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.

With the hypothesis that class IIb microcin production is a common trait among Enterobacteriaceae, we anticipated that the genes encoding for the antimicrobial and immunity peptides would exhibit a high degree of dissimilarity to already known peptides as target specificity may result in accelerated adaptive coevolution (Paterson et al., 2010). Therefore, in addition to the known microcin and immunity genes from MccE492, MccG492, MccH47, MccI47, and MccM, we included in our informatic approach the genes necessary for mature class IIb microcin biosynthesis, extending our search to longer sequences for more reliable Basic Local Alignment Search Tool (BLAST) results (Boratyn et al., 2012). Moreover, we hypothesized that the amino acid sequences of genes responsible for posttranslational modification and microcin export would be less prone to evolutionary changes, thereby maintaining the functional integrity of the gene cluster (Vassiliadis et al., 2010; Lynch et al., 2016). We then assessed their proximity in the respective genome location, because microcin genes are typically flanked by genes essential for toxin maturation (Vassiliadis et al., 2010). Further, we manually assessed and annotated small open reading frames (ORFs) upstream and downstream of the maturation genes, allowing us to also identify novel class IIb microcins without significant sequence similarity to the known antimicrobials, enabling the discovery of compounds with new molecular targets or modes of action (see Materials and methods).

Our informatics-driven analysis identified 12 promising class IIb microcin candidates from seven gene clusters with high similarity to Ec CA46 and Kp RYC492 in seven species across the Enterobacteriaceae family (Figure 1A, Figure 1—figure supplement 2): (i) Brenneria goodwinii (Bg; 2; GenBank: CP014137), (ii) Gibbsiella quercinecans (Gq; 1; CP014136), (iii) Klebsiella oxytoca (Ko; 1; CP033844), (iv) Pantoea sp. (Ps; 1; CP034363), (v) Raoultella ornithinolytica (Ro; 4; CP008886), (vi) Salmonella enterica (Se; 2; CP030220), (vii) Serratia fonticola (Sf; 1; CP033055). Although it has traditionally been a defining characteristic of class IIb microcins that all required genes are encoded within the chromosome (Sassone-Corsi et al., 2016), the gene cluster we discovered for Se is situated on a 159 kbp plasmid. Phylogenetic sequence analysis of both the antimicrobial and immunity peptide genes revealed the presence of eight different clades represented in both trees, respectively (Figure 1B and C). Regarding the well-established class IIb microcins MccH47, MccI47, MccM, MccG492, and MccE492, we identified novel members for each group, supported by nucleotide sequence similarity, amino acid identity, the closest blastp match, and domain predictions (Table 1, Figure 1—figure supplement 3). It is important to note that application of established tools for secondary metabolite identification (e.g. antiSMASH 7.0) (Blin et al., 2023) to these genomes did not yield identification of any of the old or novel microcins providing support of the relevance of our approach. In order to then ensure that these novel microcins are unique and not part of any other microcin class, we performed phylogenetic analysis for all known microcin genes from the classes I, IIa, and IIb and show distinct clustering for all newly described sequences (Figure 1—figure supplement 4). In light of this discovery, we propose a new nomenclature for class IIb microcins that includes the species initials in which they were identified (e.g. Ec, Kp), the closest relative already characterized class IIb microcin (G492, E492, H47, I47 or M), as well as the identifiers ‘A’ for antimicrobial or ‘I’ for immunity gene.

Figure 1 with 4 supplements see all

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Based on this, the novel G492 relative found in S. enterica will be called Se G492 with the antimicrobial peptide identified as Se G492A and the immunity peptide identified as Se G492I. It is worth highlighting that in the case of the G492 group, all its members have the immunity gene located downstream of the antimicrobial gene, whereas for the other clades, this arrangement is reversed. In addition to uncovering eight novel variants of the five previously characterized microcins, we have identified four additional microcins through manual curation of ORFs in proximity to the microcin maturation genes. These novel microcins, which we name microcin W (MccW), microcin X (MccX), and microcin Z (MccZ), seem to belong to three entirely new clades based on nucleotide similarity (Figure 1B and C). The two members of the microcin X group, found in B. goodwinii (Bg X) and R. ornithinolytica (Ro X), only show significant similarity between one another, but not to any of the other antimicrobial or immunity peptides. This holds true for the nucleotide similarity (Figure 1B and C) as well as amino acid identity and the closest blastp hits (Figure 1D, Table 1, Supplementary file 1). Similarly, MccW from G. quercinecans (Gq W) does not show any sequence similarity to either the known or novel antimicrobial or immunity peptides in terms of nucleotide similarity, amino acid identity, the respective blastp hits, or phylogenetic localization (Figure 1, Table 1, Supplementary file 1). Lastly, MccZ from R. ornithinolytica (Ro Z) shows insignificant amino acid similarity with Ec MA (mcmA) for the antimicrobial, whereas the immunity peptide does not have any match among the known or the novel microcins (Figure 1C, Table 1, Supplementary file 1). Crucially, the identification of MccX, and MccZ within the same gene clusters as representatives of the E492 (Bg E492), H47 (Ro H47), and I47 (Ro I47) groups, strongly implies that they are functional components of a microcin gene cluster.

To test the newly identified microcins for antimicrobial activity, we used our previously established Ec overexpression system (Mortzfeld et al., 2022; Palmer et al., 2020). All antimicrobial and immunity peptides were codon optimized, synthesized, and cloned into an inducible high copy vector (see Materials and methods). Thus, we extracted the novel microcins out of their native genomic context of siderophore biosynthesis and transferred them into a heterologous expression background optimized for microcin-monoglycosylated enterobactin (MGE) linkage (Mortzfeld et al., 2022; Palmer et al., 2020). This allowed us to create hybrid compounds that could be efficiently tested for antimicrobial activity in an E. coli background. Through static plate inhibition assays involving live-producing cells (Mortzfeld et al., 2022; Palmer et al., 2020; Palmer et al., 2018), we successfully validated robust antimicrobial activity of 11 out of the 12 newly discovered microcins (Figure 2A). Notably, antimicrobial activity was only observed in iron-depleted media (Figure 2B and C). The hybrid microcins exhibit a range of specificities, with some inhibiting targets narrowly (e.g. Ps G492AI), while others exert a broader effect against multiple bacteria (e.g. Se G492AI). Moreover, our study also provides the first evidence of inhibitory activity by Kp G492, a microcin whose existence and function have only been proposed in the scientific literature based on genetic sequence (Vassiliadis et al., 2010).

Figure 2

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To this date class IIb microcins have been only shown to be very selective and only active against different species within the Enterobacteriaceae family (Mortzfeld et al., 2022; Vassiliadis et al., 2010; Palmer et al., 2020; Palmer et al., 2018). While the activity for the novel microcins varies, we here report, for the first time, antimicrobial activity outside of the Enterobacteriaceae family utilizing hybrid antimicrobial peptides. We demonstrate that microcins Ps G492 and Se G492 have activity against Gram-negative multidrug-resistant ESKAPE pathogens with both being capable of inhibiting A. baumannii (BAA 1790), and with microcin Se G492 alone also showing activity against P. aeruginosa (PA14) (Figure 2). Specifically, compared to K. pneumoniae (BAA 1705) Se G492 is 256 times more effective against A. baumannii (BAA 1790), 128 times more effective against E. coli (BAA 196), and 8 times more effective against P. aeruginosa (PA14) (Figure 2B).

With a comprehensive analysis of publicly available bacterial genomes, we unraveled 12 previously undiscovered class IIb microcins. Among these findings, we identified three novel microcin clades, specifically MccW, MccX, and MccZ. Through heterologous expression, we showed antimicrobial activity for all but one novel microcins and are the first to demonstrate activity for the known class IIb microcin Kp G492. Hence, this research demonstrates that class IIb microcin genes exhibit a higher prevalence in Enterobacteriaceae genomes than previously reported. As a result, their impact on ecological community dynamics in natural environments, including the growth of Pseudomonadales species, might be broader than previously thought. For antimicrobial activity testing, microcin and immunity genes were overexpressed recombinantly in our Ec-derived expression system optimized for microcin-MGE production (Mortzfeld et al., 2022; Palmer et al., 2020). The common process of posttranslational modification with the siderophore consolidated the import mechanism of the hybrid microcins toward enterobactin, the most characteristic siderophore of the Enterobacteriaceae family. This allowed us to test the target-specific antimicrobial activity of the microcins irrespective of siderophore production in the native genomic background. However, it is important to note that class IIb microcin activity is dependent on active import through siderophore receptors and consequently some of these microcins might display different activity spectrums when tested in their native genomic background of siderophore biosynthesis. Furthermore, static plate inhibition assays exhibit lower sensitivity compared to purification approaches with quantitative minimum inhibitory concentration (MIC) assays. Thus, the activity spectrums of the hybrid microcins could encompass a wider range than what has been described in this study when tested as a purified product. However, historically the microcin literature proves that ideal approaches for purification and MIC testing can vary between the antimicrobials (Mortzfeld et al., 2022; Vassiliadis et al., 2010; Palmer et al., 2020; Destoumieux-Garzón et al., 2003).

We were able to expand the origins of class IIb microcins from the enteric bacteria Ec and Kp to other members of the Enterobacteriaceae family, including well-known phytopathogens (Denman et al., 2012; Brady et al., 2010; Allahverdipour et al., 2020). Specifically, B. goodwinii and G. quercinecans are associated with acute oak decline and are frequently isolated together (Denman et al., 2018) and the two strains containing microcin genes were isolated within the same research project. Notably, these bacteria grow synergistically (Brady et al., 2022), while upregulating iron transporters during co-culture (Doonan et al., 2020), hinting at class IIb microcin-related competition. We were able to show activity of the overexpressed hybrid microcins against human-derived enteric isolates, however, their native spectrum might have evolved to target more frequently encountered strains from the genus Brenneria or Gibbsiella. Further, we demonstrated activity of several class IIb microcins against the three tree pathogen genera Brenneria, Gibbsiella, as well as Rahnella (Brady et al., 2022; Brady et al., 2014; Hajialigol et al., 2023; Moradi-Amirabad and Khodakaramian, 2020; Poret-Peterson et al., 2019). Thus, treatment with potent microcins, purified or produced in live bacteria, could present a viable option to target bacteria-caused plant diseases.

In health care settings the burden by Gram-negative ESKAPE pathogens and multidrug-resistant Enterobacteriaceae weighs heavily on modern medicine and novel antimicrobials are needed to develop new treatment options (WHO, 2014; World Health Organization, 2017). In addition to enteric pathogens and pathobionts, bacteria outside of the Enterobacteriaceae family have also been shown to scavenge for and to import enterobactin, including P. aeruginosa and A. baumannii (Moynié et al., 2019; Subashchandrabose et al., 2016). Therefore, different siderophore conjugates could be a viable option to target these pathogens as well or to fine-tune the desired target range (Page, 2019; Negash et al., 2019; Rayner et al., 2023). Antimicrobial peptides and particularly microcins are promising candidates for selective eradication of enteric pathogens and have been demonstrated to potently reduce pathogen colonization in vivo, when produced by a live probiotic (Sassone-Corsi et al., 2016; Mortzfeld et al., 2022). Here, we present the most comprehensive library of class IIb microcins created so far, that is suited for heterologous expression and in vivo application for the development of novel live biotherapeutic products against drug-resistant enteric bacteria and Gram-negative ESKAPE pathogens.

In this study, we challenge the prevailing notion that class IIb microcin production is limited to Ec and Kp. Through comprehensive genomic analysis of publicly available bacterial genomes, coupled with heterologous overexpression, we unveiled a set of undiscovered class IIb microcins across Enterobacteriaceae species. Our findings not only expand the known repertoire of class IIb microcins but also hold significant implications for synthetic hybrid compounds. We demonstrate that these newly identified class IIb microcins exert remarkable inhibitory effects on ESKAPE pathogen species when expressed in a system for enterobactin-derived conjugation. This discovery underscores their potential as agents against a broader spectrum of pathogens, including those affecting humans and plants, thus opening new avenues for antimicrobial research and applications.

The genomes are accessible with the following GenBank numbers: Brenneria goodwinii (CP014137), Gibbsiella quercinecans (CP014136), Klebsiella oxytoca (CP033844), Pantoea sp. (CP034363), Raoultella ornithinolytica (CP008886), Salmonella enterica (CP030220), Serratia fonticola (CP033055). All information is included in the manuscript or supporting files. All plasmid sequences as well as annotation files to produce Figure 1A, Figure 1-figure supplement 1, Figure 1-figure supplement 2, and Figure 1-figure supplement 3 are available as supplementary material.

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Author details

1. Benedikt M Mortzfeld

   1. Program in Microbiome Dynamics, University of Massachusetts Chan Medical School, Worcester, United States
   2. Department of Microbiology, University of Massachusetts Chan Medical School, Worcester, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   benedikt.mortzfeld@umassmed.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0563-8970

2. Shakti K Bhattarai

   1. Program in Microbiome Dynamics, University of Massachusetts Chan Medical School, Worcester, United States
   2. Department of Microbiology, University of Massachusetts Chan Medical School, Worcester, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9118-9184

3. Vanni Bucci

   1. Program in Microbiome Dynamics, University of Massachusetts Chan Medical School, Worcester, United States
   2. Department of Microbiology, University of Massachusetts Chan Medical School, Worcester, United States
   3. Program in Systems Biology, University of Massachusetts Chan Medical School, Worcester, United States

   Contribution

   Conceptualization, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing

   Competing interests

   receives support from a sponsored research agreement from Vedanta Biosciences, Inc

   "This ORCID iD identifies the author of this article:" 0000-0002-3257-2922

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