Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis

The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis.


Introduction
The ability to efficiently and reliably transfer genetic circuits between different synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the main bottlenecks of the rational genome engineering. The Gram-negative E. coli and the Gram-positive B. subtilis are both well-characterized bacteria used in a number of synthetic biology and biotechnology applications [1][2][3][4]. Furthermore, they are considered to be promising chassis for the construction of the minimal cell factories [1,[5][6][7]. E. coli is easily amenable to genetic modifications by a number of DNA recombineering methods. Although E. coli was successfully engineered for the production of a number of industrially relevant products, such as biofuels, amino acids and isoprenoids [2,[8][9][10], B. subtilis is considered to be a better host for certain applications [11][12][13]. Unlike E. coli, B. subtilis is naturally competent and readily transformable with extracellular DNA which is integrated into the chromosome via RecA-mediated homologous recombination [14,15]. Furthermore, B. subtilis secretes proteins into the medium and forms durable endospores.
Novel tools combining benefits of the B. subtilis chassis with the reliable DNA recombineering in E. coli are therefore crucial for B. subtilis bioengineering efforts. Due to natural competence of B. subtilis, DNA edited in and released from the lysed E. coli cells can be readily taken up and incorporated into the B. subtilis chromosome. On the industrial scale, the conditionally-inducible cell lysis systems are preferable over the traditional methods of cell lysis, such as enzyme degradation or mechanical disruption for a number of reasons, such as lower production and product recovery costs and no need for an inducer [16]. Transfer of DNA from E. coli undergoing lambda prophage-induced lysis to B. subtilis by the co-culture method was demonstrated recently [17,18]. The co-culture method was also shown to be suitable for transferring high molecular weight DNA molecules, which are difficult to manipulate with other methods due to mechanical shearing [17,18]. However, the presence of an entire phage in the donor cell can be detrimental to the quality of the transferred DNA. This problem can be minimized by using solely the phage lysis genes.
Here we present a system for the reliable and efficient DNA transfer that utilizes the lysis genes from multiple phages to disrupt the donor E. coli cell and transfer the released DNA into B. subtilis.

Materials and Methods
Bacterial strains, BACs, plasmids, and growth conditions All bacterial strains, plasmids and BACs used in this study are listed in Table 1. Escherichia coli and Bacillus subtilis were routinely grown in Luria-Bertani broth (LB). When required, cultivation media were supplemented with chloramphenicol (30 μg/ml), ampicillin (100 μg/ml) and kanamycin (50 μg/ml) for growing E. coli, and chloramphenicol (5 μg/ml) and kanamycin (5 μg/ ml) for B. subtilis. Liquid cultures were grown in LB on a rotatory shaker at 200 r.p.m. and 30°C, 37°C or 42°C, as required. Plate cultures were incubated at 30°C, 37°C or 42°C for approximately 24 hours. Starvation medium (described below) was used to generate competent B. subtilis cells.

Competent B. subtilis generation and transformation
To generate competent B. subtilis cells, single colony was first inoculated into 10 ml minimal medium composed of 5x minimal salts solution [ammonium sulphate (10 mg/ml), potassium hydrogen phosphate (75 mg/ml), potassium dihydrogen phosphate (25 mg/ml), sodium citrate (1 mg/ml), magnesium sulphate heptahydrate (1mg/ml)], glucose (0.5% w/v), casamino acids (0.02% w/v), tryptophan (20 μg/ml), and iron ammonium citrate (2.2 μg/ml). Inoculated cells were grown at 200 r.p.m. on a rotatory shaker at 37°C for 18 hours. Then, 1.4 ml of the B. subtilis culture was inoculated into 10 ml of the fresh minimal medium and grown for another 3 hours. Subsequently, 11 ml of the starvation medium composed of 5x minimal salts solution and glucose (0.5% w/v) were added to the B. subtilis culture and cells were grown for additional 2 hours and 45 mins. 0.3 ml aliquots were transferred into 15 ml polypropylene tubes and transformed with 15 μl of BAC DNA or plasmid DNA. Transformed B. subtilis cells were incubated at 200 r.p.m. on a rotatory shaker at 37°C for 1 hour prior to addition of 700 μl LB. Cells were then continued to grow for 1.5-2 hours. 20-200 μl of this culture were plated onto selection plates and grown at 37°C for 18-24 hours. Integrations into the B. subtilis chromosome were confirmed by PCR with the flanking primers and sequencing.
supplier's instructions. Oligonucleotide primers were synthesized by Sigma-Aldrich. PCR amplified DNA fragments were assembled in a 5.2 μl final reaction volume using the modified Gibson Assembly procedure [20,23,24]. DNA assemblies were confirmed by PCR amplification and sequencing. DNA sequencing was performed by Source Bioscience. Qiaquick Gel Extraction kit (Qiagen) and Qiaprep Spin Miniprep kit (Qiagen) were used for DNA extraction and purification and plasmid isolation, respectively. BACs were isolated with Qiaquick Gel Extraction kit (Qiagen) or PhasePrep BAC DNA kit (Sigma-Aldrich), according to the manufacturer's instructions. B. subtilis genomic DNA was obtained using the GeneJET genomic DNA purification kit (Thermo Scientific).

Lambda Red recombineering for E. coli chromosomal integration
The modified Miller and Nickoloff [25] and Hanahan [26] methods were used to generate the electro-competent and chemically competent E. coli, respectively. DNA integrations into E. coli chromosome and BACs were performed using the streamlined lambda Red system-mediated method described previously [20,27,28]. Briefly, E. coli was transformed with pKM208-bourne IPTG-inducible lambda red recombinase and grown on the selective ampicillin plates at 30°C overnight. Overnight culture (1:100 dilution) was inoculated into LB with ampicillin and grown at 30°C first to OD 600 0.2. Then, IPTG (1 mM) was added and the culture was grown to the final OD 600 0.5. Cells were washed and resuspended in 100 μl of 10% glycerol per 100 ml of starting culture volume. 100 μl of the PCR amplified and gel purified DNA flanked by sequences homologous to the integration target loci were electroporated into E. coli with pKM208. Transformants were plated on selective plates and grown at 30°C overnight and then grown on LB plates at 42°C. As pKM208 is thermosensitive, growth at 42°C leads to loss of this plasmid from E. coli cells. DNA integrations were confirmed by diagnostic PCR with flanking primers and DNA sequencing. To allow repeated use of the kanamycin resistance marker, cells with DNA integrations were transformed with plasmid pCP20 encoding flippase (FLP) recombinase and grown first on selective plates at 30°C overnight and then grown on LB plates at 42°C to cure out the thermosensitive pCP20.

Culture mix method of DNA transfer from E. coli to B. subtilis
Modified culture mix method [17,18] and grown on 2 x SG plates for 72 hours to induce sporulation. Bacteria scraped off the 2 x SG plates were washed with ice-cold water and subjected to heat treatment at 90°C for 10 minutes. 100 μl were plated on LB chloramphenicol plates and grown overnight. The average numbers of CFUs and standard errors were calculated from three independent replicates.

Bacterial viability assay
The E. coli viability was measured with a Live/Dead BacLight™ bacterial viability kit (Lifetechnologies, UK) according to supplier's instructions. Briefly, cells were first grown in 5 ml LB in 15 ml Falcon conical centrifuge tubes on a rotatory shaker at 200 r.p.m. and 30°C for 6 hours prior to temperature shift to 42°C. After the temperature shift cells were grown at 42°C for another 6 hours. Then red-fluorescent nucleic acid stain propidium iodide and green-fluorescent nucleic acid stain SYTO9 were added into the medium (3 μl of the stain per ml). The numbers of the dead propidium iodide-stained bacteria and live SYTO-9 stained bacteria were determined by fluorescent microscope (Nikon Microphot-SA), 5-6 hours after the temperature shift from 30°C to 42°C. The means and standard errors were calculated from three experiments.

Results and Discussion
Lysis genes of phages MS2, ΦX174, and lambda for cell lysis Bacteriophages MS2, FX174 and lambda were chosen mainly due to their extensive characterization and different mechanisms of action [31]. In E. coli, there are 3 main barriers to phage release, namely an outer membrane (OM), a thin layer of peptidoglycan (PG), and a cell membrane (CM). The lysis gene cassette of the phage lambda consists of four genes, namely S, R, Rz and Rz1 regulated by pR promoter. Rz and Rz1 encode a class II CM protein and an OM lipoprotein, respectively, which form a complex spanning the periplasm of the host cell to promote fusion of the CM with the OM [32]. S encodes the holin and the antiholin [33], while R encodes the endolysin with lytic transglycosylase activity [34]. The endolysin and the holin accumulate in the cytoplasm and the CM, respectively. The holin-induced disruption of the CM allows endolysin-driven deterioration of the PG and cell lysis [35]. The lysis gene of the phage FX174 encodes E protein implicated in the inhibition of the PG biosynthesis. This leads to the cell wall rupture at the developing septum. As a consequence, the cell lysis induced by phage FX174 is more gradual than that of the phage lambda [36]. The lysis gene of the phage MS2 encodes L protein that binds to the adhesion zones formed by parts of the CM, the OM and the periplasm. As formation of the adhesion zones is dependent on the activation of the cell's autolysis system, the MS2-induced lysis occurs in the actively growing cells [37][38][39]. The different mechanisms of action of the lambda, FX174 and MS2 lysis genes suggests that their combination in a single cell could lead to more efficient lysis.

Engineering the inducible lysis system
To construct an inducible system for controlled E. coli lysis we chose the thermosensitive lambda repressor to regulate the expression of the phage lambda, FX174 and MS2 lysis genes. At 30°C, the expression of lysis genes from the pL promoter is inhibited by the repressor. Temperature shift to 42°C alleviates the repression and leads to cell lysis. DNA can be then taken up by other bacteria, such as B. subtilis (Fig 1).

Combining lysis genes from multiple phages improves cell lysis
To test the effect of multiple phage lysis genes on cell lysis we first used plasmids pSB1K3 (FRTL1), pSB1K3(FRTL2), pSB1K3(FRTL3), pSB1K3(FRTL12), pSB1K3(FRTL13), and pSB1K3(FRTL123), harbouring different combinations of lysis genes ( Table 1). The cell lysis was quantified by measuring absorbance of the E. coli cultures with the microplate reader. The absorbance of the bacterial cultures grown in both restrictive and permissive temperature for the thermosensitive repressor was compared with that of the controls grown in the restrictive temperature for 24 hours. The absorbance was significantly lower after the temperature shift when compared to the growth in the restrictive conditions for the whole 24 hours. The comparison between cultures harbouring plasmids with different combinations of lysis genes showed that the strain with lysis genes from all three bacteriophages lysed with the highest efficiency. Furthermore, lysis of strains harbouring two lysis genes was higher than that harbouring a single lysis gene (data not shown). The highest difference between restrictive and restrictive/permissive conditions was observed for E. coli with plasmid pSB1K3(FRTL123) encoding all three lysis genes.
In our experimental setting, the drop in the absorbance in strains Ec(RL1i), Ec(RL2i), Ec (RL3i), Ec(RL12i) and Ec(RL13i) occurred after approximately 9 hours after induction of the phage genes. Only in the strain Ec(RL123i) harbouring all three lysis genes the effect of the cell lysis on the absorbance was clearly visible after approximately 4 hours after induction. The harbouring integrated genetic circuits L1, L2, L3, L12, L13 and L123, respectively. Cells were grown at the restrictive conditions for the repressor for 24 hours (30˚C) and at the restrictive/permissive conditions (restrictive conditions for 3 hours, followed by the permissive conditions for additional 21 hours (30/42˚C)). Experiments were carried out in triplicate and the mean and standard error were calculated. T-tests were conducted to compare the differences between the absorbance at the restrictive (30˚C) and restrictive/permissive conditions (30/42˚C) at the end of measurement period for each analyzed strain. Stars show significant difference (p<0.05) between the absorbance at 30˚C and 30/42˚C for each strain. The differences were significant in all strains, except Ec(Ri) and Ec(RL2i). (B) Figure shows combinatorial effect of lysis genes on the degree of lysis calculated from the differences between the mean absorbances of cultures grown at 30˚C and 30/42˚C at the end of measurement period. T-tests were performed to compare the differences between strains Ec(RL123i) and other strains harbouring fewer lysis genes. Stars indicate significant differences (p<0.05) in the lysis efficiencies between Ec(RL123i) and other strains.
doi:10.1371/journal.pone.0165778.g002 differences in the kinetics of the cell lysis can be attributed to the variable gene components used in cell lytic systems. The previously developed systems utilized entire phages to lyse the E. coli cell [17,18]. The entire lambda phage genome has approximatelly 48 kb and in addition to lysis genes encodes a number of proteins crucial for the phage's life cycle. Furthermore, following induction, genome of the wild type lambda phage starts replicating and around 100 virions are released from each cell after lysis [42]. In comparison, our cell lytic system relies solely on individual phage lysis genes which are stably integrated into the host cell's chromosome. Thus there is only a single copy of individual lysis genes in each cell and there are no other phageencoded proteins present that contribute to the phage's life cycle.
The outcome of the absorbance measurement can be influenced by a number of factors (e.g. debris from the lysed cells). Therefore, to confirm the results of absorbance assays, lysis efficiencies were also investigated on the level of the individual cells. Bacteria grown at the restrictive and permissive conditions were stained with the BacLight Live/Dead bacterial viability stain and examined by fluorescent microscopy. The survival rates of strains Ec(RL1i), Ec (RL2i), Ec(RL3i), Ec(RL12i), Ec(RL13i), and Ec(RL123i) after temperature shift were significantly reduced when compared to the E. coli strain with no lysis genes Ec(Ri) (Fig 3). Furthermore, the statistical analysis showed that there were significant differences in the proportion of live bacteria between Ec(RL123i) and strains harbouring fewer lysis genes [Ec(RL1i) (p<0.01), Ec(RL2i) (p<0.005), Ec(RL3i) (p<0.05), Ec(RL12i) (p<0.05)], with the exception of Ec(RL13i) (p>0.05) (Fig 3). The survival of Ec(RL123i) was most affected (Fig 3), thus confirming that combining lysis genes from bacteriophages MS2, FX174, and lambda in a single E. coli cell improves lysis.

DNA transfer from E. coli to B. subtilis by induced cell lysis
To test the suitability of the engineered autolytic E. coli cells for interchassis DNA transfer we transferred plasmid pJScav and bacterial artificial chromosome iBAC(cav)-borne genetic circuits cat and ven into B. subtilis. The genetic circuits cat and ven encode Bacillus-specific chloramphenicol resistance gene and the yellow fluorescent protein-encoding gene mVenus flanked by the integration sequences homologous to amyE target site in the B. subtilis chromosome (Fig 4A). To transfer pJScav and iBAC(cav)-borne genetic circuits cat and ven we used culture mix method [17,18]. pJScav and iBAC(cav) were first introduced into E. coli strains Ec(Ri), Ec (RL1i), Ec(RL2i), Ec(RL3i), Ec(RL12i), Ec(RL13i), and Ec(RL123i) by transformation. Temperature shift led to E. coli cell lysis and release of pJScav and iBAC(cav) from Ec(RL1i), Ec(RL2i), Ec(RL3i), Ec(RL12i), Ec(RL13i), and Ec(RL123i). pJScav and iBAC(cav) were then taken up and incorporated into the B. subtilis cells cultivated in the same test tube. The resulting recipient B. subtilis colonies grew on medium containing chloramphenicol and emitted yellow fluorescent light (Fig 4B). This confirmed functionality of the integrated genetic circuits cat and ven in B. subtilis. Presence of amyE integration target sequences on pJScav and iBAC(cav) led to the integration of genetic circuits cat and ven into the amyE locus of the B. subtilis chromosome (Fig 4C). Integrations into the amyE locus were verified by diagnostic PCR using flanking primers ( Fig 4C) and sequencing. The addition of DNAse I into the culture media completely suppressed pJScav and iBAC(cav) transfer from E. coli to B. subtilis (Fig 4B), thus confirming that the transfer occurred via stable DNA in the medium. This is in line with the results of the previous study investigating DNA transfer from E. coli to B. subtilis by co-culture method [17].
The statistical T-test analysis was performed to compare the differences in the transfer of pJScav and iBAC(cav) between Ec(RL123i) and other investigated strains harbouring fewer lysis genes (Fig 4D). This analysis revealed that there were significant differences in the transfer of pJScav-encoded DNA between Ec(RL123i) and all strains with fewer than three lysis genes Notably, in addition to Ec(RL1i), Ec(RL2i), Ec(RL3i), Ec(RL12i), Ec(RL13i), and Ec (RL123i), transfer of pJScav and iBAC(cav) into B. subtilis occurred even in the absence of any , respectively. Green fluorescence shows viable bacteria with intact cell membranes, while red fluorescence indicates dead cells with damaged membranes. The numbers of the dead and live bacteria were determined 5-6 hours after the temperature shift from 30˚C to 42˚C. The survival of Ec(RL123i) was most reduced when compared to the wild type Ec(Ri). The means and standard errors were calculated from three experiments. T-tests were performed to compare the differences in the proportion of live bacteria between Ec(RL123i) and strains harbouring fewer lysis genes. Stars indicate significant difference in the percentage of live bacteria between Ec(RL123i) and the analyzed strain. The differences in the proportion of live bacteria were statistically significant (p<0.05) in all strains with the exception of Ec(RL13i).  subtilis transformants per ml of mixed E. coli and B. subtilis cultures using pJSav and iBAC(cav) 2 hours and 6 hours after temperature induced lysis. The number of transformants was highest with the strain Ec(RL123i) harbouring lysis phage gene expression using strain Ec(Ri). This is due to spontaneous lysis of the fraction of E. coli cell population [43]. DNA released from the spontaneously lysed E. coli cells can be taken up by B. subtilis cultivated in the same test tube. The number of transformants using both pJScav and iBAC(cav) was highest with strain Ec(RL123i), thus showing that combining lysis genes from phages MS2, FX174, and lambda in a single cell improves transfer of genetic circuits from E. coli to B. subtilis (Fig 4D).

Conclusions
We set out to provide the synthetic biology community with a reliable system for the transfer of genetic circuits from E. coli to B. subtilis. This is particularly important for transfer of the high molecular weight DNA molecules, such as BACs, which are prone to mechanical shearing by pipetting [17,18]. As the presence of an entire phage could be detrimental to the quality of the transferred DNA we aimed to efficiently lyse E. coli cells using solely the lysis genes from multiple bacteriophages. To this end, we investigated lysis genes from phages MS2, FX174, and lambda. Genetic circuits L1, L2, L3, L12, L13 and L123 harbouring different combinations of phage lysis genes were integrated into the E. coli chromosome using the lambda Red recombineering approach [20]. Our results show that cells harbouring lysis genes from all three bacteriophages lyse with higher efficiency than those with single or two lysis genes. This could be due to the different mechanisms of action of the three phages tested (e.g. phage lambda forms small holes across the cell surface, while MS2 forms a bigger hole at the septal area and φX174 inhibits PG biosynthesis). The engineered autolytic cells allow reliable and efficient transfer of the plasmid and BAC-encoded genetic circuits from E. coli into B. subtilis.
To our knowledge, this is the first study investigating synergistic effect of lysis genes from three different bacteriophages on the lysis of E. coli and DNA transfer from the lysed cells into the B. subtilis chromosome. Our system does not require the traditional DNA isolation and transformation techniques which are time-consuming. The advantages of our system over other cell lysis methods, such as enzyme degradation and mechanical disruption, particularly on the industrial scale, include lower production and product recovery costs. Furthermore, in our conditionally-inducible cell lysis system there is no need to add an inducer to initiate cell lysis. At the laboratory research scale, transfer of DNA from E. coli undergoing phage-induced lysis to B. subtilis by the co-culture method [17,18] used in our system mitigates against mechanical shearing, which is a serious issue particularly when manipulating the high molecular weight DNA molecules. Previous approaches utilizing an entire phage to lyse the donor E. coli cells can be detrimental to the quality of the transferred DNA, thus in our system we used solely the phage lysis genes. In the future, this system could be further enhanced by utilization of other lysis genes, such as those of bacteriophages T4, T7, and Mu. Other phage-encoded genes crucial for phage's life cycle could be integrated into the chromosome to improve the kinetics of cell lysis. Alternative promoters, such as P mgtB , P nisA , P cmp or P lac -LacI [16,44] and the visible light-sensitive lambda cI repressor [45] could be used to fine-tune expression of lysis genes.
genes from all three bacteriophages. T-tests were conducted to compare the differences in pJScav and iBAC(cav)encoded DNA transfer between Ec(RL123i) and strains harbouring fewer than three lysis genes. Stars indicate significant difference in the DNA transfer between Ec(RL123i) and the other investigated E. coli strains. The differences were significant (p<0.05) in all cases except when compared to iBAC(cav) transfer in Ec(RL3i) and Ec (RL13i) 2 hours after induction.