From b4677c649772bca6e129f33f5e71d55efbd0f902 Mon Sep 17 00:00:00 2001 From: LinaKind Date: Thu, 12 Dec 2024 13:36:01 +0000 Subject: [PATCH] Add an article with an elife assessment https://github.com/elifesciences/issues/issues/9034 --- data/articles/102912.json | 4195 +++++++++++++++++++++++++++++++++++++ 1 file changed, 4195 insertions(+) create mode 100644 data/articles/102912.json diff --git a/data/articles/102912.json b/data/articles/102912.json new file mode 100644 index 00000000..4382c935 --- /dev/null +++ b/data/articles/102912.json @@ -0,0 +1,4195 @@ +{ + "sentForReview": "2024-09-14", + "versions": [ + { + "status": "preprint", + "description": "This manuscript was published as a preprint.", + "uri": "https://doi.org/10.1101/2023.12.05.570296", + "date": "2024-08-29T00:00:00Z" + }, + { + "status": "preprint", + "description": "This manuscript was published as a reviewed preprint.", + "uri": "https://doi.org/10.7554/eLife.102912.1", + "date": "2024-11-05T00:00:00Z" + }, + { + "status": "vor", + "id": "102912", + "version": 1, + "type": "short-report", + "doi": "10.7554/eLife.102912", + "doiVersion": "10.7554/eLife.102912.2", + "authorLine": "Benedikt M Mortzfeld, Shakti K Bhattarai, Vanni Bucci", + "title": "Novel class IIb microcins show activity against Gram-negative ESKAPE and plant pathogens", + "published": "2024-12-11T00:00:00Z", + "versionDate": "2024-12-11T00:00:00Z", + "volume": 13, + "elocationId": "RP102912", + "pdf": "https://cdn.elifesciences.org/articles/102912/elife-102912-v1.pdf", + "xml": "https://cdn.elifesciences.org/articles/102912/elife-102912-v1.xml", + "figuresPdf": "https://cdn.elifesciences.org/articles/102912/elife-102912-figures-v1.pdf", + "subjects": [ + { + "id": "ecology", + "name": "Ecology" + }, + { + "id": "microbiology-infectious-disease", + "name": "Microbiology and Infectious Disease" + } + ], + "researchOrganisms": [ + "E. coli" + ], + "abstract": { + "content": [ + { + "text": "Interspecies interactions involving direct competition via bacteriocin production play a vital role in shaping ecological dynamics within microbial ecosystems. For instance, the ribosomally produced siderophore bacteriocins, known as class IIb microcins, affect the colonization of host-associated pathogenic Enterobacteriaceae species. Notably, to date, only five of these antimicrobials have been identified, all derived from specific Escherichia coli and Klebsiella pneumoniae strains. We hypothesized that class IIb microcin production extends beyond these specific compounds and organisms. With a customized informatics-driven approach, screening bacterial genomes in public databases with BLAST and manual curation, we have discovered 12 previously unknown class IIb microcins in seven additional Enterobacteriaceae species, encompassing phytopathogens and environmental isolates. We introduce three novel clades of microcins (MccW, MccX, and MccZ), while also identifying eight new variants of the five known class IIb microcins. To validate their antimicrobial potential, we heterologously expressed these microcins in E. coli and demonstrated efficacy against a variety of bacterial isolates, including plant pathogens from the genera Brenneria, Gibbsiella, and Rahnella. Two newly discovered microcins exhibit activity against Gram-negative ESKAPE pathogens, i.e., Acinetobacter baumannii or Pseudomonas aeruginosa, providing the first evidence that class IIb microcins can target bacteria outside of the Enterobacteriaceae family. This study underscores 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. Our findings hold significant promise for the development of innovative engineered live biotherapeutic products tailored to combat these resilient bacteria.", + "type": "paragraph" + } + ] + }, + "copyright": { + "license": "CC-BY-4.0", + "holder": "Mortzfeld et al.", + "statement": "This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited." + }, + "authors": [ + { + "affiliations": [ + { + "address": { + "components": { + "country": "United States", + "locality": [ + "Worcester" + ] + }, + "formatted": [ + "Worcester", + "United States" + ] + }, + "name": [ + "Program in Microbiome Dynamics, University of Massachusetts Chan Medical School" + ] + }, + { + "address": { + "components": { + "country": "United States", + "locality": [ + "Worcester" + ] + }, + "formatted": [ + "Worcester", + "United States" + ] + }, + "name": [ + "Department of Microbiology, University of Massachusetts Chan Medical School" + ] + } + ], + "competingInterests": "No competing interests declared", + "contribution": "Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing", + "emailAddresses": [ + "benedikt.mortzfeld@umassmed.edu" + ], + "name": { + "index": "Mortzfeld, Benedikt M", + "preferred": "Benedikt M Mortzfeld" + }, + "orcid": "0000-0003-0563-8970", + "type": "person" + }, + { + "affiliations": [ + { + "address": { + "components": { + "country": "United States", + "locality": [ + "Worcester" + ] + }, + "formatted": [ + "Worcester", + "United States" + ] + }, + "name": [ + "Program in Microbiome Dynamics, University of Massachusetts Chan Medical School" + ] + }, + { + "address": { + "components": { + "country": "United States", + "locality": [ + "Worcester" + ] + }, + "formatted": [ + "Worcester", + "United States" + ] + }, + "name": [ + "Department of Microbiology, University of Massachusetts Chan Medical School" + ] + } + ], + "competingInterests": "No competing interests declared", + "contribution": "Conceptualization, Resources, Data curation, Software, Formal analysis, Investigation, Methodology, Writing – review and editing", + "name": { + "index": "Bhattarai, Shakti K", + "preferred": "Shakti K Bhattarai" + }, + "orcid": "0000-0001-9118-9184", + "type": "person" + }, + { + "affiliations": [ + { + "address": { + "components": { + "country": "United States", + "locality": [ + "Worcester" + ] + }, + "formatted": [ + "Worcester", + "United States" + ] + }, + "name": [ + "Program in Microbiome Dynamics, University of Massachusetts Chan Medical School" + ] + }, + { + "address": { + "components": { + "country": "United States", + "locality": [ + "Worcester" + ] + }, + "formatted": [ + "Worcester", + "United States" + ] + }, + "name": [ + "Department of Microbiology, University of Massachusetts Chan Medical School" + ] + }, + { + "address": { + "components": { + "country": "United States", + "locality": [ + "Worcester" + ] + }, + "formatted": [ + "Worcester", + "United States" + ] + }, + "name": [ + "Program in Systems Biology, University of Massachusetts Chan Medical School" + ] + } + ], + "competingInterests": "receives support from a sponsored research agreement from Vedanta Biosciences, Inc", + "contribution": "Conceptualization, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing", + "name": { + "index": "Bucci, Vanni", + "preferred": "Vanni Bucci" + }, + "orcid": "0000-0002-3257-2922", + "type": "person" + } + ], + "reviewers": [ + { + "affiliations": [ + { + "address": { + "components": { + "country": "Colombia" + }, + "formatted": [ + "Colombia" + ] + }, + "name": [ + "CorpoGen" + ] + } + ], + "name": { + "index": "Zambrano, María Mercedes", + "preferred": "María Mercedes Zambrano" + }, + "role": "Reviewing Editor", + "type": "person" + }, + { + "affiliations": [ + { + "address": { + "components": { + "country": "United States" + }, + "formatted": [ + "United States" + ] + }, + "name": [ + "Harvard T.H. Chan School of Public Health" + ] + } + ], + "name": { + "index": "Garrett, Wendy S", + "preferred": "Wendy S Garrett" + }, + "role": "Senior Editor", + "type": "person" + } + ], + "funding": { + "awards": [ + { + "awardId": "W81XWH2020013", + "id": "fund1", + "recipients": [ + { + "name": { + "index": "Bucci, Vanni", + "preferred": "Vanni Bucci" + }, + "type": "person" + } + ], + "source": { + "funderId": "10.13039/100000090", + "name": [ + "Congressionally Directed Medical Research Programs" + ] + } + }, + { + "awardId": "1R01AG075283-01A1", + "id": "fund2", + "recipients": [ + { + "name": { + "index": "Bucci, Vanni", + "preferred": "Vanni Bucci" + }, + "type": "person" + } + ], + "source": { + "funderId": "10.13039/100000002", + "name": [ + "National Institutes of Health" + ] + } + }, + { + "awardId": "457837076", + "id": "fund3", + "recipients": [ + { + "name": { + "index": "Mortzfeld, Benedikt M", + "preferred": "Benedikt M Mortzfeld" + }, + "type": "person" + } + ], + "source": { + "funderId": "10.13039/501100001659", + "name": [ + "Deutsche Forschungsgemeinschaft" + ] + } + } + ], + "statement": "The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication." + }, + "additionalFiles": [ + { + "caption": [ + { + "text": "Red color indicates no significant match found.", + "type": "paragraph" + } + ], + "filename": "elife-102912-supp1-v1.xlsx", + "id": "supp1", + "label": "Supplementary file 1", + "mediaType": "application/xlsx", + "title": "Blastp results and closest matches to the known or novel class IIb microcins.", + "uri": "https://cdn.elifesciences.org/articles/102912/elife-102912-supp1-v1.xlsx" + }, + { + "filename": "elife-102912-mdarchecklist1-v1.pdf", + "id": "mdar", + "label": "MDAR checklist", + "mediaType": "application/pdf", + "uri": "https://cdn.elifesciences.org/articles/102912/elife-102912-mdarchecklist1-v1.pdf" + } + ], + "dataSets": { + "availability": [ + { + "text": "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.", + "type": "paragraph" + } + ], + "used": [ + { + "authors": [ + { + "name": { + "index": "Minogue, T", + "preferred": "Minogue T" + }, + "type": "person" + }, + { + "name": { + "index": "Wolcott, M", + "preferred": "Wolcott M" + }, + "type": "person" + }, + { + "name": { + "index": "Wasieloski, L", + "preferred": "Wasieloski L" + }, + "type": "person" + }, + { + "name": { + "index": "Aguilar, W", + "preferred": "Aguilar W" + }, + "type": "person" + }, + { + "name": { + "index": "Moore, D", + "preferred": "Moore D" + }, + "type": "person" + }, + { + "name": { + "index": "Jaissle, J", + "preferred": "Jaissle J" + }, + "type": "person" + }, + { + "name": { + "index": "Tallon, LJ", + "preferred": "Tallon LJ" + }, + "type": "person" + }, + { + "name": { + "index": "Sadzewicz, L", + "preferred": "Sadzewicz L" + }, + "type": "person" + }, + { + "name": { + "index": "Zhao, X", + "preferred": "Zhao X" + }, + "type": "person" + }, + { + "name": { + "index": "Vavikolanu, K", + "preferred": "Vavikolanu K" + }, + "type": "person" + }, + { + "name": { + "index": "Mehta, A", + "preferred": "Mehta A" + }, + "type": "person" + }, + { + "name": { + "index": "Aluvathingal, J", + "preferred": "Aluvathingal J" + }, + "type": "person" + }, + { + "name": { + "index": "Nadendla, S", + "preferred": "Nadendla S" + }, + "type": "person" + }, + { + "name": { + "index": "Yan, Y", + "preferred": "Yan Y" + }, + "type": "person" + }, + { + "name": { + "index": "Sichtig, H", + "preferred": "Sichtig H" + }, + "type": "person" + } + ], + "dataId": "CP033844.1", + "date": "2018", + "details": "Klebsiella oxytoca strain FDAARGOS_500 chromosome, complete genome", + "id": "dataset1", + "title": "NCBI GenBank", + "uri": "https://www.ncbi.nlm.nih.gov/nuccore/CP033844.1" + }, + { + "authors": [ + { + "name": { + "index": "Zhou, J", + "preferred": "Zhou J" + }, + "type": "person" + }, + { + "name": { + "index": "Xia, F", + "preferred": "Xia F" + }, + "type": "person" + }, + { + "name": { + "index": "Che, S", + "preferred": "Che S" + }, + "type": "person" + }, + { + "name": { + "index": "Wang, J", + "preferred": "Wang J" + }, + "type": "person" + }, + { + "name": { + "index": "Qiu, L", + "preferred": "Qiu L" + }, + "type": "person" + }, + { + "name": { + "index": "Li, G", + "preferred": "Li G" + }, + "type": "person" + }, + { + "name": { + "index": "Shao, J", + "preferred": "Shao J" + }, + "type": "person" + }, + { + "name": { + "index": "Zhang, G", + "preferred": "Zhang G" + }, + "type": "person" + }, + { + "name": { + "index": "Zhong, L", + "preferred": "Zhong L" + }, + "type": "person" + }, + { + "name": { + "index": "Liu, Q", + "preferred": "Liu Q" + }, + "type": "person" + }, + { + "name": { + "index": "Ren, B", + "preferred": "Ren B" + }, + "type": "person" + } + ], + "dataId": "CP034363.1", + "date": "2018", + "details": "Pantoea sp. CCBC3-3-1 chromosome, complete genome", + "id": "dataset2", + "title": "NCBI GenBank", + "uri": "https://www.ncbi.nlm.nih.gov/nuccore/CP034363.1" + }, + { + "authors": [ + { + "name": { + "index": "Leung, F", + "preferred": "Leung F" + }, + "type": "person" + }, + { + "name": { + "index": "Liu, L", + "preferred": "Liu L" + }, + "type": "person" + }, + { + "name": { + "index": "Jiang, J", + "preferred": "Jiang J" + }, + "type": "person" + } + ], + "dataId": "CP008886.1", + "date": "2014", + "details": "Raoultella ornithinolytica strain A14, complete sequence", + "id": "dataset3", + "title": "NCBI GenBank", + "uri": "https://www.ncbi.nlm.nih.gov/nuccore/CP008886.1" + }, + { + "authors": [ + { + "name": { + "index": "Nash, JHE", + "preferred": "Nash JHE" + }, + "type": "person" + }, + { + "name": { + "index": "Robertson, J", + "preferred": "Robertson J" + }, + "type": "person" + }, + { + "name": { + "index": "Bessonov, K", + "preferred": "Bessonov K" + }, + "type": "person" + } + ], + "dataId": "CP030220.1", + "date": "2018", + "details": "Salmonella enterica strain SA20021456 plasmid pSA20021456.1, complete sequence", + "id": "dataset4", + "title": "NCBI GenBank", + "uri": "https://www.ncbi.nlm.nih.gov/nuccore/CP030220.1" + }, + { + "authors": [ + { + "name": { + "index": "Bekkelund, AK", + "preferred": "Bekkelund AK" + }, + "type": "person" + } + ], + "dataId": "CP033055.1", + "date": "2018", + "details": "Serratia sp. 3ACOL1 chromosome, complete genome", + "id": "dataset5", + "title": "NCBI GenBank", + "uri": "https://www.ncbi.nlm.nih.gov/nuccore/CP033055.1" + }, + { + "authors": [ + { + "name": { + "index": "Doonan, J", + "preferred": "Doonan J" + }, + "type": "person" + }, + { + "name": { + "index": "Denman, S", + "preferred": "Denman S" + }, + "type": "person" + }, + { + "name": { + "index": "McDonald, JE", + "preferred": "McDonald JE" + }, + "type": "person" + } + ], + "dataId": "CP014137.1", + "date": "2016", + "details": "Brenneria goodwinii strain FRB141, complete genome", + "id": "dataset6", + "title": "NCBI GenBank", + "uri": "https://www.ncbi.nlm.nih.gov/nuccore/CP014137.1" + }, + { + "authors": [ + { + "name": { + "index": "Doonan, J", + "preferred": "Doonan J" + }, + "type": "person" + }, + { + "name": { + "index": "Denman, S", + "preferred": "Denman S" + }, + "type": "person" + }, + { + "name": { + "index": "McDonald, JE", + "preferred": "McDonald JE" + }, + "type": "person" + } + ], + "dataId": "CP014136.1", + "date": "2016", + "details": "Gibbsiella quercinecans strain FRB97, complete genome", + "id": "dataset7", + "title": "NCBI GenBank", + "uri": "https://www.ncbi.nlm.nih.gov/nuccore/CP014136.1" + } + ] + }, + "impactStatement": "12 new class IIb microcins have been structurally identified, phylogenetically assessed, and functionally tested for their antimicrobial activity, increasing the total number of these bacteriocins to 17.", + "elifeAssessment": { + "content": [ + { + "text": "This study presents important advances in the discovery and assessment of microcins that improve our understanding of their prevalence and roles. The bioinformatics analysis, expression, and antimicrobial assays are solid, although the diverging evaluations also indicated the need for additional support regarding the sequence analysis and validation to fully back some of the claims and conclusions. This study will appeal to researchers working on the discovery and analysis of novel peptide natural products.", + "type": "paragraph" + } + ], + "doi": "10.7554/eLife.102912.2.sa0", + "id": "sa0", + "significance": [ + "important" + ], + "strength": [ + "solid" + ], + "title": "eLife Assessment" + }, + "keywords": [ + "class IIb microcins", + "ESKAPE", + "antimicrobial peptides", + "acinetobacter baumannii", + "multidrug resistance", + "siderophore" + ], + "-related-articles-reviewed-preprints": [], + "body": [ + { + "content": [ + { + "text": "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).", + "type": "paragraph" + }, + { + "text": "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).", + "type": "paragraph" + }, + { + "text": "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.", + "type": "paragraph" + } + ], + "id": "s1", + "title": "Introduction", + "type": "section" + }, + { + "content": [ + { + "text": "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).", + "type": "paragraph" + }, + { + "text": "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.", + "type": "paragraph" + }, + { + "assets": [ + { + "caption": [ + { + "text": "(A) Sequence alignments of the newly identified microcin and immunity genes with the gene clusters of E. coli (Ec) CA46 and K. pneumoniae (Kp) RYC492 using Easyfig (Sullivan et al., 2011). Antimicrobial (A) and immunity (I) genes in the center are represented by darker and lighter shades, respectively. X=mchX, I=mchI, B=mchB, E=mceE, L=mceL, M=mceM. (B,C) Phylogenetic trees of antimicrobial and corresponding immunity genes using codon-aligned nucleotide sequences with general time reversible model with discrete gamma distribution (GTR+G) and the Hasegawa-Kishino-Yano model with discrete gamma distribution (HKY+G), respectively. (D) MUSCLE (Edgar, 2004) alignment of the amino acid sequence of the signal peptide sequence as well as the C-terminus of the antimicrobial peptides.", + "type": "paragraph" + } + ], + "id": "fig1", + "image": { + "alt": "", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig1-v1.tif", + "size": { + "width": 2205, + "height": 2622 + }, + "source": { + "mediaType": "image/jpeg", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig1-v1.tif/full/full/0/default.jpg", + "filename": "elife-102912-fig1-v1.jpg" + } + }, + "label": "Figure 1", + "title": "Novel class IIb microcins are found in numerous Enterobacteriaceae genomes.", + "type": "image" + }, + { + "caption": [ + { + "text": "BLAST sequence comparison of gene clusters was created using Easyfig (Sullivan et al., 2011). (A) Antimicrobial and immunity genes of each cluster are represented by darker and lighter shades, respectively. Colored polygons represent functional microcins MccE492 (pink) and MccG492 (dark blue) from K. pneumoniae as well as MccH47 (light blue), MccI47 (red), and MccM (green) from E. coli. X=mchX, I=mchI, B=mchB, E=mceE, L=mceL, M=mceM. Asterisks indicate truncated and non-functional microcin or immunity genes.", + "type": "paragraph" + } + ], + "id": "fig1s1", + "image": { + "alt": "", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig1-figsupp1-v1.tif", + "size": { + "width": 2205, + "height": 699 + }, + "source": { + "mediaType": "image/jpeg", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig1-figsupp1-v1.tif/full/full/0/default.jpg", + "filename": "elife-102912-fig1-figsupp1-v1.jpg" + } + }, + "label": "Figure 1—figure supplement 1", + "title": "Comparison of class IIb microcin gene clusters from E. coli CA46 and K. pneumoniae RYC492.", + "type": "image" + }, + { + "caption": [ + { + "text": "BLAST sequence comparison of gene clusters was created using Easyfig (Sullivan et al., 2011). (A-G) Antimicrobial and immunity genes of each cluster are represented by darker and lighter shades, respectively. Colored polygons represent novel microcins from the genome indicated on the left: MccE492 (pink), MccG492 (dark blue), MccH47 (light blue), MccI47 (red), MccM (green), MccX (black), MccW (dark red), and MccZ (dark green). X=mchX, I=mchI, B=mchB, E=mceE, L=mceL, M=mceM. Asterisks indicate truncated and non-functional microcin or immunity genes.", + "type": "paragraph" + } + ], + "id": "fig1s2", + "image": { + "alt": "", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig1-figsupp2-v1.tif", + "size": { + "width": 1402, + "height": 3031 + }, + "source": { + "mediaType": "image/jpeg", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig1-figsupp2-v1.tif/full/full/0/default.jpg", + "filename": "elife-102912-fig1-figsupp2-v1.jpg" + } + }, + "label": "Figure 1—figure supplement 2", + "title": "Comparison of class IIb microcin gene cluster from newly identified genomes with E. coli CA46 and K. pneumoniae RYC492.", + "type": "image" + }, + { + "caption": [ + { + "text": "Previously known proteins are highlighted in bold.", + "type": "paragraph" + } + ], + "id": "fig1s3", + "image": { + "alt": "", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig1-figsupp3-v1.tif", + "size": { + "width": 2164, + "height": 3031 + }, + "source": { + "mediaType": "image/jpeg", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig1-figsupp3-v1.tif/full/full/0/default.jpg", + "filename": "elife-102912-fig1-figsupp3-v1.jpg" + } + }, + "label": "Figure 1—figure supplement 3", + "title": "Region and domain prediction of antimicrobial (A) and immunity peptides (B) using SMART (Letunic et al., 2021).", + "type": "image" + }, + { + "caption": [ + { + "text": "(A) Phylogenetic tree of all known microcin genes using codon-aligned nucleotide sequences with general time reversible model with discrete gamma distribution (GTR+G). The respective signal peptides were removed to ensure alignment of the active regions with antimicrobial activity. Previously known class IIb microcins are highlighted in bold.", + "type": "paragraph" + } + ], + "id": "fig1s4", + "image": { + "alt": "", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig1-figsupp4-v1.tif", + "size": { + "width": 1378, + "height": 2599 + }, + "source": { + "mediaType": "image/jpeg", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig1-figsupp4-v1.tif/full/full/0/default.jpg", + "filename": "elife-102912-fig1-figsupp4-v1.jpg" + } + }, + "label": "Figure 1—figure supplement 4", + "title": "Phylogeny of all microcins from the classes I, IIa, and IIb.", + "type": "image" + } + ], + "type": "figure" + }, + { + "assets": [ + { + "caption": [ + { + "text": "Red color indicates no significant match found.", + "type": "paragraph" + } + ], + "id": "table1", + "label": "Table 1", + "tables": [ + "
Microcin nameSpeciesAccession no.Antimicrobial gene nameAntimicrobial closest matchIdentical/total lengthE-valueImmunity gene nameImmunity closest matchIdentical/total lengthE-value
Bg E492Brenneria goodwiniiCP014137Bg E492AKp E492A (mceA)35/1062.00E-14Bg E492IKp E492I (mceB)14/946.00E-04
Bg XBrenneria goodwiniiCP014137Bg XAKp E492A (mceA)21/986.90E-02Bg XIEc MI (mcmI)10/782.60E+00
Gq WGibbsiella quercinecansCP014136Gq WAEc I47A (mciA)7/1038.10E+00Gq WIEc MI (mcmI)8/743.30E+02
Ko H47Klebsiella oxytoxaCP033844Ko H47AEc H47A (mchB)32/771.00E-07Ko H47IEc H47I (mchI)19/692.00E-14
Ps G492Pantoea sp.CP034363Ps G492AKp G492A (mceL)43/896.00E-09Ps G492IKp G492I (mceM)37/852.00E-20
Ro ZRaoultella ornithinolyticaCP008886Ro ZAEc MA (mcmA)21/641.00E-03Ro ZIEc MI (mcmI)5/742.00E+01
Ro H47Raoultella ornithinolyticaCP008886Ro H47AEc H47A (mchB)31/792.00E-07Ro H47IEc H47I (mchI)21/694.00E-14
Ro I47Raoultella ornithinolyticaCP008886Ro I47AEc I47A (mciA)37/794.00E-26Ro I47IEc I47I (mciI)64/1351.00E-43
Ro XRaoultella ornithinolyticaCP008886Ro XAEc MA (mcmA)18/994.40E-01Ro XIEc MI (mcmI)9/611.00E-02
Sf H47Serratia fonticolaCP033055Sf H47AEc H47A (mchB)38/878.00E-11Sf H47IEc H47I (mchI)48/684.00E-22
Se G492Salmonella entericaCP030220Se G492AKp G492A (mceL)56/851.00E-11Se G492IKp G492I (mceM)38/841.00E-23
Se MSalmonella entericaCP030220Se MAEc MA (mcmA)34/901.00E-17Se MIEc MI (mcmI)38/716.00E-04
" + ], + "title": "Blastp results and closest matches to the known class IIb microcins MccE492, MccG492, MccH47, MccI47, or MccM.", + "type": "table" + } + ], + "type": "figure" + }, + { + "text": "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.", + "type": "paragraph" + }, + { + "text": "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).", + "type": "paragraph" + }, + { + "assets": [ + { + "caption": [ + { + "text": "(A) Heatmap summarizing the inhibitory potential of known and novel class IIb microcins against a library of Enterobacteriaceae, Pseudomonadales, and Gram-positive bacteria, including multidrug-resistant isolates (red) as determined by static inhibition assays with live-producing bacteria. *=activity determined through microcin purification and minimum inhibitory concentration assays (Mortzfeld et al., 2022; Palmer et al., 2020). (B) Relative minimum inhibitory concentrations for S. enterica (Se) G492 against different bacterial species. Note that Se G492 is 256 times more potent against A. baumannii (BAA 1790) compared to K. pneumoniae (BAA 1705). (C) Static inhibition assays comparing K. pneumoniae (Kp) E492, Kp G492, Ec H47, E. coli (Ec) I47, Ec M, and Se G492 activity from single colony production against multidrug-resistant A. baumannii (BAA 1790) and Pseudomonas aeruginosa (PA14). Note that iron-limited conditions (DP) are required for antimicrobial activity, confirming action of class IIb microcins. L-ara=L-arabinose, DP = 2,2-dipyridyl, scale bars: 1 cm.", + "type": "paragraph" + } + ], + "id": "fig2", + "image": { + "alt": "", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig2-v1.tif", + "size": { + "width": 2205, + "height": 2396 + }, + "source": { + "mediaType": "image/jpeg", + "uri": "https://iiif.elifesciences.org/lax:102912%2Felife-102912-fig2-v1.tif/full/full/0/default.jpg", + "filename": "elife-102912-fig2-v1.jpg" + } + }, + "label": "Figure 2", + "title": "Novel class IIb microcins are effective inhibitors of Enterobacteriaceae and Gram-negative ESKAPE pathogens.", + "type": "image" + } + ], + "type": "figure" + }, + { + "text": "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).", + "type": "paragraph" + } + ], + "id": "s2", + "title": "Results", + "type": "section" + }, + { + "content": [ + { + "text": "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).", + "type": "paragraph" + }, + { + "text": "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.", + "type": "paragraph" + }, + { + "text": "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.", + "type": "paragraph" + }, + { + "text": "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.", + "type": "paragraph" + } + ], + "id": "s3", + "title": "Discussion", + "type": "section" + }, + { + "content": [ + { + "content": [ + { + "text": "We developed a pipeline that by leveraging BLAST (Boratyn et al., 2012) enabled us to mine publicly available genome databases for novel, previously undescribed class IIb microcins. We included mchCDEF and mcmL for Ec as well as mceCDGHIJ for Kp for posttranslational modification and export, expecting more reliable hits for longer and functionally conserved proteins in close proximity to class IIb microcin and immunity genes. Thus, we first ran tblastn (Boratyn et al., 2012) against RefSeq (O’Leary et al., 2016), to screen for all genes related to biosynthesis pathways, known microcin genes, as well as immunity gene sequences exhibiting homology to the microcin gene clusters found in Ec CA46 and Kp RYC492. Homology to the microcin gene clusters were guided by BLAST parameters sseqid (genome ID), pident (percentage of identical positions) along with sstart (start of alignment in genome) and send (end of alignment position in genome). Resulting hits were concatenated by genome ID and assessed for their proximity to one another in the genome. These gene clusters should, at best, contain all the known genes required for toxin maturation, including mchCDEF and mcmL (Vassiliadis et al., 2010; Palmer et al., 2020). In addition to genomic hits to the known microcins, small ORFs of 50–150 amino acids in size close to the biosynthesis genes were screened and annotated manually using the criteria described below as well as their domains were predicted using SMART (Letunic et al., 2021). The ORFs were meticulously examined and assessed against established class IIb microcin criteria known from Ec H47, Ec I47, Ec M, Kp E492, and Kp G492: (i) a serine-rich C-terminus culminating in a final serine, (ii) the presence of fewer than two cysteine residues, (iii) a signal peptide within the initial 15 amino acids ending with GG or GA, and (iv) close proximity (≤200 bp) to an ORF featuring a predicted transmembrane domain, typically encoding an immunity peptide. The identified genes were included in the pipeline’s input to expand the scope of gene detection. We repeated this process iteratively through the pipeline until no additional genes were added to the output. Subsequently, blastp was used to assess microcin similarity as shown in Table 1 and Supplementary file 1.", + "type": "paragraph" + } + ], + "id": "s4-1", + "title": "Bioinformatic class IIb microcin identification", + "type": "section" + }, + { + "content": [ + { + "text": "For the native full-length coding sequence of the microcin and immunity genes, a codon-based sequence alignment was generated using the MUSCLE algorithm (Edgar, 2004). For phylogeny of all microcins, the nucleotide sequences without the respective signal peptides were codon-aligned. Subsequently, we determined the best fit substitution models for maximum likelihood phylogenetic analyses, resulting in the general time reversible model with discrete gamma distribution (GTR+G) and the Hasegawa-Kishino-Yano model with discrete gamma distribution (HKY+G), respectively. A bootstrap test with 1000 replicates for maximum likelihood and random seed was conducted for all trees. Alignment, model testing, and tree building were performed in MEGA11 (Tamura et al., 2021).", + "type": "paragraph" + } + ], + "id": "s4-2", + "title": "Phylogenetic analyses", + "type": "section" + }, + { + "content": [ + { + "text": "To test if similar results of class IIb microcin identification could be obtained with automated bioinformatic tools, we ran antiSMASH 7.0 (Blin et al., 2023), a widely used tool for microbial genome mining and biosynthetic gene cluster detection. As input, we utilized the seven genomes from the newly identified class IIb microcins: (i) Bg CP014137, (ii) Gq CP014136, (iii) Ko CP033844, (iv) Ps CP034363, (v) Ro CP008886, (vi) Se CP030220, (vii) Sf CP033055. As a positive control for the well-established microcins Kp E492 and Kp G492 as well as Ec H47 and Ec M, we used the accession numbers CP127839 (Kp RYC492) and CP148105 (Ec Nissle 1917), respectively. Notably, using the ‘loose’ setting, in none of the cases a class IIb microcin biosynthesis gene cluster was detected, nor were any microcin genes identified. This was the case for both, the novel microcins and the original, well-annotated, microcins.", + "type": "paragraph" + } + ], + "id": "s4-3", + "title": "antiSMASH analyses", + "type": "section" + }, + { + "content": [ + { + "text": "ORFs of identified microcin and immunity genes were codon optimized for frequent Ec codon usage without creating repetitive sequences and synthesized by Integrated DNA Technologies (Coralville, IA, USA) with 18 bp of native 5’ upstream sequence and 20 bp of native 3’ downstream sequence, respectively. Using Gibson Assembly (Gibson et al., 2009), the genes were cloned into our previously established Ec class IIb microcin expression system that results in mature class IIb microcins posttranslationally modified with an MGE (Mortzfeld et al., 2022; Palmer et al., 2020). Briefly, the antimicrobial and the immunity genes are co-expressed under the control of an arabinose-inducible pBad/araC promoter in a high copy plasmid with a pUC-derived origin of replication. All assemblies were verified using whole plasmid sequencing. DNA files for all used plasmids can be found as supplementary material.", + "type": "paragraph" + } + ], + "id": "s4-4", + "title": "Plasmids and heterologous class IIb microcin expression", + "type": "section" + }, + { + "content": [ + { + "text": "Cultures of strains with confirmed plasmid assemblies were spread on LB agar plates containing 100 µg/ml ampicillin. In addition to a pUC19 control without microcin expression, single colonies for each microcin were picked with a sterile pipet tip and all stabbed into the same solid LB agar plate containing 100 µg/ml ampicillin for plasmid retention, 0.2 mM 2,2-dipyridyl to create iron-limited conditions during the growth phase, and 0.4% L-arabinose for induction of gene expression. Plates were incubated at 37°C for up to 72 hr, before they were overlaid with the target bacterial isolates. Note that testing all microcin-expressing stains on the same plate allowed us to confidently assess differential inhibitory activity between all 17 tested microcins. For the overlay, the microcin-producing bacteria in the stabs were inactivated using chloroform vapors and 10 min under ultraviolet light. Then, target bacteria were diluted 1:2000 from overnight culture in LB media containing 100 µg/ml ampicillin and 0.2 mM 2,2-dipyridyl. Ec and Shigella flexneri strains were diluted 1:200 to acquire dense bacterial lawns. Finally, 0.5 ml of molten agar was added to 2 ml of liquid media and the resulting soft agar was spread on the plate with the inactivated bacteria and incubated for 16 hr at 37°C. The pUC19 control strain was unable to create any zone of inhibition against any of the tested target bacteria. Note that not all tested target bacteria confer resistance to ampicillin, however, placing stabs for 17 ampicillin-resistant microcin producers and the negative control evenly spaced into a single plate degrades the antibiotic within 24 hr of incubation and thus before the overlay with the target bacteria is added.", + "type": "paragraph" + } + ], + "id": "s4-5", + "title": "Static inhibition assays", + "type": "section" + }, + { + "content": [ + { + "text": "For enrichment of microcin Se G492, an MBP-microcin fusion protein was expressed from pHMT-SeG492 in E. coli BL21 cells as previously described (Mortzfeld et al., 2022; Palmer et al., 2020). Cells harvested from 6 l culture were resuspended in 50 ml column buffer (200 mM NaCl, 20 mM Tris-HCl, pH 7.5), lysed by sonication, and passed through 5 ml of high flow amylose resin (New England Biolabs, Ipswich, MA, USA) as recommended by the manufacturer. The protein was eluted with 30 ml 10 mM maltose, cleaved with Tobacco etch virus (TEV) protease, and further processed as previously reported (Mortzfeld et al., 2022; Palmer et al., 2020). After removal of the histidine-tagged TEV protease, the relative MIC assays were conducted using sterile 96-well round bottom microplates. The plates were prepared as follows: the first row contained 20 µl of 2× LB with 0.4 mM 2,2’-dipyridyl and 20 µl of Se G492 containing solution in amylose resin elution buffer (200 mM NaCl, 20 mM Tris-HCl, 10 mM maltose, pH 7.5). All other wells were filled with 20 µl of 1× LB, 0.2 mM 2,2’-dipyridyl, and 0.5× amylose resin elution buffer, and a twofold serial dilution was performed across the plate. The target bacteria were grown overnight in LB at 200 rpm and 37°C and were added to a final dilution of 10–4 into the wells. The plates were then incubated in the dark at 37°C with gentle agitation. Relative MICs were determined as the lowest concentration at which no growth was observed after 24 hr. 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"date": "2017", + "id": "bib54", + "publisher": { + "name": [ + "World Health Organization" + ] + }, + "type": "book" + } + ], + "acknowledgements": [ + { + "text": "The authors would like to thank the administrative staff in the Microbiology Department at the University of Massachusetts Chan Medical School, especially Annette Bohigian, Amy Parker, Dhruti Desai, Marie Berardi, Richard Fish, and Tracey Rae, for their support. This work was supported by the CDMRP PRMP W81XWH2020013 to VB, by the NIH NIA 1R01AG075283-01A1 to VB, and by the Deutsche Forschungsgemeinschaft (DFG) project 457837076 to BMM. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.", + "type": "paragraph" + } + ], + "authorResponse": { + "content": [ + { + "text": "We thank the anonymous reviewers very much for dedicating their time to thoroughly review our manuscript. We sincerely appreciate their thoughtful consideration and detailed assessment. Regarding the raised concerns, we acknowledge the importance of exploring the full scope of class IIb microcins, however, we believe that in depth characterization, purification, and in vivo application of the 12 novel compounds goes beyond the scope of this short report and discovery article.", + "type": "paragraph" + }, + { + "text": "At the same time, the reviewers acknowledge that the analysis, experimental design, the expression system as well as the performed assays are “sound”, “convincing”, and “corroborated by suitable controls”. In the present manuscript we sought to identify novel antimicrobials and to comprehensively verify their antimicrobial activity in E. coli irrespective of the siderophore-dependent delivery mechanism. Notably, none of the reviewers questioned that we describe new antimicrobials, the characteristics we used to find them, that they are class IIb microcins, or that they do exhibit antimicrobial activity against Gram-negative ESKAPE and plant pathogens.", + "type": "paragraph" + }, + { + "text": "We believe that our discovery study can serve as a steppingstone towards the application of bacterially produced antimicrobial compounds to target Gram-negative pathogens in numerous plant and animal species, including humans.", + "type": "paragraph" + } + ], + "doi": "10.7554/eLife.102912.2.sa4", + "id": "sa4" + }, + "publicReviews": [ + { + "content": [ + { + "text": "Summary:", + "type": "paragraph" + }, + { + "text": "Enterobacteriaceae produce microcins to target their competitors. Using informatics approaches, the authors identified 12 new microcins. They expressed them in E. coli, demonstrating that the microcins have antimicrobial activity against other microbes, including plant pathogens and the ESKAPE pathogens Pseudomonas aeruginosa and Acinetobacter baumannii.", + "type": "paragraph" + }, + { + "text": "Strengths:", + "type": "paragraph" + }, + { + "text": "Overall, this study has the merit of identifying new potential antimicrobial molecules that could be used to target important pathogens. The bioinformatics analysis, the expression system used, and the antimicrobial assays performed are solid, and the data presented are convincing. This work will set the basis for new studies to investigate the potential role of these microcins in vivo.", + "type": "paragraph" + }, + { + "text": "Weaknesses:", + "type": "paragraph" + }, + { + "text": "The work has been performed in vitro, which is a valid approach for identifying the antimicrobial peptides and assessing their antimicrobial activity. Future studies will need to address whether these new microcins exhibit antimicrobial activity in vivo (e.g., in the context of infection models), and to identify the targets (receptor and mechanisms of action) for the new microcins.", + "type": "paragraph" + } + ], + "doi": "10.7554/eLife.102912.2.sa1", + "id": "sa1", + "title": "Reviewer #1 (Public review):" + }, + { + "content": [ + { + "text": "Mortzfeld et al. describe their study of class IIb microcins. Furthering our awareness of the presence and action of microcins is an important line of research. However, several issues related to the premise, sequence analysis, and validation require attention to support the claims.", + "type": "paragraph" + }, + { + "text": "(1) Previous studies have been published on the broader distribution of microcins across bacteria. The software has been published for their identification. Comparison to this software and/or discussion of previous work should be included to place this work in the context of the field.", + "type": "paragraph" + }, + { + "text": "(2) It is not clear how immunity proteins were identified and there does not appear to be functional confirmation to show these predicted immunity proteins are real. Thus, it is premature to state that immunity genes have been found. This may also confound some of the validation studies below if proper immunity proteins have not been included.", + "type": "paragraph" + }, + { + "text": "(3) Please show the nt alignment used to generate the tree. Without seeing it, one would guess that the sequences are either quite similar (making the results from this study less novel) or there would be concerns that the phylogenetic relationship derived from the nt alignment is spurious.", + "type": "paragraph" + }, + { + "text": "(4) Figure 1 B-C: There are numerous branches that do not have phylogenetic support (values <50%). These are not statistically valid phylogenetic relationships and should be collapsed. The resulting tree should be used in the description of clades.", + "type": "paragraph" + }, + { + "text": "(5) The discovered microcins are not being directly tested since they are expressed heterologous and reliant on non-native modification systems. The results present the statement that novel microcins have been validated. This should be described accordingly.", + "type": "paragraph" + }, + { + "text": "(6) The key finding of this paper is the claim that 12 novel class IIb microcins have been validated. To substantiate this claim, original images showing evidence of antibacterial activity must be made available rather than a presence/absence chart. The negative controls for this table are unclear and should be included with the original images.", + "type": "paragraph" + }, + { + "text": "(7) Further data for the purified microcin is needed. The purification method described is standard practice and should allow for product quantification, which should be included. Standard practice includes an SDS page showing the purity of the microcin, or at least the TEV digest to show microcin has been produced, and importantly a control sample (scrambled sequence, empty vector purification, etc) to show that observed activity (Figure 2B) is not from a purification carry over. This data should be included to support that microcin has been purified and is active.", + "type": "paragraph" + } + ], + "doi": "10.7554/eLife.102912.2.sa2", + "id": "sa2", + "title": "Reviewer #2 (Public review):" + }, + { + "content": [ + { + "text": "Summary:", + "type": "paragraph" + }, + { + "text": "In this study, several novel class IIb microcin biosynthetic gene clusters have been discovered by specific homology searches and manual curation. Using a specific E. coli expression system, the microcins were expressed and conjugated to monoglycosylated enterobactin as siderophore moiety. While this synthetic biology approach cannot account for other siderophores being coupled to the microcin core peptide in the original producing strains, it nonetheless allows for a general screening for the activity of the heterologously produced compounds. Through this approach, the activity of several predicted microcins has been confirmed and three novel class IIb microcin clades were identified.", + "type": "paragraph" + }, + { + "text": "Strengths:", + "type": "paragraph" + }, + { + "text": "The experimental design is sound, the results are corroborated by suitable controls, and the findings have a high level of novelty and significance. Furthermore, the comments of the initial round of peer review have been answered satisfactorily by the authors.", + "type": "paragraph" + } + ], + "doi": "10.7554/eLife.102912.2.sa3", + "id": "sa3", + "title": "Reviewer #3 (Public review):" + } + ], + "stage": "published", + "statusDate": "2024-12-11T00:00:00Z" + } + ] +}