OmpR and LeuO positively regulate the Salmonella enterica serovar Typhi ompS2 porin gene

Abstract

The Salmonella enterica serovar Typhi ompS2 gene codes for a 362-amino-acid outer membrane protein that contains motifs common to the porin superfamily. It is expressed at very low levels compared to the major OmpC and OmpF porins, as observed for S. enterica serovar Typhi OmpS1, Escherichia coli OmpN, and Klebsiella pneumoniae OmpK37 quiescent porins. A region of 316 bp, between nucleotides -413 and -97 upstream of the transcriptional start point, is involved in negative regulation, as its removal resulted in a 10-fold increase in ompS2 expression in an S. enterica serovar Typhi wild-type strain. This enhancement in expression was not observed in isogenic mutant strains, which had specific deletions of the regulatory ompB (ompR envZ) operon. Furthermore, ompS2 expression was substantially reduced in the presence of the OmpR D55A mutant, altered in the major phosphorylation site. Upon random mutagenesis, a mutant where the transposon had inserted into the upstream regulatory region of the gene coding for the LeuO regulator, showed an increased level of ompS2 expression. Augmented expression of ompS2 was also obtained upon addition of cloned leuO to the wild-type strain, but not in an ompR isogenic derivative, consistent with the notion that the transposon insertion had increased the cellular levels of LeuO and with the observed dependence on OmpR. Moreover, LeuO and OmpR bound in close proximity, but independently, to the 5' upstream regulatory region. Thus, the OmpR and LeuO regulators positively regulate ompS2.

FIG. 1.

Southern blot hybridization of genomic DNA from S. enterica serovar Typhi (Sty), S. enterica serovar Typhimurium (Stm), and E. coli (Eco) digested with SalI restriction endonuclease, with the S. enterica serovar Typhi ompS1 gene (37) labeled with ³²P. The identities of the hybridizing bands for Salmonella genes are shown on the left, including a novel conspicuous band that corresponds to ompS2, and those for E. coli genes are shown on the right.

FIG. 2.

Nucleotide sequence of the S. enterica serovar Typhi ompS2 5′ upstream regulatory region, featuring the first five codons of the structural gene, the putative Shine-Dalgarno ribosome-binding sequences (SD), the −10 and −35 promoter sequences, and the single transcription initiation at a G residue obtained by primer extension analysis. Also shown are the boundaries for various pFM translational fusions, where the indicated segments of the regulatory region plus five codons were fused to the lacZ reporter gene. A sequence with similarity to the consensus OmpR-binding site (thick underline), the site for the binding of OmpR (thin dashed line), and two boxes for the binding of LeuO (I and II) (thick dashed lines) are also represented.

FIG. 3.

(A) Activity of the ompS2 gene depends on the length of the regulatory region and on the ompB operon. β-Galactosidase-specific activities rendered by pFM fusion plasmids, containing various lengths of the 5′ regulatory region as indicated by their numeral denomination (Fig. 2), harbored in S. enterica serovar Typhi are shown. The gray bars depict activities in the wild-type (WT) strain; black bars show activity in the isogenic S. enterica serovar Typhi 81 (ΔompB::Km) strain. (B) Mapping by primer extension of the first (+1) transcribed G residue of the ompS2 gene. The initiation of transcription is shown from pFM413, in the absence (−) and in the presence (+) of the leuO gene (cloned in ptrcleuO); from pFM97, lacking 317 bp between −413 and −97; and from pFM34. The Shine-Dalgarno (SD) sequence, the first methionine of the ORF, and the GATC sequence ladder of the complementary strand are shown.

FIG. 3.

(A) Activity of the ompS2 gene depends on the length of the regulatory region and on the ompB operon. β-Galactosidase-specific activities rendered by pFM fusion plasmids, containing various lengths of the 5′ regulatory region as indicated by their numeral denomination (Fig. 2), harbored in S. enterica serovar Typhi are shown. The gray bars depict activities in the wild-type (WT) strain; black bars show activity in the isogenic S. enterica serovar Typhi 81 (ΔompB::Km) strain. (B) Mapping by primer extension of the first (+1) transcribed G residue of the ompS2 gene. The initiation of transcription is shown from pFM413, in the absence (−) and in the presence (+) of the leuO gene (cloned in ptrcleuO); from pFM97, lacking 317 bp between −413 and −97; and from pFM34. The Shine-Dalgarno (SD) sequence, the first methionine of the ORF, and the GATC sequence ladder of the complementary strand are shown.

FIG. 4.

Effect of the OmpR D55A mutant on ompS2 activity. Activities are shown for an S. enterica serovar Typhi ompF-lacZ fusion, used as a control, and of pFM413 and pFM97 in either the IMSS-1 wild type (wt) or in the isogenic S. enterica serovar Typhi IMSS-40 ompR derivative (ΔompR::Km), not complemented (−) or complemented with either a plasmid harboring the wild-type (wt) OmpR gene or the D55A mutant gene, mutated in the codon for the main phosphorylation site (11).

FIG. 5.

Expression of the OmpS2 protein. PAGE profile of OMP preparations from wild-type (wt) S. enterica serovar Typhi, grown at either low (L) or high (H) osmolarity, and harboring either the pBR322 vector (vector), pFM413S2 (413; containing the structural ompS2 gene under the control of a 413-bp regulatory region), or pFM97S2 (97; ompS2 under a 97-bp regulatory region), S. enterica serovar Typhi ΔompB::Km, containing either no plasmid (−) or pFM97S2 (97), at low osmolarity. The major OMPs OmpC, OmpF, and OmpA are indicated as C, F, and A, respectively. The relative abundance of the OmpS2 (S2) or OmpF (F) protein with respect to OmpA (A) is shown for some lanes and was obtained by densitometric analysis.

FIG. 6.

LeuO participates in the regulation of ompS2. (A) Activity of ompS2 from pFM413, both in S. enterica serovar Typhi IMSS-1 (wild type [wt]) and in the IMSSTN103 insertion mutant (Tn10::leuO), at low (black bars) and high (gray bars) osmolarity. (B) PAGE profile of OMP preparations from S. enterica serovar Typhi IMSS-1 (wt) and from the IMSSTN103 insertion mutant (TN103) at low (L) and high (H) osmolarity, depicting the expression of OmpS2 (S2) from the chromosomal gene. The expression of OmpS2 from pFM97S2 was included as a control marker. The major OMPs OmpC, OmpF, and OmpA are indicated as C, F, and A, respectively. The relative abundance of the OmpS2 (S2) or OmpF (F) protein with respect to OmpA (A) is shown for some lanes and was obtained by densitometric analysis.

FIG. 6.

LeuO participates in the regulation of ompS2. (A) Activity of ompS2 from pFM413, both in S. enterica serovar Typhi IMSS-1 (wild type [wt]) and in the IMSSTN103 insertion mutant (Tn10::leuO), at low (black bars) and high (gray bars) osmolarity. (B) PAGE profile of OMP preparations from S. enterica serovar Typhi IMSS-1 (wt) and from the IMSSTN103 insertion mutant (TN103) at low (L) and high (H) osmolarity, depicting the expression of OmpS2 (S2) from the chromosomal gene. The expression of OmpS2 from pFM97S2 was included as a control marker. The major OMPs OmpC, OmpF, and OmpA are indicated as C, F, and A, respectively. The relative abundance of the OmpS2 (S2) or OmpF (F) protein with respect to OmpA (A) is shown for some lanes and was obtained by densitometric analysis.

FIG. 7.

Induction of ompS2 expression in the presence of cloned leuO. β-Galactosidase-specific activity from pFM413 is shown. (A) S. enterica serovar Typhi IMSS-1 (wild type [wt]) or the isogenic IMSS-40 ΔompR derivative, transformed with either the plasmid vector (pMPM-A3) or pFM800TL, harboring the cloned leuO gene with the Tn10 insertion in the upstream region, at low (black) and high (gray) osmolarity. (B) S. enterica serovar Typhi IMSS-1, transformed with the pFMTrc12 vector plasmid or with the pFMTrcleuO plasmid, carrying leuO under the control of the trc promoter, at low (black) and high (gray) osmolarity.

FIG. 8.

DNA footprinting at the 5′ regulatory region of ompS2 with LeuO and OmpR-P. On the left panel, the DNase-minus/protein-minus lane is shown with a minus sign (−) and the DNase-plus/protein-minus lane is depicted with a plus sign (+). The following lanes contained 0.03, 0.08, 0.15, 0.23 (vertical arrow), and 0.30 μM LeuO, respectively. The right panel also contains a DNase-minus/protein-minus lane (−) and a DNase-plus/protein-minus lane (+), followed by seven lanes with increasing concentrations of OmpR-P (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 μM). The next lanes contain the same increasing concentrations of OmpR-P but in the presence of 0.23 μM LeuO. Binding boxes LeuO (I) and LeuO (II) are shown, as is the binding region for OmpR. The solid black bar represents the 20-bp sequence with similarity to the OmpR-binding F1 and C1 type boxes.