scholarly journals Mutational Analysis of a Bifunctional Ferrisiderophore Receptor and Signal-Transducing Protein from Pseudomonas aeruginosa

2005 ◽  
Vol 187 (13) ◽  
pp. 4514-4520 ◽  
Author(s):  
H. Ellen James ◽  
Paul A. Beare ◽  
Lois W. Martin ◽  
Iain L. Lamont
Keyword(s):  
Signal Transduction ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Mutational Analysis ◽  
Signal Transduction Pathway ◽  
Insertion Mutagenesis ◽  
Transduction Pathway ◽  
Loss Of Function ◽  
Signaling Function ◽  
Linker Insertion Mutagenesis

ABSTRACT The FpvA protein of Pseudomonas aeruginosa strain PAO1 mediates uptake of a siderophore, ferripyoverdine. It is also a component of a signal transduction pathway that controls production of an exotoxin, a protease, pyoverdine, and FpvA itself. The purpose of the research described here was to dissect these different functions of FpvA. Signaling involves an N-terminal domain of FpvA, and it was shown that this domain is probably located in the periplasm, as expected. Short peptides were inserted at 36 sites within FpvA by linker insertion mutagenesis. The effects of these mutations on the presence of FpvA in the outer membrane, on FpvA-mediated uptake of ferripyoverdine, and on pyoverdine synthesis and gene expression were determined. Five of the mutations resulted in the absence of FpvA from the outer membrane of the bacteria. All of the remaining mutations eliminated either the transport or signaling function of FpvA and most affected both functions. Three mutations prevented transport of ferripyoverdine but had no effect on the signal transduction pathway showing that transport of ferripyoverdine is not required for the trans-membrane signaling process. Conversely, eight mutations affected pyoverdine-mediated signaling but had no effect on transport of ferripyoverdine. These data show that insertions throughout FpvA resulted in loss of function and that signaling and transport are separate and discrete functions of FpvA.

scholarly journals Further Characterization of the Signaling Proteolysis Step in the Aspergillus nidulans pH Signal Transduction Pathway

2007 ◽  
Vol 6 (6) ◽  
pp. 960-970 ◽  
Author(s):  
María M. Peñas ◽  
América Hervás-Aguilar ◽  
Tatiana Múnera-Huertas ◽  
Elena Reoyo ◽  
Miguel Á. Peñalva ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Aspergillus Nidulans ◽  
Cysteine Protease ◽  
Mutational Analysis ◽  
Signal Transduction Pathway ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Western Blots ◽  
C Terminus ◽  
Catalytic Cysteine

ABSTRACT The Aspergillus nidulans pH-responsive transcription factor PacC is modulated by limited, two-step proteolysis. The first, pH-regulated cleavage occurs in the 24-residue highly conserved “signaling protease box” in response to the alkaline pH signal. This is transduced by the Pal signaling pathway, containing the predicted calpain-like cysteine protease and likely signaling protease, PalB. In this work, we carried out classical mutational analysis of the putative signaling protease PalB, and we describe 9 missense and 18 truncating loss-of-function (including null) mutations. Mutations in the region of and affecting directly the predicted catalytic cysteine strongly support the deduction that PalB is a cysteine protease. Truncating and missense mutations affecting the C terminus highlight the importance of this region. Analysis of three-hemagglutinin-tagged PalB in Western blots demonstrates that PalB levels are independent of pH and Pal signal transduction. We have followed the processing of MYC3-tagged PacC in Western blots. We show unequivocally that PalB is essential for signaling proteolysis and is definitely not the processing protease. In addition, we have replaced 15 residues of the signaling protease box of MYC3-tagged PacC (pacC900) with alanine. The majority of these substitutions are silent. Leu481Ala, Tyr493Ala, and Gln499Ala result in delayed PacC processing in response to shifting from acidic to alkaline medium, as determined by Western blot analysis. Leu498Ala reduces function much more markedly, as determined by plate tests and processing recalcitrance. Excepting Leu498, this demonstrates that PacC signaling proteolysis is largely independent of sequence in the cleavage region.

scholarly journals Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii.

1995 ◽  
Vol 131 (2) ◽  
pp. 427-440 ◽  
Author(s):  
G J Pazour ◽  
O A Sineshchekov ◽  
G B Witman
Keyword(s):  
Signal Transduction ◽  
Chlamydomonas Reinhardtii ◽  
Insertional Mutagenesis ◽  
Mutational Analysis ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Normal Manner ◽  
Normal Light ◽  
Phototransduction Pathway

Chlamydomonas has two photobehavioral responses, phototaxis and photoshock. Rhodopsin is the photoreceptor for these responses and the signal transduction process involves transmembrane Ca2+ fluxes. This causes transient changes in flagellar beating, ultimately resulting in phototaxis or photoshock. To identify components that make up this signal transduction pathway, we generated nonphototactic strains by insertional mutagenesis. Seven new phototaxis genes were identified (ptx2-ptx8); alleles of six of these are tagged by the transforming DNA and therefore should be easily cloned. To order the mutants in the pathway, we characterized them electrophysiologically, behaviorally, and structurally, ptx5, ptx6, and ptx7 have normal light-induced photoreceptor currents (PRC) and flagellar currents (FC) but their pattern of swimming does not change in the normal manner when the intraflagellar Ca2+ concentration is decreased, suggesting that they have defects in the ability of their axonemes to respond to changes in Ca2+ concentration. ptx2 and ptx8 lack the FC but have normal PRCs, suggesting that they are defective in the flagellar Ca2+ channel or some factor that regulates it. ptx4 mutants have multiple eye-spots. ptx3 mutants are defective in a component essential for phototaxis but bypassed during photoshock; this component appears to be located downstream of the PRC but upstream of the axoneme.

scholarly journals A Ras-mediated signal transduction pathway is involved in the control of sex myoblast migration in Caenorhabditis elegans

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2823-2833 ◽  
Author(s):  
M. Sundaram ◽  
J. Yochem ◽  
M. Han
Keyword(s):  
Signal Transduction ◽  
Caenorhabditis Elegans ◽  
Growth Factor Receptor ◽  
Signal Transduction Pathway ◽  
Body Region ◽  
Transduction Pathway ◽  
Loss Of Function ◽  
Ras Pathway ◽  
Mosaic Analysis ◽  
Sex Myoblasts

Sex myoblast migration in the Caenorhabditis elegans hermaphrodite represents a simple, genetically amenable model system for studying how cell migration is regulated during development. Two separable components of sex myoblast guidance have been described: a gonad-independent mechanism sufficient for the initial anterior migration to the mid-body region, and a gonad-dependent mechanism required for precise final positioning (J. H. Thomas, M. J. Stern and H. R. Horvitz (1990) Cell 62, 1041–1052). Here, we demonstrate a role for a Ras-mediated signal transduction pathway in controlling sex myoblast migration. Loss-of-function mutations in let-60 ras, ksr-1, lin-45 raf, let-537/mek-2 or sur-1/mpk-1 cause defects in sex myoblast final positions that resemble those seen in gonad-ablated animals, while constitutively active let-60 ras(G13E) trans-genes allow fairly precise positioning to occur in the absence of the gonad. A mosaic analysis demonstrated that let-60 ras is required within the sex myoblasts to control proper positioning. Our results suggest that gonadal signals normally stimulate let-60 ras activity in the sex myoblasts, thereby making them competent to sense or respond to positional cues that determine the precise endpoint of migration. let-60 ras may have additional roles in sex myoblast guidance as well. Finally, we have also investigated genetic interactions between let-60 ras and other genes important for sex myoblast migration, including egl-15, which encodes a fibroblast growth factor receptor tyrosine kinase (D. L. DeVore, H. R. Horvitz and M. J. Stern (1995) Cell 83, 611–623). Since mutations reducing Ras pathway activity cause a different phenotype than those reducing egl-15 activity and since constitutive Ras activity only partially suppresses the migration defects of egl-15 mutants, we argue that let-60 ras and egl-15 do not act together in a single linear pathway.

scholarly journals The Global Carbon Metabolism Regulator Crc Is a Component of a Signal Transduction Pathway Required for Biofilm Development by Pseudomonas aeruginosa

2000 ◽  
Vol 182 (2) ◽  
pp. 425-431 ◽  
Author(s):  
George A. O'Toole ◽  
Karine A. Gibbs ◽  
Paul W. Hager ◽  
Paul V. Phibbs ◽  
Roberto Kolter
Keyword(s):  
Signal Transduction ◽  
Pseudomonas Aeruginosa ◽  
Carbon Metabolism ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Plastic Surface ◽  
Pilus Biogenesis ◽  
Biofilm Development

ABSTRACT The transition from a planktonic (free-swimming) existence to growth attached to a surface in a biofilm occurs in response to environmental factors, including the availability of nutrients. We show that the catabolite repression control (Crc) protein, which plays a role in the regulation of carbon metabolism, is necessary for biofilm formation in Pseudomonas aeruginosa. Using phase-contrast microscopy, we found that a crc mutant only makes a dispersed monolayer of cells on a plastic surface but does not develop the dense monolayer punctuated by microcolonies typical of the wild-type strain. This is a phenotype identical to that observed in mutants defective in type IV pilus biogenesis. Consistent with this observation, crc mutants are defective in type IV pilus-mediated twitching motility. We show that this defect in type IV pilus function is due (at least in part) to a decrease inpilA (pilin) transcription. We propose that nutritional cues are integrated by Crc as part of a signal transduction pathway that regulates biofilm development.

scholarly journals Dissecting the VanRS Signal Transduction Pathway with Specific Inhibitors

1999 ◽  
Vol 181 (2) ◽  
pp. 627-631 ◽  
Author(s):  
Andrew T. Ulijasz ◽  
Bernard Weisblum
Keyword(s):  
Signal Transduction ◽  
Pseudomonas Aeruginosa ◽  
Transcriptional Activation ◽  
Signal Transduction Pathway ◽  
Phosphoryl Transfer ◽  
Transduction Pathway ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
Specific Inhibitors

ABSTRACT The VanRS two-component signal transduction pathway fromEnterococcus faecium was reconstituted in vitro from partially purified components and shown to be inhibited by the halophenyl isothiazolone LY-266,400, inhibitor A, a compound shown previously to reduce expression of the AlgR1-AlgR2 two-component signal transduction pathway in Pseudomonas aeruginosa (S. Roychoudhury, N. A. Zielinski, A. J. Ninfa, N. E. Allen, L. N. Jungheim, T. I. Nicas, and A. M. Chakrabarty, Proc. Natl. Acad. Sci. USA 90:965–969, 1993). Inhibitor A attenuates phosphoryl transfer from VanS∼P to VanR by its action on the ability of VanR to accept. We observed an apparent stimulatory effect of inhibitor A on VanS autophosphorylation which is attributable to the accumulation of VanS∼P as an intermediate unable to transfer Pi to the inhibited VanR. Thus, inhibitor A acts on the second of two sequential steps which lead to transcriptional activation of the VanHAXYZ gene cluster and the resultant expression of vancomycin resistance.

scholarly journals Endogenous membrane stress induces T6SS activity inPseudomonas aeruginosa

2020 ◽  
Vol 118 (1) ◽  
pp. e2018365118
Author(s):  
Anne-Sophie Stolle ◽  
Bradley Thomas Meader ◽  
Jonida Toska ◽  
John J. Mekalanos
Keyword(s):  
Signal Transduction ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Signal Transduction Pathway ◽  
Polymyxin B ◽  
Transduction Pathway ◽  
Dynamic Activity ◽  
Membrane Perturbation ◽  
Tit For Tat ◽  
Outer Membrane Biogenesis

The type 6 secretion system (T6SS) is a dynamic organelle encoded by many gram-negative bacteria that can be used to kill competing bacterial prey species in densely occupied niches. Some predatory species, such asVibrio cholerae, use their T6SS in an untargeted fashion while in contrast,Pseudomonas aeruginosaassembles and fires its T6SS apparatus only after detecting initial attacks by other bacterial prey cells; this targeted attack strategy has been termed the T6SS tit-for-tat response. Molecules that interact with theP. aeruginosaouter membrane such as polymyxin B can also trigger assembly of T6SS organelles via a signal transduction pathway that involves protein phosphorylation. Recent work suggests that a phospholipase T6SS effector (TseL) ofV. choleraecan induce T6SS dynamic activity inP. aeruginosawhen delivered to or expressed in the periplasmic space of this organism. Here, we report that inhibiting expression of essential genes involved in outer membrane biogenesis can also trigger T6SS activation inP. aeruginosa. Specifically, we developed a CRISPR interference (CRISPRi) system to knock down expression ofbamA,tolB, andlptDand found that these knockdowns activated T6SS activity. This increase in T6SS activity was dependent on the same signal transduction pathway that was previously shown to be required for the tit-for-tat response. We conclude that outer membrane perturbation can be sensed byP. aeruginosato activate the T6SS even when the disruption is generated by aberrant cell envelope biogenesis.

Sign in / Sign up

Export Citation Format

Share Document