Salmonella enterica and Klebsiella pneumoniae are two major public health concerns that are responsible for millions of illnesses every year throughout the world. Much remains to explore the genes and mechanisms that are critical for their pathogenesis and antibiotic resistance. I used transposon Insertion Sequencing (INSeq), a powerful high-throughput screening technique that can link bacterial genes to phenotypes and identify genes that are essential for bacterial survival, to determine genes that are associated with survivability of Salmonella Typhimurium in Luria Bertani (LB), E-minimal medium (EMM) and under acid stress, and of K. pneumoniae under exposure to five classes of antibiotics. Growing a pool of 450,000 mutants on LB identified a total of 362 essential genes, the majority of which (90.6%) are within the S. Typhimurium core genome. The pCol1B9 replication initiation and its regulator, repZ and repY, are among the essential genes found in the strain's accessory genome. Comparing essential genes identified in LB to two earlier studies of S. Typhimurium strains and identifying genes that are conditionally essential in EMM suggest that a single growth environment and strain cannot provide a comprehensive understanding of essential genes at the species level. S. Typhimurium is hypothesized to have a special acid tolerance system in which the cytoplasm becomes more acidic. I applied INSeq to find genes that contribute to acid tolerance at pH 4.0 and pH 5.0. For which I developed a modified INSeq approach capable of identifying genes required for persistence in non-growth conditions. In addition to several known genes, this project identified novel acid tolerance genes including trxB (thioredoxin reductase), pykF (pyruvate kinase), sspA (starvation protein), and revealed that as the stress increases through time and decreasing pH, additional tolerance mechanisms are required to protect cells. Next, I used INSeq to find intrinsic resistance genes in a clinical isolate of multidrug resistant K. pneumoniae. We found pstB (ABC transporter ATP binding protein), gltA (Citrate synthase), tgt (tRNA guanosine transglycosylase), fabF (fatty acid synthase), and glycosyltransferase encoding genes: pgaptmp_000142, pgaptmp_000147, and pgaptmp_000148, each contribute to resistance across multiple classes of antibiotics. Considering all the INSeq data, healthy cell envelopes were found to be crucial for optimum growth and cell protection, regardless of the growth environment, which included laboratory conditions under acid and antibiotic stresses. In addition, numerous known genes were identified for corresponding features, such as phoP-phoQ system for acid tolerance and acrAB-tolC for multidrug resistance, confirming the effectiveness of INSeq. I next used whole genome sequencing to find genetic changes in S. Typhimurium isolates to characterize a sugar metabolism and an antibiotic resistance phenotype. First, I helped characterize how a C→T transition in the dctA promoter allows for growth at lower orotate concentrations by creating an improved binding site for the transcriptional activator CRP. Secondly, by progressively challenging cells with higher concentrations of antibiotics, I discovered an A→T transition in codon 466 of gyrB reduces ciprofloxacin sensitivity in a S. Typhimurium mutant that cannot synthesize the intracellular signaling molecule cAMP. Both Salmonella and Klebsiella are considered top priority pathogens for research and development of new antibiotics; this PhD thesis provides an improved understanding of the biology of both organisms and simultaneously identifies high-quality candidate genes that can be targeted for the development of improved antibiotics and other therapeutics.