Disruption of c-di-GMP signaling networks unlocks cryptic expression of secondary metabolites during biofilm growth in Burkholderia pseudomallei

Abstract

The regulation and production of secondary metabolites during biofilm growth of Burkholderia spp. is not well understood. To learn more about the crucial role and regulatory control of cryptic molecules produced during biofilm growth, we disrupted c-di-GMP signaling in Burkholderia pseudomallei, a soil-borne bacterial saprophyte and the etiologic agent of melioidosis. Our approach to these studies combined transcriptional profiling with genetic deletions that targeted key c-di-GMP regulatory components to characterize responses to changes in temperature. Mutational analyses and conditional expression studies of c-di-GMP genes demonstrates their contribution to phenotypes such as biofilm formation, colony morphology, motility, and expression of secondary metabolite biosynthesis when grown as a biofilm at different temperatures. RNA-seq analysis was performed at varying temperatures in a ΔII2523 mutant background that is responsive to temperature alterations resulting in hypo- and hyper- biofilm forming phenotypes. Differential regulation of genes was observed for polysaccharide biosynthesis, secretion systems, and nonribosomal peptide and polyketide synthase (NRPS/PKS) clusters in response to temperature changes. Deletion mutations of biosynthetic gene clusters (BGCs) clusters 2, 11, 14 (syrbactin), and 15 (malleipeptin) in wild-type and ΔII2523 backgrounds also reveals the contribution of these BGCs to biofilm formation and colony morphology in addition to inhibition of Bacillus subtilis and Rhizoctonia solani. Our findings suggest that II2523 impacts the regulation of genes that contribute to biofilm formation and competition. Characterization of cryptic BGCs under differing environmental conditions will allow for a better understanding of the role of secondary metabolites in the context of biofilm formation and microbe-microbe interactions.

Fostering student authorship skills in synthetic biology

“…Such practices include a discipline-specific set of technical competencies, the ability to define research questions and test hypotheses, efficacy in gathering and interpreting data, and facility with scientific communication, both oral and written. There are many mechanisms by which students develop science identities through the exercise of normative scientific practices, including coursework (Chen et al, 2020;Singer et al, 2020), laboratory courses (e.g., course-based undergraduate research experiences or CUREs) (Borlee et al, 2023;Roberts and Shell, 2023), project-based and challenge-based learning (Taconis and Bekker, 2023), mentoring programs (Remich et al, 2016;Hernandez et al, 2017), and research experiences (Remich et al, 2016). For example, increases in scientific identity were observed in undergraduate students that generated and analyzed their own data in CUREs (Cooper et al, 2020;Roberts and Shell, 2023).…”

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“…Such practices include a discipline-specific set of technical competencies, the ability to define research questions and test hypotheses, efficacy in gathering and interpreting data, and facility with scientific communication, both oral and written. There are many mechanisms by which students develop science identities through the exercise of normative scientific practices, including coursework (Chen et al, 2020;Singer et al, 2020), laboratory courses (e.g., course-based undergraduate research experiences or CUREs) (Borlee et al, 2023;Roberts and Shell, 2023), project-based and challenge-based learning (Taconis and Bekker, 2023), mentoring programs (Remich et al, 2016;Hernandez et al, 2017), and research experiences (Remich et al, 2016). For example, increases in scientific identity were observed in undergraduate students that generated and analyzed their own data in CUREs (Cooper et al, 2020;Roberts and Shell, 2023).…”