Bacillus amyloliquefaciens
Bacillus amyloliquefaciens is a species
of bacterium in
the genus Bacillus that is the source of the BamHI restriction enzyme. It also synthesizes a
natural antibiotic protein barnase,
a widely studied ribonuclease that forms a famously tight
complex with its intracellular inhibitor barstar,
and plantazolicin, an antibiotic with selective
activity against Bacillus anthracis.
It is used in agriculture, aquaculture,
and hydroponics to fight root pathogens such as Ralstonia solanacearum Pythium Rhizoctonia solani, Alternaria
tenuissima and Fusarium as
well improve root tolerance to salt stress. They are considered a
growth-promoting rhizobacteria and have the ability to
quickly colonize
Barnase
and barstar are two small soluble
proteins produced by the bacterium Bacillus amyloliquefaciens.
Barnase is a secreted ribonuclease that
is specifically inhibited by the intracellular barstar. With 110 and 89 amino
acid residues, respectively, neither protein is cross-linked by disulfide
bonds, nor do either require any metal ions or other cofactors to
fold or function. Inhibition of barnase by
barstar involves the formation of a bimolecular complex with a dissociation
constant on the order of 10−14−10−13 M,
in which the active site of the enzyme is covered and access to substrate
effectively denied. Both proteins can be reversibly unfolded in solution and
have been widely used in studies of protein
unfolding and refolding and as a model pair in protein–protein
investigations. As the genes are available and can be expressed
in Escherichia
coli, much of this work has involved protein
engineering and the effects of directed mutagenesis.
Variations on barnase and barstar are also available naturally from other
strains of Bacillus,2–6 with sequence identities
ranging down to 60% (in B. polymyxa6). More distantly
related homologs, with sequence identities below 25%, occur in Streptomyces strains.7,8
In
spite of the great difference in sequence, the enzymes of both genera have
essentially the same fold and the similarity of their active sites is indicated
by the fact that the enzymes of each group are inhibited by the inhibitors of
the other. Comparative studies with all of these proteins and with the even
more distantly related fungal ribonucleases related
to RNase T1 will be useful in determining just what elements of sequence are
most important in establishing the folds. All of the proteins mentioned here
are available from genes carried on plasmids in E. coli.
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