Browsing by Author "Hartman, Andrea H."

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    Changes in the expression of genes encoding type IV pili-associated proteins are seen when Clostridium perfringens is grown in liquid or on surfaces
    Soncini, Samantha R.; Hartman, Andrea H.; Gallagher, Tara M.; Camper, Gary J.; Jensen, Roderick V.; Melville, Stephen B. (2020-01-14)
    Background Clostridium perfringens is a Gram-positive anaerobic pathogen that causes multiple diseases in humans and animals. C. perfringens lack flagella but have type IV pili (TFP) and can glide on agar surfaces. When C. perfringens bacteria are placed on surfaces, they become elongated, flexible and have TFP on their surface, traits not seen in liquid-grown cells. In addition, the main pilin in C. perfringens TFP, PilA2, undergoes differential post-translational modification when grown in liquid or on plates. To understand the mechanisms underlying these phenotypes, bacteria were grown in three types of liquid media and on agar plates with the same medium to compare gene expression using RNA-Seq. Results Hundreds of genes were differentially expressed, including transcriptional regulatory protein-encoding genes and genes associated with TFP functions, which were higher on plates than in liquid. Transcript levels of TFP genes reflected the proportion of each protein predicted to reside in a TFP assembly complex. To measure differences in rates of translation, the Escherichia coli reporter gene gusA gene (encoding β-glucuronidase) was inserted into the chromosome downstream of TFP promoters and in-frame with the first gene of the operon. β-glucuronidase expression was then measured in cells grown in liquid or on plates. β-glucuronidase activity was proportional to mRNA levels in liquid-grown cells, but not plate-grown cells, suggesting significant levels of post-transcriptional regulation of these TFP-associated genes occurs when cells are grown on surfaces. Conclusions This study reveals insights into how a non-flagellated pathogenic rod-shaped bacterium senses and responds to growth on surfaces, including inducing transcriptional regulators and activating multiple post-transcriptional regulatory mechanisms associated with TFP functions.
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    Use of an Inducible Promoter to Characterize Type IV Pili Homologues in Clostridium perfringens
    Hartman, Andrea H. (Virginia Tech, 2012-09-13)
    Researchers of Clostridium perfringens, a Gram-positive anaerobic pathogen, were lacking a tightlyregulated, inducible promoter system in their genetic toolbox. We constructed a lactose-inducible plasmid-based system utilizing the transcriptional regulator, BgaR. Using the E. coli reporter GusA, we characterized its induction in three different strains of C. perfringens. We then used a newly-developed mutation system to create in-frame deletion mutants in three genes with homology to Type IV pilins, and we used the promoter system described above to complement the mutants. We analyzed each pilin for localization and expression, as well as tested each of the mutants for various phenotypes frequently associated with type IV pili (TFP) and type II secretion systems. PilA2, PilA3, and PilA4 localized to the poles of the cells. PilA2 was expressed in the wildtype when C. perfringens was grown on agar plates, and the PilA3 mutant lacked a von Willebrand factor A domain-containing protein in its secretome. We used our promoter system to express GFP-tagged versions of the TFP ATPase homologues and view them in cells growing on surfaces. We saw that PilB1 and PilB2 co-localized nearly all of the time, while a portion of PilT was independent of the PilB proteins. PilT appeared necessary for the localization of PilB, and it localized independently of TFP proteins in Bacillus subtilis. PilT's typical localization in Bacillus subtilis was disrupted when the GTPase and polymerization activity of cell division protein FtsZ was blocked, suggesting that PilT associates with cell division proteins.