Penicillin was discovered by accident in a messy lab, and since then its development and use has altered medicine, human experience, and the course of history. Penicillin and its derivatives have since formed an entire class of antibiotics known as β-lactams which are used readily to this day. These compounds work by inhibiting synthesis of the peptidoglycan bacterial cell wall which typically leads to the cell’s rupture and death. However, there are many instances of β-lactam treatment failure and recurrent infection after the use of such drugs, despite the bacteria lacking a known resistance factor. Previous work has found that diverse species of Gram-negative pathogens exhibit tolerance to β-lactam drugs, defined by a large proportion of a bacterial population enjoying prolonged survival in the presence of these otherwise lethal agents. β-lactam tolerance in Gram-negative bacteria is also defined by cells undergoing a drastic loss of their rod shape to endure as spheroplasts capable of reverting back to rods through successive rounds of vegetative growth when the drug is removed. To understand this phenomenon broadly, I use meropenem, a member of the carbapenem family of β-lactam drugs, to probe cell morphology, time-dependent killing dynamics, and condition of the peptidoglycan cell wall across a panel of diverse clinical isolates. I then present the results of a TnSeq screen in K. pneumoniae challenged with meropenem to identify unexplored genetic factors which positively and negatively modulate meropenem tolerance. A survey of many clinical isolates reveals that most Gram-negative opportunistic pathogens exhibit some degree of meropenem tolerance, with numerous E. coli isolates surprisingly being the exception, having little to no spheroplast-mediated tolerance to meropenem where all other isolates demonstrated meropenem tolerance. Forward genetics identified four highly conserved envelope stress response systems (OmpR/EnvZ, Rcs, Pho, and Cpx) which collectively enable a high degree of meropenem tolerance. I also identified a specific lytic transglycosylase that endogenously reduces tolerance in K. pneumoniae by accelerating the transition from rod to spheroplast. Overall, these results suggest that β-lactam tolerance is the result of many complex regulatory systems acting in concert, as well as individual cell wall modifying enzyme. These findings offer new insight into how dangerous pathogens respond to our most widely used and effective antibiotics with implications towards identifying new druggable targets or therapeutic strategies for keeping infectious bacteria sensitive to antibiotics.