Testing Hypotheses of Gene Function

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Testing Hypotheses of Gene Function

Hypotheses of gene function can have different scenarios:

1) Your favorite gene has homology but an unproven function. 
2) Your favorite gene is highly characterized with a well described function, but you still do not know it’s role in cell physiology.
3) Your favorite gene has no homology and no known function.

Biolog’s Phenotype MicroArray technology can assist in all of these scenarios.

Campylobacter jejuni Carbon Starvation Protein A (CstA) is Involved in Peptide Utilization, Motility and Agglutination, and has a Role in Stimulation of Dendritic Cells
CstA is one of many proteins antigenic to humans with campylobacteriosis and displays homology to E. coli CstA, which is involved in peptide uptake.  Phenotype MicroArrays were used to show CstA is indeed required to maintain WT levels of both di- and tripeptide utilization in C. jejuni. However, results also suggested the presence of additional systems for dipeptide uptake. CstA mutants display reduced motility and agglutination.  UV-killed CstA mutants were also defective at stimulating IL-12 production in murine dendritic cells.  The authors propose that CstA is involved in both peptide uptake and host-pathogen interactions.

H2S: A Universal Defense Against Antibiotics in Bacteria
Shatalin, Nudler, and colleagues found that cystathionine b-synthase (CBS), cystathionine g-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) contribute to H2S production in many bacterial species. They hypothesized that H2S could act as a cytoprotective gasotransmitter. Using Phenotype MicroArrays, they found that as predicted the 3MST, CBS, and CSE mutants were hyper sensitive to a wide array of diverse antibiotics. This is the second gasotransmitter (the other one being NO) involved in antibiotic resistance that these authors have discovered with the aid of Phenotype MicroArray technology.

Endogenous Nitric Oxide Protects Bacteria Against a Wide Spectrum of Antibiotics
While bacterial nitric oxide synthase (bNOS) was known to produce nitric oxide (NO), the physiological role of was largely unknown.  Nudler and colleagues employed Phenotype MicorArrays to look for novel phenotypes of a bNOS mutant strain.  The bNOS mutant was found to be relatively sensitive to a diverse array of antibiotics that act via a common mechanism targeted by NO.

A Previously Undescribed Pathway for Pyrimidine Catabolism
Nitrogen regulatory protein C controlled transcripts included genes of unknown function with no homology. Phenotype MicroArrays revealed that the knockout strains could not use uridine or uracil as a sole nitrogen source suggesting the operon is involved in uracil degradation. Radiolabled uridine and GC/MS identified novel breakdown products of uracil, confirming the hypothesis.

Phenotype MicroArray Technology
Biolog’s Phentoype MicroArray technology enables researchers to evaluate nearly 2000 phenotypes of a microbial cell in a single experiment. This integrated system of cellular assays, instrumentation and bioinformatics software provides cellular knowledge that complements molecular information, helping you interpret and find the relevant aspects in massive amounts of gene expression or proteomics data. Through comprehensive and precise quantitation of phenotypes, researchers are able to obtain an unbiased perspective of the effect on cells of genetic differences, environmental change, exposure to chemicals or drugs, and more.