scholarly journals Expression of the Moraxella catarrhalisUspA1 Protein Undergoes Phase Variation and Is Regulated at the Transcriptional Level

2001 ◽  
Vol 183 (5) ◽  
pp. 1540-1551 ◽  
Author(s):  
Eric R. Lafontaine ◽  
Nikki J. Wagner ◽  
Eric J. Hansen
Keyword(s):  
Blot Analysis ◽  
Transcriptional Start Site ◽  
Phase Variation ◽  
Nucleotide Sequence Analysis ◽  
Northern Hybridization ◽  
Transcriptional Level ◽  
Wild Type ◽  
Reading Frame ◽  
Slot Blot Analysis

ABSTRACT The UspA1 protein of Moraxella catarrhalis has been shown to function as an adhesin that mediates adherence to human epithelial cell lines in vitro (E. R. Lafontaine, L. D. Cope, C. Aebi, J. L. Latimer, G. H. McCracken, Jr., and E. J. Hansen, J. Bacteriol. 182:1364–1373, 2000). In the present study, cell lysates prepared from individual colonies of several M. catarrhalis wild-type strains were analyzed by Western blot analysis using monoclonal antibodies (MAbs) specific for the UspA1 protein. Expression of UspA1 was shown to exhibit phase variation that was correlated with both adherence ability in vitro and the number of guanine (G) residues contained within a homopolymeric [poly(G)]tract located upstream of the uspA1 open reading frame (ORF). Nucleotide sequence analysis revealed that isolates expressing relatively high levels of UspA1 had 10 G residues in theiruspA1 poly(G)tracts, whereas isolates that expressed much lower levels of UspA1 had 9 G residues. This poly(G) tract was located 30 nucleotides (nt) upstream of the uspA1 ORF and 168 nt downstream of the uspA1 transcriptional start site. Primer extension experiments, RNA slot blot analysis, and catreporter constructs were used to demonstrate that M. catarrhalis isolates with 10 G residues in theiruspA1 poly(G) tracts expressed two-to threefold moreuspA1 mRNA than did isolates which had 9 G residues in their poly(G)tracts. Northern hybridization analysis revealed that an intact uspA1 mRNA was readily detectable in RNA fromM. catarrhalis isolates that had 10 G residues in theiruspA1 poly(G) tracts, whereas no full-lengthuspA1 mRNA was observed in isolates whose poly(G)tracts contained 9 G residues. M. catarrhalis strain O35EuspA1 genes that contained wild-type and mutated poly(G) tracts were expressed in Haemophilus influenzae to demonstrate that the length and composition of the poly(G)tract affected expression of UspA1.

scholarly journals Detection of Phase Variation in Expression of Proteins Involved in Hemoglobin and Hemoglobin-Haptoglobin Binding by Nontypeable Haemophilus influenzae

2000 ◽  
Vol 68 (7) ◽  
pp. 4092-4101 ◽  
Author(s):  
Leslie D. Cope ◽  
Zbynek Hrkal ◽  
Eric J. Hansen
Keyword(s):  
Haemophilus Influenzae ◽  
Western Blot ◽  
Blot Analysis ◽  
Phase Variation ◽  
Sole Source ◽  
Nucleotide Sequence Analysis ◽  
Nontypeable Haemophilus Influenzae ◽  
Chromosomal Dna ◽  
Nthi Strain

ABSTRACT Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study (I. Maciver, J. L. Latimer, H. H. Liem, U. Muller-Eberhard, Z. Hrkal, and E. J. Hansen. Infect. Immun. 64:3703–3712, 1996) indicated that nontypeable H. influenzae (NTHI) strain TN106 expressed a protein that bound hemoglobin-haptoglobin and was encoded by an open reading frame (ORF) that contained a CCAA nucleotide repeat. Southern blot analysis revealed that several NTHI strains contained between three and five chromosomal DNA fragments that bound an oligonucleotide probe for CCAA repeats. Three ORFs containing CCAA repeats were identified in NTHI strain N182; two of these ORFs were arranged in tandem. The use of translational fusions involving these three ORFs and the β-lactamase gene from pBR322 revealed that these three ORFs, designated hgbA, hgbB, andhgbC, encoded proteins that could bind hemoglobin, hemoglobin-haptoglobin, or both compounds. Monoclonal antibodies (MAbs) specific for the HgbA, HgbB, and HgbC proteins were produced by immunizing mice with synthetic peptides unique to each protein. Both HgbA and HgbB were readily detected by Western blot analysis in N182 cells grown in the presence of hemoglobin as the sole source of heme, whereas expression of HgbC was found to be much less abundant than that of HgbA and HgbB. The use of these MAbs in a colony blot radioimmunoassay analysis revealed that expression of both HgbA and HgbB was subject to phase variation. PCR and nucleotide sequence analysis were used in conjunction with Western blot analyses to demonstrate that this phase variation involved the CCAA repeats in thehgbA and hgbB ORFs.

scholarly journals Phase Variation of Campylobacter jejuni 81-176 Lipooligosaccharide Affects Ganglioside Mimicry and Invasiveness In Vitro

2002 ◽  
Vol 70 (2) ◽  
pp. 787-793 ◽  
Author(s):  
Patricia Guerry ◽  
Christine M. Szymanski ◽  
Martina M. Prendergast ◽  
Thomas E. Hickey ◽  
Cheryl P. Ewing ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Phase Variation ◽  
Outer Core ◽  
Gene Encoding ◽  
Site Specific ◽  
Reading Frame ◽  
Specific Mutation ◽  
Normal Human ◽  
A Site

ABSTRACT The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM2 and GM3 gangliosides, with minor amounts of structures mimicking GD1b and GD2. Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM2 to GM3 ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM3 ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.

scholarly journals Acid-Inducible Transcription of the Operon Encoding the Citrate Lyase Complex of Lactococcus lactis Biovar diacetylactis CRL264

2004 ◽  
Vol 186 (17) ◽  
pp. 5649-5660 ◽  
Author(s):  
Mauricio G. Martín ◽  
Pablo D. Sender ◽  
Salvador Peirú ◽  
Diego de Mendoza ◽  
Christian Magni
Keyword(s):  
Lactococcus Lactis ◽  
Acid Stress ◽  
Nucleotide Sequence Analysis ◽  
Transcriptional Level ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
Polycistronic Mrna ◽  
Citrate Transporter ◽  
Citrate Lyase

ABSTRACT Although Lactococcus is one of the most extensively studied lactic acid bacteria and is the paradigm for biochemical studies of citrate metabolism, little information is available on the regulation of the citrate lyase complex. In order to fill this gap, we characterized the genes encoding the subunits of the citrate lyase of Lactococcus lactis CRL264, which are located on an 11.4-kb chromosomal DNA region. Nucleotide sequence analysis revealed a cluster of eight genes in a new type of genetic organization. The citM-citCDEFXG operon (cit operon) is transcribed as a single polycistronic mRNA of 8.6 kb. This operon carries a gene encoding a malic enzyme (CitM, a putative oxaloacetate decarboxylase), the structural genes coding for the citrate lyase subunits (citD, citE, and citF), and the accessory genes required for the synthesis of an active citrate lyase complex (citC, citX, and citG). We have found that the cit operon is induced by natural acidification of the medium during cell growth or by a shift to media buffered at acidic pHs. Between the citM and citC genes is a divergent open reading frame whose expression was also increased at acidic pH, which was designated citI. This inducible response to acid stress takes place at the transcriptional level and correlates with increased activity of citrate lyase. It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells (more) resistant to the inhibitory effects of the fermentation product (lactate) that accumulates under these conditions.

scholarly journals Genetic Characterization and Physiological Role of Endopeptidase O from Lactobacillus helveticus CNRZ32

1998 ◽  
Vol 64 (9) ◽  
pp. 3411-3415 ◽  
Author(s):  
Yo-Shen Chen ◽  
James L. Steele
Keyword(s):  
Genomic Library ◽  
Skim Milk ◽  
Physiological Role ◽  
Gene Replacement ◽  
Lactobacillus Helveticus ◽  
Nucleotide Sequence Analysis ◽  
Northern Hybridization ◽  
Detectable Effect ◽  
Reading Frame ◽  
Zinc Protease

ABSTRACT A previously identified insert expressing an endopeptidase from aLactobacillus helveticus CNRZ32 genomic library was characterized. Nucleotide sequence analysis revealed an open reading frame of 1,941 bp encoding a putative protein of 71.2 kDa which contained a zinc-protease motif. Protein homology searches revealed that this enzyme has 40% similarity with endopeptidase O (PepO) fromLactococcus lactis P8-2-47. Northern hybridization revealed that pepO is monocistronic and is expressed throughout the growth phase. CNRZ32 derivatives lacking PepO activity were constructed via gene replacement. Enzyme assays revealed that the PepO mutant had significantly reduced endopeptidase activity when compared to CNRZ32 with two of the three substrates examined. Growth studies indicated that PepO has no detectable effect on growth rate or acid production by Lactobacillus helveticusCNRZ32 in amino acid defined or skim milk medium.

scholarly journals Cloning and expression of human liver dehydroepiandrosterone sulphotransferase

1993 ◽  
Vol 289 (1) ◽  
pp. 233-240 ◽  
Author(s):  
K A Comer ◽  
J L Falany ◽  
C N Falany
Keyword(s):  
Molecular Mass ◽  
Bile Acids ◽  
Blot Analysis ◽  
Human Liver ◽  
Molecular Size ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Acid Protein ◽  
Liver Cdna Library

Dehydroepiandrosterone sulphotransferase (DHEA-ST) catalyses the 3′-phosphoadenosine 5′-phosphosulphate-dependent sulphation of a wide variety of steroids in human liver and adrenal tissue and is responsible for most, if not all, of the sulphation of bile acids in human liver. This report describes the isolation, characterization and expression of a cDNA which encodes human liver DHEA-ST. The DHEA-ST cDNA, designated DHEA-ST8, was isolated from a Uni-Zap XR human liver cDNA library and is composed of 1060 bp and contains an open reading frame encoding a 285-amino-acid protein with a molecular mass of approx. 33765 Da. Translation of DHEA-ST8 in vitro generated a protein identical in molecular size with that of DHEA-ST. Expression of DHEA-ST8 in COS-7 cells produces an active DHEA-ST protein which is capable of sulphating DHEA, has the same molecular mass as human liver DHEA-ST and is recognized by rabbit anti-(human liver DHEA-ST) antibodies. Northern-blot analysis of human liver RNA detects the presence of three different size transcripts; however, Southern-blot analysis of human DNA suggests that only one gene may be present in the genome. These results describe the cloning of a human ST which has an important role in the sulphation of steroids and bile acids in human liver and adrenals.

scholarly journals Flagellar Phase Variation of Salmonella enterica Serovar Typhimurium Contributes to Virulence in the Murine Typhoid Infection Model but Does Not InfluenceSalmonella-Induced Enteropathogenesis

2001 ◽  
Vol 69 (5) ◽  
pp. 3021-3030 ◽  
Author(s):  
Jack S. Ikeda ◽  
Clare K. Schmitt ◽  
Stephen C. Darnell ◽  
Patricia R. Watson ◽  
Jennifer Bispham ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Salmonella Enterica ◽  
Phase Variation ◽  
Selective Advantage ◽  
Peritoneal Macrophages ◽  
Infection Model ◽  
Wild Type ◽  
Two Phase ◽  
Serovar Typhimurium

ABSTRACT Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC+phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.

scholarly journals Evaluation of the Role of the Bvg Intermediate Phase in Bordetella pertussis during Experimental Respiratory Infection

2005 ◽  
Vol 73 (2) ◽  
pp. 748-760 ◽  
Author(s):  
Nuria Vergara-Irigaray ◽  
Alberto Chávarri-Martínez ◽  
Juan Rodríguez-Cuesta ◽  
Jeff F. Miller ◽  
Peggy A. Cotter ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Bordetella Pertussis ◽  
Wild Type Strain ◽  
Intermediate Phase ◽  
Transcriptional Level ◽  
Wild Type ◽  
Environmental Signals ◽  
Time Point

ABSTRACT The BvgAS system of Bordetella pertussis was traditionally considered to mediate a transition between two phenotypic phases (Bvg+ and Bvg−) in response to environmental signals. We characterized a third state, the intermediate (Bvgi) phase, which can be induced by introducing a 1-bp substitution into bvgS (the bvgS-I1 mutation) or by growing B. pertussis under conditions intermediate between those leading to the Bvg+ and Bvg− phases. Like B. bronchiseptica, B. pertussis displays in its Bvgi phase a characteristic colony morphology and hemolytic activity and expresses a Bvgi-phase-specific polypeptide called BipA, whose synthesis is regulated by bvgAS at the transcriptional level. Based on our results, we hypothesize that the Bvgi phase of B. pertussis may be involved in facilitating transmission between hosts. Thus, a B. pertussis mutant carrying the bvgS-I1 mutation (GMT1i) persisted at wild-type levels only in the upper murine respiratory tract. Interestingly, a bipA deletion derivative of GMT1i displayed a reduced ability to colonize the nasal cavity of mice compared with GMT1i. However, in experimental mixed infections GMT1i expressing the Bvgi phase could establish an initial colonization in the nose and trachea of mice as efficiently as GMT1, but the wild-type strain outcompeted GMT1i at a later time point at all sites of the respiratory tract, suggesting that the Bvgi phase does not serve as a phenotypic phase specialized in colonization. Finally, even though B. pertussis expresses in vitro the Bvgi phase at the human nasal temperature, anti-BipA antibodies were undetectable in a large collection of sera from pertussis patients.

scholarly journals Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

2013 ◽  
Vol 81 (4) ◽  
pp. 1121-1128 ◽  
Author(s):  
Cristine G. Campos ◽  
Luke Borst ◽  
Peggy A. Cotter
Keyword(s):  
Burkholderia Pseudomallei ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Lung Epithelial Cells ◽  
Wild Type ◽  
Passenger Domain ◽  
Content Type ◽  
Reading Frame ◽  
Type V

ABSTRACTBurkholderia pseudomalleiis a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) inB. pseudomalleistrain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3′ tobcaAis Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations ofbcaAandbcaBwere constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaAand Bp340ΔbcaBmutants to wild-typeB. pseudomalleiin vitrodemonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

scholarly journals Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori

2007 ◽  
Vol 189 (24) ◽  
pp. 8914-8921 ◽  
Author(s):  
Anna Skoglund ◽  
Britta Björkholm ◽  
Christina Nilsson ◽  
Anders F. Andersson ◽  
Cecilia Jernberg ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Restriction Enzyme ◽  
Dna Methyltransferase ◽  
Insertional Mutagenesis ◽  
Phase Variation ◽  
Restriction Enzyme Digestion ◽  
Wild Type ◽  
Gene Mutant ◽  
H Pylori

ABSTRACT A large number of genes encoding restriction-modification (R-M) systems are found in the genome of the human pathogen Helicobacter pylori. R-M genes comprise approximately 10% of the strain-specific genes, but the relevance of having such an abundance of these genes is not clear. The type II methyltransferase (MTase) M.HpyAIV, which recognizes GANTC sites, was present in 60% of the H. pylori strains analyzed, whereof 69% were resistant to restriction enzyme digestion, which indicated the presence of an active MTase. H. pylori strains with an inactive M.HpyAIV phenotype contained deletions in regions of homopolymers within the gene, which resulted in premature translational stops, suggesting that M.HpyAIV may be subjected to phase variation by a slipped-strand mechanism. An M.HpyAIV gene mutant was constructed by insertional mutagenesis, and this mutant showed the same viability and ability to induce interleukin-8 in epithelial cells as the wild type in vitro but had, as expected, lost the ability to protect its self-DNA from digestion by a cognate restriction enzyme. The M.HpyAIV from H. pylori strain 26695 was overexpressed in Escherichia coli, and the protein was purified and was able to bind to DNA and protect GANTC sites from digestion in vitro. A bioinformatic analysis of the number of GANTC sites located in predicted regulatory regions of H. pylori strains 26695 and J99 resulted in a number of candidate genes. katA, a selected candidate gene, was further analyzed by quantitative real-time reverse transcription-PCR and shown to be significantly down-regulated in the M.HpyAIV gene mutant compared to the wild-type strain. This demonstrates the influence of M.HpyAIV methylation in gene expression.

scholarly journals A C-terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein

2001 ◽  
Vol 1 (3) ◽  
pp. 122-128 ◽  
Author(s):  
Li Xiaofang ◽  
Mohammad Zafrullah ◽  
Faizan Ahmad ◽  
Shahid Jameel
Keyword(s):  
Amino Acids ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Reading Frame ◽  
Orf2 Protein ◽  
Acute Form ◽  
Open Reading Frame 2

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressedin vitroor in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms. While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585–610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585–610 of the ORF2 protein might be critical for capsid biogenesis.

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