An Easy-To-Use Simulation Program Demonstrates Variations in Bacterial Cell Cycle Parameters Depending on Medium and Temperature

Many studies are performed on chromosome replication and segregation in Escherichia coli and other bacteria capable of complex replication with C phases spanning several generations. For such investigations an understanding of the replication patterns, including copy numbers of origins and replication forks, is crucial for correct interpretation of the results

Mutagenese, ekspresjon og karakterisering av DNA polymerase I fra den varmestabile bakterien Thermotoga Maritima

Sammendrag Alle levende organismer inneholder en eller flere DNA polymeraser, og dette enzymet er helt nødvendig for videreføring av arvestoffet fra generasjon til generasjon. DNA- polymeraser har også etter hvert fått en uvurderlig rolle i flere bioteknologiske teknikker. En av disse teknikkene er DNA-sekvensering ved hjelp av kjedeterminering, det vil si ved inkorporering av dideoksynukleotider. Det er derfor interessant å forsøke å utvikle DNA polymeraser med forbedrete og tilpassede egenskaper til slike teknikker. I denne oppgaven ble det arbeidet med DNA polymerase I fra Thermotoga Maritima, Tma polymerase I. Thermotoga Maritima er en varmestabil bakterie noe som vil si at den vokser ved høyere temperaturer enn de fleste bakterier. Thermotoga Maritima har et temperaturoptimum ved 80 grader. Det å benytte en DNA polymerase fra denne bakterien er derfor en fordel i sekvensering ved kjedeterminering hvor DNA-fragmentene blir denaturert ved 95 grader i hver syklus. Hvis man da benytter varme-stabile DNA polymeraser trenger man ikke å tilsette nytt enzym for hver syklus. Det finnes allerede flere varmestabile DNA polymeraser som benyttes i sekvensering, blant annet DNA polymerase I fra Thermus Aquaticus (Taq) med mutasjonen F667Y. Denne varianten av Taq polymerase I har en forbedret evne til inkorporering av ddNTP, noe som gjør det til et bedre sekvenseringsenzym. I denne oppgaven ble denne mutasjonen innført i tilsvarende posisjon hos Tma polymerase I; Fenylalanin i posisjon 730 ble substituert med en tyrosin ved hjelp av setestyrt mutagenese. Dette skulle føre til nedsatt diskriminering mot inkorporering av ddNTP. Deretter ble både nativ og mutert Tma polymerase uttrykt i E.coli-celler og det ble laget lysat som inneholdt de aktive enzymene. Optimal ekspresjonstid, lineært område og antall enheter i lysatene ble bestemt ved hjelp av en fluorescensbasert aktivitetsanalyse. Deretter ble lysatene forsøkt renset uten særlig hell, så videre undersøkelser av enzymene ble gjort på grovlysatene. De to enzymene ble testet i sekvensering ved kjedetermineringsmetoden. Det ble da bevist at mutasjonen har den ønskede effekten, da den muterte varianten fungerte godt under betingelser som var tilpasset den muterte varianten av Taq (F667Y). Den native fungerte ikke under disse betingelsene, men krevde en veldig mye høyere konsentrasjon av dideoksynukleotider. Disse analysene ble gjort på Alfexpress; hvor det benyttes en fluorescensmerket primer for å detektere fragmentene. Det ble også på Alfexpress; gjort sammenligninger mellom nativ Taq polymerase og nativ Tma polymerase. Resultatene her tydet på at Tma er et dårligere sekvenseringsenzym enn Taq polymerase. De siste undersøkelsene som ble gjort var å benytte den muterte varianten av Tma polymerase med fluorescensmerkede dideoksynukleotider på MegaBACE-systemet. Dette ble gjort med tanke på å sammenligne Tma polymerase (F730Y) med polymerasen som benyttes i det kommersielle kitet. Da Tma polymerase ikke fungerte i dette systemet under betingelsene som ble forsøkt, var det ikke mulig å sammenligne de to polymerasene

DNA, origin and DnaA concentrations of wild type and Δ<i>fis</i> cells grown in different media.

<p> relative to the wild type.</p

Strains and plasmids used in this study.

<p><i>fis::km</i> from the CSH50 background in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083562#pone.0083562-Koch1" target="_blank">[22]</a> to MG1655 in the Messer research group.¹ WM2652 was made by P1 transduction of the </p

The effect of disruption of Fis binding site I or II on the regulation of initiation.

<p>A) An illustration of the origin region of <i>E.coli</i>. The high affinity DnaA boxes are shown in light grey and the lower affinity and ATP-DnaA boxes in dark grey. Binding sites and protected regions for the IHF and Fis protein are shown as horizontal lines. Promoters in the origin region are shown as arrows indicating the direction of transcription. B) DNA histograms of the exponential and replication run out samples of wild type cells and cells where the Fis site I had been scrambled (<i>oriC131</i>) or the Fis site II removed (<i>oriC160</i>) after growth in GluCAA medium at 37°C. The chromosome equivalents are represented on the abscissa and the number of cells on the ordinate. C) Relative values of DNA/mass and origin/mass for the two mutants relative to the wild type. The values are an average from three or more experiments and the standard deviations are given in parentheses.</p

Cellular concentration of Fis in wild type cells at different growth rates.

<p> relative to cell grown in acetate.</p

Schematic illustration of the replication pattern in wild type cells and in cells exhibiting under-initiation and asynchronous initiations.

<p><b>A</b>) In a population of wild type cells, in which initiation occurs at four origins in synchrony, the cells will end up with either four fully replicated chromosomes (representing the cells that had not initiated at the time the drugs were added) or eight fully replicated chromosomes (representing the cells that had initiated at the time where the drugs were added) after treatment with rifampicin and cephalexin (grey cells). <b>B</b>) In a population with mutant cells that are exhibiting too few and untimely initiations the cells will end up with a number of fully replicated chromosomes that is not equal to 2ⁿ or 2ⁿ⁺¹, the actual number depending on how many of the origins had been initiated at the time the drugs were added. Here, an example where the newborn cell had 4 origins, but only three is initiated in the current generation, is shown.</p

The Fis protein is necessary for timely initiations during rapid growth.

<p>Exponentially growing wild type and Δ<i>fis</i> cells and cells treated with rifampicin and cephalexin were analyzed by flow cytometry. The first (left-most) panels illustrate the replication patterns of wild type cells (see Materials and Methods for details of determination) grown at 30°C in (A) acetate medium, (B) glucose medium, (C) GluCAA medium and (D) LB medium. Cells with chromosomes (black lines) were drawn schematically to show the number of origins (red dots) initiated in the different media. In acetate grown cells initiation of replication occurred at one origin and the replication period (blue arrow) was completed within one generation (A). In the glucose, GluCAA and LB grown cells initiation of replication occurred at two, four and eight origins, respectively (B–D) and the replication spans more than one generation. The second and third panels show overlays of representative DNA histograms of exponentially and rifampicin/cephalexin treated wild type cells (dark blue) and Δ<i>fis</i> cells (light blue). Chromosome equivalents per cell are represented on the abscissa and the number of cells on the ordinate. 10000 cells were analyzed in each experiment. The values for DNA/mass, origin/mass and the origin to terminus ratio for the Δ<i>fis</i> cells relative to the wild type are shown in the bar histograms in panel four (A–D). The values are an average from three or more experiments and the error bars represent the standard deviation. *For the cells grown in acetate the origin to terminus ratio is determined from the DNA histogram of exponentially growing cells.</p

The effect of temperature on cells lacking the Fis protein.

<p>Flow cytometry DNA histograms of wild type and Δ<i>fis</i> cells grown in GluCAA medium at 30, 37 and 44°C. Chromosome equivalents per cell are represented on the abscissa and the number of cells on the ordinate. 10000 cells were analyzed in each experiment. Histograms of the replication run out samples (see Materials and Methods) are shown as small panels.</p

Simulations of DNA histograms to determine cell cycle parameters for <i>Escherichia coli</i>.

<p><i>E. coli</i> cultures were grown in four different media at three different temperatures, and samples were subjected to flow cytometry to obtain the experimental DNA distributions (green curves). By iteration of cell cycle parameters in our computer program best fit theoretical DNA histograms were found (red curves). One parallel of three is shown for each temperature for Acetate medium (A–C), Glucose medium (D–F), Glucose-CAA medium (G–I) and LB-G medium (J–L). The x-axes denote chromosome equivalents, and the scale is different from medium to medium.</p