Biology subjects

Pierce, E. C.

Publications and source records attributed to Pierce, E. C..

6 recordsLinked to original sources

bioRxiv2024

Identification of Proteins Influencing CRISPR-Associated Transposases for Enhanced Genome Editing

CRISPR-Associated Transposases (CASTs) hold tremendous potential for microbial genome editing due to their ability to integrate large DNA cargos in a programmable and site-specific manner. However, the widespread application of CASTs has been hindered by their low efficiency in diverse, non-model bacteria. In an effort to address this shortcoming, we conducted the first genome-wide screen for host factors impacting Vibrio cholerae CAST (VchCAST) activity and used the findings to increase VchCAST editing efficiency. A genome-wide loss-of-function mutant library in E. coli was screened to identify 15 genes that impact type VchCAST transposition. Of these, seven factors were validated to improve VchCAST activity and two were found to be inhibitory. Informed by homologous recombination involved effectors, RecD and RecA, we tested the {lambda}-Red recombineering system in our VchCAST editing vectors, which increased its insertion meditated-editing efficiency by 25.7-fold in E. coli while maintaining high target specificity and similar insertion arrangements. Furthermore, {lambda}-Red-enhanced VchCAST achieved increased editing efficiency in the industrially important bacteria Pseudomonas putida and the emerging pathogen Klebsiella michiganensis. This study improves understanding of factors impacting VchCAST activity and enhances its efficiency as a bacterial genome editor. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/612086v2_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@12609e5org.highwire.dtl.DTLVardef@53a128org.highwire.dtl.DTLVardef@794e63org.highwire.dtl.DTLVardef@9835f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

O-antigen biosynthesis mediates evolutionary trade-offs within a simple community

Diverse populations of bacteriophages infect and co-evolve with their bacterial hosts. Although host recognition and infection occurs within microbiomes, the molecular mechanisms underlying host-phage interactions within a community context remain poorly studied. The biofilms (rinds) of aged cheeses contain taxonomically diverse microbial communities that follow reproducible growth patterns and can be manipulated under laboratory conditions. In this study, we use cheese as a model for studying phage-microbe interactions by identifying and characterising a tractable host-phage pair co-occurring within a model Brie community. We isolated novel bacteriophage TS33 that kills Hafnia sp. JB232 (hereafter Hafnia), a member of the model community. TS33 is easily propagated in the lab and naturally co-occurs in the cheese with the Brie community, rendering it a prime candidate for the study of host-phage interactions. We performed growth assays of the Hafnia, TS33 and the fungal community members, Geotrichum candidum and Penicillium camemberti. Employing Random Barcode Transposon Sequencing (RB-TnSeq) experiments, we identified candidate host factors that contribute to TS33 infectivity, many of which are critical to the integrity of bacterial O-antigen. Notably, disruption of these genes results in decreased susceptibility to infection by phage TS33, while simultaneously exhibiting a significant negative effect on the fitness of Hafnia in the presence of the fungi. Therefore, O-antigen mutations may have pleiotropic effects on the interactions between Hafnia and the rest of the Brie community. Ongoing and future studies aim to unearth the molecular mechanisms by which the O-antigen of Hafnia mediates its interactions with its viral and fungal partners.

microbiology↗

bioRxiv2023

Metabolomics of bacterial-fungal pairwise interactions reveal conserved molecular mechanisms

Bacterial-fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial-fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecule mediated BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis spp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles (i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and their role in complex communities should continue to be a priority.

microbiology↗

bioRxiv2022

Longitudinal, Multi-platform Metagenomics Yields a High-quality Genomic Catalog and Guides an In Vitro Model for Cheese Communities

Microbiomes are intricately intertwined with human health, geochemical cycles and food production. While many microbiomes of interest are highly complex and experimentally intractable, cheese rind microbiomes have proven powerful model systems for the study of microbial interactions. To provide a more comprehensive view of the genomic potential and temporal dynamics of cheese rind communities, we combine longitudinal, multi-platform metagenomics of three ripening washed-rind cheeses with whole genome sequencing of community isolates. Sequencing-based approaches revealed a highly reproducible microbial succession in each cheese, co-existence of closely related Psychrobacter species, and enabled the prediction of plasmid and phage diversity and their host associations. Combined with culture-based approaches, we established a genomic catalog and a paired 16-member in vitro washed rind cheese system. The combination of multi-platform metagenomic time-series data and an in vitro model provides a rich resource for further investigation of cheese rind microbiomes both computationally and experimentally. ImportanceMetagenome sequencing can provide great insights into microbiome composition and function and help researchers develop testable hypotheses. Model microbiomes, such as those composed of cheese rind bacteria and fungi, then allow the testing of these hypotheses in a controlled manner. Here, we first generate an extensive longitudinal metagenomic dataset. This dataset reveals successional dynamics, yields a phyla-spanning bacterial genomic catalog, associates mobile genetic elements with their hosts and provides insights into functional enrichment of Psychrobacter in the cheese environment. Next, we show that members of the washed-rind cheese microbiome lend themselves to in vitro community reconstruction. This paired metagenomic data and in vitro system can thus be used as a platform for generating and testing hypotheses related to the dynamics within, and functions associated with, cheese rind microbiomes.

microbiology↗

bioRxiv2020

From iron to antibiotics: Identification of conserved bacterial-fungal interactions across diverse partners

Microbial interactions are major determinants in shaping microbiome structure and function. Although fungi are found across diverse microbiomes, the mechanisms through which fungi interact with other species remain largely uncharacterized. In this work, we explore the diversity of ways in which fungi can impact bacteria by characterizing interaction mechanisms across 16 different bacterial-fungal pairs, involving 8 different fungi and 2 bacteria (Escherichia coli and Pseudomonas psychrophila). Using random barcode transposon-site sequencing (RB-TnSeq), we identified a large number of bacterial genes and pathways important in fungal interaction contexts. Within each interaction, fungal partners elicit both antagonistic and beneficial effects. Using a panel of phylogenetically diverse fungi allowed us to identify interactions that were conserved across all species. Our data show that all fungi modulate the availability of iron and biotin, suggesting that these may represent conserved bacterial-fungal interactions. Several fungi also appear to produce previously uncharacterized antibiotic compounds. Generating a mutant in a master regulator of fungal secondary metabolite production showed that fungal metabolites are key shapers of bacterial fitness profiles during interactions. This work demonstrates a diversity of mechanisms through which fungi are able to interact with bacterial species. In addition to many species-specific effects, there appear to be conserved interaction mechanisms which may be important across microbiomes.

microbiology↗

bioRxiv2018

Changes in the genetic requirements for microbial interactions with increasing community complexity

Microbial community structure and function rely on complex interactions whose underlying molecular mechanisms are poorly understood. To investigate these interactions in a simple microbiome, we introduced E. coli into an experimental community based on a cheese rind and identified the differences in E. colis genetic requirements for growth in interactive and non-interactive contexts using Random Barcode Transposon Sequencing (RB-TnSeq) and RNASeq. E. colis genetic requirements varied among pairwise growth conditions and between pairwise and community conditions. Our analysis points to mechanisms by which growth conditions change as a result of increasing community complexity and suggests that growth within a community relies on a combination of pairwise and higher order interactions. Our work provides a framework for using the model organism E. coli as a probe to investigate microbial interactions regardless of the genetic tractability of members of the studied ecosystem.

microbiology↗