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

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.

Figure 1

Characterization of the S. meliloti mutant strain (GMI401) escaping FixT repressor activity. Panel A : We monitored microoxic expression of a fixK-lacZ fusion carried by the reporter plasmid pMF457 in S. meliloti. pMF10 allows constitutive expression of fixT, pMF11 is a negative control (fixT cloned in the antisense orientation). 1 : wild-type strain GMI211(pMF457)(pMF11); 2 : wild-type strain GMI211(pMF457)(pMF10); 3 : mutant strain GMI401(pMF457)(pMF10); 4 : mutant strain GMI401 (pMF457) (pMF11); 5 : transductant strain GMI401(pMF457); 6 : transductant strain GMI401(pMF457)(pMF10). Panel B : In planta phenotype of the wild-type GMI211 (pMF10) and mutant strain GMI401 (pMF10). Medicago sativa seedlings were inoculated with the bacterial strains and grown for 3 weeks on medium lacking any nitrogen source.

Figure 2

Genetic organisation of the asnO region. The genes are shown as thick arrows. The red small arrow indicates the position of the Tn5 insertion in the GMI401 mutant strain. The position and orientation of the specific primers are shown in small green arrows.

Figure 3

AsnO of S. meliloti is related to glutamine-dependent amido transferases. Alignment of amino acid sequences of AsnO and AsnB from S. meliloti, AsnO (swissprot accession number: Sp 005272) and AsnB (Sp P54420) from B. subtilis, AsnB from E. coli (Sp P22106), and LtsA from C. glutamicum (Sp BAA89484). The alignement was done by using ClustalW programme [31].

Figure 4

Complementation of the mutant phenotype by the asnO gene. RNAs isolated from free-living microoxic cultures (2% O₂) of S. meliloti strains grown in M9 minimal medium were amplified by RT-PCR using specific primers. RT-PCR products were separated on agarose gels, blotted onto a nylon membrane and hybridized with the ³²P labelled probe of the expected product. Upper panel : fixK gene (see Materials and Methods). Lower panel : hemA gene (control). Lane 1 = GMI211 (pMF10), Lane 2 = GMI401 (pMF10), Lanes 3 and 4 = GMI401 (pMF10) (pBasn2) of two independent transconjugants.

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.

Figure 6

Updated model for the regulation of respiratory and nitrogen fixation gene expression in S. meliloti.