Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes.

A genetic determinant essential for hemolysin production by Listeria monocytogenes has been inactivated by insertion of transposon Tn916 into L. monocytogenes DNA. The transposon was transferred by means of conjugation of a streptomycin-resistant L. monocytogenes recipient strain with Streptococcus faecalis CG110 on membrane filters. Among the tetracycline-resistant transconjugants, mutants were detected which had lost hemolytic activity. When tested in a mouse model, these mutants appeared to have lost the virulence that characterizes the parental strain. An extracellular protein of 58,000 apparent molecular weight was eliminated in the nonhemolytic mutants. In some of the mutants, the decrease in the production of the 58,000-dalton protein was accompanied by the production of a new protein of 49,000 apparent molecular weight. Hemolytic revertants regained the hemolytic phenotype and virulence and produced the extracellular protein that characterizes the recipient strain. Hybridization studies with Tn916 DNA indicated that the transposon is present in EcoRI and HindIII fragments of the nonhemolytic mutants. Single copies of Tn916 were detected in the chromosomal DNA of two of the three nonhemolytic mutants that were studied in detail. In hemolytic, tetracycline-sensitive revertants Tn916 appeared to be completely excised from the chromosome.     

Behavior and target site selection of conjugative transposon Tn916 in two different strains of toxigenic Clostridium difficile

The insertion sites of the conjugative transposon Tn916 in the anaerobic pathogen Clostridium difficile were determined using Illumina Solexa high-throughput DNA sequencing of Tn916 insertion libraries in two different clinical isolates: 630ΔE, an erythromycin-sensitive derivative of 630 (ribotype 012), and the ribotype 027 isolate R20291, which was responsible for a severe outbreak of C. difficile disease. A consensus 15-bp Tn916 insertion sequence was identified which was similar in both strains, although an extended consensus sequence was observed in R20291. A search of the C. difficile 630 genome showed that the Tn916 insertion motif was present 100,987 times, with approximately 63,000 of these motifs located in genes and 35,000 in intergenic regions. To test the usefulness of Tn916 as a mutagen, a functional screen allowed the isolation of a mutant. This mutant contained Tn916 inserted into a gene involved in flagellar biosynthesis.     

Molecular characterization of a STreptococcus mutans mutant altered in environmental stress responses.

A mutant defective in aciduricity, GS5Tn1, was constructed following mutagenesis of Streptococcus mutans GS5 with the conjugative transposon Tn916. The mutant grew poorly at acidic pH levels and was sensitive to high osmolarity and elevated temperatures. These properties resulted from a single insertion of Tn916 into the GS5 chromosome, and the DNA fragment harboring the transposon was isolated into the cosmid vector, charomid 9-20. Spontaneous excision of Tn916 from the cosmid revealed that Tn916 inserted into a 8.6-kb EcoRI fragment. On the basis of the restriction analyses of insert fragments, it was found that Tn916 inserted into a 0.9-kb EcoRI-XbaI fragment. Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames, ORF1 and ORF2. By using a marker rescue strategy, a 6.0-kb HindIII fragment including the target site for Tn916 insertion and the 5' end of ORF1 was isolated and sequenced. The deduced amino acid sequences of ORF1 and ORF2 showed significant homology with the diacylglycerol kinase and Era proteins, respectively, from Escherichia coli. Nucleotide sequence analysis of the Tn916 insertion junction region in the GS5Tn1 chromosome revealed that the transposon inserted near the 3' terminus of ORF1. Restoration of ORF1 to its original sequence in mutant GS5Tn1 was carried out following transformation with integration vector pVA891 containing an intact ORF1. The resultant transformant showed wild-type levels of aciduricity as well as resistance to elevated temperatures and high osmolarity. These results suggest that the S. mutans homolog of diacylglycerol kinase is important for adaptation of the organism to several environmental stress signals.     

Characterization of conjugative transposon Tn5251 of Streptococcus pneumoniae

Abstract Tn5251 belongs to the Tn916-Tn1545 family of conjugative transposons (CT) and was found integrated into CT Tn5252 , to form the composite element Tn5253 of Streptococcus pneumoniae . We show that Tn5251 is identical in structure and size to Tn916 . DNA sequence analysis of a 4,419-bp segment containing the tet(M) gene showed that only 73 nucleotides out of 4,419 were different in the the two CT. Essentially all differences (66 / 73) were clustered in a 688-bp segment of tet(M) , which was 90% identical to Tn916 and 100% identical to the tet(M) genes of Tn1545 from S. pneumoniae and pOZ101 from Neisseria gonorrhoeae . DNA sequence analysis of the Tn5251/Tn5252 junction fragments allowed us (i) to determine Tn5251 termini, (ii) to define the 6-bp coupling sequences flanking the CT, and (iii) to infer the structure of the integration site ( attB ) of Tn5251 into Tn5252 . Conjugal transfer of Tn5251 independent from Tn5253 could not be detected, even if we could show excision and formation of Tn5251 circular intermediates at a level of 5.4 copies per 10⁶ chromosomes.     

Tn916 delta E: a Tn916 transposon derivative expressing erythromycin resistance

The tetracycline resistance gene encoded within the transposon Tn916 was replaced with the gene encoding erythromycin resistance from the plasmid pVA838. The derivative transposon of Tn916 was designated Tn916 delta E and was introduced into the Streptococcus faecalis chromosome by protoplast transformation. The conjugation/transposition functions of Tn916 delta E were similar to those observed for Tn916 in S. faecalis and Tn916 delta E was capable of self-conjugation at frequencies similar to those of other S. faecalis and Group B Streptococcus. This transposon will be useful for mutagenesis studies in gram-positive organisms, especially in those species where erythromycin resistance is a more desirable selectable marker.     

阴沟肠杆菌B8拮抗活性基因‘admA’及上游调控序列的克隆与功能鉴定

为了阐明水稻白叶枯病拮抗菌阴沟肠杆菌B8的作用机理,文章采用转座子标签法和染色体步移技术克隆到突变株B8B中Tn5插入位点周边拮抗活性相关片段,并通过基因敲除验证了获得的拮抗相关片段admA’上游调控序列的功能。以转座子中Kan抗性基因为标签,克隆了B8B菌株中Tn5插入位点左侧2 608 bp序列,经两次染色体步移得到Tn5插入位点右侧的2 354 bp序列。序列拼接后获得B8菌株拮抗相关序列4 611 bp的Bcontig。生物信息学分析显示该序列含有7个ORF,分别对应于3-磷酸甘油醛脱氢酶(GADPH)基因的部分编码区、2个LysR家族转录调控因子、弧菌假设蛋白VSWAT3-20465及成团泛菌(Pantoea agglomerans)andrimid生物合成基因簇的admA、admB和部分admC基因序列。B8B菌株Tn5插入分别位于同源于弧菌假设蛋白的anrPORF及‘admA’基因上游200 bp和894 bp处。通过同源重组技术,借助敲除质粒pMB-BG,获得拮抗活性消失的突变株B-1和B-3。结果表明B8B突变株中Tn5的插入可能影响了anrP蛋白的转录和表达,进而调控拮抗物质编码基因簇的生物合成。B8菌株中拮抗物质相关基因是类似于andrimid生物合成基因簇的基因家族,其上游调控区对该抗生素的生物合成具有重要的作用。     

The capsule polysaccharide synthesis locus of streptococcus pneumoniae serotype 14: Identification of the glycosyl transferase gene cps14E.

To identify a chromosomal region of Streptococcus pneumoniae serotype 14 involved in capsule polysaccharide synthesis, two strategies were used: (i) Tn916 mutagenesis, followed by the characterization of four unencapsulated mutants, and (ii) cross-hybridization with a capsule polysaccharide synthesis gene (cps) probe from S. agalactiae, which has a structurally similar capsule. The two approaches detected the same chromosomal region consisting of two adjacent EcoRI fragments. One of these EcoRI fragments was cloned and hybridized with a cosmid library. This resulted in clone cMKO2. A similar cosmid clone was obtained from an unencapsulated Tn916 mutant, Spnl4.H. Sequence analysis of the two cosmid clones revealed that in the Tn916 mutant, a gene, cps14E, which is homologous to other bacterial genes encoding glycosyl transferases, had been inactivated. An open reading frame immediately downstream of cps14E, designated cps14F, shows no significant homology with any known genes or proteins. A functional assay showed that cps14E encodes a glycosyl transferase and that a gene-specific knockout mutant lacks this enzyme activity, whereas inactivation of cps14F does not have this effect.     

Structural characterization of Tn916-like element in Streptococcus parauberis serotype II strains isolated from diseased Japanese flounder

Aims:  To screen for the existence and determine the structure of Tn 916 -like element in Streptococcus parauberis serotype II strains isolated from cultured Japanese flounder in western Japan.
Methods and Results:  In this study, the structure of Tn 916 -like element and the flanking regions were characterized by polymerase chain reaction (PCR) and inverse PCR, followed by cloning and sequencing. The Tn 916 -like element is 18 031 bp in length and composed of 22 ORFs. Southern blot hybridization analysis showed that the Hin cII-digested internal structures of Tn 916 -like elements yielded two patterns among S. parauberis serotype II strains. The flanking sequences were identical with the corresponding region of S. parauberis serotype I strain except for the addition of 6-bp coupling sequence (ATCATA) being adjacent to the upstream of the element.
Conclusion:  The Tn 916 -like element exhibited high homology (more than 99%) with Tn 916 observed in other streptococci and enterococci and was integrated in the same site of chromosome for all of the tested S. parauberis serotype II strains.
Significance and Impact of the Study:  The results indicate that the Tn 916- like element encoding tet (M) gene is present in all of the tested S. parauberis serotype II strains, which are disseminated in the flounder-culturing areas in western Japan.     

Characterization of Tn5386, a Tn916-related mobile element

In recent work, we described excision of a large genomic region from Enterococcus faecium D344R resulting from the interaction of Tn916 and a related transposon designated Tn5386. In the present study, we present and analyze the complete sequence of Tn5386. Tn5386 is 29,451 bp in length. Fifteen of its 30 open reading frames are analogous to ORFs found in Tn916. Significant differences include a series of ORFs with homology to lantibiotic immunity genes in the same location where tetM is found in Tn916, insertion of a Group II intron and an ORF with similarities to previously described surface exposed collagen adhesion proteins. Our results indicate that Tn5386 falls within the Tn916 family of transposons, and in place of tetM encodes a novel region that may confer resistance to lantibiotics.     

Presence of chromosomal elements resembling the composite structure Tn3701 in streptococci.

Tn3701, carried by Streptococcus pyogenes A454, is the first chromosomal composite element to be described; it contains in its central region Tn3703, a transposon similar to Tn916. A comparison by DNA-DNA hybridization of Tn3701 with omega(cat-tet) and Tn3951, carried by Streptococcus pneumoniae BM6001 and by Streptococcus agalactiae B109, respectively, revealed that the two latter structures are also Tn3701-like composite elements. The DNAs of 27 other antibiotic-resistant group A, B, C, and G streptococci and of S. pneumoniae BM4200 showed sequence homologies to Tn3701 (14 strains, including BM4200), to the regions of Tn3701 outside of Tn3703 (5 strains), and to Tn916 alone (8 strains). The DNAs of five strains did not detectably hybridize with any probe. The tetM gene was identified in most chromosomal genetic elements coding for tetracycline-minocycline resistance. Since Tn3701-like elements are widely disseminated among antibiotic-resistant streptococci (47% of the 34 strains studied), we propose that Tn3701 be considered the prototype of chromosomal composite elements.     

Cloning and characterization of the genes encoding the haemolysin of Haemophilus ducreyi

We previously identified a heat- and protease-labile haemolytic activity expressed by Haemophilus ducreyi . In order to characterize the haemolysin at the molecular level, genomic DNA from H. ducreyi was probed with haemolysin genes from other Gram-negative organisms. The haemolysin genes of Proteus mirabilis hybridized to H. ducreyi DNA suggesting that the haemolysin of H. ducreyi is related to the Proteus/Serratia pore-forming family of haemolysins. Tn 916 mutagenesis was employed to isolate haemolysin-deficient mutants. Approximately 5000 Tn 916 transposon mutants were screened for the loss of haemolytic activity and two mutants were identified. One mutant, designated 35 000-1, was further characterized. Sequences flanking the Tn 916 element in strain 35 000-1 were employed to identify clones from a λDASHII library of H. ducreyi strain 35 000 DNA. A 13 kb insert from one lambda clone was selected for further study. This 13 kb fragment was able to both confer haemolytic activity to Escherichia coli and complement the haemolysin deficiency in strain 35 000-1. The haemolysin gene cluster was cloned from this 13 kb insert and two genes, designated hhdA and hhdB , were identified. The derived amino acid sequence of these genes demonstrated homology to the haemolysin and activation/secretion proteins of P. mirabilis and Serratia marcescens .     

Cloning of the gene encoding Streptococcin A-FF22, a novel lantibiotic produced by Streptococcus pyogenes, and determination of its nucleotide sequence.

Streptococcin A-FF22 (SA-FF22) is a lantibiotic produced by Streptococcus pyogenes FF22. The nucleotide sequence of the SA-FF22 structural gene (scnA) was determined and shown to encode a 51-amino-acid prepeptide. The proteolytic processing site of the SA-FF22 prepeptide differs from that which characterizes other type A lantibiotics.     

Isolation and Characterization of Pseudomonas syringae subsp. savastanoi Mutants Defective in Hypersensitive Response Elicitation and Pathogenicity

Olive strain ITM317 of Pseudomonas syringae subsp. savastanoi , the causal agent of 'Olive and Oleander knot disease' was mutagenized by random transposon (Tn5) insertion. Of the 1 400 transconjugants, four were altered in their ability to induce a hypersensitive reaction (HR) on tobacco; Southern blot analysis showed that a single copy of the Tn5 element was present in their chromosomes. In particular, mutants ITM317–69, ITM317–1010 and ITM317–1194 did not elicit HR whereas mutant ITM317–916 induced a variable response. When assayed for pathogenicity on olive, mutants ITM317–916 and ITM317–1010 induced knots comparable both in size and morphology to those caused by the parental strain. Prototrophic mutant ITM317–1194, still able to produce indole-3-acetic acid and cytokinins, did not cause any knot formation on olive; furthermore, it was unable to multiply in host tissue. Auxotrophic mutant ITM317–69 caused the formation of smaller-sized knots and its prototrophic revertant fully regained the parental phenotypes, suggesting that a single Tn5 insertion had a pleiotropic effect on the mutated phenotypes. Tn5-containing Eco RI fragments from mutants ITM317–69, ITM317–916, ITM317–1010 and ITM317–1194 were cloned into the plasmid vector pBR322. Hybridization of these clones with the hrp gene cluster of P. s. pv. syringae strain 61 was not detected. These results suggest that genes different from those of the above gene cluster might be involved in the interaction of P. s. subsp. savastanoi with olive and with the non-host plant tobacco.     

Chromosomal integration of the green fluorescent protein gene in lactic acid bacteria and the survival of marked strains in human gut simulations

An integration vector was constructed to allow introduction of the gfp gene into the chromosomes of Gram-positive bacteria. Integration depends on homologous recombination between a short 458-nt sequence of the tet(M) gene in the vector and a copy of Tn916 in the host chromosome. Strains of Lactococcus lactis IL1403, Enterococcus faecalis JH2-SS, and Streptococcus gordonii DL1 stably marked with single chromosomal copies of the gfp were readily visualised by epifluorescence microscopy. The marked L. lactis strain survived poorly in a continuous culture system inoculated with human faecal flora, while the laboratory E. faecalis strain was lost at approximately the dilution rate of the fermenter.     

Evidence that mutacin II production is not mediated by a 5.6-kb plasmid in Streptococcus mutans

Here we present evidence that the cryptic 5.6-kb plasmid found in certain strains of Streptococcus mutans is not involved in mutacin production. This evidence comes from demonstrating similarities between a plasmid-less strain T8 and a group II plasmid strain UA96. Both produce what appears to be an identical mutacin based on spectrum of activity and physiological properties. Also, T8 and UA96 are members of the same immunity group (group II). Genotypically, both strains appear similar except for plasmid content based on DNA fingerprinting profiles. T8 and UA96 exhibit identical hybridization patterns following transformation of T8 with a mutacin-negative (bac-1::Tn916) sequence from a Tn916-insertionally inactivated mutant of UA96. This transformation also resulted in the mutacin-negative phenotype in T8 transformants, showing recombination between a mutacin-associated gene in UA96 and its apparent homologous sequence in T8. Moreover, when a plasmid containing a putative repeat element from UA96 (pPC264) was used as a probe, it hybridized to the same five EcoRI fragments in both T8 and UA96. Collectively, these data, coupled with data from other sources, indicate that the plasmid resident in mutacin II strains is not involved in mutacin production.     

Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX

Tn5397 is a conjugative transposon that was originally isolated from Clostridium difficile. Previous analysis had shown that the central region of Tn5397 was closely related to the conjugative transposon Tn916. However, in this work we obtained the DNA sequence of the ends of Tn5397 and showed that they are completely different to those of Tn916. Tn5397 did not contain the int and xis genes, which are required for the excision and integration of Tn916. Instead, the right end of Tn5397 contained a gene, tndX, that appears to encode a member of the large resolvase family of site-specific recombinases. TndX is closely related to the TnpX resolvase from the mobilizable but nonconjugative chloramphenicol resistance transposons, Tn4451 from Clostridium perfringens and Tn4453 from C. difficile. Like the latter elements, inserted copies of Tn5397 were flanked by a direct repeat of a GA dinucleotide. The Tn5397 target sites were also shown to contain a central GA dinucleotide. Excision of the element in C. difficile completely regenerated the original target sequence. A circular form of the transposon, in which the left and right ends of the element were separated by a GA dinucleotide, was detected by PCR in both Bacillus subtilis and C. difficile. A Tn5397 mutant in which part of tndX was deleted was constructed in B. subtilis. This mutant was nonconjugative and did not produce the circular form of Tn5397, indicating that the TndX resolvase has an essential role in the excision and transposition of Tn5397 and is thus the first example of a member of the large resolvase family of recombinases being involved in conjugative transposon mobility. Finally, we showed that introduction of Tn916 into a strain containing Tn5397 induced the loss of the latter element in 95.6% of recipients.     

Transfer of the conjugal tetracycline resistance transposon Tn916 from Streptococcus faecalis to Staphylococcus aureus and identification of some insertion sites in the staphylococcal chromosome.

The Streptococcus faecalis pheromone-dependent conjugative plasmid pAD1::Tn916 and the membrane filter-dependent conjugative plasmid pPD5::Tn916 were used to introduce Tn916 into Staphylococcus aureus by intergeneric protoplast fusions and intergeneric membrane-filter matings. In recombinants obtained by protoplast fusion where no plasmid DNA could be detected, tetracycline resistance resulted from transposition of Tn916 from pAD1 to the S. aureus chromosome. Transformation analyses showed that S. aureus Tn916 chromosomal insertions occurred near pig, ilv, uraA, tyrB, fus, ala, and the trp operon. DNA hybridization analyses of EcoRI- and HindIII-digested chromosomal DNAs confirmed the diversity of chromosomal sites involved and demonstrated that the inserts were Tn916 insertions rather than integrations of all or part of pAD1::Tn916. Both pAD1::Tn916 and pPD5::Tn916 were transferred to S. aureus by membrane-filter matings. These plasmids remained intact and expressed tetracycline resistance in S. aureus. S. aureus strains carrying pAD1::Tn916, but not a chromosomal insert of Tn916, and any one of several conjugal gentamicin-resistance plasmids lost their ability to serve as conjugal donors of the gentamicin-resistance plasmids.