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Novel blue light-sensitive proteins from a metagenomic approach.

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Title: Novel blue light-sensitive proteins from a metagenomic approach.
Authors: Pathak, G. P.1, Ehrenreich, A.2, Losi, A.3, Streit, W. R.4 wolfgang.streit@uni-hamburg.de, Gärtner, W.1 gaertner@mpimuelheim.mpg.de
Source: Environmental Microbiology. Sep2009, Vol. 11 Issue 9, p2388-2399. 12p. 4 Diagrams, 1 Chart, 2 Graphs.
Subjects: Oligonucleotides, Photoreceptors, Genes, DNA, Blue light
Abstract: A microarray-based approach was used to screen a soil metagenome for the presence of blue light (BL) photoreceptor-encoding genes. The microarray carried 149 different 54-mer oligonucleotides, derived from consensus sequences of light, oxygen and voltage (LOV) domain BL photoreceptor genes. Calibration of the microarrays allowed the detection of minimally 50 ng of genomic DNA against a background of 2–5 μg of genomic DNA. Identification of a positive cosmid clone was still possible for an amount of 0.25 ng against a background of 10 μg of labelled DNA clones. The array could readily identify targets carrying 4% sequence mismatch. Using the LOV microarray, up to 1200 library clones in concentrations of c. 20 ng each with a c. 40 kb insert size could be screened in a single batch. After calibration and reliability controls, the microarray was probed with cosmid-cloned DNA from the thermophilic fraction of a soil sample. From this approach, a novel gene was isolated that encodes a protein consisting of several Per-Arnt-Sim domains, a LOV domain associated to a histidine kinase and a response regulator domain. The novel gene showed highest similarity to a known sequence from Kineococcus radiotolerans SRS30216 (58% identity for the LOV domain only) and to a gene from Methylibium petroleiphilum PM1 (57% identity). The gene, designated as ht-met1 (Hamburg Thermophile Metagenome 1), was isolated and fully sequenced (3615 bp). ht-met1 is followed by a second open reading frame encoding a Fe-chelatase, an arrangement quite frequent for BL photoreceptors. The LOV domain region of ht-met1 was subcloned and expressed yielding a fully functional, flavin-containing LOV domain. Irradiation generated the typical LOV photochemistry, with the transient formation of a flavin-protein photoadduct. The dark recovery lifetime was found as τREC = 120 s (20°C) and is among the fastest ones determined so far for bacterial LOV domains. [ABSTRACT FROM AUTHOR]
Copyright of Environmental Microbiology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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DbLabel: Engineering Source
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  Data: Novel blue light-sensitive proteins from a metagenomic approach.
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  Data: <searchLink fieldCode="AR" term="%22Pathak%2C+G%2E+P%2E%22">Pathak, G. P.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Ehrenreich%2C+A%2E%22">Ehrenreich, A.</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Losi%2C+A%2E%22">Losi, A.</searchLink><relatesTo>3</relatesTo><br /><searchLink fieldCode="AR" term="%22Streit%2C+W%2E+R%2E%22">Streit, W. R.</searchLink><relatesTo>4</relatesTo><i> wolfgang.streit@uni-hamburg.de</i><br /><searchLink fieldCode="AR" term="%22Gärtner%2C+W%2E%22">Gärtner, W.</searchLink><relatesTo>1</relatesTo><i> gaertner@mpimuelheim.mpg.de</i>
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  Data: <searchLink fieldCode="JN" term="%22Environmental+Microbiology%22">Environmental Microbiology</searchLink>. Sep2009, Vol. 11 Issue 9, p2388-2399. 12p. 4 Diagrams, 1 Chart, 2 Graphs.
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  Data: <searchLink fieldCode="DE" term="%22Oligonucleotides%22">Oligonucleotides</searchLink><br /><searchLink fieldCode="DE" term="%22Photoreceptors%22">Photoreceptors</searchLink><br /><searchLink fieldCode="DE" term="%22Genes%22">Genes</searchLink><br /><searchLink fieldCode="DE" term="%22DNA%22">DNA</searchLink><br /><searchLink fieldCode="DE" term="%22Blue+light%22">Blue light</searchLink>
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  Data: A microarray-based approach was used to screen a soil metagenome for the presence of blue light (BL) photoreceptor-encoding genes. The microarray carried 149 different 54-mer oligonucleotides, derived from consensus sequences of light, oxygen and voltage (LOV) domain BL photoreceptor genes. Calibration of the microarrays allowed the detection of minimally 50 ng of genomic DNA against a background of 2–5 μg of genomic DNA. Identification of a positive cosmid clone was still possible for an amount of 0.25 ng against a background of 10 μg of labelled DNA clones. The array could readily identify targets carrying 4% sequence mismatch. Using the LOV microarray, up to 1200 library clones in concentrations of c. 20 ng each with a c. 40 kb insert size could be screened in a single batch. After calibration and reliability controls, the microarray was probed with cosmid-cloned DNA from the thermophilic fraction of a soil sample. From this approach, a novel gene was isolated that encodes a protein consisting of several Per-Arnt-Sim domains, a LOV domain associated to a histidine kinase and a response regulator domain. The novel gene showed highest similarity to a known sequence from Kineococcus radiotolerans SRS30216 (58% identity for the LOV domain only) and to a gene from Methylibium petroleiphilum PM1 (57% identity). The gene, designated as ht-met1 (Hamburg Thermophile Metagenome 1), was isolated and fully sequenced (3615 bp). ht-met1 is followed by a second open reading frame encoding a Fe-chelatase, an arrangement quite frequent for BL photoreceptors. The LOV domain region of ht-met1 was subcloned and expressed yielding a fully functional, flavin-containing LOV domain. Irradiation generated the typical LOV photochemistry, with the transient formation of a flavin-protein photoadduct. The dark recovery lifetime was found as τREC = 120 s (20°C) and is among the fastest ones determined so far for bacterial LOV domains. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
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  Data: <i>Copyright of Environmental Microbiology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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      – Type: doi
        Value: 10.1111/j.1462-2920.2009.01967.x
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      – Code: eng
        Text: English
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        StartPage: 2388
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        Type: general
      – SubjectFull: Photoreceptors
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      – SubjectFull: Genes
        Type: general
      – SubjectFull: DNA
        Type: general
      – SubjectFull: Blue light
        Type: general
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      – TitleFull: Novel blue light-sensitive proteins from a metagenomic approach.
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              M: 09
              Text: Sep2009
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