Mutants Of Rhodobacter Sphaeroides Research Articles

Formation of 5-aminolevulinic acid under aerobic/dark condition by a mutant ofRhodobacter sphaeroides

A mutant strain CR-17 ofRhodobacter sphaeroides was characterized by the low activity of 5-aminolevulinic acid dehydratase (ALAD) compared to the wild-type strain. Without addition of levulinic acid (inhibitor of ALAD), the mutant secreted 5-aminolevulinic acid under anaerobic/light (64.5 μM) or under microaerobic/dark (32 μM) conditions; 82.5 μM of ALA was secreted under aerobic/dark condition with addition of 15 mM levulinic acid.

Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides

A Rhodobacter sphaeroides ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain was constructed that was complemented by plasmids containing either the form I or form II CO2 fixation gene cluster. This strain was also complemented by genes encoding foreign RubisCO enzymes expressed from a Rhodospirillum rubrum RubisCO promoter. In R. sphaeroides, the R. rubrum promoter was regulated, resulting in variable levels of disparate RubisCO molecules under different growth conditions. Photosynthetic growth of the R. sphaeroides deletion strain complemented with cyanobacterial RubisCO revealed physiological properties reflective of the unique cellular environment of the cyanobacterial enzyme. The R. sphaeroides RubisCO deletion strain and R. rubrum promoter system may be used to assess the properties of mutagenized proteins in vivo, as well as provide a potential means to select for altered RubisCO molecules after random mutagenesis of entire genes or gene regions encoding RubisCO enzymes.

Reaction centers from three herbicide resistant mutants of Rhodobacter sphaeroides 2.4.1: Kinetics of electron transfer reactions

Electron transfer rates were measured in RCs from three herbicide-resistant mutants with known amino acid changes to elucidate the structural requirements for last electron transfer. The three herbicide resistant mutants were IM(L229) (Ile-L229↦ Met), SP(L223) (Ser-L223↦ Pro) and YG(L222) (Tyr-L222↦ Gly). The electron transfer rate D(+)QA (-)QB↦D(+)QAQB (k AB) is slowed ∼3 fold in the IM(L229) and YG(L222) RCs (pH 8). The stabilization of D(+)QAQB (-) with respect to D(+)QAQB (-) (pH 8) was found to be eliminated in the IM(L229) mutant RCs (ΔG(0) ∼ 0 meV), was partially reduced in the SP(L223) mutant RCs (ΔG(0)=-30 meV), and was unaltered in the YG(L222) mutant RCs (ΔG(0)=-60 meV), compared to that observed in the native RCs (ΔG(0)=-60 meV). The pH dependences of the charge recombination rate D(+)QAQB (-)↦DQAQB (k BD) and the electron transfer from QA (-) (k QA (-)↦QA) suggest that the mutations do not affect the protonation state of Glu-L212 nor the electrostatic interactions of QB and QB (-) with Glu-L212. The binding affinities of UQ10 for the QB site were found in order of decreasing values to be native ≥IM(L229) > YG(L222)≥ SP(L223). The altered properties of the mutant RCs are used to deduce possible structural changes caused by the mutations and are dicscussed in terms of photosynthetic efficiency of the herbicide resistant strains.

1064-nm-excited Fourier transform Raman studies of bacteriochlorophyll- a in solid films and in a blue-green mutant of Rhodobacter sphaeroides

1064-nm-excited Fourier transform Raman spectra of bacteriochlorophyll- a (BChl) in various solid films and in chromatophores from a blue-green mutant of Rhodobacter sphaeroides have been obtained. The observed Raman spectra are free from high fluorescence backgrounds and sample degradation. The observed intensities seem to be enhanced because of a pre-resonant effect between the exciting radiation at 1064 nm and the Q y absorption at 770–870 nm of BChl. The spectral features are substantially different from the Soret and Q x resonance Raman spectra extensively investigated so far; several bands in the wavenumber region lower than 1200 cm −1 are particularly enhanced in the Q y pre-resonance Raman spectra. Bands due to both the C 2O and C 9O stretches appear at 1700–1620 cm −1, providing structural information on these carbonyl groups. In the CC stretching region (1620–1490 cm −1), the correlation between band positions and the co-ordination number of central magnesium, which was previously found in the Soret-excited Raman spectra, is preserved in the Q y , pre-resonance Raman spectra as well. The relative intensities of strong bands in the 1200–1000 cm −1 region appear to be useful for characterizing the BChl state. By using these advantages of the Q y , pre-resonance Raman spectra, molecular interactions and arrangements of BChl in hydrated films and in the B870 light-harvesting complex of R. sphaeroides are discussed.

Ammonia-dependent growth (Adg-) mutants of Rhodobacter capsulatus and Rhodobacter sphaeroides: comparison of mutant phenotypes and cloning of the wild-type (adg A) genes

Mutants of Rhodobacter capsulatus deficient in growth on nitrogen sources other than NH+ 4 were compared with mutants of a similar phenotype isolated from Rhodobacter sphaeroides. In addition to N2 and some amino acids (glutamate, alanine, proline, arginine), mutants of R. sphaeroides and R. capsulatus strain AD2 were unable to utilize NO3 − as sole nitrogen source for growth. Under conditions of nitrogen starvation, mutants of both species lacked the methylammonium (ammonium) uptake system, which was found in the wild-type strains under these conditions. The wild-type (adgA) genes complementing these mutants were isolated from gene banks of the respective species and localized to a 2·9 kb BamHI-SalI fragment in R. sphaeroides and to a 1·7 kb SmaI fragment in R. capsulatus. These two fragments hybridized strongly with each other, showing that they contain homologous sequences. Furthermore, the adgA gene from R. capsulatus fully restored the wild-type phenotype to Adg− mutants of R. sphaeroides and vice versa. Inactivation of the chromosomal adgA gene by insertion of an antibiotic-resistance cassette resulted in a severe inhibition of growth in rich medium and in minimal medium containing NH+ 4. This suggests that the adg A gene is required for normal growth under all growth conditions.

Energy-transfer dynamics in three light-harvesting mutants of Rhodobacter sphaeroides: a picosecond spectroscopy study

Picosecond absorption spectroscopy has been used to investigate energy-transfer dynamics within the LH1 and LH2 light-harvesting complexes of three mutants of Rhodobacter sphaeroides. We demonstrate that both complexes are inhomogeneous; each contains a specialized pigment pool which absorbs maximally at a longer wavelength. Within LH2 (mutant NF57), Bchl850 transfers energy to Bchl870 in 39 +/- 9 ps; within LH1 (mutants M21 and M2192), energy is transferred from Bchl875 to Bchl896 in 22 +/- 4 and 35 +/- 5 ps, respectively. Examination of the decay of induced absorption anisotropy indicates that each of these specialized pools exists in a state which is highly organized with respect to the remainder of the pigments. Such an arrangement may facilitate and direct energy migration toward the reaction center.

Intracytoplasmic membrane vesiculation in light-harvesting mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Intracytoplasmic membrane vesiculation in light-harvesting mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

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.

Expression of a cytochrome c2 isozyme restores photosynthetic growth of Rhodobacter sphaeroides mutants lacking the wild-type cytochrome c2 gene

Deletion of the cytochrome c 2 gene in the purple bacterium Rhodobacter sphaeroides renders it incapable of phototrophic growth (strain cycA65). However, suppressor mutants which restore the ability to grow phototrophically are obtained at relatively high frequency (1–10 in 10 7). We examined two such suppressors (strains cycA65R5 and cycA65R7) and found the expected complement of electron transfer proteins minus cytochrome c 2: SHP, c′, c 551.5, and c 554. Instead of cytochrome c 2 which elutes from DEAE-cellulose between SHP and cytochrome c′, at about 50 m m ionic strength in wild-type extracts, we found a new high redox potential cytochrome c in the mutants which elutes with cytochrome c 551.5 at about 150 m m ionic strength. The new cytochrome is more acidic than cytochrome c 2, but is about the same size or slightly smaller (13,500 Da). The redox potential of the new cytochrome from strain cycA65R7 (294 mV) is about 70 mV lower than that of cytochrome c 2. The 280 nm absorbance of the new cytochrome is smaller than that of cytochrome c 2, which suggests that there is less tryptophan (the latter has two residues). In vitro kinetics of reduction by lumiflavin and FMN semiquinones show that the reactivity of the new cytochrome is similar to that of cytochrome c 2, and that there is a relatively large positive charge (+2.6) at the site of reduction, despite the overall negative charge of the protein. This behavior is characteristic of cytochromes c 2 and unlike the majority of bacterial cytochromes examined. Fourteen out of twenty-four of the N-terminal amino acids of the new cytochrome are identical to the sequence of cytochrome c 2. The N-termini of the cycA65R5 and cycA65R7 cytochromes were the same. The kinetics and sequence data indicate that the new protein may be a cytochrome c 2 isozyme, which is not detectable in wild-type cells under photosynthetic growth conditions. We propose the name iso-2 cytochrome c 2 for the new cytochrome produced in the suppressor strains.

The construction and properties of a mutant of Rhodobacter sphaeroides with the LH1 antenna as the sole pigment protein

Recent work on the spectroscopic properties of the B875 (LH1) antenna of Rb. sphaeroides has been performed on the complex purified from membranes of the wild-type solubilised in lithium dodecyl sulphate. In order to facilitate an examination of the properties of the membrane-bound antenna free from other complexes, mutant M2192 was constructed from mutant M21, which contains reaction centres and LH1 but lacks the B800–850 (LH2)complex. This was accomplished by insertion of transposon Tn5 into the puf L gene which encodes the L polypeptide of the reaction centre. This manipulation leaves B875 as the sole pigment protein, which has been confirmed by Southern and Northern hybridisation, gel electrophoresis and fluorescence and absorbance spectroscopy. Evidence from Gaussian deconvolution of the Qy absorbance region, and from fluorescence polarisation, suggests that the long-wavelength species BChl 896 is present, and may be an inherent property of the LH1 antenna.

Expression of the Rhodobacter sphaeroides cytochrome c2 structural gene

A Rhodobacter sphaeroides mutant (CYCA1) lacking cytochrome c2 (cyt c2) was previously constructed (T. J. Donohue, A. G. McEwan, S. Van Doren, A. R. Crofts, and S. Kaplan, Biochemistry, 27: 1918-1924, 1988) by a combination of in vivo and in vitro molecular genetic techniques. CYCA1 was incapable of photosynthetic growth (PS-); in this presentation, we show that chemoheterotrophically grown CYCA1 contained significant quantities of a high potential soluble c-type cytochrome(s) with an alpha band of approximately 554 nm which had previously gone undetected under these physiological conditions in wild-type cells. In addition, the PS- phenotype of CYCA1 can be complemented in trans with stable low-copy-number (approximately 5 to 9 per R. sphaeroides genome) broad-host-range plasmids containing the wild-type cyt c2 structural gene (cycA) and upstream regulatory sequences. cyt c2 and cycA-specific mRNA levels were elevated in both the wild type and CYCA1 derivatives harboring intact cycA genes in trans, presumably as a result of increased gene dosage. Although photosynthetically grown wild-type cells contained approximately twofold more cycA-specific transcripts than chemoheterotrophically grown cells, there was an approximately four- to sevenfold increase in cyt c2 levels under photosynthetic conditions. Similarly, complemented CYCA1 strains contained between 1.3- and 2.3-fold more cycA mRNA under photosynthetic conditions than under chemoheterotrophic conditions and had 6- to 12-fold higher steady-state levels of cyt c2 under the same physiological conditions. These data are discussed in terms of possible posttranscriptional control over cyt c2.

Cloning and Oxygen-regulated Expression of the Bacteriochlorophyll Biosynthesis Genes bch E, B, A and C of Rhodobacter sphaeroides

Four mutants of the photosynthetic bacterium Rhodobacter sphaeroides were isolated which were incapable of photosynthetic growth due to inability to synthesize bacteriochlorophyll. A Rb. sphaeroides gene bank was constructed in the mobilizable vector pSUP202 and was transferred into these mutants using the helper plasmid pRK2073. Three clones that produced photosynthetic transconjugants from one or more of the bch mutants were isolated and characterized. These clones were used as probes to estimate levels of specific transcripts in cells undergoing a 100-fold increase in bacteriochlorophyll content. The maximum level of transcripts was observed at an early stage of photosynthetic membrane synthesis when only 7% of the eventual level of pigment had been synthesized.

Phenotypic and genetic characterization of cytochrome c2 deficient mutants of Rhodobacter sphaeroides.

Rhodobacter sphaeroides mutants lacking cytochrome c2 (cyt c2) have been constructed by site-specific recombination between the wild-type genomic cyt c2 structural gene (cycA) and a suicide plasmid containing a defective cyc operon where deletion of cycA sequences was accompanied by insertion of a KnR gene. Southern blot analysis confirmed that the wild-type cyc operon was exchanged for the inactivated cycA gene, presumably by double-reciprocal recombination. Spectroscopic and immunochemical measurements, together with genetic complementation, established that the inability of these mutants to grow under photosynthetic conditions was due to the lack of cyt c2. The cyt c2 deficient strains reduced photooxidized reaction center complexes approximately 4 orders of magnitude more slowly than the parent strain. The phenotype and characteristics of these mutants were restored when a wild-type cyc operon was introduced on a stable low copy number plasmid. These experiments provide the first genetic evidence for the obligatory role of cyt c2 in wild-type cyclic photosynthetic electron transport in R. sphaeroides. We have also observed that the R. sphaeroides cyt c2 deficient strains spontaneously gave rise to photosynthetically competent pseudorevertants at a frequency which suggests that the cyt c2 independent photosynthetic electron transport which suppresses the phenotype of the cyt c2 deficient strains was the result of a single mutation elsewhere in the genome.

Physiological and structural analysis of light-harvesting mutants of Rhodobacter sphaeroides.

Two mutants of Rhodobacter sphaeroides defective in formation of light-harvesting spectral complexes were examined in detail. Mutant RS103 lacked the B875 spectral complex despite the fact that substantial levels of the B875-alpha polypeptide (and presumably the beta polypeptide) were present. The B800-850 spectral complex was derepressed in RS103, even at high light intensities, and the growth rate was near normal at high light intensity but decreased relative to the wild type as the light intensity used for growth decreased. Mutant RS104 lacked colored carotenoids and the B800-850 spectral complex, as well as the cognate apoproteins. This strain grew normally at high light intensity and, as with RS103, the growth rate decreased as the light intensity used for growth decreased. At very low light intensities, however, RS104 would grow, whereas RS103 would not. Structural analysis of these mutants as well as others revealed that the morphology of the intracytoplasmic membrane invaginations is associated with the presence or absence of the B800-850 complex as well as of carotenoids. A low-molecular-weight intracytoplasmic membrane polypeptide, which may play a role in B800-850 complex formation, is described, as is a 62,000-dalton polypeptide whose abundance is directly related to light intensity as well as the absence of either of the light-harvesting spectral complexes. These data, obtained from studies of mutant strains and the wild type, are discussed in light of photosynthetic membrane formation and the abundance of spectral complexes per unit area of membrane. Finally, a method for the bulk preparation of the B875 complex from wild-type strain 2.4.1 is reported.

Construction, characterization, and complementation of a Puf- mutant of Rhodobacter sphaeroides.

A Puf- strain of Rhodobacter sphaeroides (PUFB1) was constructed by deleting a portion of the proximal region of the puf operon and inserting a kanamycin resistance gene cartridge. Southern blot analysis demonstrated that in PUFB1, the defective copy of the puf operon had replaced, through homologous recombination, the normal chromosomal copy. The Puf- phenotype was characterized by the inability of PUFB1 to grow photoheterotrophically (PS-), the lack of detectable puf-specific transcripts, the absence of the light-harvesting I complex and, by inference, the reaction center spectral complex, and greatly reduced levels of the light-harvesting II complex. The PS+ phenotype was restored to PUFB1 when a 13-kilobase BamHI restriction endonuclease fragment containing the entire puf operon and flanking regions was cloned into the broad-host-range plasmid vector RK2 derivative pRK404 and introduced by conjugation into PUFB1. In these complemented strains, there was an increased number of copies of the puf operon (four to six copies per defective chromosomal copy) as the result of plasmid copy number. However, there was no concomitant increase in either the specific bacteriochlorophyll content or the level of puf-specific transcripts when these strains were grown photoheterotrophically.

Reaction centers from three herbicide-resistant mutants of Rhodobacter sphaeroides 2.4.1: sequence analysis and preliminary characterization

Many herbicides that inhibit photosynthesis in plants also inhibit photosynthesis in bacteria. We have isolated three mutants of the photosynthetic bacterium Rhodobacter sphaeroides that were selected for increased resistance to the herbicide terbutryne. All three mutants also showed increased resistance to the known electron transfer inhibitor o-phenanthroline. The primary structures of the mutants were determined by recombinant DNA techniques. All mutations were located on the gene coding for the L-subunit resulting in these changes Ile(229) → Met, Ser(223) → Pro and Tyr(222) → Gly. The mutations of Ser(223) is analogous to the mutation of Ser(264) in the D1 subunit of photosystem II in green plants, strengthening the functional analogy between D1 and the bacterial L-subunit. The changed amino acids of the mutant strains form part of the binding pocket for the secondary quinone, Q b . This is consistent with the idea that the herbicides are competitive inhibitors for the Q b binding site. The reaction centers of the mutants were characterized with respect to electron transfer rates, inhibition constants of terbutryne and o-phenanthroline, and binding constants of the quinone UQ0 and the inhibitors. By correlating these results with the three-dimensional structure obtained from x-ray analysis by Allen et al. (1987a, 1987b), the likely positions of o-phenanthroline and terbutryne were deduced. These correspond to the positions deduced by Michel et al. (1986a) for Rhodopseudomonas viridis.

Spectroscopic properties of antenna complexes of Rhodobacter sphaeroides in vivo

Intact membranes of antenna mutants of Rhodobacter sphaeroides obtained by chemical mutagenesis containing only the B800–850 or the B875 reaction center complex were used to study the spectral properties of these antenna complexes separately in vivo. Wild-type spectral characteristics were restored to each mutant, following complementation by the relevant gene. It is shown that the absorption spectra of recombinant strains and of wild-type Rhodobacter sphaeroides can be analyzed in terms of those of the separate complexes as observed in the mutants. Distinct differences occur between the spectra of the antenna complexes isolated by means of detergent solubilization of the membrane and those of the mutants. Measurements of absorption and flash-induced absorption difference spectra and of linear dichroism and fluorescence polarization spectra at low temperature indicate that in the intact membrane the previously characterized bacteriochlorophyll Q y absorption bands near 800, 850 and 875 nm display an optical inhomogeneity and that they all contain a relatively weak transition at longer wavelength, the orientation of which is more parallel to the membrane plane than the orientation of the main transitions. Rapid and efficient energy transfer to the long-wave component (BChl 870) in the B800–850 complex could be demonstrated. Some of the long-wave transitions are also observable at room temperature. They may reflect the mode of aggregation of the complexes in their lipid environment and, by increased overlap between donor emission and acceptor absorbance, serve to facilitate energy transfer within the antenna system.