APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1992, p. 1313-1320
Vol. 58, No. 4
0099-2240/92/041313-08$02.00/0 Copyright © 1992, American Society for Microbiology
Evidence for Natural Transfer of a Tetracycline Resistance Gene between Bacteria from the Human Colon and Bacteria from the Bovine Rumen NADJA B. SHOEMAKER, GUI-RONG WANG, AND ABIGAIL A. SALYERS* Department of Microbiology, University of Illinois, Urbana, Illinois 61801 Received 18 September 1991/Accepted 5 February 1992
Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe PrevoteUla (Bacteroides) ruminicola B14. We have now shown that naturally occurring gene transfer elements in Bacteroides species and PrevoteUla ruminicola can also be transferred between these two genera. A self-transmissible chromnosomal element originally found in a clinical isolate of Bacteroidesfragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B14 and from P. ruminicola B14 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRR14) originally found in P. ruminicola 223 was transferred from P. ruminicola B14 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occuried in nature came from our finding that the tetracycline resistance (Tc') gene on the P. ruminicola plasmid pRR14 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer. contact or via ingestion of contaminated meat (3, 9, 22).
Conjugal transfer of naturally occurring plasmids among of facultative and aerobic gram-negative bacteria has been studied extensively. More recently, attention has focused on conjugative transfer of plasmids and transposons between other groups of bacteria. In fact, Bertram et al. (2) have now shown that the naturally occurring conjugative transposon Tn916 can be transferred not only between different genera of gram-positive bacteria but also from gram-positive to facultative gram-negative bacteria. Although Bertram et al. demonstrated this transfer in a laboratory setting, it seems clear that transfer of Tn916 must be occurring in nature, because tetM, the tetracycline resistance gene on Tn916, has been found in a variety of genera (16). Virtually all of the natural isolates reported to carry tetM have been human clinical isolates, and in most cases the human body was the only known habitat of the species involved. These results indicate that natural transfer among different species of bacteria is probably occurring in the human body. Although it is easy to believe that conjugative resistance elements could be transferred between bacteria that occupy the same site in the same host, it is not so obvious that conjugal transfer could occur between bacteria normally found in different hosts, e.g., between bacteria that normally colonize animals and bacteria that normally colonize humans. The question of whether such transfer between the normal microflora of different hosts could occur in nature has been raised previously in connection with the debate over the advisability of adding antibiotics to animal feed. Several studies have documented cases in which antibiotic-resistant Salmonella strains first appeared in cattle which had been fed antibiotics and then spread to humans, either through
However, these cases involved transfer of a single resistant strain of bacteria from animals to humans, rather than transfer of resistance genes between bacteria that normally colonize animals and bacteria that normally colonize hu-
genera
*
mans.
Until recently, it has not been possible to address the question of conjugal transfer between the resident microflora of animals and humans because so little was known about natural conjugative elements found in the obligate anaerobes that constitute the majority of these resident populations. Recently, Hespell and Whitehead (6) showed that Tn916 could be transferred under laboratory conditions from Enterococcus faecalis, a minor component of the human intestinal microflora, to the rumen species Butyrivibrio fibrisolvens. Also, we had shown previously that a specially constructed shuttle vector could be mobilized from Bacteroides unifornis, a species that is found in high numbers in the human colonic microflora, to Prevotella ruminicola, one of the numerically predominant species in the rumen of cattle and sheep (19). In this paper, we show that naturally occurring conjugative elements found in human colonic Bacteroides species can also be transferred to P. ruminicola. Moreover, we provide evidence that such transfer may actually have occurred between Bacteroides species and P. ruminicola in nature. Since human colonic Bacteroides species are opportunistic pathogens which are notable for their resistance to a variety of antibiotics, there has been considerable interest in the transfer of antibiotic resistance genes within this group. Although transmissible plasmids have been found in human colonic Bacteroides strains, the most widespread type of resistance transfer element appears to be a family of large (>70-kbp) chromosomal elements that appear to be transferred by conjugation (13, 15, 20). These chromosomal elements generally carry a tetracycline resistance (Tcr) gene
Corresponding author. 1313
1314
SHOEMAKER ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 1. Strains and plasmids used in this study Strain or plasmid
Characteristicsa
Source (reference)b
Bacterial strains
B. thetaiotaomicron BT4001 BT4100
Rif mutant of B. thetaiotaomicron 5482 Thy- mutants of B. thetaiotaomicron 5482
BT4107 BT4108
Rif Gnr Thy- Tpr Gnr Thy- Tpr Gnr Tcr Emr Thyr Tpr Gnr Tcr Emr
B. uniformis BU1001 BU1100 BU1104 BU1106 BU1108
Rif Gnr Thy- Tpr Gnr Thy- Tpr Gnr Tcr Thy- Tpr Gnr Tcr Emr Thy- Tpr Gnr Tcr Emr
Rif' mutant of B. unifonnis 0061 Thy- mutant of B. unifonnis 0061 BU1100 carrying the Tcr ERL element BU1100 carrying the Tcr Emr DOT element BU1100 carrying the Tcr Emr 12256 element
Rifr Gns
Rift mutant of P. ruminicola B14 (19)
pRDB5 pEG920::XBU4422
Emr (Apr Tcr) Tc (Ap Tcr) Emr (Apr) Tra+
pNFD 13-2 pRRI4
Tcr (Apr) Tcr
E. coli-Bacteroides shuttle vector pVA1l with Emr gene replaced by Tcr gene from Tcr Emr DOT (19) Cryptic conjugal chromosomal element (XBU4422) integrated into mobilizationdeficient plasmid (pEG920); homology to interior regions and ends of Tcr elements (21) 2.7-kbp Tcr clone from Tcr Emr DOT element in pFD160 (19) Plasmid isolated from P. ruminicola 23 (4)
P. ruminicola B14R
BT4100 carrying the Tcr Emr DOT element BT4100 carrying the Tcr Emr 12256 element
Plasmids
pVAL1
a Parentheses indicate that the gene is expressed in E. coli. Abbreviations: Rif, rifampin; Thy, thymidine; Tp, trimethoprim; Tc, tetracycline; Em, erythromycin; Ap, ampicillin; Gn, gentamicin; Tra, transfer. b Unless otherwise indicated, strains and plasmids were described in Valentine et al. (24).
and are thus designated Tcr elements. Many also carry an erythromycin resistance (Emr) gene and are designated Tcr Emr elements. Individual Tcr and Tcr Emr elements are designated by the name or number of the clinical isolate in which they were first found. Southern analysis of known Tcr and Tcr Emr elements has shown that elements found in different clinical isolates are closely related but not identical (20, 23). Bacteroides chromosomal elements appear to have broad host ranges because they can be transferred to a number of different colonic Bacteroides species. Tcr genes that crosshybridized with the Tcr on the colonic Bacteroides elements have also been found in gram-negative anaerobes from the resident microflora of the human mouth (5). These strains were originally classified as Bacteroides strains but have now been placed in the genus Prevotella. The finding of a similar gene in human colonic and oral anaerobes indicated that transfer between these genera has occurred in the human body. Gram-negative anaerobes that are distantly related to Bacteroides species are also found in high numbers in the rumen of cattle. These were originally classified as Bacteroides ruminicola because they had some of the metabolic traits associated with the human colonic Bacteroides species. However, the rumen strains share less than 5% DNA-DNA homology and only 25% rRNA-DNA homology with the colonic Bacteroides species and have thus been placed in the new genus Prevotella, and they are now designated P. ruminicola (17). The rumen Prevotella strains are only distantly related to the human oral Prevotella strains. In this paper, we show that one of the Bacteroides conjugative chromosomal elements (Tcr Emr 12256) is transferred from strains of B. uniformis and Bacteroides thetaiotaomicron to P. ruminicola B14. Previously, Flint et al. (4) characterized a
19.5-kbp plasmid, pRRI4, which was found in a strain of P. ruminicola (strain 223) isolated from the rumen of a sheep. pRRI4 was shown to carry a Tcr gene and to be capable of being transferred between two strains of P. ruminicola (strain 223 and strain B14). We report here that pRRI4 also is transferred from P. ruminicola B14 to a strain of B. thetaiotaomicron. Moreover, we show that the Tcr gene on pRRI4 cross-hybridizes with and has the same restriction pattern as the Tcr gene found on the Bacteroides conjugative chromosomal elements, a finding which indicates that transfer between human colonic Bacteroides strains and rumen Prevotella strains may actually have occurred in nature. MATERLILS AND METHODS Strains, plasmids, and growth conditions. The strains and plasmids used in this study are listed in Table 1. Two colonic Bacteroides species, B. uniformis and B. thetaiotaomicron, were cultivated in prereduced Trypticase-yeast extractglucose medium (TYG [7]). P. ruminicola B14 was cultivated in modified M10 medium (MM10 [19]). Mating conditions. Mating conditions for transfer from B. uniformis or B. thetaiotaomicron to Prevotella strains were the same as those described recently for transfer of a chimeric shuttle vector, pRDB5, from B. uniformis to P. ruminicola B14 (19). The donors in these matings were thymidine-requiring (Thy-) mutants of the colonic Bacteroides strains, which were also trimethoprim resistant (Tpr), and the Prevotella recipient was rifampin resistant (Rift). Selection was for Rift Thy' Tcr recipients. For matings in which P. ruminicola B14R was the donor and B. uniformis BU1100 or B. thetaiotaomicron BT4100 was the recipient, conditions were the same except that Bacteroides transconjugants were selected on TYG medium containing gentami-
NATURAL TRANSFER OF A Tcr GENE
VOL. 58, 1992
1315
TABLE 2. Transfer of Tcr Emr 12256 between P. ruminicola B14 and two human colonic Bacteroides species Donor containing Tcr Emr 12256a
B. B. P. P.
Recipient
ruminicola B14R ruminicola B14R uniformis BU1100 thetaiotaomicron BT4100 a Frequency of Tcr Emr 12256 between B. thetaiotaomicron and B. uniformis is 10-5 to 10-6 per recipient.
uniformis BU1108 thetaiotaomicron BT4108 ruminicola B14R ruminicola B14R
P. P. B. B.
Transfer frequency (per recipient)b
0.3 0.6 1.0 1.0
x 10-7-8.6 X 10-7 x 10-8-1.2 x 10-8 X 10-8-7.2 x 10-8 X 10-7-5.6 x 10-7
b Transfer frequency is expressed as transconjugants per recipient at the end of the mating. The range is given for a minimum of three separate matings. Selection was for Tcr in all cases.
cin (200 ,ug/ml), thymidine (100 ,ug/ml), and tetracycline (5 ,ug/ml). P. ruminicola is susceptible to gentamicin (Gns) and does not grow on TYG. In these matings, the donor-torecipient ratio was 3:1. Conditions for matings between B. uniformis and B. thetaiotaomicron have been described previously (24). Since P. ruminicola B14 grows more slowly than human colonic Bacteroides species and is much more sensitive to oxygen, it was important to check all putative P. ruminicola B14 transconjugants carefully to make sure that they were not spontaneous Thy' Rif' mutants of the Bacteroides donor. Thus, all P. ruminicola B14 transconjugants were checked for the following traits that characterize P. ruminicola B14 and differentiate it from the human colonic species B. uniforinis and B. thetaiotaomicron: sensitivity to oxygen, inability to grown on TYG containing thymidine, susceptibility to gentamicin, and ability to grow on MM10 containing xylan as the sole carbohydrate source. DNA isolation and analysis. Plasmids were isolated from Bacteroides species or P. ruminicola by the Ish-Horowitz modification of the Birnboim and Doly procedure (11). Chromosomal DNA was isolated by the method of Saito and Miura (14). Restriction enzyme digests were done according to manufacturers' directions. For Southern analysis, DNA restriction fragments were separated by electrophoresis on 1% agarose gels and blotted to Optibind paper (Schleicher and Schuell) by capillary blotting (11). Hybridization and wash conditions have been described previously (21). DNA probes were labeled with [32P]dCTP by random primed synthesis using the Stratagene (La Jolla, Calif.) Prime-it kit.
RESULTS AND DISCUSSION Transfer of Tcr Emr 12256 from human colonic Bacteroides species to P. ruminicola. Previously, we showed that a specially constructed shuttle vector, pRDB5, could be transferred from a strain of B. uniformis that carried a Tcr Emr element (Tcr Emr 12256) to P. ruminicola B14 (19). The Tcr Emr 12256 element was needed to mobilize pRDB5 and provide transfer functions. This particular Tcr Emr element was chosen initially because, unlike most other Tcr and Tcr Emr elements, it exhibits constitutive transfer. The transfer of most other Tcr elements is regulated by tetracycline (15, 20). Also, although many Tcr and Tcr Emr elements mobilized pRDB5 between colonic Bacteroides strains, Tcr Emr 12256 proved to be the only Tcr Emr element that transferred pRDB5 to P. ruminicola B14 (19). In earlier experiments with pRDB5, we had not detected transfer of Tcr Emr 12256 to P. ruminicola B14; that is, no Emr transconjugants were detected. Nonetheless, since Tcr Emr 12256 could mediate mating between Bacteroides strains and Prevotella strains, it seemed possible that the element itself might be transferred into P. ruminicola. This transfer would have been missed in our initial experiments if the frequency was an order of
magnitude or more lower than that of the plasmid or if the Emr gene on Tcr Emr 12256 was not expressed in P. ruminicola. To determine if Tcr Emr 12256 was transferred to P. ruminicola B14, we mated B. uniformis 1108 (Tcr Emr 12256) and B. thetaiotaomicron 4108 (Tcr Emr 12256) with P. ruminicola B14R. Transconjugants were detected in matings in which the selection was for Tcr (Table 2). The transfer frequencies reported in Table 2 are about 10-fold higher than those reported previously (19). We do not know why the frequency has increased, because the procedure is the same as that used formerly. The most likely explanation is that increased experience with the procedure has increased our success in reducing exposure of the P. ruminicola recipients to oxygen during the various steps, thus increasing the yield of P. ruminicola recipients and transconjugants. To confirm that the Tcr Emr 12256 element had been transferred, we probed EcoRV-digested DNA from several putative transconjugants with 32P-labeled p6T5 (Fig. 1A). p6T5 is a cosmid clone from another Tcr Emr element (Tcr Emr DOT) and contains approximately 30 kbp of the element, including the Tcr gene. p6T5 was shown previously to hybridize on high-stringency Southern blots with the Tcr
I
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FIG. 1. Autoradiogram of DNA from P. ruminicola B14R transconjugants containing Tcr Emr 12256. (A) EcoRV-digested DNA from P. nrminicola B14R recipient (lane 1), B. uniformis BU1108 donor (lane 2), B. uniformis BU1108(pVAL1) donor (lane 3), and P. ruminicola B14R Tcr transconjugants (lanes 4 through 9). The probe was [32P]p6T5, a cosmid clone which contains 35 kbp of the Tcr Emr DOT element, which shares extensive homology with the Tcr Emr 12256 element (23). The asterisks in lanes 2 and 3 indicate a cross-hybridizing band in B. uniformis that is due to a cryptic element, XBU4422, which is also in this strain and hybridizes with p6T5 (21). Arrows in lanes 3 and 4 indicate two bands of pVAL1 that cross-hybridize with p6T5. (B) The first four lanes of the Southern blot in panel A, which has been stripped and rehybridized with [32P]pFD160, a plasmid that hybridizes to a 9.7-kbp EcoRV band of pVAL1 (indicated by arrows in panel A) but not to Tcr Emr 12256 or XBU4422. No hybridization was detected in lanes 5 through 9 (not shown). The locations of HindlIl lambda DNA fragments (sizes indicated in kilobase pairs) are marked on the left and right of the blots.
APPL. ENVIRON. MICROBIOL.
SHOEMAKER ET AL.
1316
TABLE 3. Transfer of P. ruminicola plasmid pRRI4 between P. ruminicola B14R and two human colonic Bacteroides species, B. uniformis and B. thetaiotaomicron Donor carrying
Transfer frequency (per recipient)a
Recipient
pRRI4
B14R BT4100 BT4108
BT4107C BU1100
BU1108
BT4100 BU1100 BT4001 B14R BT4001 BU1001 B14R BT4001 B14R BU1001 B14 BU1001 B14R
pRRI4
Tc element
1 x 10-6-2 x 10-6 < 10-8 < 10-9 < 0i-9 3 x 10-5-7 x 10-5 0.3 x 10-4_1 X 1o-4 1 x 10-7-1.2 x 10-7 0.2 x 10-5_1 x 10-5 < 10-8 < 1o-9 < i0-9 1 x 10-4-3 x 10-4 6 x 10-6-7 x 10-6
NAb NA NA NA 1 x 10-6-3 x 10-6 1 X 10-5_8 X 10-5 0.3 x 10-8_1 x 10-8 3 x 10---5 x 10-6 < 10-8 NA NA 0.3 x 10-4-l X 10-4
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Vol. 58, No. 4
0099-2240/92/041313-08$02.00/0 Copyright © 1992, American Society for Microbiology
Evidence for Natural Transfer of a Tetracycline Resistance Gene between Bacteria from the Human Colon and Bacteria from the Bovine Rumen NADJA B. SHOEMAKER, GUI-RONG WANG, AND ABIGAIL A. SALYERS* Department of Microbiology, University of Illinois, Urbana, Illinois 61801 Received 18 September 1991/Accepted 5 February 1992
Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe PrevoteUla (Bacteroides) ruminicola B14. We have now shown that naturally occurring gene transfer elements in Bacteroides species and PrevoteUla ruminicola can also be transferred between these two genera. A self-transmissible chromnosomal element originally found in a clinical isolate of Bacteroidesfragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B14 and from P. ruminicola B14 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRR14) originally found in P. ruminicola 223 was transferred from P. ruminicola B14 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occuried in nature came from our finding that the tetracycline resistance (Tc') gene on the P. ruminicola plasmid pRR14 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer. contact or via ingestion of contaminated meat (3, 9, 22).
Conjugal transfer of naturally occurring plasmids among of facultative and aerobic gram-negative bacteria has been studied extensively. More recently, attention has focused on conjugative transfer of plasmids and transposons between other groups of bacteria. In fact, Bertram et al. (2) have now shown that the naturally occurring conjugative transposon Tn916 can be transferred not only between different genera of gram-positive bacteria but also from gram-positive to facultative gram-negative bacteria. Although Bertram et al. demonstrated this transfer in a laboratory setting, it seems clear that transfer of Tn916 must be occurring in nature, because tetM, the tetracycline resistance gene on Tn916, has been found in a variety of genera (16). Virtually all of the natural isolates reported to carry tetM have been human clinical isolates, and in most cases the human body was the only known habitat of the species involved. These results indicate that natural transfer among different species of bacteria is probably occurring in the human body. Although it is easy to believe that conjugative resistance elements could be transferred between bacteria that occupy the same site in the same host, it is not so obvious that conjugal transfer could occur between bacteria normally found in different hosts, e.g., between bacteria that normally colonize animals and bacteria that normally colonize humans. The question of whether such transfer between the normal microflora of different hosts could occur in nature has been raised previously in connection with the debate over the advisability of adding antibiotics to animal feed. Several studies have documented cases in which antibiotic-resistant Salmonella strains first appeared in cattle which had been fed antibiotics and then spread to humans, either through
However, these cases involved transfer of a single resistant strain of bacteria from animals to humans, rather than transfer of resistance genes between bacteria that normally colonize animals and bacteria that normally colonize hu-
genera
*
mans.
Until recently, it has not been possible to address the question of conjugal transfer between the resident microflora of animals and humans because so little was known about natural conjugative elements found in the obligate anaerobes that constitute the majority of these resident populations. Recently, Hespell and Whitehead (6) showed that Tn916 could be transferred under laboratory conditions from Enterococcus faecalis, a minor component of the human intestinal microflora, to the rumen species Butyrivibrio fibrisolvens. Also, we had shown previously that a specially constructed shuttle vector could be mobilized from Bacteroides unifornis, a species that is found in high numbers in the human colonic microflora, to Prevotella ruminicola, one of the numerically predominant species in the rumen of cattle and sheep (19). In this paper, we show that naturally occurring conjugative elements found in human colonic Bacteroides species can also be transferred to P. ruminicola. Moreover, we provide evidence that such transfer may actually have occurred between Bacteroides species and P. ruminicola in nature. Since human colonic Bacteroides species are opportunistic pathogens which are notable for their resistance to a variety of antibiotics, there has been considerable interest in the transfer of antibiotic resistance genes within this group. Although transmissible plasmids have been found in human colonic Bacteroides strains, the most widespread type of resistance transfer element appears to be a family of large (>70-kbp) chromosomal elements that appear to be transferred by conjugation (13, 15, 20). These chromosomal elements generally carry a tetracycline resistance (Tcr) gene
Corresponding author. 1313
1314
SHOEMAKER ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 1. Strains and plasmids used in this study Strain or plasmid
Characteristicsa
Source (reference)b
Bacterial strains
B. thetaiotaomicron BT4001 BT4100
Rif mutant of B. thetaiotaomicron 5482 Thy- mutants of B. thetaiotaomicron 5482
BT4107 BT4108
Rif Gnr Thy- Tpr Gnr Thy- Tpr Gnr Tcr Emr Thyr Tpr Gnr Tcr Emr
B. uniformis BU1001 BU1100 BU1104 BU1106 BU1108
Rif Gnr Thy- Tpr Gnr Thy- Tpr Gnr Tcr Thy- Tpr Gnr Tcr Emr Thy- Tpr Gnr Tcr Emr
Rif' mutant of B. unifonnis 0061 Thy- mutant of B. unifonnis 0061 BU1100 carrying the Tcr ERL element BU1100 carrying the Tcr Emr DOT element BU1100 carrying the Tcr Emr 12256 element
Rifr Gns
Rift mutant of P. ruminicola B14 (19)
pRDB5 pEG920::XBU4422
Emr (Apr Tcr) Tc (Ap Tcr) Emr (Apr) Tra+
pNFD 13-2 pRRI4
Tcr (Apr) Tcr
E. coli-Bacteroides shuttle vector pVA1l with Emr gene replaced by Tcr gene from Tcr Emr DOT (19) Cryptic conjugal chromosomal element (XBU4422) integrated into mobilizationdeficient plasmid (pEG920); homology to interior regions and ends of Tcr elements (21) 2.7-kbp Tcr clone from Tcr Emr DOT element in pFD160 (19) Plasmid isolated from P. ruminicola 23 (4)
P. ruminicola B14R
BT4100 carrying the Tcr Emr DOT element BT4100 carrying the Tcr Emr 12256 element
Plasmids
pVAL1
a Parentheses indicate that the gene is expressed in E. coli. Abbreviations: Rif, rifampin; Thy, thymidine; Tp, trimethoprim; Tc, tetracycline; Em, erythromycin; Ap, ampicillin; Gn, gentamicin; Tra, transfer. b Unless otherwise indicated, strains and plasmids were described in Valentine et al. (24).
and are thus designated Tcr elements. Many also carry an erythromycin resistance (Emr) gene and are designated Tcr Emr elements. Individual Tcr and Tcr Emr elements are designated by the name or number of the clinical isolate in which they were first found. Southern analysis of known Tcr and Tcr Emr elements has shown that elements found in different clinical isolates are closely related but not identical (20, 23). Bacteroides chromosomal elements appear to have broad host ranges because they can be transferred to a number of different colonic Bacteroides species. Tcr genes that crosshybridized with the Tcr on the colonic Bacteroides elements have also been found in gram-negative anaerobes from the resident microflora of the human mouth (5). These strains were originally classified as Bacteroides strains but have now been placed in the genus Prevotella. The finding of a similar gene in human colonic and oral anaerobes indicated that transfer between these genera has occurred in the human body. Gram-negative anaerobes that are distantly related to Bacteroides species are also found in high numbers in the rumen of cattle. These were originally classified as Bacteroides ruminicola because they had some of the metabolic traits associated with the human colonic Bacteroides species. However, the rumen strains share less than 5% DNA-DNA homology and only 25% rRNA-DNA homology with the colonic Bacteroides species and have thus been placed in the new genus Prevotella, and they are now designated P. ruminicola (17). The rumen Prevotella strains are only distantly related to the human oral Prevotella strains. In this paper, we show that one of the Bacteroides conjugative chromosomal elements (Tcr Emr 12256) is transferred from strains of B. uniformis and Bacteroides thetaiotaomicron to P. ruminicola B14. Previously, Flint et al. (4) characterized a
19.5-kbp plasmid, pRRI4, which was found in a strain of P. ruminicola (strain 223) isolated from the rumen of a sheep. pRRI4 was shown to carry a Tcr gene and to be capable of being transferred between two strains of P. ruminicola (strain 223 and strain B14). We report here that pRRI4 also is transferred from P. ruminicola B14 to a strain of B. thetaiotaomicron. Moreover, we show that the Tcr gene on pRRI4 cross-hybridizes with and has the same restriction pattern as the Tcr gene found on the Bacteroides conjugative chromosomal elements, a finding which indicates that transfer between human colonic Bacteroides strains and rumen Prevotella strains may actually have occurred in nature. MATERLILS AND METHODS Strains, plasmids, and growth conditions. The strains and plasmids used in this study are listed in Table 1. Two colonic Bacteroides species, B. uniformis and B. thetaiotaomicron, were cultivated in prereduced Trypticase-yeast extractglucose medium (TYG [7]). P. ruminicola B14 was cultivated in modified M10 medium (MM10 [19]). Mating conditions. Mating conditions for transfer from B. uniformis or B. thetaiotaomicron to Prevotella strains were the same as those described recently for transfer of a chimeric shuttle vector, pRDB5, from B. uniformis to P. ruminicola B14 (19). The donors in these matings were thymidine-requiring (Thy-) mutants of the colonic Bacteroides strains, which were also trimethoprim resistant (Tpr), and the Prevotella recipient was rifampin resistant (Rift). Selection was for Rift Thy' Tcr recipients. For matings in which P. ruminicola B14R was the donor and B. uniformis BU1100 or B. thetaiotaomicron BT4100 was the recipient, conditions were the same except that Bacteroides transconjugants were selected on TYG medium containing gentami-
NATURAL TRANSFER OF A Tcr GENE
VOL. 58, 1992
1315
TABLE 2. Transfer of Tcr Emr 12256 between P. ruminicola B14 and two human colonic Bacteroides species Donor containing Tcr Emr 12256a
B. B. P. P.
Recipient
ruminicola B14R ruminicola B14R uniformis BU1100 thetaiotaomicron BT4100 a Frequency of Tcr Emr 12256 between B. thetaiotaomicron and B. uniformis is 10-5 to 10-6 per recipient.
uniformis BU1108 thetaiotaomicron BT4108 ruminicola B14R ruminicola B14R
P. P. B. B.
Transfer frequency (per recipient)b
0.3 0.6 1.0 1.0
x 10-7-8.6 X 10-7 x 10-8-1.2 x 10-8 X 10-8-7.2 x 10-8 X 10-7-5.6 x 10-7
b Transfer frequency is expressed as transconjugants per recipient at the end of the mating. The range is given for a minimum of three separate matings. Selection was for Tcr in all cases.
cin (200 ,ug/ml), thymidine (100 ,ug/ml), and tetracycline (5 ,ug/ml). P. ruminicola is susceptible to gentamicin (Gns) and does not grow on TYG. In these matings, the donor-torecipient ratio was 3:1. Conditions for matings between B. uniformis and B. thetaiotaomicron have been described previously (24). Since P. ruminicola B14 grows more slowly than human colonic Bacteroides species and is much more sensitive to oxygen, it was important to check all putative P. ruminicola B14 transconjugants carefully to make sure that they were not spontaneous Thy' Rif' mutants of the Bacteroides donor. Thus, all P. ruminicola B14 transconjugants were checked for the following traits that characterize P. ruminicola B14 and differentiate it from the human colonic species B. uniforinis and B. thetaiotaomicron: sensitivity to oxygen, inability to grown on TYG containing thymidine, susceptibility to gentamicin, and ability to grow on MM10 containing xylan as the sole carbohydrate source. DNA isolation and analysis. Plasmids were isolated from Bacteroides species or P. ruminicola by the Ish-Horowitz modification of the Birnboim and Doly procedure (11). Chromosomal DNA was isolated by the method of Saito and Miura (14). Restriction enzyme digests were done according to manufacturers' directions. For Southern analysis, DNA restriction fragments were separated by electrophoresis on 1% agarose gels and blotted to Optibind paper (Schleicher and Schuell) by capillary blotting (11). Hybridization and wash conditions have been described previously (21). DNA probes were labeled with [32P]dCTP by random primed synthesis using the Stratagene (La Jolla, Calif.) Prime-it kit.
RESULTS AND DISCUSSION Transfer of Tcr Emr 12256 from human colonic Bacteroides species to P. ruminicola. Previously, we showed that a specially constructed shuttle vector, pRDB5, could be transferred from a strain of B. uniformis that carried a Tcr Emr element (Tcr Emr 12256) to P. ruminicola B14 (19). The Tcr Emr 12256 element was needed to mobilize pRDB5 and provide transfer functions. This particular Tcr Emr element was chosen initially because, unlike most other Tcr and Tcr Emr elements, it exhibits constitutive transfer. The transfer of most other Tcr elements is regulated by tetracycline (15, 20). Also, although many Tcr and Tcr Emr elements mobilized pRDB5 between colonic Bacteroides strains, Tcr Emr 12256 proved to be the only Tcr Emr element that transferred pRDB5 to P. ruminicola B14 (19). In earlier experiments with pRDB5, we had not detected transfer of Tcr Emr 12256 to P. ruminicola B14; that is, no Emr transconjugants were detected. Nonetheless, since Tcr Emr 12256 could mediate mating between Bacteroides strains and Prevotella strains, it seemed possible that the element itself might be transferred into P. ruminicola. This transfer would have been missed in our initial experiments if the frequency was an order of
magnitude or more lower than that of the plasmid or if the Emr gene on Tcr Emr 12256 was not expressed in P. ruminicola. To determine if Tcr Emr 12256 was transferred to P. ruminicola B14, we mated B. uniformis 1108 (Tcr Emr 12256) and B. thetaiotaomicron 4108 (Tcr Emr 12256) with P. ruminicola B14R. Transconjugants were detected in matings in which the selection was for Tcr (Table 2). The transfer frequencies reported in Table 2 are about 10-fold higher than those reported previously (19). We do not know why the frequency has increased, because the procedure is the same as that used formerly. The most likely explanation is that increased experience with the procedure has increased our success in reducing exposure of the P. ruminicola recipients to oxygen during the various steps, thus increasing the yield of P. ruminicola recipients and transconjugants. To confirm that the Tcr Emr 12256 element had been transferred, we probed EcoRV-digested DNA from several putative transconjugants with 32P-labeled p6T5 (Fig. 1A). p6T5 is a cosmid clone from another Tcr Emr element (Tcr Emr DOT) and contains approximately 30 kbp of the element, including the Tcr gene. p6T5 was shown previously to hybridize on high-stringency Southern blots with the Tcr
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FIG. 1. Autoradiogram of DNA from P. ruminicola B14R transconjugants containing Tcr Emr 12256. (A) EcoRV-digested DNA from P. nrminicola B14R recipient (lane 1), B. uniformis BU1108 donor (lane 2), B. uniformis BU1108(pVAL1) donor (lane 3), and P. ruminicola B14R Tcr transconjugants (lanes 4 through 9). The probe was [32P]p6T5, a cosmid clone which contains 35 kbp of the Tcr Emr DOT element, which shares extensive homology with the Tcr Emr 12256 element (23). The asterisks in lanes 2 and 3 indicate a cross-hybridizing band in B. uniformis that is due to a cryptic element, XBU4422, which is also in this strain and hybridizes with p6T5 (21). Arrows in lanes 3 and 4 indicate two bands of pVAL1 that cross-hybridize with p6T5. (B) The first four lanes of the Southern blot in panel A, which has been stripped and rehybridized with [32P]pFD160, a plasmid that hybridizes to a 9.7-kbp EcoRV band of pVAL1 (indicated by arrows in panel A) but not to Tcr Emr 12256 or XBU4422. No hybridization was detected in lanes 5 through 9 (not shown). The locations of HindlIl lambda DNA fragments (sizes indicated in kilobase pairs) are marked on the left and right of the blots.
APPL. ENVIRON. MICROBIOL.
SHOEMAKER ET AL.
1316
TABLE 3. Transfer of P. ruminicola plasmid pRRI4 between P. ruminicola B14R and two human colonic Bacteroides species, B. uniformis and B. thetaiotaomicron Donor carrying
Transfer frequency (per recipient)a
Recipient
pRRI4
B14R BT4100 BT4108
BT4107C BU1100
BU1108
BT4100 BU1100 BT4001 B14R BT4001 BU1001 B14R BT4001 B14R BU1001 B14 BU1001 B14R
pRRI4
Tc element
1 x 10-6-2 x 10-6 < 10-8 < 10-9 < 0i-9 3 x 10-5-7 x 10-5 0.3 x 10-4_1 X 1o-4 1 x 10-7-1.2 x 10-7 0.2 x 10-5_1 x 10-5 < 10-8 < 1o-9 < i0-9 1 x 10-4-3 x 10-4 6 x 10-6-7 x 10-6
NAb NA NA NA 1 x 10-6-3 x 10-6 1 X 10-5_8 X 10-5 0.3 x 10-8_1 x 10-8 3 x 10---5 x 10-6 < 10-8 NA NA 0.3 x 10-4-l X 10-4
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