scholarly journals The PhlA Hemolysin from the Entomopathogenic Bacterium Photorhabdusluminescens Belongs to the Two-Partner Secretion Family of Hemolysins

2002 ◽  
Vol 184 (14) ◽  
pp. 3871-3878 ◽  
Author(s):  
Julien Brillard ◽  
Eric Duchaud ◽  
Noël Boemare ◽  
Frank Kunst ◽  
Alain Givaudan
Keyword(s):  
Hemolytic Activity ◽  
Pathogenic Bacteria ◽  
Fluorescent Protein ◽  
Gene Encoding ◽  
Rich Media ◽  
The Family ◽  
Insect Interactions ◽  
Green Fluorescent

ABSTRACT Photorhabdus is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae. Bacterial hemolysins found in numerous pathogenic bacteria are often virulence factors. We describe here the nucleotide sequence and the molecular characterization of the Photorhabdus luminescens phlBA operon, a locus encoding a hemolysin which shows similarities to the Serratia type of hemolysins. It belongs to the two-partner secretion (TPS) family of proteins. In low-iron conditions, a transcriptional induction of the phlBA operon was observed by using the chloramphenicol acetyltransferase reporter gene, causing an increase in PhlA hemolytic activity compared to iron-rich media. A spontaneous phase variant of P. luminescens was deregulated in phlBA transcription. The phlA mutant constructed by allelic exchange remained highly pathogenic after injection in the lepidopteran Spodoptera littoralis, indicating that PhlA hemolysin is not a major virulence determinant. Using the gene encoding green fluorescent protein as a reporter, phlBA transcription was observed in hemolymph before insect death. We therefore discuss the possible role of PhlA hemolytic activity in the bacterium-nematode-insect interactions.

scholarly journals Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

2002 ◽  
Vol 184 (7) ◽  
pp. 1998-2004 ◽  
Author(s):  
Takako Murakami ◽  
Koki Haga ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Vegetative Growth ◽  
Fluorescent Protein ◽  
Specific Expression ◽  
Rich Media ◽  
Green Fluorescent ◽  
High Level ◽  
Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

scholarly journals Identification and Characterization of a Novel Gene Involved in the trans-Specific Nematicidal Activity of Photorhabdus luminescens LN2

2009 ◽  
Vol 75 (12) ◽  
pp. 4221-4223 ◽  
Author(s):  
Xuehong Qiu ◽  
Richou Han ◽  
Xun Yan ◽  
Mingxing Liu ◽  
Li Cao ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transposon Mutagenesis ◽  
Nematicidal Activity ◽  
Photorhabdus Luminescens ◽  
Gene Encoding ◽  
Heterorhabditis Indica ◽  
Green Fluorescent ◽  
Identification And Characterization

ABSTRACT Photorhabdus luminescens subsp. akhurstii LN2 from Heterorhabditis indica LN2 showed nematicidal activity against axenic Heterorhabditis bacteriophora H06 infective juveniles (IJs). Transposon mutagenesis identified an LN2 mutant that supports the growth of H06 nematodes. Tn5 disrupted the namA gene, encoding a novel 364-residue protein and involving the nematicidal activity. The green fluorescent protein-labeled namA mutant was unable to colonize the intestines of H06 IJs.

scholarly journals A Mig-14-like protein (PA5003) affects antimicrobial peptide recognition in Pseudomonas aeruginosa

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2647-2657 ◽  
Author(s):  
Nicholas Jochumsen ◽  
Yang Liu ◽  
Søren Molin ◽  
Anders Folkesson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Peptides ◽  
Antimicrobial Peptide ◽  
Pathogenic Bacteria ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Flow Chamber ◽  
Gene Encoding ◽  
Conventional Antibiotics ◽  
Green Fluorescent

The evolution of antibiotic resistance in pathogenic bacteria is a growing global health problem which is gradually making the treatment of infectious diseases less efficient. Antimicrobial peptides are small charged molecules found in organisms from the complete phylogenetic spectrum. The peptides are attractive candidates for novel drug development due to their activity against bacteria that are resistant to conventional antibiotics, and reports of peptide resistance are rare in the clinical setting. Paradoxically, many clinically relevant bacteria have mechanisms that can recognize and respond to the presence of cationic antimicrobial peptides (CAMPs) in the environment by changing the properties of the microbial surface thereby increasing the tolerance of the microbes towards the peptides. In Pseudomonas aeruginosa an essential component of this inducible tolerance mechanism is the lipopolysaccharide modification operon arnBCADTEF–PA3559 which encodes enzymes required for LPS alterations leading to increased antimicrobial peptide tolerance. The expression of the operon is induced by the presence of CAMPs in the environment but the molecular mechanisms underlying the cellular recognition of the peptides are poorly elucidated. In this work, we investigate the factors influencing arnB expression by transposon mutagenesis and arnB promoter green fluorescent protein reporters. We have identified a novel gene encoding a Mig-14-like protein that is required for recognition of the CAMPs colistin and Novispirin G10 by P. aeruginosa. Moreover, we show that this gene is also required for the formation of CAMP-tolerant subpopulations in P. aeruginosa hydrodynamic flow chamber biofilms.

scholarly journals Characterization of Saccharomyces cerevisiae Npa2p (Urb2p) Reveals a Low-Molecular-Mass Complex Containing Dbp6p, Npa1p (Urb1p), Nop8p, and Rsa3p Involved in Early Steps of 60S Ribosomal Subunit Biogenesis

2006 ◽  
Vol 27 (4) ◽  
pp. 1207-1221 ◽  
Author(s):  
Iván V. Rosado ◽  
Christophe Dez ◽  
Simon Lebaron ◽  
Michèle Caizergues-Ferrer ◽  
Yves Henry ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Null Allele ◽  
Fluorescent Protein ◽  
Gel Filtration ◽  
Ribosomal Subunit ◽  
Synthetic Lethal ◽  
Five Factors ◽  
Green Fluorescent

ABSTRACT We report the characterization of the yeast Npa2p (Urb2p) protein, which is essential for 60S ribosomal subunit biogenesis. We identified this protein in a synthetic lethal screening with the rsa3 null allele. Rsa3p is a genetic partner of the putative RNA helicase Dbp6p. Mutation or depletion of Npa2p leads to a net deficit in 60S subunits and a decrease in the levels all 27S pre-rRNAs and mature 25S and 5.8S rRNAs. This is likely due to instability of early pre-60S particles. Consistent with a role of Npa2p in 60S subunit biogenesis, green fluorescent protein-tagged Npa2p localizes predominantly to the nucleolus and TAP-tagged Npa2p sediments with large complexes in sucrose gradients and is associated mainly with 27SA2 pre-rRNA-containing preribosomal particles. In addition, we reveal a genetic synthetic interaction between Npa2p, several factors required for early steps of 60S subunit biogenesis (Dbp6p, Dbp7p, Dbp9p, Npa1p, Nop8p, and Rsa3p), and the 60S protein Rpl3p. Furthermore, coimmunoprecipitation and gel filtration analyses demonstrated that at least Npa2p, Dbp6p, Npa1p, Nop8p, and Rsa3p are present together in a subcomplex of low molecular mass whose integrity is independent of RNA. Our results support the idea that these five factors work in concert during the early steps of 60S subunit biogenesis.

scholarly journals The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1377
Author(s):  
Adriano M. Gigante ◽  
Francisco Olivença ◽  
Maria João Catalão ◽  
Paula Leandro ◽  
José Moniz-Pereira ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Structural Similarity ◽  
Lytic Cycle ◽  
Specific Lysis ◽  
Double Stranded Dna ◽  
The Family ◽  
N Terminus ◽  
Green Fluorescent

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

scholarly journals 20-HETE-mediated cytotoxicity and apoptosis in ischemic kidney epithelial cells

2008 ◽  
Vol 294 (3) ◽  
pp. F562-F570 ◽  
Author(s):  
Vani Nilakantan ◽  
Cheryl Maenpaa ◽  
Guangfu Jia ◽  
Richard J. Roman ◽  
Frank Park
Keyword(s):  
Caspase 3 ◽  
Fluorescent Protein ◽  
Ischemia Reperfusion ◽  
Atp Depletion ◽  
Cellular Damage ◽  
Apoptotic Pathway ◽  
Injury Model ◽  
Green Fluorescent

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK1 cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK1 cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release ( P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 μM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 μM) also inhibited cytotoxicity significantly ( P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase ( P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells ( P < 0.05). This was abolished in the presence of HET-0016 ( P < 0.05) or MnTMPyP ( P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals.

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