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Journal of Bacteriology, September 1998, p. 4344-4349, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of Genes Encoding Conjugated Bile Salt
Hydrolase and Transport in Lactobacillus johnsonii
100-100
Christopher A.
Elkins and
Dwayne C.
Savage*
Department of Microbiology, University of
Tennessee, Knoxville, Tennessee 37996-0845
Received 9 March 1998/Accepted 16 June 1998
Cytosolic extracts of Lactobacillus johnsonii 100-100 (previously reported as Lactobacillus sp. strain 100-100)
contain four heterotrimeric isozymes composed of two peptides,
and
, with conjugated bile salt hydrolase (BSH) activity. We now report
cloning, from the genome of strain 100-100, a 2,977-bp DNA segment
that expresses BSH activity in Escherichia coli. The
sequencing of this segment showed that it contained one complete and
two partial open reading frames (ORFs). The 3' partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5' and 3'
deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used
to extend and complete the 948-nucleotide sequence of the BSH gene 3'
of the cloned segment. The predicted amino acid sequence of the 5' partial ORF (651 nucleotides) was about 80% similar to the
C-terminal half of the largest, complete ORF (1,353 nucleotides), and
these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily. E. coli DH5
cells
transformed with a construct containing these ORFs, in concert with an
extracellular factor produced by strain 100-100, demonstrated levels of
uptake of [14C]taurocholic acid that were increased as
much as threefold over control levels. [14C]Cholic acid
was taken up in similar amounts by strain DH5
pSportI (control) and
DH5
p2000 (transport clones). These findings support a hypothesis
that the ORFs are conjugated bile salt transport genes which may be
arranged in an operon with BSH genes.
*
Corresponding author. Mailing address: Department of
Microbiology, M409 Walters Life Sciences Bldg., University of
Tennessee, Knoxville, TN 37996-0845. Phone: (423) 974-4015. Fax: (423)
974-4007. E-mail: DSavage1{at}utk.edu.
Journal of Bacteriology, September 1998, p. 4344-4349, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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