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 congruent to 168.3 +/- 3.5 kb. A restriction map of the congruent to 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 congruent to 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.     

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.     

Construction of a physical map of the 45 kb photosynthetic gene cluster of Rhodobacter sphaeroides

1) A number of overlapping clones have been isolated from a Rhodobacter sphaeroides gene bank. Following conjugative gene transfer from Escherichia coli these clones restore a wild type phenotype to several mutants unable to synthesise bacteriochlorophyll. 2) The insert DNA was analysed by restriction mapping and together the clones form the basis of the first restriction map of the 45 kb photosynthetic gene cluster of Rb. sphaeroides. 3) This cluster is bounded on one side by puh A encoding the reaction centre H polypeptide and on the other by the puf operon encoding reaction centre L and M apoproteins and light harvesting LH1 and polypeptides. 4) DNA fragments from the 45 kb cluster were used to probe genomic DNA from other photosynthetic bacteria. Using heterologous hybridisation conditions, a significant degree of homology is shown between Rb. sphaeroides and these other bacteria, suggesting close evolutionary links with Rb. capsulatus in particular.     

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%.     

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.     

Construction of a gene bank of Rhodopseudomonas capsulata using a broad host range DNA cloning system

A gene bank of the phototrophic bacterium Rhodopseudomonas capsulata was constructed using the binary plasmid system pRK290/pRK2013. Fragments of about 20 kb of chromosomal DNA of R. capsulata strain 37b4 were inserted into the cloning vector pRK290. The hybrid plasmids of the gene bank, maintained in Escherichia coli HB101 were transferred by conjugation to R. capsulata strains defective in the photosynthetic apparatus with frequencies of 5×10^-4 to 5×10^-2. Phototrophically growing transconjugants occurred with frequencies of 5×10^-7 to 5×10^-6. Recombination between the hybrid plasmids and the R. capsulata chromosome was shown. The hybrid plasmid pRCF1002, carrying a 25 kb insert of R. capsulata wild type DNA, was isolated from one E. coli clone of the gene bank. It reconstituted some bacteriochlorophyll- and photosynthetic negative mutants to phototrophic growth.Abbreviations Bchl Bacteriochlorophyll - RC reaction center - LH light-harvesting complex - Crt carotenoid - pho phototrophic growth - P Bchl precursor excreted, the number behind P indicates the maximum of absorption in ether (nm) - SDS sodium dodecyl sulfate - Tc tetracycline - Km kanamycin - Gm gentamicin - r resistant - kb kilo base pairs Dedicated to Hans-Günter Schlegel on occasion of his 60th birthday     

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.     

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.     

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.     

Interaction of bacteriochlorophyll with the LH1 and PufX polypeptides of photosynthetic bacteria: use of chemically synthesized analogs and covalently attached fluorescent probes

The protein components of the reaction center (RC) and core light-harvesting (LH 1) complexes of photosynthetic bacteria have evolved to specifically, but non-covalently, bind bacteriochlorophyll (Bchl). The contribution to binding of specific structural elements in the protein and Bchl may be determined for the LH 1 complex because its subunit can be studied by reconstitution under equilibrium conditions. Important to the determination and utilization of such information is the characterization of the interacting molecular species. To aid in this characterization, a fluorescent probe molecule has been covalently attached to each of the LH 1 polypeptides. The fluorescent probes were selected for optimal absorption and emission properties in order to facilitate their unique excitation and to enable the detection of energy transfer to Bchl. Oregon Green 488 carboxylic acid and 7-diethylaminocoumarin-3-carboxylic acid seemed to fulfill these requirements. Each of these probes were utilized to derivatize the LH1 β-polypeptide of Rhodobacter sphaeroides. It was demonstrated that the β-polypeptides did not interact with each other in the absence of Bchl. When Bchl was present, the probe-labeled β-polypeptides interacted with Bchl to form subunit-type complexes much as those formed with the native polypeptides. Energy transfer from the probe to Bchl occurred with a high efficiency. The α-polypeptide from LH 1 of Rb. sphaeroides and that from Rhodospirillum rubrum were also derivatized in the same manner. Since these polypeptides do not oligomerize in the absence of a β-polypeptide, reversible binding of a single Bchl to a single polypeptide could be measured. Dissociation constants for complex formation were estimated. The relevance of these data to earlier studies of equilibria involving subunit complexes is discussed. Also involved in the photoreceptor complex of Rb. sphaeroides and Rhodobacter capsulatus is another protein referred to as PufX. Two large segments of this protein were chemically synthesized, one reproducing the amino acid sequence of the core segment predicted for Rb. sphaeroides PufX and the other reproducing the amino acid sequence predicted for the core segment of Rb. capsulatus PufX. Each polypeptide was covalently labeled with a fluorescent probe and tested for energy transfer to Bchl. Each was found to bind Bchl with an affinity similar to the affinity of the LH 1 polypeptides for Bchl. It is suggested that PufX binds Bchl and interacts with a Bchlċα-polypeptide component of LH 1 to truncate, or interupt, the LH 1 ring adjacent to the location of the Q_B binding site of the RC. This revised version was published online in June 2006 with corrections to the Cover Date.     

A microbiologist's odyssey: Bacterial viruses to photosynthetic bacteria

Perspective can be defined as the relationships or relative importance of facts or matters from any special point of view. Thus, my Personal perspective reflects the threads I followed in a 50-year journey of research in the complex tapestry of bioenergetics and various aspects of microbial metabolism. An early interest in biochemical and microbial evolution led to the fertile hunting grounds of anoxygenic photosynthetic bacteria. Viewed as a physiological class, these organisms show remarkable metabolic versatility in that certain individual species are capable of using all the known major types of energy conversion (photosynthetic, respiratory, and fermentative) to support growth. Since such anoxyphototrophs are readily amenable to molecular genetic/biological manipulation, it can be expected that they will eventually provide important clues for unraveling the evolutionary relationships of the several kinds of energy conversion. I gradually came to believe that understanding the evolution of phototrophs would require detailed knowledge not only of how light is converted to chemical energy, but also of a) pathways of monomer production from extracellular sources of carbon and nitrogen and b) mechanisms cells use for integrating ATP regeneration with the energy-requiring biosyntheses of biological macromolecules. Serendipic observation of photoproduction of H₂ from organic compounds by Rhodospirillum rubrum in 1949 led to discovery of N₂ fixation by anoxyphototrophs, and this capacity was later exploited for the isolation of hitherto unknown species of photosynthetic prokaryotes, including the heliobacteria. Recent studies on the reaction centers of the heliobacteria suggest the possibility that these bacteria are descendents of early phototrophs that gave rise to oxygenic photosynthetic organisms.Abbreviations AMP adenosine monophosphate - ADP adenosine diphosphate - ATP adenosine triphosphate - ATPase adenosine triphosphatase - Bchl bacteriochlorophyll - DMSO dimethyl sulfoxide - NADH reduced nicotinamide adenine dinucleotide - nif ^– genes for dinitrogen fixation - Nif^– bacterial mutants incapable of dinitrogen fixation - O/R oxidation/reduction - P_i inorganic orthophosphate - R. capsulatus Rhodobacter capsulatus - R. sphaeroides Rhodobacter sphaeroides - Rps. Rhodopseudomonas - TMAO trimethyl amine-N-oxide Written at the invitation of Govindjee.     

Use of new strains of Rhodobacter sphaeroides and a modified simple culture medium to increase yield and facilitate purification of the reaction centre

A new gene expression system was developed in Rhodobacter sphaeroides, replacing a pRK415-based system used previously. The broad host-range IPTG-inducible plasmid pIND4 was used to create the plasmid pIND4-RC1 for expression of the puhA and pufQBALMX genes, encoding the reaction centre (RC) and light-harvesting complex 1 (LH1) proteins. The strain R. sphaeroides ΔRCLH was used to make a knockout of the rshI restriction endonuclease gene, enabling electroporation of DNA into the bacterium; a subsequent knockout of ppsR was made, creating the strain R. sphaeroides RCx lacking this oxygen-sensing repressor of the photosynthesis gene cluster. Using pIND4-RC1, LH1 levels were increased by a factor of about 8 over pRS1 per cell in cultures grown semi-aerobically. In addition, the ppsR knockout allowed for photosynthetic pigment–protein complex synthesis in the presence of high concentrations of molecular oxygen; here, LH1 levels per cell increased by 20 % when grown under high aeration conditions. A new medium (called RLB) is the E. coli medium LB supplemented with MgCl₂ and CaCl₂, which was found to increase growth rates and final cell culture densities, with an increase of 30 % of LH1 per cell detected in R. sphaeroides RCx(pIND4-RC1) grown in RLB versus LB medium. Furthermore, cell density was about three times greater in RLB compared to semi-aerobic conditions. The combination of all the modifications resulted in an increase of LH1 and RC per mL of culture volume by approximately 35-fold, and a decrease in the length of culture incubation time from about 5 days to ~36 h.     

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.     

Differentiation of the intracytoplasmic membrane of Rhodopseudomonas palustris induced by variations of oxygen partial pressure or light intensity

The photosynthetic apparatus of Rhodopseudomonas palustris contains, in addition to reaction center bacteriochlorophyll (Bchl) two spectral forms of light harvesting (LH) Bchl, i.e. LH Bchl I, characterized by an infrared absorption maximum at 880 nm (890 nm at 77°K) and LH Bchl II absorbing at 805 and 855 nm (805 and 870 nm at 77°K). LH Bchl I seems to be associated with a single protein species of an apparent mol. wt. of 13000 whereas LH Bchl II is apparently associated with two proteins of mol. wts. of 9000 and 11000.Cells in anaerobic cultures adapt to changes of light intensity 1. by variation of the size of the photosynthetic unit, i.e. the molar ratio of LH Bchl II to reaction center Bchl, 2. by variation of the number of photosynthetic units per unit of membrane area, 3. by regulation of the size of the intracytoplasmic membrane system.During adaptation of changes of oxygen partial pressure cells are able to synthesize reaction center Bchl, LH Bchl and intracytoplasmic membranes at different rates. The synthesis of reaction center Bchl and LH Bchl I are, however, coordinated with each other, while the syntheses of LH Bchl II and reaction center Bchl proceed independently.List of Non-Standard Abbreviations Bchl bacteriochlorophyll - ICM mitracytoplasmic membrane - LDAO lauryldimethyl aminoxide - R Rhodopseudomonas - RC reaction center - SDS sodium dodecylsulfate     

The early formation of the photosynthetic apparatus in Rhodospirillum rubrum

The time dependent assembly of the photosynthetic apparatus was studied in Rhodospirillum rubrum after transfer of cells growing aerobically in the dark to low aeration. While bacteriochlorophyll (Bchl) cellular levels increase continuously levels of soluble cytochrome c ₂do not change significantly. Absorption spectra of membranes isolated at different times after transfer reveal that incorporation of carotenoids lags behind incorporation of Bchl. However, a carotenoid fraction exhibiting spectral properties of spirilloxanthin isomers was isolated apart from membranes. This carotenoid fraction even was present in homogenates from Bchl-free, aerobically grown cells. Incorporation of U-¹⁴C-proteinhydrolyzate into membrane proteins showed that proteins are mainly formed which are specific for photosynthetic membranes. Although the proportion of reaction center (RC) Bchl per light harvesting (LH) Bchl does not change the proportions of membrane proteins present in RC and LH preparations change initially. But later on the proportions of the different proteins also reach constant values. Concerning proteins characteristic for cytoplasmic membranes a differential incorporation of label can be observed. The data indicate that the photosynthetic apparatus in Rhodospirillum rubrum is assembled through a sequential mechanism.Abbreviations Bchl bacteriochlorophyll - LH light harvesting - RC reaction center - R. Rhodospirillum - R. Rhodopseudomonas     

Isolation and characterization of bacteriochlorophyll-protein complexes from an aerobic bacterium,Pseudomonas radiora

Bacteriochlorophyll(Bchl)-protein complexes were isolated from a strictly aerobic and facultative methylotrophic bacterium Pseudomonas radiora strain MD-1. They were identified as the reaction center (RC)-B870 and the B870 complexes on the basis of their absorption spectra, light induced spectral changes and polypeptide compositions. The RC-B870 complex of this bacterium showed similar properties to those of typical purple photosynthetic bacteria, and contained c-type cytochrome which was oxidized upon illumination.Abbreviations Bchl bacteriochlorophyll - RC reaction center - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis