You are what you talk: quorum sensing induces individual morphologies and cell division modes in dinoroseobacter shibae

ABSTRACT _Dinoroseobacter shibae_, a member of the Roseobacter clade abundant in marine environments, is characterized by a pronounced pleomorphism. Cell shapes range from variable-sized

ovoid rods to long filaments with a high copy number of chromosomes. Time-lapse microscopy shows cells dividing either by binary fission or by budding from the cell poles. Here we

demonstrate that this morphological heterogeneity is induced by quorum sensing (QS). _D. shibae_ utilizes three acylated homoserine lactone (AHL) synthases (_luxI_1–3) to produce AHLs with

unsaturated C18 side chains. A Δ_luxI_1-knockout strain completely lacking AHL biosynthesis was uniform in morphology and divided by binary fission only. Transcriptome analysis revealed that

expression of genes responsible for control of cell division was reduced in this strain, providing the link between QS and the observed phenotype. In addition, flagellar biosynthesis and

type IV secretion system (T4SS) were downregulated. The wild-type phenotype and gene expression could be restored through addition of synthetic C18-AHLs. Their effectiveness was dependent on

the number of double bonds in the acyl side chain and the regulated trait. The wild-type expression level of _T4SS_ genes was fully restored even by an AHL with a saturated C18 side chain

that has not been detected in _D. shibae_. QS induces phenotypic individualization of _D. shibae_ cells rather than coordinating the population. This strategy might be beneficial in

unpredictably changing environments, for example, during algal blooms when resource competition and grazing exert fluctuating selective pressures. A specific response towards non-native AHLs

might provide _D. shibae_ with the capacity for complex interspecies communication. SIMILAR CONTENT BEING VIEWED BY OTHERS EVIDENCE OF TWO DIFFERENTIALLY REGULATED ELONGASOMES IN

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Article Open access 17 July 2024 IDENTIFICATION OF STRUCTURAL AND REGULATORY CELL-SHAPE DETERMINANTS IN _HALOFERAX VOLCANII_ Article Open access 15 February 2024 INTRODUCTION The term

‘quorum sensing’ (QS) refers to a form of cell-to-cell communication that involves the production, excretion and detection of small diffusible signalling molecules called autoinducers (AI).

The simplest QS systems consist of an enzyme for AI biosynthesis and a transcription factor that is activated by AI binding and induces the expression of a defined set of genes. Many

bacteria make use of more complex systems involving two or more types of AI molecules and different network architectures (Waters and Bassler, 2005). QS has first been described in the

marine bacterium _Vibrio fischeri_ colonizing the squid light organ. When cell density reaches a certain threshold, the ‘quorum’, a burst of AI synthesis coordinates the population-wide

activation of genes required for bioluminescence (Nealson and Hastings, 1979). Coordination of gene expression as a function of cell density was considered a paradigm for a long time but has

been challenged in recent years. The concept of QS activity being dependent on cell density has been extended to account for the fact that outside the shaking flask AI concentration is also

influenced by diffusion (for example, enclosures, microniches, local gradients in biofilms, microcolonies) (Kaplan and Greenberg, 1985), its stability (for example, in dependence of pH)

(Wang and Leadbetter, 2005) and various other parameters (Platt and Fuqua, 2010). One striking example comes from Schäfer et al. (2008) showing that _Rhodopseudomonas palustris_ synthesizes

an AI by incorporating a molecule from decaying plant material. They concluded that in this strain QS is dependent on both the cell density and the availability of an exogenously supplied

substrate. The concept of QS mediating the population-wide coordination of gene expression has been challenged, too, by the demonstration of a heterogeneous response towards QS signals in

several organisms (Bassler and Losick, 2006). In _Streptococcus pneumoniae_, QS through a peptide AI leads to the induction of the competent state only in a fraction of cells, whereas the

remainder of the population undergoes autolysis (Steinmoen et al., 2002), a phenomenon termed fratricide (Gilmore and Haas, 2005). A similar phenomenon was observed in _Streptococcus

mutans_. Lemme et al. (2011) used fluorescence-activated cell sorting (FACS) to separate induced and uninduced cells, identifying major transcriptome differences between both subpopulations.

For the Gram-negative bacterium _Vibrio harveyi_, it was shown that the wild type, displaying a heterogeneous QS response regarding bioluminescence, produces more biofilm than a

constitutive QS-active mutant (Anetzberger et al., 2009). Recently, a high variability of other QS-regulated traits like secretion systems and exoproteolysis was demonstrated for the same

organism (Anetzberger et al., 2012). Heterogeneous expression of _QS_ genes and controlled traits have also been shown for _Listeria monocytogenes_ (Garmyn et al., 2011) and _Vibrio

fischeri_, the model organism for coordinated gene expression through communication (Perez and Hagen, 2010; Perez et al., 2011). This paradigm shift is by far not restricted to QS. In the

last decades, it became more and more apparent that physiological heterogeneity is a fundamental characteristic of isogenic bacterial populations (but not restricted to bacteria). It is

mediated by various processes, namely stochastic gene expression, unequal distribution of molecules during cell division, ageing, and bi- or multistable gene-regulatory networks (reviewed in

Kaern et al. (2005); (Avery, 2006; Smits et al., 2006)). Population heterogeneity is thought to be a survival strategy in fluctuating and unpredictable environments (Acar et al., 2008). For

example, starved _Sinorhizobium meliloti_ differentiates into cells with low and high poly-3-hydroxybutyrate levels that have higher competitiveness for resources and long-term survival

capabilities, respectively (Ratcliff and Denison, 2010). _D. shibae_ is a representative of the Roseobacter clade, a large, diverse and ecologically important phylogenetic cluster of

Alphaproteobacteria (Wagner-Döbler and Biebl, 2006; Brinkhoff et al., 2008), abundant in temperate and polar marine habitats (Selje et al., 2004; Giebel et al., 2011). It was isolated from

the dinoflagellate _Prorocentrum lima_ (Biebl et al., 2005) and lives in symbiosis with marine algae (Wagner-Döbler et al., 2010). Under optimal growth conditions a remarkable variability in

cell size and morphology can be observed. The physiological role of the morphological heterogeneity and the mechanism by which it is controlled are unknown. _D. shibae_, like many other

Proteobacteria, relies on acylated homoserine-lactones (AHLs) for cell-to-cell communication. It produces C18en-HSL and C18dien-HSL with one and two unsaturations in the acyl side chain,

respectively, which represent novel structures not described in any other bacterial species so far (Wagner-Döbler et al., 2005). Genome analysis of _D. shibae_ revealed the presence of three

LuxI type AHL synthase genes (termed _luxI__1_, _luxI__2_, _luxI__3_). _luxI__1_ and _luxI__2_ are located on the chromosome downstream of a gene encoding a LuxR-type transcriptional

regulator, whereas _luxI__3_ is on the 86-kb plasmid without an adjacent _luxR_ gene. In addition, three orphan LuxR-type transcriptional regulator genes were found in the genome

(Wagner-Döbler et al., 2010). Here, we studied the role of the novel long-chain AHLs (C18en-HSL and C18dien-HSL) produced by _D. shibae_. We constructed a Δ_luxI__1_ knockout strain and

found that it does not produce any AHLs; thus it represents a QS signal synthesis null mutant. Without the ability to communicate, _D. shibae_ does not differentiate into morphologically

distinct cell types. We investigated the link between QS and cellular heterogeneity by comparison of the wild type and the QS null strain as well as the genetically complemented mutant using

electron microscopy, determination of chromosome copy number, time-lapse microscopy and cell-density-resolved transcriptome analysis. To study the role of distinct AHLs, we added C18-HSLs

with different numbers of unsaturations to the culture of the Δ_luxI__1_ mutant. MATERIALS AND METHODS Additional Materials and methods can be found in Supplementary Text S1. DETECTION OF

AHLS The production of AHLs was detected using the biosensor strains _E. coli_ MT102 pJBA132 and _P. putida_ F117 pKR-C12 as previously described (Wagner-Döbler et al., 2005), with the

following modifications. _D.shibae_ strains were grown in 100 ml defined minimal medium with 50 mM succinate and 2% of adsorber resins (Amberlite XAD-16, Rohm & Haas, Philadelphia, PA,

USA) for 36 h at 30 °C with agitation. Adsorber resins were removed from the culture by filtration, added to a separating funnel together with 25 ml methylene chloride and 200 ml distilled

water, and well shaken. The organic phase was removed. The methylene chloride extraction was repeated three times, with a total volume of 100 ml solvent. Extracts were concentrated to 2 ml

using a rotary evaporator (Heidolph VV2001, Kelheim, Germany) and stored at −20 °C. For bioassays, 10 μl was pipetted into polypropylene microtitre plates (PlateOne, Starlab, Hamburg,

Germany). When the methylene chloride had evaporated, 100 μl medium and 100 μl of the respective sensor strain were added. Microtitre plates were incubated at 30 °C with agitation for 30 h.

MICROARRAY ANALYSIS Processing of microarray data is described in Supplementary Text S1. For analysis of differential expression, only those genes with a false discovery rate-adjusted

_P_-value <0.01 and an absolute log2-fold change >1 under at least one condition were taken into account. Raw and processed microarray data have been deposited at the gene expression

omnibus database under the accession number GSE42013. FLOW CYTOMETRY AND FACS For flow cytometry and cell sorting, 1-ml samples were collected from cultures at the desired cell density, and

cells were fixed for 15 min by addition of 2% glutaraldehyde. Fixed samples were transferred to liquid nitrogen and stored at −20 °C. Before measurements cells were diluted 100-fold when the

optical density was below 0.4 and diluted 1000-fold when above 0.4 in sterile filtered PBS buffer (pH 7.4). For stoichiometric DNA staining, 10 μl 100 × SYBR Green solution (Molecular

Probes, Leiden, The Netherlands) was added to 1 ml sample and incubated for 20 min in the dark (Marie et al., 1997). For each sample a minimum of 50 000 cells were analyzed on a FACSCanto

flow cytometer (BD Bioscience) to follow SYBR Green fluorescence and a FACSAriaII (BD Bioscience, Heidelberg, Germany) for cell sorting. Fluorescent signals were collected logarithmically

using an FITC filter (excitation 488 nm, emission 519 nm). The sorting strategy is shown in Supplementary Figure S3. Data processing and analysis were carried out using the ‘flowCore’

package (Hahne et al., 2009) of the R BioConductor project. As the exact number of chromosomes in _D. shibae_ cells is not known, chromosome content was defined as chromosome equivalents in

relation to the SybrGreen peak with the lowest intensity. TIME-LAPSE MICROSCOPY Time-lapse microscopy was performed using an automated microscope (Zeiss Axiovert 200) using the heating

system 6 incubator and controller (Ibidi, Martinsried, Germany). For live-cell microscopy of growing microcolonies the agarose pad method was applied as described in Young et al. (2012)

using SWM medium pads in 35 mm μ-dishes (Ibidi) at an incubation temperature of 30 °C. Subsequent image analyses were performed with Axiovision (Zeiss, Jena, Germany) and the TLM-Tracker

software (Braunschweig, Germany; Klein et al., 2012). RESULTS We first investigated whether the three identified _luxI_ genes of _D. shibae_ encode functional autoinducer synthases capable

of synthesizing AHLs. The constitutive heterologous expression of each synthase in a non-AHL-producing _E. coli_ resulted in the production of long-chain AHLs (Supplementary Figure S1).

GC–MS analysis (Neumann et al., 2013) showed that when expressed in _E. coli_, the main compound produced by LuxI1 was the wild-type signal C18en-HSL. In addition, small amounts of C16-HSL,

C15-HSL and C14-HSL were found. LuxI2 expressed in _E. coli_ yielded C14-HSL variants as well as C15-HSL. LuxI3 did not produce any AHLs when cloned into pBBR1MCS-2, but synthesized C14-HSL

and 3-oxo-C14-HSL when cloned into the hyperexpression vector pTrcHis-TOPO. Compared with the wild type, the chain lengths and types of substitutions found through heterologous expression of

the AHL synthases in _E. coli_ clearly differed from those produced in _D. shibae_. For example, the main wild-type signal C18dien-HSL was not produced in _E. coli_. By contrast, C15-HSL

was never detected in _D. shibae_. These differences may be due to the different abundance of fatty acid precursors in the producing organism or lack of specific precursors. In order to

unravel the role of the autoinducer synthase LuxI1 and to identify QS-regulated traits in _D. shibae_, we constructed a Δ_luxI__1_ strain by replacing 425 bp of the coding sequence with a

gentamicin-resistance cassette via homologous recombination. Integration of the knockout cassette at the _luxI__1_ locus was verified by PCR and sequencing. The _luxI__1_ deletion was

genetically complemented with the plasmid pDP1containing the _luxI__1_ open reading frame controlled by the high-level-expression gentamicin promoter. Additionally, a control strain carrying

the empty plasmid was generated. DELETION OF _LUXI_1 COMPLETELY ELIMINATES AHL PRODUCTION IN _D. SHIBAE_ Previously it had been shown that _D. shibae_ synthesizes C18en-HSL and C18dien-HSL

in MB medium (Wagner-Döbler et al., 2005, 2010). This was now also confirmed in the minimal medium used for microarray analyses by GC–MS (Supplementary Figure S2). The previously detected

C8-HSL was not found. Extracts of the Δ_luxI__1_ strain showed no significant induction of fluorescence in the sensor strain used for the detection of long-chain AHLs (Figure 1a). GC–MS

analysis of the extracts confirmed the complete absence of AHLs in the Δ_luxI__1_ strain (data not shown). As the deletion of only one synthase leads to a complete loss of AHL production,

the contribution of LuxI2 and LuxI3 to AHL synthesis in _D. shibae_ appears to be dependent on the presence and level of the signal molecules provided by LuxI1. The wild-type signals

C18en-HSL and C18dien-HSL were strongly overproduced in the _D. shibae_ Δ_luxI__1_ strain carrying pDP1, and thus it was possible to purify these novel signals and determine their absolute

configuration (Neumann et al., 2013). Both C18en-HSL and C18dien-HSL were then chemically synthesized and their effect on the phenotype and transcriptome of the Δ_luxI__1_ deletion strain

was studied. QS AFFECTS THE GROWTH RATE OF _D. SHIBAE_ The growth of the Δ_luxI__1_ mutant differed from the wild type in various aspects: the mutant showed a shorter lag phase, higher

growth rate and higher maximum cell density than the parent strain (Figure 1b and Table 1). Genetic complementation of the _luxI__1_ deletion restored the wild-type growth behavior, whereas

the presence of the ‘empty’ vector pBBR1MCS-2 had no significant impact on the growth of the mutant. In order to study the response of the non-AHL-producing mutant Δ_luxI__1_ to AHLs,

synthetic signals C8-, C18en- and C18dien-HSL as well as C18-HSL were added to Δ_luxI__1_ cultures at final concentrations of 500 nM. The C18-HSL has not been detected in _D. shibae_,

neither in rich medium nor in minimal medium (Neumann et al., 2013). Moreover, all of the AHLs found in _D. shibae_ cultures have one or two double bonds in the acyl side chain. We therefore

assume that the saturated AHL C18-HSL is a non-native signal in _D. shibae_. In the presence of C8-HSL we did not observe any effect on the mutant’s growth behavior (Supplementary Table

S2). Growth rate and doubling time were identical to the Δ_luxI__1_ culture. The data are in accordance with the lack of detection of C8-HSL, showing that _D. shibae_ neither produces nor

responds to this signal. The growth rate was affected in different ways by the different C18-HSLs (Supplementary Table S2). The non-native signal C18-HSL did not affect the growth rate and

doubling time of the Δ_luxI__1_ mutant. By contrast, C18en-HSL and C18dien-HSL reduced the growth rate even below that of the wild type. LOSS OF QS SIGNALING RESULTS IN HOMOGENOUS CELL SIZE,

REDUCED CHROMOSOME CONTENT AND CELL DIVISION EXCLUSIVELY BY BINARY FISSION Through investigation of the Δ_luxI__1_mutant by scanning electron microscopy, we observed altered cell morphology

(Figure 2a). Wild-type cultures exhibited heterogeneous cell morphology with respect to cell shape and size. They were composed of ovoid and rod-shaped cells of different sizes and

eye-catching elongated cells reaching up to 10 μm length. Cells of _D. shibae_ Δ_luxI__1_ were homogeneous in size and morphology. Genetic complementation restored the wild-type morphotypes.

Closer examination suggested that wild-type cells were using different types of cell division (Figure 2b) that could not be observed in the mutant. To further investigate the heterogeneity

in the wild-type population and to examine whether the elongated morphotype of the subpopulation might be caused by different growth and division behavior, we determined the relative

chromosome content at various cell densities on the single-cell level using stoichiometric SybrGreen staining and subsequent flow cytometric analysis, assuming that chromosomes accumulate in

elongated cells. This technique provides an elegant tool to study the cell cycle and the DNA replication pattern (Müller, 2007) given that the chromosome content correlates with the

fluorescence intensity. As the absolute number of chromosomes per cell is not known, the term ‘chromosome equivalent’ is used. _D. shibae_ wild type showed two distinct peaks representing

two different cell fractions with one (C1n) and two chromosome equivalents (C2n) per cell, respectively (Figure 2c). However, a small fraction contained multiple chromosome equivalents per

cell (C_x_n). Sorting of the wild-type cells according to their chromosome content and subsequent microscopic investigation (Supplementary Figures S3 and S4) confirmed that the C1n fraction

consisted of small ovoid cells, the C2n fraction contained cells dividing by binary fission and the fraction containing more than two chromosome equivalents was comprised of elongated cells.

Only cells with one or two chromosome equivalents were observed in the QS null mutant Δ_luxI__1_ (Figure 2c). These data further suggest that subpopulations with different replication and

cell division patterns coexist in one wild-type culture of _D. shibae_, which are dependent on the LuxI1-produced AHLs. Flow cytometric investigation of the chromosome distribution at the

mid-exponential growth phase (OD 0.4) in mutant populations supplemented with the different long-chain AHLs revealed a graduated complementation pattern, similar to that described above for

growth. The results are presented in Figure 3. The _D. shibae_ wild-type population was composed of 30.75% cells containing one chromosome equivalent (C1n), 50.5% harboring two equivalents

(C2n) and 18.75% carrying multiple chromosome copies (C_x_n). The homogeneous mutant culture consisted of 49.85% C1n cells, 49.1% C2n cells and 1.05% cells belonging to the C_x_n fraction.

The same distribution was observed in DMSO-treated mutant cells, which served as a negative control. The addition of saturated C18-HSL resulted in a slight increase in C_x_n cells (2.4%).

However, in the presence of C18en-HSL and C18dien-HSL, the distribution of chromosome equivalents of the Δ_luxI__1_culture was shifted to the wild-type pattern almost completely. _In vivo_

analysis using time-lapse microscopy confirmed that wild-type cells employ different modes of cell division. Figure 4a demonstrates that elongated cells divide by forming one substantially

smaller daughter cell through polar growth. We define this type of cell division as budding. In contrast, small ovoid rods divide into two equally sized daughter cells; thus they employ

binary fission. In Figure 4b we highlight a cell that buds from alternating cell poles before it divides into three daughter cells. The QS mutant employs exclusively binary fission (Figure

4c). The full movies of wild-type, mutant and complemented strain can be found as Supplementary Movies S1–S3. COMPARATIVE TRANSCRIPTOME ANALYSIS OF _D. SHIBAE_ WILD-TYPE, Δ_LUXI_1 AND

Δ_LUXI_1 PDP1 To gain insights into the effects of QS on transcriptional control, two different experiments were performed: gene expression in Δ_luxI__1_ and Δ_luxI__1_ pDP1, respectively,

was compared with the wild type, with samples being taken at different culture densities (OD600 0.1, 0.2, 0.4, 0.6 and 0.8) in the exponential phase as well as in the stationary phase (6 h

after the strains reached their maximum OD600). To investigate in depth the capability of C18en-HSL and C18dien-HSL to act as signaling molecules responsible for specific regulation of gene

expression, we analyzed the transcriptome profiles of mutant cultures supplemented with those AHLs in comparison with Δ_luxI__1_and the wild type. Furthermore, the influence of non-native

C18-HSL was studied on the transcriptome level. Cultures in the mid-exponential growth phase supplemented with 500 nM of AHL were used for this experiment. Compared with the wild type, 344

genes were differentially expressed in the Δ_luxI__1_ mutant throughout growth. These genes were clustered into five groups according to their expression changes during growth (Supplementary

Table S3 and Supplementary Figure S5). Interestingly, we did not observe strong density-dependent expression profiles for most of the genes. Instead, gene expression differed between wild

type and mutant throughout exponential growth; additionally, large differences were observed in the stationary phase. One hundred and thirty-three genes showed a significant differential

regulation in both exponential and stationary phase. In all 68 genes were differentially expressed exclusively in the exponential, whereas 143 genes exclusively in the stationary phase. The

expression of only 59 genes was increased, whereas all other genes showed decreased expression in the QS null mutant. _D. shibae_ wild-type gene expression was also compared with that of the

Δ_luxI__1_ mutant complemented with pDP1 (Supplementary Table S3). Three hundred and twenty-six genes displayed a significant change in expression when all samples were taken into account.

However, major changes occurred only in the late exponential and stationary phase, possibly reflecting overexpression of the _luxI__1_ gene. The wild-type expression level of 255 out of 344

genes differentially expressed in the Δ_luxI__1_ mutant was successfully restored in the complemented strain. Eighty-nine genes showed differential expression, in most cases an inverse

regulation compared with the mutant, consistent with the overexpression of _luxI__1_. Thus, the microarray data are consistent with the observed restoration of the wild-type phenotype in the

genetically complemented strain. Two alternative sigma factors, _rpoH__I_ (Dshi_2978) and _rpoH__II_ (Dshi_2609), were downregulated in the mutant, with the minimum at the beginning and in

the stationary phase, respectively. Expression of one anti-sigma factor and its respective antagonist (Dshi_0072/73) was strongly reduced (Supplementary Table S3). Remarkably, 45% of all

genes differentially regulated in the Δ_luxI__1_ mutant encoded hypothetical proteins. This is a large fraction compared with 28% of all genes in the genome. Sixty-four genes encode proteins

with a predicted signal peptide but no transmembrane domains; thus, they might represent secreted factors. Almost all of them are hypothetical proteins. In the following sections, the four

major differentially regulated traits will be discussed in detail. _LUXI_ AND _LUXR_ TYPE QS GENES The gene expression of the cognate _luxR__1_ regulator (Dshi_0311) was not affected by

deletion of _luxI__1_, indicating that it is independent from the AHL produced by the neighboring synthase. The second _luxR__2__/I__2_ pair of genes (Dshi_2852/1) and – to a lesser extent –

the orphan synthase _luxI__3_ (Dshi_4152), however, were downregulated in the mutant, indicating that the AHLs synthesized by LuxI1 might be necessary for their activation (Figure 5a(1)).

The three orphan LuxR type transcriptional regulators (Dshi_1550/1815/1819) in contrast displayed no significant change in expression. The overexpression of _luxI__1_ in _trans_ led to a

slight overexpression of _luxR__2_/_I__2_ and restoration of _luxI__3_ wild-type expression level (Figure 5a(2)). The addition of chemically synthesized AHLs to cultures of _D. shibae_

Δ_luxI__1_ re-established the wild-type expression level of _luxR__2_/_I__2_ for all three compounds tested. In contrast, only C18dien-HSL was able to restore the expression of _luxI__3_ in

the QS null mutant (Figure 5a(3)). These microarray data for representative samples were confirmed by qRT-PCR, which additionally showed complete lack of expression of _luxI__1_ in the

mutant (Supplementary Figure S6). CELL CYCLE-RELATED GENES Cell cycle regulation has been exhaustively studied in the Alphaproteobacterium _Caulobacter crescentus_. This organism is

characterized by a dimorphic lifestyle controlled by a complex gene-regulatory network with the histidine kinases CckA and ChpT (Biondi et al., 2006) and the transcription factor CtrA as the

main components of the regulatory cascade (Purcell et al., 2008). A recent comparative genome analysis revealed that most Alphaproteobacteria share a common core set of regulators with

differing accessory elements (Brilli et al., 2010). Like in _Rodobacter sphaeroides_, _Roseobacter denitrificans_ and _Ruegeria pomeroyi_, a core of nine genes is also present in _D.

shibae_. Only five of those were affected by alterations in the QS system (Figure 5b(1)). The _luxI__1_ deletion led to a reduced expression of _cckA_ (Dshi_1644), _chpT_ (Dshi_1470) and

_ctrA_ (Dshi_1508). _DivL_ (Dshi_3346), a target gene of CtrA with unknown function in _Rhodobacterales_, was downregulated too. The transcription factor DnaA, responsible for the initiation

of DNA replication (Dshi_3373), was significantly downregulated during exponential growth and upregulated in the stationary phase; however, the log2-fold change (∼−0.7) was below the cutoff

used. The two Clp proteases controlling the protein level of CtrA in _C. crescentus_ (Dshi_1387/1388) did not change in expression in the Δ_luxI__1_mutant. The transcription factor GcrA

(Dshi_2616) and the DNA-methyltransferase CcrM (Dshi_0024) that activates DnaA promoter regionspriya through methylation were also unchanged. When _luxI__1_ was overexpressed by introducing

pDP1 into the Δ_luxI__1_ mutant, the expression of these genes was fully restored (Figure 5b(2)). The chemical complementation with different AHLs revealed a graduated response (Figure

5b(3)). Only C18dien-HSL was able to fully restore the wild-type expression level. Interestingly, only _ctrA_ and _divL_ responded to the addition of C18-HSL and C18en-HSL. In summary,

restoration of gene expression increased with the number of double bonds in the AHL side chain. To gain a better understanding how changes in cell cycle-related gene expression might act

globally, we then searched for binding motives for CtrA in the promoters of _D. shibae_ genes (Supplementary Figure S7). We identified 74 genes on the chromosome and 8 genes on the plasmids

with CtrA binding sites. However, not all of them were differentially regulated in the Δ_luxI__1_ mutant. The presence of two CtrA-binding sites in the promoter of the regulator _luxR__2_

suggests a crosstalk between the QS and cell cycle control systems. Furthermore, a CtrA binding site was present in the promoter of _rpoH_II, indicating a link between cell cycle regulation

and stress response. It seems plausible that the changes in the expression of the aforementioned genes may result in the observed differences in the chromosome content and cell division

between wild-type and mutant strain. FLAGELLAR BIOSYNTHESIS _D. shibae_ has a polar flagellum, which is encoded by three gene clusters (Dshi_3246-3268; 3358-3365, 3376-3380). Expression of

the complete flagellar biosynthesis machinery was reduced in the Δ_luxI__1_ mutant at all studied optical densities (Figure 5c(1)), with the maximum reduction occurring in the stationary

phase. Overexpression of _luxI__1_ in _trans_ restored the wild-type expression level (Figure 5c(2)). Accordingly, flagella could not be detected in the QS null mutant using flagella

staining or transmission electron microscopy, whereas flagellation was observed in wild-type cultures as well as in the genetically complemented strain (Supplementary Figure S8). Like for

the cell cycle regulation genes, the flagellar synthesis gene expression showed a graduated response to exogenous AHLs dependent on the number of double bonds in the acyl side chain (Figure

5c(3)), and flagellation of Δ_luxI__1_ was observed when AHLs were provided (Supplementary Figure S9). TYPE IV SECRETION SYSTEM _D. shibae_ contains two _vir_ gene clusters, one on the 191 

kb plasmid and the other on the 126 kb plasmid. Those two plasmids have been described as sister plasmids derived from a common ancestor (Wagner-Döbler et al., 2010). Accordingly, the two

_vir_ gene clusters are virtually identical, comprising the complete set of genes for the type IV secretion machinery for translocation of DNA or proteins (_vir_B1 to _vir_B11) (Christie et

al., 2005). The 191-kb plasmid carries altogether 198 genes; in addition to the 14 genes of the _vir_-operon, only 11 other genes were regulated in the QS null mutant. Similarly, the 126-kb

plasmid carries 136 genes. In addition to the 13 genes of the _vir_-operon, 6 other genes were differentially expressed in the mutant. All other genes on those two plasmids were unchanged,

although many of them were expressed. Thus, in _D. shibae_ not the copy number of the plasmid, but specifically the plasmid-localized _vir_ gene clusters are controlled by QS, in contrast to

the findings in _Agrobacterium tumefaciens_ (Pappas and Winans, 2003). Both _vir_ gene clusters (Dshi_3637–3650, 3972–3984) were among the only genes whose expression was constantly reduced

throughout growth as well as in the stationary phase in the QS null mutant (Figure 5d(1)). In contrast to cell division-related genes and flagellar biosynthesis, both _vir_ gene clusters

were overexpressed in _D. shibae_ Δ_luxI__1_pDP1, indicating that the response of this trait is more sensitive towards changes in AHL concentration than others (Figure 5d(2)). This

hypothesis is further confirmed by the finding that the addition of AHLs produced by _D. shibae_ caused overexpression of these genes. By contrast, the addition of non-native C18-HSL only

led to restoration of the wild-type _vir_ cluster expression level (Figure 5d(3)). This demonstrates that QS molecules from other bacteria can affect the gene expression of specific traits

in _D. shibae_. DISCUSSION The C18en-HSL and C18dien-HSL produced by _D. shibae_ represent structures whose signaling role is studied here for the first time. The data strongly suggest that

QS in _D. shibae_ controls the switch between two modes of life. In the absence of AHL signals, a fast-growing, morphologically homogenous population is found, which does not invest energy

into the synthesis of T4SS and flagella. In the presence of AHL signals, a slower-growing population of remarkable morphological and cell division heterogeneity can be observed, with some of

the cells being flagellated. We found that the QS system of _D. shibae_ is not restricted to the autoinducers produced by the organism itself; it specifically responds to a

non-self-produced structurally similar AHL by activating the T4SS. Inactivation of the autoinducer synthase LuxI1 eliminated production of AHLs in _D. shibae_ completely. This is in

accordance with the microarray and qPCR data, which showed downregulation of _luxR__2__I__2_ and _luxI__3_. Thus, a hierarchical relationship appears to be present, with expression of LuxI2

and LuxI3 depending on the signal of LuxI1. Many QS systems show a similarly hierarchical structure (Frederix and Downie, 2011). In _P. aeruginosa_, two major autoinducers are produced; the

long-chain C12-oxo-HSL (Las system) is the dominant one controlling the synthesis of the short-chain C4-HSL (Rhl system) through upregulation of the transcriptional regulator _rhlR_ (Jimenez

et al., 2012). One of the most complicated QS systems studied to date is that of _Rhizobium leguminosarium_ biovar _viciae_, a root nodule-forming soil bacterium, which has four autoinducer

synthases. They again display a hierarchical structure, with the dominant master regulator CinR being induced by the 3-OH-C14en-HSL, the product of the autoinducer synthase CinI

(Wisniewski-Dye and Downie, 2002). The Roseobacter isolate _Ruegeria_ sp. KLH11 has a QS system that is very similar to that of _D. shibae_, with two _luxI/luxR_ pairs and one orphan _luxI_

homolog. Like in _D. shibae_, knocking out its synthase _ssaI_ (sponge-associated symbiont A) gene resulted in complete loss of AHL synthesis (Zan et al., 2012). The expression of the

_luxI__1_ autoinducer synthase gene was constant throughout exponential growth in _D. shibae_ wild type, and the cognate regulator LuxR1 was highly and constitutively expressed throughout

growth. Expression of _luxI_1 under the control of a constitutive promoter restored the pleomorphic phenotype. This finding indicates that the _luxR_1_I_1 operon is involved in maintaining

morphological heterogeneity but not heterogeneously expressed itself. Bistable expression of regulators acting downstream of _luxR_1_I_1, like _ctrA_ or _luxR__2__I__2_, could be responsible

for the observed phenotype. As the hydrophobic long-chain AHLs are unlikely to diffuse freely through the membrane, variability of the transport rate could be another source of cellular

heterogeneity. The _D. shibae_ QS null mutant responded to C18-HSL, a signal that has not been detected in culture supernatants of this bacterium. C18-HSL caused upregulation of the second

QS system (_luxR__2__/luxI__2_), and thus triggered the wild-type-like QS response. However, only a selected set of genes, in particular both plasmid-encoded _vir_ operons, were re-activated

(see below). LuxR-type transcriptional regulators accept structurally similar AHLs, a phenomenon that is widely exploited by using reporter strains to detect novel AHLs. Interspecies

communication using the archetypical LuxRI system should therefore in fact be widespread. Such crosstalk has rarely been shown, one example being _Burkholderia cepacia_ and _Pseudomonas

aeruginosa_, which colonize the lung of cystic fibrosis patients (Riedel et al., 2001). To fully understand the QS network and signal integration, single-cell techniques will have to be

employed, as demonstrated for _Vibrio fischeri_ (Perez et al., 2011) and _V. harveyi_ (Long et al., 2009). Lack of AHLs affected the expression of 344 genes, representing 8% of the genome.

This is comparable to the 6% of QS-controlled genes found in _P. aeruginosa_ (Schuster et al., 2003). Interestingly, as in our study, the largest transcriptional changes occurred at the

transition to stationary phase. Thus, QS and starvation sensing converge. In _P. aeruginosa_ the QS regulon and the regulon of the alternative sigma factor RpoS controlling the general

stress response showed a strong overlap (Schuster et al., 2004; Schuster and Greenberg, 2007). This overlap may also be present in _D. shibae_, as expression of two alternative sigma factors

was reduced in the QS null mutant. In _D. shibae_ flagellar synthesis is controlled by AHL signaling. It has been calculated that marine bacteria may spend more than 10% of their total

energy budget on movement, and the smaller the cell is, the larger is the amount of energy needed to stabilize it against Brownian movement (Mitchell, 2002; Mitchell and Kogure, 2006). Thus,

the metabolic costs for flagellar synthesis are only worth spending in a diffusion-limited patchy microenvironment where motility might provide the chance to reach more optimal conditions

or nutrients, a classical condition for QS. In Roseobacters, flagella have been shown to enable chemotaxis towards dimethylsulfono-propionate (DMSP), a storage compound and osmoprotectant

synthesized by marine algae (Belas et al., 2009). Flagella mutants have been shown to be impaired in their ability to form biofilms on abiotic surfaces and were not able to attach to diatoms

(Sonnenschein et al., 2012) or dinoflagellates (Miller and Belas, 2006). The _vir_ gene clusters of _D. shibae_ encode a T4SS, which is highly conserved among Roseobacter strains

(Wagner-Döbler et al., 2010). Its physiological function has not yet been unraveled. T4SS are the only secretion systems that can translocate not only proteins but also DNA (Christie et al.,

2005). In contrast to flagellar biosynthesis and cell-cycle-related genes, the expression of the _vir_ genes could be restored by all tested C18-HSLs, even by the saturated C18-HSL. The

natural habitat of _D. shibae_, the phycosphere of marine algae, harbors microbial communities dominated by Roseobacters. They are known to produce a variety of long-chain AHLs

(Wagner-Döbler and Biebl, 2006) and _D. shibae_ may be able to respond to the prevailing AHLs in the community in a specific way. It is increasingly becoming clear that bacterial cells

within isogenic populations can display heterogeneous phenotypes. This so-called phenotypic variation can result from noise in gene expression that is most pronounced when the total number

of the involved molecules, for example, transcription factors, is small. It can also be caused by control structures of gene-regulatory networks, in particular positive feedback loops

resulting in bistability of gene expression. Variability in the phenotypic outcome of a bacterial population has been suggested to be beneficial especially in highly dynamic environments

(Acar et al., 2008). In _D. shibae_, cell morphology is the most obvious trait showing heterogeneity in the population. We could show by time-lapse microscopy that different cell division

types co-exist in this strain: binary fission and budding. The most exciting finding is that this variability is not simply the outcome of noise in the regulation of cell division but

controlled by QS. Microarray analysis confirmed the microscopic and flow cytometric investigations. Given the diversity of the cell-cycle-control mechanisms in Alphaproteobacteria and

insufficient knowledge of the control system in Rhodobacterales, it is at the moment not possible to speculate on how it functions in _D. shibae_. It remains to be elucidated if the various

types of cell division observed here are connected through a regular cell cycle. In _Rhodobacter capsulatus_ (Mercer et al., 2010) and _Silicibacter_ sp. TM1040 (Belas et al., 2009) the

growth rate of the culture was not affected by knockout of _ctrA_. However, in the latter strain the knockout leads to elongated cells. Polar growth has been described for representatives of

Rhizobiales, Caulobacterales and Rhodobacterales and may be ancient in Alphaproteobacteria (Brown et al., 2012); the core genes of cell cycle control are conserved throughout the phylum

(Brilli et al., 2010). This is the first time that the mode of cell division has been shown to be controlled by QS in Alphaproteobacteria. Strikingly, the _luxI__1_ mutant loses

morphological heterogeneity and shows a faster growth rate than the wild type. This is in contrast to previous work showing growth inhibition by an autoinducer (Gray et al., 1996). Recently,

it was observed that QS can induce gas vesicle formation in _Serratia_ sp. (Ramsay et al., 2011). Indeed, morphological differentiation processes are among the first examples that were

recognized to involve cell–cell communication. In _Bacillus subtilis_ sporulation and competence are induced by peptide pheromones through complex interconnected genetic circuits (Grossman,

1995). The frequency of sporulating cells is controlled by bistability of the isogenic population (Veening et al., 2008b). Fruiting body formation in Myxobacteria is another extremely

complex developmental process, which is controlled by autoinducers in a density-dependent way and requires polar growth. These are in fact the earliest examples of cell–cell communication,

going back to the end of the nineteenth century (Kaiser et al., 2010). CONCLUSION In _D. shibae,_ QS induces morphological heterogeneity. Moreover, QS controls flagellation and the

expression of the T4SS. It remains to be determined whether these traits are induced in a subpopulation only, and these subpopulations are distinct or overlapping. Phenotypic variability

results in a population with a reduced growth rate, thus representing a burden. We propose that QS-induced heterogeneity ensures that at least a subpopulation of cells maintains a high

fitness under constantly changing conditions. This strategy has been described as ‘risk-spreading’ or ‘bet-hedging’ (Veening et al., 2008a). It has evolved to maximize the fitness of the

population in an environment with unpredictable fluctuations (Veening et al., 2008a; de Jong et al., 2011). Such fluctuating selective pressures are likely to occur in plankton blooms and

during the seasonal succession of microbial communities in the ocean. Size-selective grazing may favor the survival of the larger cells (Gonzalez et al., 1990; Sherr et al., 1992; Hansen,

2011). Moreover, the bacteria have their ears wide open, being able to respond also to long-chain AHLs produced by neighboring cells from different species. Finally, the heterogeneity

maintained in the population by the produced QS signals calls for an in-depth investigation on the single-cell level. ACCESSION CODES ACCESSIONS GENBANK/EMBL/DDBJ * GSE42013 REFERENCES *

Acar M, Mettetal JT, van Oudenaarden A . (2008). Stochastic Switching As a Survival Strategy in Fluctuating Environments. _Nat Genet_ 40: 471–475. Article  CAS  PubMed  Google Scholar  *

Anetzberger C, Pirch T, Jung K . (2009). Heterogeneity in Quorum Sensing-Regulated Bioluminescence of _Vibrio Harveyi_. _Mol Microbiol_ 73: 267–277. Article  CAS  PubMed  Google Scholar  *

Anetzberger C, Schell U, Jung K . (2012). Single Cell Analysis of Vibrio Harveyi Uncovers Functional Heterogeneity in Response to Quorum Sensing Signals. _BMC Microbiol_ 12: 209. Article 

CAS  PubMed  PubMed Central  Google Scholar  * Avery SV . (2006). Microbial Cell Individuality and the Underlying Sources of Heterogeneity. _Nat Rev Microbiol_ 4: 577–587. Article  CAS 

PubMed  Google Scholar  * Bassler BL, Losick R . (2006). Bacterially Speaking. _Cell_ 125: 237–246. Article  CAS  PubMed  Google Scholar  * Belas R, Horikawa E, Aizawa S, Suvanasuthi R .

(2009). Genetic Determinants of Silicibacter Sp. TM1040 Motility. _J Bacteriol_ 191: 4502–4512. Article  CAS  PubMed  PubMed Central  Google Scholar  * Biebl H, Allgaier M, Tindall BJ,

Koblizek M, Lünsdorf H, Pukall R _et al_ (2005). _Dinoroseobacter Shibae_ Gen. Nov., Sp. Nov., a New Aerobic Phototrophic Bacterium Isolated From Dinoflagellates. _Int J Syst Evol Microbiol_

55: (Pt 3) 1089–1096. Article  CAS  PubMed  Google Scholar  * Biondi EG, Reisinger SJ, Skerker JM, Arif M, Perchuk BS, Ryan KR _et al_ (2006). Regulation of the Bacterial Cell Cycle by an

Integrated Genetic Circuit. _Nature_ 444: 899–904. Article  CAS  PubMed  Google Scholar  * Brilli M, Fondi M, Fani R, Mengoni A, Ferri L, Bazzicalupo M _et al_ (2010). The Diversity and

Evolution of Cell Cycle Regulation in Alpha-Proteobacteria: a Comparative Genomic Analysis. BMC. _Syst Biol_ 4: 52. Google Scholar  * Brinkhoff T, Giebel HA, Simon M . (2008). Diversity,

Ecology, and Genomics of the Roseobacter Clade: a Short Overview. _Arch Microbiol_ 189: 531–539. CAS  PubMed  Google Scholar  * Brown PJ, de Pedro MA, Kysela DT, Van der HC, Kim J, De BX _et

al_ (2012). Polar Growth in the Alphaproteobacterial Order Rhizobiales. _Proc Natl Acad Sci USA_ 109: 1697–1701. Article  CAS  PubMed  PubMed Central  Google Scholar  * Christie PJ,

Atmakuri K, Krishnamoorthy V, Jakubowski S, Cascales E . (2005). Biogenesis, Architecture, and Function of Bacterial Type IV Secretion Systems. _Annu Rev Microbiol_ 59: 451–485. Article  CAS

  PubMed  Google Scholar  * de Jong IG, Haccou P, Kuipers OP . (2011). Bet Hedging or Not? A Guide to Proper Classification of Microbial Survival Strategies. _Bioessays_ 33: 215–223. Article

  PubMed  Google Scholar  * Frederix M, Downie AJ . (2011). Quorum Sensing: Regulating the Regulators. _Adv Microb Physiol_ 58: 23–80. Article  CAS  PubMed  Google Scholar  * Garmyn D, Gal

L, Briandet R, Guilbaud M, Lemaitre JP, Hartmann A _et al_ (2011). Evidence of Autoinduction Heterogeneity Via Expression of the Agr System of _Listeria Monocytogenes_ at the Single-Cell

Level. _Appl Environ Microbiol_ 77: 6286–6289. Article  CAS  PubMed  PubMed Central  Google Scholar  * Giebel HA, Kalhöfer D, Lemke A, Thole S, Gahl-Janssen R, Simon M _et al_ (2011).

Distribution of Roseobacter RCA and SAR11 Lineages in the North Sea and Characteristics of an Abundant RCA Isolate. _ISME J_ 5: 8–19. Article  PubMed  Google Scholar  * Gilmore MS, Haas W .

(2005). The Selective Advantage of Microbial Fratricide. _Proc Natl Acad Sci USA_ 102: 8401–8402. Article  CAS  PubMed  PubMed Central  Google Scholar  * Gonzalez JM, Sherr EB, Sherr BF .

(1990). Size-Selective Grazing on Bacteria by Natural Assemblages of Estuarine Flagellates and Ciliates. _Appl Environ Microbiol_ 56: 583–589. CAS  PubMed  PubMed Central  Google Scholar  *

Gray KM, Pearson JP, Downie JA, Boboye BE, Greenberg EP . (1996). Cell-to-Cell Signaling in the Symbiotic Nitrogen-Fixing Bacterium Rhizobium Leguminosarum: Autoinduction of a Stationary

Phase and Rhizosphere-Expressed Genes. _J Bacteriol_ 178: 372–376. Article  CAS  PubMed  PubMed Central  Google Scholar  * Grossman AD . (1995). Genetic Networks Controlling the Initiation

of Sporulation and the Development of Genetic Competence in Bacillus Subtilis. _Annu Rev Genet_ 29: 477–508. Article  CAS  PubMed  Google Scholar  * Hahne F, LeMeur N, Brinkman RR, Ellis B,

Haaland P, Sarkar D _et al_ (2009). FlowCore: a Bioconductor Package for High Throughput Flow Cytometry. _BMC Bioinformatics_ 10: 106. Article  PubMed  PubMed Central  Google Scholar  *

Hansen PJ . (2011). The Role of Photosynthesis and Food Uptake for the Growth of Marine Mixotrophic Dinoflagellates. _J Eukaryot Microbiol_ 58: 203–214. Article  CAS  PubMed  Google Scholar

  * Jimenez PN, Koch G, Thompson JA, Xavier KB, Cool RH, Quax WJ . (2012). The Multiple Signaling Systems Regulating Virulence in _Pseudomonas Aeruginosa_. _Microbiol Mol Biol Rev_ 76:

46–65. Article  CAS  PubMed  Google Scholar  * Kaern M, Elston TC, Blake WJ, Collins JJ . (2005). Stochasticity in Gene Expression: From Theories to Phenotypes. _Nat Rev Genet_ 6: 451–464.

Article  CAS  PubMed  Google Scholar  * Kaiser D, Robinson M, Kroos L . (2010). Myxobacteria, Polarity, and Multicellular Morphogenesis. _Cold Spring Harb Perspect Biol_ 2: a000380. Article

  PubMed  PubMed Central  Google Scholar  * Kaplan HB, Greenberg EP . (1985). Diffusion of Autoinducer Is Involved in Regulation of the Vibrio Fischeri Luminescence System. _J Bacteriol_

163: 1210–1214. CAS  PubMed  PubMed Central  Google Scholar  * Klein J, Leupold S, Biegler I, Biedendieck R, Munch R, Jahn D . (2012). TLM-Tracker: Software for Cell Segmentation, Tracking

and Lineage Analysis in Time-Lapse Microscopy Movies. _Bioinformatics_ 28: 2276–2277. Article  CAS  PubMed  Google Scholar  * Lemme A, Gröbe L, Reck M, Tomasch J, Wagner-Döbler I . (2011).

Subpopulation-Specific Transcriptome Analysis of Competence-Stimulating-Peptide-Induced _Streptococcus Mutans_. _J Bacteriol_ 193: 1863–1877. Article  CAS  PubMed  PubMed Central  Google

Scholar  * Long T, Tu KC, Wang Y, Mehta P, Ong NP, Bassler BL _et al_ (2009). Quantifying the Integration of Quorum-Sensing Signals With Single-Cell Resolution. _PLoS Biol_ 7: 3. Google

Scholar  * Marie D, Partensky F, Jacquet S, Vaulot D . (1997). Enumeration and Cell Cycle Analysis of Natural Populations of Marine Picoplankton by Flow Cytometry Using the Nucleic Acid

Stain SYBR Green I. _Appl Environ Microbiol_ 63: 186–193. CAS  PubMed  PubMed Central  Google Scholar  * Mercer RG, Callister SJ, Lipton MS, Pasa-Tolic L, Strnad H, Paces V _et al_ (2010).

Loss of the Response Regulator CtrA Causes Pleiotropic Effects on Gene Expression but Does Not Affect Growth Phase Regulation in Rhodobacter Capsulatus. _J Bacteriol_ 192: 2701–2710. Article

  CAS  PubMed  PubMed Central  Google Scholar  * Miller TR, Belas R . (2006). Motility Is Involved in Silicibacter Sp. TM1040 Interaction With Dinoflagellates. _Environ Microbiol_ 8:

1648–1659. Article  CAS  PubMed  Google Scholar  * Mitchell JG . (2002). The Energetics and Scaling of Search Strategies in Bacteria. _Am Nat_ 160: 727–740. Article  PubMed  Google Scholar 

* Mitchell JG, Kogure K . (2006). Bacterial Motility: Links to the Environment and a Driving Force for Microbial Physics. _FEMS Microbiol Ecol_ 55: 3–16. Article  CAS  PubMed  Google Scholar

  * Müller S . (2007). Modes of Cytometric Bacterial DNA Pattern: a Tool for Pursuing Growth. _Cell prolif_ 40: 621–639. Article  PubMed  PubMed Central  Google Scholar  * Nealson KH,

Hastings JW . (1979). Bacterial Bioluminescence: Its Control and Ecological Significance. _Microbiol Rev_ 43: 496–518. CAS  PubMed  PubMed Central  Google Scholar  * Neumann A, Patzelt D,

Wagner-Döbler I, Schulz S . (2013). Identification of New _N_-Acylhomoserine Lactone Signalling Compounds of _Dinoroseobacter Shibae_ DFL-12 by Overexpression of LuxI Genes. _Chembiochem

submitted_. * Pappas KM, Winans SC . (2003). A LuxR-Type Regulator From Agrobacterium Tumefaciens Elevates Ti Plasmid Copy Number by Activating Transcription of Plasmid Replication Genes.

_Mol Microbiol_ 48: 1059–1073. Article  CAS  PubMed  Google Scholar  * Perez PD, Hagen SJ . (2010). Heterogeneous Response to a Quorum-Sensing Signal in the Luminescence of Individual Vibrio

Fischeri. _PLoS One_ 5: e15473. Article  PubMed  PubMed Central  Google Scholar  * Perez PD, Weiss JT, Hagen SJ . (2011). Noise and Crosstalk in Two Quorum-Sensing Inputs of Vibrio

Fischeri. _BMC Syst Biol_ 5: 153. Article  CAS  PubMed  PubMed Central  Google Scholar  * Platt TG, Fuqua C . (2010). What’s in a Name? The Semantics of Quorum Sensing. _Trends Microbiol_

18: 383–387. Article  CAS  PubMed  PubMed Central  Google Scholar  * Purcell EB, Boutte CC, Crosson S . (2008). Two-Component Signaling Systems and Cell Cycle Control in Caulobacter

Crescentus. _Adv Exp Med Biol_ 631: 122–130. Article  CAS  PubMed  Google Scholar  * Ramsay JP, Williamson NR, Spring DR, Salmond GP . (2011). A Quorum-Sensing Molecule Acts As a Morphogen

Controlling Gas Vesicle Organelle Biogenesis and Adaptive Flotation in an Enterobacterium. _Proc Natl Acad Sci USA_ 108: 14932–14937. Article  CAS  PubMed  PubMed Central  Google Scholar  *

Ratcliff WC, Denison RF . (2010). Individual-Level Bet Hedging in the Bacterium Sinorhizobium Meliloti. _Curr Biol_ 20: 1740–1744. Article  CAS  PubMed  Google Scholar  * Riedel K, Hentzer

M, Geisenberger O, Huber B, Steidle A, Wu H _et al_ (2001). N-Acylhomoserine-Lactone-Mediated Communication Between Pseudomonas Aeruginosa and Burkholderia Cepacia in Mixed Biofilms.

_Microbiology_ 147: (Pt 12) 3249–3262. Article  CAS  PubMed  Google Scholar  * Schäfer AL, Greenberg EP, Oliver CM, Oda Y, Huang JJ, Bittan-Banin G _et al_ (2008). A New Class of Homoserine

Lactone Quorum-Sensing Signals. _Nature_ 454: 595–599. Article  Google Scholar  * Schuster M, Greenberg EP . (2007). Early Activation of Quorum Sensing in Pseudomonas Aeruginosa Reveals the

Architecture of a Complex Regulon. _BMC Genomics_ 8: 287. Article  PubMed  PubMed Central  Google Scholar  * Schuster M, Hawkins AC, Harwood CS, Greenberg EP . (2004). The Pseudomonas

Aeruginosa RpoS Regulon and Its Relationship to Quorum Sensing. _Mol Microbiol_ 51: 973–985. Article  CAS  PubMed  Google Scholar  * Schuster M, Lostroh CP, Ogi T, Greenberg EP . (2003).

Identification, Timing, and Signal Specificity of Pseudomonas Aeruginosa Quorum-Controlled Genes: a Transcriptome Analysis. _J Bacteriol_ 185: 2066–2079. Article  CAS  PubMed  PubMed Central

  Google Scholar  * Selje N, Simon M, Brinkhoff T . (2004). A Newly Discovered Roseobacter Cluster in Temperate and Polar Oceans. _Nature_ 427: 445–448. Article  CAS  PubMed  Google Scholar

  * Sherr BF, Sherr EB, McDaniel J . (1992). Effect of Protistan Grazing on the Frequency of Dividing Cells in Bacterioplankton Assemblages. _Appl Environ Microbiol_ 58: 2381–2385. CAS 

PubMed  PubMed Central  Google Scholar  * Smits WK, Kuipers OP, Veening JW . (2006). Phenotypic Variation in Bacteria: the Role of Feedback Regulation. _Nat Rev Microbiol_ 4: 259–271.

Article  CAS  PubMed  Google Scholar  * Sonnenschein EC, Abebew SD, Grossart HP, Ullrich MS . (2012). Chemotaxis of Marinobacter Adhaerens and Its Impact on Attachment to the Diatom

Thalassiosira Weissflogii. _Appl Environ Microbiol_ 78: 6900–6907. Article  CAS  PubMed  PubMed Central  Google Scholar  * Steinmoen H, Knutsen E, Havarstein LS . (2002). Induction of

Natural Competence in Streptococcus Pneumoniae Triggers Lysis and DNA Release From a Subfraction of the Cell Population. _Proc Natl Acad Sci USA_ 99: 7681–7686. Article  CAS  PubMed  PubMed

Central  Google Scholar  * Veening JW, Smits WK, Kuipers OP . (2008a). Bistability, Epigenetics, and Bet-Hedging in Bacteria. _Annu Rev Microbiol_ 62: 193–210. Article  CAS  PubMed  Google

Scholar  * Veening JW, Stewart EJ, Berngruber TW, Taddei F, Kuipers OP, Hamoen LW . (2008b). Bet-Hedging and Epigenetic Inheritance in Bacterial Cell Development. _Proc Natl Acad Sci USA_

105: 4393–4398. Article  CAS  PubMed  PubMed Central  Google Scholar  * Wagner-Döbler I, Ballhausen B, Berger M, Brinkhoff T, Buchholz I, Bunk B _et al_ (2010). The Complete Genome Sequence

of the Algal Symbiont _Dinoroseobacter Shibae_: a Hitchhiker's Guide to Life in the Sea. _ISME J_ 4: 61–77. Article  PubMed  Google Scholar  * Wagner-Döbler I, Biebl H . (2006).

Environmental Biology of the Marine Roseobacter Lineage. _Annu Rev Microbiol_ 60: 255–280. Article  PubMed  Google Scholar  * Wagner-Döbler I, Thiel V, Eberl L, Allgaier M, Bodor A, Meyer S

_et al_ (2005). Discovery of Complex Mixtures of Novel Long-Chain Quorum Sensing Signals in Free-Living and Host-Associated Marine Alphaproteobacteria. _Chembiochem_ 6: 2195–2206. Article 

PubMed  Google Scholar  * Wang YJ, Leadbetter JR . (2005). Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils. _Appl Environ Microbiol_ 71: 1291–1299. Article  CAS 

PubMed  PubMed Central  Google Scholar  * Waters CM, Bassler BL . (2005). Quorum Sensing: Cell-to-Cell Communication in Bacteria. _Annu Rev Cell Dev Biol_ 21: 319–346. Article  CAS  PubMed 

Google Scholar  * Wisniewski-Dye F, Downie JA . (2002). Quorum-Sensing in Rhizobium. _Antonie Van Leeuwenhoek_ 81: 397–407. Article  CAS  PubMed  Google Scholar  * Young JW, Locke JC,

Altinok A, Rosenfeld N, Bacarian T, Swain PS _et al_ (2012). Measuring Single-Cell Gene Expression Dynamics in Bacteria Using Fluorescence Time-Lapse Microscopy. _Nat Protoc_ 7: 80–88.

Article  CAS  Google Scholar  * Zan J, Cicirelli EM, Mohamed NM, Sibhatu H, Kroll S, Choi O _et al_ (2012). A Complex LuxR-LuxI Type Quorum Sensing Network in a Roseobacterial Marine Sponge

Symbiont Activates Flagellar Motility and Inhibits Biofilm Formation. _Mol Microbiol_ 85: 916–933. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS

This work was funded by the German Research Foundation (DFG) within the Collaborative Research Centre Transregio 51 Roseobacter. We thank the anonymous referees for helping to improve this

manuscript. AUTHOR INFORMATION Author notes * Ina Buchholz Present address: 5Present address: Labor L+S AG, Bad Bocklet, Germany., * Diana Patzelt and Hui Wang: These authors contributed

equally to this work. * Irene Wagner-Döbler and Jürgen Tomasch: These authors contributed equally to supervision. AUTHORS AND AFFILIATIONS * Helmholtz-Centre for Infection Research (HZI),

Braunschweig, Germany Diana Patzelt, Hui Wang, Manfred Rohde, Lothar Gröbe, Irene Wagner-Döbler & Jürgen Tomasch * Leibniz Institute DSMZ-German Collection of Microorganisms and Cell

Cultures, Braunschweig, Germany Silke Pradella * Technical University of Braunschweig, Braunschweig, Germany Alexander Neumann, Stefan Schulz, Steffi Heyber, Karin Münch, Richard Münch &

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THIS ARTICLE CITE THIS ARTICLE Patzelt, D., Wang, H., Buchholz, I. _et al._ You are what you talk: quorum sensing induces individual morphologies and cell division modes in _Dinoroseobacter

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