Identification and characterization of sequence signatures in the Bacillus subtilis promoter Pylb for tuning promoter strength
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To thoroughly characterize the Pylb promoter and identify the elements that affect the promoter activity.
The sequences flanking the − 35 and − 10 box of the Pylb promoter were divided into six segments, and six random-scanning mutant promoter libraries fused to an enhanced green fluorescent protein EGFP were made and analyzed by flow cytometry. Our results showed that the four nucleotides flanking the − 35 box could mostly influence the promoter activity, and this influence was related to the GC content. The promoters mutated in these regions were successfully used for expressing the gene ophc2 encoding organophosphorus hydrolase (OPHC2) and the gene katA encoding catalase (KatA).
Our work identified and characterized the sequence signatures of the Pylb promoter that could tune the promoter strength, providing further information for the potential application of this promoter. Meanwhile, the sequence signatures have the potential to be used for tuning gene expression in enzyme production, metabolic engineering, and synthetic biology.
KeywordsBacillus subtilis Promoter Random-scanning mutant Pylb
Bacillus subtilis has been developed as a convenient host for the production of heterologous proteins and industrial enzymes (van Dijl and Hecker 2013). As the promoter is one of the key factors for efficiently expressing heterologous proteins (Blazeck and Alper 2013), a series of native promoters from B. subtilis have been identified and successfully used in B. subtilis for gene expression, such as the promoter P43 (Zhang et al. 2005), PxylA (Bhavsar et al. 2001), PsacB (Steinmetz et al. 1985) and Pglv (Ming et al. 2010). However, more endogenous promoters need to be explored for producing heterologous proteins.
Meanwhile, efforts have been made to enhance promoter strength. The most conventional strategy is modifying moderately conserved sequences of the promoter, such as the UP element, the core region (− 35 and − 10 motifs), and the 16 region (TRTG motif) within existing promoters (Cheng et al. 2016; Guan et al. 2016; Lee et al. 2010; Phan et al. 2012, 2015; Zhou et al. 2019). Furthermore, mutagenesis of the spacer region between the − 35 and − 10 regions in E. coli and Lactobacillus (Lb.) plantarum has successfully created high-coverage synthetic promoter libraries (De Mey et al. 2007). Saturation mutagenesis of a Lactococcus lactis promoter drastically modulates expression (Jensen and Hammer 1998). In addition, randomization of the spacer region of PymdA and PserA in B. subtilis has generated mostly strong to medium promoters (Guiziou et al. 2016). Thus, mutagenesis of the spacer region is considered a rational methodology to modify prokaryotic promoter strength.
Previously, we had identified a highly efficient stationary phase promoter Pylb in B. subtilis. The Pylb promoter could induce higher levels of expression of active β-galactosidase, EGFP, RFP, pullulanase, and organophosphorus hydrolase from the late log phase to the stationary phase than that of the widely used P43 promoter, demonstrating strong potential to be used for the overexpression of useful proteins in B. subtilis. By scanning site-directed mutagenesis on the Pylb promoter, we found that not only the − 35 and − 10 regions, but also the regions flanking the − 35 and − 10 regions were directly involved in the transcriptional activity of Pylb (Yu et al. 2015). In this study, we focused on the flanking sequences to thoroughly characterize the Pylb promoter and identify the elements that affect the promoter activity. Our work clarified the effect of the identified promoter sequence signatures on the activity of the Pylb promoter. Meanwhile, the promoters libraries with the mutation in the sequence signatures have a wide range of activities, and have the potential to be used for tuning gene expression in enzyme production, metabolic engineering, and synthetic biology.
Materials and methods
Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids used in this study are listed in Supplementary Table 1. The Escherichia coli strain DH5α was used as the host for gene cloning and the construction of the mutated promoter libraries. The B. subtilis strain WB600 was used for promoter screening and gene expression. All the strains were cultured in Luria–Bertani medium (LB) at 37 °C under constant shaking (200 rpm). The concentrations of antibiotics used for selection were as follow: 100 μg/mL ampicillin (Amp), 10 μg/mL kanamycin (Kan), 50 μg/mL tetracycline (Tet).
Construction of Pylb random-scanning mutagenesis libraries
Characterization of promoter libraries by flow cytometry
To measure the activity of the promoters in the six random-scanning mutagenesis libraries of Pylb, flow cytometry was used to quantify EGFP fluorescence. The overnight cultures of all the recombinant B. subtilis strains were centrifuged at 5000×g for 10 min, and the cells were resuspended in ice-cold 1 × phosphate-buffered saline (PBS) buffer (pH 7.4). Flow cytometry analysis was carried out on a FACSCalibur apparatus (Becton Dickinson Biosciences) using excitation at 488 nm. Three independent replicas were analyzed from each promoter library, and each cytometric measurement was performed on 100,000 cells. The data were acquired and analyzed using the CELLQuest software with defined gates that were measured by tight forward scatter/side scatter light to ensure a homogenous population size. The average relative fluorescence intensity (RFI) given represent the mean values of the geometric mean expression values of each replicate; the standard deviation was also calculated from the replicate geometric mean values. The value of coefficient of variation (CV) for each promoter library was obtained by dividing the standard deviation of all replicate values by the mean of all replicate values.
Screening of the T2 and T3 mutant libraries
The T2 and T3 mutant libraries were screened for Pylb mutants with respectively higher and lower activities by using a BD Influx flow cytometer. The separation gates in the dot plots of the two-parameter histogram were reset beyond the negative control (B. subtilis WB600) values and the positive control (wild type Pylb) values. P4 gate was used to collect up-regulated Pylb mutants, while the P2 gate collected the extremely weak mutants (Supplementary Fig. 1). The fluorescent intensity per OD600 (FI/OD600) of these selected mutants were measured by a SpectraMax M2 (Molecular Devices, USA) microplate reader. The excitation/emission was read at 484/507 nm. The promoters of these selected mutants were sequenced on an ABI 3730 DNA analyzer (Qingke Biotechnology Co., Ltd.) with the sequencing primers F4-cx-up and F4-cx-down (Supplementary Table 2). All the sequences were analyzed using the WebLogo online software (https://weblogo.berkeley.edu) to explore the relationship between signature sequences and the strength of Pylb.
Construction of site-directed mutagenesis promoters
The promoter Pylb in the plasmids Pylb-G-P43-R-pUBC19, Pylb-R-pUBC19, Pylb-ophc2-pUBC19 and Pylb-katA-pUBC19 were mutated into Pylb-T2G, Pylb-T2G and Pylb-T3G, respectively (Supplementary Table 1). To do this, these four plasmids were used as templates to amplify the fragments containing the promoters Pylb-T2G, Pylb-T2G and Pylb-T3G by the primer pairs P16-T2GF/V-R, P16-T2CF/V-R and P16-T3GF/V-R, and the vector fragments by the primer pairs P16-Fr-2 (P16-Fr-3)/V-F (Supplementary Table 2). The corresponding promoter and vector fragments were assembled through POE-PCR, resulting in the 12 plasmids pGT2G, pGT2C, pGT3C, pRT2G, pRT2C, pRT3C, pOT2G, pOT2C, pOT3C, pKT2G, pKT2C and pKT3C (Supplementary Table 1). The sequences of these mutated plasmids were confirmed by DNA sequencing, and then transformed into B. subtilis WB600.
Determination of the transcription levels of egfp by quantitative reverse transcription-PCR (qRT-PCR)
To determine the promoter’s activity, total RNA was isolated from 18 h cultures of the transformed B. subtilis strains EGFP-WT, EGFP-T2G, EGFP-T2C and EGFP-T3C, harboring the recombinant plasmids Pylb-G-P43-R-pUBC19, pGT2G, pGT2C, and pGT3C, using a RNAprep PureCell/Bacteria Kit (TIANGEN Biotech, Beijing, China). The first strand of cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) with the gene-specific primers egfp-rtR and BS16S-rtR (Supplementary Table 2). The qRT-PCR was performed using the SYBR Green Real-time PCR Master Mix Plus (Toyobo, Osaka, Japan) with the primers egfp-rtF and egfp-rtR (Supplementary Table 2) for the egfp gene and primers BS16S-rtF and BS16S-rtR (Supplementary Table 2) for the 16SrDNA gene.
Determination of the expression levels of green fluorescent protein EGFP and red fluorescent protein mApple
To determine the yield of fluorescent protein, the overnight cultures of the recombinant B. subtilis strains were inoculated into a 96-well microtiter plate containing 200 μL LB liquid medium with 10 μg/mL kanamycin per well, and then incubated at 37 °C with shaking at 750 rpm in an incubator 1000 (Heidolph, Germany) for 20 h. Fluorescence intensity and cell density (OD600) were measured using a SpectraMax M2 (Molecular Devices, USA) microplate reader. The excitation/emission was read at 484/507 nm for EGFP and 562/598 nm for mApple. Samples were collected from 20 h cultures for SDS-PAGE analysis.
Heterologous protein expression and enzyme activity analysis
A fresh overnight culture of the recombinant B. subtilis strains OPHC2-WT, OPHC2-T2G, OPHC2-T2C, and OPHC2-T3C, KatA-WT, KatA-T2G, KatA-T2C, and KatA-T3C were inoculated into 50 mL LB liquid medium containing 10 μg/mL kanamycin and cultivated at 37 °C, at 200 rpm, for 28 h. Organophosphorus hydrolase (OPHC2) activities were determined as described previously (Yu et al. 2015). One unit of OPHC2 activity was defined as the amount of the enzyme required to liberate 1 μmol of p-nitrophenol per minute at 37 °C. For catalase activity, the reaction was performed as described previously (Goldblith and Proctor 1950) and one unit of catalase activity was defined as the amount of enzyme required to catalyze the decomposition of 1 μmol H2O2 per minute at 30 °C.
Construction and characterization of the Pylb random-scanning mutagenesis libraries
To identify the key elements that influence the promoter strength in the regions flanking the − 35 and − 10 regions of Pylb, six mutant promoter libraries, T1–T6, were constructed (Fig. 1a) and EGFP fluorescence was quantified using flow cytometry to determine the egfp expression levels. The T2 library had the highest RFI (18.7% increase), while the T3 library had the lowest RFI (67.0% decrease), relative to the wild type Pylb (Fig. 1b). Meanwhile, the highest CV was observed in the T3 library (Fig. 1b). These data suggested that the four nucleotides flanking the − 35 box of Pylb have the most critical influence on promoter strength and randomized mutations in the T3 locus of Pylb were likely to cause more obvious changes in the promoter strength.
Characterization of Pylb mutants collected from the T2 and T3 libraries
Characterization of the effect of the identified T2 and T3 locus signatures on the promoter activity
The above results supported that the extracted signature sequences in the T2 and T3 locus could differentially regulate the activity of the Pylb promoter.
The application prospect of the signature sequences
Promoters are important regulatory elements for expressing protein. To date, some high-quality native promoters have been widely used in B. subtilis for gene expression. However, most of the endogenous promoters in B. subtilis were not well explored and comprehensively analyzed. The promoter Pylb was found and proved to be a highly active promoter of B. subtilis in our lab (Yu et al. 2015). In this study, for better application of the promoter Pylb, the elements that influence its activity were researched.
One commonly used strategy for exploring promoter element is to randomly mutate within the whole promoter sequence. It is time-consuming and easily missing information. Our precious work shown that the promoter regions I (position − 38 to − 23, relative to TSS) and II (position − 18 to − 3, relative to TSS) that contain the − 35 and the − 10 regions were particularly important for the transcriptional activity of Pylb (Yu et al. 2015). Therefore, we focused on the sequence flanking the − 35 and the − 10 regions to identify and characterize elements, apart from the core elements, which influence the promoter strength. In our study, the sequence to be interested in were divided into six groups and each group contained four nucleotides (Fig. 1a), so that each random mutagenesis library potentially contained 256 mutant promoters. Meanwhile, the transformation efficiency of the WB600 strain was 768 cfu/μg. Thus, the transformants were predicted to cover more than 99% of the possible mutants. Together with the high-throughput screening by flow cytometry, our methodological approach could thoroughly explore signature elements with higher efficiency, as compared to the traditional methods of studying promoter activity by random mutagenesis within the whole promoter sequence. Thus it can be used to systematically determine the promoter sequence features which were influenced transcriptional activity in B. subtilis.
Previous studies have demonstrated that the spacer region between the − 35 and the − 10 box within the bacterial promoter is critical to regulate the expression levels of heterologous genes (Guiziou et al. 2016; Han et al. 2017; Jensen and Hammer 1998). Our work demonstrated that the GC content in the sequence flanking the − 35 box in the T2 locus enhanced the promoter activity, while the C content in the T3 locus reduced the promoter activity. However, the rationale for this influence in transcriptional activities is obscure. As the -35 element is responsible for recruiting RNA polymerase (Hawley and McClure 1983), the GC content in the four nucleotides flanking the − 35 box may impact RNA polymerase recruiting. This could involve higher-level regulatory effects like DNA looping (Cournac and Plumbridge 2013).
A promoter with a single characteristic is unlikely to satisfy each exogenous gene since, for example, strong overexpression is not always optimal for every given gene. We can see that, the strong promoters Pylb-T2G and Pylb-T2C weren’t suitable for the expression of catalase KatA. As the catalase KatA in B. subtilis is an extracellular enzyme without known signal peptide (Naclerio et al. 1995), it may be secreted via non-classical secretion pathways. However, the mechanisms of non-classical secretion are unidentified. We speculated that the catalase KatA expressed by the strong promoter could not be folded correctly in time, which affected the secretion, resulting in the decreased extracellular expression.
The T2 and T3 libraries included mutant promoters that induced variable transcriptional levels, ranging from high to low (Supplementary Fig. 4). The randomization of the four nucleotides flanking the − 35 box gave rise to dynamic expression that covered a wide range of levels, so every promoter with differential activity in the T2 and T3 libraries could be potentially used to express genes that require the corresponding transcriptional activity. Thus, the T2 and T3 libraries have great application potentials for enzyme production, metabolic engineering, and synthetic biology.
In conclusion, our thorough characterization of the Pylb sequence provides further information for the potential application of this promoter, and contributes to the enrichment of the B. subtilis promoter library. Particularly, the T2 and T3 libraries cover a wide range of promoters with dynamic activities which have great potential to be applied for highly effective tuning of gene expression and regulation of complex metabolic pathways.
This work was supported by the National Natural Science Foundation of China (NSFC, Grant no. 31500064).
Supplementary Table 1—Plasmids and strains used in this study.
Supplementary Table 2—Primers used in this study.
Supplementary Fig. 1—Cell sorting of B. subtilis mutant strains from the T2 and T3 randomly mutated promoter libraries.
Supplementary Fig. 2—The relative fluorescent intensity of EGFP driven by selected promoter variants of Pylb from the T2 and T3 libraries.
Supplementary Fig. 3—Sequences of the Pylb mutants collected from the T2 and T3 library.
Supplementary Fig. 4—Distribution of the expression levels of EGFP driven by promoter variants from the T2 and T3 libraries.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Bhavsar AP, Zhao X, Brown ED (2001) Development and characterization of a xylose-dependent system for expression of cloned genes in Bacillus subtilis: conditional complementation of a teichoic acid mutant. Appl Environ Microbiol 67:403–410. https://doi.org/10.1128/AEM.67.1.403-410.2001 CrossRefPubMedPubMedCentralGoogle Scholar
- Phan TT, Tran LT, Schumann W, Nguyen HD (2015) Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli. Microb Cell Fact 14:72. https://doi.org/10.1186/s12934-015-0255-z CrossRefPubMedPubMedCentralGoogle Scholar
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