The distinct cell physiology of Bradyrhizobium at the population and cellular level.

The α-Proteobacteria belonging to Bradyrhizobium genus are microorganisms of extreme slow growth. Despite their extended use as inoculants in soybean production, their physiology remains poorly characterized. In this work, we produced quantitative data on four different isolates: B. diazoefficens USDA110, B. diazoefficiens USDA122, B. japonicum E109 and B. japonicum USDA6 which are representative of specific genomic profiles. Notably, we found conserved physiological traits conserved in all the studied isolates: (i) the lag and initial exponential growth phases display cell aggregation; (ii) the increase in specific nutrient concentration such as yeast extract and gluconate hinders growth; (iii) cell size does not correlate with culture age; and (iv) cell cycle presents polar growth. Meanwhile, fitness, cell size and in vitro growth widely vary across isolates correlating to ribosomal RNA operon number. In summary, this study provides novel empirical data that enriches the comprehension of the Bradyrhizobium (slow) growth dynamics and cell cycle.

Morphological and physiological properties of lupine nodule bacteria (Bradyrhizobium lupini) when grown on a typical urban soil.

In the conducted studies, the moorphological and physiological properties of nodule bacteria of lupine were studied. Lupine plants were grown under the conditions of a microfield experiment on a typical medium loamy urban soil. In the study, a pure culture of Bradyrhizobium lupini was isolated. Then, the morphological properties of nodule bacteria cells and the chemical composition of cell membranes of nodule bacteria were determined. The acid resistance and physiological properties of lupine nodule bacteria were also determined, as well as the ratio of Bradyrhizobium lupini to antibiotics. All studies were carried out according to generally accepted methods. The results of the research showed that during the cultivation of lupine on a typical urban soil, nodule bacteria Bradyrhizobium lupini were isolated, which can be characterized as gram-negative, non-spore-forming rods that do not exhibit amylolytic activity. It was revealed that the rhizobia of nodule bacteria are not acid-resistant. Nodule bacteria turned out to be the least resistant to polymyxin, then to levomycetin, and Bradyrhizobium lupini showed the greatest resistance to tetracycline.

Hopanoid lipids promote soybean-Bradyrhizobium symbiosis.

The symbioses between leguminous plants and nitrogen-fixing bacteria known as rhizobia are well known for promoting plant growth and sustainably increasing soil nitrogen. Recent evidence indicates that hopanoids, a family of steroid-like lipids, promote Bradyrhizobium symbioses with tropical legumes. To characterize hopanoids in Bradyrhizobium symbiosis with soybean, we validated a recently published cumate-inducible hopanoid mutant of Bradyrhizobium diazoefficiens USDA110, Pcu-shc::∆shc. GC-MS analysis showed that this strain does not produce hopanoids without cumate induction, and under this condition, is impaired in growth in rich medium and under osmotic, temperature, and pH stress. In planta, Pcu-shc::∆shc is an inefficient soybean symbiont with significantly lower rates of nitrogen fixation and low survival within the host tissue. RNA-seq revealed that hopanoid loss reduces the expression of flagellar motility and chemotaxis-related genes, further confirmed by swim plate assays, and enhances the expression of genes related to nitrogen metabolism and protein secretion. These results suggest that hopanoids provide a significant fitness advantage to B. diazoefficiens in legume hosts and provide a foundation for future mechanistic studies of hopanoid function in protein secretion and motility.A major problem for global sustainability is feeding our exponentially growing human population while available arable land decreases. Harnessing the power of plant-beneficial microbes is a potential solution, including increasing our reliance on the symbioses of leguminous plants and nitrogen-fixing rhizobia. This study examines the role of hopanoid lipids in the symbiosis between Bradyrhizobium diazoefficiens USDA110, an important commercial inoculant strain, and its economically significant host soybean. Our research extends our knowledge of the functions of bacterial lipids in symbiosis to an agricultural context, which may one day help improve the practical applications of plant-beneficial microbes in agriculture.

The native distribution of a common legume shrub is limited by the range of its nitrogen-fixing mutualist.

Plant-microbe mutualisms, such as the legume-rhizobium symbiosis, are influenced by the geographical distributions of both partners. However, limitations on the native range of legumes, resulting from the absence of a compatible mutualist, have rarely been explored. We used a combination of a large-scale field survey and controlled experiments to determine the realized niche of Calicotome villosa, an abundant and widespread legume shrub. Soil type was a major factor affecting the distribution and abundance of C. villosa. In addition, we found a large region within its range in which neither C. villosa nor Bradyrhizobium, the bacterial genus that associates with it, were present. Seedlings grown in soil from this region failed to nodulate and were deficient in nitrogen. Inoculation of this soil with Bradyrhizobium isolated from root nodules of C. villosa resulted in the formation of nodules and higher growth rate, leaf N and shoot biomass compared with un-inoculated plants. We present evidence for the exclusion of a legume from parts of its native range by the absence of a compatible mutualist. This result highlights the importance of the co-distribution of both the host plant and its mutualist when attempting to understand present and future geographical distributions of legumes.

Pleiotropic Effects of PhaR Regulator in Bradyrhizobium diazoefficiens Microaerobic Metabolism.

Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK2 and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia.

Soybean-mediated suppression of BjaI/BjaR1 quorum sensing in Bradyrhizobium diazoefficiens impacts symbiotic nitrogen fixation.

The acyl-homoserine lactones (AHLs)-mediated LuxI/LuxR quorum sensing (QS) system orchestrates diverse bacterial behaviors in response to changes in population density. The role of the BjaI/BjaR1 QS system in Bradyrhizobium diazoefficiens USDA 110, which shares homology with LuxI/LuxR, remains elusive during symbiotic interaction with soybean. Here this genetic system in wild-type (WT) bacteria residing inside nodules exhibited significantly reduced activity compared to free-living cells, potentially attributed to soybean-mediated suppression. The deletion mutant strain ΔbjaR1 showed significantly enhanced nodulation induction and nitrogen fixation ability. Nevertheless, its ultimate symbiotic outcome (plant dry weight) in soybeans was compromised. Furthermore, comparative analysis of the transcriptome, proteome, and promoter activity revealed that the inactivation of BjaR1 systematically activated and inhibited genomic modules associated with nodulation and nitrogen metabolism. The former appeared to be linked to a significant decrease in the expression of NodD2, a key cell-density-dependent repressor of nodulation genes, while the latter conferred bacterial growth and nitrogen fixation insensitivity to environmental nitrogen. In addition, BjaR1 exerted a positive influence on the transcription of multiple genes involved in a so-called central intermediate metabolism within the nodule. In conclusion, our findings highlight the crucial role of the BjaI/BjaR1 QS circuit in positively regulating bacterial nitrogen metabolism and emphasize the significance of the soybean-mediated suppression of this genetic system for promoting efficient symbiotic nitrogen fixation by B. diazoefficiens.IMPORTANCEThe present study demonstrates, for the first time, that the BjaI/BjaR1 QS system of Bradyrhizobium diazoefficiens has a significant impact on its nodulation and nitrogen fixation capability in soybean by positively regulating NodD2 expression and bacterial nitrogen metabolism. Moreover, it provides novel insights into the importance of suppressing the activity of this QS circuit by the soybean host plant in establishing an efficient mutual relationship between the two symbiotic partners. This research expands our understanding of legumes' role in modulating symbiotic nitrogen fixation through rhizobial QS-mediated metabolic functioning, thereby deepening our comprehension of symbiotic coevolution theory. In addition, these findings may hold great promise for developing quorum quenching technology in agriculture.

The divalent metal ion exporter IhpABC is required to maintain iron homeostasis under low to moderate environmental iron conditions in the bacterium Bradyrhizobium japonicum.

Bacterial iron export mitigates high iron stress, but a role for it under lower iron conditions has not been established. MbfA is the high iron stress exporter in Bradyrhizobium japonicum. Here, we identify the ihpABC genes in a selection for secondary site mutations that suppress the poor growth phenotype of feoAB mutants defective in iron acquisition. IhpABC belongs to the RND tripartite efflux pump family. High iron conditions that derepress the mbfA gene partially rescued the growth of an ihpC mutant but reverted the feoB ihpC mutant to the feoB growth phenotype. The ihpA mutant grown under low iron conditions accumulated higher levels of iron compared to the wild type, and it displayed aberrant iron-responsive gene expression. The mbfA mutant was more sensitive than the wild type to H2 O2 , but the ihpA mutant was not sensitive. The ihpA mutant accumulated more Zn, Co and Cd than was found in the wild type, and growth of the mutant was more sensitive to inhibition by ZnCl2 , CoCl2 and CdCl2 . The findings suggest that IhpABC is a divalent metal ion exporter that helps maintain iron homeostasis under low to moderate environmental iron levels. Thus, iron export is not limited to managing high iron stress.

Convergent gene pair dSH3 and irr regulate Pi and Fe homeostasis in Bradyrhizobium diazoefficiens USDA110 and symbiotic nitrogen fixation efficiency.

The nitrogen-fixing bacteroids inhabit inside legume root nodules must manage finely the utilization of P and Fe, the two most critical elements, due to their antagonistic interactions. While the balance mechanism for them remains unclear. A double SH3 domain-containing protein (dSH3) in the Bradyrhizobium diazoefficiens USDA110 was found to inhibit the alkaline phosphatase activity, thereby reducing P supply from organophosphates. The dSH3 gene is adjacent to the irr gene, which encodes the iron response repressor and regulates Fe homeostasis under Fe-limited conditions. Their transcription directions converge to a common intergenic sequence (IGS) region, forming a convergent transcription. Extending the IGS region through Tn5 transposon or pVO155 plasmid insertion significantly down-regulated expression of this gene pair, leading to a remarkable accumulation of P and an inability to grow under Fe-limited conditions. Inoculation of soybean with either of the insertion mutants resulted in N2-fixing failure. However, the IGS-deleted mutant showed no visible changes in N2-fixing efficiency on soybean compared to that inoculated with wild type. These findings reveal a novel regulative strategy in the IGS region and its flanking convergent gene pair for antagonistic utilization of P and Fe in rhizobia and coordination of N2-fixing efficiency.

Bradyrhizobium ottawaense efficiently reduces nitrous oxide through high nosZ gene expression.

N2O is an important greenhouse gas influencing global warming, and agricultural land is the predominant (anthropogenic) source of N2O emissions. Here, we report the high N2O-reducing activity of Bradyrhizobium ottawaense, suggesting the potential for efficiently mitigating N2O emission from agricultural lands. Among the 15 B. ottawaense isolates examined, the N2O-reducing activities of most (13) strains were approximately five-fold higher than that of Bradyrhizobium diazoefficiens USDA110T under anaerobic conditions. This robust N2O-reducing activity of B. ottawaense was confirmed by N2O reductase (NosZ) protein levels and by mitigation of N2O emitted by nodule decomposition in laboratory system. While the NosZ of B. ottawaense and B. diazoefficiens showed high homology, nosZ gene expression in B. ottawaense was over 150-fold higher than that in B. diazoefficiens USDA110T, suggesting the high N2O-reducing activity of B. ottawaense is achieved by high nos expression. Furthermore, we examined the nos operon transcription start sites and found that, unlike B. diazoefficiens, B. ottawaense has two transcription start sites under N2O-respiring conditions, which may contribute to the high nosZ expression. Our study indicates the potential of B. ottawaense for effective N2O reduction and unique regulation of nos gene expression towards the high performance of N2O mitigation in the soil.

Exploring potential soybean bradyrhizobia from high trehalose-accumulating soybean genotypes for improved symbiotic effectiveness in soybean / Exploración de bradirizobios potenciales de la soja a partir de genotipos de soja con alto contenido de trehalosa para mejorar la eficacia simbiótica en la soja

Drought is the most important factor limiting the activity of rhizobia during N-fixation and plant growth. In the present study, we isolated Bradyrhizobium spp. from root nodules of higher trehalose-accumulating soybean genotypes and examined for moisture stress tolerance on a gradient of polyethylene glycol (PEG 6000) amended in yeast extract mannitol (YEM) broth. In addition, the bradyrhizobial strains were also evaluated for symbiotic effectiveness on soybean. Based on 16S rDNA gene sequences, four bradyrhizobial species were recovered from high trehalose-accumulating genotypes, i.e., two Bradyrhizobium liaoningense strains (accession number KX230053, KX230054) from EC 538828 and PK-472, respectively, one Bradyrhizobium daqingense (accession number KX230052) from PK-472, and one Bradyrhizobium kavangense (accession number MN197775) from Valder genotype having low trehalose. These strains, along with two native strains, viz., Bradyrhizobium japonicum (JF792425), Bradyrhizobium liaoningense (JF792426), and one commercial rhizobium, were studied for nodulation, leghaemoglobin, and N-fixation abilities on soybean under sterilized sand microcosm conditions in a completely randomized design. Among all the strains, D-4A (B. daqingense) followed by D-4B (B. liaoningense) was found to have significantly higher nodulation traits and acetylene reduction assay (ARA) activity when compared to other strains and commercial rhizobia. The bradyrhizobia isolates showed plant growth promotion traits such as indole acetic acid (IAA), exopolysaccharide (EPS), and siderophore production, phosphate-solubilizing potential, and proline accumulation. The novel species B. daqingense was reported for the first time from Indian soil and observed to be a potential candidate strain and should be evaluated for conferring drought tolerance in soybean under simulated stress conditions.(AU)

Crop rotation and inoculation increase soil bradyrhizobia population, soybean grain yields, and profitability.

Crop rotation and rhizobial inoculation are strategies to increase yield by means of organic matter addition and modulation of microbial diversity. However, the extent to which these agricultural practices change soil Bradyrhizobium populations, soybean grain yield, and economic benefits to farmers is unclear. Thus, this study aimed to evaluate the interaction between crop rotation and inoculation of soybean (Glycine max) cultivated in two contrasting soils (clayey and sandy soil) on biological nitrogen fixation components, grain yields, and profits. Field experiments with a three-year crop rotation system were carried out to compare effects of inoculation and crop rotations on soil chemical attributes, bradyrhizobia most probable number (MPN) and diversity, soybean nodulation, grain yield, and economic indicators of inoculation in different crop rotations. The crop rotation did not affect the soil MPN cells of bradyrhizobia, but the inoculation and the soil sampling time did, ranging from 3.61-4.42 to 4.40-4.82 in the sandy soil, while in the clayey soil they were from 5.19-6.34 to 6.61-7.14 in Log10 per g of soil with higher population after harvest of summer crops. In the clayey soil, crop rotation influenced soybean nodulation. The grain yield of inoculated soybean in the clayey soil was higher than that in the sandy soil. Soybean inoculation with Bradyrhizobium spp. increased the profitability of agricultural production systems by up to 45% in clayey soil and up to 7% in sandy soil.

Synergistic N2-fixation and salt stress mitigation in soybean through dual inoculation of ACC deaminase-producing Pseudomonas and Bradyrhizobium.

We investigated the potential dual application of two Bradyrhizobium strains (B. diazoefficiens USDA110 and B. ottawaense SG09) and plant growth-promoting bacteria, PGPB (Pseudomonas spp.: OFT2 and OFT5), to improve nodulation and N2-fixation in soybean plants. The growth-promoting effects of dual inoculation were observed on plant growth, physiology, and nodulation of soybean under normal conditions compared with plants individually inoculated with either USDA110 or SG09. Both OFT2 and OFT5 promoted N2-fixation by 11% and 56%, respectively, when dual inoculation with USDA110 and by 76% and 81%, respectively, when dual inoculation with SG09. Salinity stress significantly reduces soybean growth, physiology, nutrient uptake, nodulation, and N2-fixation. However, these adverse effects were attenuated by the dual inoculation of PGPB and rhizobia depending on the combination of inoculants. In particular, dual inoculation of PGPB with SG09 was more effective in enhancing the salt tolerance of soybean by reducing salt-induced ethylene production and improving nutrient uptake. However, no such effect was observed with the combined inoculation of USDA110 and OFT5. An effective symbiotic association between SG09 and two Pseudomonas bacteria can be considered a beneficial approach to improving the symbiotic efficiency of nodulation and mitigating salinity stress in soybeans.

Pioneering Desmodium spp. are nodulated by natural populations of stress-tolerant alpha- and beta-rhizobia.

The rhizobia-Desmodium (Leguminosae, Papilionoideae) symbiosis is generally described by its specificity with alpha-rhizobia, especially with Bradyrhizobium. Our study aimed to isolate rhizobia from root nodules of native D. barbatum, D. incanum, and D. discolor, collected in remnants of the biomes of Atlantic Forest and Cerrado in protected areas of the Paraná State, southern Brazil. Based on the 16S rRNA phylogeny, 18 out of 29 isolates were classified as Alphaproteobacteria (Bradyrhizobium and Allorhizobium/Rhizobium) and 11 as Betaproteobacteria (Paraburkholderia). Phylogeny of the recA gene of the alpha-rhizobia resulted in ten main clades, of which two did not group with any described rhizobial species. In the 16S rRNA phylogeny of the beta-rhizobia, Paraburkholderia strains from the same host and conservation unity occupied the same clade. Phenotypic characterization of representative strains revealed the ability of Desmodium rhizobia to grow under stressful conditions such as high temperature, salinity, low pH conditions, and tolerance of heavy metals and xenobiotic compounds. Contrasting with previous reports, our results revealed that Brazilian native Desmodium can exploit symbiotic interactions with stress-tolerant strains of alpha- and beta-rhizobia. Stress tolerance can highly contribute to the ecological success of Desmodium in this phytogeographic region, possibly relating to its pioneering ability in Brazil. We propose Desmodium as a promising model for studies of plant-rhizobia interactions.

Two novel symbiovars of Bradyrhizobium yuanmingense, americaense and caribense, the symbiovar tropici of Bradyrhizobium pachyrhizi and the symbiovar cajani of Bradyrhizobium cajani are microsymbionts of the legume Cajanus cajan in Dominican Republic.

Cajanus cajan L. (guandul) is commonly cultivated in Dominican Republic where this legume is a subsistence crop. Here we identified through MALDI-TOF MS several rhizobial strains nodulating C. cajan in two Dominican locations as Bradyrhizobium yuanmingense. The phylogenetic analysis of recA and glnII housekeeping genes showed that these strains belong to a wide cluster together with the type strain of B. yuanmingense and other C. cajan nodulating strains previously isolated in Dominican Republic. The comparison of genomes from strains representative of different lineages within this cluster support the existence of several genospecies within B. yuanmingense, which is the major microsymbiont of C. cajan in Dominican Republic where it is also nodulated by Bradyrhizobium cajani and Bradyrhizobium pachyrhizi. The analysis of the symbiotic nodC gene showed that the C. cajan nodulating strains from the B. yuanmingense complex belong to two clusters with less than 90% similarity between them. The strains from these two clusters showed nodC gene similarity values lower than 90% with respect to the remaining Bradyrhizobium symbiovars and then they correspond to two new symbiovars for which we propose the names americaense and caribense. The results of the nodC gene analysis also showed that C. cajan is nodulated by the symbiovar tropici, which has been found by first time in this work within the species Bradyrhizobium pachyrhizi. These results confirmed the high promiscuity degree of C. cajan, which is also nodulated by the symbiovar cajani of Bradyrhizobium cajani in Dominican Republic.

Legume-nodulating rhizobia are widespread in soils and plants across the island of O'ahu, Hawai'i.

Legumes and their interaction with rhizobia represent one of the most well-characterized symbioses that are widespread across both natural and agricultural environments. However, larger distribution patterns and host associations on isolated Pacific islands with many native and introduced hosts have not been well-documented. Here, we used molecular and culturing techniques to characterize rhizobia from soils and 24 native and introduced legume species on the island of O'ahu, Hawai'i. We chose two of these isolates to inoculate an endemic legume tree, Erythina sandwicensis to measure nodulation potentials and host benefits. We found that all rhizobia genera can be found in the soil, where only Cupriavidus was found at all sites, although at lower abundance relative to other more common genera such as Rhizobium (and close relatives), Bradyzhizobium, and Devosia. Bradyrhizobium was the most common nodulator of legumes, where the strain Bradyrhizobium sp. strain JA1 is a generalist capable of forming nodules on nine different host species, including two native species. In greenhouse nursery inoculations, the two different Bradyrhizobium strains successfully nodulate the endemic E. sandwicensis; both strains equally and significantly increased seedling biomass in nursery inoculations. Overall, this work provides a molecular-based framework in which to study potential native and introduced rhizobia on one of the most isolated archipelagos on the planet.

Independent Component Analysis Reveals the Transcriptional Regulatory Modules in Bradyrhizobium diazoefficiens USDA110.

The dynamic adaptation of bacteria to environmental changes is achieved through the coordinated expression of many genes, which constitutes a transcriptional regulatory network (TRN). Bradyrhizobium diazoefficiens USDA110 is an important model strain for the study of symbiotic nitrogen fixation (SNF), and its SNF ability largely depends on the TRN. In this study, independent component analysis was applied to 226 high-quality gene expression profiles of B. diazoefficiens USDA110 microarray datasets, from which 64 iModulons were identified. Using these iModulons and their condition-specific activity levels, we (1) provided new insights into the connection between the FixLJ-FixK2-FixK1 regulatory cascade and quorum sensing, (2) discovered the independence of the FixLJ-FixK2-FixK1 and NifA/RpoN regulatory cascades in response to oxygen, (3) identified the FixLJ-FixK2 cascade as a mediator connecting the FixK2-2 iModulon and the Phenylalanine iModulon, (4) described the differential activation of iModulons in B. diazoefficiens USDA110 under different environmental conditions, and (5) proposed a notion of active-TRN based on the changes in iModulon activity to better illustrate the relationship between gene regulation and environmental condition. In sum, this research offered an iModulon-based TRN for B. diazoefficiens USDA110, which formed a foundation for comprehensively understanding the intricate transcriptional regulation during SNF.

Bradyrhizobium xenonodulans sp. nov. isolated from nodules of Australian Acacia species invasive to South Africa.

A genealogical concordance approach was used to delineate strains isolated from Acacia dealbata and Acacia mearnsii root nodules in South Africa. These isolates form part of Bradyrhizobium based on 16S rRNA sequence similarity. Phylogenetic analysis of six housekeeping genes (atpD, dnaK, glnII, gyrB, recA and rpoB) confirmed that these isolates represent a novel species, while pairwise average nucleotide identity (ANIb) calculations with the closest type strains (B. cosmicum 58S1T, B. betae PL7HG1T, B. ganzhouense CCBAU 51670 T, B. cytisi CTAW11T and B. rifense CTAW71T) resulted in values well below 95-96%. We further performed phenotypic tests which revealed that there are high levels of intraspecies variation, while an additional analysis of the nodA and nifD loci indicated that the symbiotic loci of the strains are closely related to those of Bradyrhizobium isolates with an Australian origin. Strain 14ABT (=LMG 31415 T = SARCC-753 T) is designated as the type strain of the novel species for which we propose the name Bradyrhizobium xenonodulans sp. nov.

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

Effects of different remediation methods on phosphorus transformation and availability.

The effects of different heavy metal pollution remediation methods on soil nutrient transformation and soil health remain unclear. In this study, the effects of phytoextraction (PE) and passivation remediation (PR) on Cd-polluted soil phosphorus transformation and availability were compared by pot experiment. The results showed that PE significantly reduced the concentrations of total and available Cd (both H2O-Cd and DTPA-Cd) in soil, PR also decreased available Cd content but had no significant effect on total Cd content. PE slightly increased soil pH and NH4+-N content, while PR significantly increased soil pH, NO3--N and AK content. PE promoted the conversion of stable P (including HCl-Pi and residual-Pt), and increased the content of labile P (including H2O-Pi, NaHCO3-Pi and NaHCO3-Po) and the proportion of moderately labile P (including NaOH-Pi and NaOH-Po), while PR showed the opposite trend. PE showed a higher soil phoC gene abundance and acid phosphatase (ACP) activity, while PR showed a higher phoD gene copies and alkaline phosphatase (ALP) activity. Soil bacteria and phoD-harboring bacteria community was significantly affected by remediation methods and soil types. Compared with PR, PE reduced phoD-harboring bacterial diversity but significantly increased the abundance of genera associated with P dissolution (Streptomyces) and P conversion (Bradyrhizobium and Frankia), both of which were significantly positively correlated with labile P or moderately labile P. In general, compared with PR, PE can effectively remove soil Cd pollution, while maintaining a higher content of labile P and a higher proportion of moderately labile P, which can be considered as a green and sustainable remediation strategy conducive to soil quality.