## 2 Results and Analysis
### sRNAs were Predicted with Potential Roles in the Adaptation to Nitrogen Starvation
Two Tn5 insertion sites were detected in FBS218, located in the RNA polymerase sigma factor RpoS gene and the phosphoribosylamine--glycine ligase gene. In order to confirm the positioning result, we designed primers targeting the flanking sequences of Tn5 insertion sites in each mutant for PCR detection independently. The results of agarose gel electrophoresis showed that the DNA bands of the PCR products of each mutant differed from those of the FBS135 wild-type by \(\sim\)1000 bp (Figure 5C-F), consistent with the length of the Tn5 transposon cassette (1066 bp). We further sequenced the PCR products, which showed complete consistency with the determined positions of Tn5. Functional Analysis of the Tn5-Disrupted Genes Revealed Their Potential in the Adaption of P. eucalyptFBS135 to Nitrogen Scarcity  
In order to understand the relationship of the disrupted genes by Tn5 to low nitrogen adaption, we analyzed their functions individually. The first attractive gene is the one interrupted by Tn5 in FBS219 (Figure 6A), which completely lost its ability to grow on a nitrogen-free solid medium. This gene is predicted to encode Ser acetyltransferase (SAT), which is responsible for the synthesis of O-acetylserine and critical for the biosynthesis of cysteine. Also, SAT is a key enzyme in the reaction process of sulfur assimilation. Through blast against the NCBI database, we found that this serine acetyltransferase of _P. eucalypt_FBS135 had 46.6% similarity to the NifP encoded in the nitrogen-fixing gene cluster of _Pseudanabena_ sp. ABRG5-3 (Figure 6B). NifP increases the synthesis rate of cysteine and methionine to optimize the expression activity of nitrogenase [43]. Although we have demonstrated that FBS135 cannot fix nitrogen, the presence of the SAT gene may contribute to its ability to adapt to extremely low nitrogen supply. The gene encoding for the host-specific protein J was also intriguing since it was the one disrupted by Tn5 in both FBS216 and FBS220 (Figure 6A). Tn5 was inserted at the front part and the middle part of this gene in FBS216 and FBS220, respectively. The full length of the gene was 3210 bp, and its specific function is not yet fully determined. At present, it is only known that the protein is usually located at the end of a phage structure. Multiple tail assembly proteins and tail fiber assembly proteins, as well as cor-protein and peptidase P60, were found to be encoded upstream and downstream of the gene. The relationship between the host-specific protein J and low nitrogen tolerance has not been investigated. Two insertion sites of Tn5 were found in the mutant strain FBS218 (Figure 6A,C). The first insertion site was in the gene for RNA polymerase sigma factor RpoS, which regulates the expression of genes in response to environmental stresses [44]. The second site in FBS218 inserted by Tn5 was the gene _purD_, which encodes phosphoribosamine-glycine ligase. This gene has an important role in the biosynthesis of IMP and thus is involved in purine metabolic processes. Notably, the _purD_ gene was not interrupted by the Tn5 transposon cassette, but the complete pSC137 plasmid sequence containing the Tn5 sequence (Figure 6A). This kind of insertion was verified via PCR and was not found in the other three mutants. Both _rpoS_ and _purD_ are important for the growth of FBS135, therefore, it remains unknown which gene is actually responsible for the failure of FBS218 to grow on nitrogen-free Ashby plates. Figure 6: Localizations of Tn5 inserts in four transposon mutants of _P. eucalypt_FBS135 and the functional analysis of the mutant FBS218. **(A)** A schematic overview of Tn5 insertion sites in the four transposon mutants; **(B)** comparison of amino acid sequences between the serine acetyltransferase (SAT) of FBS218 and NiH7 encoded in nitrogen fixation gene cluster. “**” indicates a completely identical residue; “:” indicates a particularly similar residue; and “.” indicates a weakly similar residue. **(C)** Confirmation via PCR products of dual Tn5 insertion sites in FBS218.