110 highlighting the location of proteins encoded by the acarbose and the pyochelin biosynthesis gene cluster

JPROT-02123; No of Pages 16 JOURNAL OF P ROTEOM IC S XX ( 2015) X XX–X XX

Available online at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/jprot

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Sergej Wendlera , Andreas Ottob , Vera Ortseifena,c , Florian Bonnb , Armin Neshatc , Susanne Schneiker-Bekela , Frederik Walterc,d , Timo Wolf a,c , Till Zemkee , Udo F. Wehmeier f , Michael Heckerb , Jörn Kalinowskic , Dörte Becherb , Alfred Pühlera

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Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany b Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald, F.-L. Jahnstrasse 15, 17489 Greifswald, Germany c Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany d Department of Proteome and Metabolome Research, Faculty of Biology, Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, Germany e Product Supply, Bayer Pharma AG, Friedrich Ebert Str. 217-475, 42117 Wuppertal, Germany f Department for Sportsmedicine, University of Wuppertal, Pauluskirchstr. 7, 42285 Wuppertal, Germany

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Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the pyochelin biosynthesis gene cluster

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AR TIC LE I NFO

ABSTR ACT

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Article history:

Acarbose is an α-glucosidase inhibitor produced by Actinoplanes sp. SE50/110 that is

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Received 17 December 2014

medically important due to its application in the treatment of type-2 diabetes. In this work,

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Accepted 12 April 2015

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a comprehensive proteome analysis of Actinoplanes sp. SE50/110 was carried out to

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determine the location of proteins of the acarbose (acb) and the putative pyochelin (pch) biosynthesis gene cluster. Therefore, a comprehensive state-of-the-art proteomics

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Keywords:

approach combining subcellular fractionation, shotgun proteomics and spectral counting

Actinoplanes

Comprehensive proteomics

four different proteome fractions (cytosolic, enriched membrane, membrane shaving and

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Subcellular fractionation

extracellular fraction) resulted in the identification of 1582 of the 8270 predicted proteins.

Membrane proteome acarbose

All 22 Acb-proteins and 21 of the 23 Pch-proteins were detected. Predicted membrane-

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Pyochelin

associated, integral membrane or extracellular proteins of the pch and the acb gene cluster

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to assess the relative abundance of proteins within fractions was applied. The analysis of

were found among the most abundant proteins in corresponding fractions. Intracellular biosynthetic proteins of both gene clusters were not only detected in the cytosolic, but also in the enriched membrane fraction, indicating that the biosynthesis of acarbose and putative pyochelin metabolites takes place at the inner membrane.

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Biological significance

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Actinoplanes sp. SE50/110 is a natural producer of the α-glucosidase inhibitor acarbose, a

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bacterial secondary metabolite that is used as a drug for the treatment of type 2 diabetes, a

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disease which is a global pandemic that currently affects 387 million people and accounts

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for 11% of worldwide healthcare expenditures (www.idf.org).

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The work presented here is the first comprehensive investigation of protein localization

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and abundance in Actinoplanes sp. SE50/110 and provides an extensive source of information

http://dx.doi.org/10.1016/j.jprot.2015.04.013 1874-3919/© 2015 Published by Elsevier B.V.

Please cite this article as: Wendler S, et al, Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.013

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for the selection of genes for future mutational analysis and other hypothesis driven

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experiments. The conclusion that acarbose or pyochelin family siderophores are

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synthesized at the inner side of the cytoplasmic membrane determined from this work,

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indicates that studying corresponding intermediates will be challenging. In addition to

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previous studies on the genome and transcriptome, the work presented here demonstrates

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that the next omic level, the proteome, is now accessible for detailed physiological analysis

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of Actinoplanes sp. SE50/110, as well as mutants derived from this and related species.

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© 2015 Published by Elsevier B.V.

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2. Material and methods

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Actinoplanes sp. SE50/110 is known for the production of acarbose (acarviosyl-maltose), an α-glucosidase inhibitor used as a drug in the treatment of type 2 diabetes mellitus. The medical effect of acarbose is based on a reduction of the blood glucose and serum insulin levels through a decreased release of glucose from starch- and sucrose-containing diets in the human intestine [1,2]. Actinoplanes sp. SE50/110 is a derivative of the wild type strain SE50 and the progenitor of the current industrial acarbose production strains [3,4]. The strain is characterized as a Gram-positive actinomycete displaying a slow and hyphal growth; forming sporangia and motile spores as well as containing DNA with a high G + C-content of approximately 70% [5–7]. Other acarbose producing Actinoplanes strains have also been described in literature, for example Actinoplanes sp. 56 [8,9] or Actinoplanes utahensis ZJB-08196 [10,11]. Acarbose or acarviosyl-maltose is produced in a carbon source dependent manner as one of a group of similar metabolites that consist of an invariable acarviose and a variable carbohydrate or saccharide unit [2–4,12–14]. Different acarviose metabolites as acarviosyl-maltose or acarviosyl-glucose are synthesized intracellularly [14]. In the extracellular space, the pool of acarviose metabolites can be shifted by the activity of the acarviose transferase AcbD, which transfers the acarviose unit from intracellularly synthesized metabolites to carbon sources provided in the culture medium [4,15]. The cluster responsible for the biosynthesis of acarbose (acb) was sequenced successively [16,17] and initially considered to consist of 25 genes [4,17,18]. Biochemical studies have been carried out with some of the encoded proteins [15,19–30], after which a model of the biosynthesis and function of acarbose was suggested [17] and consequently updated [4,18,31–33]. Only 22 genes are now believed to belong to the acb cluster since the putative ABC-type acarviose metabolite importer AcbGFH [4,17] was found to be a highly specific galactose importer [32]. Proteins encoded by the cluster were predicted for distinct subcellular locations including the cytosol, the membrane and the extracellular space [4]. The genome sequence of Actinoplanes sp. SE50/110 was established by pyrosequencing in 2012 [34] and subsequently improved with RNA sequencing [35], which was challenging due to the high G + C-content of the genome [36–38]. In addition to the acb cluster, four further large secondary metabolite clusters named cACPL_1-4 were detected [34]. Using RNA sequencing, it was found that the acarbose biosynthesis gene cluster is highly transcribed in maltose-containing minimal medium. Interestingly, the hybrid non-ribosomal peptide-synthetase/polyketide synthase (NRPS/PKS) gene cluster cACPL_4, referred to here as the pyochelin biosynthesis gene cluster (pch), displayed a similar

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transcription profile in maltose minimal medium [39]. For this reason, the pch cluster was selected in addition to the acb cluster and studied on the proteome level in this work. Use of the genome sequence allowed investigation of the Actinoplanes sp. SE50/110 proteome. The cytosolic and extracellular proteome of cultures grown in maltose-containing minimal medium were analyzed with 2D-PAGE MALDI-TOF-MS resulting in the identification of up to 15 Acb-proteins [14,33]. To date, gel-based studies of the Actinoplanes sp. SE50/110 proteome have omitted membrane fractions [14,33]. However, membrane proteins constitute 20% to 30% of predicted proteins of bacteria [40,41] and are responsible for many vital cellular functions such as the transport of metabolites, homeostasis of metal ions, the extrusion of noxious substances, and the generation or conversion of energy [41]. Proteins can be associated with the membrane through several transmembrane domains, lipid anchors, C-terminal anchors, different types of N-terminal anchors [42–47] or hydrophobic, ionic as well as protein–protein interactions [46]. The latter particularly applies to subunits of membrane protein complexes [48–60]. Comprehensive state-of-the-art proteomics combine subcellular fractionation with LC-MS/MS [61–63] and can thereby analyze membrane fractions with hydrophobic integral membrane and membrane-associated proteins [48,64] in addition to cytosolic and extracellular proteins. The present work uses comprehensive state-of-the-art proteomics applying subcellular fractionation and LC-MS/ MS [61–63] to study the location of proteins of Actinoplanes sp. SE50/110 in four subcellular proteome fractions, namely the cytosolic, enriched membrane, membrane shaving, and extracellular fraction. In particular, the location of proteins encoded by the acarbose and the pyochelin biosynthesis gene clusters was investigated to provide new insights into the biosynthesis of the corresponding secondary metabolites.

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1. Introduction

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115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147

2.1. Cultivation of Actinoplanes sp. SE50/110 in maltose 150 minimal medium 151 Actinoplanes sp. SE50/110 (ATCC 31044; CBS 674.73) was cultivated following a modified protocol used by Wendler et al. [14,33]. This protocol included three stages with two shake flask pre-cultures and a bioreactor main culture. Both pre-cultures were grown in baffled polycarbonate Erlenmeyer flasks (Corning, Tewksbury, MA, USA) with Silicosen C-40 plugs (Hirschmann Laborgeräte, Eberstadt, Germany) at 140 rpm and 28 °C in a GFL shaking incubator 3032 (GFL, Burgwedel, Germany). For the first

Please cite this article as: Wendler S, et al, Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.013

152 153 154 155 156 157 158 159

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189 190 191 192 193 194 195 196

t1:1 Q1 t1:2 t1:3 t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22

Table 1 – Composition of 1 l maltose minimal medium. S1 S2

S3

S4

S5 (flask) or S5 (bioreactor) S6

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172

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171

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170

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169

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168

N C O

167

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166

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For all analyzed fractions, proteomic samples were taken during the exponential growth phase after 46 h of cultivation. For this, 90 ml of the culture was harvested by centrifugation (1900 rcf, 2 min, 4 °C) and washed twice with 150 mM NaCl. During sample treatment, equal protein amounts of 14 N-unlabeled samples and 15N-labeled pools were mixed to allow relative quantification of proteins. The present study only relies on information obtained from the 14N-unlabeled mass trace of the proteome samples.

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Maltose·1 H2O Ad aqua dest. K2HPO4 KH2PO4 Ad aqua dest. (NH4)2SO4 FeCl2·4 H2O Sodium citrate·2 H2O ZnCl2 MnCl2 CuCl2 Ad aqua dest. MgCl2·6 H2O CaCl2·2 H2O Ad aqua dest. H 2O CaCO3 (100 g/l solution) Plurafac LF 1300 Inoculum solution

72.1 g 250 ml 5g 5g 600 ml 5g 184 mg 5.7 g 6.81 mg 944 μg 134 μg 63 ml 1g 2g 31 ml 26 ml 25 ml 1 ml 30 ml

197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212

2.3. LC-MS/MS measurement

213

Sample preparation and LC–MS/MS measurements were carried out according to Otto et al. [62]. Peptides of all four fractions were subjected to reversed phase C18 column chromatography operated on a Proxeon EASYnLC (Thermo Fisher Scientific, Waltham, MA, USA). MS and MS/MS data were acquired with a LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) online coupled to the LC system. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository [65] with the data set identifier PXD001497.

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2.4. Data analysis

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Data analysis was performed according to Otto et al. [62]. In short, *.dta files were searched with SEQUEST version v28 (rev.12) (Thermo Fisher Scientific) against an Actinoplanes sp. SE50/110 target-decoy protein sequence database. This database was composed of all protein sequences deduced from the genome of Actinoplanes sp. SE50/110 (GenBank accession CP003170, version of 07-Mar-2014) including reversed sequences created by Scaffold 4.3.4 (Proteome Software Inc.) and common laboratory contaminants. For the searches the following parameters were applied: enzyme type, trypsin (KR); peptide tolerance, 10 ppm; tolerance for fragment ions, 1 amu; b- and y-ion series; variable modification, methionine (15.99 Da); a maximum of three modifications per peptide was allowed. For the membrane shaving fraction the search parameters differed as follows: enzyme type, none; and variable modification, methionine (15.99 Da) and carboxyamidomethylation (57.02 Da) of cysteine. Protein false positive rates were calculated as in described by Peng et al. [66] for each analysis.

227

2.5. Relative quantification of proteins by spectral counting

245

Normalized spectral abundance factors (NSAFs) were derived to estimate the relative abundance of each protein within different fractions according to Zybailov et al. [67]. NSAF values (SpC / (L · ∑SpC)) are the number of MS/MS spectra (SpC) assigned to a protein divided by protein length (L) and

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2.2. Preparation of proteomic samples

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Preparation of the different subcellular proteome fractions was carried out as described by Otto et al. [62]. The most important steps in the preparation and analysis of the proteome samples are indicated in Fig. 1. For the cytosolic and extracellular fraction, an additional preceding step of phenol protein extraction from harvested cells or supernatant as described by Wendler et al. [33] was used. In the workflow [62], proteins of the cytosolic, the enriched membrane and the extracellular fraction were fractionated using 1D SDS-gels and were in-gel digested with trypsin. In the membrane shaving protocol, membranes were spun down by ultracentrifugation, soluble loops were digested by Proteinase K in urea (shaving), and the remaining transmembrane domains were digested with chymotrypsin in a buffer containing the MS-compatible detergent RapiGest (Waters Corporation, Milford, MA, USA).

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pre-culture, glucose complex medium [33] was inoculated from 1.75 ml glycerol cryo cultures in 250 ml flasks filled with 50 ml medium. These were grown for 48 h, harvested by centrifugation (1900 rcf, 2 min, 4 °C) and washed twice with 150 mM NaCl solution. Subsequently, cells were resuspended in 22.5 ml NaCl solution, of which 1.5 ml was used for inoculation of the second pre-culture (250 ml flasks, 50 ml medium). In the second pre-culture, cells were adapted to the minimal main culture medium. The medium was composed of solutions S1-S6 described in Table 1. After 48 h of cultivation, cells were harvested by centrifugation (1900 rcf, 2 min, 4 °C), washed twice with 150 mM NaCl solution, and resuspended in 26 ml NaCl solution (150 mM). Of the resuspended cells, 25 ml were used for the inoculation of the main culture, which was carried out in 1 l Biostat Qplus bioreactors (Sartorius AG, Göttingen, Germany) filled with 775 ml minimal medium (Table 1). For the bioreactor minimal medium, each solution was sterilized separately and combined as follows: S1 and S3 were prepared and sterile filtered separately; S2 was autoclaved in the bioreactor; S4, CaCO3 solution and antifoam were autoclaved separately. The setpoints for the cultivation parameters were pH 6.5 and 30 °C. The pH was automatically controlled through the addition of 2 M NaOH and 10% (w/v) H3PO4. The regulation of the dissolved oxygen level at 50% was ensured through a cascade. If needed, the gas flow with air was increased (minimum 0.075 to 0.75 Nl min−1) followed by an increase of the stirrer speed (minimum 600 to 1200 rpm).

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Please cite this article as: Wendler S, et al, Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.013

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Fig. 1 – Workflow illustrating the most important steps in the preparation of the different proteome fractions.

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2.6. Bioinformatics tools used for analysis of proteins

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The LocateP v.2.0 pipeline [47] was used to predict subcellular locations of proteins. Subcellular locations were assigned as follows: (1) intracellular {intracellular}; (2) integral membrane {multitransmembrane, multitransmembrane (lipid-modified N termini)}; (3) membrane-associated {lipid anchored, N-terminally anchored (no cleavage site), N-terminally anchored (with cleavage site), C-terminally anchored (with cleavage site), intracellular/ TMH start after 60}; (4) secreted {secretory (released) (with cleavage site)}. In the manual analysis of signal peptides, cleavage sites were determined with SignalP [68]. The hydrophobic H domains were determined with the ExPASy ProtScale Tool according to Kyte & Doolittle [69]. Twin arginine motifs (RRxFLk) were predicted with TatP [70]. Transmembrane helices were determined by the TMHMM [71] algorithm.

262 263 264 265 266 267 268 269

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3. Results and discussion

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3.1. Comprehensive proteome analysis of Actinoplanes sp. SE50/110

273 274 275 276 277 278 279 280 281

medium. Growth was monitored by determining the cell dry weight [14,33]. Proteomic samples were taken from the main culture during the exponential growth phase and treated according to four different protocols to obtain a cytosolic fraction (CyF), a membrane shaving fraction (MSF), an enriched membrane fraction (EMF), and an extracellular fraction (ExF) [62]. The most important steps in the preparation and analysis of the proteome samples are indicated in Fig. 1. Proteins of the CyF, the EMF and the ExF were fractionated using 1D SDS-gels and were in-gel digested with trypsin. For the MSF, exposed hydrophilic domains (soluble loops) were digested by Proteinase K and discarded, while the remaining transmembrane domains were digested for LC–MS/MS by chymotrypsin. Resulting peptides of all four fractions were subjected to reversed phase C18 column chromatography. MS and MS/MS data were acquired with an LTQ-Orbitrap mass spectrometer. The subcellular fractionation approach resulted in the identification of 1179 proteins in the cytosolic fraction (Table S1), 640 proteins in the enriched membrane fraction (Table S2), 139 proteins in the membrane shaving fraction (Table S3) and 162 proteins in the extracellular fraction (Table S4). In Tables S1-S4, the identified proteins and the calculated normalized spectral abundance factors (NSAFs) are listed for the three replicates. A list summarizing the identifications of the four fractions is provided in Table S5 (false positive rate <0.23% for all replicates). A detailed list of all identification scores is provided in Table S6. In total, 1582 different proteins were identified accounting for 19.1% of the 8270 theoretical proteins of Actinoplanes sp. SE50/110. A Venn diagram (Fig. 2A) and a bar chart (Fig. 2B) indicate the number of proteins detected in one or multiple fractions (Table S5) as total and relative values, respectively. Of the 1179 proteins detected in CyF, the majority of 63% (769 proteins) was only detected in this fraction (Fig. 2A & B). In addition, 391 proteins identified in the CyF were also detected in the EMF accounting for 33% of the proteins of the CyF (Fig. 2B). The shared protein identifications between the CyF and the

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the total number of spectra assigned to proteins (∑ SpC) within one sample. NSAFs are indicated as mean values of three replicates.

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3.1.1. Proteins identified in four subcellular proteome fractions of Actinoplanes sp. SE50/110 For the analysis, Actinoplanes sp. SE50/110 cultures were grown in a three-stage cultivation including one shake flask pre-culture in glucose complex medium, a second shake flask pre-culture in maltose minimal medium, and a bioreactor main culture in maltose minimal medium with three biological replicates. In the second pre-culture, cells were adapted to the main culture

Please cite this article as: Wendler S, et al, Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.013

282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318

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MSF or ExF, were substantially smaller with only 2% or 5%, respectively. Of the 640 proteins detected in the EMF, 29% (187 proteins) were detected only in this fraction (Fig. 2A & B). The majority of 61% (391 proteins) of proteins of the EMF was shared with the CyF (Fig. 2B), while only minor portions of proteins were shared with the MSF and the ExF, 13% and 8%, respectively. Of the 139 proteins detected in the MSF, 40% (59 proteins) were detected only in this fraction while a majority, 58% (80 proteins), was also detected in the EMF (Fig. 2A & B). The overlaps with the CyF (19%) and the ExF (4%) were substantially smaller (Fig. 2B). Of the 162 proteins detected in ExF, 59% (95 proteins) were only detected in the ExF (Fig. 2A & B). Moreover, the ExF showed substantial shared protein identifications between the CyF (36%) and the EMF (30%). The overlap with the MSF was very small and accounted only for 3% of the identified proteins (Fig. 2B). In summary, the identification of proteins in single and multiple fractions was in agreement with expectations from previous studies where a subcellular fractionation strategy was applied [62,72]. In comparison to previous gel-based studies in

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Fig. 2 – Proteins detected in subcellular proteome fractions of Actinoplanes sp. SE50/110. Proteins detected only in the cytosolic (CyF), enriched membrane (EMF), membrane shaving (MSF) and extracellular fraction (ExF) as well as in multiple fractions are indicated as total numbers in the Venn diagram in A and as percentages in the bar chart in B. Since proteins can be present in more than two fractions (A), the sum of all percentages can exceed 100% (B).

Actinoplanes sp. SE50/110 [33], in which 162 different proteins were identified in the cytosolic and 22 in the extracellular fraction, the 1582 identified proteins in this study demonstrate major progress to higher resolution protein identification.

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3.1.2. Identified cytosolic proteins of Actinoplanes sp. SE50/110 344 The analysis of the cytosolic fraction (CyF) of Actinoplanes sp. SE50/110 resulted in the identification of 1179 proteins (Table S1). Using KEGG Mapper [73], all detected proteins were parsed in the metabolic context of Actinoplanes sp. SE50/110. In total 355 proteins could be assigned to metabolic pathways and most enzymes of central metabolism pathways including glycolysis (29), tricarboxylic acid cycle (23), or pentose phosphate pathway (17) could be identified. Moreover, 73 proteins were assigned to carbon metabolism and 197 to biosynthesis pathways of secondary metabolites. A list of the 15 most abundant proteins of the CyF, disregarding proteins of unknown function and ribosomal proteins, is presented in Table 2A (complete list in Table S1). To assess the relative abundance of proteins within a sample, normalized spectral abundance factors (NSAFs) [67] were

Please cite this article as: Wendler S, et al, Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.013

345 346 347 348 349 350 351 352 353 354 355 356 357 358 359

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Table 2 – Proteins with the highest normalized spectral abundance factors disregarding proteins of unknown function and ribosomal proteins of the cytosolic (A), enriched membrane (B), membrane shaving (C) and extracellular fraction (D) of Actinoplanes sp. SE50/110.

1854 623 780 7986 858

GapA SahH Tuf GroL MstE

2.45% 2.00% 1.65% 1.32% 0.97%

In In In In MA

6 7 8 9 10 11 15 18 19 21 1

7976 3833 7260 7445 855 6152 7369 893 1855 854 858

Tst – Tsf AtpD GroES – IlvC SucC Pgk GroEL MstE

0.92% 0.86% 0.79% 0.79% 0.79% 0.74% 0.62% 0.57% 0.57% 0.55% 9.67%

In In In In In In In In In In MA

2 3 4

7449 7445 6402

AtpF AtpD MalE

4.00% 1.98% 1.62%

MA In MA

t2:25 t2:26 t2:27 t2:28 t2:29

6 7 8 9 11

1819 6150 780 623 2580

LivE – Tuf SahH AglE

1.42% 1.40% 1.30% 1.26% 1.15%

MA MA In In MA

t2:30 t2:31 t2:32 t2:33 t2:34 t2:35

12 16 17 18 19 20

1079 7447 859 3666 7986 1824

FtsE AtpA MstA AcbW GroL PaaE

1.00% 0.91% 0.91% 0.90% 0.90% 0.87%

1 3 4 5

7450 802 7451 860

AtpE SecY AtpB MstF

14.51% 3.81% 3.77% 3.58%

IM IM IM IM

3665 1650 6160

AcbX QcrA –

3.56% 3.32% 2.94%

IM IM IM

t2:36 t2:37 t2:38 t2:39

C Membrane shaving fraction

In In In In In MA

C

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t2:22 t2:23 t2:24

B Enriched membrane fraction

R

t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21

A Cytosolic fraction

7 8 10

t2:43 t2:44

11 12

6161 7234

– –

2.76% 2.64%

IM IM

13 14

3816 6400

– MalG

2.37% 2.15%

IM IM

15 16 18 22

1156 6151 1651 2578

CtaD – QcrB AglG

1.99% 1.97% 1.93% 1.19%

IM IM IM IM

1 2 3 4 6 8

3368 5091 3684 3683 3939 3663

CbpA Cgt AcbD AcbE CbpB AcbZ

21.73% 13.68% 7.20% 5.05% 4.04% 2.75%

Se IM Se Se Se Se

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t2:40 t2:41 t2:42

t2:45 t2:46 t2:47 t2:48 t2:49 t2:50 t2:51 t2:52 t2:53 t2:54 t2:55 t2:56

D Extracellular fraction

1

Function e Glyceraldehyde-3-phosphate dehydrogenase S-adenosylhomocysteine hydrolase Elongation factor Tu (GTPase) 60 kDa chaperonin ABC-type monosaccharide transporter, substrate-binding lipoprotein Thiosulfate sulfurtransferase (rhodanese-related) Putative cyclase/dehydrase Elongation factor Ts F0F1-type ATP synthase, β subunit 10 kDa co-chaperonin Aromatic-ring hydroxylase Ketol-acid reductoisomerase Succinyl-CoA synthetase, β subunit 3-Phosphoglycerate kinase 60 kDa chaperonin ABC-type monosaccharide transporter, substrate-binding lipoprotein F0F1-type ATP synthase, subunit b F0F1-type ATP synthase, β subunit ABC-type maltose/maltodextrin transporter, substrate-binding lipoprotein ABC-type BCAA transporter, substrate-binding lipoprotein FAD dependent oxidoreductase Elongation factor Tu (GTPase) S-adenosylhomocysteine hydrolase ABC-type multiple α-glucoside transporter, substrate-binding lipoprotein Cell division ATP-binding protein F0F1-type ATP synthase, α subunit ABC-type monosaccharide transporter, ATPase component ABC-type transporter, ATPase component 60 kDa chaperonin ABC-type polar amino acid transporter, substrate-binding lipoprotein F0F1-type ATP synthase, subunit c Preprotein translocase subunit SecY F0F1-type ATP synthase, subunit a ABC-type monosaccharide transporter, permease component ABC-type transporter, permease component Ubiquinol–cytochrome c reductase, iron–sulfur subunit ABC-type multidrug transporter, ATPase and permease component MFS drug resistance transporter, EmrB/QacA subfamily Putative ABC-type Na + efflux transporter, permease component ABC-type transporter, permease component ABC-type maltose/maltodextrin transporter, permease component Cytochrome c oxidase, subunit I Na+/H+ antiporter Ubiquinol–cytochrome c reductase, cytochrome b subunit ABC-type multiple α-glucoside transporter, permease component Putative carbohydrate-binding protein Small carbohydrate-binding protein Acarviose transferase Acarbose-resistant α-amylase, pullulanase Putative carbohydrate-binding protein Acarbose-resistant α-amylase, pullulanase

F

1 2 3 4 5

t2:6 t2:7 t2:8 t2:9 t2:10

pF rank d

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SCL c

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NSAF b

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Protein name

T

Gene no.

O

Rank a

t2:5

D

t2:4

E

t2:1 t2:2 t2:3

JOUR NAL OF P ROTEOM ICS XX ( 2015) X XX–XX X

3 2

222 39 125 138 4

n.r.

Please cite this article as: Wendler S, et al, Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.013

7

JOURNAL OF P ROTEOM IC S XX ( 2015) X XX–X XX

Table 2 (continued) Rank a

Gene no.

Protein name

NSAF b

SCL c

t2:57

10

1505

CpdB

1.68%

Se

t2:58 t2:59 t2:60 t2:61

11 12 13 15

3618 6399 3893 2580

PpiB PulA – AglE

1.68% 1.60% 1.56% 1.39%

Se Se Se MA

t2:62 t2:63 t2:64 t2:65

17 18 20 22

3926 4287 5172 5865

TynA Mep Apy CsdE

1.22% 1.11% 1.02% 0.79%

MA MA Se MA

pF rank d

11 574 n.r.

5′-Nucleotidase, 2′,3′-cyclic-nucleotide 2′-phosphodiesterase Putative peptidyl-prolyl cis-trans isomerase Pullulanase, α-amylase Extracellular protein containing fasciclin domain ABC-type multiple α-glucoside transporter, substrate-binding lipoprotein Cu2+-containing amine oxidase Metallopeptidase Aminopeptidase Y Cell shape determining protein

O

n.r.

Function e

F

t2:58 t2:59

a

t2:68

366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394

3.1.3. Identified membrane-associated proteins of Actinoplanes sp. SE50/110 In the analysis of the membrane proteome, two complementary sub-fractions were examined. 640 different proteins were detected in the enriched membrane fraction (EMF) (Table S2). Using the LocateP v.2.0 [47] pipeline, it was investigated whether sequence motifs or signal peptides could explain the presence of these proteins in the EMF. It was found that 33.9% of the proteins were predicted as either membrane-associated (16.9%) or integral membrane proteins (17.0%). The major group of proteins accounting for 64.2%, however, was that of predicted intracellular proteins, while the remaining 1.9% were predicted secreted proteins. This finding is in line with previous applications of the EMF protocol [62], which usually covers not only proteins with, but also without membrane-embedded (transmembrane) domains [48,64]. Similar to other Gram-positive bacteria such as Bacillus subtilis [48], Staphylococcus aureus [64] or Corynebacterium glutamicum [49], prominent classes of proteins found in the EMF in this study reflect major functions of bacterial membranes, including bioenergetics (e.g. oxidative phosphorylation proteins) or transport (e.g. substrate-binding proteins or ATPase components of ABC-type transporters). Similar to the proteins identified in the CyF, a list of the 15 most abundant proteins of the EMF, disregarding proteins of unknown function and ribosomal proteins, is presented in

Table 2B (complete list in Table S2). Some of those belong to the oxidative phosphorylation machinery such as the ATP synthase subunits AtpF, AtpD, and AtpA; to transport and metabolism of carbohydrates such as the substrate binding proteins MstE, MalE, and AglE or the ABC-type transporter ATPase subunit MstA; to transport and metabolism of amino acids such as the substrate binding proteins LivE and PaaE; or to translational processes such as ribosomal proteins, the elongation factor Tuf or the chaperonin GroL. For the subset of most abundant predicted membraneassociated proteins (Table 2B), the leader peptides were also analyzed manually (Table 3A). The five lipid anchored proteins MstE, MalE, LivE, AglE and PaaE contained corresponding signal peptides with lipobox motifs [44] and the two predicted N-anchored proteins AtpF and ACPL_6150 had distinct N-terminal transmembrane domains. Detecting abundant proteins of translational processes as in the case of Tuf and GroL in membrane fractions is a common observation [51,52,57,82–90]. This can be explained by co-translational translocation, which is a membrane-located process particularly relevant for membrane and secreted proteins (reviewed in [53–55]). Moreover, for proteins such as subunits of the ATP synthase or ABC-type transporters ATPase components an intracellular subcellular location was predicted. Their high abundance in the EMF is nevertheless obvious since they are subunits of membranelocated complexes. Particularly abundant in the EMF were also substrate-binding proteins (SBP) as for example MstE of the ABC-type monosaccharide transporter MstEAF (ACPL_858-860). A common feature of the most closely related and experimentally characterized ABC-type transporters XylFGH from Escherichia coli K12 (blastp [91] aa identity 38%, E 5e−51) [92], ChvE from Agrobacterium tumefaciens (blastp [91] aa identity 36%, E 9e−42) [93,94] and SbpA from Azospirillum brasilense (blastp [91] aa identity 36%, E 6e−40) [95] is the transport of monosaccharides. The latter two transporters were also described as relevant for chemotaxis towards sugars [95,96]. Furthermore, the SBPs MalE

E

T

C

365

E

364

R

363

R

362

calculated. The most abundant proteins of the CyF mainly belonged to the central metabolism, translational processes, and protein folding. Proteins of these central categories are commonly found among the most abundant intracellular proteins in bacteria [74–77]. Interestingly, also the aromatic-ring hydroxylase ACPL_6152 encoded in the putative pyochelin gene cluster was among the most abundant proteins. Compared to the previous gel-based study where 162 proteins were detected in cytosolic fractions [33], the LC–MS-based approach expectedly [78–81] allowed the identification over seven times more proteins (1179).

N C O

361

U

360

D

P

t2:69 t2:70

Proteins are ordered by their NSAFs. Proteins of the secondary metabolite clusters pch and acb are indicated in bold. Ribosomal proteins as well as proteins without functional information were excluded from this table (complete lists in Table S1–S4). b NSAFs are the spectral counts (SpC) of a protein divided by its length and the sum of all SpC of the experiment. c LocateP [47] was used to predict the subcellular location (SCL) for a protein from its amino acid sequence that was differentiated into an intracellular (In), a membrane-associated (MA), an integral membrane (IM) and a secreted (Se) category. d For proteins that were expected in another fraction based on the LocateP [47] predicted subcellular locations (SCL), the ranks of proteins ordered by NSAFs in the predicted fraction (pF) are indicated (n.r., no rank). e Functions were determined automatically in the Actinoplanes sp. SE50/110 genome project [34] and manually revised in this work.

R O

t2:66 t2:67

Please cite this article as: Wendler S, et al, Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.013

395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430

8 t3:1 t3:2

JOUR NAL OF P ROTEOM ICS XX ( 2015) X XX–XX X

Table 3 – Analysis of leader peptide sequences of abundant proteins of the enriched membrane and extracellular fraction of Actinoplanes sp. SE50/110.

t3:3

Gene no

Protein

Subcellular locationa

Pathwayb

Secretion signal peptidesc

MRKGLFALAAVGLLATGSMAA(◊21)CG

A) Predicted membrane associated proteins of EMF MstE

MA

Lipid anchored

Sec–(SPII)

7449

AtpF

MA

N–terminally anchored (No CS)

Sec–(SPI)

MLTY(…)ILPA19oWQEIVVGTVAFIVLCFVLMKFVF42iPQ

6402

MalE

MA

Lipid anchored

Sec–(SPII)

MRIRTAGVIAASMLCLVGAAA(◊21)CG

1819

LivE

MA

Lipid anchored

Sec–(SPII)

MKQVLARAIGGVALIGLLAGTAA(◊23)CN

6150

PrnC

MA

N–terminally anchored (No CS)

Sec–(SPI)

MNQHIAGKRDT11iYDVAILGAGMAGGMLAAVLA31oRH

2580

AglE

MA

Lipid anchored

Sec–(SPII)

MFGHSGSSRSRVALAGALSAGLIFSLAA(◊27)CG

1824

AatE

MA

Lipid anchored

Sec–(SPII)

MFRITPGRRAILGVAVAAALTVSLSA(◊26)CG

F

858

433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448

Secretory (released) (with CS)

Sec–(SPI)

MSRILRRVLLLAAAPVAALGFLAAPAAA(◊28)SP

IM

Multi–transmembrane

Sec–(SPI)

MNRTTVRAGVLATALISGVLGVAGPALA(◊28)AP

3684

AcbD

Se

Secretory (released) (with CS)

Sec–(SPI)

MQRHARHAIAAAVAVSLLPPSLPAHAA(◊27)GA

3683

AcbE

Se

Secretory (released) (with CS)

Sec–(SPI)

MDERLKSHIVRALLPVLVGAAGGLPAPRAAQA(◊32)ES

3939

CbpB

Se

Secretory (released) (with CS)

Sec–(SPI)

MPRTIAVIILASAATLAALAGPAQA(◊25)ST

3663

AcbZ

Se

Secretory (released) (with CS)

MVLSRHRDVMRRLSAVLALGLVVPLLLAPPWRASA(◊35)AP

1505

CpdB

Se

Secretory (released) (with CS)

3618

PpiB

Se

Secretory (released) (with CS)

Sec–(SPI) Possible Tat/ Sec–(SPI) Sec–(SPI)

6399

PulA

Se

Secretory (released) (with CS)

Sec–(SPI)

MAASKTRWGTAVLSLLLPLLTVPAAEASA(◊29)AS

3893

–

Se

Secretory (released) (with CS)

Sec–(SPI)

2580

AglE

MA

Lipid anchored

Sec–(SPII)

3926

TynA

MA

N–terminally anchored (No CS)

Sec–(SPI)

MTSAALGLTVPAQA(◊14)AP

4287

Apn

MA

N–terminally anchored (with CS)

Sec–(SPI)

MRRLLVATLATALTLAATGAAAQA(◊24)HP

5172

Apy

Se

Secretory (released) (with CS)

5865

Csde

MA

N–terminally anchored (No CS)

P

MTLPNGVSRRGVLAVSAAAAAAPLALTGPAQA(◊32)HG MAALTVAALVTLGAAPTPANA(◊21)AG

D

E

T

R O

Se

Cgt

MRLTRLGKKAAAVATTAVVASTLTAAPAFA(◊30)HG MFGHSGSSRSRVALAGALSAGLIFSLAA(◊27)CG

MRKRTLAVPFAAALTLALLPQPARA(◊25)VD

Sec–(SPI)

MHDT(…)GRRR40iVAAGAGAAVCLAILATMFRVVGF63oGS

C

Sec–(SPI)

a

O

R

R

E

The LocateP v.2.0 pipeline [47] was used to predict subcellular locations (SCL) of proteins. SCLs were assigned as follows: (IM) integral membrane {multitransmembrane}; (MA) membrane-associated {lipid anchored, N-terminally anchored (no cleavage site (No CS)), N-terminally anchored (with CS)}; (Se) secreted {secretory (released) (with CS)}. b The LocateP v.2.0 pipeline [47] was used to predict cleavage pathway of proteins. c Residues at positions −3 to −1 relative to the predicted cleavage sites are printed bold and underlined. The cleavage sites within the amino acid sequence are accentuated by (◊#) with subscript numbers indicating the last amino acid of the signal peptide. For predicted lipid anchored proteins the signal peptidase (SP) II cleavage pattern (lipobox) [LITAGMV]-[ASGTIMVF]-[AG]-C-[SGENTAQR] [44] is printed in bold gray script at positions +3 to −2. The hydrophobic H domains are shaded gray. Twin arginine motifs (RRxFLk) are printed bold. The positively charged amino acids arginine (R) and lysine (K) are printed in italics and underlined. Transmembrane helices are printed in italics.

and AglE of the ABC-type maltose/maltodextrin importer MalEFG (ACPL_6402-6400) and the ABC-type α-glucosidase importer AglEFG (ACPL_2580-2578) were detected in previous studies where MalEFG was suggested as the missing acarviose metabolite re-importer [97]. Interestingly, also the ABC-type transporters ATPase component AcbW of the acarbose biosynthesis gene cluster and the FAD dependent oxidoreductase ACPL_6150 of the pch cluster were found among the most abundant proteins in the EMF.

C

432

CbpA

5091

N

431

3368

U

t3:4 t3:5 t3:6 t3:8 t3:7 t3:10 t3:9 t3:11 t3:12 t3:13 t3:15 t3:14

O

B) Proteins of ExF

3.1.4. Identified integral membrane proteins of Actinoplanes sp. SE50/110 In the membrane shaving fraction (MSF), 139 different proteins were detected (Table S3). Using the LocateP v.2.0 [47] pipeline it was investigated whether the proteins contained two or more transmembrane domains which could explain their presence in the MSF. It was found that 88.5% of the proteins of the MSF contained two or more transmembrane domains and were, therefore, categorized as integral membrane proteins. This is a

known feature of the shaving protocol since it almost exclusively covers membrane-embedded peptides and strongly discriminates against exposed hydrophilic peptides [48,64]. The notable classes of proteins identified in the MSF were connected to major functions of bacterial membranes, bioenergetics or transport. A substantial proportion of proteins of the MSF had no functional annotation, which is common for membrane proteins [50,51,98,99]. As for the CyF and EMF, a list of the 15 most abundant proteins of the MSF, disregarding proteins of unknown function, is presented in Table 2C (complete list in Table S3). Here, proteins of the oxidative phosphorylation such as ATP synthase subunits AtpE and AtpB, the ubiquinol– cytochrome c reductase subunit QcrA and QcrB and the cytochrome c oxidase subunit CtaD; of transport processes such as ABC-type transporter subunits MstF, ACPL_7234, ACPL_3816, MalG, and AglG; and of translational processes such as preprotein translocase subunit SecY were particularly abundant. Remarkably, also proteins encoded by the acb cluster (ABC-type transporters ATPase component AcbX) and the pch

Please cite this article as: Wendler S, et al, Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.013

449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467

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JOURNAL OF P ROTEOM IC S XX ( 2015) X XX–X XX

481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526

F

480

527 528 529 530 531 532 533 534 535 536 537 538

3.2. Location of proteins encoded by two secondary metabolite 539 gene clusters of Actinoplanes sp. SE50/110 540

O

479

R O

478

3.2.1. Location of proteins encoded by the acarbose biosynthesis 541 gene cluster 542 The acarbose biosynthesis gene cluster acb of Actinoplanes sp. SE50/110 consists of 22 genes and is responsible for the biosynthesis of α-glucosidase inhibiting acarviose metabolites. In this work, Acb-proteins were found in the CyF, EMF, MSF, and ExF. Corresponding NSAFs and predicted subcellular locations of Acb-proteins in subcellular proteome fractions of Actinoplanes sp. SE50/110 are shown in Fig. 3. Combining the different subcellular fractions, all 22 Acb-proteins were detected, of which AcbY, AcbX, AcbW, AcbP, AcbQ, AcbM and AcbO were identified for the first time [14,33]. In the CyF, 20 of the 22 proteins of the acb gene cluster were detected (Fig. 3). All 16 predicted intracellular proteins were present in the CyF with AcbP, AcbQ, AcbO and AcbP being identified for the first time [14,33]. None of the Acb-proteins were among the most abundant proteins of the fraction. AcbK, however, was the most abundant Acb-protein of the CyF and also the only Acb-protein that has been identified under all applied conditions of previous proteome studies [14,33]. Interestingly, the ABC-type transporter ATPase AcbW was detected in the CyF. AcbW has no motifs indicating secretion or membrane location, but as a subunit of an ABC-type transporter was expected to be located at the membrane. Detecting ATPase subunits in membrane as well as cytosolic fractions is quite common [48–52,56–58,74,77,118,119] and may reflect their location at the cytosolic side of the membrane. In the EMF, 19 of the 22 Acb-proteins were detected (Fig. 3). The ABC-type transporter ATPase AcbW was expected in the EMF due to its function and indeed was one of the most abundant proteins of the EMF (Table 2B). Interestingly, also 15 predicted intracellular proteins were detected in the EMF. In particular, the proteins AcbV, AcbU, and AcbS had high NSAF values indicating a substantial abundance of these proteins at the membrane. There was evidence for membrane-location of biosynthetic proteins for several exported secondary metabolites such as xanthan [120,121], myxovirescin [122], phthiocerol dimycocerosate [123], subtilin [124], pyoverdine [125], and nisin [126]. Organizing sequential metabolic enzymes in complexes (metabolons) allows a direct transfer of intermediates from one active site to the other and is known as substrate channeling. While the complexes are joined by non-covalent interactions, the location at the membrane is considered advantageous for stabilization [127–131]. Depending

P

477

D

476

In the extracellular fraction (ExF) of Actinoplanes sp. SE50/110, 162 proteins were identified (Table S4). Using the LocateP v.2.0 [47] pipeline, it was investigated whether these proteins contained secretion signal peptides. The major groups of proteins were predicted to be secreted (39.5%) or membrane-associated proteins (33.3%). The subgroup of membrane-associated proteins includes lipid anchored proteins and N-anchored proteins with cleavage sites. Lipid anchored substrate-binding proteins of ABC transporters, for example, are commonly found in extracellular proteome fractions [33,42,76,100–109]. Accurate prediction of cleavage sites [47,110–114] and discrimination between secreted proteins with signal peptides and proteins with uncleaved N-terminal anchors for retention at the membrane is an issue and may cause false predictions [44,47,115,116]. Furthermore, it is quite common to detect intracellular proteins of the central metabolism or translational processes in extracellular proteome fractions [62,72,76,100–102,104,108], which is usually related to cell lysis. As presented for the other three fractions analyzed, a list of the 15 most abundant proteins of the ExF, disregarding proteins of unknown function, is presented in Table 2D (complete list in Table S4). Several of these proteins are involved in transport and metabolism of carbohydrates such as the putative carbohydrate-binding proteins CbpA, Cgt and CbpB, the pullulanase/α-amylase PulA and the substrate binding protein AglE. The second dominant class was affiliated to amino acid transport and metabolism such as the Cu2+-containing amine oxidase TynA and the peptidases Mep and Apy. In addition, the three proteins of the acb gene cluster predicted to be secreted, the acarviose transferase AcbD and the acarbose-resistant α-amylases/pullulanases AcbE and AcbZ were present in high abundance. For a subset of particularly abundant proteins of the ExF, a subcellular location other than secreted was predicted applying LocateP [47]. Therefore, the leader peptides of the most abundant proteins of the ExF were analyzed (Table 3B). Of the proteins with a deviating predicted subcellular location (other than secreted) Cgt, TynA and Mep had proper secretion signal peptides. In the case of the 15 kDa carbohydrate binding protein Cgt, two transmembrane domains were predicted with the TMHMM [71] algorithm. However, considering that one of these is the secretion peptide and that Cgt was also almost completely absent in the EMF and MSF, it has to be regarded as a true secreted protein. The second hydrophobic domain of Cgt might be required for the suggested oligomerization [117]. Another protein of the ExF was the substrate-binding lipoprotein AglE. This protein was predicted to be lipid anchored and was found to have a signal peptide that contained a lipobox motif [44] (Table 3B). The detection of SBPs in membrane as well as extracellular and cytosolic fractions has been observed in Gram-negative bacteria such as E. coli [100,101], Pseudomonas aeruginosa [102,103] and Xanthomonas campestris pv. campestris [104] as well as in Gram-positive bacteria such as Streptococcus

E

475

T

474

mutans [105], Alicyclobacillus acidocaldarius [106], different Bacillus species [76,107] and different Streptomyces species [108,109]. The cell shape determining protein CsdE was predicted to be membrane-associated and was found to contain a transmembrane domain (Table 3B), however, this protein was almost exclusively found in the ExF. Even though CsdE lacks a proper secretion signal, it seems to be a true secreted protein. As in a previous study [33], proteins of the acarbose biosynthesis gene cluster as well as the carbohydrate and amino acid metabolism were particularly abundant. Moreover, also all 22 proteins detected previously with a gel-based approach were among the 162 proteins detected in the ExF in this work [33].

3.1.5. Identified extracellular proteins of Actinoplanes sp. SE50/110

C

473

E

472

R

471

R

470

cluster (transporter subunits ACPL_6160, ACPL_6161 & ACPL_6151) were found under the most abundant proteins in the MSF. Many of the proteins detected in high abundance in the MSF and EMF are annotated as subunits of the same membrane protein complexes supporting the analysis at hand.

N C O

469

U

468

Please cite this article as: Wendler S, et al, Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.013

543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584

10

C

T

E

D

P

R O

O

F

JOUR NAL OF P ROTEOM ICS XX ( 2015) X XX–XX X

588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604

C

587

on the metabolite, substrate channeling (1) avoids diffusion, high concentrations of toxic intermediates and undesired or competing side reactions; (2) protects unstable intermediates; and (3) increases local intermediate pools, the reaction efficacies and turnover rates (reviewed in [128–131]). The finding of intracellular biosynthetic Acb-proteins with substantial NSAFs in the EMF strongly suggests biosynthesis of acarviose metabolites at the membrane and the associated substrate channeling. In the MSF, only the two integral membrane proteins and permease components of the ABC-type acarviose metabolite exporter AcbY and AcbX were detected (Fig. 3). In the ExF, the three proteins AcbZ, AcbD, and AcbE were detected. All three had secretion signals and were, therefore, predicted to be secreted proteins. All three were also among the most abundant proteins of the ExF, which is in accordance with previous gel-based studies on the secretome of Actinoplanes sp. SE50/110 [33]. Lastly, the high abundance of several, and the detection of all, Acb-proteins fit to results from previous gel-based proteome studies [14,33] as well as to findings of transcriptome studies [39] where the cluster was expressed as a whole.

N

586

U

585

O

R

R

E

Fig. 3 – Normalized spectral abundance factors (NSAFs) of proteins of the acarbose biosynthesis gene cluster acb in subcellular proteome fractions of Actinoplanes sp. SE50/110. Subcellular locations of gene products were predicted with LocateP [47] and are differentiated into an intracellular, a membrane-associated, an integral membrane and a secreted category as indicated in the different colors of the genes. NSAFs [67] of proteins are displayed as bubble areas for the cytosolic (CyF), enriched membrane (EMF), membrane shaving (MSF) and extracellular fraction (ExF). Colors used for bubbles in the different fractions match subcellular location colors to which they are expected to correspond. AcbW was not predicted, but due to its function it was expected to be a membrane-associated protein.

3.2.2. Location of proteins encoded by the pyochelin biosynthesis 605 gene cluster 606 The hybrid NRPS/PKS gene cluster pch was first identified during the genome sequencing project of Actinoplanes sp. SE50/110 and described to encompass three non-ribosomal peptide synthetases (NRPS), three polyketide synthases (PKS), and several other genes including a potential product exporter [34]. The metabolites produced by the pch gene cluster remain unknown. Our analysis using antiSMASH 2.0 [132,133] suggested that the metabolite of this cluster might be similar to the siderophore pyochelin [134–138]. Reinvestigating the genes and their annotation, we found many to be related to biosynthesis genes of pyochelin, which was first shown to be produced in P. aeruginosa [134] and recently also in the Gram-positive bacterium Streptomyces scabies 87-22 [137]. Interestingly, the pch cluster contains an enantio-pyochelin biosynthesis protein PchK, which was suggested to have a reductase and/or epimerase function [135] and was, so far, found in gene clusters of strains that produce enantio-pyochelin [135,136]. The proposed advantage of enantio-pyochelin is a mechanism for sequestering

Please cite this article as: Wendler S, et al, Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.013

607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624

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JOURNAL OF P ROTEOM IC S XX ( 2015) X XX–X XX

638 639 640 641 642 643

F

637

O

636

R O

635

P

634

D

633

E

632

T

631

C

630

E

629

R

628

intracellular proteins that were expected in the CyF, the TetR family transcriptional regulator ACPL_6141, the thioesterase (ACPL_6146), the AfsR family transcriptional regulator ACPL_6154, and the pyochelin biosynthetic protein PchC were not present in the obtained data set. The regulators of the TetR family are transcriptional repressors that among others were shown to regulate the biosynthesis of antibiotics (reviewed in [139]). Transcriptional regulators are mostly low abundance proteins [140–143] and are therefore difficult to detect even for sensitive gel-free LC–MS/MS approaches [56,61,144]. Interestingly, also one predicted membrane-associated protein, the FAD dependent oxidoreductase ACPL_6150, was detected in the CyF among the more abundant proteins (Table 2A), which may indicate a location of this protein at the inner side of the membrane. This may also be true for the enantio-pyochelin biosynthesis protein PchK (ACPL_6140), which was predicted to be a membrane-associated protein, but only detected in the CyF. Remarkably, 18 of the 23 proteins of the pch gene cluster were detected in the EMF (Fig. 4). The cluster contains the three

R

627

iron that avoids siderophore piracy by additionally excluding producers of pyochelin [135,136]. In this work, Pch-proteins were found in the CyF, the EMF, and the MSF. Since no secreted proteins were predicted for the cluster, proteins were neither expected nor detected in the ExF. NSAFs and predicted subcellular locations of proteins of the pch gene cluster in subcellular proteome fractions of Actinoplanes sp. SE50/110 are shown in Fig. 4. Combining the different subcellular fractions, this approach resulted in the identification of 21 of 23 proteins of the pch gene cluster. The TetR family transcriptional regulator ACPL_6141 and the pyochelin biosynthetic protein PchC were the only proteins missing in the data set. In general, it was expected to detect Pch-proteins, because the whole pch gene cluster was previously found to be transcribed in parallel to the acb gene cluster in maltose minimal medium [39]. In the CyF, 17 of the 23 Pch-proteins were detected (Fig. 4) with the aromatic-ring hydroxylase ACPL_6152 being among the most abundant proteins (Table 2A). Of the predicted

N C O

626

U

625

Fig. 4 – Normalized spectral abundance factors (NSAFs) of proteins of the putative pyochelin biosynthesis gene cluster pch in subcellular proteome fractions of Actinoplanes sp. SE50/110. Subcellular locations of gene products were predicted with LocateP [47] and are differentiated into an intracellular, a membrane-associated and an integral membrane category as indicated in the different colors of the genes. NSAFs [67] of proteins are displayed as bubble areas for the cytosolic (CyF), enriched membrane (EMF), membrane shaving (MSF) and extracellular fraction (ExF). Colors used for bubbles in the different fractions match subcellular location colors to which they are expected to correspond. Please cite this article as: Wendler S, et al, Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the..., J Prot (2015), http://dx.doi.org/10.1016/j.jprot.2015.04.013

644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662

12

677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694

699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719

C

Acknowledgments

725 724

SW acknowledges financial support from German National Academic Foundation. AN, FW, TW and VO acknowledge financial support from the Graduate Cluster Industrial Biotechnology (CLIB2021) at Bielefeld University, Germany. The Graduate Cluster is supported by the Bielefeld University and the Ministry of Innovation, Science and Research (MIWF) of the federal state North Rhine—Westphalia, Germany. AP and JK also acknowledge a grant from the MIWF entitled “Technologieplattform PolyOmics”. We also acknowledge financial support from Bayer AG (Leverkusen, Germany). The data deposition to the ProteomeXchange Consortium was supported by the PRIDE Team. We thank Kyle J. Lauersen for English proofreading.

726 Q5

F

676

E

675

R

674

R

673

O

672

C

671

N

670

U

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With 1582 detected proteins, this work represents the most comprehensive proteome analysis in Actinoplanes sp. SE50/110 to date. In addition, the subcellular fractionation approach for the first time allowed us to study the membrane proteome and to analyze the location of proteins of two secondary metabolite gene clusters. Interestingly, abundant membrane fraction proteins did not only cover major functions of bacterial membranes, but also included membrane and biosynthetic proteins of the acb and pch gene clusters. These findings indicate a biosynthesis of corresponding metabolites at the membrane and probably also substrate channeling. While acarviose metabolites of the acb gene cluster were previously characterized [2,3,13], potential metabolites of the pch gene cluster have not yet been determined. With the knowledge that the pch gene cluster is transcribed [39] and translated into proteins, the next step would be to determine the resulting metabolites. Furthermore, this work demonstrates that the proteome is now accessible for detailed physiological studies, which could focus on the metabolization of different carbon or nitrogen sources, growth phases or stress conditions. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2015.04.013.

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predicted membrane-associated proteins PchK, ACPL_6144 and ACPL_6150. Of these, only the two latter were detected in the EMF, with the FAD dependent oxidoreductase ACPL_6150 being particularly abundant (Table 2B). PchK was predicted to be lipid anchored and ACPL_6144 and ACPL_6150 were predicted to be N-terminally anchored proteins. Analyzing the secretion signal of the enantio-pyochelin biosynthesis protein & reductase/ epimerase PchK, we found that it is an N-anchored protein while the prediction as a lipid-anchored protein was incorrect. In addition to predicted membrane-associated proteins, also the four predicted integral membrane proteins (ACPL_6151 & ACPL_6159-6161) of the pch gene cluster were detected in the EMF. They were identified because the EMF protocol accesses hydrophilic domains (soluble loops) of integral membrane proteins [48]. Interestingly, 12 of the 16 predicted intracellular proteins were detected in the EMF (Fig. 4). Of these, the proteins Nrps2A, Cpm, Pks3C, ACPL_6152 and Nrps2C had high NSAF values indicating a substantial abundance at the membrane. It is known from B. subtilis that many copies of proteins of a NRPS/PKS cluster can assemble into a single organelle-like membrane-associated complex [145]. Considering the above mentioned biosynthesis of secondary metabolites at the membrane [120–126], these findings strongly suggest that the biosynthesis of the pyochelin-like metabolites takes place at the membrane, including the above mentioned substrate channeling. In the MSF, all four predicted integral membrane proteins (ACPL_6151 & ACPL_6159-6161) were found (Fig. 4), which is in accordance with expectations. Of these, the Na+/H+ antiporter (ACPL_6151), the ABC-type multidrug transporter ATPase and permease component (ACPL_6160) and the EmrB/QacA subfamily MFS drug resistance transporter (ACPL_6161) were among the most abundant proteins of the MSF (Table 2C).

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