Introduction of peptidase genes from Lactobacillus delbrueckii subsp. lactis into Lactococcus lactis and controlled expression.
Peptidases PepI, PepL, PepW, and PepG from Lactobacillus delbrueckii subsp. lactis, which have no counterparts in Lactococcus lactis, and peptidase PepQ were examined to determine their potential to confer new peptidolytic properties to lactococci. Controllable expression of the corresponding genes (pep genes) was achieved by constructing translational fusions with the promoter of the nisA gene (P(nisA)). A suitable host strain, UKLc10, was constructed by chromosomal integration of the genes encoding the NisRK two-component system into the fivefold peptidase-deficient mutant IM16 of L. lactis. Recombinants of this strain were used to analyze growth, peptidase activities, peptide utilization, and intracellular protein cleavage products. After nisin induction of P(nisA)::pep fusions, all of the peptidases were visible as distinct bands in protein gels. Despite the fact that identical transcription and translation signals were used to express the pep genes, the relative amounts of individual peptidases varied considerably. All of the peptidases exhibited activities in extracts of recombinant UKLc10 clones, but only PepL and PepG allowed the clones to utilize specific peptide substrates as sources of essential amino acids. In milk medium, induction of pepG and induction of pepW resulted in growth acceleration. The activities of all five peptidases during growth in milk medium were revealed by high-performance liquid chromatography analyses of intracellular amino acid and peptide pools.
Wegmann
U
U
Universität Kaiserslautern, Fachbereich Biologie, Abteilung Mikrobiologie, D-67653 Kaiserslautern, Germany.
Klein
J R
JR
Drumm
I
I
Kuipers
O P
OP
Henrich
B
B
D000626:Aminopeptidases / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004790:Enzyme Induction; D005798:Genes, Bacterial; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics*; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases; D000626:Aminopeptidases; C022450:prolyl aminopeptidase