Chi-Te Liu

The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571

BackgroundBiological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem.ResultsThe 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor.ConclusionThe genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.

Evaluation of Methanogenic Strains and Their Ability to Endure Aeration and Water Stress

During periods of drainage, both water stress and oxygen can cause damage to indigenous methanogens. In the present study, we evaluated the tolerance of seven methanogenic strains (Methanobrevibacter arboriphilicus, Methanobacterium formicicum, Methanococcus vannielii, Methanospirillum hungatei, Methanoculleus olentangyi, Methanoplanus limicola, and Methanosarcina mazei) to long-term exposure to air/nitrogen and drying. We found that these methanogenic strains except for M. limicola and M. olentangyi in pre-dried cells offered more tenacious resistance to desiccation and oxygen exposure than those in enriched liquid cultures. In the case of M. formicicum, the liquid culture of this strain could remain viable when mixed well with fresh or sterile soil, but not when cultured without soil, or with agar slurry. These results suggest that indigenous methanogens localize within soil compartments to protect themselves from the damage caused by gradual drying under an oxic atmosphere.

Rhizobial Factors Required for Stem Nodule Maturation and Maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 Symbiosis

ABSTRACT The molecular and physiological mechanisms behind the maturation and maintenance of N2-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N2-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N2 fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C4 metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.

Microbial indices of soil fertility

Aims:  To find the new microbial parameters explaining the soil fertility from the microbial community viewpoint.

Isolation of a novel root-determined hypernodulation mutant rdh1 of Lotus japonicus

Abstract A mutant line that develops an excess number of small nodules was found in Lotus japonicus Miyakojima MG20 during a screening for mutants defective in nodule development and nitrogen fixation. Genetic analysis revealed that the phenotype is inherited in a monogenic, recessive manner. The genes locus was mapped on chromosome 1 between 53.7 and 61.4 cM. This mutant formed 5–10-fold more nodules than the wild-type plant, and a grafting experiment revealed that the root regulated the hypernodulation. Except for the nodulations phenotype no other differences were found between the mutant and the wild-type plant with respect to growth and morphological characteristics. In the mapped locus for the mutant no nodulation genes were reported, and this fact strongly suggests that the genes locus is a new one. The gene was named root-determined hypernodulation (rdh) 1.

Comparative Genome-Wide Transcriptional Profiling of Azorhizobium caulinodans ORS571 Grown under Free-Living and Symbiotic Conditions

ABSTRACT The whole-genome sequence of the endosymbiotic bacterium Azorhizobium caulinodans ORS571, which forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, was recently determined. The sizes of the genome and symbiosis island are 5.4 Mb and 86.7 kb, respectively, and these sizes are the smallest among the sequenced rhizobia. In the present study, a whole-genome microarray of A. caulinodans was constructed, and transcriptomic analyses were performed on free-living cells grown in rich and minimal media and in bacteroids isolated from stem nodules. Transcriptional profiling showed that the genes involved in sulfur uptake and metabolism, acetone metabolism, and the biosynthesis of exopolysaccharide were highly expressed in bacteroids compared to the expression levels in free-living cells. Some mutants having Tn5 transposons within these genes with increased expression were obtained as nodule-deficient mutants in our previous study. A transcriptomic analysis was also performed on free-living cells grown in minimal medium supplemented with a flavonoid, naringenin, which is one of the most efficient inducers of A. caulinodans nod genes. Only 18 genes exhibited increased expression by the addition of naringenin, suggesting that the regulatory mechanism responding to the flavonoid could be simple in A. caulinodans. The combination of our genome-wide transcriptional profiling and our previous genome-wide mutagenesis study has revealed new aspects of nodule formation and maintenance.

Isolation and characterization of dibenzofuran-degrading Comamonas sp. strains isolated from white clover roots

Three dibenzofuran (DF)-degrading strains were newly isolated from roots of white clover (Trifolium repens L.) and poplar trees grown in DF-contaminated soil samples. These strains, designated KD2, KD7, and PD1, were characterized as Comamonas sp. on the basis of their 16S rDNA sequences and physiological characteristics. The metabolites produced when strain KD7 was incubated with DF were identified by gas chromatography–mass spectrometry (GC-MS) analysis. Interestingly, strain KD7 was found to have two pathways for DF degradation, beginning with angular dioxygenation at carbons 4 and 4a, and lateral dioxygenation at carbons 1 and 2, respectively. Furthermore, strains KD2 and KD7 not only achieved efficient root colonization in clover but also promoted clover growth. They are the first reported Comamonas sp. strains capable of utilizing DF as a sole carbon source. This provides additional information on the diversity of DF-degrading bacteria.

Root-Determined Hypernodulation Mutant of Lotus japonicus Shows High-Yielding Characteristics

Here we report the phenotypic characteristics of a novel hypernodulation mutant, Ljrdh1 (root-determined hypernodulation 1) of Lotus japonicus. At 12 weeks after rhizobial inoculation, there were no differences between the growth of Ljrdh1 and, wild-type. However, Ljrdh1 showed 2 to 3 times higher nitrogen-fixing activity, and seed and pod yields, were approximately 50% higher than the wild-type. This is the first report of a legume hypernodulation mutant showing normal growth and a high-yielding characteristic under optimal cultivation conditions.

An outer membrane autotransporter, AoaA, of Azorhizobium caulinodans is required for sustaining high N2-fixing activity of stem nodules

In this study, we investigated the function of a putative high-molecular-weight outer membrane protein, azorhizobial outer membrane autotransporter A (AoaA), of Azorhizobium caulinodans ORS571. Sequence analysis revealed that AoaA was an autotransporter protein belonging to the type V protein secretion system. Azorhizobium caulinodans forms N(2)-fixing nodules on the stems and roots of Sesbania rostrata. The sizes of stem nodules formed by an aoaA mutant having transposon insertion within this ORF were as large as those in the wild-type strain, but the N(2)-fixing activity of the nodules by the aoaA mutant was lower than that of wild-type nodules. cDNA-amplified fragment length polymorphism and reverse transcriptase-PCR analysis revealed that the expressions of several pathogen-related genes of host plants were induced in the aoaA mutant nodules. Furthermore, exopolysaccharide production was defective in the aoaA mutant under free-living conditions. These results indicate that AoaA may have an important role in sustaining the symbiosis by suppressing plant defense responses. The exopolysaccharide production controlled by AoaA might mediate this suppression mechanism.

Isolation and differential expression of β-1,3-glucanase messenger RNAs, SrGLU3 and SrGLU4, following inoculation of Sesbania rostrata

We report here the isolation and characterisation of two new β-1,3-glucanase cDNAs, SrGLU3 and SrGLU4, from a tropical legume Sesbania rostrata Bremek. & Oberm., which form N2-fixing nodules on the stem after infection by Azorhizobium caulinodans. SrGLU3 was characterised as being grouped in a branch with tobacco class I β-1,3-glucanases, where the isoforms were reported to be induced by either pathogen infection or ethylene treatment. SrGLU4 was characterised as separate from other classes, and we propose this new branch as a new class (Class VI). The SrGLU3 gene was constitutively expressed in normal stem nodules induced by the wild type strain of A. caulinodans (ORS571), and also even in immature stem nodules induced by a mutant (ORS571-C1), which could not form mature stem-nodules. In contrast, the transcript accumulation of SrGLU4 was hardly detectable in immature nodules inoculated by the ORS571-C1 mutant. We suggest that S. rostrata makes use of SrGLU4 to discriminate between symbionts and non-symbionts (mutants) in developing nodules. We propose the SrGLU4 gene as a new nodulin during nodulation.