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. Author manuscript; available in PMC: 2009 Mar 1.
Published in final edited form as: Plasmid. 2008 Jan 25;59(2):111–118. doi: 10.1016/j.plasmid.2007.12.002

Engineering of FRT-lacZ fusion constructs: induction of the Pseudomonas aeruginosa fadAB1 operon by medium and long chain-length fatty acids

Mike S Son 1, David T Nguyen 1, Yun Kang 1, Tung T Hoang 1,*
PMCID: PMC2276238  NIHMSID: NIHMS42856  PMID: 18221997

Abstract

Without prior knowledge of the promoters of various genes in bacteria, it can be difficult to study gene regulation using reporter-gene fusions. Regulation studies of promoters are ideal at their native locus, which do not require prior knowledge of promoter regions. Based on a previous study with FRT-lacZ-KmR constructs, we constructed two novel FRT-lacZ-GmR plasmids. This allows easy engineering of P. aeruginosa reporter-gene fusions, post-mutant construction with the Flp-FRT system. We demonstrate the usefulness of one of these FRT-lacZ-GmR plasmids to study the regulation of the fadAB1 operon in P. aeruginosa at its native locus. The fadAB1 operon, involved in fatty acid (FA) degradation, was significantly induced in the presence of several medium chain-length fatty acids (MCFA) and, to a lesser degree, long chain-length fatty acids (LCFA). In addition to the previous work on the FRT-lacZ-KmR tools, these new constructs increase the repertoire of tools that can be applied to P. aeruginosa or other species and strains of bacteria where kanamycin resistance may not be appropriate.

Keywords: Pseudomonas aeruginosa, FRT-lacZ fusion, fatty acid degradation

Introduction

Studying gene-regulation is the foundation of understanding cellular physiology. Specifically in bacteria, gene-expression studies have become more elaborate and the technologies more complex. Gene-expression profiles can be achieved using macro- and microarrays (Firoved et al., 2004) and quantitative real-time polymerase chain reactions (qRT-PCR) (Freeman et al., 1999; Savli et al., 2003). However, these innovative technologies can prove costly and a classical approach may be more appropriate.

Many plasmid-based systems have been developed to study gene-expression by creating reporter-gene fusions, such as lacZ (Hand and Silhavy, 2000; Hoang et al., 2000; Manoil, 2000; Schweizer, 1991; Silhavy and Beckwith, 1985; Slauch and Silhavy, 1991), xylE (Hassett, 2000), the luxAB of (Gonzalez-Flecha, 1994), and the green fluorescent protein (gfp) (Miller, 1997). However, they all suffer from inherent problems such as titration of regulators for high-copy plasmids, and incorrect representation of the natural regulation of the gene(s). Previously, lacZ fusions were introduced into P. aeruginosa using the mini-CTX system (Hoang et al., 2000) or the Tn7 integration system (Choi et al., 2005), allowing site-specific lacZ fusions to be studied as a more representative single copy. However, these existing systems require prior knowledge of the promoter region, and fusions other than at the native locus may misrepresent the more complex natural regulatory mechanism. More recently, Ellermeier et al constructed a Flp-mediated site-specific recombination system, where lacZ can be introduced into the chromosome at the native locus as a transcriptional fusion, using a Flp recombination target sequence (FRT) (Ellermeier et al., 2002). This system requires a chromosomally located FRT previously introduced via the λ-Red mutant construction method (Hand and Silhavy, 2000). A plasmid harbouring lacZ and a kanamycin resistance (KmR) cassette downstream of an FRT (FRT-lacZ-KmR) was easily constructed (Ellermeier et al., 2002). When Flp was introduced, the two FRTs recombined, integrating the plasmid-borne FRT-lacZ-KmR fusion with the chromosomally located FRT, to generate a lacZ transcriptional fusion. However, P. aeruginosa can have high resistance levels to kanamycin (≥1000 µg/ml), and a cleaner alternative selectable marker (i.e. gentamycin resistance) is more desirable.

Fatty acid degradation (Fad) is an essential pathway for bacteria to utilize lipids and other fatty acid-associated compounds. In E. coli, Fad has been well characterized (Black and DiRusso, 1994; Clark and Cronan Jr., 1996), where several of the genes, fadA, fadB, fadD, fadE and fadL, are negatively regulated by FadR in the absence of external fatty acids (FA) ≥C12:0. However, virtually nothing is known about Fad in P. aeruginosa, and the regulatory mechanism remains an enigma. With a genome of 6.3Mb (Stover et al., 2000), P. aeruginosa has 6–8 fold more genes involved in Fad than E. coli, suggesting increased complexity and redundancies in the Fad pathway. P. aeruginosa is capable of growing on short (C4:0–C8:0), medium (C10:0–C14:0) and long chain-length FAs (≥C16:0), and the involvement of one Fad operon has been identified as fadAB1 (PA1737 and PA1736, respectively) (Son et al., 2003).

Here, we report the engineering of two FRT-lacZ-GmR plasmids, one of which was utilized to generate a chromosomal PfadAB1-FRT1-lacZ transcriptional fusion. This fusion was characterized in P. aeruginosa by monitoring the regulation of the fadAB1-promoter at its native locus. P. aeruginosa containing this PfadAB1-FRT1-lacZ fusion was grown and characterized in minimal glucose media with different chain-length FAs (short chain-length FAs (SCFA), medium chain-length FAs (MCFA) and long chain-length FAs (LCFA)). These constructs add to the repertoire of FRT-based tools previously reported (Ellermeier et al., 2002) to study non-essential gene function and regulation, because these genes are often involved in virulence and pathogenesis.

Materials and Methods

Bacterial Strains and Media

Strains and plasmids used and constructed in this study are shown in Table 1. E. coli DH5α (Liss, 1987) was used for all molecular cloning steps. E. coli SM10 (Simon et al., 1983) was the donor strain for conjugation to introduce the plasmid pEX18TΔfadAB1::FRT-GmR-FRT into wild-type P. aeruginosa PAO1 to create a mutant PAO1ΔfadAB1::FRT-GmR-FRT. For tri-parental mating to introduce the FRT1-lacZ transcriptional fusion into PAO1ΔfadAB1::FRT, E. coli strains ER2566mob-pir116/pFRT1-lacZ and ER2566mob-pir116/pCD13SK-Flp-oriT were used as donors. All E. coli strains were grown in LB broth (Difco, Sparks, MD), with gentamycin (15 µg/ml) or streptomycin (25 µg/ml) (Teknova, Halfmoon Bay, CA) for plasmid maintenance. P. aeruginosa strains were grown in Pseudomonas Isolation Broth (PIB) or Pseudomonas Isolation Agar (PIA) (Difco, Sparks, MD), supplemented with gentamycin (150 µg/ml) for selection, unless otherwise stated.

Table 1.

Bacterial strains and plasmids

Genotype Reference
Strains
E. coli DH5α (ϕ80dlacZ ΔM15) Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rK−mK+) glnV44 λ− thi-1 gyrA96 relA1 (Liss, 1987)
SM10 thi-1 thr leu tonA lacY supE recA::RP4-2-Tc::Mu KmR (Simon et al., 1983)
ER2566mob-pir116 F− λ−fhuA2 [lon] ompT lacZ::T7 gene1 gal sulA11 Δ(mcrC-mrr) 114::IS10 R(mcr-73::miniTn10)2 R(zgb-120::Tn10)1 endA1 [dcm] recA:RP4-2Tc::Mu Km pir116-TetR This study
DH5α–λattB:pCD13SK-Flp (ϕ80dlacZ ΔM15) Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rK−mK+) glnV44 thi-1 gyrA96 relA1 λattB:pCD13SK-Flp SpR This study
P. aeruginosa PAO1 Prototroph (Stover et al., 2000)
PAO1ΔfadAB1::FRT-GmR-FRT GmR, PAO1 with fadAB1 operon deletion and FRT-GmR-FRT insertion
PAO1ΔfadAB1::FRT PAO1ΔfadAB1::FRT-GmR-FRT with GmR-cassette removed This study
PAO1ΔfadAB1::FRT1-lacZa GmR, PAO1 with fadAB1 operon deletion and FRT1-lacZ insertion This study
Plasmids pPS856 ApR, GmR, plasmid with FRT-GmR-FRT cassette (Hoang et al., 1998)
pEX18T ApR, oriT+, sacB+, gene replacement vector (Hoang et al., 1998)
pEX18TfadAB1 ApR, pEX18T with wild-type fadAB1 operon This study
pEX18TΔfadAB1::FRT-GmR-FRT ApR, GmR, pEX18T with ΔfadAB1 operon with FRT-GmR-FRT insertion This study
pFlp2 ApR, sacB+, plasmid with flp gene (Hoang et al., 1998)
pTZ120 ApR, lacZ operon fusion vector (Schweizer and Chuanshuen, 2001)
pXR6KΔS GmR, sacB−, suicidal vector This study
pCD13SK SpR, λattB integration vector (Platt et al., 2000)
pCD13SK-Flp SpR, pCD13SK with flp gene and cI857 This study
pCD13SK-Flp-oriT SpR, pCD13SK-Flp with oriT This study
pFRT1-lacZa GmR, FRT1-lacZ fusion containing suicidal vector This study
pFRT2-lacZa GmR, FRT2-lacZ fusion containing suicidal vector This study
pDTN100::FRT1-GmR-FRT1a GmR, pTZ120 backbone with FRT1-GmR-FRT1 This study
pDTN100::FRT2-GmR-FRT2a GmR, pTZ120 backbone with FRT2-GmR-FRT2 This study
pDTN100::FRT1-lacZa GmR, pDTN100::FRT1-GmR-FRT1 with GmR-cassette excised This study
pDTN100::FRT2-lacZa GmR, pDTN100::FRT2-GmR-FRT2 with GmR-cassette excised This study
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a

FRT1 and FRT2 refer to opposite orientations of the FRT relative to lacZ (refer to Figure 1A)

Growth curves were conducted in 1x M9 media (1x M9, 1% Brij-58, 0.5 mM MgSO4, 0.02 mM CaCl2) + 40 mM glucose ± 0.1% (w/v) of the individual saturated FAs (butyric acid, C4:0; n-caproic acid, C6:0; caprylic acid, C8:0; capric acid, C10:0; lauric acid, C12:0; myristic acid, C14:0; palmitic acid, C16:0) or an unsaturated FA (oleic acid, C18:1Δ9) (Sigma, St. Louis, MO). 3% stock solutions of FAs were made in equimolar KOH and 1% Brij-58. Cell cultures were diluted 1:4 in 4% Brij-58 prior to taking OD540 measurements.

General Molecular Methods and Reagents

All restriction enzymes, Taq DNA polymerase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA) and used according to manufacturer recommendations. Plasmids were recovered using the Zyppy Plasmid Miniprep Kit (Zymo Research, Orange, CA) and gel purification of DNA bands were performed using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, CA). All primers were from IDT Technologies (Coralville, IA).

Construction of fadAB1::FRT Mutant

The fadAB1 operon (3.7kb) was PCR amplified using forward primer 5’-CGAAAGCTTGCATGGTGCTATCTTCC-3’ and reverse primer 5’-GCGGAATTCGCCCTACCCGTGGCG-3’. PCR was assembled in 50 µl reactions containing 10 ng PAO1 chromosomal DNA, 30 pmoles of each forward and reverse primer, 0.2 mM dNTP, 1x ThermoPol reaction buffer, and 5 units of Taq DNA polymerase. After thirty-four cycles (95°C for 1 min, 58°C for 45 sec, 72°C for 3 min), a final extension at 72°C for 10 min was performed. The product was gel purified, digested with HindIII + BamHI, and cloned into the vector pEX18T, digested with the same restriction enzymes, to yield pEX18TfadAB1.

To create the final gene-replacement vector pEX18TΔfadAB1::FRT-GmR-FRT, the 1.1 kb SmaI digested FRT-GmR-FRT cassette from plasmid pPS856 (Hoang et al., 1998) was ligated into pEX18TfadAB1, previously digested with PstI + BamHI and blunt-ended. pEX18TΔfadAB1::FRT-GmR-FRT was conjugated from E. coli SM10 into PAO1 and unmarked mutations constructed as previously described (Hoang et al., 1998).

pCD13SK-Flp-oriT Helper Plasmid Construction

The 2.5 kb BamHI-SacI fragment containing flp and the λ-repressor from pFlp2 was cloned into pCD13SK cut with the same enzymes, yielding pCD13SK-Flp. The 660 bp oriT from pTZ120 was removed with NarI + NdeI, and blunt-ended. This fragment was cloned into pCD13SK-Flp at the SmaI site to give pCD13SK-Flp-oriT.

pFRT1-lacZ and pFRT2-lacZ Plasmid Construction

Plasmid pTZ120 was digested with SapI and NarI, blunt-ended and self-ligated to remove the ori1600-rep; this gave a final plasmid size of 5.7 kb. The FRT-GmR-FRT cassette (1.1 kb) was excised from plasmid pPS856 using BamHI. This fragment was then cloned into pTZ120, also digested with BamHI, yielding pDTN100::FRT1-GmR-FRT1 and pDTN100::FRT2-GmR-FRT2, which contains the GmR-cassette in the opposite orientation, along with the lacZ fusion. FRT1-lacZ and FRT2-lacZ have identical sequences but differ in the orientation of the FRT relative to the lacZ. These plasmids were transformed into a Flp expression strain (DH5α–λattB::pCD13SK-Flp), to remove the GmR-cassette, yielding pDTN100::FRT1-lacZ and pDTN100::FRT2-lacZ. The 2.3 kb FRT1-lacZ and FRT2-lacZ fragments were then excised from pDTN100::FRT1-lacZ and pDTN100::FRT2-lacZ, respectively, using XhoI + AflII and blunt-ended. These fragments were ligated with the GmR-R6K γori-T1T2 fragment (2.6 kb) from plasmid pXR6KΔS, which was digested with XmnI + PvuII. This ligation gave the final plasmids pFRT1-lacZ and pFRT2-lacZ (Figure 1A).

Figure 1. Schematic of pFRT-lacZ plasmid, helper plasmid, and chromosomal target gene of interest with FRT.

Figure 1

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(A) pFRT1-lacZ or pFRT2-lacZ is conjugated with a (B) helper plasmid, pCD13SK-Flp-oriT, into the recipient P. aeruginosa strain containing an FRT (FRT1) in the same orientation as the fusion. (C) The chromosomal ΔfadAB1::FRT is the locus where the FRT-lacZ fusion will recombine catalyzed by the Flp protein expressed on the helper plasmid. (D) The final recombined product is shown with the fusion integrated into the chromosome at the FRT site. The FRT-lacZ plasmids contain several key features that make this system appropriate for these studies: i) T1 and T2 immediately downstream of the FRT-lacZ fusion prevent any read through; ii) a suicidal R6K γori origin of replication that can only replicate in the presence of a π protein or a strain expressing the pir gene; iii) a GmR-cassette to allow for selection of positive recombinations in P. aeruginosa; iv) a Flp expressing R6K γori suicidal helper plasmid, pCD13SK-Flp-oriT. The expression of Flp is under the control of the λ-promoter, which is repressed by cI857. Abbreviations: cI857, temperature sensitive λ-repressor; flp, Flp protein driven by the λ-promoter; FRT, Flp recognition target; GmR, gentamycin resistance cassette; lacZ, β-galactosidase; oriT, origin of transfer; PfadAB1, fadAB1 promoter; R6K γori, suicidal origin of replication; SpR, streptomycin resistance cassette; T1T2, transcriptional terminators.

Conjugation and Selection

All strains were grown to mid-log phase for conjugation. Conjugation was performed by mixing 500 µl of each donor strain ER2566mob-pir116/pFRT1-lacZ and ER2566mob-pir116/pCD13SK-Flp-oriT, with 500 µl of the recipient strain PAO1ΔfadAB1::FRT1. Cells were pelleted sequentially and the media decanted leaving ~40 µl in the final pelleting step. The conjugation mixture was resuspended in the remaining media and spotted onto cellulose-acetate filters (Satorius, Goettingen, Germany) on LB plates and incubated overnight at 37°C. Filters were resuspended in 1 ml of 1x M9 (Sambrook and D.W., 2001) and plated on PIA containing gentamycin. Positive clones were screened for a 1.2kb PCR product using the GmR-specific forward primer 5’-CATACGCTACTTGCATTACAG-3’ and the fadAB1-specific reverse primer 5’-GCGGAATTCGCCCTACCCGTGGCG-3’.

Growth Curve and β-Galactosidase Assay

PAO1 was grown simultaneously, as a control, with PAO1ΔfadAB1::FRT1-lacZ. Cultures were grown for 14 hours in PIB and the cells were washed twice with 1x M9 media. A 1:100 dilution was inoculated into 50 ml 1x M9 minimal media supplemented as described in Bacterial Strains and Media. Both strains were grown in 1x M9 + 40 mM glucose as controls. Cultures were diluted as described in Bacterial Strains and Media and cell densities were measured in a Beckman-DU7500 spectrophotometer, taking dilution factors into account. Standard β-galactosidase assays were conducted in triplicate as described previously (Lederberg, 1950; Miller, 1972) and mean values determined with S.E.M.

GenBank Accession Numbers

The plasmids pFRT1-lacZ, pFRT2-lacZ and pCD13SK-Flp-oriT have been deposited in GenBank with accession numbers EU034636, EU034637 and EU034638, respectively.

Results and Discussion

When used in conjunction with the gene-replacement system (Hoang et al., 1998), the integration of the FRT-lacZ fusion makes it possible to study the regulation of non-essential genes, eliminating the arduous task of identifying promoters. Using this fusion involves two steps: first, the construction of the mutant containing a single FRT; and second, introduction of the FRT-lacZ fusion through tri-parental mating.

In this study, a fadAB1 mutant was constructed in P. aeruginosa, by inserting a GmR-cassette flanked by FRTs into the operon, as described previously (Hoang et al., 1998). The GmR-cassette and a single FRT were then excised by introducing a flp-containing plasmid, leaving behind a single FRT at the allelic replacement site. This chromosomally located FRT is the subsequent insertion site of the FRT-lacZ fusion.

The two FRT1-lacZ and FRT2-lacZ fusions were generated on the suicidal plasmids pFRT1-lacZ and pFRT2-lacZ, respectively (Figure 1A). Although two FRT-lacZ fusion plasmids were created to accommodate the two orientations of the FRTs, only pFRT1-lacZ was used in this regulation study. During tri-parental mating, the fusion plasmid pFRT1-lacZ (Figure 1A) is introduced into the P. aeruginosa recipient along with the helper plasmid pCD13SK-Flp-oriT (Figure 1B). This helper plasmid encodes Flp, which catalyzes the recombination of the FRT1 on pFRT1-lacZ fusion and the chromosomally located FRT1 within the inactivated fadAB1 operon (Figure 1C). This resulting transcriptional fusion is downstream of the fadAB1-promoter and therefore expression of the fadAB1 operon can be directly correlated to β-galactosidase activity (Figure 1D).

Using the P. aeruginosa mutant, we were able to demonstrate the utility of the FRT1-lacZ fusion to study the induction of the fadAB1 operon by the different chain-length FAs. The mutant carrying the lacZ-reporter fusion was grown in 1x M9 minimal media containing 40 mM glucose supplemented with or without FA. To address the effects of the different chain-length FAs on gene-expression levels of the fadAB1 operon, growth rates and β-galactosidase activity were measured. The growth rates of the PAO1ΔfadAB1::FRT1-lacZ mutant carrying the fusion were compared to that of wild-type PAO1 (Figure 2A and 2B), since growth rate differences can affect gene-expression. Figures 2A and 2B show that the growth rates were identical when the mutant was grown in the same media as wild-type PAO1, and that the addition of FAs both increased the length of stationary phase and decreased the rate of death. In addition, the same growth rates were observed when the mutant was grown in glucose ± individual FAs (Figure 2B). These identical growth rates allowed appropriate comparison of the fadAB1 operon expression level in response to the different FA, by measuring β-galactosidase activities during three different growth phases (early-, mid-, and late-log) for each FA chain-length (Figure 2C). In the presence of short chain-length FAs (SCFA) (C4:0–C8:0), the fadAB1 operon was not induced, as the β-galactosidase activities were relatively equal to that grown in the absence of FA (Figure 2C). The lack of gene induction by the SCFA and the absence of any growth defect in the ΔfadAB1 mutant were observed throughout all growth phases. Our laboratory has identified two other fadAB operons that are also involved in Fad, and future studies are required to show if these other two operons are involved in SCFA degradation.

Figure 2. Growth curves of wild-type PAO1 and PAO1ΔfadAB1::FRT1-lacZ fusion strain grown in glucose and various fatty acids, showing induction of the fadAB1 operon by fatty acids.

Figure 2

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Growth curves of (A) wild-type PAO1 and (B) PAO1ΔfadAB1::FRT1-lacZ fusion strain in glucose media supplemented with and without fatty acids, showing no growth defects in the mutant fusion strain. (C) The fadAB1 operon is regulated by the medium to long chain-length FAs, as indicated by the higher β-galactosidase activity of the FRT1-lacZ fusion. The three growth phases monitored were early-, mid- and late-log growth, and β-galactosidase activities measured in triplicate and displayed as an average. The points chosen at early-log (EL), mid-log (ML) and late-log (LL) for β-galactosidase measurements are indicated in (B).

The fadAB1 operon was most significantly induced when the mutant was grown in the presence of medium chain-length FAs (MCFA) (C10:0 to C14:0). In all growth phases, the effects of the MCFA were similar, with C12:0 causing slightly higher induction levels throughout. In early-log, the basal activity in glucose alone was only 7.3 Miller units, where C10:0 (59.7 Miller units), C12:0 (65.5 Miller units) and C14:0 (40.1 Miller units) induced the fadAB1 operon 8.2-, 9.0- and 5.5-folds, respectively. This trend was mirrored at slightly higher levels in mid-log, where basal activity in glucose was at 7.8 Miller units and induction levels reached 10.1-, 12.7- and 8.5-folds, for C10:0 (79.2 Miller units), C12:0 (99.1 Miller units) and C14:0 (66.4 Miller units), respectively. The most significant increase in fadAB1 operon induction was observed when going from mid- to late-log. In late-log phase, basal levels in glucose alone were at 4.1 Miller units and C10:0 induced the operon up to 26.2-folds (108 Miller units), C12:0 up to 27.6-folds (113.9 Miller units), and C14:0 up to 28.8-folds (118.5 Miller units).

Although P. aeruginosa can metabolize and reach high cell densities when grown solely on long chain-length FA (LCFA) (C16:0 and C18:1Δ9) (unpublished data), the fadAB1 operon was not induced to high levels during early-log (Figure 2C). At early-log, the β-galactosidase activity with these LCFA was 18.9 and 17.3 Miller units for C16:0 and C18:1Δ9, respectively. However, as the cells reached mid-log phase, induction rose to 45.2 Miller units and 39.9 Miller units by C16:0 and C18:1Δ9, respectively, (5.8- and 5.1-folds respectively, compared to glucose alone). As the cells reached late-log, induction rose further up to 69.5 Miller units and 60.2 Miller units by C16:0 and C18:1Δ9, respectively, (16.9- and 14.6-folds respectively, compared to glucose alone). fadAB1 is most responsive to the MCFA in all three growth phases, but also responds to LCFA to a lesser degree.

In summary, we constructed two plasmids for the simultaneous study of promoter activity in conjunction with mutational analysis using the Flp-FRT system. Furthering the existing method of creating unmarked deletions in P. aeruginosa using the gene- replacement system (Hoang et al., 1998), we capitalize on the presence of the FRT left behind after excision of the GmR-cassette with Flp as previously performed for E. coli (Ellermeier et al., 2002). We have constructed a simple FRT-lacZ fusion reporter system that allows one to study the regulation of a particular gene of interest. The utility of this reporter fusion was demonstrated using the fadAB1 operon of P. aeruginosa as a model. The regulation of this operon has not been characterized, and using the FRT1-lacZ fusion, we demonstrated its regulation by MCFA and LCFA. This study would be difficult in P. aeruginosa with the previously reported FRT-lacZ-KmR fusion (Ellermeier et al., 2002), because of high kanamycin resistance levels. However, these novel FRT-lacZ fusion constructs with the GmR-cassette add to the existing repertoire of gene fusion tools for bacteria.

Acknowledgements

This research was partially supported by an NIH grant R03 AI065852. Salary support for M.S.S. was made possible by pilot funding of a grant (P20RR018727) from the National Centre for Research Resources (NCRR), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH. Salary support for D.T.N. was funded through the Hawaii Community Foundation (HCF) grant awarded to T.T.H‥ We are very grateful to Dr. H.P. Schweizer for the generous gift of pTZ120.

Footnotes

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Competing Interests The authors declare no competing interests.

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