Accepted Manuscript

Title: Genetic engineering in Actinoplanes sp. SE50/110

development of an intergeneric conjugation system for the
introduction of actinophage-based integrative vectors

Author: Tetiana Gren Vera Ortseifen Daniel Wibberg Susanne

Schneiker-Bekel Hanna Bednarz Karsten Niehaus Till Zemke
Marcus Persicke Alfred Puhler Jorn Kalinowski

PII: S0168-1656(16)30268-1
DOI: http://dx.doi.org/doi:10.1016/j.jbiotec.2016.05.012
Reference: BIOTEC 7551

To appear in: Journal of Biotechnology

Received date: 3-4-2016

Revised date: 6-5-2016
Accepted date: 11-5-2016

Please cite this article as: Gren, Tetiana, Ortseifen, Vera, Wibberg, Daniel,
Schneiker-Bekel, Susanne, Bednarz, Hanna, Niehaus, Karsten, Zemke, Till, Persicke,
Marcus, Puhler, Alfred, Kalinowski, Jorn, Genetic engineering in Actinoplanes
sp.SE50/110 development of an intergeneric conjugation system for the
introduction of actinophage-based integrative vectors.Journal of Biotechnology
http://dx.doi.org/10.1016/j.jbiotec.2016.05.012

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
 Genetic engineering in Actinoplanes sp. SE50/110 - development of an

intergeneric conjugation system for the introduction of actinophage-

based integrative vectors

Tetiana Gren1, Vera Ortseifen2, Daniel Wibberg2, Susanne Schneiker-Bekel2, Hanna

Bednarz3, Karsten Niehaus3, Till Zemke4, Marcus Persicke1, Alfred Phler2, Jrn

Kalinowski1

Affiliations

1 Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld

University, Universittsstrae 27, 33615 Bielefeld, Germany

2 Senior Research Group in Genome Research of Industrial Microorganisms, Center

for Biotechnology, Bielefeld University, Universittsstrae 27, 33615 Bielefeld,

Germany

3 Department of Proteome and Metabolome Research, Faculty of Biology, Bielefeld

University, Postfach 100131, D-33501 Bielefeld, Germany

4 Product Supply, Bayer Pharma AG, Friedrich Ebert Str. 217-475, 42117 Wuppertal,

Germany

Corresponding author

1
 Highlights

Basic genetic engineering toolkit established for Actinoplanes sp. SE50/110

Development of the Actinoplanes sp. SE50/110 - E. coli intergeneric conjugation
Integration sites for C31, BT1 and VWB actinophage-based integration
systems sequenced and described for the first time in the Actinoplanes genus
GUS reporter system applied successfully for Actinoplanes sp. SE50/110

Abstract

The -glucosidase inhibitor acarbose is used for treatment of diabetes mellitus type

II, and is manufactured industrially with overproducing derivatives of Actinoplanes sp.

SE50/110, reportedly obtained by conventional mutagenesis.

Despite of high industrial significance, only limited information exists regarding

acarbose metabolism, function and regulation of these processes, due to the

absence of proper genetic engineering methods and tools developed for this strain.

Here, a basic toolkit for genetic engineering of Actinoplanes sp. SE50/110 was

developed, comprising a standardized protocol for a DNA transfer through E.coli-

Actinoplanes intergeneric conjugation and applied for the transfer of C31, BT1 and

VWB actinophage-based integrative vectors. Integration sites, occurring once per

genome for all vectors, were sequenced and characterized for the first time in

Actinoplanes sp. SE50/110. Notably, in case of C31 based vector pSET152, the

integration site is highly conserved, while for BT1 and the VWB based vectors

pRT801 and pSOK804, respectively, no sequence similarities to those in other

bacteria were detected.

The studied plasmids were proven to be stable and neutral with respect to strain

morphology and acarbose production, enabling future use for genetic manipulations

2
of Actinoplanes sp. SE50/110. To further broaden the spectrum of available tools, a

GUS reporter system, based on the pSET152 derived vector, was also established in

Actinoplanes sp. SE50/110.

Keywords

Acarbose, Actinoplanes sp. SE50/110, genetic engineering, integrative vectors

1. Introduction

Actinobacteria are Gram-positive filamentous soil bacteria that have a complex life

cycle, embodying several stages of morphological differentiation. They produce many

bioactive compounds as secondary metabolites, such as antibiotics, antitumor

agents, immunomodulators, anthelmintic and insect control agents (Kieser et al.,

2000).

Actinoplanes sp. SE50/110 belongs to so called rare actinomycetes and is known

for its ability to produce acarbose, which is one of its prominent secondary

metabolites. Acarbose is mainly used for treatment of type II diabetes mellitus, but

also can be regarded as a prevention drug. It helps patients with starch- and sucrose-

containing diets delaying the digestion of ingested carbohydrates in the human

intestine, thereby resulting in a smaller rise in blood glucose concentration (Puls and

Keup, 1973). Structurally, acarbose can be characterized as a pseudotetra-

saccharide, which consists of an unsaturateded cyclitol, 4-amino-4,6-dideoxyglucose

and maltose (Wehmeier, 2003).

The extensive body of data on Actinoplanes sp. SE50/110 genomics, proteomics and

transcriptomics was obtained in recent years. In 2011 the complete genome of

3
Actinoplanes sp. SE50/110 was sequenced, analyzed and annotated. It was revealed

that the genome with a high G+C content (71.32 %) consists of one circular

chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding

sequences (Schwientek et al. 2012; Schwientek et al. 2013; Schwientek et al. 2014).

In 2012, the first whole transcriptome analysis of Actinoplanes sp. SE50/110 using

RNA sequencing technology for comparative gene expression studies between cells

grown in minimal and complex growth media was conducted (Schwientek et al.,

2013).

The acb gene cluster has been sequenced, cloned and found to consist of 22 genes,

some of which were expressed and studied in heterologous conditions (Zhang et al.,

2002; Wehmeier 2003).

Also, studies on extra and intracellular proteome of the strain Actinoplanes sp.

SE50/110 (Wendler et al., 2013, 2015, 2016) as well as metabolomics studies

(Wendler and Ortseifen et al., 2014) were conducted. Based on these analyses and

other studies, several models of acarbose metabolism were proposed (Zhang et al.,

2002; Wehmeier 2003; Wendler et al., 2013; Wendler and Ortseifen et al., 2014).

Various Actinoplanes sp. SE50/110 strain derivatives, which are being used for

industrial acarbose production, were reported to be exclusively obtained via

mutagenesis and stepwise selection of overproducers. Although this strategy was

proven to be effective in the past, it is certainly limited. Genetic engineering of the

producer strain can be utilized as an alternative approach in this case. However, up

to date there is no information regarding any genetic manipulations of Actinoplanes

sp. SE50/110 and the genus Actinoplanes is described as a challenging object for

any genetic manipulation (Vobis 2006).

Regarding the DNA uptake system, a broad choice of methods, available for

Actinobacteria, exists. Among them, protoplast transformation is the route usually

4
followed for Streptomyces spp. However, conditions for protoplast formation and

regeneration have to be empirically determined in a set of laborious experiments and

are known to be species- or even strain-specific (Marcone et al., 2010a). This

approach was applied with limited success only for few non-Streptomyces strains

(Marcone et al., 2010a and b). Intergeneric transfer of plasmid DNA from E. coli to

Streptomyces spp. was reported first by Mazodier et al., 1989. Effective conjugation

systems have since been developed for many actinomycetes, in which the incoming

plasmid can be maintained episomally or integrated into the host chromosome by

site-specific or homologous recombination (Stinchi et al., 2003).

Up to now, there are only two representatives of Actinoplanaceae, Actinoplanes

friuliensis and Actinoplanes teichomyceticus, for which gene cloning systems were

successfully developed and implemented being based on intergeneric conjugation

strategies (Heinzelmann et al., 2003; Ha et al., 2008). Particularly prominent progress

in establishing the genetic system was made for A. teichomyceticus, producer of

lipoglycopeptide antibiotic teicoplanin. The procedure of intergeneric conjugation was

used to transfer a set of commonly used integrative and replicative vectors to A.

teichomyceticus cells. Later on, the above mentioned vectors were used for

overexpression of two regulatory genes in a wild type strain, which led to the increase

in teicoplanin production (Horbal et al., 2012; Ha et al., 2008).

Actinomycete bacteriophages or actinophages are frequently being used as a source

to develop integrative vectors. Among the most well studied and used temperate

actinophages are C31 (Lomovskaya et al., 1972) and BT1 (Gregory et al., 2003).

Both of them use large serine recombinases for site specific integration without

requiring any additional host or phage functions. Recombination occurs between

specific attP and attB regions, which frequently have sequence similarity, resulting in

a formation of hybrid attL and attR regions (Baltz et al., 2012). Importantly, these
5
bacteriophages use unrelated chromosomal genes for their integration: attB sites for

C31 are located in homologs of genes encoding a pirin-like protein and BT1 attB

sites are contained in the coding regions of integral membrane proteins (Baltz et al.,

2012; Combes et al., 2002).

The temperate bacteriophage VWB was originally isolated from Streptomyces

venezuelae and characterized as having a narrow host range. It integrates into the

host chromosomes by recombination with attB locus, similar to C31 and BT1. Till

nowadays, attB sites of VWB were characterized only for two strains: Streptomyces

ghanaensis and S. venezuelae (Ostash et al., 2009; Van Mellaert et al., 1998; Van

Dessel et al., 2005). However, VWB-based integrative vectors were proven to be

effective also in one representative of Actinoplanes, namely A. teichomyceticus

(Horbal et al., 2012).

Use of reporter genes gained more attention in the last decades as a promising

approach to study gene regulation and for the visualization of multiple biological

processes directly in vivo. These genes are fused to various regulatory sequences,

and introduced into a strain of interest. The signal detected from the expression of

reporters can be quantified, which gives the possibility to study the effect of various

physiological conditions, stresses and other factors (Myronovskyi et al., 2011). The -

glucuronidase enzyme (GUS) hydrolyzes several -glucuronides. The GUS reporter

system appeared to be highly sensitive due to the stability and high specific activity of

the GUS enzyme (Myronovskyi et al., 2011). It was successfully applied also in one

representative of the Actinoplanes genus, A. teichomyceticus (Horbal et al., 2013).

In this paper, we present the development of a gene cloning system for Actinoplanes

sp. SE50/110. Namely, the selection of appropriate solid media, the determination of

antibiotic resistance spectra, the development of the Actinoplanes - E.coli conjugation

6
procedure, and its application for transfer of three different integrative vectors are

described. Finally, the GUS reporter system was applied in Actinoplanes sp.

SE50/110 using a pSET152 derivative as vehicle for genetic engineering.

2. Methods

2.1 Strains, media, growth conditions and reagents

To maintain and isolate plasmid DNA, Escherichia coli DH5MCR strain was used as

a host. E. coli ET12567 pUZ8002 (dam-13::Tn9, dcm-6, hsdM, hsdS) (Kieser et al.,

2000) was used for intrageneric conjugation with Actinoplanes sp. SE50/110 (ATCC

31044). The site-specific integration vectors, pIJ6902 (7.4 kb) and pSET152 (5.7 kb),

pSOK804 (5.5 kb), pRT801 (5.2 kb), contain C31, VWB and BT1 int and attP

genetic regions respectively. All of the vectors contain and oriT of RK2, as well as an

apramycin resistance gene for selection in actinomycetes and E. coli (Bierman et al.,

1992). These plasmids do not contain the replicative functions and can be

maintained in recipient strains only in the chromosomally integrated state.

Plasmids pSET152, pSOK804, pRT801 and pIJ6902 were received from B. Ostash

(Ivan Franko National University of Lviv, Ukraine).

pSETGUS (7.7 kb) vector is based on pSET152, contains an apramycin resistance

gene for selection in actinomycetes and E. coli, C31 int and attP genetic regions

and gusA gene, cloned under tipA promoter. pSETGUS was received from A.

Luzhetskyy (Saarbrcken, Germany) (Myronovskyi et al., 2011).

All cultivations of E.coli strains were done in LB-media, prepared as described in

Kieser et al., 2000. The growth conditions were 37 C and 180 rpm in a GFL shaking

incubator 3032 (GFL, Burgwedel, Germany). All Actinoplanes strains were grown on

soy flour medium (SFM; 20 g/L soy flour, 20 g/L mannitol, 20 g/L agar, tapped water
7
to 1 L; pH adjusted to 8.0 prior to autoclaving; autoclaved twice), oatmeal medium

(OM; oatmeal flour 38g/L, agar 10 g/L; tapped water to 1 L; pH adjusted to 8.0 prior

to autoclaving; autoclaved twice). SFM plates, used for setting Actinoplanes-E.coli

conjugations, were additionally dried at 37 C before the experiment to assure

absence of water on a plate surface. Minimal, complete and R2 medium were

prepared as described in Kieser et al., 2000. Liquid cultivations were done in NBS

medium (10 g/L glucose, 4 g/L peptone, 4 g/L yeast extract, 1 g/L MgSO47H 2O, 2

g/L KH2PO4, 5.2 g/L K2HPO43H2O) and maltose minimal medium (prepared as

described in Wendler et al., 2012). Liquid cultivations of Actinoplanes strains were

performed at 28 C and 140 rpm (in a GFL shaking incubator 3032) in baffled

polycarbonate flasks (Corning, Corning, NY, USA). When needed, chloramphenicol

(25 g/mL) or kanamycin sulphate (50 g/mL) were added as a selection markers for

E. coli and apramycin sulphate (50 g/mL) was used for the selection of E. coli and

Actinoplanes sp. SE50/110.

Soy flour was manufactured by Sobo Naturkost (Cologne, Germany); oatmeal flour

was purchased from a local farmer market. Set of paper discs used for determination

of antibiotic spectra was purchased from Himedia (India). All chemicals and reagents

were purchased from Sigma-Aldrich (St. Louis, MO, USA), VWR (Radnor, PA, USA),

and Merck (Darmstadt, Germany), if not stated otherwise.

All restriction endonucleases, used for the verification of plasmids, were purchased

from NEB (Ipswich, MA, USA). All PCR reactions were set up with Phusion High-

Fidelity PCR Master Mix (GC Buffer; NEB, Ipswich, MA, USA).

2.2 Determination of a spore count

Determination of a spore count was regularly done as described in Ostash et al.,

2013 with minor modifications. Strains were grown on SFM for 6 days. Spore
8
suspensions were collected using sterile water and filtered through non-adsorbent

cotton wool. Then 10-fold dilutions of the suspensions were prepared down to 10-6

spore titer. 0.1 ml of all dilutions was usually plated. Colonies on each plate were

counted after six days of incubation.

2.3 Preparation of scanning electron images (SEM) of Actinoplanes sp. SE50/110

colonies

Single Actinoplanes colonies were grown for six days on SFM medium plates. The

colonies were cut out with the layer of agar and were mounted on glass cover slips.

These were placed in a self-build chamber over 4% osmium tetroxide solution for 12

hours. The fixed specimens were lyophilized, fixed to SEM carriers with double sided

tape and covered with a 30 nm layer of gold in a BAL-TEC sputter coater (SCD 005).

Prepared colonies were analyzed in an S-450 scanning electron microscope (Hitachi)

at 15 kV.

2.4 Determination of antibiotic resistance spectra

Antibiotic disc diffusion method was used to study the antibiotic resistance profiles of

Actinoplanes, according to the instructions of the disc manufacturer (Himedia, India).

Spore suspensions were prepared as described above. Then 10-fold dilutions of the

suspensions were prepared down to 10-6 spore titer and 0.1 ml of all dilutions was

usually plated on SFM plates. Discs were placed in sets of four on freshly plated

lawns of Actinoplanes. Growth inhibition zones were measured after five days of

incubation.

To define specific survival rates of Actinoplanes sp. SE50/110 on selected antibiotic

concentrations, freshly prepared spore suspensions were used to make stepwise

dilutions as described above and 0.1 ml of all dilutions was usually plated on SFM

plates with and without antibiotics. Colonies on each plate were counted after 6 days
9
of growth and reference (100%) survival percentage was calculated from control

plates (absence of the antibiotic).

2.5 Escherichia coli Actinoplanes conjugation

Protocols, described in Kieser et al., 2000, Horbal et al., 2012 and Ha et al., 2008

were used for development of this protocol. For setting conjugation matings, freshly

grown plates of Actinoplanes sp. SE 50/110 (SFM media, 5-6 days, 28 C), which

were showing signs of sporulation (e.g. change of a lawn colour from deep orange to

white), were used. Surface of plates was washed with 2.5 mL of sterile water or LB

and carefully scratched with sterile cotton-tipped swabs. The spore/mycelia

suspensions of all plates were mixed and separated as 1 mL of suspension per one

sterile tube. Culture of the donor E. coli ET12567 (pUZ8002), containing different

integrative vectors, was grown in the presence of 50 mg apramycin/L, 25 mg

chloramphenicol/L and 50 mg kanamycin/L to an OD600 of 0.4 0.6. To remove the

antibiotics, cells were washed once with an equal volume of sterile water or LB, and

finally resuspended in 1 mL of LB. Both host and donor cells were centrifuged shortly

at 8.000 x g for 2 minutes. The supernatant was discarded and the remained pellets

were mixed in correlation Actinoplanes:E.coli = 2:1. Mixtures were carefully

resuspended by gentle pipetting up and down and plated on fresh SFM plates. SFM

plates, used for conjugation were usually additionally dried for several hours before

usage at 37. After 20 - 22 hours of incubation, plates were overlaid with 1 mL of

sterile water, containing 1 mg of apramycin and 1 mg of fosfomycin and allowed to

dry. After 7 days of further incubation, Actinoplanes colonies were transferred to fresh

SFM plates, supplemented with apramycin. Subsequently, transconjugants were

purified from E. coli ET12567 with the help of two approaches. Single selected

Actinoplanes colonies were repeatedly re-transferred to new SFM plates until no E.

10
coli cells could be visually observed on the surface. Further, single colonies of

Actinoplanes were used for inoculation in NBS liquid medium. If still present, the

faster growing E.coli would overgrow Actinoplanes sp. SE50/110, which could be

easily detected through visual inspection. In another approach, single sporulating

Actinoplanes colonies were inoculated in liquid maltose minimal media,

supplemented with 50 mg/L of apramycin, fosfomycin and acarbose and incubated at

28 C for 48 hours (160 rpm). 0.1 mL of grown cultures was used for step wise

dilution with sterile water down to approximetely 10-6 cell count and plated on a series

of SFM plates, which were further incubated for 4-6 days. Single colonies of

Actinoplanes transconjugants were selected and inoculated in NBS media to check

the absence of E. coli cells as described above. Purified colonies of Actinoplanes

were deposited as glycerol cultures for long time storage at -80 C.

2.5 Screening of Actinoplanes colonies after conjugation and their characterization

After conjugation, selected colonies of Actinoplanes transconjugants were tested with

the help of PCR. For this purpose, genomic DNA was isolated and two sets of

primers were used, binding to internal regions of aac3(IV) gene and oriT region of all

vectors respectively (Table S1, Fig. S3).

PCR products were purified with QIAquick Gel Extraction Kit(Qiagen) and submitted

for in house sequencing.

The phenotypic characterization of Actinoplanes transconjugants was done by plating

them on SFM plates.

For determination of acarbose biosynthetic activity, all strains were grown in

triplicates in parellal (maltose minimal media, 140 rpm). Maltose minimal media was

inoculated with freshly prepared spore suspensions. For this purpose, firstly

Actinoplanes sp. SE50/110 was plated at SFM and incubated for 6 days until
11
showing clear signs of sporulation. Spore suspensions, prepared as described

above, were mixed and used for inoculation in correspondance of 1 mL per flask.

Every 48 hours, 1 mL of growing cultures was collected and centrifuged down at

20,000 x g for 2 minutes. Supernatant was carefully separated from the pellet and

stored at -20C. The rest of the pellet was washed twice with distillated water and

dried at 70C. Dried pellets were weighed and used to estimate a cell dry weight.

Acarbose measurments were carried out via high performance liquid chromatography

(HPLC) as described in Wendler et al., 2013.

The stability of plasmid inheritance was studied as described in Horbal et al., 2012

with minor modifications. After 5 passages at 28 C under nonselective conditions the

transconjugants were plated onto SFM and allowed to sporulate. The spores were

harvested and plated out in the presence and absence of apramycin. For each type

of transconjugant, 300 colonies were checked for apramycin resistance and this trait

was found to be 100% stable for all studied vectors.

2.6 Plasmid and genomic DNA Isolation

For plasmid isolation E. coli DH5 cultures, carrying respective plasmids, were

cultivated in LB media at 37 C for 12 hours. Further on, GeneJET Plasmid Mniprep

Kit (Thermo Fisher Scientific) was used accordingly to manufacturer advises. Isolated

plasmid DNA was dissolved in water and stored at -20C.

For the isolation of genomic DNA a conventional method described in Kieser et al.,

2000 was used.

2.7 DNA sequencing, sequence and insertion analysis

For library preparations of three derived Actinoplanes transconjugants, a Nextera

DNA Sample Prep Kit (Illumina, San Diego, CA, USA) was used. Resequencing was
12
performed on Illumina MiSeq System (Illumina, San Diego, CA, USA) using the

MiSeq Reagent v3 Kit (Illumina, San Diego, CA, USA). The paired-end sequencing

run produced reads with a length of 2x300 bp. In the next step, the read quality was

checked with the program FASTQC (Andrews 2010). After sequencing and

processing of the raw data, de novo assemblies were performed using the GS De

Novo Assembler Version 2.8 (Roche) with default settings. To identify the vector

sequences of pSET152, pSOK804 and pRT801, the contigs were filtered by applying

r2cat (Husemann & Stoye, 2009) and Actinoplanes sp. SE50/110 (Genbank Acc. No.

CP003170) as reference genome. Identified vector sequences were imported into the

software platform GenDB (Meyer et al., 2003) for automatic annotation. Results were

manually refined as described recently (Eikmeyer et al., 2012, Wibberg et al., 2011).

The specific integration sites of vector plasmids were determined by applying in silico

finishing (Wibberg et al., 2011). All sequencing data were deposited at the European

Nucleotide Archive (ENA) under Bioproject PRJEB12637. The vector sequences of

so far unpublished pSOK804 and pRT801 were deposited in Bioproject

PRJEB12637. For all pairwise global sequence alignments EMBOSS Needle

software was used (McWilliam et al., 2013). The alignment was created with

MUSCLE 3.8.31 program, additional analysis was done by software described in

Dereeper et al., 2008.

3. Results and discussion

3.1 Development of genetic engineering toolkit for Actinoplanes sp. SE50/110

3.1.1 Selection of appropriate culture conditions for spore formation in Actinoplanes

sp. SE50/110

As a first step in establishing the gene cloning system, different solid growth media

13
were tested for suitability for growth and sporulation of Actinoplanes sp. SE50/110. A

high level of spore formation is known to be crucial for the successful conjugation

procedure. For our tests, several different media were selected commonly being used

for streptomycetes. These were in detail: minimal, complete and R2 medium as

defined media and soya flour and oatmeal medium with a non-defined composition

(Kieser et al., 2000). Among these media, only soya flour medium (SFM) and oatmeal

medium (OM) were found suitable for strain growth. Notably, Actinoplanes sp.

SE50/110 was not able to grow on the other solid media tested.

Colonies of Actinoplanes sp. SE50/110 on SFM media are small, have a rough

structure and well-defined contours, mostly form a flower-like shape (Fig 1A).

Substrate mycelia have a dark orange coloring, while the background of the plate

exhibits dark brown pigmentation, probably due to melanin formation. Formation of a

yellow or orange pigment of unknown origin by the substrate mycelia can be

regarded as a typical and distinctive feature of the Actinoplanes genus (Parenti and

Coronelli, 1979).

Lawns of Actinoplanes sp. SE50/110 grown on SFM media showed clear signs of

spore formation such as change of colony colour after 3-5 days of incubation (from

dark orange to white) and acquired hydrophobicity. Therefore, we decided to

accompany these visual observations with electron microscopic studies. Pictures

were made from freshly grown SFM plates with different magnification. It is clearly

seen (Fig. 1C and D), that Actinoplanes sp. SE50/110 is able to form globular and

round shaped sporangia-like structures, which are tightly covering the surface of a

colony. Moreover, these structures were not detected in samples taken from liquid

media cultivations (Fig. S1). No evidences of developed aerial mycelia were found in

all samples taken. Straight, linear structures, resembling sporangiophores, are

formed directly from vegetative mycelia. These observations are in a good agreement
14
with other studies, which have shown similar structures for other members of

Actinoplanaceae (Parenti and Coronelli, 1979; Vobis 2006; Yushchuk et al., 2016). It

is expected, that members of the genus Actinoplanes form zoospores which, once

released from the submerged sporangia, are able to swim to the surface in order to

colonize distantly located environments (Vobis, 2006; Uchida et al., 2011). In the

course of this study, detection of spores was not feasible due to the resolution

limitations.

To define spore titers on various media, lawns of Actinoplanes sp. SE50/110 were

harvested and stepwise tenfold dilutions were plated for counting of colony forming

units. Estimated spore counts reached from 20.510 7 to 30.5106 spores per mL

for SFM and OM media, respectively. Since SFM media was found to be favorable

for sporulation, it was used in following experiments.

3.1.2 Determination of antibiotic resistance spectra to identify suitable markers for

gene transfer

For a gene cloning system, it is mandatory to obtain an antibiotic resistance profile of

the host strain. At first, a disc diffusion method (Ostash et al., 2013) with a standard

set of 18 antibiotics with various modes of action (e.g. -lactams, aminoglycosides,

macrolides, tetracyclines, polymyxines) was used. Detailed results of this experiment

can be found in Table S1. It can be summarized, that with such traits, as resistance to

-lactams (ampicillin), sensitivity to aminoglycosides (streptomycin, kanamycin,

gentamycin) and macrolides (erythromycin), Actinoplanes sp. SE50/110 is a typical

representative of the class Actinobacteria. Also, some specific traits were identified,

such as resistance to low concentrations of spectinomycin, cefpirome, colistin and

trimethoprim. From the tested set, several antibiotics were tested additionally, in

order to determine a specific sensitivity of Actinoplanes sp. SE50/110 for them, in

15
concentrations routinely used for selection. For this purpose, tenfold dilutions of the

strain spore suspension were grown on different antibiotic concentrations and

compared to the media without antibiotic supplement (Table 1).

Apparently, thiostrepton cannot be used for selection in case of Actinoplanes sp.

SE50/110, since the strain shows a high level of resistance towards it. With the

exception of thiostrepton, all other antibiotics studied (kanamycin, hygromycin and

apramycin) appeared to be promising as antibiotic markers for selection.

Interestingly, Actinoplanes sp. SE50/110 is sensitive to nalidixic acid, therefore this

antibiotic cannot be used for selection against Escherichia coli in conjugation

matings. As an alternative in this case, fosfomycin can be utilized, since Actinoplanes

sp. SE50/110 is resistant to high concentrations of this antibiotic.

3.1.3 Adaptation and optimization of the intergeneric conjugation system between E.

coli and Actinoplanes sp. SE50/110

As the conjugation system was already shown to be highly effective for some strains

of the genus Actinoplanes (e.g. A. teichomyceticus; Ha et al., 2008; Horbal et al.,

2012), we decided to adapt reported protocols for Actinoplanes sp. SE50/110.

In a first trial, the procedure described by Horbal et al., 2012 was followed. Later on,

several steps were modified regarding specific traits of Actinoplanes sp. SE50/110.

Further adjustments towards optimal temperature, time of incubation, amount and

correlation of donor/acceptor cells were made (data not shown). All tests towards

higher conjugation frequency were performed with the vector pSET152.

Selection of an appropriate medium was frequently reported to greatly influence the

successful conjugal transfer in Actinobacteria. It is generally expected, that medium,

supporting higher sporulation titers, results in higher transformation frequencies. In

this study, only two solid media supporting growth and sporulation were identified,
16
SFM and OM. Both of them were tested in conjugation experiments and showed

conjugation frequencies of 3.00.510-3 and 2.50.510-6 for SFM and OM,

respectively, thus confirming abovementioned assumptions.

To increase efficiency of conjugation for streptomycetes, it is generally recommended

to supplement media with 10 mM MgCl2 (Kieser et al., 2000). However, functions and

role of MgCl2 in this process are not yet fully understood (Kieser et al., 2000). For A.

teichomyceticus, an optimal concentration of MgCl2 in media was found to be 40 mM

(Ha et al., 2008). We have tested addition of 10 mM MgCl2 to SFM and OM media,

but found no influence on conjugation frequencies .

Spore suspensions, harvested for conjugation, are often being subjected to heat

shock (short incubation at 50 C - 55 C), since spore germination, which can be

induced by heating, was reported to increase conjugation rates (Mazodier et al.

1989). As it was described previously, spores of Actinoplanaceae are motile (Uchida

et al., 2011) and expected to be sensitive to a heat shock procedure (Ha et al.,

2008). For Actinoplanes sp. SE50/110, we observed that incubation of freshly

harvested spore suspension for 15 min at 50 C led to an almost halved amount of

vital spores (from 30.5107 to 20.5103). Therefore, this step was not included into

the conjugation procedure.

We observed that the incubation time for Actinoplanes-E. coli conjugation plates has

to be extended up to 20-22 hours to receive vital colonies, since no colonies were

observed after 16-18 hours of incubation (as described by Ha et al., 2008 for A.

teichomyceticus). Since Actinoplanes sp. SE50/110 was found to be sensitive to

nalidixic acid, only fosfomycin and apramycin were used as selective agents against

E. coli and added to an overlay solution. After 4-6 days of incubation, following an

overlay of the agar plates with the respective antibiotic mixture, single transconjugant

colonies were transferred to new SFM plates, supplemented with apramycin.

17
However, resulting Actinoplanes transconjugants still contained considerable

amounts of E. coli cells. In order to obtain clean cultures, we took advantage of

acarbose. As described in Brunkhorst et al., 1999, acarbose can specifically affect

growth of E. coli K-12 on maltose as the sole source of carbon and energy -

conditions that induce the maltose system. This can be explained, that due to its

structural similarity to maltotetraose, acarbose is being recognized as a substrate by

the maltose/maltodextrin system of E. coli. Single colonies of Actinoplanes were

transferred to liquid maltose minimal media, supplemented with 50 mg/L of

apramycin, fosfomycin and acarbose and cultivated for 48 hours at 28 C. After the

end of cultivations, cultures were stepwise diluted with sterile water and plated on

SFM plates. Single colonies of E. coli free Actinoplanes transconjugants were

transferred to another media and used for further tests. A general overview on the

developed Actinoplanes - E. coli intergeneric conjugation procedure is depicted in

Fig. S2; the final protocol, can be found in the Methods section.

3.2 Application of the developed gene cloning system for the genetic engineering of

Actinoplanes sp. SE50/110

3.2.1 Transfer of a set of actinophage-based integrative vectors to Actinoplanes sp.

SE50/110 and characterization of transconjugant strains

Since there was no data available on which vectors are applicable for Actinoplanes

sp. SE50/110, we decided to include different integration systems into this

experiment. Four integrative vectors: pSET152 and pIJ6902, pSOK804, pRT801

based on three phage integration systems: C31, VWB and BT1 were chosen. All

of them are widely used for the representatives of Streptomyces genus and were

shown to be applicable for A. teichomyceticus (Horbal et al., 2012). All of these

18
plasmids contain the apramycin resistance marker gene aac(3)IV, which was

selected because of the high sensitivity of Actinoplanes sp. SE50/110 to apramycin.

As donor strain in all conjugations, methylation deficient Escherichia coli ET12567

(pUZ8002) containing different single-copy integrative vectors was used. In all sets of

matings, transconjugant colonies with high conjugation frequencies were received for

all vectors tested (Table2). Authenticity of transfconjugant colonies was checked with

the help of PCR, utilizing primers to internal parts of the vector (oriT regions and

aac3(IV) gene;Fig. S3).

According to recent studies, integrative vectors are able to exist as

extrachromosomal non-replicating forms in mycelia. For example, pSET152 was

detected extrachromosomally in cells of S. ghanaensis and other Actinobacteria

(Ostash et al., 2007, Kieser et al., 2000). To verify such a possibility, total DNA of

Actinoplanes colonies bearing pSET152, pIJ6902, pSOK804 or pRT801 was used to

transform E. coli in order to check for the appearance of apramycin-resistant

colonies. Since no such colonies were received in all cases, we can conclude that

these vectors can exist only integrated in the Actinoplanes genome.

For all further implementations of a gene-cloning system such as gene

overexpression experiments, complementation of mutants, it is important to verify the

stability of integrative vectors in cells under non-selective conditions. To study the

stability of plasmid inheritance, transconjugant colonies were grown for five passages

at 28 C without application of an antibiotic. For each type of transconjugant, 300

colonies were checked for apramycin resistance and this trait was found to be 100%

stable for all studied vectors.

It is known, that some vectors can affect the morphology and/or the secondary

metabolism of certain actinomycetes. For example, the presence of pSET152 in the

cells of A. teichomyceticus was shown to significantly increase teicoplanin production

19
(Horbal et al., 2012). Therefore, colony morphology, spore titers and acarbose

production levels of all transconjugants were assessed. Regarding morphology, no

changes were observed by visual inspection in case of all received transconjugant

strains (Fig. S4). Also, sporulation capabilities of transconjugants are not changed

considerably in comparison with the wild type strain from 30.2107 for the wild type

strain to 40.5107 for the pSET152 transconjugant, respectively. Most importantly,

there was no or only little influence of all studied vectors on strain growth or on

acarbose production (Fig. 2).

With the help of DNA-DNA hybridization we were able to determine, that for each

vector there is only one unique integration site per genome of Actinoplanes (data not

shown). However, the specific sequences of attB sites in the chromosome of

Actinoplanes sp. SE 50/110 were not known. Therefore, the genomes of pSET152,

pSOK804 and pRT801 transconjugants were completely sequenced and integration

sites were determined by in silico finishing (Wibberg et al., 2013, 2011; Heinl et al.,

2012). In order to ensure, that all the possible variations of insertion sites are

included, DNA of three independently received transconjugants, for each vector

respectively, was mixed before sequencing. Colonies, containing pIJ6902, were

excluded of these analyses, because of pIJ6902 and pSET152 are both based on

C31 integrative system.

3.2.2 Characterization of pSET152, pSOK804, pRT801 integration sites in

Actinoplanes sp. SE50/110 genome

3.2.2.1 Analysis of pSET152 (C31-based) integration-site in the Actinoplanes sp.

SE50/110 chromosome

In Actinoplanes sp. SE50/110, the attBC31 site is located within gene ACPL_6602.

This gene is annotated as coding for a putative pirin homolog, likewise in other

Actinobacteria, such as S. coelicolor, A. teichomyceticus. The role of pirin in bacteria

20
is poorly understood, however it is clearly influencing diverse biological processes. In

eukaryotes, pirin was reported to act mainly as a transcriptional cofactor (Soo et al.,

2007).

Integration of vectors which have an attP site into the attB locus of recipient bacterial

genome is happening via the integrase (int) function. The efficiency of this process,

using the bacteriophage C31 att/int system, was reported to be highly dependent on

the holomogy of attB sites. Sequences of attB sites which show higher homology to

those of S. lividans, result in higher transconjugation efficiencies (Ha et al., 2008).

The nucleotide sequence of Actinoplanes sp. SE50/110 attBC31 site was found to be

most closely related to that of A. teichomyceticus (88.2% identity; 45 out of 51

identical nucleotides) and S. scabies (88.2% identity; 43 out of 51 identical

nucleotides) (Fig. 3). The Actinoplanes sp. SE50/110 attBC31 site is also highly

related to those from S. coelicolor, S. lividans, S. ambofaciens, S. aureofaciens

(80.4% identity; 41 out of 51 matches) and S. ghanaensis (82.4% identity; 42 out of

51 matches). Thus, notable accordance between attB sites across genera could be

observed. Specifically important is the conservation of an attB core sequence,

where the crossover between attB and attP takes place. This site consists of 5'-TT-3'

sequence (Combes et al. 2002).

It was reported, that S. lividans and S. coelicolor both possess three highly active

pseudoattB sites in their genome, which can serve as additional integration sites,

apart from original (Combes et al., 2002). The fourth pseudoattB site for the C31

integration system was recently identified in the genome of S. albus (Bilyk et al.,

2014). In case of Actinoplanes sp. SE50/110, no additional sites were detected.

3.2.2.2 Analysis of the pRT801 (BT1-based) integration-site in Actinoplanes sp.

SE50/110

21
The vector pRT801 is based on the attP/attB system of another temperate

bacteriophage related to C31, BT1 (Gregory et al., 2003). In S. coelicolor BT1

integrates into a gene annotated to encode an integral membrane protein that is

unrelated to the pirin-like gene used for C31 integration (Baltz et al., 2012).

However, attB sites for BT1 are comparatively poorly studied; there are no known

sequences in Actinoplanaceae or other rare actinomycetes (Gregory et al., 2003,

Baltz et al., 2012). The best studied attB site of S. coelicolor lies within gene

SCO4848, homologs of which exist in the Actinoplanes sp. SE50/110 genome.

Interestingly, in Actinoplanes sp. SE50/110 the attBBT1 site is located in an intergenic

space, between genes ACPL_7730 coding for a dihydrolipoamide dehydrogenase

and ACPL_7731 coding for a Gamma-glutamylcyclotransferase. No other pseudo-

attB sites for BT1 were detected

3.2.2.3 Analysis of the pSOK804 (VWB-based) integration-site in Actinoplanes sp.

SE50/110

To broaden the spectrum of genetic tools applicable for Actinoplanes sp. SE50/110,

the pSOK804 vector (Van Mellaert et al., 1998), that is based on the VWB

bacteriophage integration system, was applied. Previously, pSOK804 was

successfully applied for gene cloning systems in various representatives of the

actinomycetes (A. teichomyceticus, S. sioyaensis, S. venezuelae, S. ghanaensis)

(Van Mellaert et al., 1998; Ostash et al., 2009; Myronovskyy et al., 2009; Horbal et

al., 2012). However, only two attBVWB sites have been characterized so far in S.

venezuelae ETH14603, which is a natural host for VWB phage, and in S. ghanaensis

(Van Mellaert et al., 1998; Ostash et al., 2009). Nothing is known about the genomic

context and nature of attBVWB loci in other strains (Luzhetskyy et al., 2006; Ostash et

al., 2009).
22
Both S. venezuelae and S. ghanaensis attBVWB sites are embedded into a putative

tRNAArg (AGG) gene (Ostash et al., 2009). Although such gene exists in the genome

of Actinoplanes sp. SE50/110, the attBVWB site is not located in the tRNAArg gene, but

in the coding region of the hypothetical protein ACPL_7821. These results suggest a

variability among attBVWB across species, however to make any conclusions, more

information regarding attBVWB is needed.

3.2.3 Adaptation of the GUS reporter system for use in Actinoplanes sp. SE50/110

After the successful development of a genetic engineering toolkit and the application

and characterization of integration sites of different integrative vectors, we would like

to investigate the applicability of the -glucuronidase GUS reporter system for

genetic studies in Actinoplanes sp. SE50/110. In Horbal et al., 2013 utilization of

GUS reporter system for A. teichomyceticus was shown. Therefore, we decided to

transfer the pSETGUS vector, a derivative of pSET152 (Myronovskyi et al., 2011), to

Actinoplanes sp. SE50/110. It contains the gusA gene, coding for the -

glucuronidase enzyme, under the control of the thiostrepton-inducible tipA promoter.

Within a set of conjugation experiments, the pSETGUS vector was transferred to

Actinoplanes sp. SE50/110. Transconjugant strains were verified with the help of

PCR. To prove the activity of the gusA gene, transconjugant strains were grown on

SFM media together with wild type strain and Actinoplanes pSET152. On the sixth

day of growth, drops of 0.1 M XGluc solution were placed onto the surface of lawns

(Fig. 4). In all Actinoplanes pSETGUS transconjugants formation of a blue colour

(5,5-dibromo-4,4-dichloroindigo) was observed under these conditions, while colours

of the wild type strain and its pSET152 transconjugants were unchanged. This fact

not only proves the presence of transferred vectors in Actinoplanes cells, but also

23
shows an absence of any background -glucuronidase enzymatic activity in the wild

type. As it was expected from studies in A. teichomyceticus (Horbal et al., 2013), the

thiostrepton-inducible tipA promoter is suitable for gene expression experiments in

Actinoplanes sp. SE50/110 as well.

The GUS reporter system can now be used for experiments regarding gene

expression analyses in Actinoplanes sp. SE50/110 e.g. determination of promoter

strength through transcriptional or translational gene fusions or further experiments.

Conclusions

Research on acarbose, as a secondary metabolite of Actinoplanes sp. SE50/110,

acquired an extensive body of data on its transcriptomics, proteomics and

metabolomics. Several suggestions regarding possible models of acarbose

metabolism, transport and regulation were made. However, these studies could not

be completed without a suitable genetic proof, e.g. gene deletion and gene

overexpression experiments, conducted in the wild type strain itself. Untill now, such

possibility did not exist, due to the lack of suitable transformation protocol and a set

of genetic tools in Actinoplanes sp. SE50/110. In this publication, we developed a

genetic engineering toolkit and applied the gene cloning system for the transfer of

three different actinophage-based integrative vectors.to Actinoplanes sp. SE50/110.

Studies of transconjugant clones have shown, that all vectors are integrated in single

copy per-genome, are inherited stably over multiple generations and do not influence

Actinoplanes morphology or acarbose production. These results evidently show that

all of them can be used for further genetic experiments.

24
In the course of this work, integration sites for C31, VWB and BT1 bacteriophages

in the Actinoplanes sp. SE50/110 genome were described and characterized for the

first time. C31attB site was found to be homologous to one described previously in A.

teichomyceticus and possessing a typical 5'-TT-3' core sequence. In a case of BT1

and VWB integration sites, no homology to integration sites of other bacteria was

found.

To broaden the set of genetic tools, available for Actinoplanes sp. SE50/110,

applicability of GUS reporter system was studied.

Finally, we proved the applicability of this toolkit for genetic engineering by

transferring the pSETGUS vector to Actinoplanes sp. SE50/110. On pSETGUS the

gusA gene is controlled by the tipA promotor. By adding XGluc onto the lawn patches

of Actinoplanes sp. SE50/110, we could demonstrate the the gusA gene is

expressed. (1) the transfer of the gusA gene serves as an application example for the

genetic engineering of Actinoplanes sp. SE50/110 with the help of actinophage-

based integrative vectors and (2) gusA can be used as a reporter system for the

measurement of successful gene transfer and expression in Actinoplanes sp.

SE50/110.

The developed gene transfer system offers multiple possibilities to help answering

fundamental theoretical and practical questions regarding acarbose production.

Namely, formulation and verification of the complete acarbose metabolism, and its

regulation as well as the development and optimization of acarbose overproducing

strains is now possible by the use of the developed genetic engineering system.

25
 Acknowledgments

The authors wish to thank Anika Winkler and Katharina Hanuschka (CeBiTec,

Bielefeld University) for Sanger and Illumina sequencing. Dr. Udo Wehmeier

(Wuppertal University) and Timo Wolf (CeBiTec, Bielefeld University) are

acknowledged for critical reading of the manuscript. The bioinformatics support of the

BMBF-funded project Bielefeld-Gieen Center for Microbial Bioinformatics BiGi

(Grant number 031A533) within the German Network for Bioinformatics

Infrastructure (de.NBI) is gratefully acknowledged. TG and VO acknowledge financial

support from the Graduate Cluster Industrial Biotechnology (CLIB2021) at Bielefeld

University, Germany. The Graduate Cluster is supported by Bielefeld University and

the Ministry of Innovation, Science and Research (MIWF) of the federal state North

Rhine-Westphalia, Germany. We also acknowledge financial support from Bayer AG

(Leverkusen, Germany).

References

Andrews, S., 2010. FastQC: A quality control tool for high throughput sequence data.
Baltz, R.H., 2012. Streptomyces temperate bacteriophage integration systems for
stable genetic engineering of actinomycetes (and other organisms). J. Ind.
Microbiol. Biotechnol. 39, 661672. doi:10.1007/s10295-011-1069-6
Bierman, M., Logan, R., OBrien, K., Seno, E.T., Rao, R.N., Schoner, B.E., 1992.
Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to
Streptomyces spp. Gene 116, 4349. doi:10.1016/0378-1119(92)90627-2
Bilyk, B., Luzhetskyy, A. 2014. Unusual site-specific DNA integration into the highly
active pseudo-attB of the Streptomyces albus J1074 genome. Appl Microbiol
Biotechnol. 98(11), 5095-104. doi: 10.1007/s00253-014-5605-y.
Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for
Illumina sequence data. Bioinformatics 30, 21142120.
doi:10.1093/bioinformatics/btu170
Brunkhorst, C., Andersen, C., Schneider, E., 1999. Acarbose, a
26
 pseudooligosaccharide, is transported but not metabolized by the maltose-
maltodextrin system of Escherichia coli. J Bacteriol 181(8), 2612-9.
Combes, P., Till, R., Bee, S., Smith, M.C.M., 2002. The Streptomyces genome
contains multiple pseudo-attB sites for the phiC31-encoded site-specific
recombination system. J. Bacteriol. 184, 574652. doi:10.1128/JB.184.20.5746
Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard,
J.F., Guindon, S., Lefort, V., Lescot, M., Claverie, J.M., Gascuel, O. 2008.
Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucl. Acids
Res. 36 (Web Server issue):W465-9.
Eikmeyer, F., Hadiati, A., Szczepanowski, R., Wibberg, D., Schneiker-Bekel, S.,
Rogers, L.M., Brown, C.J., Top, E.M., Phler, A., Schlter, A. 2012. The
complete genome sequences of four new IncN plasmids from wastewater
treatment plant effluent provide new insights into IncN plasmid diversity and
evolution. Plasmid. 68(1), 13-24. doi: 10.1016/j.plasmid.2012.01.011.
Gregory, M.A., Till, R., Smith, M.C.M., 2003. Integration Site for Streptomyces Phage
phiBT1 and Development of Site-Specific Integrating Vectors. J. Bacteriol. 185,
53205323. doi:10.1128/JB.185.17.5320-5323.2003
Ha, H.-S., Hwang, Y.-I., Choi, S.-U., 2008. Application of conjugation using phiC31
att/int system for Actinoplanes teichomyceticus, a producer of teicoplanin.
Biotechnol. Lett. 30, 12338. doi:10.1007/s10529-008-9671-z
Heinl, S., Wibberg, D., Eikmeyer, F., Szczepanowski, R., Blom, J., Linke, B.,
Goesmann, A., Grabherr, R., Schwab, H., Phler, A., Schlter, A., 2012. Insights
into the completely annotated genome of Lactobacillus buchneri CD034, a strain
isolated from stable grass silage. J. Biotechnol. 161(2), 153-66. doi:
10.1016/j.jbiotec.2012.03.007
Heinzelmann, E., Berger, S., Puk, O., Reichenstein, B., Wohlleben, W., Schwartz, D.,
2003. A glutamate mutase is involved in the biosynthesis of the lipopeptide
antibiotic friulimicin in Actinoplanes friuliensis. Antimicrob. Agents Chemother.
47(2):447-57.
Hemker, M., Stratmann, A., Goeke, K., Schrder, W., Lenz, J., Piepersberg, W.,
Pape, H., 2001. Identification, cloning, expression, and characterization of the
extracellular acarbose-modifying glycosyltransferase, AcbD, from Actinoplanes
sp. strain SE50. J. Bacteriol. 183, 44844492. doi:10.1128/JB.183.15.4484
Hilker, R., Stadermann, K.B., Doppmeier, D., Kalinowski, J., Stoye, J., Straube, J.,

27
 Winnebald, J., Goesmann, A., 2014. ReadXplorer--visualization and analysis of
mapped sequences. Bioinformatics 30, 22472254.
doi:10.1093/bioinformatics/btu205
Horbal, L., Kobylyanskyy, A., Yushchuk, O., Zaburannyi, N., Luzhetskyy, A., Ostash,
B., Marinelli, F., Fedorenko, V., 2013. Evaluation of heterologous promoters for
genetic analysis of Actinoplanes teichomyceticus producer of teicoplanin,
drug of last defense. J. Biotechnol. 168, 367372.
doi:10.1016/j.jbiotec.2013.10.018
Horbal, L., Zaburannyy, N., Ostash, B., Shulga, S., Fedorenko, V., 2012.
Manipulating the regulatory genes for teicoplanin production in Actinoplanes
teichomyceticus. World J. Microbiol. Biotechnol. 28, 20952100.
doi:10.1007/s11274-012-1013-6
Huang, J., Shi, J., Molle, V., Sohlberg, B., Weaver, D., Bibb, M.J., Karoonuthaisiri, N.,
Lih, C-J., Kao, CM., Buttner, M.J., Cohen S.N., 2005. Cross-regulation among
disparate antibiotic biosynthetic pathways of Streptomyces coelicolor. Molecular
Microbiology. 58(5), 1276-1287. doi: 10.1111/j.1365-2958.2005.04879.x
Husemann, P., Stoye, J. 2009. r2cat: synteny plots and comparative assembly.
Bioinformatics. 26(4), 570-1. doi: 10.1093/bioinformatics/btp690.
Kieser, T.M., Bibb, J., Buttner, M.J., Chater, K.F., Hopwood, D.A., 2000. Practical
Streptomyces genetics, The John Innes Foundation, Norwich, UK.
Lomovskaya N.D., Mkrtumian N.M., Gostimskaya N.L., Danilenko V.N., 1972.
Characterization of temperate actinophage phi C31 isolated from Streptomyces
coelicolor A3(2). J. Virol. 9(2):258-62.
Luzhetskyy, A., Fedoryshyn, M., Gromyko, O., Ostash, B., Rebets, Y., Bechthold, A.,
Fedorenko, V. 2006. IncP plasmids are most effective in mediating conjugation
between Escherichia coli and streptomycetes. Genetika. 42(5), 595-601.
Marcone, G.L., Carrano, L., Marinelli, F., Beltrametti, F., 2010a. Protoplast
preparation and reversion to the normal filamentous growth in antibiotic-
producing uncommon actinomycetes. J. Antibiot. (Tokyo). 63, 8388.
doi:10.1038/ja.2009.127
Marcone, G.L., Foulston, L., Binda, E., Marinelli, F., Bibb, M., Beltrametti, F., 2010b.
Methods for the genetic manipulation of Nonomuraea sp. ATCC 39727. J. Ind.
Microbiol. Biotechnol. 37, 1097103. doi:10.1007/s10295-010-0807-5
Mazodier, P., Petter, R., Thompson, C., 1989. Intergeneric Conjugation between

28
 Escherichia coli and Streptomyces Species. J. Bacteriol. 171, 35833585.
McWilliam, H., Li, W., Uludag, M., Squizzato, S., Park, Y.M., Buso, N., Cowley, A.P.,
Lopez, R. 2013. Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids
Res. 41(Web Server issue):W597-600. doi: 10.1093/nar/gkt376.
Meyer, F., Goesmann, A, McHardy, A.C., Bartels, D., Bekel, T., Clausen, J.,
Kalinowski, J., Linke, B., Rupp, O., Giegerich, R., Phler, A. 2003. GenDB--an
open source genome annotation system for prokaryote genomes. Nucleic Acids
Res. 31(8), 2187-95.
Myronovskyi, M., Welle, E., Fedorenko, V., Luzhetskyy, A., 2011. Beta-glucuronidase
as a sensitive and versatile reporter in actinomycetes. Appl. Environ. Microbiol.
77, 537083. doi:10.1128/AEM.00434-11
Myronovskyy, M., Ostash, B., Ostash, I., Fedorenko, V., 2009. A gene cloning system
for the siomycin producer Streptomyces sioyaensis NRRL-B5408. Folia
Microbiol. (Praha). 54, 9196. doi:10.1007/s12223-009-0013-x
Ostash, B., Gren, T., Hrubskyy, Y., Tistechok, S., Beshley, S., Baranov, V.,
Fedorenko, V., 2014. Cultivable actinomycetes from rhizosphere of birch (Betula
pendula) growing on a coal mine dump in Silets, Ukraine. J. Basic Microbiol. 54,
8517. doi:10.1002/jobm.201200551
Ostash, B., Makitrinskyy, R., Walker, S., Fedorenko, V., 2009. Identification and
characterization of Streptomyces ghanaensis ATCC14672 integration sites for
three actinophage-based plasmids. Plasmid 61, 1715.
doi:10.1016/j.plasmid.2008.12.002
Parenti, F., Coronelli, C., 1979. Members of the genus Actinoplanes and their
antibiotics. Annu. Rev. Microbiol. 33, 389411.
doi:10.1146/annurev.mi.33.100179.002133
Puls, W., and U. Keup. (1973): Influence of an -amylase inhibitor (BAY d7791) on
blood glucose, serum insulin and NEFA in starch loading tests in rats, dogs and
man. Diabetologia, 9(2), pp.97-101
Sekurova, O.N., Brautaset, T., Sletta, H., Borgos, S.E., Jakobsen, M.M., Ellingsen,
T.E., Strm, A.R., Valla, S., Zotchev, S.B., 2004. In vivo analysis of the
regulatory genes in the nystatin biosynthetic gene cluster of Streptomyces
noursei ATCC 11455 reveals their differential control over antibiotic biosynthesis.
J Bacteriol. 186(5), 1345-54.
Schwientek, P., Szczepanowski, R.,; Rckert, C., Kalinowski, J., Klein, A., Selber, K.,

29
 Wehmeier, U.F., Stoye, J., Phler, A. 2012. The complete genome sequence of
the acarbose producer Actinoplanes sp. SE50/110. BMC Genomics. 13, 112.
doi: 10.1186/1471-2164-13-112
Schwientek, P., Wendler, S., Neshat, A., Eirich, C., Rckert, C., Klein, A., Wehmeier,
U.F., Kalinowski, J., Stoye, J., Phler, A. 2013. Comparative RNA-sequencing of
the acarbose producer Actinoplanes sp. SE50/110 cultivated in different growth
media. J. Biotechnol. 167(2), 166-177 doi:10.1016/j.jbiotec.2012.10.019
Schwientek, P., Neshat, A., Kalinowski, J., Klein, A., Rckert, C., Schneiker-Bekel,
S., Wendler, S., Stoye, J., Phler, A. 2014. Improving the genome annotation of
the acarbose producer Actinoplanes sp. SE50/110 by sequencing enriched 5'-
ends of primary transcripts. J. Biotechnol. 190, 85-95. doi:
10.1016/j.jbiotec.2014.03.013.
Soo, P.C., Horng, Y.T., Lai, M.J., Wei, J.R., Hsieh, S.C., Chang, Y.L., Tsai, Y.H., Lai,
H.C., 2007. Pirin regulates pyruvate catabolism by interacting with the pyruvate
dehydrogenase E1 subunit and modulating pyruvate dehydrogenase activity. J
Bacteriol 189, 109118.
Stinchi, S., Azimonti, S., Donadio, S., Sosio, M., 2003. A gene transfer system for the
glycopeptide producer Nonomuraea sp. ATCC39727. FEMS Microbiol. Lett. 225,
5357. doi:10.1016/S0378-1097(03)00490-7
Truscheit, E., Frommer, W., Junge, B., Mller, L., Schmidt, D.D., Wingender, W.,
1981. Chemistry and Biochemistry of Microbial-Glucosidase Inhibitors. Angew.
Chemie Int. Ed. English 20, 744761. doi:10.1002/anie.198107441
Uchida, K., Jang, M.-S., Ohnishi, Y., Horinouchi, S., Hayakawa, M., Fujita, N.,
Aizawa, S.-I., 2011. Characterization of Actinoplanes missouriensis spore
flagella. Appl. Environ. Microbiol. 77, 25592562. doi:10.1128/AEM.02061-10
Van Dessel, W., Van Mellaert, L., Liesegang, H., Raasch, C., DeKeersmaeker, S.,
Geukens, N., Lammertyn, E., Streit, W., Ann, J., 2005. Complete genomic
nucleotide sequence and analysis of the temperate bacteriophage VWB.
Virology 331, 325337. doi:10.1016/j.virol.2004.10.028
Van Mellaert, L., Mei, L., Lammertyn, E., Schacht, S., Ann, J., 1998. Site-specific
integration of bacteriophage VWB genome into Streptomyces venezuelae and
construction of a VWB-based integrative vector. Microbiology 144 ( Pt 1, 33518.
Vobis, G. 2006 The Genus Actinoplanes and Related Genera. In Martin Dworkin,
Stanley Falkow, Eugene Rosenberg, Karl-Heinz Schleifer, Erko Stackebrandt

30
 (Eds.): The Prokaryotes. New York, NY: Springer New York, pp. 623653
Wehmeier, U.F., 2003. The Biosynthesis and Metabolism of Acarbose in
Actinoplanes sp. SE 50/110: A Progress Report. Biocatalysis and
Biotransformation 21(4/5), 279-284
Wendler, S., Hrtgen, D., Kalinowski, J., Klein, A., Niehaus, K., Schulte, F.,
Schwientek, P., Wehlmann, H., Wehmeier, U.F., Phler, A., 2013. The cytosolic
and extracellular proteomes of Actinoplanes sp. SE50/110 led to the
identification of gene products involved in acarbose metabolism. J. Biotechnol.
167, 178189. doi:10.1016/j.jbiotec.2012.08.011
Wendler, S., Ortseifen, V., Persicke, M., Klein, A., Neshat, A., Niehaus, K.,
Schneiker-Bekel, S., Walter, F., Wehmeier, U.F., Kalinowski, J., Phler, A.,
2014. Carbon source dependent biosynthesis of acarviose metabolites in
Actinoplanes sp. SE50/110. J. Biotechnol. 191, 113120.
doi:10.1016/j.jbiotec.2014.08.019
Wendler, S., Otto, A., Ortseifen, V., Bonn, F., Neshat, A., Schneiker-Bekel, S.,
Walter, F., Wolf, T., Zemke, T., Wehmeier, U.F, Hecker M., Kalinowski, J.,
Becher, D., Phler, A.. 2015: Comprehensive proteome analysis of Actinoplanes
sp. SE50/110 highlighting the location of proteins encoded by the acarbose and
the pyochelin biosynthesis gene cluster. J. Proteomics 125, 116. doi:
10.1016/j.jprot.2015.04.013
Wendler, S., Otto, A., Ortseifen, V., Bonn, F., Neshat, A., Schneiker-Bekel, S. Wolf,
T., Zemke, T., Wehmeier, U.F., Hecker, M., Kalinowski, J., Becher, D., Phler,
A.2016 Comparative proteome analysis of Actinoplanes sp. SE50/110 grown
with maltose or glucose shows minor differences for acarbose biosynthesis
proteins but major differences for saccharide transporters. J. Proteomics. 131,
140-148. doi:10.1016/j.jprot.2015.10.023
Wibberg, D., Blom, J., Jaenicke, S., Kollin, F., Rupp, O., Scharf, B., Schneiker-Bekel,
S., Sczcepanowski, R., Goesmann, A., Setubal, JC., Schmitt, R., Phler, A.,
Schlter, A., 2011. Complete genome sequencing of Agrobacterium sp. H13-3,
the former Rhizobium lupini H13-3, reveals a tripartite genome consisting of a
circular and a linear chromosome and an accessory plasmid but lacking a tumor-
inducing Ti-plasmid. J. Biotechnol. 155(1), 50-62. doi:
10.1016/j.jbiotec.2011.01.010
Wibberg, D., Blom, J., Rckert, C., Winkler, A., Albersmeier, A., Phler, A., Schlter,

31
 A., Scharf, BE., 2013. Draft genome sequence of Sinorhizobium meliloti
RU11/001, a model organism for flagellum structure, motility and chemotaxis. J
Biotechnol 168(4), 731-3. doi: 10.1016/j.jbiotec.2013.10.015
Yushchuk, O., Horbal, L., Datsyuk, Ju., Stegmann, E., Fedorenko, V. 2016.
Peculiarities of Actinoplanes teichomyceticus NRRL-B16726 morphology and life
cycle. Visnyk of the Lviv University. Series Biology 03/2016
Zhang, C.-S., 2002. Biosynthesis of the C7-cyclitol Moiety of Acarbose in
Actinoplanes sp. SE50/110. 7-O-phosphorylation of the initial cyclitol precursor
leads to proposal of a new biosynthetic pathway. J. Biol. Chem. 277, 22853
22862. doi:10.1074/jbc.M202375200

32
Figure ledends

Fig. 1 Morphological features of Actinoplanes sp. SE50/110 colonies grown on SFM

medium (B,C and D scanning electron microphotographs). A flower shaped,

round single and joined colonies; B several tandemly joined colonies. Region,

identified by square is magnified and shown in C; C colony surface, abundantly

covered with sporangia-like structures. Region, identified by square is magnified and

shown in D; D colony surface with typical structures, presumably round sporangia

and substrate mycelia, which are indicated with arrows in a left center part and right

corner of the picture respectively

33
Fig. 2 Acarbose concentration for cultivations of Actinoplanes sp. SE50/110 wildtype

without a plasmid and the pSET152, pSOK804, pRT801 transconjugant strains,

grown in maltose minimal media. A recorded values for acarbose concentration,

g/L; B recorded values for cell dry weight, g/L. Mean values and standard

deviations are shown for three biological replicates.

34
Fig. 3 Alignment of the attB site sequences among Actinoplanes sp. SE50/110 and

other actinomycetes: setae - Kitasatospora setae KM-6054; sp. SE50/110 -

Actinoplanes sp. SE50/110; teichomyceticus - A. teichomyceticus NBRC13999;

aureofaciens S. aureofaciens ATCC10762; scabies S. scabies NRRL B-16523

B16523; ambofaciens S. ambofaciens ATCC23877; lividans S. lividans 66 TK64;

coelicolor S. coelicolor A3(2); acidiscabies S. acidiscabies; hygroscopicus S.

hygroscopicus NRRL5491; ghanaensis S. ghanaensis ATCC14672; rimosus S.

rimosus R7. The alignment was created with MUSCLE 3.8.31 program and by

software described in Dereeper et al., 2008; core sequence is marked with XX sign.

35
Fig. 4. Lawns of Actinoplanes sp. SE50/110 and its transconjugants with drops of 0.1

M XGluc solution, placed on the top center of the surface.1, 2 - Actinoplanes

pSETGUS; 3 - Actinoplanes pSET152; 4 - Actinoplanes sp. SE50/110

36
Tables

Table 1. Antibiotic resistance spectra of Actinoplanes sp. SE50/110 strain after six
days of growth on SFM plates
Concentration, Survival, Possibility of application
Name
g/l % as a marker
Ampicillin 100 0 +
Apramycin 50 0 +
Hygromycin 50 0 +
Kanamycin 50 0 +
Nalidixic acid 100 0 Not considered
50 98
Fosfomycin Not considered
100 100
Thiostrepton 50 82 -
100 69 -
Viomycin 30 0 +

Table 2. Overview of integrative vectors used in conjugation experiments

Number Vector name Integrative Transformation Reference
system frequency
1. pSET152 C31 3.30.5 10-3 Bierman et al.,
1992
2. pIJ6902 C31; contains 3.80.5 10 -3 Huang et al.,
promoter tipAp 2005
3. pRT801 BT1 4.50.2 10 -3 Gregory et al.,
2003
4. pSOK804 VWB 2.40.4 10-3 Sekurova et al.,
2004

37