Expression of the thioredoxin gene (trxA) in Rhodobacter sphaeroides Y is regulated by oxygen.

The structural gene (trxA) coding for thioredoxin in the photosynthetic bacterium Rhodobacter sphaeroides has been cloned and sequenced previously. In the present study, the role of oxygen in trxA expression in R. sphaeroides Y was investigated using mRNA analyses and plasmid-borne trxA'-lacZ+ translational and transcriptional fusions. Northern analysis revealed a trxA-specific transcript of approximately 420-460 nucleotides, indicating that trxA is transcribed as a single gene. By studying the beta-galactosidase activity in strains harboring various phi(trxA'-lacZ+) fusion constructs, the promoter region of the trxA gene was localized within a 64-bp region located 97 nucleotides upstream of the trxA initiator codon. A single trxA transcription initiation site was mapped by primer extension, 27 bp upstream of the trxA gene. Based on these results and the DNA sequence analysis, we propose that a sigma70 consensus sequence serves as a trxA promoter. Results from oxygen shift experiments, as deduced from both mRNA analysis and fusions of the trxA promoter region to lacZ indicate that transcription of the R. sphaeroides trxA gene is regulated by high oxygen tension. DNA sequences involved in this oxygen regulation were also localized in the 64-bp region containing the trxA promoter. Based on our findings the hypothetical biological function of thioredoxin from R. sphaeroides is discussed.

The Gly74-->Ser and Ser3-->Ala mutations in Rhodobacter sphaeroides Y thioredoxin: effects on active site reactivity and protein interaction.

In this study, we report the effects of two different substitutions in Rhodobacter sphaeroides thioredoxin on two regions of the protein: the N-terminus end and the hydrophobic area implicated in protein/protein interactions. We have produced by site-directed mutagenesis R. sphaeroides thioredoxin single and double mutants in which the glycine residue at position 74 is changed to a serine and the serine at position 3 is changed to an alanine; the three mutant proteins have been purified. The two substitutions are not equivalent. Substitution of serine by alanine increased the pI from 5.2 to 6.1; this pI value was the same in the double-mutated protein, which demonstrates the presence of a local conformational change. In vivo studies showed that the Gly74-->Ser substitution completely prevented phage T3/T7 growth whereas the Ser3-->Ala substitution had no effect. This finding was corroborated by the large decrease (100-fold) of polymerase activity for the double mutant in the in vitro measurement of phage T7 DNA polymerase activity with the corresponding pure proteins. Although marginal (within a factor of two), the effects of the two substitutions on the catalytic activities of the thioredoxin reductase reaction confirmed their difference. Substitution of serine by alanine had no effect on the Km and resulted in an improvement in the catalytic efficiency. In contrast, the second substitution increased the Km value, without improving the catalytic efficiency. The following can be concluded (a) glycine74 of R. sphaeroides thioredoxin has a direct role in the binding of T7 gene 5 protein and the hydrophobic area of thioredoxin; (b) the N-terminus plays a role in maintaining the conformational integrity of the active site; (c) the flexibility of Gly74 in the hydrophobic region involved in protein/protein interaction is the operative factor in the case of the activity of thioredoxin in the T7 DNA polymerase.

Cloning, nucleotide sequence, and expression of the Rhodobacter sphaeroides Y thioredoxin gene.

Synthetic oligodeoxynucleotide probes based on the known amino acid sequence of Rhodobacter sphaeroides Y thioredoxin were used to identify, clone, and sequence the structural gene. The amino acid sequence derived from the DNA sequence of the R. sphaeroides gene was identical to the known amino acid sequence of R. sphaeroides thioredoxin. An NcoI site was created by directed mutagenesis at the beginning of the thioredoxin gene, inducing in the encoded protein the replacement of serine in position 2 by alanine. The 421-base-pair NcoI-PstI restriction fragment obtained was ligated in the pKK233-2 expression vector and the resulting hybrid plasmid was used to transform Escherichia coli strains lacking functional thioredoxin. Transformants that complemented mutations in the trxA gene were identified by increased colony size on rich medium, growth on minimal medium with methionine sulfoxide, and ability to support M13 growth and T7 replication; this latter phenotype implies correct interaction between R. sphaeroides thioredoxin and the product of T7 gene 5. The presence of R. sphaeroides thioredoxin was further confirmed by enzyme assay.

Amino acid sequence determination and three-dimensional modelling of thioredoxin from the photosynthetic bacterium Rhodobacter sphaeroides Y.

The complete primary structure of thioredoxin from Rhodobacter sphaeroides Y has been determined by analysis of peptides after cleavage with cyanogen bromide, chymotrypsin and trypsin. Peptides were separated by HPLC and analyzed by liquid-phase and gas-phase sequencer degradations. The protein consists of 105 residues (Mr = 11,180); its amino acid sequence shows a clear homology to the five known thioredoxins from plant or bacterial sources, with 40-56% residue identity when the proteins are aligned at the active-site disulfide. Not only the active-site regions are conserved, but also residues which belong to the hydrophobic surface suggested to be important for binding of procaryote thioredoxins in redox interactions with other proteins (residues 75-76; 91-93 in Escherichia coli). A three-dimensional model of Rb. sphaeroides thioredoxin has been derived from the E. coli crystallographic structure with computer graphics. This model indicates that the overall structures as well as the active sites are closely similar; however, the residue substitutions allow both proteins to adopt different local folding as shown in the hydrophobic core.

Characterization of the thioredoxin system in the facultative phototroph Rhodobacter sphaeroides Y.

This paper reports the purification and characterization of a thioredoxin system (thioredoxin, thioredoxin reductase, NADPH) from the facultative phototroph Rhodobacter sphaeroides Y. Rhodobacter sph. Y thioredoxin was purified to homogeneity with an assay based on the reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by NADPH and Escherichia coli thioredoxin reductase. Rhodobacter sph. Y thioredoxin reductase was purified with the same assay using NADPH and E. coli thioredoxin. Rhodobacter sph. Y thioredoxin contained 102 amino acid residues and had a single intrachain disulfide bond. The two half-cystine residues are part of the active site made up of the sequence -Ala-Glu-Trp-Cys-Gly-Pro-Cys-Arg- which is identical to that of E. coli thioredoxin except for the presence of an Arg instead of a Lys. Rhodobacter sph. Y thioredoxin contains two tryptophan residues. The fluorescence intensity of the tryptophan residues is quenched in oxidized thioredoxin; on reduction, a much smaller increase is observed with Rhodobacter sph. Y thioredoxin than with the E. coli protein. However, the presence of 5 M guanidine X HCl results in the complete exposure of the two tryptophan residues. Rhodobacter sph. Y thioredoxin reductase has structural and functional similarities to E. coli thioredoxin reductase: it has a molecular mass of 68 kDa, and consists of two, probably identical, subunits. Each subunit has one bound FAD molecule. The enzyme is highly specific for NADPH; it is also highly specific for Rhodobacter sph. Y thioredoxin with a Km value of 3.3 +/- 0.6 microM. A kinetic study of the two thioredoxin systems shows that they have a high degree of cross-reactivity.

A photosystem II-phycobilisome preparation from the red alga, Porphyridium cruentum: oxygen evolution, ultrastructure, and polypeptide resolution.

The photosystem II-phycobilisome preparation, isolated by lauryldimethyl amine oxide treatment, had a greatly reduced chlorophyll content, with an average ratio of 90 chlorophyll a/phycobilisome as compared to approximately 1200 Chl/phycobilisome in unfractionated thylakoids. P700 was not detected in the particles. By electron microscopy the preparations were relatively homogeneous and were generally devoid of chloroplast membranes. In negatively stained preparations phycobilisome particles were seen often in clusters of two and three, probably due to retention of hydrophobic thylakoid fragments. The preparation was deficient in photosystem I chlorophyll complexes, but enriched in polypeptides of 85 to 92, approximately 43, and approximately 26 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 43- and 26-kDa polypeptides are attributable to the PS II core and the oxygen-evolving complex, respectively.

Characterization of a Purified Photosystem II-Phycobilisome Particle Preparation from Porphyridium cruentum.

Detergent preparations isolated from thylakoids of the red alga Porphyridium cruentum, in a sucrose, phosphate, citrate, magnesium chloride medium consist of phycobilisomes and possess high rates of photosystem II activity. Characterization of these particles shows that the O(2)-evolving activity is stable for several hours and the pH optimum is about 6.5 to 7.2. Response of the system to light, electron donors and acceptors, and inhibitors verify that the observed activity, measured both as O(2) evolution and 2,6-dichlorophenol-indophenol reduction, is due to photosystem II. Furthermore, photosystem II is functionally coupled to the phycobilisome in this preparation since green light, absorbed by phycobilisomes of P. cruentum, is effective in promoting both O(2) evolution and 2,6-dichlorophenol-indophenol reduction. Photosystem II activity declines when light with wavelengths shorter than 665 nm is removed. Both 3-(3,4-dichlorophenyl)-1,1-dimethylurea and atrazine inhibit photosystem II activity in this preparation, indicating that the herbicide binding site is a component of the photosystem II-phycobilisome particle.

A 4-vinylprotochlorophyllide complex accumulated by "phofil" mutant of Rhodopseudomonas spheroides. An authentic intermediate in the development of the photosynthetic apparatus.

A photosynthetically competent mutant strain of Rhodopseudomonas spheroides was isolated. In addition to bacteriochlorophyll, this organism produced particle-bound precursor 4-vinylprotochlorophyllide. The spectral characteristics of the pigment complexes(es) accumulated in the culture medium were very variable. The spectral form occurring within the bacteria was characterized from fluorescence data. Its particle weight, 130 000, was determined by Sephadex G200 filtration. The main components of the complex were protein, lipid and pigment (6.8:61, w/w). As indicated by qualitative analysis, the lipid components were characteristic constituents of the photosynthetic membrane. Kinetics of pigments synthesis showed that the total pigment synthesis was not affected by the mutation; bacteriochlorophyll content was always lower in the mutant than in the parent strain. The repigmentation process was followed by fluorescence emission. The results indicated that the mutation affected membrane component synthesis required for the bacteriochlorophyll(ide) incorporation. The pigment complex was concluded to be an authentic intermediate in photosynthetic apparatus morphogenesis. The reasons for its excretion are discussed.

5-Aminolevulinic-acid synthetases from Rhodopseudomonas spheroides Y. Comparison of the purification and properties of enzymes extracted from bacteria grown in different iron concentrations.

The two 5-aminolevulinic acid synthetases of Rhodopseudomonas spheroides Y. were extracted from cells grown in a 'low-iron' medium and purified. They have a specific activity 10-fold higher than the 'high-iron' enzymes described by us previously and have the same properties except that they do not contain any iron and have one free-SH group more per mole of enzyme (2 for E1; , for E2) Their inhibition by adenosine triphosphate and iron and their oxidation-reduction sensitivity are discussed in terms of light, oxygen and heme feed-back regulation of bacteriochlorophyll sunthesis.