Download Application of the new manP counter-selection system for B. subtilis

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Development of a markerless gene deletion system for Bacillus subtilis based
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on the mannose phosphoenolpyruvate-dependent phosphotransferase system
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Marian Wenzel and Josef Altenbuchner*
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Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart,
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Germany
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Corresponding author: Josef Altenbuchner
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E-mail: [email protected]
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Tel. ++49 711 685 67591
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Fax: ++49 711 685 66973
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Running title:
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Markerless gene deletion system for Bacillus subtilis
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Keywords:
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PTS, genome engineering, counter-selection marker, mannose degradation
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Abstract
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To optimize Bacillus subtilis as a production strain for proteins and low molecular
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substances by genome engineering, we developed a markerless gene deletion
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system. We took advantage of a general property of the phosphoenolpyruvate-
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dependent phosphotransferase system (PTS), in particular the mannose PTS.
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Mannose is phosphorylated during uptake by its specific transporter (ManP) to
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mannose 6-phosphate which is further converted to fructose 6-phosphate by the
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mannose 6-phosphate isomerase (ManA). When ManA is missing, accumulation of
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the phosphorylated mannose inhibits cell growth. This system was realized by
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deletion of manP and manA in B. subtilis 6, a 168 derivative strain with 6 large
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deletions of prophages and antibiotic biosynthesis genes. The manP gene was
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inserted into an E. coli plasmid together with a spectinomycin resistance gene for
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selection in B. subtilis. To delete a specific region, its up- and downstream flanking
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sites (each approx. 700 bps) were inserted into the vector. After transformation,
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integration of the plasmid into the chromosome of B. subtilis by single cross-over was
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selected by spectinomycin. In the second step, excision of the plasmid was selected
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by growth on mannose. Finally, excision and concomitant deletion of the target
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region was verified by Colony PCR. In this way, all 9 prophages, 7 antibiotic
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biosynthesis gene clusters, 2 sigma factors for sporulation were deleted and the
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B. subtilis genome was reduced from 4,215 to 3,640 kb. Despite these extensive
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deletions, growth rate and cell morphology remained similar to the B. subtilis 168
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parental strain.
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Introduction
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Bacillus subtilis is an important producer of industrial enzymes, such as amylases
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and proteases, with favorable features like high protein secretion capability, GRAS
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(generally recognized as safe) status, natural competence, a long tradition in
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fermentation and a deep knowledge of its genetics, physiology and biochemistry. Up-
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to-date information is available on SubtiWiki (Michna et al., 2014). Strain optimization
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to remove adverse properties like sporulation, lysogenic prophages, production of
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antibiotics and extracellular proteases (Krishnappa et al., 2013; Stein, 2005; Westers
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et al., 2003; Westers et al., 2004) or to introduce new metabolic routes, requires
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powerful tools for genetic engineering.
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An easy way for chromosomal deletions is to construct deletion cassettes comprising
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an antibiotic resistance marker flanked by sequences that normally flank the region to
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be deleted. The successful integration of the cassette via homologous recombination
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is selected by the antibiotic resistance. For multiple modifications of the
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chromosome, recognition sites for site-specific recombinases, such as Xer / dif, Flp /
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FRT or Cre / loxP (Bloor & Cranenburgh, 2006; Hoang et al., 1998; Marx & Lidstrom,
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2002), allow the precise excision of the marker gene, and the cassette can be used
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again. The risk of unintended large chromosomal deletions or inversions can be
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avoided by using chimeric recognition sites like lox71/lox66 or mroxP (Warth &
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Altenbuchner, 2013a; Warth & Altenbuchner, 2013b). However, a second
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transformation and curing of the plasmid providing the recombinase is needed. Last
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but not least, all such methods leave ‘scars’ in the chromosome.
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The development of markerless systems deals with this problem. Here, the antibiotic
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resistance gene is located outside the cloned flanking sequences on a suicide vector.
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The vector is integrated via single cross-over recombination. Spontaneous excision
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of the vector either leads to removal of just the vector or to removal of the vector and
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the target region. However, this is a rare event. By integration of a -galactosidase
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gene into the vector it is possible to detect such rare events by blue-white screening
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but this needs strains lacking -galactosidase activity and the screening of thousands
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of colonies (Leenhouts et al., 1996). Therefore, counter-selection marker systems
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have been developed, like the popular fusaric acid (tetAR), streptomycin (rpsL), and
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sucrose (sacB) sensitivity systems (reviewed by Reyrat et al., 1998). Another one
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takes advantage of the endonuclease I-SceI of Saccharomyces cerevisiae (Martínez-
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Garcia & de Lorenzo, 2011; Monteilhet et al., 1990). However, the application of
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these systems has not been proven effective or even applicable in B. subtilis due to
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natural resistances, lack of suitable vectors etc.
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A rather powerful system based on the sensitivity against 5-fluorouracil (5-FU; upp)
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has been developed for B. subtilis and successfully adapted for several other
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microorganisms (Fabret et al., 2002; Goh et al., 2009; Graf & Altenbuchner, 2011;
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Keller et al., 2009; Kristich et al., 2005; Tanaka et al., 2013). Besides the toxicity of 5-
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FU, a major disadvantage is the occurrence of diverse suppressor mutations as
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already stated by Fabret et al. (2002). Other systems based on the action of toxins
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and antitoxins such as CcdB and MazF from E. coli (Bernard et al., 1994; Zhang et
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al., 2006) require a perfect and sensitive toxin/antitoxin equilibrium. As these systems
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deal with toxic molecules, the occurrence of suppressor mutations is also very likely.
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Alternatively, temperature-sensitive plasmids allow selection of their integration at
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their non-permissive temperature. Returning to its permissive temperature, the
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plasmid starts replicating again, and excision is selected (Arnaud et al., 2004;
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Zakataeva et al., 2010). However, the cells might also survive by slow growth and
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one has to make sure that the plasmid was indeed integrated.
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Thus, the need for a less toxic, but powerful and reliable counter-selection system for
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B. subtilis in order to deal with the upcoming questions from large scale omics-data
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and analyses is obvious and necessary (Buescher et al., 2012; Goelzer et al., 2008;
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Kobayashi et al., 2001; Petersohn et al., 2001; Price et al., 2001; Yoshida et al.,
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2001). In this study, we describe the development and application of a novel,
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markerless method for multiple subsequent gene deletions in B. subtilis. We took
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advantage of a general property of the phosphoenolpyruvate-dependent
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phosphotransferase system (PTS), and particularly the mannose PTS. With this
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method we were able to construct a strain without several adverse properties and a
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reduced genome. The resulting strain was examined concerning its growth rate.
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Materials and Methods
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Plasmids, bacterial strains and growth conditions
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Relevant bacterial strains and plasmids used in this study are summarized in Table 1
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and Table S1. Standard recombinant DNA techniques were used (Sambrook et al.,
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1989). Cloning steps were performed with E. coli JM109 (Yanish-Perron et al., 1985).
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E. coli JM109 was transformed with plasmid DNA using the TSS heat shock method
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as described before (Chung et al., 1989). B. subtilis was transformed according to
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the modified “Paris method” (Harwood & Cutting, 1990). All strains were grown at
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37°C, using LB medium (Bertani, 1951) or MG1. The MG1 minimal medium used for
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the first of the two-step growth phase for competence development and for
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comparing the growth rates of B. subtilis contained per 1 liter: 2 g (NH4)2SO4, 6 g
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KH2PO4, 14 g K2HPO4, 1 g Na-citrate, 0.2 g MgSO4, 5 g glucose, 200 mg casamino
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acids, 50 mg tryptophan (if necessary) and 1 ml of a 1000-fold trace element solution
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(TES; Wenzel et al., 2011). Antibiotics were used in the following concentrations:
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ampicillin (amp), 100 µg ml-1; spectinomycin (spc), 100 µg ml-1; erythromycin (erm) 5
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µg ml-1.
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Materials
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Mannose was purchased from AMRESCO LLC (Solon, Ohio, USA). DNA modifying
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enzymes including restriction endonucleases were purchased from New England
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Biolabs GmbH (Frankfurt am Main, Germany). PCRs were run with High Fidelity PCR
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Enzyme Mix and DreamTaq DNA polymerase (Thermo Fisher Scientific GmbH,
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Schwerte, Germany) on a TPersonal Thermocycler (Biometra GmbH, Göttingen,
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Gemany). Synthetic DNA oligonucleotides (Table S2) were purchased from Eurofins
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MWG Operon GmbH (Ebersberg, Germany). DNA sequencing was performed by
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GATC Biotech AG (Constance, Germany).
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Construction of plasmids pJOE6743.1 and pJOE6577.1
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The plasmid pJOE6743.1 is derived from pJOE4786.1 (Jeske & Altenbuchner, 2010).
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The spectinomycin resistance gene was isolated from pMW168.1 (Wenzel et al.,
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2011) by endoR AgeI and EcoRI, the protruding ends of the fragment filled in by
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Klenow polymerase treatment and inserted into the SfoI site of pJOE4786.1
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(pJOE6562.4). The manP gene together with its promoter was amplified from B.
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subtilis 168 by PCR using primers s6477 and s6041, cleaved with NdeI and inserted
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into the NdeI site of pJOE6562.4 to give pJOE6743.1. The plasmid pJOE6577.1 is
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derived from pSUN346.1 (Sun & Altenbuchner, 2010). The plasmid pSUN346.1
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again is derived from pJOE4786.1 and contains the C-terminal end of manR, the
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manA gene, followed by an erythromycin resistance gene (erm), ydjF and a
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spectinomycin resistance gene (aad9). The manA gene was removed by cutting
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pSUN346.1 with BamHI and PmeI and after Klenow treatment ligated to plasmid
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pJOE6577.1.
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Protocol for the chromosomal deletion formation
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Derivatives of pJOE6743.1 with deletion cassettes were used to transform a manPA
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negative B. subtilis strain. Colonies with the integrated plasmid (via single cross-over)
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were selected on LB agar plates containing spectinomycin. Five of the transformants
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were picked and streaked on a new LB agar plate containing spectinomycin. One
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single colony obtained from each of the five transformants was used to inoculate LB
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liquid medium. After incubation at 37°C for about 8 h the cultures were diluted 10-4 in
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LB liquid medium containing 0.5% (w/v) mannose. After incubation at 37°C overnight,
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10-6 dilutions were plated on LB agar plates containing 0.5% (w/v) mannose. The
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mannose in the last two steps helps to select the cells which have lost the integrated
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vector (counter-selection). Four colonies from each plate (20 clones at total) were
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picked and streaked on LB agar plates without and with spectinomycin.
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Spectinomycin sensitive colonies were finally checked by Colony PCR.
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Colony PCR
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After selection for chromosomal plasmid excision, colonies were streaked out on LB
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agar plates. A small amount of cells was taken from these plates and resuspended in
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100 µl H2O (bidest.). After heating the suspension for 10 min at 99°C, a cold shock
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for 20 min at -70°C followed by a further heating step (10 min at 99°C) was
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performed. After centrifugation of the cell suspension, 2 µl of the supernatant
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containing chromosomal DNA was used in a 30 µl PCR preparation using the
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DreamTaq DNA polymerase. As a control, the same procedure was performed with
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two colonies still containing the integrated plasmid.
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Results
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Plasmid and strain construction for manP-based counter-selection
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In order to develop a new efficient counter-selection system for B. subtilis, we took
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advantage of the fact that mannose 6-phosphate dehydrogenase mutants (manA)
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were sensitive to the presence of mannose (Sun & Altenbuchner, 2010; Wenzel et
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al., 2011). Cells developed a bubble like morphology and stopped growing about 1 h
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after addition of mannose to the culture medium (Wenzel et al., 2011). The sensitivity
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against mannose is dependent on the presence of manP and absence of manA. A
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strain without both genes is resistant to mannose. The introduction of manP to such a
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strain would reestablish the sensitivity against mannose. Taking advantage of these
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observations, we developed the idea of using manP as a counter-selectable marker.
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To get this to work one has to delete the manPA genes in the B. subtilis chromosome
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and to develop a vector which i) carries the manP gene, ii) does not replicate in B.
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subtilis, iii) encodes an antibiotic resistance gene for selection in B. subtilis and iiii)
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has restriction sites for cloning of the deletion cassettes. The deletion cassettes, two
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sequences of about 700 bp flanking the target region, enable the integration of the
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vector into the B. subtilis chromosome via single cross-over. This is selected by the
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antibiotic resistance. After the integration, cells are cultured in the presence of
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mannose. Only cells which spontaneously have lost the vector, alone or together with
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the target region, form colonies on mannose-containing agar plates. The colonies are
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screened for loss of the antibiotic resistance and the antibiotic sensitive ones finally
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tested by Colony PCR for loss of the target region.
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To use manP for multiple deletions in the chromosome of B. subtilis the vector
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pJOE6743.1 was constructed (Fig. 1). This is a cloning vector derived from
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pJOE4786.1 (Jeske & Altenbuchner, 2010). Two long inverted repeats separated by
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a lacPOZ fragment allow a blue/white screening in strains like E. coli JM109. When
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the two repeats come together, the plasmid cannot be replicated (Altenbuchner et al.,
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1992). Therefore, a positive selection occurs on the replacement of the lacPOZ
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fragment. The vector is a pUC derivative and carries the ampicillin resistance gene
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for selection in E. coli, a spectinomycin resistance gene for selection in both B.
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subtilis and E. coli as well as the B. subtilis manP gene encoding the mannose PTS
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transporter under control of its own promoter regulated by ManR (Wenzel &
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Altenbuchner, 2013).
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Before this vector can be used in B. subtilis, the manPA genes had to be removed. B.
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subtilis 6, a derivative of B. subtilis 168 with a series of already deleted prophages
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and antibiotic biosynthesis genes was chosen as the starting strain (Westers et al.,
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2003). For an easier handling, the tryptophan auxotrophic strain was transformed
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with chromosomal DNA from B. subtilis 3NA (Michel & Millet, 1970) to replace the
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trpC2 allele present in the B. subtilis 168 derivatives (Table 1; for more details see
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Table S1). To make sure that the new strain designated IIG-Bs1 still contained the
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deletions conducted by Westers et al. (2003) PCRs were performed with primers
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shown in Table S2.
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Next, the manPA genes had to be deleted in the IIG-Bs1 chromosome. This was
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done by cloning the upstream mannose regulatory gene and the downstream yjdF
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gene to both sides of an erythromycin resistance gene in an E. coli pJOE4786.1
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derivative. This plasmid had in addition a spectinomycin resistance gene inserted
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downstream of yjdF (plasmid pJOE6577.1). This plasmid-DNA was used to transform
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B. subtilis IIG-Bs1. The resulting strain designated IIG-Bs2 had the manPA genes
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replaced by an erythromycin resistance gene and served as the initial strain for
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further deletions using the new counter-selection system. Later, the erythromycin
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resistance gene was deleted together with yjdF and several further genes
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downstream of yjdF (IIG-Bs20-1, Table 1 and Table S1).
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Development and application of the new counter-selection method
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First, the flanking regions of the target DNA to be deleted were amplified by PCR.
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The fragments were combined and cloned into the vector pJOE6743.1 in three
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different ways: i) restriction sites were added by the primers to both sides of the
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amplified fragments and the fragments cloned into the vector by a three-fragment
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ligation reaction; ii) amplified fragments were fused by overlap extension PCR using
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the outer primers (Ho et al., 1989) and inserted blunt ended into the SmaI or EcoRV
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cut vector; iii) restriction sites were added to the outer ends of the amplified
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fragments, the fragments fused by overlap PCR, cut at the corresponding restriction
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sites and inserted into the vector cleaved with the same restriction enzyme. Tables
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S1 and S2 summarize the constructed plasmids and oligonucleotides used for PCR.
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Using a pJOE6743.1 derivative with a deletion cassette, a manPA negative strain
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was transformed. In principle, the integration of the plasmid in the chromosome was
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selected by spectinomycin and in a second step, the excision was selected, first by
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growing the cells in LB liquid, in LB liquid containing mannose and on mannose
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containing LB plates. The excision and deletion formation was checked by Colony
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PCR. In theory, half of the clones should have lost the region of interest, while the
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other half should have restored the original state. In fact, we observed that on
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average, 30 - 70 % of the clones had lost the region of interest.
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Application of the new manP counter-selection system for B. subtilis genome
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engineering
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Ten large AT rich regions were identified in the B. subtilis 168 chromosome, that
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represent the temperate phage SP (Zahler et al., 1977), the defective prophage
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PBSX (Wood et al., 1990), the integrative conjugative element ICEBs1 (Auchtung et
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al., 2005), skin (sigma K intervening) integrated in sigK and 6 prophage like elements
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(Westers et al., 2003). Lysogenic phages and cryptic prophages might cause severe
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problems by spontaneous or induced phage activation during fermentation.
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Therefore, it is advantageous to remove all phage elements. In B. subtilis 6 the
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phages SB and PBSX as well as the cryptic prophages pro1, pro3 and skin have
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already been deleted. With the new counter-selection method described in the
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section above we were able to successively delete ICEBs1 together with a large
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flanking region (IIG-Bs3), pro5 (IIG-Bs4), pro6 (IIG-Bs5), pro4 (IIG-Bs20-2) and
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pro7 (IIG-Bs12) (see Table 1 and Table S1).
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About 4-5% of the B. subtilis genome is devoted to antibiotic production (Stein,
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2005). There are two lantibiotic biosynthesis gene clusters producing sublancin 168
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(Paik et al., 1998) and subtilosin A (Zheng et al., 2000). Two further gene clusters are
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responsible for the production of the non-ribosomal lipopeptides surfactin and
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plipastatin (synonymous to fengycin). The pks operon encodes the synthesis of an
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unknown polyketide (Kunst et al., 1997). Due to a mutation in sfp, the
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phosphopantetheine transferase, the non-ribosomal peptides and the polyketide are
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not produced in B. subtilis 168 (Mootz et al., 2001). Nevertheless, the pathways can
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be reactivated by simple transformation with DNA of B. subtilis wild type strains
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(Nakano et al., 1992; Tsuge et al., 1999). There are three other antibiotics produced
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by B. subtilis 168, the dipeptide bacilysin (Inaoka et al., 2003), the phospholipid
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bacilysocin (Tamehiro et al., 2002) and the amino sugar 3,3´-neotrehalosadiamine
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(Inaoka et al., 2004). The sublancin 168 gene cluster is located on phage SP and
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was already deleted in 6. The pks operon was deleted as well in 6 and replaced by
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a chloramphenicol resistance gene. To get rid of this antibiotic resistance gene, the
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original deletion was slightly extended leading to IIG-Bs8. The subtilosin A production
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genes were removed by deletion of genes sboA, sboX, albA-albG (IIG-Bs9) with the
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new method.
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In case of plipastatin the genes ppsA-ppsE (Steller et al., 1999) were deleted (IIG-
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Bs10). In case of surfactin the situation was more complicated (Marahiel et al., 1993).
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Within srfA, encoding the nonribosomal peptide synthase, there is a second small
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gene comS needed for competence. (Turgay et al., 1998). Hence, srfAA, srfAB
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(except comS), srfAC, srfAD, and additional genes up to sfp were deleted. The srf-
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promoter and comS, however, were retained (IIG-Bs20-4).
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The bacilysin production was abolished by deletion of the genes bacABCDEF (IIG-
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Bs11), the bacilysocin synthesis by deletion of ytpA (IIG-Bs13) and finally the 3,3´-
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neotrehalosadiamine biosynthesis by deletion of ntdR (yhjM), ntdA, ntdB, ntdC and
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glcP (IIG-Bs20-3). In the end, all known antibiotic biosynthesis clusters on the B.
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subtilis chromosome are deleted in strain IIG-Bs20-4.
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Endospore formation in B. subtilis is triggered by nutrient limitations. The master
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regulator for differentiation into spores is Spo0A which becomes activated by
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phosphorylation (Fujita & Losick, 2005). Activated Spo0A triggers two parallel and
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interconnected programs of gene expression, one in the forespore by activating
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sigma factor F and one in the mother cell by activating sigma factor E
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(Eichenberger et al., 2003; Molle et al., 2003; Wang et al., 2006). Deletion of spo0A
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would be the simplest way to inhibit sporulation. On the other hand, this would
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inactivate the natural transformation capability. Therefore, we deleted in one step
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sigma factor sigE and sigG together with spoGA responsible for pro-E activation and
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a gene for the extracellular protease Bpr (IIG-Bs20).
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During transition from exponential growth to sporulation some differentiating cells
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show a fratricidal behaviour (González-Pastor, 2011). They produce toxins which kill
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sibling cells. There are two independent gene clusters for toxin production: skf for
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sporulation killing factor and sdp for sporulation delaying protein (Allenby et al.,
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2006). The skf gene cluster was already removed by deleting prophage 1. Similarly
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to the skf operon, the sdp gene cluster produces a toxic peptide which is modified
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and exported by enzymes encoded by genes in two convergent operons, sdpABC
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and sdpRI (Ellermeier et al., 2006; Engelberg-Kulka et al., 2006). The two sdp
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operons were deleted together in strain IIG-Bs14 in order to get rid of upcoming killer
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cells.
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Comparison of the growth characteristics of IIG-Bs20-4 with B. subtilis 168
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The chromosomal deletions in IIG-Bs20-4 might affect the growth rate of this strain.
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Therefore we compared the growth curves of IIG-Bs20-4 with the ones from B.
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subtilis 168 in LB and minimal media. For LB, overnight cultures in LB at 37°C were
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used to inoculate 10 ml fresh LB medium in shake flasks to an optical density at 600
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nm (OD600) of 0.05. The cultures were incubated at 37°C on a rotary shaker at 150
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rpm. The OD600 was determined hourly. For minimal media, MG1 medium
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supplemented with trace elements was used. Fresh MG1 medium was inoculated by
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an overnight culture in MG1 to an OD600 of 0.1 and the growth determined
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spectroscopically like in LB.
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Independent of the used medium, both strains behaved similarly (Fig. 2). Using LB or
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MG1 the two strains showed nearly identical growth rates (Table 2). Thus, even a
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deletion of as much as 13.6 % of the B. subtilis genome did not compromise the
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viability of the bacteria suggesting that the constructed strain IIG-Bs20-4 is a suitable
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chassis for industrial applications.
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Discussion
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A counter-selection system is essential for markerless genome engineering. The
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method described in this study allows to introduce point mutations, deletions of
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various length as well as gene insertions into the B. subtilis chromosome. There is a
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shortage of reliable counter-selection systems, especially of ones which do not use
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toxic compounds. The use of the manP PTS transporter in a manPA mutant turned
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out to be a highly reproducible and efficient way to produce a series of deletions in
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the B. subtilis genome. No spontaneous resistance to mannose, for example by
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mutation of the mannose activator gene or by induction of a phosphosugar
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phosphatase was observed. The deletions from B. subtilis 6 to IIG-Bs20-4 were just
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checked by Colony PCR. Another strain derived from IIG-Bs20 with many more
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deletions made for a different purpose with this new counter-selection method clearly
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showed after sequencing that all deletions occurred exactly as planed and there were
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no DNA rearrangements or single base mutations found (D. Reuss, pers.
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communication). Since many bacteria use PTS systems for sugar uptake, it should
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be easy to adapt such a system for genome engineering in other bacterial species.
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In a series of deletions we were able to considerably reduce the B. subtilis genome
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from 4,215 to 3,640 kb. Another B. subtilis 168 strain (MGB874) with reduced
333
genome was constructed before (Morimoto et al., 2008). In MGB874, depleted of 874
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kb (20%) of its genome sequence, the purpose was to delete a large number of
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dispensable genes without altering the growth rate and other strain-specific
336
properties like sporulation or antibiotic production. Since many of the dispensable
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genes are located in regions identified as prophages or genomic islands, the strains
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MGB874 and IIG-Bs20-4 have many deleted genes in common. Nevertheless, IIG-
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Bs20-4 has the advantage that is does not produce spores, antibiotics or
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bacteriocins, properties which are desired for a production strain for enzymes or low
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molecular substances.
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The new strain provides a valuable basis for further optimization concerning protein
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production and secretion, high cell-density fermentation or metabolic engineering.
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345
Acknowledgements
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We thank Annette Schneck and Sina Blessing for their assistance in creating the
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deletion strains and Kambiz H. Morabbi for helpful discussions. The project was
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partly financed by Lonza AG and thus we would like to thank especially Christoph
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Kiziak for support and helpful discussions. We are grateful to Jan Maarten van Dijl for
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providing the strain B. subtilis 6.
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Legends to figures
580
Figure 1: Plasmid map of the positive cloning and integration vector pJOE6743.1.
581
Selected double cutters for insertion of the amplified homologous regions were
582
added to the map. Binding sites for SP6 and T7 standard primers used for
583
sequencing are shown as white boxes. Abbreviations: manP (B. subtilis mannose
584
PTS transporter); ter (transcription terminator from phage ); amp (ampicillin
585
resistance); SpcR (spectinomycin resistance); lacPOZ: (lac promoter, lac operator
586
and lacZ  fragment).
587
Figure 2: Growth curves of B. subtilis 168 and IIG-Bs20-4 in LB and MG1 liquid
588
medium at 37°C (obtained in 4 independent experiments).
589
590
Table 1: Bacterial strains used in this study.
Strain
591
592
Genotype / Properties
Deletion from bp-bp*
Reference / Source
E. coli JM109
mcrA, recA1, supE44,
endA1, hsdR17, gyrA96,
relA1, thi, (lac-proAB),
F´[traD36, proAB+, lacIq
lacZM15]
n.a.
Yanisch-Peron et al. (1985)
B. subtilis 168
trpC2
n.a.
Bacillus Genetic Stock Center
B. subtilis 3NA
spo0A3
n.a.
Bacillus Genetic Stock Center
B. subtilis 
trpC2, SP, skin,
PBSX, pro1,
pks::CmR, pro3
n.a.
Westers et al. (2003)
IIG-Bs1
trp+
n.a.
this study
IIG-Bs2
manPA::erm
1272608..1275639
this study
IIG-Bs3
pro2
529495..604658
this study
IIG-Bs4
pro5
1879831..1886598
this study
IIG-Bs5
pro6
2055447..2079118
this study
IIG-Bs8
CmlR
1781877..1859956
this study
IIG-Bs9
subtilosin
3835926..3842999
this study
IIG-Bs10
plipastin
1960195..1997960
this study
IIG-Bs11
bacilysin
3867368..3874329
this study
IIG-Bs12
pro7
2708926..2742191
this study
IIG-Bs13
bacilysocin
3121673..3124005
this study
IIG-Bs14
Bacillus toxin
3464131..3467474
this study
IIG-Bs20
protease, sporulation
(sigE, sigG)
1599076..1606455
this study
IIG-Bs20-1
manPA::erm
1272643..1279510
this study
IIG-Bs20-2
pro4
1263599..1270416
this study
IIG-Bs20-3
3,3´-neotrehalosadiamine 1125000..1130795
this study
IIG-Bs20-4
srfAA-srfX (except comS) 377215..390803
391074..408169
this study
* n.a. (not applicable)
593
594
595
596
597
Table 2: Specific growth rates of B. subtilis 168 and IIG-Bs20-4 in LB and MG1
medium. The mean values with standard deviation is given obtained from four
independent experiments.
Strain
598
Maximum specific growth rate max [h-1]
LB
MG1
B. subtilis 168
1.42  0.14
0.63  0.09
IIG-Bs20-4
1.47  0.10
0.58  0.07