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 ICE clc from Pseudomonas knackmussii B13 as model. By using self-transfer of ICE clc from strain B13 to Pseudomonas putida and Cupriavidus necator as recipients, we show that ICE clc 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 ICE clc based on the attR sequence, and we could show that ICE clc will insert with different frequencies in such traps producing brightly fluorescent cells. Starting from clonal primary transconjugants we demonstrate that ICE clc 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 .     

Evolution of a chlorobenzene degradative pathway among bacteria in a contaminated groundwater mediated by a genomic island in Ralstonia

The genetic structure of two Ralstonia spp., strain JS705 and strain JS745, isolated from the same groundwater aquifer, was characterized with respect to the degradation capacities for toluene and chlorobenzene degradation. Cosmid library construction, cloning, DNA sequencing and mating experiments indicated that the genes for chlorobenzene degradation in strain JS705 were a mosaic of the clc genes, previously described for Pseudomonas sp. strain B13, and a 5 kb fragment identical to strain JS745. The 5 kb fragment identical to both JS705 and JS745 was flanked in JS705 by one complete and one incomplete insertion (IS) element. This suggested involvement of the IS element in mobilizing the genes from JS745 to JS705, although insertional activity of the IS element in its present configuration could not be demonstrated. The complete genetic structure for chlorobenzene degradation in strain JS705 resided on a genomic island very similar to the clc element (Ravatn, R., Studer, S., Springael, D., Zehnder, A.J., van der Meer, J.R. 1998. Chromosomal integration, tandem amplification, and deamplification in Pseudomonas putida F1 of a 105-kilobase genetic element containing the chlorocatechol degradative genes from Pseudomonas sp. strain B13. J Bacteriol 180: 4360-4369). The unique reconstruction of formation of a metabolic pathway through the activity of IS elements and a genomic island in the chlorobenzene-degrading strain JS705 demonstrated how pathway evolution can occur under natural conditions in a few 'steps'.     

The Salmonella genomic island 1 is an integrative mobilizable element

Salmonella genomic island 1 (SGI1) is a genomic island containing an antibiotic resistance gene cluster identified in several Salmonella enterica serovars. The SGI1 antibiotic resistance gene cluster, which is a complex class 1 integron, confers the common multidrug resistance phenotype of epidemic S. enterica Typhimurium DT104. The SGI1 occurrence in S. enterica serovars Typhimurium, Agona, Paratyphi B, Albany, Meleagridis and Newport indicates the horizontal transfer potential of SGI1. Here, we report that SGI1 could be conjugally transferred from S. enterica donor strains to non-SGI1 S. enterica and Escherichia coli recipient strains where it integrated into the recipient chromosome in a site-specific manner. First, an extrachromosomal circular form of SGI1 was identified by PCR which forms through a specific recombination of the left and right ends of the integrated SGI1. Chromosomal excision of SGI1 was found to require SGI1-encoded integrase which presents similarities to the lambdoid integrase family. Second, the conjugal transfer of SGI1 required the presence of a helper plasmid. The conjugative IncC plasmid R55 could thus mobilize in trans SGI1 which was transferred from the donor to the recipient strains. By this way, the conjugal transfer of SGI1 occurred at a frequency of 10(-5)-10(-6) transconjugants per donor. No transconjugants could be obtained for the SGI1 donor lacking the int integrase gene. Third, chromosomal integration of SGI1 occurred via a site-specific recombination between a 18 bp sequence found in the circular form of SGI1 and a similar 18 bp sequence at the 3' end of thdF gene in the S. enterica and E. coli chromosome. SGI1 appeared to be transmissible only in the presence of additional conjugative functions provided in trans. SGI1 can thus be classified within the group of integrative mobilizable elements (IMEs).     

Genomic islands and the evolution of catabolic pathways in bacteria

Genes for the degradation of organic pollutants have usually been allocated to plasmid DNAs in bacteria or considered non-mobile when detected in the chromosome. New discoveries have shown that catabolic genes can also be part of so-called integrative and conjugative elements (ICElands), a group of mobile DNA elements also known as genomic islands and conjugative transposons. One such ICEland is the clc element for chlorobenzoate and chlorocatechol degradation in Pseudomonas sp. strain B13. Genome comparisons and genetic data on integrase functioning reveal that the clc element and several other unclassified ICElands belong to a group of elements with conserved features. The clc element is unique among them in carrying the genetic information for several degradation pathways, whereas the others give evidence for pathogenicity functions. Many more such elements may exist, bridging the gap between pathogenicity and degradation functions.     

用无启动子的GUS报告基因捕获水稻基因启动子

构建了嵌合质粒p13DGUTs,它是在Ds转座子中插入了无启动子的B．葡萄糖醛酸酶报告基因(GUS),用于分离水稻基因启动子。将p13DGUTs转化粳稻品种中花11的胚性愈伤组织,获得了496个转基因植株。抗性愈伤组织与转基因植株的GUS染色与PCR分析表明整合在水稻染色体上的Ds因子都发生了随机跳跃。转基因植株T0代与部分T1代的GUS染色结果表明,M92转基因植株中Ds转座子整合位置上游的水稻基因启动子指导GUS基因的表达及表达的特性是可遗传的。文章对此方法在分离水稻基因启动子与基因上的应用进行了讨论。     

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.     

Precise nucleotide location of the 5' ends of RNA-primed nascent light strands of mouse mitochondrial DNA

The mouse mitochondrial DNA origin of light-strand replication has been defined as a 32-nucleotide region located among five transfer RNA genes in the genomic sequence. A distinctive feature of this origin is its potential to form a perfectly complementary stem and 11-nucleotide loop structure. Previous studies have demonstrated that the 5′ ends of nascent light strands map within this region and a major trinucleotide ribosubstitution site in closed circular mouse mitochondrial DNA has been mapped within the stem sequence.Direct analysis and precise localization of the 5′ ends of nascent light strands indicate that essentially all 5′ ends are ribonucleotides mapping in the originspecific dyadic structure. The major 5′ end identified is the rG at position 5187 in the genomic sequence. Priming of replication most likely occurs within the loop portion of the potential dyad and continues for 2 to 16 nucleotides with a sharply defined switch to deoxyribonucleotide synthesis. This functional transition point is identical in map position to the trinucleotide ribosubstitution site in mature, closed circular mitochondrial DNA.