Expression Analysis of Nitrogen Metabolism Genes in Lelliottia amnigena PTJIIT1005, Comparison with Escherichia coli K12 and Validation of Nitrogen Metabolism Genes

Abstract

Escherichia coli K12 and Lelliottia amnigena PTJIIT1005 bacteria were isolated from the polluted Yamuna River (Delhi, India) site, which can remediate nitrate from groundwater media under anaerobic conditions. BV-BRC (Bacterial and Viral Bioinformatics Resource Center) information system, RAST, and PGAP servers were used to annotate the nitrogen metabolism genes from the genome sequence of these microbes. Here we compared the strains L. amnigena PTJIIT1005 with E. coli K12 in the context of nitrogen metabolism genes. Sequence alignment, similarity percentage, and phylogenetic analysis were done to find similarities between the genes. Common nitrogen genes of these strains, like respiratory nitrate reductase, nitrite reductase, nitric oxide reductase, glutamine synthetase, and hydroxylamine reductase, have found good sequence similarity (83–94%) with each other. The PATRIC tool identified N-operons, and the nitrate reductase gene clusters were also determined as per literature survey. Protein–protein interaction network was constructed using STRING 12.0 database and Cytoscape v 3.10.0 software plug-in Network analyzer. On the basis of network topological parameters NarG, NarZ, NarY, NarH, NarI, NarV, NirB, NirD, NapA, and NapB are the key genes in network of E. coli K12 strain. Nar, NirB, NirD, NasA, NasB, NasC, NasD, NasE, and GlnA are the key genes in network of L. amnigena PTJIIT1005. Among these, NarG and NirB are the superhub genes because of having highest Betweenness centrality (BC) and node degree. The functional enrichment analysis was determined using PANTHER GENE ONTOLOGY and DAVID software exhibited their role in nitrogen metabolism pathway and nitrate assimilation. Further, SWISS-MODEL was used to predict the 3D protein structure of these enzymes, and after, these structures were validated by Ramachandran plot using the PROCHECK tool. The Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) method was used to determine the N-genes expression level in both strains. This study showed that E. coli K12 and L. amnigena PTJIIT1005 have common nitrogen metabolism genes involved in the same functional role, like the denitrification pathway. Additionally, operon arrangement study and PPI network revealed that E. coli K12 has only a denitrification pathway, while L. amnigena PTJIIT1005 has both an assimilation and denitrification pathway. PCR successfully amplified selected N-metabolizing genes, and the expression level of N-genes was high in strain L. amnigena PTJIIT1005. Our previous experimental study exhibited a better nitrate remediation rate in L. amnigena PTJIIT1005 over E. coli K12. This study confirmed the presence of assimilation and denitrification process through amplified N-metabolizing genes and showed high expression of N-genes in L. amnigena PTJIIT1005, which favor the evidence of better nitrate remediation in L. amnigena PTJIIT1005 over E. coli K12.