Bacterial catabolic transposons

The introduction of foreign organic hydrocarbons into the environment in recent years, as in the widespread use of antibiotics, has resulted in the evolution of novel adaptive mechanisms by bacteria for the biodegradation of the organic pollutants. Plasmids have been implicated in the catabolism of many of these complex xenobiotics. The catabolic genes are prone to undergo genetic rearrangement and this is due to their presence on transposons or their association with transposable elements. Most of the catabolic transposons have structural features of the class I (composite) elements. These include transposons for chlorobenzoate (Tn5271), chlorobenzene (Tn5280), the newly discovered benzene catabolic transposon (Tn5542), and transposons encoding halogenated alkanoates and nylon-oligomer-degradative genes. Transposons for the catabolism of toluene (Tn4651, Tn4653, Tn4656) and naphthalene (Tn4655) belong to class II (Tn3 family) elements. Many catabolic genes have been associated with insertion sequences, which suggests that these gene clusters could be rapidly disseminated among the bacterial populations. This greatly expands the substrate range of the microorganisms in the environment and aids the evolution of new and novel degradative pathways. This enhanced metabolic versatility can be exploited for and is believed to play a major part in the bioremediation of polluted environments. Received: 13 July 1998 / Received revision: 22 September 1998 / Accepted: 26 September 1998     

The transposable elements resident on the plasmids of Pseudomonas putida strain H, Tn 5501 and Tn 5502 , are cryptic transposons of the Tn 3 family

Genes for (methyl)phenol degradation in Pseudomonas putida strain H (phl genes) are located on the plasmid pPGH1. Adjacent to the phl catabolic operon we identified a cryptic transposon, Tn5501, of the Tn3 family (class II transposons). The genes encoding the resolvase and the transposase are transcribed in the same direction, as is common for the Tn501 subfamily. The enzymes encoded by Tn5501, however, show only the overall homology characteristic for resolvases/integrases and transposases of Tn3-type transposons. Therefore it is likely that Tn5501 is not a member of one of the previously defined subfamilies. Inactivation of the conditional lethal sacB gene was used to detect transposition of Tn5501. While screening for transposition events we found another transposon integrated into sacB in one of the sucrose-resistant survivors. This element, Tn5502, is a composite transposon consisting of Tn5501 and an additional DNA fragment. It is flanked by inverted repeats identical to those of Tn5501 and the additional fragment is separated from the Tn5501 portion by an internal repeat (identical to the left terminal repeat). Transposition of phenol degradation genes could not be detected. Analysis of sequence data revealed that the phl genes are not located on a Tn5501-like transposon. Received: 21 July 1997 / Accepted: 7 July 1998     

细菌中介导多重耐药的Tn7转座子研究进展

转座子是介导细菌耐药性传播的重要可移动遗传元件。Tn7转座子与细菌耐药密切相关,其携带转座模块和Ⅱ类整合子系统。Tn7编码转座相关蛋白TnsABCDE进行“剪切-粘贴”机制转座,转座核心TnsABC也可与三链DNA或Cas-RNA复合物结合实现转座。近年来新发现了多种介导多重耐药的Tn7转座子,其在介导细菌抗生素、消毒剂和重金属抗性基因的获得、传播扩散等方面发挥了重要作用。本文综述了细菌中Tn7转座子的遗传结构、转座机制、流行以及新发现的介导多重耐药的Tn7转座子,以期为细菌中Tn7转座子的深入研究提供参考。     

Cloning and characterization of Tc1 family-derived PPTN related transposons from ridged-eye flounder (Pleuronichthys cornutus) and inshore hagfish (Eptatretus burgeri)

The Tc1 transposable element is the most widespread family among animal transposon and these elements consist of an inverted repeat (IR) sequence flanking a transposase gene that belongs to Class II type transposon, which is highly conserved in the genome of the nematode C. elegans. In order to characterize Tc1-like transposable elements from several fishes, PPTN (Tc1-like transposon was isolated from Pleuronectes platessa, marine flatfish species) IR primer-specific amplified elements were cloned from the genomic DNA of several fishes. Transposable elements were found in ridged-eye flounder (Pleuronichthys cornutus) and inshore hagfish (Eptatretus burgeri) and named as PCTN and EBTN, respectively. Amino acid sequence alignment and phylogenetic analysis confirmed that the PPTN-like transposons belonged to the Tc1 superfamily of transposons, but they comprised a unique clade of Tc1-like transposons. The IR-PCR analysis using MMTS-IR and PPTN-IR specific primers from Paralichthys olivaceus (Paralichthyidae), Paraplagusia japonica (Cynoglossidae), P. yokohamae (Pleuronectidae) and Pagurus cornutus (Pleuronectidae) (within the same order, Pleuronectiformes but different families) exhibited mutually exclusive distribution of Tc1 family-derived PPTN and MMTS-like transposons in these fish genomes. These results indicate that Tc1 family-derived PPTN and MMTS related Tc1-like transposable elements have uniquely evolved in piscine genome, and can be used as phylogenetic markers for the distribution of subfamilies of Tc1-like transposon and the involvement of horizontal and vertical transmission in the evolution of fish genome.     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

Transposition of a DNA fragment flanked by two inverted Tn1 sequences

The 32 Md fragment (derived from plasmid RP4::Tn1) carrying the Km^r gene and flanked by two inverted Tn1 elements is capable of recA-independent translocation to other plasmids. We designated this new transposon Tn1755. In various crosses, frequencies of Tn1755 transposition to plasmids Co1B-R3, R15 and F′ColVBtrp varied from 2.5 to 90% of the frequencies of Tn1 transposition. Tn1755 can integrate into various sites of the recipient plasmids. We failed to observe transposition of another RP4::Tn1 fragment flanked by two opposingly oriented Tn1 transposons and harboring the Tc^r gene. Presumably, to form a new transposable structure, other features must also be of importance.     

Heteromeric transposase elements: generators of genomic islands across diverse bacteria

Horizontally acquired genetic information in bacterial chromosomes accumulates in blocks termed genomic islands. Tn7‐like transposons form genomic islands at a programmed insertion site in bacterial chromosomes, attTn7. Transposition involves five transposon‐encoded genes (tnsABCDE) including an atypical heteromeric transposase. One transposase subunit, TnsB, is from the large family of bacterial transposases, the second, TnsA, is related to endonucleases. A regulator protein, TnsC, functions with different target site selecting proteins to recognize different targets. TnsD directs transposition into attTn7, while TnsE encourages horizontal transmission by targeting mobile plasmids. Recent work suggests that distantly related elements with heteromeric transposases exist with alternate targeting pathways that also facilitate the formation of genomic islands. Tn6230 and related elements can be found at a single position in a gene of unknown function (yhiN) in various bacteria as well as in mobile plasmids. Another group we term Tn6022‐like elements form pathogenicity islands in the Acinetobacter baumannii comM gene. We find that Tn6022‐like elements also appear to have an uncharacterized mechanism for provoking internal transposition and deletion events that serve as a conduit for evolving new elements. As a group, heteromeric transposase elements utilize diverse target site selection mechanisms adapted to the spread and rearrangement of genomic islands.     

Transposon Tn5 as an identifiable marker in rhizobia: Survival and genetic stability of Tn5 mutant bean rhizobia under temperature stressed conditions in desert soils

Five transposon Tn5 insertion mutants of a beanRhizobium strain (Rhizobium leguminosarum b. v.phaseoli) were used in an ecological study to evaluate the extent to which transposon Tn5 was stable to serve as an identifiable marker in rhizobia under a high temperature stress condition in two Sonoran Desert soils. All the mutants possessed single chromosomal insertions of the transposon. In both soils, under the temperature stress conditions that were employed (40°C), both wild type and mutant populations possessing functional transposable elements declined rapidly. After 12 days, mutant cells, when screened using the Tn5 coded antibiotic resistance markers, were significantly less in number than when they were screened using only their intrinsic antibiotic resistance markers. There were no significant differences in numbers between the mutant cell population and the wild type when the mutant cells were screened using only the intrinsic antibiotic resistance markers. DNA-DNA hybridizations using a probe indicated neither deletion nor transposition of the transposable element. The results indicate that transposon DNA sequences are present within cells under high temperature stress conditions, but kanamycin/neomycin resistance is not expressed by some of these cells, suggesting that Tn5 undergoes a possible functional inactivation under these conditions. The possible implications of these findings are discussed.     

The transposable elements resident on the plasmids of Pseudomonas putida strain H, Tn5501 and Tn5502, are cryptic transposons of the Tn3 family

Genes for (methyl)phenol degradation in Pseudomonas putida strain H (phl genes) are located on the plasmid pPGH1. Adjacent to the phl catabolic operon we identified a cryptic transposon, Tn5501, of the Tn3 family (class II transposons). The genes encoding the resolvase and the transposase are transcribed in the same direction, as is common for the Tn501 subfamily. The enzymes encoded by Tn5501, however, show only the overall homology characteristic for resolvases/integrases and transposases of Tn3-type transposons. Therefore it is likely that Tn5501 is not a member of one of the previously defined subfamilies. Inactivation of the conditional lethal sacB gene was used to detect transposition of Tn5501. While screening for transposition events we found another transposon integrated into sacB in one of the sucrose-resistant survivors. This element, Tn5502, is a composite transposon consisting of Tn5501 and an additional DNA fragment. It is flanked by inverted repeats identical to those of Tn5501 and the additional fragment is separated from the Tn5501 portion by an internal repeat (identical to the left terminal repeat). Transposition of phenol degradation genes could not be detected. Analysis of sequence data revealed that the phl genes are not located on a Tn5501-like transposon.     

Widespread occurrence of the Tc1 transposon family: Tc1-like transposons from teleost fish

We characterized five transposable elements from fish: one from zebrafish (Brachydanio rerio), one from rainbow trout (Salmo gairdneri), and three from Atlantic salmon (Salmo salar). All are closely similar in structure to the Tel transposon of the nematode Caenorhabditis elegans. A comparison of 17 Tc1-like transposons from species representing three phyla (nematodes, arthropods, and chordates) showed that these elements make up a highly conserved transposon family. Most are close to 1.7 kb in length, have inverted terminal repeats, have conserved terminal nucleotides, and each contains a single gene encoding similar poly peptides. The phylogenetic relationships of the transposons were reconstructed from the amino acid sequences of the conceptual proteins and from DNA sequences. The elements are highly diverged and have evidently inhabited the genomes of these diverse species for a long time. To account for the data, it is not necessary to invoke recent horizontal transmission.     

Genetic construction of PCB degraders

Genetic construction of recombinant strains with expanded degradative abilities may be useful for bioremedation of recalcitrant compounds, such as polychlorinated biphenyls (PCBs). Some degradative genes have been found either on conjugative plasmids or on transposons, which would facilitate their genetic transfer. The catabolic pathway for the total degradation of PCBs is encoded by two different sets of genes that are not normally found in the same organism. ThebphABCD genes normally reside on the chromosome and encode for the four enzymes involved in the production of benzoate and chlorobenzoates from the respective catabolism of biphenyl and chlorobiphenyls. The genes encoding for chlorobenzoate catabolism have been found on both plasmids and the chromosome, often in association with transposable elements. Ring fission of chlorobiphenyls and chlorobenzoates involves themeta-fission pathway (3-phenylcatechol 2,3-dioxygenase) and theortho-fission pathway (chlorocatechol 1,2-dioxygenase), respectively. As the catecholic intermediates of both pathways are frequently inhibitory to each other, incompatibilities result. Presently, all hybrid strains constructed by in vivo matings metabolize simple chlorobiphenyls through complementary pathways by comprising thebph, benzoate, and chlorocatechol genes of parental strains. No strains have yet been verified which are able to utilize PCBs having at least one chlorine on each ring as growth substrates. The possible incompatibilities of hybrid pathways are evaluated with respect to product toxicity, and the efficiency of both in vivo and in vitro genetic methods for the construction of recombinant strains able to degrade PCBs is discussed.     

Transfer of Tn5385, a Composite, Multiresistance Chromosomal Element from Enterococcus faecalis

Tn5385 is a ca. 65-kb element integrated into the chromosomes of clinical Enterococcus faecalis strains CH19 and CH116. It confers resistance to erythromycin, gentamicin, mercuric chloride, streptomycin, tetracycline-minocycline, and penicillin via β-lactamase production. Tn5385 is a composite structure containing regions previously found in staphylococcal and enterococcal plasmids. Several transposons and transposon-like elements within Tn5385 have been identified, including conjugative transposon Tn5381, composite transposon Tn5384, and elements indistinguishable from staphylococcal transposons Tn4001 and Tn552. The divergent regions of Tn5385 are linked by a series of insertion sequence (IS) elements (IS256, IS257, and IS1216) of staphylococcal and enterococcal origin. The ends of Tn5385 consist of directly repeated copies of enterococcal IS1216. Within the chromosomes of strains CH19 and CH116, Tn5385 has interrupted an open reading frame with substantial homology to previously described alkyl hydrogen peroxide reductase genes. Segments of this open reading frame in both CH19 and CH116 have been deleted, but the amount of deleted DNA differs for the two insertions. Transfer of Tn5385 from both donors into E. faecalis recipients occurs at a low frequency. Two types of transconjugants have been identified. In one type, the target alkyl hydrogen peroxide reductase open reading frame has been deleted, and sequences flanking Tn5385 in the respective donors are carried over to the transconjugants. These data suggest that the mechanism of Tn5385 insertion into the recipient chromosome in these transconjugants was recombination across flanking regions in the donors and homologous sequences in the recipients. The second type of transconjugant appears to have resulted from excision of Tn5385 from the CH19 chromosome by recombination across the terminal IS1216 elements and insertion into the recipient chromosome by recombination across Tn5381 (within Tn5385) and a previously transferred Tn5381 copy in the recipient chromosome. These data confirm that Tn5385 is a composite structure with genetic material from diverse genera and suggest that it is a functional transposon. They also suggest that chromosomal recombination is a mechanism of genetic exchange in enterococci.     

Integrons,transposons and IS elements promote diversification of multidrug resistance plasmids and adaptation of their hosts to antibiotic pollutants from pharmaceutical companies

Plasmids are important vehicles for the dissemination of antibiotic resistance genes (ARGs) among bacteria by conjugation. Here, we determined the complete nucleotide sequences of nine different plasmids previously obtained by exogenous plasmid isolation from river and creek sediments and wastewater from a pharmaceutical company. We identified six IncP/P-1ε plasmids and single members of IncL, IncN and IncFII-like plasmids. Genetic structures of the accessory regions of the IncP/P-1ε plasmids obtained implied that multiple insertions and deletions had occurred, mediated by different transposons and Class 1 integrons with various ARGs. Our study provides compelling evidence that Class 1 integrons, Tn402-like transposons, Tn3-like transposons and/or IS26 played important roles in the acquisition of ARGs across all investigated plasmids. Our plasmid sequencing data provide new insights into how these mobile genetic elements could mediate the acquisition and spread of ARGs in environmental bacteria.     

Evolutionary relationship between Tn21-like elements and pBP201, a plasmid from Klebsiella pneumoniae mediating resistance to gentamicin and eight other drugs

Summary We have characterized pBP201 one of the plasmids from a collection of 46 strains producing adenylyltransferase ANT (2) (Schmidt 1984). It confers resistance to sulphonamides and produces aminoglycoside adenylyltransferases AAD (3) and ANT (2) and -lactamase TEM-1. Plasmid pBP201 has a size of 24.8 kilobases (kb) and contains TnA and a Tn21-related element, Tn4000, with deletions in mer and the termini and a substitution at tnpR. In complementation assays with transposition-deficient mutants of Tn21 the element in pBP201 appears to be TnpA⁺ but TnpR^-. It represents a naturally occurring defective transposon. The sequence organization of pBP201 has been compared with that of Tn21-related elements such as Tn2410, Tn2603, Tn2424, Tn1696, and Tn4000. In these transposons the integration sites of resistance genes cat, bla, aacA, aacC or aadB have been identified at two preferential locations; these are at the termini of the streptomycin resistance gene aadA. Two additional sites have been localized in the Tn21 backbone to the right of the mer operon and at res (internal resolution site) and are probably involved in the evolution of these elements. Based on these results a model for the possible genealogy of class II transposons is presented.     

Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli

Summary The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).     

The phylogenetic distribution of a transposable dioxygenase from the Niagara River watershed

Horizontal gene transfer in the Bacteria has been demonstrated to occur under natural conditions. The ecological impact of gene transfer events depends on the new genetic material being expressed in recipient organisms, and on natural selection processes operating on these recipients. The phylogenetic distribution of cbaAB genes for chlorobenzoate 3,4-(4,5)-dioxygenase, which are carried within Tn5271 on the IncPβ plas-mid pBRC60, was investigated using isolates from freshwater microcosms and from the Niagara River watershed. The latter included isolates from surface water, groundwater and bioremediation reactor samples. The cbaAB genes have become integrated, through interspecific transfer, primarily into species of the β Proteobacteria (44/48 isolates). Only four isolates, identified as Pseudomonas fluorescens (3/48) and Xanthomonas maltophilia (1/48), belonged to the Γ Proteobacteria, despite the observation that pBRC60 was capable of mobilizing these genes into a wide range of β and Γ Proteobacteria in the laboratory. The natural host range correlated with the distribution of the meta-ring-fission pathway for metabolism of protocatechuates formed when the cbaAB genes were expressed (45/48 isolates). We proposed the hypothesis that natural selection has favoured recipients that successfully integrate the activity of the transferred dioxygenase with the conserved meta ring-fission pathway. The hypothesis was tested by transferring a plasmid construct containing the cbaAB genes into type strains representative of the β and γ Proteobacteria. The concept of applying mobile catabolic genes to probe the phylogenetic distribution of compatible degradative pathways is discussed.     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

Summary The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 by away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam ^– mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.     

Use of the multipurpose transposon Tn<Emphasis Type="Italic">KPK2</Emphasis> for the mutational analysis of chromosomal regions upstream and downstream of the<Emphasis Type="Italic"> sipF</Emphasis> gene in<Emphasis Type="Italic"> Bradyrhizobium japonicum</Emphasis>

The DNA regions upstream and downstream of the Bradyrhizobium japonicum gene sipF were cloned by in vivo techniques and subsequently sequenced. In order to study the function of the predicted genes, a new transposon for in vitro mutagenesis, TnKPK2, was constructed. This mutagenesis system has a number of advantages over other transposons. TnKPK2 itself has no transposase gene, making transposition events stable. Extremely short inverted repeats minimize the length of the transposable element and facilitate the determination of the nucleotide sequence of the flanking regions. Since the transposable element carries a promoterless phoA reporter gene, the appearance of functional PhoA fusion proteins indicates that TnKPK2 has inserted in a gene encoding a periplasmic or secreted protein. Although such events are extremely rare, because the transposon has to insert in-frame, in the correct orientation, and at an appropriate location in the target molecule, a direct screening procedure on agar indicator plates permits the identification of candidate clones from large numbers of colonies. In this study, TnKPK2 was used for the construction of various symbiotic mutants of B. japonicum. One of the mutant strains, A2-10, which is defective in a gene encoding a protein that comigrates with bacterioferritin (bcpB), was found to induce the formation of small and ineffective nodules.Communicated by A. Kondorosi