Defining the essential genome of bacterial pathogens is central to developing an understanding of the biological processes controlling disease. This has proven elusive for Pseudomonas aeruginosa during chronic infection of the cystic fibrosis (CF) lung. In this paper, using a Monte Carlo simulation-based method to analyze high-throughput transposon sequencing data, we establish the P. aeruginosa essential genome with statistical precision in laboratory media and CF sputum. Reconstruction of the global requirements for growth in CF sputum compared with defined growth conditions shows that the latter requires several cofactors including biotin, riboflavin, and pantothenate. Comparison of P. aeruginosa strains PAO1 and PA14 demonstrates that essential genes are primarily restricted to the core genome; however, some orthologous genes in these strains exhibit differential essentiality. These results indicate that genes with similar molecular functions may have distinct genetic roles in different P. aeruginosa strains during growth in CF sputum. We also show that growth in a defined growth medium developed to mimic CF sputum yielded virtually identical fitness requirements to CF sputum, providing support for this medium as a relevant in vitro model for CF microbiology studies.

Course-based undergraduate research experiences (CUREs) provide a promising avenue to attract a larger and more diverse group of students into research careers. CUREs are thought to be distinctive in offering students opportunities to make discoveries, collaborate, engage in iterative work, and develop a sense of ownership of their lab course work. Yet how these elements affect students’ intentions to pursue research-related careers remain unexplored. To address this knowledge gap, we collected data on three design features thought to be distinctive of CUREs (discovery, iteration, collaboration) and on students’ levels of ownership and career intentions from ∼800 undergraduates who had completed CURE or inquiry courses, including courses from the Freshman Research Initiative (FRI), which has a demonstrated positive effect on student retention in college and in science, technology, engineering, and mathematics. We used structural equation modeling to test relationships among the design features and student ownership and career intentions. We found that discovery, iteration, and collaboration had small but significant effects on students’ intentions; these effects were fully mediated by student ownership. Students in FRI courses reported significantly higher levels of discovery, iteration, and ownership than students in other CUREs. FRI research courses alone had a significant effect on students’ career intentions.

MicroRNAs (miRNAs) play essential roles in plant and animal development, but the cause and effect of miRNA expression divergence between closely related species and in interspecific hybrids or allopolyploids are unknown. Here, we show differential regulation of a miR163-mediated pathway in allotetraploids and their progenitors, Arabidopsis thaliana and Arabidopsis arenosa. miR163 is a recently evolved miRNA in A. thaliana and highly expressed in A. thaliana, but its expression was undetectable in A. arenosa and repressed in resynthesized allotetraploids. Repression of A. arenosa MIR163 (Aa MIR163) is caused by a weak cis-acting promoter and putative trans-acting repressor(s) present in A. arenosa and allotetraploids. Moreover, ectopic Aa MIR163 precursors were processed more efficiently in A. thaliana than in resynthesized allotetraploids, suggesting a role of posttranscriptional regulation in mature miR163 abundance. Target genes of miR163 encode a family of small molecule methyltransferases involved in secondary metabolite biosynthetic pathways that are inducible by a fungal elicitor, alamethicin. Loss of miR163 or overexpression of miR163 in mir163 mutant plants alters target transcript and secondary metabolite profiles. We suggest that cis-and trans-regulation of miRNA and other genes provides a molecular basis for natural variation of biochemical and metabolic pathways that are important to growth vigor and stress responses in Arabidopsis-related species and allopolyploids.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen often associated with chronic infections in the lungs of individuals with the heritable disease cystic fibrosis (CF). Previous work from our laboratory demonstrated that aromatic amino acids within CF lung secretions (sputum) not only serve as carbon and energy sources but also enhance synthesis of the cell signaling molecule Pseudomonas quinolone signal (PQS). The present study investigates the role of the aromatic amino acid-responsive regulator PhhR in mediating these phenotypes. Transcriptome analysis revealed that PhhR controls four putative transcriptional units (phhA, hpd, hmgA, and dhcA) involved in aromatic amino acid catabolism; however, genes involved in PQS biosynthesis were unaffected. The phhA, hpd, hmgA, and dhcA promoters were mapped by primer extension, and purified His 6 -PhhR was shown to bind the phhA, hpd, and dhcA promoters in vitro by use of electrophoretic mobility shift assays. Our work characterizes a transcriptional regulator of catabolic genes induced during P. aeruginosa growth in CF sputum.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes infections in the lungs of individuals with the genetic disease cystic fibrosis. Density-dependent production of toxic factors regulated by the Pseudomonas quinolone signal (2-heptyl-3-hydroxy-4-quinolone; PQS) have been proposed to be involved in P. aeruginosa virulence. PQS biosynthesis requires conversion of the central metabolite chorismate to anthranilate by anthranilate synthase. This reaction is also the first step in tryptophan biosynthesis. P. aeruginosa possesses two functional anthranilate synthases, TrpEG and PhnAB, and these enzymes are not functionally redundant, as trpEG mutants are tryptophan auxotrophs but produce PQS while mutants in phnAB are tryptophan prototrophs but do not produce PQS in minimal media. The goal of the work described in this paper was to determine the mechanism for this lack of functional complementation of TrpEG and PhnAB. Our results reveal that overexpression of either enzyme compensates for tryptophan auxotrophy and PQS production in the trpEG and phnAB mutants respectively, leading to the hypothesis that differential regulation of these genes is responsible for the lack of functional complementation. In support of this hypothesis, trpEG was shown to be expressed primarily during low-density growth while phnAB was expressed primarily at high density. Furthermore, dysregulation of phnAB expression eliminated tryptophan auxotrophy in the P. aeruginosa trpEG mutant. Based on these data, we propose a model for anthranilate sequestration by differential transcriptional regulation of the two P. aeruginosa anthranilate synthase enzymes.