scholarly journals The Caulobacter crescentus Paracrystalline S-Layer Protein Is Secreted by an ABC Transporter (Type I) Secretion Apparatus

1998 ◽  
Vol 180 (12) ◽  
pp. 3062-3069 ◽  
Author(s):  
Peter Awram ◽  
John Smit
Keyword(s):  
Abc Transporter ◽  
Transport System ◽  
Protein Transport ◽  
Caulobacter Crescentus ◽  
Sequence Similarity ◽  
Secretion System ◽  
Cell Protein ◽  
Type I ◽  
Secretion Systems ◽  
Secretion Signal

ABSTRACT Caulobacter crescentus is a gram-negative bacterium that produces a two-dimensional crystalline array on its surface composed of a single 98-kDa protein, RsaA. Secretion of RsaA to the cell surface relies on an uncleaved C-terminal secretion signal. In this report, we identify two genes encoding components of the RsaA secretion apparatus. These components are part of a type I secretion system involving an ABC transporter protein. These genes, lying immediately 3′ of rsaA, were found by screening a Tn5 transposon library for the loss of RsaA transport and characterizing the transposon-interrupted genes. The two proteins presumably encoded by these genes were found to have significant sequence similarity to ABC transporter and membrane fusion proteins of other type I secretion systems. The greatest sequence similarity was found to the alkaline protease (AprA) transport system ofPseudomonas aeruginosa and the metalloprotease (PrtB) transport system of Erwinia chrysanthemi. TheprtB and aprA genes were introduced intoC. crescentus, and their products were secreted by the RsaA transport system. Further, defects in the S-layer protein transport system led to the loss of this heterologous secretion. This is the first report of an S-layer protein secreted by a type I secretion apparatus. Unlike other type I secretion systems, the RsaA transport system secretes large amounts of its substrate protein (it is estimated that RsaA accounts for 10 to 12% of the total cell protein). Such levels are expected for bacterial S-layer proteins but are higher than for any other known type I secretion system.

scholarly journals Caulobacter crescentus Synthesizes an S-Layer-Editing Metalloprotease Possessing a Domain Sharing Sequence Similarity with Its Paracrystalline S-Layer Protein

2002 ◽  
Vol 184 (10) ◽  
pp. 2709-2718 ◽  
Author(s):  
Elizabeth Umelo-Njaka ◽  
Wade H. Bingle ◽  
Faten Borchani ◽  
Khai D. Le ◽  
Peter Awram ◽  
...  
Keyword(s):  
Caulobacter Crescentus ◽  
Sequence Similarity ◽  
Secreted Proteins ◽  
Type I ◽  
Two Dimensional ◽  
Terminal Sequence ◽  
Significant Similarity ◽  
Secretion Signal ◽  
Repeat Sequences ◽  
Secreted Proteases

ABSTRACT Strains of Caulobacter crescentus elaborate an S-layer, a two-dimensional protein latticework which covers the cell surface. The S-layer protein (RsaA) is secreted by a type I mechanism (relying on a C-terminal signal) and is unusual among type I secreted proteins because high levels of protein are produced continuously. In efforts to adapt the S-layer for display of foreign peptides and proteins, we noted a proteolytic activity that affected S-layer monomers with foreign inserts. The cleavage was precise, resulting in fragments with an unambiguous N-terminal sequence. We developed an assay to screen for loss of this activity (i.e., presentation of foreign peptides without degradation), using transposon and traditional mutagenesis. A metalloprotease gene designated sap (S-layer-associated protease) was identified which could complement the protease-negative mutants. The N-terminal half of Sap possessed significant similarity to other type I secreted proteases (e.g., alkaline protease of Pseudomonas aeruginosa), including the characteristic RTX repeat sequences, but the C-terminal half which normally includes the type I secretion signal exhibited no such similarity. Instead, there was a region of significant similarity to the N-terminal region of RsaA. We hypothesize that Sap evolved by combining the catalytic portion of a type I secreted protease with an S-layer-like protein, perhaps to associate with nascent S-layer monomers to “scan” for modifications.

scholarly journals An A/U-Rich Enhancer Region Is Required for High-Level Protein Secretion through the HlyA Type I Secretion System

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Sakshi Khosa ◽  
Romy Scholz ◽  
Christian Schwarz ◽  
Mirko Trilling ◽  
Hartmut Hengel ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Protein Secretion ◽  
Fusion Proteins ◽  
Untranslated Region ◽  
Secretion System ◽  
Type I ◽  
Secretion Systems ◽  
Enhancer Region ◽  
Ribosomal Protein S1 ◽  
Cellular Expression

ABSTRACTEfficient protein secretion is often a valuable alternative to classic cellular expression to obtain homogenous protein samples. Early on, bacterial type I secretion systems (T1SS) were employed to allow heterologous secretion of fusion proteins. However, this approach was not fully exploited, as many proteins could not be secreted at all or only at low levels. Here, we present an engineered microbial secretion system which allows the effective production of proteins up to a molecular mass of 88 kDa. This system is based on the hemolysin A (HlyA) T1SS of the Gram-negative bacteriumEscherichia coli, which exports polypeptides when fused to a hemolysin secretion signal. We identified an A/U-rich enhancer region upstream ofhlyArequired for effective expression and secretion of selected heterologous proteins irrespective of their prokaryotic, viral, or eukaryotic origin. We further demonstrate that the ribosomal protein S1 binds to thehlyAA/U-rich enhancer region and that this region is involved in the high yields of secretion of functional proteins, like maltose-binding protein or human interferon alpha-2.IMPORTANCEA 5′ untranslated region of the mRNA of substrates of type I secretion systems (T1SS) drastically enhanced the secretion efficiency of the endogenously secreted protein. The identification of ribosomal protein S1 as the interaction partner of this 5′ untranslated region provides a rationale for the enhancement. This strategy furthermore can be transferred to fusion proteins allowing a broader, and eventually a more general, application of this system for secreting heterologous fusion proteins.

scholarly journals Comparison of S-layer secretion genes in freshwater caulobacters

2004 ◽  
Vol 50 (9) ◽  
pp. 751-766 ◽  
Author(s):  
Mihai Iuga ◽  
Peter Awram ◽  
John F Nomellini ◽  
John Smit
Keyword(s):  
Protein Secretion ◽  
Caulobacter Crescentus ◽  
Sequence Similarity ◽  
Atp Binding ◽  
Type I ◽  
C Terminus ◽  
Expression Studies ◽  
Size Groups ◽  
Membrane Fusion Proteins ◽  
Atp Binding Cassette

Our freshwater caulobacter collection contains about 40 strains that are morphologically similar to Caulobacter crescentus. All elaborate a crystalline protein surface (S) layer made up of protein monomers 100–193 kDa in size. We conducted a comparative study of S-layer secretion in 6 strains representing 3 size groups of S-layer proteins: small (100–108 kDa), medium (122–151 kDa), and large (181–193 kDa). All contained genes predicted to encode ATP-binding cassette transporters and membrane fusion proteins highly similar to those of C. crescentus, indicating that the S-layer proteins were all secreted by a type I system. The S-layer proteins' C-termini showed unexpectedly low sequence similarity but contained conserved residues and predicted secondary structure features typical of type I secretion signals. Cross-expression studies showed that the 6 strains recognized secretion signals from C. crescentus and Pseudomonas aeruginosa and similarly that C. crescentus was able to secrete the S-layer protein C-terminus of 1 strain examined. Inactivation of the ATP-binding cassette transporter abolished S-layer protein secretion, indicating that the type I transporter is necessary for S-layer protein secretion. Finally, while all of the S-layer proteins of this subset of strains were secreted by type I mechanisms, there were significant differences in genome positions of the transporter genes that correlated with S-layer protein size.Key words: freshwater caulobacter, S-layer, type I secretion system, ABC transporter.

scholarly journals Protein Subassemblies of the Helicobacter pylori Cag Type IV Secretion System Revealed by Localization and Interaction Studies

2008 ◽  
Vol 190 (6) ◽  
pp. 2161-2171 ◽  
Author(s):  
Stefan Kutter ◽  
Renate Buhrdorf ◽  
Jürgen Haas ◽  
Wulf Schneider-Brachert ◽  
Rainer Haas ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Protein Interactions ◽  
Protein Transport ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Transport Systems ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Type Iv ◽  
Small Proteins

ABSTRACT Type IV secretion systems are possibly the most versatile protein transport systems in gram-negative bacteria, with substrates ranging from small proteins to large nucleoprotein complexes. In many cases, such as the cag pathogenicity island of Helicobacter pylori, genes encoding components of a type IV secretion system have been identified due to their sequence similarities to prototypical systems such as the VirB system of Agrobacterium tumefaciens. The Cag type IV secretion system contains at least 14 essential apparatus components and several substrate translocation and auxiliary factors, but the functions of most components cannot be inferred from their sequences due to the lack of similarities. In this study, we have performed a comprehensive sequence analysis of all essential or auxiliary Cag components, and we have used antisera raised against a subset of components to determine their subcellular localization. The results suggest that the Cag system contains functional analogues to all VirB components except VirB5. Moreover, we have characterized mutual stabilization effects and performed a comprehensive yeast two-hybrid screening for potential protein-protein interactions. Immunoprecipitation studies resulted in identification of a secretion apparatus subassembly at the outer membrane. Combining these data, we provide a first low-resolution model of the Cag type IV secretion apparatus.

scholarly journals Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

2020 ◽  
Author(s):  
Christopher Icke ◽  
Freya J. Hodges ◽  
Karthik Puella ◽  
Samantha A. McKeand ◽  
Jack A. Bryant ◽  
...  
Keyword(s):  
Composite System ◽  
Cysteine Residue ◽  
Secretion System ◽  
Type I ◽  
Secretion Systems ◽  
Cellular Functions ◽  
E Coli ◽  
New Class ◽  
Secretion Pathway ◽  
Bacterial Lipoprotein

AbstractProtein acylation is critical for many cellular functions including signal transduction, cell division and development. In bacteria, such lipoproteins have important roles in virulence and are therefore potential targets for the development of novel antimicrobials and vaccines. To date, all known bacterial lipoproteins are secreted from the cytosol via the Sec pathway, acylated on an N-terminal cysteine residue through the action of Lgt, Lsp and Lnt, and then targeted to the appropriate cellular location. In the case of Gram-negative bacteria, the lipoprotein trafficking Lol pathway transports the lipoproteins to the outer membrane where most substrate molecules are retained within the cell. Here we identify a new secretion pathway that displays the substrate lipoprotein on the cell surface. We demonstrate that the previously identified E. coli Aat secretion system is a composite system that shares similarity with type I secretion systems and elements of the Lol pathway. Remarkably, during secretion by the Aat system, the AatD subunit acylates the substrate CexE on a highly conserved N-terminal glycine residue (rather than the canonical cysteine). Mutations in AatD or CexE that disrupt glycine acylation interfere with membrane incorporation and trafficking. Our data suggest that CexE is the first member of a new class of glycine-acylated bacterial lipoprotein, while Aat represents a new secretion system that we propose be defined as a lipoprotein secretion system (LSS).

scholarly journals Functional Type 1 Secretion System Involved in Legionella pneumophila Virulence

2014 ◽  
Vol 197 (3) ◽  
pp. 563-571 ◽  
Author(s):  
Fabien Fuche ◽  
Anne Vianney ◽  
Claire Andrea ◽  
Patricia Doublet ◽  
Christophe Gilbert
Keyword(s):  
Legionella Pneumophila ◽  
Severe Form ◽  
Secretion System ◽  
Host Cells ◽  
Type I ◽  
Secretion Systems ◽  
Content Type ◽  
Legionnaires Disease ◽  
Infectious Cycle

Legionella pneumophilais a Gram-negative pathogen found mainly in water, either in a free-living form or within infected protozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called legionellosis or Legionnaires' disease. The involvement of type II and IV secretion systems in the virulence ofL. pneumophilais now well documented. Despite bioinformatic studies showing that a type I secretion system (T1SS) could be present in this pathogen, the functionality of this system based on the LssB, LssD, and TolC proteins has never been established. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle ofL. pneumophila. Using deletion mutants and fusion proteins, we demonstrated that therepeats-in-toxin protein RtxA is secreted through an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these results underline the active participation ofL. pneumophila, via its T1SS, in its internalization into host cells.

The Secretome of the Extremophilic Acidithiobacillus Ferrooxidans ATCC 23270

2009 ◽  
Vol 71-73 ◽  
pp. 183-186
Author(s):  
F.A. Pagliai ◽  
Carlos A. Jerez
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Extracellular Polymeric Substances ◽  
Solid Substrate ◽  
Extracellular Proteins ◽  
Classical Pathway ◽  
Type I ◽  
Secretion Systems ◽  
Extracellular Milieu ◽  
Secretion Signal ◽  
Extracellular Proteome

Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile capable of catalyzing the oxidation of ferrous iron and sulfur reducing compounds. During the dissolution of ores A. ferrooxidans adheres to the solid substrate by hydrophobic and hydrophilic interactions and also extracellular polymeric substances may be involved. Nevertheless, many of these agents that participate in the process are unknown. Therefore, the identification and characterization of the extracellular proteome of A. ferrooxidans was addressed during this work. First, by searching the genome of A. ferrooxidans ATCC 23270 the putative genes coding for proteins present in most of the different secretion systems (type I, II, IV, V systems and the Usher pathway) were found. Second, when the secretion signal prediction server SubCel 1.0 was used, 26.7 % of the total open reading frames showed possible signals that would allow these proteins to leave the cytoplasm towards the internal and outer membranes, the periplasm, or the extracellular milieu. By using 2D-NEPHGE and MS sequencing we identified the eleven most abundant proteins present in the extracellular fraction. Proteins related to the transport and binding of solutes and the folding of proteins, among others were identified. Along with the non-classical pathway, the protein secretion systems identified could constitute the secretion machinery for the proteins found in the extracellular sub-proteome of A. ferrooxidans that as a whole constitute its secretome. Further studies of the extracellular proteins from this biomining microorganism will be important to find out their possible role if any, during bacteria-mineral interactions.

Analysis of high-level S-layer protein secretion inCaulobacter crescentus

2010 ◽  
Vol 56 (6) ◽  
pp. 501-514 ◽  
Author(s):  
Janny Ho Yu Lau ◽  
John F. Nomellini ◽  
John Smit
Keyword(s):  
Protein Synthesis ◽  
Half Life ◽  
Caulobacter Crescentus ◽  
Protein A ◽  
Cell Protein ◽  
Type I ◽  
Key Factor ◽  
Multiple Copies ◽  
High Level ◽  
Quantitative Immunoblotting

Caulobacter crescentus exhibits a hexagonally arranged protein layer on its outermost surface. RsaA, the sole protein of this “S-layer”, is secreted by a type I (ABC) transporter. Few type I transporters show high-level secretion, and few bacterial S-layers have been carefully examined for the amount of protein synthesis capacity needed to maintain cell coverage. Here we determined RsaA levels by quantitative immunoblotting methods, learned that very stable mRNA is a key factor in high-level secretion, and found that the transporter was capable of still higher secretion. A propensity for RsaA to aggregate was a barrier to quantitation, but with the use of S-layer shedding mutants and methods to keep RsaA soluble, we learned that ~31% of cell protein is RsaA. When multiple copies of rsaA were introduced, the level increased to ~51% of cell protein, a higher level than we are aware of for any protein in any bacterium. Unexpectedly, in comparing normal and S-layer shedding strains, an assembled S-layer was not a significant barrier to elevated secretion. The rsaA mRNA half-life was determined by real-time PCR to be 36 min, ranking with the most stable known in bacteria. A modification of the 5′ region resulted in a shorter half-life and a reduction in maximum protein synthesis levels. If secretion was prevented by knockout of type I transporter genes, RsaA levels dropped to 10% or less of normal, but with no significant reduction in rsaA mRNA. Overall, normal levels of RsaA were unexpectedly high, and still higher levels were not limited by transporter capability, the presence of an assembled S-layer, or the capacity of the cell’s physiology to produce large amounts of one protein. The normal upper limit of RsaA production appears to be controlled only by the level of an unusually stable message. Significant down-regulation is possible and is accomplished posttranscriptionally.

scholarly journals Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Leonor García-Bayona ◽  
Monica S Guo ◽  
Michael T Laub
Keyword(s):  
Caulobacter Crescentus ◽  
Secretion System ◽  
Cell Contact ◽  
Type I ◽  
Diverse Group ◽  
Planktonic Cells ◽  
Immunity Protein ◽  
Aqueous Environments ◽  
Dependent Inhibition ◽  
Fierce Competition

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common.

scholarly journals Campylobacter fetus Surface Layer Proteins Are Transported by a Type I Secretion System

1998 ◽  
Vol 180 (24) ◽  
pp. 6450-6458 ◽  
Author(s):  
Stuart A. Thompson ◽  
Omer L. Shedd ◽  
Kevin C. Ray ◽  
Michael H. Beins ◽  
Jesse P. Jorgensen ◽  
...  
Keyword(s):  
Surface Layer ◽  
Antigenic Variation ◽  
Signal Sequence ◽  
Bacterial Species ◽  
Bacterial Pathogen ◽  
Type A ◽  
Secretion System ◽  
Type I ◽  
Secretion Systems ◽  
Campylobacter Fetus

ABSTRACT The virulence of Campylobacter fetus, a bacterial pathogen of ungulates and humans, is mediated in part by the presence of a paracrystalline surface layer (S-layer) that confers serum resistance. The subunits of the S-layer are S-layer proteins (SLPs) that are secreted in the absence of an N-terminal signal sequence and attach to either type A or B C. fetus lipopolysaccharide in a serospecific manner. Antigenic variation of multiple SLPs (encoded bysapA homologs) of type A strain 23D occurs by inversion of a promoter-containing DNA element flanked by two sapAhomologs. Cloning and sequencing of the entire 6.2-kb invertible region from C. fetus 23D revealed a probable 5.6-kb operon of four overlapping genes (sapCDEF, with sizes of 1,035, 1,752, 1,284, and 1,302 bp, respectively) transcribed in the opposite direction from sapA. The four genes also were present in the invertible region of type B strain 84-107 and were virtually identical to their counterparts in the type A strain. Although SapC had no database homologies, SapD, SapE, and SapF had predicted amino acid homologies with type I protein secretion systems (typified byEscherichia coli HlyBD/TolC or Erwinia chrysanthemi PrtDEF) that utilize C-terminal secretion signals to mediate the secretion of hemolysins, leukotoxins, or proteases from other bacterial species. Analysis of the C termini of four C. fetus SLPs revealed conserved structures that are potential secretion signals. A C. fetus sapD mutant neither produced nor secreted SLPs. E. coli expressing C. fetus sapA and sapCDEF secreted SapA, indicating that thesapCDEF genes are sufficient for SLP secretion. C. fetus SLPs therefore are transported to the cell surface by a type I secretion system.

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