The Histidine Kinase CckA Is Directly Inhibited by a Response Regulator-like Protein in a Negative Feedback Loop.

In alphaproteobacteria, the two-component system (TCS) formed by the hybrid histidine kinase CckA, the phosphotransfer protein ChpT, and the response regulator CtrA is widely distributed. In these microorganisms, this system controls diverse functions such as motility, DNA repair, and cell division. In Caulobacterales and Rhizobiales, CckA is regulated by the pseudo- histidine kinase DivL, and the response regulator DivK. However, this regulatory circuit differs for other bacterial groups. For instance, in Rhodobacterales, DivK is absent and DivL consists of only the regulatory PAS domain. In this study, we report that, in Rhodobacter sphaeroides, the kinase activity of CckA is inhibited by Osp, a single domain response regulator (SDRR) protein that directly interacts with the transmitter domain of CckA. In vitro, the kinase activity of CckA was severely inhibited with an equimolar amount of Osp, whereas the phosphatase activity of CckA was not affected. We also found that the expression of osp is activated by CtrA creating a negative feedback loop. However, under growth conditions known to activate the TCS, the increased expression of osp does not parallel Osp accumulation, indicating a complex regulation. Phylogenetic analysis of selected species of Rhodobacterales revealed that Osp is widely distributed in several genera. For most of these species, we found a sequence highly similar to the CtrA-binding site in the control region of osp, suggesting that the TCS CckA/ChpT/CtrA is controlled by a novel regulatory circuit that includes Osp in these bacteria. IMPORTANCE The two-component systems (TCS) in bacteria in its simplest architecture consist of a histidine kinase (HK) and a response regulator (RR). In response to a specific stimulus, the HK is activated and drives phosphorylation of the RR, which is responsible of generating an adaptive response. These systems are ubiquitous among bacteria and are frequently controlled by accessory proteins. In alphaproteobacteria, the TCS formed by the HK CckA, the phosphotransferase ChpT, and the RR CtrA is widely distributed. Currently, most of the information of this system and its regulatory proteins comes from findings carried out in microorganisms where it is essential. However, this is not the case in many species, and studies of this TCS and its regulatory proteins are lacking. In this study, we found that Osp, a RR-like protein, inhibits the kinase activity of CckA in a negative feedback loop since osp expression is activated by CtrA. The inhibitory role of Osp and the similar action of the previously reported FixT protein, suggests the existence of a new group of RR-like proteins whose main function is to interact with the HK and prevent its phosphorylation.

The periplasmic component of the DctPQM TRAP-transporter is part of the DctS/DctR sensory pathway in Rhodobacter sphaeroides.

Rhodobacter sphaeroides can use C4-dicarboxylic acids to grow heterotrophically or photoheterotropically, and it was previously demonstrated in Rhodobacter capsulatus that the DctPQM transporter system is essential to support growth using these organic acids under heterotrophic but not under photoheterotrophic conditions. In this work we show that in R. sphaeroides this transporter system is essential for photoheterotrophic and heterotrophic growth, when C4-dicarboxylic acids are used as a carbon source. We also found that over-expression of dctPQM is detrimental for photoheterotrophic growth in the presence of succinic acid in the culture medium. In agreement with this, we observed a reduction of the dctPQM promoter activity in cells growing under these conditions, indicating that the amount of DctPQM needs to be reduced under photoheterotrophic growth. It has been reported that the two-component system DctS and DctR activates the expression of dctPQM. Our results demonstrate that in the absence of DctR, dctPQM is still expressed albeit at a low level. In this work, we have found that the periplasmic component of the transporter system, DctP, has a role in both transport and in signalling the DctS/DctR two-component system.

Characterization of FlgP, an Essential Protein for Flagellar Assembly in Rhodobacter sphaeroides.

The flagellar lipoprotein FlgP has been identified in several species of bacteria, and its absence provokes different phenotypes. In this study, we show that in the alphaproteobacterium Rhodobacter sphaeroides, a ΔflgP mutant is unable to assemble the hook and the filament. In contrast, the membrane/supramembrane (MS) ring and the flagellar rod appear to be assembled. In the absence of FlgP a severe defect in the transition from rod to hook polymerization occurs. In agreement with this idea, we noticed a reduction in the amount of intracellular flagellin and the chemotactic protein CheY4, both encoded by genes dependent on σ28 This suggests that in the absence of flgP the switch to export the anti-sigma factor, FlgM, does not occur. The presence of FlgP was detected by Western blot in samples of isolated wild-type filament basal bodies, indicating that FlgP is an integral part of the flagellar structure. In this regard, we show that FlgP interacts with FlgH and FlgT, indicating that FlgP should be localized closely to the L and H rings. We propose that FlgP could affect the architecture of the L ring, which has been recently identified to be responsible for the rod-hook transition.IMPORTANCE Flagellar based motility confers a selective advantage on bacteria by allowing migration to favorable environments or in pathogenic species to reach the optimal niche for colonization. The flagellar structure has been well established in Salmonella However, other accessory components have been identified in other species. Many of these have been implied in adapting the flagellar function to enable faster rotation, or higher torque. FlgP has been proposed to be the main component of the basal disk located underlying the outer membrane in Campylobacter jejuni and Vibrio fischeri Its role is still unclear, and its absence impacts motility differently in different species. The study of these new components will bring a better understanding of the evolution of this complex organelle.

The Master Regulators of the Fla1 and Fla2 Flagella of Rhodobacter sphaeroides Control the Expression of Their Cognate CheY Proteins.

Rhodobacter sphaeroides is an alphaproteobacterium that has two complete sets of flagellar genes. The fla1 set was acquired by horizontal transfer from an ancestral gammaproteobacterium and is the only set of flagellar genes that is expressed during growth under standard laboratory conditions. The products of these genes assemble a single, subpolar flagellum. In the absence of the Fla1 flagellum, a gain-of-function mutation in the histidine kinase CckA turns on the expression of the fla2 flagellar genes through the response regulator CtrA. The rotation of the Fla1 and Fla2 flagella is controlled by different sets of chemotaxis proteins. Here, we show that the expression of the chemotaxis proteins that control Fla2, along with the expression of the fla2 genes, is coordinated by CtrA, whereas the expression of the chemotaxis genes that control Fla1 is mediated by the master regulators of the Fla1 system. The coordinated expression of the chemosensory proteins with their cognate flagellar genes highlights the relevance of integrating the expression of the horizontally acquired fla1 genes with a chemosensory system independently of the regulatory proteins responsible for the expression of fla2 and its cognate chemosensory system. IMPORTANCE Gene acquisition via horizontal transfer represents a challenge to the recipient organism to adjust its metabolic and genetic networks to incorporate the new material in a way that represents an adaptive advantage. In the case of Rhodobacter sphaeroides, a complete set of flagellar genes was acquired and successfully coordinated with the native flagellar system. Here we show that the expression of the chemosensory proteins that control flagellar rotation is dependent on the master regulators of their corresponding flagellar system, minimizing the use of transcription factors required to express the native and horizontally acquired genes along with their chemotaxis proteins.

The flagellar set Fla2 in Rhodobacter sphaeroides is controlled by the CckA pathway and is repressed by organic acids and the expression of Fla1.

Rhodobacter sphaeroides has two different sets of flagellar genes. Under the growth conditions commonly used in the laboratory, the expression of the fla1 set is constitutive, whereas the fla2 genes are not expressed. Phylogenetic analyses have previously shown that the fla1 genes were acquired by horizontal transfer from a gammaproteobacterium and that the fla2 genes are endogenous genes of this alphaproteobacterium. In this work, we characterized a set of mutants that were selected for swimming using the Fla2 flagella in the absence of the Fla1 flagellum (Fla2(+) strains). We determined that these strains have a single missense mutation in the histidine kinase domain of CckA. The expression of these mutant alleles in a Fla1(-) strain allowed fla2-dependent motility without selection. Motility of the Fla2(+) strains is also dependent on ChpT and CtrA. The mutant versions of CckA showed an increased autophosphorylation activity in vitro. Interestingly, we found that cckA is transcriptionally repressed by the presence of organic acids, suggesting that the availability of carbon sources could be a part of the signal that turns on this flagellar set. Evidence is presented showing that reactivation of fla1 gene expression in the Fla2(+) background strongly reduces the number of cells with Fla2 flagella.

A distant homologue of the FlgT protein interacts with MotB and FliL and is essential for flagellar rotation in Rhodobacter sphaeroides.

In this work, we describe a periplasmic protein that is essential for flagellar rotation in Rhodobacter sphaeroides. This protein is encoded upstream of flgA, and its expression is dependent on the flagellar master regulator FleQ and on the class III flagellar activator FleT. Sequence comparisons suggest that this protein is a distant homologue of FlgT. We show evidence that in R. sphaeroides, FlgT interacts with the periplasmic regions of MotB and FliL and with the flagellar protein MotF, which was recently characterized as a membrane component of the flagellum in this bacterium. In addition, the localization of green fluorescent protein (GFP)-MotF is completely dependent on FlgT. The Mot(-) phenotype of flgT cells was weakly suppressed by point mutants of MotB that presumably keep the proton channel open and efficiently suppress the Mot(-) phenotype of motF and fliL cells, indicating that FlgT could play an additional role beyond the opening of the proton channel. The presence of FlgT in purified filament-hook-basal bodies of the wild-type strain was confirmed by Western blotting, and the observation of these structures under an electron microscope showed that the basal bodies from flgT cells had lost the ring that covers the LP ring in the wild-type structure. Moreover, MotF was detected by immunoblotting in the basal bodies obtained from the wild-type strain but not from flgT cells. From these results, we suggest that FlgT forms a ring around the LP ring, which anchors MotF and stabilizes the stator complex of the flagellar motor.

A novel component of the Rhodobacter sphaeroides Fla1 flagellum is essential for motor rotation.

Here we describe a novel component essential for flagellar rotation in Rhodobacter sphaeroides. This protein is encoded by motF (RSP_0067), the first gene of a predicted transcriptional unit which contains two hypothetical genes. Sequence analysis indicated that MotF is a bitopic membrane-spanning protein. Protease sensitivity assays and green fluorescent protein (GFP) fusions confirmed this prediction and allowed us to conclude that the C terminus of MotF is located in the periplasmic space. Wild-type cells expressing a functional GFP-MotF fusion show a single fluorescent focus per cell. The localization of this protein in different genetic backgrounds allowed us to determine that normal localization of MotF depends on the presence of FliL and MotB. Characterization of a ΔmotF pseudorevertant strain revealed that a single nucleotide change in motB suppresses the Mot(-) phenotype of the motF mutant. Additionally, we show that MotF also becomes dispensable when other mutant alleles of motB previously isolated as second-site suppressors of ΔfliL were expressed in the motF mutant strain. These results show that MotF is a new component of the Fla1 flagellum, which together with FliL is required to promote flagellar rotation, possibly through MotB.

Evolutionary origin of the Rhodobacter sphaeroides specialized RpoN sigma factors.

Gene duplication and horizontal gene transfer (HGT) are two events that enable the generation of new genes. Rhodobacter sphaeroides (WS8 and 2.4.1 strains) has four copies of the rpoN gene that are not functionally interchangeable. Until now, this is the only example of specialization of this sigma factor. In this work, we aimed to determine whether the multiple copies of this gene originated from HGT or through gene duplication. Our results suggest a multiplication origin of the different rpoN copies that occurred after the Rhodobacter clade separated. Functional tests indicate that the specialization of the rpoN genes is not restricted to R. sphaeroides. We propose that the rpoN copy involved in nitrogen fixation is the ancestral gene and that the other rpoN genes have acquired new specificities.

The flagellar protein FliL is essential for swimming in Rhodobacter sphaeroides.

In this work we characterize the function of the flagellar protein FliL in Rhodobacter sphaeroides. Our results show that FliL is essential for motility in this bacterium and that in its absence flagellar rotation is highly impaired. A green fluorescent protein (GFP)-FliL fusion forms polar and lateral fluorescent foci that show different spatial dynamics. The presence of these foci is dependent on the expression of the flagellar genes controlled by the master regulator FleQ, suggesting that additional components of the flagellar regulon are required for the proper localization of GFP-FliL. Eight independent pseudorevertants were isolated from the fliL mutant strain. In each of these strains a single nucleotide change in motB was identified. The eight mutations affected only three residues located on the periplasmic side of MotB. Swimming of the suppressor mutants was not affected by the presence of the wild-type fliL allele. Pulldown and yeast two-hybrid assays showed that that the periplasmic domain of FliL is able to interact with itself but not with the periplasmic domain of MotB. From these results we propose that FliL could participate in the coupling of MotB with the flagellar rotor in an indirect fashion.

Altered expression of versican and hyaluronan in melanocytic tumors of dogs.

OBJECTIVE: To analyze the expression of versican and hyaluronan in melanocytomas and malignant melanomas of dogs, to correlate their expression with expression of the hyaluronan receptor CD44, and to identify enzymes responsible for the synthesis and degradation of hyaluronan in canine dermal fibroblasts and canine melanoma cell lines. SAMPLE POPULATION: 35 biopsy specimens from melanocytic tumors of dogs, canine primary dermal fibroblasts, and 3 canine melanoma cell lines. PROCEDURES: Versican, hyaluronan, and CD44 were detected in tumor samples by use of histochemical or immunohistochemical methods. Expression of hyaluronan-metabolizing enzymes was analyzed with a reverse transcriptase-PCR assay. RESULTS: Versican was found only in some hair follicles and around some blood vessels in normal canine skin, whereas hyaluronan was primarily found within the dermis. Hyaluronan was found in connective tissue of the oral mucosa. Versican and, to a lesser extent, hyaluronan were significantly overexpressed in malignant melanomas, compared with expression in melanocytomas. No significant difference was found between malignant tumors from oral or cutaneous origin. The expression of both molecules was correlated, but hyaluronan had a more extensive distribution than versican. Versican and hyaluronan were mainly associated with tumor stroma. Canine fibroblasts and melanoma cell lines expressed hyaluronan synthase 2 and 3 (but not 1) and hyaluronidase 1 and 2. CONCLUSIONS AND CLINICAL RELEVANCE: Versican may be useful as a diagnostic marker for melanocytic tumors in dogs. Knowledge of the enzymes involved in hyaluronan metabolism could reveal new potential therapeutic targets.

V3 versican isoform expression has a dual role in human melanoma tumor growth and metastasis.

Versican is a large chondroitin sulfate proteoglycan produced by several tumor cell types, including malignant melanoma, which exists as four different splice variants. The presence of versican in the extracellular matrix plays a role in tumor cell growth, adhesion and migration, which could be altered by altering the ratio between versican isoforms. We have previously shown that overexpression of the V3 isoform of versican in human melanoma cell lines markedly reduces cell growth in vitro and in vivo, since V3-overexpressing (LV3SN) cultured cells as well as primary tumors arising from these cells grow slower than their vector-only counterparts (LXSN). In the present work, we have extended these observations to demonstrate that the delayed cell growth is due to multiple events since differences in proliferative index as well as in apoptosis are observed in LV3SN cells and tumors compared to LXSN. For example, LV3SN melanoma cells exhibit delayed activation of MAPK in response to EGF, we have also characterized further the primary tumors originated in nude mice from V3-transduced melanoma cells to determine if other events affect the V3 tumor phenotype. For example, hyaluronan content of LV3SN tumors was higher than in LXSN tumors, whereas other related matrix components and vascularization were unaffected. Furthermore, lung metastasis in nude mice occurred only in animals carrying LV3SN tumors, indicating a dual role for this molecule, both as an inhibitor of tumor growth and a metastasis inductor.

V3 versican isoform expression alters the phenotype of melanoma cells and their tumorigenic potential.

Versican is a large chondroitin sulfate proteoglycan produced by several tumor cell types, including malignant melanoma. The expression of increased amounts of versican in the extracellular matrix may play a role in tumor cell growth, adhesion and migration. We have expressed the V3 isoform of versican in human and canine melanoma cell lines. Retroviral overexpression of V3 did not change the morphology of any of the cell lines but markedly reduces cell growth in the V3 versican expressing melanoma cells. The V3-overexpressing melanoma cells retain their diminished growth potential in vivo because primary tumors arising from these cell lines growth more slowly than their vector only counterparts. This effect was accompanied by increases in cell adhesion on hyaluronan and an enhanced ability to migrate on hyaluronan-coated transwell chambers. This enhanced migration is blocked when cells are preincubated with soluble hyaluronan, or anti-CD44 antibodies, suggesting that V3 acts by altering the hyaluronan-CD44 interaction. Hyaluronan content and CD44 expression are not altered in V3-overexpressing cells compared to vector-transduced cells. Our results show that V3 overproduction modulates the in vitro behavior of human and canine melanoma cell lines and reduces their tumorigenicity in vivo.

Differential expression of versican isoforms is a component of the human melanoma cell differentiation process.

Versican is a large chondroitin sulfate proteoglycan produced by human melanoma cell lines and malignant melanocytic lesions. In the present work, we have analyzed the expression of versican spliced variants V0, V1, V2 and V3 in human melanoma cell lines at several differentiation degrees. The isoform expression pattern depends on the degree of cell differentiation. Differentiated cell lines do not produce any of the versican isoforms as analyzed by Western blot, Northern blot and RT-PCR. All cell lines with an early or intermediate degree of differentiation (AX3, SK-mel-37, Rider, SK-mel-1.36-1-5 and SK-mel-3.44) expressed V0 and V1 transcripts, whereas V2 and V3 expression was shown only by the undifferentiated cell lines SK-mel-1.36-1-5 and Rider. Furthermore, we have analyzed the expression of versican isoforms in SK-mel-3.44 and SK-mel-1.36-1-5 cells induced to differentiate by TPA treatment. The expression of the large V0, V1 and V2 isoforms practically disappears in differentiated cells, whereas V3 remains detectable by RT-PCR analysis.

Effect of transforming growth factor-beta1, insulin-like growth factor-I, and hepatocyte growth factor on proteoglycan production and regulation in canine melanoma cell lines.

OBJECTIVE: To identify extracellular proteoglycans produced by canine melanoma cell lines and analyze the effect of transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor-I (IGF-I), and hepatocyte growth factor (HGF) on these proteoglycans. SAMPLE POPULATION: 3 canine melanoma cell lines (ie, CML-1, CML-6M, and CML-10c2). PROCEDURE: Extracellular proteoglycans were analyzed by use of metabolic labeling and western immunoblot analysis. The effect of TGF-beta1 on cell proliferation was determined by incorporation of 5-bromo-2'-deoxyuridine. RESULTS: The CML-1 and CML-6M melanoma cell lines produced 2 main extracellular proteoglycans. One of them was identified as versican, a proteoglycan found in undifferentiated human melanoma cell lines. The CML-10c2 cells produced a small amount of extracellular proteoglycans. Addition of TGF-beta1 (1.25 to 6.25 ng/ml) increased the release of sulfated proteoglycans into the medium. The TGF-beta1 had mainly a posttranslational effect, because it increased the molecular mass of the sulfated bands. Addition of IGF-I (50 ng/ml) slightly increased production of proteoglycans in the CML-6M cell line, whereas HGF (50 ng/ml) did not have any effect on proteoglycan production. CONCLUSIONS AND CLINICAL RELEVANCE: The proteoglycan content and response toTGF-beta1 treatment for CML-1 and CML-6M canine melanoma cell lines are similar to that for undifferentiated human melanoma cell lines. In contrast, CML-10c2 cells produced a low amount of proteoglycans with high molecular weight. Because these extracellular proteoglycans are involved in the control of cell adhesion, proliferation, and migration, they may play an important role in the progression of melanomas in dogs.