References

Cevallos et al. (1996) J. Bacteriol. 178, 1646-1654 Marroqui et al. (2001) J. Bacteriol. 183, 854-864

DISCOVERY OF A HEME UPTAKE SYSTEM IN THE SOIL BACTERIUM BRAD YRHIZOBIUM JAPONICUM

A. Nienaber, H. Hennecke, H.M. Fischer

Institut fur Mikrobiologie, Eidgenössische Technische Hochschule Zürich, Schmelzbergstrasse 7, CH 8092 Zürich, Switzerland

In Bradyrhizobium japonicum, the nitrogen-fixing symbiont of soybeans, we have identified a heme uptake system, Hmu, which comprises a cluster of nine open reading frames, (Figure 1; Nienaber et al. 2001). Predicted products of these genes include HmuR, a TonB-dependent heme receptor in the outer membrane, HmuT, a periplasmic heme-binding protein, and HmuUV, an ABC transporter in the inner membrane. Furthermore, we identified homologs of ExbBD and TonB, which are required for energy transduction from the inner to the outer membrane. Mutant analysis and complementation tests indicated that HmuR and the ExbBD-TonB system but not the HmuTUV transporter are essential for heme uptake or heme acquisition from hemoglobin and leghemoglobin. The TonB system seems to be specific for heme uptake since it is dispensable for the uptake of FeCitrate and ferrichrome. We therefore propose the existence of a second TonB homolog functioning in the uptake of siderophores. When tested on soybean host plants, hmuT-hmuR and exbD-tonB mutants exhibited wild-type symbiotic properties. Thus, heme uptake is not essential for symbiotic nitrogen fixation but it may enable B. japonicum to have access to alternative iron sources in its non-symbiotic state. In fact, growth on heme iron had been demonstrated for various rhizobial species (Noya et al. 1997), and a heme uptake system similar to that described here was found most recently in Rhizobium leguminosarum bv. viciae (Wexler et al. 2001).

Transcript analyses and studies with lacZ fusions showed that expression of hmuT and hmuR is induced under low-iron conditions. In B. japonicum, two regulatory proteins are known to be involved in iron homeostasis, the ferric uptake regulator (Fur) and the iron response regulator (Irr) (Hamza et al. 1998, 1999). Iron-dependent expression of hmuT and hmuR was preserved in fur or irr mutant backgrounds although maximal induction levels were decreased. We conclude either that both regulators, Fur and Irr, independently contribute to the iron control of hmuT and hmuR or that a yet unknown iron regulatory system is exerting this type of regulation. An A/T-rich element (ICE, for iron control element), located in the promoter region of the divergently transcribed hmuTUV and hmuR genes, is involved in iron control as indicated by the loss of hmu induction upon insertion of 2 nucleotides into ICE. We speculate that ICE represents a binding site for a novel iron regulatory protein.

r hmuV ■ hmuU ■ hmuT —B— hmuR -110- 167 exbli exbD tonB

Heme transport Heme receptor ? Energy transduction

Figure 1. The B. japonicum locus encoding a heme uptake system. References

Hamza I et al. (1998) J. Biol. Chem. 273, 21669-21674 Hamza I et al. (1999) J. Bacteriol. 181, 5843-5846 Nienaber A et al. (2001) Mol. Microbiol. Noya F et al. (1997) J. Bacteriol. 179, 3076-3078

NITRATE APPLICATION FROM LOWER ROOTS CAN PROMOTE NODULATION AND N2 FIXATION IN SOYBEAN

H. Yashima1, H. Fujikake1, T.I. Sato2, N. Ohtake1, K. Sueyoshi1, Y. Takahashi3, T. Ohyama1

'Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan

2Faculty of Bioresource Sciences, AkitaPref. University, Akita 010-0195, Japan

3Niigata Agricultural Experiment Station, Niigata 940-0826, Japan

It is well known that combined nitrogen, especially nitrate, strongly inhibits nodulation and N2 fixation activity in soybean. However, we have observed that a basal dressing of deep placement of controlled release nitrogen fertilizer (coated urea) did not inhibit N2 fixation, consequently, the plant growth and the seed yield were promoted compared with those in a conventional cultivation (Takahashi et al. 1991). The result indicated that a continuous supply of low level of nitrogen from lower part of roots might not inhibit nodulation and N2 fixation in the nodules on upper roots. In this paper, the effects of nitrate supply from the lower roots on the nodulation and N2 fixation in the upper nodules were investigated using two-layered pots separating the upper roots in vermiculite medium and the lower roots in hydroponics. The 0-0, 1-1, 5-5, 0-5, and 5-0 treatments were imposed where a solution containing 0 mM, 1 mM or 5 mM nitrate was supplied to the roots in the lower pot from transplanting at VI stage to R3 stage (the former number) and R3 to R7 stages (the latter number). Nitrogen-free solution was supplied to the roots in the upper pot through the experimental period. The plants were harvested at Rl, R3 and R7 stages.

At R7 stage the total plant dry weight was highest in 5-5 treatment (127 g plant"1), moderate in 1-1 (94 g), 5-0 (95 g) and 0-5 (74 g) treatments, and lowest in 0-0 treatment (51 g). At R3 stage the upper nodule dry weight was higher (0.88 g plant"1) in the plants with continuous supply of ImM nitrate (1-1 treatment) and exceeded 0-0 treatment (0.61 g) in the plants which were solely depended on N2 fixation. Similarly total ARA (acetylene reduction activity) per plant at R3 stage was higher in 1-1 treatment than in 0-0 treatment (69 and 38 pmole C2H4 h"1 plant" respectively). These results confirmed that a continuous supply of low level of nitrate from the lower roots could promote nodulation and N2 fixation in the upper nodules that were separated from the lower roots. Although the number of upper nodules were higher in 5-5 treatment than in 0-0 treatment, the nodule dry weight and ARA were depressed by continuous 5 mM nitrate supply from the lower roots (5-5 treatment). A withdrawal of 5 mM nitrate after R3 stage (5-0 treatment) enhanced greatly the nodule growth (1.44 g plant"1) and ARA per plant (40pmole C2H) h"1 plant"1) at R7 stage where ARA declined in other treatments. The nitrate concentrations in the upper nodules were very low (less than 0.1 mg N gDW"1) irrespective of the treatments, although the concentration in the lower roots were increased at 2.7 and 6.3 mg N gDW"1 by 1 mM and 5 mM nitrate supply.

The concentration and period of nitrate supply from the lower roots affected both the number and dry weight as well as N2 fixation activities (ARA) of the upper nodules. Continuous supply of low level of nitrate (ImM) from the lower roots promoted the growth of shoot and roots. Therefore the acceleration of photosynthesis and nutrient uptake might increase nodule growth and N2 fixation activity. A high level of nitrate supply to the lower roots depressed the upper nodule growth and ARA. These responses of the nodules in the upper roots may be regulated by some systemic control such as photosynthetic supply and N demand. These results support the concept that maximum soybean yield can be obtained by compatible utilization of N2 fixation in nodules and nitrate absorption from roots. The separation of N2 fixation site and nitrate absorption site, e.g. by deep placement of N fertilizer can promote nodulation, N2 fixation and seed yield of soybean.

References

Takahashi et al. (1991) Soil Sci. Plant Nutr. 37, 223-231

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