Megaplasmids in Gram-negative, moderately halophilic bacteria

FEMS Microbiology Letters 227 (2003) 81^86

www.fems-microbiology.org

Megaplasmids in Gram-negative, moderately halophilic bacteria Montserrat Argandon‹a, Fernando Mart|¤nez-Checa, Inmaculada Llamas, Emilia Quesada, Ana del Moral Departamento de Microbiolog|¤a, Facultad de Farmacia, Campus Universitario de Cartuja s/n, 18071 Granada, Spain Received 10 July 2003; accepted 18 August 2003 First published online 4 September 2003

Abstract We have discovered that many Halomonas species harbour large extrachromosomal DNA elements. Using currently available protocols it is technically very difficult to identify large plasmids in bacteria, and even more so when they are coated in mucous polysaccharide. We used culture conditions suitable for both halophilic and halophilic exopolysaccharide-producing bacteria and applied a modified gel electrophoresis method to locate and visualise the megaplasmids. Almost all the species of Halomonas studied harbour two plasmids of about 70 kb and 600 kb and some species carry other smaller extrachromosomal DNA elements. The common presence of these megaplasmids may well be related to the survival strategies of the bacteria in their special surroundings. 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Halophilic bacterium ; Halomonas; Extrachromosomal DNA; Megaplasmid

1. Introduction Moderately halophilic bacteria are to be found in a wide variety of natural habitats but thrive best in media containing 3^15% w/v NaCl [1]. In recent years it has been found that several products of these extremophilic bacteria, such as exopolysaccharides (EPSs), halophilic enzymes and compatible solutes, may have very useful applications in biotechnology [2^5] and so it is of considerable interest to learn more about their genetic make-up and characteristics. Our recent studies have led us to suspect that a considerable number of these bacteria contain large molecules of extrachromosomal DNA. These plasmids are often responsible for important biological functions produced by the bacteria, such as nitrogen ¢xation and root nodulation [6], resistance to antibiotics and heavy metals [7], induction of plant tumours and CO2 ¢xation [8], the production of ropiness in milk cultures [9] and various di¡erent metabolic transformations [10,11]. They may also determine certain aspects of their own phenotypical characteristics,

* Corresponding author. Tel. : +34 (958) 243875; Fax : +34 (958) 246235. E-mail address : [email protected] (A. del Moral).

such as mucoid colonial growth [12]. The term ‘megaplasmid’ was ¢rst used by Burkardt and Burkardt [13] when working with plasmids from Rhizobium meliloti to describe the largest of these extrachromosomal DNA elements. Although there is still no general consensus as to the minimum size at which a plasmid becomes a megaplasmid, some authors suggest that it should be 100 kb [14,15]. Whatever the precise size ¢nally agreed upon, more and more of these large plasmids are being found in a wide variety of bacteria, some of them almost as big as chromosomes [16]. Megaplasmids have tended to be overlooked until recently mainly because working with large extrachromosomal DNA molecules presents considerable technical challenges in that they cannot be separated readily from chromosomal DNA or be resolved by conventional gel electrophoresis and they are also prone to nicking and shearing in standard laboratory operations [15]. Large plasmids of this sort are known to occur widely in halophilic archaea but only a few have been physically mapped and little is known about their function [17^19]. As far as halophilic bacteria are concerned, such extrachromosomal elements have so far only been detected in Halomonas subglaciescola UQM 2927 (pHS1, 70 kb), Chromohalobacter israelensis ATCC 43985 (pH11, 48 kb) [20] and strain E-367 of Salinivibrio costicola (size not recorded) [21], but they are all smaller than 100 kb. Suspecting the presence of plasmids of this sort we de-

0378-1097 / 03 / $22.00 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(03)00651-7

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M. Argandon‹a et al. / FEMS Microbiology Letters 227 (2003) 81^86

cided to look for them in our own collection of moderately halophilic bacteria, which includes two EPS-producing species, Halomonas eurihalina and Halomonas maura. To this end, we ¢rst of all had to ¢nd the most suitable method of isolating the DNA of these large extrachromosomal elements to be able to characterise them with any accuracy. Thus we assayed several techniques, with varying degrees of success, before ¢nally deciding upon the method described by Wheatcroft et al. [22], which involves extracting the DNA inside the agarose well. We made the appropriate adaptations to Wheatcroft’s method to suit halophilic bacteria, which included culturing and resuspending the cells in a saline medium, and also modifying the medium when necessary to inhibit the excretion of polysaccharides. In this way we were able to con¢rm the presence of some previously described large plasmids and also to discover several new ones in members of the genera Halomonas, Chromohalobacter, Marinomonas, Marinobacter and Salinivibrio.

2. Materials and methods 2.1. Bacterial strains and culture media We studied the type strains of 17 species of the genus Halomonas and other species representative of Gram-negative halophilic bacteria from culture collections. To check whether these strains had undergone any changes through being kept for some years in laboratory conditions we also studied fresh isolates of seven related halophilic strains with which we are working at the moment (cf. Table 1). The bacteria were kept in MH medium supplemented with 2% agar. The EPS-producing bacteria H. eurihalina and H. maura were cultivated in a medium containing 0.2% w/v peptone protease, 0.04% w/v yeast extract and 0.057% w/w sodium thioglycolate, especially designed to reduce the production of these EPSs. The principle behind this formula was to eliminate the glucose and magnesium sulfate, reduce the rest of the nutrients and add sodium thioglycollate to create an anaerobic environment. The rest of the strains were cultivated in MH medium, as described in a previous publication [23]. Both media were prepared to a salt concentration of 7.5% w/v according to Rodr|¤guez-Valera [24]. As controls we used Escherichia coli V517, which harbours 10 extrachromosomal elements [25], and strain Sp7 of Azospirillum brasilense ATCC 29145, which carries four megaplasmids [26] seeded in LB medium [27] and TP medium [22] respectively. 2.2. Analysis of extrachromosomal DNA The bacteria were cultivated for 18 h (stirred at 150 rpm) in 5 ml of the broth medium designed for the strain in question at their optimum growth temperatures: the

Table 1 Halophilic bacterial strains studied, the plasmids detected and their approximate sizes Plasmids (kb) Bacterial species from culture collections Halomonas aquamarina DSM 30161T Halomonas desiderata DSM 9502T Halomonas elongata CECT 4279T Halomonas eurihalina ATCC 49336T Halomonas halophila ATCC 19717T Halomonas halodenitri¢cans CECT 5012T Halomonas halodurans LGM 10144T Halomonas halophila CCM 3662T Halomonas magadii NCIMB 13595T Halomonas marina ATCC 25374T Halomonas maura CECT 5298T Halomonas meridiana DSM 5425T Halomonas pantelleriensis DSM 9661T Halomonas salina CECT 5288T Halomonas subglaciescola DSM 4683T Halomonas variabilis DSM 3051T Halomonas venusta ATCC 27125T Chromohalobacter canadensis ATC 43984T Chromohalobacter marismortui DSM 6770 T Chromohalobacter israelensis CECT 5287T Marinobacter hydrocarbonoclasticus CECT 5005T Marinomonas communis CECT 5003T Marinomonas vaga CECT 5004T Salinivibrio costicola NCIMB 701T Fresh isolates of bacterial strains M15 (Halomonas maura) X8 (Halomonas eurihalina) F32 FP35 FP36 Al12 A3

600; 74 595; 88.5; 25.8 595; 70.5 610; 8.1; 5.8 595; 73.8 600 601; 75; 5.4 595; 73.8 602; 68; 7.6; 5.7 601; 71.2; 5.4 619; 70.7 597; 79.2 595; 73.4 601; 71.2 620; 68 604.8 592; 144.4; 75 620; 70.2 592; 119.3; 67.5 48 626; 68.8 627; 75.3 614 598; 71.6 620; 70 610 609 611 597 605 550; 467; 184; 140.8; 110.6; 98.2; 30.8

halophilic strains at 32‡C, E. coli at 37‡C and A. brasilense at 30‡C. 500-Wl aliquots of these cultures were then used to inoculate new tubes, which were incubated for 6^8 h until reaching the exponential growth phase. The halophilic cultures were diluted to OD600 = 0.8 and the controls to OD600 = 0.2. Aliquots of 1 ml of these dilutions were centrifuged at 13 000 rpm to harvest the cells. The halophilic bacterial cells were then resuspended in 500 Wl of 2% w/v salt solution [24] and the controls in sterile, doubly distilled water, both at 4‡C. 1 ml of 0.3% w/v sodium lauryl sarcosinate (4‡C) was added slowly and mixed gently before centrifuging at 13 000 rpm for 3 min at 4‡C. The supernatant was removed immediately and the sediment resuspended in 40 Wl bu¡er solution (Tris^HCl 10 mM, EDTA 10 mM, 20% Ficoll 400 000) and left for 15 min in ice. Electrophoresis was performed using 0.75% w/v agarose. The electrophoresis tank was levelled o¡ with 1UTBE (4‡C) until it was touching the gel, and the wells were then ¢lled with 25 Wl of sodium dodecyl sulfate (SDS; 10% w/v) mixed with xylene cyanol (1 mg ml31 ).

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The SDS was left to run for 10^15 min at 100 V with reversed polarity until it was 1 cm above the wells. 1UTBE was then added to cover the gel by 1 mm, at which time the 25-Wl samples were loaded. To prepare the samples 40 Wl of each was mixed with 10 Wl lytic solution (230 Wl of Tris^HCl, 10 mM EDTA, 10 mM RNase (0.4 mg ml31 ), xylene cyanol (1 mg ml31 ) mixed with 25 Wl of lysozyme (10 mg ml31 )). Electrophoresis was carried out in a cold chamber at 4‡C with the tank submerged in ice at 40 V for 16 h and at 100 V for a further 8 h. The gel was stained with ethidium bromide in a 1UTBE (0.5 Wg ml31 ) bu¡er for 30 min. We estimated the molecular size of the bands with the Quantity One program of the Videocamera and Imaging system. This, of course, can only be an approximate estimation because in this region of the gel the relationship between the size and mobility of the plasmids is not logarithmic. Nevertheless, we are satis¢ed that our calculations are accurate enough to be sure of the existence of megaplasmids of roughly the same size in all but one of our bacterial strains.

3. Results and discussion Several small and medium-sized plasmids have already been identi¢ed in some moderately halophilic bacteria [20,21,30^33] but larger extrachromosomal DNA molecules (megaplasmids) have remained largely undetected due to the di⁄culties involved in isolating such large DNA molecules [15]. Furthermore, their handling is complicated even further when they produce EPSs, as do H. eurihalina [28] and H. maura [29], and are consequently coated with a mucous layer. To detect the presence of megaplasmids in moderately halophilic bacteria we initially assayed several previously published protocols, including pulsed-¢eld gel electrophoresis [34,35] and the same method using S1 nuclease, which converts supercoiled plasmids into linear molecules [15]. We also tried the methods described by Kado and Liu [36] and Plazinski and colleagues [26], who lysed the bacteria inside the agarose well before applying conventional electrophoresis techniques. With some of these techniques we appeared to be getting a positive response but were far from happy with the overall results. We were ¢nally satis¢ed with the results produced by a modi¢ed version of the protocol described by Wheatcroft and colleagues [22] in his study on megaplasmids in R. meliloti. Our modi¢cations included changing the resuspension solution from one of doubly distilled water in the original method to one containing 2% w/v salts to prevent lysis of the halophilic bacteria, and also developing a new medium to inhibit the production of polysaccharides in order to be able to extract very pure DNA samples from the polysaccharideproducing species H. eurihalina and H. maura.

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Table 1 contains a list of all the bacteria assayed and the plasmids they were found to carry. This new procedure allowed us to con¢rm the existence of the small and medium-sized plasmids that had previously been described in the type strains of C. israelensis (48 kb) (data no shown) [20], H. subglaciescola (70 kb, Fig. 1B) [20] and in H. eurihalina strain F2-7 (8.1 and 5.8 kb; Fig. 1A) [31]. In the case of S. costicola we experimented with a strain (NCMIB 701) other than that studied by Mellado et al. [21], which may explain why we did not detect the plasmids of 2.95 kb, 19 kb and 21 kb that they discovered in strain E-367. This in fact is not particularly surprising because as more analyses are carried out it is becoming clear that extrachromosomal elements vary greatly not only between bacterial species but even between such close relatives as strains within the same species [16]. Our most striking discovery was that all but one of the strains we looked at harboured at least one megaplasmid of about 600 kb (cf. Fig. 1A^D) and the great majority also carried a plasmid of about 70 kb (a fact previously reported in H. subglaciescola [20]). The smaller of the two plasmids in H. desiderata was around 88 kb (Fig. 1A). Apart from these two plasmids we also found a megaplasmid of about 144 kb in H. venusta (Fig. 1B) and one of 119 kb in C. marismortui (Fig. 1D). Additionally, other smaller extrachromosomal DNA molecules were detected in H. desiderata (one plasmid), H. marina (one plasmid), H. magadii (two plasmids), H. halodurans (one plasmid) (Fig. 1A) and H. venusta (two plasmids) (Fig. 1B). The possibility always exists that the bands visualised in assays of this nature might be an artefact of the experimental technique. In our case the comparisons we have made between all our results leave us in no doubt of their veracity. Our control bacteria, E. coli and Azospirillum, were subject to the same treatment but revealed no bands at 600 kb, although all the extrachromosomal bodies which they are already known to contain, including megaplasmids [25,26], showed up perfectly. We can also rule out the idea that the bands might be a product of our modi¢cation of Wheatcroft’s method in order to accommodate halophilic bacteria because the results for strain A3 and C. israelensis, both moderate halophiles, do not show this megaplasmid whilst clearly showing other extrachromosomal elements, proving that the method in this case is in no way de¢cient (cf. Fig. 1E). Ideally of course the next step would be to characterise this group of plasmids by Southern hybridisation, above all to see whether they are the same or at least similar in their DNA sequences but, as we have mentioned above, it is extraordinarily di⁄cult to work with large DNA plasmids [15]. We have tried hard to recover the agarose gel band of the 70-kb plasmid intact to make probes and carry out hybridisations but to no avail. The chances then of being successful with the band at 600 kb are re-

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Fig. 1. Plasmid pro¢les observed in the moderately halophilic bacteria. The plasmid-containing strains V517 of E. coli and Sp7 of Azospirillum brasiliensis were used as megaplasmid size references (lanes 1 and 2). A: Lanes: (3) H. halophila, (4) H. pantellerensis, (5) H. aguamarina, (6) H. salina, (7) H. halodenitri¢cans, (8) H. desiderata, (9) H. halophila, (10) H. meridiana, (11) H. halodurans, (12) H. marina, (13) H. magadiensis, (14) H. eurihalina F2-7. B: Lanes: (3) H. variabilis, (4) H. venusta, (5) H. subglaciescola. C: Lanes: (3) H. maura, (4) H. elongata. D: Lanes: (3) C. marismortui, (4) S. costicola, (5) C. canadensis, (6) Marinomonas comunis, (7) M. vaga (8) M. hydrocarbonoclasticus. E: Lanes: (3) strain M15 (H. maura), (4) strain X8 (H. eurihalina), (5) F32, (6) FP35, (7) FP36, (8) Al12, (9) A3.

mote to say the least. Our modi¢cation of Wheatcroft’s method has allowed us to see the bands perfectly but not to purify the DNA from the gel and we are not aware of any more successful method having been published in the literature. Our ¢ndings suggest then that a great many moderately halophilic bacteria harbour largish plasmids in their genomes and that the reason for their not having been detected before may possibly be due to the fact that the

methods used to date for identifying such large plasmids have not been entirely adequate. Our approach to the problem has also allowed us to calculate fairly accurately the size of such molecules compared to the plasmids of 592, 480, 208 and 160 kb of A. brasilense ATCC 29145 and those of 339, 165, 57, 34, 7.71 and 5.87 kb and carried by strain V517 of E. coli (Fig. 1). The common occurrence of large extrachromosomal DNA elements in moderately halophilic bacterial species

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might re£ect the fact that they contain genes which encode important activities related to their adaptation to special environmental conditions. To ¢nd out more about this we are currently working on experiments to elucidate the possible functions of the plasmids found in H. maura. We have obtained EPS-de¢cient mutants by conjugation and have proved via Southern hybridisation experiments, using a transposon as probe, that the megaplasmids carried by these bacteria are probably not involved in the production of EPSs (data not shown). It remains to be seen now whether genes contained in the plasmids intervene in some way in other mechanisms such as their capacity to grow at moderately saline conditions, the resistance of some strains to heavy metal contamination and so on. As far as this is concerned, the results obtained from strain A3 are quite interesting in that it is the only halophilic strain studied, with C. israelensis, not to harbour the 600-kb megaplasmid but at the same time, after polyphasal taxonomic characterisation and on the basis of its considerable phenotypic and phylogenetic di¡erences, it is about to be proposed as a separate genus. Much work is still to be done on these plasmids and their genes and this short communication is merely intended as an alert to their common presence. Further studies and the advance of laboratory techniques will provide us with more information about what they are doing there and also contribute more to our understanding of the phylogenetic relationships between bacterial species and strains and their capacity to exchange important information quite quickly in evolutionary terms, especially with regard to survival mechanisms, via processes involving the horizontal transference of genetic material.

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Acknowledgements This research was supported by grants from the Direccio¤n General de Investigacio¤n Cient|¤¢ca y Te¤cnica (PB981315) and from Plan Andaluz de Investigacio¤n, Spain. We thank our colleague Dr J. Trout for revising our English text.

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