Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ΔtreYZΔtreS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ΔtreYZΔtreS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ΔtreYZΔtreS in trans. Surprisingly, the growth defect of the double ΔtreYZΔtreS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved “house-keeping” anabolic pathway (trehalose biosynthesis) as a potent virulence factor that allows it to replicate in the intercellular environment of a leaf.

Author Summary

Pseudomonas aeruginosa is an opportunistic human bacterial pathogen that infects a wide range of plants and animals, including the model laboratory plant Arabidopsis thaliana. P. aeruginosa utilizes many of the same virulence-related factors to infect both plants and animals. However, because plants have fundamentally different cellular architecture than animals, we hypothesized that P. aeruginosa synthesizes specific factors required for infecting plants but not animals. We found that synthesis of the sugar molecule trehalose, an unusual dimer of glucose, is required for plant but not animal pathogenesis. Although P. aeruginosa mutants defective in trehalose synthesis are non-pathogenic in Arabidopsis, Arabidopsis mutants that lack the polysaccharide xyloglucan in their cell walls can be infected by P. aeruginosa trehalose mutants. Moreover, application of ammonium nitrate overcomes the requirement for trehalose for infecting an Arabidopsis leaf. Our data suggest that trehalose promotes the acquisition of nitrogen-containing nutrients, thereby allowing P. aeruginosa to replicate in the nutrient-poor intercellular spaces in a leaf. This work shows how an opportunistic pathogen has repurposed a highly conserved “house-keeping” function (trehalose biosynthesis) as a potent virulence factor.

In bacterial communities, ‘tight economic times’ are the norm. Of the many challenges bacteria face in making a living, perhaps none are more important than generating energy, maintaining redox balance, and acquiring carbon and nitrogen to synthesize primary metabolites. The ability of bacteria to meet these challenges depends heavily on the rest of their community. Indeed, the most fundamental way in which bacteria communicate is by importing the substrates for metabolism and exporting metabolic end products. As an illustration of this principle, we will travel down a carbohydrate catabolic pathway common to many species of Bacteroides, highlighting the interspecies interactions established (often inevitably) at its key steps. We also discuss the metabolic considerations in maintaining the stability of host-associated microbial communities.

New drug development strategies are needed to combat antimicrobial resistance. The object of this perspective is to highlight one such strategy: treating infections with sets of drugs rather than individual drugs. We will highlight three categories of combination therapy: those that inhibit targets in different pathways; those that inhibit distinct nodes in the same pathway; and those that inhibit the very same target in different ways. We will then consider examples of naturally occurring combination therapies produced by microorganisms, and conclude by discussing key opportunities and challenges for making more widespread use of drug combinations.

Peptide natural products exhibit broad biological properties and are commonly produced by orthogonal ribosomal and nonribosomal pathways in prokaryotes and eukaryotes. To harvest this large and diverse resource of bioactive molecules, we introduce Natural Product Peptidogenomics (NPP), a new mass spectrometry-guided genome mining method that connects the chemotypes of peptide natural products to their biosynthetic gene clusters by iteratively matching de novo MSn structures to genomics-based structures following current biosynthetic logic. In this study we demonstrate that NPP enabled the rapid characterization of >10 chemically diverse ribosomal and nonribosomal peptide natural products of novel composition from streptomycete bacteria as a proof of concept to begin automating the genome mining process. We show the identification of lantipeptides, lasso peptides, linardins, formylated peptides and lipopeptides, many of which from well-characterized model streptomycetes, highlighting the power of NPP in the discovery of new peptide natural products from even intensely studied organisms.

We are never alone. Humans coexist with diverse microbial species that live within and upon us—our so-called microbiota. It is now clear that this microbial community is essentially another organ that plays a fundamental role in human physiology and disease. Basic and translational research efforts have begun to focus on deciphering mechanisms of microbiome function—and learning how to manipulate it to benefit human health. In this Perspective, we discuss therapeutic opportunities in the human microbiome.

Objectives

Longitudinal studies examining the baseline predictors of fatigue in SSc have not been reported. Our objectives were to examine the course of fatigue severity over time and to identify baseline clinical, demographic, and psychosocial predictors of sequentially obtained fatigue scores in early SSc. We also examined baseline predictors of change in fatigue severity over time.

Methods

We analyzed 1090 longitudinal Fatigue Severity Scale (FSS) scores belonging to 256 patients who were enrolled in the Genetics versus Environment in Scleroderma Outcomes Study (GENISOS). Predictive significance of baseline variables for sequentially obtained FSS scores was examined with generalized linear mixed models. Predictors of change in FSS over time were examined by adding an interaction term between the baseline variable and time-in-study to the model.

Results

The patients' mean age was 48.6 years, 47% were Caucasians, and 59% had diffuse cutaneous involvement. The mean disease duration at enrollment was 2.5 years. The FSS was obtained at enrollment and follow-up visits (mean follow-up time = 3.8 years). Average baseline FSS score was 4.7(±0.96). The FSS was relatively stable and did not show a consistent trend for change over time (p = 0.221). In a multivariable model of objective clinical variables, higher Medsger Gastrointestinal (p = 0.006) and Joint (p = 0.024) Severity Indices, and anti-U1-RNP antibodies (p = 0.024) were independent predictors of higher FSS. In the final model, ineffective coping skills captured by higher Illness Behavior Questionnaire scores (p<0.001), higher self-reported pain (p = 0.006), and higher Medsger Gastrointestinal Severity Index (p = 0.009) at enrollment were independent predictors of higher longitudinal FSS scores. Baseline DLco % predicted was the only independent variable that significantly predicted a change in FSS scores over time (p = 0.013), with lower DLco levels predicting an increase in FSS over time.

Conclusions

This study identified potentially modifiable clinical and psychological factors that predict longitudinal fatigue severity in early SSc.

Summary

As genetically encoded small molecules, antibiotics are phenotypes that have resulted from mutation and natural selection. Advances in genetics, biochemistry, and bioinformatics have connected hundreds of antibiotics to the gene clusters that encode them, allowing these molecules to be analyzed using the tools of evolutionary biology. This review surveys examples of convergent evolution from microbially produced antibiotics, including the convergence of distinct gene clusters on similar phenotypes and the merger of distinct gene clusters into a single functional unit. Examining antibiotics through an evolutionary lens highlights the versatility of biosynthetic pathways, reveals lessons for combating antibiotic resistance, and provides an entry point for studying the natural roles of these natural products.

Bacterial and fungal secondary metabolism is a rich source of novel bioactive compounds with potential pharmaceutical applications as antibiotics, anti-tumor drugs or cholesterol-lowering drugs. To find new drug candidates, microbiologists are increasingly relying on sequencing genomes of a wide variety of microbes. However, rapidly and reliably pinpointing all the potential gene clusters for secondary metabolites in dozens of newly sequenced genomes has been extremely challenging, due to their biochemical heterogeneity, the presence of unknown enzymes and the dispersed nature of the necessary specialized bioinformatics tools and resources. Here, we present antiSMASH (antibiotics & Secondary Metabolite Analysis Shell), the first comprehensive pipeline capable of identifying biosynthetic loci covering the whole range of known secondary metabolite compound classes (polyketides, non-ribosomal peptides, terpenes, aminoglycosides, aminocoumarins, indolocarbazoles, lantibiotics, bacteriocins, nucleosides, beta-lactams, butyrolactones, siderophores, melanins and others). It aligns the identified regions at the gene cluster level to their nearest relatives from a database containing all other known gene clusters, and integrates or cross-links all previously available secondary-metabolite specific gene analysis methods in one interactive view. antiSMASH is available at http://antismash.secondarymetabolites.org.

Streptomyces clavuligerus is an important industrial strain that produces a number of antibiotics, including clavulanic acid and cephamycin C. A high-quality draft genome sequence of the S. clavuligerus NRRL 3585 strain was produced by employing a hybrid approach that involved Sanger sequencing, Roche/454 pyrosequencing, optical mapping, and partial finishing. Its genome, comprising four linear replicons, one chromosome, and four plasmids, carries numerous sets of genes involved in the biosynthesis of secondary metabolites, including a variety of antibiotics.

Systems biology is set to go beyond single species to the study of interspecies interactions.

Systems approaches are not so different in essence from classical genetic and biochemical approaches, and in the future may become adopted so widely that the term 'systems biology' itself will become obsolete.

SUMMARY

Accumulating evidence implicates heterogeneity within cancer cell populations in the response to stressful exposures, including drug treatments. While modeling the acute response to various anti-cancer agents in drug-sensitive human tumor cell lines, we consistently detected a small subpopulation of reversibly “drug-tolerant” cells. These cells demonstrate >100-fold reduced drug sensitivity, and maintain viability via engagement of IGF-1 receptor signaling and an altered chromatin state that requires the histone demethylase RBP2/KDM5A/Jarid1A. This drug-tolerant phenotype is transiently acquired and relinquished at low frequency by individual cells within the population, implicating the dynamic regulation of phenotypic heterogeneity in drug tolerance. The drug-tolerant subpopulation can be selectively ablated by treatment with IGF-1 receptor inhibitors or chromatin-modifying agents, potentially yielding a therapeutic opportunity. Together, these findings suggest that cancer cell populations employ a dynamic survival strategy in which individual cells transiently assume a reversibly drug-tolerant state to protect the population from eradication by potentially lethal exposures.

Natural products have played a prominent role in the history of organic chemistry, and they continue to be important as drugs, biological probes, and targets of study for synthetic and analytical chemists. In this perspective, we explore how connecting Nature’s small molecules to the genes that encode them has sparked a renaissance in natural product research, focusing primarily on the biosynthesis of polyketides and nonribosomal peptides. We survey monomer biogenesis, coupling chemistries from templated and non-templated pathways, and the broad set of tailoring reactions and hybrid pathways that give rise to the diverse scaffolds and functionalization patterns of natural products. We conclude by considering two questions: What would it take to find all natural product scaffolds? What kind of scientists will be studying natural products in the future?

Objective

To measure self-reported physical and mental functioning and associated clinical features at study entry in 3 ethnic groups with systemic sclerosis (SSc).

Methods

Sixty Hispanic, 39 African American, and 104 Caucasian patients with recent-onset SSc (< 5 yrs) were assessed for perceived physical and mental functioning, using the Medical Outcomes Study Short Form-36 (SF-36) and Scleroderma-Health Assessment Questionnaire (Sclero-derma-HAQ). Socioeconomic, demographic, clinical, immunologic, immunogenetic, behavioral, and psychological variables (Interpersonal Support Evaluation List, ISEL; Illness Behavior Questionnaire, IBQ; and Arthritis Helplessness Index, AHI) were analyzed by linear regression models for associations with SF-36 and mHAQ scores as dependent variables.

Results

Perceived physical functioning scores had ethnic-specific associations with AHI > fatigue scores > IBQ > clinical variables (hypertension, skin score, and percentage predicted DLCO). Scleroderma-HAQ scores had ethnic-specific associations with IBQ > AHI scores > most clinical and laboratory variables. Decreased mental component summary (MCS) scores associated with AHI > ISEL. Ethnic-specific immunogenetic variables HLA-DQB1*0202 (Caucasian) and HLA-DRB1*11 (African American), and HLA-DQA1*0501 (Hispanic) also associated with MCS. Antinuclear autoantibodies, anti-topoisomerase I, and RNA polymerases I and III also demonstrated associations with functioning in African American and Hispanic groups.

Conclusion

Clinical, psychosocial, and immunogenetic variables had ethnic-specific associations with perceived physical and mental functioning. Consideration of ethnic-specific psychological and behavioral support in designing more personalized, relevant therapeutic interventions for the patient may improve therapeutic efficacy in SSc.

Objective

To determine human leucocyte antigen-class II (HLA-class II) (DRB1, DQB1, DQA1 and DPB1) alleles, haplotypes and shared epitopes associated with scleroderma (systemic sclerosis (SSc)) and its subphenotypes in a large multi-ethnic US cohort by a case–control association study.

Patients and methods

1300 SSc cases (961 white, 178 black and 161 Hispanic subjects) characterised for clinical skin forms (limited vs diffuse), SSc-specific autoantibodies (anticentromere (ACA), anti-topoisomerase I (ATA), anti-RNA polymerase III (ARA), anti-U3 ribonucleoprotein (fibrillarin)) and others were studied using molecular genotyping. Statistical analyses in SSc itself by ethnicity, gender, skin type and autoantibodies were performed using exact logistic regression modelling for dominant, additive and recessive effects from HLA.

Results

The strongest positive class II associations with SSc in white and Hispanic subjects were the DRB1*1104, DQA1*0501, DQB1*0301 haplotype and DQB1 alleles encoding a non-leucine residue at position 26 (DQB1 26 epi), while the DRB1*0701, DQA1*0201, DQB1*0202 haplotype and DRB1*1501 haplotype were negatively correlated and possibly protective in dominant and recessive models, respectively. These associations did not discriminate between limited and diffuse SSc. SSc in black subjects was associated with DRB1*0804, DQA1*0501, DQB1*0301 alleles. DPB1*1301 showed the highest odds ratio for ATA (OR = 14). Moreover, it showed no linkage disequilibrium or gene interaction with DR/DQ. ACA was best explained by DQB1*0501 and DQB1*26 epi alleles and ARA by DRB1*0404, DRB1*11 and DQB1*03 alleles in white and Hispanic subjects but DRB1*08 in black subjects.

Conclusion

These data indicate unique and multiple HLA-class II effects in SSc, especially on autoantibody markers of different subphenotypes.

Objective

To determine human leucocyte antigen-class II (HLA-class II) (DRB1, DQB1, DQA1 and DPB1) alleles, haplotypes and shared epitopes associated with scleroderma (systemic sclerosis (SSc)) and its subphenotypes in a large multi-ethnic US cohort by a case–control association study.

Patients and methods

1300 SSc cases (961 white, 178 black and 161 Hispanic subjects) characterised for clinical skin forms (limited vs diffuse), SSc-specific autoantibodies (anticentromere (ACA), anti-topoisomerase I (ATA), anti-RNA polymerase III (ARA), anti-U3 ribonucleoprotein (fibrillarin)) and others were studied using molecular genotyping. Statistical analyses in SSc itself by ethnicity, gender, skin type and autoantibodies were performed using exact logistic regression modelling for dominant, additive and recessive effects from HLA.

Results

The strongest positive class II associations with SSc in white and Hispanic subjects were the DRB1*1104, DQA1*0501, DQB1*0301 haplotype and DQB1 alleles encoding a non-leucine residue at position 26 (DQB1 26 epi), while the DRB1*0701, DQA1*0201, DQB1*0202 haplotype and DRB1*1501 haplotype were negatively correlated and possibly protective in dominant and recessive models, respectively. These associations did not discriminate between limited and diffuse SSc. SSc in black subjects was associated with DRB1*0804, DQA1*0501, DQB1*0301 alleles. DPB1*1301 showed the highest odds ratio for ATA (OR = 14). Moreover, it showed no linkage disequilibrium or gene interaction with DR/DQ. ACA was best explained by DQB1*0501 and DQB1*26 epi alleles and ARA by DRB1*0404, DRB1*11 and DQB1*03 alleles in white and Hispanic subjects but DRB1*08 in black subjects.

Conclusion

These data indicate unique and multiple HLA-class II effects in SSc, especially on autoantibody markers of different subphenotypes.

Objective

To investigate the clinical and genetic variables at initial presentation that predict survival in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort.

Methods

GENISOS is a prospective, observational study of a multiethnic early systemic sclerosis (SSc) cohort. To date, a total of 250 patients have been enrolled. In addition to clinical and laboratory data, electrocardiograms (EKGs), chest radiographs, and pulmonary function tests have been obtained from each patient. A modified Rodnan skin thickness score, HLA class II genotyping, and a Medsger Damage Index also have been collected. We performed multivariable analyses utilizing the Cox regression following a purposeful model building strategy.

Results

The study analyzed 122 white, 47 African American, and 71 Hispanic SSc patients with an average disease duration of 2.6 years at enrollment. At the time of analysis, 52 (20.8%) of the 250 patients had died. In the final multivariable model excluding HLA genes, 7 variables emerged as significant predictors of mortality: age ≥65 years at enrollment, forced vital capacity <50% predicted, clinically significant arrhythmia on EKG, absence of anticentromere antibodies, hypertension, chest radiograph suggestive of pulmonary fibrosis, and low body mass index (BMI). In separate modeling that included HLA genes, HLA alleles DRB1*0802 and DQA1*0501 were significant predictors of mortality in addition to the predictors mentioned above.

Conclusion

A limited number of variables collected at presentation, including BMI, are able to predict mortality in patients with early SSc. In addition, some of the HLA genes associated with SSc susceptibility are useful for predicting SSc outcome.

Objective

To investigate peripheral blood (PB) cell transcript profiles of systemic sclerosis (SSc) and its subtypes in direct comparison with systemic lupus erythematosus (SLE).

Methods

We investigated PB cell samples from 74 SSc patients, 21 healthy controls, and 17 SLE patients using Illumina Human Ref-8 BeadChips and quantitative polymerase chain reaction confirmation. None of the study participants were receiving immunosuppressive agents other than low-dose steroids and hydroxychloroquine. In addition to conventional statistical and modular analysis, a composite score for the interferon (IFN)–inducible genes was calculated. Within the group of patients with SSc, the correlation of the IFN score with the serologic and clinical subtypes was investigated, as were single-nucleotide polymorphisms in a selected number of IFN pathway genes.

Results

Many of the most prominently overexpressed genes in SSc and SLE were IFN-inducible genes. Forty-three of 47 overexpressed IFN-inducible genes in SSc (91%) were similarly altered in SLE. The IFN score was highest in the SLE patients, followed by the SSc patients, and then the controls. The difference in IFN score among all 3 groups was statistically significant (P < 0.001 for all 3 comparisons). SSc and SLE PB cell samples showed striking parallels to our previously reported SSc skin transcripts in regard to the IFN-inducible gene expression pattern. In SSc, the presence of antitopoisomerase and anti–U1 RNP antibodies and lymphopenia correlated with the higher IFN scores (P = 0.005, P = 0.001, and P = 0.004, respectively); a missense mutation in IFNAR2 was significantly associated with the IFN score.

Conclusion

SLE and SSc fit within the same spectrum of IFN-mediated diseases. A subset of SSc patients shows a “lupus-like” high IFN-inducible gene expression pattern that correlates with the presence of antitopoisomerase and anti–U1 RNP antibodies.

Objective

Birth order has been valuable in revealing the role of environmental influences on the risk of developing certain diseases such as allergy and atopy. In addition, pregnancy has profound effects on the immune system such as short-term effects that permit fetal survival as well as longer-term effects that could influence late-onset diseases. In order to better evaluate these influences, we studied the association of birth order and gravidity/parity as risk factors for systemic sclerosis (SSc; scleroderma).

Methods

Data regarding SSc cases and their unaffected sibling controls were obtained from the Scleroderma Family Registry and DNA Repository. The case-sibling design was used to minimize confounding due to differences in age, race, ethnicity, or calendar time. The gravidity/parity analysis was based on sibships with at least one SSc-affected and one unaffected sister.

Results

Birth order was examined in 974 sibships, comparing SSc cases (n = 987) with their unaffected siblings (n = 3,088). The risk of scleroderma increased with increasing birth order (odds ratio [OR] 1.25, 95% confidence interval [95% CI] 1.06–1.50 for birth order 2–5; OR 2.22, 95% CI 1.57–3.15 for birth order 6–9; and OR 3.53, 95% CI 1.68–7.45 for birth order 10–15). Gravidity/parity was analyzed in 168 sibships (256 unaffected sisters, 172 SSc cases). We found an association between a history of one or more pregnancies and SSc (OR 2.8).

Conclusion

Birth order and pregnancy were independently associated with a higher risk of developing SSc. These findings suggest that immune development in early childhood and/or pregnancy-associated events, including but not limited to microchimerism, plays a role in SSc susceptibility.

Construction of a genomic DNA library from Pantoea agglomerans strain CU0119 and screening against the plant pathogen Erwinia amylovora yielded a new family of antibiotics, dapdiamides A−E (1−5). The structures were established through 2D-NMR experiments and mass spectrometry, as well as the synthesis of dapdiamide A (1). Transposon mutagenesis of the active cosmid allowed identification of the biosynthetic gene cluster. The dapdiamide family’s promiscuous biosynthetic pathway contains two unconventional amide ligases that are predicted to couple its constituent monomers.

Antibiotic-resistant strains of pathogenic bacteria are increasingly prevalent in hospitals and the community. New antibiotics are needed to combat these bacterial pathogens, but progress in developing them has been slow. Historically, most antibiotics have come from a small set of molecular scaffolds whose functional lifetimes have been extended by generations of synthetic tailoring. The emergence of multidrug resistance among the latest generation of pathogens suggests that the discovery of new scaffolds should be a priority. Promising approaches to scaffold discovery are emerging; they include mining underexplored microbial niches for natural products, designing screens that avoid rediscovering old scaffolds, and repurposing libraries of synthetic molecules for use as antibiotics.

The present work reveals that four proteins, MceCDIJ, encoded by the MccE492 gene cluster are responsible for the remarkable post-translational tailoring of Microcin E492 (MccE492), an 84-residue protein toxin secreted by Klebsiella pneumonaie RYC492 that targets neighboring gram-negative species. This modification results in attachment of a linearized and monoglycosylated derivative of enterobactin, a nonribosomal peptide and iron scavenger (siderophore), to the MccE492m C-terminus. MceC and MceD derivatize enterobactin by C-glycosylation at the C5 position of a N-(2,3-dihydroxybenzoyl) serine (DHB-Ser) moiety and regiospecific hydrolysis of an ester linkage in the trilactone scaffold, respectively. MceI and MceJ form a protein complex that attaches C-glycosylated enterobactins to the C-terminal serine residue of both aC10 model peptide and full-length MccE492. In the enzymatic product, the terminal serine residue is covalently attached to the C4′ oxygen of the glucose moiety. Non-enzymatic and base-catalyzed migration of the peptide to the C6′ position affords the C6′ glycosyl ester linkage observed in the mature toxin, MccE492m, isolated from bacterial cultures.

For screening a pool of potential substrates that load carrier domains found in non-ribosomal peptide synthetases, large molecule mass spectrometry is shown to be an ideal, unbiased assay. Combining the high resolving power of Fourier-Transform Mass Spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A1, yersiniabactin, pyochelin and enterobactin biosynthetic pathways as proof-of-principle, preferred substrates are readily identified from substrate pools. Furthermore this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the genes pksN and pksJ, that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate.

The iroA locus encodes five genes (iroB, iroC, iroD, iroE, iroN) that are found in pathogenic Salmonella and Escherichia coli strains. We recently reported that IroB is an enterobactin (Ent) C-glucosyltransferase, converting the siderophore into mono-, di- and triglucosyl enterobactins (MGE, DGE, and TGE, respectively). Here we report the characterization of IroD and IroE as esterases for the apo and Fe3+-bound forms of Ent, MGE, DGE, and TGE, and we compare their activities with those of Fes, the previously characterized enterobactin esterase. IroD hydrolyzes both apo and Fe3+-bound siderophores distributively to generate DHB-Ser and/or Glc-DHB-Ser, with higher catalytic efficiencies (kcat/Km) on Fe3+-bound forms, suggesting that IroD is the ferric MGE/DGE esterase responsible for cytoplasmic iron release. Similarly, Fes hydrolyzes ferric Ent more efficiently than apo Ent, confirming Fes is the ferric Ent esterase responsible for Fe3+ release from ferric Ent. Although each enzyme exhibits lower kcat’s processing ferric siderophores, dramatic decreases in Km’s for ferric siderophores result in increased catalytic efficiencies. The inability of Fes to efficiently hydrolyze ferric MGE, ferric DGE, or ferric TGE explains the requirement for IroD in the iroA cluster. IroE, in contrast, prefers apo siderophores as substrates, and tends to hydrolyze the trilactone just once to produce linearized trimers. These data and the periplasmic location of IroE suggest that it hydrolyzes apo enterobactins while they are being exported. IroD hydrolyzes apo MGE (and DGE) regioselectively to give a single linear trimer product and a single linear dimer product as determined by NMR.