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Aims: Some studies have indicated that the alterations in cellular morphology induced by selenite [Se(Ⅳ)] may be attributed to its inhibitory effects on cell division. However, whether the genes associated with cell division are implicated in Se(Ⅳ) metabolism remains unclear., Methods and Results: The ftsK gene in Rahnella aquatilis HX2 was mutated with an in-frame deletion strategy. The ftsK mutation strongly reduced the tolerance to selenite [Se(Ⅳ)] and the production of red elemental selenium [Se(0)] in R. aquatilis HX2, and this effect could not be attributed solely to the inhibition of cell growth. Deleting the ftsK gene also resulted in a significant decrease in bacterial growth of R. aquatilis HX2 during both exponential and stationary phases. The deletion of ftsK inhibited cell division, resulting in the development of elongated filamentous cells. Furthermore, the loss-of-function of FtsK significantly impacted the expression of seven genes linked to cell division and Se(Ⅳ) metabolism by at least 2-fold, as unveiled by real-time quantitative PCR (RT-qPCR) under Se(Ⅳ) treatment., Conclusions: These findings suggest that FtsK is associated with Se(Ⅳ) tolerance and Se(0) generation and is a key player in coordinating bacterial growth and cell morphology in R. aquatilis HX2., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Background: Biogenic selenium nanoparticles (SeNPs) show numerous advantages including their high stability, low toxicity, and high bioactivity. While metabolism of SeNPs remains not well studied and need more investigation to reveal the process., Purpose: The objective of the study was to investigate the relationship between nitrate reductase and selenite reduction in Rahnella aquatilis HX2, characterize the properties of HX2 produced SeNPs, and explore their potential applications, particularly their anticancer activity., Procedures: Selenium species were measured by high-performance liquid chromatography coupled to inductively coupled plasma - Mass spectrometry (HPLC-ICP-MS). Transcription level of nitrate reductase was determined by Real-time quantitative PCR. Morphology, particle size, crystal structure and surface chemistry of SeNPs were determined by electron microscopy, dynamic light scattering method, Raman scattering, X-ray photoelectron spectroscopy, respectively. Anti cancer cell activity was measured by CCK-8 assay., Main Findings: SeNP production in R. aquatilis HX2 was correlated with the cell growth. The products of selenite reduction in HX2 detected by HPLC-ICP-MS included SeNPs, selenocysteine (SeCys), Se-Methylselenocysteine (MeSeCys), and 7 unknown compounds. Nitrate addition experiments suggested the involvement of nitrate reductase in selenite reduction in HX2. Both the cellular membrane and cytoplasm of HX2 exhibited selenite-reducing ability, indicating that membrane-associated nitrate reductase was not the sole selenite reductase in HX2. Characterization of the biogenic SeNPs revealed a spherical morphology and amorphous structure of them. Surface chemistry analysis implicated the binding of extracellular polymeric substances to the biogenic SeNPs, and the presence of Se 0 , Se 2- , and electron-rich Se atoms on the surface of SeNPs. Finally, the IC50 values of the biogenic SeNPs were 36.49 μM for HepG2 and 3.70 μM for HeLa cells., Conclusions: The study first revealed that the nitrate reductase is involving in selenite reduction in R. aquatilis HX2. The biogenic SeNPs coordinated with organic substances in the surface. And SeNPs produced by R. aquatilis HX2 showed excellent anticancer activities on HepG2 and HeLa cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Conflict of Interest All authors declare that there is no conflict of interest in this work., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Aims: Rahnella aquatilis HX2, a promising plant growth-promoting rhizobacterium (PGPR) in the field, contains genes homologous to the PhoP/PhoQ two-component regulatory system. Although this system regulates stress response in numerous pathogens, PhoP/PhoQ characterization in a PGPR has not received in-depth exploration., Methods and Results: The phoQ gene was mutated in strain HX2 using an in-frame deletion strategy. Compared to the wild type, the phoQ mutant exhibited increased sensitivity to acidic conditions (pH 4.0) in a chemically defined medium and in mild acidic natural soil (pH 5.7). The phoQ mutant also exhibited increased swimming motility under acidic conditions. Acid resistance was restored in the mutant by introducing the phoQ gene on a plasmid. Three acid resistance genes, add, cfa, and fur were downregulated significantly, whereas the chaperone encoding gene, dnak, was upregulated when the phoQ mutant was exposed to acid stress., Conclusions: This study suggested that the PhoP/PhoQ system positively regulates the acid resistance of R. aquatilis HX2., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Microbial transformation of selenite [Se(IV)] to elemental selenium nanoparticles (SeNPs) is known to be an important process for removing toxic soluble selenium (Se) oxyanions and recovery of Se from the environment as valuable nanoparticles. However, the mechanism of selenite uptake by microorganisms, the first step through which Se exerts its cellular function, remains not well studied. In this study, the effects of selenite concentration, time, pH, metabolic inhibitors, and anionic analogues on selenite uptake in Rahnella aquatilis HX2 were investigated. Selenite uptake by R . aquatilis HX2 was concentration- and time-dependent, and its transport activity was significantly dependent on pH. In addition, selenite uptake in R . aquatilis HX2 was significantly inhibited by the aquaporin inhibitor AgNO 3 and sulfite (SO 3 2- ), and partially inhibited by carbonyl cyanide m -chlorophenyl hydrazone (CCCP) and 2,4-dinitrophenol (2,4-DNP) treatments. Three mutants with in-frame deletions of aqp Z, glp F, and nha A genes were constructed. The transport assay showed that the water channel protein AqpZ, and not GlpF, was a key channel of selenite uptake by R . aquatilis HX2, and sulfite and selenite had a common uptake pathway. In addition, the Na + /H + antiporter NhaA is also involved in selenite uptake in R . aquatilis HX2.

Microorganisms play a critical role in the reduction of the more toxic selenite and selenate to the less toxic elemental selenium. However, the assembly process and stability of selenium nanoparticles (SeNPs) remain understudied. The plant growth-promoting rhizobacterium Rahnella aquatilis HX2 can reduce selenite to biogenic SeNPs (BioSeNPs). Two main proteins, namely flagellin FliC and porin OmpF were identified in the BioSeNPs. The fliC and ompF gene mutation experiments demonstrated that the FliC and OmpF could control the assembly of BioSeNPs in vivo. At the same time, the expressed and purified FliC and OmpF could control the assembly of SeNPs in vitro. BioSeNPs produced by R. aquatilis HX2 exhibited high stability under various ionic strengths, while the chemically synthesized SeNPs (CheSeNPs) showed a high level of aggregation. The in vitro experiments verified that FliC and OmpF could prevent the aggregation of the CheSeNPs under various ionic strengths. This work reports the preparation of highly stable BioSeNPs produced by strain R. aquatilis HX2 and verifies that FliC and OmpF both could control the assembly and stability of BioSeNPs. BioSeNPs with high stability could be suitable as nutritional supplement to remedy selenium deficiency and in nanomedicine applications., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The RNA chaperone, Hfq, is a global post-transcriptional regulator that plays an important role in regulating pleiotropic functions, such as cell growth and motility, stress tolerance, and virulence to host, in many Gram-negative bacteria. This study examined the functional roles of Hfq in Rahnella aquatilis HX2, a plant beneficial, selenium nanoparticles (SeNPs)-producing soil bacterium. A mutant HX2∆hfq with an in-frame deletion within the hfq gene in R. aquatilis HX2 was constructed and tested for various phenotypic features. Bacterial growth, motility, selenite reduction, and SeNPs production were compared between the mutant, the wild-type, and the complementation strain. The hfq gene deletion delayed the growth of strain HX2, with a lower bacterial population during the stationary phase, and significantly impaired the swimming motility of the bacterium, showing a smaller motility ring on the plate. The hfq mutation also dramatically declined microbial-induced reduction of selenite and SeNPs production in HX2, which was independent of cell growth. The introduction of a trans-expressed hfq gene into HX2∆hfq for complementation completely restored impacted phenotypes. In addition, reverse transcription real-time quantitative PCR (RT-qPCR) analysis revealed that the expression of ten genes involved in bacterial growth and survival, motility and chemotaxis, and selenite or seleno-compound metabolism were influenced by Hfq loss-of-function by at least two-fold. Six genes including two involved in SeNPs production were positively regulated by hfq, while other four genes were negatively regulated. Homolog search suggested that the rprA gene might encode a small RNA regulated by Hfq in R. aquatilis HX2. Overall, the present study provides novel information about the function of Hfq and the regulation of bacterial biosynthesis of SeNPs.

Rahnella aquatilis HX2 was isolated from Beijing vineyard soil and used as a plant growth-promoting rhizobacterium in the field. Previous studies have shown that it has a broad in vitro antimicrobial spectrum and could inhibit a variety of plant pathogenic bacteria and fungi. In this study, a gene, acdS, encoding 1-aminocyclopropane-1-carboxylic acid-deaminase was disrupted by in-frame deletion in the HX2 strain. Compared to the wild-type, the acdS-mutant had higher rates of nitrogen fixation, reduced indole-3-acetic acid production, lowered efficacy as a biological control agent against the grape crown gall pathogen Agrobacterium vitis. Under saline stress conditions, plant height, above-ground fresh weight, root fresh weight of corn plants were increased by treatment with HX2 but this increase was compromised by the disruption of acdS gene. Our data confirmed the function of HX2 on plant growth promoting and demonstrated that acdS gene plays a major role in its PGPR activities., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rahnella aquatilis HX2 (proteobacteria) shows tolerance to selenium (Se). The minimum inhibitory concentrations of selenomethionine (Se-Met), selenite [Se (IV)], and selenate [Se (VI)] to HX2 are 4.0, 85.0, and 590.0 mM, respectively. HX2 shows the ability to reduce Se (IV) and Se (VI) to elemental Se nanoparticles (SeNPs). The maximum production of SeNPs by HX2 strain is 1.99 and 3.85 mM in Luria-Bertani (LB) broth with 5 mM Se (IV) and 10 mM Se (VI), respectively. The morphology of SeNPs and cells were observed by transmission electron microscope, environmental scanning electron microscope, and selected area electric diffraction detector. Spherical SeNPs with amorphous structure were found in the cytoplasm, membrane, and exterior of cells. Morphological variations of the cell membrane were further confirmed by the release of cellular materials absorbed at 260 nm. Flagella were inhibited and cell sizes were 1.8-, 1.6-, and 1.2-fold increases with the Se-Met, Se (VI), and Se (IV) treatments, respectively. The real-time quantitative PCR analysis indicated that some of the genes controlling Se metabolism or cell morphology, including cysA, cysP, rodA, ZntA, and ada, were significantly upregulated, while grxA, fliO, flgE, and fliC genes were significantly downregulated in those Se treatments. This study provided novel valuable information concerning the cell morphology along with biological synthesis process of SeNPs in R. aquatilis and demonstrated that the strain HX2 could be applied in both biosynthesis of SeNPs and in management of environmental Se pollution.

Background: Rahnella aquatilis is ubiquitous and its certain strains have the applicative potent as a plant growth-promoting rhizobacteria. R. aquatilis HX2 is a biocontrol agent to produce antibacterial substance (ABS) and showed efficient biocontrol against crown gall caused by Agrobacterium vitis on sunflower and grapevine plants. The regulatory network of the ABS production and biocontrol activity is still limited known., Methodology/principal Findings: In this study, a transposon-mediated mutagenesis strategy was used to investigate the regulators that involved in the biocontrol activity of R. aquatilis HX2. A 366-nt noncoding RNA CsrB was identified in vitro and in vivo, which regulated ABS production and biocontrol activity against crown gall on sunflower plants, respectively. The predicted product of noncoding RNA CsrB contains 14 stem-loop structures and an additional ρ-independent terminator harpin, with 23 characteristic GGA motifs in the loops and other unpaired regions. CsrB is required for ABS production and biocontrol activity in the biocontrol regulation by a two-component regulatory system BarA/UvrY in R. aquatilis HX2., Conclusion/significance: The noncoding RNA CsrB regulates BarA-dependent ABS production and biocontrol activity in R. aquatilis HX2. To the best of our knowledge, this is the first report of noncoding RNA as a regulator for biocontrol function in R. aquatilis.

Rahnella aquatilis strain HX2 has the ability to promote maize growth and suppress sunflower crown gall disease caused by Agrobacterium vitis, A. tumefaciens, and A. rhizogenes. Pyrroloquinoline quinone (PQQ), a cofactor of aldose and alcohol dehydrogenases, is required for the synthesis of an antibacterial substance, gluconic acid, by HX2. Mutants of HX2 unable to produce PQQ were obtained by in-frame deletion of either the pqqA or pqqB gene. In this study, we report the independent functions of pqqA and pqqB genes in relation to PQQ synthesis. Interestingly, both the pqqA and pqqB mutants of R. aquatilis eliminated the ability of strain HX2 to produce antibacterial substance, which in turn, reduced the effectiveness of the strain for biological control of sunflower crown gall disease. The mutation also resulted in decreased mineral phosphate solubilization by HX2, which reduced the efficacy of this strain as a biological fertilizer. These functions were restored by complementation with the wild-type pqq gene cluster. Additionally, the phenotypes of HX2 derivatives, including colony morphology, growth dynamic, and pH change of culture medium were impacted to different extents. Our findings suggested that pqqA and pqqB genes individually play important functions in PQQ biosynthesis and are required for antibacterial activity and phosphorous solubilization. These traits are essential for R. aquatilis efficacy as a biological control and plant growth promoting strain. This study enhances our fundamental understanding of the biosynthesis of an environmentally significant cofactor produced by a promising biocontrol and biological fertilizer strain.

Rahnella aquatilis strain HX2 is a plant growth-promoting, disease-suppressive rhizobacterium that was isolated from a vineyard soil in Beijing, China. Here, we report the genome sequence of this strain, which provides a valuable resource for future research examining the mechanisms of traits associated with plant growth promotion and biocontrol.