Microbiology Commons^™

Development Of Novel Methodology For The Molecular Differentiation Of Cryptosporidium Parvum Gp60 Subtypes Via High Resolution Melting Analysis, Jennifer K. O'Leary, Liam Blake, Gerard D. Corcoran, Roy D. Sleator, Brigid Lucey

Department of Biological Sciences Publications

Cryptosporidium species subtypes are generally identified via DNA sequencing of the gp60 gene tandem repeat motif region. Due to the immunogenic nature of its glycoprotein products, gp60 is subject to host selective pressures, genetic recombination and evolutionary processes that drive extensive polymorphism at this locus. The elucidation of the polymorphic nature of this gene has led to the current mainstay in Cryptosporidium subtyping nomenclature.

This study aimed to develop a real-time polymerase chain reaction based method utilising a post-PCR application, high resolution melting (HRM) analysis, in conjunction with the abovementioned gp60 nomenclature system, in order to differentiate between Cryptosporidium parvum …

A Microbiome Analysis Of The Relationship Among Crayfish Ectosymbionts And Their Environment, John Hoverson

Senior Honors Theses

The purpose of this project was to determine if there are differences present between the α-diversities of the crayfish microbiome and its surrounding water and sediment. Furthermore, this project sought to discover if these differences hold when microbiomes are evaluated between crayfish of first and second stream orders. Finally, this project sought to determine if the presence of branchiobdellidan ectosymbionts on the crayfish caused further differences in the crayfish microbiome. While the hypothesized patterns between crayfish, ectosymbionts, and stream order were not found to exist, a significantly different microbiome was observed between water, sediment, and crayfish, and the α-diversity of …

Increasing Antibiotic Resistance In Shigella Bacteria In The United States, William J. Pharr

The Corinthian

Shigella bacteria cause half a million infections, 6,000 hospitalizations, and 70 deaths annually in the United States. These bacteria are of particular concern due to their high survivability, low infectious dose, and high adaptability. Cases of shigellosis from Shigella sonnei are becoming a more prevalent issue in the U.S. as the bacteria continues to develop higher resistance to today’s strongest antibiotics. Much of this resistance is connected to the exchange of genes between strains of Shigella due to insertion sequences (IS), intercontinental travel, and men who have sex with men (MSM). As a result of increased resistance, the use of …

Novel Small Rnas Expressed By Bartonella Bacilliformis Under Multiple Conditions Reveal Potential Mechanisms For Persistence In The Sand Fly Vector And Human Host, Shaun Wachter, Linda D. Hicks, Rahul Raghavan, Michael F. Minnick

Biology Faculty Publications and Presentations

Bartonella bacilliformis, the etiological agent of Carrión’s disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión’s disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We …

Evaluation Of The Xpert Carba-R Nxg Assay For Detection Of Carbapenemase Genes In A Global Challenge Set Of Pseudomonas Aeruginosa Isolates, Fred C. Tenover, Christian M. Gill, Tomefa E. Asempa, Isabella A. Tickler, Caitlin M. Dela Cruz, David P. Nicolau

Biology Faculty Publications

The growing prevalence and diversity of carbapenemase producers among carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates warrants an expansion of detection capabilities. The purpose of this study was to evaluate the performance of the commercially available Xpert Carba-R (Carba-R) and the research-use-only Xpert Carba-R NxG (Carba-R NxG) in a global collection of P. aeruginosa. The challenge set included 123 P. aeruginosa clinical isolates from 12 countries. Isolates were previously categorized via PCR or whole-genome sequencing. Carbapenemase classes tested include VIM, IMP, NDM, SPM, KPC, and GES. Non-carbapenemase (non-CP)-harboring isolates were also tested (negative control). Isolates were tested using the Carba-R NxG and …

Biophysical Analysis Of Pseudomonas-Phage Pap3 Small Terminase Suggests A Mechanism For Sequence-Specific Dna-Binding By Lateral Interdigitation., Marzia Niazi, Tyler J. Florio, Ruoyu Yang, Ravi K. Lokareddy, Nicholas A. Swanson, Richard E. Gillilan, Gino Cingolani

Department of Biochemistry and Molecular Biology Faculty Papers

The genome packaging motor of tailed bacteriophages and herpesviruses is a powerful nanomachine built by several copies of a large (TerL) and a small (TerS) terminase subunit. The motor assembles transiently at the portal vertex of an empty precursor capsid (or procapsid) to power genome encapsidation. Terminase subunits have been studied in-depth, especially in classical bacteriophages that infect Escherichia coli or Salmonella, yet, less is known about the packaging motor of Pseudomonas-phages that have increasing biomedical relevance. Here, we investigated the small terminase subunit from three Podoviridae phages that infect Pseudomonas aeruginosa. We found TerS is polymorphic in solution but …

Effects Of Nitrogen Fertilization And Bioenergy Crop Species On Central Tendency And Spatial Heterogeneity Of Soil Glycosidase Activities, Min Yuan, Jianjun Duan, Jianwei Li, Siyang Jian, Lahiru Gamage, Kudjo E. Dzantor, Dafeng Hui, Philip A. Fay

Agricultural and Environmental Sciences Faculty Research

Extracellular glycosidases in soil, produced by microorganisms, act as major agents for decomposing labile soil organic carbon (e.g., cellulose). Soil extracellular glycosidases are significantly affected by nitrogen (N) fertilization but fertilization effects on spatial distributions of soil glycosidases have not been well addressed. Whether the effects of N fertilization vary with bioenergy crop species also remains unclear. Based on a 3-year fertilization experiment in Middle Tennessee, USA, a total of 288 soil samples in topsoil (0–15 cm) were collected from two 15 m2 plots under three fertilization treatments in switchgrass (SG: Panicum virgatum L.) and gamagrass (GG: Tripsacum dactyloides L.) …

Spatio-Temporal Dynamics Of Fungal Communities And Their Effects On Plants, Kel Cook

Biology ETDs

Fungi perform several critical functions in the environment. Spatiotemporal distributions of fungal communities will mediate when and where these functions happen and how they vary across the landscape. I first explored tropical tree canopy fungal community variation at small spatial scales and documented near total turnover of fungi across sub-meter distances and among adjacent substrates. The second chapter analyzed fungal turnover over the course of three years, where community stability was driven primarily by abundant fungi. In the third chapter, I tested effects of the environment, including host plant and habitat, on canopy fungal communities and found only small effects, …

Environmental Influences On Expression Of Virulence- And Survival-Associated Genes And Epigenetic Modifications Of Dna In Vibrio Vulnificus, James W. Conrad

USF Tampa Graduate Theses and Dissertations

Vibrio vulnificus is an autochthonous inhabitant of warm, brackish coastal waters and is an opportunistic pathogen that causes the highest mortality of all seafood-related illnesses (1). These fatal infections are generally caused by the clinically-associated vcgC/16S rRNA type B genotypes within the biotype I group (2–5). However, the reasons for the heightened infectiousness of the clinically-associated strains over environmentally-associated ones remains elusive, as no unique clinically-associated virulence genes have been identified through genomic sequencing or other strategies. DNA methylation may contribute to regulation of virulence by affecting gene transcription, and was investigated in the highly virulent V. vulnificus strain CMCP6. …

Posttranslational Modification And Protein Disorder Regulate Protein-Protein Interactions And Dna Binding Specificity Of P53, Robin Levy

USF Tampa Graduate Theses and Dissertations

p53 is an intrinsically disordered transcription factor that suppresses tumor development by arresting the cell cycle and promoting DNA repair. p53 deletions or mutations can lead to cancer due to the inability of cells to respond to stress. The protein levels and post-translational modification state of p53 changes in response to cellular stress like DNA damage. Previous studies have shown that p53 can undergo coupled folding and binding with the E3 ubiquitin ligase, Mdm2, and the histone deacetylase, p300. In normal cells, p53 is kept at a low level by Mdm2, which marks it with ubiquitin, targeting p53 for proteasome …

Spatial And Temporal Determinants Associated With Eastern Equine Encephalitis Virus Activity In Florida, Kristi M. Miley

USF Tampa Graduate Theses and Dissertations

Eastern Equine Encephalitis virus (EEEV) is considered the most pathogenic mosquito-borne illness in the United States. Human mortality has been estimated to range between 35 to 75%, with horses experiencing mortality rates greater than 90%. A large number of EEEV cases occur in Florida. Though we have come a long way since the first human infection was identified in the 1930s, there is still much to learn regarding the virus’s ability to maintain transmission year-round in Florida. Phylogenic studies support that Florida may serve as the geographic reservoir for EEEV. This research investigated spatiotemporal and ecological variables associated with risk …

Evaluation Of Anti-Cancer Function In Natural Killer Cells Generated From Rat Hematopoietic Stem Cells, Alireza Pirahmadian

Student Works (2020-2029)

Natural killer cells (NK) cells are lymphocytes and they are the most important key constituent of mammalian immune system against cancer tumour cells. The genome of NK cells is categorically divided to inhibitory and activator genes that code ligands on the surface of NK cells. Indeed, these ligands are cancer tumour cells destroyer arms of the NK cells. On the other hand, hematopoietic stem cells (HSCs) are the origin of all blood cells, derives from BM and mature to immune cells. NK cells cannot defeat CTCs on their own. The main reason is their population (numbers are low), they cannot …

Aspergillus Niger Decreases Bioavailability Of Arsenic(V) Via Biotransformation Of Manganese Oxide Into Biogenic Oxalate Minerals, Bence Farkas, Marek Kolenčík, Miroslav Hain, Edmund Dobročka, Gabriela Kratošová, Marek Bujdoš, Huan Feng, Yang Deng, Qian Yu, Ramakanth Illa, B. Ratna Sunil, Hyunjung Kim, Peter Matúš, Martin Urík

Department of Earth and Environmental Studies Faculty Scholarship and Creative Works

The aim of this work was to evaluate the transformation of manganese oxide (hausmannite) by microscopic filamentous fungus Aspergillus niger and the effects of the transformation on mobility and bioavailability of arsenic. Our results showed that the A. niger strain CBS 140837 greatly affected the stability of hausmannite and induced its transformation into biogenic crystals of manganese oxalates—falottaite and lindbergite. The transformation was enabled by fungal acidolysis of hausmannite and subsequent release of manganese ions into the culture medium. While almost 45% of manganese was bioextracted, the arsenic content in manganese precipitates increased throughout the 25-day static cultivation of fungus. …

A Genomic Catalog Of Earth’S Microbiomes, Brian Hedlund, More Than 200 Other International Authors

Life Sciences Faculty Research

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth’s continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for …

Evaluation Of The Attitudes Of Phytotherapists Towards The Herbal Treatment Of Obesity In The Light Of Ancient And Modern Theoretical Knowledge - Field Study In Tlemcen (Algeria), Chahrazed Bessenouci

Journal of the Arab American University مجلة الجامعة العربية الامريكية للبحوث

This research aimed to reveal the phytotherapists attitudes towards medicinal plants therapy used to treat obesity through identifying the nature of these attitudes and their degree of compatibility with what is in the ancient scientific manuscripts and the research publications in the modern chemistry. To do this, a choice was made of an intentional sample of 34 Herbalists-Phytotherapists (from several regions of the province of Tlemcen). Then the researchers used a face-to-face interview to survey the attitudes using a 27-item Likert-scale test. To determine the nature of the sample's attitudes and also the level of their knowledge, a descriptive analytical …

Significance Of Viral Activity For Regulating Heterotrophic Prokaryote Community Dynamics Along A Meridional Gradient Of Stratification In The Northeast Atlantic Ocean, Kristina D.A. Mojica, Corina P.D. Brussaard

Faculty Publications

How microbial populations interact influences the availability and flux of organic carbon in the ocean. Understanding how these interactions vary over broad spatial scales is therefore a fundamental aim of microbial oceanography. In this study, we assessed variations in the abundances, production, virus and grazing induced mortality of heterotrophic prokaryotes during summer along a meridional gradient in stratification in the North Atlantic Ocean. Heterotrophic prokaryote abundance and activity varied with phytoplankton biomass, while the relative distribution of prokaryotic subpopulations (ratio of high nucleic acid fluorescent (HNA) and low nucleic acid fluorescent (LNA) cells) was significantly correlated to phytoplankton mortality mode …

Varroa Destructor Mites Vector And Transmit Pathogenic Honey Bee Viruses Acquired From An Artificial Diet, Francisco Posada-Florez, Eugene V. Ryabov, Matthew C. Heerman, Yanping Chen, Jay D. Evans, Daniel E. Sonenshine, Steven C. Cook

Biological Sciences Faculty Publications

The ectoparasitic mite Varroa destructoris one of the most destructive pests of the honey bee (Apis mellifera) and the primary biotic cause of colony collapse in many regions of the world. These mites inflict physical injury on their honey bee hosts from feeding on host hemolymph and fat body cells/cellular components, and serve as the vector for deadly honey bee viruses, including Deformed wing virus (DWV) and the related Varroa destructor virus-1 (VDV-1) (i.e., DWV-like viruses). Studies focused on elucidating the dynamics of Varroa-mediated vectoring and transmission of DWV-like viruses may be confounded by viruses present in …

Potential Virus-Mediated Nitrogen Cycling In Oxygen-Depleted Oceanic Waters, M. Consuelo Gazitúa, Dean R. Vik, Simon Roux, Ann C. Gregory, Benjamin Bolduc, Brittany Widner, Margaret R. Mulholland, Steven J. Hallam, Osvaldo Ulloa, Matthew B. Sullivan

OES Faculty Publications

Viruses play an important role in the ecology and biogeochemistry of marine ecosystems. Beyond mortality and gene transfer, viruses can reprogram microbial metabolism during infection by expressing auxiliary metabolic genes (AMGs) involved in photosynthesis, central carbon metabolism, and nutrient cycling. While previous studies have focused on AMG diversity in the sunlit and dark ocean, less is known about the role of viruses in shaping metabolic networks along redox gradients associated with marine oxygen minimum zones (OMZs). Here, we analyzed relatively quantitative viral metagenomic datasets that profiled the oxygen gradient across Eastern Tropical South Pacific (ETSP) OMZ waters, assessing whether OMZ …

Direct Binding Of Tfeα Opens Dna Binding Cleft Of Rna Polymeras, Sung-Hoon Jun, Jaekyung Hyun, Hoyoung Kim, Michael S. Bartlett, Hyun-Soo Cho, Katsuhiko S. Murakami

Biology Faculty Publications and Presentations

Opening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAPTFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix …

Investigating Microbial And Host Factors That Modulate Severity Of Clostridioides Difficile Associated Disease, Armando Lerma

Department of Food Science and Technology: Dissertations, Theses, and Student Research

Clostridioides difficile is recognized as one of the most important pathogens in hospital and community healthcare settings. The clinical outcome of infection of toxigenic C. difficile infection (CDI) ranges from asymptomatic colonization to fulminant pseudomembranous colitis and death. In recent studies, it has been suggested that a high proportion of nosocomial CDI cases are transmitted from asymptomatic carriers which might be acting as infection reservoirs. Understanding what causes the different responses to infection could lead to the development of novel prevention and treatment strategies. Although several explanations have been proposed to explain variations in susceptibility, understanding of the exact mechanisms …

Evaluating The Microbial Experience Influence On Anti-Tumor Immune Response, Jessica A. Ensing

Student Summer Scholars Manuscripts

Despite the steady increase of hygienic standards, the CDC continues to report an increase of immune-mediated diseases such as allergies and asthma. Human avoidance of microbial exposure and subsequently less experienced immune systems may be the cause for this increase. We investigated if increased microbial exposure results in increased immunity to cancer (B16 melanoma) by measuring activated lymphocytes between two groups of C57Bl/6 mice: specific pathogen free (SPF) mice (which had little microbial exposure) and cohoused (COH) mice (which were exposed to numerous microbes). Previous research shows that the CoH mouse model mimics a human adult’s immune system, while the …

Detection Of Extended Spectrum Beta-Lactamase Producing Escherichia Coli From Urinary Tract Infection In General Hospital, Minna, Nasiru U. Adabara, Naimat D. Bakinde, Sherifat O. Enejiyon, Tijani Salami, Doosuur Iorzua

Tanzania Journal of Science

Possession of virulence factors such as extended spectrum beta lactamase (ESBL) by pathogens generally exacerbates morbidity as well as increases healthcare costs. This study determined the production of ESBL among Escherichia coli isolated from the urinary tracts of subjects attending General Hospital, Minna, Nigeria. One hundred and fifty (150) urine samples were collected from subjects and cultured on CLED agar for the isolation of E. coli. Antibiotic susceptibility testing was done using Kirby Bauer disk diffusion method, while the phenotypic identification of ESBL-producing strains was carried out using double disk synergy test. All results were interpreted based on Clinical and …

Dengue Vector Distribution And Their Infection Status In Selected Regions In Tanzania, Anitha Philbert, Amina Msonga

Tanzania Journal of Science

Dengue fever is an important arboviral infection globally. Tanzania has experienced multiple dengue outbreaks since 2010 leading to mortalities and public distress. Dengue is caused by viruses which are transmitted to humans by vector mosquitoes. Nevertheless, the abundance, distribution and extent of viral infections of these vectors are poorly understood. The aim of this study was to characterize dengue vectors and determine their virus infection rates. Adult mosquitoes were collected from selected regions in Tanzania including Dar es Salaam, Tanga, Coast, and Arusha regions using CO2 baited mosquito magnet traps between December 2018 to February 2019, additional samples were collected …

Microbiological, Physicochemical And Heavy Metals Assessments Of Soils And Selected Vegetables Grown On Rumde-Doubeli Irrigated Farmland In Yola Nigeria, Mohammed Inuwa Ja’Afaru, Joel Uyi Ewansiha, Abdulwahab Dahiru, Konjerimam Ishaku Chimbekujwo

Tanzania Journal of Science

Leaf parts of vegetable plants grown on irrigated farmlands and are easily accessed by contaminants; pose greater risks of being contaminated with heavy metals. Soil, irrigation water and edible vegetable plants were collected and evaluated for concentrations of heavy metals using Atomic Absorption Spectrophotometry (AAS) and standard laboratory methods. Physicochemical parameters of irrigation water, compared with WHO limits for chloride (326.14 + 7.03 mg/L, 250 mg/L), total hardness (196.0 + 8.72 mg/L, 150 mg/L), chemical oxygen demand (286.4 ±1.35 mg/L; 150 mg/L) and alkalinity (171.0 + 4.36 mg/L, 120 mg/L) were significantly (p ˂ 0.05) higher than the control sites …

Antibacterial Activities Of The Extracts Of Allium Sativum (Garlic) And Allium Cepa (Onion) Against Selected Pathogenic Bacteria, Sherifat O. Enejiyon, Al-Hassan A. Abdulrahman, Abdulameen S. Adedeji, Ramatu Abdulsalam, Mary U. Oyedum

Tanzania Journal of Science

Although many antibiotics have been developed against bacterial diseases, the problems of antibiotic resistance and toxicity have made the continuous search for newer drugs an existential necessity. This study determined the phytochemical components and antibacterial activities of Allium sativum and Allium cepa against Salmonella typhi, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae and Escherichia coli. The phytochemical analysis was carried out using standard methods. The antibacterial activities of the aqueous, ethanol and acetone extracts of both plants at different concentrations were determined against all the test organisms using agar well diffusion method. All the extracts were found to contain important phytochemicals …

Antibacterial Action Of Graphene Oxide Towards Bacteria And Its Toxicological Effects On Human Epidermal Keratinocytes, Thiruchelvi Pulingam

Student Works (2020-2029)

The antibacterial nature of graphene oxide (GO) has stimulated wide interest in the medical field. Although the antibacterial activity of GO towards bacteria has been well studied, a deeper understanding of the mechanism of action of GO is still lacking. The objectives of the study were to characterize the physicochemical properties of GO, to determine the antibacterial and antibiofilm activity and the mechanistic action of GO against Gram-positive Staphylococcus aureus and Enterococcus faecalis and Gram-negative Escherichia coli and Pseudomonas aeruginosa, to determine the synergistic behaviour of GO with selected antibiotics against bacterial cells in suspension and biofilm and to determine …

Expanding Magnetic Organelle Biogenesis In The Domain Bacteria, Wei Lin, Wensi Zhang, Greig A. Paterson, Qiyun Zhu, Xiang Zhao, Rob Knight, Dennis A. Bazylinski, Andrew P. Roberts, Yongxin Pan

Life Sciences Faculty Research

Background: The discovery of membrane-enclosed, metabolically functional organelles in Bacteria has transformed our understanding of the subcellular complexity of prokaryotic cells. Biomineralization of magnetic nanoparticles within magnetosomes by magnetotactic bacteria (MTB) is a fascinating example of prokaryotic organelles. Magnetosomes, as nano-sized magnetic sensors in MTB, facilitate cell navigation along the local geomagnetic field, a behaviour referred to as magnetotaxis or microbial magnetoreception. Recent discovery of novel MTB outside the traditionally recognized taxonomic lineages suggests that MTB diversity across the domain Bacteria are considerably underestimated, which limits understanding of the taxonomic distribution and evolutionary origin of magnetosome organelle biogenesis. Results: Here, …

Evaluation Of Semi-Quantitative Compared To Quantitative Cultures Of Tracheal Aspirates For The Yield Of Culturable Respiratory Pathogens - A Cross-Sectional Study, Salima Rattani, Joveria Farooqi, Ghazala Jabeen, Saeeda Chandio, Qaiser Kash, Aijaz Khan, Kauser Jabeen

Department of Pathology and Laboratory Medicine

Background: Diagnosis of lower respiratory tract infections (LRTI) depends on the presence of clinical, radiological and microbiological findings. Endotracheal suction aspirate (ETSA) is the commonest respiratory sample sent for culture from intubated patients. Very few studies have compared quantitative and semi-quantitative processing of ETSA cultures for LRTI diagnosis. We determined the diagnostic accuracy of quantitative and semi-quantitative ETSA culture for LRTI diagnosis, agreement between the quantitative and semi quantitative culture techniques and the yield of respiratory pathogens with both methods.
Methods: This was a cross-sectional study conducted at the Aga Khan University clinical laboratory, Karachi, Pakistan. One hundred and seventy-eight …

The Impact Of Sample Processing And Media Chemistry On The Culturable Diversity Of Bacteria Isolated From A Cave, Katey E. Bender, Katelyn Glover, Alexander Archey, Hazel A. Barton

International Journal of Speleology

Although molecular approaches can identify members of microbial communities in the environment, genomic information does not necessarily correlate with environmental phenotype. Understanding functional roles can be done by cultivating representative species, yet the culturablility of bacteria from caves remains low, at 0.02%, limiting our understanding of microbial community interactions and processes. We have investigated several factors influencing culturability of bacteria from a single sample location in Maxwelton Sink Cave, WV, USA. Extended incubation of inoculated plates showed a significant increase in colony counts from two to four weeks, indicating that extended incubations increase culturability. There were no significant differences in …

A Mini-Tn5-Derived Transposon With Reportable And Selectable Markers Enables Rapid Generation And Screening Of Insertional Mutants In Gram-Negative Bacteria, Eric S. Nazareno, Bimala Acharya, C. Korsi Dumenyo

Agricultural and Environmental Sciences Faculty Research

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The …