Doctoral Dissertations

Date of Award

5-2001

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Dwayne C. Savage

Committee Members

Jeffrey M. Becker, Beth C. Mullin, Neil B. Quigley

Abstract

Bile salt hydrolysis (BSH) activity of Lactobacillus johnsonii strain 100-100 is catalyzed by four cytosolic homo- and heterotrimeric isozymes composed of two antigenically distinct peptides, a and p. Two separate genomic libraries of strain 100-100 were prepared with Sau3Al and EcoRl and cloned into Escherichia coli cells. The libraries were screened on MRS Lactobacillus agar medium containing 0.5 % taurodeoxycholic acid for halos of deoxycholic acid precipitate in the medium surrounding the colonies (plate assay). Clones encoding the a and P peptides were identified from the Sau3AI and EcoRl libraries, respectively. The a peptide gene, cbsHa, was encoded at a locus separate from the p peptide gene and did not share tandem arrangement with other genes. The P peptide gene, cbsHfi, shared tandem arrangement with two other genes, cbsTl and cbsT2. These genes were duplicates with sequence similarity and hydropathy profiles consistent with transporters of the major facilitator superfamily (MFS). However, they represented a new subfamily of the MFS. All three genes, cbsTl, cbsT2, and cbsHB, were coordinately regulated in stationary phase strain 100-100 cells and were present in L. acidophilus strain KS-13, a human isolate. Lactobacillus species from culture banks were screened for the BSH phenotype via plate assay and the cbsHfi genotype via the polymerase chain reaction. These properties were not always constant in isolates of the same species, which suggests a horizontal origin for the activity. E. coli cells expressing a construct containing a complete cbsT2 was assayed for capacity to accumulate [24-^14C]taurocholic acid. When an extracellular factor (EF) produced by strain 100-100 was present, uptake of [24-^14C]taurocholic acid was increased up to threefold over control levels. However, a statistically significant decrease in uptake of [24-^14C]cholic acid was also observed in these cells. Assays of net [24-^14C]taurocholic acid accumulation over time and concentration with CbsT2 and EF demonstrated that the increase is rapid (<15 sec), but saturable. Unlabeled cholic acid, if added to identical cells not exposed to EF, could produce an "EF-like" effect on uptake of [24-^14C]taurocholic acid. Studies with ionophores, 2,4dinitrophenol and CCCP did not decrease [24-^14C]taurocholic acid uptake in such cells and demonstrate that the uptake is independent of Ap. Moreover, uptake with [24-^14C]- and [^3H]taurocholic acid, labeled on the cholic acid and taurine respectively, demonstrated that [24-^14C]cholic acid leaves the cell, but [^3H]taurine remains intracellular in constructs expressing cbsT2 and cbsHB. The data support a hypothesis that CbsT2 (and CbsTl) is a facilitated taurocholic acid:cholic acid exchanger. I conclude that the known genetic determinants for BSH activity in strain 100-100 have been cloned and identified.

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