Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a heterologous host.

The entire pathway for the synthesis of a fluorescent holophycobiliprotein subunit from a photosynthetic cyanobacterium (Synechocystis sp. PCC6803) was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to the natural chromophore 3Z-phycocyanobilin, namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase, were expressed from a plasmid under control of the hybrid trp-lac (trc) promoter. Genes for the apoprotein (C-phycocyanin alpha subunit; cpcA) and the heterodimeric lyase (cpcE and cpcF) that catalyzes chromophore attachment were expressed from the trc promoter on a second plasmid. Upon induction, recombinant E. coli used the cellular pool of heme to produce holo-CpcA with spectroscopic properties qualitatively and quantitatively similar to those of the same protein produced endogenously in cyanobacteria. About a third of the apo-CpcA was converted to holo-CpcA. No significant bilin addition took place in a similarly engineered E. coli strain that lacks cpcE and cpcF. This approach should permit incisive analysis of many remaining questions in phycobiliprotein biosynthesis. These studies also demonstrate the feasibility of generating constructs of these proteins in situ for use as fluorescent protein probes in living cells.

Energy transfer cassettes for facile labeling of sequencing and PCR primers.

Fluorescence energy transfer (ET) primers and terminators are the reagents of choice for multiplex DNA sequencing and analysis. We present here the design, synthesis and evaluation of a four-color set of ET cassettes, fluorescent labeling reagents that can be quantitatively coupled to a thiol-activated target through a disulfide exchange reaction. The ET cassette consists of a sugar-phosphate spacer with a FAM donor at the 3'-end, an acceptor linked to a modified T-base at the 5'-end of the spacer and a mixed disulfide for coupling to a thiol at the 5'-end. The acceptor dye emission intensities of ET labeled primers produced in this manner are comparable to commercial ET primers. The utility of our ET cassette-labeled primers is demonstrated by performing four-color capillary electrophoresis sequencing with the M13(-21)forward primer and by generating and analyzing a set of single-nucleotide-polymorphism-specific PCR amplicons.

Recombinant phycobiliproteins. Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains.

A family of specific cloning vectors was constructed to express in the cyanobacterium Anabaena sp. PCC7120 recombinant C-phycocyanin subunits with one or more different tags, including the 6xHis tag, oligomerization domains, and the streptavidin-binding Strep2 tag. Such tagged alpha or beta subunits of Anabaena sp. PCC7120 C-phycocyanin formed stoichiometric complexes in vivo with appropriate wild-type subunits to give constructs with the appropriate oligomerization state and normal posttranslational modifications and with spectroscopic properties very similar to those of unmodified phycocyanin. All of these constructs were incorporated in vivo into the rod substructures of the light-harvesting complex, the phycobilisome. The C-terminal 114-residue portion of the Anabaena sp. PCC7120 biotin carboxyl carrier protein (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coli and 40% in wild-type Anabaena sp. His-tagged phycocyanin beta--BCCP114 constructs expressed in Anabaena sp. were >30% biotinylated. In such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avidin-coated beads. Thus, the methods described here achieve in vivo production of stable oligomeric phycobiliprotein constructs equipped with affinity purification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation.

Roger Yate Stanier, 1916-1982: a transcendent journey.

The Tenth International Symposium on Phototrophic Prokaryotes (Barcelona, 26-31 August 2000) was the latest in a series of conferences initiated by Roger Stanier in 1971 to create ties within the community of scientists working with cyanobacteria or green and purple bacteria. Consonant with Stanier's own work, the subjects of these conferences range broadly from systematics and ecology through genetics, biochemistry and physiology. The effort to define comprehensively the place of bacteria in the living world was the leitmotif in Stanier's work, the subject of one of his earliest papers (in 1941), and revisited for the final time in his autobiographical memoir of 1980. Salvador Luria noted that Stanier "...always pursued broad naturalistic interests along with chemical ones, deliberately emphasizing morphology and ecology side by side with biochemistry." Chronologically, Stanier's work addressed taxonomic and nutritional aspects of the cytophagas, enzyme induction and patterns of regulation of enzyme synthesis, aromatic degradative pathways, characterization of what would subsequently be called 70S bacterial ribosomes, the regulation of bacteriochlorophyll synthesis by nonsulfur purple bacteria, protection by carotenoids against photooxidative damage, the path of carbon in heterotrophy, the molecular basis of streptomycin dependence, the life cycle of Caulobacter, the taxonomy of pseudomonads, and, for the last 12 years of his life, wide-ranging studies of the cyanobacteria.

Design, synthesis, and spectroscopic properties of peptide-bridged fluorescence energy-transfer cassettes.

A general partial solid-phase synthetic scheme was developed for the synthesis of energy-transfer cassettes with the donor and acceptor dyes bridged by a peptide. In these cassettes, 6-carboxyfluorescein (Fam) served as a donor. For the second dye, 6-carboxy-X-rhodamine (Rox) was used as a fluorescent acceptor or erythrosin B as a quencher. Different peptides bearing Rox at the amino terminus and Fam linked through different diamines to the carboxyl terminus were synthesized to examine the effects of the chain length and rigidity on energy-transfer efficiency. The ratio of emission intensities at 605 nm of the acceptor dye (ROX) in the cassette Rox-GPPPEPPP-p-xylylenediamine-Fam versus free ROX with 488 nm excitation was approximately 14 and is similar to that obtained for optimized oligonucleotide primers bearing the same dyes [Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4347-4351].

Capillary electrophoresis chips with integrated electrochemical detection.

Capillary electrophoresis systems with integrated electrochemical detection have been microfabricated on glass substrates. Photolithographic placement of the working electrode just outside the exit of the electrophoresis channel provides high-sensitivity electrochemical detection with minimal interference from the separation electric field. Microchip electrophoretic separations of neurotransmitters in under 100 s exemplify the good resolution and attomole detection sensitivity of these devices. Using indirect electrochemical detection, high-sensitivity DNA restriction fragment and PCR product sizing has also been performed. These microdevices match the detector's size to that of microfabricated separation and reaction devices, bringing to reality the lab-on-a-chip concept.

Comparison of fluorescence energy transfer primers with different donor-acceptor dye combinations.

Fluorescence energy transfer (ET) primers are far superior to single dye-labeled primers as labels for DNA sequencing and polymerase chain reaction amplification. We compare here ET primers with different donor and acceptor dye combinations with respect to the relative acceptor fluorescence emission intensity and the amount of residual donor fluorescence emission. Primers with the following donor/acceptor pairs were synthesized: 6-carboxyfluorescein/6-carboxy-X-rhodamine (FAM-ROX), 3-(epsilon-carboxypentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine/ 6-carboxy-X-rhodamine (CYA-ROX), and the 4,4-difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene-3-propionic acid (BODIPY) derivatives, 5,7-dimethyl-BODIPY/5-(4-phenyl-1,3-butadienyl) BODIPY (BODIPY503/512-BODIPY581/591). Variables examined included the length of the 5'-amino linker arm, the number of base pairs between the donor and acceptor, and the excitation wavelength (488 or 514 nm). Of the primers examined, CYA-ROX primers offer the best combination of acceptor fluorescence emission intensity and spectral purity.

Optimization of spectroscopic and electrophoretic properties of energy transfer primers.

We have synthesized and characterized the spectroscopic properties of 56 energy transfer (ET) fluorescent dye-labeled primers differing in (i) the spacing between the donor and acceptor, (ii) the nature of the spacer (either oligonucleotide or polydideoxyribose phosphate), (iii) the primer sequence (M13 (-40), M13 (-21), M13 reverse, SP6, T3, and T7 priming sequences), and (iv) the dyes chosen as the donor (6-carboxyfluorescein, F; or 3-(epsilon-carboxypentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine, C) and acceptor (F; 5 & 6-carboxyrhodamine-110, R110; 6-carboxyrhodamine-6G, G; N,N,N',N'-tetramethyl-6-carboxyrhodamine, T; and 6-carboxy-X-rhodamine, R) chromophores. This study led to the development of two significantly improved ET primer sets for multiple-color analyses. These primers are named using the convention D-N-A, where D is the donor, A is the acceptor, and N is the number of nucleotides between the donor and the acceptor. The primer set C4R110, C4G, C4T, and C4R provides acceptor emissions of high spectral purity with donor:acceptor emission ratios of < 0.002 for C4G, < 0.004 for C4T, and < 0.005 for C4R and excellent matching in the electrophoretic mobilities of single-base extension DNA fragments. The C4R110, C4G, C4T, and C4R set is valuable for diagnostic applications where minimization of crosstalk between different labels is of particular importance. The set C10R110, C10G, C10T, and C10R, which uses only rhodamine dyes as acceptors, shows significantly improved matching in the electrophoretic mobilities of single-base extension DNA fragments over the previously described set C10F, C10G, C10T, and C10R and is the best available for sequencing.

Improved stability and electrophoretic properties of preformed fluorescent cationic dye-DNA complexes in a taps-tetrapentylammonium buffer in agarose slab gels.

High-resolution capillary electrophoresis sizing of preformed complexes of bis-intercalating fluorescent dyes with double-stranded DNA has been demonstrated using hydroxyethylcellulose and 3-[tris-(hydroxymethyl) methylamino]-1-propanesulfonic acid-tetrapentylammonium (Taps-NPe+4) buffers (S. M. Clark and R. A. Mathies, Anal. Chem. 69, 1355-1363, 1997). Such capillary electrophoresis separations were unattainable in conventional buffers containing other cations such as Tris+, Na+, and NH+4. We report here the behavior of preformed double-stranded DNA-dye complexes on agarose slab gel electrophoresis in 40 mM Taps-NPe+4, 1 mM H2EDTA, pH 8.2. Upon electrophoresis in this buffer (a) complexes formed at DNA base pairs:dye ratios ranging from 100:1 to 5:1 show the same mobility; (b) the half-lives of DNA-dye complexes with monointercalators are two- to threefold longer than those in commonly used Tris buffers; (c) there is little dye transfer between labeled and unlabeled DNA molecules; and (d) precise two-color sizing of preformed restriction fragment-dye complexes with fluorescent bisintercalators is achieved.

Characterization of cyanobacterial biliverdin reductase. Conversion of biliverdin to bilirubin is important for normal phycobiliprotein biosynthesis.

The Synechocystis sp. PCC 6803 gene (bvdR) encoding biliverdin reductase was amplified by the polymerase chain reaction, cloned, and overexpressed in Escherichia coli as the native form and as a 6-histidine-tagged amino-terminal fusion. The latter form of the enzyme was purified by affinity chromatography and shown to have the appropriate molecular weight by electrospray mass spectrometry. Both forms of the enzyme reduced biliverdin IXalpha using NADPH or NADH, with NADPH as the preferred reductant. The His-tagged enzyme has a Km for biliverdin of 1.3 microM. The pH optimum for the NADPH-dependent activity is 5.8, whereas that for rat biliverdin reductase is at pH 8.7. Absorbance spectra and high performance liquid chromatography retention times of the reaction product reaction match those of authentic bilirubin, the product of the reduction of biliverdin by the mammalian enzymes. These results provide the first evidence for the formation of bilirubin in bacteria. Fully segregated Synechocystis sp. PCC 6803 bvdR interposon mutants produce approximately 85% of the normal amount of phycobilisome cores containing allophycocyanin and other phycocyanobilin-bearing core polypeptides, but no detectable phycocyanin. Thus, surprisingly, the blockage of the conversion of biliverdin to bilirubin interferes with normal phycobiliprotein biosynthesis in cyanobacteria. Possible interpretations of this finding are presented.

Energy-transfer fluorescent reagents for DNA analyses.

Fluorescence resonance energy transfer has facilitated the development of a new class of high-performance fluorescent labeling reagents for multiplex analyses of nucleic acids. The enhanced emission of energy transfer (ET) primers has provided a decadic improvement in the performance of automated DNA sequencers. The emission spectral purity of ET primers permits the development of robust multiplex diagnostic methods for the detection of PCR products. High affinity bifunctional intercalation reagents containing ET-coupled dyes are also being used for high-performance multiplex assays of double-stranded DNA when noncovalent labeling is preferred.

Microsatellite-based cancer detection using capillary array electrophoresis and energy-transfer fluorescent primers.

The development of sensitive, rapid, and accurate methods and apparatus for high-throughput short tandem repeat (STR) analysis will be critical for the use of microsatellite alteration in cancer screening. Here we show that STR-based bladder cancer diagnosis can be performed using capillary array electrophoresis and two-color labeling with energy-transfer (ET) fluorescent primers. Rapid (< or = 35 min) separations are achieved on capillary arrays using replaceable separation matrices and the allelic ratios are quantitatively determined with a precision of +/- 10%. With this precision, a variation of 20% was considered diagnostically significant. These methods provide a significant improvement in the speed, ease, and precision of STR analyses compared to slab gel electrophoresis.

Environment-sensitive labels in multiplex fluorescence analyses of protein-DNA complexes.

Fluorescein is widely used for protein labeling because of its high extinction coefficient and fluorescence emission quantum yield. However, its emission is readily quenched by various pathways. We exploit these properties of fluorescein to examine the self-association of a DNA binding protein and determine the amount of the protein in gel-shifted complexes with specific DNA. A construct (HSFDT385SH) of the heat shock transcription factor (HSF) was expressed that contains the DNA-binding and trimerization domains, residues 192-385 of HSF, with four additional COOH-terminal residues, GMLC, and then labeled at the COOH-terminal cysteine with fluorescein 5-maleimide to form HSFDT385-Fl. The fluorescence increase accompanying the formation of heterotrimers on titration of HSFDT385-Fl with HSFDT385SH) led to an estimate of 3 x 10(-16) M2 for the equilibrium constant for trimerization of HSFDT385SH. HSFDT385-Fl fluorescence also increased 1.7-fold on binding to specific DNA, but not to nonspecific DNA. The protein and DNA content of the several gel-shifted complexes of HSFDT385-Fl (lambdamaxem 532 nm) with specific DNA labeled noncovalently with the energy transfer heterodimer TOTAB (lambdamaxem 658 nm) were accurately determined by a two-color fluorescence emission assay with 488 nm excitation.

Cyanine dyes with high absorption cross section as donor chromophores in energy transfer primers.

Energy transfer (ET) fluorescent primers are significantly superior to single dye-labeled primers for DNA sequencing and multiplex genetic analyses (Ju, J., Glazer, A. N., and Mathies, R. A. (1996) Nature Med. 2, 246-249). We describe here ET primers in which a donor chromophore with a large absorption cross section but a low fluorescence quantum yield is exploited to increase the Stokes-shifted fluorescence emission of acceptor dyes. The new ET primers have 3-(epsilon-carboxy-pentyl)-3'ethyl-5,5'-dimethyloxacarbocyanine (CYA; epsilon M488nm 142,000 M-1 cm-1) at the 5' -end as a common energy donor, and fluorescein or rhodamine derivatives (FAM, R6G, TAMRA, and ROX), attached to a modified thymidine 10 bases away within the primer sequence, as acceptors. With 488-nm excitation, the fluorescence emission intensity of these four ET primers is 1.4- to 24-fold stronger than that of the corresponding primers labeled only with the single acceptor dye. When compared with the corresponding ET primers with a fluorescein derivative (FAM; epsilon M488nm 60,000 M-1 cm-1) as donor, the fluorescence emissions of primers with CYA as donor and FAM, R6G, TAMRA, and ROX as acceptors are respectively 0.8-, 1.0-, 1.7-, and 1.7-fold as intense. The low fluorescence quantum yield of the CYA donor resulted in distinct fluorescence signals for the DNA-sequencing fragments with much lower crosstalk between the four detection channels than that seen with ET primers based on a FAM donor. With single-stranded M13mp18 DNA as the template, the CYA ET primers provided DNA sequences on a four-color capilary sequencer with 100% accuracy in the first 500 bases.

Energy transfer primers with 5- or 6-carboxyrhodamine-6G as acceptor chromophores.

Energy-transfer (ET) fluorescent primers for DNA sequencing and multiplex genetic analysis (Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351) are named according to the convention D-N-A, where D is the donor, N is the number of bases between the donor and the acceptor, and A is the acceptor. Thus, a primer that carries 6-carboxyfluorescein (FAM) at the 5'-end and 6-carboxy-4', 5'-dichloro-2',7'-dimethoxyfluorescein (JOE) attached to a modified thymidine 10 bases away is designated F10J. We describe here new ET primers, with 5- or 6-carboxyrhodamine-6G (G5 or G6) as acceptors (with FAM as the donor) in place of JOE, with improved match in the electrophoretic mobilities of the DNA fragments extended from the ET dye-labeled primers, and less overlap in the fluorescence emission of the various labeled DNA fragments. This reduced spectral overlap is most likely due to the narrower emission from G5 or G6 in F10G compared to that from JOE in F10J. With single-stranded M13mp18 DNA as the template, a typical run with F10G6 and three other ET primers on a capillary sequencer provided DNA sequences with 99% accuracy in the first 620 bases.

Ultraviolet-B photodestruction of a light-harvesting complex.

Cyanobacteria are important contributors to global photosynthesis in both marine and terrestrial environments. Quantitative data are presented on UV-B-induced damage to the major cyanobacterial photosynthetic light harvesting complex, the phycobilisome, and to each of its constituent phycobiliproteins. The photodestruction quantum yield, phi295 nm, for the phycobiliproteins is high (approximately 10(-3), as compared with approximately 10(-7) for visible light). Energy transfer on a picosecond time scale does not compete with photodestruction. Photodamage to phycobilisomes in vitro and in living cells is amplified by causing dissociation and loss of function of the complex. In photosynthetic organisms, UV-B damage to light-harvesting complexes may significantly exceed that to DNA.

Cassette labeling for facile construction of energy transfer fluorescent primers.

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.

Cryptomonad biliproteins: Bilin types and locations.

Two crytophycean phycocyanins (Cr-PCs), Hemiselmis strain HP9001 Cr-PC 612 and Falcomonas daucoides Cr-PC 69 were purified and characterized with respect to bilin numbers, types and locations. Each biliprotein carried one bilin on the α subunit and three on the ß subunit. Cr-PC 612 carried phycocyanobilin at α-Cys-18, ß-Cys-82, and ß-Cys-158, and a doubly-linked 15,16-dihydrobiliverdin at ß-DiCys-50,61. Cr-PC 569 carried phycocyanobilin at α-Cys-18 and ß-Cys-82, a singly-linked Bilin 584 at ß-Cys-158, and a doubly-linked Bilin 584 at ß-DiCys-50,61. This work, in conjunction with earlier studies on Cr-PE 545, Cr-PE 555, Cr-PE 566, and Cr-PC 645, shows that there is no conserved location for the bilin with longest wavelength visible absorption band among these proteins, and, consequently, that there is no conserved energy transfer pathway common to all native cryptophycean biliproteins. Only phycocyanobilin or phycoerythrobilin is found at ß-Cys-82; there is greater bilin variability at the other three attachment sites.

Design and synthesis of fluorescence energy transfer dye-labeled primers and their application for DNA sequencing and analysis.

We have designed and synthesized fluorescent oligonucleotide primers having improved fluorescence and electrophoretic properties by exploiting the concept of resonance fluorescence energy transfer (ET). These primers carry a fluorescein derivative at the 5' end as a common fluorescence donor and other fluorescein and rhodamine derivatives attached to a modified thymidine within the primer sequence as acceptors. These primers all have strong absorption at a common excitation wavelength (448 nm) and fluorescence emission maxima of 525, 555, and 605 nm. The fluorescence emission intensity of the ET primers increases as the spacing between the donor and acceptors is increased, and of the spacings studied the strongest fluorescence was observed when the number of nucleotides between the donor and acceptors is 10. The electrophoretic mobilities of the primers were also found to be a function of the spacing between the donor and the acceptors, and mobilities of the single base extension DNA fragments generated with primers (F10F, F10J, F10T, and F10R) is 2- to 14-fold greater than that of the corresponding primers labeled with only one dye. The increased fluorescence intensity of the ET primers and the substantially similar mobilities of the DNA fragments generated with the four ET primers allow four-color DNA sequencing on a capillary electrophoresis DNA sequencer using a single laser line at 488 nm for excitation and without applying mobility shift adjustments. With single-stranded M13mp18 DNA as the template, a typical run with the ET primers on a commercial sequencer provided DNA sequences with 99-100% accuracy in the first 500 bases using 8-fold less DNA template than that typically required using T7 DNA polymerase.