Cloning and sequencing of the genes encoding the light-harvesting B806-866 polypeptides and initial studies on the transcriptional organization of puf2B, puf2A and puf2C in Chloroflexus aurantiacus.

The genes encoding the alpha- and beta-polypeptide subunits of the B806-866 membrane-bound light-harvesting complex of Chloroflexus aurantiacus have been cloned and the nucleotide sequences determined. The gene puf2A, which encodes the B806-866 alpha-polypeptide, began 28 bases downstream of the stop codon of puf2B, which encodes the B806-866 beta gene. The gene-encoding cytochrome c-554, puf2C, was found about 250 bp downstream of puf2A. puf2A encoded a 13 amino acid extension at the C-terminus of the B806-866 alpha-polypeptide that was not present in the mature protein. These genes, unlike those of purple nonsulfur bacteria, did not form a contiguous operon with puf1L or puf1M, the genes encoding the L and M subunits of the photochemical reaction center. The occurrence of the two latter genes and of puf2B and puf2A in two separate operons has not been observed in purple bacteria. Under photoheterotrophic growth conditions, puf2B and puf2A were encoded on an abundant mRNA that was 0.5 kb long. Two monocistronic transcripts for puf2C were observed that had different 5'-ends. One transcript encoding all three genes was also detected. Nucleotide sequences very similar to the consensus promoter sequence of the Escherichia coli RNA polymerase sigma 70 subunit were found seven and eight bases upstream of the 5'-end of mRNA encoding puf2B and for one of the monocistronic mRNA encoding puf2C, respectively.

The primary structure of two chlorosome proteins from Chloroflexus aurantiacus.

The complete nucleotide sequence of two chlorosome proteins with apparent molecular weights of M(r) 18,000 and M(r) 11,000 from Chloroflexus aurantiacus have been determined. The two polypeptides were 145 and 97 amino acids long and possessed true molecular masses of 15,545 and 10,820 Da, respectively. Protein chemical sequencing was done in parallel to confirm the primary structure deduced from nucleotide sequencing. By Northern blot analysis of RNA isolated from phototrophically grown cells a transcript of 0.95 kb was detected which is the expected length for a mRNA encoding both genes.

The functional role of protein in the organization of bacteriochlorophyll c in chlorosomes of Chloroflexus aurantiacus.

The preparation of five different fractions containing bacteriochlorophyll (Bchl) c and their absorption and circular dichroic properties have been described. The fractions investigated were purified chlorosomes, proteolytically modified chlorosomes, chlorosomes treated with lithium dodecyl sulfate (LDS) which were subsequently subjected to size-exclusion chromatography, in vitro Bchl c aggregates and, additionally, the so-called GEF chlorosomes [prepared according to Griebenow and Holzwarth (1989) Biochim. Biophys. Acta 973, 235-240]. Proteolysis of chlorosomes caused a 35-40% decrease in absorption intensity, a 6-8 nm blue shift of the 740-nm peak and, in particular, a drastic increase of rotational strength as revealed by CD spectroscopy. Although oligomeric Bchl c aggregates and LDS-treated chlorosomes had absorption characteristics similar to Bchl c in vivo, the data clearly indicated that protein, perhaps the chlorosome-specific Mr-3700 polypeptide, was involved in the organization of Bchl c in chlorosomes from C. aurantiacus. Furthermore, the results showed that the LDS-treated chlorosome fraction was most likely comprised of a micellar complex of Bchl c with LDS which represented an entity entirely different from chlorosomes.

A high-yield method for the isolation of hydrophobic proteins and peptides from polyacrylamide gels for protein sequencing.

A methodological approach is described which allows the isolation of hydrophobic and hydrophilic proteins and peptides in high yield. The technique consists of (1) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) protein elution from polyacrylamide gels with an organic solvent mixture composed of formic acid/acetonitrile/isopropanol/H2O (50/25/15/10, v/v/v/v), and (3) purification of eluted proteins by size exclusion chromatography on a Superose 12 column using this organic solvent mixture as eluant. The efficiency of this technique was tested with radioactively labeled polypeptides. These proteins were reaction center from Chloroflexus aurantiacus, bacteriorhodopsin, halorhodopsin from Halobacterium halobium, bovine serum albumin, ovalbumin, alpha-chymotrypsinogen A, and cytochrome c. The elution recoveries from polyacrylamide gels were 77-95%; the final yield after chromatographic purification was still 67-76% (with one exception). Subsequent amino acid sequencing was possible without further sample treatment. The sensitivity of the method described was found to be at least 20-30 micrograms protein.

Isolation and characterization of cytoplasmic membranes and chlorosomes from the green bacterium Chloroflexus aurantiacus.

A method was developed which allows the isolation and purification of cytoplasmic membranes and chlorosomes from cells of Chloroflexus aurantiacus grown under different light conditions. The dipolar ionic detergent Deriphat (0.08%) and a sodium iodide gradient centrifugation were used in isolating cytoplasmic membranes. Chlorosomes were prepared with 0.16% of the dipolar ionic detergent Miranol and purified by a sucrose gradient centrifugation. Cytoplasmic membrane fractions prepared from either high- (3,000 W m-2), medium-(200 W m-2) or low- (7 W m-2) light-grown cells had near infrared absorption bands at 866, 808, and 755 nm in a constant characteristic absorbance ratio of 6:3.8:1. In all cytoplasmic membrane preparations, the amount of bacteriochlorophyll a (Bchl a) per cytochrome, the amount of Bchl a per reaction center, and reaction center per milligram of cytoplasmic membrane protein was found to be constant. No Bchl c was present. Five respiratory enzyme activities have been measured in the cytoplasmic membrane fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured cytoplasmic membrane showed many bands, but a major polypeptide with an apparent molecular weight of 8,000. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified chlorosomes did not contain the 8,000-molecular-weight band but revealed only three distinct protein bands with molecular weights of 15,000, 12,000, and 6,000. Isolated chlorosomes contained Bchl c and a small, yet constant, amount of Bchl a (absorbing at 790 nm) in a molar ratio of 25:1. The data indicated that the components of the photosynthetic apparatus in the cytoplasmic membrane of Chloroflexus aurantiacus remained constant and only the amount of antenna Bchl c varied with light conditions.