Biology subjects

Puri, A. W.

Publications and source records attributed to Puri, A. W..

8 recordsLinked to original sources

bioRxiv2026

A frameshift mutation drives divergent biosynthesis of metallophores in Methylobacterium extorquens

Iron is widely considered the first metallocofactor, evolving as iron-sulfur clusters in early life. While iron-chelating siderophores have been widely characterized across microbial life, the lanthanide-chelating metallophore, methylolanthanin, has only recently been described in Methylobacterium extorquens AM1. Methylolanthanin shares structural similarities to the siderophore rhodopetrobactin but contains 4-hydroxybenzoate chelating moieties in place of canonical 3,4-dihydroxybenzoates. Here we compare M. extorquens AM1, which produces methylolanthanin, and the closely related M. extorquens PA1, which produces rhodopetrobactin. We present a pathway for the biosynthesis of both metallophores and describe the unusual synthesis of methylolanthanins 4-HB moieties from tyrosine. We uncover a frameshift mutation in the predicted 3-dehydroshikimate dehydratase, mllF, that prevents production of rhodopetrobactin in AM1 through truncation of the catalytically essential N-terminus. We find that deletion of the uncharacterized gene mllG reveals a cryptic branch of the pathway, leading to production of both methylolanthanin and rhodopetrobactin. Finally, we discover that rhodopetrobactin production in this mutant is enabled through the activity of a 3-dehydroshikimate dehydratase in a separate biosynthetic gene cluster. These insights highlight an evolutionary mechanism for metallophore diversification through pseudogenization and regulation of distinct biosynthetic gene clusters with shared aromatic intermediates.

microbiology↗

bioRxiv2026

Listianol, a Carotenoid Biosynthesis Inhibitor from a Methylotrophic Bacterium, Alters the Composition of a Phyllosphere Microbial Community

The microbial communities that inhabit the phyllosphere, the above-ground portion of plants, are important for plant growth and resilience. However, the natural products that mediate interactions among these organisms and with their environment remain understudied, limiting our molecular-level understanding of how phyllosphere microbial communities are structured and maintained. Natural product-mediated microbial competition is typically associated with growth inhibition, but may also occur through other mechanisms in these communities. Many bacteria produce pigments to mitigate oxidative stress, and here we identify listianol, a previously undescribed natural product that inhibits the pigmentation of diverse bacteria. Listianol blocks carotenoid biosynthesis by targeting the desaturases CrtN and CrtI. This activity sensitizes normally pigmented bacteria to UVB radiation in vitro, and a listianol-producing strain reshapes the composition of a model bacterial community on Arabidopsis thaliana under UVB exposure. Together, these findings identify pigmentation inhibition as a potential form of competition in the phyllosphere.

microbiology↗

bioRxiv2025

Inverse stable isotope labeling (InverSIL) links predicted catecholate siderophore gene clusters to their products in diverse bacteria

Bacteria produce high-affinity iron-chelating secondary metabolites called siderophores to access insoluble Fe(III) in their environments. Genome mining has revealed many predicted siderophore biosynthetic gene clusters (BGCs) in bacterial genomes, however the structures of their siderophore products remain mostly undetermined. This limits our molecular-level understanding of how bacteria acquire iron, as well as how they interact with other taxa that may use the same siderophores within bacterial communities. Here, we apply inverse stable isotope labeling (InverSIL) to rapidly connect predicted siderophore BGCs to their products. With InverSIL, bacteria are grown on 13C-substituted carbon sources and then fed predicted biosynthetic precursors at their natural isotopic abundance to identify BGC products by mass spectrometry, which removes issues with the availability of isotopically substituted precursors. We use InverSIL to determine the structures of the siderophore products of predicted BGCs from the methylotrophic genera Methylophilus and Methylobacterium, and the siderophores produced by the opportunistic pathogen Chromobacterium violaceum, which were previously shown to be essential for virulence yet remained structurally uncharacterized. We next use this approach to reveal the unexpected production of enterobactin by the genera Kushneria and Paracoccus, which was difficult to predict from genome sequences due to the distributed nature of the biosynthetic genes within the genomes. Finally, we use InverSIL to discover new siderophores, cellulochelin A and B, from the cellulose-degrading plant symbiont Cellulomonas sp. strain Leaf334. These findings demonstrate the utility of InverSIL for functional BGC characterization and expand our molecular understanding of bacterial iron acquisition strategies. IMPORTANCEIron acquisition is essential for microbial survival, and bacteria produce secondary metabolites called siderophores to scavenge iron from the environment. While bacterial genome sequences show many predicted genes for making siderophores, most remain unlinked to their metabolic products. Understanding which siderophores bacteria produce is critical for elucidating microbial iron acquisition strategies, ecological interactions, and potential roles in host-microbe interactions. Here, we demonstrate how inverse stable isotope labeling (InverSIL) can rapidly link predicted siderophore gene clusters to their corresponding metabolites. By applying InverSIL to diverse bacterial strains, we validate known siderophore products and uncover unexpected products, highlighting the limitations of current in silico predictions. This study highlights the value of combining experimental approaches with genome mining to advance our understanding of how bacteria interact with each other and their environment.

microbiology↗

bioRxiv2023

Methanotroph phenotypic heterogeneity in a methane-oxygen counter gradient

Connecting genes to phenotypic traits in bacteria is often challenging because of a lack of environmental context in laboratory settings. Laboratory-based model ecosystems offer a means to better account for environmental conditions compared to standard planktonic cultures, and can help link genotypes and phenotypes. Here, we present a simple, cost-effective, laboratory-based model ecosystem to study aerobic methane-oxidizing bacteria (methanotrophs) within the methane-oxygen counter gradient typically found in the natural environment of these organisms. Culturing the methanotroph Methylomonas sp. strain LW13 in this system resulted in formation of a distinct horizontal band at the intersection of the counter gradient, which we discovered was not due to increased numbers of viable bacteria at this location but instead to an increased amount of polysaccharides. We also discovered that different methanotrophic taxa form polysaccharide bands with distinct locations and morphologies when grown in the methane-oxygen counter gradient. By comparing transcriptomic data from LW13 growing within and surrounding this band, we identified genes upregulated within the band and validated their involvement in growth and band formation within the model ecosystem using knockout strains. Notably, deletion of these genes did not negatively affect growth using standard laboratory conditions. This work highlights the use of a laboratory-based model ecosystem that more closely mimics the natural environment to uncover methanotroph phenotypes missing from standard planktonic cultures, and to link these phenotypes with their genetic determinants.

microbiology↗

bioRxiv2022

A widespread methylotroph acyl-homoserine lactone synthase produces an atypical quorum sensing signal

Pink pigmented facultative methylotrophs of the genera Methylorubrum and Methylobacterium are omnipresent bacteria often found associated with plants. Despite their widespread occurrence, the molecular details of how these organisms interact with each other and their environment remain understudied. We analyzed genes encoding N-acylhomoserine lactone quorum sensing signal synthases in published genomes of these bacteria and determined that the product of the largest group of signal synthases had not been characterized. We subsequently identified this N-acylhomoserine lactone product using inverse stable isotopic labeling (InverSIL), which revealed an atypical signal. We then demonstrate in one representative strain that this signal activates its cognate LuxR-family transcription factor and is produced in a positive feedback loop. These results reveal a previously undescribed yet widespread signal used by pink pigmented facultative methylotrophs, which helps us understand the chemical ecology of these important bacteria.

microbiology↗

bioRxiv2022

A conserved biosynthetic gene cluster is regulated by quorum sensing in a shipworm symbiont

Bacterial symbionts often provide critical functions for their hosts. For example, wood-boring bivalves called shipworms rely on cellulolytic endosymbionts for wood digestion. However, how the relationship between shipworms and their bacterial symbionts is formed and maintained remains unknown. Quorum sensing (QS) often plays an important role in regulating symbiotic relationships. We identified and characterized a QS system found in Teredinibacter sp. strain 2052S, a gill isolate of the wood-boring shipworm Bactronophorus cf. thoracites. We determined that 2052S produces the signal N-decanoyl-L-homoserine lactone (C10-HSL), and that this signal controls activation of a biosynthetic gene cluster co-located in the symbiont genome that is conserved among all symbiotic Teredinibacter isolates. We subsequently identified extracellular metabolites associated with the QS regulon, including ones linked to the conserved biosynthetic gene cluster, using mass spectrometry-based molecular networking. Our results demonstrate that QS plays an important role in regulating secondary metabolism in this shipworm symbiont. This information provides a step towards deciphering the molecular details of the relationship between these symbionts and their hosts. Furthermore, because shipworm symbionts harbor vast yet underexplored biosynthetic potential, understanding how their secondary metabolism is regulated may aid future drug discovery efforts using these organisms. IMPORTANCEBacteria play important roles as symbionts in animals ranging from invertebrates to humans. Despite this recognized importance, much is still unknown about the molecular details of how these relationships are formed and maintained. One of the proposed roles of shipworm symbionts is the production of bioactive secondary metabolites due to the immense biosynthetic potential found in shipworm symbiont genomes. Here, we report that a shipworm symbiont uses quorum sensing to coordinate activation of its extracellular secondary metabolism, including the transcriptional activation of a biosynthetic gene cluster that is conserved among many shipworm symbionts. This work is a first step towards linking quorum sensing, secondary metabolism, and symbiosis in wood-boring shipworms.

microbiology↗

bioRxiv2021

Methylotroph Natural Product Identification by Inverse Stable Isotopic Labeling

Natural products are an essential source of bioactive compounds. Isotopic labeling is an effective way to identify natural products that incorporate a specific precursor; however, this approach is limited by the availability of isotopically-enriched precursors. We used an inverse stable isotopic labeling approach to identify natural products by growing bacteria on a 13C-carbon source and then identifying 12C-precursor incorporation by mass spectrometry. We applied this approach to methylotrophs, ecologically important bacteria predicted to have significant yet underexplored biosynthetic potential. We demonstrate this method identifies N-acyl homoserine lactone quorum sensing signals produced by diverse methylotrophs grown on three different one-carbon compounds. We then apply this approach to simultaneously identify five uncharacterized signals produced by a methylotroph, and link these compounds to their synthases. We envision that this method can be used to identify other natural product classes synthesized by methylotrophs and other organisms that grow on relatively inexpensive 13C-carbon sources.

biochemistry↗

bioRxiv2021

GNPS Dashboard: Collaborative Analysis of Mass Spectrometry Data in the Web Browser

Access to web-based platforms has enabled scientists to perform research remotely. A critical aspect of mass spectrometry data analysis is the inspection, analysis, and visualization of the raw data to validate data quality and confirm statistical observations. We developed the GNPS Dashboard, a web-based data visualization tool, to facilitate synchronous collaborative inspection, visualization, and analysis of private and public mass spectrometry data remotely.

bioinformatics↗