Integrative Conjugative Elements (ICEs) of the SXT/R391 family drive adaptation and evolution in γ-Proteobacteria.

Integrative Conjugative Elements (ICEs) are mosaics containing functional modules allowing maintenance by site-specific integration and excision into and from the host genome and conjugative transfer to a specific host range. Many ICEs encode a range of adaptive functions that aid bacterial survival and evolution in a range of niches. ICEs from the SXT/R391 family are found in γ-Proteobacteria. Over 100 members have undergone epidemiological and molecular characterization allowing insight into their diversity and function. Comparative analysis of SXT/R391 elements from a wide geographic distribution has revealed conservation of key functions, and the accumulation and evolution of adaptive genes. This evolution is associated with gene acquisition in conserved hotspots and variable regions within the SXT/R391 ICEs catalysed via element-encoded recombinases. The elements can carry IS elements and transposons, and a mutagenic DNA polymerase, PolV, which are associated with their evolution. SXT/R391 ICEs isolated from different niches appear to have retained adaptive functions related to that specific niche; phage resistance determinants in ICEs carried by wastewater bacteria, antibiotic resistance determinants in clinical isolates and metal resistance determinants in bacteria recovered from polluted environments/ocean sediments. Many genes found in the element hotspots are undetermined and have few homologs in the nucleotide databases.

Novel Corrugated Long Period Grating Surface Balloon-Shaped Heterocore-Structured Plastic Optical Fibre Sensor for Microalgal Bioethanol Production.

A novel long period grating (LPG) inscribed balloon-shaped heterocore-structured plastic optical fibre (POF) sensor is described and experimentally demonstrated for real-time measurement of the ultra-low concentrations of ethanol in microalgal bioethanol production applications. The heterocore structure is established by coupling a 250 µm core diameter POF between two 1000 µm diameter POFs, thus representing a large core-small core-large core configuration. Before coupling as a heterocore structure, the sensing region or small core fibre (SCF; i.e., 250 µm POF) is modified by polishing, LPG inscription, and macro bending into a balloon shape to enhance the sensitivity of the sensor. The sensor was characterized for ethanol-water solutions in the ethanol concentration ranges of 20 to 80 %v/v, 1 to 10 %v/v, 0.1 to 1 %v/v, and 0.00633 to 0.0633 %v/v demonstrating a maximum sensitivity of 3 × 106 %/RIU, a resolution of 7.9 × 10-6 RIU, and a limit of detection (LOD) of 9.7 × 10-6 RIU. The experimental results are included for the intended application of bioethanol production using microalgae. The characterization was performed in the ultra-low-level ethanol concentration range, i.e., 0.00633 to 0.03165 %v/v, that is present in real culturing and production conditions, e.g., ethanol-producing blue-green microalgae mixtures. The sensor demonstrated a maximum sensitivity of 210,632.8 %T/%v/v (or 5 × 106 %/RIU as referenced from the RI values of ethanol-water solutions), resolution of 2 × 10-4%v/v (or 9.4 × 10-6 RIU), and LOD of 4.9 × 10-4%v/v (or 2.3 × 10-5 RIU). Additionally, the response and recovery times of the sensor were investigated in the case of measurement in the air and the ethanol-microalgae mixtures. The experimentally verified, extremely high sensitivity and resolution and very low LOD corresponding to the initial rate of bioethanol production using microalgae of this sensor design, combined with ease of fabrication, low cost, and wide measurement range, makes it a promising candidate to be incorporated into the bioethanol production industry as a real-time sensing solution as well as in other ethanol sensing and/or RI sensing applications.

A Review of Optical Fibre Ethanol Sensors: Current State and Future Prospects.

A range of optical fibre-based sensors for the measurement of ethanol, primarily in aqueous solution, have been developed and are reviewed here. The sensing approaches can be classified into four groups according to the measurement techniques used, namely absorption (or absorbance), external interferometric, internal fibre grating and plasmonic sensing. The sensors within these groupings can be compared in terms of their characteristic performance indicators, which include sensitivity, resolution and measurement range. Here, particular attention is paid to the potential application areas of these sensors as ethanol production is globally viewed as an important industrial activity. Potential industrial applications are highlighted in the context of the emergence of the internet of things (IoT), which is driving widespread utilization of these sensors in the commercially significant industrial and medical sectors. The review concludes with a summary of the current status and future prospects of optical fibre ethanol sensors for industrial use.

Control of expression of the ICE R391 encoded UV-inducible cell-sensitising function.

BACKGROUND: Many SXT/R391-like enterobacterial Integrative Conjugative Elements (ICEs) have been found to express an atypical, recA-dependent, UV-inducible, cell-sensitising phenotype observed as a reduction in post-irradiation cell survival rates in host cells. Characterisation of a complete deletion library of the prototype ICE R391 identified the involvement of three core ICE genes, orfs90/91 encoding a putative transcriptional enhancer complex, and orf43, encoding a putative type IV secretion system, outer membrane-associated, conjugative transfer protein. RESULTS: In this study, expression analysis of orf43 indicated that it was up-regulated as a result of UV irradiation in an orfs90/91-dependent manner. Induced expression was found to be controlled from a site preceding the gene which required functional orfs90/91. Expression of orfs90/91 was in turn found to be regulated by orf96, a λ cI-like regulator. Targeted construction of ICE R391 deletions, RT-PCR and qRT-PCR analysis confirmed a regulatory link between orfs90/91 and orf43 while site-directed mutagenesis of orf43 suggested an association with the cell membrane was a prerequisite for the cytotoxic effect. CONCLUSIONS: Because of the recA-dependence of the effect, we hypothesise that UV induction of RecA results in cleavage of the cI-like ICE-encoded repressor protein, the product of orf96. This in turn allows expression of the transcriptional enhancer complex encoded by orfs90/91, which we conclude stimulates transcription of orf43, whose product is directly responsible for the effect.

Genotypic and phenotypic diversity of Ralstonia pickettii and Ralstonia insidiosa isolates from clinical and environmental sources including High-purity Water. Diversity in Ralstonia pickettii.

BACKGROUND: Ralstonia pickettii is a nosocomial infectious agent and a significant industrial contaminant. It has been found in many different environments including clinical situations, soil and industrial High Purity Water. This study compares the phenotypic and genotypic diversity of a selection of strains of Ralstonia collected from a variety of sources. RESULTS: Ralstonia isolates (fifty-nine) from clinical, industrial and environmental origins were compared genotypically using i) Species-specific-PCR, ii) PCR and sequencing of the 16S-23S rRNA Interspatial region (ISR) iii) the fliC gene genes, iv) RAPD and BOX-PCR and v) phenotypically using biochemical testing. The species specific-PCR identified fifteen out of fifty-nine designated R. pickettii isolates as actually being the closely related species R. insidiosa. PCR-ribotyping of the 16S-23S rRNA ISR indicated few major differences between the isolates. Analysis of all isolates demonstrated different banding patterns for both the RAPD and BOX primers however these were found not to vary significantly. CONCLUSIONS: R. pickettii species isolated from wide geographic and environmental sources appear to be reasonably homogenous based on genotypic and phenotypic characteristics. R. insidiosa can at present only be distinguished from R. pickettii using species specific PCR. R. pickettii and R. insidiosa isolates do not differ significantly phenotypically or genotypically based on environmental or geographical origin.

On the bacteriostatic activity of hyaluronic acid composite films.

Biofilm-related infections and contamination of biomaterials are major problems in the clinic. These contaminations are frequently caused by Staphylococcus aureus and are a pressing issue for implantable devices, catheters, contact lenses, prostheses, and wound dressings. Strategies to decrease contamination and biofilm related infections are vital for the success of implantable biomaterials. In this context, hyaluronic acid (HA), a naturally derived carbohydrate polymer, known to be biocompatible, degradable, and immunomodulatory, has shown some antimicrobial activity effects. Due to its poor structural stability, crosslinking strategies, and the incorporation of reinforcing fibres in HA gels is required to produce tailored gels for varying applications. Whilst carbon-based reinforcing materials, such as carbon nanofibers (CNF), present some intrinsic antimicrobial activity related to their high surface area, herein, a crosslinking strategy to enhance the mechanical properties and regulate the rate of degradation of HA is presented. We utilise bis-(ß-isocyanatoethyl) disulphide (BIED) as the crosslinker with the gel reinforced using 0.25 wt% CNF. The effects of CNF and BIED on the structural, mechanical, thermal, and swelling behaviour are examined. These new HA derivatives exhibit excellent mechanical properties and are capable of withstanding physiological stresses in vivo. Antimicrobial activity of the HA derivatives were tested against Staphylococcus aureus and the results reveal antibacterial effect. These carbohydrate based materials have potential application on surfaces within clinical settings where staphylococcal contamination is currently an issue.

The Genus Ochrobactrum as Major Opportunistic Pathogens.

Ochrobactrum species are non-enteric, Gram-negative organisms that are closely related to the genus Brucella. Since the designation of the genus in 1988, several distinct species have now been characterised and implicated as opportunistic pathogens in multiple outbreaks. Here, we examine the genus, its members, diagnostic tools used for identification, data from recent Ochrobactrum whole genome sequencing and the pathogenicity associated with reported Ochrobactrum infections. This review identified 128 instances of Ochrobactrum spp. infections that have been discussed in the literature. These findings indicate that infection review programs should consider investigation of possible Ochrobactrum spp. outbreaks if these bacteria are clinically isolated in more than one patient and that Ochrobactrum spp. are more important pathogens than previously thought.

Novel Tn4371-ICE like element in Ralstonia pickettii and genome mining for comparative elements.

BACKGROUND: Integrative Conjugative Elements (ICEs) are important factors in the plasticity of microbial genomes. An element related to the ICE Tn4371 was discovered during a bioinformatic search of the Ralstonia pickettii 12J genome. This element was analysed and further searches carried out for additional elements.A PCR method was designed to detect and characterise new elements of this type based on this scaffold and a culture collection of fifty-eight Ralstonia pickettii and Ralstonia insidiosa strains were analysed for the presence of the element. RESULTS: Comparative sequence analysis of bacterial genomes has revealed the presence of a number of uncharacterised Tn4371-like ICEs in the genomes of several beta and gamma- Proteobacteria. These elements vary in size, GC content, putative function and have a mosaic-like structure of plasmid- and phage-like sequences which is typical of Tn4371-like ICEs. These elements were found after a through search of the GenBank database. The elements, which are found in Ralstonia, Delftia, Acidovorax, Bordetella, Comamonas, Acidovorax, Congregibacter, Shewanella, Pseudomonas Stenotrophomonas, Thioalkalivibrio sp. HL-EbGR7, Polaromonas, Burkholderia and Diaphorobacter sp. share a common scaffold. A PCR method was designed (based on the Tn4371- like element detected in the Ralstonia pickettii 12J genome) to detect and characterise new elements of this type. CONCLUSION: All elements found in this study possess a common scaffold of core genes but contain different accessory genes. A new uniform nomenclature is suggested for ICEs of the Tn4371 family. Two novel Tn4371-like ICE were discovered and characterised, using the novel PCR method described in two different isolates of Ralstonia pickettii from laboratory purified water.

Autothermal Thermophilic Aerobic Digestion (ATAD) for Heat, Gas, and Production of a Class A Biosolids with Fertilizer Potential.

Autothermal thermophilic aerobic digestion (ATAD) is a microbial fermentation process characterized as a tertiary treatment of waste material carried out in jacketed reactors. The process can be carried out on a variety of waste sludge ranging from human, animal, food, or pharmaceutical waste where the addition of air initiates aerobic digestion of the secondary treated sludge material. Digestion of the sludge substrates generates heat, which is retained within the reactor resulting in elevation of the reactor temperature to 70-75 °C. During the process, deamination of proteinaceous materials also occurs resulting in liberation of ammonia and elevation of pH to typically pH 8.4. These conditions result in a unique microbial consortium, which undergoes considerable dynamic change during the heat-up and holding phases. The change in pH and substrate as digestion occurs also contributes to this dynamic change. Because the large reactors are not optimized for aeration, and because low oxygen solubility at elevated temperatures occurs, there are considerable numbers of anaerobes recovered which also contributes to the overall digestion. As the reactors are operated in a semi-continuous mode, the reactors are rarely washed, resulting in considerable biofilm formation. Equally, because of the fibrous nature of the sludge, fiber adhering organisms are frequently found which play a major role in the overall digestion process. Here, we review molecular tools needed to examine the ATAD sludge consortia, what has been determined through phylogenetic analysis of the consortia and the nature of the dynamics occurring within this unique fermentation environment.

A Novel Arsenate-Resistant Determinant Associated with ICEpMERPH, a Member of the SXT/R391 Group of Mobile Genetic Elements.

ICEpMERPH, the first integrative conjugative element (ICE) of the SXT/R391 family isolated in the United Kingdom and Europe, was analyzed to determine the nature of its adaptive functions, its genetic structure, and its homology to related elements normally found in pathogenic Vibrio or Proteus species. Whole genome sequencing of Escherichia coli (E. coli) isolate K802 (which contains the ICEpMERPH) was carried out using Illumina sequencing technology. ICEpMERPH has a size of 110 Kb and 112 putative open reading frames (ORFs). The "hotspot regions" of the element were found to contain putative restriction digestion systems, insertion sequences, and heavy metal resistance genes that encoded resistance to mercury, as previously reported, but also surprisingly to arsenate. A novel arsenate resistance system was identified in hotspot 4 of the element, unrelated to other SXT/R391 elements. This arsenate resistance system was potentially linked to two genes: orf69, encoding an organoarsenical efflux major facilitator superfamily (MFS) transporter-like protein related to ArsJ, and orf70, encoding nicotinamide adenine dinucleotide (NAD)-dependent glyceraldehyde-3-phosphate dehydrogenase. Phenotypic analysis using isogenic strains of Escherichia coli strain AB1157 with and without the ICEpMERPH revealed resistance to low levels of arsenate in the range of 1-5 mM. This novel, low-level resistance may have an important adaptive function in polluted environments, which often contain low levels of arsenate contamination. A bioinformatic analysis on the novel determinant and the phylogeny of ICEpMERPH was presented.

Zymobacter palmae Pyruvate Decarboxylase is Less Effective Than That of Zymomonas mobilis for Ethanol Production in Metabolically Engineered Synechocystis sp. PCC6803.

To produce bioethanol from model cyanobacteria such as Synechocystis, a two gene cassette consisting of genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are required to transform pyruvate first to acetaldehyde and then to ethanol. However the partition of pyruvate to ethanol comes at a cost, a reduction in biomass and pyruvate availability for other metabolic processes. Hence strategies to divert flux to ethanol as a biofuel in Synechocystis are of interest. PDC from Zymobacter palmae (ZpPDC) has been reported to have a lower Km then the Zymomonas mobilis PDC (ZmPDC), which has traditionally been used in metabolic engineering constructs. The Zppdc gene was combined with the native slr1192 alcohol dehydrogenase gene (adhA) in an attempt to increase ethanol production in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 over constructs created with the traditional Zmpdc. Native (Zppdc) and codon optimized (ZpOpdc) versions of the ZpPDC were cloned into a construct where pdc expression was controlled via the psbA2 light inducible promoter from Synechocystis sp. PCC 6803. These constructs were transformed into wildtype Synechocystis sp. PCC 6803 for expression and ethanol production. Ethanol levels were then compared with identical constructs containing the Zmpdc. While strains with the Zppdc (UL071) and ZpOpdc (UL072) constructs did produce ethanol, levels were lower compared to a control strain (UL070) expressing the pdc from Zymomonas mobilis. All constructs demonstrated lower biomass productivity illustrating that the flux from pyruvate to ethanol has a major effect on biomass and ultimately overall biofuel productivity. Thus the utilization of a PDC with a lower Km from Zymobacter palmae unusually did not result in enhanced ethanol production in Synechocystis sp. PCC 6803.

Brevundimonas spp: Emerging global opportunistic pathogens.

Non-fermenting Gram-negative bacteria are problematic in clinical locations, being one of the most prevalent causes of nosocomial infections. Many of these non-fermenting Gram-negative bacteria are opportunistic pathogens that affect patients that are suffering with underlying medical conditions and diseases. Brevundimonas spp., in particular Brevundimonas diminuta and Brevundimonas vesicularis, are a genus of non-fermenting Gram-negative bacteria considered of minor clinical importance. Forty-nine separate instances of infection relating to Brevundimonas spp were found in the scientific literature along with two pseudo-infections. The majority of these instances were infection with Brevundimonas vesicularis (thirty-five cases - 71%). The major condition associated with Brevundimonas spp infection was bacteraemia with seventeen individual cases/outbreaks (35%). This review identified forty-nine examples of Brevundimonas spp. infections have been discussed in the literature. These findings indicate that infection review programs should consider investigation of possible Brevundimonas spp outbreaks if these bacteria are clinically isolated in more than one patient.

Evaluation of the ethanol tolerance for wild and mutant Synechocystis strains by flow cytometry.

Flow cytometry was used to evaluate the effect of initial ethanol concentrations on cyanobacterial strains of Synechocystis PCC 6803 [wild-type (WT), and ethanol producing recombinants (UL 004 and UL 030)] in batch cultures. Ethanol recombinants, containing one or two metabolically engineered cassettes, were designed towards the development of an economically competitive process for the direct production of bioethanol from microalgae through an exclusive autotrophic route. It can be concluded that the recombinant Synechocystis UL 030 containing two copies of the genes per genome was the most tolerant to ethanol. Nevertheless, to implement a production process using recombinant strains, the bioethanol produced will be required to be continuously extracted from the culture media via a membrane-based technological process for example to prevent detrimental effects on the biomass. The results presented here are of significance in defining the maximum threshold for bulk ethanol concentration in production media.

The orf4 gene of the enterobacterial ICE, R391, encodes a novel UV-inducible recombination directionality factor, Jef, involved in excision and transfer of the ICE.

The enterobacterial mobile genetic element R391, the prototype ICE (integrating-conjugative element) of the SXT/R391 family, shows increased conjugative transfer following UV irradiation. This is dependent on a functioning R391 orf4 gene, which is adjacent to the element encoded integrase gene, int. orf4 mutants fail to form a detectable circular transfer intermediate, do not show UV induced transfer and show a much reduced general transfer ability. The orf4 gene product, termed Jef (IncJ excision factor), shows little homology to anything currently in the nucleotide or protein databases. It is predicted to encode a 66 amino acid, 8.03 kDa, basic, DNA-binding protein with an iso-electric point of pH 8.1: these characteristics being similar to those of recombinational directionality factors involved in excision. Jef expression is up-regulated upon UV irradiation as demonstrated by real-time reverse transcriptase PCR and is controlled by two element encoded genes orf90 and orf91, which show similarity to the transcriptional activator complex FlhC and FlhD. orf4, orf90 and orf91 are conserved in all the SXT/R391-like elements sequenced to date including SXT, ICESpuPO1 and ICEVchMex1. orf4 is also conserved in other SXT/R391 family members such as R997, R392, R705 and pMERPH as shown by sequencing amplicons from these ICEs generated using orf4 specific primers.

Analysis and comparative genomics of R997, the first SXT/R391 integrative and conjugative element (ICE) of the Indian Sub-Continent.

The aim of this study was to analyse R997, the first integrative and conjugative element (ICE) isolated from the Indian Sub-Continent, and to determine its relationship to the SXT/R391 family of ICEs. WGS of Escherichia coli isolate AB1157 (which contains R997) was performed using Illumina sequencing technology. R997 context was assessed by de novo assembly, gene prediction and annotation tools, and compared to other SXT/R391 ICEs. R997 has a size of 85 Kb and harbours 85 ORFs. Within one of the variable regions a HMS-1 ß-lactamase resistance gene is located. The Hotspot regions of the element contains restriction digestion systems and insertion sequences. R997 is very closely related to the SXT-like elements from widely dispersed geographic areas. The sequencing of R997 increases the knowledge of the earliest isolated SXT/R391 elements and may provide insight on the emergence of these elements on the Indian sub-continent.

Mutagenic activities of biochars from pyrolysis.

Biochar production, from pyrolysis of lignocellulosic feedstocks, agricultural residues, and animal and poultry manures are emerging globally as novel industrial and commercial products. It is important to develop and to validate a series of suitable protocols for the ecological monitoring of the qualities and properties of biochars. The highly sensitive Salmonella mutagenicity assays (the Ames test) are used widely by the toxicology community and, via the rat liver extract (S9), can reflect the potential for mammalian metabolic activation. We examined the Ames test for analyses of the mutagenic activities of dimethylsulphoxide (DMSO) extracts of biochars using two bacterial models (S. typhimurium strains TA98 and TA100) in the presence and in the absence of the metabolic activation with the S9-mix. Tester strain TA98 was most sensitive in detecting mutagenic biochar products, and the contribution of S9 was established. Temperature and times of pyrolysis are important. Biochar pyrolysed at 400°C for 10min, from a lignocellulose precursor was mutagenic, but not when formed at 800°C for 60min, or at 600°C for 30min. Biochars from poultry litter, and manures of calves fed on grass had low mutagenicities. Biochar from pig manure had high mutagenicity; biochars from manures of cows fed on a grass plus cereals, those of calves fed on mother's milk, and biochars from solid industrial waste had intermediate mutagenicities. The methods outlined can indicate the need for further studies for screening and detection of the mutagenic residuals in a variety of biochar products.

A novel ICE in the genome of Shewanella putrefaciens W3-18-1: comparison with the SXT/R391 ICE-like elements.

A novel R391-like ICE (integrating conjugative element) has been detected in the 4.2 MB genome of Shewanella putrefaciens W3-18-1 located on three different contigs. Assembly of the ICE encoding contigs based on similarity with R391 revealed a mosaic element of plasmid, phage and transposon-like sequences typical of SXT/R391 ICE-like elements. The element, which is 110 057 bp in length, was highly similar to R391 sequences, with most related ORFs showing >96% amino acid sequence identity. The element, designated ICESpuPO1, contained a number of inserts determining resistance to copper and other heavy metals and a broad-spectrum RND efflux pump similar to antibiotic efflux systems. The element was integrated into the Shewanella prfC gene in a manner similar to related ICE-like elements. The chromosomal element junctions contained a 17-bp SXT/R391-like attL and attR site and an unannotated ORF between attL and the ICE integrase encoding a putative recombinational directional factor necessary for excision, with 100% amino acid identity to the R391 ORF4 product.

Molecular tools to detect the IncJ elements: a family of integrating, antibiotic resistant mobile genetic elements.

The IncJ group of enterobacterial mobile genetic elements, which include R391, R392, R705, R997 and pMERPH, have been shown to be site-specific integrating elements encoding variable antibiotic and heavy metal resistance genes. They insert into a specific 17-bp site located in the prfC gene, encoding peptide release factor 3, in Escherichia coli and other hosts. A key feature of known IncJ elements is the presence of a site-specific recombination module consisting of an attachment site on the element and an integrase-encoding gene of the tyrosine recombinase class, which promotes integration between the attachment site on the element and a similar site on the host chromosome. We have cloned and sequenced the integrases from a number of known IncJ elements and designed PCR primers for specific amplification of this gene. Using conserved regions of enterobacterial prfC genes upstream and downstream of the insertion site, and conserved sequences at the ends of the integrated IncJ elements, we have designed specific primers to amplify across the integrated IncJ attL and attR junction fragments. Alignment of over 30 enterobacterial prfC-like genes indicates that the primers designed to amplify attR junction would amplify IncJ element: host junctions from a wide variety of hosts. The IncJ elements have been shown to sensitise recA(+)E. coli K12 strains to UV irradiation. A simple and rapid procedure for demonstrating this effect is described. These tools should enable the rapid detection of such elements in clinical and environmental settings.

SXT/R391 Integrative and Conjugative Elements (ICEs) Encode a Novel 'Trap-Door' Strategy for Mobile Element Escape.

Integrative conjugative elements (ICEs) are a class of bacterial mobile elements that have the ability to mediate their own integration, excision, and transfer from one host genome to another by a mechanism of site-specific recombination, self-circularisation, and conjugative transfer. Members of the SXT/R391 ICE family of enterobacterial mobile genetic elements display an unusual UV-inducible sensitization function which results in stress induced killing of bacterial cells harboring the ICE. This sensitization has been shown to be associated with a stress induced overexpression of a mobile element encoded conjugative transfer gene, orf43, a traV homolog. This results in cell lysis and release of a circular form of the ICE. Induction of this novel system may allow transfer of an ICE, enhancing its survival potential under conditions not conducive to conjugative transfer.

Pre-exposure to UV irradiation increases the transfer frequency of the IncJ conjugative transposon-like elements R391, R392, R705, R706, R997 and pMERPH and is recA+ dependent.

The enteric conjugative transposon-like IncJ elements R391, R392, R705, R706 and pMERPH, all demonstrated increased conjugative transfer upon UV irradiation. The transfer frequency increased on average from its basal rate of 10(-5) to 10(-3) per recipient, upon pre-exposure to UV irradiation. However, the transfer frequency of R997, which was higher than the other IncJ elements at 10(-3) per donor, showed a smaller increase. This effect was shown to be recA+ dependent in all cases. Using PCR primers directed outwards from the ends of the integrated R391 element it was observed that a circular intermediate of the element forms within the host, which has been proposed to be a transfer intermediate. Using real-time PCR, it was determined that the amount of the circular intermediate produced increased substantially upon UV irradiation.