Dethiobiotin uptake and utilization by bacteria possessing bioYB operon.

Biotin is an essential vitamin for all organisms. Some bacteria cannot synthesize biotin and live by acquiring biotin from the environment. Bacterial biotin transporters (BioY) are classified into three mechanistic types. The first forms the BioMNY complex with ATPase (BioM) and transmembrane protein (BioN). The second relies on a promiscuous energy coupling module. The third functions independently. One-third of bioY genes spread in bacteria cluster with bioM and bioN on the genomes, and the rest does not. Interestingly, some bacteria have the bioY gene clustering with bioB gene, which encodes biotin synthase, an enzyme that converts dethiobiotin to biotin, on their genome. This bioY-bioB cluster is observed even though these bacteria cannot synthesize biotin. Azorhizobium caulinodans ORS571, a rhizobium of tropical legume Sesbania rostrata, is one of such bacteria. In this study using this bacterium, we demonstrated that the BioY linked to BioB could transport not only biotin but also dethiobiotin, and the combination of BioY and BioB contributed to the growth of A. caulinodans ORS571 in a biotin-deficient but dethiobiotin-sufficient environment. We propose that such environment universally exists in the natural world, and the identification of such environment will be a new subject in the field of microbial ecology.

Substrate recognition mechanism of tRNA-targeting ribonuclease, colicin D, and an insight into tRNA cleavage-mediated translation impairment.

Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNAArg of sensitive Escherichia coli cells. E. coli has four isoaccepting tRNAArgs; the cleavage occurs at the 3' end of anticodon-loop, leading to translation impairment in the sensitive cells. tRNAs form a common L-shaped structure and have many conserved nucleotides that limit tRNA identity elements. How colicin D selects tRNAArgs from the tRNA pool of sensitive E. coli cells is therefore intriguing. Here, we reveal the recognition mechanism of colicin D via biochemical analyses as well as structural modelling. Colicin D recognizes tRNAArgICG, the most abundant species of E. coli tRNAArgs, at its anticodon-loop and D-arm, and selects it as the most preferred substrate by distinguishing its anticodon-loop sequence from that of others. It has been assumed that translation impairment is caused by a decrease in intact tRNA molecules due to cleavage. However, we found that intracellular levels of intact tRNAArgICG do not determine the viability of sensitive cells after such cleavage; rather, an accumulation of cleaved ones does. Cleaved tRNAArgICG dominant-negatively impairs translation in vitro. Moreover, we revealed that EF-Tu, which is required for the delivery of tRNAs, does not compete with colicin D for binding tRNAArgICG, which is consistent with our structural model. Finally, elevation of cleaved tRNAArgICG level decreases the viability of sensitive cells. These results suggest that cleaved tRNAArgICG transiently occupies ribosomal A-site in an EF-Tu-dependent manner, leading to translation impairment. The strategy should also be applicable to other tRNA-targeting RNases, as they, too, recognize anticodon-loops.Abbreviations: mnm5U: 5-methylaminomethyluridine; mcm5s2U: 5-methoxycarbonylmethyl-2-thiouridine.

Diazotrophic Anaeromyxobacter Isolates from Soils.

Biological nitrogen fixation is an essential reaction in a major pathway for supplying nitrogen to terrestrial environments. Previous culture-independent analyses based on soil DNA/RNA/protein sequencing could globally detect the nitrogenase genes/proteins of Anaeromyxobacter (in the class Deltaproteobacteria), commonly distributed in soil environments and predominant in paddy soils; this suggests the importance of Anaeromyxobacter in nitrogen fixation in soil environments. However, direct experimental evidence is lacking; there has been no research on the genetic background and ability of Anaeromyxobacter to fix nitrogen. Therefore, we verified the diazotrophy of Anaeromyxobacter based on both genomic and culture-dependent analyses using Anaeromyxobacter sp. strains PSR-1 and Red267 isolated from soils. Based on the comparison of nif gene clusters, strains PSR-1 and Red267 as well as strains Fw109-5, K, and diazotrophic Geobacter and Pelobacter in the class Deltaproteobacteria contain the minimum set of genes for nitrogenase (nifBHDKEN). These results imply that Anaeromyxobacter species have the ability to fix nitrogen. In fact, Anaeromyxobacter PSR-1 and Red267 exhibited N2-dependent growth and acetylene reduction activity (ARA) in vitro Transcriptional activity of the nif gene was also detected when both strains were cultured with N2 gas as a sole nitrogen source, indicating that Anaeromyxobacter can fix and assimilate N2 gas by nitrogenase. In addition, PSR-1- or Red267-inoculated soil showed ARA activity and the growth of the inoculated strains on the basis of RNA-based analysis, demonstrating that Anaeromyxobacter can fix nitrogen in the paddy soil environment. Our study provides novel insights into the pivotal environmental function, i.e., nitrogen fixation, of Anaeromyxobacter, which is a common soil bacterium.IMPORTANCEAnaeromyxobacter is globally distributed in soil environments, especially predominant in paddy soils. Current studies based on environmental DNA/RNA analyses frequently detect gene fragments encoding nitrogenase of Anaeromyxobacter from various soil environments. Although the importance of Anaeromyxobacter as a diazotroph in nature has been suggested by culture-independent studies, there has been no solid evidence and validation from genomic and culture-based analyses that Anaeromyxobacter fixes nitrogen. This study demonstrates that Anaeromyxobacter harboring nitrogenase genes exhibits diazotrophic ability; moreover, N2-dependent growth was demonstrated in vitro and in the soil environment. Our findings indicate that nitrogen fixation is important for Anaeromyxobacter to survive under nitrogen-deficient environments and provide a novel insight into the environmental function of Anaeromyxobacter, which is a common bacterium in soils.

Localization of the reb operon expression is inconsistent with that of the R-body production in the stem nodules formed by Azorhizobium caulinodans mutants having a deletion of praR.

Azorhizobium caulinodans, a kind of rhizobia, has a reb operon encoding pathogenic R-body components, whose expression is usually repressed by a transcription factor PraR. Mutation on praR induced a high expression of reb operon and the formation of aberrant nodules, in which both morphologically normal and shrunken host cells were observed. Histochemical GUS analyses of praR mutant expressing reb operon-uidA fusion revealed that the bacterial cells within the normal host cells highly expressed the reb operon, but rarely produced R-bodies. On the other hand, the bacterial cells within the shrunken host cells frequently produced R-bodies but rarely expressed the reb operon. This suggests that R-body production is not only regulated at the transcriptional level, but by other regulatory mechanisms as well.

Identification of human immunodeficiency virus type-1 Gag-TSG101 interaction inhibitors by high-throughput screening.

The interaction between viral protein Gag and cellular protein tumor susceptibility gene 101 (TSG101) is a crucial step in the HIV-1 replication cycle. This interaction initiates the viral assembly/budding via the cellular endosomal sorting complexes required for transport (ESCRT) pathway, making it a potential target for antiviral therapy. Here we developed a simple, robust, and reliable high-throughput screening (HTS) system based on enzyme-linked immunosorbent assay (ELISA) to identify compounds that inhibit HIV-1 replication by targeting Gag-TSG101 interaction. Through screening of the 9600-compound library using the established HTS system, several hit compounds, which inhibited Gag-TSG101 interaction, were identified. Subsequent assays revealed two hit compounds, HSM-9 and HSM-10, which have antiviral activity against CD4+ T cell-tropic NL4-3 and macrophage-tropic JR-CSF HIV-1 strains. These results suggest that our established HTS system is an indispensable tool for the identification of HIV-1 Gag-TSG101 interaction inhibitors.

A Novel Regulatory Pathway for K+ Uptake in the Legume Symbiont Azorhizobium caulinodans in Which TrkJ Represses the kdpFABC Operon at High Extracellular K+ Concentrations.

Bacteria have multiple K+ uptake systems. Escherichia coli, for example, has three types of K+ uptake systems, which include the low-K+-inducible KdpFABC system and two constitutive systems, Trk (TrkAG and TrkAH) and Kup. Azorhizobium caulinodans ORS571, a rhizobium that forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, also has three types of K+ uptake systems. Through phylogenetic analysis, we found that A. caulinodans has two genes homologous to trkG and trkH, designated trkI and trkJ We also found that trkI is adjacent to trkA in the genome and these two genes are transcribed as an operon; however, trkJ is present at a distinct locus. Our results demonstrated that trkAI, trkJ, and kup were expressed in the wild-type stem nodules, whereas kdpFABC was not. Interestingly, Δkup and Δkup ΔkdpA mutants formed Fix- nodules, while the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant formed Fix+ nodules, suggesting that with the additional deletion of Trk system genes in the Δkup mutant, Fix+ nodule phenotypes were recovered. kdpFABC of the Δkup ΔtrkJ mutant was expressed in stem nodules, but not in the free-living state, under high-K+ conditions. However, kdpFABC of the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant was highly expressed even under high-K+ conditions. The cytoplasmic K+ levels in the Δkup ΔtrkA ΔtrkI mutant, which did not express kdpFABC under high-K+ conditions, were markedly lower than those in the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant. Taking all these results into consideration, we propose that TrkJ is involved in the repression of kdpFABC in response to high external K+ concentrations and that the TrkAI system is unable to function in stem nodules.IMPORTANCE K+ is a major cytoplasmic cation in prokaryotic and eukaryotic cells. Bacteria have multiple K+ uptake systems to control the cytoplasmic K+ levels. In many bacteria, the K+ uptake system KdpFABC is expressed under low-K+ conditions. For years, many researchers have argued over how bacteria sense K+ concentrations. Although KdpD of Escherichia coli is known to sense both cytoplasmic and extracellular K+ concentrations, the detailed mechanism of K+ sensing is still unclear. In this study, we propose that the transmembrane TrkJ protein of Azorhizobium caulinodans acts as a sensor for the extracellular K+ concentration and that high extracellular K+ concentrations repress the expression of KdpFABC via TrkJ.

Stringent Expression Control of Pathogenic R-body Production in Legume Symbiont Azorhizobium caulinodans.

R bodies are insoluble large polymers consisting of small proteins encoded by reb genes and are coiled into cylindrical structures in bacterial cells. They were first discovered in Caedibacter species, which are obligate endosymbionts of paramecia. Caedibacter confers a killer trait on the host paramecia. R-body-producing symbionts are released from their host paramecia and kill symbiont-free paramecia after ingestion. The roles of R bodies have not been explained in bacteria other than CaedibacterAzorhizobium caulinodans ORS571, a microsymbiont of the legume Sesbania rostrata, carries a reb operon containing four reb genes that are regulated by the repressor PraR. Herein, deletion of the praR gene resulted in R-body formation and death of host plant cells. The rebR gene in the reb operon encodes an activator. Three PraR binding sites and a RebR binding site are present in the promoter region of the reb operon. Expression analyses using strains with mutations within the PraR binding site and/or the RebR binding site revealed that PraR and RebR directly control the expression of the reb operon and that PraR dominantly represses reb expression. Furthermore, we found that the reb operon is highly expressed at low temperatures and that 2-oxoglutarate induces the expression of the reb operon by inhibiting PraR binding to the reb promoter. We conclude that R bodies are toxic not only in paramecium symbiosis but also in relationships between other bacteria and eukaryotic cells and that R-body formation is controlled by environmental factors.IMPORTANCECaedibacter species, which are obligate endosymbiotic bacteria of paramecia, produce R bodies, and R-body-producing endosymbionts that are released from their hosts are pathogenic to symbiont-free paramecia. Besides Caedibacter species, R bodies have also been observed in a few free-living bacteria, but the significance of R-body production in these bacteria is still unknown. Recent advances in genome sequencing technologies revealed that many Gram-negative bacteria possess reb genes encoding R-body components, and interestingly, many of them are animal and plant pathogens. Azorhizobium caulinodans, a microsymbiont of the tropical legume Sesbania rostrata, also possesses reb genes. In this study, we demonstrate that A. caulinodans has ability to kill the host plant cells by producing R bodies, suggesting that pathogenicity conferred by an R body might be universal in bacteria possessing reb genes. Furthermore, we provide the first insight into the molecular mechanism underlying the expression of R-body production in response to environmental factors, such as temperature and 2-oxoglutarate.

PHO8 gene coding alkaline phosphatase of Saccharomyces cerevisiae is involved in polyphosphate metabolism.

It has been argued for a long time whether alkaline phosphatase (ALP) is involved in polyphosphate (polyP) metabolism in arbuscular mycorrhizal fungi. In the present study, we have analyzed the effects of disrupting the PHO8 gene, which encodes phosphate (Pi)-deficiency-inducible ALP, on the polyP contents of Saccharomyces cerevisiae. The polyP content of the Δpho8 mutant was higher than the wild type strain in the logarithmic phase under Pi-sufficient conditions. On the contrary, the chain length of polyP extracted from the Δpho8 mutant did not differ from the wild type strain. When cells in Pi-deficient conditions were supplemented with Pi, the increase of the polyP amounts in the Δpho8 mutant was similar to that in the wild type strain. These results suggest that ALP, which is encoded by PHO8, affects the polyP content, but not the chain length, and participates in polyP homeostasis in Pi-sufficient conditions.

Effects of alteration in LPS structure in Azorhizobium caulinodans on nodule development.

The lipopolysaccharide (LPS) of Azorhizobium caulinodans ORS571, which forms N2-fixing nodules on the stems and roots of Sesbania rostrata, is known to be a positive signal required for the progression of nodule formation. In this study, four A. caulinodans mutants producing a variety of defective LPSs were compared. The LPSs of the mutants having Tn5 insertion in the rfaF, rfaD, and rfaE genes were more truncated than the modified LPSs of the oac2 mutants. However, the nodule formation by the rfaF, rfaD, and rfaE mutants was more advanced than that of the oac2 mutant, suggesting that invasion ability depends on the LPS structure. Our hypothesis is that not only the wild-type LPSs but also the altered LPSs of the oac2 mutant may be recognized as signal molecules by plants. The altered LPSs may act as negative signals that halt the symbiotic process, whereas the wild-type LPSs may prevent the halt of the symbiotic process. The more truncated LPSs of the rfaF, rfaD, and rfaE mutants perhaps no longer function as negative signals inducing discontinuation of the symbiotic process, and thus these strains form more advanced nodules than ORS571-oac2.

pH measurement of tubular vacuoles of an arbuscular mycorrhizal fungus, Gigaspora margarita.

Arbuscular mycorrhizal fungi play an important role in phosphate supply to the host plants. The fungal hyphae contain tubular vacuoles where phosphate compounds such as polyphosphate are accumulated. Despite their importance for the phosphate storage, little is known about the physiological properties of the tubular vacuoles in arbuscular mycorrhizal fungi. As an indicator of the physiological state in vacuoles, we measured pH of tubular vacuoles in living hyphae of arbuscular mycorrhizal fungus Gigaspora margarita using ratio image analysis with pH-dependent fluorescent probe, 6-carboxyfluorescein. Fluorescent images of the fine tubular vacuoles were obtained using a laser scanning confocal microscope, which enabled calculation of vacuolar pH with high spatial resolution. The tubular vacuoles showed mean pH of 5.6 and a pH range of 5.1-6.3. These results suggest that the tubular vacuoles of arbuscular mycorrhizal fungi have a mildly acidic pH just like vacuoles of other fungal species including yeast and ectomycorrhizal fungi.

Lon protease of Azorhizobium caulinodans ORS571 is required for suppression of reb gene expression.

Bacterial Lon proteases play important roles in a variety of biological processes in addition to housekeeping functions. In this study, we focused on the Lon protease of Azorhizobium caulinodans, which can fix nitrogen both during free-living growth and in stem nodules of the legume Sesbania rostrata. The nitrogen fixation activity of an A. caulinodans lon mutant in the free-living state was not significantly different from that of the wild-type strain. However, the stem nodules formed by the lon mutant showed little or no nitrogen fixation activity. By microscopic analyses, two kinds of host cells were observed in the stem nodules formed by the lon mutant. One type has shrunken host cells containing a high density of bacteria, and the other type has oval or elongated host cells containing a low density or no bacteria. This phenotype is similar to a praR mutant highly expressing the reb genes. Quantitative reverse transcription-PCR analyses revealed that reb genes were also highly expressed in the lon mutant. Furthermore, a lon reb double mutant formed stem nodules showing higher nitrogen fixation activity than the lon mutant, and shrunken host cells were not observed in these stem nodules. These results suggest that Lon protease is required to suppress the expression of the reb genes and that high expression of reb genes in part causes aberrance in the A. caulinodans-S. rostrata symbiosis. In addition to the suppression of reb genes, it was found that Lon protease was involved in the regulation of exopolysaccharide production and autoagglutination of bacterial cells.

NAD(P)+-malic enzyme mutants of Sinorhizobium sp. strain NGR234, but not Azorhizobium caulinodans ORS571, maintain symbiotic N2 fixation capabilities.

C(4)-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N(2)-fixing bacteria (bacteroids) within legume nodules. In Sinorhizobium meliloti bacteroids from alfalfa, NAD(+)-malic enzyme (DME) is required for N(2) fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont Rhizobium leguminosarum, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report that dme mutants of the broad-host-range Sinorhizobium sp. strain NGR234 formed nodules whose level of N(2) fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while single pckA and single dme mutants fixed N(2) at reduced rates, a pckA dme double mutant had no N(2)-fixing activity (Fix(-)). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix(-) phenotype of S. meliloti dme mutants may be specific to the alfalfa-S. meliloti symbiosis. We therefore examined the ME-like genes azc3656 and azc0119 from Azorhizobium caulinodans, as azc3656 mutants were previously shown to form Fix(-) nodules on the tropical legume Sesbania rostrata. We found that purified AZC3656 protein is an NAD(P)(+)-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N(2) fixation in A. caulinodans and S. meliloti, in other rhizobia this activity can be bypassed via another pathway(s).

phrR-like gene praR of Azorhizobium caulinodans ORS571 is essential for symbiosis with Sesbania rostrata and is involved in expression of reb genes.

This study focuses on the function of the gene praR that encodes a putative transcription factor in Azorhizobium caulinodans ORS571, a microsymbiont of Sesbania rostrata. The praR gene is a homolog of the phrR gene of Sinorhizobium medicae WSM419, and the praR and phrR homologs are distributed throughout the class Alphaproteobacteria. The growth and nitrogen fixation activity of an A. caulinodans praR deletion mutant in the free-living state were not significantly different from those of the wild-type strain. However, the stem nodules formed by the praR mutant showed lower nitrogen fixation activity than the wild-type stem nodules. Microscopy revealed that infected host cells with an oval or elongated shape were observed at early stages in the nodules formed by the praR mutant, but these infected cells gradually fell into two types. One maintained an oval or elongated shape, but the vacuoles in these cells gradually enlarged and the bacteria gradually disappeared. The other cells were shrunken with bacteria remaining inside. Microarrays revealed that genes homologous to the reb genes of Caedibacter taeniospiralis were highly expressed in the praR mutant. Furthermore, the stem nodules formed by an A. caulinodans mutant with a deletion of praR and reb-homologous genes showed high nitrogen fixation activity, comparable to that of the wild-type stem nodules, and were filled with oval or elongated host cells. These results suggest that PraR controls the expression of the reb-homologous genes and that high expression of reb-homologous genes causes aberrance in A. caulinodans-S. rostrata symbiosis.

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.

Comparative genome-wide transcriptional profiling of Azorhizobium caulinodans ORS571 grown under free-living and symbiotic conditions.

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.

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.

The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571.

BACKGROUND: Biological 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. RESULTS: The 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. CONCLUSION: The 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.

The molecular and physiological mechanisms behind the maturation and maintenance of N(2)-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 N(2)-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 N(2) 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, C(4) 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.

Evidence for functional differentiation of duplicated nifH genes in Azorhizobium caulinodans.

Azorhizobium caulinodans is a symbiotic diazotroph that contains duplicated nifH genes. This study focused on the biological sense behind the duplication. In-frame deletion mutants of nifH1 and nifH2 were constructed in order to analyze nitrogen fixation activity, both in symbiosis and in free-living conditions. Symbiotic nitrogen fixation activity was not affected by deletion of nifH1 or nifH2, while free-living nitrogen fixation activity was significantly decreased. Deletion of nifH1 had a significant effect in semi-aerobic condition, while deletion of nifH2 was significant in microaerobic condition, suggesting functional differences between nifH1 and nifH2. Transcriptional activity of nifH1 was higher than nifH2, both in microaerobic and semi-aerobic conditions.