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NICKEL SEQUESTERING AND STORAGE BY BRAD YRHIZOBIUM JAPONICUM

Dept of Microbiology, Univ. of Georgia, Athens, GA 30606, USA 1. Introduction

The major class of hydrogenases that facilitate energy input via H2 into respiratory metabolism are the NiFe uptake-type hydrogenases. Although important in bacterial physiology in general, these uptake hydrogenases play a specific role in maintaining the energetic efficiency of symbiotic nitrogen fixation. This is because H2 is an ample energy source readily available to diazotrophs while fixing N2, as it is made available due to its inherent production by nitrogenase. Indeed, the energetic (ATP) and reductant input into nitrogenases can in some instances favor H2 production over NH3 production. This situation can result in over 50% of the reductant through nitrogenase being in H2 rather than in ammonia. In cases where energy is the limiting factor for N2 fixation, the ability to use H2 would seem to be an important attribute for an efficient system.

The synthesis of NiFe hydrogenases is dependent on a supply of nickel and a mechanism to "sense" that the substrate H2 is available and the proper redox environment exists (see Maier, Triplett 1996). In addition to the structural proteins (which contain the nickel-containing active center and iron sulfur clusters) a remarkably diverse array of accessory proteins are needed to assemble hydrogenases. These have putative roles in nickel or iron binding, or in maintaining the correct structure for insertion/delivery of these metals or other active site ligands into the apoproteins (Drapal, Bock 1998; Maier, Bock 1996). One of the most intriguing of accessory proteins needed for B. japonicum hydrogenase expression is HypB (Fu et al. 1995; Olson et al. 1997). The HypB protein contains a histidine-rich domain at its N-terminus (Fu et al. 1995; Rey et al. 1994). These his-rich areas are present (but to a lesser extent than for B. japonicum) in some other HypB proteins and in other nickel binding/storage proteins involved in urease and carbon monoxide dehydrogenase synthesis as well (see Figure 1). For references to the origin for each of the sequences shown in the figure, the reader is referred to Olson and Maier (2000).

Bj 15 HaHdHHHdHgHdHdHgHdgHHHHHHgHdqdHHHHHdHaH 55

Rl 15 HtHevgddgHgHHHHdgHHdHdHdHdHHrgdHeHddHHHaedgsvH 60 Ss 15 H s HHHHgdgn f aH sHddHdqqeHHHHH 41

Ac 18 HHHHgydHgHHHdHafvrrpapaeaap 1 weg 1 n 1H 54

Av 18 HHHHgHdHHHHeHp fvr rp ap a e aapp a aggpn1H 53

Ms 2 6 HHHeHdHdHdHpHtHdH 42

Ka UreE 144 HgHHHaHHdHHaHsH 158

Rr Coo J 82 HspfHsHaHsHdHdHaHgHsHdHaHdHcHcHdH 114

Figure 1. Alignment of histidine (shown as H) rich regions in nickel accessory proteins.

Interestingly, the well-studied Escherichia coli protein lacks the histidine-rich domain; the protein is nevertheless very important for mobilizing nickel into the active center of the E. coli NiFe-hydrogenase (Maier, Bock 1996). Mutant strains of B. japonicum were obtained that synthesize an altered HypB lacking key components of the functional protein. Evidence for a role of HypB in nickel storage came from analysis of strain A23H (Olson et al. 1997; Olson, Maier 2000). When supplemented with nanomolar levels of nickel, hydrogenase activity of the mutant (strain A23H) was low in comparison to the wild type. However, this phenotype could be cured to wild type levels by adding uM levels of nickel to the medium. HypB also plays a role in transcription of hydrogenase, presumably because it is also likely to be involved in nickel donation to HupUV, the regulatory hydrogenase. The properties of HypB, along with the phenotypes associated with disruption of hypB, justify the conclusion that this protein (like that for E. coli) plays a key role in Ni donation to NiFe hydrogenases (Maier et al. 1995; Olson et al. 1997). We wished to use these mutant strains to study the symbiotic efficiency associated with HypB domains, and relate the function of these domains to nickel supplementation to the plants. Due to the nickel storage ability of HypB, it is sometimes referred to as nickelin.

2. Procedures

A sequence description list of the important areas within the mutant strains of B. japonicum is shown (Table 1), with histidine residues in bold. Note that the wild type contains 302 amino acids whereas the mutants A23H, AEg and K119T contain 264, 73, and 302 amino acid residues, respectively.

Table 1. Pertinent properties of mutants used.

Strain Amino Acid Sequence at the N Terminus

Wild type (X12)SIEHAHDHHHDHGHDHDHGHDGHHHHHHGHDQDHHHHHDHAHG(X247)

A23H (Xi2)SIE-----------------------------------------------------------------------------------HG(X247)

AEg First 67 a.a. like w.t.; #68-216 deleted in frame, last 6 a.a. (at C terminus) like w.t.

K119T Lysine at position 119 changed to threonine

Importantly, strains A23H and K119T were shown to synthesize detectable HypB of the expected size (determined by immunoblotting, see Olson et al. 1997). In addition to mutant strains deficient in histidine residues, a mutation in the G-binding domain was desired (strain K119T), as the Ni donation/mobilization function has been attributed to a GTPase activity.

Growth of soybeans in nickel-deficient conditions has been shown to adversely affect the symbiosis, presumably by causing deficiencies in urease and hydrogenase activities (Dalton et al. 1988). Limitation of the nickel availability to the Rhizobium leguminosarum-pea symbiosis resulted in lower hydrogenase activities of the bacteroids (Brito et al. 1994). The B. japonicum mutant strains were inoculated onto soybeans, and the plants were grown in hydroponic nickel deficient conditions in cellophane pouches; three plants were grown per pouch and they were harvested at 38 days after planting. The trace elements were provided as ultrapure salts. The nodules were harvested and the bacteroids were assayed for (whole cell) H2 oxidation activity. In addition, plant tissues, including nodules and bacteroids were assayed for nickel content by atomic absorption spectrophotometry. For most of the fractions, this was done after drying the harvested fraction, grinding it to a powder, and then acid-digestion of the powder.

3. Results and Discussion

The wild type bacteroid hydrogenase activities were affected only slightly by nickel supplementation to the plants (see Table 2), but the bacteroids of the A23H strain were deficient in hydrogenase activity when the plants were not supplemented with nickel.

Table 2. hypB mutations and symbiotic hydrogenase: effect of nickel supplementation.

Strain

Ni supplied3

Bacteroid activity

JH (wild type)

None

1.07 ±0.20

JH (wild type)

1 (iM

1.62 ±0.35

JH (wild type)

20 nM

1.16 ± 0.19

K119T

None

<0.01

K119T

1 nM

<0.01

K119T

20 iiM

<0.01

A23H

None

0.24 ± 0.07

A23H

1 nM

0.31 ±0.07

A23H

20 nM

0.99 ±0.15

aNi concentration supplied to soybeans; bHydrogenase activity in jumol H2 (hr.mg protein)"1.

At the highest Ni-supplemented level, bacteroids of this mutant approached that of the wild type. This result indicates an important role for the histidine-rich portion of HypB in storing the limited supply of nickel for hydrogenase expression. The mutant strain with a single amino acid change in the lysine residue important for GTPase activity was completely devoid of activity. This mutant is probably unable to make hydrogenase even with Ni supplementation due to an inability to incorporate the metal into hydrogenase (Olson et al. 1997). Similarly, strain AEg, containing a large in-frame deletion within nickelin had undetectable bacteroid hydrogenase activity. It lacks both the storage and the GTPase functions (Olson, Maier 2000).

Table 3. Nickel content (ng/mg protein) of acid-solubilized fractions of nodules and bacteroids.

Location

Strain

Ni supplied

Ni content

bacteroids

JH (wild type)

1 (J.M

3794 ±211

AEg

1 jiM

1761±136

A23H

1 nM

1468±136

nodules

JH (wild type)

1 jiM

454 ± 46

AEg

1 \xM

260 ± 65

A23H

1 p,M

181 ±78

bacteroids

JH (wild type)

20 pM

6301±130

AEg

20 iM

3863 ± 62

A23H

20 pM

4407± 198

nodules

JH (wild type)

20 jaM

1225±169

AEg

20 pM

1227 ±311

A23H

20 juM

1277 ±512

Experiments to analyze the amount of nickel associated with plant tissues from soybeans nodulated by the various mutant strains (Table 3) showed that the histidine rich portion of B. japonicum HypB is helpful for retaining nickel. Both bacteroids and nodules of strain JH (the wild type) had significantly more nickel than the HypB mutant strains; this was especially evident in the lower nickel (1 jiM nickel-supplied) condition. The nickel content of other plant tissues (stems, leaves, roots) increased with nickel supplementation to the nutrient solution, but we observed no differences in nickel in these tissues as a function of the different nodulating strains.

Legume nodulating B. japonicum and R. leguminosarum contain a domain very rich in histidine residues at the N-terminus of HypB. This is 24 out of 36 residues for B. japonicum and 20 out of 45 residues for R. leguminosarum. The need for a protein to store nickel for an organism that forms an intimate association with legumes could be related to the high rate of production of urease by soybeans. The latter could be considered to be a competing sink for available nickel within the same (root) tissue. The results we have obtained are consistent for a role played by nickelin in sequestering/storing (for the bacteroid) whatever pools of nickel the soybean had initially.

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