Image cytometric method for quantifying the relative amount of DNA in bacterial nucleoids using Escherichia coli.

An image cytometric method for quantifying integrated fluorescence was developed to measure the relative DNA contents of bacterial nucleoids. Image analysis was performed with newly developed macros in combination with the program Object-Image, all downloadable from http://simon.bio.uva.nl/object-image.html. Four aspects of the method were investigated. (i) Good linearity was found over a ten-fold range of fluorescence intensity in a test with a calibration kit of fluorescent latex spheres. (ii) The accuracy of the method was tested with a narrowly distributed Escherichia coli population, which was obtained by growing cells into stationary phase. The width of the image cytometric distribution was approximately 6%, in good agreement with results obtained by flow cytometry. (iii) The error contribution of manual focusing could be kept below 2%, although a strong dependency between integrated fluorescence and focus position was observed. (iv) The results were verified with a flow cytometer, which gave similar distributions for the DNA contents per cell expressed in chromosome equivalents (4.8 fg of DNA). We used the presented method to evaluate whether the DNA conformation had any effect on the total fluorescence of bacterial nucleoids. Experiments using nucleoids with the same amount of DNA in either a dispersed or a compact conformation showed no significant difference in integrated fluorescence, indicating that it is possible to determine the DNA content per nucleoid independently of the actual organization of the DNA.

Delayed nucleoid segregation in Escherichia coli.

To study the role of cell division in the process of nucleoid segregation, we measured the DNA content of individual nucleoids in isogenic Escherichia coli cell division mutants by image cytometry. In pbpB(Ts) and ftsZ strains growing as filaments at 42 degrees C, nucleoids contained, on average, more than two chromosome equivalents compared with 1.6 in wild-type cells. Because similar results were obtained with a pbpB recA strain, the increased DNA content cannot be ascribed to the occurrence of chromosome dimers. From the determination of the amount of DNA per cell and per individual nucleoid after rifampicin inhibition, we estimated the C and D periods (duration of a round of replication and time between termination and cell division respectively), as well as the D' period (time between termination and nucleoid separation). Compared with the parent strain and in contrast to ftsQ, ftsA and ftsZ mutants, pbpB(Ts) cells growing at the permissive temperature (28 degrees C) showed a long D' period (42 min versus 18 min in the parent) indicative of an extended segregation time. The results indicate that a defective cell division protein such as PbpB not only affects the division process but also plays a role in the last stage of DNA segregation. We propose that PbpB is involved in the assembly of the divisome and that this structure enhances nucleoid segregation.

Fused nucleoids resegregate faster than cell elongation in Escherichia coli pbpB(Ts) filaments after release from chloramphenicol inhibition.

The course of nucleoid movement during and upon release from protein synthesis inhibition by chloramphenicol in filaments of Escherichia coli pbpB(Ts) was analysed. Cells were grown at 42 degrees C in glucose minimal medium for two mass doublings and were treated with chloramphenicol to generate fusion (coalescence) of the nucleoids. Upon release from protein synthesis inhibition, the large distance between the border of the fused nucleoids and the cell poles immediately decreased, before full recovery of the rates of mass growth and length increase at 30 degrees C. This indicates that nucleoids can reoccupy the DNA-free cell ends independently of cell elongation. During filamentation at 42 degrees C, the pbpB cells established initial constrictions at midcell and at one-quarter and three-quarter positions. Nevertheless, divisions only started 75 min after chloramphenicol removal at 30 degrees C, when most nucleoids had moved back into the vacated cell ends. No 'guillotine-like' constrictions at the site of the nucleoids occurred. This suggests that segregating nucleoids postpone division recovery at previously established sites. The results are discussed in the light of a working model for transcription/translation-mediated chromosome segregation and nucleoid occlusion of cell division.

Effects of different carbon fluxes on G1 phase duration, cyclin expression, and reserve carbohydrate metabolism in Saccharomyces cerevisiae.

By controlled addition of galactose to synchronized galactose-limited Saccharomyces cerevisiae cultures, the growth rate could be regulated while external conditions were kept constant. By using this method, the G1 phase duration was modulated and expression of cell cycle-regulated genes was investigated. The expression of the cyclin genes CLN1 and CLN2 was always induced just before bud emergence, indicating that this event marks the decision to pass Start. Thus, G1 phase elongation was not due to a slower accumulation of the CLN1 and CLN2 mRNA levels. Only small differences in CLN3 expression levels were observed. The maximal SWI4 expression preceded maximal CLN1 and CLN2 expression under all conditions, as expected for a transcriptional activator. But whereas SWI4 was expressed at about 10 to 20 min, before CLN1 and CLN2 expression at high growth rates, this time increased to about 300 min below a particular consumption rate at which the G1 phase strongly elongated. In the slower-growing cultures, also an increase in SWI6 expression was observed in the G1 phase. The increase in G1 phase duration below a particular consumption rate was accompanied by a strong increase in the reserve carbohydrate levels. These carbohydrates were metabolized again before bud emergence, indicating that below this consumption rate, a transient increase in ATP flux is required for progression through the cell cycle. Since Start occurred at different cell sizes under different growth conditions, it is not just a certain cell size that triggers passage through Start.

Production of senescent cells of Saccharomyces cerevisiae by centrifugal elutriation.

The centrifugal elutriator has been used as a baby machine by loading the chamber with a population of mixed-generation daughter cells and allowing this population to grow, divide and age under continuous washing-out of newborn daughter cells. Clear peaks in the number of elutriated cells were reproducibly obtained for at least ten generations. The parent cells growing in the chamber continued to divide at the steady-state generation time of 95-100 min, showing no change in cycle time during aging. The washed-out daughter cells increased in volume during the first five generations from their steady-state value of 17 micro3 to a maximum of 34 micro3. As to be expected, the generation times of these large daughters, determined in a synchronous batch culture, were shorter (130 min) than that of the steady-state daughters (240 min), even when derived from 15-generation parents. No indication for a volume increase of daughter cells without bud was observed when a population was allowed to grow in the chamber without washing-out the smaller daughter cells. The 15-generation parent population, recovered from the chamber, had an average volume of 80 micro3 and consisted of: (i) 71% cells with more than ten scars, (ii) 13% cells with one to nine scars, and (iii) 17% daughter cells. The production of senescent cells by undisturbed growth in the elutriator chamber has been prolonged to 29 generations. The method is therefore suitable to examine what factors determine the life span of budding yeast.

Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry.

The pattern of volume growth of Saccharomyces cerevisiae a/alpha was determined by image cytometry for daughter cells and consecutive cycles of parent cells. An image analysis program was specially developed to measure separately the volume of bud and mother cell parts and to quantify the number of bud scars on each parent cell. All volumetric data and cell attributes (budding state, number of scars) were stored in such a way that separate volume distributions of cells or cell parts with any combination of properties--for instance, buds present on mothers with two scars or cells without scars (i.e., daughter cells) and without buds--could be obtained. By a new method called intersection analysis, the average volumes of daughter and parent cells at birth and at division could be determined for a steady-state population. These volumes compared well with those directly measured from cells synchronized by centrifugal elutriation. During synchronous growth of daughter cells, the pattern of volume increase appeared to be largely exponential. However, after bud emergence, larger volumes than those predicted by a continuous exponential increase were obtained, which confirms the reported decrease in buoyant density. The cycle times calculated from the steady-state population by applying the age distribution equation deviated from those directly obtained from the synchronized culture, probably because of inadequate scoring of bud scars. Therefore, for the construction of a volume-time diagram, we used volume measurements obtained from the steady-state population and cycle times obtained from the synchronized population. The diagram shows that after bud emergence, mother cell parts continue to grow at a smaller rate, increasing about 10% in volume during the budding period. Second-generation daughter cells, ie., cells born from parents left with two scars, were significantly smaller than first-generation daughter cells. Second- and third-generation parent cells showed a decreased volume growth rate and a shorter budding period than that of daughter cells.

A computer-aided measuring system for the characterization of yeast populations combining 2D-image analysis, electronic particle counter, and flow cytometry.

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.

Toporegulation of bacterial division according to the nucleoid occlusion model.

A model for the toporegulation of division in Escherichia coli is presented in which cell constriction is initiated by the combined action of a biochemical and a structural event. It is proposed that the biochemical event of termination of DNA replication causes a transient change in the pool of deoxyribonucleotides, which serves as a localized trigger that is converted to a diffusible, cytoplasmic activator of peptidoglycan synthesis. The second event involves the segregation of the nucleoids. Evidence is presented that the nucleoid suppresses the activity of peptidoglycan synthesis in its vicinity. It is proposed that active transcription/translation around the nucleoids produces a strong but short-range inhibitor which prohibits division (nucleoid occlusion). The combined effects of the locally produced termination-activator and of the diminished occlusion as a result of nucleoid segregation, guarantee that division is normally placed between the separated nucleoids. The model can explain the pattern of division-recovery of filaments, the majority of which constrict at sites which produce polar daughter cells containing two nucleoids. In addition, the model offers an explanation for the occurrence of mini-cells under a variety of conditions.

Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments.

Isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli were compared with their parent strain in temperature shift experiments. To improve detection of phenotypic differences in division behavior and cell shape, the strains were grown in glucose-minimal medium with a decreased osmolality (about 100 mosM). Already at the premissive temperature, all mutants, particularly the pbpB and ftsQ mutants, showed an increased average cell length and cell mass. The pbpB and ftsQ mutants also exhibited a prolonged duration of the constriction period. All strains, except ftsZ, continued to initiate new constrictions at 42 degrees C, suggesting the involvement of FtsZ in an early step of the constriction process. The new constrictions were blunt in ftsQ and more pronounced in ftsA and pbpB filaments, which also had elongated median constrictions. Whereas the latter strains showed a slow recovery of cell division after a shift back to the permissive temperature, ftsZ and ftsQ filaments recovered quickly. Recovery of filaments occurred in all strains by the separation of newborn cells with an average length of two times LO, the length of newborn cells at the permissive temperature. The increased size of the newborn cells could indicate that the cell division machinery recovers too slowly to create normal-sized cells. Our results indicate a phenotypic resemblance between ftsA and pbpB mutants and suggest that the cell division gene products function in the order FtsZ-FtsQ-FtsA, PBP3. The ftsE mutant continued to constrict and divide at 42 degrees C, forming short filaments, which recovered quickly after a shift back to the permissive temperature. After prolonged growth at 42 degree C, chains of cells, which eventually swelled up, were formed. Although the ftsE mutant produced filaments in broth medium at the restrictive temperature, it cannot be considered a cell division mutant under the presently applied conditions.