ModA

Background

Nitrogen fixation is a process in which nitrogen in the atmosphere is converted into ammonium (NH⁴⁺).No matter natural and synthetic, the process is essential for all forms of life because nitrogen is required to biosynthesize basic building blocks of plants, animals and other life forms, like nucleotides and amino acids.

All biological nitrogen fixation is done by way of nitrogenase, a metal-enzymes which contain iron, molybdenum, or vanadium. The enzyme is composed of the heterotetrameric MoFe protein that is transiently associated with the homodimeric Fe protein. Therefore, Molybdenum is an indispensable chemical element to nitrogen fixation and agriculture.

According to the Liebig's law of the minimum, which states that growth is controlled not by the total amount of resources available, but by the scarcest resource, molybdenum as a trace mineral, is identified as limiting the growth of peanuts in large field of China. So molybdenum powder is commonly used as a fertilizer in agriculture. And our project aims to establish an E.Coli strain that is able to capture Mo in the medium, directional deliver itself to root system and kill itself eventually.

ModA

The genes encoding a molybdate transport system (mod) have been isolated and sequenced from Escherichia coli[2–5] Four genes modABCD, organized in an operon, share strong structural similarity and sequence homology with genes encoding binding -protein dependent solute(or ABC-type) transport systems [2–5]. The first gene, modA, encodes a periplasmic binding protein. ModB and ModC have sequence homology with integral membrane and cytoplasmic ATP hydrolysis components of ABC transport systems, respectively. ModD does not show sequence similarity to any gene product in current databases and its role in transport is unclear. Two more genes, modEF, are also involved in molybdate transport. The modE gene encodes a regulatory protein that effects molybdenum-dependent repression at the modA promoter [6–8]. The exact role of modF is not yet clear [6].

The mod operon encodes a very high affinity(Km=15 nM) molybdate transport system. The low K values agree well with previous results obtained with the enteric diazotroph K. pneumoniae ([1]; Km=20 nM, J. Imperial, unpublished results). and adequately explain the high molybdenum scavenging capacity observed with most bacteria[11].

ModA, the periplasmic binding component of the mod system. had a K for molybdate of 3–5 mM. This is one of the highest substrate Kd values reported for any periplasmic binding protein [9,10] and contrasts with the low Kd values for molybdate uptake The Structure of modA[14]

How to bind molybdenum[15]

Stereo view of bound tungstate in the ModA binding site. The W atom is shown as a green sphere, all other elements are shown in ball-and-stick representation, and the residues are labeled. The blue mesh represents a 2Fo-Fc omit map contoured at a level of 2σ and shown only around the 5 atoms of the tungstate ion. Note that the tungstate and molybdate binding sites are indistinguishable at the current resolution, which reflects the similar ionic radii of the two ions[2,3]

Design

We cloned the ModA(sequence) into the pET21a, and translated into BL21 to expressed and purified modA, detected molybdate binding to the ModA protein based on the migration of the pure protein in native polyacrylamide gels.(protocol)

Result

Figure 1 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography.

Figure 1 Expression and purification of ModA. Expression of ModA and its purification by His-chelating chromatography. Lane 1, molecular weight standards (kDa); lane 2, total bacterial proteins before IPTG induction; lane 3, total bacterial proteins after 0.5 mM IPTG induction; lane 4, total bacterial lysate after 0.5 mM IPTG induction; lane 5,the sediment after 16,000 rpm 30 min; lane 6 the flow through of column loading; lane 7,the flow of buffer B .lane 8 The purification of ModA by size-elution chromatography.

Figure 2 Native-PAGE to provide the molybdate lane 1, the modA(acetic buffer PH 5.0); lane 2, the modA incubate with 20mM molybdate.

Discussion

Why were there 2 bands obviously in figure 1？

The two bands were in the lane 3-lane 8, so when the modA was inducted, maybe the modA has been cut, and for the pET-21a sequence, we knew that the his-tag was in the C-terminal, and the His-modA can chelate to the Ni column, so we guessed what the modA was cut was its C-terminal or pET-21a’s T7-tag(for western), what we work next maybe clone a new modA that there were few animo acid lost to see whether there were 2 bands, and we cut or use clone to cut the T7-tag to verify.

Why were there so many bands obviously in figure 2?

We actually did not know why there were few band above to lane 1, maybe the modA were polymers in the 20mM molybdate, the band below may be the modA binding molybdate that made its charge changed and got faster than others, and because we use excessive protein, not all proteins bind to the molybdate.

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