Electrotransformation of Streptococcus pneumoniae: evidence for restriction of DNA on entry

Lefrançois, Jacques; Sicard, A M

doi:10.1099/00221287-143-2-523

Cited by 9 publications

(10 citation statements)

References 25 publications

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“…This is in agreement with the demonstration that dsDNA penetrates S. pneumoniae during electroporation (Lefrangois & Sicard, 1997) and suggests that this DNA contains an intact genome ready for replica tion.…”

Section: Discussionsupporting

confidence: 91%

“…The ss form of this fragment as well as the 480-base ss s t y fragment were prepared by the same technique, using unequal amounts of primers to produce a 99% pure ssDNA. PCR amplification of the whole tetL gene was performed on pLSl DNA (Lopez et al, 1982). For all penetration experiments, except that of chromosomal DNA, DNA was labelled by PCR, adding [ c~-~, P ]~T T P to the dNTP mix, to avoid gaps in the molecules which result in natural uptake interruption (Mejean & Claverys, 1993).…”

Section: Methodsmentioning

confidence: 99%

“…It is possible to test for this activity by investigating plasmid facilitation that requires recombination (Lopez et al, 1982).…”

Section: Electrotransformation Of Naturally Competent Culturesmentioning

confidence: 99%

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Electrotransformation and natural transformation of Streptococcus pneumoniae:requirement of DNA processing for recombination

1998

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Electrotransformation has been used as a tool to introduce genes carried on replicative vectors in hundreds of bacterial species. In this study, the technique was used to try to obtain recombination of markers in the chromosome of the na tura I I y transformable bacterium Streptococcus pneumoniae . Recombination was not observed even using naturally competent cultures. Both chromosomal and cloned DNA, denatured or native, were without effect. These results suggest that it is not sufficient to introduce DNA into the cell to obtain recombinants in this bacterium. The integration of markers into the chromosome in naturally competent cells must require DNA processing during entry. Electrotransformation of replicating plasmids is red-independent but can be facilitated by a red-dependent process. This facilitation required the induction of the natural competence machinery, probably involving partial homologous pairing.

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Section: Discussionsupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

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Electrotransformation and natural transformation of Streptococcus pneumoniae:requirement of DNA processing for recombination

1998

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“…The plasmid pAUL-A Tn4001 luxABCDE Km r was electroporated into S. pneumoniae D39 as described previously (18). The transformation mix was plated on chocolate agar containing erythromycin (0.3 g/ml), and the plates were incubated for 24 to 48 h. Transformants of S. pneumoniae D39 containing pAUL-A Tn4001 luxABCDE Km r were patched onto chocolate agar plates containing erythromycin (0.3 g/ml) and incubated overnight.…”

Section: Methodsmentioning

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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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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“…Inability to transform bacteria with linear DNA using electroporation has been demonstrated in other bacteria such as Haemophilus influenzae (Mitchell et al , 1991) and Streptococcus pneumoniae (Lefrancois & Sicard, 1997; Lefrancois et al , 1998). In S. pneumoniae , double‐stranded linear DNA penetrated the cells upon electrotransformation, but was fully restricted after penetration (Lefrancois & Sicard, 1997). In contrast, Haemophilus ducreyi was easily electrotransformed with linear DNA (Hansen et al , 1992).…”

Section: Resultsmentioning

confidence: 99%

Unveiling electrotransformation ofMoraxella catarrhalisas a process of natural transformation

Meier

Troller

Heiniger

et al. 2006

FEMS Microbiology Letters

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The human respiratory tract pathogen Moraxella catarrhalis is a naturally competent microorganism. However, electrotransformation has long been used to introduce foreign DNA into this organism. This study demonstrated that electrotransformants obtained with linear or circular nonreplicating plasmid DNA originated exclusively from natural transformation processes taking place during the recovery phase after the application of current. Only replicating plasmid DNA could be introduced into M. catarrhalis by electrotransformation, in a type IV pilus-independent manner. Electrotransformation with homologous genomic DNA indicated that restriction of double-stranded DNA was independent of type III restriction-methylation systems. Nontransformability of M. catarrhalis by electrotransformation was observed using double- as well as single-stranded DNA. In addition, the study showed that natural competence is a very constant feature of M. catarrhalis.

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Electrotransformation of Streptococcus pneumoniae: evidence for restriction of DNA on entry

Cited by 9 publications

References 25 publications

Electrotransformation and natural transformation of Streptococcus pneumoniae:requirement of DNA processing for recombination

Electrotransformation and natural transformation of Streptococcus pneumoniae:requirement of DNA processing for recombination

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

Unveiling electrotransformation ofMoraxella catarrhalisas a process of natural transformation

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