scholarly journals Novel Inner Membrane Retention Signals in Pseudomonas aeruginosa Lipoproteins

2008 ◽  
Vol 190 (18) ◽  
pp. 6119-6125 ◽  
Author(s):  
Shawn Lewenza ◽  
Musa M. Mhlanga ◽  
Anthony P. Pugsley
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
Optical Sectioning ◽  
Inner Membrane ◽  
Outer Membrane Lipoprotein ◽  
Associated Proteins ◽  
And Function

ABSTRACT The ultimate membrane localization and function of most of the 185 predicted Pseudomonas aeruginosa PAO1 lipoproteins remain unknown. We constructed a fluorescent lipoprotein, CSFPOmlA-ChFP, by fusing the signal peptide and the first four amino acids of the P. aeruginosa outer membrane lipoprotein OmlA to the monomeric red fluorescent protein mCherry (ChFP). When cells were plasmolyzed with 0.5 M NaCl, the inner membrane separated from the outer membrane and formed plasmolysis bays. This permits the direct observation of fluorescence in either the outer or inner membrane. CSFPOmlA-ChFP was shown to localize in the outer membrane by fluorescence microscopy and immunoblotting analysis of inner and outer membrane fractions. The site-directed substitution of the amino acids at positions +2, +3, and +4 in CSFPOmlA-ChFP was performed to test the effects on lipoprotein localization of a series of amino acid sequences selected from a panel of predicted lipoproteins. We confirmed Asp+2 and Lys+3 Ser+4 function as inner membrane retention signals and identified four novel inner membrane retention signals: CK+2 V+3 E+4, CG+2 G+3 G+4, CG+2 D+3 D+4, and CQ+2 G+3 S+4. These inner membrane retention signals are found in 5% of the 185 predicted P. aeruginosa lipoproteins. Full-length chimeras of predicted lipoproteins PA4370 and PA3262 fused to mCherry were shown to reside in the inner membrane and showed a nonuniform or patchy distribution in the membrane. The optical sectioning of cells producing PA4370CGDD-ChFP and PA3262CDSQ-ChFP by confocal microscopy improved the resolution and indicated a helix-like localization pattern in the inner membrane. The method described here permits the in situ visualization of lipoprotein localization and should work equally well for other membrane-associated proteins.

scholarly journals The C Terminus of Substrates Is Critical but Not Sufficient for Their Degradation by the Pseudomonas aeruginosa CtpA Protease

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Sammi Chung ◽  
Andrew J. Darwin
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Outer Membrane ◽  
Content Type ◽  
C Terminus ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Carboxyl Terminal

ABSTRACT Bacterial carboxyl-terminal processing proteases (CTPs) are widely conserved and have been linked to important processes, including signal transduction, cell wall metabolism, and virulence. However, the features that target proteins for CTP-dependent cleavage are unclear. Studies of the Escherichia coli CTP Prc suggested that it cleaves proteins with nonpolar and/or structurally unconstrained C termini, but it is not clear if this applies broadly. Pseudomonas aeruginosa has a divergent CTP, CtpA, which is required for virulence. CtpA works in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. In this study, we investigated if the C termini of two nonhomologous CtpA substrates are important for their degradation. We determined that these substrates have extended C termini compared to those of their closest E. coli homologs. Removing 7 amino acids from these extensions was sufficient to reduce their degradation by CtpA both in vivo and in vitro. Degradation of one truncated substrate was restored by adding the C terminus from the other but not by adding an unrelated sequence. However, modification of the C termini of nonsubstrates, by adding the C-terminal amino acids from a substrate, did not cause their degradation by CtpA. Therefore, the C termini of CtpA substrates are required but not sufficient for their efficient degradation. Although C-terminal truncated substrates were protected from degradation, they still associated with the LbcA-CtpA complex in vivo. Therefore, degradation of a protein by CtpA requires a C terminus-independent interaction with the LbcA-CtpA complex, followed by C terminus-dependent degradation, perhaps because CtpA normally initiates cleavage at a C-terminal site. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life, but exactly how they work is poorly understood, including how they recognize substrates. Bacterial CTPs have been associated with virulence, including CtpA of Pseudomonas aeruginosa, which works in complex with the outer membrane lipoprotein LbcA to degrade potentially dangerous peptidoglycan hydrolases. We report an important advance by revealing that efficient degradation by CtpA requires at least two separable phenomena and that one of them depends on information encoded in the substrate C terminus. A C terminus-independent association with the LbcA-CtpA complex is followed by C terminus-dependent cleavage by CtpA. Increased understanding of how CTPs target proteins is significant, due to their links to virulence, peptidoglycan remodeling, and other important processes.

scholarly journals The Landscape of Pseudomonas aeruginosa Membrane-Associated Proteins

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2421
Author(s):  
Sara Motta ◽  
Davide Vecchietti ◽  
Alessandra M. Martorana ◽  
Pietro Brunetti ◽  
Giovanni Bertoni ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Protein Identification ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
Protein Domains ◽  
Host Cells ◽  
Inner Membrane ◽  
Proteomic Data ◽  
Associated Proteins

Background: Pseudomonas aeruginosa cell envelope-associated proteins play a relevant role in infection mechanisms. They can contribute to the antibiotic resistance of the bacterial cells and be involved in the interaction with host cells. Thus, studies contributing to elucidating these key molecular elements are of great importance to find alternative therapeutics. Methods: Proteins and peptides were extracted by different methods and analyzed by Multidimensional Protein Identification Technology (MudPIT) approach. Proteomic data were processed by Discoverer2.1 software and multivariate statistics, i.e., Linear Discriminant Analysis (LDA), while the Immune Epitope Database (IEDB) resources were used to predict antigenicity and immunogenicity of experimental identified peptides and proteins. Results: The combination of 29 MudPIT runs allowed the identification of 10,611 peptides and 2539 distinct proteins. Following application of extraction methods enriching specific protein domains, about 15% of total identified peptides were classified in trans inner-membrane, inner-membrane exposed, trans outer-membrane and outer-membrane exposed. In this scenario, nine outer membrane proteins (OprE, OprI, OprF, OprD, PagL, OprG, PA1053, PAL and PA0833) were predicted to be highly antigenic. Thus, they were further processed and epitopes target of T cells (MHC Class I and Class II) and B cells were predicted. Conclusion: The present study represents one of the widest characterizations of the P. aeruginosa membrane-associated proteome. The feasibility of our method may facilitates the investigation of other bacterial species whose envelope exposed protein domains are still unknown. Besides, the stepwise prioritization of proteome, by combining experimental proteomic data and reverse vaccinology, may be useful for reducing the number of proteins to be tested in vaccine development.

scholarly journals An Acidic Cluster of Human Cytomegalovirus UL99 Tegument Protein Is Required for Trafficking and Function

2004 ◽  
Vol 78 (3) ◽  
pp. 1488-1502 ◽  
Author(s):  
Thomas R. Jones ◽  
Shi-Wu Lee
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
Cytoplasmic Vacuole ◽  
Viral Growth ◽  
Reading Frame ◽  
Tegument Proteins ◽  
Green Fluorescent ◽  
And Function

ABSTRACT The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.

scholarly journals The C-terminus is critical for the degradation of substrates by the Pseudomonas aeruginosa CtpA protease

2020 ◽  
Author(s):  
Sammi Chung ◽  
Andrew J. Darwin
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Outer Membrane ◽  
Target Proteins ◽  
C Terminus ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Carboxyl Terminal

ABSTRACTBacterial carboxyl-terminal processing proteases (CTPs) are widely conserved and have been linked to important processes including signal transduction, cell wall metabolism, and virulence. However, the features that target proteins for CTP-dependent cleavage are unclear. Studies of the Escherichia coli CTP Prc suggested that it cleaves proteins with non-polar and/or structurally unconstrained C-termini, but it is not clear if this applies broadly. Pseudomonas aeruginosa has a divergent CTP, CtpA, which is required for virulence. CtpA works in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. Here, we investigated if the C-termini of two non-homologous CtpA substrates are important for their degradation. We determined that these substrates have extended C-termini, compared to their closest E. coli homologs. Removing seven amino acids from these extensions was sufficient to inhibit their degradation by CtpA both in vivo and in vitro. Degradation of one truncated substrate was restored by adding the C-terminus from the other, but not by adding an unrelated sequence. However, modification of the C-terminus of non-substrates, by adding the C-terminal amino acids from a substrate, did not cause their degradation by CtpA. Therefore, the C-termini of CtpA substrates are required but not sufficient for degradation. Although C-terminal truncated substrates were not degraded, they still associated with the LbcA•CtpA complex in vivo. Therefore, degradation of a protein by CtpA requires a C-terminal-independent interaction with the LbcA•CtpA complex, followed by C-terminal-dependent degradation, perhaps because CtpA must initiate cleavage at a specific C-terminal site.IMPORTANCECarboxyl-terminal processing proteases (CTPs) are found in all three domains of life, but exactly how they work is poorly understood, including how they recognize substrates. Bacterial CTPs have been associated with virulence, including CtpA of Pseudomonas aeruginosa, which works in complex with the outer membrane lipoprotein LbcA to degrade potentially dangerous peptidoglycan hydrolases. We report an important advance by revealing that degradation by CtpA requires at least two separable phenomena, and that one of them depends on information encoded in the substrate C-terminus. A C-terminal-independent association with the LbcA•CtpA complex is followed by C-terminal-dependent cleavage by CtpA. Increased understanding of how CTPs target proteins is significant, due to their links to virulence, peptidoglycan remodeling, and other important processes.

scholarly journals Disparate Subcellular Localization Patterns of Pseudomonas aeruginosa Type IV Pilus ATPases Involved in Twitching Motility

2005 ◽  
Vol 187 (3) ◽  
pp. 829-839 ◽  
Author(s):  
Poney Chiang ◽  
Marc Habash ◽  
Lori L. Burrows
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Distribution Patterns ◽  
Opportunistic Pathogen ◽  
Twitching Motility ◽  
Polar Localization ◽  
Type Iv ◽  
And Function

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.

scholarly journals The amino acid sequence of cytochromes c-551 from three species of Pseudomonas

1973 ◽  
Vol 131 (3) ◽  
pp. 485-498 ◽  
Author(s):  
R. P. Ambler ◽  
Margaret Wynn
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Cytochrome C ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Mitochondrial Cytochrome ◽  
Cytochromes C ◽  
Lending Library ◽  
Single Peptide

The amino acid sequences of the cytochromes c-551 from three species of Pseudomonas have been determined. Each resembles the protein from Pseudomonas strain P6009 (now known to be Pseudomonas aeruginosa, not Pseudomonas fluorescens) in containing 82 amino acids in a single peptide chain, with a haem group covalently attached to cysteine residues 12 and 15. In all four sequences 43 residues are identical. Although by bacteriological criteria the organisms are closely related, the differences between pairs of sequences range from 22% to 39%. These values should be compared with the differences in the sequence of mitochondrial cytochrome c between mammals and amphibians (about 18%) or between mammals and insects (about 33%). Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50015 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

scholarly journals Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

2008 ◽  
Vol 190 (11) ◽  
pp. 4001-4016 ◽  
Author(s):  
Wallace A. Kaserer ◽  
Xiaoxu Jiang ◽  
Qiaobin Xiao ◽  
Daniel C. Scott ◽  
Matthew Bauler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Binding Motif ◽  
Inner Membrane ◽  
E Coli ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

scholarly journals Emerging Roles for NlpE as a Sensor for Lipoprotein Maturation and Transport to the Outer Membrane inEscherichia coli

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Brent W. Simpson ◽  
M. Stephen Trent
Keyword(s):  
Outer Membrane ◽  
Copper Stress ◽  
Gram Negative Bacteria ◽  
Two Component System ◽  
Inner Membrane ◽  
Complex Process ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Outer Membrane Biogenesis ◽  
Stress Pathway

ABSTRACTOuter membrane biogenesis is a complex process for Gram-negative bacteria as the components are synthesized in the cytoplasm or at the inner membrane and then transported to the outer membrane. Stress pathways monitor and respond to problems encountered in assembling the outer membrane. The two-component system CpxAR was recently reported to be a stress pathway for transport of lipoproteins to the outer membrane, but it was unclear how this stress is sensed. May et al. [K. L. May, K. M. Lehman, A. M. Mitchell, and M. Grabowicz, mBio 10(3):e00618-19, 2019,https://doi.org/10.1128/mBio.00618-19] determined that an outer membrane lipoprotein, NlpE, is the sensor for lipoprotein biogenesis stress. The group demonstrated that CpxAR is activated by the N-terminal domain of NlpE when the lipoprotein accumulates at the inner membrane. Further, this work resolved a previously debated role for NlpE in sensing copper stress; copper was shown to inhibit acylation of lipoproteins, preventing them from being transported to the outer membrane.

scholarly journals Primary structure of OXA-3 and phylogeny of oxacillin-hydrolyzing class D beta-lactamases.

1995 ◽  
Vol 39 (4) ◽  
pp. 887-893 ◽  
Author(s):  
F Sanschagrin ◽  
F Couture ◽  
R C Levesque
Keyword(s):  
Amino Acids ◽  
Structure And Function ◽  
Amino Acid Sequences ◽  
Specific Class ◽  
Conserved Motifs ◽  
Beta Lactamases ◽  
Class A ◽  
Class D ◽  
And Function ◽  
Conserved Amino Acids

We determined the nucleotide sequence of the blaOXA-3(pMG25) gene from Pseudomonas aeruginosa. The bla structural gene encoded a protein of 275 amino acids representing one monomer of 31,879 Da for the OXA-3 enzyme. Comparisons between the OXA-3 nucleotide and amino acid sequences and those of class A, B, C, and D beta-lactamases were performed. An alignment of the eight known class D beta-lactamases including OXA-3 demonstrated the presence of conserved amino acids. In addition, conserved motifs composed of identical amino acids typical of penicillin-recognizing proteins and specific class D motifs were identified. These conserved motifs were considered for possible roles in the structure and function of oxacillinases. On the basis of the alignment and identity scores, a dendrogram was constructed. The phylogenetic data obtained revealed five groups of class D beta-lactamases with large evolutionary distances between each group.

Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

2000 ◽  
Vol 349 (1) ◽  
pp. 281-287 ◽  
Author(s):  
Patricia E. M. MARTIN ◽  
James STEGGLES ◽  
Claire WILSON ◽  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Lucifer Yellow ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

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