Expanding the molecular toolkit for the homoacetogen Clostridium ljungdahlii

Increasing interest in homoacetogenic bacteria for the production of biochemicals and biofuels requisites the development of new genetic tools for these atypical production organisms. An attractive host for the conversion of synthesis gas or electricity into multi-carbon compounds is Clostridium ljungdahlii. So far only limited achievements in modifying this organism towards the production of industrially relevant compounds have been made. Therefore, there is still a strong need for developing new and optimizing existing genetic tools to efficiently access its metabolism. Here, we report on the development of a stable and reproducible transformation protocol that is applicable to C. ljungdahlii and several other clostridial species. Further, we demonstrate the functionality of a temperature-sensitive origin of replication in combination with a fluorescence marker system as important tools for future genetic engineering of this host for microbial bioproduction.

Scientific RepoRts | 6:31518 | DOI: 10.1038/srep31518 (protocol 1; Table 1). The transformed plasmid pSOBP ptb carried a Gram-positive origin of replication (ori + ) derived from the plasmid pIMP1 (pIM13). Transformation efficiencies (E t ) were not given in this publication. Based on protocol 1 an optimized procedure was published by Leang, et al. 26 (protocol 2; Table 1). Depending on the used ori + , the authors reported E t of up to 1.7 × 10 4 transformants per μ g of plasmid DNA for plasmid pCL2 (ori + : pIP404, Table 2). With the same transformation protocol, select examples of replicable plasmids from the modular pMTL80000 plasmid system (pMTL82151, ori + : pBP1; pMTL83151, ori + : pCB102) for Clostridium spec. 28 , yielded E t of ~3 × 10 3 transformants per μ g of plasmid DNA. Protocol 2 was used in follow-up publications from the same group (without giving information about E t ) to: 1) transform a suicide plasmid; the authors reported that one single colony grew after transformation and that a single-crossover genome integration event was observed (Table 2) 29 , 2) transform different plasmids with pIP404 as ori + (Table 2) 25 , and 3) construct several strains carrying replicable plasmids and to construct several knock-out strains via either single cross-over homologous recombination or using the ClosTron system (Table 2) 24 . In the recent patent literature, a slightly modified protocol 2 was used to transform Clostridium autoethanogenum, a very closely related species to C. ljungdahlii 30 . E t between 1.3 × 10 2 and 1.3 × 10 3 transformants per μ g of plasmid DNA were reported in this patent application, depending on different methylation patterns 31 (protocol 3; Table 1). To our knowledge no other transformation procedures for C. ljungdahlii or further use of the reported protocols are published so far. Protocols 1 or 2 did not efficiently and reproducibly result in transformants in our hands. Therefore, we developed an alternative transformation procedure, which we here demonstrate to be applicable to different clostridial species. These species were not necessarily closely related to each other, showing the broad suitability of Gram + replicons and the antibiotic resistance marker catP in different clostridial species. In Table 1 the differences of the published protocols and the final version of our new procedure (protocol 4) are summarized. The most important changes are the use of 10% glycerol instead of SMP buffer for washing steps and as electroporation solution as well as a prolonged outgrowth time of 24 to 48 h after electroporation. For this outgrowth, the undefined complex medium RCM was used instead of PETC medium. In our protocol, the plasmid DNA is methylated with the Φ 3T I methyltransferase as described in Mermelstein and Papoutsakis 32 using Escherichia coli strain DH5α MCR (K-strain, Dcm + Dam + ). In contrast, in all publications that utilize protocol 2 [24][25][26]29 , the plasmid DNA is prepared from E. coli NEB Express (B-strain, Dcm − Dam + ) without external, site-specific methylation. We found robust and reproducible E t values for C. ljungdahlii of 3.23 × 10 2 -3.23 × 10 3 transformants per μ g of plasmid DNA for plasmids with different ori + s ( Table 2). There were no successful transformants with our new protocol when the plasmid DNA for transformation was prepared from E. coli NEB Express cells. We also tested how robust the protocol is to small variations. We found that a starting OD 600 ranging from 0.25 to 0.71 did not considerably change the E t . Other factors like an increased amount of plasmid DNA (3 μ g instead of 2 μ g) also had no considerable effects.
Transformation efficiencies for other tested clostridial species are given in Table 3. In these cases only plasmid pMTL82151 was used for comparison (except for C. acetobutylicum). E t values were in the same range as those found for C. ljungdahlii (4 × 10 2 -9 × 10 2 ), except for C. perfringens for which an E t of 4.5 × 10 3 was reached.
To choose the appropriate amount of antibiotics for the different Clostridia, we either used published concentrations 26,28,33 or determined a rough minimal inhibitory concentration (MIC) in liquid cultures as described in Materials and Methods. Strain-specific PCR and a PCR targeting the introduced plasmid were always used to exclude false positive results after each transformation (Fig. S1).
The anaerobic fluorescent protein system pGlow-CK XN Bs2/Pp1 is applicable in C. ljungdahlii. The fluorescence marker system evoglow ® from Evocatal (Monheim, Germany) is known to be functional in C. acetobutylicum and is available as a clostridia-optimized commercial kit. The system is based on flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) that are -in contrast to GFP -functional under anaerobic conditions 34 . Here we applied this system in C. ljungdahlii and demonstrated the functionality for this organism. C. acetobutylicum was used as a control in these experiments. The intensity of the evoglow-CK XN -Bs2 and evoglow-CK XN -Pp1 proteins was not considerably different in fluorescence microscopic images (Fig. 1).

Plasmid
Origin of replication (Clostridium) E t a (transformants/μ g DNA), mean ± SD (n) Reference Utilized protocol b pSOBP_ptb pIMP1 NI c Köpke, et al. 23    A temperature-sensitive origin of replication is a suitable tool to induce the loss of plasmids from cells. Plasmids with a conditionally suicidal origin of replication, i.e. the plasmids are only replicating under certain conditions, can be a useful tool for several applications. In these cases, it is important to maintain the functionality of the plasmid only for a given time. When the function should be shut off, the loss of the plasmid from the cells can be induced with one of several methods. For example, in C. acetobutylicum a system was established in which an inducible anti-sense RNA blocks the plasmid's origin of replication leading to low segregational stability of the plasmid 35 . More commonly, temperature-sensitive origins of replication are described for several microorganisms and some of them are functional in a broad range of microbial hosts [36][37][38][39] . For example, the temperature-sensitive ori + pWV01ts (pVE6002) derived from pWV01 from Lactococcus lactis subsp. cremoris was shown to be functional in E. coli and Bacillus subtilis 37 and also was function as the origin in pSS60, used in C. acetobutylicum 40 . In this case, the temperature sensitivity results from four clustered mutations in the repA gene 37 leading to stable maintenance at 30 °C and a non-permissive temperature of 37 °C. We tested the pWV01ts origin of replication for functionality in C. ljungdahlii. Without a selection pressure (i.e. no antibiotics in the medium), the plasmid pMTLts was only maintained at the permissive temperature of 30 °C and was lost by dilution when cells were grown at 37 °C (Figs 3 and 4).
After one transfer and growth at 37 °C for 24 h, only 3-14% of the cells maintained the plasmid and were able to grow on thiamphenicol-containing RCM plates, compared to cultures grown at a permissive temperature of 30 °C (Fig. 4).
To further analyze the temperature-sensitive origin of replication and to demonstrate its usability in a functional plasmid, it was combined with the anaerobic fluorescence protein. For this purpose, the plasmid pMTLts_Bs2 was constructed, which carries the pWV01ts ori and the fluorescence marker evoglow-Bs2-Cl under the control of the constitutive thiolase promoter (P thl ) from C. acetobutylicum. As expected, we observed the temperature-dependent decrease of fluorescence signal as the number of cells (CFU) carrying the plasmid pMTLts_Bs2 decreased (Fig. 5). The relationship was not linear, however, with ~40% reduction in fluorescence intensity vs. ~90% reduction of CFUs after incubation at 37 °C. This difference most likely results from the use of two different media. Quantitative colony counts utilized the preferred growth medium RCM during plasmid loss and plating, while fluorescence measurements of the Bs2 protein are only possible in defined PETC medium without the addition of yeast or beef extract. Hence, the growth rates were significantly different. Also, we do not have a quantitative measure for the specific growth phase or biomass correlated protein expression and fluorescence intensity of Bs2, which could be reflected in the fluorescence analysis. Compatibility with the pMTL80000 modular plasmid series. The plasmids pMTLts and pMTLts_Bs2 described here provide an additional ori + (pWV01ts) and a fluorescent marker (evoglow-CK XN -Bs2) that are functional in C. ljungdahlii and C. acetobutylicum. According to the requirements of the modular pMTL80000 system 28 , the modularity/reversibility of the system via the four reserved restriction sites AscI, FseI, PmeI and SbfI should be maintained for the integration of new modules. Further, important restriction sites in the multiple cloning site should remain exclusive and should therefore be avoided in the other modules. The fluorescent marker module that we integrated contained an additional SbfI site. Thus, we performed a conservative mutation of this site (via QuikChange mutagenesis) to maintain the modularity criteria of the pMTL80000 system. With this modification, we suggest adding these modules to the nomenclature of the pMTL system and reserve numbers for the Gram + replicon (e.g. pMTL86XXX) and the application-specific module (e.g. pMTL8XXX5).

Discussion
We developed an electroporation protocol for C. ljungdahlii that is also applicable for several other clostridial species. Intensive application of the protocol in ongoing projects in our lab have confirmed its replicability. We achieved robust E t values of 3.23 × 10 2 -3.23 × 10 3 (Table 2) for plasmids with different ori + . To date, the highest reported E t is 1.7 × 10 4 for plasmid pCL2 with ori + pIP404 26 , which was not tested with our protocol so far. Using this origin of replication might also lead to higher E t with our protocol. Alternatively, the apparently lower E t of our protocol might also be due to issues in the plating efficiency, which is highly variable for this obligate anaerobe.
One factor that seems to influence the efficiency greatly is the temperature of the molten RCM agar for the plating procedure. For plating the same volume from the same culture, we made the observation that fewer colonies arose when plates were poured at temperatures around 45 °C-50 °C, than when the agar was already cooled to approximately 40 °C. This makes it difficult to determine consistent E t values and likely explains the high standard deviation in our measurements.
Further, in our experiments with C. ljungdahlii, initially not more than about 300-400 colonies grew, independent of the estimated number of plated cells (based on OD). Besides the agar temperature, specific effects of  colony inclusion or the general fitness of cells might be influential. After a reorder of the strain from the German culture collection, significantly faster growth in liquid media and plating efficiency was observed (e.g., in the plasmid loss experiment with pMTLts_Bs2, Fig. 5). Laboratory degeneration of strains is observed frequently for solventogenic Clostridium sp. 41,42 , but this is the first report for acetogenic Clostridium strains. In our work, strain degradation probably reduced our plating efficiency and also E t (E t pMTLts_Bs2 = 3.23 × 10 3 , Table 2) for the first part of this study.
Even despite these initial limitations in plating, our transformation protocol leads to a reproducible and sufficient amount of transformants when plating ~200 μ l of a 5 ml liquid culture. For screening purposes, the number of plates can be increased to increase the number of clones.
We tested the anaerobic fluorescent marker system evoglow ® from Evocatal (Monheim, Germany) as a genetic tool for future functional engineering of C. ljungdahlii. Functionality was shown for C. ljungdahlii as well as for the control C. acetobutylicum. Different from the oxygen-dependent fluorescent proteins of the GFP family, this system is based on flavin-dependent reactions. The choice of the correct medium is therefore critical for the application of these fluorescence marker proteins. Medium containing high amounts of riboflavin (e.g., the common C. ljungdahlii media RCM and PETC) gave high background fluorescence, making it impossible to detect fluorescing cells. The utilization of PETC medium without yeast and beef extract (PETC* ) overcame this problem. Growth of C. ljungdahlii in the mineral medium without these compounds with fructose as carbon source was slower but still sufficient. A low background fluorescence in these cultures is important since the fluorescence quantum yield (Q F ) of the flavin-based fluorescent proteins naturally is lower than typical oxygen-dependent GFP-type fluorescent proteins 34,43 . Overall, we have confirmed that the anaerobic fluorescence marker system evoglow ® can be a versatile functional tool for C. ljungdahlii.
We further applied the fluorescent marker evoglow-CK XN -Bs2 to test the functionality of a third genetic toola temperature-sensitive origin of replication for controlled plasmid loss. For the construction of plasmid pMTLts from plasmid pMTL85141, only the Gram-positive origin of replication (ori + : pIM13) was exchanged against the pWV01ts ori; the high-copy number ColE1 Gram-negative origin of replication necessary for shuttle plasmid activity was maintained. Interestingly, during the cloning procedure in E. coli, it turned out that the temperature sensitive ori was dominant over the ColE1 ori. Therefore, temperature sensitivity was also observed in E. coli. In experiments with induced plasmid loss in C. ljungdahlii for pMTLts and pMTLts_Bs2 we could show a plasmid loss of ~90% through incubation at the non-permissive temperature of 37 °C for 24 h. For pMTLts_Bs2, we were also able to confirm this plasmid loss through the reduction of Bs2 fluorescence. The visible reduction in fluorescence intensity thereby was a good qualitative measure for plasmid loss, while the plate counts on RCM medium provide quantitative information. The importance of this temperature sensitive plasmid tool is mainly for applications in strain engineering, in which a temporary action of a plasmid-based element is desired, e.g., a mutagenic element or a restriction enzyme for genome editing. Once the desired action is performed, the plasmid can be easily removed from the cells. An application for bioproduction processes is less likely, since the permissive growth temperature of 30 °C results in significantly slower growth than the optimum 37 °C.
New genetic tools for C. ljungdahlii are an important prerequisite to study its physiology and for its targeted modification and application as an industrial production host. Our robust transformation protocol, the demonstration of the functionality of an anaerobic fluorescence marker system and a temperature-sensitive origin of replication broaden the spectrum of tools to study and modify C. ljungdahlii.

Materials and Methods
Details on plasmid construction and oligonucleotides can be found in the supplemental information.
Bacterial strains and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 4. E. coli was routinely grown at 37 °C or 30 °C in LB broth or on LB agar. E. coli DH5α MCR was used for plasmid construction and propagation. Clostridia were grown anaerobically in modified reinforced clostridial medium (RCM), containing (per L): 10 g peptone, 10 g beef extract, 3 g yeast extract, 5 g D(− )-fructose, 5 g NaCl, 1 g soluble starch, 0.5 g L-cysteine-HCl, 3 g sodium acetate (pH 6.8) at 37 °C or 30 °C under strictly anaerobic conditions. For spectroscopic measurements of fluorescence, cultures were grown in PETC medium (ATCC medium 1754) without yeast and beef extract. All Clostridia media were extensively flushed with N 2 (99.99999%) and residual oxygen was removed with L-cysteine-HCl as reducing agent. All manipulations of Clostridia were performed in an anaerobic glove box (Coylab, USA) under an atmosphere of 5% H 2 , 25% CO 2 and 70% N 2 . Media were supplemented with antibiotics when necessary in the following concentrations: For E. coli, chloramphenicol (30 μ g/ml); erythromycin (500 μ g/ml); carbenicillin (ampicillin) (50 μ g/ml); for C. acetobutylicum and C. ljungdahlii, thiamphenicol (5 μ g/ml), clarythromycin (4 μ g/ml), erythromycin (50 μ g/ml); for C. pasteurianum and C. perfringens, thiamphenicol (10 μ g/ml). To determine the appropriate amount of antibiotic for C. pasteurianum and C. perfringens, the cultures were inoculated to an OD 600 of 0.05 to 0.1 into 5 ml RCM supplemented with increasing amounts of antibiotics. The antibiotic concentration where clear growth inhibition was observed was doubled and used for further experiments.
For growth of Clostridia on solid media a pour-plating method was used. In detail, 20 ml freshly prepared RCM-agar (1.5% agar), cooled down to 40 °C-50 °C, were mixed with 200-1000 μ L of liquid bacterial culture and the appropriate antibiotics and poured into a petri dish. The plates were incubated at 37 °C or 30 °C until single colonies were visible (3-7 days).

Methylation of plasmids.
To prepare a plasmid for transformation into Clostridia, the desired plasmid was co-transformed into E. coli DH5α MCR together with either pAN1 32 or pANA1 (depending on the resistance cassette of the target plasmid) for methylation with the Φ 3T I methyl transferase 32  Spin Kit (Qiagen, Germany) and used to transform Clostridia. To test for a successful methylation, a digestion with the methylation-sensitive restriction endonuclease HaeIII (NEB, Germany, cuts the motif GGCC after the second guanine only when the first cytosine is not methylated) was performed and checked via agarose gel electrophoresis.
Transformation of Clostridia. All steps including centrifugation were performed in an anaerobic glove box (Coylab, USA) under a gas atmosphere of about 5% H 2 , 20% CO 2 , 75% N 2 . For a pre-culture, 50 ml RCM was inoculated to an OD 600 of 0.05 to 0.1 and incubated at 37 °C overnight. For each transformation 9 ml of cell suspension (OD 600 0.2-0.8) was stepwise harvested in a 1.5 ml reaction tube by centrifugation (2000 × g, 1 min) and cell pellets were washed twice with 1.5 ml ice-cold 10% glycerol. Subsequently, the washed cells were resuspended in 200 μ l of ice-cold 10% glycerol, 2 μ g of methylated plasmid DNA was added and the mixture was directly transferred into a precooled electroporation cuvette with a gap-size of 0.2 cm (Labomedic, Germany). Electroporation was performed at the following conditions: 2.5 kV, 600 Ω , 25 μ F. Directly after electroporation, the cells were transferred into 5 ml pre-warmed RCM and incubated for 24-48 h at 37 °C (or 30 °C for plasmids carrying pWV01ts origin of replication). After this non-selective outgrowth step, 200-1000 μ l of culture were plated using the pour-plating method described above. The plates were incubated at 37 °C (or 30 °C) until colonies were obtained, which could take between 1-2 days for C. acetobutylicum, C. perfringens, C. pasteurianum and up to a week for C. ljungdahlii. The transformants were confirmed by colony PCR with plasmid-specific primers ColEI_for/ColEI_rev (Table S1) after one restreaking step or subcultivation in liquid RCM to avoid false positives due to residual plasmid DNA or untransformed wild type cells from the transformation procedure. Additionally, the integrity of the used strain was checked by colony PCR with strain-specific primers (fdhA_for/fdhA_rev, C. ljungdahlii; ctfAB_for/ctfAB_rev, C. acetobutylicum; nifC_for/nifC_rev, C. pasteurianum; luxS_for/luxS_rev, C. perfringens; Table S1).
Transformation efficiencies E t were calculated with the following equation:  Fluorescence spectroscopic and microscopic analyses. C. ljungdahlii or C. acetobutylicum cells carrying pGlow-CK XN Pp1, pGlow-CK XN _Bs2 34 or pMTLts_Bs2 were inoculated to an OD 600 of 0.05 to 0.1 in RCM medium and incubated until exponential growth phase was reached at 37 °C or 30 °C with the addition of clarithromycin or thiamphenicol, respectively. These cultures were used to inoculate PETC medium without yeast and beef extract to an OD 600 of 0.05 to 0.1 and incubated at the required temperature before fluorescence microscopic and spectroscopic analyses. Fluorescence spectroscopic measurements in 200 μ L volume at an OD 600 of ~0.7 were performed in 96-well plates (Fluotrac 200 clear flat bottom black plates, Greiner Bio-One, Germany) in a plate reader (SynergyMX, BioTek, Germany), whereby the cultures were pelleted and diluted to adjust the OD. The emission spectra were recorded between 400 nm and 700 nm at an excitation wavelength of 450 nm.
Fluorescence microscopic pictures were captured with a fluorescence microscope (Leica DM6000B, Leica Microsystems, Germany) equipped with a fluorescence cube (LED405, Leica Microsystems, Germany) with an excitation wavelength filter of 375-435 nm and an emission wavelength filter of 445-495 nm and a monochrome digital CCD camera (Leica DFC 365 FX, Leica Microsystems, Germany).

Induced plasmid loss experiments.
A schematic description of this experiment is given in Fig. 3. In detail, C. ljungdahlii cells carrying pMTLts or pMTLts_Bs2 were inoculated in RCM with thiamphenicol to an OD 600 of 0.05 to 0.1 and incubated for 24 h at 30 °C. 200 μ l of the pre-culture was plated using the pour-plating method on selective agar. Single colonies were transferred into 5 ml liquid RCM containing thiamphenicol and the culture was incubated at 30 °C until exponential growth phase was reached. The culture was used to inoculate six new culture tubes with 5 ml RCM without antibiotics and three tubes each were incubated at 30 °C and 37 °C, respectively. For the determination of CFUs, a serial dilution of these cultures was prepared and 100 μ l of the 10 −4 , 10 −5 , and 10 −6 dilution was plated with the pour-plating method with the addition of thiamphenicol (three plates per dilution). Thereby, the starting OD for the serial dilution was comparable for the different culturing temperatures. The plates were incubated at 30 °C until colonies were obtained and counted.
To evaluate the temperature induced loss of the plasmid after cultivation at 30 °C and 37 °C in RCM, cultures carrying pMTLts_Bs2 were also transferred into PETC medium without yeast and beef extract and grown to an OD 600 of at least 0.25. Fluorescence spectrometric measurements were then performed as described above (Fig. 3 right).