An Aerobic Anoxygenic Phototrophic Bacterium Fixes CO2 Via the Calvin-Benson

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1 An Aerobic Anoxygenic Phototrophic Bacterium Fixes CO2 via the Calvin-Benson-

2 Bassham Cycle

4 Kai Tang1,2, Yang Liu1, Yonghui Zeng3, Fuying Feng1*, Ke Jin2, Bo Yuan1, 4*

6 1 Institute for Applied and Environmental Microbiology, College of Life Science,

7 Inner Mongolia Agricultural University, Hohhot 010018, China

8 2 Institute of Grassland Research, Chinese Academy of Agricultural Sciences, Key

9 Laboratory of Grassland Ecology and Restoration, Ministry of Agriculture, Hohhot

10 010010, China

11 3 Department of Environmental Science, Aarhus University, Roskilde 4000, Denmark

12 4 College of Life Science, Inner Mongolia Normal University, Hohhot 010018, China

14 *Correspondence: Fuying Feng, [email protected] or Bo Yuan,

15 [email protected]

17 Running title: An AAnPB implements the CBB cycle

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19 Abstract

20 Aerobic anoxygenic phototrophic bacteria (AAnPB) are photoheterotrophs, which use

21 light as auxiliary energy and require organic carbon (OC) for growth. Herein, we report

22 the unusual strain B3, which is a true AAnPB because it requires oxygen for growth,

23 harbours genes for cbb3- and bd-type cytochromes and acsF, and produces

24 bacteriochlorophyll. The B3 genome encodes the complete metabolic pathways for

25 AAnPB light utilization, CO2 fixation via Calvin-Benson-Bassham (CBB) cycle and

26 oxidation of sulfite and H2, and the transcriptome indicated that all components of these

27 pathways were fully transcribed. Expression of the marker genes related to

28 photosynthesis, including pufM for light harnessing and rbcL for CO2 fixation, and the

29 activity of RubisCO, the key enzyme in the Calvin-Benson-Bassham (CBB) cycle,

30 increased in response to decreased OC supply. Large amounts of cell biomass were

31 obtained in liquid BG11 medium under illumination. The strain thus likely

32 photoautotrophically grows using sulfite or H2 as an electron donor. Similar GC

33 contents between photosynthesis, the CBB cycle and 16S rRNA genes and the

34 consistency of their phylogenetic topologies implied that light harnessing and carbon

35 fixation genes evolved vertically from an anaerobic phototrophic ancestor of

36 Rhodospirillaceae in Alphaproteobacteria. In conclusion, strain B3 represents a novel

37 AAnPB characterized by photoautotrophy using the CBB cycle. This kind of AAnPB

38 may be ecologically significant in the global carbon cycle.

40 Keywords: Aerobic anoxygenic phototrophic bacteria, photoautotroph,

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41 photoheterotroph, Calvin-Benson-Bassham cycle

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43 Introduction

44 Aerobic anoxygenic phototrophic bacteria (AAnPB) are obligate aerobes and perform

45 photosynthesis without producing oxygen [1, 2]. They use the pigment

46 bacteriochlorophyll (BChl) and carotenoids to harvest solar energy and the type II

47 reaction centre (RCII) to photochemically perform electron transfer and eventually

48 drive ATP biosynthesis [1, 3]. Light may promote the growth of AAnPB. For instance,

49 the utilization of light energy prolonged the survival of Dinoroseobacter shibae, a major

50 marine AAnPB, under organic carbon (OC) starvation [4, 5]. A freshwater AAnPB

51 strain, Aquincola tertiaricarbonis L108, was induced to produce BChl a and used light

52 as an auxiliary energy source to maintain its growth only when fed at an extremely low

53 substrate rate [6]. In addition to its reported promotion of the growth of pure cultured

54 isolates, light was also found to enhance the growth rate of AAnPB species in natural

55 environments [7, 8]. The growth promotion by supplementary light resulted from the

56 absorption of light energy reducing the respiration consumption of OC for energy

57 production, which increased the nutrient supply for the anabolism of AAnPB when OC

58 was less available [9-11]. Therefore, phototrophy makes AAnPB more competitive and

59 dominant than strict heterotrophs in the open ocean, where there is little organic matter

60 available [12].

61 Carbon fixation provides the basis for all life on Earth [13], and seven carbon fixation

62 pathways have evolved in nature [14]. Among the seven pathways, the Calvin-Benson-

63 Bassham (CBB) cycle is the most widespread [15] and responsible for the most carbon

64 fixation on Earth [16]. The key catalyst of this fixation is the enzyme RubisCO, which

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65 is composed of a large subunit encoded by the rbcL gene and in some cases a small

66 subunit encoded by the rbcS gene. Photoautotrophic bacteria, including aerobic

67 cyanobacteria and some obligate or facultative anaerobic anoxygenic phototrophs, use

68 the CBB cycle for CO2 fixation, enabling autotrophic growth [17, 18]. The first clue to

69 AAnPB harbouring a complete CCB pathway was a recent marine metagenomics

70 analysis that revealed that the assembled genome of a globally distributed AAnPB

71 possessed both pufM and rbcL/S genes, demonstrating great potential for primary

72 productivity [19]. However, whether the genes associated with solar energy utilization

73 and carbon fixing are expressed and how the related physiological activities are

74 performed still need to be ascertained through multiple assays, especially those based

75 on pure cultured bacterial strains. Additionally, evidence was found that some AAnPB

76 showed the physiological activities for inorganic carbon fixation in other carbon

77 fixation pathways, but that was not substantial enough to support autotrophic growth.

78 For example, Roseobacter clade bacteria could utilize carbon monoxide as an additional

79 energy or carbon source [11, 20], and Dinoroseobacter shibae fixed CO2 using the

80 ethylmalonyl-CoA (EMC) pathway [21].

82 In arid and semiarid areas, biological soil crusts (BSCs) are widely distributed as one

83 of the major features of the Earth’s terrestrial surface and play a vital role in the global

84 carbon cycle [22]. Benefiting from supplementary light, AAnPB may occupy an

85 important niche in such organic substrate-poor terrestrial environments [23, 24].

86 Moreover, our previous work showed that AAnPB could greatly enhance organic matter

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87 content and promote the development of BSCs [25]. For these reasons, the contribution

88 of carbon accumulation by AAnPB was hypothesized to be significant in BSCs.

89 In this study, we used the spread and streak plate technique to isolate AAnPB strains

90 from BSCs. Genome and transcriptome analyses revealed that one AAnPB strain, B3,

91 possessed the genes for a full CCB cycle pathway and that all the components in this

92 pathway were transcribed. Physiological assays further ascertained that the strain

93 produced the enzyme RubisCO with high physiological activity and was

94 photoautotrophic with high biomass in media with little or no OC added. This will shed

95 light on the evolution of AAnPB and their role in the global carbon cycle.

97 Materials and Methods

98 Sampling and isolation

99 Moss-dominated biological soil crusts (BSCs) (about 2 cm of top soil) were collected

100 in October 2016 from sand dunes (42.427 N, 116.769 E, 1380 m asl) located in the

101 southeastern Hunshadake Desert in China. The sampled soils were stored in aseptic

102 sampling bags in a vehicle-mounted refrigerator and brought to the laboratory within

103 72 hours for further treatment.

104

105 Bacteria were isolated on 1/2-strength R2A agar (Table S1) using standard tenfold

106 dilution plating and streak cultivation techniques. The plates were incubated aerobically

107 at 28 °C under a light regimen of 4000 lux with a 12/12 hour cycle (light/dark).

108

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109 Identification of AAnPB strains

110 Bacterial strains possessing the pufM gene, encoding the M subunit of the type II

111 reaction centre, were tentatively considered AAnPB. The presence of the pufM gene

112 was confirmed through PCR and sequencing. The PCR template for pufM amplification

113 was prepared using a rapid cell lysis protocol for which colonies harvested from plates

114 were directly incubated in 0.05 M NaOH with 95/4 °C cycling. The 27F/1492R and

115 pufM_557F/pufM_WAW primer pairs (Table S2) were used to amplify 16S rRNA and

116 pufM genes, respectively, and PCR products were sequenced using the Sanger

117 sequencing method by Sangon Biotech Comp, Shanghai. The absorption spectra of

118 pufM-positive isolates in vivo were recorded on a Synergy H4 Hybrid Reader (BioTek)

119 with a scanning range from 300 to 900 nm. Bacteriochlorophyll pigments were

120 extracted from cells grown in 1/10-strength R2A liquid medium with pre-cooled

121 acetone/methanol (7:2) for 2 hours and determined using HPLC as described previously

122 [25]. BChl a standard was purchased from Sigma-Aldrich Fine Chemicals.

123

124 Tests of oxygen requirement for growth

125 Strain B3 was incubated on 1/2-strength R2A agar in an anaerobic atmosphere by using

126 MGC AnaeroPack-JAR and MGC AnaeroPack-Anaero (Mitsubishi Gas Chemical

127 Company, Tokyo, Japan) (Figure S1a) and agar shake tube technique [26] on Medium

128 A [27] (Figure S1b). The culture was conducted at 28 °C under illumination (4000 lux

129 of luminous intensity, 12 h/12 h light-dark cycle) and no illumination (in dark) (Figure

130 S1b). One facultative anaerobic strain, Rhodocista centenaria DSM9894T [28] was

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131 used as a reference, and three replicates were set up in these tests of oxygen requirement

132 for growth.

133

134 Photoautotrophic growth tests

135 Cell biomass (fresh and dry cell weights) was used to determine the photoautotrophy

136 of AAnPB strains grown in BG11 liquid media (Table S1). Cells grown in 1/10 R2A

137 medium were centrifuged, washed three times and resuspended in NaCl solution

138 (0.08%). The cell suspension (approximately 1.00 of OD600 nm) of 100 µL was

139 inoculated into 50 mL of BG11 liquid medium with variant trace levels of vitamin B12

140 to incubate in a shaker at 180 r/min under illumination (4000 lux of luminous intensity,

141 12 h/12 h light-dark cycle). After five days of incubation, the cells were collected by

142 centrifugation. The centrifuge tubes were placed upside-down and air-dried for 2 hours,

143 and the fresh cell biomass was determined. Dry cell weights were obtained after drying

144 at 80 ℃ for 24 hours. The levels of vitamin B12 were 0, 0.2, 0.4 and 1.0 g per 50 mL

145 of medium. Five repeats were carried out.

146 Total bacterial numbers of strain B3 were used to determine the growth curve of AAnPB

147 strains in BG11 liquid media under illumination and dark. The pH was adjusted to 7.2,

148 1.0 μg per 50 mL vitamin B12 was added, and the sample was then incubated in a shaker

149 at 180 r/min under illumination (4000 lux of luminous intensity, 12 h/12 h light-dark

150 cycle). Cell numbers were counted by hemacytometer (Qiujing XB-K-25, Shanghai,

151 China) under a phase contrast microscope (Nikon ECLIPSE Ti, Japan), and the

152 following formula was used:

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153 Cell numbers(/ml) = 4 ∗ N ∗ 106

154 the letter “N” refers to the cell number in one chamber unit.

155

156 Treatments of different organic carbon levels

157 Based on R2A, four OC treatments (labelled 5A, 2.5A, A and 0A, respectively) (Table

158 S1) were set up to determine the effect of OC on photosynthetic gene expression and

159 RubisCO enzyme activity. Cells were cultivated in a thermostatic oscillator at 180 r/min

160 at 28 ℃ under 4000 lux with a 12/12 hour (light/dark) cycle; then cells at the early

161 steady growth phase were harvested by centrifugation at 12,000 × g under 4 °C for 15

162 min.

163

164 RubisCO enzyme activity assay

165 The RubisCO enzyme activities at different OC levels were measured in a reaction

166 mixture (200 μl) containing the buffer, substrates, cofactor and enzymes prepared

167 according to Takai et al. [29] and measured by spectrophotometry as described by Yuan

168 et al. [30]. Briefly, 200 μl of reaction liquid was transferred to a 96 micro-well plate,

169 and the absorption at 340 nm was measured using the Synergy H4 Hybrid Reader

170 (BioTek). Then, after 0.1 mL of 25 mM ribulose-1,5-bisphosphate (RuBP) was added

171 to the well and reacted for 30 s, the absorption at 340 nm was measured again. For the

172 control treatment, no RuBP was added. To calculate RubisCO’s enzyme activity, the

173 following formula was used: ∆A*N*10 174 RubisCO enzyme activity (µmol/(min·g))= 6.22*2*d*∆t

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175 where ∆A is the change in OD340 nm during 30 s the change in the control, the letter “N”

176 refers to the dilution ratio (100-fold used in this study), 6.22 is the absorbency

177 coefficient of 1 mm NADH at OD340 nm, ∆t is the reaction time (30 s in this study) and

178 the letter “d” represents the colorimetric optical path, which was 0.0635 cm in this study.

179

13 180 CO2 assimilation assay

181 First, 1.59 mg/L NaH13CO3 (98 atom% 13C, Sigma-Aldrich, USA, no. 372382) was

182 added to liquid BG11 medium to test the CO2 assimilation ability of strain B3. Then

183 5.0 ml of strain B3 BG11 medium (OD=0.03) was added into 4.0 L of liquid BG11

184 medium containing NaH13CO3, which was then cultured with magnetic stirrers (IT-

185 07A3, Shanghai Yi Heng Co., Ltd, Shanghai, China) at 180 r/min at 28 °C under a light

186 regimen of 4000 lux and a 12/12 hour (light/dark) cycle. After 120 hours of culture, the

187 cells were acquired by centrifuged at 12,000 × g for 10 min, washed 3 times with 10-3

188 M H3PO4 (pH=3.0), and lyophilized using a freeze dryer (Christ Alpha 1-2 LD Plus,

189 France).

13 190 The C contents in cell matter (after combustion to CO2) and in the CO2 gas used as a

191 carbon source were determined by a Thermo Scientific MAT 253 plus mass

192 spectrometer fit with a Gasbench II-IRMS at Beijing Zhong Ke Bai Ce Testing

193 Technology, China. The δ13C value was presented as per mille deviation from the

194 Vienna Pee Dee Belemnite standard (VPDB) [31, 32] as follows:

13 13C/12C sample 3 195 δ C=( − 1) ∗ 10 (‰) 13C/12C standard

196

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197 Whole-genome sequencing and analysis

198 The genomic DNA of the B3 strain was extracted using a Bacterial Genomic DNA

199 Purification Kit (DP302-2, Tiangen Biotech Co., Beijing, China) according to the

200 manufacturer’s protocols. Genome sequencing was performed on the PacBio RSII

201 platform at Shanghai Majorbio Pharm Technology Co., Ltd. After filtering the low-

202 quality reads and adapters, clean reads were assembled into a single gap-free contig

203 using HGAP ver 3.0 [33]. The open reading frames (ORFs) of the assembled genome

204 were predicted with Glimmer ver. 3.02 [34]. The genome was annotated by the Kyoto

205 Encyclopedia of Genes and Genomes (KEGG) website (http://www.genome.jp/kegg/)

206 [35] and visualized with Circos software [36]. SWISS-MODEL was used to model the

207 RubisCO enzyme structure online based on cbbL/S/X genes online

208 (https://swissmodel.expasy.org/interactive) [37].

209

210 Assays for gene expression quantification and transcriptomics

211 Cells were grown in media with different levels of OC sources (Table S1), and the

212 expression of genes for light utilization and carbon fixation was determined and

213 compared. Cells were cultivated in a thermostatic oscillator at 180 r/min and 28 ℃

214 under natural light for five days and then collected through centrifugation at 12,000 ×

215 g for 15 min at 4 °C.

216 Total cellular RNA was extracted using the RNAprep Pure Cell/Bacteria Kit (DP430,

217 Tiangen Biotech Co, Beijing). RNA integrity was examined through agarose gel

218 electrophoresis, and RNA purity was determined with a NanoDrop spectrophotometer

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219 (Thermo Fisher Scientific). After pretreatment with RNase-free DNase I (TaKaRa,

220 Dalian, China), total RNA (1 μg) was used to synthesize first-strand cDNA by

221 employing an M-MLV Reverse Transcriptase (code no. 2641A, TaKaRa Bio Inc.,

222 Dalian, China) according to the manufacturer’s instructions. The cDNA was employed

223 as a template using SYBR Premix Ex Taq II (Takara) in a LightCycler 480 Real Time

224 PCR system (Roche, Basel, Switzerland) for qRT-PCR analysis, including the 16S

225 rRNA, pufM and rbcL genes. The primers and thermal programs are described in Table

226 S2. The standard curve of the 16S rRNA gene was created as previously described [25].

227 The 16S rRNA gene was employed as a reference gene to normalize the expression

228 levels of target genes in samples, and the 2-ΔΔCT method [38] was used to calculate the

229 relative expression levels of the target genes. Three technical replicates of qPCR were

230 performed for each reaction.

231 Total RNA sequencing was conducted by Shanghai Majorbio Pharm Technology Co.,

232 Ltd. (Shanghai, China) on a HiSeq Illumina platform. RNA-seq reads were trimmed

233 using SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (ver. 1.33). Diamond

234 (ver. 0.8.35) (https://github.com/bbuchfink/diamond) was used to ensure that the rRNA

235 ratio was lower than 5%. Based on the Burrows-Wheeler transform, Bowtie 2 (ver. 2.2.9)

236 was used to align trimmed clean reads [39]. The relative expression in transcripts per

237 million reads (TMP) was estimated by Salmon (ver. 0.8.2)

238 (https://github.com/COMBINE-lab/salmon). ORFs were annotated with the KEGG

239 database.

240

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241 Assays for putative electron donors for carbon fixation

242 After the surveys of the genome and transcriptome, potential electron donors were listed

243 and then added to cultures to observe their effects on the biomass. Compounds

244 markedly enhancing cell biomass were probable electron donors [40]. Na2SO3 and H2

245 were selected and further assayed, respectively. Na2SO3 (48 mg/L) was added into

246 liquid BG11. H2 was filled as headspace gas (20% H2 + 80% air). The cultivation

247 conditions, cells harvesting and biomass (cells dry weight) were performed as described

248 above.

249

250 Phylogenetic analyses

251 Phylogenies of the 16S rRNA, photosynthetic and carbon fixation genes were

252 constructed and compared to reveal the evolution of AAnPB and their photosynthetic

253 apparatus. The phylogenetic tree of phototrophic bacteria [41] was referred to to obtain

254 reference sequences of the 16S rRNA gene from EzBioCloud (www.ezbiocloud.net/).

255 The pufLMC-bchXYZ reference amino acid sequences were recovered from Imhoff et

256 al. [41]. Reference amino acid sequences of rbcL were obtained from the NCBI

257 (nonredundant protein database, nr) through BLASTP ver. 2.10.0+ [42]. Multiple

258 alignments were performed with the MUSCLE program, and the maximum-likelihood

259 (ML) phylogenetic trees were calculated using MEGA ver. X with 1,000 tree

260 resamplings. The Kimura 2-parameter model (16S rRNA gene) [43] and Jones-Taylor-

261 Thornton (JTT) amino acid substitution model (pufLMC-bchXYZ and rbcL gene) [44]

262 were used. The phylogenetic trees were visualized using iTOL software online.

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263

264 Statistical analyses

265 Significant differences were calculated using Tukey’s test and identified at P < 0.05 by

266 GraphPad Prism 8.0.

267

268 Results and Discussion

269 Isolate B3 is a true AAnPB of Alphaproteobacteria

270 A pink-pigmented bacterial isolate, designated B3, was obtained on 1/2 strength R2A

271 medium from moss-dominated BSCs, using aerobic spread and streak plates. No

272 growth of B3 was observed on 1/2 R2A agar plates under anaerobic conditions, even

273 for a longer culture time (31 days) (Figure S1a). Under either illumination or no

274 illumination, the strain’s growth was obvious at only the top of the tube (Figure S1b).

275 This was distinct from the growth of the reference strain Rhodocista centenaria

276 DSM9894T, which occurred in the whole tube with Medium A. Rhodocista centenaria

277 DSM9894T was identified as a facultative anaerobic purple non-sulphur bacterium

278 (PNSB) [28]. In Medium A, PNSB were characterized by growing in anaerobic

279 conditions under illumination [27]. These growth tests showed that strain B3 is obligate

280 aerobic. This was also supported by the presence of microaerobic ccb3-type and highly-

281 aerobic bd-type cytochrome oxidases and oxygen-dependent ring cyclase (acsF)

282 revealed through the strain’s genome survey and gene PCR and sequencing (Table S3).

283 Anaerobic phototrophs distinctly lack aerobic cytochrome oxidases especially ccb3-

284 type [45] and bd-type [46] cytochrome oxidases. Gene acsF encodes the aerobic form

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285 of Mg protoporphyrin IX monomethyl ester oxidative cyclase necessary for

286 bacteriochlorophyll (BChl) biosynthesis in oxic environments [47, 48], and therefore,

287 the genome’s possession if ccb3- and bd-type cytochrome oxidase and acsF genes was

288 used as key evidence for ‘Candidatus Luxescamonaceae’ having an aerobic nature and

289 thus being a true AAnPB [19].

290

291 The viable cells of strain B3 grown with vitamin B12 (VB12) supplied showed an

292 absorption peak at 870 nm (Figure 1a), which is a typical feature of type II reaction

293 centres in AAnPB [6, 12]. The HPLC elution profile of the pigment extracts showed a

294 peak at a retention time of 21 min with peak absorption at 710 nm identical to that of

295 standard BChl a (Figure 1b). This validated that strain B3 could produce BChl a under

296 aerobic conditions. However, without supplementation with VB12, BChl a was not

297 detected. VB12 regulated photosystem gene expression to affect the production of Bchl

298 a via the CrtJ antirepressor AerR [49]. In BSCs, cyanobacteria and algae are commonly

299 dominant microbes [50] and common VB12 producers [51]. When we cocultured strain

300 B3 with Microcoleus vaginatus, a cyanobacterium dominant in BSCs, Bchl a

301 production was detected (Figure S2). PCR amplification and sequencing results

302 demonstrated that strain B3 possessed the pufM gene, encoding the M subunit of the

303 type II photosynthetic reaction centre in AAnPB and, the sequence was closely related

304 to that of Rhodocista centenaria (similarity of 83.75%).

305 The 16S rRNA gene of strain B3 shared its highest similarity of 93.09% with that of

306 Oleisolibacter albus NAU-10T, which was a strictly aerobic species belonging to of the

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307 family Rhodospirillaceae of the class Alphaproteobacteria [52], suggesting that strain

308 B3 belongs in Alphaproteobacteria.

309 Collectively, these results strongly suggest that strain B3 is a true AAnPB of

310 Alphaproteobacteria.

311

312 Strain B3 grew photoautotrophically

313 AAnPB can harvest light to obtain additional energy to supplement their heterotrophic

314 growth [6, 53]; some AAnPB strains can even fix CO or CO2, but the fixed carbon

315 cannot support autotrophic growth without a substantial OC supply [12, 20, 21]. Under

316 illumination, strain B3 grew in liquid BG11 media in the presence of trace citrate (6

317 mg/L) and VB12 (0, 0.2, 0.4 and 1.0 g per 50 mL) without other OC addition (Figure

318 2a). At 0.4 g/50 mL VB12, the biomass of fresh and dry cells peaked at 110 mg and 8

319 mg per 50 mL, respectively. The production of newly synthesized biomass from CO2 is

13 320 a sign of carbon autotrophic microbes [54]. Moreover, the cells grown with CO2

321 exhibited δ13C values of 24.71 - 30.65 %, and this directly supported strain B3 having

322 the ability to fix CO2 under illumination (Table 1). Cultivated in BG11 media under

323 illumination, strain B3 showed a typical growth curve of single-celled bacteria with a

324 lag phase of 5 hours and logarithmic phase of 50 hours; in contrast, under dark, the

325 strain cell numbers were only slightly increased after 80 hours (Figure 2b). These

326 suggested that strain B3 could grow photoautotrophically. Photoautotrophy may make

327 strain B3 more competitive in OC-deficient BSCs. In fact, the inoculation of strain B3

328 effectively promoted the development of BSCs and the soil OC heavily accumulated

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329 [55], suggesting that AAnPB may make great contributions for carbon sequestration in

330 arid land. High biomass yield through carbon fixation of CO2 may help solve the global

331 warming problem [56]. Metagenomic analysis indicated that AAnPB was a potential

332 significant contributor to the carbon cycle in marine environment, because they may

333 widely distributed in CBB at high abundance [19]. Therefore, photoautotrophic AAnPB

334 may be ecologically important worldwide.

335

336 Genome of strain B3 contains complete pathways of the type II reaction centre for

337 light utilization and the CBB cycle for CO2 fixation

338 The whole genome of strain B3 was sequenced to explore the metabolic pathways

339 responsible for its photoautotrophic growth, and the GenBank accession number is

340 CP051775. The results showed that the genome of strain B3 contained one chromosome

341 with a length of 3664154 bp and a GC content of 69.26 % and four plasmids (Figure

342 3a; Table S4).

343

344 The photosynthetic genes clustered into a photosynthesis gene cluster (PGC), which is

345 a typical feature in most purple phototrophic bacteria [57], heliobacteria [58] and

346 Gemmatimonadetes [48]. The PGC identified in the B3 genome is 44.3 kb long and

347 consists of genes in the order bchODIPG, puhF-acsF-puhBA-bchMLHBA and

348 pufCMLAB-bchZYXC-crtF (Figure 3b), similar to those of proteobacterial AAnPB

349 [59]. As the presence of puf genes, which encode bacterial reaction centre subunits, and

350 genes necessary for bacteriochlorophyll biosynthesis in the PGC suggests, strain B3

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351 may carry out a phototrophic strategy with type II reaction centres.

352

353 To date, there are seven known pathways for carbon fixation, including the CBB cycle,

354 reductive tricarboxylic acid (rTCA) cycle, 3-hydroxypropionate bi-cycle (HBC),

355 Wood-Ljungdahl (WL) cycle, dicarboxylate/4-hydroxybutyrate (DH) cycle, 4-

356 hydroxypropionate cycle, and reductive glycine pathway [14]. None of pathways

357 except CBB has been identified in strain B3. Strain B3 assimilates CO2 through the

358 CBB cycle, which is complete in the genome (Figure 3c). RubisCO is a key enzyme of

359 the CBB cycle, in which CO2 serves as a substrate in organisms evolved from a non-

360 CO2-fixing and non-autotrophic ancestor [60]. The strain B3 genome possesses both an

361 rbcL and an rbcS gene, encoding large and small subunits of RubisCO, respectively.

362 The cbbX gene encoding RubisCO activase, similar to that of Rhodobacter sphaeroides

363 [61], was also identified in B3. Additionally, a complete pathway of crassulacean acid

364 metabolism (CAM) is present in B3 (Table S3), which is another possible mechanism

365 for the concentration of CO2, in addition to that through the small subunits of RubisCO,

366 and it is widely employed in C4 plants and some algae [62].

367

368 Pathways of light utilization and CO2 fixation are active in strain B3

369 To confirm whether the photosynthetic pathways for light utilization and CO2 fixation

370 are active, we determined the shifts in the transcriptome, the physiological activity of

371 the enzyme RubisCO and the quantitative expression levels of the pufM and rbcL genes

372 in response to varying OC concentrations. Transcriptome analysis showed that genes

18 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.29.441244; this version posted April 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

373 involved in the bacteriochlorophyll-dependent light utilization of the type II reaction

374 centre, CO2 fixation of the CBB cycle and CO2 condensation of CAM were all

375 transcribed at different levels (Figure 4). The expression levels of both pufM and rbcL

376 genes and RubisCO enzyme activity were significantly enhanced in strain B3 when the

377 OC concentration greatly decreased (Figure 5). The increased expression in response to

378 the OC limit implied that the AAnPB strain B3 modified its energy and carbon source

379 strategy from heavily relying on chemical energy from OC to relying more on optical

380 energy and CO2 fixation via the CBB cycle. The strategy of light utilization was

381 commonly adopted by other AAnPB as well [6-8, 10, 63, 64], and OC starvation drives

382 a few AAnPB strains to assimilate CO2 [21] or CO [29, 65], but carbon fixation via the

383 CBB cycle has not been reported. Moreover, the pufM and rbcL gene copy numbers

384 were greatly correlated with cell density (r=0.99, P=0.0034; r=0.80, P=0.198) and cell

385 biomass (r=0.97, P=0.0318; r=0.89, P=0.108) (Table S5). The strong correlation of

386 photosynthetic genes and growth features suggested that light utilization and CO2

387 fixation were connected in this strain. This may be explained as light energy is primarily

388 consumed by CO2 fixation through the CBB cycle in microbial photoautotrophy [66,

389 67].

390

391 Putative electron donors for fixation of CO2 via the CBB cycle

392 Sulfite and hydrogen could be the putative electron donors of strain B3 because of the

393 following evidence. Firstly, the strain harboured and transcribed the key genes soxYZ

394 and those for [NiFe]-H2ases responsible for sulfite [68, 69] and H2 [70] oxidation to

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395 provide electrons, respectively (Table S3). Secondly, the cell biomass (dry weight) was

396 remarkably improved by the addition of Na2SO3 or the increase in H2 as headspace gas,

397 with the maximum increases of 2.82- and 1.12-fold in the presence of Na2SO3 and H2

398 in this experiment, respectively.

399

400 Photosynthesis-related genes in strain B3 may be vertically evolved from its

401 facultative relatives

402 G+C contents, codon usage, amino acid usage, and gene position were usually used to

403 understand the gene’s origin, i.e., acquisition by inheritance or horizontal gene transfer

404 (HGT) [71]. As described above, strain B3 harbours photosynthesis-related genes,

405 including genes related to light utilization (clustered into PGCs as in other AAnPB) and

406 CO2 fixation (in the CBB and CAM cycles). These genes had similar GC contents to

407 that of the whole genome (Table S3). The similar GC content implied that the genes

408 may be inherited in the genome and not inserted via HGT [72]. Moreover, BLAST

409 analyses revealed that most of the photosynthesis-related genes of strain B3 were

410 affiliated with genes from the Rhodospirillaceae family (Table S3). Therefore, it is most

411 likely that the genes for light harnessing and the CO2 fixation pathways in strain B3 are

412 vertically inherited and evolved rather than acquired through HGT from its facultative

413 relatives.

414 The phylogenetic position of strain B3 further supported the above hypothesis, as the

415 photosynthetic gene clusters (pufMLC-bchXYZ), rbcL and rbcS coding for RubisCO,

416 and the 16S rRNA gene were most closely related to the corresponding genes in the

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417 Rhodospirillaceae family (Figure 6), which is distinct from the incongruences within

418 the phylogenetic topologies between PGCs and 16S rRNA genes that represents a

419 common incident of HGT of PGC in AAnPB [73]. The taxonomic position of strain B3

420 among purple phototrophic bacteria implied that the strain B3 probably evolved from

421 an anaerobic ancestor of Rhodospirillaceae. Anaerobic members of Rhodospirillaceae

422 had earlier differentiation times than close neighbours in Time Tree [74, 75]. During

423 the adaptation to from anoxic to oxic conditions on Earth, anaerobic anoxygenic

424 phototrophic bacteria evolved into AAnPB by modifying their photosynthetic apparatus

425 biosynthesis strategy in response to oxygen levels, such as by the loss or acquisition of

426 certain abilities [41]. However, as the cost of phototrophy is high compared to that in

427 other presently known AAnPB, light energy is auxiliary to heterotrophy based on OC

428 consumption [9]. Carbon fixation via the CCB cycle requires more energy [66], and no

429 other pure-cultured AAnPB is known to use this pathway today. Surprisingly, strain B3

430 inherited not only a light energy utilization apparatus but also CO2 fixation by the CBB

431 cycle from its anaerobic phototrophic ancestor, which suggests that the strain represents

432 a novel AAnPB lineage.

433

434 Conclusion

435 Strain B3 represents a novel aerobic anoxygenic phototrophic lineage characterized by

436 photoautotrophy using the Calvin-Benson-Bassham (CBB) cycle, in contrast to the

437 photoheterotrophy of other currently known pure-cultured AAnPB. This kind of

438 AAnPB may be ecologically significant in the global carbon cycle.

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439 Acknowledgements

440 This research was supported by grants from the National Natural Science Foundation

441 of China (No. 31560030 & 31760025 & 32001220).

442

443 Author contributions

444 KT carried out the experiments and prepared the draft of the manuscript. KT, YL, HL,

445 BY, KJ and YZ analysed the data. FF and BY designed the research and wrote the

446 manuscript with input from KT.

447

448 Compliance with ethical standards

449 The authors declare that they have no conflicts of interest.

450

451 References

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27 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.29.441244; this version posted April 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

664 Table 1 δ13C ratios in dried strain B3 cells. 665 Total mass Total 13C 12C δ 13C Samples 13C/12C (g) C (%) (mg) (mg) (‰) B3-1 0.84 7.11 0.68 59.03 1.15% 24.71 B3-2 0.75 6.28 0.54 46.59 1.16% 27.89 B3-3 0.68 6.21 0.48 41.77 1.16% 30.65

666

667 Figure legends

668 Figure 1. Cell and pigment characterization of strain B3. (a) Absorption spectrum of

669 viable cells between 300 - 900 nm; the top right shows the cell pellets. (b)

670 Chromatogram of extracted pigments; the top right shows the extract of the pigments.

671

672 Figure 2. Biomass of strain B3 in 50 mL of liquid BG11 with different levels of added

673 VB12 (a), and the growth curve of strain B3 in liquid BG11 under illumination and dark

674 (b). Letters (a, b, or c) in the superscript represent the observed significant differences

675 (Tukey’s test; P < 0.05).

676

677 Figure 3. Genome, photosynthesis gene clusters and carbon fixation metabolism of

678 strain B3. (a) Circular genome of strain B3. The first outer circle shows the size of the

679 genome; the second and third are the coding sequences (CDSs) with different colours

680 denoting various functions based on COG; the fourth circle contains photosynthesis

681 gene clusters (PGCs) and rbcL/S. PGCs are distributed in three positions in the genome.

682 The fifth circle is the GC content, and the red and blue peaks represent higher or lower

683 GC content than average; the inside is the value of GC-skew, and green and orange

684 represent the leading and lagging strands, respectively. (b) The PGCs in 6 AAnPB from 28 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.29.441244; this version posted April 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

685 Alpha-, Beta-, and Gammaproteobacteria and Gemmatimonadetes. (c) Overview of the

686 metabolic pathway of the Benson-Bassham-Calvin cycle (CBB).

687

688 Figure 4. Heatmap of the expression of genes involved in carbon fixation (a) and

689 photosynthesis gene clusters (PGCs) after five days of culture at an OC concentration

690 of 2.5A (b), including the bch, puf, puh, and crt operons and the acsF gene. The values

691 of the gapA and pufB genes are outside the defined range (TPM values are 201.54 and

692 386.06, respectively) and are represented in white. Transcripts per million reads (TPM

693 value) were used to represent differences in gene expression.

694

695 Figure 5. Relative gene expression and RubisCO enzyme activity changes in strain B3

696 at different organic carbon levels. The relative expression of pufM and rbcL genes (a),

697 RubisCO enzyme activity per cell; (b) changes in different organic carbon levels,

698 including 5A (blue), 2.5A (red), A (green) and 0A (purple). Letters (a, b, or c) in the

699 superscript represent the observed significant differences (Tukey’s test; P < 0.05).

700

701 Figure 6. Phylogenetic tree based on 16S rRNA gene (a) sequences, pufMLC-bchXYZ

702 gene clusters (b) and rbcL genes (c). A phylogenetic tree was constructed using both

703 ML methods. Numbers at nodes are bootstrap values (ML, X, no bootstrap value in ML

704 tree where nodes differ in both dendrograms; value<50% not shown). The bar

705 represents 0.01, 0.02 and 0.05 substitutions per alignment position.

706

29 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.29.441244; this version posted April 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1. Cell and pigment characterization of strain B3. (a) Absorption spectrum of viable cells between 300 - 900 nm; the top right shows the cell pellets. (b) Chromatogram of extracted pigments; the top right shows the extract of the pigments.

Figure 2. Biomass of strain B3 in 50 mL of liquid BG11 with different levels of

added VB12 (a), and the growth curve of strain B3 in liquid BG11 under illumination

and dark (b). Letters (a, b, or c) in the superscript represent the observed significant

differences (Tukey’s test; P < 0.05).

Figure 3. Genome, photosynthesis gene clusters and carbon fixation metabolism of strain B3. (a) Circular genome of strain B3. The first outer circle shows the size of the genome; the second and third are the coding sequences (CDSs) with different colours denoting various functions based on COG; the fourth circle contains photosynthesis gene clusters (PGCs) and rbcL/S. PGCs are distributed in three positions in the genome. The fifth circle is the GC content, and the red and blue peaks represent higher or lower GC content than average; the inside is the value of GC-skew, and green and orange represent the leading and lagging strands, respectively. (b) The PGCs in 6 AAnPB from Alpha-, Beta-, and Gammaproteobacteria and Gemmatimonadetes. (c) Overview of the metabolic pathway of the Benson-Bassham-Calvin cycle (CBB). bioRxiv preprint doi: https://doi.org/10.1101/2021.04.29.441244; this version posted April 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4. Heatmap of the expression of genes involved in carbon fixation (a) and photosynthesis gene clusters (PGCs) after five days of culture at an OC concentration of 2.5A (b), including the bch, puf, puh, and crt operons and the acsF gene. The values of the gapA and pufB genes are outside the defined range (TPM values are 201.54 and 386.06, respectively) and are represented in white. Transcripts per million reads (TPM value) were used to represent differences in gene expression. bioRxiv preprint doi: https://doi.org/10.1101/2021.04.29.441244; this version posted April 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 5. Relative gene expression and RubisCO enzyme activity changes in strain

B3 at different organic carbon levels. The relative expression of pufM and rbcL genes

(a), RubisCO enzyme activity per cell; (b) changes in different organic carbon levels,

including 5A (blue), 2.5A (red), A (green) and 0A (purple). Letters (a, b, or c) in the

superscript represent the observed significant differences (Tukey’s test; P < 0.05).

Figure 6. Phylogenetic tree based on 16S rRNA gene (a) sequences,

pufMLC-bchXYZ gene clusters (b) and rbcL genes (c). A phylogenetic tree was

constructed using both ML methods. Numbers at nodes are bootstrap values (ML, X,

no bootstrap value in ML tree where nodes differ in both dendrograms; value<50%

not shown). The bar represents 0.01, 0.02 and 0.05 substitutions per alignment

position.