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Comparative Study
. 2015 Feb;81(4):1502-12.
doi: 10.1128/AEM.03283-14.

Gut Symbionts From Distinct Hosts Exhibit Genotoxic Activity via Divergent Colibactin Biosynthesis Pathways

Free PMC article
Comparative Study

Gut Symbionts From Distinct Hosts Exhibit Genotoxic Activity via Divergent Colibactin Biosynthesis Pathways

Philipp Engel et al. Appl Environ Microbiol. .
Free PMC article

Abstract

Secondary metabolites produced by nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways are chemical mediators of microbial interactions in diverse environments. However, little is known about their distribution, evolution, and functional roles in bacterial symbionts associated with animals. A prominent example is colibactin, a largely unknown family of secondary metabolites produced by Escherichia coli via a hybrid NRPS-PKS biosynthetic pathway that inflicts DNA damage upon eukaryotic cells and contributes to colorectal cancer and tumor formation in the mammalian gut. Thus far, homologs of this pathway have only been found in closely related Enterobacteriaceae, while a divergent variant of this gene cluster was recently discovered in a marine alphaproteobacterial Pseudovibrio strain. Herein, we sequenced the genome of Frischella perrara PEB0191, a bacterial gut symbiont of honey bees and identified a homologous colibactin biosynthetic pathway related to those found in Enterobacteriaceae. We show that the colibactin genomic island (GI) has conserved gene synteny and biosynthetic module architecture across F. perrara, Enterobacteriaceae, and the Pseudovibrio strain. Comparative metabolomics analyses of F. perrara and E. coli further reveal that these two bacteria produce related colibactin pathway-dependent metabolites. Finally, we demonstrate that F. perrara, like E. coli, causes DNA damage in eukaryotic cells in vitro in a colibactin pathway-dependent manner. Together, these results support that divergent variants of the colibactin biosynthetic pathway are widely distributed among bacterial symbionts, producing related secondary metabolites and likely endowing its producer with functional capabilities important for diverse symbiotic associations.

Figures

FIG 1
FIG 1
(A) Comparison of the genome of F. perrara to other Orbaceae genomes. Starting from outside, the first circle shows the scale of the genome representation of F. perrara in gray and white steps of 100 kb. The second and third circles (green) depict the genes on the plus and minus strands of F. perrara. The fourth circle depicts all tRNA and rRNA genes in blue and black, respectively. The fifth circle highlights F. perrara-specific genomic islands (GIs) compared to other Orbaceae genomes: GI region 1 contains a tellurite resistance operon, GI region 2 contains genes encoding mostly hypothetical proteins and the colibactin biosynthetic gene cluster, GI regions 3 and 4 contain the type I secretion system genes, and GI region 5 contains the type VI secretion system genes. The sixth circle depicts the GC skew over the chromosome of F. perrara with positive values shown in magenta and negative values in peach. The blue circles represent orthologs identified in the genomes of G. apicola wkB1, G. apicola wkB11, G. apicola wkB30, and Orbus hercynius CN3. The blue color range denotes protein identity between these pairwise comparisons, as depicted by the scale in the center of the genome circle. (B) Presence/absence of genes of the TCA cycle (green arrows) and for fermentation (orange arrows) in the genomes of the two honey bee gut symbionts F. perrara and G. apicola wkB1. Semicircles in magenta and blue indicate presence of gene functions in the genomes of F. perrara and G. apicola wkB1, respectively. Other gene functions are either absent or could not be identified (empty semicircles).
FIG 2
FIG 2
Phylogenetic relationship of bacteria harboring variants of the colibactin (clb) genomic island (GI) and comparison of their genetic organizations. Bacteria containing the clb GI are highlighted in green (Enterobacteriaceae), magenta (Frischella perrara PEB0191), and blue (Pseudovibrio FO-BEG1). For Citrobacter koseri, Enterobacter aerogenes, and Klebsiella pneumoniae, strains 4225-83, EA1509E, and WGLW1, respectively, were analyzed. The maximum likelihood tree is based on the concatenated alignments of eight conserved housekeeping genes. Black circles denote branches with bootstraps of ≥80 (100 replicates). Orthologs are connected via gray blocks. Percentages of protein identities are depicted and reflected by the shading intensity of each block. Genes without a homolog are shown in white. The average G+C contents of the Clb GI are 40.4%, 53.7%, and 51.1% for F. perrara PEB0191, E. coli IHE3034, and Pseudovibrio FO-BEG1, respectively. Genes and gene products are depicted using the following abbreviations: clb, colibactin; IS1351, insertion sequence 1351; MobB, mobilization protein B; VgrG, valine-glycine repeat protein G; NRPS, nonribosomal peptide synthetase; PKS, polyketide synthase; AT, acyl-transferase; T, thiolation sequence of acyl/peptidyl-carrier proteins; DH, dehydrogenase; AM, amidase; EP, efflux protein; PE, peptidase; TE, thioesterase; PPT, phosphopantetheinyl-transferase; SAM, S-adenosylmethionine-binding protein; H, hydrolase.
FIG 3
FIG 3
Domain architecture of the Clb NRPS/PKS proteins of F. perrara PEB0191, E. coli IHE3034, and Pseudovibrio FO-BEG1. Predicted amino acid substrate specificities of adenylation (A) domains and residues in binding pockets are depicted. Predictions with NRPSpredictor2 confidence scores (46) of <80% are marked with a question mark. For ClbB and ClbN, the experimentally validated A domain specificities (Ala and Asn, respectively) are depicted (33, 34). For ClbN, this is consistent with the prediction, but for ClbB, the prediction suggested Val. Sequence motifs (GxSxG) of the active sites of AT domains are depicted. Relict cis-AT domains of ClbC, ClbK, and ClbO are shown in white and denoted with an asterisk. Protein identities with a sliding window size of 15 bp are shown (red depicts identity of <30%). Abbreviations of domains are as follows: C, condensation; A, adenylation; T, thiolation sequence of acyl/peptidyl-carrier proteins; KS, ketosynthase; AT, acyl-transferase; KR, ketoreductase; DH, dehydratase; ER, enoyl-reductase; Cy, condensation/cyclase; Ox, oxidase; E, epimerase.
FIG 4
FIG 4
Colibactin pathway-dependent metabolites in F. perrara (A) and proposed structures for the fatty acyl-Asn metabolites (B) and their production (C). (A) MS2 network analysis between F. perrara and E. coli strains. MOFs in square nodes are specific to F. perrara, those in diamonds are shared among F. perrara and E. coli, and those in oval nodes were detected only in wild-type E. coli strains. (B) Proposed structures for eight metabolites are shown based on network analysis, MS2 fragmentation patterns, and comparison to previously characterized colibactin metabolites. Data for the major metabolite 1 (m/z 315. 2281) support N-lauryl-d-Asn, those for metabolites 2 to 5 have previously been reported (32, 34), and those for metabolites 6 to 8 represent new metabolites produced by F. perrara. (C) Extracted ion chromatogram (EIC) of metabolites 1, 2, 3, and 8, which are produced at a 10:1.2:0.37:0.19 ratio under our experimental conditions. Metabolites 4 to 7 (not shown) were only produced as very minor constituents and were near baseline at the scale shown.
FIG 5
FIG 5
F. perrara PEB0191 causes megalocytosis (A and B) and activates a DNA damage response in HeLa cells in vitro (C). (A) Megalocytosis of HeLa cells was analyzed 48 h post-transient infection. HeLa cells were stained with Giemsa as previously described (34). Transient infections with bacteria were carried out for 4 h. Scale bars, 100 μm. (B) Quantification of megalocytosis activity was based on protein content per well using methylene blue staining 48 h postinfection, followed by methylene blue extraction and OD660 measurements as described previously (34). For each condition, three independent wells were quantified. The mean + standard deviation is shown, and P values of two-tailed t tests are indicated: **, P < 0.01; *, P < 0.05. (C) HeLa cells were infected for 4 h at an MOI of 200 for E. coli and 5,000 for F. perrara. γ-H2AX was quantified by flow cytometry after 14 h of incubation. clb+, pBAC-PKS; clb−, pBAC-control; wt, wild type.

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