The molecular relatedness among α-hemolytic plasmids from various incompatibility groups

PLASMID 4, 76-81 (1980)

The Molecular Relatedness among cr-Hemolytic Plasmids from Various Incompatibility Groups

FERNANDO DE LA CRUZ,JUAN C.~ABALA,AND Jo& M. ORTIZ

Departamento de Bioquimica, Facultad de Medicina. Universidad de Santander, Santander, Spain

Received April 11, 1980

Deoxyribonucleic acid (DNA) reassociation studies among a-hemolytic (Hly) plasmids from FVI and FIII-IV incompatibility groups showed a close similarity between the nucleotide sequences of plasmids from the same group. With respect to R plasmids from the F overgroup, they have 20-26 Mdal in common, an amount of DNA close to the amount involved in the traF operon. No more extensive sequence homology was found between pSU316 (IncFIII-IV) and the incompatible plasmids ColB-K98 (IncFIII) or RI24 (In&IV). The IncIa 12 plasmid pSU5 has only the cu-hemolytic region (5 Mdal) in common with plasmid pSU3 16 but it is much more closely related to IncFVI plasmids where the DNA in common amounts to 22 Mdal. Finally, the genetically unrelated plasmid pSU233 shares 66% of its nucleotide sequences (40 Mdal) with the IncFVI plasmids and has 16-23 Mdal in common with various F-like plasmids.

The synthesis of the a-hemolysin toxin, in some E. coli strains, is encoded by a class of extrachromosomal elements known as Hly plasmids (Smith and Halls, 1967). They are interesting in two respects: (i) genes needed for a-hemolysin expression constitute a complex system in which three clustered cistrons are involved (Noegel et al., 1979), and (ii) incompatibility studies on Hly plasmids indicate that they can be found among different incompatibility groups, some of them uncommon, or even unknown, in classifications confined to R, Cal, or Enr plasmids (de la Cruz et al., 1979; this work). Two incompatibility groups, FIII-IV and FVI, have been found to occur frequently among Hly plasmids, but plasmids which do not belong to these groups have also been found.

To answer the question of whether the genes determining cy-hemolysin synthesis on different plasmids are identical or not, re- striction fragments involved both in cY-hemolysin synthesis and extrusion, characterized from plasmid pHly152, were cloned and then hybridized to Hly plasmids by the method of Southern (Southern, 1975). It was found (de la Cruz et al., submitted for publication) that a stretch of DNA of 3 8 Mdall which con-

‘Abbreviations used: Mdal, megadaltons.

tains the hemolysin genes of plasmid pHly- 152, is common to every plasmid tested, and this suggests in turn a spread of a given (Y- hemolysin gene to plasmids of a range of incompatibility groups.

Royer-Pokora and Goebel (1976) studied various situations in wild-type strains in which Hly plasmids coexisted in the same cell with other plasmids. After conjugation, two plasmids of this type could undergo recombination, and this lead to the detection of varying degrees of homology between the plasmids which were formed by the process.

In this work we have studied the polynucleotide sequence relationships among various Hly plasmids as well as their relatedness to plasmids specifying other genetic char- acters and belonging to other incompatibility groups. Wide variations in the extent of DNA homology have been found.

MATERIALS AND METHODS

Plasmids. All Hly plasmids studied in this work have been characterized previously (de la Cruz et al., 1979) and are listed in Table 1. The representatives of plasmid incompatibility groups used are IncFI (R386), IncFII (Rl), IncFIII (ColB-K98), IncFIV (R124), IncIa (R64drdl l), IncI2 (TPl14), and IncN

0147-619Xl80@40076-06$02.00/O Copvright 0 1980 by Academic Press, Inc. Au ri&ts of repmdu&,n in any for,,, reserved.

76

RELATEDNESS IN HEMOLYTIC PLASMIDS 77

TABLE 1

PLASMIDS STUDIED AND SOMEOETHEIRRELEVANTCHARACTERISTICS

Plasmid”

psu1 psu105 psu212 pSU316 pIP240 pSU233 psus

Incompatibility group

IncFVI IncFVI IncFVI IncFIII-IV IncFIII-IV Inc? In&x12

Relevant phenotype*

Fi+(F),Tra+,Dps(MSZ),Hly+ Fi+(F),Tra+,Dps(MS2),Hly+ Fi+(F),Tra+,Dps(MS2),Hly+ Fi+(F),Tra+,Dps(MS2),Hly+ Fi+(F),Tra+,Dps(MS2),Hly+ Fi+(F),Tra+,Hly+ Fi-(F),Tra+,Dps(Ifl),Hly+

Molecular mass

(X lo6 dal)

77 77 72 48 52 60 93

a All Hly plasmids have been described previously (de la Cruz et al., 1979). b Symbols are those recommended by Novick et al. (1976).

(R113). They are described by Jacob et al. (1977).

Isolation of plasmid DNA. Minimal medium M9 (Roberts et al., 1955) supplemented with 0.2% glucose and 5 mg/ml casaminoacids (Difco) containing radioactive thymine was used to label Hly plasmid DNA in E. coli K-12 strain CR34 (F-,thy-,Bl-,Nx’). In gen- eral, cultures (15 ml) containing [3H]thymine (26.6 &i/ml), at a final concentration of 1.0 pg/ml, were inoculated with fresh unshaken overnight cultures to give about 5 x 10’ cells/ ml. They were then incubated at 37°C with shaking, until the cell titer reached about log cells/ml.

Cleared lysates were obtained by the method of Clewell and Helinski (1969). Plas- mid DNA was isolated by cesium chloride/ ethidium bromide density gradient centrifugation and recovered from gradients as described by Barth and Grinter (1974). After extraction with CsCl-saturated isopropanol (four times), DNA preparations were diluted with 2 vol of (10 mM Tris, pH 7.9, 10 mM NaCl) and one-third volume of 3 M sodium acetate and precipitated with 6 vol of ice-cold ethanol. The precipitated DNA was then dis- solved in Q.42 M NaCl and stored at -20°C.

Unlabeled plasmid DNA was obtained after polyethyleneglycol precipitation and density gradient centrifugation (Humphreys et al., 1975) of cleared lysates from cultures (1.5 liters) grown to the stationary phase in Anti-

biotic Medium 3 (Difco). The supercoiled DNA bands were recovered from gradients and treated as above.

DNA shearing and denaturation. Labeled and unlabeled plasmid DNA was sheared by ultrasonic treatment in a cell disintegrator to give DNA fragments of an approximate molecular weight of 2.5 x lo5 (Falkow and Cita- rella, 1965). Sonication was accomplished by giving bursts at 50 W of energy output for 30 s followed by pauses for cooling at 4°C for a total period of 5 min. DNA was denatured by heating for 15 min in boiling water and cooling rapidly in ice water.

DNA -DNA reassociation. Sonicated 3H- labeled plasmid DNA was diluted with 0.42 M NaCl to give 2 X lo4 cpmlml. Duplicated samples (0.1 ml) were mixed in polystyrene tubes with 8 pg of unlabeled sonicated plasmid DNA to a final volume of 0.4 ml. The DNA mixture was then denatured and incubated at 75°C for 16 to 20 h depending on plasmid size.

At the end of the reassociation period, re- sidual single-stranded DNA was hydrolyzed at 75°C with 20 units of Sl endonuclease (Sigma Chemical Co.) as described by Barth and Grinter (1975).

Calculations. The ratios of counts from each pair of tubes used in a hybridization give: s, the proportion of single-stranded DNA not hydrolyzed by the enzyme, when denatured labeled DNA was hybridized to

78 DE LA CRUZ, ZABALA, AND ORTIZ

8 pg of calf thymus DNA; d, the proportion of double-stranded DNA not hydrolyzed by the enzyme, when nondenatured labeled DNA was hybridized to 8 pg of calf thymus DNA; h, the proportion of DNA after hybridization not hydrolyzed by the enzyme. H, the proportion of DNA hybridized was calculated by the expression given by Barth and Grinter (1975):

RESULTS AND DISCUSSION

Polynucleotide Sequence Relationships among IncFVI Plasmids and Other Plasmids

The existence of a new incompatibility

H = (h - s)l(d - s).

group (IncFVI) within the F overgroup was suggested by Monti-Bragadin et al. (1975) and later confirmed in our laboratory by the finding of two Hly plasmids that were incompatible with pSU212 (de la Cruz et al ., 1979).

Homologous reassociations usually gave Usually, plasmids from the same incompat- values of around 80%. Other values were then ibility group share a considerable amount of corrected after normalizing the value for their DNA sequences in common. This was homologous reassociation to 100%. Four in- also the case for IncFVI plasmids as is shown dependent determinations were performed in Table 2. Labeled DNA from plasmid between each pair of radioactive and un- pSU 105, reassociated with plasmids pSU 1 labeled DNAs, and a mean value was cal- and pSU212, showed, respectively, 100 and culated. 88% DNA homology under the stringent con-

TABLE 2

DNA SEQUENCEHOMOLOGYBETWEEN Hly AND OTHER PLASMIDS

Unlabeled plasmid DNA

Compatibility group Plasmid

3H-Labeled plasmid DNA

psu105 pSU233 pSU316

% Mdal % Mdal % Mdal

IncFVI

IncFIII-IV

Inc?

IncFI R386 27 IncFII RI 31 IncFIII ColB-K98 34 IncFIV R124 26

IncIo IncI2

R64drd 11 TP114

IncN Rl13

psu105 100 (77)” 61 (37) psu1 100 (77) 73 w psu212 88 w-3 64 (38)

pSU3 16 pIP240

pSU233

psus

30 (23) 46 (28) 34 (26) 44 (26)

52 loo (‘30)

29 18 (11)

(21) (24)

26 (16) 37 (22) 39 (23) 29 (17)

8 0

0

(5) (0)

(0)

44 (21) -b -

100 (48) 85 (41)

56 (27)

10 (5)

45 (22) 50 (24) 53 (25) 53 (25)

7 (3) 0 (0)

0 (0)

a The values indicate the percentage of reassociation at 75°C (in brackets the equivalent data expressed in M&l) of SH-labeled plasmid DNA with unlabeled plasmid DNA related to the reassociation with DNA of the same plasmid (= 100) and with calfthymus DNA (=O). These values are the average of four independent determinations.

b Indicates that the experiment has not been done.


ditions of reassociation employed. Reasso- ciation experiments carried out between plasmid pSU105 and other F-like plasmids, including the two IncFIII-IV Hly plasmids, pSU3 16 and pIP240, indicated that there was 26-34% of homology between them all. This represents 20-26 Mdal of homologous DNA, that is close to the size of the truF operon, which is known to be common to all F-like plasmids (Moller et al., 1978). Taking this data together with the genetic evidence shown in Table 1, it is likely that IncFVI plasmids share the truF region with other F-like plasmids. IncFVI Hly plasmids do not appear to have a closer relationship with any of the other F-like groups, the FIII-IV Hly plasmids included. In contrast, little or no homology at all was found between pSU105 and plasmids of the Ia, 12, or N incompatibility groups.

The Polynucleotide Sequence Relationships of Hly Plasmids from the FM-IV Incompatibility Complex

The cw-hemolytic plasmids pSU316 and pIP240 have been described as members of an uncommon FIII-IV incompatibility complex because they are simultaneously incompatible with ColB-K98 (IncFIII) and R124 (IncFIV) (de la Cruz et al., 1979).

Plasmids pSU316 and pIP240 share 85% of their sequences in common. However, when plasmid pSU316 was allowed to re- associate with plasmids of the different F-like incompatibility groups it was found that it had 22-25 Mdal in common with all of them, similarly to what happened with the IncFVI plasmid pSU105 when it was used the same way. This degree of homology was less than expected since plasmid pSU316 is incompatible with both ColB-K98 and R124.

Similar results were obtained by Willshaw et al. (1978) for FI and FIme plasmids. Also, Palchaudhuri and Maas (1977) have examined the DNA sequence relationship between the classical F factor and the IncFI plasmids, ColV-K94 and EntP307, by heteroduplex analysis. Although all these plasmids are mutually incompatible, the extent of their

DNA sequence homology is approximately the same as that of F when tested with other compatible F-like R factors.

As discussed previously (de la Cruz et al., 1979) a plausible explanation for the double incompatibility of pSU3 16 is that this plasmid has two distinct mechanisms that give rise to incompatibility. In this study, an analogous situation has been found between F and pSU3 16: they do not share extended regions of DNA sequence homology (apart from the truF segment) with plasmids with which they are incompatible.

As with pSU105, plasmid pSU316 has practically no homology with plasmids of incompatibility groups unrelated to F (Ia, 12, or N); it has only 5 Mdal in common with the Hly plasmid pSU5 (Table 2). This probably reflects homology between the a-hemolysin regions of the plasmids (de la Cruz et ul., submitted for publication).

The Polynucleotide Sequence Relationships between Other Hly Plasmids Not Included in the Previous Groups

Plasmid pSU233 is an a-hemolytic fi+ plasmid compatible with all F-like and I-like plasmids. It is also resistant to infection by F, I, P, N, and W sex-specific bacteriophages (de la Cruz, unpublished results). When reassociated with representatives of IncF groups (Table 2), it showed 16-23 Mdal of homologous DNA. The extent of homology was higher (26-28 Mdal) when hybridized with the IncFIII-IV plasmids pSU316 and pIP240, but significantly lower (11 Mdal) when plasmid pSU5 was used. In contrast, the IncFVI plasmids showed 37-44 Mdal in common with plasmid pSU233, and this suggests that about 66% of pSU233 DNA sequences are homologous to the DNA of FVI plasmids. The plasmid pSU233 has 5 Mdal in common with R64drdll and it is nonhomologous with the other plasmids tested. These results suggest a strong phylogenetic relationship between pSU233 and the IncFVI plasmids. Moreover, the extended homology between pSU233 and all IncF plasmids pre-

80 DE LA CRUZ, ZABALA, AND ORTIZ

diets the existence of an F-like pilus structure. However, pSU233 does not propagate the F- specific phage MS2. This situation may have arisen in at least two ways: first, plasmid pSU233 (and a Tn802 derivative of it, named pSU221, which is incompatible with pSU- 233) may constitute a new incompatibility group particularly related to IncFVI. Second, plasmid pSU233 might be the result of a recombination event by which about 40 Mdal of an IncFVI plasmid (probably not including its replication region, because plasmid pSU- 233 is fully compatible with all IncFVI plasmids) have been linked to another replicon. Such an event might not be surprising, particularly in the light of the finding of Royer- Pokora and Goebel(1976) mentioned in the introduction.

Plasmid pSU5 is fi-, incompatible with R64drdll (IncIcu) and with TPl14 (IncI2). It propagates the I-specific phage If1 efficiently (de la Cruz, unpublished). Because of its large size (93 MdaI) and its high degree of homology with the IncI2 hemolytic plasmid pHly152, we suspect it to be a complex IncIaI2 replicon (de la Cruz et al., submitted for publication). Its 5 Mdal of homology with plasmid pSU- 3 16 reflects, as mentioned above, the presence of the cY-hemolytic region. It has also 11 Mdal in common with plasmid pSU233. Since plasmids R64drd 11 and pSU233 have 5 Mdal of homologous DNA, it may be expected that plasmid pSU5 contains this fragment too, since it is incompatible with R64drd 11. Thus, the total amount of homology between plasmids pSU5 and pSU233 would be about 11 Mdal: 5 Mdal corresponding to that DNA segment and 6 Mdal of known homology in their hemolytic regions. This correlates very well with the observed value. However, the high homology between pSU5 and the Inc- FVI plasmid pSU105, that amounts to 22 Mdal, cannot be explained so satisfactorily. It is supposed that these two plasmids share no more than 5.5 Mdal around their hemolytic regions because nonhomologous regions have been found at the boundaries of this DNA segment (de la Cruz et al., submitted for publication). Moreover, their homologous

region cannot be part of the traF operon, as pSU5 does not hybridize with pSU3 16 (Inc- FIII-IV) outside the hemolytic genes. Thus, this result suggests that there is a stretch of about 15 Mdal of homologous DNA between plasmids pSU5 and pSU105 apart from the hemolytic region and not contiguous with it.

All these results taken together suggest some conclusions about Hfy plasmid evolu- tion. The variable degrees of homology found among the different Hly plasmids, together with the finding that all of them encode for indistinguishable a-hemolysin determinants, suggest that active mechanisms of recombination have been involved. The transposition of the cr-hemolysin genes has not yet been proved to occur although some mechanism of illegitimate recombination has al- most certainly to be involved in the spread of this character. However, the wide limits in the extent of DNA sequence homologies (40 Mdal between pSUlO5 and pSU233, 22 Mdal between pSU105 and pSU5, but only 5 Mdal between pSU5 and pSU316) cannot solely be interpreted as the presence of an c+hemolysin gene plus or less a truF region. More likely, they indicate a variation in the amount of DNA that underwent recombination in the course of the construction of the plasmid. Some mechanism such as cointe- grate formation followed by resolution in- volving different deletions can be suspected if the results of Royer-Pokora and Goebel (1976) and the replicons analyzed in this work are anything to go by; another example of this kind of recombination among R factors has recently been reported (Nugent and Hedges, 1979). Whatever the mechanism may be, recombination of these genes and their spread to different replicons, does not elucidate the whole of the problem. One has to enquire why Hly plasmids are found mainly, if not solely, in incompatibility groups in which no R factors have been found, and why the hfy genes have never been found linked to resistance determinants. Hly plasmids seem to form a coherent group with complex relationships among themselves (see for example the ho-


mology between pSU5 and the IncFVI plasmids or between FVI plasmids and pSU- 233) but distinct, in evolutionary terms, from R factors even of the same incompatibility group.

ACKNOWLEDGMENTS

F.C. and J.C.Z. were supported by studentships of the Plan de Formation de1 Personal Investigador. We gratefully acknowledge a detailed protocol for the Sl assays received from Dr. Julian Davies. We thank Dr. M. H. Richmond for critical reading and correction of the manuscript.

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Documents

The molecular relatedness among α-hemolytic plasmids from various incompatibility groups