# Insights into evolution and coexistence of the colibactin- and yersiniabactin secondary metabolite determinants in enterobacterial populations

Haleluya Wami, Alexander Wallenstein, Daniel Sauer, Monika Stoll, Rudolf

von Bünau, Eric Oswald, Rolf Müller, Ulrich Dobrindt

► To cite this version:

Haleluya Wami, Alexander Wallenstein, Daniel Sauer, Monika Stoll, Rudolf von Bünau, et al. Insights into evolution and coexistence of the colibactin- and yersiniabactin secondary metabolite determinants in enterobacterial populations. Microbial Genomics, 2021, 7 (6), 15 p. 10.1099/mgen.0.000577.. hal-03594561

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# Insights into evolution and coexistence of the colibactin- and yersiniabactin secondary metabolite determinants in enterobacterial populations

Haleluya Wami¹†, Alexander Wallenstein¹†, Daniel Sauer², Monika Stoll³, Rudolf von Bünau⁴, Eric Oswald⁵, Rolf Müller² and Ulrich Dobrindt¹,*

## Abstract

The bacterial genotoxin colibactin interferes with the eukaryotic cell cycle by causing dsDNA breaks. It has been linked to bacterially induced colorectal cancer in humans. Colibactin is encoded by a 54 kb genomic region in *Enterobacteriaceae*. The colibactin genes commonly co-occur with the yersiniabactin biosynthetic determinant. Investigating the prevalence and sequence diversity of the colibactin determinant and its linkage to the yersiniabactin operon in prokaryotic genomes, we discovered mainly species-specific lineages of the colibactin determinant and classified three main structural settings of the colibactin-yersiniabactin genomic region in *Enterobacteriaceae*. The colibactin gene cluster has a similar but not identical evolutionary track to that of the yersiniabactin operon. Both determinants could have been acquired on several occasions and/or exchanged independently between enterobacteria by horizontal gene transfer. Integrative and conjugative elements play(ed) a central role in the evolution and structural diversity of the colibactin-yersiniabactin genomic region. Addition of an activating and regulating module (clbAR) to the biosynthesis and transport module (clbB-S) represents the most recent step in the evolution of the colibactin determinant. In a first attempt to correlate colibactin expression with individual lineages of colibactin determinants and different bacterial genetic backgrounds, we compared colibactin expression of selected enterobacterial isolates *in vitro*. Colibactin production in the tested *Klebsiella* species and *Citrobacter koseri* strains was more homogeneous and generally higher than that in most of the *Escherichia coli* isolates studied. Our results improve the understanding of the diversity of colibactin determinants and its expression level, and may contribute to risk assessment of colibactin-producing enterobacteria.

## DATA SUMMARY

DNA sequences are publicly available from the SRA and Assembly databases at the National Center for Biotechnology Information (NCBI). The corresponding accession numbers are listed in Table S2 (available in the online version of this article).

## INTRODUCTION

The non-ribosomal peptide/polyketide hybrid colibactin is a secondary metabolite found in a variety of bacterial species of the family *Enterobacteriaceae*. The colibactin biosynthetic machinery is encoded by a 54 kb polyketide synthase (pks) or clb genomic island [1], which includes 19 genes. The largest part of the island consists of a section of overlapping or closely spaced genes: clbB

Received 02 December 2020; Accepted 04 April 2021; Published 15 June 2021

**Author affiliations:** ¹Institute of Hygiene, University of Münster, Münster, Germany; ²Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Center for Infection Research, Saarland University, Campus E8 1, Saarbrücken, Germany; ³Department of Genetic Epidemiology, Institute of Human Genetics, University of Münster, Münster, Germany; ⁴Ardeypharm GmbH, Herdecke, Germany; ⁵IRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, Toulouse, France.

*Correspondence: Ulrich Dobrindt, dobrindt@uni-muenster.de

**Keywords:** high pathogenicity island; secondary metabolite; polyketide; cytopathic effect; *Escherichia coli*; *Klebsiella*; *Citrobacter*.

**Abbreviations:** DAEC, diffusely adhering *Escherichia coli*; EAEC, enteroaggregative *Escherichia coli*; EHEC, enterohemorrhagic *Escherichia coli*; EPEC, enteropathogenic *Escherichia coli*; HPI, high pathogenicity island; HTH, helix-turn-helix; ICE, integrative and conjugative element; MGE, mobile genetic element; NMEC, newborn meningitis-causing *Escherichia coli*; N-Myr-d-asparagine, N-myristoyl-d-asparagine; PAI, pathogenicity island; SAM, S'-adenosylmethionine; ST, sequence type; T4SS, type 4 secretion system; UPEC, uropathogenic *Escherichia coli*; VNTR, variable number of tandem repeat.

†These authors contributed equally to this work

**Data statement:** All supporting data, code and protocols have been provided within the article or through supplementary data files. Seven supplementary tables and ten supplementary figures are available with the online version of this article.

000577 © 2021 The Authors

<figure><img src="image_2.png" /></figure>
to *clbL* and *clbN* to *clbQ*, which are aligned on the same strand and code for components of the biosynthesis complex. The colibactin assembly line is supplemented with a dedicated transporter, encoded by *clbM*, and a resistance-conferring protein encoded by *clbS* [2, 3]. Two additional genes required for colibactin production are located ca. 400 bp upstream of the first biosynthesis gene *clbB* in the opposing reading direction: the *clbR* gene coding for an auto-activating, *pks* island-specific transcription factor and the phosphopantetheinyl transferase-encoding gene *clbA*, which is crucial for activation of polyketide biosynthesis complexes (Fig. 1) [4–6]. Between these two divergent transcription units, there is a ‘variable number of tandem repeat’ (VNTR) region, which comprises a varying number of a repeating octanucleotide sequence (5’-ACAGATAC-3’) depending on the isolate [2].

Recently, the structure of the colibactin molecule has been proposed [3, 7–9]. Yet, the biological role of colibactin is still under discussion. Colibactin can interfere with the progression of the eukaryotic cell cycle, presumably by cross-linking DNA resulting in dsDNA breaks in genomic instability in eukaryotes [1, 10, 11]. The ability to produce colibactin has been described to increase the pathogenic potential of the producing bacteria and to promote colorectal cancer development [12–17], but has also been related to beneficial effects to the host [3, 18–20].

Initially, the *pks* island has been described in extraintestinal pathogenic *Escherichia coli* to be chromosomally inserted into the *asnW* tRNA locus in close proximity to another tRNA(*Asn*) gene-associated pathogenicity island, the so-called ‘high pathogenicity island’ (HPI). The HPI harbours an additional polyketide determinant coding for the metallophore yersiniabactin biosynthetic machinery [1, 21]. As members of the *Enterobacteriaceae* are generally not known as archetypal secondary metabolite producers, the origin of the *pks* island remains to be further investigated. Interestingly, the colibactin determinant has also been detected as part of an ‘integrative and conjugative element’ (ICE) in different enterobacteria. This ICE also integrates near a tRNA(*Asn*) locus into the bacterial chromosome and commonly carries

**Impact Statement**

Colibactin can act as a bacterial genotoxin and thus promote colorectal cancer development. Little is known about the origin, diversity and prevalence of the colibactin genes (*clb*) within prokaryotes. The *clb* genes are closely associated with pathogenicity islands or integrative and conjugative elements (ICEs). We screened roughly 375000 prokaryotic genomes to analyse the diversity and evolution of such mobile genetic elements among bacterial populations. Interestingly, we only detected *clb* genes in subgroups of the order *Enterobacterales*, namely the *Enterobacter–Escherichia, Yersinia–Serratia* and *Erwinia–Pantoea* clades, mainly in *Escherichia coli, Klebsiella* species and *Citrobacter koseri*. The *clb* determinant, together with the yersiniabactin (*ybt*) gene cluster, belong to an ICE in most of the *clb*-positive enterobacteria, especially in *Klebsiella*. We show that both determinants, though in principle freely transferable within bacteria, have a mainly species-specific phylogeny, and that colibactin expression levels were species-independent. Recombination promoted the structural diversification of the ICE in different species, including its successive degeneration that led to the establishment of the colibactin and yersiniabactin islands in *Escherichia coli* phylogroup B2 strains. Our results not only illustrate differing evolutionary tracks of the *clb* and *ybt* determinants in different species of the *Enterobacterales*, but also highlight the importance of ICEs for genomic variability and the evolution of archetypal pathogenicity islands.

the yersiniabactin gene cluster [2, 22]. It has been suggested that the close linkage observed between the colibactin and yersiniabactin gene clusters results from the functional interconnection between the colibactin and yersiniabactin biosynthetic pathways via the phosphopantetheinyl transferase

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Schematic representation of the genomic architecture of the *pks* island (ca. 54 kb) present in *Escherichia coli* strain M1/5. The 19 genes within the island are coloured with respect to their function. The island codes for a phosphopantetheinyl transferase (*clbA*, in purple), a transcriptional autoactivator (*clbR*, in red), multiple core biosynthetic genes (*clbB–clbL*, *clbN*, *clbO* and *clbQ* in blue), a transporter (*clbM*, yellow), a peptidase (*clbP*, in green) and a resistance factor (*clbS*, orange).</figcaption></figure>
ClbA, which can also contribute to the biosynthesis of yersiniabactin [5, 23]. The highly conserved colibactin determinant has so far been detected in a spectrum of strains belonging to the order Enterobacteriales: most commonly among members of the family Enterobacteriaceae, including *Escherichia coli* strains of phylogroup B2, followed by *Klebsiella pneumoniae* isolates, but also in *Citrobacter koseri* and *Klebsiella aerogenes* [2, 18, 24]. Less conserved variants or homologues of the colibactin gene cluster have been described phenotypically or based on nucleotide sequence data in another member of the Enterobacterales, an *Erwinia oleae* strain, but also the honey bee symbiont *Frischella perrara*, and the marine alphaproteobacterium *Pseudovibrio* [24, 25].

Due to the low sequence similarity of the colibactin genes present in *Enterobacterales* and the two homologous polyketide determinants in *F. perrara* and *Pseudovibrio*, and because of their association with mobile genetic elements (MGEs) or at least mobility-associated genes, one can hypothesize that the *clb* gene cluster is spread by horizontal gene transfer, perhaps via an ICE-like element [2, 24]. While in most *Enterobacteriaceae* the colibactin determinant is typically associated with an ICE, the characteristic mobility and transfer features of an ICE are absent in the sequence context of the *pks* island in *Escherichia coli* phylogroup B2 strains. Nevertheless, the *pks* island in *Escherichia coli* remains mobilizable and transferable through external factors, supporting the hypothesis that former MGEs can undergo a stabilization (homing) process upon their chromosomal integration [26–28].

Studies addressing the prevalence of the colibactin genes have so far mainly focused on *Klebsiella* species or *Escherichia coli* backgrounds. The scarcity of data in other prokaryotic species regarding its distribution and the structure of the associated MGE makes it challenging to reliably further characterize the transmission and evolution of this polyketide determinant [18]. Previous data show that the prevalence varied from 5.3 to 25.6% in *Klebsiella* and from 9.5 to 58% in *Escherichia*, highlighting an enrichment of the *pks* island in specific ecological niches, whereas studies with a broader screening approach resulted in a prevalence of 14% in *Klebsiella* and 9.5% in *Escherichia* isolates, respectively [2, 29–34]. Notably, in health-related studies, a higher association of the *clb* genes was observed amongst strains with an increased virulence potential, with a prevalence of as much as 78.8% for *Klebsiella* subgroups and 72.7% for colorectal cancer-associated *Escherichia coli* isolates [29, 32, 35–37]. The colibactin genes are frequently found in hyper-virulent and multidrug-resistant *K. pneumoniae* isolates [38, 39].

The obvious prevalence of the colibactin gene cluster in specific enterobacterial species combined with the description of more distantly related homologous determinants has sparked our interest in a better understanding of the spread and evolution of the colibactin determinant and its genetic context in bacteria. Therefore, we aimed to investigate the prevalence and diversity of the colibactin determinant also in isolates outside of the family *Enterobacteriaceae*. Furthermore, we compared colibactin expression levels among

selected *Escherichia coli*, *Klebsiella* species and *C. koseri* isolates carrying different lineages of *clb* determinants as a first attempt to assess the functional context of the bacterial genetic background, pathogenicity and colibactin expression.

# METHODS

## Bacterial strains and media

For cultivation, bacteria were grown as batch cultures in lysogeny broth (LB) (per litre: 10 g tryptone, 5 g yeast extract, 5 g NaCl) at 37°C. Strains used in this study are listed in the (Table S1).

## DNA extraction and sequencing

DNA extraction of the enterobacterial strains was performed using the MagAttract HMW DNA Kit (Qiagen) according to the manufacturer's recommendations. To prepare paired-end libraries we used the Nextera XT DNA Library Preparation kit (Illumina). Libraries were sequenced on the Illumina MiSeq sequencing platform using v2 sequencing chemistry (500 cycles) or on the Illumina NextSeq500 system using v2.5 chemistry (300 cycles). Accession numbers of in-house sequences submitted to the NCBI GenBank database are included in (Table S1).

## Genome selection and phylogenetic analysis

All genome sequences not generated in this study were obtained from publicly available prokaryotic genomes (NCBI GenBank). The quality of in-house sequenced genomes was checked with FastQC v0.11.5 (https://github.com/chgibb/FastQC0.11.5/blob/master/fastqc), and low-quality reads were trimmed using Sickle v1.33 (https://github.com/najoshi/sickle). The processed reads were de novo assembled with SPAdes v3.13.1 [40] and annotated with prokka v1.12 [41]. The genomes were screened for the presence of >45 kb of the complete *pks* genomic island using standalone BLAST +v2.8.1 [42] and antiSMASH v5.0.0 [43]. The *pks* island found in the genome of the *Escherichia coli* strain M1/5 (accession no. CP053296) was used as a reference sequence. The VNTR and the sequence stretch of the *pks* island that spans between *clbJ* and *clbK* were excluded from analysis as these regions are prone to misassembly.

The contigs that align to the colibactin genes were ordered using ABACAS v1.3.1 [44] and multiple sequence alignment was generated using Kalign v3.1.1 [45]. Recombinant regions were detected and removed using Gubbins v2.4.1 [46]. The recombination filtered polymorphisms were then used to generate a maximum-likelihood phylogeny of the colibactin determinant using RAxML v8.2.11 [47] under the GTR-GAMMAX model from 9974 polymorphic sites. The branch support of the maximum-likelihood tree was estimated by bootstrap analysis of 200 replicate trees. The homologous gene cluster found in *F. perrara* was used as an outgroup. The phylogeny of the corresponding *ybt* islands was generated with a similar approach. Additionally, a core genome-based phylogeny of all strains that harboured the *clb* gene cluster was inferred using the approach described by Adeolu and
colleagues [48]. The generated trees were visualized using itolL
(https://itol.embl.de).

Phylo-grouping of *pks-positive* strains

The *Escherichia coli* and *Klebsiella* strains that harboured
the colibactin gene cluster were allocated to their corre-
sponding sequence types using mlst v2.16.1 (https://github.
com/tseemann/mlst), which detects sequence types using
the PubMLST typing schemes. The *Escherichia* strains were
further classified into their phylogenetic lineages using the
standalone tool, EzClermont v0.4.5 (https://github.com/
nickp60/EzClermont). The phylogeny of all the *clb*-positive
strains was reconstructed using the whole genome sequence-
based approach published by Adeolu and co-workers [48].

Analysis of the diversity of the colibactin and yersiniabactin
gene clusters involved virulence gene multi-locus sequence
typing (MLST) for both polyketide determinants as previ-
ously described [38]. Briefly, the allele sequences of 16 genes
of the colibactin gene cluster (*clb*ACDEFGHILMNOPQR) as
well as of 11 genes of the yersiniabactin determinant (*fyuA*,
*ybtE*, *ybtT*, *ybtU*, *irp1*, *irp2*, *ybtA*, *ybtP*, *ybtQ*, *ybtX*, *ybtS*) were
extracted from the individual genomes and analysed for allelic
variations. Each observed combination of alleles was assigned
a unique colibactin sequence type (CbST, listed in Table S5)
or yersiniabactin sequence type (YbST, listed in Table S6).

Variable number tandem repeat detection

The VNTR copy number present within the colibactin deter-
minant (upstream of *clbR*) was detected using the standalone
version of tandem repeats finder v4.09 [49]. The VNTR copy
number distribution was visualized using R v3.4.3 (https://
www.r-project.org/index.html).

Detection of *Escherichia coli* virulence markers for
pathotyping

For pathotyping, the *clb*-positive *Escherichia coli* strains were
*in silico* screened for the presence of different *E. coli* pathotype
marker genes using BLAST +v2.8.1 (Table S1). These genes
were used as markers for *Escherichia coli* pathotypes: entero-
aggregative *Escherichia coli* (EAEC), enterohaemorrhagic
*Escherichia coli* (EHEC), enteropathogenic *Escherichia coli*
(EPEC), enterotoxigenic *Escherichia coli* (ETEC), diffusely
adhering *Escherichia coli* (DAEC), uropathogenic *Escherichia
coli* (UPEC) and newborn meningitis-causing *Escherichia coli*
(NMEC).

Quantification of colibactin expression through N-
myristoyl-D-asparagine

Following an approach described by Bian and colleagues
[50], a collection of colibactin-producing strains of the
main species harbouring the colibactin determinant was
characterized for their ability to produce colibactin under
*in vitro* growth conditions. For this purpose, we quanti-
fied *N*-myristoyl-*D*-asparagine (*N*-Myr-*D*-asparagine)
a byproduct during colibactin maturation. The amount
of this intermediate extrapolates the resulting colibactin

amount produced. After growing the bacteria for 24 h at
37 °C in glass tubes in 5 ml LB supplemented with 200 µl of
a water/XAD-16-resin slurry, bacterial cells were harvested
by centrifugation. The pelleted bacteria-slurry mixes were
sedimented, filtered and dissolved three times in acetone
with increasing volume (12 ml, 100 ml and finally 200 ml).
Afterward, the solvent was exchanged by rotary evaporation
and replaced by 1.6 ml methanol. The sample was further
concentrated by centrifugation (10 min, 15000 r.p.m. at
4 °C), followed by drying 1,5 ml of the solution in a vacuum
centrifuge and subsequent resuspension in 50 µl methanol.
Then, 30 µl of these processed samples was measured by
ultra performance liquid chromatography coupled to high-
resolution mass spectrometry (UPLC-HRMS) conducted
on a Thermo Scientific Ultimate 3000 RS with a Waters
Acquity BEH 100×2.1 mm 1.7 µm 130A column (eluent
A: 0.1% formic acid in ddH₂O, eluent B: 0.1% formic acid
in acetonitrile), where a flow rate of 0.6 ml min⁻¹ followed
by a Bruker Maxis II-4G, 150–2500 *m/z* and a scan rate
of 2 Hz was applied. To enable quantification of *N*-Myr-
*D*-asparagine, we used 250 mM cinnarizine as an internal
standard and normalized peak areas based on the internal
standard and the optical density (*OD*<sub>600</sub>) of the bacterial
culture.

RESULTS

Prevalence of colibactin determinant

Of the 374754 publicly accessible prokaryotic genomes
(as of 30 June 2019) that were screened for the presence
of the colibactin gene cluster, 1969 genomes carried this
polyketide-encoding operon. An additional 198 clb-positive
enterobacterial genomes determined in-house were added
to the analysis (Tables 1, S2 and S3). The clb gene cluster was
detected in several species of the Enterobacter–Escherichia
clade of Enterobacterales, most frequently in Escherichia
coli and K. pneumoniae isolates, but also to a lesser extent
in K. aerogenes, C. koseri, Erwinia cloacae, Enterobacter
hormaechei, K. michiganensis as well as in members of
the Yersinia–Serratia clade (Serratia marcescens), and the
Erwinia–Pantoea clade (Erwinia oleae). The colibactin
determinant was, however, not detectable in 112546 Salmo-
nella enterica and 41 Salmonella bongori genomes, but in
one out of eight genomes of unspecified Salmonella isolates.
We did not detect the clb genes in 2634 Shigella species, 861
Yersinia species, 677 Serratia species, 186 Proteus species
and 69 Morganella species genomes (Table S4). A less well-
conserved homologue of the colibactin determinant was
detected in three F. perrara genomes. It should be noted that
the number of genomes of Klebsiella species and Escherichia
coli analysed in this study are markedly higher than those
of the other species and lineages due to the sequencing
bias towards Klebsiella species and Escherichia coli strains.
Accordingly, a reliable statement on the prevalence of the
colibactin determinant in the different species cannot be
made.
<table><caption>**Table 1.** Bacterial species testing positive for the presence of the colibactin determinant</caption><thead><tr><th></th><th>No. of strains screened</th><th>clb-positive strains</th><th>Predominant sequence types</th></tr></thead><tbody><tr><td>Escherichia coli</td><td>19200</td><td>1462</td><td>ST12, ST127, ST141, ST2015, ST372, ST404, ST550, ST625, ST73, ST80, ST95, ST998</td></tr><tr><td>Klebsiella pneumoniae</td><td>8038</td><td>572</td><td>ST11, ST23, ST234, ST258, ST3, ST48</td></tr><tr><td>Klebsiella aerogenes</td><td>222</td><td>101</td><td>ST4, ST93</td></tr><tr><td>Citrobacter koseri</td><td>35</td><td>27</td><td>NA</td></tr><tr><td>Enterobacter hormaechei</td><td>711</td><td>2</td><td>NA</td></tr><tr><td>Enterobacter cloacae</td><td>610</td><td>2</td><td>NA</td></tr><tr><td>Serratia marcescens</td><td>515</td><td>1</td><td>NA</td></tr><tr><td>Klebsiella michiganensis</td><td>94</td><td>1</td><td>NA</td></tr><tr><td>Salmonella sp.</td><td>8</td><td>1*</td><td>NA</td></tr><tr><td>Erwinia oleae</td><td>1</td><td>1</td><td>NA</td></tr><tr><td>Frischella perrara</td><td>3</td><td>3</td><td>NA</td></tr></tbody></table>

## Diversity of the colibactin determinant

To determine whether the prevalence of the colibactin gene cluster is restricted to specific phylogenetic lineages of *Escherichia coli* and *Klebsiella* species, the sequence types of the corresponding *Escherichia coli* and *Klebsiella* species isolates were further analysed. As shown in (Fig. S1), the *clb* gene cluster was enriched in a small subset of *Escherichia coli* STs (12 out of 11537 STs, as of 30 October 2020), *K. aerogenes* STs (two out of 214 STs, as of 30 October 2020), and *K. pneumoniae* STs (six out of 5237 STs, as of 30 October 2020), respectively. In these 12 *Escherichia coli* STs, between 58 and 94% of the allocated isolates carry the *clb* determinant. A high percentage (ca. 96%) of the *K. aerogenes* ST4 and ST93 included in our study harboured the colibactin genes. In the tested *K. pneumoniae* strains, all ST3 isolates were *clb*-positive, and more than 75% of the analysed ST23 and ST234 isolates carried the colibactin gene cluster, whereas this was only the case for a significantly lower percentage of the *K. pneumoniae* isolates allocated to ST11, ST258 and ST48. Table S2 contains a complete list of STs to which colibactin-positive *Escherichia coli* and *Klebsiella* isolates have been assigned.

The nucleotide sequences of the *clb* gene cluster extracted from the 2169 strains (Table S2) were used to generate a recombination-free phylogeny of the colibactin determinant as shown in Fig. 2a (see Figs S2 and S3 for branch support values and strain labels/assembly IDs, and Fig. S4 indicates predicted recombination events in the *clb* gene cluster). *Serratia marcescens* strain MSU97 isolated from a plant source, *Erwinia oleae* strain DAPP-PG531 isolated from olive tree knot, *Klebsiella michiganensis* strain NCTC10261 of an unknown source, and the *Escherichia coli* phylogroup E strain 14696-7 isolated from the pericardial

sac of a white-tailed deer (*Odocoileus virginianus*) harbour the most genetically distant variants of the colibactin determinant (Fig. 2a). Within the *Enterobacteriaceae*, a large group of *clb* gene clusters can be defined, which is dominated by two highly conserved clades present in *Escherichia coli* phylogenetic lineage B2 and different *Klebsiella* species isolates, respectively. The colibactin determinants detected in *Erwinia cloacae* and *Erwinia hormaechei* belong to the *Klebsiella* clades of *clb* loci, whereas the *clb* gene clusters found in *C. koseri* and in an unspecified *Salmonella* isolate represent an independent clade, i.e. *clb*6 (Fig. 3). In a few other *Escherichia coli* and *Klebsiella* species genomes, the *clb* determinant can be distinguished from the two major conserved clades of colibactin determinants observed in *Klebsiella* or *Escherichia coli*. An even more divergent group comprises the *clb* gene clusters of mainly *Escherichia coli* phylogroup A, B1 and a few B2 isolates, but also of some *K. pneumoniae* strains (Fig. 3, belonging to *clb* clades *clb*1 and *clb*2).

Although the *clb* gene clusters found in the large clade of *Escherichia coli* B2 strains are highly conserved, individual ST-specific lineages, such as *clb*10 (from *Escherichia coli* ST141 and ST2015 strains) and *clb*12 (from *Escherichia coli* ST121 strains) can still be described within this clade. Beyond that, we also observed multiple lineages per sequence type, such as *clb*2, *clb*11, *clb*15 and *clb*16 found in *Escherichia coli* strains of ST73 (Fig. 3). Three lineages of the *clb* locus were predominantly detectable in *Klebsiella pneumoniae* strains. They belong to the most distant *Klebsiella* ST3/ST380 clade (*clb*1), the remaining large and diverse ST258/ST11 clade (*clb*8), and finally the hypervirulent ST23 clade (*clb*9) (Fig. 3). A phylogeny of the colibactin gene cluster inferred from concatenated amino acid sequences

<footnote>*Unverified source organism (excluded from Refseq).</footnote>
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Maximum-likelihood-based phylogenetic analysis of the colibactin and yersiniabactin determinants. (a) Phylogenetic tree of the colibactin gene cluster (collapsed), and (b) phylogenetic tree of the corresponding *ybt* determinants (collapsed) using the genetically distant *K. michiganensis* strains as an outgroup [38]. Additionally, the yersiniabactin sequence type (YbST) as defined by Lam and colleagues [38] associated with individual bacterial clades is indicated. The branch colours in both trees depict the prominent bacterial sequence type of the clade.</figcaption></figure>

of the 17 *clb* genes (Fig. S10) was very similar to the aforementioned recombination-free nucleotide-based phylogeny (Fig. 3).

**Diversity of the yersiniabactin determinant in colibactin-positive bacteria**

The majority (>98%) of the *clb*-positive enterobacterial strains also harboured the yersiniabactin genes (*ybt*) (Fig. 3, third circle). The *Escherichia coli* strains of phylogroup A, B1 and E as well as the tested *K. michiganensis* and *Erwinia oleae* strains carrying the most genetically distant lineages of the colibactin gene cluster, together with the *F. perrara* strains used as an outgroup, are *ybt*-negative (Fig. 2b). There were no strains that carried multiple copies of the colibactin gene cluster; however, two well-separated copies of the *ybt* determinant were found in *C. koseri* strains ATCC BAA-895 and 0123A_53_520.

It should be noted that the latter strain is derived from a metagenome. The phylogenetic analysis indicated that all *ybt* operons from *Escherichia coli* clustered together (Fig. 2b). Alike the *clb* gene cluster, also the *ybt* locus of the *Escherichia coli* phylogroup B2 strains was highly conserved. In contrast, the sequence comparison of the *ybt* determinants of *Klebsiella* species resulted in different lineages, which correlate with lineages *ybt1*, 12 and 17 (ICEKp10), *ybt9* (ICEKp3), and *ybt4* (originating from a plasmid) previously described by Lam and colleagues [38].

**Congruent phylogeny of the colibactin and yersiniabactin determinants**

The strong coexistence of the colibactin and yersiniabactin determinants on the one hand and the description of different evolutionary lines of *clb* and *ybt* determinants in different
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Maximum-likelihood-based phylogeny of the colibactin gene cluster detected in 2169 enterobacterial genomes. Every leaf represents a single sequence variant of the *clb* gene cluster, which can be allocated to different lineages and clades. From innermost to outermost, the first circle indicates the species harbouring the *clb* determinant; the second circle shows the *Escherichia coli* phylogroup, the third circle shows the presence/absence of the *ybt* operon; the fourth circle shows the yersiniabactin sequence types (YbST) of the *ybt* determinant (from Fig. 1b) that correspond to the *pks* island lineage present in the individual genome. The fifth circle shows the different colibactin sequence types (CbST) of the *clb* gene cluster. The branch colours in the centre of the tree depict the prominent bacterial sequence types (Fig. 1). The large conserved *Escherichia coli* phylogroup B2 clade is separated from the large *Klebsiella* clade with a faint broken line.</figcaption></figure>

enterobacterial species on the other hand led us to analyse whether both gene clusters can predominantly be transferred individually or together. Our results indicate that the clades of the evolutionary lineages of the *clb* and *ybt* loci are chiefly species-/genus-specific. The phylogeny of *clb* and *ybt* determinants is largely congruent, with the *ybt* gene cluster being, even more, species-/genus-specific than that of the *clb* gene cluster (Fig. S5). However, in some strains we observed evidence of interspecies transfer of these genes: the *clb* and corresponding *ybt* determinants of the *C. koseri* isolates NCTC10769, ATCC BAA-895 and *Escherichia coli* strains 239A, 926, GN02370 and MOD1-EC5674/5 were allocated to the large *Klebsiella*-dominated lineage *clb8* (Fig. 3). Additionally, the *clb* gene cluster of *K. pneumoniae* strain k2265 was found within lineage *clb6*, which predominantly represents *C. koseri* isolates. However, the *ybt* determinant of *K. pneumoniae* strain k2265 belonged to the *ybt12* lineage represented by *K. oxytoca* isolates. Similarly, the *ybt* determinant of the aforementioned strains *E. coli* GN02370 and *C. koseri* ATCC BAA-895 belonged to lineage *ybt4* (plasmid originating *ybt*

loci) instead of *ybt17*. Regardless of the aforementioned exceptions, clades of the *clb* gene cluster usually correlated with the corresponding clade of *ybt* genes (Fig. 3, third and fourth circle).

## Genetic structure of the MGEs harbouring the colibactin determinant

To further investigate whether the colibactin and yersinia-bactin determinants are jointly distributed by horizontal gene transfer and to obtain clues to the underlying mechanism, we compared the chromosomal context of the two polyketide biosynthesis gene clusters and the genetic structure of associated MGEs. We observed species-specific structural differences of the chromosomal regions harbouring the *clb* and *ybt* gene clusters (Figs 4 and S6). In *Escherichia coli* phylogroup B2 strains, the *clb* and *ybt* determinants are present as part of two individual pathogenicity islands (PAIs) with their cognate integrase and different tRNA genes (class I of *clb*-harbouring MGE). Both PAIs are located neighbouring each other in the
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Structural variation of the colibactin and yersiniabactin-encoding chromosomal region in *Escherichia coli*, *Enterobacter hormaechei*, *K. pneumoniae*, *K. aerogenes* and *C. koseri*. The different genetic structures and chromosomal insertion sites of the colibactin and/or yersiniabactin determinants found within the three main structural classes are shown. The *clb* gene cluster (teal green), T4SS module (purple), *ybt* gene cluster (pink), integrase genes (green), the conserved sets of genes (Table S5) that are present up/downstream of the two polyketide determinants, classed into sets (blue boxes), and the Fe/Mn/Zn module (yellow) are shown. The number of genomes included in the tested set of genomes that harbour the different structural variants is indicated in parentheses. The colibactin-yersiniabactin chromosomal regions that do not conform to these major structures are as shown in Fig. S7.</figcaption></figure>

chromosome. Within class Ia, the two PAIs are separated by a type 4 secretion system (T4SS)-encoding operon (*virB*) and a region that includes two conserved gene sets (Set 1 and Set 2), different tRNA(*Asn*) loci and an integrase gene (see Table S3 for genes present in the different conserved gene sets). This region is shown to have been diminished in class Ib structural variants, where only gene *virB1* of the T4SS determinant is left alongside the conserved region. In the predominant *Escherichia coli* structural variant, class Ic, the complete T4SS operon (including *virB1*) has been lost together with gene set 1, the integrase gene was exchanged and a DNA stretch comprising the genes *yeeO*, tRNA(*Asn*), *clb* and *gltC* was inverted (shown in red, Fig. 4). The region separating the *pks* island and the HPI was reduced from a 40 kb (in class Ia) to a 15 kb stretch in class Ic. In contrast, within all *Klebsiella* strains, the *clb* and *ybt* gene clusters are part of an ICE, and are separated by a T4SS-encoding operon (*virB*) and the *hha* gene coding for the haemolysin expression-modulating protein. Downstream of the *ybt* gene cluster an integrase gene is located followed by a set of genes involved in Fe/Mn/Zn metabolism (structural class II of *clb*- and *ybt*-harbouring chromosomal regions) (Fig. 4). Interestingly, one type of such ICEs is located next to genes necessary for microcin E492 biosynthesis (class IIc, Fig. 4). *Enterobacter hormaechei* strains harbour a structurally

similar ICE to that of *Klebsiella* strains. In most *C. koseri* strains, however, the two polyketide determinants are separated by a large 250 kb chromosomal region. The T4SS-related genes are closely positioned to the *clb* genes while the gene set involved in Fe/Mn/Zn metabolism is located downstream of the *ybt* determinant. Only a minor fraction of enterobacterial isolates analysed displayed some variation regarding gene content and synteny of these three main classes of colibactin and yersiniabactin-encoding chromosomal regions. The structure of *clb* and *ybt* regions that do not conform to these major classes are as shown in Fig. S7. Instead of class I, several *Escherichia coli* strains carried class II-like chromosomal regions where the T4SS and the Fe/Mn/Zn metabolism genes were present. In *Escherichia coli* strain HVH128 none of the three main classes of colibactin- and yersiniabactin-encoding regions could be identified. Although both polyketide determinants are co-localized with one integrase gene each, they are widely separated on this strain's chromosome. *K. pneumoniae* strains TUM14001, TUM14002, TUM14009, TUM14126 and WCHKP13F2 harboured two T4SS-encoding gene clusters in close proximity of the *clb* and *ybt* gene clusters and lacked the Fe/Mn/Zn genes, whereas *K. pneumoniae* strain UCI110 was also missing the Fe/Mn/Zn metabolism-related genes. In contrast to the other *C. koseri* isolates, we detected a class
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** The structural organization and GC profiles of the *clb* determinants in the five most genetically distant bacterial strains (according to Fig. 2a). The genes that make up the homologous *pks* gene cluster found in *F. perrara* and the most distant *clb* determinants present in *Serratia marcescens*, *Erwinia oleae*, *K. michiganensis* and *Escherichia coli* (phylogroup E) strain 14696-7 are depicted. The GC profile of the gene cluster in the different strains is shown alongside with the colours underlining the different species. SAM genes and *clbA* homologues (*) are shown downstream of the *pks* gene cluster in *F. perrara* and upstream of the *clb* determinant in *K. michiganensis* and *Erwinia oleae*. The gaps in assembly are shown with white spaces.</figcaption></figure>

II- instead of a class III-type *clb-ybt* region in *C. koseri* isolate BAA-895.

## Organization of the colibactin gene cluster

The *clb* gene cluster is composed of 19 genes, which are required for the regulation, biosynthesis and transport of colibactin. The origin of this gene cluster is unclear. We therefore compared the structure of the gene clusters representing the homologous *clb* locus found in *F. perrara* and the phylogenetically most distant and potentially older *clb* determinants relative to the *Echerichia coli* B2 type of the *clb* determinant (Fig. 2a), which are present in *Serratia marcescens*, *Erwinia oleae*, *K. michiganensis* and *Escherichia coli* phylogroup E strain 14696-7 (Fig. 5). The various *clb* determinants correspond in terms of the structure of the genes coding for the biosynthesis machinery, transport and resistance to colibactin (*clbB-clbS*) and resemble the structure of the well-described *clb* locus in *K. pneumoniae*/*K. aerogenes*/*Escherichia coli* B2 strains. The individual *clb* determinants differ more clearly in the presence and localization of the genes involved in the regulation and activation of colibactin biosynthesis (*clbR* and *clbA*). These two genes are absent in the homologous gene cluster found in *F. perrara* and in the *clb* determinant in *Serratia marcescens*. However, in *F. perrara*, a phosphopantetheinyl transferase coding for a homologue of ClbA (43% amino acid similarity) and a radical S'-adenosylmethionine (SAM) enzyme-encoding gene are found directly downstream of *clbS*. In *Serratia marcescens*, a helix-turn-helix (HTH)-type regulatory protein homologous to *clbR* is encoded by a gene located upstream of *clbB* (78% amino acid similarity). In *Erwinia oleae* and *K. michiganensis*, a SAM enzyme-encoding gene is present directly downstream of *clbA*. Although the colibactin gene clusters in *Erwinia oleae* and *K. michiganensis* have a high nucleotide sequence similarity of ca. 99.77%, the predicted coding regions

of *clbC/D* and *clbH/I/J/K/L/M/N* are noticeably different due to multiple frameshift deletions in *K. michiganensis*. The structure of the *clb* locus found in phylogroup E *Escherichia coli* strain 14696-7 already corresponds to the structure of *Escherichia coli* strains of phylogroup B2 (Figs 1 and 5), yet this gene cluster shows the least sequence similarity of all tested *clb* gene clusters in non-B2 *Escherichia coli* isolates (Fig. 2a) to the determinant occurring in *Escherichia coli* strains of phylogroup B2.

Looking at the G+C plot of the colibactin gene clusters, it is clear that all investigated enterobacterial *clb* gene clusters show a very similar G+C plot, which has a significantly higher average G+C content and differs significantly from that of the *clb* homologous gene cluster in *F. perrara* (Fig. 5). The G+C content profile of these gene clusters indicates that there are two regions of low G+C content in the enterobacterial *clb* determinants: the region including *clbA* and *clbR* (at position ca. 1500–3000 bp of the colibactin gene cluster) and the region spanning *clbD* and *clbE* (at position ca. 15000–16500 bp of the *clb* gene cluster). The G+C content drop in the region including *clbA* and *clbR* (at position ca. 1500–3000 bp of the colibactin gene cluster) is associated with a predicted recombination site, which is located upstream of or interrupting *clbB* (Fig. S4).

The comparison of structural features of the *clb* gene cluster also included the VNTR region located upstream of *clbR* in the *clbR-clbB* intergenic region. The size of the VNTR region has been described to range from 2 to 20 copy numbers (Putze *et al.*, 2009 [2]). The VNTR copy number distribution in ca. 1300 *clb*-positive genomes demonstrated that there is a preference for VNTR regions ranging from 7 to 10 copy numbers. Copy numbers from 18 to 34 were present in only a few strains (Fig. S8). Species and/or ST-specific copy number variation was not observed.
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Comparison of colibactin production of different strains accessed by quantification of the precolibactin cleavage product *N*-Asn-D-myristol. This assay enabled us to compare the ability of different strains and different species to produce colibactin under controlled conditions *in vitro*. Measurements were conducted based on three biological replicates, and means with standard deviations are as shown.</figcaption></figure>

Comparative genomic analysis of multiple colibactin-
encoding determinants based on (draft) genome sequences
led to the observation that the homologous genes *clbJ* and
*clbK* are prone to fusion/deletion (Lam *et al.*, 2018 [38]).
We also observed that in several assemblies of the *clb* gene
cluster 625 bp from the 3' end of *clbJ* and 3540 bp from the
5' end of *clbK* including the 11 bp intergenic region are
missing, for a total of 4174 bp (Fig. S9). The assembly of our
internally generated genome sequences produced by short
read (Illumina) sequencing showed this *clbJ/K* fusion/dele-
tion. However, because assemblies of sequence data of the
same strains generated by a long-read sequencing technology
(PacBio), where the long reads covered both genes, had both
*clbJ* and *clbK* completely present, we assume that the *clbJ/K*
fusions described are artificial and result from erroneous
assemblies of short-read sequencing data.

Quantification of colibactin synthesis in selected
strains

To investigate a possible correlation between the genetic
structure of the *clb* determinant or the genetic background
of the corresponding host strain with colibactin expression,

we quantified *N*-Myr-D-asparagine levels produced *in vitro*
by selected *clb*-positive *Escherichia coli*, *Klebsiella* species and
*C. koseri* strains covering the diversity of *clb* determinants in
these species (Fig. 6). Based on the detected relative amount of
*N*-Myr-D-asparagine produced, the investigated isolates can
be roughly divided into two groups: one group included most
of the measured *Escherichia coli* strains that produced only
very low relative amounts of *N*-Myr-D-asparagine. In contrast,
the tested *C. koseri*, *K. aerogenes* and *K. pneumoniae* isolates
and the *Escherichia coli* isolates CFT073 and N1 showed a 3- to
70-fold higher *N*-Myr-D-asparagine production. Also within
the *C. koseri*, *K. aerogenes* and *K. pneumoniae* isolates, we
found differences in the relative *N*-Myr-D-asparagine levels.
However, these differences were not as strong as among the
eight *Escherichia coli* isolates studied. The observed relative
*N*-Myr-D-asparagine levels do not indicate phylogroup, ST
or species-specific differences in colibactin production. For
example, the *Escherichia coli* strain 1873, although the *clb*
gene cluster present in this strain is phylogenetically more
closely related to that of *Escherichia coli* strain N1, shows a
significantly weaker *N*-Myr-D-asparagine production than
*Escherichia coli* N1. Similarly, it should be noted that *C. koseri*
MFP3 produces less N-Myr-D-asparagine than other closely related *C. koseri*.

# DISCUSSION

## Prevalence and mobility of the colibactin gene cluster in *Enterobacterales*

The ability of MGEs to be exchanged between and within species plays a major role in the extent and speed of microbial evolution. Because MGEs are known to emerge and evolve separately from the host, it is important to explain the development and diversity of MGEs independently. In our study, we investigated on the one hand the nucleotide sequence variability of the colibactin gene cluster with the associated yersiniabactin determinants, and on the other hand the structural diversity of the MGEs hosting the colibactin and yersiniabactin determinants responsible for their horizontal distribution. Both polyketide determinants together could be detected in certain clades of the *Enterobacterales*, mainly in members of the *Enterobacter–Escherichia clade*, namely in *Escherichia coli*, *K. pneumoniae*, *K. aerogenes*, and *C. koseri*, but also in *Enterobacter cloacae*, *Enterobacter hormachei*, and possibly in uncharacterized *Salmonella* species isolates. The colibactin but not the yersiniabactin determinants could also be detected in *K. michiganensis*, members of other *Enterobacterales* clades (*Erwinia oleae*, and *Serratia marcescens*), and a few phylogroup A and B1 *Escherichia coli* isolates (Fig. 2a). Interestingly, the colibactin genes have not yet been described in not only archaeal genomes but also *Salmonella enterica* and *Salmonella bongori* genomes, although we have screened more than 112000 *Salmonella* species genomes. Overall, it is remarkable that the *clb* gene cluster was only found in extraintestinal clinical or in faecal isolates of healthy hosts, but not in enterobacterial diarrhoeal pathogens such as *Yersinia* species, *Salmonella* species, *Shigella* species as well as the various intestinal pathogenic *Escherichia coli* pathotypes. This observation is consistent with previously published data [2, 51]. In this context, it would be interesting to study whether and in which way colibactin expression supports extraintestinal pathogenicity or intestinal persistence and colonization, but is detrimental to the pathogenesis of diarrhoeal pathogens. Possibly, the genetic background also plays an important role in the horizontal distribution and establishment of MGEs carrying both polyketide determinants. The fact that the colibactin determinant has so far been preferentially distributed in only some, often highly virulent STs in *Escherichia coli* and *K. pneumoniae* or *K. aerogenes* and not more broadly within the respective species (Fig. 2) [38, 39], could also indicate that the transmission, uptake or chromosomal integration of these MGEs is restricted. It is interesting to note that the *clb* gene cluster as a whole is highly conserved and usually characteristic of the respective species or genus (Figs 3 and S5). Nevertheless, ST-specific variants have been found within a species, e.g. in *K. pneumoniae* ST3 and ST23. Several groups of sequence variants of the *clb* gene cluster can also be found within one ST, such as in the *Escherichia coli* ST73 and ST95 (Figs 3 and S5). These results show that, on the

one hand, intraspecies transfer of the colibactin determinant can happen, but on the other hand, certain adaptations of the *clb* genes to a specific genetic background can also occur at the nucleotide level. In addition, examples of interspecies transfer of the *clb* and *ybt* genes can be seen between *Klebsiella* species and *Enterobacter hormachei* strains. Similarly and in contrast to the majority of *Escherichia coli* isolates, we found *clb* and *ybt* gene clusters in some *Escherichia coli* isolates, which are assigned to the large clade of *Klebsiella/Enterobacter/Citrobacter*-specific *clb* and *ybt* variants (Figs 2a and 3). Apart from the interspecies transfer of the entire *clb*- and *ybt*-containing MGE, we also identified an example that shows that the two polyketide gene clusters can also be exchanged independently, as in the case of the *clb* gene cluster of *K. pneumoniae* strain k2265, which belongs to colibactin clade *clb6* (predominantly *C. koseri* lineage of *clb* loci), whereas the *ybt* determinant in this strain is assigned to the yersiniabactin clade *ybt12* instead of *ybt3*, which is usually associated with *C. koseri* strains carrying clade *clb6* (Fig. 3).

## Structural diversity of the colibactin-yersiniabactin region

The structural analysis of the genomic region comprising the *clb* and *ybt* determinants in their chromosomal sequence context is an important aspect to understand the evolution of these polyketide determinants and their origin. In principle, three structural constellations (classes I to III) can be described, in which the *clb* and *ybt* gene clusters are present (Fig. 4). Class I depicts the *clb* and *ybt* gene clusters found in the majority of *Escherichia coli* isolates, each associated with a tRNA(*Asn*) and an integrase gene. In class Ia, the *clb* genes are chromosomally inserted at the tRNA locus *asnV*. Our analyses suggest that in class I, the T4SS gene cluster ('mobilization module') and conserved neighbouring genes (set 1) have been lost in a stepwise process, from class Ia to Ic. A further structural modification in this region is represented by the inversion of the *yeeO*-tRNA(*Asn*)-*cbl-gltC* gene set (Fig. 4, red arrow), as a result of which in class Ic, the tRNA gene *asnW* is located closest to the *clb* genes. Taking into account that the *Escherichia coli* strains with class Ia and Ib structures are found in the potentially earlier phylogenetic clades, we hypothesize that the MGE harbouring the *clb* genes was introduced into the *Escherichia coli* chromosome separately from that carrying the *ybt* determinant. Both MGEs were then progressively modified as described above. We do not yet have an explanation for why the resulting class Ic colibactin-yersiniabactin region, which has been described as two PAIs comprising the colibactin and yersiniabactin determinants, respectively [1, 2], is only found in phylogroup B2 strains and only there has it become so successful.

Class II includes different variants of an ICE, in which the two polyketide determinants are present in association with a T4SS-encoding 'mobilization module' and a 'module' consisting of genes that contribute to Fe/Mn/Zn metabolism (Fig. 4, Table S5). This type of ICE was found in *Klebsiella* species and *Enterobacter hormachei* and in a few cases in *Escherichia coli* and *C. koseri* (Fig. S6). Unlike in class I and
III, the ICE in class II does not only have different tRNA*(Asn)* loci serving as chromosomal insertion sites, but also lacks tRNA*(Asn)* and integrase genes in between the *clb* and *ybt* genes. In a population-wide analysis of *Klebsiella* species strains, this ICE was designated ICE*Kp10* and described as being associated with different combinations of *ybt* and *clb* gene lineages [38]. In contrast to class I and II colibactin-yersiniabactin regions, the *clb* and *ybt* gene clusters are located far apart on the chromosome in most of the *C. koseri* genomes studied (class III) (Fig. 4).

The existence of an ICE that unites the *clb* and *ybt* gene clusters is the easiest way to explain the co-localization and the joint transfer of the two determinants and thus the high correlation of *clb* and *ybt* phylogenetic clades (Fig. 3, fourth circle) in *Klebsiella* species strains. Despite slight differences in the sequence context and different chromosomal insertion sites (Fig. 4), the ICEs of the four class II variants have an overall identical genetic structure (Fig. 4). The uptake of this ICE thus leads to the acquisition of both the *clb* and the *ybt* gene clusters. The presence of the Fe/Mn/Zn metabolic genes neighbouring these ICE variants with an additional integrase gene indicates recombination processes that can alter the genetic structure of the ICE. The clear separation between the *clb/T4SS* module and the *ybt-Fe/Mn/Zn* metabolism module in the *C. koseri* genomes points towards rearrangement/relocation of the *ybt* region, after ICE integration into the chromosome. The fact that *C. koseri* strain ATCC BAA-895 possesses in addition to the complete ICE (class II) a second *ybt* gene cluster (99.98% nucleotide similarity to the *ybt* genes present in the ICE) that is located far from the ICE (Fig. S7), supports the hypothesis that the individual polyketide gene clusters can also be integrated into the genome independently of each other. This state could result, for example, from the initial chromosomal integration of different ICE variants, as described by Lam and colleagues in *Klebsiella* species [38]. As a result of deletion events, through which individual modules are subsequently deleted from one of the two ICEs, the second copy of the *ybt* gene cluster remains in the genome as a fragment of the degenerated ICE. The presence of two non-identical T4SS modules in the *K. pneumoniae* strains TUM14001, TUM14002, TUM14009, TUM14126 and WCHKP13F2 associated with the *clb* or *ybt* module could be the result of such degeneration of different ICEs (Fig. 3, *clb4* and Fig. S7). In this way, our observations on the phylogeny (Fig. 2) and structure (Fig. 4) of the two polyketide determinants and their sequence context can be reconciled, which show that despite the predominant species/genus specificity, there are also ST-specific lineages of the *clb* genes, which do not necessarily have to match that of the associated *ybt* genes.

In this context, one could imagine that the arrangement of *clb* and *ybt* determinants in *Escherichia coli* strains (class I) also results from independent integration events of different MGEs, which subsequently degenerated as a result of a stabilization process of these MGEs [26, 28]. This premise is supported by the absence of *ybt* genes in the *clb*-positive *Escherichia coli* strains, which carry the phylogenetically most distant *clb* determinants compared to the *clb* genes of

phylogroup B2 isolates (Fig. 2a), along with the presence of integrase and tRNA*(Asn)* genes in close proximity to both polyketide determinants (Fig. 4, class I).

## Evolution of the colibactin determinant

Homologues of the *clb* gene cluster were detected in marine alphaproteobacteria such as various *Pseudovibrio* species (isolates AD26, FO-BEG1, POLY-S9) and *Pseudovibrio denitrificans* (isolates DSM 17465 and JCM12308) [52]. Despite the general conformity of the genetic structure of these gene clusters, their nucleotide sequence identity to the colibactin gene cluster is quite low (<26%). Therefore, it was hypothesized that these *Pseudovibrio* isolates have the potential to produce molecules related to colibactin [52]. Another homologue of the colibactin determinant with a higher (62%) amino acid sequence identity is found in *F. perrara* [24]. While the genes required for biosynthesis and transport of the polyketide are present, the genes corresponding to *clbA* and *clbR* are missing in this gene cluster (Fig. 5). The case is similar with the colibactin gene cluster in *S. marcescens*. While in *F. perrara*, a gene coding for a *clbA* homologue and a gene coding for a SAM enzyme are located immediately downstream of the *clbS* homologue, a *clbR* homologue is located upstream of *clbB* in *Serratia marcescens*. It is therefore conceivable that the *clbA* homologue in *F. perrara* and the *clbR* homologue in *Serratia marcescens* are involved in the activation or regulation of colibactin biosynthesis in these bacteria (Fig. 5). It has been described that SAM is used in NRPS modules for colibactin biosynthesis [53]. Looking at the genetic structure of the *clbB-S* homologous genes cluster in *F. perrara* and the *clb* gene cluster in *S. marcescens*, one can assume that genes involved in the regulation and activation of the biosynthetic pathway including the SAM enzyme gene as well as *clbA* and *clbR* homologues have been fused upstream to the already existing part of the island (*clbB-clbS*) to improve regulation of polyketide biosynthesis. Without having further knowledge about the origin of the colibactin biosynthetic genes themselves, the acquisition of the regulatory/activating genes is obviously among the last evolutionary steps that led to the structural organization of what we currently describe as the colibactin gene cluster. This hypothesis is supported by the abrupt decrease in G+C content and the presence of the predicted recombination site (Fig. S4) directly upstream of *clbB* in many *clb* determinants. Furthermore, a module consisting of a gene for a SAM enzyme and a *clbA* homologue is not only located directly downstream from the *clbS* homologue in *F. perrara*, but also upstream from *clbR* in *K. michiganensis* and *Erwinia oleae*, which represent evolutionarily older variants of colibactin-positive *Enterobacterales* (Fig. 5).

## Expression of colibactin in different hosts

Furthermore, we investigated the question of how differently colibactin is expressed within different genera of the *Enterobacter–Escherichia* clade or even within different lineages of the same species. In an initial approach, we compared colibactin expression in some selected model isolates. Interestingly, we observed an often lower production level
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Schematic representation of the predicted evolution of the colibactin-yersiniabactin genomic region in *Enterobacteriaceae*. The different elements of this region, i.e. the *clb* determinant (teal green), T4SS module (purple), *ybt* gene cluster (pink), integrase genes (green) and an invertible subset of genes (red arrow) are shown. Based on available genome sequence data, we suggest a development from single MGEs containing the *clb* determinant and the *ybt* gene cluster, respectively, towards the structural arrangement of both polyketide determinants, which is mainly found in enterobacterial populations. Black arrows (solid or dashed) indicate possible directions of development and DNA rearrangements. After the merge of the *clb* and *ybt* gene clusters into one MGE, represented by ICEKp10, there is evidence that three different structural variants have evolved from it: in *Klebsiella* species strains, the ICEKp10 has remained intact, whereas in *C. koseri* strains, a DNA rearrangement and re-localization of the *ybt* determinant to a different chromosomal position has taken place. In *Escherichia coli*, a gradual loss of the T4SS module and the inversion of a gene set between the two polyketide determinants led to immobilization or stabilization of the ICE, thus resulting the two PAIs known as *pks* island and HPI, respectively.</figcaption></figure>

of *N*-Myr-D-asparagine in *Escherichia coli* isolates compared to *K. aerogenes*, *K. pneumoniae* and *C. koseri* (Fig. 6), which may be expected because *Escherichia coli* is described as a non-optimal producer of complex secondary metabolites [54]. However, it is of interest that the amount of *N*-Myr-D-asparagine produced in *Escherichia coli* strains CFT073 and N1 is comparable to that of the tested *Klebsiella* species and *C. koseri* strains (Fig. 6). A species- or lineage-specific ability to produce *N*-Myr-D-asparagine could not be determined so far. We are fully aware that the data provided in Fig. 6 allow only very limited conclusions and that the comparison of larger sets of isolates will be required to generate more robust results. Future studies will have to investigate which bacterial factors are important for colibactin production and how the strain-specific differences in the expression of this polyketide come about. The systematic comparison of phenotypic colibactin production with information on the genomic context, regulatory and metabolic properties of host strains, and their classification in a phylogenetic context should help us to identify bacterial factors that affect colibactin synthesis.

## CONCLUSION

The colibactin and yersiniabactin gene clusters are highly conserved polyketide determinants present in some clades of *Enterobacteriales*. They usually coexist together in the genome and are also linked to each other at the biosynthetic level. With the exception of *Escherichia coli*, the two gene clusters are part of an ICE, which allows the horizontal transfer of

both secondary metabolite determinants usually within one species/genus. Bacteria of the genus *Klebsiella* played an important role in the evolution and distribution of both gene clusters. A large number of different ICEs has been described in *Klebsiella* species, which besides several other groups of genes include the yersiniabactin determinant [38]. Recombination and rearrangements events between different ICE types may have contributed to the evolution of the ICE variants so far identified in *Klebsiella* species and other enterobacteria as well as to the further degeneration of such MGEs leading to the colibactin and yersiniabactin-encoding PAIs present in phylogroup B2 *Escherichia coli* strains (Fig. 7).

The phylogeny of the *clb* determinants does not determine the level of phenotypic colibactin production. The underlying bacterial factors responsible for the colibactin production efficiency of individual strains need to be identified in future work.

Our investigations provide deeper insights into the evolution of the colibactin gene cluster in *Enterobacterales*. Based on our findings, we can extend the current explanation for the coexistence and genetic co-localization of both gene clusters. The combination of a PPTase-encoding gene (*clbA*) with the *clbB-S* biosynthetic gene cluster during the evolution of the *clb* determinant not only enabled the efficient activation of the colibactin biosynthesis machinery, but also linked the colibactin and yersiniabactin determinants, which are functionally connected by the activity of
PPTase ClbA. This enables the bacteria to synthesize both functionally different secondary metabolites, which leads to a stabilization of the coexistence and co-localization of the two gene clusters in the genome. Our data underpin the importance of MGEs, especially of ICEs, for genomic diversity and variability in enterobacteria as well as for the evolution of more complex bacterial phenotypes, such as the combined expression of the secondary metabolites colibactin and yersiniabactin.

## Funding information

The work of the Münster team was supported by the German Research Foundation [grant DO789/11-1 and grant 281125614/GRK2220 (EvoPAD project A3)].

## Acknowledgements

The data reported in this study appear in part in the PhD theses of A. Wallenstein and H. Wami. We thank M. K. Mammel (United States Food and Drug Administration) for providing *Escherichia coli* strain N1. We thank K. Tegelkamp and O. Mantel (Münster) for excellent technical support. We gratefully acknowledge CPU time on the high performance cluster PALMA@WWU Münster.

## Author contributions

H.W., A.W. and U.D. conceptualized the project. H.W., A.W. and D.S. ran the analyses. R.M., M.S. and E.O. contributed reagents and new tools. H.W., A.W. and U.D. analysed the data. H.W., A.W. and U.D. wrote the manuscript. H.W., A.W., M.S., R.B., E.O., R.M. and U.D. edited and revised the manuscript. All authors read, commented on and approved the final manuscript.

## Conflicts of interest

The authors declare that there are no conflicts of interest.

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