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Bacteria use Horizontal Gene Transfer (HGT) to acquire genetic material, leading to the development of novel traits, such as the spread and development of antibiotic resistance. Natural competence is one type of HGT accomplished by DNA uptake from the environment and incorporation into the genome. Bacteria from Acinetobacter are wildly distributed in the environment and are naturally competent. This propensity is a key factor to the steady increase in drug resistance in Acinetobacter, which is a cause for concern in human health as Acinetobacter is a major source of nosocomial infections. During natural competence, type IV pili (T4P) and related filaments are essential for DNA uptake. T4P are extracellular appendages composed of protein subunits (pilins) polymerized into helical fibers which can be extended into the extracellular space and retracted through depolymerization. T4P are required for diverse physiological processes, both retraction-dependent (HGT, twitching motility) and retraction-independent (biofilm formation, host cell adherence). While deletions of pilA (the primary pilin) and pilT (a cytosolic enzyme necessary for retraction) have been found to abrogate natural competence, the mechanism and DNA receptor remains unclear in Acinetobacter. In this thesis, we investigate the molecular basis for natural competence in Acinetobacter. Specific aim 1 is to identify extracellular DNA receptor(s) in Acinetobacter. To accomplish this, we proposed to identify DNA-receptors incorporated into T4P by directly measuring binding affinity of recombinantly-expressed pilin subunits to plasmid double-stranded DNA. Our data demonstrates that two pilin proteins, PilE1 and PilE2, of Acinetobacter baumannii are capable of binding plasmid DNA in vitro. Our other specific aim is to quantify the impact of allelic variation in pilA on DNA-uptake in A. baumannii. We measure natural competence using a common ΔpilA strain complemented with pilA genes from A. baumannii strains from distinct T4P subtypes to assess the impact of T4P pilA variation on natural competence. Our data shows an impact on natural competence from T4P subtype, which indicates that pilus subtype is one of the components influencing transformability in different strains of A.baumannii, potentially through T4P-subtype-depenent differences in pilus retraction. Our results provide a molecular description of HGT in Acinetobacter through natural competence.
Advisor: Kurt Piepenbrink