Acknowledgements

Purified EPS and CPS were obtained from Dr Frank Dazzo, Michigan State University.

EFFECT OF TRUNCATIONS AND CONSTITUTIVE NtrB MUTATIONS IN MOLECULAR INTERACTIONS WITHIN THE KLEBSIELLA PNEUMONIAE NITROGEN-SIGNAL TRANSDUCTION PATHWAY

I. Martinez-Argudo, P. Salinas, R. Maldonado, A. Contreras

División de Genética, Universidad de Alicante, Apartado 99, E-03080 Alicante, Spain

1. Introduction

In Klebsiella pneumoniae, signal transduction in response to nitrogen availability is mediated by the two-component regulators NtrB and NtrC. NtrB, a bifunctional histidine-kinase, modulates the activity of the response regulator NtrC by phosphorylation. The ability of NtrB of switching between opposing kinase and phosphatase activities is regulated by PII, a trimeric protein interacting with NtrB in conditions of nitrogen sufficiency. In a previous work, we showed the usefulness of the yeast two-hybrid system to probe NtrB and NtrC homodimerization and also specific interactions between NtrC receiver and NtrB transmitter domains (Martinez-Argudo et al. 2001). Here we use the same in vivo strategy to further analyze interactions of NtrB within the nitrogen signal pathway, probing NtrB determinants for interactions with itself, NtrC and PII, and testing the effect of NtrB constitutive point mutations on these interactions.

2. Results and Discussion

Protein fusions of GAL4-AD and GAL4-BD to NtrB and derived polypeptides (including truncations and a constitutive mutation at A129T) were analyzed for their ability to interact with themselves and with equivalent fusions of signal transduction proteins PII and NtrC, in appropriate pairs, using the yeast two-hybrid system. To determine the ability of two given polypeptides to interact, we determined expression of both GALl.lacZ and GALLHIS3 reporters in strains of Saecharomyces cerevisiae Y190.

Protein-protein interactions were only detected between components of the nitrogen signal pathway. Lack of signals between heterologous two-component regulators provides evidence for specific recognition between the transmitter module of NtrB and both PII and NtrC. Contacts of NtrB with NtrC and PII are mapped to the H phosphotransfer domain and to the G kinase domain, respectively. In the latter case, the integrity of the transmitter module appears important for two-hybrid signals. Taken together, our results agree with previous data on homologous two-component systems (Park et al. 1998) and with recent work on PII (Piozak et al. 2000).

In spite of the multiple evidences for dimerization of phosphotransfer domains (Tomomori et al. 1999; Jiang et al. 2000), transmitter modules from NtrB and EnvZ do not interact when paired with themselves, a result that may reflect that contacts between H domains are not very strong. This would be compatible with a model in which the helix bundle forms and dissociates during the phosphorylation circle.

Mutation A129T affects some of the interactions tested amongst NtrB derivatives, thus supporting its effect on NtrB conformation and the sensitivity of the two-hybrid system used here.

3. References

Jiang P et al. (2000) Biochem. 39, 13433-13449 Martinez-Argudo I et al. (2001) Molec. Microbiol. 40, 169-178 Park H et al. (1998) Proc. Natl. Acad. Sci. USA 95, 6728-6732 Pioszak A et al. (2000) Biochem. 39, 13450-13461 Tomomori C et al. (1999) Nature Struct. Biol. 6, 729-734

NO OR NH OR BOTH: TEMPORAL EFFECTS ON NODULE INITIATION IN THE PISUMSATIVUM!RHIZOBIUMLEGUMINOSARUM SYMBIOSIS

Dept of Plant Sci., University of Manitoba, Winnipeg, MB, Canada R3T 2N2

1. Introduction

It is generally accepted that mineral N (NO or NH) inhibits nodulation in the Iegume-rhizobia symbioses. It has been shown that low continuous concentrations of NH (< 1.0 mM), in hydroponic culture, will stimulate nodulation in pea (Pisum sativum L.) (Waterer et al. 1992). In contrast, low concentrations of NO (as low as 0.1 mM) inhibit all aspects of nodulation (Waterer, Vessey 1993).

2. Procedure

Pea plants, grown in a continuous flow hydroponic system, were exposed to one of four sources of mineral N: Zero N; 0.5 mM NO; 0.5 mM NH; or 0.25 mM NO plus 0.25 mM NH for a period of 21 days after inoculation (DAI). N concentrations in the nutrient solutions were monitored daily and kept at a relatively constant level with additions of stock solutions by a peristaltic pump. Six plants were harvested at 7, 14 and 21 DAI. Nodule numbers and dry weights were determined. A microscopy study on two secondary roots [the third distal secondary root from the crown (#1) and the secondary root 75% of the distance from the crown to the last distal secondary root with nodules (#2)] was undertaken. Nodules were rated as to stage of development using a scale developed in our lab.

3. Results

Dry matter accumulation was greatest in the combined treatment at 7 DAI, while at 14 DAI the combined treatment was greater than the two single N sources, which were greater than the control. At 7 DAI there were no nodules in the NO treatment, and by 14 DAI, the NH treatment had more nodules than any other. The number of nodules per g root DM at 7 DAI was similar in the control, NH and combined treatment, but at 14 and 21 DAI, the control and NH treated plants were higher than either treatment containing NO.

On the secondary root #1, there were fewer nodules and they developed more slowly on the NO treated plants than any other. At 14 DAI there were more nodules, and these were more mature, on the control and NH treated plants than the other two treatments. On the younger root #2, there were fewer nodules and these were at earlier developmental stages than on root #1 at each harvest date. The relative distribution in number and development stage was similar to root #1.

4. Conclusions

From the results of this study we concluded that NO inhibits nodulation in field pea, both on an absolute and specific basis. NH, on the other hand, stimulates nodulation on an absolute basis, and does not inhibit it on a specific basis. In any of the treatments containing NO, the nodules matured slowly. The nodule proliferation in the NH treatment was greatest in the younger plants, and on those roots that formed earlier in development. The combined treatment of NO plus NH resulted in nodulation patterns early in development that were more similar to the NH treatment, and later in development that were more similar to the NO treatment.

5. References

Waterer JG et al (1992) Physiol. Plant. 86, 215-220 Waterer JG, Vessey JK (1993) J. Plant Nutr. 16, 1775-1789

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