A modified two-component regulatory system is involved in temperature-dependent biosynthesis of the Pseudomonas syringae phytotoxin coronatine.
ABSTRACT: Biosynthesis of the phytotoxin coronatine (COR) in Pseudomonas syringae pv. glycinea PG4180 is regulated by temperature at the transcriptional level. A 3.4-kb DNA fragment from the COR biosynthetic gene cluster restored temperature-regulated phytotoxin production to Tn5 mutants defective in COR production. Nucleotide sequence analysis of this fragment revealed three genes, corS, corP, and corR, which encode a modified two-component regulatory system consisting of one sensor protein, CorS, and two response regulator proteins, CorP and CorR. Although only one response regulator, CorR, had a DNA-binding domain, the phosphate-receiving domains of both response regulator proteins were highly conserved. Transcriptional fusions of the corP and corR promoters to a promoterless glucuronidase gene (uidA) indicated that these two genes are expressed constitutively at 18 and 28 degrees C. In contrast, a corS::uidA fusion exhibited the temperature dependence previously observed for COR biosynthetic promoters and exhibited maximal transcriptional activity at 18 degrees C and low activity at 28 degrees C. Furthermore, glucuronidase activity for corS::uidA was decreased in corP, corR, and corS mutants relative to the levels observed for PG4180(corS::uidA). This difference was not observed for corP::uidA and corR::uidA transcriptional fusions since expression of these fusions remained low and constitutive regardless of the genetic background. The three regulatory genes functioned in a P. syringae strain lacking the COR gene cluster to achieve temperature-dependent activation of an introduced COR biosynthetic promoter, indicating that this triad of genes is the primary control for COR biosynthesis and responsible for thermoregulation. Our data suggest that the modified two-component regulatory system described in this study might transduce and amplify a temperature signal which results in transcriptional activation of COR biosynthetic genes.
Project description:Coronatine (COR) is a plasmid-encoded phytotoxin synthesized by several pathovars of phytopathogenic Pseudomonas syringae. The COR biosynthetic gene cluster in P. syringae pv. glycinea PG4180 is encoded by a 32-kb region which contains both the structural and regulatory genes needed for COR synthesis. The regulatory region contains three genes: corP, corS, and corR. corS is thought to function as a histidine protein kinase, whereas corP and corR show relatedness to response regulators of the two-component regulatory paradigm. In the present study, we investigated whether CorR is a positive activator of COR gene expression. We also studied whether CorR specifically binds the DNA region located upstream of cfl, a gene located at the 5' end of the gene cluster encoding coronafacic acid, the polyketide portion of COR. Complementation analysis with a corR mutant, PG4180.P2, and transcriptional fusions to a promoterless glucuronidase gene (uidA) indicated that CorR functions as a positive regulator of COR gene expression. Deletion analysis of the 5' end of the cfl upstream region was used to define the minimal region required for COR gene expression. A 360-bp DNA fragment located over 500 bp upstream from the cfl transcriptional start site was used in DNase I protection assays to define the specific bases bound by CorR. An area extending from -704 to -650 with respect to the cfl transcriptional start site was protected by DNase I footprinting, indicating a rather large area of protection. This area was also conserved in the promoter region for cmaA, which encodes a transcript containing genes for coronamic acid synthesis, another intermediate in the COR biosynthetic pathway. The results obtained in the current study suggest that both the coronafacic acid and the coronamic acid structural genes are controlled by CorR, a positive activator of COR gene expression.
Project description:The plant-pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 synthesizes high levels of the polyketide phytotoxin coronatine (COR) at 18 degrees C, whereas no detectable toxin is produced at 28 degrees C. Previously, we reported that the temperature-sensitive activation of three promoters within the COR biosynthetic gene cluster might explain thermoregulation of COR biosynthesis. The present study was aimed at furthering our understanding of the transcriptional as well as the posttranslational effects of temperature on expression of cmaB, which encodes an enzyme involved in COR biosynthesis. Transcriptional fusions using a promoterless glucuronidase gene and Northern blot analyses were used to monitor promoter activities and transcript abundance for the cmaABT operon during bacterial growth at 18 and 28 degrees C. Promoter activity and transcription rates were maximal when cells were incubated at 18 degrees C and sampled at mid-logarithmic phase. Transcription declined moderately during the transition to stationary phase but remained higher at 18 C than at 28 degrees C. Western blot analysis indicated that CmaB accumulated in the late stationary phase of P. syringae cultures grown at 18 degrees C but not in cultures incubated at 28 degrees C. Temperature shift experiments indicated that CmaB stability was more pronounced at 18 degrees C than at 28 degrees C. Although temperature has a strong impact on transcription of COR biosynthetic genes, we propose that thermoregulation of protein stability might also control COR synthesis.
Project description:In previous studies we have demonstrated that chlorophyllide a oxidoreductases (CORs) from bacteriochlorophyll (BChl) a-producing Rhodobacter species and BChl b-producing Blastochloris viridis show distinct substrate recognition and different catalytic hydrogenation reactions, and that these two types of CORs therefore cause committed steps for BChls a and b biosynthesis. In this study, COR genes from B. viridis were incorporated and overexpressed in a series of Rhodobacter sphaeroides mutants. We found that the following two factors are essential in making R. sphaeroides produce BChl b: the loss of functions of both intrinsic COR and 8-vinyl reductase (BciA) in the host R. sphaeroides strain; and expression of the BchYZ catalytic components of COR from B. viridis, not the complete set of COR (BchXYZ), in the host strain. In addition, we incorporated bchYZ of B. viridis into the R. sphaeroides mutant lacking BchJ and BciA, resulting in the strain accumulating both BChl a and BChl b. This is the first example of an anoxygenic photosynthetic bacterium producing BChls a and b together. The results suggest that BchJ enhances activity of the intrinsic COR. The physiological significance of BchJ in pigment biosynthetic pathways will be discussed.
Project description:Background:The soil bacterium Sinorhizobium meliloti and its allies are important nitrogen-fixing bacterial symbionts that cause N2-fixing nodules on the roots of legumes. Chromosomal ß-glucuronidase gene (uidA) transcriptional fusions are frequently used to monitor the expression of bacterial genes during the symbiosis. However, the construction of the fusions is laborious. Results:The narrow-host-range, fusion selective plasmid pVMG was constructed and used as a vector for the construction of chromosomal uidA transcriptional fusions in the S. meliloti genome. Translation termination codons were added in all three reading frames upstream of the promoterless uidA in this vector to ensure transcriptional fusions. pVMG replicated to high copy number in Escherichia coli, offering advantages for the isolation of fusion-containing plasmids and the restriction analysis. Genomic locations of uidA fusions were verified in a simple PCR experiment. All these helps reduce the sample processing time and efforts. As a demonstration of its usefulness, the N-acyl homoserine lactone (AHL) signal synthase gene promoter was fused to uidA and shown to be expressed by S. meliloti in the senescence zone of the nodule on the host plant, M. truncatula. This indicates the presence of AHL signals at the late stages of symbiosis. Conclusions:A simple, pVMG-based method for construction of chromosomal uidA transcriptional fusions has been successfully used in the model rhizobium S. meliloti. It is also applicable for other rhizobial strains.
Project description:Adverse environmental conditions limit various aspects of plant growth, productivity, and ecological distribution. To get more insights into the signaling pathways under low temperature, we identified 10 C-repeat binding factors (CBFs), 9 inducer of CBF expression (ICEs) and 10 cold-responsive (CORs) genes from Aegilops-Triticum composite group under cold stress. Conserved amino acids analysis revealed that all CBF, ICE, COR contained specific and typical functional domains. Phylogenetic analysis of CBF proteins from Triticeae showed that these CBF homologs were divided into 11 groups. CBFs from Triticum were found in every group, which shows that these CBFs generated prior to the divergence of the subfamilies of Triticeae. The evolutionary relationship among the ICE and COR proteins in Poaceae were divided into four groups with high multispecies specificity, respectively. Moreover, expression analysis revealed that mRNA accumulation was altered by cold treatment and the genes of three types involved in the ICE-CBF-COR signaling pathway were induced by cold stress. Together, the results make CBF, ICE, COR genes family in Triticeae more abundant, and provide a starting point for future studies on transcriptional regulatory network for improvement of chilling tolerance in crop.
Project description:Background:Accurate knowledge of the intervertebral center of rotation (COR) and its corresponding range of motion (ROM) can help understand development of cervical pathology and guide surgical treatment. Methods:Ten asymptomatic subjects were imaged using MRI and dual fluoroscopic imaging techniques during dynamic extension-flexion-extension (EFE) and axial left-right-left (LRL) rotation. The intervertebral segment CORs and ROMs were measured from C34 to C67, as the correlations between two variables were analyzed as well. Results:During the EFE motion, the CORs were located at 32.4 ± 20.6%, -2.4 ± 11.7%, 21.8 ± 12.5% and 32.3 ± 25.5% posteriorly, and the corresponding ROMs were 13.8 ± 4.3°, 15.1 ± 5.1°, 14.4 ± 7.0° and 9.2 ± 4.3° from C34 to C67. The ROM of C67 was significantly smaller than other segments. The ROMs were not shown to significantly correlate to COR locations (r = -0.243, p = 0.132). During the LRL rotation cycle, the average CORs were at 85.6 ± 18.2%, 32.3 ± 25.3%, 15.7 ± 12.3% and 82.4 ± 31.3% posteriorly, and the corresponding ROMs were 3.5 ± 1.7°, 6.9 ± 3.8°, 9.6 ± 4.1° and 2.6 ± 2.5° from C34 to C67. The ROMs of C34 and C67 was significantly smaller than those of C45 and C56. A more posterior COR was associated with a less ROM during the neck rotation (r = -0.583, p < 0.001). The ROMs during EFE were significantly larger than those during LRL in each intervertebral level. Conclusion:The CORs and ROMs of the subaxial cervical intervertebral segments were segment level- and neck motion-dependent during the in-vivo neck motions. The translational potential of this article:Our study indicates that the subaxial cervical intervertebral CORs and ROMs were segment level- and neck motion-dependent. This may help to improve the artificial disc design as well as surgical technique by which the neck functional motion is restored following the cervical arthroplasty.
Project description:Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.
Project description:Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-(32)PP(i) exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain L-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for L-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4'-phosphopantetheinyl group, reacts with the AMP derivative of L-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and L-[G-(3)H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the L-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid.
Project description:Despite systemic exposure to risk factors, the circulatory system develops varying patterns of atherosclerosis for unclear reasons. In a porcine model, we investigated the relationship between site-specific lesion development and inflammatory pathways involved in the coronary arteries (CORs) and distal abdominal aortas (AAs).Diabetes mellitus (DM) and hypercholesterolemia (HC) were induced in 37 pigs with 3 healthy controls. Site-specific plaque development was studied by comparing plaque severity, macrophage infiltration, and inflammatory gene expression between CORs and AAs of 17 DM/HC pigs. To assess the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in plaque development, 20 DM/HC pigs were treated with the Lp-PLA2 inhibitor darapladib and compared with the 17 DM/HC untreated pigs. DM/HC caused site-specific differences in plaque severity. In the AAs, normalized plaque area was 4.4-fold higher (P<0.001) and there were more fibroatheromas (9 of the 17 animals had a fibroatheroma in the AA and not the COR, P=0.004), while normalized macrophage staining area was 1.5-fold higher (P=0.011) compared with CORs. DM/HC caused differential expression of 8 of 87 atherosclerotic genes studied, including 3 important in inflammation with higher expression in the CORs. Darapladib-induced attenuation of normalized plaque area was site-specific, as CORs responded 2.9-fold more than AAs (P=0.045).While plaque severity was worse in the AAs, inflammatory genes and inflammatory pathways that use Lp-PLA2 were more important in the CORs. Our results suggest fundamental differences in inflammation between vascular sites, an important finding for the development of novel anti-inflammatory therapeutics.
Project description:Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally synthesized between 18°C and 20°C, while no detectable amounts are present above 28°C. The Pht cluster, involved in the biosynthesis of phaseolotoxin, contains 23 genes that are organized in five transcriptional units. The function of most of the genes from the Pht cluster is still unknown and little information about the regulatory circuitry leading to expression of these genes has been reported. The purpose of the present study was to investigate the participation of pht genes in the regulation of the operons coded into the Pht cluster. We conducted Northern blot, uidA fusions and reverse transcription-PCR assays of pht genes in several mutants unable to produce phaseolotoxin. This allowed us to determine that, in P. syringae pv. phaseolicola NPS3121, genes phtABC are essential to prevent their own expression at 28°C, a temperature at which no detectable amounts of the toxin are present. We obtained evidence that the phtABC genes also participate in the regulation of the phtD, phtM and phtL operons. According to our results, we propose that PhtABC and other Pht product activities could be involved in the synthesis of the sulfodiaminophosphinyl moiety of phaseolotoxin, which indirectly could be involved in the transcriptional regulation of the phtA operon.