Pseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (σ54). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a ß-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO∆mifS, PAO∆mifR and PAO∆mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO∆mifS and PAO∆mifR revealed that these mutants were unable to utilize C5-dicarboxylate α-ketoglutarate (α-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except α-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport. The inability of the mutants to utilize α-KG was rescued by expressing PA5530, encoding C5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, α-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

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Attention Deficit Hyperactivity Disorder (ADHD) is an increasingly prevalent neuropsychiatric disorder characterized by hyperactivity, inattention, and impulsivity. Symptoms emerge from underlying deficiencies in neurocircuitry, and recent research has suggested a role played by the gut microbiome. The gut microbiome is a complex ecosystem of interdependent taxa with an exponentially complex web of interactions involving these taxa, plus host gene and reaction pathways, some of which involve neurotransmitters with roles in ADHD neurocircuitry. Studies have analyzed the ADHD gut microbiome using macroscale metrics such as diversity and composition, and have proposed several biomarkers. Few studies have delved into the complex underlying dynamics ultimately responsible for the emergence of such metrics, leaving a largely incomplete, sometimes contradictory, and ultimately inconclusive picture. We aim to help complete this picture by venturing beyond taxa abundances and into taxa relationships (i.e. cooperation and competition), using a publicly available gut microbiome dataset from 30 Control (15 female, 15 male) and 28 ADHD (15 female, 13 male) undergraduate students. We conduct our study in two parts. We first perform the same macroscale analyses prevalent in ADHD gut microbiome literature (diversity, differential, biomarker, and composition) to observe the degree of correspondence, or any new trends. We then estimate two-way ecological relationships by producing Control and ADHD Microbial Co-occurrence Networks (MCNs), using SparCC correlations (p < 0.01). We perform community detection to find clusters of taxa estimated to mutually cooperate along with their centroids, and centrality calculations to estimate taxa most vital to overall gut ecology. We conclude by summarizing our results, and provide conjectures on how they can guide future experiments, some methods for improving our experiments, and general implications for the field.