scholarly journals Characterization of a Multigene Family Undergoing High-Frequency DNA Rearrangements and Coding for Abundant Variable Surface Proteins in Mycoplasma agalactiae

2000 ◽  
Vol 68 (8) ◽  
pp. 4539-4548 ◽  
Author(s):  
M. D. Glew ◽  
L. Papazisi ◽  
F. Poumarat ◽  
D. Bergonier ◽  
R. Rosengarten ◽  
...  
Keyword(s):  
Phase Variation ◽  
Gene Families ◽  
Surface Proteins ◽  
Dna Rearrangements ◽  
Reading Frame ◽  
Mycoplasma Agalactiae ◽  
Amino Terminal ◽  
Clonal Variant ◽  
Variable Surface

ABSTRACT A family of abundant surface proteins (Vpmas [variable proteins ofMycoplasma agalactiae]) undergoing phase variation inM. agalactiae has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An amino-terminal oligonucleotide probe identified multiple vpma genes which were clustered within a 14-kb ClaI genomic fragment. Rearrangements were found to have occurred within the vpma locus between clones which correlated with changes in their Vpma phenotype. Two neighboringvpma genes were cloned and sequenced from one M. agalactiae clonal variant expressing Vpma39. The two genes,vpmaX and vpmaY, were orientated divergently and shared highly homologous 5′ untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. ThevpmaY gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second vpmaY repeat was present, it was prematurely terminated by insertion of two nucleotides. ThevpmaX gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of vpmaY. Many of the features in common between the vpma genes were also found to be shared by thevsp genes of M. bovis, which also undergo DNA rearrangements concomitant with phenotypic changes. Since M. bovis is the closest phylogenetic relative to M. agalactiae, the vpma and vsp gene families most probably represent homologous systems.

scholarly journals Surface Diversity in Mycoplasma agalactiae Is Driven by Site-Specific DNA Inversions within the vpma Multigene Locus

2002 ◽  
Vol 184 (21) ◽  
pp. 5987-5998 ◽  
Author(s):  
Michelle D. Glew ◽  
Marc Marenda ◽  
Renate Rosengarten ◽  
Christine Citti
Keyword(s):  
Promoter Sequence ◽  
Untranslated Regions ◽  
Dna Rearrangements ◽  
Site Specific ◽  
Reading Frame ◽  
Mycoplasma Agalactiae ◽  
Variable Surface ◽  
A Site ◽  
Active Promoter ◽  
Minimal Region

ABSTRACT The ruminant pathogen Mycoplasma agalactiae possesses a family of abundantly expressed variable surface lipoproteins called Vpmas. Phenotypic switches between Vpma members have previously been correlated with DNA rearrangements within a locus of vpma genes and are proposed to play an important role in disease pathogenesis. In this study, six vpma genes were characterized in the M. agalactiae type strain PG2. All vpma genes clustered within an 8-kb region and shared highly conserved 5′ untranslated regions, lipoprotein signal sequences, and short N-terminal sequences. Analyses of the vpma loci from consecutive clonal isolates showed that vpma DNA rearrangements were site specific and that cleavage and strand exchange occurred within a minimal region of 21 bp located within the 5′ untranslated region of all vpma genes. This process controlled expression of vpma genes by effectively linking the open reading frame (ORF) of a silent gene to a unique active promoter sequence within the locus. An ORF (xer1) immediately adjacent to one end of the vpma locus did not undergo rearrangement and had significant homology to a distinct subset of genes belonging to the λ integrase family of site-specific xer recombinases. It is proposed that xer1 codes for a site-specific recombinase that is not involved in chromosome dimer resolution but rather is responsible for the observed vpma-specific recombination in M. agalactiae.

Cartilage associated protein (CASP) is a novel developmentally regulated chick embryo protein

1997 ◽  
Vol 110 (12) ◽  
pp. 1351-1359
Author(s):  
P. Castagnola ◽  
M. Gennari ◽  
R. Morello ◽  
L. Tonachini ◽  
O. Marin ◽  
...  
Keyword(s):  
Chick Embryo ◽  
Developmentally Regulated ◽  
Reading Frame ◽  
Amino Terminal ◽  
Nuclear Antigens ◽  
Cartilage Extracellular Matrix ◽  
Low Levels ◽  
Specific Rabbit ◽  
Chicken Protein

A subtracted cDNA library was generated to identify cDNAs specific for chondrocyte mRNAs preferentially expressed at the hypertrophic stage with respect to early differentiation stages. The characterization of a cDNA isolated from this library that hybridizes with a 1.8 kb mRNA is described here. This mRNA is expressed at extremely low levels in dedifferentiated chondrocytes cultured in adherent conditions, at very low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes cultured in suspension conditions. In the developing chick embryo this mRNA is detectable in RNAs extracted from several other tissues besides cartilage. The described cDNA contains a complete open reading frame coding for a polypeptide of about 33 kDa. Homology searches with known cDNA and protein sequences have revealed that the chicken protein is related to the amino-terminal half of two mammalian nuclear antigens. By immunohistochemistry with specific rabbit antisera a strong signal was detected in the cartilage extracellular matrix of selected regions of the developing skeleton. Because of this localization of the antigen we named this protein cartilage associated protein (hereafter referred to as CASP).

scholarly journals Xer1-Mediated Site-Specific DNA Inversions and Excisions in Mycoplasma agalactiae

2010 ◽  
Vol 192 (17) ◽  
pp. 4462-4473 ◽  
Author(s):  
Stefan Czurda ◽  
Wolfgang Jechlinger ◽  
Renate Rosengarten ◽  
Rohini Chopra-Dewasthaly
Keyword(s):  
High Frequency ◽  
Phase Variation ◽  
Etiologic Agent ◽  
Reporter System ◽  
Recognition Sequence ◽  
Site Specific ◽  
Phase Switching ◽  
Site Specific Recombination ◽  
Mycoplasma Agalactiae ◽  
Variable Surface

ABSTRACT Surface antigen variation in Mycoplasma agalactiae, the etiologic agent of contagious agalactia in sheep and goats, is governed by site-specific recombination within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. This high-frequency Vpma phase switching was previously shown to be mediated by a Xer1 recombinase encoded adjacent to the vpma locus. In this study, it was demonstrated in Escherichia coli that the Xer1 recombinase is responsible for catalyzing vpma gene inversions between recombination sites (RS) located in the 5′-untranslated region (UTR) in all six vpma genes, causing cleavage and strand exchange within a 21-bp conserved region that serves as a recognition sequence. It was further shown that the outcome of the site-specific recombination event depends on the orientation of the two vpma RS, as direct or inverted repeats. While recombination between inverted vpma RS led to inversions, recombination between direct repeat vpma RS led to excisions. Using a newly developed excision assay based on the lacZ reporter system, we were able to successfully demonstrate under native conditions that such Xer1-mediated excisions can indeed also occur in the M. agalactiae type strain PG2, whereas they were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1 recombinase. Unless there are specific regulatory mechanisms preventing such excisions, this might be the cost that the pathogen has to render at the population level for maintaining this high-frequency phase variation machinery.

scholarly journals Physiological Osmotic Induction of Leptospira interrogans Adhesion: LigA and LigB Bind Extracellular Matrix Proteins and Fibrinogen

2007 ◽  
Vol 75 (5) ◽  
pp. 2441-2450 ◽  
Author(s):  
Henry A. Choy ◽  
Melissa M. Kelley ◽  
Tammy L. Chen ◽  
Annette K. Møller ◽  
James Matsunaga ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Binding Activity ◽  
Surface Proteins ◽  
Microbial Pathogens ◽  
Host Proteins ◽  
Amino Terminal ◽  
Binding Domains ◽  
Multiple Ligands ◽  
Carboxy Terminal

ABSTRACTTransmission of leptospirosis occurs through contact of mucous membranes and abraded skin with freshwater contaminated by pathogenicLeptospiraspp. Exposure to physiological osmolarity induces leptospires to express high levels of the Lig surface proteins containing imperfect immunoglobulin-like repeats that are shared or differ between LigA and LigB. We report that osmotic induction of Lig is accompanied by 1.6- to 2.5-fold increases in leptospiral adhesion to immobilized extracellular matrix and plasma proteins, including collagens I and IV, laminin, and especially fibronectin and fibrinogen. Recombinant LigA-unique and LigB-unique repeat proteins bind to these same host ligands. We found that the avidity of LigB in binding fibronectin is comparable to that of theStaphylococcus aureusFnBPA D repeats. Both LigA- and LigB-unique repeats interact with the amino-terminal fibrin- and gelatin-binding domains of fibronectin, which are also recognized by fibronectin-binding proteins mediating the adhesion of other microbial pathogens. In contrast, repeats common to both LigA and LigB do not bind these host proteins, and nonrepeat sequences in the carboxy-terminal domain of LigB show only weak interaction with fibronectin and fibrinogen. A functional role for the binding activity of LigA and LigB is suggested by the ability of the recombinants to inhibit leptospiral adhesion to fibronectin by 28% and 21%, respectively. The binding of LigA and LigB to multiple ligands present in different tissues suggests that these adhesins may be involved in the initial colonization and dissemination stages of leptospirosis. The characterization of the Lig adhesin function should aid the design of Lig-based vaccines and serodiagnostic tests.

scholarly journals Characterization of membrane surface proteins of Mycoplasma agalactiae during natural infection

2006 ◽  
Vol 154 (2) ◽  
pp. 355-362 ◽  
Author(s):  
Sebastiana Tola ◽  
Daniela Manunta ◽  
Monica Cocco ◽  
Franco Turrini ◽  
Angela M. Rocchigiani ◽  
...  
Keyword(s):  
Natural Infection ◽  
Membrane Surface ◽  
Surface Proteins ◽  
Mycoplasma Agalactiae

scholarly journals Cloning, Expression, and Characterization of Aminopeptidase P from the Hyperthermophilic Archaeon Thermococcus sp. Strain NA1

2006 ◽  
Vol 72 (3) ◽  
pp. 1886-1890 ◽  
Author(s):  
Hyun Sook Lee ◽  
Yun Jae Kim ◽  
Seung Seob Bae ◽  
Jeong Ho Jeon ◽  
Jae Kyu Lim ◽  
...  
Keyword(s):  
Metal Binding ◽  
Genomic Analysis ◽  
Enzyme Characterization ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Hyperthermophilic Archaeon ◽  
Amino Terminal ◽  
Aminopeptidase P

ABSTRACT Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. strain NA1, revealed the presence of a 1,068-bp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the putative aminopeptidase P (APP) of Thermococcus kodakaraensis KOD1. Amino acid residues important for catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs were also conserved in the Thermococcus sp. strain NA1 APP. The archaeal APP, designated TNA1_APP (Thermococcus sp. strain NA1 APP), was cloned and expressed in Escherichia coli. The recombinant enzyme hydrolyzed the amino-terminal Xaa-Pro bond of Lys(N ε-Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (Km , 0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co2+-, Mn2+-, or Zn2+-dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at pH 5 and a temperature of 100°C. The APP was thermostable, with a half-life of >100 min at 80°C. This study represents the first characterization of a hyperthermophilic archaeon APP.

scholarly journals Purification, Gene Cloning, Targeted Knockout, Overexpression, and Biochemical Characterization of the Major Pyrazinamidase fromMycobacterium smegmatis

1998 ◽  
Vol 180 (22) ◽  
pp. 5809-5814 ◽  
Author(s):  
Helena I. M. Boshoff ◽  
Valerie Mizrahi
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Protein Overexpression ◽  
Reading Frame ◽  
Amino Terminal ◽  
Dna Library ◽  
Terminal Amino ◽  
Unique Specificity ◽  
Kinetics Of

ABSTRACT The pyrazinamidase from Mycobacterium smegmatis was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of M. smegmatis. An open reading frame, designatedpzaA, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the pzaA gene by homologous recombination yielded a mutant, pzaA::aph, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of M. smegmatis. Recombinant forms of the M. smegmatis PzaA and the Mycobacterium tuberculosispyrazinamidase/nicotinamidase (PncA) were produced in Escherichia coli and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparableKm values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for M. tuberculosis was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of pzaAconferred PZA susceptibility on M. smegmatis by reducing the MIC of this drug to 150 μg/ml.

scholarly journals Identification of pel, aStreptococcus pyogenes Locus That Affects both Surface and Secreted Proteins

1999 ◽  
Vol 181 (19) ◽  
pp. 6019-6027 ◽  
Author(s):  
Zhuqing Li ◽  
Darren D. Sledjeski ◽  
Bernd Kreikemeyer ◽  
Andreas Podbielski ◽  
Michael D. P. Boyle
Keyword(s):  
Streptococcus Pyogenes ◽  
Pleiotropic Effect ◽  
Surface Proteins ◽  
Insertion Mutant ◽  
Wild Type ◽  
Regulatory Factor ◽  
Reading Frame ◽  
Streptolysin S ◽  
Fibrinogen Binding

ABSTRACT A Tn917 insertion mutant of an M49 serotype, opacity factor-positive Streptococcus pyogenes, was isolated. It had the following phenotypes: decreased β-hemolysis mediated by streptolysin S, reduction in the activity of a secreted cysteine protease and streptokinase, and an altered immunoglobulin and fibrinogen-binding phenotype. The site of insertion of Tn917 into the chromosome and the surrounding sequence, thepel region (pleiotropic effect locus), was determined. Phage A25 transduction confirmed that the pleiotropic changes in phenotype could be cotransduced with Tn917. Thepel region was cloned and sequenced, and the transposon was found to be inserted upstream of a single open reading frame which led to a failure to transcribe a 500-base mRNA. The loss of this transcript decreased the transcription of emm and speBgenes and reduced the secretion of streptokinase. Enhanced Pel expression from a nisin-inducible plasmid resulted in increased message levels for emm in a wild-type organism. Characterization of the pel mutant provides evidence for the coordinated regulation of secreted and surface proteins and suggests the existence of a new global regulatory factor in S. pyogenes.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Identification of novel haplotype of a cyst nematode resistance gene, GmSNAP18 in soybean [Glycine max (L.) Merr.]

2020 ◽  
Vol 80 (03) ◽  
Author(s):  
Ik-Young Choi ◽  
Prakash Basnet ◽  
Hana Yoo ◽  
Neha Samir Roy ◽  
Rahul Vasudeo Ramekar ◽  
...  
Keyword(s):  
Soybean Cyst Nematode ◽  
Gene Families ◽  
Nematode Resistance ◽  
Cyst Nematode ◽  
Soybean Breeding ◽  
Resistant Varieties ◽  
Soybean Genotypes ◽  
Glycine Max L ◽  
Scn Resistance

Soybean cyst nematode (SCN) is one of the most damaging pest of soybean. Discovery and characterization of the genes involved in SCN resistance are important in soybean breeding. Soluble NSF attachment protein (SNAP) genes are related to SCN resistance in soybean. SNAP genes include five gene families, and 2 haplotypes of exons 6 and 9 of SNAP18 are considered resistant to the SCN. In present study the haplotypes of GmSNAP18 were surveyed and chacterized in a total of 60 diverse soybean genotypes including Korean cultivars, landraces, and wild-types. The target region of exons 6 and 9 in GmSNAP18 region was amplified and sequenced to examine nucleotide variation. Characterization of 5 haplotypes identified in present study for the GmSNAP18 gene revealed two haplotypes as resistant, 1 as susceptible and two as novel. A total of twelve genotypes showed resistant haplotypes, and 45 cultivars were found susceptible. Interestingly, the two novel haplotypes were present in 3 soybean lines. The information provided here about the haplotypic variation of GmSNAP18 gene can be further explored for soybean breeding to develop resistant varieties.

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