The Escherichia coli ssuEADCB Gene Cluster Is Required for the Utilization of Sulfur from Aliphatic Sulfonates and Is Regulated by the Transcriptional Activator Cbl

Abstract

The growth properties of an Escherichia coli strain carrying a chromosomal deletion of the ssuEADCB genes (formerly designated ycbPONME) indicated that the products of this gene cluster are required for the utilization of sulfur from aliphatic sulfonates. Sequence similarity searches indicated that the proteins encoded by ssuA, ssuB, and ssuC are likely to constitute an ABC type transport system, whereas ssuD and ssuE encode an FMNH 2 -dependent monooxygenase and an NAD(P)Hdependent FMN reductase, respectively (Eichhorn, E., van der Ploeg, J. R., and Leisinger, T. (1999) J. Biol. Chem. 274, 26639 -26646). Synthesis of ␤-galactosidase from a transcriptional chromosomal ssuE-lacZ fusion was repressed by sulfate or cystine and depended on the presence of a functional cbl gene, which encodes a LysRtype transcriptional regulator. Electrophoretic mobility shift assays with the ssu promoter region and measurements of ␤-galactosidase from plasmid-encoded ssuElacZ fusions showed that full expression of the ssu operon required the presence of a Cbl-binding site upstream of the ؊35 region. CysB, the LysR transcriptional regulator for the cys genes, was not required for expression of a chromosomal ssuE-lacZ fusion although the ssu promoter region contained three CysB-binding sites. Integration host factor could also occupy three binding sites in the ssu promoter region but had no influence on expression of a chromosomal ssuE-lacZ fusion.

Functional Dissection of the LysR-type CysB Transcriptional Regulator

“…Knowing that WT CysB is a tetramer, the trans-dominance of the multicopy CysB mutant forms may be explained by titration of WT CysB monomers and formation of inactive mixed heterooligomers. Therefore, it seems that none of the mutations altering residues 11,20,22,160,196,244, and 247 abolished the oligomerization ability of CysB. In contrast, CysB variants E41K, L44R, and I48T expressed from plasmids did not confer the Cys Ϫ phenotype to the EC1250 strain and had no effect on expression of the cysBЈ::ЈlacZ fusion in the pMH303-containing EC1250 strain (Table II).…”

“…Examples of LysR family members, NahR (7), OxyR (9), and GcvA (36), also suggest the correlation between the oligomerization defect and the inability of the mutant protein to exert a dominant-negative effect (also called a "poisoning effect") on the activity of the wild-type counterpart produced by the cell. For CysB, the negative dominance was clearly seen with some non-repressing (non-binding) variants (with mutations in region [11][12][13][14][15][16][17][18][19][20][21][22] as well as with non-inducible variants (Fig. 1).…”

“…Some CysB residues (i.e. Glu 11 and Thr 22 ) are poorly conserved in other LysR family members; these residues may therefore confer the specificity of DNA-CysB interactions. We found that a C-terminal region of CysB may be also critical for the DNAbinding function, as removal of 19 -30 C-terminal residues resulted in loss of repressing ability of the protein in vivo.…”

“…However, the structure lacking the 87 N-terminal amino acids provide little information about the overall organization of the native tetrameric protein. Third, in some promoters of E. coli, including the tau promoter (21) and the ssu promoter (22), CysB acts in conjunction with another LysR-type activator, Cbl, which itself is highly homologous to CysB (23). The molecular basis of cooperation of these two proteins is not yet understood.…”

Deletion Analysis of the Escherichia coli Taurine and Alkanesulfonate Transport Systems

“…A second set of genes, the ssuEADCB gene cluster, located at 21.4 min on the chromosome, enables E. coli to utilize aliphatic sulfonates other than taurine as a source of sulfur. Deletion of ssuEADCB caused an inability to utilize alkanesulfonates but did not affect the utilization of taurine (24). SsuD is a monooxygenase that catalyzes the desulfonation of a wide range of sulfonated substrates other than taurine, including C 2 to C 10 unsubstituted linear alkanesulfonates, substituted ethanesulfonic acids and the buffer substances HEPES, MOPS (morpholinepropanesulfonic acid), and PIPES [piperazine-N,NЈ-bis (2-ethanesulfonic acid)].…”