Bacteria use two-component system (TCS) signaling pathways to sense and respond to peptides involved in host defense, quorum sensing and inter-bacterial warfare. However, little is known about the broad peptide-sensing capabilities of TCSs. In this study, we developed an Escherichia coli display method to characterize the effects of human antimicrobial peptides (AMPs) on the pathogenesis-regulating TCS PhoPQ of Salmonella Typhimurium with much higher throughput than previously possible. We found that PhoPQ senses AMPs with diverse sequences, structures and biological functions. We further combined thousands of displayed AMP variants with machine learning to identify peptide sub-domains and biophysical features linked to PhoPQ activation. Most of the newfound AMP activators induce PhoPQ in S. Typhimurium, suggesting possible roles in virulence regulation. Finally, we present evidence that PhoPQ peptide-sensing specificity has evolved across commensal and pathogenic bacteria. Our method enables new insights into the specificities, mechanisms and evolutionary dynamics of TCS-mediated peptide sensing in bacteria.

Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyzes (p)ppGpp to counteract the synthetase activity of RelA. However, structural information about how SpoT controls the levels of (p)ppGpp is missing. Here we present the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and uncover the mechanism of intramolecular regulation of 'long'-stringent factors. In contrast to ribosome-associated Rel/RelA that adopt an elongated structure, SpoT assumes a compact τ-shaped structure in which the regulatory domains wrap around a Core subdomain that controls the conformational state of the enzyme. The Core is key to the specialization of long RelA-SpoT homologs toward either synthesis or hydrolysis: the short and structured Core of SpoT stabilizes the τ-state priming the hydrolase domain for (p)ppGpp hydrolysis, whereas the longer, more dynamic Core domain of RelA destabilizes the τ-state priming the monofunctional RelA for efficient (p)ppGpp synthesis.

Synthetic biology has shown remarkable potential to program living microorganisms for applications. However, a notable discrepancy exists between the current engineering practice-which focuses predominantly on planktonic cells-and the ubiquitous observation of microbes in nature that constantly alternate their lifestyles on environmental variations. Here we present the de novo construction of a synthetic genetic program that regulates bacterial life cycle and enables phase-specific gene expression. The program is orthogonal, harnessing an engineered protein from 45 candidates as the biofilm matrix building block. It is also highly controllable, allowing directed biofilm assembly and decomposition as well as responsive autonomous planktonic-biofilm phase transition. Coupling to synthesis modules, it is further programmable for various functional realizations that conjugate phase-specific biomolecular production with lifestyle alteration. This work establishes a versatile platform for microbial engineering across physiological regimes, thereby shedding light on a promising path for gene circuit applications in complex contexts.

Promiscuous enzymes that modify peptides and proteins are powerful tools for labeling biomolecules; however, directing these modifications to desired substrates can be challenging. Here, we use computational interface design to install a substrate recognition domain adjacent to the active site of a promiscuous enzyme, catechol O-methyltransferase. This design approach effectively decouples substrate recognition from the site of catalysis and promotes modification of peptides recognized by the recruitment domain. We determined the crystal structure of this novel multidomain enzyme, SH3-588, which shows that it closely matches our design. SH3-588 methylates directed peptides with catalytic efficiencies exceeding the wild-type enzyme by over 1,000-fold, whereas peptides lacking the directing recognition sequence do not display enhanced efficiencies. In competition experiments, the designer enzyme preferentially modifies directed substrates over undirected substrates, suggesting that we can use designed recruitment domains to direct post-translational modifications to specific sequence motifs on target proteins in complex multisubstrate environments.