Aathmaja Anandhi Rangarajan

Metals are essential co-factors that are important for the activity of many enzymes and, hence, bacterial life itself. Cyclic di-GMP (c-di-GMP) is an important signaling molecule that is prevalent across various bacterial phyla and involved in myriad physiological processes. The structure and function of diguanylate cyclase (DGCs) and phosphodiesterase (PDEs) domains that make and degrade c-di-GMP are also highly conserved. These enzymes are influenced by several signaling cues, such as temperature, oxygen, and small molecules, which determine the intracellular c-di-GMP levels and subsequent bacterial physiology. In this review, we summarize the current knowledge of the roles of divalent metals Mn ²⁺ , Mg ²⁺ , Zn ²⁺ , Ca ²⁺ , and Fe ²⁺ in modulating the activity of DGCs and PDEs and c-di-GMP-related phenotypes. We describe the role of divalent metals in modulating DGC and PDE catalysis, and then discuss the examples of divalent metals as signals that modulate c-di-GMP levels. We also discuss how metals can influence the transcription of c-di-GMP catalytic enzymes. The review highlights the underexplored question of how metal availability shapes c-di-GMP signaling across diverse environmental contexts.

The second messenger cyclic di-GMP (cdG) is indispensable for the regulation of biofilm formation, motility, and a variety of other important bacterial behaviors in the majority of bacteria. The human pathogen Vibrio cholerae has a diverse repertoire of diguanylate cyclase and phosphodiesterase (PDE) enzymes that control the intracellular cdG concentration depending on local environmental cues and its physiological state. Determining the transcriptional regulation of these enzymes and the respective environmental signals that control their activity is important to understand how and when V. cholerae switches between motile and sessile lifestyles in different environments. In some strains of the current V. cholerae seventh pandemic El Tor biotype, the horizontally acquired Vibrio seventh pandemic 2 (VSP-2) island encodes an uncharacterized PDE at the gene locus vc0515 , which we named zpdA ( z inc-inhibited p hospho d iesterase A ). We show, here, that zpdA transcription is repressed by Zur when Zn ²⁺ is abundant, as well as by the quorum sensing regulator HapR when cells grow to high density. Furthermore, we find that the PDE activity of the purified ZpdA protein is inhibited by Zn ²⁺ but is dependent on alternative divalent cations such as Mn ²⁺ , which we find are elevated in V. cholerae cells grown under zinc-limiting conditions. We conclude that ZpdA is an active metal-dependent PDE that is regulated by Zn ²⁺ availability at both the level of transcriptional and post-translational leading to elevated cdG levels when Zn ²⁺ is abundant. Our results demonstrate the important role of metal availability in modulating cdG signaling in bacteria. IMPORTANCE: Vibrio cholerae colonizes estuarine environments and human hosts, where it transitions between motile and sessile states using the second messenger cyclic di-GMP (cdG). cdG levels change in response to a variety of signals and are controlled by the activity of diguanylate cyclases and phosphodiesterase (PDEs), enzymes that make and degrade cdG. In this work, we show that Zn ²⁺ and the cell density regulator HapR repress ZpdA, the PDE present in the Vibrio seventh pandemic 2 island, at the level of transcription, and that Zn ²⁺ unexpectedly alters the PDE activity of ZpdA protein itself. Our study highlights the role of metal availability as an important signaling cue that controls V. cholerae biology.

Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger that controls the signaling pathway for the motile to sessile transition in many bacteria. Diguanylate cyclases (DGCs), characterized by a GGDEF domain, are responsible for the synthesis of c-di-GMP. c-di-GMP is degraded by EAL and HD-GYP domains that exhibit phosphodiesterase activity (PDE). In Agrobacterium tumefaciens , out of 31 predicted proteins regulating the c-di-GMP levels, this work focuses on the predicted dual-function DGC/PDE, DcpB. We show that DcpB is a cycle-dependent PDE under our experimental conditions, resulting in cell cycle-dependent control of motility and biofilm formation in A. tumefaciens . DcpB also exhibits polar and mid-cell localization. Finally, we identify genetic interactions between dcpB and a LacI-family transcriptional regulators, thuR , suggesting additional regulatory inputs for DcpB-dependent phenotypes. Importance: c-di-GMP is a universal secondary messenger known to control various biological processes in bacteria including attachment, motility, virulence, and cell cycle progression. In A. tumefaciens , there are 31 predicted c-di-GMP-metabolizing proteins predicted to affect the c-di-GMP pool, raising the question of why so many proteins are involved and how the activities of these proteins are coordinated. This work describes a cell cycle-dependent PDE activity that helps coordinate cell cycle progression and developmental phenotypes including biofilm formation and motility.

The second messenger cyclic di-GMP (cdG) in V. cholerae is indispensable for the regulation of biofilm formation, motility, and a variety of other important bacterial behaviors in the majority of bacteria. The human pathogen Vibrio cholerae has a diverse repertoire of diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that control the intracellular cdG concentration depending on local environmental cues and its physiological state. Determining the transcriptional regulation of these enzymes and the respective environmental signals that control their activity is important to understand how and when V. cholerae switches between motile and sessile lifestyles in different environments. In some strains of the current V. cholerae 7 ^th pandemic El Tor biotype, the horizontally acquired Vibrio Seventh Pandemic 2 (VSP-2) island encodes an uncharacterized PDE at the gene locus vc0515 which we named zpdA ( Z inc-inhibited P hospho d iesterase- A ). We show here that zpdA transcription is repressed by Zur when Zn ²⁺ is abundant, as well as by the quorum sensing regulator HapR when cells grow to high density. Furthermore, we find that the PDE activity of the purified ZpdA protein is inhibited by Zn ²⁺ but is dependent on alternative divalent cations such as Mn ²⁺ , which we find is elevated in V. cholerae cells grown under zinc limiting conditions. We conclude that ZpdA is an active metal-dependent PDE that is regulated by Zn ²⁺ availability at both the level of transcription and post-translation leading to elevated cdG levels when Zn ²⁺ is abundant. Our results demonstrate the important role of metal availability in modulating cdG signaling in bacteria. Importance: V. cholerae colonizes estuarine environments and human host where it transitions between motile to sessile states which are controlled by cdG levels. cdG levels change in response to a variety of signals and are controlled by the activity of DGCs and PDEs, enzymes that make and degrade cdG. In this work, we show that Zn ²⁺ and the cell density regulator HapR repress ZpdA, the PDE present in the VSP-2 island, at the level of transcription, and that Zn ²⁺ unexpectedly alters the PDE activity of ZpdA protein itself. Our study highlights the role of metal availability as an important signaling cue that controls V. cholerae biology.

The human pathogen Pseudomonas aeruginosa , a leading cause of hospital-acquired infections, inhabits and forms sessile antibiotic-resistant communities called biofilms in a wide range of biotic and abiotic environments. In this study, we examined how two global sensory signaling pathways—the RhlR quorum-sensing system and the CbrA/CbrB nutritional adaptation system—intersect to control biofilm development. Previous work has shown that individually these two systems repress biofilm formation. Here, we used biofilm analyses, RNA-seq, and reporter assays to explore the combined effect of information flow through RhlR and CbrA on biofilm development. We find that the Δ rhlR Δ cbrA double mutant exhibits a biofilm morphology and an associated transcriptional response distinct from wildtype and the parent Δ rhlR and Δ cbrA mutants indicating codominance of each signaling pathway. The Δ rhlR Δ cbrA mutant gains suppressor mutations that allow biofilm expansion; these mutations map to the crc gene resulting in loss of function of the carbon catabolite repression protein Crc. Furthermore, the combined absence of RhlR and CbrA leads to a drastic reduction in the abundance of the Crc antagonist small RNA CrcZ. Thus, CrcZ acts as the molecular convergence point for quorum- and nutrient-sensing cues. We find that in the absence of antagonism by CrcZ, Crc promotes the expression of biofilm matrix components—Pel exopolysaccharide, and CupB and CupC fimbriae. Therefore, this study uncovers a regulatory link between nutritional adaption and quorum sensing with potential implications for anti-biofilm targeting strategies. IMPORTANCE: Bacteria often form multicellular communities encased in an extracytoplasmic matrix called biofilms. Biofilm development is controlled by various environmental stimuli that are decoded and converted into appropriate cellular responses. To understand how information from two distinct stimuli is integrated, we used biofilm formation in the human pathogen Pseudomonas aeruginosa as a model and studied the intersection of two global sensory signaling pathways—quorum sensing and nutritional adaptation. Global transcriptomics on biofilm cells and reporter assays suggest parallel regulation of biofilms by each pathway that converges on the abundance of a small RNA antagonist of the carbon catabolite repression protein, Crc. We find a new role of Crc as it modulates the expression of biofilm matrix components in response to the environment. These results expand our understanding of the genetic regulatory strategies that allow P. aeruginosa to successfully develop biofilm communities.

The signaling molecule cyclic di-GMP (cdG) controls the switch between bacterial motility and biofilm production, and fluctuations in cellular levels of cdG have been implicated in Vibrio cholerae pathogenesis. Intracellular concentrations of cdG are controlled by the interplay of diguanylate cyclase (DGC) enzymes, which synthesize cdG to promote biofilms, and phosphodiesterase (PDE) enzymes, which hydrolyse cdG to drive motility. To track the complete regulatory logic of how V. cholerae responds to changing cdG levels, we followed a timecourse of overexpression of either the V. harveyi diguanylate cyclase QrgB or a variant of QrgB lacking catalytic activity (QrgB*). We find that QrgB increases cdG levels relative to QrgB* for 30 minutes after overexpression, but the effect of QrgB on cdG levels plateaus at 30 minutes, indicating tight adaptive control of cdG levels. In contrast, loss of VpsR, a master regulator activating biofilm formation upon binding to cdG, leads to higher baseline levels of cdG and continuously increasing cdG through 60 minutes after QrgB induction, revealing the existence of a negative feedback loop on cdG levels operating through VpsR. Through a combination of RNA polymerase ChIP-seq, RNA-seq, and genetic approaches, we show that transcription of a gene encoding a PDE, cdgC , is activated by VpsR at high cdG concentrations, mediating this negative feedback on cdG levels. We further identify a transcript encoded within, and antisense to, the cdgC open reading frame which we name s RNA n egative r egulator of C dgC (SnrC). RNA polymerase ChIP-seq and RNA-seq demonstrate SnrC to be expressed specifically under conditions of high cdG in the absence of VpsR. Ectopic SnrC expression increases cdG levels in a manner dependent on CdgC, demonstrating that its effect on cdG levels is likely through interference with CdgC production. Further, although cells lacking cdgC exhibit enhanced biofilm formation, these mutants are outcompeted by wild type V. cholerae in colonization assays that reward a combination of attachment, dispersal, and motility behaviors. These results underscore the importance of negative feedback regulation of cdG to maintain appropriate homeostatic levels for efficient transitioning between biofilm formation and motility, both of which are necessary over the course of the V. cholerae infection cycle.

Bacterial nucleoid-associated proteins are important factors in regulation of transcription, in nucleoid structuring, and in homeostasis of DNA supercoiling. Vice versa, transcription influences DNA supercoiling and can affect DNA binding of nucleoid-associated proteins (NAPs) such as H-NS in Escherichia coli. Here we describe genetic tools to study the interplay between transcription and nucleoid-associated proteins in E. coli. These methods include construction of genomic and plasmidic transcriptional and translational lacZ reporter gene fusions to study regulation of promoters; insertion of promoter cassettes to drive transcription into a locus of interest in the genome, for example, an H-NS-bound locus; and construction of isogenic hns and stpA mutants and precautions in doing so.

Complex carbohydrates shape the gut microbiota, and the collective fermentation of resistant starch by gut microbes positively affects human health through enhanced butyrate production. The keystone species Ruminococcus bromii (Rb) is a specialist in degrading resistant starch; its degradation products are used by other bacteria including Bacteroides thetaiotaomicron (Bt). We analysed the metabolic and spatial relationships between Rb and Bt during potato starch degradation and found that Bt utilizes glucose that is released from Rb upon degradation of resistant potato starch and soluble potato amylopectin. Additionally, we found that Rb produces a halo of glucose around it when grown on solid media containing potato amylopectin and that Bt cells deficient for growth on potato amylopectin (∆sus Bt) can grow within the halo. Furthermore, when these ∆sus Bt cells grow within this glucose halo, they have an elongated cell morphology. This long-cell phenotype depends on the glucose concentration in the solid media: longer Bt cells are formed at higher glucose concentrations. Together, our results indicate that starch degradation by Rb cross-feeds other bacteria in the surrounding region by releasing glucose. Our results also elucidate the adaptive morphology of Bt cells under different nutrient and physiological conditions.

Complex carbohydrates shape the gut microbiota and the collective fermentation of resistant starch by gut microbes positively affects human health through enhanced butyrate production. The keystone species Ruminococcus bromii ( Rb ) is a specialist in degrading resistant starch; its degradation products are used by other bacteria including Bacteroides thetaiotaomicron ( Bt ). We analyzed the metabolic and spatial relationships between Rb and Bt during potato starch degradation and found that Bt utilizes glucose that is released from Rb upon degradation of resistant potato starch and soluble potato amylopectin. Additionally, we found that Rb produces a halo of glucose around it when grown on solid media containing potato amylopectin and that Bt cells deficient for growth on potato amylopectin (Δ sus Bt ) can grow within the halo. Furthermore, when these Δ sus Bt cells grow within this glucose halo, they have an elongated cell morphology. This long-cell phenotype depends on the glucose concentration in the solid media: longer Bt cells are formed at higher glucose concentrations. Together, our results indicate that starch degradation by Rb cross-feeds other bacteria in the surrounding region by releasing glucose. Our results also elucidate the adaptive morphology of Bt cells under different nutrient and physiological conditions. Impact Statement: Dietary intake of complex carbohydrates including resistant starch benefits human health by supporting the growth of keystone species that increase the microbiome diversity via cross-feeding. For instance, Ruminococcus bromii ( Rb ) is a specialist in degrading resistant starch, and the byproducts of this degradation process are used by bacterial species. In this study, we show that Bacteroides thetaiotaomicron ( Bt ) cross-feeds on the glucose released during potato starch degradation by Rb . We also show that proximity to Rb cells that are releasing sugars is important for the growth of the cross-fed Bt in solid media. Additionally, we find a longer phenotype for Bt cells grown on solid media in high glucose conditions, indicating that, like other bacteria, human gut bacteria undergo significant nutrient-dependent morphological adaptations.