Effect of lipopolysaccharide mutations and temperature on plasmid transformation efficiency in Pseudomonas aeruginosa

1986 ◽  
Vol 32 (5) ◽  
pp. 436-438 ◽  
Author(s):  
Denise Berry ◽  
Andrew M. Kropinski
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Growth Temperature ◽  
Type Strain ◽  
Wild Type Strain ◽  
Transformation Efficiency ◽  
Wild Type ◽  
Plasmid Transformation

We have isolated, and characterized electrophoretically, two new lipopolysaccharide-defective (rough) mutants of Pseudomonas aeruginosa strain PAO. These strains, AK1401 and AK1414, together with two previously characterized isolates, AK1012 and AK1282, were used as recipients in transformation experiments with plasmid pR01614 DNA. The roughest mutant, AK1282, was not transformable, while the transformation efficiency of AK1012, and to a lesser extent the wild-type strain, was dependent upon the growth temperature. The two new isolates which are less rough than AK1012 were transformed at a frequency equivalent to that of the wild type-strain.

Identification of pilin pools in the membranes of Pseudomonas aeruginosa

1982 ◽  
Vol 152 (2) ◽  
pp. 687-691
Author(s):  
T H Watts ◽  
E A Worobec ◽  
W Paranchych
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Gel Electrophoresis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Outer Membranes

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.

scholarly journals Genetic analysis of amidase mutants ofPseudomonas aeruginosa

1974 ◽  
Vol 23 (3) ◽  
pp. 335-359 ◽  
Author(s):  
Joan L. Betz ◽  
Jane E. Brown ◽  
Patricia H. Clarke ◽  
Martin Day
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genetic Analysis ◽  
Structural Gene ◽  
Type Strain ◽  
Wild Type Strain ◽  
Relative Order ◽  
Regulator Gene ◽  
Wild Type ◽  
Growth Phenotype

SUMMARYMutants ofPseudomonas aeruginosa, which differed in amide growth phenotype from the wild-type strain, were subjected to genetic analysis using the generalized transducing phage F116. The map order of some mutational sites was determined by 3-factor crosses in which a mutation in the linked regulator geneamiRwas used as the outside marker to determine the relative order of mutations in the amidase structural geneamiE. Acetamide-positive transductants were recovered in crosses between amidase-negative strains and strains PhB3(PAC377), V2(PAC353) and V5(PAC356) producing mutant amidases which hydrolyse phenylacetamide and valeramide but not acetamide. Some recombinants carried the mutationamiE16 determining the properties of the mutant B amidase produced by strain B6(PAC351) from which both PhB and V class mutants were derived, while other recombinants produced A amidase determined by the wild-typeamiEgene.

scholarly journals The Atypical Response Regulator AtvR Is a New Player in Pseudomonas aeruginosa Response to Hypoxia and Virulence

2017 ◽  
Vol 85 (8) ◽  
Author(s):  
Gilberto Hideo Kaihami ◽  
Leandro Carvalho Dantas Breda ◽  
José Roberto Fogaça de Almeida ◽  
Thays de Oliveira Pereira ◽  
Gianlucca Gonçalves Nicastro ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Strain ◽  
Wild Type Strain ◽  
Environmental Changes ◽  
Response Regulator ◽  
Classical Pathway ◽  
Wild Type ◽  
Macrophage Infection ◽  
Content Type ◽  
Successful Infection

ABSTRACT Two-component systems are widespread in bacteria, allowing adaptation to environmental changes. The classical pathway is composed of a histidine kinase that phosphorylates an aspartate residue in the cognate response regulator (RR). RRs lacking the phosphorylatable aspartate also occur, but their function and contribution during host-pathogen interactions are poorly characterized. AtvR (PA14_26570) is the only atypical response regulator with a DNA-binding domain in the opportunistic pathogen Pseudomonas aeruginosa. Macrophage infection with the atvR mutant strain resulted in higher levels of tumor necrosis factor alpha secretion as well as increased bacterial clearance compared to those for macrophages infected with the wild-type strain. In an acute pneumonia model, mice infected with the atvR mutant presented increased amounts of proinflammatory cytokines, increased neutrophil recruitment to the lungs, reductions in bacterial burdens, and higher survival rates in comparison with the findings for mice infected with the wild-type strain. Further, several genes involved in hypoxia/anoxia adaptation were upregulated upon atvR overexpression, as seen by high-throughput transcriptome sequencing (RNA-Seq) analysis. In addition, atvR was more expressed in hypoxia in the presence of nitrate and required for full expression of nitrate reductase genes, promoting bacterial growth under this condition. Thus, AtvR would be crucial for successful infection, aiding P. aeruginosa survival under conditions of low oxygen tension in the host. Taken together, our data demonstrate that the atypical response regulator AtvR is part of the repertoire of transcriptional regulators involved in the lifestyle switch from aerobic to anaerobic conditions. This finding increases the complexity of regulation of one of the central metabolic pathways that contributes to Pseudomonas ubiquity and versatility.

scholarly journals Global Analysis of Cellular Factors and Responses Involved in Pseudomonas aeruginosa Resistance to Arsenite

2005 ◽  
Vol 187 (14) ◽  
pp. 4853-4864 ◽  
Author(s):  
Kislay Parvatiyar ◽  
Eyad M. Alsabbagh ◽  
Urs A. Ochsner ◽  
Michelle A. Stegemeyer ◽  
Alan G. Smulian ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pseudomonas Aeruginosa ◽  
Glutathione Reductase ◽  
Type Strain ◽  
Wild Type Strain ◽  
Storage Proteins ◽  
Global Analysis ◽  
Wild Type ◽  
Isogenic Mutants ◽  
The Impact

ABSTRACT The impact of arsenite [As(III)] on several levels of cellular metabolism and gene regulation was examined in Pseudomonas aeruginosa. P. aeruginosa isogenic mutants devoid of antioxidant enzymes or defective in various metabolic pathways, DNA repair systems, metal storage proteins, global regulators, or quorum sensing circuitry were examined for their sensitivity to As(III). Mutants lacking the As(III) translocator (ArsB), superoxide dismutase (SOD), catabolite repression control protein (Crc), or glutathione reductase (Gor) were more sensitive to As(III) than wild-type bacteria. The MICs of As(III) under aerobic conditions were 0.2, 0.3, 0.8, and 1.9 mM for arsB, sodA sodB, crc, and gor mutants, respectively, and were 1.5- to 13-fold less than the MIC for the wild-type strain. A two-dimensional gel/matrix-assisted laser desorption ionization-time of flight analysis of As(III)-treated wild-type bacteria showed significantly (>40-fold) increased levels of a heat shock protein (IbpA) and a putative allo-threonine aldolase (GlyI). Smaller increases (up to 3.1-fold) in expression were observed for acetyl-coenzyme A acetyltransferase (AtoB), a probable aldehyde dehydrogenase (KauB), ribosomal protein L25 (RplY), and the probable DNA-binding stress protein (PA0962). In contrast, decreased levels of a heme oxygenase (HemO/PigA) were found upon As(III) treatment. Isogenic mutants were successfully constructed for six of the eight genes encoding the aforementioned proteins. When treated with sublethal concentrations of As(III), each mutant revealed a marginal to significant lag period prior to resumption of apparent normal growth compared to that observed in the wild-type strain. Our results suggest that As(III) exposure results in an oxidative stress-like response in P. aeruginosa, although activities of classic oxidative stress enzymes are not increased. Instead, relief from As(III)-based oxidative stress is accomplished from the collective activities of ArsB, glutathione reductase, and the global regulator Crc. SOD appears to be involved, but its function may be in the protection of superoxide-sensitive sulfhydryl groups.

scholarly journals γ-Glutamyl Spermine Synthetase PauA2 as a Potential Target of Antibiotic Development against Pseudomonas aeruginosa

2012 ◽  
Vol 56 (10) ◽  
pp. 5309-5314 ◽  
Author(s):  
Xiangyu Yao ◽  
Congran Li ◽  
Jianmei Zhang ◽  
Chung-Dar Lu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Human Serum ◽  
Parental Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Strong Support ◽  
Uptake System ◽  
Wild Type ◽  
Content Type ◽  
Antibiotic Development

ABSTRACTPolyamines are absolute requirements for cell growth. When in excess,Pseudomonas aeruginosapossesses six γ-glutamylpolyamine synthetases (GPSs) encoded by thepauA1-pauA7genes to initiate polyamine catabolism. Recently, thepauA2mutant was reported to lose the capability to grow on spermine (Spm) and spermidine (Spd) as sole carbon and nitrogen sources. Although this mutant grew normally in defined minimal medium and LB broth, growth was completely abolished by the addition of Spm or Spd. These two compounds exert a bactericidal effect (Spm > Spd) on the mutants as demonstrated by MIC measurements (over 500-fold reduction) and time-killing curves. Spm toxicity in thepauA2mutant was attenuated when the major uptake system was further deleted from the strain, suggesting cytoplasmic targets of toxicity. In addition, the synergistic effect of Spm and carbenicillin in the wild-type strain PAO1 was diminished in mutants without functional PauA2. Furthermore, Spm MIC was reduced by 8-fold when the Spm uptake system was deleted from the wild-type strain, suggesting a second target of Spm toxicity in the periplasm. Experiments were also conducted to test the hypothesis that native Spm and Spd in human serum may be sufficient to kill thepauA2mutant. Growth of the mutant was completely inhibited by 40% (vol/vol) human serum, whereas the parental strain required 80%. Colony counts indicated that the mutant but not the parent was in fact killed by human plasma. In addition, carbenicillin MIC against the mutant was reduced by 16-fold in the presence of 20% human serum while that of the parental strain remained unchanged. Taking PauA2 as the template, sequence comparison indicates that putative PauA2 homologues are widespread in a variety of Gram-negative bacteria. In summary, this study reveals the importance of GPS in alleviation of polyamine toxicity when in excess, and it provides strong support to the feasibility of GPS as a molecular target for new antibiotic development.

scholarly journals Quorum-Quenching Acylase Reduces the Virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans Infection Model

2009 ◽  
Vol 53 (11) ◽  
pp. 4891-4897 ◽  
Author(s):  
Evelina Papaioannou ◽  
Mariana Wahjudi ◽  
Pol Nadal-Jimenez ◽  
Gudrun Koch ◽  
Rita Setroikromo ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Caenorhabditis Elegans ◽  
Type Strain ◽  
Wild Type Strain ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Infection Model ◽  
Wild Type ◽  
Strain Pao1

ABSTRACT The Pseudomonas aeruginosa PAO1 gene pvdQ encodes an acyl-homoserine lactone (AHL) acylase capable of degrading N-(3-oxododecanoyl)-l-homoserine lactone by cleaving the AHL amide. PvdQ has been proven to function as a quorum quencher in vitro in a number of phenotypic assays. To address the question of whether PvdQ also shows quorum-quenching properties in vivo, an infection model based on the nematode Caenorhabditis elegans was explored. In a fast-acting paralysis assay, strain PAO1(pMEpvdQ), which overproduces PvdQ, was shown to be less virulent than the wild-type strain. More than 75% of the nematodes exposed to PAO1(pMEpvdQ) survived and continued to grow when using this strain as a food source. Interestingly, in a slow-killing assay monitoring the survival of the nematodes throughout a 4-day course, strain PAO1-ΔpvdQ was shown to be more virulent than the wild-type strain, confirming the role of PvdQ as a virulence-reducing agent. It was observed that larval stage 1 (L1) to L3-stage larvae benefit much more from protection by PvdQ than L4 worms. Finally, purified PvdQ protein was added to C. elegans worms infected with wild-type PAO1, and this resulted in reduced pathogenicity and increased the life span of the nematodes. From our observations we can conclude that PvdQ might be a strong candidate for antibacterial therapy against Pseudomonas infections.

scholarly journals Controlling the hypermutation: Exploitation of the MutS protein in Pseudomonas aeruginosa

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Yue Yuan On ◽  
Martin Welch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Strain ◽  
Wild Type Strain ◽  
Resistant Mutant ◽  
Inducible Promoter ◽  
Wild Type ◽  
Cdna Analysis ◽  
In The Wild ◽  
Mismatch Recognition ◽  
Muts Gene

Pseudomonas aeruginosa infections commonly develop in individuals with cystic fibrosis (CF), and its adaptation in such an unfavourable condition is always found to be related to hypermutation. In fact, most of the hypermutation is due to the defects in mutS gene which involves in the mismatch repair mechanism, causing the acceleration of mutation rate and adaptive evolution. In order to rheostatically express the MutS protein and achieve “hypomutation” (in which the rate of mutation is lower than that of wild type strain), an exogenous mutS gene with rhamnose-inducible promoter was cloned into MPAO1 mutS::Tn mutant strain. Present findings demonstrate that this system is tightly-controlled and stable, with less rifampicin-resistant mutant frequency and more fluorescence intensity from a GFP-tagged MutS expressing cells were observed when the concentration of the inducer increases. Interestingly, the results from Western blot analysis show that less MutS protein is required to suppress hypermutation in the wild type strain, as compared to our construct that behaves similar to the wild type but obviously needs more MutS expression to achieve such state. This indicates that the exogenous MutS might be lacking of other important protein to work efficiently in mismatch recognition. Therefore, based on our cDNA analysis, we found that fdxA gene next to the mutS gene is in the same operon, which could suggest that they might be functionally related in the DNA repair machinery.

scholarly journals c-di-GMP-linked phenotypes are modulated by the interaction between a diguanylate cyclase and a polar hub protein

2017 ◽  
Author(s):  
Gianlucca G. Nicastro ◽  
Gilberto H. Kaihami ◽  
André A. Pulschen ◽  
Jacobo Hernandez-Montelongo ◽  
Ana Laura Boechat ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fine Tuning ◽  
Diguanylate Cyclase ◽  
Wild Type ◽  
Type Iv ◽  
Major Player ◽  
In The Wild ◽  
Specialized Function

Summaryc-di-GMP is a major player in the decision between biofilm and motile lifestyles. Several bacteria present a large number of c-di-GMP metabolizing proteins, thus a fine-tuning of this nucleotide levels may occur. It is hypothesized that some c-di-GMP metabolizing proteins would provide the global c-di-GMP levels inside the cell whereas others would maintain a localized pool, with the resulting c-di-GMP acting at the vicinity of its production. Although attractive, this hypothesis has yet to be proven in Pseudomonas aeruginosa. We found that the diguanylate cyclase DgcP interacts with the cytosolic region of FimV, a peptidoglycan-binding protein involved in type IV pilus assembly. Moreover, DgcP is located at the cell poles in wild type cells, but scattered in the cytoplasm of cells lacking FimV. Overexpression of DgcP leads to the classical phenotypes of high c-di-GMP levels (increased biofilm and impaired motilities) in the wild-type strain, but not in a AfimV background. Therefore, our findings strongly suggest that DgcP is regulated by FimV and may provide the local c-di-GMP pool that can be sensed by other proteins at the cell pole, bringing to light a specialized function for a specific diguanylate cyclase.

The extraction and analysis of lipopolysaccharides from Pseudomonas aeruginosa strain PAO, and three rough mutants

1979 ◽  
Vol 25 (3) ◽  
pp. 390-398 ◽  
Author(s):  
Andrew M. Kropinski ◽  
L. C. Chan ◽  
F. H. Milazzo
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Chemical Analysis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Side Chains ◽  
Wild Type ◽  
Chromatographic Resolution ◽  
Selection For

Three spontaneously arising rough mutants of Pseudomonas aeruginosa have been isolated by selection for resistance to virulent lipopolysaccharide (LPS) specific bacteriophages. In addition, the first phages specific for rough mutants of P. aeruginosa were isolated. Using these phage and autoagglutination patterns in 4% NaCl and acriflavine, these mutants could be clearly distinguished from the wild-type strain and each other. Chemical analysis of the LPS together with chromatographic resolution of the polysaccharide moieties showed alterations in both O-specific side chains and core regions.

scholarly journals Protective Role of Catalase in Pseudomonas aeruginosa Biofilm Resistance to Hydrogen Peroxide

1999 ◽  
Vol 65 (10) ◽  
pp. 4594-4600 ◽  
Author(s):  
James G. Elkins ◽  
Daniel J. Hassett ◽  
Philip S. Stewart ◽  
Herbert P. Schweizer ◽  
Timothy R. McDermott
Keyword(s):  
Hydrogen Peroxide ◽  
Pseudomonas Aeruginosa ◽  
Single Dose ◽  
Cell Viability ◽  
Catalase Activity ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Planktonic Cells

ABSTRACT The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H2O2) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibility to H2O2. Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H2O2, whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H2O2. The katB mutant, lacking the H2O2-inducible catalase KatB, was similar to the wild-type strain with respect to H2O2 resistance. The katA mutant possessed undetectable catalase activity. PlanktonickatA mutant cultures were hypersusceptible to a single dose of 50 mM H2O2, while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H2O2. Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, andlacZ reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H2O2, suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a katB::lacZtranscriptional reporter strain confirmed katB induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H2O2, while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of β-galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H2O2, particularly at high H2O2 concentrations; KatB is induced in both planktonic and biofilm cells in response to H2O2 insult, but plays a relatively small role in biofilm resistance; and KatB is important to either planktonic cells or biofilm cells for acquired antioxidant resistance when initial levels of H2O2 are sublethal.

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