Essential gene deletions producing gigantic bacteria

To characterize the consequences of eliminating essential functions needed for peptidoglycan synthesis, we generated deletion mutations of Acinetobacter baylyi by natural transformation and visualized the resulting microcolonies of dead cells. We found that loss of genes required for peptidoglycan precursor synthesis or polymerization led to the formation of polymorphic giant cells with diameters that could exceed ten times normal. Treatment with antibiotics targeting early or late steps of peptidoglycan synthesis also produced giant cells. The giant cells eventually lysed, although they were partially stabilized by osmotic protection. Genome-scale transposon mutant screening (Tn-seq) identified mutations that blocked or accelerated giant cell formation. Among the mutations that blocked the process were those inactivating a function predicted to cleave murein glycan chains (the MltD murein lytic transglycosylase), suggesting that giant cell formation requires MltD hydrolysis of existing peptidoglycan. Among the mutations that accelerated giant cell formation after ss-lactam treatment were those inactivating an enzyme that produces unusual 3->3 peptide cross-links in peptidoglycan (the LdtG L,D-transpeptidase). The mutations may weaken the sacculus and make it more vulnerable to further disruption. Although the study focused on A. baylyi, we found that a pathogenic relative (A. baumannii) also produced giant cells with genetic dependencies overlapping those of A. baylyi. Overall, the analysis defines a genetic pathway for giant cell formation conserved in Acinetobacter species in which independent initiating branches converge to create the unusual cells. Author summary Although essential genes control the most basic functions of bacterial life, they are difficult to study genetically because mutants lacking the functions die. We have developed a simple procedure for creating bacteria in which different essential genes have been completely deleted, making it possible to analyze the roles of the missing functions based on the features of the dead cells that result. When genes needed for the production of the cell wall were inactivated, the bacteria formed bizarre giant cells. It was possible to identify the functions responsible for forming the giant cells, and to formulate a model for how they form. Since cell wall synthesis is one of the most important antibiotic targets, understanding how bacteria respond to its disruption may ultimately help in developing procedures to overcome antibiotic resistant bacterial infections.