Department of Pathobiology, Box 357238, University of Washington, Seattle, WA 98195-7238, USA
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Abstract |
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Introduction |
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There are three different designations given to a group of highly related ermF genes found in various transposons: ermF (GenBank accession No. M14730) is found on Bacteroides transposons Tn4351 and Tn4400,5 ermFS (M17808) is on Tn4551,6 and ermFU (M62487) was described in Bacteroides fragilis V503. 7 There is 99% DNA identity between the ermF, ermFU and ermFS genes. On the other hand, the tetQ genes were given only one designation although they have been sequenced from three different species of bacteria, Prevotella intermedia (U73497), Bacteroides thetaiotaomicron (X58717) and Prevotella ruminicola (U10437).8,9,10 These three tetQgene sequences share > 97% DNA identity. The ORF1rteB region spans approximately 43 kb of the 150 kb conjugative transposon TcrEmrTn5030described in a few Bacteroides spp. (Figure).1,2,3 Previously,11 we found that a variety of bacteria carrying the ermF and tetQ genes also carried the ORF1 gene, which is immediately upstream of the structural ermF gene, and the region spanning the rteArteB junction, which is immediately downstream of the tetQ gene (Figure). However, absolute linkage was not established.
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To determine whether these genes are linked on the same mobile unit and transferred together, we examined three Gram-positive and 57 Gram-negative isolates, representing 17 species known to carry ermF and tetQ. We also investigated the linkage between ORF1ermF, tetQrteA and between rteArteB in order to determine if the configuration of the conjugative transposons in these species is analogous to Tn5030.
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Materials and methods |
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Three Gram-positive and 57 Gram-negative isolates, representing 17 species, which were known to carry both ermF and tetQ genes, were chosen for the study.11,12 These 60 isolates represent a wide spectrum of genera with characterized isolates that were available for study. Ten ATCC strains (33185, 8503, 29303, 33690, 35310, 43037, 29426, 49046, 25845, 33563) isolated between 1976 and 1988; 27 clinical isolates from periodontal patients seen in the 1990s from the University of Washington, Seattle, WA, USA; two isolates from periodontal patients at the University of Florida, Gainesville, FL, USA; and 21 isolates from previous studies of human urogenital and oral tract bacteria collected over the last 10 years were included.11,12
Media
Anaerobic bacteria were grown on Brucella-based blood agar (BA) (Difco Laboratories, Detroit, MI, USA) supplemented with 5% sheep blood, 5 mg/L haem and 0.05 mg/L vitamin K at 36.5°C with anaerobic gas pack (85% N2, 15% CO2) for 5 days in anaerobic jars.11 A Staphylococcus aureus isolate was streaked on BA plates as a supplement for anaerobic growth for Bacteroides forsythus.12 Enterococcus faecalis and aerobes were grown on Brain Heart Infusion agar (BHI) (Difco Laboratories) at 36.5°C with 5% CO2.11
Proteinase K treatment
Bacteria were treated with proteinase K and used as the template source for the PCRs. The proteinase K treatment was 0.1 M Tris pH 8.5, 0.05% Tween 20 and 0.05 mg/mL proteinase K in 100 µL of sterile distilled water.13 Each proteinase K-treated sample was incubated at 60°C overnight, then boiled for 10 min, aliquoted and frozen at -20°C.11
Oligonucleotides and PCR conditions for ermF and tetQ assays
The oligonucleotides and the conditions for the ermFPCR assay were as follow: F1 (5' CGG GTC AGC ACT TTA CTA TTG 3'); F2 (5' GGA CCT ACC TCA TAG ACA AG 3'); denaturing at 94°C for 30 s, annealing at 50°C for 30 s, elongation at 72°C for 2 min for 35 cycles.11 The primer sequences for the tetQ PCR were: MR6 (5' CTG TCC CTA ACG GTA AGG 3'); MR7 (5' TTA TAC TTC CTC CGG CAT CGG T 3').14 The tetQ PCR conditions were the same as previously described,12 though the annealing temperature was reduced from 52 to 46°C, which allowed for more consistent amplification of the tetQ product.
Oligonucleotides and PCR conditions for ORF1 and rteA/B junction region
The oligonucleotides for ORF1 and rteA/B were: ORF1-1 (5' GCA GAC AGG CGC AAG CAG CAA 3'); ORF1-2 (5' ACC ACG TTC CCA TGA GTG GTA TGG 3'); rteA (5' GGC AAT GAA GCC GTC ATG C 3'); rteB (5' GGC TAC CAG CCT TAT CCG G 3').11 The PCR conditions for ORF1 region and rteA/B genes were the same as previously described.11
PCR conditions for ORF1ermF and tetQrteA junction regions
New PCRs were developed to look at the linkage between ORF1 and ermF genes, and tetQand rteA genes. Each 100 µL of PCR mixture contained 200 µM deoxynucleoside triphosphate, 1x PCR buffer I (1.5 mM MgCl2), 2 U of Taq polymerase (Boehringer Mannheim Biochemicals, Indianapolis, IN, USA) and 100 ng of each primer. The oligonucleotides for amplifying the ORF1ermF junction region were ORF1-1 and F2 (see above), and the PCR product size was 1322 bp. The PCR condition was: denaturing at 96°C for 30 s, annealing at 50°C for 2 min and elongation at 72°C for 3 min for 35 cycles. The oligonucleotides for amplifying the tetQrteA junction region were tetQF (5' CCT GCT GAT TTC AGA CAG C 3' starting at base 2364); and rteAR (5' GCT CTG TAG CTG AAT GGG 3' the antisense of the sequence ending at base 3014). The PCR condition was: denaturing at 96°C for 1 min, annealing at 45°C for 2 min and elongation at 72°C for 3 min for 35 cycles. The expected size of the PCR product was 651 bp.
Plasmids
The plasmids carrying cloned ermF and tetQ were pBF411 and pNFD13.2,9 respectively. Whole plasmid pBF4 was used as a labelled probe for hybridization assays of the PCR products of ermF, ORF1 and ORF1ermF PCR assays. Plasmid pNFD13.2 was used as a labelled probe to hybridize with the products of tetQ, rteA/B and tetQrteA region PCRs.
Hybridization
Plasmid controls were labelled using the nonradioactive Genius kitTM plasmid labelling kit from Boehringer Mannheim according to the manufacturer's instructions.11 The labelled probes were hybridized to Southern blots of PCR products and Southern blots of whole-cell DNA.11 The hybridization and washes were done under the highly stringent conditions of 68°C according to the manufacturer's instructions (Boehringer Mannheim). Detection was done with CDP Star detection kit from Boehringer Mannheim according to the manufacturer's instructions.11
Mating experiments
Isolates carrying ermF and tetQ were used as the donors in the mating with E. faecalis JH2-2 as recipient. E. faecalis had previously been made chromosomally resistant to fusidic acid (25 mg/L) and rifampicin (25 mg/L),14 and had been well characterized in the matings with anaerobic donors.15,16 In addition, both the ermFand tetQgenes are expressed in E. faecalis so that the gene selection could be done directly.15,16 With anaerobic donors, the matings were done at the donor to recipient ratio of 5:1, while the matings with aerobic donors were done at the donor to recipient ratio of 1:1. The anaerobic donor and aerobic recipient mixture was incubated at 36.5°C for 48 h in anaerobic jars. The aerobic donor and recipient mixture was incubated for 24 h in a CO2 incubator (5% CO2). The transconjugants from anaerobic donors were selected aerobically on BHI plates supplemented with 10 mg/L erythromycin or 10 mg/L tetracycline. The transconjugants from aerobic donors were selected on BHI plates supplemented with 25 mg/L rifampicin and 10 mg/L erythromycin or 10 mg/L tetracycline to prevent growth of both donor and recipient. The transconjugants selected on erythromycin-supplemented plates were tested for the presence of the ermF and tetQgenes by PCRs for both ermFand tetQ, and for the ability to grow in the presence of 10 mg/L tetracycline. The transconjugants selected on tetracycline were tested for the presence of tetQ and ermF by PCR and the ability to grow in the presence of 10 mg/L erythromycin. The transconjugants were verified as E. faecalis by hybridizing with chromosomal DNA probe and biochemical tests.11,12
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Results |
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Thirty-seven (62%) out of 60 isolates were able to co-transfer ermF and tetQ to the unrelated E. faecalis recipient using either erythromycin or tetracycline for selection of transconjugants (Table I). Co-transfer of ermF and tetQ occurred from 25% of Prevotella bivia, which had the lowest percentage of isolates co-transferring both genes, and from 100% of the strains in Bacteroides distasonis, B. fragilis, Bacteroides ovatus, Bacteroides vulgatus, Clostridium butyricum, Prevotella denticola, Selenomonas dianae and Veillonella parvula isolates (Table I). However, in the last four species, only one isolate each was tested. Among the Bacteroidesspp., all B. distasonis, B. fragilis, B. ovatus and B. vulgatus isolates were able to co-transfer ermF and tetQ (Table I). In contrast, eight (62%) out of 13 B. forsythus co-transferred both antibiotic resistance genes, four (31%) isolates transferred ermFalone and one (8%) isolate did not transfer either gene (Tables I and II). No donors transferred only the tetQgene in this study. Three isolates, one B. forsythus, P. bivia and Prevotella nigrescens, did not transfer either ermF or tetQ to the E. faecalis recipient (Tables I and II), although transfer to an anaerobic recipient might have worked, but was not examined. The presence of other erm genes in the donor isolates were not tested, although all transconjugants were shown to carry the ermF gene (Table I).
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We screened 37 isolates that co-transferred the ermFand tetQ genes for the presence of ORF1, rteA and rteBas previously described.11 Twenty-nine (78%) isolates carried the ORF1 region (Tables I and II). None of the 13 B. forsythus carried the ORF1 region, though eight transferred both ermF and tetQ, and four transferred ermF (Tables I and II). We chose one isolate each of the 13 species that carried ORF1 and a B. forsythus, without ORF1, to test the linkage between the ORF1 region and the ermFgene. All 13 isolates that carried the ORF1 region had the ORF1ermF junction region as demonstrated by PCR products that hybridized with pBF4. As expected the one B. forsythus which did not carry the ORF1 gene produced no PCR product (Table II).
Thirty-three (89%) out of 37 donor isolates that transferred the ermF and tetQ genes carried the rteA gene. Only the four B. distasonis isolates examined lacked this gene (Table II). We chose one isolate from each of the 13 species carrying tetQ, rteA and rteB, and one B. distasonis (Table II) in order to examine the linkage of the tetQ and rteAgenes. All but the B. distasonis had the tetQrteA junction region, suggesting that the isolates that carried tetQ,, rteA and rteB had these three genes linked together (Table II). Four species had isolates co-transferring the ermF and tetQ genes, while the other isolates transferred the ermF gene only. P. bivia and Prevotella disiens carried the rteA gene regardless of their ability to co-transfer the ermF and tetQ genes (Table III). However, the two B. forsythus and two P. intermedia isolates that transferred the ermF gene only lacked rteA (Table III). When isolates that transferred only the ermFgene were tested, Fusobacterium nucleatum carried the rteA gene, whereas Gardnerella vaginalis lacked this gene (Table III). The tetQrteA junction region was not detected in five donor isolates (P. bivia, P. disiens, F. nucleatum) (Table III) that transferred rteA to transconjugants but not the tetQ gene (data not shown). When the 10 transconjugants that received the ermF gene only (Table III) were tested for the presence of the tetQrteA junction region, they were all negative (data not shown).
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In G. vaginalis, both the ermF and tetQ genes were present in the donor, but the ermF gene transferred alone (Table III). The G. vaginalis donor lacked the rteA and rteB genes (Table III). A different pattern was seen with the F. nucleatum donor, which carried all five genes (ORF1, ermF, tetQ, rteA and rteB). In the F. nucleatum donor, PCR did not detect the tetQrteA junction region, suggesting no linkage between these two genes (data not shown). In the F. nucleatum transconjugants ORF1, ermF, rteA and rteB genes were present and ORF1ermF and rteArteB showed linkage, but the tetQgene was not present. The tetQ gene was not transferred at a measurable frequency (<10-9/recipient) in these matings. The F. nucleatum isolate was the only one that transferred both rteA and rteB to the recipient without transferring the tetQ gene. Although the linkage between ermFrteA was not tested in the F. nucleatum donor, this region transferred as a unit to the transconjugants.
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Discussions |
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When we conducted a homology search using BLAST, the rteAgene product showed 2628% DNA identity with the genes for putative sensor proteins in E. coli. All isolates which co-transferred the ermF and tetQ genes carried rteA with the exception of B. distasonis. Whether the absence of the rteA in the B. distasonis isolates that can co-transfer both ermF and tetQ is unique to this species or more widespread is of interest for further study. It is also not known whether another gene is present in B. distasonis isolates which has the same function as rteA. There was no correlation between the presence of rteAand the ability to co-transfer ermF and tetQor transfer ermF alone. It is possible that the rteAgene is not required for co-transfer of the ermFand tetQgenes and passively transfers with the other genes.
A BLAST search showed that rteB had various degrees of DNA identity with the genes for different transcription regulatory proteins, such as hydG from E. coli (40%), algB from Pseudomonas aeruginosa (39%) and atoC from Treponema pallidum (37%). The ORF1rteB region represents approximately a 43 kb region within the 150 kb conjugative transposons previously described in Bacteroidesspp.1,2 We compared isolates from four species which included some isolates that could co-transfer ermFand tetQgenes, and some isolates that could only transfer the ermF gene. In addition, we studied two species that only transferred the ermFgene. The rteB gene was consistently present in the donors that co-transferred ermF and tetQ, but missing in donors that transferred ermF only, with one exception. This is consistent with the hypothesis that the rteB gene may be important in the transfer of elements containing the ORF1rteB region in the configuration depicted in the Figure, while rteAmay not be required. The BLAST search for the rteA and rteB genes are compatible with their previously suggested roles as putative sensor and regulator genes, respectively.17
A number of possible reasons could explain why only the ermF, ORF1ermF or ORF1rteA region was transferred in nine of 10 donors (B. forsythus, P. bivia, P. disiens, P. intermedia and G. vaginalis), all of which also carried the tetQ gene. Some of these isolates may have a different arrangement of the five genes from that illustrated in the Figure. A transposon may lack the rteB gene and have the 5' end of the rteA gene be truncated or mutated, or an insertion between the tetQ and rteA genes. This is suggested by the absence of tetQrteA and/or rteArteB junction region PCR products in these isolates. It is also possible that some donors carry a composite transposon,18 where one transposon carrying the tetQ gene is inserted within another conjugative transposon carrying ORF1, ermFand rteA or rteA/B genes. Another explanation would be that the tetQ gene resides on an independent piece of chromosomal DNA, which may either be nonmobile or transfer at a much lower frequency (<10-9/recipient) than the DNA carrying the ermF gene, and was not detectable at our assay's level of sensitivity. This is the best explanation for the F. nucleatum isolate, which carries all five genes, and can transfer all but the tetQgene to E. faecalis. The F. nucleatum isolate did not produce a PCR product from the tetQrteAassay, suggesting no linkage between the tetQ and rteA genes. We did not test for the linkage between the ermF and rteA genes because the region between these two genes is large and uncharacterized except for the tetQ gene. However, this region moved as a unit to transconjugants.
Our study indicates that a 43 kb region carrying both ermF and tetQ genes is more widely distributed than previously thought. We hypothesize that ORF1 may be a transposase, and we provide further data to support the role of rteA and rteB in transfer of the conjugative element with both ermF and tetQ.
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Acknowledgments |
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Notes |
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Present address. Department of Oral Biology, Box 357132.
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References |
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Received 18 January 1999; returned 23 March 1999; revised 12 April 1999; accepted 27 April 1999