tnpA Genes Research Articles

Prevalence of Carbapenem Resistance Genes among Acinetobacter baumannii Isolated from a Teaching Hospital in Taiwan.

The problem of antibiotic-resistant strains has become a global public issue; antibiotic resistance not only limits the choice of treatments but also increases morbidity, mortality and treatment costs. The multi-drug resistant Acinetobacter baumannii is occurring simultaneously in hospitals and has become a major public health issue worldwide. Although many medical units have begun to control the use of antibiotics and paid attention to the issue of drug resistance, understanding the transmission pathways of clinical drug-resistant bacteria and drug-resistant mechanisms can be effective in real-time control and prevent the outbreak of antibiotic-resistant pathogens. In this study, a total of 154 isolates of Acinetobacter baumannii obtained from Chia-Yi Christian Hospital in Taiwan were collected for specific resistance genotyping analysis. Ten genes related to drug resistance, including blaOXA-51-like, blaOXA-23-like, blaOXA-58-like, blaOXA-24-like, blaOXA-143-like, tnpA, ISAba1, blaPER-1, blaNDM and blaADC, and the repetitive element (ERIC2) were selected for genotyping analysis. The results revealed that 135 A. baumannii isolates (87.6%) carried the blaOXA-51-like gene, 4.5% of the isolates harbored the blaOXA-23-like gene, and 3.2% of the isolates carried the blaOXA-58-like gene. However, neither the blaOXA-24-like nor blaOXA-143-like genes were detected in the isolates. Analysis of ESBL-producing strains revealed that blaNDM was not found in the test strains, but 38.3% of the test isolates carried blaPER-1. In addition, blaADC, tnpA and ISAba1genes were found in 64.9%, 74% and 93% of the isolates, respectively. Among the carbapenem-resistant strains of A. baumannii, 68% of the isolates presenting a higher antibiotic resistance carried both tnpA and ISAba1 genes. Analysis of the relationship between their phenotypes (antibiotic resistant and biofilm formation) and genotypes (antibiotic-resistant genes and biofilm-related genes) studied indicated that the bap, ompA, ISAba1and blaOXA-51 genes influenced biofilm formation and antibiotic resistance patterns based on the statistical results of a hierarchical clustering dendrogram. The analysis of the antibiotic-resistant mechanism provides valuable information for the screening, identification, diagnosis, treatment and control of clinical antibiotic-resistant pathogens, and is an important reference pointer to prevent strains from producing resistance.

Antimicrobial resistome and mobilome in the urban river affected by combined sewer overflows and wastewater treatment effluent.

The dissemination of antimicrobial resistance in the environment is an emerging global health problem. Wastewater treatment effluent and combined sewer overflows (CSOs) are major sources of antimicrobial resistance in urban rivers. This study aimed to clarify the effect of municipal wastewater treatment effluent and CSO on antimicrobial resistance genes (ARGs), mobile gene elements, and the microbial community in an urban river. The ARG abundance per 16S-based microbial population in the target river was 0.37-0.54 and 0.030-0.097 during the CSO event and dry weather, respectively. During the CSO event, the antimicrobial resistome in the river shifted toward a higher abundance of ARGs to clinically important drug classes, including macrolide, fluoroquinolone, and β-lactam, whereas ARGs to sulfonamide and multidrug by efflux pump were relatively abundant in dry weather. The abundance of intI1 and tnpA genes were highly associated with the total ARG abundance, suggesting their potential application as an indicator for estimating resistome contamination. Increase of prophage during the CSO event suggested that impact of CSO has a greater potential for horizontal gene transfer (HGT) via transduction. Consequently, CSO not only increases the abundance of ARGs to clinically important antimicrobials but also possibly enhances potential of HGT in urban rivers.

Resistance of Klebsiella pneumoniae Strains Carrying bla NDM-1 Gene and the Genetic Environment of bla NDM-1.

ObjectiveRegional dissemination is the major cause of the widespread prevalence of a plasmid-encoding NDM-1 enzyme. We investigated the drug resistance, joint efficiency, and gene environment of a Klebsiella pneumoniae strain carrying blaNDM–1 gene.Materials and MethodsCarbapenem-non-susceptible strains were analyzed using the VITEK 2 Compact. Strains carrying blaNDM–1 were identified using polymerase chain reaction and sequencing. Antimicrobial susceptibility testing and plasmid conjugation experiments were then conducted. Strains carrying blaNDM–1 were subjected to Southern blot analysis. After the gene mapping of blaNDM–1, library construction, and sequencing, plasmids were subsequently spliced and genotyped using the software Glimmer 3.0, and then analyzed using Mauve software.ResultsAmong 1735 carbapenem-non-susceptible strains, 54 strains of blaNDM–1-positive bacteria were identified, which consisted of 44 strains of K. pneumoniae, 8 strains of Acinetobacter baumannii and 2 strains of Escherichia coli. Strains carrying blaNDM–1 had a resistance rate of more than 50% in most antibiotics. Plasmid conjugation between strains carrying blaNDM–1 and E. coli strain J53 had a success rate of 50%. Southern blot analysis indicated that each strain had multiple plasmids containing blaNDM–1. Among the five plasmids containing blaNDM–1 in K. pneumoniae for sequencing, two plasmids with complete sequences were obtained. The findings were as follows: (i) The p11106 and p12 plasmids were highly similar to pNDM-BTR; (ii) the p11106 and p12 plasmids showed differences in the 20–30 kb region (orf00032–orf00043) from the other six plasmids; and (iii) blaNDM–1 was located at orf00037, while ble was found at orf00038. Two tnpA genes were located in the upstream region, and orf00052 (tnpA) in the 36 kb region was in the downstream sequence.ConclusionblaNDM–1-containing bacteria exhibit multidrug resistance, which rapidly spreads and is transferred through efficient plasmid conjugation; the multidrug resistance of these bacteria may be determined by analyzing their drug-resistant plasmids. The presence of ble and tnpA genes suggests a possible hypothesis that blaNDM–1 originates from A. baumannii, which is retained in K. pneumoniae over a long period by transposition of mobile elements.

Inactivation of antibiotic resistant Escherichia coli and degradation of its resistance genes by glow discharge plasma in an aqueous solution

Emerging contaminants such as antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) are becoming a global environmental problem. In this study, the glow discharge plasma (GDP) was applied for degrading antibiotic resistant Escherichia coli (E. coli) with resistance genes (tetA, tetR, aphA) and transposase gene (tnpA) in 0.9% sterile saline. The results showed that GDP was able to inactivate the antibiotic resistant E. coli and remove the ARGs and reduce the risk of gene transfer. The levels of E. coli determined by 16S rRNA decreased by approximately 4.7 logs with 15 min of discharge treatment. Propidium monoazide - quantitative polymerase chain reaction (PMA-qPCR) tests demonstrated that the cellular structure of 4.8 more logs E. coli was destroyed in 15 min. The reduction of tetA, tetR, aphA, tnpA genes was increased to 5.8, 5.4, 5.3 and 5.5 logs with 30 min discharge treatment, respectively. The removal of ARGs from high salinity wastewater was also investigated. The total abundance of ARGs was reduced by 3.9 logs in 30 min. Scavenging tests indicated that hydroxyl radicals (·OH) was the most probable agents for bacteria inactivation and ARGs degradation. In addition, the active chlorine (Cl· and Cl2) which formed during the discharge may also contribute to the inactivation and degradation.

Silicon dioxide nanoparticles have contrasting effects on the temporal dynamics of sulfonamide and β-lactam resistance genes in soils amended with antibiotics

Nanoparticles (NPs) and antibiotic resistant genes (ARGs), as emerging environmental contaminants, have been reported to be accumulated in the soil environment. The use of NPs have raised increasing concerns about their environmental impacts, but the combined effect of NPs and antibiotics on ARGs remains less understood. Here, we established laboratory microcosms to explore the impacts of different concentrations of SiO2 NPs on β-lactam and sulfonamide resistance genes in soils amended with β-lactam or sulfonamide. Illumina sequencing and quantitative PCR revealed that the addition of NPs increased the bacterial community diversity but had no significant effects on the bacterial abundance. Moreover, NPs and sulfonamide jointly increased the abundances of sulfonamide resistance genes, while the exposure of NPs and β-lactam decreased β-lactam resistance genes. The detected ARGs were associated closely with two mobile genetic elements (MGEs, the tnpA and intI1 genes), indicating that MGEs may contribute to the dissemination of ARGs. Correlation analysis indicated the shifts in potential bacterial hosts and the frequency of horizontal gene transfer were important factors explaining the patterns of ARGs. Furthermore, structural equation models indicated that NPs exposure decreased the abundances of β-lactam resistance genes by driving changes in bacterial community and MGEs, whereas the increased abundances of sulfonamide resistance genes were mainly associated with the bacterial community, diversity and MGEs mediated by NPs and antibiotics. These results suggested that the combined effects of NPs and antibiotics on soil bacterial resistance were different due to the types of antibiotics.

The importance of Helicobacter pylori tnpA, tnpB, and cagA genes in various gastrointestinal diseases

The cag (cytotoxin-associated gene) pathogenicity island (cagPAI) is one of the major virulence determinants of Helicobacter pylori (H. pylori). The purpose of this study was to investigate the association of the three genes (tnpA, tnpB, and cagA) in H. pylori isolated from Azerbaijani patients with the different gastrointestinal disease. A total of 362 gastric biopsies were collected from hospitals of Tabriz University of Medical Sciences, and were cultured on Brucella agar. The tnpA, tnpB, and cagA genes were detected by PCR. Of the total 264 H. pylori isolates, tnpA, tnpB, and cagA genes were detected in 120 (45.5%), 56 (21.2%) and 172 (65.2%), respectively. A significant association between tnpA and tnpB genes and clinical outcomes were found (P < 0.05). The cagA status was not related to clinical outcomes in our subjects. The predominant genotype among cag-PAI is the cagA. The prevalence of tnpA, tnpB, and cagA genes are high in patients with gastric cancer, and a significant association is revealed between tnpA and tnpB with gastric cancer.

Prevalence of the fosfomycin-resistance determinant, fosB3, in Enterococcus faecium clinical isolates from China.

In order to investigate the prevalence of fosfomycin-resistance (fos) determinants in Enterococcus faecium, clinical strains were collected from hospitals throughout China between January 2008 and December 2009. Antimicrobial susceptibility testing was performed, after which the fos genes in all isolates and van genes in vancomycin-resistant isolates were characterized by PCR and sequencing. Conjugation experiments were carried out with fosB-positive E. faecium, DNA fragments flanking the fosB3 gene were sequenced and the genetic environment of fosB3 was analysed. Fosfomycin-resistant E. faecium (FREF) strains were characterized further by multilocus sequence typing (MLST) and PFGE. Among 145 E. faecium clinical isolates, 10 were resistant to fosfomycin with MICs>1024 mg l(-1) including six vancomycin-resistant strains of which five were vanA-positive and the sixth vanM-positive. All ten FREF strains harboured the fosB3 gene. Fosfomycin resistance and fosB3 could be transferred by conjugation from nine isolates. The fosB3 and tnpA genes were located in a circular DNA intermediate in all FREF strains and reversely inserted into vanA transposon Tn1546 in four vanA-positive FREF isolates. Ten different PFGE types and seven MLST types were found among the ten fosB3-positive isolates, while all strains belonged to the common clonal complex CC17. In conclusion, the transferable fosfomycin-resistance determinant fosB3 plays an important role in E. faecium resistance to fosfomycin in China.

Clinical relevance of the cagA, tnpA and tnpB genes in Helicobacter pylori

BackgroundNumerous proteins have been proposed as virulence factors for the gram negative gastric bacterium Helicobacter pylori but only for a few this has unequivocally been demonstrated. The aim of the current study was to evaluate the association of the putative virulence factors tnpA and tnpB (no cagA) with H. pylori associated gastroduodenal diseases.MethodsA PCR based assay was used to determine the presence of the tnpA and tnpB genes, as well as of cagA, in 360H. pylori strains isolated from H. pylori infected patients.ResultsOf 360H. pylori culture positive patients (196 men, 164 women; average age 42.1 years (range 17–73), 95 had gastritis, 92 had gastric ulcers, 108 had duodenal ulcers, and 65 had gastric cancer. Using the gastritis group as a reference a significantly aberrant gene distribution was observed for the tnpA (Relative risk: 1.45; 95% CI 1.04-1.93), the cagA (Relative risk: 1.81; 95% CI 1.44-2.29), but not the tnpB gene in the gastric cancer group.ConclusionsThe increased incidence of the tnpA gene in gastric cancer patients suggests a role of the tnpA gene in the development of H. pylori induced gastric cancer.

Fosfomycin resistance among vancomycin-resistant enterococci owing to transfer of a plasmid harbouring the fosB gene

The presence and characterisation of plasmid-mediated fosfomycin resistance determinants were investigated among 45 clinical vancomycin-resistant enterococci (VRE) isolated in Zhejiang Province, China. In total, 19 VRE were resistant to fosfomycin, of which 18 isolates had conjugative fosfomycin resistance and were positive for fosB. No reported fos genes were detected in the remaining isolate. Among the 18 fosB-carrying isolates, the fosB gene was always flanked by tnpA, suggesting the same novel fosB transposon. In 10 of the 18 fosB-carrying isolates, the fosB and tnpA genes were found reversely inserted in the vanA transposon Tn1546. In the remaining eight isolates the fosB and vanA genes were located on different plasmids. These findings indicate that acquisition of the conjugative plasmid harbouring the novel fosB transposon (ISL3-like transposon) and the Tn1546-like transposon (containing vanA and fosB) may explain, at least in part, the recent increase in fosfomycin-resistant Enterococcus faecium in China.

Novel Vancomycin-Resistance Transposon, Plasmid Replicon Types, and Virulence Factors of Vancomycin-ResistantEnterococciin Zhejiang, China

Forty-seven vancomycin-resistant Enterococcus (VRE) strains were isolated from clinical samples in 13 Zhejiang hospitals and fecal samples from ICU patients in a large teaching hospital in China. No VRE isolates were detected in healthy human subjects. CC17 was the main clonal complex in clinical Enterococcus faecium isolates but not in isolates from healthy human subjects. Novel vancomycin-resistance transposons were detected among VRE strains. This is the first report demonstrating insertion of tnpA and fosB genes in the vanRS-vanH intergenic region of Tn1546 leading to coresistance to vancomycin and fosfomycin. The four plasmid replicon types (pRUM, pRE25, pEF418, and pB82) were more common in VRE isolates, suggesting their association with vancomycin resistance and nosocomial transmission. The prevalence rate of vancomycin-resistant Staphylococcus aureus-related Inc18-like plasmid, pIP501, in VRE was 21.3%. The prevalence of the esp gene among VRE isolates was high (76.6%). In several VRE strains, the esp and hyl genes were cotransferred with the vanA gene by conjugation. Although the frequency of VRE is low in Chinese hospitals, its association with virulence determinants, the vancomycin-resistance transposon with other resistance gene insertions or plasmids may lead to multidrug resistance and the evolution of pathogenic VRE.

Bla CTX-M-65 is carried by a Tn1722-like element on an IncN conjugative plasmid of ST131 Escherichia coli

Escherichia coli clinical isolate WCE307 was found to belong to phylogroup B2, O25b and sequence type (ST) 131 and had blaCTX-M-65, which was carried by an IncN conjugative plasmid, pWCE307. On pWCE307, the ISEcp1Δ-blaCTX-M-65-IS903D-ironΔ structure was located in a Tn1722-like element flanked by 5 bp direct target repeats. This context was highly similar to that on pKC396, an IncN plasmid from E. coli isolate KC396 of ST131 from Germany. The Tn1722-like elements carrying blaCTX-M-65 on pWCE307 and pKC396 are likely to be hybrids of Tn1722 and Tn5051, resulting from double crossover at the res sites and the tnpA genes. The left end of the Tn1722-like elements was partially missing. As ISEcp1 and Tn1722 were both incomplete, blaCTX-M-65 might have been trapped in this context. A plasmid multilocus sequence typing (pMLST) scheme with five targets, repN, stbB, traI, traB and the korB-orfI spacer region, was developed for IncN plasmid typing. pMLST revealed that the five alleles of pWCE307 and pKC396 were identical, indicating that WCE307 and KC396 are likely to have originated very recently from a common strain, suggesting the international spread of blaCTX-M-65 mediated by ST131 E. coli.

Physical linkage of Tn3 and part of Tn1721 in a tetracycline and ampicillin resistance plasmid from Salmonella Typhimurium

The complete nucleotide sequence of the 12 656 bp plasmid pFPTB1 from Salmonella enterica subsp. enterica serovar Typhimurium, which mediates resistance to tetracyclines and ampicillin, was determined. The plasmid was analysed for potential reading frames and structural features indicative of transposons and transposon relics. Plasmid pFPTB1 was transformed into Escherichia coli JM109, overlapping restriction fragments were cloned into E. coli plasmid vectors and sequenced. In vitro susceptibility testing was carried out to confirm the resistance phenotype mediated by this plasmid. Plasmid pFPTB1 contains a complete Tn3-like transposon of 4950 bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, a novel gene for ampicillin resistance bla(TEM-135), and the right terminal repeat. Immediately downstream, the terminal 5215 bp at the right end of a Tn1721-like transposon, including the right terminal repeat, a truncated transposase gene, as well as the genes tet(A) and tetR for tetracycline resistance, were detected. A 5 bp direct repeat, TAAAA, was seen immediately upstream of the Tn3 part and immediately downstream of the Tn1721 part. Plasmid pFPTB1 also carries a replication region similar to that of the Klebsiella pneumoniae plasmid pJHCMW1. Plasmid pFPTB1 is one of the few completely sequenced plasmids from S. Typhimurium and harbours a novel transposon-like structure consisting of a Tn3-related part containing the bla(TEM-135) gene for ampicillin resistance and a Tn1721-related part containing the tetR-tet(A) genes for tetracycline resistance.

Tn 5044, a novel Tn 3 family transposon coding for temperature-sensitive mercury resistance

We report the discovery and characterization of the mercury resistance transposon, Tn 5044, from a Xanthomonas strain from the Kamchatka peninsula. In addition to the standard set of merRTPCAD genes, the mer operon of Tn 5044 contains a gene named sigY that encodes the RNA polymerase sigma factor-like protein. Mercury resistance determined by Tn 5044 is expressed at low (30 °C) but not at elevated temperatures (37 °C). None of the mer operon genes downstream of merA is responsible for the temperature-sensitive mercury resistance. The transposition module of Tn 5044 is closely related to those of Tn 1412 isolated from medical sources and to Tn 5563 and IS Xc5 from environmental sources. However, Tn 5044 differs from these transposons in that it has unusually long terminal inverted repeats. Sequence analysis of the transposase ( tnpA) genes places Tn 5044 and its close relatives into the Tn 3 subgroup of the Tn 3 family. However, the orientation of their resolvase and transposase genes is unusual for the Tn 3 family: tnpR is proximal to the end of the transposon, while divergently transcribed tnpA is oriented inwardly. The region between tnpA and tnpR genes is unusually large and contains two short conserved open reading frames. In addition to the complete set of sequence motifs common to true resolvases, the resolvase of Tn 5044 and its close relatives possesses a C-terminal extension showing no homology to known proteins. Despite this peculiarity, Tn 5044 resolvase can resolve cointegrates formed during Tn 5044 transposition controlled by tnpA. Genetic data suggest that the extension is essential for TnpR functioning.

Conservation of transposon structures in soil bacteria

The presence of Class II transposon genes related to Tn21 and Tn501, and their structural arrangements have been determined in a collection of 124 mercury resistant Gram-negative bacteria. Seventy-five of the 124 isolates contained a tnpA (transposase) gene related to Tn21 and Tn501 and in all 64 isolates that contained both tnpA genes and plasmids, the tnpA gene was plasmid borne. The relative orientation of the tnp genes and the mer operon (encoding mercury resistance) was also studied and revealed the presence of two distinct structural groups. The merC gene was present in 44 isolates. Five isolates were found to carry integrase genes and these contained inserted gene cassettes varying in size from 1.1 kb to 4.5 kb. The structural arrangement of the tnpA and tnpR (resolvase) genes within the isolates was determined. Sixty-nine of the 75 tnpA containing isolates had an arrangement of tnpA and tnpR genes similar to that found in the Tn21 subgroup of transposons. Four strains did not produce a PCR product using tnpR primers. The remaining two isolates had undetermined arrangements of tnpA and tnpR genes. No Tn3-like arrangements of tnpA and tnpR genes were present in these isolates, despite being detected in DNA extracted directly from the isolation sites. This suggests that Tn3-like arrangements of tnpA and tnpR genes are not commonly associated with mercury resistance genes in these environments. It was also apparent that the recombination events which have previously been observed in these strains have not significantly affected the diversity of the transposon structures within the isolates.

Conservation of transposon structures in soil bacteria

The presence of Class II transposon genes related to Tn 21 and Tn 501, and their structural arrangements have been determined in a collection of 124 mercury resistant Gram-negative bacteria. Seventy-five of the 124 isolates contained a tnpA (transposase) gene related to Tn 21 and Tn 501 and in all 64 isolates that contained both tnpA genes and plasmids, the tnpA gene was plasmid borne. The relative orientation of the tnp genes and the mer operon (encoding mercury resistance) was also studied and revealed the presence of two distinct structural groups. The merC gene was present in 44 isolates. Five isolates were found to carry integrase genes and these contained inserted gene cassettes varying in size from 1.1 kb to 4.5 kb. The structural arrangement of the tnpA and tnpR (resolvase) genes within the isolates was determined. Sixty-nine of the 75 tnpA containing isolates had an arrangement of tnpA and tnpR genes similar to that found in the Tn 21 subgroup of transposons. Four strains did not produce a PCR product using tnpR primers. The remaining two isolates had undetermined arrangements of tnpA and tnpR genes. No Tn 3-like arrangements of tnpA and tnpR genes were present in these isolates, despite being detected in DNA extracted directly from the isolation sites. This suggests that Tn 3-like arrangements of tnpA and tnpR genes are not commonly associated with mercury resistance genes in these environments. It was also apparent that the recombination events which have previously been observed in these strains have not significantly affected the diversity of the transposon structures within the isolates.

Abundance of Tn3, Tn21, and Tn501 transposase (tnpA) sequences in bacterial community DNA from marine environments.

The occurrence of the tnpA genes of the transposons Tn3, Tn21, and Tn501 was assessed in total bacterial community DNA isolated from different marine environments. The PCR technique was employed, together with most probable number statistics, to determine the abundance of the target tnpA genes. All three genes could be detected, and the Tn21 tnpA sequences predominated in all samples. The smallest amount of total community DNA in which the Tn21 tnpA sequence could be detected was 0.037 ng, and on the basis of our results, we estimated that this sequence was present in 1 of 1,000 to 10,000 bacteria. Hybridization of the PCR products with the respective tnpA probes verified the Tn21 and Tn501 tnpA sequences but only some of the Tn3 tnpA amplification products. The distribution and dissemination of transposons in natural bacterial communities are discussed.

TnMax--a versatile mini-transposon for the analysis of cloned genes and shuttle mutagenesis.

A series of Tn1721-based mini-transposons (TnMax) has been developed which are suitable for the targeting of cloned genes, shuttle mutagenesis, identification of protein export signals and the rescue of chromosomal markers. TnMax mini-Tn consist of two 38-bp inverted repeats (IR) flanking a resolution site (res), a suicide replication origin (orifd), and a gene conferring resistance to either chloramphenicol (TnMax1) or erythromycin (TnMax2). Other versions of TnMax (TnMax3 and TnMax4) carry a promoterless alkaline phosphatase-encoding gene (phoA') useful for the identification of protein export signals. The various mini-Tn cartridges are fused on the same plasmid with a common transposition control unit (TCU) comprising the tnpA (transposase) and tnpR (resolvase) genes under control of the inducible Ptrc promoter. This configuration causes a high frequency of transposition in Escherichia coli (approx. 10(-2) events/copy of target plasmid) and a minimum size of the mini-Tn. Like Tn1721, TnMax variants prefer random insertion into plasmids rather than into the E. coli chromosome, thus representing superb tools for the insertion mutagenesis of cloned genes. The TnMax-borne orifd simplifies the identification of targeted plasmids and facilitates shuttle mutagenesis, i.e., suicide delivery of a mutated gene with subsequent allelic replacement of a corresponding resident gene, in a variety of microorganisms. Rescue of such targeted chromosomal genes is easily accomplished by the excision of TnMax plus flanking segments using appropriate restriction endonucleases, ligation, and transformation of an E. coli host permissive for orifd-directed replication.

Construction of hybrid Tn501/Tn21 transposases in vivo: identification of a region of transposase conferring specificity of recognition of the 38-bp terminal inverted repeats.

In order to study the transposase enzymes of Class II prokaryotic transposable elements, we have constructed genes encoding hybrid transposase proteins. This was done by recombination in vivo between the tnpA genes of transposons Tn501 and Tn21. These hybrid genes can complement in trans a transposition-defective mutant of Tn501. The structures of the products of this complementation indicate whether the specificity of the hybrid transposase in recognising the 38 bp terminal inverted repeats is that of Tn501 or that of Tn21. The determinant of this specificity is in the N-terminal region of the transposase protein, between amino acids 28 and 216. The predicted amino acid sequences so far determined of transposases from the Class II family reveal an area of homology in this region.

Tn2501, a component of the lactose transposon Tn951, is an example of a new category of class II transposable elements.

Tn2501 is a cryptic class II transposon found as part of the lactose transposon Tn951. Insertional inactivation and nucleotide sequence analysis of Tn2501 allowed us (i) to localize the transposase (tnpA) and the resolvase (tnpR) genes as well as the resolution site (res) of Tn2501 and (ii) to compare Tn2501 with other well-known elements of the two subgroups of class II transposons (Tn3, gamma delta, Tn951, IS101; and Tn21, Tn501, Tn1721). The genetic organization of Tn2501 is similar to that of Tn3 with divergent transcription of the tnpA and tnpR genes away from the intervening res site. The tnpR gene of Tn2501 shows weak homology with that of Tn3 and even less with those of Tn21 and Tn501. However, the tnpA gene and the inverted repeat sequences of Tn2501 present more homology with those of Tn21 and Tn501 than with those of Tn3. Complementation studies showed that TnpA- mutants of Tn2501 can be complemented, at a low frequency, by the Tn21 transposase. None of the tested transposons complemented TnpR- mutants of Tn2501.

Analysis of the γδ res site : Sites required for site-specific recombination and gene expression

The γδ resolvase, product of the transposon's tnpR gene, mediates a site-specific recombination between two copies of γδ directly repeated on the same replicon. The site at which the recombination occurs, res, lies between the tnpA and tnpR genes. Within this same region lie the promoters for expression of tnpA and tnpR and the operator sites through which resolvase regulates the transcription. In order to determine the extent of the res site we have constructed in vitro a series of deletions that terminate within the tnpA- tnpR intergenic region, and have analyzed their effect on site-specific recombination. Our results indicate that a fully functional res site is about 115 base-pairs (bp) and runs from a position 15 bp to the left ( tnpA-proximal) side of the crossover point to 100 bp to the right. This segment corresponds precisely to the region defined by the three resolvase binding sites that we have demonstrated previously. Alterations of the nucleotide sequence around the crossover point indicate the importance of all or part of the central palindrome 5′ T-T-A-T-A-A within which the breakage-reunion reaction takes place. Taken together, our results strengthen our earlier conclusion that resolvase recognizes the 9 bp segment 5′ T-G-T-C-Y-N-N-T-A that occurs (in slightly degenerate form) in each half of the three binding sites. Using the deletions we have confirmed that the tnpA promoter spans the crossover site and have shown that the major tnpR promoter in vivo coincides with resolvase binding site II, although a second promoter for tnpR transcription lies across site I.