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Ann. Rev. Genet. 1976. 10:7-30 Copyright © 1976 by Annual Reviews Inc. All rights reserved

Annu. Rev. Genet. 1976.10:7-30. Downloaded from www.annualreviews.org by State University of New York - Binghamton on 05/02/13. For personal use only.

PLASMIDS IN PSEUDOMONASl

+3098

A. M Chakrabarty General Electric Research and Development Center, Schenectady, New York 12301

CONTE NTS INTRODUCTION

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DRUG-RESISTANCE PLASMIDS IN

PSEUDOMONAS

.

.

..... ... ..........................................

8 8 9

Compatibility Characteristics of Pse udomonas R Factors ........................... ......... PI Group Plas mids . . . .. ... . ... . . . . . . ... .. . . . .. . . . . . . .. .. .

10

. . ..... ..............

11

..

... .............. .

.

.. ..

.. ....... . .

Plasmids of Pl, Pl, and Other Groups . ... . .. .. . . .. .

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..

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..

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. . .............

. . .. . . . ...

.......... . . .

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...

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The Transposition of Carbenicillin-Resistance Determinant of RP4 (RPl) Plasmid . ... .. .... . ... . . ... . . ... .. . . . . . . . . .. ... ... . . ... . .. .. Translocon A, Recombination, and Evolution of Dr ug-Resistance Genes . .. ... . . .

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..

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... ..

12 13

Recombination of PI-Group Plasmids with Other Replicons and the

Chromosome . . .. .. . . .. . . ... ... . . . . . . . ... . .. .. . . . . . Molec ular Cloning with Pseudomonas R Factors . . . .. .. . . . Cryptic Plasmids ................................................... ................ ...... ............................. .

..

...... .

..

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.. .. .. ... .......... .......

.. ...................... .

SEX-FACTOR PLASMIDS IN

PSEUDOMONAS

..

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................................................... .... .....

.. . . . .

Sex-Factor Plasmids in Pseudomonas putida

. ... .. .

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14 16 16 16

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17

.... ..................

18 18

....... ............................................. ........................................... . . . . .. . . ... . . . . . .. .. . . .. . . . .

20 20 20 21

DEGRADATIVE PLASMIDS IN

PSEUDOMONAS

. ... .

The CAM Plasmid

........ ..

The SAL Plasmid The NAH Plas mid

.. . .... .............. ..

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.................

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... ... ............................

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. ..

....... .. ... ... ...... ..... ...............

.. ... ............... .. .. .....

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................ . .

. .. . .

The OCT Plasmid . . ... .. ... . . . . . . . . .. . ...... ....................... ........ ...... The XYL and TOL Plasmids . ... .. .. ... . . . . . .. .. ... :................................................ ....... ...

. .... ...........

.

RESISTANCE TO MERCURY IONS

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..

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. ...

. ...

.....

. . . Mechanism of Action ofthe Mercury-Resistance Plas mids .. . . ... ... . . . . . Enzymes Involved in Mercury Volatilization . .. .... ..... ... ... .... . ..... ... ... Biological Properties ofMercury-Resistance Plasmids .. ... .. .. . . .. ... .. .. . .

21 22

....... .. ... ......... ... ... ... ...... .... .. ... ... ... .... .. .......... .... ... .... ... .. ........ ..

25

......... ....................... . . ........................................... ..

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CONCLUDING REMARKS

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23 24

'The following abbreviations used in this review: Cb, Carbenicillin; Tc, Tetracycline; Km, Kanamycin; Su, Sulfonamides; Lv, Lividomycin; Tm, Tobramycin; Gm, Gentamicin; Sm, Streptomycin; Nm, Neomycin; Cm, Chloramphenicol; Tp, Trimethoprim; Am, Ampicillin. 7

8

CHAKRABARTY

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INTRODUCTION Plasmids are genetic elements found outside the chromosome within the cells. They can replicate autonomously independent of the chrom osome. and alth ough consid­ ered nonessential for the cell. they often perform secondary (unctions that are vital to the cell under certain conditions. E xcellent books and reviews are available on various aspects of the biology of plasmids ( 1-3). Recent ly some aspects of interac­ tion among plasmids in Pseudomonas h ave been disc ussed in detai l (4); because of that, and due to a limitation of space, I confine my review to the nature and properties of plasmids characterized in Pseudomonas. with particular emphasis on the mechanism of plasmid recombination and transposition, and on the use of p lasmids in molecular c loning in Pseudomonas. In general. the types of plasmids characterized in Pseudomonas species so far compare favorably with those in Escherichia coli and oth er ent eric bact eria . B eca use of the pathogenic nature of P. aeruginosa and its resistance t o common antibiotics, many studies h ave been directed toward developing antibiotics active against P. aeruginosa, and this in turn has led to characterization of n ewer drug-resistance factors active against such antibiotics. Characterization of R factor plasmids respon ­ sible for the inacti vation of such potent antibiotics as gentamicin, carbenicillin, and tobramycin in Pseudomonas has led to a surge of studies involving the mechanism of evolution of drugcresistance plasmids. The oth er type of plasmid f ound in Pseudomonas is the so-called sex-factor plasmid . Such p lasmids h ave been charac­ terized in both P. aeruginosa and P. putida and have contributed to our under­ standing of the organization of the Pseudomonas chromosome. The third type of plasmid available in Pseudomonas is the mercury-resistance p lasmid . Although resistance to mercury is often mediated by sex-factor or drug-resistance pla smids, it is possible to c haracterize transmissi ble or nontran smissible plasmids whose only known function is to confer resistance to toxic concentrations of mercury salt . Since the degradative plasmids, so called because they specify enzymes responsible for the biodegradation of a variety of complex organic compounds, are rather unique t o Pseudomonas, and since mercury has been shown to b e accumulat ed i n sediments rich in oily residues so that most of the Pseudomonas strains capable of uti li zin g petroleum are also mercury-resistant (5, 6) , it is perhaps imperative that Pseudomo­ nas with hydrocarbon plasmids should also possess mercury-resist ance p lasmids so that t hey can cope with the increasing concentration of mercury found in oi ly residues. In this regard associating individual plasmids specifying mercury resis­ t ance and petroleum hydrocarbon utilization to form plasmid a ggregates would be a logical step for a bacteri um to cope with mercury toxicity a ssociated with oil consumption ( 7).

DRUG-RESISTANCE PLASMIDS IN PSEUDOMONAS The genus Pseudomonas, p articularly the species P. aeruginosa, i s notorious for its resistance to m ost commonly used microbial agents. The success of this species as

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PLASM IDS IN PSEUDOMONAS

9

a common hospital pathogen stems largely from its resistance to a number o f antibiotics. This has, of course, prompted drug man ufacturers to come up with a variety of antibiotics to which most P. aeruginosa strains are sensitive. Examp les of such antibiotics active against P. aeruginosa are carbenicillin , gentamicin, tobra­ mycin, colistin . It appears that widespread use of s uch dr ugs has res ulted in the development of a variety of R factors wh ich can inactivate these antibiotics by phosphorylation, adenylation, acetylation, or by a combination of them . The emer­ gence o f R factors active against a large n umber of antibiotics in P. aeruginosa, while posing a great deal of concern for the clinicians , has never theless attrac ted the attention o f numerous workers interes ted in the evo lution of new gene functions . Thus , Sagai et al (8) have s tudied the conjugal transferability of drug resis tance in eleven P. aeruginosa s trains isolated from hospital patien ts in Frankfurt. Four R fac tors were demonstrated in three strains that were transmissible among P. aeruginosa, but not to E coli. T wo of them specified resis tance to Tc , Sm, Su, Lv, and Gm I through elaboration of aminoglycoside-inactivat ing enzymes s uch as Sm phosphotransferase I and Gm 6'- N acetyltransferase. Resistance to Tc was due to a decreased permeability toward th is drug . Similar ly epidemiological surveys con­ ducted in Japan by Mitsuhashi and his group (9) have shown tha t 40% of the resis­ tant s trains that they examined carried R fac tors active against a number of drugs . A s tudy conduc ted by Bryan et al ( 10) has demonstra ted the presence of R fac tors in 3.3% of the hospital P. aeruginosa s trains in the Alberta region . An increasing number of P. aeruginosa s trains having R fac tors active against s uch therapeutically important drugs as gentamicin, carbenicillin,and tobramycin are being isolated and studied in differen t parts of the world ( 1 1- 15). An understanding of the mechanism of emergence of new antibiotic-resistance activities carried on p lasmids is therefore vital in order to cope with the magnit ude of this medical problem. One important step in s tudying the various R fac tors foun d in hospita l P. aeruginosa isolates is to group them on the bas is o f range of resistance, nature of resistance, transmissibility among Pseudomonas species as well as to members of the Enterobacteriaceae, and their compatibility characteristics. Other characteris­ tics tha t are also importan t are their effect on sensitivity toward some phages and on production of p yocin. The latter properties are important because certain R factors appear to change host characteristics s uch as pyocin typing or bacteriophage typ ing which are used in the epidemiolog ical identification of P. aeruginosa ( 16, 17). Compatibility Characteristics of Pseudomonas R Factors One o f the major criteria in the classification of Pseudomonas R factors has been their compatibility characteristics. Since many Pseudomonas p lasmids are trans­ missible to E. coli where their compatibility characteristics can be studied in relation to o ther E. coli p lasm ids and since several E. coli p lasm ids that normally do not occur in Pesudomonas can be transferred to Pseudomonas and grouped in relation to other Pseudomonas p lasm ids that cannot be transferred to E coli, a new classifi­ cation system has been devised on the basis of their interaction ( 18, 19). Table I shows the characteristics of some typical Pseudomonas R factors that eith er occur

to

CHAKRABARTY

Table 1 Properties of some typical drug-resistance plasmids in Pseudomonas

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Plasmid RP I RK2 R702 R I 033 R931 R130 pMGl pMG2 pMG5 RPLll R151 R64 R55 S-a RI13 R5265

Compatibility group

Drug resistance characteristics

PI

Cb, Km, Tc Cb, Km, Tc Sm, Km, Tc Gm, Km,Cm, Cb Sm, Tc Sm, Su,Gm Sm, Su,Gm Sm, Su,Gm Km, Su, Tm Cb, Sm,Tc, Gm, Su Sm, Su, Gm,Tm, Cb, Am Cb, Gm, Sm, Tc Am, Cm, Su, Sm, Km Km, Gm, Tm, Sm, Su Tc Sm,Su

PI PI

PI P2 P2 P2

P2

P2 P2 P3 P3 P3 W N

P4

Molecular weight (millions)

Reference

38

25

40 46 45 25 23

28 24 19 18 18 15 15

33

36 18 39 39 15,18 18 18

naturally in Pseudomonas or else have been transferred to Pseudomonas from other mic roorganisms. In general, they can be group ed under the compatibility groups PI, P 2, P3, P4, N, or W. PI Group Plasmids The plasmids belonging to group PI constitute group P in E. coli K l 2 ( 20). These plasmids can be t ransferred to a variet y of gram- negative bacteria including E. coli ( 2 1- 23). Most PI-group plasm ids specify resistance to Cb, Tc , Km, and sometimes Sm, S u, Cm, and Tp. Until recently, no plasmid from this group was known to confer resistance to Gm, but now a plasmid has been c haracterized that confers resistance to Gm in addition to Cb and ot hers (19). Several individual plasmids such as RPI, RP4, RI8 specify resistance to Cb, Tc, Km and appear to be of the same molecular size (24-26). Alt hough some of them have been isolated from different bacteria, they are p resumably the same plasmid. In order to avoid confusion, I designate such plasm ids as RPI . The molecular size of this plasmid is about 38 megadalton, and RPI and RK2 (molecular weight 40 miHion) can transform P. pulida as well as E. coli cells to antibiotic-resistance ( 27, 28). A var iety of male and female sex-sp ecific p hages have been isolated for cells harbor ing the PI-group plasmids. At least t hree classes of p hages recognize PI­ group plasmids: the RNA p hage P RRI ( 29), the DNA p hages PRDl , PR3, and PR4 (30--3 2), and filamentous p hage pO (3 1 , 32). The t hree DNA phages are not specific for PI-group plasmids and can infect bacteria that harbor plasmids of a different compatibility group such as W or N (32, 33). This type of broad host range has so far not been demonstrated for PRRI or pD . T his might be because PRRI

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PLASMIDS IN PSEUDOMONAS

11

adsorbs to PI-grou p plasmid-coded pi li i n E. coli and presumabl y to similar struc­ tures in P. aeruginosa. In contrast P RDI,P R3, and P R4 adsorb to the cell wall o f sensitive hosts. The fil a through the cell wall (32). Not all Pseudomonas species are sensitive to P RRI or PRDI type phages even when they harbor PI-group plasmids. Thus, P putida strai n P pGI is i nsensitive to both P RRI and P RDI phages even when it harbors plasmids such as RPI , R7SI, or RK2 (34). Differential sensitivity of hosts harboring PI-group plasmids toward certai n phages has also been demonstrated . The ability of RPI to i nhibit plati ng of G 10I or RPI -I to inhibi t B39 plating by a factor of I (T6 has been used to differentiate them from R factors such as R716 which i nhibits plating o f B39 totally (26,35). Plasmids of group P2 also appear to inhibit the pro pagation of several phages such as B3,D3, E 79, G lOI, P BI, or M6 (33). Plasmids of P2, P3, and Other Groups P lasmids that are transmissible among Pseudomonas species but not to E. coli constitute th e P2 compatibi lity group. Members of thi s group are perfectly compati ­ ble with plasmids belonging to groups PI, P3, W, and N ( l8, 33). P lasmids of the P2 group can confer resistance to a wide grou p of antibiotics (Table 1), and more notably a single plasmid can confer resistance to both carbenicillin and gentamicin, two important therapeu tic agents used in treatment against P. aeruginosa infections ( 17, 36). In general, the mechanism of gentamicin resi stance by Pseudomonas R factors involves acetylation as well as adenylation ( 1 7), and P2 group plasmids such as pMGI and pMG2 have been repor ted to i nacti vate gentamicin via gentamici n acetyltransferase ( 16). P2 plasmids such as pMGS which affords resistance to Km or Tm do so by elaboration of kanamyci n acetyltransferase, which can acetylate both kanamycin as well as tobramycin. Si nce kanamycin acetyltransferase does not i nactivate all the components of the gentamicin complex, strains harboring pMG 5 appear to be gentamicin sensitive. The only PI group plasmid that affords resi stance to gentamicin (RI033) does so by gentamicin acetyltransferase I and to kanamycin and neomycin by neomycin phosphotransferase I. Although i ncapable of bei ng transferred to E coli, many P2 group R factors specify antibiotic-modifying en­ zymes similar to those specified by typical E. coli R factors. Thus, the mechanism of R93 1 -mediated streptomycin r esistanc e i s phosphoryl atio n of streptomycin (37) while that of tetracycli ne resistance by the same R factor i s diminished permeability to the drug (38). As in some of the PI-group plasmids, the physical structure, density, and genetic homology of some P2-group plasm ids h ave been studied . Iso lation of R93 1 plasmid D NA by CsCI-ethidium bromide densi ty gradient centrifugation and subsequent electron microscopy h ave revealed two kinds of covalently c losed circular duplex molecules, one with a molecular weigh t o f I X 106 and the o ther with a molecular weight of 25 X 106• Both molecular types replicate under relaxed control. The buoyant densities for R93 1 and R5265 are 1 .7 18 g/cm3 (58% G + C) which is very close to that of th e PI group plasmid RPI . However, although the densities ar e the same,RPI has a higher molecular weight (38 X 106) than R93 1 (2 5 X 106) . P2 group

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CHAKRABARTY

plasmids li ke R 931 and R 130 also exhibit considerable genetic homology not only among t hemselves, but also wit h some PI group plasmids such as RP l , as deter­ mined by DNA-DNA hybridization studies ( 18) . The plasmids included in the P3 group constitute the group C (Com 6) in E coli (39) . They are transmissible among Pseudomonas and E. coli, and li ke PI and P2 group plasmids confer resistance to a number of antibiotics i ncludi ng gentamicin. A typic al P3 plasmid such as R64 (40) confers resistance to carbenici llin t hrough /3-lactamase and gentamicin t hrough formation of gentamicin adenylyltransferase, an enzyme able t o i nactivate both gentamicin and tobramycin. Similar ly another P3-group plasmid POW l5 1 (RI5 1), i n addition to gentamicin, streptomycin, and sulfonamide resistance, also confers resistance to tobramycin and carbenicillin ( 17, 4 1) . I n addition to conferring drug r esistance, P3-group plasmids such as R64 or R55 (39) have also been reported to inhibit the fertility of sex factors like FP2 (33) . Plasmids of W and N types do not appear to occ ur naturally in Pseudomonas, although they can be transferred from other genera to Pseudomonas. Thus, t he W group plasmid S-a can be transferred from Shigella to Pseudomonas alt hough it appears to be rather unstable. I n Pseudomonas S-a produces a high level of resi s­ tance to kanamycin, gentamicin, and tobramycin. P lasmids belongi ng to incompati­ bi lity group N, such as R46, have also been transferred t o Pseudomonas (1 5). The Transposition of Carbenicillin-Resistance Determinant of RP4 (RPJ) Plasmid A wide v ariet y of R plasmids contain genes specifying /3-lactamase, the m ost com­ mon t ype of which is /3-lactamase TE M (42). The gene responsible f or conferring resistance to Cb in RPI or RP4 specifies a TE M-type /3-lactam ase. Two general classes of /3-lactamases are known. The TEM l actamase has a high absolute activity of 60 to 1700 mU of /3- lactamase against benzyl penicillin per 109 R+ bacteria. Relative to its activity on benzylpenicillin, TE M has high activity on ampicillin and cephaloridine substrates, but low activity agai nst isoxazolyl penicillins such as ox­ acillin and met hicillin. A second /3-lactamase, the so-called oxacillin-hydrol yzing enzyme (0), specified by some R factors, can be differentiated from TE M by its lower absolute activity against benzylpenicilli n, and its ability to hydrolyze oxaci llin at an appreciable rate; two broad groups are distinguished on the basis of molecular weight and enzymatic activity against methicilli n (43). The /3-lactamase gene i n the PI-group plasmid RPI ( RP4 ) has recently been shown to undergo transposition from one replicon to another . The transposition of this gene from RP4 to the I-compatibility group plasmid R64 has been reported by Datta et al (20) and onto the E. coli chromosome by Richmond & Sykes (44). I n addition to these, transposition of the carbenicillin-resistance character has been shown by Hedges & Jacob (45) onto plasmids such as JR66a (compatibility group lw) or S-a (compatibility group W). Since t here is little genetic homology bet ween S-a and RP4 or J R66a and RP4, transposition c an take place in the absence of significant genetic homology. However , not all replicons can act as transposition acceptors, since nO transposition derivatives wit h R93 1 have so far been f ound. Wherever a transposition has been shown to occ ur, the m olecul ar wei ghts of t he

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PLASMIDS IN PSEUDOMONAS

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plasmids are increased by an amount between 1 .7 and 3 .9 million daltons. Since no covalently closed circular molecules with an average molecular weight of 2-4 mil­ lion have been detected in the cell , it is concluded that transposition involves i nsertion of a D NA sequence into the continuity of the recipient replicons, and that such a sequence is incapable of replicating as a separate plasmid. The transposable carbenicillin-resist ance determinant present in RP4 has been termed transposon A (45). The insertion of transposon A onto R64 appears to require a f unctional recA product , although the absolute requirement of recA gene product in transloca­ tion of the amp gene is not clear (46) . E xt ensive hybridization analyses amon g various R factor plasmids specifying TEM /3-lactamase have shown the presence of a common D NA sequence of about 2.7 X 106 to 3.3 X 106 daltons in size, corresponding to transposon A (47) . Th is sequence is present in a large number of naturally occurring, nonhomologous R plasmids r epresenting many compatibility groups. It h as been concluded that TEM /3-lactamase-specifying R factors arose as a result of the translocation of this se­ quence of D NA from plasmid to plasmid. The size of the D NA segment suggests that it codes for six or seven proteins in addition to the TE M /3-lactamase. Heteroduplex studies w ith the D NA isolated from a ser ies of recombinant plasmids where the TE M /3-lactamase translocon (TnA) was inserted into the 5.5 X 106 dalton nonconjugat ive plasmid R SF 1010 have shown that insert ions of TnA occur at , at least , 12 distinct sites in a region corresponding t o one third of the RSF 1010 D NA molecule (48) . Plasmids that acquire TnA show in all cases a single insertion of 3 .2 ± 0.3 X 106 daltons of D NA which is bounded by inverted complementary sequences; the specifi ingly, the insertion of TnA into the left-hand end of RSF 10 10 molecule results in the loss of streptomycin resistance, and polar insertions leading to loss of both sulfonamide and streptomycin resistance in RSF 1010 have also been observed. Transloeon A, Recombination, and Evolution 0/ Drug-Resistance Genes

The loss of streptomycin resistance or the simultaneous loss of sulfonamide and streptomycin resistance because of the insertion of transl ocon A onto RSF 10 10 indicates that this element may resemble the insertion sequences (IS) found on phage, viral , and bacterial chromosomal D NA (4 9, 50). It differs from the I S sequence i n that it i s larger i n size (4500 base pairs versus 800- 1400 base pairs f or I S sequences) and has an identifiable drug-resistance char acter. The presence of TnA in a variety of heterologous R factors of different compatibility groups clearly indicates that the translocatable TnA is responsible for bringing the ampicillin resistance character into th ese R factors and must be of prime impor tance in the evolution of the gene specifying TEM /3-lactamase. The ease with which transposi­ tion of TnA occurs from one replicon to anoth er suggests that the specific recogni­ tion sites are fairly common and the inverted repeat sequences are conserved during i nsertion and excision. I n the case of t etracycline resistance, the inverted repeats flanking the Tc resistance have been found to be h omologous w ith IS3. The much shorter inverted repeat found in TnA corresponds to no know n IS sequence (48). As a general phenomenon, transposition of resistance det er minants, bounded by

14

CHAKRABARTY

inverted repeated sequences, is bel ieved to be independent of the bacter ial recombi­ nation gene functions (48, 50) and may therefore involve completely new recombina­ tional processes. A thorough understanding of the mechanism of insert ion and excision of TnA and the enz ymes, if any, invol ved in these processes woul d be extremel y val uable in assessing the evol ut ion of various drug-resist ant genetic deter­ minants.

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Recombination of Pi-Group Plasmids with Other Replicons and the Chromosome Recombination among R factor pl asmids and the chromosome is important in the evol ution of various drug-resistance genes in the plasmids. C onsiderabl e efforts have, therefore, been directed toward a st udy of the recombinational proficiency of various Pseudomonas R factors, notabl y those belonging to the incompatibil it y group PI . Among PI group pl asmids, RPI has been w idely used, mainl y because of the presence of the translocatable segment , TnA, which enables the carbenicillin­ resistance gene to be transferred from one repl icon to another by nonreciprocal recombinat ional events. TnA is know n to be able to integrate w ith the E. coli chromosome (44) and can be picked up from the chromosomal locat ion by a superinfecting plasmid such as F' (51). This allows transfer of genet ic regions adjacent to the site of Tn A integration on a plasmid or the chromosome onto RPI or other repl icons. Olsen & Gonzalez (52) have described the recombination of the E. coli histidine operon with RPI to form a stable plasmid cointegr at e wh ich transfers the histidine operon with RPI in intraspecific E. coli matings. However, in crosses w ith E. coli and Salmonella or E. coli and Pseudomonas, transfer of his genes occur s at a lower frequency than that of RP I. This differential r ate of transfer has been interpreted as due to restriction of the E. coli his operon in these bacteria (52). Another interesting example of the recombination of chr omosomal genes carried on a F' derivative w ith RPI invol ves the pr esence of a homologous TnA sequence on both plasmids. Dixon et al (51) have recently described the construction of an p' derivative carrying the his region and the nitrogen fixation (nif) genes of Klebsiella pneumoniae chromosome. They have isolated a transposition derivat ive of this p' factor which acquired resistance to carbenicillin because of the transposi­ tion of TnA. I ntroduction of this p' factor harboring chromosomal his nif genes of Klebsiella and the TnA ofRP4 to a cell harboring RP4 has resulted in the formation of a recombinant plasmid (RP41, 59 Mdal) which retains the P-group compat ibilit y, RP4 drug-resistance genes but carries in addit ion the Klebsiella chromosomal his nif genes (5 1) . Because RP4 h as a broad h ost range, this recombinant plasmid can be transferred to a variety of microorganisms such as E. coli, Salmonella, Klebsiella, Agrobacterium, Rhizobium. The recombinant plasmid (RP41) h as been transferred from E. coli to a ml his deletion strain of K. pneumoniae or other strains of E. coli where the nif genes are fulIy expressed. However, although RP41 is transferable to Agrobacterium tumefaciens or Rhizobium meliloti, extensive segr egation of some plasmid-borne markers has been observed on their transfer to these bacteria and the nif genes appear not to be ex pressed in either of these two bacteria. The transfer of RP41 to His- mutants of P. aeruginosa has not been observed. In our laboratory, we h ave been able to transfer RP41 to a His- mutant of P. putida. AlI the drug-

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PLASMIDS IN PSEUDOMONAS

15

resistance and the his genes appear to be fully expressed, although the level of nitrogenase produced by RP4 1- positive P. putida cells is extremely small. Since P. putida is strictly aerobic and h as therefore to be grown in the presence of oxygen, the absence of a functional nitrogenase migh t be due to the oxygen sensitiv ity of th is enzyme. Isolation of mutants having an oxygen-insensitive nitrogenase would be very useful in studying the expression and transferability of nif genes among aerobic microorgan isms. The recombination of RPI with other R factors has also been studied with transductionally shortened RPI derivatives and during transduction of larger R fac tors too big to be accommodated in the ph age head, provided the recipient cells harbored RPI as a resident plasmid. Shipley & Olsen (53) have desc ribed an RPI segregant, termed RPI-S2, which confers resistance to carbenicillin and tetracycline and has a molecular weight of 23 million. This plasmid was f ormed dur ing transduc­ tion of RPI in S. typhimurium LT2 with phage P 22. This transductionally short­ ened plasmid segregant has lost the transfer, ph age sensitivity, and neomycin resistance functions of RPI . Interestingly, th is shortened plasmid can recombine readily with a W group plasmid, R388, to form a transmissible carbenicillin and trimethoprim resistance plasmid termed R WPl. Recombination between parent RPI and R388 is rare and has not been observed. Whe ther the recombinant plasmid RWPI retains carbenicillin resistance because of the transposition of TnA on to R388 is not c lear. Recombination among two P-group plasmids, RPI and R26 or R527, during transductional transfer of the latter has recently been reported (54). The plasmids R26 and R527 h ave been isolated from Pseudomonas and Serratia, respectively, and both belong to the P-incompatibility group. Both of them differ from RPI in having a wider antibiotic resistance spectrum (Cb, Tc, Nm, Sm, Gm, S u, H g) and a larger size (52 Mdal). The larger size of the plasmid prevents the entire D NA segment from being packaged in the head of phage Fl 16L which has a genome size of about 40 megadaltons. It has, however, been possible to isolate recombinant plasmids when the recipients h arbor a resident plasmid such as R 18-18 ( a Cb-sensitive mutant of RPI) which lacks some of the resistance genes present on R 26 or R527. The recombinant plasm ids show an increase in size (52 Mdal) and acquisition of addi­ tional resistance determinants such as Sm, Su, Gm, H g, but not Cb. It, the refore, seems that during tran sduction a 14 megadalton D NA segment specifyin g resistance to Sm, S u, Gm, Hg was transferred to the resident plasmid RI8- 18. S ince the recombinant plasmid exhibits all the characteristics present in the parent R26 or R527 plasmid (excepting the mutation at the c arbenicillin-resistance gene), it is like ly that such parent plasmids h ave evolved afte r translocation of a 14 Mdal segment from other R factors to a P-group plasmid. Similar to the f ormation of recombinant plasm ids h aving a larger size and wider antibiotic resistance spectrum by transductional marker rescue technique , construc­ tion of recombinant plasmids has also been ach ieved during conjugal transfe r. Jacoby et al (55) have recently demonstrated recombination between plasmids of compatibility groups PI and P 2 by introducing plasmids of both groups into donor P. aeruginosa and testing f or the ability of such donor cells to transfer resistances specified

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CHAKRABARTY

as such cannot be int roduced in E. coli. selection for P2-speci fied resistance pro­ duced exconjugants which harbored recombinant PI - plasmids wit h drug-resistance genes derived f rom the former. Recombinants of RPI harboring genes from pMGI, pMG2, or RPL 1 1 and R75 1 harboring genes f rom pMG 2 or pMG5 have been constructed this way. Analyses of t he si ze distribution of various recombinant plasmids i ndicate that segments v arying from 6 megadaltons to 3Z megadaltons are involved. T he functional expression of drug-resi stance genes specified by PZ plas­ mids, when present on PI replicons, appears to suggest that the inability of PZ plasmids to be transferred stabl y in E. coli might be due to their faulty repli cation in E. coli. T he inability of another plasmid RSUZ, which is not a PZ plasmid, to be t ransferred to E coli has been shown by Hedges & Jacob (56) to be due to an inability of t he plasmid to replicate in E. coli. Molecular Cloning with Pseudomonas R Factors Molecular cloning is the process whereby D NA from a v ariety of sources, both prokaryotic and e ukaryotic, can be ligated with a bacterial pl asmid or phage D NA (57, 5S) and i nserted into the cell where the inserted D NA replicates as part of the plasmi d or . p hage D NA. Since several PI-group plasmids have a single EcoRI si te and can be used to transform E. coli and P. putida to drug-re sistance (27,28), some PI-group plasmids such as RPI and RKZ have bee n used as cloning vectors for both P. putida (34) and E. coli (ZS). Since RKZ is also capable of transforming P. putida, it can be used for cloning in Pseudomonas as well . The relative merits of RPI and RK2 are unclear, as both appear to have alm ost identical properties. Ou tsi de E. coli, molecular cloning has been shown only wit h Pseudomonas. Since t ransmissible antibiotic- resistance plasmids wit h a broad host range are not consi dered safe as vectors, known chimeric plasmids such as pVH5 (ColE l-trp) have recently been used in transforming Trp- mutants of P. putida to T rp+ (34). S uch plasmids appear to repli cate under relaxed control in P. putida and might be useful for cloning and amplification of inse rted D NA i n Pseudomonas. Cryptic Plasmids D uring physical isolation of v arious plasmids, both f rom P. aeruginosa and P. putida, small covalently closed circular molecules have bee n ide ntified for which no f unctions are known. These are known as cryptic plasmids. Small minicircular D NA (I Mdal) of unknown function has bee n isolated from a P. aeruginosa st rain harbor­ i ng the P2-group plasmid R93 1 . It does not appear to have any homology wit h R93 1 as measured by D NA-DNA hybridization (IS). At least three kinds of circular D NA m olecules (1.7, 5.S, and 9.5 mdal) of unknown f unction have been isolated from P. aeruginosa strain PAO (59). We have recently ide ntified small circular molecules (size 6 Mdal) in t he Iysates of P. putida.

SEX-FACTOR PLASMIDS IN PSEUDOMONAS Sex-factor plasmids capable of i nitiating chromosomal gene transfer from one cell to another are now known for both P. aeruginosa and P. putida. E xcellent reviews

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PLASMIDS IN PSEUDOMONAS

17

detailing the characteris tics of P aeruginosa sex-fac tors FP2 and FP39 are avail­ able (60, 6 1). Sex-factors such as FP2 or FP39 occur r ather widely in various P aeruginosa s tr ains and exhibit different phenotypic traits . Thus, out of 48 strains th at appeared to have sex-factor activity, at least four also showed mercury-resist­ ance ch aracteristics similar to FP2 (62). FP39 carries a Leu+ gene (suppressor) corresponding to the leu-38 allele which normally maps at 48 min on the P. aeruginosa chromosome. Both these sex-fac tors are known to promote transfer of the chromosome from a single site of origin . Isolation and physical charac terization of FP2 and FP39 sex factors suggests that FP2 h as a density of 1 .717 g/cm3 corresponding to a G + C content of 58%. The contour length meas urements (28.5 ±: 0 .6 f.Lm) suggest the molecular weigh t to be about 55 m illion (59). The other sex-factor FP39 h as a density of 1 . 7 19 g/cm3 (G + C content 60%) with a molecular mass of 55 X 106 dalton (59). In addition to FP2 and FP39, a th ird sex-fac tor FP5 has been described which promotes chromosomal gene transfer at frequencies comparable to those of FP2 and FP39 (63). Like FP2, FP5 also exhibits mercury-resistance . It differs from FP2 in th at FP5 X FP2 crosses are fertile . Th us, FP2 and FP5 do not appear to be closely related. Th is is also borne out by the fact that unlike FP2 or FP39, strains h arboring FP5 can be cured of the sex-factor activity on treatment with acriflavin. In addition to typical sex-fac tors s uch as FP2 or FP39,certain R factors are also known to initiate chromosomal gene tr ansfer in P. aeruginosa (64). Thus, dr ug­ resistance plasmids s uch as R9 169 and R6886 mobilize chromosomal genes at frequencies which are even greater than those medi ated by the sex-factor FP2. Sex-Factor Plasmids in Pseudomonas Putida At least four kinds of plasmids are involved in mobilizing chromosomal genes in P pulida. However, the frequency is usually low with most of them, excepting one (factor K) wh ich has been used for mapping the P PUlido chromosome from a determination of the time of entry of individual markers during interr upted mating (65). The first one described for P pulido is pfdm, a defective phage containing the host's mande late genetic region (66). Since pfdm is derived by s ubstitution of a segment of a tr ansducing phage DNA with bacteri al chromosomal DNA, its ability to transfer chromosomal genes at a low frequency is pres umably due to its occa­ sional integration with the chromosome followed by a nonreciprocal excision. Al­ though not useful as a tool in the mapping of chromosomal linkage groups, pfdm represents an interesting example of defective particles of viral or igin capable of initiating gene transfer in bacteria. The other group of plasmids capable of mobiliz­ ing chromosomal genes constitutes the hydrocarbon degradative plasm ids. Th us, plasm ids s uch as CAM or TOL can mobilize chromosomal genes in P pUlido strain PpG 1 at a low frequency (67). The third group of plasm ids that can mobilize chromosomal genes are the R factors s uch as R6844 . R6844 can mobilize a number of chromosomal genes in P. pUlido strains PPE and PPN (68). The four th group of plasmids comprise typical sex-fac tor pJ asmids s uch as f ac tor K . This plasmid c an mobilize chromosomal genes at a very high frequency (10-1 to 10-2) to a number of recipients ( 7) and c an also mobilize non transmissible plasmids . Although not pre-

18

CHAKRABARTY

Table 2

Properties of some typical degradative plasmids

Mo lecular weight

Degradative pathway

Plasmid

Naphthalene Salicylate Camphor n-octane

NAH SAL

CAM OCT

p- or m-xylene p- or m-xylene

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XYL TOL

aND

=

Transmissibility

Conj ugative Conj uga tive Conj ugative Nonconjugative Nonconjugative Conjugative

(millions)

NDa 40, 55 150

ND

10 75

Not determined.

cise ly established, factor K appears to have a single transfer origin.and gene transfer takes place in an oriented manner (6 5). Preliminary data obtained by e lectron microscopic character ization offactor K suggest that it occurs as a covalently c lose d circular duple x molec ule of an average molecular we igh t of 80 million .

DEGRADATIVE PLASMIDS I N PSEUDOMONAS The de gradative pJasmids comprise a rather unique group of plasm ids each of wh ich specifies a se t of genes involved in the biodegradation of an or ganic compound (69). These plasmids occur naturally and can be either transmissible or nontransmissible . The transmissible plasmids, although transmissible among most Pseudomonas spe­ cies, have not been sh own to be transferred to members of other genera . Another interesting a spec t ofthese plasmids is their compatibilit y. E xcepting CAM and OCT ( 70) and SAL and Ta L , most degradative plasmids a ppear to be compatible with one another . Thus, they must be long to different incom patibility gr oups. The only degradative plasmid that has been teste d in relation to compatibility characteristics with R factors is CAM, which specifies the camphor de gradative path way (Table 2). CAM has been reported to be incompatible with a n umber of P 2- group R factors and has, there fore, been assigned to group P2 ( 1 5). Because of different compatibility characteristics, several hydrocarbon de gradative plasmids can be maintained in' a single bacterial strain to form a multiplasmid strain wh ich has an enhance d capabil­ ity to utilize cr ude oil ( 7 1 ). Table 2 de picts the characteristics of some of the degradative plasm ids. In general the evidence for the plasmid nature of these pathways has been genetic, although recently several ofthese degradative plasmids have been isolate d as covalently closed circles ( 72, 73). I describe briefly the characteristics of some individual plasmids. The CAM Plasmid That the genes specifying enzymes of the camphor pathway are borne on a plasmid has been determined by the enhanced frequenc y of curing of these genes on treat­ ment with mitomycin C, their ready transfer to P. aeruginosa and other Pseudomo­ nas specie s to which chromosomal gene transfer from P. pUlida is normally

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PLASMIDS IN PSEUDOMONAS

19

extremely rare, and the incompatibility of this gene cluster with a known plasmid ( 74). Unlike most other degradative plasmids, CAM does not carry a complete pathway, but rather converts camphor to isobutyrate, so that the recipient cells would have to be isobutyrate-positive in order to be able to grow with camphor. The CAM plasmid does carry some isobutyrate genes since several chromosomal isobu­ tyrate-negative mutants can accept and express the CAM plasmid, and transduc­ tional analyses of such mutants have indicated the presence of at least two complementation groups. Mutants incapable of growing with isobutyrate and pro­ pionate usually cannot accept CAM. Th us, many of the isobutyrate genes on CAM are redundant. The chromosomal isobutyrate enzymes are induced by isobutyrate but not by camphor. In contrast, D-camphor induces the isobutyrate enzymes spe cified by the CAM plasmid. It is tem pting to speculate that the isobutyrate structural genes, but not the regulatory genes, have been translocated onto the CAM plasmid, so that they are under the regulatory control of the cam oper on . CAM is also the plasmid whose genetic circularity ( Figure I) was predicted before the isolation and physical characterization of the covalently closed circular molecules ( 75). The first marker, the hydroxylase gene, in a Cam+ X Cam- cross appears to enter at 8 m in and the last marker enters at around 14 m in ( F dehydrogenase gene). Th us it takes 6 m inutes for the entire cam genes to be transferred from the donor to the recipient. If the rate of transfer of cam genes is essentially the same as that of chromosomal genes, and assuming it takes 90 min utes for the complete transfer

Figure 1

Genetic evidence of circularity of the CAM plasmid. cam 100 and cam 101 repre­

sent mutations in the hydroxylase genes, cam 120 and cam 121 in the

F dehydrogenase

gene,

cam 133 in the ketolactonase genes, and cam206 in the step between compound XI and isobutyrate (see for example references 69, 74, and 75). Numbers above the main circle represent the transductional cotransfer frequencies while those inside represent the time differ­ ence in minutes between the entry of any two markers.

20

CHAKRABARTY

of the chromosome of an approximate size 2.3 X 109 daltons, the size of the CAM plasmid is predicte d to be of the order of 150 Mdal. The molecular weight of 150 million of CAM, as determined by electro n micr oscopy of the i solated D NA m ole­ cules (S. Palchaudhuri and W. Maas, personal communication) is in good agreement with the genetic data.

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The SAL Plasmid The salicylate plasmid specifies a complete salicylate degradative path way i ncludi ng a functi onal meta path way ( 76). Unlike CAM, the SAL plasmid is transducible by phage pf 16 and the transductants are fully capable of conjugally transferring the SAL plasmid. During conjugal transfer, two types of e xconjugants are usually see n, fast-growi ng and slow-growing v ariants. Isolation of SAL plasmid D NA and elec­ tron microscopy h ave indicated two types ofm olecules, one with a molecular weight of 40 million and the other about 55 million (72). Isolation of D NA from the slow-growing v ariants indicates both types of m olecules. The NAH Plasmid The NAH plasmid specifies a complete degradative pathway for the metabolism of naphthalene, includi ng a functi onal meta pathway (77). Like SAL , the NAH plas­ mid is also tr ansducible by phage pfl6 . Since salicylate i s an i ntermedi ate i n naph­ thalene de gradation and NAH plasmid can also confer on the host ce lls the abi li ty to me tabolize salicy late , there might be a re lationship be tween these two plasmids. Ce lls harboring SAL are unable to grow with naphthalene and do not revert on a naphthalene plate . It would be interesti ng to isolate the SAL and NAH D NA and determine their ge netic homology by D NA-DNA hybridization as we ll as by elec­ tron microscopic heteroduplex analysi s to de termine if these two plasmids migh t h ave a common ance stry . The O C T Plasmid The pre se nce of the OCT plasmid allows the host ce ll t o grow with n-alkanes such as he xane , octane , decane ( 78). E nzymatic analyses h ave shown that there is a si ngle alkane hydroxy lating enzyme complex that initiates the monoterminal oxidation of the alkane chains and the resulting fatty acids are degraded exclusively by {3oxidation through formation of acetyl- and propionyl-coenzyme A ( 78 ). Like the CAM plasmid, the OCT plasmid codes for the inducible alkane hydroxylating and primary alcohol-dehydroge nating activities, but not the e ntire alkane to fatty aci d pathway (79). The e xistence of a plasmid-code d alcohol-dehydrogenating activity again shows considerable genetic redundancy on the degradative plasmids. It is possible that such redundancies correspondi ng to k nown chrom osomal ge nes might have allowed recombinati on between CAM and OCT t o form the fused CAM-OCT plasmid (69, 70). Careful e nzymatic analyses of mutants by Shapiro and his group have shown that in addition to primary alcohols and other compounds, unoxidized alkane m olecules f unction as the inducers of alkane hydroxylase proteins coded by the OCT plasmid (80).

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PLASMIDS IN PSEUDOMONAS

21

The transfer of the OCT plasmid from P. oleovorans to P. putida h as been very useful since OCT appe ars to undergo dissociation into three separate replicons: OCT, MER, and factor K (7, 8 1). In P. putida. OCT beh aves as a nontransmissible plasmid, rendered transmissible bec ause of the presence of the transfer plasmid factor K. Factor K is not only capable of mobilizing nontr ansmissible plasmids such as OCT and XYL (82), but it acts as a potent sex factor in P. putida. This property has recently been exploited in setting up an interrupte d mating technique so that individual c hromosomal genes can now be mapped from a determinati on of their time of entry (6 5). MER is a self-transmissible plasmid that c onfers on the host cells resistance to toxic concentrations of mercury. This prope rty is dealt with in the next section. The XYL and TOL Plasm ids An inte resting e xample of two separate plasmids specifying the same degradative function is XYL and TaL . The TaL plasmid is a transmissible plasmid ch aracter­ ized in P. putida (arvilla) mt-2 strain (83, 84). The presence of the TaL plasmid allows the host cells to degrade not only p- or m-toluates, but also the corresponding xylenes (8 5). Another plasmid, termed XYL in order to distinguish it from Ta L, has been c haracterized in a strain of Pseudomonas Pxy where the xylene path way is specified by a nontransmissible plasmid (82). Sincep- or m-toluate is an interme­ diate of xylene degradation and since the degradative pathways are designated based on the nature of theprimary substrate, we have subsequently refe rred to the Ta L plasmid as XAL ( 7 1). Excepting the difference in transmissibility and molecular sizes ( 73), XY L and Ta L appear to have identical xylene degradative genes, and the regulation of the two pathways appears to be the same . In order to gain further insight as to the extent of occurrence of such plasmids specifying a single degrada­ tive pathway, Williams & Worsey (86) have analyzed the ability of 13 bac te rial cultures isolated from 9 diffe rent soil samples to metabolize m -toluate . All 13 strains appear to carry TaL plasmids similar to the one characterized in P. putida (arvilla) mt-2. Eight of the isolates can transfer their Ta L plasmids into their own cured strains and several strains appear to h arbor flontr ansmissible TaL plasmids. Many of the strains also are nonfluorescent and some may not be pseudomonads. It is thus interesting that the toluate and xylene degradative pathways appear to be specified by genes borne on plasmids in so many different types of soi l bacteria. Since the XY L and Ta L plasmids have now been c haracterized by e lectron microscopy, it would be inte resting to isolate the plasmid DNA from all the soil bacteria harboring the Ta L plasmids and determine their genetic homology by D NA-D NA hybridization as well as by electron microscope heteroduplex analysis to gain an insight into the mechanism of evolution of such plasmids.

RESISTANCE TO MERCURY IONS Various plasmids in Staphylococcus aureus as well as in the enteric bacteria c onfer on their hosts the ability to withstand toxic c oncentrations .of a variety of inorganic metal ions such as mercury, c admium, lead, bismuth, n ickel, c obalt (87, 88). Plas-

22

CHAKRABARTY

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mids specifying such resistance are also known in Pseudomonas, although an exami­ nation of the range and level of resistance pattern has sh own th at mercury is the only metal against whi ch plasmid-mediated resistance is widespread in Pseudomo­ nas. Three kinds of plasmids have been reported to carr y mercur y-resistance genetic determinants (see Table 3 ): (a) sex-factors such as FP2 and FP5 (89 ); (b) drug­ resistance plasmids such as R902 , R2 6, R3 108, pMGl, pMG2, or pMG5 (35, 90-92); and (c) plasmids wh ose only known function is pr oviding resistance to mercury. P lasmids such as Hg-r (93) or MER (7) comprise this group. Mechanism ofAction of the Mercury-Resistance Plasm ids

There is consider able evidence now that in almost all the cases so far studied (94) the mercury-resistance plasmids specify enzymes th at reduce inorganic mercury to metallic mercury which can be volatilized from the cells. The ability to reduce inorganic mercury to metallic mercury ( H g2+ to HgO) by v arious E. coli and Pseudomonas strains harboring di fferent antibiotic-resistance plasm ids h as been demonstrated by a series of elegant studies by Schottel et al (94, 95), Silver et al (9 6), and Summers et al (97). Many mercury-resistance plasmids determine re­ si stance only to inorganic mercury ( H g2+ ), while some also determine resistance to organomercurials. Thus two out of four H g-resistance plasmids studied in P. aeruginosa can confer resistance to v arious organomercurials as well (95). Plas­ mids such as FP2 and R3 108 have, for example, been shown to confer on P. aeruginosa the ability to volatilize mercury fr om meth ylmercuric ch loride ( MMC) as well as from Hg(N03)2 and phenylmercuric acetate ( PMA). Plasmids pMG 1 and pMG2 appear to h ave a more narrow range of resistances as they can volatilize mercury from HgCh, but not from organ omercurials such as P MA. In all cases, plasmids that determine resistance to organomercurials convert the organomercuri­ als to metallic mercury and the corresponding hydrocarbon, i .e . PMA � Hgo + benzene; MMC � Hgo + methane, etc. Table 3 Some typicalm ercury-resistance plasm ids in Pseudomonas Naturea

Reference

R902

Am, Sm, Su, Hg

Con

90

R26

Cb, Tc, Nm/Km, Sm , Su, Gm, Hg

Con

35

R527

Cb, Tc, Nm/Km Sm, Su, Gm, Hg

Con

35

R931

Sm, Tc, Hg

Con

10

Plasmid

Resistance characteristics

Hg-r

Hg

Non-

Plasmids in Pseudomonas.

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