Biology subjects

Giacalone, D.

Publications and source records attributed to Giacalone, D..

5 recordsLinked to original sources

bioRxiv2024

The phospho-ferrozine assay: A tool to study bacterial redox-active metabolites produced at the plant root

Soil microbial communities are pivotal to plant health and nutrient acquisition. It is becoming increasingly clear that many interactions, both among and between microbes and plants, are governed by small bioactive molecules or "secondary metabolites" that can aid in communication, competition, and nutrient uptake. Yet, secondary metabolite biogeography - who makes what, where, and why-- is in its infancy. Further, secondary metabolite biosynthesis genes are often silent or weakly expressed under standard laboratory conditions, making it incredibly difficult to study these small molecules. To begin to address these dual challenges, we focused on Redox-Active metabolites (RAMs), a specific class of small molecules, and took advantage of recent findings that many RAMs aid in acquiring phosphorus and that their production is frequently stimulated by stress for this macronutrient. We developed a screen for RAM-producing bacteria that leverages phosphorus limitation to stimulate metabolite biosynthesis and uses a colorimetric (ferrozine) iron-reduction assay to identify redox activity. We isolated 557 root-associated bacteria from grasses collected at sites across the United States (Santa Rita Experimental Range (AZ), Konza Prairie Biological Station (KS), and Harvard Forest (MA)) and from commercial tomato plants and screened them for RAM production. We identified 128 soil isolates of at least 19 genera across Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes that produced RAMs under phosphorus stress. Our work reveals that the production of RAMs under phosphorus stress is common across diverse soil bacteria and provides an approach to screen for these small molecules rapidly. ImportanceBy secreting secondary metabolites, bacteria at the plant root can defend against diseases and help acquire essential nutrients. However, the genes which synthesize secondary metabolites are typically inactive or are weakly expressed under standard laboratory conditions. This fact makes it difficult to study these small molecules and hinders the discovery of novel small molecules that may play crucial roles in agricultural and biomedical settings. Here, we focus on Redox-Active metabolites (RAMs), a class of secondary metabolites that can help bacteria solubilize phosphorus and are often produced when phosphorus is limited. We developed a screen that rapidly identifies RAM-producing bacteria by utilizing a colorimetric iron-reduction assay in combination with phosphorus limitation to stimulate biosynthesis. The screen reveals that RAM-producing bacteria are far more prevalent in soil than previously appreciated and that this approach can be used to identify RAM producers.

microbiology↗

bioRxiv2022

Single-spore germination analyses reveal that Clostridioides difficile uses parallel pathways to sense distinct classes of co-germinants during spore germination.

Clostridioides difficile infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, are essential for inducing germination, while another proposes that either co-germinant class can induce germination. The former model is based on the finding that spores defective in releasing large stores of internal Ca2+ in the form of calcium dipicolinic acid (CaDPA) cannot germinate when germination is induced with bile acid germinant and amino acid co-germinant alone. However, since the reduced optical density of CaDPA-less spores makes it difficult to accurately measure their germination, we developed a novel automated, time-lapse microscopy-based germination assay to analyze CaDPA mutant germination at the single-spore level. Using this assay, we found that CaDPA mutant spores germinate in the presence of amino acid co-germinant and bile acid germinant. Higher levels of amino acid co-germinants are nevertheless required to induce CaDPA mutant spores to germinate relative to WT spores because CaDPA released by WT spores during germination can function in a feedforward loop to potentiate the germination of other spores within the population. Collectively, these data indicate that Ca2+ is not essential for inducing C. difficile spore germination because amino acid and Ca2+ co-germinant signals are sensed by parallel signaling pathways. ImportanceClostridioides difficile spore germination is essential for this major nosocomial pathogen to initiate infection. C. difficile spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca2+ and amino acids. Prior work suggested that Ca2+ is essential for C. difficile spore germination based on bulk population analyses of germinating CaDPA mutant spores. Since these assays rely on optical density to measure spore germination and the optical density of CaDPA mutant spores is reduced relative to WT spores, this bulk assay is limited in its capacity to analyze germination. To overcome this limitation, we developed an automated image analysis pipeline to monitor C. difficile spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores.

microbiology↗

bioRxiv2022

PrrA modulates Mycobacterium tuberculosis response to multiple environmental cues and is critically regulated by serine/threonine protein kinases

The ability of Mycobacterium tuberculosis (Mtb) to adapt to its surrounding environment is critical for the bacterium to successfully colonize its host. Transcriptional changes are a vital mechanism by which Mtb responds to key environmental signals experienced, such as pH, chloride (Cl-), nitric oxide (NO), and hypoxia. However, much remains unknown regarding how Mtb coordinates its response to the disparate signals seen during infection. Utilizing a transcription factor (TF) overexpression plasmid library in combination with a pH/Cl--responsive luciferase reporter, we identified the essential TF, PrrA, part of the PrrAB two-component system, as a TF involved in modulation of Mtb response to pH and Cl-. Further studies revealed that PrrA also affected Mtb response to NO and hypoxia, with prrA overexpression dampening induction of NO and hypoxia- responsive genes. PrrA is phosphorylated not just by its cognate sensor histidine kinase PrrB, but also by serine/threonine protein kinases (STPKs) at a second distinct site. Strikingly, a STPK phosphoablative PrrA variant was significantly dampened in its response to NO versus wild type Mtb, disrupted in its ability to adaptively enter a non-replicative state upon extended NO exposure, and attenuated for in vivo colonization. Together, our results reveal PrrA as an important regulator of Mtb response to multiple environmental signals, and uncover a critical role of STPK regulation of PrrA in its function.

microbiology↗

bioRxiv2020

Role of SpoIVA ATPase Motifs During Clostridioides difficile Sporulation

The nosocomial pathogen, Clostridioides difficile, is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved coat protein, SpoIVA, and the clostridial-specific coat protein, SipL, which directly interact. Mutations that disrupt their interaction cause coat to mislocalize and decrease functional spore formation. In B. subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to help drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in C. difficiles SpoIVA ATPase motifs impair functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104 less severe than the equivalent mutations in B. subtilis. Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but retain binding to ATP enhanced SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele-specific, our data implies that SipL recognizes the ATP-bound form of SpoIVA and highlights the importance of this interaction for functional C. difficile spore formation. ImportanceThe aerotolerant spores formed by the major nosocomial pathogen Clostridioides difficile are its primary infectious particle. However, the mechanism by which this critical cell type is assembled remains poorly characterized, especially with respect to its protective coat layer. We previously showed that binding between the spore morphogenetic proteins, SpoIVA and SipL, regulates coat assembly around the forespore. SpoIVA is widely conserved among spore-forming bacteria, and its ATPase activity is essential for Bacillus subtilis to form functional spores. In this study, we determined that mutations in C. difficile SpoIVAs ATPase motifs result in relatively minor defects in spore formation in contrast with B. subtilis. Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in SipLs C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

microbiology↗

bioRxiv2017

Ligand-Mediated Biofilm Formation via Enhanced Physical Interaction Between a Diguanylate Cyclase and Its Receptor

The second messenger, cyclic dimeric GMP (c-di-GMP) regulates biofilm formation for many bacteria. The binding of c-d{inverted exclamation}-GMP by the inner-membrane protein LapD controls biofilm formation, and the LapD receptor is central to a complex network of c-di-GMP-mediated biofilm formation. In this study, we examine how c-di-GMP signaling specificity by a diguanylate cyclase (DGC), GcbC, is achieved via interactions with the LapD receptor and by small ligand sensing via GcbCs calcium channel chemotaxis (CACHE) domain. We provide evidence that biofilm formation is stimulated by the environmentally relevant organic acid citrate (and a related compound, isocitrate) in a GcbC-dependent manner through enhanced GcbC-LapD interaction, which results in increased LapA localization to the cell surface. Furthermore, GcbC shows little ability to synthesize c-di-GMP in isolation. However, when LapD is present GcbC activity is significantly enhanced ~8-fold, indicating that engaging the LapD receptor stimulates the activity of this DGC; citrate-enhanced GcbC-LapD interaction further stimulates c-di-GMP synthesis. We propose that the l-site of GcbC serves two roles beyond allosteric control of this enzyme: promoting GcbC-LapD interaction and stabilizing the active conformation of GcbC in the GcbC-LapD complex. Finally, given that LapD can interact with a dozen different DGCs of P. fluorescens, many of which have ligand-binding domains, the ligand-mediated enhanced signaling via LapD-GcbC interaction described here is likely a conserved mechanism of signaling in this network. Consistent with this idea, we identify a second example of ligand-mediated enhancement of DGC-LapD interaction that promotes biofilm formation.\n\nImportanceIn many bacteria, dozens of enzymes produce the dinucleotide signal c-di-GMP, however it is unclear how undesired crosstalk is mitigated in the context of this soluble signal, and how c-di-GMP signaling is regulated by environmental inputs. We demonstrate that GcbC, a DGC, shows little ability to synthesize c-d{inverted exclamation}-GMP in the absence of its cognate receptor LapD; GcbC-LapD interaction enhances c-di-GMP synthesis by GcbC, likely mediated by the l-site of GcbC. We further show evidence for a ligand-mediated mechanism of signaling specificity via increased physical interaction of a DGC with its cognate receptor. We envision a scenario wherein a \"cloud\" of weakly active DGCs can increase their activity by specific interaction with their receptor in response to appropriate environmental signals, concomitantly boosting c-di-GMP production, ligand-specific signaling and biofilm formation.

microbiology↗