Introduction

Photosynthesis is the single most important process developed by nature, providing all of the biological energy needed for higher forms of life to exist, because it represents a highly efficient and productive means of converting solar energy into chemical energy.

The photosynthetic apparatus of purple photosynthetic bacteria contains three major carotenoid–bacteriochlorophyll (Bchl) protein complexes, B800-850 (LHII) and B875 (LHI), which are light-harvesting complexes, and the reaction center (RC) complex, the pigment protein complexes carry out light harvesting and primary photochemistry [1, 2]. The light harvesting complex I (LHI) absorbs maximally in the near-infrared at approximately 875 nm, LHI receives energy from the peripheral light harvesting complex (LHII), which has absorption bands at 800 and 850 nm. Both types of LH complexes are composed of two membrane spanning polypeptides, α and β, that anchor bacteriochlorophyll and carotenoids in the membrane [3, 4].

Rhodovulum (Rdv.) sulfidophilum and Rhodobacter (R.) sphaeroides belong to two different genera of photosynthetic bacteria. Rdv. sulfidophilum, in contrast to R. sphaeroides, has some different characters [5].

Compared with the LHII of nonsulfur purple bacterium R. sphaeroides, the LHII of Rdv. sulfidophilum exhibits a number of unusual characteristics: (1) the primary structure of the α-subunit of the peripheral antenna complex is unusual in having a high methionine content 4 Met vs. 1 in R. sphaeroides, a cysteine residue, and a free aspartyl carboxyl group near the C-terminus; (2) The pucBs encoded-apoprotein of Rdv. sulfidophilum exhibited 82% similarity to the pucB encoded-apoprotein of R. sphaeroides. The PucA protein of Rdv. sulfidophilum undergoes C-terminal processing to remove 11 amino acid residues, resulting in a mature protein that is two amino acids shorter than PucA of R. sphaeroides, a comparison of the Rdv. sulfidophilum pucAs encoded-apoprotein and the R. sphaeroides pucA encoded apoprotein revealed 54% similarity, furthermore, 12 amino acid residues of transmembrane helices are dissimilar. The similarity level of the pucB encoded-apoprotein between R. sphaeroides and Rdv. sulfidophilum are higher than which of the pucA encoded-apoprotein. The identity and similarity between pucBA-encoded polypeptide of R. sphaeroides and the pucBsAs-encoded polypeptide of Rdv. sulfidophilum were shown in Fig. 1 [6]; (3) the reaction center and both the core LHI and peripheral LHII complexes, are synthesized and assembled even under fully aerobic conditions in the dark [7, 8]; (4) the worse ordering was found in 2D crystal of Rdv. sulfidophilum LHII, LHII from both Rdv. sulfidophilum and R. sphaeroides is nonamer, but the better ordering was found in 2D crystal of R. sphaeroides LHII. Compared to the 2D crystals obtained from Rdv. sulfidophilum LHII, the rings in the 2D crystals from R. sphaeroides LHII are packed in a different and more compact way. Only a few β-subunits of a given ring appear to make direct contact to β-subunits of adjacent rings in the 2D crystals of Rdv. sulfidophilum LHII, while in the 2D crystal of R. sphaeroides LHII, eight out of nine β-subunits are involved in direct crystal contacts [9].

Fig. 1
figure 1

a Comparison of the pucB encoded-apoprotein between R. sphaeroides and Rdv. sulfidophilum; b Comparison of the α-subunit between pucA from R. sphaeroides and mature pucAs from Rdv. sulfidophilum. The pucB polypeptide contains transmembrane helix domain between Val 21 to Ala46, the pucA polypeptide contains transmembrane helix domain between Thr13 and Thr38. The transmembrane helices are indicated by boldface type

Full size image

It was reported by Fowler et al. that LHII genes from Rubrivivax gelatinosus, Rhodopseudomonas (Rps.) palustris and Rps. acidophila were expressed in R. sphaeroides, one LHII gene from Rps. palustris are not functional in R. sphaeroides [10], and there is no evidence for energy transfer for the heterologous Rps. acidophila LHII complex in the spheroidenone background R. sphaeroides mutant DD13 [11], though they are similar in some physiological characteristics and light harvesting complexes structure.

Barbieri et al. [12] and Katsiou et al. [13] reported that the genes encoding light-harvesting complex II of Rdv. sulfidophilum were expressed in R. capsulatus, which was not concerned with their functions of energy transfer.

Therefore, we want to know whether the heterologous pucBsAs from different genera Rdv. sulfidophilum is expressed and the normal and functional LHII is assembled in R. sphaeroides? Whether energy transfer from the heterologous LHII of Rdv. sulfidophilum to the native LHI of R. sphaeroides is possible?

we undertook experiments to investigate the function of the expressed pucBsAs from Rdv. sulfidophilum in R. sphaeroides, the LHII structural genes (pucBsAs) from Rdv. sulfidophilum and the LHI structural genes (pufBA) from R. sphaeroides were amplified by PCR, and cloned into a LH expression vector, which was then introduced into LHI, LHII minus R. sphaeroides mutants; the resulting transconjugant strains synthesized LHI, LHII complexes which were examined by using RT-PCR, absorption and fluorescence spectroscopy. The results in the present article suggest that the heterologous LHII has the normal function.

Materials and methods

Bacterial strains and growth conditions

Rhodovulum sulfidophilum (wild type, Chinese Collection of Microorganisms, Beijing, China) was cultured in malate-yeast extract medium, with the addition of 0.5 M NaCl, at 32°C [12].

R. sphaeroides W1 (wild type, Chinese Collection of Microorganisms, Beijing, China). R. sphaeroides DD13 (genomic deletion of pucBAC, pufBALMX, insertion of SmR and KmR gene, Hunter) were grown aerobically or semiaerobically under the dark conditions in M22+ [14]. When required, antibiotics were added to M22+ at the final concentrations: streptomycin 20 μg ml−1, tetracycline 2 μg ml−1. Escherichia (E.) coli JM109 and S17-1 were grown in Luria–Bertani medium (LB) at 37°C, the final concentrations of antibiotics were tetracyclin 50 μg ml−1, ampicilin 100 μg ml−1 [15].

Bacterial strains, plasmids and oligonucleotides used for PCR and RT-PCR were listed in Table 1.

Table 1 Bacterial strain, plasmids and primers
Full size table

The semiaerobic dark incubation was that the bacterium were grown in 500 ml conical flask filled with 70% volume culture at 34°C under 150 rpm.

Isolation of total chromosomal DNA from Rdv. sulfidophilum and R. sphaeroides

Cultures of R. sphaeroides W1 and Rdv. sulfidophilum from the logarithmic growth phase were centrifuged at 10,000 g, resuspended in urea buffer (42% urea (W/V), 0.3 M NaCl, 50 mM Tris–HCl, 50 mM EDTA, 1% N-Lauroylsarcosine (W/V), pH 8.0), mixed with 2 vol. of phenol saturated with Tris–HCl buffer and shaken during 2 h at room temperature. The aqueous fraction was collected, washed three times with phenol–chloroform 1:1 and then precipitated with 1 vol. isopropanol [16]. The precipitated DNA was dried, resuspended in bidistilled water and stored at −20°C.

Genes amplification and plasmids construction

The chromosomal DNAs of R. sphaeroides W1 and Rdv. sulfidophilum were for PCR amplification of genes pucBA, pufBA and pucBsAs, respectively.

The PCR amplification products were cloned into pMD18-T cloning vectors in strains E. coli JM109.

Standard procedures were used for plasmid isolation, restriction endonuclease digestion, ligation, and other molecular biological techniques [17]. Sequence analyses were performed with the DNAMAN (Lynnon Biosoft).

The mobilizable plasmids used in this study were based on pRKpucC (See Fig. 2). The plasmid pRKpucC (Tcr) is derived from pRK415 encompassing puc promoter and pucC of R. sphaeroides, which was used to express the pucBA of Rdv. sulfidophilum or R. sphaeroides and pufBA of R. sphaeroides.

Fig. 2
figure 2

The structure of LH expression vector pRKpucC (derived from pRK415)

Full size image

The pucBA fragment (containing engineered SacI–BamHI ends) of R. sphaeroides was cloned into the SacI–BamHI site of pRKpucC to produce pRKpucBAC. The pucBsAs fragment gene pair (containing similarly engineered SacI-BamHI ends) of Rdv. sulfidophilum was cloned into the SacI–BamHI site of pRKpucC to produce pRKpucBsAsC. The pufBA fragments (containing engineered SacI–SacI ends) of R. sphaeroides were cloned into the SacI–SacI site of pRKpucBsAsC and pRKpucBAC to produce pRKpufBApucBsAsC and pRKpufBApucBAC, respectively, The insert orientation of pufBA fragments were screened by PCR using the screen primer and downstream primer of pufBA(pufBAR). The resulting constructions were confirmed through DNA sequencing by Shanghai Invitrogen Biotech Co Ltd

Conjugation techniques

The constructed plasmids were mobilized into strains R. sphaeroides DD13 with the mobilizing strain E. coli S17-1 by using the biparental conjugation system [11].

To select for transconjugant strains containing constructed plasmids, cells were grown aerobically in the dark on plates of medium M22+ supplemented with appropriate antibiotics: 2 μg ml−1 of tetracycline and 20 μg ml−1 of streptomycin.

Expression analysis of transconjugant strains

These strains with special plasmids were grown aerobically under the dark conditions in M22+ for 20 h, then were grown semiaerobically under the dark condition for 6 h, and collected. Total RNA was extracted with the RNA Isolation System (Promega), according to the manufacturer’s instructions. Samples were treated with DNase I (Promega) to remove contaminating genomic DNA, absence of genomic DNA contamination was checked by PCR amplification of RNA samples. The RNA samples were used for RT-PCR amplification assay with RT-PCR kit (Promega). The quantities of RT-PCR production from pucBsAs of Rdv. sulfidophilum and pucBA of R. sphaeroides were compared by ethidium–bromide-stained agarose gel electrophoresis.

Preparation of intracytoplasmic membranes

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

Rhodobacter sphaeroides strains were grown under semiaerobic conditions in the dark at 34°C. Cultures were centrifuged, washed and resuspended in 50 mM Tris–HCl (pH 8.0) containing 2.5 mM magnesium acetate and 1 mM phenyl methyl sulfonyl fluoride (PMSF). The cells were broken by disruption in a French pressure cell, followed by centrifugation at 2,000 g for 20 min. The intracytoplasmic membranes were purified by harvesting from the interface of sucrose step gradients (15%/40%, w/w) after centrifugation in a Beckman Ti45 rotor at 180,000g for 8 h [8].

Measurement of bacteriochlorophyll and LHII

BChl were extracted by a solution of acetone–methanol (7:2), the amount of BChl was determined by measuring the absorbance at 772 nm, using the extinction coefficient ε772 = 75 mM−1 cm−1 [16].

The amount of B800–850 could be measured at an optical density at 849 nm (OD849) to OD900 (ε = 96 ± 4 mM−1 cm−1) by using 3 mol of Bchl a per mol of complex. Then, the relative values of BChl contents per membrane protein and LHII contents per membrane protein in each sample were calculated [18].

Steady-state absorbance spectroscopy

The absorbance spectroscopy of transconjugant strains were scanned from 700 to 900 nm at room temperature on a Lambda-2 spectrometer (Perkin–Elmer), the control is DD13(pRK415).

Intracytoplasmic membranes were suspended in 10 mM MOPS, 50 mM KCl (pH 7.2). Absorbance spectra in the VIS/NIR spectral region at room temperature were recorded on a Lambda-2 spectrometer [10], the control is the intracytoplasmic membranes of DD13(pRK415).

Electron transfer measurement

Fluorescence emission spectra were measured at room temperature using the LS50B spectrofluorimeter(Perkin–Elmer). Intracytoplasmic membranes from semiaerobically grown cells were diluted to an absorbance of A680 1 cm = 0.05 in 50 mM Tris, pH 8.0. The sample was excited with different wavelength and measure the fluorescence on spectrofluorimeter [19–22].

Results

Expression and absorbance spectroscopy of the transconjugant strains

The expressed transconjugant strains had the similar colors of the wild type strains of R. sphaeroides W1, which is purple brown, and DD13 is pink.

The transconjugant strains were experimented by RT-PCR amplification, the pucBsAs, pucBA and pufBA in the expression vectors were transcribed in R. sphaeroides DD13. The transcript levels were compared between pucBA and pucBsAs when RT-PCR was performed. We compared the quantity of RT-PCR production between pucBsAs of Rdv. sulfidophilum and pucBA of R. sphaeroides by ethidium–bromide-stained agarose gel electrophoresis, transcript level of pucBA was higher than transcript level of pucBsAs (data not shown).

The absorption spectrum of strain DD13(pRKpucBsAsC) showed peaks at 800 and 850 nm, typical of LHII of Rdv. sulfidophilum. It was similar to the spectrum of strain DD13(pRKpucBAC), which expresses LHII of R. sphaeroides. Likewise, the absorption spectrum of strain DD13(pRKpufBApucBsAsC) showed peaks typical of LHII of Rdv. sulfidophilum at 800 and 850 nm, with a small shoulder at 870 nm due to LHI of R. sphaeroides (data not shown). It was similar to the spectrum of strain DD13(pRKpufBApucBAC), which expresses both LHII and LHI of R. sphaeroides. These observations indicate that the LHII complex of Rdv. sulfidophilum retains its characteristics when expressed in the R. sphaeroides DD13 background (data not shown).

Absorbance spectroscopy and LHII amount of membranes

After strains spectroscopy analyzed, the membranes of strains were extracted for further membrane spectroscopy analyse. The room temperature absorbance spectra of membranes prepared from transconjugant strains are shown in Fig. 3.

Fig. 3
figure 3

The absorbance spectroscopy of the membrane of transconjugant strains. (a) DD13(pRKpucBAC); (b) DD13(pRKpucBsAsC); (c) DD13(pRKpufBApucBAC); (d) DD13(pRKpufBApucBsAsC); (e, f) DD13; (g, h) DD13(pRK415)

Full size image

The absorption spectrum of the membrane from DD13(pRKpucBsAsC) and DD13(pRKpuc-BAC) showed the typical LHII absorption peaks at 800 and 850 nm. Furthermore, the absorbance properties of the heterologously synthesized LHII complexes differed from those of R. sphaeroides LHII (λmax = 800, 850 nm). The LHII complex from the pucBsAs gene pair of Rdv. sulfidophilum showed absorbance peaks in the near infrared at 801 and 853 nm.

The spectra are scaled to reflect the level of LHII complex per amount of cellular membrane as quantified by total membrane protein, and it is clear that LHII arising from the gene pucBsAs is present at comparatively low level. The amounts of LHII complex of DD13(pRKpucBAC) and DD13(pRKpucBsAsC) were 26.5 and 18.3 nmol/mg of protein, respectively. The bacteriochlorophyll contents of membrane from DD13(pRKpucBAC) and DD13(pRKpucBsAsC) were 36.3 and 23.6 nmol Bchl/mg of protein, the bacteriochlorophyll pigments concentration of membrane from Rdv. sulfidophilum is much lower than which from R. sphaeroides, it is a stringent control of the level of bacteriochlorophyll to match the level of LHII complexes.

The membrane absorption spectrum of DD13 (pRKpufBApucBsAsC) shows the LHII absorption peaks (λmax = 801, 853 nm) of Rdv. sulfidophilum with a small LHI shoulder at the 870 nm of R. sphaeroides.

It is clear that the heterologously expressed LHII does not give rise to as much as LHII of R. sphaeroides in DD13.

Energy transfer within the LHII complexes

The value of the expression of Rdv. sulfidophilum LHII complexes depends on a demonstration that the complexes are still functional, which is that it can harvest and transfer light energy.

In order to measure the ability of the 800 nm absorbing pigments to transfer energy to the 850 nm pigments within the heterologously synthesized Rdv. sulfidophilum LHII complexes, excitation spectra in the near infrared region of the spectrum were measured. This technique measures the ability of the 800 nm absorbing pigments to transfer energy to the 850 nm pigments, from which fluorescence is detected at 900 nm.

The absorption and emission spectra suggest that the LHII complexes are able to transfer energy from their B800 to their B850 pigments. It can be seen that the 800 nm absorbing pigments within the Rdv. sulfidophilum LHII are able to elicit emission at 900 nm from the B850 band. The R. sphaeroides LHII is the same.

Figure 4 shows the near infra-red region of the fluorescence excitation spectra for membrane bound complexes from the LHII-only strains DD13(pRKpucBsAsC) and DD13(pRKpucBAC). It is showed that energy is efficiently transferred from the B800 to B850 pigments in the synthesized LHII complexes.

Fig. 4
figure 4

The near infra-red region of the fluorescence excitation spectra recorded on membranes from the following strains: DD13(pRKpucBsAsC) (solid line), DD13(pRKpucBAC) (bold line). As the wavelength of excitation was varied from 750 to 850 nm, the emission of fluorescence was 900 nm

Full size image

Energy transfer between the Rdv. sulfidophilum LHII complex and the R. sphaeroides LHI complex

The membranes used in this experiment were prepared from strain DD13(pRKpufBApucBsAsC) and DD13(pRKpufBApucBAC), the membrane of DD13(pRKpufBApucBsAsC) contains the Rdv. sulfidophilum LHII complex together with the R. sphaeroides LHI complex.

The ability of the peripheral LHII antenna to transfer energy to the LHI complex was monitored by exciting the LHII complex at various wavelengths, from 750 to 900 nm, while monitoring any fluorescence emitted from the LHI complex at 910 nm. Figure 5 shows the results of such an excitation experiment on membranes from strains containing the heterologous or native LHII and native R. sphaeroides LHI complexes. The excitation spectrum of DD13(pRKpufBApucBsAsC) is a thin solid line in Fig. 5; this profile is closely matched by the excitation spectrum of DD13(pRKpufBApucBAC) (bold line), which reflects that the Rdv. sulfidophilum LHII complex is able to transfer energy to the LHI complex of R. sphaeroides, like LHII complex of R. sphaeroides.

Fig. 5
figure 5

Fluorescence excitation spectrum (λemission = 910 nm) recorded on membranes prepared from strains DD13(pRKpufBApucBsAsC) (thin solid line) and DD13(pRKpufBApucBAC) (thick solid line)

Full size image

Discussion

Photochemical reaction, the primary important redox reactions occurs in photosynthetic bacteria, the LHII is the farthest outer component in the photosynthetic apparatus, which absorbs solar radiation to LHI.

Rhodovulum sulfidophilum and R. sphaeroides belong to two different genera, their physiological characters of light and oxygen and light harvesting complexes structure are dissimilar, and the pucAs of Rdv. sulfidophilum are evidently different from pucA encoded-apoprotein of R. sphaeroides in the polypeptide sequences, especially having dissimilar 12 amino acid residues in the transmembrane helices sequences, and LHII from Rdv. sulfidophilum is different from R. sphaeroides LHII in 2D crystal structure [9, 23–25].

Rhodovulum sulfidophilum differs from the R. sphaeroides by its missing influence of oxygen tension on the rate of synthesis of the photosynthetic apparatus, and its LHII synthesis and assembly is different from the latter. Through the biochemical, spectroscopic and functional information, we believe that the pucBsAs of Rdv. sulfidophilum is normally expressed in R. sphaeroides, and the LHII is functional in it, which can availably transfer energy in itself and to the LHI of R. sphaeroides.

These transconjugant strains have their typical absorbance spectroscopy characters. The results indicate that the pucBsAs of Rdv. sulfidophilum and pufBA of R. sphaeroides can be expressed at the same time in the R. sphaeroides background strain DD13, and have their normal functions. Within the heterologously expressed Rdv. sulfidophilum LHII in R. sphaeroides, energy can be transferred from B800 to B850, likewise, energy can be transferred from Rdv. sulfidophilum LHII to R. sphaeroides LHI.

The membrane absorption spectrum of DD13(pRKpufBApucBsAsC) shows the LHII absorption peaks at 800 and 850 nm of Rdv. sulfidophilum with a small LHI shoulder at the 870 nm. However the absorbance properties of the heterologously synthesized LHII complexes in DD13 differed from which of R. sphaeroides LHII in R. sphaeroides (λ max = 800, 850 nm), the LHII complex from the pucBsAs gene pair of Rdv. sulfidophilum showed absorbance peaks in the near infrared at 801 and 853 nm, but the Rdv. sulfidophilum LHII shows the absorption peaks at 801 and 850 nm in Rdv. sulfidophilum [6, 26]. The red shifts of absorbance of Rdv. sulfidophilum LHII in R. sphaeroides are possibly for the different pigment background of R. sphaeroides [3, 8].

Furthermore, absorbance spectra, bacteriochlorophyll contents and LHII complex contents of the membrane samples clearly showed that the levels of spectral properties and assembly of the LHII complex from Rdv. sulfidophilum are lower than which from R. sphaeroides in DD13. The possible reason is that pucBsAs gene pair of Rdv. sulfidophilum were expressed under the different cell conditions which influenced their transcription, translation and assembly.

It should be noted that the pucAs used in this study is the actual processed mature sequence of mature α-subunit of Rdv. sulfidophilum LHII, not withstanding its limitation, this study does suggest investigating the expression characters of unprocessed DNA sequence which encodes the amino acid sequence of whole ORF of Rdv. sulfidophilum pucA in R. sphaeroides in nature.

The experiment data are helpful to expressing functional membrane proteins from further evolutionary relationship and different physiological species in this system.