BACKGROUND
bacteria can display a plethora of multicellular forms ; their structure and appearance depends on factors such as the presence of nutrients or neighbors.
concepts of "body" and "community", as developed for multicellular sexual eukaryots, became, however, somewhat blurred upon attempts of their application to microorganisms.
is differentiation of multicellular units in bacteria comparable to embryonic development, to the establishment of an ecosystem?
is it even the place of darwinian evolution on a micro-scale?
multicellular bacterial bodies can be viewed as ecosystems negotiated by myriads of  specialists .
each cell is understood as an individual playing its own game according to resources, energy costs, and complicated informational interactions with others.
however, patterning of multicellular bodies remains beyond interest, at the most being viewed as a passive outcome of physical forces.
a multicellular bacterial community may, however, be also perceived as the prevalent form of bacterial existence, with a genuine ontogeny.
to create and maintain such elaborated structures, a great deal of communication, regulations, mutual understanding, and cooperation takes place in bacterial morphogenesis.
differentiation in such a bacterial body  may proceed via genetically differing subclones fulfilling different roles, and appearing reproducibly at characteristic periods of cultivation  <cit> .
sophisticated networks of chemical signals  <cit> , the scaffolding of extracellular matrix  <cit>  and even cell-to-cell contacts  <cit>  may enable attaining and maintaining the integrity of the body.
research in this direction has been greatly accelerated in last two decades by the discovery of the phenomenon of quorum sensing .
bacterial populations react to such signals - and build multispecies bodies accordingly - in a context-dependent manner  <cit> .
a plethora of quorum-modulating signals, such as indole or furanole derivatives, was also described  <cit> .
the study of model monoclonal populations may contribute to understanding colony morphogenesis, providing the possibility to examine how, and even why, bacteria exert themselves towards "species-specific" appearances.
we have previously demonstrated that colonies of serratia marcescens can be viewed as multicellular bodies with genuine embryonic development  <cit> .
colonies displayed finite growth and clone-specific formative processes; even a disorganized cell slurry  could establish a regular pattern and embark on a typical developmental pathway.
under standardized culture conditions on rich semi-solid media, the final shape and patterning of bacterial bodies depended essentially on four initial settings:  amount, density, and distribution pattern of founder cells;  the configuration of surrounding free medium;  the presence and character of other bacterial bodies sharing the same niche; and  self-perception, resulting in delimitation towards other bodies.
here we further investigate the development of bacterial bodies and their interaction with close or distant neighbors of identical, or different, clonal origin.
we also propose a formal model that can account for some of our experimental results.
RESULTS
colony patterning in clonal variants of serratia rubidaea
we have chosen a wild type strain of serratia rubidaea, a gram-negative, facultatively anaerobic, rod-shaped bacterium of the enterobacteriaceae family , producing usually red glossy colonies without any distinguished structural pattern except of a slightly darker touch in the middle, as our starting material.
this strain will be further referred to as the r  strain.
the r strain occassionally segregated colonies of variant shape or colouring upon prolonged cultivation in liquid media and serial plating; from these, we selected three stable, no longer segregating clones used in this study:  w , a colorless variant of r;  f  clone named after its cross-section profile ;  fw  - a colorless, morphologically slightly different derivative of f. f and fw colonies are characterized by a typical massive rim, hence rimmed, in contrast to rimless  colonies.
colonies of the parental r strain and all daughter clones have a finite growth, their diameter being in rimmed clones about  <dig> mm, in rimless ones about  <dig> mm .
colonies ripen into final color and pattern by about 7th day upon planting, while still growing slowly, to reach their final diameter by day  <dig> .
as the f morphotype plays a central role in this study, its development deserves a closer scrutiny.
no matter how the colony was planted, in days 1- <dig> it grows as a central navel: a compact body on the agar plate only slowly propagating sideways.
this phase is followed in days 3- <dig> by spreading of the flat interstitial circle.
microscopic observations revealed a margin of extracellular material containing small swarms of bacteria at the colony periphery at this stage , a phenomenon well established in serratia sp.
.
in days 5- <dig> this lateral propagation comes to end and the peripheral rim is formed; the central navel grows red in this phase.
in following days, the rim also turns red and the growth proceeds towards a halt.
the flat interstitial ring remains colorless .
fully developed f colonies can be obtained only if bacteria are planted in densities 1- <dig> per 9-cm dish.
at the density of tens per dish, the colonies grow much smaller; below a critical distance, they tend to fuse into a confluent colony with many centers bounded by a common rim .
at densities of hundreds per dish, colonies remain very small and undifferentiated.
yet higher density of planting leads to a compact, undifferentiated body - a macula .
the scenario is similar for all four clones used in this study, except that rimless colonies  never fuse .
the development and behavior of standard colonies  were essentially independent on the way of planting , provided that the diameter of planting was ≤  <dig> mm.
similar observations have been made previously for a different serratia isolate  <cit> .
close encounters of bacterial bodies
in dense plantings, clonal rimmed colonies tend to merge into confluent colonies .
in a mix of lines differing in color , the origin of each participating colony is revealed by the color of its rim.
if colonies were dotted close  to a growing previously planted colony , the resulting fused body resembled a confluent colony, with a common rim and separate centers.
the effect was more profound with younger colonies.
if a similar protocol was followed with the rimless clones, colonies remained thoroughly delimited, whether in a single culture , or in an rw mixture : no fusions were observed, and clearly distinguishable furrows developed between bodies in contact.
similarly, dots applied close to an older colony became inhibited in growth, but kept distinct from its growing older neighbor .
upon close encounters between rimless and rimmed bodies , the r colonies grow faster and influence rimmed colonies in four ways:  if planted early enough, r colonies can engulf an immature rimmed colony; its body, however, survives and cells can be recovered from such a mixed body .
if f colonies are allowed to grow in the vicinity of an r colony as independent bodies up to 3rd day of cultivation , they develop to a new colony phenotype with a massive white rim with a thin colored ring at the inner side .
cells from x colonies restore the original f phenotype upon subsequent planting.
a rimless  dot added to a rimmed colony  tends to proliferate around the older partner and block its further growth ; the younger the rimmed colony is, the more profound the effect.
similar results were obtained with w dots .
again, the rimmed colony remains compact  and contains live cells.
the engulfment potential of the rimless colony is even more profound in a reverse arrangement, i.e.
dotting of a rimmed colony to an older rimless partner .
planting of mixed suspensions
mixed suspensions of two rimmed clones  produced varying and unpredictable colony patterns , suggesting an extreme sensitivity of such mixtures to initial conditions .
samples taken from both center and periphery of such chimeras revealed the presence of cells belonging to both clones in the central zone, and sometimes also in the periphery .
these results contrast with previous findings on a different strain  <cit> : in that case, however, both subclones tended to establish separated "areas of influence", essentially as referred below for rw mixtures.
if a colony was established from a mixture of two rimless clones rw, the center of the colony remained a mixture of both clones, sending radial monoclonal sectors as the colony grew , as if rimless clones were reluctant to cooperate towards a common end.
if a mixed suspension of rimmed  and rimless  suspension is dropped to initiate a colony, the cells of the rimmed clone remained confined to the central area, whereas the growing periphery is composed exclusively of r cells , similar to the above-described engulfment of rimmed colonies by rimless ones.
again, the inhibited strain confined to the center remains viable and can be recovered upon re-planting.
the behavior of rfw, wf and wfw colonies is analogous to the rf mixture .
effects of planting layout
the plasticity of the typical f body plan was investigated by streaking or blotting cell suspension in various geometrical settings.
if the width of the plant in one direction does not exceed a critical diameter somewhat smaller than the adult f colony diameter, the body strives to maintain the features of the colony , even if deformed to a large extent .
blotting of ring bodies using circular plastic stamps was even more informative, with results depending on the diameter of such rings .
smaller rings healed the central cavity and proceeded towards a normal  colony shape; with increasing diameter, up to the critical size, this colony phenotype was maintained, even if with a central hole in the middle.
above the critical diameter , a ring-like colony acquired an additional inner rim - resembling linear colonies  as in figure 3c, but curled.
we have also confirmed the previously described phenomenon of "ghost" colonies  <cit> , originally documented on a different strain.
briefly, colonies planted at the background of multiple  colonies became inhibited, or even "dissolved" on the background .
this is the case even in synchronous cultures if, at the beginning, the background is represented by at least about  <dig> colony-forming units.
such a background can keep at bay a plant as dense as  <dig>  <dig> cells, preventing its development towards a colony.
the effect is more profound when background colonies are older.
with this information in mind, we return to ring colonies.
a colony was planted into the center of a ring colony of greater diameter, or a ring colony was blotted around a growing f colony.
both bodies represent a "background" to each other, depending on the succession of plating.
results in figure 3d show that the synchronous planting of both structures leads to disruption of the structure of the central colony, but no change in the structure of the ring.
colonies planted on the background of older rings became inhibited.
on the other hand, when the ring is planted around an older colony, it develops into a typical structure, only with more profound reddening of the inner rim - again confirming that a developing colony can perceive the presence and layout of its neighbors.
long-distance interactions between colonies and maculae
to examine the putative long-distance signals between bacterial bodies, colonies  were planted to the vicinity of maculae of two different serratia clones  or an unrelated bacterial strain .
maculae and colonies either shared the same agar plate, or were separated by a septum.
when f colonies were planted in varying distances from an f macula , the closer was the macula to a colony, the quicker the reddening of that colony.
at the same time, the colony deviated from its typical structure to an extent inversely related to its distance from the macula.
the graph in figure 4a shows that the transition point between aberrant and standard patterns lies approximately  <dig> to  <dig> mm from the macula, corresponding roughly to the diameter of adult f colonies.
this breakdown of the colony structure was not observed with the serratia isolate characterized previously .
the fw macula exhibited weaker effects than its f counterpart, and elicited the loss of structure only when older .
the r macula, as well as a macula of e. coli, induced, again, formation of the x phenotype in colonies of the f clone .
no such x-like structures were observed when r colonies were planted in the vicinity of an e. coli macula .
communication across obstacles
if the macula and colonies have been grown on opposite sides of a septum dividing the dish, preventing diffusion in the semi-solid agar matrix but allowing gas exchange, the effect of macula was qualitatively similar to that on a shared plate, albeit the distance between the bodies appeared as if increased for simultaneously planted bodies .
if, however, the macula across the septum was at least  <dig> days old at the time of colony inoculation, colony development was similar to controls sharing a continuous plate , suggesting that older bacterial bodies produce volatiles that may be absorbed by the agar medium.
maculae of a different strain  or species  also affected development of f colonies across an obstacle; however, they never induced formation of the x structure across the septum, indicating that signals diffusing through the semi-solid substrate, distinct from those carried by the gas phase, are indispensable for the development of the x pattern.
the effect across the septum is not bound to an organized body of the macula: bacterial suspension  kept across the septum exerted an effect comparable to that of a macula .
nature of signals between bodies
in further experiments, we investigated the longevity of a putative macula-derived signal.
a macula was grown for  <dig> days on a cellulose membrane laid on the agar on one side of a septum, then removed, leaving empty macula-conditioned agar.
immediately after macula removal, colonies were dotted into the neighboring compartment containing free macula-exposed agar .
the results are indistinguishable from controls shown in figure 4a, i.e.
from the situation when the macula persisted in the neighboring compartment.
to test the obvious possibility that such free, but macula-exposed agar "took the smell" during macula growth, medium in the non-inoculated compartment was removed at the time of the macula removal, and replaced by "virgin" agar transferred from another, empty plate.
as also shown in figure 5a, the development of colonies was essentially the same as on macula-exposed agar.
thus, macula-conditioned agar can release sufficient amount of signal to influence the colony development on virgin agar.
however, macula-exposed agar alone was unable to pass the effect further, to the virgin agar in the neighboring compartment .
the effect of conditioned agar suggests that the signals between bacterial bodies are chemical rather than physical .
since the effects is transmitted in the absence of living source bacteria, the most obvious candidate is some compound soluble in the agar medium, readily evaporating , diffusing across the septum and becoming trapped in the free agar beyond.
to exclude the possibility of transmission via surface of the septum, we rendered the septum hydrophobic by medical-grade vaseline .
since this did not affect the outcome of the experiment , we are left with the hypothesis of an airborne compound playing the role of the carrier of signal  for the recipient colony.
in a preliminary experiment, we tried to remove such putative compound by placing possible absorbents into an adjacent compartment : agar , water, 20% citric acid solution, or 30% koh.
as shown in figure 5b, both citric acid and koh appeared to be powerful inhibitors of colony development, while water or agar exhibited no effect.
modeling colony ontogeny
we chose the process of development of the f colony pattern as a model case for establishing a causal scenario that might account for at least some of the processes leading to the development of intricately structured bacterial bodies.
our observations suggest involvement of volatile signals spreading through the gas phase and absorbed by the agar medium.
at the same time, production of diffusible compounds spreading through the substrate by bacterial bodies is both well documented in the literature  and convincingly demonstrated in at least some of our experiments .
we thus proposed the following model, which includes both volatile  and diffusible  signals.
it has been successfully implemented in a computer program simulating the temporal development of the f colony cross-section profile .
in the course of the f colony development, a bacterial cell enters a succession of distinct states as follows .
in state  <dig>  corresponding to freshly inoculated or "young" growing cells, the bacteria divide exponentially, resulting in a juvenile colony increasing in both its height and diameter.
cells in state  <dig> produce moderate amounts of a diffusible factor  that spreads slowly through the substrate and inhibits their own growth if above a threshold concentration .
when reaching qlim, or as a result of nutrient limitation , cells stop dividing and enter state  <dig>  corresponding to the early stationary phase and characterized by increased production of the quorum signal.
at this stage, the developing colony consists of a core of non-growing cells in state  <dig>  with a margin containing still-growing state  <dig> bacteria.
remarkably, over a range of parameters, thickness of the margin becomes soon limited by the quorum signal, while the colony keeps spreading laterally, resulting in a "hat-like" profile .
cells remain in state  <dig> for a limited time window , and then move on to state  <dig> - the mature stationary phase, where the production of the quorum signal ceases altogether but the bacteria start to emit another signaling compound - the volatile "odor" signal that is produced into the gas phase and readily absorbed into the agar across the whole dish .
both state  <dig> and state  <dig> cells respond to a limiting concentration of the odor signal  by entering state  <dig>  or a refractory growing state, where the bacteria either keep dividing  or restore division , but no longer produce any signaling compounds.
they also do not respond to the quorum signal any more, while retaining sensitivity to the odor.
finally, upon reaching either the maximum colony thickness  or a second odor threshold , state  <dig> cells cease growing and enter mature stationary phase , finishing thus colony development.
computer simulations based on these assumptions yielded often colony profiles reminiscent of the observed behavior of f colonies .
we cannot yet provide any rigorous estimate of the robustness of the f-like outcomes, as we have not systematically examined the space of model parameters; the reader is invited to do so using the provided program .
we obtained, however, "realistic" looking outcomes, though sometimes with distorted ratios of central, interstitial and peripheral colony zones, with a variety of parameters.
we thus hope that the model might adequately describe a general aspect of the colony morphogenesis rather than an fortuitous outcome of a specific combination of parameters.
moreover, we were able to generate a "rimless"  phenotype solely by modifying the quorum and odor sensitivity limits while all the other parameters have been kept constant .
simulation of specific features of rimmed colonies
while experimenting with varying layout of the initial inoculum , we have observed three worthwhile additional phenomena :  multiple inocula sharing the same dish developed into colonies of perfect shape but smaller size ;  under some circumstances, colonies initiated close to each other "developed" a common rim ;  a simulation of dropping or dotting an extended inoculum yielded "rimmed colonies" from inocula smaller than the interstitial ring of a single cell-initiated colony but maculae for larger inocula.
we have observed a similar phenomenon also in laboratory experiments aimed at estimating the area of planting that still allows cells in a slurry on the surface of semi-solid medium to coordinate their behavior.
drops of dense suspension of the f strain were planted as smears of increasing diameter.
as shown in figure 7c, up to a critical diameter, roughly corresponding to the outer diameter of the interstitial circle of a normal f colony, the cells could still coordinate their actions towards a full-fledged colony, albeit not with a full success.
if compared with the standard f pattern, the central navel always occupied the whole area of planting, leaving to the interstitial ring only the space remaining to the critical diameter.
should the diameter of planting reach  this critical diameter, no room was left for the interstitial circle, and the body turned into a macula, as predicted by our formal model.
discussion
highly structured bacterial bodies  apparently represent the "default" way of living of most bacteria  <cit> .
how do such bodies come into existence?
are they ad hoc contraptions, molded solely, or predominantly, by the external environment?
a result from an ecological succession, a game played by well-trained players?
or, finally, may an analogy of ontogeny be assumed  <cit> , similar to ontogeny in, e.g.
mycobacteria, streptomycetes, slime molds, yeasts, or even plants or animals?
our experiments with a single clone or a pair of clones, each giving well-developed colonies with finite growth, may provide initial insight into the processes of bacterial body formation.
apparently, there exists an elaborated network of communicative signals mutually affecting bacterial bodies, so the first hypothesis can be safely dismissed.
as to the "ecology" of cells: a colony, whose diversity resembles rather a carefully managed cornfield than a natural landscape, may not seem to be a good model at the first sight.
yet, our two interacting clones remind of theoretical models like "hawks-doves" or "prisoners dilemma" .
the third possibility - regular patterns of colony ontogeny - allows even genuine convergent development entering the game.
our observations suggest that development of bacterial bodies in serratia sp.
includes both events taking place within a body, and transmission of signals between distinct bodies.
signals act at a distance, i.e.
they do not require physical contact .
experiments with conditioned agar show that signals do not require simultaneous presence of living entities; hence, actively emitted light, sound, electrical or even chemical pulses of whatever nature can be excluded as carriers of the signal.
we are left with a compound or a cocktail of compounds, emitted by living entities into their environment, persisting there for some time, and being actively interpreted by recipients that happen to be present in their range.
while our observations do not provide any hints yet as to the chemical identity of these signals, they at least point towards some of their properties.
experiments with signaling across the septum suggest that the signal from the macula spreads via the gas phase.
for a different bacterial system, indole could be the carrier of a volatile signal .
ammonia appeared to be the signal carrier in yeast colonies  <cit> .
as a first step towards characterizing our signal, we demonstrated that it is readily cleared away by non-volatile acid or alkali traps.
we propose a simple model capable of simulating some aspects of our experimentally characterized examples of bacterial body morphogenesis .
this model involves two factors carrying information for both morphogenesis and mutual influencing of neighbors, generated in bodies at certain developmental stages, and diffusible to the environment.
one of the signals travels  through the substrate, the other is transmitted via the gas phase.
these bearers of the signals  are perceived by all cells, allowing their orientation and behavior in the developing colony; timing may be the second critical factor at play.
while several theoretical models of microbial colony morphogenesis have been published, they mostly focus on such aspects as kinetics of colony expansion controlled by nutrient diffusion through the colony and surrounding medium  <cit> , intra-colony spatial organization of cells  <cit>  or fine patterning of the colony margin based on interplay of nutrient and signal diffusion and, in some cases, also swarming behavior of the bacteria  <cit> .
to our knowledge, none of the published models accessible to us predicts or explains formation of "fountain" colonies of finite size , and none includes an interplay of diffusible  and volatile  signals, albeit chemotaxis or quorum sensing has been incorporated in some simulations .
so far, our model does not account for modifications of the colony's "body plan" upon interaction with different clones , where additional signals diffusible in agar  to one signal by the other), may contribute .
notably, our model includes, as one of the central parameters, some kind of cellular memory - bacteria that have recently ceased dividing behave differently from their sisters that have spent a longer time in the stationary phase.
let us suppose that in closely related bacterial clones used in our study the basic morphogenetic signals are the same, i.e.
particular clones differ in the signal interpretation.
remarkably, some combinations of quorum and odor sensitivity parameters in our model produce rimless bodies while other parameters are kept the same as for rimmed ones .
changes in the rate of lateral spreading during colony development have been observed or predicted especially for microbes exhibiting extensive swarming; however, we have not incorporated this phenomenon into our model since both our observations  and data reported by others  <cit>  document a more-less constant rate of lateral growth of serratia colonies under conditions leading to the development of compact colonies .
the present model does not yet allow simulations involving more than one "clone" .
nevertheless, the experimentally observed "aggressive" phenotype of rimless bodies upon encounter with rimmed ones is consistent with the model assuming that the rimless clone is less sensitive to the  diffusible quorum signal spreading through the substrate.
a "rimless" phenotype has been previously observed also in a s. marcescens strain capable of forming "fountain" colonies on standard media, when this strain was grown in the absence of glucose  <cit> ; the same happened also in our f clone on glucose-free media .
it is tempting to speculate that glucose  may be required to develop full sensitivity to the diffusible quorum signal.
alternatively, production of such a signal may be diminished on poor media .
the difference in "worldviews" between rimmed and rimless clones is best demonstrated when mixed suspensions or colonies planted close together are forced towards establishing a new body.
the rimless partners segregate in radial clonal sectors from a mixture, and keep separated upon close encounter.
on the other hand, two rimmed clones are much closer to each other in interpreting their morphospace than two rimless clones, as they can build a common rim when planted as a mixed suspension or upon close encounters.
we have experimentally defined several additional qualitative prerequisites for establishing and maintaining the typical "body plan" of bacterial colonies; some of them can be evaluated in the light of our model.
the presence of a bacterial body in the neighborhood of a developing colony of f clone results in its quicker ripening, i.e.
reddening.
very close encounters lead to disruption of both its growth and pattering: most profound is the effect on colonies planted close to older bodies, or inside ring-bodies.
in case of two rimmed partners, the older the neighbor was, the more profound the growth inhibition of the younger colony, which, nevertheless, remained recognizable even when overgrown by the older partner.
development of geometrically constrained bodies  can be interpreted as a conflict of two ways of recognizing the "body" across the hole: as a part of "self" , or as a neighbor.
in ring plantings up to a certain diameter, cells in the inner diameter of the ring are sufficient to produce a "virtual navel" controlling the development of the body.
in large rings, the "non-self" tendency prevails: such bodies take the inner empty space for outer space outside of their morphogenetic field.
new colonies planted into such an area are treated as foreign, and their pattern resembles those planted in the vicinity of other type of bodies.
while our model does not currently allow simulating development of multiple inocula differing in genotype , size, shape or time of planting, we could at least reproduce the faster ripening and smaller size of two colonies sharing a confined space, compared to a solitary colony.
we have also confirmed our previous results  <cit>  showing that the growth of colonies is strongly inhibited, even abolished, if the surrounding area is evenly occupied by "background" bacterial bodies - even if their total population  is much smaller than the colony inoculum.
hence, bacteria in the background emit a signal that efficiently disturbs the organizing potential of the multicellular plant, while keeping the background colonies in an underdeveloped - "dormant" - state.
experiments with effects of colonies grown in the presence of maculae , macula-exposed and "virgin" agar suggest that this effect could be identical with the gas-borne signal of our model.
finally, we have shown that the planting area necessary for the cell population to maintain the "feeling" of belonging to a single body, roughly corresponds to the outer diameter of a mature interstitial circle .
exceeding this critical diameter leads to the loss of structure and breakdown to a macula; however, even in such a case the body is self-inhibited as to lateral spreading.
this may perhaps be understood as the last remnants of its "feeling of integrity"; the results of our computer simulations suggests that even this seemingly complex effect may be produced by the interplay of mere two signals.
CONCLUSIONS
some isolates of serratia sp.
produce colonies exhibiting finite growth and clone-specific appearance, which is easily evaluated thanks to their conspicuous coloration.
the shape and patterning of developing colonies and other multicellular bodies is easily malleable by experimental conditions.
the appearance of a developing colony results from  its internal morphogenetic potential and  the character of neighbor bodies and their overall distribution on the dish.
a simple formal model is proposed, based on two morphogenetic signals generated by the bodies, one of them spreading through the substrate and the other through the gas phase.
the model can simulate some of our experimental results, namely:
<dig>  the development of colonies exhibiting finite growth and both rimmed and rimless patterns, the difference between the former and the latter being in the intensity of signal production and/or sensitivity towards the signal.
<dig>  dependence of colony size upon the number of colonies sharing common morphospace, and development of confluent colonies from closely planted inocula of a rimmed strain.
<dig>  the phenomenon of "critical planting area" which must not be exceeded should a colony develop a typical rimmed pattern.
our observations are thus consistent with bacterial colonies behaving, in some aspects, as true multicellular bodies whose patterning is controlled by positional information; the nature of the relevant signals remains to be established.

 bacteria can display a plethora of multicellular forms; their structure and appearance depends on factors such as the presence of nutrients or neighbors. concepts of "body" and "community", as developed for multicellular sexual eukaryots, became, however, somewhat blurred upon attempts of their application to microorganisms. is differentiation of multicellular units in bacteria comparable to embryonic development, to the establishment of an ecosystem? is it even the place of darwinian evolution on a micro-scale? multicellular bacterial bodies can be viewed as ecosystems negotiated by myriads of specialists. each cell is understood as an individual playing its own game according to resources, energy costs, and complicated informational interactions with others. however, patterning of multicellular bodies remains beyond interest, at the most being viewed as a passive outcome of physical forces. a multicellular bacterial community may, however, be also perceived as the prevalent form of bacterial existence, with a genuine ontogeny. to create and maintain such elaborated structures, a great deal of communication, regulations, mutual understanding, and cooperation takes place in bacterial morphogenesis. differentiation in such a bacterial body may proceed via genetically differing subclones fulfilling different roles, and appearing reproducibly at characteristic periods of cultivation. sophisticated networks of chemical signals, the scaffolding of extracellular matrix and even cell-to-cell contacts may enable attaining and maintaining the integrity of the body. research in this direction has been greatly accelerated in last two decades by the discovery of the phenomenon of quorum sensing. bacterial populations react to such signals - and build multispecies bodies accordingly - in a context-dependent manner. a plethora of quorum-modulating signals, such as indole or furanole derivatives, was also described. the study of model monoclonal populations may contribute to understanding colony morphogenesis, providing the possibility to examine how, and even why, bacteria exert themselves towards "species-specific" appearances. we have previously demonstrated that colonies of serratia marcescens can be viewed as multicellular bodies with genuine embryonic development. colonies displayed finite growth and clone-specific formative processes; even a disorganized cell slurry could establish a regular pattern and embark on a typical developmental pathway. under standardized culture conditions on rich semi-solid media, the final shape and patterning of bacterial bodies depended essentially on four initial settings: amount, density, and distribution pattern of founder cells; the configuration of surrounding free medium; the presence and character of other bacterial bodies sharing the same niche; and self-perception, resulting in delimitation towards other bodies. here we further investigate the development of bacterial bodies and their interaction with close or distant neighbors of identical, or different, clonal origin. we also propose a formal model that can account for some of our experimental results.  colony patterning in clonal variants of serratia rubidaea we have chosen a wild type strain of serratia rubidaea, a gram-negative, facultatively anaerobic, rod-shaped bacterium of the enterobacteriaceae family, producing usually red glossy colonies without any distinguished structural pattern except of a slightly darker touch in the middle, as our starting material. this strain will be further referred to as the r strain. the r strain occassionally segregated colonies of variant shape or colouring upon prolonged cultivation in liquid media and serial plating; from these, we selected three stable, no longer segregating clones used in this study: w, a colorless variant of r; f clone named after its cross-section profile; fw - a colorless, morphologically slightly different derivative of f. f and fw colonies are characterized by a typical massive rim, hence rimmed, in contrast to rimless colonies. colonies of the parental r strain and all daughter clones have a finite growth, their diameter being in rimmed clones about mm, in rimless ones about mm. colonies ripen into final color and pattern by about 7th day upon planting, while still growing slowly, to reach their final diameter by day. as the f morphotype plays a central role in this study, its development deserves a closer scrutiny. no matter how the colony was planted, in days 1- it grows as a central navel: a compact body on the agar plate only slowly propagating sideways. this phase is followed in days 3- by spreading of the flat interstitial circle. microscopic observations revealed a margin of extracellular material containing small swarms of bacteria at the colony periphery at this stage, a phenomenon well established in serratia sp.. in days 5- this lateral propagation comes to end and the peripheral rim is formed; the central navel grows red in this phase. in following days, the rim also turns red and the growth proceeds towards a halt. the flat interstitial ring remains colorless. fully developed f colonies can be obtained only if bacteria are planted in densities 1- per 9-cm dish. at the density of tens per dish, the colonies grow much smaller; below a critical distance, they tend to fuse into a confluent colony with many centers bounded by a common rim. at densities of hundreds per dish, colonies remain very small and undifferentiated. yet higher density of planting leads to a compact, undifferentiated body - a macula. the scenario is similar for all four clones used in this study, except that rimless colonies never fuse. the development and behavior of standard colonies were essentially independent on the way of planting, provided that the diameter of planting was ≤ mm. similar observations have been made previously for a different serratia isolate. close encounters of bacterial bodies in dense plantings, clonal rimmed colonies tend to merge into confluent colonies. in a mix of lines differing in color, the origin of each participating colony is revealed by the color of its rim. if colonies were dotted close to a growing previously planted colony, the resulting fused body resembled a confluent colony, with a common rim and separate centers. the effect was more profound with younger colonies. if a similar protocol was followed with the rimless clones, colonies remained thoroughly delimited, whether in a single culture, or in an rw mixture: no fusions were observed, and clearly distinguishable furrows developed between bodies in contact. similarly, dots applied close to an older colony became inhibited in growth, but kept distinct from its growing older neighbor. upon close encounters between rimless and rimmed bodies, the r colonies grow faster and influence rimmed colonies in four ways: if planted early enough, r colonies can engulf an immature rimmed colony; its body, however, survives and cells can be recovered from such a mixed body. if f colonies are allowed to grow in the vicinity of an r colony as independent bodies up to 3rd day of cultivation, they develop to a new colony phenotype with a massive white rim with a thin colored ring at the inner side. cells from x colonies restore the original f phenotype upon subsequent planting. a rimless dot added to a rimmed colony tends to proliferate around the older partner and block its further growth; the younger the rimmed colony is, the more profound the effect. similar results were obtained with w dots. again, the rimmed colony remains compact and contains live cells. the engulfment potential of the rimless colony is even more profound in a reverse arrangement, i.e. dotting of a rimmed colony to an older rimless partner. planting of mixed suspensions mixed suspensions of two rimmed clones produced varying and unpredictable colony patterns, suggesting an extreme sensitivity of such mixtures to initial conditions. samples taken from both center and periphery of such chimeras revealed the presence of cells belonging to both clones in the central zone, and sometimes also in the periphery. these results contrast with previous findings on a different strain: in that case, however, both subclones tended to establish separated "areas of influence", essentially as referred below for rw mixtures. if a colony was established from a mixture of two rimless clones rw, the center of the colony remained a mixture of both clones, sending radial monoclonal sectors as the colony grew, as if rimless clones were reluctant to cooperate towards a common end. if a mixed suspension of rimmed and rimless suspension is dropped to initiate a colony, the cells of the rimmed clone remained confined to the central area, whereas the growing periphery is composed exclusively of r cells, similar to the above-described engulfment of rimmed colonies by rimless ones. again, the inhibited strain confined to the center remains viable and can be recovered upon re-planting. the behavior of rfw, wf and wfw colonies is analogous to the rf mixture. effects of planting layout the plasticity of the typical f body plan was investigated by streaking or blotting cell suspension in various geometrical settings. if the width of the plant in one direction does not exceed a critical diameter somewhat smaller than the adult f colony diameter, the body strives to maintain the features of the colony, even if deformed to a large extent. blotting of ring bodies using circular plastic stamps was even more informative, with results depending on the diameter of such rings. smaller rings healed the central cavity and proceeded towards a normal colony shape; with increasing diameter, up to the critical size, this colony phenotype was maintained, even if with a central hole in the middle. above the critical diameter, a ring-like colony acquired an additional inner rim - resembling linear colonies as in figure 3c, but curled. we have also confirmed the previously described phenomenon of "ghost" colonies, originally documented on a different strain. briefly, colonies planted at the background of multiple colonies became inhibited, or even "dissolved" on the background. this is the case even in synchronous cultures if, at the beginning, the background is represented by at least about colony-forming units. such a background can keep at bay a plant as dense as cells, preventing its development towards a colony. the effect is more profound when background colonies are older. with this information in mind, we return to ring colonies. a colony was planted into the center of a ring colony of greater diameter, or a ring colony was blotted around a growing f colony. both bodies represent a "background" to each other, depending on the succession of plating. results in figure 3d show that the synchronous planting of both structures leads to disruption of the structure of the central colony, but no change in the structure of the ring. colonies planted on the background of older rings became inhibited. on the other hand, when the ring is planted around an older colony, it develops into a typical structure, only with more profound reddening of the inner rim - again confirming that a developing colony can perceive the presence and layout of its neighbors. long-distance interactions between colonies and maculae to examine the putative long-distance signals between bacterial bodies, colonies were planted to the vicinity of maculae of two different serratia clones or an unrelated bacterial strain. maculae and colonies either shared the same agar plate, or were separated by a septum. when f colonies were planted in varying distances from an f macula, the closer was the macula to a colony, the quicker the reddening of that colony. at the same time, the colony deviated from its typical structure to an extent inversely related to its distance from the macula. the graph in figure 4a shows that the transition point between aberrant and standard patterns lies approximately to mm from the macula, corresponding roughly to the diameter of adult f colonies. this breakdown of the colony structure was not observed with the serratia isolate characterized previously. the fw macula exhibited weaker effects than its f counterpart, and elicited the loss of structure only when older. the r macula, as well as a macula of e. coli, induced, again, formation of the x phenotype in colonies of the f clone. no such x-like structures were observed when r colonies were planted in the vicinity of an e. coli macula. communication across obstacles if the macula and colonies have been grown on opposite sides of a septum dividing the dish, preventing diffusion in the semi-solid agar matrix but allowing gas exchange, the effect of macula was qualitatively similar to that on a shared plate, albeit the distance between the bodies appeared as if increased for simultaneously planted bodies. if, however, the macula across the septum was at least days old at the time of colony inoculation, colony development was similar to controls sharing a continuous plate, suggesting that older bacterial bodies produce volatiles that may be absorbed by the agar medium. maculae of a different strain or species also affected development of f colonies across an obstacle; however, they never induced formation of the x structure across the septum, indicating that signals diffusing through the semi-solid substrate, distinct from those carried by the gas phase, are indispensable for the development of the x pattern. the effect across the septum is not bound to an organized body of the macula: bacterial suspension kept across the septum exerted an effect comparable to that of a macula. nature of signals between bodies in further experiments, we investigated the longevity of a putative macula-derived signal. a macula was grown for days on a cellulose membrane laid on the agar on one side of a septum, then removed, leaving empty macula-conditioned agar. immediately after macula removal, colonies were dotted into the neighboring compartment containing free macula-exposed agar. the results are indistinguishable from controls shown in figure 4a, i.e. from the situation when the macula persisted in the neighboring compartment. to test the obvious possibility that such free, but macula-exposed agar "took the smell" during macula growth, medium in the non-inoculated compartment was removed at the time of the macula removal, and replaced by "virgin" agar transferred from another, empty plate. as also shown in figure 5a, the development of colonies was essentially the same as on macula-exposed agar. thus, macula-conditioned agar can release sufficient amount of signal to influence the colony development on virgin agar. however, macula-exposed agar alone was unable to pass the effect further, to the virgin agar in the neighboring compartment. the effect of conditioned agar suggests that the signals between bacterial bodies are chemical rather than physical. since the effects is transmitted in the absence of living source bacteria, the most obvious candidate is some compound soluble in the agar medium, readily evaporating, diffusing across the septum and becoming trapped in the free agar beyond. to exclude the possibility of transmission via surface of the septum, we rendered the septum hydrophobic by medical-grade vaseline. since this did not affect the outcome of the experiment, we are left with the hypothesis of an airborne compound playing the role of the carrier of signal for the recipient colony. in a preliminary experiment, we tried to remove such putative compound by placing possible absorbents into an adjacent compartment: agar, water, 20% citric acid solution, or 30% koh. as shown in figure 5b, both citric acid and koh appeared to be powerful inhibitors of colony development, while water or agar exhibited no effect. modeling colony ontogeny we chose the process of development of the f colony pattern as a model case for establishing a causal scenario that might account for at least some of the processes leading to the development of intricately structured bacterial bodies. our observations suggest involvement of volatile signals spreading through the gas phase and absorbed by the agar medium. at the same time, production of diffusible compounds spreading through the substrate by bacterial bodies is both well documented in the literature and convincingly demonstrated in at least some of our experiments. we thus proposed the following model, which includes both volatile and diffusible signals. it has been successfully implemented in a computer program simulating the temporal development of the f colony cross-section profile. in the course of the f colony development, a bacterial cell enters a succession of distinct states as follows. in state corresponding to freshly inoculated or "young" growing cells, the bacteria divide exponentially, resulting in a juvenile colony increasing in both its height and diameter. cells in state produce moderate amounts of a diffusible factor that spreads slowly through the substrate and inhibits their own growth if above a threshold concentration. when reaching qlim, or as a result of nutrient limitation, cells stop dividing and enter state corresponding to the early stationary phase and characterized by increased production of the quorum signal. at this stage, the developing colony consists of a core of non-growing cells in state with a margin containing still-growing state bacteria. remarkably, over a range of parameters, thickness of the margin becomes soon limited by the quorum signal, while the colony keeps spreading laterally, resulting in a "hat-like" profile. cells remain in state for a limited time window, and then move on to state - the mature stationary phase, where the production of the quorum signal ceases altogether but the bacteria start to emit another signaling compound - the volatile "odor" signal that is produced into the gas phase and readily absorbed into the agar across the whole dish. both state and state cells respond to a limiting concentration of the odor signal by entering state or a refractory growing state, where the bacteria either keep dividing or restore division, but no longer produce any signaling compounds. they also do not respond to the quorum signal any more, while retaining sensitivity to the odor. finally, upon reaching either the maximum colony thickness or a second odor threshold, state cells cease growing and enter mature stationary phase, finishing thus colony development. computer simulations based on these assumptions yielded often colony profiles reminiscent of the observed behavior of f colonies. we cannot yet provide any rigorous estimate of the robustness of the f-like outcomes, as we have not systematically examined the space of model parameters; the reader is invited to do so using the provided program. we obtained, however, "realistic" looking outcomes, though sometimes with distorted ratios of central, interstitial and peripheral colony zones, with a variety of parameters. we thus hope that the model might adequately describe a general aspect of the colony morphogenesis rather than an fortuitous outcome of a specific combination of parameters. moreover, we were able to generate a "rimless" phenotype solely by modifying the quorum and odor sensitivity limits while all the other parameters have been kept constant. simulation of specific features of rimmed colonies while experimenting with varying layout of the initial inoculum, we have observed three worthwhile additional phenomena: multiple inocula sharing the same dish developed into colonies of perfect shape but smaller size; under some circumstances, colonies initiated close to each other "developed" a common rim; a simulation of dropping or dotting an extended inoculum yielded "rimmed colonies" from inocula smaller than the interstitial ring of a single cell-initiated colony but maculae for larger inocula. we have observed a similar phenomenon also in laboratory experiments aimed at estimating the area of planting that still allows cells in a slurry on the surface of semi-solid medium to coordinate their behavior. drops of dense suspension of the f strain were planted as smears of increasing diameter. as shown in figure 7c, up to a critical diameter, roughly corresponding to the outer diameter of the interstitial circle of a normal f colony, the cells could still coordinate their actions towards a full-fledged colony, albeit not with a full success. if compared with the standard f pattern, the central navel always occupied the whole area of planting, leaving to the interstitial ring only the space remaining to the critical diameter. should the diameter of planting reach this critical diameter, no room was left for the interstitial circle, and the body turned into a macula, as predicted by our formal model. discussion highly structured bacterial bodies apparently represent the "default" way of living of most bacteria. how do such bodies come into existence? are they ad hoc contraptions, molded solely, or predominantly, by the external environment? a result from an ecological succession, a game played by well-trained players? or, finally, may an analogy of ontogeny be assumed, similar to ontogeny in, e.g. mycobacteria, streptomycetes, slime molds, yeasts, or even plants or animals? our experiments with a single clone or a pair of clones, each giving well-developed colonies with finite growth, may provide initial insight into the processes of bacterial body formation. apparently, there exists an elaborated network of communicative signals mutually affecting bacterial bodies, so the first hypothesis can be safely dismissed. as to the "ecology" of cells: a colony, whose diversity resembles rather a carefully managed cornfield than a natural landscape, may not seem to be a good model at the first sight. yet, our two interacting clones remind of theoretical models like "hawks-doves" or "prisoners dilemma". the third possibility - regular patterns of colony ontogeny - allows even genuine convergent development entering the game. our observations suggest that development of bacterial bodies in serratia sp. includes both events taking place within a body, and transmission of signals between distinct bodies. signals act at a distance, i.e. they do not require physical contact. experiments with conditioned agar show that signals do not require simultaneous presence of living entities; hence, actively emitted light, sound, electrical or even chemical pulses of whatever nature can be excluded as carriers of the signal. we are left with a compound or a cocktail of compounds, emitted by living entities into their environment, persisting there for some time, and being actively interpreted by recipients that happen to be present in their range. while our observations do not provide any hints yet as to the chemical identity of these signals, they at least point towards some of their properties. experiments with signaling across the septum suggest that the signal from the macula spreads via the gas phase. for a different bacterial system, indole could be the carrier of a volatile signal. ammonia appeared to be the signal carrier in yeast colonies. as a first step towards characterizing our signal, we demonstrated that it is readily cleared away by non-volatile acid or alkali traps. we propose a simple model capable of simulating some aspects of our experimentally characterized examples of bacterial body morphogenesis. this model involves two factors carrying information for both morphogenesis and mutual influencing of neighbors, generated in bodies at certain developmental stages, and diffusible to the environment. one of the signals travels through the substrate, the other is transmitted via the gas phase. these bearers of the signals are perceived by all cells, allowing their orientation and behavior in the developing colony; timing may be the second critical factor at play. while several theoretical models of microbial colony morphogenesis have been published, they mostly focus on such aspects as kinetics of colony expansion controlled by nutrient diffusion through the colony and surrounding medium, intra-colony spatial organization of cells or fine patterning of the colony margin based on interplay of nutrient and signal diffusion and, in some cases, also swarming behavior of the bacteria. to our knowledge, none of the published models accessible to us predicts or explains formation of "fountain" colonies of finite size, and none includes an interplay of diffusible and volatile signals, albeit chemotaxis or quorum sensing has been incorporated in some simulations. so far, our model does not account for modifications of the colony's "body plan" upon interaction with different clones, where additional signals diffusible in agar to one signal by the other), may contribute. notably, our model includes, as one of the central parameters, some kind of cellular memory - bacteria that have recently ceased dividing behave differently from their sisters that have spent a longer time in the stationary phase. let us suppose that in closely related bacterial clones used in our study the basic morphogenetic signals are the same, i.e. particular clones differ in the signal interpretation. remarkably, some combinations of quorum and odor sensitivity parameters in our model produce rimless bodies while other parameters are kept the same as for rimmed ones. changes in the rate of lateral spreading during colony development have been observed or predicted especially for microbes exhibiting extensive swarming; however, we have not incorporated this phenomenon into our model since both our observations and data reported by others document a more-less constant rate of lateral growth of serratia colonies under conditions leading to the development of compact colonies. the present model does not yet allow simulations involving more than one "clone". nevertheless, the experimentally observed "aggressive" phenotype of rimless bodies upon encounter with rimmed ones is consistent with the model assuming that the rimless clone is less sensitive to the diffusible quorum signal spreading through the substrate. a "rimless" phenotype has been previously observed also in a s. marcescens strain capable of forming "fountain" colonies on standard media, when this strain was grown in the absence of glucose; the same happened also in our f clone on glucose-free media. it is tempting to speculate that glucose may be required to develop full sensitivity to the diffusible quorum signal. alternatively, production of such a signal may be diminished on poor media. the difference in "worldviews" between rimmed and rimless clones is best demonstrated when mixed suspensions or colonies planted close together are forced towards establishing a new body. the rimless partners segregate in radial clonal sectors from a mixture, and keep separated upon close encounter. on the other hand, two rimmed clones are much closer to each other in interpreting their morphospace than two rimless clones, as they can build a common rim when planted as a mixed suspension or upon close encounters. we have experimentally defined several additional qualitative prerequisites for establishing and maintaining the typical "body plan" of bacterial colonies; some of them can be evaluated in the light of our model. the presence of a bacterial body in the neighborhood of a developing colony of f clone results in its quicker ripening, i.e. reddening. very close encounters lead to disruption of both its growth and pattering: most profound is the effect on colonies planted close to older bodies, or inside ring-bodies. in case of two rimmed partners, the older the neighbor was, the more profound the growth inhibition of the younger colony, which, nevertheless, remained recognizable even when overgrown by the older partner. development of geometrically constrained bodies can be interpreted as a conflict of two ways of recognizing the "body" across the hole: as a part of "self", or as a neighbor. in ring plantings up to a certain diameter, cells in the inner diameter of the ring are sufficient to produce a "virtual navel" controlling the development of the body. in large rings, the "non-self" tendency prevails: such bodies take the inner empty space for outer space outside of their morphogenetic field. new colonies planted into such an area are treated as foreign, and their pattern resembles those planted in the vicinity of other type of bodies. while our model does not currently allow simulating development of multiple inocula differing in genotype, size, shape or time of planting, we could at least reproduce the faster ripening and smaller size of two colonies sharing a confined space, compared to a solitary colony. we have also confirmed our previous results showing that the growth of colonies is strongly inhibited, even abolished, if the surrounding area is evenly occupied by "background" bacterial bodies - even if their total population is much smaller than the colony inoculum. hence, bacteria in the background emit a signal that efficiently disturbs the organizing potential of the multicellular plant, while keeping the background colonies in an underdeveloped - "dormant" - state. experiments with effects of colonies grown in the presence of maculae, macula-exposed and "virgin" agar suggest that this effect could be identical with the gas-borne signal of our model. finally, we have shown that the planting area necessary for the cell population to maintain the "feeling" of belonging to a single body, roughly corresponds to the outer diameter of a mature interstitial circle. exceeding this critical diameter leads to the loss of structure and breakdown to a macula; however, even in such a case the body is self-inhibited as to lateral spreading. this may perhaps be understood as the last remnants of its "feeling of integrity"; the results of our computer simulations suggests that even this seemingly complex effect may be produced by the interplay of mere two signals.  some isolates of serratia sp. produce colonies exhibiting finite growth and clone-specific appearance, which is easily evaluated thanks to their conspicuous coloration. the shape and patterning of developing colonies and other multicellular bodies is easily malleable by experimental conditions. the appearance of a developing colony results from its internal morphogenetic potential and the character of neighbor bodies and their overall distribution on the dish. a simple formal model is proposed, based on two morphogenetic signals generated by the bodies, one of them spreading through the substrate and the other through the gas phase. the model can simulate some of our experimental results, namely: the development of colonies exhibiting finite growth and both rimmed and rimless patterns, the difference between the former and the latter being in the intensity of signal production and/or sensitivity towards the signal. dependence of colony size upon the number of colonies sharing common morphospace, and development of confluent colonies from closely planted inocula of a rimmed strain. the phenomenon of "critical planting area" which must not be exceeded should a colony develop a typical rimmed pattern. our observations are thus consistent with bacterial colonies behaving, in some aspects, as true multicellular bodies whose patterning is controlled by positional information; the nature of the relevant signals remains to be established. 
we have studied the interplay of intra-colony signaling and inter-colony influences in related clones of serratia rubidaea grown on rich media. the complex patterns of color and texture observed in serratia rubidaea colonies may be based on at least two signals produced by cells, one of them diffusing through the substrate and the other carried by a volatile compound and absorbed into the substrate. the result of development is affected by the overall distribution of neighbors in the dish. bacterial bodies can develop complex patterns of color and structure. a formal model accounting for some aspects of colony morphogenesis and inter-colony interactions is proposed. these patterns may arise as a result of both colony-autonomous developmental and regulatory processes and environmental influences, including those generated by neighbor bodies. the neighbors' presence is communicated via at least two putative signals, while additional signals may be involved in generating some unusual patterns observed upon encounters of different clones.
 bacterial bodies can develop complex patterns of color and structure. these patterns may arise as a result of both colony-autonomous developmental and regulatory processes and environmental influences, including those generated by neighbor bodies. we have studied the interplay of intra-colony signaling and inter-colony influences in related clones of serratia rubidaea grown on rich media.  colonies are shaped by both autonomous patterning and by signals generated by co-habitants of the morphogenetic space, mediating both internal shaping of the body, and communication between bodies sharing the same living space. the result of development is affected by the overall distribution of neighbors in the dish. the neighbors' presence is communicated via at least two putative signals, while additional signals may be involved in generating some unusual patterns observed upon encounters of different clones. a formal model accounting for some aspects of colony morphogenesis and inter-colony interactions is proposed.  the complex patterns of color and texture observed in serratia rubidaea colonies may be based on at least two signals produced by cells, one of them diffusing through the substrate and the other carried by a volatile compound and absorbed into the substrate. differences between clones with regard to the interpretation of signals may result from different sensitivity to signal threshold. 