scholarly journals Kanamycin Resistance Cassette for Genetic Manipulation of Treponema denticola

2015 ◽  
Vol 81 (13) ◽  
pp. 4329-4338 ◽  
Author(s):  
Yuebin Li ◽  
John Ruby ◽  
Hui Wu
Keyword(s):  
Genetic Manipulation ◽  
Periodontal Diseases ◽  
Genetic System ◽  
Kanamycin Resistance ◽  
Treponema Denticola ◽  
Oral Pathogen ◽  
Content Type ◽  
Kanamycin Resistance Cassette ◽  
Allelic Replacement ◽  
Kanamycin Cassette

ABSTRACTTreponema denticolahas been recognized as an important oral pathogen of the “red complex” bacterial consortium that is associated with the pathogenesis of endodontal and periodontal diseases. However, little is known about the virulence ofT. denticoladue to its recalcitrant genetic system. The difficulty in genetically manipulating oral spirochetes is partially due to the lack of antibiotic resistance cassettes that are useful for gene complementation following allelic replacement mutagenesis. In this study, a kanamycin resistance cassette was identified and developed for the genetic manipulation ofT. denticolaATCC 35405. Compared to the widely usedermF-ermAMcassette, the kanamycin cassette used in the transformation experiments gave rise to additional antibiotic-resistantT. denticolacolonies. The kanamycin cassette is effective for allelic replacement mutagenesis as demonstrated by inactivation of two open reading frames ofT. denticola, TDE1430 and TDE0911. In addition, the cassette is also functional intrans-chromosomal complementation. This was determined by functional rescue of a periplasmic flagellum (PF)-deficient mutant that had theflgEgene coding for PF hook protein inactivated. The integration of the full-lengthflgEgene into the genome of theflgEmutant rescued all of the defects associated with theflgEmutant that included the lack of PF filament and spirochetal motility. Taken together, we demonstrate that the kanamycin resistance gene is a suitable cassette for the genetic manipulation ofT. denticolathat will facilitate the characterization of virulence factors attributed to this important oral pathogen.

scholarly journals High-Level Production of the Industrial Product Lycopene by the Photosynthetic Bacterium Rhodospirillum rubrum

2012 ◽  
Vol 78 (20) ◽  
pp. 7205-7215 ◽  
Author(s):  
Guo-Shu Wang ◽  
Hartmut Grammel ◽  
Khaled Abou-Aisha ◽  
Rudolf Sägesser ◽  
Robin Ghosh
Keyword(s):  
Rhodospirillum Rubrum ◽  
Purple Bacteria ◽  
Dry Weight ◽  
Photosynthetic Bacterium ◽  
Downstream Processing ◽  
Kanamycin Resistance ◽  
Content Type ◽  
Light Harvesting Complex ◽  
Kanamycin Resistance Cassette

ABSTRACTThe biosynthesis of the major carotenoid spirilloxanthin by the purple nonsulfur bacteriumRhodospirillum rubrumis thought to occur via a linear pathway proceeding through phytoene and, later, lycopene as intermediates. This assumption is based solely on early chemical evidence (B. H. Davies, Biochem. J. 116:93–99, 1970). In most purple bacteria, the desaturation of phytoene, catalyzed by the enzyme phytoene desaturase (CrtI), leads to neurosporene, involving only three dehydrogenation steps and not four as in the case of lycopene. We show here that the chromosomal insertion of a kanamycin resistance cassette into thecrtC-crtDregion of the partial carotenoid gene cluster, whose gene products are responsible for the downstream processing of lycopene, leads to the accumulation of the latter as the major carotenoid. We provide spectroscopic and biochemical evidence thatin vivo, lycopene is incorporated into the light-harvesting complex 1 as efficiently as the methoxylated carotenoids spirilloxanthin (in the wild type) and 3,4,3′,4′-tetrahydrospirilloxanthin (in acrtDmutant), both under semiaerobic, chemoheterotrophic, and photosynthetic, anaerobic conditions. Quantitative growth experiments conducted in dark, semiaerobic conditions, using a growth medium for high cell density and high intracellular membrane levels, which are suitable for the conventional industrial production in the absence of light, yielded lycopene at up to 2 mg/g (dry weight) of cells or up to 15 mg/liter of culture. These values are comparable to those of many previously describedEscherichia colistrains engineered for lycopene production. This study provides the first genetic proof that theR. rubrumCrtI produces lycopene exclusively as an end product.

scholarly journals A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen

2012 ◽  
Vol 79 (4) ◽  
pp. 1102-1109 ◽  
Author(s):  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Genetic System ◽  
Content Type ◽  
Suicide Vector ◽  
Double Deletion ◽  
Clostridium Ljungdahlii ◽  
Genomic Studies

ABSTRACTMethods for genetic manipulation ofClostridium ljungdahliiare of interest because of the potential for production of fuels and other biocommodities from carbon dioxide via microbial electrosynthesis or more traditional modes of autotrophy with hydrogen or carbon monoxide as the electron donor. Furthermore, acetogenesis plays an important role in the global carbon cycle. Gene deletion strategies required for physiological studies ofC. ljungdahliihave not previously been demonstrated. An electroporation procedure for introducing plasmids was optimized, and four different replicative origins for plasmid propagation inC. ljungdahliiwere identified. Chromosomal gene deletion via double-crossover homologous recombination with a suicide vector was demonstrated initially with deletion of the gene for FliA, a putative sigma factor involved in flagellar biogenesis and motility inC. ljungdahlii. Deletion offliAyielded a strain that lacked flagella and was not motile. To evaluate the potential utility of gene deletions for functional genomic studies and to redirect carbon and electron flow, the genes for the putative bifunctional aldehyde/alcohol dehydrogenases,adhE1andadhE2, were deleted individually or together. Deletion ofadhE1, but notadhE2, diminished ethanol production with a corresponding carbon recovery in acetate. The double deletion mutant had a phenotype similar to that of theadhE1-deficient strain. Expression ofadhE1intranspartially restored the capacity for ethanol production. These results demonstrate the feasibility of genetic investigations of acetogen physiology and the potential for genetic manipulation ofC. ljungdahliito optimize autotrophic biocommodity production.

scholarly journals Molecular Toolbox for Genetic Manipulation of the Stalked Budding Bacterium Hyphomonas neptunium

2014 ◽  
Vol 81 (2) ◽  
pp. 736-744 ◽  
Author(s):  
Alexandra Jung ◽  
Sabrina Eisheuer ◽  
Emöke Cserti ◽  
Oliver Leicht ◽  
Wolfgang Strobel ◽  
...  
Keyword(s):  
Target Genes ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Genetic Manipulation ◽  
Expression System ◽  
Genetic System ◽  
High Dynamic Range ◽  
Content Type ◽  
Depth Analysis ◽  
Inducible Synthesis

ABSTRACTThe alphaproteobacteriumHyphomonas neptuniumproliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies twoH. neptuniumpromoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions inH. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species.

scholarly journals Multilocus Sequence Analysis of Phylogroup 1 and 2 Oral Treponeme Strains

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Yong-Biao Huo ◽  
Yuki Chan ◽  
Donnabella C. Lacap-Bugler ◽  
Sisu Mo ◽  
Patrick C. Y. Woo ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Periodontal Diseases ◽  
Genetic Relationships ◽  
Species Level ◽  
Multilocus Sequence Analysis ◽  
Species Differentiation ◽  
Treponema Denticola ◽  
Rrna Gene ◽  
Content Type

ABSTRACT More than 75 “species-level” phylotypes of spirochete bacteria belonging to the genus Treponema reside within the human oral cavity. The majority of these oral treponeme phylotypes correspond to as-yet-uncultivated taxa or strains of uncertain standing in taxonomy. Here, we analyze phylogenetic and taxonomic relationships between oral treponeme strains using a multilocus sequence analysis (MLSA) scheme based on the highly conserved 16S rRNA, pyrH, recA, and flaA genes. We utilized this MLSA scheme to analyze genetic data from a curated collection of oral treponeme strains (n = 71) of diverse geographical origins. This comprises phylogroup 1 (n = 23) and phylogroup 2 (n = 48) treponeme strains, including all relevant American Type Culture Collection reference strains. The taxonomy of all strains was confirmed or inferred via the analysis of ca. 1,450-bp 16S rRNA gene sequences using a combination of bioinformatic and phylogenetic approaches. Taxonomic and phylogenetic relationships between the respective treponeme strains were further investigated by analyzing individual and concatenated flaA (1,074-nucleotide [nt]), recA (1,377-nt), and pyrH (696-nt) gene sequence data sets. Our data confirmed the species differentiation between Treponema denticola (n = 41) and Treponema putidum (n = 7) strains. Notably, our results clearly supported the differentiation of the 23 phylogroup 1 treponeme strains into five distinct “species-level” phylotypes. These respectively corresponded to “Treponema vincentii” (n = 11), Treponema medium (n = 1), “Treponema sinensis” (Treponema sp. IA; n = 4), Treponema sp. IB (n = 3), and Treponema sp. IC (n = 4). In conclusion, our MLSA-based approach can be used to effectively discriminate oral treponeme taxa, confirm taxonomic assignment, and enable the delineation of species boundaries with high confidence. IMPORTANCE Periodontal diseases are caused by persistent polymicrobial biofilm infections of the gums and underlying tooth-supporting structures and have a complex and variable etiology. Although Treponema denticola is strongly associated with periodontal diseases, the etiological roles of other treponeme species/phylotypes are less well defined. This is due to a paucity of formal species descriptions and a poor understanding of genetic relationships between oral treponeme taxa. Our study directly addresses these issues. It represents one of the most comprehensive analyses of oral treponeme strains performed to date, including isolates from North America, Europe, and Asia. We envisage that our results will greatly facilitate future metagenomic efforts aimed at characterizing the clinical distributions of oral treponeme species/phylotypes, helping investigators to establish a more detailed understanding of their etiological roles in periodontal diseases and other infectious diseases. Our results are also directly relevant to various polymicrobial tissue infections in animals, which also involve treponeme populations.

scholarly journals A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

2016 ◽  
Vol 82 (14) ◽  
pp. 4421-4428 ◽  
Author(s):  
Gina L. Lipscomb ◽  
Jonathan M. Conway ◽  
Sara E. Blumer-Schuette ◽  
Robert M. Kelly ◽  
Michael W. W. Adams
Keyword(s):  
Plant Biomass ◽  
Genetic Manipulation ◽  
Optimal Growth ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Content Type ◽  
Background Strain ◽  
Caldicellulosiruptor Bescii ◽  
Kanamycin Selection ◽  
Integrating Vector

ABSTRACTCaldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in thepyrFgenome region. ThepyrFgene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand theC. besciigenetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs inC. bescii. These strains showed resistance to kanamycin at concentrations >50 μg · ml−1, whereas wild-typeC. besciiwas sensitive to kanamycin at 10 μg · ml−1. In addition, placement of the Cbhtkmarker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of thepyrEgene in wild-typeC. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. ThepyrEgene functioned as a counterselectable marker, likepyrF, and was used together with Cbhtkin the ΔpyrEbackground strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme.IMPORTANCECaldicellulosiruptor besciiis a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production. The current genetic system used withC. besciiis based upon only a single selection strategy, and this uses the gene involved in a primary biosynthetic pathway. There are many advantages with an additional genetic selection using an antibiotic. This presents a challenge for thermophilic microorganisms, as only a limited number of antibiotics are stable above 50°C, and a thermostable version of the enzyme conferring antibiotic resistance must be obtained. In this work, we have developed a selection system forC. besciiusing the antibiotic kanamycin and have shown that, in combination with the biosynthetic gene marker, it can be used to efficiently delete genes in this organism.

scholarly journals Development of a Modified Gentamicin Resistance Cassette for Genetic Manipulation of the Oral Spirochete Treponema denticola

2012 ◽  
Vol 78 (6) ◽  
pp. 2059-2062 ◽  
Author(s):  
Jiang Bian ◽  
J. Christopher Fenno ◽  
Chunhao Li
Keyword(s):  
Antibiotic Resistance ◽  
Genetic Manipulation ◽  
Targeted Mutagenesis ◽  
Treponema Denticola ◽  
Content Type ◽  
Reliable Tool ◽  
Gentamicin Resistance

ABSTRACTHerein, we report that a modified gentamicin cassette and a PCR-based method can be used for targeted mutagenesis of the oral spirocheteTreponema denticola. This approach minimizes polar effects and spontaneous antibiotic resistance. Therefore, it can serve as a reliable tool for genetic manipulation ofT. denticola.

scholarly journals Advances in Development of a Genetic System forThermoanaerobacterium spp.: Expression of Genes Encoding Hydrolytic Enzymes, Development of a Second Shuttle Vector, and Integration of Genes into the Chromosome

2000 ◽  
Vol 66 (11) ◽  
pp. 4817-4821 ◽  
Author(s):  
Volker Mai ◽  
Juergen Wiegel
Keyword(s):  
Shuttle Vector ◽  
Hydrolytic Enzymes ◽  
Genetic System ◽  
Kanamycin Resistance ◽  
Shuttle Vectors ◽  
Recent Success ◽  
Kanamycin Resistance Cassette ◽  
Expression Of Genes ◽  
Expression Host ◽  
Genes Encoding

ABSTRACT Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system,Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticumJW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of theThermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.

Bacillus cereus Fur regulates iron metabolism and is required for full virulence

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 569-577 ◽  
Author(s):  
Duncan R. Harvie ◽  
Susana Vílchez ◽  
James R. Steggles ◽  
David J. Ellar
Keyword(s):  
Bacillus Cereus ◽  
Kanamycin Resistance ◽  
Infection Model ◽  
Predicted Amino Acid Sequence ◽  
Dependent Manner ◽  
Genome Data ◽  
Kanamycin Resistance Cassette ◽  
Allelic Replacement ◽  
Cloned Gene ◽  
Null Strain

A homologue of the Bacillus subtilis fur gene was identified in Bacillus cereus and characterized. The predicted amino acid sequence of the cloned gene was found to be highly similar to other members of the Fur family of transcriptional regulators. The B. cereus fur gene was shown to partially complement an Escherichia coli fur mutant. Purified B. cereus Fur bound specifically to a 19 bp DNA sequence homologous to the B. subtilis Fur box in a metal-dependent manner. Analysis of the available B. cereus genome data identified a number of genes which contain predicted Fur box sequences in the promoter region. Many of these genes are predicted to play a role in bacterial iron uptake and metabolism, but several have also been implicated as having a role in virulence. Fur and iron regulation of a siderophore biosynthesis operon was confirmed in a β-galactosidase assay. A B. cereus fur null strain was constructed by allelic replacement of the chromosomal gene with a copy disrupted with a kanamycin resistance cassette. The Δfur mutant was found to constitutively express siderophores, to accumulate iron intracellularly to a level approximately threefold greater than the wild-type, and to be hypersensitive to hydrogen peroxide. In an insect infection model, the virulence of the fur null strain was found to be significantly attenuated, highlighting the essential role played by Fur in the virulence of this pathogen.

Comparison of Sampling Techniques For qPCR Quantification of Periodontal Pathogens

2018 ◽  
Vol 68 (12) ◽  
pp. 2853-2856 ◽  
Author(s):  
Igor Jelihovschi ◽  
Cristian Drochioi ◽  
Aida Corina Badescu ◽  
Raoul Vasile Lupusoru ◽  
Alexandra Elena Munteanu ◽  
...  
Keyword(s):  
Periodontal Disease ◽  
Periodontal Diseases ◽  
Pcr Analysis ◽  
Treponema Denticola ◽  
Subgingival Plaque ◽  
Periodontal Pathogens ◽  
Radiographic Evidence ◽  
Gingival Bleeding ◽  
As Species ◽  
Qpcr Quantification

The diagnosis of periodontal disease is mainly based on use of clinical and radiographic evidence. In this study we employed a quantitative PCR analysis of Aggregatibacter actinomycetemcomitans and Treponema denticola as species strongly involved in periodontal diseases, burden in periodontal pockets to detect the main sampling factors that interfere with qPCR results. From 22 patients with advanced periodontal disease, subgingival plaque was comparatively collected by paper points and periodontal Gracey curettes. Samples were collected from the same situs in presence of gingival bleeding and absence of bleeding. The concordance and agreement of results between samples were assessed. The present study demonstrates that subgingival plaque sampling with sterile absorbable paper points is often accompanied by gingival bleeding resulting in quantification biases of periodontal pathogens.

scholarly journals Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

2015 ◽  
Vol 81 (7) ◽  
pp. 2481-2488 ◽  
Author(s):  
Volker Winstel ◽  
Petra Kühner ◽  
Bernhard Krismer ◽  
Andreas Peschel ◽  
Holger Rohde
Keyword(s):  
Plasmid Dna ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Virulence Determinants ◽  
Coagulase Negative Staphylococci ◽  
Reliable Technique ◽  
Content Type ◽  
Research Activities ◽  
Wide Range ◽  
Genetic Barriers

ABSTRACTGenetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a uniqueStaphylococcus aureusstrain via a specificS. aureusbacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinicalStaphylococcus epidermidisisolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

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