An overlap between operons involved in carotenoid and bacteriochlorophyll biosynthesis in Rhodobacter capsulatus

A new example of superoperonal gene arrangement has been documented in the Rhodobacter capsulatus photosynthetic gene cluster. The promoter for the operon initiated by the bchI gene is embedded within an upstream operon for carotenoid synthesis. The stop codon for the crtA gene, the only gene in the first operon, overlaps the start codon of the downstream bchI gene. As a consequence of this overlap, the promoter(s) for the bch operon must be located within the crtA structural gene. The bchI gene is shown here for the first time to be required for the conversion of protoporphyrin IX to subsequent intermediates in bacteriochlorophyll biosynthesis.     

Carotenoid biosynthesis in photosynthetic bacteria. Genetic characterization of the Rhodobacter capsulatus CrtI protein

Carotenoids are photoprotective pigments present in many photosynthetic and nonphotosynthetic organisms. The desaturation of phytoene into phytofluene is an early step in the biosynthetic pathway that in the photosynthetic bacterium Rhodobacter capsulatus is mediated by the product of the crtI gene. Here we report the sequence of this gene and the identification of CrtI as a membrane protein of approximate Mr 60,000. Mutant strains with 5-fold lower or 10-fold higher levels of CrtI with respect to wild type have only small differences in their carotenoid content, indicating that the cellular concentration of CrtI is not a limiting factor in carotenoid biosynthesis. However, a correlation was found between the levels of CrtI and the formation of a photosynthetic antenna system.     

Isolation of mutants and genes involved in cytochromes c biosynthesis in Rhodobacter capsulatus.

Mutants of the photosynthetic bacterium Rhodobacter capsulatus that have combined deficiencies in the cytochrome b/c1 complex and other c-type cytochromes have been isolated. These mutants were unable to grow anaerobically in the light or dark but could grow aerobically. Cosmids with R. capsulatus wild-type DNA that complement the mutants have been used to construct genetic and physical maps of the affected genes. Complementation profiles with Tn5 and mini-Mu insertions in these cosmids and subcloned fragments from them indicated that at least three genes (called helA, helB, and helC) are involved in the defects in cytochromes c biosynthesis. The genes are clustered, and helC is transcribed away from helA and helB. Stable insertion mutants in each gene were constructed. It is postulated that helA, helB, and helC are involved in posttranslational processing during cytochromes c synthesis.     

RegA control of bacteriochlorophyll and carotenoid synthesis in Rhodobacter capsulatus

We provide in vivo genetic and in vitro biochemical evidence that RegA directly regulates bacteriochlorophyll and carotenoid biosynthesis in Rhodobacter capsulatus. beta-Galactosidase expression assays with a RegA-disrupted strain containing reporter plasmids for Mg-protoporphyrin IX monomethyl ester oxidative cyclase (bchE), Mg-protoporphyrin IX chelatase (bchD), and phytoene dehydrogenase (crtI) demonstrate RegA is responsible for fourfold anaerobic induction of bchE, threefold induction of bchD, and twofold induction of crtI. Promoter mapping studies, coupled with DNase I protection assays, map the region of RegA binding to three sites in the bchE promoter region. Similar studies at the crtA and crtI promoters indicate that RegA binds to a single region equidistant from these divergent promoters. These results demonstrate that RegA is directly responsible for anaerobic induction of bacteriochlorophyll biosynthesis genes bchE, bchD, bchJ, bchI, bchG, and bchP and carotenoid biosynthesis genes crtI, crtB, and crtA.     

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

Abstract In Chlamydomonas reinhardtii there are three glutamate dehydrogenase isozymes which can use both NADH and NADPH as cofactors and respond differently to different nitrogen sources and several stress conditions. From data of induction of isozymes in different metabolic situations, we propose a possible physiological role for each of them in algal carbon and nitrogen metabolism.     

Cytochrome c2 mutants of Rhodobacter capsulatus.

Although structurally related to other members of the class I c-type cytochromes, the cytochromes c2 have little amino acid sequence homology to the eukaryotic cytochromes c. Moreover, the cytochromes c2 exhibit distinct properties such as redox potential and an isoelectric point. In an effort to understand the differences between the cytochromes c2 and the other class I c-type cytochromes, we have developed a genetic system to study Rhodobacter capsulatus cytochrome c2 by site-directed mutagenesis. We describe here overproduction of R. capsulatus wild-type cytochrome c2 in cytochrome c2-minus strains of R. capsulatus and Rhodobacter sphaeroides. We demonstrate that R. capsulatus wild-type cytochrome c2 can transcomplement for photosynthetic growth in R. sphaeroides. Further, we describe the generation, expression, and in vivo functionality properties of nine R. capsulatus site-directed mutants. We show that mutants K12D, K14E, K32E, K14E/K32E, P35A, W67Y, and Y75F are overproduced and functional in vivo. In contrast, mutants Y75C and Y75S are expressed at low levels and exhibit poor functionality in vivo. These findings establish an effective system for the production of R. capsulatus site-directed mutants and demonstrate that interspecies complementation can be used to detect defective cytochrome c2 mutants.     

Phenotypic characterisation and genetic complementation of dimethylsulfoxide respiratory mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract Two chlorate resistant mutants of Rhodobacter sphaeroides were isolated which were deficient in dimethylsulfoxide reductase activity. Immunoblotting experiments showed that the phenotype of these mutants and that of Rhodobacter capsulatus strain DK9, a mutant unable to reduce dimethylsulfoxide, was correlated with low or undetectable levels of the dimethylsulfoxide reductase apoprotein. All three mutants were complemented by a cosmid from a library of Rhodobacter sphaeroides genomic DNA. Further genetic complementation analysis revealed that functions required for restoration of dimethylsulfoxide reductase activity in the Rhodobacter sphaeroides mutants were encoded on an 9 kb EcoR1 DNA fragment derived from this cosmid. Expression of this 9 kb DNA fragment in Escherichia coli showed that it encoded the dimethylsulfoxide reductase structural gene of Rhodobacter sphaeroides .     

Characterization of LHI- and LHI+ Rhodobacter capsulatus pufA mutants.

The NH2 termini of light-harvesting complex I (LHI) polypeptides alpha and beta of Rhodobacter capsulatus are thought to be involved in the assembly of the LHI complex. For a more detailed study of the role of the NH2-terminal segment of the LHI alpha protein in insertion into the intracytoplasmic membrane (ICM) of R. capsulatus, amino acids 6 to 8, 9 to 11, 12 and 13, or 14 and 15 of the LHI alpha protein were deleted. Additionally, the hydrophobic stretch of the amino acids 7 to 11 was lengthened by insertion of hydrophobic or hydrophilic amino acids. All mutations abolished the ability of the mutant strains to form a functional LHI antenna complex. All changes introduced into the LHI alpha protein strongly reduced the stability of its LHI beta partner protein in the ICM. The effects on the mutated protein itself, however, were different. Deletion of amino acids 6 to 8, 9 to 11, or 14 and 15 drastically reduced the amount of the LHI alpha protein inserted into the membrane or prevented its insertion. Deletion of amino acids 12 and 13 and lengthening of the stretch of amino acids 7 to 11 reduced the half-life of the mutated LHI alpha protein in the ICM in comparison with the wild-type LHI alpha protein. Under the selective pressure of low light, revertants which regained a functional LHI antenna complex were identified only for the mutant strain deleted of amino acids 9 to 11 of the LHI alpha polypeptide [U43 (pTPR15)]. The restoration of the LHI+ phenotype was due to an in-frame duplication of 9 bp in the pufA gene directly upstream of the site of deletion present in strain U43(pTPR15). The duplicated nucleotides code for the amino acids Lys, Ile, and Trp. Membranes purified from the revertants were different from that of the reaction center-positive LHI+ LHII- control strain U43(pTX35) in doubling of the carotenoid content and increase of the size of the photosynthetic unit. By separating the reaction center and LHI complexes of the revertants by native preparative gel electrophoresis, we confirmed that the higher amount of carotenoids was associated with the LHI proteins.     

Cloning and characterization of the rpoH gene of Rhodobacter capsulatus

By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified a new σ factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34?kDa with strong similarity to the RpoH (σ ³²) factors from other bacterial species. It was not possible to inactivate the R.?capsulatusrpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5′ ends of the mRNAs were mapped to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease in oxygen tension. Western analysis using a σ factor-specific antibody revealed the accumulation of a protein of about 34?kDa after heat shock, and an increase in the amounts of a protein with the same size after reduction of oxygen tension in R.?capsulatus cultures.     

Genetic manipulation of carotenoid biosynthesis and photoprotection

There are multiple complementary and redundant mechanisms to provide protection against photo-oxidative damage, including non-photochemical quenching (NPQ). NPQ dissipates excess excitation energy as heat by using xanthophylls in combination with changes to the light-harvesting complex (LHC) antenna. The xanthophylls are oxygenated carotenoids that in addition to contributing to NPQ can quench singlet or triplet chlorophyll and are necessary for the assembly and stability of the antenna. We have genetically manipulated the expression of the epsilon-cyclase and beta-carotene hydroxylase carotenoid biosynthetic enzymes in Arabidopsis thaliana. The epsilon-cyclase overexpression confirmed that lut2 (lutein deficient) is a mutation in the epsilon-cyclase gene and demonstrated that lutein content can be altered at the level of mRNA abundance with levels ranging from 0 to 180% of wild-type. Also, it is clear that lutein affects the induction and extent of NPQ. The deleterious effects of lutein deficiency on NPQ in Arabidopsis and Chlamydomonas are additive, no matter what the genetic background, whether npq1 (zeaxanthin deficient), aba1 or antisense beta-hydroxylase (xanthophyll cycle pool decreased). Additionally, increasing lutein content causes a marginal, but significant, increase in the rate of induction of NPQ despite a reduction in the xanthophyll cycle pool size.     

Purification and characterization of a 7Fe-ferredoxin from Rhodobacter capsulatus

A ferredoxin was purified anaerobically from Rhodobacter capsulatus grown photoheterotrophically with excess ammonia. This ferredoxin, called ferredoxin II (FdII), had a molecular weight of approximatively 15,000 by gel filtration and 14,000 by SDS polyacrylamide gel electrophoresis indicating that it is monomeric. Its absorption spectrum (oxidized form) exhibited maxima at 280 nm and 400 nm; the A400/A280 ratio had a calculated value of 0.55. Chemical determination of its iron and sulfur atom content, the value of the extinction coefficient at 400 nm (epsilon 400 = 26.8 mM-1 cm-1) and EPR spectra indicated that ferredoxin II contained one [3Fe-4S] and one [4Fe-4S] cluster. Upon reduction with excess dithionite only the [3Fe-4S] cluster became reduced. The reduction of both clusters was achieved by using 5-deazaflavin as photocatalyst. Ferredoxin II was also purified from bacteria grown under nitrogen limiting (nif derepressing) conditions. In in vitro assays, ferredoxin II catalyzed electron transport between illuminated chloroplasts and nitrogenase.     

FeMo cofactor biosynthesis in a nifE- mutant of Rhodobacter capsulatus.

In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20-50 microM). Moreover, in these strains a high proportion of ethane was produced from acetylene ( approximately 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.     

Isolation and characterization of an aminolevulinate-requiring Rhodobacter capsulatus mutant.

Using transposon Tn5 mutagenesis, we isolated a mutant strain of Rhodobacter capsulatus that requires aminolevulinate for growth. Southern blot analysis indicated that this strain has a single Tn5 insertion. The addition of 0.1 mM aminolevulinate to the medium allowed the mutant to grow either aerobically or photosynthetically with generation times similar to those of the parental strain. When grown photosynthetically, bacteriochlorophyll accumulation increased with increasing aminolevulinate concentration. The mutant strain had only 10% of the normal aminolevulinate synthase activity, but it had a normal level of porphobilinogen synthase activity. The requirement for aminolevulinate could be satisfied by porphobilinogen, hemin, or protoporphyrin. While the mutant grew well on agar plates containing any of these substrates, growth in liquid media containing hemin or protoporphyrin was poor. Introduction of an R' factor containing all the known R. capsulatus bch genes into the mutant strain did not relieve the requirement for aminolevulinate, suggesting that the Tn5 insertion is not within the bch region.     

Isolation and characterization of Rhodobacter capsulatus strains lacking endogenous plasmids

Phodobacter capsulatus (formerly Rhodopseudomonas capsulata) strain B10 was found to contain a single plasmid of molecular weight 86×10⁶. Strains lacking this plasmids were isolated by various methods from strains containing the mutant R plasmid, pTH10. With the exception of two strains, which were found to contain chromosomal insertions of R plasmid DNA, strains lacking the endogenous plasmid appeared to be unaffected in any of the following metabolic or genetic functions: photosynthetic, autotrophic, diazotrophic, and dark, anaerobic growth; the production of bacteriocin; homologous recombination; the restriction of foreign DNA; and the production of gene transfer agent. DNA-DNA hybridization experiments confirmed that the plasmid had been eliminated from these strains and not become integrated into the chromose. However, sequences homologous to those of the endogenous plasmid were found to be present in the chromosome of R. capsulatus B10. This suggests, among other possibilities, that the endogenous plasmid may have originated in the chromosome, and might serve to duplicate certain chromosomal functions.Abbreviations kb kilobase-pair - GTA gene transfer agent - Cma chromosome mobilizing ability     

Cobalamin (vitamin B(12)) biosynthesis in Rhodobacter capsulatus

In Rhodobacter capsulatus, cobalamin biosynthesis has been shown to occur when the bacteria are grown either aerobically or anaerobically. However, a comparison of the main cobalamin biosynthetic operon found within R. capsulatus would suggest that the encoded proteins belong to the oxygen-dependent pathway for cobalamin biosynthesis, although, significantly, no homologue of the essential mono-oxygenase CobG has yet been detected. Nonetheless, within this main cob operon is found a large open reading frame termed orf663 that is not found in any other cobalamin biosynthetic operon. When overproduced in Escherichia coli, orf663 was found to encode a 90 kDa integral membrane protein. Some of this protein is cleaved within E. coli to give a soluble N-terminal region that can easily be purified and yields a 50 kDa flavoprotein. When expressed in harness with the genes for precorrin-3a synthesis, ORF663 appears to mediate the transformation of precorrin-3a into a new chromophoric compound. Another open reading frame in close proximity to orf663 is termed orf647, and was found to encode a 2Fe-2S ferredoxin-like protein. We suggest that these two proteins may provide an alternative oxygen-independent mechanism for ring contraction within R. capsulatus.