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1 Department of Biomedical Sciences, College of Veterinary Medicine and the Molecular and Cellular Biology Program, Oregon State University, Corvallis, OR 97331, USA
2 National Animal Disease Center, USDA Agriculture Research Service, Ames, IA 50010, USA
Correspondence
Daniel D. Rockey
rockeyd{at}orst.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Pollmann et al., 2005; Wang et al., 2005). One chlamydia of veterinary concern is Chlamydia suis, a pathogen that can cause conjunctivitis, abortion and enteritis in swine (Pollmann et al., 2005). Recent work in our laboratory has identified the nature of a set of tetracycline (Tet) resistant (TetR) C. suis strains isolated from pigs in the midwestern USA (Fig. 1). These isolates are stably resistant to Tet and, in some cases, sulfadiazine (Andersen & Rogers, 1998; Lenart et al., 2001; Suchland et al., 2003). This resistance phenotype is conferred by genomic islands [tet(C) islands] that are apparently acquired through horizontal transfer (Dugan et al., 2004).
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). While there is a single genomic island within a related obligate intracellular species, the tet(C) islands represent the first example of recent horizontal acquisition of DNA in any Chlamydia spp. (Greub et al., 2004; Roberts, 2005). It is anticipated that an understanding of the mechanisms associated with acquisition of the tet(C) islands by C. suis may assist in the development of a stable transformation system in the chlamydiae.
Each of the tet(C) islands has several genes found in plasmids from Gram-negative bacteria, including tet(C) and tetR(C), which encode an efflux pump for Tet resistance and the cognate repressor protein, respectively (Chopra & Roberts, 2001). Most of these islands also carry a novel insertion element (IS), IScs605 (Dugan et al., 2004). IScs605 is homologous to the IS605 family of insertion sequence elements, which are most commonly found in Helicobacter pylori (Kersulyte et al., 1998, 2000, 2002, 2004; Ton-Hoang et al., 2005). This family of IS elements consists of two divergently oriented ORFs that are members of different families of IS elements. One of these (orfA) is related to members of the IS200 family of transposases, while the second (orfB) is similar to a family of proteins related to candidate transposases encoded within IS1341 (Bisercic & Ochman, 1993; Dugan et al., 2004; Murai et al., 1995). There is 100 % sequence identity of IScs605 from the different C. suis strains. A pentanucleotide sequence, 5'-TTCAA, is always present adjacent to the orfA end of IScs605 (Fig. 1
). The tet(C) islands integrate into the C. suis chromosome within an invasin-like gene, and the site of insertion is also 5'-TTCAA (Dugan et al., 2004).
Nucleotide sequence analysis has suggested that IScs605 may have had a role in the acquisition of the tet(C) islands by C. suis. The described experiments demonstrate that IScs605 can direct transposition in an Escherichia-coli-based mating assay, yielding transposition products consistent with those observed in the TetR C. suis strains.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). E. coli SF800 is resistant to nalidixic acid (Nal), and has a deletion in polA, which prevents the strain from supporting ColE1-based origins of replication (Gross & Gross, 1969; Heffron et al., 1977; Stachel et al., 1985). E. coli TOP10 (Invitrogen) was used for plasmid rescue, and for constructing the pUC18- and pBBR1MCS-based vectors. When indicated, particular antibiotics were added to the medium at the following concentrations: Nal, 10 µg ml–1; kanamycin (Kan), 50 µg ml–1; ampicillin (Amp), 100 µg ml–1; chloramphenicol (Chl), 100 µg ml–1; Gen, 10 µg ml–1. All antibiotics were purchased from Sigma.
General PCR conditions.
PCR reactions were performed with 0.25 mM dNTP (Fermentas), 0.4 nM forward and reverse primers (Table 1), and Taq (NEB) or Pfx polymerase (Invitrogen), as indicated. The following reaction parameters were used for all amplifications: templates were denatured for 45 s at 94 °C, the primers were annealed for 45 s at 52 °C, and products were extended for 90 s at 72 °C. All amplifications were performed for 30 cycles. Digoxigenin-labelled PCR products were made using previously described methods (Dugan et al., 2004). The digoxigenin-labelled PCR probe of aph was produced with primers JP11 and JP12, using the pCR-Blunt vector (Invitrogen) as the template. The digoxigenin-labelled PCR probe of bla was produced with primers JP13 and JP14, using pUC18 as the template. The digoxigenin-labelled PCR probe of cat was produced with primers JP15 and JP16, using pBBR1MCS as the template. The digoxigenin-labelled PCR probe of pOX38 was produced with primers JP9 and JP10, using pOX38 as the template.
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, and the details for each set of plasmids are described in the following paragraphs.
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). The PCR product was cloned into pUC18, and transformed into Top10 cells, creating pUC18 : IScs605. Since there is no antibiotic resistance marker on IScs605, a KanR cassette was constructed by placing the resistance gene (aph), flanked by approximately 150 terminal nucleotides from each end of the IS element, adjacent to full-length IScs605. The primers JP4 and JP5 were used in a long-PCR protocol to amplify the vector backbone of pUC18 : IScs605, which lacked the candidate transposase ORFs. Amplification with these primers produced a linear product containing terminal fragments of each IScs605 end (Table 1) flanking the entire pUC18 molecule, with terminal restriction sites for NotI and EcoRV. The aph sequence was then amplified from the pCR-blunt vector (Invitrogen) using Pfx polymerase with primers JP11 and JP12, and this resulted in an aph sequence with terminal NotI and EcoRV restriction enzyme sites. This product was then ligated to the amplification product from the outward PCR reaction. The resulting plasmid (pJD2) was transformed into E. coli, and selected by growth on LB agar containing Kan. The IScs605/aph fragment from pJD2 was then excised with EcoRI, and ligated into the full-length pUC : IScs605 to create the plasmid pJD1. The plasmids pJD1 and pJD2 served as experimental and control plasmids, respectively, for initial studies of the transpositional activity of IScs605.
orfA and orfB knockouts.
Each of the two ORFs in IScs605 was independently inactivated using a restriction-enzyme-based approach. A 200 bp fragment of orfA was deleted by digesting pJD1 with PsiI and PflMI, which are enzymes that each cut only once within the orfA ORF. The restricted plasmid was electrophoresed through 1 % agarose, and the vector-containing fragment was excised from the gel and purified. The DNA was then treated with T4 DNA polymerase (Fermentas) to blunt end the restriction sites. The product was then self-ligated, and transformed into E. coli TOP10. PCR was used to demonstrate that the resulting plasmid pJD3 carried an inactivation within orfA. A similar strategy was used for orfB. In this case, a 1.1 kb deletion within orfB was created by restriction digest with the enzymes NsiI and HpaI, leading to the plasmid pJD4.
Replacement of orfB with the KanR marker aph.
The orfB sequence within pJD1 was replaced with the KanR marker aph from the plasmid pCR-Blunt vector (Invitrogen) by restricting pJD1 with NsiI and HpaI, blunting the single-stranded ends, and ligating aph into the vector. The aph ORF was amplified with the primers JP11 and JP12 (Table 1). Transformed clones were identified by plating on LB agar containing Kan, and resistant colonies were screened by PCR. This resulted in the plasmid pJD5.
Deletion of the targeting sequence within IScs605.
The 5'-TTCAA found at the orfA end of IScs605 was deleted within pJD1 by performing Pfx-based PCR with the primers JP1 and JP3, using pJD5 as the template. This amplification produced an IS element identical to that found in pJD5, except for a deleted terminal 5'-TTCAA sequence. This PCR product was inserted into pUC18, creating pJD6.
IScs605 insertions into pBBR1MCS.
The medium-copy-number vector pBBR1MCS (Kovach et al., 1994) was also used to analyse IScs605 transposition activity. The IScs605 sequence from pJD5 was PCR-amplified using the primers JP1 and JP2. The PCR product was then inserted into pBBR1MCS, producing the plasmid pJD7.
Mating assay.
The mating assay was performed by individually introducing each of the plasmids (Fig. 2) into chemically competent E. coli RZ212. Following transformation, individual colonies were picked, and cultured overnight in LB broth. These cultures were diluted 50-fold into 10 ml LB broth containing 0.5 % glucose, and grown for 2–4 h to an OD600 of 0.4–0.6. The transformed donor strain was then added to exponentially growing E. coli SF800 in equal proportions. This mating mix was incubated at 37 °C for 3 h, without shaking, to allow conjugation between the donor strain and the polA-deficient recipient strain. Bacteria were then diluted and spread onto LB agar containing Nal to determine the total number of recipient bacteria, and onto LB agar containing Kan and Nal to determine the number of recipient bacteria that carried pOX38/IScs605 recombinant plasmids. The values reported represent the number of transconjugants (KanR NalR) per 109 recipient bacteria (NalR), and each experiment was repeated at least four times.
Genomic DNA purification.
Transconjugants from mating assays were grown overnight in LB at 37 °C, and then concentrated by centrifugation at 3000 g. Pellets were resuspended in 50 mM Tris, pH 7.4, 1 % SDS, and 100 units proteinase K (Fermentas). The mixture was incubated at 37 °C for 4–6 h, and extracted three times with 1 : 1 phenol/chloroform, followed by a single chloroform extraction. The DNA was precipitated with ethanol/ammonium acetate and resuspended in water to a concentration of 1 µg µl–1.
Plasmid rescue.
Genomic DNAs from individual transconjugant clones were restricted with the enzymes MluI and BssHII for 4 h. These restriction enzymes generate the same overhanging ends following digestion and cut many times within pOX38, but do not cut within pJD1, pJD5 or pJD7. Products of these digests were purified using a commercial kit (Qiagen), self-ligated, and then transformed into TOP10 cells. Individual AmpR KanR colonies were transferred to LB, and cultured overnight. Plasmids were purified from these cultures (Qiagen), and the nucleotide sequence of DNA flanking the likely insertion site was determined using primers JP6, JP7 and JP8.
Southern blot analysis.
Purified genomic DNA of transconjugants was analysed by Southern blots, as described previously (Dugan et al., 2004). Genomic DNA was digested with MluI and BssHII, PsiI or PstI, followed by electrophoresis through a 1 % agarose gel, and then passively blotted to nitrocellulose. The enzymes MluI and BssHII do not cut within any of the pJD plasmids, and thus MluI/BssHII restriction fragments analysed by Southern blotting represent DNA not digested within the original plasmid source of IScs605. In contrast, the enzymes PstI and PsiI cut once within pJD1 through pJD6 (PstI) and pJD7 (PsiI), and they were useful for identifying multimers of donor plasmids within pOX38. The blots were analysed using probes for aph, bla, IScs605 orfB, cat and pOX38.
Excision analysis.
Genomic DNA of transconjugants was used as template for PCR reactions to look for evidence of excision of IScs605 from pOX38 recombinants. Primers for these experiments (JP17 and JP18) were designed to amplify a 750 nt fragment that would be present in genomic DNA if IScs605 had deleted itself from a pJD1 construct. PCR products that were of the appropriate size were excised from gels, and the nucleotide sequence was determined.
Transposition of IScs605 versus integration of entire plasmids.
Transconjugants from both pUC18-based (pJD5) and pBBR1MCS-based (pJD7) constructs were analysed for independent transposition of IScs605 into pOX38. Standard mating assays were conducted, and colonies were selected using Kan and Nal. Transconjugants from each mating assay were then individually transferred to plates containing Kan and Nal, and either Amp (for pJD5) or Chl (for pJD7). The ratio of clones that grew on Kan and Nal only, versus those that grew on plates containing Nal and Amp, or Nal and Chl, was determined for each tested donor plasmid. Southern blotting was also conducted using PsiI-digested genomic DNAs from pJD7-based mating assays. DNAs from ChlR and ChlS transconjugants were used in these blots, and probed with aph to monitor the presence and number of IS elements in the transconjugant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2000). This assay involves transforming a donor strain with IScs605-containing plasmids (Fig. 2), and selecting for transposition into a conjugative plasmid, pOX38. Several different experiments were conducted using independent clones of donors transformed with different pJD1 plasmids, and these assays resulted in a mean of 3265 transconjugants per 109 recipient bacteria (Fig. 3). Control experiments using pJD2 resulted in a mean of <5 background colonies per 109 recipient bacteria. These data demonstrate that the presence of intact IScs605 resulted in an approximately 1000-fold increase in resistance to Kan, relative to a control vector, in this transposition system.
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ORF knockouts
The two ORFs within IScs605 are members of different families of IS elements. To assess the possible role of each ORF in the integration of pJD1 into pOX38, plasmids containing interrupted orfA or orfB (pJD3 and pJD4 respectively) were constructed and used in mating assays. The results showed that inactivation of orfA led to a reduction of transconjugants to control levels, while the inactivation of orfB had a minimal effect on the formation of AmpR NalR transconjugants (Fig. 3). Mating experiments using pJD5, in which the orfB ORF was replaced by aph, produced identical results to pJD4. This construct was also capable of facilitating integration of DNA into pOX38 (Fig. 3). These experiments supported the conclusion that orfA was essential for transposition, while inactivation of orfB had no detectable effect on transposition frequency.
Insertion-site specificity
Nucleotide sequence analysis was performed to determine the site of insertion of the IScs605 plasmids into pOX38. These data demonstrated that integration of pJD1 occurred throughout pOX38, and that insertions occurred in both orientations (Fig. 4). Certain regions appeared to lack recombined plasmid, but it is likely that integrations at these sites (i.e. critical genes for conjugation during the mating assay) may have resulted in a pOX38 : pJD1 plasmid that did not conjugate efficiently into recipient E. coli (Frost et al., 1994). These data also demonstrated that 36 out of 38 independent insertion events were adjacent to a 5'-TTCAA sequence present within pOX38 (Fig. 5), and that the two alternate target sequences (5'-GTCAA and 5'-TTTTCAG) were similar to 5'-TTCAA. This insertion-site specificity was also observed in experiments using the orfB-deletion plasmid pJD5. There was no evidence of insertion specificity at the orfB end of IScs605 (Fig. 5). The sequencing also demonstrated that there were no direct or inverted repeats at the sites of plasmid integration. These data were consistent regardless of which particular plasmid containing the IS element was used for analysis, as the plasmids pJD1 (pUC-based, intact orfA and orfB), pJD5 (pUC-based, orfB deletion) and pJD7 (pBBR1MCS-based orfB deletion) consistently targeted the 5'-TTCAA site (Fig. 5). Thus, the presence of orfA within the IS element is responsible for both the transpositional activity and insertion specificity of IScs605.
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). These experiments suggested several different scenarios: (1) more than one pOX38 recombinant may be present in a single cell, (2) secondary transposition may have occurred inside either the donor or the recipient cell, or (3) concatamers of pJD1 may be inserted into single pOX38 within the donor. As mentioned above, Southern blots were also performed using DNA digested with the enzymes MluI and BssHII, which do not have recognition sites within pJD1. These blots demonstrated that the bla-specific probe reacted with single positive restriction fragments in all tested clones (not shown). These data indicate that, in E. coli, the IScs605 element can drive the integration of entire plasmids and plasmid concatamers into a target DNA.
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). This was supported by detailed analysis of several products recovered for nucleotide sequence analysis (not shown). These observations led us to hypothesize that IScs605 may have excised itself in subpopulations from many of the primary recombinants. This was tested by PCR-based analysis of selected genomic DNAs. Oligonucleotide primers flanking IScs605 in recipient pOX38 were used to successfully amplify sequences consistent with excision events in 40 out of 40 tested transconjugants. Sequence analysis of a subset of these PCR products showed that products lacking the entire IS element were present in the population, with a signature sequence 5'-AATTCAA at the original location of the IS element (Fig. 7b). Evidence of such excision events was found in both pJD1- and pJD5-based transconjugants, suggesting that the excision event was also orfA mediated. Therefore, IScs605 can excise from cointegrates produced in this E. coli system.
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). The importance of this sequence in IScs605-mediated transposition was examined by conducting mating assays with a plasmid construct that had the 5'-TTCAA sequence deleted from the IS element (pJD6, Fig. 2). This deleted plasmid was not able to facilitate integration into pOX38 (Fig. 3). Therefore, the 5'-TTCAA sequence at the orfA end of IScs605 is essential for transposition in E. coli.
IScs605 transposition activity in pBBR1MCS
To examine the possible role of the vector in defining integration events in the mating experiments, parallel assays were conducted with plasmid pJD7, in which the medium-copy-number plasmid pBBR1MCS was the vector for delivery of orfA+ orfB IScs605 in the mating assay. Analysis of the transconjugants using pJD7 as the source of IScs605 showed both similarities and differences to experiments with the pUC-based plasmids. First, there were similar rates of transposition observed using either plasmid donor (not shown). Sequencing of the transconjugants showed that pJD7 also inserted adjacent to 5'-TTCAA (Fig. 4) and that there was evidence of concatamers in the inserted products (Fig. 7b, lanes 1–4). However, in contrast to the observations with pUC-based mating assays, transconjugants from the pBBR1MCS matings routinely resulted in only the IS element – and not the vector – being incorporated into pOX38 (Fig. 7a). This was measured using donor plasmids that contained the aph marker within the IS element (pJD5 and pJD7). Selection could then be measured first with Amp, and then transconjugants examined for a resistance marker in the vector. pUC-based mating assays universally resulted in recombinants containing both markers, while pBBR1MCS-based mating assays resulted in a high frequency of products that contained only the marker in the IS element, and not the marker in the vector. Therefore, in some plasmid backgrounds, IScs605 orfA can facilitate transposition of the IS element in the absence of the donor plasmid.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Both medium- and high-copy-number plasmids were successfully used in these assays (Fig. 7). Nucleotide sequencing showed that integration of the donor plasmids occurred at many sites within pOX38, but that the sequence 5'-TTCAA was the predominant target. Sequence analysis also demonstrated that there was no sequence duplication or evidence of repeats at the insertion site (Figs 4 and 5). Southern blotting and PCR demonstrated that IScs605 can also excise from target sequences following integration of entire plasmids (Figs 5 and 6), leaving the sequence 5'-AATTCAA at the excision site. Each of these traits is consistent with the observed organization of the tet(C) islands in C. suis.
IScs605 is structurally related to IS605, IS606, IS607 and related IS elements from H. pylori (Kersulyte et al., 1998, 2000, 2002, 2004). These relationships can be seen in a phylogenetic analysis of orfA and orfB, which have apparent homologues both within the IS605-like IS elements, and within IS elements containing only single candidate transposases (Dugan et al., 2004). The data from the mating assays demonstrate that there are also functional similarities. H. pylori IS605 integrates at chromosomal sites specifically with the orfA end adjacent to the sequence 5'-TTTAA or 5'-TTTAAC (Kersulyte et al., 1998). H. pylori IS607 requires orfA for transposition, but not orfB, and inserts adjacent to single G nucleotides (Kersulyte et al., 2000). H. pylori ISHp608 inserts at the tetranucleotide sequence 5'-TTAC, also in an orfA-dependent manner (Kersulyte et al., 2002). In no case does insertion or excision of these islands result in deleted or duplicated chromosomal sequence. Each of these traits is parallel to the insertional properties of IScs605 in E. coli, and is consistent with the sequences of IScs605 in the resistant C. suis strains.
Analysis of secondary transposition events in the mating assay demonstrated that excision of the IS element was common in this system, and these events resulted in the sequence 5'-AATTCAA remaining at the previous location of IScs605 (Fig. 5b). Such secondary events may help to explain differences in sequence within some of the tet(C) islands. In C. suis strains 130 and 132, the tet(C) island lacks the IS element, and the sequence surrounding a possible excision site is 5'-AATTCAA (Fig. 1, compare strain R19 with strain 130). A related event may also explain the small size of the R27 and H7 islands. In these strains, the genomic island has a large deletion which occurs at a 5'-AATTCAA sequence found within the other tet(C) islands. Therefore, it is likely that IScs605-mediated excision events are also responsible for production of the smaller tet(C) islands, such as those seen in C. suis strains R27 and 130.
There were, however, some differences between the behaviour of IScs605 in E. coli and the predicted events leading to insertion of the tet(C) islands into C. suis. First, we observed multiple examples of concatamer formation of plasmids associated with integration of pJD plasmids into pOX38. There are no examples of similar concatamers in any C. suis strain (Dugan et al., 2004). Additionally, when the medium-copy-number plasmid pBBRMCS was used as a source of IScs605 (i.e. pJD7), the predominant insertion event involved an actual transposition of the IS element into the pOX38 target. These results provide evidence that the IS element is also capable of true transposition into target DNA. However, this event has not yet been identified in any C. suis strain. The low-copy-number plasmid experiments were conducted with a plasmid containing aph inserted into the orfB coding sequence, and thus the transposition events were selected for using Kan. The intact IScs605 sequence has no marker, and therefore could not be directly selected for, either in our system or within the original C. suis isolates found in farmed pigs. We have previously hypothesized that feeding of Tet was responsible for selection of the TetR C. suis (Dugan et al., 2004), and that in that model, transposition of IScs605 without the accompanying plasmid sequence would not lead to selection of resistant strains.
Deletion analysis was used to test the role of the individual ORFs within IScs605. The data suggest that orfA, but not orfB, is essential for both transposition activity and insertion specificity (Fig. 3). These results parallel the tested IS605-like elements in the Helicobacter system, in which the homologous orfA sequence appears critical for transposition (Kersulyte et al., 1998, 2000, 2002). The function of the highly conserved orfB in the IS605 family of IS elements remains unclear. We have previously hypothesized that orfB of H. pylori IS605 serves to increase bacterial fitness in vivo, and this was supported by BLAST analysis, which showed that IScs605 orfB is similar to the putative E. coli virulence–factor-encoding gene ydcM (Dugan et al., 2004). Another possibility is discussed by Kersulyte et al. (2002) who proposed that ydcM might serve to complement a deletion in orfB in the mating assays. The chlamydiae do not have an apparent homologue of orfB within their genomes (not shown), and thus orfB within the IS element may have been important for the integration of the tet(C) islands in C. suis.
The described experiments demonstrate that IScs605 can direct the integration of plasmids into a target sequence with specificity that is parallel to that seen in the chlamydial tet(C) islands. It is therefore likely that IScs605 activity led to the integration of the tet(C) islands into the C. suis chromosome. IScs605 is the only known example of an IS element within any chlamydial species, and this work describes the first analyses of the activity of a horizontally acquired element within the chlamydiae. The chlamydial insertion element also may be important in practical aspects of the study of chlamydiae. The tet(C) islands have several characteristics that may be useful in the genetic transformation of chlamydiae, a technology that is currently unavailable in these organisms. The exploration of this possibility is currently under way in our laboratory.
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ACKNOWLEDGEMENTS |
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Edited by: T. P. Hatch
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Received 27 June 2006;
revised 29 August 2006;
accepted 6 September 2006.
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