Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome

1982 ◽  
Vol 152 (1) ◽  
pp. 215-222
Author(s):  
J A Tobian ◽  
F L Macrina
Keyword(s):  
Streptococcus Mutans ◽  
Blot Hybridization ◽  
Tetracycline Resistance ◽  
Southern Blot Hybridization ◽  
Vector System ◽  
Oral Streptococci ◽  
Helper Plasmid ◽  
Cloning System ◽  
Streptococcus Sanguis ◽  
High Level

A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.

scholarly journals 088 Microprojectile Bombardment-mediated Transformation of Rhododendron `Catawbiense Album' L.

HortScience ◽  
1999 ◽  
Vol 34 (3) ◽  
pp. 456D-456
Author(s):  
Jane E. Knapp ◽  
Mark H. Brand
Keyword(s):  
Microprojectile Bombardment ◽  
Blot Hybridization ◽  
Shoot Proliferation ◽  
Southern Blot Hybridization ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Selective Agent ◽  
Putative Transformants ◽  
High Level

Horticultural improvements in Rhododendron require long periods of time to produce flowering plants by traditional breeding methods. In addition, new trait development by conventional genetics is limited to existing germplasm. Genetic engineering approaches to horticultural improvement offer the possibility for introduction of new traits using foreign DNA from any source. To this end, we have developed a system for the genetic transformation of Rhododendron based on microprojectile bombardment. Leaves from in vitro-grown plantlets of R. `Catawbiense Album' L. were bombarded with the marker genes uidA (GUS) in combination with nptII or hph. Two days post-bombardment, explants were transferred to shoot iniation medium containing either 50 mg/L kanamycin or 2.5 mg/L hygromycin. After 4 weeks, proliferating tissues were transferred to media containing increased levels of selective agent (100 mg/L kanamycin or 5 mg/L hygromycin, respectively). Shoots that regenerated were then excised from necrotic tissues and transferred to shoot proliferation medium containing the high level of selective agent. PCR analysis of putative transformants revealed the presence of the transgenes. Southern blot hybridization confirmed stable transgene integration. Histochemical GUS assays of transformed tissues indicated uniform expression throughout the transgenic plant. With the development of an efficient transformation system, the introduction of genes to confer useful horticultural traits becomes feasible.

Studies on the role of IS1216E in the formation and dissemination of poxtA-carrying plasmids in an Enterococcus faecium clade A1 isolate

2020 ◽  
Vol 75 (11) ◽  
pp. 3126-3130
Author(s):  
Xinxin Shan ◽  
Xin-Sheng Li ◽  
Nannan Wang ◽  
Stefan Schwarz ◽  
Su-Mei Zhang ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Blot Hybridization ◽  
Tetracycline Resistance ◽  
Southern Blot Hybridization ◽  
Conjugative Plasmid ◽  
Fusion Event ◽  
Mobilizable Plasmid ◽  
Antimicrobial Resistance Genes ◽  
Positive Isolate

Abstract Objectives To analyse the role of IS1216E in the dissemination of the phenicol-oxazolidinone-tetracycline resistance gene poxtA in an Enterococcus faecium clade A1 isolate. Methods MICs were determined by broth microdilution. The poxtA-positive isolate was typed by MLST. The two plasmids were characterized by PCR, conjugation, S1-PFGE, Southern blot hybridization and WGS analysis. The presence of translocatable units (TUs) was examined by PCR and sequencing. Results Isolate E1077 contains the 217661 bp conjugative plasmid pE1077-217 and the 23710 bp mobilizable plasmid pE1077-23. pE1077-217 harbours erm(B), aac(A)-aph(D), aadE, spw, lsa(E), lnu(B), aphA3 and dfrG, whereas pE1077-23 carries a Tn6657-like transposon containing poxtA and fexB. pE1077-23 was apparently formed by an IS1216E-mediated composite transposon–plasmid fusion event, involving a replicative transposition process. Conjugation experiments showed that pE1077-23 is mobilizable by pE1077-217. Moreover, a novel 31742 bp plasmid, pT-E1077-31, was found in a transconjugant. WGS analysis indicated that pT-E1077-31 was formed by the integration of a Tn6657-derived, IS1216E-based translocatable unit, which carried fexB and poxtA, into a copy of pE1077-23. Conclusions This study showed the presence of two cointegrate formation events in the formation and spread of a poxtA/fexB-carrying plasmid in E. faecium. One was the integration of a transposon into a plasmid while the other was the integration of a TU into a different site of the same type of plasmid-borne transposon from which it originated. In both events, IS1216E played a major role, suggesting that IS1216E-mediated transposition and translocation processes aid the dissemination and persistence of important antimicrobial resistance genes, such as poxtA, among enterococci.

Environmental pH as a factor in the competition between strains of the oral streptococci Streptococcus mutans, S. sanguis, and "S. mitior" growing in continuous culture

1987 ◽  
Vol 33 (9) ◽  
pp. 824-827 ◽  
Author(s):  
G. H. Bowden ◽  
I. R. Hamilton
Keyword(s):  
Continuous Culture ◽  
Streptococcus Mutans ◽  
Culture Fluid ◽  
Oral Streptococci ◽  
Medium Ph ◽  
Glucose Limitation ◽  
Single Strain ◽  
Streptococcus Sanguis ◽  
Different Populations ◽  
Acidic Environments

Strains of Streptococcus mutans (biotype 1), Streptococcus sanguis, and Streptococcus mitior have been grown in mixed continuous culture in a semidefined medium under glucose limitation at a growth rate of D = 0.1 h−1. The effect of varying the environmental pH on the proportions of the different populations within the community has been determined. Initially the populations were allowed to reach steady state at pH 7.0 when S. sanguis was dominant with S. mutans and "S. mitior" maintaining similar populations. The medium pH was then lowered in steps of 0.5 pH units from pH 7.0 to 4.5, and the community was grown at each step for at least 15 generations. Viable counts of each species were made at 24-h intervals. The population ratios established at pH 7.0 remained relatively stable when the environmental pH was set at pH 6.5. However, after the medium pH was lowered to 6.0 (days 18–27), the population of S. mutans began to increase and the S. mitior population began to decline. A further change was seen at pH 5.5 (days 27–34) when S. mutans became dominant, S. sanguis declined, and S. mitior was not detectable. At pH 4.5, both S. mutans and S. sanguis were reduced in numbers, but survived until the experimental run was terminated (44 days). Samples of culture fluid were taken throughout the experiment and analyzed for the presence of the acid products of glucose metabolism. The amounts of lactic acid produced by the community increased as the environmental pH was lowered. The results show that there was variation in the ability of Streptococcus species to compete in acidic environments. Streptococcus mutans is most favoured by an environment below pH 6.0, while "S. mitior" is relatively sensitive to low pH, therefore a low environmental pH can be selective for S. mutans. Comparison of these results to two similar studies carried out recently suggests that there is variation in the ability of given strains of S. sanguis and "S. mitior" to compete with S. mutans in acid environments. Therefore, it is not valid to take data from studies of a single strain and interpret it as applicable to the species as they are presently defined.

scholarly journals Mupirocin-Resistant, Methicillin-Resistant Staphylococcus aureus Strains in Canadian Hospitals

2007 ◽  
Vol 51 (11) ◽  
pp. 3880-3886 ◽  
Author(s):  
Andrew E. Simor ◽  
Tammy L. Stuart ◽  
Lisa Louie ◽  
Christine Watt ◽  
Marianne Ofner-Agostini ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Methicillin Resistant ◽  
Pfge Typing ◽  
Laboratory Characterization ◽  
Mupirocin Resistance ◽  
Mrsa Strains ◽  
High Level ◽  
Epidemiologic Characteristics

ABSTRACT Mupirocin resistance in Staphylococcus aureus is increasingly being reported in many parts of the world. This study describes the epidemiology and laboratory characterization of mupirocin-resistant methicillin-resistant S. aureus (MRSA) strains in Canadian hospitals. Broth microdilution susceptibility testing of 4,980 MRSA isolates obtained between 1995 and 2004 from 32 Canadian hospitals was done in accordance with CLSI guidelines. The clinical and epidemiologic characteristics of strains with high-level mupirocin resistance (HLMupr) were compared with those of mupirocin-susceptible (Mups) strains. MRSA strains were characterized by pulsed-field gel electrophoresis (PFGE) and typing of the staphylococcal chromosomal cassette mec. PCR was done to detect the presence of the mupA gene. For strains with mupA, plasmid DNA was extracted and subjected to Southern blot hybridization. A total of 198 (4.0%) HLMupr MRSA isolates were identified. The proportion of MRSA strains with HLMupr increased from 1.6% in the first 5 years of surveillance (1995 to 1999) to 7.0% from 2000 to 2004 (P < 0.001). Patients with HLMupr MRSA strains were more likely to have been aboriginal (odds ratio [OR], 3.7; 95% confidence interval [CI], 1.5 to 9.4; P = 0.006), to have had community-associated MRSA (OR, 2.2; 95% CI, 1.0 to 5.0; P = 0.05), and to have been colonized with MRSA (OR, 1.7; 95% CI, 1.0 to 3.0; P = 0.04). HLMupr MRSA strains were also more likely to be resistant to fusidic acid (21% versus 4% for mupirocin-susceptible strains; P < 0.001). All HLMupr MRSA strains had a plasmid-associated mupA gene, most often associated with a 9-kb HindIII fragment. PFGE typing and analysis of the plasmid profiles indicate that both plasmid transmission and the clonal spread of HLMupr MRSA have occurred in Canadian hospitals. These results indicate that the incidence of HLMupr is increasing among Canadian strains of MRSA and that HLMupr MRSA is recovered from patients with distinct clinical and epidemiologic characteristics compared to the characteristics of patents with Mups MRSA strains.

scholarly journals Comparative Analysis of Colony Counts of Different Species of Oral Streptococci in Saliva of Dentulous, Edentulous and in those Wearing Partial and Complete Dentures

2013 ◽  
Vol 14 (4) ◽  
pp. 601-604 ◽  
Author(s):  
Praveen Kumar Madineni ◽  
Suresh Babu Ghanta ◽  
Naveen Kumar Motupalli ◽  
Mahanthesh Bembalgi ◽  
P Krishnam Raju
Keyword(s):  
Comparative Analysis ◽  
Streptococcus Mutans ◽  
Mutans Streptococci ◽  
Oral Streptococci ◽  
Complete Dentures ◽  
Streptococcus Salivarius ◽  
Streptococcus Mitis ◽  
Streptococcus Sanguis ◽  
Streptococcus Milleri ◽  
Colony Counter

ABSTRACT Objectives To study and compare the number of colony forming units of Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, Streptococcus mitis and Streptococcus milleri in dentulous, edentulous and in those wearing partial and complete dentures by using semi-quantitative culture method of saliva samples with calibrated standard loop Materials Sterile specimen collection bottles, Mitis salivarius agar plates, Standard loop, Candle jar, Incubator, Colony counter Methodology Study population consisted of 100 subjects with 25 in each group, with an age range of 40 to 80 years, who were attending the Department of Community Dentistry and Prosthodontics at MNR Dental College, Sangareddy, Hyderabad. Unstimulated saliva samples were collected from patients and inoculated on to Mitis salivarius agar plates using calibrated standard loop. The plates were then incubated anaerobically at 37°C for 24 hours and left at room temperature for further 24 hours. Using a colony counter, the number of colonies of each species was counted. Results Streptococcus mutans and Streptococcus mitis predominates in the dentulous group, Streptococcus sanguis in complete denture group, Streptococcus salivarius in edentulous group and Streptococcus milleri in removable partial denture group. Conclusion The results of our study are in accordance with the previous studies, which have sought to differentiate different groups of mutans streptococci using a simple calibrated standard loop. How to cite this article Ealla KKR, Ghanta SB, Motupalli NK, Bembalgi M, Madineni PK, Raju PK. Comparative Analysis of Colony Counts of Different Species of Oral Streptococci in Saliva of Dentulous, Edentulous and in those Wearing Partial and Complete Dentures. J Contemp Dent Pract 2013;14(4):601-604.

scholarly journals Characterization of the bla KPC-2 and bla KPC-3 genes and the novel bla KPC-15 gene in Klebsiella pneumoniae

2014 ◽  
Vol 63 (7) ◽  
pp. 981-987 ◽  
Author(s):  
Dongguo Wang ◽  
Wei Hou ◽  
Jiayu Chen ◽  
Yonghua Mou ◽  
Linjun Yang ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Gene Mapping ◽  
Southern Blot ◽  
Blot Hybridization ◽  
Carbapenem Resistance ◽  
Southern Blot Hybridization ◽  
The Novel ◽  
Plasmid Analysis ◽  
High Level

Three Klebsiella pneumoniae isolates exhibiting high-level resistance to carbapenem were analysed by PCR, PFGE, gene mapping, plasmid conjugation and Southern blot hybridization using a bla KPC probe. In addition to the frequently reported bla KPC-2 and bla KPC-3 genes, a novel bla KPC-15 gene was identified in one of the isolates. The results of plasmid analysis and Southern blot hybridization revealed that the three bla KPC genes were located on transferable plasmids exhibiting three different patterns. The patterns A, B and C were observed in the genetic makeup of each individual plasmid, and all three structures contained ISKpn6-like and ISKpn8 transposons. The results of the gene mapping and hybridization experiments performed with the bla KPC probe demonstrated that the plasmids harboured the three genes at approximately the 85.0, 54.0 and 73.0 kb positions. The study concluded that carbapenem resistance in the three isolates was primarily due to the production of carbapenem-hydrolysing β-lactamase.

scholarly journals Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

2007 ◽  
Vol 73 (23) ◽  
pp. 7542-7547 ◽  
Author(s):  
Dag Anders Brede ◽  
Sheba Lothe ◽  
Zhian Salehian ◽  
Therese Faye ◽  
Ingolf F. Nes
Keyword(s):  
Selection Marker ◽  
Multiple Cloning Site ◽  
Propionibacterium Freudenreichii ◽  
Expression Plasmid ◽  
Physiochemical Properties ◽  
Food Grade ◽  
Cloning System ◽  
Immunity Gene ◽  
Membrane Bound ◽  
High Level

ABSTRACT This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 107 transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive PpampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ∼91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.

Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 967-978 ◽  
Author(s):  
C. Viana-Niero ◽  
P. E. de Haas ◽  
D. van Soolingen ◽  
S. C. Leão
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Phospholipase C ◽  
Genetic Polymorphisms ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Clinical Isolates ◽  
Significant Similarity ◽  
Genomic Deletions ◽  
Genes Encoding ◽  
Mycobacterium Africanum

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and ‘Mycobacterium canettii’. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

scholarly journals Use of the photoaffinity cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid to characterize salivary-glycoprotein-bacterial interactions

1986 ◽  
Vol 234 (1) ◽  
pp. 43-48 ◽  
Author(s):  
E J Bergey ◽  
M J Levine ◽  
M S Reddy ◽  
S D Bradway ◽  
I Al-Hashimi
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Specific Interaction ◽  
Gel Filtration ◽  
Cross Linking ◽  
Oral Streptococci ◽  
Bacterial Surface ◽  
Acetylneuraminic Acid ◽  
Streptococcus Sanguis ◽  
Surface Examination ◽  
Inhibition Studies

The present study has utilized the iodinatable cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid (ASA) to examine the specific interaction between the proline-rich glycoprotein (PRG) of human parotid saliva and Streptococcus sanguis G9B. The binding of 125I-ASA-PRG to Streptococcus sanguis G9B displayed saturation kinetics, reversibility and was inhibited by unlabelled PRG. Inhibition studies with other glycoproteins and saccharides indicated that binding was mediated by a bacterial adhesin with specificity towards N-acetylneuraminic acid, galactose, and N-acetylgalactosamine. After cross-linking, the 125I-ASA-PRG-adhesin complex could be extracted with SDS and separated from uncoupled 125I-ASA-PRG by gel filtration on Sepharose CL-6B. Approx. 1% of the 125I-ASA-PRG was cross-linked to the bacterial surface. Examination of the 125I-ASA-PRG-adhesin complex by SDS/polyacrylamide-gel electrophoresis/fluorography on 5% -(w/v)-polyacrylamide gels revealed that PRG was bound to two bacterial components. These findings support our previous suggestion that human salivary glycoproteins can specifically interact with oral streptococci and that these interactions occur between the glycoprotein's carbohydrate units and lectin(s) on the bacterial cell surface.

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