Surfactant degradative plasmids

Introduction. T h e data about microorganisms able to utilize cationic and anionic surfactants for metabolism are insufficient. T h e genetic control of their degra­ dation has not been clearly determined yet. The plasmid localization of the genes responsible for degradation of an anionic surfactant, alkyl benzenesulfonate (ABS), was first shown by Caine et al. [1 ]. They discovered a conjugative plasmid control­ ling at least one step of ABS degradation — the cleavage of sulfonic group in the s t ra in-degrader Pseudomonas testosteronu Though the biochemical aspects of this problem had been investigated [2 ] , further studies on the genetic control and regulation of the ABS degradation were not made. This is the only report of a possible plasmid control in the case of ABS and of chromosomal localization of degradative genes for naphthalene sulfonates [3 ] . Studies on the localization of genetic determi­ nants controlling surfactant degradation expand our knowledge about microorganism-degraders. T h e data obtained may be used for construction of new s t ra insdestructors which could be perspective for the waste water t reatment . This is important for the develop­ ment of biotechnologies aimed at purifying waste water from these toxicants. We have selected and characterized highly active bacterial strains which degrade ionogenic surfactants

Introduction.The data about microorganisms able to utilize cationic and anionic surfactants for metabolism are insufficient.The genetic control of their degra dation has not been clearly determined yet.
The plasmid localization of the genes responsible for degradation of an anionic surfactant, alkyl benzenesulfonate (ABS), was first shown by Caine et al. [1 ].They discovered a conjugative plasmid control ling at least one step of ABS degradation -the cleavage of sulfonic group in the strain-degrader Pseudomonas testosteronu Though the biochemical aspects of this problem had been investigated [2], further studies on the genetic control and regulation of the ABS degradation were not made.This is the only report of a possible plasmid control in the case of ABS and of chromosomal localization of degradative genes for naphthalene sulfonates [3].
Studies on the localization of genetic determi nants controlling surfactant degradation expand our knowledge about microorganism-degraders.The data obtained may be used for construction of new strainsdestructors which could be perspective for the waste water treatment.This is important for the develop ment of biotechnologies aimed at purifying waste water from these toxicants.
We have selected and characterized highly active bacterial strains which degrade ionogenic surfactants referring to different classes [4,5].Some of them have been tested for the ability to purify waste water on the industrial scale [6,7 ].
The localization of the genetic determinants con trolling transformation of ionogenic surfactants by bacteria-degraders has been shown.
The bacterial strains-degraders of surfactants are given in Table .Bacteria were grown in LB broth and agar.The medium of the following composition (gT 1 ) was used as minimal: K 2 HP0 4 -1.0;NH 4 N0 3 -1.0;KC1 -0.5; MgCl 2 -0.1.15 g of agar was added to obtain agarized medium.Anionic and cationic sur factants (0.3 gT 1 ) were employed as the sole carbon and energy source.
To eliminate plasmids, the cells were treated with mitomycin C, using the method of Rheinwald et al. [8].
For matings, the donor and recipient cells (at the approximate density of 10 8 cell ml" 1 ) were mixed on LB plates in a ratio of 1:2.The plates were incubated for 2-4 h ( in some cases up to 18 h) at 28 °С.The cells were scrapped off the plates and suspended in 0.56 % NaCl solution and transferred onto selective medium.The transconjugants obtained were passaged thrice on the respective media.Several modifications of the alkaline lysis method were used to isolate plasmid DNA [9][10][11][12][13].The size of plasmid DNA was estimated by comparing its electrophoretic mobility with standard plasmid DNAs.pLAFR5 (21.5 kb), RP4 (55 kb), NPL-J (98 kb) and pBS2 (130 kb) which were used as marker plasmids.
The digestion of plasmid DNA was performed by EcoRI («Promega», USA) according to the supplier's protocol.
Results and Discussion.As the biodegradation of different synthetic compounds are frequently control led on the genetic level by degradative plasmids (D-plasmids) [14,15], the selected strains (Table ) were tested for the presence of plasmid DNA.The most strains of bacteria-destructors have been selec ted under the continuous-flow conditions.Continuous cultivation was carried out in bioreactor of column type with a ceramic carrier under forced aeration.The immobilization of bacteria on a ceramic carrier was made by adhesion [4,5].
We have failed to detect plasmid DNA in straindestructor of cyclimide, P. desmolytica C37, P. testosteroni TO, by screening techniques for realization of plasmid DNA such as alkaline lysis method with several modifications [10][11][12][13].
Fig. 3 demonstrates the fragments of plasmid DNA isolated from degrader strains digested by EcoRL It has been shown that the different plasmids of bacteria-degraders have different restriction pat terns.
Curing of plasmids may be induced by chemical reagents, i. e. acridine orange NO, ethidium bromide, SDS, mitomycin С (MC).For curing plasmid DNA was used MC in concentration 1,25-30.0/Ug-тГ 1 .The sublethal MC concentrations were 20//gmr 1 for anionic degrader -strain P. alcaligenes TR and strains degraders of ampholytic and cationic sur factants 30.0 and 20.0 /.g'ml' 1 , respectively.The clones which lost the ability for utilization of sur factants (Sac + phenotype) were obtained for strains P. rathonis T, P. alcaligenes TR, P. putida TO, P. stutzeri AT, P. fluorescens TR , P. putida К and P. The occurrence frequency of such variants was 0.4--1.8%.We failed to observe the reversions to Sac + phenotype in most cured mutants.Plasmid DNA was not found in clones with lost ability for utilization of surfactants, except for the strain P. fluorescens TR.The results obtained indicate the plasmid loca lization of the surfactant degradation determinants.To study whether the plasmids of strains degrading surfactants are able to conjugative transfer the expe riments on mating have been carried out.
The transconjugants obtained were selected on the media supplemented with an appropriate surfac tant as a sole carbon source.The plasmid DNA of expected molecular mass has appeared in transconjugant cells.The frequency of appearing Sac + phenotype was 10" 6 to 10~3.
The high frequency of the Alk + phenotype losing induced by mitomycin С favours the above suggestion.To elucidate the ability of the plasmid from strain P. fluorescens TR for conjugative and mobilization trans fer, we tested its ability to mobilize the non-conjugative plasmid pBS222 A-15.This plasmid was trans ferred to strain P. fluorescens TR by using a helper plasmid pRK20J3 followed by a series of matings in which the above strain was employed as a donor of plasmid pBS222 A-15, while strains of E. coli and P. putida were employed as recipients.If own plasmid of strain P. fluorescens was conjugative, the mobilization transfer of a small plasmid pBS222 A-15 to recipient cells would be observed.Yet, numerous experiments failed to reveal this plasmid conjugative transfer at a frequency less than 10" 9 .So, the question related to the resident plasmid ability for conjugation still remains open.As for this plasmid functional signi ficance, one may probably consider it to be the D-plasmid harbouring genes that control alkamone utilization.
Thus, we have shown that the degradation of anionic, cationic and ampholytic surfactants by pseudomonas is probably controlled by plasmid genes.Most plasmids found in strains-degraders of sur factants proved to be conjugative and may be used by bacteria for the degradation of ecologically hazardous compounds in the purification systems.
Acknowledgements.This work was supported by SCJW European Research Fellowships.We are very grateful to S. C. Johnson Corp. for financial help.