5.3
Secretion and Proteolytic Activation of Bacteriocin Precursors
5.3.1
ATP-Dependent Translocation and Processing
The NisP protein product encoded by the nisP gene upstream from nisR in the
nisin gene cluster showed an N-terminal signal sequence, a catalytic domain
with a high degree of similarity to that of the subtilisin-like serine proteases,
and a carboxyterminal membrane anchor [101]. Those features of its primary
structure are indicative of secretion followed by membrane anchoring resulting
in an extracellularly located catalytic N-terminal tail [40, 101]. Precursor nisin
devoid of biological activity could be activated by incubation with cell membranes from a nisP-expressing strain, while mutation of NisP resulted in the
secretion of a fully modified but unprocessed nisin precursor, indicating that
the activating NisP protease is membrane located and involved in maturation of
nisin [101].
The nisT gene encodes a 600-residue protein with strong homology to ABC
exporters [92, 100]. These ABC transporters share two main regions of homology, i.e. an ATP-binding motif in the C-terminal half and six transmembrane
domains located in the N-terminal half of the protein [105]. These data indicate
that proteolytic cleavage is a process that occurs at the extracellular face of the
cytoplasmic membrane following secretion [101]. The counterparts of NisP in
the epidermin (EpiP) and cytolysin (CylP) operons contain a signal sequence
but lack a membrane-spanning domain, suggesting that they are attached in
another way or are not membrane associated [88, 122, 190]. PepP, LasP and ElkP
involved in respectively Pep5, lactocin S and epilancin K7 proteolytic processing all lack a signal sequence, and may therefore function intracellularly, which
is in agreement with their N-terminal modification [40, 186, 187]. The subtilin
gene cluster did not contain a peptidase-like protein [87, 89, 110]. However, the
subtilin producer B. subtilis is known to contain a variety of secreted proteases
that could be involved in proteolytic activation [40].
ABC transporters encoded in the same operon or an operon adjacent to the
structural bacteriocin gene have been reported for all class II bacteriocins
genetically studied in detail [49, 62, 118, 119, 127, 130, 131, 136, 146]. Those
proteins are characterized by six transmembrane domains, a carboxy-terminal
ATP-binding cassette and an N-terminal proteolytic domain, both located in the
cytoplasm [146] (Fig. 6.). The energy needed for the translocation process is
provided by hydrolysis of ATP [146, 191, 192]. It was remarkably that the ABC
transporters involved in the secretion of the lantibiotics nisin, epidermin and
subtilin are devoid of a conserved N-terminal extension of about 150 amino
acids that is only present in the larger transporters and involved in proteolytic
processing of the prebacteriocin [146]. Among the larger ABC transporter
proteins, two conserved amino acid stretches were shown to be exclusively
found in N-terminal extensions of bacteriocin transporters and not in exporters of substrates that are not proteolytically processed during transport,
such as the haemolysin transporters AppB [193], HlyB [194], LktB [195], and
42
E. Sablon et al.
Secretion and Proteolytic Activation of Bacteriocin Precursors
5.3.1
ATP-Dependent Translocation and Processing
The NisP protein product encoded by the nisP gene upstream from nisR in the
nisin gene cluster showed an N-terminal signal sequence, a catalytic domain
with a high degree of similarity to that of the subtilisin-like serine proteases,
and a carboxyterminal membrane anchor [101]. Those features of its primary
structure are indicative of secretion followed by membrane anchoring resulting
in an extracellularly located catalytic N-terminal tail [40, 101]. Precursor nisin
devoid of biological activity could be activated by incubation with cell membranes from a nisP-expressing strain, while mutation of NisP resulted in the
secretion of a fully modified but unprocessed nisin precursor, indicating that
the activating NisP protease is membrane located and involved in maturation of
nisin [101].
The nisT gene encodes a 600-residue protein with strong homology to ABC
exporters [92, 100]. These ABC transporters share two main regions of homology, i.e. an ATP-binding motif in the C-terminal half and six transmembrane
domains located in the N-terminal half of the protein [105]. These data indicate
that proteolytic cleavage is a process that occurs at the extracellular face of the
cytoplasmic membrane following secretion [101]. The counterparts of NisP in
the epidermin (EpiP) and cytolysin (CylP) operons contain a signal sequence
but lack a membrane-spanning domain, suggesting that they are attached in
another way or are not membrane associated [88, 122, 190]. PepP, LasP and ElkP
involved in respectively Pep5, lactocin S and epilancin K7 proteolytic processing all lack a signal sequence, and may therefore function intracellularly, which
is in agreement with their N-terminal modification [40, 186, 187]. The subtilin
gene cluster did not contain a peptidase-like protein [87, 89, 110]. However, the
subtilin producer B. subtilis is known to contain a variety of secreted proteases
that could be involved in proteolytic activation [40].
ABC transporters encoded in the same operon or an operon adjacent to the
structural bacteriocin gene have been reported for all class II bacteriocins
genetically studied in detail [49, 62, 118, 119, 127, 130, 131, 136, 146]. Those
proteins are characterized by six transmembrane domains, a carboxy-terminal
ATP-binding cassette and an N-terminal proteolytic domain, both located in the
cytoplasm [146] (Fig. 6.). The energy needed for the translocation process is
provided by hydrolysis of ATP [146, 191, 192]. It was remarkably that the ABC
transporters involved in the secretion of the lantibiotics nisin, epidermin and
subtilin are devoid of a conserved N-terminal extension of about 150 amino
acids that is only present in the larger transporters and involved in proteolytic
processing of the prebacteriocin [146]. Among the larger ABC transporter
proteins, two conserved amino acid stretches were shown to be exclusively
found in N-terminal extensions of bacteriocin transporters and not in exporters of substrates that are not proteolytically processed during transport,
such as the haemolysin transporters AppB [193], HlyB [194], LktB [195], and
42
E. Sablon et al.