Analysis of promoter sequences from Lactobacillus helveticus CNRZ32 and their activity in other lactic acid bacteria.
OBJECTIVE
To clone and analyse seven putative promoter fragments (pepC, pepN, pepX, pepO, pepE, pepO2, hsp17) from Lactobacillus helveticus CNRZ32 for their expression in Lact. helveticus CNRZ32, Lact. casei ATCC334 and Lactococcus lactis MG1363.


RESULTS
Promoter fragments were fused to the promoter-less beta-glucuronidase (gusA) gene on pNZ272(RBS-) (ATG-). The resulting constructs were evaluated for their ability to drive the expression of active GusA with 0.5 mmol l(-1) 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. All promoters except P(pepN)::gusA were active in the examined strains. Northern hybridization was performed to examine the promoter strength. Sequence analysis of these promoters identified well conserved putative ribosomal binding and putative -10 hexamers sites.


CONCLUSIONS
Seven promoter fragments from Lact. helveticus CNRZ32 were recognized in the lactic acid bacteria, Lact. casei ATCC334 and L. lactis MG1363, as well as in Escherichia coli. P(pepN)::gusA could not be maintained in the strains examined because of toxicity associated with heterologous protein over-expression driven by P(pepN).


CONCLUSIONS
This study revealed that desirable levels of heterologous food-grade protein production in GRAS organisms can be obtained with the application of natural promoter fragments from closely related organisms.
Chen
Y-S
YS
Department of Food Science and Technology, Mississippi State University, Mississippi State, MS 39762, USA. yc65@ra.msstate.edu
Steele
J L
JL
D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics; D007778:Lactobacillus / Q000235:genetics*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA
D006360:Heat-Shock Proteins; C438147:heat-shock protein, 17-kDa; D010447:Peptide Hydrolases