Nucleotide sequence analysis of tetracycline resistance gene tetO from Streptococcus mutans DL5.

Streptococcus mutans DL5, isolated from the dental plaque of a pig, was resistant to high levels of streptomycin (Sm, 20 mg/ml), erythromycin (Em, 1 mg/ml), and tetracycline (Tc, greater than 100 micrograms/ml), but contained no detectable plasmid DNA. The Smr and Emr determinants were cloned from cellular DNA on the self-replicating 5-kilobase-pair (kbp) EcoRI fragment of pAM beta 1 and the 4.2-kbp cryptic plasmid pVA380-1, respectively, by transformation of Streptococcus sanguis Challis. Helper plasmid cloning, with a Challis host containing pVA380-1, was required to clone the Tcr determinant of strain DL5 on this vector. A single-colony isolate of the original Tcr clone contained a hybrid plasmid, pDL421, composed of 2.6 kbp of vector DNA and 11.4 kbp of S. mutans DNA. Plasmid pDL421 did not hybridize to plasmids containing the streptococcal Tcr determinants tetL, tetM, and tetN. A shortened derivative of this hybrid plasmid, pDL422, missing a 4.9-kbp HincII fragment from the S. mutans DNA but still encoding Tcr, was obtained by subcloning in S. sanguis Challis. The Tcr gene was located in a 1,917-base-pair open reading frame (ORF) corresponding to a 72-kilodalton protein. The ORF exhibited 99.4% sequence identity with the 1,917-base-pair tetO gene from a strain of Campylobacter coli (W. Sougakoff, B. Papadopoulou, P. Nordmann, and P. Courvalin, FEMS Microbiol. Lett. 44:153-160, 1987). A 1.67-kbp NdeI fragment, internal to the ORF from strain DL5, as well as pDL421 hybridized under stringent conditions to DNA from 10 of 10 Tcr strains of C. coli and Campylobacter jejuni from human and animal sources, but not to DNA from Tcs isolates of these two species.     

Modification of in vitro metabolism of T-2 toxin by esterase inhibitors.

A shuttle vector that can replicate in both Streptococcus spp. and Escherichia coli has been constructed by joining the E. coli plasmid pACYC184 (chloramphenicol and tetracycline resistance) to the streptococcal plasmid pGB305 (erythromycin resistance). The resulting chimeric plasmid is designated pSA3 (chloramphenicol, erythromycin, and tetracycline resistance) and has seven unique restriction sites: EcoRI, EcoRV, BamHI, SalI, XbaI, NruI, and SphI. Molecular cloning into the EcoRI or EcoRV site results in inactivation of chloramphenicol resistance, and cloning into the BamHI, SalI, or SphI site results in inactivation of tetracycline resistance in E. coli. pSA3 was transformed and was stable in Streptococcus sanguis and Streptococcus mutans in the presence of erythromycin. We have used pSA3 to construct a library of the S. mutans GS5 genome in E. coli, and expression of surface antigens in this heterologous host has been confirmed with S. mutans antiserum. A previously cloned determinant that specifies streptokinase was subcloned into pSA3, and this recombinant plasmid was stable in the presence of a selective pressure and expressed streptokinase activity in E. coli, S. sanguis (Challis), and S. mutans.     

Cloning and characterization of the tetracycline resistance determinant of and several promoters from within the conjugative transposon Tn919

Tn919 is a 15- to 16-kilobase (kb) tetracycline resistance conjugative transposon that was originally isolated from Streptococcus sanguis FC1. The tetracycline resistance determinant (tet) was found on a 4.2-kb HindII fragment by in vitro deletion analysis. This fragment was subcloned to a pWV01 origin capable of directing replication in Escherichia coli, Bacillus subtilis, and Streptococcus lactis, and expression was observed in all three genera. In all cases, expression was weaker when only the 4.2-kb cloned fragment rather than the full transposon was present. The resistance gene is of the streptococcal tetM class and codes for a protein of approximately 70 kilodaltons. The restriction map resembles that of the tetM gene of Tn1545 (P. Martin, P. Trieu-Cuot, and P. Courvalin, Nucleic Acids Res. 14:7047-7058, 1986), which codes for a protein of 72.5 kilodaltons. A number of transposon-derived promoter-bearing fragments were also cloned and sequenced. These closely resemble the consensus sequence of E. coli and B. subtilis promoters. Fusion experiments with a truncated lacZ gene indicate the possibility of an open reading frame for one of the promoters.     

Heterogeneity of tetracycline resistance determinants in Streptococcus

We found that naturally occurring tetracycline resistance in streptococci is encoded by more than one genetic determinant. Two of these distinct determinants were cloned, and the regions that are necessary and sufficient for expression of tetracycline resistance were defined by deletion analysis. These cloned determinants were further characterized by DNA-DNA hybridization experiments which also identified a third genetically unrelated tetracycline resistance determinant. Some of these genetic differences appear to represent mechanistic differences. The tetL determinant was associated with small nonconjugative plasmids and mediated resistance to tetracycline. The tetM determinant was most often "nonplasmid" associated and mediated resistance to minocycline as well as tetracycline. The tetN determinant was represented on a large conjugative plasmid and was genetically distinct from tetL and tetM, although phenotypically it resembled tetM.     

Characterization of genetic transformation in Streptococcus mutans by using a novel high-efficiency plasmid marker rescue system.

We developed a marker rescue system for study of competence development and genetic transformation in Streptococcus mutans. The system involved the recombinational rescue of a tetracycline resistance (Tcr) determinant by a homologous, inactive locus (Tcs because of a small deletion). Streptococcal cells harboring this in vitro-prepared Tcs construct (pVA1208) were restored to Tcr when plasmid (pVA981) DNA was used as donor material. pVA981 contained the intact streptococcal Tcr locus and was unable to autonomously replicate in streptococci. Marker rescue with this system followed first-order kinetics and occurred at a frequency 8- or 160-fold higher than did transformation with homologous chromosomal or plasmid DNA, respectively. By using the rescue system, we were able to confirm that competence of S. mutans appeared to be inducible. This was indicated by a sequential increase and then decrease in Tcr transformation frequencies during growth in complex medium. Also, donor DNA binding was not sequence specific, since the recovery of Tcr transformants was reduced by increasing the concentrations of heterologous DNA. We investigated the fate of donor DNA and the kinetics of plasmid establishment in the transformation of S. mutans with plasmid DNA. Monomeric plasmid molecules transformed S. mutans as a second-order process, whereas multimeric plasmid DNA and chromosomal markers were recovered as a first-order process. Approximately 50% of the initially bound donor plasmid DNA was found to remain in a trichloroacetic acid-insoluble form. Our results suggested that molecular cloning in S. mutans would be conducted most efficiently by using helper plasmid systems or shuttle vectors and that gene transfer by transformation of S. mutans occurred in a manner similar to that observed in Streptococcus sanguis.     

Phenotypic and genotypic characterization of tetracycline- and oxytetracycline-resistant Aeromonas hydrophila from cultured channel catfish (Ictalurus punctatus) and their environments

Oxytetracycline-resistant (OTcr) and tetracycline-resistant (Tcr) Aeromonas hydrophila were isolated commonly from catfish intestinal contents and the water and sediment of catfish culture ponds, but less frequently in market catfish. Isolates demonstrated two resistance phenotypes, Tcr OTcr and Tcs OTcr, when plated directly on to MacConkey agar containing 30 micrograms of tetracycline or oxytetracycline per ml. Tcs OTcr isolates expressed Tcr after induction by 1 h of growth in tryptic soy broth containing 1 microgram of tetracycline per ml. Over 90% of the resistant aeromonads hybridized with DNA probes for class A or class E Tcr determinants; class E was twice as prevalent as class A. The distribution of class A and E Tcr determinants varied with the Tcr phenotypes. Prior to induction, 86% of isolates with class A determinants were Tcr as well as OTcr, while only 16% with class E determinant were Tcr. Three of 5 Tcr aeromonads without class A or class E determinants and 1 of 14 with class E determinants transferred Tcr to Escherichia coli.     

Transposon Tn10-like tetracycline resistance determinants in Haemophilus parainfluenzae.

Tetracycline resistance (Tcr) determinants from three different strains of Haemophilus parainfluenzae expressed 10-fold higher levels of resistance when mated into Escherichia coli. No plasmid was found in any of the E. coli recipients, even in matings in which a plasmid was identified in the donor Haemophilus sp. The Tcr determinant from Haemophilus sp. caused instability of resident plasmids in the recipient E. coli: all plasmids were lost within 30 generations in antibiotic-free media. However, by serial subculture in antibiotics, stable resident plasmids were obtained which carried the Tcr determinant from Haemophilus sp. and were transferable by conjugation and transformation among E. coli strains. All Haemophilus determinants hybridized with a probe for the Tcr determinant on Tn10, which bears inducible Tcr. However, Haemophilus determinants were constitutively resistant to tetracycline in the Haemophilus donors and in the E. coli recipients. This constitutive expression was recessive to wild-type Tn10 in the same cell, indicating that the constitutive phenotype resulted from the absence of an active repressor. Restrictive enzyme analysis of various E. coli plasmid derivatives bearing a Tcr determinant from Haemophilus sp. demonstrated that the inserted DNA was of similar size (8.95 to 9.35 kilobases), close to that of Tn10. Heteroduplex analysis and DNA:DNA hybridization confirmed that the Tcr determinant from Haemophilus sp. had greater than 90% homology with the Tn10 determinant, including the DNA sequence for the repressor.     

Expression of a Streptococcus mutans glucosyltransferase gene in Escherichia coli.

Chromosomal DNA from Streptococcus mutans strain UAB90 (serotype c) was cloned into Escherichia coli K-12. The clone bank was screened for any sucrose-hydrolyzing activity by selection for growth on raffinose in the presence of isopropyl-beta-D-thiogalactoside. A clone expressing an S. mutans glucosyltransferase was identified. The S. mutans DNA encoding this enzyme is a 1.73-kilobase fragment cloned into the HindIII site of plasmid pBR322. We designated the gene gtfA. The plasmid-encoded gtfA enzyme, a 55,000-molecular-weight protein, is synthesized at 40% the level of pBR322-encoded beta-lactamase in E. coli minicells. Using sucrose as substrate, the gtfA enzyme catalyzes the formation of fructose and a glucan with an apparent molecular weight of 1,500. We detected the gtfA protein in S. mutans cells with antibody raised against the cloned gtfA enzyme. Immunologically identical gtfA protein appears to be present in S. mutans cells of serotypes c, e, and f, and a cross-reacting protein was made by serotype b cells. Proteins from serotype a, g, and d S. mutans cells did not react with antibody to gtfA enzyme. The gtfA activity was present in the periplasmic space of E. coli clones, since 15% of the total gtfA activity was released by cold osmotic shock and the clones were able to grow on sucrose as sole carbon source.     

Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria

The conjugative transposon Tn916 (15 kilobases), originally identified in Streptococcus faecalis DS16, has been cloned as an intact element on the pBR322-derived vector pGL101 in Escherichia coli. The EcoRI F' (EcoRI F::Tn916) fragment of pAM211 (pAD1::Tn916) was cloned into the single EcoRI site of pGL101 to form the chimera, pAM120, by selecting for the expression of Tn916-encoded tetracycline resistance (Tcr). Interestingly, in the absence of continued selection for Tcr, Tn916 excised from pAM120 at high frequency. This excision event resulted in a plasmid species consisting of the pGL101 vector and a 2.7-kilobase restriction fragment comigrating with the EcoRI F fragment of pAD1 during agarose gel electrophoresis. Filter blot hybridization experiments showed the 2.7-kilobase fragment generated as a result of Tn916 excision to be homologous with the EcoRI F fragment of pAD1. Analogous results were obtained with another chimera, pAM170, generated by ligating the EcoRI D' (EcoRI D::Tn916) fragment of pAM210 (pAD1::Tn916) to EcoRI-digested pGL101. Comparison of the AluI and RsaI cleavage patterns of the EcoRI F fragment isolated after Tn916 excision with those from an EcoRI F fragment derived from pAD1 failed to detect any difference in the two fragments: data in support of a precise Tn916 excision event in E. coli. Subcloning experiments showed that an intact transposon was required for Tn916 excision and located the Tcr determinant near the single HindIII site on Tn916. Although excision occurred with high frequency in E. coli, Tn916 insertion into the E. coli chromosome was a much rarer event. Tcr transformants were not obtained when pAM120 DNA was used to transform a polA1 strain, E. coli C2368.     

Characterization and mapping of regions encoding clindamycin resistance, tetracycline resistance, and a replication function on the Bacteroides R plasmid pCP1.

The Bacteroides drug resistance plasmid pCP1 encodes clindamycin resistance (Clr) and a cryptic tetracycline resistance (Tcr) determinant that is expressed in Escherichia coli cells grown aerobically, but not anaerobically, and is not expressed phenotypically in Bacteroides spp. Localization of genetic functions on pCP1 was facilitated by the construction of hybrid shuttle plasmids containing portions of pCP1 ligated to pDG5, a pBR322 derivative carrying the RK2 transfer origin. pDP1 delta 4 is a BglII deletion derivative of pCP1 linked to pDG5 and can be maintained in both E. coli and Bacteroides fragilis. By using Tn5 mutagenesis and subcloning, we localized the Clr and Tcr regions on the EcoRI B fragment between the 1.2-kilobase direct repeats of pCP1. The Clr and Tcr determinants are distinct and appear to be transcribed separately. Control of the Tcr phenotype is unusual in that expression is constitutive and is enhanced by a region encompassing the adjacent direct repeat. In addition, a region of pCP1 required for replication in Bacteroides spp. has been identified in the neighboring EcoRI A fragment.     

Identification and sequence of a tet(M) tetracycline resistance determinant homologue in clinical isolates of Escherichia coli

The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host.     

Genetic and biochemical characterization of the F-ATPase operon from Streptococcus sanguis 10904

Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species Streptococcus sanguis and Streptococcus mutans. Differences in the pH optima of the respective ATPases appears to be the main reason that S. mutans is more tolerant of low pH values than S. sanguis and hence pathogenic. We have recently reported the genetic arrangement for the S. mutans operon. For purposes of comparative structural biology we have also investigated the F-ATPase from S. sanguis. Here, we report the genetic characterization and expression in Escherichia coli of the S. sanguis ATPase operon. Sequence analysis showed a gene order of atpEBFHAGDC and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both S. sanguis and S. mutans; however, it is not induced to the same extent in the nonpathogenic S. sanguis. Expression studies with an atpD deletion strain of E. coli showed that S. sanguis-E. coli hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.     

Plasmid-mediated tetracycline resistance in Campylobacter jejuni: expression in Escherichia coli and identification of homology with streptococcal class M determinant

Plasmid pUA466, a 45-kilobase transmissible tetracycline resistance plasmid from Campylobacter jejuni was mapped with AvaI, AvaII, BclI, HincII, PstI, XhoI, and XbaI. The resistance determinant was cloned and expressed in Escherichia coli and was homologous with a class M determinant from Streptococcus spp.     

Physical and genetic analyses of streptococcal plasmid pAM beta 1 and cloning of its replication region.

Plasmid pAM beta 1, originally isolated from Streptococcus faecalis DS5, mediates resistance to the MLS (macrolide, lincosamide, and streptogramin B alpha) group of antibiotics. A restriction endonuclease map of the 26.5-kilobase (kb) pAM beta 1 molecule was constructed by using the enzymes AvaI, HpaII, EcoRI, PvuII, Kpn1, BstEII, HpaI, HhaI, and HindIII. A comparison of this map to those of four independently isolated deletion derivatives of pAM beta 1 located the MLS resistance determinant within a 2-kb DNA segment and at least one conjugative function within an 8-kb region. The 5.0-kb EcoRI-B fragment from pAM beta 1 was ligated onto the 4.0-kb Escherichia coli plasmid vector, pACKC1, and used to transform E. coli HB101. The 9.0-kb chimeric plasmid was then used to transform Streptococcus sanguis Challis with concurrent expression of the E. coli kanamycin resistance determinant. The 5.0-kb EcoRI-B fragment from pAM beta 1 was subsequently used as a vector to clone a streptomycin resistance determinant from a strain of Streptococcus mutans containing no detectable plasmid DNA. Subcloning experiments, using a HindIII partial digest of pAM beta 1 DNA, narrowed the replication region of this plasmid to a 2.95-kb fragment.     

Molecular analysis of a composite chromosomal conjugative element (Tn3701) of Streptococcus pyogenes.

The plasmid-free Streptococcus pyogenes A454 contains a conjugative element, Tn3701, encoding resistance to erythromycin (Emr), tetracycline (Tcr), and minocycline (Mnr). We have mapped a 50-kilobase (kb) chromosomal region of A454 corresponding to the internal part of Tn3701. Tn3701 includes a 19.7-kb structure, designated Tn3703, on which the Emr Tcr Mnr determinants were localized. Tn3703 was very similar in structure to Tn916. Translocation of the Emr Tcr Mnr markers from A454 onto pIP964, an Enterococcus faecalis hemolysin plasmid, yielded different pIP964 derivatives. When the inserts of four of these derivatives were aligned with the 50-kb region of Tn3701, three of them were found to result from the transposition of Tn3703 and one resulted from the insertion of a 44.0-kb portion of Tn3701, including Tn3703. Tn3701 inserted, apparently without changing its structure, in the chromosomes of various streptococcal transconjugants, as well as in one of the 12 E. faecalis transconjugants studied. Tn3703 inserted at different chromosomal sites in four E. faecalis transconjugants, and one copy of Tn3701 plus an additional copy of Tn3703 were detected in the chromosomes of seven transconjugants.     

Regions of the Streptococcus sobrinus spaA gene encoding major determinants of antigen I.

Surface protein antigen A (SpaA), also called antigen B, antigen I/II, or antigen P1, is an abundant cell envelope protein that is the major antigenic determinant of Streptococcus sobrinus and other members of the Streptococcus mutans group of cariogenic bacteria. This laboratory has previously reported the cloning and expression in Escherichia coli of a BamHI restriction fragment of S. sobrinus DNA containing most of the spaA gene (pYA726) and encoding antigen I. Regions of spaA encoding immunodeterminants of antigen I were analyzed by either deletion mapping or expressing selected restriction fragments from the trc promoter. SpaA proteins produced by mutants harboring nested deletions, constructed by BAL 31 exonuclease treatment at a unique SstI site located towards the 3' end of the gene, were examined by Western immunoblot with rabbit serum against SpaA from S. sobrinus. Only SpaA polypeptides larger than 56 kilodaltons reacted with anti-SpaA serum. Various restriction fragments of the region of spaA encoding the antigenic determinants were cloned into an expression vector. The immunoreactive properties of the polypeptides encoded by those fragments indicated that expression of the immunodominant determinant required topographically assembled residues specified by noncontiguous regions located within 0.48-kilobase PvuII-to-SstI and 1.2-kilobase SstI-to-HindIII fragments which were adjacent on the spaA map.     

Heterologous gene expression in Bacteroides fragilis.

Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.     

Host-vector system for integration of recombinant DNA into chromosomes of transformable and nontransformable streptococci.

We describe a genetic system in which transformation of Streptococcus pneumoniae and Streptococcus sanguis was used to insert recombinant DNA into the conjugative chromosomal element omega (cat tetM) 6001 (omega 6001). The element containing the recombinant DNA was then transferred by conjugation to the chromosome of transformable and nontransformable streptococci. When Escherichia coli plasmid pDP36 was used as donor in transformation, it was capable of inserting 5.9 kilobases of heterologous DNA into the chromosome of competent streptococcal strains carrying omega 6001; the transformants were scored for erythromycin resistance. Genetic analysis showed that in a fraction of the erythromycin-resistant transformants the integration via flanking homology of the heterologous DNA caused inactivation of the tetM gene of omega 6001. By analyzing the stability of the resistance markers, we found that stable integration of heterologous DNA was achieved only in the erythromycin-resistant, tetracycline-sensitive transformants. It was possible to detect conjugal transfer of the heterologous sequences from stable transformants to strains of S. pneumoniae, S. sanguis, Streptococcus pyogenes, and Streptococcus faecalis. The omega 6001-pDP36 host-vector system opens new possibilities for gene transfer in streptococci. By this method cloned streptococcal DNA (possibly mutagenized in vitro) can be returned to the original host, greatly facilitating complementation tests and fine physiological studies.