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Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor
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| Zeitschriftentitel: | Journal of Bacteriology |
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| Personen und Körperschaften: | , , |
| In: | Journal of Bacteriology, 189, 2007, 13, S. 4880-4886 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society for Microbiology
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| Schlagwörter: |
| Zusammenfassung: | <jats:title>ABSTRACT</jats:title> <jats:p> <jats:italic>Anaplasma phagocytophilum</jats:italic> , the etiologic agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium. Little is known about the gene regulatory mechanisms for this bacterium. A gene encoding a putative transcription factor, <jats:italic>tr1</jats:italic> , upstream of three tandem genes encoding outer membrane proteins, including the major outer membrane protein P44, is driven by a strong promoter. In the present study, gel mobility shift assays revealed the presence of <jats:italic>A. phagocytophilum</jats:italic> proteins that interact with the promoter region of <jats:italic>tr1</jats:italic> . These proteins interacting with the <jats:italic>tr1</jats:italic> promoter region were purified by biotin-labeled DNA affinity chromatography from a large amount of host cell-free bacteria. Mass spectrometry identified the major protein as an <jats:italic>A. phagocytophilum</jats:italic> 12.5-kDa hypothetical protein, which was named ApxR. In a DNase I protection assay, recombinant ApxR (rApxR) bound cooperatively to four 24- or 25-bp sites within 235 bp upstream of <jats:italic>tr1</jats:italic> : regions III and IV proximal to <jats:italic>tr1</jats:italic> had higher affinity than regions I and II did. Deletion assays showed that regions III and IV were essential for rApxR binding, whereas regions I and II upstream of regions III and IV were not. The primary <jats:italic>cis</jats:italic> -acting region was region IV, since region IV alone was sufficient for rApxR to strongly transactivate the downstream gene in a <jats:italic>lacZ</jats:italic> reporter assay. Addition of regions I, II, and III did not enhance transactivation. These results show that ApxR is a novel transcriptional regulator that directly regulates <jats:italic>tr1</jats:italic> . </jats:p> |
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| Umfang: | 4880-4886 |
| ISSN: |
0021-9193
1098-5530 |
| DOI: | 10.1128/jb.00318-07 |