Location
University of Nevada Las Vegas, Science and Education Building
Start Date
9-8-2011 10:15 AM
End Date
9-8-2011 12:00 PM
Description
Various species of bacteria have been reported to form an endospore, a metabolically dormant cell, during times of nutrient deficiencies and extreme stress. These said structures are outstandingly resistant to harsh chemicals, extreme temperatures, and can revert back to a metabolically active cell, through a process known as germination, when the necessary conditions are met. The rigid membrane of the endospore contains various germination (Ger) receptors which sense the external environment for necessary metabolites and germinants. Ger receptors are encoded by tricistronic operons that produce three distinct membrane proteins, the A, B, and C subunits. Although the function of the Ger receptor has been established by genetics, no information is currently available for germinant binding site. Bioinformatic and genetic approaches has predicted that the C-terminus of the B subunit is the most likely candidate to contain the germinant binding site. B. Subtilis and B. Megaterium, two species of the Bacilli genus, germinate in response to different germinants; B. Subtilis germinates in response to L-alanine by activation of the GerA receptor, while B. Megaterium germinates in response to L-leucine by the activation of its GerU receptor. The focus of this study is to construct chimeric genes in which fragments of B. Subtilis GerA receptors and B. Megaterium receptors are fused together. These B. Subtilis::B. Megaterium chimeric receptors will be introduced into the B. Subtilis genome and the mutant B. Subtilis spores will then be tested for the ability to germinate with leucine in order to establish the leucine binding site of GerUB. During the initial pilot studies, the regions coding for the Nterminus of the GerA receptor from B. Subtilis and the C-terminus of the GerU receptor from B. Megaterium were amplified using polymerase chain reaction with primer ends complementary to each other in order to further produce the desired hybrid genes without the use of restriction enzymes.
Keywords
Bacillus megaterium; Bacillus subtilis — Genetics; Bacterial spores; Genetic regulation; Germination; Stress (Physiology)
Disciplines
Bacteriology | Biochemistry | Life Sciences | Microbiology | Molecular Biology
Language
English
Synthesis of chimeric receptors essential for spore germination
University of Nevada Las Vegas, Science and Education Building
Various species of bacteria have been reported to form an endospore, a metabolically dormant cell, during times of nutrient deficiencies and extreme stress. These said structures are outstandingly resistant to harsh chemicals, extreme temperatures, and can revert back to a metabolically active cell, through a process known as germination, when the necessary conditions are met. The rigid membrane of the endospore contains various germination (Ger) receptors which sense the external environment for necessary metabolites and germinants. Ger receptors are encoded by tricistronic operons that produce three distinct membrane proteins, the A, B, and C subunits. Although the function of the Ger receptor has been established by genetics, no information is currently available for germinant binding site. Bioinformatic and genetic approaches has predicted that the C-terminus of the B subunit is the most likely candidate to contain the germinant binding site. B. Subtilis and B. Megaterium, two species of the Bacilli genus, germinate in response to different germinants; B. Subtilis germinates in response to L-alanine by activation of the GerA receptor, while B. Megaterium germinates in response to L-leucine by the activation of its GerU receptor. The focus of this study is to construct chimeric genes in which fragments of B. Subtilis GerA receptors and B. Megaterium receptors are fused together. These B. Subtilis::B. Megaterium chimeric receptors will be introduced into the B. Subtilis genome and the mutant B. Subtilis spores will then be tested for the ability to germinate with leucine in order to establish the leucine binding site of GerUB. During the initial pilot studies, the regions coding for the Nterminus of the GerA receptor from B. Subtilis and the C-terminus of the GerU receptor from B. Megaterium were amplified using polymerase chain reaction with primer ends complementary to each other in order to further produce the desired hybrid genes without the use of restriction enzymes.