Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 68 (10), 5970-8

Transcriptional Activation of the htrA (High-temperature Requirement A) Gene From Bartonella Henselae

Affiliations

Transcriptional Activation of the htrA (High-temperature Requirement A) Gene From Bartonella Henselae

S I Resto-Ruiz et al. Infect Immun.

Abstract

Bacterial htrA genes are typically activated as part of the periplasmic stress response and are dependent on the extracytoplasmic sigma factor rpoE. A putative promoter region, P1, of the sigma(E)-type heat-inducible promoters has previously been identified upstream of the htrA gene of Bartonella henselae. Further analysis of the htrA mRNA by primer extension demonstrated that transcription initiates from P1 and a second region downstream of P1. This second promoter region, termed P2, had no sequence identity to sigma(E)-type heat-inducible promoters. Promoter regions were cloned individually and in tandem into pANT3 upstream of a promoterless version of the green fluorescent protein (GFP) gene (gfpmut3) and transformed into B. henselae by electroporation. The contiguous promoter region containing both P1 and P2 were necessary for the optimal transcriptional activation of the htrA gene. Promoter activity at 37 degrees C was distinctively higher than at 27 degrees C. However, thermal induction at 47 degrees C did not increase expression of gfpmut3. Invasion of human microvascular endothelial cells (HMEC-1) by B. henselae resulted in the formation of well-defined vacuoles containing clusters of bacteria exhibiting marked expression of gfpmut3 transcribed from the P1-P2 region. In addition, a moderate yet significant increase in the ratio of bacterial GFP to DNA was detected for intracellular bacteria compared to extracellular bacteria, indicating upregulation of htrA upon invasion of HMEC-1. The activation of specific genes in the intracellular environment may help us better understand the novel pathogenic mechanisms used by this bacterium.

Figures

FIG. 1
FIG. 1
The 5′ regulatory region of B. henselae htrA. The region with extensive homology to ςE-type stress response promoters is indicated (P1) and boxed, with the corresponding direction of transcription (dotted line with arrow) and site of transcription initiation (guanine at position 61) (boldface type) as shown. A second site of transcription initiation from promoter region P2 is indicated in boldface type (adenine at position 165) and putative −35 and −10 promoter regions are overlined. The methionine codon representing the start of translation is boxed and the putative ribosome binding site is underlined and labeled (RBS). Sequences complementary to the oligonucleotides used for primer extension are underlined and labeled (PrEx1 and PrEx2). Sequence numbering is identical to that of the sequence with GenBank accession no. L20127, a description of which has been previously published (3).
FIG. 2
FIG. 2
Mapping of the transcription start site of P1. Total RNA and a 5′ end-labeled primer PrEx1 were used in primer extension reactions. The resulting cDNAs and sequencing reactions (GACT) from pBlue1-1, which harbors the htrA regulatory region, were loaded on an 8% acrylamide gel. The start of transcription was mapped to G61 and the complementary cytosine residue on the other strand is indicated (arrow). Lanes: 1, B. henselae htrA; 2, B. henselae groEL control.
FIG. 3
FIG. 3
Mapping of the transcription start site of P2. Total RNA and a 5′ end-labeled primer PrEx2 were used in primer extension reactions. The resulting cDNAs and sequencing reactions from pBlue1-1, harboring the htrA regulatory region, were loaded on an 8% acrylamide gel. The start of transcription was mapped to A165, and the complementary thymine residue on the other strand is indicated (arrow). Lanes: 1, B. henselae htrA; 2, B. henselae groEL control.
FIG. 4
FIG. 4
Thermal induction of htrA promoter regions P1, P2, or P1-P2 in B. henselae analyzed by flow cytometry. B. henselae 882str cells harboring pSIR11, pSIR12, or pSIR13 were grown at 27 or 37°C. Bacterial growth was harvested after 2 days, resuspended in PBS and analyzed in the FACSCalibur. B. henselae 882str was electrotransformed with the following plasmids: (A) pANT3, (B) pSIR11 (harboring P1), (C) pSIR12 (harboring P2), (D) pSIR13 (harboring P1+P2). Arrows indicate the mean fluorescence channel (X) at 27°C and 37°C. Data shown corresponds to one representative set of histograms.
FIG. 5
FIG. 5
Thermal induction of htrA promoter regions in the reverse orientation in B. henselae 882str by flow cytometry. Bacteria harboring pSIR14, pSIR15, or pSIR16 were grown at 27 or 37°C. Bacterial growth was harvested after two days, resuspended in PBS, and analyzed in the FACSCalibur. B. henselae 882str was electrotransformed with the following plasmids: pANT3 (A), pSIR14 (harboring P1R) (B), pSIR15 (harboring P2R) (C), and pSIR16 (harboring P1R-P2R) (D). Arrows indicate the mean fluorescence channel (X) at 27 and 37°C. One representative set of histograms is displayed.
FIG. 6
FIG. 6
Fluorescence microscopy of GFP transcribed from tandem promoter regions P1-P2 of pSIR13. (A) Expression of GFP in B. henselae pSIR13/882str by fluorescence microscopy. Green bacilli indicate uniform GFP expression throughout the bacterial surface. Overall magnification was ×20,000. (B) Invasion of HMEC-1 by B. henselae harboring pSIR13. Slides were stained with the DNA dye DAPI (blue fluorescence). Images were captured with Vysis software. The HMEC-1 nucleus is depicted in blue. Multiple intracellular bacteria are shown in the vacuole containing GFP-expressing B. henselae. Overall magnification was ×400.
FIG. 7
FIG. 7
Intracellular induction of B. henselae promoter regions P1-P2 (■) and P1R-P2R (□) in HMEC-1. The ratio of GFP to DNA was used to determine intracellular activation of the B. henselae htrA promoter regions. A moderate but significant (Pa/b = 0.002) 2.7-fold increase in the intracellular activity of P1-P2 was observed. Promoter constructs in the reverse orientation were not significantly induced (Pc/d = 0.38).

Similar articles

See all similar articles

Cited by 10 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback