A Tn4001 Delivery System for Streptococcus gordonii (Challis)

Rd, Lunsford

doi:10.1006/plas.1995.1016

Cited by 23 publications

(28 citation statements)

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“…However, due to its small size, broad host range, and genetic amenability on plasmid pMGC57 (5,21), the composite-type trans- (20,21). As far as we are aware, this is the first instance in which Tn4001 has been used for the mutagenesis of S. pneumoniae.…”

Section: Discussionmentioning

confidence: 99%

“…As far as we are aware, this is the first instance in which Tn4001 has been used for the mutagenesis of S. pneumoniae. Prior studies have shown that Tn4001 chooses its targets for insertion with a high degree of randomness and that this transposon is highly stable in streptococci (20,21). Mutagenesis of S. pyogenes with Tn4001.spc was shown to be extremely efficient using the suicide plasmid pMGC57-spc, with transposition frequencies of between 10 3 and 10 6 /g of DNA, depending on the S. pyogenes host strain used.…”

Section: Discussionmentioning

confidence: 99%

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Visualizing Pneumococcal Infections in the Lungs of Live Mice Using Bioluminescent Streptococcus pneumoniae Transformed with a Novel Gram-Positive lux Transposon

Francis¹,

Yu²,

et al. 2001

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Animal studies with Streptococcus pneumoniae have provided valuable models for drug development. In order to monitor long-term pneumococcal infections noninvasively in living mice, a novel gram-positive lux transposon cassette, Tn4001 luxABCDE Km r , that allows random integration of lux genes onto the bacterial chromosome was constructed. The cassette was designed so that the luxABCDE and kanamycin resistance genes were linked to form a single promoterless operon. Bioluminescence and kanamycin resistance only occur in a bacterial cell if this operon has transposed downstream of a promoter on the bacterium's chromosome. S. pneumoniae D39 was transformed with plasmid pAUL-A Tn4001 luxABCDE Km r , and a number of highly bioluminescent colonies were recovered. Genomic DNA from the brightest D39 strain was used to transform a number of clinical S. pneumoniae isolates, and several of these strains were tested in animal models, including a pneumococcal lung infection model. Strong bioluminescent signals were seen in the lungs of the animals containing these pneumococci, allowing the course and antibiotic treatment of the infections to be readily monitored in real time in the living animals. Recovery of the bacteria from the animals showed that the bioluminescent signal corresponded to the number of CFU and that the lux construct was highly stable even after several days in vivo. We believe that this lux transposon will greatly expand the ability to evaluate drug efficacy against gram-positive bacteria in living animals using bioluminescence.Streptococcus pneumoniae is the leading cause of invasive bacterial disease in the very young and the elderly and is the bacterium most responsible for community-acquired pneumonia in the developed world (31). It can behave as a transient commensal, colonizing the nasopharynx of 40% of healthy adults and children, with no adverse effects (2). Children carry this pathogen in the nasopharynx asymptomatically for about 4 to 6 weeks, often carrying several serotypes at a time (13, 33). Occasionally, perhaps in conjunction with a viral infection (9), one of these strains gives rise to a symptomatic pneumococcal infection, including sinusitis, otitis media, pneumonia, and meningitis (12, 13, 16).Antibiotic treatment of pneumococcal infections has been less effective in recent years with the increased occurrence of multidrug-resistant strains of S. pneumoniae. About one-third to one-half of pneumococci recovered from humans are at least partially resistant to penicillin, which may occur in addition to resistance to a number of other common antibiotics (1, 3). These factors, plus the ability of the pneumococcus to transfer genes for resistance, encapsulation, and virulence via transformation (12), make it imperative to develop a better understanding of the mechanism by which pneumococci cause disease. Probably the best way to enhance this process is to develop a better animal model.In 1995, Contag et al. (7) showed that it was possible to monitor disease processes in living animals using b...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Visualizing Pneumococcal Infections in the Lungs of Live Mice Using Bioluminescent Streptococcus pneumoniae Transformed with a Novel Gram-Positive lux Transposon

Francis¹,

Yu²,

et al. 2001

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“…Therefore, genetic characterization by Tn916-mediated mutagenesis has been of limited use with the genus Streptococcus. A recent report by Lunsford (30) indicates that the small staphylococcal transposon Tn4001 (4.5 kb) can be used for mutagenesis of two oral Streptococcus species, S. gordonii and S. mutans. As in the case of mutagenesis with Tn916, this transposon is delivered by transformation on a suicide vector, which significantly lowers the overall frequency of transposition and the efficacy of these transposons in mutagenesis.…”

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confidence: 99%

Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements

et al. 1996

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New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli(pTV21⌬2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn917, in JH1005 at the permissive temperature (30؇C) versus that at the nonpermissive temperature (45؇C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10 ؊5 to 10 ؊4) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45؇C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21⌬2TetM, a tetracycline-resistant and ampicillin-sensitive Tn917-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp. fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn917 mutagenesis in the genus Streptococcus.

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“…Both can transpose into various loci on the chromosome (12,16) and are involved in the dissemination of the resistance gene among staphylococci, enterococci, and streptococci (11,13,26). In Tn4001, IS256 was found to provide the Ϫ35 region for the promoter of the aminoglycoside resistance gene, thus constructing a hybrid promoter (23).…”

Section: Discussionmentioning

confidence: 99%

Formation of potent hybrid promoters of the mutant llm gene by IS256 transposition in methicillin-resistant Staphylococcus aureus

Maki

Murakami

1997

J Bacteriol

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From high-level methicillin-resistant Staphylococcus aureus SRM551, the low-level heterogeneously resistant mutant, SRM563, was isolated by transposon mutagenesis. The transposon insertion occurred in the 3 region of the llm gene in the mutant (H. Maki, T. Yamaguchi, and K. Murakami, J. Bacteriol. 176:4993-5000, 1994). Resistant revertants were generated from the mutant strain SRM563 on the plate containing methicillin at a concentration of 12.5 g/ml or more. In some revertants, the insertion sequence IS256 was observed to be transposed into one of five sites localized 88 to 212 bp upstream of the mutant llm at a frequency of 2.8 ؋ 10 ؊7in the bacterial population. The IS256 transposition created a new hybrid promoter in which the ؊35 region at the end of IS256 was properly arranged in relation to the ؊10-like sequence upstream of llm. The new promoters greatly enhanced the transcription of the mutant llm, as judged by blotting analysis of llm mRNA, with concomitant elevation of the methicillin resistance. Involvement of the insertion sequence in the heteroresistance characteristics of methicillin-resistant S. aureus was suggested.Various insertion sequences (ISs) are widely distributed among bacteria. They can be transposed to various loci in chromosomal DNA, which sometimes results not only in disruption of genes but also in activation of downstream genes (5). With some IS elements, such as IS10 in Escherichia coli, the activation is caused by a promoter located within the element and directed outward into the adjacent gene (3). On the other hand, some elements, such as IS1 (19) or IS2 (10), can activate the downstream genes via the formation of a hybrid promoter in which the Ϫ35 region at the end of the element is properly positioned close to the Ϫ10 region in the host DNA. Many IS elements have been shown to contain the Ϫ35-like sequence of the promoter in their terminals (5), although not all elements have been demonstrated to be involved in the formation of functional promoters. In Staphylococcus aureus, IS256 and IS257, which compose both ends of Tn4001 (14) and Tn4003 (15), respectively, belong to this category of IS elements. These elements appear to construct the hybrid promoter for the aminoglycoside resistance gene, aacA-aphD, in Tn4001 (23), and that for the trimethoprim resistance gene, dfrA, in Tn4003 (24), respectively. However, their transposition with concomitant activation of adjacent genes has not been observed.Previous studies have shown that S. aureus became methicillin resistant via the production of a low-affinity penicillinbinding protein, designated PBP2Ј or PBP2a, encoded by mecA of foreign origin (7, 30). The MIC of methicillin, however, varied from 3.1 g/ml for some strains to more than 800 g/ml for others; thus, in addition to mecA, other, unknown genetic factors seem to determine the resistance level (18, 25). We introduced transposon Tn918 with a tetracycline resistance marker into the chromosome of high-level methicillin-resistant S. aureus (MRSA) and isolated an insertional muta...

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A Tn4001 Delivery System for Streptococcus gordonii (Challis)

Cited by 23 publications

References 0 publications

Visualizing Pneumococcal Infections in the Lungs of Live Mice Using Bioluminescent Streptococcus pneumoniae Transformed with a Novel Gram-Positive lux Transposon

Visualizing Pneumococcal Infections in the Lungs of Live Mice Using Bioluminescent Streptococcus pneumoniae Transformed with a Novel Gram-Positive lux Transposon

Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements

Formation of potent hybrid promoters of the mutant llm gene by IS256 transposition in methicillin-resistant Staphylococcus aureus

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