微生物聚集：一种类似组织的细胞团体

吴随随，王洁如，缪煜轩，周 佳，冯永君

(北京理工大学生命学院，北京 100081)

Community，a crucial ecological term，had been used to describe the inter-relationship between creatures on earth for centuries.The characteristic and function of the community of ants or bees have drawn a host of attention.However，during the last two decades，scientists have changed their focus onto the community of a familiar and special life form-microorganism.

Most scientific research once had focused on viruses and bacteria independently by exploring their individualstructure and components，metabolism manners，molecular genetic principles，etc.As studies progressed，researchers began to notice that，rather than appear independently，most microbes within the environment stay in interdependent aggregation forms:some of them assemble together tightly，some of them lead a sessile lifestyle on a surface that can be vital or non-vital.All these cellular assemblages seem to be parallel with the communities of macroscopic creatures.Within such a microbial community，these tiny organisms are able to create special substances，convert their own living phenotypes，or communicate with each other by sending special signals.Many questions regarding such aggregation phenomena remain，including why they behave as assemblies，how they form such structures，and what their physiological functions are.Yet，the most remarkable feature of this particular community is that，similar to the antelope’s gathering together to fight against dangers，the community of the assembled microbes helps the members within it resist the environmental stresses.

In this review，we summarized the biochemical and molecular mechanisms that had been proposed for microorganism aggregation to withstand adversities.We also showed the sophistication of a once neglected kind of community，especially in its ecological and evolutionary aspects，and drew attentions to the intriguing aspects of these microbe colonies.

1 Mechanism for bacterial biofilms and aggregates to better adapt to the environment

Biofilm，the most prominent assembly structure of many bacteria in the natural environment，is defined as a matrix-enclosed bacterial population formed by either single or multiple species adhering to each other or to some biotic or abiotic surfaces［1］.Recently，the nomenclature of biofilms has been broadened to describe all kinds of microbial aggregations［2］.A biofilm is a highly structured and interdependent community，within which bacteria display remarkable distinctions in gene expression pattern，growth profile，and most importantly，resistance to environmentalfluctuations，as compared to single cells［3］.To survive detrimental biotic(e.g.，bio-cides)and environmental conditions(e.g.，UV light［4］，desiccation［5］，or heavy metals［6］)，the biofilm-forming bacteria embed themselves into special matrices，where they cooperate with each other to exhibit environmental adaptation activities.

1.1 The matrix shields the citizens enclosed within it from environmental threats

Aggregation triggers the bacteria to express some particular genes，including genes that contribute to the constitute of the specific intercellular matrix，e.g.，the alginate gene producing extracellular polysaccharides from Pseudomonas aeruginosa has been proven to respond to the attachment to surfaces，and its synthesis is increased significantly at the mature stage of the biofilm［7-8］.Besides extracellular polysaccharides，other major classes of macromolecules like proteins，nucleic acids and other cell components are found to present within the matrix as well［9］.

In Staphylococcus epidermidis 1585v，overexpression of a 460 kDa truncated isoform of the extracellular matrix-binding protein(Embp)is necessary for biofilm formation.It constitutes the first step of biofilm formation on conditioned surfaces［10］.Some proteins involved in motility，defense，cell envelope，and unknown functions were enriched in EPS［11］.It has proved that different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm［12］.These matrix materials form a dynamic microenvironment that can be regulated by the physiological situations of the citizens within it.Compared with other planktonic bacterial cells in the natural environment who must adapt rapidly to environmental fluctuations，assembled cells can benefit from the matrix，e.g.，in an easier access to organic nutrients and more buffered conditions.In fact，the EPS can be used by heterologous species［9］，proteins can protect cells from phagocytosis by macrophages［10］，and even DNA can serve as a source of carbon and energy sometimes［13］.

It was found that only very little UV light could be transmitted through the alginate of a biofilm［4］.It also reported that heavy metal treatment could kill most exterior cells，yet cells residing near the substratum survived［6］.Both facts indicate that the envelopelike matrix can effectively protect the biofilm-forming bacteria from environmental threats.This kind of protection can be explained by a“non-diffusion mechanism”that the adverse substances cannot penetrate the matrix.This hypothesis has been further supported by observations in piperacillin，ampicillin，or some chemical disinfectors like aminoglycoside and chlorosulfamate treatments［14-16］.For protection purpose，certain components of the matrix may react with or bind to these chemical molecules so that they can not infiltrate into the interior of the biofilm［17］.Clearly，the matrix envelop，a special property of those aggregating bacteria，plays a significant role in microbial adaptation to the fluctuating environments.

1.2 The channel system in assembly architecture of biofilms helps the bacteria overcome challenges

As mentioned above，the biofilm-forming bacteria reside within a special matrix.However，rather than being totally filled with matrix components，there is still room within the biofilm，in which channels are formed to allow nutrients and oxygen molecules to be transmitted throughout the whole structure［1］.This channel system is crucial for the sustainability of a biofilm，not just because it transports indispensable oxygen，water or hydrocarbon nutritional molecules to the cells throughout the biofilm，but also for transporting special substances［9］.Catalase is an example that has been shown to be transported to protect bacteria against hydrogen peroxide.Previous studies have demonstrated that both planktonic and bacteria in biofilm expressed catalase KatA and KatB.Despite the fact that catalase expression is reduced in biofilms compared to the planktonic cells［18］，assembly cells are apparently more resistant to the insult of hydrogen peroxide［18-19］，suggesting that there maybe other mechanisms by which biofilm-forming microorganisms overcome oxidative stress.Studies on the effects of catalase on the survival of biofilm-forming bacteria have revealed that this enzyme can prevent the penetration of hydrogen peroxide［20］.It can be conjectured that catalase released by the dead cells can pass through the channel nets in the biofilm，and mitigate the stress triggered by hydrogen peroxide to the microbe colony.In short，the wide distribution of the important protective enzymes resulting from the effective transfer in the channel system in the biofilm effectively stops the toxic substance from having a widespread influence on the biofilm.Furthermore，the channel system also facilitates the removal of toxic metabolites that may potentially exacerbate the fluctuating environment［1］.Hence，the elaborate architecture formed after aggregation ensures a more suitable surrounding for the microbial community.

1.3 The physiological hetereogenity of bacteria within a biofilm increases the resistance to biocides

When bacteria assemble into a biofilm，there is no denying that the channel system can transport oxygen，water and carbon resources.However，the amount delivered to different regions of the biofilm is variable.This variation results in differences in the metabolic and growth rates of the cells.In a study，it was found that only the top one-fifth of bacteria within the biofilm was metabolically active［21］.In addition，both the DNA replication and the protein synthesis in a biofilm were also revealed in stratified patterns and most of the members were anabolically inactive［22］.Since it has been accepted that almost all the antimicrobial agents only kill those fast-growing bacteria，this spatial heterogeneity in growth rate and metabolism may play an important part in bacterial antibiotic-resistance.Considering the contribution of a slow growth rate to chlorine resistance of mycobacteria，bacterial cells in the early exponential phase of growth(i.e.，at a faster growth rate)are more susceptible than those in the stationary phase［23］.Recent research has also demonstrated that mycobacterial cells detached from the biofilm exhibited higher resistances to chlorine than those ordinary planktonic cells［24］.Since these cells have lost the protection from the matrix，the changed physiological conditions stemming from factors induced by cellular assembly(e.g.，nutrition and oxygen limitation，cell-cell signaling)is supposed to be the main reason for their higher resistance.

1.4 The aggregation status triggers phenotype variation

Within a biofilm，besides exhibiting physiological variation，bacteria go through a variety of other changes，including alteration in gene-expression patterns，acquisition of brand-new genes，and exhibition of different phenotypes.These variations play a significant role in the ability of bacterial adaptation to the fluctuating surroundings.Using precise microarray technology，scientists have discovered that a number of genes are upregulated or repressed when bacteria form biofilms.For instance，when Streptococcus mutans forms a biofilm，2.2%of total genes are activated while 1.6%are repressed［25］.Similarly，10%of the genome of Escherichia coli K-12 shows a different expression pattern in a biofilm and about 1.9%of these genes are up-or down-regulated by more than two folds［26］.The similar results were also observed in the biofilms formed by Staphylococcus aureus，Bacillus cereus，Campylobacter jejuni，Thermotoga maritime，and P.aeruginosa［27-31］.All of them exhibit diverse gene expression patterns compared to their planktonic life styles.The up-regulated genes encode proteins including adhesion factors and enzymes involved in biosynthesis or energy generation［32-34］.Remarkably，a variety of protective and stress response genes are activated.For instance，the tolA gene whose product contributes to a reduced affinity of aminoglycoside to the outer membrane of P.aeruginosa is induced in the biofilm［30］.The biofilm-forming Vibrio cholerae can be induced to produce an antiprotozoal factor that inhibits protozoan feeding activity to protect V.cholerae from being preyed by protozoan while their planktonic counterparts are eliminated in the niches［35］.When E.coli aggregates to form a biofilm，the oxidative stress gene(e.g.，soxS)，general stress response genes(e.g.，recA and rpoS)，envelope stress genes(e.g.，cpxP，spy and psp)，and chaperones(e.g.，dnaK and dnaJ)are all activated［26］.Despite no temperature changes were concerned in a biofilm，heat shock proteins have also been found to be important as well［32］.Researchers suggest that the bacteria-bacteria interaction may trigger membrane stress that in turn induces signals to activate stress response pathways［36］.

What’s more，not only does gene expression differ between the aggregate and planktonic life styles，but also it can vary during different developmental stages of the bifilm formation process.During the development of the biofilm formed by P.aeruginosa，there are distinct gene expression patterns in each stage［37］.Specifically，comparing the 2D gel electrophoresis profile of 3-and 6-day-old biofilm of Streptococcus pneumoniae reveals a 20%change in the protein expression profile，while there are more than 40%of the genes expressing differently between 6 and 9 days of biofilm formation.Those altered proteins mainly include those involve in virulence，adhesion and resistance［38-39］.

On the other hand，phenotype variations even exist among contemporary cells within the same biofilm.They exhibit both different protein expression profiles and variant morphology which spring from the alteration in the whole genome［25］and seem to play a larger part in the adaptation process.A number of papers have related the enhanced survivals of the biofilmforming bacteria to such a self-generated diversity within the aggregates.There was suggestion that the transient phenotype changes of P.aeruginosa within a biofilm could increase their antibiotic resistance［40］.The biofilm-enhanced rugose phase variant of V.cholerae was revealed to be selected to survive protozoan grazing［35］.A significant experiment also demonstrated that the variants evolved within the biofilm of P.aeruginosa were able to escape local stress，secrete pyomelanin to tolerate environmental radiation or host defenses，and resist H2O2much better［41］.They also suggested that aggregation facilitated the degree of variation to a larger extent compared with the planktonic cells.A similar phenomenon was also found in the biofilm formed by Serratia marcescens as well［42］.A notable discovery suggested that the advent of variants was related to the general stress response of gene recA［41］.This helps to draw the conjecture that it is the stressful circumstance within the biofilm that triggers the genetic variance.

All in all，since the diversity that provides buffering effects is regarded as the guarantee for creatures to adapt to the environmental fluctuation［43］，the phenotype variations，including diverse protein expression patterns and genome alteration，seem to be another crucial strategy enhanced by aggregation so that the microbes can deal with the instable surroundings.The formation of biofilm creates a stressful microenvironment which in turn benefits a majority of the citizens.

1.5 Exchanging information among members in the biofilm

Alteration in the genome of bacteria is the major reason leading to their resistance to antimicrobials.Besides the mutations from their own genes，it is the horizontal transfer of the genes that mainly contributes to the spread of resistance and had been more emphasized as people are paying more and more attention to the multi-drug resistance［44］.Plasmids conjugation and transformation are two main mechanisms of gene transfer.Considering the juxtaposition of bacteria enclosed within a biofilm，the aggregation state seems to provide the appropriate condition for the bacteria to conjugate and transform，leading us to ratiocinate that the formation of a biofilm facilitates more efficient gene exchanges among the members.There have been a variety of experiments that illustrate the transfer of conjugative plasmids within biofilms.When mixed within a biofilm，a strain of Streptococcus sp.was able to display tetracycline resistance originally belonging to Bacillus subtilis which exclusively contains conjugative transposon Tn5397 that confers the tetracycline resistance［45］.In another significant experiment，Hausner and Wuertz built a GFP-tagged plasmid pRK415，and after enumerating the transconjugant cells within the biofilm with the help of fluorescence in situ hybridization，they concluded that the plasmid transfer rate was much higher in biofilms than in planktonic states and was independent of the help cell density or nutrition concentration［46］.It has also demonstrated that S.mutans cells could be transformed at a rate 10-600 folds higher than the planktonic cells when they aggregated in a biofilm［47］.In marine biofilms on chitinous surfaces，QS-proficient Vibrio cholerae become naturally competent to take up extracellular DNA［48］.Acquiring new genes plays a crucial role during the process of bacterial adaptation to their surroundings，and recent findings even revealed that conjugation contributes to the formation of biofilms［49］and to the stimulation of the synergistic co-cultivation of different species within the biofilm［50］.It seems that cellular aggregating can be both the consequence and cause of genome plasticity［51］.

During the last decade，a more peculiar notion was brought up that bacteria not only exchange their genes，but also communicate to each other by releasing and receiving certain chemical signals.Quorum sensing is the best studied cell-to-cell communication manner in bacteria［52-53］.Three categories of molecules are employed as signals to talk with one another:oligopeptide used by Gram-positive bacteria，AHLs by Gram-negative bacteria，and the third referred to the LuxS/AI-2 pathway［54］.The most prominent feature of quorum sensing is that bacteria would regulate their gene expression in accordance to the cell density in the environment［55］.These types of communication are more common when bacteria assemble into a biofilm because of the increased cell density within an aggregate.The communication efficiency is also enhanced as a result of the close distance between cells so that the expression of QS-target genes is better coordinated，especially those involved in resistance to environmental fluctuations.For example，mutating the genes involved in the quorum sensing process of P.aerations would lead the cells within the biofilm to be more sensitive to hydrogen peroxide and a decreased expression of superoxide dismutase［18］.Secondly，research demonstrated that tobramycin tolerance of P.aeruginosa was also quorum-sensing dependent［56］.Recently，more and more research also suggested that quorum sensing has a great impact on the formation and stability of a biofilm［57-61］.All these remarkable observations suggest that aggregation offers bacteria more and better opportunities to exchange genes and signals，which in turn ameliorate their capability to deal with the adverse situations.These special behaviors may lead bacteria to work and live collectively as a community，within which all the members cooperate to get over the environmental difficulties.

2 The Ecological significance of bacterial aggregation

We have focused mainly on the protection mechanisms brought by microbial aggregation above，which display the special ecological significance of this special cellular assemblage.Indeed，aggregation is the most widely utilized strategy for microbes to adapt to the environment.For example，when the pH in the environment is reduced，human influenza virus tends to be induced to aggregate［62］.The aggregate formation of P.syringae cells are much more capable of tolerating environmental stresses，and the preferential survival of cells in aggregates promotes a highly clustered spatial distribution of bacteria on leaf surfaces［63］.The prominent characteristic of M.xanthus is that they can be induced by nutrition limitation to assemble in the structure of fruiting bodies［64］，within which these cells would undergo different development processes and some would sporulate to resist desiccation and starvation.Aggregation seems to be able to prevent the penetration of active agent，for dispersing the aggregates by 20 Hz wave and precentrifugation clearly reduce the survivors of PFU.As the simplest life form on earth，virus aggregation cannot be deemed as complex tissues or communities as mentioned at the beginning of the review，but as an ordinary binding of the bio-molecules in some circumstances.However，this special phenomenon provides the basic indication that aggregating is a crucial strategy adopted by creatures to adapt to the environment better.

When it comes to bacteria and fungi，microbial aggregation，known as the structure so-called biofilm，plays a much larger part during their whole life course，especially at the crossroads of dying and surviving.The survival times for the biofilm-forming Acinetobacter baumannii which on dry surfaces were longer than for the non-biofilm-forming ones.Biofilm formation increases the survival rate of A.baumannii on dry surfaces and may contribute to its persistence in the hospital environment，increasing the probability of causing nosocomial infections and outbreaks［65］.Besides，it had been widely accepted that formation of biofilms can help microbes to tolerate antibiotics，and more remarkably，a recent finding revealed that subinhibitory concentration of antibiotics could induce the formation of biofilms［66］.In addition，the phagetolerant subpopulation of E.coli resulting from exposing to bacteriophages also display high tendency of biofilm formation［67］，and the prominent acid tolerance of S.mutans also relates to the formation of biofilms［68-69］.Furthermore，Lindow’s group investigated the survival of P.syringae on bean leaf surface under different humidity conditions.Under moist condition，the total number of living cells have no variance between planktonic and aggregation，yet when exposed to periodical desiccation，large aggregates were apparent and more than 90%of the living cells settled in these large aggregates［63］.

These phenomena indicate that aggregating is the main strategy adopted by microorganisms when the living conditions are adverse.Amongst the differ-ent virulence factors，biofilm formation and bacterial aggregation，often mediated by cell surface structures such as fimbriae，are common traits among uropathogens that can easier cause Catheter-associated urinary tract infection(CAUTI)［70］.About 30%to 80%of epiphytic bacteria aggregate together to reside on leaf surfaces，and numerous species，like Pseudomonas sp.and Enterobacter agglomerans，are present as aggregates or symplasmata when colonizing on root surfaces or other tissues［71-72］. Considering the protection mechanisms that biofilms may bring to their members and the fact that most bacteria form microcolonies or biofilms in their ecological environment，it is safe to suggest the crucial ecological function of aggregation:it helps bacteria to adapt the environment better.

However，while bacteria enjoy the benefit brought by colony effect，competition would raise another problem.Facing limited resources，like the community formed by macro species，some members may occupy more supplies to survive，whereas others may be washed out.This cheating phenomenon seems to tamper the benefit of aggregation since such behavior spoils cooperation among the community members.For instance，such a cheating behavior is prominent among myxobacteria［73］:a minority of members would develop into spores within the fruiting bodies and consequently survive the nutrition limitation whereas some peripheral bacteria might undergo autolysis.Besides，cell death was discovered within the biofilm formed by other species as well，and such an apoptosis-like cell death indicated important ecological advantages including generating a metabolically and phenotypically diverse state［74-76］.There is no denying that cheating will undermine the interaction between social members，and too many social conflicts may disrupt the evolution from unicellularity to multicellularity［77］.But were it not for the immolation of parts of the whole group，all of them may have perished under certain stresses.This unjustness of distinct fates of community members saves the colony to some extent，and such a temporary elusion from danger reflects the ecological effect of aggregation.It is the aggregation structure that ensures the microbial cheaters to be protected and take advantage from other altruists and reduces the waste of limited resource to a minimum.

Furthermore，living in the natural environment，symbiotic microorganismsare widespread，among which coaggregation exhibits outstanding ecological functions as well.Transfer P.fluorescens A506 to the aggregates or solitary cells formed by P.agglomerans 299R and then expose them to desiccation，the survival rate of the immigrant strain transferred to the aggregates is about twice compared to those transferred to the solitary form［78］.The synergistic interaction among four strains，Microbacterium phyllosphaerae，Shewanella japonica，Dokdonia donghaensis，and Acinetobacter lwoffii，is a cogent example.The coaggregated biofilm mass increased by more than 167%compared to the biofilm formed by a single strain and when dealt with antibacterial agents，the relative activity(biofilms exposed to the antimicrobial agents versus nonexposed ones)of the coaggregated biofilm was also significantly higher than that of the single strain biofilm［79］.In addition，high sheer forces are hypothesized to be the selective factors in nature favoring the coaggregation of multi-species biofilms，and noncoaggregating bacteria have been reported to be easily washed away when meeting these forces［80］.Indeed，coaggregation has been observed in a number of environments，such as mammalian gut，human urogenital tract，and potable-water-supply systems［80］.

Since coaggregation represents a genome-genome complementary interaction［81］，such a mutual benefit contributes to their adaptation to fluctuant niches.For instance，recent studies led people to raise the hypothesis that it is coaggregation and coadhesion that offer opportunities for bacteria to signal other species，resulting in the important physiological and metabolic interactions among different types of bacteria［80］.Oral biofilm is the best studied coaggregate which is formed by numerous genetically distinct species.Within an oral biofilm，streptococci can express sialidase and exoglycosidases that can degrade the host serum glycoproteins，producing fermentable carbohydrates for other bacteria like fusobacteria.Fusobacteria were thought to be physical bridges that promote to create an anaerobic environment，in which other strict anaerobes can live well［82］.Assembling together，a close proximity maximizes the efficiency of transferring substances unique for one species to other kinds of products that can be subsequently used by other species nearby，which makes the best use of the resources by the whole consortium.

3 The evolutionary significance of microbial aggregation

The previous parts had summarized the particularity of a widespread natural organization of most microorganisms.Besides its significant ecological functions，such a specific architecture also gives evolutionary indications.The evolutionary origin of complex life-forms had been believed to be the major transition from single cells towards multicellular organisms.However，what is the drive，in other words，what is the natural selective force of this conversion?This shift is unusual because building a multicellular organism requires not only communication among members with accurately releasing and responding to different signals，but also coordination and integration among identical and different developmental phenotypes，more importantly，because the transition offers the microbe a better ability to adapt living environment.

Not until the last two decades had scientists taken notice of the multicellular characteristics of microorganisms，since the classical microbiological study methods and research emphasis constrained researchers to the individual properties of microorganisms.Study properties of bacterial aggregation reveals their similarity to multicellular organism:aggregated cells provide a close proximity so that crucial communication can take place in cell-cell adhesion and interaction;aggregation of cells offers opportunities for members to undergo different environmental conditions，which induce cells to develop into divergent phenotypes to optimize their growth and survival in the niches［83，37］;and aggregated microorganism exhibit an prominent and sophisticated coordination among members.In addition，within biofilms，there is even cellular behavior resembling the apoptosis of multicellular creatures.Similar to the developmental significance of apoptosis，cell autolysis helps to facilitate the physiological conditions of a certain number of cells so that they can survive the adverse situations［76］.More importantly，assembling together to form a multicellular organism promotes adaptation to the natural environment.As mentioned above，forming biofilms can facilitate better utilization of nutrition and water，efficiently prevent the damage from UV light，desiccation，antimicrobials and other environmental stresses.These superiorities of microbial aggregation seem to imply that the nature favors multicellularity.It is the adaptation of multicellular organisms to their environment that drives the transition from single cell to multicellularity.

4 Acknowledgements

This work was supported by the National Natural Science Foundation of China No.30870055 and No.31170035(to F.Y.).The authors are grateful to Mark S.Miller and Becky M.Miller for their assistance in proofreading.

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