The Sequence of the Ribosomal Binding Site Controls the Gene Expression in Brucella

Brucella is an important pathogen causing Brucellosis. Vaccine strains obtained by a single knockout cannot combine low virulence and immunogenicity. Our study modied the SD sequence and spacer sequence of the RBS of Brucella to affect its protein expression. We altered the RBS of LPS-associated genes to reduce LPS-associated protein expression while retaining LPS integrity. We rst established an evaluation system based on the reporter gene red uorescent protein mCherry. The mCherry expression could be changed by altering the Shine Dalgarno sequence and spacer sequence of RBS. After optimizing the Shine Dalgarno sequence, mCherry expression was increased 4-fold in E. coli and decreased by 1/4 in Brucella. The mCherry expression was increased 1.5-fold in E. coli and decreased to 1/2 in Brucella when the length of the spacer sequence was 0. When the spacer sequence was NA (N = 4, 8, 12nt) or NG (N = 4, 8, 12nt), mCherry expression was reduced in both E. coli and Brucella. Accordingly, two mutant strains were constructed in an attempt to decrease the expression of LptA and LpxO, Brucella LPS-related genes, by 1/4. Silver staining experiments of LPS SDS-PAGE revealed an alteration in the composition of LPS in the two mutant strains. Polymyxin B experiments revealed that both mutant strains were more sensitive to Polymyxin B resistance. Conclusion: In Brucella, the expression of the target gene could be affected by changing the length or the composition of the RBS sequence. The LPS gene remained unchanged while reducing the expression of its associated protein, achieving the original goal of reducing bacterial virulence while retaining immunogenicity. It is a promising strategy to improve the safety and ecacy of vaccines. Escherichia coli; B. subtilis: Bacillus subtilis; UTR: Untranslated region; LB: Luria bertani; B. abortus: Brucella abortus; B. melitensis: Brucella melitensis; TSA: Trypticase soy agar; TSB: Trypticase soy broth; Kan r : Resistance to kanamycin; Gm r : Resistance to gentamicin; Amp r : Resistance to ampicillin; BSC: Biological safety cabinet; DNA: Deoxyribonucleic acid; PCR: Polymerase chain reaction; RFU: relative uorescence units; OD: Optical density; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis; SD: standard deviation.


Background
Brucella is a Gram-negative bacterium that causes brucellosis, a widespread zoonotic disease [1][2][3][4][5]. By expressing different virulence factors and using various strategies to escape the host's immune response, Brucella is able to survive and replicate in host cells without being completely wiped out by the host, allowing Brucellosis to progress from an acute stage to chronic infection [6].
Lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, is an important virulence factor for Brucella [7]. LPS is involved in bacterial survival in many hosts, contributing to complement and bactericidal peptide resistance and to bacterial adherence and entry into cells [8]. LPS contains a marked pathogen associated molecular pattern (PAMP) that can be recognized by immune cells and body uids. It also triggers speci c antibody responses and protective immunity against pathogens [9]. At the same time, LPS provides a permeable barrier that makes an important contribution to the structural integrity of the bacteria, which is linked to their viability. LPS is constituted by a hydrophobic lipid A linked to a short, usually negatively charged, oligosaccharide core followed (smooth LPS) or not (rough LPS) by a hydrophobic O-polysaccharide (O-chain or Oantigen) [7]. However, some species of Brucella are of weak virulence. Bacterial virulence can be altered by deleting certain genes involved in the LPS modi cation regulatory pathway [7,10]. However, this knockdown approach also reduces the immunogenicity of the strain, thus weakening the protective effect of the vaccine. Therefore, reducing virulence and retaining some immunogenicity by decreasing the protein expression of LPS-related genes would be an effective vaccine design attempt.
Gene expression requires e cient translation initiation. The initiation process is characterized by the formation of the 30S initiation complex, including the 30S ribosomal subunit, three initiation factors, the fMet-tRNA, and the mRNA at the ribosomal binding site (RBS) [11]. In bacteria, proteins are synthesized by ribosomes according to instructions encoded by mRNA sequences [12][13][14][15] . RBS affects the translation process [16]. The non-coding regions of mRNA, especially the 5'-untranslated region (5'-UTR), contribute signi cantly to the e ciency of protein synthesis [17,18]. The most important 5'-UTR sequence elements in bacterial mRNAs were Shine-Dalgarno (SD) sequences, the 16S rRNA binding sites, and the spacer between the target gene start codon and the SD sequence [19,20]. The same RBS sequence in different genetic backgrounds can also lead to large differences in protein expression levels.
The folding of transcripts into secondary structures in the RBS region is critical for the initiation of translation. Stable structure in this region may prevent ribosomes from entering the transcripts and reduce the rate of translation initiation [21,22]. The spacer between the start codon and the SD sequence is a component of the RBS and is therefore expected to affect the folding of transcripts in this region [23][24][25][26][27]. The optimum distance between the SD cassette and the start codon was 5-9 nt in E. coli [28,29] and 7-8 nt in B. subtilis [30]. It is demonstrated that the composition of the spacer sequence has a signi cant effect on the expression yield. The expression level is determined not only by the length but also by the different nucleotide composition of the spacer bars [30].
Bacterial protein synthesis systems are one of the main targets of biotechnological modi cation. The functional evaluation of protein 5'-UTR sequence elements is usually performed by monitoring the expression of their downstream reporter gene [31]. In this study, the mCherry reporter gene was introduced into Brucella genome in order to analyze how much the spacer sequence of RBS affects gene expression in Brucella, based on our experience of E. coli. It was con rmed that the number of bases and base composition of the RBS spacer in Brucella affects the expression of the downstream reporter gene mCherry. The patterns were applied to the RBS of Brucella LPS-related genes. They caused a reduction in the expression level of the Brucella LPS-related genes and thus hampered the integrity of LPS.

Results And Discussion
Construction of standard expression plasmids SecE is a non-essential protein translocase from Brucella abortus 2308 vaccine strain 104M. The expression of SecE is controlled by a strong Brucella spp. promoter called PsojA [32,33]. The RBS sequence of SecE gene is GAGGTTGTGCCTGATCAGA constituted by the SD sequence GAGGTTGTGCCTGAT and the spacer sequence CAGA. The mCherry protein is a red uorescent protein [34]. Accordingly, an RBS alteration evaluation system based on mCherry red uorescent protein was established to determine the effect of RBS alteration on Brucella gene expression. Sequentially ligate the mCherry gene, the homologous arm of SecE and Kan resistance by Overlap PCR. Clone the ligation onto the pBM16A T vector and the acquired new plasmid was named as pMCH. The 5′-anking region of SecE gene contained PsojA and RBS sequence of SecE gene. The pMCH is a shuttle plasmid. It not only contains the origin of E. coli, but also is able to recombine in Brucella. Transform pMCH into E. coli, and the new strain was named E. MCH. Integrate pMCH into the genome of Brucella 104M to replace the SecE gene, and the new strain was named B. MCH (Fig. 1a). The SD sequence of mCherry is GAGGTTGTGCCTGAT and the spacer sequence is CAGA in E. MCH and B. MCH. Due to the expression of mCherry protein, E. MCH and B. MCH were observed to be red by naked-eye, uorescence microscope and multi-function imager. Red arrow indicates one colony. (Fig. 2). B. MCH and E. MCH transformants emitted bright red uorescence under laser confocal microscopy. These uorescence were distributed throughout the cells as small red dots, whose position and size were consistent with the strain. It is noteworthy that the red uorescence of the wild strain of E. coli is brighter than that of Brucella. This may be due to the fact that mCherry is present in E. coli as a multi-copy plasmid, while in Brucella it is present as a single copy integrated into the genome.

SD sequences in uence the expression of mCherry
Replace the SD sequence GAGGTTGTGCCTGAT of mCherry with AAAGGAGGT, and the new plasmid was named as pMCH1. Transform pMCH1 into E. coli, and the new strain was named E. MCH1. Integrate pMCH1 into the genome of Brucella 104M to replace the SecE gene, and the new strain was named B. MCH1 (Fig. 1b).
We rst observed qualitatively the intensity of uorescence by confocal microscopy. The red uorescence intensity of E. MCH1 was increased compared to E. MCH (Fig. 3a). On the other hand, B. MCH1 caused a decrease in the uorescence intensity compared to B. MCH (Fig. 3b). Then the uorescence intensity of mCherry was quantitatively analyzed by a multi-functional microplate reader. The uorescence intensity of E. MCH1 increased to 432.235% of E. MCH's and B. MCH1 decreased to 74.964% of B. MCH's, respectively (Fig. 3c, 3d). This may be due to the different nature in translation levels between E. coli and Brucella. The differences of red uorescence intensity indicated the differential expression of mCherry protein. Thus, the changes of SD sequence in uenced the expression of mCherry.

Veri cation of the effect of RBS with different spacer length on gene expression
It was shown that the expression level is determined not only by the length of the spacer sequence, but also by the different nucleotide composition of the spacer sequence [35]. The preliminary results of this experiment showed that the expression level of mCherry gene was not upregulated in Brucella after changing the optimal SD sequence. To determine the effect of spacer sequence of gene expression in Brucella, subsequent experiments were designed to modify the spacer sequence of RBS. The uorescence intensities of B. MCH and E. MCH were used as negative controls.  (Fig. 1b). Their red uorescence intensities were observed qualitatively and quantitatively.
The differences in uorescence intensity were rst observed by confocal microscopy. E. MCH4G caused a decrease in the uorescence intensity and E. MCH0 was increased compared to E. MCH. Meanwhile, B. MCH0 caused a decrease in the uorescence intensity compared to B. MCH. The uorescence of other strains was not obvious (Fig. 4a, 4b).
Then the uorescence intensities of mCherry were analyzed by a multi-functional microplate reader. The uorescence intensity of E. MCH0 increased to 156.693% of E. MCH's. But E. MCH4G, E. MCH8A, and E.
MCH12A showed decreased to 82.572%, 7.772%, and 5.879% of E. MCH's, respectively. And the uorescence intensities of the other strains were reduced to 0 (Fig. 4c). What's more, the uorescence intensity of B. MCH0 and B. MCH4G decreased to 52.532% and 25.135% of B. MCH's. The uorescence intensities of the other strains were reduced to 0 (Fig. 4d).
The results of uorescence intensity observed by laser confocal microscopy and microplate reader were consistent. These experiments demonstrated that the composition and length of RBS spacer sequence had an in uence on gene expression in Brucella.

RBS in uence the expression of Brucella LPS
After obtaining the relationship between RBS and gene expression in Brucella abortus strain 104M, to further explore the role of RBS alterations in different Brucella species, the experiment was subsequently validated in a Brucella Melitensis 16M wild strain. Previous studies on Brucella related genes were performed by knockout mutations of a single gene. The strategy reduced the virulence of Brucella, but also reduced the immunoprotective effect as well. In this study, the genetic integrity of the strain was preserved, and in order to retain some immunoprotective effects while reducing some virulence, the RBS designing which retains 3/4 of protein expression, was chosen to modify Brucella LPS-related genes.
LptA and LpxO, were closely related to the synthesis of LPS lipid A. It was shown that when two Brucella LPS genes, LptA and LpxO, were knocked out separately, the mutant strain was less virulent and more sensitive to Polymyxin B as LPS integrity reduced [10]. Therefore, LptA and LpxO were chosen for this study, and the obtained mutant strains could be tested by Polymyxin B.
The gene LptA and LpxO were changed by an overlap extension PCR in the same way as described for pMCH. The RBS of the LptA gene was altered and inserted kan r and the plasmid was named pA1 (Fig.   1c). The RBS of the LpxO gene was altered and inserted Gm r and the plasmid was named pO1 (Fig. 1d).
Since LPS is the product of multiple genes, we designed single-gene mutations and double-gene combination mutations in order to study the effect of RBS alteration of genes on LPS. After the transformation of Brucella 16M, two clones were screened on resistance plates, named B. A1 and B. A1O1.
To  (Fig. 6). The mutant strains were more sensitive to Polymyxin B than wild strain B. melitensis 16M.
In summary, the results of silver staining and Polymyxin B sensitivity test showed that B. A1 and B. A1O1 strains had weaker LPS integrity and drug resistance than wild strains. Preliminarily, the B. A1 and B. A1O1 mutant strains might be weak virulent strains of Brucella 16M strain. The virulence and immunogenicity of these three mutant strains will be further demonstrated by animal immunization experiments in future studies. This will further illustrate the relationship between RBS and bacterial virulence and immunogenicity.

Conclusions
Genetic modi cation is essential for Brucella studies. Modifying RBS is an effective way to alter gene expression. Our study evaluated the spacer sequence between SD sequence and the start codon to alter Brucella gene expression [21]. In this study, mCherry red uorescent protein was used to exhibit visually the effect of RBS on Brucella gene expression. Accordingly, an RBS alteration evaluation system based on mCherry red uorescent protein was established to determine the effect of RBS alteration on Brucella gene expression. Brucella strains with altered LPS gene expression was obtained after RBS transformation of Brucella LPS-related genes. In Brucella, the expression of the target gene could be affected by changing the length or the composition of the RBS sequence. The LPS gene remained unchanged while reducing the expression of its associated protein, achieving the original goal of reducing bacterial virulence while retaining immunogenicity. It is a promising strategy to improve the safety and e cacy of vaccines.

Materials And Methods
Strains and plasmids E. coli strains were routinely cultured in Luria Bertani (LB) broth or agar at 37°C. B. abortus vaccine strain 104M and B. melitensis strain 16M were cultured in trypticase soy agar (TSA) or trypticase soy broth (TSB) (BD, USA) at 37°C for 2-3 days. When mutation screening was required, Kan r or Gm r was added to the culture medium at a nal concentration of 50 μg/ml. Other strains were obtained as shown in Table 1.
Experimental operations related to Brucella 104M and 16M were performed in a Class Biological Safety Cabinet (BSC). The plasmids used in this paper were shown in Table 1.

Prediction of RBS sequence using Bioinformatics analysis
The Ribosome Binding Site (RBS) Calculator can predict RBS sequences of a protein coding sequence in bacteria to rationally control the translation initiation rate. This tool was used to analysis the RBS sequence of Brucella. It is available at https://www.denovodna.com/software/ [36].

Construction of standard expression plasmids
To generate the standard expression plasmids, approximately 1 kbp of 5′-anking region and 3′-anking region of the SecE gene were ampli ed by PCR using the Brucella 104M genomic DNA as the template.
The Kanamycin resistance gene (Kan r ) cassette was PCR ampli ed from pMAZ-SK plasmid [37]. The red uorescence protein (mCherry) cassette was PCR ampli ed from pTREX-mCherry plasmid [38,39]. PCR was performed using PrimeSTAR high delity DNA polymerase (Takara, Japan). The PCR fragments were fused by overlap extension PCR and cloned into the pBM16A T vector Biomed, China for sequencing. The recombinant vectors were designated pMCH. The plasmids were veri ed by PCR and automated sequencing analysis. The primers are shown in Table S1. The plasmid pMCH1, pA1, and pA1O1 was constructed in the same way (primers are shown in Table S1).

Construction of different spacer sequence lengths of plasmids
The ampli ed fragment includes an RBS sequence that was ampli ed as A III / PstI fragments using the primers SP1 and SP2 (Table S1) from the pMCH1 plasmid. The spacer sequence CAGA of A III / PstI fragments was replaced by NA (N = 0, 4, 8, 12nt) or NG (N = 0, 4, 8, 12 nt)) using A III and MscI restriction enzymes with the oligos Sp3-Sp16 (Table S1) to obtain fragments with different spacer sequences.
Subsequently, the pMCH1 plasmid was hydrolyzed using the restriction enzymes A III and PstI, and fragments with different spacer sequences were ligated into the pMCH1 plasmid. The acquired plasmids series contains different spacer sequences.

Transformation of E. coli and Brucella
The target fragments were cloned into a pBM16A T vector (Biomed, China). The plasmids were transformed into chemical competent DH5α. Pick up the single colony for sequencing (Biomed, China) (The E. coli strain obtained is shown in Table 1 for comparison with Brucella). The plasmid DNA was isolated and transformed into Brucella 104M or 16M electro-transformed competent, and was screened for resistance. By using the homologous recombination function of Brucella itself, the homologous parts of the plasmid were integrated into the chromosome, and different mutants were constructed (The Brucella strain obtained is shown in Table 1).

Confocal microscope observation
Strains were harvested through centrifugation for 1 min at 12,000rpm. The sediment was suspended in 4% formaldehyde for immobilizing the cell for 20min. Subsequently, the sediment was resuspended in PBS. Preparing agarose pads and imaging bacteria using agarose pads [40,41]. Image the xed bacteria using agarose pads during the imaging process, allowing to perform color uorescence microscopy experiments. A Zeiss LSM880 laser scanning confocal microscope (Zeiss, Germany) was used to examine the red uorescent of the transformants.

Detection of uorescence intensity
This approach was designed to investigate the effect of changing RBS on the uorescence intensity of Brucella. The relative uorescence units (RFU) of bacteria were measured using a multi-functional microplate reader (Omega FLUOstar). To reduce the error caused by the concentration of the bacterial solution, the OD 600 of the bacterial solution was measured. Accordingly, the relative uorescence intensity was expressed as RFU/OD 600 for the bacterial solution. For samples, a PBS well was used as a control.
Silver staining analysis of Brucella LPS LPS was obtained by LPS Extraction kit (iNtRON Biotechnology, South Korea) and was quanti ed by LPS ELISA kit (Beijing Yanbidin technology, China). Puri ed LPS was separated in 12.5% SDS-PAGE gels, and the gels were silver stained by Protein Stains Q (Sangon Biotech, China).

Sensitivity of Brucella to Polymyxin B
Strains were harvested through centrifugation for 1 min at 12,000rpm. The sediment was resuspended in PBS. The nal concentrations of 50, 100, 200, and 500μg/mL Polymyxin B were added into bacteria liquid of Brucella cultured for 1 to 5 h at 37°C and 200 rpm. Then, 100μL of the diluted solution was plated on the TSA agar medium, and the plates were incubated at 37°C for 72 h. Calculate of the survival rates of mutant strains under the pressure of Polymyxin B.

Statistical analysis
All results were presented as mean ± standard deviation (SD). Statistical analysis was performed using Prism 9.0 (GraphPad Software, CA, USA). T student test and one-way analysis of variance (ANOVA) with Dunnett's correction were applied. P-values were indicated as ns 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicating the signi cant difference. All experiments were repeated at least three times.  Figure 1 Nucleotide composition of RBS of plasmids. (a)The expression of target gene (black) was controlled by a strong Brucella spp. promoter called PsojA (purple). SD sequence (blue), spacer (green) and start of SecE (grey) were highlighted. The mCherry gene (red) was integrated into the genome of Brucella 104M to replace the SecE gene, and the new strain was named B. MCH; (b) Nucleotide composition of RBS of plasmids pMCH, pMCH1, pMCH0, pMCH4A, pMCH4G, pMCH8A, pMCH8G, pMCH12A, pMCH12G. SD sequence (blue), spacer (green) and start of target gene (red) were highlighted. The cleavage sites of labeled A III, Msc , and Pst were used to construct RBS of different spacer sequences; (c) SD sequence (blue), spacer (green) and start of LptA (yellow) were highlighted. The SD sequence (blue) aacgccagattgaaagccc was replaced by AAAGGAGGT, and the new plasmid was named pA1; (d) SD sequence (blue), spacer (green) and start of LpxO (yellow) were highlighted. The SD sequence (blue) aaagagca was replaced by AAAGGAGGT, and the new plasmid was named pO1.   MCH12G.

Figure 5
The picture of SDS-PAGE gel of silver staining. The LPS, after extracted from the Brucella mutant strains, was visualized by silver staining. LPS consists of O-antigen, Core, and Lipid A. The breadth and color of