Cloning and expression analysis of the 28 kDa protein from Lactobacillus delbrueckii subsp. lactis ATCC 4797 hypothesized to influence lactacin B production.
OBJECTIVE
A cell wall-associated lactacin B inducer protein (IP) was purified from Lactobacillus delbrueckii subsp. lactis ATCC 4797 (Lact. lactis) by chromatofocusing and gel filtration HPLC (Barefoot et al. 1994).


RESULTS
N-terminal sequence of the purified IP was used to design an oligonucleotide (24-mer) for gene identification by Southern and colony hybridizations. Southern hybridization on Lact. lactis chromosomal DNA digested with EcoRI and PstI produced a single 4-5 kbp DNA fragment. Colony hybridizations with 6250 clones produced four positive recombinants for the proposed IP. Sequence of the DNA isolated from RU43e9 revealed a 4623 bp DNA fragment containing three open reading frames (ORF) potentially encoding enzymes that function in glycolysis. One ORF, coding for an active triosephosphate isomerase (Tpi), showed 98% homology to the N-terminal domain of the HPLC purified IP. PCR primers were designed to amplify the ORF encoding the proposed IP for subcloning, protein expression, purification and bacteriocin enhancing assays on pure cultures of Lactobacillus acidophilus N2.


CONCLUSIONS
The regions flanking the Tpi gene (data not shown) were also sequenced and it is concluded that the proposed IP reported by Barefoot et al. (1994) is located on an operon containing several glycolytic enzymes that function in glycolysis.


CONCLUSIONS
The findings of this study do not support previously published research (Barefoot et al. 1994) hypothesizing that a purified IP from Lact. lactis, homologous to a Bacillus stearothermophilus Tpi, is capable of enhancing bacteriocin synthesis in Lact. acidophilus N2.
Ulrich
R L
RL
Department of Microbiology and Molecular Medicine, Clemson University, SC 29634, USA.
Hughes
T A
TA
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001430:Bacteriocins / Q000096:biosynthesis*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D007778:Lactobacillus / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020161:Physical Chromosome Mapping; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D001430:Bacteriocins; C035728:lactocin