Diversity of rhizobia and non-rhizobia endophytes isolated from root nodules of Trifolium sp. growing in lead and zinc mine site Guelma, Algeria.

High concentrations of heavy metals in mine soil disturb the interactions between legumes and microorganisms leading to select strains adapted to these specific conditions. In this work, we analyzed the diversity of fifty strains isolated from Trifolium sp. nodules growing on Pb-Zn mine soil, in the Northeastern of Algeria and highlighted their potential symbiotic traits. The phylogeny of the 16S rRNA gene sequences revealed a high bacterial diversity with a predominance of non-rhizobial endophytes. The identified isolates belong to the thirteen following genera Cupriavidus, Pseudomonas, Bacillus, Acinetobacter, Enterobacter, Roseomonas, Paracoccus, Frondihabitans, Microbacterium, Kocuria, Providencia, Micrococcus and Staphylococcus. Regarding rhizobial strains, only isolates affiliated to Rhizobium genus were obtained. The symbiotic gene nodC and the nitrogen fixation gene nifH present showed that Rhizobium isolates belonged to the symbiovar trifolii. In addition to bacterial, one yeast strain was isolated and identified as Rhodotorula mucilaginosa by sequencing the internal transcribed spacer (ITS) region.

Novel heavy metal resistance gene clusters are present in the genome of Cupriavidus neocaledonicus STM 6070, a new species of Mimosa pudica microsymbiont isolated from heavy-metal-rich mining site soil.

BACKGROUND: Cupriavidus strain STM 6070 was isolated from nickel-rich soil collected near Koniambo massif, New Caledonia, using the invasive legume trap host Mimosa pudica. STM 6070 is a heavy metal-tolerant strain that is highly effective at fixing nitrogen with M. pudica. Here we have provided an updated taxonomy for STM 6070 and described salient features of the annotated genome, focusing on heavy metal resistance (HMR) loci and heavy metal efflux (HME) systems. RESULTS: The 6,771,773 bp high-quality-draft genome consists of 107 scaffolds containing 6118 protein-coding genes. ANI values show that STM 6070 is a new species of Cupriavidus. The STM 6070 symbiotic region was syntenic with that of the M. pudica-nodulating Cupriavidus taiwanensis LMG 19424T. In contrast to the nickel and zinc sensitivity of C. taiwanensis strains, STM 6070 grew at high Ni2+ and Zn2+ concentrations. The STM 6070 genome contains 55 genes, located in 12 clusters, that encode HMR structural proteins belonging to the RND, MFS, CHR, ARC3, CDF and P-ATPase protein superfamilies. These HMR molecular determinants are putatively involved in arsenic (ars), chromium (chr), cobalt-zinc-cadmium (czc), copper (cop, cup), nickel (nie and nre), and silver and/or copper (sil) resistance. Seven of these HMR clusters were common to symbiotic and non-symbiotic Cupriavidus species, while four clusters were specific to STM 6070, with three of these being associated with insertion sequences. Within the specific STM 6070 HMR clusters, three novel HME-RND systems (nieIC cep nieBA, czcC2B2A2, and hmxB zneAC zneR hmxS) were identified, which constitute new candidate genes for nickel and zinc resistance. CONCLUSIONS: STM 6070 belongs to a new Cupriavidus species, for which we have proposed the name Cupriavidus neocaledonicus sp. nov.. STM6070 harbours a pSym with a high degree of gene conservation to the pSyms of M. pudica-nodulating C. taiwanensis strains, probably as a result of recent horizontal transfer. The presence of specific HMR clusters, associated with transposase genes, suggests that the selection pressure of the New Caledonian ultramafic soils has driven the specific adaptation of STM 6070 to heavy-metal-rich soils via horizontal gene transfer.

Impact of seed-applied pesticides on rhizobial survival and legume nodulation.

AIMS: Compatibility of seed-applied pesticides and rhizobial inoculants is an important consideration for farmers when sowing legumes. Some of the seed-applied pesticides may influence rhizobial growth and nodulation, but there is currently little available information on the potential inhibitory effects. Therefore, common seed fungicidal and insecticidal treatments were assessed to determine adverse impacts on rhizobial inoculants both in vitro, on treated seed, and in the field. METHODS AND RESULTS: Initially, the in vitro toxicity of the seed-applied fungicides Thiram 600, P-Pickel T (PPT), their active ingredients (thiram and thiabendazole) and the insecticide Gaucho to rhizobia was measured with filter discs containing varying concentrations of the pesticides. Pea and chickpea seed was then coated with the same pesticides and inoculated with rhizobia in different inoculant substrates to determine bacterial survival and nodulation. Finally, a field trial using the fungicide PPT and commercial inoculants was conducted. Some seed fungicide treatments were found to be inhibitory to rhizobia and reduce nodulation under monoxenic conditions and in the field. SIGNIFICANCE AND IMPACT OF THE STUDY: These data provide more detailed information on the compatibility of specific rhizobial inoculants with common seed-applied pesticides. This research will provide information on the compatibility of rhizobia and seed-applied pesticides, and assist farmers to select sowing practices which reduce the risk of crop nodulation failures.

Effect of common bean seed exudates on growth, lipopolysaccharide production, and lipopolysaccharide transport gene expression of Rhizobium anhuiense.

Lipopolysaccharide (LPS) is essential for successful nodulation during the symbiosis of rhizobia and legumes. However, the detailed mechanism of the LPS in this process has not yet been clearly elucidated. In this study, the effects of common bean seed exudates on the growth, lipopolysaccharide production, and lipopolysaccharide transport genes expression (lpt) of Rhizobium anhuiense were investigated. Rhizobium anhuiense exposed to exudates showed changes in LPS electrophoretic profiles and content, whereby the LPS band was wider and the LPS content was higher in R. anhuiense treated with seed exudates. Exudates enhanced cell growth of R. anhuiense in a concentration-dependent manner; R. anhuiense exposed to higher doses of the exudate showed faster growth. Seven lpt genes of R. anhuiense were amplified and sequenced. Sequences of six lpt genes, except for lptE, were the same as those found in previously analyzed R. anhuiense strains, while lptE shared low sequence similarity with other strains. Exposure to the exudates strongly stimulated the expression of all lpt genes. Approximately 6.7- (lptG) to 301-fold (lptE) increases in the transcriptional levels were observed after only 15 min of exposure to exudates. These results indicate that seed exudates affect the LPS by making the cell wall structure more conducive to symbiotic nodulation.

Rhizobium response to sole and combined exposure to cadmium and the phytocompounds alpha-pinene and quercetin.

Soils can be contaminated with substances arising from anthropogenic sources, but also with natural bioactive compounds produced by plants, such as terpenes and flavonoids. While terpenes and flavonoids have received much less attention from research studies than metals, the effects that phytocompounds can have on soil organisms such as beneficial microorganisms should not be neglected. Herein we report the sole and combined exposure of Rhizobium to cadmium, to the monoterpene alpha-pinene and to the flavanol quercetin. A range of environmentally relevant concentrations of the phytocompounds was tested. Physiological (growth, protein content and intracellular Cd concentration), oxidative damage (lipid peroxidation, protein carbonylation) and antioxidant mechanisms (superoxide dismutase, catalase, glutathione, glutathione-S-transferases, protein electrophoretic profiles) were assessed. Results suggest that exposure to both phytocompounds do not influence Rhizobium growth, but for combined exposure to phytocompounds and Cd, different responses are observed. At low concentrations, phytocompounds seem to relieve the stress imposed by Cd by increasing antioxidant responses, but at high concentrations this advantage is lost and membrane damage may even be exacerbated. Thus, the presence of bioactive phytocompounds in soil may influence the tolerance of microorganisms to persistent toxicants, and may change their impact on the environment.

Cadmium-affected synthesis of exopolysaccharides by rhizosphere bacteria.

AIM: Study is focused on the influence of cadmium addition to growth media on production yield, their size and molecular mass of exopolysaccharides (EPS) synthesized by three rhizosphere bacteria strains. Inhibition of bacterial growth by increasing concentrations of Cd2+ was also analysed. METHODS AND RESULTS: The highest impact of Cd2+ was noticed on the growth of Arthrobacter sp. and Rhizobium metallidurans. Chryseobacterium sp. and Arthrobacter sp. produced significantly lower when compared to R. metallidurans amounts of EPS under the influence of Cd2+ . In all bacterial strains both size and molecular mass decreased after addition of Cd2+ to growth media. It causes a change in EPS conformation to more planar, which minimizes the volume of liquid in the interglobular space next to the bacterial wall. Results confirmed strong effect of Cd2+ on the structure and synthesis of bacterial EPS what can be a key factor in the interactions between rhizosphere bacteria and host plants in heavy metal polluted soils. CONCLUSION: This work proves that due to the presence of cadmium ions, the size and conformation of EPS produced by selected bacterial strains is changed to minimize their impact on cell. We suggest that shifting in EPS conformation from bigger globular particles to the smaller planar ones could be one of the probable mechanisms of Cd resistance in metallotolerant bacteria, and finally explain increased efficiency of heavy metal phytoextraction by EPS-producing plant growth-promoting micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the most promising remediation technique for Cd-contaminated areas is the phytoremediation in which rhizosphere bacteria play an important role by protecting plants' roots from toxic condition thus enhancing efficiency of intake. EPS secretion by bacteria is one of the most common mechanisms to protect the cell from impact of unpleasant environmental conditions, for example, toxicity of heavy metals like Cd.

The role of volatiles in Rhizobium tolerance to cadmium: Effects of aldehydes and alcohols on growth and biochemical endpoints.

Rhizobia have a significant agronomic and environmental role and are eminent contributors to soil fertility. However, this group of microorganisms are affected by various environmental stresses, such as Cd contamination. High Cd concentrations change bacterial metabolism. During this metabolic shift, bacteria alter their volatilome (the set of volatile metabolites synthesized by an organism). In the presence of Cd, peak areas of saturated aldehydes and alcohols were previously reported to increase, and the consequences of this increase to cells are poorly known. In this study, Rhizobium sp. strain E20-8 cells were exposed to Cd and aldehydes or their conjugated alcohols. Exposure to Cd (100 µM) inhibited cell growth and induced several biomarkers of oxidative stress. The present study also evidenced the higher toxicity of most aldehydes relatively to the corresponding alcohol in the presence of Cd, suggesting that reduction of aldehydes into alcohols may be an effective mechanism to restrain aldehydes toxicity in Rhizobium cells under Cd toxicity. Nonetheless, the protective effect was dependent on the pair aldehyde-respective alcohol considered and it differed between Cd stressed and non-stressed cells. Differences in the ability to convert aldehydes to alcohols may emerge as a new feature helping explain the oxidative tolerance variability among bacteria.

Strigolactones promote rhizobia interaction and increase nodulation in soybean (Glycine max).

Strigolactones (SLs) play an important role in controlling root growth, shoot branching, and plant-symbionts interaction. Despite the importance, the components of SL biosynthesis and signaling have not been unequivocally explored in soybean. Here we identified the putative components of SL synthesis enzymes GmMAX1a and GmMAX4a with tissue expression patterns and were apparently regulated by rhizobia infection and changed during nodule development. GmMAX1a and GmMAX4a were further characterized in soybean nodulation with knockdown transgenic hairy roots. GmMAX1a and GmMAX4a knockdown lines exhibit decreased nodule number and expression levels of several nodulation genes required for nodule development. Hormone analysis showed that GmMAX1a and GmMAX4a knockdown hairy roots had increased physiological level of ABA and JA but significantly decreased auxin content. This study not only revealed the conservation of SL biosynthesis but also showed close interactions between SL and other hormone signaling in controlling plant development and legume-rhizobia interaction.

In vitro rhizobia response and symbiosis process under aluminum stress.

Aluminum (Al) toxicity is a major problem affecting soil fertility, microbial diversity, and nutrient uptake of plants. Rhizobia response and legume interaction under Al conditions are still unknown; it is important to understand how to develop and improve legume cultivation under Al stress. In this study, rhizobia response was recorded under different Al concentrations. Al effect on rhizobial cells was characterized by combination with different two pH conditions. Symbiosis process was compared between α- and ß-rhizobia inoculated onto soybean varieties. Rhizobial cell numbers was decreased as Al concentration increased. However, induced Al tolerance considerably depended on rhizobia types and their origins. Accordingly, organic acid results were in correlation with growth rate and cell density which suggested that citric acid might be a positive selective force for Al tolerance and plant interaction on rhizobia. Al toxicity delayed and interrupted the plant-rhizobia interaction and the effect was more pronounced under acidic conditions. Burkholderia fungorum VTr35 significantly improved plant growth under acid-Al stress in combination with all soybean varieties. Moreover, plant genotype was an important factor to establish an effective nodulation and nitrogen fixation under Al stress. Additionally, tolerant rhizobia could be applied as an inoculant on stressful agroecosystems. Furthermore, metabolic pathways have still been unknown under Al stress.

Protective effects of farnesol on a Rhizobium strain exposed to cadmium.

Soil acts as a repository for many metals that human activity releases into the environment. Cadmium enters agricultural soils primarily from application of phosphate fertilizers and sewage sludge. Among soil bacteria, rhizobia have a great agronomic and environmental significance and are major contributors to a sustainable maintenance of soil fertility. However, the services that this group of microorganisms provides are affected by environmental constraints, such as Cd contamination. Bioactive compounds also influence soil microorganisms. Farnesol is a phytocompound with recognized bioactivity, inducing both beneficial and harmful effects. In this study, Rhizobium sp. strain E20-8 was exposed to sole or combined exposure to Cd and farnesol. Results showed that farnesol (25 and 200 µM) did not affect rhizobia; exposure to Cd (µM) inhibited rhizobia growth and induced several biomarkers of oxidative stress; exposure to the combination of farnesol and Cd reduced oxidative damage, and the highest concentration of farnesol tested reduced Cd accumulation and allowed a significant growth recovery. Farnesol protective effects on rhizobia exposed to Cd is novel information which can be used in the development of microbe-based environmental engineering strategies for restoration of metal contaminated areas.

Different efficiencies of the same mechanisms result in distinct Cd tolerance within Rhizobium.

Soil contamination with metals is a widespread problem posing risks to humans and ecosystems. Metal contaminated soils often hold poor microbial density and biodiversity. Among soil bacteria, rhizobia have a great agronomic and environmental significance and are major contributors to a sustainable maintenance of soil fertility. This group of microorganisms are severely affected by metals, such as cadmium (Cd), but information about metal resistance mechanisms in rhizobia is still limited. A concerted approach of the different mechanisms conferring Cd tolerance to rhizobia was conducted using two Rhizobium strains with contrasting tolerances to Cd. Results show that both strains resort to the same mechanisms (extracellular immobilization, periplasmic allocation, cytoplasmic sequestration and biotransformation of toxic products) to overcome stress, but differences in the efficiencies of some mechanisms were noticed. The ability of Rhizobium to increase glutathione in the presence of Cd emerges as a central factor in the tolerance to Cd and is as a feature to be looked for when screening or transforming microorganisms to integrate plant-microbe consortia. These could promote plant growth at contaminated sites, being more efficient for the cleanup of metals from contaminated sites and the restoration of soil quality.

The Phenylalanine Ammonia Lyase Gene LjPAL1 Is Involved in Plant Defense Responses to Pathogens and Plays Diverse Roles in Lotus japonicus-Rhizobium Symbioses.

Phenylalanine ammonia lyase (PAL) is important in the biosynthesis of plant secondary metabolites that regulate growth responses. Although its function is well-established in various plants, the functional significance of PAL genes in nodulation is poorly understood. Here, we demonstrate that the Lotus japonicus PAL (LjPAL1) gene is induced by Mesorhizobium loti infection and methyl-jasmonate (Me-JA) treatment in roots. LjPAL1 altered PAL activity, leading to changes in lignin contents and thicknesses of cell walls in roots and nodules of transgenic plants and, hence, to structural changes in roots and nodules. LjPAL1-knockdown plants (LjPAL1i) exhibited increased infection thread and nodule numbers and the induced upregulation of nodulin gene expression after M. loti infection. Conversely, LjPAL1 overexpression delayed the infection process and reduced infection thread and nodule numbers after M. loti inoculation. LjPAL1i plants also exhibited reduced endogenous salicylic acid (SA) accumulation and expression of the SA-dependent marker gene. Their infection phenotype could be partially restored by exogenous SA or Me-JA application. Our data demonstrate that LjPAL1 plays diverse roles in L. japonicus-rhizobium symbiosis, affecting rhizobial infection progress and nodule structure, likely by inducing lignin modification, regulating endogenous SA biosynthesis, and modulating SA signaling.

The Thiamine Biosynthesis Gene THI1 Promotes Nodule Growth and Seed Maturation.

Thiamine (vitamin B1) is essential for living organisms. Unlike animals, plants can synthesize thiamine. In Lotus japonicus, the expression of two thiamine biosynthesis genes, THI1 and THIC, was enhanced by inoculation with rhizobia but not by inoculation with arbuscular mycorrhizal fungi. THIC and THI2 (a THI1 paralog) were expressed in uninoculated leaves. THI2-knockdown plants and the transposon insertion mutant thiC had chlorotic leaves. This typical phenotype of thiamine deficiency was rescued by an exogenous supply of thiamine. In wild-type plants, THI1 was expressed mainly in roots and nodules, and the thi1 mutant had green leaves even in the absence of exogenous thiamine. THI1 was highly expressed in actively dividing cells of nodule primordia. The thi1 mutant had small nodules, and this phenotype was rescued by exogenous thiamine and by THI1 complementation. Exogenous thiamine increased nodule diameter, but the level of arbuscular mycorrhizal colonization was unaffected in the thi1 mutant or by exogenous thiamine. Expression of symbiotic marker genes was induced normally, implying that mainly nodule growth was delayed in the thi1 mutant. Furthermore, this mutant formed many immature seeds with reduced seed weight. These results indicate that thiamine biosynthesis mediated by THI1 enhances nodule enlargement and is required for seed development in L. japonicus.

Bioremediation model for atrazine contaminated agricultural soils using phytoremediation (using Phaseolus vulgaris L.) and a locally adapted microbial consortium.

The objective of the present study was to examine a biological model under greenhouse conditions for the bioremediation of atrazine contaminated soils. The model consisted in a combination of phytoremediation (using Phaseolus vulgaris L.) and rhizopheric bio-augmentation using native Trichoderma sp., and Rhizobium sp. microorganisms that showed no inhibitory growth at 10,000 mg L-1 of herbicide concentration. 33.3 mg of atrazine 50 g-1 of soil of initial concentration was used and an initial inoculation of 1 × 109 UFC mL-1 of Rhizobium sp. and 1 × 105 conidia mL-1 of Trichoderma sp. were set. Four treatments were arranged: Bean + Trichoderma sp. (B+T); Bean + Rhizobium sp. (BR); Bean + Rhizobium sp. + Trichoderma sp. (B+R+T) and Bean (B). 25.51 mg of atrazine 50 g-1 of soil (76.63%) was removed by the B+T treatment in 40 days (a = 0.050, Tukey). This last indicate that the proposed biological model and methodology developed is useful for atrazine contaminated bioremediation agricultural soils, which can contribute to reduce the effects of agrochemical abuse.

Deep Sequencing of the Medicago truncatula Root Transcriptome Reveals a Massive and Early Interaction between Nodulation Factor and Ethylene Signals.

The legume-rhizobium symbiosis is initiated through the activation of the Nodulation (Nod) factor-signaling cascade, leading to a rapid reprogramming of host cell developmental pathways. In this work, we combine transcriptome sequencing with molecular genetics and network analysis to quantify and categorize the transcriptional changes occurring in roots of Medicago truncatula from minutes to days after inoculation with Sinorhizobium medicae. To identify the nature of the inductive and regulatory cues, we employed mutants with absent or decreased Nod factor sensitivities (i.e. Nodulation factor perception and Lysine motif domain-containing receptor-like kinase3, respectively) and an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant (sickle). This unique data set encompasses nine time points, allowing observation of the symbiotic regulation of diverse biological processes with high temporal resolution. Among the many outputs of the study is the early Nod factor-induced, ET-regulated expression of ET signaling and biosynthesis genes. Coupled with the observation of massive transcriptional derepression in the ET-insensitive background, these results suggest that Nod factor signaling activates ET production to attenuate its own signal. Promoter:ß-glucuronidase fusions report ET biosynthesis both in root hairs responding to rhizobium as well as in meristematic tissue during nodule organogenesis and growth, indicating that ET signaling functions at multiple developmental stages during symbiosis. In addition, we identified thousands of novel candidate genes undergoing Nod factor-dependent, ET-regulated expression. We leveraged the power of this large data set to model Nod factor- and ET-regulated signaling networks using MERLIN, a regulatory network inference algorithm. These analyses predict key nodes regulating the biological process impacted by Nod factor perception. We have made these results available to the research community through a searchable online resource.

Medicago truncatula natural resistance-associated macrophage Protein1 is required for iron uptake by rhizobia-infected nodule cells.

Iron is critical for symbiotic nitrogen fixation (SNF) as a key component of multiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II.

The presence of nodules on legume root systems can alter phenotypic plasticity in response to internal nitrogen independent of nitrogen fixation.

All higher plants show developmental plasticity in response to the availability of nitrogen (N) in the soil. In legumes, N starvation causes the formation of root nodules, where symbiotic rhizobacteria fix atmospheric N2 for the host in exchange for fixed carbon (C) from the shoot. Here, we tested whether plastic responses to internal [N] of legumes are altered by their symbionts. Glasshouse experiments compared root phenotypes of three legumes, Medicago truncatula, Medicago sativa and Trifolium subterraneum, inoculated with their compatible symbiont partners and grown under four nitrate levels. In addition, six strains of rhizobia, differing in their ability to fix N2 in M. truncatula, were compared to test if plastic responses to internal [N] were dependent on the rhizobia or N2 -fixing capability of the nodules. We found that the presence of rhizobia affected phenotypic plasticity of the legumes to internal [N], particularly in root length and root mass ratio (RMR), in a plant species-dependent way. While root length responses of M. truncatula to internal [N] were dependent on the ability of rhizobial symbionts to fix N2 , RMR response to internal [N] was dependent only on initiation of nodules, irrespective of N2 -fixing ability of the rhizobia strains.

Interactions between ethylene, gibberellins, and brassinosteroids in the development of rhizobial and mycorrhizal symbioses of pea.

The regulation of arbuscular mycorrhizal development and nodulation involves complex interactions between the plant and its microbial symbionts. In this study, we use the recently identified ethylene-insensitive ein2 mutant in pea (Pisum sativum L.) to explore the role of ethylene in the development of these symbioses. We show that ethylene acts as a strong negative regulator of nodulation, confirming reports in other legumes. Minor changes in gibberellin1 and indole-3-acetic acid levels in ein2 roots appear insufficient to explain the differences in nodulation. Double mutants produced by crosses between ein2 and the severely gibberellin-deficient na and brassinosteroid-deficient lk mutants showed increased nodule numbers and reduced nodule spacing compared with the na and lk single mutants, but nodule numbers and spacing were typical of ein2 plants, suggesting that the reduced number of nodules innaandlkplants is largely due to the elevated ethylene levels previously reported in these mutants. We show that ethylene can also negatively regulate mycorrhizae development when ethylene levels are elevated above basal levels, consistent with a role for ethylene in reducing symbiotic development under stressful conditions. In contrast to the hormone interactions in nodulation, ein2 does not override the effect of lk or na on the development of arbuscular mycorrhizae, suggesting that brassinosteroids and gibberellins influence this process largely independently of ethylene.

Antibiotics Resistance in Rhizobium: Type, Process, Mechanism and Benefit for Agriculture.

The use of high-quality rhizobial inoculants on agricultural legumes has contributed substantially to the N economy of farming systems through inputs from biological nitrogen fixation (BNF). Large populations of symbiotically effective rhizobia should be available in the rhizosphere for symbiotic BNF with host plants. The rhizobial populations should also be able to compete and infect host plants. However, the rhizosphere comprises large populations of different microorganisms. Some of these microorganisms naturally produce antibiotics which are lethal to susceptible rhizobial populations in the soil. Therefore, intrinsic resistance to antibiotics is a desirable trait for the rhizobial population. It increases the rhizobia's chances of growth, multiplication and persistence in the soil. With a large population of rhizobia in the soil, infectivity of host plants and the subsequent BNF efficiency can be guaranteed. This review, therefore, puts together findings by various researchers on antibiotic resistance in bacteria with the main emphasis on rhizobia. It describes the different modes of action of different antibiotics, the types of antibiotic resistance exhibited by rhizobia, the mechanisms of acquisition of antibiotic resistance in rhizobia and the levels of tolerance of different rhizobial species to different antibiotics.

NODULE INCEPTION antagonistically regulates gene expression with nitrate in Lotus japonicus.

Legumes produce root nodules as symbiotic organs where nitrogen-fixing bacteria are accommodated. Lotus japonicus NODULE INCEPTION (NIN) is an essential factor that specifically and positively regulates nodulation processes, and has evolved from a member of the NIN-like proteins, of which Arabidopsis homologs target nitrate-responsive elements (NREs), and activate gene expression in response to nitrate. It is therefore assumed that the NIN-mediated transcriptional network overlaps with those regulated by NLPs, because of their common DNA-binding RWP-RK domains. However, nodulation is inhibited in the presence of nitrate, and involvement of NIN in nitrate responses has remained largely unknown. Here we determined a consensus of NIN-binding nucleotide sequences (NBSs) by in vitro experiments, and revealed that the sequence pattern was very similar to those of NREs. Chromatin immunoprecitiation (ChIP)-PCR analyses showed that NIN targeted NREs in L. japonicus nitrate-inducible gene promoters, including LjNIR1, LjNRT2.1 and LjNRT2.2. Affinities of NIN binding to the NREs were comparable with that to NBS-yB1a, an NBS on the symbiotic LjNF-YB1 promoter, indicating that NREs are potential targets of NIN. However, rhizobial infection did not activate LjNIR1, LjNRT2.1 and LjNRT2.2. NIN ectopic expression interfered with nitrate-dependent activation of these genes. Nitrate treatment followed by NIN activation down-regulated expression of symbiotic NIN target genes. Our results showed that NIN and nitrate antagonistically regulate expression of genes that are activated by nitrate and NIN, respectively. We propose that this antagonistic relationship prevents inappropriate activation of genes in response to nitrate and rhizobial infection.