Expression, purification and DNA-binding activities of two putative ModE proteins of Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)

Abstract

In prokaryotes molybdenum is taken up by a high-affinity ABC-type transporter system encoded by the modABC genes. The endophyte β-Proteobacterium Herbaspirillum seropedicae has two modABC gene clusters and two genes encoding putative Mo-dependent regulator proteins (ModE1 and ModE2). Analysis of the amino acid sequence of the ModE1 protein of H. seropedicae revealed the presence of an N-terminal domain containing a DNA-binding helix-turn-helix motif (HTH) and a C-terminal domain with a molybdate-binding motif. The second putative regulator protein, ModE2, contains only the helix-turn-helix motif, similar to that observed in some sequenced genomes. We cloned the modE1 (810 bp) and modE2 (372 bp) genes and expressed them in Escherichia coli as His-tagged fusion proteins, which we subsequently purified. The over-expressed recombinant His-ModE1 was insoluble and was purified after solubilization with urea and then on-column refolded during affinity chromatography. The His-ModE2 was expressed as a soluble protein and purified by affinity chromatography. These purified proteins were analyzed by DNA band-shift assays using the modA2 promoter region as probe. Our results indicate that His-ModE1 and His-ModE2 are able to bind to the modA2 promoter region, suggesting that both proteins may play a role in the regulation of molybdenum uptake and metabolism in H. seropedicae.

Coordinated regulation of nitrogen fixation and molybdate transport by molybdenum

“…For details on nitrogenase structures and activities, see recent publications (Seefeldt et al, 2013;Hu and Ribbe, 2015;Sippel and Einsle, 2017;Mus et al, 2018). (Hoffmann et al, 2016), E. coli modA and moaA (Anderson et al, 1997;McNicholas et al, 1997), H. seropedicae modA2 (Souza et al, 2008), and R. capsulatus anfA, mopA, morA, and mop (Wiethaus et al, 2006) were verified by in vitro binding studies.…”

“… Mo‐boxes (distal and proximal) of A. tumefaciens modA (Hoffmann et al , ), E. coli modA and m oaA (Anderson et al , ; McNicholas et al , ), H. seropedicae modA2 (Souza et al , ), and R. capsulatus anfA , mopA , morA and mop (Wiethaus et al , ) were verified by in vitro binding studies. Experimentally determined transcription start sites for A. tumefaciens modA (Hoffmann et al ., ), E. coli modA (Rech et al , ), and R. capsulatus anfA and mopA (Kutsche et al , ) are marked by underlining. …”

“… Mo‐boxes of A. tumefaciens modA (Hoffmann et al , ), E. coli modA and moaA (Anderson et al , ; McNicholas et al , ), H. seropedicae modA2 (Souza et al , ), and R. capsulatus anfA , mopA , morA , and mop (Wiethaus et al , ) were verified by in vitro binding studies. …”

“…This question has been extensively addressed in the photosynthetic alphaproteobacterium R. capsulatus, which is capable of synthesizing two nitrogenases, a Mo-nitrogenase and a Fe-nitrogenase (Schneider et al, 1991;Strnad et al, 2010). Both R. capsulatus modE homologs (called mopA and mopB) are part of a large nitrogen fixation gene cluster containing the structural genes of Mo-nitrogenase, nifHDK (Wang (Hoffmann et al, 2016), E. coli modA and moaA (Anderson et al, 1997;McNicholas et al, 1997), H. seropedicae modA2 (Souza et al, 2008), and R. capsulatus anfA, mopA, morA and mop (Wiethaus et al, 2006) were verified by in vitro binding studies. Experimentally determined transcription start sites for A. tumefaciens modA (Hoffmann et al, 2016), E. coli modA (Rech et al, 1995), and R. capsulatus anfA and mopA (Kutsche et al, 1996) are marked by underlining.…”

“…Overall, formation of MopA-MopB heterodimers might provide a fine-tuning mechanism regulating Mo-responsive gene expression in R. capsulatus. Since many bacteria including A. vinelandii, H. seropedicae and R. palustris have two or more modE copies (Table 1), differential formation of homo-and heterodimers might be widespread in bacteria (Larimer et al, 2004;Souza et al, 2008;Zhang and Gladyshev, 2008;Setubal et al, 2009).…”