Abstract
In the current study, a number of salt-tolerant clones previously isolated from a human gut metagenomic library were screened using Phenotype MicroArray (PM) technology to assess their functional capacity. PM's can be used to study gene function, pathogenicity, metabolic capacity and identify drug targets using a series of specialized microtitre plate assays, where each well of the microtitre plate contains a different set of conditions and tests a different phenotype. Cellular respiration is monitored colorimetrically by the reduction of a tetrazolium dye. One clone, SMG 9, was found to be positive for utilization/transport of L-carnitine (a well-characterized osmoprotectant) in the presence of 6% w/v sodium chloride (NaCl). Subsequent experiments revealed a significant growth advantage in minimal media containing NaCl and L-carnitine. Fosmid sequencing revealed putative candidate genes responsible for the phenotype. Subsequent cloning of two genes did not replicate the L-carnitine-associated phenotype, although one of the genes, a σ54-dependent transcriptional regulator, did confer salt tolerance to Escherichia coli when expressed in isolation. The original clone, SMG 9, was subsequently found to have lost the original observed phenotype upon further investigation. Nevertheless, this study demonstrates the usefulness of a phenomic approach to assign a functional role to metagenome-derived clones.
Highlights
The ability to adapt to and tolerate increases in extracellular osmolarity is an important characteristic that enables bacteria to survive in stressful environments
A total of 23,040 clones from the library were screened on LB agar supplemented with 6.5% (w/v) NaCl 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
SCREENING OF METAGENOMIC LIBRARY 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 (Culligan et al, 2012)
Summary
The ability to adapt to and tolerate increases in extracellular osmolarity is an important characteristic that enables bacteria to survive in stressful environments. During the primary response, potassium ions are rapidly accumulated within the bacterial cell to offset the detrimental effects of water loss and influx of toxic sodium and chloride ions (Sleator and Hill, 2002; Epstein, 2003). Numerous studies have shown carnitine to be important for salt tolerance, and for survival in vivo and pathogenesis of infection (Sleator et al, 2001; WemekampKamphuis et al, 2004). Carnitine is found abundantly in animal tissues and red meat and is an important compound in the host environment; for the human pathogen Listeria monocytogenes, carnitine and its uptake system OpuC are critical for infection in mice (Sleator et al, 2003). In addition to its osmoprotective properties, carnitine may be catabolised as a carbon or nitrogen source to generate energy (Wargo and Hogan, 2009)
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