Aci Actinobacteria Assemble a Functional Actinorhodopsin With

bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 acI Actinobacteria Assemble a Functional Actinorhodopsin with

2 Natively-synthesized Retinal

4 Running Title: (54 characters and spaces) Actinorhodopsin and Carotenoid Synthesis in acI

6 Jeffrey R. Dwulit-Smitha,b, Joshua J. Hamiltona, David M. Stevensona, Shaomei Hea,c, Ben O.

7 Oysermand, Francisco Moya-Floresd, Daniel Amador-Nogueza, Katherine D. McMahona,d, Katrina T.

8 Foresta,b#

10 aDepartment of Bacteriology; bProgram in Biophysics; cDepartment of Geoscience, and dDepartment of Civil

11 and Environmental Engineering, University of Wisconsin-Madison, Madison, WI, USA.

12 # Address correspondence to Katrina T. Forest, 1550 Linden Drive, Madison, WI 53706, tel. 608 265-3566,

13 email [email protected].

15 KEYWORDS

16 Freshwater, photoheterotrophy, opsin, rhodopsin, xanthorhodopsin, proton pump, carotenoid, blh

17 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

18 ABSTRACT

19 Freshwater lakes harbor complex microbial communities, but these ecosystems are often dominated by acI

20 Actinobacteria from three clades (acI-A, acI-B, acI-C). Members of this cosmopolitan lineage are proposed

21 to bolster heterotrophic growth using phototrophy because their genomes encode actino-opsins (actR).

22 This model has been difficult to experimentally validate because acI are not consistently culturable. In this

23 study, using genomes from single cells and metagenomes, we provide a detailed biosynthetic route for

24 many acI-A and -B members to synthesize retinal and its carotenoid precursors. Accordingly, these acI

25 should be able to natively assemble light-driven actinorhodopsins (holo-ActR) to pump protons, in contrast

26 to acI-C members and other bacteria that encode opsins but lack retinal-production machinery. Moreover,

27 we show that all acI clades contain genes for a complex carotenoid pathway that starts with retinal

28 precursors. Transcription analysis of acI in a eutrophic lake shows that all retinal and carotenoid pathway

29 operons are transcribed and that actR is among the most highly-transcribed of all acI genes. Furthermore,

30 heterologous expression of retinal pathway genes shows that lycopene, retinal, and ActR can be made.

31 Model cells producing ActR and the key acI retinal-producing β-carotene oxygenase formed acI-holo-ActR

32 and acidified solution during illumination. Our results prove that acI containing both ActR and retinal-

33 production enzymes have the capacity to natively synthesize a green light-dependent outward proton-

34 pumping rhodopsin.

36 IMPORTANCE

37 Microbes play critical roles in determining the quality of freshwater ecosystems that are vital to human

38 civilization. Because acI Actinobacteria are ubiquitous and abundant in freshwater lakes, clarifying their

39 ecophysiology is a major step in determining the contributions that they make to nitrogen and carbon

40 cycling. Without accurate knowledge of these cycles, freshwater systems cannot be incorporated into

41 climate change models, ecosystem imbalances cannot be predicted, and policy for service disruption

42 cannot be planned. Our work fills major gaps in microbial light utilization, secondary metabolite production,

43 and energy cycling in freshwater habitats.

2 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

45 INTRODUCTION

46 Fresh water lakes contain complex and dynamic microbial communities that influence water quality

47 by mediating biogeochemical cycling. These ecosystems are often numerically dominated by three clades

48 of acI Actinobacteria (acI-A, acI-B, acI-C), ultra-microbacteria with low GC genomes that are streamlined

49 to approximately 1.2-1.5 MBp (1–5). The ubiquity and abundance of acI raise the question, what are the

50 factors that enable their success? Major proposed advantages include scavenging resources with

51 membrane transporters (3, 6), evading protist feeding by virtue of their small size (7, 8), and supplementing

52 energy needs via photoheterotrophy. Light utilization is feasible because each clade encodes actino-opsin

53 (ActR), the putative platform protein for actinorhodopsin (holo-ActR) (3, 9–11). Validating this hypothesis

54 not only requires a demonstration that acI can synthesize or acquire sufficient retinal, the chromophore

55 needed to complete holo-ActR, but also biochemical evidence that the holo-ActR is functional. Additionally,

56 the relevant genes must be shown to be expressed in the native freshwater environments where acI is

57 dominant. That these requirements are satisfied by acI is not guaranteed; Rhodoluna lacicola holo-ActR

58 was shown to be active if, and only if, exogenous retinal was added (12). Without an encoded, retinal-

59 producing enzyme or a solution for obtaining dilute environmental chromophores, it is unclear what the

60 physiological role and ecosystem implications of ActR in such organisms are. On the other hand, the Luna1

61 bacterium Candidatus Rhodoluna planktonica encodes a putative retinal-synthesizing enzyme (13). This

62 family of Actinobacteria is radically different from acI and much less abundant and prevalent in freshwater

63 lakes (2), but this organism provides motivation to determine if acI truly use rhodopsins for

64 photoheterotrophy.

65 Rhodopsins are seven-transmembrane-helix bundles that bind retinal at an internal lysine via a

66 Schiff base (14). The retinal pocket amino acid sidechains, specifically a tuning residue (15), determine the

67 maximal absorption wavelength of the holo-protein. The captured photon energy triggers the geometric

68 isomerization of retinal, which completes a transfer pathway through the interior of the bundle and thus

69 across the membrane. Most commonly, protons are shuttled out of the cytoplasm to form a gradient that

70 can be harnessed for activities like ATP synthesis (16). The retinal that ultimately enables the gradient is

71 derived from carotenoids, colorful molecules that act as photoprotectants, cofactors, pigments, and

72 membrane stabilizers throughout nature (17, 18). The initial building blocks of carotenoids are the five-

3 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

73 carbon monomers isopentenyl diphosphate and dimethylallyl diphosphate. These units can be polymerized

74 and modified to form lycopene, β-carotene, and various intermediates (19). Moreover, more complex keto-

75 carotenoid chromophores like salinixanthin and echinenone boost rhodopsin activity by resonance energy

76 transfer to retinal in rare xanthorhodopsin-like systems (20–22). Microorganisms with the machinery to

77 produce functional, chromophore-loaded rhodopsins may have a competitive advantage in their native

78 ecosystems (23, 24).

79 Here we provide experimental evidence that acI has photoheterotrophic potential. A significant

80 challenge when these studies were initiated was the inability to purely culture acI (5, 25), although it appears

81 this hurdle may soon be overcome (26). Consequently, we pursued a multidisciplinary approach that did

82 not rely on large amounts of acI biomass, but instead focused on bioinformatic, transcriptomic, and

83 biochemical data. Specifically, we characterize a biosynthetic pathway for retinal and its carotenoid

84 precursors using heterologous expression. We then demonstrate that acI-actR is highly transcribed in the

85 freshwater acI biome, Lake Mendota, and acI-ActR can function as a retinylidene holoprotein to produce a

86 light-driven proton gradient. Most importantly, the nanomachine is active in cells that produce retinal using

87 the native acI β-carotene oxygenase. Finally, we provide evidence for a complex carotenoid that may

88 screen cells from oxidative damage and/or boost the light harvesting ability of actinorhodopsin.

89 RESULTS

90 All acI-ActR contain key amino acids for function. We first determined whether acI-ActR

91 sequences were consistent with acI-holo-ActR formation. Indeed, all contain the features for proper

92 rhodopsin structure and function (Fig. 1A). Seven predicted helices (a1-7) match those found in xantho-

93 opsin, a close homolog of acI-ActR from Salinibacter ruber (20). A conserved lysine for Schiff base

94 formation and acidic residues for proton-shuttling across the retinylidene gate are present (27), and a

95 leucine is predicted to tune the absorbance of all acI-holo-ActRs to the green region (28). We also found

96 novel features. A proline in the middle of a4 differentiates ActRs from xantho-opsins, and the residue may

97 serve as a means for better phylogenetic classification (Fig. S1). Additionally, 3D structure predictions

98 suggest cysteines on a1 and a7 in acI clades A and B form a disulfide staple (Fig. 1B). acI-ActRs also

99 contain glycine near the top of a5, a required feature for binding the ketolated antenna carotenoids (21,

4 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

100 22). This glycine replaces a bulky residue, usually tryptophan, found in most rhodopsins, including the well-

101 characterized bacteriorhodopsin.

102 Seven gene products form an actinorhodopsin pathway. Retinal is the chromophore needed

103 to form holo-ActR. We find that in addition to encoding ActR, acI single-cell amplified genomes (SAGs),

104 metagenome-assembled genomes (MAGs), and complete genomes from dilution-to-extinction cultures

105 (Table S1) appear to encode genes for enzymes that produce retinal and its carotenoid precursors (Fig. 2).

106 We assembled a plausible, complete pathway with protein assignments for forming lycopene, β-

107 carotene, retinal, and subsequently, actinorhodopsin (Fig. 3A). Lycopene synthesis in acI requires three

108 major steps: synthesis of geranylgeranyl-PP (Step 1), linkage of two geranylgeranyl-PP molecules to

109 phytoene (Step 2), and tetra-desaturation of phytoene to lycopene (Step 3), predicted to be carried out by

110 acI-CrtE, acI-CrtB, and acI-CrtI, respectively. Subsequent β-cyclization of lycopene would first produce γ-

111 carotene (Step 4) then β-carotene (Step 5). A heterodimeric enzyme composed of acI-CrtYc and acI-CrtYd

112 likely carries out these serial cyclizations, much like Myxococcus xanthus β-cyclase genes (36% and 33%

113 identity, trihelical transmembrane topology, PxE(E/D) catalytic motif) (Fig. S2) (29–32). The symmetric

114 cleavage of β-carotene by a β-carotene oxygenase, acI-Blh, would then form retinal (Step 6). acI-Blh is a

115 putative dioxygenase based on 27% sequence identity, predicted helical topology, and four histidines for

116 non-heme iron coordination that are shared with the characterized β-carotene dioxygenase from an

117 uncultured marine bacterium (Fig. S3) (30, 31, 33). To form functional holo-ActR, retinal autocatalytically

118 forms a Schiff’s base with the side chain of a conserved lysine in acI-ActR (Step 7).

119 In the genomic context, genes encoding the enzymes for chromophore production are grouped into

120 functional regions (Fig. 2). Lycopene production genes (acI-crtE, acI-crtB, acI-crtI) are found as a

121 neighborhood in the order of steps 1, 3, 2 in all acI clades. Genes for steps 4-6 (acI-crtYc, acI-crtYd, acI-

122 blh) are found at varying distances from the lycopene-synthesis cluster. In many cases, the 3’ ends of acI-

123 crtB and acI-crtYd are adjacent but encoded in the opposite sense, thus forming a β-carotene-synthesis

124 neighborhood. In other cases, the cyclase genes are instead found near acI-blh. In most cases, acI-actR is

125 widely separated from other pathway genes. An interesting exception is AAA044-D11, where acI-actR, acI-

126 crtYc, acI-crtYd, and acI-blh are contiguous (Fig. 2).

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127 A pathway for a complex carotenoid contains at least nine genes. We previously noted that

128 genes for production of a predicted carotenoid glycoside-ester may be encoded adjacent to the lycopene

129 gene neighborhood (3) (Fig. 2). In this study, we used bioinformatics and sequence homology to assign

130 functions to gene products and assemble a plausible pathway (Figs. 3B).

131 The crucial carotenoid for initiating the pathway is γ-carotene (Step 4), and it is predicted to be

132 produced in the retinal pathway. Modifications of γ-carotene are expected to include: desaturation of a

133 carbon-carbon bond at the noncyclized end by acI-CrtD, introduction of two hydroxyl groups in separate

134 steps by acI-CrtA and acI-CruF, and addition of glucose onto the terminal hydroxyl group by the glycosyl

135 transferase, acI-CruG. Transfer of a fatty acid of unknown structure to the sugar by acI-acyltransferase

136 would be the final step in the production of a novel carotenoid uniquely found in acI Actinobacteria. We

137 propose to name this molecule actinoxanthin.

138 Genes encoding enzymes for actinoxanthin synthesis (acI-crtA, acI-cruF, acI-cruG, acI-crtD) are

139 found upstream of the lycopene neighborhood as a contiguous group, which most often includes acI-

140 acyltransferase (Fig. 3). Functional assignments for acI-CrtA, acI-CruF, acI-CruG, and acI-CrtD are based

141 on sequence homology and topological predictions matching more well-characterized enzymes (Fig. S4,

142 S5, S6, S7) (30, 31, 34–40). Because many carotenoid modification proteins are related in protein sequence

143 (i.e. lycopene desaturases and carotene ketolases) (41), functional validation will be required for all of the

144 carotenoid pathway enzymes. Specifically, the acI-CrtD gene product could encode a ketolase, CrtO, which

145 would introduce a carbonyl onto the β-ionone ring rather than desaturating an additional bond (Fig. 3B).

146 The resulting carotenoid would be structured like known rhodopsin antennae, salinixanthin and echinenone

147 (Fig. 3C) (20–22). The acyltransferase identity and chemistry are also not well-defined, but the gene

148 product’s involvement in actinoxanthin synthesis is supported by database annotation as a CoA methyl

149 esterase.

150 Metatranscriptomic analysis of pathway gene transcripts in environmental acI populations.

151 For actinorhodopsin assembly in acI cells, acI-actR and retinal synthesis genes must be expressed. To

152 measure gene expression in environmental acI, four metatranscriptome samples were collected across

153 multiple time points from the surface of eutrophic Lake Mendota (Dane County, WI, USA) and sequenced.

154 The resulting transcripts were mapped to available acI SAGs and MAGs (Table S1) to quantify relative

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155 gene expression levels in acI cells. Notably, acI-actR is the most highly expressed acI-A gene, the second

156 most highly-expressed acI-B gene (11), as well as the most highly expressed gene from either pathway in

157 each of the three acI clades (Table 1). Transcription is also significant for other genes in both the retinal

158 and putative actinoxanthin pathways, but at levels 300 to 1000-fold lower than for acI-actR (Table 1). No

159 transcripts were mapped for acI-blh from acI-C because the gene is neither present in any acI-C SAG or

160 MAG genome nor the first complete acI-C genome obtained by dilution to extinction cultures (4).

161 This mapping of Lake Mendota metatranscriptome samples provides direct evidence for

162 actinoxanthin, lycopene, and lycopene cyclase operons (Fig. 4). Specifically, reads that mapped to the most

163 populous genome, ME885, overlap the intergenic regions within each of the three operons (Figs. 3, 4).

164 Synteny across acI clades, especially for the lycopene and actinoxanthin operons, similarly supports

165 assignment of the three neighborhoods as genuine operons. Reading frame overlaps with acI-crtA in acI-

166 C support acI-acyltransferase involvement in actinoxanthin synthesis.

167 acI-CrtE, acI-CrtB, and acI-CrtI produce lycopene. We sought to demonstrate whether acI-CrtE,

168 acI-CrtB, and acI-CrtI enzymes can form lycopene as predicted (Fig. 3A, Steps 1-3). Therefore, a fast,

169 reproducible method for chromophore extraction from whole E. coli cells and identification by HPLC-mass

170 spectrometry analysis was formulated. Relevant genes (acI-crtE, acI-crtB, and acI-crtI) from AAA278-O22

171 were cloned into pCDFDuet1 and expressed from the resulting acI-CrtEBI/- cassette (Table 2). Assembly

172 AAA278-O22 was chosen for carotenoid production work because it contained all relevant genes and

173 source DNA was available (3). The extracted compounds of these cells displayed absorbance maxima

174 which exactly matched a lycopene standard at expected positions, 447, 472, and 504 nm (Fig. 5A) and

175 displayed an intense red color (data not shown). HPLC-MS elution profiles depict lycopene geometric

176 isomer peaks at m/z 536.438 (Fig. 5B). The retention time of the all-trans species (39.90 min) coincides for

177 sample and standard as the peak with highest intensity. The lycopene assignment was further confirmed

178 by MS/MS fragmentation of the parent ion.

179 To further characterize the lycopene product of acI-CrtEBI enzymes, we tested whether it serves

180 as the substrate in a lycopene cyclization reaction. Pantoea ananatis genes Pa-crtE, Pa-crtB, Pa-crtI and/or

181 Pa-crtY were expressed in E. coli as positive controls (Table 2, Fig. 6A). When the Pa-crtY cyclase gene

182 and acI lycopene pathway genes were co-expressed from pCDFDuet1 acI-CrtEBI/Pa-CrtY, the cellular

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183 extract absorbance maxima were 407, 429 and 453 nm (Fig. 6B). These dramatically blue-shifted maxima,

184 compared to β-carotene, combined with the defined cyclase activity of Pa-CrtY, identify the major extract

185 product as β-zeacarotene, γ-carotene saturated between C7’ and C8’ (Fig. 6B). Thus, acI-CrtEBI produce

186 a chromophore which serves as a substrate for Pa-CrtY lycopene cyclase. Presumably, β-carotene would

187 be produced after proper desaturation and a second cyclization, which were inefficiently carried out in our

188 non-native experimental set up.

189 acI-Blh produces retinal from β-carotene. The final enzymatic step in retinal biosynthesis is the

190 symmetric cleavage of β-carotene (Fig. 3A, Step 6). This enzyme is not encoded in any acI-C genomes

191 (Fig 2). To show that acI can natively perform β-carotene cleavage, we tested the enzymatic activity of

192 AAA278-O22 Blh. This acI-blh was expressed from pCDFDuet1 Pa-CrtEBIY/acI-Blh (Table 2) to ensure a

193 large β-carotene substrate pool. Introduction of acI-Blh yielded yellow instead of intensely orange-colored

194 cells observed when β-carotene is abundant (data not shown). HPLC-MS confirmed the presence of retinal

195 at m/z 285.221 as well as a diagnostic retinol species at m/z 269.226 (Fig. 7A, 7B). As is the case for

196 lycopene, geometric isomer peak patterning is observed. Maximal all-trans-species retention times match

197 retinal and retinol standards at 23.76 min and 23.88 min, respectively. Additionally, MS/MS fragmentation

198 of the appropriate m/z species confirmed retinoid identities (Figs. 7A, 7B). Retinol was roughly 7-times more

199 abundant than retinal in the extract as judged by MS intensities. Accordingly, the UV/Vis absorbance profile

200 appears more like retinol (Fig. 7C). Retinal produced by acI-Blh is likely serving as a potent electron

201 acceptor for the E. coli alcohol dehydrogenase, ybbO (42, 43).

202 acI-ActRL06 with retinal forms an active, green light-dependent proton pump. The gold-

203 standard test of a functional rhodopsin is light-dependent activity. Given that ActR proteins contain the

204 residues for chromophore binding and proton movement, we tested an opsin from each clade (AAA278-

205 O22, AAA027-L06, MEE578) for production and chromophore binding in E. coli. Each opsin was expressed

206 from pET21b+ alongside the plasmid confirmed for retinal production. acI-ActRL06 was selected for further

207 characterization due to its high expression and efficient retinal binding, as judged from samples captured

208 by metal affinity from detergent-solubilized membranes. acI-holo-ActRL06 maximally absorbed in the green

209 region at 541 nm (Fig. 8A). The covalent attachment of retinal was confirmed by incubation with

210 hydroxylamine hydrochloride, which frees the retinal and leads to production of retinal oxime.

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211 For conclusive demonstration that acI-holo-ActRL06 outwardly pumps protons in the presence of

212 light when in a membrane environment, micro-electrode pH measurements were performed. During

213 exposure to white light, the pH of a non-buffered assay solution sharply decreases compared to periods of

214 ambient red light (Fig. 8B). To confirm protons as the source of the steep pH drop, carbonyl cyanide m-

215 chlorophenyl hydrazone (CCCP) was added to discharge the proton gradient. CCCP trials did not display

216 light-dependent proton-pumping but rather the constant downward drift of control cells. Thus, acI-holo-ActR

217 is a retinal bound, green light-dependent proton-pumping rhodopsin.

218 DISCUSSION

219 Prior to this study, there was no biochemical evidence to support the hypothesis that acI

220 Actinobacteria use opsin-based phototrophy in freshwater. We have provided the first experimental

221 exploration of the advantages that allow the acI lineage to be so abundant. We describe two favorable

222 environmental adaptations, carotenoid biosynthesis and light-utilization. The former transforms simple

223 isoprenoid precursors into carotenoids and retinal so that acI-ActR can function as a green-light-absorbing,

224 outward proton-pumping acI-holo-ActR. The latter pathway is predicted to branch from a carotenoid

225 intermediate in the acI-holo-ActR pathway to synthesize a complex carotenoid, actinoxanthin, with a yet

226 unconfirmed structure and function.

227 The pathway for retinal and rhodopsin synthesis has been experimentally confirmed using acI

228 Actinobacterial proteins. The machinery to start retinal production consists of three enzymes, acI-CrtE, acI-

229 CrtB, and acI-CrtI (Steps 1-3). The enzymes produce lycopene (Fig. 3A, 5) and cluster into the acI-crtEIB

230 operon (Figs. 2, 4). The reactions for γ-carotene and β-carotene (Steps 4, Step 5) that follow lycopene

231 synthesis are likely carried out by a heterodimeric membrane protein expressed from the acI-crtYc and acI-

232 crtYd operon (Figs. 2, 3, 4). Homologs of these gene products were found in Myxococcus xanthus and

233 confirmed to synthesize a mix of γ-carotene and β-carotene (29). Therefore, we propose that acI can also

234 synthesize these chromophores. Although the ratio of products in acI is unknown, it may be intrinsically

235 linked to intracellular concentrations of retinal and actinoxanthin. Even lycopene formation may be a

236 regulatory point for carotenoid synthesis in acI because coexpression of lycopene and lycopene cyclase

237 does not guarantee dicyclized carotenoid (Fig. 6). Unsaturated but cyclized intermediates like β-

238 zeacarotene may result from differing levels of cyclase compared to acI-CrtE, acI-CrtB, and acI-CrtI.

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239 Abundant cyclase may disrupt a substrate channeling assembly for lycopene as seen in acI-CrtEBI, Pa-

240 CrtY-producing cells. Along these lines, an efficient co-localized assembly would help explain the

241 discrepancy between extremely high levels of actR versus other pathway transcript levels in Lake Mendota

242 (Table 1). We note that a system for stable β-zeacarotene production could prove a fruitful biotechnology

243 tool, because monocyclized carotenoids are not readily available for purchase.

244 A key finding of our work is the presence, expression, and robust activity of a retinal-producing

245 oxygenase from the acI gene, acI-blh. The enzyme symmetrically cleaves β-carotene to two retinal

246 molecules (Step 6) (Figs. 3A, 7) and might additionally use alternative substrates such as a β-zeacarotene-

247 or γ-carotene-like carotenoid to yield a single retinal. Notably, the acI-blh gene is not a feature of acI-C; in

248 five acI-C genomes ranging in completeness from 25-100%, acI-blh has never been recovered (Table S1)

249 (4). This absence points towards clade-level differentiation with respect to retinal production and

250 subsequent acI-holo-ActR assembly. Specifically, we propose that tribes or populations of acI-A and acI-B

251 with the ability to synthesize retinal display population persistence because of photoheterotrophy. This is in

252 contrast to acI-C that follow a seasonal “bloom and bust” lifestyle possibly because of a reliance on

253 exogenous chromophores (e.g. from cyanobacteria) (44), for example through uptake of retinal by acI-blh-

254 lacking cells from lysed cyanobacteria. Many species of cyanobacteria encode functional b-carotene

255 oxygenases (33, 45, 46), and retinoid concentrations in eutrophic lakes during cyanobacterial blooms are

256 measurable (47). This proposal is consistent with actinorhodopsin studies in the culturable freshwater

257 organisms Rhodoluna lacicola and Candidatus Rhodoluna planktonica (12, 13). While both encode actR,

258 only the latter contains blh and thus exhibits self-sufficient rhodopsin activity in the laboratory. R. lacicola is

259 dependent on retinal supplementation. As such, it may be analogous to acI-blh-lacking organisms. Indeed,

260 recently reported complete genomes (5) indicate that even some acI-A and -B would require an alternative

261 retinal source. This heterogeneity in gene content has major implications for niche-filling by acI-A and acI-

262 B members and may dictate community-related growth patterns.

263 Regardless of where acI source retinal, all acI-ActR proteins contain the necessary features for

264 proper activity as retinal holoproteins with green maximal absorbance, and we have demonstrated that an

265 example acI-ActR functions as a retinylidene protein (Figs. 1, 8). Green absorption correlates with light

266 penetration depth for many freshwater bodies where acI thrive, and strengthens the case that acI-holo-

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267 ActR is a light-activated protein in the environment. Native production of holo-ActR would have a major

268 impact on energy availability for the acI cells. In addition to showing its proton-pumping activity, we

269 discovered interesting qualities of acI-ActR (Figs. 1, S1). A proline in helix four was determined to be a

270 phylogenetically differentiating residue between actino-opsins and other xantho-opsins, like the one in

271 Salinibacter ruber. Structurally, prolines kink helices and the residue may have broad effects on

272 photocycling times and/or intermediate photocycle structures. Similarly, a disulfide stapling of a1-a7 in acI-

273 A and acI-B could also impact activity and/or protein stability. Additionally, it is interesting that acI-C

274 organisms lack both the cysteine pair and acI-blh.

275 In addition to retinal and ActR synthesis, members of all acI clades contain an operon for other

276 carotenoid biosynthetic machinery (acI-acyltransferase, acI-crtA, acI-cruF, acI-cruG, acI-crtD) (Figs. 2, 4).

277 The gene products might produce actinoxanthin, a complex carotenoid with glycosyl and acyl modifications

278 (Fig. 3B). Although the structure and presence of the carotenoid remain to be experimentally validated, the

279 glycine void predicted to be near the top of a5 hints at possible secondary chromophore photoactivity. This

280 variance from a bulky residue is necessary although not sufficient for binding of antenna keto-carotenoids

281 (48, 49). This would be the third example of a special class of rhodopsins that we call antenna rhodopsins

282 (20–22), but others are predicted (49). We are left with the question: can acI synthesize a complex

283 carotenoid using the genes found upstream from the lycopene cluster, and does it interact with acI-ActR as

284 an antenna?

285 Two biological scenarios for carotenoid-related light utilization in acI include: acI-holo-ActR is an

286 antenna rhodopsin that augments proton-pumping via carotenoid energy transfer, or a complex carotenoid

287 protects the cell from oxidative species and excess light while the proton-pump functions (Fig. 9) (50). Both

288 situations favor production of a proton gradient to power numerous nutrient uptake transporters particularly

289 when nutrients are limited (11). Such adaptations go far toward explaining how acI Actinobacteria can reach

290 nearly 50% of the microbial cell number in freshwater lake epilimnia, and why they exist in virtually all

291 freshwater ecosystems (2, 51). Further defining the physiology of a dominant ecological force within the

292 lake biome will fill a gap in our conceptual models for freshwater carbon and energy cycling and improve

293 ecosystem behavior forecasting.

294

11 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

295 MATERIALS AND METHODS

296 acI Gene Identification and Pathway Assembly. Annotations for multiple acI SAGs (3) and MAGs

297 were analyzed for genes relating to carotenoids using the Joint Genome Institute’s Integrated Microbial

298 Genome’s Viewer (52). Translated candidate protein sequences were used to identify homologs in other

299 acI genomes, and those with consistent gene neighborhoods and known carotenoid-related functions were

300 prioritized. The gene cassette with functional assignments was grouped into two pathways for carotenoid

301 biosynthesis and use in acI Actinobacteria.

302 acI Biomass Collection and Transcriptomics. Environmental sampling, metatranscriptome

303 sequencing, and gene-expression calculations were all performed as previously described (11). Briefly, four

304 samples were collected from within the top 12m of the Lake Mendota (Dane County, WI) water column,

305 filtered through cheesecloth and onto 0.2 µm mixed cellulose filters (Whatman). Filters were immediately

306 vitrified in liquid nitrogen and stored at -80°C. Samples were subject to RNA extraction, ribosomal RNA

307 (rRNA) depletion, and sequenced on an Illumina HiSeq2500. All protocols and scripts for sample collection,

308 RNA extraction, rRNA depletion, and sequencing are on GitHub (https://github.com/McMahonLab/OMD-

309 TOIL).

310 Because this work used metatranscriptomes obtained as part of a previous study (11), raw paired-

311 end reads were trimmed, merged, subject to in-silico rRNA removal, pooled, and mapped to a single

312 reference FASTA file containing 36 high-quality acI genomes from a larger freshwater genome collection

313 (11). Reads were competitively mapped to genes using BBMap (https://sourceforge.net/projects/bbmap/)

314 and counted using HTSeq-Count (53). Raw count data were then used to compute gene expression on a

315 Reads Per Kilobase of transcript, per Million mapped reads (RPKM) basis (54). All scripts are found on

316 GitHub (https://github.com/joshamilton/Hamilton_acI_2017).

317 Plasmid construction. All cloning was performed using Phusion HF GC Master Mix

318 (ThermoFisher) and the listed primers (Table S2). A collection of stable DNA sources for amplification and

319 subcloning was first created. Plasmid containing E. coli-codon-optimized crtE, crtB, crtI, crtY from Pantoea

320 ananatis (Pa) was obtained from the International Genetically Engineered Machine (iGEM) organization

321 catalog (http://parts.igem.org/Main_Page) (Table 2). The genes were PCR-amplified from the iGEM

12 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

322 plasmid as a block. acI biosynthetic genes were PCR-amplified from AAA278-O22 genomic DNA as gene

323 clusters. The L06 opsin sequence was obtained as an E. coli-optimized gene from DNA2.0.

324 All genes were individually PCR-amplified, if needed, and added to a cloning pipeline. Primer

325 design included up to 40 base pairs of cloning site flanking regions from pCDFDuet-1 (Novagen). Genes

326 were placed into the first site between NcoI and AflII restriction sites and between NdeI and AvrII sites of

327 the second site. Appropriately restriction-enzyme-digested and gel purified plasmid backbone was

328 transformed with up to a 10-fold molar excess of PCR product into E. cloni 10G cells (Lucigen) for ligation

329 free plasmid recombination. After selection on 100µg/mL spectinomycin sulfate LB-Miller agar plates, insert

330 presence was validated by colony PCR using GoTaq Green Master Mix (ThermoFisher). Restriction

331 enzyme digestion in lab and Sanger sequencing at the UW Biotechnology Center further confirmed plasmid

332 correctness. pET21b+ (Novagen) was used in the same pipeline ensuring genes were in-frame with a C-

333 terminal hexahistidine tag with selection by 100µg/mL carbenicillin, sodium salt.

334 Chromophore Production and Extraction. For chromophore production, multiple colonies of

335 freshly transformed BL21 (DE3) Tuner E. coli (EMD Millipore) were picked into half-full flasks of LB-Miller

336 broth plus 100µg/mL spectinomycin sulfate and shaken in 37°C darkness at 250 rpm for 24 hours. Five

337 hundred or 1500 ODmL (ODmL = OD600nm* dilution * volume in milliliters) cells were centrifuged at 3300xg,

338 washed in 100mM Tris 6.8, centrifuged again, and vitrified in liquid nitrogen. For chromophore extraction,

339 cells stored at -80°C were thawed at 23°C for up to 30min. Cells were suspended at 500ODmL cells/3 mL

340 acetone (Sigma, HPLC-grade), intensely vortexed for 1 min, and iced for 5 min. The chromophore

341 containing supernatant after a 10 min 8000 x g clarification was added to 1mL/500ODmL of 23°C NaCl-

342 saturated water (Fisher, ACS-grade) and 1mL/500ODmL 23°C dichloromethane (ACROS ACS-grade) and

343 vortexed for 1 min. Further centrifugation resulted in a colorless aqueous bottom layer and a top colorful

344 organic layer. The organic layer was evaporated under nitrogen, resulting solids were suspended in

345 appropriate solvent, and filtered through a compatible 4mm 0.22µm filter.

346 UV-Vis and LC-MS/MS analysis of chromophores. Absorbance spectra were acquired on a

347 Beckman Coulter DU640B spectrophotometer. A Dionex Ultimate 3000 UHPLC coupled by electrospray

348 ionization (ESI; positive mode) to a hybrid quadrupole - high-resolution mass spectrometer (Q Exactive

349 orbitrap, Thermo Scientific) was used for detection of target compounds based on their accurate masses,

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350 mass spectra, and retention times (all matched to purified standards). Liquid chromatography (LC) was

351 based on a published protocol (55). Separation was achieved using a YMC C30 reversed-phase carotenoid

352 column (150mm x 2.5mm, 3µm particles) (SOURCE) at a flow rate of 0.2mL/minute. Solvent A consisted

353 of equal parts methanol and water with 0.5% v/v acetic acid; Solvent B was equal parts methanol and

354 methyl-tert-butyl-ether with 0.5% v/v acetic acid (55). Total run time was 58 min with the following two

355 gradients: 5 min 30% B, 20 min ramp to 100% B, 100% B (retinoids) or 5 min 30% B, 25 min ramp to 100%

356 B, 100% B (carotenoids). MS scans consisted of full positive mode scanning for m/z 200-600 from 5 min

357 onwards. In addition, MS/MS scans were obtained by isolating fractions with m/z of 537.44548 (lycopene),

358 285.22129 (retinal), and 269.22639 (retinol). MS/MS fragmentations were performed at 30 normalized-

359 collision energy (NCE) with an isolation window of 1.4 m/z and a post-fragmentation window of 50 to

360 approximately 25 m/z above the isolation mass. For all scans, mass resolution was set at 35000 m/z ppm,

361 AGC target was 1 x 106 ions, and injection time was 40ms. Settings for the ion source were: auxiliary gas

362 flow rate – 50, sheath gas flow rate – 10, sweep gas flow rate – 2, spray voltage – 3.5 kv, capillary

363 temperature – 350°C, heater temperature – 250°C, S-lens RF level – 55.0. Nitrogen was used as nebulizing

364 gas by the ion trap source. Standards for lycopene (Toronto Research Chemicals Inc., L487500, Lot 7ANR-

365 20-1) β-carotene (Sigma Aldrich, PHR1239-3x100mg, Lot LRAA6761), retinal (Sigma Aldrich, R2500-

366 25MG, Lot SLBN4199V), and retinol (Sigma Aldrich, R7632-25MG, Lot BCBP8066V) were analyzed along

367 with experimental samples. Isomer peaks within the mass window result most often from environment-

368 induced changes during sample preparation. Control cells did not display any significant sustained signal

369 within the time-mass window. Data analysis was performed using Thermo Xcalibur and visualized on

370 MAVEN (56, 57) and Thermo Xcalibur (Thermo Scientific) software.

371 Proposed gene product and actino-opsin analysis. Clustal Omega was used to align proposed

372 biosynthetic enzymes and opsin sequences for identification of protein characteristics (58–60). Opsin

373 sequences were submitted to the I-TASSER server for structure prediction using xantho-opsin (PDB

374 3DDL:A) as the template (61–63). Basic Local Alignment Search Tool (BLAST) and TM/HMM 2.0 were

375 used for identity percentage calculation and transmembrane helix estimates, respectively (30, 31). PyMOL

376 was used to visualize the resulting protein structures (64).

14 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

377 The actino-opsin phylogeny was reconstructed using opsin protein sequences from bacterial isolate

378 references, SAGS and MAGS. Opsin protein sequences were aligned with PROMALS3D multiple

379 sequence and structure alignment tool (65). The alignment was trimmed to exclude columns that contained

380 gaps for more than 30% of the included sequences. Poorly aligned positions and divergent regions were

381 further eliminated by using Gblocks (66). A maximum likelihood phylogenetic tree was constructed using

382 PhyML 3.0 (67), with the LG substitution model and the gamma distribution parameter estimated by PhyML

383 and a bootstrap value of 100 replicates. The phylogenetic tree was visualized with Dendroscope (v3.2.10)

384 with midpoint root (68). The ActR and other XR sequence subtree was extracted and displayed.

385 Actinorhodopsin enrichment and analysis. BL21 (DE3) Tuner E. coli cells co-transformed with

386 plasmid expressing Pa-CrtEBIY/acI-Blh and acI-ActRL06 were grown as during carotenoid production except

387 that carbenicillin, sodium salt was also added to 100µg/mL. All further steps were done under red light or

388 darkness at 4°C and/or on ice using 4°C buffers. 2000 ODmL cells were harvested and suspended in 5g/mL

389 Lysis Buffer (50mM Tris-HCl 8.0, 300mM NaCl, 1 50mL EDTA-free protease inhibitor tablet (Roche),

390 1mg/mL lysozyme, 20µg/mL DNase I, 5mM MgCl2, 130µM CaCl2, 4mM PMSF). Cells were lysed at 16000

391 psi by five passes through a French Pressure cell. Sequential centrifugation at 10,000xg for 15 min and

392 100,000xg for 45 min cleared debris and pelleted membranes. Membranes were loosened with Lysis Buffer

393 and transferred to a Potter-Evelhjem homogenizer. After homogenization, membranes were diluted with

394 4°C Lysis Buffer to 25mL and recentrifuged at 100,000xg for 45 min. Suspension and homogenization were

395 repeated with Solubilization Buffer (50mM Tris-HCl 8.0, 300mM NaCl, 2% m/v dodecyl-maltoside, 10mM

396 imidazole). Membranes were diluted to 15mL with Solubilization Buffer and rocked 18hr in darkness.

397 Centrifugation at 20000xg for 20min clarified the material before chromatography. The soluble fraction was

398 loaded at 0.5mL/min onto an equilibrated 1mL Ni-NTA column. The processing profile in column volumes

399 (CV) was: 3 Solubilization Buffer, 12 Wash Buffer (50mM Tris-HCl 8.0, 300mM NaCl, 0.05% m/v DDM,

400 30mM imidazole), 5 Elution Buffer (50mM Tris-HCl 8.0, 300mM NaCl, 0.05% m/v DDM, 500mM imidazole).

401 The first elution fraction was dialyzed against 1L 4°C Final Buffer (10mM HEPES 7.5, 100mM NaCl, 0.05%

402 m/v) for 2hr and then analyzed by absorbance spectroscopy. Light and 50 µL 23°C saturated hydroxylamine

403 HCl was used to selectively remove retinal from 140 µL of sample to confirm the Schiff base. The resulting

15 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

404 unbound retinal oxime absorbs at wavelength maxima between that of retinal and retinol where 247 and

405 257 nm peaks indicate 11-cis and all-trans oxime species, respectively.

406 Micro-electrode pH trace acquisition. The assay was similar to a published protocol and uses E.

407 coli for expression of holo-ActR (12). All steps were carried out under red light generated by putting red

408 cellophane over a fluorescent bulb. 500 ODmL E. coli cells were harvested by centrifugation at 3300xg for

409 15 min at 4°C. Cells were suspended in 45 mL 23°C Assay Solution (10mM NaCl, 10mM MgSO4-7H2O,

410 100uM CaCl2-2H2O) and centrifuged at 3300 x g for 10 min. The latter wash step was repeated. Final

411 suspension was in 20 mL 23°C Assay Solution to yield an OD=25. Cells were incubated for 60 min at 23°C

412 to stabilize in darkness before the onset of the assay. The assay was set up with the sample in a glass test

413 tube surrounded by a 300 mL 23°C water bath in a 400 mL beaker. A Mettler-Toledo InLab Micro probe

414 (model number 51343160) was clamped in place above the sample tube and connected to a Sper Scientific

415 Datalogger (model number 850060). The entire setup was surrounded by foil lined cardboard on four sides.

416 Two FE15T8 bulbs (15W, 700 lumens each) in an 11x45 cm housing with 90° reflectors were placed 15 cm

417 from the center of the sample tube such that light illuminated the entire length of the closest tube side. pH

418 was recorded every second after 3.5 min of equilibration time for 60 min as follows: three cycles of 10 min

419 with fluorescent lights off and 10 min fluorescent lights on. Carbonyl cyanide m-chlorophenyl hydrazone

420 (CCCP) (Alfa Aesar, L06932 Lot 10181844) was added to a final concentration of 20 μM during dark

421 stabilization after 45 min.

422 ACKNOWLEDGEMENTS and REFERENCES

423 This work was supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture

424 (2016-67012-24709 to JJH and WIS01789 to KDM), the United States National Science Foundation (NTL-

425 LTER DEB-1440297 and DEB-1344254 to KDM, MCB-1518160 to KTF) and the National Oceanic and

426 Atmospheric Administration (NA10OAR4170070 to KDM and KTF via the Wisconsin Sea Grant College

427 Program Project #HCE-25). We thank former McMahon laboratory member, Dr. Sarahi Garcia, for the initial

428 AAA0278-O22 DNA, former and current McMahon laboratory members for help obtaining transcript data,

429 Diego Yanez for assisting with RNA methodology, and Dr. James Steele for supplying plasmid iGEM DNA.

430

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25 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

598 TABLES 599 600 601 TABLE 1 Log2 average reads per kilobase of 602 transcript per million mapped read values for 603 carotenoid-related gene transcripts Gene Name acI-A acI-B acI-C acI-crtE 11.19 11.76 9.16 acI-crtB 9.33 9.64 9.23 acI-crtI 11.21 12.92 9.46 acI-crtYc 10.44 12.62 9.76 acI-crtYd NDa 10.23 7.22 acI-blh 7.30 8.48 NAb acI-actR 19.54 19.79 17.99 acI-crtD 9.39 11.98 8.29 acI-crtA 10.46 8.53 8.97 acI-cruF 9.80 10.28 9.09 acI-cruG 9.14 10.75 8.31 acI-11 9.23 9.04 7.93 recA 14.52 16.92 12.70 604 605 a Not Determined: Gene is present in clade 606 member assemblies but no transcript mapped. 607 b Not Applicable: Gene is not present in any 608 clade member assembly. 609 610 611 TABLE 2 DNA sources and utilized plasmids for this study Gene Vector Genes E. coli-codon-optimized? Single-cell acI-crtE, acI-crtB, acI-crtI No genome

acI-blh No

Plasmid

pJExpress404 acI-actRL06 Yes DNA Sources DNA Bba_K274210 Pa-crtE,Pa-crtB,Pa-crtI,Pa-crtY Yes

Backbone Cassette Shorthand Cloning Site Contents

a pCDFDuet1 -/- NAc / NAc c

acI-CrtEBI/- acI-crtE, acI-crtB, acI-crtI / NA acI-CrtEBI/Pa-CrtY acI-crtE, acI-crtB, acI-crtI / Pa-crtY Pa-CrtEBI/- Pa-crtE, Pa-crtB, Pa-crtI / NAc Pa-CrtEBIY/- Pa-crtE, Pa-crtB, Pa-crtI, Pa-crtY / NAc Pa-CrtEBIY/acI-Blh Pa-crtE, Pa-crtB, Pa-crtI, Pa-crtY / acI-blh

Utilized Plasmids Utilized b pET21b+ - NAc

pacI-ActRL06 acI-actRL06 612 613 a Plasmid contains two cloning sites with contents separated by a slash 614 b Plasmid contains one cloning site in-frame with a C-terminal hexahistidine tag 615 c Not Applicable because the cloning site is unmodified.

616

26 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

617 FIGURE LEGENDS

618 FIG 1 Features of acI-ActR from acI clades A, B, and C. (A) acI-ActR from clade A (ActRO22), clade B

619 (ActRL06), and clade C (ActRMEE578) are aligned to a homolog, xantho-opsin (XR) from Salinibacter ruber.

620 Proteins are subscripted with genome shorthand (Table S1). Features are highlighted: predicted helices

621 (gray, numbered), cysteines (yellow), main proton shuttles (orange), main absorbance tuner (green), Schiff

622 base lysine (magenta), possible antenna carotenoid residues (cyan), and proline indicative of ActR vs

623 xantho-opsin (red). Antenna carotenoid residues are based on amino acids in proximity to salinixanthin in

624 the crystal structure of xanthorhodopsin (PDB 3DDL). (B) 3-D structure prediction for ActR with key residue

625 side chains displayed according to the color coding in panel (A). The α-carbon of glycine near the top of a5

626 that would allow antenna binding is shown as a sphere, the Schiff base retinal is modeled (lime green), the

627 predicted disulfide bond of ActR from clades A and B is modeled, and the N- and C-termini are labeled.

628

629 FIG 2 acI carotenoid-related genes in the genomic context. acI genomic contigs or genomes from clades

630 A, B, and C are primarily labeled by shorthand notation (i.e. designation after “actinobacterium SCGC” or

631 “int.metabat.”). Genes are arrows pointing in the direction of transcription. A slash indicates a contig

632 boundary, which may or may not end immediately after the pictured gene, a vertical zig-zag indicates contig

633 ends that have been manually paired, and a horizontal thick bar indicates a longer region of DNA not

634 represented. Uncolored genes are neighboring genes that may or may not be functionally associated with

635 the carotenoid-related genes. Bolded labels indicate a gene source for this study. Relevant gene numbers

636 for this study from AAA278-O22 in IMG are 3540 (acI-11), 10590-10560 (acI-crtA to acI-crtD), 10530-10510

637 (acI-crtE to acI-crtB), and 7000-6980 (acI-crtYd to acI-blh), 1360 (acI-actR).

638

639 FIG 3 Predicted carotenoid-related pathways in acI. (A) The actinorhodopsin synthesis pathway requires

640 isopentenyl precursors to be assembled into carotenoids (1-3), which are modified (4-5) and cleaved to

641 produce retinal (6). A Schiff base forms between retinal and a lysine of acI-ActR to form acI-holo-ActR (7).

642 (Chemical changes are shown in red, proteins from this study with their predicted functions are shown to

643 the sides of progress arrows, and product names are shown to left of their chemical structures.) (B) A

644 complex carotenoid synthesis pathway is predicted to require γ-carotene produced in the actinorhodopsin

27 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

645 pathway with further desaturation, hydroxylation, glycosylation, and acylation of the carotenoid. Question

646 marks indicate uncertainty in enzyme naming or chemical structure, and the asterisk represents the position

647 of a carbonyl group if acI-CrtD is actually acI-CrtO, a ketolase. (C) Complex carotenoids from other

648 organisms that function as carotenoid antennae on proton-pumping rhodopsins.

649

650 FIG 4 Intergenic transcripts that map to carotenoid-related genes in genome ME885. Operons are evident

651 for actinoxanthin (acyltransferase through acI-crtD), lycopene (acI-crtE through acI-crtB), and lycopene

652 cyclase (acI-crtYc and acI-crtYd) biosynthesis. The color key in the center indicates of the number of times

653 a base was covered. Transcripts and genes may continue beyond the edges of the DNA window.

654

655 FIG 5 acI-CrtEBI catalyze lycopene formation. (A) Difference spectra for extracts from cells containing acI-

656 CrtEBI/- vs -/- (black) and lycopene vs. acetone (red). The inset depicts lycopene. (B) HPLC-MS data for

657 the same samples. The inset depicts MS/MS data from all-trans peak retention times.

658

659 FIG 6 acI-CrtEBI-produced lycopene is cyclized by Pa-CrtY. (A) Difference spectra for Pa-CrtEBI/- derived

660 lycopene vs -/- extract (red dash), standard lycopene vs acetone (red), Pa-CrtEBIY/- derived β-carotene vs

661 -/- extract (orange dash), and standard β-carotene vs acetone (orange). The insets depict lycopene and β-

662 carotene. (B) Difference spectra for synthesized β-carotene extract (orange dash) and acI-CrtEBI/Pa-CrtY

663 vs -/- extracts (black). The insets depict β-zeacarotene and β-carotene.

664

665 FIG 7 acI-Blh is a b-carotene oxygenase that catalyzes retinal formation. (A) HPLC-MS data for Pa-

666 EBIY/acI-Blh extract (black) and a retinal standard (yellow). The inset depicts MS/MS data from all-trans

667 peak retention times. (B) HPLC-MS and MS/MS (inset) data for the same experimental extract but with

668 standard retinol spectra (purple). (C) Difference spectra for Pa-CrtEBIY/acI-Blh vs -/- (black) and retinal

669 (yellow) or retinol (purple) standards vs ethanol. Insets depict retinal and retinol to highlight conjugation

670 length differences.

671

28 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Dwulit-Smith et al. Actinorhodopsin and Carotenoid Synthesis in acI

672 FIG 8 Holo-acI-ActRL06 is a light-driven proton pump. (A) Absorbance spectra for holo-acI-ActRL06 enriched

673 from cells also expressing Pa-CrtEBIY/acI-Blh without (solid) and with (dashed) hydroxylamine

674 hydrochloride. The retinal oxime that results from hydroxylamine incubation is shown. (B) Micro-electrode

675 pH traces for cells expressing Pa-CrtEBIY/acI-Blh with (black, blue) or without (yellow) acI-ActRL06. CCCP

676 is present in the blue trace. Shaded regions represent illumination by white light, and curves are from one

677 of three independent experiments.

678

679 FIG 9 Biological model for acI actinobacterial carotenoid usage. (A) acI cells may utilize a complex

680 carotenoid as an antenna that transfers light energy into the green-light-absorbing proton-pumping

681 rhodopsin. (B) acI cells may utilize a complex carotenoid as protection from excess light and oxidative

682 species to protect rhodopsin.

29 bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A B 1 2 N XR MLQELPTLTPGQYSLVFNMFSFTVATMTASFVFFVLARNNVAPKYRISMMVSALVVFIAG ActRO22 ---MSVTLDHNQWNLVYNIFSFGLISMLACTVYTLVSQSRVLPKYRNALVMSSMVTFIAG ActRL06 -MSSTIALSADQFGIVYNILSFGLVSMLACTIYTLVSQSRVLPKYRTALVMSSMVTFIAA ActRMEE578 -----MQLDASQWSTVYNILSFGLVSMLATTVYTLVSQNRVLPKYRNALVLSSMVTFIAA 3 XR YHYFRITSSWEAAYALQ----NGMYQPTGELFNDAYRYVDWLLTVPLLTVELVLVMGLPK ActRO22 YHYFRIFNSFNEASDGMAVNVS----GDQGAFNEAYRYVDWLLTVPLLLVEVIAVLALAK ActRL06 YHYMRIFNSFEDSFADGAVKVG----TGAGAFNEGYRYVDWILTVPLLLVEVVAVLALAK ActRMEE578 YHYLRIFDSFKDATKEGTFEVVTDYNVSSMAFNEGYRYVDWILTVPLLLVEVIAVLALAK 4 5 XR NERGPLAAKLGFLAALMIVLGYPGEVSENAALFGTRGLWGFLSTIPFVWILYILFTQLGD ActRO22 EVSSSLISRLVPASAAMIALGYPGEISSDK---NTAILYGVLSTIPFLYILYVLFVELGK ActRL06 AAASSLITRLVPASAAMIALGYPGEISTDN---NTKVLWGVLSTIPFLYILYVLFVELSK ActRMEE578 EAARSLITRLVPASAAMIALGYPGEVSADQ---GEKIMWGVLSTIPFLYILYVLFVELGK 6 XR TIQRQSSRVSTLLGNARLLLLATWGFYPIAYMIPMAFPEAFPSNTPGTIVALQVGYTIAD ActRO22 SLDRQPAGVGETVGRLRLLLIATWGVYPVSYILGM---NG--DPTASSFVGVQVGYTIAD ActRL06 SLDRQPAGVAATVGRLRLLLIATWGVYPIAYLLPILGQDA---LDPAAFVNRQIGYTIAD ActRMEE578 SLDRQPEGVAATIGRLRLLLIATWGVYPIAYLLPIIDADG--AASSGAFVGRQVGYTIAD 7 XR VLAKAGYGVLIYNIAKAKSEEEGFNVSEMVEPATASA ActRO22 ILAKCVFGLTILKIARMKSHAEGMPADH------C ActRL06 VLAKCVFGLTILKIARMKSVAEGMKDDH------ActRMEE578 VAAKCVFGLTILKIARMKSIAEGMKDDH------

FIG 1 Features of acI-ActR from acI clades A, B, and C. (A) acI-ActR from clade A (ActRO22), clade B

(ActRL06), and clade C (ActRMEE578) are aligned to a homolog, xantho-opsin (XR) from Salinibacter ruber. Proteins are subscripted with genome shorthand (Table S1). Features are highlighted: predicted helices (gray, numbered), cysteines (yellow), main proton shuttles (orange), main absorbance tuner (green), Schiff base lysine (magenta), possible antenna carotenoid residues (cyan), and proline indicative of ActR vs xantho-opsin (red). Antenna carotenoid residues are based on amino acids in proximity to salinixanthin in the crystal structure of xanthorhodopsin (PDB 3DDL). (B) 3-D structure prediction for ActR with key residue side chains displayed according to the color coding in panel (A). The α-carbon of glycine near the top of α5 that would allow antenna binding is shown as a sphere, the Schiff base retinal (lime green) and predicted disulfide bond in clades A and B are modeled, and the N- and C-termini are labeled. bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

acI-acyltransferase?acI-crtAacI-cruFacI-cruGacI-crtDacI-crtEacI-crtIacI-crtBacI-crtYc,YdacI-blh acI-actR AAA024-D14

AAA023-J06

MMS-IA-53 A AAA028-I14

AAA028-E20

AAA278-O22

AAA028-A23

AAA027-L06

MMS-21-122 B AAA027-J17

AAA278-I18

AAA044-D11

IMCC26077

ME885

MEE578 C BIN_10

ME3864

FIG 2 acI carotenoid-related genes in the genomic context. acI genomic contigs or genomes from clades A, B, and C are primarily labeled by shorthand notation (i.e. designation after “actinobacterium SCGC” or “int.metabat.”). Genes are arrows pointing in the direction of transcription. A slash indicates a contig boundary, which may or may not end immediately after the pictured gene, a vertical zig-zag indicates contig ends that have been manually paired, and a horizontal thick bar indicates a longer region of DNA not represented. Uncolored genes are neighboring genes that may or may not be functionally associated with the carotenoid-related genes. Bolded labels indicate a gene source for this study. Relevant IMG database gene numbers for this study from AAA278-O22 are 3540 (acI-acyltransferase), 10590-10560 (acI-crtA to acI-crtD), 10530-10510 (acI-crtE to acI-crtB), 7000-6980 (acI-crtYd to acI-blh), and 1360 (acI-actR). bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A Dimethylallyl-PP B (4) γ-Carotene Isopentenyl-PP PP PP

Geranyl-PP acI-CrtD γ-carotene 3’,4’-desaturase acI-CrtO* 4-ketolase PP

Precursors Farnesyl-PP * acI-CrtA spheroidene mono-oxygenase PP OH (1) Geranylgeranyl-PP acI-CrtE geranylgeranyl-PP synthase acI-CruF γ-carotene 1’-hydroxylase PP OH

(2) Phytoene acI-CrtB phytoene synthase OH Intermediates

acI-CruG glycosyltransferase OH

(3) Lycopene acI-CrtI lycopene synthase O HO

O OH acI-Acyltransferase? acyltransferase HO Actinoxanthin OH OH (4) γ-Carotene acI-CrtYc, acI-CrtYd lycopene β-cyclase

O HO

O ??? OH O OH (5) β-Carotene acI-CrtYc, acI-CrtYd lycopene β-cyclase O C Salinixanthin

OH acI-Blh β-carotene 15,15’-dioxygenase HO (6) Retinal O

O O O OH O O OH Echinenone O (7) acI-Holo-ActR acI-ActR actino-opsin actinorhodopsin + acI-ActR N H O

FIG 3 Predicted carotenoid-related pathways in acI. (A) The actinorhodopsin synthesis pathway requires isopentenyl precursors to be assembled into carotenoids (1-3), which are modified (4-5) and cleaved to produce retinal (6). A Schiff base forms between retinal and a lysine of acI-ActR to form acI-holo-ActR (7). Chemical changes are shown in red, proteins from this study with their predicted functions are shown to the sides of progress arrows, and product names are shown to left of their chemical structures. (B) A complex carotenoid synthesis pathway is predicted to require γ-carotene produced in the actinorhodopsin pathway with further desaturation, hydroxylation, glycosylation, and acylation of the carotenoid. Question marks indicate uncertainty in enzyme naming or chemical structure, and the asterisk represents the position of a carbonyl group if acI-CrtD is actually acI-CrtO, a ketolase. (C) Complex carotenoids from other organisms that function as carotenoid antennae on proton-pumping rhodopsins. bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

5’ ... G T T A C A A A A T G A T C C T A G T T ... 3’ acyltransferase? acI-crtA

5’ ... G T T A T A A G G C A C A A T T A A G C C A T G T C G A T ... 3’ acI-crtA acI-cruF

5’ ... G A G A T T A A G G G A A A A T G A C T T T ... 3’ acI-cruF acI-cruG

5’ ... A G A A A C T A T G A G C C A T C A C ... 3’ acI-cruG acI-crtD

2 4 6 8 10 12 14

5’ ... G T C T T T A A G A T G A G A A A ... 3’ acI-crtE acI-crtI

5’ ... T A A A G T A A A G A T G G A T G A ... 3’ acI-crtI acI-crtB

5’ ... A T G A G C G A T G A G T T A T A C C ... 3’ acI-crtYc acI-crtYd

FIG 4 Intergenic transcripts that map to carotenoid-related genes in genome ME885. Operons are evident for actinoxanthin (acI-acyltransferase through acI-crtD), lycopene (acI-crtE through acI-crtB), and lycopene cyclase (acI-crtYc and acI-crtYd) biosynthesis. The color key in the center indicates of the number of times a base was covered. Transcripts and genes may continue beyond the edges of the DNA window. bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A 0.5 B 1.6E+7 417.85

320.92

0.4 358.84 435.87 1.2E+7

536.43 0.3 250 350 450 550 m/z 8.0E+6

0.2 lycopene

4.0E+6 0.1 Relative Intensity at m/z 536.438 Normalized Scaled Absorbance 0.0 0.0E+0 400 450 500 550 600 20 30 40 50 Wavelength (nm) Retention Time (min)

FIG 5 acI-CrtEBI catalyze lycopene formation. (A) Difference spectra for extracts from cells containing acI-CrtEBI/- vs -/- (black) and lycopene vs. acetone (red). The inset depicts lycopene. (B) HPLC-MS data for the same samples. The inset depicts MS/MS data from all-trans peak retention times. bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A 0.5 B 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1 Normalized Scaled Absorbance Normalized Scaled Absorbance 0.0 0.0 400 450 500 550 600 400 450 500 550 600 Wavelength (nm) Wavelength (nm)

FIG 6 acI-CrtEBI-produced lycopene is cyclized by Pa-CrtY. (A) Difference spectra for Pa-CrtEBI/- derived lycopene vs -/- extract (red dash), standard lycopene vs acetone (red), Pa-CrtEBIY/- derived β-carotene vs -/- extract (orange dash), and standard β-carotene vs acetone (orange). The insets depict lycopene and β-carotene. (B) Difference spectra for synthesized β-carotene extract (orange dash) and acI-CrtEBI/Pa-CrtY vs -/- extracts (black). The insets depict β-zeacarotene and β-carotene. bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A B 1.4E+8 161.10 175.16 3.5E+8 93.07 269.23 119.09 81.07 107.09 69.07 119.09 285.22 2.8E+8 1.1E+8 50 100 150 200 250 300 50 100 150 200 250 300 m/z m/z

2.1E+8 7.0E+7 1.4E+8

3.5E+7 7.0E+7 Relative Intensity at m/z 285.221 Relative Intensity at m/z 269.226

0.0E+0 0.0E+0 20 25 30 20 25 30 Retention Time (min) Retention Time (min) C 0.5

0.4

O 0.3

0.2 OH

0.1 Normalized Scaled Absorbance

0.0 300 400 500 600 Wavelength (nm)

FIG 7 acI-Blh is a β-carotene oxygenase that catalyzes retinal formation. (A) HPLC-MS data for Pa-EBIY/a- cI-Blh extract (black) and a retinal standard (yellow). The inset depicts MS/MS data from all-trans peak retention times. (B) HPLC-MS and MS/MS (inset) data for the same experimental extract but with standard retinol spectra (purple). (C) Difference spectra for Pa-CrtEBIY/acI-Blh vs -/- (black) and retinal (yellow) or retinol (purple) standards vs ethanol. Insets depict retinal and retinol to highlight conjugation differences. bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A 0.5 B Time (min) 10 20 30 605040 OH N 0.02 0.4 0.00

0.3 -0.02 -0.04

0.2 Δ pH -0.06

Scaled Absorbance -0.08 0.1 -0.10

0.0 -0.12 300 350 400 450 500 550 600 650 Wavelength (nm)

FIG 8 Holo-acI-ActRL06 is a light-driven proton pump. (A) Absorbance spectra for holo-acI-ActRL06 enriched from cells also expressing Pa-CrtEBIY/acI-Blh without (solid) and with (dashed) hydroxylamine hydrochloride. The retinal oxime that results from hydroxylamine incubation is shown. (B) Micro-electrode pH traces for cells expressing Pa-CrtEBIY/acI-Blh with (black, blue) or without

(yellow) acI-ActRL06. CCCP is present in the blue trace. Shaded regions represent illumination by white light, and curves are from one of three independent experiments. bioRxiv preprint doi: https://doi.org/10.1101/367102; this version posted July 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A B

Light Light

Cell Wall Cell Wall

+ + + + + + H H H H H H

Antenna Retinal Retinal Carotenoid Protector Rhodopsin Carotenoid Rhodopsin

Plasma Membrane Plasma Membrane

FIG 9 Biological model for acI actinobacterial carotenoid usage. (A) acI cells may utilize a complex carotenoid as an antenna that transfers light energy into the green-light-absorbing proton-pumping rhodopsin. (B) acI cells may utilize a complex carotenoid as protection from excess light and oxidative species to protect rhodopsin.