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Siderophore Synthesis Ability of the Nitrogen-Fixing Bacterium (NFB) GXGL-4A is Regulated at the Transcriptional Level by a Transcriptional Factor (trX) and an Aminomethyltransferase-Encoding Gene (amt)

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Abstract

Kosakonia radicincitans GXGL-4A, a gram-negative nitrogen-fixing (NF) bacterial strain is coated with a thick capsulatus on the surface of cell wall, which becomes a physical barrier for exogenous DNA to enter the cell, so the operation of genetic transformation is difficult. In this study, an optimized Tn5 transposon mutagenesis system was established by using a high osmotic HO-1 medium combined with the electroporation transformation. Eventually, a mutant library containing a total of 1633 Tn5 insertional mutants were established. Of these mutants, the mutants M81 and M107 were found to have an enhanced capability to synthesize siderophore through the CAS agar plate assay and the spectrophotometric determination. The bacterial cells of two mutants were applied in cucumber growth-promoting experiment. Cucumber seedlings treated with M81 and M107 cells had a significant increase in biomass including seedling height, seedling fresh weight, root fresh weight, and root length. The whole genome sequencing of the mutants M81 and M107 showed that the integration sites of Tn5 transposon element were located in MmyB-like helix-turn-helix transcription regulator (locus tag: A3780_19720, trX) and aminomethyltransferase-encoding genes (locus tag: A3780_01680, amt) in the genome of GXGL-4A, respectively. The ability of siderophore synthesis of the target mutants was improved by Tn5 insertion mutagenesis, and the mutants obtained showed a good plant growth-promoting effect when applied to the cucumber seedlings. The results suggest that the identified functional genes regulates the biosynthesis of siderophore in azotobacter GXGL-4A, and the specific mechanism needs to be further investigated.

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Data Availability

The whole genomic sequence of nitrogen-fixing bacterium GXGL-4A has been deposited in GenBank database, and the nucleotide sequences of trX and amt genes mentioned in this study are available under the genomic accession number CP015113. The mutants M81 and M107 have been stored in the China General Microbiological Culture Collection Center (CGMCC) with the preservation numbers CGMCC 24400 and CGMCC 24401, respectively.

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Acknowledgements

The authors thank professor Gong-You Chen (Shanghai Jiao Tong University) for his constructive discussion on experimental design.

Funding

This work was financially supported by the National Natural Science Foundation of China (Grant No. 31870496).

Author information

Authors and Affiliations

  1. Department of Resources and Environment, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China

    Bao-Yun Feng, Meng-Ting Zhang, Guo-Xun Su & Yun-Peng Chen

  2. School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, China

    Yu-Qing Bao

  3. College of Geography and Environmental Sciences, Zhejiang Normal University, Jinhua, 321004, China

    Yu Xu

  4. Shanghai Yangtze River Delta Eco-Environmental Change and Management Observation and Research Station, Ministry of Science and Technology, Shanghai, 200093, China

    Yun-Peng Chen

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Contributions

The establishments of Tn5 transposon mutagenesis system and mutant library were carried out by BYF. The screening and identification of mutants were conducted by MTZ and GXS, and the qRT-PCR and Phylogenetic analysis were completed by YQB. The plant growth promotion experiment, CAS agar plate assay and data analysis were performed by YX and BYF. YPC conceived, designed, and supervised the whole project.

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Correspondence to Yun-Peng Chen.

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Supplementary Information

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Supplementary file1 (DOCX 21 KB)

Fig. S1

Morphology of the NFB GXGL-4A cultured in different media (17500×). a LB (10 g/L NaCl); b LB (15 g/L NaCl); c HO-2 (7.5 g/L CaCl2). Supplementary file2 (TIF 1595 KB)

Fig. S2

Quantitative PCR (qRT-PCR) analysis of the target genes in mutants M81 a and M107 b The rpoB gene of GXGL-4A was used as an internal reference gene. Relative transcriptional levels of the target genes were calculated relative to rpoB using the ΔΔCT method, where CT is the threshold cycle. Double asterisks indicate that there is an extremely significant difference in transcription level between two samples (p<0.01), and a single asterisk denotes a significant difference in transcription level between two samples (p<0.05). The bars in diagrams mean standard deviations (SD). Analysis of variance (ANOVA) was conducted based on the data from three replicates using the software IBM SPSS Statistics (version 17.0). Supplementary file3 (PNG 14 KB)

Supplementary file4 (PNG 12 KB)

Fig. S3

Transcriptional levels of four functional genes, sdm a, acps b, bks c and atpbp d flanking the Tn5 transposon integration sites of the mutants M81 and M107. The bars mean standard deviations (SD) in these diagrams, and the analysis of variance (ANOVA) was carried out based on the data from three duplications using the software IBM SPSS Statistics (version 17.0). Supplementary file5 (TIF 11 KB)

Supplementary file6 (TIF 11 KB)

Supplementary file7 (TIF 10 KB)

Supplementary file8 (TIF 10 KB)

Fig. S4

PCR cloning of the trX and amt genes in the genome of GXGL-4A. M: DNA marker DL2000; Lane 1, PCR amplicon of the trX gene; Lane 2, PCR amplicon of the amt gene. Supplementary file9 (TIF 437 KB)

Fig. S5

CAS agar plate test in the functional complementarity and gene overexpression experiments. a the mutant M81 and original strain GXGL-4A; b the mutant M107 and original strain GXGL-4A; c the diameters of siderophore rings produced by the cells of GXGL-4A, 4A::trX and M81::trX (cm); d the diameters of siderophore rings produced by the cells of GXGL-4A, 4A::amt and M107::amt (cm). All the experiments were duplicated three times. Supplementary file10 (TIFF 2320 KB)

Supplementary file11 (TIFF 2320 KB)

Supplementary file12 (PNG 14 KB)

Supplementary file13 (PNG 14 KB)

Fig. S6

The phylogenetic relationship between K. radicincitans GXGL-4A and other Kosakonia or Escherichia coli strains. The phylogenetic trees were constructed based on the nucleotide acid sequences of trX a and amt b genes by using the Neighbor-Joining method. The bootstrap consensus tree inferred from 2000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) are shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Evolutionary analyses were conducted in MEGA7. T: type strain; E: the genus Escherichia; K: the genus Kosakonia. Supplementary file14 (PNG 27 KB)

Supplementary file15 (PNG 26 KB)

Fig. S7

Growth curve comparison of the mutants M81, M107 and the wild-type strain GXGL-4A. The experiment was repeated three times. Supplementary file16 (PNG 21 KB)

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Feng, BY., Zhang, MT., Su, GX. et al. Siderophore Synthesis Ability of the Nitrogen-Fixing Bacterium (NFB) GXGL-4A is Regulated at the Transcriptional Level by a Transcriptional Factor (trX) and an Aminomethyltransferase-Encoding Gene (amt). Curr Microbiol 79, 369 (2022). https://doi.org/10.1007/s00284-022-03080-4

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