JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Nov. 1999, p. 6607–6614 Vol. 181, No. 21

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

On the Origin of Branches in Escherichia coliBJORN GULLBRAND,1 THOMAS ÅKERLUND,2 AND KURT NORDSTROM1*

Department of Cell and Molecular Biology, Biomedical Center, Uppsala University,S-751 24, Uppsala,1 and Department of Bacteriology, Swedish Institute

for Infectious Disease Control, S-171 82, Solna,2 Sweden

Received 14 April 1999/Accepted 26 July 1999

Some Escherichia coli strains with impaired cell division form branched cells at high frequencies duringcertain growth conditions. Here, we show that neither FtsI nor FtsZ activity is required for the developmentof branches. Buds did not form at specific positions along the cell surface during high-branching conditions.Antibiotics affecting cell wall synthesis had a positive effect on branch formation in the case of ampicillin,cephalexin, and penicillin G, whereas mecillinam and D-cycloserine had no substantial effect. Altering the cellmorphology by nutritional shifts showed that changes in morphology preceded branching, indicating that thecell’s physiological state rather than specific medium components induced branching. Finally, there was noincreased probability for bud formation in the daughters of a cell with a bud or branch, showing that budformation is a random event. We suggest that branch formation is caused by abnormalities in cell wallelongation rather than by aberrant cell division events.

Some strains of Escherichia coli form branched cells duringcertain growth conditions. The mechanism(s) for branch for-mation is not known, although an understanding of this phe-nomenon might help in understanding how the rod shape ofE. coli cells is maintained. Branching occurs at high frequen-cies in one type of the so-called intR1 strains (6), in whichchromosome replication starts from an integrated R1 plasmid(8). Branched cells have also been observed in min mutants (6)during blockage of chromosome replication by antibiotics (48)and in thymine-requiring strains starved for thymine (48, 54,55). The frequency of branching has also been shown to be de-pendent on the type of medium (6).

Branching strains often display a disturbed nucleoid distri-bution (6, 8, 26, 36) and produce filaments and/or minicells (4,10, 22). One report has indicated that branches develop fromcell poles formed by asymmetric cell division events (53). Inthis report, we have investigated whether branches result fromaberrant cell division events or, alternatively, whether branchesare formed as outgrowths from small asymmetries arising atlow frequencies during cell wall elongation.

MATERIALS AND METHODS

Bacterial strains, growth conditions, and shift of medium. The E. coli strainsused in this study are listed in Table 1. The intR1 strain EC::71CC is a derivativeof EC1005 in which part of oriC has been deleted and replaced by the R1replicon, which controls replication in this strain (8). Strain EC1005ptac-ftsZ wasobtained by transduction of EC1005 with a P1 lysate of VIP205 (19) and selec-tion for kanamycin resistance on Luria agar (LA) plates containing 20 mM IPTG(isopropyl-b-D-thiogalactopyranoside). Strains EC1005ftsZ84 and MG1655ftsZ84were obtained by transduction of EC1005 and MG1655, respectively, with a P1lysate of VIP183 and selection for tetracycline resistance on LA plates.VIP183 was obtained from Miguel Vicente and carries the ftsZ84(Ts) allele(31) and leu::Tn10. Strains were grown in Luria broth medium containing 0.2%glucose (LBglu) or in M9 minimal medium (43) supplemented with 0.2% glucose(M9glu) or 0.2% sodium acetate and 0.5% Casamino Acids (Difco) (M9caace).For EC1005 and for strains derived from EC1005, M9glu was supplemented withmethionine (50 mg/ml). Drugs were added to the following concentrations: am-picillin, 20 mg/ml; cephalexin, 10 mg/ml; kanamycin, 20 mg/ml (M9caace) or 50mg/ml (LA plates, for selection of P1 transductants); and tetracycline, 10 mg/ml.The cells were incubated in shaker baths as follows: EC1005, MG1655, EC1005

ptac-ftsZ, and EC1005DminB at 37°C; EC::71CC and MG::71CC at 34°C; andEC1005ftsZ84 and MG1655ftsZ84 at 30°C (permissive temperature) or 42°C(nonpermissive temperature).

For the shift of growth medium, 1 ml of culture in exponential growth phase(optical density of ,0.3, measured at 550 nm) was centrifuged at about 4,000 3g for 7 min, the supernatant was removed by aspiration, and the cells wereresuspended in 10 ml of prewarmed growth medium.

Microscopic studies. To measure cell length and the frequency of branchedcells in exponentially growing cultures, cells were first fixed in 70% ethanol. Cellswere then resuspended in 0.9% NaCl, and 10 ml was put on microscope slidescovered with thin 1% agar layers. For visualization of nucleoids, 0.5 mg of DAPI(49,6-diamidino-2-phenylindole) per ml was included in the agar. To monitorchanges in the frequency of branched cells after the shift of growth medium,10-ml samples were put directly onto microscope slides with agar layers, and thecells were studied immediately. The microculture technique (5) was used tomonitor the growth of individual cells and to study microcolonies: 5 to 20 ml ofcell culture was added to microscope slides covered with thin 1% agar layerscontaining the same growth medium. The slides were incubated at 37°C or on athermostatically controlled heating plate connected to the microscope, where thetemperature was monitored by using a small probe inserted directly into the agar.

Immunofluorescence staining was performed essentially as described byHiraga et al. (22), except that we used 10-well multitest slides from ICN Bio-medicals, Inc. FtsZ-specific antisera was a gift from Joe Lutkenhaus, and fluo-rescein isothiocyanate-labelled goat anti-rabbit immunoglobulin G antibody waspurchased from Southern Biotechnology Associates, Inc.

Cells were analyzed with a Nikon Optiphot-2 microscope, and the images weredigitized by using a cooled charge-coupled device camera (Meridian) connectedto a computerized image analysis system (soft- and hardware were from Berg-strom Instrument AB). The digitized images were printed by using a SonyUP-860/CE videoprinter or were processed in Adobe Photoshop and submittedin digital form.

RESULTS

Bud formation in the wild-type and intR1 and minB mutantstrains. We first investigated whether branches develop at spe-cific positions along the cell surface, e.g., related to putativedivision sites (midcell) or cell poles. E. coli strains giving low(wild-type), moderate (minB), and high (intR1) branching fre-quencies were grown exponentially in LBglu and/or M9caace(Table 2 and Materials and Methods). The microculture tech-nique was used to monitor the growth of individual cells (5).

In LBglu, the frequency of branched wild-type (EC1005)cells was ,1% (Table 2). Four bud formation events wereobserved by monitoring the growth of microcolonies, and all ofthese buds were formed at cell poles (Fig. 1A). The cells thatformed buds were of wild-type length and continued to grownormally after bud formation. Interestingly, all four poles at

* Corresponding author. Mailing address: Department of Cell andMolecular Biology, Biomedical Center, Uppsala University, Box 596,S-751 24, Uppsala, Sweden. Phone: (46) 18-4714526. Fax: (46) 18-530396. E-mail: Kurt.Nordstrom@icm.uu.se.

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which bud formation was observed were present at the start ofthe microculture experiments, and the buds were formed be-tween one and three cell generations later, i.e., branching oc-curred at the old cell poles. Similarly, buds were located at orclose to cell poles in fixed cells of the intR1 strains EC::71CC(21 of 23) and MG::71CC (25 of 29).

In M9caace, the frequency of branching was higher than inLBglu (Table 2), and three polar and seven nonpolar branchformation events were observed in the wild type. The branchedcells were of wild-type length, and the polar buds appearedfrom cell poles present at the start of the microculture exper-iments after about one cell generation. Six of the seven non-polar buds were formed between the cell pole and the center ofthe cell (Fig. 1B), whereas one bud formed at midcell. In theintR1 strain (EC::71CC), most buds were localized along fila-ments (Fig. 2A, 0 min, arrows) and grew slowly, sometimesresulting in a kink of the cell (Fig. 2A, 210 min, left daughter

cell; and Fig. 2B, 210 min). Cell division sometimes occurredclose to buds, resulting in dumbbell-shaped cell poles (Fig. 2A,160 min, right daughter cell), and new buds developed at var-ious positions along the cell surface (Fig. 2A, 210 min, arrows;and Fig. 2B, 95 min, arrow). Some newly formed cells did notgrow, indicating that chromosome-less cells were formed (Fig.2B, 100 min). Strikingly, buds appeared to rotate around thecell cylinder (cf. Fig. 2B, 60 and 95 min). The slow develop-ment and growth of the buds (Fig. 3A, arrow; Fig. 3C; and Fig.3D, 200 min, arrow), as well as the formation of dumbbell-shaped cells (Fig. 3B, 40 min, left daughter cell), was alsoobserved in a minB mutant.

In conclusion, the positions at which buds appeared weremedium dependent. In LBglu, branching preferentially initi-ated from the old cell poles, whereas in M9caace, a majority ofbranches appeared along the length axis of the cells. The budsdeveloped and grew slowly, and cell division sometimes oc-

TABLE 1. E. coli strains used in this study

Strain Parent Relevant features Source orreference(s)

EC1005 metB1 nalA relA1 spoT1 lr F2 20EC1005DminB EC1005 EC1005 DminB Kmr 5EC::71CCb EC1005 EC1005 DoriC::pOU71 Apr 8EC1005ptac-ftsZ EC1005 EC1005 ftsA::kan T4 lacIq

ptac::ftsZaThis study; 19

EC1005ftsZ84 EC1005 EC1005 ftsZ84(Ts) leu::Tn10 This studyMG1655 lr F2 7, 27MG1655ftsZ84 MG1655 MG1655 ftsZ84(Ts) leu::Tn10 This studyMG::71CCb MG1655 MG1655 DoriC::pOU71 Apr 6

a The insert between ftsA and ftsZ was introduced into EC1005 with a P1 lysateof VIP205 (17) and was selected for by kanamycin.

b The initiation frequency of chromosome replication in EC::71CC andMG::71CC is regulated by temperature, such that growth at 35°C or higherresults in overreplication (see Bernander et al. [8]).

TABLE 2. Effects on branch formation of cephalexin or growthat the nonpermissive temperature of strains

carrying the ftsZ84(Ts) allele

Strain Medium,temp (°C)

Cephalexin(10 mg/ml)

%Frequency

of branchedcellsa

Avg celllength(mm)b

No. ofbranches/

mm

EC1005 LBglu, 37 2 0.1 4.3 0.21 0.2 15 0.2

M9caace, 37 2 3.3 3.1 111 23 13 18

MG1655 LBglu, 37 2 0.4 4.6 0.91 1.3 16 0.9

M9caace, 37 2 2.8 3.0 9.41 26 10 25

EC::71CC M9caace, 34 2 20 6.0 431 53 29 47

EC1005ftsZ84 M9caace, 30 2 1.3 (1.5)c 2.5 (2.2) 5.0 (6.9)1 15 (6.2) 11 (9.8) 14 (6.3)

M9caace, 42 2 4.7 (0.99) 9.6 (3.0) 4.9 (3.3)1 9.0 (5.3) 13 (9.5) 6.8 (5.5)

MG1655ftsZ84 M9caace, 30 2 ,0.1 (0.2) 2.3 (2.1) ,0.5 (1.0)1 4.3 (3.0) 10 (8.4) 4.2 (3.5)

M9caace, 42 2 1.1 (0.1) 8.7 (3.1) 2 (0.32)1 1.8 (1.4) 11 (8.0) 1 (1.7)

a Values were obtained by counting more than 2,000 cells from each culturegrown in LBglu and more than 500 cells for cultures grown in M9caace.

b The average cell lengths were obtained by measuring 50 cells of EC1005 andMG1655 grown in M9caace and 100 cells from each of the other cultures.

c Numbers in parentheses are values obtained with the corresponding wild-type under the same conditions. Note that the effect of cephalexin on branchingfor EC1005 at 30 and 42°C was much smaller than what was typically observedat 37°C.

FIG. 1. Formation of branched cells in EC1005. Cells were grown exponen-tially in LBglu medium (A) or M9caace (B) at 37°C, whereupon 10 ml of theculture was transferred to a flat agar surface containing the same growth me-dium. Bar, 2 mm.

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curred close to buds. However, no clear correlation betweenthe positioning of nonpolar buds and the putative cell divisionsites (at midcell) was observed.

Bud formation does not require FtsI. Septum synthesis inE. coli cells requires the action of the ftsI gene product, FtsI,also known as penicillin-binding protein 3 (PBP3) (11, 45, 47).FtsI is a cytoplasmic membrane protein which is specificallyinvolved in peptidoglycan synthesis of the septum. Cephalexin

inhibits FtsI activity, which results in elongated cells and even-tual cell death (44, 46). If FtsI activity is required throughoutthe early parts of bud formation, the addition of cephalexinshould stop the appearance of new buds and the frequency ofbranched cells should remain constant or decrease. To test thisidea, exponentially growing cultures were divided into twoparts, and cephalexin was added to one part to a final concen-tration of 10 mg/ml. After a fourfold increase in optical density

FIG. 2. Growth and division pattern of branched cells in the intR1 strain EC::71CC. Cells were grown exponentially in M9caace medium at 34°C, whereupon 5 to10 ml of the culture was transferred to a flat agar surface containing the same medium, and the growth of two cells (A and B) was monitored. Small buds on the cellsurface are indicated (arrows).

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(untreated culture), the cells were fixed in 70% ethanol and thefrequency of branched cells was measured (Table 2).

For EC1005 (wild-type) grown in LBglu, the frequency ofbranched cells was low both in cultures grown with and withoutcephalexin. In a screen for branched cells in a cephalexin-treated culture, all six buds were located at or close to the polesof the filamented cells. For MG1655 the frequency of branchedcells increased 3.4- and 4-fold (two experiments) after theaddition of cephalexin (Table 2). Again, most (18 of 21) of thebuds were located at or close to cell poles. The number of budsper millimeter (cell length) was similar before and after theaddition of cephalexin, suggesting that new buds appearedduring filamentation of the cells.

In M9caace, the frequency of branched cells in the wild-typestrains increased from 2 to 3% to 20 to 30% after the additionof cephalexin (Table 2). In a microculture experiment, a ma-jority (13 of 14) of the buds developed at nonpolar positions (incontrast to that in LBglu cells [see above]). In the intR1 strain(EC::71CC), the frequency of branched cells increased from 20to 53% after the addition of cephalexin (measured after twomass doublings for the untreated culture). A significant pro-portion of the branched cells had more than one branch, andthe average number of branches per cell was ca. 1.4. For thewild-type strains, the number of branches per millimeter of celllength had increased after two mass doublings in the presenceof cephalexin, whereas the total increase in cell length re-mained fairly constant (Table 2).

The results suggest that FtsI activity is not required forbranch formation. Instead, blockage of FtsI by cephalexin in-creased the number of buds and branches per millimeter of celllength. This observation argues against the idea that the celldivision process is involved in branch formation.

Bud formation is not dependent on FtsZ. One of the earlieststeps in cell division in E. coli is the assembly of the FtsZ

protein to a ring around the cytoplasm at the future divisionsite (9, 12, 30, 35). FtsZ is essential for cell division, and FtsZring formation is a prerequisite for the localization of manyother cell division proteins to the cell division site, includingFtsI (1, 3, 51). Severe over- or underexpression of FtsZ inE. coli inhibits cell division (14, 52).

To test whether there is any correlation between FtsZ andbud formation, FtsZ was visualized by immunofluorescencemicroscopy, and the localization of FtsZ structures was com-pared to the localization of buds in strains EC1005 andMG1655. When grown exponentially in M9caace, ca. 40% ofthe cells in populations of EC1005 and 45% of the cells inpopulations of MG1655 had a distinct, central fluorescent bandor, in cells with a deep constriction at midcell, a dot. A few cellsshowed diffuse fluorescence at one or both poles. Buds did notspecifically colocalize with FtsZ structures. As with the cellpoles, however, buds sometimes contained diffuse fluores-cence, and it is possible that FtsZ rings or structures wereassembled and depolymerized before the bud became visible.We next added cephalexin to increase the number of branchesper unit cell length (above) and analyzed the distribution ofFtsZ rings by immunofluorescence microscopy. No structuresother than a central fluorescent band or, in some cells taken ata later time point, one band at each cell quarter position weredetected. Thus, we did not find any colocalization of FtsZstructures and buds. It should be noted, however, that any FtsZstructures involved in bud formation might be transient andtherefore difficult to detect.

To further investigate any correlation between FtsZ and budformation, we tested the capability of bud formation in cellswith low and high levels of FtsZ and in strains producing atemperature-sensitive FtsZ protein after shifting the cells tothe nonpermissive temperature.

FtsZ levels were altered by using a construct in which chro-

FIG. 3. Growth and division pattern of branched cells in the EC1005DminB strain. Cells were grown exponentially and treated as described in the legend of Fig.2, except that the temperature was 37°C. Four different cells (A to D) are shown. Small buds on the cell surface are indicated (arrows).

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mosomal ftsZ gene expression is under the control of the tacpromoter (19). This construct also contains four copies of astrong transcriptional terminator, uncoupling ftsZ from its nat-ural promoters, the lacIq gene and a kanamycin resistancegene. The construct was introduced into strain EC1005, result-ing in EC1005ptac-ftsZ, and IPTG was used to set the level offtsZ gene expression. Growth of EC1005ptac-ftsZ in M9caaceat 0 or 100 mM IPTG yielded populations with broad celllength distributions, whereas at 5 mM IPTG, the cell size dis-tribution was similar to that of EC1005. No effect of IPTG onthe cell size distribution of EC1005 was evident. As observedby immunofluorescence microscopy, the amount of FtsZ inEC1005ptac-ftsZ increased with the concentration of IPTG. At0 mM IPTG, most cells had one or no centrally localized FtsZband, and the fluorescence signals from these bands were sig-nificantly weaker than for those in cells grown at 5 mM IPTG.At 100 mM IPTG, most cells had a broad band of FtsZ at theircenter with a marked increase in fluorescence intensity, andmany cells had additional long regions with an equally highfluorescence intensity. At 5 mM IPTG, the frequency ofbranched cells was ca. 0.5%. At 0 and 100 mM IPTG, thefrequency was ca. 2 to 3%. Thus, there was no proportionalitybetween FtsZ levels and the frequency of branched cells.

Strains producing a temperature-sensitive FtsZ protein weremade by introducing the ftsZ84(Ts) allele (31) into EC1005and MG1655, yielding EC1005ftsZ84 and MG1655ftsZ84, re-spectively. The strains were grown continuously in M9caace at30°C (permissive temperature) and then shifted to 42°C (non-permissive temperature). Cells were fixed after two doublingsin optical density, and the frequency of branched cells and thenumber of branches per millimeter of cell length were mea-sured (Table 2). The results clearly showed that branches wereformed in both strains at both the permissive and nonpermis-sive temperatures. Buds were also formed at the nonpermissivetemperature in the presence of cephalexin. Shifting EC1005and MG1655 from 30 to 42°C indicated that the increasedtemperature had a slight negative effect on branch formation,both in the absence and in the presence of cephalexin (Table2).

In conclusion, inactivation or alteration of the levels of FtsZdid not inhibit branch formation. Together with the findingthat branch formation continues after inactivation of FtsI bycephalexin, these results strongly suggest that the cell divisionprocess is not required for branch formation.

Buds can form in cells with both normal and disturbednucleoid distribution. Branched cells have been reported to

form in cells affected in chromosome replication (48, 54, 55),and we have previously shown that strains with a disturbednucleoid distribution have an elevated frequency of branchedcells (6). Mulder and Woldringh (37) showed that in a dnaX(Ts)mutant grown at the restrictive temperature, the peptidoglycansynthesis rate is slower at the central part of the filamentouscells containing the nucleoid than at the nucleoid-free cellends, suggesting a negative effect of the nucleoid on peptido-glycan synthesis rate. Thus, an irregular nucleoid distributionmay lead to asymmetric peptidoglycan synthesis, thereby caus-ing the formation of buds on the cell surface. As stated above,the frequency of branched cells was significantly higher inM9caace than in LBglu (Table 2), and we therefore investi-gated whether the nucleoid distribution was different in cellsgrown in M9caace compared to those grown in LBglu. In thewild-type strains, growth in both media in the presence ofcephalexin resulted in an essentially regular distribution ofnucleoids (Fig. 4). Buds in these cells were found in regionsoccupied by nucleoids, as well as in nucleoid-free regions. Instrain EC::71CC, in which the branching frequency was higher,the nucleoid distribution was similarly disturbed in both LBgluand M9caace. Thus, a disturbed nucleoid distribution ap-peared not to be required for bud formation.

Branch formation correlates with cell physiology ratherthan with medium components. The frequency of branchedcells varies with the growth medium. This might correlate withthe different cell morphologies obtained in the different media(Fig. 5) and/or with specific components in the medium. In anattempt to distinguish between these possibilities, we shiftedcells (EC1005 and MG1655) from M9glu (low branching) toM9caace (high branching) and monitored the changes in cellmorphology and the frequency of branched cells (Fig. 6A andC). An increase in branch formation before any change inmorphology would suggest a direct effect of some mediumcomponent on branching. A change in morphology before anincrease in branch formation, on the other hand, would suggestthat the medium effect on branching comes by an overallchange in cell morphology. EC1005 had a slower growth rateduring the first 3 h after the shift from M9glu to M9caace(doubling time of 80 min) than when grown continuously inM9caace, whereas MG1655 reached its new growth rate inabout 1 h. (The doubling times during balanced growth forEC1005 and MG1655 in M9caace were ca. 40 and 60 min,respectively, and in M9glu were ca. 40 and 50 min, respective-ly.) The frequency of branched cells did not start to increaseuntil the cells had become microscopically indistinguishable

FIG. 4. Nucleoid distribution in EC1005 and MG1655 grown in the presence of cephalexin (10 mg/ml) for about two mass doublings. Panels: A, EC1005 grown inLBglu; B, EC1005 grown in M9caace; C, MG1655 grown in LBglu; D, MG1655 grown in M9caace. All cultures were grown at 37°C, and cephalexin was added toexponentially growing cultures. Bar, 2 mm.

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from cells growing continuously in M9caace (one to three massdoublings [indicated by arrows in Fig. 6]). In one experiment,the frequencies of branched cells in M9glu were 4 and 3% forEC1005 and MG1655, respectively. (These high frequencies inM9glu were only observed once and could not be reproduced.It should also be noted that the branching frequencies ob-tained here with EC1005 grown continuously in M9caace werehigher than we have reported previously [6]. We have no ex-planation for these differences.) When these cultures wereshifted to M9caace, there was a drastic initial decrease in thefrequency of branched cells. This decrease was followed by anincrease, which again came well after the observable changes incell morphology were complete.

During a shift from M9caace to M9glu, there was a longperiod (several hours) of metabolic adaptation with no or verylittle mass growth. After a shift from M9caace to LBglu, thecells became larger (Fig. 5A and B), and a drastic, initial de-crease in the frequency of branched cells was observed (Fig. 6Band D). This decrease was consistent with low or no branch-forming activity, i.e., it does not imply the disappearance ofalready existing buds and branches. A shift from LBglu toM9caace was followed by a long period with very little or nomass growth, as observed with the shift from M9caace toM9glu.

In conclusion, changes in cell morphology preceded changesin branching frequencies in the shifts from M9glu to M9caace,suggesting that branching correlates with cell physiology ratherthan with specific medium components. The drastic initial de-crease in branching after shifts from M9caace to LBglu couldbe due to branch formation being sensitive to changes in cellphysiology.

Effect on branching of antibiotics affecting cell wall synthe-sis. Our results so far do not suggest any involvement of thecell division process or a disturbed nucleoid distribution inbranch formation. An alternative mechanism for branch for-mation would be that branches form from small asymmetries inthe cell wall that arise during elongation. We therefore inves-

tigated whether branch formation was affected by antibioticsaffecting cell wall synthesis in different ways. Antibiotics wereadded at a series of different concentrations to cultures inbalanced growth, and cells were fixed after two doublings inoptical density (as measured for the untreated culture). Thefrequencies of branched cells and branches per millimeter ofcell length in the absence or presence of antibiotics are sum-marized in Table 3. Values are given for the antibiotic concen-trations yielding the largest observed effect on branching or, inthe case of no observed effect, for the highest concentration atwhich no substantial cell lysis or rounded cells were observed.

Like cephalexin, ampicillin and penicillin G have a highaffinity for PBP3 (45). As opposed to cephalexin, however,ampicillin and penicillin G also bind to PBP2 with relatively

FIG. 6. Changes in branching frequency after a shift of growth medium.Exponentially growing cells were collected by centrifugation and resuspended inthe same growth medium or in another growth medium. The cultures were di-luted repeatedly to maintain exponential growth. The frequency of cells withbuds and branches was estimated by putting cells directly onto an agar surface,and at least 500 cells were counted. Arrows indicate the first time sample in whichcells shifted to the new growth medium had become microscopically indistin-guishable from cells grown continuously in the new growth medium. Panels: Aand C, EC1005 and MG1655, respectively, shifted from M9glu to M9caace; Band D, EC1005 and MG1655, respectively, shifted from M9caace to LBglu.

FIG. 5. Cell morphology of EC1005 and MG1655 in different growth media.Panels: A, C, and E, EC1005; B, D, and F, MG1655; A and B, LBglu; C and D,M9glu; E and F, M9caace. Bar, 2 mm.

TABLE 3. Effects of different antibiotics on branch formation

Straina AntibioticAntibiotic

concn(mg/ml)

%Frequency

of branchedcellsb

Avg celllength(mm)c

No. ofbranches/

mm

EC1005 Ampicillin 2 14 (3.1)d 9.6 (2.9) 15 (10)MG1655 2 3.9 (0.7) 8.9 (2.7) 4.4 (2.6)EC1005 Penicillin G 10 15 (3.1) 9.2 (2.9) 16 (10)MG1655 10 5.2 (0.7) 9.3 (2.7) 5.7 (2.6)EC1005 Mecillinam 0.01 2.1 (2.7) 2.5 (2.7) 8.5 (10)EC1005 D-Cycloserine 6 1.8 (1.4) 2.5 (2.5) 7.2 (5.8)MG1655 6 1 (1.2) 2.7 (2.6) 3.7 (4.7)

a Strains were grown in M9caace at 37°C.b Values were obtained by counting at least 500 cells.c The average cell lengths were obtained by measuring at least 100 cells.d Numbers in parentheses refer to the untreated culture in each experiment.

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high affinities (45). PBP2 is required for cell wall elongation(44). The addition of ampicillin or penicillin G at 2 or 10 mg/ml, respectively, yielded filaments with an increased number ofbranches per millimeter similar to that obtained with cepha-lexin (Tables 2 and 3), thus showing that any effect on PBP2 byampicillin and penicillin G had no extra effect on branch for-mation. Also, the addition of mecillinam, an antibiotic highlyspecific for PBP2 (44), did not have any substantial effect onbranching at concentrations not causing substantial roundingof the cells (Table 3) (at higher concentrations, buds weredifficult to distinguish due to the round shape of the cells).

To disturb cell wall synthesis at an earlier step than thetranspeptidation reactions carried out by PBP2 and PBP3 (23,24), we used the antibiotic D-cycloserine that inhibits formationof D-alanine dipeptides (51), required for cell wall elongation.D-Alanine dipeptides are added to tripeptide side chains inpeptidoglycan precursors to form pentapeptide side chains(17, 25). Pentapeptides are then required for the subsequenttranspeptidation reactions. We could find no major effect ofD-cycloserine on branch formation.

In conclusion, cephalexin, ampicillin, and penicillin G had apositive effect on branching, whereas mecillinam and D-cy-closerine had no substantial effect. Cephalexin, ampicillin, andpenicillin G all bind several penicillin-binding proteins (45),and the effect on branching could be due to the binding to oneor more of these proteins.

Branch formation is a random event. The frequency ofbranched cells was qualitatively the same in most experimentsperformed with the same strain and the same growth condi-tions. In the shift experiments described above, cells resus-pended in the same medium after centrifugation maintainedapproximately the same frequency of branched cells for severalgenerations. Cells shifted from M9glu to M9caace showed anincrease in bud-forming activity after a certain time periodafter the shift. These observations suggest that bud formationdoes not take place primarily in a subpopulation with an ele-vated bud-forming activity, which would lead to variations inthe frequency of branched cells during the course of an exper-iment. It is still conceivable, however, that once a bud hasformed on a cell, there is an increased probability that itsdaughter cells will also form a bud. If this increase in proba-bility is sufficiently small and/or if branched cells have a suffi-ciently decreased growth rate or viability, this kind of “inher-itance” would not give rise to large subpopulations with anelevated bud-forming activity.

In the time-lapse experiments we found one example of acell with a bud that gave rise to a daughter cell that also formeda bud. To complement this observation with a larger number ofcells, cells were grown as in the time-lapse experiments for3.5 h, and the total number of cells and the number of cellswith a bud were then counted for each of at least 100 micro-colonies (Table 4). In the two samples from the same culture ofEC1005 grown in M9caace, the total frequencies of branchedcells on the agar slide were ca. 2.4 and 1.9%. The averagenumbers of cells per microcolony were ca. 10.5 and 11.1. Only

one microcolony in one of the samples was found to containtwo cells with a bud, and no microcolony was found that con-tained three or more cells with a bud. These results do notindicate any increased probability for daughter cells to a cellwith a bud to form a new bud.

Cultures of EC1005 and MG1655 grown in M9caace in thepresence of cephalexin for about two mass doublings containedmore than 20% branched cells. However, only ca. 1% of thecells had two buds (cells with more than two buds were veryrare). This is significantly less than what would be expected ifbuds formed independently of each other in the same cell.Thus, there appears to be some physiological constraint in thecephalexin-treated cells on the formation of a second bud.Since only one microcolony contained two branched cells (Ta-ble 4), it is also possible that, in a culture not treated withcephalexin, this constraint exists for the daughter cells of a cellthat has formed a bud.

DISCUSSION

Previous reports have indicated that an aberrant cell divisionprocess could lead to branch formation. We have previouslyreported an increased branching frequency in the filamentingintR1 strains EC::71CC and MG::71CC and in strains with theminB operon deleted (6). Branched cells have also been ob-served after depletion of the cell division protein FtsL (21) andFtsZ spirals have been shown to cause spiral invaginations (2),suggesting a potential of FtsZ to alter cell wall morphology. Inthe present study, we found no correlation between the celldivision process and branch formation. Branch formation wasnot confined to putative cell division sites, and no FtsZ struc-tures were found to colocalize preferentially to buds. In addi-tion, separate or simultaneous inactivation of the cell divisionproteins FtsI and FtsZ did not inhibit branch formation.

A correlation between a disturbed chromosome replicationor nucleoid morphology is indicated in previous reports (6, 48,54, 55). Also, nucleoids have been shown to affect the pepti-doglycan synthesis rate (37) and might therefore contribute toputative asymmetries in the cell wall that cause branch forma-tion. Here, branch formation was shown to occur in normallyreplicating wild-type strains with a normal nucleoid distribu-tion.

An alternative mechanism to account for branch formationwould be that branches form from small asymmetries in thecell wall. In support of this idea, interference with cell wallsynthesis by growing cells in the presence of cephalexin, am-picillin, or penicillin G had a positive effect on branching. Thiseffect cannot (solely) be due to filamentation, since cephalexinhad a positive effect on branching in filaments induced bygrowth at the nonpermissive temperature of strains carryingthe ftsZ84(Ts) allele.

Many bacterial species are able to change the direction ofgrowth, resulting in branched cells or hypha-like networks offilamented cells, but the mechanisms that lead to the formationof new cell poles and branching are unknown. In Rhizobiummeliloti, inhibition of cell division by addition of cephalexin,nalidixic acid, or mitomycin C caused branching at the cellpoles, whereas overexpression of either of the bacterium’s twoFtsZ proteins caused branching at midcell (28). Also, consti-tutive expression of FtsZ in Caulobacter crescentus caused theformation of a bifurcated stalk (41). However, neither ftsZ norftsQ is required for branch formation in Streptomyces coelicolor(33, 34), and it is possible that the extended C terminus of FtsZin R. meliloti FtsZ1 and C. crescentus is causing the differences(32, 40).

TABLE 4. Number of branched cells in microcoloniesof EC1005 grown on M9caace

Sample

No. of microcoloniescontaininga: Avg no. of cells/

microcolony

Total %frequency of

branched cells0 BC 1 BC 2 BC

1 82 25 1 10.5 2.42 97 26 0 11.1 1.9

a BC, branched cell(s).

VOL. 181, 1999 BRANCHING IN E. COLI 6613

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ACKNOWLEDGMENTS

We thank Joe Lutkenhaus for providing FtsZ-specific antiserum andMiguel Vicente for providing strains VIP183 and VIP205.

This work was supported by the Swedish Natural Science ResearchCouncil and the Swedish Cancer Society. A grant from the Knut andAlice Wallenberg Foundation enabled us to purchase the microscopeand the image analysis equipment.

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