAAGG |
| PcwlX-R | CCTCCGTGCAAACTATATCTAGCCAT |
表选项
1.2 CcpA表达菌株构建 根据GenBank中Bt HD73菌株(GenBank登录号：CP004069)的ccpA (HD73_4987)基因序列及pET21b质粒的酶切位点，设计扩增ccpA基因ORF的引物ccpA-F和ccpA-R (表 2)，以HD73基因组为模板扩增ccpA基因，PCR产物纯化后经BamH Ⅰ和Sal Ⅰ双酶切，连接含有His标签的pET21b质粒BamH Ⅰ和Sal Ⅰ双酶切片段上，转化E. coli TG1菌株，获得重组质粒pETccpA。重组质粒经PCR、酶切和测序鉴定，转化至E. coli BL21 (DE3)菌株，获得表达菌株BL21 (pETccpA)。
1.3 CcpA蛋白的表达与纯化 将BL21 (pETccpA)菌株在含有100 μg /mL氨苄青霉素的新鲜LB培养基中，37 ℃、220 r/min培养至OD600=0.5–0.6，加入终浓度为0.5 mmol/L IPTG (isopropyl-β-D-thiogalactopyranoside)，18 ℃、150 r/min诱导12 h。8000 r/min低温离心15 min收集菌体，用50 mmol Tris-HCl缓冲液(pH 8.0)悬浮菌体，将悬浮液置于冰上超声破碎6 min (CP750 COLE-PARMER，超声功率40%，超声3 s，暂停5 s)。12000 r/min低温离心10 min分离上清和沉淀，上清中含有可溶性CcpA蛋白。
将上清置于平衡好的镍亲和层析柱中，使含有His标签的CcpA蛋白与柱料充分结合，弃去流穿的液体；用5倍柱体积的平衡缓冲液(20 mmol/L Tris-HCl pH 8.5，0.5 mol/L NaCl，20 mmol/L咪唑)洗脱杂蛋白；用5倍柱体积的洗脱缓冲液(20 mmol/L Tris-HCl pH 8.5，0.5 mol/L NaCl，250 mmol/L咪唑)洗脱目的蛋白并收集；SDS-PAGE检测洗脱的蛋白样品。对纯化的蛋白用pH 8.0 Tris-HCl进行透析。
1.4 凝胶迁移实验 以苏云金芽胞杆菌HD73为模板，用带有FAM (羧基荧光素)标记的引物(表 2)扩增带有cre序列的DNA片段。凝胶阻滞实验(EMSA，electrophoresis mobility shift assays)确定DNA片段与蛋白的结合：20 μL反应体系中含有0.05 μg或0.1 μg带标记的DNA、不同浓度的CcpA蛋白、结合缓冲液(10 mmol/L Tris-HCl，0.5 mmol/L dithiothreitol (DTT)，50 mmol/L NaCl，500 ng poly (dI:dC)，pH 7.5和4% (V/V) glycerol)，25 ℃反应20 min。用5% (W/V)非变性聚丙烯酰胺胶在TBE缓冲液(90 mmol/L Tris-base，90 mmol/L硼酸，2 mmol/L EDTA，pH 8.0)中电泳检测反应产物(Mini-PROTEAN system (Bio-Rad)，160 V，4 ℃，1 h)。用荧光凝胶成像系统(Fujifilm，FLA Imager FLA-5100)对非变性胶进行扫描(Laser：473 nm，电压：900 V，Filter：526-000/01)。
2 结果和分析 2.1 Sigma54和CcpA共同调控的基因分析 转录组测序分析表明，Bt HD73菌株中有121个基因在sigL (编码Sigma54因子)突变体中上调表达，255个基因下调表达[28]；Bc ATCC 14579菌株中，有173个基因在ccpA突变体中上调表达，80个基因下调表达[34]。根据Bc ATCC 14579菌株CcpA缺失突变体的转录组，在HD73基因组中找到同源基因(一致性90%以上)，与Bt HD73菌株Sigma54缺失突变体的转录组进行比较，共发现31个基因同时出现在CcpA缺失突变体和Sigma54缺失突变体的转录组数据中(表 3)。在ccpA突变体中上调表达的基因中，有11个基因在sigL突变体中上调表达，16个基因下调表达；在ccpA突变体中下调表达的基因中，有3个基因在sigL突变体中上调表达，2个基因下调表达。对Sigma54与CcpA共同调控的31个基因的启动子区域(ATG上游500 bp到ATG下游100 bp)在DBTBS数据库(http://dbtbs.hgc.jp/)进行序列分析，发现14个基因(或基因所在的操纵子)的启动子序列具有保守的cre序列(图 1)。
表 3. Sigma54与CcpA共同调控的基因 Table 3. Sigma54 and CcpA regulated genes
| Bt HD73 | Bc ATCC 14579 | Identify/% | In sigL mutant | In ccpA mutant | Annotation |
| HD73_0460 | BC0378 | 97 | Up-regulated | Up-regulated | 5-Methylthioribose kinase |
| HD73_0493 | BC0410 | 99 | Up-regulated | Up-regulated | Crp family transcriptional regulator |
| HD73_0742 | BC0664 | 97 | Down-regulated | Up-regulated | RbsB (Ribose ABC transporter) |
| HD73_1144 | BC0999 | 94 | Down-regulated | Up-regulated | Hypothetical protein |
| HD73_1238 | BC1082 | 99 | Up-regulated | Up-regulated | Acetyltransferase |
| HD73_1239 | BC1083 | 99 | Up-regulated | Up-regulated | Transcriptional regulator LacI |
| HD73_1412 | BC1181 | 99 | Down-regulated | Up-regulated | Oligopeptide transport system permease oppC |
| HD73_1413 | BC1182 | 99 | Down-regulated | Up-regulated | Oligopeptide ABC transporter ATP-binding protein |
| HD73_1416 | BC1185 | 99 | Up-regulated | Up-regulated | Peptide/nickel transport system substrate-binding protein |
| HD73_1455 | BC1224 | 99 | Down-regulated | Up-regulated | Acetyltransferase |
| HD73_1456 | BC1225 | 99 | Down-regulated | Up-regulated | Hypothetical protein |
| HD73_1467 | BC1235 | 97 | Down-regulated | Up-regulated | Indole-3-glycerol phosphate synthase |
| HD73_1469 | BC1237 | 94 | Down-regulated | Up-regulated | Tryptophan synthase beta chain |
| HD73_1488 | BC1251 | 98 | Down-regulated | Up-regulated | Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex |
| HD73_2025 | BC1776 | 98 | Down-regulated | Up-regulated | Branched-chain amino acid aminotransferase |
| HD73_2063 | BC1822 | 98 | Down-regulated | Up-regulated | Pyrimidine-nucleoside phosphorylase |
| HD73_3012 | BC2961 | 90 | Up-regulated | Up-regulated | AI-2 transport system permease protein |
| HD73_3011 | BC2962 | 94 | Up-regulated | Up-regulated | AI-2 transport system permease protein |
| HD73_3010 | BC2963 | 99 | Up-regulated | Up-regulated | ABC transporter |
| HD73_3901 | BC3627 | 96 | Down-regulated | Up-regulated | Acetyl-CoA C-acetyltransferase |
| HD73_4119 | BC3834 | 99 | Down-regulated | Up-regulated | Succinyl-CoA synthetase beta subunit |
| HD73_4205 | BC3921 | 98 | Up-regulated | Up-regulated | Putative N-acetyltransferase |
| HD73_4466 | BC4161 | 98 | Down-regulated | Up-regulated | Branched-chain-fatty-acid kinase |
| HD73_4468 | BC4163 | 99 | Down-regulated | Up-regulated | Phosphate butyryltransferase |
| HD73_4889 | BC4594 | 99 | Up-regulated | Up-regulated | Citrate synthase 2 |
| HD73_4900 | BC4606 | 99 | Down-regulated | Up-regulated | Hypothetical protein |
| HD73_5854 | BC5439 | 99 | Up-regulated | Up-regulated | Murein hydrolase exporter |
| HD73_0668 | BC0595 | 99 | Up-regulated | Down-regulated | Cadmium efflux system accessory protein |
| HD73_1412 | BC1181 | 99 | Up-regulated | Down-regulated | Oligopeptide transport system permease protein |
| HD73_3222 | BC2771 | 88 | Up-regulated | Down-regulated | Hypothetical protein |
| HD73_4302 | BC4017 | 98 | Down-regulated | Down-regulated | Hypothetical protein |
| HD73_5768 | BC5359 | 99 | Down-regulated | Down-regulated | Aminopeptidase YwaD |
表选项
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| 图 1 启动子的cre序列 Figure 1 Consensus sequence of cre in promoter region. The underline indicated the conserved base. |
| 图选项 |
2.2 CcpA蛋白的表达与纯化 为了进一步验证14个具有cre位点的启动子在体外是否与CcpA蛋白结合，构建Bt HD73菌株的CcpA表达纯化载体，Bt HD73 ccpA基因ORF区全长为999 bp，氨基酸序列与Bc ATCC 14579菌株的CcpA相似性为100%。PCR扩增ccpA基因全长并与含有His标签的pET21b载体连接，转化E. coli TG1菌株，获得重组质粒pETccpA，经PCR鉴定得到999 bp的条带(图 2-A)，BamH Ⅰ和Sal Ⅰ双酶切鉴定得到5.4 kb大小的载体条带和1000 bp左右大小的目的片段(图 2-A)，将重组质粒送生物公司进行测序分析，序列比对表明重组质粒pETccpA构建正确。重组质粒转化至E. coli BL21菌株，获得BL21 (pETccpA)表达菌株，经IPTG诱导表达，超声破碎细胞，离心收集可溶性组分，对CcpA-His融合蛋白进行Ni2+螯合琼脂糖亲和纯化，SDS-PAGE结果表明所得蛋白纯度较高，分子量大小约为37 kDa (图 2-B)。
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| 图 2 CcpA表达载体构建及纯化 Figure 2 Construction and purification of CcpA. A: identification of pETccpA plasmid; M1: DNA marker. B: purification of CcpA-His fusion protein; M2: protein marker |
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2.3 CcpA蛋白与启动子的结合 利用FAM标记的引物(表 2)扩增14个基因(操纵子)的启动子片段，与纯化的CcpA-His蛋白进行体外结合实验，结果表明(图 3)，凝胶底部的条带为带标记的自由DNA，上层为DNA与蛋白结合的条带，随着蛋白浓度的增加，底部自由DNA条带浓度越来越低，上层条带浓度逐渐升高，说明14个启动子都与CcpA蛋白结合，而对照DNA条带(cwlX基因的启动子，360 bp)不与CcpA蛋白结合。并且不同浓度的启动子与CcpA蛋白结合的亲和力不同。这些结果表明具有cre序列的14个启动子受CcpA的直接调控。
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| 图 3 CcpA与启动子的结合 Figure 3 Binding of CcpA and promoter. Lane 1: FAM-labeled DNA probe; lanes 2–5: incubation of the probe with increasing concentrations of purified CcpA indicated at the top of the figure. |
| 图选项 |
3 讨论 苏云金芽胞杆菌是昆虫病原菌，它与人类病原菌炭疽芽胞杆菌(Bacillus anthracis)、机会致病菌蜡样芽胞杆菌(Bacillus cereus)同属于蜡样芽胞杆菌族，它们的基因组有90%以上的相似性。本研究根据Bt HD73菌株Sigma54缺失突变体的转录组和Bc ATCC 14579菌株CcpA缺失突变体的转录组数据，在Bt HD73基因组中找到31个共同受Sigma54和CcpA调控的基因，并对其中14个具有cre序列的启动子进行体外结合验证，结果表明CcpA蛋白可以与这14个启动子结合。根据基因组的注释，这14个基因及其所在的基因簇参与的生理功能多样，如碳氮源代谢相关(HD73_0460、HD73_1467、HD73_1469、HD73_1488、HD73_4466、HD73_4468、HD73_5768)、转录调控因子(HD73_0493、HD73_1239)、运输系统(HD73_0742、HD73_1416、HD73_5854、HD73_0668)等。对这些启动子的序列进行分析发现，只有HD73_4468基因的启动子有保守的与Sigma54结合的–12/–24区域，而其它启动子无保守序列，说明这些基因的转录并不直接受Sigma54的控制，可能存在间接的调控作用。本实验室前期研究表明，HD73_4468基因所在的bkd基因簇的转录受Sigma54的控制[28, 37]，并受BkdR的调控，本研究证明CcpA蛋白可以与HD73_4468的启动子结合，说明bkd基因簇受CcpA的直接调控。这与枯草芽胞杆菌中的报道相似，bkd基因簇在枯草芽胞杆菌中的转录也是受Sigma54控制，并同时受CcpA的调控[38]，此外还受CodY的负调控[18]。枯草芽胞杆菌中的bkd操纵子编码的酶系统参与支链氨基酸的代谢，在细胞抵御冷刺激条件中发挥作用，Bt和Bs中bkd基因簇的转录模式具有相似性，因此它们的功能也可能相似，Bt菌株可能会通过bkd基因簇的转录使自身具有抵御冷刺激的功能，这也为我们在低温环境下寻找新的Bt菌株资源提供了依据。
细菌中受Sigma54控制的基因大多数参与碳氮源的代谢，CcpA主要调控碳源分解代谢，二者调控的基因也参与了细菌毒力作用、生物膜形成、芽胞形成等重要生理过程，它们共同调控的基因参与的生理功能对菌株的生长具有重要意义。目前已报道艰难梭状芽胞杆菌(Clostridium difficile)的半胱氨酸代谢同时受Sigma54和CcpA调控[39]，枯草芽胞杆菌受Sigma54控制的启动子(参与多糖代谢的lev操纵子[40]、3-羟基丁酮的aco操纵子[13]、精氨酸代谢的rocG基因[41])也同时受CcpA的调控。在Bt HD73菌株中，已经证明同源的lev和aco操纵子的转录受Sigma54的控制[42, 28]，并且在这些基因的启动子序列中也存在cre结合位点，但在Bc ATCC 14579菌株CcpA的转录组数据中并没有发现这些基因，可能与转录组测序样品的培养条件有关。通过本研究发现的共同受Sigma54和CcpA调控的基因参与的生理功能不尽相同，这些基因的发现丰富了对Bt的代谢调控网络的认识。
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