The Reaction Center H Subunit Is Not Required for High Levels of Light-Harvesting Complex 1 in Rhodospirillum rubrum Mutants

Abstract

The gene (puhA) encoding the H subunit of the reaction center (RC) was deleted by site-directed interposon mutagenesis by using a kanamycin resistance cassette lacking transcriptional terminators to eliminate polar effects in both the wild-type strain Rhodospirillum rubrum S1 and the carotenoid-less strain R. rubrum G9. The puhA interposon mutants were incapable of photoheterotrophic growth but grew normally under aerobic chemoheterotrophic conditions. Absorption spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the RCs were absent. In minimal medium and also in modified medium containing succinate and fructose, the light-harvesting 1 complex (LH1) levels of the S1-derived mutants were about 70 to 100% of the wild-type levels in the same media. The correct assembly of LH1 in the membrane and the pigment-pigment interaction were confirmed by near-infrared circular dichroism spectroscopy. LH1 formation was almost absent when the carotenoid-less G9-derived puhA mutants were grown in standard minimal medium, suggesting that carotenoids may stabilize LH1. In the fructose-containing medium, however, the LH1 levels of the G9 mutants were 70 to 100% of the parental strain levels. Electron micrographs of thin sections of R. rubrum revealed photosynthetic membranes in all mutants grown in succinate-fructose medium. These studies indicate that the H subunit of the RC is necessary neither for maximal formation of LH1 nor for photosynthetic membrane formation but is essential for functional RC assembly.It is well established that in phototrophic purple bacteria the photosynthetic unit is expressed in a specialized intracytoplasmic membrane (ICM) (16,18) and is composed of a reaction center (RC) surrounded by a light-harvesting complex (1,17,44). Although most phototrophic bacteria contain two kinds of light-harvesting complexes, light-harvesting complex 1 (LH1) and light-harvesting complex 2 (LH2), only one of these, LH1, is in intimate contact with the RC. Recent structural studies (6,20,22,25,31,36,37,43,(59)(60)(61) have indicated that the component ␣␤ dimers of LH1, each of which binds two bacteriochlorophyll (BChl) molecules and at least one molecule of carotenoid, form a ring around the RC. For Rhodospirillum rubrum (33,37,59,61) and Blastochloris viridis (previously called Rhodopseudomonas viridis) (60), both of which contain only a single light-harvesting complex, LH1 appears to completely surround the RC. In Rhodobacter sphaeroides, however, recent electron micrographic evidence (36) has suggested that the LH1 in this organism may not be completely closed, although the low resolution of the data in this study may not have been sufficient to allow a definite conclusion to be drawn. In all cases, however, it appears that the assembly of RCs and LH1 in vivo is always perfectly coordinated, and neither biochemical nor spectroscopic evidence has indicated the presence of empty LH1 rings or incorrectly assembled RCs. A continuing puzzle, therefore, concerns the mechanism of assembly of...

High-Level Production of the Industrial Product Lycopene by the Photosynthetic Bacterium Rhodospirillum rubrum

“…3) showed, as reported previously for the thspx-expressing mutant, ST4 (27), that the near-infrared (near-IR) Q y absorption maximum of the LH1 complex is present at 882 nm, which is characteristic for crt-containing LH1 complexes (11,60). The LH1 peak maximum at 882 nm is easily distinguishable from that of LH1 complexes lacking carotenoids, which occurs at 874 nm (11,17,32,60). The absorption maxima of the observable RC at 750 and 802 nm, corresponding to the RC-bound bacteriopheophytin (BPh) and accessory BChla, respectively, are unchanged in both position and relative intensity for all three strains.…”

“…In fact, the thspx-LH1 complex shows a somewhat higher photochemical stability than that from S1 (data not shown). Second, the position of the near-IR peak maximum of the SLYC18 LH1 at 882 nm, which is a well-established diagnostic feature of LH1 containing bound carotenoid (11,17,32,60), is identical to those of S1 and ST4. The prediction that the lycopene is indeed bound specifically to LH1 complexes was confirmed unambiguously by the analysis of purified LH1 complexes obtained from SLYC18.…”

“…The very strong similarity between the ST4 and SLYC18 cell spectra suggests strongly that, in SLYC18, lycopene is also bound to pigmentprotein complexes. Fractionation of SLYC18 cells after breakage with a French press-type apparatus, followed by differential centrifugation (17,32), showed the lycopene to be located exclusively in the membrane fraction, of which the ICM, containing the LH1 and RC, comprises ca. 80%.…”

“…2). Subsequently, a 1.2-kb BstEII fragment was replaced with a 1.5-kb HindIII/SalI blunt-ended fragment, derived from Tn5, which contains the npt gene encoding neomycin phosphotransferase (32), yielding plasmids pBsSGE5K1 and pBsSGE5K2, with the npt gene in the opposite and same orientation as the crtD promoter, respectively. The BstEII fragment deletion simultaneously eliminates large regions of both the crtC and crtD genes, thus leading to their functional arrest.…”

“…The BstEII fragment deletion simultaneously eliminates large regions of both the crtC and crtD genes, thus leading to their functional arrest. Finally, the inserts of pBsSGE5K1 and pBsSGE5K2 were subcloned into the conjugable suicide vector pSUP202 (50) to yield the plasmids pSUPSGE5K1 and pSUPSGE5K2, respectively, which were subsequently triparentally conjugated with R. rubrum S1 (12,32). Selection of doubly recombinant npt interposon mutants was determined by their Kan r Tet s phenotypes.…”