Abstract
Background
Integrative Conjugative Elements (ICEs) are important factors in the plasticity of microbial genomes. An element related to the ICE Tn
4371
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 Tn
4371
-like ICEs in the genomes of several β and γ- 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 Tn
4371
-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 Tn
4371
- 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 Tn
4371
family. Two novel Tn
4371
-like ICE were discovered and characterised, using the novel PCR method described in two different isolates of
Ralstonia pickettii
from laboratory purified water.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A novel approach to the intensification of renewable and sustainable production of photoautotrophic ethanol from microalgae was investigated using periodic ultrasonication. The effect of utilizing ...ultrasonic pulsing during ethanol production using a metabolically engineered ethanol producing cyanobacterium Synechocystis PCC6803 strain NAV001 was analysed with an ultrasonic frequency of 20 kHz. Ultrasonic treatment resulted in enhancement of ethanol yields. The ethanol yield was found to be higher when ultrasonic pulsing was initiated during the exponential phase of growth. The optimum ultrasonic conditions for enhanced yield of ethanol within this model system was found to be 30 °C, 15% of power input (97.5 W) and 10 min pulse time. The cytotoxic effect of ultrasonic pulsation was investigated by analysing the survival percentage and auto-fluorescence of the cyanobacterium via imaging by confocal laser scanning microscopy. The overall effect of ultrasonic dose on ethanol production and growth rate of microalgae was analysed by using Haldane inhibition kinetics.
•Introduction of ultrasonic intensification on photoautotrophic ethanol production.•Analysis on ultrasonic effect on growth and ethanol production rate.•Implementation of Haldane model for ultrasonic inhibition analysis.•Analysis on cyto-inhibitory effect of ultrasonication.
The mechanisms of metal ion transport in thermophilic organisms are poorly understood. Phage display-based screening of a Thermus thermophilus genomic library in Escherichia coli led to the ...identification of a novel metal cation efflux protein. The Thermus protein showed extensive sequence and putative structural conservation to Czr and Czc proteins in mesophilic bacterial and mammalian species. Expression of the gene in E. coli led to increased resistance to zinc and cadmium ions, but not to cobalt, in an effect that was apparently caused by increased efflux of metals from the cell. This increased resistance was inducible by zinc and cadmium and, to a lesser extent, by cobalt. Furthermore, E. coli cells containing the Thermus gene exhibited improved cell physiology and delayed cell lysis during recombinant protein production, leading to accumulation of higher levels of recombinant protein. The molecular basis and potential application of the findings are discussed.
Abstract
The IncJ group of mobile elements have not been extensively studied until recently, due to the inability to isolate extrachromosomal DNA from IncJ-strains. Sequence analysis of the prototype ...IncJ element, R391, revealed it to be a mosaic structure, integrated into the prfC gene in E. coli. Using inverse PCR (iPCR), we localised the other available IncJ elements (R392, R705, R997 and pMERPH) site of insertion to a 17-bp sequence, within the 5′ end of prfC at 99.31 min on the E. coli chromosome, and confirmed this for R391. Despite disrupting prfC, the IncJ's encode novel promoter and 5′ sequences, restoring function of the disrupted prfC. Sequence analysis of the elements ends revealed that they contain integrase genes, which share extensive homologies among the group, despite being isolated from broad geographic locations. The elements excise from the host chromosome by recombination between their attL and attR sites, with subsequent recombination between the attP sites on the circular forms and the attB sites in the host genomes. The attB site is highly conserved and found in many different bacteria, suggesting a possible broad host range.
Abstract
The integration site(s) of the IncJ element, R391, was localised to a specific region of the Escherichia coli chromosome, between the uxuA and serB loci (98.0–99.5 min), using classical Hfr ...mapping techniques. F-prime plasmid hosts, diploid for regions spanning the E. coli chromosome, were used as recipients in R391 and R997 conjugal transfer assays. Analysis of transconjugants revealed the integration of R391 and R997 into specific F-primes that contain the uxuA to serB region, but not F-primes that contain other regions of the chromosome. A comparison of the electrophoretic mobility of the original F-primes with those containing inserts demonstrated the integration of large elements, in excess of 85 kb. Linear integration of the IncJ elements into chromosomal DNA was demonstrated in recombination-deficient (recA) backgrounds in the absence of detectable autonomous stages. These observations account for the inability to isolate plasmid DNA from IncJ hosts, and suggests that the elements exhibit a conjugative transposon-like biology in E. coli.
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 sensitisation function which results in stress induced killing of bacterial cells harbouring the ICE. This sensitisation 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.
The IncJ group of mobile elements have not been extensively studied until recently, due to the inability to isolate extrachromosomal DNA from IncJ-strains. Sequence analysis of the prototype IncJ ...element, R391, revealed it to be a mosaic structure, integrated into the
prfC gene in
E. coli. Using inverse PCR (iPCR), we localised the other available IncJ elements (R392, R705, R997 and pMERPH) site of insertion to a 17-bp sequence, within the 5
′ end of
prfC at 99.31 min on the
E. coli chromosome, and confirmed this for R391. Despite disrupting
prfC, the IncJ's encode novel promoter and 5
′ sequences, restoring function of the disrupted
prfC. Sequence analysis of the elements ends revealed that they contain integrase genes, which share extensive homologies among the group, despite being isolated from broad geographic locations. The elements excise from the host chromosome by recombination between their
attL and
attR sites, with subsequent recombination between the
attP sites on the circular forms and the
attB sites in the host genomes. The
attB site is highly conserved and found in many different bacteria, suggesting a possible broad host range.
A novel, low cost and highly sensitive optical fibre probe sensor for concentration measurement of ethanol solvent (C2H5OH) corresponding to bio-ethanol production rate by an algae is reported. The ...principle of operation of the sensor is based on inter-fibre light coupling through an evanescent field interaction to couple the light between two multimode fibres mounted parallel to each other at a minimum possible separation i.e. ~ <; 1mm. The sensor was fabricated using a low cost 1000um plastic optical fibre (POF) and was characterized for real time measurement in the broadband spectrum including visible and near infra-red. The wavelength dependency of this sensor design was also investigated by post processing analysis of real time data and hence the optimum wavelength range determined. The proposed sensor has shown significant response in the range of 0.005 - 0.1 %v/v (%volume/volume or volume concentration) which depicts the high sensitivity for monitoring very minute changes in concentration corresponding refractive index changes of the solution. Numerically, sensor has shown the sensitivity of 21945 intensity counts/%v/v or 109.7 counts per every 0.0050 %v/v.
Abstract
The incompatibility between the chromosomally integrating, conjugative transposon-like, IncJ elements R997 (ampicillin resistant) and R391 (kanamycin resistant) was examined by constructing ...strains harbouring both elements. Unusually, recA+ strains harbouring the resistance determinants of both elements could be isolated but all strains lacked detectable extrachromosomal DNA. The phenotypic characteristics and transfer patterns observed suggested the formation of recombinant hybrids rather than strains harbouring both elements independently. Formation of strains harbouring two IncJ elements in a recA background was thus examined and resulted in the visualisation of extrachromosomal DNA. When R391 was transferred to a recA strain containing integrated R997, both elements co-existed stably and resulted in the isolation of a plasmid of 93.9 kb. When R997 was transferred to a recA strain harbouring an integrated R391, a plasmid of 85 kb was isolated. Comparison of restriction patterns for both elements revealed many common and several distinct fragments indicating a close physical relationship. These data suggest that although IncJ elements normally integrate at a unique site in the Escherichia coli chromosome, they possess the ability for autonomous replication which becomes manifest in a recA background when this site is occupied. This observation has implications for the nature of the incompatibility associated with IncJ elements and also provides a reliable method for isolating IncJ elements for molecular characterisation.
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. 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. 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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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