Identification of a gene required for the oxygen-regulated formation of the photosynthetic apparatus of Rhodobacter capsulatus

The pigment-binding proteins of Rhodobacter capsulatus are encoded by the polycistronic puf and puc operons. Both operons show higher expression under low oxygen tension than under high oxygen tension in the wild-type strain. The Tn5 mutant strain AH2 shows only low levels of puf and puc mRNA under high and low oxygen tension, indicating that it lacks a gene product required for stimulation of puf and puc gene expression under low oxygen tension. The formation of wild-type levels of photosynthetic complexes and normal oxygen regulation could be restored by the expression in trans of a 1.7 kb fragment of the R. capsulatus wild-type chromosome or by addition of 10μg I^-1 vitamin B₁₂ to the growth medium. An open reading frame of 798 nucleotides containing the Tn5 insertion was identified on the 1.7kb fragment. This open reading frame shows no homology to known genes and has a remarkably high GC content of 76%.     

Thioredoxin can influence gene expression by affecting gyrase activity

The expression of many genes of facultatively photosynthetic bacteria of the genus Rhodobacter is controlled by the oxygen tension. Among these are the genes of the puf and puc operons, which encode proteins of the photosynthetic apparatus. Previous results revealed that thioredoxins are involved in the regulated expression of these operons, but it remained unsolved as to the mechanisms by which thioredoxins affect puf and puc expression. Here we show that reduced TrxA of Rhodobacter capsulatus and Rhodobacter sphaeroides and oxidized TrxC of R.capsulatus interact with DNA gyrase and alter its DNA supercoiling activity. While TrxA enhances supercoiling, TrxC exerts a negative effect on this activity. Furthermore, inhibition of gyrase activity strongly reduces puf and puc expression. Our results reveal a new signaling pathway by which oxygen can affect the expression of bacterial genes.     

Coregulation of the syntheses of bacteriochlorophyll and pigment-binding proteins in Rhodobacter capsulatus

We quantified the expression of the puf and puc operons, which encode pigment binding proteins, in a number of strains of Rhodobacter capsulatus that have defects in genes affecting different steps of bacteriochlorophyll biosynthesis. Our results show that these mutations have a very similar effect on puf and puc expression. This suggests that the reduced expression of genes encoding pigment-binding proteins is due neither to the accumulation of a specific intermediate of bacteriochlorophyll synthesis nor to the lack of an early intermediate, but is rather the result of the lack of a very late intermediate or the bacteriochlorophyll molecule itself. Received: 14 September 1998 / Accepted: 29 December 1998     

A powerful hybrid <Emphasis Type="Italic">puc</Emphasis> operon promoter tightly regulated by both IPTG and low oxygen level

Rhodobacter sphaeroides has been intensively studied and provides an excellent model for studying both photo-synthesis and membrane development. The photosynthetic apparatus (LH2 and LH1-RC complexes) can be synthesized in large scale and integrated into the intracytoplasmic membrane system under specific conditions, which thus provides us insight to utilize the puc or(and) puf operon to heterologously express recombinant proteins in the intracytoplasmic membrane using Rb. sphaeroides as a novel expression system. However, basal level of expression of puc and puf promoter is uncontrolled. We report the construction of LH2 polypeptide expression vector that contains a reengineered lacI ^q-puc promoter-lac operator hybrid promoter, which allows the puc operon to be regulated by both IPTG and low oxygen level. Synthesis of LH2 complexes was completely repressed in the absence of isopropyl β-D-thiogalactoside (IPTG), and the degree of induction was controlled by varying the concentration of IPTG. The optimal concentration of IPTG was determined. SDS-PAGE and Western blot were employed for further analysis. Our results suggest that the reengineered hybrid promoter is efficient to tightly regulate the expression of the puc operon, and our strategy can open up a new approach in the study of the membrane protein expression system.     

DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1

This paper describes the DNA sequence of the photosynthesis region of Rhodobacter sphaeroides 2.4.1^T. The photosynthesis gene cluster is located within a ~73 kb AseI genomic DNA fragment containing the puf, puhA, cycA and puc operons. A total of 65 open reading frames (ORFs) have been identified, of which 61 showed significant similarity to genes/proteins of other organisms while only four did not reveal any significant sequence similarity to any gene/protein sequences in the database. The data were compared with the corresponding genes/ORFs from a different strain of R.sphaeroides and Rhodobacter capsulatus, a close relative of R.sphaeroides. A detailed analysis of the gene organization in the photosynthesis region revealed a similar gene order in both species with some notable differences located to the pucBAC=cycA region. In addition, photosynthesis gene regulatory protein (PpsR, FNR, IHF) binding motifs in upstream sequences of a number of photosynthesis genes have been identified and shown to differ between these two species. The difference in gene organization relative to pucBAC and cycA suggests that this region originated independently of the photosynthesis gene cluster of R.sphaeroides.     

Effect of light and oxygen and adaptation to changing light conditions in a photosynthetic mutant in which the LHII complex of Rhv. sulfidophilum was heterologously expressed in a strain of Rb. capsulatus whose puc operon was deleted

In this paper we show the effect of oxygen and light on the expression of the photosynthetic apparatus of a mutant heterologously expressing the puc operon. This mutant was obtained by introducing in trans an expression plasmid, bearing the puc A, B, and C genes of Rhv. sulfidophilum, as well as its own promoter, in an LHII⁻ mutant of Rb. capsulatus. The results showed that oxygen and light repressed LHII expression. Even low-light intensities lowered the LHII content to undetectable levels by spectrophotometry or by SDS-PAGE. In high-light grown cells, where the relative ratios of LHI and LHII complexes were significantly diminished, we were able to detect LHII complexes. Under the latter condition, the absorption spectrum showed that some pigment accumulated in the membrane even in the absence of cell division. These pigments were used in a later step to assemble LHII complexes, when the high-light grown cells were transferred to semiaerobiosis in the dark. Transition of high-light grown cells to low-light conditions allowed us to study the adaptability of these heterologous mutant cells. We observed that adaptation never occurred, in part probably owing to energy limitation. Received: 20 November 2001 / Accepted: 31 December 2001     

Bacterial regulatory networks include direct contact of response regulator proteins: interaction of RegA and NtrX in Rhodobacter capsulatus

The formation of photosynthetic complexes in facultatively photosynthetic bacteria is controlled by the oxygen tension in the environment. In Rhodobacter capsulatus the two-component system RegB/RegA plays a major role in the redox control of photosynthesis genes but also controls other redox-dependent systems. The response regulator RegA is phosphorylated under low oxygen tension and activates the puf and puc operons, which encode pigment binding proteins, by binding to their promoter regions. Data from a yeast two-hybrid analysis as well as an in vitroanalysis indicate that RegA interacts with the NtrX protein, the response regulator of the NtrY/NtrX two-component system which is believed to be involved in regulation of nitrogen fixation genes. Our further analysis revealed that NtrX is indeed involved in the regulation of the puf and puc operons. Furthermore, we showed that an altered NtrX protein, which is predicted to adopt the conformation of phosphorylated NtrX protein, binds within the puf promoter region close to the RegA binding sites. We conclude that a direct interaction of two response regulators connects the regulatory systems for redox control and nitrogen control.     

Genetic and physical mapping of the Rhodobacter sphaeroides photosynthetic gene cluster from R-prime pWS2

Plasmid pWS2 is an R68.45 chimera originally isolated as an R-prime which complemented the Rhodobacter sphaeroides bch-420 allele. Our experiments have shown that pWS2 is also able to complement a wide range of R. sphaeroides pigment and photosynthetic mutants employing nitrosoquanidine, transposon or insertion-generated mutations effecting puhA, puc, puf, cycA, bch, and crt genes. A combination of orthogonal-field-alternation gel electrophoresis, transverse alternating field gel electrophoresis, and conventional electrophoresis have been used to estimate the size of pWS2 at -168.3 ± 3.5 kb. A restriction map of the -109 kb of R. sphaeroides insert DNA was generated by partial and complete restriction endonuclease digestion coupled with Southern hybridization analysis using either gene-specific or junction fragment probes. Genes encoding bacteriochlorophyll (Bchl)-binding proteins (pufBALMX, pucBA, and puhA), cytochrome c₂ (cycA), and enzymes involved in Bchl (bch) and carotenoid (crt) biosynthesis have been shown to reside within a contiguous 53-kb region of the R. sphaeroides DNA present on pWS2. The puf operon lies at one end of the 53-kb segment, while the genes puhA, cycA, and pucBA, the latter two of which are located within -12.0 kb of each other, define the other end of this 53-kb region. The genetic and physical mapping data provided in this paper are discussed in terms of the similarities and differences in the organization of the photosynthetic gene cluster between R. sphaeroides and other photosynthetic bacteria as well as highlighting the use of pWS2 in studies of photosynthetic gene structure and function.     

Oxygen and light effects on the expression of the photosynthetic apparatus in Bradyrhizobium sp. C7T1 strain

Photosynthetic bradyrhizobia are nitrogen-fixing symbionts colonizing the stem and roots of some leguminous plants like Aeschynomene. The effect of oxygen and light on the formation of the photosynthetic apparatus of Bradyrhizobium sp. C7T1 strain is described here. Oxygen is required for growth, but at high concentration inhibits the synthesis of bacteriochlorophyll (BChl) and of the photosynthetic apparatus. However, we show that in vitro, aerobic photosynthetic electron transport occurred leading to ADP photophosphorylation. The expression of the photosynthetic apparatus was regulated by oxygen in a manner which did not agree with earlier results in other photosynthetic bradyrhizobia since BChl accumulation was the highest under microaerobic conditions. This strain produces photosynthetic pigments when grown under cyclic illumination or darkness. However, under continuous white light illumination, a Northern blot analysis of the puf operon showed that, the expression of the photosynthetic genes of the antenna was considerable. Under latter conditions BChl accumulation in the cells was dependent on the oxygen concentration. It was not detectable at high oxygen tensions but became accumulated under low oxygen (microaerobiosis). It is known that in photosynthetic bradyrhizobia bacteriophytochrome photoreceptor (BphP) partially controls the synthesis of the photosystem in response to light. In C7T1 strain far-red light illumination did not stimulate the synthesis of the photosynthetic apparatus suggesting the presence of a non-functional BphP-mediated light regulatory mechanism.     

Heterologous synthesis and assembly of functional LHII antenna complexes from <Emphasis Type="Italic">Rhodovulum sulfidophilum</Emphasis> in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> mutant

The light harvesting complexes, including LHII and LHI, are the important components of photosynthetic apparatus. Rhodovulum (Rdv.) sulfidophilum and Rhodobacter (R.) sphaeroides belong to two genera of photosynthetic bacteria, and they are very different in some physiological characteristics and light harvesting complexes structure. The LHII structural genes (pucBsAs) from Rdv. sulfidophilum and the LHI structural genes (pufBA) from R. sphaeroides were amplified, and cloned into an expression vector controlled by puc promoter from R. sphaeroides, which was then introduced into LHI and LHII-minus R. sphaeroides mutants; the transconjugant strains synthesized heterologous LHII and native LHI complexes, which played normal roles in R. sphaeroides. The Rdv. sulfidophilum LHII complex from pucBsAs had near-infrared absorption bands at ~801–853 nm in R. sphaeroides, and was able to transfer energy efficiently to the native LHI complex. The results show that the pucBsAs genes from Rdv. sulfidophilum could be expressed in R. sphaeroides, and the functional foreign LHII and native LHI were assembled into the membrane of R. sphaeroides.     

Photosynthetic reaction center mutagenesis via chimeric rescue of a non-functional Rhodobacter capsulatus puf operon with sequences from Rhodobacter sphaeroides

Photosynthetically active chimeric reaction centers which utilize genetic information from both Rhodobacter capsulatus and Rb. sphaeroides puf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. sphaeroides donor puf operon. Following photosynthetic selection, three revertant classes were recovered: 1) those which used Rb. sphaeroides donor sequence to repair the Rb. capsulatus host operon without modification of Rb. sphaeroides puf operon sequences (conversions), 2) those which exchanged sequence between the two operons (inversions), and 3) those which modified plasmid or genomic sequences allowing expression of the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random and could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct characteristic(s). The minimum region required for chimeric rescue is the D-helix and half of the D/E-interhelix of M. When puf operon sequences 3 of nucleotide M882 are exchanged, significant impairment of excitation trapping is observed. This region includes both the 3 end of pufM and sequences past the end of pufM.     

Characterization of a mutant strain of Rhodovulum sulfidophilum lacking the pufA and pufB genes encoding the polypeptides for the light-harvesting complex 1 (B 870)

Contradictory results on the effectiveness of energy transfer from the light harvesting complex 2 (LH2) directly to the reaction center (RC) in mutant strains lacking the core light-harvesting complex 1 (LH1) have been obtained with cells of Rhodobacter capsulatus and Rhodobacter sphaeroides. A LH1⁻ mutant of Rhodovulum sulfidophilum, named rsLRI, was constructed by deletion of the pufBA genes, resulting in a kanamycin resistant photosynthetically positive clone. To restore the wild type phenotype, a complemented strain C2 was constructed by inserting in trans a DNA segment containing the pufBA genes. Light-induced FTIR difference spectra indicate that the RC in the rsLRI mutant and in the C2 complemented strains are functionally and structurally identical with those in the wild type strain, demonstrating that the assembly and the function of the RC is not impaired by the LH1 deletion. The photosynthetic growth rate of the rsLRI strain increased with decreasing light intensity. At 50 W m⁻² no photosynthetic growth was observed. These results indicate that the light energy harvested by the LH2 complex was not or inefficiently transferred to the RC; thus most of the energy necessary for photosynthetic growth is in the LH1⁻ strain directly absorbed by the RC. It is supposed that in the mutant strain, RC and LH2 cannot interact in an efficient way.