Abstract
The elucidation of the cellular processes involved in vitamin and cofactor biosynthesis is a challenging task. The conventional approaches to these investigations rely on the discovery and purification of the products (i.e proteins and metabolites) of a particular transport or biosynthetic pathway, prior to their subsequent analysis. However, the purification of low-abundance proteins or metabolites is a formidable undertaking that presents considerable technical challenges. As a solution, we present an alternative approach to such studies that circumvents the purification step. The proposed approach takes advantage of: (1) the molecular detection capabilities of a riboswitch-based sensor to detect the cellular levels of its cognate molecule, as a means to probe the integrity of the transport and biosynthetic pathways of the target molecule in cells, (2) the high-throughput screening ability of fluorescence-activated cell sorters to isolate cells in which only these specific pathways are disrupted, and (3) the ability of next-generation sequencing to quickly identify the genes of the FACS-sorted populations. This approach was named “RiboFACSeq”. Following their identification by RiboFACSeq, the role of these genes in the presumed pathway needs to be verified through appropriate functional assays. To demonstrate the utility of our approach, an adenosylcobalamin (AdoCbl)-responsive riboswitch-based sensor was used in this study to demonstrate that RiboFACSeq can be used to track and sort cells carrying genetic mutations in known AdoCbl transport and biosynthesis genes with desirable sensitivity and specificity. This method could potentially be used to elucidate any pathway of interest, as long as a suitable riboswitch-based sensor can be created. We believe that RiboFACSeq would be especially useful for the elucidation of biological pathways in which the proteins and/or their metabolites are present at very low physiological concentrations in cells, as is the case with vitamin and cofactor biosynthesis.
Highlights
The products of vitamin and cofactor biosynthetic pathways are essential for the survival of all eukaryotic and bacterial microorganisms, whether they are produced by the organism or consumed from the environment
To validate that the AdoCbl-Rb-sfGFP sensor can detect the transport and biosynthesis of AdoCbl in E. coli, the fluorescence response of the sensor to a variety of vitamin B12 (VB12) nutrient conditions was examined in WT E. coli BW25113 strain carrying this construct (Fig 2)
We have previously demonstrated the effectiveness of the E. coli AdoCbl-responsive riboswitch sensor as an intracellular tool for monitoring the physiologically relevant concentrations of its cognate molecule
Summary
The products of vitamin and cofactor biosynthetic pathways are essential for the survival of all eukaryotic and bacterial microorganisms, whether they are produced by the organism or consumed from the environment. The elucidation of biosynthetic pathways classically relies on the coordinated use of multiple chemical and biochemical approaches, which typically include isotopic labeling, recombinant overexpression of proteins, and the complementary use of genetic screens [2,3] They have certain drawbacks: (1) crucial to the success of these approaches is the serendipitous detection and isolation of auxotrophic mutants that lack enzymes involved in the pathway under investigation, especially when little to no information is available on the pathway [2]; (2) the comprehensive analysis of a pathway relies on the purification of target proteins and/or their metabolites through the conventional method of fractionation, which often requires multiple steps, specialized equipment (e.g. LC/GC-MS, HPLC, MS-MS, NMR, FT-IR) and trained personnel [4,5]. The traditional methods of investigation lack broad applicability, necessitating a more sensitive and comprehensive method for the systematic elucidation of bacterial transport and biosynthetic pathways
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