Additional Material and Methods
Strains, growth media, and cultivation conditions
Strains, plasmids and primers used in this study are listed in Table S1. Dinoroseobacter
shibae DFL-12T strains were grown in defined minimal medium (see below). Precultures
were inoculated from fresh half-concentrated Marine Broth plates (MB, Difco 2216). For
selective conditions, 80-150 µg/mL gentamicin or 500 µg/mL kanamycin were added to the
MB agar. Precultures were grown over night and then transferred to fresh minimal medium.
Cultures were incubated at 30°C in the dark, liquid cultures were shaken at 180 rpm.
Escherichia coli strains were cultured in Luria-Bertani broth (LB, Roth), supplemented with
50 µg/mL aminoluvic acid, 25 µg/mL gentamicin, 25 µg/mL tetracycline or 50 µg/mL
kanamycin if required, at 37°C and 180 rpm. Pseudomonas putida was cultivated at 30°C in
LB supplemented with 25 µg/mL gentamicin. AHLs were purchased from Cayman Chemicals
or synthesized by Neumann et al. (manuscript in preparation), and prepared as 1 mM DMSO
stock solutions. For chemical complementation the respective AHL was added to the culture
at a final concentration of 500 nM. DMSO alone was used as a control.
Composition of the minimal medium used for cultivation of Dinoroseobacter shibae.
All chemicals used were of pro analysi grade. Deionised water for all solutions was purified
through a 0.22 µm membrane filter (MilliPore). For 1 L of medium the following chemicals
were dissolved in 900 ml deionised H2O: 4.0 g NaSO4, 0.2 g KH2PO4, 0.25 g NH4Cl, 20.0 g
NaCl, 3.0 g MgCl2×6H2O, 0.5 g KCl and 0.15 g CaCl2×2H2O. In addition, 0.19 g NaHCO3
was dissolved in separate 100 ml deionised H2O. Both solutions were autoclaved separately
and mixed after cooling. Then, trace elements (1 ml stock solution per litre medium), vitamins
(10 ml stock solution per litre medium) and Na-Succinate (20ml 2.5 M stock solution, pH 7.5
per litre, final concentration: 5 mM) were added. The trace element stock solution contained
per litre: 2.1 g FeSO4×7H2O, 13.0 ml 25% HCl, 5.2 g Titriplex-(III) (Na2-EDTA), 30.0 mg
H3BO3, 100.0 mg MnCl2×4H2O, 190.0 mg CoCl2×6H2O, 24.0 mg NiCl2×6H2O, 2.0 mg
CuCl2×2H2O, 144.0 mg ZnSO4×7H2O and 36.0 mg Na2MoO4×2H2O. The vitamin solution
for D. shibae contained the following components per litre: 2 mg biotin, 20 mg nicotinic acid
and 8 mg 4-aminobenzoic acid.
Construction of D. shibae DFL-12T luxI1 deletion mutant and complementation strain
The luxI1 gene (Dshi_0312) was replaced by a gentamicin resistance via double-homologous
recombination. For construction of the knock-out vector the gentamicin resistance cassette
was amplified from pBBR1MCS-5 (primer Gm_F and Gm_R) using Pfu DNA-polymerase
(Promega). Flanking sequences 819 bp upstream (primer 1ups_F_SacI and 1ups_R_SalI) and
654 bp downstream (primer pair 1dos_F_NheI and 1dos_R_NheI) of luxI1 were amplified
from D. shibae DFL-12T genomic DNA. The purified gentamicin cassette flanked by the two
homologous regions was cloned into the multiple cloning site of the suicide vector pJB5603.
The knock-out vector pJB5603∆luxI1::Gmr was subcloned in E. coli ST18 and introduced into
DFL-12T via biparental mating as described before (Piekarski et al., 2009). Selected
transconjugants were confirmed by PCR and sequencing. For genetic complementation a
plasmid expressing luxI1 was constructed. The luxI1 open reading frame was amplified from
D. shibae DFL-12T genomic DNA (primer luxI1_F_NdeI and luxI1_R) and the gentamicin
promoter was amplified from pBBR1MCS-5 (GmPromF and GmPromR_NdeI). Fragments
were digested and ligated. The ligated luxI1 open reading frame controlled by the upstream
non-native promoter was cloned into the blunt-end digested expression vector pBBR1MCS-2
yielding pDP1. The complementation plasmid pDP1 was conjugated into D. shibae DFL12
∆luxI1 via biparental mating as described (Piekarski et al., 2009).
Growth curve analysis
For growth curves, cell materials from streak plates were inoculated into 20 ml of SWM + 5
mM succinate and were incubated overnight in the dark at 30°C and 160 rpm. Optical density
at 600 nm (OD600) was measured with spectrophotometer Ultrospec 3100 pro (Biochrom Ltd).
For the growth curve predicted from optical density, cultures were diluted to an initial OD600
of 0.01 and then 200 µl were placed to each well of Honeycomb 2 plate. OD600 of all strains
were monitored every 30 min for 36 hours in an automated microbiology growth analysis
system Bioscreen C (Oy Growth Curves Ab Ltd). Growth curves were plotted using R
program. For the growth curve obtained from cell counts, 100 ml culture with an initial OD600
of 0.01 was prepared from overnight culture, placed in an Erlenmeyer flask and incubated
under the same conditions mentioned above for 36 hours. Samples for cell counting were
taken at various time intervals, which were then fixed at a final concentration of 2%
glutaraldehyde and stained with SYBR Green I (Molecular Probes). Cell counts were assessed
by flow cytometry (BD FACS Canto analyzer) using procedures that have been previously
described (Marie et al., 2005).
Scanning electron microscopy
Samples (20 µl) were fixed with 2% glutaraldehyde, immobilized on polylysine treated
coverslips and washed with TE buffer (pH 7). Samples were dehydrated and gold covered as
described (Lünsdorf et al., 2001). Cells were examined with a Zeiss DSM 982 Gemini (LEO)
equipped with a field emission gun, in the magnification range from 500x to 10,000x at 5 kV
and 8 mm working distance.
Microarray design and experimental design
The design of the custom made Agilent microarray for D. shibae (AMADID 026232) has
been previously described (Tomasch et al., 2011). Information on layout and probe annotation
can be downloaded from the gene expression omnibus (GEO) database under accession
number GPL11243 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL11243).
Differential gene expression in D. shibae ΔluxI1 and D. shibae ΔluxI1pDP1 compared to the
wild-type strain was determined for two independent cultures at five different culture
densities measured as optical density at 600 nm (OD600) during exponential growth and 6 h
after OD600 had reached a maximum (stationary phase). The samples from the wild-type strain
were labelled with Cy3, the RNA from the ΔluxI1 deletion mutant and the complementation
strain ΔluxI1pDP1 was labelled with Cy5. In addition to the genetic complementation, a
chemical complementation was carried out whereby the ΔluxI1 deletion mutant culture was
provided with synthetic autoinducer signals. For the comparison of the wild-type with the
ΔluxI1-strain complemented with synthetic AHLs cultures in the mid-exponential growth
phase were analysed, using a loop-design with three biological replicates for D. shibae ΔluxI1
and the complementation with C18-dien-HSL and two biological replicates for the wild-type
strain and the complementation with C18- and C18-en-HSL.
RNA isolation, labelling and microarray experiment
For isolation and purification the RNeasy-kit (Qiagen) was used according to the
manufacturer’s manual. Modifications are listed below. Cells lysis was performed
enzymatically using 15 mg/mL lysozyme in Tris-EDTA-buffer (pH 8.0) and mechanically by
vortexing for 3 min with acid washed glass beads. A first digestion of genomic DNA was
performed on the column, using RNase-free DNase I (Qiagen) according to the
manufacturer’s protocol. Total RNA was eluted in 88 µL RNase-free H2O and a second
DNase I-digestion of genomic DNA was performed in solution, followed by a second RNeasy
purification step. In this second purification an additional washing step with 80 % ethanol was
performed prior to elution with 30 µl RNase-free water. RNA integrity was determined on the
Agilent Bioanalyzer. Total RNA (2 µg) was labelled with Cy3 or Cy5 using the ULS-system
(Kreatech, Amsterdam, The Netherlands) according to the manufacturer's manual. Cy5 and
Cy3-labelled RNA (500 ng each) was fragmented and hybridized to the microarray according
to Agilent's two-colour microarray protocol.
Microarray Analysis
Microarray slides were scanned using the Agilent DNA microarray Scanner. Median spot
intensities and background signals of the Cy3 and Cy5 channel were loaded into the R
environment (http://www.R-project.org/) and processed using the LIMMA package (Smyth,
2005). Spots were down weighted 25% per flag if 2 or more quality flags were set by the
scanner software. Background signals were subtracted using the normexp method (Ritchie et
al., 2007), Cy3 and Cy5 signals were Loess normalized and finally quantile normalisation was
performed on all microarrays from one dataset. Signals from replicate probes for single genes
were averaged. The linear model fitted for each comparison of interest is described in (Smyth,
2004) and allows for indirect comparisons across microarrays. The obtained p-values were
adjusted for false discovery rate (fdr) using the method by Benjamini and Hochberg
(Benjamini et al., 1995).
Clustering of microarray data
The genes in the filtered datasets were further clustered by their Euclidean distance using the
fuzzy c-means algorithm from the Bioconductor package mfuzz. Clustering parameters and
the number of clusters were optimised using the corresponding functions. Increasing the
cluster number and subsequent grouping of similar clusters by their distance on a twodimensional PCA-plot further optimised the clustering output. Genes with a maximum
membership value of 0.25 were treated as outliers.
Identification of transcription factor binding sites (TFBS)
For the identification of binding sites for the transcription factor CtrA, promotor-regions
ranging from 300 bp upstream to 50 bp downstream of the translation start were searched for
matches with a position weight matrix obtained from a comparative phylogenetic analysis of
cell cycle regulation in Alphaproteobacteria (Brilli et al., 2010). A TFBS was considered as
identified if its score reached at least 85% of the maximum weight of the PWM.
Flagella staining
Cells were harvested by centrifugation (10 min, 376 g). The supernatant was decanted and the
cells were suspended in the remaining medium. An aliquot (1 µl) of the suspension was
spotted on a microscopic slide and 1 µL staining solution were soaked beneath the cover slip.
The staining solution contained two components: (A) 10 ml 5% aqueous phenol-solution with
2 g tannic acid was mixed with 10 ml of a saturated KAl(SO4)2 x 12 H2O solution. (B) 12%
cristal violet solution in 95% ethanol. 1/10 volume of B was added to A. Flagella were
observed using an Olympus BX60F-3 microscope, equipped with a 100x/1.3 oil objective and
a Colorview II camera.
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