The role of the γ subunit in the photosystem of the lowest-energy phototrophs

Abstract

Purple phototrophic bacteria use a ‘photosystem’ consisting of light harvesting complex 1 (LH1) surrounding the reaction centre (RC) that absorbs far-red–near-infrared light and converts it to chemical energy. Blastochloris species, which harvest light >1000 nm, use bacteriochlorophyll b rather than the more common bacteriochlorophyll a as their major photopigment, and assemble LH1 with an additional polypeptide subunit, LH1γ, encoded by multiple genes. To assign a role to γ, we deleted the four encoding genes in the model Blastochloris viridis. Interestingly, growth under halogen bulbs routinely used for cultivation yielded cells displaying an absorption maximum of 825 nm, similar to that of the RC only, but growth under white light yielded cells with an absorption maximum at 972 nm. HPLC analysis of pigment composition and sucrose gradient fractionation demonstrate that the white light-grown mutant assembles RC–LH1, albeit with an absorption maximum blue-shifted by 46 nm. Wavelengths between 900–1000 nm transmit poorly through the atmosphere due to absorption by water, so our results provide an evolutionary rationale for incorporation of γ; this polypeptide red-shifts absorption of RC–LH1 to a spectral range in which photons are of lower energy but are more abundant. Finally, we transformed the mutant with plasmids encoding natural LH1γ variants and demonstrate that the polypeptide found in the wild type complex red-shifts absorption back to 1018 nm, but incorporation of a distantly related variant results in only a moderate shift. This result suggests that tuning the absorption of RC–LH1 is possible and may permit photosynthesis past its current low-energy limit.

Introduction

Chlorophototrophic organisms use chlorophyll (Chl) and/or bacteriochlorophyll (BChl) pigments to capture solar radiation and convert it to chemical energy to power cellular metabolism [1]. Oxygenic chlorophototrophs, such as plants, algae and cyanobacteria, use Chls to primarily absorb photons of visible wavelengths, which have sufficient energy to drive the thermodynamically challenging extraction of electrons from water, liberating molecular oxygen as a by-product [2]. Anoxygenic phototrophic bacteria are generally found below oxygenic organisms in water columns and microbial mats, and use BChls to capture lower-energy wavelengths in the far-red and near-infrared region of the spectrum that have not been utilised by the Chl-containing organisms above [3]. These bacteria use alternative sources of electrons than water, and thus do not generate O₂. The vast majority of anoxygenic phototrophs use BChl a to harvest in the 780–900 nm range, while a few phototrophs found within the Proteobacteria use BChl b, and harvest wavelengths greater than 1000 nm [4].

BChl b was discovered in 1963 as the sole BChl extracted from an organism tentatively identified as a species of Rhodopseudomonas; the extracted pigment displayed a Q_y maximum red-shifted by 23 nm compared with that of BChl a [5]. An additional ‘Rhodopseudomonas’ isolate containing this pigment was found to have a whole-cell absorption maximum at ∼1020 nm, 142 nm further into the near-infrared than the BChl a-containing proteobacterium Rhodospirillum rubrum to which it was compared [6]. This strain was subsequently named Rhodopseudomonas viridis due to its intense green colour [7]. Phylogenetic analysis of this strain, and closely related species synthesising BChl b, led to transferral to the novel Blastochloris genus; the isolate described above being designated Blastochloris (Blc.) viridis [8].

The reaction centre (RC), the site of charge separation that initiates photosynthetic electron transfer, is encircled by an antenna known as light-harvesting complex 1 (LH1) to form the ‘core’ RC–LH1 supercomplex, the key functional unit for phototrophy in Proteobacteria. RC–LH1 complexes contain five universal components: the L, M, and H subunits of the RC, and the α and β polypeptides of LH1. Most proteobacterial RCs, including that of Blc. viridis, have a bound cytochrome subunit, C, containing four haem cofactors. The RC from Blc. viridis was the first membrane protein to have its structure solved, earning the 1988 Nobel Prize in Chemistry [9,10]. Some LH1 antennas also contain additional subunits; the Rhodopseudomonas palustris LH1 ring contains a single transmembrane helix known as protein W [11,12], LH1 from Rhodobacter spp. contains a PufX polypeptide [13–15], and a further subgroup of these organisms additionally contain PufY [16–18]. These polypeptides create a channel in the LH1 ring allowing quinone/quinol exchange between the RC and the cytochrome bc₁ complex [19,20]. An additional LH1 subunit was also identified in Blc. viridis [21]. However, unlike those mentioned above, this LH1γ polypeptide was found to be in apparent equimolar ratio with the α and β polypeptides [22]. A recent cryo-electron microscopy structure of the RC–LH1 complex from Blc. viridis revealed the location of γ, packing between β polypeptides on the outside of the ring (Figure 1) [16]. The α : β : γ ratio was found to be 17 : 17 : 16; in this case the ‘missing’ γ subunit creates the channel for quinone diffusion. We proposed that the role of the γ subunit in this complex is to tighten packing of the BChls in LH1, increasing excitonic coupling of these pigments, which results in the extreme ‘red-shift’ of the complex past 1000 nm to the current low-energy limit for photosynthesis on Earth.

Figure 1. Cryo-EM structure of the Blc. viridis RC–LH1 complex, displaying γ subunit locations.

(A) Side-on view of the RC–LH1 complex, with the periplasmic side facing up. The individual components are indicated in text of the respective colour. The gap created by the ‘missing’ LH1γ polypeptide is at the anterior of the complex. (B) View of the RC–LH1 complex from above the periplasmic surface of the membrane, with the gap in LH1 at the bottom of the ring. (C) View as in previous with only LH1γ subunits displayed around the LH1 BChls (green). The route for diffusion of quinone/quinol (tan) is indicated by the orange arrow.

Peptide analysis of γ isolated from Blc. viridis indicated that the polypeptide is 36 amino acids in length, and variance at position 34 was detected with threonine and valine residues identified in a 2 : 1 ratio, respectively [22]. This suggested that multiple copies of LH1γ-encoding genes were present in the Blc. viridis genome. Subsequent genome sequencing has revealed that Blc. viridis has four genes encoding putative γ polypeptides, three of which are clustered and share high sequence identity (LH1γ_1–3), and the fourth being more divergent (LH1γ₄) [23,24] (Figure 2).

Figure 2. Amino acid sequence alignment of translated LH1γ-encoding genes from Blc. viridis.

Genetic loci for each ORF are listed in parentheses. Full-length sequence comparisons between clustered LH1γ polypeptides 1–3 (blue box), and with the additional, divergent LH1γ₄ (red box); identical, highly similar and similar amino acids are indicated by asterisks, colons, and periods, respectively. The sequences of the polypeptides found in the purified complex, identified by Edman degradation [22], and those resolved in the cryo-EM structure [16], are indicated by light and heavy underlining, respectively.

In the present study, we constructed mutants of Blc. viridis that do not produce LH1γ, permitting the confirmation of the role of this unusual core complex component. Our results indicate that the loss of γ results in large blue-shifts in the absorption maxima of whole cells and of the isolated RC–LH1 complex to a region of the solar spectrum within which photons poorly transmit through the atmosphere, providing an evolutionary rationale for the recruitment of this additional subunit into the bacterial photosystem.

Results

Deletion of LH1γ-encoding genes prevents absorption of light >1000 nm

To determine the role of the LH1γ polypeptides in the Blc. viridis RC–LH1, the open reading frames encoding LH1γ₁, LH1γ₂, and LH1γ₃, which are clustered together in the genome, were replaced with a spectinomycin resistance cassette, generating the mutant strain ΔLH1γ_1–3 (Figure 3A). Subsequently, the more divergent paralog LH1γ₄ was replaced in the ΔLH1γ_1–3 background with an ampicillin/carbenicillin resistance cassette, generating strain ΔLH1γ_1–4 lacking all copies of LH1γ-encoding genes (Figure 3B). The presence of LH1γ₄ has not been detected in the RC–LH1 of Blc. viridis, thus was not removed from the genome of the WT strain.

Figure 3. Replacement of LH1γ-encoding genes.

Cartoon representations of the arrangement of (A) LH1γ_1–3, and (B) LH1γ₄-encoding genes (left), and their replacement with antibiotic resistance cassettes. Arrows represent binding sites of primers used for screening via colony PCR, scale bars are shown, and resulting agarose gels confirming replacement are shown (right).

BChl-dependent anoxygenic phototrophs have classically been cultured with illumination provided by incandescent bulbs, which emit a broad spectrum of light from the visible deep into the infrared [26]. Since incandescent light bulbs have been banned in many parts of the world (e.g. phased out in the European Union by 2012), many researchers working on these organisms switched to halogen bulbs [27,28], which have now also been banned for production but can still be sourced. Therefore, our standard laboratory conditions for culturing anoxygenic phototrophic proteobacteria is illumination at 100 µmol photons m⁻² s⁻¹ provided by halogen bulbs (see Supplementary Figure S1 for bulb emission spectra). The ΔLH1γ_1–3 and ΔLH1γ_1–4 mutants, along with the WT, were cultured under these conditions, and their whole-cell absorption spectra were measured (Figure 4). The WT displayed the characteristic absorption band with a maximum at 1018 nm. Interestingly, the spectra obtained for the ΔLH1γ_1–3 and ΔLH1γ_1–4 strains displayed a prominent absorption peak at 825 nm, and a small absorption feature at ∼970 nm, consistently observed through multiple passages. This absorption profile is similar to that of the purified RC from Blc. viridis [29], and to membranes isolated from a mutant of BChl a-synthesising Rhodobacter sphaeroides containing RCs but lacking any antenna complexes, albeit with absorption maxima blue-shifted from those of the BChl b-containing mutants described here [30]. In the context of these studies, our results indicate that mutants ΔLH1γ_1–3 and ΔLH1γ_1–4 are unable to assemble LH1 in the absence of LH1γ polypeptides, when grown under our standard laboratory conditions. This suggested that loss of LH1γ may severely affect the stability of the LH1 ring, or prevents its assembly altogether.

Figure 4. Whole-cell absorption spectra of Blc. viridis strains.

Spectra of cultures of WT (green traces), ΔLH1γ1–3 (orange traces), and ΔLH1γ1–4 (black traces), grown under halogen light (solid lines) or white light (dashed lines) were recorded. Major absorption bands in the near-IR region of the spectrum are indicated.

The absorption features in the 400–550 nm range of the whole-cell spectra of the ΔLH1γ_1–3 and ΔLH1γ_1–4 mutants grown under halogen light indicate that the cells accumulate carotenoids, accessory pigments that absorb in this range and also play roles in pigment–protein complex stability and quenching of triplet excited states of (B)Chls [31]. The ΔLH1γ_1–3 and ΔLH1γ_1–4 mutants, along with the WT, were cultured under white light from fluorescent tubes in order to provide carotenoid-specific wavelengths that would not be captured by the BChls in the RC. Under these conditions the strains were phototrophically viable and the whole-cell absorption spectra were measured (Figure 4). As under halogen light, the WT displayed characteristic absorption with a maximum at 1018 nm. Surprisingly, ΔLH1γ_1–3 and ΔLH1γ_1–4 both displayed spectra with absorption maxima at 972 nm, 46 nm blue-shifted with respect to the WT maximum. These mutant spectra displayed an absorption profile more similar to the WT and unlike those of the RC alone, suggesting that these strains assemble the LH1 ring around the RC, albeit without LH1γ. Interestingly, strain ΔLH1γ_1–3, which still contains LH1γ₄, displays the same absorption spectra as the mutant lacking all encoding genes; the ΔLH1γ_1–4 strain completely lacking LH1γ was subsequently used for all experiments.

LH1 assembles around the RC without LH1γ only under white light

To analyse the pigment–protein complexes assembled in the described strains, they were cultured under halogen light and white light, the membranes from these cells were solubilised with detergent, and the resulting membrane complexes were subjected to rate-zonal centrifugation on continuous sucrose density gradients (Figure 5). The WT grown under both halogen light and white light accumulates a sole pigmented complex, each displaying identical densities and absorption spectra. The mutant lacking LH1γ grown under halogen light accumulates a single complex at greatly reduced density compared with that of the WT; the absorption spectrum of this band displays the characteristic profile of an isolated BChl b-containing RC. When this mutant is grown under white light, RCs can also be identified in the sucrose gradients, but a second pigmented complex, with a density slightly less than that of the WT RC–LH1, is also present as the major band. The absorption spectrum of this band displays a maximum at 965 nm, slightly blue shifted from its whole-cell absorbance maximum, as is common for isolated complexes.

Figure 5. Sucrose density gradients of solubilised membranes from WT and LH1γ-lacking strains of Blc. viridis.

Absorption spectra of relevant isolated bands are indicated by dashed lines, and the presumed structures of these complexes are shown to the right of their respective spectra for illustrative purposes. HL, halogen light; WL, white light.

The major pigmented band from each of the gradients was isolated and buffer-exchanged in a spin concentrator to remove sucrose. The complexes were denatured in SDS sample buffer and the subunits separated via Tris–tricine gel electrophoresis (Supplementary Figure S2). The stained gel indicates that the major complex from the LH1γ-lacking mutant grown under halogen light does not contain any LH1 components, including LH1β and LH1α. However, the major complex isolated from this strain grown under white light contains LH1β and LH1α, but as expected lacks LH1γ, confirming that this blue-shifted complex is an intact RC–LH1.

Loss of LH1γ slows the phototrophic growth rate under halogen light

To assess the effect of deletion of the genes encoding LH1γ, the WT and ΔLH1γ_1–4 strains were cultured under white light conditions to mid exponential phase, standardised by OD₇₀₀ and used to inoculate biological triplicates in fresh medium incubated under halogen light and white light. When grown under halogen light, the doubling time of the mutant was almost twice that of the WT (Table 1). However, the growth rates measured under white light, when both strains assemble intact RC–LH1, were comparable, albeit much slower than under halogen light, and each strain displayed inconsistent growth across the triplicate cultures.

Table 1. Phototrophic growth rates of Blc. viridis strains under different light regimes.

Strain	Doubling time (h)
	Halogen light	White light
WT	12.8 ± 0.38	94.8 ± 18.3
ΔLH1γ1–4	25.3 ± 0.63	90.1 ± 20.4

Doubling times were calculated from biological triplicates under each condition.

Pigment analysis of the LH1γ mutant

The RCs of purple bacteria assemble with four BChls, two bacteriopheophytin (BPhe) molecules (demetallated analogues of their parent BChl, which are also the primary breakdown products of BChls) and a ‘kinked’ cis-carotenoid [2,32]. The 17 LH1 subunits of the Blc. viridis core complex (16 αβγ trimers and an αβ pair) each co-ordinate two BChl b pigments and a linear ‘all-trans’ carotenoid [16], meaning that the total pigment content of its RC–LH1 is 38 BChl b, 2 BPhe b, and 18 carotenoids (Supplementary Figure S3). The RC of Blc. viridis is known to contain 15-cis-1,2-dihydroneurosporene, but the cells also accumulate a range of neurosporene- and lycopene-type carotenoids [10,33]. To analyse the pigment composition of WT and ΔLH1γ_1–4 cultured under both light regimes, biological triplicates of strains grown to late exponential phase were collected, standardised by cell number, and pellets of these were subjected to pigment extraction with an excess of organic solvent. The standardised extracts were subjected to analysis by HPLC. Representative traces from one of each triplicate sample, normalised to major peak height for clarity, demonstrating separation of BChl b and BPhe b are shown in Figure 6A, and those demonstrating the separation of carotenoid species are shown in Figure 6B. Figure 6C shows the mean contents of each pigment calculated from the areas under HPLC peaks for all replicates, relative to those found in the WT under our standard growth conditions (hereafter the ‘control’ sample) where the total for each in the control is arbitrarily set to 100% for ease of comparison.

Figure 6. HPLC analysis of pigments accumulated by described strains under halogen and white light.

Representative traces of (A) BChl and BPhe separation, monitored at 367 nm, and (B) carotenoid species separation, monitored at 470 nm. Traces are normalised to major peak height for clarity. (C) The cellular content of each pigment from three biological replicates, relative to those found in the WT grown under standard laboratory conditions (100 µmol photons m⁻² s⁻¹ provided by halogen bulbs). Standard deviations for each pigment are shown by the error bars. 15-cis-DHN; 15-cis-1,2-dihydroneurosporene, DHN, 1,2-dihydroneurosporene; DHL; 1,2-dihydrolycopene, N; neurosporene, DH-3,4-DDHL, 1,2-dihydro-3,4-didehydrolycopene, L; lycopene.

When grown under white light, the WT accumulates a very similar amount of BChl b to when grown under halogen light (1.3% less), but 45.4% more BPhe b (Figure 6C); the lack of an RC band in the sucrose density gradient from this condition (see Figure 5) indicates that there may be greater turnover of BChl b when the cells are grown under white light. When the ΔLH1γ_1–4 strain is grown under halogen light it displays a considerably higher BPhe:BChl ratio than when grown under white light, and when compared with the WT grown under either condition (Figure 6A,C), this equates to 22.9% of the BChl b content and 369.3% of the BPhe b content of the control (Figure 6C). This is unsurprising since the mutant grown under halogen light solely assembles RCs without LH1 (see Figure 5). When grown under white light, ΔLH1γ_1–4 accumulates 8.2% more BChl b but more than three times the BPhe b content of the control, and 9% more BChl b and 2.15 times more BPhe b than the WT under white light; despite primarily assembling RC–LH1 under this condition, some RCs lacking the antenna are found in the sucrose gradient (see Figure 5), which may explain this increase in the BPhe b content.

The contents of each carotenoid species differ considerably in both strains from those in the control. Using 15-cis-1,2-dihydroneurosporene as a marker for the RC, the mutant displays increases of 82.4% and 155.6% in the content of this carotenoid under halogen and white light, respectively, compared with the control, which correlates with the presence of RCs in their sucrose gradients (see Figure 5). The WT grown under white light accumulates 29.7% less of this carotenoid than when grown under halogen light, despite displaying a large amount of variance. This implies that the overall cellular RC–LH1 content may be lower than the control, perhaps as a result of a smaller surface area of internal membrane in which to house it. The carotenoid 1,2-dihydro-3,4-didehydrolycopene accumulates in both strains when grown under white light, the WT displaying 227.4% of the control content, and the mutant having 2.5 times that of the control and 6.4 times more than when it is grown under halogen light. The data shown in Figure 6C indicates that the synthesis of Blc. viridis carotenoids, and perhaps the regulation of this process, is dependent on the source of light used for growth. However, the slow doubling times and large variations displayed by both WT and mutant grown under white light may also play a role in the complicated carotenoid profiles of these strains.

Complementation of the mutant lacking LH1γ in trans restores absorption past 1000 nm

To determine if the addition of native Blc. viridis genes encoding LH1γ are able to restore the in vivo absorption maximum of the ΔLH1γ_1–4 mutant to that of the WT, genes encoding LH1γ₁ (which is identical to LH1γ₂, and differs by a single amino acid to LH1γ₃) and divergent LH1γ₄ (see Figure 2), were cloned into the purple bacterial expression vector pBBRBB-Ppuf_843–1200 [34], in place of DsRed. These constructs were conjugated into ΔLH1γ_1–4, and the transconjugants were cultured under halogen light and white light. The whole-cell absorption spectra of these strains were unchanged from that of the untransformed mutant strain (data not shown). These plasmids contain the strong puf promoter from the Rhodobacterales member Rhodobacter sphaeroides, which may not drive expression in a distantly related member of the Rhizobiales such as Blc. viridis [35]. To overcome this, the Rhodobacter sphaeroides promoter was replaced with the native puf promoter, which has been shown to drive expression of genes in Blc. viridis from a replicating plasmid [36]. These constructs were conjugated into ΔLH1γ_1–4, and the whole cell spectra of these strains grown under halogen light were recorded (Figure 7). The transconjugant harbouring the plasmid-borne gene encoding LH1γ₁ displayed a whole-cell absorption maximum at 1018 nm, identical to that of the WT. Interestingly, the strain expressing LH1γ₄ displayed an absorption maximum red-shifted from that of the mutant lacking LH1γ, absorbing maximally at 1003 nm, but this maximum is 15 nm shorter than that of the WT or the mutant complemented with LH1γ₁. This indicates that the amino acid sequence of LH1γ influences the interaction with the neighbouring LH1β polypeptides and determines the extent of the red-shift in the absorption profile of RC–LH1.

Figure 7. Whole-cell absorption spectra of complemented Blc. viridis strains.

Spectra of cultures of WT (green trace), ΔLH1γ_1–4 (black trace), ΔLH1γ_1–4 + pBBRBB-Ppuf^Bv[LH1γ₁] (pink trace), and ΔLH1γ_1–4 + pBBRBB-Ppuf^Bv[LH1γ₄] (blue trace). Cultures were grown under halogen light (solid lines) or white light (dashed line). Major absorption bands in the near-IR region of the spectrum are indicated.

Discussion

The vast majority of anoxygenic phototrophs in diverse bacterial phyla discovered to date use BChl a as the RC pigment for phototrophy [37]. Only a few purple bacteria use BChl b [4]. Of these, only Blastochloris spp. have orthologs of LH1γ. In each case, the common components of the photosystem are encoded by single ORFs within the photosynthesis gene cluster (PGC), which also contains genes encoding pigment biosynthesis enzymes and photosystem assembly factors [38]. However, each sequenced strain contains multiple LH1γ genes that are located distant from the PGC [24,25,39,40]. This suggests that these genes evolved separately from the other PGC genes, and that assembly of RC–LH1 without LH1γ is safeguarded against by the presence of multiple paralogs in the genome. The evolutionary rationale for this is provided by our results; our mutant strains assembling RC–LH1 lacking LH1γ absorb maximally at 972 nm, which sits in a range of the solar spectrum that poorly transmits through the atmosphere due to absorption by water vapour (Figure 8). In this range, few photons reach the surface of Earth, and those that do also poorly transmit through liquid water at the surface [41]. Therefore, it is likely that Blastochloris spp. evolved or recruited an additional LH1 subunit to influence absorption of the complex, shifting to a region of the spectrum where photons are of lower energy but are more abundant.

Figure 8. Absorption spectra of RC–LH1 complexes relative to available solar energy.

The purified complex from Rsp. rubrum containing BChl a (blue trace) is blue-shifted in comparison the BChl b-containing complexes from WT Blc. viridis (green trace) and the ΔLH1γ_1–4 mutant (black trace). The overlayed red trace displays the spectrum of solar radiation that reaches the surface of the Earth [42]. Absorption bands from O₂ and H₂O in the atmosphere are labelled, and the corresponding spectral regions from which photons poorly transmit are highlighted in grey.

LH1γ polypeptides 1 and 2 are identical, and LH1γ₃ differs by a single amino acid (see Figure 2); their encoding genes are clustered in the genome. The original proteomic analysis of Blc. viridis demonstrated that highly similar polypeptides containing threonine or valine at position 34 could be isolated from its photosystem and are present at a 2 : 1 ratio, while a polypeptide equivalent to LH1γ₄ was not detected [22]. This indicates that the genes encoding LH1γ_1–3 are transcribed together, and that LH1γ₄ is not produced when the cells are grown under full-spectrum light from incandescent bulbs. Similarly, we could not detect any phenotypic change in the cells of the ΔLH1γ_1–3 mutant when compared with the mutant lacking all γ-encoding genes under any condition tested that we could ascribe to the production of LH1γ₄. Our finding that expression of the LH1γ₄ gene from a plasmid in the ΔLH1γ_1–4 mutant leads to the cells having an absorption maximum of 1003 nm indicates that incorporation of γ₄ into RC–LH1 induces a moderate red-shift, which may provide more favourable absorption under certain environmental conditions (e.g. when in competition with other BChl b phototrophs such as Halorodospira spp. displaying similar absorption at 1018 nm, see below). It may be that incorporation of LH1γ₄ into the WT RC–LH1 can be induced by growth under specific LED regimes in the laboratory, which we intend to explore further.

We found that the ΔLH1γ_1–3 and ΔLH1γ_1–4 mutants grown under our standard laboratory conditions, with illumination provided by halogen bulbs, resulted in the assembly of RCs, but no intact RC–LH1 complexes. However, growth under white-light fluorescent tubes that would be routinely used for cultivation of oxygenic cyanobacteria permitted assembly of RC–LH1, with an absorption maximum far outside the range of emission from the light source. This appears counterintuitive, but it is possible that the emission from the halogen bulbs, which reduces sharply at ∼850 nm (see Supplementary Figure S1), provides wavelengths that efficiently excite the BChls contributing the major absorption feature of the RC at 831 nm (see RC band spectrum in Figure 5). This RC contains only a single 15-cis-1,2-dihydroneurosporene carotenoid, providing limited absorption in the visible range of the spectrum. The observation that RC–LH1 lacking γ only assembles when the mutants are grown under white light might be explained by the carotenoid content of LH1; the 17 all-trans pigments bound in the ring provide strong absorption in the 400–550 nm range, which overlaps with the narrow, intense emission band of the fluorescent tubes in this range (see Supplementary Figure S1). Taking this observation together with the very slow growth rate of WT and ΔLH1γ_1–4 under white light (see Table 1), an alternative explanation might be that Blc. viridis senses this regime as very low light and so may compensate by increasing expression of the LH1 α and β genes. A complete elucidation of this phenomenon will form the basis of a future study. Our results will be noteworthy to those studying anoxygenic phototrophy; although it is possible to purchase LED panels that mimic ‘photosynthetically active radiation’ for oxygenic phototrophs, panels emitting wavelengths to at least 1200 nm that more accurately mimic full-spectrum natural sunlight do not yet exist. As it becomes more difficult to source incandescent and halogen bulbs there may be a need for researchers to assemble broad-spectrum LED regimes themselves, to accurately study phototrophic bacteria under conditions similar to those experienced in nature.

The mutants lacking LH1γ assembling RC–LH1 in white light also accumulate RCs, as can be seen in the sucrose density gradients, while the WT does not (Figure 5). This suggests that the presence of the 16 LH1γ subunits provide a stabilising role; the interaction with the adjacent β polypeptides may fix the complex in place in addition to imparting the red-shift in absorption, and the presence of γ may orient the RC in such a position that efficient quinone/quinol shuttling can occur at the ‘missing’ 17th γ position [16]. This stabilising effect may be analogous to the role that PufX plays in the primarily dimeric Rhodobacter sphaeroides RC–LH1 [43]. Deletion of the pufX gene leads to formation of solely monomeric RC–LH1, and the recent cryo-EM structure of this complex was solved at much lower resolution than the PufX-containing dimer or monomer, attributed to PufX's role in orienting the RC within LH1 [43,18]. It is interesting that we do not observe assembly of RCs without LH1 in the WT under either white light or halogen light conditions. This could be due to the stability imparted by γ; it is likely that the formation of RC–LH1 is a highly co-ordinated process and that γ is incorporated into LH1 at the same time as the α and β polypeptides by the assembly machinery, and this may not be easily reversed once γ has stabilised the complex. Turnover and recycling of light-harvesting and RC complexes is a well-studied process; photosystem II of oxygenic phototrophs is damaged by the reaction it catalyses so is constantly reassembled [44], and the metabolically diverse purple bacteria using BChl a can assemble and disassemble RC–LH1 depending on whether conditions suit aerobic respiration [45]. However, Blastochloris spp. are unable to grow in the presence of O₂ and they generally inhabit anoxic ecological niches such as the bottom of microbial mats, where rapid turnover of the photosystem is not required [46]. Here, extremely red-shifted absorption provided by the γ-stabilised complex affords a competitive advantage over the abundant BChl a-containing phototrophs in the mat where wavelengths are highly filtered, and thus a route to survival and growth.

Halorhodospira (Hrs.) halochloris and Hrs. abdelmalekii (formerly Ectothiorhodospira halochloris and Ectothiorhodospira abdelmalekii) are additional BChl b-containing purple bacteria isolated from hypersaline and alkaline soda lakes in Wadi El Naturn, Egypt, that also display whole-cell absorption maxima at 1018 nm [47,48]. The isolated RC–LH1 complexes from these organisms were found to contain three low molecular weight components, and the LH1 absorption at 1018 nm could be reversibly blue-shifted to 964 nm when titrated with acid in the pH range of 7.5–5.7 [49]. The small polypeptide of a similar size to Blc. viridis LH1γ was isolated from the RC–LH1 complex of Hrs. halochloris and was assigned to be analogous, although its sequence was not presented in the publication [50]. The recent complete genome sequence of Hrs. halochloris has revealed than an ortholog of a Blastochloris spp. gene encoding LH1γ is absent [51]. The inability to find LH1γ orthologs in the genome of Hrs. halochloris — as well as in other non-Blastochloris BChl b-containing organisms — via BLAST search is unsurprising since the polypeptide is a small, single transmembrane helix (e.g. the sequences of the single transmembrane PufX polypeptides from Rhodobacter spp. display low sequence identity across the genus [52]). Hrs. halochloris was found to have an unusual complement of genes encoding RC–LH1 subunits [51]. This organism has 2 pufBALMC operons, and an additional two pairs of pufBA genes found elsewhere in the genome, one pair of which (pufB₃A₃) is more divergent, sharing only 41% and 42% identity with LH1β₁ and LH1α₁, respectively, which may lead to assembly of LH1 containing multiple forms of α- and/or β-polypeptides, as was found in the RC–LH1 of BChl a-containing Thiorhodovibrio strain 970 [53]. A recent study demonstrates that desalting the purified Hrs. halochloris RC–LH1 complex results in the same blue-shift described previously with a decrease in pH, which can be reversed by supplementation with salts [54]. Taking all of these results together, it is likely that Halorhodospira spp. have evolved an alternative approach to accessing photons of wavelengths >1000 nm to that taken by members of the Blastochloris genus; the red-shift displayed by these organisms appears to be strongly influenced by electrostatic charge.

This study elucidates the role of the γ polypeptide in the RC–LH1 of Blc. viridis. The presence of the highly conserved γ isoform stabilises the complex and shifts its absorption 46 nm further into the near-infrared. This extreme red-shift allows Blastochloris spp. to access the more abundant photons >1000 nm that are not absorbed by water in the atmosphere. The discovery that plasmid-borne expression of a divergent isoform of LH1γ not found in the WT complex results in only a moderate red-shift of the complex upon assembly provides a route to explore the extent to which polypeptide sequence influences the absorption shift, and whether the low energy limit of natural photosynthesis can be surpassed by rational design of the LH1γ subunit.

Materials and methods

Growth of described strains

Blc. viridis DSM 133 and mutant strains were grown phototrophically at 30°C in sodium succinate medium 27 (N27 medium) [55] under illumination (100 µmol photons m⁻² s⁻¹) provided by 100W Bellight halogen bulbs or a bank of 24W fluorescent tubes as previously described [56]. Immediately after autoclaving, bottles containing hot growth medium were tightly sealed and transferred to an anoxic chamber (Coy Laboratories) maintained with an atmosphere of 95% N₂, 2.5% H₂, 2.5% CO₂. Once inside, lids were loosened and incubated for at least 48 h prior to use to ensure complete deoxygenation. All culture transfers were performed in this chamber to prevent introduction of O₂. Where required, medium was supplemented with antibiotics at the following concentrations: kanamycin (30 µg ml⁻¹), spectinomycin (30 µg ml⁻¹), carbenicillin (100 µg ml⁻¹). E. coli strains JM109 [57] and ST18 (DSM 22074) [58] transformed with pK18mobsacB [59] and pBBRBB-derived plasmids [34] were grown in a rotary shaker at 37°C in LB medium supplemented with 30 µg ml⁻¹ kanamycin. ST18 cells were supplemented with 50 µg ml⁻¹ 5-aminolevulinic acid (ALA). All strains and plasmids used in the present study are listed in Supplementary Table S1.

Construction of deletion mutants of Blc. viridis

Blc. viridis open reading frames were replaced with genes conferring antibiotic resistance such that their expression is driven by the promoter of the targeted genes, as previously described [60]. Sequences ∼600 bp up- and downstream of genes encoding LH1γ polypeptides were amplified with relevant UpF and UpR, and DownF and DownR primer pairs, respectively; sequences of the primers used in the present study can be found in Supplementary Table S2. The aadA gene from pSRA81 conferring streptomycin/spectinomycin resistance, and the bla gene from pET3a conferring resistance to ampicillin/carbenicillin, were amplified from purified plasmids. Resulting amplicons were fused by overlap extension PCR, digested with relevant restriction enzymes, and ligated into similarly digested pK18mobsacB. The resulting plasmids were verified by DNA sequencing and conjugated into Blc. viridis via E. coli ST18. Transconjugants in which the plasmid had integrated into the genome by a single homologous recombination event were selected on N27 medium supplemented with kanamycin. A second recombination event was then promoted by sacB-mediated counterselection on N27 medium supplemented with 5% (w/v) sucrose, containing either spectinomycin or carbenicillin, but lacking kanamycin. Sucrose- and spectinomycin/carbenicillin-resistant, kanamycin-sensitive colonies had excised the allelic exchange vector through the second recombination event, and replacement of targeted open reading frames with antibiotic resistance genes was confirmed by colony PCR and sequencing using relevant CheckF and CheckR primers.

Construction of a replicating expression plasmid for Blc. Viridis

The puf promoter, found upstream of the genes encoding the majority of the LH1 and RC subunits, was amplified from Blc. viridis genomic DNA with Ppuf^BvF and Ppuf^BvR primers. The resulting amplicon was digested with XbaI and BglII, and cloned in place of the Rhodobacter sphaeroides puf promoter in pBBRBB-Ppuf_843–1200-DsRed [34], a broad-host range plasmid containing the pBBR1 origin of replication, generating pBBRBB-Ppuf^Bv.

Complementation of Blc. viridis mutants in trans

The genes encoding LH1γ₁ and LH1γ₄ were amplified from Blc. viridis genomic DNA with the relevant primer pairs, and the resulting amplicons were digested with BglII and SpeI and cloned in place of DsRed-Express2 in pBBRBB-Ppuf^Bv. The resulting plasmids were verified by DNA sequencing and conjugated into the Blc. viridis mutant lacking LH1γ from E. coli ST18. Transconjugants harbouring the plasmids were selected on N27 medium supplemented with kanamycin, and the presence of the plasmids was confirmed by colony PCR using the primers used to amplify LH1γ₁ and LH1γ₄.

Cell breakage, membrane isolation and solubilisation

All steps were carried out at 4°C under dim light. Cultures were centrifuged at 4000×g for 30 min. Cell pellets were resuspended in in 20 mM Tris, 5 mM sodium ascorbate pH 8.0 (hereafter referred to as ‘working buffer’). Ascorbate is routinely added to buffers when handling complexes containing BChl b to limit photoisomerisation and photooxidation of the pigment under oxic conditions [61]. Cells were broken by two passages through a cell disruptor (Constant Systems) at 18 000 psi. Unbroken cells were removed by centrifugation at 33 000×g for 15 min. The supernatant was centrifuged at 113 000×g for 2 h in a Beckman Type 45 Ti rotor. Pelleted membranes were resuspended in working buffer and homogenised until no aggregates remained. Resulting membrane suspensions were solubilised by the addition of n-dodecyl β-d-maltoside (β-DDM) to a final concentration of 3% (w/v) with constant gentle stirring for 1 h in the dark, followed by further centrifugation at 150 000×g for 1 h to remove insoluble material.

Isolation of RC/RC–LH1 complexes

Solubilised membranes were gently layered on top of continuous 15–25% (w/w) sucrose density gradients made up in working buffer containing 0.03% (w/v) β-DDM. Gradients were centrifuged in a Beckman SW41 Ti rotor at 90 000×g for 19 h at 4°C. Gradients were briefly illuminated while photographs were taken. Pigmented bands containing photosynthetic complexes were collected using a fixed needle and a peristaltic pump.

Absorption spectroscopy

UV/vis/near-IR absorption spectra were collected on a Cary 3500 spectrophotometer (Agilent Technologies) scanning between 300 and 1100 nm at 1 nm intervals with a 0.1 s integration time.

Measurement of lightbulb emission

Emission spectra from halogen bulbs and fluorescent tubes were measured on a FlouroMax-4 spectrofluorometer (Horiba). Bulbs were directed into the open sample chamber in a dark room and emission measurements were taken between 365 and 1150 nm at 1 nm intervals with the excitation source switched off.

Pigment extraction

Bacterial cultures were washed in 50 mM Tris–HCl pH 8.0 and pelleted by centrifugation. Total pigments were extracted by adding 10 pellet volumes of 7 : 2 (v/v) acetone/methanol, immediately vortex-mixed for 30 s, and the resulting suspension incubated on ice for 15 min. Extracts were clarified by centrifugation and the supernatants were filtered through 0.22 µm PVDF membrane filters and immediately analysed. For the preparation of carotenoids, a drop of 5 M NaCl and 10 pellet volumes of hexane were added to the clarified acetone/methanol extract. The sample was mixed and the phases allowed to partition. The upper hexane phase was transferred to a glass vial, dried in a vacuum concentrator at 30°C, and reconstituted in a small volume of acetonitrile immediately prior to analysis by reversed-phase high-performance liquid chromatography (HPLC). Centrifugation steps were performed at 15 000g for 5 min at 4°C.

Pigment analysis

Extracted pigments were separated on an Agilent 1100 HPLC system maintained at 40°C with a flow rate of 1 ml min⁻¹. BChls and BPhe pigments were separated on a ThermoFisher Acclaim 120 C18 column (3 µm particle size, 120 Å pore size, 150 × 3 mm) using a 20 min isocratic gradient of 80 : 20 (v/v) methanol/acetone. Elution of BChl b and bacteriopheophytin b (BPhe b) was monitored by checking the absorbance at the Soret and Qy absorption maxima of the relevant pigments. Carotenoids were separated on a Supelco Discovery HS C18 column (5 µm particle size, 120 Å pore size, 250 × 4.6 mm) using a method modified from Magdaong et al. [62]. Pigments were eluted on a 50 min isocratic gradient of 58 : 35 : 7 (v/v/v) acetonitrile/methanol/tetrahydrofuran. Elution of carotenoid species was monitored at 470 nm and 505 nm.

Abbreviations

BChl

bacteriochlorophyll

BPhe

bacteriopheophytin

DDM

n-dodecyl β-d-maltoside

DHN

1,2-dihydroneurosporene

LH1

light harvesting complex 1

PGC

photosynthesis gene cluster

RC

reaction centre

Data Availability

All original data and bacterial strains described in this paper are available from the authors upon reasonable request. Structural and genomic data used are already published on universally accessible websites.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by grants from Wellcome Trust [ISSF 204822/Z/16/Z], the Royal Society [RGS\R2\192311], and University of Liverpool start-up funds to D.P.C. D.N. is supported by a Mahidol–Liverpool PhD Scholarship. N.M.R. is supported by a Doctoral Training Partnership from the Biotechnology and Biological Sciences Research Council (BBSRC). D.P.C. acknowledges support from BBSRC grant BB/W008076/1.

Open Access

Open access for this article was enabled by the participation of University of Liverpool in an all-inclusive Read & Publish agreement with Portland Press and the Biochemical Society under a transformative agreement with JISC.

Author Contributions

D.P.C. conceived the study and designed the experiments with significant input from D.N. and N.M.R. All authors conducted experiments and analysed the results. D.P.C wrote the manuscript with contributions from D.N. and N.M.R.

CRediT Author Contribution

Daniel Patrick Canniffe: Conceptualization, Resources, Supervision, Investigation, Writing — original draft, Project administration. Dowrung Namoon: Data curation, Formal analysis, Investigation, Writing — review and editing. Nicola Mary Rudling: Data curation, Formal analysis, Investigation, Writing — review and editing.