Open AccessTranscriptional Activation of the htrA (High-Temperature Requirement A) Gene from Bartonella henselae
Author(s) -
Sandra Resto-Ruiz,
Debra Sweger,
Raymond Widen,
Nikola Valkov,
Burt Anderson
Publication year - 2000
Publication title -
infection and immunity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.508
H-Index - 220
eISSN - 1070-6313
pISSN - 0019-9567
DOI - 10.1128/iai.68.10.5970-5978.2000
Subject(s) - biology , promoter , bartonella henselae , microbiology and biotechnology , reporter gene , gene , transcription (linguistics) , gene expression , periplasmic space , regulation of gene expression , green fluorescent protein , escherichia coli , genetics , linguistics , philosophy , serology , antibody
BacterialhtrA genes are typically activated as part of the periplasmic stress response and are dependent on the extracytoplasmic sigma factorrpoE . A putative promoter region, P1, of the ςE -type heat-inducible promoters has previously been identified upstream of thehtrA gene ofBartonella henselae . Further analysis of thehtrA 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 ςE -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 intoB. henselae by electroporation. The contiguous promoter region containing both P1 and P2 were necessary for the optimal transcriptional activation of thehtrA gene. Promoter activity at 37°C was distinctively higher than at 27°C. However, thermal induction at 47°C did not increase expression ofgfpmut3 . Invasion of human microvascular endothelial cells (HMEC-1) byB. henselae resulted in the formation of well-defined vacuoles containing clusters of bacteria exhibiting marked expression ofgfpmut3 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 ofhtrA 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.