The photosynthetic deficiency due to puhC gene deletion in Rhodobacter capsulatus suggests a PuhC protein-dependent process of RC/LH1/PufX complex reorganization

Optimal photosynthetic reaction centre (RC) and core antenna (LH1) levels in the purple bacterium Rhodobacter capsulatus require the puhC gene. Deletion of puhC had little effect on RC and LH1 assembly individually, but significantly inhibited the photosynthetic growth of RC+ LH1- strains, suggesting that maximal RC catalytic activity is PuhC-dependent. Consistent with post-assembly reorganization of the RC/LH1/PufX core complex by PuhC to include latecomer proteins, spatial separation of pufX from the RC/LH1 genes inhibited PufX accumulation and photosynthetic growth only in PuhC- strains. Photosynthetic activity improved to different degrees when PuhC homologues from three other species were expressed in PuhC- R. capsulatus, indicating that PuhC homologues function similarly but may interact inefficiently with a heterologous core complex. Anaerobic photosynthetic growth of PuhC- strains was affected by the duration of prior semiaerobic growth, and by two genes that modulate bacteriochlorophyll production: pufQ and puhE. These observations agree with a speculative model in which reorganization of the core complex is an important regenerative process, accelerated by PuhC.     

The puhE gene of Rhodobacter capsulatus is needed for optimal transition from aerobic to photosynthetic growth and encodes a putative negative modulator of bacteriochlorophyll production

A conserved orf of previously unknown function (herein designated as puhE) is located 3' of the reaction centre H (puhA) gene in purple photosynthetic bacteria, in the order puhABCE in Rhodobacter capsulatus. Disruptions of R. capsulatus puhE resulted in a long lag in the growth of photosynthetic cultures inoculated with cells grown under high aeration, and increased the level of the peripheral antenna, light-harvesting complex 2 (LH2). The amount of the photosynthetic reaction centre (RC) and its core antenna, light-harvesting complex 1 (LH1), was reduced; however, there was no decrease in expression of a lacZ reporter fused to the puf (RC and LH1) promoter, in RC assembly in the absence of LH1, or in LH1 assembly in the absence of the RC. In strains that lack LH2, disruption of puhE increased the in vivo absorption at 780 nm, which we attribute to excess bacteriochlorophyll a (BChl) pigment production. This effect was seen in the presence and absence of PufQ, a protein that stimulates BChl biosynthesis. Expression of puhE from a plasmid reduced A(780) production in puhE mutants. We suggest that PuhE modulates BChl biosynthesis independently of PufQ, and that the presence of excess BChl in PuhE(-)LH2(+) strains results in excess LH2 assembly and also interferes with the adaptation of cells during the transition from aerobic respiratory to anaerobic photosynthetic growth.     

Investigation of Rhodobacter capsulatus PufX interactions in the core complex of the photosynthetic apparatus

The photosynthetic apparatus of purple bacteria in the genus Rhodobacter includes a core complex consisting of the reaction centre (RC), light-harvesting complex 1 (LH1), and the PufX protein. PufX modulates LH1 structure and facilitates photosynthetic quinone/quinol exchange. We deleted RC/LH1 genes in pufX ⁺ and pufX ⁺⁺ (merodiploid) strains of Rhodobacter capsulatus, which reduced PufX levels regardless of pufX gene copy number and location. Photosynthetic growth of RC-only strains and independent assembly kinetics of the RC and LH1 were unaffected by pufX merodiploidy, but the absorption spectra of strains expressing the RC plus either LH1 α or β indicated that PufX may influence bacteriochlorophyll binding environments. Significant self-association of the PufX transmembrane segment was detected in a hybrid protein expression system, consistent with a role of PufX in core complex dimerization, as proposed for other Rhodobacter species. Our results indicate that in R. capsulatus PufX has the potential to be a central, homodimeric core complex component, and its cellular level is increased by interactions with the RC and LH1.     

The PufX protein of Rhodobacter capsulatus affects the properties of bacteriochlorophyll a and carotenoid pigments of light-harvesting complex 1

A pufX gene deletion in the purple bacterium Rhodobacter capsulatus causes a severe photosynthetic defect and increases core light-harvesting complex (LH1) protein and bacteriochlorophyll a (BChl) levels. It was suggested that PufX interrupts the LH1 alpha/beta ring around the reaction centre, allowing quinone/quinol exchange. However, naturally PufX(-) purple bacteria grow photosynthetically with an uninterrupted LH1. We discovered that substitutions of the Rhodobacter-specific LH1 alpha seryl-2 decrease carotenoid levels in PufX(-)R. capsulatus. An LH1 alphaS2F mutation improved the photosynthetic growth of a PufX(-) strain lacking the peripheral LH2 antenna, although LH1 BChl absorption remained above wild-type, suggesting that Rhodobacter-specific carotenoid binding is involved in the PufX(-) photosynthetic defect and LH1 expansion is not. Furthermore, PufX overexpression increased LH1-like BChl absorption without inhibiting photosynthetic growth. PufX(+) LH1 alphaS2-substituted mutant strains had wild-type carotenoid levels, indicating that PufX modulates LH1 carotenoid binding, inducing a conformational change that favours quinone/quinol exchange.     

The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens

Background  

The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences.     

The PuhB protein of Rhodobacter capsulatus functions in photosynthetic reaction center assembly with a secondary effect on light-harvesting complex 1

The core of the photosynthetic apparatus of purple photosynthetic bacteria such as Rhodobacter capsulatus consists of a reaction center (RC) intimately associated with light-harvesting complex 1 (LH1) and the PufX polypeptide. The abundance of the RC and LH1 components was previously shown to depend on the product of the puhB gene (formerly known as orf214). We report here that disruption of puhB diminishes RC assembly, with an indirect effect on LH1 assembly, and reduces the amount of PufX. Under semiaerobic growth conditions, the core complex was present at a reduced level in puhB mutants. After transfer of semiaerobically grown cultures to photosynthetic (anaerobic illuminated) conditions, the RC/LH1 complex became only slightly more abundant, and the amount of PufX increased as cells began photosynthetic growth. We discovered that the photosynthetic growth of puhB disruption strains of R. capsulatus starts after a long lag period, which is due to physiological adaptation rather than secondary mutations. Using a hybrid protein expression system, we determined that the three predicted transmembrane segments of PuhB are capable of spanning a cell membrane and that the second transmembrane segment could mediate self-association of PuhB. We discuss the possible function of PuhB as a dimeric RC assembly factor.     

Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor

The mechanisms for Fe(III) oxide reduction in Geobacter species are of interest because Fe(III) oxides are the most abundant form of Fe(III) in many soils and sediments and Geobacter species are prevalent Fe(III)-reducing microorganisms in many of these environments. Protein abundance in G. sulfurreducens grown on poorly crystalline Fe(III) oxide or on soluble Fe(III) citrate was compared with a global accurate mass and time tag proteomic approach in order to identify proteins that might be specifically associated with Fe(III) oxide reduction. A total of 2991 proteins were detected in G. sulfurreducens grown with acetate as the electron donor and either Fe(III) oxide or soluble Fe(III) citrate as the electron acceptor, resulting in 86% recovery of the genes predicted to encode proteins. Of the total expressed proteins 76% were less abundant in Fe(III) oxide cultures than in Fe(III) citrate cultures, which is consistent with the overall slower rate of metabolism during growth with an insoluble electron acceptor. A total of 269 proteins were more abundant in Fe(III) oxide-grown cells than in cells grown on Fe(III) citrate. Most of these proteins were in the energy metabolism category: primarily electron transport proteins, including 13 c-type cytochromes and PilA, the structural protein for electrically conductive pili. Several of the cytochromes that were more abundant in Fe(III) oxide-grown cells were previously shown with genetic approaches to be essential for optimal Fe(III) oxide reduction. Other proteins that were more abundant during growth on Fe(III) oxide included transport and binding proteins, proteins involved in regulation and signal transduction, cell envelope proteins, and enzymes for amino acid and protein biosynthesis, among others. There were also a substantial number of proteins of unknown function that were more abundant during growth on Fe(III) oxide. These results indicate that electron transport to Fe(III) oxide requires additional and/or different proteins than electron transfer to soluble, chelated Fe(III) and suggest proteins whose functions should be further investigated in order to better understand the mechanisms of electron transfer to Fe(III) oxide in G. sulfurreducens.     

Comparative genomic analysis of Geobacter sulfurreducens KN400, a strain with enhanced capacity for extracellular electron transfer and electricity production

ABSTRACT: BACKGROUND: A new strain of Geobacter sulfurreducens, strain KN400, produces more electrical current in microbial fuel cells and reduces insoluble Fe(III) oxides much faster than the wildtype strain, PCA. The genome of KN400 was compared to wildtype with the goal of discovering how the network for extracellular electron transfer has changed and how these two strains evolved. RESULTS: Both genomes were re-annotated, resulting in 14 fewer genes (net) in the PCA genome; 28 fewer (net) in the KN400 genome; and ca. 400 gene start and stop sites moved. 96% of genes in KN400 had clear orthologs with conserved synteny in PCA. Most of the remaining genes were in regions of genomic mobility and were strain-specific or conserved in other Geobacteraceae, indicating that the changes occurred post-divergence. There were 27,270 single nucleotide polymorphisms (SNP) between the genomes. There was significant enrichment for SNP locations in non-coding or synonymous amino acid sites, indicating significant selective pressure since the divergence. 25% of orthologs had sequence differences, and this set was enriched in phosphorylation and ATP-dependent enzymes. Substantial sequence differences (at least 12 non-synonymous SNP/kb) were found in 3.6% of the orthologs, and this set was enriched in cytochromes and integral membrane proteins. Genes known to be involved in electron transport, those used in the metabolic cell model, and those that exhibit changes in expression during growth in microbial fuel cells were examined in detail. CONCLUSIONS: The improvement in external electron transfer in the KN400 strain does not appear to be due to novel gene acquisition, but rather to changes in the common metabolic network. The increase in electron transfer rate and yield in KN400 may be due to changes in carbon flux towards oxidation pathways and to changes in ATP metabolism, both of which indicate that the overall energy state of the cell may be different. The electrically conductive pili appear to be unchanged, but cytochrome folding, localization, and redox potentials may all be affected, which would alter the electrical connection between the cell and the substrate.