The oral microbiome is home to some of the most diverse and vital bacteria. It is important to understand how it works in its home environment and in laboratory settings to see if any discrepancies come from the different settings. It is also important to see how different bacteria interact with each other to either support or hinder different functions of all the bacteria.

The need for new tuberculocidal drugs is crucial with drug resistance on the rise as the tuberculosis epidemic rages on. One new potential drug target is the PrrAB two component system (TCS) since it does not exist in humans and is essential to viability in Mycobacterium tuberculosis. This project examines Mycobacterium smegmatis, and this nonpathogenic and fast-growing organism possesses two full length PrrAB orthologs, in addition to an orphaned PrrB sensor histidine kinase. While it was determined that PrrAB1 and PrrAB2 are nonessential, the lone PrrB3 is not yet characterized for essentiality. To confirm individual dispensability of PrrAB1 and PrrAB2 and investigate the essentiality of PrrB3 and the full M. smegmatis PrrAB multiplex, we utilized CRISPRi dCas9 to repress the expression (knockdown) of prrAB1 (MSMEG_5662-5663), prrAB2 (MSMEG_0244-0246), and the lone prrB3 (MSMEG_2793) in M. smegmatis independently and simultaneously. Repression of prrAB1 resulted in the greatest growth defect, with a lag of 17 cellular division cycles compared to the control, a strain generated with an empty vector. However, the knockdown of prrAB1 was not lethal to M. smegmatis. The inhibition of all three prrAB orthologs simultaneously, also known as a multiplex knockdown, lagged the control by 13 cellular division cycles. At the 48-hour point, both the single ortholog repression of prrAB1 as well as the whole prrAB system knockdown had a growth defect of 13 replication cycles behind the control. However, the multiplex knockdown stabilized growth at 48 hours, revealing a possible compensatory mechanism in M. smegmatis. Conclusively, we show that the PrrAB TCS is globally inessential for viability in M. smegmatis.

Peptidyl-prolyl cis-trans isomerases (PPIases) are a class of chaperone proteins that catalyze isomerization between the cis and trans configuration of a peptide bond. Flavobacterium johnsoniae, a model organism for the study of bacterial gliding motility and the Type 9 Secretion System (T9SS), has six different proteins that are predicted to encode PPIases. Loss of one putative PPIase (GldI) results in a complete lack of gliding motility. However, loss of another putative PPIase encoded by fjoh_4997 has no impact on motility. In this study, genes fjoh_2367 and fjoh_2368, which are immediately downstream of gldI and have protein products homologous to GldI, were deleted. We found that the mutants exhibited a decrease in speed as compared with the wild type (1.62 ± 0.77 μm/s for mutant 5 and 1.60 ± 0.67 μm/s for mutant 6 vs 2.02 ± 0.62 μm/s for wild type). The slope of the mean square displacement of single cells was lower for mutants as compared with the wild type (1.08 for mutant 5, and 1.13 for mutant 6 vs 1.48 for wild type). Additionally, mutant colonies exhibited less spreading on PY2 agar plates than wild type, as shown by their measured colony diameters, which were 22.91 ± 2.18 mm for wild type, 16.75 ± 2.17 mm for mutant 5, and 15.97 ± 1.12 mm for mutant 6.