PubMed Health⌕ Search

Biomedical subjects

F L Macrina

Publications and source records attributed to F L Macrina.

At least 55 records · Page 3Linked to original sources
PubMed1988

Inter- and intrageneric transfer of Tn916 between Streptococcus faecalis and Clostridium tetani.

We report that the streptococcal resistance transposon, Tn916, is conjugally transferred to Clostridium tetani (Utrecht) in intergenic matings. Streptococcus faecalis CG180, harboring a 41-kb plasmid (pAM180) containing Tn916 (15 kb), transferred the transposon-associated tetracycline resistance (Tcr) to C. tetani in filter matings at a frequency of about 10(-4)/donor. An erythromycin resistance marker carried by pAM180 was not transferred, indicating lack of plasmid conjugation or stable inheritance of plasmid sequences. DNA extracted from C. tetani transconjugants was probed with radiolabeled Tn916 using Southern blot analysis and these results indicated that the transposon integrated at multiple host genomic sites. Tn916-carrying C. tetani strains were able to transfer Tcr to suitable recipient strains of C. tetani as well as to S. faecalis recipients. These results indicate that this transposon is able to be disseminated and expressed in obligately anaerobic gram-positive bacteria. Moreover, this system opens avenues for the implementation of transposon mutagenesis in this important pathogenic species.

Clostridium tetani↗
PubMed1987

Extrachromosomal systems and gene transmission in anaerobic bacteria.

Obligately anaerobic bacteria are important in terms of their role as medical pathogens as well as their degradative capacities in a variety of natural ecosystems. Two major anaerobic genera, Bacteroides and Clostridium, are examined in this review. Plasmid elements in both genera are reviewed within the context of conjugal transfer and drug resistance. Genetic systems that facilitate the study of these anaerobic bacteria have emerged during the past several years. In large part, these developments have been linked to work centered on extrachromosomal genetic systems in these organisms. Conjugal transfer of antibiotic resistance has been a central focus in this regard. Transposable genetic elements in the Bacteroides are discussed and the evolution and spread of resistance to lincosamide antibiotics are considered at the molecular level. Recombinant DNA systems that employ shuttle vectors which are mobilized by conjugative plasmids have been developed for use in Bacteroides and Clostridium. The application of transmission and recombinant DNA genetic systems to study these anaerobes is under way and is likely to lead to an increased understanding of this important group of procaryotes.

Bacteroides↗
PubMed1987

Expression of Streptococcus sanguis antigens in Escherichia coli: cloning of a structural gene for adhesion fimbriae.

Chromosomal DNA from Streptococcus sanguis FW213 was partially digested with EcoRI and ligated into the positive-selection cloning vector pOP203(A2+). The ligation mixture was used to transform Escherichia coli K-12, and 4,500 transformants were examined. The tetracycline-resistant colonies had inserts averaging 3.2 kilobases. The entire colony bank was screened by colony immunoassay with polyclonal rabbit serum raised against S. sanguis FW213 whole cells. Thirty recombinant colonies produced stable positive reactions of various intensities, indicating that S. sanguis antigens could be expressed in E. coli. Restriction endonuclease digestion of these clones suggested that 26 of the clones were unique. Only two clones, VT616 and VT618, gave positive reactions with fimbria-specific antisera. That the gene coding for the antigen was located on the plasmid was confirmed by demonstrating that the presence of the plasmid was linked to antigen production. Western immunoblot analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that both clones produced a fimbrial peptide of Mr 30,000. The two recombinant plasmids were shown by Southern analysis and restriction mapping to contain the same 6-kilobase EcoRI fragment inserted in opposite orientations. Southern hybridization confirmed that this fragment is present in S. sanguis genomic DNA. The Mr 30,000 protein gene was expressed in both orientations, suggesting that the fimbrial promoter is located on the 6-kilobase fragment. These results show that at least one streptococcal fimbrial gene can be cloned and expressed in E. coli.

Antigens, Bacterial↗
PubMed1987

Molecular cloning and characterization of the glucosyltransferase C gene (gtfC) from Streptococcus mutans LM7.

A glucosyltransferase (GTF) gene, designated gtfC, was cloned from Streptococcus mutans LM7. Its gene product was detected by screening a bacteriophage lambda library with rabbit antiserum raised against S. mutans LM7 extracellular proteins. DNA isolated from the immunopositive recombinant phage revealed two S. mutans chromosomal EcoRI fragment inserts, 8.1 and 4.7 kilobase pairs in size. Escherichia coli minicell analyses revealed the approximate position and direction of transcription of the gtfC gene. The gene product was determined to be a polypeptide of about 150 kilodaltons which synthesized a water-soluble glucan. Restriction endonuclease mapping and DNA hybridization indicated a repeated region of DNA corresponding to a portion of the coding region of gtfC immediately downstream from the intact gtfC locus on the chromosome. A 300-base-pair gtfC-specific probe showed that the gene and the putative duplicated sequence were present in S. mutans serotypes c, e, and f, but not in other related oral streptococci which had GTF activity. In addition, the gtfC determinant displayed homology to sequences corresponding to the carboxy-terminal coding region of a gene (gtfB) encoding a GTF activity that synthesized water-insoluble glucans. These data suggest that at least one class of GTF genes may be present in multiple copies in S. mutans and, further, that GTF genes may contain conserved sequences internal to their coding regions.

Chromosome Mapping↗
PubMed1987

Molecular cloning and characterization of a Streptococcus sanguis DNase necessary for repair of DNA damage induced by UV light and methyl methanesulfonate.

We developed a method for cloning cellular nucleases from streptococci. Recombinant lambda gt11 bacteriophage containing streptococcal nuclease determinants were identified by the production of pink plaques on toluidine blue O DNase plates. We used this technique to clone a 3.2-kilobase-pair EcoRI fragment with DNase activity from the chromosome of Streptococcus sanguis. The locus was designated don (DNase one) and could be subcloned and stably maintained on plasmid vectors in Escherichia coli. Minicell analyses of various subclones of the don locus allowed us to determine the coding region and size of the Don nuclease in E. coli. The don gene product had an apparent molecular mass of 34 kilodaltons and degraded native DNA most efficiently, with lesser activity against denatured DNA and no detectable activity against RNA. S. sanguis don deletion mutants were constructed by transformation of competent cells with in vitro-prepared plasmid constructs. S. sanguis don deletion mutants retained normal transformation frequencies for exogenously added donor DNA. However, when compared with Don+ wild-type cells, these mutants were hypersensitive to DNA damage induced by UV light and methyl methanesulfonate. An S. sanguis don-specific DNA probe detected homology to chromosomal DNA isolated from Streptococcus pneumoniae and Streptococcus mutans Bratthall serogroups d and g. Our results suggested that the don locus was the S. sanguis allele of the previously described S. pneumoniae major exonuclease and was involved in repair of DNA damage. Furthermore, hybridization studies suggested that the don locus was conserved among species of oral streptococci.

Bacterial Proteins↗
PubMed1987

Complete nucleotide sequence of insertion element IS4351 from Bacteroides fragilis.

The nucleotide sequence and genetic analyses of one of the directly repeated sequences flanking the macrolide-lincosamide-streptogramin B drug resistance determinant, ermF, from the Bacteroides fragilis R plasmid, pBF4, suggested that this region is an insertion sequence (IS) element. This 1,155-base-pair element contained partially matched (20 of 25 base pairs) terminal-inverted repeats, overlapping, anti-parallel open reading frames, and nine promoterlike sequences, including three that were oriented outward. Analysis of this sequence revealed no significant nucleotide homology to 13 other known IS elements. Inasmuch as Southern blot hybridization analysis detected homologous sequences in chromosomal DNA and its G+C content (42 mol%) was similar to that of B. fragilis, the data suggested that this element is of Bacteroides origin. Transposition promoted by this element was demonstrated in recA E. coli. Recombinants were recovered by selecting for the activation of a promoterless chloramphenicol resistance gene on the plasmid pDH5110 and were characterized by restriction endonuclease mapping and Southern blot hybridization. We propose that this IS element be designated IS4351.

Bacteroides fragilis↗
PubMed1986

Human serum antibody response against Streptococcus mutans antigens.

Antigens from Streptococcus mutans were examined to identify specific polypeptides that may have stimulated antibody responses and possibly play some role in caries immunity. A group of 10 adult human subjects was screened for serum antibodies reactive with antigens from S. mutans. Extracellular and cellular protein preparations from S. mutans LM7 (Bratthall serotype e) and V403 (biotype c) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western electrophoretic transfer and immunoblotting analysis. Antibodies reactive with polypeptides ranging from 34 to 400 kilodaltons in apparent molecular mass were detected by these means. Radioimmunoassay competition experiments revealed that the cellular and extracellular antigens did not compete with each other for serum antibodies. Preabsorption of sera with extracellular proteins from other oral streptococcal species prior to immunoblotting indicated that the antigens unique to S. mutans have molecular masses greater than 100 kilodaltons, and each individual produced antibodies against different antigens of high molecular mass. Examination of sera from young children also indicated heterogeneous responses against S. mutans LM7 antigens.

Animals↗
PubMed1986

Molecular organization and expression of the gtfA gene of Streptococcus mutans LM7.

The Streptococcus mutans LM7 gene gtfA was cloned in Escherichia coli along with flanking regions of the chromosome as a fragment representing 10.3 kilobases (kb) of streptococcal DNA. Restriction endonuclease mapping revealed that the cloned DNA consisted of four EcoRI fragments with gtfA sucrase activity localized to one fragment, EcoRI-B (2.4 kb). Subsequent analysis with E. coli minicells indicated that three polypeptides were encoded on the 10.3-kb insert (55 [GtfA], 45, and 35 kilodaltons). Neither the 45- nor 35-kilodalton polypeptide exhibited any detectable sucrase activity. The approximate positions and directions of transcription of the two larger proteins were determined from minicell protein profiles displaying truncated versions of these polypeptides. The restriction endonuclease data for the cloned gtfA gene were used to develop a strategy for insertional inactivation of this locus in vivo. An internal HincII fragment of the gtfA gene was removed and replaced with a DNA fragment containing a tetracycline resistance determinant. This new recombinant plasmid was linearized and then transformed into S. mutans GS5 and S. mutans V403 where it was incapable of replication. It was predicted that Tcr colonies would result from double-crossover recombinational events involving homologous regions flanking the gtfA gene. This was verified by Southern DNA hybridization analyses. The inactivation of the gtfA gene in both S. mutans GS5 and S. mutans V403 resulted in a decrease of water-soluble exopolysaccharide but no detectable changes in the amounts of water-insoluble polymers.

Chromosome Mapping↗
PubMed1986

Molecular cloning and characterization of scrB, the structural gene for the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system sucrose-6-phosphate hydrolase.

A DNA fragment encoding the sucrose-6-phosphate hydrolase component of the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system has been recovered from a plasmid-based genomic library of strain GS5. The locus, designated scrB, was found to reside within a 2.9-kilobase-pair restriction fragment present on the chimeric molecule pVA1343 (7.3 kilobase pairs). Minicell analysis of pVA1343-directed translation products revealed that the scrB product synthesized in Escherichia coli V1343 was a single peptide of Mr 57,000. This polypeptide was reactive with antiserum prepared against S. mutans intracellular invertase, which has been previously shown to have an Mr of 43,000 to 48,000. The basis of this difference in Mr was not established but may represent a posttranslational proteolytic event which occurred in S. mutans but not in recombinant V1343. Sucrose-6-phosphate hydrolase purified to homogeneity from V1343 exhibited Michaelis constants of 180 mM for sucrose and 0.08 mM for sucrose-6-phosphate. Deletion analysis of pVA1343 facilitated the assignment of a coding region for the hydrolase within the insert, as well as an orientation for the transcription of scrB. scrB-defective strains of S. mutans constructed by additive integration of an insertionally inactivated scrB locus exhibited the sucrose sensitivity characteristic of this mutant class. Similar loci were detected by DNA-DNA hybridization in additional strains of S. mutans and two strains of Streptococcus cricetus, but not in single strain representatives of S. rattus, S. sobrinus, S. sanguis I and II, S. salivarius, or S. mitis.

Animals↗
PubMed1986

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.

DNA Restriction Enzymes↗
PubMed1986

Complete nucleotide sequence and transcription of ermF, a macrolide-lincosamide-streptogramin B resistance determinant from Bacteroides fragilis.

DNA sequence analysis of a portion of an EcoRI fragment of the Bacteroides fragilis R plasmid pBF4 has allowed us to identify the macrolide-lincosamide-streptogramin B resistance (MLSr) gene, ermF. ermF had a relative moles percent G + C of 32, was 798 base pairs in length, and encoded a protein of approximately 30,360 daltons. Comparison between the deduced amino acid sequence of ermF and six other erm genes from gram-positive bacteria revealed striking homologies among all of these determinants, suggesting a common origin. Based on these and other data, we believe that ermF codes for an rRNA methylase. Analysis of the nucleotide sequences upstream and downstream from the ermF gene revealed the presence of directly repeated sequences, now identified as two copies of the insertion element IS4351. One of these insertion elements was only 26 base pairs from the start codon of ermF and contained the transcriptional start signal for this gene as judged by S1 nuclease mapping experiments. Additional sequence analysis of the 26 base pairs separating ermF and IS4351 disclosed strong similarities between this region and the upstream regulatory control sequences of ermC and ermA (determinants of staphylococcal origin). These results suggested that ermF was not of Bacteroides origin and are discussed in terms of the evolution of ermF and the expression of drug resistance in heterologous hosts.

Amino Acid Sequence↗
PubMed1985

Genetics of clindamycin resistance in Bacteroides.

Results presented in this paper have shown that a widely distributed LM-resistance determinant is present on at least 3 distinct Bacteroides R-plasmids. In fact these plasmids bear no homology outside of the defined regions implicated in the LM resistance. This resistance determinant on pBF4, pBFTM10, and pBI136 is located within DNA segments bounded on each side by a directly repeated sequence of more than 500 bp. The intervening sequences of these 3 elements are variable, and range in size from about 3.7 kb to 7.2 kb (Fig. 7). Apart from the EcoRI/AvaI restriction sites which characterize the repeated sequence, there is a notable lack of common restriction sites within these elements. These results suggest that the elements do possess a certain degree of structural similarity but significant evolutionary divergence has occurred. The presence and location of the directly repeated sequences, their association with specific deletions, and their association with an antibiotic-resistance determinant, are features common to many antibiotic-resistance transposons described for other prokaryotes. In addition, these elements are highly mobile, being found on a number of R-plasmids. The unique relationship between pBF4, pBI106, and pBI136 described here is a clear indication of the potential for these DNA sequences to move from one molecule to another. Given the extensive dissemination and the genetic and structural characteristics described above, it seems likely that the LM-resistance determinant is carried on transposon-like elements present in pBF4, pBFTM10, and pBI136. However, further experimentation will be necessary to document the transposition event. Bacteroides strains such as B. fragilis V503, possess a transmissible LM-resistance determinant which does not appear to be associated with detectable extrachromosomal elements (5,9,10). Presently, a number of strains of this type have been found over a wide geographic area. The LM-resistance genes associated with these strains are apparently similar to the one carried on the Bacteroides R-plasmids because homology between the 2 has been observed. However, it is important to note that within the limits of Southern filter blot hybridization, neither V503 nor its transconjugants possess the directly repeated sequence found on the LM-resistance plasmids (Fig. 8). The elusive nature of the V503 LM-resistance elements presents an intriguing problem. One model that has been proposed is that these resistance determinants reside on a conjugative transposon similar to Tn916 of Streptococcus faecalis (3).(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteroides↗
PubMed1985

Molecular cloning of a pIP501 derivative yields a model replicon for the study of streptococcal conjugation.

We have previously constructed a derivative of the broad host range streptococcal plasmid pIP501, a conjugative plasmid designated pVA797, that confers chloramphenicol resistance and contains a unique EcoRI site in a non-essential region of the plasmid molecule. pVA797 (30.7 kb) when cloned in toto as an EcoRI fragment into the positive selection vector pOP203(A2+) gave a recombinant, pVA904 (37.7 kb), which was able to replicate in Escherichia coli and in streptococcal species. It can be phenotypically monitored in either genus by specific drug resistance markers (chloramphenicol resistance in streptococci, tetracycline resistance in E. coli). pVA904 segregates into E. coli minicells where it specifies the production of at least 13 polypeptides. Many of the polypeptides are missing in minicells containing a transfer-defective, deletion derivative of pVA904. pVA904 is an ideal model replicon for the study of streptococcal conjugation because it is a shuttle plasmid thus enabling manipulation using procedures established for E. coli. Specifically, it should be possible to define the genetic basis of streptococcal conjugation by coupling mutagenesis protocols and minicell protein analyses in E. coli with evaluation of transfer function in streptococci.

Bacterial Proteins↗
PubMed1985

Cloned gtfA gene of Streptococcus mutans LM7 alters glucan synthesis in Streptococcus sanguis.

Streptococcus mutans LM7 (Bratthall serotype e) chromosomal DNA was partially digested with EcoRI and ligated into the positive-selection plasmid vector pOP203(A2+). The ligation mixture was transformed into Escherichia coli, and transformants were selected for tetracycline resistance. Recombinant-bearing clones were screened for their ability to ferment raffinose, using the procedure of Robeson et al. (J. Bacteriol. 153:211-221, 1983). One raffinose-fermenting clone was isolated and found to contain a plasmid with an insert consisting of four EcoRI fragments totalling approximately 10.3 kilobases (kb). This strain was capable of growth on defined medium plus raffinose or sucrose and generated reducing sugars from a sucrose substrate. Southern hybridization analysis of the four EcoRI fragments revealed homology not only to S. mutans LM7 chromosomal DNA but also to S. mutans serotypes b, c, and f. Subcloning of this fragment array into a streptococcal E. coli shuttle vector indicated that a 2.4-kb EcoRI fragment was essential for sucrase activity. E. coli minicell experiments revealed a gene product of 55 kilodaltons. These data along with restriction endonuclease analysis and Southern hybridizations suggested that the cloned S. mutans LM7 gene was closely related to the gtfA gene cloned by Robeson et al. from S. mutans PS13 (Bratthall serotype c). The shuttle plasmid containing the 2.4-kb fragment was transformed into Streptococcus sanguis, which subsequently displayed increased sucrase activity in both intracellular and extracellular fractions. Elevated levels of synthesis of alcohol-insoluble and water-insoluble glucans were observed with crude extracellular fractions of the S. sanguis strain bearing the 2.4-kb fragment. An isolate cured of the shuttle plasmid plus the 2.4-kb fragment displayed wild-type S. sanguis glucan synthesis. In S. sanguis, this gtfA allele may play a role in glucan synthesis by interacting with extant high-molecular-weight glucosyltransferases.

Cloning, Molecular↗
PubMed1984

Disseminated tetracycline resistance in oral streptococci: implication of a conjugative transposon.

A DNA sequence specifying tetracycline resistance (Tcr) has been previously cloned from a clinical isolate of Streptococcus mutans designated U202 (J. A. Tobian and F. L. Macrina, J. Bacteriol. 152:215-222, 1982). We used this sequence as a molecular probe in studying the dissemination of Tcr among oral streptococcal species isolated from patients treated with tetracycline. Eleven strains (including S. sanguis I, S. sanguis II, S. mitis, and S. salivarius) from seven patients were examined by Southern blot analysis. Seven strains showed strong hybridization to the Tcr probe, two showed weak hybridization, and two did not display detectable hybridization. Based on previous characterization of the cloned sequence, our data suggest the dissemination of the tetM class of resistance determinants among these oral streptococci. One of the clinical S. sanguis I isolates studied was able to transfer its Tcr phenotype to other oral streptococci and to enteric streptococci in the absence of plasmid DNA. This transfer appeared to be conjugation-like on the basis of its insensitivity to DNase and its dependence on intimate cell-to-cell contact. Using the cloned Tcr sequence, we were able to study the progeny of the matings. Our data suggest that this resistance transfer element occupies a chromosomal location in streptococcal cells and that it strongly resembles the conjugative transposon Tn916 in its behavior.

Cloning, Molecular↗
PubMed1984

Large transmissible clindamycin resistance plasmid in Bacteroides ovatus.

Bacteroides ovatus IB106 contained two plasmids, pBI106 (46 kilobases) and pBI136 (82 kilobases). Transmissible clindamycin-erythromycin resistance (Ccr) was mediated by pBI136 , whose Ccr determinant was closely related to the determinant on the Bacteroides R plasmids pBF4 and pBFTM10 . Hybridization studies showed that pBI106 was not involved in Ccr transfer, but it shared extensive homology to pBF4 with the exception of the pBF4 region implicated in Ccr.

Bacteroides↗
PubMed1984

Characterization and expression of a cloned tetracycline resistance determinant from the chromosome of Streptococcus mutans.

A chromosomal tetracycline resistance (Tcr) determinant previously cloned from Streptococcus mutans into Streptococcus sanguis (Tobian and Macrina, J. Bacteriol. 152:215-222, 1982) was characterized by using restriction endonuclease mapping, deletion analysis, and Southern blot hybridization. Deletion analysis allowed localization of the Tcr determinant to a 2.8-kilobase region of the originally cloned 10.4-kilobase sequence. This cloned determinant hybridized to a representative of the tetM class of streptococcal Tcr determinants but not to representatives of the tetL and tetN classes and, like other tetM determinants, mediated high-level resistance to tetracycline and low-level resistance to minocycline. A portion (approximately 3 kilobases) of the isolated streptococcal fragment was subcloned into Escherichia coli, where it conferred resistance to tetracycline and minocycline. Two proteins with apparent molecular weights of 33,000 and 35,000, encoded by the S. mutans DNA, were synthesized in E. coli minicells. Insertion of DNA into a unique SstI site of the cloned S. mutans fragment resulted in inactivation of Tcr expression in E. coli and S. sanguis, as well as loss of production of both the 33,000- and 35,000-dalton proteins in E. coli minicells. Incubation of minicells in subinhibitory concentrations of tetracycline did not result in changes in the levels of synthesis of either protein. Our data suggest that at least one of these proteins is involved in the expression of Tcr.

Chromosome Deletion↗