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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 |
|---|---|
| Personen und Körperschaften: | , , |
| In: | Journal of Bacteriology, 189, 2007, 13, S. 4880-4886 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society for Microbiology
|
| Schlagwörter: |
| author_facet |
Wang, Xueqi Kikuchi, Takane Rikihisa, Yasuko Wang, Xueqi Kikuchi, Takane Rikihisa, Yasuko |
|---|---|
| author |
Wang, Xueqi Kikuchi, Takane Rikihisa, Yasuko |
| spellingShingle |
Wang, Xueqi Kikuchi, Takane Rikihisa, Yasuko Journal of Bacteriology Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor Molecular Biology Microbiology |
| author_sort |
wang, xueqi |
| spelling |
Wang, Xueqi Kikuchi, Takane Rikihisa, Yasuko 0021-9193 1098-5530 American Society for Microbiology Molecular Biology Microbiology http://dx.doi.org/10.1128/jb.00318-07 <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> Proteomic Identification of a Novel <i>Anaplasma phagocytophilum</i> DNA Binding Protein That Regulates a Putative Transcription Factor Journal of Bacteriology |
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| title |
Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| title_unstemmed |
Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| title_full |
Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| title_fullStr |
Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| title_full_unstemmed |
Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| title_short |
Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| title_sort |
proteomic identification of a novel
<i>anaplasma phagocytophilum</i>
dna binding protein that regulates a putative transcription factor |
| topic |
Molecular Biology Microbiology |
| url |
http://dx.doi.org/10.1128/jb.00318-07 |
| publishDate |
2007 |
| physical |
4880-4886 |
| description |
<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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| author | Wang, Xueqi, Kikuchi, Takane, Rikihisa, Yasuko |
| author_facet | Wang, Xueqi, Kikuchi, Takane, Rikihisa, Yasuko, Wang, Xueqi, Kikuchi, Takane, Rikihisa, Yasuko |
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| description | <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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| imprint_str_mv | American Society for Microbiology, 2007 |
| institution | DE-D275, DE-Bn3, DE-Brt1, DE-Zwi2, DE-D161, DE-Gla1, DE-Zi4, DE-15, DE-Rs1, DE-Pl11, DE-105, DE-14, DE-Ch1, DE-L229 |
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| physical | 4880-4886 |
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| series | Journal of Bacteriology |
| source_id | 49 |
| spelling | Wang, Xueqi Kikuchi, Takane Rikihisa, Yasuko 0021-9193 1098-5530 American Society for Microbiology Molecular Biology Microbiology http://dx.doi.org/10.1128/jb.00318-07 <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> Proteomic Identification of a Novel <i>Anaplasma phagocytophilum</i> DNA Binding Protein That Regulates a Putative Transcription Factor Journal of Bacteriology |
| spellingShingle | Wang, Xueqi, Kikuchi, Takane, Rikihisa, Yasuko, Journal of Bacteriology, Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor, Molecular Biology, Microbiology |
| title | Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| title_full | Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| title_fullStr | Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| title_full_unstemmed | Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| title_short | Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| title_sort | proteomic identification of a novel <i>anaplasma phagocytophilum</i> dna binding protein that regulates a putative transcription factor |
| title_unstemmed | Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor |
| topic | Molecular Biology, Microbiology |
| url | http://dx.doi.org/10.1128/jb.00318-07 |