A Protease-Resistant Catalase, KatA, Released upon Cell Lysis during Stationary Phase Is Essential for Aerobic Survival of a Pseudomonas aeruginosa oxyR Mutant at Low Cell Densities

ABSTRACT A Pseudomonas aeruginosa oxyR mutant was dramatically sensitive to H2O2, despite possessing wild-type catalase activity. Oxygen-dependent oxyR phenotypes also included an inability to survive aerobic serial dilution in Luria broth and to resist aminoglycosides. Plating the oxyR mutant after serial dilution in its own spent culture supernatant, which contained the major catalase KatA, or under anaerobic conditions allowed for survival. KatA was resistant to sodium dodecyl sulfate, proteinase K, pepsin, trypsin, chymotrypsin and the neutrophil protease cathepsin G. When provided in trans and expressed constitutively, the OxyR-regulated genes katB,ahpB, and ahpCF could not restore both the serial dilution defect and H2O2 resistance; only oxyR itself could do so. The aerobic dilution defect could be complemented, in part, by only ahpB andahpCF, suggesting that the latter gene products could possess a catalase-like activity. Aerobic Luria broth was found to generate ∼1.2 μM H2O2 min−1via autoxidation, a level sufficient to kill serially dilutedoxyR and oxyR katA bacteria and explain the molecular mechanism behind the aerobic serial dilution defect. Taken together, our results indicate that inactivation of OxyR rendersP. aeruginosa exquisitely sensitive to both H2O2 and aminoglycosides, which are clinically and environmentally important antimicrobials.

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Identification of pilin pools in the membranes of Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.152.2.687-691.1982 ◽

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.

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Cytoplasmic p53 polypeptide is associated with ribosomes.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3146 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3146-3154 ◽

Cited By ~ 39

Author(s):

B M Fontoura ◽

C A Atienza ◽

E A Sorokina ◽

T Morimoto ◽

R B Carroll

Keyword(s):

Sodium Dodecyl ◽

Proteinase K ◽

Mutant P53 ◽

Tumor Suppressor P53 ◽

Wild Type ◽

Mcf7 Cells ◽

Gapdh Mrna ◽

5.8S Rrna ◽

Proteinase K Treatment ◽

Covalently Linked

Our previous finding that the tumor suppressor p53 is covalently linked to 5.8S rRNA suggested functional association of p53 polypeptide with ribosomes. p53 polypeptide is expressed at low basal levels in the cytoplasm of normal growing cells in the G1 phase of the cell cycle. We report here that cytoplasmic wild-type p53 polypeptide from both rat embryo fibroblasts and MCF7 cells and the A135V transforming mutant p53 polypeptide were found associated with ribosomes to various extents. Treatment of cytoplasmic extracts with RNase or puromycin in the presence of high salt, both of which are known to disrupt ribosomal function, dissociated p53 polypeptide from the ribosomes. In immunoprecipitates of p53 polypeptide-associated ribosomes, 5.8S rRNA was detectable only after proteinase K treatment, indicating all of the 5.8S rRNA in p53-associated ribosomes is covalently linked to protein. While 5.8S rRNA linked to protein was found in the immunoprecipitates of either wild-type or A135V mutant p53 polypeptide associated with ribosomes, little 5.8S rRNA was found in the immunoprecipitates of the slowly sedimenting p53 polypeptide, which was not associated with ribosomes. In contrast, 5.8S rRNA was liberated from bulk ribosomes by 1% sodium dodecyl sulfate, without digestion with proteinase K, indicating that these ribosomes contain 5.8S rRNA, which is not linked to protein. Immunoprecipitation of p53 polypeptide coprecipitated a small fraction of ribosomes. p53 mRNA immunoprecipitated with cytoplasmic p53 polypeptide, while GAPDH mRNA did not. These results show that cytoplasmic p53 polypeptide is associated with a subset of ribosomes, having covalently modified 5.8S rRNA.

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Overexpression of Methanococcus voltaeFlagellin Subunits in Escherichia coli and Pseudomonas aeruginosa: a Source of Archaeal Preflagellin

Journal of Bacteriology ◽

10.1128/jb.181.14.4146-4153.1999 ◽

1999 ◽

Vol 181 (14) ◽

pp. 4146-4153 ◽

Cited By ~ 18

Author(s):

Douglas P. Bayley ◽

Ken F. Jarrell

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Sodium Dodecyl ◽

Leader Peptide ◽

Fusion Product ◽

Peptidase Activity ◽

Wild Type ◽

Sds Page ◽

In The Wild ◽

Prepilin Peptidase

ABSTRACT Methanococcus voltae is a flagellated member of theArchaea. Four highly similar flagellin genes have previously been cloned and sequenced, and the presence of leader peptides has been demonstrated. While the flagellins of M. voltae are predicted from their gene sequences to be approximately 22 to 25 kDa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified flagella revealed flagellin subunits with apparent molecular masses of 31 and 33 kDa. Here we describe the expression of a M. voltae flagellin in the bacteria Escherichia coli and Pseudomonas aeruginosa. Both of these systems successfully generated a specific expression product with an apparently uncleaved leader peptide migrating at approximately 26.5 kDa. This source of preflagellin was used to detect the presence of preflagellin peptidase activity in the membranes of M. voltae. In addition to the native flagellin, a hybrid flagellin gene containing the sequence encoding theM. voltae FlaB2 mature protein fused to the P. aeruginosa pilin (PilA) leader peptide was constructed and transformed into both wild-type P. aeruginosa and a prepilin peptidase (pilD) mutant of P. aeruginosa. Based on migration in SDS-PAGE, the leader peptide appeared to be cleaved in the wild-type cells. However, the archaeal flagellin could not be detected by immunoblotting when expressed in thepilD mutant, indicating a role of the peptidase in the ultimate stability of the fusion product. When the +5 position of the mature flagellin portion of the pilin-flagellin fusion was changed from glycine to glutamic acid (as in the P. aeruginosa pilin) and expressed in both wild-type and pilD mutant P. aeruginosa, the product detected by immunoblotting migrated slightly more slowly in the pilD mutant, indicating that the fusion was likely processed by the prepilin peptidase present in the wild type. Potential assembly of the cleaved fusion product by the type IV pilin assembly system in a P. aeruginosaPilA-deficient strain was tested, but no filaments were noted on the cell surface by electron microscopy.

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Assembly of XcpR in the Cytoplasmic Membrane Is Required for Extracellular Protein Secretion in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.181.2.382-388.1999 ◽

1999 ◽

Vol 181 (2) ◽

pp. 382-388 ◽

Cited By ~ 46

Author(s):

Geneviève Ball ◽

Virginie Chapon-Hervé ◽

Sophie Bleves ◽

Gérard Michel ◽

Marc Bally

Keyword(s):

Pseudomonas Aeruginosa ◽

Secretory Pathway ◽

Cytoplasmic Membrane ◽

Extracellular Proteins ◽

Membrane Association ◽

Stoichiometric Ratio ◽

Gene Products ◽

Type Ii ◽

Wild Type ◽

The Relationship

ABSTRACT A broad range of extracellular proteins secreted byPseudomonas aeruginosa use the type II or general secretory pathway (GSP) to reach the medium. This pathway requires the expression of at least 12 xcp gene products. XcpR, a putative nucleotide-binding protein, is essential for the secretion process across the outer membrane even though the protein contains no hydrophobic sequence that could target or anchor it to the bacterial envelope. For a better understanding of the relationship between XcpR and the other Xcp proteins which are located in the envelope, we have studied its subcellular localization. In a wild-type P. aeruginosa strain, XcpR was found associated with the cytoplasmic membrane. This association depends on the presence of the XcpY protein, which also appears to be necessary for XcpR stability. Functional complementation of an xcpY mutant required the XcpY protein to be expressed at a low level. Higher expression precluded the complementing activity of XcpY, although membrane association of XcpR was restored. This behavior suggested that an excess of free XcpY might interfere with the secretion by formation of inactive XcpR-XcpY complexes which cannot properly interact with their natural partners in the secretion machinery. These data show that a precise stoichiometric ratio between several components may be crucial for the functioning of the GSP.

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Identification of a baculovirus polyhedron formation mutant with a novel phenotype

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166440 ◽

1996 ◽

Vol 54 ◽

pp. 796-797

Author(s):

James M. Slavicek ◽

Melissa J. Mercer ◽

Mary Ellen Kelly

Keyword(s):

Viral Gene ◽

Gene Products ◽

Type Number ◽

Infection Cycle ◽

Wild Type ◽

Protein Matrix ◽

Insect Midgut ◽

Viral Particles ◽

Budded Virus ◽

Viral Form

Nucleopolyhedroviruses (NPV, family Baculoviridae) produce two morphological forms, a budded virus form and a viral form that is occluded into a paracrystalline protein matrix. This structure is termed a polyhedron and is composed primarily of the protein polyhedrin. Insects are infected by NPVs after ingestion of the polyhedron and release of the occluded virions through dissolution of the polyhedron in the alkaline environment of the insect midgut. Early after infection the budded virus form is produced. It buds through the plasma membrane and then infects other cells. Later in the infection cycle the occluded form of the virus is generated (reviewed by Blissard and Rohrmann, 1990).The processes of polyhedron formation and virion occlusion are likely to involve a number of viral gene products. However, only two genes, the polyhedrin gene and 25K FP gene, have been identified to date that are necessary for the wild type number of polyhedra to be formed and viral particles occluded.

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XYLANASE, AN ENVIRONMENTALLY FRIENDLY BLEACHING AGENT FOR PULP AND PAPER: Characterization And Stabilization Study Of Bacillus licheniformis I-5 CRUDE Xylanase

Jurnal Rekayasa Lingkungan ◽

10.29122/jrl.v7i3.1961 ◽

2018 ◽

Vol 7 (3) ◽

Author(s):

Budiasih Wahyuntari., dkk

Keyword(s):

Thermal Stability ◽

Bacillus Licheniformis ◽

Hot Spring ◽

Sodium Dodecyl ◽

Luria Broth ◽

Crude Enzyme ◽

Half Time ◽

Triton X100 ◽

Sds Page ◽

Xylanolytic Enzymes

Isolate I-5 was isolated from Ciseeng hot spring, West Java and was identified as Bacillus licheniformis I-5. The isolate produces extracellular xylanolytic enzymes on Oatspelt containing Luria broth agar medium. Optimal activity of the crude enzyme was Â observed at 50ÂºC and pH 7. The effect of sodium dodecyl sulphate, b-mercaptoethanol and Triton-X100 were observed. Incubating the crude enzyme in 1.5% SDS and 1.5% b-mercaptoethanol at 50oC for 90 minutes then adding Triton-X100 at final concentration of 3.5% for 45 minutes only reduced 5.75% of the initial enzyme activity. SDS/PAGE and zymogram analysis showed that at least two xylanolytic enzymes presence in the crude enzyme. The molecular weight of the enzyme was estimated about 127 and 20kD. The enzyme hydrolysed xylan into xylobiose, xylotriose and other longer xylooligosaccharides. Thermal stability of the crude enzyme was observed at 50, 60, and 70oC and pH 7 and 8. The results showed that the half time of the crude enzyme incubated at 50, 60, and 70oC pH 7 was 2 hours 55 minutes; 2 hours 33 minutes and 1 hour 15 minutes respectively. The half time at 50, 60 and 70oC, pH 8 was 2 hours 48 minutes; 1 hour 22 minutes and 1 hour 9 minutes respectively.keywords: Xilanase, Bacillus licheniformis I-5, thermal stability

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Faculty Opinions recommendation of Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1011135.192160 ◽

2003 ◽

Author(s):

Leo Eberl

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Type Iv Pili ◽

Wild Type ◽

Type Iv

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Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants

Molecular Microbiology ◽

10.1046/j.1365-2958.2003.03525.x ◽

2003 ◽

Vol 48 (6) ◽

pp. 1511-1524 ◽

Cited By ~ 581

Author(s):

Mikkel Klausen ◽

Arne Heydorn ◽

Paula Ragas ◽

Lotte Lambertsen ◽

Anders Aaes-Jørgensen ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Type Iv Pili ◽

Wild Type ◽

Type Iv

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ParB deficiency in Pseudomonas aeruginosa destabilizes the partner protein ParA and affects a variety of physiological parameters

Microbiology ◽

10.1099/mic.0.024661-0 ◽

2009 ◽

Vol 155 (4) ◽

pp. 1080-1092 ◽

Cited By ~ 28

Author(s):

A. A. Bartosik ◽

J. Mierzejewska ◽

C. M. Thomas ◽

G. Jagura-Burdzy

Keyword(s):

Pseudomonas Aeruginosa ◽

Fluorescence Microscopy ◽

Bacterial Growth ◽

Complete Loss ◽

Physiological Parameters ◽

Wild Type ◽

Partial Removal ◽

Partner Protein ◽

The One ◽

Mutant Phenotypes

Deletions leading to complete or partial removal of ParB were introduced into the Pseudomonas aeruginosa chromosome. Fluorescence microscopy of fixed cells showed that ParB mutants lacking the C-terminal domain or HTH motif formed multiple, less intense foci scattered irregularly, in contrast to the one to four ParB foci per cell symmetrically distributed in wild-type P. aeruginosa. All parB mutations affected both bacterial growth and swarming and swimming motilities, and increased the production of anucleate cells. Similar effects were observed after inactivation of parA of P. aeruginosa. As complete loss of ParA destabilized its partner ParB it was unclear deficiency of which protein is responsible for the mutant phenotypes. Analysis of four parB mutants showed that complete loss of ParB destabilized ParA whereas three mutants that retained the N-terminal 90 aa of ParB did not. As all four parB mutants demonstrate the same defects it can be concluded that either ParB, or ParA and ParB in combination, plays an important role in nucleoid distribution, growth and motility in P. aeruginosa.

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