Cell Reproduction and Morphological Changes in Mycoplasma … · The cell reproduction of Mycoplasma capricolum was studied. The velocity of DNA replication fork progres-sion was

JOURNAL OF BACTERIOLOGY,0021-9193/98/$04.0010

Jan. 1998, p. 256–264 Vol. 180, No. 2

Copyright © 1998, American Society for Microbiology

Cell Reproduction and Morphological Changes inMycoplasma capricolum

SHINTARO SETO AND MAKOTO MIYATA*

Department of Biology, Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka 558, Japan

Received 7 July 1997/Accepted 13 November 1997

The cell reproduction of Mycoplasma capricolum was studied. The velocity of DNA replication fork progres-sion was about 6 kb/min, which is 10 times slower than that of Escherichia coli. The time required for one roundof DNA replication accorded with the doubling time. The origin/terminus ratio was 2.0. M. capricolum cellmorphology was classified into two types, rod and branched. In the ordinary-growth phase, the rod cellsaccounted for about 90% of the total population, with branched cells comprising the remaining 10%. Theproportion of branched cells increased to 90% following inhibition of DNA replication by nucleoside starvation.An increase in the proportion of branched cells was induced by transfer of a temperature-sensitive mutantdeficient in DNA replication to the restrictive temperature. The rod cells had a regular structure, a fixed celllength, and constrictions in the center. The DNA contents of individual rod cells were distributed with astandard deviation of 0.40 of average. The branched cells had irregular structures and a wide distribution ofDNA contents. Counting of viable cells revealed that the cells ceased division upon cell type conversion;however, branched cells maintained a reproductive capacity. A model for the reproduction process is proposed.

Mycoplasmas are parasitic bacteria that have extremely lowG1C contents and small genomes (9). Their morphology isirregular because of the lack of a peptidoglycan layer.

In Escherichia coli, initiation of chromosomal DNA replica-tion occurs once during the cell’s replicative cycle, and thenucleoids partition before cell division (13). The chromosomalreplication of E. coli initiates in a small region and proceeds inboth directions. It is mainly controlled by the timing and fre-quency of initiation, while the velocity of replication is con-stant.

In mycoplasmas, chromosome replication also starts at afixed site, followed by bidirectional progression (19–21, 25, 40).As in many eubacteria (36), the dnaA gene is expressed andplays important roles in the initiation of replication (35). Theseobservations suggest that the outline of chromosome replica-tion of mycoplasmas is similar to that of E. coli. However, theprocess of mycoplasma cell reproduction has not been clari-fied. Moreover, the cell division cycle of E. coli cannot besimply applied to mycoplasmas because of their irregular cellmorphology (4). A model has been suggested for the cell cycleof Mycoplasma mycoides (6, 30, 31), which is closely related toMycoplasma capricolum (39). According to this model, an el-ementary rounded body grows into a filamentous form andthen new elementary rounded bodies are developed within thisfilament and released, but this model has not been adequatelysubstantiated.

In this study, we analyzed the process of DNA replication,cell morphology, and viability under various conditions of M.capricolum and proposed a model of cellular reproduction forthis bacterium.

MATERIALS AND METHODS

Cultivation. M. capricolum ATCC 27343 was grown at 37°C unless otherwisespecified. Modified Edward medium (MEM) (28) was used with some modifi-cations, i.e., 5% horse serum was replaced by 2 mg of bovine serum albumin/ml,

20 mg of cholesterol/ml, 10 mg of palmitic acid/ml, and 12 mg of oleic acid/ml,according to the synthetic medium recipe (29). For supplementation with nucleo-sides, 40 mg (each) of adenosine, guanosine, and uridine/ml and 20 mg of thy-midine/ml were added to the medium. Frozen cultures were inoculated into themedium and grown overnight to reach an optical density at 600 nm (OD600)around 0.05. The cultures were diluted as the OD600 became 1024 and were usedfor assays after several hours.

Titration of total DNA. DNA content in cultures was assayed by Southernhybridization (7). Cells were lysed by mixing the growing culture with a solventcomposed of 50% phenol, 48% chloroform, and 2% isoamyl alcohol. To nor-malize the yield of DNA extraction, 1-ml aliquots of late-growth-phase culturegrown in the presence of 14 mM [14C]thymine (2.1 TBq/mol) were added to eachcell sample just before cell lysis. DNA was isolated by the phenol extractionmethod (33), and the yield was determined from the radioactivity of 14C in thetrichloroacetic acid-insoluble fraction. A series of DNA dilutions were heattreated, dot blotted on uncharged nylon sheets, and subjected to hybridizationanalysis. The chromosomal DNA prepared from a late-growth-phase culture wasused as the template for synthesis of probes. The sheets were exposed to a Fujiimaging plate, and the radioactivity was measured with a Bio Image AnalyzerBAS 1000. The radioactivity of [14C]thymine incorporated into the chromosomalDNA was much less than that of 32P-labeled probe hybridized to the blottedDNA. Standard radioactivity was determined by using a dilution set of thechromosomal DNA.

Titration of protein contents. Cells were collected by centrifugation at15,000 3 g at 4°C for 5 min and were washed once with solution A, consisting of20 mM Tris-HCl (pH 7.6), 0.25 M NaCl, and 10 mM EDTA. Washed cells wereresuspended in solution A and lysed by addition of 0.1% sodium dodecyl sulfate.The cell lysate was diluted to the appropriate concentration, and the total proteinwas titrated by the Bradford method.

Radiolabeling of DNA and analysis of replication intermediates. [32P]dAMPwas prepared from [a-32P]dATP as described previously (21). Radiolabeling ofmycoplasmal DNA was carried out by addition of 92.5 KBq of [32P]dAMP per ml(16 nM) to each culture. Two minutes later, 1 mM cold dAMP was added to eachculture. For analysis on alkaline agarose gels, DNA synthesis was stopped bymixing aliquots of cultures with a solvent composed of phenol, chloroform, andisoamyl alcohol. The chromosomal DNA was prepared by the phenol methodand subjected to 1% alkaline agarose gel electrophoresis (33). The fractionatedDNA was transferred onto charged nylon sheets, and each sheet, with thehalf-dried gel attached, was exposed to the imaging plate, followed by analysiswith the image analyzer. For analysis by field inversion gel electrophoresis(FIGE), DNA synthesis was stopped by mixing aliquots of cultures with an equalvolume of fixing solution composed of 75% ethanol, 2% phenol, 21 mM sodiumacetate (pH 5.3), and 2 mM EDTA (12). The chromosomal DNA was isolated bythe agarose block method as previously described (22, 26). The following pro-cedures were performed as described previously (21).

Measurement of origin/terminus ratio. Cells were lysed by mixing the cultureswith a solvent composed of phenol, chloroform, and isoamyl alcohol. DNA wasisolated by the phenol method. Dilution sets of the chromosomal DNA were heattreated, dot blotted on uncharged nylon sheets, and subjected to hybridizationanalysis (7). The plasmid pUNH119 was used as the standard for the origin

* Corresponding author. Mailing address: Department of Biology,Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka 558,Japan. Phone: 81 (6) 605 3157. Fax: 81 (6) 605 2522. E-mail: [email protected].

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titration. This plasmid harbors a 1,949-bp fragment extending in the origin regionfrom nucleotide 2691 to 4639 (numbered according to previous reports [19, 20]).The plasmid used as the standard for terminus titration was clone 4 of the gyrasegene reported previously (34). These plasmids were digested by single-cutterendonucleases, diluted appropriately, heat treated, and dot blotted onto thesheets. Radiolabeled probes were made by using DNA fragments of about 500 bpcomplementary to the insertion sequences of the standard plasmids. The amountof plasmid on each sheet was normalized by hybridization using a probe com-plementary to the ampicillin resistance gene, which is carried by both of thestandard plasmids. Hybridization was performed sequentially with the samesheets. The results did not depend on the probing order.

Microscopic observation. M. capricolum cells were collected by centrifugationat 10,000 3 g at 4°C for 3 min, suspended in phosphate-buffered saline (PBS)containing 3% glutaraldehyde and 10 mM EDTA, and incubated for 30 min atroom temperature for fixation. The fixed cells were collected, washed with PBS,and resuspended in PBS. For light microscopic observation, cell suspensionswere dropped onto glass slides and covered with coverslips. For observation byfluorescence microscopy, an equal volume of 20-mg/ml 49,6-diamidino-2-phenyl-idole (DAPI) solution was mixed with the fixed cell suspension. Fluorescencemicroscopic images were photographed with Fuji super G 400 or TriX pan 400(Kodak) film, captured by using Quickscan 35 (Minolta), and analyzed withNIH-Image. The deviation in image intensity among films was confirmed to beless than 10% with the fluorescence intensity of calibration beads for a flowcytometer (Bio-Rad). For electron microscopic observation, fixed cells wereplaced on a 180-Å grid covered by a collodion membrane. Grids were allowed todry, negatively stained with 2% ammonium molybdate for 1.5 min, and observedwith a transmission electron microscope (8).

Examination of cell viability. The viability of individual cells was examined byusing thin-layer solid medium (37). The cultures were mixed with an equalvolume of fresh medium not supplemented with nucleotides containing 2%low-melting-temperature agarose at 37°C. Aliquots of 100 ml were spread onslide glasses. The cell mixtures on the slide glasses were incubated at 37°C in amoist chamber. For microscopic observation, cells were fixed with 100% ethanol,

followed by two washes with PBS. For fluorescence observation, cells werestained by addition of 10 ml of DAPI solution and were covered with a coverslip.Total CFUs in each culture were counted by inoculating cultures onto MEMplates as previously described (35).

RESULTS

DNA and protein content of culture. The coupling of DNAand protein syntheses was examined by monitoring the netcontents. The protein contents of cultures grown in MEMfollowed the OD600 through growth phase (Fig. 1C). However,the DNA content did not increase after the OD600 reached 0.1,while it increased in parallel with OD600 in the early-growthstage (Fig. 1A). We searched for factors capable of preventingthe reduction of DNA synthesis and found that the nucleosidemix used for a synthetic medium (29) was effective. The DNAcontent in the supplemented cultures increased almost in par-allel with OD600, even in the later-growth stage (Fig. 1B). Theprotein synthesis and increase in OD600 were not affected bythis supplementation (Fig. 1D). These results showed thatDNA replication is coupled with protein synthesis, if DNAreplication is not inhibited by nucleoside starvation.

Replication fork velocity. To determine the time for oneround of chromosome replication, we assayed the time re-quired for completion of replication of a pulse-labeled endo-nuclease fragment. We used [32P]dAMP, which can be incor-porated into the chromosome (21, 24). We tested whether

FIG. 1. DNA and protein contents of batch cultures. Mycoplasma cells were cultured without (A and C) or with (B and D) nucleoside supplementation. DNA (Aand B) and protein (C and D) contents in the cultures are shown by open circles. OD600 is shown by solid circles.

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[32P]dAMP taken up into a mycoplasma cell can be rapidlydiluted by cold dAMP by monitoring the polymerization pro-cess of Okazaki fragments (Fig. 2). Mycoplasma cultures werelabeled with 16 nM [32P]dAMP for 2 min; then 1 mM colddAMP was added, and DNA was isolated after various incu-bation periods and analyzed by denaturing gel electrophoresis.Okazaki fragments were widely distributed in size. The label-ing of nascent Okazaki fragments was stopped at 10 s after theaddition of cold dAMP, and the sizes of labeled small frag-ments started to shift. This result showed that [32P]dAMP wasavailable for the pulse-labeling of the chromosomal DNA.

Mycoplasma cultures were pulse-labeled with [32P]dAMPfor 2 min, and DNA was isolated after various incubationperiods, digested with BamHI, and subjected to FIGE. Theradioactivity of each fragment was detected by autoradiogra-phy and quantified (Fig. 3). The band intensities increased withthe incubation period and became saturated at times, whichdepended on the fragment size. We used the largest twoBamHI fragments, Bm1 and Bm2, because of their separationfrom other fragments in the gel. The band intensities of Bm1and Bm2 increased linearly with the incubation period andbecame saturated at 48.5 and 42.2 min, respectively. The forkvelocities were calculated to be 6.4 and 6.0 kb/min, respec-tively, from the results of Bm1 and Bm2, and the times re-quired for one round of chromosome replication were esti-mated to be 91 and 97 min, respectively (Table 1). These valuesdid not depend on the growth phase if the medium was sup-plemented with the nucleoside mix. The fork velocity in thenonsupplemented cultures was similar to that in the supple-mented cultures until the OD600 reached 0.1. However, amarked reduction was observed after the OD600 reached 0.1(data not shown).

Origin/terminus ratio. The ratio of replicating intermediatesin the chromosome molecules was monitored by titrating theorigin/terminus copy number ratio. The origin/terminus ratiowas estimated by the hybridization method. The origin regionhas been defined (19), and a DNA fragment expanding in thisregion was used for titrating the origin. The region, which isreplicated last, has not been identified. Therefore, we used aDNA region expanding on the gyrase gene which was reportedto be in the position opposite the origin on the chromosomemap (34). The origin/terminus ratio was estimated to be 2.0.

This value did not change through the growth phase, and it didnot depend on nucleoside supplementation.

Definition of cell types. Transmission electron microscopyrevealed that the morphology of M. capricolum cells was clas-sified into two types, i.e., rod and branched types (Fig. 4A andB, respectively). Most cells in the ordinary-growth phase had acomparatively regular rod-like structure. A small fraction ofcells had an irregular branched structure, with tubes radiatingout from the center of the cell body. These cell types could alsobe distinguished by light microscopy (Fig. 4C and D).

Cell type conversion. The occurrence of branched cells wasexamined by phase-contrast microscopy (Fig. 5A). In culturessupplemented with nucleosides, the proportion of branchedcells did not change significantly until the OD600 reached 0.17,after which it started to increase, and the branched cells finallyaccounted for 37% of the total cell number. In cultures withoutnucleoside supplementation, the proportion increased in the

FIG. 2. Polymerization of labeled Okazaki fragments. Mycoplasma cells werelabeled with [32P]dAMP. After 2 min, labeled dAMP was diluted with 1 mM colddAMP. DNA was isolated at 0, 10, 20, 30, 60, 90, and 120 s after dilution and wasanalyzed by alkaline agarose gel electrophoresis in lanes 1 through 7, respec-tively. Fragment sizes are indicated on the left.

FIG. 3. Completion of replication of the chromosomal fragments. Myco-plasma cells were labeled with [32P]dAMP for 2 min, and 1 mM cold dAMP wasadded. The chromosomal DNA isolated at each time point was digested withBamHI and subjected to FIGE followed by autoradiography. (A) The bandintensities of Bm1 and Bm2 fragments are shown as the saturation extent by solidand open circles, respectively. The saturation extents until 40 min were fittedwith the dashed line. (B) Autoradiogram of Bm1 and Bm2 fragments at 0, 5, 10,15, 20, 25, 30, 40, 50, 60, and 70 min (lanes 1 through 11, respectively).

TABLE 1. Replication fork velocity

Fragment Time forsaturationa (min)

Sizeb

(kb)Fork velocity

(kb/min)Time for oneroundc (min)

Bm1 48.5 309.5 6.38 90.5Bm2 42.2 252 5.97 96.8

a Estimated from Fig. 3.b Estimated by FIGE (22).c Time required for one round of chromosome replication was calculated as

the reactions proceed in both directions with equivalent velocity.

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earlier stage. The proportion started to increase after theOD600 reached 0.10, and the branched cells finally accountedfor 90% of the total cell number. The starting point of theincrease in the proportion of branched cells corresponded tothe cessation of DNA synthesis due to nucleoside starvation,with subsequent protein synthesis (Fig. 1). These results sug-gest that nucleoside starvation induced cell type conversion.

Cell type conversion in a temperature-sensitive mutant de-ficient in DNA replication. We examined the cell type conver-sion of a temperature-sensitive mutant strain in which theelongation reaction of DNA replication is specifically inhibitedat the restrictive temperature (21). Mutant and wild-type cellswere grown at 33°C until the OD600 reached 0.05, and then theincubation temperature was shifted to the nonpermissive tem-

FIG. 4. Images of M. capricolum cells. The left and right columns show images of cells at OD600s of 0.05 and 0.4, respectively. (A and B) Electron microscopy. (Cand D) Phase-contrast microscopy. (E and F) DAPI-stained cell images. Bars below panels B and F represent 2 and 5 mm, respectively. Arrowheads point to the positionof constriction.


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perature of 41°C (Fig. 5B). The OD600 of mutant cells contin-ued to increase for more than 180 min after the temperatureshift. The proportion of branched cells started to increase justafter the temperature shift, so that they accounted for 54% ofthe total cell population at 120 min after the temperature shift,while it did not increase significantly in wild-type cultures.

Characterization of rod and branched cells. The rod typecells in cultures supplemented with the nucleosides at anOD600 of 0.05 were analyzed by electron microscopy. Theaverage length of rod cells was 0.941 mm, with a standarddeviation (SD) of 0.231 mm, and constriction sites were foundaround the middle in 21.8% (113 of 519) of the rod cells (Fig.4). The average ratio of the distance from the constriction siteto the furthest cell pole, relative to the cell length, was 0.576,with an SD of 0.051. The cells stained with DAPI were ana-lyzed by fluorescence microscopy, and the DNA contents inindividual cells were estimated from fluorescence intensity

(Fig. 6). The average DNA content at an OD600 of 0.05 wasnormalized to 1 U. The SD of DNA content in rod cells was0.397 U. These results were not considerably different in theother growth stages or in cultures without nucleoside supple-mentation (data not shown).

The branched cells had irregular cell shapes (Fig. 4) and awider distribution of DNA content than rod cells.

Viability of branched cells. To determine the correlationbetween cell division and cell type, CFU was monitored withrespect to culture OD600 (Fig. 7). The rate of increase throughtime paralleled that of the OD600 until the latter reached 0.1.Thereafter, the relative rate of increase diverged and the via-bility as measured by CFUs was no longer reflected by theOD600 unless the medium was supplemented with nucleosides.The point at which the rates of increase in CFUs and theOD600 diverged corresponded to the starting point of the in-crease in the population of branched cells. This uncoupling was

FIG. 5. Cell type conversion. (A) Proportions of branched cells for cultures without and with nucleoside supplementation are shown by solid and open circles,respectively. OD600s for cultures without and with nucleoside supplementation are shown by solid and open squares, respectively. (B) Proportions of branched cellsin temperature-sensitive-mutant and wild-type cultures are shown by open and solid circles, respectively. The shift to the restrictive temperature was performed at timezero. OD600s for temperature-sensitive mutant and wild-type cultures are shown by open and solid squares, respectively.

FIG. 6. DNA contents of individual cells. DNA contents of rod cells at an OD600 of 0.05 (A) and of branched cells at an OD600 of 0.4 (B) are shown. The amountof individual fluorescence was measured as DNA content in a cell. The average of the values shown in panel A was normalized to 1 U.


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not observed in the nucleoside-supplemented cultures. Theseresults indicate that cells ceased division upon cell type con-version. To examine the viability of branched cells, cultures atan OD600 of 0.4 were inoculated onto a thin solid medium onglass slides and were incubated at 37°C (Fig. 8). Light andfluorescence microscopic observation at 3 and 6 h after theinoculation revealed that all cells formed microcolonies, indi-cating the ability of branched cells to reproduce.

DISCUSSION

We showed that M. capricolum cells were starved of nucleo-sides in MEM, which is widely used for cultivation of myco-plasmas (28). MEM contains animal DNA, which is thought tobe used as the source of nucleotides essential for growth (18).We examined the effects of DNA in the medium on the growthand incorporation of dAMP into the chromosomal DNA butdid not find any effects (data not shown). Further studies arenecessary to determine if this starvation is specific to M. capri-colum and if there is a need for the specific DNA supplemen-tation.

The DNA content of M. capricolum in cultures increasedwith the protein content without nucleoside starvation, and theDNA contents in individual rod cells did not change through-out the growth phase. These observations suggest that theinitiation frequency of DNA replication agrees with the rate ofincrease of protein content, although it is unclear if the fre-quency can be modified according to the growth conditions, ashas been shown for E. coli (13).

The polymerization process of Okazaki fragments in myco-plasma cells was examined in this study. We found a signal thatmigrated rapidly in the denaturing gel, the intensity of whichincreased with the chase time. It is unlikely that this signal wascaused by host exonuclease activity present at the time of lysis,because the signal intensity did not depend on the time fromcell lysis to the first centrifugation (data not shown). The signalwas presumably derived from pseudo-Okazaki fragmentscaused by excision of uracil incorporated into DNA (17). A

homolog of uracil N-glycosylase has also been found in genomeanalyses of mycoplasmas (10, 14).

Monitoring of the progression of DNA replication in myco-plasmas has been reported previously (19–21, 25). However,the replication fork velocity has not been studied, because ofthe difficulty of achieving a synchronous reproductive cycle inmycoplasmas. We used pulse-labeling coupled with FIGE andexamined the fork velocity without synchronization (Fig. 3).The absolute velocity of the replication fork in M. capricolumwas about 10 times slower than that in E. coli (13) (Table 1).This slow progression of DNA replication may be related tothe slow growth of mycoplasmas. An absence of replicationmachinery has not been observed in the genetic components ofmycoplasmas (10, 14). The time for one round of chromosomereplication was estimated to be around 94 min. This valueroughly corresponded to the doubling time, suggesting thatDNA replication occurs in an interval between two cell divi-sions. This assumption was supported by the origin/terminusratio. The value of 2.0 can be explained by the assumption thatthe replication procedure takes most of the time of one divi-sion interval, and consequently most DNA molecules in theculture are replicating intermediates.

M. capricolum cells were morphologically classified into twotypes, i.e., rod and branched (Fig. 4). The rod cells are assumedto be the reproductive form under normal-growth conditionsbecause (i) the majority of cells in normal-growth cultures wererod type, (ii) constrictions were found in 22% of the rod cells,(iii) the rod cells had cell length and DNA content distribu-tions suitable for reproduction by division, (iv) the DNA con-tents of rod cells did not change during the normal-growthphase, which agrees with the constant increase in the DNAcontents of the cultures, and (v) the branched cells cannot bea stage of the ordinary cell division cycle, because the DNAcontents of branched cells were not significantly larger thanthose of rod cells (Fig. 6). If the rod cells became branchedbefore division, the DNA contents of branched cells should besignificantly larger than that of the rod cells.

Cell type conversion was induced by starvation of nucleo-sides and by transfer of a temperature-sensitive DNA replica-tion mutant to the nonpermissive temperature. These resultssuggest that the inhibition of DNA replication with subsequentprotein synthesis induced the cell type conversion. In the con-version, cells did not divide; i.e., the increase in CFUs wasextensively reduced at the beginning of the conversion, and noanucleate minicells were observed in the microscopic field(data not shown). It is likely that mycoplasma cells whosedivision system is ready convert to the branched type whendivision is inhibited by the nonreplicated chromosomal DNA.

In ordinary growing cultures, a small proportion of cells wasfound as the branched type, and the proportion did not dependon the growth stage. Presumably, a small fraction of rod cellsoccasionally converts to the branched type and then returns tothe rod type. The reproductive capability of branched-typecells was confirmed by microplate observation (Fig. 8).

We propose a model for the reproductive cycle of M. capri-colum (Fig. 9). In ordinary growth, the rod cells divide into twonascent cells. DNA replication occurs in a cell division interval.On the other hand, a cell whose DNA replication is inhibitedby nucleoside starvation cannot undergo cell division, and itmakes new projections due to the excess potential of cell divi-sion. In ordinary growth, a small fraction of rod cells whichhave some delay in completion of chromosome replication alsoconvert to the branched type.

M. mycoides is closely related to M. capricolum (39), and theappearance of this species is also similar to that of M. capri-colum (30). Buxton and Fraser (6) reported that M. mycoides

FIG. 7. CFUs in cell type conversion. Solid and open circles, CFU numbersfor cultures without and with nucleoside supplementation, respectively. Solid andopen squares, OD600s of cultures without and with supplementation, respec-tively.


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develops from an elementary rounded body into a branchedand filamentous form, and then new rounded elementary bod-ies develop within these filaments and are released when theyare mature. Our conclusion is not in agreement with this hy-

pothesis. Models of cell division into two equivalent cells werealso proposed for Mycoplasma gallisepticum (23), Mycoplasmamobile (32), and Spiroplasma citri (11), which are also relatedto M. capricolum (39). The growth phase dependency of the

FIG. 8. Growth of branched cells in thin-layer solid medium. Cultures at an OD600 of 0.4 were mixed with low-melt agarose, spread on glass slides, and analyzedafter 0 (A), 3 (B), and 6 (C) h of incubation at 37°C. In the left and right columns are images of DAPI-stained cells observed by phase-contrast and fluorescencemicroscopy, respectively. Bar, 5 mm.


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morphological change of Mycoplasma pneumoniae is similar tothat in M. capricolum. Cells of M. pneumoniae are spherical inyoung cultures, become branched and filamentous with thegrowth phase, and then change to asymmetrical rounded formsin declining cultures (3, 16). The morphological change in M.pneumoniae was suggested to respond to the change in growthmedium components. The cell type conversion of M. pneu-moniae may be caused by a process similar to that in M.capricolum.

A morphological change induced by nucleoside starvation,whereby cells became filamentous with thymine starvation, wasalso reported for E. coli. This process was independent of theSOS response (15). However, no branching was seen in thiscase. Akerlund et al. (2) showed that 5% of E. coli cells formedbranches in nutrient-poor medium when chromosome replica-tion or nucleoid segregation was genetically disturbed. Thesephenomena may be related to branch formation in mycoplas-mas.

Since mycoplasmas lack the peptidoglycan layer, changes inthe cytoplasm and cell membrane can be directly reflected inthe appearance of the cells. Therefore, branch formation bymycoplasmas is probably coupled with abnormal assembly ofproteins that play roles in cell division or maintenance of cellshape. This is supported by the observation that cell type con-version required subsequent protein synthesis (data notshown). FtsZ protein is known to form a Z ring at the positionof septation prior to cell division and to play key roles in celldivision in E. coli and other walled bacteria (1). Homologs ofthe ftsZ gene have been identified in some mycoplasmas, in-cluding M. capricolum (5, 10, 14, 38). FtsZ may be related tothe branch formation of mycoplasmas. It has been reportedthat overexpression of FtsZ protein induces a branch at thestalk in Caulobacter crescentus (27).

ACKNOWLEDGMENTS

We thank R. D’Ari of Universite Paris for supplying the detailedprotocol of the slide culture for E. coli.

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Cell Reproduction and Morphological Changes in Mycoplasma … · The cell reproduction of Mycoplasma capricolum was studied. The velocity of DNA replication fork progres-sion was