Abstract
Cytophaga hutchinsonii is an important Gram-negative bacterium belonging to the Bacteroides phylum that can efficiently degrade cellulose. But the promoter that mediates the initiation of gene transcription has been unknown for a long time. In this study, we determined the transcription start site (TSS) of C. hutchinsonii by 5′ rapid amplification of cDNA ends (5′RACE). The promoter structure was first identified as TAAT and TATTG which are located -5 and -31 bp upstream of TSS, respectively. The function of -5 and -31 regions and the spacer length of the promoter Pchu_1284 were explored by site directed ligase-independent mutagenesis (SLIM). The results showed that the promoter activities were sharply decreased when the TTG motif was mutated into guanine (G) or cytosine (C). Interestingly, we found that the strong promoter was accompanied with many TTTG motifs which could enhance the promoter activities within certain copies. These characteristics were different from other promoters of Bacteriodes species. Furthermore, we carried out genome scanning analysis for C. hutchinsonii and another Bacteroides species by Perl6.0. The results indicated that the promoter structure of C. hutchinsonii possessed more unique features than other species. Also, the screened inducible promoter Pchu_2268 was used to overexpress protein CHU_2196 with a molecular weight of 120 kDa in C. hutchinsonii. The present study enriched the promoter structure of Bacteroidetes species and also provided a novel method for the highly expressed large protein (cellulase) in vivo, which was helpful to elucidate the unique cellulose degradation mechanism of C. hutchinsonii.
Key points
• The conserved structure of strong promoter of C. hutchinsonii was elucidated.
• Two novel regulation motifs of TTTG and AATTATG in the promoter were discovered.
• A new method for induced expression of cellulase in vivo was established.
• Helpful for explained the unique cellulose degradation mechanism of C. hutchinsonii.
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Acknowledgements
We sincerely thank Professor Mark J McBride (University of Wisconsin-Milwaukee, Milwaukee, WI) for providing the C. hutchinsonii ATCC 33406 strains. We thank Dr. Yizhao Chang (State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao, China) for the bioinformatic analysis. We also give thanks to Dr. Edward C. Mignot (Shandong University) for the linguistic advice.
Funding
This work was supported by the National Natural Science Foundation of China (Grant numbers 31770080 and 31371262).
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XM L and GQ F developed the article initial concepts. GQ F and WX S performed the experiments under the supervision of XM L. GQ F, WX S, ZW G, and WC Z analyzed the results and reviewed the manuscript.
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Fan, G., Song, W., Guan, Z. et al. Some novel features of strong promoters discovered in Cytophaga hutchinsonii. Appl Microbiol Biotechnol 106, 2529–2540 (2022). https://doi.org/10.1007/s00253-022-11869-3
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DOI: https://doi.org/10.1007/s00253-022-11869-3