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Remediation and Control Technologies

Linking exoproteome function and structure to anammox biofilm development Zijian Chen, Yabing Meng, Binbin Sheng, Zhongbo Zhou, Chao Jin, and Fangang Meng Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04397 • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

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Page 1 of 31

Environmental Science & Technology

1 2

Linking exoproteome function and structure to anammox

3

biofilm development

4 5

Zijian Chena, b, Yabing Menga, b, Binbin Shenga, b, Zhongbo Zhoua, b, Chao Jina,

6

Fangang Menga, b*

b

and

7

School of Environmental Science and Engineering, Sun Yat-sen University,

8

a

9

Guangzhou 510275, PR China Guangdong Provincial Key Laboratory of Environmental Pollution Control and

10

b

11

Remediation Technology (Sun Yat-sen University), Guangzhou 510275, China

12 13 14 15 16

Corresponding author.

17

*

18

Fangang MENG, Ph.D.; E-mail: [email protected]

19 20

ACS Paragon Plus Environment


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21

Abstract: Extracellular proteins are of paramount importance in the cell-cell

22

interactions of anammox biofilms. However, the inherent aggregation mechanisms

23

of anammox have largely remained elusive. Herein, using a quartz sand extraction

24

protocol and follow-up iTRAQ-based quantitative proteomics, we identified 367

25

extracellular proteins from initial colonizer, mature and detached biofilms. The

26

extracellular proteins were mainly membrane-associated. Most of the recovered

27

proteins (226, 72.5%) originated from the phylum Planctomycetes. In summary, 251

28

and 190 out of the 367 proteins recovered were up and/or downregulated at least

29

1.2-fold during the biofilm formation and detachment periods, respectively. These

30

differentially expressed proteins were dominantly involved in metal ion binding,

31

which was regarded as strong evidence for their abilities to enhance ionic bridges in

32

extracellular polymeric substances (EPS). SEM-EDX analysis of the biofilms further

33

showed substantial levels of calcium and iron minerals. Critically, representative

34

Sec-dependent

35

rod-shaped Proteobacteria and filamentous Chloroflexi (11, 4 and 2 with differential

36

expression, respectively) were found to have typical and abundant inner β-sheet

37

structures, wherein hydrophobic moieties can promote anammox aggregation.

38

Overall, these findings highlight links between differentially expressed protein

39

functions and morphologic traits of anammox consortia during biofilm

40

development.

secretory

proteins

affiliated

with

41 42 43 44 45 46 47


coccoid

Planctomycetes,

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48 49 50

Table of Contents:

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66



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67 68

Introduction

69

Nitrogen pollution has become increasingly frequent, most egregiously in

70

inland lakes or offshore marine areas, where practitioners, researchers and

71

specialists all face great challenges and opportunities. Anaerobic ammonium

72

oxidation (anammox)-based processes largely contribute to the elimination of

73

nitrogen in engineered facilities1-3 and natural ecosystems.4 In practice, anammox

74

technologies show greater superiority over conventional nitrification-denitrification

75

processes because of their eliminated organic carbon consumption, much lower

76

greenhouse gas emissions and much lower sludge production irrespective of the

77

slow growth rate of anammox bacteria.5

78

Microorganisms form compact particle-based biofilms, which facilitate the

79

design and operation of wastewater treatment systems.6, 7 Extracellular polymeric

80

substances (EPS), presenting outside of microbial cells and within bio-aggregates,

81

are involved in the cohesion and adhesion of cells during biofilm formation.8,

82

Unlike conventional bacterial cultures, the EPS in anammox consortia have much

83

higher protein contents than carbohydrates,10 which would contribute toward

84

anammox bacteria aggregation.11 A prior study documented the presence of

85

considerable levels of hydrophobic amino acids; the exposure of inner hydrophobic

86

groups of protein led to increased hydrophobic interactions, thereby enhancing

87

anammox bacteria aggregation.12 A recent study also revealed that glycoproteins

88

comprised a major fraction of EPS and played an important role in the granulation

89

of anammox bacteria.13 These results implied that various proteins in EPS may

90

participate in cell-cell interactions during anammox biofilm development. Thus,

91

identifying the functional attributes and structural traits of recovered extracellular

92

proteins is essential for elucidating anammox aggregation and biofilm formation

93

mechanisms. More significantly, to the best of our knowledge, no one has reported


9

Page 5 of 31


94

the isolation and authentication of extracellular proteins in different morphotypic

95

anammox biofilms, such as initial colonizers, mature biofilm and detached biofilm.

96

Quantitative proteomics analysis using isobaric tags for relative and absolute

97

quantification (iTRAQ) allows simultaneous protein identification and (relative)

98

quantification from peptide fragmentations and low-mass reporter ions,

99

respectively.14 iTRAQ can perform multiplex analyses in a high-throughput

100

manner15 and possesses the potential for extreme sensitivity.16 Currently, iTRAQ

101

proteomics analysis has been applied exclusively to mammals, plants and microbial

102

communities (i.e., hamsters, diatoms, and waste-activated sludge).17-20 It can be

103

expected that iTRAQ technology is highly promising for identifying and quantifying

104

extracellular proteins in anammox consortia.

105

A prerequisite for successful protein characterization is the use of a proper

106

extraction method that can extract EPS with both high extraction efficiency and low

107

protein damage. Traditional extraction protocols (e.g., heat, formaldehyde, sodium

108

hydroxide (NaOH), sonication, and resin-based methods) can inevitably result in

109

protein denaturation, chemical perturbation, or cell damage.21,

110

alkaline solutions can break disulfide bonds in proteins and damage microbial

111

cells.23 These problems could potentially decrease the analysis accuracy of

112

proteomics. To ensure the efficiency of iTRAQ analysis, we developed a new physical

113

extraction method using mechanical scouring without requiring the addition of

114

chemicals or harsh treatment.

22

For instance,

115

The objective of the present study was to find potential linkages between the

116

functions and structures of exoproteomes and macro morphologies of anammox

117

consortia. EPS was extracted by a quartz sand protocol, and protein expression

118

levels were compared using an integrated shotgun proteomics analysis with iTRAQ.

119

The bacterial community compositions and diversities of various biofilms were

120

evaluated by 16S rRNA gene sequencing. All Sec-dependent secretory proteins were



121

structure modeled through homology models and ab initio calculation methods.

122

This study provides means for exploring extracellular protein functions in

123

aggregation mechanisms from an iTRAQ-based extracellular proteome perspective,

124

which will be of paramount importance to understanding the colonization,

125

development and dispersal of anammox biofilm.

126 127

Materials and methods

128

Bioreactor operation and anammox biofilm collection

129

An 8-L bench-scale biofilm reactor (with nonwoven fabric attachment carrier) was

130

inoculated with biomass (ca. 4 g dry weight) from an anammox-based membrane

131

bioreactor (MBR) operated in our lab, achieving stable anammox performance. The

132

tested biofilm reactor was operated in mesophilic (31~35 °C), alkalescent (7.9~8.2

133

pH) and low dissolved oxygen (Do < 0.05 mg/L) conditions for approximately 300

134

days. The hydraulic retention time (HRT) of the biofilm reactor was set to 8 h. Feed

135

was provided by mixing (NH4)2SO4 and NaNO2 at a molar ratio of 1:1 with a total

136

nitrogen (TN) content of ca. 100 mg/L, together with approximately 420 mg

137

NaHCO3/L, 50 mg KH2PO4/L, 150 mg MgSO4·7H2O/L, 68 mg CaCl2/L, and 1 mL trace

138

elements/L. From biofilm formation to detachment, the nitrogen loading rate (NLR)

139

of the biofilm reactor at steady operation ranged from 0.75-0.85 kg-N m-3 d-1, and

140

the corresponding nitrogen removal rates (NRRs) reached 0.52 ± 0.04 kg-N m-3 d-1.

141

Colonized biofilm cells and mature/dispersed biofilm aggregates were separately

142

collected between days 10-20 and 150-170, respectively. This strategy was adopted

143

to highlight proteins that were similarly expressed during a specific biofilm

144

development stage. For more details on the biofilm reactor operation and biofilm

145

sampling, refer to Supplementary Information (SI) Figure S1 and Tables S1 and S2.

146 147

16S rRNA gene sequencing


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148

Genomic DNA was extracted from the anammox biofilms (n = 3 per morphotype

149

according to distinct development stages; 9 samples total) using a MoBio Soil DNA

150

Isolation Kit (http://www.mobio.com/) according to manufacturer instructions. The

151

16S rRNA gene fractions spanning the V4 region were amplified utilizing

152

methodology

153

(http://press.igsb.anl.gov/earthmicrobiome/empstandard-protocols/16s/)

154

original

155

(5’-GGACTACHVGGGTWTCTAAT-3’). The amplicons were sequenced on an Illumina

156

HiSeq2500 platform (http://www.illumina.com/). The paired-end raw reads were

157

merged utilizing FLASH24 and quality filtered for capturing clean tags. Effective tags

158

were ultimately acquired via chimera detection and removal.25 Operational

159

taxonomic units (OTUs) were obtained via UPARSE26 with a 97% similarity

160

threshold followed by taxonomy assignment using a Ribosomal Database Project

161

(RDP) classifier. To visualize the microstructure and determine the elemental

162

composition of the anammox biofilms, scanning electron microscopy combined with

163

energy dispersive X-ray detector (SEM–EDX) was employed. Detailed methodologies

164

can be found in SI Texts SI.

developed

primers

515F

by

the

Earth

Microbiome

(5’-GTGCCAGCMGCCGCGGTAA-3’)

and

Project with 806R

165 166

EPS analysis and protein preparation

167

To warrant minimal anammox cell lysis and maximally eliminate the interference or

168

masks of chemicals, as previously proposed cation exchange resins27 and grinding,28

169

methods, a physically based quartz sand protocol (friction stripping) was adopted to

170

separate and recover EPS fractions. Briefly, 2 g of homogenized wet anammox

171

biofilm and 7 g of quartz sand (approximately 0.5 mm particle size, 140 g/g-VSS)

172

were transferred into a glass beaker with 30 mL of a 0.05% NaCl (w/v) solution. A

173

magnetic stirrer equal in size to that of the glass-bottom beaker diameter was used

174

to stir the mixture at 600 rpm for 3 h at 4 °C to ensure felicitous mixing and milling.



Page 8 of 31

175

The liquid mixture was then diverted to a centrifuge tube and centrifuged at 10,000

176

g for 20 min at 4 °C. The supernatant was filtered through a 0.22-µm

177

polytetrafluoroethylene membrane before collecting the EPS. The recovered

178

proteins, polysaccharides and DNA were quantified by the modified Lowry

179

method,29 the phenol-sulfuric acid method30 and the diphenylamine colorimetric

180

method,31 respectively. Total biomass content (in terms of VSS) of the biofilm

181

sample (2 g wet anammox consortia) was determined by standard methods32 prior

182

to EPS extraction. To enhance the reliability of the EPS quantification, triplicate

183

samples in each growth stage were collected for each biofilm culture.

184 185

Protein separation, digestion and iTRAQ labeling

186

Phenylmethanesulfonyl fluoride (PMSF) was added to the EPS solution (n = 2 per

187

morphotype; 6 samples total) at a final concentration of 1 mM to prevent protein

188

pellet degradation and stored at −80 °C for further analysis. Treated proteins were

189

concentrated to approximately 500 µL using a 10-kDa filter (regenerated cellulose

190

membranes)33 at 5,000 g for 1 h at 4 °C. Concentrated protein solutions were

191

measured by the Bradford assay with BSA as the standard.34 Next, 10 µg of protein

192

from each sample were pooled into 2× gel loading buﬀer (10% SDS, 250 mM

193

Tris-HCl, pH 6.8) and boiled for 5 min. Protein lysates were further separated by

194

12% SDS-PAGE as previously described35 to degrade fragment complexity and

195

warrant the generation of differential protein profiles.36

196

Trypsin digestion was modified as follows:37 200 µg of protein from each

197

anammox sample were reduced with tris-(carboxyethyl) phosphine hydrochloride

198

(TCEP), alkylated with methyl methane thiosulfate (MMTS), and digested with

199

trypsin (Wtrypsin: Wprotein = 1: 50) at 37 °C overnight. Secondary digestion (Wtrypsin:

200

Wprotein = 1: 100) was further conducted at 37 °C for 4 h to improve the hydrolysis

201

efficiency.

Sufficiently

digested

peptides

were


reconstituted

in

0.5

M

Page 9 of 31


202

triethylammonium bicarbonate (TEAB), incorporating the two previously digested

203

peptides and the reconstituted peptides amounting to 100 µg.

204

iTRAQ labeling was implemented with iTRAQ® Reagents–8plex according to

205

manufacturer instructions (Applied Biosystems, Sciex, USA). Three different

206

morphologic anammox biofilms were labeled for 2 h at room temperature as

207

follows: tag116-initial colonizers, tag117-initial colonizers, tag113-mature biofilm,

208

tag115-mature biofilm, tag119-detached biofilm, and tag121-detached biofilm.

209

Duplicates of each anammox biofilm were considered to increase the confidence of

210

differential proteins. Following the labeling reaction, equal volumes of labeled

211

peptides from individual biological replicates were pooled together and

212

vacuum-dried. The dried peptides were stored at −80 °C until MS analysis.

213 214

Liquid chromatography (LC)-MS/MS proteomic measurements

215

High-pH reversed-phase liquid chromatography was used for fractionating complex

216

peptides. LC-MS/MS was directed at matching the proteins in question. As

217

previously described,38 95% solvent A (20 mM HCOONH4, pH 10) and 5% solvent B

218

(20 mM HCOONH4, 80% acetonitrile (ACN), pH 10) were loaded onto a

219

reversed-phase column (Phenomenex columns; Gemini-NX 3u C18 110 Å; 150*2.00

220

mm) in advance for 30 min. The dried peptides were redissolved in 200 µL of

221

solvent A. Then, 100 µL of the resuspended peptides were subjected to gradient

222

elution at 200 µL/min. The 85-min linear gradient consisted of 5~37% solvent B

223

over 75 min, 37~95% solvent B over 5 min, and a hold at 95% solvent B for 5 min.

224

Eluted segments were obtained every 50 s and compiled into 24 segments based on

225

peak intensities at 214 and 280 nm (focusing mainly on 214 nm). Subsequently, the

226

obtained segments were suspended in solvent C (0.1% formic acid (FA) and 2%

227

ACN) and examined on a Q Exactive mass spectrometer (Thermo Fisher Scientific,

228

Bremen, Germany) operating in data-dependent mode. The dissolved peptides were



Page 10 of 31

229

eluted with an effective linear gradient comprising 5~35% solvent D (0.1% FA and

230

80% ACN) at 300 nL/min for 40 min. Full MS1 scans were identified by the Q

231

Exactive instrument over a mass range of 350 to 1800 m/z set to a 70,000

232

resolution; 20 data-dependent selective MS/MS scans were obtained per full scan

233

set to a 17,500 resolution and a 60-ms maximum injection time.

234 235

iTRAQ proteomic functional and statistical analyses

236

iTRAQ raw data were selected using ProteinPilot™ Software 5.0 (AB Sciex). Only

237

proteins characterized by unused scores > 1.3 (i.e., greater than a 95.0% confidence

238

level) were considered. Up and downregulated proteins were stipulated based on

239

many previous studies.19,

240

proteins was a ratio > 1.2 or < 0.83 coupled with a P value < 0.05. A custom

241

SEARCH_DB_UniProt.2017.1.7.fasta database was created to match concerned

242

proteins based on 16S rRNA gene sequences and a few previous reports,41-43 which

243

comprised

244

http://www.uniprot.org/taxonomy/203682),

245

sequences, http://www.uniprot.org/taxonomy/1224), Chlorobi (47,018 sequences,

246

http://www.uniprot.org/taxonomy/1090), Bacteroidetes (2,740,782 sequences,

247

http://www.uniprot.org/taxonomy/976),

248

http://www.uniprot.org/taxonomy/200795), Acidobacteria (57,786 sequences,

249

http://www.uniprot.org/taxonomy/57723), Armatimonadetes (20,565 sequences,

250

http://www.uniprot.org/taxonomy/67819), Actinobacteria (7,126,231 sequences,

251

http://www.uniprot.org/taxonomy/201174), and Nitrospirae (81,411 sequences,

252

http://www.uniprot.org/taxonomy/40117) phyla. Necessarily, the sequences of

253

common contaminating proteins (ABSciex_ContaminantDB_20070711.fasta) were

254

also added to the custom protein database. Recovered proteins were annotated by

255

the Gene Ontology (GO) database (http://www.geneontology.org) in terms of

the

39, 40

The typical criterion for differentially expressed

Planctomycetes

(190,807 Proteobacteria

Chloroflexi


(154,458

sequences, (21,563,853

sequences,

Page 11 of 31


256

molecular function, cellular components and biological processes. Quality control

257

information and SDS-PAGE analysis are provided in SI Figures S2 and S3 and Texts

258

S2 and S3.

259

All original data and the ProteinPilot output tables were uploaded to iProX

260

(http://www.iprox.org) under the accession number IPX0001034001. Graphical

261

and statistical analyses were carried out using GraphPad Prism 7 (GraphPad

262

Software Inc., San Diego, CA, USA). P < 0.05 was deemed significant.

263 264

Modeling of secreted protein 3D structures

265

Secreted proteins from eukaryotes to the phylum Planctomycetes (anammox signal

266

peptides are more akin to those of eukaryotes than those of prokaryotes)44 and from

267

Gram-negative bacteria to the phyla Proteobacteria and Chloroflexi were predicted

268

using the SignalP 4.1 server (www.cbs.dtu.dk/services/SignalP/).45 Best-fit 3D

269

models of target proteins were identified pluralistically utilizing a combination of

270

SWISS-MODEL

271

(https://swissmodel.expasy.org/))46,

272

structure

273

(http://zhanglab.ccmb.med.umich.edu/I-TASSER/)).48

274

SWISS-MODEL homology modeling, to sieve out higher confidence target proteins,

275

the GMQE-score was considered the principal criterion (values between 0 and 1,

276

where higher GMQE scores represent higher confidences in the predicted structure).

277

Because all information came from the same family, sequence identities of

278

approximately 30% or higher and excellent QMEAN scoring functions were

279

considered. Remaining unmatched target proteins were additionally determined by

280

I-TASSER based on the lowest (Gibbs) free energy principle (C-score is typically in

281

the range of [-5, 2]; a C-score > -1.5 indicates a correct global topology model).

and

(an

online

function

platform 47

prediction

for

protein

homology

modeling

and I-TASSER (an automated protein server

originating


In

from

ab

initio

the

web-based


282

Eventually, 3D structure figures of authoritative target proteins were generated by

283

UCSF Chimera.49

284 285

Results

286

Microbial community structure

287

16S rRNA gene sequences of microbial taxa from initial colonizers and mature and

288

detached biofilms yielded 83,070 sequences, representing 795 OTUs on average.

289

Additionally, rarefaction curves of all biofilms nearly reached apices, indicating that

290

the dominant and prevalent species had been effectively captured (SI Figure S4).

291

Taxonomic abundance scenarios of the three anammox biofilms demonstrated

292

general differences in bacterial community composition and diversity (Figure 1a).

293

Expectedly, the phylum Planctomycetes was dominant, with a high abundance of

294

49.1%-60.5% in various biofilm aggregates. Highly enriched anammox bacteria

295

comprised genera Candidatus Jettenia (28.9%-51.1%), Candidatus Brocadia

296

(0.1%-0.2%) and Candidatus Kuenenia (0.6%-1.6%) (Supplementary Data 1).

297

Strikingly, the mature and detached biofilms comprised higher abundances of the

298

phylum Proteobacteria (18.8% and 17.1%, respectively) than the initial colonizers

299

(12.0%, P < 0.05). In comparison, there were no significant differences in the

300

phylum Chlorobi, ranging from 10.7-13.0%, among the three biofilm aggregates.

301

Interestingly, the abundance of Chloroflexi was significantly higher in mature and

302

dispersed biofilms (4.0% and 3.8%, respectively) compared with that in initial

303

colonizers (1.8%, P < 0.05; SI Figure S6). Simpson and ACE indices (Figure 1b)

304

revealed that mature biofilm harbored a more diverse microbial community than

305

initial and dispersal biofilms (P < 0.05). Conversely, beta diversity patterns showed

306

community composition converged in the mature and detached biofilms (Figure 1c).

307

SEM observations also supported the result that considerable filamentous and

308

rod-shaped bacteria coexisted in the two biofilms, which was very different from


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Page 13 of 31

initial colonizers (Details were presented in SI Text S4 and Figure S5).

Actinobacteria Cyanobacteria Acidobacteria Nitrospirae Chloroflexi Armatimonadetes Firmicutes Chlorobi Bacteroidetes Proteobacteria Planctomycetes

0.75

0.50

0.25

0.00 In itia

l c o lo

n

ize r s

M a tu

io f re b

ilm D e ta

ched

b io f

ilm

Alpha diversity 1000

1.0

0.9

0.40 0.38

*

850 800

0.7

0.6

Beta diversity

950

*

900 0.8

c

Unweighted Unifrac

b

1.00

ACE

Relative abundance%

a

Simpson

309


0.36 0.34 0.32

750

0.30

700

0.28

s s er film film er film film niz bio bio niz bio bio oloure hed oloure hed c c l t l t tia a tac tia a tac Ini MDe Ini MDe

*

rs lm lm ize iofi iofi lonre bed b o l c tu h tia a tac Ini MDe

310 311 312 313 314 315 316 317 318 319

Figure 1. Community composition and diversity in various anammox biofilms. (a) Relative abundances of microbial phyla in initial colonizers, mature biofilm and dispersed biofilm. (b) Bacterial alpha diversity comparisons among different anammox biofilms were based on Simpson and ACE metrics, and (C) bacterial beta diversities were based upon unweighted UniFrac metrics. Significant pairwise differences were measured by the Wilcoxon test (* P < 0.05, where * indicates an anammox biofilm form significantly different from the other two). OTUs abundance was normalized using 59,337 reads per biofilm (the minimum amount of sequences as the standard). Subsequent diversity analyses were all performed based on this output normalized 16S data.

320 321

Chemical composition of EPS

322

Initial colonizers (46.2 ± 1.6 mg/g-VSS) had a higher extracted EPS content than

323

mature (30.0 ± 1.4 mg/g-VSS) and detached biofilms (39.2 ± 2.0 mg/g-VSS). The

324

proteins of colonizing biofilm and dispersed aggregates accounted for up to 82.6%

325

(38.2 ± 3.1 mg/g-VSS) and 76.5% (30.0 ± 3.4 mg/g-VSS), respectively. The

326

polysaccharides in the two biofilms were both less than 20%. In comparison,

327

extracellular proteins and polysaccharides were harvested from sessile biofilm at

328

proportions of 55.0% (16.3 ± 0.3 mg/g-VSS) and 31.7% (9.6 ± 2.5 mg/g-VSS),

329

respectively. All three biofilm aggregates contained much less DNA (1.7-4.1

330

mg/g-VSS), which can act as a cell integrity marker (Details can be found in SI Figure

331

S7). In all, these results suggested that the dominant key component in anammox



332

EPS is proteins (over 70%, especially in initial and detached biofilms).10

333 334

Origin and localization of the recovered extracellular proteins

335

In total, 15,545 spectra, 5,476 distinct peptides, 39,757 proteins before grouping

336

and 396 detected proteins (unused cutoff > 1.3) were obtained (SI Table S3 and Text

337

S5). After reverse database searching, 367 trusted proteins were ultimately

338

identified, most of which (pI ranging from 4.0 to 7.0, 75.3%) were negatively

339

charged under environmentally relevant pH levels (SI Text S6, Figure S8). As

340

expected, a majority of these identified proteins (226, 72.5%) originally affiliated

341

with the phylum Planctomycetes (Figure 2a), followed by those from the phyla

342

Proteobacteria (45, 12.3%) and Actinobacteria (36, 9.8%). Other microbes including

343

the Chloroflexi, Bacteroidetes and Chlorobi phyla contributed a small proportion of

344

the identified proteins (5.4% in total). Moreover, Planctomycetes-affiliated proteins

345

were deeply branched in Candidatus Jettenia-dominated species at the genus level.

346

According to GO-Slim subcellular localization, 107 proteins (Planctomycetes,

347

accounting for 69.2%) annotated for 195 cellular components in total (Figure 2b).

348

Of these, up to 50.8% of the annotated proteins possessing GO terms originated

349

from the membrane-associated and extracellular regions, including the cell

350

membrane (20.0%), integral components of the membrane (14.4%), cell outer

351

membrane, periplasmic space, and extracellular matrix. These features suggest that

352

membrane-associated proteins were dominant in the EPS matrix. Meanwhile, some

353

intracellular leakage substrates, such as the cytoplasm and ribosomes, were also

354

detected as a potential source of EPS matrix proteins.


Page 14 of 31

Page 15 of 31


355 356 357 358 359

Figure 2 ． Overview of iTRAQ proteomics and distribution of extracellular proteins. (a) Complete constitution of proteins in different morphotypic anammox biofilms at the phylum level. (b) Localization of extracellular proteins in initial colonizers, mature biofilm and detached biofilm. The exploded sections represent potential extracellular components.

360 361

Functional potentials and activities of differential proteins in anammox

362

biofilms

363

Pairwise comparisons were conducted based on anammox biofilm growth

364

dynamics, including mature biofilm vs. initial colonizers (biofilm formation phase),

365

detached biofilm vs. mature biofilm (biofilm detachment phase) and detached

366

biofilm vs. initial colonizers. Overall, 215 (58 upregulated, 157 downregulated), 190

367

(134

368

downregulated) out of the 367 identified proteins were at least 1.2-fold

upregulated,

56

downregulated)

and

226


(100

upregulated,

126


Page 16 of 31

369

differentially expressed in these three comparisons (based on P2 Folder change

1.2-1.5 Folder change 1.5-2 Folder change

50

6.1% 13 Actinobacteria 13.0% 28 Proteobacteria

100

2.6% 5 Chloroflexi

12.1% 23 Proteobacteria

100

0.9% 2 Chlorobi

75.8% 163 Planctomycetes

Total=215

150

c

1.6% 3 Bacteroidetes

7.4% 14 Actinobacteria 0.5% 1 Chlorobi


Down-regulated

Total=190

150

NO.of Differentially expressed proteins 200

NO.of indicator GOs

Up-regulated

200

NO.of Differentially expressed proteins

d n mucle ox t eta oti id ran l iode or s A n bi ed fe T bi nd ur P n i iro ctase bi dinng a n nu n i se ac ding cl he on activ g hyeic me bintivity el dr ac b d ity p ec ep ol id in in tr ca tidas bi ding nu on ta a e nd g cl eo calyt se act ing tid rr ic ac ivit i a t y yl tra lyaer actiivity st 4 ru ir iro trans RNse ctivity fe A a vi ct on n n ur , -s is sp ra b cti ty al 4 ulf om o se in vit co su ur e rte a diny in ns lfu cl ras r activ g te gr tit r c us e ct ity ue lu te ac iv al nt ster b tivity co i m ki of rr bindi ity po na ib n ng se os din ne in tra nt a omg ce of me ctiv e pl llu as memb ity la rr ouma cy mbran ib te m top ra e o D nu r mem la ne N A cle embr sm po op r b an smlymper rot intribo ra e al er iplaein ac somne ox l id ribas smcoellu e at ose I ic mp lar io ce omII c sp le nllu r al om ac x la d ed ra e su pl e uc buex m fen t in se m io ni nu o r n cl ac e eta p celt ei s id p bo t ro l c bi on lic rance ac c os se p p s ss id ar yn to ro ro po p h bo c r os h y prthe bateo es t ph dr ot tic ctelys s e p i odate ph t in rocriums i m e el st e o ra fo e ec e ta sp ns ld ss tror b bo ho la in re D n on lic ry tiog m N tra d p la n ov A nshydroction al b of ios DN mpor rol ess su yn A et t c ys pe th re hy ha is r e p la i pr oxitic lication ot de pr ti n ei r oc on n ad e re ic ss fo a ld ls in g

Mature biofilm vs Initial colonizers

NO.of indicator GOs

a

el ec tr on h c e ru ox trme ar me ct id an ta rie b ur or s l i r in al e f o a d co duera n b cti ing ns ca ct se in vit tit ta as ac din y ue ly e t g nt tic ac ivi o a tiv ty R f rib cti ity rRNA os vity o n in uc ANA bin me te gr hy leo TP bindin g al irodro tide binding in c tra om n las bindin ce io e d g po n ac in llu ne bi tiv g la n it nt rr ib of me din y on mm g uc embr D s leo c b an N m p A a ro ytoprane te rib la e po ll i la lymribo inn c oso sm rg e so t om m e ra m ra p e rib se a ce le os II l s llu x I u l pl om co bu ar as al m ni ox su ple t c om id at ell utea m bu x n ou r e io nt m m c it re er membra ell du b n re ct em rane m e b ov le m io r e al ct e n p ane nu of ron tab tra ro cl su tr ol ns ce e pe an ic la ss phic a p ro sp ro tio os cid xi or c n de t es ph p c or ho D p ra ha s el sp NA se cys ay h m rotedic in p a o e b le no tein su sig die ios DNrote thyolysls io cys e b per nal ste ynt A in lat is no ox tr r b he rep fol ion io t e tro in syn id an on tic lic din yl pi th e msd d pr at g -t c gl RN etic etaucti hyd oceion ut ca A o am rb (S pro ph bo n sroly ss at oh ec) ce os lic p ys sis t e yd b s p re ra io s f ho roc em ce te sy ro ryl es pt m nt m ati s or e he tra se on si tab tic ns rin gn o p p e al lic ro or in p c e t g ro s pa ce s th ss w ay st


b 30


Down-regulated

Detached biofilm vs Mature biofilm 30

Up-regulated

-30

411 ACS Paragon Plus Environment

Page 18 of 31

Strikingly higher in Initial Colonizers

Strikingly higher in Mature Biofilm

20

Molecular function

Strikingly higher in Detached Biofilm Strikingly higher in Mature Biofilm

Molecular function

Mature biofilm vs Initial colonizers

10

0

-10

-20

-30 Cellular component

Cellular component

Biological process

Detached biofilm vs Mature biofilm

20

10

0

-10

-20

Biological process

Page 19 of 31


e

f

Detached biofilm vs Initial colonizers

30

Strikingly higher in Detached Biofilm Strikingly higher in Initial Colonizers

Detached biofilm vs Initial colonizers

20

Up-regulated

>2 Folder change 1.5-2 Folder change


NO.of indicator GOs

3.1% 7 Chloroflexi

0.4% 1 Chlorobi

6.6% 15 Actinobacteria

1.2-1.5 Folder change

12.8% 29 Proteobacteria


Total=226 Down-regulated

10

0

-10

-20

-30 50

100

150

200

NO.of Differentially expressed proteins

g

Detached Biofilm vs Mature Biofilm 38

Cellular component

Biological process

GO annotations

33

52

Molecular function

ox m id et or al traedu ion ns cta b fe s in n ca rase a din st el ucletaly e activ g ru ec t c i ct tro otidic a tiv ty ur n AT e b ctivity al ca P in it co y ns herrie bindin r d g tit in ue Rme ac ing te gr hy nt NA bintivit of b d y al d iro ro ri in ing in co tra n las bos din m ce io e o g po n ac m llu ne bi tiv e la n it nt rr ou i b of me din y te on rm m g m uc embr em le a sm op i rib br ne br an al rotentra os ane lr eo i b i n ce m bo un pla oso c comllulae ox s m yt d r id ed per ma al opl plex at pe ipl m su as io a e m n- ripl sm mbbun as ic ra it re du m s n ct ic space de m ion pace fe et ns ce ab tr pro e e a llu p r h oli ns ce e la o r a n ele spo ospc p lat ss m e-c ctr ns ho roc ion in a on e pr ry es o rb ce trato bote lati s a o ll re cecid n m ns ac oly on gu ula p te si ll b e la r a tio m ula ios ta ort riu s n tr ino r mynt bol tra ch m of an a et he ic p ns ain nu tra sc ci ab tic ro po d cl ns rip m p ol p ce rt ei c i r io cysc a arb cription eta rot c p ocess te cid re oh t tio , bo ein roc ss br not an ro ine ph mo yd ran n, DN lic fo es A p s r D ch ic b o v a l - p ld s ed gl ios sphal ote matio NA temroc ing -c uta yn od f s e na -te pl es ha m th ie u ta l e m at s p e b in at lo pl e t s e am e r ic pter roxolic ng ated in ece rocbon ide proatiod o p e d ra ce n ac to s id r s s frhyd dic ss o r a bi ig os na m m olysls e yn lin th se is th g yl rin et pa at e ic th io pr w n oc ay es s

0

103

M atu

412 413 414 415 416 417 418

re B io f i l m

20

vs In

14 40

i ti a l

C o lo

n iz e

rs

De

ed tach

B iof

il m v

ti a l s Ini

C o lo

n iz e

rs

Figure 3．The functional potentials and activities of differentially expressed proteins in pairwise comparisons (i.e., mature biofilm vs. initial colonizers, detached biofilm vs. mature biofilm and detached biofilm vs. initial colonizers). (a, c, e) Numbers of upregulated and downregulated proteins and the proportion of dominant bacteria phyla. (g) Venn diagram showing shared and unique differentially expressed proteins. (b, d, f) Numbers of upregulated and downregulated proteins assigned to GO terms that are strikingly different in each comparison group. Only the top ten GO terms with a quantity ≥ 2 are shown. Annotated proteins with different GO terms may originate from one identified protein. See Supplementary Data 2-7 for details regarding differentially expressed proteins.



Page 20 of 31

419 420

Structural attributes of representative secreted proteins in the extracellular

421

regime

422

Bacterial proteins harbor signal peptides of the general-ubiquitous Sec secretion

423

pathway that target them into the extracellular milieu.52 Of the 57 identified

424

extracellular proteins based on the GO-Slim subcellular localization analysis (Figure

425

2b),

426

Chloroflexi-affiliated proteins with differential expression retained signal peptides.

427

Intriguingly, these Sec pathway-dependent proteins that interacted with the

428

aqueous environment had lower pI values (5.7±1.4) or electronegativity.

429

Furthermore, they were mostly involved in transport processes (e.g., ion, substrate

430

and polysaccharide), metal ion-binding functions and outer membrane cellular

431

components (Supplementary Data 8).

11

Planctomycetes-affiliated,

4

Proteobacteria-affiliated

and

2

432

Figure 4a shows the representative secreted protein expression profiles and

433

structure information across different anammox biofilm communities. For the

434

phylum Planctomycetes, ammonium transporter protein (I3IPX5), which mainly

435

participates in ammonium uptake, has three dominant α-helical subunits. This

436

up-and-down amphipathic helix bundle structure of I3IPX5 exhibits a continuous

437

hydrophobicity when contacting a lipid surface, which was downregulated and

438

upregulated during biofilm formation and detachment stages, respectively. Putative

439

cytochrome c (I3IH53) related to electron transfer, a key member of the respiratory

440

chain,53 displayed an open-face sandwich structure. The structure consists of

441

β-sheets laid in a relatively hydrophobic core regime, with amphipathic α-helices

442

distributed to the periphery or margins. TonB-dependent receptor (I3IK03), which

443

binds and mediates the passage of iron-chelating compounds into the periplasm,54

444

has an anti-parallel β-barrel structure with a band of hydrophobic side embedded in

445

the lipid bilayer. PQQ-dependent dehydrogenase (H1KGZ5), which triggers the


Page 21 of 31


446

formation of a proton motive force and ATP synthesis and is affiliated with the

447

phylum Proteobacteria,55 has an α2β2 subunit structure and was estimated to be

448

underexpressed approximately 7.1-fold in mature biofilm vs. initial colonizers.

449

Similarly,

450

(A0A0M8JPU0) was overexpressed 2.2-fold in the detached biofilm vs. initial

451

colonizers comparison group. In their structural domains, amphipathic α-helices

452

that can interact with water flank the central hydrophobic β-sheets. Collectively,

453

although there was no significant discrepancy in the proportions of secondary

454

structures (i.e., higher abundance of a-helices or β-sheets (Supplementary Data 8)),

455

the hydrophobic cores of representative secreted proteins in the tertiary structures

456

were dominated by β-sheets. This suggests that under certain conditions the inner

457

β-sheets can fulfill a pivotal role in cell aggregation via hydrophobic forces, and the

458

external hydrophilic α-helices (charged and uncharged residues) help solubilize the

459

proteins in aqueous solutions.56

Chloroflexi-associated

ATP-binding

cassette

460


(ABC)

transporter


461 462 463 464 465 466 467 468 469 470 471 472 473 474

Page 22 of 31

Figure 4 ． The representative secreted proteins were displayed as ribbons with secondary structure elements rainbow-colored from blue at the N-terminus to red at the C-terminus. The color intensity represents protein differential expression based on iTRAQ-based proteomics mapping. The three pairwise comparison groups: mature biofilm vs. initial colonizers (left), detached biofilm vs. mature biofilm (middle) and detached biofilm vs. initial colonizers (right). A scale (log2-fold change) is shown at the center. The targeted structural models were identified pluralistically using SWISS-MODEL and I-TASSER prediction. The vast majority of protein conformation displayed the “oil drop model” under UCSF Chimera observation because the core of a protein is relatively hydrophobic. For example, the secretion pathway protein I3IQK8 was affiliated with the phylum Planctomycetes utilizing the SWISS-MODELI prediction server. The best-fit template was 5wq7.1.A (circular associations of β-sandwiches, α-helixes were embedded on the surface of the β-barrel). More modeling and expression details of the secreted proteins are shown in SI Figures S12 and S13 and Supplementary Data 8.

475 476

Discussion

477

To ensure the natural traits of EPS matrices and to achieve effective separations of

478

extracellular proteins by SDS-PAGE, a physically based quartz sand method was

479

adopted to extract exoproteomes from anammox biofilms dominated by Candidatus

480

Jettenia (based on Illumina sequencing data). Our data supported the idea that cell

481

integrity of different biofilms is maintained without excessive leakage of

482

intracellular substrates. Lower absolute DNA release (1.7-4.1 mg/g-VSS) within the

483

extracted EPS was detected than in previous reports.22,

484

extracellular DNA (eDNA) released from autolysis or cell demise should be

485

considered.59 As such, the trade-offs between high extraction yields and low

486

biomass lysis were satisfying and better than those reported by prior studies.20, 60

487

The majority of the extracted components were membrane-associated and

488

extracellular matrix proteins (50.8%).

57, 58

Furthermore,

489

Proteins in vital and dominant fractions have been observed in different

490

anammox biofilms. Correspondingly, iTRAQ extracellular proteomics allowed us to

491

link the functional attributes and structural roles of differentially expressed


Page 23 of 31


492

proteins to anammox biofilm morphologies. Planctomycetes-affiliated proteins

493

accounted for the majority of the recovered 367 proteins. Remarkably, a

494

considerable number of differentially expressed proteins in colonizing anammox

495

biofilm were highly active in oxidation-reduction (e.g., ammonium oxidation

496

(Q1PVE0, Q1PW30, and Q1PX48), nitrate oxidoreductase and reductase

497

(A0A136LTX9, Q1PZD8)), metabolic processes (e.g., ammonium substrate uptake

498

and transport (I3IPX5)) and translation. To a certain extent, these data indicated

499

that the initial anammox colonizers exhibited higher metabolic potentials and

500

functional activities than mature and detached biofilms, which is likely related to the

501

rapid proliferation of anammox bacteria. More importantly, numerically dominant metal ion-binding proteins (e.g., Fe3+-

502 503

and

Ca2+-binding),

primarily

originating

from

504

Proteobacteria and Actinobacteria, exhibited strikingly different expressions in the

505

various anammox biofilm development phases. These ion-binding proteins

506

(especially secreted proteins) in the anammox consortia had lower negative

507

charges. Thus, the presence of metal ion-binding proteins may capture metals ions

508

in the reactor bulk, which potentially triggers cell–cell aggregation or biofilm

509

formation.61-63 The protein-induced precipitation of metal ions can increase biofilm

510

density and help maintain mechanical strength.64 A previous attempt has shown

511

that mineral complexes (e.g., hydroxyapatite) can accumulate in anammox consortia

512

under relatively higher pH, NH4+ and PO3-4 levels.65 In line with prior studies,

513

SEM-EDX revealed the presence of calcium and iron minerals in different anammox

514

biofilms. To a great extent, our current results may explain why anammox cells

515

always have a higher cell density than other bacteria. These results also potentially

516

showed that the high abundance of metal ion-binding proteins bridged with metal

517

ions from wastewater is an important driver for anammox biofilm development.


the

phyla

Planctomycetes,


Page 24 of 31

518

Planctomycetes-affiliated species were found to yield more Sec-dependent

519

secretory proteins, most of which contained inner hydrophobic β-sheets and/or

520

α-helices, than heterotrophic bacteria (e.g., Proteobacteria and Chloroflexi). Once

521

temperature or entropy increases (e.g., the binding process between metal ions and

522

EPS),66 the inner hydrophobic amino acid moieties are more accessible because of

523

the twist of β-sheets; this results in increased hydrophobicity.12,

524

anammox-based processes take place at mesophilic temperatures, which aids in

525

increasing hydrophobic interactions between anammox cells. Additionally, previous

526

studies have attributed the aggregation or flocculation behavior of bacteria cells to

527

the presence of α-helices.68,

528

calculation showed the presence of amphipathic α-helices on the outsides of the

529

central hydrophobic β-sheets. Therefore, the interactive impacts between α-helices

530

and β-sheets are expected to take part in cell aggregations. Further work is

531

recommended to explore their respective contributions to the cell aggregation of

532

anammox.

69

67

Normally,

In this study, homology modeling and ab initio

533

In addition to the Planctomycetes-affiliated species, microbes associated with

534

the phyla Proteobacteria and Chloroflexi (significantly higher abundance in mature

535

and detached biofilms) can yield Sec-dependent secretory proteins. For instance, the

536

Chloroflexi produced an ATP-binding cassette (ABC) transporter (A0A0M8JPU0),

537

which mediates substrate movement for heterotrophic bacteria to obtain carbon

538

sources 70. Previous studies have experimentally shown that filamentous Chloroflexi

539

offers a stabilizing backbone or core in three-dimensional frameworks of sludge

540

particles.71 Moreover, Proteobacteria-affiliated species with rod-shaped phenotypes

541

(e.g., Aeromonas genus,72 Oceanibulbus indolifex,73 Methylobacterium species74 and

542

denitrifying Hyphomicrobium bacteria75 (Supplementary Data 8)), particularly with

543

flagellar-like adhesive apparatus,76 were expected to contribute to biofilm

544

development attributable to their capability to swim. Overall, the proteomics


Page 25 of 31


545

characterization together with Illumina sequencing as conducted in our study can

546

greatly corroborate these hypotheses at molecular levels.

547

Taken together, iTRAQ proteomics provides a new avenue for exploring the

548

mechanism of microbe assembly during anammox biofilm growth and development.

549

A comprehensive and robust analysis of the functional and structural properties of

550

exoproteomes

551

Proteobacteria and Chloroflexi) was effectively established. Our study illustrated

552

that anammox aggregation and biofilm formation are multifactorial consequences,

553

e.g., protein-metal ion interactions and protein-induced hydrophobic interactions

554

can potentially contribute to the formation of biofilms. This proteome-based study

555

also reinforced a previous hypothesis of the positive roles of filamentous Chloroflexi

556

and rod-shaped Proteobacteria in anammox bacteria cell aggregation. Thus, the

557

functional attributes and structural traits analysis regarding representative

558

extracellular proteins will provide a valuable groundwork for future studies. While

559

further studies are required to detect how metal ions bind to proteins and to

560

elucidate the mechanisms underlying interactions between anammox bacteria and

561

heterotrophic microbes, the proteomic characterization herein advances our insight

562

on anammox aggregation.

underlying

several

representative

taxa

(Planctomycetes,

563 564 565

ASSOCIATED CONTENT

566

Supporting Information

567 568 569 570 571 572 573

Figure S1. Biofilm reactor operation and biofilm sampling. Figure S2. Quality control evaluation of mass spectrometry dataset. Figure S3. SDS-PAGE analysis images. Figure S4. Rarefaction curve of the different biofilm samples. Figure S5. Microscale structures of the different anammox biofilms by SEM observation. Figure S6. Statistically significant differences between two groups in bacterial phylum level. Figure S7. EPS composition from the three different anammox biofilms through quartz sand extraction protocol. Figure S8. Physicochemical characteristics of extracellular proteins. Figure S9. SEM images of inorganic precipitates in the



574 575 576 577 578 579 580 581

different anammox biofilms. Figure S10. Numbers and relative abundances of secreted proteins based on signal peptide prediction. Figure S11. The 3D structure information of secreted proteins. Table S1. The contents of synthetic wastewater. Table S2. Performance data from the anammox biofilm reactor around the time of sampling. Table S3. MS/MS Spectra Researching Databases. Table S4. The EDX analysis of the different anammox biofilms. Text S1. SEM-EDX mapping. Text S2. Quality control analysis. Text S3. SDS-PAGE analysis. Text S4. The morphological trait of the different biofilms in microscale. Text S5. Sequence database choice on exoproteome. Text S6. Physicochemical characteristics of extracellular proteins.

582 583 584 585

AUTHOR INFORMATION

586

Corresponding author

587

Email: [email protected] (Fangang MENG)

588 589

Notes

590

The authors declare no competing financial interests.

591 592

Conflicts of Interest

593

The authors declare no conflicts of interest.

594 595

Acknowledgments

596

This project was supported by funding from the National Natural Science

597

Foundation of China (no. 51622813) and the China Postdoctoral Science Foundation

598

(funded project 2018T110910).

599 600

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