Dataset Information

A Novel Module Promotes Horizontal Gene Transfer in Azorhizobium caulinodans ORS571.

ABSTRACT: Azorhizobium caulinodans ORS571 contains an 87.6 kb integrative and conjugative element (ICE^Ac) that conjugatively transfers symbiosis genes to other rhizobia. Many hypothetical redundant gene fragments (rgfs) are abundant in ICE^Ac, but their potential function in horizontal gene transfer (HGT) is unknown. Molecular biological methods were employed to delete hypothetical rgfs, expecting to acquire a minimal ICE^Ac and consider non-functional rgfs as editable regions for inserting genes related to new symbiotic functions. We determined the significance of rgf4 in HGT and identified the physiological function of genes designated rihF1a (AZC_3879), rihF1b (AZC_RS26200), and rihR (AZC_3881). In-frame deletion and complementation assays revealed that rihF1a and rihF1b work as a unit (rihF1) that positively affects HGT frequency. The EMSA assay and lacZ-based reporter system showed that the XRE-family protein RihR is not a regulator of rihF1 but promotes the expression of the integrase (intC) that has been reported to be upregulated by the LysR-family protein, AhaR, through sensing host's flavonoid. Overall, a conservative module containing rihF1 and rihR was characterized, eliminating the size of ICE^Ac by 18.5%. We propose the feasibility of constructing a minimal ICE^Ac element to facilitate the exchange of new genetic components essential for symbiosis or other metabolic functions between soil bacteria.

SUBMITTER: Li M

PROVIDER: S-EPMC9601964 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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A Novel Module Promotes Horizontal Gene Transfer in Azorhizobium caulinodans ORS571.

Li Mingxu M Chen Qianqian Q Wu Chuanhui C Li Yiyang Y Wang Sanle S Chen Xuelian X Qiu Bowen B Li Yuxin Y Mao Dongmei D Lin Hong H Yu Daogeng D Cao Yajun Y Huang Zhi Z Cui Chunhong C Zhong Zengtao Z

Genes 20221019 10

Azorhizobium caulinodans ORS571 contains an 87.6 kb integrative and conjugative element (ICEAc) that conjugatively transfers symbiosis genes to other rhizobia. Many hypothetical redundant gene fragments (rgfs) are abundant in ICEAc, but their potential function in horizontal gene transfer (HGT) is unknown. Molecular biological methods were employed to delete hypothetical rgfs, expecting to acquire a minimal ICEAc and cons ...[more]

PMID: 36292780

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Project description:The genome of Azorhizobium caulinodans ORS571 encodes two chemotaxis response regulators: CheY1 and CheY2. cheY1 is located in a chemotaxis cluster (cheAWY1BR), while cheY2 is located 37 kb upstream of the cheAWY1BR cluster. To determine the contributions of CheY1 and CheY2, we compared the wild type (WT) and mutants in the free-living state and in symbiosis with the host Sesbania rostrata Swim plate tests and capillary assays revealed that both CheY1 and CheY2 play roles in chemotaxis, with CheY2 having a more prominent role than CheY1. In an analysis of the swimming paths of free-swimming cells, the ΔcheY1 mutant exhibited decreased frequency of direction reversal, whereas the ΔcheY2 mutant appeared to change direction much more frequently than the WT. Exopolysaccharide (EPS) production in the ΔcheY1 and ΔcheY2 mutants was lower than that in the WT, but the ΔcheY2 mutant had more obvious EPS defects that were similar to those of the ΔcheY1 ΔcheY2 and Δeps1 mutants. During symbiosis, the levels of competitiveness for root colonization and nodule occupation of ΔcheY1 and ΔcheY2 mutants were impaired compared to those of the WT. Moreover, the competitive colonization ability of the ΔcheY2 mutant was severely impaired compared to that of the ΔcheY1 mutant. Taken together, the ΔcheY2 phenotypes are more severe than the ΔcheY1 phenotype in free-living and symbiotic states, and that of the double mutant resembles the ΔcheY2 single-mutant phenotype. These defects of ΔcheY1 and ΔcheY2 mutants were restored to the WT phenotype by complementation. These results suggest that there are different regulatory mechanisms of CheY1 and CheY2 and that CheY2 is a key chemotaxis regulator under free-living and symbiosis conditions.IMPORTANCEAzorhizobium caulinodans ORS571 is a motile soil bacterium that has the dual capacity to fix nitrogen both under free-living conditions and in symbiosis with Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis to chemoattractants derived from host roots promotes infection and subsequent nodule formation by directing rhizobia to appropriate sites of infection. In this work, we identified and demonstrated that CheY2, a chemotactic response regulator encoded by a gene outside the chemotaxis cluster, is required for chemotaxis and multiple other cell phenotypes. CheY1, encoded by a gene in the chemotaxis cluster, also plays a role in chemotaxis. Two response regulators mediate bacterial chemotaxis and motility in different ways. This work extends the understanding of the role of multiple response regulators in Gram-negative bacteria.

Project description:BackgroundSwitchgrass (Panicum virgatum L.) is an important biofuel crop that may contribute to replacing petroleum fuels. However, slow seedling growth and soil salinization affect the growth and development of switchgrass. An increasing number of studies have shown that beneficial microorganisms promote plant growth and increase tolerance to salinity stress. However, the feasibility of inoculating switchgrass with Azorhizobium caulinodans ORS571 to enhance the growth and salt tolerance of its seedlings is unclear. Our previous study showed that A. caulinodans ORS571 could colonize wheat (Triticum aestivum L.) and thereby promote its growth and development and regulate the gene expression levels of microRNAs (miRNAs).ResultsIn this study, we systematically studied the impact of A. caulinodans ORS571 on switchgrass growth and development and the response to salinity stress; we also studied the underlying mechanisms during these biological processes. Inoculation with A. caulinodans ORS571 significantly alleviated the effect of salt stress on seedling growth. Under normal conditions, A. caulinodans ORS571 significantly increased fresh plant weight, chlorophyll a content, protein content, and peroxidase (POD) activity in switchgrass seedlings. Under salt stress, the fresh weight, dry weight, shoot and root lengths, and chlorophyll contents were all significantly increased, and some of these parameters even recovered to normal levels after inoculation with A. caulinodans ORS571. Soluble sugar and protein contents and POD and superoxide dismutase (SOD) activities were also significantly increased, contrary to the results for proline. Additionally, A. caulinodans ORS571 may alleviate salt stress by regulating miRNAs. Twelve selected miRNAs were all upregulated to different degrees under salt stress in switchgrass seedlings. However, the levels of miR169, miR171, miR319, miR393, miR535, and miR854 were decreased significantly after inoculation with A. caulinodans ORS571 under salt stress, in contrast to the expression level of miR399.ConclusionThis study revealed that A. caulinodans ORS571 increased the salt tolerance of switchgrass seedlings by increasing their water content, photosynthetic efficiency, osmotic pressure maintenance, and reactive oxygen species (ROS) scavenging abilities and regulating miRNA expression. This work provides a new, creative idea for improving the salt tolerance of switchgrass seedlings.

Dataset Information

A Novel Module Promotes Horizontal Gene Transfer in Azorhizobium caulinodans ORS571.

Publications

A Novel Module Promotes Horizontal Gene Transfer in <i>Azorhizobium caulinodans</i> ORS571.

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