Morphological and functional characterization of the C-terminal two-thirds of the autotransporter Ag43 alpha domain via site-directed mutagenesis in Escherichia coli DH5α

Abstract

The role of Antigen 43 in bacterial aggregation and biofilm formation has been extensively studied, emphasizing its significance in bacterial resilience, antibiotic resistance, and infection persistence. Despite this knowledge, the specific influence and function of the alpha domain of Antigen 43 on bacterial colony morphology remains a poorly understood aspect. Antigen 43 is composed of an N-terminal signal sequence, alpha domain, autotransporter, and C-terminal beta domain. In this investigation, we employed site-directed mutagenesis to precisely delete the C-terminal two-thirds of the alpha domain of Antigen 43 in Escherichia coli DH5α cells. We aimed to obtain a more nuanced understanding of the specific regions of the alpha domain responsible for aggregation and the characteristic ‘frizzy’ colony morphology. While our results indicate the deletion of amino acids 193-551 did not eliminate aggregation, we surprisingly observed a modified aggregative behavior and altered colony morphology. Further validation through Western blot analysis and whole plasmid sequencing confirmed the deletion while also underscoring differences in Ag43 expression in the mutant bacteria. In summary, our study provides insights into the relationship between the alpha domain of Ag43 and bacterial behavior, suggesting that specific deletions within Ag43 may not entirely disrupt the aggregation process but rather modify it, prompting further exploration into the functional consequences of such deletions.