Tannerella forsythensis is a Gram-negative anaerobe closely associated with adult periodontitis, and is also detected with high frequency from the root canals with periradicular lesions. The virulence of a pathogenic bacterium like this could be modulated with two-component regulatory systems. The type strain ATCC43037 has at least 15 open reading frames encoding two-component regulators (RR) in the genome. Of these, TF0022 has a unique domain organization with a fusion of a sensor kinase and a RR. The purpose of this study was to identify the genes regulated by TF0022.
Methods:
Cells were grown in trypticase soy broth supplemented with 2.5% Fildes extract (Oxoid) and 10 ìg/ml N-acetyl muramic acid. TF0022 mutants with an insertion of the ermF-ermAM cassette were generated using an allelic exchange procedure. Utilization of carbon sources was analyzed with a BIOLOG AN MicroPlate. Total protein samples from 5-day cultures of each strain were separated in a rehydrated Immobiline DryStrip (GE), followed by SDS-PAGE. The CBB-stained gels were scanned and analyzed with ImageMaster 2D Platinum software (GE). The protein spots were cut out from the gels and digested with trypsin, then analyzed with a 4800 MALDI TOF/TOF Analyzer (Applied Biosystems). RT-PCR analyses were conducted with SuperScript III (Invitrogen), random primers, and the primer sets designed for the genes encoding differentially produced proteins.
Results:
Disruption of the TF0022 locus caused a decreased growth rate and a change in utilization of carbon sources. Comparative proteomic analyses revealed that the production levels of at least four proteins were increased, whereas one protein was decreased in the TF0022 mutant. RT-PCR analyses showed that some of them were regulated at the transcriptional level.
Conclusion:
TF0022 could act as both a positive and a negative transcriptional regulator under the standard culture conditions.