Sakiko Nagashima

Horizontal Transfer of the Photosynthesis Gene Cluster and Operon Rearrangement in Purple Bacteria

Abstract. A 37-kb photosynthesis gene cluster was sequenced in a photosynthetic bacterium belonging to the β subclass of purple bacteria (Proteobacteria), Rubrivivax gelatinosus. The cluster contained 12 bacteriochlorophyll biosynthesis genes (bch), 7 carotenoid biosynthesis genes (crt), structural genes for photosynthetic apparatuses (puf and puh), and some other related genes. The gene arrangement was markedly different from those of other purple photosynthetic bacteria, while two superoperonal structures, crtEF–bchCXYZ–puf and bchFNBHLM–lhaA–puhA, were conserved. Molecular phylogenetic analyses of these photosynthesis genes showed that the photosynthesis gene cluster of Rvi. gelatinosus was originated from those of the species belonging to the α subclass of purple bacteria. It was concluded that a horizontal transfer of the photosynthesis gene cluster from an ancestral species belonging to the α subclass to that of the β subclass of purple bacteria had occurred and was followed by rearrangements of the operons in this cluster.

Photocurrent and electronic activities of oriented-His-tagged photosynthetic light-harvesting/reaction center core complexes assembled onto a gold electrode.

A polyhistidine (His) tag was fused to the C- or N-terminus of the light-harvesting (LH1)-α chain of the photosynthetic antenna core complex (LH1-RC) from Rhodobacter sphaeroides to allow immobilization of the complex on a solid substrate with defined orientation. His-tagged LH1-RCs were adsorbed onto a gold electrode modified with Ni-NTA. The LH1-RC with the C-terminal His-tag (C-His LH1-RC) on the modified electrode produced a photovoltaic response upon illumination. Electron transfer is unidirectional within the RC and starts when the bacteriochlorophyll a dimer in the RC is activated by light absorbed by LH1. The LH1-RC with the N-terminal His-tag (N-His LH1-RC) produced very little or no photocurrent upon illumination at any wavelength. The conductivity of the His-tagged LH1-RC was measured with point-contact current imaging atomic force microscopy, indicating that 60% of the C-His LH1-RC are correctly oriented (N-His 63%). The oriented C-His LH1-RC or N-His LH1-RC showed semiconductive behavior, that is, had the opposite orientation. These results indicate that the His-tag successfully controlled the orientation of the RC on the solid substrate, and that the RC produced photocurrent depending upon the orientation on the electrode.

Complete Genome Sequence of Phototrophic Betaproteobacterium Rubrivivax gelatinosus IL144

Rubrivivax gelatinosus is a facultative photoheterotrophic betaproteobacterium living in freshwater ponds, sewage ditches, activated sludge, and food processing wastewater. There have not been many studies on photosynthetic betaproteobacteria. Here we announce the complete genome sequence of the best-studied phototrophic betaproteobacterium, R. gelatinosus IL-144 (NBRC 100245).

Transcription of three sets of genes coding for the core light-harvesting proteins in the purple sulfur bacterium, Allochromatium vinosum

The nucleotide sequence of the puf operon coding for the subunits of the photosynthetic reaction center and the core light-harvesting complex (LH1) of the purple sulfur bacterium, Allochromatium (A.) vinosum (formally Chromatium vinosum), was completely determined. Unlike other known puf operons, which contain only one set of genes coding for the LH1 apoproteins, pufB and pufA, the A. vinosum puf operon included three sets of pufB and pufA genes with a gene order of pufB1A1LMCB2A2B3A3. Northern hybridization analysis suggested that all of the nine puf genes are co-transcribed as a 4.43 kb mRNA. Three small mRNAs corresponding to pufB2A2B3A3, pufB2A2B3, and pufB2A2 were detected, as well as two small mRNAs covering pufB1A1. Analysis of the nucleotide sequence of the puf operon, including the flanking regions and 5′-ends of the six mRNAs, suggested that the transcription of the A. vinosum puf operon is initiated at 74 bp downstream from the bchZstop codon (295 bp upstream from the pufB1 start codon), and regulated by a promoter located at its direct upstream. The possible promoter is overlapped with a binding motif of a repressor protein for pigment-biosynthesis genes, PpsR or CrtJ, known in other purple bacteria. No other possible promoters were found within the puf genes. These findings indicate that three sets of pufA and pufB genes of A. vinosum are co-transcribed as a long mRNA containing all the puf genes, and, from this long mRNA, the five short mRNAs are possibly derived by post-transcriptional modifications.

Chapter Five - Comparison of Photosynthesis Gene Clusters Retrieved from Total Genome Sequences of Purple Bacteria

Abstract Studies on some purple photosynthetic bacteria have shown that the genes required for photosynthesis form a large cluster. This is often called a photosynthesis gene cluster (PGC) and has been described as a unique feature of purple photosynthetic bacteria. Here, photosynthesis genes were retrieved from the rapidly increasing genomic data of purple bacteria. Arrangements of these genes on the genomes were compared among 24 species and strains were selected from the wide distribution of alpha, beta, and gamma classes of purple bacteria. The presence of PGC was confirmed in the all of these, although it is divided into two or several islands in some species. Gene arrangements in several subclusters possibly forming an operon or a superoperon are well conserved while the directions and locations of these subclusters in the PGC vary according to the strains. Phylogenetic trees based on the sequences of the suspected products of the genes in the PGCs consistently place the species of the beta and gamma classes in a cluster within the branch of the alpha class, showing that a horizontal transfer of PGC had occurred in the process of evolution. Information obtained from the increasing genome projects on bacteria is very useful for the study on evolution of photosynthesis and even for its application in metabolic engineering.

Photo-induced electron transfer in intact cells of Rubrivivax gelatinosus mutants deleted in the RC-bound tetraheme cytochrome: insight into evolution of photosynthetic electron transport.

Deletion of two of the major electron carriers, the reaction center-bound tetrahemic cytochrome and the HiPIP, involved in the light-induced cyclic electron transfer pathway of the purple photosynthetic bacterium, Rubrivivax gelatinosus, significantly impairs its anaerobic photosynthetic growth. Analysis on the light-induced absorption changes of the intact cells of the mutants shows, however, a relatively efficient photo-induced cyclic electron transfer. For the single mutant lacking the reaction center-bound cytochrome, we present evidence that the electron carrier connecting the reaction center and the cytochrome bc(1) complex is the High Potential Iron-sulfur Protein. In the double mutant lacking both the reaction center-bound cytochrome and the High Potential Iron-sulfur Protein, this connection is achieved by the high potential cytochrome c(8). Under anaerobic conditions, the halftime of re-reduction of the photo-oxidized primary donor by these electron donors is 3 to 4 times faster than the back reaction between P(+) and the reduced primary quinone acceptor. This explains the photosynthetic growth of these two mutants. The results are discussed in terms of evolution of the type II RCs and their secondary electron donors.

The cytochrome c8 involved in the nitrite reduction pathway acts also as electron donor to the photosynthetic reaction center in Rubrivivax gelatinosus

The purple photosynthetic bacterium Rubrivivax gelatinosus has, at least, four periplasmic electron carriers, i.e., HiPIP, two cytochromes c₈with low- and high-midpoint potentials, and cytochrome c₄ as electron donors to the photochemical reaction center. The quadruple mutant lacking all four electron carrier proteins showed extremely slow photosynthetic growth. During the long-term cultivation of this mutant under photosynthetic conditions, a suppressor strain recovering the wild-type growth level appeared. In the cells of the suppressor strain, we found significant accumulation of a soluble c-type cytochrome that has not been detected in wild-type cells. This cytochrome c has a redox midpoint potential of about +280 mV and could function as an electron donor to the photochemical reaction center in vitro. The amino acid sequence of this cytochrome c was 65% identical to that of the high-potential cytochrome c₈of this bacterium. The gene for this cytochrome c was identified as nirM on the basis of its location in the newly identified nir operon, which includes a gene coding cytochrome cd₁-type nitrite reductase. Phylogenetic analysis and the well-conserved nir operon gene arrangement suggest that the origin of the three cytochromes c₈ in this bacterium is NirM. The two other cytochromes c₈, of high and low potentials, proposed to be generated by gene duplication from NirM, have evolved to function in distinct pathways.

Probing structure–function relationships in early events in photosynthesis using a chimeric photocomplex

Significance Phototrophic bacteria have provided fundamental insight into the biological mechanism of solar energy conversion. Early events in photosynthesis are carried out by the antenna apparatus for light-harvesting (LH) and the reaction center (RC) for charge separation. Here we describe a system for expressing a chimeric LH1-RC complex from two phylogenetically distant phototrophic purple bacteria: the LH1 from Thermochromatium tepidum and the RC from Rhodobacter sphaeroides. This system is exploited to definitively localize Ca2+ within the Tch. tepidum LH1 complex. The hybrid photocomplexes also provide powerful new tools for probing photosynthetic energy transfer, identifying intrinsic LH1–RC interactions, monitoring altered behavior of carotenoids in a nonnative environment, and linking specific amino acid residues to the specific spectroscopic properties of different phototrophic organisms. The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex.