scholarly journals Oms38 Is the First Identified Pore-Forming Protein in the Outer Membrane of Relapsing Fever Spirochetes

2008 ◽  
Vol 190 (21) ◽  
pp. 7035-7042 ◽  
Author(s):  
Marcus Thein ◽  
Ignas Bunikis ◽  
Katrin Denker ◽  
Christer Larsson ◽  
Sally Cutler ◽  
...  
Keyword(s):  
Amino Acid ◽  
Outer Membrane ◽  
Signal Sequence ◽  
Amino Acid Identity ◽  
Amino Acid Sequences ◽  
Relapsing Fever ◽  
Small Water ◽  
Membrane Spanning

ABSTRACT Relapsing fever is a worldwide, endemic disease caused by several spirochetal species belonging to the genus Borrelia. During the recurring fever peaks, borreliae proliferate remarkably quickly compared to the slow dissemination of Lyme disease Borrelia and therefore require efficient nutrient uptake from the blood of their hosts. This study describes the identification and characterization of the first relapsing fever porin, which is present in the outer membranes of B. duttonii, B. hermsii, B. recurrentis, and B. turicatae. The pore-forming protein was purified by hydroxyapatite chromatography and designated Oms38, for outer membrane-spanning protein of 38 kDa. Biophysical characterization of Oms38 was done by using the black lipid bilayer method, demonstrating that Oms38 forms small, water-filled channels of 80 pS in 1 M KCl that did not exhibit voltage-dependent closure. The Oms38 channel is slightly selective for anions and shows a ratio of permeability for cations over anions of 0.41 in KCl. Analysis of the deduced amino acid sequences demonstrated that Oms38 contains an N-terminal signal sequence which is processed under in vivo conditions. Oms38 is highly conserved within the four studied relapsing fever species, sharing an overall amino acid identity of 58% and with a strong indication for the presence of amphipathic β-sheets.

scholarly journals Ponticulin is an atypical membrane protein.

1994 ◽  
Vol 126 (6) ◽  
pp. 1421-1431 ◽  
Author(s):  
A L Hitt ◽  
T H Lu ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Plasma Membranes ◽  
Signal Sequence ◽  
Metabolic Labeling ◽  
Cortical Actin ◽  
Intact Cells ◽  
Membrane Spanning

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

scholarly journals Molecular characterization of three newly recognized rat parvoviruses

2002 ◽  
Vol 83 (8) ◽  
pp. 2075-2083 ◽  
Author(s):  
Cho-Hua Wan ◽  
Maria Söderlund-Venermo ◽  
David J. Pintel ◽  
Lela K. Riley
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Molecular Characterization ◽  
Amino Acid Sequences ◽  
Minute Virus ◽  
Genomic Nucleotide Sequence ◽  
Kilham Rat Virus

Rodent parvoviruses have been documented to interfere with both in vivo and in vitro research. In this study, three rat parvoviruses distinct from previously characterized rodent parvoviruses were identified from naturally infected rats obtained from four discrete sources. These three newly recognized parvoviruses were designated rat minute virus (RMV)-1a, -1b and -1c. In this study, the genomic nucleotide sequence and the predicted amino acid sequences of proteins for each of the three RMV-1 variants and Kilham rat virus (KRV) were determined and compared with previously characterized rodent parvoviruses. The three RMV-1 variants were shown to be closely related to each other, to be distinct from but closely related to KRV and H-1 virus, and to be significantly different from the previously identified rat parvovirus isolate, RPV-1a.

scholarly journals Characterization of a novel flavivirus isolated from Culex (Melanoconion) ocossa mosquitoes from Iquitos, Peru

2013 ◽  
Vol 94 (6) ◽  
pp. 1266-1272 ◽  
Author(s):  
Julio Evangelista ◽  
Cristhopher Cruz ◽  
Carolina Guevara ◽  
Helvio Astete ◽  
Cristiam Carey ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Amazon Basin ◽  
Yellow Fever Virus ◽  
Amino Acid Identity ◽  
Amino Acid Sequences ◽  
Newborn Mice ◽  
Isolation And Characterization ◽  
Indirect Immunofluorescent Assay

We describe the isolation and characterization of a novel flavivirus, isolated from a pool of Culex (Melanoconion) ocossa Dyar and Knab mosquitoes collected in 2009 in an urban area of the Amazon basin city of Iquitos, Peru. Flavivirus infection was detected by indirect immunofluorescent assay of inoculated C6/36 cells using polyclonal flavivirus antibodies (St. Louis encephalitis virus, yellow fever virus and dengue virus type 1) and confirmed by RT-PCR. Based on partial sequencing of the E and NS5 gene regions, the virus isolate was most closely related to the mosquito-borne flaviviruses but divergent from known species, with less than 45 and 71 % pairwise amino acid identity in the E and NS5 gene products, respectively. Phylogenetic analysis of E and NS5 amino acid sequences demonstrated that this flavivirus grouped with mosquito-borne flaviviruses, forming a clade with Nounané virus (NOUV). Like NOUV, no replication was detected in a variety of mammalian cells (Vero-76, Vero-E6, BHK, LLCMK, MDCK, A549 and RD) or in intracerebrally inoculated newborn mice. We tentatively designate this genetically distinct flavivirus as representing a novel species, Nanay virus, after the river near where it was first detected.

scholarly journals Genetic Diversity of the 28-Kilodalton Outer Membrane Protein Gene in Human Isolates of Ehrlichia chaffeensis

1999 ◽  
Vol 37 (4) ◽  
pp. 1137-1143 ◽  
Author(s):  
Xue-Jie Yu ◽  
Jere W. McBride ◽  
David H. Walker
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Dna Sequence ◽  
Outer Membrane Protein ◽  
Signal Sequence ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
Amino Acid Sequences ◽  
Ehrlichia Chaffeensis

The Ehrlichia chaffeensis 28-kDa outer membrane protein (p28) gene was sequenced completely by genomic walking with adapter PCR. The DNA sequence of the p28 gene was nearly identical to the previously reported sequence (N. Ohashi, N. Zhi, Y. Zhang, and Y. Rikihisa, Infect. Immun. 66:132–139, 1998), but analysis of a further 75 bp on the 5′ end of the gene revealed DNA that encoded a 25-amino-acid signal sequence. The leader sequence was removed from the N terminus of a 30-kDa precursor to generate the mature p28 protein. A monoclonal antibody (MAb), 1A9, recognizing four outer membrane proteins of E. chaffeensis (Arkansas strain) including the 25-, 26-, 27-, and 29-kDa proteins (X.-J. Yu, P. Brouqui, J. S. Dumler, and D. Raoult, J. Clin. Microbiol. 31:3284–3288, 1993) reacted with the recombinant p28 protein. This result indicated that the four proteins recognized by MAb 1A9 were encoded by the multiple genes of the 28-kDa protein family. DNA sequence alignment analysis revealed divergence of p28 among all five human isolates of E. chaffeensis. The E. chaffeensis strains could be divided into three genetic groups on the basis of the p28 gene. The first group consisted of the Sapulpa and St. Vincent strains. They had predicted amino acid sequences identical to each other. The second group contained strain 91HE17 and strain Jax, which only showed 0.4% divergence from each other. The third group contained the Arkansas strain only. The amino acid sequences of p28 differed by 11% between the first two groups, by 13.3% between the first and third groups, and by 13.1% between the second and third groups. The presence of antigenic variants of p28 among the strains of E. chaffeensis and the presence of multiple copies of heterogeneous genes suggest a possible mechanism by which E. chaffeensismight evade the host immune defenses. Whether or not immunization with the p28 of one strain of E. chaffeensis would confer cross-protection against other strains needs to be investigated.

scholarly journals Signal Sequence Mutations as Tools for the Characterization of LamB Folding Intermediates

2002 ◽  
Vol 184 (24) ◽  
pp. 6918-6928 ◽  
Author(s):  
Amy Rizzitello Duguay ◽  
Thomas J. Silhavy
Keyword(s):  
Amino Acid ◽  
Outer Membrane ◽  
Biochemical Analysis ◽  
Positive Charge ◽  
Signal Sequence ◽  
Wild Type ◽  
Inner Membrane ◽  
Folding Intermediates ◽  
Sequence Processing

ABSTRACT lamBA23DA25Y and lamBA23YA25Y tether LamB to the inner membrane by blocking signal sequence processing. We isolated suppressors of lamBA23DA25Y and lamBA23YA25Y, all of which mapped within the LamB signal sequence. Most interesting were mutations that changed an amino acid with a strong positive charge to an amino acid with no charge. Further characterization of two such suppressors revealed that they produce functional LamB that is localized to the outer membrane with its entire signal sequence still attached. Biochemical analysis shows that mutant LamB monomer chases into an oligomeric species with properties different from those of wild-type LamB trimer. Because assembly of mutant LamB is slowed, these mutations provide useful tools for the characterization of LamB folding intermediates.

scholarly journals Identification and Characterization of thefis Operon in Enteric Bacteria

1998 ◽  
Vol 180 (22) ◽  
pp. 5932-5946 ◽  
Author(s):  
Michael B. Beach ◽  
Robert Osuna
Keyword(s):  
Amino Acid ◽  
Genomic Sequence ◽  
Erwinia Carotovora ◽  
Enteric Bacteria ◽  
Integration Host Factor ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
E Coli

ABSTRACT The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage λ genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, andProteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens,E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence forH. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) precedingfis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from −53 to +27 and the region around −116 containing an ihfbinding site. Both β-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.

scholarly journals Small Mass but Strong Information: Diagnostic Ions Provide Crucial Clues to Correctly Identify Histone Lysine Modifications

Proteomes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 18
Author(s):  
Alaa Hseiky ◽  
Marion Crespo ◽  
Sylvie Kieffer-Jaquinod ◽  
François Fenaille ◽  
Delphine Pflieger
Keyword(s):  
Amino Acid ◽  
Small Mass ◽  
Mass Region ◽  
Amino Acid Sequences ◽  
Lysine Modification ◽  
Lysine Residues ◽  
Two Factors ◽  
Low Mass ◽  
Diagnostic Ions

(1) Background: The proteomic analysis of histones constitutes a delicate task due to the combination of two factors: slight variations in the amino acid sequences of variants and the multiplicity of post-translational modifications (PTMs), particularly those occurring on lysine residues. (2) Methods: To dissect the relationship between both aspects, we carefully evaluated PTM identification on lysine 27 from histone H3 (H3K27) and the artefactual chemical modifications that may lead to erroneous PTM determination. H3K27 is a particularly interesting example because it can bear a range of PTMs and it sits nearby residues 29 and 31 that vary between H3 sequence variants. We discuss how the retention times, neutral losses and immonium/diagnostic ions observed in the MS/MS spectra of peptides bearing modified lysines detectable in the low-mass region might help validate the identification of modified sequences. (3) Results: Diagnostic ions carry key information, thereby avoiding potential mis-identifications due to either isobaric PTM combinations or isobaric amino acid-PTM combinations. This also includes cases where chemical formylation or acetylation of peptide N-termini artefactually occurs during sample processing or simply in the timeframe of LC-MS/MS analysis. Finally, in the very subtle case of positional isomers possibly corresponding to a given mass of lysine modification, the immonium and diagnostic ions may allow the identification of the in vivo structure.

scholarly journals Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (15) ◽  
pp. 5067-5074 ◽  
Author(s):  
Daisuke Kasai ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Ring Cleavage ◽  
Sphingomonas Paucimobilis ◽  
Acid Protein ◽  
Demethylation Pathway ◽  
Dioxygenase Gene ◽  
Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

Functional and Structural Characterization of Acquired Pan-Aminoglycoside Resistance 16S rRNA Methyltransferase NpmB1

2021 ◽  
Author(s):  
Akito Kawai ◽  
Masahiro Suzuki ◽  
Kentaro Tsukamoto ◽  
Yusuke Minato ◽  
Yohei Doi
Keyword(s):  
Amino Acid ◽  
16S Rrna ◽  
Amino Acid Identity ◽  
Single Amino Acid ◽  
Aminoglycoside Resistance ◽  
E Coli ◽  
The United Kingdom ◽  
Amino Acid Variant ◽  
A Site

Post-translational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such function. Here, we present function and structure of NpmB1 whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single amino acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the β6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

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