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Copyright 0 1984 by the Genetics Society of America

IDENTIFICATION O F MUTATIONS ASSOCIATED W I T H

MACROFIBER FORMATION IN

BACILLUS SUBTZLIS

CHARLES L. SAXE, III*.

'

AND NEIL H. MENDELSON*'

*Graduate Committee on Genetics and *Department of Cellular and Developmental Biology, University of Arizona, Tucson, Arizona 85721

Manuscript received August 26, 1983 Revised copy accepted April 16, 1984

ABSTRACT

A search was made for the genes responsible for the production of helical macrofibers in the original collection of macrofiber-producing strains of B. subtilis. Two loci were identified: jibA, located between hisA and tag-I, and fibB, linked to cysB. jibA governs a short-lived division suppression phenomenon associated with the production of rudimentary fibers, whereas j b B appears to be responsible for a persistent division suppression and a more highly organized helical macrofiber. Both mutations are recovered from each of the original macrofiber-producing strains which also carried the diu N-BI mutation respon- sible for minicell production. The latter mutation by itself is not sufficient, however, for the production of macrofibers. Other known mutations leading to division suppression that map in the same region are shown not to be allelic to jibA or j 3 B . Neither j i b locus appears to be responsible for helix hand determination.

H E L I C A L 'macrofiber-producing strains of Bacillus subtilis were originally

described by MENDELSON (1 976, 1978, 1982a). These strains produce

long division-suppressed filaments that progress through a series of morpho-

logical states resulting in highly organized helical macrofibers. A typical de-

velopmental sequence would begin following spore outgrowth with the pro- duction of a helical double-stranded cellular structure. Growth and repeated folding of such structures result in the formation of multistranded macrofibers. Eventually, cell separation leads to the release of individual cells and the dis- ruption of organized structure.

T h e production of macrofibers is not restricted to the strains described.

Strains carrying the lyt mutation, previously associated with division suppres-

sion, have also been found to produce helical macrofibers (ROGERS and THUR-

MAN 1978; FEIN 1980). More recently ZARITSKY and MACNAB (1981) have

shown that similar structures arise when wild-type B. subtilis strains are grown

at very low cell densities. In all cases regulation of autolytic activity appears to be involved.

T h e present communication describes two mutations, $bA and $bB, that are

associated with macrofiber production. Map locations and the phenotypes gov- erned by these mutations are reported.

' Present address: Department of Biology, University of California at San Diego, La Jolla, California 92093.

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552 C. L. SAXE, 111 AND N. H. MENDELSON

TABLE 1

Strains of B. subtilis used in this study

~~

Genotype

Strain Auxotrophic markers Other markers Origin

QB917 QB944 QB928 QB934 QB943 QB935 QB936 BlS

R H X l l S

C6D C6@R4 KS27 KS27-200B Dts5-5 Dts7-1

hisA1, thrA5, trpC2 purAl6, cysA1, trpC2 aro1906, purB33, d a l l , trpC2 trel2, metC3, glyBl33, trpC2 pyrDI, iluA1, thyA1, thyB1, trpC2 aroD, lys

leuA8, aroG932, a l d l , trpC2

ura, metB diuIV-Bl, j b A , P B

ura, metB diulV-Bl, jb A , P B

ura, metB diUIV-Bl, j b A , j b B

ura, metB

hisAI, cysB3 hisA1, cysB3 thr5, trpC2

diuN-Bl, j b A , j b B , gtaCr4

tag- I diu11 5-5(ts)

thyAI, thyB1, trpC2 diuV 7-1(ts)

DEWNDER et al. (1977) DEWNDER et al. (1 977) DEDONDER et al. (1 977) DEWNDER et al. (1977) DEDONDER et al. (1977) DEWNDER et al. (1977) DEWNDER et al. (1977)

MENDELSON (1976); right-handed original macrofiber- producing strain

MENDELSON (1978); helix hand reversible derivative of B 1 S

MENDELSON (1 978); left-handed derivative of R H X l l S

(1 984); bacteriophage- resistant derivative of C6D This study This study VAN ALSTVNE and

SIMON (1971) VAN ALSTYNE and

SIMON (1971) SAXE and MENDEWN

MATERIALS AND METHODS

Strains of B. subtilis: Strains used in this study are listed in Table 1. All are derived from B. subtilis 168. Strains RHXl lS, C6D and C66R4 are derivatives of the original B. subtilis macrofiber strain, B1S.

Media: Growth of bacterial macrofibers was carried out by the procedure of MENDELSON (1978). Macrofibers were cultured in 0.10-0.15 ml drops of media dispensed on the inside of a Petri dish lid (100 X 15 mm). The Petri dish base was then used as a cover. Each dish could accommodate 36 drops. The drops were incubated either at room temperature (ca. 24") or at 20°, in a covered plastic box (26.5 X 35.5 cm) used as a moisture chamber (MENDELSON 1978; SAXE 1979). Growth in drop culture involved inoculation by stabbing a toothpick containing cells into a drop of medium (TB) (MENDEWN 1978). A small volume of the drop was transferred on the toothpick to a second drop as a dilution. Macrofibers were consistently produced in the second drop. Nutrient medium was tryptose blood agar base (TBAB, Difco, Detroit, Michigan).

Transformation: Transformation was carried out according to the method of Erickson and Cope- land as described by B ~ Y L A N et al. (1972). Because macrofiber-producing strains develop compe- tence very poorly, a modification of the procedure was made that improved the transformation efficiency (SAXE and MENDELSON 1984).

(3)

B. SUBTILIS MACROFIBER GENES 553

TABLE 2

Mapping offibA by three-factor crosses

Recombinants % cotransformation

No. using No. u i n g

limiting saturating Limiting Saturating

Donor" ClaSS DNA DNA DNA DNA

B1S his+ cys-fibA+

his+ cys-fibA his+ cys+fibA+ his+ cys+fibA

R H X l l S his+ cys-fibA+ his+ cys-fibd his+ cys+fibA+ his+ cys+jibA

C6D his+ cys-fibA+

his+ cys-fibA his+ cys+fibA+ his+ c y s + j b A

C64R4 his+ cys-jibA+

his+ cys-fibA his+ cys+fibA+ his+ cys+fibA

225 222

23 25

10 13

0 ND

250 210

8 29

2 21

0 ND

367 212

15 27

6 21

1 ND

245 200

25 42

1 1 15

0 ND

9" 10

4' 5

Ob

3 1 1

1 8

0

4 10

2 8

14

8 16

3 6

0

Transformations were performed with either limiting (0.03-0.04 rg/108 cells) or saturating (3-6 pg/ml) DNA concentrations. Transformed cells were initially screened for his+ on minimal medium containing cysteine. Cells were picked and subcloned on fresh minimal plus cysteine and then replica plated on minimal medium without cysteine and onto TBAB. The colonies that grew on minimal medium were scored at his+-cys+. The colonies on TBAB were picked into drops of TB, grown overnight at room temperature (approximately 20") and screened microscopically for macrofiber production. ND = values not determined.

The recipient strain was KS27 (hisAI cysB3).

Percentage of his+-cys+ cotransformants also producing macrofibers.

Percentage of total number of his+ transformants also producing macrofibers.

RESULTS

Using a subsaturating concentration of DNA (0.03-0.04 &lo8 cells) ex-

tracted from macrofiber-producing strain C64R4, we obtained prototrophic

transformants of 19 different markers. These markers circumscribe the entire

B. subtilis chromosome (DEDONDER et al. 1977). T h e prototrophs were screened for macrofibers. Such transformants were only found among His' and T h r + prototrophs of strain QB9 17, indicating a gene (or genes) governing macrofi-

ber production in the vicinity of map positions 290 and 305 (HENNER and

HOCH 1980).

T h e relevant loci were further localized as follows. Strain KS27 carrying

hisA1 and cysB3 alleles was constructed and used as a recipient in transforma-

tion with macrofiber-producing donor strains, B1 S, RHX 1 1 S, C6D and

C64R4. Four sets of transformations were performed with each donor, two

using saturating and two subsaturating concentrations of DNA. Table 2 illus-

(4)

554 C. L. SAXE, I11 AND N. H. MENDELSON

TABLE 3

Analysis of three-factor crosses with hisA, tag-1 undfibA

~

Recombinants

No. of % cotrans-

Donor' Class recombinants formationb

Selection for his+

C66R4 his+ tag-fibA' 469

his+ tag-fibA 31 6'

his+ tag+fibA+ 12 2'

his+ tag+fibA 11 47d

Selection for tag+

tag' his-fibA+ 205

tag" his-$bA 13 6'

tag' his+fibA 10 50d

tag' his+fibA+ 10 4"

Transformations were performed using limiting DNA concentrations (0.03-0.04 pg/ 10' cells). Transformed cells were initially screened for his+ on minimal medium or for

tug' on TBAB at 45". Cells were picked, subcloned and screened for cotransformants after the methods described in Table 2.

a The recipient strain was KS27-200B (hisAI tag-1).

'

The suggested order is hisA-fibA-tag-1.

Percent of total number of his+ transformants.

Percent of his+-tag+ cotransformants also producing macrofibers.

* Percent of total number of tag+ transformants.

subsaturating concentrations of DNA, linkage between hisA and a gene gov-

erning macrofiber production, termed fibA, was indicated. Histidine-independ- ent transformants that also became cysteine independent did not, however,

produce macrofibers, suggesting that j b A does not reside between the former

markers.

T h e linkage relationships of t h e j b A gene in strain C64R4 to hisA and the

closely linked tug-] are shown in Table 3. Many of the transformants that

obtained his and tug from the donor also obtained fibA, using subsaturating

concentrations of DNA. This too supports the conclusion thatJibA is linked to

hisA.

T h e morphological phenotype of strains carrying a JibA mutation is illus- trated in Figure 1. T h e macrofibers consist of loosely packed arrays of division- suppressed filaments in which the parallel alignment of filaments creates a

"swirled" appearance. Such structures persist at 20" in T B medium for only

15-18 hr, at which time they decay by liberation of individual cells. T h e

isogenic j b A + strain, KS27, grown under identical conditions produces only

individual cells of normal rod-shaped morphology.

Transformations involving the j b A gene revealed that the number of co- transformants found among His+ isolates was increased when saturating

amounts of DNA were used (Table 2). This would be expected if an additional

(5)

R. SURTILIS MACROFlRER GENES 555

FIGURE I . - l ' t i o l o i i i i ( , r o ~ i . ~ i i ) t i \ of ii j h A iiiiit;irit ni;icrofitwr. 2'he structure was prcwluced by the drop culture nirttiod. a. 1,ow niagnification phase-contrast micrograph of an entire structure; size bar = I O pm. b, High magnification oFa region of the structure shown in a; size bar = I pm.

independently, although simultaneously, with the f i b A mutation. Such events,

termed congression (ROYLAN et al. 1972), have been described in other systems

using saturating concentrations of D N A in transformation.

T h e results of transformations involving the nearby cysB marker a r e shown

in Table 4. In all cases linkage was found between a gene governing macrofiber

formation and the cysB marker. This gene is termed fibB. Cys+ transformants

that simultaneously became His+ when subsaturating concentrations of DNA

w e r e used infrequently produce macrofibers. ThefibB locus appears, therefore,

(6)

556 C. L. SAXE, I11 AND N. H. MENDELSON

TABLE 4

Mapping offibB by three-factor crosses

No. of % cotrans- DonolC Class recombinants formation'

BIS cys+ his-fibB+

cys+ his-fibB cys+ his+fibB+ cys' his'fibB

RHXl IS cys+ his-fibB+

cys+ his-fibB

cys+ his+fibB+ cys+ his+fibB

C6D cys+ his-fibB+

cys+ his-fibB cys+ his+jibB+ cys+ his+fibB

C64R4 cys+ his-fibB+

cys+ his- fibB cys+ his+ j b B +

cys+ his+fibB

287

15 5'

9 3"

1 1 o d

232

17 7

7 3

2 22

322

7 2

8 3

1 1 1

316

12 3

20 6

2 9

Procedures were the same as in Table 2. Initial selection was for cys' with

a The recipient strain was KS27 (hisAI cysB3).

'

The suggested order isfibB-cysB-hisA. ' Percent of cys+ transformants. subsequent selection for his+ andfibB.

Percent of cys+

-

his+ cotransformants also producing macrofibers.

Figure 2 illustrates the morphological phenotype of strains that carry a F b B

mutation. T h e macrofibers consist of organized helical structures in which division-suppressed filaments are arranged as in the original donor macrofiber-

producing strains, although less tightly so. These structures persist at 20" in

the standard T B medium for 25-28 hours. Upon decay long filaments equiv-

alent to six- to eight-cell lengths are liberated from the structures. In contrast,

parental C6D structures grown in the same manner retain their organization

for 36-38 hours.

Tables 2 and 4 indicate that all of the original macrofiber-producing strains

studied carry both the JibA and JibB mutations. Table 5 illustrates that the

structures produced by strains that carry both jibA and JibB mutations most closely resemble those found in the original macrofiber-producing strains.

We have explored the relationship of the JibA and JibB alleles present in

strain C64R4 to other mutations that lead to division suppression that are

known to lie in the same region of the linkage map (VAN ALSTYNE and SIMON

1971). divZZ and divZV are located at map positions 320 and 285, respectively.

Table 6 illustrates that wild-type alleles of both of these markers reside in

strain C64R4. In addition it is shown that strain C64R4 also retains the original

diu IV-BI mutation present in the original B l S strain from which C64R4 was

(7)

R. SURTIIJS MACROFIBER GENES 557

T h u s far n o mention has been made of the helix hand of macrofibers studied or of the inheritance of helix handedness. T h e helix hand phenotype of donor

strains used throughout a r e shown in Table 1. We have, therefore, examined

the helix hand phenotype of macrofiber-producing transformants derived from

donors that produced either ( 1 ) right-handed macrofibers, (2) left-handed mac-

rofibers or (3) either hand depending upon growth environment (MENDELWN

1982a; MENDELFON and KARAMATA 1982). Table

7

illustrates that all classes can be found among transformants originating from each of the three cate-

gories. It appears, therefore, that neither the j b A nor the j b B gene controls

helix hand phenotype. O t h e r experiments to be reported elsewhere will de-

scribe additional loci concerned with helix hand determination (D. FAVRE and

(8)

558 C. L. SAXE, 111 AND N. H. MENDELSON

TABLE 5

Crosses demonstrating the fiber-producing genotypes of representative C6D-derived strains

No. showing

Selected superior macrc- Donor Recipient marker fiber structures

C6D" c l 240b his+ 2513 12

h1432' cys+ 211312

h1432' c1240 his+ 131312

c 1240' h1432 cys+ 613 10

BlS" c1240 his+ 2013 12

h1432

v+

2/312

a Transformations were performed that involved the use of saturating DNA concentrations (3-6 rgjml). Transformants were screened initially for his+ or

cys', and then drop cultures of T B were made to determine the nature of the macrofibers made. A fiber was determined to be superior if it was observed to be more organized and elaborate than the recipient strain and if it retained a significant structure after more than 30 hr. An inferior structure was less or nized and decayed faster than the recipient.

&train cl 240 derived from a limiting DNA concentration transformation with C6D and KS27 the donor and recipient, respectively. The strain was initially screened for cys* and was found to produce macrofibers. It is a presumedfibB mutant. Its phenotype is like that seen in Figure 2.

' Strain h1432 derived from a limiting DNA concentration transformation with C6D and KS27 the donor and recipient, respectively. The strain was initially screened for his+ and was found to produce macrofibers. It is a presumedfibA mutant. Its phenotype is like that seen in Figure 1.

*

Crosses were performed like those in footnote "; however, limiting concen- trations (0.03-0.04 pg/108 cells) of donor DNA were used.

DISCUSSION

Previous publications have described the structure of helical macrofibers and

the dynamics that are associated with their formation (MENDELSON 1982a, b).

We have attempted to identify genes that control various aspects of this com- plex dynamic phenotype. T w o loci, $bA and$bB, were discovered. T h e former governs the transient suppression of cell separation during the initial stages of growth. T h e resulting structures are poorly organized and short lived. Else- where it has been argued that the helical shape deformation is brought about as a result of the blockage of rotation normally associated with cell elongation during growth. If so, then mutations that govern helical macrofiber production

may do so not by a fundamental alteration of normal growth pattern but rather

by bringing about conditions that prevent daughter cell separation and inhibit cellular rotation. Such properties appear to be associated with a reduction in

autolytic enzyme activity. T h e $bA mutant phenotype is consistent with only a

partial block in cell rotation and reduction in autolytic activity.

T h e second mutationfibB governs the production of a more highly organized and longer lived macrofiber structure than those found in$bA mutants. T h e multicellular length of filaments liberated upon breakdown of$& macrofibers

suggests that this locus may control some aspect of cell septation as well as cell

(9)

B . SUBTZLZS MACROFIBER GENES 559

TABLE 6

Genetic relation of C66R4 to divll, diulVBI and divV

~~

Selected Unselected X cotrans- Donor Recipient marker marker formation"

(thyA, B, metB, divlVBI) met+ d i v W 0 (0/717)

[thr, trpC, d i v l l 5 - 5 (ts)] trp+ d i v l r 6 (30/512)

[ t h y , trpC, diuV 7-1 (ts)] trp+ divV+ 8 (40/520)

C66R4 CU403divIVBI

Dts 5-5

Dts 7-1

Crosses involved transformations using saturating concentrations of DNA (3-6 Pg/ml). Transform- ants were initially screened for met+ or trp' on appropriate minimal medium and then picked onto selective minimal medium again. Strains screened for d i v W were replica plated onto TBAB and analyzed microscopically for any strain no longer producing minicells. Transformants screened for d i v W or d i v V were replica plated onto duplicated TBAB plates. One plate was incubated at 30" and the other at 45". After 3 hr, cells at 45" were screened for ability to divide normally. Cells at 30" were screened as controls.

Numbers in parentheses are data from which the percent cotransformation was derived.

TABLE 7

Inheritance of helix hand in macrofiber transformants of KS27

cys+ transformants his' transformants

Limiting DNA Saturating DNA Limiting DNA Saturating DNA

Donor LH P RH LH P RH LH P RH LH P RH

BIS 1 1 14 1 1 21 1 5 17 0 9 10

R H X l l S 0 2 17 0 1 23 0 2 6 5 3 21

C6D 0 2 6 5 0 7 2 6 8 11 2 14

C66R4 0 0 1 4 5 2 4 0 6 1 9 1 7 0 2 5

Macrofiber transformants used in this study are derived from experiments described in Tables 2

and 4. LH = left-handed helix; RH = right-handed helix; P = parallel arrays.

Both mutations, j b A and j b B , have been mapped, and they appear to r e p

resent new genes concerned with some aspect of growth regulation. They are

located in a map region around 300 that contains at least six other genes

concerned with cell growth or division phenotypes: divZZ, divV, gtaA, gtaB, tag-

I and rodC (HENNER and HOCH 1980). Mutations in these six genes have not

been associated, however, with either helical growth or the production of

macrofibers. Neither fibA nor fibB control phenotypes as drastic as those pro-

duced by mutations in the Zyt gene, a gene that controls cell wall turnover,

autolysis and the production of highly ordered helical macrofibers (FEIN 1980;

MENDELSON 1982a). ThefibA andfibB mutations appear, however, to be the

primary genes responsible for helical macrofiber production in the original strains in which such structures were discovered. Reconstructed strains carrying both mutations were found to closely resemble the helical macrofiber pheno- type of the original strains.

(10)

560 C. L. SAXE, 111 AND N. H. MENDELSON

strains can form helical macrofibers when cultured at very low cell densities. It appears, therefore, that there are both genetic and physiological mechanisms leading to helical macrofiber formation. Their observations are compatible with the idea that a basic helical growth pattern is present in all bacilli and that conditions leading to macrofiber formation are concerned primarily with mechanisms directed at cell separation events and cell wall turnover properties

rather than basic growth geometry. ZARITSKY and MACNAB’S observations also

underline the necessity of determining in all genetic experiments concerned with helical macrofiber production whether or not the recipient strains them- selves are capable of structure formation when cultured at low cell density. Such precautions have been taken in all experiments reported in this com- munication.

Finally, we wish to mention that, although both genetic and physiological

factors are known to contribute to the determination of helix hand in B. subtilis

macrofibers (FAVRE, BRIEHL and MENDELSON 1983), no evidence could be

found to link either of the genes described here to helix handedness. It ap- pears, therefore, that, although macrofiber formation and helix hand deter- mination must be closely related processes developmentally, their genetic reg- ulation must be distinct.

Discussions with D. F. SAXE are gratefully acknowledged. This work was supported by a research grant from the National Institutes of General Medical Sciences to N.H.M. C.L.S. was supported by a traineeship from the Public Health Service, grant 5 T 32 GM07239-04.

LITERATURE CITED

BOYLAN, R. J., N. H. MENDELSON, D. BROOKS and F. E. YOUNG, 1972 Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid. J.

Bacteriol. 110 281-290.

DEDONDER, R. A., J. A. LEPESANT, J. LEPESANT-KEJELAVROVA, A. BILLAULT, M. STEINMETZ and F. KUNST, 1977 Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168. Appl. Environ. Microbiol. 33: 989-993.

FAVRE, D., M. M. BRIEHL and N. H. MENDELSON, 1983 Protein and peptidoglycan synthesis requirements for helix hand inversion of Bacillus subtilis macrofibers (Abstr.). Annual Meeting of the American Society of Microbiology, 524, p. 175.

FEIN, J. E., 1980 Helical growth and macrofiber formation of Bacillw subtilis 168 autolytic enzyme deficient mutants. Can. J. Microbiol. 26: 330-337.

HENNER, D. J. and J. A. HOCH, 1980 The Bacillus subtilis chromosome. Microbiol. Rev. 44: 57- 82.

MENDELSON, N. H., 1975 Cell division suppression in the Bacillus subtilis divN-AI minicell-pro-

Helical growth of Bacillus subtilis: a new model of cell growth. Proc.

Helical Bacillus subtilis macrofibers: morphogenesis of a bacterial mul-

Bacterial growth and division: genes, structures, forces and clocks.

Dynamics of Bacillus subtilis helical macrofiber morphogenesis: writh- ducing mutant. J. Bacteriol. 121: 1166-1 172.

Natl. Acad. Sci. USA 7 3 1740-1744. MENDELSON, N. H., 1976

MENDELSON, N. H., 1978

ticellular macroorganism. Proc. Natl. Acad. Sci. USA 75: 2478-2482. MENDELSON, N. H., 1982a

Microbiol. Rev. 4 6 341-375. MENDELSON, N. H., 1982b

(11)

B. SUBTILIS MACROFIBER GENES 561

MENDELSON, N. H. and D. KARAMATA, 1982 Inversion of helix orientation in B a d u s subtilis macrofibers. J. Bacteriol. 151: 450-454.

ROGERS, H. J. and P. F. THURMAN, 1978 Double mutants of Bacillus subtilis growing as helices. J. Bacteriol. 131: 1508-1509.

SAXE, C. L., 111, 1979 Mutations affecting morphogenesis in helical macrofibers of Bacillus sub-

tilis. Ph.D. Thesis, University of Arizona, Arizona.

SAXE, C. L., 111 and N. H. MENDELSON, 1984 Morphological and genetic characterization of a bacteriophage-resistant Bacillus subtilis macrofiber-producing strain. J. Bacteriol. 157: 109- 114.

Division mutants of Bacillus subtilis: isolation and PBSl

transduction of division-specific markers. J. Bacteriol. 10% 1366-1 379.

iological properties of Bacilluc subtilis. J. Bacteriol. 147: 1054-1062.

VAN ALSTYNE, D. and M. I. SIMON, 1971

ZARITSKY, A. and R. M. MACNAB, 1981 Effects of lipophilic cations on motility and other phys-

Figure

TABLE 2 Mapping offibA by three-factor crosses
TABLE 3 Analysis of three-factor crosses with hisA, tag-1 undfibA
FIGURE I bar drop culture nirttiod. .-l'tioloiiii(,ro~i.~ii)ti\ of ii j h A  iiiiit;irit ni;icrofitwr
TABLE 4 Mapping offibB by three-factor crosses
+2

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