Biology subjects

Hammond, M. C.

Publications and source records attributed to Hammond, M. C..

6 recordsLinked to original sources

bioRxiv2026

Glycine detection with a nuclease-stable L-RNA sensor

Glycine is a vital extracellular signal in bacteria, plants, and the brain. Although RNA-based sensors detect glycine in cells, their extracellular application in native biological environments is limited by enzymatic degradation from nucleases. Mirror-image RNA is nuclease-resistant and preserves the tertiary structure required for RNA function, but synthesizing long L-RNAs such as the 170-nt glycine sensor (glyS) remains challenging. Here, we applied cross-chiral ligation with natural D-RNA ribozymes to assemble a mirror-image L-RNA glycine sensor (L-glyS). Optimization of the ligation conditions enabled up to 68% conversion to the full-length sensor. L-glyS displayed nuclease resistance and maintained glycine-dependent fluorescence in serum, where the original D-glyS lost function. These results establish cross-chiral ligation as a strategy for constructing long, functional L-RNAs and broaden the possible applications of RNA-based sensors to extracellular detection of small molecules.

synthetic biology↗

bioRxiv2026

Development of a Fluorescent RNA Biosensor for Dual Detection of cGAMP and c-di-GMP Signals in Live Bacteria

Cyclic dinucleotide (CDN) signaling molecules, such as cyclic di-GMP (c-di-GMP) and 3,3 cyclic GMP-AMP (cGAMP), are second messengers that play critical roles in phenotypic regulation, such as biofilm formation, host colonization, and bacterial virulence. Recently, hybrid promiscuous (Hypr) GGDEF proteins have been identified in certain bacteria to produce both cyclic dinucleotides. One such enzyme, Bd0367, from the predatory Bdellovibrio bacteriovorus, switches between synthesizing c-di-GMP and cGAMP to regulate the bacterial predation cycle and prey exit. However, the molecular mechanism controlling this switch remains unknown. Here, we introduce an RNA-based ratiometric, dual metabolite biosensor that enables simultaneous detection of c-di-GMP and cGAMP in live cells. This sensor integrates a Pepper-based biosensor for c-di-GMP detection and a Spinach2-based biosensor for cGAMP detection into a single transcript, producing distinct fluorescent outputs. In E. coli, the dual metabolite sensor reliably reported shifts in c-di-GMP/cGAMP production ratios from various CDN synthases, including Bd0367. Additionally, a histidine kinase was discovered as the probable regulatory partner of Bd0367. These findings demonstrate the sensors capacity to assess relative CDN levels and to uncover complex signaling pathways. Together, this ratiometric dual metabolite biosensor provides a foundation for broader applications of fluorogenic RNA biosensors in dissecting bacterial signaling networks, microbial ecology, and host-pathogen interactions.

synthetic biology↗

bioRxiv2024

A Conditional Degron Tag for RNA Sensing in Bacteria

Here we report the development of a conditional degron system for use in bacteria in which a tagged fluorescent protein (FP) reporter, mVenus-S1, is stabilized only in the presence of TAR RNA. The functional design takes advantage of the bipartite nature of the ssrA degron sequence, so that TAR RNA binding to the inserted Tat peptide sequence blocks degron recognition to rescue protein activity. Several fluorescent proteins and a chemiluminescent enzyme can be tagged and activated using this system, and our results reveal that chromophore maturation time correlates with fold change, with up to [~]60-fold turn-on. Finally, we designed a functional biosensor for oxyS sRNA and integrated it into a single transcript in which the FP and RNA-based biosensor are encoded on the same mRNA. To our knowledge, this is the first proof-of-concept for a novel biosensor design that expresses both protein and RNA components from a single transcript. The streamlined system is fully genetically encoded, does not require an exogenous fluorophore, and permits both components to be coordinated by expression from the same promoter.

synthetic biology↗

bioRxiv2019

Ratiometric bioluminescent sensors towards in vivo imaging of bacterial signaling

Second messenger signaling networks allow cells to sense and adapt to changing environmental conditions. In bacteria, the nearly ubiquitous second messenger molecule cyclic di-GMP coordinates diverse processes such as motility, biofilm formation, and virulence. In bacterial pathogens, these signaling networks allow the bacteria to survive changing environment conditions that are experienced during infection of a mammalian host. While studies have examined the effects of cyclic di-GMP levels on virulence in these pathogens, it has previously not been possible to visualize cyclic di-GMP levels in real time during the stages of host infection. Towards this goal, we generate the first ratiometric, chemiluminescent biosensor scaffold that selectively responds to c-di-GMP. By engineering the biosensor scaffold, a suite of Venus-YcgR-NLuc (VYN) biosensors is generated that provide extremely high sensitivity (KD < 300 pM) and large BRET signal changes (up to 109%). As a proof-of-concept that VYN biosensors can image cyclic di-GMP during host infection, we show that the VYN biosensors function in the context of a tissue phantom model, with only [~]103-104 biosensor-expressing cells required for the measurement. Furthermore, the stable BRET signal suggests that the sensors could be used for long-term imaging of cyclic di-GMP dynamics during host infection. The VYN sensors developed here can serve as robust in vitro diagnostic tools for high throughput screening, as well as genetically encodable tools for monitoring the dynamics of c-di-GMP in live cells, and lay the groundwork for live cell imaging of c-di-GMP dynamics in bacteria during host infection, and other complex environments.

synthetic biology↗

bioRxiv2019

Second messengers and divergent HD-GYP enzymes regulate 3’,3’-cGAMP signaling

3,3-cyclic GMP-AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have appeared narrowly distributed based upon the characterized synthases, DncV and Hypr GGDEFs. Here we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di-GMP, which provides evidence for cross-regulation between different CDN pathways. Finally, we reveal that the HD-GYP enzyme PmxA is a cGAMP-specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non-canonical HD-GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling.

biochemistry↗

bioRxiv2018

cGAMP signaling controls a transient surface-associated lifestyle

A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3,3-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 [A]-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.

biochemistry↗