Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections.
Lactococcus lactis subsp. lactis INIA 415, a strain harboring the structural genes of bacteriocins nisin Z and lacticin 481, was used as adjunct culture in the manufacture of Hispánico cheese with a mesophilic starter and a thermophilic starter of high aminopeptidase activity. Addition of the bacteriocin producer promoted early lysis of mesophilic and thermophilic starter bacteria. Extracellular aminopeptidase activity in 7-day-old cheese made using mesophilic and thermophilic starters plus bacteriocin producer was 3.0-fold the level reached in cheese made without the bacteriocin producer. Proteolysis in cheese made with mesophilic and thermophilic starters plus bacteriocin-producing adjunct culture after 25 days of ripening was 1.5-fold the level reached in cheese made without the bacteriocin producer, and the level of total free amino acids was 2.9-fold the level found in cheese made without the bacteriocin producer. Cheese made with mesophilic and thermophilic starters plus bacteriocin producer received the highest scores for flavor quality and flavor intensity and reached in 25 days the flavor intensity score of a 75-day-old cheese made without the bacteriocin producer.
S. Hispa! nico cheese, a semi-hard Spanish variety, was manufactured from a mixture of pasteurized cows' and ewes' milks (4 : 1) using a commercial mesophilic LD-type starter comprising Lactococcus lactis subsp. cremoris, Lc. lactis subsp. lactis, Lc. lactis subsp. lactis var diacetylactis and Leuconostoc mesenteroides subsp. cremoris. Varying amounts (0-1n0 g\kg) of an Enterococcus faecalis INIA 4 culture in milk were added as a bacteriocin-producing adjunct. Differences in pH between cheeses manufactured with and without the bacteriocin producer did not exceed 0n11 pH units. Starter lactococci lost viability more rapidly in cheeses made with the bacteriocin producer, which reached counts of up to 6i10( cfu\g during ripening. Aminopeptidase activity in 1-d-old cheese made from milk inoculated with 1n0 g bacteriocin-producing culture\kg was twice that in control cheese. Degrees of overall proteolysis and levels of total free amino acids in 45-d-old cheese made with 1n0 g bacteriocin-producing culture\kg were 1n80-fold and 2n17-fold those in control cheese of the same age. Inoculating milk with 1n0 g\kg bacteriocin-producing culture reduced the level of hydrophobic peptides in the resultant cheese, increased the concentrations of 3-methyl-1-butanal, diacetyl and acetoin, and resulted in the highest scores for flavour quality and flavour intensity throughout ripening.
Hydrophobic and hydrophilic peptides in the water-soluble fraction
of 20
batches of Hispanico cheese made from pasteurized and raw milk were determined
by reversed-phase HPLC, with detection at 214 and 280 nm. Cheese flavour
characteristics were evaluated by a sensory panel, and
regressions of bitterness scores
on levels of hydrophobic and hydrophilic peptides and their ratio were
calculated.
The best fitting relationship for pasteurized milk cheese was
the linear regression of
mean panel bitterness scores on hydrophobic peptides at 280 nm
(r2=0·791). The
determination coefficient for the regression of hydrophobic peptides at
280 nm on
panellist bitterness scores (r2=0·356)
was lower, owing to individual differences in
the perceived intensity of bitterness. In the case of
raw milk cheese, the respective
determination coefficients were 0·203 for panel scores and
0·034 for individual panellist scores.
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