ST6GAL1 inhibits metastasis of hepatocellular carcinoma via modulating sialylation of MCAM on cell surface.

The poor prognosis of hepatocellular carcinoma (HCC) is mainly because of its high rate of metastasis. Thus, elucidation of the molecular mechanisms underlying HCC metastasis is of great significance. Glycosylation is an important post-translational modification that is closely associated with tumor progression. Altered glycosylation including the altered sialylation resulting from aberrant expression of ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1) has long been considered as an important feature of cancer cells. However, there is limited information on the roles of ST6GAL1 and α2,6 sialylation in HCC metastasis. Here, we found that ST6GAL1 and α2,6 sialylation were negatively correlated with the metastatic potentials of HCC cells. Moreover, ST6GAL1 overexpression inhibited migration and invasion of HCC cells in vitro and suppressed HCC metastasis in vivo. Using a metabolic labeling-based glycoproteomic strategy, we identified a list of sialylated proteins that may be regulated by ST6GAL1. In particular, an increase in α2,6 sialylation of melanoma cell adhesion molecule (MCAM) inhibited its interaction with galectin-3 and decreased its expression on cell surface. In vitro and in vivo analysis showed that ST6GAL1 exerted its function in HCC metastasis by regulating MCAM expression. Finally, we found the relative intensity of sialylated MCAM was negatively correlated with tumor malignancy in HCC patients. Taken together, these results demonstrate that ST6GAL1 may be an HCC metastasis suppressor by affecting sialylation of MCAM on cell surface, which provides a novel insight into the roles of ST6GAL1 in HCC progression and supports the functional complexity of ST6GAL1 in a cancer type- and tissue type-specific manner.

The ß-galactoside α2,6-sialyltranferase 1 (ST6GAL1) inhibits the colorectal cancer metastasis by stabilizing intercellular adhesion molecule-1 via sialylation.

Background: Colorectal cancer (CRC) is one of the most frequent malignancies of the digestive system. Elevated expression of ß-galactoside α2,6-sialyltranferase 1 (ST6GAL1) has been observed in multiple cancers. But the mechanism of how ST6GAL1 might affect cancer cells remains to be clarified. Our previous study recognized intercellular adhesion molecule-1(ICAM-1) as a probable substrate of ST6GAL1 through mass spectrometry (MS) analysis. ICAM-1 is related to tumor metastasis in various cancers. Methods: First, ST6GAL1 was overexpressed and knocked down to perform transwell and wound healing assays, and the results were further confirmed in vivo. Based on the results of MS, GO and KEGG analysis were applied to reveal the connection between ST6GAL1 and ICAM-1. Immunoblot and tissue microarrays were administered to investigate the expression of ICAM-1 in different stages of CRC. Next, PCR, lectin precipitation and cycloheximide (CHX) were used to demonstrate the mechanism of ST6GAL1 on ICAM-1. Moreover, we investigated the sialylation on soluble ICAM in serum and its connection to tumor staging. Results: Overexpression of ST6GAL1 inhibited the migratory ability, while knockdown of ST6GAL1 cells had the reverse effect. Moreover, nude mice injected with ST6GAL1-knockdown cells harvested more liver metastases. Based on the GO and KEGG analysis, data from TCGA database showed a positive correlation between ST6GAL1 and ICAM-1. ICAM-1 also demonstrated a significant decrease in stage III/IV compared with stage I/II tumors. Our results revealed that ST6GAL1 could increase the stability of ICAM-1 through sialylation but had little influence on transcriptional level. Additionally, results of serum lectin precipitation revealed a correlation between the level of sialylation on soluble ICAM and CRC staging. Conclusion: This study illustrated that ST6GAL1 inhibited the metastatic ability of CRC by stabilizing ICAM-1 via sialylation and demonstrated a correlation between CRC staging and the sialylation on soluble ICAM-1 in serum.

Polypeptide N-acetylgalactosaminyltransferase 18 non-catalytically regulates the ER homeostasis and O-glycosylation.

Mucin-type O-glycosylation plays important roles in various biological processes. It is initiated by a family of 20 conserved UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). Unlike most ppGalNAc-Ts localized to the Golgi apparatus, ppGalNAc-T18 is predominantly distributed on the endoplasmic reticulum (ER) and exhibits no ppGalNAc-T catalytic activity in vitro. Herein, we found that ppGalNAc-T18 silencing in cells decreased O-glycosylation levels and activated ER stress leading to apoptosis. After treatment with chemical chaperone 4-phenylbutyric acid (PBA) or forced expression of ppGalNAc-T18 in the ppGalNAc-T18 knockdown cell, these defects could be significantly alleviated, suggesting that ppGalNAc-T18 is important for ER homeostasis and protein O-glycosylation. Furthermore, we found that ppGalNAc-T18 exerts its functions in O-glycosylation and ER stress via a non-catalytic mechanism. These results reveal a novel molecular role of ppGalNAc-Ts that the ER-localized ppGalNAc-T18 could regulate the O-glycosylation and ER homeostasis in a non-catalytic manner.

Synthesis of a Novel Fluorescent Ruthenium Complex with an Appended Ac4GlcNAc Moiety by Click Reaction.

The O-linked ß-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells, which plays a fundamental role in the activity of many cells and is associated with pathologies like type II diabetes, Alzheimer's disease or some cancers. However, the precise connexion between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Confocal microscopy is a powerful and effective tool for in-cell elucidation of the function of biological molecules. Chemical labeling of non-ultraviolet or non-fluorescent carbohydrates with fluorescent tag is an essential step that makes intra-cellular microscopic inspection possible. Here we report a strategy based on the 1,3-dipolar cycloaddition, called click chemistry, between unnatural N-acetylglucosamine (GlcNAc) analogues Ac4GlcNAc (substituted with an azido group) and the corresponding fluorescent tag Ru(bpy)2(Phen-alkyne)Cl2 (4) to synthesize the fluorescent dye Ru(bpy)2(Phen-Ac4GlcNAc)Cl2 (5) under mild and neutral reaction conditions. Moreover, 5 showed good stability, desirable fluorescence characteristics, and exhibited rather low levels of cytotoxicity against sensitive MCF-7 cells. Additionally, we have achieved successful fluorescent imaging of 5 transported in living MCF-7 cells. Cell images displayed that proteins are potentially labelled with 5 in the cytoplasm.

The polypeptide N-acetylgalactosaminyltransferase 4 exhibits stage-dependent expression in colorectal cancer and affects tumorigenesis, invasion and differentiation.

The aberrant expression of mucin-type O-glycosylation plays important roles in cancer malignancy. The polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) are a family of conserved enzymes that initiate the mucin-type O-glycosylation in cells. In human, consistent up- or down-regulation of ppGalNAc-Ts expression during cancer development has been frequently reported. Here, we provide evidence that ppGalNAc-T4 shows a stage-dependent expression at the different stages of colorectal cancer (CRC) in the 62 pair-matched tumor/normal tissues. In detail, ppGalNAc-T4 expression is significantly induced at stage I and II but not at stage III and IV. Overexpression of ppGalNAc-T4 in CRC cells enhances colony formation and sphere formation suggesting an important role of ppGalNAc-T4 in tumorigenesis. Conversely, knockdown of ppGalNAc-T4 in CRC cells increases the cell migration and invasion, and leads to an epithelial-mesenchymal transition-like transition. Further analysis suggests that loss of ppGalNAc-T4 contributes to the dedifferentiation of CRC and high expression of ppGalNAc-T4 correlates to a good prognosis of patients. Taken together, our results not only demonstrate a stage-dependent expression of ppGalNAc-T4 in CRC progression, but also suggest that such stage-dependent expression may contribute to the tumorigenesis at the early stage and promote cell migration and invasion at the advanced stage.

The small molecule luteolin inhibits N-acetyl-α-galactosaminyltransferases and reduces mucin-type O-glycosylation of amyloid precursor protein.

Mucin-type O-glycosylation is the most abundant type of O-glycosylation. It is initiated by the members of the polypeptide N-acetyl-α-galactosaminyltransferase (ppGalNAc-T) family and closely associated with both physiological and pathological conditions, such as coronary artery disease or Alzheimer's disease. The lack of direct and selective inhibitors of ppGalNAc-Ts has largely impeded research progress in understanding the molecular events in mucin-type O-glycosylation. Here, we report that a small molecule, the plant flavonoid luteolin, selectively inhibits ppGalNAc-Ts in vitro and in cells. We found that luteolin inhibits ppGalNAc-T2 in a peptide/protein-competitive manner but not promiscuously (e.g. via aggregation-based activity). X-ray structural analysis revealed that luteolin binds to the PXP motif-binding site found in most protein substrates, which was further validated by comparing the interactions of luteolin with wild-type enzyme and with mutants using 1H NMR-based binding experiments. Functional studies disclosed that luteolin at least partially reduced production of ß-amyloid protein by selectively inhibiting the activity of ppGalNAc-T isoforms. In conclusion, our study provides key structural and functional details on luteolin inhibiting ppGalNAc-T activity, opening up the way for further optimization of more potent and specific ppGalNAc-T inhibitors. Moreover, our findings may inform future investigations into site-specific O-GalNAc glycosylation and into the molecular mechanism of luteolin-mediated ppGalNAc-T inhibition.

Differential expression of ST6GAL1 in the tumor progression of colorectal cancer.

Elevated expression of ß-galactoside α2,6-sialyltranferase 1 (ST6GAL1) has been observed in colorectal cancer (CRC) and demonstrated to be important for its tumorigenesis. Here, we found that ST6GAL1 expression was significantly higher in non-metastatic tumors (stage I and II) than that in metastatic tumors (stage III and IV) using 62 pair-matched tumor/normal tissues. To elucidate the molecular mechanisms of how ST6GAL1 affected the CRC progression, we performed a global identification of the substrates of ST6GAL1 in the colon adenocarcinoma cell line SW480. A total of 318 membrane proteins were identified differentially affected by ST6GAL1 overexpression using metabolic labeling and proteomic analysis. Subsequent bioinformatic analysis revealed a list of potential substrates that might mediate the different functions of ST6GAL1 in CRC including cell movement, cell death and survival. Taken together, these results indicate a dynamic change in the expression of ST6GAL1 during the CRC progression and provide a list of sialylated proteins potentially relevant to the different functions of ST6GAL1 in CRC.

Polypeptide N-Acetylgalactosaminyltransferase 13 Contributes to Neurogenesis via Stabilizing the Mucin-type O-Glycoprotein Podoplanin.

Mucin-type O-glycosylation is initiated by an evolutionarily conserved family of polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). Previously, it was reported that ppGalNAc-T13 is restrictively expressed at a high level in the brain. Here we provide evidence for the critical role of ppGalNAc-T13 in neural differentiation. In detail, we show that the expression of ppGalNAc-T13 was dramatically up-regulated during early neurogenesis in mouse embryonic brains. Similar changes were also observed in cell models of neuronal differentiation by using either primary mouse cortical neural precursor cells or murine embryonal carcinoma P19 cells. Knockout of ppGalNAc-T13 in P19 cells suppressed not only neural induction but also neuronal differentiation. These effects are at least partly mediated by the mucin-type O-glycoprotein podoplanin (PDPN), as knockdown of PDPN led to a similar inhibition of neuronal differentiation and PDPN was significantly reduced at the posttranscriptional level after ppGalNAc-T13 knockout. Further data demonstrate that PDPN acts as a substrate of ppGalNAc-T13 and that the ppGalNAc-T13-mediated O-glycosylation on PDPN is important for its stability. Taken together, this study suggests that ppGalNAc-T13 contributes to neuronal differentiation through glycosylating and stabilizing PDPN, which provides insights into the regulatory roles of O-glycosylation in mammalian neural development.

Importance of membrane-proximal N-glycosylation on integrin ß1 in its activation and complex formation.

N-Glycosylation of integrin α5ß1 plays important roles in cell biologic functions; however, the mechanisms that underlie those roles remain poorly understood. Here, we present evidence that the membrane-proximal N-glycosylation on integrin ß1 could positively regulate cell migration by promoting ß1 activation. The S4-6 ß1 mutant contains only 3 N-glycosylation sites, which are essential for α5 and ß1 heterodimer formation, and despite only a small difference in expression levels of α5ß1 between wild-type and S4-6 mutant, cell spreading and migration of the S4-6 mutant was significantly decreased compared with that of control. Consistent with these phenotypes, ß1-mediated cellular signaling and its activation were clearly suppressed in the S4-6 mutant. Of note, these developments could be rescued by restoration of N-glycosylation sites in the membrane-proximal domain. Further study on the regulatory mechanisms suggested that membrane-proximal N-glycosylation is critical for intermolecular interactions between integrin ß1 and other cell membrane proteins, such as syndecan-4 and epidermal growth factor receptor. Moreover, α2,6-sialylation is required for ß1 activation. These data suggest a novel regulatory mechanism wherein N-glycosylation near the cell membrane on ß1 may serve as a platform that facilitates its complex formation on the cell membrane, thereby affecting integrin-mediated functions.-Hou, S., Hang, Q., Isaji, T., Lu, J., Fukuda, T., Gu, J. Importance of membrane-proximal N-glycosylation on integrin ß1 in its activation and complex formation.

Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation, and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels.

N-Acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of the bisecting GlcNAc branch on N-glycans, is usually described as a metastasis suppressor. Overexpression of GnT-III inhibited migration in multiple types of tumor cells. However, these results seem controversial to the clinical observations for the increased expression of GnT-III in human hepatomas, glioma, and ovarian cancers. Here, we present evidence that these inconsistencies are mainly attributed to the different expression pattern of cell sialylation. In detail, we show that overexpression of GnT-III significantly inhibits α2,3-sialylation but not α2,6-sialylation. The migratory ability of cells without or with a low level of α2,6-sialylation is consistently suppressed after GnT-III overexpression. In contrast, the effects of GnT-III overexpression are variable in tumor cells that are highly α2,6-sialylated. Overexpression of GnT-III promotes the cell migration in glioma cells U-251 and hepatoma cells HepG2, although it has little influence in human breast cancer cell MDA-MB-231 and gastric cancer cell MKN-45. Interestingly, up-regulation of α2,6-sialylation by overexpressing ß-galactoside α2,6-sialyltranferase 1 in the α2,6-hyposialylated HeLa-S3 cells abolishes the anti-migratory effects of GnT-III. Conversely, depletion of α2,6-sialylation by knock-out of ß-galactoside α2,6-sialyltranferase 1 in α2,6-hypersialylated HepG2 cells endows GnT-III with the anti-migratory ability. Taken together, our data clearly demonstrate that high expression of α2,6-sialylation on the cell surface could affect the anti-migratory role of GnT-III, which provides an insight into the mechanistic roles of GnT-III in tumor metastasis.

Significance of ß-Galactoside α2,6 Sialyltranferase 1 in Cancers.

Altered glycosylation is a common feature of cancer cells. It takes a variety of forms, which includes loss of expression or excessive expression of some structures, the accumulation of precursors, the appearance of novel structures, etc. Notably, these changes in glycan structure do not occur as a random consequence of disorder biology. Only a limited subset of oligosaccharides is found frequently enriched on the tumor cell surface and implicated in different tumor phenotypes. Among these, altered sialylation has long been associated with metastatic cell behaviors such as invasion and enhanced cell survival and accumulating evidence points to the alteration occurring in the sialic acid linkage to other sugars, which normally exists in three main configurations: α2,3, α2,6, and α2,8, catalyzed by a group of sialyltransferases. The aberrant expression of all three configurations has been described in cancer progression. However, the increased α2,6 sialylation catalyzed by ß-galactoside α2,6 sialyltranferase 1 (ST6Gal I) is frequently observed in many types of the cancers. In this review, we describe the findings on the role of ST6Gal I in cancer progression, and highlight in particular the knowledge of how ST6Gal I-mediated α2,6 sialylated glycans or sialylated carrier proteins regulate cell signaling to promote the malignant phenotype of human carcinoma.

Loss of α1,6-fucosyltransferase inhibits chemical-induced hepatocellular carcinoma and tumorigenesis by down-regulating several cell signaling pathways.

Up-regulation of core fucosylation catalyzed by α1,6-fucosyltransferase (Fut8) has been observed in hepatocellular carcinoma (HCC). Here, to explore the role of Fut8 expression in hepatocarcinogensis, we established the chemical-induced HCC models in the male wild-type (WT; Fut8(+/+)), hetero (Fut8(+/-)), and knockout (KO; Fut8(-/-)) mice by use of diethylnitrosamine (DEN) and pentobarbital (PB). In the Fut8(+/+) and Fut8(+/-) mice, multiple large and vascularized nodules were induced with an increased expression of Fut8 after DEN and PB treatment. However, the formation of HCC in Fut8(-/-) mice was suppressed almost completely. This potent inhibitory effect of Fut8 deficiency on tumorigenesis was also confirmed by the abolished tumor formation of Fut8 KO human hepatoma cell line cells by use of a xenograft tumor model. Furthermore, loss of the Fut8 gene resulted in attenuated responses to epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in the HepG2 cell line, which provides the possible mechanisms for the contribution of Fut8 to hepatocarcinogensis. Taken together, our study clearly demonstrated that core fucosylation acts as a critical functional modulator in the liver and implicated Fut8 as a prognostic marker, as well as a novel, therapeutic target for HCC.

Loss of α1,6-fucosyltransferase suppressed liver regeneration: implication of core fucose in the regulation of growth factor receptor-mediated cellular signaling.

Core fucosylation is an important post-translational modification, which is catalyzed by α1,6-fucosyltransferase (Fut8). Increased expression of Fut8 has been shown in diverse carcinomas including hepatocarcinoma. In this study, we investigated the role of Fut8 expression in liver regeneration by using the 70% partial hepatectomy (PH) model, and found that Fut8 is also critical for the regeneration of liver. Interestingly, we show that the Fut8 activities were significantly increased in the beginning of PH (~4d), but returned to the basal level in the late stage of PH. Lacking Fut8 led to delayed liver recovery in mice. This retardation mainly resulted from suppressed hepatocyte proliferation, as supported not only by a decreased phosphorylation level of epidermal growth factor (EGF) receptor and hepatocyte growth factor (HGF) receptor in the liver of Fut8(-/-) mice in vivo, but by the reduced response to exogenous EGF and HGF of the primary hepatocytes isolated from the Fut8(-/-) mice. Furthermore, an administration of L-fucose, which can increase GDP-fucose synthesis through a salvage pathway, significantly rescued the delayed liver regeneration of Fut8(+/-) mice. Overall, our study provides the first direct evidence for the involvement of Fut8 in liver regeneration.

ß-Galactoside α2,6-sialyltranferase 1 promotes transforming growth factor-ß-mediated epithelial-mesenchymal transition.

ß-Galactoside α2,6-sialyltranferase 1 (ST6GAL1) catalyzes the addition of terminal α2,6-sialylation to N-glycans. Increased expression of ST6GAL1 has been reported in diverse carcinomas and highly correlates with tumor progression. Here, we report that St6gal1 transcription and α2,6-sialylated N-glycans are up-regulated during TGF-ß-induced epithelial-mesenchymal transition (EMT) in GE11 cells, requiring the Sp1 element within the St6gal1 promoter. Knockdown of St6gal1 strongly suppressed TGF-ß-induced EMT with a concomitant increase in E-cadherin expression, a major determinant of epithelial cell adherens junctions. Conversely, overexpression of ST6GAL1 increased the turnover of cell surface E-cadherin and promoted TGF-ß-induced EMT. Overexpressing ß-galactoside α2,3-sialyltranferase 4 had little influence on EMT, indicating specificity for α2,6-sialylation. The basal mesenchymal phenotype of MDA-MB-231 human breast cancer cells was partially reversed by ST6GAL1 silencing. Moreover, ST6GAL1 knockdown inhibited the phosphorylation of Akt, but not Smad2, suggesting that ST6GAL1 contributes to EMT through a non-Smad signaling pathway. Taken together, our data indicate that ST6GAL1 promotes TGF-ß-dependent EMT as well as maintenance of the mesenchymal state by growth signaling, providing a plausible mechanism whereby up-regulated ST6GAL1 may promote malignant progression.

An oncogenic protein Golgi phosphoprotein 3 up-regulates cell migration via sialylation.

Recently, the Golgi phosphoprotein 3 (GOLPH3) and its yeast homolog Vps74p have been characterized as essential for the Golgi localization of glycosyltransferase in yeast. GOLPH3 has been identified as a new oncogene that is commonly amplified in human cancers to modulate mammalian target of rapamycin signaling. However, the molecular mechanisms of the carcinogenic signaling pathway remain largely unclear. To investigate whether the expression of GOLPH3 was involved in the glycosylation processes in mammalian cells, and whether it affected cell behavior, we performed a loss-of-function study. Cell migration was suppressed in GOLPH3 knockdown (KD) cells, and the suppression was restored by a re-introduction of the GOLPH3 gene. HPLC and LC/MS analysis showed that the sialylation of N-glycans was specifically decreased in KD cells. The specific interaction between sialyltransferases and GOLPH3 was important for the sialylation. Furthermore, overexpression of α2,6-sialyltransferase-I rescued cell migration and cellular signaling, both of which were blocked in GOLPH3 knockdown cells. These results are the first direct demonstration of the role of GOLPH3 in N-glycosylation to regulate cell biological functions.

Proteoglycans and glycosaminoglycans improve toughness of biocompatible double network hydrogels.

Based on the molecular stent concept, a series of tough double-network hydrogels (St-DN gels) made from the components of proteoglycan aggregates - chondroitin sulfate proteoglycans (1), chondroitin sulfate (2), and sodium hyaluronate (3) - are successfully developed in combination with a neutral biocompatible polymer. This work demonstrates a promising method to create biopolymer-based tough hydrogels for biomedical applications.

Alg14 organizes the formation of a multiglycosyltransferase complex involved in initiation of lipid-linked oligosaccharide biosynthesis.

Protein N-glycosylation begins with the assembly of a lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER) membrane. The first two steps of LLO biosynthesis are catalyzed by a functional multienzyme complex comprised of the Alg7 GlcNAc phosphotransferase and the heterodimeric Alg13/Alg14 UDP-GlcNAc transferase on the cytosolic face of the ER. In the Alg13/14 glycosyltransferase, Alg14 recruits cytosolic Alg13 to the ER membrane through interaction between their C-termini. Bioinformatic analysis revealed that eukaryotic Alg14 contains an evolved N-terminal region that is missing in bacterial orthologs. Here, we show that this N-terminal region of Saccharomyces cerevisiae Alg14 localize its green fluorescent protein fusion to the ER membrane. Deletion of this region causes defective growth at 38.5°C that can be partially complemented by overexpression of Alg7. Coimmunoprecipitation demonstrated that the N-terminal region of Alg14 is required for direct interaction with Alg7. Our data also show that Alg14 lacking the N-terminal region remains on the ER membrane through a nonperipheral association, suggesting the existence of another membrane-binding site. Mutational studies guided by the 3D structure of Alg14 identified a conserved α-helix involved in the second membrane association site that contributes to an integral interaction and protein stability. We propose a model in which the N- and C-termini of Alg14 coordinate recruitment of catalytic Alg7 and Alg13 to the ER membrane for initiating LLO biosynthesis.

The distinct quorum sensing hierarchy of las and rhl in Pseudomonas sp. M18.

Pseudomonas sp. M18 is a rhizosphere isolate capable of producing two kinds of antifungal agents: phenazine-1-carboxylic acid (PCA) and pyoluteorin. Recently, the two well-studied quorum sensing (QS) systems of Pseudomonas aeruginosa, LasR/LasI and RhlR/RhlI, have also been identified in this strain. However, in this study, through the use of lacZ translational fusion expression analysis and acyl-homoserine lactone thin-layer chromatography (TLC) bioassays, we clearly display a more complex and distinctive hierarchy of the las and rhl QS systems in strain M18. In this QS cascade, expression of rhlI was negatively controlled by the LasR/LasI QS system. In contrast with lasI, which negatively regulated the rhlR induction, lasR exerted a positive influence on rhlR expression during the log-phase. This interrelationship indicated that the response regulators (LasR and RhlR) of the QS system are expressed independently of their cognate synthases (LasI and RhlI). Furthermore, the las system also modulated the timing and magnitude of the rhlI and rhlR maximal expression. In addition, our data imply that the lasR gene exerts its negative control on PCA production through modulation of rhlI expression. Thus, interactions between the two QS systems are strain specific.

LysR family transcriptional regulator PqsR as repressor of pyoluteorin biosynthesis and activator of phenazine-1-carboxylic acid biosynthesis in Pseudomonas sp. M18.

The biocontrol rhizobacterium Pseudomonas sp. M18 can produce two different types of antibiotics, pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA), which are inhibitory to a number of soil-borne plant pathogens. The pqsR gene, identified in Pseudomonas sp. M18, encodes a LysR-type transcriptional regulator in the Pseudomonas quinolone signal (PQS)-mediated quorum-sensing (QS) system. Here we investigated the regulatory mechanisms of PqsR in PCA and Plt biosyntheses. The results clearly suggest that PqsR functions as a double-duty transcriptional regulator, either as a repressor of Plt biosynthesis or as an activator of PCA biosynthesis. The chromosomal inactivation of pqsR resulted in significant enhancement of Plt production and its genes expression, while almost full inhibition of PCA production and its genes expression. This was further confirmed by multiple pqsR gene dosage experiments, lacZ fusion reporter analysis, and semi-quantitative RT-PCR. Furthermore, PqsR had little effect on expression of the plt pathway-specific activator PltR, indicating that PqsR does not exert its negative regulation on Plt biosynthesis through the mediator PltR. In addition, the pqsR mutation did not have any obvious influence on production of RhlI directing N-acylhomoserine lactones (C4 and C8-HSLs). This result shows PqsR functions as a crucial transcriptional regulator independently of the rhl QS system.