Mutational analysis of LtgC, a lytic transglycosylase required for cell separation inNeisseria gonorrhoeae

Abstract

Lytic transglycosylases function to degrade peptidoglycan strands that comprise the bacterial cell wall. Degradation of peptidoglycan at the septum following cell division is necessary for cell separation, and a deletion ofltgCin Neisseria gonorrhoeae results in growth in clusters of around 6-20 cells rather than as normal diplococci or monococci.N. gonorrhoeaeLtgC is a homolog ofEscherichia coliMltA, and comparison of the two proteins shows that LtgC has an extra domain not found in MltA, referred to as domain 3. To better understand the function of LtgC, we characterizedN. gonorrhoeaemutants with substitutions in amino acids predicted to be necessary for enzymatic activity or amino acids predicted to be on the surface of domain 3, and we characterized a mutant lacking domain 3. All the mutants showed defects in cell separation, and the bacteria failed to release peptidoglycan-derived disaccharides into the medium. Purified LtgC proteins with the amino acid substitutions had reduced peptidoglycan degradation activity. LtgC was found to bind AmiC in bacterial 2-hybrid assays, and domain 3 mutations reduced binding. In human blood, anltgCmutant showed decreased survival, suggesting the cell wall defects in the mutant make the bacteria more sensitive to innate immune system components.