Muricauda ruestringensis Has an Asymmetric Cell Cycle

Muricauda ruestringensis Has an Asymmetric Cell Cycle

MÜLLER1, S., KIESEL1, B., BERTHE-CORTI2∗, L.

1 UFZ – Umweltforschungszentrum Leipzig–Halle GmbHSektion UmweltmikrobiologiePermoserstr. 1504318 Leipzig, Germany

2 Carl von Ossietzky Universität Oldenburg * Corresponding authorFachbereich BiologieGeo- und Umweltwissenschaften Phone: + 49 441 798 3290Postfach 2503 Fax: + 49 441 758 325026111 Oldenburg, Germany E-mail: [email protected]

Summary

Muricauda ruestringensis B1 is a GRAM-negative, marine bacterium and a member of the Flavobac-teriaceae family. It is characterized by long appendages, which appear at different stages of growth.At the outer end of these appendages there is a bulbous structure. Investigating the cell morphologyof strain B1 during batch growth revealed a high diversity of cell types and sizes. Apart from smallrod-shaped cells and rods with appendages, there were large rods and spherical cells of different sizesas well as spherical cells which had fimbriae. To be able to study the cell cycle events, it was essen-tial to monitor the population dynamics of the involved individuals. For this purpose, fluorochromi-sing techniques, multi-parametric flow cytometry, image analysis and fluorescence microscopy wereused. It was demonstrated that all cell types displayed a broad variation in DNA content; the precisenumber of chromosomes varied depending on the growth phase. The assortment was testified to hold16S rDNA sequence identity. The cultures consisted of subpopulations whose density within aPercoll gradient varied considerably, ranging from 1.028 to 1.070. Consolidating the results of themorphological data, the chromosome content and the density of the subpopulations at differentgrowth stages enabled us to construct an asymmetric cell cycle for the growth of strain B1 under thespecific culture conditions of our experiments.

Introduction

Muricauda ruestringensis is a newly described bacterium within the family of Flavo-bacteriaceae, which has been isolated from sediment suspension-cultures containing

© WILEY-VCH Verlag Berlin GmbH, 13086 Berlin, 2001 0138-4988/01/0411-0343 $ 17.50+.50/0

Acta Biotechnol. 21 (2001) 4, 343–357

intertidal sediment from the German North Sea Bay [1]. The type strain B1T is depos-ited at the DSMZ, Braunschweig, Germany, as DSM13258T and at the BCCM/LMG,Gent, Belgium, as LMG 19739T. The bacterium will be validly described in the Inter-national Journal of Systematic and Evolutionary Microbiology [1]. This bacterium is arod-shaped bacterium showing appendages with a bulb at their end. The function ofthese appendages has not yet been determined. Within the phylum “Cytophaga-Flavobacterium-Bacteroides” only very few species are known to have appendages(Flavobacterium aquatile strain ATCC 1947T, Flexibacter strain BH3, Cytophagajohnsoneae [johnsonii] and Polaribacter irgensii). In contrast to the function of theappendages of other prosthecate or budding bacteria, the function of the appendages ofthe bacteria in the CFB phylum is not clear. HUMPHRY et al. [2] demonstrated that theappendages of Flexibacter HB3 consist of extruded material derived from thelipopolysaccharide membrane. They conclude that the extruded material is related tothe gliding motility of this species. GOSINK et al. [3] showed that the appendages ofPolaribacter irgensii strain 23-P are true flagella, although motility in this genus hasnever been observed. The nature of the appendages of Flavobacterium aquatile has notyet been characterized [4].There are several genera in which the formation of true appendages is common [5],especially within the genera Caulobacter [6, 7], Rhodomicrobium [8] and Asticcacaulis[9]. The formation of appendages is generally related to an asymmetric cell cycle [9].The regulation mechanism of an asymmetric cell differentiation, mainly investigated inCaulobacter crescentus, is well known [10–13]. Two cell types have been described, aswarmer and a stalked cell. The swarmer cell must first change into a stalked cellbefore replication initiation can occur. This stalked cell then divides into a swarmercell, which is unable to replicate, and a stalked cell, which is able to perform additionalreplications [12]. In this paper we demonstrate that the appendages of the newlydescribed bacterium Muricauda ruestringensis strain B1 are associated with its repro-ductive cycle and that the bacterium has an asymmetric cell cycle.

Materials and Methods

Cultivation

Muricauda ruestringensis was grown in batches of artificial seawater medium (SWB-X) contain-ing per litre: 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 × 6 H2O, 5.94 g MgSO4 × 7 H2O, 1.3 g CaCl2× 2 H2O, 43 mg NaHPO4 × 2 H20 and 220 mg NaNO3. The medium was supplemented with 3 ml of adefined vitamin solution [14] and 1 ml of a defined mineral salts solution (medium No. 124,DSMZ catalogue of strains, 1993). The pH was adjusted with NaHCO3 to 7.2. 1 g/l pyruvate, 4.16 g/lNa-acetate and 1g/l casamino acids were applied as carbon sources. The cells were precultivated onagar plates (SWB-X-medium with 1.5% agar agar). A single colony was used for inoculation ofstarter cultures (50 ml). The cultures were inoculated with 15 ml of a 7-day-old starter culture; grownin 300-ml ERLENMEYER flasks containing 100 ml medium, incubated at 100 rpm and a temperature of25 °C.To monitor the growth of Muricauda strain B1, 15 ml samples were taken at regular intervals. Opticaldensity (OD) was determined at λ = 700 nm (d = 0.5 cm). Protein content was analyzed according toBRADFORD’s method [15]. Cells were harvested by sedimentation during different growth phases at10,100 × g (10 min, 20 °C). The cells were then preserved in an aqueous NaN3 solution (10%) andstored at 4 °C until the flow cytometric analysis or Percoll gradient separation.

344 Acta Biotechnol. 21 (2001) 4

Preparation and Staining of the Cells

Determination of the DNA content

The DNA content was determined by measuring the fluorescence intensity of the cells after stainingthem with the dye 4´,6-diamidino-2´-phenylindole (DAPI). The preserved cells were centrifuged at10,100 × g (10 min), washed in sodium chloride-phosphate buffer (pH 7.2) and diluted to an OD of0.035 (λ = 700 nm) in sodium chloride-phosphate buffer. Samples of these preparations (2 ml) werethen centrifuged a second time and stained, using a modification of a standard procedure according toOTTO [16].

Viewing Lipid Structures

In this study, the dye Nile red was used to make lipid structures visible, using the following method:40 µl of a Nile red stock solution (1 mg/ml acetone) were added to a diluted sample with an OD of0.035 (λ = 700 nm); 10 min were needed to reach optimum alignment. Due to the Nile red'shydrophobic nature, the equilibrium between the free and the intracellularly bound Nile red isdisturbed by dye crystallization; for this reason, the time interval allowed for reaching the stainingequilibrium must be kept constant.

Flow Cytometry

In order to measure the chromosome content, a modular flow cytometer was used with a stream-in-airflow chamber and an argon-ion-laser (Innova 304, COHERENT) as the light source (as describedelsewhere, [17]). Parameters were forward light-scatter (3.5°–9.6°) and fluorescence emission at a90° direction (DAPI: λexc = 333.6–363.8 nm, λexc = 400–580 nm; power output: 370–400 mW).DAPI fluorescence was measured with a band-pass filter combination from 390 nm to 540 nm formaximum detection sensitivity. Light-scatter analysis was applied principally to examine cell sizeand to trigger the system. The pulse heights of all the fluorescence and scatter signals were recordedlinearly in all channels. A total of 32,000 cells were analyzed in each sample, at a rate of250–350 cells per second. Optimum alignment was based on the optimized signal from 0.5 µmdiameter fluorescent beads (POLYSCIENCES Inc., Ref. YG 17152, BB 18339). Data were evaluatedusing WinList 2.01 and IsoContour 3.2.

Image Analysis

In order to analyze and count the different morphological forms of single bacterial cells, the imageanalysis system Optimas 4.1 was used (Optical Measurement and Analysis Software; STEMMERPC-SYSTEME GmbH; Germany).

Microscopy

Samples were routinely viewed using a phase-contrast and fluorescence microscope (BH-2,OLYMPUS, Japan). Excitation of the cells was effected using ultraviolet light from a 100 W mercuryarc lamp. Blue fluorescence was passed through a UG1 filter (band pass 360). Red fluorescence wasmonitored using a BP545 filter. Microphotographs were performed using a reflex camera(OLYMPUS, Japan) and KODAK film (ISO 200).

MÜLLER, S. et al., Muricauda ruestringensis 345

Extraction of Chromosomal DNA

Cells were harvested by centrifugation (30 min, 10,100 × g, 4 °C ). The pellet was washed twice withTE-buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Chromosomal DNA was extracted using theC+T Kit (MACHEREY & NAGEL, Germany) and was prepared according to the supplier’s recom-mendations. To extract the DNA from the supernatant, PEG precipitation was carried out using themethod for preparation of DNA from single stranded bacteriophages [18].

Characterization of DNA by 16S rDNA Analysis

PCR Amplification of 16S rDNA: The PCR amplification of 16S rDNA was carried out with the TaqPCR Master Mix Kit (QIAGEN, Germany) using forward-primer 27F and reverse-primer 1525R (an-nealing position of 16S rDNA of E. coli) [19]; and the DNA prepared from the cell pellet or from thesupernatant. A negative control without the template DNA was always included. Amplification wasperformed in a Thermocycler PTC-200 (MJ RESEARCH Inc., USA). The cycle programme includesan initial denaturation step at 95 °C for 5 min, followed by 30 cycles at 95 °C for 45 s, 48 °C for 45 s.and 72 °C for 75 s, followed by a 5 min elongation at 72 °C. To provide an initial assessment, theRestriction Fragment Length Polymorphism (RFLP) patterns of 16S rDNA amplification productswere analyzed. These patterns had been created by the 4 base cutting restriction enzymes Sau3A,AluI and HhaI (NEW ENGLAND BIOLABS, Germany). Prior to sequencing, the PCR products werepurified using the Nucleospin Extraction Kit (MACHEREY & NAGEL, Germany).

16S rDNA Sequencing: The purified PCR products were used as templates for sequencing, employingthe ABI PRISM BigDye Cycle Sequencing Ready Reaction Kit (APPLIED BIOSYSTEMS, USA),according to the manufacturer’s recommendations. The following eight sequencing primers, 27F,357F, 519R, 530F, 926F, 1100R, 1114F and 1525R (annealing position of 16S rDNA of E. coli),were used in the sequencing reactions (25 cycles: 96 °C for 30 s and 60 °C for 4 min 15 s). Theproducts were purified using Centrisep columns (QIAGEN, Germany).The entire 16S rDNA operon was sequenced by automatic cycle sequencing, using the ABI PRISM310 Genetic Analyzer (APPLIED BIOSYSTEMS, USA) with the polymer POP6.The 16S rDNA sequences were aligned with the Sequence Navigator software and assembled fromcontigs with the auto assembler. The results were compared with the Muricauda ruestringensis B1sequence of the GenBank database (accession number AF 218782) using the programmeBLASTN 2.0 (NCBI).

Fractionation of Cells with Percoll

Various gradient media have been developed for specific applications of Percoll [20]. In our experi-ments, discontinuous gradients with Percoll in 1x seawater bouillon were used, according to thesupplier’s recommendations (PHARMACIA BIOTECH, Germany). To form the gradients, stockisotonic Percoll (SIP) was diluted to a series of seven densities, ranging from 1.080; 1.070; 1.068;1.066; 1.064; 1.062 to 1.04. The gradient was constructed in a 15-ml Corex tube with 1.5 ml of eachdensity, after which it was underlaid with 1 ml of a mixture of fresh or fixed cells. In order to do this,10 ml samples were taken at regular intervals, the cells were harvested by centrifugation, and wereresuspended in 0.475 ml medium SWB-X and 0.833 ml SIP for cell separation. For reference pur-poses, 1 ml of a mixture of 0.833 ml SIP, 0.475 ml medium SWB-X and 0.1 ml of density markerbeads 1-9 (PHARMACIA BIOTECH, Germany) was prepared. These beads served as references inadditional control tubes. Centrifugation was carried out in an angle-head rotor at 45 ° for 60 min at20 °C, 10,100 × g. The distances of density marker bands were estimated, and the gradient shapeswere plotted. The density of the bands which developed as a result of the morphologically diverseforms from Muricauda ruestringensis B1, were calculated using a density marker plot. The fractionswere collected from the top of the tube and analyzed by microscopy as well as by flow cytometry.


Results

Cell Morphology

In batch cultures, strain B1 showed an extended lag phase of more than 16 h followedby a short but intense increase (≥ factor 5) in OD and in protein concentration.Microscopic observations, which were performed parallel to the flow cytometric analy-sis of the DNA content, showed cells with very different morphological types andsizes. The data were obtained by staining the DNA of the cells with DAPI and, parallelto this, staining the lipid structures of the cells with Nile red (Fig. 1). The culturescontained rods of different types and sizes, in addition to spherical cells that also var-ied significantly in size. The cells showed varying intensity in DAPI fluorescence;therefore one has to assume that the cells contained different amounts of DNA (Fig. 1,No. 6–7).The Nile red staining makes lipid structures, such as cell membranes, visible. Thestaining showed basically the same cell types as the DAPI staining did. However, thereare some important differences. The Nile red staining also reveals thread-like filamentswhich connect the spherical and rod-like structures (Fig. 1, No. 8–9). There are alsostructures composed of one large red-stained spherical cell which, when stained withDAPI (Fig. 1, No. 12–14), were shown to contain two clear, parallel DNA areas.The counting of the different cell types over the course of the experiment demonstratedthat the frequency distribution of single cell types changed with the cultivation time(Fig. 2).The concentration of each of the cell types demonstrated a general tendency to increaseor decrease. The number of rod-shaped cells decreased within the first 24 h of cultiva-tion but increased at the end of the experiments. This seemed to be negativelycorrelated with the appearance of rods with spheres. Furthermore, large fluctuationswere observed in the concentration of spherical cells with rods. After seven days, alarge subpopulation consisting of mainly large spheres was detected (above 50%),suggesting that considerable changes in replication and cell division had occurred.

DNA Content and Cell Size

Using flow cytometry, the DNA content as well as the light-scatter behaviour of thecells was analyzed (the light-scatter behaviour reveals information about cell size).Looking at these two parameters, a clear multiphasic chromosome content of the cellswas observed (Fig. 3). Calculation of the number of cells with a specific size and chro-mosome content was carried out by dot-plot gating. A comparison of the light-scatterdata of the inoculum with the data of the 16.5-h-old cultures demonstrated an increasein the size of the cells. The cells also started DNA replication, as is indicated by thedecrease (35.01% to 13.07%) of the cell fraction having only one chromosome (Fig. 3,black area) and the increase (17.38% to 42.29%) in the cells containing at least two ormultiple copies of chromosomes (Fig. 3, dark grey area). In the following four to fivehours, in which the OD rose sharply, rapid changes were observed regarding thedistribution of the subpopulations of the cultures. The size of the cells alteredconsiderably, due to chromosome segregation and cell division.


Fig. 1. Fluorescent photomicrographs of M. ruestringensis, harvested during different phases of batchgrowthThe cells were stained simultaneously with DAPI and Nile red (upper row: red fluorescence of Nilered stained lipids; bottom row: blue fluorescence of DAPI-stained DNA). Cells with different mor-phologies were presented.The numbers indicate the different phases of the life cycle: Small rods grow to larger rods (1) whichthen undergo cell division (2). Some of these larger rods begin to form a vesicle containing no DNA(3). Afterwards, the DNA is transferred into the vesicle (4), which then matures to a sphere (5). Somespheres show higher fluorescence intensity in comparison with the rods (6, 7). Then, a threaddevelops between a sphere and a rod (8, 9). Afterwards, the sphere detaches from the rod (10) losingits thread (11). Some of them most likely release a small rod (11a, b), while others grow larger.Separation of the chromosomes occurs within these large spheres (12, 13). At the end of the batchcultivation, cell aggregates were sporadically observed, which consisted of rods and spheres withmultiple copies of DNA (14).


Fig. 2. Cell concentrations of batch-cultivated M. ruestringensis in SWB-X-mediumTypes of cells were discriminated using fluorescence microscopy. The upper diagram portrays therods which have just synthesized a vesicle or which already possess a sphere (�) as well as the singlespheres (small and large ones; l). The lower diagram shows the rods (both single and double; È) andthe spheres with a rod (n). About two hundred cells (amounting to a total of 100%) were analyzedevery hour within the first 24 h of cultivation; afterwards, samples were taken at larger intervals.


Fig. 3. Flow cytometric characterization of M. ruestringensis, from batch cultures overa time span of 165 hScatter signals (cell size) are shown on the left side; fluorescence intensity (DNA con-tent) is shown on the right side. The black, pale grey and dark grey histograms represent


the following subpopulations: one chromosome; two chromosomes; or three and fourchromosomes. Sampling times are indicated in the left row of histograms. Thevariation in cell number of the subpopulations is shown in the right row of histogramsin percentages.


Cells having only one chromosome presented low scatter signals, whereas cells withmultiple chromosome content clearly scattered a higher amount of light, indicatingtheir larger size (compare Fig. 3, 20–26 h). These changes equalised following another24-h period, after which the cell size and chromosome distribution remained relativelyconstant. The main part of the cells contained two chromosomes; only two small cellfractions contained either one chromosome (black area) or three and four chromo-somes (dark grey area). In the late stationary phase, all the cells increased in size, eventhose with one chromosome. This was accompanied by a change in the subpopulationdistribution, which at 165 h was very similar to the cell distribution in the inoculum.

Density of Cell Fractions and DNA Content

Each sample, which was harvested at different growth stages, was separated by cen-trifugation into a pellet containing the cells, and a supernatant containing non-sedimentary particles. These two fractions were examined to determine 16S rDNAsequence identity. Both parts contained chromosomal DNA, verified by 16S rDNAamplification. The RFPL pattern, which was generated with three different restrictionenzymes (Sau3A, AluI, HhaI), resulted in identical DNA patterns of the cell pellet andthe non-sedimentary particles. The results of the DNA sequencing analysis of theentire 16S rDNA of both fractions showed 100% similarity to each other as well as tothe sequence of Muricauda ruestringensis strain B1 deposited under the accessionnumber AF218782. This indicates that the pellet and supernatant contained geneticallyidentical B1-cells with very different sedimentation behaviours.These differences in the sedimentation behaviour of the B1 population were alsodemonstrated using a Percoll density gradient. The sedimentation pattern varied withinthe cultivation time. During the first 20 h of cultivation, only one band, with a densityof 1.062, was visible. Four hours later, population differentiation began, as could beseen by the generation of two additional diffuse bands (1.042 and 1.051), which alsoappeared in 48-h-old cultures. After 68 h, the clearly marked 1.062 density band waseither no longer visible or it had spread out over 0.8 cm, encompassing the 1.051 andthe 1.036 density bands. Cell samples of a 92-h-old culture revealed three density frac-tions: 1.036-1.041, 1.051-1.06 and 1.070. In the late stationary phase (165 h), the1.036 density band had decreased to a density of 1.028-1.036. A band of 1.06, whichhad been visible with young cultures, reappeared as well as a new large density band(1.064-1.07). The diversity of these bands was reproducible and is not an experimentalartefact. Furthermore, by separating the different density bands, analyzing the cell sizeand the chromosome content by flow cytometry, and then quantifying the different celltypes by microscopic counting, each density band could be assigned to a specific pat-tern of cell sizes, chromosome numbers of cells and cell types (Fig. 4). This waspossible even though there was no sharp separation of the different cell types withinthe bands but a clear statistical distribution.The results allowed us to compare the morphology of the cells with the number ofchromosomes in the cells as well as with the density and size of the different cell types.This clearly indicated the existence of an asymmetric cell cycle of Muricauda ruestrin-gensis strain B1.


Fig. 4. Separation of different cell types of M. ruestringensis, using a high density Percoll gradientCells were harvested after growing for 92 h on SWB-X-medium. Three different density fractionswere found. Samples from each fraction were analyzed by flow cytometry (left row) and microscopy(right row). Left row: The histograms represent subpopulations with one chromosome (black), twochromosomes (pale grey), or three and four chromosomes (dark grey). The number of cells within thesubpopulations is given in percentages. Right row: Two hundred cells were morphologically differ-entiated within each band of the Percoll gradient using microscopy. The different cell types and theirquantities are shown in the diagrams in percent. It is clearly demonstrated that a subpopulation wasfound in Fraction 1, which contained mainly spherical cells (above 60%). Furthermore, the samefraction contained the cells with the highest DNA content. Spherical cells were not found in Frac-tions two and three.


Discussion

Strain B1 is a GRAM-negative, marine bacterium and a member of the Flavobac-teriaceae family. The 16S rDNA sequence is deposited at the GenBank as AF 218782.Strain B1 produces long appendages with a bulbous structure at the end. These append-ages, which emerge during growth, are a continuum of the outer membrane and havebeen shown to sometimes have fimbriae; the function of these appendages has not yetbeen clarified [1].BRUNS and her co-workers cultivated strain B1 on the same medium as was describedin this paper but instead of a mixture of acetate, pyruvate and casamino acids they usedpeptone, yeast extract and hexadecane as carbon sources [1]. Monitoring the cellmorphology of strain B1 during its growth on the medium which contained acetate,pyruvate and casamino acids revealed a high diversity of cell types and sizescomprising the following: non-motile small rods (0.5–1 µm) with and withoutappendages; large rods; small and large spherical cells; and large spherical cells withfimbriae, which might be motile. Motility could not be demonstrated unequivocally asthe cells with fimbriae had a very short life span. This diversity in the cell morphologywas not observed when the strain was grown on nutrient agar according to BRUNS’method [1], but this is not uncommon for appendaged cells. Caulobacter crescentusalso shows a variety of different morphological cell types. It produces a non-replicativeswarmer cell with pili and a replicative stalked cell during growth [9]; in addition, itundergoes morphological changes when it is grown for extended periods in a complexmedium [21].An examination of the different growth stages of strain B1 in a batch culture reveals apopulation which is characterized by a predominant fraction of cells containing at leasttwo fully replicated chromosomes. Only two stages seem to constitute an exception.This is the late stationary phase (165 h), in which a significant number of the cellscontain only a single chromosome and a very short phase, in which maximum DNAsynthesis and cell division occurred, visible clearly as altered population distributions(compare Fig. 3, 16.5–26 h). This indicates a DNA replication and division patternwhich is very different from fast growing GRAM-negative bacterial populations [23].The well-known replication behaviour of fast-growing strains shows uncoupled DNAsynthesis during the exponential growth phase, which results in a large variety ofdissimilar chromosome contents. Strain B1, however, shows mainly cells with one,two or four chromosomes. This replication behaviour is more similar to slow-growingbacterial cells or bacteria which grow under starvation conditions [23]. In addition,strain B1 shows a few cells with three chromosomes, mainly in the phase of maximumgrowth, especially between 16 and 26 h (compare Fig. 3). This indicates asymmetricnucleoid segregation, which may result in a progeny with an unequal number ofchromosomes. Such segregation and division behaviour has also been suggested forMethanococcus jannaschii, a methanogenic archeon [24].Comparing the cell morphology with the number of chromosomes in the cells led us toconclude that asynchronous cell division occurred. Consolidating all the data, one canconstruct a cell cycle during the growth of strain B1 in batch cultures (Fig. 5 andcompare Fig. 1). The cycle occurs repeatedly over the span of a batch culture. Whenthe cycle starts, the predominant cells in the culture are small and rod-shaped.


Subsequently, these cells increase in volume to approximately 2 to 3 times the volumeof the small ones. This is the phase in which the cells show a bright and intenseDAPI signal, indicating that they contain high amounts of DNA. The large rodsgenerate one of two different cell types: the first consists of two small rods, the secondof a rod and a spherical cell. The development of the spherical cell begins as a vesiclewithout DNA. Later on in the cell cycle, it has a high DNA fluorescence intensity. Thespherical cells are fixed to the primary cell by a lipophilic thread, which becomesvisible when stained with Nile red but contains no DNA. Later on, the thread breaks,and the spherical cell is released. These cells show different fluorescence intensitiesand, we suggest, are the origin of a new (small) rod. An additional morphological type,large spherical cells with fimbriae, appears in old cultures (≥ 7 days). These cells thatare visible with Nile red staining, develop two clearly demarcated fluorescent areaswhen stained with DAPI. Since these large spheres burst easily, one may speculate thatthey are the origin of new (small) rods.

Fig. 5. Schematic illustration of the hypothetical cell cycle of strain B1The fleshes illustrate possible ways of cell development, which can proceed in parallel.In the upper part of the scheme, the development of small and big rods is demon-strated. In the middle part, the development of the spherical cell begins as a vesicle(sketched as a rod with a spherical appendage). The spherical cell is fixed to theprimary cell by a thread (sketched as a line). Later on, the thread breaks and thespherical cell is released. An additional morphological type, large spherical cells withfimbriae appear (see lower part right of the scheme).

Several previously described appendaged bacteria are known to have an asymmetriccell cycle. The mechanism of a comparable cycle was analyzed in the model organismCaulobacter crescentus [10]. However, up to now there have been no published reportsof an asymmetric cell cycle within the family of Flavobacteriaceae, to which the strainB1 belongs.The model of the cell cycle presented in this paper is only a first step in analyzing thegrowth and reproduction mechanism of strain B1. Further studies, especially regardingthe molecular mechanism of the reproductive process, are needed.

Acknowledgement

The authors wish to acknowledge the skilful assistance of Christine SÜRING in the laboratory work.


Received 16 August 2001Received in revised form 10 September 2001Accepted 12 September 2001

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Book Review

COPELAND, R. A.

EnzymesA Practical Introduction to Structure, Mechanism, and Data Analysis

New York, Chichester, Weinheim, Brisbane, Singapore, Toronto: John Wiley & Sons, Inc., Publica-tion, 2000397 pages, 172 figures, 34 tables, 263 references; hardcover; £ 64.50ISBN 0-471-35929-7

Progress in biochemical engineering and molecular biology is developing rapidly. Enzymes becomemore significant in this field and remain the most common targets for therapeutic purposes in thepharmaceutical industry. The author's main intention is to give an up-to-date introduction thatsupports the understanding of both theoretical and practical aspects of enzymology. The book isdirected to graduate and medical students, as well as research scientists and technicians who areactively involved in enzyme studies.Compared to the first edition, a new chapter on protein-ligand binding equilibria has been added. Thechapters on chemical mechanisms in enzyme catalysis and on experimental measures of enzymeactivity have been expanded. The subjects of enzyme inhibitors and multiple substrate reactions havebeen refined. The second edition was completed in such a way that the introductory nature of thebook, which is subdivided into twelve sections, is maintained.The chapters are– A Brief History of Enzymology– Chemical Bonds and Reactions in Biochemistry– Structural Components of Enzymes


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