The puc2BA operon of Rhodobacter sphaeroides is highly similar to the original puc1BA operon. Genetic, biochemical and spectroscopic approaches were used to investigate the function of puc2BA; the puc1BA and puc2BA structural genes were amplified and cloned into the pRK415 vector controlled by the puc promoter from R. sphaeroides, which was then introduced into R. sphaeroides mutant strains. The results indicated that puc2BA was normally expressed and puc2BA-encoded polypeptides were assembled into membrane LHII (light-harvesting II) complexes, although the puc2A-encoded polypeptide was much larger than the puc1A-encoded polypeptide. Semi-quantitative RT-PCR (reverse transcription-PCR) and SDS/PAGE indicated that puc1BA and puc2BA were expressed in R. sphaeroides when integrated into the genome or expressed from vectors. Furthermore, the polypeptides from the puc1BA and puc2BA genes were both involved in LHII assembly, and pucC is also necessary to assemble LHII complexes. Nevertheless, the LHII complexes synthesized from puc2BA in R. sphaeroides have blue-shift absorption bands at 801 and 846 nm.

INTRODUCTION

Photosynthesis is the single most important process developed by Nature, as it provides all of the biological energy needed for higher forms of life to exist. The photosynthetic apparatus of the purple bacterium Rhodobacter sphaeroides is composed of three membrane-spanning photopigment protein complexes, the RC (reaction centre), B875 LHI (light-harvesting I) and B800–850 LHII (light-harvesting II) antenna complexes; the light-harvesting complexes are responsible for the absorption of most of the light energy that irradiates the cells [1]. LHII is composed of two membrane-spanning polypeptides, α and β, that anchor bacteriochlorophyll and carotenoids in the membrane, which have absorption bands at 800 and 850 nm [2,3].

It has been reported previously that the puc operon of R. sphaeroides consists of the pucBA (designated puc1BA in the present study) structural genes, whch encode the LHII α- and β-polypeptides, and an additional pucC gene. PucC affects the post-transcriptional expression and assembly of the LHII α- and β-polypeptides [4,5].

In a study of R. sphaeroides mutant construction performed previously, it was observed that following deletion of the puc1BAC genes, there was a second highly homologous transcript present, as determined by hybridization. However, no LHII complexes were found in the mutant strain (strain DD13 used in the present study), despite the presence of the second transcript.

Sequence analysis of the R. sphaeroides genome revealed that there is a second copy of the puc1BA genes (designated puc2BA in the present study) present in chromosome I, some distance from the photosynthesis gene cluster [6,7].

The length of puc2BA mRNA (963 bp) is much longer than the corresponding length of puc1BA mRNA (337 bp). The puc2BA-encoded polypeptides are similar to the polypeptides encoded by puc1BA; the level of similarity between the puc1B-encoded β-apoprotein and the puc2B-encoded polypeptide is 95%, with a difference of only three amino acid residues between puc1B and puc2B.

The puc2A-encoded polypeptide comprises 263 amino acid residues, which is 209 amino acid residues longer than the puc1A-encoded α-apoprotein. A comparison of puc1A with the first 54 amino acid residues of the N-terminal of puc2A (designated puc2AM in the present study) revealed 68% similarity between the proteins.

The DAS (dense alignment surface) transmembrane prediction server (http://www.sbc.su.se/∼miklos/DAS/) predictions suggested that the puc2B polypeptide contains only one transmembrane helix domain between Val31 and Ala44, and the puc2A polypeptide contains two transmembrane helix domains, one between Leu14 and Thr37 and the other between Glu201 and Ala215.

The three-dimensional structure predicted using the SwissModel (http://swissmodel.expasy.org/SWISS-MODEL.html) using the automatic modelling mode revealed that the model templates of puc2B and puc2A were automatically based on β- and α-apoprotein of LHII from Rhodopseudomonas acidophila 10050 (RA 10050), for which the three-dimensional crystal structure has been determined with a high degree of resolution [8], and their sequence identities were 66% and 50% respectively. There are some common characteristics shared between the puc1BA operon and puc2BA operon [913].

A previous study reported that puc2BA of R. sphaeroides was transcribed at apparently normal levels, and that puc2BA-encoded polypeptides can enter the membrane, but LHII complexes were not observed in the puc1BA-containing mutant strains. However, a 30% decrease in the level of LHII complexes in the absence of a functional puc2BA operon revealed that this operon contributes to the abundance of LHII complexes. It was concluded that puc2B and puc2A can be transcribed and translated normally, and the puc2B polypeptide enters LHII complexes, but the puc2A polypeptide does not assemble into discernible LHII complexes [14], which seems contradictory. What is the actual function of the puc2BA operon in R. sphaeroides? Are the puc2BA-encoded polypeptides involved in the assembly of LHII complexes? What is the role of the presumed polypeptide extension of the puc2A-encoded polypeptide?.

In the present study, we undertook experiments to investigate the expression and function of puc2BA of R. sphaeroides, using genetic, biochemical and spectroscopic approaches. The results from the present study suggest that the puc2BA demonstrates normal function in R. sphaeroides.

MATERIALS AND METHODS

Bacterial strains and growth conditions

R. sphaeroides W1 (wild-type) (Chinese Collection of Micro-organisms, Beijing, China), R. sphaeroides DD13 (genomic deletion of pucBAC, pufBALMX, insertion of small-multidrug resistance and kanamycin-resistance genes respectively) [a gift from Professor C. Neil Hunter (Department of Molecular Biology, University of Sheffield, Sheffield, U.K.)] and R. sphaeroides DD13R (DD13 genomic deletion of puc2BA and insertion of ampicillin-resistance gene) strains were grown aerobically or semi-aerobically in the dark in M22+ medium. When required, antibiotics were added to M22+ medium at the following final concentrations: 50 μg·ml−1 ampicillin, 20 μg·ml−1 streptomycin and 2 μg·ml−1 tetracycline [15]. For aerobic incubation conditions, the bacteria were grown in a 500 ml conical flask containing 150 ml of medium at 34°C and shaking at 250 rev./min. For semi-aerobic incubation conditions, the bacteria were grown in a 500 ml conical flask containing 350 ml of medium at 34°C with shaking at 150 rev./min [16].

Escherichia coli strains JM109 and S17-1 were cultivated at 37°C in LB (Luria–Bertani) medium containing 50 μg·ml−1 tetracycline and 100 μg·ml−1 ampicillin (final concentrations). The bacterial strains, plasmids and oligonucleotides used for PCR amplification are listed in Table 1.

Table 1
Bacterial strains, plasmids and primers used in the present study

AmpR, ampicillin-resistance; F, forward primer; R, reverse primer; SmR, small-multidrug resistance; TcR, tetracycline-resistance.

DescriptionReference/source
Plasmids  
 pMD18-T AmpR, 2.8 kb PCR product cloning vector TaKaRa 
 pMD18-T-puc1B AmpR, pMD18-T with 172 bp PCR product containing puc1B Present study 
 pMD18-T-puc1A AmpR, pMD18-T with 182 bp PCR product containing puc1A Present study 
 pMD18-T-puc2B AmpR, pMD18-T with 179 bp PCR product containing puc2B Present study 
 pMD18-T-puc2BHis AmpR, pMD18-T with 176 bp puc2B fragment no termination codon Present study 
 pMD18-T-puc2AM AmpR, pMD18-T with 208 bp PCR product containing puc2AM Present study 
 pMD18-T-puc2A AmpR, pMD18-T with 815 bp PCR product containing puc2A Present study 
 pRKpucC, pRK415 containing the puc promoter and pucC of R. sphaeroides, TcR Chen laboratory 
 pRK10HispucC, pRKpucC containing 10His tag, TcR Chen laboratory 
 pRK1B1AC, pRKpucC containing the fragment of puc1B and puc1A, TcR Present study 
 pRK1B2AMC, pRKpucC containing the fragment of puc1B and puc2AM, TcR Present study 
 pRK2B1AC, pRKpucC containing the fragment of puc2B and puc1A, TcR Present study 
 pRK1B2AC, pRKpucC containing the fragment of puc1B and puc2A, TcR Present study 
 pRK2B2AMC, pRKpucC containing the fragment of puc2B and puc2AM, TcR Present study 
 pRK2B2AC, pRKpucC containing the fragment of puc2B and puc2A, TcR Present study 
 pRK2BHis2AMC, pRKpucC containing the fragment of puc2BHis and puc2AM, TcR Present study 
 pRK2BHis2AC, pRKpucC containing the fragment of puc2BHis and puc2A, TcR Present study 
Bacterial strains  
R. sphaeroides W1 (wild-type strain) Chinese Collection of Micro-organisms 
R. sphaeroides DD13, genomic deletion of puc1BAC, pufBALMX, insertion of SmR Professor C. Neil Hunter (Department of Molecular Biology, University of Sheffield, Sheffield, U.K.) 
 DD13R, genomic deletion of puc1BAC, puc2BA, pufBALMX, insertion of SmR and AmpR Chen laboratory 
Transconjugant strains  
 DD13R (pRK415), DD13R containing pRK415 Present study 
 DD13R (pRKpuc1B1AC), DD13R containing pRK1B1APresent study 
 DD13R (pRK1B2AMC), DD13R containing pRK1B2AMC Present study 
 DD13R (pRK2B1AC), DD13R containing pRK2B1AC Present study 
 DD13R (pRK1B2AC), DD13R containing pRK1B2AC Present study 
 DD13 (pRKpucC), DD13 containing pRKpucC Present study 
 DD13R (pRK2B2AMC), DD13R containing pRK2B2AMC Present study 
 DD13R (pRK2B2AC), DD13R containing pRK2B2AC Present study 
 DD13R (pRK2BHis2AMC), DD13R containing pRK2BHis2AMC Present study 
 DD13R (pRK2BHis2AC), DD13R containing pRK2BHis2AC Present study 
Escherichia coli strains  
 JM109 recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 Δ(lac-proAB) TaKaRa 
 S17-1 TpR SmRhsdR pro recA RP4-2-Tc::Mu-Km::Tn7 in chromosome TaKaRa 
Primer sequences  
puc1BF: 5′-AAGAGCTCCATATGACTGACGATCTGAACAAAG-3′ Present study 
puc1BR: 5′-TTTCTAGATCAGCCGAGCCACGGGGTCGCAGCGGCG-3′ Present study 
puc1AF: 5′-TTTCTAGACATGACCAACGGCAAAATCTGGCTCG-3′ Present study 
puc1AR: 5′-AAGGATCCTTACTCGGCCGCGACCGCAGCCGAGCCTTG-3′ Present study 
puc2BF: 5′-CACAGGTACCAGTTGGGAGACGACACAGAGCTCATGACCGATGATCCGAAAAAGG-3′ Present study 
puc2BR: 5′-ATTATCTAGATCAGCCGAGCCACGGCGTCG-3′ Present study 
puc2BHisR: 5′-ATTAGAATTCGCCGAGCCACGGCGTCG-3′ Present study 
puc2AF: 5′-ATTATCTAGAGAGCTCGGATCCGGAGAGGACTGACATGAACAACTCGAAGATGTGGC-3′ Present study 
puc2AMR: 5′-AAAGGATCCTTACGCGACCGGCCACGGCTCCG-3′ Present study 
puc2AR: 5′-AATTGGATCCTTATTGCGCGGCCGGAACGAACG-3′ Present study 
rpozF: 5′-ATCGCGGAAGAGACCCAGAG-3′ Present study 
rpozR: 5′-GAGCAGCGCCATCTGATCCT-3′ Present study 
DescriptionReference/source
Plasmids  
 pMD18-T AmpR, 2.8 kb PCR product cloning vector TaKaRa 
 pMD18-T-puc1B AmpR, pMD18-T with 172 bp PCR product containing puc1B Present study 
 pMD18-T-puc1A AmpR, pMD18-T with 182 bp PCR product containing puc1A Present study 
 pMD18-T-puc2B AmpR, pMD18-T with 179 bp PCR product containing puc2B Present study 
 pMD18-T-puc2BHis AmpR, pMD18-T with 176 bp puc2B fragment no termination codon Present study 
 pMD18-T-puc2AM AmpR, pMD18-T with 208 bp PCR product containing puc2AM Present study 
 pMD18-T-puc2A AmpR, pMD18-T with 815 bp PCR product containing puc2A Present study 
 pRKpucC, pRK415 containing the puc promoter and pucC of R. sphaeroides, TcR Chen laboratory 
 pRK10HispucC, pRKpucC containing 10His tag, TcR Chen laboratory 
 pRK1B1AC, pRKpucC containing the fragment of puc1B and puc1A, TcR Present study 
 pRK1B2AMC, pRKpucC containing the fragment of puc1B and puc2AM, TcR Present study 
 pRK2B1AC, pRKpucC containing the fragment of puc2B and puc1A, TcR Present study 
 pRK1B2AC, pRKpucC containing the fragment of puc1B and puc2A, TcR Present study 
 pRK2B2AMC, pRKpucC containing the fragment of puc2B and puc2AM, TcR Present study 
 pRK2B2AC, pRKpucC containing the fragment of puc2B and puc2A, TcR Present study 
 pRK2BHis2AMC, pRKpucC containing the fragment of puc2BHis and puc2AM, TcR Present study 
 pRK2BHis2AC, pRKpucC containing the fragment of puc2BHis and puc2A, TcR Present study 
Bacterial strains  
R. sphaeroides W1 (wild-type strain) Chinese Collection of Micro-organisms 
R. sphaeroides DD13, genomic deletion of puc1BAC, pufBALMX, insertion of SmR Professor C. Neil Hunter (Department of Molecular Biology, University of Sheffield, Sheffield, U.K.) 
 DD13R, genomic deletion of puc1BAC, puc2BA, pufBALMX, insertion of SmR and AmpR Chen laboratory 
Transconjugant strains  
 DD13R (pRK415), DD13R containing pRK415 Present study 
 DD13R (pRKpuc1B1AC), DD13R containing pRK1B1APresent study 
 DD13R (pRK1B2AMC), DD13R containing pRK1B2AMC Present study 
 DD13R (pRK2B1AC), DD13R containing pRK2B1AC Present study 
 DD13R (pRK1B2AC), DD13R containing pRK1B2AC Present study 
 DD13 (pRKpucC), DD13 containing pRKpucC Present study 
 DD13R (pRK2B2AMC), DD13R containing pRK2B2AMC Present study 
 DD13R (pRK2B2AC), DD13R containing pRK2B2AC Present study 
 DD13R (pRK2BHis2AMC), DD13R containing pRK2BHis2AMC Present study 
 DD13R (pRK2BHis2AC), DD13R containing pRK2BHis2AC Present study 
Escherichia coli strains  
 JM109 recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 Δ(lac-proAB) TaKaRa 
 S17-1 TpR SmRhsdR pro recA RP4-2-Tc::Mu-Km::Tn7 in chromosome TaKaRa 
Primer sequences  
puc1BF: 5′-AAGAGCTCCATATGACTGACGATCTGAACAAAG-3′ Present study 
puc1BR: 5′-TTTCTAGATCAGCCGAGCCACGGGGTCGCAGCGGCG-3′ Present study 
puc1AF: 5′-TTTCTAGACATGACCAACGGCAAAATCTGGCTCG-3′ Present study 
puc1AR: 5′-AAGGATCCTTACTCGGCCGCGACCGCAGCCGAGCCTTG-3′ Present study 
puc2BF: 5′-CACAGGTACCAGTTGGGAGACGACACAGAGCTCATGACCGATGATCCGAAAAAGG-3′ Present study 
puc2BR: 5′-ATTATCTAGATCAGCCGAGCCACGGCGTCG-3′ Present study 
puc2BHisR: 5′-ATTAGAATTCGCCGAGCCACGGCGTCG-3′ Present study 
puc2AF: 5′-ATTATCTAGAGAGCTCGGATCCGGAGAGGACTGACATGAACAACTCGAAGATGTGGC-3′ Present study 
puc2AMR: 5′-AAAGGATCCTTACGCGACCGGCCACGGCTCCG-3′ Present study 
puc2AR: 5′-AATTGGATCCTTATTGCGCGGCCGGAACGAACG-3′ Present study 
rpozF: 5′-ATCGCGGAAGAGACCCAGAG-3′ Present study 
rpozR: 5′-GAGCAGCGCCATCTGATCCT-3′ Present study 

DNA preparation and PCR amplification

Cultures of R. sphaeroides W1 and DD13 strains were grown until reaching the exponential growth phase, harvested and chromosomal DNA was extracted as described previously [17].

The puc1BA and puc2BA genes from R. sphaeroides were isolated using PCR, cloned into the pMD18-T plasmid and DNA sequenced to check their authenticity.

Construction of plasmids

The mobilizable plasmids used in the present study were based on pRKpucC (see Figure 1), which was used to express puc1BA and puc2BA from R. sphaeroides. The plasmid pRKpucC (TcR) was derived from pRK415 encompassing the puc promoter and pucC from R. sphaeroides. Standard procedures were used for plasmid isolation, restriction endonuclease digestion, ligation and other molecular biology techniques [18].

The structure of the light-harvesting complex expression vectors pRKpucC derived from pRK415 (A) and pRK10HispucC derived from pRKpucC (B)

Figure 1
The structure of the light-harvesting complex expression vectors pRKpucC derived from pRK415 (A) and pRK10HispucC derived from pRKpucC (B)
Figure 1
The structure of the light-harvesting complex expression vectors pRKpucC derived from pRK415 (A) and pRK10HispucC derived from pRKpucC (B)

The puc1B fragments (containing engineered SacI–XbaI ends) and puc1A fragments (containing engineered XbaI–BamHI ends) from R. sphaeroides were cloned into the SacI–XbaI and XbaI–BamHI sites of pRKpucC to produce pRKpuc1B1AC. The puc2B fragments (containing engineered SacI–XbaI ends) and puc2A fragments (containing engineered XbaI–BamHI ends) from R. sphaeroides were cloned into the SacI–XbaI and XbaI–BamHI sites of pRKpucC to produce pRKpuc2B2AC.

A series of expression plasmids that encompass chimaeric structural genes from the two operons were constructed. The puc2B fragments from R. sphaeroides were cloned into the SacI–XbaI sites of pRKpuc1B1AC in place of the puc1B gene to produce pRK2B1AC, and the puc2A fragments from R. sphaeroides were cloned into the XbaI–BamHI sites of pRKpuc1B1AC in place of the puc1A gene to produce pRK1B2AC, and the resulting constructs were confirmed by DNA sequencing.

To purify the LHII complexes, plasmids to express His-tagged LHII complexes were constructed. The puc2B fragments without termination codons (containing engineered SacI–EcoRI ends) and puc2AM or puc2A fragments (containing engineered XbaI–BamHI ends) from R. sphaeroides were cloned into the SacI–EcoRI and XbaI–BamHI sites of pRK10HispucC to produce pRKpuc2B10His2AMC and pRKpuc2B10His2AC respectively [19]. The constructs generated are listed in Table 1.

Conjugation technique

The constructs were introduced into R. sphaeroides by conjugative transfer. The mobilizable plasmids to be introduced into R. sphaeroides were first transformed into E. coli strain S17-1 and matings were then performed as described by Fowler et al. [20]. Transconjugants were grown aerobically in the dark on plates containing medium M22+ supplemented with appropriate antibiotics [21].

RNA extraction and semi-quantitative RT-PCR (reverse transcription-PCR)

In order to investigate the mRNA transcript levels of puc1BA and puc2BA in the strains, semi-quantitative RT-PCR was performed. R. sphaeroides W1, DD13 and DD13R strains with expression plasmids were grown aerobically or semi-aerobically in the dark and collected. Total RNA was extracted using the RNA Isolation System (Promega) following the manufacturer's instructions. Samples were treated with DNase I (Promega) to remove contaminating genomic DNA, and the absence of genomic DNA contamination was checked by PCR amplification of RNA samples.

The RNA samples were used for semi-quantitative RT-PCR amplification using an RT-PCR kit (Promega), with 25 ng/ml RNA template and 10 pmol of each primer used per reaction. The reaction products were separated and compared by agarose-gel electrophoresis and ethidium bromide staining. The band intensities were quantified using a CCD camera on the GelDoc-It Imaging Station (UVP). The intensities of puc1- and puc2-specific PCR products were normalized to the intensity of the rpoZ-specific PCR product. The primers used are listed in Table 1.

Preparation of intracytoplasmic membranes

Following conjugative transfer, antibiotic-resistant strains were further screened for the presence or absence of light-harvesting complexes by absorbance spectroscopy.

R. sphaeroides strains were grown under semi-aerobic or aerobic conditions in the dark at 34 °C. Cultures were centrifuged at 180000 g for 20 min, washed and resuspended in 50 mM Tris/HCl (pH 8.0) containing 2.5 mM magnesium acetate and 1 mM PMSF. Cells were disrupted using a French pressure cell, followed by centrifugation at 2000 g for 20 min. The intracytoplasmic membranes were purified by harvesting membranes from the interface of sucrose-step density gradients [15%/40% (w/w)] after centrifugation at 27000 rev./min for 8 h (Beckman Ti45 rotor).

Measurement of LHII and protein concentrations

The amount of LHII complex present in the membranes was measured as described previously by Meinhardt et al. [22] and the protein concentrations of the membranes was determined as described previously by Markwell et al. [23]. The relative values of LHII content per membrane protein in each sample were calculated.

Steady-state absorbance spectroscopy

The absorbance spectroscopy of the transconjugant strains was performed at wavelengths of 700–900 nm at room temperature (23°C) using a Lambda-2 spectrometer (PerkinElmer), with the DD13R (pRK415) strain used as a control. Intracytoplasmic membranes were resuspended in 10 mM Mops and 50 mM KCl (pH 7.2). Absorbance spectra in the visible/near IR spectral region were recorded at room temperature on a Lambda-2 spectrometer (PerkinElmer), with intracytoplasmic membranes extracted from DD13R (pRK415) strain used as a control [24].

Purification of LHII complexes and SDS/PAGE

The intracytoplasmic membranes from DD13R (pRK2B10His-2AM) and DD13R (pRK2B10His2A) strains were used for the purification of LHII complexes by HiTrap™ chelating HP columns (GE Lifesciences).

The intracytoplasmic membranes were isolated as described above and dissolved in a binding buffer containing 20 mM Tris/HCl (pH 7.9), 150 mM NaCl, 5 mM imidazole and 0.1% LDAO (N,N-dimethyldodecylamine-N-oxide) (1 ml/min), and washed with 10 column vol. of 20 mM Tris/HCl (pH 7.9), 150 mM NaCl, 10 mM imidazole and 0.1% LDAO (3 ml/min). The LHII complexes were eluted using a buffer containing 20 mM Tris/HCl (pH 7.9), 150 mM NaCl, 500 mM imidazole and 0.1% LDAO at 0.1 ml/min.

SDS/PAGE was then performed using minigels (16% gels), which were then stained with Coomassie Brilliant Blue R250.

RESULTS

Analysis of mutants by absorption spectroscopy

Except for the DD13, DD13R (pRK415) and DD13 (pRKpucC) (aerobic) strains, the strains showed the typical absorption spectrum of the LHII complex (results not shown).

All of the mutants and the wild-type strains were grown semi-aerobically or aerobically in the dark, and intracytoplasmic membranes were isolated on a discontinuous sucrose density gradient. The membranes of the various strains were extracted for further membrane spectroscopy analysis.

The distinctive wild-type absorption spectrum of the W1 strain (Figure 2a) contains three easily distinguishable peaks in the near-IR region; the peaks at 800 and 850 nm are attributable to the LHII complex, and the absorbance at approx. 875 nm, which appears as a shoulder on the 850 nm peak, arises from the LHI complex [25]. Absorption spectra of the membranes from DD13 and DD13 (pRKpucC) (aerobic) strains show that they lack the two peaks of the LHII complex.

Room temperature absorbance spectra of membranes from various R. sphaeroides strains

Figure 2
Room temperature absorbance spectra of membranes from various R. sphaeroides strains

Spectra have been scaled to reflect the levels of the LHII complex on the basis of the amount of LHII complex per mg of total membrane protein. (a) Strain W1 (λmax=800 and 850 nm); (b) DD13R (pRK1B1AC) strain (λmax=800 and 850 nm); (c) DD13R (pRK1B2AMC) strain (λmax=801 and 846 nm); (d) DD13R (pRK2B1AC) strain (λmax=801 and 849 nm); (e) DD13R (pRK1B2AC) strain (λmax=801 and 846 nm); (f) DD13 (pRKpucC) strain (λmax=801 and 846 nm); (g) DD13R (pRK2B2AMC) strain (λmax=801 and 846 nm); (h) DD13R (pRK2B2AC) strain (λmax=801 and 846 nm); (i) DD13 (pRKpucC) (aerobic) strain (no LHII absorbance peak); (j) DD13R (pRKpucC) strain (no LHII absorbance peak); (k) DD13R (pRK415) strain (no LHII absorbance peak).

Figure 2
Room temperature absorbance spectra of membranes from various R. sphaeroides strains

Spectra have been scaled to reflect the levels of the LHII complex on the basis of the amount of LHII complex per mg of total membrane protein. (a) Strain W1 (λmax=800 and 850 nm); (b) DD13R (pRK1B1AC) strain (λmax=800 and 850 nm); (c) DD13R (pRK1B2AMC) strain (λmax=801 and 846 nm); (d) DD13R (pRK2B1AC) strain (λmax=801 and 849 nm); (e) DD13R (pRK1B2AC) strain (λmax=801 and 846 nm); (f) DD13 (pRKpucC) strain (λmax=801 and 846 nm); (g) DD13R (pRK2B2AMC) strain (λmax=801 and 846 nm); (h) DD13R (pRK2B2AC) strain (λmax=801 and 846 nm); (i) DD13 (pRKpucC) (aerobic) strain (no LHII absorbance peak); (j) DD13R (pRKpucC) strain (no LHII absorbance peak); (k) DD13R (pRK415) strain (no LHII absorbance peak).

The room temperature absorbance spectra of membranes prepared from other transconjugant strains are shown in Figure 2, and they clearly demonstrate the presence of appreciable amounts of the LHII complex in these strains.

The spectral profile of the membrane from the DD13R (pRK1B1AC) strain displayed typical LHII absorption peaks (λmax=800 and 850 nm; Figure 2b). The LHII complex of other transconjugant strains showed altered absorbance properties, with a blue shift in LHII absorbance; the absorption spectra of the DD13R (pRK1B2AMC) and DD13R (pRK2B2AC) strains contained LHII absorption peaks at approx. 801 and 846 nm (Figures 2c and 2h), and the LHII complex from the puc2BA gene pair showed blue shift in LHII absorbance. The absorption spectra of the membrane from DD13R (pRK2B10His2AM) and DD13R (pRK2B10His2A) strains were similar to those of the membrane from DD13R (pRK2B2AM) and DD13R (pRK2B2A) strains (results not shown).

The spectra are scaled to reflect the level of LHII complex proportional to the amount of cellular membrane protein as quantified by total membrane protein, so it is clear that none of the transconjugants give rise to as much LHII complex as the R. sphaeroides W1 strain does, and that the LHII complex arising from the puc2BA gene pair is present at a comparatively low level.

Transcript analysis and LHII assembly of strains

In order to investigate whether these genes were transcribed in the strains examined, mRNA transcript levels of puc1BA and puc2BA were examined by semi-quantitative RT-PCR. Total RNA was prepared from semi-aerobic or aerobic cultures.

Semi-quantitative RT-PCR analysis does show that the transcripts were present in R. sphaeroides W1 and DD13 strains cultured under semi-aerobic dark conditions, and not present in the R. sphaeroides W1 and DD13 strains cultured under aerobic dark conditions (Figure 3).

Gene expression in R. sphaeroides W1, DD13 and DD13R strains with various plasmids was quantified by semi-quantitative RT-PCR

Figure 3
Gene expression in R. sphaeroides W1, DD13 and DD13R strains with various plasmids was quantified by semi-quantitative RT-PCR

The gels used for quantification are shown. (A) Semi-quantitative RT-PCR using the R. sphaeroides W1 strain. (B) Semi-quantitative RT-PCR using the DD13 and DD13R strains expressing various plasmids.

Figure 3
Gene expression in R. sphaeroides W1, DD13 and DD13R strains with various plasmids was quantified by semi-quantitative RT-PCR

The gels used for quantification are shown. (A) Semi-quantitative RT-PCR using the R. sphaeroides W1 strain. (B) Semi-quantitative RT-PCR using the DD13 and DD13R strains expressing various plasmids.

The puc1BA and puc2BA genes in the expression vectors were also transcribed in the transconjugant strains under semi-aerobic dark conditions. The puc2BA genes integrated into the genome were not transcribed in DD13 and DD13 (pRKpucC) strains under aerobic dark conditions (Figure 3B).

Semi-quantitative RT-PCR revealed that the mRNA levels of puc1B and puc1A were higher than those of puc2B and puc2A in the R. sphaeroides W1 strain, but there was no apparent difference in mRNA levels between puc1BA and puc2BA transcribed from the expression vectors (Figure 3B).

LHII complexes were not detected in the DD13 and DD13 (pRKpucC) (aerobic) strains. The amount of the LHII complex revealed the synthesis and assembly of LHII complex in the other strains. In agreement with spectroscopic and transcripts analysis, the LHII genes are transcribed in the mutants in which LHII complexes are present, as spectroscopic analysis has shown that the mutants contain LHII complexes. But the amounts of LHII complex detected in these strains are different (see Table 2); the level of the LHII complex detected in the R. sphaeroides W1 strain (18.6 nmol/mg of protein) is relatively high compared with the other strains, and the amount of LHII complex in the DD13R (pRK2B2AC) strain is the lowest level which was detected in the present study (4.1 nmol/mg of protein). Thus it is clear that LHII complexes arising from the puc2BA gene are present at a comparatively low level.

Table 2
Light-harvesting complex LHII in various R. sphaeroides strains

Cells were grown under aerobic conditions, then semi-aerobically in the dark. Results are means for three independent experiments, and the SD was within ±8%. ND, not detectable.

R. sphaeroides strainAmount of LHII complex (nmol/mg of protein)
W1 18.6 
DD13R (pRK1B1AC14.3 
DD13R (pRK1B2AMC8.1 
DD13R (pRK2B1AC7.6 
DD13R (pRK1B2AC6.8 
DD13 (pRKpucC5.0 
DD13R (pRK2B2AMC4.3 
DD13R (pRK2B2AC4.1 
DD13 (pRKpucC) (aerobic) ND 
DD13R (pRK415) ND 
DD13 ND 
R. sphaeroides strainAmount of LHII complex (nmol/mg of protein)
W1 18.6 
DD13R (pRK1B1AC14.3 
DD13R (pRK1B2AMC8.1 
DD13R (pRK2B1AC7.6 
DD13R (pRK1B2AC6.8 
DD13 (pRKpucC5.0 
DD13R (pRK2B2AMC4.3 
DD13R (pRK2B2AC4.1 
DD13 (pRKpucC) (aerobic) ND 
DD13R (pRK415) ND 
DD13 ND 

Purification of LHII complex from DD13R (pRK2B10His2AM) and DD13R (pRK2B10His2A) strains

SDS/PAGE revealed the presence of two bands which belong to the LHII complexes. SDS/PAGE of the LHII complex from DD13R (pRK2B10His2A) showed the presence of puc2B and puc2A-encoded polypeptides (Figure 4). The larger peptide, puc2A, migrated with an apparent molecular mass of 25.3 kDa, and the small peptide, puc2B–10His (decahistidine), migrated with an apparent molecular mass of 6.8 kDa when stained with Coomassie Brilliant Blue. Two bands of 6.8 and 5.7 kDa were assigned to the subunits puc2B–10His and puc2AM of the LHII complex from DD13R (pRK2B10His2AM), as shown by SDS/PAGE in Figure 4. Their apparent molecular masses were quite consistent with the values derived from the corresponding gene sequences.

SDS/PAGE of LHII complexes isolated from the DD13R (pRK2B10His2AM) and DD13R (pRK2B10His2A) strains

Figure 4
SDS/PAGE of LHII complexes isolated from the DD13R (pRK2B10His2AM) and DD13R (pRK2B10His2A) strains

Lane 1, molecular-mass markers (in kDa). Lane 2, LHII complexes from the DD13R (pRK2B10His2AM) strain. Lane 3, LHII complexes from the DD13R (pRK2B10His2A) strain. The polypeptides are indicated on the left-hand side of the gel.

Figure 4
SDS/PAGE of LHII complexes isolated from the DD13R (pRK2B10His2AM) and DD13R (pRK2B10His2A) strains

Lane 1, molecular-mass markers (in kDa). Lane 2, LHII complexes from the DD13R (pRK2B10His2AM) strain. Lane 3, LHII complexes from the DD13R (pRK2B10His2A) strain. The polypeptides are indicated on the left-hand side of the gel.

DISCUSSION

Through sequence analysis and structural, genetic, and biochemical characterization of the LHII complex encoded by puc2BA, we believe that puc2BA of R. sphaeroides has a normal function in its natural environment. It is clear that pucC is required for the normal function of puc2BA, as when puc2BA is transcribed in the DD13 strain under semi-aerobic dark conditions no LHII complex can be detected, as there is no pucC gene present in the in puc2BA operon. When pRKpucC was introduced into the DD13 strain, the resulting transconjugant strain DD13 (pRKpucC) synthesized the LHII complex under semi-aerobic dark conditions. However, the amount of LHII synthesized was at a comparably low level (5.0 nmol/mg of protein), so pucC does affect the post-transcriptional expression and assembly of LHII from puc2BA in membranes [5], indicating that the expression characteristics of puc2BA are similar to those of puc1BA. The absorbance spectroscopy of the LHII complex assembled from puc2BA-encoded polypeptides has blue-shift characteristics (λmax=801 and 846 nm) for the different amino acid residues [26,27].

In the present study, the transcription and translation of puc2BA has been proved to be normal, as normal LHII complexes assembled from puc2BA-encoded polypeptides were detected. Therefore the transcription of puc2BA and the assembly of the LHII complex are independent of the expression of puc1BA, and are only dependent upon the expression of pucC [28]. Zeng et al. [14] reported that puc2B-encoded polypeptides are involved in the assembly of LHII complexes, and the puc2A-encoded polypeptides do not take part in LHII assembly. A possible reason for this observation is that the normal function of puc2BA is entirely dependent upon the presence of functional pucC, which affects the assembly of LHII complexes, as no functional pucC was present at the same time in that experiment when puc2BA-encoded polypeptides were synthesized.

From the results described above, it seems clear that puc2BA does influence the the amount of LHII present in R. sphaeroides, as a 23% decrease in the level of LHII complexes in the absence of a functional puc2BA operon revealed that this operon contributes to LHII complex abundance (see Table 2). The level of LHII complex assembly from puc2BA is comparatively lower than that from puc1BA, as the amount of LHII complex synthesized in the DD13R (pRK1B1AC) strain is nearly three times the amount of LHII complex synthesized in the DD13 (pRKpucC) strain.

Semi-quantitative RT-PCR revealed that the mRNA levels of puc2B and puc2A were lower than those of puc1B and puc1A in the R. sphaeroides W1 strain. Also, there was no apparent difference in mRNA levels between puc1BA and puc2BA transcribed from the expression vectors, but the amounts of the LHII complex detected from puc2BA did not correspond to the amount of the LHII complex detected from puc1BA in the R. sphaeroides mutant strain DD13R (Table 2 and Figure 3B). The puc2BA-encoded polypeptides were not as efficient at assembling LHII complexes compared with puc1BA-encoded polypeptides, suggesting that the puc2AB-encoded polypeptides merely supplement the assembly of the LHII complex in R. sphaeroides.

Experiments aimed at determining the relationship between puc1BAC and puc2BA indicated that the chimaeric structural genes from puc1BA and puc2BA were expressed and the relevant polypeptides were involved in the assembly of LHII spectral complexes.

These results raise an obvious question: as both the puc2AM and puc2A-encoded polypeptides are involved in the assembly of the LHII complex, is the puc2A-encoded polypeptide possibly processed in response to exposure to particular conditions? The puc2A-encoded polypeptide contains two transmembrane helix domains, and perhaps the additional transmembrane helix domain is beneficial to the stability of LHII complexes in the membrane of R. sphaeroides, and the functional domain is located in the N-terminal of the polypeptide.

Abbreviations

     
  • 10His

    decahistidine

  •  
  • LDAO

    N,N-dimethyldodecylamine-N-oxide

  •  
  • LHI

    light-harvesting I

  •  
  • LHII

    light-harvesting II

  •  
  • RT-PCR

    reverse transcription-PCR

We thank Professor C. Neil Hunter (Department of Molecular Biology, University of Sheffield, Sheffield, U.K.) for the gift of the DD13 strain, and also thank our colleagues in the laboratory for their intellectual contribution.

FUNDING

This work was supported by the Ministry of Science and Technology of the People's Republic of China [grant numbers 2006AA02Z138, 2003CB716601)] (Programme ‘863’ and 973 project respectively), and by the Natural Science Foundation of the Chongqing Science and Technology Commission [grant number CSTC, 2006BA5006].

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