(Received for publication, August 13, 1996, and in revised form, November 2, 1996)
From the Centre de Génétique Moléculaire du CNRS (UPR 9061) Associé à l'Université Pierre et Marie Curie, Bâtiment 24, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France
Rubrivivax gelatinosus mutants affected in the carotenoid biosynthesis pathways were created by interposon mutagenesis within the puf operon. Genetic and biochemical analysis of several constructed mutants suggest that at least crtC is localized downstream of the puf operon and that it is cotranscribed with this operon. Sequence analysis confirmed the genetic data and showed the presence of crtD and crtC genes downstream of the puf operon, a localization different from that known for other purple bacteria. Inactivation of the crtD gene indicated that the two crt genes are cotranscribed and that they are involved not only in the hydroxyspheroidene biosynthesis pathway as in Rhodobacter sphaeroides and R. capsulatus, but also in the spirilloxanthin biosynthesis pathway. Carotenoid genes implicated in the spirilloxanthin biosynthesis pathway were thus identified for the first time. Furthermore, analysis of carotenoid synthesis in the mutants gave genetic evidence that crtD and crtC genes are cotranscribed with the puf operon using the oxygen-regulated puf promoter.
The photosynthetic apparatus in many purple bacteria contains three types of pigment-protein complexes. The two light-harvesting LHII1 and LHI antenna capture light and transfer energy to the third complex, the reaction center (RC) in which charge separation occurs. The three complexes contain pigments, bacteriochlorophyll and carotenoids, which play an important role in the photosynthetic process.
Carotenoids have three functions in bacterial photosynthesis. They act in photoprotection, they function as accessory light-harvesting pigments by absorbing light in the 450-570 nm region (1, 2), and, in addition, they participate in the assembly of the light-harvesting antenna (3, 4).
Biosynthetic pathways of carotenoids in some purple bacteria are now
well known (5-7). Spectroscopic and chemical studies of the processes
of carotenoid biosynthesis have been performed in different mutants, in
particular in R. sphaeroides and R. capsulatus (8-11). In these bacteria the end products of the biosynthetic pathway
are spheroidene and spheroidenone; in other bacteria such as
Rhodospirillum rubrum and Rubrivivax gelatinosus,
in addition to the spheroidene pathway, another pathway leading to
biosynthesis of spirilloxanthin and derivatives was described (5) (Fig. 1).
The genes encoding many carotenoid biosynthetic enzymes have now been mapped (12). The arrangement of carotenoid genes was first described for R. capsulatus (13). As in R. sphaeroides, the carotenoid genes are clustered and flanked by bacteriochlorophyll genes within a 45-kb region of the chromosome (14, 15). In these bacteria, the first enzyme assigned to carotenoid biosynthesis is the geranylgeranyl-pyrophosphate synthase encoded by crtE gene (16, 17). The condensation of two geranylgeranyl pyrophosphate by an enzyme encoded by crtB lead to the synthesis of phytoene (16, 17), which is transformed to neurosporene by sequential desaturations which involved crtI (18, 19). In R. sphaeroides and R. capsulatus, a hydratase encoded by crtC catalyzes hydration of neurosporene to hydroxyneurosporene (8), which is then transformed to spheroidene by the products of crtF and crtD (8). The crtA and crtC products catalyze the transformation of spheroidene to spheroidenone and hydroxyspheroidene, respectively (8), and finally crtA product catalyzes oxygenation of hydroxyspheroidene to hydroxyspheroidenone (11). In R. rubrum and R. gelatinosus, neurosporene is also transformed to lycopene. A hydratase catalyzes the formation of rhodopin from lycopene, and subsequent reactions (desaturations and methylations) lead to the formation of spirilloxanthin, the end product of this second pathway. The genes involved at each step of this second pathway have not yet been identified.
The expression of pigment biosynthesis genes has been studied mainly in R. sphaeroides and R. capsulatus. Bacteriochlorophyll and carotenoid genes are proposed to be organized in transcription units (operons) (20). The existence of such an organization is supported by the results of RNA mapping (20) and interposon mutagenesis experiments (21). In particular, crtEF, bchCXYZ, and puf operons are organized in a "superoperon" (22-24). In these bacteria, the cotranscription of the three operons has been found to be phenotypically significant for adaptation to changes in environmental conditions (25). Up to now, nothing is known about the relevance of the genes downstream of the puf operon.
R. gelatinosus is a facultative phototrophic non-sulfur
bacterium belonging to the subclass of purple bacteria. This
strain, as R. sphaeroides and R. capsulatus and
unlike Rhodopseudomonas viridis, can grow very easily under
aerobic conditions in the dark as well as under photosynthetic
conditions. These trophic characteristics are very useful for studies
of the photosynthetic processes using mutants. The genes coding for the
subunits of LHI and RC and the polypeptides of LHII are organized in
operons. The nucleotide sequence of the puf operon has been
determined for two strains of R. gelatinosus, strain IL144
(26, 27) and strain S1 (28, 29); it contains two open reading frames
(ORFs) in addition to five photosynthetic genes which have been
reported in species belonging to the
subclass. These genes include
pufB, -A, -L, -M, and
-C coding for the
and
subunits of the B875 light-harvesting protein and for the L, M, and cytochrome subunits of
the RC complex, respectively. The localization and the organization of
the pigment genes are unknown. Recently, gene transfer systems in
R. gelatinosus have been established (29), and genetic
evidence for an organization of the puf operon and upstream
bch genes in a superoperon has been obtained (29).
In this study we report the construction and the analysis of interposon and insertion R. gelatinosus mutants, in which carotenoid biosynthesis has been affected. The accumulation of neurosporene and lycopene in some mutants suggested that at least the crtC gene could be localized downstream of the puf operon. Sequence analysis of the downstream region of the puf operon revealed the presence of crtD and crtC genes. Inactivation of crtD gene indicated that the two carotenoid genes are cotranscribed. Analysis of mutants gave evidence of the involvement of both genes in the hydroxyspheroidene and the spirilloxanthin carotenoid biosynthesis pathways. Furthermore, analysis of the accumulation of carotenoid intermediates in the different constructed mutants showed that crtD and crtC genes are cotranscribed with puf operon using the oxygen-regulated puf promoter.
Escherichia coli strains were grown at 37 °C on LB medium (30). R. gelatinosus strain S1 (31) and the constructed strains were grown anaerobically (photoheterotrophic conditions) or aerobically (nonphotosynthetic conditions) at 32 °C in malate (ML) medium (32). Antibiotics were used at the following concentrations for E. coli and R. gelatinosus: spectinomycin, 50 µg/ml; streptomycin, 50 µg/ml; ampicillin, 100 µg/ml; kanamycin, 50 µg/ml; tetracycline, 10 µg/ml. Bacterial strains and plasmids used in this work are listed in Table I.
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Plasmid DNA was introduced into R. gelatinosus cells using the electroporation system described in Ref. 29. Following the electric pulse, cells were diluted in 10 ml of ML medium. After incubation at 32 °C for 6 h in darkness, serial cell dilutions were plated on nonselective ML plates to assess cell survival and on selective ML plates to select transformants. Two different antibiotic resistance markers were used to distinguish a double crossover event from a single crossover event, the first one being located on the vector and the second one as the cartridge inserted in the gene to be inactivated.
Chromatophore Isolation and Carotenoid Extraction; Spectrophotometric MeasurementsChromatophores from R. gelatinosus were prepared by differential centrifugation after disruption of cells with a French press and were resuspended in 10 mM Tris HCl pH 8 buffer. Carotenoids were extracted from chromatophores with acetone/methanol (7:2, v/v) and separated by thin-layer chromatography in a acetone/petroleum ether (1:9) solvent, as described in Ref. 3. Each carotenoid spot was recovered, eluted from silica by a small volume of acetone, and analyzed by spectroscopy. Spectral analysis was carried out on a Cary 2300 spectrophotometer interfaced with a computer.
Molecular Biology TechniquesStandard methods were
performed, if not otherwise indicated, according to Ref. 30. Plasmid
DNAs were purified using Qiagen columns (Diagen). DNA was treated with
restriction enzymes and other nucleic acid modifying enzymes (Klenow
fragment, alkaline phosphatase, T4 DNA ligase) according to
manufacturer specifications. DNA fragments were analyzed on agarose
gel, and different restriction fragments were purified using a
Geneclean kit (Bio 101). Genomic DNA was purified as described in Ref.
29. Southern hybridization analysis of genomic DNA was performed as
indicated by Amersham; probes were labeled with
[-32P]CTP by nick translation.
Construction of pMP8 and pSO8 has
been described previously in Ref. 29. Plasmid pMP8 was linearized with
BstBI restriction enzyme and treated with the Klenow
fragment to create blunt ends. The resulting plasmid was ligated with
the SmaI or Km cartridge to construct pSM2 and PSO6,
respectively. For constructing pSO14 and pSO15, the BamHI
or Km cartridge was inserted in the unique BglII site of
pufL in pMP8 plasmid. To inactivate the crtD
gene, pSOX plasmid was linearized with BglII restriction
enzyme and ligated with the BamHI
or Km cartridge to
create pSO20 and pSO21, respectively.
To clone the region downstream of the puf operon, genomic DNA from the SIC strain was digested with different restriction endonucleases and ligated. The ligation product was used to transform E. coli. Transformants were analyzed and several plasmids were obtained. The crt genes were subcloned in plasmid pSOX and pSO24. Sequencing was performed on both strands using the dideoxy chain termination method of Sanger with the Sequenase version 2.0 kit, Amersham.
A
puf operon deletion strain called SP was described
previously (29). A pleiotropic effect of the deletion was a change in
the color of the cells which became green (the wild type being purple).
Absorption spectra of chromatophores of this strain showed that the
three main absorption peaks of carotenoids in the wild type (455, 481, and 512 nm) were shifted to the blue, giving a shoulder at about 425 nm
and two peaks at 458 and 487 nm in the mutant. This phenotype was due
neither to spontaneous mutations nor to the
cartridge itself, but
to the disruption of the puf operon. Indeed, transformants
with pSO8, a replicative plasmid bearing the
cartridge, were
purple, but the complemented strain CS
P, bearing the puf
operon on a replicative plasmid, was also green. Furthermore, we
isolated revertants from the complemented strain CS
P which became
purple and in which the correct chromosome structure was restored as a
result of recombination between the chromosome and the complementing
plasmid (29).
Other interposon strains, SIL1 and SC1 (Table I), in
which the
cartridge was inserted in pufL and in
pufC genes, respectively, were constructed (Fig.
2A). Spr and Aps
transformants resulting from double crossover events were selected. The
inactivation of the genes with the drug cartridges was confirmed by
Southern blot analyses. Fig. 2B shows an example of Southern analysis. Genomic DNAs from wild type and two different clones of
S
C1 strains were digested either by SacI or by
SalI and probed with the SalI fragment containing
a large part of pufC. The 4.9-kb SacI band seen
in the wild type was replaced, as expected, by a 6.9-kb band resulting
from the 2-kb
cartridge insertion in the gene. The 1.1-kb
SalI band seen in the wild type was replaced, as expected,
by two small 0.7- and 0.4-kb bands because of the two SalI
sites of the polylinkers flanking the
cartridge. The same type of
analysis, using other restriction enzymes, was performed with the SIL1
strain (data not shown) to confirm the inactivation of the
pufL gene. The S
C1 and SIL1 strains were also green,
substantiating the polar effect of the puf deletion.
Spectroscopic analysis carried out on chromatophores indicated a
modification in the carotenoid absorption peaks. It was assumed that
the green phenotype reflected a lesion in the carotenoid
biosynthesis.
Analysis of the Constructed Strains: Carotenoid Contents
Pigment modifications in the mutants SIL1 and SC1
were checked by recording absorption spectra of acetone/methanol
extracts from chromatophores of these strains and comparing them with
that of the wild type (Fig. 3). In the wild type, the
three main carotenoid absorption peaks are located at 430, 455, and 485 nm, and there is a minor peak at 530 nm. In both mutants, the three
major peaks were shifted toward the blue giving maxima at 415, 440, and
470 nm, with a minor peak at 500 nm. This indicated that the green color was due to a change in the carotenoid composition of the cells
and that the mutants were blocked presumably at the same step in the
carotenoid biosynthesis pathway. To confirm this result, the carotenoid
extracts of the mutants and of the wild type were analyzed by thin
layer chromatography (3), and the major carotenoids were identified by
their relative mobilities and by their absorption spectra (Table
II). In the wild type, the major carotenoids were identified as hydroxyspheroidene, spheroidene, and spirilloxanthin, as
in Ref. 5. In the mutants, two major carotenoids were identified as
neurosporene and lycopene (Table III). Since the next
steps in the carotenoid biosynthesis pathway for transforming,
respectively, neurosporene to hydroxyneurosporene and lycopene to
rhodopin are hydrations (Fig. 1), our results suggested that in the
mutants the synthesis of a hydratase encoded by crtC gene
was blocked and that at least this gene could be located downstream of
the puf operon.
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To check the localization of the crtC gene and to see whether it is the only crt gene localized downstream of the puf operon, cloning of this region was achieved by insertional cloning using SIC and SID3 strains in which recombinant plasmids were inserted downstream of the puf operon (Table I). Two plasmids pSOX and pSO24 were obtained, and the cloned DNA was sequenced on both strands. The complete nucleotide sequence of a 3-kb SacI fragment downstream of the puf operon (Fig. 2A) was determined. This fragment was found to contain two ORFs immediately downstream of the pufC gene. The initial assignment of the ORFs was based on the alignment with R. capsulatus and R. sphaeroides photosynthetic gene cluster, which showed that they correspond to crtD and crtC carotenoid genes. The crtD and crtC genes were in the same direction as the puf operon. The nucleotide and deduced amino acid sequences were deposited in the GenBankTM Data Library under the accession number U73944[GenBank].
Characteristics of the Nucleotide SequenceThe crtD and crtC genes have the same transcriptional orientation, and no transcriptional terminator was found between them, suggesting that they could be cotranscribed. For both genes, an ATG initiating codon is proposed. The crtC gene initiation codon was found within the crtD gene (overlap of 372 nucleotides). No alternative initiation codon could be found. Putative translation starts of these genes were based on the position of a Shine-Dalgarno ribosome binding site evaluated by comparison with other sequenced genes from R. gelatinosus known to encode proteins and comparison with genes from R. capsulatus (10). The first third of the crtC gene shows atypical codon usage. In R. gelatinosus, the codon usage deduced from the pufBALMC genes is characterized by the presence of a majority (89%) of C and G at the third position. The first 126 codons of crtC have only 64% CG at the third position, whereas the rest of the sequence has a value of 93% comparable with the mean value. No putative promoter sequence with significant homology to the known puf promoter or to the crt promoters from R. capsulatus and R. sphaeroides was found for the two genes; this is in agreement with genetic data concerning their expression (see below).
Cotranscription of crtD and crtC GenesWe disrupted
crtD gene either with an cartridge which contains a
strong transcriptional terminator or with a Km cartridge devoid of the
transcriptional terminator (Fig. 2A). If the two genes were
cotranscribed, inactivation of crtD with an
cartridge would lead to a crtC-less phenotype, and cells would
accumulate neurosporene and lycopene since CrtC operates upstream of
CrtD in the carotenoid biosynthetic pathway, whereas inactivation with a Km cartridge leads to a crtD-less phenotype, and cells
accumulate other carotenoid intermediates.
Plasmids pSO20 and pSO21 were used to transform R. gelatinosus leading to SID1 and SID2 strains, in which the
crtD was inactivated with the and the Km cartridge,
respectively (Table I, Fig. 2A). All the obtained
transformants were green. Southern blot analyses were performed and
confirmed the inactivation of crtD by a double crossover
event in the transformants (data not shown). Carotenoids were extracted
and identified by their relative mobilities in thin layer
chromatography and their visible absorption spectra (Table III). In
SID1, the major carotenoids corresponded to neurosporene and lycopene,
a phenotype of a crtC
genotype, whereas in
SID2 the carotenoid intermediates corresponded to neurosporene,
methoxyneurosporene, hydroxyneurosporene, lycopene, and rhodopin, a
phenotype of a crtD
genotype. When the whole
plasmid pSO21 was inserted into the chromosome, the resulting strain
SID3 was green and had the same phenotype and pigment composition as
SID1. These results indicated that crtD and crtC
are cotranscribed.
The crtD gene is 1572 bp long and encodes a
protein with a predicted molecular mass of about 56 kDa. The CrtD
protein from R. gelatinosus is highly similar (53%) to the
corresponding protein from R. capsulatus (Fig.
4A). Particularly, a stretch of residues in
the C-terminal region which is highly conserved in the carotenoid dehydrogenases (CrtD and CrtI) from R. capsulatus (10) is
also found in R. gelatinosus (underlined in Fig.
4A).
The crtC gene is 1218 bp long and encodes a protein with a predicted molecular mass of about 44 kDa. The CrtC protein from R. gelatinosus presents less similarity (42%) than crtD to the corresponding protein from R. capsulatus (Fig. 4B). This is owing to the extension of 120 residues in the N-terminal region of the R. gelatinosus protein.
It is noteworthy that in R. gelatinosus, the first part of the CrtC protein, which corresponds to the region with atypical codon usage, was enriched in proline (10.5%) whereas the rest of the sequence has the usual percentage of proline, i.e. about 6-7%. The hydropathy plots of both proteins were determined according to Kyte and Doolittle (33), and they did not reveal any putative transmembrane domain.
Involvement of CrtC and CrtD in the Spirilloxanthin Biosynthesis PathwayEvidence of the involvement of the CrtC and CrtD proteins
in the spirilloxanthin biosynthesis pathway arises from the carotenoid content of SID1 and SID2 mutants. In this biosynthesis pathway, lycopene is converted to rhodopin by a hydration reaction. In SID1
strain (crtC), this conversion does not occur,
implying that the crtC gene product catalyzes the
transformation of lycopene to rhodopin in R. gelatinosus.
The formation of anhydrorhodovibrin from rhodopin needs a
dehydrogenase. Inactivation of crtD gene in SID2 strain
(crtD) results in the accumulation of
rhodopin. Thus, in R. gelatinosus, the crtD gene
product catalyzes the formation of anhydrorhodovibrin at the expense of
rhodopin.
Analysis of the interposon strains indicated that there is no promoter for carotenoid genes within the puf operon (see above). One possible promoter could be the puf operon promoter itself. To examine this hypothesis, two insertion strains called SIO and SIC (Table I) were used (29). In the SIO strain, a recombinant plasmid has been inserted between the bch genes and the puf operon and thus the puf operon remained linked to the carotenoid genes. In the SIC strain, the recombinant plasmid has been inserted between the puf operon and the carotenoid genes (29). Because of this chromosome organization, if the crt genes are transcribed using the puf promoter (which had been shown to be repressed under aerobic conditions), we expected the SIC strain to be green independently of the growth conditions. On the contrary, the SIO strain would be green in darkness under aerobiosis, but would become purple when grown under photosynthetic conditions (light and anaerobiosis).
In darkness under aerobiosis, the SIC and SIO strains were green.
Pigment analysis indicated that both strains accumulated neurosporene
and lycopene (Table III). When transferred in photosynthetic conditions, the SIC cells remained green, whereas the SIO cells became
progressively purple. Carotenoid were extracted from cells after 2, 5, and 7 days of growth in photosynthetic conditions. For the SIC strain,
no difference was observed (not shown). For SIO, the spectra of the
carotenoid extracts from cells grown during 2 days and during 7 days in
photosynthetic conditions were different (Fig. 5) and
the difference spectrum, computed between these two culture extracts,
showed the same carotenoid absorption peaks as in the wild type.
Analysis of carotenoids by thin layer chromatography showed the
progressive disappearance of neurosporene and lycopene and the
progressive appearance of spheroidene, hydroxyspheroidene, and
spirilloxanthin (Table III). These results indicated that the carotenoid genes are cotranscribed with the puf operon using
the oxygen-regulated puf promoter. To confirm these results,
we constructed two other interposon strains SIL2 and SC2 in which
the pufL or pufC, respectively, were disrupted by
the Km cartridge which is devoid of a transcriptional terminator (Table
I, Fig. 2A). The resulting strains were purple, and they had
the same pigment composition as the wild type. However, when the whole
plasmids (pSO15 and pSO6) were inserted into the chromosome, the
resulting strains SIL3 and SIC2 were green and had the same phenotype
and pigment composition as SIL1 and S
C1.
The cartridge has been proved to be an efficient polar mutagen
(34). We have used it to construct puf inactivations and deletions. The resulting strains exhibited a green phenotype, suggesting a localization of carotenoid genes downstream from the
puf operon (29). In this work we demonstrate that the
localization of the crtD and crtC carotenoid
genes in R. gelatinosus is different from that described up
to now in the other purple bacteria. Indeed, crtD and
crtC were found downstream of the puf operon in
R. gelatinosus, whereas in R. capsulatus and
R. sphaeroides the two genes are located in a carotenoid
cluster including all the crt genes and flanked by the
bch genes, upstream of the puf operon.
The polarity effects arising from the interposon mutagenesis within crtD showed that crtD and crtC are cotranscribed and in the crtD-crtC order. More interestingly, interposon mutagenesis within puf operon also had polar effects on the carotenoid genes; this indicated that the crt genes are cotranscribed with the puf operon. Unlike the situation in R. capsulatus and R. sphaeroides, no promoter sequence was found for the transcription of the crtD and crtC genes in R. gelatinosus, and we have genetic evidences that under photosynthetic conditions the crtD and crtC genes are cotranscribed with puf using its oxygen-regulated promoter. Thus, one may wonder how the crt genes are transcribed under nonphotosynthetic conditions. Since in darkness and under aerobiosis R. gelatinosus is still pigmented, we suppose that these genes are transcribed from an oxygen-insensitive promoter. We have already shown that under nonphotosynthetic conditions the puf operon is transcribed from an upstream promoter insensitive to oxygen regulation, and that cells under these conditions produced a reduced amount of LHI and RC proteins (29). We propose that the carotenoid genes localized downstream of the puf operon could be also transcribed from this oxygen-insensitive promoter. In R. capsulatus, it was proposed that crtEF, bchCXYZ, and puf operons were cotranscribed under nonphotosynthetic conditions using the crt promoter (35) and that bchFBNHLM-F1696 and puhA are cotranscribed using the bch promoter (22). In R. gelatinosus, we have to assume that bch, puf, and crt genes could be cotranscribed under the nonphotosynthetic conditions using the bch oxygen-insensitive promoter; this hypothesis will be confirmed by mRNA analysis (under investigation).
A -independent terminator-like structure was found between
pufC and crtD genes of R. gelatinosus.
Similar structures were found in R. capsulatus, R. sphaeroides, R. rubrum, and R. viridis (36).
We would suggest that these structures could have a regulatory function
and serve to control the stability of the transcript. The carotenoid
genes which encode enzymatic proteins would be required in low amounts,
in contrast to puf genes encoding structural polypeptides
which require a higher level of expression, and, accordingly, the
hairpin structures could protect puf mRNA from exonuclease digestion to increase its lifetime. Beatty (20) speculated
that in spite of the two
-independent terminator-like structures
found downstream of pufX in R. capsulatus, a
read-through transcription downstream of the puf operon was
possible; here we confirm this hypothesis for R. gelatinosus.
In R. capsulatus and R. sphaeroides, only one carotenoid biosynthesis pathway has been identified, leading to the synthesis of spheroiden(on)e; the reactions and the involved genes are now well known (6). In R. gelatinosus, the diversity of carotenoids is greater and two carotenoid biosynthesis pathways lead, respectively, to synthesis of hydroxyspheroidene and spirilloxanthin (5). The reaction steps of both pathways are known (5), but the involved genes have not been identified.
The biosynthesis of hydroxyspheroidene, in R. gelatinosus, might involve the same enzymatic reactions as in R. capsulatus and R. sphaeroides. Here we report the identification of the crtD and crtC genes, and we confirm the functions of their products in the spheroidene branch.
The biosynthesis of spirilloxanthin via rhodopin in R. gelatinosus and in R. rubrum includes several known reactions (5). In this work, we propose another function to crtC and crtD gene products in R. gelatinosus. The crtC gene, encoding a hydratase, and the crtD gene, encoding a dehydrogenase, are, respectively, involved in the formation of rhodopin from lycopene and the formation of anhydrorhodovibrin from rhodopin (Fig. 1). It implies that the crt genes involved in the hydroxyspheroidene pathway are also involved in the spirilloxanthin one.
To complete the scheme, we propose the participation of crtI, crtC, crtD, and crtF in the final steps of spirilloxanthin synthesis (Fig. 1). Indeed, transformation of neurosporene to lycopene requires a "specific" desaturation catalyzed by a dehydrogenase which could be encoded by crtI. It is interesting to note that in R. capsulatus and R. sphaeroides, this CrtI enzyme is present but its catalytic function stops at the level of neurosporene (7). This difference in the functioning of the dehydrogenase suggests that in R. gelatinosus, the crtI gene could be different from the corresponding gene in R. capsulatus and R. sphaeroides. Conversion of anhydrorhodovibrin to rhodovibrin requires a hydration which could be catalyzed by crtC gene product. The crtD gene encodes a dehydrogenase which could catalyze the formation of monodemethyl spirilloxanthin from rhodovibrin. Finally, formation of the spirilloxanthin requires the methylation of the monodemethyl spirilloxanthin which is catalyzed by an O-methyltransferase. In the hydroxyspheroidene pathway, the O-methyltransferase is encoded by the crtF gene; thus, crtF might also be implicated in the spirilloxanthin pathway. This has been also suggested by Yildiz et al. (37) as a crtF mutant of R. capsulatus has been complemented by DNA from R. centenum which synthesizes spirilloxanthin rather than spheroidene. Further genetic experiments have to be performed to confirm these hypotheses.