Bacterial strains, plasmids, and oligonucleotide primers (Eurofins, MWG Operon, Germany) used in this study are listed in Table 1. Escherichia coli EPI300::pCC1FOS (Epicentre Biotechnologies, Madison, WI, USA) was grown in Luria-Bertani (LB) medium containing 12.5 μg/ml chloramphenicol (Cm). E. coli MKH13 was grown in LB medium and in LB medium supplemented with 20 μg/ml Cm for strains transformed with the plasmid pCI372. LB media was supplemented with 1.5% w/v agar when required. All overnight cultures were grown at 37°C with shaking.

A previously constructed fosmid clone library (Jones and Marchesi, 2007; Jones et al., 2008), created from metagenomic DNA isolated from a fecal sample from a healthy 26 year old Caucasian male was used to screen for salt-tolerant clones. The library was screened as outlined previously (Culligan et al., 2012). Briefly, a total of 23,040 clones from the library were screened on LB agar supplemented with 6.5% (w/v) NaCl (a concentration which inhibits the growth of the cloning host, E. coli EPI300) and 12.5 μg/ml Cm using a Genetix QPix 2 XT colony picking/gridding robotics platform to identify clones with an increased salt tolerance phenotype compared to the cloning host (E. coli EPI300) carrying an empty fosmid vector (pCC1FOS). Identification of any salt tolerant clones will therefore most likely be due to a gene (or genes) present on the metagenomic insert from the human gut microbiota. Clones were gridded onto Q-trays (Genetix) using the robotics platform. Q-trays were incubated at 37°C for 3 days and checked twice daily for growth of likely salt-tolerant clones. Salt-tolerant clones were subsequently replica plated onto LB agar containing 12.5 μg/ml Cm and 6.5% NaCl and onto LB containing 12.5 μg/ml Cm, but without 6.5% NaCl (which represented a positive control plate). Each salt tolerant clone identified was streaked on LB agar + 12.5 μg/ml Cm to ensure a pure culture and all clones were maintained as glycerol stocks at −80°C.

The PM9 osmolytes microplate was used to compare the cellular phenotypes of SMG 9 and the cloning host, E. coli EPI300::pCC1FOS (containing an empty fosmid vector) under 96 different conditions. Preparation of the different IF (Inoculating Fluids; proprietary formulation supplied by BIOLOG) solutions and inoculation of the PM plates was performed according to the BIOLOG PM protocol for E. coli and other Gram negative bacteria. Briefly, IF-0 solution was prepared by adding 25 ml of sterile water to 125 ml of 1.2× IF-0. IF-0 + dye mix A solution was prepared by adding 1.8 ml of dye mix A and 23.2 ml of sterile water to another bottle containing 125 ml of 1.2× IF-0. IF-10 solution was prepared by adding 1.5 ml of dye mix A and 23.5 ml of sterile water to a bottle containing 125 ml of 1.2× IF-10. E. coli strains were grown overnight on LB agar at 37°C by streaking from a frozen stock. Cells were sub-cultured by streaking again on LB agar and grown overnight again. Isolated colonies were removed from the agar plate using a sterile swab and added to a tube containing 16 ml of IF-0 solution until a cell suspension of 42% T (transmittance) was achieved using the BIOLOG Turbidimeter. 15 ml of this 42% T solution was diluted in 75 ml of IF-0 + dye mix A to achieve 85% T. 600 μl of the 85% T cell suspension solution was added to 120 ml of IF-10 + dye mix A. Finally, 100 μl of the final cell suspension was inoculated to each well of the PM 9 microplate. Plates were incubated at 37°C for 24 h in the Omnilog plate reader (BIOLOG).

Fosmid DNA was isolated from SMG 9 as described above to a concentration >200 ng/μl (approximately 5 μg in total). Sequencing of the full fosmid insert of SMG 9 was performed by GATC Biotech (Konstanz, Germany) using 454-pyrosequencing on a titanium mini-run of the Roche GS-FLX platform, achieving approximately 65-fold coverage. Sequencing reads were assembled into a single contig by GATC Biotech. The retrieved sequence was analyzed using the FGENESB software program (Softberry) to identify putative open reading frames and translated nucleotide sequences were subjected to BLASTP analysis to assign putative functions to the encoded proteins and identify homologous sequences from the National Centre for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The full fosmid insert sequence of SMG 9 was submitted to GenBank and assigned the following accession number; KJ524644.

Extraction of fosmids containing metagenomic DNA: 5 ml of bacterial culture was grown overnight with 12.5 μg/ml Cm. One millilitre of culture was used to inoculate 4 ml of fresh LB broth. To this, 5 μl of 1000× Copy Control Induction solution (Epicentre Biotechnologies) and 12.5 μg/ml Cm were added. The mixture was incubated at 37°C for 5 h with vigorous shaking (200–250 rpm) to ensure maximum aeration. Cells were harvested from the whole 5 ml of induced culture by centrifuging at 2100 × g for 12 min. Qiagen QIAprep Spin mini-prep kit was used to extract fosmids as per manufacturer's instructions. PCR products were purified with a Qiagen PCR purification kit and digested with XbaI and PstI (Roche Applied Science) for mfsT and with SalI and XbaI for sdtR, followed by ligation using the FastLink DNA ligase kit (Epicentre Biotechnologies) to similarly digested plasmid pCI372. Electrocompetent E. coli MKH13 and E. coli EPI300 were transformed with the ligation mixture and plated on LB agar plates containing 20 μg/ml Cm for selection. Colony PCR was performed on all resistant transformants using primers across the multiple cloning site (MCS) of pCI372 to confirm the presence and size of the insert.

Growth experiments were performed in defined M9 minimal media (M9MM) (Fluka) to confirm the observed phenotype. Single isolated colonies of SMG 9 and EPI300::pCC1FOS were grown overnight in M9MM (containing final concentrations of; D-glucose (0.4%), Bacto™ casamino acids (w/v 0.2%) (Becton, Dickinson and Co, Sparks, MD, USA), magnesium sulfate (MgSO₄) (2 mM), calcium chloride (CaCl₂) (0.1 mM) and 12.5 μg/ml Cm). Reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Cells were harvested by centrifugation, washed in ¼ strength Ringers solution and resuspended in fresh M9MM. A 2% v/v inoculum was sub-cultured in fresh M9MM containing various concentrations of sodium chloride (0–8% w/v NaCl) and 1 mM of L-carnitine when required. Triplicate wells of a 96-well micro-titre plate were inoculated with 200 μl of the appropriate cell suspension. Plates were incubated at 37°C for 24–48 h in an automated spectrophotometer (Tecan Genios) which recorded the optical density at 595 nm (OD_{595 nm}) every hour. After 48 h the data was retrieved and analyzed using the Magellan 3 software program and graphs were created with Sigma Plot 10.0 (Systat Software Inc, London, UK). Results are presented as the average of triplicate experiments, with error bars being representative of the standard error of the mean (SEM).

Approximately 23,000 clones from a metagenomic fosmid library from the human gut microbiome were screened previously and resulted in the identification of 53 salt tolerant clones which could grow on LB agar supplemented with 6.5% NaCl (a concentration which inhibits growth of the cloning host, E. coli EPI300::pCC1FOS) (Culligan et al., 2012). The salt tolerant clones identified were annotated as SMG 1-53 (Salt MetaGenome 1–53).

A number of clones initially identified from the metagenomic library were chosen at random and phenotypically screened using PM 9 osmolytes plate from BIOLOG. The layout and contents of PM9 can be viewed at http://www.biolog.com/pdf/pm_lit/PM1-PM10.pdf. One clone, SMG 9, gave a positive result under one of the 96 different conditions tested. It was found that SMG 9 had an increased metabolic response (causing a reduction of the tetrazolium dye to generate a purple color, Figure 2A) compared to the cloning host containing an empty fosmid vector, E. coli EPI300::pCC1FOS (Figure 2B) in 6% w/v NaCl supplemented with L-carnitine. The color formation within each well was measured by BIOLOG's Omnilog machine, which produces a color-coded graph. A comparison of the kinetic data output from SMG 9 and EPI300::pCC1FOS can be seen in Figure 3.

In an attempt to confirm and replicate the result from the PM analysis, SMG 9 and EPI300::pCC1FOS were grown in M9MM containing various concentrations of NaCl (0–6% w/v) and supplemented with 1 mM L-carnitine when appropriate. Figure 4A shows growth of both clones in M9MM in the presence and absence of 1 mM L-carnitine. In the presence of L-carnitine, SMG 9 displays a growth defect, while growth is similar under all other conditions. The growth defect is alleviated when grown at 4% w/v NaCl + 1 mM L-carnitine and there is no difference in growth between clones either in the presence or absence of L-carnitine (Figure 4B). At 5% w/v NaCl however, SMG 9 has a significant growth advantage compared to EPI300::pCC1FOS both in the presence and absence of 1 mM L-carnitine (Figure 4C). The positive effect of L-carnitine on the growth of SMG 9 is evident with cells entering logarithmic phase growth sooner and reaching a much higher final optical density (OD_{595 nm}). A similar, positive effect for L-carnitine on growth of SMG 9 is also seen at 6% w/v NaCl (Figure 4D).

Fosmid SMG 9 was fully sequenced (454-pyrosequencing) and assembled by GATC Biotech. This generated a total of 2.3 × 10⁶ base pairs of sequence data in 6939 sequencing reads. The average read length was 334 base pairs and coverage of 64.5× was achieved. Following vector trimming the length of the insert was approximately 36.5 kb and the %G+C content was 49.43%. Twenty-four putative open reading frames were predicted using Softberry's FGENESB, bacterial operon and gene prediction software (www.softberry.com) (Mavromatis et al., 2007). Translated nucleotide sequences were functionally annotated by homology searches using the BLASTP program to identify homologous sequences and determine their taxonomic origin. All but two of the genes encoded proteins with high identity (98–100% at the amino acid level) to Bacteroides sp. CAG:545. A list of the genes on SMG 9, their encoded functions and putative domains are presented in Table 2. The full fosmid insert sequence of SMG 9 has been submitted to GenBank and assigned the accession number, KJ524644.

Following initial inspection of the encoded proteins on SMG 9, the presence of an L-carnitine or general osmoprotectant transporter, nor indeed any protein with a functional link to carnitine metabolism was not immediately obvious. Transposon mutagenesis was attempted in order to create a knock-out mutant; this however, proved unsuccessful. Two genes (gene 5 and gene 18), which we felt may be likely to have a possible role in L-carnitine utilization based on bioinformatic analysis were cloned in isolation to further examine the phenotype. Genes 5 and 18 were annotated sdtR for sigma-dependent transcriptional regulator and mfsT for major facilitator superfamily transporter, respectively.

Both mfsT and sdtR were cloned in the vector pCI372 and expressed in both E. coli EPI300 and the osmosensitive strain E. coli MKH13 (Haardt et al., 1995). The effect of each gene on the growth of each strain under salt stress in the presence and absence of L-carnitine was assessed. The mfsT gene had no effect on growth under any of the conditions tested (data not shown). The sdtR gene on the other hand conferred a significant salt tolerance phenotype to E. coli MKH13 when grown in media supplemented with both 3 and 4% w/v NaCl (Figures 5C,D, respectively), while growth was similar in media lacking NaCl and in media supplemented with 2% w/v NaCl (Figures 5A,B, respectively). Cloning and expression of sdtR in EPI300 resulted in an increase in salt tolerance compare to wild-type EPI300 carrying an empty copy of the plasmid pCI372. Addition of 1 mM L-carnitine increased the growth rate and final optical density of both strains, but its effect on the sdtR⁺ strain was not significant relative to the EPI300::pCI372 control (Figures 6A,B).

Although sdtR did confer a salt tolerance phenotype, neither of the cloned genes replicated the original phenotype related to L-carnitine. We re-examined clone SMG 9 and carried out further studies, however these revealed that SMG 9 had lost the carnitine-associated phenotype seen originally and unfortunately, it was therefore not possible to identify the gene(s) responsible.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.