Transient infection of eukaryotic cells with commensal and extraintestinal pathogenic Escherichia coli of phylogenetic group B2 blocks mitosis and induces megalocytosis. This trait is linked to a widely spread genomic island that encodes giant modular nonribosomal peptide and polyketide synthases. Contact with E. coli expressing this gene cluster causes DNA double-strand breaks and activation of the DNA damage checkpoint pathway, leading to cell cycle arrest and eventually to cell death. Discovery of hybrid peptide-polyketide genotoxins in E. coli will change our view on pathogenesis and commensalism and open new biotechnological applications.

A genomic island encoding the biosynthesis and secretion pathway of putative hybrid nonribosomal peptidepolyketide colibactin has been recently described in Escherichia coli. Colibactin acts as a cyclomodulin and blocks the eukaryotic cell cycle. The origin and prevalence of the colibactin island among enterobacteria are unknown. We therefore screened 1,565 isolates of different genera and species related to the Enterobacteriaceae by PCR for the presence of this DNA element. The island was detected not only in E. coli but also in Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter koseri isolates. It was highly conserved among these species and was always associated with the yersiniabactin determinant. Structural variations between individual strains were only observed in an intergenic region containing variable numbers of tandem repeats. In E. coli, the colibactin island was usually restricted to isolates of phylogenetic group B2 and inserted at the asnW tRNA locus. Interestingly, in K. pneumoniae, E. aerogenes, C. koseri, and three E. coli strains of phylogenetic group B1, the functional colibactin determinant was associated with a genetic element similar to the integrative and conjugative elements ICEEc1 and ICEKp1 and to several enterobacterial plasmids. Different asn tRNA genes served as chromosomal insertion sites of the ICE-associated colibactin determinant: asnU in the three E. coli strains of ECOR group B1, and different asn tRNA loci in K. pneumoniae. The detection of the colibactin genes associated with an ICE-like element in several enterobacteria provides new insights into the spread of this gene cluster and its putative mode of transfer. Our results shed light on the mechanisms of genetic exchange between members of the family Enterobacteriaceae.

The recently described hybrid nonribosomal peptide-polyketide colibactin, found in various Escherichia coli strains, invokes a cytopathic effect in HeLa cells upon cocultivation with these bacteria. However, not much is known so far about the transcriptional organization of the colibactin genes (clb) or the regulation of their transcription. Here, the operon structure of the colibactin gene cluster of E. coli strain Nissle 1917 was investigated by means of reverse transcriptase (RT)-PCR and seven transcripts were found of which four are transcribed polycistronically. The polycistrons comprise the genes clbC to clbG, clbI to clbN, clbO to clbP, and clbR to clbA and span 6.3, 23.3, 3.9, and 0.9 kb, respectively. Furthermore, transcript levels for different cultivation conditions were determined by RT-PCR of the whole cluster as well as by luciferase reporter gene assays of the genes clbA, clbB, clbQ, and clbR. RT-PCR revealed an overall increased transcription in shaking cultures as well as of the genes clbA to clbH in general. Luciferase reporter gene fusions indicated an influence of the carbon source on clb gene expression.

The nonribosomal peptide/polyketide hybrid colibactin can be considered a bacterial virulence factor involved in extraintestinal infection and also a procarcinogen. Nevertheless, and despite its genotoxic effect, colibactin expression can also inhibit bacterial or tumor growth and correlates with probiotic anti-inflammatory and analgesic properties. Although the biological function of this natural compound has been studied extensively, our understanding of the regulation of colibactin expression is still far from complete. We investigated in detail the role of regulatory elements involved in colibactin expression and in the growth conditions that promote colibactin expression. In this way, our data shed light on the regulatory mechanisms involved in colibactin expression and may support the expression and purification of this interesting nonribosomal peptide/polyketide hybrid for further molecular characterization.

Arthrobacter nitroguajacolicus Rü 61a, which utilizes quinaldine as sole source of carbon and energy, was shown to contain a conjugative linear plasmid of approximately 110 kb, named pAL1. It exhibits similarities with other linear plasmids from Actinomycetales in that it has proteins covalently attached to its 59 ends. Southern hybridization with probes for the genes encoding quinaldine 4-oxidase and N-acetylanthranilate amidase indicated that pAL1 contains the gene cluster encoding the degradation of quinaldine to anthranilate. A mutant of strain Rü 61a that had lost pAL1 indeed could not convert quinaldine, but was still able to grow on anthranilate. Conjugative transfer of pAL1 to the plasmid-less mutant of strain Rü 61a and to Arthrobacter nicotinovorans DSM 420 (pAO1) occurred at frequencies of 5?4610 "4 and 2?0610per recipient, respectively, and conferred the ability to utilize quinaldine. Five other quinaldine-degrading Gram-positive strains were isolated from soil samples; 16S rDNA sequence analysis suggested the closest relationship to different Arthrobacter species. Except for strain K2-29, all isolates contained a pAL1-like linear plasmid carrying genes encoding quinaldine conversion. A 478 bp fragment that on pAL1 represents an intergenic region showed 100 % sequence identity in all isolates harbouring a pAL1-like plasmid, suggesting horizontal dissemination of the linear plasmid among the genus Arthrobacter. INTRODUCTIONBacteria of the genus Arthrobacter are considered to be ubiquitous in soil and have been found to be among the predominant members of culturable communities from several terrestrial subsurface environments (Crocker et al., 2000). Among the explanations advanced for their ubiquity or even predominance in soil are their resistance to desiccation and nutrient depletion, and their nutritional versatility. Arthrobacter spp. utilize a wide and varied range of natural as well as xenobiotic compounds and thus may play a significant role in the mineralization of organic matter in the environment (Cacciari & Lippi, 1987).Arthrobacter nitroguajacolicus strain Rü61a (formerly assigned to the species Arthrobacter ilicis) utilizes quinaldine (2-methylquinoline), a constituent of coal tar, as sole source of carbon and energy (Hund et al., 1990). Degradation via the anthranilate pathway ( Fig. 1) is initiated by the oxidation of quinaldine to 1H-4-oxoquinaldine, catalysed by quinaldine 4-oxidase (Qox). 1H-4-oxoquinaldine 3-monooxygenase subsequently generates 1H-3-hydroxy-4-oxoquinaldine, which undergoes 2,4-dioxygenolytic ring cleavage to form carbon monoxide and N-acetylanthranilate. This unusual mode of ring cleavage is catalysed by a cofactor-less 2,4-dioxygenase that does not share any similarity with aromatic ring cleavage dioxygenases, but seems to belong to the a/b-hydrolase fold superfamily of proteins (Fetzner, 2002). In the next step, an amidase (Amq) catalyses the hydrolysis of N-acetylanthranilate to anthranilate. We have characterized the gene cluster encoding this 'upper part' of th...

Secondary metabolite expression is a widespread strategy among bacteria to improve their fitness in habitats where they constantly compete for resources with other bacteria. The production of secondary metabolites is associated with a metabolic and energetic burden.

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