A toolbox to measure changes in the cell wall glycopolymer composition during differentiation of Streptomyces coelicolor A3(2)

    A toolbox to measure changes in the cell wall glycopolymer composition during differentiation of Streptomyces coelicolor A3(2) Steffen Sigle, Nadja Steblau, Wolfgang Wohlleben, G¨unther Muth PII: DOI: Reference:

S0167-7012(16)30170-1 doi: 10.1016/j.mimet.2016.07.002 MIMET 4948

To appear in:

Journal of Microbiological Methods

Received date: Accepted date:

1 July 2016 1 July 2016

Please cite this article as: Sigle, Steffen, Steblau, Nadja, Wohlleben, Wolfgang, Muth, G¨ unther, A toolbox to measure changes in the cell wall glycopolymer composition during differentiation of Streptomyces coelicolor A3(2), Journal of Microbiological Methods (2016), doi: 10.1016/j.mimet.2016.07.002

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ACCEPTED MANUSCRIPT A toolbox to measure changes in the cell wall glycopolymer composition during differentiation of Streptomyces coelicolor A3(2).

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Steffen Sigle, Nadja Steblau, Wolfgang Wohlleben and Günther Muth* Interfakultäres Institut für Mikrobiologie und Infektionsmedizin Tübingen IMIT,

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Mikrobiologie/Biotechnologie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 28,

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72076 Tübingen, Germany

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* for correspondence +4970712974637

Fax:

+497071295979

E-mail:

[email protected]

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Phone:

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Keywords: teichulosonic acid, polydiglycosylphosphate, colorimetric assay, Kdn

Highlights: •

Establishment of colorimetric assays for the quantification of S. coelicolor cell wall glycopolymers

•

Development of a PAGE system for separation of polydiglycosylphosphate fragments

•

Characterization of the cell wall glycopolymer composition during differentiation of S. coelicolor A3(2)

Abbreviations: PG: peptidoglycan, GlcNAc: N-acetylglucosamine, CWG: cell wall glycopolymer, PDP: polydiglycosylphosphate, LPDP: long polydiglycosylphosphate, Kdn: 2-keto-3deoxy-D-glycero-D-galacto-nononic acid 1

ACCEPTED MANUSCRIPT Abstract Cell wall glycopolymers (CWG) represent an important component of the Grampositive cell envelope with many biological functions. The mycelial soil bacterium coelicolor

A3(2)

incorporates

two

distinct

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Streptomyces

CWGs,

polydiglycosylphosphate (PDP) and teichulosonic acid, into the cell wall of its

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vegetative mycelium but only little is known about their role in the complex life cycle

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of this microorganism. In this study we established assays to measure the total amount of CWGs in mycelial cell walls and spore walls, to quantify the individual CWGs and to determine the length of PDP. By applying these assays, we discovered

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that the relative amount of CWGs, especially of PDP, is reduced in spores compared to vegetative mycelium. Furthermore we found that PDP extracted from mycelial cell

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walls consisted of at least 19 repeating units, whereas spore walls contained

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1. Introduction

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substantially longer PDP polymers.

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Beside peptidoglycan (PG), cell wall glycopolymers (CWGs) are the second major component of the cell wall of most Gram-positive bacteria. In some species they can account for up to 70% of the cell wall dry weight under specific growth conditions (Ellwood, 1970). CWGs show a very high chemical variety (Weidenmaier and Peschel, 2008) and can even differ in strains of the same species (Lazarevic et al., 2002). In response to altered environmental conditions specific CWG can be completely exchanged by a different type (Ellwood, 1970). Best understood in terms of biosynthesis, composition and biological role is the class of wall teichoic acids (WTA), which consist of phosphodiester linked polyol subunits and a disaccharide as PG linkage unit (Neuhaus and Baddiley, 2003). For these polymers many different cellular functions, in particular in morphogenesis, ion homeostasis, antibiotic resistance and in host interaction have been demonstrated (summerized in Brown et al., 2013). Moreover, their role in horizontal gene transfer by serving as phage receptor was recently shown (Winstel et al., 2013; Xia et al., 2011). For the actinomycetal model organism Streptomyces coelicolor A3(2), grown in rich liquid culture, no WTA but two distinct CWGs were described (Fig. 1A,B). The teichulosonic acid [-6)-ß-D-Galp-(1
9)-α-Kdn-(2-] is a phosphate free polymer of two to seven 2

ACCEPTED MANUSCRIPT repeating units composed of galactose (Gal) and the sialic acid 2-keto-3-deoxy-Dglycero-D-galacto-nononic acid (Kdn), often methylated or substituted with Nacetylglucosamine (GlcNAc). The second polymer polydiglycosylphosphate (PDP)

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consists of [-6)-a-Galp-(1
6)-a-GlcpNAc-(1-P-] subunits (Shashkov et al., 2012)

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Polydiglycosylphosphate Phosphate

Mycelium: n ≤ 19, spores: n > 19

Teichulosonic acid

Galactose

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GlcNAc

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Galactose

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A

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whereby the chain length is unknown.

n = 2-7

Kdn

R = H, CH3, GlcNAc

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Figure 1. Structure of the S. coelicolor cell wall glycopolymers polydiglycosylphosphate (A) and teichulosonic acid (B), as determined by Shashkov et al., (2012).

While first efforts to elucidate the biosynthesis of these polymers have been made

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(Kleinschnitz et al., 2011b; Ostash et al., 2014), only little is known about their functions and relative prevalence during different phases of the complex life cycle of S. coelicolor. The identification of putative CWG biosynthetic enzymes in the spore wall building machinery SSSC suggested a crucial role of these molecules during sporulation (Kleinschnitz et al., 2011b). But until now, a methodology to investigate the roles of the different CWGs was not available. In this study we adapted techniques well established for the analysis of WTAs from B. subtilis and S. aureus and combined them with colorimetric assays from other fields of glycobiology. This enabled us to compare mycelial cell walls and spore walls of S. coelicolor with respect to: i. their total CWG content, ii. the relative amounts of both individual polymers described for S. coelicolor (Shashkov et al., 2012), iii. and the polymer lengths of PDP. We compared the two developmental stages of S. coelicolor as proof of principle for the reliability of the described methods. In future studies the toolbox could be applied to elucidate CWG biosynthesis by characterizing CWG compositions of specific deletion mutants.

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ACCEPTED MANUSCRIPT 2. Material and Methods 2.1.

Growth conditions

To isolate mycelial cell walls, two liters HA medium were inoculated with about 105

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spores/ml and cultivated for 3 days at 30°C and 120 rpm. Spores were isolated after 8 days of growth at 30°C from about 80 MS agar plat es. To further purify the spores

2.2.

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from mycelial contamination they were filtered through a sterile wet cotton pad.

Isolation of mycelial cell walls and spore walls and determination

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of the PG proportion

Mycelial cell walls as well as spore walls were isolated according to a protocol

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described previously (Schäberle et al., 2011). For the determination of the CWG content, 10 mg mycelial cell walls or spore walls, respectively, were hydrolyzed using 600 µl 50 mM HCl at 90 °C. After 90 min the samples were neutralized by the

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addition of 30 µl 1 M NaOH and the insoluble PG-sacculi were separated from the

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soluble CWG fraction by centrifugation (21,000 g, 30 min, 4 °C). The PG was washed twice, lyophilized and the dry weight was compared to the initial weight. The CWG

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fraction was lyophilized, resuspended in 100 µl H2O and stored for chemical analysis. As a control whether the HCl hydrolysis was sufficient to remove all PG bound CWGs, we compared its efficiency with that of concentrated hydrofluoric acid (HF), the standard purification method used in PG analytics. 500 µl HF were added to 10 mg mycelial cell walls and incubated under continuous stirring. After 48 hours at 4 °C the PG was precipitated by centrifugation (21,000 g, 30 min, 4 °C) and washed 10 times with 1 ml ultrapure H2O before lyophilization. The pure PG was then hydrolyzed again using HCl, as described above. After both steps the PG dry weight was determined and compared to the starting dry weight.

2.3.

Determination of the phosphate content in the cell wall

glycopolymer fraction The amount of phosphate in the polymer fraction was measured according to Chen et al. (1956) with some modifications. Briefly, 10 µl of the CWG containing supernatants were transferred into ultrapure HPLC glasses and dried at 100 °C. After adding 75 µl 70 % perchloric acid, the glasses were closed and the samples were incubated at 4

ACCEPTED MANUSCRIPT 100 °C for two hours to release the phosphate from the polymers. Successively, 250 µl H2O, 100 µl 1.25 % ammonium molybdate and 100 µl 5 % L-ascorbic acid were added. After additional 10 min at 100 °C and coolin g down to room temperature the

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absorbance at 750 nm was measured in a photometer, compared to a KH2PO4 standard curve and normalized against the used starting dry weight before hydrolysis

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of the mycelial cell walls or spore walls, respectively.

2.4.

Determination of the hexosamine content in the CWG fraction

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To quantify the amino sugar GlcNAc in the CWG fraction, the hexosamine content was measured according to Smith and Gilkerson (1979). The absorbance was determined at 650nm, compared to a GlcNAc (Serva) standard curve and normalized

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to the starting dry weight before hydrolysis of the mycelial cell walls or spore walls,

Determination of the Kdn content in the CWG fraction

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2.5.

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respectively.

To quantify the amount of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn) in

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the CWG fraction, a method originally used for the quantification of sialic acids in glycoproteins (Matsuno and Suzuki, 2008) was adapted. In detail, 5 mg mycelial cell wall or spore wall was hydrolyzed for 90 min at 90 °C using 600 µl 50 mM HCl. After neutralization (30 µl, 1 M NaOH) the soluble CWG fraction was separated from the insoluble PG fraction by centrifugation (21,000 g, 30 min, 4 °C), freeze dried and hydrolyzed with 100 µl 2 M HCl to release Kdn from the teichulosonic acid glycan chains. After 3 hours at 100 °C the samples were ne utralized, freeze dried and resolved in 200 µl H2O. 10 µl of each sample was mixed with 90 µl ice cold H2O and 10 µl of 10 mM sodium periodate in ultrapure HPLC glasses. The oxidation process was stopped after 45 min by adding 50 µl sodium thiosulfate. After adding 250 µl 4 M ammonium acetate (pH 7.5) and 200 µl of an ethanolic solution of 100 mM acetoacetanilide, the mixture was incubated for 10 min at room temperature in darkness. The fluorescence intensity was determined in a fluorometer at 471 nm after excitation with 388 nm, compared to a Kdn (Santa Cruz Biotechnology) standard curve and normalized to the starting dry weight before hydrolysis of the mycelial cell walls or spore walls, respectively. 5

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PAGE of anionic glycopolymers Polymer samples for PAGE were generated by partial hydrolysis (0 – 90 min, 600 µl

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50 mM HCl, 90 °C) of 5 mg cell wall or spore walls followed by neutralization (30 µl 1 M NaOH) and separation from PG through centrifugation (21,000 g, 4 °C, 30 min).

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The soluble polymer fraction was lyophilized and resuspended in 100 µl H2O. 10 µl of

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each sample was mixed with 2 µl loading buffer (2 M sucrose, 0.2 M Tris, 0.2 M boric acid, 2 mM EDTA; pH 8.3, bromophenol blue) and applied to polyacrylamide gel electrophoresis. TBE Gels (16x20x0.1 cm or 40x20x0.1 cm for high resolution PAGE)

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with a final concentration of 20 % acrylamide (37.5:1) were polymerized over night at 7°C. The electrophoresis was performed with constan t 30 mA in a Protean II xi Cell

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(Bio-Rad) or a Base Ace Sequencer (Stratagene) for high resolution PAGE. Polymer fragments were visualized by staining with 0.005 % (w/v) alcian blue in EAW (40 % ethanol, 5 % acetic acid, 55 % H2O), followed by silver staining as described

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previously (Blum et al., 1987). The presence of phosphate in the separated polymer

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fragments was verified through recovery of the polymer fragments out of a gel

2.6.

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followed by the phosphate quantification assay described above (see also Fig S1).

Statistical analysis

Assays were performed with four independent mycelial cell wall preparations and six independent spore wall preparations. For statistical analysis GraphPad Prism version 6 was used.

3. Results and Discussion 3.1.

Relative PG and CWG content in cell walls isolated from vegetative

mycelium and spores To study the prevalence of CWGs during the life cycle of S. coelicolor, cell walls of vegetative mycelium, grown in liquid culture and cell walls of mature spores, harvested from agar plates were isolated according to Schäberle et al. (2011). The procedure includes the purification from all potentially misleading cellular components as proteins, RNA, and DNA, ending up with a nearly pure mixture of PG and attached CWGs. To determine the relative amount of these components, they had to be 6

ACCEPTED MANUSCRIPT separated. A well-established method, widely used in muropeptide analytics (de Jonge et al., 1992) to purify PG from the attached CWGs is the hydrolysis of the linking phosphodiester bonds using concentrated hydrofluoric acid (48 % HF)

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followed by the precipitation of the insoluble PG. Unfortunately, this treatment has the disadvantage that the detached CWGs are dissolved in the toxic HF, preventing their

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subsequent chemical analysis under normal lab conditions. Kühner et al. (2014) recently showed that HF could be replaced in PG purification by 2M HCl. Also,

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teichuronic acids could be detached from cell walls of Micrococcus luteus with 50 mM HCl (Wolters et al., 1990). To separate S. coelicolor CWGs from PG, we hydrolyzed

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lyophilized mycelial cell walls or spore walls, respectively, in 50mM HCl for 90 min at 90 °C. The insoluble PG was precipitated by centrif ugation and washed twice ahead

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of a second lyophilization step. The dry weight, representing the PG proportion within the mycelial cell walls/spore walls was determined and compared to the initial weight

100 90 80 70 60 50

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Prop ortio n of PG in cell/ spor e wall (%)

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Cell/spore wall composition

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prior to the hydrolysis (Fig. 2).

myc elial cell wall

s p or e w all

Fig. 2. Relative PG content of mycelial cell walls and spore walls of S. coelicolor. Purified mycelial cell walls (n=4) and spore walls (n=6) were hydrolyzed under mild acidic conditions to detach CWGs from the PG. The dry weight of the insoluble PG was set in relation to the initial weight prior to hydrolysis. Results are presented as mean with sd. Mann-Whitney test, * = p ≤ 0.05.

Due to the elaborate cell wall/spore wall isolation protocol, the weight difference could be interpreted as the proportion of detached CWGs. Accordingly, cell walls of vegetative mycelium contained 64% PG (sd: 4.5%; n=4) and 36% attached CWGs. In contrast, S. coelicolor spore walls were composed of 78% PG (sd: 6.9%; n=6) and only 22% CWGs.

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ACCEPTED MANUSCRIPT To confirm that the above described hydrolysis conditions were sufficient to completely detach all CWGs from S. coelicolor PG, we compared the efficiency of HCl-hydrolysis with that of the established HF protocol (de Jonge et al., 1992).

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58.25% (sd: 3.6%; n=4) of the initial dry weight (Fig. 3).

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Cell wall hydrolysis (HF/HCl)

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HF HF purif purif ied ied PG PG +HC l

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Fig. 3. Percentage of PG in mycelial cell walls determined after HF hydrolysis and HF/HCl hydrolysis. Purified mycelial cell walls (n=4) were hydrolyzed with hydrofluoric acid (HF). Subsequently, the HF purified PG was hydrolyzed a second time with 50mM HCl. The dry weight of the insoluble PG was set in relation to the initial weight prior to hydrolysis. The unchanged amount of PG demonstrates that the HCl hydrolysis did not result in PG degradation. Results are presented as mean with sd. Mann-Whitney test, * = p ≤ 0.05.

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P 120 ell et dr 100 y w 80 ei g 60 ht ( % 40 myc ) elial cell wall

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Following HF hydrolysis of mycelial cell walls, the dry weight (PG) was reduced to

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Considering the extensive washing steps (about 10 times), required to dilute the toxic HF to an acceptable concentration, this value underestimated the PG proportion than overestimating it, implying that the 64 % PG proportion determined by HCl hydrolysis (Fig. 2) was quite accurate and represented pure PG. HCl hydrolysis of HF purified PG did not cause a further reduction in the dry weight (Fig. 3), since CWGs had been already completely removed during HF treatment. Moreover, this demonstrates that only the phosphodiester bonds within the CWGs and those linking the CWGs to PG, but neither the peptide- nor the glycosidic bonds within the PG were cleaved during the used HCl hydrolysis conditions. The relative increase of the PG portion in spore walls implies that either CWGs are removed from the spore wall during differentiation or that the reduced CWG proportion in spore walls was caused by an increased relative amount of PG. This indicates that S. coelicolor remodels its cell wall during differentiation to meet the distinct requirements of the envelope during different stages of its life cycle. Whereas vegetative mycelium depends on the efficient and fast uptake of nutrients to penetrate the substrate, spores have to withstand detrimental environmental conditions (Flardh and Buttner, 2009). Previously, we showed active PG biosynthesis 8

ACCEPTED MANUSCRIPT at the lateral walls of maturating spores by staining with fluorescent vancomycin (Sigle et al., 2015), suggesting that PG-thickening is the cause of the reduced relative CWG content of S. coelicolor spores. Also, the finding of at least four putative

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penicillin binding proteins involved in the SSSC (Kleinschnitz et al., 2011a) suggests active PG synthesis during the remodeling of the mycelial cell wall into the exospore

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wall.

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We cannot fully exclude that the different media used for cultivation of vegetative mycelia in liquid culture (HA) and spores (MS-agar) could have influenced the cell wall composition. Unfortunately, different media were necessary for the isolation of

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mycelial cell walls and spore walls because the required amounts of S. coelicolor spores could only be obtained from 80 MS-agar plates. However, MS medium was

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not appropriate for liquid culture due to the large content of insoluble soy flour particles, which made it impossible to harvest pure mycelial biomass without misleading contaminations. Nevertheless, the method worked reliable well for the

3.2.

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alterations in CWG/PG ratio.

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quantification of S. coelicolor PG and can be used in further studies to characterize

Assay development to quantify the distinct CWGs

With the aim to confirm the relative CWG reduction in spore walls with independent methods, we intended to directly determine the amounts of the CWGs in the supernatant fraction after cell wall hydrolysis. To differentiate the two CWGs of S. coelicolor, teichulosonic acid and PDP, colorimetric assays to quantify characteristic CWG building blocks were established. As teichulosonic acid is a phosphate free polymer, determination of the phosphate concentration is indicative for the amount of PDP (Fig. 1A). Since Kdn is only present in teichulosonic acid, but not in PDP (Shashkov et al., 2012), Kdn measurement specifically indicates the teichulosonic acid content (Fig. 1B). A valid method to measure phosphate was originally described by Chen et al. (1956) and since then widely used for the quantification of phosphate containing WTAs (e.g. Lazarevic and Karamata, 1995). It is based on the formation of phosphomolybdic acid

followed

by

a

reduction

by

ascorbic

acid.

The

resulting

reduced

phosphomolybdate complex can be colorimetrically detected due to its intense blue 9

ACCEPTED MANUSCRIPT color. The advantages of this method are the high sensitivity and the possibility to perform the assay in parallel for many replicates in microliter scales. Kdn belongs to the family of sialic acids and is widely distributed in nature. Since its

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first isolation from polysialoglycoprotein of rainbow trout eggs (Nadano et al., 1986) it was found in many biological samples including capsular polysaccharides of Gram-

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negative bacteria (Knirel et al., 1989) and human blood and ovarian cancer cells.

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Besides in S. coelicolor, Kdn containing teichulosonic acids were also found in other Streptomyces species like S. albus and S. albidoflavus (Shashkov et al., 2016). Several methods were established to quantify sialic acids, most with disadvantages

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for the quantification of teichulosonic acid, as time-consuming, poor sensitivity, or requirement of expensive equipment (Aminoff, 1961; Hara et al., 1989; Jourdian et

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al., 1971; Warren, 1959).

Here, we adapted a method developed by Matsuno and Suzuki (2008) to measure

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the amount of sialic acids in glycoproteins to quantify Kdn of teichulosonic acid. The

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assay is based on the indirect detection of sialic acids by the quantification of formaldehyde, generated through periodate oxidation of the carbon-9 (C-9) sialyl

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residues. The released formaldehyde can be determined after condensation with acetoacetanilide and ammonia to a fluorescent dihydropyridine derivative by measuring the fluorescence intensity (388/471 nm) and comparison to a Kdn standard curve. For the analysis of glycoconjugates with terminal sialic acid, typical for glycoproteins, this assay does not involve an initial hydrolysis step. However, quantification of Kdn from the 1
9 linked galactose-Kdn teichulosonic acid of S. coelicolor required hydrolysis of the polymer to release Kdn. The method has several advantages. Beside its high sensitivity, it is very simple to use and can be performed in many replicates in less than one hour. In addition, a third method, originally described to analyze hexosamines present in glycosaminoglycans (Smith and Gilkerson, 1979) was tested for the reliable quantification of GlcNAc. The method is based on the interaction between deaminated hexosamines and 3-methyl-2-benzothiazolone hydrazine hydrochloride, resulting in an intense blue colored complex. It can be performed with glycan chains as substrate without previous hydrolysis.

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ACCEPTED MANUSCRIPT To validate the described assays regarding their sensitivity and specificity, we performed each assay with all CWGs building blocks, namely phosphate, galactose, GlcNAc, and Kdn (Fig. 4A-C). All three methods worked reproducible well with high

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specificity and no cross reactions could be observed. Also the sensitivity was very high up to a low nmol range and a linear relationship between concentration and

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signal was given in the preferred measuring range between 1 and 100 nmol for all

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Phosphate 0.5

0.4 O D6 0.3

O 0.4 D7

50

0.3

nm

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50

Hexosamine

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A

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assays.

nm

0.2

0.2

0.1

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0.1

0.0

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20 40 60 80 100 nmol

20 40 60 80 100 nmol

C

Kdn assay 10000

Fluo resc enc e inte 5000 nsit y 388/ 471 nm 0

20 40 60 80 100 nmol

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Fig. 4. Sensitivity and specificity of colorimetric assays to differentiate the S. coelicolor cell wall glycopolymers (CWG) polydiglycosylphosphate (PDP) and teichulosonic acid. Colorimetric assays for the quantification of the PDP components phosphate (A) and GlcNAc (B) and Kdn (C), a building block of teichulosonic acid were tested with all CWG components (galactose, GlcNAc, phosphate and Kdn). All assays were sensitive and highly specific. The results of 3 independent measurements are presented as means with sd.

3.3.

Quantification of CWGs in cell walls of vegetative mycelium and

spore walls To compare the CWG composition of mycelial cell walls and spore walls, we indirectly quantified the amounts of PDP and teichulosonic acid in the cell wall hydrolysates, using the assays described in 3.2. In accordance to the findings of Shashkov (Shashkov et al., 2012), mycelial cell walls contained high levels of all measured molecules (phosphate, Kdn, and hexosamines) indicative for the described CWGs. Interestingly, also in the spore samples substantial amounts of phosphate, Kdn and hexosamines were detected, suggesting that S. coelicolor spores contain 11

ACCEPTED MANUSCRIPT GWGs identical or similar to those found in mycelial cell walls. This finding is not obvious for a quite dormant developmental state, considering the various CWG functions in other bacteria, which more or less are all linked to active cellular

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processes (Swoboda et al., 2010). Nevertheless, the relative amount of all tested molecules was reduced in spore walls (Fig. 5A-C). The phosphate content decreased

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from 172 nmol/mg in cell walls isolated from vegetative mycelium to 88 nmol/mg in spore walls (Fig. 5A). This reduction (-49%) indicated a strong decrease in the

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relative amount of PDP in spores and correlated with the decrease of detachable CWGs (-39 %) in spore walls measured by PG dry weight determination after acidic

0

150 Kdn per mg cell/ 100 spor e wall (nmo 50 l)

Kdn content

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250 Phos phat e per 200 mg cell/ 150 spor e 100 wall (nmo 50 l)

B

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Phosphate content

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A

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hydrolysis (Fig. 2).

myc elial cell wall

sp or e w all

0 myc elial cell wall

sp or e w all

C Hexo sami ne per mg cell/ spor e wall (nmo l)

Hexosamine content 500 400 300 200 100 0 myc elial cell wall

sp or e w all

Fig. 5. Quantification of cell wall glycopolymers in vegetative mycelium and spores of S. coelicolor. Purified mycelial cell walls (n=4) and spore walls (n=6) were hydrolyzed under mild acidic conditions. The key components phosphate (A), indicative for PDP and Kdn (B), indicative for teichulosonic acid, and GlcNAc (C, hexosamine) present in both polymers, were measured in the CWG fraction. Compared to mycelial cell walls, the amount of phosphate and GlcNAc was stronger reduced in spore walls (Mann-Whitney test, ** = p ≤ 0.01) than that of Kdn, suggesting spore wall specific changes in the PDP content. All results are presented as mean with sd.

The Kdn content only showed a slight decrease from 95 nmol in mycelial walls to 80 nmol in spore walls (Fig. 5B), indicating an almost stable relative amount of teichulosonic acid in both developmental stages. So far only little is known about the 12

ACCEPTED MANUSCRIPT function of teichulosonic acid. Deletion of genes involved in the synthesis of Kdn did not cause a detectable phenotype (Ostash et al., 2014), suggesting a less relevant role in the cell envelope. Since in our assays, the measured amount of phosphate

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was always higher than that of Kdn, irrespective whether mycelial walls or spore walls were analyzed, the previous classification of teichulosonic as the “major” CWG

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of S. coelicolor (Shashkov et al., 2012) might be questioned.

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Hexosamines in the polymer fraction can be derived from mainly three different sources, namely PDP subunits, modification of Kdn in teichulosonic acid, and hexosamine-containing linkage units. Contamination of the samples with PG derived

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GlcNAc in considerable amounts is unlikely, since the used hydrolysis conditions did not affect PG (no reduction of PG dry weight after hydrolysis of purified PG (Fig. 3).

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The hexosamine content in spore walls (119 nmol) was significantly reduced in comparison to that of mycelial cell walls (356 nmol; Fig. 5C). Even after subtracting the part which can be assigned to PDP based on the one to one ratio with phosphate

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and the theoretical supposition that all Kdn molecules were modified with GlcNAc, a

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considerable amount of additional hexosamines remained. These additional hexosamines were most likely derived from the until now non-described linkage units,

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which are present in all CWGs (Coley et al., 1978). In spore walls the ratio between phosphate and hexosamine was much smaller. A reason could either be a less modified teichulosonic acid or the replacement of short polymers, each one with a linkage unit, by fewer polymers with a longer chain length.

3.4.

Determination of the chain length of PDP in mycelial cell walls and

spore walls Wall teichoic acids are long polymers often consisting of more than 50 glycerol phosphate or ribitol phosphate subunits (Neuhaus and Baddiley, 2003). The teichulosonic acid of S. coelicolor was characterized as a polymer composed of only up to seven subunits, while no length was described for the PDP polymer (Shashkov et al., 2012). To get more insight into the structural properties of PDP, we established a protocol for polyacrylamide gel electrophoresis (PAGE) of partially hydrolyzed PDP fragments. At the used conditions (pH 8.3), electrophoresis was possible due to the evenly distributed negative charge provided by the phosphate groups of PDP. We 13

ACCEPTED MANUSCRIPT first tried a Tris-Tricin buffered system (data not shown), often used in WTA-analytics (Meredith et al., 2008). However, better separation results were achieved after switching to TBE buffered conditions. To elucidate the number of repeating subunits

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via PAGE, the polymer had to be partially hydrolyzed, ending up with a mixture of fragments statistically representing all intermediates from single subunits to the entire

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full length polymer. We already showed that mild acidic hydrolysis was sufficient to remove the teichulosonic acid (Fig. 5B) as well as the PDP (Fig. 5A) from PG. In a

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next step, we hydrolyzed cell walls isolated from vegetative mycelium in a time dependent manner to check whether also the phosphodiester bonds between PDP

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subunits can be cleaved by 50mM HCl at 90°C. After 5 min hydrolysis already a spectrum of bands was visible in alcian blue/silver stained gels (Fig. 6A), although

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the absolute majority of bands comprised relative long fragments, as indicated by the short migration distance. Up to 30 min of hydrolysis the overall signal increased, meaning that further polymers were detached from the PG. At longer incubation

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times (60 – 90 min), the polymers got degraded more and more (Fig. 6A), indicated

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by the disproportionately increase in shorter fragment bands (long migration distance). The optimal hydrolysis time to obtain differently sized fragments was

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between 15 min to 30 min.

Several lines of evidence suggested that the PAGE-separated fragments were derived from PDP and not from teichulosonic acid. First, clearly more bands than the seven repeating units described for teichulosonic acid were observed. Second, cleavage of teichulosonic acid into separable subunits would require the hydrolysis of glycosidic bonds. As demonstrated by the absence of PG degradation during acidic hydrolysis, glycosidic bonds were not attacked under the used conditions. To finally prove whether the separated fragments were derived from PDP, we recovered them from gel slices and quantified the phosphate content (explained in detail in Fig. S1A). Whereas the negative control samples did not contain any measurable amount of phosphate, the samples derived from hydrolyzed vegetative cell walls (Fig. S1B) and spore walls (Fig. S1C) contained substantial amounts of phosphate. This confirmed that the fragments separated by PAGE were derived from PDP and not from the phosphate-free teichulosonic acid.

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ACCEPTED MANUSCRIPT

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60 min .

90 min .

B

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15

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10

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b

19

a 5

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10

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15

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5

1

My celi um

1 Spo res

Fig. 6. Structural analysis of S. coelicolor cell wall glycopolymers by PAGE. A: Mycelial cell walls were hydrolyzed in a time dependent manner (0-90 min). With increasing time, CWGs hydrolyzed into fragments of different length, which could be separated by PAGE according to their molecular weight (A). To study the structural composition of the separated polymer in more detail, the 15 min sample was investigated by high resolution PAGE (B). A minimum of 19 regular spaced bands was detected. To reveal structural differences between mycelial cell wall and spore wall polymers both were separated next to each other (C). The polymer from vegetative cell walls consisted of a minimum of 19 subunits (a). The polymer from spore walls was clearly longer (a,b), although separation of the high-molecular fragments by PAGE was not satisfying.

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ACCEPTED MANUSCRIPT For a more reliable determination of the polymer length, the cell walls of vegetative mycelium were hydrolyzed for 15 min and separated in a Stratagene Base Ace Sequencer, which provides a longer separation distance with better resolution. At

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least 19 regularly spaced bands were counted (Fig. 6B), implying that PDP (Fig. 1A) of vegetative mycelium is composed of at least 19 uniform repeating units. To

next

to

each

other

(Fig.

6C).

This

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compare PDP of vegetative mycelium and spore walls, hydrolysates were loaded revealed

the

presence

of

longer

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polydiglycosylphosphate polymers (LPDP) in spore walls (Fig. 6C, a+b) compared to those of vegetative mycelium (Fig. 6C, a), although the separation quality of the long

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fragments was not completely satisfying. This indicates that during the development of spores, longer spore-specific polydiglycosylphosphate polymers (LPDPs) are

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synthesized and incorporated into the maturating spore wall. It further implies that the altered proportions of PG and CWG (Fig. 2 and Fig. 5) are not simply the result of active peptidoglycan biosynthesis, but also reflect changes in PDP.

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Here, we developed a toolbox for the analysis of S. coelicolor CWGs. We described a

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mild hydrolysis procedure for the efficient separation of CWGs from PG. We showed for the first time that the sialic acid assay developed by Matsuno and Suzuki (2008) is

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suitable for the quantification of S. coelicolor teichulosonic acid. By the additional determination of the phosphate content we were able to differentiate the two CWGs of S. coelicolor. Establishment of a PAGE procedure for the separation of PDP fragments demonstrated changes in the PDP structure during differentiation of S. coelicolor. These assays will be valuable for future applications, eg. tracking CWG alterations over the lifecycle or identifying genes involved in PDP and teichulosonic acid synthesis (Sigle et al., submitted).

Acknowledgement We thank der DFG (SFB766) for financial support.

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de Jonge, B.L., Chang, Y.S., Gage, D., and Tomasz, A. (1992). Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of

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Matsuno, K., and Suzuki, S. (2008). Simple fluorimetric method for quantification of sialic acids in glycoproteins. Anal. Biochem. 375, 53-59.

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Agents Chemotherapy 55, 4283-4289. Shashkov, A.S., Ostash, B.E., Fedorenko, V.A., Streshinskaya, G.M., Tul’skaya,

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E.M., Senchenkova, S.y.N., Baryshnikova, L.M., and Evtushenko, L.I. (2012). Novel teichulosonic acid from cell wall of Streptomyces coelicolor M145. Carbohydr. Res.

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albidoflavus

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Streptomyces pathocidini. Antonie van Leeuwenhoek, 1-14. Sigle, S., Ladwig, N., Wohlleben, W., and Muth, G. (2015). Synthesis of the spore

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envelope in the developmental life cycle of Streptomyces coelicolor. Int. J.Med.

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Microbiol.: IJMM 305, 183-189.

Smith, R.L., and Gilkerson, E. (1979). Quantitation of glycosaminoglycan hexosamine

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using 3-methyl-2-benzothiazolone hydrazone hydrochloride. Anal. Biochem. 98, 478-

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ACCEPTED MANUSCRIPT Wolters, P.J., Hildebrandt, K.M., Dickie, J.P., and Anderson, J.S. (1990). Polymer length of teichuronic acid released from cell walls of Micrococcus luteus. J. Bacteriol. 172, 5154-5159.

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Xia, G., Corrigan, R.M., Winstel, V., Goerke, C., Grundling, A., and Peschel, A. (2011). Wall teichoic acid-dependent adsorption of staphylococcal siphovirus and

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myovirus. J. Bacteriol. 193, 4006-4009.

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ACCEPTED MANUSCRIPT s a m pl e 2

n e g. co nt ro l2

s a m pl e 3

n e g. co nt ro l3

B Phosphate recovered from gel

C Phosphate recovered from gel

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n e g. co nt ro l1

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SC

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A

Bro mo ph en ol blu e sa mp le

s a m pl e 1

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Mycelial polymer

Spore wall polymer

Negative

Negative control

control

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Supplementary Figure. S1. Identification of the PAGE-separated CWG as PDP. PAGE was performed as illustrated (A). 40 µl samples of vegetative CWG (B) or spore CWG (C) were mixed with 8 µl of loading buffer without bromophenol blue to avoid any interference with subsequent colorimetric assays. These samples were loaded to the gel next to control samples (bromophenol blue: CWG with loading dye, and water as a negative control). After electrophoresis, the left part (bromophenol blue sample) of the gel was stained with alcian blue and silver to visualize the separated polymer fragments. The stained gel was then used as a reference to cut out corresponding slices from the other lanes (as indicated in A). The gel pieces were incubated for 48h hours in 10 ml of H2O to recover polymer fragments by diffusion. All gel remains were removed by centrifugation and the samples were lyophilized and resuspended in 100µl H2O. Whereas, nearly no phosphate was detected in the lanes of the water control, high amounts of phosphate were recovered from lanes containing the CWG hydrolysates of either vegetative cell walls or spore walls. This indicated that the PAGE-separated polymer fragments represented PDP. Results are presented as mean with sd.

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ACCEPTED MANUSCRIPT Highlights •

Establishment of colorimetric assays for the quantification of S. coelicolor cell wall glycopolymers Development of a PAGE system for separation of polydiglycosylphosphate

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•

fragments

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Characterization of the cell wall glycopolymer composition during

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differentiation of S. coelicolor A3(2)

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•

22