Investigation of Protein Export in Bifidobacterium
breve
UCC2003

ABSTRACT The molecular interactions between the bifidobacterial cell and its natural environment, namely, the gastrointestinal tract of its host, are particularly important in understanding the presumed positive effects of Bifidobacterium on the health status of the host. In this study an export-specific reporter system, designed for use in gram-positive organisms and based on the use of the staphylococcal nuclease (Nuc) as a reporter, was employed to identify exported proteins in Bifidobacterium breve UCC2003. A B. breve genomic library of translational fusions to the Nuc-encoding gene devoid of its own export signal was established in the shuttle vector pFUN (I. Poquet, S. D. Ehrlich, and A. Gruss, J. Bacteriol. 180:1904-1912, 1998) and screened for bifidobacterial export signals. Sequence analysis of the fusion proteins obtained that displayed a nuclease-producing phenotype in both Lactococcus lactis and B. breve predicted the presence of a classical signal peptide and/or single or multiple transmembrane domains, thus indicating that some of the export signals in B. breve are comparable to those used in L. lactis. Cell fractionation studies, zymograms, nuclease assays, and Western blotting were employed to confirm the function of the predicted signals and to determine the location and activity of the exported fusion proteins in B. breve and/or L. lactis.

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An Export-Specific Reporter Designed for Gram-Positive Bacteria: Application to Lactococcus lactis

Journal of Bacteriology ◽

10.1128/jb.180.7.1904-1912.1998 ◽

1998 ◽

Vol 180 (7) ◽

pp. 1904-1912 ◽

Cited By ~ 74

Author(s):

Isabelle Poquet ◽

S. Dusko Ehrlich ◽

Alexandra Gruss

Keyword(s):

Lactococcus Lactis ◽

Genomic Library ◽

Shuttle Vector ◽

Nuclease Activity ◽

Protein Export ◽

Cell Fractionation ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Membrane Protein Topology ◽

Export Signal

ABSTRACT The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of theStaphylococcus aureus secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called ΔSPNuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in Lactococcus lactis, thus demonstrating the suitability of ΔSPNuc to report protein export. The shuttle vector pFUN was designed to construct ΔSPNuc translational fusions whose expression signals are provided by inserted DNA. The capacity of ΔSPNuc to reveal and identify exported proteins was tested by generating anL. lactis genomic library in pFUN and by screening for Nuc activity directly in L. lactis. All ΔSPNuc fusions displaying a strong Nuc+ phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in L. lactis. Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding. Our results with L. lactis show that ΔSPNuc is well suited to report both protein export and membrane protein topology.

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Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.23.11322 ◽

1993 ◽

Vol 90 (23) ◽

pp. 11322-11326 ◽

Cited By ~ 20

Author(s):

S Björklund ◽

K Hjortsberg ◽

E Johansson ◽

L Thelander

Keyword(s):

Ribonucleotide Reductase ◽

Genomic Library ◽

Protein Complexes ◽

Specific Binding ◽

Transcriptional Start Site ◽

Large Subunit ◽

Mobility Shift ◽

Direct Role ◽

Encoding Gene ◽

R1 Gene

Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two nonidentical subunits, proteins R1 and R2, both required for enzyme activity. The structure of the genomic mouse ribonucleotide reductase R1 gene was compiled from a number of overlapping lambda clones isolated from a Charon 4A mouse sperm genomic library. The R1-encoding gene covers 26 kb and consists of 19 exons. All exon-intron boundaries were located by dideoxynucleotide sequencing, showing that intron 7 starts with the variant GC instead of GT. About 3.5 kb of DNA from the 5'-flanking region of the R1-encoding gene were cloned and sequenced, and the transcriptional start site was determined by nuclease S1 mapping of RNA. DNase I footprinting assays on the R1 promoter identified two nearly identical 23-bp-long protein-binding regions. Three protein complexes binding to one of the 23-mer regions were resolved and partially identified by using gel-retardation mobility-shift assays and UV crosslinking. One complex most likely contained Sp1, and another complex showed S-phase-specific binding, suggesting a direct role in the cell-cycle-dependent R1 gene expression.

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Recognition of RNA Branch Point Sequences by the KH Domain of Splicing Factor 1 (Mammalian Branch Point Binding Protein) in a Splicing Factor Complex

Molecular and Cellular Biology ◽

10.1128/mcb.21.15.5232-5241.2001 ◽

2001 ◽

Vol 21 (15) ◽

pp. 5232-5241 ◽

Cited By ~ 29

Author(s):

Hadas Peled-Zehavi ◽

J. Andrew Berglund ◽

Michael Rosbash ◽

Alan D. Frankel

Keyword(s):

Branch Point ◽

Fusion Proteins ◽

Rna Binding ◽

Binding Protein ◽

Splicing Factor ◽

Splice Sites ◽

Reporter System ◽

Kh Domain ◽

Tat Fusion Proteins ◽

Splicing Factor 1

ABSTRACT Mammalian splicing factor 1 (SF1; also mammalian branch point binding protein [mBBP]; hereafter SF1/mBBP) specifically recognizes the seven-nucleotide branch point sequence (BPS) located at 3′ splice sites and participates in the assembly of early spliceosomal complexes. SF1/mBBP utilizes a “maxi-K homology” (maxi-KH) domain for recognition of the single-stranded BPS and requires a cooperative interaction with splicing factor U2AF65 bound to an adjacent polypyrimidine tract (PPT) for high-affinity binding. To investigate how the KH domain of SF1/mBBP recognizes the BPS in conjunction with U2AF and possibly other proteins, we constructed a transcriptional reporter system utilizing human immunodeficiency virus type 1 Tat fusion proteins and examined the RNA-binding specificity of the complex using KH domain and RNA-binding site mutants. We first established that SF1/mBBP and U2AF cooperatively assemble in our reporter system at RNA sites composed of the BPS, PPT, and AG dinucleotide found at 3′ splice sites, with endogenous proteins assembled along with the Tat fusions. We next found that the activities of the Tat fusion proteins on different BPS variants correlated well with the known splicing efficiencies of the variants, supporting a model in which the SF1/mBBP-BPS interaction helps determine splicing efficiency prior to the U2 snRNP-BPS interaction. Finally, the likely RNA-binding surface of the maxi-KH domain was identified by mutagenesis and appears similar to that used by “simple” KH domains, involving residues from two putative α helices, a highly conserved loop, and parts of a β sheet. Using a homology model constructed from the cocrystal structure of a Nova KH domain-RNA complex (Lewis et al., Cell 100:323–332, 2000), we propose a plausible arrangement for SF1/mBBP-U2AF complexes assembled at 3′ splice sites.

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Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

Applied and Environmental Microbiology ◽

10.1128/aem.06127-11 ◽

2011 ◽

Vol 78 (2) ◽

pp. 560-567 ◽

Cited By ~ 7

Author(s):

Denise Knobloch ◽

Kai Ostermann ◽

Gerhard Rödel

Keyword(s):

Cell Surface ◽

Bacillus Megaterium ◽

Fusion Proteins ◽

Shuttle Vector ◽

Surface Display ◽

Fluorescent Labeling ◽

Content Type ◽

Sporosarcina Ureae

ABSTRACTMonomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report thatBacillus megateriumis an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA fromSporosarcina ureaeATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into theEscherichia coli-Bacillus megateriumshuttle vector pHIS1525. After transformation of the respective plasmids intoBacillus megateriumprotoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemblein vitrointo highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope ofBacillus megaterium, indicating that the cell surface can servein vivoas a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

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Identification of P1 gene domain containing epitope(s) mediating Mycoplasma pneumoniae cytoadherence.

Journal of Experimental Medicine ◽

10.1084/jem.167.2.718 ◽

1988 ◽

Vol 167 (2) ◽

pp. 718-723 ◽

Cited By ~ 44

Author(s):

S F Dallo ◽

C J Su ◽

J R Horton ◽

J B Baseman

Keyword(s):

Amino Acids ◽

Mycoplasma Pneumoniae ◽

Synthetic Peptide ◽

Fusion Proteins ◽

Genomic Dna ◽

Expression Vector ◽

Genomic Library ◽

Vaccine Candidate ◽

Single Copy

A genomic library of Mycoplasma pneumoniae was constructed by cloning sheared genomic DNA into the expression vector lambda gt11. Recombinant clones were screened using anti-M. pneumoniae mAbs reactive with adhesin P1 epitopes that mediate cytadherence. 10 clones with different size inserts were isolated. These clones possessed P1 sequences localized to the COOH terminus of the P1 gene. All clones produced fusion proteins that reacted with acute and convalescent sera of patients infected with M. pneumoniae. Interestingly, one clone, P1-7, contained an epitope that was confined to a region of 13 amino acids present in the M. pneumoniae genome as a single copy. The identification of this cytadherence-related epitope permits the production of a synthetic peptide that can be used as a rational vaccine candidate and serodiagnostic probe.

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An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies and gene therapy

Gene ◽

10.1016/j.gene.2012.01.011 ◽

2012 ◽

Vol 496 (2) ◽

pp. 103-109 ◽

Cited By ~ 1

Author(s):

John Fuesler ◽

Yasunori Nagahama ◽

Joseph Szulewski ◽

Joshua Mundorff ◽

Stephanie Bireley ◽

...

Keyword(s):

Gene Therapy ◽

Genomic Library ◽

Association Studies ◽

Shuttle Vector ◽

Genome Wide Association ◽

Genome Wide Association Studies ◽

Genome Wide ◽

Human Genomic

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Signal-sequence-independent secretion of the staphylococcal nuclease in Mycobacterium smegmatis

Microbiology ◽

10.1099/00221287-148-2-529 ◽

2002 ◽

Vol 148 (2) ◽

pp. 529-536 ◽

Cited By ~ 7

Author(s):

Chiara Recchi ◽

Jean Rauzier ◽

Brigitte Gicquel ◽

Jean-Marc Reyrat

Keyword(s):

Mycobacterium Smegmatis ◽

Mutational Analysis ◽

Signal Sequence ◽

Staphylococcal Nuclease ◽

Secreted Protein ◽

Bacterial Survival ◽

Reporter System ◽

Secretion Pathway ◽

Hybrid Proteins ◽

Translocation Pathway

Staphylococcus aureus nuclease is a small, secreted protein which has been successfully used as a reporter system to identify exported products in Lactococcus lactis. Here, biochemical evidence is provided that the nuclease is exported by Mycobacterium smegmatis in the presence, but also in the absence of a signal sequence, and thus probably independently of the Sec translocation pathway. This implies that the nuclease should not be used as a reporter system in mycobacteria for the identification of exported products, despite what has been reported previously in the literature. The nuclease can be extended to create hybrid proteins that remain compatible with its secretion, whereas some other shorter fusions are not tolerated. This suggests that correct folding is required for efficient export. Extensive mutational analysis did not identify a specific secretion pathway. This suggests that the nuclease may be exported by different redundant systems or that components of this alternative Sec pathway are essential for bacterial survival.

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Structural and Functional Analysis of the Gene Cluster Encoding the Enzymes of the Arginine Deiminase Pathway ofLactobacillus sake

Journal of Bacteriology ◽

10.1128/jb.180.16.4154-4159.1998 ◽

1998 ◽

Vol 180 (16) ◽

pp. 4154-4159 ◽

Cited By ~ 93

Author(s):

Manuel Zúñiga ◽

Marie Champomier-Verges ◽

Monique Zagorec ◽

Gaspar Pérez-Martínez

Keyword(s):

Genomic Library ◽

Arginine Deiminase ◽

Degenerate Primers ◽

Pcr Screening ◽

Lactobacillus Sake ◽

Arginine Catabolism ◽

Carbamate Kinase ◽

Encoding Gene ◽

Ornithine Transcarbamoylase ◽

Encoding Genes

ABSTRACT Lactobacillus sake can use arginine via the arginine deiminase (ADI) pathway. We designed degenerate primers based on an alignment of known sequences of ornithine transcarbamoylase (OTC)-encoding genes in order to amplify the L. sakecounterpart sequences by PCR. Screening a genomic library of L. sake in λEMBL3 allowed us to isolate a clone containing a 10-kbL. sake genomic DNA insert. Sequence analysis revealed that the genes involved in arginine catabolism were clustered and encoded ADI (arcA), OTC (arcB), carbamate kinase (arcC), and a putative carrier with high similarity to the arginine/ornithine antiporter of Pseudomonas aeruginosa(arcD). Additionally, a putative transaminase-encoding gene (arcT) was located in this region. The genes followed the order arcA arcB arcC arcT arcD, which differs from that found in other microorganisms. arcA, arcB,arcC, and arcD mutants were constructed, and the ADI pathway was impaired in all of them. Transcriptional studies indicated that arcA gene is subject to catabolite repression, and under the conditions used, several transcripts could be detected, suggesting the existence of different initiation sites or processing of a larger mRNA.

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Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination

BMC Biotechnology ◽

10.1186/s12896-020-00650-x ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Sanum Bashir ◽

Tu Dang ◽

Jana Rossius ◽

Johanna Wolf ◽

Ralf Kühn

Keyword(s):

Fusion Proteins ◽

Mammalian Cells ◽

Gene Editing ◽

Hek293 Cells ◽

Histone H2a ◽

Traffic Light ◽

Reporter System ◽

Binding Domains ◽

And Shifts ◽

Endogenous Loci

Abstract Background Precise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathway and depends on the presence of repair templates its efficiency is often limited and demands optimized methodology. Results For the enhancement of HDR we redirect the DSB repair pathway choice by targeting the Ubiquitin mark for damaged chromatin at Histone H2A-K15. We used fusions of the Ubiquitin binding domain (UBD) of Rad18 or RNF169 with BRCA1 to promote HDR initiation and UBD fusions with DNA binding domains to attract donor templates and facilitate HDR processing. Using a traffic light reporter system in human HEK293 cells we found that the coexpression of both types of UBD fusion proteins promotes HDR, reduces NHEJ and shifts the HDR/NHEJ balance up to 6-fold. The HDR enhancing effect of UBD fusion proteins was confirmed at multiple endogenous loci. Conclusions Our findings provide a novel efficient approach to promote precise gene editing in human cells.

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Cloning of the Gene Encoding a 22-Kilodalton Cell Surface Antigen of Mycobacterium bovisBCG and Analysis of Its Potential for DNA Vaccination against Tuberculosis

Infection and Immunity ◽

10.1128/iai.68.3.1040-1047.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1040-1047 ◽

Cited By ~ 25

Author(s):

Philippe Lefèvre ◽

Olivier Denis ◽

Lucas De Wit ◽

Audrey Tanghe ◽

Paul Vandenbussche ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Genomic Library ◽

Signal Sequence ◽

Interleukin 2 ◽

Dna Vaccination ◽

Mouse Strains ◽

Mycobacterial Antigen ◽

Cell Fractionation ◽

Dna Encoding ◽

Gene Encoding

ABSTRACT Using spleen cells from mice vaccinated with liveMycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687–2693, 1993). These monoclonal antibodies were used to screen an M. bovis BCG genomic library made in phage λgt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contained a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosis challenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosis challenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test.

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