Transformation by Extracellular DNA Produced by Pseudomonas aeruginosa

Abstract

Most Pseudomonas aeruginosa strains are capable of producing extracellular DNA. Very closely linked chromosomal markers (leu+ and trp+) were cotransferred to P. aeruginosa PAO1819 (leu9001, trp9008) by the extracellular DNA produced by P. aeruginosa strains IFO3445 and PAO1 at a frequency of 10-7 to 10-8. Treatment of the extracellular DNA with DNase, heating at 95 C or sonication completely destroyed its transforming ability. The R plasmid in the extracellular DNA produced by P. aeruginosa IFO3445 (RP4) or PAO2142 (RLb679) could be transferred to Escherichia coli ML4901 or P. aeruginosa PA01819. The resultant transformants showed identical resistance patterns in the respective donors, and the sizes of the DNAs of RLb679 and RP4 isolated from the transformants were the same as those in the respective donors. These results demonstrate that the extracellular DNA contains both chromosomal DNA and plasmid DNA, and that it exhibits transforming ability. This implies that transformation by the extracellular DNA produced by P. aeruginosa may occur in nature and this seems to be of clinical importance in view of the spread of R plasmids among pathogens.Pseudomonas aeruginosa is well known as an opportunistic pathogen. In addition, the organism has the unique biological character of producing large amounts of DNA which accumulates extracellularly (7, 16). The occurrence of extracellular DNA has also been reported for Neisseria (2), Bacillus subtilis (15), Pseudomonas fluorescens (7), Vibrio parahaemolyticus (7), Staphylococcus aureus (3), Staphylococcus epidermidis (3), and Alcaligenes faecalis (3). Moreover, transformation by extracellular DNA has been demonstrated in Neisseria (2), Streptococcus (12), and B. subtilis (14).The mean size of the extracellular DNA produced by P. aeruginosa is estimated to be about 4 Mdal (16), hence it is possible that the extracellular DNA of P. aeruginosa and other bacteria has transforming ability. Hara et al (8) reported that a chromosomal marker (his+) in P. aeruginosa KYU-1 was transferred by intra-and extracellular DNA.Therefore, we investigated whether the extracellular DNA produced by P. aeruginosa can transfer chromosomal markers in P. aeruginosa by using two very closely linked selective markers. We also examined whether any plasmid DNA is contained in the extracellular DNA by searching for gene expression of the 621

A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms

“…The PCR and Southern analysis presented here suggested that the extracellular DNA present in P. aeruginosa late‐log phase cultures is similar to whole‐genome DNA. In agreement, it has previously been shown that different markers, including his + , leu + and trp + , could be transferred by transformation of CaCl 2 ‐treated P. aeruginosa cells with extracellular DNA, at the same frequencies as when transformation was performed with an equivalent amount of purified intracellular DNA (Hara et al ., 1981; Muto and Goto, 1986). In accord with the suggestion that the extracellular DNA is similar to whole‐genome DNA, our experiments with lacZ ‐containing P. aeruginosa strains suggested that DNA release occurs through cell lysis, or through lysis of released DNA‐containing membrane vesicles.…”

“…Many strains of P. aeruginosa , including PAO1, have previously been shown to produce large amounts of extracellular DNA (e.g. Goto et al ., 1971; Murakawa, 1973a,b; Hara and Ueda, 1981; Muto and Goto, 1986), which may function as a matrix component in biofilms (Whitchurch et al ., 2002; Nemoto et al ., 2003). In the present report we provide evidence that this extracellular DNA is organized in distinct patterns in P. aeruginosa biofilms.…”

Antimicrobial and antibiofilm efficacy of a copper/calcium hydroxide-based endodontic paste against <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i> and <i>Candida albicans </i>

“…This last result can be interpreted considering the contribution of extracellular DNA (eDNA). Notoriously, eDNA is abundantly accumulated during P. aeruginosa biofilm formation by lysis of a subpopulation of bacteria 23,24) , by mechanisms dependent on acyl homoserine lactone (autoinducer 1) and quinolone signalling during quorum sensing attainment 45) . Thus, eDNA may initially facilitate bacterial adhesion and cell-to-cell aggregation; then, as an important component of biofilm matrix, eDNA enhances stability of the sessile community and strengthens its resistance to antibiotics or detergents 46) .…”

“…Notoriously, once structured as biofilm, microbial agents have enhanced resistance to antibiotics and disinfectants, given their complex and heterogeneous arrangement as a microbial sessile population embedded into an extracellular, minimally permeable polymeric matrix [18][19][20] . Among the numerous soluble factors involved in biofilm formation/maturation, quorum sensing molecule, including pyoverdine 21,22) and eDNA 23,24) , have been described. Because of such a complex scenario, antibiotic therapy as well as intracanal irrigants treatment have to be focused towards the eradication of root canals infections and possibly endodontic biofilm disruption 19) .…”

Current Status of In Vitro Models and Assays for Susceptibility Testing for Wound Biofilm Infections

“…DNA is another component of the ECM mixture. P. aeruginosa produces extracellular DNA (eDNA) composed of both chromosomal DNA and plasmid DNA that can be transferred among the various microorganisms comprising the biofilm [82]. Biofilms could incorporate human DNA from lysate leucocytes into the ECM [72].…”