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The carotenoids accumulated by a mutant Rhodospirillum rubrum ST4, containing a single Tn5 lesion in the pathway for carotenoid biosynthesis, were analyzed by HPLC, 1H NMR spectroscopy, and field desorption mass spectrometry. The main carotenoid was identified as 3,4,3',4'-tetrahydrospirilloxanthin, and the four minor carotenoids were identified as rhodopin, 3,4-dihydroanhydrorhodovibrin, 3', 4'-dihydrorhodovibrin, and 1,1'-dihydroxylycopene. The C-3,4 and C-3',4' bonds of all 5 carotenoids are saturated, and they have 11 conjugated double bonds. With the exception of rhodopin, which is a normal intermediate of the wild-type pathway, all of the carotenoids are not naturally occurring. The Tn5 lesion was assigned to rhodopin 3,4-desaturase which is proposed to catalyze dehydrogenation at both ends of the symmetrical spirilloxanthin derivative. An unexpected finding was that the enzymes following rhodopin 3,4-desaturase are still able to end-modify the 3,4-, and 3',4'-saturated precursors and that the order of methylation and hydroxylation is not obligatory. It is proposed that the observed nonnatural carotenoids can be explained by the inclusion of a cryptic branch, unmasked by the absence of rhodopin 3,4-desaturase, in the established linear pathway for spirilloxanthin biosynthesis. This is the first example of latent branching of the carotenoid biosynthesis pathway exhibited by a carotenoid mutant of a phototrophic bacterium.

Although isopentenyl diphosphate is a precursor of isoprenoids in Escherichia coli, the genes and enzymes involved in its biosynthesis have not been identified. Thus, we tried to isolate E. coli mutants deficient in the biosynthesis and their complementary genes by use of an artificial phenotypic screening system employing three carotenoid biosynthetic genes, crtE, crtB, and crtI. Cells were mutagenized with ethylmethanesulfonate, then transformed with a plasmid for expression of the carotenogenic genes. Mutants deficient in biosynthesis of isopentenyl diphosphate were expected to form white colonies, because they are unable to produce enough lycopene, whereas wild-type cells form red colonies. Among large numbers of red colonies, we identified 117 white colonies. Next, we transformed each mutant with an E. coli genomic library. Twenty-nine complementary genes that restore red color of host colonies were isolated. A homology search and further complementation study using subcloned genes revealed that the true complementary genes encode isopentenyl diphosphate isomerase, subunits of ATP synthase, enzymes of the Krebs cycle, some aldehyde dehydrogenases, phosphate acetyltransferase, and enzymes which relate to the biosynthesis of ubiquinones and menaquinones. Two unknown genes were also found, designated elb1 and 2, which may be involved in the early steps of isoprenoid biosynthesis.

Escherichia coli cells transformed with several carotenogenic genes to mediate the formation of zeta-carotene, neurosporene, lycopene, beta-carotene, and zeaxanthin were exposed to UV-B radiation. Short-term kinetics revealed that endogenous levels of neurosporene and beta-carotene protected E. coli against irradiation with UV-B. Zeaxanthin protected against only the photosensitized UV-B treatment. All other carotenoids were ineffective.

Carotenoid synthesis in Escherichia coli, when transformed with plasmid containing a carotenoid gene cluster from Erwinia herbicola (pPL376), is regulated by RpoS. When the plasmid was transformed into E. coli mutants that were oxyR minus, the intracellular carotenoid concentration dramatically increased from that observed in an oxyR plus allele. The higher carotenoid concentration in these mutants correlated with an increase in rpoS transcription as indicated by beta-galactosidase activity from a rpoS::lacZ promoter fusion. This indication of a higher concentration of carotenoids correlated with an increased resistance to hydrogen peroxide and near-ultraviolet radiation (310-400 nm; near-UV).

Carotenoids are essential to protection against photooxidative damage in photosynthetic and non-photosynthetic organisms. In a previous study, we reported the disruption of crtD and crtC carotenoid genes in the purple bacterium Rubrivivax gelatinosus, resulting in mutants that synthesized carotenoid intermediates. Here, carotenoid-less mutants have been constructed by disruption of the crtB gene. To study the biological role of carotenoids in photoprotection, the wild-type and the three carotenoid mutants were grown under different conditions. When exposed to photooxidative stress, only the carotenoid-less strains (crtB-) gave rise with a high frequency to four classes of mutants. In the first class, carotenoid biosynthesis was partially restored. The second class corresponded to photosynthetic-deficient mutants. The third class corresponded to mutants in which the LHI antenna level was decreased. In the fourth class, synthesis of the photosynthetic apparatus was inhibited only in aerobiosis. Molecular analyses indicated that the oxidative stress induced mutations and illegitimate recombination. Illegitimate recombination events produced either functional or non-functional chimeric genes. The R. gelatinosus crtB- strain could be very useful for studies of the SOS response and of illegitimate recombination induced by oxidants in bacteria.

We have identified a major 32-kDa protein in the dinoflagellate Gonyaulax polyedra as a peridinin-chlorophyll a-binding protein (PCP), based on micro-sequence data and immunological cross-reaction with antibodies raised against PCP from another dinoflagellate species. A cDNA for this protein, identified by a PCR-based cloning strategy, encoded all 68 of the amino acids microsequenced, thus confirming the identity of the clone. The PCP gene is highly expressed at both the mRNA and protein levels, and only PCP transcripts corresponding in size to the cDNA sequence were detected. Slot blot analyses show that there are roughly 5000 copies of the PCP gene in Gonyaulax, making this gene one of the most highly repeated protein-coding genes ever reported, yet the sequence of the different gene copies in the genome appears extraordinarily well conserved as judged by Southern blot analyses. The gene, as indicated by Southern blot and PCR data, is suggested to be present in 5000 intronless copies arranged head to tail in the genome, separated by conserved 1-kb spacers. Based on the conserved sequence of the spacer region, its presence next to each of the PCP coding sequences, and the uniform size of the PCP transcript, we propose that this region represents a dinoflagellate transcriptional promoter. This putative promoter region contains none of the sequence elements for DNA-binding proteins involved in transcriptional initiation reported in other organisms.

Two chromosome walks covering 420 and 110 kb on the left arm of linkage group VI (LGVIL) of Neurospora crassa were purscrooued with the goal of cloning carotenogenic loci. Complementation analysis with clones isolated in the 420-kb walk allowed identification of the yellow-1 (ylo-1) gene which is essential for Neurospora carotenogenesis. We have physically located a second gene, unknown-13 (un-13), between the cross-pathway control-1, (cpc-1) and ylo-1 loci. Cloning of a second potential carotenogenic locus, vivid (vvd), from our walks was attempted using screening of Northern blots with radiolabeled DNA fragments from walk clones to identify gene transcripts. The radiolabeled DNA fragments were used to clone complementary DNA isolates representing an additional four genes in the two walks.

Two genes which encode the enzymes lycopene cyclase and phytoene desaturase in the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 have been cloned and sequenced. The gene for lycopene cyclase, designated crtY, was expressed in a strain of Escherichia coli which contained the crtE, B, I and Z genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and beta-carotene hydroxylase, respectively. As a result, zeaxanthin production was observed in E. coli transformants. In addition, expression of the E. longus gene crtI for phytoene desaturase in E. coli containing crtE and B resulted in the accumulation of lycopene in transformants. Zeaxanthin and lycopene were also determined by mass spectrum. Nucleotide sequence similarities between E. longus crtY gene and other microbial lycopene cyclase genes are 40.2% (Erwinia herbicola), 37.4% (Erwinia uredovora) and 22.9% (Synechococcus sp.), and those between phytoene desaturase genes are 50.3% (E. herbicola), 54.7% (E. uredovora) and 39.6% (Rhodobacter capsulatus).

The Gram-negative bacterium Flavobacterium sp. strain R1534 is a natural producer of zeaxanthin. A 14 kb genomic DNA fragment of this organism has been cloned and a 5.1 kb piece containing the carotenoid biosynthesis genes sequenced. The carotenoid biosynthesis cluster consists of five genes arranged in at least two operons. The five genes are necessary and sufficient for the synthesis of zeaxanthin. The encoded proteins have significant homology to the crtE, crtB, crtY, crtI and crtZ gene products of other carotenogenic organisms. Biochemical assignment of the individual gene products was done by HPLC analysis of the carotenoid accumulation in Escherichia coli host strains transformed with plasmids carrying deletions of the Flavobacterium sp. strain R1534 carotenoid biosynthesis cluster.

Carotenoids represent one of the most widely distributed and structurally diverse classes of natural pigments, with important functions in photosynthesis, nutrition, and protection against photooxidative damage. In the eubacterial community, yellow, orange, and red carotenoids are produced by anoxygenic photosynthetic bacteria, cyanobacteria, and certain species of nonphotosynthetic bacteria. Many eukaryotes, including all algae and plants, as well as some fungi, also synthesize these pigments. In noncarotenogenic organisms, such as mammals, birds, amphibians, fish, crustaceans, and insects, dietary carotenoids and their metabolites also serve important biological roles. Within the last decade, major advances have been made in the elucidation of the molecular genetics, the biochemistry, and the regulation of eubacterial carotenoid biosynthesis. These developments have important implications for eukaryotes, and they make increasingly attractive the genetic manipulation of carotenoid content for biotechnological purposes.

The action of the food dyes E160e (20% beta-apo-8'-carotinale in oily suspension) and E160e (20% beta-carotene in oily suspension) on the clastogenic effects of the indirect alkylating mutagen cyclophosphamide (CP) and the prooxidative mutagen dioxidine (D) was examined by the using the chromosomal aberrations tests on the bone marrow cells from C57B1/6 mice. E160e in a dose of 50 mg/kg caused a significant decrease in the count of aberrant cells damaged by D or CP. When given in a dose 5 mg/kg, the dye reduced the effects of these mutagens only during preadministration. Moreover, E160e in a dose of 0.5 mg/kg substantially lowered the clastogenic effect of D after its 5-day use in combination with the mutagen E160a. Pretreatment of the animals with E160a in doses of 5 und 50 mg/kg caused a significant reduction in the cytogenetic effect of D. When used in combination with the mutagens, this dye in doses of 5 and 50 mg/kg reduced the clastogenic action of CP, when given in doses of 0.5, 5, and 50 mg/kg, it decreased the count of cells damaged by D. In other variants of the experiment E160a was inactive.

The genes for geranylgeranyl diphosphate synthase (crtE) and phytoene synthase (crtB) from the epiphytic bacterium Erwinia uredovora and the phytoene desaturase gene from the photosynthetic bacterium Rhodobacter capsulatus (Rc-crtI) were introduced into Escherichia coli, which resulted in the accumulation of the acyclic carotenoid, neurosporene. Further introduction of the lycopene cyclase gene from E. uredovora (crtY) or the higher plant Capsicum annuum (Icy) resulted in the production of a bicyclic carotenoid, 7,8-dihydro-beta-carotene, via monocyclic beta-zeacarotene. zeta-Carotene was also found to be cyclized to bicyclic 7,8,7',8'-tetrahydro-beta-carotene by the Erwinia cyclase. These results indicate that both lycopene cyclases can cyclize a 7,8-dihydro-psi end group to a 7,8-dihydro-beta end group, in addition to the usual cyclization of the psi end group to the beta end group. Furthermore, beta-carotene hydroxylase from Erwinia (CrtZ) was able to add a hydroxyl group to the 7,8-dihydro-beta end group and the beta end group.

Peridinin-chlorophyll a-proteins (PCPs) have been purified by combination of ammonium sulphate precipitation and cation exchange chromatography. The amino acid sequences of several of the most abundant forms have been deduced by direct protein sequencing and from DNA and indicate a highly conserved multi-gene family. At least two of the PCP genes are tandemly arranged. A novel form of the protein was also obtained in low yield with fewer peridinins (six vs eight) per chlorophyll a and with a different molecular mass (34 kDa vs 32 kDa) of its apoprotein. It had only 31% sequence identity with any of the more abundant PCP forms but retained a two-domain structure.

Lutein, a dihydroxy beta, epsilon-carotenoid, is the predominant carotenoid in photosynthetic plant tissue and plays a critical role in light-harvesting complex assembly and function. To further understand lutein synthesis and function, we isolated four lutein-deficient mutants of Arabidopsis that define two loci, lut1 and lut2 (for lutein deficient). These loci are required for lutein biosynthesis but not for the biosynthesis of beta, beta-carotenoids. The lut1 mutations are recessive, accumulate high levels of zeinoxanthin, which is the immediate precursor of lutein, and define lut1 as a disruption in epsilon ring hydroxylation. The lut2 mutations are semidominant, and their biochemical phenotype is consistent with a disruption of epsilon ring cyclization. The lut2 locus cosegregates with the recently isolated epsilon cyclase gene, thus, providing additional evidence that the lut2 alleles are mutations in the epsilon cyclase gene. It appears likely that the epsilon cyclase is a key step in regulating lutein levels and the ratio of lutein to beta,beta-carotenoids. Surprisingly, despite the absence of lutein, neither the lut1 nor lut2 mutation causes a visible deleterious phenotype or altered chlorophyll content, but both mutants have significantly higher levels of beta, beta-carotenoids. In particular, there is a stable increase in the xanthophyll cycle pigments (violaxanthin, antheraxanthin, and zeaxanthin) in both lut1 and lut2 mutants as well as an increase in zeinoxanthin in lut1 and beta-carotene in lut2. The accumulation of specific carotenoids is discussed as it pertains to the regulation of carotenoid biosynthesis and incorporation into the photosynthetic apparatus. Presumably, particular beta, beta-carotenoids are able to compensate functionally and structurally for lutein in the photosystems of Arabidopsis.

High level expression of the functional beta-carotene ketolase gene bkt from Haematococcus pluvialis occurred in Escherichia coli transformants producing beta-carotene or zeaxanthin as a result of the presence of additional carotenoid genes from Erwinia uredovora. Requirement of molecular oxygen for the insertion of the keto group was demonstrated. The final product of this two-step ketolase reaction from beta-carotene is canthaxanthin (4,4'-diketo-beta-carotene) with the 4-monoketo derivative echinenone as an intermediate. A reaction sequence for the formation of astaxanthin from beta-carotene was established based on kinetic data on astaxanthin formation in E. coli transformants carrying the hydroxylase gene crtZ from Erwinia along with bkt. We conclude that the carotenoids zeaxanthin and adonixanthin which accumulate in addition to astaxanthin in this transformant are products of side reactions rather than direct precursors of astaxanthin. The possible mechanisms for the formation of the keto derivatives are discussed.

Carotenoids occur universally in photosynthetic organisms but sporadically in nonphotosynthetic bacteria and eukaryotes. The primordial carotenogenic organisms were cyanobacteria and eubacteria that carried out anoxygenic photosynthesis. The phylogeny of carotenogenic organisms is evaluated to describe groups of organisms which could serve as sources of carotenoids. Terrestrial plants, green algae, and red algae acquired stable endosymbionts (probably cyanobacteria) and have a predictable complement of carotenoids compared to prokaryotes, other algae, and higher fungi which have a more diverse array of pigments. Although carotenoids are not synthesized by animals, they are becoming known for their important role in protecting against damage by singlet oxygen and preventing chronic diseases in humans. The growth of aquaculture during the past decade as well as the biological roles of carotenoids in human disease will increase the demand for carotenoids. Microbial synthesis offers a promising method for production of carotenoids.

In the bacterium Myxococcus xanthus, several genes for carotenoid synthesis lie together at the carA-carB chromosomal locus and are co-ordinately activated by blue light. A 12-kb DNA stretch from wild-type M. xanthus has been sequenced that includes the entire carA-carB gene cluster. According to sequence analysis, the cluster contains 11 different genes. Intergenic distances are very short or nil (implying translational coupling), giving further support to previous evidence indicating that most (or all) of the genes in the cluster form a single operon. At the promoter region, a potential -35 site for the binding of sigma factors is found. However, the -10 region shows little similarity with analogous sites in other bacterial promoters. Five (possibly six) genes in the carA-carB operon code for enzymes acting on early or late steps of the pathway for carotenoid synthesis. Other genes in the operon show no overall similarity with previously known genes. However, peptide stretches in the predicted products of two genes exhibit strong similarity with the DNA binding domain of the MerR family of transcriptional regulators. At least one of the predicted DNA-binding domains is altered in a mutant strain affected in light-regulation of the car genes.

For most species of purple photosynthetic bacteria, the presence of molecular oxygen represses synthesis of carotenoids and bacteriochlorophyll. In this study we characterize a strain of Rhodobacter capsulatus, DB469, which contains a genomic disruption of an open reading frame in the photosynthesis gene cluster termed ORF469. Characterization of the steady-state level of bacteriochlorophyll synthesis demonstrates that disruption of ORF469 results in a 2.5-fold increase in aerobic synthesis of bacteriochlorophyll over that observed with the parent strain. Utilizing reporter plasmids that contain transcriptional fusions of lacZ to various carotenoid and bacteriochlorophyll biosynthesis genes, we also demonstrate that disruption of ORF469 leads to an approximate twofold increase in bacteriochlorophyll and carotenoid gene expression under anaerobic growth conditions. Similar analysis with reporter plasmids that contain translational fusions of lacZ to the puf, puh, and puc operons demonstrates that disruption of ORF469 leads to elevated levels of aerobic transcription of light harvesting-II genes (puc), without affecting light harvesting-I or reaction center gene expression (puf and puh, respectively). Gel mobility analysis demonstrates that DB469 cells lack a DNA-binding protein that interacts with a palindromic sequence in the bchC promoter region. The results of this study indicate that ORF469 codes for a DNA-binding protein that acts as an aerobic repressor of promoters for bacteriochlorophyll, carotenoid, and light harvesting-II gene expression.

The carotenoid biosynthesis genes form a cluster within the genome of Rhodobacter sphaeroides, lying in the middle of a larger cluster and 45 kb in length, which contains genes for bacteriochlorophyll biosynthesis and for the reaction center and light-harvesting apoproteins. The positions and approximate limits of the carotenoid genes were determined previously by localized transposon Tn5 mutagenesis and by comparison with the closely related Rhodobacter capsulatus carotenoid gene cluster. In this report, analysis of the DNA and deduced amino acid sequences of the carotenoid genes in R. sphaeroides are presented. Twenty-five Tn5 insertion mutants were used to produce a base-specific Tn5 insertion map of this region, and carotenoid gene assignment was supported by spectroscopic, ultrastructural, and high-pressure liquid chromatography analyses of these mutants. A region in the 3' end of crtD which affects bacteriochlorophyll biosynthesis was discovered, and CrtA was found to possess a proline-rich C-terminal region containing a repeated (Ala-Pro)n motif. CrtF also showed a high degree of sequence conservation with eukaryotic O-methyltransferases. This study provides gene sequences and assignments based upon a comprehensive structural, spectroscopic, and biochemical analysis of a range of carotenoid biosynthetic mutants; in each mutation, the point of Tn5 insertion is determined accurate to 1 bp on the gene cluster.

The carotenoid biosynthesis operon of Erwinia herbicola Eho13 consists of five genes, which are organized in the order crtE-crtX-crtY-crtI-crtB. These genes, with the exception of crtX, encode functions of beta-carotene biosynthesis and give an orange-coloured phenotype in Escherichia coli. Since crtX is not involved in the biosynthesis of beta-carotene, deletion of this gene does not alter the phenotype of pigmented cells. On the other hand, insertion of Tn1000 into crtX or into the upstream untranslated region of the operon resulted in a light-yellow, rather than an unpigmented phenotype, indicating that Tn1000 does not exert a strong polar effect when inserted in this operon. RNA analysis revealed that the sequence downstream from the insertion site was transcribed at a low level. Primer extension showed that the "-35"-like sequence in the terminal inverted repeats was not responsible for the transcription of the downstream sequence. Furthermore, primer extension and polymerase chain reaction (PCR) studies revealed that RNA transcribed from the promoters inside of Tn1000 was extended through the terminal inverted repeats into the adjacent sequences. In addition Tn1000, in either orientation, was able to generate fusion transcripts when placed upstream of a promoter-less tetracycline-resistance gene and resulted in cells resistant to the drug. These results showed that Tn1000 insertion transcriptionally activates the DNA sequences adjacent to the transposon.

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, beta-carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for beta-carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase.

The Gram-negative bacterium Myxococcus xanthus responds to blue light by producing carotenoid pigments (Car+ phenotype). Genes for carotenoid synthesis lie at two unlinked chromosomal sites, the carC and the carBA operon, but are integrated in a single "light regulon" by the action of common trans-acting regulatory elements. Three known regulatory genes are grouped together at the (light-inducible) carQRS operon. By screening the Car phenotype of a large collection of transposon-induced mutants, we have identified a new car locus that has been named carD (carD1 for the mutant allele). The carD gene product plays a critical role in the light regulon, as it is required for activation of the carQRS and carC promoters by blue light. The carD1 mutant is impaired in the (starvation-induced) developmental process that allows M. xanthus cells both to form multicellular fruiting bodies and to sporulate. Our results indicate that the carD gene product is also required for the expression of a particular set of development-specific genes that are normally activated through the action of intercellular signals.

Reconstitution of the photoelectric responses involved in photosensory transduction in "blind" cells of Chlamydomonas reinhardtii carotenoid-deficient mutants was studied by means of a recently developed population method. Both the photoreceptor current and the regenerative response can be restored by addition of all-trans-retinal, 9-demethyl-retinal, or dimethyl-octatrienal, while the retinal analogs prevented from 13-cis/trans isomerization, 13-demethyl-retinal and citral, are not effective. Fluence dependence, spectral sensitivity, and effect of hydroxylamine treatment on retinal-induced photoelectric responses are similar to those found earlier in green strains of Chlamydomonas, although an alternative mechanism of antenna directivity in white cells of reconstituted "blind" mutants (likely based on the focusing effect of the transparent cell bodies) leads to the reversed sign of phototaxis in mutant cells under the same conditions. The results obtained indicate that both photoreceptor current and regenerative response are initiated by the same or similar rhodopsins with arhaebacterial-like chromophore(s) and prove directly the earlier suggested identity of the photoreceptor pigment(s) involved in photomotile and photoelectric responses in flagellated algae.

In Thermus thermophilus HB27, the crtB gene encoding phytoene synthase was found to exist on the large plasmid, pTT27. One of the carotenoid under-producing mutants, Crt31, carried a derivative of pTT27 (pTT27') in which deletion and inversion were observed near the crtB gene. T. thermophilus HB8 also contained a large plasmid which showed homology to pTT27 and the crtB gene. These results suggested that genes for carotenoid biosynthesis occurred as a cluster on a large plasmid in Thermus thermophilus. This is the first report to show directly that carotenogenesis is plasmid-encoded in microorganisms.

mRNA from the dinoflagellate Symbiodinium sp. isolated from the staghorn coral Acropora formosa was used for the construction of cDNA libraries. A cDNA clone was identified which encoded the precursor of peridinin-chlorophyll a-binding protein (PCP), including a 52 amino acid transit peptide and the 313 amino acid mature protein. The deduced amino acid sequence clearly contains an internal duplication, implying that amongst dinoflagellates the M(r) 35,000 form of PCP has arisen by duplication and fusion of genes encoding the M(r) 15,000 form. This is the first reported sequence of a dinoflagellate light-harvesting protein. The anatomy of the mature protein and the transit peptide are discussed.

When a wild-type strain of Neurospora crassa is transformed with different portions of the carotenogenic albino 1 or albino 3 genes, up to 30-35% of the transformants show an albino phenotype. The albino transformants presented a variety of phenotypes ranging from white or yellow to dark yellow colour. The ectopically integrated sequences provoke a severe impairment of the expression of the endogenous al-1 or al-3 genes. This phenomenon, that has been termed quelling, is found to be spontaneously and progressively reversible. In fact, all of the albino transformants have an unstable phenotype and revert progressively to wild type or intermediate phenotypes over a prolonged culturing time. The phenotypic reversion is characterised by a progressive release of the transcriptional inhibition and seems to correlate with the reduction of the number of the ectopic integrated sequences. However, there is no strict correlation between the copy number of the ectopic sequences and the intensity of quelling, as indicated by the existence of albino transformants containing only 1-2 ectopic sequences. The nature of the molecular events determining the onset of quelling is unclear, in any event, these are likely to involve some kind of interaction between the resident genes and ectopically integrated exogenous sequences. Recent evidences on a possible mechanism are presented.

The plethora of natural organic chemicals contrasts with the relative scarcity of genes and the apparent difficulty to evolve new ones. The genetical analysis of metabolism may be reviewed with this paradox in mind. The terpenoids constitute a particularly varied group of natural compounds; many of them are dispensable to the cell and their biosynthesis is amenable to mutational analysis and other genetical and chemical methods. The production of carotene and gibberellins by the fungi Phycomyces blakesleeanus and Gibberella fujikuroi, respectively, seems to require an unexpectedly small number of genes. A number of gene-saving devices are detected that may have general validity for other cases of secondary metabolism. The most important one is versatile genes whose products are specific for a chemical reaction but not for the substrate. This versatility allows a combinatorial use that increases chemical and behavioral diversity. Physical separation of cellular functions in compartments or enzyme aggregates not only makes processes more efficient but helps avoid some deleterious consequences of enzyme versatility.

The carR region encodes a light-inducible promoter, a negative regulator of the promoter and a trans-acting activator that controls the light-inducible Myxococcus xanthus carotenoid biosynthesis regulon. DNA sequence analysis revealed, downstream of the promoter, three translationally coupled genes, carQ, carR and carS. Sequencing of mutations demonstrated that carR encoded the negative regulator and was an integral membrane protein. Mutant construction and sequencing revealed that carS was the trans-acting activator and that carQ was a positive regulator of the promoter. Neither gene encodes proteins with known sequence-specific DNA-binding motifs. The sequence of the light-inducible promoter region, identified by primer extension analysis, showed similarity to the consensus sequence of the Escherichia coli stress response ('heat-shock') promoters.

We have cloned and sequenced a 1.5-kb chromosomal fragment of Thermus thermophilus which promoted the overproduction of carotenoids in T. thermophilus. An open reading frame (ORF-A) coding for a polypeptide with 289 amino acids was responsible for carotenoid overproduction. The putative ORF-A protein showed significant homology with the amino acid sequences of crtB gene products (phytoene syntheses) of other microorganisms. The clone containing the ORF-A on a multicopy plasmid produced about three times as much carotenoid as that produced by the host strain, suggesting that the crtB gene product is a rate-limiting enzyme for carotenoid biosynthesis in T. thermophilus.

Characterizations are presented for RNA, 2 cDNA libraries, and 2 full-length cDNA sequences encoding for photosynthetic light-harvesting peridinin-chlorophyll a-protein (PCP) in the dinoflagellate Heterocapsa pygmaea. Subsequent analyses of the PCP system also indicate that (1) it is represented by multiple nuclear encoded genes, (2) a subset of mRNAs encoding for PCP apoproteins are regulated by growth irradiance, (3) PCP preproteins are larger than the mature apoproteins, and (4) PCP cDNA clones sequenced thus far contain a conserved region but are not identical. Results are discussed in the context of photoadaptation in dinoflagellates.

The temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus. Accumulation of crtA, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus. The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen. The regulation patterns of promoters for the crtA and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns. We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants.

Phytoene synthase (Psy) and phytoene desaturase (Pds) are the first dedicated enzymes of the plant carotenoid biosynthesis pathway. We report here the organ-specific and temporal expression of PDS and PSY in tomato plants. Light increases the carotenoid content of seedlings but has little effect on PDS and PSY expression. Expression of both genes is induced in seedlings of the phytoene-accumulating mutant ghost and in wild-type seedlings treated with the Pds inhibitor norflurazon. Roots, which contain the lowest levels of carotenoids in the plant, have also the lowest levels of PDS and PSY expression. In flowers, expression of both genes and carotenoid content are higher in petals and anthers than in sepals and carpels. During flower development, expression of both PDS and PSY increases more than 10-fold immediately before anthesis. During fruit development, PSY expression increases more than 20-fold, but PDS expression increases less than threefold. We concluded that PSY and PDS are differentially regulated by stress and developmental mechanisms that control carotenoid biosynthesis in leaves, flowers, and fruits. We also report that PDS maps to chromosome 3, and thus it does not correspond to the GHOST locus, which maps to chromosome 11.

Staphylococcus aureus mutants lacking pigment, or expressing only low levels of pigment, were more sensitive to oleic acid than were the parent strain and mutants making more pigment than the parent. One class of mutants (colour index 5), although possessing significant levels of pigment, were nevertheless quite sensitive to oleic acid. This suggested that only certain carotenoids in the biosynthetic pathway were capable of imparting resistance to fatty acids. The phenotypic expression of pigment also affected the sensitivity of a strain to oleic acid. The parent S. aureus strain 18Z, when grown to express its maximal pigment potential, was more resistant to oleic acid than when it was grown to express minimal levels of pigment.

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.

Light, chemicals, and mutations that affect the carotene content of the fungus Phycomyces blakesleeanus had practically no effect on the ergosterol content. Lovastatin, a specific inhibitor of hydroxymethylglutaryl coenzyme A reductase, blocked growth at 1 microM; sodium DL-mevalonate (10 mM) fully reversed this inhibition. In the presence of [14C]mevalonate, a carS mutant accumulated 16 times more beta-carotene than the wild-type with a specific radioactivity five times lower. The specific radioactivity of ergosterol was different from that of beta-carotene, even when calculated in terms of the constituent isoprene units, and unaffected by the carS mutation. The carotene and sterol pathways of Phycomyces are independently regulated and physically separated in different subcellular compartments.

The photosynthetic apparatus of Rhodobacter sphaeroides is comprised of three types of pigment-protein complex: the photochemical reaction centre and its attendant LH1 and LH2 light-harvesting complexes. To augment existing deletion/insertion mutants in the genes coding for these complexes we have constructed two further mutants, one of which is a novel double mutant which is devoid of all three types of complex. We have also constructed vectors for the expression of either LH1, LH2 or reaction-centre genes. The resulting system allows each pigment-protein complex to be studied either as part of an intact photosystem or as the sole complex in the cell. In this way we have demonstrated that reaction centres can assemble independently of either light-harvesting complex in R. sphaeroides. In addition, the isolation of derivatives of the deletion/insertion mutants exhibiting spontaneous mutations in carotenoid biosynthesis provides an avenue for examining the role of carotenoids in the assembly of the photosynthetic apparatus. We show that the LH1 complex is assembled regardless of the carotenoid background, and that the type of carotenoid present modifies the absorbance of the LH1 bacteriochlorophylls.

We have previously shown that heat-shock in the dark evokes photomorphogenesis-like effects and circadian rhythmicity at the level of mRNAs when applied to emerging pea plantlets during several consecutive days [15]. Here we extend these findings by showing that a temperature shift to 10 degrees C above average and a single heat-shock are sufficient for induction of circadian rhythmicity and changes in morphogenesis. The maximum response to a single heat-shock occurs at days 2 to 3 after sowing indicating additional developmental control of the response. An increasing number of heat-shock treatments intensifies the morphogenetic effect. The heat-shocked plantlets have an elevated level of the xanthophyll lutein in the dark. Upon illumination of heat-shocked plantlets accumulation of chloroplast pigments as well as that of individual thylakoid membrane proteins and their corresponding mRNAs occur much faster than in the etiolated controls. This is reflected in an accelerated formation of grana stacks. Therefore, heat-shock seems to evoke a responsiveness of plantlets similar to that obtained earlier by other authors using pre-illumination. The working hypothesis is put forward that induction or synchronization of circadian rhythmicity by either light or heat-shock might be sufficient to explain the observed morphogenetic changes.

The synthesis of carotenoids in strain 2.4.1 of the phototrophic bacterium Rhodobacter sphaeroides is spontaneously turned on and off at a high frequency (10(-5) per cell per generation) giving rise alternatively to red (wild type) and green (mutant) clones. The crtD gene is not functional in green mutants as a consequence of the spontaneous addition of a guanosine in a stretch of seven guanosines located in the 5'-terminal coding region of this gene originating a frameshift. All spontaneous wild-type revertants isolated from green mutants had recovered the crtD gene function by loss of one of these reiterated guanosines. The transition Crt(+)----Crt(-)----Crt+, is strain-dependent, since Crt+ clones were not detected in ethyl methane sulphonate (EMS)-induced CrtD- mutants of two other strains of R. sphaeroides (WS22 and RS630) which harbour a recombinant plasmid containing the crtD gene from a spontaneous CrtD- mutant of strain 2.4.1 of R. sphaeroides.

This report describes the first successful transfer and complete expression of clustered mycobacterial genes controlling a biosynthetic pathway (carotenogenesis) in a homologous system. A genomic library of pigmented Mycobacterium aurum A+ (yellow-orange) DNA was constructed in shuttle vector pHLD-69. The colourless mutant A11 and the brick-red mutant NgR9 derived from M. aurum A+ were electroporated with the plasmid library. Among the transformants, colonies different in colour from the recipient mutants were detected, and were cloned. One of the clones from the transformed A11 mutant had a yellow-orange phenotype, and was designated A11T; one of the clones from the NgR9 (brick-red) mutant had a yellow-orange phenotype and was designated NgR9T. The carotenoid pigments from the A11T and NgR9T clones were analyzed and in both the end product of carotenogenesis in M. aurum (leprotene) was detected. A11T and NgR9T harboured the same recombinant plasmid (Cl) containing a 11-kb M. aurum fragment. pCl was used to transform the colourless Mycobacterium smegmatis MC2-155 strain. All the transformants were pigmented. A colony (MC2-T) was arbitrarily chosen and leprotene was detected. It was therefore concluded that M. aurum genes involved in carotenogenesis had been cloned, and were expressed not only in M. aurum mutants, but also in M. smegmatis.

Codon usage in Rhodobacter was evaluated and found to be strikingly different from that in Escherichia coli. While codon usage for genes concerned with nitrogen utilization and carotenoid biosynthesis corresponded to expectation, based on codon usage for Rhodobacter in general, that for the fructose utilization (fru) operon and for the photosynthetic genes encoding the reaction centre and light harvesting proteins exhibited significant deviation from expectation and from each other for specific amino acids. The differences in codon usage for the fru operon versus the photosynthetic genes may reflect different proportions of the various tRNA specific for certain amino acids when cells are grown under heterotrophic versus phototropic conditions. In addition, preferential use of the initiation codon, GTG, was found for the first cistrons of Rhodobacter operons.

A genetic system has been developed for studying bacterial photosynthesis in the recently described nonsulfur purple photosynthetic bacterium Rhodospirillum centenum. Nonphotosynthetic mutants of R. centenum were obtained by enrichment for spontaneous mutations, by ethyl methanesulfonate mutagenesis coupled to penicillin selection on solid medium, and by Tn5 transposition mutagenesis with an IncP plasmid vector containing a temperature-sensitive origin of replication. In vivo and in vitro characterization of individual strains demonstrated that 38 strains contained mutations that blocked bacteriochlorophyll a biosynthesis at defined steps of the biosynthetic pathway. Collectively, these mutations were shown to block seven of eight steps of the pathway leading from protoporphyrin IX to bacteriochlorophyll a. Three mutants were isolated in which carotenoid biosynthesis was blocked early in the biosynthetic pathway; the mutants also exhibited pleiotropic effects on stability or assembly of the photosynthetic apparatus. Five mutants failed to assemble a functional reaction center complex, and seven mutants contained defects in electron transport as shown by an alteration in cytochromes. In addition, several regulatory mutants were isolated that acquired enhanced repression of bacteriochlorophyll in response to the presence of molecular oxygen. The phenotypes of these mutants are discussed in relation to those of similar mutants of Rhodobacter and other Rhodospirillum species of purple photosynthetic bacteria.

The complete sequence has been determined for the A2 subunit of crustacyanin, an astaxanthin-binding protein from the carapace of the lobster Homarus gammarus. The polypeptide chain is 174 residues long and is similar to proteins of the retinol-binding protein superfamily. Some regions of the sequence are most similar to the retinol-binding protein, beta-lactoglobulin subgroup, while the disulphide bonding pattern is more akin to that seen in the porphyrin binding proteins insecticyanin and bilin-binding protein. It is beginning to appear as though this superfamily of proteins, characterized by a similar gross structural framework, may be further subdivided into interrelated subclasses. Model building based on the coordinates of the known structure of human plasma retinol-binding protein and on empirical prediction algorithms has allowed the putative identification of side-chains which line the binding cavity. This pocket is larger than in retinol binding protein and beta-lactoglobulin but does not allow the carotenoid to adopt a folded conformation. The amino acid composition of the pocket does not support a 'charge-shift'-type hypothesis to support the bathochromic shift phenomenon which takes place on interaction of the chromophore with the protein. Instead aromatic side-chains may play a prominent role.

A unique case-control study design including family members of cases and controls was used to assess the association between serum beta-carotene and cancer. The cases (n = 389) were incident cancer cases diagnosed between 1981 and 1984 in Wellington, New Zealand, and the controls (n = 391) were hospital patients without cancer. Both cases and controls were on a home diet at the time beta-carotene levels were measured. Since findings concerning patients who have already been diagnosed with cancer may reflect changes that occurred subsequent to development of cancer, family members of cancer patients (n = 618) and control patients (n = 675) were included, giving a total of 2,073 study participants. Low levels of beta-carotene were observed for individuals with a number of cancers, including cancers of the lung stomach, esophagus, small intestine, cervix, and uterus. Low levels of beta-carotene were also found in the relatives of these cancer patients. These differences persisted after stratification by current cigarette smoking. The strongest findings were those for lung cancer. Excluding adenocarcinoma, lung cancer patients had average serum beta-carotene levels of 40.2 micrograms/dl, 25.9 micrograms/dl lower than those of controls, adjusted for age, sex, and length of sample storage (p less than 0.01). Family members of the lung cancer patients also had lower values than family members of control patients, with an adjusted difference of 10.8 micrograms/dl (p less than 0.01). Odds ratio estimates for lung cancer by quartiles of beta-carotene residuals ranged from 3.6 (90% confidence interval (CI) 1.1-12.2) for the second-highest quartile to 6.6 (90% CI 1.9-23.0) for the lowest quartile (test for trend, p less than 0.001). Patients with cancers of the breast, colon, prostate, and skin did not have lower levels of serum beta-carotene than expected. Family members of individuals with these cancers also did not have lower levels of serum beta-carotene. The cancer sites that were associated with serum beta-carotene are, in general, sites for which smoking is a strong risk factor, and the sites that were not associated with beta-carotene do not have smoking as a risk factor.

In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light. Here we report the characterization of the albino-3 (al-3) gene of N. crassa, which encodes the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthetase. This is the first geranylgeranyl-pyrophosphate synthetase gene isolated. Nucleotide sequence comparison of al-3 genomic and cDNA clones revealed that the al-3 gene is not interrupted by introns. Transcription of the al-3 gene has been examined in dark-grown and light-induced mycelia. The analysis revealed that the al-3 gene is not expressed in the dark and that its transcription is induced by blue light (Nelson, M. A., Morelli, G., Carattoli, A., Romano, N., and Macino, G. (1989) Mol. Cell. Biol. 9, 1271-1276). The al-3 gene encodes a polypeptide of 428 amino acids. Comparison of the deduced amino acid sequence of al-3 with the sequences of prenyltransferases of other species, from bacteria to humans, showed three highly conserved homologous regions. These homologous regions may be involved in the formation of the catalytic site of the prenyltransferases.

Six genes have been mapped and identified by hybridization or by carotenoid analysis of deletion mutants on a 9 kb chromosomal fragment originating from Erwinia herbicola Eho 10. These genes include crtB, E and I which have been formerly described for Rhodobacter, and three new ones: crtZ encoding lycopene cyclase, crtH for beta-carotene hydroxylase, and crtG for zeaxanthin glycosilase. Their arrangement on the plasmid has been elucidated.

The carotenoid pigment astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) is an important component in feeds of aquacultural animals. It is produced as a secondary metabolite by the yeast Phaffia rhodozyma, and the isolation of rare mutants that produce increased quantities is limited by the lack of genetic selections. As a model system for enriching mutants increased in production of secondary metabolites, we have used quantitative flow cytometry/cell sorting (FCCS) to isolate astaxanthin hyperproducing mutants of the yeast. Experimental conditions were developed that gave a quantitative correlation of fluorescence and carotenoid content. In mutated populations, a 10,000-fold enrichment of carotenoid-overproducing yeasts was obtained. Distinctive differences were detected by FCCS in fluorescence and forward scatter values of mutant and wild-type populations of yeasts. Comparison of wild-type and mutant clones by fluorescence confocal laser microscopy showed that the mutants had more intense fluorescence throughout the cell than the wild-type. Quantitative FCCS is a sensitive method to isolate and characterize carotenoid overproducing mutants and should be useful as a general method for the isolation of mutants increased in other fluorescent metabolites.