Characterization of the immunity factor in producer self protection against Leucocin A.
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Date
2008
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Abstract
Lactic acid bacteria produce pediocin-like bacteriocins designated as Class Ha. These
antimicrobial peptides are antagonistic against Listeria monocytogenes and other closely
related Gram-positive bacteria Self-protection of the producer organism is attributed to
the immunity proteins, encoded by genes that are eo-transcribed with the structural gene
that encode the bacteriocin. The lactic acid bacterium, Leuconostoc gelidum UAL 187-22
is immune to its own bacteriocin, leucocin A. This is accredited to its immunity protein
and the possible absence of a receptor on its cytoplasmic membrane. Leucocin A was
purified from the supernatant of 1. gelidum to 90% purity by ion-exhange chromatography
and C18 reverse phase High Pressure Liquid Chromatography (RP-HPLC) eluted with an
acetonitrile, 0.1% Triflouroacetic acid (TFA) gradient. The immunity gene was isolated
from the same producer using the polymerase chain reaction from the recombinant plasmid
pJF 5.5 using primers EAL-2 and EAL-3. The amplicon was truncated into versions A and
B by removing the C- and N-terminals, with HaeIII and ClaI restriction enzymes,
respectively. The amplicon and the truncated fragments A and B were cloned into pMALc2
to construct recombinant plasmids pKPl, pKPIA and pKPIB, correspondingly, which
were transformed into Escherichia coli (E. coli) strain JMI03. Clones were confirmed by
colony PCR and Southern blot hybridization. The recombinant clones were subsequently
expressed as MBP-IP, MBP-IPA and MBP-IPB fusion proteins that were verified by
Western blot using the anti-MBP antibody. Factor Xa protease was used to cleave MBP
from the proteins of interest. The resulting pure immunity protein versions had an
approximate molecular weight of slightly more that 10 kDa. The binding interactions of
the purified immunity protein constructs and leucocin A were compared on the Biacore
2000 instrument with surface plasmon resonance. None of the immunity constructs
interacted with leucocin A, however, the N-terminal region of the immunity protein
interacted with the cytoplasmic extract.
Description
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.
Keywords
Bacteriocins., Bacteriocins--Genetic aspects., Cellular immunity., Cellular immunity--Genetic aspects., Genetics., Lactic acid bacteria., Immunogenetics., Theses--Biochemistry.