A large body of theoretical and experimental work has shown that dynamics of microbiomes are shaped by the network of interbacterial interactions (Stein et al., 2013; Bucci and Xavier, 2014; Coyte et al., 2015; Hromada and Venturelli, 2023). These cooperative and competitive interactions are often achieved via the secretion of cross-feeding metabolites (Culp and Goodman, 2023), antimicrobial peptides (Heilbronner et al., 2021), and bacterially produced small molecules (Hibbing et al., 2010; Donia and Fischbach, 2015) and are crucial to ecological properties including stability and ability to respond to external perturbations (Coyte and Rakoff-Nahoum, 2019; Coyte et al., 2021). Among the competitive interactions, bacteriocin production is proposed to be a prominent mediator of microbiome dynamics (Niehus et al., 2021) and, specifically, several reports including ours have shown that a bacteriocin subclass, class IIb microcins, mediates Enterobacteriaceae dynamics in vivo (Sassone-Corsi et al., 2016; Mortzfeld et al., 2022; Cherrak et al., 2024).
Class IIb microcins are ribosomally synthesized bacteriocins between 5 kDa and 10 kDa in size with activity against closely related strains or species (Mortzfeld et al., 2022; de Lorenzo, 1984; Vassiliadis et al., 2010; Palmer et al., 2020; Baquero et al., 2019). Unlike all other microcins, they carry a serine-rich C-terminal motif for a posttranslational modification with a siderophore, here an enterobactin or an enterobactin derivative, before they are secreted into the extracellular space (Azpiroz et al., 2001). Siderophores are iron-chelating molecules commonly employed by various bacteria to scavenge free iron to compete with other bacteria, particularly in resource-scarce environments such as the gastrointestinal tract (Vassiliadis et al., 2010; Palmer et al., 2020; Patzer et al., 2003) and are often associated with increased pathogenicity or virulence (Miethke and Marahiel, 2007; Wilson et al., 2016; Khasheii et al., 2021). The iron-chelating moiety of these posttranslationally modified antimicrobial peptides is recognized by high-affinity receptors and functions as a Trojan Horse key to susceptible bacteria as it triggers import into the periplasmic space, where the peptide inhibits the molecular target of susceptible bacteria (Patzer et al., 2003; Bieler et al., 2006; Destoumieux-Garzón et al., 2003; Rodríguez and Laviña, 2003). Because of these features, delivery of class IIb microcins by wildtype and engineered probiotics has been recently proposed as a strategy to combat drug-resistant enteric bacteria (Sassone-Corsi et al., 2016; Mortzfeld et al., 2022; Palmer et al., 2020; Palmer et al., 2018), which is in line with a growing body of work from the past decade that explores siderophore conjugation, including with enterobactin, to specifically deliver antibiotics and other small molecules to drug-resistant Gram-negative pathogens (Page, 2019; Negash et al., 2019; Rayner et al., 2023).
To date only five class IIb microcins have been described and only four have been characterized in terms of their antimicrobial activity. Specifically, the class IIb microcins MccE492 and MccG492 (uncharacterized) are solely present in Klebsiella pneumoniae (Kp), whereas MccH47 is specific for Escherichia coli (Ec) (Vassiliadis et al., 2010). Additionally, truncated versions of mciA (MccI47) and mcmM (MccM) are present in Kp RYC492, whereas they are intact in the Ec CA46 genome (Vassiliadis et al., 2010). Interestingly, while the genes encoding for microcin posttranslational modifications are highly conserved between Ec and Kp, suggesting a conserved pathway for microcin maturation, the toxin and corresponding immunity genes are significantly more variable (Figure 1—figure supplement 1). We hypothesized that class IIb microcin production extends beyond these specific compounds and organisms and identified a total of 12 novel class IIb microcins in seven additional Enterobacteriaceae species. Utilizing heterologous expression of these compounds in our Ec system optimized for enterobactin conjugation, we show potent antimicrobial activity by the encoded toxins against a library of bacteria, including Gram-negative ESKAPE and plant pathogens. This demonstrates that class IIb microcin genes are more prevalent in the microbial world than previously recognized and that synthetic hybrid microcins can be a viable tool to target clinically relevant drug-resistant pathogens.