Status des Manuskripts: in Bearbeitung zur Veröffentlichung in Journal of Bacteriology
Eigenbeitrag:
− Anfertigung des Manuskripts;
− Alignment von PilS und PilR;
− Charakterisierung der PilR-Bindestelle mittels Punktmutationen;
− Kohlenstoffmangel-Experimente mit Wildtyp und pilS-Deletionsmutante (Reportergenanalysen sowie Northern Blot);
− Nachweis des pilAB-gfp Genexpressionslevels in der pilS-Deletionsmutante in verschiedenen Wachstumsphasen;
− Nachweis von AHL-Produktion bei Azoarcus sp. BH72 und der Positivkontrolle Rhizobium sp. NGR234 mit den Sensorstämmen E. coli (pJBA89) und P. putida (pKR-C12);
− Reproduktion der Dichlormethanextraktion;
− Inaktivierung der potentiellen in Quorum Sensing involvierten Genen mittels Insertionsmutagenese und anschließender Untersuchung, ob eine Beeinflussung der pilAB-induzierende Aktivität vorliegt mittels Reportergenfusion (gusA);
− Biochemische Charakterisierung des unbekannten Quorum Sensing-Signals;
Regulation of the Type IV Pilin Gene Expression in the Grass Endophyte Azoarcus sp. Strain BH72 is affected by Carbon
Starvation and Cell Density
Melanie Böhm1, Juliane Dörr2, Jörg Plessl2, and Barbara Reinhold-Hurek* 1, 2
1 Laboratory of General Microbiology, University of Bremen, P. O. Box 33 04 40, D- 28334, Bremen, Germany; 2 former address Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Str.,
D-35043 Marburg, Germany
Keywords: transcriptional regulation, quorum sensing, autoinducer, carbon starvation, pili.
*Corresponding author. Tel.: +49-(421)-218-2001; fax: +49-(421)-218-9058; E-mail:
breinhold@uni-bremen.de
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Type IV pili play a key role in the interaction between the grass endophyte Azoarcus sp. strain BH72 and rice plants. Therefore, the transcriptional regulation of the structural genes pilAB was studied. Primer extension analyses revealed that the pilAB operon was transcribed from a sigma 54-type promoter. Furthermore we identified a two component regulatory system PilSR that shows high sequence similarity to the system of P. aeruginosa and contains the relevant domains for a sensor kinase and the corresponding response regulator. Mutational analyses showed that PilR functions as the transcriptional activator and binds upstream of the σ54 promoter sequence. The sensor kinase PilS is stimulated by carbon starvation, leading to an elevated pilAB gene expression. As far as we know, this is the first described environmental stimulus of PilS for the regulation of type IV pilin gene expression. Monitoring the pilAB expression with the reporter genes gusA and gfp at various time points revealed also an induction of the pilAB gene expression in a cell density dependent manner. This up-regulation was more pronounced in a ΔpilS deletion mutant, which may be due to an inhibitory effect of PilS.
Experiments with different AHL sensor strains and genome based analyses led to the assumption that Azoarcus does not use an AHL (N-acyl-homoserine lactone) based quorum sensing system for pilAB expression, although AHLs are a widely used in plant-associated bacteria. We propose that Azoarcus sp. strain BH72 uses a novel autoinducer molecule (HSF, hydrophilic signal factor) for intercellular communication.
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INTRODUCTION
Azoarcus sp. BH72 is a Gram-negative N2 fixing β-proteobacterium which is able to infect Kallar grass and rice plants systemically in an apathogenic way (34, 53). Azoarcus also interacts with a rhizosphere fungus, an asexual ascomycete originating from Kallar grass roots (35, 36). The type IV pili of Azoarcus are essential in the colonization process of plant and fungal host surfaces (18). They are flexible thin filaments with a length of 5-7 µm, located at poles of the bacterial cell (44). A functional type IV pilus structure of Azoarcus depends on the presence of the pilAB operon. The pilA gene is coding for a short prepilin with only 59 amino acids showing 100% sequence similarity to the conserved N-terminus of the Pseudomonas aeruginosa type IV pilin. It is cotranscribed with pilB whose gene product is an exported protein which is also obligatory for functional pilus structure (18).
Important sensory transduction systems are two-component regulatory systems (33). In the human pathogen Pseudomonas aeruginosa, PilR and PilS form a classical RpoN-dependent two-component system, which is required for the transcriptional regulation of the pilA gene encoding the major pilin (32). Two-component regulatory systems are typically involved in the activation of gene expression in response to environmental signals. A sensor component recognizes an environmental signal and autophosphorylates a conserved histidine residue. The phosphate is transferred onto a conserved aspartate residue in the response regulator that is then able to recognize a binding motif upstream of the target promoter and activates transcription from a promoter that requires a RNA polymerase containing the alternative sigma factor σ54 (33).
In Pseudomonas aeruginosa the pilS gene encodes a sensor kinase that is located at the pole of the cell (8), but the signal to which it responds is unknown (22). PilR, as a transcriptional regulator of the type IV pilin gene, binds upstream of the pilin promoter to four binding sites, three of them with the pattern 5` N(4-6)C/GTGTC3, which are necessary for a PilS/R mediated pilin gene expression (38). In Myxococcus xanthus the pilA gene expression requires PilR, but not the sensor kinase PilS, which probably functions as a negative rather than a positive regulator (74).
The cell density-dependent regulation of gene expression, referred to as quorum sensing, allows bacterial populations to communicate and respond collectively to changing environmental conditions. Many bacteria produce and release chemical signal molecules called autoinducers to detect their population density and regulate a wide range of physiological processes. These processes include e.g. symbiosis, virulence, competence,
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conjugation, antibiotic production, sporulation, motility and biofilm formation (48). In general, Gram-negative bacteria use membrane-permeable N-acyl-L-homoserine lactones (AHLs) based quorum sensing systems, while gram-positive bacteria use linear or cyclic oligopeptides as signalling molecules (66, 73). The biosynthesis of AHLs depends on synthetases belonging to two classes: the LuxI homologues and the AinS homologues (28).
Parallel to the increasing cell density, the concentration of the autoinducer increases, and a LuxR homologue
perceives the signal and controls the expression of the quorum sensing regulated genes as a transcriptional regulator (27). In peptide-mediated quorum sensing systems, the peptide is secreted via an ATP-binding cassette (ABC) transporter. Gram-positive bacteria are using two-component regulatory systems to detect the autoinducer and pass the signal to gene expression level (66). The Pseudomonas aeruginosa quorum sensing response is influenced by another chemical signal distinct from the AHLs, 3,4-hydroxy-2-heptylquinolone, called PQS (49) that probably links the two AHL based quorums sensing systems las and rhl (46).
Cell-cell communication occurs not only within a bacterial population, there are also autoinducers that may represent a language between bacterial species. The interspecies communication in both bacterial groups is mediated by a furanone-based quorum sensing signal (autoinducer-2) which was first described for the marine bacterium Vibrio harveyii (11, 23). The synthesis is dependent on the LuxS enzyme (67).
Since type IV pili play a key role in the interaction between Azoarcus and rice plants, it is important to gain more insights in the regulation process of type IV pilin gene expression.
Here we describe the identification of a two component regulatory system PilSR that regulates the pilin gene expression in Azoarcus sp. strain BH72. PilR functions as the transcriptional activator and binds upstream of a σ54 promoter sequence. The sensor kinase PilS is stimulated by carbon starvation. We also show evidence that the pilAB operon is quorum sensing regulated. First biochemical and mutational analyses suggest that Azoarcus sp. communicates using an unusual quorum sensing signal.
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MATERIAL AND METHODS
Bacterial strains, growth conditions and DNA manipulations. The bacterial strains, plasmids and primers used in this study are listed in Table 1.Azoarcus sp. BH72 was grown aerobically at 37°C on Ethanol medium (52) and for electroporation/conjugation on VM-medium with malate instead of ethanol (39) and on SM VM-medium (51) without a carbon and nitrogen source (starvation medium). Escherichia coli and Chromobacterium violaceum were grown aerob on Luria-Bertani-medium (LB) at 37°C or 30°C (4, 45) and Pseudomonas putida on modified LB medium with 4 g NaCl/liter at 30°C (64). Rhizobium sp. NGR234 was grown on TY medium (6) at 30°C as described elsewhere. Antibiotics used for E. coli or Azoarcus strains were tetracycline (12.5 µg ml-1), chloramphenicole (12,5 µg ml-1), ampicilline (150 or 30 μg ml-1), kanamycine (30 μg ml-1) and (25 µg ml-1) for Chromobacterium violaceum. Gentamycine (25 µg ml-1) was used in the cultivation of Pseudomonas putida.
DNA manipulations, plasmid and genomic DNA extractions, transformation, Southern hybridization, and DIG labelling of probe DNA were performed by using standard molecular biology techniques (4). Restriction enzymes, T4 DNA Ligase, and Pfu Turbo DNA Polymerase were purchased from New England Biolabs (Hertfordshire, England UK), Fermentas (St. Leon-Rot, Germany) and Stratagene (La Jolla, CA, USA). Extractions of DNA from agarose gels were performed using the Bio 101 GeneClean III Kit (MP Biomedicals, Heidelberg, Germany). Oligonucleotides were purchased from Invitrogen (Karlsruhe, Germany) and Operon Biotechnologies (Cologne, Germany).
DNA sequence analysis. DNA sequencing was performed using the didesoxynucleotide chain termination method (57) with the ALFexpress automated sequencer (GE Healthcare, Freiburg, Germany) by standard procedures. Gene annotation was done by GenDB 2.0 system (47) and sequence comparisons were analysed using Blast program (2). Protein domains were predicted using Pfam (5) and SMART (42), transmembrane helices were determined by were determined by the DAS method (dense alignment surface method) (13).
Construction of mutants and reporters. The plasmids used in this study are listed in Table 1 and were generated using standard procedures (4). Plasmids were constructed as follows.
pJBLP1 (18) is carrying the chromosomal part of pilR and pilAB genes. The HincII-BglII fragment of pJBLP1 was cloned into the HincII-BamHI restriction site of the cloning vector pUC19 to create pJMB2. To create reporter gene fusions with the target genes pilAB, the gene gusA, coding for the reporter enzyme β-glucuronidase was cloned into the MfeI site. The
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reporter gene construct pJMB2GUS was subcloned with EcoRI-HindIII into pLAFR3. The plasmid construct pJLAMB2 was conjugated into Azoarcus sp. BH72 by triparental mating using E. coli (pRK2013) as helper strain (24). The PstI-BglII fragment of pJBLP1 was cloned into the PstI/BamHI restriction sites of the cloning vector pUC18 to create pJMB3. This plasmid construct was used to perform the site directed point mutations in the upstream region of the sigma54 promoter of the pilAB operon using Stratagene QuickChange® Site Directed Mutagenesis kit (La Jolla, CA, USA). Two bases in each of the four palindromic sequences were exchanged (pJMB3a, b, c, d) (Fig.3). All oligonucleotides used to perform point mutations are listed in Table 1. The constructs pJMB3 a, b, c, d were controlled by sequencing and restriction digests (new restrictions site arised within the mutated sequences).
To create reporter gene fusions with the target genes pilAB, the gene gusA, coding for the reporter enzyme β-glucuronidase was cloned into the MfeI restriction site. The reporter gene constructs were subcloned with EcoRI-HindIII into pLAFR3. The plasmid constructs pJLAMB3a, b, c, d were transformed into Azoarcus sp. BH72 wild type by triparental mating using E. coli pRK2013 as helper strain (24).
The insertional mutations of the azo0390, azo1746, azo3178 and azo3379 ORFs (open reading frame) were performed by the insertion of truncated versions of the target genes with a STOP codon at the 5`end. An interruption of the ORFs were performed by polymerase chain reaction (PCR) using primers (Table 1) amplifying only a part of the ORFs and carrying a STOP codon at the 5`end of the forward primer. The PCR products were cloned into the pPCR-Script™ AmpSK(+) vector (Stratagene, La Jolla, CA, USA) and subcloned in the mobilizing vector pK18mob2 (68) with HindIII-XbaI. The final constructs were conjugated into Azoarcus by triparental mating with the helper strain E. coli (pRK2013) (24) and the correct integrations were confirmed by Southern blot analysis.
In frame deletion of pilS gene was performed by using the sucrose selection system (62). To construct BHΔpilS, the 0.9 kb BsaBI-NruI fragment of pJBLP231 was deleted, resulting in pJBLP2311. The deletion was consistent of amino acid 107-407 of PilS. The 1.8 kb SmaI fragment of pJBLP23 was exchanged by the 0.9 kb SmaI fragment of pJBLP2311, which contains the pilS deletion (pJBLP232). The 3.2 kb insert of pJBLP232 was subcloned into the EcoRI-HindIII restriction site of pK18mobsacB. The final construct pJBLP234 was conjugated into the Azoarcus genome by triparental mating with the helper strain E. coli (pRK2013) (24). Following conjugation, transconjugants were selected on 6% sucrose media.
PCR and Southern blot analysis were performed to proof the deletion mutant of pilS.
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To perform an insertional knock out mutant of pilR, the 1.3 kb HincII fragment of pUC4K was subcloned into the SmaI restriction site of pJBL24 to interrupt the pilR gene. The final construct pJBL241 was inserted into Azoarcus sp. BH72 by electroporation and the selection for double homologous recombinants took place via antibiotic resistance Kmr+ and Ampr-.
Mutant strain BHΔpilR was genotypically confirmed by Southern blot analysis.
pilAB::gusA or pilAB::gfp transcriptional fusion was constructed by placing 1.8 kb gusA or 0.7 kb gfp gene in the MfeI site of the pJBLP1 plasmid, resulting in pJBLP14 or pJBLP1gfp.
The transcriptional fusion of pilSR::gusA was performed by inserting the XhoI-Asp718 fragment of pJBLP2 containing pilSR in pSKGUS, resulting in pJBLP21. The reporter gene contructs were introduced into Azoarcus sp. BH72 and BHΔpilS by electroporation. Single-crossover homologous recombination events to incorporate the plasmids into the chromosome were confirmed by southern blot analyses.
RNA methods. Total RNA was isolated from Azoarcus wild type cells using hot phenol method (4). For hybridization analysis, RNA samples were denatured, separated by electrophoresis on 1% agarose gel with formaldehyde, and transferred to Hybond-N+
(GE Healthcare, Freiburg, Germany) by capillar blotting. The blots were hybridized with DIG labelled pilAB probe. RNA hybridizations were performed at 65°C as described by Ausubel et al. (4).
Primer extension. Primer extension analysis was performed by standard procedure described by Sambrook et al. (56). The primer (5`-GCAGTTTCTTCATTTCAATTCTCC-3`) complementary to the sequence of pilA 42 bp downstream of the predicted transcription start site was prepared. The primer was radioactively labeled by 35S-α-ATP, mixed with 30 µg RNA, and extended using avian-myleoblastosis-virus reverse transcriptase (Boehringer, Mannheim, Germany). The RNA transcript was purified via extraction with 50% phenol, 49%
chloroform and 1% isoamylalcohol followed by ethanol precipitation. A DNA sequencing reaction prepared with USB 70770 Sequenase version 2.0 DNA Sequencing Kit (GE Healthcare, Freiburg, Germany) with the same oliconulceotide primer was then separated alongside the primer extension reaction products on a standard denaturating gel electrophoresis and the autoradiography was detected on a x-ray film.
PAGE and Western blot analysis. For detection of the proteins, SDS-soluble cellular proteins from equal volumes and optical density (OD578) of bacterial liquid cultures were extracted (40) and protein digests were subjected to sodium dodecyl sulfate SDS- or Tris-Tricin- polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting. Blots were incubated with rabbit polyclonal antibodies (1:5000) raised against PilA custom-synthesized
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peptides (aa29-54 of PilA) (Eurogentec, Seraing, Belgium). Antigen detection was performed using peroxidase-conjugated swine anti-rabbit antibodies (Dakocytomation, Celostrup, Denmark) and a chemiluminescence reaction (ECL, Pierce, Erembodgem, Belgium). Details of the methods used for Tris-Tricin or SDS-PAGE, Western blotting, and antigen detection are described elsewhere (34, 60).
Determination of the β-glucuronidase activities and relative GFP fluorescence intensities. Activity of ß-glucuronidase was measured in quantitavely using the method described by Jefferson et al. (37) according to Egener et al. (20) and expressed in Miller Units defined as E420 x 1000 /(t (min) x OD600).
To determine the relative GFP fluorescence intensity, samples were incubated for 1 h at 4°C and green fluorescence was measured using the Typhoon 8600 Variable Mode Imager (GE Healthcare, Freiburg, Germany). The total average value (average sample / average media) was normalised by using the optical density (578 nm) of the sample measured photometrically.
Carbon starvation assay. For the carbon starvation assay, exponentially grown cells were washed twice by centrifugation for 10 min at 16.200 x g and 37°C in SM medium without any carbon and nitrogen source (starvation medium) and incubated for 1 h in starvation medium with an without a carbon source at 37°C on a rotary shaker at 200 rpm (CH-4103 Firma Infors AG, Bottmingen, Switzerland). ß-glucuronidase activity was measured before and after the washing and again after 1 h of incubation.
Production of conditioned supernatant. Azoarcus preculture was cultured in VM-Ethanol medium (52) at 37°C shaking (190 rpm, CH-4103 Firma Infors AG, Bottmingen, Switzerland) for 24 h. The main culture was inoculated with 17% of the preculture and was further incubated shaking at 37°C for 24 h to stationary growth phase (OD578 > 1.4). During production of conditioned supernatant, it was obligate to keep the fillrate of the Erlenmeyer flask at 12% maximum, to ensure the aeration of the culture, otherwise the inducing activity decrease. Bacterial cells were pelleted by centrifugation (6.300 x g; 10 min) and the cell free supernatant was tested for its pilAB inducing activity.
Quorum sensing bioassay. The reporter strain Azoarcus sp. BHΔpilS::pJBLP14 (pilAB::gusA) was cultured in VM-Ethanol medium until reaching exponentiell growth phase.
Test sample supernatants were supplied with ethanol (0,6%), potassium phosphate buffer (8.68 mM; pH 6.8) and for HPLC fractions additionally with 200 mg of MgSO4 x 7 H2O, 6,4 mg of CaCl2 x 2 H2O, 10 mg of MnSO4 x H2O, 2 mg of Na2MoO4 x 2 H2O per liter and yeast extracts (0,1%) to establish medium concentration. The uninduced culture was diluted
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1:5 with fresh medium as a negative control or with test samples and allowed to grow while shaking (180 rpm, CH-4103 Firma Infors AG, Bottmingen, Switzerland) for 4 h at 37°C.
β-glucuronidase activity was measured as described above.
Cross-streak experiments. On VM-Ethanol plate, the test strain Azoarcus sp. BH72 or Rhizobium sp. NGR234 (71) as a positive control were streaked close to the gfp(ASV)-based AHL sensor strains E. coli JM105 (pJBA89) (3, 75) or P. putida IsoF (pKR-C12) (54, 64) to form a T. Following 24 h of incubation at 30/37°C, the green fluorescent phenotype of the AHL sensor strains were visualized by exciting the plates with blue light and the results were documented with the Hamamatsu Color Chilled 3CCD camera mounted on a binocular (Olympus SZX12) (3).
AHL extraction. AHL molecules were extracted with 1 volume dichlormethane from culture supernatants (grown in VM-Ethanol medium to stationary phase). The extracts were evaporated by 40°C to dryness. Residues were dissolved in 1 ml acetonitrile following volume reduction to 0,1 ml by rotational evaporation. The liquid phase and the extracts were then applied to the AHL plate detection assay and/or to the quorum sensing bioassay (described above).
AHL plate detection assay. Single colonies of AHL monitor strains P. putida IsoF (pKR-C12) or E. coli JM105 (pJBA89) were resuspended in 0.9 % sodium chloride and plated via Drigalski spatula on LB agar plates. If C. violaceum CV026 was used, 100 µl of the reporter culture was mixed with 3 ml semi solid LB softagar and poured on LB agar plates.
The test samples were filled up in holes, which were produced by punching the blunt end of a Pasteur pipette into the agar. The plates were incubated at 30/37°C for 24 h. The analysis occured either by detecting the GFP fluorescence via scanning the plates with the Typhoon 8600 Variable Mode Imager (GE Healthcare, Freiburg, Germany), or by documenting the violacein production using a MPEG MOVIE HQX, Digital Still Camera, DSC-F717.
Biochemical characterisation of the autoinducer. The conditioned supernatant was treated with chymotrypsine (3 µg/ml) for 18 h at 25°C and subtilitsin (150 µg/ml) supplemented with potassium phosphate buffer (8.68 mM; pH 7.5) for 2 h at 25°C. As a negative control samples were also treated with inactive enzymes. To confirm the protease activity under the experimental conditions, control digests with BSA and α-lactalbumine were performed and analysed by 12% Tris-Tricin PAGE.
To purify the autoinducer molecule, conditioned supernatant was passed through ultrafiltration membrane YM1 (Amicon, Milipore Corporation, Bedford, USA) and was loaded onto a carboxymethyl sepharose cation exchanger cartridge equilibrated with
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potassium phosphate buffer (20 mM; pH 6), washed with potassium phosphate buffer (10 mM; pH 6) and eluted in a batch method with sodium chloride (500 mM). Active ion exchange fraction containing pilAB inducing activity was applied to a HPLC RP-C18 column (Merck, Darmstadt, Germany) and eluted by using a 0-100% acetonitrile gradient or a 0-15%
acetonitrile gradient. Medium was always treated in parallel to the conditioned supernatant to function as a control for side effects on the pilAB expression level in the quorum sensing bioassay.
Statistical analysis. The GraphPad InStat software package (GraphPad software, San Diego, CA) was used for statistical analysis.
10 RESULTS
Identification of a putative two component regulatory system encoded upstream of pilAB. Sequence analysis of the region upstream of the pilAB operon (Fig. 1A) revealed two genes (pilSR) that showed high similarity to genes encoding two-component-regulatory systems of the NtrC family. The putative response regulator PilR (458 amino acids) showed 57% or 55% amino acid sequence identity to the type IV pilin gene activators PilR of Pseudomonas aeruginosa (acc. no. AAG07935) or Xylella fastidiosa (NP_780109). It also exhibited 44% sequence identity to PehR, a transcriptional activator of virulence genes in Ralstonia solanacearum (ZP_00943861) (Fig. S1A, Supplementary material). PilR of Azoarcus sp. contained at amino acid residues 13 to 124 a CheY-homologous receiver domain (Smart SM00448, E-value 2.06e-30), at 165 to 308 an AAA_ATPase domain (Smart SM00382, E-value 7.60e-08) and at amino acid residues 416 to 455 a Helix_turn_Helix motif (Pfam PF02954, E-value 1.20e-12).
PilS showed similarities to sensor kinases of two component regulatory systems. The deduced amino acid sequence had 31%, 28% or 27% identity to PilS of P. aeruginosa (AAP81268), of X. fastidiosa (NP_780110) or to PehS of R. solanacearum (NP_520929), respectively (Fig. S1B). The putative sensor kinase PilS of Azoarcus (517 amino acids) consisted of 6 predicted transmembrane helices, and a DUF1109 domain of unknown function at amino acid residues 4 to 141 (Pfam 06532, E-value 1.30e-02). Furthermore PilS contained a PAS domain (Smart SM00091, E-value 2.28e-0.0) in the cytoplasmic linker region at positions 193 to 261 aa, a conserved kinase region, including the invariant histidine residue characterized by a His kinase A domain at amino acid residues 304 to 369 (Smart SM00388, E-value 4.06e-14), and at position 410 to 517 aa the histidine-kinase like ATPase (Smart SM00387, E-value 1.15e-27).
Mapping of the pilAB promoter and the putative binding site of the transcriptional activator PilR. To elucidate the role of PilR as a transcriptional activator for expression of the pilAB operon, a knockout mutant of pilR was constructed by insertion of a kanamycine resistance cartridge. In order to quantify pilAB expression, a pilAB::gusA transcriptional fusion was integrated into the chromosome of the wild type (BH72) and the pilR mutant strain (BHΔpilR1) (Table 1). The mutant strain showed a strong, statistically significant (P< 0001 in unpaired t-test) reduction in pilAB::gusA expression in comparison to the wild type in the exponential growth phase (Fig. 2A). This indicated that PilR is necessary for activation of the pilAB gene expression.
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Examination of the nucleotide sequence upstream of pilA (18) revealed a putative σ54-promoter sequence (Fig. 1A). To determine the transcriptional start site of pilAB, primer extension analysis was performed (Fig. 1B). It indicated a transcriptional start site at the C12 downstream of the putative σ54 dependent promoter which confirmed the utilization of this promoter sequence.
In order to map the putative binding site of PilR more precisely, deletion and point mutation analyses were carried out. Sequence deletions (PstI-HincII fragment, Fig. 1A) in the upstream region of pilAB caused a 17-fold decrease of GUS activity from a transcriptional pilAB::gusA fusion in trans, which suggests that the transcriptional activator PilR binds in this region (control strain BH72 (pJLA): 4143 ± 30,4 Miller units; strain BH72 (pJLAMB2) with the sequence deletion in the upstream region of pilAB: 244 ± 2,8 Miller units, respectively). Three short inverted repeats were found in this region (1-3 in Fig. 2B). Since regulatory proteins often prefer these typical sites, site-directed point mutations were constructed. Mutagenized fragments were linked to the pilAB::gus fusion in trans. Only a slight decrease in GUS activities was observed in mutants a (GT to TG) and d (CA to TC) (P< 0.01) (parametric analysis by Tukey-Kramer test) (Fig. 2B). However, GUS-activities decreased drastically (65-fold, P< 0.001) in comparison to the wild type control when two bases within the second part of the binding motif 1 (mutant b) had been exchanged (AC to CA) (Fig. 2B).
Expression of pilAB is enhanced by carbon starvation. To identify environmental signals that influence pilAB transcription, we tested the expression level under different growth conditions (not shown). Conditions of carbon starvation revealed significant differences: the chromosomal pilAB::gusA fusion strain (BH72::pJBLP14) showed a 2-fold (P< 0.0001) increase of GUS-activity within one hour of incubation without C- and N-source in comparison to cells incubated with potassium malate as carbon source (Fig. 3A). Northern blot analysis with a pilAB probe confirmed the elevated expression level under carbon starvation (Fig. 3B). In order to exclude osmotic effects, the experiment was repeated with addition of potassium citrate, which cannot be metabolized as a carbon source (53). As for carbon starvation, a 2-fold induction was obtained (data not shown). The addition of ammonium did not affect expression levels (not shown), indicating that the expression depended on the availability of carbon and not nitrogen sources. In contrast, control experiments with a different transcriptional fusion, a pilRS::gusA reporter strain, did not show significant differences under carbon starvation conditions (P > 0.05) (Fig. 3C).
To elucidate whether this effect was mediated by the sensor kinase PilS, an in frame-deletion mutant of pilS was constructed that contained the chromosomal pilAB::gusA fusion
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(BHΔpilS::pJBLP14). The expression of pilAB in the pilS deletion background did not increase significantly upon carbon starvation, however was slightly elevated constitutively in comparison to the wild type (Fig. 3C). Thus PilS appeared to be involved in sensing the absence of carbon sources in the medium.
Cell-density dependent regulation of the type IV pilin genes. We also monitored the expression of pilAB during growth to various optical densities in batch culture with transcriptional gusA or gfp reporter gene fusions. In the wild type, the expression increased significantly (2.6-fold, P< 0.0001) at high cell density (Fig. 4A). However, in the pilS mutant the expression increased more strongly, 10- to 11-fold (P< 0.0001), as measured by GUS activity (Fig. 4B) or GFP fluorescence (Fig. 4C). This suggested that PilS had a strong negative effect on the pilAB expression under these conditions. These results were confirmed by Western blot analysis. Immuno-detection of PilA showed a dramatic increase in whole-cell pilin production in the ΔpilS mutant (Fig. 4D). In order to analyze whether the increased pilin production led to an enhanced surface piliation, we examined Azoarcus sp. strain BH72 and BHΔpilS mutant cells by transmission electron microscopy by negative staining with uranyl acetate. Consistent with the results of the Western blot analyses, the pilS mutant showed a slight hyperpiliation with 2-3 pili visible per cell in contrast to the wild type with only one pilus per cell (data not shown).
Cell-density dependent induction suggested that pilin gene expression in Azoarcus sp. strain BH72 is under the control of quorum sensing. Therefore, we tested if cell free supernatants of stationary phase cultures (conditioned supernatants) could induce pilin gene expression in exponentially-growing test cells at low cell densities. Indeed, the conditioned supernatant of stationary phase cultures induced the pilAB::gusA expression in the pilS mutant significantly (P < 0.0001) 3,69 ± 0,06-fold in comparison to cells incubated for the same time (4 h) in fresh medium. This indicated that the conditioned supernatant contained soluble autoinducer molecules leading to elevated pilAB-expression. Based on these results we developed a quorum-sensing bioassay for Azoarcus sp.; briefly, an aliquot of cells of the reporter strain BHΔpilS::pJBLP14 growing exponentially in liquid VM-ethanol medium was transferred to either fresh VM-ethanol medium, or to conditioned supernatant produced by the pilAB::gfp reporter strain BHΔpilS::pJBLP1gfp and supplemented with 6 ml ethanol per l. After 4 h of aerobic growth, pilAB::gusA expression of washed cells was quantified in GUS assays.
Azoarcus does not use an AHL-based quorum sensing system for pilAB expression. In Gram negative bacteria AHLs are widespread as autoinducer molecules. To test whether Azoarcus is using homoserine lactones to communicate, we performed cross streaking
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experiments with GFP-based AHL sensor strains that are able to respond to different spectra of AHL molecules. Pseudomonas putida IsoF harbouring plasmid pKR-C12 responds to 3-oxo-C12-HSL, 3-oxo-C10-HSL, C10-HSL, and to C12-HSL (64). To monitor short AHL molecules we used Escherichia coli JM105 harbouring plasmid pJBA89 which senses 3-oxo-C6-HSL, C6-HSL, C8-HSL, 3-oxo-C12-HSL and C4-HSL (3). In contrast to the respective control strain Rhizobium sp. NGR234, an AHL producer (3-oxo-C8-HSL) (30), no GFP based fluorescence was observed in Azoarcus sp. strain BH72 (Fig. 5).
To exclude the influence of AHL in the cell density-dependent induction of the pilAB genes, we extracted conditioned culture supernatants with dichlormethane and tested the extracts in an AHL plate detection assay and in a quorum sensing bioassay. The AHL-producing strain Chromobacterium violaceum CV017 was used as a positive control. For the AHL detection assay three different AHL monitor strains were used: the GFP-based sensor strains described above and C. violaceum CV026 as reporter for short chain AHL (C4-C8) and cyclic dipeptides. As expected only the dichlormethane extracts of the positive control strain yielded a strong signal for short chain AHLs in both reporter strains. In contrast, the extracts of Azoarcus yielded no signals (data not shown).
Furthermore, conditioned supernatants were also used for the Azoarcus quorum sensing bioassay. The inducing effect of the untreated conditioned supernatant (c. s.), the c.s. after extraction with dichlormethane, and the dichlormethane extract were tested for induction of pilAB::gusA expression in comparison to fresh medium. Inducing activity was not found in the dichlormethane extract, but remained in the conditioned culture supernatant (Table 2).
Thus the autoinducer was apparently not sufficiently hydrophobic to be extracted by the solvent, unlike the case for AHLs.
Genome-based search for proteins putatively relevant for autoinducer production. The bioinformatic analysis of the whole genome sequence of Azoarcus sp. BH72 revealed no evidence for genes encoding proteins for known quorum sensing systems, such as homologues of LuxIR/LasIR, LuxS (41). Therefore, the genome was screened for predicted protein domains that are relevant in known systems. A predicted conserved hypothetical protein (azo3178) was found to contain an autoinducer synthethase (LasI) domain (Pfam PF00765) at amino acid 18 to 223 (E-value 9.40e-03). LasI, a LuxI homologue, is one of the AHL synthetases of the Pseudomonas aerguinosa quorum sensing system (28). For the conserved hypothetical protein azo1746, a peptidase C39 motif (Pfam PF03412) was predicted at amino acid residues 15 to 134 (E-value 5.10e-01). This domain is found in ABC-type bacteriocine exporters, that are responsible for the export of signaling peptides (16). In
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the human pathogen Pseudomonas aeruginosa, quinolone (PQS) is an integral component of the quorum sensing circuitry. In the Azoarcus genome, two genes encoding conserved hypothetical proteins (azo0390/azo3379) were found with a predicted lactamase B domain at amino acid residues 40 to 254 for azo0390 (E-value 2.00e-29) and at amino acid residues 35 to 240 (E-value 6.40e-24) for azo3379; this domain is also present in the PqsE protein of P. aeruginosa which takes probably part in the signaling response to the PQS (29).
These four genes were separately inactivated in strain BH72 by directed insertional mutagenesis. The mutants were used for the production of conditioned supernatant, and the accumulation of autoinducer was tested in the Azoarcus quorum sensing bioassay. The pilAB::gusA expression was still significantly induced (P< 0.01, unpaired t-test) by supernatants of all mutants (Table 2), indicating that autoinducer production was not affected by any of the mutations in any of the candidate proteins that might be related to autoinducer production.
Characterization of the signaling molecule HSF (hydrophilic signal factor) in Azoarcus sp. strain BH72. To test whether the autoinducer might be a peptide, the conditioned supernatant of Azoarcus sp. was treated with subtilisin and chymotrypsin. In the quorum sensing bioassay, pilAB::gusA expression was still significantly induced after protease treatment (Table 2). To test the stability of the autoinducer towards pH changes, conditioned supernatant was adjusted to pH 5-9, incubated for 15 min, and then neutralized. The inducing activity in the bioassay was not significantly reduced between pH 5-7 (Table 2). In order to estimate the size of the autoinducer molecule, conditioned supernatant was passed through ultrafiltration membranes with a molecular mass cutoff of approximately 1000 Da. The filtrate was still inducing pilAB::gusA expression (Table 2), indicating a size of ≤ 1 kDa. To purify the autoinducer molecule, conditioned supernatant and media as a control were loaded onto a cation exchange cartridge. Fractions were collected and assayed in the quorum sensing bioassay. The autoinducer activity was already detected in the wash fraction (Table 2), suggesting that it is weakly binding and positively charged at pH6. This active wash fraction was further fractionated by HPLC with a C18 reversed-phase capillary column. The HPLC fractions were collected and analyzed in a quorum sensing bioassay. The pilAB::gusA-inducing fraction eluted already at 2-7 % acetonitrile (Table 2). This further corroborated the assumption that the autoinducer was a rather hydrophilic molecule, and was thus termed HSF (hydrophilic signaling factor).
15 DISCUSSION
The type IV pilus plays a key role in the interaction of Azoarcus with the rice plant and fungal mycelium (18). In this study we have begun to analyse the mechanisms which are involved in the regulation of the type IV pilin genes in the grass endophyte Azoarcus.
Upstream of the pilAB operon, we identified a two component regulatory system which show high similarity to the sensor kinase PilS and the response regulator PilR of the human pathogen P. aeruginosa (32) and to systems of the plant pathogens Ralstonia solanacearum (1) and Xylella fastidiosa (12, 26). The deduced amino acid sequence of PilS and PilR show the characteristic motifs of a two component sensor kinase and its corresponding transcriptional activator, like it is described for Pseudomonas aeruginosa (32).
Primer extension analysis confirmed the σ54 dependent promoter sequences as the responsible type IV pilin gene promoter in Azoarcus, like it is typical for the type IV pilin genes of the subclass A (65). An insertional knockout mutant of PilR revealed a drastic reduction in the pilAB gene expression which identified PilR as the transcriptional activator. This is also the case in P. aeruginosa and M. xanthus where PilR was characterised as the activator protein of the type IV pilin gene transcription (38, 74).
The binding motif of PilR in P. aeruginosa consists of four binding sites, three of them with the pattern 5` N(4-6) C/GTGTC3` (38). This suggests that PilR binds as a tetramer upstream of the pilin promoter. In contrast NifA, the transcriptional activator of the nitrogenase structural genes, binds as a dimer on the 5`TGT-N10-ACA3` motif. Early studies showed that transversions in the conserved G or C residues of the binding motif both reduced activation by NifA in Klebsiella pneumoniae, confirming that the two-fold rotational symmetry of the upstream activator sequence is important for its function (9). In Azoarcus we could observe a drastic reduction in the pilAB expression level after the deletion of 142 bp upstream of the σ54 promoter sequence. This suggests that the binding motif of PilR is within this deleted region. Site directed point mutations in the potential NifA binding sequence and in two further palindroms revealed that a mutation in the second part of the NifA binding motif causes a dramatic decrease in the pilAB transcriptional level. Mutations in the first part of the potential NifA binding site leads only to a slight reduction in the pilAB transcriptional level.
These results suggest that probably the binding motif of PilR has similarities to the one of NifA. We expected that the mutation of the first part of the NifA binding site leads to the same drastic reduction like it was observed for the second part. It was surprising that only a slight reduction was determined, which probably indicates that the base exchanges in the first
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part still allowed the binding of PilR to the DNA. An explanation can be that the PilR motif in Azoarcus is composed of direct repeats instead of inverted repeats like it was shown for Pseudomonas aeruginosa (38). We didn`t find the motif of Pseudomonas PilR, but nevertheless the use of direct repeats in the PilR binding site can`t be excluded in case of Azoarcus. To determine the exact sequence of the PilR binding site of Azoarcus DNase I foot print analyses should be performed.
A two-component regulatory system, as identified in this study, is typically involved in the activation of genes in response to environmental signals (33). The signal sensed by PilS which affects the expression of the fimbrial subunit is yet unknown (22). In Azoarcus we observed an enhanced pilin expression under carbon starvation, while the expression of pilSR genes did not change. To test if this response is mediated by PilS we constructed a pilS deletion mutant and studied the pilAB gene expression. In the pilS mutant background the type IV pilin gene expression was not enhanced under carbon starvation. This indicates that PilS is able to sense the absence of carbon in the environment. For the fruiting body developing soil bacterium Myxococcus xanthus was shown that the sensor kinase PilS is negatively regulating the pilA expression while in Pseudomonas the PilS is required for the pilin gene expression. However overexpression of pilS leads also to the inhibition of the pilA expression in Pseudomonas (7, 74).
We monitored the pilAB expression in wild type and pilS mutant backgrounds during growth to various optical densities. Interestingly in stationary growth phase the pilAB expression starts to increase. In the wild type only 2.6-fold, but in the pilS mutant background 10 to 11-fold, which leads to the suggestions that the pilAB operon in Azoarcus is under the control of quorum sensing and that PilS inhibits the induction under these conditions. This negative effect of PilS was further demonstrated by western blot analyses and electron microscopy.
PilS seems to have also a dual function in Azoarcus but under different conditions than in Pseudomonas, where the relative amounts of pilS and pilR affects pilA expression (7). It was proposed from Wu and Kaiser that the stochiometric balance between the two components could be another kind to modulate pilin synthesis (74). Therefore it will be interesting to analyse overexpressing mutants of pilS and pilR in Azoarcus, to test if probable another way of regulating the pilAB gene expression is determined by balancing the amounts of the two components.
The pilAB inducing activity of conditioned supernatant suggests the presence of a quorum sensing signal in adequat concentration. Azoarcus as a gram-negative bacteria was tested first for the utilization of an AHL based quorum sensing system. Cross streaking experiments with
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different AHL sensor strains and also the analysis of the AHL extraction fraction exclude the use of an AHL or the use of cyclic dipeptides as the autoinducer signal. Furthermore the pilAB inducing activity was found in the liquid phase which is characteristic for hydrophilic molecules.
Although many plant-associated bacteria are found to be AHL producers (10, 21, 55, 61, 70), 10-20% of cultivable bacteria in soil and rhizosperic environments are producing AHL as quorum sensing signals (14, 15, 50, 64). The plant-symbiont Bradyrhizobium japonicum (43) and the plant-pathogens Ralstonia solanacearum (25), Xanthomonas campestris (72) and probable also Xylella fastidiosa (58) have only, or next to an AHL autoinducer, other kinds of quorum sensing signals, which suggests that they have an advantage of an uncommon intraspecies communication in better preventing the infiltration of their system by AHL mimics or signal degrading (17, 69).
The genome sequence of Azoarcus sp. BH72 showed no genes which are coding for components of known quorum sensing systems (41). That is why we screened the genome for probable quorum sensing relevant predicted protein domains and performed insertional mutagenesis of the potential candidates. We found a conserved hypothetical protein with an LasI AHL synthetase domain (28) and one conserved hypothetical protein with a predicted Peptidase C39 motif, which is found in peptide autoinducer exporting ABC-type transporter (16). Another two conserved hypothetical proteins contained predicted Lactamase B motifs, which are also found in the pqsE gene product. A component probably involved in transducing the signal response to the PQS molecule (29). Non of the insertional mutants showed a decrease in the pilAB inducing activtiy. These findings together with the circumstance that Azoarcus uses non AHL based quorum sensing system suggests the presence of a new kind of autoinducer.
The quorum sensing signal of Azoarcus is not protease sensitive, which excludes the participation of a peptide component. The pH stability at pH 5-7 and the binding of the cation exchanger suggests an cationic charged autoinducer at pH 6. Finding the pilAB-inducing activity in the filtrate after passing the conditioned supernatant through an ultrafiltration membrane (1 kD mass cut off) indicates a size of the autoinducer probably smaller than 1 kD.
Purifications by HPLC supported a hydrophilic character of the autoinducer molecule, the pilAB inducing fraction eluted from the C18 reverse phase capillary column at 2-7%
acetonitrile.
The grass endophyte Azoarcus lives in a diverse bacterial community. Quorum sensing offers bacteria the possibility to act as an unit, which gives advantages in many ecological niches