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17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Title Genome sequence of Mesorhizobium huakuii 7653r which establishes a highly specific symbiosis with Astragalus sinicus in China Author Youguo Li, Shanming Wang, Jieli Peng, Baohai Hao & Liu Liu Poster Board Number 49 State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, 430070, P. R. China M. huakuii 7653R, isolated from a rice-growing field in southern China, can establish a highly specific and effective symbiosis with A. sinicus via forming indeterminate-type nitrogen-fixing nodules. To date, there is very limited information on the mechanism of its bacteroid development, nodulation specificity and molecular interactions with Chinese milk vetch. In the present work, the complete genome sequence of M. huakuii 7653R has been established and the genome structure and phylogeneticas signment of the organism was analysed. For de novo sequencing of the M. huakuii 7653R genome, a combined strategy comprising Solexa-sequencing on the Illumina GAIIx platform and PCR-based amplicon sequencing for gap closure was applied. The finished genome consists of three replicons and comprises 6,952,365 bases. The genome sequence of M. huakuii 7653R totals 6.95 Mbp with a GC content of 62.9%. It contains 6,792 predicted open reading frames (ORFs), located on a chromosome and two plasmids. The 6.2 Mbp circular chromosome encodes 6,150 ORFs with a mean GC content of 63.1%. The 320,051-bp pSym pMh7653Rb comprises 282 ORFs with a mean GC content of 62.6%. The plasmid pMh7653Ra (170,165 bp) has a relatively lower GC content (59.5%). Comparative analysis of the assembled genome sequence of M. huakuii 7653R revealed high homology and extensive synteny with M. loti MAFF303099, and partly to other rhizobial genomes, in particular to Sinorhizobium meliloti 1021 and to Rhizobium sp. NGR234. 164 2011
17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Title Identification of the regulatory genes required for the acid activation of the low pH inducible gene lpiA in Sinorhizobium medicae Author Rui Tian 1 , Ravi Tiwari 1 , Lambert Bräu 1 , John Howieson 1 , Graham O‟Hara 1 & Wayne Reeve 1 . Poster Board Number 50 1 Center for Rhizobium studies, Murdoch University, South Street, Murdoch, Western Australia, 6150. The acid tolerance response of Sinorhizobium medicae (S. medicae) WSM419 enables cell adaptation to lethal acid after cell exposure to mild acidity. The expression of lpiA (low pH induced gene A) gene is critical for this response and is acid-activated at least 20-fold in mild acidic conditions. Inactivation of an upstream gene fsrR (fused sensor regulator) reduces lpiA expression to just 3-fold of its maximum level. This observation suggested that other regulatory proteins are involved in complete activation of the lpiA gene in acidic conditions. Inactivation of putative regulators of lpiA located upstream (tcrA and tcsA) or downstream (acvB) genes and the sigma-54 factor encoded by rpoN was achieved by single crossover mutation in WSM419. Expression studies (GUS-linked assays and qRT-PCR) revealed that the two component system TcsA/TcrA and a fused sensor regulator FsrR (encoded by a gene directly upstream from lpiA) are controlling expression of lpiA and demonstrated that RpoN is essential for acid activation. A putative RpoN enhancer binding protein (Smed_5956) was located upstream of tcsA. RACE analysis located the transcriptional start site 14 bp downstream of a classical -24 and -12 RpoN binding motif upstream of the lpiA start codon demonstrating that lpiA and acvB are co-transcribed as an operon. The expression of acvB was determined by qRT-PCR and was found to be induced 18-fold by acid which is consistent with the finding that both lpiA and acvB share the same transcription start site. In contrast, fsrR, tcrA, tcsA, and rpoN were constitutively expressed with respect to pH. While mutations in tcsA, tcrA, fsrR and acvB affect acid induction of lpiA, these genes are not essential for stress tolerance or symbiotic nitrogen fixation. In contrast, rpoN is essential for symbiotic nitrogen fixation with Medicago. This study has therefore identified for the first time an alternative sigma factor which is essential for pH response as well as symbiosis in S. medicae. 165 2011
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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 Genome sequence of Mesorhizobium huakuii 7653r which establishes a highly specific<br />
symbiosis with Astragalus sinicus in China<br />
Author Youguo Li, Shanming Wang, Jieli Peng, Baohai Hao & Liu Liu<br />
Poster Board Number 49<br />
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan,<br />
Hubei, 430070, P. R. China<br />
M. huakuii 7653R, isolated from a rice-growing field in southern China, can establish a highly specific and<br />
effective symbiosis with A. sinicus via forming indeterminate-type nitrogen-fixing nodules. To date, there is very<br />
limited information on the mechanism of its bacteroid development, nodulation specificity and molecular<br />
interactions with Chinese milk vetch. In the present work, the complete genome sequence of M. huakuii 7653R<br />
has been established and the genome structure and phylogeneticas signment of the organism was analysed.<br />
For de novo sequencing of the M. huakuii 7653R genome, a combined strategy comprising Solexa-sequencing<br />
on the Illumina GAIIx platform and PCR-based amplicon sequencing for gap closure was applied. The finished<br />
genome consists of three replicons and comprises 6,952,365 bases. The genome sequence of M. huakuii 7653R<br />
totals 6.95 Mbp with a GC content of 62.9%. It contains 6,792 predicted open reading frames (ORFs), located on<br />
a chromosome and two plasmids. The 6.2 Mbp circular chromosome encodes 6,150 ORFs with a mean GC<br />
content of 63.1%. The 320,051-bp pSym pMh7653Rb comprises 282 ORFs with a mean GC content of 62.6%.<br />
The plasmid pMh7653Ra (170,165 bp) has a relatively lower GC content (59.5%).<br />
Comparative analysis of the assembled genome sequence of M. huakuii 7653R revealed high homology and<br />
extensive synteny with M. loti MAFF303099, and partly to other rhizobial genomes, in particular to Sinorhizobium<br />
meliloti 1021 and to Rhizobium sp. NGR234.<br />
164<br />
2011
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