Download Insertional inactivation studies of the csmA and csmC genes of the

FEMS Microbiology Letters 164 (1998) 353^361
Insertional inactivation studies of the csmA and csmC genes of the
green sulfur bacterium Chlorobium vibrioforme 8327:
the chlorosome protein CsmA is required for viability
but CsmC is dispensable
Soohee Chung
a
1;a
, Gaozhong Shen a , John Ormerod b , Donald A. Bryant a; *
Department of Biochemistry and Molecular Biology, S-234 Frear Building, The Pennsylvania State University, University Park,
PA 16802, USA
b
Department of Biology, University of Oslo, Blindern, N-0316 Oslo, Norway
Received 12 May 1998 ; accepted 23 May 1998
Abstract
Targeted mutagenesis was used to investigate the roles of the CsmA and CsmC proteins of the chlorosomes of the green
bacteria Chlorobium tepidum and Chlorobium vibrioforme 8327. Under the photoautotrophic growth conditions employed,
CsmA is required for the viability of the cells but CsmC is dispensable. The absence of CsmC caused a small red shift in the
near-infrared absorption maximum of bacteriochlorophyll d in whole cells and chlorosomes, but chlorosomes were assembled
in and could be isolated from the csmC mutant. The doubling time of the csmC mutant was approximately twice that of the
wild-type strain. Fluorescence emission measurements suggested that energy transfer from the bulk bacteriochlorophyll d to
another pigment, perhaps bacteriochlorophyll a, emitting at 800^804 nm, was less efficient in the csmC mutant cells than in
wild-type cells. These studies establish that transformation and homologous recombination can be employed in targeted
mutagenesis of Chlorobium sp. and further demonstrate that chlorosome proteins play important roles in the structure and
function of these light-harvesting organelles. z 1998 Federation of European Microbiological Societies. Published by
Elsevier Science B.V. All rights reserved.
Keywords : Chlorosome ; Green sulfur bacterium; Interposon mutagenesis ; Bacteriochlorophyll ; Photosynthesis; Light-harvesting antenna
1. Introduction
Chlorosomes are photosynthetic light-harvesting
* Corresponding author. Tel.: +1 (814) 865 1992;
Fax: +1 (814) 863 7024; E-mail: [email protected]
1
Present address: Department of Biological Sciences, and
Basic Science Research Institute, Ewha Womans University,
Seoul 120-750, South Korea.
complexes found only in green bacteria [1,2]. These
sac-like organelles, which occur tightly appressed to
the inner surface of the cytoplasmic membrane, contain highly aggregated, rod-shaped arrays of bacteriochlorophyll (Bchl) c, d, or e dependent upon the
source organism, and additionally contain proteins,
carotenoids, glycolipids, and quinones as major constituents. Electron microscopy, protease susceptibility mapping, and agglutination experiments using a
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 2 3 8 - 9
FEMSLE 8244 9-7-98
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S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361
variety of antisera suggest that chlorosomes are surrounded by a protein- and lipid-containing, monolayer envelope [3^7]. Highly puri¢ed chlorosomes
from Chlorobium tepidum have been shown to contain 10 di¡erent proteins [8^10], and the genes for
most of these have been cloned and characterized [6^
10]. However, the speci¢c functional roles of these
proteins in chlorosome structure, function, and biogenesis remain unknown.
In studies of phycobilisomes, the light-harvesting
antenna structures of cyanobacteria, targeted or interposon mutagenesis was a very e¡ective method for
the elucidation of the functional roles of the component proteins of these structures (for reviews, see
[11,12]). In this report we describe the ¢rst application of targeted mutagenesis to the study of chlorosome structure and function and describe the properties of a csmC mutant lacking the 14-kDa protein of
the chlorosomes. These experiments establish the occurrence of homologous recombination in Chlorobium sp. and the feasibility of using targeted mutagenesis to study the roles of proteins in the structure,
function, and biogenesis of chlorosomes.
2. Materials and methods
Chlorobium vibrioforme strain 8327 and Chlorobium tepidum were grown as previously described
[13,14]. All DNA manipulations were performed in
Escherichia coli strain DH5K (Gibco-BRL, Gaithersburg, MD). E. coli strains were grown in Luria-Bertani medium [15]; when appropriate, the medium
was supplemented with ampicillin (100 Wg ml31 ), kanamycin (40 Wg ml31 ) or streptomycin (30 Wg ml31 ).
Plasmids pUC18 or pUC19 were employed for subcloning, interposon mutagenesis, and DNA sequencing. The cloning and characterization of the csmCA
operons of Cb. vibrioforme and Cb. tepidum have
been described [8]. DNA fragments encoding resistance to streptomycin/spectinomycin (the 6 cassette
[16]) or to kanamycin (the aphII gene that encodes
aminoglycoside 3P-phosphotransferase II [17]) were
used to perform insertional inactivation of the
csmA and csmC genes. Southern blot hybridization
analyses were performed as described [8].
Transformation of Cb. vibrioforme was performed
as described [13,18]. Cells were mixed with a loopfull of either plasmid or linearized DNA fragment
suspension and plated on a non-selective plate as a
thick patch and incubated in the light for 1^3 days.
After the cells had grown, transformants were selected on agar plates (Chlorobium medium containing 1.5% (w/v) Bacto agar including antibiotics.
Antibiotic resistant transformants were selected on
agar plates containing 15 Wg ml31 streptomycin
and 15 Wg ml31 spectinomycin or 40 Wg ml31 kanamycin. The plates were incubated under the light for
1^3 days in an anaerobic controlled environment
chamber in an atmosphere of 85% N2 , 10% CO2 ,
and 5% H2 . Growth curves for liquid cultures were
generated by monitoring the optical density of cultures at 650 nm.
Optical density and absorption measurements
were performed with a Cary 14 spectrophotometer
that was modi¢ed for computerized data acquisition
by On-Line Instruments Systems (Bogart, GA). Fluorescence emission spectra at 77 K were obtained
with an SLM-Aminco 8000C £uorometer with excitation at 449 nm. Protein concentrations were determined by the method of Bradford [19] using the
BCA reagents purchased from Pierce Chemical Co.
(Rockford, IL). Bchl concentrations were determined
by extraction with aqueous acetone (80% v/v) using
the speci¢c absorption coe¤cient 92.6 l (g cm)31 for
Bchl c or 98.0 l (g cm)31 for Bchl d [20]. Protein
compositions were analyzed by polyacrylamide gel
electrophoresis as described [7,21]. Immunoblotting
C
Fig. 1. Interposon mutagenesis of the csmA gene of Cb. vibrioforme. A: Physical maps of the constructions employed in insertional inactivation of the csmA gene. The antibiotic resistance cassettes were inserted into the NcoI site within the coding sequence of the csmA gene.
Arrows indicate the direction of transcription. Abbreviations for restriction enzymes : H, HindIII; N, NcoI; Sp, SphI. B and C: Southern
blot hybridization analyses. Total genomic DNA was digested with HindIII. Lane 1, wild-type ; lane 2, transformant strain 336; lane 3,
transformant strain 337; lane 4, transformant strain 337 grown in the presence of 600 Wg ml31 streptomycin. The blot shown in panel B
was probed with a 0.27-kb NdeI-BamHI fragment, which encodes the csmA gene and which was derived from expression plasmid
pET11a/CsmA [7]. The blot shown in panel C was probed with the 6 cassette encoding resistance to streptomycin/spectinomycin (a 2.0kb BamHI fragment from plasmid pHP45 [16]).
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S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361
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356
S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361
Fig. 2. Interposon mutagenesis of the csmC gene of Cb. vibrioforme. A: Physical maps of the constructions employed in insertional inactivation of the csmC gene. The antibiotic resistance cassettes were inserted into the AvaI site within the coding sequence of the csmC gene.
Arrows indicate the direction of transcription. Abbreviations for restriction enzymes : A, AvaI ; N, NcoI; Sa, SalI; Sp, SphI. B and C:
Southern blot hybridization analyses. Total genomic DNA in lanes 1^3 was digested with NcoI and SalI, and the DNA in lanes 4 and 5
was digested with HindIII. Lane 1, wild-type ; lane 2, csmC mutant strain 633 ; lane 3, csmC mutant strain 634; lane 4, wild-type ; lane 5,
csmC mutant strain 626. The blot shown in panel B was probed with a 0.5-kb HinfI fragment speci¢c for the csmC gene. Lanes 1^3 of
panel C were probed with the 6 cassette encoding resistance to streptomycin/spectinomycin (a 2.0-kb BamHI fragment from plasmid
pHP45 [16]) and lanes 4 and 5 were probed with the aphII gene encoding resistance to kanamycin (a 1.32-kbp BamHI fragment from
plasmid pRL161 [17].
C
with antisera prepared against recombinant chlorosome proteins was performed as described [6,7].
3. Results and discussion
To investigate the roles of CsmA and CsmC, the
csmA and csmC genes were insertionally inactivated
by interposon mutagenesis with the 6 cassette [16],
encoding resistance to streptomycin and spectinomycin, or the aphII gene, encoding aminoglycoside 3Pphosphotransferase and conferring resistance to kanamycin [17] (see Figs. 1A and 2A). In plasmids
pCB336 and pCB337, the csmA gene of Cb. vibrioforme is insertionally inactivated by the introduction
of the 6 cassette in either orientation into the unique
NcoI site within the coding sequence of that gene
(Fig. 1A). Similarly, plasmids pCB633 and pCB634
contain the 6 cassette inserted in either orientation
in an AvaI site within the coding sequence of the
csmC gene of Cb. vibrioforme; plasmid pCB626 contains the aphII gene inserted into the same site (Fig.
2A). These plasmids were used to transform Cb. vibrioforme cells, and transformants resistant to streptomycin/spectinomycin or kanamycin as appropriate
were selected. Transformants selected for detailed
studies were identi¢ed by the number of the plasmid
number used to create them.
Southern blot hybridization analyses with DNA
isolated from Cb. vibrioforme transformants 336
and 337 con¢rmed that homologous recombination
of the appropriate linearized plasmid and the chromosomal csmA locus had occurred (Fig. 1B,C), but
that segregation of the csmA and csmA: :6 alleles
had not occurred. As shown in Fig. 1A,B, lane 1,
the csmA gene is encoded on a 1.57-kb HindIII fragment. Insertion of the 6 cartridge at the unique NcoI
site within the csmA coding sequence introduced two
additional HindIII sites as indicated in Fig. 1A. For
all transformants tested, the hybridization pattern
showed the presence of both the 1.57-kb HindIII
fragment wild-type fragment as well as the two
smaller HindIII fragments of 0.85-kb and 0.72-kb
that arise from the introduced HindIII sites that
£ank the 6 cartridge in the insertionally inactivated
csmA gene. Even after repeated restreaking and
growth on up to 600 Wg ml31 streptomycin (Fig.
1B,C, lanes 4), no segregation of alleles occurred.
To show that the antibiotic resistance of the mutants
was not the result of a spontaneous mutation, Southern hybridization experiments were also performed
with a probe speci¢c for the 6 cartridge. As shown
in Fig. 1C, lanes 2^4, a 2.0-kb HindIII fragment in
the DNA isolated from each of the transformants
hybridized to this probe, con¢rming that homologous recombination had occurred and that the antibiotic resistance was due to the 6 cassette. A similar
set of experiments was performed with the csmA
gene of Cb. tepidum, and identical results were obtained (data not shown). In other organisms the failure of alleles to segregate has been taken to be strong
presumptive evidence for the essentiality of the product of the gene in question [22]. Thus, we conclude
that, under the photoautotrophic growth conditions
employed in these experiments, CsmA is required for
viability of Cb. vibrioforme and Cb. tepidum.
The requirement of the csmA gene product for
viability was unexpected, since in other photosynthetic bacteria, photosynthesis and photoautotrophic
growth can still occur in the complete absence of
light-harvesting antenna complexes, as long as su¤cient light is provided. For example, a cyanobacterial
mutant lacking detectable phycobiliproteins was still
capable of photoautotrophic growth [23], and many
mutants lacking various light-harvesting proteins in
purple bacteria have been described [24]. Foidl et al.
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S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361
[25] have recently shown that chlorosomes and
chlorosome proteins are present even in chemotrophically grown cells of Chloro£exus aurantiacus. The
357
ratio of these proteins to the speci¢c Bchl c content
of the cells changed approximately 8^10-fold when
cells were shifted from aerobic/chemotrophic to
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S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361
anaerobic/phototrophic growth conditions. These
studies show that the synthesis of chlorosome polypeptides is largely independent of Bchl c synthesis. In
Cb. vibrioforme anaesthetic gases, such as N2 O,
acetylene, and ethylene, can be used to inhibit the
synthesis of Bchl d and chlorosomes [26], yet the
chlorosome proteins are still present in such cells in
large amounts (V. Nguyen and J.G. Ormerod, unpublished results). These observations are consistent
with the idea that chlorosomes and/or speci¢c
chlorosome proteins may have an important cellular
function(s) other than light-energy harvesting. Since
reaction center preparations of green sulfur bacteria
typically contain substantial amounts of the FMO
protein, which forms the baseplate of the chlorosome
[2,27], one intriguing possibility is that components
of the chlorosome envelope, including CsmA, are
required for the proper structural organization of
the proteins on the acceptor side of the reaction center complex. Alternatively, biogenesis of the photosynthetic apparatus in Chlorobium species may be
dependent upon the assembly of the chlorosome envelope. However, further experiments on the localization and functional role(s) of CsmA will be required to elucidate its function.
In contrast to the results obtained with attempts to
inactivate the csmA genes, Southern blot hybridization experiments with DNA isolated from Cb. vibrioforme transformants 633 and 634 showed that the
csmC and csmC: :6 alleles had segregated completely
(Fig. 2B,C). As shown in Fig. 2B, when DNA the
Fig. 3.
100 Wg
probed
mutant
transformant strains was digested with NcoI and
SalI, no evidence for the presence of the 1.45-kb
wild-type fragment (Fig. 2B, lane 1) was observed
in these strains (compare Fig. 2B, lanes 1^3). Similarly, Cb. vibrioforme transformant 626, in which the
csmC gene was insertionally inactivated with the
aphII gene, also segregated completely (compare
Fig. 2B, lanes 4 and 5). For each transformant, hybridization experiments performed with DNA fragments encoding the appropriate drug resistance
marker con¢rmed that the antibiotic resistance was
due to the presence of the appropriate DNA cassette
(Fig. 2C, lanes 2, 3 and 5). These experiments again
con¢rm that homologous recombination occurs in
Cb. vibrioforme and that CsmC is not essential under
the photoautotrophic growth conditions employed
during the isolation of these mutant strains. These
results additionally show that neither the aphII nor
6 cassette DNA fragments exerts a polar e¡ect on
the expression of the essential downstream csmA
gene. Since the 6 cassette contains strong transcription termination signals £anking the gene encoding
drug resistance [16], these results suggest that at least
some transcription of csmA occurs from a promoter
located between csmC and csmA. The 5P endpoint of
the major csmA transcript of Cb. tepidum has been
mapped to a position 52 nucleotides upstream from
the translational start site for this gene [8]. Thus, this
endpoint may not arise from a processing event as
originally suggested [8], and a strong promoter possibly occurs upstream from it.
Immunoblot analysis of the wild-type and csmC mutant strains of Cb. vibrioforme. Whole cell extracts (proteins associated with
of Bchl d) were separated by SDS-PAGE, electrophoretically transferred to Immobilon membranes (Millipore, Bedford, MA) and
with polyclonal rabbit antibodies against the CsmC protein [7]. Lane 1, extract from wild-type cells; lane 2, extract from csmC
strain 633; lane 3, extract from csmC mutant strain 626.
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S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361
359
To con¢rm that the csmC mutant strains of Cb.
vibrioforme were missing CsmC, total proteins from
whole cell extracts were separated by SDS-PAGE
and immunoblotted with antibodies speci¢c for
CsmC. As indicated in Fig. 3, the immunoreactive
14.2-kDa CsmC protein observed in extracts of the
wild-type cells (Fig. 3, lane 1) is missing extracts of
mutant strains 633 (Fig. 3, lane 2) and 626 (Fig. 3,
lane 3). Chlorosome proteins other than CsmC were
veri¢ed to be present in approximately normal
amounts by both SDS-PAGE and immunoblot analyses of isolated chlorosomes (data not shown).
To determine whether the absence of CsmC had
Fig. 4. Absorption spectra of whole cells and chlorosomes for
the wild-type (solid lines) and csmC mutant strain 633 (dashed
lines) of Cb. vibrioforme. A: Absorption spectra of whole cells.
The maximum absorbance in the near infrared region of the
spectrum was 728 nm for the wild-type and 734 nm for the
csmC mutant strain. B: Absorption spectra of puri¢ed chlorosomes. The maximum absorbance was at 728 nm for the wildtype and at 730 nm for the csmC mutant.
Fig. 5. Fluorescence emission spectra at 77 K of whole cells and
chlorosomes for the wild-type (solid lines) and csmC mutant
strain 633 (dashed lines) of Cb. vibrioforme. The excitation wavelength was at 449 nm which is the maximum absorption peak of
Bchl d in the blue region of the spectrum (see Fig. 4). Spectra
were measured at identical cell densities for whole cells
(OD650nm = 0.2) and identical concentrations of Bchl d (20 Wg
ml31 ) for chlorosomes. A: Fluorescence emission spectra of
whole cells. The two emission peaks occurred at 750 nm and 804
nm. B: Same as panel A but the spectra have been normalized
at 804 nm. C: Fluorescence emission spectra of puri¢ed chlorosomes. The two emission peaks occurred at 750 nm and 800 nm.
any physiological e¡ect on the growth rate of Cb.
vibrioforme strains 626 and 633, doubling times
were monitored by optical density measurements at
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S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361
650 nm. Doubling times were measured for cultures
growing in 300-ml bottles with a diameter of 8 cm at
33³C at a light intensity of 250 WE m32 s31 provided
by cool-white £uorescent tubes. Under these conditions, the doubling time for the wild-type was about
1.75 h, while the doubling times for csmC mutant
strains 626 and 633 were about twice as long: 3.25
h and 3.5 h, respectively. Thus, the absence of CsmC
had a quite noticeable e¡ect on the doubling times of
the mutant organisms.
Fig. 4A shows a comparison of the absorption
spectra of whole cells of the wild-type and csmC
mutant strain 633 of Cb. vibrioforme. As shown in
Fig. 4A, the absorbance maximum of the csmC mutant strain in the near infrared region of the spectrum was shifted about 6 nm to the red compared to
that of the wild-type. The absorption maximum of
the wild-type strain occurred at 728 nm while that of
the csmC mutant 633 occurred at 734 nm. However,
the total Bchl content of the csmC mutant and wildtype cells were similar. In the absorption spectra of
isolated chlorosomes (Fig. 4B), the absorbance maximum for the wild-type strain was 728 nm, but the
absorbance maximum of chlorosomes isolated from
csmC mutant strain 633 was shifted 2 nm to the red
to 730 nm. These observations suggest that the loss
of the CsmC polypeptide resulted in some changes in
the environment or conformation of Bchl d in the
chlorosomes.
To investigate the e¡ect of the csmC mutation on
energy transfer, steady-state £uorescence emission
measurements were made at 77 K with whole cells
(Fig. 5A,B) and isolated chlorosomes (Fig. 5C) and
compared to the results obtained for the wild-type
strain. Both the wild-type and csmC mutant strain
633 exhibited two emission peaks at about 750 nm
and 804 nm. As shown in Fig. 5A, the steady-state
£uorescence emission of the csmC mutant strain 633
was much greater than that from the wild-type, implying that energy transfer in chlorosomes might be
impaired in the absence of CsmC. As seen in Fig.
5B,C, even when the £uorescence emission maxima
of whole cells (Fig. 5B) or isolated chlorosomes (Fig.
5C) were normalized at the 804 nm maximum, the
amplitude of the emission peak at about 750 nm was
signi¢cantly greater for the csmC mutant than for
the wild-type.
The light-harvesting energy transfer pathway in
Cb. vibrioforme is believed to be: antenna Bchl d
(728 nm)Cantenna Bchl a (794 nm)Cbaseplate
Bchl a/FMO protein (808 nm)Creaction center
Bchl a (840 nm) [1,2]. The cellular content, assembly
and absorption properties of chlorosomes of the
csmC mutant strain were not signi¢cantly di¡erent
from those of wild-type cells (Fig. 4). Nevertheless,
the absence of the CsmC protein had demonstrable
e¡ects on the £uorescence emission properties of
whole cells and isolated chlorosomes, and these
data suggest that CsmC may play at least two roles
in chlorosome function. Firstly, the £uorescence
emission data for whole cells in Fig. 5A suggest
that overall energy transfer from chlorosomes to
the reaction centers is less e¤cient in the csmC mutant than in the wild-type strain. Thus, the absence
of CsmC appears to a¡ect the coupling of chlorosomes to the FMO protein and to reaction centers in
some manner that causes total £uorescence emission
at both 750 and 804 nm to increase substantially.
Secondly, energy transfer from Bchl d to the species
emitting at about 804 nm was also less e¤cient in the
mutant than in the wild-type, as can be seen in the
normalized spectra for whole cells (Fig. 5B) and isolated chlorosomes (Fig. 5C). Therefore, CsmC might
also play a role in the organization of the bulk Bchl
d, might be involved in the organization of the Bchl
a of the chlorosome, or might be involved in the
organization of some `special' Bchl d molecules or
oligomers that might serve as intermediary acceptors
of light energy between the bulk Bchl d and Bchl a of
chlorosomes. Although it is not possible at this time
to distinguish among these possibilities, the e¡ects on
both the growth rate and the £uorescence emission
properties are consistent with the idea that the absence of CsmC causes a decrease in the overall e¤ciency of light-energy harvesting. This in turn
strongly supports the view that chlorosome proteins
have speci¢c and important roles in the structure and
function of chlorosomes. Finally, the studies reported here establish the feasibility of using targeted
mutagenesis to study the roles of proteins in the
structure, function, and biogenesis of chlorosomes.
Acknowledgments
This work was supported by DOE Grants DE-
FEMSLE 8244 9-7-98
S. Chung et al. / FEMS Microbiology Letters 164 (1998) 353^361
FG02-94ER20137
D.A.B.
and DE-FG02-97ER20137
to
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