Phycoerythrins of the oxyphotobacterium Prochlorococcus marinus ...

Plant Molecular Biology 40: 507–521, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Phycoerythrins of the oxyphotobacterium Prochlorococcus marinus are
associated to the thylakoid membrane and are encoded by a single large
gene cluster
Wolfgang R. Hess 1,∗ , Claudia Steglich, Christiane Lichtlé 2 and Frédéric Partensky 3
1 Humboldt-University, Department of Biology, Chausseestrasse 117, 10115 Berlin, Germany ( ∗ author for
correspondence); 2 Laboratoire de Photorégulation et Dynamique des Membranes Végétales, Ecole Normale
Supérieure, CNRS URA 1810, 46 rue d’Ulm, Paris Cedex 05, France; 3 Station Biologique, CNRS, INSU et
Université Pierre et Marie Curie, BP 74, 29682 Roscoff Cedex, France
Received 18 December 1998; accepted in revised form 22 March 1999
Key words: cyanobacteria, immunogold labelling, light-harvesting complexes, photosynthesis, phycobilins,
phytoplankton
Abstract
An intrinsic divinyl-chlorophyll a/b antenna and a particular form of phycobiliprotein, phycoerythrin (PE) III,
coexist in the marine oxyphotobacterium Prochlorococcus marinus CCMP 1375. The genomic region including
the cpeB/A operon of P. marinus was analysed. It encompasses 10 153 nucleotides that encode three structural
phycobiliproteins and at least three (possibly five) different polypeptides analogous to cyanobacterial or red algal
proteins involved either in the linkage of subunits or the synthesis and attachment of chromophoric groups. This
gene cluster is part of the chromosome and is located within a distance of less than 110 kb from a previously
characterized region containing the genes aspA-psbA-aroC. Whereas the Prochlorococcus phycobiliproteins are
characterized by distinct deletions and amino acid replacements with regard to analogous proteins from other
organisms, the gene arrangement resembles the organization of phycobiliprotein genes in some other cyanobacteria,
in particular marine Synechococcus strains. The expression of two of the Prochlorococcus polypeptides as recombinant
proteins in Escherichia coli allowed the production of individual homologous antisera to the Prochlorococcus
α and β PE subunits. Experiments using these sera show that the Prochlorococcus PEs are specifically associated
to the thylakoid membrane and that the protein level does not significantly vary as a function of light irradiance or
growth phase.
Abbreviations: Chl, chlorophyll; IPTG, iso-propyl-thiogalactoside; PC, phycocyanin; PE, phycoerythrin; PBS,
phycobilisome; PUB, phycourobilin
Introduction
In higher plants and green algae, the major
light-harvesting complexes consist of membraneassociated
chlorophyll (Chl) a/b-binding proteins,
whereas in cyanobacteria and red algae, the lightharvesting
function is fulfilled by an extrinsic macromolecular
complex, the phycobilisome (Grossman
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession number AJ001230.
507
et al., 1995). The major antenna complex of
the Chl b-possessing oxyphototrophic prokaryotes
Prochlorothrix, Prochloron and Prochlorococcus belongs
to a third type consisting of an intrinsic Chl
a/b-binding protein (Pcb) with no phylogenetic relatedness
to the previous two antenna systems (Laroche
et al., 1996). Although these three genera, unproperly
called ‘prochlorophytes’, all belong to the cyanobacterial
phylum (Palenik and Haselkorn, 1992; Urbach
et al., 1992; Hess et al., 1995), electron microscopy
studies show that their thylakoids are closely ap-

508
pressed (Bullerjahn and Post, 1993) and, therefore,
that they probably do not contain any phycobilisomes
in addition to their Pcb antenna complexes. Yet, in
Prochlorococcus marinus CCMP 1375 (or SS120)
genes coding for the α and β chains of phycoerythrin
(PE) have been identified (Hess et al., 1996).
This organism is the type species of a genetically
diverse group of unicellular oxyphototrophic bacteria
(Chisholm et al., 1992; Urbach et al., 1998),
which contains divinyl-chlorophyll a and b (Chl a2
and b2; Goericke and Repeta, 1992) and which dominates
the photosynthetic biomass in most temperate
and intertropical oceanic ecosystems (for reviews, see
Whitman et al., 1998; Partensky et al., 1999a, b).
P. marinus cpeA and cpeB genes, encoding PE α and β
subunits, have been shown to be functional, although
weakly transcribed (Hess et al., 1996). Furthermore,
the presence of PE in a water-soluble extract of P. marinus
cells was demonstrated by cross-reaction of a
21 kDa protein with a heterologous serum to PE. At
last, the fluorescence emission spectra of this PE were
shown to resemble those of marine Synechococcus
(Ong et al., 1984) with dominance of the 496 nm
peak due to phycourobilin over the 550 nm peak of the
phycoerythrobilin (Hess et al., 1996). Since, in P. marinus
CCMP 1375, the photosynthetic antenna system
is made up of the Chl a2/b2-Pcb complexes (Partensky
et al., 1997), the exact function, intracellular localization
and mode of regulation of these PE molecules
remained obscure. In particular, it is not clear whether
they fulfil a light-harvesting function, are involved in
a sensor function in light-perception processes as are
phytochromesknown from other cyanobacteria (Kehol
and Grossman, 1996; Hughes et al., 1997) or are simply
function-less. Their phylogenetic origin is problematic,
too, and one can wonder whether these genes
represent evolutionary remnants of ancestral phycoerythrins
or have been more recently acquired by
horizontal gene transfer from marine Synechococcus,
a genus that often co-occurs with Prochlorococcus in
natural assemblages (Partensky et al., 1999a).
So far, it has neither been investigated whether
the P. marinus cpeA and cpeB genes are located on
extrachromosomal genetic elements nor if there are
additional phycobiliprotein genes. In cyanobacteria,
genes for phycobiliproteins are frequently clustered
together or with genes encoding other components
of the phycobilisomal apparatus. Therefore analysis
of the genome region adjacent to P. marinus cpeB
and cpeA could be informative about the possible
presence of further phycobiliproteins in this organ-
ism. We show here that the cpeB and cpeA genes of
P. marinus are part of a larger gene cluster comprising
altogether six, possibly eight, different genes either
coding for phycobiliproteins or being involved in the
linkage of subunits or the biosynthesis of phycobilins.
Furthermore, we have obtained homologous antisera
against Prochlorococcus PE α and β subunits in order
to perform an immunocytochemical study of PE
intracellular localization and to study their expression
under different light conditions and in different growth
phases.
Materials and methods
Cultures
Cultures of P. marinus clone CCMP 1375, obtained by
courtesy of Prof. S.W. Chisholm and Dr L.R. Moore,
were grown at 21 ± 1 ◦ C in PCR S11 medium
(Partensky et al., 1999b) under continuous blue light at
either 8 µmol photons m −2 s −1 for immunocytochemical
studies, 15 µmol photons m −2 s −1 for biomass
production or a range of irradiances for expression
studies.
Cloning and DNA sequence analysis
Total cellular DNA was isolated by suspending cells
from 2.4 l culture in 4 ml of DNA isolation buffer
(50 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM EDTA,
0.5% w/v SDS). After addition of proteinase K (final
concentration 100 µg/ml), cells were incubated for
4hat50 ◦ C. DNA was extracted by gentle inversion
with an equal volume of phenol/chloroform/isoamyl
alcohol (25:24:1) for 5 min at room temperature. After
centrifugation, high-molecular-weight DNA was
spooled out from the upper phase by addition of 3
volumes of ethanol/NaOAc (96%/100 mM) using a
glass rod. The DNA was washed once in 70% ethanol,
air-dried and dissolved in 0.5 ml TE buffer (10 mM
Tris-HCl, 1 mM EDTA pH 8.0). A 100 µg portion
of high-molecular-weight genomic DNA was partially
cut by the restriction endonuclease Sau3AI in a volume
of 1000 µl. Quality of restriction digests was
evaluated by size fractionation of DNA fragments on
a CHEF mapper pulsed-field gel electrophoresis (Bio-
Rad, Richmond, CA). The majority of DNA fragments
was in the desired size range of 30–42 kb. About
5 µg of the partially digested chromosomal DNA was
dephosphorylated using calf intestine alkaline phosphatase,
extracted once with phenol/chloroform then

once with chloroform, ethanol-precipitated and resuspended
at a concentration of 1 µg/µl. Exactly 2.5 µg
of the partially digested dephosphorylated chromosomal
DNA was ligated to 2.5 µg XbaI/BamHI-digested
SuperCos1 vector DNA at 4 ◦ C for 16 h. The ligation
products were packaged into lambda particles
using Gigapack III Gold packaging extracts (Stratagene,
La Jolla, CA). E. coli XL1-Blue MR served
as host cells. Cosmid clones were grown on plates,
individually picked on a clone grid and hybridized
to radioactively labelled probes. Identified cosmids
clones were grown in liquid medium. DNA was purified
and cut by EcoRI. EcoRI subclones were prepared
in plasmid vector pBluescript. Additionally, orientation
and arrangement of subclones was verified by
PCR and further compared to fragments obtained by
PCR amplification from genomic DNA.
For sequence determination we used the dideoxy
chain termination method throughout. Sequences were
obtained from both strands of the DNA by primer
walking on an ABI 373 sequencer (Applied Biosystems,
Perkin Elmer, Fullerton, CA) after individual
sequence reactions using the dye terminator and dye
primer cycle sequencing kits from Applied Biosystems.
Production of recombinant proteins in E. coli
The P. marinus phycoerythrin genes cpeA and cpeB
were selected for expression as recombinant proteins
in E. coli. The genes were subcloned in vector
pMAL-c2 (NEB, Beverly, MA) by PCR using
a proof-reading DNA polymerase (KlenTaq; Clontech,
Palo Alto, CA) and the following primers (the
restriction sites inserted for construction of the expression
plasmids are underlined): CPEAMEXF: 5 ′ -
GCTGCAAATGGGATCCACAGTCACCACAG-3 ′
(cpeA gene forward primer); CPEAMEXR: 5 ′ -
AAGTTCTCTGCAGGTTTTGATCAAGCCAAGGC-
3 ′ (cpeA gene reverse primer); CPEBMEXF: 5 ′ -
CCAGATGCTTGGATCCTTCTCAAGAGCAG-3 ′
(cpeB gene forward primer); CPEBMEXR: 5 ′ -
TAATTCGTCGACATGGCCATCAATTTAAAGC-3 ′
(cpeB gene reverse primer). Total E. coli protein extracts
were prepared from cultures induced by the
addition of 0.3 mM IPTG for 3 h. The cells were collected
by centrifugation and disrupted by 15 strokes
of ultrasonic power for 15 s each at 4 ◦ C. The fusion
proteins consisting of the maltose-binding protein
part from E. coli and the respective phycoerythrin
were purified by affinity chromatography on amylose
509
columns. The collected fractions containing the purified
protein were obtained after elution with column
buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA
pH 7.4) complemented by 10 mM maltose.
Immunology and electron microscopy
Polyclonal antisera were raised in two rabbits each by
a commercial producer (Biogenes, Berlin). After a basic
immunization, three boosts with new recombinant
protein were done and the antibody titre was followed
by ELISA tests. For the β PE, an ELISA signal could
still be detected at a dilution of >1:200 000 and for the
α PE at >1:300 000. The antisera were tested on blots
and further purified by protein A-chromatography.
Immunocytochemistry was performed as previously
described (Lichtlé et al., 1995) with the following
modifications: cells of Prochlorococcus were
centrifuged and fixed at 4 ◦ C for 60 min in 2%
glutaraldehyde in 0.1 M phosphate buffer pH 7.2
plus 0.25 M sucrose. After dehydratation, they were
embedded in LRWhite medium grade resin and immunological
reactions were performed as previously
reported (Lichtlé et al., 1992) with antibodies diluted
1:1000 (1 h) and 10 nm gold particles (Bio Cell Gold
Conjugates). Sections were examined with an electron
microscope Jeol CX2.
Western blots were prepared from total protein
extracts separated on SDS-polyacrylamide gels and
blotted onto Hybond-C extra (Amersham Pharmacia
Biotech, Freiburg, Germany) or PVDF membrane
(NEN Life Sciences, Boston, MA). Lanes of gels were
loaded either with the same amount of proteins, as
measured by the BioRad protein assay, or with a quantity
of proteins adjusted to have the same amounts of
chlorophyll. Incubation with antisera was performed
at titres of 1:200 to 1:1000, secondary antisera were
conjugated with alkaline phosphatase and blots were
developed using the chromogenic substrates nitro-blue
tetrazolium and bichlorindophenol. For control, heterologous
antisera against the thylakoid membrane
proteins CP43 from Synechocystis PCC 6803 (courtesy
of Prof. R. Barbato) or D1 from pea chloroplasts
(courtesy of Dr P.J. Nixon), were used.
Pulsed-field gel electrophoresis (PFGE)
Cells from exponentially growing cultures were pelleted
by centrifugation and embedded in 0.6% lowmelting-point
agarose in 50 mM EDTA, pH 8.0. After
digestion by proteinase K (0.1% w/v) for 16 h at
55 ◦ C in 0.5 M EDTA, pH 8.0, the agarose plugs were

510
Figure 1. A. Overview of a 10 153 bp region containing the P. marinus CCMP 1375 phycoerythrin gene cluster. Genes encoding structural
phycobiliproteins are shown as black boxes. Dark grey boxes indicate genes, the products of which have significant similarity to phycobiliprotein-associated
open reading frames in other species. Light grey denotes an open reading frame with homology to a particular group
of light-harvesting complex-associated genes of rhodobacteria. Other open reading frames are displayed as white boxes. Dotted lines indicate
possible alternative start codons. Arrows show the direction of transcription. Restriction sites used for subcloning and sequence analysis (EcoRI)
or physical mapping (MluI) are indicated. For details of the sequenced region, see EMBL accession number AJ001230. Sequence analysis of
cosmid ends showed the presence of an S-adenosylmethionine synthetase gene (metK) about 3 kb downstream (right end) and of a putative
Mg-protoporphyrin chelatase gene (chlD about 20–22 kb upstream (left end) the PB protein gene cluster. B. Comparison to the corresponding
region of Synechococcus WH 8020 (Wilbanks and Glazer, 1993a).
washed three times in 1× TE (10 mM Tris-HCl, 1 mM
EDTA pH 8.0) and twice in 1× TE supplemented
with fresh phenylmethylsulfonylfluoride for 30 min
each. After three further wash steps in 1× TE, the
gel slices were equilibrated in the appropriate restriction
buffer. Samples were then incubated overnight
with restriction enzymes at 37 ◦ C(NotI, MluI) or at
25 ◦ C(SmaI). Separation of restriction fragments was
performed with a CHEF (contour-clamped homogeneous
field electrophoresis) system in 1% agarose gels
in 0.5× TBE at 14 ◦ C, at a field strength of 6 V/cm
and using the auto-algorithm modus. Separated DNA
fragments were transferred onto positively charged nylon
membranes (GeneScreen, NEN Life Sciences) by
capillary blotting and hybridized with gene probes
radioactively labelled by random priming using the
Rediprime kit (Amersham). Composition of the hy-
bridization buffer was 7% SDS (w/v), 250 mM NaCl
and 250 mM sodium phosphate pH 7.2.
Results
Prochlorococcus phycoerythrin genes are part of a
cluster which includes various genes implicated in
phycobiliprotein structure and biosynthesis
In P. marinus CCMP 1375, genes for several phycobiliproteins
or polypeptides with known or supposed
functions in the stabilization of subunits or
chromophore biosynthesis and assembly, are tightly
clustered in a region of about 10 kb on both strands
of the DNA (Figure 1A and Table 1). On one side,
this cluster is adjacent to a gene with 55% identity to
uvrD, a gene encoding a DNA helicase that plays an
important role in prokaryotic nucleotide (nt) excision

Figure 2. Similarity of the protein encoded by orf431/462 to proteins of rhodobacteria that are involved in the assembly of light-harvesting
systems. The deduced amino acid sequence of this protein (PMorf431) has been aligned with the corresponding sequences from the cyanobacterium
Synechocystis PCC 6803 (sll1906) and two different rhodobacterial proteins (rhocapucc, R. capsulatus PucC protein (Tichy et al., 1991;
LeBlanc and Beatty, 1996); rhocaF1696, R. capsulatus LhaA protein (Young and Beatty, 1998; Young et al., 1998)). Putative membrane
spanning regions are indicated by a bar above the sequences. Translation of orf431 may occur from an alternative UUG initiation codon,
resulting in the indicated length of 462 amino acids.
repair, mismatch repair and DNA replication (Oeda
et al., 1982; Bierne et al., 1997; Petit et al., 1998).
On the opposite end, this region is neighboured by
about 1 kb of non-coding sequence. Sequence analysis
of cosmid ends revealed the presence of a gene
for S-adenosylmethionine synthetase, metK, atadistance
of about 3 kb downstream and of a gene for a
subunit of Mg-protoporphyrin chelatase, chlD, about
20–22 kb upstream (Figure 1A). The overall G+C
content in the investigated region is 34.1%. The structural
genes cpeB and cpeA, encoding phycoerythrin β
511
and α subunits, respectively, are co-transcribed and
have been described previously (Hess et al., 1996).
It is not known if these genes constitute an operon
together with the adjacent gene cpeZ. An mRNA with
a size of about 1.3 kb previously detected for cpeBcpeA
(Hess et al., 1996) would be too short to include
also cpeZ. The arrangement of genes and the overall
organization of this region resembles that of a gene
cluster for the major phycobiliprotein rod components
of Synechococcus WH 8020 (Figure 1B and Wilbanks
and Glazer, 1993a).

Table 1. Summary of genes and open reading frames from the P. marinus phycobiliprotein gene cluster (c, complementary strand). Their similarity to related genes from other
organisms is indicated: description of the one or two best database matches is listed together with its accession number and the organism. The percentage of amino acid identity is
given together with the length (number of amino acids) of the compared match in parenthesis. Abbreviations: S., Synechocystis; Syn, Synechococcus; sll/slr, designations of genes
from the Synechocystis PCC 6803 total genome project.
Gene/ORF Position in Function of analogous genes in other organisms Accession number of Source Amino acid Reference
Figure 1 best matches identity
uvrD 1–1557 (c) DNA helicase II (UvrD) sll1143/D90906 S. PCC 6803 55% (512) Kaneko et al., 1996
cpeB 2084–2632 PE β subunit (CpeB) Q08087 Syn. WH 7803 63% (184) Newman et al., 1994
cpeA 2682–3149 PE α subunit (CpeA) Q02179 Syn. WH 8020 55% (164) De Lorimier et al., 1993
cpeZ 3197–3805 Phycobiliprotein CpeZ C45045 Syn. WH 8020 29% (184) Wilbanks and Glazer, 1993a
mpeX 3809–4705 (c) Bilin biosynthesis protein MpeV Q02178 Syn. WH 8020 43% (296) Wilbanks and Glazer, 1993a
Bilin biosynthesis protein MpeU Q02177 45% (284)
cpeY 4783–6102 Bilin biosynthesis protein CpeY Q02174 Syn. WH 8020 33% (404) Wilbanks and Glazer, 1993a
orf195 6121–6708 (c) Hypothetical protein encoded by orf198 located 3 ′ from rpcA Q02426 Syn. WH 8103 32% (180) De Lorimier et al., 1993
orf181 6705–7250 (c) Hypothetical protein Slr2049 encoded adjacent to rod D90903 S. PCC 6803 29% (155) Kaneko et al., 1996
core linker
ppeC 7441–8253 (c) PE II γ subunit (MpeC) Q02181 Syn. WH 8020 43% (171) Wilbanks and Glazer, 1993a, b
orf431/462 8266–9561 (c) Putative bacteriochlorophyll synthase sll1906 D90910 S. PCC 6803 37% (446) Kaneko et al., 1996
Assembly protein LhaA P26176 R. capsulatus 25% (412) Young and Beatty, 1998;
Young et al., 1998
512

The cpeY and cpeZ genes are homologous (28–
33% identical and 50–55% similar residues) to the
corresponding genes found in the phycobiliproteinrelated
operons of Synechococcus WH 8020 (cf. Table
1), Pseudanabaena PCC 7409 and Fremyella
diplosiphon (Mazel et al., 1986; Dubbs and Bryant,
1991; Wilbanks and Glazer, 1993a). A role in
cyanobacterial phycoerythrin biosynthesis has recently
been described for these proteins (Kahn
et al., 1997). Noteworthily, the ‘modified E-Z motif’
(LVYI)X(RE)X(AS)(AV)(KR)(ASGT)L(GANT),
a sequence that is normally found in all phycobiliprotein-associated
open reading frames (Wilbanks and
Glazer, 1993a), is, in P. marinus, only present in the
gene product of cpeZ (aa 55–64).
The product of another gene of the cluster has
about the same degree of similarity with regard to
two different Synechococcus WH 8020 proteins which
are encoded by mpeU and mpeV (43 and 45% identical,
and 65 and 61% similar residues, respectively)
and which might have a function in bilin biosynthesis
(Wilbanks and Glazer, 1993a), possibly as lyases (De
Lorimier et al., 1993). Since Prochlorococcus possesses
only one such gene, we have called it mpeX
in Figure 1A and Table 1. Translation of this reading
frame might occur from an UUG start codon
because the first encoded Met is preceded by a region
potentially encoding 95 additional residues with
pronounced similarity to the 5 ′ end of mpeV or mpeU.
The putative products of two open reading frames
with a calculated length of 181 and 195 amino
acids, respectively, orf181 and orf195, can be aligned
with ease with two hypothetical proteins encoded by
slr2049 in Synechocystis PCC 6803 and by orf198 in
Synechococcus WH 8103 (Table 1). There are no data
on the function of the putative proteins encoded by
these open reading frames yet. The protein encoded
by orf195 and the related proteins in other cyanobacteria
are very hydrophilic and acidic. The genes
homologous to orf195 in Synechococcus WH 8020
(orf197; Wilbanks and Glazer, 1993a) and WH 8103
(orf198; De Lorimier et al., 1993), and in Synechocystis
PCC 6803 (orf192 or slr2049; Kaneko et al., 1996)
are in the immediate vicinity of rpc genes, which encode
R-phycocyanins (WH 8020 and WH 8103) or
phycobilisome (phycocyanin) rod core linker proteins
(PCC 6803). Therefore, the proteins encoded by these
orfs likely have a function related to phycocyanin or
another phycobiliprotein. However, no homologues of
rpc genes have been found so far in P. marinus.The3 ′
end of orf181, including the stop codon TGA, overlaps
513
Figure 3. Phylogenetic analysis of the P. marinus ppeC gene product.
For this analysis, only the most conserved region was considered,
corresponding to ppeC aa 101 to 270. The other linker proteins
are the Synechococcus WH 8020 PE II γ subunit (SwissProt accession
number Q02181), two PC-associated linker peptides, of
Mastigocladus laminosus (P11398) and Synechococcus elongatus
(P50034), three PE-associated linker polypeptides of Calothrix
PCC 7601 (P18543, P18542, A43323), and one PC-linker polypeptide
each from Anabaena PCC 7120 (P07123) and Synechocystis
PCC 6803 (P73203). The tree was obtained by a maximum likelihood
estimation of phylogenies using the Jones model of evolution
and the quartet puzzling search for the best tree as part of the
PUZZLE 4.0 program package (Strimmer and von Haeseler, 1997).
Numbers at the nodes indicate QP support values for the branch
distal to it. From 126 quartets analysed in 1000 puzzling steps, 15
remained unresolved.
with the begin of orf195 including the ATG start codon
by 4 nt (cf. Figure 1A).
A major difference between the P. marinus phycobiliprotein
gene cluster and that of other cyanobacteria
is the presence of the most distally located putative
gene coding for a protein of 431 amino acids. Translation
of this orf might occur alternatively from an
UUG initiation codon, which would result in 462
residues. Localization and sequence conservation of
this orf431/462 are interesting. The distance of only
13 nt between the stop codon of this orf and the start
codon of ppeC suggests their possible cotranscription.
In database searches, the gene sll1906 from Synechocystis
PCC 6803 (Table 1) turned out to be the
most similar match to orf431/462. However, the annotation
(‘bacteriochlorophyll synthase’) of sll1906 may
be erroneous. Both proteins are less similar (BLASTP
scores of 120 and 125, respectively) to the Rhodobacter
capsulatus bacteriochlorophyll synthase (SwissProt
accession number P26171) than to members of
the LhaA/PucC family of proteins (BLASTP scores of
151 and 146). Furthermore, there is over their whole
length strong structural similarity between the pre-

514
dicted membrane-spanning regions of the hypothetical
gene products from orf431/462 and sll1906 and equivalent
regions in the LhaA or PucC polypeptides in
R. capsulatus and other rhodobacteria (Figure 2). The
degree of similarity is comparable to the relatedness
that exists between rhodobacterial reaction centre proteins
(L- and M-chains) and the D1 proteins of PSII
reaction centres. Hence the putative proteins encoded
by orf431/462 and sll1906 might have a role analogous
but not necessarily identical to the LhaA/PucC
family of proteins. Intriguingly, the latter have a function
in the assembly of light-harvesting complexes
B875 (LHI) and B800–B850 (LHII), respectively, or
the delivery of pigment (bacteriochlorophyll) molecules
(Tichy et al., 1991; LeBlanc and Beatty, 1996;
Young and Beatty, 1998; Young et al., 1998). Evidence
has recently been reported for factors involved
in the stability or assembly of PSI (Boudreau et al.,
1997; Ruf et al., 1997) and PSII (Meurer et al., 1998).
Nothing is known about the identity of putative proteins
implicated in the assembly of light-harvesting
systems. Within the cyanobacterial radiation, proteins
encoded by orf431/462 and sll1906 might be candidates,
despite the dissimilar light-harvesting systems
of rhodobacteria, cyanobacteria and Prochlorococcus.
The amino acid sequence deduced from the ppeC
gene characterizes a 31.5 kDa protein consisting of
270 residues. Physico-chemical characteristics (pI of
10.11) and phylogenetic analysis (Figure 3) suggest
this protein as a putative phycoerythrin linker polypeptide.
In contrast to most cyanobacterial phycoerythrinand
phycocyanin-associated linker polypeptides, the
P. marinus ppeC gene product is characterized by a
long N-terminal extension and an approximately similar
number of residues lacking at the C terminus. This
characteristic is only shared by the phycoerythrin γ
subunit [or γ (L 32 R )] of Synechococcus WH 8020, encoded
by mpeC (Wilbanks and Glazer, 1993b). However,
the latter differs from other cyanobacterial linker
polypeptides associated with phycoerythrin in that it
bears a single phycourobilin bound via a thioether to
γ -Cys-49. In the Prochlorococcus protein, there is
no cysteine at that position nor at the position corresponding
to the non-bilin bearing γ -Cys-64 of the
Synechococcus sequence. It is therefore most probably
colourless. Nevertheless, in phylogenetic analyses,
the Prochlorococcus protein was consistently placed
on the same branch as the mpeC gene product from
Synechococcus WH 8020 (cf. Figure 3). This strongly
suggests a common origin for the two proteins. This is
why we introduced the designation ‘ppeC’ for the gene
encoding this putative Prochlorococcus phycoerythrin
gamma subunit here and in Figure 1 and Table 1.
The phycobiliprotein genes of Prochlorococcus are
physically part of the chromosome
Horizontal gene transfer is a major factor in bacterial
genome evolution, frequently mediated by mobile genetic
elements such as plasmids, bacteriophages, IS elements,
transposons or combinations thereof (Cassier-
Chauvat et al., 1997; Koonin and Galperin, 1997;
Lawrence and Ochman, 1998). One known example in
the Synechococcus marine A-Prochlorococcus clade
is the rbcL gene which is of gamma-proteobacterial
type in Prochlorococcus GP2 (Shimada et al., 1995)
and Synechococcus WH 7803 (Watson and Tabita,
1996), whereas all other Prochlorococcus and Synechococcus
strains examined so far possess the more
widespread cyanobacterial rbcL type (Reichelt and
Delaney, 1983; Pichard et al., 1997). This may
raise questions about the origin of Prochlorococcus
PE genes and their physical integration within the
genome. We addressed this problem by localizing the
phycobiliprotein-coding region within the genome by
partial mapping. Prochlorococcus DNA was cleaved
by the rare cutting restriction enzymes NotI, SmaIand
MluI and the resulting fragments were separated by
PFGE (Figure 4). In hybridization experiments, the
probe p2500 (cf. Figure 1) recognized fragments in
the size range of about 1.4 Mb for NotI (Figure 4A),
110 kb for MluI and 43 kb for SmaI (Figure 4B). A
probe to the previously characterized aspA-psbA-aroC
coding region (Hess et al., 1995; Hess, 1997) hybridized
to fragments of 1.4 Mb, 110 kb and 58 kb for
NotI, MluI andSmaI, respectively (Figure 4). Hence
both regions are at the genome level physically linked
to each other. There is a single MluI recognition site
in the region investigated in this paper, at position
8153 within ppeC (cf. Figure 1). Therefore, with regard
to Figure 1A, the aspA-psbA-aroC coding region
can be localized to the left of cpeB within a distance
of not more than 110 kb. Both probes did not
show significant hybridization to DNA from another
Prochlorococcus strain, MED4. Absence of hybridization
of the probe to the aspA-psbA-aroC coding region
is the result of the stringency of hybridization and not
an indication of the absence of this gene cluster in
MED4.

Figure 4. Physical mapping of the phycobiliprotein gene cluster to a chromosomal region in P. marinus CCMP 1375. A. A DNA probe to the
phycobiliprotein gene cluster hybridizes to a 1.4 Mb NotI fragment also containing other genes as aspA. B. Fine mapping: both the genomic
region coding for aspA-psbA-aroC and the phycoerythrin coding region map to an identical 110 kb MluI fragment. Total cellular DNA of
P. marinus CCMP 1375 (S) was digested by the restriction enzymes NotI, MluI orSmaI as indicated and separated by PFGE. In subsequent
Southern hybridizations, the gels were hybridized with a probe for cpeA-Z-Y-mpeX (probe P25 in Figure 1) and aspA (Hess, 1997). The MluI
cleavage was done in duplicate and, to be absolutely sure about the identity of this hybridizing band, DNA of Prochlorococcus MED4 (M)
was included for comparison. Concatemeric lambda DNA (L), mid range size marker from New England Biolabs (MR) or yeast complete
chromosomes (YC) were used as molecular weight standards. Sizes of marker DNAs are given in kb, the location of the compression zone is
indicated by an arrow and labelled ‘comp’.
The amount of α and β phycoerythrin does not
change significantly with decreasing light intensity
and in different growth phases
In western blots, the anti-α subunit serum immunodecorated
a polypeptide with an apparent molecular
mass of about 15 kDa in the case of P. marinus total
proteins, whereas two major polypeptides of about 18
and 12 kDa, respectively, were recognized in the case
of Chamaesiphon PCC 6605 total proteins taken as a
control (Figure 5A). This strain is a cyanobacterium in
which PE are very abundant proteins (Bryant, 1982).
Whereas the identity of the 12 kDa band remains unknown,
18 kDa corresponds well to the molecular
mass expected for an α PE. The smaller size of the
P. marinus α PE is in agreement with the fact that it
515
lacks 9 amino acids compared to other cyanobacterial
α-PE’s (Hess et al., 1996). According to the gene
sequence, its absolute molecular mass is 17.3 kDa.
Batch cultures were kept at different light conditions
of 8, 17 and 38 µmol m −2 s −1 and total proteins
were extracted. As expected from previous studies
(Partensky et al., 1993; Moore et al., 1995), the
Chl a2/b2 ratio changed with the different light irradiances,
causing variations in the relative heights of
the 440 and 480 nm or 660 and 670 nm peaks of
absorption spectra, corresponding to Chl a2 and b2,respectively
(Figure 5B). Analysis of the extracted total
proteins by western blotting was done on the basis of
identical amounts of chlorophyll (Figure 5C) or protein
(not shown). The amounts of proteins loaded per
lane, the separation by SDS-PAGE and the transfer to

516
Figure 5. Analysis of the amounts of phycoerythrin under different light intensities by western blots. A. Control experiment using anti-α PE.
PM1 and PM2, two different samples of 5 µg ofP. marinus CCMP 1375 total protein extracts; Cha, 0.5 µg total protein from Chamaesiphon
PCC 6605; rec, recombinant protein. Molecular weight standard is shown to the left. B. Absorption spectra of P. marinus CCMP 1375 cultures
grown under the light conditions indicated (in µmol photons m −2 s −1 ). C. Comparison of the amounts of α and β PE at light intensities ranging
from 8 to 38 µmol m −2 s −1 or of the D1 protein for control.
membranes by electroblotting was further compared
by immunodecoration with antisera to other proteins,
namely CP43 or D1. The antiserum obtained against
the β PE recognized a broad band with an apparent
molecular mass of 19.5–21 kDa (Figure 5C). This is
slightly larger than the deduced molecular mass of
19.3 kDa (Hess et al., 1996), but might be explained
in part by the presence of chromophoric groups. In all
our western blots the β PE band looked broader than
the α PE band. This indicates the presence of β PEs
of slightly different molecular masses, for example
differently chromophorylated iso-forms or otherwise
modified polypeptides. We can clearly exclude the
possibility of cross-reactivity of the anti-β PE serum
with α PE (not shown).
Although the experiment was done on two separate
batches of acclimated cultures, there was no
significant change in the relative amount of α or β phycoerythrin
with decreasing or increasing light intensity
(Figure 5C).
In a second set of experiments, we tested if cells
taken from different growth phases of a batch culture
would show a regulation of PE content. The growth
curve and results of western bloting with both sera
are presented in Figure 6. There is slightly less PE
detectable with both sera at the beginning of the experiment,
however this probably does not translate
to variation in the relative content of PE, because a
very similar variation can be seen for the D1 protein,
used as a control. The amount of phycoerythrin also
decreases in the post-stationary phase at day 10. However,
since there was a large proportion of dying cells
in this sample (not shown), this decrease is rather an
indication of a drastically lowered stability of PE under
these conditions than of a regulated expression.
Consequently, it can be concluded that the P. marinus
cpeB and cpeA genes were mainly expressed
constitutively under the conditions tested.
Prochlorococcus β phycoerythrin is attached to the
thylakoid membrane
Immunogold labelling of Prochlorococcus cross sections
was performed to study the intracellular localization
of PE. As expected, the electron micrographs
did not show the phycobilisome structure typical of

Figure 6. Correlation between growth phase and amount of β PE.
A. Growth curve. Days 0–2, lag phase; 3–6, exponential phase; 7–8
stationary phase; and 10, post-stationary phase. The experiment was
done in duplicate. B. Western blots using the serum against α and β
PE, respectively, as indicated. An antiserum against D1 protein was
taken as control. Selected size markers are shown to the right.
cyanobacteria (see e.g. Stanier, 1988). The cells contain
two to four closed circular layers of thylakoid
membranes parallel to the inner side of the cell membrane,
a feature that is characteristic of this strain
of Prochlorococcus (Chisholm et al., 1992; Lichtlé
et al., 1995). In our cell preparations for immunocytochemistry,
thylakoids were very tighly appressed,
so that the limit between two adjacent thylakoids
was not clearly visible, but they displayed a (probably
artefactually) wide lumen (33 nm) compared to
photographs obtained by others using classical TEM
microscopy (see e.g. Chisholm et al., 1988, 1992;
Partensky et al., 1999b). Clearly, the majority of gold
particles is distributed over the thylakoid membranes
although in rare cases, labelling of cytoplasm also
occurred (Figure 7). More precisely, the statistically
frequent occurrence of gold particles in the lumen suggests
that the structural elements containing PE are
located in this part of the thylakoid. This hypothesis
must however be viewed with care, since an anti-
517
body molecule is about 8 nm long (Roth, 1982) and
the gold particles are 10 nm in diameter. Considering
the size of complex primary antibody-secondary
antibody-gold particle, the distance between the antigen
and the centre of a gold particle is about 21 nm.
Thus, it cannot be said with certainty whether the PE is
rather located within the lumen or at the external side
of the thylakoids. The tight appression of thylakoids is
however an additional element in favour of the first hypothesis.
The relatively small number of gold particles
per cell is probably related to the comparatively low
cellular concentration of PE in P. marinus (for comparison,
see staining of Prochlorococcus cells with an
anti-D2 antibody; Lichtlé et al., 1995).
Discussion
The molecular phylogeny of the Prochlorococcus
genus has been studied with a variety of genes including
rrn (encoding 16S rRNA (Urbach et al., 1992,
1998; Moore et al., 1998), psbA (Hess et al., 1995),
rpoc1 (Palenik and Haselkorn, 1992), psbB (Urbach
et al., 1998) and portions of the petB/D operon (Urbach
et al., 1998; Urbach and Chisholm, 1999).
These studies have demonstrated that Prochlorococcus
(1) belongs to the cyanobacterial radiation, (2) has
evolved independently to the only other two prokaryotes
possessing a Pcb-Chl a/b complex as the major
antenna system, Prochloron and Prochlorothrix
(Laroche et al., 1996) and (3) is closely related
to marine Synechococcus species. The latter are
characterized by their well-developed phycobilisomal
apparatus, which is the most efficient lightharvesting
structure known among photosynthetic organisms
(Sidler, 1994). Despite their striking differences
in light-harvesting systems, the relatedness
between Prochlorococcus and Synechococcus became
further supported by the discovery of functional genes
for phycoerythrin α and β subunits in the Prochlorococcus
type strain, P. marinus CCMP 1375 (Hess
et al., 1996).
In the present study, we show that the phycoerythrin
genes cpeA and CpeB are not isolated but are
part of a larger gene cluster that includes at least
four more phycobiliprotein-related genes. Three proteins
encoded by these genes (CpeZ, CpeY, MpeX)
are most likely required for biosynthesis and attachment
of chromophoric groups to PE whereas one gene,
ppeC, might code for a linker polypeptide. Thus, these
six genes possibly represent the minimal set of fac-

518
Figure 7. A. Electron micrograph of immunogold-labelled P. marinus
CCMP 1375 cells using anti-β PE and 10 nm gold particles.
The gold particles are localized over the thylakoids (arrows), the
central cytoplasm of the cell is unlabelled. B. Detail of the labelling
of the thylakoids, the gold particles are preferentially localized in
the tylakoid lumen, near the internal membrane of the thylakoid.
C. Control with pre-immune serum and 10 nm gold particles. No
gold particles are observed within the cell. tl, thylakoid lumen; tm,
thylakoid membrane. Scale bar: 0.2 µm.
tors necessary to form a functional light-harvesting
structure. Indeed, we show that, despite its relatively
low cellular concentration, the phycoerythrin
in Prochlorococcus is predominantly localized within
the thylakoid membranes. Small rod-shaped phycobiliprotein
aggregates, significantly smaller than phycobilisomes,
have recently been described for another
marine prokaryote, Acaryochloris marina (Marquardt
et al., 1997). It is conceivable that even smaller structures
exist in P. marinus, at the extreme consisting
only of a phycoerythrin (α, β) monomer bound via
a γ -PE linker polypeptide to the thylakoids or more
probably an (α,β)(α,β) PE dimer comparable to the
(α1, β)(α2, β) PC-645 of the cryptophyte Chroomonas
sp. (Sidler, 1994). Our immunocytochemistry results
using an homologous β-PE antibody further suggest
that these structures might be located in the lumen
of thylakoids. If this hypothesis holds true, the localization
of PE in Prochlorococcus would be similar to
what has been observed previously for the PE or PC in
Cryptophyceae (Gantt et al., 1971; Rhiel et al., 1989;
Lichtlé et al., 1995) and therefore very different from
what is observed for PBS in typical cyanobacteria.
Although the genes for phycobiliprotein α and β
subunits are always organized in an operon in both
pro- and eukaryotes (Apt et al., 1995), clustering of
this operon with a large number of other genes involved
in the biosynthesis and attachment of bilins,
and possibly phycobiliprotein regulation, as observed
in P. marinus CCMP 1375, is not always found. The
most similar example, but for the presence of several
phycocyanin genes, is provided by the gene cluster of
Synechococcus WH 8020 (Figure 1B). Nevertheless,
the possibility of a recent acquisition of these genes by
horizontal gene transfer from a marine Synechococcus
cyanobacterium resembling WH 8020 appears unlikely
for several reasons. First, we have shown that
this region is physically part of the Prochlorococcus
genome and not of any episomal genetic element and
there is no evidence for inverted repeats or similar
elements that might have facilitated the integration
of an external genetic element into the genome of
Prochlorococcus. Second, the GC content of this region
is not significantly different from the average
value of 36.82% (mol/mol) G+C determined from
all sequences of genes and intergenic regions known
to date in P. marinus CCMP 1375 (Partensky et al.,
1999b). Third, there is evidence for a very similar set
of genes in three other strains of Prochlorococcus that
were isolated in the Pacific Ocean (Hess and Campbell,
unpublished) and in the Sargasso Sea (Ting et al.,

1998). These facts are neither consistent with an undirected
mutational pressure aiming at eliminating genes
acquired during a recent gene transfer nor with a situation
that might be expected for remmants of evolution
on a way to total degeneration. Instead, the situation
observed in Prochlorococcus can better be explained
as the result of a series of recombination and deletion
events progressively modifying an ancestral gene cluster
and resulting in the conservation of a minimal set
of PE genes that still allow function. This view is fully
in agreement with current models on the phylogeny of
Prochlorococcus. These models suggest a rapid diversification
of the Prochlorococcus and Synechococcus
groups from a common ancestor (Urbach et al., 1998).
ThepresenceofPEinsomeProchlorococcus strains
but not in other ones most likely reflects different retention
and not transfer. Thus the similarity of both
the individual gene sequences and of the overall gene
organization in P. marinus compared to Synechococcus
is merely an indication of their close evolutionary
relatedness.
If so, what is the function of P. marinus phycoerythrins?
Their affiliation to thylakoid membranes
clearly supports a light-harvesting function (Lokstein
et al., 1999). Furthermore, all three Prochlorococcus
genotypes for which there is molecular evidence
for phycoerythrin (CCMP 1375, this paper and Hess
et al., 1996; PAC1, Hess and Campbell, unpublished;
MIT9303, Ting et al., 1998), have been isolated from
low-light conditions. If these genes were, for example,
induced under very low light conditions, as is
suggested by the increase of the orange fluorescence
signal of natural Prochlorococcus populations at depth
(Hess et al., 1996), a role as an additional antenna
would receive strong support. However, under the culture
conditions tested in this study, such a light effect
was not detectable, which does not exclude that an
induction of gene expression occurs under natural conditions
where other factors such as variable nutrient
availability might also play a role in the PE regulation.
Clearly, this question merits further attention as do the
mechanisms of energy transfer (Lokstein et al., 1999)
and the in vivo structural arrangement of P. marinus
phycobiliproteins. A further part of the puzzle might
be provided by the extensive identification of genotypes
among Prochlorococcus that possess a similar
set of genes.
Acknowledgements
519
This work was supported by grant HE 2544/1-2 and
by SFB429 from the Deutsche Forschungsgemeinschaft,
Bonn and by the European Union program
PROMOLEC (MAS3-CT97-0128). We thank G.W.M.
van der Staay, Köln, for providing helpful technical
hints with the western blot experiments, R. Rippka,
Paris for providing a live culture of Chamaesiphon
PCC 6605, J. Ressler and S. Penno for support in some
of the initial cloning work and T. Hübschmann, Berlin,
for critical reading of the manuscript.
References
Apt, K.E., Collier, J.L. and Grossman, R. 1995. Evolution of the
phycobiliproteins. J. Mol. Biol. 248: 79–96.
Bierne, H., Seigneur, M., Ehrlich, S.D. and Michel, B. 1997. uvrD
mutations enhance tandem repeat deletion in the Escherichia
coli chromosome via SOS induction of the RecF recombination
pathway. Mol. Microbiol. 26: 557–567.
Boudreau, E., Takahashi, Y., Lemieux, C., Turmel, M. and Rochaix,
J.D. 1997. The chloroplast ycf3 and ycf4 open reading frames of
Chlamydomonas reinhardtii are required for the accumulation of
the photosystem I complex. EMBO J. 16: 6095–6104.
Bryant, D.A. 1982. Phycoerythrocyanin and phycoerythrin: properties
and occurrence in cyanobacteria. J. Gen. Microbiol. 128:
835–844.
Bullerjahn, G.S. and Post, A.F. 1993. The prochlorophytes: are they
more than just chlorophyll a/b-containing cyanobacteria? Crit.
Rev. Microbiol. 19: 43–59.
Cassier-Chauvat, C., Poncelet, M. and Chauvat, F. 1997. Three
insertion sequences from the cyanobacterium Synechocystis
PCC6803 support the occurrence of horizontal DNA transfer
among bacteria. Gene 195: 257–66.
Chisholm, S.W., Olson, R.J., Zettler, E.R., Waterbury, J., Goericke,
R. and Welschmeyer, N. 1988. A novel free-living prochlorophyte
occurs at high cell concentrations in the oceanic euphotic
zone. Nature 334: 340–343.
Chisholm, S.W., Frankel, S.L., Goericke, R., Olson, R.J., Palenik,
B., Waterbury, J.B., West-Johnsrud, L. and Zettler, E.R. 1992.
Prochlorococcus marinus nov. gen. nov. sp.: an oxyphototrophic
marine prokaryote containing divinyl chlorophyll a and b. Arch.
Microbiol. 157: 297–300.
De Lorimier, R., Wilbanks, S.M. and Glazer, A.N. 1993. Genes
of the R-phycocyanin II locus of marine Synechococcus spp.,
and comparison of protein-chromophore interactions in phycocyanins
differing in bilin composition. Plant Mol. Biol. 21:
225–237.
Dubbs, J.M. and Bryant, D.A. 1991. Molecular cloning and transcriptional
analysis of the cpeBA operon of the cyanobacterium
Pseudanabaena species PCC7409. Mol. Microbiol. 5: 3073–
3085.
Gantt, E., Edwards, M.R. and Provasoli, L. 1971. Chloroplast structure
of the Cryptophyceae. Evidence for phycobiliproteins within
intrathylakoidal spaces. J. Cell. Biol. 48: 280–290.
Goericke, R. and Repeta, D.J. 1992. The pigments of Prochlorococcus
marinus: the presence of divinyl chlorophyll a and b in a
marine prochlorophyte. Limnol. Oceanogr. 37: 425–433.

520
Grossman, A.R., Bhaya, D., Apt, K.E. and Kehoe, D.M. 1995.
Light-harvesting complexes in oxygenic photosynthesis: diversity,
control, and evolution. Annu. Rev. Genet. 29: 231–288.
Hess, W.R. 1997. Localization of an open reading frame with homology
to human aspartoacylase upstream from psbA in the
prokaryote Prochlorococcus marinus CCMP 1375. DNA Sequence
7: 301–306.
Hess, W.R., Weihe, A., Loiseaux-de Goër, S., Partensky, F. and
Vaulot, D. 1995. Characterization of the single psbA gene of
Prochlorococcus marinus CCMP 1375 (Prochlorophyta). Plant
Mol. Biol. 27: 1189–1196.
Hess, W.R., Partensky, F., van der Staay, G.W.M., Garcia-
Fernandez, J.M., Börner, T. and Vaulot, D. 1996. Coexistence
of phycoerythrin and a chlorophyll a/b antenna in a marine
prokaryote. Proc. Natl. Acad. Sci. USA 93: 11126–11130.
Hughes, J., Lamparter, T., Mittmann, F., Hartmann, E., Gartner,
W., Wilde, A. and Börner, T. 1997. A prokaryotic phytochrome.
Nature 386: 663.
Kahn, K., Mazel, D., Houmard, J., Tandeau de Marsac, N.
and Schaefer, M.R. 1997. A role for cpeYZ in cyanobacterial
phycoerythrin biosynthesis. J. Bact. 179: 998–1006.
Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E.,
Nakamura, Y., Miyajima, N., Hirosawa, M., Sugiura, M.,
Sasamoto, S., Kimura, T., Hosouchi, T., Matsuno, A., Muraki,
A., Nakazaki, N., Naruo, N., Okumura, S., Shimpo, S., Takeuchi,
C., Wada, Z., Watanabe, A., Yamada, M., Yasuda, M. and Tabata,
S. 1996. Sequence analysis of the genome of the unicellular
cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence
determination of the entire genome and assignment of potential
protein-coding regions. DNA Res. 3: 109–136.
Kehoe, D.M. and Grossman, A.R. 1996. Similarity of a chromatic
adaptation sensor to phytochrome and ethylene receptors.
Science 273: 1409–1412.
Koonin, E.V. and Galperin, M.Y. 1997. Prokaryotic genomes: the
emerging paradigm of genome-based microbiology. Curr. Opin.
Genet. Dev. 7: 757–763.
Laroche, J., van der Staay, G.W.M., Partensky, F., Ducret, A.,
Aebersold, R., Li, R., Golden, S.S., Hiller, R.G., Wrench,
P.M., Larkum, A.W.D. and Green, B.R. 1996. Independent
evolution of the prochlorophyte and green plant chlorophyll
a/b light-harvesting proteins. Proc. Natl. Acad. Sci. USA 93:
15244–15248.
Lawrence, J.G. and Ochman, H. 1998. Molecular archaeology of the
Escherichia coli genome. Proc. Natl. Acad. Sci. USA 95: 9413–
9417.
LeBlanc, H.N. and Beatty, J.T. 1996. Topological analysis of
the Rhodobacter capsulatus PucC protein and effects of Cterminal
deletions on light-harvesting complex II. J. Bact. 178:
4801–4806.
Lichtlé, C., McKay, R.M. and Gibbs, S.P. 1992. Immunogold localization
of photosystem I and photosystem II light-harvesting
complexes in cryptomonad thylakoids. Biol. Cell 74: 187–194.
Lichtlé, C., Thomas, J.C., Spilar, A. and Partensky, F. 1995.
Immunological and ultrastructural characterization of the photosynthetic
complexes of the prochlorophyte Prochlorococcus
(Oxychlorobacteria). J. Phycol. 31: 934–941.
Lokstein, H., Steglich, C. and Hess, W.R. 1999. Light-harvesting
antenna function of phycoerythrin in Prochlorococcus marinus.
Biochim. Biophys. Acta 1410: 97–98.
Marquardt, J., Senger, H., Miyashita, H., Miyachi, S. and Mörschel,
E. 1997. Isolation and characterization of biliprotein aggregates
from Acaryochloris marina, aProchloron-like prokaryote
containing mainly chlorophyll d. FEBS Lett. 410: 428–432.
Mazel, D., Guglielmi, G., Houmard, J., Sidler, W., Bryant, D.A. and
Tandeau de Marsac, N. 1986. Green light induces transcription of
the phycoerythrin operon in the cyanobacterium Calothrix 7601.
Nucl. Acids Res. 14: 8279–8290.
Meurer, J., Plüchken, H., Kowallik, K.V. and Westhoff, P. 1998. A
nuclear-encoded protein of prokaryotic origin is essential for the
stability of photosystem II in Arabidopsis thaliana. EMBO J. 17:
5286–5297.
Moore, L.R., Goericke, R. and Chisholm, S.W. 1995. Comparative
physiology of Synechococcus and Prochlorococcus: influence of
light and temperature on growth, pigments, fluorescence and
absorptive properties. Mar. Ecol. Prog. Ser. 116: 259–275.
Moore, L.R., Rocap, G. and Chisholm, S.W. 1998. Physiology and
molecular phylogeny of coexisting Prochlorococcus ecotypes.
Nature 393: 464–467.
Newman, J., Mann, N.H. and Carr, N.G. 1994. Organization and
transcription of the class I phycoerythrin genes of the marine
cyanobacterium Synechococcus sp. WH7803. Plant Mol. Biol.
24: 679–683.
Oeda, K., Horiuchi, T. and Sekiguchi, M. 1982. The uvrD gene of
E. coli encodes a DNA-dependent ATPase. Nature 298: 98–100.
Ong, L.J., Glazer, A.N. and Waterbury, J.B. 1984. An unusual phycoerythrin
from a marine cyanobacterium. Science 224: 80–83.
Palenik, B. and Haselkorn, R. 1992. Multiple evolutionary origins
of prochlorophytes, the chlorophyll b-containing prokaryotes.
Nature 355: 265–267.
Partensky, F., Hoepffner, N., Li, W.K.W., Ulloa, O. and Vaulot, D.
1993. Photoacclimation of Prochlorococcus sp. (Prochlorophyta)
strains isolated from the North Atlantic and the Mediterranean
Sea. Plant Physiol. 101: 285–296.
Partensky, F., LaRoche, J., Wyman, K. and Falkowski, P.G. 1997.
The divinyl-chlorophyll a/b-protein complexes of two strains
of the oxyphototrophic marine prokaryote Prochlorococcus:
characterization and response to changes in growth irradiance.
Photosynth. Res. 51: 209–222.
Partensky, F., Blanchot, J. and Vaulot, D. 1999a. Differential distribution
of Prochlorococcus and Synechococcus: a review. In: L.
Charpy and A.W.D. Larkum (eds.), Marine Cyanobacteria. Bull.
Inst. Océanogr., Monaco, in press.
Partensky, F., Hess, W.R. and Vaulot, D. 1999b. Prochlorococcus,
a marine photosynthetic prokaryote of global significance.
Microbiol. Mol. Biol. Rev. 63: 106–127.
Petit, M.A., Dervyn, E., Rose, M., Entian, K.D., McGovern, S.,
Ehrlich, S.D. and Bruand, C. 1998. PcrA is an essential DNA
helicase of Bacillus subtilis fulfilling functions both in repair and
rolling-circle replication. Mol. Microbiol. 29: 261–273.
Pichard, S.L., Campbell, L. and Paul, J.H. 1997. Diversity of
the ribulose bisphosphate carboxylase/oxygenase Form I gene
(rbcL) in natural phytoplankton communities. Appl. Environ.
Microbiol. 63: 3600–3606.
Reichelt, B.Y. and Delaney, S.F. 1983. The nucleotide sequence for
the large subunit of ribulose 1,5-bisphosphate carboxylase from
a unicellular cyanobacterium, Synechococcus PCC6301. DNA 2:
121–129.
Rhiel, E., Kunz, J. and Wehrmeyer, W. 1989. Immunocytochemical
localization of phycoerythrin-545 and of a chlorophyll a/c light
harvesting comlex in Cryptomonas maculata (Cryptophyceae).
Bot. Acta 102: 46–53.
Roth, J. 1982. The protein A-gold (pAg) technique: a qualitative
and quantitative approach for antigen localization on thin
sections. In: G.R. Bullock and P. Petruz (Eds.), Techniques in
Immunocytochemistry, Vol. I, Academic Press, New York, pp.
107–133.

Ruf, S., Kössel, H. and Bock, R. 1997. Targeted inactivation of a
tobacco intron-containing open reading frame reveals a novel
chloroplast-encoded photosystem I-related gene. J. Cell. Biol.
139: 95–102.
Shimada, A., Kanai, S. and Maruyama, T. 1995. Partial sequence of
ribulose-1,5-bisphosphate caboxylase/oxygenase and the physiology
of Prochloron and Prochlorococcus (Prochlorales). J. Mol.
Evol. 40: 671–677.
Sidler, W.A. 1994. Phycobilisome and phycobiliprotein structures.
In: D.A. Bryant (ed.), The Molecular Biology of Cyanobacteria,
Kluwer Academic Publishers, Dordrecht, Netherlands, pp. 139–
216.
Stanier, R.Y. 1988. Fine structure of cyanobacteria. Meth. Enzymol.
167: 157–172.
Strimmer, K. and von Haeseler, A. 1997. Likelihood-mapping: a
simple method to visualize phylogenetic content of a sequence
alignment. Proc. Natl. Acad. Sci. USA 94: 6815–6819.
Tichy, H.V., Albien, K.U., Gadon, N. and Drews, G. 1991. Analysis
of the Rhodobacter capsulatus puc operon: the pucC gene plays
a central role in the regulation of LHII (B800–850 complex)
expression. EMBO J. 10: 2949–2955.
Ting, C., Rocap, G., King, J. and Chisholm, S.W. 1998. Characterization
of phycoerythrin genes in the chlorophyll a2/b2containing
prokaryote Prochlorococcus sp. MIT9303. Abstracts
11th International Photosynthesis Congress, Budapest, p. 29.
Urbach, E. and Chisholm, S.W. 1999. Genetic diversity in uncultured
Prochlorococcus populations flow cytometrically sorted
from the Sargasso Sea and the Gulf Stream. Limnol. Oceanogr.,
in press.
Urbach, E., Robertson, D.L. and Chisholm, S.W. 1992. Multiple
evolutionary origins of prochlorophytes within the cyanobacterial
radiation. Nature 355: 267–269.
521
Urbach, E., Scanlan, D.J., Distel, D.L., Waterbury, J.B. and
Chisholm, S.W. 1998. Rapid diversification of marine picophytoplankton
with dissimilar light harvesting structures inferred from
sequences of Prochlorococcus and Synechococcus (cyanobacteria).
J. Mol. Evol. 46: 188–201.
Watson, G.M.F. and Tabita, F.R. 1996. Regulation, unique gene
organization, and unusual primary structure of carbon fixation
genes from a marine phycoerythrin-containing cyanobacterium.
Plant Mol. Biol. 32: 1103–1116.
Whitman, W.B., Coleman, D.C. and Wiebe, W.J. 1998. Prokaryotes:
the unseen majority. Proc. Natl. Acad. Sci. USA 95: 6578–6583.
Wilbanks, S.M. and Glazer, A.N. 1993a. Rod structure of a phycoerythrin
II-containing phycobilisome I. Organization and sequence
of the gene cluster encoding the major phycobiliprotein
rod components in the genome of the marine Synechococcus sp.
WH 8020. J. Biol. Chem. 268: 1226–1235.
Wilbanks, S.M. and Glazer, A.N. 1993b. Rod structure of a phycoerythrin
II-containing phycobilisome. II. Complete sequence and
bilin attachment site of a phycoerythrin γ subunit. J. Biol. Chem.
268: 1236–1241.
Young, C.S. and Beatty, J.T. 1998. Topological model of the
Rhodobacter capsulatus light-harvesting complexI assembly
protein LhaA (previously known as ORF1696). J. Bact. 180:
4742–4755.
Young, C.S., Reyes, R.C. and Beatty, J.T. 1998. Genetic complementation
and kinetic analyses of Rhodobacter capsulatus
ORF1696 mutants indicate the ORF1696 protein enhances assembly
of the light-harvesting I complex. J. Bact. 180: 1759–
1765.