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A glutamine-amidotransferase-like protein modulates FixT anti-kinase activity in Sinorhizobium meliloti.

Bergès H, Checroun C, Guiral S, Garnerone AM, Boistard P, Batut J - BMC Microbiol. (2001)

Bottom Line: The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti.We provide evidence that asnO is active during symbiosis.Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, UMR215 CNRS-INRA, BP27, 31326 Castanet-Tolosan Cedex, France. hberges@toulouse.inra.fr

ABSTRACT

Background: Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown.

Results: We have isolated by random Tn5 mutagenesis a S. meliloti mutant strain that escapes repression by FixT. Complementation test and DNA analysis revealed that inactivation of an asparagine synthetase-like gene was responsible for the phenotype of the mutant. This gene, that was named asnO, encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis.

Conclusions: Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti. Our data suggest that asnO might control activity of the FixT protein, in a way that remains to be elucidated. A proposed role for asnO might be to couple nitrogen fixation gene expression in S. meliloti to the nitrogen needs of the cells.

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asnO gene expression RT-PCR analysis of RNAs isolated from S. meliloti GMI211 wild-type strain (wt) or GMI5704 fixJ mutant strain (fixJ) grown in minimal medium M9 in either oxic (+) or microoxic (2% O2) conditions (-). RT-PCR were performed with either asnO or hemA specific primers and the products were separated on agarose gels, blotted on a nylon membrane and hybridized with the corresponding 32P labelled PCR product and analysed on a Phosphorimager.
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Figure 5: asnO gene expression RT-PCR analysis of RNAs isolated from S. meliloti GMI211 wild-type strain (wt) or GMI5704 fixJ mutant strain (fixJ) grown in minimal medium M9 in either oxic (+) or microoxic (2% O2) conditions (-). RT-PCR were performed with either asnO or hemA specific primers and the products were separated on agarose gels, blotted on a nylon membrane and hybridized with the corresponding 32P labelled PCR product and analysed on a Phosphorimager.

Mentions: We have monitored by RT-PCR experiments expression of the asnO gene in a wild-type strain grown in minimal medium, in either oxic or microoxic conditions. The results showed that asnO gene expression was slightly enhanced in microoxic conditions as compared to oxic conditions (Figure 5). No effect of a fixJ mutation was observed on asnO expression using a GMI5704 fixJ mutant strain [18].


A glutamine-amidotransferase-like protein modulates FixT anti-kinase activity in Sinorhizobium meliloti.

Bergès H, Checroun C, Guiral S, Garnerone AM, Boistard P, Batut J - BMC Microbiol. (2001)

asnO gene expression RT-PCR analysis of RNAs isolated from S. meliloti GMI211 wild-type strain (wt) or GMI5704 fixJ mutant strain (fixJ) grown in minimal medium M9 in either oxic (+) or microoxic (2% O2) conditions (-). RT-PCR were performed with either asnO or hemA specific primers and the products were separated on agarose gels, blotted on a nylon membrane and hybridized with the corresponding 32P labelled PCR product and analysed on a Phosphorimager.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC32199&req=5

Figure 5: asnO gene expression RT-PCR analysis of RNAs isolated from S. meliloti GMI211 wild-type strain (wt) or GMI5704 fixJ mutant strain (fixJ) grown in minimal medium M9 in either oxic (+) or microoxic (2% O2) conditions (-). RT-PCR were performed with either asnO or hemA specific primers and the products were separated on agarose gels, blotted on a nylon membrane and hybridized with the corresponding 32P labelled PCR product and analysed on a Phosphorimager.
Mentions: We have monitored by RT-PCR experiments expression of the asnO gene in a wild-type strain grown in minimal medium, in either oxic or microoxic conditions. The results showed that asnO gene expression was slightly enhanced in microoxic conditions as compared to oxic conditions (Figure 5). No effect of a fixJ mutation was observed on asnO expression using a GMI5704 fixJ mutant strain [18].

Bottom Line: The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti.We provide evidence that asnO is active during symbiosis.Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, UMR215 CNRS-INRA, BP27, 31326 Castanet-Tolosan Cedex, France. hberges@toulouse.inra.fr

ABSTRACT

Background: Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown.

Results: We have isolated by random Tn5 mutagenesis a S. meliloti mutant strain that escapes repression by FixT. Complementation test and DNA analysis revealed that inactivation of an asparagine synthetase-like gene was responsible for the phenotype of the mutant. This gene, that was named asnO, encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis.

Conclusions: Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti. Our data suggest that asnO might control activity of the FixT protein, in a way that remains to be elucidated. A proposed role for asnO might be to couple nitrogen fixation gene expression in S. meliloti to the nitrogen needs of the cells.

Show MeSH
Related in: MedlinePlus