Genetic organization of the sgrR sgrS region. The directions of transcription of the sgrR and sgrS genes are indicated at the top. Both strands of the sgrR-sgrS intergenic region are shown below the gene map. The +1 at right denotes the start of sgrS transcription, the −70 position at left is with respect to sgrS transcription. The −35 and −10 promoter elements are indicated by horizontal lines above the nucleotide sequence. The shaded base pairs represent conservation of putative regulatory sequences upstream of the −35 element (21). On the lower strand, the ATG start codon for sgrR is circled. The boxed nucleotides show the positions of conserved sequences where a 5-bp substitution was constructed. The substituted sequences marked “5 bp sub” are shown below the wild-type sequence.

The same SgrR binding site is required for activation of sgrS and autorepression of sgrR. β-Galactosidase assays were performed as described for Fig. 2A. Host strains were wild type (sgrR⁺); lacZ transcriptional fusions had either a wild-type (wt) sequence in the sgrR-sgrS promoter region (shown in Fig. 1) or the 5 bp substitution mutation (mut) (also as shown in Fig. 1) in the P_sgrS, P_sgrR1, or P_sgrR2 fragments. Strains are as follows (see Table 1): CV5200, P_sgrS (wt); CV5500, P_sgrS (mut); CV9000, P_sgrR1 (wt); CV9100, P_sgrR1 (mut); CV9200, P_sgrR2 (wt); and CV9201, P_sgrR2 (mut). Gray and black bars represent activities of fusions with the wild-type or mutant 5-bp site, respectively. The presence or absence of the stress signal αMG is indicated below the graph. Error bars indicate standard deviations.

Titration of endogenous SgrR by DNA fragments from the sgrR-sgrS-setA region. The relative sizes and orientations of transcription of the sgrR, sgrS, and setA genes are represented by the gene map at top. The limits of the original titrating clone pBRlib15 and all subclones are represented by horizontal lines below the gene map. The relative position of the mutation in clones pBRCV18 and pBRCV3 is denoted by the box over the horizontal line. For these two plasmids, the “sub” mutation is the 5-bp substitution described in the text and shown in detail in Fig. 1. All plasmids were transformed to a wild-type host strain carrying the P_sgrS-lacZ fusion (strain CV5200 [Table 1]) (21); activity of the fusion in each strain was measured after 45 min of exposure to αMG. β-Galactosidase activity was quantitated and normalized to the activity of cells carrying the vector control, which was set at 100%. Error bars indicate standard deviations.

Mlc affects activation of sgrS by an undefined mechanism. A. The relative sizes and directions of transcription of the clcB, ynfK, mlc, and ynfL genes are indicated by the gene map at top. The limits of clones and the location of 3-bp substitution mutations are indicated as described for Fig. 5. The host strain carrying the clones is CV5200 (Table 1), which contains the P_sgrS-lacZ fusion. Cells were streaked on MacConkey lactose indicator plates containing αMG at 0.05%; cells were grown for 18 h and the Lac phenotype observed. The vector control-containing cells were red, or Lac⁺, since αMG strongly induces P_sgrS-lacZ expression. Strains carrying plasmids that downregulated the fusion had a Lac⁻ phenotype (white). B. Western blotting for EIICB^Glc (PtsG) protein was performed as described in Materials and Methods. The ΔptsG::cat strain (CV105) lacks EIICB^Glc and is included for comparison. The two bands above the PtsG band are proteins that cross-react with the αIIB^Glc antibody and were used as loading controls. The wild-type host carries P_sgrS-lacZ and ptsG under the control of the native promoter elements (CV5200). Strain CV5282 also has P_sgrS-lacZ, but ptsG is under the control of the constitutive P_bla promoter. The pmlc plasmid is pBRCV10 as described in panel A. Relative levels of activity from P_sgrS-lacZ for each strain are indicated below the Western blot. C. Alignment of the P_sgrS and P_mlc sequences. Conserved residues are indicated by asterisks below the sequence alignment. The positions of the 3-bp substitution mutations constructed in plasmids pBRCV24 and pBRCV25 are indicated by boxes. For sub1, “5′-AAG-3′” was changed to “5′-TTC-3′”; for sub2, “5′-AGG-3′” was changed to “5′-TCC-3′” by QuikChange mutagenesis as described in Materials and Methods.

Negative autoregulation by SgrR. A. The relative limits of the short (P_sgrR1) and long (P_sgrR2) sgrR-lacZ transcriptional fusions are represented at the top. Wild-type (+; CV9000 and CV9200) (Table 1) and ΔsgrR::cat mutant (−; CV9030 and CV9201) (Table 1) host strains carrying the P_sgrR1-lacZ or P_sgrR2-lacZ transcriptional fusion (as indicated below the graph) were grown to mid-log phase, split, and cultured with αMG (+αMG) or without αMG (−αMG). After an additional 45 min, samples were harvested and assayed for β-galactosidase activity. Gray bars represent activity of wild-type strains; black bars represent activity of sgrR mutant strains. The presence or absence of αMG is indicated below the graph. B. The ΔsgrR::cat host strain (CV9030) carrying the P_sgrR1-lacZ transcriptional fusion and a low-copy plasmid with sgrR under control of the heterologous P_lac promoter (pWSKCV1) was grown to mid-log phase, split, and cultured with or without IPTG to induce overexpression of sgrR. The graph of β-galactosidase activity over time begins at the point where the culture was split. Circles represent activity of the culture that was exposed to IPTG; triangles show activity of cells cultured in the absence of IPTG. For both panels A and B, the results reported are representative of at least three experimental trials. Error bars indicate standard deviations.

SgrR binds sgrS promoter DNA in a site-specific manner. Gel mobility shift assays were carried out using purified SgrR-His protein as described in Materials and Methods. Wild-type P_sgrS is a 74-bp fragment encompassing nucleotides −70 to +4 with respect to sgrS. Mutant P_sgrS contains the 5-bp substitution as pictured in Fig. 1.

Titration of endogenous SgrR by DNA fragments from the yfdZ-ypdA region. A. The relative sizes and directions of transcription of the yfdZ and ypdA genes are indicated by the gene map at top. The limits of titrating clones and location of a 3-bp substitution mutation are indicated as described for Fig. 5. The host strain carrying plasmid clones and relative levels of β-galactosidase activity are as described for Fig. 5. B. Alignment of the P_sgrS and P_yfdZ sequences. Conserved residues are indicated by asterisks below the sequence alignment. The position of the 3-bp substitution mutation constructed in plasmid pBRCV20 is indicated by the box. “5′-AGG-3′” was changed to “5′-TCC-3′” by QuikChange mutagenesis as described in Materials and Methods. C. Cells were cultured and β-galactosidase assays were performed as described for Fig. 2A. Host strains carried P_ypdA-lacZ (LW1000 and LW1010 [Table 1]) or P_yfdZ-lacZ (LW2000 and LW2010 [Table 1]) transcriptional fusions in the wild-type or ΔsgrR::cat backgrounds, as indicated. Gray and black bars represent activities of cultures without and with αMG, respectively.