Transcriptome analysis of ionic-liquid tolerant Bacillus amyloliquefaciens CMW1 and identification of a novel efflux pump

Abstract Bacteria that exhibit ionic-liquid (IL) tolerance are useful in chemical industries using renewable carbon sources pre-treated by ILs to produce biofuels and fine chemicals. An IL, 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), has a remarkable ability to solubilize wood components, whereas [BMIM]Cl inhibits the growth of various bacterial hosts useful for bioconversion. We previously isolated a 10% [BMIM]Cl-tolerant bacterium Bacillus amyloliquefaciens CMW1. Here we report novel mechanisms of tolerance to [BMIM]Cl in strain CMW1 and a novel major facilitator superfamily transporter coded by an ionic-liquid tolerance (ILT) gene. First, using CMW1 cells grown in the presence or absence of 10% [BMIM]Cl, whole-transcriptome analysis and differentially expressed gene analysis were performed. Probable mechanisms of tolerance to [BMIM]Cl include the uptake of osmoprotectants from the culture medium toward CMW1 cells and the export of [BMIM] cations that accumulated in CMW1 cells. The finding represents a first step in elucidation of the mechanisms of IL resistance in Gram-positive bacteria. Second, we conferred tolerance to 5% [BMIM]Cl on [BMIM]Cl-susceptible Brevibacillus choshinensis using ILT gene. This finding provides a notable basis for engineering IL-tolerant bacterial hosts that are applicable for the effective and sustainable production of industrially important chemicals.


Introduction
Ionic liquids (ILs) are a great variety of organic-molten salts that are composed of a bulky asymmetric cation and a small anion. Cation and anion combinations can be modified, and hydrophilic and hydrophobic ILs can be prepared [1,2]. Unlike conventional organic solvents applied for biocatalytic reactions, ILs remain liquid over a wide range of temperatures and possess no vapour pressure [3]. An IL, 1-butyl-3-methylimidazolium chloride ( [BMIM]Cl), improved the digestibility of various biomasses (e.g. cellulose, chitin and keratin). But [BMIM]Cl was toxic to the bacteria that are used in the subsequent production step of valuable compounds (e.g. liquid fuels and fine chemicals) [4][5][6]. Therefore, there exist demands for a bacterial host that is tolerant to [BMIM]Cl and elucidation of the resistance mechanisms to [BMIM]Cl for the development of an efficient production step.
The toxicity of ILs against microorganisms is considered to be due to the accumulation of ILs, which have hydrophobic and hydrophilic structures, toward the cell membrane and cytoplasm [7,8]. Nonetheless, several IL tolerant microorganisms have been identified recently. Fungi, such as Aspergillus sp. and Saccharomyces sp. [9,10], several Gram-positive bacteria, such as Nocardiopsis sp., Brevibacterium sanguinis, and Rhodococcus erythropolis [11,12], and a Gram-negative bacterium, such as Pluralibacter lignolyticus SCF1 [8,13], exhibit various levels of tolerance to various ILs. The fatty acid composition and transcriptional responses of the Gram-negative P. lignolyticus SCF1 were analyzed [8]. It has been reported that the increase of cyclopropane fatty acids in the cell membrane and up-regulation of osmoprotectant transporters and drug efflux pumps are responsible for the resistance to 1-ethyl-3-methylimidazolium chloride ([EMIM]Cl). The recombinant Escherichia coli expressing the multidrug efflux pump gene, eilA gene, of P. lignolyticus SCF1 grew in the presence of 410 mmol/L [EMIM]Cl [14]. It was suggested that EilA transports [EMIM] cations out of the cells and confers resistance to [EMIM]Cl on the recombinant cells. EilA belongs to the major facilitator superfamily (MFS) transporter and plays a critical role in the tolerance to [EMIM]Cl.
MFS is composed of secondary transporters that use the electrochemical gradient of protons across the cell membrane to catalyze the export of substrates, such as drugs, toxic compounds and antibiotics [15][16][17]. MFS is classified into 82 families according to the transporter classification system (http://www.tcdb. org). Drug: H þ antiporter families (DHA) 1, 2 and 3 are the largest drug exporter families in MFS. Although the properties of the efflux pump EilA of P. lignolyticus SCF1 were elucidated [8,14], those of the efflux pumps of other bacteria that exhibit IL tolerance have not yet been investigated. There are many unknowns in the effects of bacterial MFS against various ILs.
We previously isolated a [BMIM]Cl-tolerant Grampositive bacterium, Bacillus amyloliquefaciens CMW1 [6]. Strain CMW1 is a moderately halotolerant bacterium that grows in the presence of >10% (v/v, 631 mmol/L) [BMIM]Cl, which is toxic to most bacteria [11,18]. Strain CMW1 produces an extracellular protease that exhibits tolerance to ILs, halo, alkaline and organic solvents and the bioactive peptide that exhibits antibacterial activity against Staphylococcus aureus [18,19]. Strain CMW1 was transformed by electroporation using a plasmid pHY300PLK, a shuttle vector for E. coli and Bacillus subtilis (Kurata, unpublished data). The transformation efficiency was 2.8 Â 10 3 transformants per 1 mg of plasmid DNA. Although strain CMW1 has many utilities for industrial applications, the resistance mechanism of strain CMW1 against [BMIM]Cl has remained unclear. Therefore, to understand the molecular basis for the tolerance of strain CMW1 against [BMIM]Cl, we performed whole-genome gene expression analysis using RNA-sequencing and identified differentially regulated genes in the presence or absence of 10% [BMIM]Cl. Additionally, using the transporter gene that was upregulated in the presence of 10% [BMIM]Cl, we showed the responsibility of the transporter gene for the tolerance of the bacterial cell to [BMIM]Cl using a heterologous gene expression system. The novel transporter gene was designated as the ionic-liquid tolerant (ILT) gene. Our finding represents an important advance in understanding the mechanisms of IL tolerance of bacteria and provides a foundation for engineering IL-tolerant strains.

Library construction, sequencing and bioinformatic analysis
Using the total RNA samples (7.4 mg for þ10% [BMIM]Cl and 9.2 mg for À [BMIM]Cl), each library was constructed and RNA-seq was carried out at Hokkaido System Science (Hokkaido, Japan). After the elimination of rRNA using a Ribo-zero rRNA removal kit (Illumina, San Diego, CA), each cDNA library was prepared using the TruSeq RNA sample preparation kit (Illumina). Each cDNA was sequenced using paired-end methods (100 bp) by a HiSeq System (Illumina) to obtain reads. The functional annotation of the strain CMW1 genome sequence was previously carried out [6]. In Table 1, Annotation was performed by D-FAST (https:// dfast.nig.ac.jp). The obtained reads were mapped to the reference CMW1 genome sequence (GenBank Accession No. DF836084 through DF836091) using the software tools Tophat (version 2.0.14) and Bowtie (version 1.1.1). The read abundances mapping to the reference CMW1 genome sequence was normalized by the FPKM calculation (fragments per kilobase of transcript per million RNA-seq reads) using the software tool Cufflinks (version 2.2.1). Subsequently, differentially expressed gene analysis was performed. Gene expression change due to the addition of 10% [BMIM]Cl is indicated by the value of log 2 (fold change) that refers to the ratio of expression levels in the presence of 10% [BMIM]Cl against that in the absence of [BMIM]Cl. The false discovery rate is indicated by the p-value. The classification criteria for differentially expressed genes were p value < .05 and absolute value of jlog 2 (fold change)j > 1 [20,21]. In Table 1, 'Locus' indicates the location of the gene at genome sequence of B. amyloliquefaciens CMW1.

Phylogenetic analysis of ILT and homologous proteins
The phylogenetic tree was constructed using amino acid sequences of ILT (GenBank accession number LC554416) from B. amyloliquefaciens CMW1, homologous proteins from B. subtilis strain 168 (NP_388149, NP_388189, NP_388461, NP_388672, NP_390281, NP_390536, NP_391167, NP_391637, and NP_391957), Bacillus cereus ATCC 14579 (AAP10250), Staphylococcus aureus (WP_000170403 and WP_001041274), Streptococcus pneumoniae (VPM23664 and WP_000136147), Streptococcus pyogenes (WP_000417517) and Clostridium perfringens (YP_001967742), and lactose permease (NP_414877) from E. coli strain K-12 as an out group. Amino acid substitution rates were determined, and a distance matrix tree was constructed by using the neighbour-joining (NJ) method with the CLUSTALX program. Alignment gaps and unidentified base positions were not taken into consideration for the calculations. The topology of the phylogenetic tree was evaluated by performing a bootstrap analysis with 1000 replications. NJ plot software (http://pbil.univ-lyon1.fr/software/njplot. html) was used to prepare a graphical view of the phylogenetic tree.

Evaluation of [BMIM]Cl tolerance with B. choshinensis harbouring pBIC-ILT
The growth of recombinant cells was compared with that of non-recombinant cells, and the [BMIM]Cl tolerance contributed by the ILT gene was investigated. Recombinant B. choshinensis harbouring pBIC-ILT and non-recombinant B. choshinensis were individually inoculated in the 5% (v/v, 316 mmol/L) [BMIM]Cl medium described above. Subsequently, each inoculated medium was shaken at 37 C for 24 h, and each cell growth was confirmed by measuring OD 600 . The measurement of cell growth was carried out using three independent cell cultures with 5% [BMIM]Cl.

RNA sequencing and differentially expressed gene analysis
We previously reported that the [BMIM] cations are not degraded by strain CMW1 and the [BMIM] cations do not accumulate in CMW1 cells [18]. Therefore, we investigated the molecular mechanisms of tolerance of B. amyloliquefaciens CMW1. B. amyloliquefaciens CMW1 grew until the late-exponential phase in the presence of 10% [ Table S1). Transcriptional analysis revealed that gene expression levels of 359 genes were changed in the presence of 10% [BMIM]Cl using the parameters p value < .05 and jlog 2 fold-changej > 1. Of these, 277 genes were up-regulated (>twofold increase) and 82 genes were down-regulated (>two-fold decrease) in the presence of 10% [BMIM]Cl.
Expression changes of genes related to uptake of osmoprotectants and genes related to drug efflux pumps It was reported that P. lignolyticus SCF1 grows in the presence of 8.2% (v/v, 562 mmol/L) [EMIM]Cl [8]. P. lignolyticus SCF1 probably resists the toxic effect of [EMIM]Cl by taking up osmoprotectants (sugars and amino acids) from the environment and pumping the [EMIM] cations out of the cells. We reported that B. amyloliquefaciens CMW1 has genes associated with the uptake of osmoprotectants [6]. Under the 10% [BMIM]Cl-added condition, genes for uptake of osmoprotectants, such as glycine betaine, L-carnitine and choline, from the culture medium were up-regulated in strain CMW1 (Table 1). In Bacillus sp. bacteria, proline and analogous compounds are considered to be effective osmoprotectants [22,23]. In the metabolism of proline and analogous compounds in strain CMW1 under the 10% [BMIM]Cl-added condition, the gene expression levels of amino acid permease in the 4hydroxyproline catabolic gene cluster (Gene No. 60) and proline dehydrogenase (Gene No. 93) were up-regulated with 6.04036-and 2.987-fold increases, respectively (Supplemental Table S1). The permease and dehydrogenase probably aid the intercellular accumulation of osmoprotectants or their precursors to offset the osmotic pressure generated by exposure to [BMIM]Cl. The result shows that the osmotic-stressresistance genes contributed to the IL tolerance in strain CMW1 as in P. lignolyticus SCF1.
We showed the expression levels for the genes of drug efflux pumps in strain CMW1 (Table 1). Although the drug resistance transporter gene (Gene No. 95) was down-regulated with a À 2.17571-fold decrease in the presence of 10% [BMIM]Cl, the multidrug resistance protein B gene (Gene No. 175) was up-regulated with a 2.80335-fold increase. Amino acid sequence analysis predicted that multidrug resistance protein B (Gene No. 175) belongs to a membrane transporter. Although we identified at least 29 membrane transporter genes in the genome sequence of strain CMW1 [6], significant changes of expression levels of other genes, except for the multidrug resistance protein B (Gene No. 175), were not detected by classification using the parameters p value < .05 and jlog 2 foldchangej > 1. Therefore, it was suggested that the membrane transporter coded by the gene (Gene No. 175) functions as a specific efflux pump against [BMIM] cations in strain CMW1.

Conservation of ILT gene in grampositive bacteria
We found the gene (Gene No. 175) that probably catalyzes the transport of [BMIM] cations out of the cells and designated it the ionic-liquid tolerant (ILT) gene. Amino acid sequence analysis using the BlastP program in the transporter classification system (http:// www.tcdb.org) classified ILT as a multidrug efflux transporter of MFS. We show the phylogenetic relationship of the amino acid sequence of ILT to those of representative MFS transporters (namely, Drug: H þ antiporter family (DHA) 1 through 4) of Gram-positive bacteria (Figure 2, Bar, 0.05 amino acid substitutions per site). ILT clearly belongs to DHA1. It was reported that transporter EilA of P. lignolyticus SCF1 is involved in the tolerance to [EMIM] cations and is an MFS transporter by amino acid sequence analysis [14]. BlastP analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) found no high homology between the amino acid sequence of ILT and that of EilA, indicating that ILT is different from EilA. Therefore, we found that ILT is a novel transporter belonging to DHA1 of MFS and potentially transports [BMIM] cations.

Multiple sequence alignment of ILT and its homologous transporters
Sequence analysis using BlastP showed that ILT exhibits an amino acid sequence similarity to multidrug efflux pump EmrD (NP_418129), MFS transporter AraJ (P23910), and sugar efflux transporter YdeA (P31122) from E. coli K-12 with 21.84%, 19.85% and 19.00% amino acid identities, respectively. In Figure 3, we indicated multiple alignment of amino acid sequence of ILT with those of MFS transporters. Conserved amino acid residues in the transporters are indicated by black boxes.
Transmembrane (TM) helices 1 through 12 of ILT and those of EmrD are indicated by dashed and solid lines, respectively. The TM helices of EmrD were determined by X-ray structure analysis [24]. The TM helices of ILT were predicted using the HMMTOP program (http://www.enzim.hu/hmmtop/). The sequence analysis indicated that ILT is a homologue of proton antiporters, possessing 12 TM helices that span the inner membrane. The predicted TM 1-8 of ILT overlapped well with TM1-8 of EmrD. The predicted TM 9-12 of ILT partially overlapped with TM 9-12 of EmrD.
Sequence alignment revealed that amino acid sequence motifs characteristic for MFS transporters, namely motif B (L-x-x-x-R-x-x-x-G at TM4, x indicating any amino acid) and motif C (G-x-x-x-G-P-x-x-G-G at TM5), were conserved in ILT. Motifs B and C are indicated under the aligned amino acid sequences. Motif B contains a basic amino acid residue (Arg105 of ILT) that plays a role in proton transfer, and motif C is conserved only in exporters and not importers [25,26]. The functional and structural characterizations of motifs B and C support the probable efflux function of ILT.
In the case of EmrD, the hydrophobicity of Val17, Ile28, Tyr52 and Ile217 play a critical role in recognition of the hydrophobic structure of substrates [24], and the hydrophobicity was conserved by Phe20, Ile30, Phe54 and Phe225 in ILT. It suggests that these amino acids recognize the hydrophobic domain of the [BMIM] cation. In Figure 3, the amino acids are indicated by closed triangles. Glu227 of EmrD, which is involved in import of protons, is conserved in ILT as Glu235. In Figure 3, the amino acid is indicated by an open circle. Asp123, which is involved in the topology of EmrD, is conserved in ILT as Asp125. In Figure 3, the amino acid is indicated by an open triangle. Consequently, amino acid sequence analysis indicated that ILT would probably function as a novel proton antiporter that localizes at the cell membrane and exports the [BMIM] cation that accumulated in the cytosol.  [14]. Therefore, we investigated whether the ILT gene confers resistance to 5% (v/v, 316 mmol/L) [ Figure 4(B), the growth of recombinant B. choshinensis expressing ILT was promoted in the medium containing 5% [BMIM]Cl, compared with that of non-recombinant B. choshinensis. The results suggested that ILT of B. amyloliquefaciens CMW1 contributes to the bacterial resistance against [BMIM]Cl by pumping [BMIM] cations as in EilA of P. lignolyticus SCF1.

Expression of ILT in
Generally, it is investigated that various biomasses are dissolved in ILs and subsequently converted to valuable compounds by microorganisms [27][28][29]. There is a potential economic incentive for unifying the process by using an IL-tolerant bacterium that would carry out degradation of pre-treated biomass and production of valuable compounds. We conferred tolerance to 5% [BMIM]Cl on [BMIM]Cl-susceptible B. choshinensis using ILT gene. We plan to examine what concentration of [BMIM]Cl the transformant can grow at.

Conclusions
We reported the first phylogenetic and functional characterization of ILT as a member of the DHA1 in MFS. When [BMIM] cations accumulate in the CMW1 cell, ILT would use the electrochemical gradient of protons across the cell membrane to export the accumulated [BMIM] cations out of cells. Thus, ILT potentially functions as a specific transporter for an imidazoliumic cation of IL. In future studies, we will purify ILT from B. amyloliquefaciens CMW1 and investigate the characteristics of ILT, such as, transporter activity, substrate specificity and so on. Transcriptional analysis of IL tolerance microorganisms that are identified recently and elucidation of the properties of IL efflux pumps are urgently required for development of useful compounds production systems using ILs and biocatalysts.

Disclosure statement
No potential conflict of interest was reported by the authors.

Data availability
The data that support the findings reported in this study are available at https://datadryad.org/stash/dataset/doi:10.5061/ dryad.fj6q573st.