Hybrid Two-component Regulator GppX Regulates Peptidoglycan Recycling in Tannerella forsythia

Objectives: The periodontal pathogen Tannerella forsythia is unable to synthesize the peptidoglycan precursor amino-sugars and thus scavenges on peptidoglycan fragments (muropeptides) released by the cohabiting oral bacteria. In Gram-negative bacteria, AmpG-like permeases transport muropeptides from the periplasmic space into the cytosol. We identified a functional homolog of AmpG (Tanf_08365) in T. forsythia by trans complementation strategy in E. coli. It was demonstrated that a mutant deficient in a hybrid two-component system GppX had reduced transcript levels of peptidoglycan recycling (including Tanf_08365) and protein glycosylation associated genes. Whether these genes are regulated via indirect effects of GppX signaling or direct binding of GppX to their promoter elements is unknown. In this study, we focused on defining the function of GppX in the regulation of peptidoglycan recycling in T. forsythia.

Methods: The growth of T. forsythia was measured in TSB broth supplemented with muropeptide prepared from F. nucleatum. An E. coli based reporter system was generated to assess GppX-mediated regulation of peptidoglycan recycling operon. After determining the transcription start site by 5’-RACE the putative promoter region (-400 to +1) was fused upstream of a promoterless lacZ gene in pRS414 plasmid. In parallel, a synthetic lac promoter fused to a DNA fragment coding for T. forsythia GppX protein (lac-gppX chimera) was cloned into pACYC184. Both constructs were placed in E.coli and b-galactosidase expression was determined with O-nitrophenyl-b-D-galatoside (ONPG) chromogenic substrate.
Results: (a) Muropeptide supplementation did not efficiently support the growth of the T. forsythia gppX-deletion mutant compared to the wild-type strain. (b) E. coli reporter strain with GppX induction compared to no GppX induction showed an increase in b-galactosidase activity.
Conclusions: The results show that the promoter activity of Tf ampG operon is under the direct control of GppX which is important for peptidoglycan uptake since muropeptides can sustain wild-type T. forsythia growth.