- Email: [email protected]
Suppression of the thermosensitive replication phenotype of the derivative plasmid of pI9789::Tn552 in Staphylococcus aureus may involve integration of the plasmid into the host chromosome
MICROBIOLOGY LETTERS
ELSEVIER
FEMS Microbiology
Letters I36 (1996) I29- 136
Suppression of the thermosensitive replication phenotype of the derivative plasmid of ~19789: :Tn 5.52 in Staphylococcus aureus may involve integration of the plasmid into the host chromosome Muhammad Sohail, Keith G.H. Dyke Microbiology
Unit, Department
of Biochemistry,
Soulh Parks Road, Unirersig
Received 8 November 1995; accepted 3 December
*
of O_Fford, Oxford
OX1 3Qll,
fJK
1995
Abstract Plasmid-chromosome co-integration was found to be the mechanism of choice to overcome thermosensitivity of replication of the plasmid pS1 in PS8Od and RN4220 strains of Sruphylococccrs UUWUS.The integration of the plasmid was sometimes accompanied by deletion of a specific section of the plasmid pS1 in PSSOd. Growth of bacteriophage on strains containing the integrated plasmid and the subsequent use of the phage in transduction gave transductants containing plasmids that had regained their replication thermosensitivity. These plasmids had not acquired any detectable chromosomal DNA. The 16-kb EcoRI fragment of the PS80d chromosome that hybridizes to pS1 is the target for recombination in many cases, but apparently other sites are also used. This fragment contains sequence homologous to parts of the transposon Tn552 and it is probable that site-specific recombination is involved in the integration. The possible mechanisms for the integrations and the deletions are discussed. Kep~ords:
Staphylococcus
aureus;
pI9789::Tn552;
Thermosensitivity;
1. Introduction Staphylococcus aureus can be a problem organism in nosocomial and community-acquired infections because it often possesses multiple antimicrobial resistances. The mechanisms of homologous recombination, site-specific recombination and transposition seem to have played a key role in the evolution of these multiresistant S. aureus [l-3]. Although some chromosomally specified resistance determinants may arise by mutation (e.g. resistance
_ Corresponding author. Tel.: +44 (1865) 275 293; Fax: +44 (1865) 275 297; E-mail: [email protected]. 0378.1097/96/$12.00 0 1996 Federation SSDI 0378- 1097(95)00488-2
of European
Microbiological
Plasmid-chromosome
integration
to nalidixic acid and rifampicin; [4]), not all chromosome-borne resistances are due to mutation. Evidence suggests that some, originally known to be present on plasmids, are now accumulating on the chromosome. In the majority of recently isolated multiresistant S. aureus strains, the determinants for resistance to erythromycin, tetracycline, streptomycin, spectinomycin. penicillin via a p-lactamase, etc., and to cadmium and mercury are found on the chromosome. Evidence from some more recent Australian epidemic staphylococcal strains suggests that the chromosomally located determinants for tetracycline, penicillin and mercury resistances are homologous to determinants which were plasmid-borne in Societies. All rights reserved
M. Sohuil, K.G.H.
130
D~kr / FEM.5 Microbio/oCq
isolates prior to 1970 151. Although accumulation of the resistance determinants on the chromosome is disadvantageous since the genes occur in a low copy number in the population and are less easily transferable to other bacteria, such accumulation provides a greater stability to the resistance determinants in the absence of selective pressure. It is thus of clinical importance to study the mechanisms involved in the accumulation of resistance determinants on the chromosome and hence plasmid integration into the chromosome. A thermosensitive derivative of the large, natustaphylococcal plasmid occurring rally pI9789::Tn_552, called pS1. was used in this study. This plasmid carries genes that confer resistance to metal ions such as cadmium, mercury, arsenate and also to penicillins. For a detailed account of the plasmid pI9789::Tn5.52 and its derivatives see [6,7].
2. Materials 2.1.
and methods
Bacterial
propagation
strains,
growth
conditions,
phage
and transduction
Bacterial strains used here and some of their characteristics are listed in Table 1. S. aureus strains containing a plasmid with thermosensitive replication were grown and maintained for routine use at 30°C on CY (yeast extract, 10 g l- ’ ; casein hydrolysate, 10 g I-‘; and MgSO,, 0.25 g l- ‘1, unless otherwise stated. Long-term storage was at -70°C in broth containing 25% (v/v) glycerol. Cadmium acetate was used at 26.6 wg ml ’ Phage propagation and transductions were done according to [8].
Lettrm
136 (IYY6I
/2Y-136
2.2. DNA preparation
and manipulation
Large-scale plasmid DNA from S. aureus was prepared according to [9]. Small-scale plasmid preparation was carried out by the alkaline lysis method [lo]. 7 ~1 of lysostaphin (10 mg ml-’ stock) was added per 100 ~1 of the cell suspension before the addition of SDS/NaOH lysis solution and the mixture was incubated at 42°C for 5-10 min. The phenol/chloroform purification step was replaced with two cleaning steps with StrataClean Resin’“’ from Stratagene which was used according to the manufacturer’s instructions. Total DNA from S. aureus was prepared as follows. Cells were harvested from 1.5 ml of an overnight culture and resuspended in 400 ~1 of TE (IO mM Tris . HCl pH 8.0. I mM EDTA) containing heat-treated RNase A at 10 pg mll ’ 6 ~1 of lysostaphin (10 mg mll’ stock) was added and the mixture was incubated at 42°C for 7 min. This was followed by addition of 20 ~1 of 10% SDS (v/v). 2 ~1 of proteinase K (10 mg ml ’ stock) and incubation at 37°C for 7 min. 70 ~1 of 5 M NaCl and 50 ~1 of CTAB (lo%, w/vl/NaCl (0.7 M) solution were then added and the mixture was incubated at 65°C for 3 min. After incubation, the sample was cooled slowly and extracted twice with phenol/chloroform/isoamyl alcohol (25/24/l, v/v/v) and once with chloroform/isoamyl alcohol (24/ 1, v/v). DNA was precipitated with 0.6 volumes of isopropanol at room temperature for 15 min, pelleted by centrifugation at 12 000 X g for 10 min and washed with 70% ethanol (v/v). The DNA pellet was suspended in 50 ~1 of sterile distilled water. Restriction enzyme digestions and agarose gel
Table I Bacterial strains Strain
Remarks
Reference
Chromosome carries genes for /3-lactamase production P-Lactamase negative derivative of PS9789 Propagating strain of S. ~~ureu.s phages 53 and 80 Restriction minus derivative of NCTC 8325 strain
[I II iI41
Primary host for pBR322- and pOX7-based
1101
s. aldrl?llS PS80 PS8Od 1054 RN4220
1161
1121
E. coli
JM107
procedures
clones
M. Sohail, K.G.H. Dyke/ FEMS Microbiology Letters 136 (1996) 129-136
electrophoresis, etc. were carried out according to [IO]. For Southern hybridization, DNA was blotted onto a nylon membrane [lo]. The labelling of the probes and the detection of hybridization were carried out using the ECL’” (Amersham) labelling/detection systems according to the manufacturer’s instructions. 2.3. Determination of thermosensitivity suppression The 5. aureus strain containing a thermosensitive plasmid was grown at 30°C for 18 h in 10 ml CY broth in a 50-ml flask. Optical density of the culture was measured at 675 nm and lo6 cfu were plated onto CY-Cd (26.6 pg ml-‘) agar plates. The plates were dried with lids off at 42°C for 30 min. The lids were then replaced and plates were incubated for another 17.5 h at 42°C. The number of cadmium-resistant colonies appearing on the plates was used as a measure of the thermostability of replication of the plasmid DNA. All experiments were performed in triplicate and repeated at least three times.
131
was grown on PS8Od(pSl) and the phage used to transduce pS1 to 1054 to give rise to 1054(pS 1). The plasmid pS1 was then transduced to RN4220 using phage 53 grown on 1054(pSl) to obtain RN422O(pSl). The presence of the correct transduced plasmid in RN4220 was verified by restriction enzyme analysis and testing for the expression of the phenotypic traits (e.g. /3-lactamase production, resistance to cadmium and mercury ions) conferred by the plasmid. The thermosensitivity suppression of the plasmid pS1 was then determined in both strains and the results show that the suppression frequency is influenced by the host strain. It is higher in PS80d (0.33 X 10m4 per bacterium) than in RN4220 (0.04 X 10m4 per bacterium). Twenty independently isolated thermostable colonies from each of the PS8Od(pSl) and RN422O(pSl) types were subjected to the procedure for preparation of plasmid DNA. No plasmid DNA was detected in any of these colonies and so it was assumed that at least the cadmium gene and probably the whole plasmid had integrated into the host chromosome. This assumption was investigated using bacteriophage transductions and Southem hybridization.
3. Results and discussion 3.2. Evidence of integration from phage transduc3. I. The suppression of thermosensitivity of the plasmid pS1 is strain-specific
The increase in the suppression frequency of pS1 in the presence of the transposon Tn551 on the chromosome of PS80d (i.e. in PS80d::Tn551) suggested that the composition of the host chromosome can influence the suppression and that there was some interaction between the host chromosome and the plasmid [8]. The effect of the host strain on the thermosensitivity suppression was thus investigated. Two strains of 5. aureus, PS80d [l I] and RN4220 [ 121 were used to study the suppression. The plasmid pS1 was transferred from PS80d to RN4220 using phage transduction. Two phages, phage 80 and phage 53 of the International Phage Typing System of 5. aureus [13] were used. The strain PS80d is susceptible to phage 80 only and RN4220 to phage 53 only. Another strain 1054 [14] of S. aureus is susceptible to both phage 80 and phage 53 and so served as a ‘bridging’ or ‘intermediate’ host strain in the transfer of the plasmid from PS80d to RN4220. Phage 80
tions and restriction endonuclease mapping
On excision of the integrated plasmid from the chromosome, covalent linkage between the plasmid DNA and some chromosomal DNA may be expected (e.g. see [ 153) and in the case of phenotypically cryptic DNA, restriction enzymes could be used to detect this linkage. The strain PS8Od was chosen for further work because of the higher frequency of the suppression of thermosensitivity obtained in this strain. Phage 80 was grown separately on 20 of the thermostable colonies of PS8Od(pSl) type and then used to transduce cadmium resistance to a new PS80d host. Two transductants were picked from each transduction and they were all found to contain a plasmid. Analysis of the uncut plasmid DNA revealed predominantly three sizes in the population of 40 plasmids. A total of 12 transductants (four from each of the three sets of the plasmids) were picked and their plasmid DNA analysed after digestion with EcoRI and BgZII. This allowed the grouping of the
into three types: Type I indistinguishable of about 3.2 kb in the large 10.2-kb ELwRI fragment (EcoA fragment): and Type III had a deletion of about 6.5 kb in the 10.2-kb EcvRI fragment of’ pS 1 (see Fig. I ). However, no non-psi DNA was detected in these three types. The positions of the deletion in the Type II and III plasmids were investigated more precisely. The deletions found in Type II and III plasmids were shown to be within the EcoA fragment and so replasmids
from PSI: Type II had a deletion
striction enzymes known to cut within this fragment were chosen. The restriction enzyme maps of the two types of the plasmids thus obtained are given in Fig. I. For both types, the deletions have removed most of the invertible region, itlr,. and appear to start at or near one W.S site. For Type II, the 3.2-kb deletion has deleted DNA between the A~c.1 site (2.05) and the MKI (5.7) that is situated in the MO region of Tn552. For Type III, the 6.5-kb deletion has remo\,ed
DNA
between
the Ac,c,l site (2.05)
and the
pS1 and Type I
Type III
PI
E’
EV
7
i
I 35 ,
Sn, 3.lli
37
1 :\_____________--________._______;I
4 rrs 3. Deletion m
irw
(-INegative
orientation
of inv
(+)Positive
orientation
of inv
Fig. I. Restriction of the Type
enqme
I plasmid
map5 4~owing dclrtion\
is indi\tingui\hahle
irzd on the plasmid PSI is capable of inverting B. &III:
C. Clol: EI. f?oRI:
Au.1 \ites at co-ordinate
M. Mvcl:
L\ ithtn the EcoA Ira~mcnr of the Types II and 111plauni&.
rrom that of pS I and i\ no1 shown separately.
The region tlanletl
and can occur in either of the two orientations.
N. N~kl:
PI. PI ul: PII.
2.05 i\ generated when the i/rl
Key to restriction
Pi 011: Sl. So/l: Sn. .S~~~tBI.Various
is in the negatl\c
orientation.
The restriction
ewymr
h! w.$ Gtes (invertible
co-ordinate\
enLyme name\:
map
region orA. Awl.
are shown. Note that the
M. Sohail. K.G.H.
D,vkr / FEMS Micmhiolog~
proximal Sal1 site (9.15) situated outside the transposon Tn.552, resulting in the deletion of the whole of Tn552. Although, inr was apparently involved in all of the deletions, none of the deletions were a precise deletion of the ins from the plasmid. Also, the deletion in the Type III plasmids is not a precise deletion of Tn552, since the CM site (8.8) of pS1 is absent and it is not within Tn552. Deletions were reported to occur in the plasmid pE194 when chromosomally integrated pE194 is excised from the chromosome [15]. The deletions recorded for pS1 during this study could be regarded as a result of imprecise excision during transduction.
(4
Letters
136 (IYY6)
129-136
133
However, the deletions in pS1 occur only when the plasmid is transduced from a thermostable derivative but not when pS1 was transduced from cells grown at 30°C. All three types of plasmid were found to be thermosensitive for replication and so it was concluded that the suppression was not due to the reversion of the mutation conferring thermosensitivity on the plasmid. The thermosensitivity suppression frequency of the Type I plasmids was approx. 0.35 X IO-” per bacterium, which is similar to that for the plasmid pS I, and that of Types II and III was approx. 0.1 X IO-’ per bacterium. However, one
(B)
2
Fig. 2. (A) Agarose gel stained with ethidium bromide. Total DNA was digested with EcoRI and electrophoresedon a 0.7% agarose gel for 14 h. The DNA was transferred to a nylon membrane and hybridized to the pS I probe (plus pUC I8 to illuminate the Raoul ladder; pUC I8 does not hybridize to PS80d, RN4220 or pS I DNA; data not shown). (B) An autolumigraph of the membrane showing hybridization of the plasmid pS I to the total DNA isolated from PSBo(pS 1) thermostable colonies. The plasmids pS I, Type II and Type III are also shown. For the plasmid pS I, in addition to the four EcoRI bands, there are fainter hybridizing bands at about 13.5 kb and 7 kb that do not correspond to any band seen in the corresponding lane on the ethidium bromide stained photograph. These bands have been ignored as either artifacts or minor contaminants.
134
M. Sohail. K. G.H. Dyke / FEMS Microbinlogy Letters 136 f 19961 12% 136
Type
11
multimer
EcoRI b
X
Recombination
EcoRl 4
PS80 chromosome
16kb
Chromosome of the Type II parent
I
Fig. 3. A possible route for the generation of the cadmium-resistant transduction. The drawings are not to scale. See text for details.
thermostable
bacteria
giving
rise to some Type II plasmids
on
M. Sohail, K.G.H. Dyke/FEMS MicrobiologyLetters136 (1996) 129-136
example of the Type I showed a higher suppression frequency (approx. 1.O X 1O-4 ), although indistinguishable from other members of the group on restriction analysis. 3.3, Evidence of plasmid integration from the Southem hybridization analysis
The chromosomes of the bacteria with integrated plasmids, which gave rise to the two types of deletions in the plasmid pS1 on transduction, were analysed using Southern hybridization in order to discover whether the deletions found in the two types of transductant plasmids produced during transduction were the consequence of integration/excision into/from the chromosome. The plasmid pS1 was digested with EcoRI and then used to probe total DNA derived from three thermostable colonies that gave rise to Type II plasmids and three that gave rise to Type III plasmids (Fig. 2). The pS1 probe hybridizes to a single approx. 16-kb EcoRI fragment of the PSSOd chromosome. Analysis of the PSSOd chromosome using more specific probes derived from within the transposon Tn.552 (e.g. binL, res and orf 271) showed that this 16-kb fragment contains most of Tn552 (data not presented). The chromosome of the P-lactamase-producing S. aureus strain PS9789, which has Tn552 on its chromosome and which gave rise to the plactamase-negative derivative PS80d [ 11,16,17], also showed a hybridization pattern similar to that of PSSOd. However, the chromosomal DNA of strain RN4220 did not show any homology to the pS1 probe (not shown). The thermostable colonies that gave rise to the Type II plasmids all contain the 16-kb hybridizing chromosomal fragment of PS80d and all possess the four EcoRI bands corresponding to those found in the Type II plasmid, thereby suggesting that the deletion found in the plasmids occurred at or before integration. All’three also show bands at 13.5 kb and 9.7 kb which could be the sum of the 16-kb fragment from the chromosome and 7.0 kb from the plasmid. Thus, a possible explanation is that growth at 42°C first caused or selected a deletion of 3.2 kb from within the 10.2-kb EcoRI fragment of pS1 to give the 7.0-kb piece. The integration may be unstable and reversible, thereby producing a mixture of bacte-
135
ria, some with an intact 16-kb fragment and the others without. There is a slightly lower intensity of hybridization of the 7.0-kb fragment as compared to the 7.9-kb and the 6.5-kb fragments, which is consistent with this hypothesis (Fig. 3). The hybridization patterns from the total DNA of the three thermostable colonies that gave rise to the Type III transductant plasmids are very similar and indicate recombination between the plasmid pS 1 and the PSSOd chromosome. Two EcoRI fragments, one each from the chromosome (16 kb) and the plasmid pS1 (10.2 kb) are replaced by two EcoRI fragments of 11 kb and 5.7 kb. The two new fragments may be a result of the recombination between PSI and the chromosome accompanied by a deletion in both the plasmid and the chromosome, since 16 kb + 10.2 kb (= 26.2 kb) generate 11 kb + 5.7 kb (= 16.7 kb). Moreover, the Type III plasmid has neither the 11-kb nor the 5.7-kb fragment, but does contain a 3.6-kb fragment (Fig. 2). Hybridization data and the ancestry of the strain PS80d [11,16,17] suggest the presence of a Tn552like structure on the 16-kb EcoRI fragment of the chromosome which is important in the ‘plasmid integration’ by providing a region of homology between the plasmid and the chromosome. The generation of the P-lactamase-negative phenotype in PSSOd may have been the result of a point mutation or a small deletion rather then deletion of the whole or most of the bla region from the chromosome as was previously suggested [ 111. Overall, the results can be summarized as below: 1. Plasmid-chromosome co-integration seems to be the most common mechanism to overcome thermosensitivity in both S. aureus PS8Od and RN4220. Such events may also have occurred in the natural populations of bacteria under conditions of stress and starvation or otherwise to promote integration of plasmids carrying genes for antibiotic resistances into the host chromosome. 2. The deletions observed in the transductant plasmids are not phage-mediated and occur on integration of the plasmid into the chromosome. The deletions always reside in the EcoA fragment of pS1 and one end of the deletions is near the res site, suggesting that this site is probably involved in forming co-integrates with the host chromo-
some. The EcoA fragment of PSI seems to be contain important DNA sequences which provide regions for recombination with other genomes. This fragment also contains a recombination site rsY789 at which it can form stable co-integrates with another plasmid pox1054 [IS] and a site which is a ‘hot spot’ for site- and orientationspecific transposition of Tn5.51 from the chromosome [8]. The functional analysis of such regions may help in the long run to design drugs/methods to control the spread of resistance determinants on introduction of a new antibiotic.
Acknowledgements M.S. was the recipient of the S&T Scholarship f rom the Government of Pak-
;;ay-89/G(5)3065)
References [II Sayre. P. and Miller. R. (1091) Bacterial
mobile genetic elements: importance in assessing the environmental fate of genetically engineered sequences. Plasmid 26, I5 I - I7 I. [21 Lyon, B.R. and Skurray, R.A. (1987) Antimicrobial resistance of Sruphylococcw ~I~IWUS: genetic basis. Microbial. Rev. 51, 88-134. [31 Cohen. S.N. (1976) Transposable genetic elements and plasmid evolution. Nature 263, 73 l-738. [41 Saunders. J.R. (1984) Genetics and evolution of antibiotic resistance. Br. Med. Bull. 40, 54-60. 151 Skurray, R.A.. Rouch. D.A.. Lyon, B.R., Gillespie. M.T., Tennent, J.M., Bryne, M.E.. Messerotti, L.J. and May. J.W. (1988) Multiresistant Sruphdococcus UUMUS: genetics and evolution of epidemic Australian strains. J. Antimicrob. Chemother. 21 (Suppl. C). 19-38.
[h] Rowland. S.-J. and Dyke, K.G.H. (1989) Characterization of the staphylococcal p-lactamase transposon Tn552. EMBO J. 8. 2761-2773. [7] Rowland. S.-J. and Dyke, K.G.H. (1990) Tn552. a novel transposable clement from Stcll~h~lococrus UY~US. Mol. Microhiol. 4. 961-975. [8] Sohail. M.. Oldridge, M. and Dyke. K.G.H. (1995) Interaction of the chromosomal Tn5.51 with two thermosensitive derivatives. pS I and p-1D. of the plasmid ~19789 in .Stcqh\~loc~oc~c~us LIUT~I~.\. FEMS Microbial. Lett. 127. I65- 170. [9] Novick, R.P. and Bouanchaud, D. (I 97 I ) Extrachromosomal nature of drug resistance in Stryhylococcus CIUWU.F.Annals N.Y. Acad. Sci. 182. 279-294. [IO] Sambrook, J., Fritach, E.F. and Maniatis. T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. [I I] Johnston, L.H. and Dyke, K.G.H. (1971) Stability of penicillinase plasmids in Stclph~kjc,occ.lrs nureus. J. Bacterial. 107. 68-73. [I21 Kreiswirth. B.H., LBfdahl, S., Betley. M.J., O’Reilly, M.. Schleivert. P.M., Bergdoll. M.S. and Novick. R.P. (1983) The toxic shock syndrome exotoxin is not detectably transmitted by a prophage. Nature (Land.) 305. 709-7 12. [ 131 Kloos. W.E. and Schleifer. K.H. (1986) Staphylococcus. In: Bergey’s Manual of Systematic Bacteriology (Williams. S.T.. Sharpe, M.E. and Holt. J.G., Eds.). Vol. 2. pp. 1013-1035. Williams & Wilkins, Baltimore, MD. [IA] Dyke, K.G.H. and Richmond, M.H. (1967) Occurrence of various types of penicillinase plasmids among hospital staphylococci. J. Clin. Pathol. 20. 75-79. [IS] Conrad. B.. Bashkirov, V.I. and Hofemeister, J. (1992) Imprecise excision of plasmid pEl94 from the chromosomes of Btrcillus suhti/i.\ pEl94 insertion strains. J. Bacterial. 174, 6997-7002. [ 161 Asheshov. E.H. (I 969) The genetics of penicillinase production in Srcrphylococcus curreu.s strain PSSO. J. Gen. Microbiol. 59, 289-301. [ 171 Johnston, L.H. and Dyke, K.G.H. (1974) Staphylococcal penicillinase plasmids: studies on the reversion of a temperature-sensitive replication mutant to temperature stability. J. Gen. Microbial. 82, 309-3 17. [ 181 Sohail, M. and Dyke. K.G.H. (1995) Sites for co-integration of large staphylococcal plasmids. Gene 162, 63-68.
ELSEVIER
FEMS Microbiology
Letters I36 (1996) I29- 136
Suppression of the thermosensitive replication phenotype of the derivative plasmid of ~19789: :Tn 5.52 in Staphylococcus aureus may involve integration of the plasmid into the host chromosome Muhammad Sohail, Keith G.H. Dyke Microbiology
Unit, Department
of Biochemistry,
Soulh Parks Road, Unirersig
Received 8 November 1995; accepted 3 December
*
of O_Fford, Oxford
OX1 3Qll,
fJK
1995
Abstract Plasmid-chromosome co-integration was found to be the mechanism of choice to overcome thermosensitivity of replication of the plasmid pS1 in PS8Od and RN4220 strains of Sruphylococccrs UUWUS.The integration of the plasmid was sometimes accompanied by deletion of a specific section of the plasmid pS1 in PSSOd. Growth of bacteriophage on strains containing the integrated plasmid and the subsequent use of the phage in transduction gave transductants containing plasmids that had regained their replication thermosensitivity. These plasmids had not acquired any detectable chromosomal DNA. The 16-kb EcoRI fragment of the PS80d chromosome that hybridizes to pS1 is the target for recombination in many cases, but apparently other sites are also used. This fragment contains sequence homologous to parts of the transposon Tn552 and it is probable that site-specific recombination is involved in the integration. The possible mechanisms for the integrations and the deletions are discussed. Kep~ords:
Staphylococcus
aureus;
pI9789::Tn552;
Thermosensitivity;
1. Introduction Staphylococcus aureus can be a problem organism in nosocomial and community-acquired infections because it often possesses multiple antimicrobial resistances. The mechanisms of homologous recombination, site-specific recombination and transposition seem to have played a key role in the evolution of these multiresistant S. aureus [l-3]. Although some chromosomally specified resistance determinants may arise by mutation (e.g. resistance
_ Corresponding author. Tel.: +44 (1865) 275 293; Fax: +44 (1865) 275 297; E-mail: [email protected]. 0378.1097/96/$12.00 0 1996 Federation SSDI 0378- 1097(95)00488-2
of European
Microbiological
Plasmid-chromosome
integration
to nalidixic acid and rifampicin; [4]), not all chromosome-borne resistances are due to mutation. Evidence suggests that some, originally known to be present on plasmids, are now accumulating on the chromosome. In the majority of recently isolated multiresistant S. aureus strains, the determinants for resistance to erythromycin, tetracycline, streptomycin, spectinomycin. penicillin via a p-lactamase, etc., and to cadmium and mercury are found on the chromosome. Evidence from some more recent Australian epidemic staphylococcal strains suggests that the chromosomally located determinants for tetracycline, penicillin and mercury resistances are homologous to determinants which were plasmid-borne in Societies. All rights reserved
M. Sohuil, K.G.H.
130
D~kr / FEM.5 Microbio/oCq
isolates prior to 1970 151. Although accumulation of the resistance determinants on the chromosome is disadvantageous since the genes occur in a low copy number in the population and are less easily transferable to other bacteria, such accumulation provides a greater stability to the resistance determinants in the absence of selective pressure. It is thus of clinical importance to study the mechanisms involved in the accumulation of resistance determinants on the chromosome and hence plasmid integration into the chromosome. A thermosensitive derivative of the large, natustaphylococcal plasmid occurring rally pI9789::Tn_552, called pS1. was used in this study. This plasmid carries genes that confer resistance to metal ions such as cadmium, mercury, arsenate and also to penicillins. For a detailed account of the plasmid pI9789::Tn5.52 and its derivatives see [6,7].
2. Materials 2.1.
and methods
Bacterial
propagation
strains,
growth
conditions,
phage
and transduction
Bacterial strains used here and some of their characteristics are listed in Table 1. S. aureus strains containing a plasmid with thermosensitive replication were grown and maintained for routine use at 30°C on CY (yeast extract, 10 g l- ’ ; casein hydrolysate, 10 g I-‘; and MgSO,, 0.25 g l- ‘1, unless otherwise stated. Long-term storage was at -70°C in broth containing 25% (v/v) glycerol. Cadmium acetate was used at 26.6 wg ml ’ Phage propagation and transductions were done according to [8].
Lettrm
136 (IYY6I
/2Y-136
2.2. DNA preparation
and manipulation
Large-scale plasmid DNA from S. aureus was prepared according to [9]. Small-scale plasmid preparation was carried out by the alkaline lysis method [lo]. 7 ~1 of lysostaphin (10 mg ml-’ stock) was added per 100 ~1 of the cell suspension before the addition of SDS/NaOH lysis solution and the mixture was incubated at 42°C for 5-10 min. The phenol/chloroform purification step was replaced with two cleaning steps with StrataClean Resin’“’ from Stratagene which was used according to the manufacturer’s instructions. Total DNA from S. aureus was prepared as follows. Cells were harvested from 1.5 ml of an overnight culture and resuspended in 400 ~1 of TE (IO mM Tris . HCl pH 8.0. I mM EDTA) containing heat-treated RNase A at 10 pg mll ’ 6 ~1 of lysostaphin (10 mg mll’ stock) was added and the mixture was incubated at 42°C for 7 min. This was followed by addition of 20 ~1 of 10% SDS (v/v). 2 ~1 of proteinase K (10 mg ml ’ stock) and incubation at 37°C for 7 min. 70 ~1 of 5 M NaCl and 50 ~1 of CTAB (lo%, w/vl/NaCl (0.7 M) solution were then added and the mixture was incubated at 65°C for 3 min. After incubation, the sample was cooled slowly and extracted twice with phenol/chloroform/isoamyl alcohol (25/24/l, v/v/v) and once with chloroform/isoamyl alcohol (24/ 1, v/v). DNA was precipitated with 0.6 volumes of isopropanol at room temperature for 15 min, pelleted by centrifugation at 12 000 X g for 10 min and washed with 70% ethanol (v/v). The DNA pellet was suspended in 50 ~1 of sterile distilled water. Restriction enzyme digestions and agarose gel
Table I Bacterial strains Strain
Remarks
Reference
Chromosome carries genes for /3-lactamase production P-Lactamase negative derivative of PS9789 Propagating strain of S. ~~ureu.s phages 53 and 80 Restriction minus derivative of NCTC 8325 strain
[I II iI41
Primary host for pBR322- and pOX7-based
1101
s. aldrl?llS PS80 PS8Od 1054 RN4220
1161
1121
E. coli
JM107
procedures
clones
M. Sohail, K.G.H. Dyke/ FEMS Microbiology Letters 136 (1996) 129-136
electrophoresis, etc. were carried out according to [IO]. For Southern hybridization, DNA was blotted onto a nylon membrane [lo]. The labelling of the probes and the detection of hybridization were carried out using the ECL’” (Amersham) labelling/detection systems according to the manufacturer’s instructions. 2.3. Determination of thermosensitivity suppression The 5. aureus strain containing a thermosensitive plasmid was grown at 30°C for 18 h in 10 ml CY broth in a 50-ml flask. Optical density of the culture was measured at 675 nm and lo6 cfu were plated onto CY-Cd (26.6 pg ml-‘) agar plates. The plates were dried with lids off at 42°C for 30 min. The lids were then replaced and plates were incubated for another 17.5 h at 42°C. The number of cadmium-resistant colonies appearing on the plates was used as a measure of the thermostability of replication of the plasmid DNA. All experiments were performed in triplicate and repeated at least three times.
131
was grown on PS8Od(pSl) and the phage used to transduce pS1 to 1054 to give rise to 1054(pS 1). The plasmid pS1 was then transduced to RN4220 using phage 53 grown on 1054(pSl) to obtain RN422O(pSl). The presence of the correct transduced plasmid in RN4220 was verified by restriction enzyme analysis and testing for the expression of the phenotypic traits (e.g. /3-lactamase production, resistance to cadmium and mercury ions) conferred by the plasmid. The thermosensitivity suppression of the plasmid pS1 was then determined in both strains and the results show that the suppression frequency is influenced by the host strain. It is higher in PS80d (0.33 X 10m4 per bacterium) than in RN4220 (0.04 X 10m4 per bacterium). Twenty independently isolated thermostable colonies from each of the PS8Od(pSl) and RN422O(pSl) types were subjected to the procedure for preparation of plasmid DNA. No plasmid DNA was detected in any of these colonies and so it was assumed that at least the cadmium gene and probably the whole plasmid had integrated into the host chromosome. This assumption was investigated using bacteriophage transductions and Southem hybridization.
3. Results and discussion 3.2. Evidence of integration from phage transduc3. I. The suppression of thermosensitivity of the plasmid pS1 is strain-specific
The increase in the suppression frequency of pS1 in the presence of the transposon Tn551 on the chromosome of PS80d (i.e. in PS80d::Tn551) suggested that the composition of the host chromosome can influence the suppression and that there was some interaction between the host chromosome and the plasmid [8]. The effect of the host strain on the thermosensitivity suppression was thus investigated. Two strains of 5. aureus, PS80d [l I] and RN4220 [ 121 were used to study the suppression. The plasmid pS1 was transferred from PS80d to RN4220 using phage transduction. Two phages, phage 80 and phage 53 of the International Phage Typing System of 5. aureus [13] were used. The strain PS80d is susceptible to phage 80 only and RN4220 to phage 53 only. Another strain 1054 [14] of S. aureus is susceptible to both phage 80 and phage 53 and so served as a ‘bridging’ or ‘intermediate’ host strain in the transfer of the plasmid from PS80d to RN4220. Phage 80
tions and restriction endonuclease mapping
On excision of the integrated plasmid from the chromosome, covalent linkage between the plasmid DNA and some chromosomal DNA may be expected (e.g. see [ 153) and in the case of phenotypically cryptic DNA, restriction enzymes could be used to detect this linkage. The strain PS8Od was chosen for further work because of the higher frequency of the suppression of thermosensitivity obtained in this strain. Phage 80 was grown separately on 20 of the thermostable colonies of PS8Od(pSl) type and then used to transduce cadmium resistance to a new PS80d host. Two transductants were picked from each transduction and they were all found to contain a plasmid. Analysis of the uncut plasmid DNA revealed predominantly three sizes in the population of 40 plasmids. A total of 12 transductants (four from each of the three sets of the plasmids) were picked and their plasmid DNA analysed after digestion with EcoRI and BgZII. This allowed the grouping of the
into three types: Type I indistinguishable of about 3.2 kb in the large 10.2-kb ELwRI fragment (EcoA fragment): and Type III had a deletion of about 6.5 kb in the 10.2-kb EcvRI fragment of’ pS 1 (see Fig. I ). However, no non-psi DNA was detected in these three types. The positions of the deletion in the Type II and III plasmids were investigated more precisely. The deletions found in Type II and III plasmids were shown to be within the EcoA fragment and so replasmids
from PSI: Type II had a deletion
striction enzymes known to cut within this fragment were chosen. The restriction enzyme maps of the two types of the plasmids thus obtained are given in Fig. I. For both types, the deletions have removed most of the invertible region, itlr,. and appear to start at or near one W.S site. For Type II, the 3.2-kb deletion has deleted DNA between the A~c.1 site (2.05) and the MKI (5.7) that is situated in the MO region of Tn552. For Type III, the 6.5-kb deletion has remo\,ed
DNA
between
the Ac,c,l site (2.05)
and the
pS1 and Type I
Type III
PI
E’
EV
7
i
I 35 ,
Sn, 3.lli
37
1 :\_____________--________._______;I
4 rrs 3. Deletion m
irw
(-INegative
orientation
of inv
(+)Positive
orientation
of inv
Fig. I. Restriction of the Type
enqme
I plasmid
map5 4~owing dclrtion\
is indi\tingui\hahle
irzd on the plasmid PSI is capable of inverting B. &III:
C. Clol: EI. f?oRI:
Au.1 \ites at co-ordinate
M. Mvcl:
L\ ithtn the EcoA Ira~mcnr of the Types II and 111plauni&.
rrom that of pS I and i\ no1 shown separately.
The region tlanletl
and can occur in either of the two orientations.
N. N~kl:
PI. PI ul: PII.
2.05 i\ generated when the i/rl
Key to restriction
Pi 011: Sl. So/l: Sn. .S~~~tBI.Various
is in the negatl\c
orientation.
The restriction
ewymr
h! w.$ Gtes (invertible
co-ordinate\
enLyme name\:
map
region orA. Awl.
are shown. Note that the
M. Sohail. K.G.H.
D,vkr / FEMS Micmhiolog~
proximal Sal1 site (9.15) situated outside the transposon Tn.552, resulting in the deletion of the whole of Tn552. Although, inr was apparently involved in all of the deletions, none of the deletions were a precise deletion of the ins from the plasmid. Also, the deletion in the Type III plasmids is not a precise deletion of Tn552, since the CM site (8.8) of pS1 is absent and it is not within Tn552. Deletions were reported to occur in the plasmid pE194 when chromosomally integrated pE194 is excised from the chromosome [15]. The deletions recorded for pS1 during this study could be regarded as a result of imprecise excision during transduction.
(4
Letters
136 (IYY6)
129-136
133
However, the deletions in pS1 occur only when the plasmid is transduced from a thermostable derivative but not when pS1 was transduced from cells grown at 30°C. All three types of plasmid were found to be thermosensitive for replication and so it was concluded that the suppression was not due to the reversion of the mutation conferring thermosensitivity on the plasmid. The thermosensitivity suppression frequency of the Type I plasmids was approx. 0.35 X IO-” per bacterium, which is similar to that for the plasmid pS I, and that of Types II and III was approx. 0.1 X IO-’ per bacterium. However, one
(B)
2
Fig. 2. (A) Agarose gel stained with ethidium bromide. Total DNA was digested with EcoRI and electrophoresedon a 0.7% agarose gel for 14 h. The DNA was transferred to a nylon membrane and hybridized to the pS I probe (plus pUC I8 to illuminate the Raoul ladder; pUC I8 does not hybridize to PS80d, RN4220 or pS I DNA; data not shown). (B) An autolumigraph of the membrane showing hybridization of the plasmid pS I to the total DNA isolated from PSBo(pS 1) thermostable colonies. The plasmids pS I, Type II and Type III are also shown. For the plasmid pS I, in addition to the four EcoRI bands, there are fainter hybridizing bands at about 13.5 kb and 7 kb that do not correspond to any band seen in the corresponding lane on the ethidium bromide stained photograph. These bands have been ignored as either artifacts or minor contaminants.
134
M. Sohail. K. G.H. Dyke / FEMS Microbinlogy Letters 136 f 19961 12% 136
Type
11
multimer
EcoRI b
X
Recombination
EcoRl 4
PS80 chromosome
16kb
Chromosome of the Type II parent
I
Fig. 3. A possible route for the generation of the cadmium-resistant transduction. The drawings are not to scale. See text for details.
thermostable
bacteria
giving
rise to some Type II plasmids
on
M. Sohail, K.G.H. Dyke/FEMS MicrobiologyLetters136 (1996) 129-136
example of the Type I showed a higher suppression frequency (approx. 1.O X 1O-4 ), although indistinguishable from other members of the group on restriction analysis. 3.3, Evidence of plasmid integration from the Southem hybridization analysis
The chromosomes of the bacteria with integrated plasmids, which gave rise to the two types of deletions in the plasmid pS1 on transduction, were analysed using Southern hybridization in order to discover whether the deletions found in the two types of transductant plasmids produced during transduction were the consequence of integration/excision into/from the chromosome. The plasmid pS1 was digested with EcoRI and then used to probe total DNA derived from three thermostable colonies that gave rise to Type II plasmids and three that gave rise to Type III plasmids (Fig. 2). The pS1 probe hybridizes to a single approx. 16-kb EcoRI fragment of the PSSOd chromosome. Analysis of the PSSOd chromosome using more specific probes derived from within the transposon Tn.552 (e.g. binL, res and orf 271) showed that this 16-kb fragment contains most of Tn552 (data not presented). The chromosome of the P-lactamase-producing S. aureus strain PS9789, which has Tn552 on its chromosome and which gave rise to the plactamase-negative derivative PS80d [ 11,16,17], also showed a hybridization pattern similar to that of PSSOd. However, the chromosomal DNA of strain RN4220 did not show any homology to the pS1 probe (not shown). The thermostable colonies that gave rise to the Type II plasmids all contain the 16-kb hybridizing chromosomal fragment of PS80d and all possess the four EcoRI bands corresponding to those found in the Type II plasmid, thereby suggesting that the deletion found in the plasmids occurred at or before integration. All’three also show bands at 13.5 kb and 9.7 kb which could be the sum of the 16-kb fragment from the chromosome and 7.0 kb from the plasmid. Thus, a possible explanation is that growth at 42°C first caused or selected a deletion of 3.2 kb from within the 10.2-kb EcoRI fragment of pS1 to give the 7.0-kb piece. The integration may be unstable and reversible, thereby producing a mixture of bacte-
135
ria, some with an intact 16-kb fragment and the others without. There is a slightly lower intensity of hybridization of the 7.0-kb fragment as compared to the 7.9-kb and the 6.5-kb fragments, which is consistent with this hypothesis (Fig. 3). The hybridization patterns from the total DNA of the three thermostable colonies that gave rise to the Type III transductant plasmids are very similar and indicate recombination between the plasmid pS 1 and the PSSOd chromosome. Two EcoRI fragments, one each from the chromosome (16 kb) and the plasmid pS1 (10.2 kb) are replaced by two EcoRI fragments of 11 kb and 5.7 kb. The two new fragments may be a result of the recombination between PSI and the chromosome accompanied by a deletion in both the plasmid and the chromosome, since 16 kb + 10.2 kb (= 26.2 kb) generate 11 kb + 5.7 kb (= 16.7 kb). Moreover, the Type III plasmid has neither the 11-kb nor the 5.7-kb fragment, but does contain a 3.6-kb fragment (Fig. 2). Hybridization data and the ancestry of the strain PS80d [11,16,17] suggest the presence of a Tn552like structure on the 16-kb EcoRI fragment of the chromosome which is important in the ‘plasmid integration’ by providing a region of homology between the plasmid and the chromosome. The generation of the P-lactamase-negative phenotype in PSSOd may have been the result of a point mutation or a small deletion rather then deletion of the whole or most of the bla region from the chromosome as was previously suggested [ 111. Overall, the results can be summarized as below: 1. Plasmid-chromosome co-integration seems to be the most common mechanism to overcome thermosensitivity in both S. aureus PS8Od and RN4220. Such events may also have occurred in the natural populations of bacteria under conditions of stress and starvation or otherwise to promote integration of plasmids carrying genes for antibiotic resistances into the host chromosome. 2. The deletions observed in the transductant plasmids are not phage-mediated and occur on integration of the plasmid into the chromosome. The deletions always reside in the EcoA fragment of pS1 and one end of the deletions is near the res site, suggesting that this site is probably involved in forming co-integrates with the host chromo-
some. The EcoA fragment of PSI seems to be contain important DNA sequences which provide regions for recombination with other genomes. This fragment also contains a recombination site rsY789 at which it can form stable co-integrates with another plasmid pox1054 [IS] and a site which is a ‘hot spot’ for site- and orientationspecific transposition of Tn5.51 from the chromosome [8]. The functional analysis of such regions may help in the long run to design drugs/methods to control the spread of resistance determinants on introduction of a new antibiotic.
Acknowledgements M.S. was the recipient of the S&T Scholarship f rom the Government of Pak-
;;ay-89/G(5)3065)
References [II Sayre. P. and Miller. R. (1091) Bacterial
mobile genetic elements: importance in assessing the environmental fate of genetically engineered sequences. Plasmid 26, I5 I - I7 I. [21 Lyon, B.R. and Skurray, R.A. (1987) Antimicrobial resistance of Sruphylococcw ~I~IWUS: genetic basis. Microbial. Rev. 51, 88-134. [31 Cohen. S.N. (1976) Transposable genetic elements and plasmid evolution. Nature 263, 73 l-738. [41 Saunders. J.R. (1984) Genetics and evolution of antibiotic resistance. Br. Med. Bull. 40, 54-60. 151 Skurray, R.A.. Rouch. D.A.. Lyon, B.R., Gillespie. M.T., Tennent, J.M., Bryne, M.E.. Messerotti, L.J. and May. J.W. (1988) Multiresistant Sruphdococcus UUMUS: genetics and evolution of epidemic Australian strains. J. Antimicrob. Chemother. 21 (Suppl. C). 19-38.
[h] Rowland. S.-J. and Dyke, K.G.H. (1989) Characterization of the staphylococcal p-lactamase transposon Tn552. EMBO J. 8. 2761-2773. [7] Rowland. S.-J. and Dyke, K.G.H. (1990) Tn552. a novel transposable clement from Stcll~h~lococrus UY~US. Mol. Microhiol. 4. 961-975. [8] Sohail. M.. Oldridge, M. and Dyke. K.G.H. (1995) Interaction of the chromosomal Tn5.51 with two thermosensitive derivatives. pS I and p-1D. of the plasmid ~19789 in .Stcqh\~loc~oc~c~us LIUT~I~.\. FEMS Microbial. Lett. 127. I65- 170. [9] Novick, R.P. and Bouanchaud, D. (I 97 I ) Extrachromosomal nature of drug resistance in Stryhylococcus CIUWU.F.Annals N.Y. Acad. Sci. 182. 279-294. [IO] Sambrook, J., Fritach, E.F. and Maniatis. T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. [I I] Johnston, L.H. and Dyke, K.G.H. (1971) Stability of penicillinase plasmids in Stclph~kjc,occ.lrs nureus. J. Bacterial. 107. 68-73. [I21 Kreiswirth. B.H., LBfdahl, S., Betley. M.J., O’Reilly, M.. Schleivert. P.M., Bergdoll. M.S. and Novick. R.P. (1983) The toxic shock syndrome exotoxin is not detectably transmitted by a prophage. Nature (Land.) 305. 709-7 12. [ 131 Kloos. W.E. and Schleifer. K.H. (1986) Staphylococcus. In: Bergey’s Manual of Systematic Bacteriology (Williams. S.T.. Sharpe, M.E. and Holt. J.G., Eds.). Vol. 2. pp. 1013-1035. Williams & Wilkins, Baltimore, MD. [IA] Dyke, K.G.H. and Richmond, M.H. (1967) Occurrence of various types of penicillinase plasmids among hospital staphylococci. J. Clin. Pathol. 20. 75-79. [IS] Conrad. B.. Bashkirov, V.I. and Hofemeister, J. (1992) Imprecise excision of plasmid pEl94 from the chromosomes of Btrcillus suhti/i.\ pEl94 insertion strains. J. Bacterial. 174, 6997-7002. [ 161 Asheshov. E.H. (I 969) The genetics of penicillinase production in Srcrphylococcus curreu.s strain PSSO. J. Gen. Microbiol. 59, 289-301. [ 171 Johnston, L.H. and Dyke, K.G.H. (1974) Staphylococcal penicillinase plasmids: studies on the reversion of a temperature-sensitive replication mutant to temperature stability. J. Gen. Microbial. 82, 309-3 17. [ 181 Sohail, M. and Dyke. K.G.H. (1995) Sites for co-integration of large staphylococcal plasmids. Gene 162, 63-68.