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Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

Summary: Mother’s milk contains diverse bacterial communities, although their impact on microbial colonization in very-low-birth-weight (VLBW,

Abstract Functional redundancy is a key ecosystem property representing the fact that different taxa contribute to an ecosystem in similar ways through the expression of redundant functions. The redundancy of potential functions (or genome-level functional redundancy $${{{{{{\rm{FR}}}}}}}_{g}$$ FR g ) of human microbiomes has been recently quantified using metagenomics data. Yet, the redundancy of expressed functions in the human microbiome has never been quantitatively explored. Here, we present an approach to quantify the proteome-level functional redundancy $${{{{{{\rm{FR}}}}}}}_{p}$$ FR p in the human gut microbiome using metaproteomics. Ultra-deep metaproteomics reveals high proteome-level functional redundancy and high nestedness in the human gut proteomic content networks (i.e., the bipartite graphs connecting taxa to functions). We find that the nested topology of proteomic content networks and relatively small functional distances between proteomes of certain pairs of taxa together contribute to high $${{{{{{\rm{FR}}}}}}}_{p}$$ FR p in the human gut microbiome. As a metric comprehensively incorporating the factors of presence/absence of each function, protein abundances of each function and biomass of each taxon, $${{{{{{\rm{FR}}}}}}}_{p}$$ FR p outcompetes diversity indices in detecting significant microbiome responses to environmental factors, including individuality, biogeography, xenobiotics, and disease. We show that gut inflammation and exposure to specific xenobiotics can significantly diminish the $${{{{{{\rm{FR}}}}}}}_{p}$$ FR p with no significant change in taxonomic diversity.

BackgroundProcess evaluations conducted alongside clinical trials can improve understanding of treatment fidelity and provide contextual knowledge to aide interpretations of trial outcomes. We adopted a multiple-goals perspective to investigate treatment fidelity in two related pilot clinical trials of an adjuvant treatment for pediatric-onset Inflammatory Bowel Disease. This included a focus on barriers and enablers of performing trial-specific activities and of integrating those activities into daily life.MethodsWe conducted one-time semi-structured interviews with a sub-sample of participants of the Resistant Starch in Pediatric Inflammatory Bowel Disease (NCT04522271) and Optimized Resistant Starch in Inflammatory Bowel Disease pilot trials (NCT04520594) and their caregivers (N = 42). The trials examined the effects of personalized food-derived resistant starches as an adjuvant therapy on intestinal microbiome functioning. Interviews were conducted within 3-months of participants completing or withdrawing from the trials. Interview guides with age-appropriate language were developed and pilot tested. Codes were identified inductively though conventional content analysis and then mapped to personal projects analysis, to explore how participants navigated between activities.ResultsThree themes were identified. The first described the potential impact of living with inflammatory bowel disease and taking prescribed medications. The second described characteristics of trial-specific activities that might impact on their enactment, including perceived difficulty, and challenges following procedures or using trial materials. The third described the integration of trial-specific activities with school, work, household demands, and social, and extracurricular activities.ConclusionsAdjusting to living with inflammatory bowel disease and managing its treatment can impact trial participation. Integrating trial-related activities into daily life can be challenging, which could heighten perceptions of goal conflict. Findings can inform interpretations of trial outcomes and development of strategies for trial optimization and implementation of the adjuvant therapy into clinical practice.

ABSTRACTThe human gut virome has been increasingly explored in recent years. However, nearly all virome-sequencing efforts rely solely on fecal samples and few studies leverage multiomic approaches to investigate phage–host relationships. Here, we combine metagenomics, metaviromics, and metatranscriptomics to study virome-bacteriome interactions at the colonic mucosal-luminal interface in a cohort of three individuals with inflammatory bowel disease; non-IBD controls were not included in this study. We show that the mucosal viral population is distinct from the stool virome and houses abundant crAss-like phages that are undetectable by fecal sampling. Through viral protein prediction and metatranscriptomic analysis, we explore viral gene transcription, prophage activation, and the relationship between the presence of integrase and temperate phages in IBD subjects. We also show the impact of deep sequencing on virus recovery and offer guidelines for selecting optimal sequencing depths in future metaviromic studies. Systems biology approaches such as those presented in this report will enhance our understanding of the human virome and its interactions with our microbiome and our health.

Abstract Background During gut colonization, the enteric pathogen Campylobacter jejuni must surmount the toxic effects of reactive oxygen species produced by its own metabolism, the host immune system, and intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology. Results The mechanisms of oxidative stress defense in C. jejuni were characterized by transcriptional profiling and phenotypic analysis of wild-type and mutant strains. To define the regulon of the peroxide-sensing regulator, PerR, we constructed an isogenic ΔperR mutant and compared its transcriptome profile with that of the wild-type strain. Transcriptome profiling identified 104 genes that belonged to the PerR regulon. PerR appears to regulate gene expression in a manner that both depends on and is independent of the presence of iron and/or H2O2. Mutation of perR significantly reduced motility. A phenotypic analysis using the chick colonization model showed that the ΔperR mutant exhibited attenuated colonization behavior. An analysis of changes in the transcriptome induced by exposure to H2O2, cumene hydroperoxide, or menadione revealed differential expression of genes belonging to a variety of biological pathways, including classical oxidative stress defense systems, heat shock response, DNA repair and metabolism, fatty acid biosynthesis, and multidrug efflux pumps. Mutagenic and phenotypic studies of the superoxide dismutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed a role for these proteins in oxidative stress defense and chick gut colonization. Conclusion This study reveals an interplay between PerR, Fur, iron metabolism and oxidative stress defense, and highlights the role of these elements in C. jejuni colonization of the chick cecum and/or subsequent survival.

Proteases control plant growth and development by limited proteolysis of regulatory proteins at highly specific sites. This includes the processing of peptide hormone precursors to release the bioactive peptides as signaling molecules. The proteases involved in this process have long remained elusive. Confirmation of a candidate protease as a peptide precursor–processing enzyme requires the demonstration of protease-mediated precursor cleavage in vitro. In vitro cleavage assays rely on the availability of suitable substrates and the candidate protease with high purity. Here, we provide a protocol for the expression, purification, and characterization of tomato (Solanum lycopersicum) phytaspases as candidate proteases for the processing of the phytosulfokine precursor. We also show how synthetic oligopeptide substrates can be used to demonstrate site-specific precursor cleavage.Graphical abstract

Biobanking of live microbiota is becoming indispensable for mechanistic and clinical investigations of drug–microbiome interactions and fecal microbiota transplantation. However, there is a lack of methods to rapidly and systematically evaluate whether the biobanked microbiota maintains their cultivability and functional activity. In this study, we use a rapid ex vivo microbiome assay and metaproteomics to evaluate the cultivability and the functional responses of biobanked microbiota to treatment with a prebiotic (fructo-oligosaccharide, FOS). Our results indicate that the microbiota cultivability and their functional responses to FOS treatment were well maintained by freezing in a deoxygenated glycerol buffer at −80°C for 12 months. We also demonstrate that the fecal microbiota is functionally stable for 48 hours on ice in a deoxygenated glycerol buffer, allowing off-site fecal sample collection and shipping to laboratory for live microbiota biobanking. This study provides a method for rapid evaluation of the cultivability of biobanked live microbiota. Our results show minimal detrimental influences of long-term freezing in deoxygenated glycerol buffer on the cultivability of fecal microbiota.

Abstract Background The consumption of resistant starches is a promising adjuvant therapy for patients with inflammatory bowel disease. Rigorous evaluation of resistant starches in this setting depends on the intervention being delivered, received, and enacted as intended, that is, with fidelity. As part of a planned pilot trial, participants will be randomized to ingest resistant starches or a placebo. They will also be asked to collect stool samples and keep symptom and dose diaries to inform trial outcomes. We aim to identify potential factors impacting fidelity to the receipt and enactment of trial intervention and data collection activities from the perspective of patients and caregivers in the trial. Identifying fidelity barriers and enablers at the pilot trial phase of a clinical intervention may help to determine optimization processes when expanding to multiple sites in future trials. Methods We will conduct 15-30 semi-structured interviews with pilot trial participants (aged 8-17) and their caregivers. Trial participants will be approached for interviews approximately 6 months after the start of their trial participation. Personal projects analysis, a tool for understanding how individuals manage competing demands in their daily lives, will guide an in-depth exploration of how trial participants engage in activities related to intervention and data collection fidelity (ingesting resistant starches or placebo, collecting stool samples, keeping a symptom and dose diary) amidst the complexities of daily living. Discussion The present study will seek to explore and demonstrate how theory-informed fidelity assessments can be conducted alongside pilot trials to inform future multisite trials. Study results will clarify what factors may affect fidelity to trial intervention and data collection activities. Results may suggest what to modify to optimize the design and conduct, and ensure the integrity, of future multisite trials. Conducting process evaluations alongside clinical trials has the potential to improve our understanding of trial participant experiences. Results will provide a better understanding of how trial participants manage to engage in necessary trial activities along with other priorities.

Background: Process evaluations conducted alongside clinical trials can improve understanding of treatment fidelity and provide contextual knowledge to aide interpretations of trial outcomes. We adopted a multiple-goals perspective to investigate treatment fidelity in two related pilot clinical trials of an adjuvant treatment for pediatric-onset Inflammatory Bowel Disease. This included a focus on barriers and enablers of performing trial-specific activities and of integrating those activities into daily life. Methods: We conducted one-time semi-structured interviews with a sub-sample of participants of the Resistant Starch in Pediatric Inflammatory Bowel Disease (NCT04522271) and Optimized Resistant Starch in Inflammatory Bowel Disease pilot trials (NCT04520594) and their caregivers (N = 42). The trials examined the effects of personalized food-derived resistant starches as an adjuvant therapy on intestinal microbiome functioning. Interviews were conducted within 3-months of participants completing or withdrawing from the trials. Interview guides with age-appropriate language were developed and pilot tested. Codes were identified inductively though conventional content analysis and then mapped to personal projects analysis, to explore how participants navigated between activities. Results: Three themes were identified. The first described the potential impact of living with inflammatory bowel disease and taking prescribed medications. The second described characteristics of trial-specific activities that might impact on their enactment, including perceived difficulty, and challenges following procedures or using trial materials. The third described the integration of trial-specific activities with school, work, household demands, and social, and extracurricular activities. Conclusions: Adjusting to living with inflammatory bowel disease and managing its treatment can impact trial participation. Integrating trial-related activities into daily life can be challenging, which could heighten perceptions of goal conflict. Findings can inform interpretations of trial outcomes and development of strategies for trial optimization and implementation of the adjuvant therapy into clinical practice. [ABSTRACT FROM AUTHOR]

Abstract Background Inflammatory bowel disease (IBD) is a debilitating chronic disease with limited treatment options. Resistant starches may represent a novel treatment for IBD. However, its efficacy and safety remain unclear. Our objective was to perform a systematic review to summarize the preclinical and clinical effects of resistant starch, which may help guide future studies. Methods Medline, EMBASE, and the Cochrane Central Register were searched. Included studies investigated the use of resistant starch therapy in in vivo animal models of IBD or human patients with IBD. Articles were screened, and data extracted, independently and in duplicate. The primary outcomes were clinical remission (clinical) and bowel mucosal damage (preclinical). Results 21 preclinical (n = 989 animals) and seven clinical (n = 164 patients) studies met eligibility. Preclinically, resistant starch was associated with a significant reduction in bowel mucosal damage compared to placebo (standardized mean difference − 1.83, 95% CI − 2.45 to − 1.20). Clinically, five studies reported data on clinical remission but clinical and methodological heterogeneity precluded pooling. In all five, a positive effect was seen in patients who consumed resistant starch supplemented diets. The majority of studies in both the preclinical and clinical settings were at a high or unclear risk of bias due to poor methodological reporting. Conclusions Our review demonstrates that resistant starch is associated with reduced histology damage in animal studies, and improvements in clinical remission in IBD patients. These results need to be tempered by the risk of bias of included studies. Rigorously designed preclinical and clinical studies are warranted. Trial registration The review protocols were registered on PROSPERO (preclinical: CRD42019130896; clinical: CRD42019129513).

The understanding of the effects of compounds on the gut microbiome is limited. In particular, it is unclear whether structurally similar compounds would have similar or distinct effects on the gut microbiome. Here, we selected berberine (BBR), an isoquinoline quaternary alkaloid, and 16 structural analogs and evaluated their effects on seven individual gut microbiomes cultured in vitro. The responses of the individual microbiomes were evaluated by metaproteomic profiles and by assessing butyrate production. We show that both interindividual differences and compound treatments significantly contributed to the variance of metaproteomic profiles. BBR and eight analogs led to changes in proteins involved in microbial defense and stress responses and enrichment of proteins from Verrucomicrobia, Proteobacteria, and Bacteroidetes phyla. It also led to a decrease in proteins from the Firmicutes phylum and its Clostridiales order which correlated to decrease proteins involved in the butyrate production pathway and butyrate concentration. Three of the compounds, sanguinarine, chelerythrine, and ethoxysanguinarine, activated bacterial protective mechanisms, enriched Proteobacteria, increased opacity proteins, and markedly reduced butyrate production. Dihydroberberine had a similar function to BBR in enriching the Akkermansia genus. In addition, it showed less overall adverse impacts on the functionality of the gut microbiome, including a better maintenance of the butyrate level. Our study shows that ex vivo microbiome assay can assess differential regulating effects of compounds with subtle differences and reveals that compound analogs can have distinct effects on the microbiome.

Intestinal microbiota is increasingly reported to influence human health, but little is known on how its functions are regulated. Here the authors characterize microbiome protein acetylation and demonstrate its potential roles in shaping gut microbial functions and the onset of Crohn’s disease.

Abstract Background Few studies have examined how maternal body mass index (BMI), mode of delivery and ethnicity affect the microbial composition of human milk and none have examined associations with maternal metabolic status. Given the high prevalence of maternal adiposity and impaired glucose metabolism, we systematically investigated the associations between these maternal factors in women ≥20 years and milk microbial composition and predicted functionality by V4-16S ribosomal RNA gene sequencing (NCT01405547; https://clinicaltrials.gov/ct2/show/NCT01405547 ). Demographic data, weight, height, and a 3-h oral glucose tolerance test were gathered at 30 (95% CI: 25–33) weeks gestation, and milk samples were collected at 3 months post-partum (n = 113). Results Multivariable linear regression analyses demonstrated no significant associations between maternal characteristics (maternal BMI [pre-pregnancy, 3 months post-partum], glucose tolerance, mode of delivery and ethnicity) and milk microbiota alpha-diversity; however, pre-pregnancy BMI was associated with human milk microbiota beta-diversity (Bray-Curtis R2 = 0.037). Women with a pre-pregnancy BMI > 30 kg/m2 (obese) had a greater incidence of Bacteroidetes (incidence rate ratio [IRR]: 3.70 [95% CI: 1.61–8.48]) and a reduced incidence of Proteobacteria (0.62 [0.43–0.90]) in their milk, compared to women with an overweight BMI (25.0–29.9 kg/m2) as assessed by multivariable Poisson regression. An increased incidence of Gemella was observed among mothers with gestational diabetes who had an overweight BMI versus healthy range BMI (5.96 [1.85–19.21]). An increased incidence of Gemella was also observed among mothers with impaired glucose tolerance with an obese BMI versus mothers with a healthy range BMI (4.04 [1.63–10.01]). An increased incidence of Brevundimonas (16.70 [5.99–46.57]) was found in the milk of women who underwent an unscheduled C-section versus vaginal delivery. Lastly, functional gene inference demonstrated that pre-pregnancy obesity was associated with an increased abundance of genes encoding for the biosynthesis of secondary metabolites pathway in milk (coefficient = 0.0024, P FDR

Abstract Background Human-targeted drugs may exert off-target effects or can be repurposed to modulate the gut microbiota. However, our understanding of such effects is limited due to a lack of rapid and scalable assay to comprehensively assess microbiome responses to drugs. Drugs and other compounds can drastically change the overall abundance, taxonomic composition, and functions of a gut microbiome. Results Here, we developed an approach to screen compounds against individual microbiomes in vitro, using metaproteomics to both measure absolute bacterial abundances and to functionally profile the microbiome. Our approach was evaluated by testing 43 compounds (including 4 antibiotics) against 5 individual microbiomes. The method generated technically highly reproducible readouts, including changes of overall microbiome abundance, microbiome composition, and functional pathways. Results show that besides the antibiotics, the compounds berberine and ibuprofen inhibited the accumulation of biomass during in vitro growth of the microbiota. By comparing genus and species level-biomass contributions, selective antibacterial-like activities were found with 35 of the 39 non-antibiotic compounds. Seven of the compounds led to a global alteration of the metaproteome, with apparent compound-specific patterns of functional responses. The taxonomic distributions of altered proteins varied among drugs, i.e., different drugs affect functions of different members of the microbiome. We also showed that bacterial function can shift in response to drugs without a change in the abundance of the bacteria. Conclusions Current drug-microbiome interaction studies largely focus on relative microbiome composition and microbial drug metabolism. In contrast, our workflow enables multiple insights into microbiome absolute abundance and functional responses to drugs. The workflow is robust, reproducible, and quantitative and is scalable for personalized high-throughput drug screening applications.

Resistant starches (RS) are dietary compounds processed by the gut microbiota into metabolites, such as butyrate, that are beneficial to the host. The production of butyrate by the microbiome appears to be affected by the plant source and type of RS as well as the individual’s microbiota. In this study, we used in vitro culture and metaproteomic methods to explore individual microbiome's functional responses to RS2 (enzymatically-resistant starch), RS3 (retrograded starch) and RS4 (chemically-modified starch). Results showed that RS2 and RS3 significantly altered the protein expressions in the individual gut microbiomes, while RS4 did not result in significant protein changes. Significantly elevated protein groups were enriched in carbohydrate metabolism and transport functions of families Eubacteriaceae, Lachnospiraceae and Ruminococcaceae. In addition, Bifidobacteriaceae was significantly increased in response to RS3. We also observed taxon-specific enrichments of starch metabolism and pentose phosphate pathways corresponding to this family. Functions related to starch utilization, ABC transporters and pyruvate metabolism pathways were consistently increased in the individual microbiomes in response to RS2 and RS3. Given that these taxon-specific responses depended on the type of carbohydrate sources, we constructed a functional ecological network to gain a system-level insight of functional organization. Our results suggest that while some microbes tend to be functionally independent, there are subsets of microbes that are functionally co-regulated by environmental changes, potentially by alterations of trophic interactions.

Abstract The gut microbiome has emerged as an important factor affecting human health and disease. The recent development of –omics approaches, including phylogenetic marker-based microbiome profiling, shotgun metagenomics, metatranscriptomics, metaproteomics, and metabolomics, has enabled efficient characterization of microbial communities. These techniques can provide strain-level taxonomic resolution of the taxa present in microbiomes, assess the potential functions encoded by the microbial community and quantify the metabolic activities occurring within a complex microbiome. The application of these meta-omics approaches to clinical samples has identified microbial species, metabolic pathways, and metabolites that are associated with the development and treatment of human diseases. These findings have further facilitated microbiome-targeted drug discovery and efforts to improve human health management. Recent in vitro and in vivo investigations have uncovered the presence of extensive drug-microbiome interactions. These interactions have also been shown to be important contributors to the disparate patient responses to treatment that are often observed during disease therapy. Therefore, developing techniques or frameworks that enable rapid screening, detailed evaluation, and accurate prediction of drug/host-microbiome interactions is critically important in the modern era of microbiome research and precision medicine. Here we review the current status of meta-omics techniques, including integrative multi-omics approaches, for characterizing the microbiome’s functionality in the context of health and disease. We also summarize and discuss new frameworks for applying meta-omics approaches and microbiome assays to study drug-microbiome interactions. Lastly, we discuss and exemplify strategies for implementing microbiome-based precision medicines using these meta-omics approaches and high throughput microbiome assays.

While the prevalence of metabolic syndrome (MetS) is steadily increasing worldwide, no optimal pharmacotherapy is readily available to address its multifaceted risk factors and halt its complications. This growing challenge mandates the development of other future curative directions. The purpose of the present study is to investigate the efficacy of cranberry proanthocyanidins (PACs) in improving MetS pathological conditions and liver complications; C57BL/6J mice were fed either a standard chow or a high fat/high sucrose (HFHS) diet with and without PACs (200 mg/kg), delivered by daily gavage for 12 weeks. Our results show that PACs lowered HFHS-induced obesity, insulin resistance, and hyperlipidemia. In conjunction, PACs lessened circulatory markers of oxidative stress (OxS) and inflammation. Similarly, the anti-oxidative and anti-inflammatory capacities of PACs were noted in the liver in association with improved hepatic steatosis. Inhibition of lipogenesis and stimulation of beta-oxidation could account for PACs-mediated decline of fatty liver as evidenced not only by the expression of rate-limiting enzymes but also by the status of AMPKα (the key sensor of cellular energy) and the powerful transcription factors (PPARα, PGC1α, SREBP1c, ChREBP). Likewise, treatment with PACs resulted in the downregulation of critical enzymes of liver gluconeogenesis, a process contributing to increased rates of glucose production in type 2 diabetes. Our findings demonstrate that PACs prevented obesity and improved insulin resistance likely via suppression of OxS and inflammation while diminishing hyperlipidemia and fatty liver disease, as clear evidence for their strength of fighting the cluster of MetS abnormalities.

SUMMARY: The intercellular space or apoplast constitutes the main interface in plant–pathogen interactions. Apoplastic subtilisin‐like proteases—subtilases—may play an important role in defence and they have been identified as targets of pathogen‐secreted effector proteins. Here, we characterise the role of the Solanaceae‐specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post‐translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D‐deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum. Significance Statement: The Solanaceae‐specific P69 subtilase family participates in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. [ABSTRACT FROM AUTHOR]

The authors present an in vitro gut microbiome model that maintains the functional and compositional profiles of individual gut microbiomes, as assessed by metaproteomics. Gut microbiome responses to metformin in this model correlate with those shown in mice fed a high-fat diet.

Resistant starch, microbiome, and precision modulation. Mounting evidence has positioned the gut microbiome as a nexus of health. Modulating its phylogenetic composition and function has become an attractive therapeutic prospect. Resistant starches (granular amylase-resistant α-glycans) are available as physicochemically and morphologically distinguishable products. Attempts to leverage resistant starch as microbiome-modifying interventions in clinical studies have yielded remarkable inter-individual variation. Consequently, their utility as a potential therapy likely depends predominantly on the selected resistant starch and the subject’s baseline microbiome. The purpose of this review is to detail i) the heterogeneity of resistant starches, ii) how resistant starch is sequentially degraded and fermented by specialized gut microbes, and iii) how resistant starch interventions yield variable effects on the gut microbiome.

Campylobacter jejuni NCTC11168 does not produce any endogenous siderophores of its own yet requires the CfrA enterobactin transporter for in vivo colonization. In addition, the genome of C. jejuni NCTC11168 contains three distinct TonB energy transduction systems, named TonB1, TonB2, and TonB3, that have not been tested for their role in siderophore uptake or their functional redundancy. We demonstrate that C. jejuni NCTC11168 transports ferric-enterobactin in an energy dependent manner that requires TonB3 for full activity with TonB1 showing partial functional redundancy. Moreover C. jejuni NCTC11168 can utilize a wide variety of structurally different catechol siderophores as sole iron sources during growth. This growth is solely dependent on the CfrA enterobactin transporter and highlights the wide range of substrates that this transporter can recognize. TonB3 is also required for growth on most catechol siderophores. Furthermore, either TonB1 or TonB3 is sufficient for growth on hemin or hemoglobin as a sole iron source demonstrating functional redundancy between TonB1 and TonB3. In vivo colonization assays with isogenic deletion mutants revealed that both TonB1 and TonB3 are required for chick colonization with TonB2 dispensable in this model. These results further highlight the importance of iron transport for efficient C. jejuni colonization.