Publications

Manuscripts

  • Levent, A. Schlochtermeier, K. N. Norman, S.E. Ives, S. D. Lawhon, G. H. Loneragan, R. C. Anderson, J. Vinasco, H. M. Scott. Population Dynamics of Salmonella enterica within Beef Cattle Cohorts followed from Single-Dose Metaphylactic Antibiotic Treatment until Slaughter. (manuscript has been accepted by AEM/ASM Journal in September 2019 and currently in publication process)
  • Levent G, R.B. Harvey, G. Ciftocioglu, R.C. Beier, K.J. Genovese, H.L. He, R.C. Anderson, and D.J. Nisbet In vitro effects of thymol-beta-D-glucopyranaside on Salmonella enterica serovar Typhimurium and Escherichia coli K88. J. Food Prot., 79 (2016), pp. 299–303 https://www.ncbi.nlm.nih.gov/pubmed/26818992

Other

  • Translator of “Horses and Ponies Sticker Book” , The Scientific and Technological Research Council of Turkey (TUBITAK, 2012)

Education & Experience

EDUCATION

Ph.D. Candidate | Texas A&M University| college station, tx, usa | 2015-2020

Major: Biomedical Sciences   Current GPA: 3.7

College/Department: College of Veterinary Medicine and Biomedical Sciences/Veterinary Pathobiology

Related coursework: Applied epidemiology, risk analysis, disease detection and surveillance, epidemiologic data analysis, statistics in research I, epidemiologic methods I, epidemiologic methods II & data analysis, microbial genetics, bioinformatics command line, metagenomics data analysis, foundations of biomedical science education, scientific ethics

Ph.D. Student | Istanbul University | istanbul, turkey| 2013-2015

Major: Health sciences

College/Department: Faculty of Veterinary Medicine/Food hygiene and Technology 

Related coursework: Meat and meat production hygiene techniques, food legislation, advanced food chemistry, advanced food microbiology, advanced food hygiene, advance food technology, milk and dairy products hygiene and technology, slaughtering science and meat inspection, advanced food control and analysis techniques

DVM | Ankara University |ankara, turkey| 2006-2012

Major: Doctor of Veterinary Medicine 

College: Faculty of Veterinary Medicine

EXPERIENCES

Governmental Visitor | USDA-ARS| cOLLEGE STATION, tx, usa | 2014-2015

I was a research scholar and governmental visitor at the United State Department of Agriculture, Southern Plains Agricultural Research Center in College Station in Texas for one year.  My research focused on the alternatives to antibiotics to reduce foodborne pathogen carriage in swine GIT.  I worked under the supervision of Dr. Robin C. Anderson to test thymol compounds on Salmonella enterica serovar Typhimurium DT104 and Escherichia coli K88.  My research involved both in vitro, in vivo experiments and microbiology.

Intern | United Nations- Food and Agriculture Organization|ankara, turkey | 2012-2013

I worked as an intern under the supervision of FAO/UN Sub-regional Coordinator in Turkey.  I was under the direct supervision of the International administrative officer at FAO for 6 months in Ankara, Turkey.  I was responsible for the procurement of goods, services, and maintaining vendor databases and documentation.  

Summer School Attendee| Brno Veterinary Medicine and Pharmaceutical Sciences University|brno, czech republic| 2012 

In the summer of 2012, I participated in professional level education in the Food Hygiene and Technology Department at the University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic.  During this summer school, I took 120 hours of training including various areas, such as microbiology, food safety, and EU regulations.

Intern | Universitat Autònoma de Barcelona| barcelona, spain | 2011 

In the summer of 2011, I visited Universitat Autònoma de Barcelona, Spain.  I was an intern for three months at Centre Especial de Recerca Planta de Tecnologia dels Aliments (Food Safety and Technology Unit) founded by Erasmus Vocational Training Programme.  I gained expertise in measuring the effect of high hydrostatic pressure treatments (HPT) on seafood and dairy products.  I performed analyses for the quantification of microbial inactivation after the HPT and the evolution during refrigerate storage under the supervision of Josep Yuste Puigvert, Martin Buffa, and Ramón Gervilla.

Trainee | Veterinary University of Vienna| vienna, austria| 2010 

I was accepted as a trainee in the Milk Hygiene and Technology Department of the Veterinary University of Vienna, Austria.  I spent one month being trained on dairy product testing, sample preparation, and the molecular biological detection of pathogens, especially for the contamination of Listeria monocytogenes, under the supervision of Dr. Martin Wagner.

Exchange Student | University of Bologna |bologna, italy| 2008-2009

In the third year of my Doctor of Veterinary Medicine study, I studied at the Faculty of Veterinary Medicine, University of Bologna, as an exchange student supported by the Erasmus Mundus Scholarship. 

EILC Student | Perugia University for Foreigners |perugia, italy| 2008 

I enrolled in an Italian EILC (Erasmus Intensive Language Courses) for a month in Perugia University for Foreigners in Italy to be eligible to study as an exchange student at the University of Bologna.

WORKSHOPS

The Computational Molecular Evolution Training at Wellcome Genome Campus |Cambridge, UK | 2019.

With this training I am now capable of understanding phylogenetics tool at levels of: 1) data retrieval and assembly, 2) alignment techniques, 3) phylogeny reconstruction (including maximum likelihood and Bayesian methods), 4) hypothesis testing, 5) population genetics approaches, 6) protein and nucleotide sequence analysis.

Workshop for GMP in Food Hygiene, Finland Leonardo project organised by Finnish– Turkish Businessmen Association |hELSINKI, fINLAND |2013

Field and bench performance   

  • Utilizing proper bench sterilization and bench-work principles
  • Keeping regular records of experiments and maintaining lab notebooks
  • Modifying the current standard protocols and generating new standard protocols
  • Collecting samples from farm animals and the environment
  • Sample processing and preparation (e.g., animal tissues, fecal, environmental samples, and food products) for microbial detection
  • Performing microbiology techniques for bacterial isolation and quantification using non-specific and specific enrichment methods, and media preparation
  • Using MALDI-TOF and serum agglutination tests for confirmatory assays
  • Determining phenotypic antibiotic resistance using micro-broth dilution and disc-diffusion methods
  • Utilizing PCR and real-time Q-PCR methods
  • Ability to extract bacterial DNA using QIAcube HT Platform and QIAgen DNA extraction kits
  • Ability to prepare sequencing libraries for next- generation sequencing (NGS) using Miseq Platform and Illumina Nextera (XT and Flex) library preparation kits
  • Assessing DNA and library qualities using Nano-Drop Spectrophotometer, Qubit and Fragment Analyzer platforms
  •  Understanding and performing phylogenetics tools at levels of:  1) data retrieval and assembly,  2) alignment techniques,  3) phylogeny reconstruction (including maximum likelihood and Bayesian methods), 4) hypothesis testing, 5) population genetics approaches, and 6) protein and nucleotide sequence analysis.

Data analysis and bioinformatics skills

  • Managing and maintaining big data, and storing results properly
  • Analyzing run metrics on Miseq platform and troubleshooting when necessary
  • Performing advanced epidemiological statistical analysis using STATA 15.1
  • Managing data on a high-performance computing cluster, using command-line tools for data science (sed, grep, etc.), Finding scripts online for specialized tasks, and submitting job scripts to high-performance research computers
  • Determining sequencing data quality metrics, bacterial antibiotic resistance genes, plasmids, serotype, sequence type, gene annotations, SNPs and phylogenetic tree using command line and web-tools
  • Having familiarity to QIIME-2 and R programming for metagenomics analysis  
  • Ability to work with databases and software in command-line such as: ResFinder, CARD, PlasmidFinder, PATRIC, RAST, ParSNP, McOutbryk, ABRicate, Figtree, FastTree, IQ-TREE, Model-test NG, iTOL, GrapeTree, FastQC, MultiQC, SeqSero, MLST, SRST2  
  • Ability to use basic office software such as Excel, Word and PowerPoint          
  • Ability to use Endnote reference management software

Personal aspects  

  • Written and oral communication in English
  • Troubleshooting in any of the fields performed
  • Understanding problems and focusing on solutions by trying different approaches
  • Being creative on the bench and data analysis stages
  • Being self-motivated, initiative and innovative
  • Fast learning and developing new techniques and skills
  • Strong work ethics, ability to work within a team
  • Excellent time management, delivery before due dates
  • High work capacity and enthusiasm in the field of interest

Memberships

  • American Society for Microbiology, 2015-current
  • Graduate Student Association, CVM, Texas A&M University , 2015-current
  • Graduate Academic Appeals Panel, Texas A&M University, 2018-current
  • Graduate and Professional Student Council, Texas A&M University, current
  • Turkish- American Scientists & Scholars, 2016-current
  • Co- coordinator of incoming volunteers by European Voluntary Service (EVS) S&G Association –System and Generation Association, 2011
  • IVSA International Veterinary Student Association, Ankara University, 2006 – 2010
  • Ankara University Swimmer, Swimming Team, 2006 – 2008

Oliver and Beckwith

Escherichia coli Mutant Pleiotropically Defective in the Export of Secreted Proteins

Donald B. Oliver and Jon Beckwith

Background information

There are 6 main secretary pathways in gram-negative bacteria:

  • Type I; Chaperone-dependent secretion system, the process begins as a leader sequence HlyA is recognized and binds HlyB on the membrane. This signal sequence is extremely specific for the ABC (ATP-binding cassette) transporter. It is also called ISP( type I secretory pathway)
  • Type II; Proteins secreted through the type II system, or main terminal branch of the general secretory pathway (SEC), depend on the Sec or Tat system for initial transport into the periplasm. It is also called IISP( type II secretory pathway)
  • Type III; It is homologous to the basal body in bacterial flagella. It is like a molecular syringe through which a bacterium can inject proteins into eukaryotic cells. It is also called IIISP( type III secretory pathway.
  • Type IV; it is homologous to conjugation machinery of bacteria. It is capable of transporting both DNA and proteins. It is also called IVSP( type IV secretory pathway)
  • Type V; also called the autotransporter system, type V secretion involves the use of the Sec system for crossing the inner membrane. . It is also called VSP( type V secretory pathway)
  • Type VI; identified in 2006

There are many outer and inner membrane proteins that cause channel-forming in gram-negative bacteria. Gram-negative bacterial inner membrane channel-forming translocases

  1. ATP-binding cassette translocase (ATP(ISP))
  2. General secretary translocase (SEC(IISP))
  3. Flagellum/ virulence-related translocase (Fla/ Path (IIISP))
  4. Conjugation related translocase (Conj(IVSP))
  5. Twin-arginine targeting translocase (Tat (IISP))
  6. Cytochrome oxidase biogenesis family (Oxa1(YidC))
  7. Large conductance mechanosensitive channel family (MscL)
  8. Holing functional superfamily (Holins)

Gram-negative bacterial outer membrane channel- forming translocases;

  1. Main terminal branch of the general secretory translocase (MTB(IISP))
  2. Fimbrial usher protein autotransporter-1 (FUP)
  3. Autotransporter –2 (AT-2)
  4. Outer membrane factor (OMF (ISP))
  5. Two-partner secretion (TPS)
  6. Secretin (IISP and IIISP)
  7. Outer membrane insertion porin (OmpIP)

Sec-dependent protein secretion (also called general secretion pathway)

The Sec machinery is an ensemble of proteins that facilitates the translocation of proteins and pre-proteins into and across biological membranes. This pathway is also similar in eukaryotes and archaea. This pathway translocates the proteins from the cytoplasm across or into the plasma membrane. Secreted proteins are initially synthesized as “preprotein” which contain an amino-terminal signal peptide. The translocase pathway comprises 7 proteins;

  • SecA; a motor protein that uses ATP as an energy source and threads the unfolded polypeptide through the channel.
  • SecB; chaperone protein, a highly acidic protein that exists in the bacterial cytoplasm. SecB maintains preproteins in an unfolded state after translation and targets these to the peripheral membrane protein ATPase SecA for secretion.
  • SecY, SecE, and SecG; integral membrane complex, the structure of the Escherichia coli SecYEG assembly revealed a sandwich of two membranes interacting through the extensive cytoplasmic domains.
  • SecD and SecF; additional membrane proteins that promote the release of the mature peptide into the periplasm.

Image result for sec pathway

Oliver and Beckwith research

Back at the 1970s most of the studies were about the proteins in the cytoplasm and inner membrane of E.coli. The further information was unknown about outer membrane or periplasm proteins and their translocation. According to the signal hypothesis proposed by Blobel; there was an N-terminal sequence at the end of the periplasmic and outer membrane proteins’ sequence which determine whether the protein will be secret or not. Later by this hypothesis was supported by Smith et al.

The study of genetic and protein translocation began in the late 1970s with the Beckwith group. These studies played an important role to identify the protein components that comprise the machinery and providing that the information for export is located within the signal sequence. This signal sequence was important for protein secretion but Oliver and Beckwith wanted to go further and see whether other regions of the peptide chain has a role in the  localization of secreted proteins. Briefly, they wanted to isolate and characterize the mutants that change the cell’s secretion machinery and result in pleiotropic effects on protein localization. They used an E.coli which harbors a hybrid β-galactosidase protein localized in the cytoplasmic membrane. This hybrid protein had different enzymatic properties when compared with the same protein which is located in the cytoplasm of E. coli. Beckwith and friends exploited fusion of the preprotein maltose binding protein (MBP) with cytosolic reporter β- galactosidase encoded by the lacZ gene which, when active, allows the cell to use lactose as a carbon source. PreMBP encoded by the malE gene is a periplasmic protein and is needed for the cell to use maltose as a carbon source. The MalE-LacZ fusion protein is toxic when expressed by the addition of maltose in E.coli; therefore, the cells were maltose sensitive. They used this as a discriminative method.

Isolation of mutants impaired in protein localization

They used E.coli strain (MM18) which harbors malE gene (this gene encodes maltose binding proteins) and fused to a lacZ gene which encodes β -galactosidase. This strain made a hybrid protein which changed the N-terminus of β-galactosidase to the large N terminus of maltose binding protein (MBP) but still retained the β -galactosidase activity. β -galactosidase was normally cytoplasmic and MBP was periplasmic. This hybrid protein was found in the cytoplasmic membrane. They assumed that this hybrid protein was bigger to pass through the cytoplasmic membrane and somehow blocked the secretion process which caused the cytoplasmic accumulation of the hybrid protein.

They found a significant enzymatic difference of the hybrid β-galactosidase when they are cytoplasm (MM7) or membrane associated (MM18). To discriminate β-galactosidase activity of such mutants (MM7 and MM18) they used lactose tetrazolium agar and MM7 gave red (Lac+), MM18 (Lac -) gave colorless cells. Red MM7 cells were picked, purified and tested for their phenotypes on various media at 30°C and 40°C. Two mutants out of 80 were screened MM49 and MM51 were temperature sensitive (ts). They applied malE-lacZ fusion, all derivatives kept being ts. So, they concluded that being ts was not related to the fusion. By several maltose-containing agar growth experiments for all mutants, they concluded that the mutants maintained their sensitivity to maltose while having intermediate β- galactosidase activities. They also determined whether the ts and β-galactosidase activity were due to a single mutation.

A defect in secretion of maltose-binding protein

They tasted the old and newly synthesized MBP by radioactively labeled (pulse labeled) methionine at different temperatures (30°C, 37°C and 42°C) and time intervals (fig.1 and fig.2). By the several experiments they run, they found that MM52 codes for a ts processing enzyme worked slower or persistent in reduced amount of permissive temp which cause the conclusion of MBP precursor present after a short pulse of 37°C is stable for up to 20 min during which time of the half of the newly synthesized MBP precursor was matured during a 20 sec pulse (fig.3) MM52 synthesized some stable MBP which is not subsequently processed. Moreover, MM51 was found in the cytoplasm after pulse-labelling of the protein at 37°C.

A defect of secretion of other exported proteins; they identified a precursor of another outer membrane protein (an ompF gene product) accumulating in MM52. Since MM52 accumulates several periplasmic and outer membrane proteins the thought it seemed possible it was defective in the transport of all secreted proteins. To understand this, they pulse labeled periplasmic fraction of mutant and wild type by cold osmotic shock method and EDTA-lysozyme method. They saw that many proteins were in common for both types except 3-4 proteins were only found in wildtype especially the amount of MBP was more than founded the mutant type.

Genetic mapping of the TS locus; they used Hfr strains to determine the location of ts51 mutation. After determining its locus, they wanted to map it by using Tn5 and Tn10 insertions. As a result, of this experiment, they concluded that this mutant gene was in the 2 to 4 min region of E. coli chromosome. At that suspected location there were ftsQ, ftsA, ftsZ and envA genes with similar phenotypes. However the one that they found was lambda specialized transducing phage which is a complement the ts51 mutation, and this locus was not previously described. They called this gene as secA.

 

Gizem Levent

Mechanism of action of streptomycin in E. coli: interruption of the ribosome cycle at the initiation of protein synthesis

MECHANISM OF ACTION OF STREPTOMYOCIN IN E. COLI: INTERAPTION OF THE RIBOSOME CYCLE AT THE INITATION OF PROTEIN SYNTHESIS.

By L. Luzzatto

There are several kind of antibiotics with a different antimicrobial mechanism which can be used against gram positive and negative bacteria. There are also many kinds of classifications can be used in order to classify the antibiotics. Some of these antibiotics have a broad-spectrum bacterial effect whereas others have narrow- spectrum bacterial effect. Some of them have bactericidal (they kill the bacteria) impact and others have bacteriostatic (they inhibit the bacterial growth or replication) impact. Some antibiotics can be both bactericidal and bacteriostatical.

Different antibiotics have different modes of action and target sites within bacterial cells. There are five basic mechanisms of antibiotic action against bacterial cells;

  • Inhibition of cell wall synthesis
  • Inhibition of protein synthesis (translation)
  • Alteration of cell membranes
  • Inhibition of nucleic acid synthesis
  • Antimetabolite activity

Inhibition of cell wall synthesis is the most common mechanism of antibiotics. Second largest class antibiotics are showing their antimicrobial effect by inhibiting the translation mechanism in the cell. In the paper of Luzzatto and colleagues, they proved the antimicrobial mechanism of streptomycin on Escherichia coli by the paper published on 1968.

There are a couple of ways of antibiotics inhibiting translation in the cell. These are;

  1. tRNA mimicry
  2. Inhibitors of peptide-bone formation
  3. Inhibitors of binding of tRNA to the A site
  4. Inhibitors of translocation
  5. Binding to 23S RNA
  6. Binding to the 30S ribosome

Streptomycin belongs to aminoglycoside class antibiotic and it is known as a protein synthesis inhibitor.

Back at 1964, in a study called “Streptomycin, Suppression and the Code” conducted by Julian Davies and Walter Gilbert, they were aware of the protein synthesis inhibiting the power of streptomycin, however, the mechanism was unknown. Previous studies of this study had suggested that streptomycin blocked the protein synthesis by strongly binding to nucleic acids. Julian Davies had concluded that streptomycin had done some alterations in the coding properties. Moreover provided the evidence that the ribosomes control the accuracy of the reading and may have a role in suppression. After that study, in 1968 Luzzatto lightened the unknown mechanism of streptomycin inhibiting the translation by this research.

In the paper of Luzzatto, they found that a certain concentration (lethal concentration) of streptomycin cause the accumulation of 70S ribosomes in E. coli cells. These ribosomes are incapable of doing translation in the cell. These 70S ribosomal units that accumulated were called “streptomycin monosomes”. Moreover, they found that these units consist of a complex of 30S and 50S subunits, tRNA, mRNA, and streptomycin. They observed that the 70S subunits which consist streptomycin had abnormal initiation complexes that cannot elongate; therefore they accumulate. So, they conclude that streptomycin molecules somehow “freeze” the protein initiation in E. coli.

According to their findings; Streptomycin blocks bacterial protein synthesis at initiation. After intact bacteria are exposed to streptomycin, polysomes become rapidly depleted and 70S particles. “The streptomycin monomers” build up. Although the formation of initiation complex is not affected, the complex formed in the presence of streptomycin cannot synthesize protein and remains fixed in the position. It is proposed that ribosome beyond the initiation stage are able to continue their movement and detachment so that a 70S ribosomal complex of mRNA and 50S and 30S units with bound streptomycin results. In effect, the initiation complex is frozen.

The Experiment

In their experiment, they used Escherichia coli mutant sud 24 and grow it in fragile form to be able to lyse and analyze during the translations. To trace the RNA during translations; they used radioactively labelled RNAs. They measured the speed of the molecules by using sucrose gradient analysis to determine the size distribution.

They used a streptomycin resistant derivative (N21) and susceptible AB301 strain, labelled ala-transfer tRNA, and labeled F-met tRNA, natural mRNA (f-met dependent), synthetic mRNA (poly AUG, f-met independent) and also they used a phage protein called R17.

Their results were;

  • Polyribosome metabolism in cultures treated with streptomycin

At the moment they add streptomycin the ribosome was the 30S and the 50S form as well as bound to mRNA. After 20 and 40 min intervals; they released (a) a decrease in large polyribosomes and in free 30S and 50S particles. (b) Accumulation of 70S monomers. (fig.1)

  • Streptomycin blocks the function of natural mRNA.

The protein synthesis directed by the phage R17 was observed alone with streptomycin and streptomycin was observed alone without the phage R17. Their finding had shown that streptomycin blocked the function of the natural mRNA (fig. 2)

  • Streptomycin blocks normal initiation of protein synthesis

Streptomycin blocked the RNA synthesis directed by natural mRNA but not with the synthetic mRNA. The natural mRNA initiates the protein synthesis by using f-met however the synthetic mRNA doesn’t require the f-met to start the translation. Thereby, they released that the streptomycin was blocking somehow the f-met boundary on the 30S ribosomal subunit to and block the F-met to come and attached in order to initiate the translation process. However, the synthetic RNA experiment did not block by adding the streptomycin. The amount of S35 f-met-tRNA was reduced by streptomycin and H3-ala-tRNA bound to ribosomes.

They realized that the initiated protein synthesis with 70S ribosomes was not blocked and continue when streptomycin added but only new translation processes did not initiate. So,            by using the synthetic mRNA and natural and also by using a radioactive label with the knowledge of f-met requirement for the translation they were able to understand this mode of action of streptomycin was at the initiation right after ribosomal subunits association.

 

Gizem Levent

Luria and Delbruck

MUTATIONS OF BACTERIA FROM VIRUS SENSITIVITY TO VIRUS RESISTANCE

S.E LURIA and M. DELBRUCK

Background information

Mutation refers to any heritable change in the DNA sequence. The errors and damages that repaired by DNA are not mutations. This genetic changes needs to be permanent and should transfer to the second generation.

There are three main types of mutants;

  1. Auxotrophic mutants; a mutant strain of microorganism that will proliferate only when the medium is supplemented with some specific substance not required by wild-type organisms.
  2. Conditional lethal mutants; these mutants can cause lethality under one condition (the restrictive or non-permissive condition) but not another (the permissive condition).
  • Temperature sensitive mutants; mutants which are able to grow at certain low temperatures(32°C), which is referred to as the permissive temperature, but are unable to grow at higher temperatures (39°C), which is referred to as the nonpermissive temperature.
  • Cold-sensitive mutants; mutant cells with proteins that fail to function at lower temperatures
  • Nonsense mutations; mutations that changed the three nonsense codons (UGA, UAG or UAA)
  1. Resistant mutants; mutants that develop resistance mechanisms against to a substance (drugs, phages, antimicrobials) that kills or inhibits the growth of a bacterium.

Mutation rate; it is a measure of the rate at which various types of mutations occur over time.

Bacteriophage; a virus that infects and replicates within a bacterium.

Luria and Delbruck Experiment

Back at that time, many microbiologists believed that bacteria were carrying genetic heritance and having mutations as a result of the adaption to the new environment whereas some others believed these mutations were accruing randomly. However in the year of 1943, Luria and Delbruck wondered about the nature of mutations. Are mutations spontaneous? Or do they occur in response to environmental conditions?

In the early 1990s, it was a well-known fact that bacterial viruses had antimicrobial effects. Some of the bacterial colonies that survive from this attack were called “resistant” whereas the succumbed ones called “sensitive”. The mechanism beyond this selection was unknown, however In the 1920s there was two hypothesis suggested regarding this issue;

Hypothesis one was the direct action of the virus reduces induced the resistant variants. The second hypothesis was; the resistant bacterial variants are produced by mutation in the culture before the virus was added. However, none of them was proved at that time. Basically, in terms of the experiment, a bacterial culture from a single cell plated with the virus in access and upon incubation, a small fraction of would survive. So, the attack of the virus prevailed the development of small numbers of colonies. By discovering these little survival resistant colonies;

Luria and Delbrück suggested two main hypothesis; this resistance was occurring due to mutations which are independent of a virus or there was an acquired immunity (hereditary predisposal individuals or after the infection they gained immunity) in the resistant cells. The first hypothesis had nothing to do with the virus since the mutation supposed to happen sometimes randomly there, every offspring of mutants supposed to be resistant unless a reverse mutation occurs. The second hypothesis which had a smaller chance; had something to do with the virus since it was the reason for the resistance. So for both hypothesis each offspring of a tested bacterium would survive after the virus exposure. However they would be distinguished by two main differences;

  1. A single colony would be expected on the mutation hypothesis while a random distribution of resistant would be expected on the acquired hereditary immunity hypothesis.
  2. If the resistance was due to mutation, the proportion of the resistant bacteria supposed to increase by time.

They observed some fluctuations (thereby their experiment also named the fluctuation test experiment) about the number of resistant cells after a couple of basic experiments and they said the controlled conditions were the same for all experiments. They couldn’t understand these fluctuations at the beginning. And they assumed that the large fluctuations might be the result of the mutation hypothesis.

The clues that they achieved about the growth of the resistance cells; they wanted to predict the number of the mutant cells will be achieved by time. To have such probability analyzes they needed to use a binomial distribution because the result of this virus exposure could be only; survive or die. However, since the survival chance is not equal, as a matter of fact it is much lesser than the death chance therefore, they used poisson distribution instead of the binomial. At the end, they achieve an equation for the mutation rate by grouping mutations according to the bacterial generations.

They used Escherichia coli B cells and bacterial virus α, they used nutrient broth and agar plates and asparagine-glucose synthetic medium. The division time for the medium was 35 min whereas for the broth it as 19 min. In the synthetic medium that they used the pH had increased from 7 to 5 by the incubation time. They also used only nutrient agar plates for plating the bacterial cells and exposing them to the virus. Since the lysis was very fast on the plate due to the virus, it was very easy to detect the resistant colonies. They also run a test to see how reliable the plating method by parallel plating is. The results were as expected. They also test the number of resistant bacteria in different samples from the same cultures. The hypothesis was; if the resistance was due to the mutation the variance supposed to be much greater from the average whereas if it is due to immunity the variance supposed to close to the mean. They run 100 sample by using aerated broth, regular broth, and synthetic medium and compared the results with their expected numbers. They inoculated a small number of bacteria (Escherichia coli) into separate culture tubes. After a period of growth, they plated equal volumes of these separate cultures onto agar containing the virus. If resistance to the virus in bacteria were caused by an induced activation in bacteria if resistance were not due to hereditary immunity, then each plate supposed to contain roughly the same number of resistant colonies. Assuming a constant rate of mutation, they hypothesized that if mutations occurred after and in response to exposure to the selective agent, the number of survivors would be distributed according to a Poisson distribution with the mean equal to the variance. However what they observed was; instead, the number of resistant colonies on each plate varied drastically: the variance was considerably greater than the mean. This could be caused by many simple reasons that would cause such results e.g. t0, physiological state of the bacteria or sudden transition from sensitivity to resistance. However of these were the case, they wouldn’t expect to see a very high portion of cultures to be different, in the fig. 2 the number of jackpots* (>9 resistant bacteria) far exceeded that expected by chance.

Luria and Delbruck’s experiments showed unequivocally that mutations were spontaneous. By these experiments, they proved that the resistance occurred due to mutations independently of the action of the virus and with the math they did, they found how to calculate/ estimate the mutation rate.

*They called this difference “jackpot” cause they say” The situation is similar to the operation of a (fair) slot machine, where the average return on a limited number of plays is probably considerably less than the input, and improbably, when the jackpot is hit, the return is much bigger than the input.

 

Gizem Levent

Meselson and Stahl Experiment

nm1004-1034-f1

THE REPLICATION OF DNA IN ESCHERICHIA COLI

MESELSON AND STAHL

 

See the paper here. 

The DNA and nucleic acid structure had been modelled but was not proven by Watson and Crick in 1953. Previous studies about DNA has shown that the DNA carries hereditary information and it was capable to replicate itself. However, the replication mechanism was unknown.

Back in time, there were three models suggested for the DNA replication; conservative, semi- conservative and dispersive models. According to the conservative theory which was proposed by David P. Bloch; the entire DNA molecules act as template and synthase itself like as a photocopy machine. Based on this theory; the daughter molecules were newly synthesized from the parental DNA. The other theory was the semi-conservative theory which was proposed by Watson and Crick in a paper published called “Genetical implications of the structure of Deoxyribonucleic acid”. The paper was about the possible replication of the DNA which is called “semi- conservative replication theory”.   According to this theory the DNA strands break and each strand was used as a template. According to this theory, the daughter molecule would have one newly synthesized strand and one old strand. The third theory was the dispersive theory proposed by Max Delbruck. According to this model, the DNA backbones are chopped into the little pieces and each piece acts as template, DNA is synthesized in short pieces where the daughter molecules would carry the mixture of one old and one newly synthesized pieces of the DNA.

Meselson and Stahl wanted to answer this question via measuring density and mass difference of the DNA molecules with the method called density gradient centrifugation by labeling the DNA. They decided to run an experiment which might prove that the DNA replication mechanism. They used Escherichia coli which was easy and rapid to grow.

2000px-meselson-stahl_experiment_diagram_en-svg

Meselson and Stahl wanted to label the DNA of Escherichia coli with something heavy which would give a chance to separate the DNA molecules based on their density and weight.

Nucleic acids are the main and required elements of the DNA molecules to replicate itself. Each nucleotide has three components: a 5-carbon sugar, a phosphate group, and a nitrogenous base. These nitrogen containing bases are organic molecules with a nitrogen (N) atoms. The natural N has 14 isotopes (7 protons and 7 neutrons) with the isotopic mass of 14 atomic mass unit (amu). Meselson idea was to use heavy isotopes to determine the replication model of the DNA. He wanted to use radioisotopic labels thereby they used the isomer called N15 which has 7 protons and 8 neutrons. The atomic mass of this isomer was 15 amu which makes it heavier than N14.

So, they hypnotized that; The DNA which replicates itself under the heavy N15 presence would have a density increase which might permit the analysis the difference of the DNA mass and density. Based on this knowledge, Meselson and Stahl grew Escherichia coli in heavy N isotope (N15) containing medium and removed a sample of Escherichia coli colonies lysed and DNA was extracted and ultra-centrifuged. They moved some of the colonies from N15 into the N14 containing media and later they extracted the DNA from these various generations of colonies and ultra-centrifuged them too.

For the centrifugation; they added the extracted DNA molecules into the solution of cesium chloride (CsCL) and measured the band weights and tried to determine separation via density- gradient centrifugation method which allows to determine the small density differences between micro-molecules. The cesium chloride helped them to separate the DNA with different molecular weight if there were any. Based on this approach, DNA would form a band form at the place where the CsCL is equal to buoyant density. By doing this, they expected to see the DNA which was labeled with heavy N (N15) would separate from the light one which was labeled with N14. Based on this theory; they were able to observe the separation of the DNA molecules according to their density and mass. They were expecting to see some bands which are the belong to N15 labeled DNA molecules would possibly move closer to the bottom whereas some of the light (harboring N14) one would be on the top of that.

UV photograph of the DNA bands were taken during the centrifugation processes and analysis were done by microdensitometer.

They process 2 different experiments. The second experiment was like a confirmation of the first one. They published the graph of the DNA band from different generations of E.coli and compare them by mixing the generations. The picture was revealing that there were different bands located in various segments. Only semi- conservative model could cause such difference.

According to the semi- conservative model; the parental strands which were labeled with N15 would divide and match itself with light N14 nitrogen containing nucleotides, when they were transferred from N15 media to N14 media. Thereby in the second generation, the expected results were to see the entire daughter DNA molecules would have one old and one newly synthesized strands. This would lighten the DNA weight when compared with the parental strands.  After the second generation. In the third generation; 50% of the daughter molecules would harbor only N14 where as other 50% would have one old and one newly synthesized strands. As a result of these replications and generations by time; the number of newly synthesized DNA molecules with the N14 would increase and the heavy N15 labeled DNA strands number would be stable. Since those could not synthesized themselves with the N15 anymore.

That was the results that Meselson and Stahl found. These finding were supporting the hypothesis of Watson and Crick. They were able to photograph these bands by the time of the generations and prove this hypothesis.

The finding were revealed 3 key points;

  1. The original parental molecules contain N15 because they initially grown into N15 rich media.
  2. Replicated daughter molecules will have N14
  3. DNA with different densities can be separated by centrifugation where they have a difference in mass.

In addition to these finding that I mentioned above, they also analyzed the heat effect on the denaturation of the DNA. When they heated the DNA of the labeled (N15) and unlabeled (N14) DNA in the CsCL centrifuging medium, they realized that the heating decreased the density of the DNA almost half that of unheated material. Upon these findings, they concluded that the two molecular sub-units were disassociated upon heating. When they tried to compare the molecular density of the salmon sperm DNA with E. coli DNA by heating at 100 °C, 30 min. The salmon sperm DNA weight did not decreased by heating. These findings they achieved revealed two conclusions. Their first suggestion was; the salmon DNA strands might had bound more tightly when compared with E. coli DNA. The second conclusion was; if the salmon DNA does not have the subunits that the E. coli DNA has, the salmon sperm DNA might be more complex than E. coli DNA.

As a result of these studies that they conducted in the California Research Institute of Technology, they had proven that the DNA uses the semi-conservative model to replicate itself and heat had an effect on the DNA structure.

 

Based on their conclusion of the heating effect on salmon sperm DNA and E. coli DNA, it was reasonable to conclude such ideas however, they could change the heating time and the temperature too see how various heating conditions might affect the salmon sperm DNA and E. coli DNA.

Gizem Levent

 

The Famous Hershey and Chase Experiment

 

Iportrait-hersheychase-600wINDEPENDENT FUNCTIONS OF VIRAL PROTEIN AND NUCLEIC ACID IN GROWTH OF BACTERIOPHAGE

By A. D. HERSHEY AND MARTHA CHASE

Background Information

The viruses that infect bacteria are called bacteriophages. Phages have often a head and a tail. The tail structure allows them to penetrate to the host cell membranes and cell walls to inject their DNA into the cell. Their heads are containing protein. And their nucleic acid can be DNA or RNA depending on the type of phage wrapped in a protein coat for protection. They cannot multiply without benefit of a host cell. To start the infection the phage binds to the receptors on the cell surface and absorbs the bacterial cell. In general speaking; the next step is, the phage injects its entire DNA into the cells where transcription of RNA begins almost immediately.

Bacteriophages may have a lytic cycle or a lysogenic cycle, and a few viruses are capable of carrying both. With lytic phages such as the T4 phage, bacterial cells are lysed and destroyed after immediate replication of the virion. As soon as the cell is destroyed, the phage progeny can find new hosts to infect. In contrast, the lysogenic cycle does not result in immediate lysis of the host cell. Those phages able to undergo lysogeny are known as temperate phages.

 

Hershey and Chase Experiment

The Hersey- Chase experiment was based on the biology of the bacteriophage T2 (a virus of bacteria) which attacks to a bacterium. Viruses were known to be composed of a protein shell and DNA. A Part but not all of the virus enters to the bacterial cell and in about 20 min later, the cell bursts and releases dozens of the DNA which are belong to the virus from that virus infected the cell. They were curious about which part of that bacteriophage were staying outside and which part were being injected. Was it the DNA or the protein?

Back at that time from previous researches what they knew was that; the phages attach to bacteria by their tails, osmotic shock ruptures the phage producing an empty- headed phage ‘ghost’ that is non-infectious and agitation of phage and bacteria in a Waring blender prevents the infection.

They ran set of experiments by gathering these information. Basically; they chose to uniquely label each DNA and protein of the phage with a different elemental isotope. This method allowed them to observe and analyze the protein and the DNA separately. They used P32 to label for the DNA. The two strands of DNA have a sugar- phosphate backbone that contains phosphorus atoms. Phosphorus is not present in most of the proteins. They used S35 to label the proteins which contain sulfur. Because the sulphur is found in amino acids cysteine and methionine and not present in the DNA.

So, they grew the virus in two different media to label the bacteriophage. One was containing labelled sulphur whereas the other media was containing the labeled phosphorus. After labeling them by growing in these media, they added these phage into a fresh culture where E. coli hosts were present. They gave enough time to the virus to infect these cells and after that, they dethatched these virus particles from the cell via vigorous shaking with a blendor. Samples then place into the tubes and centrifuged to force the bacterial cell to move to the bottom of the tube whereas the virus would stay in the supernatant. Centrifugation allowed for the separation of the phage coats from the bacteria. For each of these samples, the labelled components would me in different fractions, either in the pellet or the supernatant.

hersheychaseexpt

They found that most of the labeled phosphorus were in the pellet whereas the most of the labeled sulphur were in the supernatant fluid with the viruses. So, they conclude that the DNA enters to the host cell and multiply itself not the protein.

Hershey and Chase also showed that the introduction of deoxyribonuclease (referred to as DNase), an enzyme that breaks down DNA, into a solution containing the labeled bacteriophages did not introduce any P32 into the solution. This demonstrated that the phage is resistant to the enzyme while intact. Additionally, they were able to plasmolyze the bacteriophages so that they went into osmotic shock, which effectively created a solution containing most of the P32 and a heavier solution containing structures called “ghosts” that contained the S35 and the protein coat of the virus. It was found that these “ghosts” could adsorb to bacteria that were susceptible to T2, although they contained no DNA and were simply the remains of the original bacterial capsule. They concluded that the protein protected the DNA from DNAse, but that once the two were separated and the phage was inactivated, the DNAse could hydrolyze the phage DNA. At the end they concluded that T2 phage were consist of ~50% protein and 50% DNA, the phage tail was the part that binds to E. coli cells, the progeny viruses produced in the infected cell and the bacterium lysed to release progeny virus.

As a result, Hershey and Chase concluded that protein was not likely to be the hereditary genetic material. However, they did not make any conclusions regarding the specific function of DNA as hereditary material and only said that it must have some undefined role.

Gizem Levent