Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms.     

Improved Blue,Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae

Fluorescent protein fusions are a powerful tool to monitor the localization and trafficking of proteins. Such studies are particularly easy to carry out in the budding yeast Saccharomyces cerevisiae due to the ease with which tags can be introduced into the genome by homologous recombination. However, the available yeast tagging plasmids have not kept pace with the development of new and improved fluorescent proteins. Here, we have constructed yeast optimized versions of 19 different fluorescent proteins and tested them for use as fusion tags in yeast. These include two blue, seven green, and seven red fluorescent proteins, which we have assessed for brightness, photostability and perturbation of tagged proteins. We find that EGFP remains the best performing green fluorescent protein, that TagRFP-T and mRuby2 outperform mCherry as red fluorescent proteins, and that mTagBFP2 can be used as a blue fluorescent protein tag. Together, the new tagging vectors we have constructed provide improved blue and red fluorescent proteins for yeast tagging and three color imaging.     

Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants

A series of versatile cloning vectors has been constructed that facilitate the expression of protein fusions to the Aequorea victoria green fluorescent protein (GFP) in plant cells. Amino-terminal- and carboxy-terminal protein fusions have been created and visualized by epifluorescence microscopy, both in transgenic Arabidopsis thaliana and after transient expression in onion epidermal cells. Using tandem dimers and other protein fusions to GFP, we found that the previously described localization of wild-type GFP to the cell nucleus is most likely due to diffusion of GFP across the nuclear envelope rather than to a cryptic nuclear localization signal. A fluorescence-based, quantitative assay for nuclear localization signals is described. In addition, we have employed the previously characterized mutants GFP–S65T and GFP–Y66H in order to allow for the expression of red-shifted and blue fluorescent proteins, respectively, which are suitable for double-labeling studies. Expression of GFP-fusions was controlled by a cauliflower mosaic virus 35S promoter. Using the Arabidopsis COP1 protein as a model, we confirmed a close similarity in the subcellular localization of native COP1 and the GFP-tagged COP1 protein. We demonstrated that COP1 was localized to discrete subnuclear particles and further confirmed that fusion to GFP did not compromise the activity of the wild-type COP1 protein.     

Optimization of Fluorescent Tools for Cell Biology Studies in Gram-Positive Bacteria

The understanding of how Gram-positive bacteria divide and ensure the correct localization of different molecular machineries, such as those involved in the synthesis of the bacterial cell surface, is crucial to design strategies to fight bacterial infections. In order to determine the correct subcellular localization of fluorescent proteins in Streptococcus pneumoniae, we have previously described tools to express derivatives of four fluorescent proteins, mCherry, Citrine, CFP and GFP, to levels that allow visualization by fluorescence microscopy, by fusing the first ten amino acids of the S. pneumoniae protein Wze (the i-tag), upstream of the fluorescent protein. Here, we report that these tools can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus and Bacillus subtilis, possibly due to optimized translation rates. Additionally, we have optimized the i-tag by testing the effect of the first ten amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found that manipulating the structure and stability of the 5′ end of the mRNA molecule, which may influence the accessibility of the ribosome, is determinant to ensure the expression of a strong fluorescent signal.     

Dynamic Localization of Tat Protein Transport Machinery Components in Streptomyces coelicolor

The Tat pathway transports folded proteins across the bacterial cytoplasmic membrane and is a major route of protein export in the Streptomyces genus of bacteria. In this study, we have examined the localization of Tat components in the model organism Streptomyces coelicolor by constructing enhanced green fluorescent protein (eGFP) and mCherry fusions with the TatA, TatB, and TatC proteins. All three components colocalized dynamically in the vegetative hyphae, with foci of each tagged protein being prominent at the tips of emerging germ tubes and of the vegetative hyphae, suggesting that this may be a primary site of Tat secretion. Time-lapse imaging revealed that localization of the Tat components was highly dynamic during tip growth and again demonstrated a strong preference for apical sites in growing hyphae. During aerial hypha formation, TatA-eGFP and TatB-eGFP fusions relocalized to prespore compartments, indicating repositioning of Tat components during the Streptomyces life cycle.     

Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling

Since their discovery, fluorescent proteins have been widely used to study protein function, localization or interaction, promoter activity and regulation, drug discovery or for non-invasive imaging. They have been extensively modified to improve brightness, stability, and oligomerization state. However, only a few studies have focused on understanding the dynamics of fluorescent proteins expression in bacteria. In this work, we developed a set plasmids encoding 12 fluorescent proteins for bacterial labeling to facilitate the study of pathogen-host interactions. These broad-spectrum plasmids can be used with a wide variety of Gram-negative microorganisms including Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia, Bordetella bronchiseptica, Shigella flexneri or Klebsiella pneumoniae. For comparison, fluorescent protein expression and physical characteristics in Escherichia coli were analyzed using fluorescence microscopy, flow cytometry and in vivo imaging. Fluorescent proteins derived from the Aequorea Victoria family showed high photobleaching, while proteins form the Discosoma sp. and the Fungia coccina family were more photostable for microscopy applications. Only E2-Crimson, mCherry and mKeima were successfully detected for in vivo applications. Overall, E2-Crimson was the fastest maturing protein tested in E. coli with the best overall performance in the study parameters. This study provides a unified comparison and comprehensive characterization of fluorescent protein photostability, maturation and toxicity, and offers general recommendations on the optimal fluorescent proteins for in vitro and in vivo applications.     

Use of mCherry Red Fluorescent Protein for Studies of Protein Localization and Gene Expression in Clostridium difficile

Fluorescent proteins are powerful reporters in biology, but most require O₂ for chromophore maturation, making them inherently difficult to use in anaerobic bacteria. Clostridium difficile, a strict anaerobe with a genomic GC content of only 29%, is the leading cause of hospital-acquired diarrhea in developed countries, and new methods for studying this pathogen are sorely needed. We recently demonstrated that a cyan fluorescent protein called CFP_opt that has been codon optimized for production in low-GC bacteria can be used to study protein localization in C. difficile provided the cells are fixed prior to exposure to air. We describe here a codon-optimized variant of mCherry (mCherryOpt) that exhibits faster acquisition of fluorescence and a better signal-to-noise ratio than CFP_opt. We utilized mCherryOpt to construct plasmids for studying protein localization (pRAN473) and gene expression (pDSW1728) in C. difficile. Plasmid pRAN473 is an mCherryOpt fusion vector with a tetracycline-inducible promoter. To document its biological utility, we demonstrated septal localization of two cell division proteins, MldA and ZapA. Plasmid pDSW1728 is designed for cloning a promoter of interest upstream of mCherryOpt. As proof of principle, we studied the expression of the pdaV operon, which is required for lysozyme resistance. In confirmation and extension of previous reports, we found that expression of the pdaV operon requires the alternative sigma factor σ^v and that induction by lysozyme is dose dependent and uniform across the population of lysozyme-treated cells.     

PSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions

Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.     

Expression of Green Fluorescent Protein Fused to Magnetosome Proteins in Microaerophilic Magnetotactic Bacteria

The magnetosomes of magnetotactic bacteria are prokaryotic organelles consisting of a magnetite crystal bounded by a phospholipid bilayer that contains a distinct set of proteins with various functions. Because of their unique magnetic and crystalline properties, magnetosome particles are potentially useful as magnetic nanoparticles in a number of applications, which in many cases requires the coupling of functional moieties to the magnetosome membrane. In this work, we studied the use of green fluorescent protein (GFP) as a reporter for the magnetosomal localization and expression of fusion proteins in the microaerophilic Magnetospirillum gryphiswaldense by flow cytometry, fluorescence microscopy, and biochemical analysis. Although optimum conditions for high fluorescence and magnetite synthesis were mutually exclusive, we established oxygen-limited growth conditions, which supported growth, magnetite biomineralization, and GFP fluorophore formation at reasonable rates. Under these optimized conditions, we studied the subcellular localization and expression of the GFP-tagged magnetosome proteins MamC, MamF, and MamG by fluorescence microscopy and immunoblotting. While all fusions specifically localized at the magnetosome membrane, MamC-GFP displayed the strongest expression and fluorescence. MamC-GFP-tagged magnetosomes purified from cells displayed strong fluorescence, which was sensitive to detergents but stable under a wide range of temperature and salt concentrations. In summary, our data demonstrate the use of GFP as a reporter for protein localization under magnetite-forming conditions and the utility of MamC as an anchor for magnetosome-specific display of heterologous gene fusions.     

Cellular localization of choline-utilization proteins in Streptococcus pneumoniae using novel fluorescent reporter systems

The molecular mechanisms underlying cell growth, cell division and pathogenesis in Streptococcus pneumoniae are still not fully understood. Single-cell methodologies are potentially of great value to investigate S. pneumoniae cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the S. pneumoniae chromosome. We have also established a zinc-inducible system for the fine control of gfp -fusion gene expression and for protein depletion experiments in S. pneumoniae . Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of S. pneumoniae teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.     

Libraries of green fluorescent protein fusions generated by transposition in vitro

Two artificial transposons have been constructed that carry a gene encoding Green Fluorescent Protein and can be used for generating libraries of GFP fusions in a gene of interest. One such element, AT2GFP, can be used to generate GFP insertions in frame with the amino acid sequence of the protein of interest, with a stop codon at the end of the GFP coding sequence; AT2GFP also contains a selectable marker that confers trimethoprim resistance in bacteria. The second element, GS, can be used to generate tribrid GFP fusions because there is no stop codon in the GFP transposon, and the resulting fusion proteins contain the entire amino acid sequence encoded by the gene. The GS element consists of a gfp open reading frame and a supF amber suppressor tRNA gene; the supF portion of the GS transposon can be utilized as a selectable marker in bacteria. Its sequence contains a fortuitous open reading frame, and thus it can be translated continuously with the gfp amino acid sequence. As a target for GFP insertions, we used a plasmid carrying the native Ty1 retrotransposon of the yeast Sacharomyces cerevisiae. The resulting multiple GFP fusions to Ty1 capsid protein Gag and Ty1 integrase were useful in determining the cellular localization of these proteins. Libraries of GFP fusions generated by transposition in vitro represent a novel and potentially powerful method to study the cell distribution and cellular localization signals of proteins.     

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.     

Use of fluorescent protein tags to study nuclear organization of the spliceosomal machinery in transiently transformed living plant cells

Although early studies suggested that little compartmentalization exists within the nucleus, more recent studies on metazoan systems have identified a still increasing number of specific subnuclear compartments. Some of these compartments are dynamic structures; indeed, protein and RNA-protein components can cycle between different domains. This is particularly evident for RNA processing components. In plants, lack of tools has hampered studies on nuclear compartmentalization and dynamics of RNA processing components. Here, we show that transient expression of fluorescent protein fusions of U1 and U2 small nuclear ribonucleoprotein particle (snRNP)-specific proteins U1-70K, U2B", and U2A ', nucleolar proteins Nop10 and PRH75, and serine-arginine-rich proteins in plant protoplasts results in their correct localization. Furthermore, snRNP-specific proteins also were correctly assembled into mature snRNPs. This system allowed a systematic analysis of the cellular localization of Arabidopsis serine-arginine-rich proteins, which, like their animal counterparts, localize to speckles but not to nucleoli and Cajal bodies. Finally, markers for three different nuclear compartments, namely, nucleoli, Cajal bodies, and speckles, have been established and were shown to be applicable for colocalization studies in living plant protoplasts. Thus, transient expression of proteins tagged with four different fluorescent proteins is a suitable system for studying the nuclear organization of spliceosomal proteins in living plant cells and should therefore allow studies of their dynamics as well.     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum , like Mycobacterium tuberculosis , is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200–1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2–20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis , Mycobacterium bovis BCG and Mycobacterium tuberculosis . These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.     

Fluorescent and bimolecular-fluorescent protein tagging of genes at their native loci in Neurospora crassa using specialized double-joint PCR plasmids

The double-joint polymerase chain reaction (DJ-PCR) is a technique that can be used to construct vectors for targeted genome integration without laborious subcloning steps. Here we report the availability of plasmids that facilitate DJ-PCR-based construction of Neurospora crassa tagging vectors. These plasmids allow the creation of green or red fluorescent protein (GFP or RFP) tagging vectors for protein localization studies, as well as split-yellow fluorescent protein (YFP) tagging vectors for bimolecular fluorescence complementation (BiFC) analyses. We have demonstrated the utility of each plasmid with the tagging of known meiotic silencing proteins. Microscopic analysis of the tagged strains indicates that SMS-2 and QIP form macromolecular complexes in the perinuclear region during meiosis.     

Monomeric red fluorescent protein as a reporter for macromolecular localization in Streptomyces coelicolor

The enhanced green fluorescent protein (eGFP) is widely used to investigate cell type specific gene expression and protein localization in the filamentous streptomycetes. To broaden the scope of cell biological investigation in these organisms, we have adapted shuttle vectors for the construction of gene fusions to the monomeric red fluorescent protein (mRFP1) and have tested them in Streptomyces coelicolor. Using fusions of mRFP1 to the cell division proteins DivIVA and FtsZ, we show that mRFP1 is comparable to eGFP for cell biological research in this organism and suggest that this paves the way for the future use of two-color imaging and FRET.     

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.     

Green fluorescent protein functions as a reporter for protein localization in Escherichia coli

The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.