## 2 Results and Analysis
### sRNAs were Predicted with Potential Roles in the Adaptation to Nitrogen Starvation  
The transcriptome sequencing reads can be assembled and compared with the annotated gene model to determine new unknown transcript regions, untranslated regions (UTRs), regulatory small non-coding RNAs (sRNAs), and single nucleotide polymorphisms (SNPs). Compared with the Nr library by Blastx (e-value = 1 \(\times\) 10\({}^{-5}\)), a total of 458 new transcripts were predicted in the FBS135 genome (Supplementary Table S2). Based on the transcription information, 2936 5'-UTRs and 2915 3'-UTRs, and 120 sRNAs (50-492 bp) were predicted (Supplementary Table S3). In addition, the analysis revealed 18 sites with SNPs events and 74 sites with InDels events which were detected in the six samples (Supplementary Table S4). When NH4 Cl was added, approximately half the number (59) of sRNAs were differentially expressed, with 36 of them upregulated and 23 downregulated (Supplementary Table S5). Among the differentially expressed sRNAs, sRNA00124 had  
Figure 3: Different expression patterns of _P. eucalypt_FBS135 DEGs without (A) and with (AN) NH4\({}^{+}\) addition. (**A–D**) Expression patterns of samples in different clusters. The samples from the A and the AN group harvested at 36, 48, and 60 h were numbered A_1, A_2, A_3, and AN_1, AN_2, AN_3, respectively. the highest upregulation of 194-fold, and sRNA00261 had the highest downregulation of 65-fold. However, both of them remain unknown in function. So far non-coding regulatory RNAs have not been fully studied in most prokaryotes. These differentially expressed sRNAs in FBS135 may play an important role in the adaptation to nitrogen limitation and deserve further investigation in the future. Figure 4: GO and KEGG enrichment analysis for the DEGs of _P. eucalypt_FBS135 under nitrogen deficiency. (**A**) 30 most enriched GO terms in the group of biological processes (BP), cellular component (CC), and molecular function (MF) under nitrogen deficiency. (**B**,**C**) 20 most enriched KEGG pathways of the upregulated (**B**) and downregulated (**C**) genes under nitrogen deficiency. Gene Ratio represents the ratio of the number of DEGs annotated to the KEGG pathway to the total number of DEGs. Five Tn5 Insertion Mutants of P. eucalyptpti FBS135 Were Identified to Be Associated with Nitrogen Utilization  
In order to identify key genes related to the adaption of _P. eucalypt_ FBS135 to nitrogen scarcity, we constructed a transposon mutant library using the method of three-parent hybrid transformation. After screening 5500 transport mutant strains, we discovered five strains that could not grow or grew poorly on nitrogen-free Ashby plates. We named them FBS216, FBS217, FBS218, FBS219, and FBS220. Similar to the FBS135 wild-type, the five mutant strains grew normally on LB agar plates forming yellow smooth colonies (Figure 5A). By contrast, FBS219 could not grow on nitrogen-free Ashby plates at all (Figure 5B). FBS216, FBS217, FBS218, and FBS220 could grow on the nitrogen-free plates after continuous streaking over 10 generations, however, their colonies were significantly reduced. Among the four of them, FBS218 grew the weakest, forming barely visible colonies (Figure 5B). Figure 5: Five Tn5 insertion mutants of _P. eucalypt_ FBS135 were defective in growth upon nitrogen scarcity. The growth status of the FBS135 wild-type and its Tn5 insertion mutants were grown on (**A**) LB medium plates and (**B**) nitrogen-free Ashby medium plates. (**C–F**) Confirmation via PCR of the products of the Tn5 insertion sites in four mutants. Lane M is the DNA marker DL2501 (Generay Biotech Co., Ltd., Shanghai, China). Using reverse PCR and random primer PCR technology, we successfully determined the Tn5 insertion sites in four mutants (FBS216, FBS218, FBS219, and FBS220) but failed to determine the Tn5 position in FBS217. In FBS219, Tn5 was inserted in the gene encoding serine acetyltransferase (SAT); in FBS216 and FBS220, Tn5 was inserted in the different positions of the gene for the host-specific protein J.