www.nature.com/scientificreports OPEN The development of fluorescent protein tracing vectors for multicolor imaging of clinically Received: 16 November 2016 isolated Staphylococcus aureus Accepted: 20 April 2017 Published: xx xx xxxx Fuminori Kato, Motoki Nakamura & Motoyuki Sugai Recent advances in fluorescent protein technology provide a wide variety of biological imaging applications; however current tools for bio-imaging in the Gram-positive bacterium Staphylococcus aureus has necessitated further developments for fluorescence intensity and for a multicolor palette of fluorescent proteins. To enhance the expression of multicolor fluorescent proteins in clinical S. aureus strains, we developed new fluorescent protein expression vectors, containing the blaZ/sodp promoter consisting of the β-lactamase gene (blaZ) promoter and the ribosome binding site (RBS) of superoxide dismutase gene (sod). We found S. aureus-adapted GFP (GFP ) driven by the blaZ/sodp sa promoter was highly expressed in the S. aureus laboratory strain RN4220, but not in the clinical strains, MW2 and N315, harboring the endogenous blaI gene, a repressor of the blaZ gene promoter. We therefore constructed a constitutively induced blaZ/sodp promoter (blaZ/sodp(Con)) by introducing substitution mutations into the BlaI binding motif, and this modification allowed enhanced expression of the multicolor GFP variants (GFP , EGFP, mEmerald, Citrine, Cerulean, and BFP) as well as codon- sa optimized reef coral fluorescent proteins (mCherry and AmCyan) in the S. aureus clinical strains. These new fluorescent probes provide new tools to enhance expression of multicolor fluorescent proteins and facilitate clear visualization of clinical S. aureus strains. Fluorescent proteins are widely used as biological markers that enable visualization of subcellular protein locali- 1–3 zation, gene expressions, protein-protein interactions, and in vivo monitoring of bacterial infection . Currently available uo fl rescent proteins are primarily derived from either the green uo fl rescent protein (GFP) originating 4, 5 5–7 in the jellys fi h Aequorea victoria , or reef coral uo fl rescent proteins (RCFP) derived from Discosoma sp . The wild type A. victoria GFP exhibits poor fluorescent brightness in Escherichia coli and mammalian cell lines; and many of the wild type GFP have a strong tendency to be expressed as an insoluble protein, showing cytotoxicity 8, 9 in E. coli . To date, extensive studies have reported numerous types of A. victoria GFP variants that provide sig- 4, 5, 9 nic fi ant improvements in brightness, protein solubility, stability, pH-sensitivity, and yield . Further, numerous variants of GFP and RCFP with distinct colors have been engineered using a combination of random mutagenesis and directed evolution. This has enabled co-visualization of several proteins in a single cell, selective identification of particular cells in co-culture systems, and detection of protein-protein interactions based on a measurement of 1–5 uo fl rescence resonance energy transfer (FRET) . Staphylococcus aureus is a low-GC Gram-positive bacterium that causes a variety of diseases, e.g., abscess, bullous impetigo, toxic shock syndrome, pneumonia, sepsis, and food poisoning. Multidrug-resistant strains such as methicillin-resistant S. aureus (MRSA) cause severe hospital-acquired infections such as pneumonia and sepsis; and the resistance makes the treatment increasingly difficult. The development of molecular genetic tools including gene deletion, controllable gene expression, and bio-imaging is essential for our understanding of the 10, 11 mechanisms underlying the pathogenesis of S. aureus infections . Currently, for bio-imaging numerous GFP and RCFP variants are commercially available from many distributors; however, these FPs were not optimized for less common bacterial strains (e.g., clinical strains of S. aureus). Bio-imaging tools based on fluorescent proteins have also been widely used in the studies of S. aureus to visualize subcellular proteins or for biofilm formation as Department of Bacteriology, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima, 734-8551, Japan. Correspondence and requests for materials should be addressed to M.S. (email: sugai@ hiroshima-u.ac.jp) Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 1 www.nature.com/scientificreports/ Figure 1. Replacement of RBS sequence and introduction of Cycle3 mutations into GFPmut3. (a) Sequence alignment of the blaZp and its derivative blaZ/sodp promoters. Blue bold face indicates the surrounding sequence containing RBS derived from the sod gene. Underlined sequences indicate −35 and −10 elements of the blaZ promoter. Double underlines indicate the RBS sequence. Initiation codons are shown in uppercase. (b) The fluorescing colonies were photographed under UV excitation. S. aureus RN4220 containing pS1GFP, pFK51, and pFK52 were grown on TSB agar plate containing chloramphenicol. (c) The fluorescent intensities of GFPmut3b, and GFP in S. aureus RN4220. Cell homogenate of S. aureus containing pS1GFP, pFKS1 sa and pFK52 was prepared and the fluorescent intensities at 513 nm were measured with a microplate reader, λ = 490 nm. The data represent mean values ± standard deviation. (d) The expression efficiency of pS1GFP, ex Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 2 www.nature.com/scientificreports/ pFK51, and pFK52 in S. aureus RN4220. SDS-PAGE and Western blot analysis show the relative quantities of the GFP in the whole cell lysates. The Western blot gel was cropped and the full-length image is included in Supplemental Fig. 5(a). (e) Excitation and (f) Emission spectra for GFPmut3b and GFP in S. aureus RN4220. sa Each spectrum was normalized to a maximum value of 1. Excitation spectra were recorded with emission at 540 nm. Emission spectra were recorded with excitation at 460 nm. GFPmut3b and GFP were depicted by a sa solid line and dotted line, respectively. (g) S. aureus RN4220 containing pMK4blaZ/sodpGFP (pFK52) was sa grown on TSB containing chloramphenicol and visualized using an FV1000 confocal scanning laser microscope (Olympus). Panels show (1) DAPI, (2) GFP, (3) overlay of DAPI and GFP images, (4) differential interference contrast (DIC), (5) overlay of DAPI, GFP, and DIC images. well as to establish a S. aureus infection model using fluorescent proteins, in which optimization of codon usage and replacement of the region surrounding the ribosome binding sequence (RBS) have been reported to enhance 12–16 the expression of the fluorescent proteins in S . aureus . However, the current methods often entail several limitations as to color palette and its brightness and therefore necessitated the development of new fluorescent vectors that can efficiently enhance fluorescence intensity and the multicolor palette in S . aureus strains. Many proteins are generally difficult to highly express in heterologous host organisms. In E. coli, extensive research has established many die ff rent types of promoters and a great number of engineered laboratory strains for heterol - ogous protein production . However, a well-established method for the heterologous protein expression is still lacking in S. aureus, much less clinically isolated strains. Previous studies have focused on the β-lactamase gene (blaZ) promoter P for the g fp expression in S. aureus. Notably, the GFPmut2 was constitutively expressed in blaZ 18–20 the laboratory strain RN4220 . This study therefore developed fluorescent protein expression vectors with the P promoter to highly express GFP and RCFP variants in clinically isolated S. aureus. blaZ Here, we describe novel fluorescent protein expression vectors, which were shown to exhibit greater fluores - cence intensity in clinical S. aureus strains. To improve the expression of an exogenous fluorescent protein in S . aureus clinical strains, we used the blaZ/sodp(Con) promoter and codon-optimized fluorescent protein genes, and showed that the fluorescence intensity in the series of GFP and RCFP variants were significantly enhanced in the clinical strains. These new tools efficiently expressing fluorescent proteins in clinical S . aureus strains are valuable for understanding the pathogenic mechanism of S. aureus. Results Adaptation of the green fluorescent protein to S. aureus. We aimed to develop fluorescent pro- tein expression vectors that can produce sufficient fluorescence intensity to visually identify fluorescing colonies of S. aureus on agar plates with the naked eye. S. aureus strain RN4220 colonies containing pS1GFP in which GFPmut3b was expressed under the control of the β-lactamase gene (blaZ) promoter exhibited faint fluorescence on TSB agar plates (Fig. 1b). When we replaced the 13-bp sequence containing RBS sequence of the blaZ gene with the corresponding region of the superoxide dismutase gene (sod) (Fig. 1a) that has been reported to enhance the expression of fluorescent protein in S. aureus , the fluorescent activity of the colonies expressing GFPmut3b was significantly improved on the agar plates (Fig. 1b). Further, the fluorescence intensity increased by an approx- imately 10-fold using the microplate assay reader (Fig. 1c). The high expression of GFPmut3b was confirmed as a major protein band corresponding to its molecular weight, 26.8 kDa, in SDS-PAGE (Fig. 1d). Although the GFPmut3b is one of the faster folding GFP variants that has been optimized for bacteria and has minimal toxic- ity , overexpression of GFPmut3b in S. aureus showed adverse effects such as cell growth inhibition, producing smaller colonies, and frequent co-occurrence of larger colonies exhibiting no u fl orescence signal (Fig. S1a). To overcome these adverse effects, GFPmut3b was modified by introducing Cycle3 mutations (F99S/M153T/V163A) 9, 22 that are known to improve the solubility and reduce the toxicity , yielding S. aureus-adapted GFPmut3b, GFP sa (S65G/S72A/F99S/M153T/V163A). As a result, the mutations antagonized the appearance of non-fluorescent colonies and maintained high levels of the fluorescent intensity (Figs 1b,c and S1b) and GFP expression (Fig. 1d) sa without ae ff cting its excitation and emission wavelengths (Fig. 1e,f ). Further, confocal laser scanning microscopic analysis revealed that almost all of S. aureus cells expressed GFP protein and were clearly visualized (Fig. 1g). sa Introducing suppressor mutations into the blaZ gene promoter. S. aureus-adapted GFP demon- sa strated higher expression under the control of the hybrid blaZ/sodp promoter in laboratory strain RN4220. However, in clinical strains, MW2 and N315, with the same vector, the expression of GFP was very low, and the sa 23, 24 uo fl rescence intensity was poor (Fig. 2b,c,d). BlaZ plasmid is prevalent in clinical strains , and the blaIRZ gene 25–27 operon is natively present on a plasmid in both clinical strains, MW2 and N315, but not in RN4220 . The BlaI and MecI specifically bind to the same dyad symmetry (TACA/TGTA) sequence and repress the blaZ promoter activity (Fig. 2a) . To minimize the possible negative effect by the endogenous BlaI/MecI on the blaZ /sodp pro- moter activity, we introduced suppressor mutations into the BlaI/MecI binding motif (TACA/TGTA), yielding a constitutively induced blaZ/sodp(Con) promoter (Fig. 2a). As a consequence, GFP expression was significantly sa elevated, and the resulting fluorescence intensity was highly improved in both N315 and MW2, whereas the uo fl rescence intensity was slightly reduced in strain RN4220, compared with the parental sequence (Fig. 2b,c,d). Taken together, the data demonstrated the modified blaZ/sodp promoter, (blaZ/sodp(Con)) can effectively enhance the expression of GFP in S. aureus clinical strains. sa Expression of multicolor GFP variants in clinical strains. A. victoria GFP and its variants contribute to multicolor imaging with spectral profiles ranging in color from blue to yellow, and have been engineered by 29–34 site-directed mutagenesis . Using GFP as the standard, we next constructed these GFP color variants (EGFP, sa Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 3 www.nature.com/scientificreports/ Figure 2. Site-directed mutagenesis into the BlaI/MecI binding sequence. (a) Sequence alignment of the blaZ/sodp and its constitutively induced blaZ/sodp(Con) promoter region. Asterisks indicate the sequence exchanged by site-directed mutagenesis. Bold face indicates the BlaI/MecI binding motif (TACA/TGTA) within the larger palindromes, the R1 dyad and Z dyad are indicated by arrows. Up arrows with the tip to right indicates the transcription initiation site. Underlined sequences indicate −35 and −10 elements. Double underline indicates the RBS sequence. Initiation codons are shown in uppercase. (b) The fluorescing colonies were photographed under UV excitation. S. aureus RN4220, MW2 and N315 containing either pFK52, or pFK54 were grown on TSB agar plate containing chloramphenicol. (c) The comparison of fluorescence intensity among S. aureus strain RN4220, MW2, and N315 containing either pFK52, or pFK54. The fluorescent intensities at 513 nm were measured with a microplate reader, λ = 490 nm. The data represent mean ex values ± standard deviation. (d) The comparison of GFP expression efficiency among S. aureus strain RN4220, sa MW2, and N315 containing either pFK52, or pFK54. SDS-PAGE and Western blot analysis show the relative quantities of GFP in the whole cell lysates. The arrow indicates the position of GFP in gel. The Western blot sa sa gel was cropped and the full-length image is included in Supplemental Fig. 5(b). Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 4 www.nature.com/scientificreports/ Figure 3. Detection of multicolor GFP variants in the clinical strain, MW2. (a) The fluorescing colonies were photographed using UV excitation. S. aureus strain MW2 expressing multicolor GFP variants (GFP , EGFP, sa mEmerald, Citrine, Cerulean, and BFP) were grown on TSB agar plate containing chloramphenicol. (b) The whole cell lysates of S. aureus MW2 expressing multicolor variants used for SDS-PAGE and Western blot analysis were photographed under UV excitation. (c) The comparison of expression efficiency among multicolor GFP variants in S. aureus strain MW2. SDS-PAGE and Western blot analysis show the relative quantities of the multicolor GFP variants in the whole cell lysates. The arrow indicates the position of GFP variants in gel. The Western blot gel was cropped and the full-length image is included in Supplemental Fig. 5(c). mEmerald, Citrine, Cerulean, and BFP) using amino acid substitutions considering the codon usage patterns in S. aureus. Since several BFP variants were engineered using substitution of the T66H mutation; we constructed three BFP variants, EBFP (F64L/S65T/Y66H/Y145F), 1EMF (F64L/Y66H/V163A), and P4–3E (F64L/Y66H/Y145F/ V163A) and evaluated which BFP variants would be more suitable for S. aureus MW2 and N315. Fluorescence assay showed that homogenates of P4-3E and EBFP exhibited somewhat greater fluorescence intensity than 1EMF when S. aureus MW2 were grown in TSB medium (Fig. S2a), while only S. aureus expressing P4-3E was visually identie fi d as uo fl rescing colonies on agar plates by the naked eye (Fig. S2b). Each strain expressing the multicolor GFP variant exhibited sufficient fluorescence intensity to visually identify fluorescing colonies by the naked eye (Fig. 3a), and SDS-PAGE and Western blot analyses showed that like GFP all of these multicolor variants were sa expressed with high yield in S. aureus MW2 harboring the corresponding vectors with the blaZ/sodp(Con) pro- moter (Fig. 3b,c). The multicolor GFP variant genes driven by the blaZ /sodp(Con) promoter also exhibited high uo fl rescence intensity in another clinical strain, N315 (Fig. S3a). Further, fluorescence excitation and emission spectrum analysis showed these strains expressing the multicolor GFP variants reflected the previously published spectrum profiles (Fig. S4). This data indicated that multicolor GFP variants were highly expressed in clinical strains MW2 and N315 with the blaZ/sodp(Con) promoter. Codon optimization of AmCyan and mCherry for S. aureus. Like Aequorea GFP, reef coral uo fl rescent proteins (RCFP) and their variants also contribute to multicolor imaging with spectral profiles ranging in color 1, 5 from cyan to far red . We constructed the RCFP expression vectors for S. aureus clinical strains, in which the amCyan or mCherry gene was expressed under the control of the hybrid blaZ/sodp(Con) promoter. Unexpectedly, S. aureus expressing the commercially available amCyan or mCherry gene formed faintly fluorescent colonies on agar plates (Fig. 4a), we therefore optimized the codon usage of amCyan and mCherry genes by replacing rare GC- rich codons with AT-rich ones. As a consequence, the codon-optimized AmCyan (AmCyan(S.a)) and mCherry (mCherry(S.a)) exhibited sufficient fluorescence intensity to visually identify fluorescing colonies on agar plates by the naked eye (Fig. 4a), and the corresponding proteins were confirmed to their molecular weights, 25.2 kDa and 26.7 kDa, respectively (Fig. 4b) and detected by Western blot (Fig. 4c). The fluorescence assay shows the codon adapted genes resulted in increased fluorescence intensity of AmCyan and mCherry (Fig. 4d,e). The amCyan and mCherry genes driven by the blaZ/sodp(Con) promoter also exhibited a significant uo fl rescence intensity in strain N315 (Fig. S3b). Further, fluorescence excitation and emission spectrum analysis showed these strains expressing AmCyan or mCherry reflected previously published spectrum profiles (Fig. S4). Taken together, the expression of AmCyan and mCherry were significantly improved in S. aureus clinical strains. Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 5 www.nature.com/scientificreports/ Figure 4. Codon usage optimization of the amCyan and mCherry genes. (a) The fluorescing colonies were photographed under UV excitation. S. aureus strain MW2 containing pKAT (control), pFK62 (AmCyan), pFK64 (AmCyan(S.a)), pFK63 (mCherry), and pFK65 (mCherry(S.a)) were grown on TSB agar plates containing chloramphenicol. (b) SDS-PAGE analysis showed the relative quantities of AmCyan and mCherry in the whole cell lysates. Arrows indicate the position of AmCyan and mCherry in gel. (c) Western blot analysis of AmCyan or mCherry in the whole cell lysates. The Western blot gels were cropped and the full-length image are included in Supplemental Fig. 5(d). The comparison of fluorescent intensities of pKAT (control), pFK62 (AmCyan), and pFK64 (AmCyan(S.a)) in S. aureus MW2. The fluorescent intensities at 489 nm were measured with a microplate reader, λ = 458 nm. The data represent mean values ± standard deviation. (e) The ex comparison of fluorescent intensities of pKAT (control), pFK63 (mCherry), and pFK65 (mChaerry(S.a)) in S. aureus MW2. The fluorescent intensities at 610 nm were measured with a microplate reader, λ = 586 nm. e Th ex data represent mean values ± standard deviation. Identification of S. aureus cells expressing a specific fluorescent protein in co-culture sys- tems. We next aimed to discriminate S. aureus cells expressing a specific fluorescent protein from the co-existence of bacteria expressing different fluorescent proteins in a co-culture experiment. Confocal laser scan- ning microscopic analysis revealed that each S. aureus clinical strain expressing different fluorescent proteins was selectively detected in the co-culture system in which Cerulean, Citrine, and mCherry(S.a) were differently expressed in the strain N315, TY34 , and MW2, respectively (Fig. 5a). Further, S. aureus N315 cells expressing mCheery(S.a) were clearly distinguished from E. coli DH5α cells expressing EGFP (Fig. 5b). These results demon- strated that our fluorescent protein expression vectors have the potential to sensitively detect particular S. aureus cells in co-culture with different strains or other bacteria. Discussion In this study, we developed new fluorescent protein vectors to express a bright fluorescence intensity enough to visually identify fluorescent colonies of S . aureus macroscopically on agar plates. Practically available high-level expression of fluorescent protein has not been achieved in previous studies in which fluorescent proteins were 10–14 detected only with fluorescence microscopy . Our findings provide new applications to enhance the expression Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 6 www.nature.com/scientificreports/ Figure 5. Confocal laser microscopic analysis in co-culture systems. (a) Co-culture of three clinically isolated S. aureus strains. S. aureus strain N315 containing pFK60 (Cerulean), strain TY34 containing pFK56 (Citrine), and strain MW2 containing pFK65 (mCherry(S.a)) were co-cultured in BHI broth. Panels show (1) Cerulean (N315), (2) Citrine (TY34), (3) mCherry(S.a) (MW2), (4) overlay of Cerulean, Citrine, and mCherry images, (5) DIC, (6) overlay of Cerulean, Citrine, mCherry, and DIC images. (b) Co-culture of S. aureus with E. coli. S. aureus N315 containing pFK65 (mCherry(S.a)) and E. coli DH5α containing pFK55 (EGFP) were co-cultured in BHI broth. Panels show (1) DAPI, (2) EGFP, (3) mCherry(S.a), (4) DIC, (5) overlay of DAPI and DIC images, and (6) overlay of EGFP, mCherry(S.a), and DIC images. Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 7 www.nature.com/scientificreports/ of multicolor GFP variants and would allow us to sensitively detect particular S. aureus cells in bacterial popula- tions and in animal infection models. To enhance fluorescent brightness, we replaced the sequence from the Shine-Dalgarno (SD) sequence to the start codon with sod that was previously reported with the sod RBS to lead to the highest fluorescence intensity with the sarA promoter . Consequentially, we not only modified the SD sequence of blaZ gene to an optimal SD sequence (AGGAGG), but also altered the distance from the SD sequence to the start codon. The distance between the RBS and the start codon is also known to ae ff ct the efficiency of translation initiation . Our results support the concept that the improvement of the sequence from the SD sequence to the start codon sequence significantly enhances the expression of the GFP variant, although the exact mechanism responsible for this effect remains unclear (Fig. 1b). However, recent studies suggest the interaction among the RBS, the initiation codon, the 5′-coding region in translation initiation, and RNA secondary structure at the 5′ terminus ae ff cts the protein 37, 38 expression in bacteria . Therefore, the enhancement of GFP production may be accounted for by the decrease of free fold energy of the 5′ end of mRNA transcripts. At the beginning of this study, we evaluated GFP expression vectors with the blaZ/sodp promoter in a lab- sa oratory strain, RN4220. This strain is easily genetically manipulated; however, it carries a number of genetic 20, 27 mutations that may ae ff ct the virulence of the strain . Like RN4220, laboratory strains may not be suitable for the evaluation of pathogenesis, because laboratory strains oe ft n lack important pathophysiological characters . Hence, clinical strains should be used to properly evaluate the pathogenesis and virulence of S. aureus. S. aureus harboring the bla gene locus appeared in the 1940s aer t ft he introduction of penicillin; and at present, most clini- 23, 40, 41 cal isolates carry the blaIRZ gene on plasmids or on the chromosome . Because the GFP expression driven sa by the blaZ/sodp promoter was not detectable in S. aureus clinical strains, MW2 and N315 (Fig. 2), we hypoth- esized the endogenous BlaI may inhibit the transcription of the blaZ/sodp promoter in trans; and we therefore generated the constitutively induced blaZ/sodp(Con) promoter to overcome the limitation of the clinical strains. The blaZ/sodp(Con) promoter can be adapted to enable expression of not only fluorescent proteins but also vari- ous exogenous proteins (toxins) from clinical S. aureus strains. 37, 42 Codon optimization frequently plays a key role in exogenous protein expression . The GC contents of human codon-optimized amCyan and mCherry are 46.8% and 62.5%, respectively, showing strong preferences for G + C at the third codon position is distinct from the codon usage patterns in S. aureus with 33.5% GC content. Exchanging the 92 nucleotides of amCyan gene and 76 nucleotides of mCherry gene, respectively, both fluores- cence intensities were significantly improved overcoming the codon usage bias in S . aureus. However, SDS-PAGE analysis showed the level of RCFPs production did not reach the level of GFP variants (Figs 3c and 4b). These results suggest the possibility that further investigation with these vectors could improve the expression level of RCFPs by further adapting of codon usage or decreasing the free folding energy of the initial 5′-coding region. In summary, we have developed new multicolor fluorescent protein vectors that efficiently enhance fluores- cence intensity in S. aureus clinical strains where the greater fluorescence intensity of multicolor fluorescent proteins may facilitate clear visualization of S. aureus clinical strains. Ultimately, these findings may help in better understanding the pathogenic mechanisms of S. aureus. Methods Bacterial strains, plasmids and growth conditions. The bacterial strains and plasmids used in this study are in Table 1. S. aureus and E. coli were cultured at 37 °C with shaking at 140 rpm in test tube (25 mm × 150 mm) containing 3 ml of trypticase soy broth (TSB) (Becton, Dickinson and Company) or 3 ml of lysogeny broth (5 g yeast extract, 10 g polypeptone, 10 g NaCl per liter; pH 7.2), respectively. Ampicillin (Amp, 100 µ g/ml) and chloramphenicol (Cp, 10 µ g/ml) were added to the medium if necessary. The plasmids encoding AmCyan or mCherry were purchased from Clontech, TaKaRa Bio Inc., Japan. Improvement of GFP expression vector using site-directed mutagenesis. To increase expression of fluorescent proteins in S . aureus clinical strains, we improved a GFP expression vector based on pS1GFP car- rying GFPmut3b gene (a kind gift from Prof. M. Krönke) . The hybrid blaZ/sodp promoter was constructed by replacing the sequence containing the RBS in the blaZ gene in the sod gene with blaZPR and GFP-F primers using the inverse PCR method with KOD Plus Neo DNA polymerase (TOYOBO, Japan). The PCR product was phosphorylated with T4 Polynucleotide Kinase (TaKaRa Bio Inc, Japan), and circularized by self-ligation with Ligation high Ver.2 (TOYOBO, Japan); and then the circular DNA was transformed into E. coli DH5α. Plasmid TM DNA was extracted from the transformed E. coli DH5α using FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd. Japan) and the resultant plasmid was verified using ABI 3130 DNA sequencer (Applied Biosystems). To express the fluorescent protein in S . aureus clinical strains, the substitution of the repressor BlaI/MecI binding motif (TACA/TGTA) was performed with the blaZ-mutF and blaZ-mutR primers using inverse PCR as above, yielding the constitutively induced blaZ/sodp(Con) promoter. Construction of the multicolor GFP variants using amino acid substitution. Amino acid substi- tution was performed using inverse PCR with pMK4blaZ/sodp(Con)GFPmut3b (pFK53) as the initial template with the KOD Plus Neo DNA polymerase (TOYOBO, Japan). The primers described in Table 2 were used to con- struct the GFP variants (Table 3). The resulting plasmids were confirmed using DNA sequencing. Construction of codon-optimized RCFP expression vectors. The amCyan and mCherry genes were combined with the hybrid blaZ/sodp(Con) promoter using overlap extension PCR and cloned into the pKAT 43 44 vector . Plasmid pKAT derived from pND50 is an E. coli-S. aureus shuttle vector containing the replication origins of pUB110 (S. aureus) and the pUC19 (E. coli) lacZ(α) gene from pUC19. This enables a simple blue-white screening for clones in E. coli, and enables the cat gene conferring resistance to chloramphenicol in both E. coli Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 8 www.nature.com/scientificreports/ Bacterial strain or Source or plasmid Relevant characteristic(s) reference E. coli − − + F , ϕ80dlacZΔM15, Δ(lacZYA−argF)U169, deoR, recA1, endA1, hsdR17(rk , mk ), phoA, DH5α TaKaRa supE44, λ , thi-1, gyrA96, relA1 S. aureus - + RN4220 NCTC8325-4, r m 20 N315 hospital-aquired MRSA 25 MW2 community-aquired MRSA 26 TY34 clinical isolate MRSA from patient with impetigo 35 Plasmids r r pMK4 Shuttle vector between E. coli and S. aureus, Am in E. coli, Cm in S. aureus 46 pS1GFP pMK4 containing GFPmut3b gene fused to the blaZp promoter 19 pND50 Shuttle vector between E. coli and S. aureus, Cm 44 pKAT pND50 derivative containing the lacZ(α) gene from pUC19, Cm 43 pAmCyan Plasmid encoding AmCyan gene, Amp TaKaRa Clontech pmCherry Plasmid encoding mCherry gene, Amp TaKaRa Clontech pFK51 pMK4 containing GFPmut3b gene fused to the blaZ/sodp promoter This study pFK52 pMK4 containing GFPsa gene fused to the blaZ/sodp promoter This study pFK53 pMK4 containing GFPmut3b gene fused to the blaZ/sodp(Con) promoter This study pFK54 pMK4 containing GFPsa gene fused to the blaZ/sodp(Con) promoter This study pFK55 pMK4 containing EGFP gene fused to the blaZ/sodp(Con) promoter This study pFK56 pMK4 containing Citrine gene fused to the blaZ/sodp(Con) promoter This study pFK57 pMK4 containing EBFP gene fused to the blaZ/sodp(Con) promoter This study pFK58 pMK4 containing BFP(P4-3E) gene fused to the blaZ/sodp(Con) promoter This study pFK59 pMK4 containing BFP(1EMF) gene fused to the blaZ/sodp(Con) promoter This study pFK60 pMK4 containing Cerulean gene fused to the blaZ/sodp(Con) promoter This study pFK61 pMK4 containing mEmerald gene fused to the blaZ/sodp(Con) promoter This study pFK62 pKAT containing AmCyan gene fused to the blaZ/sodp(Con) promoter This study pFK63 pKAT containing mCherry gene fused to the blaZ/sodp(Con) promoter This study pFK64 pKAT containing codon-optimized AmCyan(S.a) gene fused to the blaZ/sodp(Con) promoter This study pFK65 pKAT containing codon-optimized mCherry(S.a) gene fused to the blaZ/sodp(Con) promoter This study Table 1. Bacterial strains and plasmids used in this study. and S. aureus. All restriction enzyme sites in the multiple cloning site (MCS) located in the lacZ(α) gene can be used for cloning into pKAT. In the first PCR, the hybrid blaZ /sodp(Con) promoter region, amCyan gene, and mCherry gene were amplified from pFK53, pAmCyan (Clontech, TaKaRa Bio Inc., Japan), and pmCherry (Clontech, TaKaRa Bio Inc., Japan) with the following primer sets (AmCyan: blaZp-F and blaZPR-Cyan and blaZP-CyanF and pUC-RH; mCherry: blaZp-F and blaZPR-Cherry and blaZP-mCherryF and pUC-RH), respec- tively. The second PCR was performed with the mixture of two PCR fragments as the template using the primer set (blaZp-F and pUC-RH). The resulting PCR products were digested with HindIII and cloned into the same site in pKAT. Codon optimization was then repeatedly performed using inverse PCR as mentioned above with the primers described in Table 2 that were designed to optimize the codon usage of amCyan and mCherry gene using the Kazusa Codon Usage Database (http://www.kazusa.or.jp/codon/). The DNA sequences of codon-optimized amCyan and mCherry genes have been submitted to the GenBank and are available under accession numbers LC088723 (amCyan) and LC88724 (mCherry). Transformation of S. aureus and quantification of fluorescence intensity. In brief, S. aureus was individually transformed using electroporation as described previously . Each plasmid was first transformed into S. aureus RN4220 and selected as chloramphenicol-resistant colonies, then the resulting modified plas- mids were isolated and electroporated into S. aureus MW2 and N315. S. aureus was grown as described in the growth conditions. Bacterial cells from overnight cultures were washed with phosphate-buffered saline (PBS) and re-suspended to an optical density at 660 nm of 0.2. The cell suspension was then dispensed into triplicate wells (100 µ l/well) of a U-bottom 96 well cell culture plate (Greiner Bio-One). The fluorescence intensity was measured using a Varioskan Flash Multimode Reader (ThermoFisher Scientific Inc.) with two independent samples. Quantification of fluorescent proteins using SDS-PAGE and Western blot analysis. Fluorescent proteins were detected from whole cell lysates. S. aureus was cultured overnight as described in the growth con- ditions. e Th pre-cultured cells were adjusted to an optical density at 660 nm of 0.02 with TSB with chloramphen- icol, and 3 ml of the culture were transferred into test tube (25 mm × 150 mm) and then incubated at 37 °C with shaking at 140 rpm for 16 h. The bacterial cells from 1 ml cultures were harvested by centrifugation, and then the whole cell lysates were prepared as follows: cells were re-suspended in 200 μl CS bue ff r (100 mM Tris-HCI, Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 9 www.nature.com/scientificreports/ Name primer sequence (5' to 3') Purpose blaZPR AAATAATCATCCTCCTATTACAGTTGTAA replacement of RBS sequence GFP-F ATGAGTAAAGGAGAAGAACTTTTCAC blaZPmut-F TTTGTAAAAATATACACTTGAATAGGAGGATGAT destruction of BlaI motif blaZPmut-R TCAATAATATTACCATTATGATATTGATG F64LS65T-R TTGAACACCATATGTTAAAGTAGTAACAAG construction of Emerald and EGFP construction of BFP(P4-3E) and F64LY66H-R TTGAACACCATGAGATAAAGTAGTAACAAG BFP(1EMF) F64LS65TY66H-R TTGAACACCATGTGTTAATGTAGTAACAAG construction of EBFP F64LS65TY66W-R TTGAACACCCCATGTTAAAGTAGTAACAAGTGTTG construction of Cerulean S65GV68LQ69M-R CATTAAACCATAACCAAATGTAGTAACAAG construction of Citrine S72-F TGTTTTTCAAGATATCCAGATCATATG construction of EGFP and BFP construction of Cerulean, Emerald, S72A-F TGTTTTGCAAGATATCCAGATCATATG and Citrine F99S-F TTCAAAGATGACGGTAACTACAAGAC construction of GFPsa F99S-R AGATATAGTTCTTTCCTGTACATAACC construction of GFPsa Y145F-R GATATATACATTATGTGAGTTAAAGTTATATTC construction of EBFP, P4-3E(BFP) Y145AN146IH148D-R AATATATACATTGTCTGAGATAGCGTTATATTCCAA construction of Cerulean N149KM153T-R CTTTTGTTTGTCTGCTGTAATATATACTTTATGTGAG construction of Emerald construction of GFPsa and I152-R AATATATACATTATGTGAATTATAGTTATATTCC BFP(1EMF) M153-F ATGGCAGACAAACAAAAGAATGGAATC construction of EBFP, P4-3E(BFP) M153TV163A-F ACAGCAGACAAACAAAAGAATGGAATCAAAGCTAAC construction of GFPsa and Cerulean V163A-F ATGGCAGACAAACAAAAGAATGGAATCAAAGCTAAC construction of 1EMF(BFP) I167T-F AATGGAATTAAAGTTAACTTCAAAACAAGACAC construction of Emerald T203Y-F TATCAATCTGCATTATCAAAAGATCCAAAC construction of Citrine T203Y-R TGACAAATAATGATTGTCTGGTAAAAGAAC construction of Citrine A206K-F ACACAATCTAAATTATCAAAAGATCCAAACG monomerization gapRF AGAGAGGATCCTTAAATAGTTAGTTG amplification of gapR promoter amplification of gapR promoter gapRGFPR GAAAAGTTCTTCTCCTTTACTCATTACTACCTCCTCCTTATATTTATA fused to GFP amplification of gfp gene fused to gapRGFPF TATAAATATAAGGAGGAGGTAGTAATGAGTAAAGGAGAAGAACTTTTC gapR promoter GFPRB GTCTAGATCTTTATTTGTATAGTTCATC amplification of gfp gene blaZPF ACAAAAGCTTACTATGCTCATTATTAA amplification of blaZ gene promoter amplification of blaZ gene promoter blaZPR-Cyan AACTTGTTTGAAAGAGCCATAAATAATCATCCTCCTATTA fused to AmCyan amplification of amCyan gene fused blaZP-CyanF TAATAGGAGGATGATTATTTATGGCTCTTTCAAACAAGTT to blaZ promoter amplification of blaZ gene promoter blaZPR-mCherry TCCTCGCCCTTGCTCACCATAAATAATCATCCTTCCTATTA fused to mCherry amplification of mCherry gene fused blaZP-mCherryF TAATAGGAGGATGATTATTTATGGTGAGCAAGGGCGAGGA to blaZ promoter amplification of amCyan and pUC-RH AATGGAAGCTTCCGGCGCTCAGTTGG mCherry Cyan1F CATATGAAGGTACACAAACATCAACTTTTAAAGTTACAATGGCAAACGGTGGTCCACTTGCATTCTCATT codon optimization for amCyan Cyan1R GTTTACCACTACCTTCACCTTTAACTGTAAAATAATGACCGTTAACACAACCATCCATATGATATGT Cyan2F ATGCCAGATTATTTTAAACAAGCATTTCCTGATGGTATGTCATATGAACGTACTTTTACA codon optimization for amCyan Cyan2R ACTTGTAGGATATGCAGTAAAACAACGATTACCATACATAAAAACTGTTGATAGAATAC Cyan3F GAACATAAATCAACATTTCATGGAGTTAACTTTCC codon optimization for amCyan Cyan3R AAAACAGTTACCTTTAAGACTTATTTCCCAACTTGC Cyan4F CAAGGAGGTGGTAATTATAGATGTCAATTTCATACTTCTTATAAGACA codon optimization for amCyan Cyan4R TAACATTAAAAATGCTGTAACATCACCCTTCAATATTCCATCACAAACAGTC Cyan5F AAGGTGGTAATAGTGTTCAATTAACAGAACATGCTGTTGCACATATAACATCTGTTGTTCC codon optimization for amCyan Cyan5R TATCTAAATCTGTTCTTGCAATACGATGTTCAACTGCATGGTTTGGTGGCATTGTAACTGGTTT Cherry1F AGTTAATGGTCATGAATTCGAAATCGAGGGCGAGGGCGAGGGTCGTCCATATGAGGGCACACAAACAGC codon optimization for mCherry Cherry1R GAACCTTCCATATGAACTTTAAAACGCATGAACTCCTTGATGATTGCCATGTTATCCTCCTCACCCTTAC Cherry2F TATGGTTCAAAAGCATATGTTAAGCATCCAGCAGACATCCCAGACTATTTGAAGTTGTCATTCCCAGAGG codon optimization for mCherry Cherry2R CATAAATTGAGGTGATAAGATGTCCCATGCGAATGGCAATGGACCACCCTTTGTCACCTTCAACTTTGC Cherry3F TATTTATAAAGTTAAGTTGCGTGGTACAAACTTCCCATCAGACGGCCCAGTAATGCAGAAGAAGACAATG codon optimization for mCherry Cherry3R AATTCACCATCTTGCAATGATGAGTCTTGTGTCACTGTAACCACACCGCCGTCCTCGAAGTTCATCACAC Table 2. Oligonucleotide primers. Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 10 www.nature.com/scientificreports/ GFP variant Mutations relative to wtGFP Reference GFPmut3b S65G, S72A 21 GFPsa S65G, S72A, F99S, M153T, V163A This study EGFP F64L, S65T 4 mEmerald F64L, S65T, S72A, N149K, M153T, I167T, A206K 4 Citrine S65G, S72A, V68L, Q69M, T203Y 30 Cerulean F64L, S65T, Y66W, S72A, Y145A, N146I, H148D, M153T, V163A 31 1EMF(BFP) F64L, Y66H, V163A 32 EBFP F64L, S65T, Y66H, Y145F 33 P4-3E(BFP) F64L, Y66H, Y145F, V163A 34 Table 3. GFP variants used in this study. 150 mM NaCl, 100 mM EDTA, pH7.5) containing 1 μg of lysostaphin (Wako Pure Chemical Industries, Co., Ltd, Japan) and incubated at 37 °C for 30 min. Ten microliters of cell lysates were separated on SDS–PAGE and stained with Coomassie Brilliant Blue (CBB), and the fluorescent proteins were detected using Western blot with the following antibodies: Anti-GFP-HRP-Direct T (MBL Co., Ltd.), Living colors Anti-RCFP Polyclonal Pan Antibody (Clontech Laboratories) and DsRed Polyclonal Antibody (Clontech Laboratories) as the primary anti- body, and HRP-conjugated goat antibodies against rabbit IgG (MP Biomedicals, LLC-Cappel Products) as the secondary antibody. Immuno-detection of protein was performed on Pierce Western Blot Substrate (Thermo Fisher Scientific Inc.) with X-ray film. Confocal laser scanning microscopic analysis. S. aureus strains and E. coli were cultured in test tube (25 mm × 150 mm) containing 3 ml of Brain Heart Infusion (BHI) broth with chloramphenicol or ampicillin at 37 °C with shaking at 140 rpm overnight. 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Krönke (University of Cologne) for the gift of plasmid pS1GFP; Dr. Junzo Hisatsune for his assistance with the confocal laser scanning microscope; and Dr. Wakako Ikeda-Ohtsubo for critical reading of the manuscript. This study was supported by Grant-in-Aid for Young Scientists (B) Grant Number JP25861744 and Grant-in-Aid for Scientific Research (C) Grant Number JP25460533 from the Japan Society for the promotion of Science (JSPS). A confocal laser scanning microscopy was performed at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University. Author Contributions F.K. conceived and designed the experiments, performed the experiments, and wrote the main manuscript. M.K. constructed the multicolor-GFP variants. M.S. conceived the experiments and critically revised the manuscript. All authors reviewed approved the manuscript. Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-02930-7 Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 12 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 Scientific Repo R ts | 7: 2865 | DOI:10.1038/s41598-017-02930-7 13
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