## 2 Results and Analysis
### P. eucalypti FBS135 Modulates Its Gene Transcription Adapting to Nitrogen Scarcity
Notably, in this group \(\sim\)18% of genes (64 out of 358) were enriched in the ABC transporter KEGG pathways, suggesting an enhanced transportation of substrates by _P. eucalypt_ FBS135 responding to nitrogen starvation. Cluster 2 contained 25 genes, and most of the genes were also related to ABC transporters. They had low expression in the absence of NH4\({}^{+}\) but high expression in the presence of NH4\({}^{+}\). Unlike cluster 1, in the absence of NH4\({}^{+}\) nitrogen, the expression level of the genes in this cluster showed an upward trend as culture time continued (Figure 3B). Cluster 3 contained 913 genes. In contrast to cluster 1, in this cluster the genes had low expression in the presence of nitrogen but high expression in the absence of nitrogen. However, similar to cluster 1, the expression under either condition showed a similar level across the three time points (Figure 3C). In cluster 3, \(\sim\)23.5% of genes (65 out of 276) were enriched in the ribosome KEGG pathway. Ribosomes are the cellular factories responsible for producing proteins. Therefore, a large portion of downregulated genes were related to this pathway, which indicated a strongly reduced protein-making activity due to nitrogen limitation. Cluster 4 contained 166 genes, which were expressed at a similar level in the absence of NH4\({}^{+}\) nitrogen. But in the presence of NH4\({}^{+}\), their expression level increased by approximately 30 times at 36 h and 48 h, and then sharply dropped at 60 h to a similar level as under the condition of no NH4\({}^{+}\) (Figure 3D). In general, it could be seen that when FBS135 was starved of a nitrogen source, the expression of half of its genes changed significantly, showing expression patterns over time. Nitrogen Utilization-Related Genes of P. eucalypt FBS135 Were Significantly Downregulated in Expression upon Nitrogen Starvation  
Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to understand the functions of DEGs of _P. eucalypt_ FBS135 adapting to nitrogen limitation. With \(p_{\text{adj}}\)! 0.05 as the threshold, there were 1841 DEGs enriched in different GO groups, including 780 (42.4%) categorized in Biological Process (BP), 733 (39.8%) categorized in Molecular Function (MF), and 328 (17.8%) categorized in Cellular Component (CC). When there was no NH4\({}^{+}\) nitrogen, of the 30 most enriched GO terms, most were downregulated (Figure 4A), while only three GO terms in the BP group (gene expression, organonitrogen compound metabolic process, and organonitrogen compound biosynthetic process) and one term in the MF group (transport activity) contained more than 20 upregulated DEGs. Moreover, when there was no nitrogen, 80 genes were upregulated and 27 genes were downregulated in the process of organonitrogen compound biosynthesis; while 99 genes were upregulated and 49 genes were downregulated in the process of organonitrogen compound metabolism (Figure 4A). The result of the GO enrichment analysis showed that in the absence of a nitrogen source, the expression levels of FBS135 genes related to nitrogen source metabolism and nitrogen source utilization were changed significantly, mostly downregulated. With \(p_{\rm adj}<0.05\) as the threshold of significant enrichment, 696 DEGs were enriched in the KEGG pathways. The 20 most enriched KEGG pathways included the pathways for biosynthesis of secondary metabolites, ABC transporters, microbial metabolism in diverse environments, two-component system, and ribosome each containing more than 50 DEGs (Figure 4B,C). When deprived of nitrogen, the DEGs of FBS135 enriched in peptidoglycan biosynthesis and cationic antimicrobial peptide resistance were significantly downregulated (Figure 4B); whereas, the expression of ribosome-related genes was most significantly downregulated (Figure 4C), accounting for 24% of the total downregulated genes. The results were consistent with the findings of the GO enrichment analysis.