Transcriptional activation of the htrA (High-temperature requirement A) gene from Bartonella henselae.
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.
Resto-Ruiz
S I
SI
Department of Medical Microbiology and Immunology, College of Medicine, University of South Florida 33612, USA.
Sweger
D
D
Widen
R H
RH
Valkov
N
N
Anderson
B E
BE
D016917:Angiomatosis, Bacillary / Q000382:microbiology; D018416:Bartonella henselae / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D001483:Base Sequence; D002460:Cell Line; D018274:Electroporation / Q000379:methods; D004730:Endothelium, Vascular / Q000166:cytology / Q000382:microbiology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006360:Heat-Shock Proteins; D006358:Hot Temperature; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D033903:Periplasmic Proteins; D011401:Promoter Regions, Genetic; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation; D014774:Virulence
D006360:Heat-Shock Proteins; D008164:Luminescent Proteins; D033903:Periplasmic Proteins; D049452:Green Fluorescent Proteins; C061682:DegP protease; D012697:Serine Endopeptidases