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Keywords:

  • Serratia liquefaciens;
  • Exoenzyme;
  • Flagella;
  • Surface motility;
  • Quorum sensing

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Differential control of growth phase-dependent production of extracellular enzymes
  5. 3Involvement of two different regulatory systems in control of growth phase-dependent gene expression
  6. 4Surface motility
  7. 5Communication and interactions between bacterial cells: towards multicellularity
  8. Acknowledgements
  9. References

Serratia liquefaciens secretes a broad spectrum of hydrolytic enzymes to the surrounding medium and possesses the ability to differentiate into specialized swarmer cells capable of rapid surface motility. Control of exoenzyme production and swarming motility is governed by similar regulatory components, including a quorum-sensing mechanism and the flagellar master operon flhDC.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Differential control of growth phase-dependent production of extracellular enzymes
  5. 3Involvement of two different regulatory systems in control of growth phase-dependent gene expression
  6. 4Surface motility
  7. 5Communication and interactions between bacterial cells: towards multicellularity
  8. Acknowledgements
  9. References

Serratia liquefaciens is an opportunistic pathogen which is capable of colonizing a wide variety of surfaces in water, soil, the digestive tract of rodents, plants, insects, fish and humans [1]. Like most Serratia species, S. liquefaciens secretes a broad spectrum of hydrolytic enzymes to the surrounding medium [2]. S. liquefaciens is generally motile, by means of peritrichous flagella and one strain, designated MG1, also possesses the ability to differentiate into specialized swarmer cells capable of rapid surface motility [3]. Control of exoenzyme production and the formation of an expanding swarming culture relies on similar regulatory components, namely a quorum-sensing mechanism that employs two diffusible pheromones and the flagellar master operon flhDC[3–5]. Since swarming motility has to be viewed as a form of multicellular behaviour, S. liquefaciens MG1 is an interesting model organism for studying procaryotic cell interactions. This review summarizes the current knowledge of environmental stimuli and the genetic control elements involved.

2Differential control of growth phase-dependent production of extracellular enzymes

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Differential control of growth phase-dependent production of extracellular enzymes
  5. 3Involvement of two different regulatory systems in control of growth phase-dependent gene expression
  6. 4Surface motility
  7. 5Communication and interactions between bacterial cells: towards multicellularity
  8. Acknowledgements
  9. References

2.1Expression profiles

In the course of a search for bacteria that produce extracellular hydrolytic enzymes we isolated a strain from a softened cucumber that was subsequently identified as S. liquefaciens and was denoted MG1 [6]. Like most Serratia species the strain produces various extracellular enzymes including two proteases, at least one chitinase, a lipase, a phospholipase, and a nuclease [2, 6, 7]. In a growing liquid culture, production of these extracellular enzymes is growth phase-dependent showing a characteristic burst in the transition between exponential growth and the stationary phase. We speculated that common mechanisms may be responsible for control of synthesis and secretion of the enzymes; however, experimental evidence did not support this hypothesis. A detailed analysis of the temporal pattern of exoenzyme production revealed that in rich medium the phospholipase was expressed earlier (induced at on OD450 of ∼0.3 coinciding with a slight reduction in growth rate) than the other hydrolytic enzymes which are induced at a 10-fold higher cell density (unpublished observations and [8]). After entry into stationary phase the production of the enzymes ceases and the cells initiate a precisely timed and coordinated program that transforms them into a stress resistant state enabling the strain to survive carbon starvation for many months (unpublished observation). These changes in response to nutrient limitation are reminiscent of the situation in other Gram-negative bacteria [9–12].

2.2Production of exoenzymes

Like all Serratia species, S. liquefaciens MG1 is capable of hydrolyzing chitin. It has been shown that S. marcescens produces five proteins with chitinolytic activity, whereas S. liquefaciens 1–141 produces three proteins with such activity [13]. The chitin utilization regulon of S. liquefaciens 1–141 contains two genes, chiA and chiB, encoding chitinases and another gene, chiC, encoding a chitobiase [14]. Two regulatory elements have also been identified: chiD which encodes a trans-acting repressor of chitinase and chitobiase expression and chiE whose product is thought to be involved in the synthesis of an inducer molecule.

Grimont et al. [15] identified Serratia strains by the electrophoretic profiles of their various proteases. S. liquefaciens MG1 produces several proteases of which the synthesis of at least two is regulated by a density-dependent quorum-sensing mechanism employing diffusible signal molecules ([5] and unpublished observations).

The fact that virtually all Serratia species produce an extracellular nuclease has been exploited for the development of a selective medium for the isolation of members of the genus [16]. S. liquefaciens MG1 also produces a very powerful nuclease [6]. In S. marcescens expression of nucleolytic activity has been demonstrated to be inducible by the lexA-dependent SOS system [17], and we have found that mitomycin treatment similarly leads to induction of nucleolytic activity in MG1 (unpublished observation).

We have concentrated on the identification of factors affecting phospholipase production. Expression of the phospholipase encoding gene, phlA, is regulated at the level of transcription initiation from one or both of two separate promoters which respond to oxygen content, temperature, catabolite repression and changes in the growth phase of the culture [8]. One promoter (pX) which is positioned approximately 600 bp upstream from the phlA gene almost perfectly matches the consensus binding site of the Fnr protein [18]. Expression studies of a phlA–lacZ fusion combined with primer extension analysis under oxygen limited as well as under oxygen saturated growth conditions, strongly suggested that this promoter is mainly responsible for phospholipase expression under anaerobic conditions. The other promoter (pA) which is positioned 50 bp upstream from the phlA gene is subject to catabolite repression and up-regulated during the transition from exponential to late exponential growth [8]. Furthermore, the activities of both promoters are highly responsive to temperature changes. pA is highly homologous to σF-controlled E. coli promoters and is completely silent in flhDC backgrounds of both E. coli and S. liquefaciens[4, 8]. In E. coli the genes flhD and flhC constitute a master operon known to control expression of flagellin, chemotaxis and motility genes through the flagellar sigma factor σF [19, 20]. In contrast to the nuclease and the chitinases which are induced upon partial SOS induction [17], we have not observed any stimulation of phospholipase synthesis in response to addition of the SOS inducer, mitomycin (unpublished observation).

In contrast to cloned S. marcescens metalloprotease and cloned Erwinia chrysanthemi metalloproteases B and C [21, 22] which require the presence of specific secretion functions, the phospholipase is secreted efficiently from most E. coli strains. However, closer examination revealed the participation of specific, yet unidentified, helper proteins allowing true secretion of the enzyme. The process of secretion of the phospholipase is dependent on the presence of a functional flhDC operon suggesting that genes encoding the putative secretion components of the exoenzyme are in fact present in E. coli and contained within the flagellar regulon of both E. coli and S. liquefaciens[23]. In contrast, synthesis and secretion of the other S. liquefaciens extracellular enzymes remain entirely unaffected by the presence or absence of a functional flhDC operon [4].

Phospholipase is co-transcribed with a downstream gene phlB[6]. phlB encodes an intracellular immunity protein, PhlB, which when complexed with the phospholipase neutralizes its enzymatic cell lysis activity [23]. This protein plays a key protective role in situations where phospholipase secretion is limited. In the absence of a functional secretion mechanism phospholipase is able to make its own way through the membranes the result of which is cell lysis. In situations were phlA is mainly expressed from the pX promoter, PhlA and PhlB are synthesized in what seems to be a correct molar ratio [23]. As a result enzymatic inactive complexes are formed within the cells. A mutational analysis has pointed to a DNA region positioned upstream of the pA promoter as being involved in post-transcriptional regulation of PhlA and PhlB co-expression. Genes encoding such intracellular enzyme inhibitors of metalloproteases have been identified in S. marcescens and Erwinia chrysanthemi[22, 24] and of E. coli colicin M [25]. Interestingly, the genes encoding the Serratia metalloprotease and the protease inhibitor are organized in a two gene operon as the phlA and phlB genes.

2.3Biological significance of exoenzymes

S. marcescens, S. liquefaciens, and S. proteamaculans are considered potential insect pathogens [26]. They cause a lethal septicemia after penetration into the hemocoel. Pathogenicity appears to be correlated with the production of phospholipase, proteinase, and chitinase. Purified Serratia protease or chitinase is very toxic when injected into the hemocoel [27, 28]. S. marcescens is known as an opportunistic human pathogen causing infections in immunocompromised patients. The proteases produced by S. marcescens play a major role in the phatogenesis of experimental pneumonia [29], and keratitis [30].

3Involvement of two different regulatory systems in control of growth phase-dependent gene expression

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Differential control of growth phase-dependent production of extracellular enzymes
  5. 3Involvement of two different regulatory systems in control of growth phase-dependent gene expression
  6. 4Surface motility
  7. 5Communication and interactions between bacterial cells: towards multicellularity
  8. Acknowledgements
  9. References

3.1The flagellar master operon flhDC controls phospholipase and flagellar synthesis

Growing cells of S. liquefaciens MG1 have the capability to develop extensive flagellation which is growth-phase dependent and follows the same pattern as synthesis of the phospholipase [4]. In E. coli synthesis of flagella is controlled by a hierarchy at the transcriptional level [31]. The top of this hierarchy consists of only one operon, the flhDC operon. In E. coli activation of this operon is under catabolite repression and requires binding of the cAMP/CRP complex for its expression [32]. In addition, the heat shock proteins, DnaK, DnaJ and GrpE, are involved in expression of the flhDC operon [33]. Recently, acetyl phosphate has been demonstrated to be involved in the inhibition of flagellum formation at elevated temperatures [34]. This inhibition is mediated by OmpR, the response regulator of the osmoregulation system [35], the phosphorylated form of which is able to bind to the promoter region of the flhDC operon [36].

Expression of both phospholipase and flagella genes in S. liquefaciens MG1 is controlled by an operon that is highly homologous to the flhDC operon of E. coli[4]. Controlled expression of the flhDC operon from an IPTG-inducible Ptac promoter results in cells that are not only highly flagellated but also produce phospholipase during the entire growth phase suggesting that a putative inducing stimulus leading to growth phase-dependent expression of target genes is channeled through this key regulator. Overexpression of flhDC not only induces hyperflagellation but also leads to filamentous, multinucleate cells indicating that the flhDC operon may also be involved in the regulation of cell division [3]. In E. coli, overexpression of flhDC causes inhibition of growth, and a connection between synthesis of flagella and cell division has been suggested [37]. It is noteworthy that differentiated S. liquefaciens cells strongly resemble swarm cells that are produced by certain strains of Serratia and Proteus on appropriate surfaces (see below).

In S. liquefaciens, expression of the phospholipase is tightly coupled to the synthesis of flagella. Likewise, in E. coli the expression of the microcin B12 (mcbA) gene is positively regulated by flhDC[37]. These observations, together with the involvement of flhDC in the regulation of cell division, strongly support the idea that the flhDC operon is involved in global gene regulation rather than being a specific regulator solely controlling synthesis of flagella.

3.2The quorum-sensing mechanism

In recent years it has become evident that N-acyl homoserine lactones (AHL) are widely used by Gram-negative bacteria as diffusible communication signals. Such regulatory circuits function as quorum-sensing systems that allow bacteria to sense and express target genes in relation to their culture density. AHL/response regulator systems homologous to the LuxR–LuxI signaling system in V. fischeri have been identified in various bacteria, and it has been demonstrated that they are involved in the regulation of diverse physiological processes including bioluminescence, antibiotic production, and conjugal transfer of Ti plasmids [38, 39]. The fact that these molecules often control the synthesis of exoenzyme virulence factors in plant and animal pathogens in a cell density-dependent manner prompted us to investigate whether such a system could also be involved in exoenzyme production in S. liquefaciens MG1. Indeed, using high-performance liquid chromatography (HPLC), high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy in conjunction with chemical synthesis, we were able to identify two signal molecules, N-butanoyl-l-homoserine lactone (BHL) and N-hexanoyl-l-homoserine lactone (HHL) in cell-free S. liquefaciens culture supernatants [5]. BHL and HHL are present in a molar ratio of approximately 10:1. A gene, designated swrI, the predicted translation product of which exhibits homology to the LuxI family of putative AHL synthases was found to be responsible for directing synthesis of both BHL and HHL. In a swrI mutant strain, extracellular proteolytic activity is reduced when compared with the wild-type [5]. A more detailed analysis revealed that two proteolytic enzymes, with molecular weights of approximately 51 and 57 kDa, were specifically down-regulated in the mutant strain (unpublished observation). However, expression of the other extracellular enzymes was unaffected by the mutation [5].

DNA sequencing has revealed the presence of an open reading frame next to the swrI gene with homology to the LuxR family of AHL-binding transcriptional regulators (unpublished results). The biological function of this gene, which has been denoted swrR, remains to be elucidated.

4Surface motility

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Differential control of growth phase-dependent production of extracellular enzymes
  5. 3Involvement of two different regulatory systems in control of growth phase-dependent gene expression
  6. 4Surface motility
  7. 5Communication and interactions between bacterial cells: towards multicellularity
  8. Acknowledgements
  9. References

Exposure of S. liquefaciens MG1 to different agar concentrations affects cell morphology and motility dramatically. When inoculated in the centre of plates containing agar concentrations below 0.4% the cells swim in the water-filled channels producing classical chemotactic rings of dense growth. If the agar concentration is elevated and kept in the range of 0.4–1.2% the cells are no longer able to move within the agar. Instead a regular colony is formed at the inoculation point. Thereafter the cells at the rim of the colony initiate a differentiation process resulting in long (up to 50 μm), multinucleate, aseptate, hyperflagellated swarm cells, which have the unique ability to move on top of the agar surface. This type of behaviour is referred to as swarming by analogy to the phenomenon observed in swarming bees. The rapid outward movement of the swarm cells at the rim of the swarm colony is accompanied by bacterial growth inside the colony resulting in an extremely fast colonization of all available surface space. We have recorded velocities of colony expansion of up to 10 mm/h. Microscopic inspection revealed that swarm cells migrate in groups or rafts in a coordinated fashion. Individual swarm cells are unable to swarm. This is in sharp contrast to swimming cells which move separately in periods of smooth runs interrupted by short tumbles in an apparently unorganized manner.

Rich media generally support swarming motility. Swarming is also observed on minimal medium supplemented with a mixture of amino acids (such as Casamino acids), but no single amino acid is able to promote swarming migration [3, 5]. The doubling time of S. liquefaciens grown in liquid minimal medium is significantly higher in the presence of even low amounts of Casamino acids suggesting that the requirement of amino acids for swarming can be attributed to the stimulation of growth. Swarming motility of Proteus mirabilis is strongly promoted by media supporting high growth rates [40]. Furthermore, only poor cell elongation is observed in minimal medium [3]. The apparently indispensable requirement of amino acids may reflect a high demand for both building blocks and energy to synthesize and operate the hundreds of flagella produced during swarming differentiation.

4.1Swarm cell differentiation is dependent on flhDC

The flhDC operon plays a key role in the swarmer differentiation process. A flhDC null mutant strain of S. liquefaciens is neither able to swim nor to swarm [3]. Controlled expression of the flhDC operon from an IPTG-inducible Ptac promoter restores both forms of motility. Moreover, overexpression of flhDC in liquid medium results in filamentous, multinucleate, and hyperflagellated cells that are indistinguishable from swarm cells isolated from the edge of a swarm colony [3]. Thus, artificial stimulation of flhDC can overcome the otherwise obligatory requirement of surface contact. This indicates that at least the sensing of surface contact, which is the major stimulus for swarm cell differentiation, is channeled through the flagellar master regulon flhDC. Since the S. liquefaciens phospholipase is a member of the flagellar regulon and the phospholipase was found to be differentially up-regulated in swarm cells [3] we speculate that the flhDC operon may link swarm cell differentiation with the production of virulence factors. Interestingly, synthesis of the virulence factor hemolysin in swarm cells of P. mirabilis is also highly stimulated [41].

4.2Colony expansion on the surface is dependent on quorum sensing

Genetic analysis demonstrated the importance of a functional swrI gene for swarming motility. In an swrI null mutant, the formation of a swarming colony is abolished but can be restored by the addition of exogenous AHL (therefore swrI, swarmer initiation). However, inactivation of swrI neither affects the growth rate in a liquid culture or swimming motility, nor the development of hyperflagellation and cell elongation [5, 42].

We recently observed that S. liquefaciens MG1 conditions the culture medium in a way that lowers the surface tension. Work currently in progress aims at a structural identification of a putative extracellular surface active compound. S. marcescens produces large amounts of extracellular biosurfactants, and so far three different wetting agents, named serrawettin W1, W2, and W3, have been isolated [43]. S. rubidaea produces two surface-active lipids rubiwettins R1 and RG1 [44]. In S. liquefaciens MG1, reduction of the surface tension of the medium appears to be dependent on the presence of a functional swrI gene. Interestingly, rhamnolipid biosurfactant production in Pseudomonas aeruginosa is controlled by a protein, RhlR, that belongs to the LuxR family of transcriptional regulators suggesting regulatory similarities [45]. In S. liquefaciens MG1 the medium conditioning was found to be indispensable for the colony expansion (unpublished observation). The requirement of biosurfactants for swarming motility of S. marcescens has been demonstrated previously [46].

4.3Interference with quorum sensing

The seaweed Delisea pulchra produces a number of halogenated furanones, which are structurally similar to the bacterial AHLs and have strong biological activity, including antifouling and antimicrobial properties [42, 47]. As a consequence this seaweed is rarely fouled in nature. Addition of purified furanone compounds to the growth medium inhibits swarming motility of S. liquefaciens as well as a number of other bacteria including P. mirabilis[7, 42]. Competition experiments suggest that at least two of the compounds are competitive inhibitors of AHL-mediated regulation processes. Although these strains do not encounter D. pulchra in the field, swarming in general is likely to play an important role in bacterial colonization of submerged surfaces. Swarm cells have been observed in biofilms [48], and an initial screening has revealed that a number of the bacteria, which live as discrete colonies on the algal surface, are in fact capable of swarming motility [42]. Similar to the model strain S. liquefaciens the furanone compounds produced by the alga abolish this process, and it might be speculated that this functions as a mechanism that affects the abundance, species composition and phenotypes of associated bacteria [42].

4.4Signal integration

In S. liquefaciens MG1 the formation of a swarm colony is not controlled by a single event or signal, but rather requires the sensing and integration of a variety of signals including metabolic potential, culture density and cell–cell and cell–surface recognition. At the genetic level at least two regulatory systems, FlhDC and SwrI/SwrR, are involved in the integration and interpretation of the diverse signals. A model of the control of swarm colony formation in which the various inputs are transduced through these two regulatory pathways is shown in Fig. 1.

image

Figure 1. Summary of the control systems involved in exoenzyme production, cell differentiation and formation of a swarming culture. The horizontal arrows indicates stimulation of gene expression. A? indicates that the compound or the biological function remains to be elucidated. Phl (phospholipase), Prt (protease), Fla (flagella) and W (wetting agent).

Download figure to PowerPoint

Swarm cell differentiation clearly depends on the gene products encoded by the flhDC operon whose concentration or activity status determines whether cells undergo a differentiation process that leads to filamentous, multinucleate, profusely flagellated cells. In our model, external signals sensing medium viscosity (surface recognition) as well as internal signals reflecting the physiological and biochemical makeup (nutritional status) of the cell are channeled through FlhD–FlhC. Thus, FlhD–FlhC seems to be integrating inputs from multiple converging signal-transduction pathways. Although unproven, the quorum-sensing (SwrI/SwrR) is likely to control the production of an extracellular wetting agent, which enables already differentiated swarm cells to move on top of a suitable substratum. Since the autoinducer cascade is only triggered on reaching a critical threshold concentration, expression will only occur when a certain culture density has been attained. Thus, the quorum-sensing circuit provides the cell with a system for signaling cell–cell interactions. The extent, however, to which these two regulatory systems interact remains to be elucidated.

5Communication and interactions between bacterial cells: towards multicellularity

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Differential control of growth phase-dependent production of extracellular enzymes
  5. 3Involvement of two different regulatory systems in control of growth phase-dependent gene expression
  6. 4Surface motility
  7. 5Communication and interactions between bacterial cells: towards multicellularity
  8. Acknowledgements
  9. References

The process of surface-induced swarm cell differentiation as well as cell–cell communication is dispensable for cells that are maintained in typical liquid laboratory cultures. Such cultures are considered to develop in a homogeneous way, all cells grow at the same period and they all exhaust the growth limiting nutrient at the same time and subsequently they simultaneously differentiate into a starvation resistant state. In contrast, a swarming colony represents a dynamic bacterial culture. Continuously the swirling motion of the swarm creates new zones of growth, the biomass of the culture increases and as a consequence the culture will spread and ultimately colonize all the available surface. This has consequences in nature since microbial activity is often associated with surfaces and it appears that one of the most remarkable characteristics of bacteria is their ability to form structured and co-operative consortia. It has also been appreciated for a long time that certain bacteria exhibit coordinated behavioural patterns that can be viewed as a primitive form of multicellularity. This form of surface translocation can thus be viewed as an example of organized bacterial behaviour in which expression of a range of extracellular products is linked to motility.

In S. liquefaciens MG1, cell–cell interactions are at least in part coordinated by diffusible AHL signal molecules, which enable the cells to communicate with each other. These observations strongly support a concept in which AHL-mediated cell–cell communication plays an integral role for the development of cooperative multicellular behaviour.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Differential control of growth phase-dependent production of extracellular enzymes
  5. 3Involvement of two different regulatory systems in control of growth phase-dependent gene expression
  6. 4Surface motility
  7. 5Communication and interactions between bacterial cells: towards multicellularity
  8. Acknowledgements
  9. References

This work was supported by grants from the Danish National Research Council, Danish Centre for Microbial Ecology, the Carlsberg Foundation, Løvens Kemiske Foundation, the Biotechnology and Biological Sciences Research Council UK, and the Australian Research Council.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Differential control of growth phase-dependent production of extracellular enzymes
  5. 3Involvement of two different regulatory systems in control of growth phase-dependent gene expression
  6. 4Surface motility
  7. 5Communication and interactions between bacterial cells: towards multicellularity
  8. Acknowledgements
  9. References
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