Identification of an ArgR-controlled promoter within the outermost region of the IS 10 R mobile element

Abstract

The transposon Tn10 is a prevalent composite element often detected in enteric bacteria, including those obtained from clinical samples. The Tn10 is flanked by two IS10 elements that work together in mediating transposition. IS10-right (IS10R) promotes transposition, while IS10-left lacks a functional transposase and cannot transpose independently. IS10R contains a weak promoter crucial for transposase transcription (pIN), along with two outward-oriented promoters, pOUT and OUTIIp, which may influence the expression of adjacent genes flanking the transposition site. Here, we report the identification of a novel outward-facing promoter, pOUT70, and a functional translation initiation region (TIR) within the last 70 nucleotides of IS10R. Furthermore, we show that pOUT70 is negatively regulated by ArgR and positively controlled by IHF, and we demonstrate that pOUT70 enables growth phase-dependent expression of a truncated yet constitutively active version of the histidine kinase BarA. These findings underscore the significance of IS elements in enhancing downstream gene expression, and highlights the role of outward-facing promoters in derepressing virulence factors or acquiring antibiotic resistance.

Importance: Mobile genetic elements are small DNA fragments that can relocate within the genome, causing either gene inactivation or enhanced gene expression. Our research identified a new functional promoter and mRNA translation region within the IS10R element, which is part of the widely distributed Tn10 transposon. We found that the global regulators ArgR and IHF control the activity of this promoter. Additionally, insertion of this mini-Tn10 derivative into the barA gene resulted in the expression of a truncated but constitutive active form of the BarA sensor kinase. Overall, our work sheds light on how mobile genetic elements could impact the physiology and virulence of opportunistic pathogenic bacteria.

Keywords: ArgR; BarA histidine kinase; IHF; Tn10; Transposon; outward-facing promoter.

Fig 1

The barA’ mutant allele results in growth phase-dependent expression of a truncated version of BarA, the activity of which is independent of CsrA. (A) Overnight cultures of the isogenic strains KSB837 (WT) (diamonds), IFC5010 (csrA^-) carrying the uvrY-expressing plasmid pMX543 (squares), IFC6029 (csrA^- barA’ harboring pMX543) (triangles), and IFC6031 (csrA^- barA’) (circles), all carrying the csrB-lacZ transcriptional fusion, were diluted to an OD₆₀₀ of ≈ 0.01 in LB medium, and after the OD₆₀₀ reached 0.15, the β-galactosidase activity was followed for 240 min. (B) Cultures of IFC6030 (barA’) (circles) and IFC6032 (uvrY^- barA’) (squares) were grown in LB medium, and their β-galactosidase activity was followed. To facilitate comparisons, the β-galactosidase activity during growth of the wild-type (KSB837) strain is included (diamonds, dashed line). (C) Extracts from strains KSB837 (WT), IFC5036 (barA^-), and IFC6030 (barA’) were loaded in lanes 1, 2, and 3, respectively. BarA protein (102 KDa as full-length protein) was detected by using BarA polyclonal antibodies. DnaK (70 KDa), detected using DnaK polyclonal antibodies, was used as a loading control. (D) Levels of BarA protein (102 KDa) in KSB837 (wt) and of BarA^249-918 (77 KDa) in IFC6030 (barA’) cells harvested throughout the growth curve (OD₆₀₀ of 0.3 to 3.0) as determined by Western blot analyses using BarA polyclonal antibodies. DnaK was used as a loading control.

Fig 2

Identification of the functional promoter (pOUT70) and translation initiation region (TIR) located within the outermost 70 base pairs of IS10R. (A) Schematic depiction of the IS10R and mini-Tn10 arrangement, and sequence of the outermost 70 base pairs of IS10R containing the pOUT70 promoter and TIR. The sequence alterations introduced in IS10R-70^-10mut, IS10R-70^RBSmut, and IS10R-70^ATCmut variants are shown (highlighted in red). (B) Cultures of the isogenic strains IFC6033 (IS10R-70’-lacZ) (circles) and IFC6034 (IS10R-70^-10mut’-lacZ) (squares) were grown in LB medium, and β-galactosidase activity was followed. (C) Cultures of strains IFC6035 (IS10R-70′-TIR^-lacZ) (circles), IFC6036 (IS10R-70^ATCmut’-TIR^-lacZ) (triangles), and IFC6037 (IS10R-70^RBSmut’-TIR^-lacZ) (squares) were grown aerobically in LB medium, and β-galactosidase activity was monitored. For comparison, the β-galactosidase activities of strain IFC6033 (IS10R-70’-lacZ) grown under the same conditions are depicted (inverted triangles, dashed line).

Fig 3

Disruption of the argR gene leads to a derepression of the pOUT70 activity in exponential phase growth growing cells. (A) Strains IFC6033 (WT) and IFC6039 (argR^-), both carrying the transcriptional IS10R-70’-lacZ fusion, were grown aerobically in LB medium to an OD₆₀₀ of 0.7 (white bars) or 2.1 (black bars), and β-galactosidase activity was measured and expressed in Miller Units. (B) Strains KSB837 (WT), IFC6047 (argR^-), IFC5036 (barA^-), IFC6048 (barA^- argR^-), IFC6030 (barA’), and IFC6043 (barA’ argR^-), all of which carrying the UvrY-P activatable csrB-lacZ reporter, were grown aerobically in LB medium to an OD₆₀₀ of 0.7 (white bars) or 2.1 (black bars), and β-galactosidase activity was measured and expressed in Miller Units. (C) Levels of BarA protein in KSB837 (WT), IFC6047 (argR^-), IFC6030 (barA’), and IFC6043 (barA’ argR^-) cells harvested at an OD₆₀₀ of 0.7 or 2.1 were determined by Western blot analyses using BarA polyclonal antibodies. DnaK was used as a loading control.

Fig 4

Overproduction of ArgR results in repression of the pOUT70 activity. (A) The cells of strain IFC6033 (WT, IS10R-70’-lacZ), without plasmid or carrying either a pACT3 derivative plasmid that harbor the argR gene under the araP promoter or the empty plasmid backbone, were grown in LB to midexponential growth phase (OD₆₀₀ of 0.7) (white bars) or to an OD₆₀₀ of 2.1 (black bars) in the presence or absence of l-arabinose as inducer, and the β-galactosidase activity was measured. (B) The cells of strain IFC6030 (barA’), carrying the UvrY-P activatable csrB-lacZ reporter, without plasmid or carrying either a pACT3 derivative plasmid that harbor the argR gene under the araP promoter or the empty plasmid backbone, were grown in LB to midexponential growth phase (OD₆₀₀ of 0.7) (white bars) or to an OD₆₀₀ of 2.1 (black bars) in the presence or absence of l-arabinose as inducer, and the β-galactosidase activity was measured. (C) Levels of BarA protein in KSB837 (WT) and IFC6030 (barA’), without plasmid or carrying either a pACT3 derivative plasmid that harbor the argR gene under the araP promoter or the empty plasmid backbone, were grown in LB in the presence or absence of l-arabinose as inducer to an OD₆₀₀ of ~2.1 were analyzed by Western blot using BarA polyclonal antibodies. DnaK was used as a loading control.

Fig 5

ArgR regulates the pOUT70 activity by binding to the ARG operator within the IS10R-70 sequence. (A) Predicted ARG boxes within the outermost 70 base pairs of IS10R containing the pOUT70 promoter (enclosed in the upper sequence). The sequence alterations introduced in IS10R-70^-ARGmut-lacZ transcriptional reporter are highlighted in red (bottom sequence). (B) Cells of strain IFC6038 (IS10R-70^-ARGmut-lacZ), without plasmid or carrying a pACT3 derivative plasmid that harbor the argR gene under the araP promoter, and of strain IFC6033 (IS10R-70’-lacZ), were grown in LB to an OD₆₀₀ of 0.7 (white bars) or to an OD₆₀₀ of 2.1 (black bars) in the presence or absence of l-arabinose as inducer, and the β-galactosidase activity was measured.