Differential assembly of polypeptides of the light-harvesting 2 complex encoded by distinct operons during acclimation of Rhodobacter sphaeroides to low light intensity

In order to obtain an improved understanding of the assembly of the bacterial photosynthetic apparatus, we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced incident light intensity. Photoheterotrophically growing cells were shifted from 1,100 to 100?W/m(2) and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)-LH1 complexes were excised and subjected to in-gel trypsin digestion followed by liquid chromatography (LC)-mass spectroscopy (MS)/MS. The results revealed that the LH2 band contained distinct levels of the LH2-α and -β polypeptides encoded by the two puc operons. Polypeptide subunits encoded by the puc2AB operon predominated under high light and in the early stages of acclimation to low light, while after 24?h, the puc1BAC components were most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in Puc1A, was a protein of major abundance. A predominance of Puc2A components in the LH2 complex formed at high light intensity is followed by a >2.5-fold enrichment in Puc1B levels between 3 and 24?h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This indicates that the puc1BAC operon is under more stringent light control, thought to reflect differences in the puc1 upstream regulatory region. In contrast, elevated levels of Puc2 polypeptides were seen 48?h after the gratuitous induction of ICM formation at low aeration in the dark, while after 24?h of acclimation to low light, an absence of alterations in Puc polypeptide distributions was observed in the upper LH2-enriched gel band, despite an approximate twofold increase in overall LH2 levels. This is consistent with the origin of this band from a pool of LH2 laid down early in development that is distinct from subsequently assembled LH2-only domains, forming the LH2 gel band.     

Characterization and complementation of a mutant of Rhodobacter sphaeroides with a chromosomal deletion in the light-harvesting (LH2) genes

An LH2- strain of Rhodobacter sphaeroides, DBC1, has been constructed by deleting the puc operon, which encodes the LH2 alpha and beta polypeptides, from the chromosome and replacing it with a kanamycin resistance gene. Southern blot analysis indicates that the 950 bp BamHI restriction fragment which contains the puc operon has been lost and has been replaced by the 1.25 kb Km(R) cassette derived from Tn903. Strain DBC1 lacked the LH2 complex, as shown by loss of the characteristic absorbance bands at 800 and 850 nm. The LH2 polypeptides were also found to be absent after SDS-PAGE. The wild-type phenotype was restored to DBC1 by the transfer of a 3.8 kb BscI fragment containing the puc operon in plasmid pMA81. Transconjugants possessed a wild-type absorbance spectrum and LH2 polypeptides.     

The accumulation of the light-harvesting 2 complex during remodeling of the Rhodobacter sphaeroides intracytoplasmic membrane results in a slowing of the electron transfer turnover rate of photochemical reaction centers

A functional proteomic analysis of the intracytoplasmic membrane (ICM) development process was performed in Rhodobacter sphaeroides during adaptation from high-intensity illumination to indirect diffuse light. This initiated an accelerated synthesis of the peripheral light-harvesting 2 (LH2) complex relative to that of LH1-reaction center (RC) core particles. After 11 days, ICM vesicles (chromatophores) and membrane invagination sites were isolated by rate-zone sedimentation and subjected to clear native gel electrophoresis. Proteomic analysis of gel bands containing the RC-LH1 and -LH2 complexes from digitonin-solubilized chromatophores revealed high levels of comigrating electron transfer enzymes, transport proteins, and membrane assembly factors relative to their equivalent gel bands from cells undergoing adaptation to direct low-level illumination. The GroEL chaperonin accounted for >65% of the spectral counts in the RC-LH1 band from membrane invagination sites, which together with the appearance of a universal stress protein suggested that the viability of these cells was challenged by light limitation. Functional aspects of the photosynthetic unit assembly process were monitored by near-IR fast repetition rate analysis of variable fluorescence arising from LH-bacteriochlorophyll a components. The quantum yield of the primary charge separation during the early stages of adaptation showed a gradual increase (variable/maximal fluorescence = 0.78-0.83 between 0 and 4 h), while the initial value of ~70 for the functional absorption cross section (σ) gradually increased to 130 over 4 days. These dramatic σ increases showed a direct relation to gradual slowing of the RC electron transport turnover rate (τ(QA)) from ~1.6 to 6.4 ms and an ~3-fold slowing of the rate of reoxidation of the ubiquinone pool. These slowed rates are not due to changes in UQ pool size, suggesting that the relation between increasing σ and τ(QA) reflects the imposition of constraints upon free diffusion of ubiquinone redox species between the RC and cytochrome bc(1) complex as the membrane bilayer becomes densely packed with LH2 rings.     

Thorax closure in Drosophila: involvement of Fos and the JNK pathway.

Dorsal closure, a morphogenetic movement during Drosophila embryogenesis, is controlled by the Drosophila JNK pathway, D-Fos and the phosphatase Puckered (Puc). To identify principles of epithelial closure processes, we studied another cell sheet movement that we term thorax closure, the joining of the parts of the wing imaginal discs which give rise to the adult thorax during metamorphosis. In thorax closure a special row of margin cells express puc and accumulate prominent actin fibres during midline attachment. Genetic data indicate a requirement of D-Fos and the JNK pathway for thorax closure, and a negative regulatory role of Puc. Furthermore, puc expression co-localises with elevated levels of D-Fos, is reduced in a JNK or D-Fos loss-of-function background and is ectopically induced after JNK activation. This suggests that Puc acts downstream of the JNK pathway and D-Fos to mediate a negative feed-back loop. Therefore, the molecular circuitry required for thorax closure is very similar to the one directing dorsal closure in the embryo, even though the tissues are not related. This finding supports the hypothesis that the mechanism controlling dorsal closure has been co-opted for thorax closure in the evolution of insect metamorphosis and may represent a more widely used functional module for tissue closure in other species as well.     

Adaptation of intracytoplasmic membranes to altered light intensity in Rhodobacter sphaeroides

The model photosynthetic bacterium Rhodobacter sphaeroides uses a network of bacteriochlorophyll (BChl)-protein complexes embedded in spherical intracytoplasmic membranes (ICM) to collect and utilise solar energy. We studied the effects of high- and low-light growth conditions, where BChl levels increased approximately four-fold from 1.6×10(6) to 6.5×10(6) molecules per cell. Most of this extra pigment is accommodated in the proliferating ICM system, which increases from approximately 274 to 1468 vesicles per cell. Thus, 16×10(6)nm(2) of specialised membrane surface area is made available for harvesting and utilising solar energy compared to 3×10(6)nm(2) under high-light conditions. Membrane mapping using atomic force microscopy revealed closely packed dimeric and monomeric reaction centre-light harvesting 1-PufX (RC-LH1-PufX) complexes in high-light ICM with room only for small clusters of LH2, whereas extensive LH2-only domains form during adaptation to low light, with the LH2/RC ratio increasing three-fold. The number of upper pigmented band (UPB) sites where membrane invagination is initiated hardly varied; 704 (5.8×10(5) BChls/cell) and 829 (4.9×10(5) BChls/cell) UPB sites per cell were estimated under high- and low-light conditions, respectively. Thus, the lower ICM content in high-light cells is a consequence of fewer ICM invaginations reaching maturity. Taking into account the relatively poor LH2-to-LH1 energy transfer in UPB membranes it is likely that high-light cells are relatively inefficient at energy trapping, but can grow well enough without the need to fully develop their photosynthetic membranes from the relatively inefficient UPB to highly efficient mature ICM.     

Phosphatidylcholine is required for the efficient formation of photosynthetic membrane and B800-850 light-harvesting complex in Rhodobacter sphaeroides

No phosphatidylcholine (PC) was detected in the membrane of Rhodobacter sphaeroides pmtA mutant (PmtA1) lacking phosphatidylethanolamine (PE) N-methyltransferase, whereas PE in the mutant was increased up to the mole % comparable to the combined level of PE and PC of wild type. Neither the fatty acid composition nor the fluidity of membrane was altered by pmtA mutation. Consistently, aerobic and photoheterotrophic growth of PmtA1 were not different from wild type. However, PmtA1 showed an extended lag phase (15 h) after the growth transition from aerobic to photoheterotrophic conditions, indicating the PC requirement for the efficient formation of intracytoplasmic membrane (ICM). Interestingly, the B800-850 complex of PmtA1 was decreased more than twofold in comparison with wild type, whereas the level of the B875 complex comprising the fixed photosynthetic unit was not changed. Since puc expression is not affected by pmtA mutation, PC appears to be required for the proper formation of the B800-850 complex in the ICM of R. sphaeroides.     

Characterization of expressed pigmented core light harvesting complex (LH 1) in a reaction center deficient mutant of Blastochloris viridis

The utility of photosynthetically defective mutants in the purple photosynthetic bacterium Blastochloris viridis (formerly Rhodopseudomonas viridis)was demonstrated with construction of a reaction-center deficient mutant, LH 1-H. This LH 1-H mutant has a photosynthetic apparatus in which most of the puf operon genes were deleted, resulting in an organism containing only the genes for the light harvesting polypeptides and the H subunit of the reaction center. This B. viridisstrain containing a truncation of the puf operon was characterized by gel electrophoresis, lipid-to-protein ratio analysis, optical spectroscopy, electron paramagnetic resonance and transmission electron microscopy. Optical and electron paramagnetic resonance spectroscopies revealed no photoactivity in this LH 1-H mutant consistent with the absence of intact reaction centers. Electron paramagnetic resonance evidence for assembled LH 1 complexes suggested that the interactions between light harvesting polypeptide complexes in membranes were largely unchanged despite the absence of their companion reaction center cores. The observed increase in the lipid-to-protein ratio was consistent with modified interactions between LH 1s, a view supported by transmission electron microscopy analysis of membrane fragments. The results show that B. viridis can serve as a practical system for investigating structure-function relationships in membranes and photosynthesis through the construction of photosynthetically defective mutants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase.

Two new loci, prrB and prrC, involved in the positive regulation of photosynthesis gene expression in response to anaerobiosis, have been identified in Rhodobacter sphaeroides. prrB encodes a sensor histidine kinase that is responsive to the removal of oxygen and functions through the response regulator PrrA. Inactivation of prrB results in a substantial reduction of photosynthetic spectral complexes as well as in the inability of cells to grow photosynthetically at low to medium light intensities. Together, prrB and prrA provide the major signal involved in synthesis of the specialized intracytoplasmic membrane (ICM), harboring components essential to the light reactions of photosynthesis. Previously, J. K. Lee and S. Kaplan (J. Bacteriol. 174:1158-1171, 1992) identified a mutant which resulted in high-level expression of the puc operon, encoding the apoproteins giving rise to the B800-850 spectral complex, in the presence of oxygen as well as in the synthesis of the ICM under conditions of high oxygenation. This mutation is shown to reside in prrB, resulting in a leucine-to-proline change at position 78 in mutant PrrB (PRRB78). Measurements of mRNA levels in cells containing the prrB78 mutation support the idea that prrB is a global regulator of photosynthesis gene expression. Two additional mutants, PRRB1 and PRRB2, which make two truncated forms of the PrrB protein, possess substantially reduced amounts of spectral complexes. Although the precise role of prrC remains to be determined, evidence suggests that it too is involved in the regulatory cascade involving prrB and prrA. The genetic organization of the photosynthesis response regulatory (PRR) region is discussed.     

Transforming growth factor-beta 1 stimulates fibronectin production in bovine adrenocortical cells in culture.

Transforming growth factor-beta (TGF-beta 1) suppresses cortisol production when added to cultured bovine adrenocortical (BAC) cells while concomitantly increasing fibronectin synthesis and assembly into extracellular fibrils. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of gelatin-Sepharose-treated media from BAC cells demonstrated a 2-fold stimulation of fibronectin production by TGF-beta 1 in both the presence and absence of serum. Indirect immunofluorescence studies revealed that TGF-beta 1 caused a striking increase in the fibronectin content of BAC extracellular matrix. TGF-beta 1 caused a 4-fold increase in deoxycholate-insoluble fibronectin after 12 h and a 7-fold increase after 24 h over that of control BAC cultures. Northern hybridization analyses indicated that TGF-beta 1 stimulated levels of fibronectin poly(A)+ RNA 2.3-fold. We found that cultured BAC cells express TGF-beta 1 mRNA, suggesting a possible autocrine role for TGF-beta 1 in the adrenal.