Deletion analysis of the Escherichia coli taurine and alkanesulfonate transport systems

Abstract

The Escherichia coli tauABCD and ssuEADCB gene clusters are required for the utilization of taurine and alkanesulfonates as sulfur sources and are expressed only under conditions of sulfate or cysteine starvation. tauD and ssuD encode an alpha-ketoglutarate-dependent taurine dioxygenase and a reduced flavin mononucleotide-dependent alkanesulfonate monooxygenase, respectively. These enzymes are responsible for the desulfonation of taurine and alkanesulfonates. The amino acid sequences of SsuABC and TauABC exhibit similarity to those of components of the ATP-binding cassette transporter superfamily, suggesting that two uptake systems for alkanesulfonates are present in E. coli. Chromosomally located in-frame deletions of the tauABC and ssuABC genes were constructed in E. coli strain EC1250, and the growth properties of the mutants were studied to investigate the requirement for the TauABC and SsuABC proteins for growth on alkanesulfonates as sulfur sources. Complementation analysis of in-frame deletion mutants confirmed that the growth phenotypes obtained were the result of the in-frame deletions constructed. The range of substrates transported by these two uptake systems was largely reflected in the substrate specificities of the TauD and SsuD desulfonation systems. However, certain known substrates of TauD were transported exclusively by the SsuABC system. Mutants in which only formation of hybrid transporters was possible were unable to grow with sulfonates, indicating that the individual components of the two transport systems were not functionally exchangeable. The TauABCD and SsuEADCB systems involved in alkanesulfonate uptake and desulfonation thus are complementary to each other at the levels of both transport and desulfonation.

FIG. 1

Organization of the ssuEADCB (A) and tauABCD (B) operons, showing the approximate positions of the oligonucleotide primers used for the construction of in-frame deletion inserts and the plasmids used for complementation analysis of deletion mutants.

FIG. 2

Active components of the TauABC and SsuABC transport systems in the deletion mutants.

FIG. 3

Growth of E. coli EC1250 and deletion mutants. Cells were grown in a microtiter plate as described in Materials and Methods (150-μl cultures). The optical density at 600 nm (OD_{600 nm}) was recorded with a microtiter plate reader every 5 min over 24 h. Every fifth measurement is shown. Only the growth profiles obtained with mutants grown on 1,3-dioxo-2-isoindolineethanesulfonate (A), butanesulfonate (B), PIPES (C), and MOPS (D) are shown. The optical densities obtained ranged from 0.25 to 0.5 and corresponded to optical densities of 1.0 to 2.0 when measured with a 1-cm light path in a Uvikon P-810 spectrophotometer.

FIG. 4

Substrate specificities of the E. coli TauABC and SsuABC transporters.