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1 Type II photosynthetic reaction center genes of avocado (Persea americana Mill.) bark
2 microbial communities are dominated by aerobic anoxygenic Alphaproteobacteria
3
4 Eneas Aguirre-von-Wobeser
5
6
7 CONACYT – Centro de Investigación y Desarrollo en Agrobiotecnología Alimentaria, Centro de
8 Investigación y Desarrollo, A.C., Blvd. Sta. Catarina s/n, Col. Santiago Tlapacoya, 42110, San Agustín
9 Tlaxiaca, Hidalgo, Mexico
10
11
12 Correspondance:
13 E. Aguirre-von-Wobeser
15 52-55-25-24-86-26
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16 Abstract
17
18 The tree bark environment is an important microbial habitat distributed worldwide on thrillions of
19 trees. However, the microbial communities of tree bark are largely unknown, with most studies on plant
20 aerial surfaces focused on the leaves. Recently, we presented a metagenomic study of bark microbial
21 communities from avocado. In these communities, oxygenic and anoxygenic photosynthesis genes
22 were very abundant, especially when compared to rhizospheric soil from the same trees. In this work,
23 Evolutionary Placement Algorithm analysis was performed on metagenomic reads orthologous to the
24 PufLM gene cluster, encoding for the bacterial type II photosynthetic reaction center. These
25 photosynthetic genes were found affiliated to different groups of bacteria, mostly aerobic anoxygenic
26 photosynthetic Alphaproteobacteria, including Sphingomonas, Methylobacterium and several
27 Rhodospirillales. These results suggest that anoxygenic photosynthesis in avocado bark microbial
28 communities functions primarily as additional energy source for heterotrophic growth. Together with
29 our previous results, showing a large abundance of cyanobacteria in these communities, a picture
30 emerges of the tree holobiont, where light penetrating the trees canopies and reaching the inner stems,
31 including the trunk, is probably utilized by cyanobacteria for oxygenic photosynthesis, and the far-red
32 light aids the growth of aerobic anoxygenic photosynthetic bacteria.
33
34 Keywords: Bark microbial communities, Metagenomics, Aerobic Anoxygenic Photosynthesis, PufLM,
35 Persea americana (avocado)
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36 Introduction
37
38 There are trillions of trees growing worldwide [1], constituting an important habitat for epiphytic
39 bacteria. However, most of the attention to tree aerial habitat has been centered on the leaves, with stem
40 bark as a microbial habitat largely neglected [2]. Recently, 16S microbiome surveys of bark from
41 different woody plants have revealed bark surfaces as important microbial environments, usually more
42 diverse than the leaves [3-7].
43 Recently, a bark metagenomic study was conducted on bark from avocado trees, revealing highly
44 diverse microbial communities dominated by bacteria, with the presence of archaea, fungi, algae and
45 other eukaryotic groups [8]. Functional analysis of these communities revealed the presence of many
46 oxygenic and anoxygenic photosynthetic genes, which were absent from rhizospheric soil from the
47 same trees [9]. The presence of these genes in avocado bark suggests active phototrophic communities.
48 However, a detailed phylogenetic analysis is needed to better understand the bacterial groups
49 potentially capable of anoxygenic photosynthesis in these communities.
50 Phylogenetic analysis of metagenomic sequences is challenging, as many non-overlapping fragments
51 are found of a potentially diverse collection of othologs for a given gene. This complicates the correct
52 alignment of reads, and can result in biases during tree reconstruction. Atamna-Ismaeel et al. [10]
53 reconstructed phylogenetic trees from the genes encoding for the type II photosynthetic reaction center,
54 PufL and PufM for a collection of different metagenomic data-sets, including several from plant leaves.
55 By using several datasets, and focusing in a fragment from each gene, they managed to get enough
56 coverage for alignment and three inference. However, this approach proved difficult to implement for a
57 single metagenomic dataset.
58 Recently, Evolutionary Placement Algorithms (EPA) have been developed, in which a reference tree is
59 constructed from known sequences of the gene of interest, and the most likely position of each query
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60 metagenomic read is found in the branches of that tree [11]. These algorithms have many advantages,
61 as any number of reads can be mapped (or placed, as it is formally termed) to the tree. Furthermore,
62 reads are not only placed at the branches of the tree, but they can find their position at the inner
63 branches. These inner branch placements can be interpreted as phylogenetic relations with the taxa at
64 the tips, equivalent to new bifurcating branches in a traditional phylogenetic tree. The deeper a
65 placement is observed, the more ancestral its relation with the taxa at the tips.
66 In this work, the diversity and phylogenetic affiliation of anoxygenic photosynthetic bacteria utilizing
67 type II reaction centers in avocado bark microbial communities is explored. EPA analysis was
68 performed on the avocado bark metagenomic dataset presented previously [8, 9] of the PufLM gene
69 cluster, encoding for a type II photosynthetic reaction center. A dominance of aerobic anoxygenic
70 photosynthetic bacteria was clearly observed among PufLM-carrying bacteria of the avocado bark
71 environment.
72
73 Methods
74
75 Metagenomic dataset
76
77 The analysis presented in this work is built on a database of metagenomic data from Hass variety
78 avocado tree bark, which was presented before [8]. Here, I present an analysis performed on DNA
79 sequences from an organic-managed orchard located near Malinalco, Mexico. Details on the orchard
80 can be found at Aguirre-von-Wobeser et al., [8]. Approximately 5 x 5 cm incisions were collected at
81 about 1 m from the ground on the main stem (trunk) of avocado trees. The periderm was carefully
82 removed and subjected to DNA extraction using a PowerSoil extraction kit (Qiagen, USA). Three bark
83 samples from the organic orchard yielded high-quality DNA, and where used for this analysis.
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84 Extracted DNA was used for library preparation with a DNA Flex kit (Illumina, USA) and sequenced
85 on a nextSeq machine (Illumina).
86
87 Identification of PufLM reads
88
89 PufLM fragments were identified in the metagenomic reads from avocado bark using the blastx
90 algorithm, run with Diamond [12], using a e-value threshold of 0.001. Two collections of PufLM
91 sequences were used as references for this step. First, a comprehensive PufLM database from Imhoff et
92 al. [13] was used, which contains well-characterized PufLM genes from genome-sequencing projects,
93 most of which were derived from type strains. This database contains 197 full-length sequences of
94 concatenated PufL and PufM genes, and is representative of the current knowledge on the diversity of
95 these genes, covering strains with a wide range of phylogenetic affiliations and environmental origins.
96 Second, a database which was originally derived from environmental samples through metagenomics
97 from phyllospheres of trees was used [10]. This database has fractions of the pufL and pufM genes in
98 separate files. Therefore, the reads identified with blastx using these references correspond to matches
99 restricted to these fractions.
100
101 Phylogenetic placement
102
103 All reads identified as fractions of the PufL and PufM reads were placed on a phylogenetic tree of the
104 Imhoff et al. [13] database, for which they were previously put in the adequate reading frame (reverse
105 complement as necessary) and translated to amino acids. To build the reference tree, the PufLM
106 sequences were re-aligned using Muscle [14], resulting in an alignment almost identical as in the
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107 original database. This alignment was used for phylogenetic tree reconstruction with RAxML [15]. The
108 most adequate substitution model for these sequences was determined by RAxML to be LG.
109 The metagenomic reads were aligned to the reference alignment using PaPaRa [16], resulting in a
110 single file with the reference and query sequences aligned. This alignment, and the reference
111 phylogenetic tree, were used as inputs for the placement algorithm, which was run with the option -f v
112 of RAxML [17]. The resulting placement was explored and visualized with functions from the Gappa
113 package [18].
114
115 Data availability
116
117 All sequences analyzed in this study have been previously presented and are available at NCBI Short
118 Read Archive (BioProject PRJNA656796, accession SUB7881803) and KBase
119 (https://kbase.us/n/69195/32/).
120
121 Results
122
123 Localization of PufLM genes in Bark metagenomic data
124
125 Metagenomic reads corresponding to bacterial reaction center type II (pufLM genes) were located in
126 metagenomic reads from avocado bark samples using blast methods. Two data-sets were used as
127 references for this purpose. The Imhoff et al. [13] reference contained genes from carefully selected
128 strains, including type strains when available, representatives of other strains from groups lacking type
129 materials, and some strains of interest for the study of prokaryotic photosynthesis. The Atamna-Ismaeel
130 et al. [10] reference contained PumL and PufM genes recovered from strains from the leaves of several,
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131 mostly herbaceus plants. We recovered a total of 2400 pufLM metagenomic reads from avocado bark
132 using the Imhoff et al. [13] reference and 4082 using the Atamna-Ismaeel et al. [10] reference (Table
133 1).
134
135 Table 1. PufML reads recovered from the avocado bark metagenomic dataset
Sample Imhoff et al. (2018) Atamna-Ismaeel et al. Atamna-Ismaeel et al.
reference (PufLM) (2012) reference (PufL) (2012) reference (PufM) A1 580 439 497 A4 862 701 789 A5 958 767 889 136
137
138 Phylogenetic placement of PufLM reads
139
140 Evolutionary phylogenetic placement was used to map the short metagenomic reads from avocado bark
141 to the branches of a reference phylogenetic tree. In order to obtain well-characterized phylogenetic
142 affiliations of the avocado bark anoxygenic photosynthetic bacteria, the Imhoff et al. [13] phylogenetic
143 tree was used as a reference for placement. First, the Imhoff et al. [13] sequences were re-aligned, and
144 the phylogenetic tree was reconstructed using RAxML. The obtained tree had essentially the same
145 topology as the published tree from these sequences [13], indicating a robust reconstruction of the
146 phylogenetic relationships of these genes (Figure 1). Evolutionary phylogenetic placement indicated
147 the affiliation of the avocado bark reads to known PufLM reads. The obtained placements were
148 essentially the same with reads collected using the Imhoff et al. [13] reference (Figure 1) and the
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149
150 Fig. 1. Evolutionary Placement of metagenomic reads from avocado (Persea americana) bark on a
151 PufLM tree reconstructed from the sequences presented in Imhoff et al. (2018). The metagenomic reads
152 where selected with blast methods using the PufL and PufM sequences presented in Atamna-Ismaeel et
153 al. (2012). Placements are shown as read counts in the branches of the trees, according to the scale. For
154 the placement, reads from three avocado bark samples were pooled.
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155 Atamna-Ismaeel reference (Figure S1). Most avocado bark PufLM genes were affiliated to a restricted
156 set of branches from the known PufLM phylogeny, including the Sphingomonadales, different groups fo
157 Rhizobiales, and specific branches of Rhodospirillales and Chloroflexales.
158
159 Avocado bark Sphingomonadales
160
161 PufLM genes belonging to the Sphingomonadales were very abundant in bark microbial communities
162 (Figure 1). All of these genes were affiliated to the Sphingomonadaceae family, with no placements
163 mapping to the Erythrobacteraceae. Particularly, most of the genes fragments from this groups mapped
164 to the tip of the branch corresponding to Sphingomonas sanxanigenens, and to a much lower extent to
165 S. lacus. Although the exact species assignation of these reads is not certain, as many species are not
166 represented in the reference, placement analysis indicates that close relatives of S. sanxanigenens
167 dominate the photosynthetic Sphingomonadales on avocado bark.
168 Other PufLM gene fragments from the Sphingomonadaceae family mapped to the base of the
169 Novosphingobium genus branch, including Erythrobacter marinus, which is probably misclassified
170 [13]. This suggests the presence of photosynthetic bacteria related to Novosphingobium, belonging to
171 species not included in the reference sequences.
172
173 Avocado bark Rhizobiales
174
175 Many reads from avocado bark microbial communities where mapped to different Rhizobiales groups
176 by the placement algorithm. Of these, one branch of Methylobacterium, which included M. oryzae, M.
177 populi and Methylobacterium radiotolerans had many placements at the tips, as well in internal
178 branches. This shows that avocado bark communities include a diversity of species related to this
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179 group. Two of these species, M. oryzae and M. populi where originally isolated from plants [19-20],
180 suggesting that they are common inhabitants of plant surfaces. Curiously, PufLM genes from another
181 Methylobacterium branch, including M. aquaticum, M. platani, M. tarhaniae and M. variabile were
182 absent from avocado bark in this study. From these, M. platani was isolated from a leaf of a Platanus
183 orientalis tree [21](Kang et al., 2007), and the other three from water or soil. Only the basal branch
184 leading to this Methylobacterium cluster showed placements, indicating the presence of a related, yet
185 independent group in avocado bark.
186 Other Rhrizobiales groups had PufLM genes in the bark envrionment. These genes do not form a
187 cohesive phylogenetic group [13], and are found in different branches of the PufLM grene phylogeny
188 (Figure 1). The Blastochloris genus had placements in a long branch starting deep in the phylogenetic
189 tree, but not in the tips. The separation of Blastochloris from other Rhizobacteria has been explained
190 [13], since this genus uses bacteriochlorophyll b instead of bacteriocholophyll a in its reaction center
191 [22]. The placement of metagenomic reads at the basal branch of this group suggests that other
192 lineages, related to the Blastochloris references, but with different PufLM genes, exist in the bark
193 communities of avocado. Methagenomic reads were also mapped to Methylocella sylvestris, both at the
194 tip and at a branch connecting this species with Prosthecomicrobium hirschii. Methylocella sylvestris is
195 a methanotrophic bacterium [23]. Fulvimarina pelagi, antoher rhizobacterium, and Caenispirillum
196 salinarum of the order Rhodospirillales, which PufLM genes clustered tightly [13], were also
197 represented by placements at both tips and at the branch connecting them. Placements were also
198 observed at two inner branches of the Bradyrhizobium/Rhodopseudomonas cluster, suggesting the
199 presence of PufLM genes from different species of those genera, or genera related to them.
200 Genes for the photosynthtetic type II reaction center were also found for the order Rhodospirillales.
201 The group containing Roseococcus, Rubritepida, Skermanella. Paracraurococcus, Rhodocista and
202 Acidisphaera had many PufLM placements, both at the tips and at internal branches.
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203 Another group that showed many PufLM genes in the avocado bark samples included an internal
204 branch joining two species of the genus Roseiflexus (Chloroflexales). In the reads recovered with the
205 Atamna-Ismaeel references, his group also had many more placements in internal branches and at the
206 tips (Figure S1).
207
208 Discussion
209
210 Metagenomic read placement of avocado bark PufLM reads was highly concentrated on the
211 Sphingomonas sanxanigenens tip of the phylogenetic tree. Sphingomonas is a highly diverse genus,
212 which includes photosynthetic and non-photosynthetic bacteria. Sphingomonas was one of the most
213 abundant and diverse genera in avocado bark microbial communities overall [8]. Moreover, this genus
214 has been found to dominate on bark surfaces of other trees [4, 7], as well as on leafs [24]. The high
215 abundance of PufLM genes detected for Sphingomonas, particularly mapping to S. sanxanigenens,
216 indicates that this species, or closely related species, are major contributors to the anoxygenic
217 photosynthetic potential in this environment. Photosynthetic Sphingomonas are among the bacteria
218 capable of performing anoxygenic photosynthesis in the presence of oxygen [13], and they typically
219 require organic carbon substrates for growth. The capability of performing photosynthesis in the
220 presence of oxygen, as an additional source of energy, could be an advantage on plant surfaces
221 including leafs and bark, as they are not restricted to finding and/or creating anoxic niches for
222 photosynthesis. Interestingly, the type strain of S. sanxanigenens was isolated from soil from a wheat
223 plantation [25]. Given the high plant biomass found on wheat plantations, and the dominance of S.
224 saxanigenes in the bark environment, an association of this species with plants seems likely.
225 Remarkably, the absence fo PufLM genes in soil in this study suggests that the main habitat of these
226 bacteria is the surfaces of plants, rather than soil.
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227 Bacteria from the Methylobacterium genus have been observed in 16S microbiome studies of bark
228 surfaces from different trees [7], and are very common on leaf surfaces [24]. In a previous analysis, it
229 was shown that Methylobacterium is one of most abundant genera in avocado bark microbial
230 communities [8]. PufLM genes from Methylobacterium were observed mainly in one group of species
231 from the references available for this genus. Within this group, however, reads were placed at branches
232 at many different levels, suggesting a high diversity of PufLM genes, as compared to other abundant
233 taxa, like Sphingomonas. This suggests a more flexible photosynthtetic apparatus, possibly with more
234 structural diversity in this genus. A characteristic feature of members of Methylobacterium is their
235 ability to utilize methanol and other one-carbon molecules for growth. Furthermore, some
236 Methylobacterum species can degrade complex organic molecules, including refractory compounds
237 [26, 27]. In the bark environment, they could access difficult to degrade plant-derived molecules as
238 carbon sources. Complete sets of photosynthetic genes are commonly found in Methylobacterium [28],
239 and bacteriochlorophyll production in strains isolated from leaves has been demonstrated by far-red
240 light absorption in vivo [29]. Therefore, photosynthetic activity in this group is almost certain, however,
241 its physiological role is still being elucidated. A metabolic model of Methylobacterium extorquens AM1
242 found all the genes necessary for carbon fixation through the Calvin Cycle, suggesting the possibility of
243 autotrophic growth [30]. However, it is also possible that CO2 fixation is utilized as a mechanism to
244 dispose excess electrons from reduced cofactors [30], as has been shown for other bacteria [31].
245 The presence of Blastochloris reads in bark microbial communities, different from the reference
246 sequences, is interesting, as only four species from this genus are known [22]. Mapping of reads from
247 the avocado bark metagenome to Methylocella species PufLM suggests the posibility of methanotrophy
248 in this environment. However, no methane monoxygenase genes were fund in any bark sample [9],
249 which casts doubt on the presence of methanotrophic activity in avocado bark.
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250 A group of Rhodospirillales was well represented in the PufLM sequences of the avocado bark
251 metagenome. It included many placements in the whole branch, including the tips and internal
252 branches. From these, reads from Paracraurococcus ruber and Acidisphaera rubrifaciens were
253 especially abundant. There is not much information about these bacterial gropus, and the presence,
254 diversity and significance of these genes in the bark environment should be explored in future studies.
255 The only group of bacteria with abundant PufLM genes in avocado bark outside of the class
256 Alphaproteobacteria, was a branch Chloroflexales containing the genus Roseiflexus. These species are
257 known from extreme environments [32], and the presence of related genes in the bark environment is
258 interesting. Roseiflexus perform photosynthesis under anaerobic light conditions, and can also thrive
259 chemoheterotrophically when oxygen is present. To test whether close relatives of Roseiflexus were
260 found in the bark environment, a search for metagenomic reads was conducted in our dataset using the
261 genome of Roseiflexus RS-1 as a reference. Only a few matches were found (data not shown),
262 indicating that the homology is restricted to the PufLM genes.
263 Most of the bacterial type II reaction centers found in this study belonged to Aerobic Anoxygenic
264 Photosynthetic Alphaproteobacteria, with a dominance from Sphingomonas, Methylobacterium, and a
265 diversity of Rhodospirillales. This suggests that the role of anoxygenic photosynthesis in the bark
266 environment is mainly as an additional energy source for heterotrophic growth. In a previous analysis
267 of the avocado bark metagenome dataset, many genes for cyanobacterial photosynthesis were found
268 [9]. The present study completes the picture of photosynthetic activity in the tree holobiont. Light not
269 harvested in the canopy, and reaching the inner stems of the trees, could be enriched in wavelengths left
270 unused by the leaves. This light seems to support phtotrophic communities, where Cyanobacteria
271 utilize remaining light in the visible spectrum, absorbed by phycobilisomes for carbon fixation [9], and
272 Aerobic Anoxygenic Photosynthetic bacteria utilize far-red light as an additional energy source for
273 photoheterotropic growth.
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274 Conclusions
275
276 Genes for the bacterial anoxygenic type-II photosynthetic apparatus found in avocado bark were
277 mainly from Aerobic Anoxygenic Photosynthetic bacteria. From these, Sphingomonas PufLM genes
278 had low diversity as compared to Methylobacterium, which where more abundant. PufLM genes from
279 other bacterial groups were present in the avocado bark environment, including Rhodospirillales,
280 Blastochloris, and bacteria from the phylum Chloroflexia. In many cases, metagenomic reads mapped
281 to internal branches, indicating the presence of PufLM genes from phyla not represented in the
282 reference sequences. This suggests that unknown groups of photosynthetic bacteria might be present in
283 this microbial environment. The present study confirms that the avocado bark environment is an
284 important habitat for photosynthetic bacteria, which probably thrive on light unutilized by the canopy
285 leaves.
286
287 Acknowledgments
288
289 I thank the following people and institutions: Ulricke Wiegel and Jesús Torres for allowing sampling of
290 their avocado trees, and the Mexican Council of Science and Technology (CONACyT) and the
291 Mexican Ministry of Education (SEP) for providing financial support, through grant CB-2014-01-
292 242956.
293
294 Declarations
295
296 Conflicts of interest/Competing interests – Non declared.
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297 References
298
299 1. Crowther TW, Glick HB, Covey KR, Bettigole C, Maynard DS, Thomas SM, Smith JR, Hintler G,
300 Duguid MC, Amatulli G et al (2015) Mapping tree density at a global scale. Nature 525:201-205.
301 https://doi.org/10.1038/nature14967
302
303 2. Stone BW, Weingarten EA, Jackson CR (2018) The role of the phyllosphere microbiome in plant
304 health and function. Annual Plant Reviews 1:1-24. https://doi.org/10.1002/9781119312994.apr0614
305
306 3. Lambais MR, Lucheta AR, Crowley DE (2014) Bacterial community assemblages associated with
307 the phyllosphere, dermosphere, and rhizosphere of tree species of the Atlantic forest are host taxon
308 dependent. Microb Ecol 68:567-574. https://doi.org/10.1007/s00248-014-0433-2
309
310 4. Leff JW, Del Tredici P, Friedman WE, Fierer N (2015) Spatial structuring of bacterial communities
311 within individual Ginkgo biloba trees. Environ Microbiol 17:2352-2361. https://doi.org/10.1111/1462-
312 2920.12695
313
314 5. Arrigoni E, Antonielli L, Pindo M, Pertot I, Perazzolli M (2018) Tissue age and plant genotype affect
315 the microbiota of apple and pear bark. Microbiol Res 211:57-68.
316 https://doi.org/10.1016/j.micres.2018.04.002
317
15 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.12.295014; this version posted September 13, 2020. 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.
318 6. Vitulo N, Lemos WJF, Calgaro M, Confalone M, Felis GE, Zapparoli G, Nardi T (2019) Bark and
319 grape microbiome of Vitis vinifera: influence of geographic patterns and agronomic management on
320 bacterial diversity. Front Microbiol 9:3203. https://doi.org/10.3389/fmicb.2018.03203
321
322 7. Arrigoni E, Albanese D, Longa CMO, Angeli D, Donati C, Ioriatti C, Pertot I, Perazzolli M (2020)
323 Tissue age, orchard location and disease management influence the composition of fungal and bacterial
324 communities present on the bark of apple trees. Environ Microbiol 22:2080-2093.
325 https://doi.org/10.1111/1462-2920.14963
326
327 8. Aguirre-von-Wobeser E, Alonso-Sanchez A, Mendez-Bravo A, Espino LAV, Reverchon F (2020)
328 Bark from avocado trees of different geographic locations have consistent microbial communities.
329 BioRxiv. https://doi.org/10.1101/2020.08.21.261396
330
331 9. Aguirre-von-Wobeser E (2020) Functional metagenomics of bark microbial communities from
332 avocado trees (Persea americana Mill.) reveals potential for bacterial primary productivity. BioRxiv.
333 https://doi.org/10.1101/2020.09.05.284570
334
335 10. Atamna-Ismaeel N, Finkel O, Glaser F, von Mering C, Vorholt JA, Koblížek M, Belkin S, Béjà O
336 (2012) Bacterial anoxygenic photosynthesis on plant leaf surfaces. Env Microbiol Rep 4:209-216.
337 https://doi.org/10.1111/j.1758-2229.2011.00323.x
338
339 11. Matsen FA, Hoffman NG, Gallagher A, Stamatakis A (2012) A format for phylogenetic placements.
340 PLoS One 7:e31009. https://doi.org/10.1371/journal.pone.0031009
341
16 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.12.295014; this version posted September 13, 2020. 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.
342 12. Buchfink B, Xie C, Huson DH (2015) Fast and sensitive protein alignment using DIAMOND. Nat
343 Methods 12:59-60. https://doi.org/10.1038/nmeth.3176
344
345 13. Imhoff JF, Rahn T, Künzel S, Neulinger SC (2018) Photosynthesis is widely distributed among
346 Proteobacteria as demonstrated by the phylogeny of PufLM reaction center proteins. Front Microbiol
347 8:2679. https://doi.org/10.3389/fmicb.2017.02679
348
349 14. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput.
350 Nucleic Acids Res 32:1792-1797. https://doi.org/10.1093/nar/gkh340
351
352 15. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large
353 phylogenies. Bioinformatics 30:1312-1313. https://doi.org/10.1093/bioinformatics/btu033
354
355 16. Berger SA, Stamatakis A (2011) Aligning short reads to reference alignments and trees.
356 Bioinformatics 27:2068-2075. https://doi.org/10.1093/bioinformatics/btr320
357
358 17. Czech L, Barbera P, Stamatakis A (2019) Methods for automatic reference trees and multilevel
359 phylogenetic placement. Bioinformatics 35:1151-1158. https://doi.org/10.1093/bioinformatics/bty767
360
361 18. Czech L, Barbera P, Stamatakis A (2020) Genesis and Gappa: processing, analyzing and visualizing
362 phylogenetic (placement) data. Bioinformatics 36:.3263-3265.
363 https://doi.org/10.1093/bioinformatics/btaa070
364
17 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.12.295014; this version posted September 13, 2020. 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.
365 19. Van Aken B, Peres CM, Doty SL, Yoon JM, Schnoor JL (2004) Methylobacterium populi sp. nov., a
366 novel aerobic, pink-pigmented, facultatively methylotrophic, methane-utilizing bacterium isolated from
367 poplar trees (Populus deltoides× nigra DN34). Int J Syst Evol Micr 54:1191-1196.
368 https://doi.org/10.1099/ijs.0.02796-0
369
370 20. Madhaiyan M, Kim BY, Poonguzhali S, Kwon SW, Song MH, Ryu JH, Go SJ, Koo BS, Sa TM
371 (2007) Methylobacterium oryzae sp. nov., an aerobic, pink-pigmented, facultatively methylotrophic, 1-
372 aminocyclopropane-1-carboxylate deaminase-producing bacterium isolated from rice. Int J Syst Evol
373 Micr 57:326-331. https://doi.org/10.1099/ijs.0.64603-0
374
375 21. Kang YS, Kim J, Shin HD, Nam YD, Bae JW, Jeon CO, Park W (2007) Methylobacterium platani
376 sp. nov., isolated from a leaf of the tree Platanus orientalis. Int J Syst Evol Micr 57:2849-2853. https://
377 doi.org/10.1099/ijs.0.65262-0
378
379 22. Kyndt JA, Salama DM, Meyer TE (2020) Genome Sequence of the Alphaproteobacterium
380 Blastochloris sulfoviridis DSM 729, Which Requires Reduced Sulfur as a Growth Supplement and
381 Contains Bacteriochlorophyll b. Microbiology Resource Announcements 9: 00313-20.
382 https://doi.org/10.1128/MRA.00313-20
383
384 23. Dedysh SN, Knief C, Dunfield PF (2005) Methylocella species are facultatively methanotrophic. J
385 Bacteriol 187:4665-4670. https://doi.org/10.1128/JB.187.13.4665-4670.2005
386
387 24. Vorholt JA (2012) Microbial life in the phyllosphere. Nat Rev Microbiol 10:828-840.
388 https://doi.org/10.1038/nrmicro2910
18 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.12.295014; this version posted September 13, 2020. 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.
389
390 25. Huang HD, Wang W, Ma T, Li GQ, Liang FL, Liu RL (2009) Sphingomonas sanxanigenens sp.
391 nov., isolated from soil. Int J Syst Evol Micr 59:719-723. https://doi.org/10.1099/ijs.0.000257-0
392
393 26. Pasternak G, Kołwzan B (2013) Surface tension and toxicity changes during biodegradation of
394 carbazole by newly isolated methylotrophic strain Methylobacterium sp. GPE1. Int Biodeter Biodegr
395 84:143-149. https://doi.org/10.1016/j.ibiod.2012.07.021
396
397 27. Li X, Wang J, Wu W, Jia Y, Fan S, Hlaing TS, Khokhar I, Yan Y (2020) Co-metabolic
398 biodegradation of quizalofop-p-ethyl by Methylobacterium populi YC-XJ1 and identification of
399 QPEH1 esterase. Electronic Journal of Biotechnology 46:38-49.
400 https://doi.org/10.1016/j.ejbt.2020.05.003
401
402 28. Miroshnikov KK, Belova SE, Dedysh SN (2019) Genomic Determinants of Phototrophy in
403 Methanotrophic Alphaproteobacteria. Microbiology 88:548–555.
404 https://doi.org/10.1134/S0026261719050102
405
406 29. Stiefel P, Zambelli T, Vorholt JA (2013) Isolation of optically targeted single bacteria by application
407 of fluidic force microscopy to aerobic anoxygenic phototrophs from the phyllosphere. Appl Environ
408 Microb 79:4895-4905. https://doi.org/10.1128/AEM.01087-13
409
410 30. Peyraud R, Schneider K, Kiefer P, Massou S, Vorholt JA, Portais JC (2011) Genome-scale
411 reconstruction and system level investigation of the metabolic network of Methylobacterium
412 extorquens AM1. BMC Syst Biol 5:189. https://doi.org/10.1186/1752-0509-5-189
19 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.12.295014; this version posted September 13, 2020. 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.
413
414 31. McKinlay JB, Harwood CS (2010) Carbon dioxide fixation as a central redox cofactor recycling
415 mechanism in bacteria. P Natl Acad Sci USA 107:11669-11675.
416 https://doi.org/10.1073/pnas.1006175107
417
418 32. Van Der Meer MT, Klatt CG, Wood J, Bryant DA, Bateson MM, Lammerts L, Schouten S, Damsté
419 JSS, Madigan MT, Ward DM (2010) Cultivation and genomic, nutritional, and lipid biomarker
420 characterization of Roseiflexus strains closely related to predominant in situ populations inhabiting
421 Yellowstone hot spring microbial mats. J Bacteriol 192:3033-3042.
422 https://doi.org/10.1128/JB.01610-09
20