Pseudomonas sp. strain B13 is a bacterium known to degrade chloroaromatic compounds. The properties to use 3-and 4-chlorocatechol are determined by a self-transferable DNA element, the clc element, which normally resides at two locations in the cell's chromosome. Here we report the complete nucleotide sequence of the clc element, demonstrating the unique catabolic properties while showing its relatedness to genomic islands and integrative and conjugative elements rather than to other known catabolic plasmids. As far as catabolic functions, the clc element harbored, in addition to the genes for chlorocatechol degradation, a complete functional operon for 2-aminophenol degradation and genes for a putative aromatic compound transport protein and for a multicomponent aromatic ring dioxygenase similar to anthranilate hydroxylase. The genes for catabolic functions were inducible under various conditions, suggesting a network of catabolic pathway induction. For about half of the open reading frames (ORFs) on the clc element, no clear functional prediction could be given, although some indications were found for functions that were similar to plasmid conjugation. The region in which these ORFs were situated displayed a high overall conservation of nucleotide sequence and gene order to genomic regions in other recently completed bacterial genomes or to other genomic islands. Most notably, except for two discrete regions, the clc element was almost 100% identical over the whole length to a chromosomal region in Burkholderia xenovorans LB400. This indicates the dynamic evolution of this type of element and the continued transition between elements with a more pathogenic character and those with catabolic properties.

Genomic islands (GEI) comprise a recently recognized large family of potentially mobile DNA elements and play an important role in the rapid differentiation and adaptation of bacteria. Most importantly, GEIs have been implicated in the acquisition of virulence factors, antibiotic resistances or toxic compound metabolism. Despite detailed information on coding capacities of GEIs, little is known about the regulatory decisions in individual cells controlling GEI transfer. Here, we show how self-transfer of ICEclc, a GEI in Pseudomonas knackmussii B13 is controlled by a series of stochastic processes, the result of which is that only a few percent of cells in a population will excise ICEclc and launch transfer. Stochastic processes have been implicated before in producing bistable phenotypic transitions, such as sporulation and competence development, but never before in horizontal gene transfer (HGT). Bistability is instigated during stationary phase at the level of expression of an activator protein InrR that lays encoded on ICEclc, and then faithfully propagated to a bistable expression of the IntB13 integrase, the enzyme responsible for excision and integration of the ICEclc. Our results demonstrate how GEI of a very widespread family are likely to control their transfer rates. Furthermore, they help to explain why HGT is typically confined to few members within a population of cells. The finding that, despite apparent stochasticity, HGT rates can be modulated by external environmental conditions provides an explanation as to why selective conditions can promote DNA exchange.integrase ͉ integrative and conjugative element ͉ regulation ͉ 3-chlorobenzoate ͉ clc element

Conjugative transfer of the integrative and conjugative element ICE clc in the bacterium Pseudomonas knackmussii is the consequence of a bistable decision taken in some 3% of cells in a population during stationary phase. Here we study the possible control exerted by the stationary phase sigma factor RpoS on the bistability decision. The gene for RpoS in P. knackmussii B13 was characterized, and a loss-of-function mutant was produced and complemented. We found that, in absence of RpoS, ICE clc transfer rates and activation of two key ICE clc promoters (P int and P inR ) decrease significantly in cells during stationary phase. Microarray and gene reporter analysis indicated that the most direct effect of RpoS is on P inR , whereas one of the gene products from the P inR -controlled operon (InrR) transmits activation to P int and other ICE clc core genes. Addition of a second rpoS copy under control of its native promoter resulted in an increase of the proportion of cells expressing the P int and P inR promoters to 18%. Strains in which rpoS was replaced by an rpoS-mcherry fusion showed high mCherry fluorescence of individual cells that had activated P int and P inR , whereas a double-copy rpoS-mcherry –containing strain displayed twice as much mCherry fluorescence. This suggested that high RpoS levels are a prerequisite for an individual cell to activate P inR and thus ICE clc transfer. Double promoter–reporter fusions confirmed that expression of P inR is dominated by extrinsic noise, such as being the result of cellular variability in RpoS. In contrast, expression from P int is dominated by intrinsic noise, indicating it is specific to the ICE clc transmission cascade. Our results demonstrate how stochastic noise levels of global transcription factors can be transduced to a precise signaling cascade in a subpopulation of cells leading to ICE activation.

SummaryGenomic islands are DNA elements acquired by horizontal gene transfer that are common to a large number of bacterial genomes, which can contribute specific adaptive functions, e.g. virulence, metabolic capacities or antibiotic resistances. Some genomic islands are still self-transferable and display an intricate life-style, reminiscent of both bacteriophages and conjugative plasmids. Here we studied the dynamical process of genomic island excision and intracellular reintegration using the integrative and conjugative element ICEclc from Pseudomonas knackmussii B13 as model. By using self-transfer of ICEclc from strain B13 to Pseudomonas putida and Cupriavidus necator as recipients, we show that ICEclc can target a number of different tRNA Gly genes in a bacterial genome, but only those which carry the GCC anticodon. Two conditional traps were designed for ICEclc based on the attR sequence, and we could show that ICEclc will insert with different frequencies in such traps producing brightly fluorescent cells. Starting from clonal primary transconjugants we demonstrate that ICEclc is excising and reintegrating at detectable frequencies, even in the absence of recipient. Recombination site analysis provided evidence to explain the characteristics of a larger number of genomic island insertions observed in a variety of strains, including Bordetella petri, Pseudomonas aeruginosa and Burkholderia.

BackgroundIntegrative and conjugative elements (ICE) form a diverse group of DNA elements that are integrated in the chromosome of the bacterial host, but can occasionally excise and horizontally transfer to a new host cell. ICE come in different families, typically with a conserved core for functions controlling the element's behavior and a variable region providing auxiliary functions to the host. The ICEclc element of Pseudomonas knackmussii strain B13 is representative for a large family of chromosomal islands detected by genome sequencing approaches. It provides the host with the capacity to degrade chloroaromatics and 2-aminophenol.ResultsHere we study the transcriptional organization of the ICEclc core region. By northern hybridizations, reverse-transcriptase polymerase chain reaction (RT-PCR) and Rapid Amplification of cDNA Ends (5'-RACE) fifteen transcripts were mapped in the core region. The occurrence and location of those transcripts were further confirmed by hybridizing labeled cDNA to a semi-tiling micro-array probing both strands of the ICEclc core region. Dot blot and semi-tiling array hybridizations demonstrated most of the core transcripts to be upregulated during stationary phase on 3-chlorobenzoate, but not on succinate or glucose.ConclusionsThe transcription analysis of the ICEclc core region provides detailed insights in the mode of regulatory organization and will help to further understand the complex mode of behavior of this class of mobile elements. We conclude that ICEclc core transcription is concerted at a global level, more reminiscent of a phage program than of plasmid conjugation.

Our data highlight the structure and evolution of IncHI1 from equine E. coli. A plasmid-mediated sugar metabolic element could play a key role in strain fitness, contributing to the successful dissemination and maintenance of these plasmids in the intestinal microflora of horses.

Resveratrol is among the best-known secondary plant metabolites because of its antioxidant, anti-inflammatory and anticancer properties. It also is an important allelopathic chemical widely credited with protection of plants from pathogens. The ecological role of resveratrol in natural habitats is difficult to establish rigorously because it does not seem to accumulate outside of plant tissue. It is likely that bacterial degradation plays a key role in determining the persistence, and thus the ecological role, of resveratrol in soil. Here we report the isolation of an species that can use resveratrol as a sole carbon source from the rhizosphere of peanut plants. Both molecular and biochemical techniques indicate that the pathway starts with the conversion of resveratrol to 3,5-dihydroxybenzaldehyde and 4-hydroxybenzaldehyde. The aldehydes are oxidized to substituted benzoates that subsequently enter central metabolism. The gene that encodes the enzyme responsible for the oxidative cleavage of resveratrol was cloned, and expressed in to establish its function. Its physiological role in the resveratrol catabolic pathway was established by knockouts and by RT-qPCR demonstration of expression during growth on resveratrol. The results establish the presence and capabilities of resveratrol degrading bacteria in the rhizosphere of the peanut plants and set the stage for studies to evaluate the role of the bacteria in plant allelopathy. In addition to its antioxidant properties, resveratrol is an example of a broad array of allelopathic chemicals produced by plants to inhibit competitors, herbivores and pathogens. Bacterial degradation of such chemicals in the rhizosphere would reduce the effects of the chemicals. Therefore, it is important to understand the activity and ecological role of bacteria that biodegrade resveratrol near the plants that produce it. This study describes the isolation from the peanut rhizosphere of bacteria that can grow on resveratrol. Characterization of the initial steps in the biodegradation sets the stage for investigation of the evolution of the catabolic pathways responsible for biodegradation of resveratrol and its homologs.

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