Genomic analysis of qnr-harbouring IncX plasmids and their transferability within different hosts under induced stress.

BACKGROUND: Conjugative plasmids play a major role in the dissemination of antibiotic resistance genes. Knowledge of the plasmid characteristics and behaviour can allow development of control strategies. Here we focus on the IncX group of plasmids carrying genes conferring quinolone resistance (PMQR), reporting their transfer and persistence within host bacteria of various genotypes under distinct conditions and levels of induced stress in form of temperature change and various concentrations of ciprofloxacin supplementation. METHODS: Complete nucleotide sequences were determined for eight qnr-carrying IncX-type plasmids, of IncX1 (3), IncX2 (3) and a hybrid IncX1-2 (2) types, recovered from Escherichia coli of various origins. This data was compared with further complete sequences of IncX1 and IncX2 plasmids carrying qnr genes (n = 41) retrieved from GenBank and phylogenetic tree was constructed. Representatives of IncX1 (pHP2) and IncX2 (p194) and their qnrS knockout mutants, were studied for influence of induced stress and genetic background on conjugative transfer and maintenance. RESULTS: A high level of IncX core-genome similarity was found in plasmids of animal, environmental and clinical origin. Significant differences were found between the individual IncX plasmids, with IncX1 subgroup plasmids showing higher conjugative transfer rates than IncX2 plasmids. Knockout of qnr modified transfer frequency of both plasmids. Two stresses applied simultaneously were needed to affect transfer rate of wildtype plasmids, whereas a single stress was sufficient to affect the IncX ΔqnrS plasmids. The conjugative transfer was shown to be biased towards the host phylogenetic proximity. A long-term cultivation experiment pointed out the persistence of IncX plasmids in the antibiotic-free environment. CONCLUSIONS: The study indicated the stimulating effect of ciprofloxacin supplementation on the plasmid transfer that can be nullified by the carriage of a single PMQR gene. The findings present the significant properties and behaviour of IncX plasmids carrying antibiotic resistance genes that are likely to play a role in their dissemination and stability in bacterial populations.

Dissecting the in vivo dynamics of transcription locking due to positive supercoiling buildup.

Positive supercoiling buildup (PSB) is a pervasive phenomenon in the transcriptional programs of Escherichia coli. After finding a range of Gyrase concentrations where the inverse of the transcription rate of a chromosome-integrated gene changes linearly with the inverse of Gyrase concentration, we apply a LineWeaver-Burk plot to dissect the expected in vivo transcription rate in absence of PSB. We validate the estimation by time-lapse microscopy of single-RNA production kinetics of the same gene when single-copy plasmid-borne, shown to be impervious to Gyrase inhibition. Next, we estimate the fraction of time in locked states and number of transcription events prior to locking, which we validate by measurements under Gyrase inhibition. Replacing the gene of interest by one with slower transcription rate decreases the fraction of time in locked states due to PSB. Finally, we combine data from both constructs to infer a range of possible transcription initiation locking kinetics in a chromosomal location, obtainable by tuning the transcription rate. We validate with measurements of transcription activity at different induction levels. This strategy for dissecting transcription initiation locking kinetics due to PSB can contribute to resolve the transcriptional programs of E. coli and in the engineering of synthetic genetic circuits.

The precision of the symmetry in Z-ring placement in Escherichia coli is hampered at critical temperatures.

Cell division in Escherichia coli is morphologically symmetric due to, among other things, the ability of these cells to place the Z-ring at midcell. Studies have reported that, at sub-optimal temperatures, this symmetry decreases at the single-cell level, but the causes remain unclear. Using fluorescence microscopy, we observe FtsZ-GFP and DAPI-stained nucleoids to assess the robustness of the symmetry of Z-ring formation and positioning in individual cells under sub-optimal and critical temperatures. We find the Z-ring formation and positioning to be robust at sub-optimal temperatures, as the Z-ring's mean width, density and displacement from midcell maintain similar levels of correlation to one another as at optimal temperatures. However, at critical temperatures, the Z-ring displacement from midcell is greatly increased. We present evidence showing that this is due to enhanced distance between the replicated nucleoids and, thus, reduced Z-ring density, which explains the weaker precision in setting a morphologically symmetric division site. This also occurs in rich media and is cumulative, i.e. combining richer media and critically high temperatures enhances the asymmetries in division, which is evidence that the causes are biophysical. To further support this, we show that the effects are reversible, i.e. shifting cells from optimal to critical, and then to optimal again, reduces and then enhances the symmetry in Z-ring positioning, respectively, as the width and density of the Z-ring return to normal values. Overall, our findings show that the Z-ring positioning in E. coli is a robust biophysical process under sub-optimal temperatures, and that critical temperatures cause significant asymmetries in division.

Resveratrol as a Growth Substrate for Bacteria from the Rhizosphere.

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 the 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 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 Acinetobacter 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 Escherichia coli to establish its function. Its physiological role in the resveratrol catabolic pathway was established by knockouts and by the reverse transcription-quantitative PCR (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.IMPORTANCE In addition to its antioxidant properties, resveratrol is representative of a broad array of allelopathic chemicals produced by plants to inhibit competitors, herbivores, and pathogens. The 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. The characterization of the initial steps in the biodegradation process sets the stage for the investigation of the evolution of the catabolic pathways responsible for the biodegradation of resveratrol and its homologs.

Complete sequences of IncHI1 plasmids carrying blaCTX-M-1 and qnrS1 in equine Escherichia coli provide new insights into plasmid evolution.

OBJECTIVES: To determine the structure of two multidrug-resistant IncHI1 plasmids carrying blaCTX-M-1 in Escherichia coli isolates disseminated in an equine clinic in the Czech Republic. METHODS: A complete nucleotide sequencing of 239 kb IncHI1 (pEQ1) and 287 kb IncHI1/X1 (pEQ2) plasmids was performed using the 454-Genome Sequencer FLX system. The sequences were compared using bioinformatic tools with other sequenced IncHI1 plasmids. RESULTS: A comparative analysis of pEQ1 and pEQ2 identified high nucleotide identity with the IncHI1 type 2 plasmids. A novel 24 kb module containing an operon involved in short-chain fructooligosaccharide uptake and metabolism was found in the pEQ backbones. The role of the pEQ plasmids in the metabolism of short-chain fructooligosaccharides was demonstrated by studying the growth of E. coli cells in the presence of these sugars. The module containing the blaCTX-M-1 gene was formed by a truncated macrolide resistance cluster and flanked by IS26 as previously observed in IncI1 and IncN plasmids. The IncHI1 plasmid changed size and gained the quinolone resistance gene qnrS1 as a result of IS26-mediated fusion with an IncX1 plasmid. CONCLUSIONS: 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.

Cellular variability of RpoS expression underlies subpopulation activation of an integrative and conjugative element.

Conjugative transfer of the integrative and conjugative element ICEclc 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, ICEclc transfer rates and activation of two key ICEclc 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 ICEclc 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 ICEclc 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 ICEclc 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.

Transcriptome analysis of the mobile genome ICEclc in Pseudomonas knackmussii B13.

BACKGROUND: Integrative 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. RESULTS: Here 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. CONCLUSIONS: The 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.

Intracellular excision and reintegration dynamics of the ICEclc genomic island of Pseudomonas knackmussii sp. strain B13.

Genomic 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.

Stochasticity and bistability in horizontal transfer control of a genomic island in Pseudomonas.

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.

The clc element of Pseudomonas sp. strain B13, a genomic island with various catabolic properties.

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.