scholarly journals The Azotobacter vinelandii Response Regulator AlgR Is Essential for Cyst Formation

1999 ◽  
Vol 181 (1) ◽  
pp. 141-148 ◽  
Author(s):  
Cinthia Núñez ◽  
Soledad Moreno ◽  
Gloria Soberón-Chávez ◽  
Guadalupe Espín
Keyword(s):  
Electron Microscopy ◽  
Pseudomonas Aeruginosa ◽  
Biosynthetic Pathway ◽  
Azotobacter Vinelandii ◽  
Response Regulator ◽  
Cyst Formation ◽  
Sensor Kinase ◽  
Alginate Production ◽  
Key Enzyme

ABSTRACT Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for the encystment process. InPseudomonas aeruginosa, as well as in A. vinelandii, the ςE factor encoded byalgU is required for transcription of algD, which encodes a key enzyme of the alginate biosynthetic pathway. TheP. aeruginosa response regulator AlgR activates transcription of algD. fimS, located upstreamalgR, is proposed to encode the AlgR cognate sensor kinase. We have cloned and characterized the A. vinelandii algRgene; the deduced amino acid sequence of the protein encoded by this gene shows 79% identity with its P. aeruginosa homolog. Sequence analysis around the algR gene revealed the absence of a fimS homolog. Inactivation of A. vinelandii algR diminished alginate production by 50%, but did not affectalgD transcription, and completely impaired the capacity to form mature cysts. Electron microscopy of the cyst structures formed by the algR mutant revealed that the encystment process is blocked at the step of exine formation. The transcriptional regulation of the A. vinelandii algR gene and the role of AlgR in alginate production differ significantly from those of its P. aeruginosa counterparts. These differences could be due to the fact that in A. vinelandii, alginate plays a role in encystment, a function not found in P. aeruginosa.

scholarly journals The GacS Sensor Kinase Regulates Alginate and Poly-β-Hydroxybutyrate Production in Azotobacter vinelandii

2000 ◽  
Vol 182 (9) ◽  
pp. 2624-2628 ◽  
Author(s):  
Miguel Castañeda ◽  
Josefina Guzmán ◽  
Soledad Moreno ◽  
Guadalupe Espín
Keyword(s):  
Biosynthetic Pathway ◽  
Azotobacter Vinelandii ◽  
Response Regulator ◽  
Extracellular Polysaccharide ◽  
Predicted Amino Acid Sequence ◽  
Sensor Kinase ◽  
Alginate Production ◽  
Gene Coding ◽  
Key Enzyme ◽  
Cognate Response Regulator

ABSTRACT Azotobacter vinelandii produces two polymers: the extracellular polysaccharide alginate and the intracellular polyester poly-β-hydroxybutyrate (PHB). A cosmid clone (pSMU588) from anA. vinelandii gene library diminished alginate production by A. vinelandii mucoid strain ATCC 9046. The nucleotide sequence and predicted amino acid sequence of the locus responsible for the mucoidy suppression revealed 65% identity toPseudomonas GacS, a transmembrane sensor kinase of the two-component regulators, whose cognate response regulator, GacA, is a global activator regulating several products and virulence factors. Plasmid pMC15, harboring gacS, and a strain carrying agacS nonpolar mutation were constructed. Either pMC15 or the gacS mutation significantly reduced alginate production and transcription of algD, the gene coding for the key enzyme GDP-mannose dehydrogenase of the alginate biosynthetic pathway. We found that the gacS mutation also reduced PHB accumulation and impaired encystment. Taken together, these data indicate that in A. vinelandii the gacSA global system regulates polymer synthesis.

scholarly journals The NtrC Family Regulator AlgB, Which Controls Alginate Biosynthesis in Mucoid Pseudomonas aeruginosa, Binds Directly to the algD Promoter

2007 ◽  
Vol 190 (2) ◽  
pp. 581-589 ◽  
Author(s):  
Andrew J. Leech ◽  
April Sprinkle ◽  
Lynn Wood ◽  
Daniel J. Wozniak ◽  
Dennis E. Ohman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Response Regulator ◽  
Mobility Shift ◽  
Alginate Production ◽  
Mobility Shift Assay ◽  
Exponential Enrichment ◽  
Microarray Analyses ◽  
Phosphorylation Domain

ABSTRACT Alginate production in mucoid (MucA-defective) Pseudomonas aeruginosa is dependent upon several transcriptional regulators, including AlgB, a two-component response regulator belonging to the NtrC family. This role of AlgB was apparently independent of its sensor kinase, KinB, and even the N-terminal phosphorylation domain of AlgB was dispensable for alginate biosynthetic gene (i.e., algD operon) activation. However, it remained unclear whether AlgB stimulated algD transcription directly or indirectly. In this study, microarray analyses were used to examine a set of potential AlgB-dependent, KinB-independent genes in a PAO1 mucA background that overlapped with genes induced by d-cycloserine, which is known to activate algD expression. This set contained only the algD operon plus one other gene that was shown to be uninvolved in alginate production. This suggested that AlgB promotes alginate production by directly binding to the algD promoter (PalgD). Chromosome immunoprecipitation revealed that AlgB bound in vivo to PalgD but did not bind when AlgB had an R442E substitution that disrupted the DNA binding domain. AlgB also showed binding to PalgD fragments in an electrophoretic mobility shift assay at pH 4.5 but not at pH 8.0. A direct systematic evolution of ligands by exponential enrichment approach showed AlgB binding to a 50-bp fragment located at bp −224 to −274 relative to the start of PalgD transcription. Thus, AlgB belongs to a subclass of NtrC family proteins that can activate promoters which utilize a sigma factor other than σ54, in this case to stimulate transcription from the σ22-dependent PalgD promoter.

scholarly journals Inactivation of the ampDE Operon Increases Transcription of algD and Affects Morphology and Encystment of Azotobacter vinelandii

2000 ◽  
Vol 182 (17) ◽  
pp. 4829-4835 ◽  
Author(s):  
Cinthia Núñez ◽  
Soledad Moreno ◽  
Luis Cárdenas ◽  
Gloria Soberón-Chávez ◽  
Guadalupe Espín
Keyword(s):  
Escherichia Coli ◽  
Biosynthetic Pathway ◽  
Azotobacter Vinelandii ◽  
Transmembrane Protein ◽  
Nucleotide Sequencing ◽  
Insertion Mutant ◽  
Gene Encoding ◽  
Alginate Production ◽  
Key Enzyme

ABSTRACT Transcription of algD, encoding GDP-mannose dehydrogenase, the key enzyme in the alginate biosynthetic pathway, is highly regulated in Azotobacter vinelandii. We describe here the characterization of a Tn5 insertion mutant (AC28) which shows a higher level of expression of analgD::lacZ fusion. AC28 cells were morphologically abnormal and unable to encyst. The cloning and nucleotide sequencing of the Tn5-disrupted locus in AC28 revealed an operon homologous to the Escherichia coli ampDEoperon. Tn5 was located within the ampD gene, encoding a cytosolicN-acetyl-anhydromuramyl-l-alanine amidase that participates in the intracellular recycling of peptidoglycan fragments. The ampE gene encodes a transmembrane protein, but the function of the protein is not known. We constructed strains carryingampD or ampE mutations and one with anampDE deletion. The strain with a deletion of theampDE operon showed a phenotype similar to that of mutant AC28. The present work demonstrates that both alginate production and bacterial encystment are greatly influenced by the bacterial ability to recycle its cell wall.

scholarly journals Role of Azotobacter vinelandii mucA andmucC Gene Products in Alginate Production

2000 ◽  
Vol 182 (23) ◽  
pp. 6550-6556 ◽  
Author(s):  
Cinthia Núñez ◽  
Renato León ◽  
Josefina Guzmán ◽  
Guadalupe Espı́n ◽  
Gloria Soberón-Chávez
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Regulation ◽  
Sigma Factor ◽  
Azotobacter Vinelandii ◽  
Bacterial Species ◽  
Alternative Sigma Factor ◽  
Gene Products ◽  
Alginate Production ◽  
Negative Role

ABSTRACT Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor ςE regulates the expression of functions related to the extracytoplasmic compartments. In A. vinelandii and Pseudomonas aeruginosa, the ςE factor (AlgU) is essential for alginate production. In both bacteria, the activity of this sigma factor is regulated by the product of the mucA, mucB, mucC, and mucD genes. In this work, we studied the transcriptional regulation of the A. vinelandii algU-mucABCD gene cluster, as well as the role of themucA and mucC gene products in alginate production. Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production.

scholarly journals Regulation of Alginate Biosynthesis inPseudomonas syringae pv. syringae

1999 ◽  
Vol 181 (11) ◽  
pp. 3478-3485 ◽  
Author(s):  
Mohamed K. Fakhr ◽  
Alejandro Peñaloza-Vázquez ◽  
Ananda M. Chakrabarty ◽  
Carol L. Bender
Keyword(s):  
Sigma Factor ◽  
Promoter Region ◽  
Biosynthetic Pathway ◽  
Response Regulator ◽  
Consensus Sequence ◽  
Gene Encoding ◽  
Environmental Signals ◽  
Alginate Production ◽  
Key Enzyme ◽  
Alginate Biosynthesis

ABSTRACT Both Pseudomonas aeruginosa and the phytopathogenP. syringae produce the exopolysaccharide alginate. However, the environmental signals that trigger alginate gene expression in P. syringae are different from those inP. aeruginosa with copper being a major signal in P. syringae. In P. aeruginosa, the alternate sigma factor encoded by algT (ς22) and the response regulator AlgR1 are required for transcription of algD, a gene which encodes a key enzyme in the alginate biosynthetic pathway. In the present study, we cloned and characterized the gene encoding AlgR1 from P. syringae. The deduced amino acid sequence of AlgR1 from P. syringae showed 86% identity to its P. aeruginosa counterpart. Sequence analysis of the region flankingalgR1 in P. syringae revealed the presence ofargH, algZ, and hemC in an arrangement virtually identical to that reported in P. aeruginosa. An algR1 mutant, P. syringaeFF5.32, was defective in alginate production but could be complemented when algR1 was expressed in trans. ThealgD promoter region in P. syringae(PsalgD) was also characterized and shown to diverge significantly from the algD promoter in P. aeruginosa. Unlike P. aeruginosa, algR1was not required for the transcription of algD in P. syringae, and PsalgD lacked the consensus sequence recognized by AlgR1. However, both the algD andalgR1 upstream regions in P. syringae contained the consensus sequence recognized by ς22, suggesting thatalgT is required for transcription of both genes.

scholarly journals Role of Alternative ς Factor AlgU in Encystment of Azotobacter vinelandii

1998 ◽  
Vol 180 (10) ◽  
pp. 2766-2769 ◽  
Author(s):  
Soledad Moreno ◽  
Rebeca Nájera ◽  
Josefina Guzmán ◽  
Gloria Soberón-Chávez ◽  
Guadalupe Espín
Keyword(s):  
Wild Type Strain ◽  
Azotobacter Vinelandii ◽  
Strain Atcc ◽  
Consensus Sequences ◽  
Alginate Production ◽  
Naturally Occurring ◽  
Key Enzyme ◽  
In The Wild ◽  
P2 Promoter

ABSTRACT Alginate is essential for encystment in Azotobacter vinelandii. Transcription of the algD gene, which codes for GDP-mannose dehydrogenase, a key enzyme in the alginate biosynthetic pathway, is initiated at two promoters, one of which, p2, has ςE consensus sequences. AlgU is the A. vinelandii alternative ςE factor. In this study, we constructed an algU mutant (SMU88) which, as expected, is impaired in alginate production, encystment, and transcription of thealgD gene from the p2 promoter. Plasmid pJMSAT1, carrying the A. vinelandii algU gene, restored alginate production and encystment to SMU88 and to strain UW136, a naturally occurringalgU mutant. Plasmid pSMU865, carrying the A. vinelandii mucABCD genes coding for negative regulators of AlgU activity and previously shown to diminish alginate production in the wild-type strain, ATCC 9046, was shown here to impair encystment and transcription of the algD gene from the p2algU-dependent promoter. Since nonencysting strain ATCC 9046/pSMU865 produced more alginate than some encysting strains, such as UW136/pJMSAT1, we propose an AlgU role in encystment, independent of the structural role that alginate plays in mature cysts.

scholarly journals The Pseudomonas aeruginosa Sensor Kinase KinB Negatively Controls Alginate Production through AlgW-Dependent MucA Proteolysis

2009 ◽  
Vol 191 (7) ◽  
pp. 2285-2295 ◽  
Author(s):  
F. Heath Damron ◽  
Dongru Qiu ◽  
Hongwei D. Yu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Response Regulator ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Cystic Fibrosis Mutation ◽  
Strain Pao1 ◽  
Alginate Production ◽  
Mucoid Conversion ◽  
Σ Factor ◽  
Lung Infections

ABSTRACT Mucoidy, or overproduction of the exopolysaccharide known as alginate, in Pseudomonas aeruginosa is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-σ factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of P. aeruginosa have a wild-type (wt) mucA. Here, we describe a loss-of-function mutation in kinB that causes overproduction of alginate in the wt mucA strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of kinB was correlated with high expression at the alginate-related promoters P algU and P algD . Deletion of alternative σ factor RpoN (σ54) or the response regulator AlgB in kinB mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the kinB algB double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when kinB was inactivated. The inactivation of the DegS-like protease AlgW in the kinB mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the kinB mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required algB and rpoN in the kinB mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt mucA strain PAO1.

scholarly journals Mutational Analysis of RetS, an Unusual Sensor Kinase-Response Regulator Hybrid Required for Pseudomonas aeruginosa Virulence

2006 ◽  
Vol 74 (8) ◽  
pp. 4462-4473 ◽  
Author(s):  
Michelle A. Laskowski ◽  
Barbara I. Kazmierczak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mutational Analysis ◽  
Response Regulator ◽  
Acute Infection ◽  
Opportunistic Pathogen ◽  
Sensor Kinase ◽  
Chronic Infections ◽  
Reciprocal Regulation ◽  
Wide Range

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in a wide range of hosts. Expression of the type III secretion system (T3SS) proteins is correlated with virulence in models of acute infection, while downregulation of the T3SS and upregulation of genes important for biofilm formation are observed during chronic infections. RetS, a hybrid sensor kinase-response regulator protein of P. aeruginosa, plays a key role in the reciprocal regulation of virulence factors required for acute versus chronic infection and is postulated to act in concert with two other sensor kinase-response regulator hybrids, GacS and LadS. This work examines the roles of the putative sensing and signal transduction domains of RetS in induction of the T3SS in vitro and in a murine model of acute pneumonia. We identify distinct signaling roles for the tandem receiver domains of RetS and present evidence suggesting that RetS may serve as a substrate for another sensor kinase. Phenotypes associated with RetS alleles lacking periplasmic and/or transmembrane domains further indicate that the periplasmic domain of RetS may transmit a signal that inhibits RetS activity during acute infections.

scholarly journals Alginate synthesis by Pseudomonas aeruginosa: a key pathogenic factor in chronic pulmonary infections of cystic fibrosis patients.

1991 ◽  
Vol 4 (2) ◽  
pp. 191-206 ◽  
Author(s):  
T B May ◽  
D Shinabarger ◽  
R Maharaj ◽  
J Kato ◽  
L Chu ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Biosynthetic Pathway ◽  
Pulmonary Infection ◽  
Regulatory Mechanism ◽  
Regulatory Proteins ◽  
Host Immune System ◽  
Pathogenic Factor ◽  
Pulmonary Infections

Pulmonary infection by mucoid, alginate-producing Pseudomonas aeruginosa is the leading cause of mortality among patients suffering from cystic fibrosis. Alginate-producing P. aeruginosa is uniquely associated with the environment of the cystic fibrosis-affected lung, where alginate is believed to increase resistance to both the host immune system and antibiotic therapy. Recent evidence indicates that P. aeruginosa is most resistant to antibiotics when the infecting cells are present as a biofilm, as they appear to be in the lungs of cystic fibrosis patients. Inhibition of the protective alginate barrier with nontoxic compounds targeted against alginate biosynthetic and regulatory proteins may prove useful in eradicating P. aeruginosa from this environment. Our research has dealt with elucidating the biosynthetic pathway and regulatory mechanism(s) responsible for alginate synthesis by P. aeruginosa. This review summarizes reports on the role of alginate in cystic fibrosis-associated pulmonary infections caused by P. aeruginosa and provides details about the biosynthesis and regulation of this exopolysaccharide.

scholarly journals RscA, a Member of the MDR1 Family of Transporters, Is Repressed by CovR and Required for Growth of Streptococcus pyogenes under Heat Stress

2006 ◽  
Vol 188 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Tracy L. Dalton ◽  
Julie T. Collins ◽  
Timothy C. Barnett ◽  
June R. Scott
Keyword(s):  
Streptococcus Pyogenes ◽  
Abc Transporters ◽  
Group A Streptococcus ◽  
Response Regulator ◽  
Transcriptional Analysis ◽  
Sensor Kinase ◽  
Two Component System ◽  
Transcription Profiles ◽  
Group A

ABSTRACT The ability of Streptococcus pyogenes (group A streptococcus [GAS]) to respond to changes in environmental conditions is essential for this gram-positive organism to successfully cause disease in its human host. The two-component system CovRS controls expression of about 15% of the GAS genome either directly or indirectly. In most operons studied, CovR acts as a repressor. We previously linked CovRS to the GAS stress response by showing that the sensor kinase CovS is required to inactivate the response regulator CovR so that GAS can grow under conditions of heat, acid, and salt stress. Here, we sought to identify CovR-repressed genes that are required for growth under stress. To do this, global transcription profiles were analyzed by microarrays following exposure to increased temperature (40°C) and decreased pH (pH 6.0). The CovR regulon in an M type 6 strain of GAS was also examined by global transcriptional analysis. We identified a gene, rscA (regulated by stress and Cov), whose transcription was confirmed to be repressed by CovR and activated by heat and acid. RscA is a member of the MDR1 family of ABC transporters, and we found that it is required for growth of GAS at 40°C but not at pH 6.0. Thus, for GAS to grow at 40°C, CovR repression must be alleviated so that rscA can be transcribed to allow the production of this potential exporter. Possible explanations for the thermoprotective role of RscA in this pathogen are discussed.

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