© ELSEVIER Par is ~988

Ann. Inst. Pasteur/Microbiol. 1988, 139, 79-103

VARIATION OF ~-GALACTOSIDASE EXPRESSION

FRONa i:~;iDLAC ELEMENTS DURING TtIE DEVELOPMENT

OF ESCHERICHIA COLI COLONIES

J.A. Shapiro (I) and N.P. Higgins (2)

(1) Department of Biochemistry and Molecular Biology, University of Chicago, 920 E. 58th Street, Chicago, IL, 60637 (USA) and

(2) Department of Biochemistry, University of Alabama at Birmingham, Birmingham, AL. 35294 (USA)

SUMMARY

Bacterial colonies reveal the action of systems for multicellular regulation of genetic activity during growth and development. Colonies prnduced on agar indicator medium by Escherichia co!i strains carrying the transposable genetic fusion element Mudlac displayed organized patterns of differential ~-galacto~qdase expression, One feature of these patterns was the presence of phenotypically distinct concentric rings containing cells that were not genetically distinct. A second feature of the patterns was the aFPearance of sectorial populations with novel phenotypes, frequently displaying coincident variation in several characters, such as enzyme activity, multicellular aggrega- tion and rate of spread over the agar substrate. Subcloning analysis of sec- tors with more expansive growth phenotypes revealed that they contained bacteria expressing novel developmental sequences on more than one kind of medium. These novel developmental sequences could be transmitted to pro- geny bacteria but were reversiNe during growth in liquid medium. More stable c!onal variation in patterns of .B-galactosidase expression arose during storage in liquid medium. Most of these changes correlated wi ' -anspositions or rearrangements of Mudlac sequences. Some changes in ,alactesidase ex- pression involved interactions between Mudlac elements and unlinked Mu derivatives. These results revealed the operation of novel control systems regulating ~-ga!actosidase expression from the lacZ sequences in a

Received October 9, 1987.

Send corresponden:e to James A. Shapiro.

80 J.A. SHAPIRO A N D N.P. HIGGINS

chromosomal Mudlac element and demonstrated at least two different kinds of clonal variation events which affected pattern formation in bacterial colonies.

KEY-WORDS: Escherichia coli, Colony, [3-Galactosidase; Clonal variations, Mudlac sequences.

INTRODUCTION

In 1920, a remarkable paper entitled <~ t~tat organis6 des colonies bact6rien- nes>> by R. Legroux and J. Magrou was published in these Annales [5]. Legroux and Magrou had observed regular morphological changes as bacterial cultures grew on agar surfaces. They consequently carried out histological examinations of the structures of colonies at different stages of development. They observed a h~gh degree of differentiation and order within colonies and that order changed in a predictable manner over time. Although little noti- ced in the general bacteriological literature, these results posed basic ques- tions about the functional significance and morphogenetic control of bacterial colony organization. There are many ways that organized multicellular growth can be useful to bacteria in permitting biochemical specialization of certain cells, providing protection against chemical and biological threats [7] and facilitating the utilization of resources that would not be available to isolated cells [1]. Although laboratury studies of colony development on regular agai substrates are certainly artificial in some senses, they do reveal the intrinsic morphogenetic capabilities of bacteria. Thus, examining colcnies in thc laboratory can help us to understand the tools bacteria use in their many roles as geochemists, symbionts and pathogens.

A few years ago, a chance observation connected the ,~henomena descri- bed by Legroux and Magrou with the use of genetic fusion techniques to study the expression of specific DNA sequences. The introduction of transposable Mudlac elements into Pseudomonas putida and Escherichia coli strains and growth of the resulting genetically engineered bacterial cultures on indicator medium provided a syst ~- n. for vital staining of colonies [8, 9]. One particular Mudlac element, MudII1681 (fig. 1), was used to place ~-galactosidase ex- pression under nc;vel controls when appropriately inserted into the genome. This element was designed so that the lacZYA sequences could only be transcribed from a promoter outside the right Mu extremity, and lacZ could only be translated as a hybrid protein originating at an initiation codon b~t- ween the outside promoter and the Mu terminus [3]. Although such ~'~udiac fusion elements have been very helpful in assaying transcriptiun and transla-

CFU = colony-forming unit. TYE = tDp tone yeast-extrac~ medium.

XGal 5-bromo- 3-chloro-3-indolyt-~-D-galac- toside (a chromogenic substrate for ~-galactosidase)_

0 Mu left end

737 TaqI 1001 HindIII

1307 EcoRV

1850 HpaI/HincII

4219 Hpal/HincTI (Tn5/IS50) 4555 HpaI/HincII

4824 SacII 4855 XhoI

5565 Hind!II 5895 BglII (ZS50)

7054 SalI/HincIZ 7205 Xhol 7293 HincI[ (Tn5) 7420 BamHI ("iac"7840) 7975 Sa!I/HincZI (lac7304) 7995 BstEZI (lac7285) 8220 S~cll (lac7036) 8490 Bs~EZZ (lac6769)

II,000 Hincll (Iac4006) 11,130 EcoRl (!ac4134)

12, 19~ SacZ (:.ac3066)

13,020 EcoRV (lac2241} 13,080 HpaZ/Hinc~Z (iac2178) 13,305 TaqI (Iac1954)

13,705 Hpal/HincZZ(iac1554)

14,120 BamHZ(lacl139=codonS) 14,240 ~ right end

9.} C) N - <

(%

0")

~ . . ~ , " .O " 0 ' ~ 0 . ~

= ' : : ~ ~ = -,~ ~ .

. _ . . 0 0 2 . . C - - , . C -T..~7,'G

"CI ,., ~ ~ : : :3~ ¢) , .= 0

~..~ ~. -~. = ~ o ~ ~ . ~ O ~ ,..-,L-~ 0 = ~ ~) 0 0 0 ~ ' - f./']

• - ~ " 1 3 - , n . _ ~ ~ . . ~ , . . E : ' , , O E :

0 ~ ~ ~ " ~ ~ . ~ - ~ 0 0 ~

i ~ . ~ . ~ - ~ ~ ~ ~ ~ ~ "~' ~ O 0 ~ P ' ~ ~ * -, ~'~ ~ ~ "~_..~ ~ --~

0-) ~ J - J , ;~ • 9.) ::~

> ~,-~.~ ~,~ ~ u ~ ~ ~- - - .~ .~

0

,.= H,

82 J . A . S r t A P I R O A N D N . P . H I G G I N S

FIG. 2. - - Two MS1534 colonies.

When bacteria were picked from the darker sectors and tested, ~hey were found to have lost the trimethoprim-resistance marke: of R388 and to conta,_'n no ;alasmid DNA, and their descendants showed no R388- MudlI 1681 junction fragments. The particular sector which was picked as MS1534.04 is indicated by an arrow. Photographed after 14 days incuba- tion on XGal indicator agar (all incubations on agar were at 32°C).

FIG° 3. - - The efj~ct of a plasmid-carried Muc+pApl prophage on lacZ expression fi,om a ~ hromosomal Mudli1681 element.

The top panel shows streakings of the gentamycin-resistant exconjugants obtained by cros- sing bacteria freshly isolated from a dark MS1534 sector with donors for either the pOX- Gen (top left) or pOXGen::Muc+pAp~ p~aamid (bottom right); photographed after 16 days. The middle panel shows a similar str~ aking of MS1534.04705353 f@OXGen) ex- conjugants, and the bottom panel a streaking o : MS1534.04705353 (pOXGen::Muc-pApl) exconjugants ; both photographed after 8 days The pOXGen plasmid was constructed in the Beckwith laboratory as a derivative of F ~eleted for all IS elements and containing a gentamycin-resistance cassette (M.J. Casadaban, personal comm~mication). The dark spots on these colonies were papillae containing cell~ with iptense lacZ express;en.

©

> O0

84 J.A. SHAPIRO A N D N.P, HIGGINS

tion of many regions of the bacterial genome, there have been frequent anec- dotal reports from investigator's using this method that expression was sub- ject to unexpected variations. Some of these variations may have resulted from genetic instability, and others may have reflected the operation of unknown regulatory systems affecting genetic expression. Our results showed that both explanations are correct and that expression of the lac sequences in Mudlac elements can be subject to a number of different controls, some of which operate at the level of the entire colony.

By following patterns of {3-galacte~idase expression in colonies containing Mudlac elements and correlating those patterns with other features of colony structure, it has been possible to show that differential biochemical activity is a normal feature of bacterial development on agar. Examination of stai- ned colonies on medium containing the chromogenic ~-galactosidase substrate XGal revealed two basic elements of biochemical organization in P. putida and E. coli colonies: sectorial organization in clonal populations and con- centric organization in non-clonal populations [9]. Subsequent examination of the cellular composition of colonies with the scanning Aectron microscope confirmed these basic organizational elements by demonstrating sectorial and concentric differentiation into zones of cells with characteristic sizes, shapes and multiceliular patterns of alignment [10, 12].

The appearance of sectors and of subclones with novel patterns provided a means to begin studying the control of colony differentiation by providing populations of bacteria with specific differences in merphogenetic regulation. A study of pattern changes in P. putida (Mudlac) cultures revealed three aspects of the variational process that were not initially ~.xpected [11]. I) There was an unexpectedly high rate of morphological variation between sibling clones from a single culture, especially when the parental culture was more than a couple of weeks old. 2) Within a single sector or subclone, there was often coincident variation of several characters, such as [3-galactosidase expression, multicellular aggregation, growth regulation and nutritional markers. 3) There was clonal specificity to the extent and typ~s of variation that occ~Jrred, in the sense that sectoring was more frequent (or less frequent) in the colonies produced by particular clones, and certain nutritional markers were variable in some lineages and stable in others. These results pointed to the operation of an integrated system regulating many aspects of :olony morphogenesis and patterping.

Fl¢~,. 4. - - Colonies produced by dilutions o f bacteria picked f rom different concentric rings o f a well-isolated MS1534.031 colony.

The top panel was picked from the dark central region; the middle panel was picked from the lighter ring just outski.~ the central region ; and the bottom panel was picked from the zone of intermediate pigmentation located between the colony edge and the outer dark ring seen in the best-isolated colonies. Photographed after 8 days incubation.

• ~

ii ~

86 J.A. SIJA PIRO /! N!) N~ P. III(;(;IN£'

l o c~e t i d the f', put ida ob~crvaliow~ and io ~akc adva~ltatc of more ,a~pllisticated molecular genetics, a similar ;maly~is was m~cfct'iakcn ~'f pa~, lcra variat ion in an E'. col;, (Mudluc) lineage. Our results pointed to .,~ mimbcr o1:' facior,, a[i 'ccling tacZ exprcs~;iol~ f rom tim Mud! t t681 elcmen! in cotonh:~ which were aot a~l ic ipaied in lhe iiiiliat Co!lstrttclioll {}f this atid similar {u~ ~,i~m clement,, 1:~, 31, l h e s c [a<:t{~ts iachided diffcrcnl ia t expi'e',~dcm in con,, t:cnfiic Iint'~', of getlctically similar bacter ia , inhibi t ion of cx|)re,,~,io~ by add~lir)i'lM M!)(Ill t 681 cieme)lbl <~)" or!let Mu dcriv.:ilives amt reversible chm_al val~i;~!it'm',, altcr'ir|g l)olh !ac;Z dw, l g rowlh phellolypes.

R ES U i ; l S

The MSi534 lh',ea:ge: ctian~es in mull ipie phenotypes ,

Wlten ,~tt R , ig8: :Mudl l t681 pla:;mid was in t roduced by c(nl iugal ion into atl l;', COH ~]hlC ~,{|'~|ill, characteristic sectoring patterns were observed in cokmi;:s on X(;al iadicator agar [9, 13]. The sectors were wtliie witll deeply p igmented borders and conla ined plasmid-free bacteria. Whet | R388: :Mudl 11681 st raim, were gencra led by t r ans fo rma i ion with puril 'ied plasmid 1)NA rathcl' than by coIljttgatioIt, novel sectoring pat terns were somet imes d,,:lecied, One t r a n s f o r m a m cttt tuie, labelled MS153,t gave rise 1o light cokmies conta in ing deeper blue sectors with deeply p igmented borders (fig. 2). These blue sec- tors conla ined plasmid-free bacteria w h i c h h a d a Mudl11681 element inserted into the t h r o b ",',ome.

The more intense LacZ activity in tim plasmid, free sectors o f MS t 534 co]- oitie'~ appeared to restllt f rom a release of inhibi t ion by plasmid Mu func- litms {m chromosomal Mud[! 1681 expression. To demonst ra te such inhibition, lhc l"dcrived pOXGcn : :Muc ~ pApl plasmid was transferred inlo celiq freshly i:~olated f rom stich a sector. ' l he resuhil lg cxconju,:,ants did not display thc iypical conccni r ic patterri of LacZ activity, while similar exconjugants which had received the p ( ) X G e , plasmid wi lhout a Muc + pApl insert retained the l a c Z rings (fig, 31.

I:i(;. 5, -- Pediyfeex of some cultures descended from MSI534,

Suc:cecdmg ,;ubclom.>~ were dc,@,aatcd by adding a non-zcio digit to lhe decimal expansion. Each m:w cohmm h~dicalc~ a ~,ubclonmg step, and sibling subclones are either listed below each olhc~ o~ indicated by hyphenating the last two digits of the culture designations (e.g..047(i122-25 indicatc~> lbur sibling subdones of culture .04702 with similar phenotypes). Cultures established by picking expansive sectors are indicated by large bold type (123456"/8901, Single-colony subclone~ exhibiting a rcstricied spobcolony phenotype are indicaied by ~lnall type (1234567890) and si~lgk: colony suhclones exhibiting an expansive sire! cokmy phenolype arc indicated by small shadowed italic type (1234567890L MS1534.04 was picked from a non- e~,pan~ivt~ darker sector on one of the MS! 534 colonic~ ilhistrated in figure 2. The numbers in~ide the arrows between cohmms indicate the number of days which elapsed between the establishmenl of a parental culture by picking, a sector or colony into TYE broth and the establishment of its subclonal culture by a similar operation. Unless preceeded i0y an arrow, subclones following eacl, other in a column were established after the same delay.

0 4 ~ Z ~6 , .0472~

0 ~ 3

04~4 04741

0477

0478

.a4 :. ,0470'i f~ ~. 0470~

0 4 7 0 2 04/0~2

0-~ z02~

047022 2'~,,

.04703 .8. ~ . 0 4 7 0 3 1

, 0 ~ t 0 3 5 .04704

,04105

3 2 , > 04t2~1 t3

, 0 ~ 7 2 t 4 047215

~ > 0~72t0~C~'~ 2 t , . 0 4 7 2 1 0 t !

104721012

0 4 7 2 1 0 1 3

3? ,, 0474'~1 15

~3 ~ 047,,',1t0t 04

. 0 4 1 4 t O 5

fk34 ,, 047011

04 /0 t17

-32 > Oa/0,~"~0t

~} > 047020109

--~ ~ ,047021011 .047021012 .047021013

. 0 ~ 7 0 5 t - -32 .> 04;05~ t;' 2~ > , 0 4 7 0 5 ! 6 1

.0470 ~ t 71 . 0 4 7 0 5 1 ~ t

r04705~ 9

.04,7 '0 $ 2 047052t-29 - 2 t , > .04705241

. 0 4 7 0 5 2 9 t • 0 , 4 7 0 5 3 0470531.39 .... 61[~° > 04/0~;311 t 2

047054

,~ 4 7 0 5 5

• ..?fJ > ~0470501 ~ 0 4 7 0 5 0 2

.0470~03

.0470~4

047050509 .04706 - 8 - > .047061

. C t ,¢062

.0470,G3

. 0 ~ 7 0 6 4

047065

.047081

. 0 4 7 0 8 2

. 0 4 ~ 'n83

. 0 4 7 0 8 4

047085

.04707

.04708

.04709

04705341.42

,21-> .04705351

6 1 ~ > .04705352-53 .04705361-62

0410533! 2? .0470541~4g

• 04705,51-53 . 0 4 Z 0 5 ~ 4

. 0 4 7 0 5 . ~ 5

04705f~-59 ;~"f-> .04705591

--;~--> .04705041

88 J.A. SHAPIRO A N D N.P. HIGGINS

When bacteria from a blue MS1534 sector were diluted and plated on XGal agar, a uniform field of colonies displayed concentric rings of differential [3-galactosidase expression. When the bacteria from any of the various rings were picked and streaked on XGal agar, a similar field of colonies appeared with the same ring pattern (fig. 4). This result showed that there was no hereditary difference between the CFU in phenotypically different zones of the colony and consequently, that the viable bacteria in these zones were not undergoing a succession of genetic changes. It was, of course, possible that the observed phenotypic differences in lacZ expression reflected the activities in each ring of a subpopulation of terminally differentiated bacteria that syn- thesized [3-galactosidase, but had lost the ability to proliferate into colonies. Future stucie~ will have to determine the ,zellular basis for macroscopic biochemica! Jifferentiation in bacterial colo~Aeso

Bacteria t om blue plasmid-free MS1534 sectors were particularly suitable as lineage pr~,genitors for studying pattern variation, because they retaiped the Mudlac e~ement for vital staining, yet would not undergo sectoring due to plasmid instability which might obscure other kinds of clonal variation events. Accordingly, a number of deeply pigmented sectors from MS1534 colonies were carefully picked into broth suspensions, and the resulting cultures were labellec~ MS i 534.01-1534.09. To keep track of various cultures and their subclones, the. following nomenclature convention was used [11] : each subclo- ning event was recorded by adding a non-zero digit to the decimal expansion following MS~534 (iig. 5).

In the first stages of establishing different lineages, particulm- attention was paid to sectors which grew beyond the normal limits of colony expan- sion. When inoctflated with equal spacing, E. coli colonies tend to grow to a uniform size which depends upon the number of colonies per plate (diameter inversely proportional to density). Certain cultures produced colonies with , expansive, sectors that grew farther onto the agar surface than the rest of the colony, indicating some alteration in sensitivity to the factors which nor- mally restricted colony spread. Several of these expansive sectors were picked for subculturing and subcloning. For example, MS1534.047 was e~;tablished by picking a dark expansive sector on an MS1534.04 colony.

Figure 6 illustrates the sectoring colonies produced on XGal indicator agar by spotting 1 ;~1 aliquots of the original .047 culture and its derivatives. Four subcultures were obtained by ,diluting .047 into TYE broth, and eight subciones were obtained by suspending isolated single colonies that grew after plating a dilution of the .047 culture on TYE agar. As can be seen, the mbclonal cultures from single colonies (.0471-.0478) produced restricted colonies with occasional dark expansive sectors that resembled tt:e original .047 sector. The spots of .047 and it~ subcultures were much more f lamboyant and contained a variety of expansive sectors with different LacZ phenotypes. These were picked as indicated to establish the .04701-.04709 subclonal cultures. The dif- ference in colony types seen in figure 6 was ass~:med to reflect (at least in part) the different ages of the cultures when spotted [11]. The .0471-.0478

E. COLI (MUDLAC) COLONIES 89

cultures were 3-days old when plated, whereas the .047 culture was over a month old before subculturing and thus had had time to produce the variety of cell types which gave rise to the different sectors. Consistent with this assumption was the observation that the .047 colonies produced by differem subcultures contained repetitions of the same kinds of sectors. For example, sectors .04701, .04704, and .04707 all came from different colonies but had the same phenotype characterized by a smooth round periphery and a low level of 13-galactosidase activity concentrated in a faint ring just inside the sector edge.

FIG. 6. ~ Colony patterns produced by MS1534.047 and its derivatives.

These colonies art~se from 1-~1 spot inoculations (about 105 bacteria) of the original MS1534.047 culture (top right), dilution subc'~lmres (remainder of top two rows) and 8 single-colony ~ubclonal cultures (.047-.0478, bt.~:tom three rows), The subcultures were prepared by diluting the original culture either 10--' or 10 -4 in TYE broth followed by growth to saturation at re, ore temperature. The subclo~al cultures were prepared from isolated colonies produced by spreading_ a small a!iql,m_ . . . . . . . . . . m" ,~,~ 10-~ ,~.u,o,'~:"-'" on TYE agar. Note the similaridc~ in L~,; expansfve sectors on the colonies produced by the original MS1534.047 culture and ks four subcultures, except for the one it. ~:he middle of the se- cond row, where intercolonial inhibitory effects appear to have pro ~ented sectorial pro- liferation. Y~e expansive ~;ectors which were picked from .047 colonies to establish the lineages schematized in figure 5 are indicated by the last tw~ digits of the designation. Photographed after 13 days.

90 J.A. ,SHAPIRO A N D N.P. HIGGINS

instability of the expansive growth phenotype.

When expansive sectors were picked into broth, grown to saturation and then spotted on XGal indicator medium, the resulting colonies generally displayed a more expansive growth phenotype than those produced by the parental culture. Nonetheless, as illustrated in figure 6, it was frequently observed that many of the individual bacteria isolated from such an expan- sive sector suspension produced cultures that grew into colonies with the restricted parental growth phenotype. This observation suggested reversibili- ty of the clonai variation events which eriginally had led to the formation of sectors with a more expansive growth phenotype. However, heterogeneity of the original sectorial popvladon could not be excluded.

To examine the hereditary transmission of the expansive phenotype, resuspended sectorial populations were subjected to two sequential subclo- nings by single colony isolation. This procedure made it possible to examine the progeny of single CFU from an expansive sector. One well-studied exam- ple was sector .04705 (fig. 5 and 6). When this sector was resuspended and ailuted for platflig, many of the resulting colonies gave rise to cultures which had retained tile expansive growth phenotype. Thus, the new phenotype could be transmiLted through individual CFU. When these e×pansive single-colony cultures were dilu:ed and plated m turn, the majority of the resulting colonies gave rise to cultures which had returned to the restricted growth phenotype. Thus, tile new phenotype was lost in the progeny of single CFU and was therefore reversible. It is important to note that phenotypic shifts occurred in both directions because these restricted colonies frequently gave rise to ex- par~sive sectors which could then be picked and subjected to the same analysis with similar results.

During the analysis of bacteria undergoing changes from expressing ex- pansive to expressing restricted growth phenotypes when spotted on XGal indicator agar, an important additional difference betweer~ the two types of bacteria was noted. When an expansive sector was suspended, grown to satura- tion, diluted and plated on TYE agar, two colony size3 could be seen after about 16-h incubation (fig. 7). However, after an additional day's incuba- tion, colonies which were originally in different size classes had grown to become indistinguishable. To see if this difference in the sizes of young colonies reflected some basic differentiation between two classes of bacteria or, alter- natively, was merely the result of accidental variations in colony growth kinetics, coloifies of each size were picked into brotL , ; ter overnight incuba-

Flo. 7. - - Colonies produced on T Y E agar by spreading an aliquot o f a 10 -4 dilution o f the MSt534.04705171 culture.

The two panels show a single field of colonies photographed after 16 h (top) or 40 h (bot- tom). Note the colonies over the dash and above and to the left of the number 4 which were almost imperceiztible at 16 h and have become full-size at 40 h.

0 2 r"

,,>

0

92 J.A. S H A P I R O A N D N.P° HIGGINS

tion ~,.nd allowed to grow to saturation. The two classes of colonies were also marked and subsequently picked after an additional day's incubation. When the resu!ting <<large colony >> and << small colony >> cultures were spotted on XGal indicator agar, there was a systematic difference in the phenotypes of the resulting colonies (fig. 8). bma!! colony cultures produc~_d expansive col- onies and large colony cmtures p;oduced restricted colc, nies. While there were a few exceptions to this rule, the degree of correlation w':-~ quite high (table I). A difference was also evident in the phenotypes of ti~e expansive colonies depending on whether they were produced by small colony suspensions established from i6 h or from 40-h colonies. The a0-h << small colony >~ cultures produced spot colonies with a uniform outer fringe of expansive growth,

TABLE I. - - Morphogenet ic analysi, s o f large and small colonies .

Large colonies - , Small colonies MS1534 subclone Restr icted spot colonies Expans ive spot colonies

.04721013 9 /12 12/12 • 04705041 1 0 / ! 2 12/12 .04705171 12/12 1! /11 .04705241 11/12 9 /12 .04705291 i 2 / 1 2 12/12 .04705351 11/12 11/12

Subclonal populations were diluted and plated on TYE agar at 32°C. After 16-22 h, colonies were scored as large or small and picked into TYE broth either immediately or after an additional day's in- cubation. After one or two days' incubation at morn temperature, 1 ~tl of each saturated broth suspen- sion was spotted on indicator agar and the plates were incubated at 32°C for 5-15 days, at which time the colonies were scored as restricted (with, at most, two sectors) or expansive (with three or more sec- tors). As illustrated in figure 8, the scoring was usually quite clear. The numbers indicate colonies scoring as expected out of those tested.

FIG. 8. - - Expansive and restricted colony phenotypes displayed by ~ large colorly ~; and ~ small colony ~ cultures.

The MS 1534.04705171, .04705291 and .04705041 cultures were established by picking expan- sive sectors from the restricted .0470517, .0470529 and .0470504 spot colonies into TYE broth (cf. fig. 5). These cultures were diluted and plated on TYE agar, incubated, and the resulting (<large~) and <(small>> colonies (cf. figure 7) picked to establish single-colony subclonal cultures, which were grown to saturation at room temperature and then spotted on XGal indicator abar. The too, middle, and bottom plates display colonies from subcloncs of .04705041, .04705171, and .04705291, respectively. Each plate was divided into four rows, and the cultures were spotted as follows: top row with those from small colonies picked at 16 h; second row with those from large colonies picked at 16 h; third row with those from previously small colonies picked at 40 h; and bottom row with those from previously large colonies picked at 40 h. Note the partial, sectorial nature of the flamboyant fringes on some of the colonies in the top row of each plate and the one putative large- colony culture giving flamboyant growth in the bottom row of the top plate. Photogra- phed after 9 days.

94 J.A. S H A P I R O A N D N.P. HIGGLNS

whereas the 16-h colony cultures often produced restricted spot colonies with a number of independent expansive sectors. Sometimes adjacent expansive sectors fused to give the appearance of a continuous expansive growth ring.

Two conclusions about the expansive growth phenotype could be drawn from these results. One was that ttze initial stages of growth on TYE agar were markedly different for CFU which gave rise to cultures that produced expansive spot colonies when compared to CFU which gavc rise to cultures that produced restricted spot colonies. The second conclusion was that the protocol used here to establish subclonal cultures from <~ expansive >> CFU yiel- ded mixed cell populations so that the expansive colonies produced by these populations were actually restricted colonies with many expansive sectors. The proportion of cells pro&~cing expansive sectors was inversely proportional to the amount of growth that occurred in broth after picking. This suggested that cells destined to produce expansive colonies frequently lost this character and gave rise to progeny with the parental morphogenetic potential during growth in liquid medium. Experiments with liquid cultures inoculated with single CFU supported this idea: growth to between 8 and 16 CFU often pro- duced mixtures of the two colony types when plated on TYE agar.

Changes in Mudlac DNA associated with pattern variation.

To study genomic changes associated with some of the LacZ variation observ- ed in the MSi534 lineage, the Mudlac sequences were examined in DNA ex- tracted from a number of related cultures descended from MS1534.047. Particular attention was given to subclones of the .047053 culture for two reasons: (1) this single-colony culture displayed the expansive growth phenotype but yielded nine subclones with the restricted phenotype, and (2)the nine subclones displayed three different LacZ phenotypes (fig. 9). The cultures for DNA preparation were obtained by streaking old suspensions (600 + days) of .047011, .C470531, .0470534, .0470535 and .0470538 on XGal indicator medium and picking individual colonies for analysis. These cultures produced colonies with more than one phenotype, and sibling subclones with high and low levels of LacZ activity were chosen from all of them except for .0470535 (fig. 10). In the case of .0475341, the LacZ phenotype was unstable (going from high to low levels of expression), and four sibling subclones were also picked (.047053411/53412 with high LacZ and .047053413/53414 with low LacZ).

Figure 11 shows the results of Southern hybridization on DNA digested with EcoRI plus HindIII using two different probes: (a) an oligonucleotide probe complementary to the Mu c repressor sequence and (b)an oligo- nucleotide (MuR26) complementary to the right end of Mu. The repressor seqoe~ce iies to the left of the HindlII site at coordinate 1 kb (fig. 1)o Thus, the probe revealed junction fragments of the ~.zft end of the Mudlac element joined to adjacent sequences. The right-end probe also lay outside restriction sites for the two enzymes used and so revealed junction fragments of the right end joined to adjacent sequences.

E. COLI (MUDLAC) COLONIES 95

The Southern hybridization results shown in figure 11 were consistent with the strain pedigrees and demonstrated ongoing MudH1681 genetic activity in the MS1534.047 lineage. All of the cultures examined contained common HindlII-EcoRI junction fragments measuring = 1.4 kb at the left end and = 7 kb at the right end. Since both .04701 and .04705 descendants shared these fragments, we assume that they were present in the genome of MS1534.047 and represent the two ends of a single MudlI1681 in the ch~omosome. Most of the cultures contained additional bands, consistent with MudlI1681 transpositions to new sites in the genome. Both .0470111 and .0470112 had bands that were different from those of the .047053 line. Among the .047053 progeny, the two .0470535 descendants only had the common bands, while .04705311 had different additional bands than did the .0470534 and .0470538 descendants. Data from Southern hybridization experiments

FIG. 9. - - Colonies produced by MS1534.047053 and its descendants.

The top row shows colonies produced by spotting the original .047053 culture (right) and two dilution subcultures. The bottom three rows show the colonies produced by si~gle-colony subclonal cultures. From right to left, these cultures were .0470531, .0470532, .0479533 (second row); .0470534, .0470535, .0470536 (third row); .0470537, .0470538, .0470539 (third row). Nofe the three distinct phenotypes in the subclcnes. The one displayed by .0470535/536 was closest to that of the .047 subclones (fig. 6). The .0470531/537 phenotype had a lower overall LacZ level and slightly more extensive colony spread. The .0470532/533/534/538/539 phenotype showed very little LacZ activity (detectable mostly in the colony center) and slightly more extensive colony spread. Photographed after 16 days.

96 J.A. S H A P I R O A N D N.P. H I G G I N S

with Taqi or H m c l I + H i n d l I I + E c o R I digests confirmed the results "- 111

figure 11 and yielded additional information about the °04705312 culture which did not show up in that experiment and about the .047011 subclones whose larger HindIII + EcoRI fragments displayed faint hybridization. The .04705312 culture displayed only a single junction fragment with each probe, and these fragments were shared with all the other cultures. The .0470ii1 culture : 'qplayed three junction fragments with each probe, and two of t lese were present in .0470112 DNA.

The .0470534 and .0470538 cultures produced spot colonies with similar phenotypes displaying little LacZ activity (fig. 9), and their descendants shared almost all the same new junction fragments" 3 kb and 4 kb at the left end and 4 kb and > 8 kb at the right end. The .04705341 culture displayed a distinct 6-kb band at the right end, while the .04705381 culture lacked the 4-kb band but appeared to have an extra > 8-kb fragment at the right end. Densitometer tracings showed that the 3-kb left-end fragment was present in do,lble molar ~r,,,-,,,,,,~ in the DNA of 0a7 .n~:~A1 ~.~vn¢"~11 and .047053412.

In addition to very clear clonal patterns of variation in the distribution of MudI11681 junction fragments~ the data indicated certain interesting cor- relations between the number of these elements and the LaeZ phenotypes. In the .0470111/.0470112 and .04705311/.04705312 pairs, it was the culture with higher LacZ activity which had an additional MudII1681 insert in the genome. Thus, it is possible in these cases that the higher expression can be attr;buted to the additional element, perhaps due tc fusion of lacZ to an ac- tive outside promoter. The .0470534 and .0470538 descendants presented a more intrigui~.g picture. The original phenotypes of these cultures matched those of the .04705342 and .04705382 subclones with low LacZ activity (fig. 9 and 10), and these two cultures shared all MudlI1681 junction fragments (fig. 11). Thus, it appeared that .0470534 and .0470538 were sibling descen- dants of a variant with two new MudII1681 insertions and, further, that the .04705341 and .04705381 subclones with higher LacZ activities arose as novel variants during the 600 + days that passed before the 0470534 and .0470538 cultures were streaked to obtain single-colony cultures for DNA extraction. In the case of .04705341, the change leading to higher expression correlated with the appearance of a new 6-kb junction fragment at the right end and duplication of the 3-kb fragment at the left end. The correlation between these

FIG. 10. - - Colony phenotypes displayed by the cultures used for DNA extractions.

Single-colony cultures of .047011 and .0470531/534/535/538 subclones were grown overnight as confluent lawns on TYE agar and suspended in buffer for DNA extractions. An aliquot of each culture was diluted into TYE broth and plated on XGal indicator agar. Two dark and two light colonies from .0475341 were aiso picked and subjected to the same process. Photographed after 4 days (.047011 and .047534 descendants) or after 3 days (.0470531/535/538 descendants).

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98 J.A. SHAPIRO AND N.P. HIGGL1VS

changes and the more highly expressed LacZ phenotype was confirmed by isolating two variant subclones with lower levels (.047053413/53414)and showing that they had returned to the .04705342 MudlI1681 structure (two sibling subclones wb.ich retained tile higher level LacZ phenotype, .047053411/53412, also retained the additional junction fragments). The case of .04705381 was different from that of .04705341 at both the DNA and phenotypic levels (fig. 9 and 10). No change at the left end of the MudlI !681 elements was detectable in DNA from .04705381 by the methods we used, but at the right end there was a loss of the 4-kb fragment and possible amplificatien of the > 8-kb fragment. Thus, at least two different kinds of hereditary cl~an~es were revolved in increasing Mudlac expression in bacteria with the .0470534/538 DNA structure. We do not yet know the com- plete structures of the MudlI1681 elements in tile .0470534/538 descendants, but the instability of .04705341 and the possible amplifications of some fragments suggested that there may have been recombinational interactions between them.

One additional feature of LacZ expression in these cultures was revealed by testing the effect of a pOXGen::Muc+pAp! plasmid on XGal staining patterns in exconjugants. LacZ activity was not affected by the Muc + pAp l prophage in exconjugants of .0470111, .04705341 and .04705381, but expression was strongly inhibited in exconjugants of .04705352 and .04705353 (fig. 3). Thus, expression from the original MudII1681 insert remained sensitive to Mu functions after five subclonings, l~iJt expression from .047 descendants with additional inserts was not inhibited. It is interesting to note here that .0470534 and .0470538 displayed reduced expression compared to .0470535 (fig. 9), indicating an inhibitory effect of the additional MudI11681 elements.

FIG. 11. - - Southern hybridizations of DNA from MS1534.047 subclones with probes for the extremities o f the MudII1681 element.

The top panel shows the results with the MuR26 probe for the right end, and the bottom panel shows the results with the Mu c probe for the left end (fig. 1). The last four or five digits of the subclonal designation are given above each lane, and the positions of size standards (BRL l-kb ladder) are indicated at the right of each panel ; 10 ~g DNA from each culture was digested with HindIII + EeoRI, phenol-extracted and ethanol precipitated, and each lane was loaded with 2.5 lxg of cleaved DNA before electrophoresis in 1 % agarose for 14 h at 10V/cm at room temperature. The gels were trimmed, denatured in 0.4N NaOH, anci DNA was transferred directly to Zeta-probe nylon membranes. The oligonucleotide probes were labelled at the 5' end by polynucleotide kinase reactions [6], applied to NACS columns (BRL) in 10 mM Tris-HC1, 100 mM NaCI, 1 mM EDTA pH 7.5, and eluted in 10 mM Tri~-HCI, 1 M NaCI, 1 mM EDTA pH 7,5. Hybridization was carried out with standard methods [6l in 4 x S S C , 0 . 1 % SDS at 45°C. Filters were stripped for rehybridization by washing in 0.2 x SSC, 0 . 1 % SDS above the T m of the particular oligonucleotide.

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100 J.A. SHAPIRO A N D N.P~ HiGGINS

DISCUSSION

To account for the specificity of E. coli colony patterns, it is necessary to assume that there are systems for integrating when many kinds of biochemical activity take place during colony development. Changes in these integrating systems were detected by observing the formation of sectors with novel phenotypes. Since these novel phenotypes included altered lacZ expres- sions, it appeared that the integrating systems also regulated expression from the MudlI1681 elements. Defining the nature of the cellular events which resulted in the appearance of bacteria expressing new phenotypes remains a major challenge. At least two distinct types of events were observed in these studies.

i) One class of events produced expansive sectors v~lth novel LacZ expres- sions. Some of the bacteria derived from these sectors produced expansive colonies when grown into cultures and spotted on XGal indicator medium, while some produced restricted colonies indistinguisbab!e from those of the ancestral culture. The bacteria producing expansive colonies also followed a novel morphogenetic pathway with characteristic colony growth kinetics when plated on TYE agar (.fig. 7). The eveWs occurring at the origins of these expansive sectors did not permanently alter MudI11681 because descendants of such expansive sectors (e.g..04705352/5353) retained the original inser- tion which was still sensitive to Muc+pApl inhibition. Thus, after the first elonal variation event, both phenotype and Mudlac genotype could return to their prior status during further growth. It will be interesting to see if such events involved either reversible changes in DNA sequence organization, as in phase variations of pathogenic bacteria, or chemical modifications of Mudlac DNA, similar to what has been documented in eukaryotes.

2) The second type of clonal variation event occurred during prolonged storage in liquid medium and produced variant subclones which generally displayed rather stable novel phenotypes (fig. 10). In these cases, char~ges in the MudII1681 element DNA could be correlated with the new pattern of LacZ activity. The Mudlac elements were designed to connect lacZ to transcription and translation signals outside the right end of Mu (fig. 1 ; 2, 3). Such linear connectim?s to novel control sequences along the bacterial chromosome will help to explain the observed differences in lacZ expression in those cases where increased LacZ activity correlated with the presence of additional Mudl11681 insertions (e.g. °0470111 compared to .0470112 and .04705311 compared to .04705312). However, there must have been other factors involving interac- tions between different regions of the genome which regulated lacZ expres- sion. These interactions were seen most clearly in the correlation between changes of lacZ expression from the initial MS 1534 chromosomal Mud II 1681 insert and the presence or absence of unlinked Mu derivatives: (a) the loss of R388::MudII1681 relieved inhibition of LacZ (fig. 1); (b) the introduc- tion of ,~OXGen::Muc+pApl inhibited LacZ (fig. 2); and (c) the appearance of additional MudII1681 insertions in the progenitor of the .0470534/538

E. COLI (~V/UDLAC) COLONIES 101

subclones inhibited LacZ (fig. 9). These inhibitory effects were not unique to the particular chromosomal MudI11681 insertion in MS1534; a similar ef- fect was exerted by chromosomal Muc + pApl prophages on lacZ expression from a plasmid MudlI1681 element [13]. Whether the inhibition was due to the action of Mu repressor or of some other Mu function will have to be deter- mined b~ a genetic analysis of piasmids carrying Mu derivatives !t will also be necessary to explain why expression from some Mudlac elements is inhibited but expression from others is not.

The finding of at least two different kinds of clonal variation events af- fecting colony morphogenesis on agar is reminiscent of some of the earliest studies of hereditary changes in bacterial cultures [4]. The classical bacteriologists were much concerned with coincident changes in multiple pro- perties, such as virulence, antigenicity, capsule synthesis, fermentation specifici- ty and colony morphology. These complex variations were of great practical importance 0ecause they affected vaccine production and the diag=osis of infectious disease. Today, when we wish to produce genetically engineered bacteria for bioteehnological and environmental tasks, understanding the systems which control multicellular phenotypes is equally usef,.l. Since the study of bacterial colony development is a subject that will teach us about rules governing the coordinated behaviours of large numbers of bacteria, this line of research continues the Pasteurian tradition of a single microbiological science in which theoretical advances and practical applications move for- ward together.

RI~SUMI~

VARIATION DANS L'EXPRESSION DE LA ~3-GALACTOS1DASE

,~ PARTIR D't~LEMENTS Mudlac PENDANT LE DI2VELOPPEMENT

DES COLONIES DE ESCHERICHIA COLI

Au cours de leur d6veloppement, les colonies bact6riennes r6v61ent Faction de syst6mes de r6gulation multicellulaire de l'activit6 g6n6tique. Les colonies produites, sur un milieu indicateur g61os6, par des souches de Escherichia coil portan* i'616ment de fusion g6n6tique Madlac, font apparaTtre des aspects orga- nis6~ de i'expression dfff6rentielle de la t3-galactosidase. L'un de ces aspects cst la pr6sence d'anneaux concentriques de ph6notypes distincts, contenant des cellules g6n6tiquement semblables. Un autre aspect est l 'apparition de secteurs de population de ph6notypes nouveaux, montrant fr6quemment des variations de plusieurs caract6res tels l'activit6 enzymatique, l'agr6gation mul- ticellu!aire, la vitesse d'6ta!ement sur la g61ose. L'analyse par sous-clonage des secteurs montrant une croissance importante r6v6le qu'ils contiennent des bact6ries exprimant des s6quences morphog6n6tiques nouvelles sur plus d'un milieu. Ces s6quences peuvent &re transmises aux g6n6rations ult6rieures mais le caract~re est r6versible pour les bact6ries en cours de croissance en milieu

102 J .A . Sh'API.;,!O A N D N.P . H i G G I N S

liquide. Des variations clonales plus s~ables de I'expres~]on de Ia ~-galactosidase surviennent au cours du stokage des bactdries en milieu liquide. La p!upart de ces changements sont correles avec des transpositions ou des rearrange- ments de sequences Mudlac. Certaines de ces modifications imptiquent des interactions entre les elements Mudlac et des derivds non lies du phage ~x!u.

Ces rdsultats mettent en evidence t ' intervention ~e nouveaux syst+mes de contrele pour la regulation de i'expression de la ~-galactosidase, a i,,r~ir des sequences lacZ dans un element Mudlac chromosomique, et demontrent qu'il existe au moins deux sortes de variations clonales qui affectent la morpho- gen6se des colonies bacteriennes.

MOTS-CLES: Esckelqchia coil, Colonie, ~-Ga!actosidase; Variations clo- nales, Sequence Mudlac.

ACKNOWLEDGMENTS

We wish to thank Pamela Brinkley and Nancy Cole for technical assistance. This research was supported uy grants from the National Science Fouvd,t~un

(DCB-8416998) to J.A.S. and from the National Institutes of Health to J.A.S. (CA-19265) and to N.P.H. (GM-33143).

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[2] CASADABAN, M.J. & CHou, J., In vivo formation of gene fusions encoding hybrid ~-galactosidase proteins in one step with a transposable Mu-lac t~ansducing phage. Proc. nat. Acad. Sci. (Wash.), 1984, 81, 535.

[3] CASTILHO, B.A., OLFSON, P. & CASDABAN, M., Plasmid insertion mutagenesis and !ac gene fusion with Mini-Mu bacteriophage transposons. J. Bact., t984, 158, 488-495.

[4] HAD'A2Y, P., Microbic dissociation. J. infect. Dis., 1927, 46, 1-312 (also publish- ed as a monograph).

[51 LEGRObX, R. & MAGROU, J., I~tat organis6 des colonies bacteriennes. Ann. h~st. Pasteur, 1920, 34, 417-431.

[61 MANbXrIs, T., FRrrSCH, E.F. & SAMBROOK, J., ¢¢ Molecular cloning: A laboratory manual~>. Cold Spring Harbor Laboratory, New York, 1982.

[7] MARRIE, T.J., NELLIGAN, J. & COSTERTON, J.W., A scanning and transmission electron microscope study of an infected endocardial pacemaker lead. Cir- culation, 1982, 66, 1339-1341.

[8] SHAPIRO, J.A., Transposable elements, genuine reorganization and cellular dif- ferentiation in Gram-negative bacteria. Symp. Soc. gen. ?ficrobioi., 1984, 36, 169-193.

[9] SHAPIRO, J.A., The use of Mudlac transposons as tools for vital staining to visualize clonal and non-clonal patterns of organization in bacteri: l growth on agar surfaces. J. gen. Microbiol., 1984, 130, 116%1181.

E. COLI ( M U D L A C ) C O L O N I E S t03

[10] SH.~PiRO, J.A., Scanning electron microscope study of Pseudomona's p~:lida colonies. J. Bact., 1985, 164, 117i-i t81.

{11] SHM,I.RO, J.A., Control of Pseudomonas putida growth on agar surf'aces, in ,,,The Bacteria, (J.R. Sokatch), voI. X (pp. 27-69). Academic Press, New York, London, 1986.

[12] SHAPIRO, J.A., Organization of developing E. coli colonies viewed by scanning electron microscopy. J. Bact., 1987, i97, 142-156,

[i3] SHAPrRO, J.A. & BRtNKLEV, P.M., Progl-amming of DNA rearrangements involving Mu prophages. CoM Spr. ttarb. Sym!2. quanL Biol., 1984, 49, 313-320.