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JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010
Oct. 2000, p. 5440–5447 Vol. 182, No. 19
Copyright © 2000, American Society for Microbiology. All Rights
Reserved.
The orf162b Sequence of Rhodobacter capsulatus Encodes aProtein
Required for Optimal Levels of Photosynthetic
Pigment-Protein ComplexesMUKTAK AKLUJKAR,1 ANDREA L. HARMER,1
ROGER C. PRINCE,2 AND J. THOMAS BEATTY1*
Department of Microbiology and Immunology, University of British
Columbia, Vancouver, British Columbia, Canada,1
and ExxonMobil Research and Engineering Company, Annandale, New
Jersey2
Received 26 April 2000/Accepted 10 July 2000
The orf162b sequence, the second open reading frame 3* of the
reaction center (RC) H protein gene puhA inthe Rhodobacter
capsulatus photosynthesis gene cluster, is shown to be transcribed
from a promoter located 5*of puhA. A nonpolar mutation of orf162b
was generated by replacing most of the coding region with
anantibiotic resistance cartridge. Although the mutant strain
initiated rapid photosynthetic growth, growthslowed progressively
and cultures often entered a pseudostationary phase. The amounts of
the RC and lightharvesting complex I (LHI) in cells obtained from
such photosynthetic cultures were abnormally low, but
thesedeficiencies were less severe when the mutant was grown to a
pseudostationary phase induced by low aerationin the absence of
illumination. The orf162b mutation did not significantly affect the
expression of a pufB::lacZtranslationally in-frame gene fusion
under the control of the puf promoter, indicating normal
transcription andtranslation of RC and LHI genes. Spontaneous
secondary mutations in the strain with the orf162b
disruptionresulted in a bypass of the photosynthetic growth
retardation and reduced the level of light harvesting complexII.
These results and the presence of sequences similar to orf162b in
other species indicate that the Orf162bprotein is required for
normal levels of the photosynthetic apparatus in purple
photosynthetic bacteria.
Purple nonsulfur photosynthetic bacteria such as Rho-dobacter
capsulatus are capable of aerobic respiratory and an-aerobic
photosynthetic growth. The photosynthetic apparatusincludes three
membrane-bound pigment-protein complexes:the reaction center (RC),
where light-dependent electrontransfer is initiated; light
harvesting (LH) complex I, which isadjacent to and perhaps forms a
ring encircling the RC as partof the so-called core complex; and
the LHII complex, which isthought to be present in multiple copies
of a ring-shaped struc-ture that interconnect core complexes (20).
These complexesare located within differentiated invaginations of
the cytoplas-mic membrane called the intracytoplasmic membrane
system(ICM), which is formed upon oxygen deprivation of
cultures(12). The presence of the various photosynthetic
complexescan be evaluated by their characteristic light absorption
spec-tra, which reflect the protein environments around
bacterio-chlorophyll a (Bchl). For example, the Bchl’s of the
LHIIcomplex of R. capsulatus absorb light of 800 and 850 nm,whereas
the Bchl’s of the less abundant LHI complex absorbapproximately
870-nm light (13).
Two of the three protein subunits of the RC, designated RCL and
RC M, and both protein subunits of LHI (LHI a andLHI b), are
encoded by the puf operon (2). The third subunitof the RC, called
RC H, is encoded by the puhA gene, which istranscribed as part of
the bchFNBHLM-lhaA-puhA super-operon from two promoters, one 59 of
bchF and the otherwithin the lhaA gene (3). As shown in Fig. 1,
several openreading frames (ORFs) located 39 of puhA and in the
sametranscriptional orientation have been identified on the basis
ofDNA sequence analysis (2). A previous publication reported
that disruptions of orf214, located immediately 39 of the
puhAgene, resulted in reduced amounts of the RC and LHI com-plexes
and abolished photosynthetic growth, and it was sug-gested that the
Orf214 protein is an RC assembly factor (28).
The orf162b sequence immediately follows orf214 as thesecond ORF
39 of puhA. ORFs similar to orf162b have beenfound in four other
species of purple photosynthetic bacteria:orf153 in Rhodobacter
sphaeroides (M. Choudhary and S.Kaplan, personal communication),
orf154 in Rubrivivax gelati-nosus (K. Nagashima, personal
communication), orfI3087 inRhodospirillum rubrum (6), and orf168 in
Rhodopseudomonaspalustris (genome sequence made available by the
Joint Ge-nome Institute at
http://spider.jgi-psf.org/JGI_microbial/html/rhodo_homepage.html).
In all five species the ORF similar toorf162b is immediately 39 of
a homologue of orf214, whichimmediately follows the puhA gene, just
as in R. capsulatus (2,6, 9a; K. Nagashima, personal communication;
M. Aklujkar,analysis of the R. palustris genome). The predicted
proteinsequences are only 43% (R. sphaeroides), 15% (R.
gelatinosus),14% (R. rubrum), and 17% (R. palustris) identical to
Orf162b,but they have similar hydropathy profiles (using the
Goldman-Engelman-Steitz algorithm and the TOPPRED program [10])with
a transmembrane segment near the amino terminus.None of these
predicted proteins has significant sequence sim-ilarity to proteins
of known function.
In this paper we present evidence that orf162b encodes aprotein
that is required for optimal photosynthetic growth, andthat a
disruption of orf162b reduces the amounts of RC andLHI in
oxygen-deprived and photosynthetically grown cells.This phenotype
is complemented in trans by a plasmid-bornecopy of orf162b. We also
demonstrate that transcription oforf162b is abolished by insertion
of a transcription terminationsequence into the puhA gene, and that
the orf162b mutantphenotype is suppressed by spontaneous mutations
that reducethe amount of LHII.
* Corresponding author. Mailing address: Department of
Microbi-ology and Immunology, University of British Columbia, 300 -
6174University Blvd., Vancouver, British Columbia, Canada V6T
1Z3.Phone: (604) 822-6896. Fax: (604) 822-6041. E-mail:
[email protected].
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MATERIALS AND METHODS
Bacterial strains and plasmids. The photosynthetically wild-type
R. capsulatusstrain SB1003 (25), the gene transfer agent (GTA)
overproducer strain DE442(29), and the puhA polar mutant DW1 (28)
have been described previously.Escherichia coli C600 r2 m1 (7) and
DH5a (Life Technologies, GIBCO BRL)were used for the construction
and maintenance of plasmids; strains HB101(pRK2013) and TEC5 (11,
26) were used in E. coli-to-R. capsulatus conjugations.Plasmids
pXCA601::935, pUC13, and pRR5C have been described previously
(1,19, 30). Plasmid pUC4KIXX was from Pharmacia Biotech, Inc.
Bacterial strainsand plasmids produced in this study are described
below.
Growth conditions and media. E. coli strains were grown in
Luria-Bertani(LB) medium (22) or on LB agar plates. Highly aerated
and oxygen-deprivedcultures of R. capsulatus were grown at 30°C
without illumination in RCVmedium (5), in Erlenmeyer flasks filled
to 20 or 80% of nominal capacity,respectively, and shaken at 300 or
150 rpm, respectively. Highly aerated culturesdid not enter the
stationary phase until reaching about 300 Klett units (seebelow),
whereas the growth of oxygen-deprived cultures began to slow at
about80 Klett units. Photosynthetic cultures were grown
anaerobically in screw-captubes (20 ml) or Roux bottles (800 ml)
inoculated from oxygen-deprived culturesand filled with RCV, or on
RCV agar plates placed in BBL GasPak anaerobicjars (Becton
Dickinson & Co.). Photosynthetic cultures were incubated at
30°Cin an aquarium filled with water and illuminated by tungsten
filament incandes-cent lamps at a light intensity of 150
microeinsteins/m2/s, measured with aphotometer equipped with the
LI-190SB quantum sensor (LI-COR Inc.). Cul-ture turbidity was
monitored with a Klett-Summerson photometer equipped witha red (no.
66) filter (100 Klett units 5 3.3 3 108 CFU/ml).
Antibiotic-resistant E.coli and R. capsulatus strains were selected
with 25 and 10 mg of kanamycin/ml,10 and 0.5 mg of tetracycline/ml,
and 10 and 2 mg of gentamicin/ml, respectively.
DNA techniques. Recombinant DNA procedures were carried out
essentiallyas described previously (22). Plasmid DNA was isolated
from cells and fromagarose gels with kits from QIAGEN. Conjugative
transfer of plasmids from E.coli to R. capsulatus was performed by
mixing of overnight cultures of donor,helper, and recipient cells
in a 1:1:1 ratio by volume; centrifugation at 15,000 3g for 2 min;
resuspension in an equal volume of RCV; and incubation of a
10-mlaliquot adsorbed onto an RCV agar plate overnight at 30°C.
Donor cells wereabsent from the negative controls. Cells from each
spot were streaked onto RCVagar plates containing the appropriate
antibiotics. Transconjugant colonies werestreaked onto YPS agar
(22) plates to ensure the absence of E. coli donors.
Construction of the nonpolar orf162b mutant SBK1. A 2-kbp DNA
fragmentfrom BamHI digestion of the R9 plasmid pBLM2 (26)
containing the orf162bgene and flanking sequences (Fig. 1) was
inserted into the BamHI site of pUC13,and 63% of the coding
sequence of orf162b was eliminated by digestion withBsaBI and
PflMI. T4 DNA polymerase was used to resect single-stranded
pro-truding 39 ends from this deletion, and a NotI site in the
linker (59 AGCGGCCGCT 39) was inserted by linker tailing (22); the
resultant plasmid was cut at thisNotI site, and the ends were
filled in with the Klenow fragment. A 1.3-kbp SmaIfragment
containing the kanamycin resistance gene and the first 153 bp of
thebleomycin resistance gene from pUC4KIXX was ligated into the
filled-in NotIsite such that the transcriptional orientation of
these genes is the same as inorf162b. This fragment does not
usually terminate transcription (8), and itsnonpolar effect was
confirmed by genetic complementation (see Results). E. coliTEC5 was
transformed with the plasmid carrying the deleted, kanamycin
resis-tance-marked orf162b gene. Homologous recombination of this
pUC-derivedplasmid with the conjugative plasmid pDPT51 in E. coli
TEC5 permitted con-
jugative transfer of kanamycin resistance from TEC5 to R.
capsulatus strainDE442. DE442 overproduces phage-like GTA particles
containing random 4.6-kbp linear fragments of genomic DNA (29),
which were used to replace thewild-type orf162b gene in SB1003 by
transduction, producing strain SBK1.
Construction of the complementation plasmid pAH8. A pUC13
constructcontaining the ClaI-to-EcoRI segment including orf162b
(Fig. 1) was digestedwith PstI, and an EcoRI site in the linker (59
CCGAATTCGG 39) was ligatedinto this site (made blunt with T4 DNA
polymerase) by linker tailing. The 748-bpEcoRI fragment containing
orf162b was excised from the resultant plasmid andligated into the
vector pRR5C (30). The resultant plasmid, called pAH8,
whichcontains the orf162b gene transcribed from the puf promoter,
was used to restoreexpression of orf162b in mutant backgrounds.
RNA blots. Aerobic and oxygen-limited cultures were grown to a
density of 100Klett units, and RNA was isolated from 25 ml of each
culture using the RNeasykit (QIAGEN). Samples were treated with 30
U of DNase I in 100 mM sodiumacetate–5 mM MgSO4 (pH 5.0) for 30 min
at room temperature, followed byphenol-chloroform extraction and
ethanol precipitation. Seven micrograms ofeach RNA sample was used
for formaldehyde gel electrophoresis (16) andelectroblotted onto a
nylon membrane (ICN) for 2 h at 80 V in 0.53 Tris-borate-EDTA
(TBE). The membrane was baked at 80°C for 2 h, placed on a
nylonmesh, and prehybridized in 10 ml of 50% formamide–10% dextran
sulfate–5.8%NaCl–1% sodium dodecyl sulfate (SDS)–0.2% bovine serum
albumin (BSA)–0.2% Ficoll–0.2% polyvinylpyrrolidone–0.1 sodium
pyrophosphate–50 mM TrisHCl (pH 7.5) with 0.1 mg of sheared salmon
sperm DNA (Sigma)/ml at 42°C for3 h with rotation (in an oven from
BIO/CAN Scientific). The probe was agel-purified DNA fragment
extending from the BsaBI site in orf162b to theEcoRI site in orf55
(Fig. 1), labeled with 32P by using the Redi-Prime kit(Amersham
Pharmacia Biotech) for 2 h. After 16 h of hybridization, the
mem-brane was washed with agitation twice with 100 ml of 23 SSC (13
SSC is 0.15 MNaCl plus 0.015 M sodium citrate) for 10 min at room
temperature, twice with100 ml of 23 SSC containing 1% SDS for 15
min at 60°C, and once with 100 mlof 0.13 SSC for 15 min at room
temperature. Hybridization signals were de-tected with film
(Kodak).
b-Galactosidase assays. Cells from oxygen-deprived R. capsulatus
cultures at80 or 165 Klett units, and photosynthetic cultures at
125 Klett units, wereharvested by centrifugation. Pellets from 40
ml of culture were stored at 280°Cbefore assay as previously
described (18).
Chromatophore isolation. Oxygen-deprived and photosynthetic
culturesgrown to 150 Klett units were centrifuged, and the cell
pellets were stored at280°C. The cells were resuspended in 20 ml of
chromatophore buffer [20 mM3-(N-morpholino)propanesulfonate (MOPS),
100 mM KCl, 1 mM MgCl2 (pH7.2)] and passed through a French press
three times. Cell debris was removed bycentrifugation at 12,000 3 g
for 15 min, and the supernatant was centrifuged at412,000 3 g for
14 min to pellet membrane vesicles (chromatophores), whichwere
resuspended in chromatophore buffer. An aliquot stored at 4°C was
usedfor flash spectroscopy, and the remainder was stored at 280°C
and used forprotein electrophoresis.
Spectroscopy. Absorption spectroscopy of intact cells was
performed as de-scribed previously (17), and data were collected
with the Spectra Calc softwarepackage (Galactic Industries
Corporation). Light scattering at 650 nm was usedto normalize the
spectra. Low-temperature absorption spectroscopy used a Hi-tachi
557 double-beam spectrophotometer. Chromatophores in
chromatophorebuffer (see above) were mixed with an equal volume of
anhydrous glycerol andfrozen in liquid nitrogen. Spectra were
obtained with the samples chilled by, but
FIG. 1. The orf162b locus and flanking regions. The arrow
indicates the direction of transcription from a promoter
immediately 59 of puhA. The 2-kbp BamHIfragment was cloned in
pUC13, and the restriction sites shown were used to disrupt
orf162b, construct a complementation plasmid, and produce a probe
for RNA blothybridization. The solid horizontal bar shows the
extent of this BsaBI-to-EcoRI probe.
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not immersed in, liquid nitrogen. Flash spectroscopy was carried
out as previ-ously described (17).
SDS-PAGE. The amount of protein in each chromatophore
preparation wasdetermined by a modified Lowry method, with bovine
serum albumin as thestandard (21). Samples containing 40 mg of
protein were mixed with loadingbuffer, heated at 50°C for 10 min,
and used in a Tricine-SDS-polyacrylamide gelelectrophoresis (PAGE)
system (23). Gels were stained in a solution of 0.025%Coomassie
brilliant blue G-250 in 40% methanol and 10% acetic acid and
weredestained in the same solution lacking the dye.
RESULTS
orf162b is transcribed as part of the puhA
superoperon.Hybridization of RNA blots with an orf162b-specific
probe(Fig. 1) showed that orf162b is transcribed to produce
mes-sages at higher levels in oxygen-deprived than in highly
aeratedSB1003 cells (Fig. 2); transcripts were also detected in
photo-synthetically grown cultures (data not shown). The largestRNA
species detected was approximately 2.7 kb in size and wasdistinct
from a smear of less than 1.0 kb, indicating that deg-radation of
the primary transcript is rapid. The probe alsobound to molecules
of 1.5 kb from SBK1 cultures (attributed totranscription
originating at the kanamycin resistance cartridgepromoter, which
would be complementary to the 39 end of theprobe [Fig. 1]). There
was nonspecific hybridization of theprobe to rRNA in all six
samples (sizes of 1.6 and 1.0 kb) (31).However, no 2.7-kb species
or low-molecular-weight smearswere detected in lanes containing RNA
from strain DW1, inwhich transcription is terminated at the V
cartridge insertedinto puhA (28). Therefore, orf162b is
cotranscribed with thepuhA and orf214 genes and is part of a
bchFNBHLM-lhaA-puhA-orf214-orf162b superoperon (4).
Photosynthetic growth kinetics of the orf162b mutant
andcomplemented strains. The partial deletion and KIXX disrup-tion
of orf162b in strain SBK1 impaired photosynthetic growth,as
evidenced by a progressive slowing of growth and often apremature
stationary phase compared to the growth of SB1003(Fig. 3).
Complementation of strain SBK1 with the orf162bgene borne on
plasmid pAH8 resulted in a photosyntheticgrowth pattern similar to
that of the parental strain. Thus itseems that orf162b transcripts
are translated, the resultantOrf162b protein is required to sustain
rapid photosyntheticgrowth, and the wild-type allele is dominant
over the disruptedorf162b allele.
Evaluation of the presence and function of RC and LHcomplexes by
SDS-PAGE and spectroscopy. The levels of RCand LH proteins in
chromatophores isolated from oxygen-
deprived (dark) and anaerobic photosynthetic
(illuminated)cultures of strains SBK1 and SB1003 were compared by
SDS-PAGE (Fig. 4). In both modes of growth, the orf162b
mutantstrains had lower levels of the RC proteins H, M and L,
andwas deficient in the LHI a protein. The level of the LHI
bprotein could not be ascertained due to its comigration withthe
LHII b protein. The levels of the LHII a and b proteinswere not
greatly affected by the orf162b mutation.
Room temperature absorption spectra of SBK1 intact
cellsindicated a decrease in the 870-nm LHI shoulder on the850-nm
LHII peak (not shown). Absorption spectra at 77 K(liquid N2) of
chromatophores isolated from oxygen-deprivedand photosynthetic
cultures of SBK1 and SB1003 clearly re-vealed a significant
decrease in the LHI 870-nm absorption(shoulder of the LHII 850-nm
peak in Fig. 5). These spectrashow that the level of the LHI
complex is lower in the orf162bmutant than in the parental strain
SB1003, and that the differ-ence is more pronounced in
photosynthetically grown cells.The LHII peaks at 800 and 850 nm
were marginally increasedby the orf162b mutation in these samples,
which were normal-ized on the basis of Bchl content.
The magnitude of the SBK1 RC Bchl special pair bleachingin
response to a train of eight flashes of actinic light was
greatlyreduced in this orf162b mutant relative to SB1003 (Fig.
6a).These and other experiments indicate that strain SBK1 con-tains
approximately 33% (in oxygen-deprived cells) or 3% (incells from
photosynthetically grown cultures in premature sta-tionary phase)
of the level of RCs in SB1003 cells grown to thesame cell density
under identical conditions, based on theextent of RC bleaching
after the eighth flash.
The carotenoid bandshift of R. capsulatus, like that of
R.sphaeroides, is an intrinsic monitor of transmembrane poten-tials
that change in response to light-driven electron and pro-ton
translocation (15). In concert with the measurements of
FIG. 2. Blot of R. capsulatus RNA hybridized with the orf162b
probe (Fig. 1).RNA was isolated from highly aerated and
oxygen-deprived cultures of DW1,SB1003, and SBK1. The approximate
sizes of relevant signals (in kilobases) aregiven on the left.
FIG. 3. Effect of orf162b mutation and trans complementation
with pAH8 onthe photosynthetic growth of R. capsulatus strains.
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the RC, the carotenoid bandshift after a single flash wassmaller
in the orf162b-disrupted strain SBK1 than in SB1003,both in the
absence (Fig. 6b) and in the presence (Fig. 6c) ofantimycin, which
inhibits the cytochrome b/c1 complex (27). Allthree phases of the
bandshift were apparent in the single-flashabsorbance changes of
the deletion strain in the absence ofantimycin (Fig. 6b),
indicating that functional RCs were con-nected appropriately to the
cytochrome b/c1 complex. Multipleflashes resulted in successive
turnovers of the RC and progres-
sively greater transmembrane potentials, but to a lesser
extentin SBK1 than in SB1003, and the difference was more
pro-nounced in chromatophores from cells grown photosyntheti-cally
(Fig. 6d).
In summary, the flash-induced spectral changes indicate thatthe
RC of SBK1 cells is present in only small amounts, but thatneither
the catalytic activity of the RC nor the quinone transferfrom the
RC to the b/c1 complex is impaired. The spectro-scopic data, like
the SDS-PAGE data, show that this mutationreduces the amounts of
the RC and LHI complexes, and reveala more severe effect of the
orf162b disruption after an initialperiod of rapid growth under
photosynthetic conditions.
pufB (LHI b) gene fusion expression in the orf162b mutantstrain
SBK1. To evaluate whether the reduction in LHI andRC levels in SBK1
might be due to decreased expression of thepuf genes that encode
LHI and two of the three RC proteins,the b-galactosidase activities
expressed from a translationallyin-frame pufB::lacZ gene fusion,
which is transcribed from thepuf promoter in plasmid pXCA601::935,
in strains SBK1(pXCA601::935) and SB1003(pXCA601::935) were
determined.During oxygen-deprived growth, the
SBK1(pXCA601::935)b-galactosidase activities ranged from 84 to 95%
of the SB1003(pXCA601::935) values, and during photosynthetic
growth therelative activity was 155%. We conclude that neither
initiationof transcription from the puf promoter nor initiation of
trans-lation of the LHI b protein is significantly decreased by
theorf162b mutation, and that the Orf162b protein functions
post-translationally to yield appropriately large amounts of RC
andLHI complexes.
Suppression of the orf162b phenotype. An agar plate spreadwith
about 106 SBK1 cells that had reached premature station-ary phase
during photosynthetic growth was incubated underphotosynthetic
growth conditions, and about 150 coloniesformed within 2 to 4 days.
Four strains in which the orf162bphotosynthetic growth phenotype
was thus suppressed (desig-nated SBK18, SBK20, SBK21, and SBK23)
were isolated fromthese colonies. When grown photosynthetically in
liquid cul-ture, these suppressor strains did not exhibit a
premature slow-ing of growth, in contrast to the SBK1 parental
strain (Fig. 7a).During growth under oxygen-deprived conditions,
all four sup-pressor strains were found to contain less of the LHII
complex
FIG. 4. Effects of the orf162b mutation on RC and LHI protein
levels inchromatophores isolated from oxygen-deprived or
photosynthetic cultures ofSBK1 (orf162b mutant) compared to SB1003
(wild type), as revealed by SDS-PAGE.
FIG. 5. Low-temperature absorption spectra of chromatophores
from SB1003 and SBK1 cultures grown under either oxygen-deprived or
photosynthetic condi-tions.
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FIG. 6. Flash spectroscopy analysis of chromatophores from
SB1003 and SBK1 strains. (a) The amount of functional RC. (b)
Single-flash carotenoid bandshiftwithout antimycin. (c)
Single-flash carotenoid bandshift with antimycin. (d) Carotenoid
bandshift over eight flashes without antimycin. The vertical bars
on the leftrepresent 0.00435 absorbance units at the wavelength
pairs indicated.
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than did SBK1 and the parental strain SB1003, as indicated
bydecreases in the 800- and 850-nm peaks of room
temperatureabsorption spectra of intact cells (Fig. 7b; for
brevity, onlySBK23 data are shown). When grown photosynthetically
to the
density at which SBK1 enters the characteristic
prematurestationary phase, SBK18 and SBK21 contained less LHII
thanSBK1 and SB1003; the level of LHII in SBK20 and SBK23
wascomparable to that in SB1003 but less than that in SBK1
(Fig.7c). In the stationary phase of photosynthetic growth, all
foursuppressor strains had approximately the same level of LHII
asSB1003 (not shown). The spectra in Fig. 7b and c also showthat
SBK1 contains more LHII than SB1003 when samples arenormalized to
equivalent cell numbers. The frequency of sup-pressor mutations
(ca. 1.5 3 1024) and the different effects onLHII levels indicate
that the orf162b mutant phenotype issuppressed by several types of
second-site mutations.
DISCUSSION
As noted above, in the five species of purple
photosyntheticbacteria for which DNA sequence data are available,
the puhAgene is followed by orf214-like and orf162b-like ORFs in
thesame transcriptional orientation as puhA. In R.
capsulatus,genetic experiments indicated that puhA and orf214 are
co-transcribed with at least one 39 gene (28), and similar
conclu-sions were reached as a result of puhA disruption
experimentson R. sphaeroides (9, 24). Our RNA blots provide
biochemicalevidence that synthesis of orf162b messages depends on
tran-scription that initiates 59 of puhA from either or both of
thepromoters located within the lhaA gene and 59 of the bchFgene.
Such a long transcript was not detected (Fig. 2), and sowe suggest
that degradation of segments of these primary tran-scripts is so
rapid that such long molecules are undetectable inblot
hybridizations, by analogy to the R. capsulatus
crtEF-bchXYZ-pufQBALMX superoperon (1, 4). This rapid degrada-tion
may explain the extremely small amounts of the orf162b2.7-kb RNA
segment detected with the orf162b probe, and whyBauer et al. (3)
did not detect an orf162b precursor RNAmolecule in blots probed
with a puhA-specific probe. Thisinterpretation implies that the
bchFNBHLM-lhaA-puhA-orf214 superoperon of R. capsulatus (28)
includes orf162b,which suggests a physiological connection among
the puhA,orf214, and orf162b genes.
Hybridization of the orf162b probe to a 1.5-kb transcript inSBK1
suggests that the 39 region of the disrupted orf162b geneis
transcribed from the kanamycin resistance cartridge pro-moter. The
reason why this transcript was more abundant inhighly aerated SBK1
cultures is not known. This transcriptionmay or may not cause
overexpression of downstream ORFs,which have no known function at
present. However, such hy-pothetical overexpression could not be
responsible for the im-paired photosynthetic growth of strain SBK1,
because plasmidpAH8 (which expresses orf162b from the puf promoter)
re-stores normal growth, indicating that the Orf162b protein
isproduced in wild-type R. capsulatus and is required for
optimalphotosynthetic growth.
Disruption of orf162b leads to impaired photosyntheticgrowth,
typically exhibited as a premature stationary phase.Our
spectroscopic and SDS-PAGE data demonstrate thatSBK1 is deficient
in the RC and LHI proteins. These findingsindicate that the
function of Orf162b is to promote the expres-sion, assembly, or
stabilization of these pigment-protein com-plexes. The presence or
absence of the Orf162b protein did notsignificantly reduce
initiation of transcription from the pufpromoter or translation of
a pufB::lacZ fusion, and so the effectof Orf162b on the LHI and the
RC complexes appears to bemanifested posttranslationally. We
propose that Orf162b en-hances the assembly or stability of RC-LHI
core complexes.
The impaired photosynthetic growth of SBK1 was overcomeby
frequent spontaneous secondary mutations that led to a
FIG. 7. (a) Photosynthetic growth of suppressor strains SBK18,
SBK20,SBK21, and SBK23, compared to growth of SB1003 and SBK1. (b
and c) Ab-sorption spectra (normalized to light scattering at 650
nm) of intact cells fromoxygen-deprived (b) and photosynthetic (c)
cultures of the representative sup-pressor strain SBK23 against
those of the wild-type SB1003 and orf162b mutantSBK1 strains. The
vertical axes give absorbance units, and the horizontal axesgive
wavelengths (in nanometers).
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decrease in the amount of LHII. This might indicate that
thepremature slowing of growth is due to lower levels of the RCand
LHI complexes caused by an increase in the ratio of LHIIto LHI-RC
core complexes, rather than a simple decrease inthe amounts of the
core complex. It is conceivable that theOrf162b protein provides an
optimal arrangement of com-plexes within the ICM of R. capsulatus
such that, in the absenceof Orf162b, LHII mixes with the core
complex and interfereswith assembly. Alternatively, Orf162B may
function to removea growth-inhibiting factor associated with excess
LHII. Re-gardless, the orf162b and suppressor mutant phenotypes
sug-gest that the function of Orf162b relates to all three
complexesof the photosynthetic apparatus. Because an orf162b-like
ORFis present in R. rubrum, which lacks LHII, and because we
haveobserved an orf162b mutant phenotype in an LHII2 R. capsu-latus
background (14a), we suggest that the effects of orf162bdisruption
on LHII are secondary to a primary effect on theRC and LHI.
The core complex deficiencies of SBK1 were more pro-nounced when
photosynthetic growth slowed after a brief pe-riod of rapid cell
division than when growth of this mutant hadslowed due to oxygen
deprivation, and so this low-oxygengrowth condition appears to
compensate in some measure forthe loss of Orf162b function. We
entertained the possibilitythat the presence of oxygen allows the
oxidation of a cofactorrequired for photosynthesis and that Orf162b
catalyzes thisoxidation under anaerobic conditions; however,
inclusion ofthe alternative electron acceptor dimethyl sulfoxide
(14) in themedium of photosynthetic cultures did not compensate
fororf162b disruption (unpublished data). We favor an
alternativehypothesis, that the low rate of cell division (and ICM
synthe-sis) in the pseudostationary phase of oxygen-limited
culturesallows the inefficient accumulation of core complexes in
theICM, whereas cell division upon transfer to anaerobic,
illumi-nated conditions of growth is too rapid for
commensurateaccumulation of core complexes, leading to a gradual
decreasein the number of core complexes per cell so that rapid
growthcannot be sustained. This model will be investigated in
futureexperiments.
We suggest that the ORFs in R. sphaeroides, R. gelatinosus,R.
rubrum, and R. palustris that resemble orf162b also encodeproteins
which optimize the levels of RC-LHI core complexesand that these
ORFs are genes that are cotranscribed withpuhA. If these
Orf162b-like sequences found in different spe-cies are indeed
homologous, they exhibit remarkable evolu-tionary divergence. A
predicted transmembrane domain com-mon to the N-terminal regions of
all five sequences suggeststhat membrane attachment and hence the
proximity of Orf162bto the membrane-bound photosynthetic apparatus
may be im-portant for the accumulation of RC-LHI core complexes in
theICM. This function may require Orf162b and related proteinsto
associate intimately with other proteins that have
evolveddivergently in different species, but in parallel with them
withineach species. Further studies of Orf162b function will be
ac-companied by analyses of the orf153 locus in R. sphaeroides,
theorf154 locus in R. gelatinosus, the orfI3087 locus in R.
rubrum,and the orf168 locus in R. palustris to assess whether
theseORFs are functionally equivalent to the R. capsulatus
orf162bgene.
In conclusion, our analyses of the orf162b locus revealed thatit
encodes a protein that affects the relative levels of the RC,LHI,
and LHII complexes and is required for optimal photo-synthetic
growth of R. capsulatus. The transcriptional coregu-lation of
orf162b with the puhA gene in R. capsulatus and thepresence of ORFs
similar to orf162b at the same chromosomallocation relative to puhA
in other species suggest that Orf162b-
like proteins perform a similar function in a variety of
purplephotosynthetic bacteria.
ACKNOWLEDGMENTS
We thank W. Collins for construction of the pUC13::BamHI
plas-mid and C. Harwood, S. Kaplan, and K. Nagashima for provision
ofunpublished data.
M.A. was supported in part by a fellowship from UBC, and
thisresearch was funded by NSERC (Canada) grant 5-82796 to
J.T.B.
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