1. Characterisation of Pseudomonas putida FNR proteins
- Author
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Ibrahim, Susan, Green, Jeffrey, and Melanie, Stapleton
- Subjects
572.8 - Abstract
Pseudomonas putida KT2440 is a Gram-negative obligate aerobic bacterium. It is found in very diverse habitats such as soil, plants and water. The Escherichia coli Fumarate-Nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2- sensing transcription factors. However, unlike E. coli, Pseudomonas putida KT2440 possesses three FNR proteins (ANR, PP_3233, and PP_3287). The functions and properties of these multiple FNR proteins are poorly understood, but it has been suggested that they have evolved to fulfill distinct roles. Under anaerobic conditions, all three P. putida FNR proteins possessed iron-sulfur clusters. The iron-sulfur cluster of ANR reacted rapidly with O2 and was converted from a [4Fe-4S] to a [2Fe-2S] cluster, similar to that of E. coli FNR. Furthermore, during cluster conversion sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Activities of P. putida FNR proteins in vivo were consistent with those observed in vitro. Transcription from an FNR-dependent promoter indicated that all three FNR proteins were O2-senstive transcription factors in vivo. Overall, the experimental results suggested that the amino acid substitutions flanking the cysteine residues that coordinate the iron sulfur clusters of P. putida PP_3233 and PP_3287 influence reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2. Reconstituted ANR, PP_3233 and PP_3287 were capable of specifically binding at an FNR box under anaerobic conditions, whereas the apo-proteins were not. Thus, it was concluded that the binding activities of three FNR proteins were enhanced by the iron-sulfur cluster under anaerobic conditions. The recognition of an FNR box by all P. putida FNR proteins indicated that the expressions of all three proteins are likely to be either spatially or temporally separated and they could control identical/overlapping regulons. Similar to P. putida, Desulfovibrio desulfuricans possesses multiple FNR proteins. Desulfovibrio desulfuricans is sulfate-reducing bacterium that has the ability to utilize nitrate and sulfate as electron acceptors. Desulfovibrio desulfuricans possesses two regulatory proteins associated with the response to nitrosative stress HcpR and HcpR2. It is shown that HcpR2 is a transcription factor that responds to O2 and nitric oxide, via a [4Fe-4S] cluster. Holo-HcpR2 reacted with O2 via assembly and disassembly of its iron-sulfur cluster similarly to E. coli FNR.
- Published
- 2016