CesH Represses Cereulide Synthesis as an Alpha/Beta Fold Hydrolase in Bacillus cereus
Abstract
:1. Introduction
2. Results
2.1. The Negative Effect of cesH on Cereulide Production
2.2. Prediction and Characterization of CesH as a Membrane Associated Esterase
2.3. The Effect of CesH on Cereulide Analogue–Valinomycin In Vitro and In Vivo
3. Discussion
4. Materials and Methods
4.1. Strains, Plasmids, Primers, and Growth Condition
4.2. Extraction and Quantification of Cereulide via High Performance Liquid Chromatography (HPLC)
4.3. Bioinformatic Analysis of CesH
4.4. RT-PCR and DNA Manipulations
4.5. Growth Measurements
4.6. Activity Assays of CesH
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Strains and Plasmids | Characteristics | Reference |
---|---|---|
B. cereus AH187 | Wild cereulide-producing isolate | Ehling-Schulz, 2005 |
B. cereus AH187 ΔcesH | a kanamycin gene fragment was taken the place of cesH in AH187; KanaR | This study |
B. cereus AH187 Cm.ΔcesH | AH187ΔcesH complemented with pHT315-cesH; KanaR EryR | This study |
B. cereus AH187 gfp | AH187 containing pHT315-gfp | This study |
B. cereus AH187 cesH-gfp | AH187 containing pHT315-cesH-gfp | pHT315-cesH-gfp |
B. subtilis 168 | Wild isolate | Spizizen, 1958 |
B. subtilis 168H | B. subtilis 168 containing pHT315-cesH for expression; EryR | This study |
B. subtilis S86A | B. subtilis 168H containing an alanine substitution at serine 86 of CesH; EryR | This study |
B. subtilis H189A | B. subtilis 168H containing an alanine substitution at histidine 189 of CesH; EryR | This study |
B. subtilis E197A | B. subtilis 168H containing an alanine substitution at glutamate 197 of CesH; EryR | This study |
B. subtilis E199A | B. subtilis 168H containing an alanine substitution at glutamate 199 of CesH; EryR | This study |
B. subtilis H227A | B. subtilis 168H containing an alanine substitution at histidine 227 of CesH; EryR | This study |
E. coli BL21 (DE3) pET28a-cesH | E. coli BL21 (DE3) containing pET28a-cesH for protein expression | This study |
pMD18T-Kana-gfp | pMD18T plasmid containing a kanamycin resistance cassette and a gfp gene | This study |
pHT315ts-cesH::kana | Shutter vector containing the flank region of cesH and kanamycin resistance fragment, and a temperature sensitive replicon | This study |
pHT315-cesH | Shutter plasmid containing the intact cesH fragment and its promoter | This study |
pHT315-gfp | Shutter plasmid containing the intact gfp fragment and the promoter of cesH | This study |
pHT315-cesH-gfp | Shutter plasmid containing the intact cesH with a gfp fragment fused in the c-terminal | This study |
pET28a-cesH | Expression vector containing the complete sequence of cesH | This study |
Order | Sequence | Features |
---|---|---|
1 | GCGGGATCCGCAATCCCCCCTAGCTATG | Forward primer for cloning up stream of cesH (KpnI) |
2 | GCGGGTACCTCTAACACATTCATATAGTA | Reverse primer for cloning up stream of cesH (BamHI) |
3 | GCGAAGCTTTATTTCAATTTCATACGGGTA | Forward primer for cloning down stream of cesH (SalI) |
4 | GCGGTCGACAATTTTAGCTCTTTAGTTCC | Reverse primer for cloning down stream of cesH (HindIII) |
5 | CGGGGATCCAGCGAACCATTTGAGGTGATAG | Forward primer for cloning kanamycin resistance cassette (BamHI) |
6 | CGGGTCGACCTAGGTACTAAAACAATTCATCCAG | Reverse primer for cloning kanamycin resistance cassette (SalI) |
7 | GCGGGTACCCAAACAAATTAGATAAGTGGATAGAGAGACA | Forward primer for cloning promoter (KpnI) |
8 | GCGGTTCTTCTCCTTTACTCATGCAATCCCCCCTAGCTATG | Reverse primer for gfp element overlapping pcr |
9 | GCGGTGGCAATAGGTTTCGCTTTAGGATCCCAAGTA | Forward primer for generating mutation at residue 86 |
10 | GCGTACTTGGGATCCTAAAGCGAAACCTATTGCCAC | Reverse primer for generating mutation at residue 86 |
11 | GCGCATTCAGGGAATACTCAGCTAATATATTAGTTACTGT | Forward primer for generating mutation at residue 189 |
12 | GCGACAGTAACTAATATATTAGCTGAGTATTCCCTGAATG | Reverse primer for generating mutation at residue 189 |
13 | GCGATATTAGTTACTGTTGGTGCTAAAGAGAAAAAAATAATG | Forward primer for generating mutation at residue 197 |
14 | GCGCATTATTTTTTTCTCTTTAGCACCAACAGTAACTAATAT | Reverse primer for generating mutation at residue 197 |
15 | GCGACTGTTGGTGAAAAAGCTAAAAAAATAATGAAGGAT | Forward primer for generating mutation at residue 199 |
16 | GCGATCCTTCATTATTTTTTTAGCTTTTTCACCAACAGT | Reverse primer for generating mutation at residue 199 |
17 | GCGATTCCTAAAATTGGTGCTGGGATACCTTTAGCA | Forward primer for generating mutation at residue 277 |
18 | GCGTGCTAAAGGTATCCCAGCACCAATTTTAGGAAT | Reverse primer for generating mutation at residue 277 |
19 | GCGCCCGCGCTGCCACTATTAGATACCAATTTCACTTCATC | Reverse primer for gfpligation |
20 | GCGGAATTCATGTATTATACAGAATTTGGAACGGATC | Forward primer for cloning cesH (EcoRI) |
21 | GCGAAGCTTTCAACTATTAGATACCAATTTCACTTC | Reverse primer for cloning cesH (HindIII) |
22 | GCGCATAGCTAGGGGGGATTGCATGAGTAAAGGAGAAGAAC | Forward primer for gfp element overlapping pcr |
23 | GCGGTACCTTATTTGTAGAGCTCATCCATGC | Reverse primer for cloning gfp element (KpnI) |
24 | AAGCCTGATGAATTAGTTATTG | Forward primer for ilvB in RT-PCR assay |
25 | CTGGTTGACACGATAGTAA | Reverse primer for ilvB in RT-PCR assay |
26 | GATTACGTTCGATTATTTGAAG | Forward primer for cesA in RT-PCR assay |
27 | CGTAGTGGCAATTTCGCAT | Reverse primer for cesA in RT-PCR assay |
28 | TGCTTAGTTCTTGACCTA | Forward primer for cesH in RT-PCR assay |
29 | CACAACAGACTTACCTTC | Reverse primer for cesH in RT-PCR assay |
30 | GGAGGAAGGTGGGGATGACG | Forward primer for 16s rrn in RT-PCR assay |
31 | ATGGTGTGACGGGCGGTGTG | Reverse primer for 16s rrn in RT-PCR assay |
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Tian, S.; Xiong, H.; Geng, P.; Yuan, Z.; Hu, X. CesH Represses Cereulide Synthesis as an Alpha/Beta Fold Hydrolase in Bacillus cereus. Toxins 2019, 11, 231. https://doi.org/10.3390/toxins11040231
Tian S, Xiong H, Geng P, Yuan Z, Hu X. CesH Represses Cereulide Synthesis as an Alpha/Beta Fold Hydrolase in Bacillus cereus. Toxins. 2019; 11(4):231. https://doi.org/10.3390/toxins11040231
Chicago/Turabian StyleTian, Shen, Hairong Xiong, Peiling Geng, Zhiming Yuan, and Xiaomin Hu. 2019. "CesH Represses Cereulide Synthesis as an Alpha/Beta Fold Hydrolase in Bacillus cereus" Toxins 11, no. 4: 231. https://doi.org/10.3390/toxins11040231
APA StyleTian, S., Xiong, H., Geng, P., Yuan, Z., & Hu, X. (2019). CesH Represses Cereulide Synthesis as an Alpha/Beta Fold Hydrolase in Bacillus cereus. Toxins, 11(4), 231. https://doi.org/10.3390/toxins11040231