scholarly journals Role of Ornibactin Biosynthesis in the Virulence ofBurkholderia cepacia: Characterization of pvdA, the Gene Encoding l-OrnithineN5-Oxygenase

1999 ◽  
Vol 67 (9) ◽  
pp. 4443-4455 ◽  
Author(s):  
P. A. Sokol ◽  
P. Darling ◽  
D. E. Woods ◽  
E. Mahenthiralingam ◽  
C. Kooi
Keyword(s):  
Burkholderia Cepacia ◽  
Respiratory Infections ◽  
Cosmid Library ◽  
Gene Encoding ◽  
Transposon Insertion ◽  
Peptide Synthetase ◽  
Transposon Insertions ◽  
Siderophore Activity

ABSTRACT Burkholderia cepacia is a frequent cause of respiratory infections in cystic fibrosis patients. B. cepacia has been shown to produce at least four siderophores which may play a role in the virulence of this organism. To characterize genes involved in the synthesis of siderophores, Tn5-OT182 mutants were isolated in strain K56-2, which produces two siderophores, salicylic acid (SA) and ornibactins. Two mutants were characterized that did not produce zones on Chrome Azurol S agar in a commonly used assay to detect siderophore activity. These mutants were determined to produce sevenfold more SA than K56-2 yet did not produce detectable amounts of ornibactins. These mutants, designated I117 and T10, had a transposon insertion in genes with significant homology to pyoverdine biosynthesis genes of Pseudomonas aeruginosa. I117 contained an insertion in a pvdA homolog, the gene for the enzymel-ornithine N 5-oxygenase, which catalyzes the hydroxylation of l-ornithine. Ornibactin synthesis in this mutant was partially restored when the precursorl-N 5-OH-Orn was added to the culture medium. T10 contained an insertion in a pvdDhomolog, which is a peptide synthetase involved in pyoverdine synthesis. β-Galactosidase activity was iron regulated in both I117 and T10, suggesting that the transposon was inserted downstream of an iron-regulated promoter. Tn5-OT182 contains alacZ gene that is expressed when inserted downstream of an active promoter. Both I117 and T10 were deficient in uptake of iron complexed to either ornibactins or SA, suggesting that transposon insertions in ornibactin biosynthesis genes also affected other components of the iron transport mechanism. The B. cepacia pvdA homolog was approximately 47% identical and 59% similar tol-ornithine N 5-oxygenase fromP. aeruginosa. Three clones were identified from a K56-2 cosmid library that partially restored ornibactin production, SA production, and SA uptake to parental levels but did not affect the rate of 59Fe-ornibactin uptake in I117. A chromosomalpvdA deletion mutant was constructed that had a phenotype similar to that of I117 except that it did not hyperproduce SA. ThepvdA mutants were less virulent than the parent strain in chronic and acute models of respiratory infection. A functionalpvdA gene appears to be required for effective colonization and persistence in B. cepacia lung infections.

scholarly journals Characterization of RelA in Acinetobacter baumannii

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
María Pérez-Varela ◽  
Aimee R. P. Tierney ◽  
Ju-Sim Kim ◽  
Andrés Vázquez-Torres ◽  
Philip Rather
Keyword(s):  
Acinetobacter Baumannii ◽  
Galleria Mellonella ◽  
Multidrug Resistant ◽  
Cell Physiology ◽  
Content Type ◽  
Content Model ◽  
Transposon Insertion ◽  
Downstream Effectors

ABSTRACT In response to nutrient depletion, the RelA and SpoT proteins generate the signaling molecule (p)ppGpp, which then controls a number of downstream effectors to modulate cell physiology. In Acinetobacter baumannii strain AB5075, a relA ortholog (ABUW_3302) was identified by a transposon insertion that conferred an unusual colony phenotype. An in-frame deletion in relA (ΔrelA) failed to produce detectable levels of ppGpp when amino acid starvation was induced with serine hydroxamate. The ΔrelA mutant was blocked from switching from the virulent opaque colony variant (VIR-O) to the avirulent translucent colony variant (AV-T), but the rate of AV-T to VIR-O switching was unchanged. In addition, the ΔrelA mutation resulted in a pronounced hypermotile phenotype on 0.35% agar plates. This hypermotility was dependent on the activation of a LysR regulator ABUW_1132, which was required for expression of AbaR, a LuxR family quorum-sensing regulator. In the ΔrelA mutant, ABUW_1132 was also required for the increased expression of an operon composed of the ABUW_3766-ABUW_3773 genes required for production of the surfactant-like lipopeptide acinetin 505. Additional phenotypes identified in the ΔrelA mutant included (i) cell elongation at high density, (ii) reduced formation of persister cells tolerant to colistin and rifampin, and (iii) decreased virulence in a Galleria mellonella model. IMPORTANCE Acinetobacter baumannii is a pathogen of worldwide importance. Due to the increasing prevalence of antibiotic resistance, these infections are becoming increasingly difficult to treat. New therapies are required to combat multidrug-resistant isolates. The role of RelA in A. baumannii is largely unknown. This study demonstrates that like in other bacteria, RelA controls a variety of functions, including virulence. Strategies to inhibit the activity of RelA and the resulting production of ppGpp could inhibit virulence and may represent a new therapeutic approach.

scholarly journals Identification of a Siderophore Receptor Required for Ferric Ornibactin Uptake in Burkholderia cepacia

2000 ◽  
Vol 68 (12) ◽  
pp. 6554-6560 ◽  
Author(s):  
P. A. Sokol ◽  
P. Darling ◽  
S. Lewenza ◽  
C. R. Corbett ◽  
C. D. Kooi
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Burkholderia Cepacia ◽  
Parent Strain ◽  
Respiratory Infections ◽  
Infection Model ◽  
Protein Profiles ◽  
Outer Membrane Receptor ◽  
Gene Encoding

ABSTRACT Ornibactins are linear hydroxamate siderophores produced byBurkholderia cepacia with peptide structures similar to that of pyoverdines produced by the fluorescent pseudomonads. The gene encoding the outer membrane receptor (orbA) was identified, sequenced, and demonstrated to have significant homology with hydroxamate receptors produced by other organisms. The orbAprecursor was predicted to be a protein with a molecular mass of 81 kDa. An orbA mutant was constructed and demonstrated to be unable to take up 59Fe-ornibactins or to grow in medium supplemented with ornibactins. Outer membrane protein profiles from the parent strain, K56-2, revealed an iron-regulated outer membrane protein of 78 kDa that was not detectable in the K56orbA::tp mutant. When this mutant harbored a plasmid containing the orbA gene, the 78-kDa protein was present in the outer membrane protein profiles and the mutant was able to utilize ornibactin to acquire iron. The orbA mutant was less virulent in a chronic respiratory infection model than the parent strain, indicating that ornibactin uptake and utilization are important in the pathogenesis of B. cepacia respiratory infections.

scholarly journals Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase (lpd), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants

2006 ◽  
Vol 74 (2) ◽  
pp. 931-939 ◽  
Author(s):  
P. Hudson ◽  
T. S. Gorton ◽  
L. Papazisi ◽  
K. Cecchini ◽  
S. Frasca ◽  
...  
Keyword(s):  
Insertion Site ◽  
Mycoplasma Gallisepticum ◽  
Dihydrolipoamide Dehydrogenase ◽  
Gene Encoding ◽  
Transposon Insertion ◽  
Transposon Mutants ◽  
Genomic Regions ◽  
Transposon Insertions ◽  
Biologic Function

ABSTRACT To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.

scholarly journals Role of flm Locus in MesophilicAeromonas Species Adherence

2001 ◽  
Vol 69 (1) ◽  
pp. 65-74 ◽  
Author(s):  
Ioannis Gryllos ◽  
Jonathan G. Shaw ◽  
Rosalina Gavı́n ◽  
Susana Merino ◽  
Juan M. Tomás
Keyword(s):  
Epithelial Cells ◽  
Aeromonas Caviae ◽  
Human Epithelial Cells ◽  
Putative Operon ◽  
Adherence Mechanism ◽  
Transposon Insertions ◽  
Flagella Assembly ◽  
Insertion Mutants

ABSTRACT The adherence mechanism of Aeromonas caviae Sch3N to HEp-2 cells was initially investigated through four mini-Tn5 mutants that showed a 10-fold decrease in adherence. These mutants lost motility, flagella, and their lipopolysaccharide (LPS) O antigen (O-Ag). Three genes,flmB-neuA-flmD, were found to be interrupted by the transposon insertions; additionally, two other genes, one lying upstream (flmA) and one downstream (neuB), were found to be clustered in the same operon. While the flmAand flmB genes were present in all mesophilicAeromonas spp. (A. hydrophila, A. caviae, A. veronii bv. veronii, andA. veronii bv. sobria) tested, this was not the case for the neuA-flmD-neuB genes. Construction and characterization of flmB insertion mutants in five other mesophilic Aeromonas strains revealed the loss of motility, flagella, and adherence but did not alter the LPS composition of these strains. Taking the above findings into consideration, we conclude (i) that flagella and possibly the LPS O-Ag are involved in the adherence of the mesophilic Aeromonas to human epithelial cells; (ii)flmA and flmB are genes widely distributed in the mesophilic Aeromonas and are involved in flagella assembly, and thus adherence; and (iii) in A. caviae Sch3N the flmA and flmB genes are found in a putative operon together with neuA, flmD, andneuB and are involved in LPS O-Ag biosynthesis and probably have a role in flagellum assembly.

scholarly journals Construction and Characterization of aHelicobacter pylori clpB Mutant and Role of the Gene in the Stress Response

1998 ◽  
Vol 180 (2) ◽  
pp. 426-429 ◽  
Author(s):  
Elaine Allan ◽  
Peter Mullany ◽  
Soad Tabaqchali
Keyword(s):  
Helicobacter Pylori ◽  
High Temperature ◽  
Stress Response ◽  
Structural Features ◽  
High Temperature Stress ◽  
Gene Encoding ◽  
Increased Sensitivity ◽  
H Pylori

ABSTRACT Antiserum raised against whole Helicobacter pyloricells identified a novel 94-kDa antigen. The nucleotide sequence of the gene encoding the 94-kDa antigen was determined, and analysis of the deduced amino acid sequence revealed structural features typical of the ClpB ATPase family of stress response proteins. An isogenic H. pylori clpB mutant showed increased sensitivity to high-temperature stress, indicating that the clpB gene product functions as a stress response protein in H. pylori.

scholarly journals MFα1, the Gene Encoding the α Mating Pheromone of Candida albicans

2003 ◽  
Vol 2 (6) ◽  
pp. 1350-1360 ◽  
Author(s):  
Sneh L. Panwar ◽  
Melanie Legrand ◽  
Daniel Dignard ◽  
Malcolm Whiteway ◽  
Paul. T. Magee
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Null Mutant ◽  
Mating Pheromone ◽  
Gene Encoding ◽  
Human Fungal Pathogen ◽  
Isolation And Characterization ◽  
Α Pheromone

ABSTRACT Candida albicans, the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus (MTL). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene (KEX2) that is involved in the processing of the α mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the α pheromone gene. Here we report the isolation and characterization of the gene MFα1, which codes for the precursor of the α mating pheromone in C. albicans. Two active α-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans. To examine the role of this gene in mating, we constructed an mfα1 null mutant of C. albicans. The mfα1 null mutant fails to mate as MTLα, while MTLa mfα1 cells are still mating competent. Experiments performed with the synthetic α-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTLα kex2 mutant strain when added exogenously, thereby confirming their roles as α mating pheromones.

scholarly journals Production and Characterization of a Thermostable Alcohol Dehydrogenase That Belongs to the Aldo-Keto Reductase Superfamily

2006 ◽  
Vol 72 (1) ◽  
pp. 233-238 ◽  
Author(s):  
Ronnie Machielsen ◽  
Agustinus R. Uria ◽  
Servé W. M. Kengen ◽  
John van der Oost
Keyword(s):  
Alcohol Dehydrogenase ◽  
Pyrococcus Furiosus ◽  
Enantiomeric Excess ◽  
Physiological Role ◽  
Secondary Alcohols ◽  
Gene Encoding ◽  
Ph Optimum ◽  
Reduction Of Ketones

ABSTRACT The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a molecular mass of 32 kDa. The catalytic activity of the enzyme increases up to 100°C, and a half-life value of 130 min at this temperature indicates its high thermostability. AdhD exhibits a broad substrate specificity with, in general, a preference for the reduction of ketones (pH optimum, 6.1) and the oxidation of secondary alcohols (pH optimum, 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl-acetoin (22.5 U/mg) in the oxidative and reductive reactions, respectively. Gas chromatrography analysis indicated that AdhD produced mainly (S)-2-pentanol (enantiomeric excess, 89%) when 2-pentanone was used as substrate. The physiological role of AdhD is discussed.

scholarly journals σB Modulates Virulence Determinant Expression and Stress Resistance: Characterization of a Functional rsbU Strain Derived from Staphylococcus aureus 8325-4

2002 ◽  
Vol 184 (19) ◽  
pp. 5457-5467 ◽  
Author(s):  
Malcolm J. Horsburgh ◽  
Joanne L. Aish ◽  
Ian J. White ◽  
Les Shaw ◽  
James K. Lithgow ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Minor Role ◽  
Gene Encoding ◽  
Phenotypic Properties ◽  
Relative Level ◽  
General Stress Response ◽  
A Minor ◽  
Derivatives Of

ABSTRACT The accessory sigma factor σB controls a general stress response that is thought to be important for Staphylococcus aureus survival and may contribute to virulence. The strain of choice for genetic studies, 8325-4, carries a small deletion in rsbU, which encodes a positive regulator of σB activity. Consequently, to enable the role of σB in virulence to be addressed, we constructed an rsbU + derivative, SH1000, using a method that does not leave behind an antibiotic resistance marker. The phenotypic properties of SH1000 (8325-4 rsbU +) were characterized and compared to those of 8325-4, the rsbU mutant, parent strain. A recognition site for σB was located in the promoter region of katA, the gene encoding the sole catalase of S. aureus, by primer extension analysis. However, catalase expression and activity were similar in SH1000 (8325-4 rsbU +), suggesting that this promoter may have a minor role in catalase expression under normal conditions. Restoration of σB activity in SH1000 (8325-4 rsbU +) resulted in a marked decrease in the levels of the exoproteins SspA and Hla, and this is likely to be mediated by reduced expression of agr in this strain. By using Western blotting and a sarA-lacZ reporter assay, the levels of SarA were found to be similar in strains 8325-4 and SH1000 (8325-4 rsbU +) and sigB mutant derivatives of these strains. This finding contrasts with previous reports that suggested that SarA expression levels are altered when they are measured transcriptionally. Inactivation of sarA in each of these strains resulted in an expected decrease in agr expression; however, the relative level of agr in SH1000 (8325-4 rsbU +) remained less than the relative levels in 8325-4 and the sigB mutant derivatives. We suggest that SarA is not likely to be the effector in the overall σB-mediated effect on agr expression.

scholarly journals Cytochrome cM downscales photosynthesis under photomixotrophy in Synechocystis sp. PCC 6803

2019 ◽  
Author(s):  
Daniel Solymosi ◽  
Dorota Muth-Pawlak ◽  
Lauri Nikkanen ◽  
Duncan Fitzpatrick ◽  
Ravendran Vasudevan ◽  
...  
Keyword(s):  
Photosynthetic Apparatus ◽  
Wild Type ◽  
Gene Encoding ◽  
Transport Chain ◽  
Similar Phenotype ◽  
Photosynthetic Microorganisms ◽  
Photosynthesis And Respiration ◽  
Pcc 6803

AbstractPhotomixotrophy is a metabolic state, which enables photosynthetic microorganisms to simultaneously perform photosynthesis and metabolism of imported organic carbon substrates. This process is complicated in cyanobacteria, since many, including Synechocystis sp. PCC 6803, conduct photosynthesis and respiration in an interlinked thylakoid membrane electron transport chain. Under photomixotrophy, the cell must therefore tightly regulate electron fluxes from photosynthetic and respiratory complexes. In this study, we show via characterization of photosynthetic apparatus and the proteome, that photomixotrophic growth results in a gradual reduction of the plastoquinone pool in wild-type Synechocystis, which fully downscales photosynthesis over three days of growth. This process is circumvented by deleting the gene encoding cytochrome cM (CytM), a cryptic c-type heme protein widespread in cyanobacteria. ΔCytM maintained active photosynthesis over the three day period, demonstrated by high photosynthetic O2 and CO2 fluxes and effective yields of Photosystem II and Photosystem I. Overall, this resulted in a higher growth rate than wild-type, which was maintained by accumulation of proteins involved in phosphate and metal uptake, and cofactor biosynthetic enzymes. While the exact role of CytM has not been determined, a mutant deficient in the thylakoid-localised respiratory terminal oxidases and CytM (ΔCox/Cyd/CytM) displayed a similar phenotype under photomixotrophy to ΔCytM, demonstrating that CytM is not transferring electrons to these complexes, which has previously been suggested. In summary, the obtained data suggests that CytM may have a regulatory role in photomixotrophy by reducing the photosynthetic capacity of cells.One sentence summaryThe cryptic, highly conserved cytochrome cM completely blocks photosynthesis in Synechocystis under three days of photomixotrophy, possibly by suppressing CO2 assimilation.

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