The photosensor protein Ppr of Rhodocista centenaria is linked to the chemotaxis signalling pathway
© Kreutel et al; licensee BioMed Central Ltd. 2010
Received: 28 March 2010
Accepted: 9 November 2010
Published: 9 November 2010
Rhodocista centenaria is a phototrophic α-proteobacterium exhibiting a phototactic behaviour visible as colony movement on agar plates directed to red light. As many phototrophic purple bacteria R. centenaria possesses a soluble photoactive yellow protein (Pyp). It exists as a long fusion protein, designated Ppr, consisting of three domains, the Pyp domain, a putative bilin binding domain (Bbd) and a histidine kinase domain (Pph). The Ppr protein is involved in the regulation of polyketide synthesis but it is still unclear, how this is connected to phototaxis and chemotaxis.
To elucidate the possible role of Ppr and Pph in the chemotactic network we studied the interaction with chemotactic proteins in vitro as well as in vivo. Matrix-assisted coelution experiments were performed to study the possible communication of the different putative binding partners. The kinase domain of the Ppr protein was found to interact with the chemotactic linker protein CheW. The formation of this complex was clearly ATP-dependent. Further results indicated that the Pph histidine kinase domain and CheW may form a complex with the chemotactic kinase CheAY suggesting a role of Ppr in the chemotaxis signalling pathway. In addition, when Ppr or Pph were expressed in Escherichia coli, the chemotactic response of the cells was dramatically affected.
The Ppr protein of Rhodocista centenaria directly interacts with the chemotactic protein CheW. This suggests a role of the Ppr protein in the regulation of the chemotactic response in addition to its role in chalcone synthesis.
Rhodocista centenaria, first described as Rhodospirillum centenum is a thermotolerant phototrophic purple bacterium of the α-proteobacteria group isolated from hot springs in Wyoming 1985. The slightly spiroid or vibrioid shaped cells are motile by means of a single long flagellum, their intracellular photosynthetic membranes are lamellar and their in vivo absorption spectra show features almost indistinguishable from those of Rhodospirillum rubrum. However, 16S rRNA analysis elucidated considerable differences between the species, hence Rhodocista was separated into a new genus , now consisting of three species [4, 5]. R. centenaria is closely related to the plant-associated genus Azospirillum. As virtually all phototrophic organisms, R. centenaria exhibits a sensory response to light originally described as "Schreckbewegung" . Engelmann and also Manten  found that R. rubrum cells accumulated in the most intense area of light gradients between wavelengths 800 and 900 nm. R. centenaria shows a particularly unique form of macroscopic phototactic behaviour, first described in 1994 by Gest and coworkers . On solid media, the phototactic colonies move towards longwave light and away from light with wavelengths less than 650 nm . R. centenaria develops lateral flagella in viscous media or on solidified surfaces. These flagella consist of a distinct flagellin whose expression is controlled by specific mot and fli genes .
For R. centenaria, a close relationship between chemotaxis and the phototactic response has been found . As seen with many other photosynthetic bacteria, R. centenaria has multiple chemotaxis operons with distinct functions [13–15]. The chemotaxis gene cluster has been well characterized and most of the genes are similar to those of other Gram negative bacteria like Escherichia coli. In brief, the histidine kinase CheA is linked to the chemotactic receptors (MCPs) by the CheW protein . This trimeric receptor complex controls the phosphorylation level of the response regulator CheY. Activated CheY (CheY-P) interacts directly with the motor of the flagella to control swimming direction. The dephosphorylation of CheY-P occurs spontaneously, only in enterobacteria this reaction is accelerated by the phosphatase CheZ. For adaptation, CheB and its antagonist CheR remove or add methyl groups to the receptors, respectively.
In R. centenaria, the two central components of the chemotactic signal transduction cascade, namely CheA and CheY, are present as the fusion protein Rc-CheAY located in the first chemotactic operon , a situation that is also observed in Helicobacter. Whereas the role of the CheY-domain of the CheAY protein in H. pylori seems to be a phosphate sink, in R. centenaria, the function of Rc-CheAY remains still unclear. While Che proteins are generally involved in chemotactic responses, they were also shown to affect the phototactic response in R. centenaria as demonstrated by the analysis of many che mutants .
In the last decade, bacterial photoreactive proteins like phytochromes, previously thought to be a unique feature in plants, have been identified as photoactive yellow proteins (Pyp) and have now been extensively studied in a variety of eubacterial species (for review see [20, 21]). For R. centenaria, a Pyp-like protein, Ppr, was described in 1999 by Bauer and colleagues . The large fusion protein Ppr consists of three functional domains, an N-terminal Pyp domain with the cinnamic acid chromophore, the central phytochrome-like bilin attachment domain Bbd and the C-terminal histidine kinase domain Pph which autophosphorylates an essential histidine residue . Although some Pyp proteins have been crystallized and biophysically characterized in great detail (reviewed by ), no distinct physiological role could be attested to these unique proteins. A Ppr-deletion mutant lacking amino acid residues 114-750 did not show any alterations in phototactic behaviour, instead exhibited a strongly deregulated expression of the chalcone synthase gene suggesting a regulatory function in the polyketide synthesis . Although there is no obvious direct involvement of Ppr in the phototactic or scotophobic reaction, an interaction with the chemotactic signal transduction components is plausible to regulate general phosphorylation levels or transduce phosphoryl groups to a yet unknown light-dependent signal transducing protein. We therefore analysed whether the Ppr protein and in particular its phosphorylating kinase domain Pph interacts with the Rc-Che proteins.
The chemotactic response of E. coli is inhibited by the expression of Ppr
The Pph protein interacts with Rc-CheW in an ATP-dependent manner
The histidine kinase Pph is present in a complex with Rc-CheW and Rc-CheAY
Taken together, the results give preliminary evidence that the C-terminal histidine kinase domain Pph of the photosensor protein Ppr assembles in vitro into a trimeric complex of Pph, Rc-CheW and Rc-CheAY.
The oligomeric state of the histidine kinase domain Pph
The Pph protein expressed in R. centenaria is found in a complex with Rc-CheW
Since photosynthetic bacteria have to locate their habitat with optimal light conditions, specialized sensor systems and signal transduction cascades involving different chromophores arose during evolution (for review see ). The blue light sensitive Ppr protein of R. centenaria consists of three distinct domains, the Pyp domain containing a cinnamic acid chromophore, the phytochrome-like bilin binding domain and the histidine kinase domain Pph (Figure 1; ). The structural organization suggests that the protein is involved in a light-dependent signaling pathway similar to chemotaxis. Since R. centenaria exhibits a strikingly obvious phototactic behavior it is compelling to assume that the Ppr protein is involved in this reaction. Light with a wavelength of above 650 nm is attractive, whereas light with less than 650 nm acts as a repellent . The absorption maximum of a prototypical cinnamic acid chromophore in a Pyp light sensor is at about 450 nm , whereas the phytochrome-linked biliverdin absorbs red light, suggesting that the latter could function as an attractant sensor. Recently, Cusanovich and co-workers showed that the holo-Ppr of R. centenaria has absorption maxima at 425 nm (Pyp), 400, 642 and 701 nm (phytochrome)  corresponding to the typical absorption spectrum of Pyp  and phytochromes . The phytochromes TaxD1, Cph2 and PlpA were found to be involved in the phototactic reaction of Synechocystis sp. PCC 6803, a finding that supports the idea of a participation of the Ppr sensor in the phototactic response of R. centenaria[42, 43].
The data presented here show that the histidine kinase Pph domain of the Ppr receptor is found in a complex with Rc-CheW when isolated from R. centenaria (Figure 8). The interaction of Pph and Rc-CheW was also observed in vitro with purified components showing a strong affinity with a KD of about 130 nM (Figure 5). In contrast to the trimeric Tsr-CheA-CheW complex that is formed in E. coli with an affinity of about 3 μM  we observed that the complex formation of Pph and Rc-CheW is clearly ATP-dependent (Figure 4B). It is likely that the Pph-CheW complex is capable to bind Rc-CheAY (Figure 6) consistent with the idea that the chemotactic network is functioning in the presence of Pph. However, the function of the Rc-CheAY fusion protein in this signaling cascade remains unclear. Preliminary transphosphorylation experiments that we perfomed indicate that the CheY domain of the Rc-CheAY protein acts as a phosphate receiver domain and that the CheY domain acts as a phosphate sink similar as it has been described for the chemotactic system in Rhizobium meliloti and Helicobacter pylori[44, 45]. The involvement of Ppr in chemotaxis is also supported from the experiments we performed with E. coli. The heterologous expression of Pph has a strong inhibitory effect on chemotaxis as demonstrated by the swarm assay (Figure 2) and the capillary assay (Figure 3). Both assays showed that upon expression of Ppr or Pph the chemotaxis of E. coli is turned off whereas expression of the R. centenaria histidine kinase KdpE had no effect. This suggests that the Ppr protein interacts with Ec-CheW although the CheW proteins of E. coli and R. centenaria show a homology of only about 59% and an identity of 28% . However, the structural analysis suggests that all CheW proteins of different species share common features [46, 47]. We propose that the binding of the Ppr protein results in a non-functional Ec-CheW-Ppr complex that is inhibitory for chemotaxis (Figures 2 and 3) due to the inactivation of Ec-CheW. Remarkedly, a mutant of the predicted phosphorylation site of Pph with the histidine at position 670 being changed to an alanine residue had a less inhibitory effect on chemotaxis, suggesting that the kinase activity of Pph has a functional role in CheW binding. Similar inhibitory effects on chemotaxis have been observed for E. coli when Ec-CheW, Ec-CheA or the MCP-receptors were overproduced [23, 25, 27]. In addition, such an inhibitory effect was also observed when chemotactic proteins from other organisms like Rhodobacter capsulatus or Leptospira interrogans were heterologously expressed in E. coli.
We found that the histidine kinase domain Pph was mainly present as a monomer when expressed in E. coli (Figure 7) and only a minor fraction was found as dimers. Most other bacterial histidine kinases that have been investigated so far were found to be homodimers . Accordingly, when the plasmid encoded Pph protein was isolated from R. centenaria it appeared in a complex consisting of CheW and most likely a dimer of Pph (Figure 8).
Working in a network together with the perception of chemical substrates by the chemotactic signal transduction cascade, Ppr could very well be a part of a complex sensory machinery to tune metabolic and phototrophic processes in phototrophic bacteria. The perception of light may only be an oblique indicator for the metabolic state of a R. centenaria cell as is suggested by its influence on cyst formation [13, 22]. Therefore, Ppr could work in parallel with the photosynthetic electron transport sensor Ptr of R. centenaria to specifically regulate cellular motility and sense the metabolic state of the cell.
Bacterial strains and culture conditions
All genetic manipulations were performed according to standard methods in E. coli XL1-Blue (recA1 thi supE44 endA1 hsdR17 gyrA96 relA1 lac F′ (pro AB+lacIqlacZ ΔM15 Tn10) as described . For expression of Rc-CheW and Pph, E. coli C41  was used. For genetic transfer into R. centenaria, E. coli RR28  and in the swarm assays, E. coli MM500  was used. For E. coli, antibiotics were added at final concentrations of 200 μg/ml ampicillin, 10-50 μg/ml kanamycin and 5 μg/ml gentamycin and for R. centenaria 5 μg/ml gentamycin, 10 μg/ml kanamycin. All E. coli strains were cultured in LB medium at 37°C if not indicated otherwise. R. centenaria (ATCC 43720) was obtained from the culture collection. (For anaerobic photosynthetic growth R. centenaria was cultured in screw cap bottles filled to the top with PYVS medium  and illuminated by an 80 W tungsten bulb (Concentra, Osram, Germany) at 42°C.
Construction of Pph and Che Plasmids
Constructs and plasmids used in this study
Ampr, pET16b, His10-Ppr
Ampr, araC, PBAD, pBR322 ori, expression vector
Ampr, pBAD18, His10-PprΔ1-601-StrepTag II
Ampr, pBAD18, His10-PprΔ1-601; H670A-StrepTag II
Ampr, pBAD18, His10-Ppr-StrepTag II
Ampr, pBAD24, Rc-KdpE
Ampr, T7 promoter, N-terminal deca histidyl tag
Ampr, pET16b, His10-PprΔ1-601-StrepTag II
Ampr, pET16b, His10-PprΔ1-601; H670A-StrepTag II
Ampr, pET16b, His10-Ppr-StrepTag II
Kanr, pET28a(+), His6-CheAY
Kanr, Tra, helper plasmid
Ampr, pT7-7, PprΔ1-601-StrepTag
Ampr, T7 promoter, expression vector
Ampr, pT7-7, His6-Rc-CheW
Ampr, Kanr, lac promoter, lacZ
Genr, pRK2 derived plasmid, lacZ
A ppr-strep tag II fusion gene was constructed as follows. pET16b containing the entire ppr gene (pNB10), as well as pET16b-Pph were cut by Nco I and the resulting fragments (~6.0 kb and ~2.5 kb) were ligated. The orientation of the ppr-insert was checked by DNA-sequencing and the resulting plasmid was named pET16b-Ppr. To construct an arabinose inducible full length ppr, the gene was excised by Xba I and Hin dIII from pET16b-Ppr and ligated into the pBAD18 vector.
The putative phosphorylation site (the histidine at position 670 in the Ppr protein) was changed to an alanine (CAC→GCG) using site directed mutagenesis with the primers (5'-CTGGCGAACATGAGCGCG GAGCTGCGGACTCCG-3') and (5'-CGGAGTCCGCAGCTCCGC GCTCATGTTCGCCAG-3') and pSK4 as a template. The resulting mutant was digested by Nde I and Bam HI and subcloned into the pET16b vector generating pET16b-PphH670A. Then the pph H670A mutant was excised by Xba I and Hind III and the fragment was inserted into the pBAD18 vector to create pBAD-PphH670A.
To express the histidine kinase domain Pph with an N-terminal his10-tag and a C-terminal strep-tag II in R. centenaria, the plasmid pZJD11 (kindly provided by C. Bauer) was used . We used the oxygen regulated puc promoter and the puh A Shine Dalgarno sequence from Rhodobacter capsulatus to initiate translation. Therefore, a PCR reaction with the primers (5'-TACGTAGGGCCCTAAGCTAAAGGAGGACTAACATGGGCCATCATCAT-3') and (5'-TACGTA GGCGCGAATTCGGCTTGATCAGGC-3') and pET16b-Pph as a template was conducted. Simultaneously, a Sna BI restriction site was introduced at the 3' end of the gene. The resulting fragment was subcloned into pGEM T-easy vector (Promega) and verified by DNA sequencing. This plasmid was used as a template to insert the puc promoter via a second PCR. The primers (5'-GGTAACC TTGATCGCCGACACTTGGGCTCCCA TAGTGGAGCTCGGGCCCTAAG-3') and (5'-TACGTA GGCGCGAATTCGGCTTGATCA GGC-3') were used to introduce a Bst EII site at the 5' end. The resulting fragment was inserted into pGEM T-easy vector. After sequencing, the pph construct was excised by Bst EII and Sna BI and ligated into the corresponding sites of pZJD11 to generate pSK10.
To express the Rc-CheW protein in E. coli, the cheW gene was amplified by PCR from the R. centenaria genome using the primers (5'CATATGCATGCCCGCCTGCCCGTTCCC-3') and (5'GGGAATCGTTCATTGCGATCAGTTTCCGG-3'), respectively. The resulting fragment was first cloned into pT-Adv. Then the cheW gene was excised by Nde I and Eco RI and ligated into the corresponding restriction sites of a pT7-7 derivative containing a decahistidine sequence to create the IPTG inducible expression vector pT7-7-CheW.
Swarm agar assays
TB swarm agar plates (1% bacto-tryptone, 0.8% NaCl; 0.35% bacto-agar) containing 0.2% arabinose or 0.2% fructose, respectively, were inoculated with a single colony of E. coli MM500 or MM500 harbouring one of the plasmids pBAD-Ppr, pBAD-Pph, pBAD-PphH670A, pBADKdpE and pBAD, respectively. The plates were incubated for 6 hours at 37°C.
Chemotaxis assay using a chemotactic chamber
2 ml minimal medium A (MMA)  containing an amino acid mixture (threonine, leucine, histidine, methionine), vitamin B1 (final concentration 10 μg/ml each), 200 μg/ml ampicillin and 0.2% fructose were inoculated with an overnight culture of E. coli MM500 or cells harbouring pBAD-Pph, pBAD-PphH670A, pBAD-KdpE or pBAD18, respectively. When the cultures reached an OD600 = 0.6 the cells were washed twice with MMA without sugar and finally either 0.2% arabinose to induce protein expression or 0.2% fructose (as a control) were added. The cultures were incubated for 60 min at 37°C. For the kinetic analysis the incubation times are indicated in Figure 3B. Again, the cells were washed twice with MMA without carbon source and were back diluted to an OD600 = 0.6. The chemotactic assays were performed as follows. 300 μl of the cell suspension were filled in each drilling of the chamber and a capillary containing either 2 μl 1 mM aspartate or 2 μl H2O as a control was placed into the channel between the two cylindrical compartments. The chamber was incubated at 37°C for 30 minutes. The outside of the capillary was washed extensively with sterile water and the content of the capillary was blown out and a dilution series was streaked on agar plates. After overnight incubation at 37°C the colonies were counted and the chemotactic inhibition (CI) was calculated as the ratio of colonies of the water containing capillary to the colonies from the aspartate containing capillary. Therefore, a low CI indicates an undisturbed chemotactic response whereas a high CI reflects an inhibition of the E. coli chemotactic system.
Expression and purification of Pph protein from inclusion bodies
E. coli strain C41  harbouring the plasmid pET16b-Pph were grown at 37°C in 1 l LB medium containing 200 μg/ml ampicillin. When cells reached the midlogarithmic phase, IPTG was added at a final concentration of 1 mM and the cells were grown for an additional 4 hours at 37°C. Then the cells were harvested by centrifugation. The resulting pellets were resuspended in 100 mM Tris-HCl pH 8.0, 150 mM NaCl (buffer W) and lysed by a French Press. Inclusion bodies were precipitated by centrifugation and resuspended in buffer W containing 0.5% N-lauroylsarcosine. The inclusion bodies were solubilized overnight at 4°C with gentle shaking. To the filtered extract 10 mM imidazole was added and applied to a Sepharose 6b (GE Healthcare) column precharged with Cu(II) ions. Unbound proteins were removed by washing the column with 15 column volumes of buffer W containing 0.5% N-lauroylsarcosine and 10 mM imidazole. The bound protein was eluted by a linear gradient up to 500 mM imidazole in buffer W + 0.5% N-lauroylsarcosine. The Pph protein containing fractions were pooled, diluted 1:40 with buffer W (final detergent concentration = 0.01%) and applied to a streptactin-sepharose column (IBA, Göttingen, Germany) to remove contaminating proteins. After washing the column with five column volumes buffer W + 0.01% N-lauroylsarcosine, the protein was eluted with buffer W + 0.01% N-lauroylsarcosine containing 2.5 mM desthiobiotin. The protein was dialyzed against buffer W + 0.01% N-lauroylsarcosine and the purity was checked by SDS-PAGE analysis as described . Protein marker SM0431 and SM0441(Fermentas) were used.
Expression and purification of Rc-CheW
1 Liter of LB medium containing 200 μg/ml ampicillin was inoculated with a freshly transformed single colony of E. coli C41 harbouring the plasmid pT7-7-CheW. The cells were grown to a cell density of 2 × 108 cells per ml at 37°C, then IPTG was added to a final concentration of 1 mM. The cells were incubated for an additional 4 hours and harvested by centrifugation. The pellet was resuspended in TBS (50 mM Tris-HCl pH 7.4, 150 mM NaCl) and lysed by a French Press. Cell debris was removed by centrifugation and a final concentration of 10 mM imidazole was added. This crude extract was applied to a Cu(II)-charged Sepharose 6b column and unbound proteins were washed out with 10 column volumes of TBS + 10 mM imidazole. The protein was eluted with a linear gradient from 10 to 500 mM imidazole and fractions containing Rc-CheW were dialyzed against TBS-buffer. The homogeneity of the protein was monitored by SDS-PAGE.
Expression of the Pph protein in R. centenaria
The plasmid pSK10 was transferred to wild type R. centenaria by triparental conjugation using E. coli RR28 , the helper plasmid pRK2013  and the filter-mating technique as described previously . After conjugation, about 109 T7 phages were added, and the mixture was incubated for 30 minutes at 37°C to eliminate remaining E. coli cells. Finally, conjugants were selected on the basis of gentamycin resistance on PYVS plates containing 5 μg/ml gentamycin under anaerobic conditions. 2L PYVS media containing 5 μg/ml gentamycin and 10 μg/ml kanamycin (R. centenaria is naturally resistant to kanamycin ) was inoculated with a culture of pSK10 containing R. centenaria cells. The cells were grown under anaerobic and illuminated conditions for 96 h and harvested by centrifugation, resuspended in 100 mM Tris pH 8.0, 150 mM NaCl (buffer W) and lysed by a French Press. The cell debris and the photosynthetic membranes were removed by centrifugation. The cleared extract was applied to a streptactin-sepharose column (IBA). The unbound proteins were removed by extensively washing the column with buffer W and the bound proteins were eluted with buffer W containing 2.5 mM desthiobiotin. The success of the purification was verified by SDS-PAGE, silver staining and Western blot analysis with the antibodies raised against the his-tag or the strep-tagII, respectively.
Determination of the dissociation constant of Pph and Rc-CheW by resonant mirror spectroscopy
The Pph protein was purified from inclusion bodies as described above and the aminosilane cuvette was activated as described by the manufacturer (Iasys, Biosensors). 200 μl of the purified Pph protein (50 μg/ml) was added to the activated cuvette and the immobilization was recorded for 30 minutes. The unbound protein was removed by extensive washing and increasing amounts of purified Rc-CheW (see above) were added. After 30 minutes of incubation the free Rc-CheW was washed out and the amount of bound Rc-CheW was determined for each experiment. The fractional saturation was calculated and depicted against the amount of the added Rc-CheW concentration. The resulting Scatchard Plot is illustrated as the inlet of Figure 5.
In vitro transcription and translation
The histidine kinase domain Pph as well as Rc-CheAY were transcribed in vitro from the plasmids pSK4 and pET28-CheAY, respectively, using a T7 transcription kit (Fermentas) according to the manufacturers manual. The translation reaction was performed as described previously  by using an E. coli based cell free expression system The proteins were labeled with 10 μCi of [35S]methionine (ICN) in each experiment. The high speed supernatant (S-135) was prepared as described from E. coli MRE600 .
50 μg of the purified his6-Rc-CheW protein was mixed with 25 μl of the in vitro translated Pph protein and 25 μl Rc-CheAY when indicated. The protein mixture was incubated overnight at 37°C. Then, the his6-Rc-CheW protein was bound to a column containing 50 μl Sepharose 6b (GE Healthcare) charged with Cu(II) ions and pre-equilibrated with buffer I (20 mM sodium phosphate pH 7.7, 200 mM NaCl, 50 mM imidazole pH 8.0). After 30 minutes at room temperature, the unbound proteins were removed by washing the column five times with 500 μl buffer I followed by an elution with 1.5 ml buffer II (20 mM sodium phosphate pH 7.7, 200 mM NaCl, 500 mM imidazole pH 8.0). All fractions were TCA precipitated and analyzed by SDS-PAGE. The gels were stained with coomassie brilliant blue and the radiolabeled bands were quantified using a Fuji BAS 1500 phosphorimager.
1L terrific broth  in a Fernbach flask was inoculated with an overnight culture of E. coli C41 (DE3) harbouring pET16b-Pph. The cells were incubated at 18°C with gentle shaking for 48 hours. This procedure prevents the formation of inclusion bodies . Then the cells were harvested by centrifugation and resuspended in 20 mM Tris pH 7.4, 40 mM NaCl, 20% glycerol. The cells were lysed by 3 passages through a French Press and the cell debris was removed by centrifugation. The filtered crude protein extract was applied on a Sephadex G-200 gelfiltration column (GE Healthcare) and separated according to the manufacturer's manual. The resulting fractions were analyzed by SDS-PAGE and Western blotting with an antibody to strep tag II (IBA, Göttingen, Germany).
We thank Dr. Robin Ghosh for his generous support and scientific input and Dr. Birgit Scharf for critical reading of the manuscript.
- Favinger J, Stadtwald R, Gest H: Rhodospirillum centenum, sp. nov., a thermotolerant cyst-forming anoxygenic photosynthetic bacterium. Antonie van Leeuwenhoek. 1989, 55: 291-296. 10.1007/BF00393857.View ArticlePubMed
- Nickens D, Fry CJ, Ragatz L, Bauer CE, Gest H: Biotype of the nonsulfur purple photosynthetic bacterium Rhodospirillum centenum. Arch Microbiol. 1996, 165: 91-96. 10.1007/s002030050302.View Article
- Kawasaki H, Hoshino Y, Kuraishi H, Yamasato K: Rhodocista centenaria gen. nov., sp. nov., a cyst-forming anoxygenic photosynthetic bacterium and its phylogenetic position in the proteobacteria alpha group. J Gen Appl Microbiol. 1992, 38: 541-551. 10.2323/jgam.38.541.View Article
- Zhang D, Yang H, Zhang W, Huang Z, Liu SJ: Rhodocista pekingensis sp. nov., a cyst-forming phototrophic bacterium from a municipal wastewater treatment plant. Int J Syst Evol Microbiol. 2003, 53: 1111-1114. 10.1099/ijs.0.02500-0.View ArticlePubMed
- Do TT, Tran VN, Kleiner D: Physiological versatility of the genus Rhodocista. Z Naturforsch. 2007, 62c: 571-575.
- Stoffels M, Castellanos T, Hartmann A: Design and application of new 16S rRNA-targeted oligonucleotide probes for the Azospirillum-Skermanella-Rhodocista-cluster. Syst Appl Microbiol. 2001, 24: 83-97. 10.1078/0723-2020-00011.View ArticlePubMed
- Engelmann TW: Bacterium photometricum - Ein Beitrag zur vergleichenden Physiologie des Licht- und Farbsinnes. Arch Physiol. 1883, 30: 95-124. 10.1007/BF01674325.View Article
- Manten A: Phototaxis in the purple bacterium Rhodospirillum rubrum and the relation between phototaxis and photosynthesis. Antonie van Leeuwenhoek. 1948, 14: 65-86. 10.1007/BF02272681.View ArticlePubMed
- Ragatz L, Jiang ZY, Bauer CE, Gest H: Phototactic purple bacteria. Nature. 1994, 370: 104-10.1038/370104a0.View Article
- Ragatz L, Jiang ZY, Bauer CE, Gest H: Macroscopic phototactic behaviour of the purple photosynthetic bacterium Rhodospirillum centenum. Arch Microbiol. 1995, 163: 1-6. 10.1007/BF00262196.View ArticlePubMed
- McClain J, Rollo DR, Rushing BG, Bauer CE: Rhodospirillum centenum utilizes separate motor and switch components to control lateral and polar flagellum rotation. J Bacteriol. 2002, 184: 2429-2438. 10.1128/JB.184.9.2429-2438.2002.PubMed CentralView ArticlePubMed
- Jiang ZY, Bauer CE: Analysis of a chemotaxis operon from Rhodospirillum centenum. J Bacteriol. 1997, 179: 5712-5719.PubMed CentralPubMed
- Berleman JE, Bauer CE: Involvement of a che-like signal transduction cascade in regulating cyst cell development in Rhodospirillum centenum. Mol Microbiol. 2005, 56: 1457-1466. 10.1111/j.1365-2958.2005.04646.x.View ArticlePubMed
- Berleman JE, Bauer CE: A che-like signal transduction cascade involved in controlling flagella biosynthesis in Rhodospirillum centenum. Mol Microbiol. 2005, 55: 1390-1402. 10.1111/j.1365-2958.2005.04489.x.View ArticlePubMed
- Lu YK, Marden J, Swingley WD, Mastrian SD, Chowdhury SR, Hao J, Helmy T, Kim S, Kurdoglu A, Matthies H, Rollo D, Stothard P, Blankenship RE, Bauer CE, Touchman JW: Metabolic flexibility revealed in the genome of the cyst-forming α-1 proteobacterium Rhodospirillum centenum. BMC Genomics. 2010, 11: 325-10.1186/1471-2164-11-325.PubMed CentralView ArticlePubMed
- Gegner JA, Graham DR, Roth AF, Dahlquist FW: Assembly of an MCP receptor, CheW, and kinase CheA complex in the bacterial chemotaxis signal transduction pathway. Cell. 1992, 70: 975-982. 10.1016/0092-8674(92)90247-A.View ArticlePubMed
- Jiang ZY, Gest H, Bauer CE: Chemosensory and photosensory perception in purple photosynthetic bacteria utilize common signal transduction components. J Bacteriol. 1997, 179: 5720-5727.PubMed CentralPubMed
- Foynes S, Dorrell S, Ward SJ, Stabler RA, McColm AA, Rycroft AN, Wren BW: Helicobacter pylori possesses two CheY response regulators and a histidine kinase sensor, CheA, which are essential for chemotaxis and colonization of the gastric mucosa. Infect Immun. 2000, 68: 2016-2023. 10.1128/IAI.68.4.2016-2023.2000.PubMed CentralView ArticlePubMed
- Jiang ZY, Rushing BG, Bai Y, Gest H, Bauer CE: Isolation of Rhodospirillum centenum mutants defective in phototactic colony motility by transposon mutagenesis. J Bacteriol. 1998, 180: 1248-1255.PubMed CentralPubMed
- van der Horst MA, Laan W, Yeremenko S, Wende A, Palm P, Oesterhelt D, Hellingwerf KJ: From primary photochemistry to biological function in the blue-light photoreceptors PYP and AppA. Photochem Photobiol Sci. 2005, 4: 688-693. 10.1039/b418442b.View ArticlePubMed
- Imamoto Y, Kataoka M: Structure and photoreaction of photoactive yellow protein, a structural prototype of the PAS domain superfamily. Photochem Photobiol. 2007, 83: 40-49. 10.1562/2006-02-28-IR-827.View ArticlePubMed
- Jiang ZY, Swem LR, Rushing BG, Devanathan S, Tollin G, Bauer CE: Bacterial photoreceptor with similarity to photoactive yellow protein and plant phytochromes. Science. 1999, 285: 406-409. 10.1126/science.285.5426.406.View ArticlePubMed
- Sanders DA, Mendez B, Koshland D: Role of the CheW protein in bacterial chemotaxis: overexpression is equivalent to absence. J Bacteriol. 1989, 171: 6271-6278.PubMed CentralPubMed
- Studdert CA, Parkinson JS: Insights into the organization and dynamics of bacterial chemoreceptor clusters through in vivo crosslinking studies. Proc Natl Acad Sci USA. 2005, 102: 15623-15628. 10.1073/pnas.0506040102.PubMed CentralView ArticlePubMed
- Conley PM, Wolfe AJ, Blair DF, Berg HC: Both CheA and CheW are required for reconstitution of chemotactic signaling in Escherichia coli. J Bacteriol. 1989, 171: 5190-5193.PubMed CentralPubMed
- Liu JD, Parkinson JS: Role of CheW protein in coupling membrane receptors to the intracellular signaling system of bacterial chemotaxis. Proc Natl Acad Sci USA. 1989, 86: 8703-8707. 10.1073/pnas.86.22.8703.PubMed CentralView ArticlePubMed
- Zhang P, Khursigara CM, Hartnell LM, Subramaniam S: Direct visualization of Escherichia coli chemotaxis receptor arrays using cryo-electron microsopy. Proc Natl Acad Sci USA. 2007, 104: 3777-3781. 10.1073/pnas.0610106104.PubMed CentralView ArticlePubMed
- Cardozo MJ, Massazza DA, Parkinson JS, Studdert CA: Disruption of chemoreceptor signaling arrays by high level of CheW, the receptor-kinase coupling protein. Mol Microbiol. 2010, 75: 1171-1181. 10.1111/j.1365-2958.2009.07032.x.View ArticlePubMed
- Kim D, Forst S: Genomic analysis of the histidine kinase family in bacteria and archaea. Microbiology. 2001, 147: 1197-1212.View ArticlePubMed
- Palleroni NJ: Chamber for bacterial chemotaxis experiments. Appl Environ Microbiol. 1976, 32: 729-730.PubMed CentralPubMed
- Gegner JA, Dahlquist FW: Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA. Proc Natl Acad Sci USA. 1991, 88: 750-754. 10.1073/pnas.88.3.750.PubMed CentralView ArticlePubMed
- Francis NR, Wolanin PM, Stock JB, DeRosier DJ, Thomas DR: Three-dimensional structure and organization of a receptor/signaling complex. Proc Natl Acad Sci USA. 2004, 101: 17480-17485. 10.1073/pnas.0407826101.PubMed CentralView ArticlePubMed
- Li M, Hazelbauer GL: Cellular stoichiometry of the components of the chemotaxis signaling complex. J Bacteriol. 2004, 186: 3687-3694. 10.1128/JB.186.12.3687-3694.2004.PubMed CentralView ArticlePubMed
- Kentner D, Thiem S, Hildenbeutel M, Sourjik V: Determinants of chemoreceptor cluster formation in Escherichia coli. Mol Microbiol. 2006, 61: 407-417. 10.1111/j.1365-2958.2006.05250.x.View ArticlePubMed
- Baker MD, Wolanin PM, Stock JB: Signal transduction in bacterial chemotaxis. Bioessays. 2006, 28: 9-22. 10.1002/bies.20343.View ArticlePubMed
- Kyndt JA, Fitch JC, Meyer TE, Cusanovich MA: The photoactivated PYP domain of Rhodospirillum centenum Ppr accelerates the recovery of the bacteriophytochrome domain after white light illumination. Biochemistry. 2007, 46: 8256-8262. 10.1021/bi700616j.View ArticlePubMed
- Chung YH, Masuda S, Bauer CE: Purification and reconstitution of PYP-phytochrome with biliverdin and 4-hydroxycinnamic acid. Methods Enzymol. 2007, 422: 184-189. full_text.PubMed CentralView ArticlePubMed
- Hennecke H, Günther I, Binder F: A novel cloning vector for the direct selection of recombinant DNA in E. coli. Gene. 1982, 19: 231-234. 10.1016/0378-1119(82)90011-7.View ArticlePubMed
- Falciatore A, Bowler C: The evolution and function of blue and red light photoreceptors. Curr Top Dev Biol. 2005, 68: 317-350. 10.1016/S0070-2153(05)68011-8.View ArticlePubMed
- Genick UK, Borgstahl GE, Ng K, Ren Z, Pradervand C, Burke PM, Srajer V, Teng TY, Schildkamp W, McRee DE, Moffat K, Getzoff ED: Structure of a protein photocycle intermediate by millisecond time-resolved crystallography. Science. 1997, 275: 1471-1475. 10.1126/science.275.5305.1471.View ArticlePubMed
- Hughes J, Lamparter T, Mittmann F, Hartmann E, Gärtner W, Wilde A, Börner T: A prokaryotic phytochrome. Nature. 1997, 386: 663-10.1038/386663a0.View ArticlePubMed
- Wilde A, Fiedler B, Börner T: The cyanobacterial phytochrome Cph2 inhibits phototaxis towards blue light. Mol Microbiol. 2002, 44: 981-988. 10.1046/j.1365-2958.2002.02923.x.View ArticlePubMed
- Ng WO, Grossman AR, Bhaya DJ: Multiple light inputs control phototaxis in Synechocystis sp. strain PCC6803. J Bacteriol. 2003, 185: 1599-1607. 10.1128/JB.185.5.1599-1607.2003.PubMed CentralView ArticlePubMed
- Sourjik V, Schmitt R: Phosphotransfer between CheA, CheY1, and CheY2 in the chemotaxis signal transduction chain of Rhizobium meliloti. Biochemistry. 1998, 37: 2327-2335. 10.1021/bi972330a.View ArticlePubMed
- Jiménez-Pearson MA, Delany I, Scarlato V, Beier D: Phosphate flow in the chemotactic response system of Helicobacter pylori. Microbiology. 2005, 151: 3299-3311. 10.1099/mic.0.28217-0.View ArticlePubMed
- Li ZH, Dong K, Sun JC, Yuan JP, Hu BY, Liu JX, Zhao GP, Guo XK: Characterization of cheW genes of Leptospira interrogans and their effects in Escherichia coli. Acta Biochim Biophys Sin. 2007, 38: 79-88. 10.1111/j.1745-7270.2006.00137.x.View Article
- Li Y, Hu Y, Fu W, Xia B, Jin C: Solution structure of the bacterial chemotaxis adaptor protein CheW from Escherichia coli. Biochem Biophys Res Commun. 2007, 360: 863-867. 10.1016/j.bbrc.2007.06.146.View ArticlePubMed
- Porter SL, Warren AV, Martin AC, Armitage JP: The third chemotaxis locus of Rhodobacter sphaeroides is essential for chemotaxis. Mol Microbiol. 2002, 46: 1081-1094. 10.1046/j.1365-2958.2002.03218.x.View ArticlePubMed
- Stock AM, Robinson VL, Goudreau PN: Two-component signal transduction. Ann Rev Biochem. 2000, 69: 183-215. 10.1146/annurev.biochem.69.1.183.View ArticlePubMed
- Jiang ZY, Bauer CE: Component of the Rhodospirillum centenum photosensory apparatus with structural and functional similarity to methyl-accepting chemotaxis protein receptors. J Bacteriol. 2001, 183: 171-177. 10.1128/JB.183.1.171-177.2001.PubMed CentralView ArticlePubMed
- Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. 1982, Cold Spring Harbor: Cold Spring Harbor Press
- Miroux B, Walker JE: Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol. 1996, 260: 289-298. 10.1006/jmbi.1996.0399.View ArticlePubMed
- Abouhamad WN, Manson M, Gibson MM, Higgins CF: Peptide transport and chemotaxis in Escherichia coli and Salmonella typhimurium: characterization of the dipeptide permease (Dpp) and the dipeptide-binding protein. Mol Microbiol. 1991, 5: 1035-1047. 10.1111/j.1365-2958.1991.tb01876.x.View ArticlePubMed
- Tabor S, Richardson CC: A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad SciUSA. 1985, 82: 1074-1078. 10.1073/pnas.82.4.1074.View Article
- Guzman LM, Belin D, Carson MJ, Beckwith J: Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter. J Bacteriol. 1995, 177: 4121-4130.PubMed CentralPubMed
- Cold Spring Harbor Laboratory, Miller J: Experiments in Molecular Genetics. 1972, Cold Spring Harbor Laboratory
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685. 10.1038/227680a0.View ArticlePubMed
- Figurski DH, Helinski DR: Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA. 1979, 76: 1648-1652. 10.1073/pnas.76.4.1648.PubMed CentralView ArticlePubMed
- Willetts N: Conjugation. Methods Microbiol. 1984, 17: 33-59. full_text.View Article
- Kiefer D, Hu X, Dalbey R, Kuhn A: Negatively charged amino acid residues play an active role in orienting the Sec-independent Pf3 coat protein in the Escherichia coli inner membrane. EMBO J. 1997, 16: 2197-2204. 10.1093/emboj/16.9.2197.PubMed CentralView ArticlePubMed
- T D, Kuhn A: Hydrophobic forces drive spontaneous membrane insertion of the bacteriophage Pf3 coat protein without topological control. EMBO J. 1999, 18: 6299-6306. 10.1093/emboj/18.22.6299.View Article
- Tartoff KD, Hobbs CA: Improved media for growing plasmid and cosmid clones. Bethesda Res Lab Focus. 1987, 9: 12-
- Schkölziger S: Klonierung und Expression des ppr-Gens aus Rhodospirillum centenum. Diploma-thesis. 2000, University of Hohenheim, Institute of Microbiology