IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
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17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Title Identification of the regulatory genes required for the acid activation of the low pH inducible<br />
gene lpiA in Sinorhizobium medicae<br />
Author Rui Tian 1 , Ravi Tiwari 1 , Lambert Bräu 1 , John Howieson 1 , Graham O‟Hara 1 & Wayne Reeve 1 .<br />
Poster Board Number 50<br />
1 Center for Rhizobium studies, Murdoch University, South Street, Murdoch, Western<br />
Australia, 6150.<br />
The acid tolerance response of Sinorhizobium medicae (S. medicae) WSM419 enables cell adaptation to lethal<br />
acid after cell exposure to mild acidity. The expression of lpiA (low pH induced gene A) gene is critical for this<br />
response and is acid-activated at least 20-fold in mild acidic conditions. Inactivation of an upstream gene fsrR<br />
(fused sensor regulator) reduces lpiA expression to just 3-fold of its maximum level. This observation suggested<br />
that other regulatory proteins are involved in complete activation of the lpiA gene in acidic conditions.<br />
Inactivation of putative regulators of lpiA located upstream (tcrA and tcsA) or downstream (acvB) genes and the<br />
sigma-54 factor encoded by rpoN was achieved by single crossover mutation in WSM419. Expression studies<br />
(GUS-linked assays and qRT-PCR) revealed that the two component system TcsA/TcrA and a fused sensor<br />
regulator FsrR (encoded by a gene directly upstream from lpiA) are controlling expression of lpiA and<br />
demonstrated that RpoN is essential for acid activation. A putative RpoN enhancer binding protein (Smed_5956)<br />
was located upstream of tcsA. RACE analysis located the transcriptional start site 14 bp downstream of a<br />
classical -24 and -12 RpoN binding motif upstream of the lpiA start codon demonstrating that lpiA and acvB are<br />
co-transcribed as an operon. The expression of acvB was determined by qRT-PCR and was found to be induced<br />
18-fold by acid which is consistent with the finding that both lpiA and acvB share the same transcription start site.<br />
In contrast, fsrR, tcrA, tcsA, and rpoN were constitutively expressed with respect to pH.<br />
While mutations in tcsA, tcrA, fsrR and acvB affect acid induction of lpiA, these genes are not essential for stress<br />
tolerance or symbiotic nitrogen fixation. In contrast, rpoN is essential for symbiotic nitrogen fixation with<br />
Medicago. This study has therefore identified for the first time an alternative sigma factor which is essential for<br />
pH response as well as symbiosis in S. medicae.<br />
165<br />
2011