Defining the gate domain of the filamentous phage secretin pIV
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Date
2010
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Massey University
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Abstract
Secretins are a family of large outer membrane proteins with large-diameter
lumens (5-10 nm). This allows them to transport bulky substrates, including
folded proteins, or assembled macromolecular structures – filamentous phages
and type IV pili. Many proteins exported by secretins are essential for virulence
of Gram-negative pathogens. Such a large channel would ordinarily sensitize
the bacterial cell to noxious agents. However, secretins do not - the presence of
a mobile septum, or gate, across the lumen of the channel prevents access by
noxious agents. Despite the importance of the gate in secretin function, the
sequence identity of the gate residues is unknown. In this study, in vivo random
mutagenesis was used to map amino acid residues involved in gating the
filamentous phage secretin - pIV. This approach has identified 34 residues that
are involved in the gating mechanism. These residues are predominantly
located within the secretin homology domain and organised into two clusters;
GATE1 (39 residues) and GATE2 (14 residues). A number of isolated point
mutants sensitised Escherichia coli to bile salts and antibiotics. These findings
allowed the construction of a site-directed deletion mutant of GATE2, confirming
the gate function.
This thesis mapped, for the first time, a secretin gate. Given the success of the
mutagenesis approach used in this thesis, the method here will be applicable to
secretins of pathogenic bacteria and other outer membrane channels whose
gate regions have not been determined as yet. Knowing the gate regions of
“pathogenic” secretins in turn will help design secretin-targeting antimicrobials.
Description
Appendix 5 removed due to copyright restrictions. Spagnuolo, J., Opalka, N., Wen, W. X., Gagic, D., Chabaud, E., Bellini, P.,...Rakonjac, J. (2010). Identification of the gate regions in the primary structure of the secretin pIV. Molecular microbiology, 76(1), 133-150. doi:10.1111/j.1365-2958.2010.07085.x
Keywords
Secretin gates, Secretins of pathogenic bacteria