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1 Genome sequencing and functional genes comparison between Sphingopyxis
2 USTB-05 and Sphingomonas morindae NBD5
3
4 Chao Liu1, Qianqian Xu1, Zhenzhen Zhao1, Shahbaz Ahmad1, Haiyang Zhang1, Yufan
5 Zhang1, Yu Pang1, Abudumukeyiti Aikemu1, Yang Liu1,* Hai Yan1,*
6
7 1 School of Chemistry and Biological Engineering, University of Science and
8 Technology Beijing, Beijing 100083, China
9 *Corresponding author: Prof Hai Yan; mail: [email protected]. Yang Liu; mail:
11
12 Keywords: Sphingomonadaceae; Genome; Lutein; Hepatotoxin; Microcystins;
13 Biodegradation
14
15
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25
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26 ABSTRACT
27 Sphingomonadaceae has a large number of strains that can biodegrade hepatotoxins or
28 environmental pollutants. The latest research reported that certain strains can also
29 produce lutein. Based on the third-generation sequencing technology, we analyzed the
30 whole genome sequence and compared related functional genes of two strains of
31 Sphingomonadaceae isolated from different habitats. The genome of Sphingopyxis
32 USTB-05 was 4,679,489 bp and contained 4312 protein coding genes. The 4,239,716
33 bp nuclear genome of Sphingomonas morindae NBD5, harboring 3882 protein coding
34 genes, has two sets of chromosomes. Both strains had lutein synthesis metabolism
35 pathway sharing identical synthetic genes of crtB, crtE, crtI, crtQ, crtL, crtR, atoB, dxs,
36 dxr, ispD, ispE, ispDF, gcpE, ispG, ispH, ispA, ispB and ispU. Sphingopyxis USTB-05
37 had hepatotoxins microcystins and nodularin metabolic pathways related to 16 genes
38 (ald、ansA、gdhA、crnA、phy、ocd、hypdh、spuC、nspC、speE、murI、murD、
39 murC、hmgL、bioA and glsA), while these genes were not found in Sphingomonas
40 morindae NBD5. The unique protein sequences of strain NBD5 and strain USTB-05
41 were 155 and 199, respectively. The analysis of whole genome of the two
42 Sphingomonadaceae strains provides insights into prokaryote evolution, the new
43 pathway for lutein production and the new genes for environmental pollutant
44 biodegradation.
45
46 IMPORTANCE
47 Understanding the functional genes related to the special functions of strains is essential
48 for humans to utilize microbial resources. The ability of Sphingopyxis USTB-05 to
49 degrade hepatotoxins microcystins and nodularin has been studied in depth, however
50 the complete metabolic process still needs further elucidation. Sphingomonas morindae
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51 NBD5 can produce lutein, and it is necessary to determine whether there is a new
52 pathway of lutein. In this study, the whole genome sequencing of Sphingopyxis USTB-
53 05 and Sphingomonas morindae NBD5 were performed for the first time. Lutein
54 synthesis metabolic pathways and synthetic genes were discovered in
55 Sphingomonadaceae. We predicted the existence of new lutein synthesis pathways and
56 revealed most of the genes of the new synthesis pathways. A comparative analysis of
57 the functional genes of the two strains revealed that Sphingopyxis USTB-05 contains a
58 large number of functional genes related to the biodegradation of hepatotoxins or
59 hexachlorocyclohexane. Among them, the functional genes related to the
60 biodegradation and metabolism of hexachlorocyclohexane had not been previously
61 reported. These findings lay the foundation for the biosynthesis of lutein using
62 Sphingomonas morindae NBD5 or Sphingopyxis USTB-05 and the application of
63 Sphingopyxis USTB-05 for the biodegradation of hepatotoxins microcystins and
64 nodularin or environmental pollutants.
65
66 INTRODUCTION
67 Lutein is a kind of carotenoid, which widely exits in vegetables, fruits and other plant.
68 It is also the main pigment in the macular area of human's eyes (1), and cannot be
69 synthesized by the body itself. Although it must be obtained from daily food, most
70 people's daily intake is seriously insufficient. The latest research showed that daily
71 intake of 10 mg lutein and 2 mg zeaxanthin could improve visual function and delay
72 the development of age-related macular degeneration (AMD) (2). Many researches
73 focused on the production of lutein by eukaryotes, and its synthesis pathway had been
74 clarified already. However, the production of lutein by prokaryotes was reported rarely.
75 The prokaryotic strains were only confined to some specific strains, for examples,
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76 Erwinia, Agrobacterium and Rhodobacter capsulatus (2). Few research was reported
77 about Sphingomonas strains in lutein production. In our previous research,
78 Sphingomonas morindae NBD5 had the function of producing lutein (3). Phylogenetic
79 analysis showed that they belonged to two closely related genera of
80 Sphingomonadaceae (4) (5).
81
82 Sphingomonadaceae has a large number of strains that can biodegrade hepatotoxins or
83 environmental pollutants. In the 1990s, Sphingomonas wittichii RW1 was reported to
84 biodegrade dibenzo-p-dioxin (DD) polychlorinated derivatives under aerobic condition
85 in contaminated soil and water (6). Other types of Sphingomonas could also biodegrade
86 a series of intractable compounds (biphenyl, herbicide dichlorohaloperidin, γ-
87 hexachlorocyclohexane, aromatic hydrocarbons, chlorophenol, pentachlorophenol,
88 naphthalenesulfonic acid, N,N-dimethylaniline, diphenyl ether, dibenzofuran) that were
89 harmful to the environment (7). Hepatotoxins microcystins (MCs) and nodularin (NOD)
90 are derived from algae and have high toxicity and potential harm to humans and aquatic
91 animals. The World Health Organization (WHO) stipulated that the concentration of
92 MCs in drinking water should not be higher than 1.0 μg/L (8). Biodegradation is a very
93 promising method to remove hepatotoxins. At present, many strains of
94 Sphingomonadaceae family have the function of biodegrading hepatotoxins. However,
95 in order to fully clarify the metabolic process of biodegradation, new hepatotoxins
96 biodegradation genes need to be discovered.
97
98 Sphingomonas morindae NBD5 was a new species that was identified as this genus
99 only a few years ago (4). It could produce high yield of lutein, and the metabolic genes
100 needed further study. Sphingopyxis USTB-05 was isolated from Dianchi Lake in China
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101 and could biodegrade MCs and NOD (5) (9) (10). These functional genes USTB-05-A,
102 USTB-05-B, and USTB-05-C of Sphingopyxis sp. USTB-05 had been verified by
103 heterologous expression in Escherichia coli to biodegrade MCs (11)(12). The purified
104 first recombinant enzyme was found to have a strong ability to catalyze hepatotoxins in
105 Sphingopyxis sp. USTB-05 (13). Because of the characteristics of lutein production and
106 hepatotoxin biodegradation of these two strains, the relationship between gene and
107 function was found through searching for functional genes. Here, the method of whole
108 genome combined with biometric analysis was used to compare the similar and unique
109 characteristics of Sphingomonas morindae NBD5 and Sphingopyxis USTB-05, and
110 analyze their functional genes, especially those associated with lutein synthesis and
111 hepatotoxin biodegradation.
112
113 RESULTS
114 General features of the nuclear genome
115 The Sphingomonas morindae NBD5 genome contained two circular chromosomes and
116 two circular plasmids (Figure 1). Polychromosomes were common in some genera, but
117 rare in Sphingomonas. Two circular chromosome of 4,239,716 bases was finally
118 obtained with a G + C content of 70%, and 3882 protein coding sequences (CDSs) were
119 predicted, accounting for 62.39% of the total coding sequence. The 16S rRNA of strain
120 NBD5 had 3 complete copies (Table 1). The Sphingopyxis USTB-05 genome contained
121 one chromosome (Figure 2). Its circular chromosome of 4,679,489 bases was finally
122 obtained with a G + C content of 64%, and 4312 CDSs were predicted, accounting for
123 62.39% of the total coding sequence. The 16S rRNA of strain USTB-05 was a single
124 copy (Table 1) without CRISPR site. Compared with strain USTB-05, the genome of
125 strain NBD5 was much richer in GC and contained plasmids, which indicated that some
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126 genetic transfer events happened in strain NBD5 (Table 1). The number of functional
127 genes annotated in strain NBD5 was much more than that in strain USTB-05, but the
128 chromosome length of strain USTB-05 was longer than that of strain NBD5.
129
130 131 Figure 1 Genome circle of Sphingomonas morindae NBD5
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132 133 Figure 2 Genome circle of Sphingopyxis USTB-05 134 135 136 137 Table 1 Comparison of genome characteristics between Sphingomonas morindae NBD5 and 138 Sphingopyxis USTB-05 category Sphingomonas morindae NBD5 Sphingopyxis USTB-05 bases 4239716 4679489 tmRNA 1 1 tRNA 61 48 CDS 3882 4312 GC(%) both 70% 64% plasmid 2 0 139 140 Genome annotation and genome wide comparative analysis
141 GO annotation
142 The distribution of genes in different Gene Ontology (GO) terms can intuitively reflect
143 the distribution of target genes on the secondary level of GO terms. A total of 798 genes
144 were annotated in the Sphingomonas morindae NBD5 genome through the GO
145 database (Figure 3). Under the classification of biological process, the number of genes
146 annotated to metabolic process was up to 43.8%, and the number of genes for
147 intracellular processes was 40.8%. Response to stimulus, biological regulation,
148 localization, regulation of biological process, multi-organism process, cellular 7 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
149 component organization or biogenesis, signaling, and growth accounted for 15%, 14%,
150 11.8%, 11.8%, 7.3%, 6.3%, 4.1%, and 3%, respectively. Under the classification of
151 cellular component, the number of genes annotated to cell was up to 38%, and the
152 number of genes for cell part was 37.9%. Membrane, membrane part, protein-
153 containing complex, organelle, extracellular region, organelle part, nucleoid, and other
154 organism accounted for 22.6%, 14.8%, 6.2%, 3.2%, 1.9%, 1.5%, 0.4%, and 0.2%,
155 respectively. Under the classification of molecular function, the number of genes
156 annotated to catalytic activity was up to 1623 (40.6%), and the number of genes for
157 binding was 37.5%. Transporter activity, transcription regulator activity, structural
158 molecule activity, molecular transducer activity, obsolete signal transducer activity,
159 antioxidant activity, molecular function regulator, and molecular carrier activity
160 accounted for 6.9%, 4.1%, 1.7%, 1.6%, 1.4%, 0.7%, 0.4%, and 0.1%, respectively.
161
162 A total of 786 genes were annotated in the Sphingopyxis USTB-05 genome through the
163 GO database (Figure 3). Under the classification of biological process, the number of
164 genes annotated to cellular process was up to 14.5%, and the number of genes for
165 metabolic process was 13.9%. Response to stimulus, cellular component organization
166 or biogenesis, biological regulation, regulation of biological process, growth,
167 localization, negative regulation of biological process, and positive regulation of
168 biological process accounted for 4.1%, 3.2%, 3.2%, 2.6%, 2.4%, 1.6%, 0.9%, and 0.8%,
169 respectively. Under the classification of cellular component, the number of genes
170 annotated to cell was up to 14.4%, and the number of genes for cell part was 14.4%.
171 Membrane, protein-containing complex, organelle, organelle part, membrane part,
172 extracellular region, membrane-enclosed lumen, and extracellular region part
173 accounted for 5.5%, 3.4%, 2.2%, 1.8%, 1.6%, 0.4%, 0.2%, and 0.1%, respectively.
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174 Under the classification of molecular function, the number of genes annotated to
175 catalytic activity was up to 11.9%, and the number of genes for binding was 7.9%.
176 Structural molecule activity, transporter activity, transcription regulator activity,
177 molecular function regulator, and antioxidant activity accounted for 1.3%, 0.9%, 0.5%,
178 0.2%, and 0.1%, respectively.
179
180 Although Sphingopyxis USTB-05 had more bases in its genome than Sphingomonas
181 morindae NBD5, Sphingomonas morindae NBD5 had significantly more genes in most
182 GO classifications than Sphingopyxis USTB-05 (Figure 3). Sphingomonas morindae
183 NBD5 had 66 unique genes in carbohydrate utilization (GO:0009758): 11 and obsolete
184 signal transducer activity (GO:0004871): 55. But there were also some genes in
185 behavior (GO:0007610): 1, cell proliferation (GO:0008283): 2, multicellular
186 organismal process (GO:0032501): 2, obsolete transcription factor activity, protein
187 binding (GO:0000988): 4, obsolete transcription factor activity, transcription factor
188 binding (GO: 0000989): 2, translation regulator activity (GO: 0045182): 1, these genes
189 in Sphingopyxis USTB-05 were unique, representing behavior; cell proliferation;
190 multicellular organismal process; obsolete transcription factor activity, protein binding;
191 obsolete transcription factor activity, transcription factor binding; translation regulator
192 activity, respectively.
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193
194 Figure 3 Comparison of GO functional classification of Sphingomonas morindae
195 NBD5 and Sphingopyxis USTB-05
196 COG annotation
197 Cluster of Orthologous Groups (COG) is a database based on the systematic evolution
198 of bacteria, algae and eukaryotes. The assembled Sphingomonas morindae NBD5 gene
199 was analyzed in the COG database, and the results showed that 62.39% of the genes in
200 COG were annotated (Figure 4). In the 25 COG functional categories, 3668 genes had
201 been classified. These categories were mainly: transcription (COG category K) (7.66%,
202 as a percentage of all functional allocation genes), cell wall/membrane/envelope
203 biogenesis (M) (7.06%), carbohydrate transport and metabolism (G) (6.82 %), amino
204 acid transport and metabolism (E) (6.73%), signal transduction mechanisms (T)
205 (6.05%), inorganic ion transport and metabolism (P) (5.62%), energy production and
206 conversion (C) (5.37%), translation, ribosomal structure and biogenesis (J) (4.80%),
207 replication, recombination and repair (L) (4.77%), and lipid transport and metabolism
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208 (I) (4.58%). The rate of COG classification as transcription (K) was high (7.66%),
209 which was consistent with the fact that this strain contained two circular chromosomes
210 and complex gene expression.
211
212 Analysis of the assembled Sphingopyxis USTB-05 gene in the COG database showed
213 that 62.39% of the genes were annotated in COG. Among the 25 functional categories
214 of COG, a total of 4,040 genes had been classified (Figure 4). These categories were
215 mainly: amino acid transport and metabolism (E) (8.71%), transcription (K) (7.65%),
216 lipid transport and metabolism (I) (6.76%), inorganic ion transport and metabolism (P)
217 (6.14%), energy production and conversion (C) (5.87%), cell wall/membrane/envelope
218 biogenesis (M) (5.50%), secondary metabolite biosynthesis, transport and catabolism
219 (Q) (5.05%), carbohydrate transport and metabolism (G) (4.85%), translation,
220 ribosomal structure and biogenesis (J) (4.65%), replication, recombination and repair
221 (L) (4.38%), and posttranslational modification, protein turnover, chaperones (O)
222 (4.06%).
223
224 Most COGs showed similar distributions among Sphingomonas morindae NBD5 and
225 Sphingopyxis USTB-05 (Figure 4). Among them, Sphingopyxis USTB-05 had the
226 largest number of genes in most COGs categories; however, the number of genes in the
227 COG G, M, and T categories for the Sphingopyxis USTB-05 was lower. The COG G,
228 M, and T categories for Sphingomonas morindae NBD5 represented carbohydrate
229 transport and metabolism, cell wall/membrane/envelope biogenesis and signal
230 transduction mechanisms, respectively. These data suggested that Sphingomonas
231 morindae NBD5 had significant differences in carbohydrate-related metabolic
232 synthesis and signal transduction. These reflected the fact that Sphingomonas morindae
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233 NBD5 could produce more lutein than Sphingopyxis USTB-05, and the synthesis of
234 lutein was related to the metabolic synthesis of carbohydrates. The number of genes in
235 the COG C, E, I, and Q categories for Sphingopyxis USTB-05 was higher; they
236 represented energy production and conversion; amino acid transport and metabolism;
237 lipid transport and metabolism and secondary metabolites biosynthesis, transport and
238 catabolism, respectively. These data suggested that Sphingopyxis USTB-05 had
239 significant differences in catabolism. These reflected the fact that Sphingopyxis USTB-
240 05 had the molecular basis for decomposing MCs that a cyclic polypeptide composed
241 of seven amino acids in the environment.
242
243 Figure 4 Comparison of COG functional classification of Sphingomonas morindae
244 NBD5 and Sphingopyxis USTB-05
245 KEGG annotation
246 Biological functions usually require coordination of different genes. Therefore, in order
247 to identify the representative biological pathway of strain NBD5, single genes were 12 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
248 annotated. A total of 1,839 genes were annotated as 112 metabolic pathways, some of
249 which could be matched with multiple metabolic pathways. In the Kyoto Encyclopedia
250 of Genes and Genomes (KEGG) database, genes annotated as 23 major pathways
251 accounted for more than half of all annotated genes. The top ten pathways were: amino
252 acid biosynthesis, carbon metabolism, two-component system, purine metabolism,
253 flagella assembly, oxidative phosphorylation, ribosomes, ABC transporter, bacterial
254 chemotaxis, and pyrimidine metabolism (Figure 5). These annotations provided
255 important information about the specific biological processes and pathways of strain
256 NBD5.
257
258 A total of 1927 genes of strain USTB-05 had been annotated to 121 metabolic pathways,
259 some of which could be matched with multiple metabolic pathways. In the KEGG
260 database, genes annotated to 28 major pathways accounted for more than half of all
261 annotated genes. The top ten pathways were: amino acid biosynthesis, carbon
262 metabolism, two-component system, ribosomes, purine metabolism, oxidative
263 phosphorylation, Pyruvate metabolism, glyoxylate and dibasic acid metabolism,
264 quorum sensing, glycine, and serine and threonine metabolism (Figure 5). These notes
265 provided important information about the specific biological processes and pathways
266 of strain USTB-05.
267
268 Comparative analysis of KEGG function of the two strains of Sphingomonadaceae was
269 did as well. Among the top ten pathways, two-component system (ko02020), purine
270 metabolism (ko00230), flagella assembly (ko02040) (correlation of the neatness of
271 colony edges on the plate), bacterial chemotaxis (ko02030)(correlation of the neatness
272 of colony edges on the plate) genes strain NBD5 was obviously more than strain USTB-
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273 05; and amino acid biosynthesis (ko01230)(synthesis of enzymes related to algal toxin
274 degradation), carbon metabolism (ko01200)(synthesis of enzymes related to algal toxin
275 degradation), pyruvate metabolism (ko00620), glyoxylic acid and dibasic acid
276 metabolism (ko00630), quorum sensing (ko02024), glycine, serine and threonine
277 metabolism (ko00260)(synthesis of enzymes related to the degradation of algal toxins).
278 The process gene strain USTB-05 was obviously more than strain NBD5. In addition,
279 the KEGG function annotated that the unique metabolic processes of strain NBD5
280 included other polysaccharide degradation (ko00511), plant-pathogen interaction
281 (ko04626) (from endophytes in plant noni), life regulation pathways-multi-species
282 (ko04213), FoxO signaling pathway (ko04068), sphingolipid metabolism (ko00600),
283 life regulation pathway (ko04211); the unique metabolic processes of strain USTB-05
284 included β-alanine metabolism (ko00410), degradation of chlorinated alkanes and
285 chloroalkenes (ko00625), taurine and hypotaurine metabolism (ko04626), xylene
286 degradation (ko00622), caprolactam degradation (ko00930), dioxin degradation
287 (ko00621), limonene and pinene degradation (ko00903), chlorocyclohexane and
288 chlorobenzene degradation (ko00361), PPAR signaling pathway (ko03320), apoptosis
289 (ko04214), D-glutamine and D-glutamate metabolism (ko00471), naphthalene
290 degradation (ko00626), styrene degradation (ko00643), toluene degradation (ko00623).
291 This was consistent with the fact that the strain NBD5 was an endophyte and lutein-
292 producing functional bacteria from the plant Noni, while the strain USTB-05 was a
293 functional bacteria that could biodegrade complex organic matter in the environment.
294
295 Figure 5 Comparison of KEGG pathway distribution of Sphingomonas morindae 14 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
296 NBD5 and Sphingopyxis USTB-05
297 Comparison of the biosynthesis pathways and genes of lutein
298 Lutein contains two ionone rings in its chemical formula and is a carotenoid with
299 vitamin A activity. Sphingomonas morindae NBD5 genome COG analysis shows that
300 its core carbon skeleton is completed by carbohydrate transport and metabolism (G)
301 (6.82%). Among them, the ratio of cell wall/membrane/envelope biogenesis (M)
302 (7.06%) related to compound endocytosis and exocytosis is significantly higher. In
303 addition, energy production and conversion (C) (5.37%) also plays an important role
304 (Figure 4).
305
306 The corresponding genes of the terpenoid backbone biosynthesis pathway and the
307 carotenoid biosynthesis pathway of Sphingomonas morindae NBD5 and Sphingopyxis
308 USTB-05 were the same. The corresponding enzyme genes for lutein synthesis were
309 also the same. Both strains found the presence of β-carotene 3-hydroxylase, which was
310 involved in the last step of lutein synthesis, and its corresponding code gene was CrtR-
311 b. This is a fact that lycopene existed as a synthetic intermediate, which is synthesized
312 through glycolysis/gluconeogenesis pathway (Figure 6), terpenoid backbone
313 biosynthesis pathway (Figure 7) and carotenoid biosynthesis pathway (Figure 8). But
314 in the metabolic pathway of lutein production, no complete synthesis pathway had been
315 found. Combined with the mass spectrometry results (3), the two strains of
316 Sphingomonadaceae could synthesize lutein, which suggest that they could probably
317 find new pathways of lutein synthesis.
318
319 In eukaryotes, especially higher plant, lutein is primarily synthesized from terpenoids.
320 Two molecules of geranylgeranyl diphosphate (GGPP) are used to synthesize phytoene.
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321 Phytoene is then converted to ζ-carotene, after which ζ-carotene is converted into
322 lycopene. Lycopene is then converted to α-carotene through cyclization reaction
323 catalysed by lycopene cyclase (εLCY /βLCY). Finally, the formation of lutein from α-
324 carotene is catalysed by α-carotene hydroxylase (βCHX /εCHX) (14). In the carotenoid
325 biosynthesis pathway of the two Sphingomonadaceae strains, enzymes related to
326 lycopene synthesis have been found (Figure 8), but the cyclases from lycopene to lutein
327 synthesis are lacking. Therefore, it inferred that there were new cyclase genes for the
328 lutein metabolism pathways in these two bacteria.
329
330 The lutein synthesis genes in Sphingomonas morindae NBD5 and Sphingopyxis USTB-
331 05 genomes mainly existed in the terpenoid backbone biosynthesis pathway and the
332 carotenoid biosynthesis pathway, which are completely the same. These synthetic genes
333 are crtB, crtE, crtI, crtQ, crtL, crtR, atoB, dxs, dxr, ispD, ispE, ispDF, gcpE, ispG, ispH,
334 ispA, ispB and ispU. Only these genes ackA, pgm, gpmI, and pckA in the glycolysis &
335 gluconeogenesis pathway in Sphingomonas morindae NBD5 are unique. These genes
336 porB, meh, and fldA in the glycolysis & gluconeogenesis pathway in Sphingopyxis
337 USTB-05 are unique (Figure 9).
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338
339 Figure 6 The metabolic pathway of glycolysis/gluconeogenesis of Sphingomonas
340 morindae NBD5 and Sphingopyxis USTB-05. The enzyme or gene in the orange box
341 in the figure indicates that two strains co-exist, the enzyme or gene in the blue box
342 indicates that neither of the two bacteria exist, the enzyme or gene in the pink box
343 indicates that it only exists in Sphingomonas morindae NBD5, and the enzyme or gene 17 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
344 in the bright yellow box indicates that it only exists in Sphingopyxis USTB-05.
345
346 Figure 7 The biosynthetic pathway of terpenoid skeleton of Sphingomonas morindae
347 NBD5 and Sphingopyxis USTB-05. The enzyme or gene in the orange box in the figure
348 indicates that two strains co-exist, and the enzyme or gene in the blue box indicates that
349 neither of the two bacteria exist. 18 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
350
351 Figure 8 The biosynthetic pathway of carotenoid of Sphingomonas morindae NBD5
352 and Sphingopyxis USTB-05. The enzyme or gene in the orange box in the figure
353 indicates that two strains co-exist, and the enzyme or gene in the blue box indicates that
354 neither of the two bacteria exist.
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355
356 Figure 9 Comparison of genes related to lutein synthesis in the genomes of
357 Sphingomonas morindae NBD5 and Sphingopyxis USTB-05. The gray box in the figure
358 indicate that the gene exists in the strain, and the white indicate that the gene does not
359 exist in the strain.
360
361 Comparison of the pathways and genes of hepatotoxin biodegradation
362 Nowadays, the mlr gene cluster encoding functional hydrolase have been identified in
363 many hepatotoxin biodegrading bacteria (15). The mlr gene cluster contained mlrC,
364 mlrA, mlrB and mlrD, corresponding to the enzymes MlrC, MlrA, MlrB and MlrD that
365 biodegraded hepatotoxin. By comparing the genomes of these two strains, it was found
366 that Sphingopyxis USTB-05 contained the high homology genes mlrC, mlrA, mlrB and
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367 mlrD, while Sphingomonas morindae NBD5 did not have the mlr gene cluster (Suppl
368 Table S1). It was consistent with the fact that Sphingopyxis USTB-05 was MCs
369 biodegrading strain, while Sphingomonas morindae NBD5 was not.
370
371 Since hepatotoxin MCs and NOD are a class of monocyclic heptapeptide and
372 pentapeptide compounds, some amino acid metabolism processes may be involved in
373 MCs and NOD biodegradation. According to the general chemical molecular structure
374 of MCs and NOD, these structures are D-alanine, variable L-amino acid, D-isoleucine,
375 D-erythro-β-methylaspartic acid, N-dehydrogenation alanine, L-arginine, D-glutamic
376 acid, Adda. Adda is the particular C20 β-amino acid: (2S, 3S, 8S, 9S) 3-amino-9-
377 methoxy-2, 6, 8-trimethyl-10-phenyldeca-4(E), 6(E)-dienoic acid (10). Among them,
378 the variable L-amino acids are leucine and arginine. Sphingopyxis USTB-05 had the
379 following metabolic processes: alanine, aspartate and glutamate metabolism; arginine
380 and proline metabolism; degradation of aromatic compounds; valine, leucine and
381 isoleucine biosynthesis; D-glutamine and D-glutamate metabolism.
382
383 These MCs and NOD biodegradation genes in the genome of Sphingopyxis USTB-05
384 mainly existed in ABC transporters; alanine, aspartate and glutamate metabolism;
385 arginine and proline metabolism; D-glutamine and D-glutamate metabolism; and valine,
386 leucine and isoleucine degradation. These genes ald, ansA, and gdhA in the metabolic
387 pathways of alanine, aspartate and glutamate were unique to Sphingopyxis USTB-05.
388 These genes crnA, phy, ocd, hypdh, spuC, nspC, and speE in the metabolic pathways
389 of arginine and proline were unique to Sphingopyxis USTB-05. These genes murI,
390 murD, and murC in the metabolic pathways of D-glutamine and D-glutamate were
391 unique to Sphingopyxis USTB-05. These genes hmgL, bioA, and glsA in the degradation
21 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
392 pathway of valine, leucine and isoleucine may be involved in the biodegradation of
393 MCs (Figure 10).
394
395 Figure 10 Comparison of genes related to microcystin degradation in the genomes of
22 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
396 Sphingomonas morindae NBD5 and Sphingopyxis USTB-05. The gray box in the figure
397 indicate that the gene exists in two strains, and the white indicate that the gene does not
398 exist in two strains.
399
400 Genome sequencing data comparison and proteins prediction
401 Based on the genome sequencing data of Sphingomonas morindae NBD5 and
402 Sphingopyxis USTB-05, the protein sequences were predicted. The same protein
403 sequences were combined into a cluster, and then the unique protein sequence was
404 annotated. 1983 of protein sequences were predicted in Sphingopyxis USTB-05, and
405 1939 protein sequences were predicted in Sphingomonas morindae NBD5. Among
406 them, there were a total of 1784 protein sequences showed homology, 199 unique
407 protein sequences for Sphingopyxis USTB-05, and 155 unique protein sequences for
408 Sphingomonas morindae NBD5 (Figure 11).
409
410 Figure 11 Venn diagram of comparison of genomic predicted difference proteins
411 Sphingomonas morindae NBD5 and Sphingopyxis USTB-05
412 DISCUSSION
413 The two strains were found in different habitats, but they were similar or different in
414 some functions. Sphingomonas morindae NBD5 had two genomes, which were
415 relatively rare in Sphingomonas. The genome and plasmid prediction showed two 23 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
416 genomes and two plasmids, indicated that Sphingomonas morindae NBD5 was a
417 special strain of Sphingomonas. According to the description in the Berger’s Handbook
418 of Bacteria, the GC mole percentage of Sphingomonas was 59-68% [7]. The GC content
419 of the genome of Sphingomonas morindae NBD5 was 70%.
420
421 Genes exchanged between various species occurs frequently. The GC content of both
422 genomes of Sphingomonas morindae NBD5 was 70%, and the GC content of its
423 plasmids was 63%. The results indicated that the plasmids of strain NBD5 may have
424 been obtained from other species during evolution. In addition, the GC content of
425 Sphingopyxis USTB-05 was 64%, which was close to the GC content of plasmids of
426 strain NBD5.
427
428 Erwinia uredovora was a representative of carotenoid production by prokaryotes, and
429 its carotenoid synthesis coding genes had been studied. The carotenoid biosynthesis
430 gene clusters with GGPP as the precursor contain 6 open reading frames (ORFs), which
431 are crtE, crtX, crtY, crtI, crtB and crtZ. α-carotene is converted into zeaxanthin by β-
432 carotene 3-hydroxylase, which could be derived from the crtZ of Erwinia uredovora
433 and the CrtR-b of the two strains of this study (16). In addition, there was a protein-
434 coding gene CrtL-b that catalyzed the mutual conversion of different types of carotene
435 in the lutein synthesis and metabolism pathway of the two strains in this study (Figure
436 8).
437
438 Combining the synthetase genes were found in the terpenoid backbone biosynthesis
439 pathway (Figure 7) and carotenoid biosynthesis pathway (Figure 8), the most probable
440 lutein synthesis pathway was predicted. In these two strains of Sphingomonadaceae, 1-
24 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
441 deoxy-D-xylulose 5-phosphate (DXP) is synthesized from pyruvate and D-
442 glyceraldehyde 3-phosphate via the 2-Cmethyl-D-erythritol 4-phosphate (MEP)
443 pathway. After seven steps of enzyme reactions, geranyl diphosphate (GPP) or
444 isopentenyl pyrophosphate (IPP) is generated. Taking GGPP as the precursor, lutein is
445 synthesized by nine steps of enzyme reactions. These reaction products are prephytoene
446 pyrophosphate, phytoene, phytofluene, ξ-carotene, neurosporene, lycopene. Then, δ-
447 carotene or α-carotene is reacted with zeaxanthin or α-cryptoxanthin, and hydroxylase
448 is hydroxylated to form lutein. Among them, the metabolic process of synthesizing
449 GGPP from GPP or IPP is lack. However, the molecular structures of zeaxanthin and
450 lutein are only different in the position of the double bond of the left six-membered
451 carbon ring, and they can be synthesized via the 2-Cmethyl-D-erythritol 4-phosphate
452 (MEP) pathway. The synthesis pathway of zeaxanthin can provide an example. The
453 condensation of IPP with dimethylallyl diphosphate (DMAPP) to form GPP is catalysed
454 by geranyl diphosphate synthase (GPS). GPP is condensed with one molecule of IPP to
455 form farnesyl diphosphate (FPP) by FPP synthase (FPS). One molecule of FPP
456 condenses with one molecule IPP to form GGPP under the catalysis of GGPP synthase
457 (CrtE) (17)(18). Last but not least, whether there were new enzymes in the metabolic
458 process of synthesizing carotene from lycopene was worthy of further investigation.
459
460 Hepatoxin MCs degrading bacterium were found in Arthrobacter spp., Brevibacterium
461 sp. and Rhodococcus sp. (19). However, the presence of biodegradable hepatoxins MCs
462 in Sphingomonadaceae has been reported more frequently (15). Through cloning and
463 gene library screening, the gene clusters for biodegrading MC-LR (mlr A, mlr B, mlr C
464 and mlr D) were identified preliminarily by Bourne et al (20). It was further discovered
465 that all or part of these four genes existed in many MCs biodegrading strains. Molecular
25 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
466 research found that Sphingopyxis USTB-05 contained USTB-05-A, USTB-05-B, USTB-
467 05-C genes with high homology to mlr A, mlr B, mlr C, respectively (21). Hashimoto
468 et al. (22) speculated that the genes involved in the degradation of MCs were far more
469 than these four genes. Through the KEGG database metabolic pathway annotation, the
470 following genes may be involved in the biodegradation process of hepatotoxins MCs
471 and NOD by Sphingopyxis USTB-05. These genes ald, ansA and gdhA in the metabolic
472 pathways of alanine, aspartate and glutamate are involved in the biodegradation of D-
473 alanine, D-erythro-β-methylaspartate and D-glutamate. These genes crnA, phy, ocd,
474 hypdh, spuC, nspC and speE in the metabolic pathway of arginine and proline are
475 involved in the biodegradation of L-arginine. These genes murI, murD and murC in the
476 metabolic pathways of D-glutamine and D-glutamate are involved in the
477 biodegradation of D-glutamate. These genes hmgL, bioA and glsA in the metabolic
478 pathways of valine, leucine and isoleucine are involved in the biodegradation of D-
479 isoleucine (Figure 10). Sphingopyxis USTB-05 has the function of biodegrading MCs,
480 but Sphingomonas morindae NBD5 does not. Sphingopyxis USTB-05 has 199 unique
481 protein sequences, including MCs degradation-related unique aspartate-type
482 endopeptidase activity (GO: 0004190), metallopeptidase activity (GO: 0004181) and
483 carboxylate hydrolase activity (GO: 0052689).
484
485 The biodegradation pathway of Sphingopyxis USTB-05 for MC-YR and NOD has been
486 clarified. The first step is to convert the cyclic structure to the linear structure by the
487 same enzyme. Adda is produced as the final product by their last enzyme reaction.
488 However, the further biodegradation of Adda by Sphingopyxis USTB-05 has not been
489 reported yet. Recently, these genes and transposable elements that may be involved in
490 the biodegradation of phenylacetate have been observed near the mlr gene cluster (23).
26 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
491 Sphingopyxis sp. YF1, which relies on the mlr biodegradation pathway, can biodegrade
492 Adda through phenylacetic acid metabolism (24). Through the KEGG database
493 metabolic pathway annotations, the evidence of further biodegradation of Adda is found.
494 The biodegradation of Adda is related to some degradation genes in the metabolic
495 pathways of styrene, toluene and xylene (Suppl Figure S1-3).
496
497 The results of Sphingobium indicum B90A, Sphingobium japonicum UT26 and
498 Sphingobium francense Sp+ show that they are able to transform β- and δ-
499 hexachlorocyclohexane (β- and δ-HCH, respectively), the most recalcitrant
500 hexachlorocyclohexane isomers, to pentachlorocyclohexanols, but only Sphingobium
501 indicum B90A can further transform the pentachlorocyclohexanol intermediate to the
502 corresponding tetrachlorocyclohexanediols (25). The linB gene of Sphingobium
503 indicum B90A heterologously expressed protein was incubated with γ- and β-
504 hexachlorocyclohexane, the pentachlorocyclohexanol product was further transformed
505 and eventually disappeared from the culture medium (25). The linB gene was also found
506 in the annotation of chlorocyclohexane and chlorobenzene metabolism of Sphingopyxis
507 USTB05 (Suppl Figure S4). In addition, Sphingopyxis USTB05 also found many genes
508 related to the degradation of environmental pollutants, such as: dioxins, toluene, xylene,
509 chlorocyclohexane and chlorobenzene, chloroalkane and chloroalkene, styrene,
510 naphthalene and other degradation genes (Suppl Figure S5-7). Although the functions
511 of these genes in the Sphingopyxis USTB05 genome still need to be verified, it still
512 shows that Sphingopyxis USTB05 has great potential as an environmental pollutant
513 degradation bacteria.
514
515 MATERIALS AND METHODS
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516 Bacterial strains and culturing conditions
517 Sphingopyxis sp. USTB-05 was isolated and identified from the sediment of Dianchi
518 Lake in China (5). Sphingomonas morindae NBD5 was isolated and identified from
519 Noni (Morinda citrifolia L.) branch (4). They were grown in LB media at 30 ℃.
520
521 DNA extraction, identification and sequencing
522 Glycerol stocks of the original strains were initially used as inoculum for regrowth on
523 the original solid isolation media at 30 °C. Single colonies were picked and cultured in
524 LB media. Genomic DNA was extracted using the Bacterial Genomic DNA Kit (CoWin
525 Biosciences, China) according to the manufacturer’s instructions. DNA quality and
526 integrity were checked by NanoDrop Nucleic Acid Quantification (Thermo Fisher
527 Scientific, USA) and gel electrophoresis.
528
529 To confirm the identity of the strains, 16S ribosomal RNA (rRNA) gene amplicons were
530 generated by PCR using primers 27F (5´-AGAGTTTGGATCMTGGCTCAG-3´) and
531 1492R (5´-GGTTACCTTGTTACGA CTT-3´). The PCR reaction mixture contained 25
532 μL PCR Mix, 2 μL primer 27F (10 mM), 2 μL primer 1492R (10 mM) and 1μL of the
533 extracted DNA. Nuclease-free water was added to reach a total reaction volume of 50
534 μL. The following conditions were used for the bacterial 16S rRNA gene amplification:
535 initial denaturation at 94 ℃ for 5 min followed by 30 cycles of denaturation at 94 ℃
536 for 40 s, annealing at 55 °C for 40 s, elongation at 72 °C for 45 s and a final extension
537 step at 72 °C for 10 min. PCR products were purified using 1% gel electrophoresis. The
538 purified PCR products were sent for DYY-8C DNA sequencer sequencing. The
539 sequencing results was put in EZ biocloud Alignment to determine the homology
540 relationship with the known sequence.
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541
542 The genomic DNA of two strains was constructed by nanopore single molecule
543 sequencing library according to the standard protocol provided by Oxford Nanopore
544 Technologies (ONT). Large fragments of DNA were recovered by using BluePippin
545 (Sage Science, USA) automatic nucleic acid recovery system (26). DNA damage repair
546 and end repair, magnetic beads purification and linker connection were processed by
547 using the official SQK-LSK 109 ligation kit (Oxford Nanopore, UK). The Qubit library
548 quantification were transferred to computer sequencing. A second-generation
549 sequencing library for the genomic DNA and plasmid DNA of the two strains was
550 constructed. Genome sequencing of strains USTB-05 and NBD5 was performed using
551 the Illumina MiSeq platform (paired end, 2 × 300 bp reads) (27).
552
553 Genome assembly and quality control
554 For genome assembly, the subreads used second-generation sequencing technology
555 were first filtered with fastp software to obtain high-quality reads. The sequences
556 containing the linker fragment were deleted. The sequences with mass value Q <25 and
557 low mass base number length more than half of the sequence length were deleted.
558
559 For nanopore data, the original fastq format was obtained by base calling fast 5 file
560 through Albacore software in MinKNOW software package. In order to obtain more
561 accurate assembly of results, it was necessary to filter these impurities to obtain reliable
562 subreads, including filtering out Polymerase Reads with a length less than 1000 bp.
563
564 Genome annotation and comparative genomic analysis
565 The online NMPDR-rust server was used to predict the gene and coding sequence (CDs)
29 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
566 region of the assembly sequence. The predicted protein sequences were searched
567 against KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Cluster of
568 Orthologous Groups), GO (Gene ontology) to predict gene functions and metabolic
569 information through Blastall (28). Circos software was used to integrate the COG
570 annotation results, methylation results, RNA annotation results, GC content, and GC-
571 skew to map the entire genome of the bacterial strain. In addition, CRISPRFinder
572 software was used to predict the clustered regularly interspaced short palindromic
573 repeats (CRISPR) structure of the genome (29). The coding sequences of the two
574 genomes were aligned using MUMmer and analyzed in conjunction with the results of
575 the genome annotation (30).
576
577 Drawing tool
578 The heat map was generated by using the online software Hiplot (https://hiplot.com.cn).
579 Adobe Illustrator CS6 was used to generate other figures.
580
581 ABBREVIATIONS
582 AMD: age-related macular degeneration; MCs: microcystins; NOD: nodularin; WHO:
583 world health organization; DD: dibenzo-p-dioxin; LPS: lipopolysaccharide; CDSs:
584 coding sequence; CRISPR: clustered regularly interspaced short palindromic repeats;
585 GO: gene ontology; COG: cluster of orthologous groups; KEGG: kyoto encyclopedia
586 of genes and genomes; VFDB: virulence factors database; CARD: comprehensive
587 antibiotic resistance database; ORF: open reading frame; NMEP: non-mevalonate
588 pathway; GPP: geranyl pyrophosphate; IPP: isoprene pyrophosphate; GGPP: geranyl
589 geranyl pyrophosphate.
590
30 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
591 ACKNOWLEDGMENTS
592 This work was supported by the National Natural Science Foundation of China
593 (21677011) and the Fundamental Research Funds for the Central Universities (FRF-
594 TP-20-044A2; FRF-BR-19-003B; FRF-TP-18-012A1; FRF-TP-17-009A2).
595
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685
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34 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
691 Tables
692
693 Table 1. Comparison of genome characteristics between Sphingomonas morindae NBD5 and
694 Sphingopyxis USTB-05
695
696
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716 35 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
717 Figure Legends
718
719 Figure 1: Genome circle of Sphingomonas morindae NBD5
720
721 Figure 2: Genome circle of Sphingopyxis USTB-05
722
723 Figure 3: Comparison of GO functional classification of Sphingomonas morindae NBD5 and
724 Sphingopyxis USTB-05
725
726 Figure 4: Comparison of COG functional classification of Sphingomonas morindae NBD5 and
727 Sphingopyxis USTB-05
728
729 Figure 5: Comparison of KEGG pathway distribution of Sphingomonas morindae NBD5 and
730 Sphingopyxis USTB-05
731
732 Figure 6: The metabolic pathway of glycolysis/gluconeogenesis of Sphingomonas morindae
733 NBD5 and Sphingopyxis USTB-05
734 The enzyme or gene in the orange box in the figure indicates that two strains co-exist, the enzyme
735 or gene in the blue box indicates that neither of the two bacteria exist, the enzyme or gene in the
736 pink box indicates that it only exists in Sphingomonas morindae NBD5, and the enzyme or gene in
737 the bright yellow box indicates that it only exists in Sphingopyxis USTB-05.
738
739 Figure 7: The biosynthetic pathway of terpenoid skeleton of Sphingomonas morindae NBD5
740 and Sphingopyxis USTB-05
741 The enzyme or gene in the orange box in the figure indicates that two strains co-exist, and the
742 enzyme or gene in the blue box indicates that neither of the two bacteria exist.
743
744 Figure 8: The biosynthetic pathway of carotenoid of Sphingomonas morindae NBD5 and
36 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
745 Sphingopyxis USTB-05
746 The enzyme or gene in the orange box in the figure indicates that two strains co-exist, and the
747 enzyme or gene in the blue box indicates that neither of the two bacteria exist.
748
749 Figure 9: Comparison of genes related to lutein synthesis in the genomes of Sphingomonas
750 morindae NBD5 and Sphingopyxis USTB-05
751 The gray box in the figure indicate that the gene exists in the strain, and the white indicate that the
752 gene does not exist in the strain.
753
754 Figure 10: Comparison of genes related to microcystin degradation in the genomes of
755 Sphingomonas morindae NBD5 and Sphingopyxis USTB-05
756 The gray box in the figure indicate that the gene exists in two strains, and the white indicate that the
757 gene does not exist in two strains.
758
759 Figure 11: Venn diagram of comparison of genomic predicted difference proteins
760 Sphingomonas morindae NBD5 and Sphingopyxis USTB-05
761
762
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764
765
766
767
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769
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37 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
772 Table 1. Comparison of genome characteristics between Sphingomonas morindae NBD5 and
773 Sphingopyxis USTB-05
category Sphingomonas morindae NBD5 Sphingopyxis USTB-05 bases 4239716 4679489 tmRNA 1 1 tRNA 61 48 CDS 3882 4312 GC(%) both 70% 64% plasmid 2 0 774
775
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793 38 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
794
795
796
797
798
799
800
801
802 Figure 1: Genome circle of Sphingomonas morindae NBD5
803
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814 39 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
815
816
817
818
819
820
821
822
823
824
825 Figure 2: Genome circle of Sphingopyxis USTB-05
826
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40 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
835
836
837
838
839
840
841 Figure 3: Comparison of GO functional classification of Sphingomonas morindae NBD5 and
842 Sphingopyxis USTB-05
843
844
845
846
847
848
849
850
851
41 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
852
853
854
855
856
857
858 Figure 4: Comparison of COG functional classification of Sphingomonas morindae NBD5 and
859 Sphingopyxis USTB-05
860
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868 42 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
869
870
871
872
873
874
875 Figure 5: Comparison of KEGG pathway distribution of Sphingomonas morindae NBD5 and
876 Sphingopyxis USTB-05
877
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43 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
887
888 Figure 6: The metabolic pathway of glycolysis/gluconeogenesis of Sphingomonas morindae
889 NBD5 and Sphingopyxis USTB-05
890 The enzyme or gene in the orange box in the figure indicates that two strains co-exist, the enzyme
891 or gene in the blue box indicates that neither of the two bacteria exist, the enzyme or gene in the
892 pink box indicates that it only exists in Sphingomonas morindae NBD5, and the enzyme or gene in
893 the bright yellow box indicates that it only exists in Sphingopyxis USTB-05. 44 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
894
895
896 Figure 7: The biosynthetic pathway of terpenoid skeleton of Sphingomonas morindae NBD5
897 and Sphingopyxis USTB-05
898 The enzyme or gene in the orange box in the figure indicates that two strains co-exist, and the
899 enzyme or gene in the blue box indicates that neither of the two bacteria exist.
900
901
902
45 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
903
904
905
906 Figure 8: The biosynthetic pathway of carotenoid of Sphingomonas morindae NBD5 and
907 Sphingopyxis USTB-05
908 The enzyme or gene in the orange box in the figure indicates that two strains co-exist, and the
909 enzyme or gene in the blue box indicates that neither of the two bacteria exist.
910
911
912
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46 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
920
921 Figure 9: Comparison of genes related to lutein synthesis in the genomes of Sphingomonas
922 morindae NBD5 and Sphingopyxis USTB-05
923 The gray box in the figure indicate that the gene exists in the strain, and the white indicate that the
924 gene does not exist in the strain.
925
926
927
928
47 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
929
930 Figure 10: Comparison of genes related to microcystin degradation in the genomes of
931 Sphingomonas morindae NBD5 and Sphingopyxis USTB-05
932 The gray box in the figure indicate that the gene exists in two strains, and the white indicate that the
933 gene does not exist in two strains. 48 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
934
935
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937
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939
940
941
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946
947 Figure 11: Venn diagram of comparison of genomic predicted difference proteins
948 Sphingomonas morindae NBD5 and Sphingopyxis USTB-05
949
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955 49 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
956 Supplementary Tables and Figures
957
958 Suppl Table S1. The sequences of USTB-A, USTB-B, USTB-C, and USTB-D in the
959 Sphingopyxis USTB-05 genome are similar to mlr A, mlr B, mlr C and mlr D, respectively
960
961 Suppl Figure S1. The metabolic pathway of styrene degradation of Sphingopyxis USTB-05
962
963 Suppl Figure S2. The metabolic pathway of toluene degradation of Sphingopyxis USTB-05
964
965 Suppl Figure S3. The metabolic pathway of xylene degradation of Sphingopyxis USTB-05
966
967 Suppl Figure S4. The metabolic pathway of chlorocyclohexane and chlorobenzene
968 degradation of Sphingopyxis USTB-05
969
970 Suppl Figure S5. The metabolic pathway of dioxin degradation of Sphingopyxis USTB-05
971
972 Suppl Figure S6. The metabolic pathway of chloroalkane and chloroalkene degradation of
973 Sphingopyxis USTB-05
974
975 Suppl Figure S7. The metabolic pathway of naphthalene degradation of Sphingopyxis USTB-
976 05
977
978
979
980
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982
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50 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
984
985
986
987
988
989
990
991
992
993
994
995 Suppl Table S1. The sequences of USTB-A, USTB-B, USTB-C, and USTB-D in the
996 Sphingopyxis USTB-05 genome are similar to mlr A, mlr B, mlr C and mlr D,
997 respectively
998
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51 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
1009
1010
1011
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1016
1017
1018 Suppl Figure S1. The metabolic pathway of styrene degradation of Sphingopyxis
1019 USTB-05. The enzyme or gene in the red box in the figure indicates that two strains
1020 co-exist, and the enzyme or gene in the blue or white box indicates that neither of the
1021 two bacteria exist.
1022
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1024
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52 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
1030
1031
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1033
1034
1035 Suppl Figure S2. The metabolic pathway of toluene degradation of Sphingopyxis
1036 USTB-05. The enzyme or gene in the red box in the figure indicates that two strains
1037 co-exist, and the enzyme or gene in the blue or white box indicates that neither of the
1038 two bacteria exist.
1039
1040
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53 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
1045
1046
1047
1048
1049
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1051
1052 Suppl Figure S3. The metabolic pathway of xylene degradation of Sphingopyxis
1053 USTB-05. The enzyme or gene in the red box in the figure indicates that two strains
1054 co-exist, and the enzyme or gene in the blue or white box indicates that neither of the
1055 two bacteria exist.
1056
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1059
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54 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
1065
1066
1067
1068
1069 Suppl Figure S4. The metabolic pathway of chlorocyclohexane and chlorobenzene
1070 degradation of Sphingopyxis USTB-05. The enzyme or gene in the red box in the
1071 figure indicates that two strains co-exist, and the enzyme or gene in the blue or white
1072 box indicates that neither of the two bacteria exist.
1073
1074
1075
1076
55 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
1077
1078 Suppl Figure S5. The metabolic pathway of dioxin degradation of Sphingopyxis
1079 USTB-05. The enzyme or gene in the red box in the figure indicates that two strains
1080 co-exist, and the enzyme or gene in the blue or white box indicates that neither of the
1081 two bacteria exist.
1082
1083
1084
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56 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
1086
1087 Suppl Figure S6. The metabolic pathway of chloroalkane and chloroalkene
1088 degradation of Sphingopyxis USTB-05. The enzyme or gene in the red box in the
1089 figure indicates that two strains co-exist, and the enzyme or gene in the blue or white
1090 box indicates that neither of the two bacteria exist.
1091
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57 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
1099
1100 Suppl Figure S7. The metabolic pathway of naphthalene degradation of
1101 Sphingopyxis USTB-05. The enzyme or gene in the red box in the figure indicates that
1102 two strains co-exist, and the enzyme or gene in the blue or white box indicates that
1103 neither of the two bacteria exist.
1104
1105
1106
1107
58 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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: Genome circle of Sphingomonas morindae NBD5 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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 2: Genome circle of Sphingopyxis USTB-05 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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 3: Comparison of GO functional classification of Sphingomonas morindae NBD5 and
Sphingopyxis USTB-05 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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: Comparison of COG functional classification of Sphingomonas morindae NBD5
and Sphingopyxis USTB-05 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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: Comparison of KEGG pathway distribution of Sphingomonas morindae NBD5 and
Sphingopyxis USTB-05 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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 6: The metabolic pathway of glycolysis/gluconeogenesis of Sphingomonas morindae NBD5 and Sphingopyxis USTB-05
The enzyme or gene in the orange box in the figure indicates that two strains co-exist, the enzyme or gene in the blue box indicates that neither of the two bacteria exist, the enzyme or gene in the pink box indicates that it only exists in Sphingomonas morindae NBD5, and the enzyme or gene in the bright yellow box indicates that it only exists in Sphingopyxis USTB-05. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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 7: The biosynthetic pathway of terpenoid skeleton of Sphingomonas morindae NBD5 and Sphingopyxis USTB-05
The enzyme or gene in the orange box in the figure indicates that two strains co-exist, and the enzyme or gene in the blue box indicates that
neither of the two bacteria exist. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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 8: The biosynthetic pathway of carotenoid of Sphingomonas morindae NBD5 and Sphingopyxis USTB-05
The enzyme or gene in the orange box in the figure indicates that two strains co-exist, and the enzyme or gene in the blue box indicates that neither of the two bacteria exist. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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 9: Comparison of genes related to lutein synthesis in the genomes of Sphingomonas morindae NBD5 and Sphingopyxis USTB-05
The gray box in the figure indicate that the gene exists in the strain, and the white indicate that the gene does not exist in the strain. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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 10: Comparison of genes related to microcystin degradation in the genomes of Sphingomonas morindae NBD5 and Sphingopyxis
USTB-05
The gray box in the figure indicate that the gene exists in two strains, and the white indicate that the gene does not exist in two strains. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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 11: Venn diagram of comparison of genomic predicted difference proteins Sphingomonas morindae
NBD5 and Sphingopyxis USTB-05 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
Tables
Table 1. Comparison of genome characteristics between Sphingomonas morindae NBD5 and
Sphingopyxis USTB-05
bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437629; this version posted March 31, 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.
Table 1. Comparison of genome characteristics between Sphingomonas morindae NBD5 and
Sphingopyxis USTB-05
category Sphingomonas morindae NBD5 Sphingopyxis USTB-05
bases 4239716 4679489
tmRNA 1 1
tRNA 61 48
CDS 3882 4312
GC(%) both 70% 64%
plasmid 2 0