scholarly journals Infection-Blocking Genes of a Symbiotic Rhizobium leguminosarum Strain That Are Involved in Temperature-Dependent Protein Secretion

2003 ◽  
Vol 16 (1) ◽  
pp. 53-64 ◽  
Author(s):  
M. R. Bladergroen ◽  
K. Badelt ◽  
H. P. Spaink
Keyword(s):  
Nitrogen Fixation ◽  
Protein Secretion ◽  
Rhizobium Leguminosarum ◽  
Signal Sequence ◽  
Gene Clusters ◽  
Secreted Proteins ◽  
Type I ◽  
Secretion Systems ◽  
Temperature Dependent ◽  
Animal Pathogens

Rhizobium leguminosarum strain RBL5523 is able to form nodules on pea, but these nodules are ineffective for nitrogen fixation. The impairment in nitrogen fixation appears to be caused by a defective infection of the host plant and is host specific for pea. A Tn5 mutant of this strain, RBL5787, is able to form effective nodules on pea. We have sequenced a 33-kb region around the phage-transductable Tn5 insertion. The Tn5 insertion was localized to the 10th gene of a putative operon of 14 genes that was called the imp (impaired in nitrogen fixation) locus. Several highly similar gene clusters of unknown function are present in Pseudomonas aeruginosa, Vibrio cholerae, Edwardsiella ictaluri, and several other animal pathogens. Homology studies indicate that several genes of the imp locus are involved in protein phosphorylation, either as a kinase or dephosphorylase, or contain a phosphoprotein-binding module called a forkhead-associated domain. Other proteins show similarity to proteins involved in type III protein secretion. Two dimensional gel electrophoretic analysis of the secreted proteins in the supernatant fluid of cultures of RBL5523 and RBL5787 showed the absence in the mutant strain of at least four proteins with molecular masses of approximately 27 kDa and pIs between 5.5 and 6.5. The production of these proteins in the wild-type strain is temperature dependent. Sequencing of two of these proteins revealed that their first 20 amino acids are identical. This sequence showed homology to that of secreted ribose binding proteins (RbsB) from Bacilus subtilis and V. cholerae. Based on this protein sequence, the corresponding gene encoding a close homologue of RbsB was cloned that contains a N-terminal signal sequence that is recognized by type I secretion systems. Inoculation of RBL5787 on pea plants in the presence of supernatant of RBL5523 caused a reduced ability of RBL5787 to nodulate pea and fix nitrogen. Boiling of this supernatant before inoculation restored the formation of effective nodules to the original values, indicating that secreted proteins are indeed responsible for the impaired phenotype. These data suggest that the imp locus is involved in the secretion to the environment of proteins, including periplasmic RbsB protein, that cause blocking of infection specifically in pea plants.

scholarly journals Characterization of Rhizobium leguminosarum Exopolysaccharide Glycanases That Are Secreted via a Type I Exporter and Have a Novel Heptapeptide Repeat Motif

1998 ◽  
Vol 180 (7) ◽  
pp. 1691-1699 ◽  
Author(s):  
Christine Finnie ◽  
Angeles Zorreguieta ◽  
Nigel M. Hartley ◽  
J. Allan Downie
Keyword(s):  
Protein Secretion ◽  
Rhizobium Leguminosarum ◽  
Repeat Motif ◽  
Type I ◽  
Secretion Systems ◽  
Pectate Lyases ◽  
New Family ◽  
Repeat Units ◽  
Bacterial Exopolysaccharide ◽  
Symbiotic Properties

ABSTRACT The prsDE genes encode a type I protein secretion system required for the secretion of the nodulation protein NodO and at least three other proteins from Rhizobium leguminosarum bv. viciae. At least one of these proteins was predicted to be a glycanase involved in processing of bacterial exopolysaccharide (EPS). Two strongly homologous genes (plyA and plyB) were identified as encoding secreted proteins with polysaccharide degradation activity. Both PlyA and PlyB degrade EPS and carboxymethyl cellulose (CMC), and these extracellular activities are absent in aprsD (protein secretion) mutant. The plyA gene is upstream of prsD but appears to be expressed at a very low level (if at all) in cultured bacteria. AplyB::Tn5 mutant has a very large reduction in degradation of EPS and CMC. Cultures of plyBmutants contained an increased ratio of EPS repeat units to reducing ends, indicating that the EPS was present in a longer-chain form, and this correlated with a significant increase in culture viscosity. Thus, PlyB may play a role in processing of EPS. Analysis of the symbiotic properties of a plyA plyB double mutant revealed that these genes are not required for symbiotic nitrogen fixation and that nodulation was not significantly affected. PlyA and PlyB are similar to bacterial and fungal polysaccharide lyases; they contain 10 copies of what we propose as a novel heptapeptide repeat motif that may constitute a fold similar to that found in the family of extracellular pectate lyases. PlyA and PlyB lack the Ca2+-binding RTX nonapeptide repeat motifs usually found in proteins secreted via type I systems. We propose that PlyA and PlyB are members of a new family of proteins secreted via type I secretion systems and that they are involved in processing of EPS.

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 The Road Less Traveled? Unconventional Protein Secretion at Parasite–Host Interfaces

2021 ◽  
Vol 9 ◽  
Author(s):  
Erina A. Balmer ◽  
Carmen Faso
Keyword(s):  
Protein Secretion ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Cell Model ◽  
Signal Sequence ◽  
Model Organism ◽  
Secreted Proteins ◽  
Current Status ◽  
Secretory Proteins ◽  
Secretion Pathways

Protein secretion in eukaryotic cells is a well-studied process, which has been known for decades and is dealt with by any standard cell biology textbook. However, over the past 20 years, several studies led to the realization that protein secretion as a process might not be as uniform among different cargos as once thought. While in classic canonical secretion proteins carry a signal sequence, the secretory or surface proteome of several organisms demonstrated a lack of such signals in several secreted proteins. Other proteins were found to indeed carry a leader sequence, but simply circumvent the Golgi apparatus, which in canonical secretion is generally responsible for the modification and sorting of secretory proteins after their passage through the endoplasmic reticulum (ER). These alternative mechanisms of protein translocation to, or across, the plasma membrane were collectively termed “unconventional protein secretion” (UPS). To date, many research groups have studied UPS in their respective model organism of choice, with surprising reports on the proportion of unconventionally secreted proteins and their crucial roles for the cell and survival of the organism. Involved in processes such as immune responses and cell proliferation, and including far more different cargo proteins in different organisms than anyone had expected, unconventional secretion does not seem so unconventional after all. Alongside mammalian cells, much work on this topic has been done on protist parasites, including genera Leishmania, Trypanosoma, Plasmodium, Trichomonas, Giardia, and Entamoeba. Studies on protein secretion have mainly focused on parasite-derived virulence factors as a main source of pathogenicity for hosts. Given their need to secrete a variety of substrates, which may not be compatible with canonical secretion pathways, the study of mechanisms for alternative secretion pathways is particularly interesting in protist parasites. In this review, we provide an overview on the current status of knowledge on UPS in parasitic protists preceded by a brief overview of UPS in the mammalian cell model with a focus on IL-1β and FGF-2 as paradigmatic UPS substrates.

scholarly journals Genetic Characterization of Pseudomonas fluorescens SBW25 rsp Gene Expression in the Phytosphere and In Vitro

2005 ◽  
Vol 187 (24) ◽  
pp. 8477-8488 ◽  
Author(s):  
Robert W. Jackson ◽  
Gail M. Preston ◽  
Paul B. Rainey
Keyword(s):  
Pseudomonas Fluorescens ◽  
Ectopic Expression ◽  
Gene Clusters ◽  
Regulatory Genes ◽  
Regulatory Control ◽  
Lag Phase ◽  
Secretion Systems ◽  
General Role ◽  
Animal Pathogens

ABSTRACT The plant-colonizing Pseudomonas fluorescens strain SBW25 harbors a gene cluster (rsp) whose products show similarity to type III protein secretion systems found in plant and animal pathogens. Here we report a detailed analysis of the expression and regulation of the P. fluorescens rsp pathway, both in the phytosphere and in vitro. A combination of chromosomally integrated transcriptional reporter fusions, overexpressed regulatory genes, and specific mutants reveal that promoters controlling expression of rsp are actively transcribed in the plant rhizosphere but not (with the exception of the rspC promoter) in the phyllosphere. In synthetic medium, regulatory (rspL and rspR) and structural (rspU, plus the putative effector ropE) genes are poorly expressed; the rspC promoter is subject to an additional level of regulatory control. Ectopic expression of regulatory genes in wild-type and mutant backgrounds showed that RspR controls transcription of the alternate sigma factor, rspL, and that RspL controls expression of gene clusters encoding structural genes. Mutation of rspV did not affect RspR-mediated expression of rspU. A search for additional regulators revealed two candidates—one with a role in the conversion of alanine to pyruvate—suggesting that expression of rsp is partly dependent upon the metabolic status of the cell. Mutations in rsp regulators resulted in a significant reduction in competitive colonization of the root tips of sugar beet seedlings but also caused a marked increase in the lag phase of laboratory-grown cultures, indicating that rsp regulatory genes play a more significant general role in the function of P. fluorescens SBW25 than previously appreciated.

scholarly journals Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants

1998 ◽  
Vol 62 (2) ◽  
pp. 379-433 ◽  
Author(s):  
Christoph J. Hueck
Keyword(s):  
Host Cell ◽  
Protein Secretion ◽  
Type Iii Secretion ◽  
Plant Pathogens ◽  
Host Cells ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Animal Pathogens ◽  
Type Iii Protein Secretion

SUMMARY Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli, and Chlamydia spp. and the plant pathogens Pseudomonas syringae, Erwinia spp., Ralstonia solanacearum, Xanthomonas campestris, and Rhizobium spp.

Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins

Science ◽  
1993 ◽  
Vol 261 (5121) ◽  
pp. 600-603 ◽  
Author(s):  
K Tashiro ◽  
H Tada ◽  
R Heilker ◽  
M Shirozu ◽  
T Nakano ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Signal Sequence ◽  
Secreted Proteins ◽  
Type I ◽  
Cloning Strategy

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.

scholarly journals Molecular characterization and symbiotic importance of prsD gene of Rhizobium leguminosarum bv. trifolii TA1.

1998 ◽  
Vol 45 (4) ◽  
pp. 1067-1073 ◽  
Author(s):  
A Mazur ◽  
J Wielbo ◽  
J Król ◽  
J Kopcińska ◽  
B Lotocka ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Abc Transporters ◽  
Rhizobium Leguminosarum ◽  
Trifolium Pratense ◽  
Microscopic Analysis ◽  
Type I ◽  
Secretion Systems ◽  
Sds Page ◽  
The Family ◽  
Culture Supernatants

The prsD, prsE and orf3 genes of Rhizobium leguminosarum bv. trifolii strain TA1 encode the proteins which are significantly related to the family of bacterial ABC transporters type I secretion systems. The prsD:Km(r) mutant of strain TA1 induced non-nitrogen-fixing nodules on Trifolium pratense. Microscopic analysis of the nodules induced by prsD mutant did not reveal major abberations in the bacteroid appearance. The exopolysaccharide of prsD mutant was produced in increased amount and its level of polymerization was changed. SDS/PAGE of the proteins from the culture supernatants showed a lack of the 47-kDa protein in the culture of prsD mutant. Thus, PrsD may play a role in the export of this protein.

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