FEMS MicrobiologyLetters 93 (1902) 75-82 .~ 1992 Federation of European MicrobiologicalSocieties11378-11)97/92/$115.1X) Published by Elsevier

FEMSLE (14878

The introduction of colonic-Bacteroides shuttle plasmids into Porphyromonas gingicalis: Identification of a putative P. gingivalis insertion-sequence element J a n e t t e M a l e y ", N a d j a B. S h o e m a k c r b and l a n S. R o b e r t s " " Department of Microbioh~gy, Uniter.~ity of I.ciccstcr. Lcict'.~ter, UK. and t, Dcln:rnncn t t~]"Microhioh~hs', Ut~icersity c~l"Illinois, Urbana. IL, USA

Received 13 FebruaD'1992 Accepted 25 Februat)' 1992

Key words: Porphy~t,,nonas gingicalis: Plasmid transfer; Insertion sequence

!. S U M M A R Y Two Escherichia coli-Bacterokles plasmid-shuttie vectors pNJR5 and pNJR12 .~ere introduced for the first time into Porphyromonas gingiralis W83 by conjugal transfer from EL coli. The transfer frequencies were comparable to those obtained when using colonic BacteroMes as recipients. Both plasmids were maintained in P. gingicalis W83 and could be isolated and introduced back into E. co6. Plasmid D N A extracted from one P. gingiralis W83 p N J R I 2 transconjugant had an additional 1.5 kb of inserted DNA. Southern-blot analysis of P. gingicalis W83 chromosomal D N A using this inserted D N A as a probe revealed the presence of multiple copies of this sequence on the chromosome. We propose that this D N A represents a P. ghtgicalis insertion sequence (IS) element and should be referred to

Corrt~7,~tmttt'nce to: I.S. Roberts, Department of Microbiology, University of Leicester, P.O. Box 138, Medical Sciences Building, University Road, Leicester, LEI 9ttN, UK.

as IS1126. This is the first IS element to be isolated from a Gram-negative oral anaerobic bacterium.

2. I N T R O D U C T I O N

Porphyromonas gingicalis, is a bl':,:k pigmented, Gram-negative anaerobe implicated as a major aetiologic agent in the pathogenesis of certain forms of human periodontal disease [1-4]. A number of putative virulence factors of P. gingit'alis have been proposed, including a polysaccharide capsule, fimbriai adhesins and an array of hydrolytic enzymes [5-7]. Recently genes encoding for a number of these putative virulence factors have been cloned from P. gingicalis and expressea in Lb'cherichia coil [8-10]. However. these studies have been limited by the lack of suitable genetic systems in P. gingicalis. In colonic Bacteroides a genetic system has been developed by exploiting naturally occurring cryptic plasmids and transposons [11-16]. This includes plasmidshuttle vectors which can be transferred by conju-

gation b e t w e e n E. coli and colonic Bacteroides species as well as suicide vectors for t r a n s p o s o n m u t a g e n e s i s [12-14]. As no such cryptic plasmids have b e e n isolated from P. gingicalis on which to begin to develop such a genetic system [17], the exploitation of the plasmid vectors from colonic Bacteroides may r e p r e s e n t a sensible starting point from which to develop a genetic system in P. gingicalis and o t h e r oral G r a m - n e g a t i v e anaerobes. In this p a p e r we r e p o r t the introduction at high frequency of two Bacteroides-E. coli shuttle plasmids p N J R 5 and p N J R 1 2 into P. gingit,alis and the isolation o f a putative insertion s e q u e n c e (IS) e l e m e n t from ,a. gingicalis.

3. M A T E R I A L S A N D M E T H O D S

3.1. Bacterial strahts and plasmids Bacterial strains and plasmids used in this study are listed in Table 1. T h e E. coli strains were routinely grown in L-broth s u p p l e m e n t e d

with 5 0 / x g o f kanamycin or 100 ~ g t r i m e t h o p r i m p e r mi w h e r e appropriate. P. gingit'alis and Bacteroides uniformis were grown on Oxoid blood agar base n u m b e r 2 containing 7 % horse blood or in BM b r o t h [18] s u p p l e m e n t e d with 5 /~g o f h a e m i n p e r ml and 0.5 p.g o f m e n a d i o n e p e r mi in an a t m o s p h e r e o f 80% nitrogen, 10% hydrogen and 10% carbon dioxide.

3.2. Bacterial matings Both d o n o r and recipient cells w e r e grown to earl,., logarithmic phase, ranging from 1.0 × 104 to 1.5 × 108 colony forming units p e r mi. For e a c h mating, 1 ml o f P. gingit,alis W83 o r /3. uniformis was mixed with 200 p.l o f E. coli SF8 (R751 p N J R 5 ) o r (R751 p N J R I 2 ) and t h e cells harvested by centrifugation for 2 min at 9400 x g. T h e resulting pellet was r e s u s p e n d e d in 100 p.I o f L-broth and s p o t t e d o n t o Millipore H A W G filters (Millipore, U K ) on blood agar plates. C o n trols o f the d o n o r and recipient cells alone w e r e t r e a t e d similarly. T h e plates w e r e t h e n i n c u b a t e d anaerobically at 37°C for 72 h. Bacterial cells

Table I Bacterial strains and !alasmids Strain or plasmid E. coli DH5a SF8

Relevant phenotype "

Description or source

RecA RecB. RecC. Smr

D. Hanahan W. Reznikoff

P. gingicalis W83

Gen ~

H. Shah. Naturally resistant to gentamycin.

Bacteroides uniformis BUI001

Rif r

Spontaneous Rif' mutant of BU0061 [15].

Plasmids R751

Tpr Tra *

lncP/3 E. coli plasmid. Mobilises shuttle vectors from E. coli to BacteroMes [141 and to P. gingicalis; this study. lncQ RSFI010 based E. coli-Bacteroides shuttle vector [13].

pNJR5 oNJR12 pNJRI2-1

Kinc, Mob ÷ (Em r, Rep +, Mob- ) Kmr, Mob + (Tc r, Rep +, Mob + ) Kmr, Mob + (Tc r. Rep +. Mob- )

Same RSFI010 based vector as pNJRS. Contains the Bacteroides plasmid pB8-51 and a 2.7-kb Sstl fragment encoding Tc r from a conjugal Bacteroides element (Fig. l). pNJRI2 with the 1.5 kb P. gingicalis IS element. This study.

a Abbreviations for bacterial phenotypes; Smr. streptomycin resistance; Gen r, gentamyein resistance, Rif r, rifampiein resistance. Abbreviations for plasmids; Tp r, trimetboprim resistance; Tra +, capable of self transfer; Kmr, kanamycin resistance; Mob +, either can ( + ) or cannot ( - ) be mobilised; Em r, erythromycin resistance; Rep ~ can replicate; Tc r, tetracycline resistance. The abbreviations within parentheses are plasmid phenotypes that are expressed in P. gingicalis.

were then recovered from the plates with a sterile cotton swab, resuspended in 500 p,I of L-broth and plated onto selective blood agar plates containing gentamycin at 200 p,g per ml an~d either clindamycin at 0.5 p,g per ml of tetracycline at 10 p,g per ml, depending on the plasmid used. All plates were then incubated anaerobically for 1014 days.

3.3. DNA procedures Plasmid DNA was purified from both E. colt and P. gingivalis by the method of Birnboim and Doly [19]. Chromosomal DNA was extracted from both E. colt and P. gingit'alis W83 essentially as described by Saito and Muira [20]. Restriction endonucleases were purchased from Bethesda Research Laboratories, and used according to the manufacturers' instructions.

3.4. DNA-DNA hybridization Restriction e n d o n u c l e a s e - g e n e r a t e d D N A fragments were transferred to nitrocellulose as described previously [21]. Radiolabelled probes were generated by the method of Feinberg and Vogelstein [22].

4. RESULTS The colonic Bacteroides-E. colt shuttle plasmid pNJR5 [13] was initially used as a vector to be

introduced into P. gingicalis W83. This plasmid confers resistance to both clindamycin (Cc ~) and erythromycin (Em r) in colonic Bacteroides and kanamycin resistance in E. colt [13] and has the highest mobilisation frequency of the available shuttle vectors between E. colt and colonic Bacteroides [12]. Filter matings were performed as described in MATt-RIALS AND METI'IODS using a range of donor : recipient ratios (Table 2) and Cc r P. gingicalis W83 transconjugants selected. The highest transfer frequency was 4.5 × 10 --4 l ~ r P. gingicalis recipient (Table 2). A comparable transfer frequency of 8 . 8 × 10 -3 was obtained when pNJR5 was mobilised from E. colt into B. uniformis using the same mating conditions but with a reduced d o n o r : r e c i p i e n t ratio (Table 2). Plasmid DNA was extracted from twelve Cc ~ pNJR5 P. gingicalis transconjugants and analysed by restriction endonuclease digestion (data not shown). Comparison to the known restriction map of pNJR5 showed no discernable differences. However, the pNJR5 Ce r P. gingicalis transconjugants were difficult to subculture and maintain. In order to overcome this problem another E. coli-Bacteroides shuttle vector p N J R l 2 (Fig. 11 was used which confers resistance to tetracycline (Tc r) in colonic Bacteroides but not E. coil Again filter matings were performed using a range of donor : recipient ratios (Table 2) with P. gingicalis transconjugants being selected on media containing 10/.tg m l - t tetracycline (Tc). The

Table 2 Conjugal transfer of plasmids pNJR5 and pNJRI2 from E. colt strain SF8 into P gmgicalis W83 and B. nnifimnis BUll101 Plasmid

Recipient

Donor: recipient ratio

Transfer frequency ~'

pNJR5

P. gingil'alis W83

1211: I 12:1 1:3846 4:1 1:17 262: I 26: I 1:1.25

4.5× 10 4 2.5× Ill 5 1.3× I(1-7 3.2x 10 - 7 8.8x II1-3 3.9X 10 5 2.2 × 10- * 2.5× 10-5 3.1×10 t,

1:2 1:21

3.0X 10 - 7 6 . 9 × l0 - s

10: l 1:111

4.2× l0 -3 4.5×10 -5

1:3

B. unifonnis BUI001 pNJRI2

P. gmgicalis W83

B. uniformis BU1001

Frequencies are expressedas transconjugantsper recipient at the end of the mating.

EcoRI Sstl ~

EcoRV EcoRI

/

-. ~.s+,, ?.... :Y

EcoRV ~'-'-'--~

I

cated the insertion within the 0.6-kb E c o R I - S s t l fragment of pNJRI2 (Fig. 1). The acquisition of 1.5 kb of DNA into pNJR12-1 might indicate that this additional DNA represents a P. gingiz'alis insertion sequence (IS) element that has transposed onto p N J R I 2 from the chromosome of P. gingit'alis W83. To test this, the 3.6-kb :;st I fragment of pNJRI2-1 containing the majority of the putative IS element was used as a radiolabelled probe in Southern-blot experiments to chromosomal DNA of P. ghtgit'alis W83 cleaved with a number of restriction endonucleases. As a control

\ SSI

Fig. I. Res',riction endonuclease cleavage map of plasmid pNJR 12. The box labelled TET denotes the tetracyclineresistance gene cloned from the Ba~'te,'oides thetaiotamicron conjugal element [24] while the box labelled KN denotes the kanamycin-resistanc¢ gent of the RSFI01I) '.rector [24]. The arrows indicate the direction of transcription of these two genes. The filled box denotes the cryptic Bacteroides plasmid pBS-51. In pNJRI2-1 the putative IS element had inserted between the /:2'oRI and Sstl restriction enzyme sites near to the promoter of the tetracyclineresistancegene.

highest transfer frequency was 2.2 × 10 -4 per P. gingit'alis recipient (Table 2) which is similar to the transfer frequency obtained when p N J R I 2 was mobilised from /::. coil to Bacteroides uniformis using the same mating conditions albeit with a reduced d o n o r : r e c i p i e n t ratio (Table 2). Plasmid DNA was extracted from six Tc r P. gingit'alis transconjugants and analysed by digestion with the restriction erl.donuclease Sstl which cleaves p N J R I 2 to generate three fragments of 2.7 kilobases (kb), 4.1 kb and 10.2 kb (Fig. 1). Plasmid DNA from five of the transconjugants gave the predicted fragment sizes, whilst plasmid DNA from one of the transconjugants had an altered profile. Digestion of this plasmid DNA with Sstl generated the 4.l-kb and 10.2-kb D N A fragments as predicted but the 2.7-kb fragment had an increased size of 3.6 kb and there was an additional Sstl fragment of 0.6 kb (data not shown). This plasmid was termed pNJR2-1. To define more accurately the site of insertion of the additional 1.5 kb of DNA this plasmid was cleaved with restriction e n d o n u c l e a s e s E c o R i and E c o R V in single and double digestions. This Io-

ABCDEFGH 12

O

Fig. 2. Southern-blotanalysisof E. coil and P. gbzgit'alis W83 chromosomal DNA using the 2.7-kb Sst I fragmentof pNJRI2 as a radiolabelled probe. E, coil chromosomal DNA was digested with restriction endonuclease Ssti (lane B) and P. gingit'alis chromosomal DNA was cleaved with the following restriction endonucleases: Sstl (hme C), Hindill (lane D), EcoRV (lane E), EcoRI (lane F) and BamHI (lane G). Plasmid pNJRI2 DNA (hme A) ::nd pNJRI2-1 DNA (lane H) were digested with the restriction endonuelease Sstl. The sizes ;.re in kb.

ABCDEFGH 12

2 1.6

Fig. 3. Southern-blot analysis of E. coli and P. gb~gicalis chromosomal DNA using the 3.8-kb Sstl fragment of pNJRI2-1 as a radiolabelled probe. E. coil chromosomal DNA was digested with restriction endonuclcasc S~tI (lane B) and P. gingicalis chromosomal DNA was cleaved with the following restriction endonuclease,~: Sstl (lane C), Hindlil (hme D). EcoRV (lane E), EcoRI (lane F) and BamHI (lane G). Plasmid pNJRI2 DNA (lane A) and pNJRI2-1 DNA (lane H)were digested with the restriction endonucleasc Sst I. The sizes are in kb.

the corresponding 2.7-kb Ssti fragment from p N J R I 2 was also used in a similar Southern-blot experiment. While this 2.7-kb fragment of pNJR12 failed to detect any homologous sequences within the chromosomal D N A of P. gingit'alis W83 (Fig. 2), the 3.6-kb fragment of pNJR12-1 hybridised to a large n u m b e r of fragments (Fig. 3). No homology was detected with E. coli chromosomal D N A (Figs. 2, 3).

5. DISCUSSION in this paper we describe the introduction of the E. coli-Bacteroides shuttle plasmids pNJR5 and p N J R I 2 into P. gmgicalis W83 by anaerobic filter matings with E coli. Both plasmids were maintained in P. gingit'aiis W83 and could be purified by standard plasmid-extraction procedures. The transfer frequencies of 4.5 × 10 -3 and 2.2 × l0 -4 were comparable to those obtained when these plasmids were introduced using the same procedure into B. uniformis. These transfer frequencies are a significant improvement on the transfer frequency of 2 × 10 -7 previously reported for the transfer of plasmid pE5-2 from E. coli to P. gingicalis [23]. This increase in transfer frequency may reflect differences in .both the plasmid vectors used and in the mating conditions. Notably, in those experiments using pE5-2 the matings were not performed on filters but involved mixing the donor and recipient on the surface of an agar plate. The highest transfer frequencies were achieved when there was an excess of donor to recipient (Table 2), while an increase in recipient relative to donor caused a marked decrease in transfer frequency by a factor of up to l04 (Table 2). It has been reported that aerobic filter matings between E. coli and colonic BacteroMes result in a 50-fold increase in transfer frequency compared to identical matings performed anaerobically [24]. Attempts to perform aerobic filter matings using P. gingiralis W83 as a recipient consistently failed due to the lack of viability of P. gingicalis W83 under aerobic conditions. The high transfer frequencies obtained in the anerobic filter matings described here may he due to the longer incubation period of 72 h rather than the 17 h previously used [24], which may have allowed greater conjugation to occur. The transfer frequencies obtained using these mating conditions are high enough to attempt to generate transposon insertion mutants using colonic Bacteroides transposon suicide vectors and to undertake allelic replacement experiments in P. gingicalis W83. Such studies should prove invaluable in dissecting out the roles of individual virulence factors.

The observation that pNJR5 transconjugants grew slowly and were difficult to maintain on selective media may be due to ) o p t expression of the chndamycin resistance gene (ermF) in P. gingiralis W83. The presence ol pNJR5 in P. gingit'alis conferred resistance to 0.5/xg ml-~ of Cc compared to l0 /.tg ml -~ when present in colonic Bacteroides [13]. Similar results have been found in the rumen anaerobe Prevotella ruminicola in which the ermF is also poorly expressed [25]. No such problems were encountered with the tetracycline resistance gene on pNJR12. This gene cloned from a T C conjugal element from Bacteroides thetaiotaomicron [13] would appear to be a more useful selectable marker in P. gingi-

t'alis. The acquisition of 1.5 kb of D N A into pNJRI2-1 might indicate that this additional D N A represents a P. gingivalis insertion sequence (IS) element that has transposed onto pNJR12 from the P. gingivalis W83 chromosome. Southern-blot analysis using the 3.6-kb fragment of pNJR12-1 as a probe identified multiple copies of sequences homologous to the probe on the P. gingivalis W83 chromosome. This pattern of hybridisation is typical of an IS element which is often present in multiple copies on the chromosome [27]. Failure to detect homology to E. coli chromosomal D N A in these experiments indicated that the putative IS did not originate from the E. coli chromosome. From the preliminary restriction endonuclease mapping of pNJRI2-1 it was known that the putati~,e IS did not have an EcoRl cleavage site. Therefore the" n u m b e r of fragments of EcoRl-cleaved P. gim,,ivalis W83 chromosomal D N A which hybridised to the 3.6-kb probe is the minimum n u m b e r of copies of the putative IS element. The probe hybridised to at least 12 fragment (Fig. 3). This may be an underestimate since some large D N A fragments may have more than one copy of this putative IS element. This n u m b e r is comparable to the number of copies of ISI in E. coli which range from 6 to 17 depending on the particular isolate [26]. !S elements associated with transposons have been described in colonic Bacreroides species [27-29]. Both Tn4351 and Tn4400 isolated trom Bacteroides fragilis have termini consisting of di-

rectly repeated sequences (DRS) termed 154351 [27-29]. The putative IS element identified in this study is the first such element to be isolated in P. gingit'alis and we propose to designate this element IS1126 in keeping with the nomenclature of the plasmid reference centre. This putative IS element may prove useful as an epidemiological tool in Southern-blot experiments for differentiating between different isolates of P. gingivalis. Already such an approach has proven successful with other pathogenic bacteria [30]. Likewise once the putative IS element has bgen characterised further it may be possible to exploit it in molecular genetic studies of P. gingivalis. Experiments are how in progress to characterise this IS element more fully.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the generous gift of P. gingivalis s~rain W83 as well as invaluable advice from Dr. H. Shah. This work was supported by a grant from the Medical Research Council of the United Kingdom to ISR.

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The introduction of colonic-Bacteroides shuttle plasmids into Porphyromonas gingivalis: identification of a putative P. gingivalis insertion-sequence element.

Two Escherichia coli-Bacteroides plasmid-shuttle vectors pNJR5 and pNJR12 were introduced for the first time into Porphyromonas gingivalis W83 by conj...
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